Cancer of the Nervous System 9781451165463, 9780781737319

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Cancer of the Nervous System

Cancer of the Nervous System SeCoNd editioN

Peter M. Black, M.d., Ph.d. Frances d. ingraham Professor of Neurosurgery Harvard Medical School Chairman department of Neurosurgery Brigham and Women’s Hospital Boston, MA

Jay S. Loeffler, M.d. Herman and Joan Suit Professor of Radiation oncology Harvard Medical School Chairman department of Radiation oncology Massachusetts General Hospital Boston, MA

Acquisitions editor: Jonathan Pine developmental editor: Louise Bierig Marketing Manager: Adam Glazer Supervising Production editor: erica Woods tucker Production editor: Print Matters, inc. Manufacturing Manager: Ben Rivera Compositor: Compset, inc. Printer: Maple Press © 2005 by LiPPiNCott WiLLiAMS & WiLKiNS 530 Walnut Street Philadelphia, PA 19106 USA LWW.com All rights reserved. this book is protected by copyright. No part of this book may be reproduced in any form or by any means, including photocopying, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. 1st edition Published 1997 by Blackwell Science, inc. Printed in the USA Library of Congress Cataloging-in-Publication data Cancer of the nervous system/[edited by] Peter M. Black, Jay S. Loeffler.—2nd ed. p. ; cm. includes bibliographical references and index. iSBN 0-7817-3731-1 (HC) 1. Central nervous system—Cancer. i. Black, Peter McL. ii. Loeffler, Jay S. [dNLM: 1. Central Nervous System Neoplasms. WL 358 C2148 2004] RC280.N43C37 2004 616.99’48—dc22 2004048611 Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of this information in a particular situation remains the professional responsibility of the practitioner. the authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. this is particularly important when the recommended agent is a new or infrequently employed drug. Some drugs and medical devices presented in this publication have Food and drug Administration (FdA) clearance for limited use in restricted research settings. it is the responsibility of the health care provider to ascertain the

CoNteNtS

FdA status of each drug or device planned for use in their clinical practice. 10  9  8  7  6 5  4  3  2  1

Contributors  ix Preface  xvii

i. ePideMioLoGy, PAtHoLoGy, ANd iMAGiNG oF BRAiN tUMoRS  1 1

epidemiology of Brain tumors  1 tracy t. Batchelor, Molly V. dorfman, and david J. Hunter

2

Neuropathology of Central Nervous System tumors  15 Umberto de Girolami and thomas W. Smith

3

Clinical Presentation of Patients with Brain tumors 47 Alison M. o’Neill

4

imaging in Central Nervous System tumors 53 John W. Henson and R. Giliberto González

5

Single Photo emission Computed topography in the evaluation of Adult Primary Brain tumors  89 Richard B. Schwartz

6

Positron emission tomography for the Management of Brain tumors  95 Mark Levivier, david Wikler, olivier de Witte, Nicolas Massager, Serge Goldman, and Jacques Brotchi

7

Magnetic Resonance Spectroscopy and its Use in Brain tumor diagnosis and therapy 109 Andrea Pirzkall

8

Functional Magnetic Resonance imaging for Neurosurgery 123 Steven Stufflebeam and Bruce L. Rosen

ii. PRiNCiPLeS oF BRAiN tUMoR MANAGeMeNt ANd tHeRAPy  135 9

Principles of Pediatric Brain tumor Surgery 135 Liliana C. Goumnerova

10 Principles of Brain tumor Surgery in Adults 141 Raymond e. Sawaya and Jeffrey S. Weinberg

11

image-guided Surgery 155

vi

Contents

Mark A. Spicer and Michael L. J. Apuzzo

12 Principles of Brain tumor Radiotherapy 167 Annie W. Chan

13 Biologic-based Brain tumor therapy 179 Howard A. Fine

14 Chemotherapy for Central Nervous System tumors 193 Ravi d. Rao and Jan C. Buckner

15 immunotherapy for Malignant Gliomas 209 John S. yu, Moneeb ehtesham, and Keith L. Black

16 Gene therapy 225 Manish Aghi and ennio Antonio Chiocca

17 Management of Non-Neoplastic Problems in Brain tumor Patients 243 Patrick y. Wen, Lisa M. Macdonald, and debra Conrad Gigas

18 Psychiatric Management of Brain tumor Patients 255 Malcolm P. Rogers and Alejandro y. Mendoza

Schwannomas 383 John C. Flickinger, douglas Kondziolka, and L. dade Lunsford

28 Fractionated Stereotactic Radiotherapy in the treatment of Acoustic Neuromas 389 Stephanie e. Weiss, Lawrence Kleinberg, Moody Wharham, and daniele Rigamonti

29 Vestibular Schwannomas in the Setting of Neurofibromatosis type 2 403 Mia MacCollin and Anat Stemmer-Rachamimov

30 Medical Management of Pituitary Adenomas 411 erik Alexander and Anne Klibanski

31 Surgery for Pituitary Adenoma 427 K. Michael Webb, Jeffrey J. Laurent, edward R. Laws, Jr., and John A. Jane, Jr.

32 Fractionated Conformal Radiotherapy in the treatment of Pituitary tumors 439 John M. Buatti, Sanford L. Meeks, and timothy R. Ryken

33 Craniopharyngiomas 445 A. Leland Albright

iii. SPeCiFiC tUMoR tyPeS ANd tHeiR tReAtMeNt  279 19 Meningiomas: Natural History, diagnosis, and imaging 279 Ross A. Goldstein, Michelle A. Jorden, and Griffith R. Harsh iV

20 Meningioma: Surgery 315 ian F. dunn and Peter McL. Black

21 Meningioma: Skull Base Surgery 329 Badih Adada and ossama Al-Mefty

22 Radiosurgery in the treatment of Meningiomas 343 Michael W. Mcdermott, Alfredo Quinones-Hinojosa, and david A. Larson

23 Radiotherapy in the treatment of Meningiomas 357 dennis C. Shrieve and Jay S. Loeffler

24 Medical Management of Meningiomas 363 Uwe Schlegel

25 Vestibular Schwannoma: overview of Natural History and diagnosis 367 Robert G. ojemann

26 Surgery in the treatment of Vestibular Schwannoma 371 Marc S. Schwartz and William e. Hitselberger

27 Radiosurgery in the treatment of Vestibular

34 Nerve Sheath tumors of the Spine 453 Paul Klimo, Jr., Meic H. Schmidt, and Richard G. Fessler

35 Low-Grade Supratentorial Glioma 465 Glenn S. Bauman and edward G. Shaw

36 Pediatric Low-Grade Gliomas of the Chiasm and Cerebellum 491 Henry e. Aryan, derek A. Bruce, Hal S. Meltzer, and Michael L. Levy

37 Malignant Gliomas 497 ian F. Parney, david A. Larson, eric Christopher Burton, and Susan M. Chang

38 Sarcomas 521 Fred G. Barker ii

39 ependymomas 533 Joel W. Goldwein and Leslie N. Sutton

40 Medulloblastoma and other Primitive Neuroectodermal tumors 543 Sridharan Gururangan and Henry S. Friedman

41 Hemangioblastoma 563 Mika Niemelä

42 tumors of the Pineal Region 573 Jeffrey N. Bruce, Michael Richard Fetell, and Casilda M. Balmaceda

43 Central Nervous System Lymphoma in AidS and Non-AidS Patients 589 Joachim M. Baehring and Fred H. Hochberg

Contents

44 Gliomas of the Spine 605 Khalid M. Abbed and Lawrence F. Borges

45 Vertebral Body tumors 615 eric Woodard

46 Rare Malignant Brain tumors 627 Michael Schulder and Pallavi S. Uthukuri

47 Surgery for Brain Metastases 645 tze-Ching tan and Peter M. Black

vii

48 Radiotherapy and Radiosurgery for Brain Metastases 657 Minesh Mehta and Rakesh R. Patel

49 epidural disease 673 thomas N. Byrne

50 Neoplastic Meningitis 683 eric t. Wong

51 Paraneoplastic disorders of the Nervous System 691 edward J. dropcho

iV. BASiC ANd APPLied BioLoGy oF BRAiN tUMoRS  715 52 Hereditary Brain tumor Syndromes 715 Christopher d. turner and Christine A. Chordas

53 Growth Factors in Brain tumors 733 Johann C. Micallef, Gelareh Zadeh, and Abhijit Guha

54 Angiogenesis in Brain tumors 761 Kewal K. Jain

55 invasion in Brain tumors 779 Katharine J. drummond

56 Molecular Genetics of Brain tumors 793 Jianhong Zhu, Helen Li Zhang, and eric t. Wong

57 Predicting Medulloblastoma outcome 821 Stephanie Greene and Scott L. Pomeroy

58 Predictive Markers in Human Gliomas 827 Arnab Chakravarti

59 Molecular Neuropathology 837 Catherine L. Nutt and david N. Louis

60 Molecular Markers of Metastatic disease 849 George Perides and Julian K. Wu

61 Stem Cell Biology and Brain tumors 855 Kadir erkmen and Peter McL. Black

62 issues Regarding death and dying in Brain tumor Patients 859 Malcolm P. Rogers, Bernadino Vaccaro, and Peter McL. Black

index  869

CoNtRiBUtoRS

Khalid M. Abbed Resident in Neurosurgery Massachusetts General Hospital Boston, MA Badih Adada, M.d. department of Neurosurgery University of Arkansas for Medical Sciences Little Rock, AR Manish Aghi, M.d., Ph.d. department of Neurosurgery Massachusetts General Hospital Boston, MA A. Leland Albright, M.d. Children’s Hospital, Pittsburgh Pittsburgh, PA erik Alexander, M.d. department of endocrinology Brigham and Women’s Hospital Boston, MA ossama Al-Mefty, M.d. department of Neurosurgery University of Arkansas for Medical Sciences Little Rock, AR Michael L J. Apuzzo, M.d. department of Neurological Surgery University of Southern California School of Medicine Los Angeles, CA Henry e. Aryan, M.d. Neurosurgeon department of Neurosurgery University of California—San diego School of Medicine University of California—San diego Medical Center San diego, CA Joachim M. Baehring, M.d. department of Neurosurgery yale University School of Medicine New Haven, Ct

Casilda M. Balmaceda, M.d. department of Neurology College of Physicians and Surgeons of Columbia University New york, Ny Fred G. Barker ii, M.d. department of Neurosurgery Massachusetts General Hospital Boston, MA tracy t. Batchelor, M.d., M.P.H. executive director Brain tumor Center Massachusetts General Hospital Boston, MA Glenn S. Bauman, M.d. Head department of Radiation oncology University of Western ontario London Health Services Centre London, oN Keith L. Black, M.d. director Maxine dunitz Neurosurgical institute Cedars-Sinai Medical Center Los Angeles, CA Peter M. Black, M.d., Ph.d. Frances d. ingraham Professor of Neurosurgery Harvard Medical School Chairman department of Neurosurgery Brigham and Women’s Hospital Boston, MA Lawrence F. Borges, M.d. department of Neurosurgery Massachusetts General Hospital Boston, MA Jacques Brotchi, M.d. department of Neurosurgery erasme Hospital Brussels, Belgium

x

Contributors

derek A. Bruce, M.d. Pediatric Neurosurgeon department of Pediatric Neurosurgery University of California—San diego School of Medicine Children’s Hospital of San diego San diego, CA Jeffrey N. Bruce, M.d. Attending Neurosurgeon department of Neurosurgery Columbia University New york Presbyterian Hospital—Columbia University Medical Center New york, Ny John M. Buatti, M.d. Chairman department of Radiation oncology University of iowa Hospital and Clinics iowa City, iA Jan C. Buckner, M.d. the Mayo Clinic Rochester, MN eric Christopher Burton, M.d. department of Neurological Surgery/Neuro oncology University of California—San Francisco Medical Center San Francisco, CA thomas N. Byrne, M.d. department of Neurology, Neurosurgery and internal Medicine yale University School of Medicine yale-New Haven Hospital New Haven, Ct Arnab Chakravarti, M.d. director, Laboratory of Molecular Radiation oncology department of Radiation oncology Harvard Medical School Massachusetts General Hospital Boston, MA Annie W. Chan, M.d. department of Radiation oncology Harvard Medical School Massachusetts General Hospital Boston, MA Susan M. Chang, M.d. Vice Chair department of Neurological Surgery University of California at San Francisco San Francisco, CA

ennio Antonio Chiocca, M.d., Ph.d. Brain tumor Center Massachusetts General Hospital Boston, MA Christine Chordas, PNP, MSN, RN Nurse Practitioner department of Pediatric oncology dana-Farber Cancer institute Boston, MA Umberto de Girolami, M.d. department of Pathology Children’s Hospital Boston, MA olivier de Witte department of Neurosurgery Université Libre de Bruxelles erasme Hospital Brussels, Belgium Molly V. dorfman, B.A. department of Neurology Harvard Medical School Massachusetts General Hospital Boston, MA edward J. dropcho, M.d. department of Neurology indiana University Medical Center indianapolis, iN Katharine J. drummond, MBBS, FRACS Neurosurgeon department of Neurosurgery University of Melbourne the Royal Melbourne Hospital Melbourne, Australia ian F. dunn, M.d. department of Neurosurgery Brigham and Women’s Hospital Boston, MA Moneeb ehtesham, M.d. department of Neurological Surgery Vanderbilt University School of Medicine Vanderbilt University Medical Center Nashville, tN Kadir erkmen, M.d. department of Neurosurgery Brigham and Women’s Hospital Boston, MA

Contributors

Richard G. Fessler, M.d., Ph.d. Chief department of Neurosurgery University of Chicago Chicago, iL Michael Richard Fetell, M.d. Vice President of oncology and CNS ivax Research Miami, FL Howard A. Fine, M.d. director, Neurooncology Branch National institute of Health/National Cancer institute Bethesda, Md John C. Flickinger, M.d. Joint Radiation oncology Center Presbyterian University Hospital Pittsburgh, PA Henry S. Friedman, M.d. Co-director department of Surgery duke University Medical Center the Brain tumor Center at duke durham, NC debra Conrad Gigas, R.N., o.C.N. Program Nurse Center for Neuro-oncology dana-Farber Cancer institute Boston, MA Serge Goldman Pet/Biomedical Cyclotron Unit Université Libre de Bruxelles erasme Hospital Brussels, Belgium Ross A. Goldstein, M.d. department of Radiology Section of Neuroradiology Stanford University School of Medicine Stanford, CA

Liliana C. Goumnerova, M.d. department of Neurosurgery Children’s Hospital Boston, MA Stephanie Greene, M.d. department of Neurosurgery Children’s Hospital Boston, MA Abhijit Guha, M.d., M.Sc., FRCSC Co-director & Senior Scientist and Staff Neurosurgeon division of Neurosurgery the Arthur and Sonia Labatt Brain tumour Research Centre the Hospital for Sick Children toronto Western Hospital toronto, oN Sridharan Gururangan, MRCP (UK) director of Pediatric Clinical Services department of Pediatrics duke University Medical Center department of Surgery the Brain tumor Center at duke durham, NC Griffith R. Harsh, iV, M.d. department of Neurosurgery Stanford University School of Medicine Stanford, CA John W. Henson, M.d. Brain tumor Center Massachusetts General Hospital Boston, MA William e. Hitselberger, M.d. Neurosurgeon House ear Clinic Los Angeles, CA

Joel W. Goldwein, M.d. University of Pennsylvania Medical Center Philadelphia, PA

Fred H. Hochberg, M.d. department of Neurology Brain tumor Center Massachusetts General Hospital Boston, MA

R. Giliberto González, M.d., Ph.d. department of Neuroradiology Massachusetts General Hospital Boston, MA

david J. Hunter, M.d., Sc.d. department of epidemiology Harvard School of Public Health Boston, MA

xi

xii

Contributors

Kewal K. Jain, M.d., F.R.C.S.C., F.R.A.C.S., F.i.C.S., F.F.P.M. Jain Pharmabiotech institute Basel, Switzerland John A. Jane, Jr., M.d. department of Neurosurgery University of Virginia School of Medicine Charlottesville, VA Michelle A. Jorden, M.d. department of Pathology Stanford University School of Medicine Stanford, CA Lawrence Kleinberg, M.d. department of Radiation oncology Johns Hopkins Sidney Kimmel Cancer Center Baltimore, Md Anne Klibanski, M.d. Neuroendocrine Unit Harvard Medical School Massachusetts General Hospital Boston, MA Paul Klimo, Jr., M.d. department of Neurosurgery University of Utah Salt Lake City, Ut

erasme Hospital Brussels, Belgium Michael L. Levy, M.d., Ph.d. Chief, Pediatric Neurosurgeon department of Pediatric Neurosurgery Children’s Hospital of San diego San diego, CA Jay S. Loeffler, M.d. Herman and Joan Suit Professor of Radiation oncology Harvard Medical School Chairman department of Radiation oncology Massachusetts General Hospital Boston, MA david N. Louis, M.d. Associate Chief of Pathology department of Pathology Harvard Medical School Massachusetts General Hospital Boston, MA L. dade Lunsford, M.d. departments of Radiation oncology, Neurological Surgery, and Radiology the Center for image-Guided Neurosurgery University of Pittsburgh School of Medicine Pittsburgh, PA

douglas Kondziolka, M.d. departments of Radiation oncology, Neurological Surgery University of Pittsburgh School of Medicine Pittsburgh, PA

Mia MacCollin, M.d. department of Neurology Massachusetts General Hospital Boston, MA

david A. Larson, M.d., Ph.d. department of Neurological Surgery University of California at San Francisco Program Member, UCSF Comprehensive Cancer Center San Francisco, CA

Lisa M. Macdonald, APRN, BC, oCN Nurse Practitioner Center for Neuro-oncology dana-Farber Cancer institute Boston, MA

Jeffrey J. Laurent, M.d. department of Neurosurgery University of Virginia School of Medicine Charlottesville, VA edward R. Laws, Jr., M.d., FACS department of Neurosurgery University of Virginia School of Medicine Charlottesville, VA

Nicolas Massager, M.d. department of Neurosurgery Gamma Knife Center Université Libre de Bruxelles erasme Hospital Brussels, Belgium

Marc Levivier, M.d., Ph.d. Associate editor Radiosurgery department of Neurosurgery and Gamma Knife Center Université Libre de Bruxelles

Michael W. Mcdermott, M.d., FRCSC director, Patient Care Services department of Neurosurgery University of California—San Francisco Moffitt Hospital San Francisco, CA

Contributors

Sanford L. Meeks, Ph.d. director of Physics department of Radiation oncology University of iowa Hospitals and Clinics iowa City, iA Minesh Mehta, M.d. Chairman department of Human oncology University of Wisconsin School of Medicine Madison, Wi Hal S. Meltzer, M.d. Pediatric Neurosurgeon department of Pediatric Neurosurgery Children’s Hospital of San diego San diego, CA Alejandro y. Mendoza, M.d. North easton, MA Johann C. Micalleff, BScH department of Medical Biophysics University of toronto the Arthur and Sonia Labatt Brain tumour Research Centre the Hospital for Sick Children toronto, oN Mika Niemelä, M.d., Ph.d. Attending Neurosurgeon department of Neurosurgery Helsinki University Central Hospital Helsinki, Finland Catherine L. Nutt, Ph.d. department of Pathology Harvard Medical School Molecular Pathology Unit Massachusetts General Hospital—east Charlestown, MA Robert G. ojemann, M.d. department of Neurosurgery Massachusetts General Hospital Boston, MA Alison M. o’Neill, M.d. Massachusetts General Hospital Boston, MA ian F. Parney, M.d., Ph.d. division of Neurosurgery, department of Clinical Neurosciences University of Calgary Calgary, AB

Rakesh R. Patel, M.d. department of Human oncology University of Wisconsin School of Medicine Madison, Wi George Perides, Ph.d. Beth israel deaconess Medical Center Boston, MA Andrea Pirzkall department of Radiation oncology University of California—San Francisco School of Medicine San Francisco, CA Scott L. Pomeroy, M.d., Ph.d. department of Neurology Children’s Hospital Boston, MA Alfredo Quinones-Hinojosa, M.d. Neurosurgical Resident department of Neurological Surgery University of California—San Francisco School of Medicine San Francisco, CA Ravi d. Rao, M.d. department of oncology the Mayo Clinic Rochester, MN daniele Rigamonti, M.d. department of Neurosurgery Johns Hopkins Sidney Kimmel Cancer Center Baltimore, Md Malcolm P. Rogers, M.d. department of Psychiatry Brigham and Women’s Hospital Boston, MA Bruce L. Rosen, M.d., Ph.d. Massachusetts General Hospital—east Charlestown, MA timothy R. Ryken, M.d. department of Neurosurgery University of iowa Hospitals and Clinics iowa City, iA Raymond e. Sawaya, M.d. Md Anderson Cancer Center Houston, tX

xiii

xiv

Contributors

Uwe Schlegel, M.d. Vice-Chairman department of Neurology University Hospital Bonn Bonn, Germany Michael Schulder, M.d. Vice Chairman Attending Neurosurgery department of Neurosurgery New Jersey Medical School University Hospital Newark, NJ Meic H. Schmidt, M.d. department of Neurosurgery University of Utah Salt Lake City, Ut Marc S. Schwartz, M.d. department of Neurosurgery House ear Clinic Los Angeles, CA Richard B. Schwartz, M.d. department of Radiology Boston, MA edward G. Shaw, M.d. Chairman department of Radiation oncology Wake Forest University Baptist Medical Center Winston-Salem, NC dennis C. Shrieve, M.d., Ph.d. department of Radiation oncology Professor and Chairman University of Utah School of Medicine Salt Lake City, Ut thomas W. Smith, M.d. department of Pathology University of Massachusetts Medical School Worcester, MA Mark A. Spicer, Ph.d., M.d. department of Neurological Surgery University of Southern California School of Medicine Los Angeles, CA Anat Stemmer-Rachamimov, M.d. Molecular Neuro-oncology Laboratory Charlestown, MA Steven Stufflebeam, M.d. Martinos Center for Biomedical imaging Charlestown, MA

Leslie N. Sutton, M.d. Chief, Neurosurgical Services the Children’s Hospital of Philadelphia Philadelphia, PA tze-Ching tan, M.d., FRCS Senior Medical officer department of Neurosurgery Queen elizabeth Hospital Kowloon, Hong Kong Christopher d. turner, M.d. director, Pediatric Neuro-oncology outcomes Research department of Pediatrics department of Pediatric oncology Harvard Medical School dana-Farber Cancer institute Boston, MA Bernardino Vaccaro, M.d. department of Surgery Harvard Medical School Brigham and Women’s Hospital Boston, MA K. Michael Webb, M.d. department of Neurosurgery University of Virginia School of Medicine Charlottesville, VA Jeffrey S. Weinberg, M.d. department of Neurosurgery Houston, tX Stephanie e. Weiss, M.d. department of Radiation oncology John Hopkins Sidney Kimmel Cancer Center Baltimore, Md Patrick y. Wen, M.d. Clinical director, Center for Neuro-oncology director, division of Neuro-oncology department of Neurology Harvard Medical School dana-Farber Cancer institute Brigham and Women’s Hospital Boston, MA Moody Wharam, M.d. department of Radiation oncology Johns Hopkins Sidney Kimmel Cancer Center Baltimore, Md david Wikler, M.d. department of Neurosurgery

Contributors

Pet/Biomedical Cyclotron UnitUniversité Libre de Bruxelles erasme Hospital Brussels, Belgiumeric Woodard, M.d. Spine Center Brigham and Woman’s Hospital Boston, MA eric t. Wong, M.d. department of Neurology Harvard Medical School Brain tumor Center Beth israel deaconess Medical Center Boston, MA Julian K. Wu, M.d. Chief of Neurosurgery department of Surgery division of Neurosurgery Harvard Medical School Beth israel deaconess Medical Center Boston, MA John S. yu, M.d. Head of Surgical Neuro-oncology

Co-director, Comprehensive Brain tumor Program Maxine dunitz Neurosurgical institute Cedars-Sinai Medical Center Los Angeles, CA Gelareh Zadeh, M.d., Ph.d. the Arthur and Sonia Labatt Brain tumor Research Centre the Hospital for Sick Children toronto, oN Helen Li Zhang, M.d. Staff Scientist, Harvard Gene therapy initiative department of Genetics Harvard Medical School Boston, MA Jianhong Zhu, M.d., Ph.d. deputy director department of Neurosurgery National Key Laboratory for Medical Neurobiology Fudan University Shanghai Medical College Fudan University Huashan Hospital Shanghai, China

xv

PReFACe

B

enign and malignant tumors of the nervous system are relatively uncommon. But they are important both for the disability they produce and for the understanding of the nervous system and cancer biology they engender. they are the second most common cause of cancer death in children and a major cause of cancer disability in adults. the disabling effects they can produce are out of proportion to their incidence. the first edition of this book was one of the first texts to provide a comprehensive description of central nervous system tumors and their treatment. Since the publication of the first edition, there has been growing scientific and clinical information about the molecular genetics, imaging, and treatment of these tumors.

the current edition of our book provides a comprehensive approach to understanding these tumors and the treatment of our patients. Chapters on specific tumor histologies are written by multidisciplinary teams of physicians who have significant experience caring for patients with these diagnoses. We believe this approach will provide a comprehensive and coordinated reference textbook for healthcare providers who treat and care for patients with tumors of the nervous system. We hope we have been successful in our effort to bring together the art and science of human brain tumor management. We further hope this edition will continue to foster clinical and basic investigation of these tumors and enhance the care of our patients P.M.B. J.S.L.

Epidemiology, Pathology, and Imaging of Brain Tumors C h a p t e r 1

w Epidemiology of Primary Brain Tumors Tracy T. Batchelor, Molly V. Dorfman, David J. Hunter Primary brain tumors are a diverse group of neoplasms arising from different cells of the central nervous system. These tumors are classified by light microscopy, according to predominant cell type, and graded for malignancy based on the presence or absence of standard pathologic features. Gliomas are the most common brain tumors and may arise from astrocytes (astrocytomas), oligodendrocytes (oligodendrogliomas), or ependymal cells (ependymomas), with astrocytomas accounting for approximately 80% of all malignant brain tumors. Despite a large number of epidemiologic studies, the etiology of brain tumors remains unknown, and this type of cancer is associated with a unique set of challenges for observational study designs (Table 1-1). Despite a slow start, observational studies in brain tumors may yet yield important clues in the pathogenesis of this cancer.

CLASSIFICATION Histopathologic The different cellular origins of brain tumors have contributed to the difficulty in achieving a single classification system with wide acceptance. Historical attempts at developing a classification system for brain tumors date back to the 1830s, and the German pathologist Rudolf Virchow introduced the term “glioma” in 1860. Virchow was also the first to attempt a correlation of microscopic to macroscopic features of central nervous system tumors (1). Bailey and Cushing (2) devised the first major classification system for brain tumors in 1926 and proposed that this type of cancer arose from primitive neuroectoderm. Their system consisted of 14 distinct tumor types, each

1

2

Cancer of the Nervous System

w TABLE 1-1

Challenges in Brain Tumor Epidemiology Studies

Uncommon form of cancer Diversity of histologic brain tumor types Lack of uniformly accepted classification system Changing methods of ascertainment (clinical, radiographic, histologic) Variable reliability of exposure assessment (cognitive impairment of subjects, remote exposures)

arising from a cell arrested at a different stage of neuronal development and morphologically different from its normal counterpart. In 1912, Tooth (3) argued that the biologic characteristics of brain tumors were determined by their histologic features. Yet it was not until 1949 that Kernohan and colleagues (4) suggested that different histopathologic appearances do not represent separate tumor types but rather degrees of tumor differentiation. The Ringertz system of 1950 was based on the notion that different brain cells give rise to different histologic types of brain tumors. In addition, Ringertz proposed that astrocytoma consisted of three different grades: astrocytoma, astrocytoma with anaplastic foci, and glioblastoma multiforme (GBM). In 1981, DaumasDuport and Szikla (5) organized a tumor grading system, the St. Anne-Mayo system, based on the absence or presence of four criteria: nuclear atypia, mitoses, endothelial cell proliferation, and necrosis. In 1979, the World Health Organization (WHO) published a classification system that encompassed all central nervous system tumors. The WHO classification system was subsequently revised in 1993 and 2000 (6) and included 10 major categories and 126 subcategories of brain tumors. In this system, the most common type of glioma, astrocytoma, is assigned different grades of malignancy (I–IV) based on the presence or absence of the criteria described above. Malignant astrocytomas (anaplastic astrocytoma [III/IV] and GBM [IV/IV]) are the most common subtypes and account for 80% of all malignant brain tumors. A comparison of the major classification systems currently in use for astrocytoma is seen in Table 1-2.

w TABLE 1-2

Molecular Interest in a genetic classification of gliomas is based on studies demonstrating a predictable set of genetic changes in the progression from low-grade to high-grade gliomas and the observation that certain genetic changes better predict prognosis than histopathologic characteristics for certain types of brain tumors. Formation of brain tumors involves an accumulation of lesions in genes important for the regulation of cell proliferation, differentiation, and death (Table 1-3). As with other types of cancer, both oncogenes and tumor suppressor genes play critical roles in glioma pathogenesis. Oncogenes are dominantly acting genes coding for protein products that accelerate cell growth and proliferation. Typically, genetic alterations of oncogenes involve amplification and activating mutations. Conversely, protein products coded by tumor suppressor genes repress cell growth. These genes are most commonly altered by physical elimination (deletions) or inactivating mutations, leading to apoptosis. Low-grade astrocytoma (WHO grades I/IV and II/IV) are characterized by a high frequency of mutations in the p53 tumor suppressor gene (approximately 50%) and amplification of the oncogene, platelet-derived growth factor (approximately 60%) (7). Progression to anaplastic astrocytoma is associated with loss of heterozygosity (LOH) on chromosome 19q and alterations in the retinoblastoma tumor suppressor gene. Progression to GBM (approximately 80%) correlates with LOH on chromosome 10q and amplification of epidermal growth factor receptor (7). Two studies examining these genetic alterations have suggested possible prognostic significance in GBM. Leenstra et al. reported that GBM patients with LOH on chromosome 10 (60% to 85% of GBMs) and epidermal growth factor receptor amplification (40%) experienced shorter survival than those without such alterations (8,9). Similarly, Lin et al. (10) reported LOH at the PTEN/MMAC1 (25% of cases) locus was more common in high-grade astrocytomas and was predictive of shorter survival for these patients. Another study demonstrated the age-dependent prognostic effects of specific genetic alterations. For example, epidermal growth factor receptor amplification was found to be a negative prognostic

Pathologic Grading Systems for Astrocytomas

World Health Organization

Modified Ringertz

— Astrocytoma (grade II) Anaplastic astrocytoma (grade III)

— Astrocytoma (grade 1) Anaplastic astrocytoma (grade 2)

Glioblastoma multiforme (grade IV)

Glioblastoma multiforme (grade 3)

University of California, San Francisco Mildly anaplastic astrocytoma Moderately anaplastic astrocytoma Highly anaplastic astrocytoma Gemistocytic astrocytoma Glioblastoma multiforme

Adapted from Wen PY, Black PMcL. Brain tumors in adults. Neurol Clin 1995;13:875–900, with permission.

St. Anne-Mayo Astrocytoma grade 1 Astrocytoma grade 2 Astrocytoma grade 3 Astrocytoma grade 4

1. Epidemiology of Primary Brain Tumors

w TABLE 1-3

3

Gene and Chromosomal Alterations in Gliomas

Gene

Gene Function

Chromosome

Comment

p53

Tumor suppressor

17p13.1

MDM2 p15 and p16 CDK4 and CDK6 Retinoblastoma — — Epidermal growth factor receptor Platelet-derived growth factor receptor — —

Tumor suppressor Tumor suppressor Promoter of cell proliferation Tumor suppressor — — Oncogene

12q14.3-q15 9p21 12q13–14 and 7q21–22 13q14 19 22q 7

Oncogene

—

33% of all grades of astrocytomas, 65% of low-grade astrocytomas 10% of glioblastoma multiforme Deleted in 67% of glioma cell lines Amplified in 15% of cases without p15 or p16 mutations 33% of high-grade astrocytoma Frequent in high grade astrocytomas 20–30% of all grades of astrocytomas 33–50% of high grade astrocytomas, always associated with 10 loss Expressed in all grades of astrocytomas

Tumor suppressor —

10q 1p

marker in younger patients, whereas it was associated with a better prognosis in elderly patients (11). Future classification will undoubtedly incorporate many of these genetic markers of prognosis.

DESCRIPTIVE EPIDEMIOLOGY Incidence The traditional source of descriptive data on brain tumors has been the Surveillance, Epidemiology and End Results program sponsored by the National Cancer Institute. This program collects population-based cancer data on approximately 26% of the U.S. population (including 12 state cancer registries) to gauge national trends in cancer incidence and survival (12). However, these data encompass only malignant tumors. In contrast, the Central Brain Tumor Registry of the United States (CBTRUS) includes both benign and malignant brain tumors. The CBTRUS was established in 1992 and is the nation’s largest population-based registry of primary brain tumors, compiling information from 14 state cancer registries (13). Incidence estimates differ according to the inclusion or exclusion of benign brain tumors. Although Surveillance, Epidemiology and End Results program data estimated approximately 17,200 new primary brain tumor cases in the United States for 2002 (14), CBTRUS data estimated approximately 35,519 new cases of both malignant and benign brain tumors for the same year (13). The annual incidence of malignant brain cancer for all races from 1993 to 1997 was 6.8 per 100,000 person-years, compared with 4.2 per 100,000 person-years for primary benign brain tumors (12,13).

80% of all glioblastomas Nearly all oligodendrogliomas; usually expressed with 19q loss

Mortality and Prognostic Factors Although brain tumors account for only 2% of all cancers and are one-fifth as common as breast or lung cancer, these neoplasms result in a disproportionate share of cancer morbidity and mortality. The 5-year survival rates for brain tumors are the sixth lowest among all types of cancer (following pancreas, liver, esophagus, lung, and stomach, respectively) (14). The 5-year survival rates for the most common histologic subtypes, anaplastic astrocytoma and GBM, are 28.2% and 2.9%, respectively. The U.S. mortality rate for malignant brain cancer was 4.7 per 100,000 persons for 1999 and represented a 46% increase in mortality since 1950 (12). There will be approximately 13,100 deaths attributed to malignant brain and nervous system tumors in 2002, and this number has been increasing for the last 30 years (12). Age-specific mortality rates among all races demonstrate gradual increases in mortality with each decade of age until the age of 55, after which there is a dramatic increase in mortality rates from brain tumors. When stratified by race or gender, these trends are present, although whites exhibited greater mortality (5.1 per 100,000 persons) than African Americans (2.8 per 100,000 persons) from 1995 to 1999 (Fig. 1-1). Finally, for all primary brain tumors, there has been a significant rise in 5-year survival rates over the past 30 years (from 22% to 32%) (12). Young age, high Karnofsky performance status, and lower pathologic grade are favorable prognostic factors for these tumors. Less significant predictors of favorable prognosis include long duration of symptoms, absence of mental changes at the time of diagnosis, cerebellar location of tumor, small preoperative tumor size, and completeness of surgical resection (15).

4

Cancer of the Nervous System

FIGURE 1-1. Invasive brain tumor mortality rates for U.S. whites and blacks, stratified by age, 1995–1999. (From Ries LAG, Eisner MP, Kosary CL, et al., eds. SEER cancer statistics review, 1973–1999. Bethesda, MD: National Cancer Institute, 2002, with permission.)

Gender and Race

Temporal Trends

There is a slight male predominance in the incidence of malignant brain tumors (7.7 per 100,000 for men vs. 5.4 per 100,000 for women) (12). However, when all tumor types are examined, the disparity between the sexes is less apparent (11.8 per 100,000 vs. 11.0 per 100,000, respectively) (13). This is partially explained by the predominance of one particular type of benign brain tumor, meningioma, in women (4.24 per 100,000 person-years) compared with men (2.22 per 100,000 person-years) (13). Figure 1-2 compares the incidence rates of malignant brain tumors among white and African American individuals. Whites display a higher incidence of malignant brain tumors compared with African Americans for both genders: White males have an incidence rate of 9.1 per 100,000 person-years compared with African American males at 5.1 per 100,000 person-years, whereas white females have an annual incidence of 6.1 per 100,000 person-years versus an annual incidence rate of 2.8 per 100,000 person-years among African-American females (12). For benign lesions such as meningioma, there is an excess of cases among whites (3.28 per 100,000 personyears) compared with African Americans (3.04 per 100,000 person-years). Comparisons between other racial groups within the United States are difficult because of the small numbers of cases, but malignant brain tumor incidence rates among Asian Americans, Latinos, and Native Americans are lower than those of either whites or African Americans.

Multiple studies have documented rising incidence rates for brain tumors in several industrialized countries. These increases seem to be confined mainly to the elderly population with no clear ethnic, gender, or geographic differences. Overall, there was an increased incidence of 18% from 1973 to 1994 and 80% from 1950 to 1994 (white population only) (12,16,17). However, other population-based data suggest that incidence rates have recently stabilized among all age groups (18). From 1992 to 1999, the incidence of all malignant brain tumors decreased by ⫺0.4%. This reduction in incidence was observed in both males (⫺0.7%) and females (⫺0.1%) and was associated with a reduction in mortality due to brain tumors across all races (⫺0.5%) (14). These data support the concept that the increasing incidence observed before 1992 may have been the result of better ascertainment. The increases in incidence observed in the United States for all age strata and racial groups concur with observations in other parts of the world. Although factors responsible for this increase in incidence remain unclear, there is agreement that at least part of this increase is the result of more complete case ascertainment with improved diagnostic technology, access to health care, and clinical specialization. Some investigators have argued the increase in brain tumor incidence correlates with the introduction of noninvasive diagnostic technology, including computed tomography (CT) in the 1970s and magnetic resonance imaging (MRI) in the 1980s (19). Medicare data

1. Epidemiology of Primary Brain Tumors

5

FIGURE 1-2. Invasive brain tumor incidence rates for U.S. whites and blacks, stratified by age, 1995–1999. (Ries LAG, Eisner MP, Kosary CL, et al., eds. SEER cancer statistics review, 1973–1999. Bethesda, MD: National Cancer Institute, 2002, with permission.)

from 1986 to 1994 suggest that whereas the use of CT and MRI has stabilized among those aged less than 85 years, the use of CT continues to increase among those older than 85 years. This may reflect a more aggressive diagnostic approach to the very elderly and may account for the finding mentioned previously, that although the incidence rates of brain tumors have stabilized for those less than 85 years, the rates have continued to increase among those older than 85 years (18). Other contributing factors include better health care access for the elderly population and increased availability of neurologic physicians. From 1970 to 1987, the number of neurologists in the United States increased 3.6-fold (20). However, other investigators have concluded that improved diagnostic capability does not fully account for the magnitude of the observed increase and that changing environmental exposures may play a role (21).

Geographic Trends and Migrant Studies Increasing incidence rates of brain tumors have been observed in many other countries of the world over the last 30 years. The highest rates are noted in industrialized nations such as the United States, Canada, Australia, and the United Kingdom, whereas developing nations have lower incidence rates. Generally, it appears brain cancer incidence is associated with level of economic development, and concomitant differences in the availability of diagnostic methodology (CT, MRI, neurosurgical technology) may account for some of the observed disparities.

Even within the United States, there is a significant variation in the incidence of brain tumors between the states. Hawaii has the lowest rate for males (3.9 per 100,000 person-years), whereas Mississippi has the lowest rate for females (3.3 per 100,000 person-years) (22). Maine has the highest rate (8.5 per 100,000 person years) for males and North Dakota (7.1 per 100,000 person-years) has the highest rate for females. In contrast to international trends, there does not appear to be a clear relationship between these rates and economic conditions. Despite sparse data from developing countries, it appears most migrants adopt the brain cancer incidence rates of their host country. Migrant studies have typically exhibited elevated incidence rates in the host country compared with the country of origin, with increases in brain cancer incidence and mortality developing among migrants. This suggests environmental factors may influence the development of brain tumors. However, these studies are complicated by disparities in case ascertainment in the countries under study.

Primary Central Nervous System Lymphoma The descriptive epidemiology of primary central nervous system lymphoma (PCNSL), a type of non-Hodgkin lymphoma, deserves special comment. There has been a dramatic increase in the incidence of this brain tumor over the past few decades. This parallels a doubling of the incidence rate of systemic non-Hodgkin lymphoma over the past four decades (20). The peak of this incidence

6

Cancer of the Nervous System

occurred in the early-1990s but has since decreased. One of the primary forces behind this rising incidence is the acquired immunodeficiency syndrome (AIDS) epidemic, as 2% to 6% of persons with AIDS develop PCNSL at some point during their disease course (23). The incidence of AIDS has decreased along with the rate of PCNSL since the beginning of the 1990s. However, AIDS does not fully account for all the increase in the incidence rate. In a population-based study that excluded never-married males, a group previously considered at highest risk for human immunodeficiency virus infection, a nearly threefold increase in the incidence rate of PCNSL from the 1970s to the 1980s was shown (24). This rise in incidence has been reported among both younger and elderly populations with clear predilections toward males (71%) and whites (83%) (25,26). There has been some speculation that this may become the most common primary brain tumor in the future, although such forecasts may be premature as some cancer centers have reported decreases since the early 1990s (27). Clearly, only updated population-based data will be able to address this speculation.

ANALYTIC EPIDEMIOLOGY Methodologic Challenges Because brain tumors are an uncommon and heterogeneous form of cancer, prospective cohort studies have not been feasible and most case-control studies have been characterized by small numbers of cases. Despite these limitations, numerous case-control studies have examined putative risk factors for brain tumors. In addition to the usual problem of selection bias with the case-control study design, recall bias may be an especially important limitation in retrospective case-control studies of this patient population. Persons with brain tumors often have cognitive and language difficulties, and reliable exposure assessment is therefore difficult to achieve. Another shortcoming of observational studies in this patient population has been the tendency to group all brain tumors together as one entity. Although this may identify risk factors common to all brain tumors, it will potentially miss important exposures for specific histopathologic types of brain tumors (28). Although exceptions exist, most exposures analyzed have been characterized by small effects and marked inconsistencies across studies. The major categories of exposures are reviewed below.

Occupational Studies A large number of occupational studies of brain tumor risk have been conducted in cohorts of workers presumed to share common exposures. Most of these studies report standardized incidence or mortality ratios using expected rates

from the general population. Detection bias is a risk in these studies because working adults with health insurance may be more likely to have diagnostic tests, leading to more complete ascertainment of brain tumors in the worker cohort compared with the general population. Hence, overestimation of the true association may occur (21). In general, the data on occupational exposures and brain tumors have been inconsistent. Small numbers of cases, imprecise methods of exposure assessment, potential confounding, and bias have complicated the interpretation of many of these studies. In addition, the lack of biologic measures of exposure and specific chemical identities as well as the tendency to group all types of brain tumors into one category characterize most of these studies and weaken their validity. However, moderate risks cannot be excluded for most of these occupational exposures, and well-designed case-control studies are needed to further address this issue. Observational studies have found workers in “white collar” professions have higher rates of brain tumors. Some of these occupations involve chemical exposures of some type, such as laboratory researchers and health professionals. However, the existence of detection bias must be considered because more members of these professions are more likely to have health insurance and seek medical attention (28–30). McLaughlin et al. (29) analyzed the potential effect of detection bias in a study based in Sweden, where health care is free and available to all citizens. Despite universal access, higher rates of brain tumors were reported among professionals, suggesting that detection bias does not fully account for this observation (29). Electrical workers have been the subject of a number of occupational cohort studies based on possible exposures to electromagnetic fields (EMFs). A review of 17 studies concluded few were of sufficient size and design to accurately ascertain EMF exposure (31). Despite the limitations of these studies, most have failed to detect a significant association between EMF exposure and risk of brain tumors. However, a study of Swiss railway workers found that exposure to low-frequency EMFs among shunting yard engineers may account for the higher mortality of brain tumors in this particular population. Yet, the study failed to yield a dose-response relationship between brain tumor mortality and similar exposure to lowfrequency EMFs (32). At least 10 occupational cohort studies of workers in the oil refinery industry have reported an increased risk of brain tumors. However, a working group from the International Agency for Research on Cancer (IARC) concluded that only 1 of these 10 studies was without significant methodologic or statistical limitations. This single study reported an increased risk of brain tumors only among oil refinery workers who had been employed in the industry for a short duration of time, raising doubts

1. Epidemiology of Primary Brain Tumors

about the validity of the results. No specific chemical exposure has been identified as a risk factor in these studies, and the weight of the evidence to date does not establish employment in this industry as a risk for the development of brain tumors (33). Agricultural workers are exposed to a large number of chemicals in the form of pesticides, herbicides, and fungicides. However, each of these categories consists of many different combinations of chemicals. Similar to other occupational cohort studies, one limitation of studies among these workers is the use of job classification as a proxy for nonspecific chemical exposures. Biologic markers of exposure are lacking in most of these studies. Farming as an occupation and residence on a farm have been associated with an increased risk of brain tumors. An increased risks of brain tumors has been reported among Italian farmers exposed to fungicides and copper sulfate (relative risk [RR] = 2.0; 95% confidence interval [CI], 1.22–3.23) (34), Swedish horticulturists (standardized mortality ratio = 3.2; 95% CI, 1.6–5.7) (35), and Chinese farming women exposed to pesticides (standardized incidence ratio = 3.6; 95% CI, 1.2–8.5) (36). However, no consistent evidence exists to date to indicate any one chemical exposure responsible for the apparent increased risk of brain tumors among agricultural workers. Increased risks have also been reported for workers in the vinyl chloride, petrochemical, and rubber industries. Other studies have found that aircraft pilots, firefighters, welders, glass manufacturers, tile makers, and metal cutters develop brain tumors at a higher rate than the general population. However, these findings have been inconsistent over time (21). Despite a large number of studies in numerous occupational settings, an increased risk of developing a brain tumor based on occupational status has not been established. The studies conducted to date, characterized by significant design flaws, have yielded conflicting results.

Radiation Three different types of radiation have been studied in the etiology of brain tumors: ionizing radiation and two forms of nonionizing radiation, electromagnetic radiation (EMR) and radiofrequency (RF) radiation. Ionizing radiation exposure from radiation therapy has been established as a cause of certain types of brain tumors, especially meningiomas and nerve sheath tumors. Biologic studies have suggested meningeal cells may be more susceptible to radiation than other parts of the body. A cohort of 10,000 children treated with 1.5 Gy of cranial radiation for tinea capitis had elevated risks of nerve sheath tumors (RR = 33.1; 95% CI, 9.4–116.5) and meningiomas (RR = 9.5; 95% CI, 3.5–25.7) but less elevation of risk for malignant gliomas (RR = 2.6; 95% CI, 0.8–8.6) (37). Ionizing radiation has also been associated with an in-

7

creased risk of pituitary adenoma, in another study of patients with tinea capitis who received scalp irradiation at doses ranging from 1 to 2 Gy (mean, 1.4 Gy) (38). Less dramatic but still significant elevations of risk for meningiomas and nerve sheath tumors have been reported in cohorts of individuals treated with variable doses of ionizing radiation for thymic enlargement, enlarged tonsils and adenoids, and thyroid and nasopharyngeal conditions (39–42). In a mutagen sensitivity assay, peripheral blood lymphocytes from glioma subjects were more prone to chromosomal damage when exposed to gamma radiation. In this study, mutagen sensitivity of lymphocytes was associated with an increased risk of glioma (odds ratio = 2.09; 95% CI, 1.43, 3.06) (43). Exposure to diagnostic x-rays is less established as a risk factor for brain tumor development. Dental x-rays have been the subject of at least 10 studies, with odds ratios of brain tumor risk ranging from 0.4 to 4.0 (20). Three U.S. studies and an Australian study have demonstrated an increased risk of meningioma associated with frequent dental x-rays. This effect was strongest for younger age of exposure and exposure in the remote past when doses were higher (exposures before 1956, when fast speed film was introduced, were associated with exposures orders of magnitude higher than today) (21). All these studies demonstrated only modest elevations in risk for gliomas. Since an initial report in 1979 suggesting electric wiring configurations may be associated with increased risks for leukemia and brain tumors, occupational cohort studies have also suggested an increased risk of brain tumors among electric utility workers, possibly due to the effects of EMR (44,45). Two large occupational cohort studies of electric utility workers with direct measurements of magnetic fields demonstrated elevated risks of brain tumors with apparent dose-response relationships (RR = 2.6; 95% CI, 1.4–4.9; and RR = 3.0; 95% CI, 1.0–8.8 in highest exposure groups) (36). However, the role of other occupational carcinogens cannot be excluded in these studies, and other investigations did not find an excess risk of brain tumors among electrical workers (46). Twenty-one studies of EMF exposure of varying methodology starting as early as 1948 reported odds ratios from 0.3 to 13.1 and standardized mortality ratios ranging from 114 to 340 (20). Other observations have cast doubt on the possibility that EMR causes brain tumors. Biologic plausibility has yet to be established, subsequent studies have reported conflicting results, and the probable roles of bias and confounding in these earlier studies have been emphasized. Studies using more rigorous methodology, including direct in-home measurement of EMR, concluded that a large effect of EMR on the risk of adult and childhood brain tumors can be excluded (21). RF radiation exposures mainly involve microwave and radar equipment and occupational exposures (seal-

8

Cancer of the Nervous System

ers, plastic welders, amateur radio operators, medical personnel, and telecommunications workers). Biologic plausibility of RF radiation as a risk factor for cancer has not been established. The limited data available are equivocal with respect to any cancer risk. Cellular telephones are also sources of RF exposure and have received attention as potential risk factors for brain tumors. This is presumably based on exposure of the head of the user to RF energy. Unlike mobile cellular phones, portable cellular phones include an antenna as part of the headset, theoretically increasing cranial exposure to RF energy. Despite limited biologic plausibility and little human data, coverage in the popular media has caused concern among the public. A number of epidemiologic studies have been conducted to address this controversial issue. Rothman and colleagues (47) reiterated that exposures to RF energy are difficult to quantify, even under laboratory conditions. Therefore, proper assessment of exposure will involve a proxy measure such as phone billing records. Studies aiming to validate billing records as an accurate measure of exposure to cellular phone use have been published. In a cohort of 250,000 portable and mobile cellular phone customers, the age-specific rates of mortality were similar between the groups (portableto-mobile mortality ratio of 0.86; 95% CI, 0.47–1.53) (48). A large case-control study found no evidence for an increased risk of astrocytomas, meningiomas, or acoustic neuromas among those subjects who used these devices for an extended period of time (49). Additionally, among the cases there was no correlation between the side of the tumor and the side of the cranium that was generally exposed to use. These data do not support cell phone use as an important risk factor for primary brain tumors. In summary, biologic and epidemiologic studies have established ionizing radiation as a cause of brain tumors in animal models and humans. Credible evidence exists for the causation of meningiomas and nerve sheath tumors, whereas support for causation of the most common type of primary brain tumor, glioma, is equivocal. Although there is some support from occupational cohort studies for increased risk of brain tumors among electric utility workers, presumably due to EMR, the weight of evidence does not support EMF/EMR as an important causal factor. There is less evidence supporting RF radiation as a risk factor for cancer. Biologic plausibility has yet to be established for these nonionizing forms of radiation. Despite the evidence supporting ionizing radiation as a cause of brain tumors, this exposure is responsible for only a tiny fraction of brain tumors in the United States.

Trauma A number of observations have implicated head trauma as a potential risk factor for the development of brain tumors. The evidence is strongest for meningiomas and

less convincing for gliomas (50,51). Anecdotal reports of brain tumors arising after head trauma date back to the reports of Harvey Cushing in 1922 (52). Cushing and Eisenhardt (53) reported the presence of a head scar or skull depression in 8% of meningioma patients. Experimental studies have also implicated physical trauma as a cocarcinogen. Certain occupations that convey a higher risk of head trauma (i.e., farmers, carpenters) are associated with a possible excess of brain tumors. Three casecontrol studies reported an excess risk of meningiomas in persons with a history of serious or repetitive head trauma (54–56). Childhood brain tumors may be more common in firstborn children (higher risk of birth trauma) and in children with a documented history of birth trauma (forceps delivery, prolonged labor, and cesarian section). Other studies of childhood brain tumors have not confirmed increased risk among these groups (21). Studies of head trauma and brain tumors may be confounded by ionizing radiation because individuals with a history of head trauma are more likely to have had exposure to ionizing radiation in the form of skull x-rays. Recall bias is another factor complicating interpretation of casecontrol studies of head trauma and risk of brain tumors. Persons with brain tumors may be more likely to recall minor and major episodes of head injury than control subjects. One study reporting a positive association between head trauma and risk of brain tumors found no association when the definition of head trauma was restricted to episodes that required medical attention. Future studies should standardize the definition of head trauma to include episodes requiring medical attention and should also search for and document concurrent x-ray studies. One particular type of brain tumor, acoustic neuroma (nerve sheath tumor of the VIII/auditory cranial nerve), has been associated with acoustic trauma of 10 years duration in a case-control study (odds ratio = 2.2; 95% CI, 1.12–4.67) in Los Angeles (57). Noise exposure was based on a blinded review of job histories. A dose-response relationship was observed in this study as 20 or more years of exposure and was associated with an odds ratio of 13.2 (95% CI, 2.01–86.98) (20). Experimental studies of tissue destruction and repair after acoustic trauma support the biologic plausibility of this association.

N-Nitroso Compounds N-nitroso compounds (NOCs) are broadly acting and potent carcinogens in animal models (58,59). It has been known for 40 years that NOCs are present in foods treated with sodium nitrite (59). Although there have been declines in NOC levels in many foods over the last 20 years, it has been suggested that even small amounts of these compounds are important given the possibility that humans are

1. Epidemiology of Primary Brain Tumors

more sensitive to the carcinogenic effects of NOCs than are laboratory rodents (59). A prevalent and longstanding hypothesis in the epidemiology of gliomas is that exposure to NOCs may increase the risk of this type of brain tumor. NOCs include nitrosamines, which require metabolic activation to a carcinogenic form, and nitrosamides, which do not. Transplacental exposure to ethylnitrosourea, a nitrosamide, results in formation of brain tumors, including gliomas, in rodents and primates (60). The addition of vitamin C to the diet prevents the formation of tumors in this model (61). Human exposure to NOCs is estimated to be equally divided between exogenous and endogenous sources. The exogenous, or environmental, sources of population exposures to NOCs are best known for nitrosamines and include tobacco (mainly sidestream smoke), cosmetics, automobile interiors, and cured meats (62). Other sources include rubber products (baby pacifiers, bottle nipples) and certain drugs, including antihistamines, diuretics, oral hypoglycemic agents, antibiotics, tranquilizers, and narcotics. N-nitrodiethanolamine, a carcinogen in animal models, occurs mainly as a contaminant in cosmetic products, soaps, shampoos, and hand lotions (63). It is possible that some of these environmental sources contain both nitrosamines and nitrosamides (59,63). Endogenous formation of NOCs is a complex process occurring in the stomach. This process depends on the presence of NOC precursors, gastric pH, the presence of bacteria, and other physiologic parameters (64). Measurement of NOC exposure is difficult given both exogenous and endogenous sources of NOCs. Thus, exposure misclassification is a major limitation for any study of this topic. However, because processed and cured meats are sources of exogenous NOC exposure, dietary assessment may serve as a useful surrogate marker of at least this form of NOC exposure. These foods contain nitrates and nitrites that can react with secondary amines or amides to form NOCs. The NOC hypothesis is controversial, with some studies reporting an increased risk of gliomas in persons exposed to environmental sources of NOCs, whereas others do not. Epidemiologic support for NOC exposure as a risk factor for brain tumors comes mainly from nine studies of pediatric brain tumors and childhood and maternal diet. Studies in children present fewer methodologic challenges because dietary exposures are relatively recent or characterized by a fixed period of interest (e.g., gestation) (64). An elevated risk of brain tumors in children has been reported for increased maternal consumption of nitritecured or processed meats (e.g., cooked ham, processed pork, corned beef, and fried bacon) (65,66). Studies of dietary NOC exposures and risk of gliomas in adults have yielded inconsistent results. Retrospective case-control studies in adults are complicated by inaccurate measurement of remote diet (67). Moreover, many studies of diet and risk of brain tumors have not used valid reli-

9

able instruments to measure dietary intake over time. A population-based, retrospective, case-control study in Germany (115 gliomas, 418 control subjects) reported a significantly increased risk of glioma in adults with higher levels of dietary meat, processed pork, fried bacon, and cooked ham (68). In a smaller case-control study in Los Angeles County in the United States (94 gliomas, 94 control subjects), an increased risk of glioma was associated with consumption of cured meats, especially bacon (69). In a case-control study in the San Francisco Bay Area, higher consumption of cured foods was associated with an increased risk of glioma in men but not in women (70). Case-control studies of glioma risk and other environmental sources of NOCs, like drinking water, tobacco, and medications, have yielded inconsistent but mainly negative results (71). Despite the existence of both experimental and observational data that NOC exposure may increase the risk of glioma, this hypothesis remains tenuous. In addition to the problem of inaccurate measurement of the exposure in these studies, all these retrospective case-control studies are subject to recall bias. It has been shown that the magnitude and direction of errors in dietary recall data may be influenced by disease status, casting doubt on the results of many of these retrospective studies (72). This has led some authorities to call for studies within prospective cohorts as the best method to definitively address the NOC hypothesis (72,73). Vitamins and Fruit Juices Indirect support for the NOC hypothesis includes the observation that certain modulators of the nitrosation process, such as vitamins (C, E) and fruit juices, appear to reduce brain tumor risk in adults and children (74–76). Dietary studies have demonstrated a reduced risk of brain tumors in children and meningiomas in males who consume increased amounts of fruits and fruit juices (74). Tobacco and Alcohol The presence of nitrosamines in tobacco smoke has stimulated interest in tobacco exposure as a potential risk factor for brain tumors. Studies of active and passive smoking and risk of brain tumors have been inconclusive. A case-control study in 1970 suggested a protective effect of smoking but was flawed because exposure was assessed at the time of the interview (after diagnosis) and likely reflected persons who quit after learning of their diagnosis (77). A series of subsequent case-control studies in Los Angeles found no association of smoking and risk of glioma or meningioma (20). Studies of maternal smoking (active and passive) and risk of brain tumors in offspring have also resulted in conflicting results. There is no conclusive evidence at present that either active or passive smoking is a risk factor for brain tumors (78,79).

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Cancer of the Nervous System

Because beer and liquor contain nitrosamines, there has been speculation that consumption of alcoholic beverages may increase the risk of brain tumors. In one study a correlation was found between childhood brain tumors and paternal use of hard alcohol (white distilled alcohol up to 60% by volume) (RR = 3.72; 95% CI, 1.91–7.26) for less than 15 years of hard liquor consumption and an even more elevated risk of 4.06 (95% CI, 1.09–15.21) for greater than 16 years of hard liquor use (80). However, no consistent association between consumption of different types of alcoholic beverages and risk of gliomas or meningiomas in childhood (maternal consumption) or adulthood has been demonstrated (20,21).

Infections Although a number of reports have implicated certain infectious agents in the development of different types of brain tumors, these associations have been inconsistent. Viruses and virus-like particles have been detected in brain tumor specimens with speculation that viral infection may be a risk factor for development of brain tumors. Attention to simian virus 40 (SV40), a polyoma virus, has been stimulated by animal studies documenting brain tumor development after intracerebral inoculation with SV40 and human studies that have isolated SV40 from brain tumor tissue. Poliomyelitis vaccine administered between 1955 and 1962 was contaminated with SV40, and this cohort has been the subject of study over subsequent decades (21). Although these subjects did not experience an overall increase in brain tumors over the ensuing 20 years, some uncommon brain tumor types (medulloblastoma and spongioblastoma) did appear to occur more often than expected (81,82). Other nonpolyoma viruses (adenovirus, cytomegalovirus, other herpes viruses, arboviruses, and retroviruses) are known to induce brain tumors in experimental animal models. Most of these viruses have not been subjected to rigorous epidemiologic study as possible risk factors for brain tumors. Human cytomegalovirus is a ␤-herpes virus that has been implicated in glioma development. In one study, human cytomegalovirus gene products were found in 27/27 malignant glioma specimens but

w TABLE 1-4

were not found in other types of central nervous system tumors or non-neoplastic neurologic diseases (83). The investigators raised the possibility that human cytomegalovirus could play a causative factor in glioma growth. However, one cannot exclude the possibility that infection with human cytomegalovirus could have occurred after glioma formation. Studies of maternal infection have generally found no increased risk of brain tumors in offspring of mothers infected with varicella, rubella, or mumps during pregnancy (84). One large case-control study reported an increased risk (RR = 2.4; 95% CI, 1.5–4.0) of all types of brain tumors after different neonatal infections (85). Infection with Toxoplasma gondii has been associated with an increased risk of meningioma and astrocytoma in two case-control studies (86,87). Although this parasite has a propensity to infect the nervous system, it has not been established as a risk factor for the development of brain tumors. Despite evidence that different types of viruses may induce formation of brain tumors in animal models and the isolation of viruses and virus-like particles from human brain tumors, there is limited epidemiologic evidence implicating infectious agents as important factors in the etiology of brain tumors.

Genetic Syndromes Approximately 1% to 5% of brain tumors are due to genetic syndromes that confer an increased risk of tumors of the nervous system (20,88). Neurofibromatosis type 1 (NF1) occurs in 1 in 3,000 persons and is linked to a gene on chromosome 17. Approximately 5% to 10% of persons with NF1 develop brain tumors, primarily nerve sheath tumors, astrocytomas, and meningiomas. Neurofibromatosis type 2 (NF2) is defined by the presence of bilateral acoustic neuromas and a corresponding link to a gene on chromosome 22. Astrocytomas and other brain tumor types are also encountered in persons with NF2. Other inherited syndromes associated with an increased risk of nervous system tumors are reviewed in Table 1-4.

Genetic Syndromes Associated with Nervous System Tumors

Syndrome

Chromosome

Inheritance

CNS tumors

NF1 NF2 Tuberous sclerosis von Hippel-Lindau

17q1 22q 9q32–34 3p13–14 3p25–26

AD AD AD AD

Glioma, meningiomas Acoustic neuroma, optic glioma, meningioma Ependymoma, astrocytoma, ganglioneuroma Hemangioblastoma

AD

Choroid plexus papilloma

Sturge-Weber

AD, autosomal dominant; CNS, central nervous system; NF1 and 2, neurofibromatosis, types 1 and 2.

1. Epidemiology of Primary Brain Tumors

w TABLE 1-5

11

Metabolic Polymorphisms and Risk of Astrocytoma and Meningioma

Tumor

Enzyme System

Polymorphism

Astrocytoma Meningioma Astrocytoma Meningioma Gliomas

CYP450 CYP450 Glutathione S-transferase Glutathione S-transferase HRAS

CYP2D6 PM CYP2D6 PM GSTT1 Null GSTT1 Null “Rare” HRAS1

Cases

Control Subjects

Odds Ratio [95% Confidence Intervals]

109 48 109 47 52

412 412 494 494 109

4.17 [1.57, 11.09] 4.90 [1.39, 17.26] 2.67 [1.53, 4.65] 4.52 [2.18, 9.34] 2.72 [1.17–6.32]

HRAS, Harvey RAS Oncogene.

A number of familial cancer syndromes are associated with an increased risk of brain tumors. The Li-Fraumeni syndrome, an autosomal dominantly inherited condition, may involve a mutation in the tumor suppressor gene p53, conferring an increased risk of brain tumors, sarcomas, and breast cancer (89). Persons with nevoid basal cell carcinoma (Gorlin syndrome) and Wilms tumor have an increased risk of medulloblastoma. Turcot syndrome is associated with adenomatous polyps and increased risk of medulloblastoma and GBM. Familial clustering of brain tumors is also recognized outside of these defined syndromes. The chance of having a family member with a brain tumor is twice as high in adult brain tumor patients (8%) as in control subjects (4%). This familial clustering is even more pronounced for children and certain histologic types of brain tumors (medulloblastoma). Although genetic syndromes account for only a small fraction of brain tumors in the United States, further study of the affected genes may provide insight into the molecular pathogenesis of sporadic brain tumors.

Genetic Polymorphisms Genetic polymorphisms in specific detoxification/metabolic enzymes have been implicated in the pathogenesis of certain neoplasms. Despite conflicting results from the small number of studies addressing this category of potential risk factors for primary brain tumors, there is a sound rationale for further investigation (90–92). In addition to the possible association of NOCs and gliomas, other xenobiotics have been shown to induce brain tumors in animal models, including polycyclic aromatic hydrocarbons (93). These associations between chemical carcinogens and brain tumors in experimental models raise the possibility that susceptibility to these tumors may be related to genetic polymorphisms at loci encoding phase I cytochrome P-450 and phase II glutathione S-transferase enzymes because a number of potential neurocarcinogens (polycyclic aromatic hydrocarbons, nitrosoureas, and methyl halide) are substrates for these enzymes. A number of case-control studies of brain tumors have assessed other polymorphic enzymes (GSTT1, GSTM1, GSTP1, CYP2D6, NAT2, HRAS1) implicated in the activation or detoxification of

potential neurocarcinogens. In most of these studies, an elevated risk of glioma and other types of primary brain tumors has been associated with polymorphisms in these enzymes (Table 1-5). The study of genetic polymorphisms in different detoxification/metabolic enzymes as potential risk factors for brain tumors is a new and promising area of investigation.

CONCLUSION Brain tumors are an uncommon but especially lethal form of cancer, and the incidence of these tumors has increased dramatically over the past five decades. The only firmly established environmental risk factor, ionizing radiation, accounts for only a fraction of incident cases, and genetic predisposition exists in only a small percentage of cases. The study of brain tumors has been complicated by a number of factors. Brain tumors are an uncommon form of cancer, making centralized reporting and multiinstitutional collaboration essential for effective observational study. Such reporting and collaboration are recent developments and promise improved study design and execution in the future. Because brain tumors are a heterogeneous group of cancers arising from different cell types, perhaps by different molecular mechanisms, future studies of brain tumors should focus on biologically distinct tumor types such as astrocytoma and meningioma. Molecular classification of these tumors may allow even greater refinement of tumor classification. The WHO system is now the most widely accepted histopathologic classification system, with high interrater reliability (94%), and serves as the basis of reporting to the CBTRUS. Use of WHO criteria supplemented with genetic classification will improve case definition in future studies. Future epidemiologic studies of brain tumors need to explore more fully the role of dietary NOCs, physical trauma, and viral infection. Collaboration and centralized reporting will improve these studies. Ongoing prospective cohort studies are likely to accumulate enough cases in the future for more detailed assessment of diet, comorbid illnesses, and drug exposures as potential risk factors. Understanding the molecular pathology of gliomas may also allow

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correlation of candidate exposures with specific molecular subtypes of tumors that may shed light on the molecular mechanisms of disease causation and progression.

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1. Epidemiology of Primary Brain Tumors 47. Rothman KJ, Chou CJ, Morgan R, et al. Assessment of cellular telephone and other radio frequency exposure for epidemiologic research. Epidemiology 1996;7:291–298. 48. Rothman KJ, Loughlin JE, Funch DP, et al. Overall mortality of cellular telephone customers. Epidemiology 1996;7: 303–305. 49. Inskip PD, Tarone RE, Hatch EE, et al. Cellular-telephone use and brain tumors. N Engl J Med 2001;344:79–86. 50. Wrensch M, Miike R, Lee M, et al. Are prior head injuries or diagnostic x-rays associated with gliomas in adults? The effects of control selection bias. Neuroepidemiology 2000;19: 234–244. 51. Nygren C, Adami J, Ye W, et al. Primary brain tumors following traumatic brain injury—a population based cohort study in Sweden. Cancer Causes Control 2001;12:733–737. 52. Cushing H. The meningiomas (dural endotheliomas). Their source and favored seats of origin. Brain 1922;45:282–316. 53. Cushing H, Eisenhardt L. Meningiomas, their classification, regional behavior, life history and surgical end results. Springfield, IL: Charles C Thomas, 1938. 54. Morantz RA, Shain W. Trauma and brain tumors: an experimental study. Neurosurgery 1978;3:181–186. 55. Preston-Martin S, Pogoda JM, Schlehofer B, et al. An international case-control study of adult glioma and meningioma: the role of head trauma. Int J Epidemiol 1998;27:579–586. 56. Preston-Martin S, Paganini-Hill A, Henderson BE, et al. Case-control study of intracranial meningiomas in women in Los Angeles county, California. J Natl Cancer Inst 1980;65:67–73. 57. Preston-Martin S, Henderson BE, Yu MC. Epidemiology of intracranial meningiomas: Los Angeles Country, California. Neuroepidemiology 1983;2:164–178. 58. Lijinsky W. N-nitroso compounds in the diet. Mutat Res 1999;443:129–138. 59. Bogovski P, Bogovski S. Animal species in which N-nitroso compounds induce cancer. Int J Cancer 1981;27:471–474. 60. Rice JM, Ward JM. Age dependence of susceptibility to carcinogenesis in the nervous system. Ann N Y Acad Sci 1982;381:274–289. 61. Mirvish SS. Inhibition of the formation of carcinogenic Nnitroso compounds induce cancer. Int J Cancer 1981;27: 471–474. 62. Preston-Martin S, Mack WJ. Neoplasms of the nervous system. In: Schottenfeld D, Fraumeni JF, eds. Cancer epidemiology and prevention, 2nd ed. New York: Oxford University Press, 1996:1231–1281. 63. Batchelor T, Piscatelli N, Alderson L. Brain tumors. In: Batchelor T, Cudkowicz MC, eds. Principles of neuroepidemiology. Boston: Butterworth-Heinemann, 2001. 64. Davis FG, Preston-Martin S. Epidemiology, 6th ed. London: Arnold, 1998. 65. McCredie M, Maisonneuve P, Boyle P. Perinatal and early postnatal risk factors for malignant brain tumours in New South Wales children. Int J Cancer 1994;56:11–15. 66. Bunin GR. Maternal diet during pregnancy and risk of brain tumors in children. Int J Cancer 1998;11[Suppl]:23–25. 67. Giles GG, McNeil JJ, Donnan G, et al. Dietary factors and the risk of glioma in adults: results of a case-control study in Melbourne, Australia. Int J Cancer 1994;59:357–362. 68. Beohing H, Schlehofer B, Blettner M, et al. Dietary carcinogens and the risk of glioma and meningioma in Germany. Int J Cancer 1993;53:561–565. 69. Blowers L, Preston-Martin S, Mack WJ. Dietary and other lifestyle factors of women with brain gliomas in Los Angeles County (California, USA). Cancer Causes Control 1997;8:5–12. 70. Lee M, Wrensch M, Mike R. Dietary and tobacco risk factors for adult onset glioma in the San Francisco Bay area. Cancer Causes Control 1997;8:13–24.

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71. Steindorf K, Schlehofer B, Becher H, et al. Nitrate in drinking water. A case control study on primary brain tumours with an embedded drinking water survey in Germany. Int J Epidemiol 1994;23:451–457. 72. Wilkins JR, Bunn JY. Comparing dietary recall data for mothers and children obtained on two occasions in a casecontrol study of environmental factors and childhood brain tumours. Int J Epidemiol 1997;26:953–963. 73. Blot WJ, Henderson BE, Boice JD. Childhood cancer in relation to cured meat intake: review of the epidemiological evidence. Nutr Cancer 1999;34:111–118. 74. Burch JD, Craib KJ, Choi BC, et al. An exploratory casecontrol study of brain tumors in adults. J Natl Cancer Inst 1987;78:601–609. 75. Preston-Martin S, Mack W. Gliomas and meningiomas in men in Los Angeles County: investigation of exposures to Nnitroso compounds. IARC Sci Publ 1991;105:197–203. 76. Bartsch H, Ohshima B, Pigntatelli B. Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention. Mutat Res 1988;202:307–324. 77. Choi NW, Schuman LM, Gullen WH. Epidemiology of primary central nervous system neoplasms. II. Case-control study. Am J Epidemiol 1970;91:467–485. 78. Filippini G, Maisonneuve P, McCredie M, et al. Relation of childhood brain tumors to exposure of parents and children to tobacco smoke: the Search International Case-Control Study. Int J Cancer 2002;100:206–213. 79. Zheng T, Cantor KP, Zhang Y, et al. Risk of brain glioma not associated with cigarette smoking or use of other tobacco products in Iowa. Cancer Epidemiol Biomark Prevent 2001; 10:413–414. 80. Hu J, Mao Y, Ugnat A-M. Parental cigarette smoking, hard liquor consumption and the risk of childhood brain tumors. Acta Oncol 2000;39:979–984. 81. Geissler E, Staneczek W. SV40 and human brain tumors. Arch Geschwulstforsch 1988;58:129–134. 82. Klein G, Powers A, Croce C. Association of SV40 with human tumors. Oncogene 2002;21:1141–1149. 83. Cobbs CS, Harkins L, Samanta M, et al. Human cytomegalovirus infection and expression in human malignant glioma. Cancer Res 2002;62:3347–3350. 84. Adelstein AM, Donovan JW. Malignant disease in children whose mothers had chickenpox, mumps or rubella in pregnancy. Br Med J 1972;4:629–631. 85. Linet MS, Gridley G, Cnattingius S, et al. Maternal and perinatal risk factors for childhood brain tumors (Sweden). Cancer Causes Controls 1996;7:437–448. 86. Schuman LM, Choi NW, Gullen WH. Relationship of central nervous system neoplasms to Toxoplasma gondii infection. Am J Public Health 1967;57:848–856. 87. Ryan P, Hurley SF, Johnson AM, et al. Tumours of the brain and presence of antibodies to Toxoplasma gondii. Int J Epidemiol 1993;22:412–419. 88. Bondy ML, Lustbader ED, Buffler PA, et al. Genetic epidemiology of childhood brain tumors. Epidemiology 1991; 8:253–267. 89. Chompret A. The Li-Fraumeni syndrome. Biochimie 2002; 84:75–82. 90. Elexpuru-Camiruaga J, Buxton N, Kandula V, et al. Susceptibility to astrocytoma and meningioma: influence of allelism at glutathione S-transferase (GSTT1 and GSTM1) and cytochrome P-450 (CYP2D6) loci. Cancer Res 1995; 55: 4237–4239. 91. Kelsey KT, Wrensch M, Zuo ZF, et al. A population-based case-control study of the CYP2D6 and GSTT1 polymorphisms and malignant brain tumors. Pharmacogenetics 1997; 7:463–468. 92. Trizna Z, de Andrade M, Kyritsis AP, et al. Genetic polymorphisms in glutathione S-transferase mu and theta, N-acetyltransferase, and CYP1A1 and risk of gliomas. Cancer

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2

w Neuropathology of Central Nervous System Tumors

Umberto De Girolami, Thomas W. Smith The term “brain tumor” is often used clinically to mean any neoplasm arising within the cranial vault, whether it originates within or outside the parenchyma of the brain. In this chapter we consider the pathology of brain tumors that originate from the specific cellular constituents of the brain and the pathology of tumors of adjacent or distant tissues that are usually considered within the realm of brain tumors, such as meningioma and cerebral metastasis. Readers interested in a more extensive discussion of the various tumors described herein are referred to the several excellent general references (1–5) or selected papers cited in the bibliography listed at the end of this chapter.

CLASSIFICATION Modern schemes of brain tumor classification are derived from the clinicopathologic correlations of Bailey and Cushing, who based their classification on cell type, degree of differentiation, and prognosis (1,2). Although central nervous system (CNS) tumors are still primarily classified on basis of their morphologic appearance, it is becoming clear that information derived from cytogenetics and molecular genetics will play an increasingly important role, particularly with respect to providing more precise diagnostic and prognostic information about a particular tumor. Underlying most histology-based classification schemes has been an implicit assumption that the phenotypic appearance of a particular tumor accurately reflects its cellular derivation (e.g., low-grade astrocytomas are derived from mature astrocytes, etc.). Recent evidence, however, suggests that at least some gliomas might be derived from glial progenitor cells that may persist throughout adult life (6). Also, as in other human cancers, CNS tumors may arise when alterations occur in growth regulatory genes, such as oncogenes and tumor suppressor genes. Thus, it is paramount that any classification scheme be flexible enough to allow for the

inclusion of new diagnostic categories as well as the modification and even removal of prior categories on the basis of information derived from newer methodologies. The classification scheme adapted by the World Health Organization (WHO) in 2000 (4,7) has received general approval and is used in this chapter (Table 2-1).

SPECIAL CHARACTERISTICS OF CENTRAL NERVOUS SYSTEM TUMORS Nervous system tumors have features that set them apart from somatic neoplasms. The localization and control of somatic function within the CNS dictate the type of neurologic deficit a given patient will experience as a result of a tumor’s anatomic location. The rate of clinical progression and the degree of a surgical resection are related to a tumor’s histologic elements and its anatomic relationship within the nervous system. All intracranial tumors, whether histologically well differentiated and relatively “benign” or undifferentiated and malignant, are potentially lethal because of the mass effect of their growth within the confines of the cranium. Although the pathologist may designate a tumor as histologically “malignant,” a clinician is concerned with the overall clinical outcome and may refer to a histologically benign tumor that shortens a patient’s life as malignant (e.g., an ependymoma located in the fourth ventricle). In communicating with patients and other physicians, it is important to define how the word malignant is used. The concept of “invasiveness,” so important in somatic epithelial tumors, has limited application to parenchymal CNS neoplasms: Neuroglial cells have no basement membrane or other defined boundary through which to invade, so the transition from “in situ” to “invasive” phenotype cannot be recognized. From this viewpoint, brain tumors are comparable with soft tissue neoplasms, some of which are notoriously difficult to distinguish from normal mesenchymal tissue and whose behavior cannot be predicted 15

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w TABLE 2-1

World Health Organization Classification of Tumors of the Nervous System

Tumors of neuroepithelial tissue Astrocytic tumors Diffuse astrocytoma Fibrillary astrocytoma Protoplasmic astrocytoma Gemistocytic astrocytoma Anaplastic astrocytoma Glioblastoma Giant cell glioblastoma Gliosarcoma Pilocytic astrocytoma Pleomorphic xanthoastrocytoma Subependymal giant cell astrocytoma Oligodendroglial tumors Oligodendroglioma Anaplastic oligodendroglioma Mixed gliomas Oligoastrocytoma Anaplastic oligoastrocytoma Ependymal tumors Ependymoma Cellular Papillary Clear cell Tanycytic Anaplastic ependymoma Myxopapillary ependymoma Subependymoma Choroid plexus tumors Choroid plexus papilloma Choroid plexus carcinoma Glial tumors of uncertain origin Astroblastoma Gliomatosis cerebri Chordoid glioma of the third ventricle Neuronal and mixed neuronal—glial tumors Gangliocytoma Dysplastic gangliocytoma of cerebellum (Lhermitte-Duclos) Desmoplastic infantile astrocytoma/ganglioglioma Dysembryoplastic neuroepithelial tumor ganglioglioma Anaplastic ganglioglioma Central neurocytoma Cerebellar liponeurocytoma Paraganglioma of the filum terminale Neuroblastic tumors Olfactory neuroblastoma (esthesioneuroblastoma) Olfactory neuroepithelioma Neuroblastomas of the adrenal gland and sympathetic nervous system Pineal parenchymal tumors Pineocytoma Pineoblastoma Pineal parenchymal tumor of intermediate differentiation Embryonal tumors Medulloepithelioma Ependymoblastoma Medulloblastoma Desmoplastic medulloblastoma

Large cell medulloblastoma Medullomyoblastoma Melanotic medulloblastoma Supratentorial primitive neuroectodermal tumor Neuroblastoma Ganglioneuroblastoma Atypical teratoid/rhabdoid tumor Tumors of peripheral nerves Schwannoma (Neurilemmoma, Neurinoma) Cellular Plexiform Melanotic Neurofibroma Plexiform Perineurioma Intraneural perineurioma Soft tissue perineurioma Malignant peripheral nerve sheath tumor (MPNST) Epithelioid MPNST with divergent mesenchymal and/or epithelial differentiation Melanotic Melanotic psammomatous Tumors of the meninges Tumors of meningothelial cells Meningioma Meningothelial Fibrous (fibroblastic) Transitional (mixed) Psammomatous Angiomatous Microcystic Secretory Lymphoplasmacyte rich Metaplastic Clear cell Chordoid Atypical Papillary Rhabdoid Anaplastic meningioma Mesenchymal, nonmeningothelial tumors Lipoma Angiolipoma Hibernoma Liposarcoma (intracranial) Solitary fibrous tumor Fibrosarcoma Malignant fibrous histiocytoma Leiomyoma Leiomyosarcoma Rhabdomyoma Rhabdomyosarcoma Chondroma Chondrosarcoma Osteoma Osteosarcoma (continued)

2. Neuropathology of Central Nervous System Tumors

w TABLE 2-1

17

World Health Organization Classification of Tumors of the Nervous System (Continued)

Osteochondroma Hemangioma Epithelioid hemangioendothelioma Hemangiopericytoma Angiosarcoma Kaposi sarcoma Primary melanocytic lesions Diffuse melanocytosis Melanocytoma Malignant melanoma Meningeal melanomatosis Tumors of uncertain histogenesis Hemangioblastoma Lymphomas and hemopoietic neoplasms Malignant lymphomas Plasmacytoma Granulocytic sarcoma

Germ cell tumors Germinoma Embryonal carcinoma Yolk sac tumor Choriocarcinoma Teratoma Mature Immature Teratoma with malignant transformation Mixed germ cell tumors Tumors of the sellar region Craniopharyngioma Adamantinomatous Papillary Granular cell tumor Metastatic tumors

From Kleihues P, Cavenee WK, eds. Pathology and genetics of tumours of the nervous system. Lyon, France: IARC Press, 2000, with permission.

with certainty on a cytologic basis. Many brain tumors have typical growth and spread characteristics. Astrocytomas generally infiltrate the surrounding brain, often along white matter tracts. It is uncommon for these neoplasms to grow and spread along the cerebrospinal fluid (CSF) pathway. Medulloblastomas, in contrast, commonly spread via the CSF route. The absence of a welldeveloped system of lymphatics in the brain is perhaps an explanation why it is rare for primary CNS tumors to metastasize to regional lymph nodes. Hematogenous spread to other regions of the body is likewise rare, occurring most commonly after surgery or through a shunt. In contrast, tumors of somatic organs such as breast and lung commonly metastasize to the brain. One of the most important features determining the length of survival of a patient with a brain tumor is the age of the individual; younger patients live longer than older ones, even when they are affected with a primary brain tumors of comparable histologic grade (8). From these considerations, it should be clear that the prognosis of an individual patient with a brain tumor must be based on an integrated evaluation of a tumor’s general characteristics, such as location, growth, spread, and histologic findings, correlated with clinical and radiologic data.

INCIDENCE Tumors of the CNS, dura, and meninges represent about 9.2% of all primary neoplasms in human beings (1). The incidence of primary brain tumors increases up to the seventh decade, with over 17,000 new cases in the United States per year (9). In adults, approximately 70% of all

brain tumors occur supratentorially (i.e., within the cerebral hemispheres or coverings) and include, in descending order of frequency, metastases followed by gliomas, meningiomas, and schwannomas. In children, brain tumors are the second most common neoplasm encountered. In contrast to adults, approximately 70% of childhood brain tumors are infratentorial in location (e.g., cerebellum, brainstem) and are of neuroectodermal origin, the most common types being pilocytic astrocytoma, medulloblastoma, and ependymoma, in descending order of frequency. Spinal cord tumors constitute about 15% of all primary CNS tumors and include schwannomas, meningiomas, and gliomas (ependymoma, astrocytoma), in descending order of frequency.

TUMORS OF NEUROEPITHELIAL TISSUE Astrocytic tumors Astrocytic tumors are frequently divided into two main categories: diffusely infiltrating astrocytomas and circumscribed (or localized) astrocytomas (2). The diffusely infiltrating astrocytomas as a group are more often seen in adults and are characterized by their striking propensity for diffuse infiltration of adjacent and often distant brain structures, tendency for progression to anaplasia over time, and occurrence in almost all regions of the brain and spinal cord. In the WHO classification (4), these tumors include diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma. By contrast, circumscribed astrocytomas occur more often in children and are characterized by minimal infiltration of tumor cells into adjacent structures, slow growth with little or no tendency toward

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anaplasia, and occurrence within more restricted regions of the brain and spinal cord. These tumors include pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and subependymal giant cell astrocytoma. All astrocytic tumors must be evaluated for their degree of differentiation and anaplasia. Differentiation of an astrocytoma is determined by how closely its neoplastic cells resemble normal and reactive astrocytes. Anaplasia is assessed by nuclear density, pleomorphism, hyperchromasia, mitotic index of the tumor cells, the presence or absence of necrosis and vascular proliferation, and in some cases may be aided by the use of immunohistochemical markers for cell proliferation such as Ki-67 (MIB-1). It is common practice to convey the degree of anaplasia within an astrocytoma by assigning it a numerical grade. A number of histologic grading schemes have been used for diffusely infiltrating astrocytomas; however, the Ste. Anne-Mayo grading system (10) and its adaptation to the current WHO classification has proved to be the most reproducible and predictive of tumor behavior. The Ste. Anne-Mayo criteria are based on the presence or absence of four easily recognizable histologic features: nuclear pleomorphism, mitoses, vascular proliferation, and necrosis. Although the St. AnneMayo system recognizes a grade I diffuse astrocytoma lacking all the above features, this has proved to be such a rare (and possibly controversial) entity that the WHO scheme uses modifications of the St. Anne-Mayo criteria for only the three higher grades of diffuse astrocytoma (Table 2-2). The WHO grade I category is presently reserved for certain circumscribed astrocytomas (e.g., pilocytic astrocytoma and subependymal giant cell astrocytoma). It is important to be aware that diffusely infiltrating astrocytomas are not always uniform in histologic appearance and often show variable degrees of differentiation and/or anaplasia in different regions of the same tumor (11). The tumor in one region may be well differentiated with low cellularity and in another region show extreme pleomorphism and hypercellularity. Conventional

w TABLE 2-2

II III IV a

Diffuse Astrocytoma (World Health Organization Grade II) These tumors account for about 10% to 15% of all astrocytic neoplasms (Figs. 2-1 and 2-2). They can affect all age groups but are mainly tumors of adults, with 25% occurring between the ages of 30 and 40 years. They most commonly occur in the cerebral hemispheres, followed by brainstem and spinal cord, and least often in the cerebellum. Most low-grade diffuse astrocytomas will progress to a higher grade tumor, with an average interval to malignant change of about 4 to 5 years. Grossly, these tumors enlarge and distort involved brain structures, although their exact margins are often difficult to identify. Small cysts and focal calcifications may be present. Microscopically, these tumors are of low to moderately cellularity and composed of well-differentiated astrocytes. Some degree of nuclear atypia is almost always present, which should help distinguish the neoplastic cells from reactive astrocytes. Mitoses are extremely rare or absent (12). Vascular proliferation and necrosis are never present. The background matrix may be loose, vacuolated, or even microcystic. The Ki-67/MIB-1 labeling index (an indicator of tumor cell proliferation) is usually less than 1% to 2% (2,13). Three major histologic variants of diffuse astrocytoma are recognized, although in practice most have a mixture

Grading of Diffuse Astrocytoma

World Health Organization Grade I

practice dictates that a particular tumor be assigned a single grade, which is based on areas showing the highest degree of anaplasia. However, assigning a “correct” grade may be difficult if not impossible in small biopsy samples (especially those obtained from stereotactic procedures) because of the inherent histologic variability within the tumor. For this reason, a clear description of histologic findings is recommended, which also may help to describe more accurately the features of anaplasia identified within a single CNS tumor. Clearly, correlation with the clinical and radiographic findings is of paramount importance.

Designation

Histologic Criteria (Ste. Anne-Mayo)

Pilocytic astrocytoma; subependymal giant cell astrocytoma Diffuse astrocytoma Anaplastic astrocytoma Glioblastoma

Not applicable One criterion: usually nuclear pleomorphism Two criteria: usually nuclear pleomorphism and mitosesa Three or four criteria: nuclear pleomorphism, mitoses, vascular proliferation, and/or necrosisb

The presence of a single mitosis in an adequately sampled diffuse astrocytoma that only exhibits nuclear pleomorphism is not sufficient to reclassify it as a WHO grade III tumor (11). b Necrosis is not required for the diagnosis of glioblastoma as long as vascular proliferation is present, although it is almost always present. Adapted from Kleihues P, Cavenee WK, eds. Pathology and genetics of tumours of the nervous system. Lyon, France: IARC Press, 2000, with permission.

2. Neuropathology of Central Nervous System Tumors

A

19

B

FIGURE 2-1. A and B. Diffuse fibrillary astrocytoma with a poorly defined margin, enlargement of the hemisphere, and effacement of the adjacent cortex.

of cell types (4). By far the most common variant is the fibrillary (diffuse) astrocytoma, which is composed of neoplastic cells having relatively scant perikaryal cytoplasm but elongated processes that form a loose but consistently positive glial fibrillary acidic protein (GFAP) fibrillary matrix. Gemistocytic astrocytoma (14) is defined as a tumor in which at least 20% of the neoplastic cells resemble gemistocytic astrocytes, that is, have abundant

eosinophilic cytoplasm and peripherally displaced nuclei and show strong expression for GFAP. Although gemistocytic astrocytomas are highly associated with progression to anaplastic astrocytoma and glioblastoma, they should not automatically be assigned a higher grade unless the appropriate histologic criteria are fulfilled. Gemistocytic astrocytomas must be differentiated from giant cell glioblastoma, subependymal giant cell astrocytoma, and gangliocytoma. The protoplasmic astrocytoma is the least common (and most controversial) variant. It is an astrocytic tumor composed mainly of small round cells with scant minimally GFAP-reactive processes in a prominent mucoid or microcystic background matrix. This pattern bears a striking resemblance to the loose (spongy) tissue of pilocytic astrocytomas and may also be seen focally in some other glial tumors (e.g., oligodendroglioma, dysembryoplastic neuroepithelial tumor). Molecular changes that are often present in diffuse astrocytoma (WHO grade II) include mutations of the TP53 tumor suppressor gene in about 50% of cases, overexpression of the platelet-derived growth factor and its receptor, and loss of portions of chromosome 22 (3,4). Anaplastic Astrocytoma (World Health Organization Grade III)

FIGURE 2-2. Fibrillary astrocytoma composed of loosely arranged neoplastic cells having round to spindle-shaped nuclei and a variable amount of cytoplasm.

These tumors most often arise from a preexisting lowgrade diffuse astrocytoma but can also present de novo without clear evidence of a less malignant precursor (Fig. 2-3). The average age of presentation is about 10 years

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Cancer of the Nervous System

FIGURE 2-4. Glioblastoma multiforme with a variegated cut surface at the level of the corpus striatum.

FIGURE 2-3. Anaplastic brainstem glioma with diffuse spread into cerebellum and pons.

older than low-grade diffuse astrocytoma; however, their location and gross appearance are otherwise similar. Microscopically, this tumor is a diffusely infiltrating astrocytoma that shows features of anaplasia, such as increased cellularity, nuclear atypia, and mitotic activity, but without the vascular proliferation or necrosis characteristic of glioblastoma. Most tumor cells express GFAP, but this can be variable. Ki-67/MIB-1 labeling indices are almost always greater than 3% and can overlap with glioblastoma (2). Glioblastoma (World Health Organization Grade IV) Glioblastoma (glioblastoma multiforme [GBM]) is the most malignant astrocytic neoplasm (Figs. 2-4 and 2-5). It is the most common primary brain tumor, accounting for approximately 10% to 15% of all intracranial tumors and 40% to 50% of all glial tumors (1). The tumor may arise de novo in the absence of a preexisting astrocytic tumor (primary GBM) or develop from a less-malignant diffuse astrocytoma (secondary GBM) (3,4). GBM occurs in all age groups, but most arise in adults with a peak incidence between the ages of 45 and 70 years. It can occur in any region of the CNS; however, in adults the cerebral hemispheres (especially the frontal and temporal lobes, basal ganglia, and commissural pathways) are sites of predilection. In children, GBM usually occurs in the brainstem and may infiltrate into surrounding structures such as the

cerebellum. Glioblastomas of the spinal cord or those arising solely within the cerebellum are rare. Grossly, GBM may appear as a relatively well-defined mass lesion, although there is almost always microscopic infiltration of tumor into the surrounding parenchyma. A classic macroscopic appearance is the “butterfly” pattern due to spread of the tumor across the corpus callosum into the opposite hemisphere. On cut section, GBM typically has a variegated appearance, with solid gray-pink tissue at the periphery and yellow zones of central necrosis. In some cases, old and recent hemorrhage is seen. In common with other diffuse astrocytomas, GBM can widely infiltrate adjacent tissue and extend for long distances within fiber tracts. It may sometimes form new masses at distant sites, creating the impression of a multifocal or “multicentric” glioma. True multifocal gliomas do occur, although their exact frequency has been difficult to establish (estimated range, 2.4% to 7.5%). These tumors would by definition be polyclonal and presently can only be proven by the use of molecular markers (4). Some GBMs may extend into the subarachnoid space or ventricles with the potential for CSF dissemination, although this is relatively infrequent. Extracranial extension and/or hematogenous dissemination (metastatic GBM) is a very rare occurrence. All GBMs share in common the histologic features of high cellularity, marked nuclear atypia, mitoses, vascular proliferation, and necrosis. However, their microscopic appearance can be highly variable with considerable regional heterogeneity. In some GBMs the tumor cells may show considerable nuclear and cytoplasmic pleomorphism with multinucleated giant cells, whereas others may consist mainly of small undifferentiated cells with scant cytoplasm and often poor GFAP expression. This small cell pattern is said to be more characteristic of de novo (primary) GBMs (15). Many GBMs also contain zones having better differentiated fibrillary and gemistocytic astrocytes. Other cell types that may be infrequently

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21

A

B

C

FIGURE 2-5. Glioblastoma multiforme with (A) nuclear pleomorphism, (B) high cellularity and prominent vascular proliferation with glomeruloid formation, and (C) increased cellularity surrounding a region of necrosis, pseudopalisading.

present include cells with glandular or epithelioid features, oligodendrocyte-like cells, periodic acid–Schiff-positive granular cells, and heavily lipidized cells. Proliferative activity is prominent in GBM, and both typical and atypical mitoses are found. Ki-67/MIB-1 labeling indices are likewise high, usually averaging 15% to 20% (2). Proliferative activity is usually greatest in tumors composed predominantly of small undifferentiated cells. Necrosis is a characteristic feature of GBM and can consist of either

large confluent foci of coagulative necrosis and/or small band-like or serpiginous “geographic” necrotic foci surrounded by a rim of densely packed tumor cells imparting the characteristic and highly diagnostic pseudopalisading pattern. Vascular proliferation (also referred to as microvascular or capillary endothelial proliferation) is defined as the presence of abnormal small vessels with walls composed of two or more layers of mitotically active endothelial (and/or smooth muscle/pericytic) cells.

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Cancer of the Nervous System

Some of these abnormal vessels resemble renal glomeruli and are commonly known as glomeruloid structures. Abnormal vessels of both large and small caliber often undergo thrombosis within a GBM. Occasional GBMs may show considerable connective tissue reaction, which may due to meningeal invasion by tumor, organization of zones of necrosis, or as a response to marked vascular proliferation. In some cases, a diagnosis of GBM may be made in the absence of necrosis if the other three requisite Ste. AnneMayo histologic features (pleomorphism, mitoses, vascular proliferation) are present (Table 2-1), although it is our practice to secure the diagnosis by identifying foci of necrosis. This may be particularly relevant when evaluating small stereotactically guided biopsies of a suspected GBM that are usually targeted to the contrast-enhancing rim where viable tumor and vascular proliferation will likely be seen. The origin of GBM remains controversial. The traditional explanation is that the tumor arises from differentiated adult astrocytes, but more recently it was suggested that at least some GBMs may arise from glial progenitor cells (6). Molecular studies have shown that GBMs nearly always have inactivation of the cell cycle control pathway that includes the CDKN2A/p16/ARF, CDK4, and RB genes as well as loss of parts of the long arm of chromosome 10, which may be associated with mutations of the PTEN tumor suppressor gene (3,4). EGFR gene amplification/overexpression is also frequently present, especially in de novo (primary) or small cell GBMs (4,15), whereas GBMs arising from lower grade precursors more often have a sequential series of genetic alterations, the most common of which is T53 gene mutation (3,4). GBMs arising de novo in children (particularly those in the brainstem) differ genetically from their adult counterparts, with approximately 40% having TP53 gene mutations and loss of chromosome 17p (3,4). Two histologic variants of glioblastoma are recognized: gliosarcoma and giant cell glioblastoma. Gliosarcomas comprise approximately 2% of all GBMs (1,2). Grossly, they may resemble a typical GBM but can often present as circumscribed masses resembling a meningioma. Their histologic diagnosis rests on establishing the presence of unequivocally malignant glial and mesenchymal elements within the tumor. The sarcomatous regions usually consist of malignant spindle cells arranged in a fascicular, herringbone, or sometimes storiform pattern and may rarely show other types of mesenchymal differentiation, including cartilage, bone, skeletal, and smooth muscle. Rare examples have shown keratinpositive epithelial and adenoid structures. GFAP immunohistochemistry is very helpful in distinguishing between the glial and mesenchymal components; likewise, a reticulin stain will show abundant reticulin fibrils in the sarcomatous but not glial component. The origin of the sarcomatous component has been traditionally

ascribed to malignant transformation of the proliferating blood vessels in GBM; however, recent molecular studies have shown that the glial and mesenchymal components have a clonal origin (16,17). Giant cell glioblastomas account for less than 5% of all GBMs (1). Histologically, they demonstrate all the features of a GBM but in addition contain giant and multinucleated cells whose morphology well exceeds that of a typical GBM. GFAP expression in the giant cells may be variable. Many examples also have an abundant stromal reticulin network. The presence of anaplastic features distinguishes them from the morphologically similar pleomorphic xanthoastrocytoma (2). Pilocytic Astrocytoma (World Health Organization Grade I) This tumor is a well-circumscribed, slow-growing, often cystic glioma that predominantly occurs in children and young adults (Figs. 2-6 and 2-7) (1–3). They are the most common glioma in children but are much less frequently encountered in adults, being rare after age 50. The most common sites are the cerebellum followed by hypothalamus/third ventricular region, optic nerves, brainstem, cerebral hemispheres, and spinal cord. Depending on their location, they may present grossly as either cystic or solid circumscribed masses. The cerebellar examples are more frequently cystic, often with a mural nodule, whereas the hypothalamic and optic tumors are more likely solid. The current WHO classification does not distinguish between so-called adult and juvenile variants of pilocytic astrocytoma (4). The histologic features of pilocytic astrocytoma are highly distinctive and classically consist of a biphasic pattern of compact and loose (“spongy”) tissue, although the proportion of these two patterns may vary within a given tumor. The compact zones consist of dense aggregates of elongated bipolar astrocytes with variable numbers of strongly eosinophilic irregular Rosenthal fibers; whereas the loose or spongy areas consist of small round multipolar astrocytes in association with microcysts and eosinophilic granular bodies. Some tumors may consist almost exclusively of one histologic pattern. Adding to the diagnostic difficulty is the presence of other tissue patterns (e.g., foci of oligodendrocyte-like cells) that can form part of the histologic spectrum of pilocytic astrocytoma (2). Pilocytic astrocytomas may also show vascular proliferation (including glomeruloid vessels), nuclear atypia, and meningeal infiltration; however, none of these features alone should be interpreted as evidence of anaplastic change. Mitoses are rare, and most examples have a very low Ki-67/MIB-1 labeling index (usually around 1%) (12). True malignant transformation of a pilocytic astrocytoma is very rare, and most examples occur after prior radiotherapy. No specific or diagnostically useful cytogenetic or molecular genetic alterations

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23

FIGURE 2-6. Pilocytic astrocytoma of the cerebellar hemisphere with a large cyst and an irregular mural nodule.

A

B

FIGURE 2-7. Pilocytic astrocytoma with (A) small uniform nuclei associated with regions composed of a fine loose fibrillary meshwork and microcysts and (B) more closely packed neoplastic cells with bipolar cytoplasmic processes with Rosenthal fibers.

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Cancer of the Nervous System

have been identified in pilocytic astrocytomas (2,3); however, a possible role for the NF1 gene is suggested by the well-established association between neurofibromatosis type 1 (NF1) and pilocytic astrocytomas, especially those that occur in the optic nerve pathways. Pleomorphic Xanthoastrocytoma (World Health Organization Grade II) Pleomorphic xanthoastrocytoma is a low-grade slowgrowing astrocytoma that occurs primarily in children and young adults (1,2). It characteristically involves the superficial cortex with extensive involvement of the leptomeninges and may be solid or cystic; the latter may have a mural nodule. Almost all are supratentorial in location and have a predilection for the temporal lobes. Microscopically, pleomorphic xanthoastrocytoma has a varied mix of cell types, including more typically astrocytic cells with fibrillary processes intermingled with often strikingly bizarre giant cells with single or multiple pleomorphic nuclei and variable xanthomatous change in the cytoplasm. Mitoses are usually rare or absent, and necrosis and vascular proliferation are not present. The tumor cells show consistent but often variable GFAP immunoreactivity, and some examples also express neuronal markers, including synaptophysin and neurofilament protein. Other histologic features include the presence of a conspicuous reticulin network (possibly reflecting a putative origin from subpial astrocytes), lymphocytic infiltrates, and eosinophilic granular bodies similar to those found in pilocytic astrocytomas and ganglion cell tumors; the latter are almost always found in pleomorphic xanthoastrocytoma and are considered an important diagnostic feature (2). Pleomorphic xanthoastrocytoma generally has a good prognosis, with recurrence-free 10-year survival rates of greater than 60%. Recurrences may show the same histology as the original tumor or, rarely, show evidence of anaplasia with significantly increased mitotic activity (five or more mitoses per 10 high-power field), necrosis, and vascular proliferation (18). Subependymal Giant Cell Astrocytoma (World Health Organization Grade I) These are benign, slow-growing, intraventricular tumors that are characteristically associated with tuberous sclerosis and in some cases may be the presenting feature of this disease (4). It is unresolved whether they occur in the absence of tuberous sclerosis. They typically occupy the wall of one of the lateral ventricles and may lie in close juxtaposition to the hamartomatous subependymal nodules (“candle gutterings”) of tuberous sclerosis from which these tumors may be derived. They can cause obstruction of the foramen of Monro with resulting hydrocephalus and may rarely undergo massive hemorrhage. These tumors are composed of large cells resembling gemistocytic astrocytes

but often having ganglioid nuclei with prominent nucleoli (2). Spindle-shaped cells with elongated fibrillar processes may also be encountered. Some tumor cells may show considerable nuclear pleomorphism and occasional mitoses may be present; however, these features do not indicate anaplastic change. Calcifications may be present. Immunohistochemically, the tumor cells may express either or both glial and neuronal-associated antigens, which may reflect their putative origin from dysplastic bipotential cells in the subependymal region. The histologic differential diagnosis includes gemistocytic astrocytoma and giant cell glioblastoma (2). Most of these tumors have genetic alterations in the two genes implicated in tuberous sclerosis: the TSC1 gene on chromosome 9q, which encodes the hamartin protein, and the TSC2 gene on 16p, which encodes the tuberin protein (3,4).

Oligodendroglial Tumors Oligodendroglioma (World Health Organization Grade II) Oligodendrogliomas are diffusely infiltrating gliomas composed of cells morphologically resembling mature oligodendrocytes (Fig. 2-8) (1–3). They account for approximately 5% of all intracranial gliomas, and most occur in adults with a peak incidence between the ages of 30 and 60. They are most often found in the cerebral hemispheres but have been reported in the cerebellum, brainstem, and spinal cord and even in the leptomeninges as a primary site. Grossly, these tumors are usually well circumscribed, grayish pink and often include areas of mucoid change, which may result in a gelatinous consistency, and may also contain zones of cystic degeneration, focal hemorrhage, and calcification. The histologic appearance of oligodendroglioma is uniform and usually easily recognizable. In paraffin sections the tumor cells are closely packed and appear swollen, consisting of a small round nucleus (usually slightly larger than a normal oligodendrocyte) surrounded by a clear halo. This imparts a very characteristic “honeycomb” or “fried egg” appearance to the tumor; however, this pattern is not apparent in smear preparations or frozen sections and is often absent in paraffin sections made from previously frozen material. These tumors also characteristically contain a network of thin-walled branching capillaries, often described as a “chicken-wire” vascular pattern. The presence of occasional mitoses and nuclear atypia (which may be marked in some cases) is still compatible with a low-grade (WHO grade II) tumor; however, the presence of significant mitotic activity, vascular proliferation, or necrosis indicates anaplastic transformation (WHO grade III). Most low-grade oligodendrogliomas have a Ki-67/MIB-1 labeling index less than 5% (2). Some oligodendrogliomas may contain nodules of increased cellularity; careful attention to these

2. Neuropathology of Central Nervous System Tumors

25

B

A

FIGURE 2-8. Oligodendroglioma. A. Coronal section showing diffuse infiltration of temporal lobe. B. Microscopic section showing uniform round neoplastic cells with perinuclear halos.

areas may reveal other anaplastic features within these nodules. Small calcifications (calcospherites) are a characteristic histologic feature but are only seen in 20% of cases and are not specific for oligodendroglioma. Another diagnostically useful feature is the presence of perineuronal, perivascular, or subpial tumor aggregates (secondary structures) when the tumor infiltrates cortex. Because there is yet no commercially available immunohistochemical marker specific for neoplastic oligodendrocytes, the histologic diagnosis of oligodendroglioma rests on its characteristic hematoxylin and eosin appearance. GFAP immunostaining can be problematic because gemistocytic astrocytes in the tumor as well as some neoplastic oligodendrocytes may express GFAP. These cells often resemble small gemistocytic astrocytes and are referred to as mini(or micro-) gemistocytes. The presence of these cells has no specific prognostic significance; they may be seen in anaplastic oligodendrogliomas and oligoastrocytomas. Oligodendrogliomas generally have a favorable outcome, with a median postoperative survival of 3 to 5 years. Some patients develop extensive postoperative recurrences with progression to frank anaplasia. Metastases through the cerebrospinal pathways may also occur. Molecular genetic analysis has shown that approximately 80% of histologically typical oligodendrogliomas have concurrent loss of both the 1p and 19q chromosome arms (3,19). Anaplastic Oligodendroglioma (World Health Organization Grade III) The exact incidence of anaplastic oligodendroglioma has been difficult to determine, largely because of the lack of clear-cut histopathologic criteria in prior studies. The age at onset, location, and macroscopic features are similar to those of low-grade oligodendroglioma. These tumors

retain the general histologic appearance of an oligodendroglioma but in addition demonstrate focal or diffuse features of anaplasia, including increased cellularity, cytologic atypia, frequent mitoses, and often (but not always) vascular proliferation and/or necrosis with or without pseudopalisading (20). GFAP-positive mini-gemistocytes and gliofibrillary oligodendrocytes are often present. Although the presence of a clearly identifiable oligodendroglial pattern is a prerequisite for the histologic diagnosis of this tumor, it is acknowledged that some examples may be difficult to distinguish from GBM (2). Anaplastic oligodendrogliomas share in common with low-grade oligodendrogliomas the characteristic loss of chromosome arms 1p and 19q and when present show a remarkable response to combination chemotherapy with procarbazine, chloroethyl-cyclohexyl-nitrosourea, and vincristine and a median survival of over 10 years (3,21). However, some anaplastic oligodendrogliomas have genetic alterations more akin to those found in anaplastic astrocytomas or GBMs with a poor response to chemotherapy with procarbazine, chloroethyl-cyclohexyl-nitrosourea, and vincristine and median survival of less than 2 years (3).

Mixed Gliomas Oligoastrocytoma (World Health Organization Grade II) These tumors are composed of admixtures of astrocytoma and oligodendroglioma. Their exact incidence has been difficult to determine because of the lack of uniformity of the histologic criteria used to define this tumor; it probably varies between 2% and 10% of all gliomas. The age and sex distribution, location, clinical presentation, and macroscopic appearance are all similar to low-grade

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Cancer of the Nervous System

oligodendroglioma. The current WHO classification (4) recognizes two histologic variants: a “compact” or biphasic variant and a “diffuse” or intermingled variant. Molecular studies have shown oligoastrocytomas to be monoclonal tumors having genetic alterations characteristic of either oligodendrogliomas or astrocytomas (3,22). Anaplastic Oligoastrocytoma (World Health Organization Grade III) These are mixed tumors in which either or both glial components show clearly malignant histology (4). Some examples may be difficult to distinguish from GBM because of the presence of common histologic features, including GFAP-positive cells, vascular proliferation, and necrosis. If GBM-like histologic features predominate, it may be preferable to classify these tumors as “glioblastoma with oligodendroglioma component.” Molecular markers may eventually be very useful to help make the diagnosis.

Ependymal Tumors Ependymoma (World Health Organization Grade II) Ependymomas (Fig. 2-9) account for approximately 6% of intracranial gliomas (1). They may occur at any level of the ventricular system and at any age; however, most occur in the posterior fossa in childhood and adolescence. In the spinal cord, ependymomas are the most common neuroepithelial tumor, accounting for approximately 60% of spinal gliomas. They most often occur during midlife, predominantly in the lumbosacral seg-

A

ments and region of the filum terminale/cauda equina. Grossly ependymomas are gray-red, lobulated, and usually well-demarcated tumors that usually but not always have a relationship to a ventricular cavity, where they may cause ventricular obstruction with hydrocephalus. Infratentorial tumors may extend into the cerebellopontine angle or within the cisterna magna along the medulla. In the spinal cord they usually present as circumscribed intramedullary tumors. Some ependymomas, especially those arising infratentorially, may spread widely throughout the CSF and rarely outside the CNS. Ependymomas have a characteristic and easily recognizable microscopic appearance (2,3). The typical tumor is moderately cellular and composed of polygonal cells having uniform nuclei. Two diagnostically important but often inconstant features include the presence of perivascular pseudorosettes and true ependymal rosettes or tubules. Perivascular pseudorosettes are the common pattern and consist of tumor cells arranged radially around a central vessel with a clear region composed of slender cytoplasmic processes oriented perpendicular to the vessel wall. The ependymal rosettes (tubules) are composed of ependymal cells lining central lumens. In general, the tumor cells show minimal atypia and mitoses are rare or absent; however, some WHO grade II ependymomas may have foci of necrosis without pseudopalisading. Occasional examples show myxoid degeneration, focal hemorrhage, and bone and cartilage formation. GFAP immunoreactivity is almost always present in the cytoplasmic process surrounding the perivascular pseudorosettes but is more variable elsewhere; the tumor cells in ependymal canals may also express epithelial membrane antigen. Ultrastructurally, the tumor cells show features of ependymal differentiation,

B

FIGURE 2-9. Ependymoma. A. Tumor fills the fourth ventricle and is associated with hemorrhage. B. Microscopic section shows true ependymal canals and perivascular pseudorosettes.

2. Neuropathology of Central Nervous System Tumors

including cilia, blepharoplasts, surface microvilli, and sometimes microrosettes. Several histologic variants of ependymoma are recognized (4). Cellular ependymomas show high cellularity without increased mitotic rate or other anaplastic features; perivascular rosettes and ependymal tubules are uncommon. Papillary ependymomas have well-formed papillary structures and are distinguished from choroid plexus papillomas by the presence of GFAP-positive processes abutting central vessels. Clear cell ependymomas have tumor cells with prominent clear perinuclear halos; immunohistochemistry or electron microscopy may be needed to distinguish this variant from oligodendroglioma or central neurocytoma. Tanycytic ependymomas have tumor cells arranged in fascicles with illdefined perivascular pseudorosettes and rare ependymal tubules; they most often occur in the spinal cord and may appear similar to astrocytomas or schwannomas. Ependymomas are generally considered to be slowgrowing benign tumors. However, it has been difficult to precisely correlate tumor histology with prognosis, partly because of the inability to define reliable indicators of anaplasia. CSF dissemination is associated with a poorer prognosis. Molecular studies have shown a role for the NF2 tumor suppressor gene (located on chromosome 22q12) in ependymomas associated with neurofibromatosis type 2 (NF2) and possibly in some sporadic spinal ependymomas (23). Anaplastic Ependymoma (World Health Organization Grade III) Anaplastic ependymomas are characterized histologically by increased cellular density and cytologic atypia, frequent mitoses, and usually vascular proliferation and necrosis, although as noted above tumor morphology and clinical outcome do not often correlate well (2). Of the anaplastic features mentioned, only high cell density, a high mitotic index, and a Ki-67/MIB-1 labeling index more than 4% appear to independently predict a poorer outcome (24). The presence of necrosis alone does not indicate anaplastic change. Anaplastic ependymoma must be distinguished from ependymoblastoma, a rare embryonal neuroectodermal tumor composed of densely packed small cells resembling medulloblastoma and having small primitive ependymal rosettes (see below). Myxopapillary Ependymoma (World Health Organization Grade I) This tumor almost exclusively occurs in the region of the conus medullaris and cauda equina where it is thought to arise from ependymal cells located within the filum terminale. Its clinical and pathologic features are distinct, and it must be thought of as an entity separate from

27

other ependymomas (1,2). Myxopapillary ependymomas may occur at any age but are generally more common in early middle age. Grossly, they are well-circumscribed lesions but sometimes may erode into adjacent bone and soft tissues. Some examples may show cystic change or hemorrhage. They have a distinctive histologic appearance, consisting of GFAP-positive cuboidal tumor cells arranged around vascularized stromal cores, which exhibit variable amounts of mucoid material or fibrous tissue. Mitoses are very rare or absent, and other features of anaplasia are not present. This tumor has an extremely favorable outcome after surgical resection. Glial tumors histologically similar to myxopapillary ependymomas can also occur in the subcutaneous tissues of the sacrococcygeal or presacral region where they are considered to arise from ectopic ependymal remnants. This variant has a somewhat more aggressive clinical behavior, with a greater rate of regrowth and potential for metastatic dissemination (3,25). Subependymoma (World Health Organization Grade I) Subependymomas are well-demarcated, slow-growing, benign tumors composed of cells resembling subependymal glia (1,2). They are typically attached to the ventricular wall and project into the lumen. They may occur at any site but are most often encountered in the fourth ventricle. Most subependymomas are clinically silent, but occasionally some are symptomatic due to ventricular obstruction, increased intracranial pressure, or rarely, spontaneous hemorrhage. Microscopically, they consist of small nests of glial cell nuclei embedded in a hypocellular GFAP-positive fibrillary matrix. Mitoses are rare or absent. Additional histologic features include the presence of microcysts, calcifications, focal hemorrhage, abnormal vasculature, and Rosenthal fibers. Some tumors, especially those occurring in the fourth ventricle in children, may have histologic features of both ependymoma and subependymoma. Their clinical behavior appears to be more aligned with ependymomas, and they probably should be classified as such (2).

Choroid Plexus Tumors Choroid plexus tumors (papilloma and carcinoma) account for 0.5% of all brain tumors and for 2% of the tumors of the glioma group. They are encountered most often in the first decade, with 10% to 20% presenting in the first year of life. The ratio of choroid plexus papilloma to carcinoma is approximately 5:1; however, 80% of the carcinomas arise in children. They may occur in any region of the ventricular system where choroid plexus is normally found. Most lateral ventricular tumors occur in individuals less than 20 years old, whereas those in the

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Cancer of the Nervous System

fourth ventricle are more evenly age distributed. Patients present clinically with signs of hydrocephalus and increased intracranial pressure but only rarely do they produce excessive amounts of CSF. They are treated primarily by surgical excision. The prognosis is excellent for choroid plexus papilloma (nearly 100% 5-year survival rate) but much less favorable (40%) for carcinoma. Choroid Plexus Papilloma (World Health Organization Grade I) Grossly, these tumors are well-defined friable papillary masses that originate from the choroid plexus and protrude into the surrounding ventricle, which may be distended (Fig. 2-10). On section, they are often gritty because of calcium deposits. Their histology resembles normal choroid plexus except for having slightly more crowded and elongated papillae. The individual papilla consist of a simple columnar or cuboidal epithelium with a vascular connective tissue core (in contrast to the gliovascular cores of papillary ependymoma). Prominent mitotic activity, necrosis, and brain invasion are absent. Most choroid plexus papillomas are immunoreactive for transthyretin as well as vimentin, cytokeratin, and S-100 protein but not epithelial membrane antigen (2,3,26,27). Focal staining for GFAP may also be present. Choroid Plexus Carcinoma (World Health Organization Grade III) These tumors show clear histologic evidence of malignancy, including frequent mitoses, nuclear and cytoplasmic atypia, more solid growth pattern with at least some loss of papillary architecture, and often brain invasion. The diagnosis of choroid plexus carcinoma is more confi-

dently made in children, whereas in adults the major differential diagnosis is that of metastatic carcinoma, which would be distinctly more common in this age group. Immunohistochemistry may be helpful, especially if the tumor expresses markers that are considered more characteristic of choroid plexus; however, some of these markers (e.g., transthyretin) are not always expressed in choroid plexus carcinoma or conversely may also be expressed by some metastatic carcinomas (2).

Glial Tumors of Uncertain Origin Astroblastoma This is a rare glial neoplasm of uncertain histogenesis (2,28,29). They most often occur in the first three decades of life. Grossly, they are well-circumscribed solid or cystic masses that occur mainly in the cerebral hemispheres but may be found anywhere in the CNS. Astroblastomas histologically have perivascular pseudorosettes composed of GFAP-positive tumor cells whose processes radiate to a central blood vessel that is often thick and hyalinized. Mitotic activity and other anaplastic features are variable, and both low- and high-grade examples have been described. The major histologic differential diagnoses include ependymoma, papillary meningioma, and especially astrocytomas and glioblastomas that may have focal astroblastoma-like histology. Gliomatosis Cerebri This tumor is defined as a diffusely infiltrating glioma that involves more than two lobes of the brain (2,4). It is frequently bilateral and may extend into posterior fossa structures and even the spinal cord. Its histogenesis has been controversial, although it is now thought to represent the most infiltrative example of a clonal diffuse astrocytoma. Its microscopic appearance can be variable, with some neoplastic cells resembling astrocytes and others having more indeterminate features or rarely oligodendroglial histology. GFAP immunostaining can likewise be variable. The tumor nuclei usually show enough atypia to facilitate their identification as neoplastic, although mitoses may be minimal or even absent. Vascular proliferation and necrosis are usually absent. A diagnosis of gliomatosis cerebri cannot be made histologically from a small biopsy specimen but may be suggested if warranted by the imaging studies in a particular case (2). Chordoid Glioma of the Third Ventricle

FIGURE 2-10. Choroid plexus papilloma with complex fronds containing fibrovascular stalks.

This is a recently described glioma that exclusively occurs within the rostral third ventricle (30). It consists of nests and cords of GFAP-positive epithelioid cells within a mucinous stroma, often containing lymphoplasmacytic

2. Neuropathology of Central Nervous System Tumors

infiltrates with prominent Russell bodies. They are lowgrade tumors treated by surgical excision.

Neuronal and Mixed Neuronal–Glial Tumors These tumors are much less common than the pure glial tumors described above and as a rule tend to occur most often in children and young adults. They are made up either entirely or partly of neoplastic cells that have a neuronal phenotype. The degree of neuronal differentiation exhibited by these cells is variable depending on the type of tumor. Some tumors (e.g., gangliocytoma) consist almost entirely of mature ganglion cells that are usually easy to recognize by light microscopy. Other tumors (e.g., neuroblastoma) are composed mainly of small poorly differentiated cells that may require electron microscopy or immunostaining for proteins such as neurofilament or synaptophysin for positive identification of their neuronal phenotype. Some tumors (e.g., ganglioglioma) are composed of both neoplastic neuronal and glial elements. Gangliocytoma and Ganglioglioma Gangliocytomas and gangliogliomas may be found in almost any region of the brain and spinal cord (2,3,31). A considerable number arise in the temporal lobe, where they are often associated with a history of seizures. Their hamartoma-like histologic features and frequent location within or adjacent to foci of cortical dysplasia suggest a possible etiologic association. Grossly, they are circumscribed homogeneous masses having a uniform granular cut surface and occasionally small cysts or calcifications. Gangliocytomas (WHO grade I) consist entirely of mature ganglion cells without the presence of a neoplastic glial component. The neoplastic cells may resemble entirely normal neurons; however, various cytoarchitectural abnormalities are more often encountered, which may include the presence of binucleated or cytologically bizarre forms, loss of normal polarity, and abnormal clustering of the neoplastic cells. Calcifications are often present, and blood vessels may show lymphocytic cuffing. These tumors do not show anaplastic features, including mitoses, necrosis, or vascular proliferation. Gangliogliomas (WHO grade II or III) have both neoplastic neuronal and glial components; the latter are usually astrocytic, although oligodendroglial differentiation has been described. In some cases, the glial component may overshadow the neuronal component, whose identification may require immunohistochemical staining for neuronal markers such as neurofilament proteins or synaptophysin. Care must be taken that “entrapped” neurons (e.g., cortex infiltrated by astrocytoma) are not mistakenly identified as neoplastic cells. The behavior of

29

gangliogliomas is determined by the degree of anaplasia of the glial component. Most gangliogliomas are lowgrade slow-growing tumors treated by surgical excision. Rarely, the glial component may show anaplastic features, including mitotic activity, vascular proliferation, and even necrosis. Depending on the degree of anaplasia, these tumors would be classified as WHO grade III (anaplastic ganglioglioma) or very rarely grade IV (glioblastoma) (4). Dysplastic Gangliocytoma of the Cerebellum (Lhermitte-Duclos Disease) This is a unique form of gangliocytoma that occurs exclusively in the cerebellum (2,3). It has been long debated whether this lesion should be classified as hamartomatous or neoplastic. The malformation-like histology, very low or absent proliferative activity, and lack of progression favor hamartoma; however, it may occasionally recur after surgical resection in the manner of a true neoplasm. About half of the cases are associated with Cowden syndrome, which is a constellation of multiple verrucous skin lesions, facial trichilemmomas, fibromas of the oral mucosa, hamartomatous polyps of the gastrointestinal tract, and thyroid and breast tumors (both benign and malignant). Cowden syndrome arises from mutations in the PTEN gene on chromosome 10q; thus, it is likely that altered PTEN function is related to the pathogenesis of dysplastic cerebellar gangliocytoma (32). Grossly, the lesion is characterized by diffuse enlargement of cerebellar folia, often with a coarse gyral pattern, that almost always affects only one hemisphere. Microscopically, the dysmorphic cerebellar folia consist of a superficial layer of abnormally myelinated axons and an expanded inner layer of dysplastic and disorganized neurons, a pattern often referred to as inverted cerebellar cortex. The neuronal component consists of cells similar to granular neurons and a smaller number of large cells that morphologically and immunohistochemically resemble Purkinje cells. The histologic differential diagnosis includes ganglioglioma, which may also occur in the cerebellum but contains a population of neoplastic glia in addition to a ganglion cell component. Desmoplastic Infantile Ganglioglioma (World Health Organization Grade I) This tumor is a rare variant of ganglioglioma, which is largely restricted to patients in their first year of life (2,33). They characteristically involve the cortical surface, most often in the frontal and parietal regions, where they may be quite large and adhere to the overlying meninges and are often associated with one or more deep cysts. Histologically, these tumors are characterized by their abundant connective tissue stroma

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(desmoplasia) and intermixture of astrocytic and ganglion cell components, whose recognition is greatly facilitated by the use of appropriate immunostains. The ganglion cells are usually less frequent compared with the astrocytes and may even be entirely absent, in which case the tumor is designated desmoplastic astrocytoma of infancy. Some examples may contain aggregates of small poorly differentiated neuroepithelial cells, although their presence does not appear to alter the overall favorable behavior and prognosis of this tumor. These tumors are slow growing and treatable by surgery. Dysembryoplastic Neuroepithelial Tumor (World Health Organization Grade I) Dysembryoplastic neuroepithelial tumors are low-grade tumors that predominantly occur in children and are nearly always associated with a history of partial complex seizures (2,3,34). They have been described in a wide variety of sites but are most often seen in the temporal lobe. Deformity of the overlying skull is common and supports a longstanding chronic process. Their cellular composition and slow growth as well as their frequent association with cortical dysplasia suggest a possible developmental or hamartomatous origin for this tumor. Histologically, dysembryoplastic neuroepithelial tumors have a distinctive multinodular architecture, the individual nodules consisting of a variable admixture of oligodendroglial and/or astrocyte-like cells and neuronal elements, including small neurocytic cells and larger neurons. The nodules are typically intracortical, but most examples also have subcortical nodules. Some cortical nodules may have a distinctive cytoarchitecture referred to as the specific glioneuronal element of dysembryoplastic neuroepithelial tumor in which the small round oligodendroglial- or neurocyte-like cells are admixed with axon bundles bounding mucinous microcystic spaces containing larger ganglion cells (“floating neurons”). Other nodules may have histologic features more closely resembling typical oligodendroglial, astrocytic, or mixed tumors. Cytologic atypia may be observed in some examples, but other features of anaplasia, including mitoses, vascular proliferation, and necrosis, are rarely if ever present. Surgical excision is usually considered curative. Central Neurocytoma (World Health Organization Grade II) Central neurocytoma is a low-grade neuronal tumor that usually arises in the third or lateral cerebral ventricle in the region of the foramen of Monro, most commonly in young adults (2,3,35). Grossly, they are well-circumscribed intraventricular masses that may show focal calcification. Microscopically, they are composed of small

cells with uniform round nuclei, fine chromatin, and occasional nucleoli within a neuropil-like background. The neuronal character of the tumor is readily ascertained by immunohistochemistry for markers such as synaptophysin or neurofilament. This tumor is treated by surgical resection, and most patients have good long-term survival. Atypical and malignant phenotypes have been recognized, however, that are characterized by dense cellularity, vascular proliferation, mitotic activity, infiltration of adjacent brain, and a tendency to recur. Central neurocytomas share a similar histologic appearance with both oligodendrogliomas and ependymomas. A diagnosis of oligodendroglioma may be suggested by the presence of perinuclear halos, which are often present because of fixation artifact. In fact, central neurocytomas were initially thought to be unusual intraventricular oligodendrogliomas until their neuronal character was established by electron microscopy. Likewise, a diagnosis of ependymoma may be suggested by the intraventricular location of the tumor, presence of structures resembling perivascular pseudorosettes, and perinuclear halos that are seen in the clear cell variant of ependymoma. In most cases, immunohistochemistry for neuronal markers and GFAP and, if necessary, electron microscopy will allow a distinction to be made among these tumors. Cerebellar Liponeurocytoma (World Health Organization Grade I/II) This is an uncommon tumor composed of small neoplastic neurocytes and lipidized cells that resemble adipocytes, both of which express neuronal markers such as synaptophysin and MAP-2 (3,4,7). The tumor usually shows no mitotic activity and has a Ki-67/MIB-1 proliferative index of 1% to 3%. In keeping with its low-grade character, this tumor has a favorable prognosis. Paraganglioma (World Health Organization Grade I) Tumors of extraadrenal chromaffin tissue (paragangliomas) analogous to pheochromocytomas of the adrenal gland may arise within the cranial vault and spinal canal (2,3). Most often they are circumscribed nodules in the filum terminale, although cranial and spinal nerve root masses extending into the skull or vertebral foramina have been noted (e.g., extension of a middle ear “glomus jugulare” tumor into the posterior fossa in the cerebellopontine angle). All these tumors are identical histologically to paragangliomas occurring elsewhere in the body and consist of nests or “zellballen” of plump neuroendocrine cells separated by fine fibrovascular septa. The tumor cells typically are immunoreactive for chromogranin and synaptophysin with interspersed sustentacular cells that are S-100 protein positive. They

2. Neuropathology of Central Nervous System Tumors

only rarely produce catecholamines, instead causing symptoms as a result of local compression. The biologic behavior of these neoplasms is determined more by their anatomic extent at the time of presentation than by histologic features.

Neuroblastic Tumors Olfactory Neuroblastoma (Esthesioneuroblastoma) Olfactory neuroblastomas are small “blue-cell” tumors that arise from the olfactory epithelium of the upper nasal cavity (2–4). They occur in late childhood through adulthood (mean age, between 40 and 60 years) and present clinically with symptoms of nasal obstruction or headache. The tumor may extend through and destroy the cribriform plate, allowing growth of tumor into the anterior cranial fossa, meninges, and frontal lobes of the brain. They have a characteristic broad nodular growth pattern. Histologically, the neoplastic cells show a neuronal immunohistochemical phenotype and contain neurosecretory granules by electron microscopy. The tumor cells are arranged in a lobular configuration with varying degrees of neurofibrillary background (sometimes forming Homer-Wright or Flexner-Wintersteinertype rosettes), nuclear atypia, mitoses, and necrosis. The tumor lobules also characteristically have a peripheral rim of S-100–positive cells similar to sustentacular cells. Hyams et al. (36) proposed a four-level grading scheme based on the degree of differentiation, cellular anaplasia, and mitotic rate, which has been simplified into lowgrade (Hyams grade I and II) and high-grade (Hyams grade III and IV) categories; they have been found to correlate fairly well with disease outcome (4). Some highgrade examples may have nuclear and architectural features as well as coexpression of cytokeratin and neural markers that favor their classification as neuroendocrine carcinomas. Peripheral Neuroblastic Tumors These tumors arise within the neural crest–derived adrenal gland or sympathetic nervous system (4). Almost all of them occur in the first or second decades of life. These tumors most often present as abdominal or thoracic masses, which may cause spinal cord compression if the tumor extends through the spinal foramina. Grossly, they can become very large (up to 10 cm), are encapsulated, and may contain hemorrhages, calcifications, or cysts. Histologically, these tumors consist of neuronal cells (ranging from neuroblasts to mature ganglion cells depending on degree of differentiation) and schwannian stroma. Tumors composed mainly of small undifferentiated cells may require immunohistochemistry

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or electron microscopy to demonstrate the neuronal phenotype. The morphologic classification of peripheral neuroblastic tumors is based on the modified Shimada scheme (37). The major categories are: (a) neuroblastoma, the most primitive, that consists almost entirely of densely packed small blue cells, sometimes forming Homer-Wright rosettes and neuropil stroma, but with little or no schwannian stroma; (b) ganglioneuroblastomas (intermixed and nodular subtypes), consisting of mixtures of neuroblasts, “differentiating” neuroblasts, and mature neurons with variable schwannian stroma; and (c) ganglioneuroma, consisting entirely of schwannian stroma with scattered mature ganglion cells.

Pineal Tumors The most common tumors involving the pineal gland, in decreasing order of frequency, are germ cell tumors, tumors of pineal parenchymal cells (pineocytes), and glial tumors (1–5,38,39). (Although pineal germ cell tumors are not derived from neuroepithelial cells, they will be included in this section.) Germ Cell Tumors Intracranial germ cell tumors are thought either to arise from the neoplastic transformation of an otherwise normal resident population of germ cells lying in the midline, between the cerebral hemispheres, or to derive from ectopic germ cells (Fig. 2-11). The histologic appearance of intracranial germ cell tumors is identical to that of germ cell tumors occurring in other parts of the body, such as the gonads. Germinoma is the most commonly encountered tumor of the pineal gland. The tumor may arise in the pineal or posterior third ventricle region, the suprasellaranterior third ventricle area, or both sites, either through seeding (ordinarily from back to front) or from independent neoplastic transformation. Macroscopically, the tumor is gritty, granular, and gray. Commonly, the neoplasm extends into neighboring tissues along the walls of the third ventricle and midbrain tectum, the ventricular system (with aqueductal obstruction), and the subarachnoid space with implantation along the spinal cord. Microscopically, they consist of two cell populations. First, the neoplastic cells are clustered in nests or sheets and have a polygonal shape with sharply defined cell borders, fluffy periodic acid–Schiff-positive cytoplasm, and a centrally placed nucleus with moderately compact chromatin, and an unusually prominent nucleolus. The other cell type is composed of mature nonneoplastic lymphocytes scattered or clumped together between the tumor cells. Mitoses, necrosis, and nuclear pleomorphism, all features evidencing rapid growth, are often present in and around the tumor cells. Germinoma may occur as a

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A

B

FIGURE 2-11. Germinoma of pineal region. (A) Sagittal section. (B) Microscopic section showing tumor cells with large nucleus and nucleoli and vesicular cytoplasm. A second population, lymphoid cells, is also shown.

“pure” lesion or as a mixed tumor containing components of cystic or solid teratoma, choriocarcinoma, embryonal carcinoma, or endodermal sinus tumor. Other germ cell neoplasms have macroscopic and microscopic features identical to those seen at extracranial sites. Intracranial teratomas may attain enormous size, replacing or greatly compressing midline structures. Immunoperoxidase methods have helped to sharpen the diagnostic criteria for germ cell neoplasms. The commonly used battery of reactions includes placental alkaline phosphatase, which is typically positive in germinoma and often in other germ cell tumors; ␣-fetoprotein, which is typically positive in endodermal sinus tumors; and human chorionic gonadotropin, which is typically positive in choriocarcinoma. Immunostaining for leukocyte common antigen (CD45) highlights the lymphoid infiltrates in germinoma. Pineal Parenchymal Tumors Pineocytoma (WHO grade II) occurs in young to middleaged adults and represents about 45% of all pineal parenchymal tumors. Microscopically, it has a lobular architecture recalling the normal structure of the pineal gland and is composed of cells with uniform round to oval nuclei, occasional fine nucleoli, and fibrillary or clubshaped eosinophilic processes, which may converge in the center of rosettes. Variable amounts of neuropil may be present in the background. The tumor cells and their processes are characteristically immunoreactive for neural markers such as synaptophysin, chromogranin A, and neurofilament, and special silver impregnation techniques may bring out the characteristic club-shaped

expansions at the terminal ends of the cell processes (40). Some examples may show evidence of photoreceptor differentiation (e.g., immunoreactivity for retinal S-antigen and rhodopsin). Ultrastructurally, the tumor shows features of neural differentiation (synaptic vesicles, microtubules) and sometimes photoreceptor differentiation (cytoplasmic annulate lamellae, cilia). The tumor is histologically low grade and lacks mitotic activity or pleomorphism. Pineocytomas are well circumscribed and slow growing but not easily resectable because of their central location. Nonetheless, 5-year survival rates of up to 90% may be expected. Pineoblastoma (WHO grade IV) presents primarily in children and young adults as a rapidly growing mass that may show hemorrhage, necrosis, and cystic degeneration. Microscopically, it is a densely cellular blue-cell tumor (resembling medulloblastoma) consisting of cells with oval hyperchromatic nuclei and little visible cytoplasm, resulting in “molding” of nuclear contours. Some cells may have recognizable eosinophilic processes or form rosettes of Homer-Wright or Flexner-Wintersteiner type. Pineoblastoma has an immunohistochemical profile similar to pineocytoma, although expression may be more variable. Evidence of malignancy is manifested by invasion of adjacent brain structures, high mitotic activity, and necrosis. Pineoblastoma can gain access to the CSF and spread within the ventricles and subarachnoid space to other sites in the neuraxis. Pineal parenchymal tumor of intermediate differentiation can occur at all ages and is rare, comprising about 10% of all pineal parenchymal tumors. Microscopically, it has more sheet-like architecture and atypical cytology than pineocytoma but does not have the overt small cell

2. Neuropathology of Central Nervous System Tumors

malignant phenotype of pineoblastoma. Biologic behavior and clinical survival are intermediate between those of the aggressive pineoblastoma and more indolent pineocytoma. Because the normal pineal gland contains glial cells and some neurons in addition to the pineal parenchyma cells, it is not surprising that gliomas (astrocytoma, ependymoma, GBM) and rarely ganglion cell tumors may occur as primary pineal tumors. The pineal gland may also be secondarily invaded by tumors arising in other sites (e.g., glioma, meningioma, hemangiopericytoma, sarcoma, melanoma, metastases).

Embryonal Tumors These are tumors composed largely (but not always exclusively) of immature small blue cells resembling primitive neuroepithelium, the origin of the precursor cells of the nervous system (1–5). Tumors made up of these cells may retain the capability to differentiate along neural, glial, and sometimes even mesenchymal lines, as demonstrated by immunohistochemical or ultrastructural means. Most of these tumors arise predominantly in infants and young children, and all are mitotically active clinically aggressive neoplasms that tend to grow to a relatively large size and have a characteristic predilection for spread (“seeding”) along CSF pathways. It has been proposed that all tumors in this group, regardless of their location or origin within the CNS, should be classified as primitive neuroectodermal tumors (PNETs) (41,42). This was largely based on the assumption that they all arose from common neuroepithelial progenitor cells located in the subependymal germinal matrix layer and would thus have similar morphologic, biologic, and clinical features. However, evidence from recent molecular genetic studies as well as differences in clinical behavior and response to adjuvant therapy among these tumors suggest that this may be an overly simplistic approach. For this reason, we believe that it is best to avoid use of the generic term PNET for these tumors, especially when the site of origin of the tumor is well established. This would also avoid confusion with the extracranial Ewing/PNET group of tumors, whose molecular genetic and clinical characteristics clearly differ from their intracranial counterparts. Medulloepithelioma (World Health Organization Grade IV) This is a rare tumor arising in infants and young children. It may occur throughout the CNS but is most often encountered in the cerebral hemispheres in a periventricular location. Grossly, these tumors are often quite large and circumscribed and may have foci of necrosis and hemorrhage. Histologically, the tumor mimics the embryonic neural tube, with a distinctive papillary or tubular (“glan-

33

dular”) pattern of immature neuroepithelial cells resting on an external basement membrane. These cells exhibit a very high mitotic rate and are immunoreactive mainly for nestin and vimentin. Regions of the tumor away from the neuroepitheliomatous areas will often show divergent differentiation to neural, glial, and even mesenchymal elements. Medulloepitheliomas are rapidly growing aggressive tumors that usually lead to death within a year, often with evidence of CSF dissemination (43). Ependymoblastoma (World Health Organization Grade IV) This tumor arises primarily within the cerebral hemispheres of infants and young children. Grossly, they often present as a large circumscribed mass bordering a ventricle. Microscopically, it is a small blue-cell tumor characterized by the presence of multilayered rosettes with a true lumen bounded by ciliated neuroepithelial cells. The rosettes may be scattered or form back-to-back arrangements within a population of small undifferentiated cells. As in other CNS embryonal tumors, the neoplastic cells may show immunohistochemical evidence of both glial and neuronal differentiation. Ependymoblastoma is distinguished from anaplastic ependymoma by the presence of multilayered rosettes and the lack of perivascular pseudorosettes that characterize the latter tumor. It should be noted that medulloepithelioma (see above) may also contain ependymoblastoma-type rosettes. Ependymoblastoma behaves in a malignant fashion, with rapid growth and the capacity for CSF dissemination and occasionally extraneural metastasis. Medulloblastoma (World Health Organization Grade IV) The medulloblastoma (Fig. 2-12) has been considered the “prototypical” embryonal CNS tumor, and indeed it is the most common tumor in this category (1–5). Among the primary CNS tumors occurring in children, it is second in frequency only to pilocytic astrocytoma; however, a second peak age incidence for medulloblastoma occurs in early adulthood. Medulloblastoma by definition arises in the cerebellum, usually the vermis in children, where it may cause ataxia, headache, and vomiting due to acute obstruction of the fourth ventricle with hydrocephalus. In adults, the tumor more often arises in the lateral hemispheres. Neuroimaging studies reveal enhancing lobulated masses, and up to one-third of patients will have leptomeningeal enhancement representing CSF seeding at presentation. Recent advances in the diagnosis and treatment of medulloblastoma have resulted in improved 5-year survival rates in the range of 50% to 70%. There has been debate over the years as to the putative cell or site of origin of medulloblastoma. There is now fairly convincing evidence, supported by molecular

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A

B

C

FIGURE 2-12. Medulloblastoma. A. Sagittal section shows tumor extending from cerebellum into fourth ventricle and brainstem. B. Microscopic sections of desmoplastic variant shows high cellularity with some regions showing a tendency to form regions that are loose with fibrillarity (“spongioblastic’’). C. Surrounded by reticulin.

studies, that at least one medulloblastoma subtype (desmoplastic medulloblastoma [see below]) arises from primitive neuroepithelial cells comprising the external granular layer of the neonatal cerebellar cortex (44,45). Other probable sites of origin include the posterior medullary velum or cerebellar vermis. Histologically, medulloblastomas are densely cellular small blue-cell tumors composed of small dark round to oval cells with intensely hyperchromatic nuclei, usually but not always with conspicuous mitoses and single cell necrosis (apoptosis). Larger areas of necrosis and sometimes vascular proliferation may be present. In keeping with their origin from primitive neuroepithelial cells, medulloblastomas can exhibit neuronal or glial differentiation, although this may not always be apparent. In our experience, medulloblastomas more often show neuroblastic differentiation, indicated by the presence of Homer-Wright rosettes with central neurofibrillary processes, which will show immunoreactivity for neuronal markers such as synaptophysin or neurofilament protein. Glial (“spongioblastic”) differentiation does occur but is often more difficult to identify with certainty, because GFAP-immunoreactive processes in the tumor may also originate from reactive astrocytes in addition to neoplastic cells. The prognostic significance of cellular differentiation in medulloblastoma remains controversial (4). There are several well-characterized histologic variants of medulloblastoma. Desmoplastic medulloblastoma tends to occur more often in adults in a lateral location and is firm because of the presence of a collagenous stroma. It always has a nodular architecture consisting of pale reticulin-free neuropil-rich islands surrounded by densely cellular actively proliferating cells enmeshed in a dense reticulin-collagen network. A similar-appearing but much rarer variant is the medulloblastoma with extensive nodularity and advanced neuronal differentiation (“cerebellar neuroblastoma”) (46). It also has a conspicuous nodular architecture but differs from the desmoplastic subtype by less conspicuous collagen stroma and greater evidence of neuronal differentiation in the form of central neurocytoma-like areas and occasional presence of mature ganglion cells. Both the desmoplastic and nodular subtypes may have a slightly better prognosis than classic medulloblastoma. A more recently described subtype is the large cell (anaplastic) medulloblastoma, which represents about 4% of all medulloblastomas (47,48). They are usually located in the vermis and have a very aggressive behavior, with early CSF dissemination. Microscopically, they are composed of closely packed tumor cells with large round or pleomorphic nuclei with prominent nucleoli. Mitoses and apoptotic cells are abundant, and they often show large areas of necrosis. This variant also shows immunohistochemical staining for synaptophysin, although Homer-Wright rosettes are not observed. The medullomyoblastoma contains tumor cells representing

2. Neuropathology of Central Nervous System Tumors

rhabdomyoblasts in addition to the typical histologic features of medulloblastoma (49). The rhabdomyoblastic cells are best demonstrated by immunohistochemistry for myoglobin, desmin or fast myosin, or by electron microscopy showing thick and thin myofilaments and Z bands. The melanotic medulloblastoma has scattered tubular clusters of pigmented melanosome-laden tumor cells. These latter two variants are quite rare. The most common cytogenetic alteration in medulloblastoma is isochromosome 17q, which is a chromosomal abnormality characterized by loss of all or most of the short arm of chromosome 17, replaced by a duplicated long arm (3). Medulloblastomas are also associated with two hereditary tumor syndromes. Gorlin syndrome (nevoid basal carcinoma syndrome), which also has cutaneous tumors (basal cell carcinomas, epidermoid cysts) and ovarian fibromas, is due to a mutation in the PTCH gene on chromosome 9q (3). Some sporadic medulloblastomas have also been found to have mutations of the PTCH gene (50). Turcot syndrome has two components, one due to germline mutations in the APC gene on chromosome 5q and characterized by the association of medulloblastoma and colonic polyposis (3), and the other due to defects in DNA mismatch repair genes and characterized by non-polyposis colon cancer and malignant gliomas, but not medulloblastoma. The genetic abnormalities in these two syndromes have implicated the SHH-PTCH-SMO and the Wnt-ß-catenin pathways, respectively, in medulloblastoma tumorigenesis (3).

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posterior fossa, the latter with a predilection for the cerebellopontine angle (51,52). It grossly resembles other embryonal CNS tumors and shares a similar propensity for CSF dissemination. The characteristic histologic feature of AT/RT is the presence of rhabdoid cells, which are medium-sized round to oval cells having distinct borders, an eccentric nucleus with a prominent nucleolus, and homogeneous cytoplasm, sometimes having an ill-defined eosinophilic “inclusion.” The rhabdoid cells are almost always immunoreactive for vimentin and epithelial membrane antigen but are more variably reactive for other markers, including smooth muscle actin, cytokeratin, GFAP, and neurofilament protein; they do not react for germ cell markers or desmin. Most AT/RTs also have a primitive small cell component resembling medulloblastoma, and some examples may also have malignant mesenchymal or epithelial elements. The proportion of the various components within a given tumor can be quite variable, and the rhabdoid cells may be either extremely numerous or scarce. Abundant mitoses and necrosis are usually present. The overall prognosis of AT/RT is very poor, with most patients dying within a year of diagnosis. AT/RTs are nearly always associated with allelic loss of one copy of chromosome 22 and mutation in the INI1/SNF5 gene on the remaining copy of 22q (53,54). Molecular testing for these two genetic changes can be helpful in diagnosing difficult cases of AT/RT.

TUMORS OF PERIPHERAL NERVES Supratentorial Primitive Neuroectodermal Tumor (World Health Organization Grade IV) Supratentorial PNET is an uncommon embryonal neoplasm that arises in the cerebrum or suprasellar region in infants and young children (4). Grossly and histologically they resemble other primitive small blue-cell CNS tumors such as medulloblastoma and can similarly show immunohistochemical or ultrastructural evidence of divergent differentiation along glial, neuronal, ependymal, muscle, or melanocytic lines. Supratentorial PNETs with evidence of clear-cut neuronal differentiation are termed cerebral neuroblastoma. These tumors may have a nodular growth pattern, Homer-Wright rosettes, and a neuropil background that is immunoreactive for synaptophysin or neurofilament protein. Rare examples containing a combination of undifferentiated cells and mature neurons are designated ganglioneuroblastoma. Atypical Teratoid/Rhabdoid Tumor (World Health Organization Grade IV) Atypical teratoid/rhabdoid tumor (AT/RT) is a unique embryonal neoplasm that occurs predominantly in infants and young children within the cerebral hemispheres or

These tumors arise from cells of the peripheral nerve, including Schwann cells, perineurial cells, and fibroblasts (55,56). Many express Schwann cell characteristics, including the presence of S-100 antigen and the potential for melanocytic differentiation. As nerves enter and exit the brainstem and spinal cord, there is a transition between myelination by oligodendrocytes and myelination by Schwann cells. This occurs within several millimeters of the substance of the CNS (with notable exceptions, i.e., the eighth cranial nerve); thus, peripheral nerve tumors can arise within the dura and may cause changes in adjacent brain or spinal cord. Tumors of comparable histogenesis and biologic behavior also arise along the peripheral course of nerves.

Schwannoma (World Health Organization Grade I) These benign tumors arise from the neural crest-derived Schwann cell and are associated with NF2 (see below). Symptoms are referable to local compression of the involved nerve or to compression of adjacent structures (such as brainstem or spinal cord).

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Schwannomas are well-circumscribed encapsulated masses that are attached to the nerve but can be separated from it. Tumors are firm gray masses but may also have areas of cystic and xanthomatous change. On microscopic examination, tumors show a mixture of two growth patterns. In the Antoni A pattern of growth, elongated cells with cytoplasmic processes are arranged in fascicles in areas of moderate to high cellularity with little stromal matrix; the “nuclear-free zones” of processes that lie between the regions of nuclear palisading are termed Verocay bodies. In the Antoni B pattern of growth, the tumor is less densely cellular with a loose meshwork of cells along with microcysts and myxoid changes. In both areas, the cytology of the individual cells is similar, with elongated cell cytoplasm and regular oval nuclei. Electron microscopy shows basement membrane deposition encasing single cells and long-spacing collagen. Because the lesion displaces the nerve of origin as it grows, silver stains or immunostains for neurofilament proteins demonstrate that axons are largely excluded from the tumor, although they may become entrapped in the capsule. The Schwann cell origin of these tumors is borne out by their S-100 immunoreactivity. A variety of degenerative changes may be found in schwannomas, including nuclear pleomorphism, xanthomatous change, and vascular hyalinization. Sporadic schwannomas are associated with mutations in the NF2 gene on chromosome 22; there is usually absence of the NF2 gene product by Western blotting or immunostaining, even if there is not evidence of a mutation in the gene (57).

Neurofibroma (World Health Organization Grade I) Two histologically, and perhaps biologically, distinct lesions have been termed neurofibromas (55,56). The most common form occurs in the skin (cutaneous neurofibroma) or in peripheral nerve (solitary neurofibroma). These arise sporadically or in association with NF1 (see below). The skin lesions are evident as nodules, sometimes with overlying hyperpigmentation; they may grow to be large and become pedunculated. The second type is the plexiform neurofibroma, which is considered by some to occur only in patients with NF1. Plexiform neurofibromas arising in individuals with NF1 show loss of the wild-type copy of the NF1 gene (58). Cutaneous neurofibroma occurs in the dermis and subcutaneous fat. These well-delineated but unencapsulated masses are composed of spindle cells. Although they are not invasive, the adnexal structures are sometimes enwrapped by the edges of the lesion. The stroma of these tumors is highly collagenized and contains little myxoid material. Lesions within peripheral nerves are of identical histologic appearance. Plexiform neurofibromas may arise anywhere along the extent of a nerve, although the large nerve trunk is the most

common site. They are frequently multiple. At the site of each lesion, the host nerve is irregularly expanded, as each of its fascicles is infiltrated by the neoplasm. Unlike the case with schwannomas, it is not possible to separate the lesion from the nerve. The proximal and distal extremes of the tumor may have poorly defined margins, as fingers of tumor and individual cells insert themselves between the nerve fibers. On microscopic examination, the lesion has a loose myxoid background with a low cellularity. A number of cell phenotypes are present, including Schwann cells with typical elongated nuclei and extensions of pink cytoplasm, larger multipolar fibroblastic cells, and scattered inflammatory cells, often including mast cells. Although the myoid appearance dominates the picture, there are often areas of collagen bundles, which have a “shredded carrot” appearance. In contrast to schwannomas, axons can be demonstrated within the tumor. Various ultrastructural and immunohistochemical studies have identified the neoplastic cells as showing markers of diverse lineages, including Schwann cells, perineurial cells, and fibroblasts. Type 1 Neurofibromatosis This autosomal dominant disorder is characterized by neurofibromas (plexiform and solitary), gliomas of the optic nerve, pigmented nodules of the iris (Lisch nodules), and cutaneous hyperpigmented macules (café-au-lait spots). It is one of the more common genetic disorders, having a frequency of 1 in 3,000 (58). Except for plexiform neurofibromas, the tumors that occur in NF1 are histologically comparable with those that occur sporadically. In patients with NF1, there is a propensity for the neurofibromas to undergo malignant degeneration at a higher rate than that observed for comparable tumors in the general population. This is especially true for plexiform neurofibromas. The gene, located at 17q11.2, has been identified and encodes a protein termed neurofibromin. The protein contains a region homologous to the Ras family of GTPase-activating proteins, and it is presumed that neurofibromin plays a role in regulating signal transduction. The protein is widely expressed, with the highest levels found in neural tissue. The NF1 gene is a tumor suppressor gene, based on evidence of loss of heterozygosity in tumors from NF1 patients. Mutations involving the NF1 gene are of a wide variety of types, and there do not appear to be specific “hot spots” for changes. The clinical phenotype does not appear to correlate with the type or location of the NF1 mutation. The course of the disease is highly variable: Some individuals carry a mutated gene and have no symptoms, whereas others develop progressive disease with spinal deformities, disfiguring lesions, and compression of vital structures, including the spinal cord. Type 2 Neurofibromatosis This is an autosomal dominant disorder in which patients develop a range of tumors, most commonly bilateral acous-

2. Neuropathology of Central Nervous System Tumors

37

tic schwannomas and multiple meningiomas (57). Gliomas, typically ependymomas of the spinal cord, also occur in these patients. Many individuals with NF2 also have non-neoplastic lesions, which include nodular ingrowth of Schwann cells into the spinal cord (schwannosis), meningioangiomatosis (a proliferation of meningeal cells and blood vessels that grows into the brain), and glial hamartia (microscopic nodular collections of glial cells at abnormal locations, often in the superficial and deep layers of cerebral cortex). This disorder is much less common than NF1, having a frequency of 1 in 40,000 to 50,000. The NF2 gene is located on chromosome 22q12, and the gene product, merlin, shows structural similarity to a series of cytoskeletal proteins. The protein is widely distributed throughout tissues, and its function remains uncertain. There is some correlation between the type of mutation and clinical symptoms, with nonsense mutations usually causing a more severe phenotype than missense mutations. The NF2 gene is commonly mutated in sporadic meningiomas and schwannomas as well.

cells, with elongated nuclei and prominent bipolar processes. Fascicle formation may be present. Mitoses, necrosis, and extreme nuclear anaplasia are common. Some but not all malignant peripheral nerve sheath tumors are immunoreactive for S-100 protein. In addition to the basic appearance of these tumors, a wide variety of “divergent” histologic patterns may be admixed, including epithelial structures, rhabdomyoblastic differentiation (termed Triton tumors), cartilage, and even bone. Epithelioid malignant schwannomas are aggressive variants derived from nerve sheaths and contain tumor cells having visible cell borders and epithelial type nests. They are immunoreactive for S-100 but not for keratin, the latter differentiating them from epithelial tumors. Genetic alterations in malignant peripheral nerve sheath tumors include mutations of the NF1 gene; because this alteration is also found in benign plexiform neurofibromas, additional genetic alterations must be involved. There is evidence for involvement of TP53 and p16, as well as probably other loci.

Perineurioma

TUMORS OF THE MENINGES

This is a benign tumor composed of perineurial cells that accounts for less than 1% of all tumors of the peripheral nerve (56,59,60). Intraneural perineuriomas most commonly present in adolescence and involve the extremities. Microscopically, the lesion consists of concentric whorls of perineurial cells forming pseudo-onion bulbs; silver stains and immunoperoxidase reactions for neurofilament proteins demonstrate axons within some of these. Perineuriomas are strongly positive for epithelial membrane antigen, and electron microscopy demonstrates the complex whorling of perineurial cells to best advantage. Monosomy of chromosome 22 has been reported.

Meningiomas The term meningioma, coined by Cushing, was defined as a primary tumor originating from the cellular constituents of meninges, thereby encompassing all meningeal-based neoplasms (Figs. 2-13 and 2-14) (61). The term has evolved over time to mean those tumors that arise from meningothelial or arachnoidal cap cells, and this category of tumors encompasses a complex array of neoplastic expression. Despite the long list of descriptive terms corresponding to various subtypes of meningioma, such as syncytial, fibroblastic, transitional, and psammomatous,

Malignant Peripheral Nerve Sheath Tumor (World Health Organization Grades III/IV) These are highly malignant sarcomas that are locally invasive, frequently leading to multiple recurrences and eventual metastatic spread (56). These tumors do not arise from malignant degeneration of schwannomas. Instead, they arise de novo or from transformation of a plexiform neurofibroma and therefore are strongly associated with NF1. These tumors may also follow radiation therapy. The lesions are poorly defined tumor masses with frequent infiltration along the axis of the parent nerve as well as invasion of adjacent soft tissues. Associated with the malignant nature of the neoplasm, necrosis is commonly present. On microscopic examination, a wide range of histologic findings can be encountered. Patterns reminiscent of fibrosarcoma or malignant fibrous histiocytoma may be found. In other areas, the tumor cells resemble Schwann

FIGURE 2-13. Meningioma arising from the inner aspect of the parasagittal dura mater with a well-defined bed indenting the underlying cortex.

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Cancer of the Nervous System

B

A

FIGURE 2-14. Meningioma. A. Whorls and psammomatous calcification. B. Smear preparation showing typical whorl.

there are only a few histologic subtypes that are associated with distinctive clinical behavior. In this section, the typical meningioma is discussed, followed by description of its anaplastic variants and other meningeal-based tumors of uncertain classification. Meningioma (World Health Organization Grade I) Meningiomas as a group are most prevalent in middle age, although they can occur throughout the entire span of life. In adults, there is a striking predilection for women, with an occurrence of 2:1 for intracranial meningiomas and up to 9:1 for intradural spinal meningiomas (1–5). Symptomatic meningiomas constitute about 15% of all intracranial tumors. In children, there is an increased incidence of spinal epidural meningiomas having a male predominance, although the overall incidence of meningiomas in children is low. Patients with breast carcinoma may have meningiomas, noted before or after the carcinoma is diagnosed (61). These cases, the prevalence of meningiomas in women, and the apparent exacerbation of growth of meningiomas during pregnancy led to the discovery of steroid receptors in some meningiomas (62). The location of meningiomas parallels the distribution of the pacchionian granulations and arachnoidal cap cells with prevalent sites such as the cerebral convexities and midlines of the cerebral hemispheres, especially near the major venous sinuses of the brain, the falx cerebri, the subfrontal region, particularly the olfactory groove, the sphenoid ridge, and the tuberculum sellae. In the posterior fossa, they arise in the cerebellopontine angle, the clivus, and the foramen magnum. Within the spinal canal, the thoracic region is a frequent site of involve-

ment. Intraventricular meningiomas are uncommon and occur more frequently in children. Meningiomas rarely grow in extracranial locations, such as the nasal cavity, paranasal sinus, oral cavity, carotid, ear, neck, mediastinum, skin, and peripheral nerves. The orbit may be the site of meningioma, either as an extension from an intracranial tumor or from an intraorbital primary. The growth of meningiomas generally is confined to the location in which they arise, and their enlargement may compress adjacent structures, such as the cortex with an accompanying astrocytosis in its bed. Meningiomas occasionally infiltrate surrounding tissues, such as connective tissue and bone with lysis or hyperostosis, and sometimes brain. Non-CNS tissue infiltration is usually not associated with a worse prognosis; other than that it may preclude complete resection of the tumor, especially at the base of the skull. In contrast, invasion of the brain is associated with a worse prognosis, as are features of anaplasia. Distant spread, whether by CS seeding or via lymphohematogenous metastasis, is rare. In most patients with meningiomas that have spread beyond the local confines, repeated surgical resections with more than one recurrence is the rule. Atypical, anaplastic, and metastatic meningioma is most often seen in males, which is somewhat surprising given the overall predilection of meningiomas to occur in adult female patients. The most common sites of metastases in order of frequency include the lungs, liver, lymph nodes, bone, and kidney. Meningiomas may grow as round lobulated masses or as a plate-like formation (meningioma en plaque). They usually are well circumscribed, with a cleavage plane between the tumor and the adjacent CNS. On cut surface, the tumor may be gritty, especially when containing large numbers of psammoma bodies. The microscopic features

2. Neuropathology of Central Nervous System Tumors

of meningiomas are quite diverse and also varied within a given tumor. The three appearances most frequently encountered in meningiomas are meningotheliomatous (syncytial), transitional, and fibroblastic. Pure examples of meningotheliomatous and fibroblastic types are less common than those in the transitional category. Meningotheliomatous meningiomas consist of epithelial-like cells with indistinct cytoplasmic borders and well-defined nuclei usually containing several small nucleoli. Cells may be arranged in sheets or in ill-defined lobules divided by blood vessels. Neoplastic cells may be arranged in whorls. Frequently, circular, concentric, lamellated calcifications are present in the center of these whorls and are called psammoma bodies. Reticulin and collagen fibers generally are limited to perivascular regions. Fibroblastic meningiomas, in contrast, are composed of tapered spindle cells with oval to narrow nuclei. These cells often are arranged in swaths or interlacing bundles and may resemble ill-defined palisades. Reticulin and collagen fibers frequently surround neoplastic cells. Whorls and psammoma bodies may be present. Transitional meningiomas form a morphologic spectrum between the meningotheliomatous and fibroblastic variants. Whorls are most prominent in this form of meningioma, and the psammoma bodies often are conspicuous. Other variants, such as the psammomatous and microcystic variants, are recognized by the predominance of one histologic feature. It is widely accepted that these forms are not separate clinicopathologic entities, with variable clinical behaviors based on histologic pattern. Rather, they represent a spectrum of histologic variations or patterns. Histologic features that have been correlated with aggressive biologic behavior of meningiomas are papillary architecture, pattern-less sheets of tumor cells, high cellularity, nuclear pleomorphism, prominent nucleoli, small cells with a high nuclear-to-cytoplasmic ratio, mitotic activity, micronecrosis, and single-cell invasion of brain tissue. When papillary growth is present, the tumor may be designated as a papillary meningioma, usually with a more aggressive behavior. When several of the other histologic features are present, the term atypical meningioma is recommended, and when obvious cytologic malignancy is present, the term malignant meningioma is recommended. WHO grading in these instances is designated as grade II (atypical) or grade III (anaplastic/malignant) (4). Occasionally, distinction of a meningioma from other tumor types, such as schwannoma, glioma, paraganglioma, neurofibroma, metastasis, or lymphoma, can be difficult, requiring specialized studies. Immunohistochemistry often is sufficient to confirm the initial microscopic impression of meningeal differentiation when immunoreactivity for epithelial membrane antigen is present. Immunostaining for S-100 protein usually is

39

strongly positive in nerve sheath tumors, and immunostaining for cytokeratins, a class of intermediate filaments present in many epithelial cells and carcinomas, is variable, though typically very limited, in meningiomas. GFAP is typically negative in meningiomas, in distinction to glial tumors. As with the light microscope, the electron microscope reveals many characteristics in meningiomas that are identical to normal arachnoid cells, such as elaborate interdigitating cell borders that are connected by many intercellular junctions, including desmosomes, gap junctions, and zonulae adherents. Tonofilaments may be present in the cytoplasm. Historically, meningiomas with a prominent vascularity were grouped under the category of angioblastic meningioma, a classification that included three distinct tumor types: angiomatous meningioma, hemangioblastoma, and hemangiopericytoma. It now seems clear that the angiomatous meningioma represents a pacchionian-related type of meningioma with abundant vasculature. The use of this designation is purely descriptive and reserved to indicate a prominent vascular component in an otherwise typical meningioma. The cell of origin of the hemangioblastic and hemangiopericytic meningeal-based tumors is uncertain. Opinion differs, and controversy revolves around whether arachnoid cells may differentiate into these histologic patterns or whether other cellular constituents in the meninges, such as pericytes and endothelial cells, give rise to such neoplasms. Evidence exists for both points of view. Some investigators have found these tumors without any arachnoid cell elements, which suggested that the hemangioblastic and hemangiopericytic tumors do not arise from arachnoid cells. Other investigators report a transition between classic meningothelial cells and pericytes or hemangioblastic elements, which suggests that these tumors share an arachnoid stem cell origin. Because the biologic behavior and clinical prognosis in these two tumors are very different from each other and from other forms of meningioma, it is our opinion that the category of “angioblastic meningioma” should be retired to an historical note, with reassignment of these meningeal-based tumors to categories that reflect their clinicopathologic characteristics, namely, hemangioblastoma and hemangiopericytoma (see below). Metastasis of other tumors to meningiomas can occur. Glioblastoma or anaplastic astrocytoma may rarely develop adjacent to a meningioma, either benign or malignant. Occasionally, a meningioma arises after irradiation, often in patients treated with radiotherapy at a young age. These tumors characteristically occur at the site of radiotherapy after a long latent period and tend to pursue an aggressive clinical course. The most common cytogenetic abnormality is loss of chromosome 22, especially the long arm (22q) (63). The deletions include the region at 22q12 that harbors the

40

Cancer of the Nervous System

NF2 gene (see above). Indeed, 50% to 60% of meningiomas not associated with NF2 have mutations in the NF2 gene; most of these mutations are predicted to result in absence of functional protein. These genetic abnormalities are more common in meningiomas with fibroblastic or transitional histologic appearance (64–66). Investigation of chromosomal patterns associated with meningiomas has been the subject of a number of studies. These have shown a high incidence of hypoploidia, with complete or partial loss of one chromosome 22 (63). Complex karyotypes are seen in atypical (malignant) types and primarily involve chromosomes 1 and 14 with, to a lesser extent, chromosomes 6, 11, 13, 18, 19, X, and Y. Investigations for specific antigens in meningioma have revealed expression of cytokeratin and epithelial membrane antigen. Familial and multiple metachronous meningiomas have been reported, some being associated with NF2 and occurring with tumors such as neurofibromas, schwannomas, and gliomas. The NF2 gene has been suggested to be the meningioma locus on chromosome 22, especially in fibroblastic and transitional types of meningiomas. Meningeal-based Hemangiopericytoma (World Health Organization Grade II/III) The cellularity of hemangiopericytomas is frequently homogeneously dense, although sometimes variable, and they are sometimes arranged in lobules (1–5). Neoplastic cells have only a scant amount of cytoplasm and contain nuclei that may appear spindled and thin to fat and oval. Cellular lobules or sheets of neoplastic cells are associated with flat endothelial cells lining “staghorn” vascular channels. One of their most characteristic features is the arrangement of reticulum fibers into a net that encases individual neoplastic cells. Mitoses, when present, are an indication of the aggressive rapidly growing behavior of this tumor. Hemangiopericytomas may be subdivided into differentiated and anaplastic. Anaplastic forms show necrosis or more than five mitoses per 10 high-power microscopic fields associated with hemorrhage, moderate to high cellularity, and moderate to high nuclear atypia. Such a distinction has prognostic value, with anaplastic tumors recurring and metastasizing more rapidly and lowering overall patient survival. Other Meningeal-based Tumors Melanocytomas, pigmented neurofibromas, lymphomas, chondromas, osteochondromas, osteomas, and their malignant counterparts, such as chondrosarcoma, have been reported to arise in the meninges. Solitary fibrous tumor is a meningeal-based spindle cell neoplasm occurring in adult individuals; most cases on record have been

intracranial, but the tumor has also been seen along the spine. The tumor lacks the whorling pattern of most meningiomas and has a fairly distinctive immunohistochemical profile: positive for CD34 and vimentin but often negative for epithelial membrane antigen. In most cases the tumor has not recurred after complete surgical resection.

Tumors of Uncertain Histogenesis Hemangioblastoma (World Health Organization Grade I) Hemangioblastomas are characterized by abundant vascular channels resembling capillaries that reside in stroma containing cells with a foamy appearance (1–5). Hemangioblastomas in the CNS most frequently originate in the cerebellum, although other sites such as the spinal cord and the floor of the fourth ventricle are known. The incidence of this tumor is stated to be between 1% and 2.5% of all intracranial neoplasms and 7.3% of tumors originating within the structures of the posterior fossa. Many patients with capillary hemangioblastomas are members of a family with von Hippel-Lindau disease (see below). More than one hemangioblastoma may be present in patients with von Hippel-Lindau disease. Although patients with von Hippel-Lindau disease comprise onethird of patients with hemangioblastoma, many cases of hemangioblastoma do have a mutation of the von HippelLindau gene. Secondary polycythemia that usually disappears after resection of the hemangioblastoma has been observed. This phenomenon is due to elaboration of erythropoietin by the tumor, which may be detected in serum, CSF, and tumor cyst fluid. Hemangioblastomas rarely grow rapidly, and the prognosis is favorable when the tumor is completely resected. Grossly, hemangioblastomas are well-defined circumscribed neoplasms that are usually cystic with a mural nodule, but they may be solid. Cysts are usually glistening and smooth. On cut surface, solid hemangioblastomas frequently contain small cysts and are spongy, with brown, red, or yellow regions. Microscopically, two elements are conspicuous: thin-walled vascular channels lined by endothelial cells and the interstitial or stromal cells. Stromal cells are somewhat large and polygonal with foamy lipid-laden cytoplasm and prominent nuclei located either centrally or eccentrically. Neither the endothelial cells nor the stromal cells show anaplastic features, although it must be pointed out that the nuclei of the stromal cell may be hyperchromatic. This appearance does not need to be interpreted as indicative of rapid growth or malignancy. Mitoses are usually absent. Reticulin fibers characteristically are arranged around blood vessels and ramify between neoplastic cells.

2. Neuropathology of Central Nervous System Tumors

By light microscopy, hemangioblastomas should be distinguished from metastatic clear cell carcinomas, which they may resemble. Hemangioblastoma stromal cells show immunoreactivity to erythropoietin, renin, keratin, S-100 protein, Leu M1, Leu 7, actin, neuron-specific enolase, and GFAP, whereas metastatic clear cell carcinomas are usually positive for epithelial membrane antigen. von Hippel-Lindau disease is an autosomal dominant inherited disease in which affected individuals develop tumors (capillary hemangioblastomas) within the cerebellar hemispheres, retina, and less commonly the brainstem and spinal cord (67 ). Patients may also have cysts involving the pancreas, liver, and kidneys and have a high propensity to develop renal cell carcinoma of the kidney. The disease frequency is 1 in 30,000 to 40,000. The gene for von HippelLindau disease, a tumor suppressor gene, is located on chromosome 3p25-26 and encodes a protein (pVHL) that appears to play a role in regulation of several aspects of cellular function. The protein can serve as a component of a protein complex that functions in the ubiquitination pathway, contributing to protein degradation. This link may explain the vascular component of various von Hippel-Lindau-associated tumors. Other roles for pVHL in regulation of cell cycle proteins have been found as well. Missense mutations, but not other types of mutations, are highly likely to result in a phenotype that includes, in addition to the vascular tumors, adrenal pheochromocytoma.

LYMPHOMAS Lymphomas (Fig. 2-15) involving the brain may originate systemically and spread secondarily to the CNS (secondary lymphomas), or they may actually arise primarily within the brain and lack systemic involvement (primary cerebral lymphomas) (1–5). Characteristically, primary cerebral lymphomas involve the parenchyma of the brain, in contrast to secondary lymphomas, which have a tendency particularly to involve the meninges and adjacent brain, especially around the Virchow-Robin spaces. In this section, we discuss only primary brain lymphomas. The cell that gives rise to primary lymphomas of the CNS has been a source of much controversy. Once a rare disease, the primary CNS lymphoma was variably known as the reticulum cell sarcoma, histiocytic lymphoma, and microglioma until the lymphoid nature of the tumor was appreciated. Primary CNS lymphoma has become more common since the early 1980s, the beginning of the era of acquired immunodeficiency syndrome (AIDS) and of the increasing utilization of immunosuppressive drugs, after bone marrow and solid-organ transplantation. There also has been a well-documented though poorly explained rise in the incidence of this form of lymphoma in nonimmunosuppressed individuals during approximately

41

the same time period (68–70). Primary CNS lymphoma in any setting represents up to 6.6% of all brain tumors and at least 1% of all non-Hodgkin lymphomas (71). The relationships between these lymphomas arising in immunosuppressed persons, in immunocompetent persons (73,74), and in association with the Epstein-Barr virus (EBV) are complex and only now are being clarified. It is clear that the incidence of EBV infection is significantly greater in primary CNS lymphoma in AIDS patients as compared with immunocompetent patients and is significantly increased in primary CNS lymphoma compared with systemic lymphoma in immunocompromised patients (75). Furthermore, it has been suggested that AIDSrelated CNS lymphoma has a profile distinct from AIDS-related systemic lymphoma, the former being characterized by EBV positivity, monoclonality, and absence of c-myc rearrangements (76). Correlation between EBV positivity and histomorphologic subtype of primary CNS lymphoma, usually expressed according to the guidelines of the Working Formulation for Systemic Non-Hodgkin Lymphomas, has not been found. In addition to EBV infection, AIDS patients with primary CNS lymphoma have been found to harbor other infectious agents, including varicella-zoster virus, cytomegalovirus, polyoma virus, and toxoplasma and candida species (77). EBV-associated lymphoproliferative disorders—which are not clearly neoplastic, have been found to be polyclonal, and, at least in other sites, may regress with discontinuation of immunosuppressive medications—have been noted to occur in the CNS of transplant patients (78). In virtually all cases, the lymphoproliferative disorder was generalized, also involving extra-CNS sites. Demographically, primary CNS lymphoma associated with AIDS differs significantly from CNS lymphoma in non-AIDS patients (79). In AIDS, the mean age is 30.8 years, compared with 55.2 years in non-AIDS patients, and the male-to-female ratio is 7.38 compared with 1.35. Survival after treatment is better among non-AIDS patients with CNS lymphoma than in AIDS patients with CNS lymphoma. Most primary CNS lymphomas are B cell in origin in both patient groups; however, AIDS patients have a greater incidence of high-grade phenotype than do non-AIDS patients (5). Rarely, a primary CNS lymphoma may be of T-cell phenotype (80,81). Grossly, primary lymphomas of the CNS in non-AIDS patients are usually well circumscribed. On cut surface, they have a gray opalescent appearance and may obliterate the architectural detail of the region in which they arise. In AIDS patients, the incidence of multifocality of lesions and of tumor necrosis is increased. Necrotic lesions grossly have hyperemic rims and gritty centers. Microscopically, they comprise deeply staining cells with a very scant amount of cytoplasm reminiscent of systemic lymphomas. Neoplastic cells frequently are

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Cancer of the Nervous System

A

B

C

FIGURE 2-15. Primary central nervous system lymphoma, highgrade non-Hodgkin B-cell type. A. Basal view of brain showing diffuse expansion of infundibular area and optic nerve. B. Diffuse infiltration of white matter. C. Neoplastic lymphoid cells showing typical perivascular arrays and single-cell involvement in the brain parenchyma. D. Cerebrospinal fluid cytology showing nucleus and cytoplasmic characteristics.

found within blood vessel walls and grow in a concentric arrangement around these blood vessels. Characteristically, reticulin fibers are arranged among the neoplastic cells in concentric perivascular hoops. Neoplastic cells also may be scattered throughout the parenchyma of the

D

brain. In contrast to systemic lymphomas, CNS lymphomas do not have nodular or follicular forms, but otherwise all subtypes of lymphoma may be encountered. In AIDS patients, rapid diagnoses may be made on the basis of a smear preparation, which allows the cytologic

2. Neuropathology of Central Nervous System Tumors

43

detail of the lymphoid cells to be appreciated and which minimizes risk of exposure to unfixed tissue in contrast to a frozen section. As with systemic lymphomas, formalin- or B5-fixed specimens of primary CNS lymphoma can be examined for B- and T-cell markers, ␬ and ␭ light chains, and other markers. Gene rearrangement studies may be helpful in classifying individual tumors. Examination of CSF generally is not useful in making the diagnosis of primary CNS lymphoma. A rare variant of lymphoma, known as intravascular large cell lymphoma, may involve the CNS, along with other organs of the body. Many of these patients develop fluctuating occlusion of vessels by malignant cells, most often B-cell types (82,83).

METASTATIC TUMORS TO THE CENTRAL NERVOUS SYSTEM Some of the neoplastic entities involving the CNS we discussed in this chapter are quite rare. Metastatic neoplasm to the CNS (Fig. 2-16), however, is an everyday occurrence and a common cause of neurologic impairment. There is a broad range of primary somatic tumors that metastasize to the brain, although incidence figures vary depending on the clinic from which they originate (1–5). Common sites of origin include the lung, breast, melanoma, kidney, and gastrointestinal tumors. Other somatic neoplasms almost never metastasize to the brain, such as the prostate and ovary. One of the most important complications of metastatic tumor is the development of edema and increased intracranial pressure. The amount of edema is variable among different neoplastic types and may vary surprisingly among different metastatic nodules from a single tumor within the same brain. Metastatic melanoma is particularly prone to induce edema. Other metastatic tumors have distinguishing characteristics, such as the proclivity to develop hemorrhage, as in choriocarcinoma. Grossly, metastatic tumors, most notably carcinomas, have a sharp border that pushes against the adjacent host tissue. Foci of necrosis and hemorrhage may be present. On cut surface, tumors may have distinguishing features, such as melanin. For the most part, they appear firm and somewhat granular. Microscopically, metastatic tumors often recapitulate the pattern of the primary neoplasm, although they may be quite undifferentiated with little clue as to their site of origin.

ACKNOWLEDGMENT We acknowledge with pleasure the contributions made by Drs. D. C. Anthony, R. D. Folkerth, and W. C. Schoene in the earlier editions of this chapter. We are also grateful to Ms. Diane Brown for preparing the final manuscript.

A

B

FIGURE 2-16. Metastatic carcinoma. A. Basal view of brain showing multiple metastatic foci in breast carcinoma. B. Several coronal sections showing hemorrhagic foci of malignant melanoma.

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3. Ironside JW, Moss TH, Louis DN, et al. Diagnostic pathology of nervous system tumours. London: Churchill Livingstone, 2002. 4. Kleihues P, Cavenee WK, eds. Pathology and genetics of tumours of the nervous system. Lyon, France: IARC Press, 2000. 5. Lantos PL, Louis DN, Rosenblum M, et al. Tumours of the nervous system. In: Graham DI, Lantos P, eds. Greenfield’s neuropathology. Vol. 2. London: Arnold, 2002:767–1052. 6. Shoshan Y, Nishiyama A, Chang A, et al. Expression of oligodendrocyte progenitor cell antigens by gliomas: implications for the histogenesis of brain tumors. Proc Natl Acad Sci USA 1999;96:10361–10366. 7. Kleihues P, Louis DN, Scheithauer BW, et al. The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002;61:215–225. 8. Burger PC, Vogel FS, Green SB, et al. Glioblastoma multiforme and anaplastic astrocytoma: pathologic criteria and prognostic implications. Cancer 1985;56:1106–1111. 9. Wingo PA, Tong T, Bolden S. Cancer statistics, 1995. Cancer J Clin 1995;45:8–30. 10. Daumas-Duport C, Scheithauer BW, O’Fallon J, et al. Grading of astrocytomas: a simple and reproducible method. Cancer 1988;62:2152–2165. 11. Burger PC, Heinz ER, Shibata T, et al. Topographic anatomy and CT correlations in the untreated glioblastoma multiforme. J Neurosurg 1988;68:698–704. 12. Giannini C, Scheithauer BW, Burger PC, et al. Cellular proliferation in pilocytic and diffuse astrocytomas. J Neuropathol Exp Neurol 1999;58:46–53. 13. McKeever PE, Strawderman MS, Yamini B, et al. MIB-1 proliferation index predicts survival among patients with grade II astrocytoma. J Neuropath Exp Neurol 1998;57:931–936. 14. Krouwer HG, Davis RL, Silver P, et al. Gemistocytic astrocytomas: a reappraisal. J Neurosurg 1991;74:399–406. 15. Burger PC, Pearl DK, Aldape K, et al. Small cell architecture—a histological equivalent of EGFR amplification in glioblastoma multiforme? J Neuropathol Exp Neurol 2001; 60:1099–1104. 16. Biernat W, Aguzzi A, Sure U, et al. Identical mutations of the p53 tumor suppressor gene in the gliomatous and the sarcomatous components of gliosarcomas suggest a common origin from glial cells. J Neuropathol Exp Neurol 1995;54: 651–656. 17. Boerman RH, Anderl K, Herath J, et al. The glial and mesenchymal elements of gliosarcomas share similar genetic alterations. J Neuropathol Exp Neurol 1996;55:973–981. 18. Giannini C, Scheithauer BW, Burger PC, et al. Pleomorphic xanthoastrocytoma: what do we really know about it? Cancer 1999;85:2033–2045. 19. Bigner SH, Rasheed BK, Wiltshire R, et al. Morphologic and molecular genetic aspects of oligodendroglial neoplasms. Neuro-Oncol 1999;1:52–60. 20. Giannini C, Scheithauer BW, Weaver AL, et al. Oligodendrogliomas: reproducibility and prognostic value of histologic diagnosis and grading. J Neuropathol Exp Neurol 2001;60:248–262. 21. Cairncross JG, Ueki K, Zlatescu MC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Inst 1998;90:1473–1479. 22. Mueller W, Hartmann C, Hoffmann A, et al. Genetic signature of oligoastrocytomas correlates with tumor location and denotes distinct molecular subsets. Am J Pathol 2002;161:313–319. 23. Goussia AC, Kyritsis AP, Mitlianga P, et al. Genetic abnormalities in oligodendroglial and ependymal tumours. J Neurol 2001;248:1030–1035. 24. Prayson RA. Clinicopathologic study of 61 patients with ependymoma including MIB-1 immunohistochemistry. Ann Diagn Pathol 1999;3:11–18. 25. Ilhan I, Berberoglu S, Kutluay L, et al. Subcutaneous sacrococcygeal myxopapillary ependymoma. Med Pediatr Oncol 1998;30:81–84.

26. Herbert J, Cavallaro T, Dwork AJ. A marker for primary choroid plexus neoplasms. Am J Pathol 1990;136:1317–1325. 27. Albrecht S, Rouah E, Becker LE, et al. Transthyretin immunoreactivity in choroid plexus neoplasms and brain metastases. Mod Patho1 1991;4:610–614. 28. Bonnin JM, Rubinstein LJ. Astroblastomas: a pathological study of 23 tumors, with a postoperative follow-up in 13 patients. Neurosurgery 1989;25:6–13. 29. Brat DJ, Hirose Y, Cohen KJ, et al. Astroblastoma: clinicopathologic features and chromosomal abnormalities defined by comparative genomic hybridization. Brain Pathol 2000; 10:342–352. 30. Brat DJ, Scheithauer BW, Staugaitis SM, et al. Third ventricular chordoid glioma: a distinct clinicopathologic entity. J Neuropathol Exp Neurol 1998;57:283–290. 31. Blümcke I, Wiestler OD. Gangliogliomas: an intriguing tumor entity associated with focal epilepsies. J Neuropathol Exp Neurol 2002;61:575–584. 32. Koch R, Scholz M, Nelen MR, et al. Lhermitte-Duclos disease as a component of Cowden’s syndrome. Case report and review of the literature. J Neurosurg 1999;90:776–779. 33. VandenBerg SR. Desmoplastic infantile ganglioglioma and desmoplastic cerebral astrocytoma of infancy. Brain Pathol 1993;3:275–281. 34. Daumas-Duport C. Dysembryoplastic neuroepithelial tumours. Brain Pathol 1993;3:283–295. 35. Hassoun J, Soylemezoglu F, Gambarelli D, et al. Central neurocytoma: a synopsis of clinical and histological features. Brain Pathol 1993;3:297–306. 36. Hyams VJ, Batsakis JG, Michaels L. Tumors of the upper respiratory tract and ear. In: Hartmann WH, Sobin LH, eds. Atlas of tumor pathology, 2d series, fascicle 25. Bethesda, MD: Armed Forces Institute of Pathology, 1988:240–248. 37. Shimada H, Ambros IM, Dehner LP, et al. Terminology and morphologic criteria of neuroblastic tumors: recommendations by the International Neuroblastoma Pathology Committee. Cancer 1999;86:349–363. 38. Scheithauer BW. Pathobiology of the pineal gland with emphasis on parenchymal tumors. Brain Tumor Pathol 1999; 16:1–9. 39. Jouvet A, Saint-Pierre G, Fauchon F, et al. Pineal parenchymal tumors: a correlation of histological features with prognosis in 66 cases. Brain Pathol 2000;10:49–60. 40. DeGirolami U, Zvaigzne O. Modification of the AchúcarroHortega pineal stain for paraffin-embedded formalin-fixed tissue. Stain Technol 1973;4S:48–50. 41. Rorke LB. The cerebellar medulloblastoma and its relationship to primitive neuroectodermal tumors. J Neuropathol Exp Neurol 1983;42:1–15. 42. Becker LE, Hinton D. Primitive neuroectodermal tumors of the central nervous system. Hum Pathol 1983;14:538–550. 43. Molloy PT, Yachnis AT, Rorke LB, et al. Central nervous system medulloepithelioma: a series of eight cases including two arising in the pons. J Neurosurg 1996;84:430–436. 44. Buhren J, Christoph AH, Buslei R, et al. Expression of the neurotrophin receptor p75NTR in medulloblastomas is correlated with distinct histological and clinical features: evidence for a medulloblastoma subtype derived from the external granule cell layer. J Neuropathol Exp Neurol 2000;59: 229–240. 45. Marino S, Vooijs M, van Der Gulden H, et al. Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev 2000;14:994–1004. 46. Giangaspero F, Perilongo G, Fondelli MP, et al. Medulloblastoma with extensive nodularity: a variant with favorable prognosis. J Neurosurg 1999;91:971–977. 47. Giangaspero F, Rigobello L, Badiali M, et al. Large-cell medulloblastomas. A distinct variant with highly aggressive behavior. Am J Surg Pathol 1992;16:687–693. 48. Brown HG, Kepner JL, Perlman EJ, et al. “Large cell/ anaplastic” medulloblastomas: a Pediatric Oncology Group Study. J Neuropathol Exp Neurol 2000;59:857–865.

2. Neuropathology of Central Nervous System Tumors 49. Bergmann M, Pietsch T, Herms J, et al. Medullomyoblastoma: a histological, immunohistochemical, ultrastructural and molecular genetic study. Acta Neuropathol (Berl) 1998; 95:205–212. 50. Raffel C, Jenkins RB, Frederick L, et al. Sporadic medulloblastomas contain PTCH mutations. Cancer Res 1997;57: 842–845. 51. Rorke LB, Packer RJ, Biegel JA. Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood: definition of an entity. J Neurosurg 1996;85:56–65. 52. Burger PC, Yu IT, Tihan T, et al. Atypical teratoid/rhabdoid tumor of the central nervous system: a highly malignant tumor of infancy and childhood frequently mistaken for medulloblastoma: a Pediatric Oncology Group study. Am J Surg Pathol 1998;22:1083–1092. 53. Biegel JA, Fogelgren B, Zhou JY, et al. Mutations of the INI1 rhabdoid tumor suppressor gene in medulloblastomas and primitive neuroectodermal tumors of the central nervous system. Clin Cancer Res 2000;6:2759–2763. 54. Packer RJ, Biegel JA, Blaney S, et al. Atypical teratoid/rhabdoid tumor of the central nervous system: report on workshop. J Pediatr Hematol Oncol 2002;24:337–342. 55. Harkin JC, Reed RJ. Tumors of the peripheral nervous system. Washington, DC: Armed Forces Institute of Pathology, 1969. 56. Scheithauer BW, Woodruff JM, Erlandson RA. Tumors of the peripheral nervous system. Washington, DC: Armed Forces Institute of Pathology, 2000. 57. Louis D, Stemmer-Rachamimov AD, Wiestler OD. Neurofibromatosis type 2. In: Kleihues P, Cavanee WK, eds. Pathology and genetics of tumours of the nervous system. Lyon, France: IARC Press, 2000:219–222. 58. Von Deimling A, Foster R, Krone W. Neurofibromatosis type I. In: Kleihues P, Cavenee WK, eds. Pathology and genetics of tumours of the nervous system. Lyon, France: IARC Press, 2000:216–218. 59. Anthony DC, Ward WA, McAlister JA, et al. Ultrastructural comparison of perineurioma (“localized hypertrophic neuropathy”) and hereditary and hypertrophic neuropathy. J Neuropathol Exp Neurol 1990;149:296. 60. Bilbao JM, Khouri NJS, Hudson AB, et al. Perineurioma (localized hypertrophic neuropathy). Arch Pathol Lab Med 1984;108:557–560. 61. Cushing H, Eisenhart L. Meningiomas: their classification, regional behavior, life history and surgical end results. Springfield, IL: Charles C Thomas, 1938. 62. Schoenberg BS, Christine BW, Whisnant JP. Nervous system neoplasms and primary malignancies of other sites: the unique association between meningiomas and breast cancer. Neurology 1975;25:705–712. 63. Lekanne Deprez RH, Riegman PH, van Drunen E, et al. Cytogenetic, molecular genetic and pathological analyses in 126 meningiomas. J Neuropathol Exp Neurol 1995;54:224–235. 64. Wellenreuther R, Kraus JA, Lenartz D, et al. Analysis of the neurofibromatosis 2 gene reveals molecular variants of meningioma. Am J Pathol 1995;146:827–832. 65. Louis DN, Scheithauer BW, Budka H, et al. Meningiomas. In: Kleihues P, Cavanee WK, eds. Pathology and genetics of tumours of the nervous system. Lyon, France: IARC Press, 2000:176–184.

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66. Souffer D, Gomori JM, Siegal T, et al. Intracranial meningiomas after high-dose irradiation. Cancer 1989;63:1514–1519. 67. Böhling T, Plate KH, Haltia MS, et al. Von Hippel-Lindau disease and capillary hemangioblastoma. In: Kleihues P, Cavenee WK, eds. Pathology and genetics of tumours of the nervous system. Lyon, France: IARC Press, 2000:223–226. 68. Eby NL, Grufferman S, Flannelly CM, et al. Increasing incidence of primary brain lymphoma in the U.S. Cancer 1988; 62:2461–2465. 69. Hochberg FH, Miller DC. Primary central nervous system lymphoma. J Neurosurg 1988;68:835–853. 70. Miller DC, Hochberg FH, Harris NL, et al. Pathology with clinical correlations of primary central nervous system nonHodgkin’s lymphoma: the Massachusetts General Hospital experience 1958–1989. Cancer 1994;74:1383–1397. 71. O’Neill BP, Illig JJ. Subject review: primary central nervous system lymphoma. Mayo Clin Proc 1989;64:1005–1020. 72. Bashir R, Freedman A, Harris N, et al. Immunophenotypic profile of CNS lymphoma: a review of eighteen cases. J Neuro-Oncol 1989;7:249–254. 73. De Angelis LM, Wong E, Rosenblum M, et al. Epstein-Barr virus in acquired immune deficiency syndrome (AIDS) and non-AIDS primary central nervous system lymphoma. Cancer 1992;70:1607–1611. 74. Rouah E, Rogers BB, Wilson DR, et al. Demonstration of Epstein-Barr virus in primary central nervous system lymphomas by the polymerase chain reaction and in situ hybridization. Hum Pathol 1990;21:545–550. 75. Morgello S. Epstein-Barr and human immunodeficiency viruses in acquired immunodeficiency syndrome-related primary central nervous system lymphoma. Am J Pathol 1992;141:441–450. 76. Meeker T, Shiramizu B, Kaplan L, et al. Evidence for molecular subtypes of HIV-associated lymphoma: division into peripheral monoclonal and central nervous system lymphoma. AIDS 1991;5:669–674. 77. Morgello S, Petito CK, Mouradian JA. Central nervous system lymphoma in the acquired immunodeficiency syndrome. Clin Neuropathol 1990;9:205–215. 78. Kaplan MA, Ferry JA, Harris NL, et al. Clonal analysis of posttransplant lymphoproliferative disorders, using both episomal Epstein-Barr virus and immunoglobulin genes as markers. Am J Clin Pathol 1994;101:590–596. 79. Fine HA, Mayer RJ. Primary central nervous system lymphoma. Ann Intern Med 1993;119:1093–1104. 80. Grant JW, von Deimling A. Primary T-cell lymphoma of the central nervous system. Arch Pathol Lab Med 1990;114:24–27. 81. Hardwidge C, Diengdoh JV, Husband D, et al. Primary cerebral lymphoma: a clinicopathological study. Clin Neuropathol 1990;9:217–223. 82. Domizio P, Hall PA, Cotter F, et al. Angiotropic large cell lymphoma (ALCL): morphological, immunohistochemical and genotypic studies with analysis of previous reports. Hematol Oncol 1989;7:195–206. 83. Sheibani K, Battifora H, Winberg CD, et al. Further evidence that “malignant angioendotheliomatosis” is an angiotropic large-cell lymphoma. N Engl J Med 1986;314:943–948.

C h a p t e r

3

w Clinical Presentation of

Patients with Brain Tumors

Alison M. O’Neill In this chapter we introduce the general principles of diagnosing brain tumors and review basic presenting clinical features and diagnostic testing strategies. Detailed discussions of features relevant to distinct tumor types are presented in subsequent chapters, and detailed discussions of imaging techniques and pathologic evaluation occur in chapters dedicated to these topics. The diagnosis of tumors affecting the central nervous system (CNS), whether benign or malignant, primary or metastatic, requires careful attention to the patient’s history, physical, and especially neurologic examination. A past medical history of prior malignancy raises the index of suspicion for metastatic disease, whereas a family history of neurofibromatosis or other recognized genetic predispositions to CNS malignancy, such as von Hippel-Lindau or Turcot syndrome, triggers greater concern about primary nervous system tumors. The history and physical examination findings guide the appropriate selection of diagnostic imaging and other testing. The continued evolution of magnetic resonance imaging (MRI) techniques in particular has greatly improved the early identification of brain neoplasms, although pathologic evaluation of diagnostic tissue remains the cornerstone of accurate diagnosis and subsequent treatment planning.

CLINICAL PReSeNTATION The clinical symptoms and signs of CNS tumors are as varied as their anatomic distribution and share common manifestations related to the limited ways in which the nervous system responds to disruption in function. Historically, the cardinal feature of a brain tumor, or any CNS mass lesion, is focal neurologic dysfunction that progresses subacutely over time. Other common presentations include new or progressive headaches, seizures, or more global cognitive or personality changes (1–3). The specific constellation of symptoms and signs relates to the region of the brain affected by the tumor or tumors. Symptoms and neurologic findings re-

flect dysfunction produced by the tumor mass itself plus any surrounding edema. The rate of progression in symptoms may reflect the growth rate of the neoplasm or, in some cases, the anatomic limitations to accommodating increases in tumor mass or edema, such as in the posterior fossa (Table 3-1).

Focal Neurologic Dysfunction Reflecting the principles of neurologic localization, there are certain common constellations of symptoms and signs related to the anatomic localization of brain tumors (1–3). Patients with frontal lobe tumors may present with personality changes ranging from disinhibition and impaired judgment to apathy and abulia. Often, anterior frontal lobe tumors are relatively clinically silent unless there is bilateral involvement or a tumor has become quite large in size. In the posterior frontal lobes, tumors often present with progressive contralateral motor findings manifest as hemiparesis or motor seizures. In the dominant hemisphere, speech and language function are sensitive to disruption by tumors in the region of the Sylvian fissure. In the frontal lobes, as elsewhere in the brain, even small tumors may present with dramatic and profound neurologic impairment if they interfere with function in highly eloquent regions such as the motor strip. Temporal lobe tumors frequently present with seizure symptoms related to the tendency for hippocampal irritation to produce seizures, particularly complex partial seizures. Memory disturbances are also common in patients with tumors in this region. With involvement of the optic radiations, quadrantic visual field impairments are frequent. Less commonly, patients report tinnitus or auditory hallucinations with temporal lobe tumors. In the dominant hemisphere, often there is associated aphasia if the highly eloquent regions subserving speech and language are affected. Parietal tumors typically present with alterations in higher level sensory discrimination or disturbances in visual–spatial processing. Contralateral sensory seizures can be seen with cortical involvement near the postcentral 47

48

Cancer of the Nervous System

w TABLe 3-1

Symptoms of Brain Tumors Tumor Type

Symptom

Low-grade glioma

Malignant glioma

Meningioma

Primary CNS Lymphoma

(percent of patients with symptom) Headache Seizure Hemiparesis Mental status abnormalities

40 65–95 5–15 10

50 15–25 30–50 40–60

36 40 22 21

35 17 24 61

CNS, central nervous system. Adapted from DeAngelis LM. Brain tumors. N engl J Med 2001;44:114–123, with permission.

gyrus. The optic radiations can also be affected with characteristic homonymous visual field impairments. In the dominant hemisphere, alexia and aphasias with a receptive component reflect involvement of Wernicke’s area or underlying white matter tracts. Nondominant parietal lobe dysfunction is frequently associated with dressing apraxia in particular. Because of the prevalence of neglect symptoms in nondominant parietal lesions, patients with tumors in this location may be relatively unaware of deficits that family members have noted. Tumors affecting the occipital lobes are associated with visual symptoms, particularly homonymous hemianopic defects, and more varied disorders of visual processing. Focal seizures arising from cortical irritation by tumors in this region produce visual images ranging from abstract patterns to well-formed visual hallucinations. Thalamic tumors may present with subtle contralateral hemisensory or motor impairment and cognitive or gait changes, reflecting the importance of the deep midline structures in multiple neurologic systems. Tumors of the hypothalamic region may manifest with changes in appetite, emotional control, and endocrine abnormalities. Additionally, visual changes related to invasion or pressure on the optic tracts and chiasm are common in tumors of the hypothalamic and pituitary regions. Midline tumors have an increased tendency to present with symptoms related to increased intracranial pressure due to disruption of the cerebrospinal fluid (CSF) pathways and resultant hydrocephalus. The hallmarks of posterior fossa dysfunction (ataxia, cranial neuropathies, diplopia, nausea, vertigo) are the clinical features associated with tumors of the brainstem and cerebellum (4). The posterior fossa is particularly unforgiving of expansion in tumor size or brain edema; relatively small tumors can have devastating effects, and compression of the fourth ventricle may lead to the rapid development of hydrocephalus.

Multifocal or global Neurologic Dysfunction Not all brain tumors present with well-defined focal findings. Tumors, particularly metastatic tumors, may be multifocal and thus present with a mixture of signs and symptoms. Alternatively, solitary tumors may become symptomatic only when there is significant mass effect, producing global alterations in mental status. Such changes can range from subtle alterations in concentration and executive functioning to rapidly progressive alterations in mental status associated with herniation syndromes or acute hydrocephalus. Hydrocephalus results from disruption of the normal CSF pathways due to compression produced by the tumor mass or surrounding edema. The third and fourth ventricles are most often affected by midline tumors of the hypothalamic, thalamic, and pineal regions, whereas the lateral ventricles can become entrapped by large temporal lobe tumors. Patients may present with acute headache, nausea, and depression of consciousness as intracranial pressure rises (Fig. 3-1). Herniation of one CNS compartment into another is related to the development of pressure gradients caused by enlarging mass lesions, including tumors (5). Subfalcine herniation refers to herniation of the cingulate gyrus under the falx cerebri typically associated with large frontal tumors. Resultant compression of the anterior cerebral artery by the cingulate gyrus can cause acute infarction in the anterior cerebral artery distribution. Central transtentorial herniation is caused by increased supratentorial pressure from large bihemispheric or diencephalic mass lesions producing downward pressure on the brainstem. In this syndrome there is progressive depression of consciousness associated with rostral to caudal impairment in brainstem function, leading to death if not corrected. Upward transtentorial herniation occurs when increased pressure of the posterior fossa contents produces upward compression of the midbrain through the tentorium into the diencephalon. early signs include the loss of upgaze and impairment of con-

3. Clinical Presentation of Patients with Brain Tumors

49

tendency for headache to be most severe at night or upon awakening, improving over the course of the day with upright posture, association with nausea, and worsening of headache with cough, strain, bending over, or Valsalva maneuver. In any patient with headache associated with focal neurologic signs or papilledema, imaging to exclude a structural lesion is mandatory (9). In patients with longstanding headache disorders, a change in the headache severity or symptomatology should likewise trigger consideration of imaging investigation.

Seizures

FIgURe 3-1. Magnetic resonance imaging of a large central neurocytoma producing hydrocephalus.

sciousness. Uncal herniation refers to the sequence of events noted when a mass lesion of the frontal or temporal lobe causes the uncus of the medial temporal lobe to herniate over the tentorial surface, producing pressure on the ipsilateral third nerve as an early sign. With progressive uncal herniation there is pressure on the midbrain, producing contralateral hemiparesis and depression of consciousness. If uncorrected, progressive loss of brainstem function and death can ensue. Tonsillar herniation occurs when tumors of the posterior fossa produce enough mass effect to cause downward herniation of the brainstem and cerebellar tonsils through the foramen magnum. This syndrome may also produce rapid respiratory depression, leading to death, and is often associated with nuchal rigidity early in the course. Because herniation syndromes are associated with grave outcomes if uncorrected, and the brain tumor patient population is particularly at risk, early identification of these syndromes and appropriate intervention is critical.

Headache Headache is a common, though by no means universal, presenting symptom of brain tumor. Roughly half of patients with primary and metastatic tumors of the brain report headache as a presenting symptom (3,6,7). In brain tumor patients, headache symptoms are most often related to increased intracranial pressure, though in some situations headache appears to reflect direct involvement of pain-sensitive structures, such as the vasculature of the meninges, by tumor (6,8). No absolute criteria allow for the distinction of headache related to increased intracranial pressure from benign tension-type headache; however, there are features that are more commonly seen in increased intracranial pressure: the

Seizures are the presenting symptom of brain tumor in 20% to 40% of patients, with reports of seizures occurring at some point in the illness in approximately 50% of patients (10). The specific semiology of the seizure event correlates with tumor location and may be focal or secondarily generalized. Seizures are more common in certain histologies of brain tumor, with multiple series demonstrating an increased frequency of seizures in slower growing glial tumors. Tumor localization also affects the risk of seizures, with cortically based lesions demonstrating an increased seizure risk over tumors located in the deep or periventricular regions. Patients with new onset seizures, particularly with focal features or abnormalities on neurologic examination, should have diagnostic imaging to exclude brain tumor.

DIAgNOSTIC TeSTINg Imaging The clinical presentation of a patient with progressive focal neurologic deficits has a large differential diagnosis, and the presence or absence of additional clinical features such as headache or seizure does not limit the differential meaningfully. Any lesion producing mass effect in the CNS, from abscess to demyelinating lesions, may present in a similar way. Additional testing beyond the clinical examination is essential, but the information gained from careful review of the patient’s presenting symptoms and neurologic examination will guide the cost-effective use of diagnostic imaging modalities and other testing. The diagnostic imaging test of choice when a brain tumor is considered in the differential diagnosis is MRI with and without gadolinium. This is currently the most sensitive noninvasive modality in the assessment of brain lesions. In cases where patients are unable to have MRI, computed tomography (CT) with and without contrast should be performed. Because of the more rapid scan times, greater availability, and lower cost of CT, it is widely used in emergency departments as a screening tool, although it will miss smaller lesions. In some instances, CT augments MRI by identifying areas of calcification, which are common in oligo-

50

Cancer of the Nervous System

dendrogliomas and craniopharyngiomas or bony abnormalities associated with base of skull tumors, but CT is less sensitive than MRI in assessment of soft tissue lesions. The limitations of CT are particularly evident in the evaluation of nonenhancing and posterior fossa lesions. MRI provides information about the size, number, and distribution of mass lesions; the presence of blood–brain barrier breakdown; and the extant of edema. Several tumor types have characteristic signal attributes and enhancement patterns, allowing for refinement of the differential diagnosis before definitive pathologic diagnosis (11). This has important consequences in surgical planning because biopsy alone is preferable to resection in some tumor types, such as CNS lymphoma. In certain instances imaging characteristics in conjunction with the clinical presentation establish the diagnosis with enough certainty that surgical intervention is avoided or deferred. Infiltrating brainstem glioma falls in this category; treatment is often initiated on the basis of characteristic clinical and imaging features because the risk of biopsy in this region is high (Fig. 3-2). Newer applications of MRI continue to improve our ability to noninvasively assess the CNS microenvironment and by extension the biologic behavior of tumors. MR spectroscopy is making inroads in functional imaging of brain tumor metabolism with correlates to tumor grade as well as response to therapy (12). Changes in diffusion weighted MRI are also being explored as surrogate markers of tumor response to treatment (13,14). MR and CT angiography have progressed rapidly in the past few years, allowing for noninvasive evaluation of the blood supply to hemorrhagic tumors and the ve-

FIgURe 3-2. Magnetic resonance FLAIR image of brainstem glioma.

nous sinuses and reducing the need for conventional angiography in surgical planning. Although positron emission tomography and single photon emission CT provide additional noninvasive techniques to investigate tumor metabolism, they are expensive and not widely available. Studies have shown correlation between positron emission tomography activity and tumor grade, though clinically this test is most often used to distinguish radiation necrosis from recurrent tumor (15,16). Imaging is also critical in the evaluation of patients presenting with metastatic disease. A patient without a known primary cancer but with multiple metastatic brain lesions requires CTs of the chest, abdomen, and pelvis to assess for a primary site of cancer that may be more amenable to biopsy than the brain lesions. Such staging evaluations are also critical to treatment planning and prognostication. In patients with systemic cancer who present with cranial neuropathies or multifocal radiculopathies, MRI of the brain and spine is needed to evaluate for bulky leptomeningeal disease that may be amenable to palliative radiation therapy (17). Similarly, CNS staging evaluations are required to appropriately plan for treatment of patients with primary brain tumors that tend to disseminate through the CSF pathways. All patients found to have medulloblastoma, primitive neuroectodermal tumor, ependymoma, or CNS lymphoma should receive MRI of the spine to evaluate for leptomeningeal spread of disease.

Cerebrospinal Fluid evaluation examination of the CSF is critical in diagnosing leptomeningeal spread of tumor, whether primary or metastatic, and in the evaluation of infectious or inflammatory conditions that may confound the diagnosis of tumor. The risk of lumbar puncture in the patient population with CNS mass lesions needs to be weighed carefully; in many instances lumbar puncture will need to be deferred if there is a risk of herniation. Routine CSF studies should include protein, glucose, and cell counts, with additional studies tailored to the differential diagnosis. In cases of known metastatic disease and when staging CNS neoplasms, cytologic evaluation is critical, and multiple taps will improve the diagnostic accuracy (18). When there is a high index of suspicion for leptomeningeal disease, up to three separate lumbar punctures should be performed. The diagnosis of CNS lymphoma can be made based on CSF alone in about 25% of patients (19). Flow cytometry enhances cytologic evaluation in patients suspected of having lymphoma. Though not widely available, gene rearrangement studies can also be performed on CSF to identify a monoclonal lymphoid population. In the case of germ cell tumors, CSF measurements of ␣-fetoprotein and human chorionic gonadotropin ␤ are helpful in the diagnosis and long-term monitoring of these patients.

3. Clinical Presentation of Patients with Brain Tumors

Serologic evaluation Serologic studies should be targeted either to further narrow the differential diagnosis in the patient with an undiagnosed brain mass or to aid in the staging and future monitoring of response to therapy in patient with an identified malignancy. Indiscriminate testing for tumor markers is seldom helpful. Serologic markers such as ␣-fetoprotein and human chorionic gonadotropin ␤ should be checked in germ cell tumors, because this provides a useful way to monitor response to therapy if there are elevations. Unfortunately, no serologic markers are helpful in the diagnosis of glial neoplasms. In the case of tumors affecting the sella or hypothalamic regions, initial evaluation of endocrine function, particularly the pituitary axis, is vital. Moreover, in patients who have received radiation to the brain, late effects on endocrine function are common and evaluation of endocrine function should be revisited if symptoms of hypopituitarism develop.

Ophthalmologic evaluation Formal ophthalmologic testing is helpful in a number of patients with brain tumors. Perimetry to precisely map the visual fields is useful in patients who present with visual field defects or in patients with tumors near the chiasm who may develop subtle visual field changes with disease progression. Patients with CNS lymphoma should also have slitlamp examination to evaluate for ocular involvement. Approximately 20% of patients with CNS lymphoma have ocular involvement at presentation, and in cases where brain biopsy is nondiagnostic or excessively risky, vitrectomy may yield definitive diagnostic tissue (3).

Audiometry Audiometry is useful in the evaluation of patients with cerebellopontine angle tumors, particularly vestibular schwannoma. In many cases, changes in auditory acuity will influence treatment decisions, and such changes may occur independently of obvious changes in tumor size on imaging.

Swallowing evaluation In patients with lower brainstem tumors or multiple cranial neuropathies, early evaluation of aspiration risk and instruction in proper swallowing techniques can reduce the risk of aspiration and its complications. Prompt recognition of patients at risk may reduce morbidity and have significant quality of life benefits.

SUMMARY Although patients with CNS tumors present with a wide spectrum of neurologic complaints and findings, clinical

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evaluation and appropriate diagnostic testing in the MRI era is quite successful in narrowing the differential diagnosis. However, it is still a minority of tumors that can be reliably diagnosed based on clinical and imaging characteristics alone. Pathologic evaluation of tissue is still required for accurate diagnosis and staging of nearly all CNS neoplasms. In some instances, pathologic diagnosis can be based on CSF analysis, but surgery remains the primary means of obtaining tissue for diagnosis, in many cases effecting a therapeutic reduction in tumor mass. The recent progress in molecular characterization of tumor subtypes holds great promise in expanding our understanding of tumor biology and ability to target therapy more specifically.

ReFeReNCeS 1. Berger MS, Leibel, SA, Bruner JM, et al. Primary central nervous system tumors of the supratentorial compartment. In: Levin, VA, ed. Cancer of the nervous system. Churchill Livingstone, 1996:57–126. 2. Ironside JW, Moss TH, Louis DN, et al. Diagnostic pathology of nervous system tumors. Churchill Livingstone, 2002:1–17. 3. DeAngelis LM. Brain tumors. N engl J Med 2001;344:114–123. 4. Packer RJ, Nicholson S, Venzina g, et al. Brainstem gliomas. Neurosurg Clin 1992;3:863–880. 5. Plum F, Posner J. The diagnosis of stupor and coma, 3rd ed. Philadelphia: FA Davis, 1980. 6. Forsyth PA, Posner JB. Headaches in patients with brain tumors. Neurology 1993;43:1678–1683. 7. Peterson K. Brain tumors. Neurol Clin 2001;19:887–902. 8. Newton HB, Turowski RC, Stroup TJ, et al. Clinical presentation, diagnosis, and pharmacotherapy of patients with primary brain tumors. Ann Pharmacother 1999;33:816–832. 9. American Academy of Neurology. Practice parameter: the utility of neuroimaging in the evaluation of headache in patients with normal neurological examinations. Neurology 1994;44:1353–1354. 10. glanz MJ, Cole BF, Forsyth PA, et al. Practice parameter: anticonvulsant prophylaxis in patients with newly diagnosed brain tumors. Neurology 2000;54:1886–1893. 11. Ricci Pe. Imaging of adult brain tumors. Neuroimag Clin 1999;9:651–669. 12. Castillo M, Kwock L. Proton MR spectroscopy of common brain tumors. Neuroimag Clin 1998;4:733–752. 13. Chenevert TL, Stegman LD, Taylor JM, et al. Diffusion magnetic resonance imaging: an early surrogate marker of therapeutic efficacy in brain tumors. J Natl Cancer Inst 2000;92: 2029–2036. 14. Chenevert TL, Meyer CR, Moffat BA, et al. Diffusion MRI: a new strategy for assessment of cancer therapeutic efficacy. Mol Imaging 2002;1:336–343. 15. Sasaki M, Kuwabara Y, Yoshida T, et al. A comparative study of thallium-210 SPeCT, carbon-11 methionine PeT and fluorine18 fluorodeoxy-glucose PeT for the differentiation of astrocytic tumors. eur J Nucl Med 1998;25:1261–1269. 16. De Witte O, Levivier M, Violon P, et al. Prognostic value of positron emission tomography with [18] fluoro-2-deoxy-D-glucose in low grade glioma. Neurosurgery 1996;39:470–6. 17. National Cancer Center Network. Central nervous system cancer guidelines 2002. National Cancer Center Network 18. Kesari S, Batchelor TT. Leptomeningeal metastases. Neurol Clin 2003;21:25–66. 19. Deangelis LM. Primary central nervous system lymphoma. Curr Opin Neurol 1999;12:687–691.

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C h a p t e r

4

w Imaging in Central Nervous System Tumors

John W. Henson, r. Gilberto Gonzalez The introduction of computed tomography (CT) and magnetic resonance imaging (mrI) into clinical practice is among the most important of all advances in the care of patients with brain tumors. Neuroimaging has many crucial roles in neurooncology, including refinement of preoperative differential diagnosis based on a combination of tissue imaging characteristics, anatomic site, and patient age; precise anatomic localization for operative planning; detection of response to therapy and of tumor progression; and recognition of treatment-related side effects. Gadolinium-enhanced mrI has replaced CT as the imaging modality of choice because of the sensitivity of mrI to pathologic alterations in brain tissue, its markedly superior anatomic resolution, and its ability to differentiate between the varying types of lesional tissue within tumors (1). Despite the value of mrI and CT in the management of patients with nervous system tumors, however, there are still significant limitations surrounding the lack of specificity inherent in an in vivo gross anatomic analysis of a mass lesion. In the same sense that neuropathology would be limited in its ability to refine gross anatomic findings without the availability of histopathology, and more recently immunohistochemistry and genetic analysis, current neuroimaging has limitations with respect to specificity. These limitations are changing, however, with the development of functional and spectroscopic mr techniques. These techniques herald a new era in neuroimaging in which tissue characteristics can be probed in ways that will add to diagnostic specificity as well as opening new windows into tumor physiology. In this chapter we present a descriptive approach to the neuroimaging features of brain tumors. The figures demonstrate typical features of biopsy-proven tumors.

SupraTeNTorIal prImary TumorS low-Grade Diffuse Fibrillary astrocytomas This tumor is characterized by formation of a lesion with mildly increased cellularity, infiltration of tumor cells into surrounding brain, and the absence of vascular endothelial proliferation, mitotic figures, and necrosis (Fig. 4-1). There is expansion, often without anatomic distortion, of involved structures. The average age at diagnosis is 34 years, although the range of ages is quite wide (2). However, astrocytomas of low-grade histopathology become progressively more uncommon with increasing age, such that by age 45 a nonenhancing mass lesion is more likely to be high grade than low grade (3). on imaging, the lesions demonstrate hyperintense T2-weighted signal in the white matter and cortex with varying degrees of mass effect and ill-defined margins. There is little, if any, vasogenic edema. linear enhancement may be seen along the periphery of the lesion, but the presence of enhancement within the mass suggests the presence of high-grade histology. It should be reemphasized, however, that the absence of gadolinium enhancement is not reliable evidence of low-grade histology. Cyst formation is common, and large cysts often correlate with the presence of gemistocytes, a cell type whose presence implies a tendency to aggressive behavior. Calcification may be seen but is less common than with oligodendroglioma. There is no evidence of restricted diffusion, cerebral blood volume maps do not show increased cerebral blood volume, and mr spectroscopy shows a normal or mildly increased cholineto-creatine ratio and decreased N-acetylaspartate. Serial follow-up imaging is invaluable to detect the presence of stability, gradual enlargement, or anaplastic progres-

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FIGure 4-1. low-grade astrocytoma. a 43-year-old man had sudden onset of confusion followed by a secondarily generalized seizure. a. axial T1-weighted image before gadolinium administration demonstrates a slightly hypointense mass lesion within the medial left temporal lobe, with effacement of the left ambient cistern and compression of the left cerebral peduncle. B. There is no evidence of enhancement after contrast administration. C. axial FlaIr images reveal a poorly circumscribed hyperintense lesion expanding cortex and white matter of the medial temporal lobe. There is no evidence of vasogenic edema. Serial follow-up magnetic resonance imaging revealed an enhancing nodule and extension of the T2-weighted signal abnormality 6 months after diagnosis (not shown). In middle-aged and older adults, low-grade astrocytomas should be followed closely for evidence of early anaplastic progression.

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sion to a higher grade tumor. In addition to a comparison with a most recent prior study, it is helpful to compare each new study to a defined earlier baseline posttreatment study, because gradual growth may otherwise go undetected. The development of a focus of contrast enhancement is indicative of anaplastic progression, but transient radiation-induced enhancement and radiation necrosis must be included in the differential diagnosis in the subset of patients who have received irradiation.

anaplastic astrocytoma and Glioblastoma multiforme Together, anaplastic astrocytoma (Fig. 4-2) and glioblastoma multiforme (Fig. 4-3) constitute high-grade diffuse fibrillary astrocytomas and are characterized by high cell density, mitotic figures, vascular endothelial proliferation, and, in the case of glioblastoma multiforme, areas of necrosis. multifocality is seen in 5% of cases. The peak age incidence is 41 years for anaplastic astrocytoma and 53 years for glioblastoma multiforme (2). These lesions have imaging

4. Imaging in Central Nervous System Tumors

characteristics that are more complex than with low-grade astrocytomas (4). There is more mass effect, and areas of hemorrhage may produce heterogeneous signal on T1weighted and T2-weighted images. Dense cellularity may produce relative hypointensity on T2-weighted images. enhancement with gadolinium may be homogeneous or heterogeneous. Irregular ring enhancement implies the presence of necrosis and is thus suggestive of glioblastoma multiforme. Smooth linear enhancement around a cystic-appearing structure can be seen in the absence of necrosis.

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extensive areas of hyperintense T2-weighted signal within the white matter surrounding the enhancing portion of the tumor represent a combination of vasogenic edema and infiltrating tumor cells (5,6). High-grade gliomas occasionally do not demonstrate enhancement after administration of gadolinium. Serial imaging studies are performed in patients with astrocytomas to detect tumor response or progression. Highgrade astrocytomas typically do not show a marked re-

b

FIGure 4-2. anaplastic astrocytoma. a 36-year-old man developed brief episodes of aphasia. T1-weighted images before (a) and after (B) gadolinium administration demonstrate a small area of minimal hypointensity in the left frontal lobe that exhibits several foci of enhancement. There is no evidence of an area of ring enhancement. T2-weighted image (C) shows a poorly circumscribed area of hyperintensity centered within the white matter but also involving cortex. Single voxel spectroscopy (continued) C

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e

FIGure 4-2. (D, e) demonstrates an elevated choline-to-creatine ratio and decrease in N-acetylaspartate within the lesion.

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FIGure 4-3. Glioblastoma multiforme. a 64-year-old man had episodes of olfactory hallucinations and confusion with amnesia. a. T1-weighted image before gadolinium shows a hypointense mass lesion within the right temporal lobe that has a central cystic-appearing region of even lower intensity. There is effacement of the right ambient cistern and compression of the right cerebral peduncle. B. after administration of gadolinium, irregular ring enhancement is seen. C. T2-weighted image demonstrates that the cystic-appearing component of the lesion is very hyperintense, representing a large area of necrosis, as demonstrated at the time of surgery. a surrounding region of milder hyperintensity with ill-defined margins within the white matter is produced by a combination of vasogenic edema and infiltrating tumor.

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sponse to treatment, and enlarging areas of contrast enhancement are common findings on follow-up studies. The differential diagnosis includes tumor progression, transient radiation-associated enhancement, and radiation necrosis. Changes in the intensity of contrast enhancement, changes in the size of cystic or necrotic areas, and intratumoral hemorrhage are not reliable as indicators of tumor growth. Initiation of glucocorticoids for peritumoral edema can lead to a significant decrease in the size of both the contrast-enhancing component and peritumoral edema and in some instances can be of sufficient degree to masquerade as a partial response (7).

low-Grade oligodendroglioma low-grade oligodendrogliomas (Fig. 4-4) are tumors of low to moderate cell density that usually have more well-defined margins with adjacent normal brain than do astrocytomas. There is abundant proliferation of capillaries and absence of necrosis. These tumors demonstrate prominent involvement of cortex in addition to white matter, often show microscopic spread into the subarachnoid space, and frequently calcify. The frontal and temporal lobes are the most common location. peak age incidence is 42 years (2). These tumors are T1-weighted hypointense, T2-weighted hyperintense mass lesions that usually have well-defined margins and expand the cortex prominently (8). Calcification may be seen as hyperdense foci on CT and variable signal intensity on mrI. areas of contrast enhancement result from abnormal blood–brain barrier formation within tumor capillaries and are not as strong an indicator of high grade as this finding is

57

FIGure 4-3. (CoNTINueD)

with astrocytomas. Cyst formation may occur. These tumors may show gradual enlargement over a number of years, and for this reason, it is important to have for comparison a baseline study from initial diagnosis, in addition to the most recent prior study. In patients who require treatment with irradiation or chemotherapy, marked shrinkage of oligodendroglioma may be seen during or after treatment.

anaplastic oligodendroglioma These tumors share many characteristics with low-grade oligodendrogliomas but in addition usually show more contrast enhancement and may have areas of necrosis (Fig. 45). The frontal and temporal lobes are common locations (2). The mass lesion is seen as a T1-weighted hypointense, T2-weighted hyperintense lesion that may be well circumscribed and typically expands the cortex in addition to involvement of white matter. Cystic areas and calcification are common. lesions that show ring enhancement are less likely to exhibit a major response to treatment than are tumors that show more homogeneous enhancement (9). oligodendrogliomas are unique among the gliomas because they exhibit marked shrinkage in size during treatment in 75% of cases. Tumors that show this prominent response to therapy have loss of heterozygosity on the short arm of chromosome 1p. These tumors may also demonstrate a fairly benign natural history, compared with anaplastic astrocytomas and those oligodendrogliomas that have maintained heterozygosity of 1p.

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FIGure 4-4. low-grade oligodendroglioma. a 50-year-old woman had a 10-month history of episodes of aphasia and right hemiparesis. axial FlaIr image (a) and T1-weighted image (B) demonstrate a well-circumscribed lesion within the left frontal insular region that has prominent cortical expansion as well as involvement of white matter. There is effacement of sulci in the left frontal region but no evidence of significant surrounding vasogenic edema or mass effect on the left lateral ventricle. There is a small focus of hypointensity in the medial aspect of the lesion at the site of the biopsy. after administration of gadolinium, there is no evidence of enhancement (C). low-grade oligodendrogliomas often demonstrate some enhancement. Computed tomography showed no evidence of calcification (not shown). b

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FIGure 4-4. (CoNTINueD)

FIGure 4-5. anaplastic oligodendroglioma. a 58-year-old man had a single seizure 22 years before presenting with a 2-month history of episodes of confusion. T1-weighted images before (a) and after (continued)

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b

FIGure 4-5. (B) administration of gadolinium demonstrate solid and cystic-appearing components of a right frontal lobe mass lesion. areas of hyperintensity before gadolinium likely represent foci of hemorrhage. The enhancing portion of the tumor is expanding the cortex and appears to abut the dura. Histopathologic examination demonstrated prominent subarachnoid invasion. The right lateral ventricle is effaced, and the left lateral ventricle demonstrates enlargement, suggesting obstruction. There is subfalcine herniation. C. axial T2-weighted image shows clear delineation of the solid tumor (mildly hyperintense to brain), cystic component (markedly hyperintense), and moderately hyperintense vasogenic edema and infiltrating tumor in the adjacent white matter.

optic pathway Glioma This childhood tumor (Fig. 4-6) is most commonly a pilocytic astrocytoma and thus is of low malignant potential. In adults, however, the tumors may be high grade and have a poor prognosis. These tumors may involve the optic nerves, optic chiasm, and hypothalamus. Bilateral optic nerve gliomas are seen in patients with neurofibromatosis type 1 (2). on imaging these tumors demonstrate enlargement of the affected optic pathway structure and usually show contrast enhancement. T2-weighted bright signal may be seen to extend posteriorly along the optic tracts, even to the visual cortex, and may represent tumor infiltration or edema. Cysts and calcification are not common within the optic nerve and chiasm, but cysts may be seen in a hypothalamic component, if present (10).

pleomorphic Xanthoastrocytoma This rare tumor is diagnosed in children usually before age 18 years and contains lipid-laden neoplastic astrocytes. The mass is most commonly seen in the temporal lobes and is attached to the meninges in addition to a parenchyma compo-

nent (2). Gadolinium enhancement is seen, cyst formation is common, and calcification is uncommon (11).

Subependymal Giant Cell astrocytoma Subependymal giant cell astrocytoma is a tumor arising at the foramen of monroe in the setting of tuberous sclerosis. The tumor occurs by age 20 and develops in approximately 10% of patients with tuberous sclerosis. a family history of tuberous sclerosis is present in only one-half of patients, indicating a high rate of spontaneous mutation. Calcification is common, and there may be a propensity to hemorrhage (2). Because most patients with tuberous sclerosis are now easily diagnosed in childhood with a combination of clinical features and mrI, serial imaging is recommended to detect the development of subependymal giant cell astrocytoma. The tumor may arise in an enlarging subependymal calcified nodule that demonstrates increasingly dense contrast enhancement, but in the stereotyped location at the foramen of monroe (12). Neurologic complications usually result from attendant hydrocephalus. These tumors are benign and can be eradicated with surgical removal. on imaging, in addition to the features described above, the other feature of tuber-

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FIGure 4-6. optic pathway anaplastic astrocytoma. a 54-year-old woman complained of several weeks of progressive blurring of vision in both eyes. a. T1-weighted image before gadolinium shows an isointense mass centered in the optic chiasm with extension along bilateral optic nerves and tracts. B. There is mildly heterogeneous contrast enhancement. The mass fills most of the suprasellar cistern. C. a FlaIr image shows T2-weighted hyperintensity within the lesion and hyperintense signal in the optic tracts and the medial right temporal lobe. Involvement of the adjacent temporal lobe occurs by direct extension from the optic tracts. Tumor may sometimes be seen to spread to the lateral geniculate nuclei and even along the optic radiations.

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ous sclerosis are also seen, including cortical tubers and subependymal nodules.

Ganglioglioma This tumor contains a combination of dysplastic large neurons and neoplastic astrocytic cells (Fig. 4-7). When the glial component appears to have more aggressive features, the tumor is designated anaplastic ganglioglioma. Tumors composed of cells with predominantly neuronal features are

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called gangliocytomas. Calcification and cystic changes are common. age at diagnosis ranges widely from childhood into adulthood. The most common location for these tumors is in the temporal lobes, although they can be found in any intraaxial location (2). on mrI, these lesions are solid or cystic and often show nodular areas of contrast enhancement (13). Calcification may be seen on CT.

b

FIGure 4-7. Ganglioglioma. a 21-year-old man had a history of seizures since age 18 months, following an episode of bacterial meningitis. a and B. T1-weighted images after administration of gadolinium demonstrate a small nodule of enhancement within the right temporal lobe. C. FlaIr and T2-weighted image. C

4. Imaging in Central Nervous System Tumors

Dysembryoplastic Neuroepithelial Tumor Dysembryoplastic neuroepithelial tumors (Fig. 4-8) contain glial and neuronal elements and are benign, with little tendency to regrow after total or subtotal surgical resection.

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They are located in a supratentorial cortical location, most commonly in the medial aspect of the temporal lobes, although any lobe may be involved. The peak age incidence at diagnosis is the second and third decades (2). on imaging, these tumors are well-demarcated lesions expanding

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FIGure 4-8. Dysembryoplastic neuroepithelial tumor. a 19-year-old woman had intractable simple partial seizures since age 3 after an episode of meningitis. a. T1-weighted images before gadolinium administration demonstrate a hypointense mass lesion within the right posterior frontotemporal region. The trigone of the right lateral ventricle is effaced. linear foci of hyperintensity are secondary to dense calcification. B. There is minimal gadolinium enhancement. C. The tumor has heterogeneous T2-weighted signal. D. Computed tomography shows areas of dense calcification.

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the cortex and subcortical white matter. remodeling of the adjacent calvarium is common. after administration of contrast, small rings of enhancement may be seen. There is minimal vasogenic edema. Calcification is common, whereas macroscopic cyst formation is uncommon (14).

Central Neurocytoma This tumor was described in 1982 as an intraventricular mass arising from the region of the foramen of monroe (i.e., within the lateral and third ventricles), densely calcified, and containing homogenous small round cells histologically indistinguishable from oligodendroglioma (Fig. 4-9). However, the cells show immunohistochemical and ultrastructural features of neuronal cells. It is likely that most cases once diagnosed as “intraventricular oligodendroglioma” were in fact central neurocytomas. average age at diagnosis is 29 years, with a wide range (2). The left lateral ventricle is preferentially involved. on CT and mrI, there is evidence of calcification, enhancement, and occasionally cyst formation. Hydrocephalus is common. These tumors have a low potential for aggressive behavior (15). leptomeningeal spread is uncommon, despite the intraventricular location of the tumor, and thus staging of the spine with mrI is usually not indicated.

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primary Central Nervous System lymphoma primary central nervous system lymphoma (pCNSl; Fig. 410) is a non-Hodgkin B-cell lymphoma that arises in the brain and eye (intraocular lymphoma). This tumor has become much more common over the past decades, in part because of an increase in cases in immunocompetent patients of older age. peak age incidence is in the sixth and seventh decades. In patients with a typical presentation of pCNSl, synchronous systemic (extra–central nervous system) sites of disease are uncommon. The tumor arises as a mass of densely packed neoplastic lymphocytes, without necrosis. Tumor masses favor deep cerebral and periventricular structures and are multifocal in 25% of cases (2). These masses are often hyperdense on CT. The lesions may be hypointense on T2-weighted images and demonstrate dense homogeneous enhancement with gadolinium and extensive surrounding vasogenic edema (16). pCNSl commonly show restricted diffusion. pCNSl may shrink markedly or even disappear after administration of glucocorticoids such as dexamethasone. Because glucocorticoids are often initiated on an urgent basis because of the presence of significant vasogenic edema, the radiologic picture of a rapidly disappearing brain mass in

b

FIGure 4-9. Central neurocytoma. a 45-year-old man had several weeks of headache, episodic lightheadedness, a feeling of heaviness in his legs, and decreased visual acuity. a. on T1-weighted image, the tumor is seen as an isointense mass lesion within the left lateral ventricle. areas of hyperintensity likely represent calcification. B. after administration of gadolinium, there is mild heterogeneous enhancement. The lateral ventricle are dilated. C. on T2-weighted image, the tumor is mildly hyperintense to brain. Small areas of hyperintensity may be due to cystic change. a small amount of postbiopsy air is present within the frontal horn of the right lateral ventricle. D. Computed tomography shows foci of dense calcification.

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FIGure 4-9. (CoNTINueD)

this setting is suggestive of pCNSl. Biopsy in this situation is usually nondiagnostic, showing only inflammatory cells. once glucocorticoids are discontinued, however, the tumor typically regrows rapidly over a period of several weeks. It is a rare circumstance in which glucocorticoids cannot be de-

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layed until immediately after an urgent diagnostic brain biopsy, and thus when pCNSl is suspected on neuroimaging grounds, withholding glucocorticoids can expedite early diagnosis and appropriate therapy.

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FIGure 4-10. primary central nervous system lymphoma. a 65-year-old man had 3 weeks of progressive cognitive changes and gait ataxia. a. T1-weighted image before gadolinium shows a single minimally hypointense mass lesion in the region of the right caudate head, producing marked midline shift and subfalcine herniation. B. There is strong homogeneous contrast enhancement. (continued)

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FIGure 4-10. C. on T2-weighted image, the tumor is mildly hyperintense to brain and is surrounded by a large area of vasogenic edema. D. Diffusion-weighted images demonstrate markedly abnormal diffusion, suggesting high cell density.

pCNSl is also unique among brain tumors in that a complete response, as detected by resolution of contrastenhancing tumor on mrI, is seen in one-half of cases. Because leptomeningeal dissemination is common, gadolinium-enhanced imaging of the entire spine is required.

Germinoma Intracranial germinomas occur in two midline sites: the suprasellar and pineal regions. These are densely cellular tumors that probably arise in an extraaxial site (i.e., suprasellar and pineal regions) and invade adjacent brain structures. pure germinomas are highly radiosensitive lesions that do not secrete tumor markers, are more common in the suprasellar location, and may be more common in girls. mixed germinomas (also known as nongerminomatous germinomas) are resistant to treatment, may secrete ␣-fetoprotein or ␤-human chorionic gonadotropin, and are more common in the pineal region and in boys. Tumors are multifocal in 5% of cases. peak age incidence is 10 years (2). on imaging, germinomas may be hyperdense on CT and show robust enhancement on mrI after administration of gadolinium (Fig. 4-11) (17). Brain invasion is common. Calcification and fat may be seen in teratomas (a type of mixed germinoma). In distinguishing between germinoma and pineal parenchymal tumor, germinomas tend to surround a calcified pineal gland, whereas pineal tumors tend to disperse the calcification into multiple small foci. Because of the high incidence of leptomeningeal dissem-

ination, gadolinium-enhanced imaging of the entire spine is usually performed.

Choroid plexus papilloma and Carcinoma These tumors (Fig. 4-12) arise from the choroid plexus and are distributed in the lateral ventricles (50%), third ventricle (5%), and fourth ventricles (40%). This is usually a tumor of early childhood, although tumors arising in the fourth ventricle do not show a clear age preference (2). Imaging demonstrates a lobulated, well-circumscribed, enhancing, intraventricular lesion, usually with hydrocephalus due to cerebrospinal fluid (CSF) overproduction. Calcification is not common. Choroid plexus carcinoma is much less common than papilloma and may show areas consistent with necrosis and brain invasion (18).

Colloid Cyst This rare cystic lesion of the anterior aspect of the third ventricle occurs in young middle-aged adults due to chronic or intermittent symptoms of hydrocephalus (Fig. 413). The cystic component contains high concentrations of protein and lipid and may show gradual enlargement in size (2). Calcification is not seen. on mrI the cyst has a typical location in the third ventricle adjacent to the fora-

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FIGure 4-11. pineal region germinoma. a 19-year-old man had 3 weeks of intermittent lightheadedness and vertical diplopia. a and B. Gadolinium-enhanced T1-weighted images show a homogeneously enhancing lesion within the walls of the posterior third ventricle and pineal region. There is clear evidence of invasion of the medial thalami. C. The tumor is mildly hyperintense on T2-weighted image.

men of monroe and has variable signal intensity on T1weighted and T2-weighted images depending on the concentration of protein and cholesterol (19). peripheral enhancement may or may or not be present.

INFraTeNTorIal prImary TumorS Juvenile pilocytic astrocytoma Juvenile pilocytic astrocytoma (Fig. 4-14) is a World Health organization grade I/IV astrocytoma that most commonly arises as a well-circumscribed cystic mass lesion with a mural nodule within the posterior fossa in children. These tumors do not show aggressive growth or brain invasion. Complete surgical resection is often curative (2). The imaging characteris-

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FIGure 4-12. Choroid plexus papilloma. a 4-year-old boy underwent neuroimaging after a concussion. T1-weighted images before gadolinium (a, C) show an isointense mass lesion within the trigone of the right lateral ventricle. The border of the lesion appears lobulated. There is homogenous enhancement (B). The ventricles are not enlarged. The tumor is isointense to certex on T2-weighted image (D). There is no evidence of brain invasion or vasogenic edema. multivoxel spectroscopy shows a markedly elevated ratio of choline to creatine and decreased N-acetylaspartate (e, F).

tics reflect the above features. The lesions are well-demarcated, hypointense on T1-weighted images, and hyperintense on T2-weighted images (20). The cystic component may follow CSF signal or demonstrate evidence of elevated protein with signal differing slightly from CSF. a nodule or

mass lesion demonstrating dense homogenous enhancement is almost always present. occasional tumors are solid without a cystic component. Hemorrhage and calcification may be seen but are not common.

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FIGure 4-12. (CoNTINueD)

Brainstem Glioma Brainstem gliomas of childhood are divided into three types. Diffuse pontine astrocytomas are the most common, have the poorest prognosis, and demonstrate diffuse enlargement of

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the pons, indistinct margins, and no enhancement, cystic change, or calcification. astrocytomas of the cervicomedullary junction are much less common and are seen as nonenhancing well-circumscribed lesions with an exophytic component. These tumors are less aggressive than the pontine type. Tec-

b

FIGure 4-13. Colloid cyst. a 35-year-old woman had severe headache, nausea, and vomiting for 2 weeks. a spherical centrally hypointense structure with mild peripheral enhancement is present in the region of the foramen of monroe on gadolinium-enhanced axial (a) and coronal (B) (continued)

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FIGure 4-13. T1-weighted images. The lateral ventricles are dilated. The lesion is hyperintense on axial T2-weighted image (C). T1weighted and T2-weighted image signal intensities of colloid cysts are variable.

tal astrocytomas are the rarest of the three and are nonenhancing lesions enlarging the tectal plate, often expanding this structure into the supracerebellar cistern. Hydrocephalus is an associated finding with tectal gliomas and is usually the cause of the patient’s symptoms. This tumor is the least aggressive

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of the three types. The presence of enhancement in a brainstem glioma is often an indication of high-grade histology (2). In adults, brainstem gliomas are divided into low-grade diffuse lesions and high-grade lesions (21). The former present in young adults, are usually in the pons or medulla, and

b

FIGure 4-14. Juvenile pilocytic astrocytoma. an 8-year-old girl had a 7-month history of progressive headache, clumsiness, and irritability. a. precontrast axial T1-weighted image shows a well-circumscribed hypointense lesion with internal signal heterogeneity. There is deviation of the fourth ventricle and dilation of the temporal horns of the lateral ventricles. B. Irregular ring enhancement is seen after administration of gadolinium on axial images. (continued)

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FIGure 4-14. There is thin, smooth enhancement of the medial, multicystic appearing structures. on T2WI, the bulk of the tumor (lateral aspect of the mass) is mildly hyperintense to brain. The medial, cystic areas are markedly hyperintense.

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demonstrate no enhancement (Fig. 4-15). High-grade lesions occur throughout the brainstem in older patients and demonstrate contrast enhancement (Fig. 4-16). The latter tumor has a poor prognosis.

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ependymoma This tumor arises from the ependymal cells lining the surfaces of the ventricles or the central canal of the spinal cord. The order of frequency of location is fourth ventricle,

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FIGure 4-15. Brainstem glioma. a 54-year-old man had a 2-month history of progressive gait ataxia and slurred speech. a hyperintense mass lesion with ill-defined margins is seen to expand the pons on serial axial FlaIr images (a). after administration of gadolinium, there is no evidence of enhancement on axial (B) (continued)

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FIGure 4-15. and sagittal (C) T1-weighted images within the pontomedullary tumor.

spinal, lateral ventricle, and extraventricular supratentorial. The fourth ventricle is overwhelmingly the most common location in children, whereas the other locations, in addition to the fourth ventricle, are seen in adults. The tumor may arise as a low-grade lesion (ependymoma; Fig. 4-17) or as

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a higher grade tumor (anaplastic ependymoma; Fig. 4-18) (2). on imaging, these lesions expand the fourth ventricle, whereas medulloblastomas compress the fourth ventricle. The tumors are well-circumscribed lesions that show variable degrees of enhancement (22). Calcification and cystic

b

FIGure 4-16. anaplastic astrocytoma of the brainstem. a 49-year-old woman with a 19-year history of a stable pontomedullary mass developed progressive brainstem signs over a 1-year period. a and B. Gadolinium-enhanced T1-weighted images show a hypointense pontomedullary mass with a small focus of enhancement and effacement of the fourth ventricle. C. The tumor has heterogeneous T2-weighted hyperintensity. D. Fourteen months later the mass is larger, and there is a ring-enhancing central component.

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FIGure 4-16. (CoNTINueD)

changes are not common. products of hemorrhage may be seen. ependymomas arising in the region of the conus medullaris and cauda equina are of the myxopapillary variety.

A

Subependymoma This tumor arises as a nodular mass lesion along the ventricular surface in middle to late adulthood (Fig. 4-19). The fourth ventricle is a somewhat more common location

b

FIGure 4-17. ependymoma. a 5-year-old boy had a 5-month history of worsening nausea and vomiting. a. T1-weighted image shows a hypointense mass lesion within the fourth ventricle that has well-circumscribed margins. B. after gadolinium, the lesion shows strong enhancement. (continued)

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Cancer of the Nervous System

FIGure 4-17. C. a sagittal, gadolinium-enhanced, T1-weighted image in another patient clearly demonstrates expansion of the fourth ventricle. medulloblastomas typically compress the fourth ventricle. C

than are the lateral ventricles. The lesions are usually smaller than 2 cm in size, may be calcified, and may or may not demonstrate enhancement (22). These tumors are rarely symptomatic and have a very benign prognosis.

A

medulloblastoma medulloblastomas are aggressive tumors of the cerebellar vermis arising in childhood and showing a tendency for spread via the leptomeningeal spaces. Histologically, the tumor demon-

b

FIGure 4-18. anaplastic ependymoma. a 38-year-old woman had a 4-week history of morning nausea and vomiting. a. T1-weighted image shows an isointense mass lesion within the fourth ventricle. B and C. The tumor shows heterogeneous enhancement after gadolinium administration. on the sagittal image (C), the tumor is seen to extend through the foramen of luschka. The tumor is hyperintense on T2-weighted image (D). It would be difficult to differentiate with magnetic resonance imaging between the low-grade lesion in Figure 4-17 from the anaplastic tumor seen here.

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d

C

FIGure 4-18. (CoNTINueD)

strates densely packed cells with little cytoplasm. peak age at diagnosis is 7 years. When this tumor occurs in adults, it is more likely to arise in a lateral cerebellar hemisphere location (2). These lesions compress the fourth ventricle rather than expanding it, and they may be hyperdense on CT and relatively hy-

A

pointense on T2-weighted images, likely due to high cell density (23). Strong enhancement is the rule (Fig. 4-20), although tumors may occasionally be nonenhancing (Fig. 4-21). postoperative imaging and gadolinium-enhanced spinal imaging is important for staging.

b

FIGure 4-19. Subependymoma. a 65-year-old man had a 3-year history of tingling and sensory loss in all four extremities and headache when coughing. an isointense mass lesion is present in the fourth ventricle on pregadolinium axial T1-weighted image (a). There is heterogeneous enhancement as seen on axial (B) (continued)

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Cancer of the Nervous System

d

FIGure 4-19. and sagittal (C) T1-weighted images. The lesion appears exophytic, and there is compression of the medulla. The tumor is mildly hyperintense to brain on T2-weighted image (D), and small foci of hyperintense cystic change are present.

A

b

FIGure 4-20. medulloblastoma. a 15-year-old boy had a 2-week history of gait ataxia, nausea, and vomiting. a. Sagittal T1-weighted image before contrast administration demonstrates a well-demarcated hypointense mass lesion in the fourth ventricle. B. There is moderate heterogeneous gadolinium enhancement, as seen on sagittal image. C. The tumor is slightly hyperintense on T2-weighted image. D and e. Single-voxel magnetic resonance spectroscopy shows extreme elevation of the choline-to-creatine ratio and absence of N-acetylaspartate.

4. Imaging in Central Nervous System Tumors

C

e

Hemangioblastoma Hemangioblastomas (Fig. 4-22) are cystic tumors that occur in the posterior fossa, although rarely supratentorial lesions are encountered. When solitary, the patient may or may not have the autosomal dominant von Hippel-lindau syndrome. multiple hemangioblastomas, however, occur as a component of von Hippel-lindau syndrome (2). The cystic component may follow CSF signal or demonstrate evidence of elevated protein with signal differing slightly from CSF. an

77

d

FIGure 4-20. (CoNTINueD)

associated nodule or mass lesion demonstrating dense homogenous enhancement is almost always present and usually can be shown to be in contact with the pial surface of the cerebellum (24). The wall of the cyst may or may not enhance. Smaller tumors can be solid without a cystic component. Despite the vascular nature of this tumor, hemorrhage is not seen, even on susceptibility images. When a diagnosis of hemangioblastoma is entertained, the cervical spine should be examined for lesions of the spinal cord (Fig. 423). These lesions cause morbidity by mass effect due to

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Cancer of the Nervous System

A

b

FIGure 4-21. Nonenhancing medulloblastoma. a 46-year-old man had a 6-month history of progressive truncal ataxia. a. FlaIr image reveals an ill-defined hyperintense mass lesion within the superior cerebellar vermis, with effacement of the basilar cistern. B. There is no evidence of contrast enhancement after the administration of gadolinium. C. The tumor shows abnormal diffusion, which was confirmed on apparent diffusion coefficient maps (data not shown). C

enlargement of the cystic component or growth of the tumor nodule. lesions may start to grow after years of quiescence, and new lesions may appear, making surveillance imaging a lifelong necessity. In patients found to have von Hippel-lindau syndrome, the entire neuraxis should be imaged with gadolinium-enhanced mrI.

lhermitte-Duclos This lesion is also called dysplastic cerebellar gangliocytoma and is thought to represent a hamartoma of neuronal cells rather than a true neoplasm. lhermitteDuclos is diagnosed in adults due to either hydrocephalus or cerebellar dysfunction (2). on mrI, a cerebellar mass lesion is seen without evidence of contrast enhancement

4. Imaging in Central Nervous System Tumors

A

C

or vasogenic edema (Fig. 4-23). There is a characteristic striated (“tiger-striped”) appearance (25).

Vestibular Schwannoma Vestibular schwannoma (Fig. 4-24) may occur as a sporadic unilateral lesion or as bilateral lesions as part of the neurofibromatosis type 2 syndrome (2). Schwannomas are mildly hypointense on T1-weighted images, show heterogeneous mild hyperintensity on T2-weighted images, and

79

b

FIGure 4-22. Hemangioblastoma. a 35-year-old woman with von Hippel-landau syndrome complained of 2 weeks of severe headache. a and B. Gadolinium-enhanced T1-weighted images show multiple cystic lesions within the cerebellum and cervical spinal cord, with associated nodules of densely enhancing hemangioblastoma. There is minimal enhancement of the cyst walls. C. a T2-weighted image shows regions of the mural nodule to be isointense to brain.

dense or heterogeneous enhancement. Cystic changes may occur, but hemorrhage and calcification are rare. This tumor often produces enlargement of the porus acusticus (meatus of the internal auditory canal). on mrI, high-resolution, postgadolinium, fat-saturated axial and coronal T1weighted images are valuable for monitoring these lesions. Constructive interference in steady state (CISS) sequences provide excellent demonstration of the seventh and eighth nerve complexes within the internal auditory canals (26).

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Cancer of the Nervous System

b

C

FIGure 4-23. lhermitte-Duclos. a 52-year-old woman had a 2-year history of gait difficulty. a. T1weighted image shows a large, well-circumscribed, hypointense mass lesion within the cerebellum, producing mass effect upon the fourth ventricle and effacing the basilar cistern. linear foci of hypointensity are seen, and the adjacent occipital bone is scalloped, suggesting chronicity. B. There is no evidence of enhancement after administration of gadolinium. C. on T2-weighted image there is linear heterogeneous signal. There is no evidence of surrounding vasogenic edema. The lesion was calcified on computed tomography.

4. Imaging in Central Nervous System Tumors

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b

C

d

FIGure 4-24. Vestibular schwannoma. a 58-year-old man gave a history of several years of left-sided tinnitus and hearing loss. a. T1-weighted image shows an isointense mass lesion within the left cerebellopontine angle, with mass effect upon the adjacent brainstem and middle cerebellar peduncle and extension into the left internal auditory canal. B and C. The tumor demonstrates heterogeneous gadolinium enhancement and extends to the infundibulum of the internal auditory canal. D. T2-weighted image shows heterogeneous signal intensity. Vestibular schwannomas often show cystic changes.

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Cancer of the Nervous System

epidermoid epidermoids and dermoids are rare nonneoplastic masses that are thought to represent ectodermal heterotopias. The most common location for epidermoids is the cerebellopontine angle, with parasellar second most common; dermoids are more often midline in location. epidermoids often come to attention because of cranial nerve dysfunction due to envelopment by the gradually enlarging cyst (2). on mrI, epidermoids demonstrate T1- and T2-weighted signal similar to CSF (Fig. 4-25) (27). The cyst may be hyperintense to CSF on flair images and very hyperintense on diffusion weighted images due primarily to T2-weighted shine through.

oTHer TumorS meningiomas (Fig. 4-26) are common tumors that arise from the leptomeninges and therefore may occur in supratentorial, infratentorial, or spinal locations. In addition to these sites, meningiomas are rarely seen within the trigone of a

A

lateral ventricle, where they arise from meningothelial cells in the choroid plexus and along the optic nerve sheath (2). These tumors are dural-based mass lesions that are relatively isointense to brain on T1-weighted and T2-weighted images. after administration of gadolinium, the lesions demonstrate strong homogeneous enhancement unless there is dense calcification, in which case the enhancement may appear heterogeneous. a dural tail may be seen, which consists of tapering of the enhancing mass lesion along the dura adjacent to the mass. However, dural tails are a nonspecific characteristic of many dural-based tumors. Calcification is common. mass effect on adjacent brain is often seen, and vasogenic cerebral edema may be minimal or extensive (28). evidence of brain invasion suggests the presence of a malignant meningioma. Sellar region meningiomas (Fig. 4-24) invade the cavernous sinuses, encasing adjacent arteries and growing into the sella without associated sellar enlargement. pituitary adenomas (Fig. 4-27) are slow-growing tumors that arise from the adenohypophysis and are located within

b

FIGure 4-25. epidermoid. a 23-year-old man had neuroimaging after a concussion. a. T1-weighted image shows a hypointense extraaxial mass lesion in the left cerebellopontine angle. B. There is no evidence of gadolinium enhancement. There is a suggestion of soft tissue intensity within the lesion. C. on T2-weighted image, the lesion is isointense to cerebrospinal fluid. D. Diffusion-weighted images show markedly hyperintense signal.

4. Imaging in Central Nervous System Tumors

FIGure 4-25. (CoNTINueD)

d

C

the gland according to the distribution of normal cells. prolactinoma (laterally located) and nonfunctioning tumors comprise 50% of cases, and thyroid-stimulating hormone producing tumors are the least common. microadenomas (less than 10 mm in greatest diameter) are much more com-

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83

mon than are macroadenomas (10 mm or larger) and typically present with endocrine disturbances. macroadenomas come to attention through compression of parasellar structures. The tumors present across a wide range of ages. on mrI, microadenomas show subtle hypointensity to the nor-

b

FIGure 4-26. meningioma. a 77-year-old patient had a 2-year history of diplopia. a. T1-weighted image shows an isointense mass lesion, centered in the left cavernous sinus, with extension into the sella, right cavernous sinus, and middle cranial and posterior fossas. There is mass effect upon adjacent brain without evidence of brain invasion. There may be extension into the left superior orbital fissure, and multiple vascular structures are encased. B (continued)

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Cancer of the Nervous System

d

FIGure 4-26. and C. Homogeneous enhancement is seen after gadolinium administration. multiple dura tails are present. D. on T2-weighted image, the mass is mildly hyperintense to brain.

A

b

FIGure 4-27. pituitary macroadenoma. an asymptomatic 42-year-old man participated in a magnetic resonance imaging study. a. T1-weighted image demonstrates a large isointense mass lesion expanding the sella turcica filling the suprasellar cistern and the sphevoid suice. There is a focus of hyperintensity in the superior right aspect of the tumor, possibly representing a small hemorrhage. The tumor is constricted into a waistline by the diaphragma sella. B and C. after administration of gadolinium, the tumor shows heterogeneous enhancement. There is encasement of the right internal carotid artery, and mass effect upon the floor of the third ventricle. The optic chiasm is deviated superiorly. D. T2-weighted image shows heterogeneous signal intensity.

4. Imaging in Central Nervous System Tumors

C

85

d

FIGure 4-27. (CoNTINueD)

mal gland on T1-weighted images and are often more difficult to detect on T2-weighted images (29). Immediately after administration of gadolinium, adenomas show less gadolinium enhancement than the normal adjacent gland. With delayed views, the tumor enhances more than normal gland. Inden-

A

tation of the floor of the sella, deviation of the infundibulum, and mass effect upon adjacent structures provide secondary evidence of the presence of tumor. Craniopharyngioma is a suprasellar region cystic epithelial tumor arising from rathke’s pouch (2). Two forms of cranio-

b

FIGure 4-28. Suprasellar adamantinomatous craniopharyngioma. a 4-year-old boy developed headache, lethargy, and vomiting. a and B. Gadolinium-enhanced T1-weighted images show a mass lesion within the suprasellar cistern that has an inferior, enhancing, nodular component and a superior cystic-appearing structure that produces mass effect upon the floor of the third ventricle. The cyst has a smooth rim of peripheral enhancement. (continued)

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Cancer of the Nervous System

d

FIGure 4-28. C. T2-weighted images show hyperintensity in the cystic component. D. Computed tomography demonstrates dense calcification in the region of the enhancing nodule.

A

b

FIGure 4-29. Third ventricular papillary craniopharyngioma. a 37-year-old man had a 1-year history of fatigue, problems with memory and concentration, anorexia, and vomiting. a. T1-weighted images reveal a slightly hypointense mass lesion within the third ventricle. Small foci of hyperintense signal may represent calcification. B. after gadolinium administration there is heterogeneous enhancement, but no evidence of a significant cystic component. There is hydrocephalus. C. T2-weighted signal is heterogeneous. D. Computed tomography shows tiny foci of high attenuation, likely representing a small amount of calcification.

4. Imaging in Central Nervous System Tumors

C

87

d

FIGure 4-29. (CoNTINueD)

pharyngiomas are seen. adamantinomatous craniopharyngiomas usually arise in children but are also seen in adults, are located in the suprasellar cistern, and are calcified and cystic (Fig. 4-28). papillary craniopharyngiomas usually arise in adults, are located within the suprasellar region or third ventri-

cle, and are heterogeneously enhancing solid lesions with little calcification or cyst formation (Fig. 4-29) (30). Brain metastasis (Fig. 4-30) may arise from almost any primary tumor in the body but are rare with prostate cancer and skin tumors other than melanoma. The most common

A

b

FIGure 4-30. Brain metastasis. a 79-year-old woman with metastatic ovarian adenocarcinoma had 3 weeks of occipital headache, gait ataxia, and vomiting. a. Gadolinium-enhanced T1-weighted images show a septated cystic-appearing lesion with a posterior mural nodule. There is mass effect on the fourth ventricle and medulla. The T2-weighted image demonstrates hyperintense signal within the cystic portion of the lesion (B), whereas the solid portion of the lesion is isointense.

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primary tumors in patients with brain metastasis are lung, breast, and melanoma. The latter site is notable because melanoma comprises only 1% of all cancers. Brain metastases, like most neurologic complications of cancer, usually occur in the setting of advanced systemic metastatic disease, making the radiologic diagnosis more straightforward in patients with this history. on the other hand, 10% of lung cancer patients present with neurologic symptoms of brain metastasis as the first sign of cancer. multiple lesions are seen in 50% of patients, with the distribution to various brain regions correlating with blood flow. on imaging, brain metastasis favor the gray-white junction but commonly occur elsewhere. The lesions are well circumscribed, demonstrate contrast enhancement, and usually are associated with a substantial degree of vasogenic edema. multiplanar gadolinium-enhanced imaging is crucial for the detection of subcentimeter lesions.

aCKNoWleDGmeNT This work was supported in part by the Brain Tumor research Fund.

reFereNCeS 1. atlas SW, lavi e, Fisher pG. Intraaxial brain tumors. In: atlas SW, ed. magnetic resonance imaging of the brain and spine, 3rd ed. philadelphia: lippincott, Williams & Wilkins, 2002:565–693. 2. Kleihues p, Cavanee W, eds. Tumours of the nervous system. WHo classification. oxford: oxford university press, 2000. 3. Barker FG, Chang Sm, Huhn Sl, et al. age and the risk of anaplasia in magnetic resonance-nonenhancing supratentorial cerebral tumors. Cancer 1997;80:936–941. 4. Graif m, Bydder Gm, Steiner re. Contrast-enhanced mr imaging of malignant brain tumors. am J Neuroradiol 1985;6:855–862. 5. Burger pC, Heinz er, Shibata T, et al. Topographic anatomy and CT correlations in the untreated glioblastoma multiforme. J Neurosurg 1988;68:698–704. 6. Kelly p, Daumas-Duport C, Kispert D. Imaging-based stereotactic serial biopsies in untreated intracranial glial neoplasms. J Neurosurg 1987;66:865–874. 7. Cairncross JG, macdonald Dr, pexman JHW, et al. Steroid-induced CT scan changes in patients with recurrent malignant glioma. Neurology 1988;38:724–726. 8. lee yp. VT intracranial oligodendrogliomas: imaging findings in 35 untreated cases. am J Neuroradiol 1989;10:119–127. 9. Cairncross JG, ueki K, Zlatescu mC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Inst 1998;90:1473–1479. 10. Kornreich l, Blaser S, Schwarz m, et al. optic pathway glioma: correlation of imaging findings with the presence of neurofibromatosis. am J Neuroradiol 2001;22:1963–1969. 11. lipper m, eberhard D, phillips C. pleomorphic xanthoastrocytoma, a distinctive astroglial tumor: neuroradiologic and pathologic features. am J Neuroradiol 1993;14:1397–1404.

12. Nishio S, morioka T, Suzuki S, et al. Subependymal giant cell astrocytoma: clinical and neuroimaging features of four cases. J Clin Neurosci 2001;8:31–34. 13. Castillo m, Davis pC, Takei y. Intracranial ganglioglioma: mr imaging, CT, and clinical findings in 18 patients. am J Neuroradiol 1990;11:109–114. 14. Kuroiwa T, Bergery GK, rothman mI, et al. radiologic appearance of the dysembryoplastic neuroepithelial tumor. radiology 1995;197:233–238. 15. Sgouros S, Jackowski a, Carey mp. Central neurocytoma without intraventricular extension. Surg Neurol 1994;42: 335–339. 16. Buhring u, Herrlinger u, Krings T, et al. mrI features of primary central nervous system lymphomas at presentation. Neurology 2001;57:393–396. 17. liang l, Korogi l, Sugahara T, et al. mrI of intracranial germcell tumors. Neuroradiology 2002;44:382–388. 18. pencalet p, Sainte-rose C, lellouch-Tubiana a, et al. papillomas and carcinomas of the choroid plexus in children. J Neurosurg 1998;88:521–528. 19. armao D, Castillo m, Chen H, et al. Colloid cyst of the third ventricle: imaging-pathologic correlation. am J Neuroradiol 2000;22:1632. 20. lee VT, Bruner Jm. Juvenile pilocytic astrocytoma: CT and mr imaging characteristics. aJr am J roentgenol 1989;152: 1263–1270. 21. Guillamo J-S, Doz F, Delattre J-y. Brainstem gliomas. Curr opin Neurol 2001;14:711–715. 22. Spoto G, press Ga, Hesselink Jr. Intracranial ependymoma and subependymoma: mr manifestations. am J Neuroradiol 1990;11:83–91. 23. Tortori-Donati p, Fondelli mp, rossi a, et al. medulloblastoma in children: CT and mrI findings. Neuroradiology 1996;38:352–359. 24. Conway Je, Chou D, Clatterbuck re, et al. Hemangioblastomas of the central nervous system in von Hippel-lindau syndrome and sporadic disease. Neurosurgery 2001; 48:55–62. 25. Klisch J, Juengling F, Spreer J. lhermitte-Duclos disease: assessment with mr imaging, positron emission tomography, single photo emission CT, and mr spectroscopy. am J Neuroradiol 2001;22:824–830. 26. Somers T, Casselman J, de Ceulaer G, et al. prognostic value of magnetic resonance imaging findings in hearing preservation surgery for vestibular schwannoma. otol Neurotol 2001;22:87–94. 27. annet l, Duprez T, Grandin C, et al. apparent diffusion coefficient measurements within intracranial epidermoid cysts in six patients. Neuroradiology 2002;44:326–328. 28. Nakano T, asano K, miura H, et al. meningiomas with brain edema: radiological characteristics on mrI and review of the literature. Clin Imag 2002;26:243–249. 29. rand T, Kink e, Sator m, et al. mrI of microadenomas in patients with hyperprolactinemia. Neuroradiology 1996;38: 744–746. 30. Sartoretti-Schefer S, Wichmann W, aguzzi a, et al. mr differentiation of adamantinomas and squamous-papillary craniopharyngiomas. am J Neuroradiol 1997;18:77–87.

C h a p t e r

5

w Single Photon Emission Computed Tomography in the Evaluation of Adult Primary Brain Tumors

Richard B. Schwartz One of the ironies of neurosurgical practice is that gliomas, which tend to be solitary and rarely metastasize—and which therefore should be amenable to local control—are extremely difficult to control. Low-grade astrocytomas tend to dedifferentiate into a higher grade, and even after aggressive debulking and radiotherapy, high-grade neoplasms almost always recur. Treatment approaches are limited by the potential of damage to surrounding normal brain and recurrence usually at the margins of the treated tissue. It is therefore of importance to accurately delimit the extension of tumor through the brain. Conventional imaging techniques, which include computed tomography (CT) and magnetic resonance imaging, are the primary modalities for the diagnosis and characterization of primary intracranial tumors. Such factors as the location, size, mass effect, associated edema, and, in many cases, tumor type and grade can be deduced accurately via these imaging modalities. However, the growth potential of these brain tumors at diagnosis and after treatment often cannot be accurately elucidated with conventional imaging techniques alone. In these circumstances, functional imaging techniques are required to provide the information necessary for the accurate analysis of tumor behavior before and after therapy. Functional imaging techniques make use of the wellknown association between regional delivery of energy substrates to the brain in accordance with metabolic needs, first quantified by Kety and Schmidt at the University of Pennsylvania in 1945 (1). This relationship has been exploited in the development of techniques that measure regional activity in normal brain and brain tumors. Of particular importance to neurosurgeons and neuro-oncologists is the need to determine the presence of residual or recurrent tumor after highdose radiotherapy. High-grade astrocytomas (anaplastic astrocytomas or glioblastoma multiforme) are treated with brachytherapy or radiosurgery after surgical debulking and conventional external beam radiotherapy (2,3). These treatments are designed to deposit very high radiation doses within the tumor mass while minimizing damage to the adja-

cent brain. Nevertheless, radiation-induced reactive changes and gliosis may occur in otherwise healthy tissue within weeks to months after radiotherapy (4). This is manifested by expanding often centrally necrotic masses with peripheral enhancement, surrounded by edema, that mimic recurrent tumor growth clinically and by standard radiographic techniques. The differentiation of radiation necrosis and recurrent glioma is important to design and implement further therapy. The most widely used nuclear medicine technique for imaging brain tumor is single photon emission CT (SPECT). SPECT makes use of equipment and isotopes that are widely available in any nuclear medicine facility. There are two general classes of SPECT agents: perfusion and metabolic. Perfusion agents, isotopes that have “microsphere-like” properties, were the first SPECT agents to be developed. Such agents are injected intravenously and then transported to the brain in proportion to the regional blood flow, cross the blood–brain barrier, and are retained in brain tissue long enough to be imaged. Most agents now in use incorporate technetium-99m (Tc-99m), the standard radioisotope of nuclear medicine. Tc-99m hexamethylpropyleneamine oxime (HMPAO) is a lipophilic compound that crosses the blood–brain barrier and is transformed into a polar form that remains trapped within the neurons and glial cells, allowing scintillation cameras to record beta emissions from the Tc99m moiety (5). A dedicated head scanner routinely can produce perfusion images of 6 to 8 mm of resolution in 30 minutes. Perfusion agents are ideal for evaluating regional changes in brain perfusion resulting from deficits in blood flow or variations in activity. However, brain tumors are not well evaluated by this technique because brain tumors, especially those of lower grade, may not show much difference in perfusion compared with normal brain. However, the use of perfusion agents as an adjunct to radioisotopes that are more specific for brain tumors may be helpful (see below). Radioisotopes that are specific for the imaging of brain tu89

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Cancer of the Nervous System

mors are generally agents that are taken up in proportion to the increased metabolic activity of abnormal tumor cells. Examples include agents that are taken up because of increased activity of the energy cycle or via the increased availability of transport systems. Thallium-201 (Tl-201) is a potassium analogue that does not normally enter into the brain. However, once it has entered the brain in regions with a disrupted blood–brain barrier, Tl-201 is taken up in proportion to the activity of the sodium-potassium ATPase pump (6). A major advantage of Tl-201 over 2-fluoro-2-deoxy-d-glucose positron emission tomography (PET) and other measures of metabolic activity is the excellent tumor-to-background contrast due to the absence of Tl-201 uptake in normal brain, and direct comparison between these methods showed Tl-201 SPECT to be superior to 2-fluoro-2-deoxy-d-glucose PET in the analysis of brain tumor activity (7). Tl-201 uptake is determined relative to the contralateral hemisphere or to the scalp, and counts are expressed as the average counts per volume or maximal counts in the volume. A comparison between various Tl-201 uptake indices showed that the use of maximal count rates relative to the average count rates in the contralateral hemisphere correlated best with clinical outcome and provided the best sensitivity and sensitivity (92% and 88%, respectively) in differentiating recurrence from radiation change (8). The uptake of Tl-201 in brain tumors correlates roughly with tumor grade in untreated patients (9), but this technique is most useful in patients who have received radiation therapy in differentiating between recurrent tumor and radiation change. Black et al. (10) and Kahn et al. (11) compared SPECT and PET in the same patients and showed that these methods are equivalent in their ability to detect recurrent glioma. Tl-201 also compares favorably with magnetic resonance spectroscopy in the evaluation of tumor recurrence (12). Because Tl-201 requires a disturbed blood–brain barrier to gain access to brain tissue, it stands to reason that Tl-201 can differentiate low-grade (World Health Organization grades I and II) tumors, which do not enhance, from highgrade tumors (World Health Organization grades III and IV), which do enhance, because of blood–brain barrier breakdown (13). However, this distinction is also made with relative ease on conventional imaging studies, and Tl-201 imaging has little role to play in the analysis of low-grade astrocytomas, either before or after radiotherapy. Tl-201 uptake in high-grade neoplasms has been found to correlate strongly with agents that indicate cells are engaged in the proliferative phase of the cell cycle within brain tumors; these agents include bromodeoxyuridine, proliferating nuclear antigen, or MIB-1, which recognizes the Ki-67 antigen (14–16). These studies have been performed in untreated tumors; the efficacy of these markers has not been established in patients after radiotherapy. It should also be noted that due to the marked vascularity and absent blood–brain barrier of meningiomas, Tl-201

uptake may be very high in these tumors (16). Therefore, Tl201 imaging should always be performed in concert with conventional imaging studies for diagnostic purposes. Tl-201, however, is not an ideal isotope for SPECT imaging because of its low-energy gamma emission and the poor photon flux associated with the limited dose that can be safely administered. Tc-99m compounds are potentially more sensitive than Tl-201 because of the higher gamma ray energy and high photon flux of the Tc-99m moiety (17,18). Therefore, there has been a continuous search for a Tc-99m–based moiety that is specifically taken up by brain tumors. Because it is not yet possible to relate uptake characteristics to absolute regional perfusion, Tc-99m agents provide only relative values of blood flow, but accurate results can be achieved by comparing regional values of activity with internal standards, such as the cerebellum. Tc-99m hexakis-2-methoxyisobutylisonitrile (MIBI), an agent developed for myocardial perfusion imaging, recently has been applied to the study of primary brain tumors. Like Tl-201, MIBI is not taken up by normal brain and therefore has a high tumor-to-background ratio. However, MIBI is taken up by the choroid plexus, and this limits its efficacy in the investigation of deep lesions (19). Tc-99m MIBI is believed to be taken up in accordance with transmembrane potentials in the mitochondrial membranes; thus, this agent is expressed in proportion to the increased mitochondrial density in tumor cells. Tc-99m MIBI has been shown to be of use in differentiating low-grade from high-grade tumors (18) and appears to correlate better than Tl-201 with proliferative activity of gliomas as measured by MIBI (20). This agent has been used to diagnose vertebral body metastases from glioblastoma multiforme (21). Another Tc-99m radiopharmaceutical, Tc-99m-tetrofosmin, also used for myocardial evaluation, has been applied to brain tumor physiology (18). Like MIBI and Tl-201, this agent requires a disturbance in blood–brain barrier and is taken up according to blood flow and cellular metabolic activity. Soricelli et al. (22) found that the uptake of Tc-99m-tetrofosmin is significantly correlated with Tl-201 uptake in benign and malignant tumors. They also found that the Tc-99m moiety is somewhat better visualized than Tl-201 because of lower background activity and better count statistics; however, these authors also noted that because of its uptake by choroid plexus, Tc-99m-tetrofosmin has limited utility in the analysis of deep tumors. Both Tc99m-tetrofosmin and MIBI are also strongly expressed in the scalp, which might limit the evaluation of peripheral lesions (18). The use of Tl-201 with Tc-99m HMPAO (6,23) or MIBI (19) agents has been shown to provide more accurate information than using either alone. This technique has proved to be very accurate in predicting the presence of necrosis or recurrent tumor after high-dose interstitial radiotherapy to malignant gliomas, as well as in distinguishing infiltrating tumor from solid tumor recurrence (Fig. 5-1). We found that in glioma patients imaged after high-dose radiotherapy, in-

5. SPECT in the Evaluation of Adult Primary Brain Tumors

tense thallium uptake (greater than 3.5 times that of the scalp in our experience) usually reflects the presence of solid actively proliferating recurrent tumor and a poor prognosis (median survival of less than 6 months) (24). Low thallium uptake (less than two times that of scalp) usually indicates the absence of recurrent tumor growth and correlates with good survival (greater than 21 months median survival time). However, intermediate Tl-201 uptake (between 2 and 3.5 times that of the scalp) is by itself indeterminate; TcHMPAO imaging can provide important additional information. In these patients, low perfusion (less than 50% that of the cerebellar cortex) is associated with the presence of infiltrating tumor and intermediate survival (median survival of approximately 15 months); a subgroup of patients with low Tl-201 uptake and high Tc-99m HMPAO also fell into this category. An intermediate Tl-201 uptake associated with high perfusion was associated with the presence of solid tumor and poor prognosis. The use of Tc-99m also helps to militate against false-positive uptake of Tl-201 as may occur with abscesses (25) or within cystic or necrotic portions of tumor beds; in those circumstances, Tl-201 may passively diffuse into the fluidic cavities, but Tc-99m HMPAO will show those regions to be poorly perfused and therefore not of concern for active tumor growth. Tc-99m ethyl cysteinate dimer (Tc-99m-ECd) is a more recently developed agent that is taken up with greater avidity for normal brain tissue than HMPAO and is now being used as an adjunct to Tl-201 in the analysis of high-grade neoplasms. However, this agent, in and of itself, has not been shown to be useful in the

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analysis of malignant brain tumors; in the arena of cerebral neoplasms, Tc-99m-ECd has found its greatest utility in the investigation of low-grade astrocytomas and meningiomas (26). The most promising radiopharmaceuticals for SPECT are the synthetic amino acid analogues, 123I-␣-methyl-tyrosine (IMT) (27) and p-123I-iodo-L-phenylalanine (IPA) (28). SPECT using these radioisotopes yields results similar to PET with the labeled amino acid L-11C-methionine (MET); however, IMT and IPA do not require an on-site cyclotron for production. Also, unlike MET (and the more commonly used PET isotope, 18F-fluorodeoxyglucose), these SPECT agents are not taken up by normal brain (29). Both IMT and IPA use neutral amino acid carrier systems to transport them across the intact blood–brain barrier, like MET, but are not widely incorporated into normal proteins. Thus, background uptake is very low, and because blood–brain barrier breakdown is not required for these agents to enter into brain tissue, these isotopes are taken up by all gliomas, regardless of grade. Weckesser et al. (30), Weber et al. (31), and Kuwert et al. (32) showed that low IMT uptake correlated with good longterm survival, but the degree of uptake did not correlate with histologic grade. Thus, IMT uptake as measured by SPECT appears to be a good marker for tumor growth but not necessarily tumor aggressiveness. In this respect, IMT uptake is similar to magnetic resonance spectroscopy, which is sensitive to the presence, but not necessarily the grade, of brain tumors. This is in contradistinction to Tl-201, which is a poor marker for low-grade neoplasms but can distinguish between

FIGURE 5-1. Thallium-201 (Tl-201)/technetium-99m (Tc-99m) hexamethylpropyleneamine oxime (HMPAO) single photon emission computed tomography (SPECT) in a 63-year-old woman treated for glioblastoma multiforme with high-dose radiotherapy approximately 1 year earlier. An axial SPECT image using Tl-201 (left) demonstrates a well-defined focus of intense uptake in the left posterior frontal lobe. This corresponds to a region of preserved uptake of Tc-99m HMPAO in the axial scan that shows decreased perfusion elsewhere in the radiated field (center). An axial computed tomography through the same level (right) demonstrates nonspecific enhancement in the left posterior frontal lobe with surrounding edema. Biopsy of this area showed recurrent high-grade neoplasm.

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anaplastic astrocytomas and glioblastomas (33). However, IMT appears to be more accurate than gadolinium enhancement in determining the extent of tumor before resection (27). In patients after resection, IMT has been found to be accurate for differentiating tumor recurrence from radiation change; Kuwert et al. (32) showed sensitivity of 78% and 100% specificity for the diagnosis of recurrent glioma. Schmidt et al. (34) found no correlation between survival and IMT uptake in patients upon diagnosis with glioma, but in patients with recurrent gliomas after therapy, they found a tendency for longer survival in patients with lower IMT uptake. However, Weckesser et al. found no correlation between tumor recurrence and IMT uptake. One disadvantage of amino acid imaging is that these agents have relatively low uptake into the brain. For greatest accuracy, IMT imaging must be completed within 40 minutes after injection, and this limits the counts that can be obtained for imaging. IPA has a significantly longer retention in brain tumors (especially low-grade tumors) than IMT; this allows longer scanning time, which helps to counteract the low count rate statistics of amino acids in the brain (35). Finally, SPECT data should be registered with CT or magnetic resonance imaging whenever possible (36). The superimposition of functional and anatomic information allows biopsy sites or resection trajectories to be chosen on the basis of tumor activity, thereby minimizing sampling error and increasing the yield of diagnostic tissue. More important, coregistration of SPECT and conventional imaging also allows radiotherapy to be planned on the basis of regional tumor activity. In summary, there are now a variety of SPECT agents that can be used to aid in the diagnosis and treatment of brain tumors, each with their benefits and drawbacks. Tl-201 remains the most widely used SPECT agent for the evaluation of high-grade brain tumors before and after treatment. The intratumoral uptake of this agent correlates with proliferation indices and broadly correlates with tumor grade before therapy and also correlates with tumor recurrence and survival in patients after surgery and radiotherapy. However, this agent requires blood–brain barrier breakdown to enter brain tissue, and this limits its potential application in the evaluation of low-grade astrocytomas and other nonenhancing neoplasms. The Tc-99m agents MIBI and tetrofosmin, like Tl-201, also require blood–brain barrier breakdown and correlate with proliferation indices, but they have a higher signal-to-background ratio than Tl-201. However, these agents are also taken up by the choroid plexus and show intense uptake in the scalp, which limits their potential application in deep and superficial lesions. The perfusion agents Tc-99m HMPAO and Tc-99m-ECd are taken up in proportion to cerebral blood flow by tumors as well as normal brain, resulting in a relatively low lesion-to-background ratio. Therefore, these agents by themselves are not ideal for the analy-

sis of brain tumors. However, they may be useful when used as an adjunct to Tl-201 imaging. The amino acid analogues IMT and IPA have several advantages. They are taken up specifically by both low-grade and high-grade neoplasms and do not require blood–brain barrier breakdown to do so. Thus, these agents are likely to be useful in the determination of recurrent tumor growth of low-grade and high-grade primary brain tumors after therapy. However, as opposed to Tl-201 imaging, the degree of uptake of amino acid analogues has not been shown to correlate with either tumor grade or survival. More important, at the time of this writing, these two agents are not approved by the Federal drug Administration for use in the United States. There is therefore no single ideal SPECT agent for all brain tumor applications. Based on the above review, the analysis of high-grade tumors is perhaps best achieved by the use of Tl-201, especially if used in conjunction with Tc99m HMPAO or Tc-99m-ECd, whereas low-grade astrocytomas may be best followed using IMT or IPA, where available. Continued investigation of these various SPECT agents will further elucidate their efficacy in the evaluation of primary brain tumors before and after therapy.

REFERENCES 1. Kety SS, Schmidt CF. The determination of cerebral blood flow in man by the use of nitrous oxide in low concentrations. Am J Physiol 1945;143:53–66. 2. Loeffler JS, Alexander EA III, Hochberg EH, et al. Clinical patterns of failure following stereotactic interstitial irradiation in malignant gliomas. Int Radial Oncol Biol Phys 1990;19:1455–1462. 3. Leibel SA, Gutin PH, Sneed PK, et al. Interstitial irradiation for the treatment of primary and metastatic brain tumor. Princip Pract Oncol 1989;3:1–11. 4. Loeffler JS, Siddon RL, Wen PY, et al. Stereotactic radiosurgery of the brain using a standard linear accelerator: a study of early and late effects. Radiother Oncol 1989;17:311–321. 5. Masdeu JC, Brass LM, Holman BL, et al. Brain single-photon emission computed tomography. Neurology 1994;44: 1970–1977. 6. Schwartz RB, Carvalho PA, Alexander EA III, et al. Radiation necrosis vs high-grade glioma: differentiation by using dual-isotope SPECT with 201-Tl and 99m-Tc HMPAO. Am J Neuroradiol 1991;12:1187–1192. 7. Sasaki M, Kuwabara Y, Yoshida T, et al. A comparative study of thallium-201 SPET, carbon-11 methionine PET and fluorine-18 fluorodeoxyglucose PET for the differentiation of astrocytic tumours. Eur J Nuc Med 1998;25:1261–1269. 8. Zhang JJ, Kim SM, Andrews d, et al. Comparative assessment of four different Tl-201 tumor uptake indices in the evaluation of brain tumors. Clin Nucl Med 1998;23:691–694. 9. Burkard R, Kaiser KP, Wieler H, et al. Contribution of thallium201 SPECT in the grading of tumorous alterations of the brain. Neurosurg Rev 1992;15:265–273. 10. Black KL, Emerick T, Hoh C, et al. Thallium-201 SPECT and positron emission tomography are equal predictors of glioma grade and recurrence. Neurol Res 1994,16:93–96. 11. Kahn d, Follett KA, Bushnell dI, et al. diagnosis of recurrent brain tumor: value of 201TI SPECT vs 18F-fluorodeoxyglucose PET. AJR Am J Roentgenol 1994;163: 1459–1465. 12. Kallen K, Burtscer IM, Holtas S, et al. Thallium-201 and 1HMRS compared with MRI in chemotherapy monitoring of high-

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2000;176:180–185. Samnick S, Hellwig d, Bader JB, et al. Nucl Med Commun 2002;23:121–130. Sonoda Y, Kumabe T, Takahashi T, et al. Clinical usefulness of C11-MET PET and Tl-201 SPECT for differentiation of recurrent glioma from radiation necrosis. Neurol Med Chir 1998;38:342–348. Weckesser M, Matheja P, Schwarzrock A, et al. Prognostic significance of amino acid transport imaging in patients with brain tumors. Neurosurgery 2002;50:958–965. Weber WA, dick S, Reidl G, et al. Correlation between postoperative 3-I123-iodo-L-alpha-methyltyrosine uptake and survival in patients with gliomas. J Nucl Med 2001;42: 1144–1150. Kuwert T, Woesler B, Morgenroth C, et al. diagnosis of recurrent glioma with SPECT and iodine-123-alpha-methyl tyrosine. J Nucl Med 1998;39:23–27. Weckesser N, Matheja P, Rickert C, et al. Evaluation of the extension of cerebral gliomas by scintigraphy. Strahlenther Onkol 2000;176:180–185. Schmidt d, Gottwald U, Langen K-J, et al. 3-I123 alpha-methylL-tyrosine uptake in cerebral gliomas: relationship to histological grading and prognosis. Eur J Nucl Med 2001; 28:855–861. Samnick S, Hellwig d, Bader JB, et al. Nucl Med Commun 2002;23:121–130. Holman BL, Zimmerman RE, Carvalho PA, et al. Computer-assisted superimposition of magnetic resonance and high resolution Tc-99m HMPAO and TI-201 SPECT images of the brain. J Nucl Med 1991;32:1478–1482.

C h a p t e r

6

w Positron Emission Tomography for the Management of Brain Tumors

Marc Levivier, David Wikler, Olivier De Witte, Nicolas Massager, Serge Goldman, Jacques Brotchi The management of brain gliomas remains a challenge and a frustration despite new diagnostic tools such as magnetic resonance imaging or technical progress like navigation and perioperative electrophysiology. Postoperative life expectancy for malignant gliomas was 12 months in 1960 and is still 12 months 40 years later (1). In the 1960s, we already knew that a patient with an operated glioblastoma surviving more than 12 months had a secondary glioblastoma (2). We have also learned that low-grade gliomas are not always slow-growing tumors, because of anaplastic focus that we were unable to see before postmortem pathology. For these reasons, new approaches through some metabolic aspects are of interest in that they may change our perception of brain tumors. Through the use of a cyclotron and easy access to positron emission tomography (PET) in the early 1990s, we developed a new trend toward the use of metabolic data in the diagnosis and management of brain gliomas, starting with PET-guided stereotactic biopsies. As described below, that has enhanced the accuracy of histologic diagnosis that in turn has allowed applying more logical adjunct treatments. Thereafter, new combinations of PET with navigation and radiosurgery have been developed. In the meantime, routine use of PET in the follow-up of gliomas has widened our understanding of the evolution of these tumors and our perspective to develop better treatment approaches.

ASSESSMENT OF ThE METABOLISM OF BRAIN TuMORS WITh POSITRON EMISSION TOMOGRAPhy RADIOTRACERS The potential of PET to investigate accurately in vivo pathophysiology has opened new perspectives for the management of neurologic disorders, especially brain tumors. PET is a computed tomographic imaging technique that allows accurate and sensitive in vivo visualization of metabolic pro-

cesses and measurement of the local concentration of radiotracers. Therefore, PET may play a critical role in improving diagnostic and therapeutic procedures in many areas of medicine. The integration of PET in the management of brain tumors represents a unique way to provide metabolic and functional information that can be used for diagnosis, guidance of therapies, and treatment monitoring. Among the different types of cancer, brain tumors have been the first to be extensively studied by PET because their localization in a heterogeneous organ protected by the skull makes them poorly accessible for histologic and biochemical in vitro examinations. Furthermore, brain tumors do not produce markers that are accessible in biologic fluids that may be helpful for diagnosis or follow-up. This led to the first attempts to study brain tumor metabolism using tracers developed for the metabolic investigation of the normal or diseased brain. Different types of radiotracers have been used with PET to help in the management of patients with brain tumors, especially glial tumors (3,4). These tracers may be classified into three groups: markers of energetic metabolic pathways, markers of protein and nucleic acid synthetic pathways, and radioligands for receptor imaging. Most investigators use tracers from the two first classes, such as 18F-2-fluoro-2deoxy-D-glucose (18F-FDG), which assays glucose metabolism, and 11C-methionine (11C-MET), or other amino acid tracers, which assays amino acid transport and metabolism (5,6). The tracer choice depends on the goal, which may involve diagnosis, lesion delineation, grading, prognosis estimation, and evaluation or prediction of response to treatment. For instance, it has been claimed that for the definition of tumor limits, PET with 11C-MET (PET-MET) is better than PET with 18F-FDG (PET-FDG) or other modalities such as computed tomography (CT) and magnetic resonance (MR) (7). In neuro-oncology, the largest PET experience has been acquired with 18F-FDG, a radiotracer derived from 2-deoxyD-glucose, which is used to study the first steps of glucose

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metabolism, that is, its uptake by the cells and its phosphorylation under the effects of the hexokinase activity (5,8). Malignant tumors are characterized by an increase in glucose consumption, but the mechanisms leading to this glucose hypermetabolism are not fully elucidated. Part of this phenomenon is derived from the increased energetic demand related to the proliferative process. Other causes may be found either in the increased expression of glucose transporters in response to oncogenes expression or in the dysregulation of the hexokinase enzymatic activity (9). Quantification of the uptake of 18F-FDG makes use of methods that vary from simple scales based on visual analysis of the images (10,11) to simple mathematical methods, such as graphic or autoradiographic methods or more exhaustive mathematical modeling (3,10). For 11C-MET and the other amino acid tracers, their accumulation in tissues seems to reflect the transmembrane transport of the amino acid. The uptake of the tracer is therefore influenced by multiple factors, such as its specific activity in plasma (which depends on the concentration of endogenous amino acids in plasma), the transfer of the tracer at the level of the blood–brain barrier, the intracellular metabolism of the tracer, and finally their incorporation into proteins, which is the actual process of interest (12). The uptake of amino acid tracers somehow correlates with tissue proliferation and the degree of malignancy. however, because of their complex metabolism in both the brain and peripheral tissues, the quantification of amino acid incorporation is difficult and less accurate than the quantification of glucose metabolism. In neuro-oncology, the major advantage of PET-MET in comparison with PETFDG is that the uptake of 11C-MET in tumors is much higher than in the surrounding brain tissue. This may be critical for the delineation of brain tumors in some neurosurgical procedures, as described below. Other PET tracers have been developed or are under investigation (13). These include tracers for the in vivo study of the blood-brain barrier (e.g., 68Ga-EDTA), the metabolism of nucleic acids (e.g., 11C-thymidine, 18F-fluorodeoxyuridine), the glial cells (e.g., 11C-PK 11195), the pharmacokinetics of therapeutic agents such as drugs for chemotherapy (e.g., radiolabeled carmustine), or new agents designed to allow specific assessment of particular gene therapy protocols applied to brain tumors. For example, tracers of this last type have been developed for the quantification of the expression of thymidine kinase when the herpes simplex thymidine kinase gene is transferred in tumor cells to render those sensitive to the lethal effects of antiviral agents such as ganciclovir (14,15).

uSE OF POSITRON EMISSION TOMOGRAPhy INFORMATION FOR ThE MANAGEMENT OF BRAIN TuMORS Neurosurgical management remains an essential step for the treatment of most brain tumors. Over the last few years, major improvements have been obtained in this matter, mainly due to the development of image-guided neurosurgery. This new neurosurgical concept takes advantage of the computing speed and capacities of present workstations to allow on-line correlation of archived imaging data with the actual space during various neurosurgical procedures. Planning of stereotactic biopsy, resection, or irradiation may therefore occur on the imaging data acquired beforehand and may be directed at precise targeted areas. The value of PET images in the delineation of tumor areas and in the detection of zones of higher malignancy prompted us to incorporate PET in the imaging data set used in the planning of stereotactic procedures. This allows routine use of PET data for the planning of frame-based stereotactic biopsy, frameless neuronavigation, and radiosurgery procedures, as we describe later in the chapter. Evaluation of prognostic factors in brain tumors is complex when interactions between multiple factors are taken into account. Whereas several factors influencing survival have been identified in patients with high-grade astrocytoma, the prognostic factors in adult low-grade gliomas remain poorly defined. One of the main clinical applications of PET in neuro-oncology has been its use in the follow-up of patients treated for brain tumors, because PET can differentiate between tumor recurrence and necrosis induced by radiotherapy and chemotherapy (16,17). Its use to predict tumor response to particular treatment remains limited to a few studies. This application of PET, however, appears promising in conjunction with new therapeutic strategies that should hopefully improve the prognosis of brain tumors and has led to the development of new algorithms, including more aggressive therapeutic strategies, in patients with poorer prognosis.

INTEGRATION OF POSITRON EMISSION TOMOGRAPhy IMAGES WITh OThER IMAGING MODALITIES Registration of PET images to other modalities or to the patient space are considered either in the context of framebased high precision procedures, such as for biopsy, framebased open surgery, or radiosurgery, or in the context of frameless procedures, such as in neuronavigation or radiotherapy. The choice of one or the other approach is a trade-off between accuracy and reliability against minimal invasiveness and clinical routine feasibility.

6. PET for the Management of Brain Tumors

For stereotactic PET, imaging fiducial systems defining the stereotactic coordinate space of a particular frame attached to the patient are with no doubt the most accurate and reliable way of registering PET images with the patient treatment space. Nevertheless, this requires careful solutions in addressing the various technical challenges associated with PET acquisition. The procedure of PET requires two acquisitions. Traditionally, a first scan, the transmission scan, is conducted before the injection of the radiotracer to the patient. This scan images the attenuation of the scanned object using an external radio-emitting source (either 68Ge or 18F, depending on the scanner). Then, a second scan, the emission scan, will image the detected positron-electron annihilation events after the injection and metabolic equilibrium of a positron emitter radiotracer. The final PET volume, the emission scan, reconstructs images corrected for attenuation using the transmission scan. The proposed procedure to image the fiducials is to scan the patient’s head with an adapted fiducial system for both the transmission and emission steps. The fiducial system is based on fiducials used during stereotactic MR images acquisition, where markers are tubes filled with a copper-sulfate solution. The PET adaptation uses tubing that can be filled between the transmission and emission scans with a positron radio-emitting solution with minimum radiation dose delivered to the staff. hence, a unique tube with openings on both sides equipped with an easy locking system is a convenient solution, because the solution can be filled and flushed quickly, minimizing the amount of air bubbles. This will, however, distort the configuration of the fiducials near the extremity of the fiducial system, which usually has a V or N shape, depending on the stereotactic system. The diameter of the tube should be similar to the size of the detectors to provide maximum accuracy with minimum partial volume effect. The connection paths of the tubing located between the fiducial plates should be kept as far away as possible from the field of view. Similarly, the locking system should be kept away from the field of view, because its larger diameter will produce hot spots in the image, reducing the image contrast. The activity of the radiotracer solution should be chosen to give similar signal as the maximum estimated voxel activity expected in the brain tissue. This will guarantee maximum signal-to-noise ratio for the fiducials, with no contrast losses in the imaged volume. This will depend on the radioisotope used for the solution filling the fiducials system, the diameter of the tube, the radioisotope of tracer compound injected into the patient, the weight of the patient, the acquisition parameters, and eventually a prediction of the abnormal tumor uptake of the radiotracer. When using radioactive fiducials, a typical 18F-fluoride dose for filling 1.5mm diameter tubes for a two-dimensional FDG acquisition scan of a 70-kg patient is 20 ␮Ci/mL. (A two-dimensional

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methionine scan with the same conditions would require 10 ␮Ci/mL.) Obviously, these values are illustrative, because they depend on the local setting. Defining the stereotactic transform based on the imaged fiducials is conducted with standard automatic fiducial segmentation tools that estimate rigid body transform using the Procruste algorithm (18). The most accurate results will be obtained using a large contrast window to minimize error contribution of partial volume effect and omitting volume extreme slices where linear progression of the fiducial geometry is invalidated by the tube curve. Typical fiducial registration error, defined as the root mean square error between measured and expected fiducial position, is around 0.2 mm mean for the volume with maximum value around 0.6 mm for higher error slice. Acquisition characteristics and reconstruction processes of PET scanners do not present dramatic geometric distortion as long as proper detector dimensions and arc correction are used by the scanner software and signal-to-noise ratio is sufficient. It is important to note that low resolution of PET acquisition with a point spread function full width at half maximum between 4 and 5 mm is not an indication that PET will produce inaccurate data for treatment planning, as long as the previously mentioned criteria are met. Adequate figures for mean and maximum fiducial registration error will provide a valid indicator for stereotactic PET accuracy. Phantombased validation has been conducted to verify the application accuracy of the procedure estimating the target registration error (19,20). A low-distortion CT was used as a gold standard and phantom-simulated target distance were measured between stereotactic CT and PET. When using a new generation PET camera, submillimetric target registration error is obtained; it was also shown that two-dimensional PET is more accurate than three-dimensional PET. For frameless PET, the noninvasiveness of the procedure and the organization problems related to multiple scans over a very short period require a solution to register PET without frame fiducials yet with high accuracy and reliability. Frameless registration techniques are numerous, and those that could apply to neurosurgical procedures can be divided in extrinsic registration, using external screw-based or skin-based landmarks, and intrinsic registration, using patient information such as anatomic landmarks, segmentation of surfaces, or voxel properties (21). External markers are very reliable and accurate, but although they are less invasive than a frame, they still require a dedicated scan for the planning purpose and hence will not match the clinical routine constraints. Anatomic landmarks and surface registration methods are generally the techniques used for intraoperative image to patient registration but show weaknesses in multimodality image matching. Although less reliable, voxel-based methods were validated in terms of accuracy and could provide a good alternative, provided that the physicians are aware of the potential caveats and re-

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FIGuRE 6-1. Frameless mutual information-based registration of magnetic resonance (MR) and positron emission tomography with 11 C-methionine (PET-MET) used for neuronavigation. The patient presented with a pilocytic astrocytoma that was diagnosed by stereotactic biopsy and was scheduled for resection under neuronavigation. The lesion was ill-defined on gadolinium-enhanced T1weighted MR (left), and a clear area of 11C-MET uptake on PET is shown (right). Image fusion (center) was used to optimize the definition of the resection target for neuronavigation.

spect careful quality assurance procedures (22). These registration techniques rely on the intensities of each voxel, looking for the geometric transform that maximizes a statistical similarity measures function between the two images. The most commonly used similarity measure for PET-MR registration is mutual information (23,24), because it presents more immunity to the difference between the anatomic and functional information of these modalities (Fig. 6-1). Good results were, however, reported recently using other similarity measures, such as correlation ratio. however, if validated in terms of accuracy onto a small population, their clinical validation is still under investigation and will probably show constraints for the choice of such methods.

STEREOTACTIC POSITRON EMISSION TOMOGRAPhy DATA ACQuISITION Stereotactic PET data acquisition is the gold standard for the routine management and the development of new approaches for the neurosurgical management of brain tumors. Therefore, we review in more detail the technical considerations related to this approach. The routine use of PET in stereotactic conditions was originally developed using the Fischer ZD frame (Stryker Leibinger, Freiburg, Germany) (25). We have established a 1-day procedure for the placement of the stereotactic head ring, the acquisition of images, including stereotactic PET, and the surgical planning and procedure (19). We use the Leksell Gamma Knife® C (Elekta AB, Stockholm, Sweden) and validate the acquisition of stereotactic PET with the Leksell model G® frame (Elekta AB) (20). After stereotactic CT or MR images are obtained, the patient is transferred to the PET/Biomedical Cyclotron unit, which is directly connected to the main hospital building. The unit was first equipped

with a CTI/Siemens ECAT 933 tomograph (Knoxville, TN, uSA) that allows simultaneous acquisition of 15 slices about 6.5 mm thick and that was used for the procedures performed with the ZD frame. More recently, we use a CTI/ Siemens ECAT 962 (hR+) two-dimensional and threedimensional tomograph, allowing the simultaneous acquisition of 63 planes with a slice thickness of 2.4 mm. This high precision PET imager is now used for all stereotactic procedures. During PET acquisition, the stereotactic head frame is secured in a clamp used for CT that also fits into the PET Siemens couch with a custom-made adapter. This allows a fast and easy comparable positioning of the patient’s head during CT or MR and PET. The fiducial reference system compatible with PET is based on the stereotactic MR localizers system with minor modifications, as described above. The tubes are filled either with saline for the transmission scan or with an 18F-fluoride solution for the emission scan. Transmission scan is always performed to correct the emission scan for attenuation. The patient is then injected intravenously with 6 to 9 mCi 18F-FDG and maintained in the darkened room with no verbal stimulation. Images used for stereotactic calculation are acquired between 40 and 60 minutes after injection of the tracer. We have also combined the use of PET-MET and PET-FDG in stereotactic conditions to compare the information provided by both tracers (26). The technique for PET data acquisition is similar except that after the transmission scan, the patient is first injected intravenously with 10 to 15 mCi of 11C-MET. Images used for the stereotactic planning are acquired between 20 and 40 minutes after injection of the tracer. Eighty minutes after 11CMET injection, the patient is then injected with 6 to 9 mCi of 18 F-FDG and images used for stereotactic calculation are acquired between 40 to 60 minutes later when residual 11C radioactivity from the 11C-MET injection is negligible (half-life of 11C = 20 minutes). This study evaluates the relative contribution of each type of tracer, allowing a better choice a priori of which tracer should be used when stereotactic PET is performed in patients with brain tumors (26). Currently, we only use one radiotracer for routine stereotactic procedures. Based on the accumulated experience acquired with PETguided stereotaxy, the choice of 18F-FDG or 11C-MET is based on the goal of the neurosurgical procedure applied to the management of a specific brain tumor. Although originally stereotactic PET images had to be managed separately using the PET processing software and a customized personal computer spreadsheet software (19), we are now able to routinely transfer stereotactic PET images via Ethernet to the various workstations used for the planning of the neurosurgical procedures. PET images can be transferred in the unix-based computer that runs the STP software licensed for frame-based stereotactic procedures (Stryker Leibinger) and for neuronavigation (Carl

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Zeiss, Oberkochen, Germany). This is made possible thanks to the development of a specific PET import module that permits automated integration and correlation of PET with other stereotactic image modalities. The PET image files in the CTI ECAT 5.x proprietary format are transferred to the hard disk of the STP computer where they are converted in the STP format via the PET import module. For further integration of PET data in the neuronavigation planning, we developed a specific PET modality. This permits STP to take into account the intrinsic characteristics of PET images, such as resolution, slice thickness, and the management of the color maps. This system allows rapid and easy target calculation and surgical planning using the combined information provided by PET and MR or CT. With the introduction of Leksell Gamma Knife® radiosurgery in our department, we now use the Leksell model G® frame on a regular basis, not only for radiosurgery but also for stereotactic biopsy procedures. Thus, we have established a collaboration with Elekta allowing the integration of PET images both in the software used for radiosurgery (GammaPlan®, Elekta AB) and for stereotactic neurosurgery (SurgiPlan®, Elekta AB). The approach is very similar. Registration fiducials are obtained with a dedicated PET indicator box developed by Elekta. To import PET data into GammaPlan® or SurgiPlan®, we wrote a custom software that converts PET data file format to Elekta file format. The PET volume is then handled as a CT or MR volume. To visualize PET with a high-contrast pseudocolor lookup table, we replace the grayscale lookup table file with our own PET color lookup table file when planning radiosurgery or biopsy on PET. The study on the spatial accuracy of stereotactic PET with the Leksell model G® frame provided highly accurate values, allowing the routine use of stereotactic PET as an imaging modality with this frame (20). In all stereotactic procedures used in brain tumors (biopsy, open neurosurgery, and LGK radiosurgery), planning always begins with the analysis of the stereotactic PET images. Areas of abnormal metabolism used for the procedure are either areas of tracer uptake that are higher than the surrounding normal-appearing brain or foci of relative increase of the tracer in a hypometabolic lesion. A three-dimensional volumetric PET contour delineating this area is drawn on a visual basis or using the software-based segmentation algorithm and is projected onto the corresponding MR or CT volume. The final target is defined on the MR or CT taking into account the PET-defined volume, the various aspects of the tumor (e.g., density, contrast-enhancement, infiltration), and the anatomic location of the tumor and of functional areas at risk. In specific cases, this may be refined by integrating information of other imaging modalities, such as those of functional MR.

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STEREOTACTIC POSITRON EMISSION TOMOGRPAhy FOR ThE NEuROSuRGICAL MANAGEMENT OF BRAIN TuMORS From a 10-year clinical experience with this approach, we confirm that PET data acquisition in stereotactic conditions is easily performed and does not cause any major additional discomfort to the patient. Thus far, no patient has experienced complications related to the technique. The approach is accurate enough, in that the precision of target calculation is within the PET camera resolution. Also, when performing the surgical planning, we always found a tight correspondence between PET- and CT- or MR-based targets. Actually, in our hands, data acquisition and target selection with PET have always appeared as straightforward as with CT or MR. This is facilitated by a close relationship between the neurosurgeon and the nuclear physician who set the leveling and windowing of stereotactic PET images and define together the PET volume of interest. This collaboration is very important and limits the risk of misinterpretation (which may lead to over- or underestimation of the abnormal metabolic area) of PET images by physicians inexperienced in PET methodology.

Positron Emission Tomographyguided Stereotactic Biopsy The first step toward the clinical application of PET-guided neurosurgery was provided by our experience with the use of PET for the guidance of stereotactic brain biopsy. The rationale for developing PET-guided stereotactic biopsy was based on the observation that although CT and MR were commonly used to guide stereotactic procedures, this approach fails, even in experienced hands, to provide a pathologic diagnosis in about 5% of the cases (27). Although anatomically very accurate, CT- and MR-guided stereotactic biopsy may also be responsible for inaccurate diagnosis or underestimation of histologic grading. Thus, because brain tumors, especially gliomas, are histologically heterogeneous, there is always a risk that an insufficient number or an inadequate localization of the stereotactic biopsy specimens may demonstrate tissue irrelevant to their identification. Because PET provides independent metabolic information, we have routinely integrated the use of PET data in the planning of stereotactic procedures in an attempt to improve the diagnostic yield of stereotactic brain biopsy. With the technique described above, the selection of targets on stereotactic PET-generated images can be accurately compared with those of other imaging modalities (e.g., CT or MR) and allows the neurosurgeon to direct biopsies accurately in the abnormal metabolic regions of brain tumors (Fig. 6-2). The type of information obtained with PET mostly depends on the radiotracer used. Initially, we use PET with 18F-

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FDG because of the large core of information available on the use of this marker for tracing brain tumors. Indeed, PETFDG is helpful in assessing the degree of malignancy and the prognosis of brain neoplasms independently of the histologic grading, and it can also help in differentiating between the effects of treatments and in assessing tumor persistence, progression, or recurrence. We found that PET-FDG guidance improves the diagnostic yield of stereotactic brain biopsy (28). We also found a statistically significant difference in the distribution of histologic diagnoses between PET- and CT-guided trajectories, suggesting that PET may help in selecting targets for biopsy that are the most representative of the actual diagnosis or grading of a brain tumor (28). Although, in our hands, PET-guided biopsy using 18F-FDG as a tracer appeared to be beneficial, it had, nonetheless, some limitations. Indeed, choosing a target for biopsy on PET images was not feasible when there was no or minor 18F-FDG uptake. In most instances, this happened in cases of low-grade tumors. Also, when a hypermetabolic lesion was in close relationship with the cortical or subcortical gray matter, tumor and normal 18F-FDG uptake were difficult to differentiate. When we compared the respective contribution of 18F-FDG and 11C-MET in patients who underwent PET-guided stereotactic brain biopsy with both tracers, we found that all tumors sampled had an area of abnormal 11 C-MET uptake (26). In 50% of the cases, a target could not easily be defined on PET-FDG, and PET-MET was used for target selection. For example, in tumors located in the gray matter, where tumor and normal 18F-FDG uptake were difficult to differentiate, PET-MET delineates better the areas of tumor tissue. Also, all low-grade gliomas, whether astrocytomas or oligodendrogliomas, had no distinct tumor 18FFDG uptake, but all cases disclosed a high 11C-MET uptake

FIGuRE 6-2. Planning with Leksell SurgiPlan® (Elekta AB, Sweden) of a stereotactic biopsy using combined positron emission tomography with 18F2-fluoro-2-deoxy-D-glucose (PET-FDG) (right) and gadolinium-enhanced T1-weighted magnetic resonance (MR) (left). The area of 18F-FDG uptake on PET is larger that the area of MR contrast enhancement. Serial biopsies targeting the areas of high 18F-FDG uptake, both inside (posterior temporal) and outside (anterior temporal) the area of contrast enhancement on MR, revealed tumoral blocks of a glioblastoma.

that could be used for target selection, even in ill-defined lesions on MR. The experience with PET-guided stereotactic biopsy is further confirmed in our continuous routine use of the technique, which now surpasses 150 cases. The radiotracer (18F-FDG or 11C-MET) is chosen a priori on the basis of the suspected or known diagnosis of the tumor. In many cases, we find that PET is helpful to provide a more accurate diagnosis and that when PET is not available, there is an increased risk of misdiagnosis or even failure in providing a histologic diagnosis with stereotactic biopsy. Also, increased diagnostic accuracy provided by PET-guided stereotactic biopsy improves the confidence of the neurosurgeon in its selection of targets for biopsy and contributes decreased number of targets or trajectories to perform without impairing the diagnostic yield of the procedure. This is exemplified in the management of brainstem lesions, where the place of stereotactic biopsy remains debatable (29). We showed that combined MR and PET imaging improves radiologic interpretation of a mass lesion in the brainstem but does not accurately replace histologic diagnosis provided by a stereotactic biopsy. In this latter approach, the combined information provided by MR and PET in stereotactic conditions improves the accuracy of targeting and the diagnostic yield of the stereotactic biopsy (30). The use of stereotactic PET does not only allow defining targets for biopsy, it provides a unique tool for accurate correlation between the tumor metabolism and its histopathologic characteristics. PET-guided stereotactic biopsy specifically relates a metabolic area with further pathologic analysis of the sample corresponding to the same stereotactic coordinates and has, therefore, improved our knowledge of the interpretation of structural and metabolic images (31).

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Analysis of the metabolic (e.g., quantification of tracer uptake) and histologic characteristics of the stereotactic biopsy specimen provide additional evidence that gliomas are histologically heterogeneous and that regional increase of both 18 F-FDG uptake (10) and 11C-MET uptake (32) significantly correlates with the presence of anaplasia.

Positron Emission Tomographyguided Neuronavigation We took advantage of our experience with the use of PET in stereotactic conditions, included in some open-guided stereotactic procedures (33), and integrated this image modality in the microsurgical neuronavigation procedure performed with the MKM microscope (Carl Zeiss), optimizing the resection of brain tumors. PET in stereotactic conditions, using either 18F-FDG or 11C-MET, was obtained in 43 patients presenting with a brain tumor intended for resection. The stereotactic PET images, together with stereotactic MR, were imported and correlated in the neuronavigation software. The tumor resection was planned taking into account the PET-generated metabolic and MR-generated anatomic data. This information was transferred to the computer-based neuronavigation system and displayed in the eyepiece of the microscope during the neurosurgical procedure (Figs. 6-3 and 6-4). Thus, PET data, when projected on the MR images, provided specific metabolic information on the tumor heterogeneity or extension that were useful for planning the surgery. The intraoperative availability of metabolic information was a significant aid during tumor resection, because it provided more accurate information on the location of the tumor and its extension. There was no complication specifically related to the technique. Based on combined PET and MR data, maximal tumor resection was accomplished in each case, with the intention to remove the entire abnormal metabolic area comprised in the surgical planning. As for PET-guided stereotactic biopsy, PET data acquisition and planning for neuronavigation are straightforward. These data show that metabolic neuronavigation based on the integration of PET data can be routinely used to guide the resection of brain tumors and may be used to optimize the neurosurgical management of these patients. The prognosis of brain tumors may depend on the extent of the neurosurgical resection (34,35). Consequently, because we showed that PET may help to define better tumor residues that affect the patient’s prognosis (36), one can anticipate that a neurosurgical planning integrating PET data may optimize the resection of brain tumors directed by neuronavigation, a factor that may ameliorate the prognosis. Another example can be found in the management of low-grade gliomas. The resection of these tumors is not always advisable or easy to perform because of their infiltrating nature and their possible location in highly functional regions. We showed that an area of high 18F-FDG uptake in low-grade gliomas is correlated with a poorer prognosis

FIGuRE 6-3. Neuronavigation planning with STP (StrykerLeibinger, Freiburg, Germany) for tumor resection in a patient presenting an anaplastic oligodendroglioma. Frame-based stereotactic positron emission tomography with 11C-methionine (PET-MET) (left) shows a well-defined area of increased 11C-MET uptake that was outlined and secondarily projected onto the corresponding magnetic resonance (MR) slice. This volume was used as the final target volume for resection, because it is larger than the contrast enhancement on gadolinium-enhanced T1-weighted MR and it projects in front of the motor cortex, as defined using functional MR images (not shown). These two volumes of interest are visible in the eyepiece of the microscope, as illustrated in FIGuRE 6-4.

(11). Therefore, when surgery is advisable, one should take into account the glucose metabolism status of the tumor and use neuronavigation based on PET-FDG to aim at the resection of any hypermetabolic focus to improve their prognosis. Alternatively, the margins of low-grade infiltrating lesions, which are usually ill defined on MR or CT, might be better visualized with PET-MET (26) and integrated into the neuronavigation planning. Preliminary data on the prognosis of patients treated more aggressively look promising. The analysis of longer and more detailed follow-up of these pa-

FIGuRE 6-4. Neurosurgical view from the eyepiece of the MKM microscope (Carl Zeiss, Oberkochen, Germany) in a patient with a right prerolandic anaplastic oligodendroglioma. The contours represent the projection of the volumes of interest, as defined in FIGuRE 6-3 (delineation of the tumor; delineation of the motor cortex).

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tients should provide valuable information on the potential benefits of this approach.

Positron Emission Tomographyguided Radiosurgery with LGK The integration of PET image modality in GammaPlan®, as described above, is an ongoing project in collaboration with the Elekta research and development department. The current PET software module is not a commercially available version, and its clinical evaluation is performed in the framework of a study approved by the Ethical Committee of our institution (20). There are two goals of this approach: first, to improve the targeting in recognized indications of radiosurgery (e.g., recurrent metastasis, recurrent pituitary adenoma) and, second, to provide an appropriate targeting technique for lesions that are not classic indications of radiosurgery but may benefit from this treatment (i.e., infiltrative and primary tumors of the brain, more specifically glial tumors). Between December 1999 and September 2002, 46 patients with recurrent tumors were treated with LGK using the combined guidance of PET and MR for the dosimetry planning with GammaPlan®. This represents 10% of the total number of patients treated with LGK in our center during the same period of time. There were 33 primary brain tumors, including 30 glial tumors (5 pilocytic astrocytomas, 8 low-grade astrocytomas or oligodendrogliomas, 7 anaplastic astrocytomas or ependymomas, 10 glioblastomas), 2 neurocytomas, and 1 ganglioglioma; five patients with metastases (single or multiple lesions); and eight patients with pituitary adenomas. Thus, most treated indications (65%) corresponded to recurrent glial tumors that received LGK radiosurgery in the framework of multimodality therapies. PET-FDG was used in 24 cases, mostly (21 cases) for the identification of specific areas of high metabolic activity within malignant primary tumors and in metastases. PET-MET was used in 22 cases, mostly (20 cases) to define the tumor limits in benign primary tumors and in pituitary adenomas. The planning begins with analysis of the PET images in GammaPlan®. A three-dimensional volumetric contour delineating the area of increased radiotracer uptake is defined and is projected onto the corresponding MR. The final volume treatment is defined on the MR image, taking into account the gadolinium contrast enhancement as well as other MR characteristics and the PET-generated metabolic data (Fig. 6-5). Multiple isocenters with collimators of different sizes are used to fit the prescription isodose to the target volume. When PET uptake is partially shifted outside of the MR-defined volume, it was comprised in the final volume, only in relatively safe brain regions. When PET uptake is smaller and comprised in the MR volume, we try to focus the highest spot of the delivered dose in the area of PET uptake; however, when the MR volume is too large and incompatible with radiosurgery, we limit the target volume to the area of abnormal PET uptake. The

strategy of target selection also takes into account the nature of the tumor, the clinical condition, and the prognosis. PET provided contributive data in 43 cases (93%), and the information was specifically used to adapt and alter the target volume in 35 cases (76%). Preliminary analysis of these data shows that this approach is accurately applicable in clinical routine and that it provides additional information that may be of significant benefit for the patients with primary or metastatic recurrent brain tumors. Also, new indications for PET are emerging, such as the use of PET-MET to guide LGK in recurrent pituitary adenomas, in which PET-MET helps to differentiate between the normal residual pituitary gland, scar tissue, and residual or recurrent adenoma. PET is also useful for the pretreatment and follow-up evaluation of these patients. The use of stereotactic PET in these patients accurately compares PET-based metabolic data with MR-based anatomic data and, when combined with sequential PET and MR, will enable a better understanding of the metabolic changes after radiosurgery.

PROGNOSTIC VALuE OF POSITRON EMISSION TOMOGRAPhy IN NEuROONCOLOGy The prognosis of gliomas is highly dependent on their histologic grades. PET allows obtaining, before surgery, information on the glucose and amino acid metabolisms of these tumors. Although this information is correlated with the degree of malignancy of gliomas (32), our experience shows that PET also represents a prognostic factor that is independent of histology. Consequently, PET may play a role not only in the planning of neurosurgical procedures, but to a larger degree in optimizing the treatment of gliomas.

Prognostic Value of 18F-2-Fluoro-2Deoxy-D-Glucose uptake in Glioma Glucose uptake in tumor cells has been reported in various neoplasms more than 50 years ago and depends on several factors, such as glycolysis originating from increased hexokinase activity (increased expression or changes in the regulatory process) and changes in glucose transporter (37). The cellular causes of this increase in uptake are not yet completely clarified. Low-grade gliomas (World health Organization [WhO] grade II) generally have a glucose metabolism that is lower than high-grade gliomas (WhO grades III and IV) (38). The uptake of 18F-FDG is statistically correlated with the histologic grade and therefore with the length of survival. Low-grade gliomas generally have a low uptake of 18 F-FDG and are hypometabolic on PET images (Fig. 6-6). Di Chiro (39) described a high uptake of 18F-FDG within the limits of low-grade gliomas in four cases but did not provide clinical follow-up of these patients. In 1996, we reported our clinical results in patients with low-grade gliomas assessed

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FIGuRE 6-5. Dosimetry planning with Leksell GammaPlan® (Elekta AB, Sweden) using combined positron emission tomography with 18F-2fluoro-2-deoxy-D-glucose (PET-FDG) and magnetic resonance (MR) images, for Leksell Gamma Knife® (Elekta AB, Sweden) radiosurgery in a patient with a recurrent anaplastic astrocytoma, after multiple surgical resections, fractionated radiation therapy, and chemotherapy. The large area of 18FFDG uptake (left) is contoured and projected onto the corresponding MR slice. This volume has been combined with the area of contrast enhancement on gadolinium-enhanced T1-weighted MR to define the target volume for the prescription isodose volume.

with PET-FDG (11). Patients had an area of high 18F-FDG uptake on PET images (Fig. 6-6B). The histologic diagnosis of low-grade glioma was always confirmed, because PETguided stereotactic biopsy was performed in the area of hypermetabolism (19,28). A statistical analysis of survival revealed that if PET showed hypermetabolism, 75% of those patients are expected to die during the first year, whereas if there is no hypermetabolism, 95% are expected to survive to the end of the first year. This metabolic feature, detectable with a noninvasive procedure, may provide a clue to cellular changes, announcing malignant transformation in a tumor that retains the histologic features of a low-grade glioma. The survival time of high-grade gliomas is short. Information obtained with PET studies showed a correlation between survival and the uptake of 18F-FDG (40). PETFDG-guided stereotactic biopsies demonstrated that the presence of anaplasia is associated with a higher level of glucose metabolism (10). high uptake of 18F-FDG is also attributed to an increased glycolytic rate in rapidly growing brain tumors cells. In another study (39), prenecrotic cells were hypermetabolic, probably because anaerobic glycolytic pathways were activated with an increased glucose uptake. Altogether, these data provide information of the aggressiveness of the tumor and help to explain the correlation between survival and glucose uptake. In patients with recurrent high-grade gliomas, adjuvant chemotherapy will benefit only a few patients (41). PET-FDG could predict the response of malignant gliomas to chemotherapy and therefore help to select the nonresponder patient for another treatment (42–45). In our experience, an increase of 18F-FDG uptake after the first course of carmustine is correlated with a longer survival (44). Other studies have shown that the response to treatment is correlated with a decrease of the 18F-FDG uptake. The discrepancy be-

tween those data may reflect a difference in the selection of patients and the timing of the PET analysis.

Prognostic Value of uptake in Glioma

11

C-Methionine

Tumor uptake of 11C-MET was first described more than 20 years ago, but mechanisms and biologic significance of this phenomenon remain incompletely understood (46). 11CMET and other large neutral amino acids are transported across the blood–brain barrier and cell membranes by a particular carrier system (47). In the normal brain and in gliomas, this carrier system is stereospecific and saturable. Therefore, 11C-MET uptake can be competitively inhibited by an unlabeled amino acid. uptake of 11C-MET in gliomas is attributed to the activation of the carrier-mediated transport and not to increased diffusion at the blood–brain barrier level. Indeed, low-grade glioma, for which no contrast enhancement is demonstrated on CT or MR, may present with high uptake of 11C-MET. This uptake does not directly reflect protein synthesis, but it represents the cell avidity for amino acids, which relates to the activity of the transport system and incorporation into proteins (48). The correlation between 11C-MET uptake and the histologic grade or anaplasia in glioma has been reported (32,49). We found that the presence of 11C-MET uptake in gliomas also represents a prognostic factor that is independent of histology (50). The survival was significantly shorter for patient presenting a high uptake of 11C-MET on visual or semiquantitative scale, including when the different grades of the tumors are considered separately. More importantly, lowgrade gliomas with a ratio (ratio between the count rate determined in the regions of interest drawn in the tumor area of highest uptake of 11C-MET and the mean count rate in three

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FIGuRE 6-6. A. Patient with a left frontoparietal astrocytoma, grade II. A huge area of hypometabolism is visible on positron emission tomography with 18F-2-fluoro-2-deoxy-D-glucose (PET-FDG), illustrating the usual 18F-FDG metabolic findings in low-grade gliomas. B. Patient with a left frontal astrocytoma, grade II. A spot of high uptake of 18F-FDG is observed within the hypometabolic limits of the tumor. The histology has been confirmed with PET-guided stereotactic biopsy aiming at the hypermetabolic focus. This patient died 6 months after diagnosis.

regions of interest drawn in the contralateral cortical areas) threshold of 2.2 are statistically associated with a shorter survival (Fig. 6-7). The finding is similar for anaplastic tumors but with a ratio threshold of 2.8. Expression of growth factors and their receptors (particularity vascular endothelial growth factor) may play a role in the relationship between the evolution of the tumor and 11CMET uptake. The angiogenic process initiated by the vascular endothelial growth factor system induces an increase in carrier-mediated large amino acid transport. Interestingly, the relationship between 11C-MET uptake and Ki-67 labeling in WhO grade III gliomas (relation between cellular proliferation and 11C-MET uptake) has been reported (51). An upregulation of amino acid transport may anticipate the phase of rapid cell proliferation in glioma. An in vitro study on glioma cell lines demonstrated that the L-like neutral amino acid transport is up-regulated in parallel with the expression of the tumor marker 4F2 antigen in the lag phase preceding the exponential growth phase (52). That could explain why for the same histologic grade the uptake of 11C-MET could be different and represent the expression of changes in gene expression, which may be an early sign of tumor evolution.

Interest in Positron Emission Tomography Data for the Management of Brain Tumor The knowledge gained from PET analysis of brain tumors has completely changed our management of gliomas. The prognostic value of PET-FDG and PET-MET in low-grade gliomas allows earlier detection of patients with a rapid and deleterious evolution. Therefore, since 1996 we proposed an aggressive therapeutic strategy to patients with a lowgrade glioma presenting a high uptake of 18F-FDG and 11CMET on PET. Some patients refused treatment (control group), whereas other patients had aggressive treatment, including resective surgery under PET-guided neuronavigation (33), chemotherapy (carmustine for grade II astrocytoma, procarbazine, CCNu, vincristine for pure or mixed oligodendroglioma), and radiation therapy. Survival curves are significantly different between patients who were and were not treated (Fig. 6-8). For patients with high-grade gliomas, PET information does not change the management. however, at the time of recurrence, the metabolic data of PET are routinely integrated in the neuronavigation system (33) or in the planning of radiosurgery (20) to identify and focus the treatment on the active part of the tumor. A similar strategy is now proposed in the early phase of surgery and radiation therapy when residual tumor is still visible on PET images. More specifically, PET-guided radiosurgery

6. PET for the Management of Brain Tumors

FIGuRE 6-7. Survival curves for patients presenting with a lowgrade glioma and different uptake of 11C-methionine (11C-MET) on positron emission tomography. Patients with a higher uptake of 11 C-MET (ratio between the count in the tumor area and the count in the contralateral cortex, R > 2.2) are statistically associated with a shorter survival.

is proposed as an early adjunct therapy for residual tumor that could not be resected. Further studies must be performed for the evaluation of tumor progression with those treatments.

CONCLuSION AND PERSPECTIVES PET has widened our perspective for better treatment of low-grade gliomas and probably also for high-grade gliomas. This technology may be used in research and development projects with new markers but is already in use for the benefit of many patients. Detecting an anaplastic focus in a low-grade glioma can offer the patient the most appropriate treatment. Removing surgically or destroying that anaplastic focus with LGK radiosurgery offers a longer survival time with a better quality of life. Following the effect of chemotherapy can predict the benefits of treatment. Differentiating radionecrosis from tumor recurrence makes our strategy more logical and efficacious. We know that surgery or radiosurgery, although playing a major role in management, is not a curative approach. The future may be in immunotherapy or gene therapy or perhaps in new forms of chemotherapy that will target the infiltrative process. The need to better understand the biology of gliomas is obvious if we wish to adapt our strategy with the greatest hope of success. In the future, the cooperation between neurosurgeons and other specialists, like neuroradiologists, nuclear physicians, and immunologists, and nonmedical specialists, like engineers, physicists, chemists, computer scientists, and biologists, will bring together all their knowledge to invent new tools and new treatments and will help neurosurgeons in deciding which is the best strategy for patients suffering from a brain glioma.

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FIGuRE 6-8. Survival curves in patients presenting with lowgrade gliomas and a high uptake of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and 11C-methionine (11C-MET) (positron emission tomography positive [PET+]). Patients receiving aggressive treatment, including surgery, chemotherapy, and radiation therapy (PET+/treatment), have a statistically significant longer survival than patients who refused treatment at the time of diagnosis (PET+/No treatment): p = 0.0017.

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14. Goldman S, Monclus M, Cool V, et al. A novel PET tracer for evaluation of gene therapy. J Nucl Med 1996;37:53P. 15. Monclus M, Luxen A, Cool V, et al. Development of a positron emission tomography radiopharmaceutical for imaging thymidine kinase gene expression: synthesis and in vitro evaluation of 9[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine. Bioorg Med Chem Lett 1997;7:1879–1882. 16. Patronas NJ, Di-Chiro G, Brooks RA, et al. Work in progress: [18F] fluorodeoxyglucose and positron emission tomography in the evaluation of radiation necrosis of the brain. Radiology 1982;144:885–889. 17. Di Chiro G, Oldfield E, Wright DC, et al. Cerebral necrosis after radiotherapy and/or intraarterial chemotherapy for brain tumors: PET and neuropathologic studies. Am J Radiol 1988;50:189–197. 18. Kelly PJ. Tumor stereotaxis. Philadelphia: WB Saunders, 1991. 19. Levivier M, Goldman S, Bidaut LM, et al. Positron emission tomography-guided stereotactic brain biopsy. Neurosurgery 1992;31:792–797. 20. Levivier M, Wikler D, Goldman S, et al. Integration of the metabolic data of positron emission tomography in the dosimetry planning of radiosurgery with gamma knife: early experience with brain tumors. J Neurosurg 2000;93[Suppl 3]:233–238. 21. Maintz JB, Viergever MA. A survey of medical image registration. Med Image Anal 1998;2:1–36. 22. West J, Fitzpatrick JM, Wang My, et al. Retrospective intermodality registration techniques for images of the head: surface-based versus volume-based. IEEE Trans Med Imag 1999;18:144–150. 23. Maes F, Collignon A, Vandermeulen D, et al. Multimodality image registration by maximization of mutual information. IEEE Trans Med Imag 1997;16:187–198. 24. Wells WM 3rd, Viola P, Atsumi h, et al. Multi-modal volume registration by maximization of mutual information. Med Image Anal 1996;1:35–51. 25. Levivier M, Goldman S, Pirotte B, et al. First year experience using the Fischer ZD-Neurosurgical Localizing unit with particular reference to the development of PET-guided stereotactic biopsy. Acta Neurol Belg 1993;93:5–22. 26. Pirotte B, Goldman S, David P, et al. Stereotactic brain biopsy guided by positron emission tomography with [18F]fluorodeoxyglucose and [11C]methionine. Acta Neurochir 1997; 68[Suppl]:133–138. 27. Black PM. Brain tumors. N Eng J Med 1991;324:1471–1476, 1555–1564. 28. Levivier M, Goldman S, Pirotte B, et al. Diagnostic yield of stereotactic brain biopsy guided by positron emission tomography with [18F]fluorodeoxyglucose. J Neurosurg 1995; 82:445–452. 29. Levivier M, Massager N, Brotchi J. Management of mass lesions of the brainstem. Crit Rev Neurosurg 1998;8:338–345. 30. Massager N, David P, Goldman S, et al. Combined MR and PET imaging in brain mass lesions: diagnostic yield in a series of 30 stereotactically biopsied patients. J Neurosurg 2000;93:951–957. 31. Levivier M, Becerra A, De Witte O, et al. Radiation necrosis or recurrence. J Neurosurg 1996;84:148–149. 32. Goldman S, Levivier M, Pirotte B, et al. Regional methionine and glucose uptake in high grade gliomas: a comparative study on PET-guided stereotactic biopsy. J Nucl Med 1997; 38:1–4. 33. Levivier M, Wikler D, Goldman S, et al. Positron emission tomography in stereotactic conditions as a functional imaging technique for neurosurgical guidance. In: Alexander EI, Maciunas RJ, eds. Advanced neurosurgical navigation. New york: Thieme, 1999:85–99. 34. Albert FK, Forsting M, Sartor K, et al. Early postoperative magnetic resonance imaging after resection of malignant glioma— objective evaluation of residual tumor and its influence on regrowth and prognosis. Neurosurgery 1994;34: 45–61. 35. Nitta T, Sato K. Prognostic implications of the extent of surgical resection in patients with intracranial malignant gliomas. Cancer 1995;75:2727–2731.

36. De Witte O, Levivier M, Violon P, et al. Quantitative imaging study of extent of surgical resection and prognosis of malignant astrocytomas. Neurosurgery 1998;43:398–399. 37. Warburg O. On the origin of cancer cells. Science 1956; 123:309–314. 38. Delbeke D, Meyerowitz C, Lapidus RL, et al. Optimal cutoff levels of (18F) fluorodeoxyglucose uptake in the differentiation of low grade from high grade brain tumors with PET. Radiology 1995;195:47–52. 39. Di Chiro G. Positron emission tomography using (18F) fluorodeoxyglucose in brain tumors: a powerful diagnostic and prognostic tool. Invest Radiol 1986;22:360–371. 40. De Witte O, Lefranc F, Levivier M, et al. FDG-PET as a prognostic factor in high-grade astrocytoma. J Neuro-Oncol 2000;49:157–163. 41. Walker MD, Green SB, Byar DP, et al. Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 1978;303:1323–1329. 42. Rozental JM, Cohen JD, Metha MP, et al. Acute changes in glucose uptake after treatment: the effect of carmustine (BCNu) on human glioblastoma multiforme. J Neuro-Oncol 1993;15:57–66. 43. Rozental JM, Levine RL, Nickles RJ, et al. Glucose uptake by gliomas after treatment: a positron emission tomographic study. Arch Neurol 1989;46:1302–1307. 44. Valk PE, Budinger TF, Levin VA, et al. PET of malignant cerebral tumors after interstitial brachytherapy: demonstration of metabolic activity and correlation with clinical outcome. J Neurosurg 1988;69:830–838. 45. De Witte O, hildebrand J, Luxen A, et al. Acute effect of carmustine on glucose metabolism in brain and glioblastoma. Cancer 1994;74:2836–2842. 46. Comar D, Cartron J, Mazière M, et al. Labeling and metabolism of methionine-methyl-11C. Eur J Nucl Med 1976;1: 11–14. 47. Pardridge WM. Brain metabolism a perspective from the bloodbrain barrier. Physiol Rev 1983;63:1481–1535. 48. Derlon JM, Bourdet C, Bustany P, et al. (11C)L-Methionine uptake in gliomas. Neurosurgery 1989;25:720–728. 49. Kaschten B, Stevenaert A, Sadzot A, et al. Preoperative evaluation of 54 gliomas by PET with fluorine-18-fluorodeoxyglucose and/or carbon-11-methionine. J Nucl Med 1998;39:778–785. 50. De Witte O, Goldberg I, Wikler D, et al. Positron emission tomography with injection of methionine as a prognostic factor in glioma. J Neurosurg 2001;95:746–750. 51. Kaschten B. Contribution à l’étude du métabolisme et de la croissance des tumeurs cérébrales. Définitions d’indices pronostiques. Academic Thesis, university of Liège, Belgium, 1997:159–162. 52. Langen KJ, Bonnie R, Mühlensiepen h, et al. 3-(123I)-a-methylL-tyrosine transport and 4F2 antigen expression in human glioma cells. Nucl Med Biol 2001;28:5–11.

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w Magnetic Resonance Spectroscopy and Its Use in Brain Tumor Diagnosis and Therapy

andrea pirzkall any targeted therapeutic intervention for gliomas, be it surgery or radiation therapy (RT), carries an increased incidence of side effects if the therapy is not delivered accurately to the target and surrounding tissues spared. Magnetic resonance imaging (MRI) has become the imaging technique of choice for interventional treatment planning for gliomas. MRI is believed to be clearly superior to computed tomography for most brain tumors because of its improved soft tissue contrast, providing much better visualization of anatomic relationships (1,2). In addition, there is the potential to use MRI for detecting subtle physiologic or structural changes by evaluating parameters such as diffusion, perfusion, and/or flow (3). There are several problems with basing therapy on MRI alone, however. The gadolinium-enhancing lesion as seen on T1-weighted MRI may not always correspond to the region of active disease because of nonenhancing tumor tissue and of contrast-enhancing necrosis. nonspecific processes, such as inflammation or reactive edema formation, also may appear hyperintense on T2-weighted MRI, making it difficult to determine what is and what is not tumor. In addition, serial biopsy studies show tumor cells extending in a variable pattern beyond the enhancement region, in the area of edema, and even in normal-appearing brain adjacent to the region of T2weighted hyperintensity (4). Serial biopsy studies also have shown that tumor infiltration tends to follow white matter fiber tracts (5). Information that can improve the definition of the spatial extent of tumor may improve the ability to define the volumes to which treatment should be directed. Functional or metabolic imaging has been proposed for this purpose, and a variety of techniques, such as positron emission tomography (peT) and single photon emission computed tomography (SpeT), has been explored. one of the most promising techniques for delineating tumor is magnetic resonance spectroscopy (MRS). For in vivo systems it is possible to combine

the chemical specificity of MRS with localization techniques that have been developed for MRI to obtain signals from one or more different regions of tissue. Recent improvements in hardware and software have made it possible to obtain routinely spectra from patients with brain tumors and to correlate these with anatomic MR images. In this chapter we examine the use of multivoxel proton (1H) MRS for diagnosing and planning focal therapy in patients with gliomas.

MagneTIc ReSonance SpecTRoScopy: geneRal aSpecTS What Is Magnetic Resonance Spectroscopy? MRS makes use of the fact that certain atomic nuclei, such as 1H, 31p, 19F, and 13c, have inherent spin properties, allowing them to acquire discrete amounts of energy in the presence of a static magnetic field. The application of electromagnetic fields (nonionizing radiofrequency radiation) at right angles to the static magnetic field causes these nuclei to jump to states of higher energy levels. after removal of the electromagnetic fields, the nuclei subsequently drop back to their original spin states by emitting electromagnetic radiation at a certain rate that is measured by T1-weighted (spinlattice) and T2-weighted (spin-spin) relaxation times. a receiver coil detects the emitted radiation and records the time domain MR signal. This signal must be processed using a Fourier transformed to reveal the spectrum of intensities and frequencies of the nuclei from different chemical species within the excited volume. The location of peaks in the spectrum defines the chemicals within the sample, and the peak intensity reflects their concentrations and relaxation times. MRI uses the properties of the protons from water to obtain information about the spatial distribution of different tissues. Specialized radiofrequency pulses and magnetic field gradients are used to label the water signal as a function of space and, after appropriate postprocessing, provide an an109

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atomic image of the changes in proton density and relaxation properties. By modifying the data acquisition procedures for MRI to detect signals at a frequency corresponding to 31p, 19F, and 13c or by suppressing the signal from water protons (1H), it is possible to obtain data that reflect variations in spatial and frequency information at the same time. postprocessing of such spectroscopic imaging (magnetic resonance spectroscopy imaging [MRSI]) data reveals the “hidden” information on the chemical composition of tissues of interest within each spatial element and can be used to generate maps of the spatial distribution of cellular metabolites. although early attempts at in vivo MRSI focused on phosphorus (31p) spectroscopy (6), the higher sensitivity of 1H-MRSI has made it more accessible to patients with brain tumors and has been used to demonstrate significant differences in spectra between tumor and normal brain tissue. a significant advantage of 1H-MRSI over other metabolic imaging techniques is that the data can be obtained as part of a conventional MRI examination and hence directly correlated with anatomic images.

Magnetic Resonance Spectroscopy Imaging Techniques in Brain a number of techniques have been developed for obtaining proton spectra from selected regions within the brain. These techniques provide either a single spectrum or a multidimensional array of spectra from the region of interest (7,8). Many of the initial studies applied the single-voxel technique to provide a spectrum from a defined volume of tissue as large as 4 ⫻ 4 ⫻ 4 cm that included the entire tumor (9–12). although the spatial resolution has improved and singlevoxel MRS studies are easy to implement for routine clinical applications, they often suffer from improper or imprecise localization because they are located based on MR images that may not differentiate between tissue types. In addition, the acquisition of metabolic data within a fairly large volume compartment introduces partial volume effects. other limitations of single-voxel techniques are the inability to assess spatial distribution of disease and to observe metabolic changes that would provide information for follow-up studies that assess response to therapy and degree of local control. advances in MRS techniques that were proposed as early as 1982 (13) allow the excitation and recording of a multidimensional array of spectra from hundreds of contiguous voxels of 1 ml or less in size (10,14,15). phase-encoding techniques are applied to obtain localized spectra from arrays of multiple contiguous voxels. although two-dimensional, or single slice, MRSI can provide data from within a chosen plane (10,15), the extent and distribution of potential tumor or suspicious spectra outside of the selected slice remains unknown. In addition, follow-up examinations are complicated by the need for reproducible patient positioning and slice selection. These problems can be overcome by the application of the point-resolved spectral selection (pReSS) or

stimulated echo acquisition mode (STeaM) spectroscopy localization to select a region of interest covering the tumor and surrounding tissue and then phase encoding in three spatial dimensions to yield data that can be reconstructed to correspond to volumetric arrays of spectral voxels. This multivoxel or three-dimensional MRSI technique provides spectra throughout the defined volume of interest and allows the spatial discrimination of different tissues based on their molecular characteristics. The increase in spatial resolution, and the associated decrease in partial volume effects, makes three-dimensional MRSI of potential value in target delineation and monitoring response to therapy.

Magnetic Resonance Spectroscopy Metabolites in the Brain a wide array of brain metabolites has been studied using in vivo 1H-MRS. Most of these have been performed at the standard clinical field strength of 1.5 T, but there are an increasing number of institutions that have access to 3 T or 4 T whole body systems. The most common metabolites considered at an echo time of 144 ms are n-acetylaspartate (naa), choline, creatine, alanine and other amino acids, lipid, and lactate. at shorter echo times and with stronger magnets, additional peaks can be identified, including myoinositol, ␥-aminobutyric acid, glutamine, and glutamate. In normal brain tissue, the largest signal arises from naa (2.0 ppm, Fig. 7-1a), shown by labeled antibody studies to be confined to neurons and absent in glial cells (16). In brain tumors, naa is diminished (or absent, Fig. 7-1B) because of the infiltration of tumor cells that replace functional neurons (10,17,18) or because of the loss of functional activity (19). an increase in the choline signal (3.2 ppm, Fig. 7-1B) also is characteristic of neoplastic lesions (15,20,21). The choline signal is caused by free choline and choline containing compounds such as phosphocholine and glycerophosphocholine. choline is a neurotransmitter, and as a component of the cell membrane it is involved in its synthesis and degradation (22). an increase in choline may indicate rapid cell division in a growing tumor or cellular hyperplasia, neuronal degeneration such as phospholipid metabolism and myelin degradation, and/or an early necrotic process (23). The creatine signal (3.0 ppm) is indicative of cellular bioenergetic processes: creatine and phosphocreatine (total creatine) are essential for the adenosine diphosphate and adenosine triphosphate energy cycle (22). It is used typically as a reference for estimating changes in other metabolites. Some brain tumors also may show a lactate signal (1.3 ppm) that is normally inverted at an echo time of 144 ms. lactate is an end product of anaerobic metabolism that occurs when a rapidly growing tumor does not get a sufficient oxygen supply from its neovasculature (24); its presence can therefore represent cellular breakdown. Quantification of the lactate signal is complicated by the fact that it may be masked by a positive signal from lipids. lipid peaks

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FIgURe 7-1. patient with left temporoparietal glioblastoma multiforme. axial T1-weighted postcontrast magnetic resonance image shows typical contrast-enhancing ring around central necrosis. Three-dimensional magnetic resonance spectroscopy imaging performed within area of excitation (point resolved spectral selection box, represented by gridlines) reveals metabolic signature of examined brain tissue. Three examples of spectral patterns are displayed: (a) normal brain tissue marked by high peak of n-acetylaspartate (naa) and low peak of choline (cho). The resulting cho-to-naa ratio is therefore low (about 1:2). (B) Tumor spectrum characteristic by an increase of cho and a decrease in naa as compared with the normal tissue voxel. The cho-to-naa ratio is high (about 1:0.5). note that the spectrum is derived from a single voxel in the area of contrast enhancement. (c) loss of all metabolite signals (flat signal) characteristic for necrosis in a voxel within the contrast enhancement. (necrotic spectra might also exhibit lactate and/or lipid peaks.)

(1.3 and 0.9 ppm) also are associated with necrosis. necrosis can also be detected as a loss of all metabolite signals (Fig. 7-1c). less frequently studied metabolites may be disease specific. For instance, alanine is an amino acid that is sometimes observed in meningiomas (25). Myoinositol may be increased in hemangiopericytomas, thus differentiating it from meningiomas (26).

Magnetic Resonance Spectroscopy Imaging protocol at University of california San Francisco Three-dimensional MRSI has been applied and continuously improved at the Magnetic Resonance Science center at the University of california San Francisco (UcSF) since 1990. our current protocol uses a three-dimensional, chemical shift imaging (cSI), multivoxel MRSI sequence that is usually prescribed at the conclusion of a diagnostic MRI study. all MR examinations to date have been performed on a 1.5-T Signa echospeed clinical scanner (general electric Medical Systems, Milwaukee, WI, USa). The imaging component of each study includes the acquisition of (a) an axial fluid-attenuated inversion recovery (FlaIR) sequence and/or a fast spin echo (FSe) sequence as T2-weighted se-

quences with 3-mm slice thickness and (b) T1-weighted preand postgadolinium contrast-enhanced spoiled gradient (SpgR) echo volume sequence with 1.5-mm slice thickness. The acquisition parameters used in these studies have been reported elsewhere (27,28). The total MR examination time is approximately 90 minutes, of which 17 minutes are used for MRSI acquisition. For MRSI, is applied via pReSS with volume localization with spectral and spatial-selective pulses for water suppression. Because of acquisition time constraints, the size of the region of interest from which MRSI data are obtained is usually restricted. Typical acquisitions consist of a 16 ⫻ 8 ⫻ 8 cm or 12 ⫻ 12 ⫻ 8 cm matrix (“pReSS box”) with a nominal spatial resolution of 1 ml (1 ml/voxel). The pReSS box is positioned so as to extend beyond the suspected disease to include normal brain from the contralateral hemisphere to allow the recording of control spectra (presumed normal brain tissue) even if this means that the entire region of suspected disease cannot be covered. The pReSS box also is positioned to avoid areas of subcutaneous lipid and varying magnetic susceptibility that might compromise the quality of the spectra. In addition, spatially selective saturation bands, custom designed to eliminate signals from subcutaneous lipid, are applied as necessary (29).

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The raw spectral data are reconstructed using software developed at the Magnetic Resonance Science center (27). a variety of approaches has been used to analyze the spectroscopic data. These have included a comparison of absolute values for peak height, width, and area in the involved areas with similar values derived from normal volunteers or from unaffected brain tissue as well as an examination of the ratios of various metabolites. Studies from our group comparing the MRSI pattern and histology at specific brain locations in patients with both treated and untreated glioma showed that a combined increase in choline and a decrease in naa was highly indicative of tumor (30,31). another significant finding was that 100% of the biopsies taken from regions where choline was elevated more than 2 standard deviations above normal and where naa was decreased at least 2 standard deviations below normal contained tumor (31). a third finding was that for spectra acquired with a long echo time, the ratio of choline to naa was a more robust parameter than other metabolic ratios, such as choline to creatine and choline to normal choline, for distinguishing tumor from nontumor in patients with new or recurrent glioma. Based on these findings, initial studies at UcSF assessing the clinical value of MRSI in evaluating gliomas have focused on characterizing the spectra based on the relative levels of choline and naa. The peak parameters (height, width, area) for choline and naa are estimated on a voxel by voxel basis within the excited region (Fig. 7-2). an automated statistical analysis identifies normal and abnormal voxels based on an iterative procedure that selectively removes outliers from a linear regression of choline to naa and simultaneously identifies a control population of spectra acquired from normal tissue from the contralateral hemisphere included within the image volume from the studied patient (32). The degree of spectra abnormality is determined on a voxel by voxel basis, quantified as the cholineto-naa index (cnI) defined by its residual or z-score (labeled previously as the abnormality index) (33). The cnI values are displayed as contour maps overlaid on the appropriate MR image (Fig. 7-2). our current working assumption uses a cnI of 2 as the cutoff or threshold for abnormal brain tissue (control voxels have a choline-to-naa ratio that falls reliably within 2 standard deviations for each patient and therefore has a cnI ⬍ 2). preliminary studies correlating the cnI with histopathology assessed a cnI of 2 as 96% sensitive and 57% specific (34). other metabolite indices, such as choline to creatine, creatine to naa, and lactate ⫽ lipid, can be calculated using a similar regression analysis to investigate their spatial distribution within the suspicious lesion (35). The metabolic indices can be displayed as contour maps superimposed on the MR image, a process that involves spatial interpolation. We plan to investigate these metabolic ratios in relation to their prognostic value. However, our recent research has mainly focused on the evaluation of the spatial extent of cnI

values of 2, 3, 4, and the absolute maximum value, assuming that the highest cnI might correlate to the highest degree of abnormality. although we are confident that a cnI higher than 2 implies malignancy, we do not know yet the significance of progressively higher values of cnI. It seems intuitive that such demarcated areas within a lesion might correspond to tumor areas that are more active or have a higher tumor cell density, and preliminary observations confirm such an assumption. For instance, in low-grade gliomas with naturally no or only very little ce, the highest cnI values were spatially correlated to the small islands of ce (36). However, further correlation studies are required before these results can be considered conclusive.

SpecIFIc applIcaTIonS oF MagneTIc ReSonance SpecTRoScopy FoR BRaIn TUMoRS Support for Diagnosis and Tumor grading Before Surgery although MRS has been found to be of value in differentiating between neoplastic and non-neoplastic processes such as abscess, infarction, and demyelinating disease (i.e., multiple sclerosis) (18,22,37,38), most research to date has been in the diagnosis and noninvasive grading of tumors (10,39,40). MRS techniques using single-voxel MRS and two-dimensional spectroscopic imaging have been applied to investigate the usefulness of MRS in this setting. although these initial studies reported a clear trend for higher mean choline and lower mean naa levels in high-grade tumors, large standard deviations in metabolite ratios and substantial overlap in values were found that precluded accurate tumor grading. More recently, through the use of advanced data analysis techniques, a higher degree of accuracy for in vitro and in vivo MRS has been achieved (12,41). one important study by preul and colleagues (10) demonstrated a significant improvement in the accurate diagnosis of supratentorial brain tumors in 91 adults using 1H-MRS, with a pattern recognition analysis yielding a correct diagnosis 99% of the time compared with the primary preoperative diagnosis based on MRI alone of 77%. another study from the same group showed further that the distinction between brain tumors and isolated demyelinating lesions of the type seen in multiple sclerosis is possible with MRS even though more research is necessary to determine the definite metabolic profile for nonacute lesions (42). a study by li et al. (35), examining a variety of metabolic indices (cnI, choline to creatine index (ccrI), creatine to naa index (gnI), and lactate to lipid index (llI) in grades II to IV brain gliomas in 49 patients, showed that the tumor burden measured with either the volume of the metabolic abnormalities or the maximum magnitude of the metabolic indices was correlated

7. MRS and Brain Tumor Diagnosis and Therapy

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FIgURe 7-2. patient with left temporooccipital glioblastoma multiforme after subtotal resection. Top: axial T1-weighted postcontrast magnetic resonance (MR) image shows small area of contrast enhancement (ce) along the posterior and lateral aspect of the resection cavity, but T1-weighted precontrast revealed mainly blood residue in the remaining parts. Superimposed is the extension of the point resolved spectral selection box (solid line) and a subregion (broken line) enlarged to the right. Bottom left: Magnetic resonance spectroscopy imaging (MRSI) spectral pattern and calculated choline-to-n-acetylaspartate index (cnI) as acquired in contiguous 1-ml voxels. Display of cnI contours after interpolation and sampling to match the resolution of the MR image. Bottom right:. Incorporation of the MRSI derived cnI contours into the treatment plan for fractionated intensity modulated radiation therapy (IMRT). The gross tumor volume (gTV) encompassed ce and cnI 3, 4; the clinical target volume (cTV) was defined based on the extension of T2-weighted hyperintensity and cnI 2. a dose of 72 and 60 gy were prescribed to the gTV and cTV, respectively, in 30 fractions, therefore delivering a daily dose of 2.5 and 2.0 gy, respectively. (The isodose lines represent, from the inside to the outside, 76, 72, 60, 54, and 30 gy.)

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with the degree of malignancy. In particular, the volumes of cnI abnormality seemed to be useful for distinguishing lowfrom high-grade gliomas, and the volume of llI index correlated not only with the presence of necrosis but also with the volume of ce in grade IV gliomas. The authors also looked for so-called hot spots (voxels with highest metabolic index, i.e., cnI or cgI) and found that the levels measured in these voxels increased with grade, with a significant difference between grade II and grade IV patients and between grade III and grade IV patients. Despite these encouraging studies, the ability to use MRSI data to predict the histologic grade of individual brain tumors remains controversial. a study from Sweden found that such discrimination is impossible on a case by case basis because of the considerable overlap between the metabolite levels seen in the different tumor grades (43). In addition, although McKnight et al. (32) showed in an early study of newly diagnosed and recurrent gliomas that the maximum cnI value was higher in grade III than in grades II gliomas, a subsequent study that was restricted to newly diagnosed gliomas found no differences between the cnI values of biopsies containing low-grade (grade II) or highgrade (grades III and IV) glioma and that the cnI value was very variable in grade IV gliomas, with a median higher than the corresponding grade III population (34). The variation in metabolic index within newly diagnosed grade IV lesions is not totally unexpected, given the heterogeneity within these lesions. a more direct approach would be to correlate metabolic parameters with specific physiologic measures of tumor aggressiveness such as proliferative index and tumor cell density. Recent studies have successfully demonstrated the correlation between choline (or relative choline) and naa levels with tumor cell density (44), degree of tumor infiltration (45), and proliferative activity (46). our group is currently exploring the relationship between the cnI, cell density, and MIB-1 proliferation index of grades II and III glioma with the hopes of identifying intratumoral regions that have a more aggressive phenotype irrespective of the overall histopathologic grade. Based on the current state of the art, it is unlikely that MRS will replace tissue histology for diagnosis of brain tumors. However, it is reasonable to assume that anatomic and metabolic imaging will be used together to identify the regions most appropriate for tissue sampling during MRIguided surgery or stereotactic biopsy.

Mapping the extent of Brain Tumors (Microscopic Infiltration and Spatial Distribution) Mapping the extent of infiltrating brain tumors is of great importance for deciding on and guiding therapy. as mentioned above, conventional anatomic imaging techniques are unable to discriminate sufficiently between areas of actively growing tumor, microscopic invasion, and necrotic changes.

Studies on serial biopsies in patients undergoing craniotomy for gliomas found tumor cells up to 3 cm distant from the contrast-enhancing margin and outside the hyperintensity on T2-weighted images for malignant lesions and sporadically even in low-grade gliomas beyond the hyperintensity on T2-weighted images (4,47). MRS might be of value in mapping out this “invisible” tumor cell infiltration. our group has performed several studies comparing the spatial extent of metabolic (MRSI) and anatomic (MRI) information in patients with newly diagnosed gliomas, exploring the value MRSI might have for the target delineation for RT in high-grade and low-grade gliomas, respectively (33,36). For high-grade gliomas, there were significant differences in the spatial relation of anatomic and metabolic extent of the neoplastic lesion with considerable, but not complete, overlap with the hyperintensity on the T2-weighted image. There were also extreme differences relative to the volume of ce. In 12 patients with grade IV gliomas, the following results were found. First, the cnI of 2, as the measure for tumor suggestive spectra, was smaller than and mostly contained within the T2-weighted hyperintensity in 83% of patients, although it extended beyond the T2weighted hyperintensity in 75% of patients by up to 9 mm. Second, the cnI of 2, 3, and 4 were greater than the volume of ce in 92%, 52%, and 42%, respectively, whereas extending beyond the ce in 100%, 100%, and 75% by up to 18, 14, and 10 mm, respectively. Similar but more pronounced findings were observed for grade III gliomas (33). Taken together, these findings suggest that MRI-derived volumes may misestimate the location and volume of microscopic and actively growing disease when compared with MRSI. a similar study was conducted in 20 patients with lowgrade gliomas (36). The microscopic measurable extension beyond T2-weighted images was not only detected in only 45% of patients but it was also found to be much less in volume compared with high-grade gliomas. all gliomas, regardless of grade, had in common a tendency for the greatest degree of cnI extension beyond the T2-weighted hyperintensity to correspond to white matter fiber tracts. This indicates a preferred path of travel for tumor cells not evident on MRI alone. These reported differences in spatial extent of measurable tumor extent potentially have an enormous impact on the management of gliomas in general and malignant ones in particular. any type of local or targeted nonsystemic therapy relying solely on anatomic information may suffer from insufficient information about the extent of tumor, resulting in potential over- and/or undertreatment of involved regions.

7. MRS and Brain Tumor Diagnosis and Therapy

Distinction Between Tumor and necrosis in Brain Tumors posttreatment patients with brain tumors are usually followed by imaging and clinical examinations posttreatment to assess treatment response and to guide the need for further treatment options. evaluation of new or enlarged areas of ce is often difficult, especially after RT, because they can be interpreted as either progression or reoccurrence of the tumor or radiation injury (necrosis). numerous studies have investigated the applica-tion of MRS to this difficult diagnostic problem (48–53) and have shown that MRS is able to distinguish recurrent/progressive tumor from necrosis via characteristic spectral patterns. a decrease in choline levels can be interpreted as a successful treatment response, indicative of the transformation from tumor pattern to necrosis. a retention or increase in choline, especially when coupled with a poor radiologic response, is suggestive of tumor recurrence. However, it is important to interpret any results in the context of subsequent examinations; transient increases in choline and reductions in naa have been reported after RT, especially in areas exposed to a lower dose (51,54). Spectral changes also are often accompanied by a first occurrence or increase in the peak corresponding to lactate/lipid, which might be important for evaluating the extent of necrosis (or contrast-enhancing areas in transition, i.e., micronecrosis) in high-grade brain tumors. Wald et al. (53) performed a study of response to brachytherapy and external beam RT. They followed 12 patients with glioblastoma multiforme with serial MRS and MRI. all 12 patients showed regions with abnormal spectra before and after seed implantation, with at least one voxel with elevated choline found beyond the region of ce on T1weighted MRI. Spectra from the regions implanted with I125 seeds demonstrated a dramatic reduction in choline levels within 10 weeks, whereas the region of tumor that received only external beam RT showed a much slower reduction in choline levels. This observation reflects the much more aggressive delivery of dose with brachytherapy versus fractionated RT using standard fractionation schemes. In 9 of 12 cases an increase in choline was observed in areas that appeared either normal or necrotic before treatment, suggesting unsuspected tumor spread. an interesting related observation from a study by graves et al. (48) evaluating MRSI in patients undergoing radiosurgical treatment for recurrent gliomas was that the development of spectral abnormality preceded a coincident increase in ce by 1 to 2 months.

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MagneTIc ReSonance SpecTRoScopy IMagIng IncoRpoRaTeD DIagnoSIS anD TReaTMenT guiding Biopsy and Surgery in Brain Tumors The ultimate goal of a preoperative diagnostic study is to provide the physician with the most comprehensive information possible to help guide the decision-making process. although the acquisition of an MRI before intervention is considered the current standard of care, information from additional imaging modalities, such as SpecT, peT, or MRS, can be coregistered with the MR anatomic data to enhance localization beyond that offered by MRI alone. liu and colleagues (55) used MRS and other functional MRI techniques intraoperatively in a combined MR operating room suite equipped with a 1.5-T MR scanner. They used a single-voxel (1 to 8 ml) 1H-MRS technique able to provide information on the neurochemistry within the examined volume within less than 9 minutes. Specifically, the choline map was used to guide the biopsy. Twenty-five neurosurgical procedures have been reported to date in which an increased choline signal was found invariably to be consistent with rapid tumor cell proliferation in the corresponding biopsy location. The authors believed that the intraoperative application of MRS could ease and improve targeting biopsy in heterogeneous lesions and provide valuable information on the optimal site, especially in nonenhancing lesions (56). In cases where there is a wide range of cnIs distributed throughout the MRI T2-weighted hyperintense lesion, the cnI shows promise as a tool for targeting the region of highest histologic grade. ongoing studies at UcSF are exploring the potential benefit of using MRSI for determining the biopsy location in patients with suspected brain gliomas, mainly in presumed low-grade gliomas where the chance of missing a focal area with a higher grade component could yield to misdiagnosis and therefore inappropriate or insufficient therapy. patients scheduled for biopsy undergo an MRSI examination on the day of, or a few days before, surgery. The voxels with highest cnI values are selected as desired targets for biopsy, achieved by means of the surgical navigation system. These features make the cnI a particularly useful in vivo parameter for directing biopsies and targeting focal therapy to the nonenhancing regions of glial tumors. a potential future use of MRSI is in the selection of surgical candidates. Surgical removal of the entire lesion is often not achievable because of size and location of the neoplasm. If a gross total removal is believed by the surgeon to be potentially achievable as determined by preoperative MRI, MRSI might be considered to validate such a determination and/or alter the extent or appropriateness of the surgical removal.

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For instance, we showed in a study population of 30 patients who underwent MRI/MRSI after surgical resection of their high-grade glioma that MRSI was superior in determining the presence of residual disease compared with MRI alone (57). Ten of 30 patients were presumed to have received a gross total resection by MRI alone given the absence of ce; metabolic activity, however, was detected in essentially all patients but was measurable with a cnI 2 more than 1.5 ml in 8 of these patients, averaging 20 ml and extending as much as 11 to 36 mm beyond the resection cavity. The remaining 20 patients had contrast-enhancing residual lesions, indicating a subtotal resection. However, substantial variation between the ce and cnI 2 volume was found, with most metabolic activity falling outside the ce by as much as 8 to 33 mm in 19 patients, averaging 21 ml. preliminary evaluation of MRSI follow-up examinations performed after RT revealed that, particularly in the non-ce cases, there was a predictive value of MRSI for focal recurrence (57). We were able to establish a spatial correspondence of areas of new ce, developed during follow-up, with areas of MRSI abnormality as assessed after surgery whereby confirming the presence of metabolically active residual disease after presumed gross total resection.

application in Radiation Therapy for Brain gliomas Regardless of the tumor type, it can be postulated that the greatest clinical value of a targeted therapeutic intervention will be achieved only if “all” the active disease is addressed and if normal tissue is spared as much as possible. accurate target definition thus becomes of prime importance. The variability in tumor cell distribution makes it difficult to define an effective yet safe margin for purposes of RT treatment planning. a uniform margin can be expected to cover either too much noninfiltrated brain or to leave small areas of tumor infiltration out of the treatment volume. The latter is undesirable because it will increase the likelihood of local recurrence. The former is equally unacceptable because it is clear that radiation-induced central nervous sytem toxicity is related not only to dose but to volume as well (58). Because focal RT appears to decrease neuropsychologic sequelae when compared with large-volume RT (59,60) and because the goal of any revised treatment protocol is to prolong survival, a margin that is tailored to tumor extension, rather than one that is uniform, could be presumed to improve quality of survival as well as local control. MRSI may be of value in helping to achieve this goal by better defining the extent of the tumor and the degree of heterogeneity within the tumor. The metabolic heterogeneity demarcated by MRSI might be of particular interest when deciding on “differential” therapy to delineated regions within brain gliomas. Specifically in relation to RT, MRSI might offer valuable information on the relative radiosensitivity or ra-

dioresistance of certain tissue types within a neoplastic lesion. For instance, the presence of lactate can be considered an indicator of anaerobic metabolism or suggestive of poor blood supply and therefore the presence of hypoxic cells within the tumor. Targeting these areas with focal higher doses may be appropriate. In contrast, regions with increased creatine relative to naa reflect high cellularity and active bioenergetic processes (phosphorylation of adenosine diphosphate). Therefore, regions of tumor with a creatine to naa index of at least 2 may demarcate radiosensitive areas that may need a lower dose. In fact, the difference in spatial extent of gliomas as seen on MRSI versus MRI and the spatial heterogeneity within gliomas as assessed on MRSI in patients newly diagnosed with a brain glioma is forcing a reassessment of the targeting and dosing concepts for the delivery of RT to malignant gliomas. Modern radiotherapy approaches using intensity modulated RT are capable of delivering differential doses to different user-defined regions. To garner the greatest benefit from such techniques, it is critical that the regions identified for special attention be defined accurately; areas of active tumor (suitable for high dose) must be identified separately (as gross tumor volume) from areas suspicious for tumor extension that are appropriate to receive a lower dose (classified as clinical target volume). Therefore, what is required is the ability to determine the most appropriate dose for each region. In the treatment of high-grade gliomas with RT, the standard definition of the target volume is the contrast-enhancing area, as determined based on a contrast-enhanced T1weighted MRI or a computed tomography, plus a margin of 1 to 4 cm to account for “invisible” tumor infiltration (61–64). Dose escalation protocols deliver an additional dose to the contrast-enhancing area itself; a lower dose may be delivered to the T2-weighted region of hyperintensity plus a variable margin (65,66). In a preliminary study of 12 newly diagnosed grade IV gliomas, the regions with cnI 2, 3, and 4 were compared with the MRI enhancing volume for the purposes of investigating how the use of the metabolic data might modify the radiation target(s) (33). This analysis showed that adding the volume of cnI 2, 3, and 4 that extended beyond the volume of ce to the volume of ce would increase the size of the dose escalation target by 150%, 60%, and 50%, respectively, whereby the metabolic abnormality defined by the cnI of at least 3 would reside virtually disjoint, just beyond the MRI enhancing lesion. The median distances that the cnI regions extended outside the enhancing volume were 1 to 2 cm. In addition, the cnI 2 volume was on average only about 50% of the volume of T2weighted hyperintensity. although it did extend beyond the T2-weighted hyperintensity in some cases, it would only have extended the T2-weighted lesion by about 10% if it had been included for targeting purposes. Figure 7-2 shows an example of a patient with high-grade gliomas where the

7. MRS and Brain Tumor Diagnosis and Therapy

MRSI derived metabolic information on the tumor extent has been incorporated into the treatment planning process. The analysis of metabolic lesions for 22 grade III gliomas showed even more dramatic results than for the grade IV gliomas (33). adding the volume of cnI extending outside the volume of ce would increase the size of the target by 500%, 300%, and 150% for cnI 2, 3, and 4, respectively. The median distances that the cnI regions extended outside the enhancing volume were 2 to 3 cm. The cnI 2 volume was on average about 70% of the T2-weighted lesion, and although it did extend beyond the T2-weighted lesion, it would have only extended the target by about 15%. The estimated numbers on an increase of the gross tumor volume are obviously only estimates because they were derived from newly diagnosed gliomas and would apply to patients undergoing RT after biopsy only. a subsequent study investigating this same issue for patients with high-grade gliomas after surgical resection is currently underway and will be reported soon. The definition of target volumes for low-grade gliomas is equally difficult. Margins are defined usually for the purpose of encompassing suspected microscopic spread of the tumor (clinical target volume). The size of the margin required to define the clinical target volume remains problematic because these tumors are poorly demarcated with respect to normal surrounding tissue and are infiltrative in nature; the differentiation between normal brain, edema, and tumor infiltration on MRI is therefore quite difficult. In a study similar to the one reported for high-grade gliomas, analysis of the cnI contours for 20 grade II gliomas showed that the metabolic abnormality was usually within the T2-weighted lesion; when it extended outside the T2-hyperintensity (in 45% of patients) the extension was relatively small and usually directed along white matter tracks (36). In the two grade II patients who had gadolinium enhancement, the maximum cnI corresponded with the small volume of patchy enhancement. If the treatment volume was modified from the usual T2-weighted hyperintensity plus a 2- to 3-cm margin (67) to include the T2weighted lesion plus the region with cnI greater than 2, there would have been a substantial reduction in the target volume and hence, presumably, in the radiation damage to normal brain tissue. although it is not clear how planning margins might be adjusted for high-grade gliomas based on MRSI findings, it is clear that MRSI would provide a different estimate of gross, clinical, and boost target volumes, depending on how those are defined for a given patient. We believe that our results should be taken as a justification to consider a reduction in treatment planning margins for patients with lowgrade gliomas, paying careful attention to preferential avenues of spread of disease. It is also interesting to note that even low-grade gliomas demonstrate regions of varying metabolic activity within their volume. The possible differentiation of more or less radiosensitive/resistant areas, as de-

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fined by metabolic measures, may play an important role in the future diagnosis and treatment of low-grade gliomas.

Value in Recurrent gliomas (patient Selection, Radiosurgery Treatment planning) The idea of combining intensity modulated radiotherapy and MRSI data to deliver extremely sophisticated treatment plans with a range of doses delivered across the target volume is still quite “theoretical” for the treatment of brain gliomas. However, its value in radiosurgery for recurrent malignant gliomas appears to stand on a firmer ground. although the prognosis for recurrent gliomas is poor in general, focal retreatment has been shown to prolong survival. Radiosurgery, although seemingly counterintuitive for an infiltrating disease such as recurrent gliomas, offers an effective and noninvasive treatment option, with median survival times after radiosurgery on the order of 8 to 13 months (6870). Recurrence pattern, however, is predominantly local and marginal in up to 80% (69,71). graves et al. (72) studied the prognostic value of MRSI in gamma knife radiosurgery of recurrent malignant gliomas (72). Thirty-six patients with recurrent gliomas were retrospectively divided into two groups depending on whether the metabolic lesion was confined to the radiosurgical target or whether it extended well outside the target. In the subset of patients with recurrent gB, median survival was significantly shorter with 36 weeks in patients with metabolic extension versus 96 weeks for patients with no metabolic abnormality beyond the radiosurgical target at the time of treatment. In addition, a significant difference was found between the two groups in the volume of ce as assessed throughout the follow-up period, with an increase in ce in the patient group with metabolic abnormality beyond the region of treatment. In some cases the metabolic lesion outside the treatment area would have been small enough to be included within the gamma knife target, but in others the lesion would have been too large to be treated adequately. as an extension of this study, we looked at the potential impact MRSI would have on patient selection and treatment plans for radiosurgery of recurrent gliomas (73). eighteen patients with recurrent glioma (4 anaplastic astrocytomas and 14 glioblastoma multiforme) were treated at the time of progression after initial RT (mean time to progression, 9 months) with gamma knife radio-surgery at UcSF. an MRSI examination was acquired at the conclusion of the treatment planning MRI. The target volume was outlined according to common standards—ce plus 1- to 2-mm margins—and was compared with the cnI volumes derived from the MRSI as described above. The cnI 2 volume was found to extend beyond the ce to some degree in all patients. This extension was less than 1.5 ml (potentially within the error of interpolation) in 7 patients (39%) but more than 6 ml in 11 patients (61%); in 4 of these 11 patients the metabolic abnormality was com-

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CE

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FIgURe 7-3. Volumetric analysis for patients with recurrent glioma; volumes of contrast enhancement (ce) in relation to the volumes that choline-to-nacetylaspartate index 2 extends beyond the ce.

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pletely outside the ce. The individual relationship of ce and cnI 2 is shown in Fig. 7-3, whereas Fig. 7-4 summarizes graphically the average relative sizes of these volumes. Figure 7-5 illustrates sample cases. To estimate the impact MRSI might have on the definition of target volumes, it was assumed that the cnI 2 metabolic abnormality outside the ce plus the region of ce, rather than the cnI volume itself, would be the preferred way of targeting at this point. Such a volume definition would increase the standard target volume by 9% to 277% (0.5 to 27 ml), resulting in a change in the dose prescription according to the gamma knife dose-volume relationship in 12 of 18 patients (67%). In fact, four patients (22%) would have ended up with a treatment volume exceeding 20 ml and would therefore not be considered eligible for radiosurgery in most institutions. The authors of both studies reached the same conclusion: a pretreatment MRSI provides information that may improve the selection, planning, and treatment process for glioma patients. as a result of these two studies, UcSF now uses MRSI on an active basis to define treatment volumes for gamma knife radiosurgery (74).

and have provided up to eightfold higher signal-to-noise ratio near the surface as compared with a conventional head coil. Resolutions as small as 0.2 ml have been achieved for brain tumors and other focal lesions near the cortex at 1.5 T. The other limitations on the data acquisition are the magnetic field strength and echo times used for clinical applications of 1HMRSI. Improvements in spectral dispersion and spatial resolution can be achieved using newly released 3-T clinical scanners. Much work remains to be done to determine how levels of metabolic activity can be used to guide sequential dose delivery with new treatment techniques such as intensity modulated RT. However, it is the belief of the authors that strong consideration should be given to the incorporation of functional imaging into the treatment process for focal treatments

CE

8 mm conclUSIon It is too early to advise the use of biologic imaging as the only determinant of target volumes for surgical and RT intervention. However, the incorporation of metabolic imaging into a treatment planning process that currently relies solely on anatomic imaging seems worthwhile, especially for infiltrating tumors. Such incorporation would be expected to improve treatment effectiveness and patient outcome. It also suggests that the previous assessment of the effectiveness of a specific therapeutic approach may have been flawed because of a suboptimal appreciation of tumor extent and heterogeneity. Developing MRSI as a tool for routine use in RT and surgical planning and follow-up requires improvements in coverage of the lesion and in the signal-to-noise ratio of the spectral data. one way to achieve higher spatial resolution is to use radiofrequency coils with higher sensitivity. a number of coils have been developed for high resolution MRI and MRSI

CNI 2 = CE outside CNI 2 = CNI 2 outside CE = CNI 2 overlap CE = CNI 2 max extension FIgURe 7-4. graphic summary of magnetic resonance spectroscopy imaging results for patients with recurrent gliomas. The average relative size of the total volumes, conjoint and disjoint volumes, and the maximum extension of the choline-to-n-acetylaspartate index 2 volume beyond the contrast enhancement volume are displayed.

7. MRS and Brain Tumor Diagnosis and Therapy

FIgURe 7-5. Magnetic resonance spectroscopy imaging data acquired in patients with recurrent glioma before radiosurgery. Top: Superimposed choline-to-n-acetylaspartate index (cnI) contours (cnI 2, 3) suggest considerable tumor spread beyond the actual contrast enhancement (ce). Incorporation of the cnI 2 into the target volume definition (ce + cnI 2) would have increased the target volume by 217%. Bottom: cnI 2 and 3 are restricted to the area of ce; no tumor suggestive voxels beyond the ce. (But there was little extension of cnI 2 outside the ce in the superior aspect of the tumor.)

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or boost techniques for high-grade gliomas. In addition, the results of controlled dose escalation trials that used MRI-derived target volumes should be reevaluated given the possibility that these volumes may have been suboptimally defined. The incorporation of MRSI before surgery and other types of focal therapy, such as brachytherapy, radiosurgery, and gene therapy, might help to improve patient selection and therefore guide the decision making in patients with brain gliomas.

acKnoWleDgMenTS Special thanks to the departments of Radiology, MRSc (S. J. nelson, T. R. McKnight, e. e. graves, X. li, a. antiniw, J. oh, R. Srinivasan, and W. p. Dillon), Radiation oncology (D. a. larson, p. K. Sneed, and l. J. Verhey), and neurological Surgery (S. M. chang, M. W. McDermott, and M. S. Berger). also, special thanks to M. p. carol for editing. Supported in part by national Institutes of Health grants ca79719 and ca 59880.

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55. liu H, Hall Wa, Martin aJ, et al. MR-guided and MR-monitored neurosurgical procedures at 1.5 T. J comput assist Tomogr 2000;24:909–918. 56. liu H, Hall Wa, Martin aJ, et al. an efficient chemical shift imaging scheme for magnetic resonance-guided neurosurgery. J Magn Reson Imag 2001;14:1–7. 57. pirzkall a, li X, oh J, et al. MR-Spectroscopy imaging for resected high-grade gliomas prior to radiation therapy: tumor extent according to metabolic activity in relation to MRI. Int J Radiat oncol Biol phys 2004;59:126–137. 58. Marks Je, Baglan RJ, prassad Sc, et al. cerebral radionecrosis: incidence and risk in relation to dose, time, fractionation and volume. Int J Radiat oncol Biol phys 1981;7:243–252. 59. Maire Jp, coudin B, guérin J, et al. neuropsychologic impairment in adults with brain tumors. am J clin oncol 1987; 10:156–162. 60. Hochberg FH, pruitt a. assumptions in the radiotherapy of glioblastoma. neurology 1980;30:907–911. 61. Bleehen nM, Stenning Sp. a Medical Research council trial of two radiotherapy doses in the treatment of grades 3 and 4 astrocytoma. The Medical Research council Brain Tumour Working party. Br J cancer 1991;64:769–774. 62. garden aS, Maor MH, yung WK, et al. outcome and patterns of failure following limited-volume irradiation for malignant astrocytomas. Radiother oncol 1991;20:99–110. 63. liang Bc, Thornton aF Jr, Sandler HM, et al. Malignant astrocytomas: focal tumor recurrence after focal external beam radiation therapy. J neurosurg 1991;75:559–563. 64. Wallner Ke, galicich JH, Krol g, et al. patterns of failure following treatment for glioblastoma multiforme and anaplastic astrocytoma. Int J Radiat oncol Biol phys 1989; 16:1405–1409. 65. nakagawa K, aoki y, Fujimaki T, et al. High-dose conformal radiotherapy influenced the pattern of failure but did not improve survival in glioblastoma multiforme. Int J Radiat oncol Biol phys 1998;40:1141–1149. 66. Fitzek MM, Thornton aF, Rabinov JD, et al. accelerated fractionated proton/photon irradiation to 90 cobalt gray equivalent for glioblastoma multiforme: results of a phase II prospective trial. J neurosurg 1999;91:251–260. 67. Morris De, Bourland JD, Rosenman Jg, et al. Three-dimensional conformal radiation treatment planning and delivery for low- and intermediate-grade gliomas. Semin Radiat oncol 2001;11:124–137. 68. chamberlain Mc, Barba D, Kormanik p, et al. Stereo tactic radiosurgery for recurrent gliomas. cancer 1994;74: 1342–1347. 69. Hall Wa, Djalilian HR, Sperduto pW, et al. Stereotactic radiosurgery for recurrent malignant gliomas. J clin oncol 1995;13:1642–1648. 70. larson Da, gutin pH, McDermott M, et al. gamma knife for gliomas: selection factors and survival. Int J Radiat oncol Biol phys 1996;36:1045–1053. 71. Shrieve Dc, alexander e 3rd, Wen py, et al. comparison of stereotactic radiosurgery and brachytherapy in the treatment of recurrent glioblastoma multiforme. neurosurgery 1995;36:275282, discussion 282–274. 72. graves ee, nelson SJ, Vigneron DB, et al. a preliminary study of the prognostic value of proton magnetic resonance spectroscopic imaging in gamma knife radiosurgery of recurrent malignant gliomas. neurosurgery 2000;46:319–328. 73. pirzkall a, graves ee, lau a, et al. gamma knife (gK) radiosurgery (RS) for recurrent high-grade gliomas: what does magnetic resonance spectroscopy have to add? proceedings of the 5th International Stereotactic Radiosurgery Society congress, las Vegas, nevada, 2001, p. 44. 74. McDermott MW, chang SM, Keles ge, et al. gamma knife radiosurgery for primary brain tumors. In: germano IM, ed. lInac and gamma knife radiosurgery. park Ridge: american association of neurological Surgeons, 2000:189–202.

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Functional Magnetic Resonance Imaging for Neurosurgery Steven Stufflebeam and Bruce L. Rosen

In this chapter we review functional magnetic resonance imaging (fMRI) applications intended for the presurgical planning of patients with cancer of the nervous system. The chapter is organized into three parts: (a) justification of using fMRI for presurgical mapping; (b) practical issues of image acquisition, image analysis, and paradigm design; and (c) applications of fMRI for the neurosurgeon.

FuNCTIONaL MaGNETIC RESONaNCE IMaGING IN ThE WORkup OF paTIENTS WITh BRaIN TuMORS fMRI of the brain is the application of MRI techniques to investigate cerebral physiology while preserving anatomic specificity. The umbrella term of fMRI includes imaging of microscopic water mobility (diffusion imaging), microvascular hemodynamics (cerebral blood flow [CBF] and volume [CBV] imaging), and blood oxygenation–sensitive imaging. although these techniques are as yet unable to image neural activity directly, underlying normal or pathologic tissue function can be assessed through the use of the wellestablished relationship between neuronal activity, cellular metabolism, and closely linked parameters of CBV, CBF, the oxygenation state of blood or water diffusion. fMRI provides crucial information in the treatment of patients with brain tumors. We briefly discuss the use of diffusion and perfusion measures but focus on the use of blood oxygenation–sensitive techniques necessary for mapping eloquent areas of the brain. Task-activated fMRI has gained acceptance at some medical centers as a valid procedure for the mapping of eloquent cortex in brain tumor patients as part of surgical planning or radiation treatment planning. In particular, task-activated fMRI provides a convenient and noninvasive map of the neural activity of the brain. Maximizing the resection boundaries of a tumor while minimizing the loss of essential cortex is a well-established goal of the surgical treatment

of brain tumors. Localization of eloquent cortex improves the efficacy of surgical resection of brain tumors (1). Before the establishment of functional neuroimaging and interoperative mapping, functional localization was done by recognizing specific anatomic landmarks and correlating them to know functional areas. This is, of course, limited by such factors as the distortion of brain tissue due to mass effect or tumor infiltration and by patient-to-patient variability of the site of function and perhaps by plasticity due to reorganization of cortex. Most neurosurgeons require some type of functional mapping before or during central nervous system tumor resection. This has led to the development of invasive mapping procedures, such as stimulation arrest language mapping (2) or use of grid electroencephalography intra-cranial EEG (iEEG). These methods are the current clinical gold standard and are in widespread use, but they carry some risk to the patient. fMRI is a natural noninvasive alternative to the surgical methods. Can fMRI reliably replace invasive functional measurements? This remains somewhat of a controversy. Several reports over the past decade have confirmed that the spatial correlation of the blood oxygen level–dependent (BOLD) signal and that of intraoperative methods is accurate enough so that fMRI can be used to replace, or at least supplement, the invasive methods (3,4). Some recent reports, however, dispute this claim, particularly in the case of language regional mapping (5). The limits of fMRI lie both in the technical ability of available clinical scanners and in the underlying physiologic mechanism of fMRI, detailed below (6). Therefore, the exact role of fMRI in the treatment planning of a brain tumor patient continues to evolve. In this chapter we hope to show that fMRI provides a preoperative assessment and can replace invasive mapping or reduce the time needed for invasive mapping.

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phySIOLOGIC MEChaNISM OF FuNCTIONaL MaGNETIC RESONaNCE IMaGING This chapter focuses on task-activated fMRI used as presurgical mapping of eloquent cortex. First, a brief introduction to some other types of fMRI: MRI diffusion and MRI perfusion imaging.

Diffusion Diffusion is the random motion of all molecules driven by thermal energy, also known as Brownian motion. MRI can be made to be sensitive to molecular diffusion of the primary contributor to the MRI signal, molecular water (7,8). Because diffusion in the brain has only recently been measurable in a clinical setting, its clinical utility and indications beyond acute stroke are still being investigated. It has already become a mainstay in the diagnosis and treatment of stroke. For the neurosurgeon its greatest potential may be in the estimation of the location and connectivity of the white matter tracks, known as MRI tractography (9–12). Molecules diffuse in all three dimensions. however, the local environment does influence diffusion; for example, in areas of white matter there is greater diffusion along the myelin sheaths than across them. Therefore, the diffusion of water in some tissues is anisotropic. That is, there are different apparent diffusion coefficients in each direction, whereas in other tissues it is quite isotropic. The mathematical description of diffusion for each voxel is termed a tensor. Complete characterization of the molecular diffusion tensor requires at least seven measurements. New methods use many more measurements in an attempt to characterize crossing white matter fibers by determining the entire diffusion function, such as diffusion spectral imaging (10,11). This has led to the development of tractography, which can show the relationship of major white matter tracks to tumor masses (Fig. 8–1).

Magnetic Resonance Imaging perfusion Traditionally, tissue perfusion has been assessed using a variety of radiologic techniques, from conventional catheter angiography to positron emission tomography (pET) and single photon emission computed tomography. MRI has unique features that add to its value in assessing tissue perfusion in the brain: It can be relatively sensitive to tissue microvasculature, is minimally invasive, and has higher spatial resolution than radionuclide-based techniques. Over the past 10 years, these techniques have demonstrated their utility in a wide variety of neuroradiologic applications; any disease process with microvascular alterations may potentially benefit from hemodynamic imaging.

FIGuRE 8-1. Magnetic resonance imaging (MRI) tractography mapping motor fibers displaced by tumor mass effect. how diffusion tensor imaging can be used to map the corticospinal tracts is demonstrated (D), relative to the occipital hemangioblastoma, high T2 signal in the axial MRI turbo spin echo image (a) and FLaIR (B). Note that the right-sided corticospinal tracts are displaced somewhat anteriorly due to mass effect of the occipital-parietal tumor that enhances dramatically (C). Transhemispheric white matter tracts are shown in red (D) traversing the interhemispheric fissure via the corpus callosum. The diffusion tensor images were acquired with a 3-T MRI scanner using high angular resolution pulse sequence.

Magnetic susceptibility contrast phenomena can be coupled with rapid imaging to resolve the first-pass tissue transit of intravenously administered contrast materials, thereby providing an index with which to measure CBF and CBV. To determine quantitatively CBV or CBF, however, it is necessary to convert regional changes in MR signal intensity with respect to time into contrast agent tissue concentration–time curves. perfusion is the volume of blood flowing through per unit time, with a normal value of approximately 50 mL/100 g/min. These measurements are difficult to make in practice but can be estimated with perfusion MRI methods to estimate whole brain blood flow (CBF), blood volume (CBV), and mean transit time. MRI perfusion imaging can be used to aid in the surgical biopsy of suspected tumors, either primary or recurrent. although low-grade lesions have a typical appearance on conventional MRI, on occasion lesions that appear low grade and uniform on conventional pre- and postcontrast MRI studies can have focal areas of malignant de-differentiation. Figure 8–2 demonstrates how CBV maps can be used to aid in determining where to biopsy or treat such lesions. Conventional MRI demonstrated a relatively homogenous appearance, even after the administration of intravenous contrast. however, CBV maps demonstrate focal areas of increase signal corresponding to higher grade tumor. These maps cannot only be used to direct the surgical biopsy, but

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FIGuRE 8-2. perfusion case demonstrating cerebral blood volume (CBV) maps aiding in biopsy. a 57-year-old man presented 7 years after resection of an oligodendroglioma. a. post-gadolinium T1-weighted images demonstrate slight enhancement in the area of prior surgery. B. Functional magnetic resonance CBV mapping of the tumor demonstrates an area of increased uptake approximately 2 cm anterior to the central sulcus. Biopsies from anterior and posterior to the area of highest CBV demonstrated low-grade tumor, and a biopsy in the lesion in the area of highest CBV demonstrated grade 3/4 astrocytoma (From Sorensen aG, Rosen BR. Functional MRI of the brain. In: atlas SW, ed. Magnetic resonance imaging of the brain and spine. philadelphia: Lippencott-Raven, 1996:1051–1545, with permission.)

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served by Linus pauling, because deoxyhemoglobin is paramagnetic, whereas oxyhemoglobin loses its paramagnetism and is thus diamagnetic (16). The paramagnetic effect of deoxyhemoglobin was shown by Thulborn et al. in 1982 to shorten T2 (17,18). This property is familiar to neurosurgeons as the cause for the drop in T2-weighted signal seen in some stages of acute hemorrhage. The intrinsic contrast effects of deoxygenated paramagnetic hemoglobin with a gradient echo technique were used by Ogawa et al. (19) to study the influence of intravascular susceptibility perturbation on signal arising from blood vessels. The MRI signal of brain tissue on T2-, T2*-, and T2⬘-weighted images is strongly influenced by the oxygenation state of the blood (19, 20). kwong et al. (21,22) first established the link between these earlier observations and changes in the MRI signal during neuronal activation. Why does blood oxygenation state, and hence MRI signal, change during conditions of increased flow seen with neuronal activation? pET and other studies have shown that oxygen delivery, CBF, and CBV increase by about a factor of 2 (23,24). however, there is a mismatch between oxygen delivery (governed by CBF) and cerebral metabolic rate of oxygen consumption (the CMRO2) under at least most activation paradigms, with oxygen utilization rising only slightly with cerebral activation (15,25). The combination of increased oxygen delivery with-

occasionally can aid the surgical pathologist. The case from Figure 2 had an original pathology reading based on the superficial samples taken outside the regions of increased CBV and was reported as low-grade astrocytoma. after reviewing the CBV map, which highlighted regions of intense microvascular proliferation suggesting greater malignancy, additional tissue specimens were located from surgical samples localized to the CBV “hot” areas. These samples subsequently documented the lesion as high grade. This case demonstrates that perfusion fMRI adds information to conventional MRI and has the capability to change the clinical management of brain cancer patients.

Blood Oxygen Level–dependent Imaging as early as 1890, local cerebral hemodynamics was proposed to be closely linked to local cerebral activity (13). although fMRI is not yet sensitive to neuronal activity directly, cerebral hemodynamic changes can be used as a surrogate marker for cerebral activity (14,15). One of the first MRI techniques to take advantage of this physiologic process was the BOLD contrast mechanism that links changes in deoxyhemoglobin content to MRI signal changes. The effect of states of hemoglobin was first ob-

FIGuRE 8-3. physiology of task-activated blood oxygen level– dependent (BOLD) functional magnetic resonance imaging (fMRI). Cortical activation is associated with a localized increase in Cerebral Metabolic Rate of Oxygen consumption (CMRO2), which in turn causes an increase in cerebral blood flow and cerebral blood volume and blood oxygenation. Local oxyhemoglobin content increases at the same time that deoxyhemoglobin concentration decreases. Deoxyhemoglobin, in turn, links neuronal activity and the fMRI signal change. Deoxyhemoglobin is paramagnetic and disturbs the local magnetic field in its environment, which when it decreases in concentration leading to an increase in the BOLD signal on MRI.

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FIGuRE 8-4. preoperative functional magnetic resonance imaging (fMRI) activation mapping of motor activity in a patient with a low-grade glioma. Images show statistically significant changes in fMRI blood oxygen level–dependent signal (scale as on the right) as tested by the kolmogorov-Smirnov test, a nonparametric statical test, overlayed on a T2-weighted structural MRI. Note the displacement of the locus of activity of the right foot, presumably due to tumor. This was verified at surgery using intraoperative electrophysiologic cortical mapping.

out concomitant increase in oxygen extraction, coupled with a more modest increase in blood volume, leads to a decrease in the local tissue deoxyhemoglobin content during states of increased neuronal activity (Fig. 8–3). This drop in paramagnetic deoxyhemoglobin leads to increase signal on susceptibility sensitive T2- and T2*- weighted pulsed sequences. Especially when coupled with high-speed imaging, these techniques have become a powerful tool in assessing changes in regional blood, and hence tissue, oxygenation, and flow (26). These allow direct visualization of brain activity using task-activated fMRI (Figs. 8–4 and 8–5).

TaSk-aCTIVaTED FuNCTIONaL MaGNETIC RESONaNCE IMaGING: aNaLySIS aND VISuaLIzaTION Experimental Design of Task-activated Functional Magnetic Resonance Imaging The classic task-activated fMRI stimulus presentation paradigm is the block design. The block design paradigm consists of acquiring a series of images during the time that stimuli are presented continuously over a period of several seconds to minutes, followed by periods of rest (or some other type of control stimulus). The major advantage of the block design is that it allows for the hemodynamic response to summate over multiple stimuli presented rapidly, which results in a large steady-state response within each stimulus block. a potentially important advancement for functional brain mapping is the ability to image the response of single evoked responses, termed event-related fMRI, and is anal-

FIGuRE 8-5. Three-dimensional (3D) rendering of task-activated functional magnetic resonance imaging (fMRI). a. Structural MRI with a 3D rendering. B. fMRI using self-paced finger tapping. Gradient echoes blood oxygen level–dependent fMRI activation. C. Tumor segmented relative to functional activation. D. Gadoliniumenhanced fMRI showing 3D “roadmap” of activation and tumor. E. Bilateral activation maps (showing right-hand finger tapping and left-hand finger tapping). Notice medial frontal lobe activation indicating supplementary motor activation. F. Somatosensory activation of hand and tongue. (Courtesy Dr. Gordon harris of the MGh Radiology 3D Imaging Service and Dr. a. Gregory Sorensen at Massachusetts General hospital.)

ogous to the stimulus presentation paradigm used in electrical potential evoked responses (27). Single trials are separated by several seconds and averaged selectively based on the stimulus type (Fig. 8–6). This allows visualization of the time course of activation in response to each stimulus type. The major disadvantage of an event-related design is a decrease in the sensitivity of the measurement, so that the block design is generally used for presurgical mapping, with the exception of retinoptic mapping, which requires an event-related design.

Task-activated Functional Magnetic Resonance Imaging pulse Sequences after immobilizing the head and ensuring patient comfort, the choice of which MRI pulse sequence can have important implications in the quality of the fMRI study. Most fMRI sequences that measure neuronal activity are sensitive to the hemodynamics changes induced by the underlying activity

8. Functional Magnetic Resonance Imaging for Neurosurgery

FIGuRE 8-6. Task-activated functional magnetic resonance imaging (fMRI) design types: blocked vs. event-related paradigms. The block design has rapid presentation of several stimuli with intervening blocks of rest, whereas the event-related technique presents stimuli less rapidly but does not have rest blocks. Both can use a similar imaging pulse sequences. The blocked design allows for a larger buildup of the hemodynamic response, giving an overall larger signal-to-noise ratio. The event-related design allows for measurement of individual responses, which can be deconvolved in the postprocessing.

in the brain. Two broad classes of task-activated fMRI pulse sequences are available: measure the CBF, as in arterial spin labeling sequences (Fig. 8–7), and signal is modulated by changes in the blood oxygenation state (i.e., BOLD). Gradient echo pulse BOLD sequences are the mainstay of task-activated fMRI at most medical centers but have the

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distinct disadvantage of biasing the BOLD signal toward large draining veins, which are often several millimeters from the true center of activation. This is particularly true of fMRI studies at 1.5 T. as the static magnetic field strength increases, the signal from the capillaries and venules also increases, which is more congruent with the underlying neural activity. at lower field strengths, the spin echo sequence increases the signal derived from smaller vessels but has a long acquisition time and boasts only a modest signal-tonoise ration (SNR). The asymmetric spin echo sequence has a good compromise between the gradient echo and the spin echo sequence and is the preferred fMRI sequence at some medical centers for field strengths of 1.5 T. The disadvantage of the asymmetric spin echo pulse sequence is that it has an overall lower SNR, potentially increasing the amount of false activation. Other more advanced fMRI imaging techniques are currently under development that may lessen the effect of large draining veins on spatial resolution (28–32). The details and implementation of fMRI varies among medical centers. Most imaging centers typically use a 1.5-T scanner equipped with echo planar imaging. The generic presurgical planning protocol typically includes (a) a sagittal T1-weighted spin echo localizer, (b) an echo planar imaging T1- and T2-weighted anatomic image set, (c) a series of echo planar imaging asymmetric spin echo functional images coincident with the anatomic reference images, (d) a three-dimensional T1-weighted gradient recalled image (Spoiled Gradient Recalled acquisition), and (e) MR venography for venous anatomy (See Fig 8–5). paradigm Selection

FIGuRE 8-7. Motor mapping with gradient echo blood oxygen level–dependent (BOLD) and arterial spin labeling (aSL) imaging techniques. The BOLD and aSL pulse sequences are shown in the same patient during that same imaging session, using a 3-T MRI scanner, with a 1-minute block of 2-hz self-paced finger tapping followed by a block of rest. Notice that the BOLD shows two areas of activation, but the aSL image shows activation near the presumed hand area of the precentral sulcus. In this subject, there is no evidence of activity in the premotor activation but some supplementary motor activation. Note that the temporal profiles over the motor cortex are similar. (Courtesy of Dr. Rick hoge, Martinos Center for Biomedical Imaging, Massachusetts General hospital, Boston.)

paradigms should be selected to activate the eloquent areas in close proximity to the lesion. unfortunately, given our ignorance of brain function, it is only possible to activate selected areas of the brain that are thought to be important. Even given the immense research into brain activation, only a few paradigms have been systematically evaluated in patient populations. For example, auditory or visual verb generation is a common method for generating robust language activation on fMRI. a category or stem-completion task or a visual or auditory word comprehension also produces lateralized activation of the language system. Control tasks may include listening to complex tones, visualizing or listening to nonsense words, or to simply have no auditory or visual stimulation (rest). Details regarding the exact paradigm to select are described below.

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aNaLySIS OF BLOOD OxyGEN LEVEL–DEpENDENT FuNCTIONaL MaGNETIC RESONaNCE IMaGING The statistical analysis of fMRI is used to identify voxels in the image that correlate with the task paradigm. Many commercial and academic methods are readily available, most based on a corrected t-test, on a voxel-by-voxel basis. Despite the large number of statistical tests—including t-test, kolmogorovSmirnov, cross-correlation, Fourier analysis, and analysis of variance—no universal consensus has been reached. For most presurgical mapping purposes, the voxel-by-voxel t-test is adequate. No matter which statistic is chosen, head motion during the examination is common yet detrimental to fMRI studies. If the head motion is small, a computer algorithm can be used to correct it across the functional images. For example, the automated image registration technique (33) is commonly used by a variety of academic and commercial software packages for fMRI analysis. additional processing of the image data is required to fuse the structural and functional images. Typically, the activation is presented in color scale showing the level of statistical significance on the individual slices of the T1 volume acquisitions (Magnetization prepared Rapidly acquired Gradient Echo or Spoiled Gradient Recalled acquisition). Three-dimensional renderings are also convenient for neurosurgical purposes to clearly show the location of the overlying structures, such as head, dura, and venous anatomy (See Figure 8–5).

pRESuRGICaL MappING Language Mapping In patients with brain tumors in the perisylvian region, lateralization and localization of language processing are critical. although fMRI is routine at some medical centers, the intracarotid injection of amobarbital, known as the Wada test, is still considered the gold standard for presurgical determination of hemispheric dominance. The Wada test yields a lateralization index (LI) based on the number of correct answers during the barbiturate administration of one hemisphere compared with the other hemisphere. Despite a long history and general acceptance of the results, the Wada test has been criticized because of potential cross-flow to the contralateral hemisphere, the lack of evaluation of the territory supplied by the posterior circulation, and the lack of any data regarding reproducibility. There are several reports of fMRI being used successfully in determining both hemispheric dominance for language and regional language mapping of individual language areas (2,5,34–42). FitzGerald et al. (38) found a good correspondence between fMRI and electrocorticostimulation for presurgical language mapping purposes. Roux et al. (5) recently compared the results of fMRI language mapping using a verb generation task with

FIGuRE 8-8. Regional language mapping using a neuronavigational system and functional magnetic resonance imaging (fMRI), compared with intraoperative mapping. a. activated fMRI area (verb generation task) in a patient with a left anterior temporal meningioma. B. Integration of fMRI data in the neuronavigational system (here the 3D patient model). It should be noted that in these 3D reconstructions, the cortex seems flattened by the tumor edema. Dotted white line indicates the limit of craniotomy. C. Intraoperative view. The sulcal and gyral anatomic features of the patient were compared with the 3D reconstructions, and correlations were guided with the neuronavigational probe. Each cortical site tested intraoperatively was compared with the fMRI data (correlation/no correlation). N, negative sites. (From Roux FE, Boulanouar k, Lotterie Ja, et al. Language functional magnetic resonance imaging in preoperative assessment of language areas: correlation with direct cortical stimulation. Neurosurgery 2003;52:1335–1345, discussion 1345–1347, with permission.)

stimulation arrest language. They found, however, an imperfect correlation of fMRI with intraoperative mapping ( 8–8). Determining regional language processing in a brain tumor patient requires choosing a task that best activates the desired stream of language processing. Specific language processes include phonologic, lexical, and syntactic processing. Concurrent memory storage and retrieval occur with any language paradigm. Supporting processes include attention, motor planning (speech), and basic visual or auditory processing. Semantic decision tasks are probably the most popular because they require a response from the patient, such as a forced binary decision, allowing for one to monitor the quality of the patient’s responses. Some consider covert responses desirable, because overt (spoken) responses can lead to unacceptable motion artifacts. Still, some passive sensory paradigms requiring no patient response are widely reported to be successful and circumvent the potential of movement-related artifacts. For example, a verb generation task consists of having a noun read out load while the patient is asked to think of an action verb to go with it (i.e., car/drive). a semantic decision task might consist of having a series of nouns presented on a video monitor while

8. Functional Magnetic Resonance Imaging for Neurosurgery

the patient decides whether it is “abstract” or “concrete” (i.e., love/abstract or book/concrete). hemispheric Dominance for Language Determination of the language-dominant hemisphere is critical in the presurgical workup of tumors near language processing areas (43). Wada and Rasmussen determined that over 93% of patients are left language dominant and that over 96% of right-handed patients are as well, although more recent studies indicate that many patients have more bilateral representation of language than the original studies (44,45). In left-handed patients, only about 70% of patients demonstrate left hemispheric dominance for language, with about 15% of patients demonstrating bilateral language lateralization. The laterality of language is usually achieved with fMRI by activating both Broca and Wernicke language areas and comparing the relative strength of activation between the right and left hemispheres (Fig. 8–9) (35,41, 43). The LI, analogous to the Wada LI, is computed by the following formula (35,43,46):

FIGuRE 8-9. Language lateralization. The stimulus was generating verbs corresponding to objects that were projected onto to screen in the scanner during imaging. The control task was fixation on a dot. Increased activity is noted in Brodmann areas 47 and 45 on this comparison, and also when comparing the verb generation with viewing the objects passively. This localizes the language activity in this subject to the left side (LI = 0.72). Of note, this patient was left handed and also had an arachnoid cyst on the left, which was thought to increase the chance of language lateralization on the right. however, Wada testing confirmed that this patient’s language process localized to the left. Lower left shows the p value scale used for functional color maps registered with T2 anatomic images.

LI =

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Lh ⫺ Rh Lh ⫹ Rh

where LI is the lateralization index, Lh is the number of activated pixels in the left hemisphere, and the Rh is the number of activated pixels in the right hemisphere. Therefore, when LI = 1, the patient is purely left-hemispheric dominant; when LI = ⫺1, the patient is largely right dominant; and when LI = 0, language function is bilaterally represented. Multiple groups report a strong correlation with both intraoperative mapping techniques and the results of Wada test (5,34,35,39–42,47). Regional Language Mapping Task-activated fMRI can locate, with a high spatial resolution, both receptive and productive brain areas. This has led to a revolution in the understanding of the basic neuroscience principles involved in where and how the brain processes information. The practical needs for the presurgical workup of brain tumor patients, however, and the needs of basic neuroscience are fundamentally different. First, the neurosurgical application requires precise localization in the individual patient, whereas the neuroscientist can average the response over several subjects to increase the SNR of small activations. Second, the neurosurgeon usually requires mapping the essential language areas, not just the participating areas. Essential language areas are ones that when removed result in a language deficit. participating areas are activated during language paradigms but do not result in a postoperative language deficit after resection, either because there are areas of redundant processing or because other areas learn to take over the same functions. Currently, there is no way to distinguish essential from participating areas with noninvasive imaging, and improving the detection of essential areas is a major goal of clinical functional imaging. Four distinct language areas are strongly lateralized to the left hemisphere and are readily found with a variety of language tasks (36): (a) prefrontal (inferior frontal gyrus, superior frontal gyrus, and the anterior cingulated); (b) angular gyrus, excluding the supramarginal gyrus; (c) ventrolateral temporal lobe (superior temporal, middle temporal, inferior temporal, and fusiform gyri); and (d) retrosplenial cortex (see Fig. 8–9). These areas of activation are seemingly at odds with the neurologic dogma of language processing. Binder et al. (35,36,46) suggests that the imaging findings may not be so different from these prior theories. The differences may be because lesion studies of anterior lateral prefrontal cortex (Broca’s area) may be overemphasized with respect to its role in Broca aphasia, because pure Broca lesions cause apraxic deficits of articulation with only temporary disruptions of language function. a permanent Broca aphasia requires a more extensive lesion—involving the an-

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terior inferior frontal gyrus, middle frontal gyrus, and pericentral gyri—than simply a lesion involving Broca’s area. Thus, the historical lesion studies and fMRI activation areas may not be so divergent as originally thought, because both suggest a wide area of frontal lobe participation in language processing outside the classic confines of Broca’s area. Lesions studies also suggest that people with large frontal infarctions often present with a receptive aphasia, which later evolve into the so-called expressive aphasias. Task-activated fMRI can provide evidence of cortical reorganization of language areas, due to mass lesions or after surgical resection (40).

Central Sulcus Currently, the central sulcus can be identified by invasive means during surgery, often by using sensory evoked potentials with a phase reversal technique. This technique, sometimes combined with intraoperative ultrasound, makes it possible to successfully remove tumors in or near the central motor cortex (48,49). It is quite time consuming, however, and must be done in the operating room while the patient is under anesthesia. hence, it is advantageous to locate the central sulcus with a preoperative noninvasive fMRI. This is accomplished by localizing the sensorimotor cortex, typically by using a tactile sensory and/or a motor task during the fMRI scanning session. Identification of the central sulcus with fMRI has been validated by several groups using intraoperative measurements (50–57). Even with histologically low-grade tumors, the central sulcus can be distorted by tumor infiltration and/or mass effect, obscuring it on an anatomic MRI. although the central sulcus is usually identifiable on anatomic grounds and the somatotopic organization of the motor and sensory cortex is well established, fMRI can play a role when the normal anatomy is distorted or as a method to exclude normal variation in the functional architecture. abnormal vascular anatomy is common in vascularized lesions, such as with an arteriovenous malformation, with enlarged draining veins making the use of normal venous anatomy impossible. Further, some neurosurgeons prefer to have the precise localization of a well-known area, such as the hand area, as a reference during tumor resection. Motor Mapping using self-paced finger-tapping mapping of the sensorimotor or somatomotor cortex is one of the oldest and most common methods of cortical brain mapping (see Figures 8–4 and 8–7). Sensorimotor mapping using fMRI has been evaluated by several groups, with the overall goal of localizing the central sulcus (37,55,56,58–68). Clinically useful motor tasks include repetitive hand clenching, sequential finger–thumb opposition, and squeezing an object (sponge or rubber ball). Typically the control task is no movement

(rest), using a block paradigm. The timing can be done either via the MRI scanner intercom or a video monitor. Simple motor tasks preferentially activate the contralateral hemisphere involving not only the primary motor cortex (MI) but also the somatosensory cortex (SI). This occurs because of inevitable activation of tactile receptors on the skin as well as proprioceptive mechanisms. The MI activation occurs in Brodmann area 4, located along the anterior bank of the central sulcus and the posterior portion of the precentral gyrus. SI activation occurs along the anterior bank of the central sulcus, mainly in Brodmann area 3b. higher magnetic field strengths, surface coils, and a combination with other methods are making it possible to more precisely decompose the areas of activity. Supplementary and premotor activity tends to be more variable among patients. Ipsilateral cerebellar activation is also common. The amplitude of the functional activation increases with frequency and force of movement (51,69), and thus it is important to encourage patients to increase the intensity (force used in finger–thumb opposition) as much as possible and the increase the frequency of movement (1 to 5 hz) as is tolerable. Several groups have validated fMRI with other functional mapping techniques, including Intra-cranial EEG (64,65,70), pET (70), and transcranial magnetic stimulation (71–73).

Somatosensory Mapping using a tactile stimulator or vibrotactile stimulator, the somatosensory cortex homunculus can be easily mapped by successively stimulating finger digits, foot digits, and lip while performing an fMRI. alternatively, an electrical nerve stimulator can be used to map the median nerve, tibial nerve, and lip representative areas. If an electrical nerve stimulator is used, the electrodes are placed and the intensity set, such that thumb twitching or toe twitching is elicited. It has been found that regardless of tumor grade, intraaxial brain tumors may border on or invade the somatosensory cortex. Interestingly, low-grade tumors were more likely than high-grade tumors to involve the functionally viable cortex. Low-grade tumors, due to slow growth, more often demonstrate functional activity within the radiologically abnormal areas than high-grade tumors. high-grade tumors, on the other hand, often show functional activity at the margin of the contrast-enhancing area. These findings are usually caused by physical displacement of the functional tissue due to mass effect of the high-grade tumor.

Visual Cortex and auditory areas In patients with tumors lying near visual eloquent areas, mapping of the visual cortex may be valuable. The primary visual cortex can be mapped from nearly any strong visual stimulus. Retinotopic maps are of more interest for the clinical neuroscientist (Fig. 8–10) than for surgical planning but

8. Functional Magnetic Resonance Imaging for Neurosurgery

FIGuRE 8-10. Sensory areas that can be routinely imaged with task-activated functional magnetic resonance imaging (fMRI). Inflated cortex that is cut and topographically flattened is shown as are the gyri and the sulci. Functional activation areas are shown as well. Motor (shown as Brodmann areas 4/6), somatosensory (areas 1/2, 3b, SII), and visual (V1, V2, V3/a, Vp, V4, LOR, MT SpO, and pFa) areas can be routinely imaged with well-established retinotopic fMRI paradigms.

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mercially available and academic software packages overcome some of the common problems with coregistration of functional data and structural data. The 3D Slicer, developed at the Brigham and Women’s hospital, allows the registration of functional data onto structural MRIs (Fig. 8–11). (50). When used in combination with an interventional MRI, it is possible to overcome the problem of brain shift during the procedure itself. The compensation for any change in the brain morphology is done by calculating a mathematical transformation of the anatomic MRI during the coarse of the neurosurgical procedure. The 3D Slicer requires the use of an interventional MRI to acquire new intraoperative structural images (see Figure 8.2). The transformation is created by comparing the intraoperative scans with the preoperative scans. Once the transformation is computed, it can be applied to the preoperative functional scans, which are then fused with the intraoperative scans.

can be used in patients with tumors located in and near the primary visual areas. The auditory areas themselves are rarely of interest to the neurosurgeon, but in the context of language mapping auditory cortex mapping can be useful.

INTEGRaTION INTO ThE OpERaTIVE SuITE: NEuRONaVIGaTION The widespread use of frameless stereotactic systems makes it possible to locate small deep-seated tumors during neurosurgery, with a precision exceeding 1 mm. Gumprecht et al. (39) reported that fMRI provided important information that affected the management of patients with lesions adjacent to eloquent cortex. They believed that the fMRI data used in combination with neuronavigation reduced morbidity. They cautioned, however, that the accuracy of the display and registration of functional data into the neuronavigational system continues to evolve. Errors in localization in the operative suite include precision of fiducial markers, registration of functional and structural MRI, shift of brain contents, and inaccuracy in the inverse solutions. accurate coregistration of the images with skin fiducial markers requires markers that delimit the volume containing the lesion and the surgical field (74–76). To overcome the errors associated with shift of brain contents during an operation, it is often necessary to use the sulcal landmarks on three-dimensional reconstructions to aid in localizing fMRI activity on the patient’s brain. Several com-

FIGuRE 8-11. Interventional magnetic resonance imaging (MRI): integration of functional data into operating room suite. This is an example of a program, 3D-Slicer, used to integrate functional and structural information. The functional data include both taskactivated and diffusion information as well as structural information into an operating theater, including the functional activation from an auditory language task shown are the pre-resection tumor mass, the functional activation, the venous system, and the lateral ventricles. The source images acquired during the operation are displayed at the bottom of the image, demonstrating the left frontal resection cavity with posterior brain shift. (Courtesy of Dr. Ion-Florin Talos, MD, and Dr. Steve pieper, phD, of the Surgical planning Laboratory, Brigham and Women’s hospital, Boston, Massachusetts, uSa.)

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COMBINING FuNCTIONaL MaGNETIC RESONaNCE IMaGING WITh OThER IMaGING TEChNOLOGIES To improve the temporal resolution of fMRI, the activation maps from fMRI can be combined with the temporal information provided from other sources, such as magnetoencephalography (MEG), EEG, and optical imaging. Both MEG and EEG, unlike other hemodynamic techniques fMRI and pET, directly measure neural activity. MEG is similar to EEG; in fact, the neural generators of the measured signals are identical. The difference is that MEG preferentially detects activity in superficial nonradial areas of cortex (i.e., the fissural cortex of the cerebral hemispheres). This is particularly advantageous if the area of activity is in the walls of the sulci, such as in the motor cortex, somatosensory, and the auditory cortices. Further, nearly all activity measured by MEG is related to postsynaptic activity in the pyramidal cells of the cerebral cortex. The recently developed methods of statistical combination of structural MRI, fMRI, with EEG/MEG may provide the greatest benefit to the neurosurgeon in the near future (77,78). The combination of fMRI and MEG can be particularly illuminating for regional language mapping. The mapping of equivalent current dipoles of the late auditory evoked fields can be used for both posterior temporal and frontal operculum mapping (79,80). Temporal maps of activation have similar profiles as determined by invasive electrocorticography. The latency of Wernicke’s area is typically between 210 and 420 ms and Broca’s area between 400 and 1,100 ms, depending on the individual subject and the particular language paradigm (80–82). Generally, the peak activation of Wernicke’s area precedes Broca’s area, although occasionally other temporal profiles have been reported (83). Thus, maps based on both the activity latency and the spatial location can be used to identify language related areas, increasing the accuracy of the functional map. a disadvantage of fMRI is that, especially at lower field strengths (3.0 T and below), significant activity is detected because of large draining veins, which can be some distance from the cortical sites, leading to confusion in the localization of eloquent cortex. MEG is useful in cases of a compromise to the hemodynamic response mechanisms, for example, in situations of abnormal blood flow response, such as arteriovenous malformations and, importantly, tumors (6,84). This is especially true if the functional cortex is located within the radiologically defined extent of the tumor, such as those of a slow-growing low-grade nature. Inoue et al. (60) found two examples where the tumor and edema mass effect led to disruption of normal hemodynamic response that caused an incorrect localization. holodny et al. (85,86) found decreased BOLD activation of the motor and somatosensory cortices adjacent to brain tumors, despite normal neurologic function. Thus, abnormal vascular supply may decrease the hemodynamic response measured by

fMRI. MEG, on the other hand, is a direct measure of neural activity and is immune to the constraints imposed by the vascular system (84). The future of functional brain mapping will require the intelligent integration of various imaging modalities into a single composite image, which encompasses the strength of each imaging modality.

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84. 85.

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Principles of Brain Tumor Management and Therapy

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C h a p t e r 9

w Principles of Pediatric Brain Tumor Surgery Lilly C. Goumnerova Brain tumors in children are the most common solid neoplasm and the second most common neoplasm in childhood after hematologic malignancies. Their pathology, presentations, and surgical management are different from the management of similar tumors in adults. In addition, the management of tumors in the pediatric population poses certain challenges unique to children in the domains of clinical evaluations, perioperative management, and postoperative care. The care of the children includes not only the patient but also the entire family, and this requires unique skills and treatment teams that include many members to deal with such issues as schooling, psychosocial issues due to tumors in the teenage population, and neuropsychologists to assess the effect on cognitive development by the tumor and its treatment. With the evolution of subspecialization in all aspects of medicine and also in neurosurgery, pediatric neurosurgeons and children’s hospitals are becoming the prime caregivers for children with brain tumors. This allows the coordinated care by a number of subspecialists who are attuned to the unique clinical features of pediatric neuro-oncology and also provides long-term follow-up that can address many of the persistent issues that arise as a result of the disease and its therapy. With better surgery and adjuvant therapy, including

chemotherapy and radiotherapy, resulting in improved surgical survival and improved overall survival of children with brain tumors, it is now recognized that improving quality of life is the next goal in the overall treatment of children with brain tumors. This has led to the creation of pediatric brain tumor programs in many institutions and is becoming the model for the management of pediatric brain tumors. In this chapter, I discuss some of the unique features that are relevant to all children with brain tumors and then address issues that are specific to certain subtypes of pediatric brain tumors.

GeneraL PrInCIPLeS of neuroLoGIC SurGery In ChILdren Clinical evaluation The clinical presentation, including history and examination, in children depends on the age of the patient and on the location of the tumor. Commonly, children will exhibit minimal or no symptoms yet have large tumors for which they have compensated, largely because of the plasticity of the young brain. This is especially true for visual loss, which is rarely 135

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reported at time of presentation but often identified on examination in patients with large craniopharyngiomas or optic pathway tumors. In young children, the parent’s observation of their child’s behavior is invaluable because they may be the only people to detect minor changes that indicate an underlying problem. In neonates and infants under the age of 1 year, presentation is often of a large mass that can be localized to its site or frequently the presentation is that of rapid decompensation secondary to increased intracranial pressure and herniation. There are nevertheless clinical syndromes that are considered pathognomonic of tumors in certain locations. The triad of headaches, ataxia, and nausea and vomiting is almost always present in children with posterior fossa tumors, independent of their pathology. Subtle differences in the duration of symptoms and signs may sway the physician to one diagnosis or another. a short duration of symptoms lasting up to 4 weeks is more typical of a rapidly growing tumor such as a medulloblastoma, whereas patients with pilocytic astrocytomas of the cerebellum will frequently report longstanding headaches or the parents will comment that their child has always been “somewhat clumsy and uncoordinated.” a short history of ataxia, hemiparesis, and cranial nerve deficits (typically extraocular muscle palsies and facial paresis) almost always indicates tumors involving the pons (diffuse brainstem glioma). examination in children is also influenced by their age. It is important to know the appropriate developmental milestones of children until they reach school age so that one can separate abnormal neurologic findings and behaviors from those normally expected of a child of a certain age. This also influences the ability of the examiner to perform a comprehensive neurologic examination, and therefore neuroimaging is extremely valuable in children. head circumference measurements are a sensitive and simple way to assess for the presence of hydrocephalus or an intracranial mass. head growth follows a steady growth curve, and deviations away from it should alert the examiner and prompt appropriate investigations. The examination of the head should also include an assessment of the shape of the head, the sutures and fontanelles, presence of a head tilt, and accomplishment of head control in young infants. forehead bossing or prominence, splaying of the sutures, and a tense or full fontanelle are indicators of increased intracranial pressure. These are often seen in conjunction with downward deviation of the eyes (so-called sun setting) and indicate the presence of hydrocephalus or a tumor around the tectal plate. a head tilt is often seen with tumors in the cerebellopontine angle such as ependymomas of the fourth ventricle or cervicomedullary tumors. Parinaud syndrome is classically seen in patients with tumors in the pineal gland area. a unique presentation is of the diencephalic syndrome where young children (most often under the age of 1 year) will not gain weight despite apparently adequate oral intake.

These children will often undergo extensive gastrointestinal investigations, including invasive testing, and finally a magnetic resonance imaging (MrI) of the brain will identify a tumor in the hypothalamic area. This is almost always an astrocytoma, either fibrillary or pilocytic, and its treatment will often halt and reverse the wasting identified upon presentation. See-saw nystagmus is another typical finding in children with tumors involving the optic chiasm and tracts. This is often the first and only symptom and sign on examination because formal visual assessment in children is extremely difficult and both patient and examiner dependent. formal visual field testing cannot be reliably performed until children reach the age of 3 years and older if there is any developmental delay, which is often seen in young children with brain tumors. Sensory and motor examinations are certainly age dependent. In young children, observation of gross motor movements and a history of accomplishment of gross motor skills will identify problems in the motor system. Sensory examination, once it is possible, is performed in a similar manner as in adults and can provide useful information regarding the location of the lesion. another unique aspect of pediatric examination is assessment of school performance. Seizures, which may be unrecognized, may impair a child’s school performance and lead to deteriorating grades and failure. This is not uncommonly seen with supratentorial tumors affecting the frontal or temporal lobes, gangliogliomas, or other low-grade gliomas. ultimately, however, as in all neurosurgery, imaging has become the gold standard for making the diagnosis of a brain tumor.

Imaging MrI has become the standard for assessment of pediatric brain tumors. however, ultrasonography, computed tomography (CT), and myelography continue to play a role, although not dominant, in pediatric imaging. In infants, an ultrasound can be performed that will identify both the presence of hydrocephalus and on occasion the tumor causing it. however, it only provides a gross assessment and is only used as a rare screening tool. Its advantage is the relative ease of obtaining the study and the lack of sedation that is frequently, if not always, needed for CTs or MrIs. CTs are used primarily as a screening tool in patients who are suspected to harbor a brain tumor. They are also the imaging of choice in the patient who presents in extremis where rapid imaging is necessary before initiation of therapy. CTs are a better modality for evaluation of skull tumors and are also used for image-guided surgery (either biopsy or craniotomy). CTs are frequently fused to images from MrI in the treatment planning for radiotherapy (radiosurgery and fractionated radiotherapy).

9. Principles of Pediatric Brain Tumor Surgery

If a brain tumor is identified on CT, the patient should proceed to MrI to identify the exact location of the brain tumor. MrI provides the best information regarding the location of the tumor and its relationship to the surrounding structures. any child who is identified to have a posterior fossa tumor or a potentially seeding tumor in the supratentorial space should also have a screening spine MrI at that time to assess for potential dissemination/cerebrospinal fluid (CSf) spread. Mr angiography is also useful, especially in tumors that are in the suprasellar area and may involve the vessels at the skull base. Certain Mr characteristics have been identified that are becoming reliable in the diagnosis of brain tumors. The appearance of diffuse expansion of the pons with patchy enhancement and extension around the basilar artery is typical for a diffuse pontine glioma, and MrI in conjunction with the clinical picture is sufficient to make a diagnosis and has supplanted the biopsy for tissue analysis (1). The distinct appearance of T2-weighted bright signal with enhancement upon contrast administration is classic of a pilocytic astrocytoma. In addition, newer techniques are being developed within the field of MrI such as Mr spectroscopy and functional MrI, which are starting to play a significant role in brain tumor imaging and to provide information regarding the biology of the tumors, their functional role and impact on the brain, and their response to therapy. Mr spectroscopy has been able to separate gliomas from other intracranial tumors and also has been used to grade gliomas and assess response to therapy (2). Positron emission tomography (PeT) is still being evaluated for its role in the management of pediatric brain tumors. It is a modality used primarily after treatment to assess the metabolic activity of the tumor and to judge its response to therapy. however, few, if any, treatment decisions are made solely on the information from PeT. rather, its information is collated with the clinical and MrI assessment. a number of trials have incorporated PeT and the newer Mr techniques in their schema, trying to answer the questions about the role of these new imaging modalities in diagnosis and follow-up of pediatric brain tumors. The disadvantage of these techniques, however, is their long scanning time, increased radiation exposure with PeT, and the need for sedation and general anesthesia in some cases. With the increased use of MrI in pediatric neuro-oncology and general imaging, sedation policies have been developed, and these issues are being addressed. Interventional neuroradiology is also useful as an adjunct in the perioperative management of certain tumors (preoperative embolization, functional testing). This is mostly performed as a planned procedure under general anesthesia and in the semielective or elective setting and requires coordination with the planned surgery.

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Perioperative Management Preoperative assessment includes evaluations by the neurosurgeon and by a number of additional subspecialists. Tumors around the sellar and suprasellar area frequently involve abnormalities of the pituitary gland-hypothalamic axis and optic pathways and require the preoperative assessment by an endocrinologist and an ophthalmologist to document any visual abnormalities. recognition of the multiple organ system involvement is therefore essential before any planned surgical procedure. The involvement of a neuroanesthesiologist is essential because they recognize the unique features of the infant physiology, especially for surgeries that may involve significant blood loss, as is the case in a number of infant brain tumors. This allows careful planning and preoperative preparation of the child, including assessment of hydration, blood volume, and other associated illnesses that may affect neuroanesthesia. for certain tumors, such as tumors in the pineal area, the preoperative evaluation should also include serum markers (␣-fetoprotein and ␤-human chorionic gonadotropin) that may in some instances provide a diagnosis obviating the need for biopsy of the tumor. a distinct subgroup of patients is the neurofibromatosis type 1 population. These children, in addition to learning disabilities and other neurologic problems, often have brain tumors affecting the optic pathways and may also develop gliomas in other locations. The close coordination of their care with a geneticist is important because the biology of these tumors appears to be different. as well, they also have multiple plexiform neurofibromas that may not require treatment, and again careful discussion with the family and the geneticist assists in the perioperative evaluation.

operative Management The involvement of a dedicated team including anesthesia and nursing is ideal and crucial. experienced neuroanesthesiology teams are able to recognize sudden changes in the child’s condition that are unusual in the adult. Precipitous drops in oxygenation occur in pediatrics with positioning that require immediate correction. Because of the smaller blood volume in children, hypotension can rapidly result from hypovolemia, and therefore hemostasis and fluid replacement are important and require vigilant monitoring and prompt replacement with fluids and blood products (3). Most infants and children undergoing craniotomies for brain tumors will have at least one large-bore intravenous line and an arterial line for fluid management throughout the surgery. on rare occasions, central lines are placed if peripheral venous access cannot be established. Children have a rich emissary venous network in the skull and large venous dural sinuses, and any craniotomy is associated with the potential for air

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embolism. for that reason, almost all craniotomies are carried out with precordial doppler monitoring (4). hypothermia is another consideration in pediatric brain tumor surgery. This is also important in adult neurosurgery, but even more so in the young infant who can lose heat while obtaining intravenous access or while positioning. for that reason, the room temperature is frequently raised before bringing the child into the room, and the use of heating blankets and lamps is liberal and routine. Positioning of the patient is performed with the surgical approach in mind. This requires careful padding of all exposed areas and ensuring that there is no obstruction of the airway in the prone position or pressure on the face. for infants undergoing surgery in the prone position, a cerebellar head rest is used and the face is protected by placing foam on the cheeks and protecting the exposed pressure points. The same is done for any other pressure points identified after the patient is placed in the final position. flexion of the neck is performed to allow adequate exposure but with protection of the patency of the airway/tube. nasotracheal intubation is the usual for prone cases although endotracheal intubation is also used. flexion of the neck should also take into account the possibility of obstruction of the venous drainage system, which may lead to venous congestion and edema during the surgery with its inherent complications. Placement of pneumatic boots to prevent thromboembolism is not routinely performed. however, with the increased number of overweight children and the recognition of thromboembolic events, pneumatic boots are being placed for cases that will take several hours or where postoperative immobilization is expected. Mannitol and Lasix are not routinely used and are reserved for cases where there is significant cerebral swelling. Steroids are used routinely, and we wean them over a period of 1 to 2 weeks after surgery. anticonvulsants are only used if there is a history of seizures before the diagnosis of the brain tumor and if there is a cortical injury. The preferred medication is phenytoin in older children and phenobarbital in infants under 1 year of age. This is usually administered either preoperatively or after the induction of anesthesia. Most procedures are performed in the prone or supine position with the head turned in the appropriate direction. The sitting position, which used to be a common position for posterior fossa and pineal tumors, is no longer used. frameless navigation is routinely used to plan the surgical incision, and this is usually performed after completion of anesthetic preparation. This can be very helpful for deep intraparenchymal lesions and for mesial temporal tumors to plan approach and assess for extent of surgical resection. This requires the use of rigid immobilization of the skull. In children under the age of 5 years, pediatric pins are used, and generally the torque pressure is lower than 50 and adjusted to the thickness of the skull to prevent accidental perforation through the skull or skull fractures. rigid immobilization may be difficult and is potentially dangerous in

children younger than 1 year old and those under 2 years with a fairly thin skull. In those children, positioning is accomplished on a cerebellar head rest or gel donuts, which provide some rigidity to immobilization. hair shaving along the suture line is performed routinely in our institution, although it is not necessary. The skin incisions are handled with care because infant skin is very thin and easily traumatized. We use coagulating blades and infiltration of the skin with local anesthetic with a long acting agent (0.25% Sensorcaine with 1:200,000 epinephrine) to provide hemostasis. raney clips are generally not necessary if bleeding is controlled while the skin incision is made. If necessary, they can be applied in older children but should not be used in young infants or those under the age of 1 year because they can cause necrosis along the skin edges. Stripping the periosteum can also lead to significant blood loss in the young child and infants and should be readily controlled with irrigation to prevent air embolism and with application of bone wax. The craniotomy is performed applying standard techniques with high-speed drills using pediatric bits for the burr holes and the craniotomy cuts. Whenever exposure of the venous sinuses occurs, it is important to anticipate potential tears and bleeding. These can be controlled by application of “sinus packs,” which are made of surgical patties and Gelfoam soaked in thrombin and should be ready before the beginning of the bony removal. The sinuses in the posterior fossa can be oversewn to avoid placement of metal clips, which will affect postoperative imaging, although vascular clips should also be available to control bleeding, if necessary. Immobilization of the bone flap is performed with either absorbable or metal plates. absorbable plates are used in children under the age 2 years where the metal plates may inhibit bone growth. These plating systems allow immediate immobilization and rigid fixation. The bone flap is replaced in all cases, including posterior fossa craniotomies. We do not apply full head dressings to any of the patients undergoing craniotomies for brain tumors. a dressing of Xeroform is applied to the surgical incision followed by an occlusive dressing that is kept in place until the sutures are removed. recently, we have adopted absorbable suture material for most or our craniotomies and use nylon for selected cases where there are concerns about the wound healing and the possibility of a CSf leak. Intraoperative pathologic (frozen section) diagnosis is obtained in all cases. This is especially important for posterior fossa tumors where knowledge of the diagnosis can modify the surgical plan. This also requires discussion with the patient’s family before the surgery as to the possible pathologic diagnoses and their implication for resection and the potential for neurologic morbidity. Intraoperative monitoring and frameless navigation techniques are new modalities that have impacted our practice significantly. although we do not perform monitoring for brainstem tumors routinely, this is performed in some cen-

9. Principles of Pediatric Brain Tumor Surgery

ters but has not made a significant impact on extent of resection and ultimately on surgical management. The decision to use or not to use intraoperative monitoring requires discussion with the patient’s family and clear understanding of what the goals of the operation are and what the anticipated complications may be. frameless navigation is used to assist in planning of the surgical approach and to a limited extent to assess for extent of resection (5). This is particularly valuable for temporal lobe tumors where the signal abnormality extends further than the visible tumor and is thought to represent tumor. Most of the supratentorial and temporal lobe tumors in children are gliomas, and complete surgical resection is the goal so as to accomplish the best result. It is also helpful in midline tumors to assess extent of resection because there is not much brain shift with resection in those cases. This may be useful in pineal area tumors that extend into the thalamus or for deep intraparenchymal tumors. The use of ultrasound in the operating room is helpful to assist with cyst localizations and catheterizations/aspirations. Color doppler can also assist in assessing the vascularity of the lesions. This modality has the advantage of providing real-time imaging as the lesion is aspirated or biopsied and to monitor for extent of resection and blood clot formation. however, it is dependent on the experience of the operator and understanding the imaging characteristics of the lesions. neurophysiologic monitoring is used in patients who have a seizure disorder and a brain tumor. Intraoperative electroencephalographic monitoring is performed and can localize additional foci after resection of the tumor. awake surgery cannot be reliably performed in children under the age of 10 and requires a cooperative and mature teenager for the best results.

Postoperative Management The postoperative care of the pediatric brain tumor patient follows standard practices. all patients are monitored in the intensive care unit overnight or until stable and then transferred to a neurosurgical ward. We obtain postoperative imaging with MrI on all patients within 24 to 48 hours to document extent of resection and determine subsequent therapy, including the need for additional surgery. Postoperative spine imaging is usually deferred until at least 2 weeks after the surgery to clear the CSf of blood products that may give the appearance of metastatic disease. a unique complication encountered in pediatric neurosurgery is the posterior fossa mutism syndrome. This typically occurs after resection of large midline tumors, most often medulloblastomas, and is thought to be related to injury to the dentate nucleus and its tracts. It usually develops hours to days after the operation has ended and consists of mutism, agitation, and hemiparesis and may take up to sev-

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eral months to a year to clear. Its pathophysiology is poorly understood, and therefore therapy is directed toward symptoms because clear surgical causes have not been identified. It is important to discuss the possibility of this complication with the patient’s family before surgery. This syndrome requires early recognition and extensive rehabilitation as well as continued support to the patient’s family throughout this very trying period. Postoperative CSf collections in the subcutaneous tissues after surgery in the posterior fossa require close observation because they may be an indicator of developing hydrocephalus, even in the presence of complete resection of the tumor.

SPeCIfIC CondITIonS/ TuMorS In ChILdren Posterior fossa Tumors Posterior fossa tumors will frequently have significant hydrocephalus in addition to the mass. The hydrocephalus usually does not require treatment before the treatment of the tumor, and prompt removal of the tumor with clearing of the aqueduct will usually resolve the problem. rarely, a preoperative frontal external ventricular drain is placed for control of intracranial pressure, and that is removed soon after the operation. We do not place occipital burr holes at the time of posterior fossa craniotomies. The surgical management consists of an attempt at complete surgical resection for all tumors, but especially so for astrocytomas and ependymomas because long-term survival is significantly affected by the presence of residual tumor. In medulloblastoma cases we will leave tumor that is adherent to the floor of the fourth ventricle or to the cerebellar peduncles because it does not impact survival to the same extent as in ependymomas. Preoperative imaging should include a spine MrI. If this is not obtained preoperatively, it can be done 2 weeks after surgery. Second-look surgeries for residual tumor are becoming more common in an attempt to accomplish a complete resection.

diencephalic Syndrome, hypothalamic Tumors, and Craniopharyngiomas These tumors are unique to the pediatric population, and their classic presentation and location are almost pathognomonic. nevertheless, surgery is performed for pathologic diagnosis. however, our surgical approach is not aggressive because these tumors are very responsive to chemotherapy, and the complications of radical surgery are significant and include hypothalamic dysfunction, hypoactivity, eating disorders, and visual abnormalities.

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Pineal area Tumors Tumors in the pineal region are frequently germ cell tumors (either germinoma or mixed germ cell tumors). These can often be diagnosed via serum markers and treated with chemotherapy before surgery. an endoscopic third ventriculostomy is usually performed at presentation to deal with the hydrocephalus, obtain CSf for markers and cytology, and possibly obtain a biopsy. endoscopic third ventriculostomy is also the procedure of choice for tectal plate tumors that present with hydrocephalus alone. These tumors do not require biopsy and can generally be followed.

new Techniques endoscopic techniques have been used in brain tumor surgery routinely for the past 15 years. The greatest impact has been in the management of hydrocephalus, either primary aqueductal stenosis or secondary to tumors. With the development of better endoscopes, it is now possible to perform limited procedures on brain tumors, and endoscope-assisted surgery is slowly being used in pediatric brain tumor surgery. This technique is promising, and refinements in technology and training will make it an even more useful procedure. however, there is a steep learning curve with endoscopic surgery that is clearly operator dependent. MrT is a new modality that has been used in adult neurosurgery for approximately 10 years. With its ability to image and confirm complete resections of tumors, it will be important to assess its role in pediatric neurosurgery where longterm survival is clearly linked to complete surgical resection. In summary, pediatric brain tumor surgery is unique because of the patient’s age, the types of tumors observed in that population, and the differences in the surgical management of these tumors. It requires the coordinated team effort of a number of specialists dedicated to the care of children with brain tumors to recognize their unique features and provide the best possible care to our small patients.

referenCeS 1. albright aL, Packer rJ, Zimmerman r, et al. Magnetic resonance scans should replace biopsies for the diagnosis of diffuse brain stem gliomas: a report for the Children’s Cancer Group. neurosurgery 1993;33:1026–1029. 2. Tzika aa, Vajapeyam S, Banes Pd. Multivoxel proton Mr spectroscopy and hemodynamic Mr imaging of childhood brain tumors: preliminary observations. aJnr am J neuroradiol 1997;18:203–218. 3. anonymous. evaluation and preparation of pediatric patients undergoing anesthesia. american academy of Pediatrics. Section on anesthesiology. Pediatrics 1996; 98:502–508. 4. harris Mh, yemen Ta, Stafford Ma, et al. Venous air embolism during craniectomy in supine infants. anesthesiology 1987;67:643. 5. Berger MS. The impact of technical adjuncts in the surgical management of cerebral hemispheric low-grade gliomas of childhood. J neuro-oncol 1996;28:129–155.

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w Principles of Brain Tumor Surgery in Adults

Raymond E. Sawaya, Jeffrey S. Weinberg Brain tumors represent a debilitating disease that often is rapidly fatal, particularly when the tumors are malignant. The reported incidence of brain tumors, including those that are metastatic, is 16.7 per 100,000 persons (1). generally, there are two age peaks for the development of a brain tumor: within the first decade of life and during the sixth decade of life, when gliomas and metastases predominate and most commonly are located in the supratentorial compartment. Since the advent of modern diagnostic and surgical techniques, as well as anesthesia techniques that allow most patients to tolerate brain tumor resection (2), neurosurgery has played an increasingly critical role in the management of brain tumors. Appropriate neurosurgical intervention often offers the patient not only an improved quality of life but also prolonged survival, even in cases of extremely aggressive tumors. Recent technical advances have enhanced the efficacy of neurosurgery. We discuss in this chapter the basic principles related to the goals of neurosurgery, the factors that determine the decision to perform surgery, the standard craniotomy procedure, the use of intraoperative monitoring devices, complications, and techniques for the future of neurosurgery.

goAlS of NEURoSURgERy once a positive diagnosis of tumor has been established, the decision to perform surgical intervention is determined by the potential advantages of accomplishing cytoreduction and/or decompression and, when possible, a cure (3). Tissue samples are used to determine the type of tumor and the presence of malignant features, other tumor components (e.g., mixed germ cell tumors of the pineal region), or metastasis. Special scrutiny (e.g., ki-67) of the tissue also can indicate the rate of tumor growth and aggressiveness; once histologic features are determined, decisions can be made for adjuvant therapy, intraoperative management, and postoperative follow-up.

Cytoreduction purportedly increases the effectiveness of adjuvant therapies by apparently decreasing the number of tumor cells that must be treated, altering cell kinetics, removing the radioresistant hypoxic cells, and removing areas of the tumor inaccessible to chemotherapy (3,4). Until recently, its efficacy remained controversial (5) because no study had shown a clear correspondence between the extent of resection and the length of survival of adults with astrocytic tumors. Recent studies do suggest a correlation (6–8). Surgical decompression may be necessary when size or hydrocephalus causes significant neurologic deficit or when the perilesional edema is too severe to treat with steroids alone. The main goal of neurosurgery is a long-term diseasefree interval or even cure, either of which often can be obtained for benign primary tumors. Recurrent tumors pose additional problems, and these patients have poorer prognoses. Patients with metastatic tumors generally have very poor prognoses as well. Nevertheless, surgery has been shown to be more effective than radiation alone, providing both extended survival and functional independence (9,10).

DECiSioN MAkiNg iN NEURoSURgiCAl iNTERvENTioN Determining the advisability of surgery for patients with brain tumors is based on both tumor and patient factors. The former include mass effect, location, size, vascularity, composition, and number; the latter involve neurologic status, age, surgical risks, anesthesia risks, prior therapy, extent of systemic disease in cases of metastasis, and quality of life that can be expected. The neurosurgeon considers all these factors to weigh the advantages of surgery against the potential neurologic deficits and complications that may result from resection.

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Tumor factors The two primary tumor considerations are location and size. Superficial lesions that are easy to locate and remove through a craniotomy are, obviously, better candidates for surgical success than deeper lesions or lesions in an area of the brain that controls an important aspect of neurologic function. Because in the latter instances additional neurologic dysfunction may result, other therapies (e.g., stereotactic radiosurgery) may be advisable. The size of the tumor is another major consideration (11). A large mass effect that causes distortions of adjacent normal intracranial structures and those that produce extensive edema causing a marked midline shift can predispose the patient to a herniation syndrome and to significant neurologic deficits. Surgery may be urgent in either of these situations. However, large masses that do not distort adjacent brain have, in all probability, infiltrated normal brain regions irreversibly and replaced the normal brain tissue. if so, surgery is unlikely to relieve the neurologic deficits and indeed may even precipitate new ones. A positive response to corticosteroids indicates that the neurologic deficits are from local mass effect, whereas a lack of response may indicate loss of functional integrity of the underlying and infiltrated brain. The vascularity of the tumor, which can be estimated by computed tomography (CT) or magnetic resonance imaging (MRi), and its composition also affect the choice of a surgical intervention. for instance, a highly vascularized tumor has an increased risk of bleeding during stereotactic biopsy. Tumor that receives its blood supply from a vessel or vessels that do not supply adjacent normal brain areas can be embolized with particulate matter or glue preparations to decrease the vascularity of the tumor and to minimize intraoperative blood loss. finally, the number of tumors figures significantly in determining the most efficacious therapeutic program. The surgeon must determine which, if not all, of the tumors can be removed safely and what adjuvant therapy should be administered.

figURE 10-1. Magnetic resonance imaging of the upper cervical area showing an intramedullary cystic mass expanding the medulla and causing difficulty swallowing and imbalance of gait.

Until recently, patients with multiple metastatic tumors were not considered surgical candidates (12), but in 1993 a landmark study by Bindal et al. (9) revealed that surgical excision of multiple metastatic lesions is as efficacious as that of single metastases. in patients with significant systemic disease, removal of intracranial metastases may afford limited benefit.

Patient factors A patient’s candidacy for surgical intervention is based mainly on his or her neurologic status, which can be assessed by neurologic examination (13) and standardized grading scores, such as the karnofsky performance scale. This scale ranks patients with scores ranging from 0 to 100 depending on the extent of self-sufficiency the patient is experiencing (0 = moribund; 100 = normal with no complaints and no evidence of disease) (14). Age is a significant consideration for patients with malignant gliomas (15), whereas it has less relevance for patients with meningiomas (16). for patients who harbor recurrent tumors, a common occurrence with malignant brain tumors, the patient’s previous therapeutic program greatly affects the potential advocacy of surgery. other factors are the patient’s mental or emotional condition and the quality of life that surgery will afford. in patients with significant systemic disease, removal of intracranial metastases may afford limited benefit.

BASiC CRANioToMy PRoCEDURE A basic craniotomy begins with certain preoperative measures, namely evaluation of the patient’s presentation; neurologic examination; diagnostic studies, of which MRi is of utmost importance for localization and definition of the lesion (fig.  10-1); review of the blood work, particularly coagulation studies; and institution of a regimen of steroids and anticonvulsants. Consent for surgery and for any investigational studies planned is obtained from the patient or family

10. Principles of Brain Tumor Surgery in Adults

(3). We also frequently obtain a formal neuropsychological evaluation before treatment to establish a baseline against which future evaluations will be compared to determine the effect the treatment has on the patient’s cognitive function. The operation is performed under general anesthesia except in rare cases, such as when intraoperative monitoring requires the patient to be awakened to monitor eloquent cortex (e.g., resecting tumors near Broca or Wernicke areas). Prophylaxis to prevent deep venous thrombosis is performed by placing sequential compression boots on the patient’s legs before general anesthesia induction. The actual craniotomy begins with the operative field being washed with noncaustic soap the night before surgery; in the operating room, the patient’s head is shaved, and the incision site is disinfected with a solution containing iodine. The type of incision is determined by the location of the tumor and the scalp blood supply, cosmetic factors (including position of the patient’s hairline), and considerations regarding possible future operations. The patient is usually positioned with the tumor at the highest point in the field, and then the patient’s head is frequently immobilized in a head clamp. imageguided surgical systems (see below), which display a preoperative MRi, may be used to minimize the skin incision and bone opening. This places the minimum brain at risk during the opening, tumor dissection, and removal. once the bone is removed, the dura is opened with an incision that provides optimal exposure. Care is taken during the dural opening to avoid injury to venous sinuses and to veins that are adherent to or run in the dura before draining into a sinus. Ultrasound is used to determine the location and extent of the tumor. To access the tumor, a decision is made to proceed through the brain (e.g., transcortical, transcallosal), through a sulcus (e.g., transsulcal), or between hemispheres (e.g., interhemispheric). once the tumor is encountered, it is circumscribed if possible. if the margins are not well defined, by direct vision or through textural differences, other intraoperative devices (discussed below) are used to better differentiate tumor from the surrounding brain. The extent of the resection is based on the pathologic diagnosis, the tumor’s location, and the patient’s preoperative deficits. We favor aggressive resection—gross-total removal—whenever possible. The area of edema visualized on MRi is not resected; for soft tumors such as gliomas and some metastases, simple bipolar cautery and suction are usually sufficient for resection. Most firm tumors can be resected with the aid of an ultrasonic aspirator. once the tumor has been resected, hemostasis is obtained, the dura is closed with suture, and the bone flap is replaced and secured with miniplates and screws. if the dura or bone has been invaded by tumor, it is removed and a duraplasty or cranioplasty, respectively, is performed. Patients are usually mobilized by the following morning and can be discharged as early as the third postoperative day (3). Postoperative imaging is performed within 48 hours to document extent of resection and presence of hematoma or ischemia. it is cru-

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cial to perform the scan in this acute period because enhancement second to postoperative gliosis is visible after 48 hours and can be confused with residual enhancing tumor. Decisions concerning the management of patients with brain metastases can be especially difficult, mainly because of the poor prognosis that accompanies disseminated cancer. Because most patients (at least 50%) have uncontrollable systemic cancer, control of the metastasis for maintaining or improving quality of life, rather than survival, is often the most appropriate end point for analyzing the efficacy of treatment. in such cases, the success of therapy is determined by the improvement of specific neurologic symptoms, a factor that depends on the patient’s neurologic function at the time of presentation with brain metastases. Although radiotherapy has been considered the preferred treatment and until recently surgical therapy has not been recommended for most patients, improvements in neurosurgical and anesthetic techniques and perioperative care have increased the benefits of surgery such that it is becoming a routine consideration for many patients. The median survival times of heterogeneous groups of patients have been extended to 10 to 14 months for patients treated surgically for a single metastasis (17–20) or multiple metastases (9). The techniques for surgical removal of metastases are the same as those performed for primary intracranial lesions, although the tendency of a cerebral metastasis to be well delineated or even encapsulated facilitates their resection. Advantages of resection include extended survival times, improved control of neurologic problems, and expanded diagnostic determinations. Both uncontrolled surgical series and retrospective studies have confirmed the greater efficacy of surgery combined with whole-brain irradiation over whole-brain irradiation alone (21–31).

iNTRAoPERATivE MoNiToRiNg DEviCES Among the advances that have contributed to the continuing strides being gained by neurosurgery are intraoperative monitoring devices, among which are ultrasound, cortical mapping, and intraoperative imaging.

intraoperative Ultrasound gaining wide acceptance and applicability, intraoperative ultrasound (ioUS) (10,11,32–36) imaging has been shown to offer significant benefits during neurosurgery (31,34,37), particularly in its ability to provide the surgeon with real-time feedback and to yield images that aid in both the diagnosis and maximal resection of the tumor (38–41). intracranial ultrasound imaging is usually performed using a real-time sector scanner, which is a self-contained portable unit with a 9-inch television monitor on which the image produced by the transducer is viewed in real time. The transducer can be

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of different shapes and frequencies (3, 5, 7.5, or 10 MHz). low-frequency transducers usually scan deep-seated lesions better, whereas small lesions near the surface are detected better with high-frequency transducers (7.5 MHz). once the bone has been removed, the transducer head may be placed either on the dura or directly on the exposed brain surface that is moistened with saline solution. Although investigation is preferentially performed with the dura closed so as not to exert pressure on the underlying cortex, scanning through the exposed brain surface gives better images in some cases, especially when the dura is thickened or partially calcified. our customary procedure is to scan the entire area of the bone opening in two perpendicular planes to confirm that the entire lesion can be accessed through the exposed dura. After the dura is opened, gentle scanning over the brain surface is then performed to locate the shortest and safest route to the tumor. Before resection, ultrasound can accurately identify a sulcus that can be used to approach a tumor. for deeply located tumors, it gives real-time updates regarding the distance until the tumor is resected. During tumor removal, ioUS is used to evaluate the progress being made and to detect any residual tumor in cases where complete excision is the aim. After the excision has been completed, the cavity is filled with saline and ioUS is used, preferably with the dura closed, to determine the extent of resection and to detect the presence of any developing hematoma. ioUS has various uses, the principal of which is to localize the tumor. in addition to readily identifying both low-grade and high-grade gliomas, which are typically echogenic relative to the surrounding brain (11,25,32,42,43), ultrasound is particularly useful for imaging small subcortical lesions as well as deep-seated ones. localizing lesions in this manner helps the surgeon develop better intraoperative strategies and design an opening of the dura that allows for smaller flaps. ioUS can be used to define the borders of tumors, especially those of gliomas. low-grade gliomas may appear much like normal brain during surgery and can be extremely difficult to localize and define. likewise, although the appearance and consistency of high-grade gliomas are usually different from those of normal tissue at the time of surgery, the actual borders of these tumors cannot be ascertained absolutely. ioUS has become an especially valuable adjuvant because it delineates both gliomas and their transition toward normal tissue, regardless of their CT or MRi patterns (34,40,44). it also can differentiate edema from solid tumor and normal brain. Sonographically, peritumoral edema is either not visible or, if visible, is less echogenic than the tumor and therefore does not interfere with tumor delineation (31,40,45). Ultrasound imaging is also used for characterizing the tumor and for guidance during tumor biopsy (44,46). it has been shown to be superior to CT in differentiating between viable tumor, necrosis, and cysts (40,43,44). This differenti-

ation is especially important if the tumor is located in a deep portion of the brain and hence subtotal resection is anticipated or if the tumor is cystic and can be aspirated with minimal disruption of the overlying brain parenchyma. guidance by real-time ioUS during biopsy provides reliable knowledge of the position of the needle tip with respect to the tumor and allows multiple biopsies to be taken from different areas of the tumor for more accurate histopathologic diagnoses. finally, ioUS is helpful in detecting residual tumor toward the end of the operation after complete resection has been attempted. Residual masses that might exist after the course of tumor resection can be evaluated with ioUS and a decision made as to whether or not further resection is appropriate (38–40) (fig. 10-2). There are several complications or difficulties with ioUS. first, confluence of the cortical sulci may appear like tumors and may be confused with a true mass lesion. Second, in patients who have undergone previous surgery and have received radiation therapy, the echogenicity of peritumoral edema may be similar to that of the tumor and radiation necrosis, with the result that the tumor margin is indistinct, and hence the surgeon may make an excision that is wider than necessary. Third, a hyperechogenic rim, which is called acoustic enhancement (47) and is related to the penetration of sound through two different media (i.e., tumor tissue before excision versus a saline-filled cavity after it), appears on ioUS images after complete excision and may be confused with residual tumor. finally, a small hematoma in the surgical bed cavity may be confused with residual tumor, particularly when hemostatic agents such as oxycel or Surgicel are left in the tumor bed. in the first instance, the problem can be avoided by intraoperative comparison of the location of this false lesion, called a sulcar pseudomass (35), with that of the true mass seen on preoperative CT or MRi; the second problem usually can be avoided by careful inspection of the images in different planes during surgery to distinguish between tumor and edema. The results that we have obtained using ioUS indicate that it is not only helpful in localizing and defining the margins of gliomas but also accurate in determining the extent of resection, as confirmed by postoperative MRi, except in cases where the lesion represents radiation effect. ioUS appears to be a promising tool for the achievement of maximal surgical resection of gliomas.

functional Mapping functional mapping of the brain can be performed preoperatively using functional MRi (fMRi) to demonstrate cortical function and diffusion-weighted imaging (48) to demonstrate functional neural pathways. it can also be performed intraoperatively using one of two stimulation procedures, direct cortical stimulation or somatosensory evoked potentials (SSEPs). These brain-mapping techniques are especially efficacious when the tumor is located in discrete brain areas.

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figURE 10-2. Patient is a 45-year-old man who presented with headaches and a change in vision. A. Axial T1–weighted gadolinium-enhanced magnetic resonance (MR) image demonstrates a heterogeneously contrast-enhancing lesion in the deep right temporal—occipital lobe with perilesional edema. The entire lesion is encircled while the falx cerebri is demonstrated by a line. There is a small component of tumor that has herniated under the falx. Mass effect on the surrounding brain is demonstrated by the choroid plexus being displaced anteriorly. B. Postoperative axial T1–weighted gadolinium-enhanced MR image demonstrates complete resection of the contrast-enhancing lesion as well as the subfalcine component. The resection cavity is smaller than the size of the original tumor, and the mass effect on the choroid plexus has decreased significantly. C_E. Dynamic and real-time imaging and therefore usefulness of ultrasound during tumor resection is demonstrated. C. intraoperative ultrasound before the resection and performed after durotomy demonstrates the hyperechogenicity of the tumor as well as the normal anatomy of the falx cerebri and the subfalcine component of the tumor. D. intraoperative ultrasound performed during the resection demonstrates residual hyper-echogenic tumor at the base of the resection cavity. With resection, the mass effect has decreased and a portion of the subfalcine component has retracted back to the ipsilateral side. E. The resection cavity has collapsed. The subfalcine component has been resected, and only a minimal amount of hyper-echogenic lesion remains to be resected.

functional Magnetic Resonance imaging A new technique, fMRi, can be used to perform preoperative mapping of motor, sensory, and speech areas of the brain. This technique is based on the assumption that there is increased blood flow to regions of the brain that are metabolically active. Thus, as one asks a patient to perform an action such as speaking or tapping fingers, there is increased blood flow to the regions of the brain that are being used to perform this activity. As a result, there is local vasodilation in these ac-

tivated areas resulting in an increase in oxyhemoglobin concentration (due to increased cerebral blood flow). Without an equal increase in oxygen extraction, the result is a net decrease in the local deoxyhemoglobin concentration. Because deoxyhemoglobin is paramagnetic, a decrease in its concentration yields increased signal intensity on both T1- and T2weighted images. By overlaying this area of increased T2 signal on the brain MRi, an anatomic localization of this area of activation is obtained. it provides excellent spacial resolution to the order of 1 to 2 mm. By identifying the area of activation,

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a surgeon can plan the approach to a tumor through a sulcus or gyrus that is found not to contain the particular function being studied. A correlation between fMRi and intraoperative mapping has determined that localization of function concurred between both fMRi and intraoperative electrophysiology, including SSEPs and direct cortical stimulation (see below) (49). The information gained from the fMRi can also be overlaid with a preoperative MRi and transferred to an image-guided surgical workstation. in this manner, a pointing device can be used to point out not only the tumor and normal anatomy but the functional anatomy as well (50). one of the more recent imaging studies developed to be used in the preoperative period is that of diffusion tensor imaging or tractography. This is the first noninvasive method that can be used to generate images of the functional white matter pathways. This technique is based on water diffusion that is dependent on multiple local factors, including geometry and orientation of the local environment. Diffusion tensor

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imaging can measure this directionality, and the information can be displayed as a picture with color-coded images of the tracts based on the direction of water diffusion. This technique is currently being used in the preoperative period to determine the relationship between tumor and the descending tracts. it is believed that this technique can be used to demonstrate mass effect from tumor versus tumor cell infiltration. Thus, planning an approach to a tumor and determining the amount of tumor resection that can be performed before reaching these tracts can be estimated in the preoperative period. The use of both fMRi and tractography allows a surgeon to plan the safest operation for a patient (fig. 103). Direct Cortical Stimulation four major steps have played a part in the development of direct cortical stimulation for brain surgery: identification of

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figURE 10-3. The patient is a 53-year-old man with a right posterior frontal glioblastoma multiforme previously treated with resection, radiation, and chemotherapy who now presents with mild left hemiparesis. A. Axial T1-weighted gadolinium-enhanced magnetic resonance (MR) image shows heterogeneously contrast-enhancing lesion in the right posterior frontal lobe with significant perilesional hypointensity consistent with edema. B. functional MR image. left-hand sensorimotor task superimposed on T1-weighted nonenhanced sagittal image demonstrates the proximity of the signal activation to the lesion. C. Axial diffusion tenor image demonstrates mild medial displacement and decreased thickness of posterior limb of the ipsilateral internal capsule (single arrow) as compared with the contralateral (dotted arrow). D. Axial T1weighted gadolinium-enhanced MR image demonstrates complete resection of contrast-enhancing portion of the tumor and decreased perilesional edema. E. Axial diffusion tensor image performed postoperatively demonstrates that the medial extent of the resection cavity did not violate the posterior limb of the internal capsule, which is less displaced and more substantial than in the preoperative period (compare with C). f. Same axial image as E; however, the white matter tracts have been grey-scale coded according to direction of water diffusion. The posterior limb of the internal capsule (still present) is vertically oriented as compared with the corpus callosum, which runs from side to side, and the anterior limb of the internal capsule, which runs anteriorly—posteriorly.

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figURE 10-3. (CoNTiNUED)

the motor cortex by fritsch and Hitzig in 1870 (51); demonstration of the electrical excitability of the human motor cortex 4 years later by Bartholow (52); identification of the motor cortex during standard craniotomies 10 years afterward by Sir victor Horsley, who was using faradic stimulation and local anesthesia (53); and a landmark study by Penfield and Boldrey (54) showing that electrical stimulation of the cortical surface evoked responses in patients undergoing resection of epileptogenic tissue. The achievements accomplished by Penfield and Boldrey in 126 patients provided the foundation for practical intraoperative localization of the sensory motor

cortex, which is necessary for minimizing sensory or motor deficits when the area of intended resection lies in or adjacent to the sensorimotor region. After these initial developments, the methods of eliciting movement by applying electrical stimulation to the cortex have been improved considerably. in 1991, a report on 270 patients with intrinsic tumors of the brain and spinal cord noted that direct stimulation methods allowed for greater resection of the tumor and the infiltrated brain adjacent to the tumor nidus, improved seizure control, and lessened permanent operative morbidity. one of the noted advantages is that direct stimulation

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figURE 10-4. A. Magnified view of the cortical surface showing a discolored gyrus with prominent vasculature. B. Cortical mapping using a flexible and transparent grid has identified the abnormal gyrus that is infiltrated with tumor (T) as representing the motor cortex (M) and the adjacent cortex to the left as presenting the sensory cortex (S).

provides the surgeon with the means to map descending motor pathways during the actual tumor resection (55). The procedure has two distinct disadvantages, however. first, movements may accompany stimulation of the sensory cortex and thereby yield ambiguous identification of the precentral and postcentral gyri, and, second, having to use local rather than general anesthesia poses difficulties for the patient and the surgical team alike and may even be hazardous with certain mass lesions. Somatosensory Evoked Potentials Because it avoids the problem of ambiguous feedback and is compatible with general anesthesia, the method using SSEPs (fig. 10-4) is considered by some surgeons to be superior to that using cortical stimulation. in 1987, king and Schell (56) reported that of 35 patients on whom both median nerve stimulation and direct cortical stimulation of the motor cortex were performed with the patients under general anesthesia, none of the patients who were free of neurologic deficits preoperatively demonstrated neurologic deficits in the immediate postoperative period. Most of the patients with preoperative neurologic deficits showed improvement in the immediate postoperative period. in 1988, Wood et al. (57) compared (a) the results obtained in 18 patients who were operated on under local anesthesia and in whom the sensorimotor cortex was independently localized by electrical stimulation mapping with (b) the results obtained using SSEPs in an additional 27 patients who were operated on under general anesthesia without electrical stimulation mapping. Their findings demonstrated that hand area localization was equally effective regardless of whether local or general anesthesia was used. Subsequently, Ebeling et al. (59) reported the results in 50 patients with lesions located in or adjacent to the motor

strip that were microsurgically removed with the use of intraoperative electrophysiologic mapping of the sensorimotor cortex: 21 (42%) had motor paresis preoperatively and experienced some postoperative recovery (30%) (12,58). Those with no or mild paresis preoperatively had good outcomes, with no new deficits or recovery of a preexisting deficit; patients with severe or complete motor paralysis preoperatively had improvement in the motor paresis; and a few patients with lesions located within the motor area experienced full recovery, a result consistent with other reports (12,58–60). By locating the motor strip, which can be achieved only by intraoperative mapping of the pre- and postcentral gyri, the surgeon can identify the sensorimotor region and thereby not only preserve the motor strip and the pyramidal tract but also achieve a more complete resection of the lesion (61–64). our own experience has involved using intraoperative cortical mapping in 81 patients with primary or metastatic paracentral tumors who underwent craniotomy under general anesthesia. Mapping showed that 49 lesions (60%) were just anterior to the motor strip, 15 (19%) were just posterior to the sensory strip, and 13 (16%) were in the sensorimotor cortex. Despite the critical locations of these tumors, the postoperative neurologic outcomes revealed improvement from the preoperative baselines in 23 patients (28%), no change in 50 patients (62%), and deterioration in only 8 patients (10%). Patients with metastatic disease had better results than did those with primary lesions. A positive correlation was found between the extent of parenchymal pathology and the distortion of the cortical tracings. A drawback of cortical mapping is that it allows localization of only the central area. Even if an impairment to the cortical surface is avoided, motor deficits can occur if the efferent nerve fibers from the motor cortex are injured deep in the white matter during surgical intervention (65).

10. Principles of Brain Tumor Surgery in Adults

ADvANCES iN NEURoSURgERy The safety of intracranial surgery has been enhanced significantly by many developments, the most important of which are the surgical microscope and microsurgical instruments. The former provides a wide range of magnification and enhanced illumination, which, with the addition of developed long bayonet-shaped microsurgical instruments, provides the surgeon with the means to approach lesions in any intracranial location while maintaining good visualization of the vascular supply and critical areas of the central nervous system (fig. 10-5). The bipolar coagulator provides efficacious hemostasis and eliminates the danger of spreading current encountered with older monopolar coagulators. The self-retaining brain retractor simultaneously improves the surgical exposure, maintains a constant pressure force on the brain, and frees the surgeon’s hand. Modern neuroanesthesia, advanced methods of managing increased intracranial pressure, and more precise control of fluids and electrolyte balance also have improved the field of neurosurgery. The Cavitron ultrasonic aspirator, which uses ultra–high-frequency vibrations to shatter tumor cells with sound waves, inflicts minimal damage on surrounding normal structures and blood vessels and is especially effective on fibrous tumors such as meningiomas and acoustic neuromas. interventional neuroradiology uses techniques that aim to aid the management of intracranial vascular lesions such as meningiomas, glomus tumors, and chemodectomas through the use of gelfoam, silicone or acrylic polymer, metallic pellets, and polyvinyl alcohol sponges for embolization. This decreases tumor vascularity and minimizes intraoperative blood loss. Preoperative functional imaging (see above) has greatly enhanced our ability to plan an operation

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that minimizes the risk to eloquent brain and maximizes the chance of the best functional outcome. Magnetic resonance spectroscopy (MRS) is a new imaging technique that has been gaining popularity. This technique measures proton-containing metabolites from small regions of tissue after suppressing signals from both water and lipids (12). The metabolites that are most importantly identified are that of N-acetylaspartate, which is a marker in normal functioning neurons; choline, which is present in cell membranes; creatine, which is important in energy metabolism; and lactate, which is a by-product of anaerobic metabolism and is usually found in regions of necrosis. Preliminary data have suggested that MRS can help differentiate tumor histology in the preoperative period (66) and can help differentiate pure recurrent tumor from pure radiation necrosis (67). in addition, MRS has been suggested to be able to predict the response of gliomas to tamoxifen chemotherapy (68). Analysis of a tumor with MRS in a patient who is to undergo a stereotactic biopsy can help the surgeon target the most malignant part of the tumor (69). in patients with gliomas undergoing stereotactic radiosurgery, time to recurrence and survival is decreased in those with MR spectra consistent with tumor outside the treated volume (70). finally, serial imaging with MRS may help identify tumor recurrence or clinical progression (71). image-guided surgical tools have greatly enhanced the safety of neurosurgery. There are multiple systems that are either frame based or frameless. They augment a surgeon’s ability to identify the location of tumors more precisely, enabling one’s ability to plan a small skin incision, a small craniotomy, and put the minimal amount of brain (which is not to be resected) at risk for injury during surgery. Stereotactic procedures are discussed in an earlier chapter of this book. finally, the use of an MRi in the operating room allows a surgeon to obtain accurate imaging during tumor resection, identify nonre-

figURE 10-5. A. Preoperative (left) and postoperative (right) magnetic resonance image of the same patient as in figURE 6-1 showing a cystic mass with an enhancing mural module representing a hemangioblastoma. The tumor was totally resected. (continued)

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figURE 10-5. B. intraoperative picture showing a tumor mass through an incision in the medulla. Several lower cranial nerves and upper cervical roots are seen exiting on the right side. The patient recovered fully within 4 days of the operation.

sected tumor that may not be directly visible, and obtain new images that can be stored in the stereotactic apparatus. This reregistration eliminates loss of accuracy secondary to brain shift that occurs because of tumor resection or release of cerebrospinal fluid, a main problem with image-guided surgical tools. intraoperative MRi is discussed in another chapter.

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lished since 1980. The complication rate appeared to be more dependent on the location of the tumor than on the histology. The authors also emphasized that the advent of corticosteroids, modern neuroanesthesia, and microneurosurgery had lowered the operative mortality rate from 20% to 40% to 5% or less for metastatic tumors, to less than 3% for glial tumor surgery, and to 0% for stereotactic resection (30,54,72,73).

CoMPliCATioNS Surgical management of brain tumors involves both medical and surgical perioperative complications. The former are related to anesthesia (0.2%) (1), electrolyte imbalances (especially hyponatremia), infection, and venous thromboembolism (due to immobility of some postoperative patients and tumor-related hypercoagulable states). Postoperative brain swelling, one of the most serious surgical complications, may result from manipulation of the surrounding brain tissue, changes in regional blood flow because of tumor resection, or injury due to excessive retraction. other complications may be postoperative hematoma at the operative site caused by postoperative hypertension, incomplete tumor resection, or coagulation abnormalities (4.8%); a transient increase in neurologic deficit immediately after surgery due to swelling, retraction, or resection (10%); seizures; postoperative hydrocephalus caused by tumor, periventricular swelling, or intraventricular blood; stroke (30,72); and postoperative deterioration in mental status or the appearance of a new deficit. A recent study that examined complications in a single surgeon’s series of 207 consecutive adult patients undergoing first craniotomy for intraaxial brain tumors (73) found no difference in complication rates for surgery of gliomas and metastases from selected series pub-

TECHNology of THE fUTURE Cutting-edge technology that has been applied to neurosurgery includes the fields of robotics and virtual reality.

Robotics The concept of robotics was first introduced by karel Capek in a 1923 play in which his mechanical men performed robota, a Czech term for compulsory servitude. Some 70 years later, the term has taken on new meanings: According to the Robotic industries Association, a robot is a “reprogrammable, multifunctional manipulator designed to move materials, parts, tools, or specialized devices through variable programmed motions to perform a variety of tasks” (74). Robots perform some human task that is dangerous or repetitive or one that requires superhuman precision, accuracy, and/or strength (75). one of the first robotic systems to be used clinically was developed by kelly while at the Mayo Clinic (76). This system encircles the patient’s head while CT, MRi, and angiographic images are obtained and then used to create computer-generated three-dimensional maps of the tumor area. During surgery, the head-holder reference system of the ro-

10. Principles of Brain Tumor Surgery in Adults

botic device is placed precisely as it was during the imaging studies. it functions during this state of the procedure to position the patient’s head so that the surgical field precisely conforms to the computer-generated slice images of that field. The surgeon operates with excising instruments. New robotic equipment is being introduced into surgery. The Evolution 1 precision robot, which is a holding and positioning device for an endoscope, can perform slow and precise movements with the use of an image-guided surgical system (77). The NeuRobot system, which consists of a micromanipulator, the manipulator supporting device, the operation-input device, and a three-dimensional display monitor (78), enhances the dexterity and the distance from which the surgeon can operate.

virtual Reality one of the most recent advances in technology has been in the area of virtual reality, once a fantasy limited to science fiction or comic books. Having entered the computer technology world, virtual reality is becoming recognized as a viable venue for the development of dramatic new medical applications (79–83). virtual reality and micromachine technology combined with remote control are becoming increasingly important for minimally invasive surgery, and future techniques may include the use of miniature motors and instruments that can be introduced into body cavities to perform operations that currently are impossible (83). The process involves turning traditional CT or MRi of the patient’s tumor and brain into three-dimensional models, and thus the patient’s actual anatomy becomes the virtual anatomy. By means of a head-mounted device, the physician is able to enter visually and interact spatially with the virtual world of the tumor. virtual arrows or beams of radiation allow the physician to explore the tumor and its surroundings from all angles. This ability is particularly advantageous for directing radiation beams in positions deemed the least likely to damage healthy tissue (82). The possibilities that the future may hold for virtual reality will become more evident once some of the needed advances are achieved, for example, when a second camera can be placed on the head-mounted display to produce stereoscopic imagery, improved head mounted display resolution, a better sensor tracking system, and better and faster imagery. other promising surgical techniques include microengineering, which, like microelectronics, is based on crystal technology. it is now possible to etch tiny gears and mechanisms on a silicon crystal by photoreduction. The process leaves a minute train of gears and levers that rotate around fixed pivot points and can be combined with electronic controls to make microscopic electric motors that are less than 1 mm in size. This technology offers the possibility of designing scissors or forceps with blade lengths of 1 ␮m or less that can be controlled by remote means and actually in-

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troduced into body cavities or organs to perform operations that are currently impossible. All these advances also present new problems and difficulties as well as challenges and suggest that the future for brain surgery may require totally new principles and considerations.

SUMMARy Each surgical case presents with different problems, requiring that management be determined on the bases of individual tumor and patient considerations. fortunately, with the significant advances in neurosurgical, anesthesia, and imaging techniques, today almost all brain tumor patients can benefit from appropriate and timely neurosurgical intervention. Considerably better results and prognoses than were once the case can now be expected, and patients are living longer and having a better quality of life after surgical intervention. These advances, along with the ones that are now being developed, allow the surgeon to perform more accurate and extensive surgical procedures and promise brighter futures for patients.

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39. Quencer RM, Montalvo BM. intraoperative cranial sonography. Neuroradiology 1986;28:528–550. 40. Chandler Wf, knake JE. intraoperative use of ultrasound in neurosurgery. Clin Neurosurg 1983;31:550–563. 41. gooding gA, Edwards MS, Rabkin AE, et al. intraoperative real-time ultrasound in the localization of intracranial neoplasms. Radiology 1983;146:459–462. 42. leRoux PD, Winter TC, Berger MS, et al. A comparison between preoperative magnetic resonance and intraoperative ultrasound tumor volumes and margins. J Clin Ultrasound 1994;22:29–36. 43. Chandler Wf, knake JE, Mcgillicuddy JE, et al. intraoperative use of real-time ultrasonography in neurosurgery. J Neurosurg 1982;57:157–163. 44. knake JE, Chandler Wf, gabrielsen To, et al. intraoperative sonographic delineation of low-grade brain neoplasms defined poorly by computed tomography. Radiology 1984;151:735–739. 45. Enzmann DR, Wheat R, Marshall WH, et al. Tumors of the central nervous system studied by computed tomography and ultrasound. Radiology 1985;154:393–399. 46. Sutcliffe JC. The value of intraoperative ultrasound in neurosurgery. Br J Neurosurg 1991;5:169–178. 47. Rubin JM, Carson Pl. Physics and techniques. in: Rubin JM, Chandler Wf, eds. Ultrasound in neurosurgery. New york: Raven, 1990:1–66. 48. Unsgaard g, gronningsaeter A, ommedal S, et al. Preoperative assessment of motor cortex and pyramidal tracts in central cavernoma employing functional and diffusion-weighted magnetic resonance imaging. Surg Neurol 2002;58:302–307. 49. Ruge Mi, victor J, Hosain S, et al. Concordance between functional magnetic resonance imaging and intraoperative language mapping. Stereotact funct Neurosurg 1999;72:95–102. 50. gumprecht H, Ebel gk, Auer DP, et al. Neuronavigation and functional MRi for surgery in patients with lesion in eloquent brain areas. Minim invasive Neurosurg 2002;45:151–153. 51. fritsch g, Hitzig E. Ueber die elektrische Erregbarkeit des grosshirns. Arch Anat Physiol Wiss Med 1870;37:300–332. 52. Bartholow R. Experimental investigations into the functions of the human brain. Am J Med Sci 1874;67:305–313. 53. Northfield DWC. Sir victor Horsley: his contributions to neurological surgery. Surg Neurol 1973:131–134. 54. Penfield D, Boldrey E. Somatic motor and sensory representation in the cerebral cortex of man as studied by electrical stimulation. Brain 1937;60:398–443. 55. Berger MS, ojemann gA. intraoperative brain mapping techniques in neuro-oncology. Stereotact funct Neurosurg 1992;58:153–161. 56. king RB, Schell gR. Cortical localization and monitoring during cerebral operations. J Neurosurg 1987;67:210–219. 57. Wood CC, Spencer DD, Allison T, et al. localization of human sensorimotor cortex during surgery by cortical surface recording of somatosensory evoked potentials. J Neurosurg 1988;68:99–111. 58. Witt TC, kondziolka D, Baumann SB, et al. Preoperative cortical localization with functional MRi for use in stereotactic radiosurgery. Stereotact funct Neurosurg 1996;66:24–29. 59. Ebeling U, Schmid UD, ying H, et al. Safe surgery of lesions near the motor cortex using intra-operative mapping techniques: a report on 50 patients. Acta Neurochir (Wien) 1992;119:23–28. 60. gregorie EM, goldring S. localization of function in the excision of lesions from the sensorimotor region. J Neurosurg 1984;61:1047–1054. 61. Witzmann A, Beran H, Bohm-Jurkovic H, et al. The prognostic value of somatosensory evoked potential monitoring and tumor data in supratentorial tumor removal. J Clin Monit 1990;6:75–84. 62. Rostomily RC, Berger MS, ojemann gA, et al. Postoperative deficits and functional recovery following removal of tumors involving the dominant hemisphere supplementary motor area. J Neurosurg 1991;75:62–68. 63. Berger MS, kincaid J, ojemann gA, et al. Brain mapping techniques to maximize resection, safety, and seizure control in children with brain tumors. Neurosurgery 1989;25:786–792.

10. Principles of Brain Tumor Surgery in Adults 64. Berger MS, ojemann gA, lettich E. Neurophysiological monitoring during astrocytoma surgery. Neurosurg Clin N Am 1990;1:65–80. 65. Taniguchi M, Cedzich C, Schramm J. Modification of cortical stimulation for motor evoked potentials under general anesthesia: technical description. Neurosurgery 1993;32:219–226. 66. Meyerand ME, Pipas JM, Mamourian A, et al. Classification of biopsy-confirmed brain tumors using single-voxel MR spectroscopy [comment]. AJNR Am J Neuroradiol 1999;20:117–123. 67. Rock JP, Hearshen D, Scarpace l, et al. Correlations between magnetic resonance spectroscopy and image-guided histopathology, with special attention to radiation necrosis. Neurosurgery 2002;51:912–919. 68. Preul MC, Caramanos Z, villemure Jg, et al. Using proton magnetic resonance spectroscopic imaging to predict in vivo the response of recurrent malignant gliomas to tamoxifen chemotherapy. Neurosurgery 2000;46:306–318. 69. Preul MC, Cendes f, Just N, et al. intracranial aneurysms and sickle cell anemia: multiplicity and propensity for the vertebrobasilar territory. Neurosurgery 1998;42:971–977, discussion 977–978. 70. graves EE, Nelson SJ, vigneron DB, et al. A preliminary study of the prognostic value of proton magnetic resonance spectroscopic imaging in gamma knife radiosurgery of recurrent malignant gliomas. Neurosurgery 2000;46:319–326, discussion 326–328. 71. Tedeschi g, lundbom N, Raman R, et al. increased choline signal coinciding with malignant degeneration of cerebral gliomas: a serial proton magnetic resonance spectroscopy imaging study. J Neurosurg 1997;87:516–524. 72. Raabe A, kirshnan R, Wolff R, et al. laser surface scanning for patient registration in intracranial image-guided surgery. Neurosurgery 2002;50:797–801, discussion 802–803. 73. Cabantog AM, Bernstein M. Complications of first craniotomy for intra-axial brain tumour. Can J Neurol Sci 1994;21:213–218. 74. Azimov i, frenkel kA. The origin of robots: myth and reality. in: Robots: machines in man’s image. Ny: Harmony Books, 1985;15–21. 75. Drake JM, Joy M, goldenberg A, et al. Computer- and robot-assisted resection of thalamic astrocytomas in children. Neurosurgery 1991;29:27–33. 76. kelly PJ. Computer-assisted stereotaxis: new approaches for the management of intracranial intra-axial tumors. Neurology 1986;36:535–541. 77. Zimmerman M, krishnan R, Raabe A, et al. Robot-assisted navigated neuroendoscopy. Neurosurgery 2002;51: 1446–1451. 78. kelly PJ. Comment on article NeuRobot: telecontrolled micromanipulator system for minimally invasive microneurosurgery preliminary results. Neurosurgery 2002;51:988. 79. Wickham JE. Minimally invasive surgery. future developments. Br Med J 1994;308:193–196. 80. larijani lC. The virtual reality primer. New york: Mcgraw-Hill, 1994. 81. Anonymous. Being and believing: ethics of virtual reality. lancet 1991;338:283–284.

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C h a p t e r

1 1

w Image-guided Surgery Mark A. Spicer and Michael L. J. Apuzzo The ability of the modern neurosurgeon to navigate the intracranial contents arises from a detailed knowledge of neuroanatomy coupled with an ability to directly visualize the living brain and its pathology in near real time (1). Startling advances afforded by the modern era of technical achievements in mathematics, physics, and material science have driven phrenology and a murky interpretation of the localized function of the brain into obsolescence. The result has been a better understanding of the submillimeter resolution of cerebral structures and precise delineation of neuronal centers subserving specific neuronal functions. Classically, localization of intracranial pathology relied chiefly on the clinicopathologic correlations obtained from observations of seizure activity. Early preoperative planning and intraoperative guidance were derived from hand-drawn illustrations in anatomy texts that often provided only approximate anatomic guidance. These depictions did not achieve any meaningful measure of accuracy until the advent of routine human dissection in the early renaissance and the rapid global dissemination and subsequent refinement of this information. However, even in the best hands, the localization of intracranial pathology with absolute exactness remained elusive, primarily due to the lack of technologies and methods affording the means by which to image the living brain. This profound limitation prompted Sir William Gowers to comment in 1886 that “The nervous system is almost entirely inaccessible to direct examination. The exceptions to this are trifling.” Dittmar may be credited as having published the first description of a functional stereotactic procedure when in 1873 he described a device for the guided insertion of probes into the medulla oblongata in animals. This procedure likely had a strong basis in sound scientific principles rather than dogma and superstition and represents the beginnings of the concatenation of neurophysiology and functional neuroanatomy. Images produced as a result of the development of penetrating imaging modalities allowed neurosurgeons to visualize the pathology responsible for their patients’ neurologic deficits. These likenesses, however convincing, lacked an essential component for successful neuronavigation, namely the ability to reference the image to and register it in

three-dimensional space. Such studies therefore remained open to subjective interpretation, and the skill and experience of the operating neurosurgeon remained the major determinant in the successful localization and completeness in extirpation of the lesion. Although this represented a significant leap from the craniometric and other empiric methods previously used in the preoperative determination of the lesions spatial relations, synthesis of the amalgam allowing interactive (real-time) image-guided intracranial neurosurgery would have to await the development of magnetic resonance imaging (MRI). Early efforts at preoperative planning for the surgical removal of intracranial tumors were largely focused on the localization of the neoplasm before the performance of a trephination, craniectomy, or craniotomy. Little or no interactive intraoperative neuronavigation was possible given the practicalities of cost limitations and the limited availability of high technology (2). The first widely recognized resection of a primary brain tumor was that performed by Rickman J. Godlee on November 25, 1884 at The Hospital for Epilepsy and Paralysis, Regents Park, London, England. Although the patient subsequently died as a result of postoperative infectious complications, this operation is regarded as marking the advent of modern neurosurgery (2). Although invented by Horsley and Clarke in 1908, the stereotactic frame was not used for surgery on human subjects until 1946 when pneumoencephalography was used to relate landmarks within the brain to anatomic targets (3). Subsequent developments related to the introduction of contrast agents for ventriculographies, the creation of stereotactic anatomic atlases and frames, and improvements in surgical techniques were responsible for dramatic improvements in morbidity and mortality rates related to functional neurosurgery. Thus, the treatment of parkinsonian tremor and rigidity by a nonstereotactic transventricular sectioning of pallidofugal fibers was replaced by Spiegel and Wycis with a stereotactic approach, with a corresponding fall in mortality from 15.7% to 2%. Advances in medical treatments soon began to replace many of the functional neurosurgical interventions. However, the introduction of commercially available computed 155

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tomographic scanners revolutionized the localization of intracranial neoplasms. It was only as a result of technical improvements made in diagnostic imaging, particularly with reference to spatial fidelity, that functional neurosurgery enjoyed a renaissance, most notably in the 1970s and 1980s with the advent of computed tomography (CT) and MRI (4). These revolutionary inventions allowed the use, for the first time, of patient-specific anatomic data, thereby avoiding the problems associated with often-distorted individual anatomy that could not be accounted for using standardized atlases (5). Anatomic atlases were now replaced with patientderived, and therefore anatomically specific, scan slices that also eliminated the prerequisite for the presence of normal anatomy in the region of interest. The introduction of modern computational methods and rapidly expanding capacities for data acquisition and manipulation coupled with the astounding advances in electronic and materials engineering have culminated in an equally rapid development, refinement, and sophistication of approaches to the localization of intracranial tumors. Landmark events in this progression are noted briefly in Table 11-1. This technology has been naturally carried forward in the arena of neuronavigation to enhance the surgeon’s ability to navigate the operative substrate accurately and define and minimize corridors of surgical approach. Classically, even these advances have been somewhat constrained by the limitations of singlemodality imaging and of intraoperative brain shift. Localization technologies have resulted in a combinatorial approach both in the preoperative planning stages and during intraoperative tumor resection.

w TABLE 11-1

ACCuRACY AS THE GoAL oF IMAGE-GuIDED SuRGERY The ultimate goal of image-guided surgery is improvement of both accuracy and precision. Although accuracy is defined as correlating the localization of a point in space as determined by the localizing device with the actual position of that point in space, precision is the ability to return to the same point in space repeatedly and reproducibly within acceptable margins of error. Accuracy may be affected by one or a combination of several factors, some of which will depend on the method (i.e., magnetic vs. optical localization), apparatus (i.e., frameless vs. framed), or imaging modality (i.e., ultrasound vs. CT) used. Reduction in accuracy may be seen as a result of errors in registration. This could be caused either by poor correlation between the position of a fiducial marker on loose skin overlying the skull or by initial inaccurate registration of the fiducial marker. The utility of stereotactic neurosurgery lies in the maintenance of spatial correspondence between the operative instrument and images of the surgical substrate. Traditionally, this was achieved by the use of a stereotactic reference frame being rigidly affixed to the head of the patient. The frame was designed to ensure that the lesion would be contained within the volume defined by the dimensions of the frame. Definition of reference points common to the intracranial contents and the reference frame allows spatial correspondence between the two systems. Applying spherical polar coordinates to the volume of the brain allows the discretization of all intracranial points. The coordinate system is transferred between brain volume and image. Such images may be two- or three-dimensional depictions using a single imaging modality (CT, MRI, angiography) or may be derived from fusions of image data sets. Newer techniques based on image coregistration, fusion, and segmentation for

Landmark Events in the Approaches to the Localization of Intracranial Tumors

Year

Event

1889 1895 later. 1918 1927 1940 1940s 1951 1957 1963 1965 1971 London. 1973

Zernov describes first stereotactic frame in Revue Generale de Clinique et de Thérapeutique. Wilhelm Roentgen discovers x-rays, the “Medical Miracle of the Modern Age.” Fluoroscopy appears about 1 month Walter Dandy introduces air contrast ventriculography. Egas Moniz describes intraarterial contrast cerebral angiography. Gabriel Frank patents back projection computed tomography (CT). Takahashi reconstructs images from sinograms. Wrenn postulates the application of positron emission tomography (PET) to medical imaging. Report of the construction of a medical CT scanner in Kiev. Kuhl and Edwards introduce single photon emission computed tomography (SPECT). Kuhl performs transmission CT. Modern era of CT. Geoffrey Hounsfield develops the EMI scanner, which is used at Atkinson Morley Hospital, Lauterbur demonstrates the generation of two-dimensional images using magnetic field gradients across a sample.

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delineation of neuronal substructures augments the utility of image-guided surgery in that not only are anatomic relationships defined, but functional specialization and activity may also be represented. Chen and colleagues (6,7) pointed out that developmental trends imply realization of the three major directions of technical neurosurgery (8): precise preoperative simulation (9), minimization or avoidance of transcranial operative corridors (8), and increased refinement of technical adjuvants— both physical and molecular (10). A significant body of literature focused on substantiating the early promises of intraoperative neuronavigation, addressing not only the clinical aspects of this issue, but also those related to economic considerations, is beginning to emerge. As an appreciation for the utility of this now almost ubiquitously applied surgical modality is forming, subsets of data concerning comparisons with microsurgical results are being reported (11–14). Radical resection of high-grade gliomas has been shown to have a significant beneficial effect on progression-free and overall survival rates (15–18). Wirtz et al. (19) similarly demonstrated prolonged survival times in a subgroup of patients undergoing radical tumor resection but pointed out that shorter follow-up times in the neuronavigation case subset precludes a final analysis of the data. Many studies have concluded that although not reaching statistical significance, surgical times are almost uniformly reduced (20). However, exceptions to this general rule may occur such as in the instance of complex image segmentation and spatial integration required for the reconstruction of complex neural structures (21). Closer inspection of time savings in neuronavigational cases shows that the added setup time is almost always negated by the reduced surgical time. If personnel trained in the setup of the neuronavigational system are used, the time load of the neurosurgeon does not necessarily increase. Considerable anesthesia time savings may be realized in the case of image-guided biopsy (9,22,23).

PRINCIPLES oF NEuRoNAvIGATIoN WITH FRAMES AND ARMS Frame-based systems allow a point on a two-dimensional image to be translated into a three-dimensional target lesion in the patient. The goal of all frame-based systems is to allow the surgeon a wide range of trajectories from which to access a lesion accurately and reproducibly. The stereotactic frame is usually a rigid metallic platform usually secured to the outer table of the skull with three or four screws. The aiming device most commonly used consists of a high-precision multiply articulated arm that can be moved in various directions so that a sterile probe located at the end of the arm can be pointed at a point of interest that is then mapped to, and displayed on, a (usually) preoperative image of the same region.

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Spatial correspondence between physical objects in three-dimensional space and computer-generated images displayed in a two-dimensional medium was first made possible in the late 1970s and early 1980s (6). This was largely the result of the continued development of the microprocessor that was able, for the first time, to handle the large computational burdens placed upon it by the calculation of the transformation matrices that are required to register physical fiducial markers with image space. Watanabe and coworkers (24) are widely credited as having developed the first example of such a device for use in intracranial navigation. Their system incorporated prototypical elements, refinements of which are now commonly found in modern neuronavigational devices. Generally, this includes a neuronavigator arm with the ability to move with six degrees of freedom (x, y, and z translational movement plus yaw, pitch, and roll), which is then used to define the physical point of interest with concomitant representation in image space. Accuracy of localization depends on considerations related to the fiducial markers as well as those related to the mechanics of the neuronavigational arm. Specifically, accuracy will be increased with type of fiducial, the number of markers used, and their placement. The neuronavigational arm itself is responsible for translating its movement via the use of potentiometers or optical digitizers. To increase the utility of this approach to the surgeon, the neuronavigational arm must be either counterbalanced (much like the operating microscope) or dampened in some way so as not to constrain the natural movements of the surgeon. Although specific neuronavigational arms are not reviewed here, the literature contains many examples of the use of counterweights or springs or pneumatic, hydraulic, or electromagnetic dampers to achieve this goal. The most modern and sophisticated systems use articulated arms with interchangeable bayonet-shaped probes that further minimize cranial and dural exposures (25). Watanabe et al. (26) reported their use of a multiarticulated arm in open cranial surgeries with the adjunctive use of multimodality imaging studies to further enhance the surgeon’s ability to navigate. Most neuronavigational systems based on the use of a framed device with an attached articulated arm are being rapidly replaced by systems that do not require attachment of a frame to the patient’s head. This followed the short-lived attempt to reduce the invasiveness of frame systems. Chief among these were the Gill/Thomas (27) and Laitinen frames. The former used an anodized aluminum frame attached to the maxillary teeth of the patient via a customized dental splint and was further stabilized by two straps that passed over the back of the patient’s head to attach to a plate at the occipital protuberance. The Laitinen frame was positioned on the patient’s head by pressing two earplugs against the external suitor meatus and a nasion support against the bridge of the nose. The position of the stereoadapter for the individual patient is recorded from millimeter

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scales on the nasion support arms and on the connector plate. A CT is obtained, and the stereoadapter can be refitted to the Laitinen frame at the time of a biopsy.

Gamma Radiosurgery The number of cases of intracranial upper cervical diseases treated using stereotactic radiosurgery has increased at a dramatic rate over the last decade. This has been made possible by simultaneous advances in imaging technology, affordability of high-end computational abilities, and better understanding of the response of various intracranial pathologic processes to gamma radiation therapy. Radiosurgery of intracranial lesions is currently accomplished using a wide range of treatment paradigms, including LINAC-based dynamic conformal treatments, static conformal methods, and those based on cobalt (60Co à photon) disintegration. various radiation sources have been used in this regard and include charged particles (e.g., protons) as well photons. These differ in their properties of physics in that uncharged particles such as photons (used in gamma knife radiosurgery) do not produce a single peak of interaction with the pathologic tissue, unlike photons. Thus, treatments relying on uncharged particles as the source of radiation require a superposition of multiple convergent beams. Also, the falloff rate from the dosage isocenter is far sharper with charged particle therapy than with the use of photons.

The Model C Leksell Gamma Knife The model C Leksell (Elekta) gamma knife provides precise irradiation from cobalt 60 disintegration 201 beams converge of intracranial pathology. This has found wide use in the treatment of tumors, ranging from highly microscopically invasive tumors to benign primary lesions, as well as metastatic disease. vascular pathologies are also remediable using this technology (i.e., arteriovenous malformations). This system allows the surgeon to base dosimetry planning on imaging studies (MRI, CT or angiography) obtained with an external reference frame attached to the patient’s head with skull screws. The fiducial rods contained within the frame are then spatially coregistered with reference to the intracranial pathology. Targeting and dosage planning are conducted at a computer console before the patient is taken into the gamma unit for treatment. Highly sophisticated automatic positioning software is used to move the patient around with respect to the position of the treatment beam. Treatment time will vary depending on the size, location, and type nature of the lesion being treated but is usually on the order of 40 minutes. Although still finding broad use and acceptance in the area of stereotactic surgery, framed systems are rapidly being replaced by frameless systems.

PRINCIPLES oF FRAMELESS NEuRoNAvIGATIoN Frameless systems offer several advantages over the framed counterparts. First, possible complications, such as hemorrhage, pain, infection at the pin sites, and intraprocedural slippage of the frame, associated with the methods used for rigid fixation of the frame to the patient’s head are eliminated. other advantages include less encumbered surgical access, ease with which adjunctive imaging devices such as ultrasonic and transcranial Doppler (TCD) probes may be brought into the operative field, and ease with which an operative microscope may be maneuvered. Frameless devices may be either linked or nonlinked. Linkage refers to the means by which the positional probe communicates with positional detectors. The neuronavigator system represents an example of a frameless linked system (due to the attachment of the articulated arm to the reference arc), whereas the vectorvision BrainLab® system is a nonlinked system.

Frameless Linked Systems The NeuroNavigator is a multijointed articulated arm capable of translating the location of its arm tip within the surgical field to imaging coordinates. It is made up of a personal computer, multijoint sensing arm, and image scanner. The sensing arm has six joints with six degrees of freedom, each of which is equipped with a high-resolution potentiometer. The three-dimensional coordinates of the arm tip are calculated using the angle of each joint and length of each arm segment. Preoperative calibration by touching three points on the patient’s head with their corresponding points on CT and MR images using the arm tip (point matching technique) is crucial for proper functioning and accuracy. Multiple models, including the Watanabe potentiometer, Codman Acustar, and oulu neuronavigator, have been in development since the 1980s. Watanabe first described the use of a six-joint sensing arm that was attached to a Mayfield pin headrest. The pointing tip could be shortened to a specified length, creating a “virtual tip” that still indicated its original location. Because this point exists only in virtual space, it can be introduced into the tissue before actual tip introduction. If the virtual tip has reached the tumor, this means that the tumor will be found at a specified known length ahead of the actual tip. The surgeon can then easily track the tumor edge with the virtual tip while designing the proper position and size of the craniotomy. The system is also capable of interacting with other imaging modalities, making it useful in locating blood vessels using angiograms and MR scans. Watanabe reported this instrument’s use in the treatment of 35 patients with gliomas. The efficacy with regard to patient outcome has not been analyzed. Real-time localization may be obtained somewhat in this system by a combination of ultrasound imaging and TCD. An ultrasound probe may be attached to the tip of the pointer, allowing comparison of echo-

11. Image-guided Surgery

graphic and CT images. This has helped in determining whether significant intracranial structure shifting has occurred secondary to the craniotomy. The addition of TCD helps to locate intracranial vessels that are not well demonstrated on the preoperative CT images. Localization of a vessel segment in this way has demonstrated accuracy within 2 mm. The ISG viewing Wand System (ISG Technologies) is a currently available u.S. Food and Drug Administration approved device that also makes use of an articulated arm, acting as a three-dimensional digitizer, and sophisticated computer graphics workstation. The three-dimensional images may be represented in several different ways to give a comprehensive display of the involved anatomy. This system also relies on preoperative imaging; however, external fiducial markers are not necessary. Rather, the computer uses reproducible anatomic landmarks such as the eyes and nose to process targeting information. The average error of the system in testing has been between 1.5 and 2.5 mm. A component of real-time imaging is provided by combining an ultrasound probe with the tip of the viewing wand. various instruments, including an endoscope, may be introduced through the modified tips of the articulated arm (13). Koivukangas et al. (28) introduced a similar articulated arm system. However, this system makes use of ultrasound in a routine arrangement to make real-time confirmations and comparisons between the open intracranial and the preoperatively digitized intracranial environment. This system provides an improved sense of real-time information. The articulated arm also can be fitted with various types of adaptors at the end of the probe to attach various instruments. The MKM model microscope produced by Zeiss has stereotactic capability. The principle of operation is much the same as with the neuronavigator, with the microscope head functioning in a similar manner to the neuronavigator tip. Again, preoperative acquisition of imaging data using fiducial markers placed on the patient’s head is necessary to synchronize the microscope to the imaging obtained. The computer sends a signal to the microscope that translates into a correction of the microscope trajectory so that it remains fixed on any preselected target within the intracranial volume. Thus, the microscope acts as its own navigator, always keeping the surgeon directed toward the target volume along a selected trajectory no matter how the surgeon adjusts the microscope. Although the articulated arm has been the most well-developed frameless system, it has several disadvantages. It is often cumbersome to use, and its tip position is limited by the length of the arm segments. Also, it is impossible to adapt the arm so that it has the flexibility and feel of an instrument. Finally, increasing miniaturization and accuracy of the arm will increase its cost of production significantly.

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Frameless Nonlinked Systems Roberts was the first to use a sonic-based principle for his operating microscope. This system uses spark gap generators to produce an ultrasonic frequency signal that is received by a receiver panel mounted above the microscope. The signals are then converted into spatial information regarding the relationship of the microscope to the patient’s head. The three-dimensional information from the preoperative CT or MR scans is then spatially registered with the operating microscope. outlines of the tumor margin are directed via a beam splitter back through the microscope to be superimposed on the operative field. This provides the surgeon with a comparison between the actual tissue appearance and the boundaries of enhancement or to the imaging characteristic as seen on preoperative studies. This arrangement, although demonstrated to be reasonably accurate, does not compensate for any gross shifts of the intracranial contents that may occur intraoperatively. From Roberts’ initial sonic design for the operating microscope, Barnett and coworkers developed a frameless, armless, sonic wand, with four components: a hand-held probe with two ultrasonic emitters, a microphone array rigidly fixed to the operating room table in proximity to the surgical field, hardware to control and detect timing of signal production and reception, and a color graphics computer workstation with software to calculate and present the location of the probe tip on reconstructed neuroimaging studies. The localization works on the premise that ultrasonic “pulses” are sequentially emitted by two emitters on the probe. Spatial localization of the probe tip is based on the time delay between pulse emission and detection by at least three microphones, enabling the calculation of the distance between the wand and microphones. Although Barnett cited reproducibility in the operating room of ⫾0.6 mm, accurate localization of the sonic wand depends on unobstructed “line of sight” between emitters and detectors, local temperature changes and humidity, sonic echoes and reflections (i.e., suctioning that may hinder performance of the sonic wand), and positioning of emitters in the zone of optimal detector sensitivity. These considerations aside, the wand is lightweight, unencumbered, and hand held. Another type of frameless nonlinked system uses infrared light in optical digitizers. Both Zamorano and Bucholz and colleagues designed optical digitizers using infrared light. In this system, light-emitting diodes (LEDs) are attached to an instrument. A camera array is used to detect the position of the LEDs. Infrared light is frequently used for its maximal accuracy, decreased sensitivity to ambient light, and minimal emitter size. Multiple LEDs also can be placed on different instruments, each with unique intraemitter spacing, allowing the software to recognize not only the location of the instrument but also the type of instrument being used. Disadvantages of an optical system include a clear “line of sight” between the diode emitters and the camera system and interference with

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operating room infrared sources such as lights. Zamorano cited accuracy in locating the spatial position of an LED of 0.1 mm with a resolution better than 0.01 mm. vectorvision represents the MRI-based interactive and real-time neuronavigational tracking system marketed by BrainLab. This system is based on an improved wireless implementation of the optical digitizer method presented above. Now widely in use, Gumprecht and Lumenta (29) and Muacevic et al. (30) reported their early success using this system in endoscopic procedures, stereotactic biopsies, and tumor resections with a reported average accuracy approaching 4 ⫾ 2 mm. No complications were reported. use of the Stealth NeuroStation (Medtronic-Sofamor Danek) by Fahlbusch et al. (31) illustrated application of intraoperative neuronavigation with subsequent intraprocedural updating to account for positional inaccuracies. Such inaccuracies are derived not only from those inherent to the instrument itself, 0.55 ⫾ 0.64 mm (32), but also those resulting from intraoperative brain shift (33). Systems based on the use of magnetic digitizers represent the final major class of nonlinked frameless neuronavigation systems. Kato and colleagues designed a small magnetic field sensor that is attached to various instruments, including a resin probe, a triangle-shaped pointer, or a suction tube. A three-dimensional digitizer using a low-frequency magnetic field is used to detect the three-dimensional coordinates of the small magnetic field sensor. A major drawback with magnetic digitizers is the need for a precise magnetic field to maintain accuracy. Ferromagnetic substances, aluminum, and electromagnetic radiation may all distort the magnetic fields. Kato reported an error of ⫾4 mm. Sonic and ultrasonic digitizers mark the early beginnings of the use of armless intraoperative neuronavigation systems. Both systems rely on the detection of an emitted waveform with subsequent determination of emitter position by the relative delay between the emitter and each of the detectors. In the former case a piezoelectric emitter was typically used to produce a sonic waveform, which was detected by at least three microphones, whereas in the latter case, the position of an ultrasonic source was localized by ultrasonic range finders. Both of these systems suffered from considerable positional errors given the dependence of time of flight (and therefore localization accuracy) on various physical parameters, including air temperature, thermal convection, sonic/ultrasonic echoes, and ambient noise levels, but have undergone significant improvements since their initial introduction with marked improvement in accuracy and precision. Magnetic fields overlaid on the operative field avoided the problem of having a clear line of site between the emitter source and the detector. However, static field inhomogeneities created by the proximity of ferromagnetic substances to the field were noted to produce significant errors in positional determination. Surgical instruments, operating

room tables, and machinery could cause such field inhomogeneities (34), and this has resulted in the limited development and use of this type of system. optical digitizers based on an infrared system track LEDs using an array of charge coupled detector triangles that triangulate the position of the diodes. Although these offer significantly improved positional accuracy (in the absence of stray intraoperative infrared radiation), this is accomplished at the expense of having to use more complex and therefore costly equipment. Registration of the localizing device is achieved by its registration within the confines of a fixed reference frame or arc. All these methods have shown greatest utility when used for preoperative planning to minimize proposed craniotomy size while optimizing the radicality of tumor excision. The results obtained both with and without external frame preregistration do not support the use of digitized localizers for positional reference as an intraoperative standalone. The utility of these systems is maximized when used in conjunction with diagnostic imaging studies and appears to be optimal in endoscopic surgery of intraventricular or intraparenchymal cysts and for third ventriculostomies. High spatial fidelity between frameless neuronavigation and those using frames was demonstrated by Hopf et al. (35) and particularly in patients in which the ventricular anatomy is distorted or unusual, where the foramina of Monroe are small, in conditions of poor visibility, and where there needs to be precise control of the endoscope trajectory for the completion of an atraumatic procedure (29,36). Gronningsaeter et al. (37) reported their experience using a frameless and armless system (SonoWand) with both MRI and ultrasonic guidance. They found that the accuracy achieved with intraoperative ultrasound guidance was similar to that found with preoperative MRI but acknowledge the need for further refinement of their pioneering work.

Linear Accelerator-based Systems Novalis incorporated features of the Leksell gamma radiosurgical unit, such as the ability to perform image-guided stereotactic irradiation of pathologic tissue. However, instead of attempting to treat the lesion with multiple spheroid or ellipsoid shaped doses, the Novalis allows beam shaping. A principal advantage of this technology is that it allows greater dose conformation (shaped beam) and accurate dose delivery at a depth with a relatively low scatter factor. This approach theoretically maximizes the tumor treatment volume while minimizing irradiation of nonpathologic tissue. The Cyberknife, manufactured by Accuray, represents a new approach to image-guided radiosurgery in that it incorporates the use of a lightweight linear accelerator mounted on an arm that is able to move with multiple treatment degrees of freedom. For most cases the bony landmarks of the patient’s body provide the reference fiducial markers, thereby obviating the need for the placement of an external

11. Image-guided Surgery

reference frame. Another advantage conferred by this system is its ability to treat pathology in parts of the body that may not be accessible using other stereotactic radiosurgical devices (10,38–41), thus allowing the treatment of intracranial and extracranial lesions up to 6 cm in size. The Cyberknife uses a proprietary Image Processing System that evaluates patient position during treatment and is able to account for patient motion and can track the lesion, thereby maintaining spatial registration.

SCoPE oF APPLICATIoN Image-guided surgery is a now widely accepted adjunct to surgical planning and intraoperative guidance and has found application in almost all forms of surgery, particularly in major academic centers. Surgical subspecialties making extensive use of image guidance in the preoperative and/or intraoperative arenas and well represented in the current literature include craniofacial, plastic, orthopedic, neurosurgery, and general surgery, among others (42–47). As well as the surgical subspecialties being well represented, individual procedures specific to each specialty comprise several thousand literature citations. An exhaustive review of these is beyond the scope of this chapter.

SYSTEM LIMITATIoNS Preoperative Image Registration Although affording a higher degree of confidence in the localization of intracranial pathology, whether intraoperative or preoperatively, image-guided surgical techniques suffer from a number of potential disadvantages. Among these are the time required for preoperative registration and patient preparation, time to set up the equipment in the operating room, and intraoperative brain shift. Most image-guided systems require the preoperative placement of fiducial markers on the patient’s skin near the region of operative interest before the acquisition of localizing imaging studies. These may be in the form of the Z-bar configuration found in most stereotactic frame systems or may take the form of markers adherent to the skin. In the former case, the patient is taken to the operating room before being lightly sedated. After preparation of pin sites on the patient’s forehead and occiput, a suitable amount of local anesthesia is given and the skull screws are then applied, thereby rigidly securing the frame/fiducial system to the patient’s skull. This procedure usually adds about 30 minutes to the overall procedure. Fiducial markers may also be applied directly to the skin and should be positioned in such a fashion as to delineate the region of surgical interest in three dimensions. Application of the markers requires shaving the patient’s hair at positions

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where the markers are to be placed. Compared with the placement of a stereotactic frame, the latter method has several advantages. The use of the operating room or sedation/anesthesia is not required, and application of adherent fiducial markers is painless and may be done well in advance of the proposed surgical procedure. Care should be taken to mark the position of the skin-applied fiducial markers so that in the event they should be inadvertently removed after the localizing MRI, their position on the scalp may be easily determined. Many image guidance systems are multicomponent arrangements requiring set up in the operating room over and above that required for the surgical procedure itself. Workers have reported that the time added to surgical cases because of the additional setup time required ranges from being negligible to about 20 minutes and tended to decrease with increased experience of the people setting up the equipment. Wirtz et al. (48) compared three neuronavigational devices, viewing Wand, SPoCS and a microscope integrated system (MKM), and found that the mean time for preoperative registration was 23 ⫾ 13 minutes for the viewing Wand, 21 ⫾ 16 minutes for the SPoCS system, and 27 ⫾ 22 minutes for the microscope integrated system.

Intraoperative Brain Shift As described in the preceding section, many neuronavigational systems use the skull (or fiducials placed in close proximity to it) as a reference for translation of image data to physical space. This requires that a constant relationship be maintained between the time the localizing image data are acquired to the time the intracranial procedure is complete. It is well known that the degree of brain shift may vary significantly depending on the presence of edema, depth of the tumor, lesion volume, preoperative midline shift, and the magnitude of shift of the cortex on dural opening. Dorward and colleagues (49,50) conducted a study to quantify brain shifts during open cranial surgery to determine correlations between these shifts and image characteristics. They found that the mean shift of the cortex after dural opening was 4.6 mm, shift of the deep tumor margin was 5.1 mm, and shift of the cortex at completion was 6.7 mm. Meningiomas displayed significantly greater shift at depth compared with gliomas with significantly less shift in skull base cases than other groups and in many cases could be neglected making potential value of neuronavigational devices in the latter group uncertain (51). ostertag and Warnke (52) pointed out that alternative therapies with proven and equivalent efficacy and reduced morbidity are available for the treatment of skull base tumors and that the anatomy of the tumor makes neuronavigation unnecessary. Given that radicality of resection and complication avoidance related to resection of nonpathologic structures depends largely on the intraoperative accuracy of any neuronavigational system used, it is of paramount importance that the problem of brain shift be minimized. Two principal methods do this: intraoperative referencing using specific image

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modalities with real-time updating of neuronavigational data, thereby accounting for brain shift, and utilization of spatial corrections based on computational algorithms. The greater spatial fidelity afforded by CT when compared with that afforded by MR images has been well described. These inaccuracies are usually the result of magnetic field distortions caused by susceptibility artifacts, static field inhomogeneities, and peripheral magnetic field warping. MRI, however, unquestionably delivers a far greater level of tissue discrimination in most instances than that afforded by CT. Given the dichotomy of these properties, but the recognized need for the optimization of both, software algorithms have been developed allowing combination of the anatomic definition of MRI with the spatial precision of CT (53). Although this combination approach optimizes the initial spatial discretization, problems with intraoperative accuracy are still encountered as a result of brain shift. The benefits of intraoperative image guidance include the ability to update data sets for the navigational system, control of the radicality of brain tumor resection, and image guidance of secondary procedures such as placement of brachytherapy or time-release chemotherapeutic devices. of the diagnostic imaging techniques available, MRI undoubtedly provides the greatest tissue discrimination. Its use in the operating environment, however, is somewhat limited by the presence of a magnetic field. Traditionally, acquisition of intraprocedural images often meant lengthy transport of the patient to the site of the MR machine. This has more recently been replaced by emerging techniques that depend on MRI of the patient in the operating room. This in situ method, however, usually requires the use of surgical instruments constructed of specialized nonferromagnetic substances (e.g., ceramics). Another limitation is that of space, as both the surgeon and the patient must occupy the volume enclosed by the magnet. An elegant solution to both of these problems has been reported by Nimsky et al. (36,54) and consists of placing the patient with their head approximately 1.5 m from the center of the magnet, fixed in an MRI-compatible ceramic head holder. Intraoperative data acquisition is achieved by sliding the patient into the magnet’s core field, thus allowing updating of intraoperative guidance images. Advantages of this system include minimization of the need for specialized equipment, use of an operating microscope in the fringe magnetic field, and no requirement for intraoperative transport of the patient. In addition to finding application in the determination of displacement of the nonpathologic intracranial contents by lesional processes, ultrasound is finding relevance as a means by which to provide dynamic tracking of intraoperative brain shift (55–60). Progressive corruption of intraoperative spatial data obtained by coregistration of fiducial markers with preoperative imaging data has been well documented. Traditionally, attempts to overcome or minimize this source of overall inaccuracy centered on noncom-

putational methods such as avoidance of postimaging osmotic diuresis, positioning of the craniotomy in the horizontal plane, and tracing intraoperative brain shift by tracking of radiopaque markers placed within the brain tissue. With the ever-increasing spatial fidelity provided by imaging modalities such as CT, ultrasonography, and MRI, there has been renewed effort directed at solving this problem based on high-speed computational algorithms. Although correlate cortical shift measurements with model predictions does not currently account for factors other than gravity, which may be responsible for intraoperative cortical shift (diuresis, altered blood flow, intracranial air), they report significantly less misregistration error from an average of 5.7 mm in the direction of gravity to an average of 1.2 mm. In a study designed to determine the influence of patient head positioning on the degree of intraoperative localization error, Reinges et al. (61) acquired preoperative MR scans on 24 patients who later underwent pterional craniotomy. Positional correlation between points on previously selected surface cortical features was carried out intraoperatively with the patient’s head constrained in a Mayfield head holder. It was found that the positioning of the patient’s head was not a significant factor when applied to intraoperative brain shift, although the authors conceded that further work is needed in this area.

Image Segmentation, Multimodality Imaging, and Data Set Fusion The presence of intracranial pathology, more often than not, will cause distortion of the normal anatomic relationships. This is of particular significance proximal to the pathology where spatial displacement is likely to be greatest and therefore potential consequences are maximized when the lesion is adjacent to eloquent neural structures. An obvious example of this would be a posterior frontal lobe meningioma causing displacement of the motor strip. Preoperative planning in such cases will often rely on functional MRI to delineate the location of the displaced motor strip. This approach, however, is limited in that the location of the deeper white matter of the corticospinal tract cannot be appreciated. Additional information may be derived from three-dimensional representation of such tracts and is undoubtedly useful and has been reported (21,62). Jannin et al. (63) used a technique relying on the derivation of anatomic detail from multimodal imaging. Thus, the intracranial arterial vasculature was defined from MR angiography images, whereas brain cortex, parenchyma, and lesional areas were delineated with MRI, and functional areas were defined using MEG and functional MRI. Subsequent image fusion allowed the construction of three-dimensional computer models in the neuronavigational system and was found to be a useful adjunct in the surgical procedure. Disadvantages of this approach were the length of time required for the segmentation

11. Image-guided Surgery

procedure, the lack of the ability to interactively (and intraoperatively) update the position of the pyramidal tract, and brain shift. Consideration of the combined data provided by MRI and CT studies is adequate in most neurosurgical procedures for completion of the surgical procedure and allows good to excellent intraoperative guidance. However, a higher level of safety and confidence in the proposed surgical remediation of intracranial pathology in which there must be consideration of the location of eloquent neural structures (such as the motor or sensory cortices) may be realized by using multimodality imaging techniques. Imaging data fall principally into two major subsets, structural (MRI, CT, DSA) and functional (positron emission tomography [PET], single photon emission CT [SPECT], MEG, MR spectroscopy), giving rise to three nonredundant combinations, namely structural–structural, structural–functional, and functional–functional. All three combinations have been used in the identification, quantification, and treatment of surgically remediable and nonsurgically remediable disease processes. Notable among the latter are the use of MRI/SPECT/CT in the quantification of substantia nigra neuronal degeneration in Parkinson disease (64), MRI/SPECT in Alzheimer disease (65), and MRI/MR spectroscopy in the correlation between neuronal loss and clinical disability in multiple sclerosis (66). MRI and MR angiography/MRv are best suited to imaging of brain tissue and intracranial vasculature, respectively, whereas bony structures are best imaged by x-ray–based techniques. To optimize the preoperative assessment of many intracranial lesions, a combinatorial approach to this imaging data is required. Most processes amenable to surgical intervention use imaging combinations of structural–structural data. Gandhe et al. (67) provided an early example of this in their 1994 report of eight patients with “cranial base lesions” who underwent MRI, MR angiography, and CT investigations with subsequent coregistration of the images to internal anatomic landmarks rather than an external frame. They reported that their technique resulted in more accurate intraoperative interpretation of the anatomic structures and their intracranial relationships. Determination of the location of intracranial arterial vasculature was achieved by Watanabe et al. (26) using a TCD flow probe mounted to the end of a multiarticulated arm. This arm, together with a personal computer and imaging scanner, forms a device known as the neuronavigator. When the TCD probe is placed at the patient’s head, the system then translates the measuring point of the TCD flowmeter into imaging coordinates, and the navigator immediately displays the location of the measuring probe and projects it on the corresponding CT/MRI image. In their series of 68 patients, they reported a detection error of 2.5 mm when the neuronavigator was used.

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Further, the localization of intracranial pathology may not be apparent using standard MRI or CT techniques. Levin et al. (68) reported their use of combined MR and PET in the construction of a three-dimensional computer model of the brain surface. Localization of the precentral, postcentral, Broca, and Heschl gyri were determined from the MR data and were fused with averaged PET data depicting metabolic abnormalities. The predicted relationships were confirmed intraoperatively by means of visual inspection of the brain and by electroencephalography. Schreckenberger et al. (69) used 18 F-fluorodeoxyglucose PETs overlayed on MR images in their resection of a right precentral chondroma without postoperative neurologic deficit, whereas Braun et al. (70) described the coregistration of data obtained from T1-weighted MRI with that of functional MRI and PET in a patient with a left precentral oligodendroglioma not well defined on MRI alone. The data sets were integrated by landmark matching with reference to skin fiducials with an overall navigation accuracy of 1.2 mm. Intraoperative somatosensory evoked potentials were used intraoperatively to verify the location of the precentral gyrus. Resection using this combinatorial approach yielded gross total resection of the tumor without postoperative functional deficits.

BENEFITS A review of the literature demonstrates the almost ubiquitous benefits to be derived from image-guided surgery when compared with non-neuronavigational standard surgical techniques. These benefits are realized in the areas of cost savings, reduction in surgical morbidity, greater radicality in resection (particularly of glial tumors) and increased survival (18), decreased intensive care unit days, decreased overall length of stay in the hospital, and reduced surgical time. These benefits apply not only to adult procedures, but also to those performed on pediatric patients (23). Neuronavigation allows intraoperative localization of eloquent structures as well as the identification of structures that if seen early in the course of surgery allow the avoidance of complications. This is reported by Muacevic and Steiger (43), who described the identification of feeding arteries and draining veins in their resection of an arteriovenous malformation. Definition of a suitable operative corridor was determined using preoperative imaging data, affording an optimal approach to the feeding arteries while avoiding eloquent areas of brain. Elias et al. (71), in a multifactorial examination of the benefits afforded by the use of neuronavigation in the performance of frameless stereotaxy for transsphenoidal craniotomies, found an overall additional cost saving to the patient of uS$318.00. The cost savings due to overall reduction in total case time were offset by the additional cost of setup time in the operating room and by the cost of the preoperative computed tomographic study required for fiducial registration. They

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found no complications attributable to inaccurate localization from the neuronavigational system. others reported similar findings for pituitary surgical techniques (72). Although they noted an increase in setup time (12 minutes), the overall case time was reduced from 133 minutes to 127 minutes (not statistically significant). In a study comparing conventional surgery to image-guided surgery to the resection of meningiomas, Paleologos et al. (20) reported a group of 100 patients undergoing image-guided surgery for meningioma resection compared with a group of 170 patients in which standard surgery was performed; an overall cost savings of 20% per patient was shown. This financial benefit was gained from a consideration of the reduced operative time, reduced number of days in the intensive care unit (1.7 vs. 1 day), reduced hospital stay (13.5 vs. 8.5 days), and lower complication rate (14% vs. 6%) in the image-guided surgery group. In an analysis of the impact of image-guided neuronavigation on efficiency and workflow (73), it was reported that cost savings are further increased by using a “closed system” in which a computed tomographic scanner located in the operating room was draped and used for intraoperative image guidance with updating to account for brain shift. This approach obviates the need for transportation of the patient intraoperatively and was associated with a savings of 55 hours per operating room over a 6-month period. others have reported image-guided neurosurgery to be time neutral, particularly as applied to general neurosurgical procedures (22); however, time savings in anesthesia (not overall surgical) time in the performance of stereotactic procedures such as biopsy were reported. There are currently no reports in the scientific and technical literature implicating the use of neuronavigational devices in increased cost, increased surgical time, or complication rates exceeding those associated with standard surgery.

SuMMARY AND PRoSPECTuS Just as phrenology served as a precursor to stereotactic methods that in turn gave rise to intraoperative imageguided surgery, so have the latest techniques of neuronavigation begun to serve as a foundation for real-time interactive neurosurgical, simulation allowing not only more detailed preoperative assessment but also procedural and operative rehearsal (74–76). Framed systems, once almost ubiquitously used, gradually have given way to less intrusive and less cumbersome frameless systems. Stereotactic surgery remains the notable exception to this observation and still widely uses frame-based systems. The transition from frame-based to frameless systems has been greatly accelerated by concomitant advances in both imaging and computational technologies. These developments have allowed more accurate and precise localization of pathologic and normal intracranial, and now extracranial, entities. As processor speeds continue to increase and imaging modalities

offer higher resolution images from physically smaller MRI, CT, and ultrasound scanners, real-time interactive updating of intraoperative localization will become more commonplace (77–80). That the developmental trend toward immersive virtual reality surgical simulators has already begun is evidenced by the emergence of systems allowing for the placement of virtual ventricular catheters using frameless neuronavigation (81) as well as the early development of virtual dissection programs (82). These systems remain in the early stages of development and require substantial improvements in graphic rendering of tissues, haptic (tactile) feedback, artificial intelligence coaching functions, and interactivity. Nonetheless, given the continued exponential rate of advancement in technologic and computational arenas over the last couple of decades, patients and doctors will likely soon derive further benefits from virtual reality expert-system based environments.

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c h a p t e r

1 2

w Principles of Brain Tumor radiotherapy Annie W. chan

Important advancements have been made in the field of brain tumor radiotherapy in the past decade. These advancements are a result of the discoveries and refinements of various stereotactic irradiation techniques; advances in neuroimaging and dose-planning software, which maximize target delineation and minimize dose to normal tissues; and availability of treatment outcome and toxicity data in defining the type of radiation techniques and their respective doses and fractionation schedules. Although there are radiobiology concepts and guidelines that govern the current treatment of brain tumors, the concepts and practice of brain tumor radiotherapy are constantly evolving. In this chapter, we review the principles and rationales of brain radiotherapy. Emphasis is on the relative indications of different radiation therapy techniques: conventional radiation techniques, stereotactic radiosurgery, stereotactic radiotherapy, and proton beam therapy. Important and recent clinical studies on the treatment of benign and malignant tumors are summarized in this chapter. Toxicity related to radiation is also discussed.

SElEcTIon of IrrAdIATIon TEchnIquES General goals of brain tumor radiotherapy, regardless of the types of radiation modalities, are to achieve tumor control and minimize acute and long-term toxicities. Selection of the appropriate irradiation techniques and dose fractionation schedules are critical to achieving these goals. Table 12-1 provides a general guideline for the use of radiotherapy for some of the common brain tumors. The type and the technique employed depend on the type and behavior of the specific tumor. In general, fractionated stereotactic radiotherapy is recommended for most benign brain tumors. Stereotactic radiosurgery is used for well-demarcated malignant tumors, such as brain metastasis, and for benign tumors, such as functioning pituitary tumors, that require high biological dose. conventional radiotherapy is used for malignant tumors with infiltrative borders, such as glioblastoma multiforme.

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w TABlE 12-1

common radiation Techniques used in the Treatment of Brain Tumors concepts and Techniques

Indications

comments

conventional radiotherapy

daily small dose over weeks Broad dose gradient no limit in the size of target

high-grade glioma Whole-brain radiation

Many years of experience dose and normal tissue tolerance are well characterized A larger amount of normal tissue received high dose of radiation compared to stereotactic radiotherapy

Stereotactic radiosurgery

A single large dose Sharp dose gradient Small volume of target Prototype: Gamma knife, lInAc, proton, and Brainlab

Arteriovenous malformation Boost after whole-brain radiotherapy for brain metastasis functioning pituitary adenoma Small vestibular schwannoma Small meningioma

lesions involved with or intrinsic to critical structures, such as optic apparatus or brainstem, are contraindicated lesions that are greater than 3 cm in diameter are contraindicated

Stereotactic radiotherapy

dose and fractionation are the same as conventional radiation Sharper dose gradient compared to conventional radiotherapy Small to intermediate volume of target Prototype: lInAc, Brainlab, proton, and cyberknife

Vestibular schwannoma nonvestibular schwannoma Meningioma craniopharyngioma functioning and nonfunctioning pituitary adenoma

A larger amount of normal brain tissue received low dose of radiation compared to conventional radiotherapy techniques

hypofractionated Several fractions of moderate Stereotactic dose of radiation radiotherapy dose and fractionation schedules between those of stereotactic radiosurgery and radiotherapy techniques

high-grade glioma Brain metastasis

limited clinical experience use in the treatment of benign brain tumors such as vestibular schwannomas is investigational

conventional radiotherapy

Stereotactic radiosurgery

conventional radiotherapy is the delivery of fractionated daily radiation, usually for 6 to 6.5 weeks, generated by highenergy linear accelerator. Modern radiotherapy requires accurate and reproducible immobilization for treatment planning and delivery of radiation. for conventional radiotherapy, this is achieved through the use of customized aquaplastic facemasks, made with the patient in the treatment position. In the past, two-dimensional image sets as defined by bony anatomic landmarks were generated using fluoroscopy and plain film radiographs. Tumor volumes were then manually transferred from diagnostic imaging studies to simulator radiographs. With the availability of computed tomography (cT), three-dimensional data sets of the areas of interest are generated, and radiation portals are developed through the use of the beam-eye-view approach. Treatment films are generated as digitally reconstructed radiographs. The cT images also contain an electron density data set, allowing the accurate generation of radiation dose distribution data by the treatment planning software.

Stereotactic radiosurgery (SrS) is the delivery of a stereotactically directed, highly focal, and large single dose of radiation to a small target. SrS can be done using interstitial techniques such as brachytherapy, intraoperative electrons, or low-energy X-rays. It can also be done using external stereotactic techniques such as Gamma Knife© (1–3), charged particles such as protons (4,5), linear accelerator (lInAc-) based SrS (6–8), or miniature linear accelerator with robotic arm such as cyberknife (9). Invasive stereotactic head rings (figure 12-1a) are used for treatment because impeccable accuracy is necessary. The concept of stereotaxis is involved in most of these techniques, which relates a patient to an image data set with a mathematic coordinate system. When radiosurgery is limited to lesions that are 3 cm or less, dose falloff is sharp, and a small amount of normal brain tissue receives a high dose of radiation. Gamma knife and most lInAc-based SrS use multiple arcs to treat the target volume. for a circular target, a single isocenter with a single circular collimator allows high conformity. for irregularly shaped lesions, multiple isocenters are necessary to improve conformity.

12. Principles of Brain Tumor radiotherapy

A

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b

fIGurE 12-1. A. A patient undergoing stereotactic computed tomography (cT) imaging. The cT localizer is attached to the BrW head ring, which is attached to the patient’s skull through four pins. B. Gill-Thomscosman relocatable noninvasive head ring for stereotactic radiotherapy (SrT).

In contrast to Gamma knife and most lInAc-based SrS, the depth-dose distribution of proton beams allowed the use of a relatively small number of fixed beams to produce dose distributions similar to those of Gamma knife or lInAc radiosurgery. The lowered dose received by the normal brain, as a result of the use of a few beams in proton, may be of significance in reducing acute and late toxicities as well as the incidence of second cancers. With longer follow-up, second cancers, particularly malignant tumors, have been reported in patients treated with radiosurgery. Malignant tumors, mainly glioblastoma multiforme, have been reported as early as 6 years after radiosurgery (10). Benign tumors have been reported to develop between 16 and 19 years later. Tumors developed both within the fulldose prescription volume and in the lower-dose periphery (11). The overall incidence seems to be quite low. however, continued surveillance of treated patients is necessary.

Stereotactic radiotherapy Stereotactic radiotherapy (SrT), also known as fractionated stereotactic radiotherapy, can be thought of as a hybrid between conventional radiotherapy and SrS. It is the delivery

of stereotactically directed and highly focal conventionally fractionated irradiation (1.8 to 2.0 Gy per day for a full course of 5 to 6 weeks). SrT provides similar precision in target localization, patient immobilization, and dose distribution as is obtained in SrS. SrT combines the precision of stereotactic positioning with the radiologic biologic advantage of fractionation. The biologic effectiveness of conventionally fractionated stereotactic radiotherapy is believed to be the same as conventional radiotherapy. however, the toxicity and complication rates are significantly lower because there is decreased volume of normal tissue in the moderateto-high dose range. figure 12-2 demonstrates the conformal superiority of SrT technique compared to conventional irradiation techniques, thereby sparing the temporal lobes and other critical structures from high-dose radiation, in a patient with benign meningioma of the right cavernous sinus. The lInAc-based system (6–8), charged particles (4,5), and cyberknife (9) all have the capability to perform SrT. relocatable noninvasive stereotactic head rings are used for daily treatments (figure 12-1b). Some institutions employ frameless stereotactic devices by the use of an opticguided system. SrT is not feasible with Gamma knife because of the impracticality of daily insertion of an invasive head ring and the limitation of small field sizes. The immobi-

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lization errors for the current relocatable frames are reported to be in the range of 0.5 to 3.0 mm.

hypofractionated Stereotactic radiotherapy Whereas SrT uses conventionally fractionated fractionation schemes (i.e., 1.8 to 2.0 Gy per day for a full course of 5 to 6

weeks), hypofractionated stereotactic radiotherapy (h-SrT) delivers the total dose in an unconventionally fractionated schedule. A variety of fractionation schedules are currently employed, such as 25 Gy in five consecutive daily fractions (12), 20 Gy in weekly doses of 4 to 5 Gy (13), 24 Gy given three times a day in one day (14), and so on. The rationales for different fractionation schedules employed are based on biomathematic models. Because the comparison and quan-

A

b

c

d

fIGurE 12-2. dosimetric comparison of three different irradiation techniques in a patient with benign meningioma of the right cavernous sinus. (A, d) Two-dimensional radiation treatment with a pair of parallelopposed lateral fields. (B, E) Three-dimensional conformal radiation with four noncoplanar fields. (c, f) Stereotactic radiotherapy (SrT) with six noncoplanar arcs. The optic nerves, chiasm, orbits, and brainstem are outlined. The percentage isodose lines are illustrated. note that the parallel-opposed lateral fields, three-dimensional conformal radiation, and stereotactic radiotherapy result in maximal dose to the temporal lobe of 122%, 30%, and 5% of the prescribed dose, respectively (arrows).

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12. Principles of Brain Tumor radiotherapy

e

f

fIGurE 12-2. (conTInuEd)

tification of the biologic effectiveness of these different fractionation schedules are not feasible, one must rely on clinical data to support the use of h-SrT. however, there is limited clinical experience in the use of h-SrT. This technique, therefore, should be considered somewhat investigational for the treatment of benign tumors and inoperable vascular malformation. for malignant tumors that are too large for radiosurgery boost (Table 12-1), the use of this technique allows good palliation of symptoms with limited acute toxicity.

IMPorTAncE of frAcTIonATIon In order to minimize toxicity, one must understand radiobiology of the nervous system, the radiation tolerance of normal structures, and the appropriate use of radiation techniques and fractionation. Ionizing radiation is the type of radiation used in radiation treatment. It interacts with water, which produces reactive species causing changes and injuries at the cellular and molecular levels. one of the most important targets is believed to be the deoxyribonucleic acid (dnA). The difference in cellular sensitivities of normal tissue and tumors form the basis of the principle and the important concept of fractionation. In general, normal tissue at low doses can repair dnA damage more than tumors. normal brain tissue is therefore very sensitive to the size of fraction of radiation dose. Therefore, the use of fractionation and smaller dose per fraction would allow more radiation damage repair of normal brain tissue. Tumor cells in general undergo less radiation-induced sublethal damage repair and therefore are less affected by fractionation. fractionation also allows tumor cells to reassort

into the more radiosensitive phases of cell cycle between fractions as compared to slowly cycling normal brain tissue. Because radiation damage is mediated through the production of oxygen radicals, fractionation allows tumor cells that are usually hypoxic to become oxygenated between fractions, thus increasing radiation sensitivity. These “4 rs” (i.e., repair, reassortment, reoxygenation, and repopulation), which occur in tumors but not in normal tissue, form the basic principles of fractionated radiotherapy. unlike conventional radiotherapy that employs fractionation, radiosurgery cannot exploit the biologic differences between normal brain tissue and tumors. however, the distinctive physical properties of radiosurgery in properly selected cases allow high biologic dose of radiation to be delivered to the tumors and at the same time exclude normal brain tissue from high dose. This can only be achieved when the target is of small volume, because the volume of normal tissue receiving high dose is exponentially correlated to the size of the target. Stereotactic radiotherapy combines the precision of stereotactic positioning with the radiologic biologic advantage of fractionation. This technique is particularly useful in the treatment of benign brain tumors such as vestibular schwannoma and meningioma, in which high biologic doses are not necessary to control the lesions and normal brain tissue will be spared from high dose. historically, the late injury from radiation therapy from conventionally fractionated radiotherapy has been reported as the tolerance dose, either 5% or 50% risk level at 5 years (Td 5/5 or Td 50/5). for example, the Td 5/5 and the Td 50/5 values for the brain are 54 Gy and 70 Gy, respectively. for radiosurgery, the radiation tolerance of the normal tissue is much less because the relative biologic impact of a single dose is

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considerably greater than for conventionally fractionated radiotherapy. The tolerance of the optic structures and brainstem surface for radiosurgery is reported to be 8 Gy or less and 12 Gy or less, respectively.

SElEcTIon of doSE dose selection is of extreme importance in SrS because dose is strongly correlated with local control and complications. for SrT, acute and long-term complications are rare because of the use of conventional dose and fractionation schedules and the use of stereotactic techniques. The selection of the appropriate technique (SrS versus radiotherapy) and dose prescribed requires a thorough knowledge of neuroanatomy and individual disease process. Stereotactic irradiation is a rapidly evolving treatment modality. As more long-term data and results of prospective randomized trials become available, the indications and the doses that are currently employed may change. The current general guidelines for radiosurgery dose selection are as described in the following sections.

Size of lesion The relationship between size and dose is one of the most important determinants of complication. As the size of the lesion increases, the dose prescribed must be decreased in order to avoid the development of symptomatic brain injury. The volume of the nontarget tissue in the high-dose regions increases exponentially as the volume of the lesion increases. In general, lesions that are greater than 3 cm are not targets for radiosurgery because the dose has to be lowered to a level that would not be biologically effective. The 12 Gy volume of the brain has been shown to correlate with complications (15).

location of lesion The dose used in radiosurgery needs to be adjusted according to the location of the lesion. radiation-induced injury may not result in any symptoms in noneloquent regions of the brain, whereas it can cause significant symptoms in eloquent areas. flickinger and colleagues constructed a scoring system known as Significant Post-radiosurgery Injury Expression (SPIE) location-risk scores that was based on rates of permanent neurologic sequelae following radiosurgery to arteriovenous malformation (AVM). AVM locations in order of increasing risk and SPIE score (from 0 to 10) were frontal, temporal, intraventricular, parietal, cerebellar, corpus callosum, occipital, medulla, thalamus, basal ganglia, and pons/midbrain (15). In our routine practice, the indication and the dose prescribed in radiosurgery are strongly affected by the location of the lesion. lesions in the

frontal lobe are generally treated with higher doses than lesions of corresponding sizes in the motor strip.

Type of lesion The aggressiveness of treatment and the dose employed in radiosurgery are based in part on the risk analysis of competing causes of death. Except for AVMs, benign tumors that are treated with radiosurgery utilizing the currently used dose range do not appear to have clear dose-response curves. caution must be taken not to use unnecessarily high doses, because patients will live long enough to develop long-term complications. for patients with malignant gliomas and brain metastases with expected survival of less than one year, a dose should be employed that is likely to achieve palliative control without causing significant acute toxicity.

Presence of neighboring critical Structures lesions that are intrinsic or abutting the optic apparatus are contraindicated for radiosurgery. Special sensory nerves (cn II, VII, VIII) are more radiosensitive than sensory and motor cranial nerves in the cavernous sinus (cn III, IV, V, VI) and lower cranial nerves. Extreme efforts in dose planning are required to minimize the dose to the optic apparatus and brainstem. dose constraint to the chiasm and optic nerves is limited to less than 8 Gy and to the surface of the brainstem is limited to less than 12 Gy (16). It has been shown that for patients who received higher than 8 to 10 Gy to the optic nerve or chiasm, the risk of developing optic neuropathy is significant (17,18).

Preexisting neurologic Symptoms, co- Morbidities, and Previous Surgical resections In general, patients with preexisting neurologic deficits are at higher risk of developing complications. These patients should be considered for treatment with lower radiosurgery dose or in case of benign brain tumors with SrT. In patients with previous surgical resection(s) or diabetes mellitus, the tolerance of the critical structures could be lower, most likely as a result of vascular compromise (19). for patients with benign tumors such as vestibular schwannoma, meningioma, or craniopharyngioma in whom a high rate of preservation of cranial nerve functions is desired, we employ SrT (i.e., 54 Gy in 1.8 Gy per fraction over a 6-week course) (20–24). for patients with vestibular schwannomas with no useful hearing and who are not at risk for cranial nerve toxicities, a dose of 12 to 15 Gy at 90% is employed (16,25,26). for meningiomas that are treated with radiosurgery, a dose of 12 to 15 Gy is usually prescribed

12. Principles of Brain Tumor radiotherapy

(27,28). for patients with functioning pituitary adenomas, the dose to the chiasm and optic structures is minimized significantly by the use of proton radiosurgery. In general, a dose of 20 Gy is used, with the dose to the optic apparatus always less than 8 Gy (29,30). We currently employ a boost of 12 to 18 Gy and 14 to 18 Gy for patients with malignant glioma and brain metastasis, respectively, depending on the lesion and patient characteristics.

TrEATMEnT rESulTS Benign Brain Tumors The local control rate of radiotherapy of benign brain tumors is very high. Extreme efforts must be taken to reduce the dose to the nontarget tissues to reduce long-term complications such as cranial nerve toxicity, neurocognitive dysfunction, and development of second cancer. This makes stereotactic irradiation an attractive treatment for most patients with benign brain tumors.

173

surgery reported in most recent series is still significant (25, 32–34). fractionated irradiation has been used in the treatment for vestibular schwannomas since 1945. Wallner et al. reported one of the first studies on the use of fractionated radiotherapy in the treatment of vestibular schwannomas (35). In a retrospective analysis of 33 patients who had undergone subtotal resection, the local control rate was reported to be 85% for patients who received postoperative conventional radiotherapy (45 Gy in 1.8 Gy per fraction) compared to 54% for those who did not receive postoperative radiation. The high rate of complication seen in radiosurgery has led to increased interest in the use of SrT in recent years. With conventionally fractionated SrT, facial and trigeminal toxicities are extremely uncommon (22,23,36,37). our current policy is to recommend conventionally fractionated SrT, 54 Gy in 1.8 Gy per fraction, to patients with functional hearing or large lesions. for patients with no useful hearing and/or with very small lesions, we currently employ radiosurgery, 12 Gy normalized to 90%.

Pituitary Adenoma Vestibular Schwannoma (Also Known as Acoustic neuroma) The treatment goals of vestibular schwannoma are to (a) achieve local control, (b) preserve trigeminal and facial nerve function, and (c) preserve hearing. Advances in microsurgical resection and intraoperative cranial nerve monitoring have significantly improved the outcome of surgical treatment of vestibular schwannoma. The risk of cranial nerve dysfunction is related to the size of the lesion. for lesions that are 2 cm or less, the risk of greater than grade 2 facial weakness and functional hearing loss is 4% and 52%, respectively. however, for lesions that are greater than 4 cm, the risk is 62% and 100%, respectively (31). The significant impact of hearing loss and facial paralysis on the quality of life of patients with this curable disease has led to the exploration of other treatment options. radiosurgery has been used in the treatment of vestibular schwannoma for about 30 years. foote et al. from the university of florida provided an excellent risk factor analysis of lInAc radiosurgery for vestibular schwannomas (16). In their retrospective study of 149 cases of patients treated between 1988 and 1998, they found the significant factors that predicted post-treatment cranial neuropathies were maximum dose to the brainstem, prior resection, and treatment era. They also found that dose reduction was the most important factor in reducing the complications and that prescription dose of 12.5 Gy to the tumor margin resulted in the best combination of maximum tumor control and minimum complications in their series. radiosurgery offers several advantages over surgery in that it is noninvasive, a one-day procedure, less costly, and as effective as surgery. however, the risk of toxicity of radio-

The goals of radiotherapy treatment for pituitary adenoma are to control growth and to improve visual and neurologic symptoms in both functioning and nonfunctioning tumors. Another important goal for radiotherapy, either alone or as an adjunctive treatment, in the treatment of functioning adenoma is to achieve biochemical remission. Because of the proximity of the lesions to the critical structures, such as the chiasm, optic nerves, hypothalamus, and temporal lobes, stereotactic irradiation plays a very important role in treatment for patients with pituitary adenomas.

Acromegaly The therapeutic goals in acromegaly are to reduce circulating insulin-like growth factor I (IGf-I) levels to normal for age and sex, and to reduce growth hormone (Gh) levels to less than one microgram per liter after an oral glucose load. Surgery is the primary treatment modality because the hormonal hypersecretion can be normalized rapidly with surgery as opposed to the time delay that occurs after fractionated radiotherapy or radiosurgery. radiotherapy is an important adjuvant therapy used alone or in combination with medical therapy in unsuccessfully operated cases. using normalization of IGf-I as a definition of cure, several studies showed that fractionated radiotherapy normalized IGf-I levels in 40% to 65% of cases 5 to 10 years after treatment (38,39). The slow time course of biochemical response of fractionated radiotherapy necessitates the continuation of medical treatment until complete normalization of hormonal levels. Preliminary data from landolt et al. (40) and Mitsumori et al. (41) suggested that the average time to normalization of both Gh and IGf-I was shorter in patients treated with radiosurgery than fractionated radiotherapy. The earlier efficacy of radiosurgery compared with fraction-

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cancer of the nervous System

ated radiotherapy needs to be confirmed with more studies and longer follow-up periods.

cushing’s disease Transsphenoidal surgery is the treatment of choice for cushing’s disease (adrenocorticotropic hormone–producing pituitary adenoma). Pituitary irradiation is widely considered the most appropriate treatment for patients with cushing’s disease for whom transsphenoidal surgery has been unsuccessful. Estrada et al. showed that fractionated radiotherapy is an effective treatment for patients with cushing’s disease in whom transsphenoidal microsurgery had been unsuccessful (42). With a median dose of 50 Gy given in 25 fractions, 83% of patients achieved clinical and biochemical remission. The median time to remission was 18 months (6 to 60 months). Sheehan et al. showed that normal 24-hour urinary free cortisol levels were achieved in 27 patients (63%) at an average time from treatment of 12.1 months (range 3 to 48 months) after radiosurgery (43). With the use of radiosurgery, it seems that biochemical remission occurs earlier than that of conventional radiotherapy. Treatment-induced hypopituitarism with radiosurgery also appears to occur at a lower rate than with larger-field conventional radiotherapy. A longer follow-up period is necessary to confirm this advantage of radiosurgery. longterm complication data after radiosurgery are also not available.

nonfunctioning Pituitary Adenomas nonfunctioning pituitary adenomas are usually larger than functioning adenomas. The patients commonly present with visual symptoms caused by local mass effect on the optic nerve and chiasm. Stereotactic irradiation is indicated for incompletely resected nonfunctioning adenomas. Because of the size and irregularity of the typical lesion, and also the proximity of the lesion to the optic apparatus, SrT with a conventional fractionated schedule is a very attractive approach. Milker-Zabel et al. (44) demonstrated that SrT resulted in a local control rate of 93% with minimal complications.

Meningiomas The goals of surgery are to reduce acute symptoms or mass effect and to obtain tumor control by maximal debulking of tumor while minimizing short- and long-term morbidities. The goals of stereotactic irradiation, either in the form of radiosurgery or fractionated radiotherapy, are maintenance of local control and preservation of neurologic function. The preferred treatment for meningioma, whether surgical resection, SrS, SrT, or a combination of these treatments, depends on the location and size of the lesion. Meningiomas in the base of the skull, such as the cavernous sinus and petroclival areas, commonly surround or

infiltrate critical structures such as the brainstem, cranial nerves, chiasm, or carotid arteries. Surgical access to these tumors remains a challenge despite tremendous advances in microsurgical techniques. radical resection can rarely be achieved without causing significant morbidity. Stereotactic irradiation, therefore, plays an important role in the management of base of skull meningioma. radiosurgery is indicated for lesions that are small, well-localized, and away from the optic nerves and chiasm. In a retrospective study of 56 patients treated with lInAc radiosurgery at the Brigham and Women’s hospital, the local control rate was reported to be 95% with a median dose of 15 Gy. neurologic symptoms were improved in 34% of patients. nine percent of patients developed new or worsened symptoms (27). for lesions that are large and close to critical normal structures, SrT has been shown to result in high local control and minimal morbidity. debus et al. reported the outcome of 189 patients with large base of skull meningiomas treated with conventionally fractionated SrT at the university of heidelberg (19). With a median follow-up of 35 months, the local control rate was 97% at 5 years. Preexisting neuropathy resolved completely in 28% of the patients. only 1.6% of patients developed new neurologic deficits. for surgically accessible meningiomas, such as the convexity, falx, and parasagittal meningiomas, complete resection can generally be performed with minimal morbidity, and radiotherapy can cause significant peritumoral edema. radiotherapy should be reserved for patients who are of poor medical risk. for paraorbital meningiomas that are encroaching on the optic nerve, radiosurgery is contraindicated because the dose required to achieve local control is higher than the tolerance of the optic nerve. The role of surgery is to relieve acute visual symptoms or mass effect. We recommend SrT to a total dose of 54 Gy in 1.8 Gy per fraction. for meningiomas in the parasagittal sinuses that are small (175 27 57 >57 162 266 66 >84 125 >78 31 237 58 41

dose (gy) MPd 18 MPd 18 MPd 20 MCd 21.5 MPd 21.4 MPd 29 MPd 20 MPd 25 MPd 17.5 MPd 26.1 MPd 15 MCd 50 MPd 18.9 MPd 30 MPd 18.7 MPd 26.6 Med 20 MPd 16 MPd 18.5 MCd 25 MPd 17 MPd 16.2 MPd 14–40 MPd 6–50 MPd 22 — MPd 18 MPd 16 MPd 25 — MPd 20–25 MPd 20 MPd 18 MPd 19 MPd 18 MPd 15–24 MPd 16.2 MPd 20 MPd 12–20 MPd 15–35

RR (%)

lC (%)

Ms (mo)

86 71 100 55 72 n/a 53 47 n/a n/a n/a 86 53 88 n/a n/a n/a n/a 47 92 81 53 25.7 n/a n/a 78 n/a 81 n/a 96.5 77 n/a 32 63 n/a 52 29.6 81.4 n/a 87

93 82 n/a 95 92 94 85 25 85 99 85 n/a 97 n/a 60 96 n/a 25 77 84 73 71 87.5 80 83 97 75 95 68.4 n/a n/a 94 91 n/a n/a 40 85 95 93 100

6.5 6.5 5.0 6.0 n/a n/a 6.0 6.0 11 8.5 9.4 13.5 6.4 15 8 8.0 6.5 11 12 8.7 8 8.4 7 7.8 9 8 11 9 9.3 10 11 6 6.7 7 6.8 8.8 15 9 13 40

g, gamma Knife; l, linear accelerator; lC, local control (when actuarial, the 1-year figure is used); MCd, median/mean central dose; MPd, median/mean peripheral dose; Ms, median survival from radiosurgery; n/a, data not available; RR, response rate.

survival was 14 months. a report from the university of California, san Francisco (uCsF) also analyzed the survival of patients comparable with the series of Patchell et al. and found a rather impressive median survival of 70 weeks (84). Randomized trials to date, results from three randomized radiosurgery trials are available. the first of these was a small trial of WBRt versus WBRt plus radiosurgery in patients with two to four brain metastases (all no more than 25 mm diameter and

known primary tumor type) (83). twenty-seven patients were randomized to initial brain tumor management with WBRt alone (30 gy in 12 fractions) or WBRt plus radiosurgery. the study was stopped at an interim evaluation after 60% accrual at which time 14 patients had received WBRt alone and 13 had additional radiosurgery. the groups were well matched to age, sex, tumor type, number of tumors, and extent of extracranial disease. the rate of local failure at 1 year was 100% after WBRt alone but only 8% in patients who had boost radiosurgery. the median time to local failure was

48. Radiotherapy and Radiosurgery for Brain Metastases

6 months after WBRt alone in comparison with 36 months after WBRt plus radiosurgery. the median time to any brain failure improved in the radiosurgery group. Patients who received WBRt alone lived a median of 7.5 months, whereas those who received WBRt plus radiosurgery lived 11 months (83). although the small trial size is a concern, the outcomes are promising. Chougule et al. (85) reported a three-arm, prospective, randomized, clinical trial comparing gamma Knife radiosurgery alone, WBRt alone, or both. One hundred and four patients received protocol therapy: radiosurgery alone (n = 36), WBRt plus radiosurgery (n = 37), and WBRt (n = 31). Fifty-one patients underwent surgical resection of large symptomatic lesions before randomization. the distribution of these participants was not even among the three treatment arms. the overall median survival was 7, 5, and 9 months for the sRs alone, WBRt plus radiosurgery, and WBRt arms, respectively. the local control was 87%, 91%, and 62%, respectively, for the three arms. the occurrence of new brain lesions was lower (43%, 19%, and 23%, respectively) in the two arms receiving WBRt. the authors concluded that the local control was superior with the radiosurgery arms. although the risk of developing new brain lesions was higher for patients not receiving WBRt, there was no difference in overall survival between the three arms. the major variable limiting the interpretation of these data is the uneven distribution of surgical resection in the various arms, introducing a possible treatment bias. in the largest randomized trial conducted to date, sperduto et al. (86) reported the data from the randomized phase iii trial, RtOg 9508. Between January 1996 and June 2001, these investigators randomized 333 patients from 34 institutions to receive WBRt + sRs or WBRt alone. they analyzed survival, local control, treatment technique (linaC vs. gamma Knife), cause of death, toxicity, and quality of life. Quality of life indicators included the mini-mental status examination, KPs, neurologic Function score, and steroid dose. there was a statistically significant survival advantage with WBRt + sRs for the following patients: solitary brain metastases (Mst 6.5 vs 4.9 months, p = 0.04), RPa class i (median survival time [Mst] 11.6 vs 9.6 months, p = 0.05), age less than 50 years (9.9 vs 8.3 months, p = 0.04), and patients with non–small cell lung cancer or any squamous cell carcinoma (5.9 vs 3.9 months, p = 0.05). also, patients with initial KPs of 90 to 100 appeared to benefit (10.2 vs 7.4 months) without reaching statistical significance (p = 0.07). Furthermore, all patients in the WBRt + sRs group were more likely to have a stable or improved performance status (KPs) at 3 months (50% vs. 33%, p = 0.02) and at 6 months (43% vs. 27%, p = 0.03). there was no significant difference in cause of death, but there was a significant difference in time to progression (18% and 29% at 1 year for the WBRt + sRs group vs. WBRt alone, respectively; p = 0.01). toxicities were comparable between the two treatment groups. therefore, patients treated with both WBRt and radiosurgery had a statisti-

663

cally significant survival benefit and were more likely to have a stable or improved performance status than those treated with WBRt alone. in summary, both prospective and retrospective data confirm that radiosurgery enhances local control of brain metastases. Consequently, it decreases neurologic death, increases steroid dependance, and enhances quality of life as measured by the neurologic Function score and KPs. in well-selected patients, a survival advantage has been demonstrated in retrospective and a large multiinstitutional, randomized, controlled, clinical trial. this survival benefit is contingent on appropriate patient selection and thereby is a function of identifying appropriate prognostic variables. Prognostic Factors several studies have attempted to perform detailed univariate and multivariate analysis of prognostic factors. a summary of some of these variables is presented in table 48-2. influence of number of intracranial Metastases

several trials consistently found that patients with one or two lesions fare equally well, but those with three or more metastases have a uniformly poor outcome in terms of survival (53,58,60,66). the presence of three or more metastases at the time of radiosurgery was significantly associated with decreased survival with a relative risk of 1.69 in the study by alexander et al. (53). Perhaps due to the different methodology used across studies, it is not surprising that some investigators have found no difference in median survival after treatment of single or multiple brain metastases and no prognostic value (74,77,86,88). it appears that a KPs higher than 70 and absence or controlled systemic disease account at least partially for this lack of difference in survival. Wronski et al. (87) used fractionated radiosurgery on patients with 5 to 10 brain metastases from different tumor histologies, including lung (n = 31), melanoma (n = 7), and breast (n = 15). the median survival was 18.4 weeks, comparable with the median survival obtained from series with single intracranial metastases. in the stanford study, multivariate analysis was done to test the independence of the number of metastasis (one to two vs. three to four) as a prognostic factor (58). the best-fit model showed independent prognostic value for the number of lesions (p = 0.0001). in addition, the results of this study showed that patients treated with radiosurgery with up to two brain metastases identified on computed tomography or magnetic resonance imaging have a median survival time identical to that of patients with a solitary lesion. similarly, Breneman et al. (60) found that patients with one or two lesions had identical survival after radiosurgery (44 weeks), but patients with more than two lesions had significantly shorter survival (p = 0.02).

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w taBle 48-2

Prognostic Variables for survival

Variable no. of metastases eCd KPs ⱕ 70 Histology age gender size dose WBRt

auchter

-ve -ve ± none none

engenhart

alexander

Joseph

Breneman

none ±

ⱖ3 -ve -ve

ⱖ3 -ve -ve

none

none ⬎60 -ve none

ⱖ3 -ve ± -ve none none none

none none none

Flickinger

Breast none none none none

shu none -ve -ve none ⬎70 -ve none -ve

shirato

-ve none none none none none

eCd, extracranial disease; KPs, Karnofsky performance score; none, variable is not significant; -ve, variable predicts significantly for decreased survival; WBRt, whole brain radiation therapy; ±, variable has borderline significance; blank cells denote variables not applicable

One report suggested that the overall total volume of intracranial metastatic burden rather than the number of metastases was a prognostic variable for survival (42). in this retrospective review, 55 patients with single or multiple intracranial melanoma metastases were treated at the uCsF, 16 for recurrence after previous radiation therapy, 11 receiving radiosurgery as a boost to WBRt, and 28 treated with radiosurgery alone for initial management of brain metastases. the median total target volume per patient was 6.1 ml (range, 0.25 to 28.3 ml). With a median follow-up of 75 weeks in living patients, the median survival was 35 weeks overall, 35 weeks for patients with solitary metastases versus 33 weeks for those with multiple metastases. in uni- and multivariate analysis of survival, total target volume treated remained a significant prognosticator. influence of active extracranial disease

Of the eight studies summarized in table 48-2, one did not analyze this factor, five found the presence of active extracranial disease to be a negative prognostic factor for survival, and in the remaining two it reached borderline significance, suggesting that this factor is probably one of the most important prognostic variables to consider. in the study by alexander et al. (53), the presence of known systemic disease at the time of radiosurgery was markedly unfavorable for survival with a relative risk of death of 4.43. Breneman et al. (60) found that patients without active extracranial disease at the time of radiosurgery had a superior survival (45 vs. 35 weeks; p = 0.03). in the study by shirato et al. (62), the actuarial survival rate was higher in patients without active extracranial disease (p = 0.041). in the report by shu et al. (84), absence of known extracranial disease at the time of radiosurgery was the second most significant factor associated with improvement in overall survival (p = 0.006). subsequent studies on prognostic factors reinforce extracranial disease as one of the most robust prognosticators for outcome (73,76,77,80,81).

influence of Karnofsky score

in the stanford report by Joseph et al. (58), when several clinical factors potentially associated with a prolonged survival after radiosurgery were examined by univariate and multivariate analysis, a pretreatment KPs of at least 70 was significant as a continuous variable. this is in agreement with our finding in the multiinstitutional analysis (39). in the uCsF study, a decreasing KPs was the third most important factor predicting for decreased survival (p = 0.009) (84). Other studies have highlighted the importance of the KPs as a consistent robust prognostic factor for treatment response and survival (63). in general, a KPs of at least 70 is a favorable factor in the assessment of prognosis. influence of Histologic type

although “conventional wisdom” categorizes tumors into radiosensitive or radioresistant, there are no significant differences in survival with respect to the primary tumor histologic types in any of the major trials. there was initially some speculation that radioresistant tumors such as melanoma would be less responsive. in a volumetric analysis, Voges et al. (49) showed that the mean relative volume reduction was 23% in melanoma, 27% in renal cell, 53% in breast, 60% in adenocarcinomas, and 77% in squamous cell. in one of our trials, we evaluated complete response rates in terms of histology. Whereas 100% of lymphomas achieved a complete response, the rates for other histologic types were 67% for melanoma/sarcoma, 50% for non–small cell lung cancer, 33% for breast cancer, and 11% for renal cell carcinoma (44). a more detailed analysis of this phenomenon shows some intriguing results. exquisitely radiosensitive tumors such as small cell carcinoma indeed show a marked radiographic regression in comparison with radioresistant tumors such as melanoma, which are typically slower to respond and tend not to undergo complete radiographic resolution. these response rates, however, do not necessarily correlate with local control. in fact, it appears that the so-called radioresistant tumors may have better control than the radiosensitive lesions. somaza et al. (89) reported 32 mela-

665

48. Radiotherapy and Radiosurgery for Brain Metastases

noma metastases in 23 patients treated radiosurgically, with local control in 31 of 32 lesions (97%) despite an imaging response rate of 13 of 32 (41%). in Breneman et al.’s series (60), all 12 melanoma patients remained locally controlled through the duration of the study. loeffler and alexander (90) reported one of the largest linaC–based series, with 330 metastatic lesions treated in 217 patients and a local control of 94%. the most common histologic subtypes were lung (n = 134), small cell (22), melanoma (60), renal cell (25), breast (44), sarcoma (18), colon (10), germ cell (7), and others (10). the so-called radioresistant tumors actually had higher local control rates. in the more recent update by alexander et al. (53), this trend continued without statistical significance. When analyzed by univariate and multivariate methods, there were no significant differences in survival with respect to the primary tumor histologic types in several studies (58,60,62,76). in contrast, Flickinger et al. (51) found breast histology to be the only significant predictor for survival (p = 0.0002). Patients with breast cancer had a median survival of approximately 18 months in comparison with less than 12 months for other histologic types. in terms of local control, however, multivariate analysis revealed improved control for melanoma and renal cell carcinoma, as opposed to other histologies (p = 0.0006). Other investigators have independently reported good local control rates in patients with brain metastases from melanoma using radiosurgery alone (65,66). influence of age and gender

the most relevant studies in which age has had prognostic importance are those of alexander et al. (53), shu et al. (84), and Fernandez-Vicioso et al. (63). in the first two studies, age greater than 60 years at the time of radiosurgery was significantly associated with decreased survival, with a relative risk increasing by 18% per decade. there were no significant differences in survival with age in any of the other major series. similarly, gender was not prognostic in any of the trials. influence of dose

intuitively, one would expect dose to have a major influence on local control and possibly survival. However, there is no uniform agreement about the doses to use for lesions of various sizes. in general, larger tumors receive lower doses, resulting in some size-dependent loss of local control. alexander et al. reviewed the effect of dose and found that it did not influence local control. shirato et al. reported no effect of dose on survival (54,62). Flickinger et al. (51) did not find a correlation between dose and survival. However, Breneman et al. (60) found a significant improvement in local control with a radiation dose of at least 1,800 cgy (median time to failure, 52 weeks vs. 25 weeks; p = 0.008). the RtOg completed a phase i dose-seeking trial and recommended a dose prescription based on tumor size as follows: 24 gy for

tumors no more than 20 mm, 18 gy for tumors 21 to 30 mm, and 15 gy for tumors 31 to 40 mm (91). in a separate report analyzing the uCsF data, shiau et al. (61) specifically evaluated the effect of dose and found that increased doses led to improved local control and freedom from progression, with lesions treated to at least 18 gy being controlled for substantially longer than those treated to less than 18 gy. Breneman and colleagues (60) reported on 84 patients undergoing sRs for brain metastases, 79 of whom (93%) were treated at recurrence after previous WBRt. Patients had between one and six lesions treated with a median minimum tumor dose of 16 gy. local control was significantly improved for radiosurgery doses of more than 18 gy. influence of lesion size

in a volumetric analysis of 54 recurrent and newly diagnosed brain metastases treated with linaC–based sRs from one of our earlier studies, some insight into the relationship between size and response can be discerned (44). as expected, the rate of complete responses declined dramatically with increasing tumor size (table 48-3). Kida and colleagues (54) from Japan also noted that smaller tumors tend to respond faster than larger tumors. in alexander et al. (53), a volume greater than 3 ml approached borderline significance in a multivariate analysis for local control. no significant differences were found in survival with respect to size of primary tumor (29,62). shu et al. (84) found that tumor volume was the most important predictive factor for survival (p = 0.0005). therefore, whereas smaller tumors may be more effectively controlled, there are no clear data to suggest this variable influences survival (table 48-2). influence of Whole Brain Radiation therapy

a retrospective review of patients with newly diagnosed brain metastases by Chidel et al. (92,93) showed a trend toward longer survival among the 78 patients managed initially with radiosurgery alone versus 57 patients treated with radiosurgery plus upfront WBRt (median survival times, 10.5 vs. 6.4 months). similarly to the study by sneed et al. (69), intracranial disease-free survival was significantly worse for patients managed initially with radiosurgery alone versus radiosurgery plus WBRt (60% vs. 34% at 2 years).

w taBle 48-3

tumor size vs. Response analysis

size (ml) (%)

n

Mean Volume

Mean dose (gy)

⬍2 ⬎2, ⬍6 ⬎6, ⬍10 ⬎10

23 16 5 10

0.97 3.27 7.89 19.94

22.4 17 13.2 13.2

CRa 14 (61) 7 (43) 1 (20) 1 (10)

PRa 4 (17) 5 (32) 2 (40) 4 (40)

CR + PR 78 75 60 50

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Cancer of the nervous system

However, data collected from our 10-institution analysis permitted a more in-depth analysis of this question. We reviewed these data to compare survival probabilities of patients with newly diagnosed brain metastases managed initially with radiosurgery alone versus radiosurgery plus WBRt. We created a database with raw data submitted from these 10 institutions on patients treated with radiosurgery for brain metastases. Of the 660 assessable patients, 272 had radiosurgery alone initially (24% of whom ultimately had salvage WBRt) and 388 had radiosurgery plus upfront WBRt. the median survival times for patients treated with radiosurgery alone initially versus radiosurgery plus WBRt were 16.3 versus 16.2 months for RPa class i patients, 8.6 versus 7.9 months for class ii, and 5.1 versus 5.5 months for class iii. With adjustment by RPa class, there was no survival difference comparing radiosurgery alone initially with radiosurgery plus upfront WBRt. Omission of upfront WBRt did not appear to compromise survival of patients treated with radiosurgery for newly diagnosed brain metastases. an important caveat is that this concept has not been adequately tested in a randomized context. influence of technique

there are two primary technical radiosurgery modalities, gamma Knife and linaC systems. the gamma Knife consists of multiple cobalt sources (201) focused to a central point using tiny collimators ranging in size from 8 to 18 mm. typically, multiple such “shots” are stacked together to produce a heterogenous distribution of dose within the tumor with a very large gradient, commonly approaching 50%, that is, the central portion of the tumor could easily have twice the radiation dose as the periphery. this could have both theoretical advantages and disadvantages. linaC systems typically have a larger range of collimator size choices (e.g., 4 to 40 mm), and hence typically require fewer shots to conform the radiation dose around a target, resulting in a more homogenous dose distribution. these different dose distributions have become the subject of much discussion without any reasonable data to support one point of view versus the other. at times, these debates take on commercial rather than scientific undertones. a direct randomized comparison of the two approaches has never been undertaken. a report (78) has spurred considerable attention to this issue. in a phase i RtOg trial (90–05), primarily designed to establish the maximum tolerated dose of radiosurgery, a post-facto data analysis suggested improved local control (mostly for recurrent glioma) with the gamma Knife technique (78). an accompanying editorial identified the major methodologic pitfalls that flawed the study conclusion. First, the trial did not have the design, power, or stratification to answer such a question. For example, the gamma Knife technique contributed only 37 patients compared with 131 from the linaC technique, a 3.5-fold discrepancy. the number of institutions contributing linaC cases was 15 (average case/institution = 8.7), compared with the 2 gamma Knife users (average

case/institution = 18.5, a 2.1-fold variance). there was considerable imbalance between the tumor types (primary vs. metastatic). Clearly, these major clinical imbalances cannot be statistically convolved into a meaningful conclusion regarding comparative efficacy. additional concerns raised by the editorial addressed the lack of central review for what is essentially a neuroradiologic end point: local control. However, the actual reported methodology of central review by two non-neuroradiologists, who parenthetically represented one of the two gamma Knife centers and at least one of which was intimately involved with the study, negates objectivity and eliminates the likelihood of necessary “blinding” for interpretation (94). data from much larger trials do not support the contention that technique makes a difference. Both in the randomized phase iii RtOg trial (n = 139) and our 10-center review (n = 541), technique was not prognostically predictive for superior outcome, and in fact, results were identical. these findings validate the well-known scientific observation that individual cells are unable to determine the source (gamma Knife or linaC) when faced with a stream of photons. the concept of “differential boosting” engendered by dose heterogeneity needs far more proof. Radiosurgery Complications acute complications of radiosurgery may occur within hours of treatment and include nausea, vomiting, and, rarely, seizures in the case of cortically based lesions. late complications develop in less than 10% of patients (19,39). in particular, symptomatic radiation necrosis may become a problem, requiring prolonged steroid treatment or surgical resection. the likelihood of this occurring is related to radiation dose and prior exposure to radiation (39). acute toxicity

there are very few significant acute complications reported within the first week after radiosurgery. loeffler and alexander (90) reported 22 of 196 episodes (11.2%) of nausea in the first 24 hours (patients received more than 2.75 gy to the area postrema), 12 of 196 seizures (6.1%) within 24 hours, and transient motor weakness in 4 patients (2%) within 36 hours, all of who had motor cortex lesions. alexander et al.’s update (53) suggests that to reduce or eliminate radiosurgery-induced nausea/vomiting, all patients who receive more than 375 cgy to the area postrema should receive antiemetic therapy before treatment. ten patients experiencing postradiosurgery seizures had a history of seizure disorder and, in retrospect, had subtherapeutic levels of anticonvulsants. since 1990, the Boston group recommends that all patients with cortical lesions should be prophylactically premedicated for seizures before the radiosurgery procedure, regardless of their seizure history. in a report of the 30-day postradiosurgery adverse event rate in 195 consecutive patients treated with radiosurgery,

48. Radiotherapy and Radiosurgery for Brain Metastases

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incidence of complications and their severity was analyzed and classified as mild, moderate, and severe as defined earlier by Werner-Wasik. immediate adverse events of any grade occurred in 35% of patients after radiosurgery. Most were mild or moderate (91%). Headache, nausea, and seizures were the most common adverse events and were usually self-limiting. severe adverse events include a cerebrospinal fluid leak, infection, and swelling, causing neurologic change such as aphasia, hemiparesis, and diplopia (95,96). Joseph et al. (58) reported a low incidence of severe headaches or nausea and a 2.3% incidence (3 of 120) of seizures within 12 hours of completing the treatment. each of these patients had a subtherapeutic anticonvulsant level at the time of treatment. it is prudent to consider premedicating patients with lesions near the posterior fossa with antiemetics and to routinely check serum anticonvulsant levels in those with a seizure history.

Only about 1% of patients (2 of 196) in the Boston series developed permanent cranial nerve palsies (90). this phenomenon was observed at 7 and 8 months after treatment. One involved the fifth nerve, which received 16.5 gy, and the other involved the eighth nerve, which received 15 gy. another potential complication ascribed to radiosurgery is persistent edema requiring chronic steroid usage, with all of its attendant complications. in the university of Wisconsin experience, the need for steroids past 6 months was documented in 4 of 54 patients (7%), a figure very comparable with the 8% steroid dependence at 12 months in the Boston experience (44,89). in the report by Flickinger et al. (51), the 2-year actuarial rate of developing delayed symptomatic edema was 10.8%. Radiosurgery can result in dramatic resolution of edema rather than persistence of this phenomenon. Jokura et al. (52) provided further confirmatory evidence, finding “dramatic improvement of perifocal edema with regression of tumors.”

subacute toxicity

Radiosensitizing agents

Complications occurring within the first 6 months are considered subacute. the data from the major trials suggest the following as possible subacute complications (51,53,61,90). alopecia was seen in 11 patients (5.6%), all of who received at least 4.4 gy to the scalp, as reported in the Joint Center for Radiation therapy (JCRt) series. steroid reinstitution and/or continuation were necessary in 21% of survivors at 6 months in the JCRt series, probably reflecting vascular change rather than tumor recurrence. in the multicenter gamma Knife report by Flickinger et al. (51), steroid reinitiation at less than 6 months was necessitated in less than 3% of patients. neurologic deterioration was reported in six cases in a uCsF series between 2 weeks and 3 months after radiosurgery, including four cases of increased hemiparesis and or confusion treated with steroids and two of necrosis documented by magnetic resonance spectroscopy/autopsy. Chronic sequelae

the major chronic complications of radiosurgery are necrosis, cranial nerve palsies, and chronic steroid dependence. in the Boston experience, symptomatic radiation necrosis was present in 17 of 217 patients (8%) from 2 to 22 months after therapy, all of which eventually required resection. ten of these patients had also received methotrexate, which could have contributed to the necrosis (53,90). delayed local necrosis within the treated volume was the major complication in 16.6% of patients (20 of 120) in the stanford study (58). the risk of necrosis was a function of tumor volume and prior or concurrent WBRt. symptomatic and pathologically confirmed necrosis with no viable tumor developed in 1 of 116 patients, 11 months after radiosurgery in Flickinger et al.’s report (51), yielding a 2-year actuarial risk of developing necrosis of 4%. However, reoperation was required in 10 of 116 patients (8.6%) because of hemorrhage, necrosis, or recurrence.

Radiosensitizers enhance the effects of radiation therapy. two radiosensitizing agents are currently under investigation: motexafin-gadolinium, a redox modulator that enhances radiotherapy-induced apoptosis by depleting intracellular reducing agents, and RsR-13, an allosteric effector of hemoglobin that releases oxygen to hypoxic tissues (97,98). a prospective, randomized, phase iii trial of motexafin-gadolinium and WBRt versus WBRt was recently completed in which patients were stratified by tumor type and RPa class. Preliminary results show that motexafin-gadolinium did not confer an overall advantage in overall survival but improved time to neurologic progression (99). the administration of RsR-13 in conjunction with radiation therapy has resulted in radiation dose enhancements of up to 1.7-fold in preclinical studies (100). Retrospective evaluation of a phase ii trial suggests some clinical efficacy (101). a phase iii study has recently been completed, and results are awaited.

ROle OF COMBinatiOn CHeMORadiOtHeRaPy traditionally, chemotherapy has had a minimal role in the treatment of patients with brain metastases. For a treatment to be effective in the brain, chemotherapeutic agents must be both active against the tumor and cross the blood–brain barrier. Many currently available chemotherapeutic agents are ineffective for treating primary brain tumors or have limited ability to cross the blood–brain barrier when administered systemically. this may potentially be less of an issue for the treatment of some brain metastases, because recent data suggest that the integrity of the blood–brain barrier is im-

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paired or absent in large areas of the tumor vasculature. Brain metastases originating from chemosensitive tumors, such as small cell lung cancer, may respond as well to chemotherapy as do extracranial metastases. temozolomide is a novel second-generation alkylating agent that is structurally similar to dacarbazine and has essentially 100% bioavailability when given orally (102). Because of its small molecular size and lipophilic properties, temozolomide is able to cross the blood–brain barrier, reaching concentrations in the cerebrospinal fluid that are approximately 40% of plasma concentrations (103). it has demonstrated in vitro activity against a broad range of primary brain tumors, including anaplastic astrocytoma, glioblastoma multiforme, and low-grade gliomas, and has shown early promise in tumors outside the central nervous system (33,104–107). Furthermore, temozolomide has demonstrated additive or synergistic activity when used in combination with other chemotherapeutic agents or radiation therapy (108–111). at least four studies have examined the effects of concurrent treatment with temozolomide plus radiation therapy in patients with newly diagnosed brain metastases, on the basis that the two together maybe additive or even potentially synergistic (108,109,112,113). the data supporting the possible radiosensitizing effects of temozolomide are sparse (114,115). dardoufas et al. (109) conducted a single-arm study to investigate the efficacy of concurrent temozolomide plus radiation therapy in patients with newly diagnosed brain metastases. the 20 patients enrolled in the study had both single (n = 6) and multiple (n = 14) brain metastases, and most patients (55%) had lung cancer as their primary tumor type. the overall response rate was 55% after six cycles of chemotherapy. the overall response rate in patients with lung cancer was 86%. in an ongoing spanish trial, Verger et al. (112) reported 3-month response rates of 38% versus 14%, favoring the WBRt + temozolomide combination over WBRt alone. antonadou et al. (108) conducted a study to investigate the efficacy of concurrent temozolomide plus radiation therapy (temozolomide + WBRt) in 48 patients who were randomly assigned to temozolomide plus radiation therapy versus radiation therapy alone. the overall response rate was 95% for temozolomide + WBRt, compared with 66% for WBRt alone. in a subsequent randomized confirmatory trial, the same authors confirmed a higher 3-month response rate (58% vs. 30%), higher 3-month steroid independence (85% vs. 49%), lower brain death (13% vs. 28%), and a trend toward favorable median survival (7.9 vs. 4.3 months) in non–small cell lung cancer patients with brain metastases treated with temozolomide + WBRt compared with WBRt alone (113). Overall, temozolomide was well tolerated in these four clinical trials and exhibited clinical activity, particularly in combination with WBRt for patients with newly diagnosed brain metastases. Collectively, the results from the trials merit more extensive clinical testing of the agent. in particu-

lar, the high response rates observed in combination with WBRt provide the rationale for larger well-controlled clinical trials.

ROle OF suRgeRy surgical resection is reserved primarily for patients with a single brain metastasis who have minimal systemic dissemination and a KPs higher than 70. Clear indications for resection include craniotomy or stereotactic biopsy to establish the diagnosis when in doubt and resection in those patients where removal of the tumor mass is likely to provide immediate palliation. such considerations lead to a very high level of selectivity in surgical series. it is common practice in some institutions to select patients with single brain metastases who are otherwise clinically stable for surgical resection. the goals of surgery are mainly two: complete eradication of the brain metastasis and the effective and prompt relief of symptoms. Retrospective series published in the 1980s suggested that in highly selected patients, surgical resection followed by WBRt may result in useful and prolonged survival ranging from a median of 16 to 26 months. three randomized clinical trials were conducted to compare surgical resection plus WBRt with WBRt alone (15,40,116). two of these studies concluded that the addition of surgical resection to WBRt provided greater benefits than WBRt alone, specifically in terms of median survival and functional independence and particularly in patients younger than 65 years of age, with a KPs higher than 70, and controlled systemic disease. in the study reported by Patchell et al. (15), patients treated with surgery plus WBRt had longer median overall survival, fewer recurrences, and maintained functional independence for a longer period of time. noordjik et al. (40) also reported improvement in overall and functionally independent survival with combined treatment. Median overall survival was significantly longer for combination therapy compared with patients treated with WBRt alone. Hence, these two studies support the concept that improved local control improves overall clinical outcome. a third study conducted by Mintz and colleagues (116), however, did not demonstrate a survival advantage when surgery was added to WBRt. in this study, median overall survival in the two treatment groups was not significantly different. Furthermore, no differences were noted between the two groups in terms of quality of life and KPs scores. the inconsistent results may be attributed in part to the use of different eligibility requirements for the study and illustrate the effects of eligibility criteria on study outcome. For example, this study (116) included patients with prognostically poor KPs as well as those with active systemic disease. effects on tumor recurrence, death, and survival time of surgical resection of brain metastasis without the addition of postoperative WBRt have also been evaluated in a ran-

48. Radiotherapy and Radiosurgery for Brain Metastases

domized controlled study. Patchell and colleagues (14) tested whether or not surgical resection was more efficacious when followed by WBRt than when used alone. after a median follow-up of 43 and 48 weeks (surgery alone vs. surgery plus WBRt, respectively), significant differences in favor of combined treatment were noted in the proportion of patients experiencing tumor recurrence (18% vs. 70% in surgery alone; p < 0.001), median time to tumor recurrence (226 weeks vs. 38 weeks; p < 0.001), and fewer deaths due to neurologic causes (14% vs. 44%; p = 0.003). However, no differences were noted between groups with respect to overall length of survival and duration of functional independence, underscoring the importance of adequate control of systemic disease.

COnClusiOns Brain metastases remain a major clinical problem, characterized by poor survival, limited palliation, and high rates of neurocognitive deficits and hence represent a major complication of cancer. the most common treatment modality is WBRt. For select patients, surgery or radiosurgery may prolong survival by a few months. there is a critical need for better treatment options with the potential to influence survival and maintain or enhance quality of life. the investigation of newer radiosensitizers and combination chemoradiotherapy currently form a significant component of the research strategy directed at improving survival for this problem.

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therapy in vitro and in vivo. Cancer Chemother Pharmacol 1998;42:24–30. Mehta MP, Rodrigus P, terhaard C, et al. Results from the phase iii trial of motexafin gadolinium (Mgd) in brain metastases. Proc am soc Clin Oncol 2002;21:72a(abst 286). Kavanagh Bd, Khandelwal sR, schmidt-ullrich RK, et al. a phase i study of RsR13, a radiation-enhancing hemoglobin modifier: tolerance of repeated intravenous doses and correlation of pharmacokinetics with pharmacodynamics. int J Radiat Oncol Biol Phys 2001;49:1133–1139. shaw e, scott C, stea B, et al. survival improvement with RsR13 plus cranial radiation therapy in patients with brain metastases: comparison to the radiation therapy oncology group recursive partitioning analysis brain metastases database. Proc am soc Clin Oncol 2000;19:159a. agarwala ss, Kirkwood JM. temozolomide, a novel alkylating agent with activity in the central nervous system, may improve the treatment of advanced metastatic melanoma. Oncologist 2000;5:144–151. stupp R, Ostermann s, leyvraz s, et al. Cerebrospinal fluid levels of temozolomide as a surrogate marker for brain penetration [abstract]. Proc am soc Clin Oncol 2001;20: 59a. Chinot Ol, Honore s, dufour H, et al. safety and efficacy of temozolomide in patients with recurrent anaplastic oligodendrogliomas after standard radiotherapy and chemotherapy. J Clin Oncol 2001;19:2449–2455. Van den Bent MJ, Keime-guibert F, Brandes aa, et al. temozolomide chemotherapy in recurrent oligodendroglioma [abstract]. neurooncology 2000;2:298. yung WK, albright Re, Olson J, et al. a phase ii study of temozolomide vs. procarbazine in patients with glioblastoma multiforme at first relapse. Br J Cancer 2000;83:588–593. yung WK, Prados Md, yaya-tur R, et al. Multicenter phase ii trial of temozolomide in patients with anaplastic astrocytoma or anaplastic oligoastrocytoma at first relapse. J Clin Oncol 1999;17:2762–2771. antonadou d, Paraskevaides M, Coliarakis n, et al. temozolomide enhances radiation treatment efficacy in brain metastases: a randomized phase ii study. J Clin Oncol 2002;17:3644–3650. dardoufas C, Miliadou a, skarleas C, et al. Concomitant temozolomide (tMZ) and radiotherapy (Rt) followed by adjuvant treatment with temozolomide in patients with brain metastases from solid tumours [abstract]. Proc am soc Clin Oncol 2001;20:75b. Prados M. temozolomide in combination with other cytotoxic agents. semin Oncol 2001;28:24–33. stupp R, newlands e. new approaches for temozolomide therapy: use in newly diagnosed glioma. semin Oncol 2001;28:19–23. Verger e, gil M, yayar, et al. Proc am soc Clin Oncol 2002; 21: abstract 309. antonadou d, et al Confirmatory phase iii trial. schering Plough global investigator Meeting, Monte Carlo, July 2002. Van Rijn J, Heimans J, Van deberg J, et al. int J Radiat Oncol Biol Phys. 2000;47:779–784. Wedge sR, Porteous JK, glaser Mg, et al. anticancer drugs 1997;8:92–97. Mintz aH, Kestle J, Rathbone MP, et al. a randomized trial to assess the efficacy of surgery in addition to radiotherapy in patients with a single cerebral metastasis. Cancer 1996;78:1470–1476.

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C h a p t e r

4 9

w Epidural Disease Thomas N. Byrne Spinal neoplasms can be classified according to location, that is, epidural, intradural–extramedullary (leptomeningeal), and intramedullary (Fig. 49-1). Although the histology of primary spinal tumors that occur in these locations reflects the tissues found in each of these sites, metastases from any primary elsewhere may grow in the epidural, leptomeningeal, or intramedullary locations. In this chapter we discuss the diagnosis and management of epidural metastases. Symptomatic metastatic epidural spinal cord compression (MESCC) occurs in approximately 5% to 10% of patients dying from cancer and appears to be increasing (1,1a). Left untreated, MESCC ultimately leads to paraplegia and sphincter paralysis. Alternatively, if treated before neural injury develops, most patients maintain neurologic function. (For brevity in this chapter, MESCC is used to include cauda equina compression.) In approximately 85% of cases of MESCC (2), the metastasis begins in the vertebral column and extends into the epidural space to cause neural compression. Vertebral metastases have been reported to occur in 15% to 41% (3) of patients dying from malignancy. Thus, among the 400,000 patients dying annually from cancer in the United States, between 60,000 and 160,000 harbor vertebral metastases and 20,000 suffer from MESCC.

PAThogENESIS oF SPINE METASTASES The pathogenesis of cancer metastasis has been long debated. Paget emphasized the “seed and soil” hypothesis in which metastases develop in locations where the metabolic environment supports the growth of these cells. Ewing, on the other hand, maintained that hemodynamic and anatomic factors were the key determinants in defining the sites of metastases. In the hemodynamic model, the metastatic cascade is based on the anatomy of the draining veins. Thus, metastases pass to the lung before spreading to bone. Clinical experience reveals, however, many exceptions to this predicted cascade (3).

In an attempt to explain the discrepancy that spine metastases occur so frequently in the absence of lung and other visceral metastases, Batson (4) studied the epidural venous plexus that now bears his name. Using human cadavers, he showed that this valveless system acts as a source of collateral circulation for veins draining the chest, abdomen, pelvis, and breast. In living primates, he demonstrated that the epidural plexus did not fill after injection of the dorsal vein of the penis unless the abdomen was compressed, mimicking a Valsalva maneuver. Batson predicted that in the human, this vertebral venous plexus of low intraluminal pressure could be filled from venous circulation draining breast, intrathoracic, and intraabdominal organs during coughing, sneezing, and straining. Furthermore, because the vertebral system is valveless, blood may flow in a rostral or caudal direction unimpeded. Coman and DeLong (5) confirmed Batson’s hypothesis by injecting cancer cells into the femoral veins of animals either with (experimental group) or without (control) external abdominal pressure. Although in most of the control group only lung metastases were seen, spine metastases occurred in most of the experimental group. The significance of these finding has been challenged by Arguello et al. (6), who found that intracardiac injection of tumor cells resulted in metastases to the subchondral regions of vertebral bodies. They attributed this localization to the fact that bone marrow contains hematopoietic growth factors that have been shown to stimulate cancer cells to grow in vitro. Thus, both Paget’s seed and soil hypothesis and Ewing’s hemodynamic mechanism may offer explanations for the high incidence of spine metastases.

Location of Tumor in Relation to Spinal Cord MESCC arises from metastases to one of three locations: the vertebrae in 85% of cases, the paravertebral tissues in 10% to 15%, or, rarely, the epidural space itself. Although the vertebral body is the most common site of metastasis, the pedicle and posterior arch also may be involved. Compression of the spinal cord by tumor or, less frequently, bone 673

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Spinal Cord Pathology and Pathophysiology of Neurologic Manifestations

FIgURE 49-1. Anatomic locations of spine metastases are shown. A. Intramedullary metastases are located within the spinal cord. B. Leptomeningeal metastases are in the subarachnoid space and are extramedullary and intradural. Epidural metastases arise from the extension of metastases located in the adjacent (C) vertebral column, (D) the paravertebral spaces via the intervertebral foramina, or, rarely, (E) the epidural space itself. As these epidural metastases grow, they compress adjacent blood vessels, nerve roots, and spinal cord, resulting in local and referred pain, radiculopathy, and myelopathy. (From Byrne TN. Spinal cord compression from epidural metastases. N Engl J Med 1992;327:614–619, with permission.)

fragments develop as tumor destroys the vertebra. Tumor is usually anterior or anterolateral to neural structures, which has implications for surgical therapy. Plain x-rays and radionuclide bone scans are typically positive in these cases. Paravertebral tumors may extend into the vertebral canal through the intervertebral foramina. Approximately two-thirds of cases of MESCC due to lymphoma and pediatric neoplasms occur via this mechanism. Plain x-rays of the spine and radionuclide bone scans are usually negative in such cases. Alternatively, computed tomography (CT) and magnetic resonance imaging (MRI) can visualize the paravertebral regions.

The spinal cord pathology in cases of MESCC includes edema, demyelination, hemorrhage, and cystic necrosis. McAlhany and Netsky (7) undertook a clinicopathologic study among 19 patients with extramedullary spinal cord compression. There was no correlation between the axial location of the neoplasm and the presenting clinical manifestations or pathologic findings that were present both ipsilateral and/or contralateral to the mass. The white matter was more severely affected than the gray matter, but the distribution of demyelination and infarction in the axial plane did not conform to the arterial supply. More recent autopsy studies (8) have reported “pencilshaped softenings” of the spinal cord at the level of epidural tumors that may extend over several segments of the spinal cord in a cephalad or, less frequently, caudad direction. The necrotic cavity is usually located in the ventral portion of the posterior column or dorsal horn. This corresponds to the region involved in cases of venous infarction but is also considered a watershed zone for arterial circulation (9). These cystic intramedullary lesions have been imaged using delayed CT myelography and MRI (10). Both circulatory disturbance and direct neural compression have been cited as causes of neurologic injury. Ushio et al. (11) injected Walker 256 carcinoma into the epidural space of the rat and demonstrated that vasogenic edema of the spinal cord was an early pathologic finding. The marker horseradish peroxidase, which is normally excluded from the spinal cord, entered the cord at the site of compression, suggesting a breakdown of the blood–spinal cord barrier as a cause of edema. Diminution of vasogenic edema occurred after the administration of corticosteroids, and this was associated with improvement in neurologic function of the animals. In another animal model of MESCC, Siegal and colleagues (12) examined the role of prostaglandins and somatosensory evoked responses in the evolution of pathologic changes and myelopathy. These authors found that abnormalities in spinal somatosensory evoked responses precede neurologic signs of myelopathy. Furthermore, electron microscopic studies revealed that demyelination was caused by both mechanical compression and ischemia. Vasogenic cord edema is accompanied by an increase in prostaglandin estradiol synthesis, which is inhibited by steroidal and nonsteroidal antiinflammatory agents (13). These investigators also found that cyproheptadine, a serotonin antagonist, reduces prostaglandin estradiol synthesis, vascular permeability, and cord edema and delays the development of paraplegia in rats. These experimental results may offer new therapeutic approaches in the future.

49. Epidural Disease

Primary Malignancies Epidemiologic studies of MESCC generally report that breast, lung, and prostate cancer comprise nearly 50% of all primaries (14). other frequent primary neoplasms include lymphoma, renal carcinoma, melanoma, multiple myeloma, and sarcoma (2,3). Among children with MESCC, the most common tumors are sarcoma, neuroblastoma, and lymphoma (15). MESCC usually occurs in the setting of disseminated disease, although it can be the presenting manifestation of malignancy or the sole site of recurrence. In a study from the Mayo Clinic, 20% of cases of spinal epidural metastasis occurred as the initial manifestation of malignancy (15a). Carcinoma of the lung, cancer of unknown primary site, nonhodgkin lymphoma, and multiple myeloma accounted for 78% of cases. Alternatively, MESCC can be a manifestation of progression or recurrence of previously established malignancy years after the diagnosis. In a series from Memorial Sloan-Kettering Cancer Center (MSKCC), the interval between the diagnosis of cancer and spinal cord compression was 0 to 19 years (2).

Level of Spinal Cord Compression Several studies have confirmed that the thoracic spine is the most common level of spinal metastasis. The spinal level of MESCC is thoracic in approximately 70%, cervical in 10%, and lumbar in 20% (2,16). MESCC occurs at multiple noncontiguous levels in 10% to 38% of cases (2,16). Most epidural masses are located in the anterior or anterolateral spinal canal, which has implications for surgical therapy, as discussed below (2).

CLINICAL PRESENTATIoN oF METASTATIC EPIDURAL SPINAL CoRD CoMPRESSIoN The major presenting clinical signs and symptoms of MESCC are pain, weakness, sensory loss, and autonomic disturbance. Progressive truncal, referred, and/or radicular pain, the most common initial complaint of both vertebral metastasis and MESCC, is found in approximately 95% of adults (2) and 80% of children (15) with MESCC. In a study of patients with symptoms of spinal metastases who were suspected of MESCC, Bernat and colleagues (17) found that the character of pain did not distinguish between vertebral metastasis alone and MESCC. Thus, each patient with pain due to spine metastasis must be considered as being at risk for having MESCC. In the MSKCC series of MESCC (2), pain was the first symptom in 96% of 130 cases of MESCC (Table 49-1). The pain was radicular in 79% of cervical lesions, 55% of thoracic lesions, and 90% of lumbosacral lesions. Radicular pain typi-

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w TABLE 49-1 Signs and Symptoms of Metastatic Epidural Compression in 130 Patients Sign/Symptom Pain Weakness Autonomic dysfunction Sensory complaints Ataxia herpes zoster Flexor spasms

First Symptom

Symptoms at Diagnosis

125 (96) 2 (2) 0 0 2 (2) 0 0

125 (96) 99 (76) 74 (57) 66 (51) 4 (3) 3 (2) 2 (1)

Values are total number of instances, with percents in parentheses. From gilbert RW, Kim Jh, Posner JB. Epidural spinal cord compression from metastatic tumor: diagnosis and treatment. Ann Neurol 1978;3:40–51, with permission.

cally was bilateral when it occurred in the thoracic region. Vertebral tenderness was reported in only 42 of 130 patients (32%). The pain of MESCC is frequently misdiagnosed as due to degenerative joint disease (DJD). Both types of pain may be aggravated by movement, Valsalva maneuver, straight-leg raising, and neck flexion. however, DJD rarely occurs outside the low cervical or lumbar spine, and the pain of DJD is usually familiar to the patient. Another unusual characteristic that serves to distinguish the two sources of pain is the response to recumbency. The pain of DJD is alleviated by bedrest, whereas that of MESCC is commonly exacerbated by recumbency such that patients may sleep in a chair (2,3). Finally, one should be cautious in attributing symptoms or signs due to DJD in cancer patients, because plain films or bone scan usually will confirm incidental DJD in patients over 50 years of age (3). The duration of pain before the diagnosis of spinal cord compression may vary with different primary tumors (16). Among all tumors in the London hospital series, pain was present on average for 5 months (range, 3 days to 3.8 years) before diagnosis. This duration was significantly shorter for spine metastases from lung cancer (mean, 4 months) than for those secondary to breast cancer (mean, 7 months). In the MSKCC series, the median duration of pain was 2 months for all patients irrespective of their primary tumor. Weakness is nearly always accompanied by pain in MESCC and is rarely the sole manifestation. As shown in Table 49-1, only 2 of 130 patients in the MSKCC series had weakness as the initial manifestation of cord compression. Alternatively, at the time of diagnosis, subjective weakness was found in over 76% and objective signs of weakness in 87% of patients. As with weakness, sensory loss is rare as a sole presenting manifestation of MESCC. Although sensory disturbance was not the presenting complaint in any of the 130 patients from MSKCC, numbness and paresthesias were reported at the time of diagnosis by 51% of patients (Table 49-1). on

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examination, sensory loss was found in 78% of patients in the MSKCC group. As the somatotopically arranged spinothalamic tracts are progressively compressed, an ascending sensory level may occur. This has clinical implications, because a sensory level may mislead the examiner and underscores the need to radiographically examine the entire spinal canal in patients with symptoms or signs of MESCC. Sphincter disturbances are rare sole presenting manifestations of MESCC unless the lesion is located at the conus medullaris or cauda equina. Among the series of 600 patients reported by Constans et al. (18), sphincter disturbances were the sole presenting complaint in only 2%. In the MSKCC series, no patients presented with sphincter dysfunction alone. At the time of diagnosis, however, sphincter disturbances were present in 57% (Table 49-1). Sphincter disturbance was a poor prognostic indicator for continued ambulation after therapy. Alternatively, patients with caudal tumors may present with bladder difficulties and impotence. In such cases, large volumes of urine may be retained with secondary overflow incontinence. Rare clinical presentation of MESCC include the BrownSequard syndrome, zoster, and truncal ataxia (1–3). In the MSKCC series, 3 of 130 patients had a zoster eruption at the site of cord compression. Ataxia was the sole presenting symptom in 2% of patients in the MSKCC series and was present in an additional seven patients on examination. The mechanism of gait ataxia was not secondary to position sense abnormalities. gait ataxia may be secondary to compression of the spinocerebellar tracts and, when not associated with any pain or signs of myelopathy, may suggest cerebellar or cerebral disease. once a neurologic deficit appears, it can evolve rapidly to paraplegia over a period of hours to days (2).

LABoRAToRy STUDIES Cerebrospinal Fluid Cerebrospinal fluid (CSF) abnormalities in cases of MESCC are nonspecific, with the protein content typically elevated and occasionally a mild CSF pleocytosis. The CSF glucose is typically normal in cases of MESCC. Although not all authors have had similar experiences, some reports suggest that lumbar puncture may result in neurologic deterioration in patients with extramedullary neoplasms. In a retrospective series, hollis and colleagues (19) found that 14% of 50 patients had “significant neurologic deterioration” after lumbar puncture. No deterioration was seen in patients undergoing myelography via a cervical (C1–2) puncture. The mechanism for neurologic deterioration in such patients is uncertain but has been thought to be secondary to impaction of the spinal cord tumor, also known as spinal coning. Despite these occasional reports, the quantitative risk is difficult to establish. For example, among several hundred accumulated patients, no neurologic deterioration was reported

after myelography in several series (3). Because there is no specific information to be gained from CSF analysis alone that assists in the diagnosis of MESCC, a lumbar puncture should not be performed to “rule in” or “rule out” this diagnosis. Alternatively, if CSF analysis is indicated to diagnose infectious or neoplastic meningitis, close neurologic observation after lumbar puncture is indicated, as well as neurosurgical consultation when necessary. Furthermore, although MRI precludes the need for myelography in most patients, it is not available in a timely fashion in many centers, and patients with pacemakers or claustrophobia are not candidates for MRI. In this setting, myelography should not be delayed or avoided if it is needed to confirm the diagnosis of MESCC and plan therapy (see below) (20).

Diagnostic Imaging Because plain films are commonly performed in cancer patients with pain, the limitations of plain films that predict the presence or absence of MESCC are important to recognize. Approximately 50% of bone must be destroyed before plain x-ray abnormalities are seen. Despite this, the most characteristic appearances include a lytic or blastic metastasis, erosion of a pedicle, or vertebral body collapse that has been reported to be present in approximately 85% of adult patients with MESCC (1–3,16). The tumor type determines the frequency of bony abnormalities. Spinal metastases arising from carcinoma of the breast were much more likely to demonstrate abnormalities on plain x-ray (94%) than those arising from carcinoma of the lung (74%). Alternatively, plain films are abnormal in only approximately one-third of cases due to lymphoma and pediatric neoplasms because the tumor extends into the epidural space via the intervertebral foramina from paravertebral locations typically (15,21,22). Rodichok et al. (23,24) reported the frequency of epidural disease in cancer patients with back pain, no neurologic deficit, and metastasis on radiographs to be greater than 60%. In a study of 41 cancer patients with pain, weakness, and/or sensory loss undergoing myelography, Portenoy et al. (25) retrospectively determined the frequency of epidural tumor when clinical and plain x-ray findings were combined. The three segments of spine–cervical, thoracic, and lumbar were each identified as symptomatic or asymptomatic. The plain films of each segment were identified as showing the presence or absence of vertebral metastases. When plain films were positive at symptomatic segments, epidural disease was found on myelography in 86%, whereas epidural tumor was present in 8% of symptomatic segments with normal radiographs. At asymptomatic segments with abnormal plain films, epidural tumor was found in 43% and epidural disease was present in 3% of asymptomatic segments with normal radiographs. The appearance of the vertebral metastasis correlated with the risk of MESCC, according to a study by graus et al. (26). They found epidural lesions in 7% of vertebrae with tumor limited to the vertebral body without collapse, in 31%

49. Epidural Disease

with pedicle erosion, and in 87% with more than 50% collapse. Radionuclide bone scans are more sensitive than plain films in detecting bone metastases (except in multiple myeloma). Rodichok et al. (24) found that 54% of cancer patients with back pain and a positive myelogram had a normal bone scan, underscoring the fact that a normal bone scan in a symptomatic patient does not exclude MESCC. Alternatively, the development of a new spinal abnormality on bone scan in cancer patients has been found to be due to metastases in 72% of cases using plain films and CTs (27,28). The study also demonstrated that among 18 patients with abnormal bone scans and normal plain films, CT demonstrated metastases in 12 (67%), among whom 3 had epidural extension. This has led to the recommendation that new bone scan abnormalities in cancer patients should undergo plain films and CT or MRI to differentiate benign from metastatic disease (28). Although Portenoy et al. (25) found that a high frequency of false-positive results limited the usefulness of a positive bone scan, they did find that adding a negative study in an asymptomatic region with negative plain films reduced the risk from 0.02 to 0.001 using decision analysis. Spinal CT is more sensitive and specific than plain films or radionuclide scanning for identifying and distinguishing benign (e.g., spondylosis) from malignant disease and visualizing paravertebral masses (27–29). Spinal CT can be used to investigate unexplained pain or abnormalities on plain films and radionuclide studies in the cancer patient with a normal

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neurologic examination (27). Alternatively, in patients with clinical manifestations of MESCC, the entire spinal axis must be visualized, making spinal CT impractical, and without myelography, the sensitivity has not been adequately validated. Smoker et al. (30), Carmody et al. (31), and others (32,33) found unenhanced MRI equivalent to myelography in detecting MESCC and superior in detecting vertebral metastases and paravertebral masses. This has led to MRI becoming the preferred imaging method in centers where MRI is readily available. Alternatively, myelography should be performed when management is delayed by inability to schedule MRI in a timely fashion, in patients unable to undergo MRI (e.g., those with pacemakers and pain precluding recumbency), or when a technically adequate MRI cannot be obtained. Contrast-enhanced MRI is superior to unenhanced studies for detecting leptomeningeal metastases (34,35) and intramedullary tumors (36) and may provide additional information regarding epidural disease. Sze et al. (37) found that contrast enhancement may provide additional information in cases of MESCC. These authors found that regions of vertebral enhancement may correspond to areas of active tumor proliferation and may be useful for directing biopsies (Fig. 49-2).

FIgURE 49-2. Thoracic spine magnetic resonance image of a patient with spine metastasis from plasmacytoma. Sagittal (A) and axial (B) images show partial collapse of T-10 vertebral body and replacement of normal bone marrow with low-intensity tumor (solid arrows). A poorly defined hypointense focus in the T-8 vertebral body is present (open arrow, A). C. Both lesions are seen on the T2-weighted images. After administration of gadolinium-DTPA, the T1-weighted sagittal (D) and axial (E) scans show enhancement of the T-10 lesion (solid arrows) that extends to impinge on the spinal canal. Alternatively, the T-8 lesion is poorly seen because it is isointense with the remainder of the vertebral body (open arrow). E. The axial scan shows more enhancement (arrows) on the left (L) than on the right side of the vertebral body. Although tumor may permeate the entire vertebral body, multiple biopsies on the right were negative, whereas biopsy of the enhanced region on the left revealed tumor. (From Sze g, Krol g, Zimmerman RD, et al. gadolinium-DTPA: malignant extradural spinal tumors. Radiology 1988;167:217–223, with permission.)

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CLINICAL APPRoACh To ThE PATIENT SUSPECTED oF SPINAL METASTASES Cancer patients with clinical evidence of spinal metastases fall into four groups: patients with axial pain, normal neurologic examination, and abnormal plain radiographs; those with axial pain, normal neurologic examination, and normal radiographs; those with radiculopathy; and those with clinical manifestations of MESCC.

Neck/Back Pain, Normal Neurologic Examination, and Positive Plain Films The management of the cancer patient with neck or back pain, normal neurologic examination, and metastatic disease on conventional radiographs that explain the pain is controversial. Although myelography or MRI is recommended by many authors (38,39) to define radiotherapy ports, many physicians (24,40) irradiate such symptomatic vertebral metastases without further imaging. The arguments for and against definitive canal imaging have been discussed elsewhere (20). Ultimately, if definitive canal imaging is not performed, a greater onus is placed on the physician, initially to be confident of a normal neurologic examination and subsequently to be clinically vigilant to recognize early symptoms of MESCC.

Pain, Normal Neurologic Examination, and Normal Plain Films In cancer patients who experience unexplained axial or radicular pain, have a normal neurologic examination, and have negative plain films, MRI of the involved region is indicated to distinguish benign from malignant disease. If MRI is unavailable, CT and myelography should be considered.

Radiculopathy The manifestations of radiculopathy include pain, weakness, sensory loss, and/or reflex loss in a single nerve root distribution. Because MESCC is present in a large proportion of such cancer patients, MRI is indicated to identify adjacent levels of vertebral metastases that could cause confounding referred or radicular pain and to define the extent of epidural tumor to better plan radiotherapy ports or surgery. If MRI is unavailable, CT and myelography should be considered.

below) and undergo an emergent total spine MRI or myelography. In cases where the noncontrast spinal MRI is negative but the clinical suspicion of a spinal tumor remains, contrastenhanced MRI or myelography should be considered.

ThERAPy The management of metastatic epidural compression usually consists of corticosteroids and radiotherapy, but recent excellent results with anterior decompression in selected patients has renewed surgical interest.

Corticosteroids Corticosteroids alleviate pain acutely and improve neurologic function; they should be administered promptly to patients with clinical manifestations of metastatic epidural compression confirmed by diagnostic imaging or when strongly suspected on clinical grounds pending confirmation by diagnostic imaging (41,42). The dosage of dexamethasone, the most commonly reported corticosteroid used, remains controversial. Although some authors recommend 4 mg four times a day (41,43), laboratory studies have shown a dose-related benefit with dexamethasone (11) leading to the clinical use of a loading dose of 100 mg followed by 24 mg four times a day (38). A clinical trial found that high-dose glucocorticoid therapy is superior to no corticosteroids (42). In an effort to provide evidence-based guidelines for the treatment of MESCC, Loblaw and Laperriere found few studies of high methodologic quality (49a). They did conclude that there was “good evidence” to support the use of high-dose dexamethasone (96 mg/d) but “inconclusive evidence” for the use of 16 mg/d in combination with radiotherapy. They also concluded that there was “fair evidence” for not using steroids in patients who are nonparetic and ambulatory pretreatment. They also found “fair evidence” for administering radiotherapy to patients with subclinical spinal cord compression. Steroid administration is usually continued throughout radiation therapy at a tapering dose. one tapering schedule that has been useful in many patients is an approximately one-half to one-third reduction in dose every 2 to 4 days. A trial of escalated dose followed by taper may be attempted if tapering is not tolerated and neurologic deterioration occurs. The toxicities of high-dose corticosteroids must be considered.

Clinical Manifestations of Metastatic Epidural Spinal Cord Compression

Radiotherapy and Surgical Treatment

Cancer patients with mild, stable, or equivocal clinical manifestations of MESCC should undergo total spine MRI or myelography by the next day (20,38). Patients with significant neurologic signs or rapidly progressive neurologic deterioration should be given intravenous corticosteroids (see

Before the late 1970s, surgical laminectomy was commonly performed in patients with MESCC. In 1978, a retrospective study from MSKCC (2) demonstrated no benefit of laminectomy over radiotherapy. Since that time, other retrospective studies (44) and one small prospective study (45) failed to

49. Epidural Disease

demonstrate a difference in neurologic outcome between radiotherapy versus laminectomy followed by radiotherapy. Furthermore, laminectomy has been associated with a significant increase in morbidity, including spinal instability, in patients with MESCC (44). Thus, radiotherapy alone has become the primary definitive treatment for most patients with metastatic epidural compression (20,46). Neurologic prognosis primarily depends on the level of neurologic function at the initiation of radiotherapy. Studies from MSKCC (2) and the United Kingdom (44) conclude that the proportion of patients who are ambulatory after radiotherapy declines from more than 80% to less than 50% to less than 10% for patients who are ambulatory, paraparetic, and paraplegic, respectively, at the initiation of treatment. The efficacy of radiotherapy has more recently been further elucidated. In a prospective study of 139 male veterans with MESCC, Zaidat and Ruff (1a) reported that the likelihood of regaining ambulation increased if radiotherapy began within 12 hours of loss of ambulation and if bowel and bladder function were preserved. Patients were treated initially with 100 mg of dexamethasone intravenously followed by a tapering schedule (24 mg four times daily for 2 days, 12 mg four times daily for 2 days, 8 mg four times daily for 2 days, 4 mg four times daily for 2 days, 2 mg four times daily for 2 days, 1 mg four times daily for 2 days, and then 1 mg twice daily for 2 days) and radiotherapy extending two vertebral bodies beyond the upper and lower borders of the spinal epidural metastasis. The authors found that treatment reduced pain. The median survival for ambulatory patients was 104 weeks and that for nonambulatory patients was 6 weeks. Nearly one-half of nonambulatory patients died from complications of myelopathy and immobility. The study underscored the importance of early diagnosis and treatment. Because most cases of metastatic epidural compression begin as metastases to the vertebral body, it is the most common location for the initiation of cord compression. The principle of relieving the compression of the spinal cord from the effects of metastatic epidural compression by removing tumor or bone from the area of compression rather than just enlarging the spinal canal via laminectomy appears to be more rational. This has led to the procedure of anterior decompression (vertebral body resection) and stabilization (47–49). Nearly two decades ago, harrington (47) reported the neurologic outcome in 40 patients with major neurologic deficits preoperatively who underwent vertebral body resection followed by stabilization. of these patients, 21 had complete neurologic recovery postoperatively. In addition, of 13 plegic patients, 7 had no neurologic deficit postoperatively. he stressed, however, that many patients are too debilitated from their underlying disease to permit major surgery even in the presence of vertebral collapse or instability with neurologic compromise, and the procedure is hazardous. Moreover, the long-term benefit of surgery has been questioned (50).

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More recently, the advent of increasingly sophisticated spinal reconstructive procedures and instrumentation has led to the use of en bloc spondylectomy and other aggressive surgical procedures (51,52). The rationale for the use of these procedures generally fall into two categories, spinal and oncologic. The spinal indications are usually directed at achieving spinal stability, prevention of neurologic injury, and alleviation of pain. The oncologic indications that are considered are tumor control. Scoring systems have been studied in an attempt to select the patients who will most benefit from surgical intervention (51,53). To be valid, these scoring systems need to incorporate several issues, including spinal stability issues, neurologic status, primary tumor type, and extent of disease. No consensus has been reached as to the optimal criteria for selecting patients for surgery and, moreover, which surgical procedure should be used (51,54). Selection bias is always a concern when interpreting such studies. As harrington recognized more than a decade ago, the realization that many patients with spine metastasis are extremely ill and poor surgical candidates with a limited life expectancy is a significant problem in interpreting many of these studies. The large number of variables that impact on outcome is a major obstacle in the design and performance of a randomized controlled trial. Although many surgical indications still need to be defined, it has been suggested that surgery be considered in the following circumstances (20,38): (a) in patients without a diagnosis, (b) in patients who deteriorate neurologically due to metastatic epidural compression at a previously irradiated level, (c) in patients with progressive neurologic deterioration during radiotherapy despite large doses of corticosteroids, or (d) when symptomatic spinal instability or bone compression of neural structures occurs. Surgery is also considered in patients with radioresistant tumors and intractable pain. Patients with MESCC often receive chemotherapy and/or hormonal therapy in conjunction with radiotherapy or surgery. Whether symptomatic MESCC can be successfully treated with these agents alone is unknown. Recently, a case was reported of a patient with MESCC from prostate cancer presenting with back pain, leg weakness, and hypesthesia and urinary retention. The patient could not undergo radiotherapy or surgery and had clinical and radiologic improvement with hormonal therapy alone (55).

REFERENCES 1. Barron KD, hirano A, Araki S, et al. Experiences with metastatic neoplasms involving the spinal cord. Neurology 1959;9:91–106. 1a. Zaidat oo, Ruff RL. Treatment of spinal epidural metastasis improves patient survival and functional state. Neurology 2002;58:1360–1366. 2. gilbert RW, Kim Jh, Posner JB. Epidural spinal cord compression from metastatic tumor: diagnosis and treatment. Ann Neurol 1978;3:40–51.

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3. Byrne TN, Benzel E, Waxman Sg. Diseases of the spine and spinal cord. New york: oxford University Press, 2000. 4. Batson oV. The function of the vertebral veins and their role in the spread of metastases. Ann Surg 1940;112:138–148. 5. Coman DR, DeLong RP. The role of the vertebral venous system in the metastasis of cancer to the spinal column. Cancer 1951;4:610–618. 6. Arguello F, Baggs RB, Duerst RE, et al. Pathogenesis of vertebral metastasis and epidural spinal cord compression. Cancer 1990;65:98–106. 7. McAlhany hJ, Netsky Mg. Compression of the spinal cord by extramedullary neoplasms: a clinical and pathological study. J Neuropathol Exp Neurol 1955;14:276–287. 8. hashizume y, Iljima S, Kishimoto h, et al. Pencil-shaped softening of the spinal cord: pathologic study in 12 cases. Acta Neuropathol (Berl) 1983;61:219–224. 9. henson RA, Parsons M. Ischaemic lesions of the spinal cord: an illustrated review. Q J Med 1967;36:205–222. 10. Al-Mefty o, harkey Lh, Middleton Th, et al. Myelopathic cervical spondylotic lesions demonstrated by magnetic resonance imaging. J Neurosurg 1988;68:217–222. 11. Ushio y, Posner R, Posner JB, et al. Experimental spinal cord compression by epidural neoplasms. Neurology 1977; 27:422–429. 12. Siegal T, Siegal TZ, Sandbank U, et al. Experimental neoplastic spinal cord compression: evoked potentials, edema, prostaglandins, and light and electron microscopy. Spine 1987;12:440–448. 13. Siegal T, Siegal TZ. Participation of serotonergic mechanisms in the pathophysiology of experimental neoplastic spinal cord compression. Neurology 1991;41:574–580. 14. grant R, Papadopoulos SM, greenberg hS. Metastatic epidural spinal cord compression. Neurol Clin 1991;9: 825–841. 15. Lewis DW, Packer RJ, Raney B, et al. Incidence, presentation, and outcome of spinal cord disease in children with systemic cancer. Pediatrics 1986;78:438–442. 15a. Schiff D, o’Neill BP, Suman VJ. Spinal epidural metastasis as the initial manifestation of malignancy: clinical features and diagnostic approach. Neurology 1997;49:452–456. 16. Stark RJ, henson RA, Evans SJW. Spinal metastases: a retrospective survey from a general hospital. Brain 1982;105: 189–213. 17. Bernat JL, greenberg ER, Barrett J. Suspected epidural compression of the spinal cord and cauda equina by metastatic carcinoma. Cancer 1983;51:1953–1957. 18. Constans JP, Divitiis ED, Donzelli R, et al. Spinal metastases with neurological manifestations: review of 600 cases. J Neurosurg 1983;59:111–118. 19. hollis Ph, Malis LI, Zappulla RA. Neurological deterioration after lumbar puncture below complete spinal subarachnoid block. J Neurosurg 1986;64:253–256. 20. Byrne TN. Spinal cord compression from epidural metastases. N Engl J Med 1992;327:614–619. 21. Rodriguez M, Dinapoli RP. Spinal cord compression: with special reference to metastatic epidural tumors. Mayo Clin Proc 1980;55:442–448. 22. haddad P, Thaell JF, Kiely JM, et al. Lymphoma of the spinal extradural space. Cancer 1976;38:1862–1866. 23. Rodichok LD, harper gR, Ruckdeschel JC, et al. Early diagnosis of spinal epidural metastases. Am J Med 1981;70: 1181–1188. 24. Rodichok LD, Ruckdeschel JC, harper gR, et al. Early detection and treatment of spinal epidural metastases: the role of myelography. Ann Neurol 1986;20:696–702. 25. Portenoy RK, galer BS, Salamon o, et al. Identification of epidural neoplasm: radiography and bone scintigraphy in the symptomatic and asymptomatic spine. Cancer 1989;64: 2207–2213. 26. graus F, Krol g, Foley KM. Early diagnosis of spinal epidural metastases (SEM): correlation with clinical and radiological findings. Proc Am Soc Clin oncol 1985;4:269.

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C h a p t e r

5 0

w Neoplastic Meningitis Eric T. Wong Neoplastic meningitis is a multifaceted disorder increasingly seen in cancer patients. Typically, multiple levels of the neuraxis are affected at presentation. For example, some patients present with multifocal cranial neuropathies, whereas others are encephalopathic and incontinent. The proper diagnosis of this condition requires localization of neurologic impairment in the neuraxis and selecting the appropriate neuroimaging and cerebrospinal fluid (CSF) studies. Once neoplastic meningitis is diagnosed, treatment may consist of chemotherapy, either administered intrathecally or given at high doses systemically; radiation therapy; or both. The judicious use of these treatment modalities depends on a patient’s primary malignancy and the tempo of neurologic progression. For neuro-oncologists and oncologists at the front line, this chapter provides a conceptual framework for the proper diagnosis and management of this disorder.

NATuRAL HISTORy OF NEOpLASTIC MENINgITIS The overall incidence of neoplastic meningitis is less than 1% in general autopsy series (1) and 5% to 8% (1–3) among patients with a known diagnosis of cancer. However, these figures probably underestimate the true incidence of this disorder. As treatments for systemic cancer improve, relapse in the central nervous system (CNS), including neoplastic meningitis, has become increasingly common. This phenomenon can be traced to a number of factors, including the blood–brain barrier that excludes efficacious chemotherapies from the CNS, lack of physician awareness, and the increasing incidence of systemic malignancies, such as melanoma and lymphoma, that have a high propensity of brain metastases. Although neoplastic meningitis is mostly found as a late complication of systemic malignancies, about 6% of these patients are presented at initial diagnosis of cancer (4). In addition, patients with this disorder often have a high rate of concurrent parenchymal brain metastases (12% to 18%), epidural metastases (16%), or both (1%) (5,6). A high index of suspicion is often the first step in detecting this disorder. It is useful to think of neoplastic meningitis with respect to a patient’s primary malignancy, particularly in the categories

of solid tumors, hematologic malignancies, and primary brain neoplasms, because of differences in biologic behaviors and response to chemotherapy or radiation. The solid tumors that are frequently seen in patients with a known diagnosis of neoplastic meningitis are breast cancer (12% to 34%), lung cancer (10% to 26%), and melanoma (17% to 25%) (4). Small cell lung cancer, in particular, has a high propensity of leptomeningeal spread (11%), in addition to its high tendency of metastasizing to the brain parenchyma (7). Those with an unknown primary malignancy comprise less than 10% of the population. In hematologic malignancies, neoplastic meningitis in Hodgkin disease is rare. This risk is variable in non-Hodgkin lymphoma depending on the histology. Those with high-grade Burkitt’s type lymphoblastic lymphoma or immunoblastic lymphoma have high risk of neoplastic meningitis (8), and for this reason their CNS is prophylactically treated with intrathecal chemotherapy. Leptomeningeal spread in intermediate-grade non-Hodgkin lymphoma is variable, but diffuse histology, as in diffuse large cell lymphoma, has increased risk. In addition, disease staging is important, because patients with primary lymphoma in the orbits, paranasal sinus, testes, and bone marrow often have secondary lymphoma in the CNS in the form of neoplastic meningitis (9). In contrast, myeloid malignancies spreading to the CSF and subarachnoid space are less common. But the factor predictive of leptomeningeal infiltration is often cytogenetic abnormality rather than cellular morphology. Translocation of chromosome 8 to 21 (10), inversion or deletion of chromosome 16 (11,12), translocation of chromosomes 9 to 11 (13), and deletion of chromosome 11q23 (14) have been associated with neoplastic meningitis in myeloid malignancies. In the category of primary brain neoplasms, patients with primitive neuroectodermal tumors, including those with medulloblastoma, trilateral retinoblastoma, and supratentorial primitive neuroectodermal tumors, and are at risk of developing neoplastic meningitis. Similarly, there is a high propensity of initial spread of leptoinehingeal spread in patients diagnosed with nongerminomatous germ cell tumors, particularly with yolk sac, embryonal, and choriocarcinoma components. They should routinely undergo spinal magnetic resonance imaging (MRI) and lumbar puncture for disease staging in the CNS. Any pa683

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tient with high-grade astrocytoma is at risk of spreading into the leptomeninges, but this risk varies according to histology and duration of the disease. It is often seen in young patients, probably because they tend to live longer than older patients. Likewise, neuraxis dissemination can be a serious complication of ependymomas, particularly for anaplastic ependymomas and myxopapillary ependymomas (15). On rare occasions, even benign brain neoplasms, like juvenile pilocytic astrocytoma (16) and central neurocytoma (17), can spread to the leptomeningeal space, but routine staging of the neuraxis in patients with these tumors is not warranted.

DIAgNOSIS The presence of malignant cells in the CSF is considered a diagnostic gold standard and has high specificity for neoplastic meningitis (18). However, CSF cytology has low sensitivity (5). In patients with persistently negative CSF cytology for malignant cells, a clinical diagnosis of neoplastic meningitis can still be established based on a combination of clinical findings, neuroimaging features, and other CSF results. The clinical deficits in these patients often involve multiple levels in the neuraxis, probably because malignant cells can cause damage to any neuroanatomic structures within the subarachnoid space. In a carefully conducted clinical study of patients with leptomeningeal metastases from solid tumors, Wasserstrom et al. (5) noted headache, double vision, and a lower motor neuron pattern of weakness as three of the most frequent neurologic complaints, whereas delirium, oculomotor palsy, and reflex asymmetry were three common signs detected on examination. A key pattern found in these patients is the multifocality of signs and symptoms. For example, a patient may have a right facial palsy and a dropped reflex in the contralateral knee. Therefore, those having a malignancy with high propensity of CNS metastasis and a pattern of multifocal neurologic deficits should undergo further diagnostic evaluations for neoplastic meningitis. gadolinium-enhanced MRI is the diagnostic neuroimaging modality of choice for neoplastic meningitis. It is more sensitive than contrast-enhanced computed tomography in detecting subtle abnormalities, such as small nodules in the leptomeninges or ependyma and linear subarachnoid enhancements (19). It is important to distinguish subarachnoid enhancement from pachymeningeal enhancement of the dura (Fig. 50-1). Although not completely diagnostic based on neuroimaging alone, neoplastic meningitis is commonly associated with subarachnoid enhancement, either in linear or nodular forms. pachymeningeal enhancement of the dura has a much broader differential diagnoses in addition to neoplastic meningitis, including chronic meningeal infection, noninfectious inflammatory disorder, and neoplasms like en plaque meningiomas (20). Although the sensitivity and specificity of MRI for neoplastic meningitis is not available, about one-third of patients with cytologically negative CSF would

FIguRE 50-1. Subarachnoid enhancement (arrowhead) and pachymeningeal enhancement (arrow). (From Wong ET, O’Brien S. Leptomeningeal disease in pre-leukemic syndrome: Cytogenetic abnormality versus cellular morphology. Oncol Rep 1998;5:493–495, with permission.)

have MRI abnormalities consistent with neoplastic meningitis (21,22). The CSF offers important diagnostic and prognostic information in patients with neoplastic meningitis. But CSF cytology has a high false-negative rate, and the sensitivity from an initial lumbar puncture is only 54% (5). The yield of a positive cytology, however, can be improved by getting at least 10.5 mL of CSF, taking CSF from a site near the neurologic deficits or MRI abnormality, processing the CSF immediately, and performing a second spinal tap (23). In particular, a second spinal tap could increase the yield of positive malignant cells in the CSF to 82% (5), whereas a third tap would increase the yield further to 90% range (24). Another method to increase the diagnostic yield is a cistern puncture at C1-2 level, especially when cerebral or cranial nerve abnormalities are detected on neurologic examination or on neuroimaging (25). However, even with diligent evaluation of CSF, one-third of patients with neoplastic meningitis would have persistently negative CSF cytology (21). Biochemical markers in the CSF, such as protein level, lactate dehydrogenase, ␤2-microglobulin, ␤-glucuronidase, and carcinoembryonic antigen, typically have high sensitivity for neoplastic meningitis but have low specificity. These markers are often elevated in other CNS diseases, such as bacterial infection or encephalitis from viral causes. A high protein level in the CSF, in particular, is a poor prognostic marker for patients with neoplastic meningitis from solid tumors and from lymphoma (24,26). It is important to mention that

50. Neoplastic Meningitis

human chorionic gonadotropin-␤ and ␣-fetoprotein levels are often elevated in nongerminomatous germ cell tumors of the CNS as well as in brain metastases from testicular cancer and choriocarcinoma, and they are good markers for detecting early disease recurrence. CSF markers detected by polymerase chain reaction may be useful clinically because this technique is sensitive (27), but its specificity is unclear.

pATHOLOgy Neoplastic meningitis, by definition, is defined by the presence of neoplastic cells in the subarachnoid space of the neuraxis. Because there is no convincing demonstration of actively migrating tumor cells in human or animal studies, how the neoplastic cells get into the CSF and subarachnoid space remains open to debate. However, postmortem observations suggest multiple routes in which tumor cells could penetrate to the CSF and subarachnoid space. First, neoplastic cells can reach there by way of the penetrating arteries to the brain, as neoplastic cells are found aggregated in choroids plexus from autopsy specimens and from tumor deposits on the dura with local extension to the subarachnoid space (4). Another hematogenous route is the venous plexus of the spine, like the extensive Batson plexus at the lumbar and thoracic spinal levels (28,29). The perivascular lymphatics in the spine and skull can also harbor malignant cells, and these lymphatic channels are found near intervertebral foramina. A third route could originate from spinal and paraspinal metastases, as tumor cells can mi-

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grate to the intradural space by propagating proximally along the perineurial tumor deposits compressing surface vasculatures on nerve roots, cranial nerves, and cauda equina could give rise to similar ischemic neuropathy while direct consequence of compression would result in demyelimaningraphy (4) of spinal nerve roots (30). Likewise, skull base metastases can migrate along the cranial nerves to the leptomeninges. There are multiple mechanisms in which tumor cells in the subarachnoid space can cause neurologic deficits. First, these cells can penetrate the Virchow-Robin spaces and choke off blood supply to the underlying brain parenchyma, resulting in focal cerebral infarction (31). Neuropathy can also occur as a result of direct compression of spinal nerve roots and cranial nerves by neoplastic cells causing demyelination (4). Furthermore, neoplastic meningitis can cause hydrocephalus, manifesting clinically as encephalopathy, lethargy, or apathy (Fig. 50-2). In the case of obstructive hydrocephalus, the blockage of CSF outflow by tumor aggregates occurs at the foramina of Magendie and Luschka (32). In communicating hydrocephalus, the obstruction is most likely at the arachnoid granulation responsible for CSF absorption in communicating hydrocephalus. It is important to note that marked dilation of the lateral ventricles or the temporal horns may not occur in patients who had cranial irradiation, probably from radiation-induced stiffening of the ventricles (33).

FIguRE 50-2. Obstructive hydrocephalus on computed tomography (left) and papanicolaou staining of malignant cells (right). (From Wong ET, Joseph JJ. Meningeal carcinomatosis in lung cancer. J Clin Oncol 2000;18:2926–2927, with permission.)

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FIguRE 50-3. Ommaya reservoir in ventricle. (From Ratcheson RA, Ommaya AK. Experience with the subcutaneous cerebrospinalfluid reservoir: preliminary report of 60 cases. N Engl J Med 1968; 279:1025–1031, with permission.)

TREATMENT Neurosurgical intervention in patients with neoplastic meningitis is limited to placement of a ventriculoperitoneal shunt to relieve obstructive or communicating hydrocephalus and insertion of a ventricular access device (VAD), like an Ommaya or Leory reservoir (Fig. 50-3), for intrathecal administration of chemotherapy. Although the complication rate is low in experienced hands, care must be taken during surgical placement of the catheter to the frontal horn anterior to the third ventricle and during reservoir usage. The incidence of reservoir catheter malfunction ranges from 7% to 23% (34,35), and it is primarily due to catheter misplacement. No chemotherapy should be injected into the reservoir when the catheter is misplaced; otherwise, chemotherapyinduced neurotoxicity may occur. Furthermore, bacterial infection can occur in 15% of patients with a VAD (34), and the risk may be increased in patients with a weakened immune system or those having poor scalp hygiene. The organisms isolated from the CSF are frequently Staphylococcus epidermidis or Corynebacterium species. The infection may clear after treatment with systemic antibiotics. If it does not clear, the reservoir should be removed and the patient placed on antibiotics for at least 1 month. Hair or scalp washing with chlorhexidinegluconate (Hibiclens) at home on the day of the procedure and meticulous sterilization with Betadine followed by sterile techniques during access may help to lower the risk of bacterial infection.

For those having communicating hydrocephalus and neoplastic meningitis from chemoresponsive malignancies, a VAD connected to a shunt catheter via an on–off valve is feasible for both intrathecal chemotherapy and shunting of CSF; an important step is to turn off the valve for 1 hour after administration of chemotherapy so that it can equilibrate in the CSF. For patients with neoplastic meningitis, the judicious use of chemotherapy and radiation may offer the best clinical outcome and minimum neurotoxicity. How to combine the two treatment modalities depends on the tempo of neurologic progression, the sensitivity of the primary malignancy to chemotherapy, and whether or not a block in CSF flow exists. For solid tumors, radiation is frequently used in a palliative fashion, with the exception of the more chemoresponsive breast cancer, testicular cancer, and small cell lung cancer. Because these three primary malignancies are significantly more chemoresponsive than other solid tumors, chemotherapy may be considered first and radiation used in a consolidative fashion (7) if a patient’s neurologic deficits are not progressing rapidly. In contrast, those with rapidly deteriorating neurologic signs should undergo palliative whole brain irradiation or selected spinal irradiation to halt further neurologic deterioration. For neoplastic meningitis from leukemia and lymphoma, chemotherapy should be considered first because these malignancies have a high rate of response to intrathecal and systemically administered high-dose chemotherapy. Radiation in these cases should be given in a consolidative fashion if possible. For primary brain neoplasms such as primitive neuroectodermal tumors, nongerminomatous germ cell tumors, and high-grade gliomas, intrathecal chemotherapy is used in a palliative fashion because these patients have poor prognosis to begin with. Radiation therapy for bulky leptomeningeal disease may be necessary before intrathecal administration of chemotherapy. It has been shown in primates that chemotherapy administered into the ventricles can only penetrate the brain parenchyma up to 3 mm from the ependymal surface (36). As a result, intrathecal chemotherapy is expected to have no impact on parenchymal brain metastasis or nodular leptomeningeal metastasis with a diameter of 3 mm or greater. In these situations, stereotactic radiosurgery, whole brain radiation, or involved-field radiation therapy should be considered. Another situation in which radiation may help the delivery of intrathecal chemotherapy is by relieving a CSF flow block (Fig. 50-4). This phenomenon of CSF flow block occurs because tumor cells are lodged in the septations of the subarachnoid space. It may not be seen on gadolinium-enhanced MRI of the brain or spine. But when indium-111 or technetium-99 is injected into the subarachnoid space, areas of impaired CSF flow can be detected (37,38). Therefore, to ensure that intrathecally administered chemotherapies are distributed evenly in the neuraxis, a CSF flow study is necessary and local-field radiation may be needed to relieve a block.

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FIguRE 50-4. Cerebrospinal fluid flow block. In the CSF flow study performed 6 hours after indium-111 injection into VAD (left), most of the radionuclide signal is located in the lateral ventricles and fourth ventricle. When the flow study was repeated 48 hours after injection (right), no detectable radionuclide signal (arrow heads) was noted in the left convexity of the brain, suggesting a flow block.

The efficacy of chemotherapy against neoplastic meningitis can be maximized by intraventricular administration and by increasing the duration of drug exposure using a concentration ⫻ time approach. Intraventricularly administered chemotherapy via a VAD has several advantages: (a) it has better CSF distribution compared with intralumbar injection (39,40), resulting in improved efficacy (41); (b) the chemotherapy is always in the CSF, whereas an intralumbar approach may result in injection of chemotherapy to the subdural or epidural space (42); and (c) it is relatively pain free. Another important consideration is extending the exposure time to intrathecally administered chemotherapy. This can be accomplished by administering chemotherapy in a concentration ⫻ time approach, in which lower doses of chemotherapy are given at increased frequency to establish a basal concentration of chemotherapy in the CSF. When methotrexate is given in this fashion at a dose of Img every 12 hours for 3 days, it has the same efficacy as the singledose approach while patients experienced less neurotoxicity (43). This concentration ⫻ time approach can be simulated by an extended-release chemotherapy like liposomal cytarabine. For lymphomatous meningitis, liposomal cytara-

bine is superior to cytarabine in effecting a cytologic response (44). Drugs routinely used for intrathecal administration include methotrexate (45), cytarabine (44), thiotepa (46), and liposomal cytarabine (44,47,48). For solid tumors, the efficacy of methotrexate has been compared with thiotepa for neoplastic meningitis, and neither drug is superior to the other (49). In another trial testing combination intrathecal chemotherapies, the efficacy of methotrexate ⫹ cytarabine is not superior to methotrexate alone (50). These data suggest that conventional drugs for intrathecal administration all have similar efficacy against neoplastic meningitis, and combination intrathecal chemotherapies is not better than single-agent chemotherapy. Methotrexate is the most extensively studied drug for neoplastic meningitis, and its pharmacokinetics is best understood. When administered systemically at 8 g/m2, peak CSF methotrexate is detectable at cytotoxic levels of more than 1 ⫻ 10⫺6 molar concentration (51). Likewise, cytotoxic concentrations of cytarabine in the CSF can be achieved with the systemic administration of high-dose cytarabine at 1 to 3 g/m2 (52,53). At a dose of 3 g/m2, cytarabine administered systemically can be efficacious against neoplastic meningitis from myeloid leu-

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kemia (54). Thiotepa is too myelosuppressive to be administered systemically at high doses.

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22. Freilich RJ, Krol g, DeAngelis LM. Neuroimaging and cerebrospinal fluid cytology in the diagnosis of leptomeningeal metastasis. Ann Neurol 1995;38:51–57. 23. glantz MJ, Cole BF, glantz LK, et al. Cerebrospinal fluid cytology in patients with cancer, minimizing false-negative results. Cancer 1998;82:733–739. 24. Balm M, Hammack J. Leptomeningeal carcinomatosis: presenting features and prognostic factors. Arch Neurol 1996;53: 626–632. 25. Rogers LR, Duchesneau pM, Nunez C, et al. Comparison of cisternal and lumbar CSF examination in leptomeningeal metastasis. Neurology 1992;42:1239–1241. 26. Blay J-y, Conroy T, Chevreau C, et al. High-dose methotrexate for the treatment of primary cerebral lymphomas: analysis of survival and late neurologic toxicity in a retrospective series. J Clin Oncol 1998;16:864–871. 27. gleissner B, Siehl J, Korfel A, et al. CSF evaluation in primary CNS lymphoma patients by pCR of the CDR III IgH genes. Neurology 2002;58:390–396. 28. Batson OV. The role of the vertebral veins and their role in the spread of metastases. Ann Surg 1940;112:138–149. 29. Batson OV. The role of the vertebral veins in metastatic process. Ann Intern Med 1942;16:38–45. 30. Kokkoris Cp. Leptomeningeal carcinomatosis: how does can reach the pia-arachnoid. Cancer 1983;51:154–160. 31. Klein p, Haley EC, Wooten gF, et al. Focal cerebral infarctions associated with perivascular tumor infiltrates in carcinomatous leptomeningeal metastases. Arch Neurol 1989;46:1149–1152. 32. Wong ET, Joseph JJ. Meningeal carcinomatosis in lung cancer. J Clin Oncol 2000;18:2926–2927. 33. Thiessen B, DeAngelis DM. Hydrocephalus in radiation encephalopathy: results of ventriculoperitoneal shunting. Arch Neurol 1998;55:705–710. 34. Ratcheson RA, Ommaya AK. Experience with the subcutaneous cerebrospinal-fluid reservoir: preliminary report of 60 cases. N Engl J Med 1968;279:1025–1031. 35. Bleyer WA, pizzo pA, Spence AM, et al. The Ommaya reservoir: newly recognized complications and recommendations for insertion and use. Cancer 1978;41:2431–2437. 36. Blasberg Rg, patlak C, Fenstermacher JD. Intrathecal chemotherapy: brain tissue profile after ventriculo-cisternal perfusion. J pharmacol Exp Ther 1975;195:73–83. 37. Mason Wp, yeh SDJ, DeAngelis LM. 111Indium-diethylenetriamine pentaacetic acid cerebrospinal fluid flow studies predict distribution of intrathecally administered chemotherapy and outcome in patients with leptomeningeal metastases. Neurology 1998;50:438–443. 38. Chamberlain MC, Corey-Bloom J. Leptomeningeal metastases: 111 indium-DTpA CSF flow studies. Neurology 1991;41: 1765–1769. 39. Shapiro WR, young DF, Mehta BM. Methotrexate: distribution in cerebrospinal fluid after intravenous, ventricular and lumbar injections. N Engl J Med 1975;293:161–166. 40. Chamberlain MC, Kormanik p, Howell SB, et al. pharmacokinetics of intralumbar DTC-101 for the treatment of leptomeningeal metastases. Arch Neurol 1995;52:912–917. 41. Bleyer WA, poplack Dg. Intraventricular versus intralumbar methotrexate for central nervous system leukemia: prolonged remission with the Ommaya reservoir. Med pediatr Oncol 1979;6:207–213. 42. Rieselbach RE, DiChiro g, Freireich EJ, et al. Subarachnoid distribution of drugs after lumbar injection. N Engl J Med 1962;267:1273–1278. 43. Bleyer WA, poplack Dg, Simon RM, et al. “Concentration ⫻ time” methotrexate via a subcutaneous reservoir: a less toxic regimen for intraventricular chemotherapy of central nervous system neoplasms. Blood 1978;51:835–842. 44. glantz MJ, LaFollette S, Jaeckle KA, et al. Randomized trial of a slow-release versus a standard formulation of cytarabine for the intrathecal treatment of lymphomatous meningitis. J Clin Oncol 1999;17:3110–3116.

50. Neoplastic Meningitis 45. Ongerboer BW, Somers R, Nooyen WH, et al. Intraventricular methotrexate therapy of leptomeningeal metastasis from breast carcinoma. Neurology 1983;33:1565–1572. 46. gutin pH, Weiss HD, Wiernik pH, et al. Intrathecal N, N’, N”-triethylenethiophosphoramide [thio-tepa (NSC 6396)] in the treatment of malignant meningeal disease: phase I-II study. Cancer 1976;38:1471–1475. 47. glantz MJ, Jaeckle KA, Chamberlain MC, et al. A randomized controlled trial comparing intrathecal sustained-release cytarabine (DepoCyt) to intrathecal methotrexate in patients with neoplastic meningitis from solid tumors. Clin Cancer Res 1999;5:3394–3402. 48. Jaeckle JA, phuphanich S, van den Bent MJ, et al. Intrathecal treatment of neoplastic meningitis due to breast cancer with a slow-release formulation of cytarabine. Br J Cancer 2001;84:157–163. 49. grossman SA, Finkelstein DM, Ruckdeschel JC, et al. Randomized prospective comparison of intraventricular methotrexate and thiotepa in patients with previously untreated neoplastic meningitis. J Clin Oncol 1993;11:561–569. 50. Hitchens R, Bell D, Wood R, et al. A prospective randomized trial of single-agent versus combination chemotherapy in meningeal carcinomatosis. J Clin Oncol 1987;5:1655–1662. 51. glantz MJ, Cole BF, Recht L, et al. High-dose intravenous methotrexate for patients with nonleukemic leptomeningeal cancer: is intrathecal chemotherapy necessary? J Clin Oncol 1998;16:1561–1567. 52. Slevin ML, piall EM, Herne gW, et al. Effect of dose and schedule on pharmacokinetics of high-dose cytosine arabinoside in

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C h a p t e r

5 1

w Paraneoplastic Disorders of the Nervous System

Edward J. Dropcho Paraneoplastic disorders are rare compared with metastases, neurotoxicities of chemotherapy and radiotherapy, and other neurologic complications of systemic cancer, but they have clinical importance for several reasons: 1. Paraneoplastic disorders often cause severe and permanent neurologic morbidity. 2. in most patients with paraneoplastic disorders the neurologic symptoms are the presenting feature of an otherwise undiagnosed tumor. 3. Among patients with a known diagnosis of cancer, the paraneoplastic syndromes are an important part of the differential diagnosis of neurologic dysfunction. 4. Prompt recognition of a paraneoplastic disorder maximizes the likelihood of successful tumor treatment and a favorable neurologic outcome. For each paraneoplastic neurologic disorder there is an overrepresentation of one or more particular neoplasms. Small cell lung carcinoma is the tumor most often associated with paraneoplastic phenomena in adults, although the actual incidence of paraneoplastic disorders among patients with this tumor is no more than 1% to 3% (1,2). other tumors overrepresented among patients with paraneoplastic syndromes include breast carcinoma, ovarian carcinoma, hodgkin’s lymphoma, germ cell tumors, and thymoma. Evidence gathered over the last 20 years indicates that several neurologic paraneoplastic disorders are autoimmune diseases. the central theory of autoimmunity for paraneoplastic disorders postulates that tumor cells express “onconeural” antigen(s) identical or antigenically related to molecules normally expressed by neurons and that in rare instances an autoimmune response initially arising against the tumor subsequently attacks neurons expressing the same or related antigen(s). Since the mid-1980s the list of antineuronal autoantibodies identified in the sera of patients with paraneoplastic disorders has grown steadily (tables 51-1 and 51-2). the autoantibodies that are most specifically linked to a particular

neurologic clinical syndrome are the voltage-gated calcium channel antibodies associated with Lambert-Eaton myasthenic syndrome, the anti-Yo antibodies associated with cerebellar degeneration, and the antiretinal antibodies associated with retinal degeneration. Most of the other antibodies show “pan-neuronal” reactivity and are associated with a variety of clinical syndromes or with a multifocal encephalomyeloneuritis. Conversely, a given clinical syndrome may be associated with one of a number of different autoantibodies. Antineuronal antibodies are a useful, though not infallible clinical tool, both for diagnosing a patient’s condition as paraneoplastic and for guiding the search for specific underlying tumor types. With a few exceptions, antineuronal antibodies are believed to be markers for autoimmunity but probably do not directly mediate neuronal injury. For some central nervous system (CNS) syndromes recent evidence implicates cellular-mediated autoimmune mechanisms, but the exact immunopathogenetic mechanisms underlying CNS paraneoplastic syndromes remain unclear (25).

ENCEPhALoMYELitiS/SubACutE SENSorY NEuroNoPAthY Clinical Features A large proportion of patients with paraneoplastic disorders can be grouped into the clinicopathologic entity of paraneoplastic encephalomyelitis (PEM). Small cell lung carcinoma is by far the tumor most commonly associated with PEM, with a scattering of patients with a variety of other neoplasms. PEM is characterized clinically and pathologically by patchy, multifocal involvement of any or all areas of the cerebral hemispheres, limbic system, cerebellum, brainstem, spinal cord, dorsal root ganglia, and autonomic ganglia. Neuronal loss is accompanied by a variable degree of perivascular and leptomeningeal infiltration of mononuclear cells, including t and b lymphocytes and plasma cells (26,27).

691

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Cancer of the Nervous System

w tAbLE 51-1

Paraneoplastic Disorders and Autoantibodies

Clinical Syndrome

Associated tumor(s)

Autoantibodies*

Multifocal Encephalomyelitis/sensory neuronopathy

SCLC

Anti-hu (ANNA-1) Anti-CV2 (CrMP-5) Anti-amphiphysin ANNA-3 Anti-Ma, anti-hu, anti-CV2

Various carcinomas Cerebellar degeneration

breast, ovarian, others SCLC, others hodgkin’s lymphoma

Limbic encephalopathy

SCLC

testicular, breast thymoma

Anti-Yo, anti-Ma, anti-ri (ANNA-2) Anti-hu, anti-CV2, PCA-2, ANNA-3 Anti-tr, anti-mGlur1 Anti-hu, anti-CV2, PCA-2, ANNA-3 Anti-amphiphysin Anti-ta Anti–voltage-gated potassium channel

opsoclonus–myoclonus

breast, ovarian SCLC Neuroblastoma testicular, others

Anti-ri, anti-Yo Anti-hu, anti-amphiphysin Anti-hu Anti-ta

Extrapyramidal syndrome

SCLC

Anti-CV2, anti-hu

Stiff-person syndrome

breast, SCLC

Anti-amphiphysin breast Anti-GAD

retinal degeneration

SCLC, others Melanoma

Anti-recoverin Anti-bipolar cell

Neuromyotonia

thymoma

Anti–voltage-gated potassium channel

Sensorimotor polyneuropathy

SCLC, others

Anti-hu, anti-CV2, ANNA-3

Autonomic insufficiency

SCLC

Anti-hu

Lambert-Eaton myasthenic syndrome

SCLC

Anti–voltage-gated calcium channel

Abbreviations: SCLC, small cell lung carcinoma. *For each of the clinical syndromes, there are patients who are antibody-negative or who have an unnamed “atypical” autoantibody that does not fit the well-characterized patterns listed.

in most patients with PEM the neurologic syndrome is the presenting feature of an otherwise occult tumor and precedes the discovery of the neoplasm by an average of several months (28–31). the most common clinical manifestation of PEM is subacute sensory neuronopathy (SSN) reflecting involvement of the dorsal root ganglia (32). Early symptoms are patchy or asymmetric numbness and paresthesias, often involving face, trunk, or proximal limbs. the symptoms eventually spread to involve all limbs. burning dysesthesias and severe aching or lancinating pain are common. Examination reveals severe sensory ataxia, predominant impairment of vibration sense and proprioception, frequent pseudoathetosis, and hypoactive or absent

muscle stretch reflexes. Most patients cannot walk unassisted because of pain and profound loss of proprioception. SSN is not clinically evident or is a minor component in approximately 20% to 30% of patients with PEM (28–31). these patients have a clinical diagnosis of subacute cerebellar degeneration, limbic encephalitis, brainstem encephalitis, motor neuron disease, extrapyramidal movement disorder (33,34), or epilepsia partialis continua (35). regardless of individual patients’ predominant clinical manifestations, nearly all display signs and symptoms of multifocal involvement of the CNS and dorsal root ganglia. Patients may also show involvement of the peripheral nervous system, including sensorimotor poly-

51. Paraneoplastic Disorders of the Nervous System

w tAbLE 51-2

693

CNS Paraneoplastic Antibodies and Antigens

Antibody Anti-hu (ANNA-1) (3,4) Anti-Yo (PCA-1) (5–7) Anti-ri (ANNA-2) (8,9)

Anti-CV2 (CrMP-5) (10–12)

immunocytochemical Staining Pattern

Protein Antigen(s)

Nucleus > cytoplasm of all neurons in CNS, dorsal root ganglia, myenteric ganglia

Group of closely related 35- to 40-kDa rNA-binding proteins, including huD, huC, hel-N1

Purkinje cell cytoplasm and proximal dendrites

55- to 60-kDa cdr2 protein

Nucleus ⬎ cytoplasm of all neurons in CNS (not peripheral neurons)

two groups of closely related 50- to 55kDa and 70- to 80-kDa rNA-binding proteins including Nova-1 and Nova-2 66 kDa CV2 protein, 62 kDa CrMP-5 protein

Anti-tr (13, 14)

Cytoplasm of neurons and a subpopulation of oligodendrocytes

Anti-Ma (15,16)

Purkinje cell cytoplasm and dendritic trees

unknown

Anti-ta (16,17)

Nuclei and nucleoli of all neurons in CNS, dorsal root ganglia, myenteric ganglia

37-kDa and 40-kDa Ma1 and Ma2 proteins

PCA-2 (18)

Nuclei and nucleoli of all neurons

Antiamphiphysin (19–21)

Cytoplasm and dendrites of Purkinje cells, cytoplasm of neurons in granular layer and dentate nuclei

ANNA-3 (22)

Diffuse neutrophil staining in cerebrum and cerebellum

Anti-recoverin (23,24)

neuropathy, mononeuritis multiplex, autonomic system failure, or Lambert-Eaton syndrome (see later discussion). brain Mr scans in patients with PEM/SSN are usually normal, except in patients with predominant cerebellar involvement or limbic encephalopathy (see later). Most patients have abnormal cerebrospinal fluid (CSF), including elevated protein, mild mononuclear pleocytosis, elevated igG index, and/or oligoclonal bands, although at least 10% of patients have normal CSF (28). Electrophysiologic studies in paraneoplastic SSN show severely reduced amplitude or complete absence of sensory nerve potentials, with normal or only slightly reduced sensory nerve conduction velocities if a response is elicitable (32). Motor nerve conduction studies may be abnormal, but to a much lesser degree (see later). by far the most common clinical course of PEM/SSN is deterioration over a period of weeks to months, and then stabilization at a level of severe neurologic disability, regardless of treatment. Subsequent stepwise or gradual neurologic deterioration is less common and tends to occur in patients with less than complete response of the associated small cell lung cancer to treatment (30). Sudden death is not rare and is presumed to be caused by acute dysautonomia. Conversely, a few patients with minimal CNS manifestations and a sensory

40-kDa Ma2 protein 280-kDa protein

125-kDa amphiphysin associated with synaptic vesicles

neuronopathy takes a relatively indolent course independent of any treatment (36).

Autoimmunity Some patients with PEM/SSN have circulating antineuronal autoantibodies, the most common of which are polyclonal igG anti-hu antibodies (also called ANNA-1 antibodies) (28–31) (tables 51-1 and 51-2). More than 90% of reported patients with anti-hu antibodies and PEM/SSN have small cell lung carcinoma, with individual case reports of patients with neuroblastoma, non–small cell lung carcinoma, or carcinoma of the breast or prostate. in a disproportionately high proportion of patients with PEM/SSN and anti-hu antibodies, the associated small cell lung carcinoma is limited to the lung and mediastinum, as compared with neurologically unaffected small cell cancer patients (37). Patients with anti-hu antibody-associated PEM/SSN appear to have a longer survival than those with small cell lung cancer but no paraneoplastic syndrome (30). this is circumstantial evidence supporting an antitumor immune response, but an alternative explanation is that the occurrence of neurologic symptoms leads to early tumor diagnosis and treatment.

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Cancer of the Nervous System

Anti-hu antibodies react with a group of closely related 35to 40-kDa rNA-binding proteins, several of which have been cloned (4,25). Alternative splicing of mrNA transcripts produces multiple isoforms of the autoantigen proteins, which probably differ in their neuronal distribution and antigenicity (38,39). Expression of one or more hu autoantigens is common but is not universal among small cell lung carcinomas, including tumors from patients with PEM/SSN and anti-hu antibodies as well as tumors from neurologically unaffected patients (40,41). No definite genetic mutations or antigenic differences have been identified between hu autoantigen(s) expressed by tumors from PEM/SSN patients and the proteins from neurologically unaffected small cell carcinoma patients (38). occasionally, patients are false-positive or false-negative for anti-hu antibodies. high-titer anti-hu antibodies have been reported in a few patients with encephalomyeloneuritis in whom no tumor was detected, even at autopsy (28). Depending on the methodology used, low titers of anti-hu antibodies can be detected in the sera of 15% to 40% of patients with small cell lung carcinoma but no overt clinical paraneoplastic disorder (42,43). there is currently no definite evidence that anti-hu antibodies directly cause neuronal injury. Animals actively immunized with recombinant hu antigens produce anti-hu antibodies, but they do not develop the clinical or pathologic features of PEM/SSN (44). recent studies show cytotoxic anti-hu t lymphocytes in patients and other evidence for cellular immune mechanisms (45–48), but the exact immune-mediated mechanism(s) of neuronal injury in PEM/SSN remain unknown. A minority of patients with PEM/SSN and small cell lung carcinoma or other tumor either has no detectable antineuronal autoantibodies (49) or has antibodies with patterns of immunoreactivity distinct from anti-hu antibodies (tables 51-1 and 51-2). these include antibodies against the synaptic vesicle-associated protein amphiphysin (20,21,50), antiCV2 (CrMP-5) antibodies directed against a group of proteins expressed by neurons and oligodendrocytes (10,12), anti-Ma antibodies against a 40-kDa neuronal protein (15), ANNA-3 antibodies (22), and other unnamed antibodies. the clinical features of PEM/SSN in anti-hu antibodynegative patients do not reliably differ from the spectrum of signs and symptoms seen in patients with anti-hu antibodies.

Patient Management Patients with encephalomyelitis or sensory neuronopathy, with or without anti-hu antibodies, should undergo a workup looking for small cell lung carcinoma. Chest computed tomography (Ct) or magnetic resonance imaging (Mri) is clearly more sensitive than “plain” chest X-rays in detecting a neoplasm (51). Fluorodeoxyglucose positron emission tomography (PEt) may detect lung neoplasms in patients with suspected paraneoplastic syndromes but unrevealing or equivocal chest

Ct or Mr scans (52,53). it is not uncommon for patients’ initial evaluation for an occult lung tumor to be unrevealing; in these patients the workup should be repeated every several months and should include workup for other tumors, including lymphoma and carcinoma of the breast or prostate. Patients with PEM/SSN rarely show significant neurologic improvement despite successful tumor treatment and/or a variety of immunosuppressive therapies, including prednisone, cyclophosphamide, intravenous immunoglobulin (iVig), or plasmapheresis (28,30,31,54,55). it is difficult to interpret reports of “stabilization” of PEM/SSN with immunosuppression, considering that the disease in most patients stabilizes spontaneously. there are a few patients with PEM/SSN and anti-hu antibodies, fewer than 10%, who do in fact have significant neurologic improvement with prednisone and/or iVig. there is nothing distinctive about the clinical presentation of the few responders (54,56). Patients who receive early tumor treatment and immunosuppressive therapy do not seem to fare significantly better than patients with a longer lag between onset of neurologic symptoms and initiation of therapy. it is likely that, at the time of diagnosis, many patients with PEM/SSN have already suffered irreversible neuronal damage or loss. PEM/SSN associated with anti-hu antibodies may rarely spontaneously improve without any specific treatment (57). there is theoretical concern that if the paraneoplastic disorder arises from an immune response directed against the tumor, attempts to treat the neurologic disorder with immunosuppression may adversely affect the evolution of the tumor. there is no evidence that immunosuppressive treatment in patients with PEM/SSN and anti-hu antibodies is associated with a worse tumor outcome (30).

LiMbiC ENCEPhALitiS Clinical Features Approximately 50% to 60% of reported patients with paraneoplastic limbic encephalitis have small cell lung carcinoma (58,59). other associated neoplasms include testicular germ cell tumors (60), thymoma (61–63), hodgkin’s lymphoma (64), and other carcinomas of the lung, colon, breast, kidney, and genitourinary system. in most patients with small cell lung cancer the limbic encephalitis is part of multifocal PEM. Some patients with small cell lung cancer, and most patients with other tumors, have clinically “pure” limbic system involvement. in the majority of patients the neurologic symptoms are the presenting feature of the associated neoplasm. Limbic encephalitis presents as a subacute amnestic syndrome or affective disorder; most patients eventually develop features of both (59). the memory loss includes short-term anterograde amnesia and a variable period of retrograde amnesia. Denial of the deficit and confabulation are common. the affective disorder usually includes some combination of depression, anxiety,

51. Paraneoplastic Disorders of the Nervous System

emotional lability, and personality change. hallucinations and paranoid delusions may occur. Generalized or partial complex seizures are fairly common and may be the initial neurologic feature. Less common manifestations of limbic or diencephalic dysfunction include abnormal sleep–wake cycles, disturbed temperature regulation, labile blood pressure, inappropriate secretion of antidiuretic hormone, and elements of the Klüverbucy syndrome, such as hyperphagia and hypersexuality. Mr imaging in paraneoplastic limbic encephalitis may be normal but usually shows increased signal on t2-weighted images in the medial temporal lobes and amygdala, and less commonly in the hypothalamus and basal frontal cortex (59). the lesions may enhance with gadolinium. in some patients, the Mr abnormalities show spontaneous regression with or without concomitant clinical improvement. At some time during the course of limbic encephalitis, the CSF in most patients shows a mild lymphocytic pleocytosis and/or slight elevation of protein. Some patients also have oligoclonal bands and an elevated CSF igG index. Patients with subacute neurologic symptoms referable to the temporal lobes or limbic system may come to a brain biopsy to rule out herpes encephalitis or other infectious processes, only to be subsequently diagnosed with paraneoplastic limbic encephalitis. biopsies show a variable degree of nonspecific changes, including neuronal loss, astrogliosis, or perivascular and leptomeningeal mononuclear cell infiltrates. At autopsy, the most consistent and severe neuropathologic abnormalities in limbic encephalitis are neuronal loss, gliosis, and microglial nodules in the hippocampus and amygdala (59). Similar but less severe changes are often present in the parahippocampal gyrus, cingulate gyrus, insular cortex, orbital frontal cortex, basal ganglia, and diencephalon.

Autoimmunity Among patients with limbic encephalitis and small cell lung carcinoma, approximately one-half have anti-hu antibodies, a few have other antibodies, and the remainder have no identifiable antibodies (58,59). Anti-hu-positive patients usually show additional signs and symptoms of multifocal PEM, whereas patients without anti-hu antibodies are more likely to have clinically “pure” limbic system involvement. Some patients with seminomatous or nonseminomatous testicular cancer or breast cancer, and paraneoplastic limbic encephalitis and/or brainstem encephalitis have anti-ta antineuronal antibodies that react with the 40-kDa Ma2 protein (17,59,65). there are reports of patients with paraneoplastic limbic encephalitis and other autoantibodies (table 51-1), including antiamphiphysin antibodies (21), anti-CV2 antibodies (10), PCA-2 antibodies (18), ANNA-3 antibodies (22), antibodies against voltage-gated potassium channels (62), and several different unnamed “atypical” antibodies (61,63). A high percentage of patients with paraneoplastic limbic en-

695

cephalitis, regardless of tumor association, do not have identifiable autoantibodies.

Patient Management Middle-aged patients with a smoking history who present with limbic encephalitis should be suspected of harboring a small cell lung carcinoma. Anti-hu antibodies in these patients greatly raise suspicion for small cell lung carcinoma, while the absence of antibodies does not rule out a neoplasm (58,59). in young adults or nonsmokers presenting with limbic encephalitis the most likely neoplasms to consider are thymoma, hodgkin’s lymphoma, and testicular cancer. these patients should have Ct of the chest and abdomen. there are reports of young men in whom a testicular tumor was discovered by ultrasonography even after negative clinical examinations by a urologist (66). Serum anti-ta antibodies are a marker for testicular germ cell tumors, although a negative assay does not rule out a tumor (17). the course of limbic encephalitis is variable and unpredictable. Limbic encephalitis is unusual among CNS paraneoplastic disorders in that a significant proportion of patients has major neurologic improvement after treatment of the associated tumor. Approximately 50% of patients with limbic encephalitis and small cell lung cancer improve after tumor treatment (58,59,67). Patients with small cell lung cancer but without antineuronal antibodies are more likely to improve than those with anti-hu antibodies. Among patients with anti-hu antibodies in whom limbic encephalitis is a component of PEM, the “limbic” features may improve after tumor treatment, whereas the other neurologic features rarely do so. of the small number of reported cases, most patients with limbic encephalitis and thymoma, and some patients with limbic encephalitis, anti-ta antibodies, and germ cell tumors have significant neurologic improvement following successful tumor treatment (59,61). there are also reports of partial or complete reversal of limbic encephalitis after treatment of other tumors, including hodgkin’s lymphoma (64) and teratoma (68). the responsiveness of paraneoplastic limbic encephalitis to immunosuppressive therapy is difficult to judge from the literature, because many patients received immunosuppression concurrently with tumor treatment. Some patients with limbic encephalitis associated with small cell lung carcinoma or other tumors do show neurologic improvement after immunosuppressive treatment; there do not seem to be any factors that would predict a significant response. roughly one-half of patients with paraneoplastic limbic encephalitis, regardless of tumor association, fail to improve with tumor treatment, with or without immunosuppressive therapy (59). these patients usually plateau at a level of moderate or severe neurologic disability. Less commonly, patients may become progressively demented with eventual obtundation and fatal coma.

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CErEbELLAr DEGENErAtioN Clinical Features Ninety percent of patients with paraneoplastic cerebellar degeneration (PCD) have small cell lung carcinoma, hodgkin’s lymphoma, or carcinomas of the breast, ovary, or female genital tract (69,70). the neurologic symptoms are usually the presenting feature of cancer and antedate discovery of the neoplasm by up to 2 years. in association with hodgkin’s lymphoma, the onset of PCD frequently occurs after diagnosis of the tumor, sometimes in patients with apparent remission from the lymphoma. the most striking and consistent neuropathologic finding in PCD is a severe, diffuse loss of Purkinje cells throughout the cerebellar cortex. the remaining Purkinje cells often show nonspecific degenerative changes and are surrounded by a reactive proliferation of bergmann astroglial cells (71,72). there may also be some neuronal loss in the granular cell layer and deep cerebellar nuclei. Some patients have perivascular cuffing and mononuclear cell infiltrates in the cerebellum and overlying leptomeninges. up to one-half of autopsied patients with predominant cerebellar signs and symptoms have a multifocal PEM. the clinical features of PCD are similar regardless of the associated tumor type. the onset is typically fairly abrupt. Patients display signs and symptoms reflecting diffuse dysfunction of the cerebellum, including dysarthria and severe appendicular and gait ataxia. Abnormalities of oculomotor function are common and include nystagmus, particularly downbeat nystagmus, disruption of smooth pursuit movements, ocular dysmetria, and opsoclonus. Superimposed on the cerebellar deficits, many patients develop symptoms or signs of a multifocal PEM, including lethargy, cognitive deterioration, bulbar palsy, long-tract signs, sensory neuronopathy, and limb weakness. PCD may also occur in conjunction with paraneoplastic peripheral neuropathy or LambertEaton myasthenic syndrome, usually in patients with small cell lung carcinoma (72,73). brain Mr scans in patients with PCD are usually normal early in the course of the illness, and later show nonspecific diffuse cerebellar atrophy (70). CSF in at least two-thirds of patients shows some combination of mildly elevated protein, mild mononuclear pleocytosis, increased igG index, or oligoclonal bands. Normal CSF does not exclude the diagnosis. the neurologic deficits in PCD generally worsen over a period of several weeks to months and then stabilize at a level of severe disability (74). only about one-third of patients can walk independently, and many cannot sit up or feed themselves. Significant neurologic improvement, either spontaneously or after successful treatment of the associated tumor, is distinctly unusual (69,70,72).

Autoimmunity Virtually all paraneoplastic antibodies react with antigens in the cerebellum and can be associated with clinical cerebellar dysfunction, either in isolation or as part of a multifocal encephalomyelitis (tables 51-1 and 51-2). Anti-Yo antibodies were the first paraneoplastic antibodies to be well characterized (5,6). Anti-Yo antibodies are almost entirely restricted to patients with PCD and carcinomas of the breast, ovary, or female genital tract, and to a few women and men with other adenocarcinomas (75,76). the only reported false positives for anti-Yo antibodies are a few patients in whom no tumor can be detected after extended follow-up (70,74) and a very small percentage of patients with ovarian cancer but no neurologic symptoms (77). Anti-Yo antibodies have not been found in patients with a wide variety of cerebellar disorders unrelated to a neoplasm. Anti-Yo antibodies react with the cdr2 neuronal protein, whose normal cellular function is unknown (7,78). the cdr2 protein is expressed by some but not all breast and ovarian carcinomas (79). there have been several unsuccessful attempts to establish an experimental model for PCD by passive transfer of patients’ anti-Yo antisera into animals (80,81). Mice immunized with recombinant cdr2 protein produce high-titer anti-Purkinje cell antibodies but do not develop the clinical or pathologic features of PCD (82,83). recent studies implicate possible cellular immune effectors. Patients with PCD have circulating cytotoxic t lymphocytes that react with epitopes on the cdr2 protein (84,85). Many patients with PCD do not have anti-Yo antibodies (tables 51-1 and 51-2). Anti-tr antibodies are present in some patients with PCD and hodgkin’s lymphoma; these antibodies have not been found in association with other tumors or other clinical syndromes (13,14). A few patients with hodgkin’s lymphoma have antibodies against the mGlur1 glutamate receptor (86). Anti-ri antibodies are present in some patients with a predominantly cerebellar syndrome that often but not always includes opsoclonus and myoclonus, occurring in patients with breast carcinoma or small cell lung carcinoma (8). Anti-CV2 (CrMP-5) antibodies are present in some patients with PCD and lung carcinoma (10,12,87). Anti-Ma antibodies are present in a few patients with predominant cerebellar or brainstem syndromes associated with a number of carcinomas, including lung, breast, and colon (15,16). Some patients with PEM and small cell lung carcinoma, with or without anti-hu antibodies, have relatively selective involvement of the cerebellum (28,31,72). A significant proportion of these patients develops concomitant Lambert-Eaton myasthenic syndrome, with antibodies against P/Q-type voltage-gated calcium channels (see later). Some patients with PCD have anti-voltage-gated calcium channel antibodies but no clinical or electrophysiologic evidence for Lambert-Eaton syndrome, raising the possibility that an autoimmune response against cerebellar neuronal calcium channels has a pathogenetic role in PCD (72,88).

51. Paraneoplastic Disorders of the Nervous System

Patients with small cell lung cancer may have antiamphiphysin antibodies (50), PCA-2 antibodies (18), or ANNA-3 antibodies (22). A few PCD patients have one of a number of atypical antineuronal antibodies in association with a number of different tumors, including hodgkin’s lymphoma (69) and adenocarcinoma of the lung, colon, uterus, or prostate (72,89). Many patients who develop PCD in association with hodgkin’s lymphoma, non–small cell lung cancer, gastrointestinal carcinomas, and a number of other neoplasms do not have any demonstrable antineuronal antibodies (69,90). there are no major clinical differences among PCD patients with anti-Yo antibodies, those with other antineuronal antibodies, and those without autoantibodies, including abruptness of neurologic onset, tempo of progression, additional CNS involvement, and ultimate neurologic outcome (90). one possible exception to this is that antibody–negative patients, especially those with hodgkin’s lymphoma, may be slightly more likely to show neurologic improvement, either spontaneously or after antitumor treatment, than patients with anti-Yo antibodies (69).

Patient Management the search for a tumor in patients with suspected PCD should be guided by the clinical features and type of autoantibodies if present. the workup for women with antiYo or anti-ri antibodies should include mammography, thorough pelvic examination, imaging of the pelvis, serum CEA, and CA 125 antigen. there are reports of postmenopausal women with anti-Yo antibodies and negative mammograms in whom a “blind” exploratory laparotomy discovered a small ovarian carcinoma (70,91). Whether this should be performed for all such patients is not clear. Patients with anti-hu or other antibodies associated with small cell lung carcinoma should have chest Ct or Mr scans. Whole-body PEt may reveal an occult neoplasm undetectable by other means (52,53). if the tumor workup is unrevealing, it should be repeated at regular intervals. Some patients with PCD and hodgkin’s lymphoma and a few patients with carcinomas improve neurologically after successful tumor treatment, but these patients are clearly exceptions (69,87). Fewer than 10% of reported patients with PCD show significant neurologic improvement after plasmapheresis, iVig, corticosteroids, or cyclophosphamide (alone or in combination), despite reductions in serum autoantibody titers (31,54,55,70,91). occasional patients do improve with immunotherapy (73,92–95). there are no apparent differences in the clinical presentation of the few responders versus the large majority of patients in whom immunotherapy is ineffective.

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oPSoCLoNuS-MYoCLoNuS Clinical Features Approximately one-half of the reported cases of the opsoclonus–myoclonus syndrome in children occurred in association with neuroblastoma, although this is probably an overestimate of the true frequency of paraneoplastic opsoclonus (PoM) due to reporting bias. PoM occurs in approximately 2% to 3% of children with neuroblastoma (96). in nearly all these patients it is the neurologic syndrome that leads to discovery of an otherwise occult neoplasm. the peak incidence of PoM is around 18 months of age, with a slight overrepresentation of females. PoM typically has an abrupt onset, and most children develop irritability, vomiting, and ataxia of the trunk and limbs in addition to opsoclonus and myoclonus (97). opsoclonus as a remote effect of cancer is less common in adults than in children. Small cell lung carcinoma and breast carcinoma together account for approximately 70% of reported adult cases (98,99). the syndrome has been reported in association with other tumors, including thymoma, lymphoma, melanoma, non–small cell lung carcinoma, and carcinoma of the ovary, uterus, stomach, kidney, bladder, pancreas, and thyroid (100–104). the neurologic symptoms and signs that accompany PoM in adults are heterogeneous. in some patients the opsoclonus occurs in the setting of pure or relatively pure pancerebellar dysfunction. in other patients the opsoclonus is part of a multifocal PEM, with or without truncal or limb ataxia, myoclonus, or altered mental status (98). Ataxia and myoclonus vary in severity from mild to incapacitating. Most children and adults with PoM have mild elevation of CSF protein, lymphocytic pleocytosis, oligoclonal igG bands, and an elevated igG index. brain Mr scans are usually normal but occasionally show small, nonspecific lesions in the brainstem (8). there are no clinical, neuroimaging, or CSF findings that reliably differentiate PoM from “postinfectious” or idiopathic opsoclonus–myoclonus syndrome in children or adults. there are no distinctive or uniformly present neuropathologic abnormalities in PoM. Approximately one-half of autopsied cases show diffuse dropout of Purkinje cells, ranging from mild to nearly complete (105). Some patients have neuronal loss in the inferior olivary nuclei and perivascular mononuclear cell infiltrates in the cerebellum, brainstem, and/or leptomeninges, with or without Purkinje cell loss (98,104). in a significant proportion of autopsied children and adults with PoM there are no identifiable histopathologic abnormalities either in the cerebellum or brainstem (105,106). Disturbance of the tonic inhibitory control of saccadic burst neurons by the “omnipause” neurons in the pontine reticular formation has been postulated as the key pathophysiologic event in producing opsoclonus, but the pons may not show any histologic changes (105,107).

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Autoimmunity

Patient Management

the tumors of a disproportionately high percentage of children with PoM and neuroblastoma show intratumoral mononuclear cell infiltrates and/or evidence for maturation to ganglioneuromas (97,108,109). the occurrence of PoM in a child with neuroblastoma generally carries a good prognosis for survival independent of patient age, tumor site, or tumor stage (96,110), although a good oncologic outcome is not universal (111). these observations support the theory that PoM occurs when an antineuroblastoma immune response causes tumor regression or differentiation, but simultaneously attacks cross-reacting neuronal antigens. there are a few reports of anti-hu antibodies in children with PoM and neuroblastoma (112–114). Circulating antineuronal antibodies with differing immunocytochemical reactivity are demonstrable in some children with PoM, as well as in a lower percentage of children with neuroblastoma but no neurologic symptoms (115,116). Except for antineurofilament antibodies in some of these sera, no definite target antigen has yet been identified. Some adults with PoM have antineuronal autoantibodies in serum and CSF. Anti-ri antibodies (ANNA-2 antibodies) were initially described as specifically associated with paraneoplastic opsoclonus in adults with carcinoma of the breast, ovary, lung, or female genital tract (8,102). in addition to opsoclonus, these patients usually have limb and gait ataxia and myoclonus. Anti-ri antibodies react with a group of closely related rNA-binding “Nova” proteins believed to be involved in the regulation of mrNA splicing (9,25,117,118). the Nova proteins are differentially expressed by various subgroups of CNS neurons. recent studies show that anti-ri antibodies are not absolutely specific, either for opsoclonus or for a paraneoplastic condition. A few patients with breast carcinoma and antiri antibodies have features of multifocal PEM or brainstem encephalitis, but no opsoclonus (119,120). reports exist of women with opsoclonus and high-titer anti-ri antibodies in whom no tumor was detected after extended periods of follow-up (8,121,122). Patients with PoM may have antineuronal antibodies other than anti-ri antibodies (table 51-1). opsoclonus may occur in patients with anti-Yo antibody-associated PCD (70) and with anti-hu antibody-associated PEM (106). there are individual reports of patients with PoM and anti-ta antibodies associated with testicular cancer (17) or with non–small cell lung cancer (104), antiamphiphysin antibodies associated with small cell lung carcinoma (50), and one of several unnamed “atypical” antibodies with different patterns of reactivity associated with small cell lung cancer or other neoplasms (123,124). A fairly high percentage of patients with PoM has no identifiable antineuronal antibodies (99).

Children who present with opsoclonus should have a workup for neuroblastoma, including chest radiograph, abdominal Ct scan, and a 24-hour urine collection for vanillylmandelic acid (VMA) and metanephrine (97). Nuclear medicine imaging with the norepinephrine analog metaiodobenzylguanidine (MibG) may demonstrate a tumor in the absence of a radiographic lesion. if initially unrevealing, the workup should be repeated at regular intervals. Some children with PoM show neurologic improvement after neuroblastoma resection (114). With or without tumor treatment, adrenocorticotropic hormone (ACth) produces rapid and dramatic neurologic improvement in at least twothirds of children with PoM and neuroblastoma, independent of the status of the tumor. oral or intravenous corticosteroids are probably not as efficacious as ACth (125). A sizable proportion of children becomes dependent on ACth or steroids, or suffers exacerbations of neurologic symptoms during febrile illnesses (97,108,126). there are reports of improvement with iVig (127). Most children are left with residual motor deficits, speech delay, learning disability, or behavior problems despite an initial good response to ACth and tumor treatment (96,110,114,125). Children who receive chemotherapy may have a better longterm neurologic outcome (110). Women with opsoclonus in whom serum anti-ri antibodies or anti-Yo antibodies are discovered should undergo mammography, thorough pelvic examination, imaging of the pelvis, and measurement of serum carcinoembryonic antigen and CA 125 antigen levels. Given the association between opsoclonus and breast carcinoma, it is probably worthwhile to obtain a mammogram on women without autoantibodies in whom there is no obvious alternative explanation for opsoclonus. Adults with a history of cigarette smoking should have chest Ct or a Mr scan. Discovery of anti-hu antibodies in these patients greatly raises the index of suspicion for an underlying small cell lung carcinoma. As a group, adults with PoM have a better neurologic outcome than patients with PCD or PEM. Some patients show spontaneous neurologic improvement (100) or improvement following corticosteroids (103) or successful tumor treatment (98,99,101). Some patients, however, suffer progressive encephalopathy leading to coma and death despite tumor treatment, corticosteroids, or plasmapheresis (98,99,105). occasionally, the opsoclonus and other neurologic signs improve and worsen in association with regression and recurrence of the associated tumor (98). Approximately one-half of the small number of reported patients with paraneoplastic opsoclonus and anti-ri antibodies (most of whom had breast carcinoma) showed significant improvement following antitumor chemotherapy or prednisone (8,102). one patient with anti-ri antibodies and steroid-responsive exacerbations of nonparaneoplastic opsoclonus–ataxia benefited from the addition of cyclophospha-

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mide (121). Patients with opsoclonus and breast or lung carcinoma but no antineuronal antibodies may also benefit from corticosteroids, with or without iVig. A few patients with small cell lung cancer or breast carcinoma and PoM improved following immunoadsorption therapy with a protein A column (94,123).

EXtrAPYrAMiDAL SYNDroMES Chorea, athetosis, dystonia, or parkinsonism are rare manifestations of paraneoplastic encephalitis, occurring most often in association with small cell lung carcinoma (33,34,128,129) and with hodgkin’s lymphoma (130), breast carcinoma (131), or other tumors (129,132). the extrapyramidal features may occur with or without other signs of multifocal encephalomyelitis. Mr scans in some patients show abnormal signal in the striatum and/or basal ganglia. Associated antibodies include anti-hu (33) and anti-CV2 (CrMP-5) (129). Some patients show neurologic improvement after tumor treatment or immunosuppression.

ViSuAL SYNDroMES Carcinoma-Associated retinopathy More than 75% of reported patients with paraneoplastic retinopathy have small cell lung carcinoma (133–135). other patients have one of a variety of other tumors, including non–small cell lung cancer or carcinoma of the breast, ovary, prostate, stomach, and uterus (136–138). in nearly all patients with carcinoma-associated retinopathy (CAr), the visual symptoms are the presenting feature of the tumor, preceding discovery of the tumor by intervals ranging from several months up to 2 years or more. the signs and symptoms nearly always involve both eyes, but asymmetry is the rule, especially early in the course. the initial symptoms are most often a painless dimming or blurring of vision (139–141). Night blindness is common and may be the sole initial complaint. Many patients additionally report episodic obscurations or “positive symptoms” described as distortions, “sparkles,” “shimmering,” or bizarre images. Some patients report visual glare or photosensitivity. in most patients the visual symptoms worsen over several weeks to months, either in a steady or a stepwise course. the bedside examination of patients with CAr usually shows severely impaired vision. Color vision is affected less often. Approximately one-half of patients have afferent pupillary defects. Funduscopic exam usually shows mild or moderate narrowing of retinal arterioles but is otherwise unremarkable except for occasional vitreous cells. the most common visual field deficits are asymmetric central or ring scotomas and concentric constriction. the electroretinogram in almost all patients is flat or nearly so, reflecting diffuse dysfunction of both rod and cone photoreceptor cells. the electroretino-

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gram can be severely abnormal in the face of relatively preserved visual acuity. the typical histopathologic features of CAr are severe, sometimes total loss of inner and outer segments of rods and cones, and widespread degeneration of the outer nuclear layer (141,142). there may be patchy, mild infiltration of mononuclear cells around retinal arterioles. Ganglion and bipolar cells are preserved in the great majority of cases; exceptional patients show diffuse loss of ganglion cells and atrophy of the optic nerves and chiasm, with preservation of photoreceptor cells (143). Most but not all patients with CAr have circulating antiretinal autoantibodies, the most prevalent being igG antibodies against the 23-kDa calcium-binding protein recoverin (table 51-2). Nearly all patients with CAr and antirecoverin antibodies have small cell lung carcinoma (24,144,145), with individual case reports of gynecologic neoplasms or other tumors (133). Some patients with antirecoverin antibodies also have antibodies against a 65kDa heat shock protein (146). recoverin is expressed by retinal rods, cones, and bipolar cells. it functions in the phototransduction cascade by modulating the phosphorylation of rhodopsin. recoverin is expressed by some but not all small cell lung carcinomas and cell lines (145,147,148). there are reports of patients with retinal degeneration and antirecoverin antibodies in whom no tumor was found after 2 or more years (149). With these rare exceptions, antirecoverin antibodies have not been detected in a variety of patients with retinal disorders unrelated to cancer (144). Features of CAr have been successfully reproduced in experimental models. Patients’ antirecoverin antibodies exert a cytotoxic effect and induce apoptosis in rat retinal cells in vitro (150–152). rats immunized with recoverin develop antirecoverin antibodies, uveoretinitis with cellular infiltrates, and degeneration of photoreceptors (145,153). the same histopathologic changes can be reproduced by passive transfer of stimulated lymphocytes from rats immunized with recoverin into naive animals. intravitreous injection of antirecoverin antibodies into rats produces abnormal electroretinograms and thinning of the inner and outer nuclear retinal layers (137,148). these effects are enhanced when antibodies against heat-shock protein are injected into the vitreous together with antirecoverin antibodies. A significant minority of patients with CAr either has no identifiable antiretinal antibodies or has autoantibodies that react with retinal target antigens distinct from recoverin (154). these include antibodies against retinal enolase (135,136), neurofilaments (134), retinal S-antigen (141,155), carbonic anhydrase (138), a 78-kDa retinal protein that belongs to the “tubby” gene family (156), and antibodies against one of several unidentified retinal proteins (157,158). the clinical course of CAr is usually one of deterioration over several weeks or months in a gradual or stepwise fashion, to a level of severe visual impairment. there are no

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well-documented instances of significant spontaneous improvement. Most patients treated with prednisone show mild to moderate improvement in visual acuity and visual fields within days to weeks of beginning treatment, often before discovery of the underlying neoplasm (159). in some patients the improvement is maintained after discontinuation of the steroids, whereas other patients have a fluctuating steroid-dependent course. there does not seem to be a difference in the response to prednisone between patients with antirecoverin antibodies and patients with other antiretinal antibodies or no antibodies. there are no definite reports of visual improvement following surgery or chemotherapy of small cell lung cancer without concomitant corticosteroid therapy. intravenous immunoglobulin may also be beneficial (135).

Melanoma-Associated retinopathy Paraneoplastic retinopathy is unusual among neurologic remote effects of cancer in its association with melanoma (160–164). the clinical features of melanoma-associated retinopathy differ somewhat from those of carcinomaassociated retinopathy. Nearly all reported patients developed visual symptoms after the diagnosis of melanoma, with intervals of up to 10 years. in some patients visual symptoms herald previously unsuspected systemic metastases. Subacute night blindness is a universal feature of melanoma-associated retinopathy. Most patients also report floaters or shimmering lights. Visual acuity, visual fields, color vision, and funduscopic exam are usually normal, though some patients have an afferent pupillary defect, constricted visual fields, arcuate scotomas, or central scotomas. uveitis or retinal “vasculitis” manifesting as leaky vessels on fluorescein angiography have also been reported (165,166). A few reported autopsies of patients with melanoma-associated retinopathy showed preservation of the photoreceptor cell layers but marked depletion of cell nuclei in the inner nuclear and bipolar layers (167). the electroretinogram in patients with melanomaassociated retinopathy shows a selective reduction in the amplitude of the dark-adapted b-wave (160,162). this resembles the abnormalities in patients with “congenital stationary night blindness” and indicates a severe transmission defect in the rod pathway. Cone pathways may also be affected in severe cases (163,165). Some patients with melanoma-associated retinopathy have serum antibodies that stain a subset of approximately 30% of retinal bipolar cells and, to a lesser degree, outer rod segments (161,163,165,166). there is a single reported patient with retinal degeneration and antibipolar cell antibodies associated with colon carcinoma (168). in retinal immunoblots the antibodies react with a 33-kDa membraneassociated protein. injection of igG from patients with melanoma-associated retinopathy into the vitreous of mon-

keys produces electroretinographic changes similar to those seen in human patients (169). of the handful of reported patients with melanomaassociated retinopathy who were treated with prednisone, approximately one-half showed partial improvement in vision (160,161,165,166).

optic Neuritis optic neuropathy is a rare complication of breast carcinoma, small cell lung carcinoma, or other tumors. Visual symptoms may occur with or without other signs and symptoms of multifocal paraneoplastic encephalomyelitis (135,170–172). Nothing is clinically distinctive about the optic neuritis in these patients, who have decreased visual acuity, afferent pupillary defects, cecocentral scotomas, and disc edema. Some of these patients have anti-CV2 or other antineuronal antibodies (87), but not antirecoverin or other antibodies associated with paraneoplastic retinal degeneration.

MYELoPAthY Necrotizing myelopathy is clinically and pathologically distinct from the multifocal patchy involvement of the spinal cord in cases of PEM (173). this rare syndrome may occur in association with a variety of carcinomas and lymphoid tumors, without a clear preponderance of any specific tumor type. Patients present with subacute bilateral symptoms involving motor, sensory, and sphincter function with little or no pain. Physical examination may initially demonstrate a brown-Sequard syndrome, but more commonly reveals a specific level of transverse spinal cord dysfunction. Spine Mri may be normal or show intramedullary signal abnormalities. the differential diagnosis of subacute myelopathy in cancer patients includes intramedullary spinal metastases, radiation myelopathy, or neurotoxicity of intrathecal chemotherapy. there are anecdotal reports of neurologic improvement after intrathecal corticosteroids (174,175), but most patients suffer rapid deterioration of function and a progressively ascending level of flaccid paralysis and numbness, often leading to death from respiratory failure, sepsis, or other medical complications. At autopsy there is typically a region, most often in the thoracic cord, of massive transverse necrosis and nearly total loss of architectural features extending over several cord segments, with the severity of involvement tapering off gradually in more rostral and caudal segments (173). both white and gray matter are involved without predilection for specific tracts, cell groups, or vascular territories. there is little if any inflammatory reaction. A single patient with necrotizing myelopathy and hodgkin’s lymphoma had acute, localized arteritis and fibrinoid necrosis in affected areas (176).

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Motor NEuroN DiSEASE Motor neuron dysfunction as a paraneoplastic phenomenon occurs in a variety of different settings. Lower motor neuron signs and symptoms are among the presenting or predominant manifestations in up to 25% of patients with multifocal PEM (28,31,177). As with other manifestations of PEM, motor neuron involvement in these patients tends to progress independently of the status of the underlying neoplasm and does not usually improve with treatment. Autopsy demonstrates patchy loss of anterior horn cells and variable inflammatory infiltrates within the spinal cord. A more problematic issue is how often, if ever, isolated motor neuron disease or amyotrophic lateral sclerosis (ALS) is a paraneoplastic syndrome (178). Several epidemiologic studies of ALS from various parts of the world report an incidence of neoplasms ranging from zero to 7.8%. there is no convincing epidemiologic evidence that nonhematologic neoplasms occur in ALS patients any more frequently than would be expected in an age-matched control population. these data argue against performing an extensive search for an occult carcinoma as part of the routine workup for all patients with clinically typical ALS. Despite the absence of a clear epidemiologic link between motor neuron disease and neoplasia, the relationship in small subsets of patients is probably more than coincidental. First, the presence of serum anti-hu antibodies in a few patients is a strong though not absolute marker for a paraneoplastic process and an etiologic link between the tumor and the neurologic disorder. Second, there are several welldescribed patients with a lower motor neuron syndrome or combined upper and lower motor neuron syndrome who had significant neurologic improvement after resection of lung or renal carcinomas (179,180). third, there are associations between “atypical” motor neuron syndromes and particular tumor types that seem to be more than fortuitous. A pure or nearly pure upper motor neuron syndrome resembling primary lateral sclerosis was the presenting feature of breast carcinoma in several women (181). the neurologic syndrome in these patients was chronic and progressive, and did not respond to tumor treatment. there is a single report of a lower motor neuron syndrome in a patient with breast carcinoma and serum antibodies reacting with axons and nodes of ranvier (182). there appears to be a special association between motor neuron disorders and lymphomas or plasma cell dyscrasias. the prevalence of plasma cell dyscrasias among patients with motor neuron disease is higher than that in age-matched control populations, but the vast majority of these patients have “monoclonal gammopathy of undetermined significance,” and the etiologic association between the two conditions remains unclear. the clinical syndrome and neuropathologic findings in reported patients with motor neuron disease and igG or igA paraproteinemias closely resemble classical ALS. in most cases this is probably a coincidental

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association, although there are reports of neurologic improvement after plasma exchange, corticosteroids, and alkylating agents such as melphalan or cyclophosphamide (183). there are more than 50 published reports of patients with concurrent ALS or “progressive spinal muscular atrophy” and lymphoproliferative disease, including hodgkin’s or nonhodgkin’s lymphoma and chronic lymphocytic leukemia (184). the frequency of lymphoproliferative disease in patients with motor neuron disorders has been estimated as high as 2% to 5%. in roughly half of patients the neurologic symptoms precede diagnosis of the lymphoproliferative disease. Some patients have no “systemic” signs or symptoms, and the lymphoma is discovered only by bone marrow biopsy. up to one-third of patients have an associated monoclonal igA, igG, or igM paraprotein. there is nothing clinically distinctive about the motor neuron disease in patients with lymphoproliferative disease, except for an elevated CSF protein without pleocytosis in up to 50%. Patients with an ALS-like syndrome generally have progressive weakness and often die a “neurologic” death despite treatment of the associated lymphoproliferative disease. the association of motor neuron disease and lymphoproliferative disease includes the syndrome of “subacute motor neuronopathy” described in patients with hodgkin’s or nonhodgkin’s lymphoma, or thymoma (185). Patients develop a subacute lower motor neuron syndrome predominantly involving the legs, without pain, significant sensory loss, or upper motor neuron findings. the neurologic deficits generally worsen over weeks to months independent of the course of the malignancy, but usually stabilize or improve (sometimes dramatically) after a period of months to years. the few cases examined at autopsy have shown patchy degeneration and loss of anterior horn cells, occasional inflammatory infiltrates, secondary thinning of ventral nerve roots, and widespread patchy segmental demyelination of spinal roots and of brachial and lumbosacral plexuses.

StiFF-PErSoN SYNDroME Muscle stiffness or rigidity may occur in the setting of several different paraneoplastic disorders, reflecting either central or peripheral nervous system dysfunction. A syndrome of muscle rigidity and spasms that clinically resembles the “stiff-person syndrome” is associated with a variety of neoplasms, including small cell lung carcinoma, thymoma, hodgkin’s lymphoma, and carcinoma of the breast or colon (19,186–188). in at least some of these patients the rigidity is probably caused by multifocal PEM affecting spinal cord interneurons. Patients develop progressive aching and rigidity of the axial and proximal limb musculature, usually asymmetric at onset. there are superimposed painful and sometimes violent spasms, either occurring spontaneously or

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triggered by voluntary movement, passive movement, or sensory stimuli. Patients may eventually develop fixed flexion of the limbs or even opisthotonos and respiratory difficulty. Needle EMG shows abnormally continuous firing of otherwise normal motor unit potentials in agonist and antagonist muscles. high doses of diazepam, clonazepam, or baclofen may provide partial relief. Some patients with paraneoplastic rigidity or stiff-person syndrome have serum and CSF igG antibodies directed against the synaptic vesicle-associated protein amphiphysin (table 51-2). breast carcinoma and small cell lung cancer are the most commonly associated tumors, with reports of patients with colon carcinoma, ovarian carcinoma, and hodgkin’s lymphoma (19,20,50). Antiamphiphysin antibodies are not specific for paraneoplastic rigidity, as they have also been identified in patients with a variety of clinical presentations, including PEM/SSN, limbic encephalopathy, PCD, myoclonus, or Lambert-Eaton syndrome (20,21,50). there are reports of patients with stiff-person syndrome and antiamphiphysin antibodies in whom no tumor was discovered after 2 or more years of follow-up (189). Some patients with paraneoplastic rigidity and antiamphiphysin antibodies show neurologic improvement following tumor treatment and prednisone (19,20,190). Patients with breast carcinoma and stiff-person syndrome may have antiglutamic acid decarboxylase (antiGAD) antibodies (190,191). there is a single reported patient with stiff-person syndrome, carcinoma of unknown origin, and antibodies against the protein gephyrin that is associated with inhibitory synapses (192).

NEuroMYotoNiA Patients with small cell lung carcinoma, thymoma, hodgkin’s lymphoma, or plasmacytoma may develop a syndrome of diffuse muscle stiffness, cramps, and myokymia similar to “neuromyotonia” or “continuous muscle fiber activity” (isaacs’ syndrome) (193–196). Patients with thymoma may concurrently have myasthenia gravis, and some patients have a confusional state, dysautonomia, and/or CSF oligoclonal bands suggesting CNS involvement (termed Morvan’s fibrillary chorea) (197,198). Needle EMG shows repetitive bursts of rapidly firing motor unit discharges (myokymic potentials) and/or very-high-frequency trains of discharges. Fibrillations and fasciculations may also be present. Some patients with thymoma have serum antibodies against voltage-gated potassium channels, which have also been identified in patients with nonparaneoplastic neuromyotonia (199). the antibodies are postulated to cause prolonged motor neuron depolarization leading to abnormal spontaneous muscle activity. Successful tumor treatment may bring about significant neurologic improvement. Patients may also benefit from phenytoin, carbamazepine, plasmapheresis, and/or other immunosuppressive treatment.

DEMYELiNAtiNG NEuroPAthiES AND CArCiNoMA or LYMPhoMA Paraneoplastic neuropathies may be classified either by their associated neoplasm or by their clinical pattern of presentation (table 51-3). there are several reports of an acute, predominantly motor polyradiculoneuropathy occurring in the setting of a number of primary neoplasms, particularly lymphomas. the clinical features, electrophysiologic findings, CSF, and peripheral nerve pathology in these patients are indistinguishable from Guillain-barré syndrome (200). improvement may follow steroid therapy or tumor resection, but many of these patients undergo dramatic spontaneous remission of the neuropathy independent of the status of their underlying tumor. it is not clear whether the acute polyneuropathy in these patients is coincidental or a true “paraneoplastic Guillain-barré syndrome.” the incidence in patients with hodgkin’s lymphoma seems to be greater than would be predicted by chance alone. there are also several well-documented cases of patients with lymphomas or a variety of carcinomas who develop a sensorimotor polyneuropathy that fulfills the diagnostic criteria for chronic demyelinating polyneuropathy (CiDP) (201–204). the neurologic syndrome may precede discovery of the neoplasm by up to several years. Electrophysiologic studies show abnormal Fwaves, conduction blocks, reduced nerve conduction velocities, and abnormal temporal dispersion. Nerve biopsies demonstrate segmental demyelination and mononuclear cell infiltrates. Neurologic improvement may follow tumor resection, iVig, or corticosteroid therapy. Several recently reported patients with CiDP and melanoma had vitiligo or serum antibodies against gangliosides and other antigens shared by tumor cell and peripheral nerve (205,206).

DEMYELiNAtiNG NEuroPAthY AND ANti-MAG ANtiboDiES A significant proportion of patients presenting with an “idiopathic” polyneuropathy are discovered to have a monoclonal gammopathy. Most of these patients have monoclonal gammopathy of undetermined significance (MGuS), and a minority has or will eventually develop multiple myeloma, plasmacytoma, Waldenstrom macroglobulinemia, nonhodgkin’s lymphoma, chronic lymphocytic leukemia, or Castleman syndrome (207). the paraprotein may be igA, igG, or igM, and the clinical and electrophysiologic features may indicate an axonal, demyelinating, or “mixed” pattern (208,209). in some patients there is a clear etiologic link between the neuropathy and the monoclonal gammopathy, whereas in others the association is probably fortuitous. in most patients with an igM monoclonal gammopathy and neuropathy the igM paraprotein reacts with specific components of peripheral nerve. in 50% to 60% of these patients the paraprotein reacts with an oligosaccharide determinant

51. Paraneoplastic Disorders of the Nervous System

wtAbLE 51-3

Paraneoplastic Neuropathies

Clinical Syndrome

Associated tumors

Sensory neuronopathy/ carcinoma, ataxic neuropathy Acute demyelinating polyneuropathy

Small cell lung

Chronic demyelinating myeloma, polyneuropathy other Multifocal motor neuropathy Mononeuritis multiplex Small fiber painful/ autonomic neuropathy

plasma cell dyscrasias hodgkin’s lymphoma, variety of solid tumors (rare) osteosclerotic PoEMS syndrome, plasma cell dyscrasias Plasma cell dyscrasias Variety of solid tumors, cryoglobulinemia Amyloidosis, plasma cell dyscrasias

shared by myelin-associated glycoprotein (MAG) and the glycolipids sulfoglucuronyl paragloboside (SGPG) and sulfoglucuronyl lactosaminyl paragloboside (SGLPG) (210–214). in some patients the igM paraprotein crossreacts with a similar determinant shared by the myelin proteins Po and PMP-22. Approximately 75% of patients with an anti-MAG paraprotein have MGuS (214). there is a male predominance, with onset usually after 60 years of age. Patients with anti-MAG antibodies generally develop a slowly progressive, predominantly sensory neuropathy that may be present for several years before the diagnosis is established. Proprioception and vibratory sense are selectively affected. intention tremor and ataxia are each present in up to one-half of patients at initial presentation. Muscle stretch reflexes are diminished or absent. romberg test may be positive. Cranial nerves and autonomic functions are usually unaffected. Approximately 20% of patients have a predominantly motor neuropathy. Even though the progression is slow, the neuropathy is frequently debilitating. CSF protein concentration is almost always elevated. Electrophysiologic studies are consistent with demyelinating neuropathy, showing prolonged distal latencies, slow nerve conduction velocities, and dispersion of compound muscle action potentials (212). Sensory nerve action potentials are frequently unobtainable. Conduction blocks are relatively uncommon. biopsied nerves typically show loss of myelinated fibers, segmental demyelination, thinned myelin sheaths, and onion bulbs (210,215). the anti-MAG igM and complement are deposited on myelin sheaths. in most cases there is a characteristic widening of the myelin lamellae at the minor dense lines. treatment of patients with anti-MAG neuropathy is directed at lowering the paraprotein concentrations, usually with chemotherapy such as chlorambucil, cyclophosphamide, or fludarabine (210,216,217). improvement or stabilization of the

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neuropathy does not always correlate with a reduction in paraprotein levels (218). Plasmapheresis or iVig also benefit some patients, whether alone or in combination with chemotherapy (219–221). there are reports of neurologic improvement after treatment with a monoclonal antibody against the CD20 blymphocyte antigen (222).

DEMYELiNAtiNG NEuroPAthY AND oStEoSCLErotiC MYELoMA osteosclerotic myeloma constitutes fewer than 3% of all myelomas, with a younger average age of onset than multiple myeloma. Approximately one-half of individuals with osteosclerotic myeloma have a predominantly motor polyneuropathy (as compared with a 3% to 5% incidence of neuropathy among patients with osteolytic multiple myeloma) (223). Patients with osteosclerotic myeloma and neuropathy usually present with distal weakness, which progresses proximally. Examination also shows disproportionate loss of vibration sense and proprioception. Muscle stretch reflexes are diminished or absent. Pain or autonomic involvement are rare. the neuropathy is usually slowly progressive but can be severely disabling. Electrophysiologic studies show changes indicating demyelination, including prolonged distal motor latencies, abnormal dispersion of compound muscle action potentials, and slowed nerve conduction velocities. Nearly all patients have elevated CSF protein. Sural nerve biopsy shows a combination of segmental demyelination and axonal degeneration. these patients often fulfill the clinical and electrophysiologic criteria for CiDP. Approximately 80% of patients with neuropathy and osteosclerotic myeloma have a paraprotein, usually of the igG or igA lambda type, which is often at a low level and only detectable by immunofixation. radiographic skeletal survey reveals the sclerotic myeloma (single in about one-half of patients) most often in the axial or proximal appendicular bones (223). in patients without an identifiable paraprotein the diagnosis can be made by biopsy of a suspicious bone lesion. in the great majority of patients the osteosclerotic myeloma is only discovered through investigation of the neuropathy and is otherwise asymptomatic. Some patients with neuropathy and osteosclerotic myeloma have the multisystem PoEMS syndrome (Polyneuropathy, organomegaly, Endocrinopathy, M-protein, and Skin changes) (224–227). PoEMS syndrome is disproportionately more common in Japan but occurs worldwide. Peak age of onset is between 40 and 60 years. Men are more commonly affected than women. organomegaly most often involves the liver, spleen, and lymph nodes. Endocrine dysfunction includes diabetes mellitus in 50% of patients and/or amenorrhea, hypothyroidism, gynecomastia, impotence, and testicular atrophy. Skin changes include diffuse thickening, hirsutism, hyperpigmentation, finger clubbing, and sclerodermatous features. other features include thrombocytosis, anasarca, and papilledema.

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Some patients have Castleman disease (angiofollicular lymph node hyperplasia) instead of or in addition to osteosclerotic myeloma. in the great majority of patients the neuropathy is the presenting and clinically most disabling feature. the pathogenesis of neuropathy and of the varied multisystemic manifestations of osteosclerotic myeloma is not yet understood. the paraprotein, if present, does not react with peripheral nerve antigens. PoEMS patients have elevated serum levels of vascular endothelial growth factor, and of “pro-inflammatory” cytokines, including interleukin-6, interferon-gamma, and tumor necrosis factor-␣ (228,229), although the actions by which these cytokines may bring about the clinicopathologic features are unknown. treatment of patients with osteosclerotic myeloma or PoEMS syndrome includes some combination of surgical resection of the myeloma, radiation, prednisone, or chemotherapy (usually including cyclophosphamide or melphalan) (225–227,230). Plasmapheresis or iVig alone does not appear to be beneficial. When the myeloma responds to treatment patients usually have improvement of the neuropathy as well as all other multisystemic elements of the syndrome. there do not seem to be clinical features that reliably predict response to therapy, except that patients with a solitary osteosclerotic myeloma seem to have a better outcome. treatment approaches aimed at altering cytokine levels include tamoxifen or retinoic acid (231). there are reports of improvement after myeloablative chemotherapy in patients who failed other treatments (232).

SENSorY NEuroPAthY AND ANtiDiSiALoSYL GANGLioSiDE ANtiboDiES the syndrome of sensory neuropathy and antidisialosyl ganglioside antibodies is rare relative to other neuropathies associated with plasma cell dyscrasias. Patients usually have an igM paraprotein in the setting of “monoclonal gammopathy of undetermined significance” (MGuS). the paraprotein binds to GD1b and to several other gangliosides that bear disialosyl groups. Patients present with progressive numbness, paresthesias, and gait ataxia (288–290). Examination shows severe loss of proprioception and vibratory sense, with relative sparing of pain and temperature sensations. Patients are frequently unable to walk due to severe sensory ataxia. Weakness, if any, is mild. Autonomic involvement may occur but is not severe. Some patients have ophthalmoparesis or other cranial nerve/bulbar involvement, including respiratory insufficiency. Muscle stretch reflexes are usually absent. this syndrome resembles subacute sensory neuronopathy associated with small cell lung cancer and antihu antibodies.

Electrophysiologic studies in patients with anti-GD1b paraprotein usually show low amplitude or absent sensory nerve potentials. Motor nerve conduction studies are usually normal or slightly abnormal, although some patients have temporal dispersion and conduction block. CSF protein may be elevated. Sural nerve biopsy shows variable loss of large myelinated fibers and segmental demyelination, without deposition of the igM paraprotein. the anti-GD1 paraproteins from affected patients immunocytochemically bind to dorsal root ganglia neurons, and immunization of rabbits with GD1b induces an ataxic neuropathy, supporting a direct immunopathogenetic role for the antibodies. Some but not all of the small number of published patients responded to treatment with corticosteroids, cyclophosphamide, iVig, or plasmapheresis.

VASCuLitiC NEuroPAthY AND CrYoGLobuLiNEMiC NEuroPAthY Peripheral nerve microvasculitis may occur in association with lymphomas or with carcinoma of the lung, prostate, uterus, kidney, or stomach (233,234). Neurologic symptoms usually precede discovery of the tumor. the clinical presentation is either that of mononeuritis multiplex or of an asymmetric distal sensorimotor neuropathy. Pain is common. Patients usually have an elevated sedimentation rate but rarely have cutaneous vasculitis or other systemic symptoms. Nerve conduction studies show a variable degree of axonal degeneration in motor and sensory nerves that reflects the asymmetric clinical involvement. Sural nerve biopsy or autopsy demonstrate focal mononuclear cell infiltration of epineurial vessel walls and active nerve fiber degeneration. there may additionally be arteriolar fibrinoid necrosis with obliteration of lumens. Patients with peripheral nerve vasculitis and small cell lung cancer may also have clinical and pathologic features of PEM/SSN (233,235). Patients with small cell lung cancer may have anti-hu antibodies, with or without overt CNS involvement (236). there are reports of neurologic improvement after tumor treatment and/or cyclophosphamide; corticosteroids alone do not seem to have much effect. Neuropathy occurs in approximately 20% of patients with cryoglobulinemia. Patients present either with a subacute mononeuritis multiplex or with a chronic distal symmetric sensorimotor polyneuropathy (237,238). Nerve biopsy shows axonal degeneration and necrotizing vasculitis in epineural blood vessels. A variable proportion of patients shows neurologic improvement after treatments aimed at reducing the paraprotein or cryoglobulin levels.

51. Paraneoplastic Disorders of the Nervous System

othEr NEuroPAthiES As described earlier, the most common peripheral nervous system syndrome associated with anti-hu antibodies is sensory neuronopathy reflecting dorsal root ganglionitis. Nerve conduction studies show, however, that only a minority of patients with clinically “pure” or predominantly sensory neuronopathy have conduction abnormalities totally restricted to sensory nerves. rather, most patients have at least some electrophysiologic abnormalities in motor nerves (239). A minority of patients with anti-hu antibodies has a clinical presentation suggestive of mixed sensorimotor polyneuropathy with a mixed axonal-demyelinating electrophysiologic pattern. A few patients with anti-hu antibodies have what appears to be a primary demyelinating polyneuropathy superimposed on sensory neuronopathy (240), or a mononeuritis multiplex with biopsy-proven nerve vasculitis (233,236). Patients with anti-CV2 antibodies (most of whom have small cell lung carcinoma) may develop a sensorimotor polyneuropathy with mixed axonal-demyelinating electrophysiologic features (241). Some of these patients have both anti-hu and anti-CV2 antibodies. Anti-CV2 antibodies immunocytochemically label peripheral nerve axons. Sensorimotor polyneuropathy may also occur in patients with ANNA-3 antibodies (22). Distal, slowly progressive axonal sensorimotor polyneuropathy occurs in patients with a wide variety of carcinomas and hematologic neoplasms (204,242,243). the reported incidence of “paraneoplastic polyneuropathy” among cancer patients varies from 10% to 40% or more, varying with the specific clinical and electrophysiologic diagnostic criteria used, and with the stage and duration of illness of cancer patients studied. Most affected patients have suffered significant weight loss and are in an advanced or preterminal phase of their illness; in these patients the neuropathy itself is generally not a major source of disability. Electrophysiologic studies and limited pathologic data are consistent with a primary distal axonopathy. the exact etiology of the polyneuropathy in these patients is usually unclear and is probably multifactorial. the frequent occurrence of neuropathy secondary to chemotherapy drugs or to nutritional deficiencies also makes it difficult to determine the incidence of “pure” paraneoplastic neuropathy.

AutoNoMiC iNSuFFiCiENCY Paraneoplastic autonomic dysfunction most commonly occurs as a part of PEM in patients with small cell lung carcinoma. in some patients the autonomic symptoms overshadow other manifestations of PEM/SSN. these patients may develop severe and progressive gastrointestinal dys-

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motility, including gastroparesis, chronic intestinal pseudo-obstruction, and severe constipation/ obstipation, presenting several months before discovery of the small cell lung tumor (244–246). orthostatic hypotension and other features of sympathetic dysfunction may also be present. Some of these patients have anti-hu antibodies. the pan-neuronal reactivity of anti-hu antibodies includes staining of neurons in the sympathetic ganglia and myenteric plexus. improvement in autonomic function follows tumor resection or chemotherapy in some patients with isolated dysautonomia or PEM. Acute or subacute dysautonomia involving cardiovascular, urinary, and/or gastrointestinal systems may rarely occur in association with other tumors, including bronchial carcinoid, thymoma, and hodgkin’s lymphoma (247,248).

LAMbErt-EAtoN MYASthENiC SYNDroME Clinical Features the reported prevalence of an associated neoplasm among patients with Lambert-Eaton syndrome (LEMS) ranges from 40% to 70% (249–251). on average, patients with nonparaneoplastic LEMS are younger and are more often female than the paraneoplastic patients. Small cell lung carcinoma is the associated tumor in more than 90% of well-documented cases of paraneoplastic LEMS. the prevalence of LEMS among small cell lung carcinoma patients is approximately 1% to 3% (1). LEMS in patients with small cell lung cancer appears to convey a relatively favorable survival outcome, supporting the theory that an autoimmune response in these patients slows tumor growth (252). LEMS may also occur in patients with other neoplasms, including prostate carcinoma, non–small cell lung carcinoma, thyroid carcinoma, small cell carcinoma of the cervix, lymphoma, thymoma, germinoma, and leukemia (253,291,292). in most patients with paraneoplastic LEMS, the neurologic symptoms precede discovery of the associated neoplasm; this interval may be as long as 5 years, but in the majority of reported cases is less than 12 months (249,251,254). the clinical features of LEMS are indistinguishable between paraneoplastic and nonparaneoplastic patients. Most patients have an insidious and gradual onset of weakness and fatigue. Early in the course there is often a discrepancy between patients’ subjective weakness and easy fatigability and the relatively minor abnormalities seen on neurologic exam. Symmetric weakness predominantly affects pelvic and to a lesser extent, shoulder girdle muscles. Muscle aches or distal paresthesias are not uncommon. Muscle stretch reflexes are diminished or absent. transient improvement in strength and reflexes following sustained voluntary contraction is a classic feature but cannot always

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be demonstrated. Some patients have a positive edrophonium test. Approximately 75% of patients have symptoms of autonomic dysfunction, including dry mouth, impotence, blurred vision, constipation, difficulty with micturition, and reduced sweating (255). bulbar functions are relatively spared, but up to one-third of patients have dysphagia, ptosis, or diplopia. respiratory failure requiring ventilatory support is uncommon but can occur, either spontaneously or induced by drugs, and may rarely be the presenting or predominant feature (256,257). one feature that distinguishes paraneoplastic from nonparaneoplastic LEMS is that some patients with a neoplasm have concomitant cerebellar degeneration, encephalomyelitis, sensorimotor polyneuropathy, or other “overlap” syndromes (73,258). there appears to be a particular association between LEMS and PCD among patients with small cell lung cancer, some of whom have anti-hu antibodies (72). in these patients either weakness or CNS symptoms may dominate the clinical presentation. Drugs that interfere with neuromuscular transmission may exacerbate weakness in LEMS. these agents include anesthetic neuromuscular blocking agents, aminoglycoside antibiotics, ofloxacin or other fluoroquinolone antibiotics, procainamide, quinidine, magnesium, beta-adrenergic blockers, calcium channel blockers, and iodinated contrast agents. Electrophysiologic testing remains the gold standard for diagnosing LEMS. the characteristic electrophysiologic profile includes (a) reduced amplitude of muscle action potentials evoked by a supramaximal stimulus, sometimes to as little as 10% of normal; (b) a greater than 100% increase in compound muscle action potentials after several seconds of maximal voluntary contraction (postexercise facilitation); (c) a decremental response of greater than 7% at 2 hz to 5 hz repetitive nerve stimulation; (d) an incremental response (of up to 2,000%) at 20- to 50-hz stimulation; and (e) increased jitter and impulse blocking on single-fiber EMG testing. the jitter and blocking often decrease at rapid stimulation rates (249,259,260). Many laboratories test for facilitation after maximal voluntary contraction rather than performing 20- to 50-hz stimulation (261). the electrophysiologic abnormalities are often detected in clinically unaffected muscles, though not all muscles are equally affected. No single one of these features will absolutely distinguish LEMS from myasthenia gravis, nor can electrophysiologic tests distinguish between the paraneoplastic and nonparaneoplastic forms of LEMS.

Autoimmunity the primary pathophysiologic abnormality in LEMS is a reduction of the quantal release of acetylcholine triggered by a nerve impulse. ultrastructural studies of muscle from LEMS patients show a marked depletion of presynaptic active zones (the sites of synaptic vesicle exocytosis), paucity and

disorganization of active zone intramembrane particles, and aggregation of the active zone particles into clusters (262). Active zone particles contain P/Q-type voltage-gated calcium channels that mediate the quantal release of acetylcholine in response to nerve impulses. Passive transfer and in vitro experiments using LEMS patients’ igG demonstrate that LEMS is caused by autoantibodies cross-linking the voltage-gated calcium channels, with subsequent aggregation and internalization of the active zone particles (263–266). Expression of different types of voltage-gated calcium channels is a common if not universal feature of small cell lung carcinomas, from patients with or without LEMS (267). Serum antibodies against P/Q-type voltage-gated calcium channels are found in more than 90% of LEMS patients (88,268,269). Anti–voltage-gated calcium channel antibody titers do not clearly differentiate between LEMS patients with or without an associated neoplasm, and do not correlate well with clinical or electrophysiologic disease severity among patients. Longitudinal studies within individual patients may show falling titers in concert with clinical response to therapy (269,270). in most laboratories low titers of anti-P/Q-type voltage-gated calcium channel antibodies are detected in 10% or fewer patients with small cell lung carcinoma but without neurologic symptoms (270,271). Approximately 15% of patients with small cell lung cancer and PEM/SSN but no clinical evidence for LEMS also have anti–voltage-gated calcium channel antibodies (271).

Patient Management in patients with confirmed LEMS the search for a tumor should focus on small cell lung cancer. there are reports of patients in whom “blind” bronchoscopy in the absence of a definite radiographic pulmonary lesion discovered a small cell lung carcinoma (272). Management of patients with LEMS includes pharmacologic facilitation of neuromuscular transmission, removal of autoantibodies, suppression of autoantibody production, and treatment of the underlying tumor (249,273). the clinical and electrophysiologic features of paraneoplastic LEMS generally improve with successful treatment of small cell lung carcinoma or other associated tumors (251). tumor recurrence is not usually accompanied by a relapse of LEMS (254). rarely, the course of the neurologic syndrome fluctuates independently of the status of an individual patient’s tumor. one of the major reasons for the successful treatment of LEMS, relative to other paraneoplastic disorders, is that several agents enhance neuromuscular transmission independent of suppression of the autoimmune response. Pyridostigmine in doses effective for myasthenic gravis also improves strength in the majority of patients with LEMS, although most require additional treatment.

51. Paraneoplastic Disorders of the Nervous System

3,4-Diaminopyridine blocks potassium channels and prolongs the action potential at motor nerve terminals, thereby increasing the release of acetylcholine by a nerve impulse. Diaminopyridine produces some degree of symptomatic improvement in nearly all patients with LEMS, with or without an associated neoplasm (274–276). the most serious side effect is seizure, which usually, but not always, occurs at daily doses of greater than or equal to 100 mg. the addition of pyridostigmine often enhances and prolongs the effect of diaminopyridine and permits dose reduction (251), although patients may have cholinergic gastrointestinal side effects with combined treatment. Diaminopyridine is an orphan drug and is not available for general use in the united States. Guanidine facilitates neuromuscular transmission and has been used in treatment of LEMS for more than 20 years. the addition of guanidine to a maximally effective dose of pyridostigmine may permit a lower dose of both drugs, with reduction in the risk of serious guanidine toxicity (277). the side effects of guanidine are roughly dose related and include myelosuppression, renal tubular acidosis, interstitial nephritis, cardiac arrhythmias, hepatotoxicity, ataxia, and confusion. For patients with paraneoplastic LEMS receiving tumor treatment, it is usually reasonable to use pyridostigmine, diaminopyridine, and/or guanidine, and to defer immunotherapy, since many of these patients will improve with successful tumor treatment. if this is not an option or patients still have severe weakness, there are several therapeutic options for removal of autoantibodies or suppression of autoantibody production. Prednisone or azathioprine is generally effective, but improvement may not be apparent for several weeks or longer. Some patients benefit from combined treatment with both agents (273). Cyclosporine has been occasionally used for treatment of patients who do not respond to or tolerate corticosteroids or azathioprine (273). Plasma exchange produces an improvement in strength and electrophysiologic parameters in most patients with LEMS, usually beginning within 1 to 3 weeks and persisting for as long as 2 to 3 months. iVig is also effective (278,279), with peak clinical improvement within 2 to 4 weeks, and with beneficial effects usually lasting from 2 to 3 months.

MYoPAthiES there has been a long-standing uncertainty over how often polymyositis (PM) or dermatomyositis (DM) occurs as a paraneoplastic syndrome. Although every published study has methodologic flaws, the weight of evidence indicates a higher than expected incidence of cancer among patients with PM or DM (280–283). in most studies the risk ratio is higher for DM than for PM. the associated neoplasms include hodgkin’s lymphoma or carcinoma of the lung, breast, ovary, genitourinary tract, or gastrointestinal system. there is not a clear-cut overrepresentation of any particular tumor

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type compared with an age-matched control population. Patients with paraneoplastic DM have a higher average age of onset than DM patients in general. in most patients the PM/DM and the associated neoplasm are diagnosed within a short time of each other. there is nothing distinctive about the neurologic symptoms, EMG findings, muscle pathology, clinical course, or response to immunotherapy in patients with paraneoplastic PM/DM that would reliably distinguish them from nonparaneoplastic patients. A possible exception may be more frequent elevation of erythrocyte sedimentation rate and a higher incidence of cutaneous necrosis in patients with paraneoplastic DM compared with nonparaneoplastic DM (284). A few published patients with PM or DM had significant neurologic improvement after treatment of the associated tumor, without immunosuppressive therapy. it is not clear how extensive the search for a tumor should be in patients presenting with PM or DM and no previous cancer diagnosis (280,282,285). Chest X-ray, mammogram, digital rectal exam, pelvic exam in women, and prostate-specific antigen in men seem reasonable. Whether all patients presenting with PM/DM should also undergo chest and abdominal Ct scans, colonoscopy, and gastrointestinal barium studies is controversial. Several reports of severe necrotizing myopathy are associated with lung carcinoma or other neoplasms (286,287). Patients develop severe, rapidly progressive weakness with marked elevation of serum creatine kinase. Muscle biopsy or autopsy show diffuse, extensive muscle fiber degeneration and necrosis with minimal or no inflammatory reaction. A few patients improved after tumor resection and corticosteroids, whereas others were severely disabled or died of bulbar and respiratory weakness.

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Basic and Applied Biology of Brain Tumors C h a p t e r 5 2

w Tumor Hereditary Brain Syndromes Christopher D. Turner and Christine A. Chordas Virtually every time a clinician makes the diagnosis of a new central nervous system (CNS) tumor, the patient or a family member will at some point ask the inevitable question, “Why did this happen?” The answer in most cases, “We don’t know,” has been as ritualistic as the question asked. Environmental factors such as diet, chemical exposure, power line exposure, microwaves, and cell phone use continue to be raised as causative agents of CNS tumors, even though little, if any, causal evidence exists. Other than ionizing radiation–induced tumors and CNS lymphomas arising in immunosuppressed individuals, patients who have hereditary brain tumor syndromes are the rare but important component of the population where the answer to “Why me?” can be provided (1). Of the thousands of primary brain tumors diagnosed each year, only a small fraction can be linked to a genetic predisposition syndrome (⬍4% in pediatric tumors and even less in adults) (Table 52-1) (2). Advances in cytogenetics and molecular biology over the last decade have clearly demonstrated that cancer is a genetic disease, even if only a small number are hereditary. Many of the advances that have been made in our understanding of the biology of CNS tumors have resulted from the careful study of known hereditary brain tumor syndromes described within this chapter. An understanding of the genes involved, the products they produce, the pathways

they participate in, the tumors that develop, and the patterns of inheritance of the syndromes is likely to stimulate new treatment approaches and to lead basic scientists closer to helping the clinician and the patient.

THE NEUROFIBROMATOSES Neurofibromatosis type 1 (NF1) and neurofibromatosis type 2 (NF2) are two diverse genetic autosomal dominant disorders. NF1 is associated with tumors of astrocytes and neurons, whereas NF2 is associated with tumors of the meninges and Schwann cells. Neurofibromatosis is referred to as one of the phakomatoses (a Greek term referring to multiple spotty tumors of the lens of the eye). Other related disorders presenting with similar features of neurofibromatosis include Proteus syndrome, Noonan syndrome, and Watson syndrome.

Neurofibromatosis Type I Neurofibromatosis type 1 is one of the most common genetic disorders in humans and accounts for a variety of CNS and extraneural tumors. NF1 was initially known as von Recklinghausen disease, after Friedrich von Recklinghausen. In 1882 he provided the first description of the 715

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w TABLE 52-1

Autosomal Dominant Inherited Syndromes Associated with Brain Tumors

Syndrome

CNS Manifestations

Gene/Function

Neurofibromatosis type 1 (NF-1), von Recklinghuasen disease

Neurofibrosarcoma, rhabdomyosarcoma, optic glioma, astrocytoma, GBM

NF-1 on 17q11 Negative regulation of RAS-mediated cell proliferation

Neurofibromatosis type 2 (NF-2), familial schwannomatosis

Neurofibromas, meningioma, ependymoma, glioma, neurofibromosarcoma

NF2 on 22q12 Maintain cell structure

Tuberous sclerosis (TSC), Bourneville–Pringle syndrome

Subependymal giant cell astrocytoma/nodules, cortical tubers, astrocytoma, GBM oligodendroglioma, ganglioneuroma

TSC1 on 9q34; TSC2 on 16p13 Regulation of cell function and cell growth

Von Hippel-Lindau syndrome

Retinal/spinal hemangioblastoma, ependymoma

VHL on 3p25–p26 Ubiquitin ligase complex targeting HIF-1 for degradation

Nevoid basal cell carcinoma syndrome (NBCCS), Gorlin–Goltz syndrome

Medulloblastoma, astrocytoma

PTC on 9q22.3 Negative regulation of cell division

Li-Fraumeni syndrome

Astrocytomas, medulloblastoma, PNET, choroid plexus carcinoma GBM

TP53 on 17q13 Transcription factor, cell cycle regulation, apoptosis

Brain tumor–polyposis syndrome (BTPS), Turcot syndrome

Medulloblastoma, GBM ganglioglioma

Unclear autosomal dominant vs recessive pattern/APC gene on 5q21 Regulation of multiple cell functions

Multiple endocrine neoplasia type 1 (MEN1), Wermer syndrome

Prolactinoma, astrocytoma

MEN 1 on 11q13 Tumor suppressor gene activity

Trilateral retinoblastoma (TRb)

PNET of the pineal gland (pineoblastoma)

pRB on 13q14 Tumor suppressor gene activity

Rhabdoid predisposition syndrome

AT/RT

Familial pattern of inheritance hSNF5/INI1 on 22q11.2 Truncating mutations

Abbreviations: AT/RT, atypical teratoid/rhabdoid tumor; GBM, glioblastoma multiforme; PNET, primitive neuroectodermal tumor.

w TABLE 52-2

Diagnostic Criteria for NF1

Two or more need to be present for the diagnosis of NF1: Six or more café-au-lait spots: 1/5 cm or larger in postpubertal individuals 0.5 cm or larger in prepubertal individuals Two or more neurofibromas (any type) One or more plexiform neurofibromas Axilla or groin freckling Optic glioma Two or more Lisch nodules A distinctive osseous lesion Dysplasia of the sphenoid bone Dysplasia or thinning of long bone cortex A first-degree relative with NF1 Adapted from Gutmann DH, Aylsworth A, Carey JC, et al. The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and

characteristic fibrous tumors designated neurofibromas (benign peripheral nerve sheath tumors) (3). NF1 primarily affects multiple organs, including the skin and nervous system, with features that include café-au-lait spots, freckling in skin folds, Lisch nodules (asymptomatic pigmented hamartomas of the iris), and neurofibromas at peripheral nerve endings or alongside nerves (Table 52-2). Other features include macrocephaly, short stature, and learning disabilities (4,5). Symptoms aid in diagnosis but are not associated with morbidity. Less common but more severe symptoms that may be progressive and result in significant morbidity or mortality include plexiform neurofibromas, bone abnormalities (scoliosis and pseudoarthrosis), seizures, and neoplasms (6). Five percent of individuals with NF1 develop malignancies, including neurofibrosarcoma, malignant peripheral nerve sheath tumor (MPNST), astrocytoma, pheochromocytomas, melanoma, rhabdomyosarcoma, and

52. Hereditary Brain Tumor Syndromes

w TABLE 52-3

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Screening Recommendations for Patients with NF1 Asymptomatic NF-1 Followed Regularly in a Comprehensive NF Clinic

NF-1 with Symptomatic Optic Pathway Glioma

MRI screening

Not routinely recommended. Not shown to improve outcome.

At diagnosis and 3, 9, 15, 24, then yearly throughout puberty. Insufficient data to recommend intervals after first 2 years of diagnosis but presumably intervals can be lengthened if no evidence of progression.

Ophthalmologic examination

At diagnosis. Yearly in children ⱖ 6 years. Condensed exam at 8, 13, 20 years Full exam at 10, 16, 25 years or at any age with new signs/symptoms.

At diagnosis. Every 3 months for 36 months, then yearly.

Adapted from Listernick R, Louis DN, Packer RJ, et al. Optic pathway gliomas in children with neurofibromatosis 1: consensus statement from the NF-1

chronic myeloid leukemia (7–10). Despite the relatively chronic nature of NF1, the lifespan of affected individuals is often reduced, with a mean survival of 61.2 years (11), because of the increased risk of malignancy (12,13) and complications of hypertension-associated renal artery stenosis or pheochromocytomas (11,14). NF1 is an autosomal dominant disorder with an incidence of approximately one in 3,500 births (15–17). It is estimated that 50% of newly diagnosed patients have no previous family history, indicating that approximately one in 10,000 individuals harbors a new mutation (18). Thus, the use of DNA testing for diagnostic purposes is hampered (19). Genotype–phenotype testing has not been particularly instructive, except for large deletional mutations that appear to lead to a more severe phenotype and more severe learning disabilities. Age of onset and expression of symptoms is variable, with presentations occurring at different stages throughout life. Most patients have characteristic skin findings in childhood (18). Penetrance is just under 100% by 6 years of age. Variability exists among related individuals who carry the same mutations, suggesting that modifier genes may contribute to the severity of symptoms (4,20). Because of the accuracy of clinical diagnosis (Table 52-2) at a relatively young age and difficulties of analyzing the NF1 gene, molecular diagnostics do not play a significant part in the diagnosis or management of this syndrome other than to assist in problematic cases. Currently revised diagnostic criteria for NF1 originally established by the NIH Consensus Development Conference are listed in Table 52-3 (21,22). Increased signal intensities identified on magnetic resonance imaging (MRI), often referred to as unidentified bright objects (UBOs), are identified in 30% to 60% of pediatric patients with NF1 (5,23). These findings occur in the basal ganglia, thalamus, cerebellum, and brainstem regions; exert no mass effect; and do not enhance with

contrast. They are not related to clinical manifestations, focal neurologic deficits, or macrocephaly. Recent studies report an association between the presence of these findings and learning disabilities or low IQ scores in children with NF1 (24,25). The nature of these imaging abnormalities is still unknown. The isolation of the NF1 gene, located on chromosome 17q11.2, was achieved in 1990 with subsequent identification of its protein product, neurofibromin (15). Neurofibromin functions as a tumor suppressor via the downregulation of the Ras protein by increasing GTPase activity. Tumors are believed to arise by the loss of function of the NF1 protein. The role of Ras in the pathogenesis of tumors in NF1 has suggested an approach to treatment using Ras inhibitors, and clinical investigations are ongoing (26,27). Inherited and de novo NF1 are caused by a wide spectrum of mutations affecting the NF1 gene. Most de novo NF1 mutations occur on paternally derived chromosomes (28,29). Patients with this disease require attention to other potential medical complications associated with NF1, such as hypertension, scoliosis, focal neurologic deficits, visual loss or field defects, proptosis, developmental disabilities, short stature, and signs of precocious puberty (21). First- and second-degree relatives should be screened by slit-lamp examination to look for Lisch nodules to assist in determining if the case is familial or sporadic (30). In uncomplicated asymptomatic NF1 patients, yearly clinical assessment and physical examination to the completion of puberty is warranted (30,31). Yearly ophthalmologic examination is indicated in all children up to 8 years of age and thereafter, if clinically warranted. The UBOs that appear on MRI examinations do not behave like tumors. Thus therapeutic procedures and routine MRI screening should not be instituted (32). Routine screening audiology is not indicated (33). Table 52-3 out-

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lines recommended screening guidelines. Counseling for all patients and family members is critical, recognizing the variable expressivity of the disease throughout the lifespan of the patient and genetic implication for their offspring. Optic Pathway Gliomas in NF1 Low-grade astrocytic tumors of the optic pathway, known as optic pathway gliomas (OPGs), are the most common neoplasms associated with NF1. They occur in approximately 15% to 20% of individuals with NF1 (31,34).When OPGs are biopsied, the majority are pilocytic astrocytomas on pathologic review. Low-grade astrocytomas of the cerebral hemispheres, brainstem, and cerebellum, as well as glioblastoma multiforme and meningiomas, have also been reported in individuals with NF1 (35–38). Rare cases of NF1 have been associated with pleomorphic xanthoastrocytoma (PXA) (39). The detailed discussion of lowgrade astrocytomas is discussed elsewhere in this book; however, a brief discussion is warranted here because patients with NF1 have different clinical manifestations and unique biologic properties that influence treatment considerations and convey a more favorable clinical outcome than similar tumors in patients without NF1 (40,41). Impaired function of nervous system cells via tumor suppressor genes and oncogenes contributes to the development of astrocytomas (42). Increased p21-ras activity is a common feature of sporadic malignant astrocytomas and is a direct result of altered signaling through the epidermal growth factor receptor (EGF-R)(43). In NF1 individuals, reduced neurofibromin expression acts as a loss of growth regulator for astrocytes with increased cerebral astrocytes and astrocyte proliferation (43). Astrocyte proliferation may be enough to account for the increased astrocyte-rich white matter regions in children with NF1 and may be sufficient to result in increased astrocytes observed in the brains of patients with NF1 (43,44). However, it is not felt that proliferation is sufficient for tumor formation; rather, consistent with the Knudson two-hit hypothesis, it is thought that gliomas require an additional genetic event to the remaining wild-type allele (43). Specific point mutations in the NF1 gene have not yet been well characterized in pilocytic astrocytomas of the optic pathway (31). However, subpopulations of OPGs have a propensity for aggressive behavior. These subpopulations can be identified by a Ki-67 labeling index of 2% to 3% and a p53 labeling index of less than 1% (43,45). Optic gliomas associated with NF1 can expand the optic nerve and chiasm with or without subarachnoid spread (30,31,46,47). The lesions are typically slow growing and indolent (48). Overall the outcome is good for patients with OPGs. However, patients with extra-optic tumors, particularly adults, have an increased mortality rate (49). Most OPGs arise during the preschool years, with a

peak incidence between 4 and 6 years (21,34,48,50). The incidence and natural history of gliomas of a higher grade, arising in adults or in other locations, are uncommon in NF1 (49). A large number of OPGs remain stable for long periods of time, and in some cases they can even undergo spontaneous regression (51). Despite this, abnormal vision can develop in up to 52% of cases, depending on extent and location of the tumor (41,52). Proptosis and signs of precocious puberty are less common presenting symptoms (53,54). The decision on whether patients with OPGs require treatment is most often made based on symptoms and not radiographic findings alone (55). Children with non-NF1-associated OPGs frequently present earlier and with a more severe clinical presentation than those associated with NF1 (56). MRI is the imaging modality of choice for diagnosing optic pathway gliomas. Management decisions are based on tumor location, presumed or known histology, patient symptoms, and evidence of clinical or neuroimaging progression. All patients with OPGs require a formal ophthalmologic evaluation with visual field testing by a neuro-ophthalmologist at the time of diagnosis and at regular follow-up intervals. Expectant observation is frequently used, since some tumors spontaneously regress (57,58) and nearly 50% progress without requiring intervention (48,59). Consequently, neuroimaging screening in asymptomatic NF1 patients is unwarranted (55,59). When treatment is warranted because of progressive disease on imaging and/or progressive vision loss, therapeutic considerations involve multidisciplinary management that can include surgery, radiation, or chemotherapy. Treatment should focus on maximizing quality of life, preserving visual and endocrine function, and avoiding late effects of therapy (60). Chemotherapy is the most commonly used modality for OPGs in NF1 patients, since the location of these tumors frequently prohibits the use of surgery unless it is to relieve acute symptomatic compression of the optic pathway or other critical structures and the use of radiation therapy is associated with an increased risk of secondary malignancy, endocrinopathies, and vasculopathies (61). Although many different chemotherapy treatments are available, the standard approach used by most centers of vincristine and carboplatin will achieve disease stabilization or response in the majority of patients for an extended period of time (61–66). Prospective studies are ongoing, with varying administration schedules and coadministration of additional agents. Although chemotherapy will often delay the need for radiation therapy, a number of patients will experience progression. However, the outcome of patients with NF1 who have OPGs is considerably better than that of those without NF1, as progressive disease is seen in only 12% of patients with NF1 OPGs, compared with 63% of those with non-NF1 OPGs (41). Regardless of the better outcome, careful surveillance is required.

52. Hereditary Brain Tumor Syndromes

Neurofibromatosis Type 2 Neurofibromatosis type 2 (NF2) is a rare tumor suppressor gene syndrome predisposing affected individuals to lesions derived from the neural crest. Vestibular neurofibroma-like tumors, known as schwannomas (reviewed elsewhere in Chapter 29), are the hallmark of the syndrome. The incidence of NF2 is one in 50,000 (22). Affected individuals are also at increased risk for nervous system and skin tumors. Diagnostic criteria for NF2 include having a bilateral vestibular schwannoma (VS) or having a family history of NF2 plus unilateral VS diagnosed earlier than 30 years of age or having two of the following: meningioma, schwannoma, glioma, juvenile posterior subcapsular lens opacities, juvenile cortical cataract (21,22, 67). Most patients manifest symptoms in the second or third decade of life; however, in 10% of cases, individuals with NF2 become symptomatic before 10 years of age (21). Penetrance is 95% by 50 years of age. Two different types of NF2 exist, differentiated by time of onset and severity of symptoms (68). The diagnosis of NF2 in children should be considered in individuals with multiple central nervous system or skin tumors without café-aulait spots or Lisch nodules (69). Hemispheric, brainstem, cerebellar astrocytomas, glioblastomas, meningiomas, and ependymomas have been identified in individuals with NF2 (13,70,71). Reduced or absent MERLIN expression has been found in sporadic meningiomas and ependymomas, suggesting that the NF2 gene may be involved in the pathogenesis of the sporadic forms of some of these tumors as well (72–76). Meningiomas represent the most common brain lesions seen in NF2 patients. Meningiomas in NF2 generally occur at an earlier age and are often multiple, compared with meningiomas occurring sporadically (77). Spinal tumors have also been identified in patients with NF2 (78). The gene for NF2 is located on chromosome 22q12 and was cloned in 1993 (79,80). The product of the NF2 gene is a protein identified as MERLIN (moesin-ezrinradixin-like) or schwannomin. MERLIN exhibits tumor suppressor activity through interaction with CD 44 to maintain cell structure and inhibit cell growth (81–83). Mutations in the NF2 gene that result in truncated proteins are associated with earlier onset and more severe disease (19). De novo disease or mosaicism exists in approximately half of NF2 patients (84,85). Patients with NF2 require regular, thorough audiologic, neurologic, and dermatologic examinations that include periodic MRIs of the brain and spine (69). Similar surveillance should be applied in a child presenting with meningioma, since a higher fraction is genotypically and phenotypically more aggressive than with adult presentation (86). Audiologic examinations are critical with the presence of VS. In patients at high risk of developing

719

NF2, a formal ophthalmologic evaluation during the first or second year of life is recommended. Screening for family members of patients with known NF2 should begin at 10 years of age with MRI screening continued in asymptomatic family members every 2 years, then every 3 years at age 30 years (87). DNA linkage analysis provides more than 90% certainty of affected status but is not a practical screening tool at this time in all centers. The management for vestibular schwannomas is reviewed in detail in Chapter 29 and will not be reviewed here. Of note, similar to NF1 patients, there are reports of increased second malignant neoplasms in the irradiated field in patients with NF2. Thus, irradiation is reserved for only the most aggressive tumors (88). Preliminary research in the development of directed treatment for schwannomas and meningiomas is ongoing (89).

TUBEROUS SCLEROSIS Tuberous sclerosis (TS) is an autosomal dominant systemic disorder and was first reported by Bourneville in 1880 as “tuberous sclerosis of the cerebral convolutions” (90). Like neurofibromatosis, TS is one of the phakomatoses. The hallmark of TS is the development of multiple hamartomas that can occur anywhere but are most commonly seen in the brain, kidneys, heart, lungs, and skin. Although a vast majority of tumors associated with TS are classified histologically as benign, progression to malignant neoplasms does occur, particularly in the kidney, and even the benign tumors in the brain can cause serious disability and even death (91–94). Classically, TS was associated with the triad of seizures, mental retardation, and facial angiofibromas. However, fewer than 50% of TS patients have this classical triad (95). Currently, the diagnostic features of TS can be divided into major and minor features. The major features include facial angiofibromas, subependymal giant cell astrocytomas, subependymal nodules (“candle gutterings”), cortical tubers, multiple retinal hamartomas (“mulberry lesions”), a shagreen patch, ash-leaf spots, nontraumatic ungula or periungual fibromas, cardiac rhabdomyomas, renal angiomyolipomas, and pulmonary lymphangiomyomatosis. The minor features of TS include gingival fibromas, multiple randomly distributed dental enamel pits, bone cysts, “confetti” skin lesions, retinal achromic patches, hamartomatous rectal polyps, and nonrenal hamartomas (96). The diagnosis of TS is made based on the presence of two major features or one major and two minor features. The genetic basis of TS has been isolated to mutations in two genes. TSC1 produces a protein known as hamartin and is located on chromosome 9q34 (97). TSC2 produces a protein named tuberin and is located on chromosome 16p13 (98). Mutations in both of these genes are

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transmitted in an autosomal dominant manner, although approximately 60% of patients present with new sporadic mutations (99). A comprehensive review of the pathogenesis of TS and the functions of hamartin and tuberin was performed by Narayanan (90). Briefly, hamartin is a transmembrane protein that binds to ezrin and other moesin–ezrin–radixin proteins to link the cell membrane to the cytoskeleton. Tuberin appears to function as a signaling protein and has homology to a guanosine triphosphatase-activating protein rap1GAP. Both appear to interact directly with each other to regulate specific cell functions, including cell growth (100). Studies have shown that the loss or reduction tuberin can be seen in giant cell astrocytomas (101–103). Based on this and other studies, there is evidence to indicate that TSC1 and TSC2 function as tumor-suppressor genes. The central nervous system (CNS) involvement of TS consists of three distinct lesions: cortical tubers, subependymal nodules, and subependymal giant cell astrocytomas (SEGAs). The cortical tubers are areas of cortical dysplasia that frequently occur within the frontal lobe along the suci and gyri and frequently appear as potatolike growths (104). These lesions are best visualized with MRI of the brain. Despite their benign histology, cortical tubers can cause significant morbidity and mortality. Up to 80% of TS patients experience seizures thought to be due to the presence of cortical tubers (104). Adequate control of infantile spasms and generalized seizures seems to be a key factor for maximizing neurocognitive development (105). Large cortical tubers, especially near the third ventricle, can lead to obstructive hydrocephalus and death if not resected. Subependymal nodules or “candle gutterings” are of glial origin, but typically accumulate calcium with the first years of life and are best visualized with computed tomography (CT). At least one study reports that increased numbers of subependymal nodules (greater than eight) may be a prediction of the severity of the cerebral dysfunction in TS with an increased incidence of infantile spasms and lower global intelligence quotient (IQ) scores (⬍70) (106). Subependymal giant cell astrocytomas (SEGAs) are considered low-grade or benign glial tumors that occur in 5% to 15% of individuals with TS. SEGAs are particularly problematic because they occur at a young age (mean age of 13 years) and are usually located near the foramen of Monro, where their growth produces obstructive hydrocephalus (107). These tumors have traditionally represented a significant cause of death in TS patients, with mortality rates reaching up to 10% even when emergency surgery was attempted (107,108). Although these numbers are likely lower in the modern era of MRI and improved neurosurgical intervention, clinicians caring for TS patients need to remain vigilant in their screening and monitoring for SEGAs. There have been rare reports of malignant brain tumors such as glioblastoma multiforme

arising either from a TS harmartoma or following the treatment of a TS SEGA with radiation therapy (109–111). Neurosurgical intervention is the primary treatment modality for TS patients with symptomatic or potentially symptomatic CNS lesions. SEGAs have not been shown to be chemoresponsive, and radiation therapy should only be considered when there is no surgical alternative. Cranial MRI screening is recommended at the time of initial diagnosis and then every 1 to 3 years through adolescence (112). Children with TS who have nodules located near the foramen of Monro should have yearly MRIs with gadolinium enhancement until the age of 10 years (107). The use of cranial CT scans may sometimes be preferable in asymptomatic patients due to cost and specificity (96). Additional screening should include neurodevelopmental testing and ophthalmologic, dermatologic, cardiac, and renal evaluations as clinically indicated. TS patients and their families should also be referred for genetic counseling, although molecular testing is not commercially available at this time.

VON HIPPEL-LINDAU DISEASE Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited cancer syndrome characterized by multiple benign and malignant vascular-rich tumors that affect multiple organ systems. The most frequent neoplasms of VHL disease include central nervous system (CNS) hemangioblastoma, retinal hemangioblastoma, clear cell renal carcinoma (CCRC) (113), and pheochromocytomas (114). Pancreatic neuroendocrine tumors, epididymal cystadenomas, endolymphatic sac tumors, and carcinoid tumors are other, less common manifestations of VHL (115–118). Affected individuals are also at increased predisposition for multiple cysts of the kidney, pancreas, lungs, bone, and epididymis. The disease affects one in 36,000 births (114,119). Seventy-five percent of index cases have a positive family history of VHL (115); however, de novo mutations do exist (120). The median age of onset is 30 years for most presentations, with near 95% penetration by 65 years of age (115). Tumor presentations vary and are classified according to genotype– phenotype association (115). Type I manifestations include CCRC and CNS hemangioblastomas without any pheochromocytomas. Type II manifestation include all those in type I plus pheochromocytomas. Type II is further classified into type IIA, with a low risk for CCRC; type IIB, with a high risk for CCRC; and type IIC, which presents with pheochromocytoma only. In 1988 the tumor suppressor gene responsible for VHL was mapped to chromosome 3p25-p26 (121) with formal identification of the VHL gene in 1993 (114). Subsequent heterogeneity in type and location of mutations

52. Hereditary Brain Tumor Syndromes

within the VHL gene have been detected in nearly 100% of documented affected families (122,123) and are listed in a VHL mutation database at http://www.umd.necker.fr. VHL patients without detectable mutations may be mosaic (123). Tumorigenesis arises in affected individuals that inherit a mutated tumor suppressor gene and develop neoplastic disease in association with an additional loss of expression or mutational inactivation of the remaining wild-type VHL allele (124,125). The mechanisms leading to disease involve the VHL protein (pVHL) encoded by the VHL tumor suppressor gene. The pVHL joins with other proteins to form a ubiquitin ligase complex that targets hypoxia-inducible factors (HIF) for ubiquitin degradation (126). Absent or abnormal VHL protein leads to HIF-1 and HIF-2 degradation, resulting in abnormal overexpression of angiogenic, growth, and mitogenic factors (VEGF, GLUT-1, PDGF, TGF) and tumor formation (113,124,127,128). Other mechanisms of tumorigenesis caused by absent or abnormal VHL protein include disruption of normal cell cycle, increased angiogenesis, and abnormalities in the fibronectin extracellular matrix assembly (129–132). CNS hemangioblastomas account for 80% of the lesions in VHL patients (119,133,134) and can arise anywhere along the craniospinal axis: cerebellum, 37%; brainstem, 10%; spinal cord, 50%; lumbosacral nerve roots, less than 1%; and supratentorial region, less than 1% (135). The most common VHL tumor, CNS hemangioblastoma, is still one of the rarest tumors of the CNS, accounting for less than 2% of all CNS tumors (115,136). Contrastenhanced T1-weighted MRI best reveals isolated or multiple lesions, and T2-weighted or Fluid-Attenuated Inversion Recovery (FLAIR) best reveals edema and cysts. Arteriography reveals intense persistent signal intensity, arteriovenous shunting, and early draining veins (124). Pathologic examination reveals a well-defined capillary network surrounded by lipid-laden cells (stromal cells) with abundant mast cells and few astrocytes at the tumor margin. Stromal cells stain positive for vimentin. Loss of heterozygosity has been identified in stromal cells of hemangioblastoma, suggesting their neoplastic nature (137). CNS hemangioblastomas evolve slowly through periods of growth and periods of quiescence, and may be the only manifestation of the disease, but more frequently, multiple tumors are present involving both the posterior fossa and spinal cord (136). Clinical presentation is related to the tumor size and location, the mass effect on surrounding structures, and rapidly growing cysts that can occur with 30% to 80% of hemangioblastomas (124). Symptoms can include gait ataxia, dysmetria, headaches diplopia, vertigo, emesis, hyperesthesia, dysphagia, hyperreflexia, dysmetria weakness, pain, and incontinence. Despite their benign histopathologic designation, hemangioblastomas are a major cause of death in VHL disease.

w TABLE 52-4

721

Screening Recommendation for Individuals at Risk for VHL

Test

Start Age (frequency)

Vision Plasma or 24-hr urinary catecholamines and metanephrines MRI of CNS axis * Auditory CT and MRI * Abdominal ultrasound Abdominal CT Audiology

Infancy (yearly) 2 years (yearly)** 11 years of age (yearly)** Onset of symptoms 8 years (yearly) 18 years (yearly)** When clinically indicated

Adapted from Lonser RR, Glenn GM, Walther M, et al. Von Hippel-Lindau disease. Lancet 2003;361:2059–2067. *Pre- and postcontrast imaging techniques recommended.

Clinical diagnosis for VHL disease is definitive in patients with a positive family history and a CNS or retinal hemangioblastoma and in those patients with no family history but with two or more CNS hemangioblastomas, or one CNS hemangioblastoma and a visceral tumor (115). De novo cases present more of a diagnostic challenge (123). Molecular genetic analysis in families and patients identifies individuals who do not meet clinical diagnostic criteria. Surveillance and presymptomatic diagnosis of VHL tumors reduces morbidity and mortality in VHL disease and has improved prognosis. In relatives that are determined not to carry the gene, surveillance is discontinued. Recommended surveillance (Table 52-4) in at-risk individuals continues throughout the seventh decade of life and comprises multiorgan screening of CNS and visceral organs (124,138). The clinical management of VHL disease is challenging and requires a multidisciplinary approach. Management strategies are tailored to the type and extent of tumor and cyst involvement, and can include surgery, radiation, and chemotherapy. Novel therapies inhibiting angiogenesis and blocking vascular endothelial growth factor (VEGF) signaling have been reported in the literature (139–142). Surgery is the standard treatment for most symptomatic hemangioblastomas of the CNS axis with or without preoperative embolization and outcome usually is favorable (143,144).

NEVOID BASAL CELL CARCINOMA SYNDROME (GORLIN-GOLTZ SYNDROME) In 1960, Gorlin and Goltz were the first to describe an autosomal dominant syndrome in patients who had multiple nevoid basal-cell epitheliomas, odontogenic jaw cysts, and bifid ribs (145). An association of Gorlin-Goltz syndrome with medulloblastoma was made by Herzberg shortly

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w TABLE 52-5

Major and Minor Diagnostic Criteria for NBCCS or Gorlin’s Syndrome

Major Criteria First degree relative with NBCCS One BCC under the age of 20 or Multiple BCCs at any age Bilamellar calcification of the falx cerebri Biopsy proven odontogenic keratocysts of the jaw Bifid, fused or markedly splayed ribs Three or more palmar or plantar pits Minor Criteria Medulloblastoma Ovarian fibroma Macrocephaly Congenital malformations (cleft lip or palate, frontal bossing, severe hypertelorism, or “coarse face”) Skeletal abonormalities (syndactyly, Sprengel deformity, pectus deformity) Radiologic abnormalities (Bridging of the sella turcica, fused vertebra or hemivertebrae, modeling defects or flame shaped lucenies of the hands or feet) Adapted from Kimonis VE, Goldstein AM, Pastakia B, et al. Clinical manifestations in 105 persons with nevoid basal cell carcinoma syndrome. Am J Med Genet 1997;69:299–308.

thereafter (146). Although many still refer to this syndrome as Gorlin syndrome, it is currently known as nevoid basal cell carcinoma syndrome (NBCCS), in recognition of its most common characteristic, the early development of basal cell carcinomas (BCC). It is estimated that the prevalence of NBCCS is about one per 60,000, with 35% to 50% of diagnosed patients representing new mutations (147). The neoplasms or hamartomas associated with NBCCS include the development of multiple BCCs, medulloblastoma, ovarian fibromas and carcinomas, fetal rhabdomyomas, lymphomesenteric cysts, and at least one report of an astrocytoma (148–151). Other characteristics of NBCCS include odontogenic keratocyst of the jaws, plantar or palmar pits, congenital anomalies (including short metacarpal bones and bifid or fused ribs or vertebra, microphthalmia, cataracts, cleft lip or palate, polydactyly), and calcification of the falx cerebri (147,151,152). Criteria for the diagnosis of NBCCS are divided into major and minor criteria (Table 52-5). Diagnosis of NBCCS is made with the presence of two major criteria or one single major criterion and two minor criteria (152). Basal cell nevi occur frequently in NBCCS and often begin around the time of puberty and can increase to enormous numbers. Although basal cell nevi and BCCs are one of the most common findings in white and fair skin individuals, they are less common in people with increased skin pigmentation, such as African-Americans or those of Mediterranean descent (153,154). However, while increased skin pigmentation may be relatively protective of ultraviolet (UV) radiation from forming BCCs, it does not afford one any increase in protection from ionizing radiation.

Medulloblastoma is the most common brain tumor associated with NBCCS. A study of 105 patients seen at the National Institutes of Health over 10 years identified four patients diagnosed with medulloblastoma. All were children and the mean age of diagnosis of medulloblastoma was 2.3 years (152). One estimation is that of all children diagnosed with medulloblastoma less than 3 years of age, 10% will have NBCCS (155). Therefore, all children with newly diagnosed medulloblastoma, especially those less than 7 years of age, should be carefully screened for signs of NBCCS. The genetic basis for NBCCS has been shown to be linked to inactivating mutations in the tumor-suppressor gene patched (PTCH), located on chromosome 9q22.3 (156,157). Some have argued that PTCH acts as a classic tumor suppressor gene and follows Knudson’s two-hit mechanism for neoplasia, even suggesting that the associated developmental defects may also be explained by the two-hit mode (158), although this theory is not universally accepted. Patched is the transmembrane protein receptor for Sonic hedgehog and plays a central role in the Sonic hedgehog-Patched signaling pathway, an important regulating pathway for cerebellar granule cell precursors proliferation and a major site of mutations in medulloblastoma formation, BCC development, and a variety of other cancers (159). Inhibitors of this pathway have been identified and are being investigated as potential “mechanism-based” therapeutic agents for treatment of these tumors (160). Because of the risk of UV radiation, all NBCCS patients need to be counseled about avoiding sun exposure. To detect medulloblastomas early in young NBCCS patients, annual screening MRIs of the brain and careful neurologic examinations every 6 months are recommended for children up to the age of 7 (152). All individuals with NBCCS treated with radiation therapy are at extremely high risk of developing multiple BCCs within the radiation field 1 to 4 years after radiation treatment (151,153,161). Additionally, postradiation meningiomas have been documented years after treatment in patients with NBCCS who were treated with cranial irradiation (152). Whenever possible, protocols that utilize chemotherapy in lieu of radiation therapy should be considered in NBCCS patients with malignancies. Since virtually all medulloblastoma patients require some type of radiation therapy, however, late surveillance for secondary intracranial tumors such as meningiomas and astrocytomas, in addition to skin surveillance for BCCs, is especially important in NBCCS patients.

LI-FRAUMENI SYNDROME Li-Fraumeni syndrome (LFS) is a dominantly inherited cancer predisposition characterized by familial aggregations of rare tumors first described by Li and Fraumeni in 1969 (162,163). The most prevalent tumors observed in LFS include soft tissue sarcomas, premenopausal breast

52. Hereditary Brain Tumor Syndromes

cancer, brain tumors, bone leukemias, and adrenocortical carcinoma (164). Consequently, it is commonly referred to by the acronym SBLA syndrome (162). Approximately 23% of cancer presentations associated with LFS are at risk for cancers outside the classical realm and include melanoma, germ cell tumors, lung and gastric carcinoma, pancreatic tumors, ovarian tumors, and Wilms tumor (165,166). The definition of LFS includes a proband diagnosed with sarcoma before 45 years of age, a first-degree relative with cancer before this same age, and another first- or second-degree relative in the lineage with any cancer before this age or sarcoma at any age (167,168). In subsequent years the Li-Fraumeni-like syndrome (LFL) has been identified as a proband with any childhood tumor, or a sarcoma, brain tumor, or adrenocortical tumor aged under 45 years plus a first- or second-degree relative in the same lineage with a typical LFS tumor at any age, and an additional first- or second- degree relative in the same lineage with any cancer under the age of 60 years (169). Presentations of these syndromes typically occur at a younger age than with presentations of sporadic disease. In LFS the mean age of tumor presentation varies: 5 years for adrenocortical carcinomas, 16 years for sarcomas, 25 years for brain tumors, 37 years for breast cancer, and nearly 50 years for lung cancer (164). Early onset of adrenocortical carcinoma and breast cancer prompts suspicion of LFS and warrants further investigation. Families within the LFS have been demonstrated to have multiple primary cancers, with 15% of theses patients developing a second cancer, 4% a third cancer, and 2% a fourth cancer (170). Studies have shown the occurrence of radiation-induced second tumors in carriers of the p53 mutation, indicating avoidance of radiation therapy when possible (171–173). Members of families with this syndrome develop multiple primary cancers via an autosomal dominant gene transmission mode. Germ-line mutations in the p53 gene discovered in 1979 (174) are the primary factor in malignancy development (175) and have been described in 50% to 70% of cases of LFS (162). Similar p53 mutations are seen in sporadic tumors (176). The p53 gene acts as a transcription factor for genes that induce apoptosis, mediating arrest in the G1 phase of the cell cycle (170,177). Introduction of wild-type p53 into cell lines that have lost functional p53 causes growth arrest or induces apoptosis; however, it remains unclear how the wild-type p53 allele is functionally inactivated. More than one single genetic predisposition may be involved (170,178). Heterozygous germ-line mutations of hCHK2, a regulator of TP53, have been identified in LFS (178). Most epidemiologic searches and case studies of nervous system tumors demonstrate familial aggregation of gliomas independent of a tumor syndrome (179–182) and fewer incidences of gliomas in LFS (183,184). The genetic mutations have been demonstrated to be similar in sporadic and hereditary malignancies involving the

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p53 gene. Astrocytes appear to be at more risk for malignant transformation than any other cell type of the nervous system (164). In low-grade astrocytomas, p53 mutations are the earliest detectable genetic alteration, whereas progression to anaplastic astrocyomas and glioblastomas is associated with additional genetic alterations (164). An excess of germ-line p53 mutations is reported in patients with multifocal gliomas, gliomas in association with a second primary tumor, and gliomas found as an integral lesion in families with diverse cancers. This is not seen in patients with unifocal gliomas without a family history of cancer or another primary malignancy (184). Medulloblastomas, primitive neuroectodermal tumors (PNETs), and choroid plexus carcinoma are less prevalent in LFS families (185,186). Surveillance for the p53 germ-line mutation is complicated by several factors. The diversity and unpredictability of tumors in LFS make it difficult to predict who should be screened and what areas should be screened (187,188). Additionally, the lack of effective intervention for mutation carriers may preclude screening for the p53 mutation. Finally, the lack of a standard uniform testing procedure allows for significant variability in the screening process. While the ethical, psychological, social, and medical consequences of screening results are not clearly understood, genetic counseling and individualized screening practices ought to be considered in those at most risk, prompting evaluation of any mass or unusual symptoms (170,184).

BRAIN TUMOR-POLYPOSIS SYNDROME (TURCOT SYNDROME) Turcot described the association of malignant tumors of the central nervous system with familial polyposis of the colon in 1959 in two teenaged siblings whose parents were third cousins (189). Both had numerous adenomatous polyps of the colorectum. One developed medulloblastoma and the other developed a cerebral glioblastoma muliforme and a pituitary adenoma. A similar case involving medulloblastoma and adenomatous polyposis was first described by Crail 10 years earlier (190). However, the syndrome of colorectal polyposis and a primary central nervous system tumor is referred to as Turcot syndrome or brain tumor–polyposis syndrome (BTPS). Initially, many believed BTPS is a variant of familial adenomatous polyposis (FAP), an autosomal and dominant disease linked to mutations of the APC gene where hundreds of adenomatous colonic polyps develop at an early age and virtually always progress to cancer unless a colonectomy is performed (191). However, more recent reports using molecular genetics and the type of CNS neoplasm at presentation have helped to delineate two clinical subtypes of BTPS (192–197) (Table 52-6).

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w TABLE 52-6

Summary of Features of BTPS or Turcot Syndrome

Other name Type of brain tumor Number of colonic polyps Skin involvement Family history Siblings affected Consanguinity Mode of inheritance Genetic mutation Related genetic disorder

BTPS Type 1

BTPS Type 2

Random Brain Tumor Polyposis Cases

Turcot syndrome Gliomas ⬍100 Frequent None Usually Frequent Mixed? Mismatch repair genes HNPCC

FAP variant Medulloblastomas ⬎100 Occasional History of polyposis Usually None Autosomal dominant Adenomatous polyposis coli (APC) FAP, Gardner syndrome

Independent diseases Variable Variable None Possible history of polyposis No history of brain tumors None Not inherited None or somatic tumor-specific alterations None

Adapted from Paraf F, Jothy S, Van Meir EG. Brain tumor–polyposis syndrome: two genetic diseases? J Clin Oncol 1997;15:2744–2758.

BTPS type 1 is often recognized as true Turcot syndrome and is associated with hereditary nonpolyposis colorectal cancer and the development of an astrocytoma or glioblastoma multiforme at a young age (a majority are less than 20 years old) (192). One patient has also recently been reported to have this type of BTPS and had a ganglioglioma as the primary CNS tumor (198). This type of BTPS has been described as having an autosomal recessive inheritance by some authors (192,197) and autosomal dominant by others (194). Type 1 BTPS is caused by germline mutations in genes involved in DNA nucleotide mismatch repair (MLH1, MSH2, PMS1, and PMS2) (194,197). BTPS type 2 is not considered true Turcot syndrome and is more likely a variant of FAP. These patients have a predisposition to develop medulloblastomas in addition to the numerous adenomatous colorectal polyps that they develop. Occasional cases of gliomas or ependymomas have been reported with BTPS type 2. However, it is unclear whether these represent true type 2 cases or whether they happen to be random brain tumor–polyposis (BTP) associations (192,199). These patients have an autosomal dominant germ-line mutation of APC that significantly increases the relative risk of developing a brain tumor, especially medulloblastoma (194). The treatment for all CNS tumors of both type 1 and type 2 BTPS is unchanged from those without an associated hereditary cancer syndrome. There does not appear to be any increased risk of second malignancies over the general population when either chemotherapy or radiation therapy is used. Routine colonoscopy is recommended for all family members of patients with BTPS. If a brain tumor develops in a patient who has a family history of FAP, the patient should undergo a diagnostic and future screening colonoscopies to look for polyposis, since the brain tumor may procede the development of colonic polyps in BTPS (199). Patients with BTPS type 1 are at increased risk for malignancies other than brain and colon, such as ovarian, gastric, urologic tract, small bowel, and hepatobiliary tract cancers (200). Genetic counseling and testing, when appropriate, are recom-

mended for all patients diagnosed with BTPS to help provide better prognostic advice and to increase the known genetic data for this syndrome.

MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 (WERMER SYNDROME) Multiple endocrine neoplasia type 1 (MEN1), or Wermer syndrome, is a disorder that is characterized by a patient having at least two of the following three tumors: parathyroid adenomas, enteropancreatic endocrine tumors, and pituitary tumors (201). Familial MEN1 is defined when at least one first-degree relative of an index case is diagnosed with at lease one of the three major MEN1 tumors. Approximately one-third of MEN1 patients have tumors of the anterior pituitary, and a majority of these measure less than 1 centimeter (microadenomas) (201). The most common anterior pituitary tumors seen in MEN1 patients are prolactinomas, followed by somatotrophinomas, corticotropinomas, and nonfunctioning pituitary tumors (202). The MEN1 gene is found on chromosome 11q13, and its loss demonstrates that it represents a tumor suppressor gene consistent with Knudson’s model for tumor development (202). Its gene product, menin, has been proposed to interact with glial fibrillary acidic protein (GFAP) within the brain, and in rare cases, bi-allelic inactivation of MEN1 genes has been associated with the development of grade II astrocytomas (203). MEN1 pituitary adenomas are treated the same as sporadic pituitary tumors, depending on the type. Patients should be monitored by regular cranial MRIs and periodic serum testing of prolactin and IGF-I (201).

MULTIPLE ENDOCRINE NEOPLASIA TYPE 2A (SIPPLE SYNDROME) Multiple endocrine neoplasia type 2a (MEN 2a), or Sipple syndrome, is characterized by medullary thyroid car-

52. Hereditary Brain Tumor Syndromes

cinomas, pheochromocytomas, and parathyroid adenomas. The syndrome is caused by germ-line mutations in the RET gene, and it is inherited in an autosomal dominant pattern (201). There is no strong evidence linking MEN 2a to primary CNS tumors.

TRILATERAL RETINOBLASTOMA The association of bilateral retinoblastoma in conjunction with a primary intracranial neoplasm is called trilateral retinoblastoma (TRb). The intracranial neoplasm is virtually always a PNET in the region of the pineal gland (i.e., pineoblastoma). However, PNETs of the suprasellar, parasellar, and even posterior fossa have also been associated with TRb (204–206). Most cases of TRb have hereditary bilateral retinoblastoma, an autosomal dominant trait with almost total penetrance. The retinoblastoma gene (RB) is located on chromosome 13q14 and is a classic tumor suppressor gene (207). TRb is a disease of early childhood, with the median age of diagnosis of retinoblastoma of 5 months of life and the median time to diagnosis of the intracranial lesion 21 months later, with a poor overall prognosis (208). Conventional therapy is typically used to treat the retinoblastomas, whereas the intracranial lesions are treated with therapy directed at high-risk supratentorial PNETs. Five percent of patients with hereditary retinoblastoma will develop TRb, so a screening MRI of the brain is recommended at the time of retinoblastoma diagnosis and yearly for at least 3 to 5 years (209).

RHABDOID PREDISPOSITION SYNDROME Bonnin et al. were the first to describe the association of renal embryonal neoplasms in infants under 2 years of age, with an embryonal primary tumor originating in the CNS (210). These embryonal tumors were located within the posterior fossa, were associated with poor outcome, and were most often grouped with classic PNETs based on standard clinical, radiographic, and pathologic criteria. However, Rourke et al. reported on the clinical and morphologic features of 32 cases of unique, biologically aggressive tumors in children less than 2 years of age and classified them as a distinct neoplasm named atypical teratoid/rhabdoid tumors (AT/RTs), based on the appearance of rhabdoid cells, and, less frequently, malignant mesenchymal and/or epithelial tissue in tumors that otherwise look like typical PNETs (211). Cytogenetic and molecular analyses linked AT/RTs to the deletion of chromosome 22q11.2 and the creation of truncating mutations of hSNF5/INI1 gene (212,213). Germ-line mutations in INI1 have been identified and thought to predispose infants to the development of both renal and CNS tumors (214–216). The term rhabdoid predisposition syndrome is used to describe

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the familiar pattern on inheritance of these malignant rhabdoid tumors. Although most of these patients have loss of a portion of chromosome 22, the loss of other chromosomes such as 1p and 8p has also been reported (217). Clinically, patients diagnosed with CNS AT/RTs have a dismal prognosis, with an average survival following diagnosis of less than 1 year, even with gross total resection and aggressive multiagent therapy (218). There are fewer than 10 published survivors of CNS AT/RT. A number of international mulitcenter clinical trials are under way that incorporate multimodality therapy for these young patients in an attempt to improve outcome.

OTHER RARE HEREDITARY BRAIN SYNDROMES Carney complex is an extremely rare autosomal dominant familial tumor syndrome that is associated with myxomas, mucocutaneous spotty pigmentation, pituitary tumors, and nerve sheath tumors. Since patients with Carney complex frequently have Cushing’s syndrome and/or cardiac myxomas, neurosurgeons and neurooncologists should be aware of this syndrome in order to perform appropriate family screening and to recognize the potential for surgical co-morbidity (219). Alexander disease is a rare disorder caused by mutations in the gene that codes for glial fibrillary acid protein (GFAP) and is associated with the development of glial tumors. The childhood form is caused by de novo mutations. However, an autosomal dominant adult variant has been described that is clinically distinct from the childhood de novo mutations (220). Several authors have reported on multiple primary brain tumors (most often cerebral gliomas) within a family without a well-recognized hereditary disorder (221,222). There are undoubtedly a number of yet-to-be-described genetic mutations and brain cancer syndromes that remain to be investigated and classified. The completion of the mapping of the human genome and an increasing understanding of the pathogeneses of CNS tumors will, it is hoped, assist in this endeavor.

GENETIC TESTING AND ETHICAL CONSIDERATIONS This chapter would not be complete without at least a brief mention of some of the methods used to screen and test patients suspected of having hereditary brain tumor syndromes and other ethical and legal implications of diagnosing patients with a hereditary cancer syndrome. Because of scientific advances, societal concerns, financial repercussions, issues of privacy, and implications associated with diagnosis for those other than just the patient, the field of genetic testing for cancer susceptibility continues to evolve.

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Who should be tested and how testing should be performed continues to receive much discussion and scrutiny. These issues are amplified even more when one begins to talk about testing minors. To help the oncology community deal with these issues, the American Society of Clinical Oncology (ASCO) issued a policy statement to clarify the indications for genetic testing. They recommend “genetic testing be offered when 1) the individual has personal or family history features suggestive of a genetic cancer susceptibility condition, 2) the test can be adequately interpreted, and 3) the results will aid in diagnosis or influence the medical or surgical management of the patient or family members at hereditary risk of cancer” (223). Essential to ASCO’s genetic testing recommendation is the need for preand posttest counseling to discuss the risks and potential benefits and advise of any prevention strategies. The ASCO policy statement also addresses the issue of performing genetic testing in children. In general, genetic testing should be delayed until the patient has reached an age sufficient to make an independent informed decision for himself or herself, unless such delay is likely to increase the risk of missing a potentially treatable childhood malignancy (223). An interesting corollary to the issue of genetic testing of an individual patient is the physician’s duty to warn either the patient or a third party about potential risks of developing a malignancy. The American Society of Human Genetics (ASHG) issued a policy statement concluding that physicians have a firm duty to disclose familial genetic risks to their patients (224). The issue of whether physicians have a duty to warn a third party (e.g., a firstdegree relative who is not under the care of the physician) is a much more complicated one that has been the subject of several lawsuits against physicians and is beyond the scope of this chapter (225). A final area of controversy that is worth mentioning is the emerging technology of preimplantation genetic testing. A recent paper reported that couples undergoing standard in vitro fertilization procedures who had a predisposition for hereditary cancer syndromes (all of which were hereditary brain tumor syndromes) were able to successfully have the embryos tested for cancer-causing mutations prior to being implanted in the uterus (226). Studies such as this highlight some of the complicated social issues associated with these disorders and the potential for conflict that arises between scientific, ethical, and moral principles.

CONCLUSION The hereditary brain tumor syndromes discussed within this chapter represent a small but critically important number of patients diagnosed with CNS tumors. Cancer has clearly been shown to be a genetic disease, and although most CNS tumors diagnosed are the result of somatic mutations, it is the study of the germ-line mutations

that has proven most informative in uncovering the biology and pathways involved in malignant transformation. Many of the newer therapeutic agents currently being developed and tested in early clinical trials target specific genes or their products that are responsible for the disorders described within this chapter (i.e., farnesyltransferase inhibitors for NF1 and sonic hedgehog antagonists for BCCS). With the rapid advances in molecular genetics and the increased understanding of the genetic basis for human malignancy, the next decade promises to shed new light on our understanding of an individual’s predisposition to develop malignancies.

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182. Hemminki K, Li X. Familial risks in nervous system tumors. Cancer Epidemiol Biomarkers Prev 2003;12(11 Pt 1):1137–1142. 183. Li YJ, Sanson M, Hoang-Xuan K, et al. Incidence of germline p53 mutations in patients with gliomas. Int J Cancer 1995;64:383–387. 184. Kyritsis AP, Bondy ML, Xiao M, et al. Germline p53 gene mutations in subsets of glioma patients. J Natl Cancer Inst 1994;86:344–349. 185. Yuasa H, Tokito S, Tokunaga M. Primary carcinoma of the choroid plexus in Li-Fraumeni syndrome: case report. Neurosurgery 1993;32:131–133; discussion, 133–134. 186. Garber JE, Burke EM, Lavally BL, et al. Choroid plexus tumors in the breast cancer–sarcoma syndrome. Cancer 1990;66:2658–2660. 187. Varley JM. Germline TP53 mutations and Li-Fraumeni syndrome. Hum Mutat 2003;21:313–320. 188. Friend SH, Iggo R, Ishioka C, et al. Overcoming complexities in genetic screening for cancer susceptibility. Cold Spring Harb Symp Quant Biol 1994;59:673–676. 189. Turcot J, Despres JP, St Pierre F. Malignant tumors of the central nervous system associated with familial polyposis of the colon: report of two cases. Dis Colon Rectum 1959;2:465–468. 190. Crail HW. Multiple primary malignancies arising in rectum, brain, and thyroid: report of a case. US Naval Med Bull 1949;49:123–128. 191. Burt RW, Bishop DT, Cannon-Albright L, et al. Hereditary aspects of colorectal adenomas. Cancer 1992;70[5 Suppl]: 1296–1299. 192. Paraf F, Jothy S, Van Meir EG. Brain tumor–polyposis syndrome: two genetic diseases? J Clin Oncol 1997;15: 2744–2758. 193. Mastronardi L, Ferrante L, Lunardi P, et al. Association between neuroepithelial tumor and multiple intestinal polyposis (Turcot’s syndrome): report of a case and critical analysis of the literature. Neurosurgery 1991;28:449–452. 194. Hamilton SR, Liu B, Parsons RE, et al. The molecular basis of Turcot’s syndrome. N Engl J Med 1995;332:839–847. 195. Kikuchi T, Rempel SA, Rutz HP, et al. Turcot’s syndrome of glioma and polyposis occurs in the absence of germ line mutations of exons 5 to 9 of the p53 gene. Cancer Res 1993;53:957–961. 196. Mori T, Nagase H, Horii A, et al. Germ-line and somatic mutations of the APC gene in patients with Turcot syndrome and analysis of APC mutations in brain tumors. Genes Chromosomes Cancer 1994;9:168–172. 197. De Rosa M, Fasano C, Panariello L, et al. Evidence for a recessive inheritance of Turcot’s syndrome caused by compound heterozygous mutations within the PMS2 gene. Oncogene 2000;19:1719–1723. 198. Tamiya T, Hamazaki S, Ono Y, et al. Ganglioglioma in a patient with Turcot syndrome. Case report. J Neurosurg 2000;92: 170–175. 199. Mullins KJ, Rubio A, Myers SP, et al. Malignant ependymomas in a patient with Turcot’s syndrome: case report and management guidelines. Surg Neurol 1998;49:290–294. 200. Watson P, Lynch HT. Cancer risk in mismatch repair gene mutation carriers. Fam Cancer 2001;1:57–60. 201. Brandi ML, Gagel RF, Angeli A, et al. Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab 2001;86:5658–5671. 202. Thakker RV. Multiple endocrine neoplasia—syndromes of the twentieth century. J Clin Endocrinol Metab 1998;83: 2617–2620. 203. Karges W, Karges B, Ludwig L, et al. Bi-allelic inactivation of the MEN1 tumor suppressor gene in human grade II astrocytoma. Cancer Lett 2003;196:23–27. 204. Elias WJ, Lopes MB, Golden WL, et al. Trilateral retinoblastoma variant indicative of the relevance of the retinoblastoma tumor-suppressor pathway to medulloblastomas in humans. J Neurosurg 2001;95:871–878. 205. Chang YW, Yoon HK, Shin HJ, et al. Suprasellar retinoblastoma in a 5-month-old girl. Pediatr Radiol 2002;32:869–871.

52. Hereditary Brain Tumor Syndromes 206. Moll AC, Imhof SM, Schouten-Van Meeteren AY, et al. Trilateral retinoblastoma: is the location of the intracranial tumor important? Cancer 2000;88:965–967. 207. Kivela T, Tuppurainen K, Riikonen P, et al. Retinoblastoma associated with chromosomal 13q14 deletion mosaicism. Ophthalmology 2003;110:1983–1988. 208. Kivela T. Trilateral retinoblastoma: a meta-analysis of hereditary retinoblastoma associated with primary ectopic intracranial retinoblastoma. J Clin Oncol 1999;17:1829–1837. 209. Moll AC, Imhof SM, Schouten-Van Meeteren AY, et al. Second primary tumors in hereditary retinoblastoma: a register-based study, 1945–1997: is there an age effect on radiation-related risk? Ophthalmology 2001;108:1109–1114. 210. Bonnin JM, Rubinstein LJ, Palmer NF, et al. The association of embryonal tumors originating in the kidney and in the brain. A report of seven cases. Cancer 1984;54:2137–2146. 211. Rorke LB, Packer RJ, Biegel JA. Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood: definition of an entity. J Neurosurg 1996;85:56–65. 212. Versteege I, Sevenet N, Lange J, et al. Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 1998;394:203–206. 213. Sawyer JR, Goosen LS, Swanson CM, et al. A new reciprocal translocation (12;22)(q24.3;q11.2-12) in a malignant rhab-

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C h a p t e r

5 3

w Growth Factors in Brain Tumors

Johann Micallef, Gelareh Zadeh, and Abhijit Guha Growth factors, their cognate receptors, and the intracellular proteins they utilize to transmit their signals to the nucleus have pleotropic functions in cellular proliferation, differentiation, and apoptosis. Normal orchestration of these signals, in terms of their temporal, spatial, and quantitative expression, is essential for the development and maintenance of a multicellular organism. Abnormalities in expression or regulation of these pathways are responsible for both benign and neoplastic nervous system disorders. In this chapter we will focus on the role of main growth factors in the pathogenesis of astrocytomas, which are the most common primary central nervous system tumor in adults. Most growth factors are secreted soluble polypeptides, which activate specific membrane-bound receptors. The list of growth factors and receptors implicated in the biology of nervous system cancer is expanding, with at least 19 families based on their structural and functional similarities (Table 53-1). Specificity of growth factor–receptor interactions depends on structural recognition sites on both the ligand and the extracellular domain of the receptor. However, significant crossover, and hence functional overlap, does exist, especially between related growth factor and receptor families. Growth factors can modulate receptors on similar or different neighboring cells by paracrine stimulation, or on the surface of the secreting cell itself, leading to autocrine stimulation. Occasionally, the growth factor–receptor interaction does not require secretion of the factor into the extracellular space, but occurs within cytosolic compartments, giving rise to an internal– autocrine loop (1–3). Many growth factors have been implicated in the pathogenesis of astrocytomas, by demonstrating aberrant expression of the growth factor and/or its specific wild-type or sometimes mutant receptor. Phosphorylation of proteins by kinases is a major mechanism by which their functions are modulated. Most protein kinases phosphorylate serine or threonine residues. However, those that act on tyrosines are the most important in cellular signal transduction. These kinases can be

broadly divided into receptor and nonreceptor protein tyrosine kinases (RPTKs). Most (but not all) of the growth factors bind to the extracellular domains of RPTKs, which have intrinsic kinase activity when bound to their specific ligands. The ligand-binding extracellular domains are quite distinctive among the various RPTKs, containing different patterns of cysteine residues, immunoglobulin, fibronectin, and cadherin-like domains, as well as varying glycosylation patterns (Fig. 53-1). The functions of these variable motifs in the extracellular domain are not fully known, but they may serve purposes other than ligand recognition, which requires only a small portion of the extracellular domain. In addition to the extracellular domain, which is preceded by a N-terminal signal peptide facilitating secretion, RPTKs contain a single hydrophobic transmembrane domain (compared with the seven transmembrane serpentine receptors, which lack intrinsic protein kinase activity and are coupled to G proteins), and an intracellular domain. The intracellular domain consists of a juxtamembrane domain followed by the kinase domain and the C-terminal tail. The kinase domain, where the phosphor-transfer reaction is catalyzed, is the most highly conserved region among the 19 subfamilies of RPTKs, which are classified based on structural similarities (Table 53-1). For example, the platelet-derived growth factor receptor (PDGFR) family, which consists of receptors not only for PDGF but also for vascular endothelial growth factor (VEGF), both of which are relevant in the pathogenesis of astrocytomas as discussed later, are characterized by a split kinase domain with an intervening kinase insert domain. The C-terminal tail is quite variable between the RPTK families. A variable number of tyrosine residues are found dispersed throughout the intracellular domain of RPTKs (Fig. 53-2A). On ligand binding to the extracellular domain, receptor dimerization, or in some cases oligomerization, occurs, leading to activation of the kinase domain and transphosphorylation of the tyrosine residues (4–7). The kinase domain serves to phosphorylate tyrosines on various intracellular signal transduction and 733

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w TABLE 53-1

Families of Receptor Protein Tyrosine Kinases (RPTKS)

ALK

AXL

DDR

ALK LTK

AXL MER TYRO3

DDR1 DDR2

EGFR

EPH

FGFR

EGFR ERBB2 ERBB3 ERBB4

EPHA1 EPHA2 EPHA3 EPHA4 EPHA5

FGFR1 FGFR2 FGFR3 FGFR4

INSR IGF1R INSR INSRR

MUSK EPHA6 EPHA7 EPHA8 EPHB1 EPHB2

Met

MUSK CSF1R

PDGFR

MET RON EPHX

EPHB3 EPHB4 EPHB6

FLT3 KIT PDGFR␣ PDGFR␤

PTK7

RET

ROR

PTK7

RET

ROR1 ROR2

ROS

RYK

TIE

ROS1

RYK

TEK TIE

TRK

VEGFR

NTRK1 NTRK2 NTRK3

VEGFR1 VEGFR2 VEGFR3

AATYK

Uncharacterized

AATYK AATYK2 AATYK3

DKFZp761P1010

cytoskeletal proteins. Specificity of these receptor phosphotyrosines–intracellular signaling protein interactions is through protein–protein binding domains located on the intracellular proteins (Fig. 53-2B). Some of these domains, such as the PTB (phosphotyrosine binding) or src homology-2 (SH2) domains, recognize specific phosphotyrosines and their N-terminal (in the case of PTB domains) or C-terminal (in case of SH2 domains) flanking amino acids. For example, the three amino acids

C-terminal to a phosphorylated tyrosine on the activated receptor specifies which SH2 domain containing intracellular protein will bind to that site and thereby activate a particular downstream signaling pathway (8–10). SH2 domains are stretches of approximately 100 amino acids found in a variety of intracellular proteins, which act to link one protein to another, thereby altering their function and/or subcellular localization, to ultimately transmit the signal to the nucleus to modify gene regulation. Many of the SH2, PTB, and other protein–protein interacting domains are present in proteins such as enzymes, whose function is regulated by similar interactions. However, other proteins do not harbor additional functions, such as a catalytic domain, with their only function being to bring two or more relevant proteins together and facilitate their interactions and functional modulation. These important signal transduction proteins are called adaptor proteins, an example of which is Grb2, composed of one SH2 and two SH3 domains. The SH2 domain recognizes specific phosphotyrosines on activated receptors, such as phosphotyrosine 716 on PDGFR-␤, with its C-terminal flanking amino acid sequence of Y-S-N-A (11). The SH3 (src homology-3) protein domains are composed of about 60 amino acids, recognizing specific proline-rich regions of approximately 10 amino acids in length on other proteins (12–14). In the case of Grb2, its SH3 domain is constitutively bound to the nucleotide exchange factor mSos, which is involved in GDP to GTP exchange and activation of the important signaling pathway mediated by p21-Ras, as also discussed later. Activated RPTKs, such as PDGFR-␤, recruits the Grb2-mSos complex to the inner cell membrane by the specific Grb2-SH2:phosphotyrosine interactions, where mSos can interact with inactivated p21-Ras:GDP (also located in the inner cell membrane) and activate to p21-Ras:GTP. These signaling modules or domains are not just restricted to protein–protein interactions as described, but may also involve protein–lipid interactions, such as through Pleckstrin homology (PH) domains, found in important signaling molecules involved in the PI3–kinase pathway as described in brief here, although a full discussion cannot be undertaken in this chapter (13) (Fig. 53-2B). Phosphorylation of tyrosine residues induces activation of several signaling pathways by interaction with specific protein modules, as discussed earlier. Although often simplified to be running in parallel, these downstream signaling pathways have significant cross-talk, with redundancy being the rule rather than the exception. Some of the signaling pathways that are implicated in astrocytomas, such as the protein kinase-C (PKC), p21-Ras, PI3-K, and Jak/STAT pathway, will be discussed in further detail in this chapter. Dysregulation of any component of the signal transduction cascade, starting from the ligand, the RPTKs they activate, the intracellular proteins that transduce the signal to the nucleus, and the genes whose transcription they modulate, have been shown to have oncogenic potential.

53. Growth Factors in Brain Tumors

735

FIGURE 53-1. Receptor protein tyrosine kinases (RPTKs) and their ligands discussed in this chapter. RPTKs are grouped into several families based on structural and functional similarities (Table53-1). RPTKs consist of a variable extracellular domain, composed of different types of domains, some of which are listed here. The unique extracellular domain confers specificity to ligand binding, though some RPTKs can recognize several members of a growth factor family, but with different affinities. The transmembrane, juxtamembrane, and intracellular catalytic or kinase domains are the least variable portions of RPTKs. One exception to this general structure of RPTKs is the IGF-IIR, which is really not a RPTK but a type-1 integral membrane glycoprotein. Details of each of the RPTKs and their ligands, with emphasis on their aberrant expression and functional role in astrocytomas, are discussed in the text. RPTKs: EGFR, epidermal growth factor receptor; PDGFR, platelet derived growth factor receptor; Tie, tunica interna endothelial cell kinase; IGFR, insulin-like growth factor receptor.VEGFR, vascular endothelial growth factor receptor. Ligands: AR, amphiregulin; BTC, betacellulin; EPR, epiregulin; HB-EGF, heparin binding EGF-like growth factor; HGF/SF, hepatocyte growth factor/scatter factor; M6P, mannose 6-phosphate; TGF, transforming growth factor.

These aberrancies, which alter normal growth factor regulated homeostasis leading to transformation, may involve both gain-of-function or oncogenes and loss-of-function or tumor suppressor gene alterations at the level of the gene, RNA, or protein. Understanding these aberrancies and how they contribute to the tumorigenic process in astrocytomas and other neoplasms not only fosters scientific knowledge, but, it is hoped, will yield novel biological therapeutic targets. Signal transduction is at the frontiers of oncologic research, and the discussion in this chapter is cursory at best. Several excellent reviews are cited, and the reader is referred to the specific references for more detailed discussion of each topic.

EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) The most common karyotypic abnormality with a gain of genetic material in GBMs is an increased number of copies or amplification (double minutes or homogeneously staining repeats) of chromosome 7 (15). The proto-oncogene c-erbB1 encoding the EGFR located on

this chromosome is amplified at the DNA level in about 50% of GBMs but is expressed at much lower levels in low-grade astrocytomas (16–19). However, long-term malignant astrocytoma cultures do not retain the DNA amplifications, though they may still overexpress EGFR at the protein level. The mature human wild-type (wt) EGFR (Her1, c-erbB1) (Fig. 53-1) is a 170-kDa glycoprotein, containing 1,186 amino acid residues, that is encoded by a gene located on chromosome 7 (20). The extracellular domain contains two cysteine-rich regions, which can bind a number of ligands, including epidermal growth factor (EGF), transforming growth factor-␣ (TGF-␣), Amphiregulin (AR), Betacellulin (BTC), heparin binding EGF-like growth factor (HB-EGF), Epiregulin (EPR), and Epigen (reviewed in ref. 21) (Fig. 53-1). EGF and especially TGF-␣ are both expressed by astrocytoma cell lines and tumor specimens. Ligand binding to EGFR leads to the formation of receptor homo- and heterodimers, where ErbB2 (a member of the EGFR family) is the preferred heterodimerization partner of EGFR (22). As discussed previously, ligand binding leads to activation of intrinsic kinase activity with recruitment of cytoplasmic proteins to phosphotyrosine residues.

736

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FIGURE 53-2. A: Schematic representation of EGFR. The five “known” cytoplasmic domain phosphotyrosine sites, their local sequence, and interacting proteins are indicated. In EGFRvIII a portion of the extracellular domain (amino acids 6–273) is missing and replaced by a unique glycine-containing epitope. EGFR-C958 mutant contains the first 958 amino acids of EGFR but is missing the “tail” region. B: Examples of domains involved in protein–protein interactions in signal transduction. These protein domains consisting of 35 to 40 amino acids bind to a variety of target proteins consisting of specific sequences of short conserved amino acid cores. The PTB (protein tyrosine binding) and SH2 (src homology 2) domains are discussed in greater detail in the text, as they relate to aberrant signal transduction pathways in astrocytomas.

Amplification of EGFR (found in 50% of GBMs) is frequently associated with intragenic rearrangements and/or deletions (23) that leads to expression of several mutant EGFRs (24) (Fig. 53-2A). The most common mutant receptor, found in approximately 40% of GBMs that have EGFR gene amplification, is EGFRvIII (25,26), which arises from a genomic deletion of exons 2 to 7 of the wtEGF-R gene and encodes for a truncated protein lacking amino acids 6 to 273 of the extracellular domain (25,27). The truncation prevents ligand binding, creation of a novel glycine splice site, a target for toxin or radio-labeled antibody mediated therapy, and leaves the receptor constitutively activated (25,28) (review on drug therapy in refs. 29 and 30). Several experiments have shown that EGFRvIII-expressing tumors are more tumorigenic than wtEGFR-expressing tumors (28,31). Compared with wtEGFR-expressing tumors, EGFRvIII-expressing tumors show an increase in proliferation, invasion, and angiogenesis (28,31–33). These differences may be modulated through activation of different signaling pathways. For example, compared with wtEGFR-expressing cells, EGFRvIII-expressing cells have higher levels of constitutively activated PI3-K (34), which leads to a plethora of cellular effects, including cell survival, proliferation, and cytoskeletal organization, as discussed later in this chapter.

EGFRvIII is believed to be involved in the malignant progression of gliomas rather than in their initiation. Initial evidence was based on the observation that high-grade but not low-grade gliomas are associated with amplification and mutations of EGFR, such as EGFRvIII. Work done in our lab has shown that GFAP-regulated expression of wtEGFR and EGFRvIII transgenic mice do not develop gliomas (32). When these mice were crossed to GFAPregulated V12Ha-Ras transgenic mice, which develop and die from diffuse astrocytomas by approximately 12 to 16 weeks, embryonic expression of EGFRvIII but not wtEGFR potentiated glioma formation. In addition to dying at about 3 to 4 weeks, these double transgenic mice also developed mainly malignant oligodendroglial (approximately 80%) or oligoastrocytoma (10% to 15%) lineage gliomas (35). Our lab has also examined the effects of postnatal expression of EGFRvIII in normal and gliomasusceptible mice. AdEGFRvIII or AdGFP (Ad=adenovirus) injection into the frontal lobe of normal mice did not result in glioma formation. However, frontal lobe injection of AdEGFRvIII into GFAP-V12Ha-Ras transgenic mice caused the development of large, confluent, high-grade gliomas, which were mainly astrocytic in nature (about 70%), with a few being malignant mixed oligoastrocytomas (approximately 30%) (unpublished data). This is in

53. Growth Factors in Brain Tumors

contrast to the predominance of oligodendroglial lineage tumors (about 80%) with embryonal expression of EGFRvIII (35). These experiments in transgenic models are congruent to human molecular-epidemiologic data, suggesting that EGFRvIII is a progression factor and not an initiation factor. Second, it is consistent with the thesis that the type and grade of gliomas (or any other cancers) are functions of the nature of the genetic alterations and the time point at which they occur during development. Several other mutant EGFRs have been identified. EGFR-C958 is the second most common mutant, found in 20% of GBMs that have EGFR amplification and express EGFRvIII (24). EGFR C958 results from a genomic deletion of exons 23 to 25 of the cytoplasmic domain of wtEGFR, resulting in a transcript encoding the first 958 amino acids of wtEGFR followed by Asn and Thr residues before a stop codon (36) (Fig. 53-2A). EGFRC958 has an intact kinase domain, is activated by ligand, but lacks the major tyrosine phosphorylation sites for signal generators associated with wtEGFR. Our group and others have shown that the signaling properties of this mutant EGFR differ compared with wtEGFR signaling in GBMs. For example, whole-cell phosphotyrosine blots of EGF-activated EGFR-C958 demonstrate receptor phosphorylation up to approximately 4 hours after EGF stimulation, compared with approximately 5 minutes for wtEGFR. This may be a consequence of decreased receptor internalization and turnover in EGFR-C958, perhaps due to the missing internalization domain (mapped to residues 973-1022 in wt-EGFR) (37,38). Other examples of mutant EGFRs with constitutive activation exist. For example, the retroviral v-erbB1 oncogene encoding the transforming viral protein of the avian erythroblastosis virus (AEV), originated from the protooncogene c-erbB or EGFR (39,40). The v-erbB1 oncogene (74 kDa) differs from the normal receptor by the deletion of nearly the entire extracellular domain of EGFR, a much larger deletion than EGFRvIII found in GBMs. This results in constitutive activation and transforming capacity of v-erbB1 (39). The neu oncogene is another example of a member of the EGFR family that can be oncogenic. Neu was first described in 1984 (41,42) as a transforming gene extracted from neuroblastomas that developed in the fetuses of pregnant rats injected with ethylnitrosurea. The normal proto-oncogenic counterpart of neu is c-erbB2 (also referred to as Her2 or p185), a 185-kDa transmembrane glycoprotein for which the natural ligand is yet to be definitively identified. (Thus it is an “orphan receptor.”) The overall amino acid homology between c-erbB2 and EGFR is about 50%, with 82% similarity in the kinase domain (43,44); however, the two proteins are serologically distinct, with the human c-erbB2 gene mapped to chromosome 17 (41). A single base mutation in the transmembrane domain where a valine residue is replaced by glutamic acid (44) converts c-erbB2

737

into the neu oncogene. This point mutation induces spontaneous dimerization and tyrosine kinase activity of c-erbB2 (46), thus sending an uncontrolled, ligandindependent proliferative signal continuously to the nucleus. Amplification of c-erbB2 has been detected in a variety of human neoplasia, such as breast and ovarian carcinomas (47). Expression of c-erbB2 has also been found to occur with high frequency among high-grade astrocytomas (47–49). In medulloblastomas, overexpression of c-erbB2 has been associated with poor prognosis (50–53) and a metastatic phenotype (54). Because of the potential role of EGFR in the pathogenesis of a variety of neoplasms, including astrocytomas, there is considerable interest in developing tyrosine kinase inhibitors of EGFR and related family members. However, several clinical trials to date have demonstrated only limited efficacy. Gefitinib (Iressa, ZD1839) is an orally active drug that reversibly inhibits the tyrosine kinase activity of EGFR. Clinical studies have shown Gefitinib to have low toxicity (phase I) (55–57) and therapeutic activity (phase II) in several cancers (58,59). Gefitinib has also been used to evaluate the therapeutic effect in brain metastases (60–62) and GBMs (63). A recent phase II trail of Gefitinib in recurrent GBMs (63) has shown this drug to be well tolerated, but only shows modest activity against these tumors. Other EGFR tyrosine kinase inhibitors include Tarceva (erlotinib), a reversible tyrosine kinase inhibitor (64–66); CI 1003, a irreversible pan ErbB inhibitor (67): EKB 569, an irreversible inhibitor of EGFR and erbB2 (68); GW572016, an inhibitor for both EGFR and Her2 (69,70); PKI 166, an inhibitor of EGFR and Her2 (71). Several other therapies, in addition to targeting EGFR tyrosine kinase, also exist. For example, antibodies or vaccines have been designed against the novel glycine residue of EGFRvIII. However, these macromolecule targeting strategies have current limitations because of poor delivery across the blood–brain barrier and ability to reach the invasive tumor cells, areas of ongoing research activity.

PLATELET-DERIVED GROWTH FACTOR Platelet-derived growth factor (PDGF) is a dimeric growth factor composed of four individually nonfunctional peptides, the classical ligands termed PDGF-A and PDGF-B, and the recently discovered novel ligands PDGF-C and PDGF-D (72–74) (Fig. 53-1). Mature forms of PDGF-A and PDGF-B are 60% homologous at the amino acid level, with conservation of all eight cysteine residues crucial in dimerization (75,76). PDGF-C and PDGF-D share an overall homology of 43% and show conservation of all eight cysteine residues (77). In addition, four extra cysteine residues have been found in the growth domain of PDGF-C, and two extra cysteine residues are seen in the

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growth factor domain of PDGF-D. Although not confirmed, it has been suggested that the extra cysteine residues found in PDGF-C may be involved in disulfide bond formation and thus dimerization (77). In humans, PDGF-A, -B, -C, and -D map to chromosomes 7p22, 22q12.3–q13.1, 4q32, and 11q22.3, respectively (72,73,78–80). The active growth factor is formed from homo- or heterodimeric combinations of these four peptides, yielding five types of PDGF dimers: AA, BB, CC, and DD homodimers, and the heterodimer AB. All five types of PDGF are known to be active in a variety of normal and neoplastic cells (81–86), though to differing extents. Coexpression of PDGF-A and PDGF-B is seen in all tissues that express PDGF-C and PDGF-D. The highest levels of mRNA expression of the novel PDGF-C and PDGF-D are in the ovary, kidney, pancreas, and heart, with PDGF-C expression at higher levels than PDGF-D (77). Although the link between PDGF ligands and receptors in normal glial development and gliomas is best recognized with the established PDGF-A and PDGF-B, as discussed in detail later, the more recently discovered PDGF-C and PDGF-D ligands are expressed in GBMs, medulloblastomas, and Ewing family tumor cell lines (87–89). Many different normal functions of PDGF have been suggested, including its role in the development of the CNS. For example, in the rat optic nerve PDGF-A produced by type-1 astrocytes influences the differentiation and proliferation of the O-2A bipotential progenitor cells, which gives rise to type-2 astrocytes and oligodendrocytes (90). Second, PDGF-A and the PDGFR-␣ have been found in mouse embryos and likely have profound effects on the developing CNS (91,92). PDGF-B and the PDGFR-␤ have been demonstrated in neurons throughout the CNS, suggesting an as yet undefined neurotrophic role for PDGF (93). PDGF is a mitogen to human fetal astrocytes in culture, whereas in adult normal astrocytes the mitogenic role of PDGF is still unclear, although there is evidence that adult astrocytes do express PDGF receptors (94). Another normal function of PDGF in the CNS may be in the formation of a glial scar in response to PDGF released from injured neurons (93,95). The observation that PDGF-B is almost identical to the viral oncoprotein encoded by v-sis (96–98) represented the first link of aberrant PDGF in oncogenesis. v-sis, isolated from the simian sarcoma virus (SSV), induces a variety of tumors, including astrocytomas in primates (99). Like other retroviral oncoproteins, the SSV retrovirus had incorporated and modified the normal cellular proto-oncogene c-sis, which codes for PDGF-B, to become the oncogene v-sis. This landmark discovery of the link between v-sis and PDGF-B set the stage for future links between oncogenic gene products exerting their transforming capabilities by qualitative or quantitative aberrations of growth factors, their receptors, or intracellular signaling pathways (100,101). Unlike PDGF-B,

PDGF-A is not directly involved in transformation (102). PDGF-C may have a role in tumorigenesis, as its overexpression has been shown to increase the rate of tumor formation through autocrine and paracrine signaling (102). PDGF-D has also been shown to act as a strong transforming growth factor (103,104), and it can increase proliferation and the expression of angiogenic factors such as vascular endothelial growth factor (VEGF), as well as tumor formation in nude mice. The expression profile of PDGFs and PDGFRs in human gliomas of varying subtypes and grades suggests that aberrant PDGF signaling may be an early or initiating event in the formation of low-grade astrocytomas and/or oligodendrogliomas (105). The overexpression of PDGFs and PDGFRs is most commonly seen in low-grade astrocytomas with a loss of function of the tumor suppressor gene p53, as well as in secondary GBMs that arise from these low-grade astrocytomas. In support of PDGF being involved in glioma initiation, transgenic mice that overexpress PDGF-B in neural progenitors develop oligodendrogliomas, with oligodendrogliomas or mixed oligoastroctyomas formed when PDGF-B was transfected into differentiated normal astrocytes (106). Other studies also suggest a role for PDGF in the early stages of tumor development. For example, primates injected intracranially with SSV develop astrocytomas, which are pathologically quite similar to those naturally found in humans (98). Most established malignant astrocytoma cell lines overexpress a combination of PDGF-A or PDGF-B ligands and PDGFR-␣ or PDGFR-␤ genes and protein, which could in principle form an autocrine/paracrine loop (108). Work done by our group and others shows increased expression of both PDGF-A and PDGF-B in malignant astrocytomas (AA and GBMs), with lower expression levels in low-grade astrocytomas. PDGFR␣ was overexpressed in all astrocytoma grades compared with non-neoplastic glia, suggesting that expression of PDGFR-␣, capable of binding both PDGF subunits, may be an early event in the transformation with subsequent progression to a malignant astrocytoma cell, depending on increased expression of the PDGF ligands and other genetic alterations. Amplification or rearrangements of the two PDGF subunits (A and B) or PDGFRs (␣ and ␤) genes are not found in most in vivo astrocytomas. Southern analysis of 50 GBM specimens demonstrated amplification in only 8% and in no specimens for the PDGFR-␣ and PDGFR-␤ genes, respectively, with no evidence of genetic rearrangements in either gene (109). Overexpression of PDGFR-␣ protein, as determined by western-blot analysis, was demonstrated in 24% of the GBMs examined. One group did find amplification of a rearranged PDGFR-␣ gene in a GBM (110). This mutant PDGFR-␣, PDGFR-␣⌬8,9, has an in-frame deletion of 243 base pairs coding for 81 amino acids in the extracellular domain, resulting in complete loss of exons 8 and 9. PDGFR-␣⌬8,9 is predominantly localized to the cytoplasm, where it is constitutively activated and causes in-

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creased phosphorylation of c-Jun in the absence of ligand (111). This mutant PDGFR-␣⌬8,9 enhances cell growth and survival when transfected into a fibroblast cell line as well as when inducing tumor growth in nude mice. Further studies are necessary to understand the mechanisms by which this receptor signals. Several groups have shown that survival of GBMs cells depends on autocrine signaling of PDGF-A and PDGF-B (112–114). To test the possibility that the PDGF autocrine/ paracrine loops are essential contributors of astrocytoma formation, we created PDGF dominant-negative mutants by site-directed mutagenesis of the mouse PDGF-A cDNA, to inactivate both human PDGF-A and PDGF-B by forming unstable heterodimers (115,116). We found that both in vitro and in vivo growth in nude mice was significantly diminished with cells transfected with the PDGF dominant-negative mutant. Overall, these results suggest that PDGF is not only highly expressed in malignant astrocytomas, but also an important and relevant mitogen for astrocytoma cells. In addition to being a mitogen for astrocytomas, PDGF-B and PDGFR-␤ likely have important angiogenic roles in glioma development, perhaps by induction of VEGF mRNA, the major angiogenic stimulant in tumor angiogenesis (117). In conclusion, experimental evidence suggests that PDGFs are important in astrocytoma initiation, progression, and overall growth by both mitogenic and angiogenic stimulation, leading to it being sought after as a biological target in gliomas and other relevant human cancers. Delivery of genetic material, such as the PDGF dominant-negative mutant or antisense constructs to block activation and signaling of relevant RPTKs in astrocytoma cells, is appealing, but limitations of gene therapy remain formidable. Pharmacologic means of blocking PDGF stimulation holds promise, with development of several specific small-molecule tyrosine kinase inhibitors similar to those described for EGFRs. Ideally, these PDGFR antagonists are able to revert PDGF-induced transformed cells (118), with some existing candidates demonstrating promise against astrocytoma xenograft models. Some of these have completed clinical trials in salvage therapy for recurrent GBMs, with little efficacy demonstrated to date as single agents. STI571 (Gleevec) is a small-molecule kinase inhibitor that was designed to target the activated Abl oncoprotein in CML and was found to have activity against various RPTKs such as c-kit and PDGFRs. Growth of astrocytoma cells stimulated by PDGF can be selectively inhibited by STI571 in tissue culture and nude mice (119), through cell cycle arrest rather than apoptosis. STI571 is currently under clinical trials in recurrent GBMs, though as a single agent its efficacy is likely to be marginal, at best. Whether these biologically targeted inhibitors are effective as combinatorial agents, or in combination with nontargeted chemo- or radiation therapies, needs to be properly evaluated.

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INSULIN-LIKE GROWTH FACTORS The insulin-like growth factors (IGFs), or somatomedins, are mitogenic polypeptides of approximately 75 kDa that are similar in structure to pro-insulin (Fig. 53-1). IGFs engage in diverse metabolic activities, including promoting oligodendroglial differentiation and mitogenic stimulation of a variety of mesenchymal cells (120–122). There are two forms of IGFs, IGF-I and IGF-II, which were isolated from human tissues and serum and have about 62% amino acid sequence homology (120). IGF-I, also called somatomedin-C, is a basic peptide that mediates many of the growth-promoting effects of growth hormone, including stimulation of skeletal growth (123). The IGF-I gene contains six exons, which may undergo alternative splicing to yield multiple mRNA transcripts, the functional significance of which is still unclear (124). IGF-II is a neutral peptide that is expressed in many tissues during fetal development, with the gene mapped to the short arm of chromosome 1 (125). In the CNS, IGF-II is expressed only in the leptomeninges and choroid plexus, but not in the brain parenchyma itself (126). The physiologic actions of the somatomedins are mediated through two distinct receptors (Fig. 53-1). The type-I IGF receptor (IGF-IR) is a transmembrane dimeric protein structurally related to the insulin receptor. It consists of an extracellular ligand-binding domain containing two ␣-subunits, plus an intracellular tyrosine kinase domain containing two ␤-subunits (127,128). The amino acid sequences of the IGF-IR and the insulin receptor are highly homologous, with their tyrosine kinase domain having partial homology with the v-ros oncogene (123). IGF-IR has a high affinity for IGF-I and a somewhat lower affinity for IGF-II and insulin. The type II receptor (IGF-IIR) is a dimeric protein with a large extracellular ligand-binding domain, which is able to bind IGF-II with a much greater affinity than IGF-I. It does not seem to possess any intrinsic tyrosine kinase activity, with the suggestion that IGF-IIR plays a role in cellular uptake and subsequent degradation of IGF-II (123,129). Although insulin and the IGFs are known to play a role in normal neural growth and differentiation, they have also been implicated as autocrine growth stimulators of several tumor types, such as Wilms tumor, colon carcinomas, human breast carcinoma cells, and small cell lung tumors (125,130–132). Gliomas and meningiomas can also be included in this list, as they demonstrate elevated expression of the IGFs and their receptors (129). Initial studies using radioimmunoassay techniques showed that patients with glial tumors expressed elevated levels of somatomedins in their tumor cyst fluid, CSF, and tumor cytosol, compared with normal adult brains (133). Although it does not appear that insulin or insulin receptor levels are elevated in glial tumors, exogenous insulin is able to bind to IGF-IR and may exert some positive

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growth effect on those tumor cells that overexpress IGF-IR. Several roles for IGF-I/IGF-IR in tumorigenesis have been suggested, including a role in angiogenesis, invasion, and proliferation in astrocytomas (134–136). In addition, activation of IGF-IR may regulate apoptosis, since the extent of apoptosis of C6 rat GBM cells treated with antisense IGF-IR oligodeoxynucleotides correlates to the decrease in IGF-1R levels (137). IGF-IR may also be important in tumor resistance in GBMs, since GBM cell lines expressing high levels of IGF-IR are more resistant to therapies directed at other receptors, such as EGFR, that may also be expressed by that tumor (135). Given these direct and indirect regulatory roles postulated for IGFs and IGFRs in astrocytomas, they have become targets for experimental antitumor therapy, involving a variety of strategies. These involve receptor blocking monoclonal antibodies to IGF-IR, which inhibits growth of human breast cancer cells and Wilms tumor cells, both in vitro and in vivo (131,138,139). Antisense RNA directed against IGF-I has shown promising results in C6 rat glioma xenograft models, with the interesting result that antisense C6 transfectants result in marked inhibition of C6 nontransfected tumors also, suggestive of an additional immunomodulatory role. IGF-I analogs that inhibit autophosphorylation of IGF-IR have been shown to be capable of inhibiting proliferation of prostate carcinoma cells and SV40-transformed cells, but not that of human glioma cells (140). Another interesting approach is the use of triple-helix technology to target IGF-1. This approach uses triple-helix-forming oligonucleotides (TFOs) that inhibit gene expression by forming triple-helical structures on DNA, thereby blocking RNA polymerase and thus transcription (141–143). Rat C6 glioblastoma cells transfected with a IGF-I triple-helix plasmid reduced tumorigenic growth in nude mice (144,145). In summary, IGFs and IGFRs are expressed in glioma cell lines and tissues, with preclinical evidence that targeting this system may be efficacious in gliomas; however, there are no clinical trials that are in support of this thesis currently.

HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR The hepatocyte growth factor (HGF) and scatter factor (SF) are identical molecules but have different names because of their initial method of isolation (146,147) (Fig. 53-1). HGF was first identified as a mitogen for hepatocytes (148), whereas SF was identified through its ability to induce dissociation (scattering) of epithelial cells (149). The gene for HGG/SF is found on chromosome 7q21.1, encoding for a 6-kb transcript that gives rise to an inactive 728–amino acid pro-polypeptide (pro-HGF/SF) (150). The active form of HGF/SF is produced through cleavage of the

propolypeptide by proteases such as uPA and tPA, to give rise to a heterodimeric molecule consisting of an ␣ and a ␤ subunit held together by a disulfide bond (148,151,152) (Fig. 53-1). The ␣-subunit (62 kDa) contains an N-terminal hairpin loop domain, followed by four kringle domains, which are involved in protein–protein interactions. The hairpin loop and the second kringle domain provide a lowaffinity-binding site for heparin sulfate proteoglycans that are associated with the membrane. Through the first kringle domain, HGF/SF binds with high affinity to its receptor, c-met. The ␤-subunit (34 kDa) is similar to the catalytic domain of serine proteases. However, it is devoid of enzymatic activity due to the replacement of two amino acids within the active site (S to Y and H to Q) (reviewed in ref. 153). HGF/SF induces a wide spectrum of biological events, including proliferation, angiogenesis, invasion, branching morphogenesis, and transformation, through binding of its receptor c-met located on chromosome 7q21-31 (154) (Fig. 53-1). The c-met gene encodes for a polypeptide that is glycosylated and cleaved into two subunits, an ␣-chain and a ␤-chain that are linked by disulfide bonds. HGF/SF binding to c-met induces receptor dimerization; transphosphorylation of the catalytic domain, thereby binding; and phosphorylation of several intracellular proteins, including the intracellular multifunctional docking site. Among these proteins are p120Ras-GAP, PLC-␥, Srcrelated tyrosine kinase, Grb2-mSos complex, Gab-1, and the p85 subunit of PI3-K. Binding of these proteins can in turn activate several cell signaling pathways, including Ras-MAPK and PI3-K, as discussed later and recently reviewed (155). HGF/SF is predominantly produced by mesenchymal cells, whereas its receptor c-met is primarily expressed by cells of epithelial or endothelial origins (156), invoking c-met activation via endocrine or paracrine mechanisms. Aberrant signaling through c-met–HGF/SF interactions has been detected in a variety of human nervous system tumors, including meningioma, schwannomas, and gliomas (157). Mechanisms leading to aberrant c-met signaling include overexpression of c-met and/or HGF/SF, activating mutations in c-met and concomitant expression of HGF/SF and c-met by the same cell leading to autocrine stimulation. Autocrine stimulation has been implicated in glioma progression, with expression levels of both HGF/SF and c-met increasing with malignancy grade of human gliomas, with the highest levels seen in GBMs (158). HGF/SF transcription and secretion is increased through a positive feedback loop (159). Autocrine stimulation of c-met, by HGF/SF produced by U87 GBM cells, results in activation of Raf, PKC, MEK, and p38 MAPK pathway, resulting in increased HGF/SF transcription and secretion. In a similar fashion, HGF/SF regulates c-met receptor expression, as it is induced in both U-373 MG and T98G

53. Growth Factors in Brain Tumors

GBM cell lines by activation of signaling pathways mediated by p21-Ras, MAPK, PI3-K, PKC, and the transcription factor AP-1 (160). Aberrant c-met activation may contribute to glioma growth through various mechanisms: 1. Several studies have implicated HGF/SF-mediated c-met activation in tumor angiogenesis, mediated by enhanced VEGF mRNA and protein expression (161). Levels of VEGF induction by c-met activation were even higher than those secondary to activation of EGFR in gliomas. 2. c-met activation may also be a direct tumor mitogen, as it stimulates DNA synthesis of glioma cells (160). 3. HGF/SF can also stimulate the chemotactic migration of glioma cells on cultured surfaces coated with ECM molecules such as fibronectin, collagen IV, and laminin, suggesting a role in glioma migration and invasion (162). This may occur through regulation of matrix degradation enzymes and cascades such as the matrixmetalloproteases (MMPs) and TIMP. For more detailed investigations please refer to ref. 163. 4. c-met activation may also contribute to the wellrecognized resistance of gliomas to radiation and chemotherapy. Activated c-met protected GBM cells from a wide range of DNA-damaging agents, including ionizing radiation, cisplatin, camptothecin, taxol, and adriamycin. Protection against such a wide range of DNA-damaging agents with different mechanisms of action suggests that the cytoprotective effects of activated c-met occur through pathways involving DNA repair and antiapoptosis, perhaps involving the pro-survival PI3-K–Akt signaling pathway (160,164). Because of the various potential mechanisms by which HGF/SF is involved in the pathogenesis of astrocytomas there has been a considerable interest in developing inhibitors against c-met, HGF/SF, and the various signaling components of this pathway. One such inhibitor is the HGF-antagonist NK4, intratumoral injections of which inhibit GBM cell growth in an orthotopic xenograft mouse model (165). NK4 reduced intratumoral microvessel density, with increased apoptosis of the GBM cells. In addition, in vitro studies with NK4 prevented cell migration and proliferation following HGF/SF treatment, in a dose-dependent manner (165). Another group used chimeric U1snRNA/ribozymes to inhibit glioma growth, also by inhibiting angiogenesis plus promoting tumor cell apoptosis in glioma xenografts models (164). Finally, a combination of monoclonal antibodies designed against HGF/SF was shown to inhibit the subcutaneous growth of GBMs in mice (166). These preclinical studies all support the use of inhibiting this pathway as an effective therapeutic approach to gliomas, leading to planned clinical studies in the future.

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ANGIOGENIC FACTORS It is well accepted that solid tumor growth requires blood supply in order to maintain growth and malignant progression. Since the introduction of the concept of tumor angiogenesis there has been an explosion of research and interest in this field with the hopes that manipulating regulators of angiogenesis may provide biological therapies against disease processes that involve insufficient or exuberant vascularization in their pathogenesis. Normal blood vessel formation results from a highly complex process, involving a large number of growth factors that are precisely regulated under temporal, spatial, and environmental influences. In tumor angiogenesis these processes are aberrant, as tumor vessels are structurally and functionally abnormal. To date, most antiangiogenic agents have targeted either a known or unknown single pathway or growth factor, and although they have demonstrated preclinical efficacy for the most part, they have not shown clinical benefit, highlighting the need for combinatorial strategies. To date a large number of molecular regulators of angiogenesis have been identified; however, a unified concept is still evolving, and the present focus of research in this field is directed toward improving our understanding of the mechanism of interplay among these regulators of angiogenesis. An in-depth discussion of angiogenesis is a large subject that is beyond the scope of this chapter. The reader is referred to a number of comprehensive review articles on the subject (167–174). Here we will discuss two endothelial cell-specific growth factors (VEGF and angiopoietins), plus two growth factors with pleotropic functions but closely linked with angiogenesis (ephrins and fibroblast growth factor [FGF]), in terms of their biology and what is known of their role in astrocytoma angiogenesis.

VASCULAR ENDOTHELIAL GROWTH FACTOR Vascular endothelial growth factor (VEGF)—vascular permeability factor (VPF), or vasculotropin—was initially isolated as a 40- to 46-kDa protein in the early 1980s from tumor ascitic fluid and the conditioned medium of several tumor cell lines (175–177). It was initially known as VPF because of its ability to increase microvascular permeability (175,178). Independently, a factor was isolated from the conditioned medium of cultured bovine pituitary folliculostellate cells, shown to produce profound and specific mitogenic stimulation of endothelial cells, and termed VEGF (179). Structural characterization of VPF and VEGF eventually showed them to be identical (175). Since the initial discovery, several other members of the VEGF superfamily of growth factors have been

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discovered, with discussions here restricted to VEGF-A, commonly known simply as VEGF (Fig. 53-1). VEGF is a dimeric growth factor with 20% homology with the PDGF-A and PDGF-B subunits (175). VEGF normally exists as a dimer with two of the eight conserved cysteine residues, characteristic of the PDGF family of growth factors, forming disulfide bonds. Utilizing in situ hybridization, high levels of VEGF mRNA are expressed in the ventricular neuroectoderm during the embryonic and postnatal period, with a marked reduction in the mature animal. Thus during embryologic development, high levels of VEGF are found in areas of active angiogenesis, such as the periventricular region, with lower levels of VEGF seen in other areas such as the cerebral cortex (180,181). VEGF contains a consensus secretory signal sequence of 26 amino acids found immediately 5’ to the N-terminal (175). Four isoforms of VEGF with lengths of 121, 165, 189, and 206 amino acids exists, because of alternative splicing of a single gene. The predominant isoform is VEGF165, which, along with VEGF121, are the isoforms most efficiently secreted (182). The VEGF189 and VEGF206 isoforms are not highly secreted and in addition are not as mitogenically active on endothelial cells. The isoforms arise because of alternative splicing of exons 6 and 7, with the full-length human VEGF gene containing eight exons. All VEGF isoforms can increase microvascular permeability and act as an endothelial cell mitogen and stimulate angiogenesis. The ability to increase vascular permeability has been shown to be direct, independent of mast cells or other inflammatory mediators. As an angiogenic factor, VEGF stimulates endothelial cell growth in culture, increases their numbers, and can induce the entire sequence of angiogenesis in the chick chorioallantoic membrane assay (179,183). These highly specific and potent (picomolar concentrations) effects on endothelial cells are mediated by direct interaction of VEGF with its specific RPTKs (see later). The effects of VEGF on endothelial cells are specific, as similar mitogenic response with endothelial cells is not seen in a variety of other cell types, such as vascular smooth muscle cells, fibroblasts, sarcoma cells, and so on (183). This specificity toward endothelium is one of the most important and unique properties of VEGF, in contradistinction to other nonspecific angiogenic growth factors, such as the FGFs. The effects of VEGF on endothelial cells are dependent on activation of RPTKs (184). Several VEGFRs have been identified, with VEGFR1 (also known as Flt-1) and VEGFR2 (also known as Flk-1 or KDR) being the receptors most closely associated with modulating angiogenesis mediated by VEGF-A. Furthermore, VEGFR2 seems to be the dominant receptor involved in VEGF-mediated physiologic and aberrant angiogenesis. Specificity of VEGF being mainly a modulator of angiogenesis lies in the fact that both VEGFR1 and VEGFR2 are almost exclusively expressed on endothelial cells. Quantitative

audioradiographic studies utilizing iodinated VEGF165 have shown a wide distribution of endothelial binding sites in the adult rat, with the highest density in the brain and spinal cord, adrenal cortex, lung, stomach, spleen, and pancreas (176), correlating to the distribution of the VEGFRs mRNA. VEGFR mRNA expression by endothelial cells is prominent during the early embryonic and postnatal active angiogenic period, with mature cells such as in the normal adult CNS showing minimal expression (181). These expression and binding studies support the concept that VEGF and its receptor function as a major regulator of angiogenesis and endothelial proliferation during embryologic development, a fact that is also supported by embryonic lethality of knockout mice for both VEGF and VEGFR1 and VEGFR2 (185–188). Many transformed cells are associated with increased secretion of VEGF, whereas nontransformed counterparts secrete little or no VEGF. In situ hybridization localizes VEGF mainly to tumor, whereas immunohistochemistry detects VEGF in both the tumor cells and the targeted endothelial cells (189–191). Since tissue culture and in situ studies have shown that endothelial cells do not produce VEGF, this represents secondary binding and uptake of the highly secreted protein acting in a paracrine manner (190). There is also significant regional intratumoral variation in VEGF production, with increased levels adjacent to necrotic foci (189,192). This is due to hypoxia, which is the major physiologic regulator of VEGF transcription through binding of hypoxiainduced transcription factors to the hypoxia regulatory elements (HRE) of the VEGF promoter. Normal quiescent astrocytes express little to no VEGF, although reactive astrocytosis in response to a variety of nontumorigenic stimuli induces robust VEGF expression (193). The importance of angiogenesis in astrocytoma progression is vividly demonstrated by the fact that florid and aberrant angiogenesis is an essential histologic feature of malignant astrocytomas (176,186,194–196). In low-grade astrocytomas there is increased, but still relatively little, VEGF, whereas there is 20- to 50-fold increase in malignant astrocytomas. However, whether VEGF-induced angiogenesis by itself results in malignant progression of glial tumors is less certain, with experimental evidence suggesting it does not (197). GBM cell lines produce and secrete large amounts of VEGF (188,198,199), but not VEGFRs (191,200,201), which is not due to amplification or other alterations at the level of the VEGF gene. This increased basal VEGF level may be secondary to other cytokines known to be overexpressed by GBMs, such as EGFs, PDGFs, and FGFs, whose receptors when activated result in induction of VEGF production (199). In addition, signaling pathways stimulated by these predominant mitogenic factors, such as p21-Ras or PI3-K, have been shown by our group and others to result in VEGF induction (202). In GBM specimens, VEGF immunopositivity is

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greatest in tumor cells surrounding abnormal blood vessels and regions of tumor necrosis (i.e., perinecrotic pseudopalisading cells), and also detected in the tumorassociated blood vessels (191). However, VEGF mRNA was not detected in the tumor vessels and restricted to the GBM cells, similar to other human cancers as discussed earlier, and consistent with paracrine stimulation of VEGFRs located on the vessel endothelium. It must be noted that VEGF may be relevant in several benign CNS tumors (191,200). A recent study has shown high levels of VEGF mRNA in 100% of hemangioblastomas, as well as a significant number of meningiomas (200). Hemangioblastomas, although benign, are exceedingly vascular tumors, and as such VEGF may play a significant pathogenetic role. Meningiomas, although usually histologically benign, can be quite vascular and are sometimes associated with prominent peritumoral edema (203). The crucial role of angiogenesis in neoplasia and the specific and potent effects of VEGF on endothelial cells make this growth factor system an ideal candidate to undergo therapeutic manipulation in attempts to abrogate angiogenesis and tumor growth. Like all growth factors, potential interventions could utilize antibodies directed to VEGF protein or VEGFRs, antisense oligonucleotides to VEGF mRNA, gene therapy utilizing dominant-negative receptor mutants, and pharmacologic therapy to interrupt the receptor and postreceptor signal transduction pathways (204). Experimentally, antibodies directed to both the N- and C-termini of the VEGF molecule significantly block the permeability and the endothelial mitogenic activity, with combined antibodies to both termini producing nearly 100% reduction in these biological activities. A monoclonal antibody against VEGF significantly reduces tumor growth of human GBM cell line in nude mice by up to 80%, with a marked reduction in tumor vessel density (205). These kinds of preclinical strategies have led to VEGF-neutralizing antibody therapy in human cancer clinical trials, such as Avastin (Genentech, CA), which has shown encouraging results in recurrent colorectal cancers and awaits evaluation in CNS tumors. Pharmacologic therapies as discussed for EGFRs and PDGFRs, aimed at blocking VEGFR2 activation, are of high interest. Several clinical trials have been conducted in non-CNS cancers, with little efficacy demonstrated as single agents. Combinatorial therapy and/or development of agents with efficacy not only against VEGFRs, but also against other relevant RPTKs in gliomas and other cancers such as PDGFR or EGFR are of promise for future evaluation.

ANGIOPOIETINS AND TIE2 Like VEGF, angiopoietins are important modulators of angiogenesis and are the second class of angiogenic factors to have their cognate RPTK expressed exclusively on

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endothelial cells (174,206,207). The two main angiopoietin family members are Ang1 and Ang2, and their endothelial cell-specific receptor is Tie2/Tek (Tunica Interna endothelial cell kinase; Tek-human homolog) (174,206,207) (Fig. 53-1). Ang1 is a naturally occurring agonist, composed of 498 amino acid with an amino-terminal secretory signal sequence (208). Four isoforms of Ang1 have been identified by one group (1.3 kb, 1.5 kb, 0.9 kb, and 0.7 kb), with two of the isoforms shown to act potentially as dominant negatives to the full-length Ang1. Ang2 is the naturally occurring antagonist of Ang1-mediated activation of Tie2/Tek, composed of 496 amino acid, also with a secretory signal peptide and approximately 60% similar identity to Ang1 (209). Alternative splicing of Ang2 results in an Ang2 isoform, whose expression pattern and biological function is not yet precisely known. In addition to Ang1 and Ang2, homology-based cloning approaches have deciphered two additional members of the angiopoietin family. Ang4 is similar to Ang1 in that it activates Tie2, whereas Ang3, similar to Ang2, acts as an antagonist to Tie2/Tek (210,211). Ang1 knockout mice are embryonally lethal at E12.5d, because of an immature endocardium with loss of trabeculation and an immature vasculature that is devoid of complex branching with dilated vessels in the forebrain. Endothelial cells in Ang1 knockout mice are rounded and separated from the extracellular matrix (ECM) as a result of poor associated smooth muscle cells or pericytes (212). Of further interest, Tie2/Tek knockout mice are also embryonally lethal, because of vascular and cardiac developmental abnormalities similar to the Ang1 knockouts (207). This similarity suggested that Ang1 is the natural ligand for Tie2/Tek, which was later confirmed biochemically. Cumulative evidence suggests that Ang1, through activation of Tie2/Tek, mediates interaction of mesenchymal perivascular supportive cells in the ECM (such as smooth muscles and pericytes) with endothelial cells, leading to maturation of the vasculature. In contrast to VEGF, activation of Tie2/Tek by Ang1, is not mitogenic to the endothelial cells. Other mechanisms by which angiopoietins direct endothelial cell biology are gradually being deciphered, and perivascular cells remain unknown. Ang1 has been found to facilitate interactions between fibronectin and integrin receptors, however, at higher doses than is required for its modulation of Tie2/Tek (213). Regulation of endothelial cell tight-junctions has also been linked to angiopoietins, since Ang1 knockout embryos have leaky vessels and VEGFmediated permeability can be inhibited by Ang1 (214,215). A possible mechanism to explain this may involve Ang1 regulation of Platelet-EC-Adhesion-Molecule-1 (PECAM-1) and vascular endothelial cadherins, both of which are required to maintain endothelial cell tight-junctions and vessel integrity (216). Transgenic mice overexpressing Ang2 die embryonally, with a phenotype similar to that of the Ang1- and

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Tie2/Tek-deficient mice described earlier (209). This suggested that Ang2 was one of the only examples of a natural occurring antagonist for a RPTK, by inhibiting Ang1-mediated Tie2/Tek activation in endothelial cells (174,207,217,218). The biological effects of Ang2, however, are not as simple as one would predict solely on inhibiting Ang1-mediated Tie2/Tek activation. At high nonphysiologic doses, Ang2, similar to Ang1, activates the PI3-K:Akt pathway by phosphorylation of Tie2/Tek (219). Ang2 can also induce tubule formation of endothelial cells in vitro, which is in contrast to its predicted role as an Ang1 antagonist (220). Additionally, Ang2 deletion in knockout mice shows a phenotype that implicates Ang2 in regulation of lymphangiogenesis (174,217) and demonstrates phenotypes that are suggestive of a role for this Ang2 distinct from the Tie2/Tek receptor (174,217,221). The Tie family of RPTKs is the second family of endothelial cell-specific receptors, with two family members, Tie1 and Tie2/Tek (222,223) (Fig. 53-1). Tie1 and Tie2/Tek are 117- and 140-kDa polypeptide proteins, respectively, with high conservation in their cytoplasmic region but a highly divergent extracellular ligand-binding domain. Although both are endothelial cell specific, the Tie receptor family is structurally distinct from VEGFRs, especially in the extracellular domain, where Tie receptors consist of two Ig-like loops separated by three tandem epidermal growth factor–like cysteine repeats followed by three fibronectin type III-like motif (174,217,224) (Fig. 53-1). In contrast, the intracellular domains are similar to VEGFRs and in fact PDGFRs with two tyrosine kinase domains, split by a short stretch of amino acids (174, 217,224). Although it is now well established that angiopoietins are the ligands, which bind and modulate activity of Tie2/Tek, the ligand for Tie1 remains unknown; hence it remains an “orphan” receptor. Little is known about the signaling pathways activated by Tie2/Tek receptors. Initial studies demonstrated association of the signaling molecules Grb2 and Shp2 to activated Tie2/Tek (224,225). However, since both of these molecules are involved in regulation of the mitogenic p21-Ras:MAPK signaling pathway and activated Tie2/Tek does not lead to endothelial cell proliferation, their functional role in Tie2/Tek signaling remains to be clarified. The p85 subunit of PI3-K can directly associate with phosphorylated Tie2/Tek, resulting in activation of PI3-K:Akt survival pathway (224,225). Two antiapoptotic genes have been described related to this pathway, with survivin mRNA levels increased by Ang1 stimulation, whereas levels of bcl2 do not seem to be modulated by Ang1 (224,225). PI3-K activation by Ang1 also seems to regulate endothelial cell migration and motility, by phosphorylation of focal adhesion kinase (FAK) (219,226). Another signaling protein associated with activated Tie2/Tek is Dok-R, a novel docking protein that on phos-

phorylation can bind additional signal transducers such as Ras-GAP or Nck, the latter involved in Ang1-mediated endothelial cell migration (224,225). Further understanding of these Tie2/Tek-mediated signaling pathways will be important in understanding vascular biology and identifying potential antiangiogenic therapeutic targets. An obvious question is how these two angiogenesisspecific growth factors, VEGF and angiopoietins, and their receptors, VEGFRs and Tie2/Tek, interact to regulate normal and pathologic angiogenesis. A paradigm incorporating this crucial interaction between VEGF and angiopoietins has been proposed (227). VEGF and VEGFRs are essential for the formation of the primitive vascular network, whereas angiopoietin and Tie2/Tek interactions signal maturation of the primitive vessel. The quiescent mature vasculature expresses minimal VEGF, with some basal Ang1-mediated Tie2/Tek activation. During normal and pathologic angiogenesis, a relative increase in Ang2 expression by the endothelial cells inhibits Tie2/Tek activation, thereby destabilizing the vessels and sensitizing endothelial cells to VEGF. VEGF expression is increased likely as a result of microphysiologic hypoxia and cross-modulation by other cytokines, as previously described. Activation of VEGFRs on the endothelial cells results in endothelial cell proliferation, sprouting, and neo-angiogenesis. However, this paradigm is an oversimplification, as we are gradually deciphering the multiple aspects of endothelial cell biology that angiopoietins and Tie2/Tek are involved in. Transgenic mice have been used to better decipher the interactive roles of angiopoietins and VEGF. This work suggests a highly VEGF-dependent role for both Ang1 and Ang2. Biochemical data also demonstrate that angiopoietins may have a dual role, pro- or antiangiogenic, depending on the level of VEGF present in the environment (214,228). Factors in addition to VEGF interactions that add to the complexity of the angiopoietin-Tie2/Tek pathway are the identified isoforms of angiopoietins and the regulatory role(s) that angiopoietin-related proteins (ARP) may play. In addition, angiopoietin-mediated regulation of the vasculature may depend on its interactions with other nonspecific angiogenic factors, such as FGFs, PDGFs, and so on. The role of angiopoietins and Tie2/Tek in tumor angiogenesis is not as well established as that of VEGF. We and others have documented that both angiopoietins and Tie2/Tek expression and activation increase with astrocytoma grade and compared with normal brain (217, 229–232). Increased activation of Tie2/Tek, similar to the status of VEGFR in the GBM vasculature, suggests that both of these angiogenesis-specific factors play a role in the characteristic florid tumor angiogenesis of these tumors. Ang1 is overexpressed by the astrocytoma cells, but unlike VEGF, it is not induced by hypoxia and does not show increased perinecrotic expression, because it lacks a

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HRE element in its promoter (217,229–232). In fact, we and others have shown Ang1 expression to be downregulated in GBM cell lines after 8 hours of hypoxia, a time of maximal VEGF induction (230). Ang2 expression is mainly in the endothelial cells of the tumor vessels, though the astrocytoma cells also express it to a degree (230–232). In contrast to Ang1, Ang2 expression is increased by hypoxia, with increased expression by endothelial cells in the perinecrotic hypervascular regions of GBMs, similar to VEGF and VEGFRs. However, since VEGF induces Ang2 expression, whether Ang2 in GBMs is induced directly by hypoxia or by VEGF is not yet clear (231,233). With the background of the potential mechanism(s) of interaction of angiopoietins and VEGF in normal angiogenesis as discussed briefly earlier, a paradigm of astrocytoma vessel development may be proposed. Astrocytomas initiate their growth by co-opting existing host vessels, subsequent to which the host mounts a defensive response and chokes off the tumor’s vascular supply (234). This in turn elicits a hypoxic response that induces increased expression of VEGF and Ang2, which together induce endothelial cell proliferation, sprouting, and migration, leading to neo-angiogenesis (232,234). Ang1 expression is also induced by non–hypoxia-mediated stimulants, which are currently not well deciphered, leading to recruitment of perivascular cells and maturation of the newly formed vessels. This of course is a dynamic and evolving process, with questions regarding how the relative levels of both angiopoietins and VEGF modulate tumor angiogenesis needing further study. Taking several approaches, our work clearly shows that activation of Tie2/Tek by angiopoietins is of functional importance in GBM vascularization and growth. First, inhibition of Tie2/Tek activation by a soluble kinase-dead Tie2 construct, which acts as a dominant-negative mutant, leads to disruption of GBM xenograft angiogenesis and growth, both in subcutaneous explant and in intracranial cell line models (235). These tumor vessels were not lined with smooth muscle cells or pericytes, resulting in abnormally dilated vessels. Second, overexpression of Ang1promoted GBM angiogenesis and growth (unpublished data), which is closely linked to presence of VEGF by the xenografts. We postulate that in the presence of abundant VEGF, which along with Ang2 induces neo-angiogenesis, Ang1 is pro-angiogenic by promoting development of a mature vasculature with more efficient blood supply to the tumor. These results are the first to support the functional role of angiopoietin-mediated Tie2/Tek activation in brain tumor angiogenesis and growth. In addition, it emphasizes the highly intricate and co-modulatory impact of angiogenic factors in regulating tumor angiogenesis and highlights the need for targeting crucial and perhaps multiple key growth factors involved in tumor angiogenesis. These are very recent and promising results

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that support a significant regulatory role for these growth factors in astrocytomas. A number of pharmaceutical agents, small molecule inhibitors to Tie2 in particular, have been proposed to be tested at a preclinical level. The outcome of these studies will be exciting to see whether a combinatorial inhibition of VEGF/VEGFRs and angiopoietins/Tie2 will provide a more efficacious antiangiogenic therapy for astrocytomas.

EPHRINS Ephrins are the most recent of all endothelial cell-specific growth factors, with their role in tumor angiogenesis in particular astrocytomas not yet investigated. Until recently, factors that govern arterial and venous assignment and differentiation were thought to be related to hemodynamic and physiologic factors, such as blood flow, blood pressure, and nutritional needs. However, recent data indicate that certain genes are involved in this process of vessel differentiation, with perhaps hemodynamic factors modulating the expression of these genes. Implicated genes regulating this differentiation belong to the Eph receptor family, the largest family of RPTKs (236–241). Eph receptors, together with their ligands, EphrinA and EphrinB plus their many isoforms, were first identified in the nervous system in regulating neuronal patterning and axonal guidance. However, their role has been expanded to include regulation of arterial-venous differentiation. EphrinB2 is an early marker of arterial endothelial cells, and EphB4 is an early marker of venous endothelial cells (236–241). Knockout mice of EphrinB2 are embryonally lethal, as a result of a lack of appropriate orchestration of arterial and venous endothelial cells. EphrinB2 interacts with EphB2, EphB3, and EphB4 receptors. However, only the EphB4 knockout mice show embryonal lethality and phenotypic alterations with vascular changes identical to the EphrinB2 knockout mice. Overexpression of EphrinB2 demonstrates a role in endothelial cell and smooth muscle cell interactions, suggesting a role in vessel maturation similar to Ang1, with the additional function of maintaining distinct arterial–venous beds. The developmental and physiologic functions of Ephrins and Eph receptors known to date suggest that this family of growth factors and receptors also plays a critical role in normal and pathologic angiogenesis, an area of research that is young and in need of ongoing investigation.

FIBROBLAST GROWTH FACTORS In the early 1970s, two growth factors strongly mitogenic for fibroblasts were isolated from brain and pituitary extracts. One had an acidic isoelectric point and

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was termed acidic fibroblast growth factor (aFGF); the other was basic and therefore termed basic FGF (bFGF) (242,243). Purification of these FGFs and their subsequent sequencing became possible after taking advantage of their strong affinity for binding to heparin columns (244). The pleotropic functions of the FGFs became apparent when amino acid sequence analysis led to the realization that 20 to 30 other growth factors named after various tissues of origin (e.g., retinal derived growth factor, tumor angiogenesis factor) or target cells (e.g., astroglial growth factor, endothelial cell growth factor) were actually the same as one of the two FGFs (245). The FGFs perform several important functions in the development and maintenance of the CNS. For example, in situ hybridization has shown widespread CNS expression of aFGF prenatally in rats, with postnatal localization in the hippocampus and cerebellum (246), suggesting a role in the early development of the CNS. In the developing rat optic nerve, bFGF increases the number of PDGFR-␣ in O-2A progenitor cells, thereby making them more responsive to the mitogenic effects of PDGF-A released by type 1 astrocytes and blocking their differentiation to oligodendrocytes (247). In adults, focal animal brain injury models have demonstrated that reactive astrocytes express bFGF (248). Both aFGF and bFGF have two heparin-binding domains (249,250) that allow them to bind proteoglycans and glycosaminoglycans (e.g., heparin and chondroitin sulfate) found in basement membranes and other ECMcontaining heparin-like molecules (251). Interaction with heparin changes the tertiary structure of FGFs and is crucial to its function, with an increase in its potency between 10 and 100 times (249,252,253). The exact mechanism(s) by which heparin interacts with FGFs to affect potency is not known, but several possibilities exist: 1. Heparin binding may stabilize FGF by making it less susceptible to protease degradation, hence prolonging its biological effects (254–256). 2. Heparin-bound FGF may bind to its receptor(s) more avidly than FGF alone (257). 3. Sequestering of FGFs in the ECM by heparin and subsequent degradation by heparinase-like enzymes may provide a mechanism of FGF action in a variety of normal and pathologic conditions. For example, a model of normal or tumor angiogenesis involves breakdown of ECM by secreted enzymes from endothelial cells, thus releasing bound FGFs (250,258–260), which are mitogens for the endothelial cells, thereby inducing formation of new blood vessels. Whether these models are relevant in the CNS ECM, which differs from other tissues, remains to be examined (261). Using the characteristic of heparin binding, several other related growth factors have subsequently been identified and

are collectively called the heparin binding growth factors (HBGF) (262). FGFs bind to receptors on target cells with both low and high affinity. The low-affinity binding probably represents binding to heparin-like compounds in the basement membrane around the cells or the ECM. Highaffinity RPTKs have been identified for FGFs, which are also capable of binding other members of the HBGF family (263–266). FGF-R1 is encoded by c-flg (fms-like gene), which was initially identified using low-stringency hybridization conditions with a proto-oncogene c-fms (263,266) and is localized in humans to chromosome 8. A second RPTK belonging to this family, FGF-R2, has homology between the mouse tyrosine kinase gene (bek) and a recently identified tyrosine kinase from a human tumor cDNA library named TK-14 (264,265). At least two other human receptors (FGF-R3, FGF-R4) have been cloned, and nine other related members exist, secondary to alternate splicing. These receptors bind with different efficiencies to the two FGFs and other members of the HBGF family. bFGF is highly expressed in most cell cultures (i.e., GBM cell lines [267,268]), highly vascularized organs (i.e., brain [243,246]) and tumors (i.e., GBMs [269–271]). In contrast, aFGF expression is more limited with high expression in certain areas of the brain, retina, bone matrix, and some tumors, such as osteosarcomas (245,272). The role of FGFs in angiogenesis has been demonstrated in vitro using cultured endothelial cells and in vivo using the chick chorioallantoic membrane or rabbit cornea models (258). Although implicated in tumorigenesis, neither aFGF nor bFGF has been linked to any presently identified oncogene, unlike some other members of the HBGF family, such as Hst/K-FGF isolated from both a human stomach cancer and Kaposi sarcoma cell line and FGF-5 from a bladder carcinoma cell line (273,274). Another member of HBGF that can be oncogenic is Int-2, which is normally expressed in embryonic mouse, but when expression is enhanced under control of the mouse mammary tumor virus it becomes oncogenic (275). For bFGF itself to become oncogenic a signal peptide must be added, allowing efficient secretion (276,277). Transformation without a signal peptide is not efficient, requiring large amounts of bFGF expression and perhaps involving intracellular autocrine mechanisms (277). In contrast, Hst/K-FGF, which contains a signal sequence, transforms at a much higher efficiency than bFGF (278). These experiments suggest that overexpression of aFGF or bFGF is probably not a primary event in the genesis of neoplasia. However, they may significantly influence tumor growth by mechanisms such as angiogenesis. Cytogenetic studies in astrocytomas do not show any consistent gross changes in the chromosomes encoding the FGFs or their presently identified receptors. Astrocytoma cells in culture express mRNA for both aFGF and

53. Growth Factors in Brain Tumors

bFGF, and synthesize the active growth factors (267,270). Several reports using immunohistochemistry or in situ hybridization techniques on operative specimens also suggest that aFGF and bFGF are expressed by astrocytomas (269–271). GBMs express higher levels of FGF (both in the astrocytes and in endothelial cells) than the peritumoral region or lower-grade astrocytomas, which may reflect the higher degree of vascularity in GBMs rather than implicating FGFs as a progression factor. In support of this interpretation is the observation that bFGF expression has been also found in high levels in benign but vascular intracranial tumors such as meningiomas or schwannomas (270,279). The potential functional importance of FGFs in the growth of astrocytomas was illustrated by antisense bFGF oligonucleotides, causing a significant reduction in the growth rate of the astrocytoma cells (280). Interestingly, blocking bFGF had no effect on the growth rate of cells derived from non-neoplastic astrocytes. In addition, small-molecule FGF receptor inhibitors have shown some

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efficacy in preclinical GBM xenograft models, though not entered in clinical trials.

PROTEIN KINASE C The protein kinase C (PKC) family is a large family of serine-threonine protein kinases, involved in signal transduction with many isoforms identified in mammalian tissues (281). The PKC isoforms differ in their enzymatic properties, tissue expression, and intracellular localization. However, all consist of a C-terminal kinase domain and a N-terminal regulatory domain, the latter region acting to inhibit the kinase activity (Fig. 53-3A). This inhibition can be relieved by proteolytic cleavage of the N-terminal domain or addition of co-factors, including calcium, anionic phospholipid, diacylglycerol (DAG), or tumor-promoting phorbol esters (TPA). The PKC isoforms are subdivided into three groups, designated by

FIGURE 53-3. A: Schemata of PKC families, consisting of a N-terminal regulatory domain and a C-terminal kinase domain. B: Simplified diagram of PKC activation secondary to activated RPTK. A major pathway involves activation of PLC␥ and generation of diacyl-glycerol (DAG) and inositoltriphosphate (IP3), which in turn leads to release of intracellular calcium and with DAG the activation of PKC. Several families of PKC exist, classified according to mechanism(s) of activation/regulation, with some of these isoforms implicated in astrocytomas, as discussed further in the text.

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their physiologic activating substrate (282). Group 1 requires both calcium and DAG for activation and consists of the ␣, ␤1, ␤2, and ␥ isoforms. Group 2 requires only DAG (⌬, ␧, ␯, ␩, ␪, ␶). Group 3 requires neither (␨, ␭). The repertoire of PKC isoforms in a particular cell is not static but alters with development, transformation, differentiation, and senescence (282). Little is known, however, about the mechanism(s) that determines what set of PKC isozymes will be expressed and the substrates for these enzymes. Interest in PKC in oncology came to the forefront with the observation that TPA exerts its tumorigenic effect by activating PKC. The mechanism involves TPA acting as a DAG analog to activate PKC, with the difference that TPA is metabolically stable and the binding is irreversible, leading to uncontrolled activation of PKC (281). However, normal activation of PKC involves an activated receptor leading to activation of various intracellular phospholipases, resulting in degradation of membrane phospholipids, the

products of which lead to activation of the PKC (Fig. 533B). Phospholipase C␥ (PLC␥) is one such enzyme, activated by binding through its specific SH2 binding site to RPTKs such as PDGFR-␤, leading to hydrolysis of phosphatidylinositol 4,5 biphosphate (PIP2) located in the cell membrane, with the release of inositol 3,4,5-triphosphate (IP3) and DAG into the intracellular compartment. IP3 also mobilizes calcium from the endoplasmic reticulum, which with DAG activates PKC (Fig. 53-3B). The downstream substrates and mechanisms of cellular control by PKC are not entirely clear, though we know they interact and cross-talk with canonical mitogenic pathways, such as those mediated through the p21-Ras: Raf:MAPK (Fig. 53-4). Activated PKC does not directly activate p21Ras-GTP but is able to stimulate Raf kinase activity, thereby modulating nuclear DNA methyltransferase and transcription factors such as jun and fos (283). PKC is expressed at high levels in the brain and is important for its normal development and maintenance,

FIGURE 53-4. p21-Ras signaling pathway and some of its cross-talk with other signaling pathways: RPTKs activate p21-Ras:GDP associated to the inner cell membrane, through the intermediate adaptor proteins Grb2 and Shc, which recruits the nucleotide exchange factor mSos to the activated RPTK in inner cell membrane. Activated p21-Ras:GTP recruits the effector protein Raf, which sequentially activates the MAPKKs (MEK1 and MEK2) and in turn the MAPKs (ERK1 and ERK2). Other effectors of p21-Ras:GTP like p120GAP and PI3-K activate signaling proteins such as Rac1, resulting in activation of the Jun N-terminal kinases (JNK) and other key signaling pathways. In this typical manner of cross-talk between signaling pathways the signal is amplified and diversified to alter a large number of cellular processes. Besides RPTKs, various stressors (such as heat shock and ultraviolet rays) or inflammatory molecules (such as TNF-␣) can activate MAPKK/MAPK-like pathways, resulting in the activation of p38MAPK. Cross-talk enables convergent and divergent signaling between all these pathways. This figure also demonstrates that transcriptional activation is occasionally dependent on the cooperative signaling of multiple pathways; c-Jun, and c-Fos together form heterodimers in the Activator Protein-1 (AP-1) transcriptional complex. Whereas c-fos transcription is activated by ERK, the transcription of c-Jun is predominantly regulated by Jun N-terminal kinase (JNK).

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including being a neonatal but not an adult glial mitogen (284,285). In fetal astrocytes, in addition to being a mitogen, PKC induces differentiation features such as outgrowth of cellular processes and increased glutamate synthetase production (286,287). The role of TPA and PKC mediated tumorigenesis along with this high neonatal and fetal CNS expression, sparked interest to investigate the role of PKC in the pathogenesis of astrocytomas. Malignant astrocytoma cell lines and surgical specimens were found to have increased expression of PKC, similar to fetal astrocytes, perhaps as a result of dedifferentiation (288,289). In addition, stimulation of the EGFRs in these cell lines resulted in activation of PKC along with p21-Ras, with increased proliferation, migration, and invasion of GBM cells (289). Attempts at correlating PKC and specific isoform levels to astrocytoma grade remain unclear, with several groups reporting increased PKC␣ levels, which if decreased with antisense PKC␣ results in growth inhibition of human GBM cell lines (290). However, other reports suggest an inverse relationship of PKC expression with astrocytoma grade and a different pattern of isoform expression in these tumors (291,292). Pharmacologic inhibitors of PKC have led to promising preclinical results in GBM cell lines, with inhibition of proliferation by nonspecific PKC inhibitors such as polymyxin-B, tamoxifen, and more specific agents, such as staurosporine (288, 289,293–296). However, clinical trials with tamoxifen, with its acceptable side effects and established clinical use in breast cancer, have shown minor beneficial effects (293). Lack of specificity and toxicity are major drawbacks to the presently available PKC inhibitors, as most, including staurosporine, act on a variety of protein kinases, including PKC. Development of more suitable inhibitors by the pharmaceutical industry is required, to determine if inhibition of the PKC signaling pathway will have any clinical use in management of astrocytomas (297).

P21-RAS The three human p21-Ras genes encode for four proteins (Ha, N, K4A, K4B) and belong to the important small-G protein-mediated signaling family (Fig. 53-4). Activating mutations (residue 12,13,61) of p21-Ras are prevalent in greater than 30% of all human cancers, making this the most common oncogene (298–300). Activating p21-Ras mutations are biochemically characterized by inability to bind to a family of proteins termed Ras-GAPs (i.e., p120GAP and neurofibromin), which hydrolyze and inactivate p21-Ras:GTP to p21-Ras:GDP. Much is known of how activation of p21-Ras is regulated by activated RPTKs and its downstream effectors, leading to alterations in cell behavior (9,10,301,302) (Fig. 53-4). p21-Ras

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activation requires posttranslational addition of hydrophobic isoprenyl groups to its C-terminal end, which contains a specific sequence of amino acids termed a CAAX (cysteine–aliphatic–aliphatic–variable) box, by farnesyl or gerynyl-gerynyl transferase. This allows p21-Ras to bind to the inner cell membrane, where exchange of GDP for GTP can occur by nucleotide exchange factors, such as mSos (mammalian homolog of the Son Of Sevenless) gene product identified as a p21-Ras activator in drosophilla (303,304). The proline-rich regions of mSos are bound to the SH3 domains of Grb2, with the Grb2:Sos complex recruited to the inner membrane near p21-Ras, by binding of the SH2 domain of Grb2 directly to specific phosphotyrosines in activated RPTKs. Indirectly, this Grb2:Sos complex can also bind to activated RPTK through another SH2-containing adapter protein called Shc (305–307). Inactivation of p21-Ras:GTP to p21-Ras:GDP requires binding of a family of enzymes called GAPs (GTPase Activating Protein). Two Ras:GAPs called p120GAP and neurofibromin (the gene product of neurofibromatosis 1-NF1) have been identified (308–317). Decreased levels of these p21-Ras:GAPs such as neurofibromin would in theory lead to elevated levels of active p21-Ras:GTP, as documented in NF1-associated peripheral nerve tumors and astrocytomas by our group (318). In oncogenic p21Ras mutations, these Ras-GAPs cannot bind to p21-Ras, leading to constitutive activation of downstream mitogenic pathways. Of the several downstream substrates of p21-Ras, activation of Raf which is translocated to the cell membrane by p21-Ras and subsequent activation of MAPKinase (ERK1,2) by phosphorylation on threonine and tyrosine residues, leading to its translocation to the nucleus is presently best understood (300,308,319-324) (Fig. 53-4). Many signaling proteins bind and are activated by p21-Ras:GTP including PLC␥ and PKC, as demonstrated in Fig. 53-4. Although oncogenic p21-Ras mutations are not prevalent in malignant astrocytomas, data initially from our laboratory and subsequently confirmed by others suggest that levels of activated p21-Ras:GTP are elevated in astrocytomas from signals utilized by overexpressed and/or aberrantly activated RPTKs in these tumors, as discussed previously. The functional importance of activated p21-Ras:GTP in astrocytomas was demonstrated by several techniques, including neutralizing p21-Ras antibody, p21-Ras dominant-negative mutants, and inhibitors of farnesylation, which as described earlier is critical for processing of p21-Ras, allowing it to be activated by RPTKs in the inner cell membrane (325,326). Farnesyl transferases are most effective against Ha-Ras and have shown promise in preclinical studies on GBM xenografts, with a few clinical studies initiated. Gerynyl-gerynyl transferases have a wider spectrum of activity, especially against the more human-tumor-prevalent K-Ras mutations.

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Hence although they are of greater toxicity, they are being developed for potential clinical applications.

PI3-K PATHWAY The PI3-Kinase pathway is another major and pleotropic signaling pathway implicated in gliomagenesis. PI3-K can be activated either through p21-Ras-dependent or -independent mechanisms, with downstream activation of AKT/PKB, which in turn activates a multitude of downstream effecter pathways leading to cell survival, proliferation, and cytoskeletal organization (327–331) (Fig. 53-5). The importance of the PI3-K pathway in malignant astrocytomas is not only from its activation of upstream RPTKs, but also because it is negatively regulated by the tumor suppressor gene PTEN/MMAC located on chromosome 10q23, the most commonly lost chromosomal region in GBMs. Loss of expression, either through mutation, deletion, or gene

inactivation of PTEN, is one of the most common genetic aberrations of GBMs (332,333). PTEN does not directly act on PI3-K, but it does act as an inositol phospholipid phosphatase, resulting in dephosphorylation of the 3⬘ phosphate of PtdInsP3 (PIP3) to PtdInsP2 (PIP2), thereby inhibiting activation of the key pro-survival protein AKT/PKB (330,334) (Fig. 53-5). In addition to this lipid phosphatase activity, PTEN is a protein phosphatase, the implication of which in cancer biology is being slowly understood. PTEN/MMAC1 mutations have not been detected in low-grade astrocytomas and are rare in anaplastic astrocytomas; however, both the so-called primary and secondary GBMs harbor PTEN/MMAC1 mutations. PTEN/MMAC1 mutations in primary GBMs are more common (about 32%) and are associated with amplifications/mutations of EGFR, compared with mutations in secondary GBMs (about 4%) (335). Loss of PTEN protein expression is seen in approximately 70% to 95% of GBMs, suggesting that in addition to inactivating mutations, another mechanism of

FIGURE 53-5. PI3-Kinase signaling pathway: PI3-K can be activated by p21-Ras-dependent (not shown here) or p21-Ras-independent (shown here) mechanisms. Signaling through PI3-K leads to phosphorylation of PIP2 to PIP3, which in turn leads to activation of several downstream effector pathways involved in cell survival, proliferation, and cytoskeletal organization. This figure highlights a few of the downstream signaling events of the complex PI3-K pathway, the main one of which is mediated through activation of Akt/PKB. Activation of PI3-K can lead to regulation of the actin cytoskeleton through Rac signaling, protein synthesis through mTOR or GSK-3, and apoptosis through regulation of Bad or Caspase 9. PTEN, a dual lipd and protein phosphatase, is a negative regulator of the PI3-K pathway, by dephosphorylating PIP3 to PIP2. As detailed in the text, loss of expression of PTEN, one of the most common genetic aberrations in GBMs, leads to an increase in cell proliferation and migration and a decrease in apoptosis.

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PTEN loss, such as gene inactivation, is also present (330,335–337). The functional relevance of PTEN loss in GBMs has been demonstrated by reintroduction of wildtype PTEN, leading to G1 cell cycle arrest (330–332). Of note, PTEN/MMAC1 is not likely the only tumor suppressor gene in that region with relevance to gliomas, with other candidate genes such as DMBT, which is deleted in a subset of GBMs and low-grade astrocytomas (338). Of interest, we and other groups have attempted to develop mouse glioma models based on deletion of PTEN/MMAC1. However, PTEN loss by itself most likely does not suffice to induce gliomas, similar to our findings with EGFRvIII, and embryonic loss in astroglial cells leads to early perinatal death from gross CNS developmental abnormalities. Strategies to delete in PTEN/MMAC1 in adult astrocytes in the background of other relevant genetic alterations are currently being used in mouse glioma modeling. Aberrant activation of AKT/PKB leads to activation of several downstream signaling molecules and pro-survival pathways (reviewed in ref. 339) (Fig. 53-5). Among these is mTOR (mammalian Target Of Rapamycin), which phosphorylates 4EBP1 and prevents it from forming a complex with eIF4E, thereby inhibiting cap-dependent translation. Another downstream target of mTOR is activation of S6, involved in 5’TOP mRNA translation. Since the PI3-K:AKT:mTOR pathway is commonly activated in GBMs, there is considerable interest in designing specific drugs against these molecules (340). PI3-K inhibitors, such as LY294002, demonstrate in vitro and

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in vivo efficacy in GBMs, with inhibition of tumor angiogenesis and growth (341). Clinical interest is somewhat limited by bioavailability and toxicity issues, though an area of interest. Pharmacologic inhibitors of AKT/PKB are still at the preclinical stage, while mTOR inhibitors based on rapamycin and its analogs CCI-779 and RAD001 are being evaluated in early clinical trials in recurrent GBMs (339).

JAK/STAT PATHWAY Activation of the Jak (Janus tyrosine kinases)/STAT (Signal Transducers and Activators of Transcription) signaling pathway by various cytokine receptors is important in cellular regulation (reviewed in (342)) (Fig. 53-6). The Jak family of proteins ranges in size from 120 to 130 kDa and consists of cytoplasmic proteins with four members, Jak1 Jak2, Jak3, and Tyk2. With the exception of Jak3, which is only expressed in cells of the lymphoid and myeloid lineages, all are ubiquitously expressed. There are seven regions of high homology between the Jaks, known as Jak homology regions (JH1-JH7). The C-terminal JH-1 domain encodes the catalytic kinase; the JH2 region is a pseudokinase domain and may be important for JH1 catalytic activity or may act as a docking site for STAT proteins. The remaining domains, JH3-JH7, located at the N-terminus, have been implicated in receptor association.

FIGURE 53-6. Jak/STAT signaling pathway. Cytokine (or growth factor) binding to their specific receptor leads to receptor dimerization and activation/phosphorylation of Jaks. In turn, STAT monomers are recruited to the receptor complex, where they are phosphorylated by the Jaks, dimerize, translocate to the nucleus, and bind to specific sequence elements, activating gene transcription. Alternatively, it has been proposed that STATs do not exist as monomers but as a complex of proteins termed Statosome. These Statosomes consist of novel accessory scaffolding proteins, including the chaperone GRP53, and may play a role in recruitment of STATs to the activated receptor as well as transit to the nucleus.

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Seven STAT proteins, ranging in size from 80 to 115 kDa, have been identified in mammals, including STAT1-4, STAT5a, STAT5b, and STAT6 (also reviewed in ref. 342). Structural and functional analysis between all members of STAT proteins reveal several common domains: 1. N-terminal domain involved in protein–protein interactions, nuclear translocation, and deactivation. 2. Coiled-coil domain involved in protein–protein interactions, receptor binding, nuclear export, and tyrosine phosphorylation. 3. DNA-binding domain. 4. Linker domain that links the DNA-binding domain with the SH2 domain. 5. SH2 domain involved in association with activated Jaks and cytokine receptors, as well as homo- and heterodimerization of STAT proteins. 6. Transcriptional activation domain (TAD). The transcriptional activation domain of STATs, located in the C-terminal domain, can be alternatively spliced in STAT 1, 3, 4, and 5 and may function as dominantnegative regulators. The alternatively spliced form is known as ␤, whereas the full-length form is known as ␣ (342). Jak/STAT signaling follows recruitment of Jaks to cytokine receptor and subsequent activation either by auto- or transphosphorylation. This in turn results in the phosphorylation of downstream substrates such as STATs, which are latent cytoplasmic transcription factors that on phosphorylation become activated and form homo- or heterodimers. These dimers then translocate to the nucleus, where they bind to specific sequence elements and regulate gene transcription (reviewed in ref. 343). It has recently been shown that STATs can also be a part of a heteromeric complex, termed statosome, consisting of chaperone proteins that may shuttle activated STATs to the nucleus or accessory proteins that may be important in recruitment of STATs to the plasma membrane–receptor complex (344). In addition to the STATs, Jaks can also recruit other molecules to the receptor, to activate the MAPK or PI3-K pathways (reviewed in ref. 343). Several studies have looked at the expression levels of the various Jaks and STATs in brain tumors. One group found that Jak1 and STAT3 were more elevated in lowgrade gliomas than in high-grade gliomas. The other STAT proteins were equally expressed, except for STAT1, which was slightly higher in low-grade gliomas. Another group found that STAT3 was constitutively activated in glioma and medulloblastoma tumors (345). Analysis of these gliomas found that activated STAT3 was mainly localized to the endothelial cells. Activated STAT3␣ induced transcription of VEGF promoter, suggesting a possible role of STAT3␣ signaling in glioma angiogenesis. Preclinical studies suggest that targeting the Jak/STAT pathway may be

useful in gliomas, with further in vivo studies required to support the efficacy of targeting this pathway.

CONCLUSIONS For the proper development and maintenance of the multicellular organism, cellular proliferation, differentiation, and programmed cell death (apoptosis) has to be closely regulated. Communication between cells involving growth factors or cytokines, regulated activation of their cognate receptors, and transduction of the message from the activated receptor to the nucleus are vital for this regulation. Breakdown of this regulated intercellular communication at any of these junctions has the capability of inducing neoplasia. In this chapter we have outlined the evidence based on both in vitro and in vivo experiments that implicate several of these growth regulatory pathways in the pathogenesis of astrocytomas. Cancer is a multistep process. Hence, further experimentation is required to decipher the interactions and relative importance of these growth regulatory aberrations. Such efforts have the potential to yield a molecular classification of these tumors, which would have tremendous clinical implications because of the heterogeneity present in astrocytomas. Our knowledge of these molecular regulators has advanced considerably in the last two decades. However, our understanding of the intricacies of the various signaling pathways is far from complete. Therefore, caution must be observed before becoming discouraged from the failure of therapeutic trials seen to date and before making broad conclusions that result in discarding important observations, which if pursued more rigorously may lead to therapeutic breakthroughs. A critical step toward exploiting our knowledge of growth factors involved in the development and progression of malignant astrocytomas is to better understand how the multitude of factors interact and collaborate in an orchestrated manner. Therapeutic inroads that may come from the molecular understanding of astrocytomas are the ultimate goal. Several junctions of potential therapeutic alterations are present with growth factors and receptor signaling that contributes to the development of astrocytomas. As outlined in this chapter, these involve antibody, gene therapy, and pharmacologic strategies that may be directed at the growth factor, the receptor, or the cytoplasmic signaltransducing pathways. It is important to emphasize that the growing body of evidence suggests that the most effective therapy will ultimately encompass and combine a number of different targets and modalities. The best results probably will come from a combination of various pathways, angiogenic and other signal transduction pathways, relevant to the individual disease process. Mouse models that more closely and accurately mimic spontaneous human diseases based on genetic aberrations

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that are known to be involved in CNS pathologies are essential to increase our knowledge of the molecular pathogenesis of gliomas and provide better preclinical models to test novel biological therapies. Another area of exciting research that is rapidly advancing is in use of small animal anatomic and biological imaging. Within the last two decades the application of MRI in the study of human disease using small animals has experienced major advances. These imaging technologies allow in vivo assessment of growth and other biological properties, such as angiogenesis in spontaneous and orthotopic xenograft models of brain tumors, plus how they respond to biological targeted therapies. Combinatorial therapies, which are most likely required, can then be evaluated in spontaneous small-animal models of gliomas, with imaging in addition to the traditional survival and pathologic end-points. Slow advances in improving patient survival are likely with this bedside-to-bench approach, which we hope will ultimately remove malignant astrocytomas from being one of the most lethal human cancers, which they currently are.

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317. Xu G, et al. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell 1990;62:599–608. 318. Feldkamp MM, Angelov L, Guha A. Neurofibromatosis type 1 peripheral nerve tumors: aberrant activation of the Ras pathway. Surg Neurol 1999;51:211–218. 319. Roberts TM. A signal chain of events. Nature 1992;360:534. 320. Lange-Carter CA, Johnson GL. Ras-dependent growth factor regulation of MEK kinase in PC12 cells. Science 1994; 265:1458–1461. 321. Warne PH, Viciana PR, Downward J. Direct interaction of ras and the amino-terminal region of raf-1 in vitro. Nature 1993;364:352–356. 322. Zhang X-F, et al. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-raf-1. Nature 1993;364:308–334. 323. Dickson B, et al. Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature 1992;360: 600–603. 324. Moodie, SA, et al. Complexes of RasGTP with Raf–1 and mitogen–activated protein kinase kinase Science 1993;260: 1658–1660. 325. Feldkamp MM, Lau N, Guha A. Astrocytomas are growth inhibited by farnesyl transferase inhibitors through a combination of anti-proliferative and anti-angiogenic activities. Cancer Res 1998;submitted. 326. Feldkamp MM, Lau N, Guha A. Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects. Oncogene 1999;18:7514–7526. 327. Franke T, et al. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphotidylinositol 3-kinase. Cell 1995;81:727–736. 328. Stambolic V, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998;95: 29–39. 329. Haas-Kogan D, et al. Protein kinase B (PKB/Akt) activity is elevated in glioblastoma cells due to mutation of the tumor suppressor PTEN/MMAC. Curr Biol 1998;8:1195–1198.

330. Furnari FB, et al. Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc Natl Acad Sci U S A 1997;94:12479–12484. 331. Sun H, et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci U S A 1999;96:6199–6204. 332. Li L, et al. A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast. J Biol Chem 1997;272:29403–29406. 333. Steck PA, et al. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q233 that is mutated in multiple advanced cancers. Nat Genet 1997;15: 356–362. 334. Cantley L, Neel B. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A 1999;96:4240–4245. 335. Maher EA, et al. Malignant glioma: genetics and biology of a grave matter. Genes Dev 2001;15:1311–1333. 336. Myers MP, et al. The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proc Natl Acad Sci U S A 1998;95:13513–13518. 337. Von Deimling A, et al. Subsets of glioblastoma multiforme defined by molecular genetic analysis. Brain Pathol 1993;3: 19–26. 338. Mollenhauer J, et al. DMBT1, a new member of the SRCR superfamily, on chromosome 10q253-261 is deleted in malignant brain tumours. Nat Genet 1997;17:32–39. 339. Huang S, Houghton PJ. Targeting mTOR signaling for cancer therapy. Curr Opin Pharmacol 2003;3:371–377. 340. Choe G, et al. Analysis of the phosphatidylinositol 3’-kinase signaling pathway in glioblastoma patients in vivo. Cancer Res 2003;63:2742–2746. 341. Su JD, et al. PTEN and phosphatidylinositol 3’-kinase inhibitors up-regulate p53 and block tumor-induced angiogenesis: evidence for an effect on the tumor and endothelial compartment. Cancer Res 2003;63:3585–3592. 342. Kisseleva T, et al. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 2002;285:

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w Angiogenesis in Brain Tumors Kewel K. Jain Angiogenesis (formation of blood vessels) is fundamental to reproduction, development, and repair. During human embryonic growth, vessels develop to deliver adequate nourishment and oxygen from the maternal circulation. Angioblasts of extraembryonic mesoderm give rise to primitive vascular channels, and angiogenesis originates from these structures. After the developing embryo has formed a primary vascular plexus by a process termed vasculogenesis, further blood vessels are generated by both sprouting and nonsprouting angiogenesis, which are progressively pruned and remodeled into a functional adult circulatory system. In infants, angiogenesis is proportional to the proliferation of the tissue in which it takes place and declines in childhood. In healthy adults, the turnover of endothelial cells is extremely low, but they are not postmitotic and can form new blood vessels under pathologic conditions, such as in wound healing, ophthalmologic disorders, and tumors. Angiogenesis can be triggered by various humoral stimuli and occurs in some diseases, such as retinopathy of prematurity and hemangiomas. Angiogenesis is also a normal component of regeneration and helps in the revascularization, for example, after ischemic brain infarction. In contrast to spontaneously occurring angiogenesis in tumors, “therapeutic angiogenesis” is induced for the treatment of ischemic disorders. Tumor angiogenesis has been known for more than 100 years, but its mechanism has remained obscure. It was identified as an active process induced by humoral tumor-derived stimuli only in the 1960s. In 1971, Judah Folkman proposed the hypothesis that tumor growth is angiogenesis dependent and that endothelial cells may be switched from a resting state to a rapid growth phase by a “diffusible” chemical signal from tumor cells (1). This formed the basis of the concept for antiangiogenesis therapy. There is now considerable evidence that angiogenesis is essential for tumor growth and progression. This applies to malignant brain tumors, particularly glioblastoma multiforme, although angiogenesis occurs in some benign tumors as well. An understanding of the mechanism of angiogenesis and its inhibition by pharmacologic

means has opened up another possibility for the treatment of brain tumors.

ROLE OF ANGIOGENESIS IN TUMOR GROWTH Tumor angiogenesis resembles developmental angiogenesis, with the important difference that it ceases once development is completed, but tumor angiogenesis continues uncontrolled. The tumor vasculature consists of vessels from the preexisting network of host vasculature and the angiogenic response of the host vessels to cancer and stroma cells. Various steps in angiogenesis are (2) 1. Dissolution of the basement membrane and interstitial matrix of the vessel. 2. Migration of the endothelial cells toward the stimulus. 3. Proliferation of endothelial cells trailing behind the leading cells. 4. Formation of lumen in the endothelial sprout. 5. Formation of branches and loops to allow blood flow. 6. Development of pericytes and basement membrane around the immature vessels. New vessels can also be formed by the insertion of interstitial tissue column into the lumen of preexisting vessels. Regardless of the mechanism of formation, tumor vessels lose the normal anatomic structural arrangements and can be leaky and fragile, leading to edema and hemorrhage. An overview of some key steps in tumor angiogenesis is shown in Fig. 54-1. Various brain tumors associated with angiogenesis are listed in Table 54-1. The most significant manifestation of angiogenesis is in primary solid malignant tumors. Angiogenesis plays an important role, not only in the growth of primary tumors, but also in metastases. Primary or metastatic tumors that are not angiogenic do not enlarge to a size that is clinically detectable. The onset of angiogenic activity leads to a rapid expansion of the tumor and increases the risk of metastases. This is relevant to the topic as brain is a frequent site for metastases from tumors of other organs. 761

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FIGURE 54-1. An overview of some key steps in tumor angiogenesis. Tumor cells release proangiogenic factors such as vascular endothelial growth factor (VEGF), which diffuse into nearby tissues and bind to receptors on the endothelial cells of the preexisting blood vessels, leading to their activation. This leads to secretion and activation of various proteolytic enzymes such as matrix metalloproteinases (MMPs), which degrade the basement membrane and extracellular matrix, allowing activated cells to migrate toward the tumor. Integrin molecules help to pull the sprouting new blood vessels forward. The endothelial cells deposit a new basement membrane and secrete growth factors such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel. (Abbreviations: PDGFR, PDGF receptor; VEGFR, VEGF receptor.) (Reproduced from Cristofanilli M, et al. Nature reviews drug discovery 2002;1:415–426. By permission of the publisher, Macmillan Publishers Ltd. Copyright 2002.)

w TABLE 54-1

Brain Tumors Associated with Angiogenesis

Primary brain tumors Malignant Anaplastic astrocytoma Glioblastoma multiforme Hemangioblastoma Medulloblastoma Pituitary carcinoma Benign Craniopharyngiomas Meningiomas Neoplasms associated with tuberose sclerosis Pituitary adenomas Metastatic tumors

FACTORS THAT INFLUENCE TUMOR ANGIOGENESIS Angiogenesis is a complex multistep process involving extensive interplay between cells, soluble factors, and extracellular matrix components. Several angiogenic peptides have been discovered. Some stimulate vascular endothelial cells to proliferate, whereas others act indirectly by mobilizing host cells to release endothelial growth factors. Endogenous inhibitors of angiogenesis counteract the activity of these angiogenic factors. The switch to neoplastic angiogenesis is a tilt in the balance in favor of positive regulators (enhancers) of microvessel growth over the negative regulators (inhibitors).

54. Angiogenesis in Brain Tumors

w TABLE 54-2

Factors that Enhance Angiogenesis in Brain Tumors

Growth factors and cytokines Angiogenin Basic fibroblast growth factor (bFGF) Granulocyte colony-stimulating factor (G-CSF) IL (interleukin)-8 Insulin-like growth factor (IGF)-1 Hepatocyte growth factor (HGF) Placental growth factor (PlGF) Platelet-activating factor (PAF) Platelet-derived epithelial cell growth factor (PD-ECGF) Transforming growth factor ␣ (TGF-␣) Tumor necrosis factor ␣ (TNF-␣) Vascular endothelial growth factor (VEGF) Signaling molecule Inducible nitric oxide synthase (iNOS) Enzymes Matrix metalloproteinases (MMPs) Adhesion molecules Integrins: ␣V␤3, ␣V␤5

Angiogenesis- Enhancing Factors Many of the growth factors for tumors may also act as angiogenic factors, causing a response of enzyme production, migration, and/or proliferation in endothelial cells. Table 54-2 lists various enhancers of angiogenesis that are relevant to brain tumors. The important ones are discussed in the following text. The action of hypoxia is mediated through several of the factors listed. Growth Factors/Cytokines Angiogenin is an important factor of angiogenesis during different stage of tumor development and exists widely in various tumors. Angiogenin may contribute to the malignant transformation of gliomas. It is secreted by both tumor and normal cells, and interacts with endothelial and smooth muscle cells to induce a wide range of cellular responses, including cell migration and invasion, proliferation, and formation of tubular structures. Angiogenin is rapidly endocytosed and translocated to the cell nucleus, where it accumulates in the nucleolus and binds to DNA. This suggests that the nuclear function of angiogenin relates to its capacity to induce rRNA synthesis. Because rRNA transcription is essential for the synthesis of new ribosomes that are necessary for protein translation and cell growth, inhibition of angiogenin-stimulated transcription of rRNA may preclude angiogenesis and therefore serve as a molecular target for therapeutic intervention (3). bFGF, the most potent angiogenic peptide, is distributed widely in normal as well as neoplastic tissues. Astrocytes exhibit significant changes in FGF receptor (FGFR) gene

763

expression during malignant progression. These changes include induction of FGFR1 and concomitant loss of FGFR2 expression (4). FGFR1 antisense oligonucleotides markedly reduced expression of both FGFR1 mRNA and highaffinity bFGF binding sites, whereas FGFR1 reverse antisense control oligonucleotide had no effect. Growth of human malignant gliomas is stringently dependent on an angiogenic process that probably involves VEGF. In a rodent malignant glioma model, tumor vascular permeability and tumor growth can be inhibited by neutralization of endogenous vascular endothelial growth factor (VEGF) and suggest that angiogenesis with the maintenance of endothelial hyperpermeability requires the presence of VEGF within the tissue microenvironment (5). Glioblastoma multiforme expresses ubiquitously VEGF-D, which colocalizes with glial fibrillary acidic protein (6). This study also reveals that an up-regulation of AP-1 factors may be a hallmark of glioblastoma multiforme. Because VEGF-D activates VEGF receptor 2 and 3, receptors important for tumor angiogenesis, it may represent an X-linked/AP-1-regulated oncoangiogen in human glioblastoma multiforme. The VEGF-D system and AP-1 activity appear to be very attractive targets for new molecular diagnostics and rational molecular anticancer therapies. A new splice variant of VEGF-B (VEGF-B155) that lacks exons 5 and 6 is described (7). Expressions of VEGF mRNA in cultured glioblastoma cells were up-regulated by hypoxia, but the sensitivity of the cells to hypoxia was reduced as compared with normal rat astrocytes. Up-regulation of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and tenascin-C (TN-C) is spatially and temporally related to neovascularization. Spatially, VEGF/VPF and TN-C are both found at the site of neovascularization, but they are not detected in areas of normal brain or in areas without neovascularization. Temporally, VEGF/VPF and TN-C are found at the peak of angiogenesis and are not detected when angiogenesis had ceased (8). Neuropilin-1 (NRP1), a cell-surface receptor for VEGF, regulates angiogenesis through a VEGF-dependent pathway (9). Hepatocyte growth factor (HGF) has a stimulatory effect on the synthesis of matrix metalloproteinase-2, which is involved in glioma invasion (10). Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic cytokine that has been implicated in glioma invasion and angiogenesis. The SF/HGF receptor, MET, is expressed in neoplastic astrocytes as well as in endothelial cells of the tumor vasculature. Both SF/HGF and MET expression have also been described to correlate with the malignancy grade of human gliomas. HGF is not an active cytokine with regards to in vitro glial cell proliferation and may stimulate glioma cellular invasion in vivo through binding to its receptor and by activating tyrosine kinase secondary messengers (11). Hypoxia plays an important role in angiogenesis. Microvascular hyperplasia is spatially and temporally associated with pseudopalisading necrosis in GBM and is

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believed to be driven by hypoxia-induced expression of proangiogenic cytokines such as vascular endothelial growth factor (12). Integrins Integrins are proteins consisting of ␣- and ␤-subunits that connect the interior of a cell to the extracellular matrix. Antibodies that bind to integrins and block their activity have been shown to inhibit the formation of new blood vessels in animal models. But there are many different types of integrin, and defining precisely which of these proteins are involved is crucial to developing an effective drug. The hypothesis goes that if integrins are important to the growth of new blood vessels, then angiogenesis should be impeded in their absence. Mice lacking ␤3 integrins or both ␤3 and ␤5 integrins not only grow properly, but also develop new tumors with more enhanced angiogenesis than observed in normal mice (13). This obviously suggests that these integrins are not essential to angiogenesis. NG2 Proteoglycan During embryogenesis, the NG2 proteoglycan is expressed on immature capillary vessels, but as the vessels mature they lose this expression. NG2 is up-regulated in high-grade gliomas, but it is not clear to what extent it contributes to malignant progression. An overexpression of NG2, demonstrated by functional magnetic resonance imaging and histopathologic analyses, increases tumor initiation and growth rates, neovascularization, and cellular proliferation, which predisposes to a poorer survival outcome (14). Confocal microscopy and cDNA gene array expression profiles show that NG2 tumors express lower levels of hypoxia-inducible factor-1a, vascular endothelial growth factor, and endogenous angiostatin in vivo compared with wild-type tumors. Moreover, the authors demonstrate that NG2-positive cells bind, internalize, and coimmunoprecipitate with angiostatin. These results indicate a unique role for NG2 in regulating the transition from small, poorly vascularized tumors to large, highly vascular gliomas in situ by sequestering angiostatin. Matrix Metalloproteinases Matrix metalloproteinases (MMPs) are a family of enzymes responsible for normal turnover and remodeling of the extracellular matrix. MMPs are required for tumor angiogenesis and invasion. Degradation of basement membrane is one of the crucial steps in tumor angiogenesis and is performed by MMPs. Among many other factors, gliomas express elevated levels of matrix metalloproteinases (MMPs), which have been implicated to play an important role in tumor invasion as well as neovascularization. The

investigation of the use of MMP inhibitors to gliomas as an adjunct to chemotherapy was started in an attempt to control the tumor growth. MMPs have a dual function in tumor angiogenesis: MMP-2 and MT1-MMP are required in breaking down basement membrane barriers in the early stage of angiogenesis, whereas other MMPs are involved in the generation of an angiogenic inhibitor, angiostatin. Understanding the role of MMP-mediated proteolysis in the brain relies heavily on advances in other areas of molecular neuroscience, most notably an understanding of extracellular matrix (ECM) composition and the function of cell adhesion molecules such as integrins, which communicate knowledge of ECM composition intracellularly. Recently, protease expression and function have been shown to be strongly influenced by the functional state and signaling properties of integrins. Membrane-type MMPs (MT-MMPs) constitute a growing subclass of recently identified matrix metalloproteinases (MMPs). Besides the highly conserved MMP functional domains, the MT-MMPs have additional insertion sequences (IS) that confer unique functional roles. Although most of the MMPs are secreted, the MT-MMPs are membrane associated, and a number of these have cytoplasmic domains, which may be important in cellular signaling. This membrane localization leads to focal areas of receptor recruitment and subsequent activity, thereby enhancing pericellular proteolysis in specific areas of contact within the brain interstitium. MT1-MMP is the bestcharacterized MT-MMP, the measure against which subsequently cloned homologs are compared. MT1-MMP activates proMMP2 via its interaction with TIMP2, which serves as an intermolecular bridge for proMMP2 binding to MT-MMPs. In addition to activation of proMMP2, MTMMPs display intrinsic proteolytic activity toward extracellular matrix molecules (ECM), which is independent of MMP2 activation. The increased expression levels of several members of the MMP family have been shown to correlate with high-grade gliomas, including MTI-MMP. Despite improvements in the diagnosis and treatment of patients with glial tumors, they remain the most common and least curable brain cancer in adults. The ability of glioma cells to infiltrate surrounding brain tissue, and ultimately escape current therapeutic modalities, could be minimized using antiinvasive therapies. Proteolysis is a necessary part of the invasion process, within which the MT-MMPs appear to play a central role. The development of pharmaceutical approaches that target expression and regulation of MT-MMPs may prove beneficial in targeting invading glioma cells (15). The Role of Hypoxia Cells undergo a variety of biological responses when placed in hypoxic conditions, including activation of signaling

54. Angiogenesis in Brain Tumors

pathways that regulate proliferation, angiogenesis, and death. Cancer cells have adapted these pathways, allowing tumors to survive and even grow under hypoxic conditions, and tumor hypoxia is associated with poor prognosis and resistance to radiation therapy (16)). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates adaptive responses to reduced O2 availability, including angiogenesis and glycolysis. Expression of the O2-regulated HIF-1␣ subunit and HIF-1 transcriptional activity are increased dramatically in hypoxic cells. Recent studies indicate that many common tumor-specific genetic alterations also lead to increased HIF-1␣ expression and/or activity (17). HIF-1␣ plays an important role in the vascularization of glioblastomas, and the up-regulation is at least partly at a transcriptional level (18). Thus, genetic and physiologic alterations within tumors act synergistically to increase HIF-1 transcriptional activity, which appears to play a critical role in the development of invasive and metastatic properties.

Endogenous Factors That Inhibit Angiogenesis Inhibitory factors, listed in Table 54-3, are endogenous factors that inhibit angiogenesis and not therapeutically administered substances. The phenomenon by which tumor-bearing hosts are capable of inhibiting secondary tumor implants or metastases, known as concomitant antitumoral resistance (CAR), is due to antiangiogenesis at places distant from the primary tumor. This is due to differential angiostatin generation by CAR-inducing cancer cells (19). p53, in addition to its function as a tumor suppressor gene, may also act as an angiogenesis suppressor gene. Glioblastoma multiforme is distinguished from its less malignant astrocytoma precursors by intense angiogenesis and frequent loss of tumor suppressor genes on chromosome 10. This is partly due to release of constraints on angiogenesis that are maintained by thrombospondin in lower-grade tumors (20). Establishment and growth of metastases are influenced by soluble factors secreted from the originating solid tumor. Among these factors are so-called endogenous inhibitors of angiogenesis, which keep metastasis in

w TABLE 54-3

Endogenous Factors that Inhibit Angiogenesis

Angiostatin Circulating endothelial cell inhibitors: platelet factor 4 Endostatin P53 Thrombospondin-1 Tissue inhibitors of metalloproteinases (TIMP) Tumor-derived endothelial cell inhibitors

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a nonproliferating quiescent state. Removal of inhibiting antiangiogenic factors leads to growth of dormant metastases (21). A number of endogenous inhibitors have been identified and have shown success in experimental therapeutic trials. This might be of special interest for the treatment of cerebral metastases. Similar to the spread of metastases, it is known that single glioma cells can be found in distant parts of the brain.

DEVELOPMENT OF ANTIANGIOGENIC THERAPIES Angiogenesis has become a hotbed of pharmacologic research as well as industrial drug discovery. Its attractiveness stems from the fact that this approach avoids the formidable hurdles of other anticancer therapeutic modalities, such as the diversity of cancer types, heterogeneity within a given tumor, genetic or chromosomal instability, and drug resistance. Tumor angiogenesis is, therefore, thought to be comparable for a large variety of solid cancers. If this is correct, in most situations, elements such as the activated endothelial cell, remodeling of the basement membrane, and pericyte function can be approached in a systematic fashion, and therapeutic approaches can be designed that are based on a rapidly increasing body of evidence regarding the biology of tumor angiogenesis. Approaches developed for cancers of other organs can be applied to brain tumors. More than 40 biotechnology companies are involved in developing antiangiogenesis drugs, but to date none is close to obtaining market approval.

Classification of Antiangiogenic Agents It is estimated that more than 300 drug candidates are currently in various stages of testing, but there is still no drug on the market that achieves clinical benefit based on a selective modulation or inhibition of angiogenesis alone. Most of the antiangiogenic therapies are considered as adjuvant to radiation or chemotherapy. Rather than listing all the antiangiogenic compounds, the emphasis in this chapter is on projects that are either in preclinical or clinical development and are relevant to the treatment of brain tumors. A classification of antiangiogenic therapies is shown in Table 54-4.

Development of Angiogenesis Assays The challenge for the discovery and characterization of antiangiogenic targets remains in developing efficient in vitro or in vivo preclinical angiogenesis screening assays to assess and compare antiangiogenic activity. Several semiquantitative or quantitative angiogenesis assays exist, including in vitro endothelial cell systems and ex vivo or

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w TABLE 54-4

A Classification of Antiangiogenic Therapies

Vasculostatic agents that interfere with the process of forming new blood vessels. They can be divided further into Antagonists of angiogenic growth factors (e.g., vascular endothelial growth factor receptor [VEGF-R] tyrosine kinase inhibitors) Inhibitors of endothelial cell proliferation/migration (e.g., Endostatin and Angiostatin [EntreMed, Inc.]) Antibodies with vasculostatic properties (e.g., AngioMab [Antisome plc]) Matrix metalloproteinase inhibitors (e.g., Marimostat [British Biotech]) Integrin inhibitors (e.g., Vitaxin) Miscellaneous small-molecule vasculostatic agents Vasculotoxins that use elements of newly formed blood vessels to target toxic principles, destroying them and thereby producing antitumor effects. VEGF-mediated targeting of a diphtheria toxin conjugate to tumor endothelial cells. Vascular targeting agents that thrombose the newly formed vessels, depriving the tumor of blood supply Monoclonal antibody targeting (e.g., Cotara [Peregerine Pharmaceuticals]) Hyperthermia Drugs that mediate their action through induction of tumor necrosis factor (TNF)-␣ (e.g., 5,6-dimethylxanthenone-4-acetic acid [DMXAA]) Tubulin-interfering agents (e.g., combretastatin) Antisense therapy (e.g., c-myc antisense phosphorothioate oligonucleotides) Gene therapy (e.g., targeted adenoviral vectors that are antiangiogenic) Miscellaneous drugs developed primarily for other disorders but discovered to have antiangiogenic properties (e.g., thalidomide)

in vivo neovascularization models utilizing mouse, rat, or human tissues. More common and cost-effective angiogenesis assays currently in use, with their unique advantages and disadvantages, have been reviewed elsewhere (22). Among the simplest antiangiogenic screening assays are endothelial cell assays, which look at proliferation, migration, and tube formation as indicators of a test compound’s antiangiogenic potential. Cell proliferation assays use cell counting, staining, and thymidine incorporation to measure endothelial cell growth or death. The endothelial cell migration assay, which is amenable to a moderately high-throughput approach, measures the ability of cells to cross a synthetic barrier. BioCoat Angiogenesis System for Endothelial Cell Invasion (BD Biosciences), for example, uses fluorometric detection of migration to measure endothelial cells invading a Matrigel barrier in the presence of angiogenesis inhibitor candidates. Finally, the tube-formation assay scores the ability of endothelial cells to produce tubelike structures. The aortic ring assay is another widely used in vitro assay. Rat aortas are sliced crosswise and placed in Matrigel. After 3 to 5 days, microvessel sprouting can be roughly quantified by manual counting or by using image analysis. The aortic ring assay is low-cost, is easy to use, and offers many assays from a single animal, but it can be difficult to quantitate. A relatively new variant uses embryonic chick aortic arches. The human saphenous vein is an excellent ex vivo model for angiogenesis. The vein is sectioned in 2-mm-thick cross-sections and embedded into 12-well plates coated with Matrigel. The rings are cultured for 14 days before growth analysis. The limitations to this model are the amount of tissue available and donor-to-donor variation.

The mouse corneal angiogenesis assay offers a vascularization model in a normally avascular tissue. Pellets are inserted into surgically created micropockets in the cornea. This model has the distinct advantage of measuring only new vessel growth, which can be easily visualized. But because the cornea is normally avascular, it may not be an ideal tested, as it lacks many of the blood-borne substances that modulate endothelial cell growth. In the chick embryo chorioallantoic membrane (CAM) assay, a window is cut into the eggshell, exposing the vessels below. Alternatively, the embryo can be transferred to a petri dish, where the CAM grows as a flat membrane, thus allowing several test sites. This assay’s caveats include critical timing for observing effects, confounding inflammatory and vasodilatory effects, and an inability to assess drugs requiring metabolic activation.

Molecular Diagnostic Studies MMP profiling studies in glioma tissues and cell lines by RT-PCR and Western blotting. Co-expression of MMPs and certain integrins substantiates the gathering evidence of a functional intersection between the two, and inhibition studies using recombinant TIMP-1 and integrin antisera demonstrate significant inhibition of glioma invasion in vitro.

In vivo Imaging of Angiogenesis in Brain Tumors Angiogenesis in glioblastoma can be assessed by contrast enhancement of computerized tomography (CT). Magnetic resonance imaging (MRI) corresponds more directly

54. Angiogenesis in Brain Tumors

with tumor angiogenesis and provides an attractive tool for in vivo analysis of the basic biology of angiogenesis. MRI can be used for preclinical evaluation of the activity of a number of potential antiangiogenic agents, as well as for clinical detection, diagnosis, and prognosis (23). Changes in tumor vessel permeability and tumor volumes as measured by contrast-enhanced MRI provide an assay that could prove useful for clinical monitoring of antiangiogenic therapies in brain tumors. Vascular hyperpermeability of tumor vessels for macromolecular solutes yields a proteinaceous exudate within tumor interstitium that is a favorable medium for the ingrowth of new capillary buds. One of the features of MRI is the wide range of physiologic parameters by which angiogenesis can be imaged. For example, one can detect angiogenesis in vivo using MRI and a paramagnetic contrast agent targeted to the angiogenic vasculature. Another technique for in vivo imaging of tumor angiogenesis is genetic engineering of tumor cells to express the jellyfish green fluorescent protein (GFP). When the genetically fluorescent tumors are transplanted to mouse models, the tumor blood vessels originating from them can be easily detected when blue light is shined on them. The tumor glows bright green and the vessels appear black (24). This tumor model can be used for drug discovery and to test agents that are effective against tumor angiogenesis.

Animal Models for Glioblastoma Preclinical studies need an assortment of models, which should enable investigation of a variety of antiangiogenic strategies (25). Basic endothelial cell (EC) characteristics can adequately be studied in vitro using EC monolayer assays. Three-dimensional spheroid techniques evaluate the more complex cell–cell and cell–environment interplay within a three-dimensional culture. To optimize the imitation of the crucial interaction of human gliomas with host endothelial cells, immunologic cells and extracellular matrix, animal models are mandatory. An essential rule is to utilize an orthotopic model, since tumor–host interaction is organ-specific. To avoid alloimmunogenic responses, it

w TABLE 54-5

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is desirable to use weakly or not immunogenic glioma grafts, which is best accomplished in a syngeneic model. However, since rat gliomas poorly resemble human glioma growth patterns, human glioma xenografting into immunocompromised animals should be considered. In vivo monitoring techniques like videoscopy via a cranial window or MRI enable functional studies and improve the validity of the model employed. Finally, it is essential to recognize the limitations of each model considered and to select that model, which seems to be most appropriate for the objectives to be investigated.

ANTIANGIOGENIC APPROACH TO THE TREATMENT OF BRAIN TUMORS An antiangiogenic approach is designed to reduce the neovasculature of the tumor. Sometimes a distinction is drawn between antiangiogenic strategy and antivascular strategy, but there is considerable overlap between the two. For example, VEGF withdrawal has antiangiogenic effect as manifested by vascular regression, but it can also lead to apoptosis and tumor necrosis (26). The dosing schedule may be more relevant to the effect. A single dose is effective for vascular targeting and may shut down the vascular system, whereas chronic dosing over a period of time is required for antiangiogenic effect. Numerous antiangiogenic approaches are in development for the treatment of cancer, but only selected products that either have been used for brain tumors or have the potential for this application will be described. Table 54-5 lists those that are being or have been tested in clinical trials for brain tumors. (Two of the clinical trials had already been discontinued at the time of writing.)

Inhibitors of Endothelial Proliferation Angiostatin Angiostatin, a multiple kringle- (1 to 4 of 5) containing fragment of plasminogen, is one of the highly effective natural cryptic angiogenesis inhibitors. The therapeutic

Clinical Trial Status of Antiangiogenic Agents for Brain Tumors

Drug

Sponsor

Mechanism

Status

AG3340 AngioMab

Agouron, Inc. Antisoma Ltd

Phase II discontinued Phase I/II

AP 12009 Cotara

Antisense Pharma Peregerine Pharmaceuticals

Endostatin Marimastat Metastat

EntreMed British Biotech Collagenex Pharmaceuticals

MMP inhibitor Monoclonal antibody that specifically targets a signature protein produced by new blood vessels Antisense action inhibits the translation of TGF-␤2 in glioma cells Radiolabeled monoclonal antibody vascular targeting endothelial cell growth factor Endogenous inhibitor of angiogenesis MMP inhibitor MMP inhibitor

Phase II Phase II Phase II Phase II discontinued Phase I/II

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efficacy of nonglycosylated and small molecular size recombinant kringles 1 to 3 (rPK1–3) was studied in the treatment of brain tumors generated by stereotactic intracerebral implantation of human glioma cells in nude mice (27). Their results indicate that the nonglycosylated, small-molecular-size rPK1-3 is an efficient tumoristatic agent for the treatment of intracranial human glioma xenografts in mice and might provide new strategies for the treatment of brain tumors. Endostatin Endostatin, the 20-kDa C-terminal fragment of collagen XVIII, has previously been shown to inhibit growth and induce regression of different experimental tumors in rodents. Recombinant murine and human endostatin, respectively, inhibit ectotopic as well as orthotopic growing BT4Cn gliosarcomas in rats (28). Tumors were not observed to increase in size or regrow after their shrinkage and cessation of therapy. Furthermore, endostatin-treated rats with intracranial tumors had significantly longer survival time than did untreated controls. In the treated rats, endostatin therapy resulted in a reduced tumor blood vessel volume and an increased tumor cell density with an increased apoptotic index within a given tumor volume, as verified by flow cytometry and by staining. This study verifies the general antiangiogenic and antitumor effects of endostatin and indicates that the protein may also be considered as a treatment strategy for malignant brain tumors. Clinical trials have been conducted in various cancers, including neuroendocrine tumors. TNP-470 TNP-470 (TNP), a synthetic analog of fumagillin isolated from Aspergillus fumigatus, was used as an antiangiogenic agent in a study in a nude mouse model with a subcutaneously implanted fragment of medulloblastoma (29). TNP inhibited the growth of brain tumor dosedependently and induced various ischemic changes within the tumor tissue. Therefore, TNP may be effective for the treatment of malignant brain tumors such as medulloblastoma. However, some glioma cell lines are resistant to TNP.

Monoclonal Antibodies with Vasculostatic Properties

or indirectly to cause its death. The BC-1 antibody is being humanized to allow repeat dosing. Since essentially all solid tumors—including those of the brain, lung, breast, colon, and prostate—need their own blood supply, products based on this antibody have the potential to treat many different cancers. The first product candidate is likely to be a radiolabeled antibody to treat glioblastoma multiforme in combination with RACER technology. Glioblastomas produce high levels of the protein targeted by BC-1. Preclinical studies have been completed and clinical trials are planned.

Vascular Targeting Agents Combretastatin Combretastatin A-4 (CA-4) and A-1 (CA-1) are tubulinbinding agents, structurally related to colchicine, that induce vascular-mediated tumor necrosis in animal models. CA-1 and CA-4 were isolated from the African bush willow, Combretum caffrum, and several synthetic analogs are also now available, such as the AVE8062 (Aventis Pharma). More soluble, phosphated forms of CA-4 are commonly used for in vitro and in vivo studies. These are cleaved to the natural forms by endogenous phosphatases and are taken up into cells. The lead compound, CA-4 phosphate, is currently in phase I clinical trial as a tumor vascular targeting agent. In animal models, CA-4-P causes a prolonged and extensive shutdown of blood flow in established tumor blood vessels, with much less effect in normal tissues (30). CA-4 disodium phosphate induces a gradual reduction in tumor blood flow, which can be exploited to sensitize the BT4An rat glioma to hyperthermia (31). Cotara Cotara (Peregerine Pharmaceuticals) is a radiolabeled monoclonal antibody with a vascular targeting action. One of the targets is vascular endothelial cell growth factor, a prominent marker within tumor blood vessels. The drug was shown in preclinical studies to possibly have broad application for a number of different tumor types. It was also shown to become more effective with successive use. The current phase II Cotara brain trial is a multicenter, open-label study designed to treat patients with newly diagnosed and recurrent glioblastoma multiforme and recurrent anaplastic astrocytoma.

AngioMab AngioMab (Antisoma Ltd) is a mouse monoclonal antibody BC-1 that specifically targets a signature protein produced by new blood vessels. Therapies based on BC1, acting alone or carrying a killing agent, are in development to interrupt the tumor’s blood supply and directly

VEGF121/rGelonin Chimeric Fusion Toxin One study has demonstrated that a chimeric fusion construct (VEGF121/rGel) containing VEGF and the plant toxin gelonin is selectively toxic to dividing endothelial cells overexpressing the KDR (kinase domain receptor)/Flk-1

54. Angiogenesis in Brain Tumors

receptor (32). Nondividing (confluent) endothelial cells were almost 60 times more resistant than were dividing cells to the fusion construct and were more resistant to free rGel. The fusion construct localized selectively to PC-3 tumor vessels and caused thrombotic damage to tumor vessels with extravasation of red blood cells into the tumor bed. This study demonstrates the successful use of VEGF121/rGel fusion construct for the targeted destruction of tumor vasculature in vivo. The tumor model used was melanoma, but this technique is applicable to glioma models as well.

Matrix Metalloproteinase Inhibitors Because matrix metalloproteinases (MMPs) levels are raised during tumor growth, MMP inhibitors were developed to treat patients with cancer. A critical review of MMPs indicates that (33) n n

n

Most MMPs in tumors are made by stromal cells, not carcinoma cells. Cancer cells induce stromal cells to synthesize MMPs using extracellular matrix metalloproteinase inducer and cytokine stimulatory mechanisms. MMPs promote cell migration and the release of growth factors sequestered in the extracellular matrix.

Marimastat Marimastat (British Biotech), a MMP inhibitor, was tested in phase II clinical trials for glioblastoma, but the results were not encouraging. Despite some controversy, the drug is still undergoing clinical trials, mainly in cancers of other organs and in combination with antineoplastic drugs. The combination of temozolomide and Marimastat was studied in patients with recurrent glioblastoma multiforme in a phase II study and resulted in a progression-free survival at 6 months that exceeded the literature target by 29% (34). One adverse effect of this therapy was joint pain. Further studies may be justified. Metastat (COL-3) Metastat (Collagenex Pharmaceuticals) belongs to a group of compounds called IMPACS (Inhibitors of Multiple Proteases And CytokineS) that are based on the four-ring structure of the antibiotic tetracycline and are often referred to as chemically modified tertracyclines. IMPACS compounds can directly suppress the activity of MMPs believed to arise via a variety of mechanisms, including direct enzyme inhibition, inhibition of enzyme activation, and inhibition of enzyme synthesis. Metastat has been shown to significantly inhibit malignant tumor cell invasion into normal lung tissue in rat and mouse models of metastasis. Other studies have suggested that Metastat

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may inhibit tumor-induced angiogenesis in animal models. A phase I/II study in glioblastoma multiforme is currently recruiting patients. Ag3340 The effect of AG3340 (Agouron, Inc.), a novel synthetic MMP inhibitor, was tested on the growth of a human malignant glioma cell line (U87) in SCID-NOD mice (35). AG3340 produced a profound inhibition of glioma tumor growth and invasion and markedly increased survival in this in vivo glioma model. Treatment with AG3340 was considered to be potentially useful in patients with malignant gliomas. AG3340 was tested in clinical trials for a variety of cancers. Development was discontinued because of lack of efficacy. SI-27 SI-27, a MMP inhibitor, was used at noncytotoxic concentrations (1 to 100 ␮mol/L), and its effect on nonmitogenic vascular endothelial growth factor (VEGF)-enhanced cell motility and in vitro angiogenesis by human umbilical vein endothelial cells was determined (36). The activity of MMP-1, MMP-2, and tissue inhibitor of metalloproteinase 1 was determined by enzyme-linked immunosorbent assay. The effect of SI-27 on in vitro angiogenesis stimulated by supernatants of human glioma cell lines also was examined. Angiogenesis was detected with variable vacuum scanning electron microscopy. Cell motility and in vitro angiogenesis by human umbilical vein endothelial cells were significantly increased by VEGF. SI-27 inhibited in vitro tumor angiogenesis by suppression of MMP. This agent may be anticipated to prevent tumor growth through an angiosuppressive effect. Recommendations for Future Studies Despite considerable recent progress in identifying multiple roles of MMPs in disease, our understanding of MMP function in cancer is far from complete. Based on accumulated data, it is recommended that future MMPI trials focus on (a) patients with early-stage cancer; (b) the use of MMPIs along with chemotherapy; (c) the measurement of MMPs in tumor tissue and blood as a means of identifying patients who are more likely to respond to MMPI therapy; and (d) identification of biomarkers that reflect activation or inhibition of MMPs in vivo.

1,3,6-Naphthalenetrisulfonate 1,3,6-Naphthalenetrisulfonate (NTS) can inhibit the proliferation in vitro of cells of various origins, including glioma. A study of the effects of NTS on intratumoral angiogenesis and tumor growth in the rabbit cornea after

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implantation of C6 rat glioma cells showed that neovascularization and glioma growth were abolished by topical administration of NTS (37). This effect could be mediated by both induction of programmed cell death and inhibition of growth, in endothelium and in tumor cells, most likely as a consequence of the disruption of the autocrine and paracrine effects of FGF released from endothelial and tumor cells. The results suggest that NTS is a promising candidate to lead the development of new angiogenesis inhibitors for the treatment of cancer and other diseases whose progression is dependent on the development of new blood vessels. Toxin Fusion Protein Diphtheria toxin amino-terminal (DTAT) is a novel recombinant toxin fusion protein that consists of a fragment of urokinase-type plasminogen activator (uPA), enabling it to bind to glioblastoma cells, which are known to overexpress the uPA receptor (38). DTAT also has the catalytic portion of diphtheria toxin. DTAT destroys glioblastoma cells in culture and reduces the size of such tumors in a murine cancer model. The protein also destroyed uPA-receptor-expressing endothelial cells, which could be beneficial in limiting a tumor’s vascular supply. Thus this compound has the ability to target tumor cells and tumor vasculature simultaneously. Toxicity testing in mice without tumors revealed that the agent had minimal effect on kidney, liver, heart, lung, and spleen. One problem with immunotoxins is that they usually have severe side effects when administered systemically. DTAT demonstrated minimal toxicity in this study, but because mice don’t express the same uPA receptor that humans do, the effects of systemic administration are uncertain. However, the plan is to deliver DTAT to human patients intracranially rather than systemically. Clinical testing is anticipated in 2003. Antiangiogenic Effect of Conventional Anticancer Drugs Taxol, vincristine, and vindesine behave similarly in their ability to reduce the capillary network formation by human umbilical vein endothelial cells cultured on Matrigel (39). These antiangiogenic effects appear at noncytotoxic concentrations. In contrast, vinblastine and vinorelbine produce apparent antiangiogenic effects by direct cytotoxicity. Microtubule-affecting agents are also able to significantly reduce the level of migration of tumor cells at noncytotoxic concentrations. Some of these effects may occur via modifications to the actin cytoskeleton organization. Several types of microtubuleaffecting agents could be used as antiangiogenic agents by administering them at noncytotoxic concentrations, and some microtubule-affecting agents abandoned in

pharmacologic assays could turn out to be potent antimigratory drugs acting on tumor cells, though without being too cytotoxic. Glial tumor cell–conditioned medium induces angiogenesis in large-vessel endothelial cells in vitro via mechanisms, which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis (40).

Antiangiogenic Antisense Therapy Antisense oligodeoxynucleotides (ODNs) have been proposed as a new therapy for patients with cancer, including malignant brain tumors. Antisense ODNs are taken up by tumor cells and selectively block gene expression. Use of ODNs for brain tumors is attractive because of their theoretical specificity and relative ease of production.

Antisense Protein Kinase C-␣ The protein kinase C (PKC) family represents several closely related genes that are involved in signal transduction for processes such as cell proliferation, angiogenesis, and immune response. PKC has been proposed as a target for anticancer drug development. Antisense PKC-␣ phorsphorothioate oligodeoxynucletide (ISIS 3521, Isis Pharmaceuticals, Inc.) given by intraperitoneal injection has been shown to suppress growth in mice bearing subcutaneous implants of U-87 glioblastoma (41). This is a potential therapy for glioblastoma multiforme in humans. ISIS 3521, under the proprietary name of Affinitac, is currently in phase III clinical trials for non–small-cell lung cancer. AP 12009 AP 12009 (Antisense Pharma), a synthetic phosphorothioate oligonucleotide, is an innovative therapeutic approach to treat malignant brain tumors and other solid tumors. The antisense oligonucleotide targets the transforming growth factor (TGF)-␤2 mRNA to inhibit its overexpression. TGF-␤2 is the most potent immunosuppressor known to date and plays a key role in tumorigenesis, tumor proliferation, and simulation of angiogenesis. Compared with monoclonal antibodies or ribozymes, the smaller size of AP 12009 allows it to penetrate into the cell matrix and directly reach its target in the tumor cells. AP 12009 inhibits the translation of TGF-␤2 in glioma cells and thus blocks tumor cell growth, reduces cell migration, and enhances the host’s immune response. The drug exhibits a good tolerability and safety profile. Phase I/II clinical studies were initiated in October 2000 for the treatment of recurrent anaplastic astrocytoma and glioblastoma (WHO grades III and IV, respectively). The FDA has granted the orphan drug designation for AP 12009 in the treatment of malignant glioma.

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Targeting SF/HGF and C-Met Expression Glioma The multifunctional scatter factor/hepatocyte growth factor (SF/HGF) and its receptor c-met have been implicated in the genesis, malignant progression, and chemo/radioresistance of multiple human malignancies, including gliomas. The antitumor effects of targeting SF/HGF and c-met expression in preestablished glioma xenografts was examined by using novel chimeric U1snRNA/ribozymes (42). Treatment of animals bearing intracranial glioma xenografts with anti-SF/HGF and anti-c-met U1snRNA/ribozymes by either intratumoral injections of adenoviruses expressing the transgenes or intravenous injections of U1snRNA/ribozyme–liposome complexes substantially inhibited tumor growth and promoted animal survival. Thus SF/HGF and/or c-met expression can be targeted in vivo to inhibit tumor growth. Histologic analysis of tumors treated with U1snRNA/ribozymes showed a significant decrease in blood vessel density, an increase in activation of the pro-apoptotic enzyme caspase-3, and an increase in tumor cell apoptosis. Antisense VEGF Therapy Saleh et al. have transfected rat C6 glioma cells with eukaryotic expression vector bearing an antisense VEGF cDNA (43). When transplanted subcutaneously into nude mice, the growth of antisense-VEGF cell lines was greatly inhibited as compared with control cells. Analysis of the tumors revealed that the reduced tumor size was due to necrosis and paucity of blood vessels. Antisense VEGF provides the basis for the development of antiangiogenic gene therapy for brain tumors. A gene therapy strategy has been used for efficiently delivering an antisense cDNA molecule of the vascular endothelial growth factor (VEGF) to glioma cells (44). The recombinant adenoviral vector Ad5CMV-␣ VEGF carried the coding sequence of wild-type VEGF165 cDNA in an antisense orientation. Infection of U-87 MG malignant glioma cells with the Ad5CMV-␣ VEGF resulted in reduction of the level of the endogenous VEGF mRNA and drastically decreased the production of the targeted secretory form of the VEGF protein. Treatment of subcutaneous human glioma tumors established in nude mice with intralesional injection of Ad5CMV-␣ VEGF inhibited tumor growth. Taken together, these findings indicate that the efficient down-regulation of the VEGF produced by tumor cells using antisense strategies has an antitumor effect in vivo. These results suggest that this system may have clinical and therapeutic utility.

Antiangiogenic Gene Therapy Gene therapy strategies developed to interfere with the normal function of vascular endothelial growth factor

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receptors have been successfully used in different experimental models to block tumor angiogenesis and to inhibit tumor growth. One of the explanations of the “bystander effect” in “suicide gene therapy” of glioblastoma multiforme with HSV-tk (thymidine kinase) is tk expression in vascular endothelial cells made mitotically active by tumor angiogenesis factors with consequent killing of these cells by acquisition of ganciclovir susceptibility leading to ischemia of the tumor mass (45). Some of the antiangiogenic gene therapies are described here briefly. Delivery of Natural Antiangiogenic Factors to Inhibit Angiogenesis Ciafre et al. have constructed a tricistronic retroviral vector encoding two inhibitors of angiogenesis expressed in mammals: the N-terminal fragment of rat prolactin (16KrPRL) and a secreted form of human platelet factor 4 (sPF4) (46). When transduced by this retroviral vector, a rat glioblastoma cell line loses its ability to promote endothelial cell locomotion, the initial step of angiogenesis, and to form an endothelial cell tube network. Despite this encouraging in vitro result, the antiangiogenic vector cannot block glioblastoma progression in animal models. These results suggest that therapeutic strategies aiming to block tumor progression through the inhibition of tumorassociated angiogenesis should not only provide large numbers of angiogenesis inhibitors, but also target the angiogenic factors produced by tumor cells. Retroviral Vector-Mediated VEGFR Inhibition Survival time of rats with intracerebral tumors was significantly prolonged in a dose-dependent manner when retroviruses carrying a VEGFR-receptor 2 mutant were cotransplanted with tumor cells (47). After implantation of tumor cells stably transfected with a truncated form of VEGFR-2, rats exhibited a rate of survival similar to that of animals treated with high numbers of virus-producing cells encoding the truncated form of VEGFR-2. Morphologically, tumors showed signs of impaired angiogenesis, such as extensive necrosis and reduced tumor vascular density. These results suggest a dual mode of function of truncated VEGFR-2, namely, dominant-negative inhibition of VEGFR-2 function and VEGF depletion by receptor binding. These results indicate that VEGFR-2 inhibition is useful for the treatment of malignant gliomas. MMI Gene Therapy Several studies have been conducted to show that in vivo gene therapy with MMIs leads to excellent prevention as well as treatment of established primary tumors and metastases in animal models (48). Various methods of gene transfer have been used, including adenoviral vectors and

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liposomes. For optimal effect a high concentration of the MMI was required at the tumor site. None of the studies were conducted in brain tumor models, but this is a promising method for adjuvant treatment of residual tumor in combination with chemotherapy. Further research is required to determine how and which MMI can exert optimal effects on malignant brain tumors. MMAC/PTEN-Based Gene Therapy for Gliomas The tumor suppressor gene mutated in multiple advanced cancers/phosphatase and tensin homolog (MMAC/PTEN) has been shown to inhibit cell migration, spreading, and focal adhesion. In one study, reintroduction of the MMAC/ PTEN gene into human glioma U251 and U87 cells modified their phenotype and growth characteristics (49). The ability of MMAC/PTEN to induce anoikis in U251 cells was accompanied by a significant inhibition of in vitro invasion (70%). Expression of MMAC/PTEN in U251 and U87 cells inhibited MMP-2 enzymatic activity as determined by zymography. Furthermore, MMAC/PTEN expression strongly decreased MMP-2 mRNA levels, which correlated well with the inhibition of invasion capacity in these cells. Concomitant with MMP-2 expression and activity, MMP-2 promoter activity was also reduced in MMAC/PTEN–expressing cells. These observations suggest that MMAC/PTEN inhibits tumor cell invasion in part by regulating MMP-2 gene transcription and thereby its enzymatic activity. Further characterization of this regulation will facilitate the development of MMAC/PTEN–based gene therapy for gliomas. Delivery of Endostatin by Gene Therapy In one study, the subcutaneous implantation of engineered C6 cells that endogenously express mouse endostatin in athymic mice reduced tumor growth rate (90% inhibition) compared with control cell lines (50). Tumor inhibition was associated with a 50% reduction in the number of vessels, which were also smaller in morphology. However, endostatin-C6 tumors were no more necrotic than control tumors. The implantation of endostatin-C6 cells into immunocompetent Wistar rat brains also resulted in reduced tumor volumes (71% inhibition) when compared with controls. These findings demonstrate that the endogenous expression of endostatin by C6 glioma cells results in a reduced tumor growth rate in vivo that is associated with an inhibition of tumor angiogenesis. These data suggest that endostatin should be developed as an adjuvant gene therapy for the effective treatment of gliomas. A recombinant adeno-associated viral (AAV) vector carrying the angiostatin gene has been used as an antiangiogenesis strategy to treat the malignant brain tumor in a C6 glioma/Wistar rat model (51). The AAV

vector has been proven to be able to deliver sustained and high-level gene expression in vivo, and therefore is well suited to such a purpose. In addition, the authors investigated the combined gene therapy of an adenoviral vector carrying the suicidal thymidine kinase gene along with the AAV-angiostatin vector. The combined therapy offered the best tumor-suppressive effects and increased long-term survival to 55% in the treated rats. This study has demonstrated the potential of using AAV as a safe and effective vector for antiangiogenic gene therapy of brain tumors.

Nonviral Vector Targeting of Endothelium A nonviral vector can deliver genes specifically to endothelial cells of angiogenic blood vessels. Integrin ␣v␤3, an endothelial cell receptor, potentiates tumor cell invasion and proliferation, and this protein also promotes internalization of several pathogenic viruses. Based on those characteristics, they synthesized a cationic polymerized lipid-based particle to which they coupled a ␣v␤3-binding ligand. Coupling this vector to a mutant Raf gene and injecting it into mice causes apoptosis of tumor-associated endothelium and sustained regression of established primary and metastatic tumors (52). After demonstrating that the particles could selectively deliver genes to angiogenic tumor-associated blood vessels, the researchers complexed particles with a mutant Raf gene that blocks endothelial signaling and angiogenesis in response to growth factors. They first targeted the gene to melanomas that intrinsically lack the ␣-v ␤-3 receptor. Three days after injecting the gene-vector particle into mice with established melanomas, microscopic examination showed apoptosis among the new blood vessels and in rings of tumor cells surrounding the affected vessels. After 6 days, there was 95% to 100% tumor regression, which was sustained for more than 250 days. These results suggest that the antitumor effect is based on the antiangiogenic effects, not a direct effect on the tumor. The lipid-based nanoparticles are not as immunogenic as viral vectors. Therefore, it may be feasible to deliver therapeutic genes repeatedly to angiogenic blood vessels for sustained treatment of diseases that depend on angiogenesis and vascular remodeling.

Miscellaneous Drugs with Antiangiogenic Properties Various drugs and other agents having a secondary antiangiogenic effect in brain tumors are listed in Table 54-6. These compounds are of interest, as some of them are approved for other indications. Some of these are used as antineoplastic agents, and antiangiogenic action was discovered later.

54. Angiogenesis in Brain Tumors

w TABLE 54-6

Agents with Secondary Antiangiogenic Effect in Brain Tumors

Doxorubicin encapsulated in liposome Interferon ␣ Nonsteroidal antiinflammatory agents: indomethacin Paclitaxel Polysulfated heparinoids: pentosan polysulfate Somatostatin Squalamine Suramin Thalidomide Thiram

Doxorubicin Long-circulating liposomes extravasate in tumors such as the rat 9L gliosarcoma and accumulate in perivascular areas. Functional MRI imaging was used for noninvasive, serial evaluation of intracranial 9L tumor responses to repetitive doses of liposome-encapsulated doxorubicin (53). The breakdown of tumor vasculature induced by the latter might have arisen from the perivascular accumulation of liposomes in tumor and cytotoxic effects on tumor vascular endothelium. Interferon ␣ Interferon ␣-2b is commercially available for the treatment of multiple sclerosis. It inhibits bFGF by downregulating its mRNA, and angiogenesis inhibition has been clearly demonstrated in the treatment of a hemangioendothelioma (54). Interferon ␣ acts synergistically with valproic acid, an antiepileptic drug with antiangiogenic effect, to inhibit cell growth of a well-established model of neuroblastoma differentiation (55). A recent phase III clinical trial showed that interferon ␣ does not appear to improve time to disease progression or overall survival in patients with high-grade glioma and appears to add significantly to toxicity (56). Suramin Suramin and its analogs are polyanionic naphthylureas with known antineoplastic action, which is considered to be due to antiangiogenic effect. This may be due to suramin’s ability to inhibit growth factor binding to their target receptors and cellular enzymes. Suramin has been used in patients with glioblastoma multiforme usually in conjunction with other treatment modalities such as radiation. Toxicity of suramin is a problem with the use of high doses required to achieve antiangiogenic effect. Therefore, use of suramin has been limited in patients with glioblastoma multiforme.

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Paclitaxel A review of published studies evaluating paclitaxel alone or in combination with other chemotherapeutic agents suggests that paclitaxel alone is not highly active against newly diagnosed or recurrent glioblastoma multiforme. However, paclitaxel inhibits angiogenesis and tumor invasion. Additional prospective trials are warranted to evaluate the efficacy of paclitaxel plus conventional cranial irradiation or stereotactic radiosurgery (57). Polysulfated Heparinoids Polysulfated heparinoids exert a selective inhibitory effect on heparin binding angiogenesis factors such as bFGF. Pentosan polysulfate, a compound of this class, is an effective inhibitor of heparin-binding growth factors in vitro and can effectively inhibit the establishment and growth of tumors in nude mice. It has been tested in clinical trials for some cancers but not for glioblastoma multiforme. It has some toxic effects and hemorrhagic complications. Thalidomide Thalidomide (N-phthalylglutamic acid amide) is a wellknown old drug. In the 1950s, thalidomide was banned as a sedative because it caused birth defects when taken during pregnancy. In these cases, thalidomide had stopped new blood growth, a process critical to humans in the first 3 months of embryonic development. Among its many new uses discovered in recent years is its effect on malignant glioma of the brain based on its antiangiogenic properties. A proprietary preparation is Thalomid (Celgene), which is approved for the treatment of cutaneous form of leprosy and is in phase II clinical trials for cancer in collaboration with the National Cancer Institute. Preliminary results showed that the tumor size was reduced by 50% in half of the patients. Results of phase II clinical trials showed that thalidomide is a well-tolerated drug that may have some activity in the treatment of recurrent glioblastoma (58,59). The FDA has designated this drug for fast-track development. Thalidomide is contraindicated in pregnant women and women capable of becoming pregnant. Even a single capsule taken by a pregnant woman can cause severe birth defects or death to an unborn baby. Other adverse drug reactions known to be associated with thalidomide therapy include peripheral neuropathy, a common, potentially severe side effect that may be irreversible; drowsiness/somnolence; dizziness/orthostatic hypotension; neutropenia; hypersensitivity reactions; and increased HIV-viral load. Thiram Thiram-tetramethylthiuram disulfide is a chelator of heavy metals. It also inhibits DNA synthesis and induces

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apoptosis in cultured bovine capillary endothelial cells, which are 10 to 60 times more sensitive than other cell types to thiram. In one study, oral administration of thiram to mice caused significant inhibition of C6 glioma tumor development (60%) and marked reduction (by a factor of three to five times) in metastatic growth of Lewis lung carcinoma (60). These data establish thiram as a potential inhibitor of angiogenesis and raise the possibility for its use as therapy in tumors in which neovascularization is involved. Squalamine Squalamine (Genaera Pharmaceuticals) belongs to a new class of naturally occurring molecules called aminosterols. It was previously shown to have antibiotic and anticancer activity but later studies showed that squalamine inhibits the growth of gliomas implanted in the flanks of rats by antiangiogenesis (61). This effect is mediated, at least in part, by blocking mitogen-induced proliferation and migration of endothelial cells, thus preventing neovascularization of the tumor. Squalamine was also found to have remarkable effects on the primitive vascular bed of the chick chorioallantoic membrane, which has striking similarities to tumor capillaries. Squalamine may thus be well suited for treatment of tumors and other diseases characterized by neovascularization in humans. Squalamine is currently being evaluated in multiple phase II clinical studies for the treatment of solid tumors. Clinical studies are ongoing in non–small-cell lung cancer, ovarian cancer, and glioblastoma multiforme.

Dietary Restriction for Antiangiogenesis in Brain Tumors Dietary restriction may shift the tumor microenvironment from a proangiogenic to an antiangiogenic state through multiple effects on the tumor cells and the tumorassociated host cells. Moderate dietary restriction of 30% to 40% significantly inhibited the intracerebral growth of the CT-2A syngeneic malignant mouse astrocytoma by almost 80% (62). These findings suggest that moderate dietary restriction may be an effective antiangiogenic therapy for recurrent malignant brain tumors.

ANTIANGIOGENIC THERAPY AGAINST CEREBRAL METASTASES The goal of antiangiogenic therapy of other organs is to markedly retard, or even prevent, the growth of metastases, particularly those to the brain. Metastases that are less than 1 mm or so in diameter may survive by diffusion of nutrients and waste without the development of their own vascular network. However, growth beyond

1 mm requires a network of blood vessels to provide vital support functions. Antiangiogenic therapy may prevent the growth of metastases. An important theoretical advantage of the antiangiogenic approach over chemotherapy is the probable nontoxic nature of antiangiogenic therapy. Although any of the antiangiogenic drugs might be used for this purpose, some have been studied noting their effect on metastases. Troponin I (Boston Life Sciences, Inc.) inhibits endothelial cell proliferation and angiogenesis, both in vivo and in vitro by blocking interaction with the cell’s VEGF and FGF receptors. It also inhibits metastasis of a wide variety of tumors in vivo. Troponin I inhibits both bFGFstimulated and basal levels of endothelial cell proliferation, and this inhibition occurs, at least in part, via an interaction of troponin with the cell-surface bFGF receptor on capillary endothelial cells (63). Troponin 1 is in a preclinical phase and is being studied for the antiangiogenic potential in cancers of various organs, including the brain, as well as for its ability to reduce metastases.

DELIVERY OF ANTIANGIOGENIC AGENTS TO THE BRAIN Various methods of delivery of antiangiogenic agents to the brain are shown in Table 54-7.

Therapies Targeting Tumor Vasculature Several innovative approaches are being developed for neovasculature-specific drug delivery to brain tumors. Novel PRP-containing peptides can home in to the phagepresented peptide containing the amino acid sequence “PRPGAPLAGSWPGTS” and found in angiogenic vessels in tumors such as glioblastoma (64). Liposome modified with APRPG peptide, isolated from a phage-displayed random peptide library, showed high accumulation in murine tumor xenografts, and APRPG-modified liposome

w TABLE 54-7

Methods of Delivery of Antiangiogenic Agents to the Brain

Systemic delivery orally or by injection and targeting to tumor vasculature Selective intraarterial injection into the arteries supplying the tumor Injection into the cerebral ventricles Injection or deposition into the tumor cavity after resection of the tumor Injection of encapsulated or coated cells releasing angiostatic substances into the tumor Tumor-specific drug delivery by genetically engineered organisms (e.g., clostridium spores)

54. Angiogenesis in Brain Tumors

encapsulating adriamycin effectively suppressed experimental tumor growth. DPP-CNDAC (dipalmitoylphosphatidyl 2’-C-cyano-2’-deoxy-1-␤-D-arabino-pentofuranosylcytosine), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, is quite useful for cancer therapy when modified with APRPG (65).

Cell-Coated Microcarrier Technology for Brain Tumors Cell-coated microcarrier technology (Titan, Inc.) was shown to significantly enhance the therapeutic benefit of two different cell types for the treatment of glioblastoma multiforme, according to results of studies conducted in animal models of glioma. These results were presented on 28 May 2002 at the International Society for Cellular Therapy meeting held in Barcelona, Spain. Both studies confirmed the ability of cell-coated microcarrier technology to greatly improve cell survival and function after implantation to the brain, thereby paving the way for potential new cell therapy approaches to treating this deadly cancer. In one study, endostatin-producing cells attached to microcarriers and delivered to the brain reduced tumor size and significantly improved survival. In a four-arm controlled study, animals with glioma were treated with either endostatin cells attached to microcarriers, endostatin cells alone, nonendostatin cells alone, or control saline solution. Results showed that the increase in overall survival of animals treated with endostatin-producing cells on microcarriers was statistically significant as compared with the control groups. In addition, tumor volume was reduced in treated animals. Necrosis (tumor cell death) was also observed in the active arm, and release of endostatin was detected both in the tumor core and at a distance from injection site. Blood vessel distribution showed fewer vessels in treated tumors as compared with controls, indicating the inhibition of angiogenesis.

Encapsulated Cells for Brain Tumors This strategy involves the use of genetically engineered producer cells that secrete a protein with therapeutic potential. The cells are encapsulated in an immunoisolating material that makes them suitable for transplantation. The capsules, or bioreactors, permit the release of recombinant proteins that may assert their effects in the tumor microenvironment. The polysaccharide alginate is the most commonly used material for cell encapsulation and is well tolerated by various tissues. A wide spectrum of cells and tissues has been encapsulated and implanted, both in animals and in humans, indicating the general applicability of this approach for both research and medical purposes, including brain tumors. To provide local and sustained drug delivery, the bioreactors can be implanted in the brain parenchyma or in the ventricular system (66).

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Scientists at the University of Bergen, Norway, are using this approach to genetically modified Chinese hamster ovary (CHO) cells to overproduce endostatin (inhibitor of tumor vasculature) and then encapsulate them in a matrix of alginate. Sealing the endostatin-producing cells in the inert matrix protects them from immune attack. In experiments on rats, the CHO-alginate beads and a quantity of glioma cells were injected simultaneously into the brain, so that the two were half a millimeter apart. After 6 weeks, histologic examination of brains taken from some of the rats showed that the tumor cells were indeed dying, both around the beads and at distant sites throughout the tumor. They are currently involved in experiments to see if angiostatin, another antiangiogenic agent, works in the same way.

Clostridium Spores for Tumor-Specific Drug Delivery Hypoxia is a unique feature of cancer that is exploited by the use of nonpathogenic clostridia to deliver toxic agents to the tumor cells. Following systemic administration, these strictly anaerobic, Gram-positive, spore-forming bacteria selectively colonize hypoxic/necrotic areas within the tumor. Moreover, they can be genetically modified to secrete therapeutic proteins like cytosine deaminase or tumor necrosis factor-␣ (67). This method can also be combined with radiotherapy.

CONCLUSIONS AND FUTURE PROSPECTS Although no angiogenesis inhibitor has entered the market yet, a few are expected to do so by the year 2005. The role of this therapy in the overall management of the cancer patient, and specifically of the patients with malignant brain tumors, remains to be defined. One approach is to consider a tumor as containing two cell compartments that interact with each other: the endothelial cell compartment and the tumor cell compartment. For optimal anticancer therapy, each compartment should be treated by drugs that selectively target each cell type. The mutational rate in the endothelial cell compartment is lower than that in the tumor cell compartment. Thus, it may be possible to employ antiangiogenic therapy for the long term, either alone or together with conventional chemotherapy, and subsequently in the postchemotherapy period. Although antiangiogenic therapy alone can suppress the growth of established tumors, it can also potentiate the effects of radiation and chemotherapy. Because the latter treatments depend on adequate blood flow to the tumor to deliver oxygen and drugs, respectively, antiangiogenic therapy to reduce the tumor blood supply would interfere with delivery. It is recommended

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that the abnormal vasculature be pruned by antiangiogenic treatment to normalize the circulation for adjunctive treatment (68). Antiangiogenic therapy should be given during the angiogenic phase of glioblastoma multiforme. There is an avascular phase during the growth of glioblastoma multiforme. This might explain why some antiangiogenic compounds have no effect on avascular phase of the growth of the tumor. Antiangiogenic therapy still has many drawbacks. The production of antiangiogenic agents as functional recombinant proteins has been proven to be difficult. In addition, high dosages of these inhibitors are required to suppress tumor growth in animal studies. Other disadvantages of the antiangiogenic protein therapy include repeated injections, prolonged treatment, transmission of toxins and infectious particles, and high cost for manufacturing large amounts of protein molecules. Thus, alternative strategies need to be developed to improve the clinical settings of antiangiogenic therapy. It is still unclear what percentage of tumor-induced angiogenesis must be blocked before tumor growth is inhibited, nor is it known if endothelial cells can become “resistant” to angiogenesis inhibitors. Developments of these strategies are ongoing and include identification of more potent inhibitors, antiangiogenic gene therapy, improvement of protein/compound half-lives in the circulation, and increase of their concentrations at the disease location (69). In the ongoing clinical trials, results have been impressive, and some patients have shown tumor regression. However, some recent studies question the utility of native endostatin for controlling human lineage acute lymphoblastic leukemia in an animal model despite effective delivery by gene therapy and several-fold increase in endostatin serum levels in transplanted recipients (70). Another study of endostatin, in a human neuroblastoma xenograft model in nude mice, showed that tumor growth was only slowed down in endostatin-treated mice when compared with control mice, and no statistically significant difference in serum levels of endostatin was observed between endostatin-treated and control groups (71). Endostatin will not be accepted as mainstream cancer therapy until studies have clarified its mechanisms and resolved the inconsistencies in results. Nevertheless, the great therapeutic potential of these agents has yet to be realized, which could, in part, be because the traditional strategies that are used in clinical trials for anticancer therapies are not appropriate for assessing the efficacy of agents that modulate angiogenesis. Better methods are needed for monitoring the biological activity of angiogenic modulators and innovative approaches to trial design that might facilitate the integration of these agents into anticancer therapy (72). Evaluation of antiangiogenic treatments for brain tumors would require different and more stringent protocols than those for some other cancers.

For cure of glioblastoma multiforme, there is another, more stringent requirement—complete eradication— otherwise, recurrence is inevitable. Antiangiogenic therapy could prevent the growth of metastatic lesions in the brain if they are detected early and treated aggressively in combination with radiotherapy and/or chemotherapy along with eradication of the primary lesion. Detection of gliomas at a stage when they are 1 mm3 is not yet possible. At the time of diagnosis, most tumors are much larger, with both vascular and tumor cell components. Even if antiangiogenic therapy could eradicate the vascular component, adjunctive therapies are needed that could destroy the tumor cell component completely. There is a dire need to develop such therapies.

REFERENCES 1. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182–1186. 2. Jain RK, Schlenger K, Hockel M, et al. Quantitative angiogenesis assays: progress and problems. Nat Med 1997;3:1203–1208. 3. Xu ZP, Tsuji T, Riordan JF, et al. The nuclear function of angiogenin in endothelial cells is related to rRNA production. Biochem Biophys Res Commun 2002;294:287–292. 4. Yamada SM, Yamaguchi F, Brown R, et al. Suppression of glioblastoma cell growth following antisense oligonucleotidemediated inhibition of fibroblast growth factor receptor expression. Glia 1999;28:66–76. 5. Gossmann A, Helbich TH, Kuriyama N, et al. Dynamic contrast-enhanced magnetic resonance imaging as a surrogate marker of tumor response to anti-angiogenic therapy in a xenograft model of glioblastoma multiforme. J Magn Reson Imaging 2002;15:233–240. 6. Debinski W, Slagle-Webb B, Achen MG, et al. VEGF-D is an X-linked/AP-1 regulated putative onco-angiogen in human glioblastoma multiforme. Mol Med 2001;7:598–608. 7. Gollmer JC, Ladoux A, Gioanni J, et al. Expression of vascular endothelial growth factor-b in human astrocytoma. Neuro-oncol 2000;2:80–86. 8. Zagzag D, Capo V. Angiogenesis in the central nervous system: a role for vascular endothelial growth factor/vascular permeability factor and tenascin-C. Common molecular effectors in cerebral neoplastic and non-neoplastic “angiogenic diseases.” Histol Histopathol 2002;17:301–321. 9. Lee P, Goishi K, Davidson AJ, et al. Neuropilin-1 is required for vascular development and is a mediator of VEGFdependent angiogenesis in zebrafish. Proc Natl Acad Sci U S A 2002;99:10470–10475. 10. Yano H, Hara A, Murase S, et al. Expression of hepatocyte growth factor and matrix metalloproteinase-2 in human glioma. Brain Tumor Pathol 2001;18:7–12. 11. Welch WC, Kornblith PL, Michalopoulos GK, et al. Hepatocyte growth factor (HGF) and receptor (c-met) in normal and malignant astrocytic cells. Anticancer Res 1999;19:1635–1640. 12. Brat DJ, Castellano-Sanchez A, Kaur B, et al. Genetic and biologic progression in astrocytomas and their relation to angiogenic dysregulation. Adv Anat Pathol 2002;9:24–36. 13. Reynolds LE, Wyder L, Lively JC, et al. Enhanced pathological angiogenesis in mice lacking 3 integrin or 3 and 5 integrins. Nat Med 2001;8:27–34. 14. Chekenya M, Hjelstuen M, Enger PO, et al. NG2 proteoglycan promotes angiogenesis-dependent tumor growth in CNS by sequestering angiostatin. FASEB J 2002;16:586–8. 15. Fillmore HL, VanMeter TE, Broaddus WC. Membrane-type matrix metalloproteinases (MT-MMPs): expression and function during glioma invasion. J Neurooncol 2001;53:187–202.

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w Invasion in Brain Tumors Katharine J. Drummond Invasion—the passage of motile cells through a tissue—is a normal physiologic process, vital to embryogenesis, morphogenesis, wound healing, and inflammation. However, invasion may be inappropriate or uncontrolled, such as occurs in the destructive invasion of normal parenchyma by malignant cells, or the invasion of normal organs by inflammatory cells in autoimmune disease. Invasion may also be inadequate (such as the failure of many tumors to be invaded by immune effector cells). Thus, an understanding of the features and mechanisms of invasion has become important in many clinical and basic science fields. The invasive nature of gliomas and other brain tumors has become an area of intense interest. Diffuse brain invasion is the property of gliomas that most prevents their effective primary treatment. Invading cells that escape treatments are the nidus for local and distant tumor recurrence. From the early 20th century, patterns of invasion in central nervous system (CNS) tumors have been recognized and raised many questions. n

n

How is the capacity for invasive growth related to histologic malignancy? Benign gliomas can infiltrate widely through normal brain (1,2). Benign meningiomas can invade through dura, bone, and scalp, and even metastasize, but only malignant meningiomas will invade the brain (3–5). What is the pathophysiologic basis of these patterns of invasive growth?

This chapter will describe the histopathologic, anatomic, and radiologic features of brain tumor invasion and its clinical consequences. The pathophysiologic mechanisms for invasive growth will be reviewed as well as the models available to investigate them. Finally, the growing interest in therapies for brain tumor invasion will be discussed. Although all types of CNS tumors have some invasive characteristics, the following discussion will largely center on gliomas, and in particular benign and malignant astrocytoma, as these are the lesions in which invasive growth is the greatest clinical problem.

HISTOPATHOLOGIC AND ANATOMIC FEATURES OF INVASION Studies looking at the entire brain of patients with untreated gliomas have given the best overview of the histologic appearance of invasion (6,7). Extensive histology has shown gliomas to be variable in geometry, extent, and in the characteristics of the peripheral invading margin. These studies also noted that the detection of single invading tumor cells with routine histologic examination is inadequate due to morphologic characteristics similar to those of normal or reactive astrocytes (8,9). Thus, even in these careful examinations, the true extent of the tumor may be underestimated. In general terms, gliomas consist of a central region of necrosis, a cellular rim of tumor, and a peripheral zone of infiltrating cells. Infiltration may be into normal brain, or into a region of low-grade tumor, as a significant proportion of malignant tumors arise within low-grade tumors (7). Only a small proportion of tumors grow entirely as a solid mass and these are almost always pilocytic astrocytomas (9). The infiltrative region may extend only a short distance from the macroscopic tumor margins and consist of an intensely cellular zone. This can be seen in gliomas of all grades but is particularly common in oligodendrogliomas, making them somewhat more surgically amenable. The infiltrative zone may also be limited by certain CNS structures; the gray-white junction, the ventricular ependyma, the pia, or tightly packed myelinated white matter traveling perpendicular or tangentially to the direction of the tumor expansion (see Fig. 55-2A). The infiltrative zone may also be more diffuse; extending widely beyond the macroscopic tumor borders. Invading tumor utilizes many pathways and substrates for invasion, including neurons and their processes, white matter and the basement membranes (BM) of blood vessels, ependyma, and the pia. Further proliferation of these cells leads to smaller tumor deposits, known as “secondary structures of Scherer” (7). These structures precede the zone of massive infiltration and, thus, the distinction between local metastasis and true multicentric tumor 779

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growth may be difficult or impossible to make. In up to 25% of gliomas, particularly low-grade tumors and those in the brainstem, but also occasionally in malignant glioma, there is no tumor mass or destruction of normal tissue, and the entire tumor consists of an infiltrative zone. Widespread invasion is not necessarily associated with destruction of normal brain or loss of function (7,9). Invasion into the meninges and skull is uncommon. Invasion into the ventricles commonly occurs in a circumscribed fashion where nodules of the tumor expand spatially into the ventricular cavity but are still covered by ependyma. Diffuse intraventricular invasion, carpeting or filling of the ventricles, and distant ventricular metastases are uncommon (less than 10% of patients) and often a late event (7,8). In addition to characteristic histopathologic features of invasion, gliomas have characteristic anatomic patterns of invasion. Most commonly, spread is via white matter fiber pathways, including the corpus callosum, fornix, optic radiation, associational fibers, anterior and posterior commissures, and the cerebral peduncles. Tumors arising close to these fiber tracts tend to invade more rapidly and widely than those that are distant to such a conduit or bordered by limiting structures (8). Importantly, these studies of whole brains showed heterogeneity of the appearance and extent of invasion. A single tumor may have different appearances at different borders and gliomas of comparable histologic features may show quite different patterns of invasive growth (7). This may be due to seemingly minor regional anatomic or biochemical differences in the brain, or biologic differences in the tumor not detected by routine histology.

RADIOLOGIC FEATURES OF INVASION Over the last few decades, radiologic detection and characterization of tumors of the CNS has made rapid advances. The detection of invading brain tumor cells with accuracy is however, still not possible. Comparison of computed tomography (CT) scans and stereotactic biopsy of malignant glioma (6,10) showed that tumor was present well beyond the contrast-enhancing tumor rim and the lowdensity zone of peritumoral edema. In fact, even densely cellular regions of tumor extension could sometimes be missed on CT scanning, making it a poor tool to determine the true extent of tumor. With the advent of magnetic resonance imaging (MRI), the radiologic abnormality seen for a given tumor was much larger in comparison with the CT scan of the same patient. However, even with this more accurate imaging modality, invading tumor cells were found to extend at least to the edges of the abnormality seen on T2-weighted images and in some cases beyond. In low-grade lesions, tumor tissue may not be distinguishable from normal

brain on MRI or CT (10). Imaging studies also confirmed the previously noted common pathways of invasion along white matter tracts (11).

CLINICAL CONSEQUENCES OF BRAIN TUMOR INVASION The clinical picture of the patient presenting with a brain tumor is variable. Brain tumor invasion is not necessarily associated with neurologic abnormalities. Widely infiltrated brain can function normally, and this is particularly seen in patients with low-grade gliomas. Rapidly expanding, infiltrating, destructive growth is more often the cause of neurologic abnormalities, the specific nature of which is determined by the position of the lesion. As described above, brain tumors have typical patterns and pathways of invasion that give rise to regional differences within the tumor and have different clinical sequelae. The central necrotic and cellular region of the tumor and the intensely cellular infiltrative edge is associated with the rapid destruction and replacement of brain by tumor tissue and neovasculature. The blood–brain barrier is disrupted and there is cerebral edema. Clinically, this region relates to symptoms of raised intracranial pressure and neurologic deficit caused by pressure on, or destruction of, eloquent brain. Further from the main tumor mass, in the diffusely infiltrating zone, single cells invade the surrounding brain, which often remains intact and functioning. These cells are usually not proliferating and the blood–brain barrier around them is intact. Clinically, the immediate effect of these invading cells is not clear, but if in total they contribute a large volume, raised intracranial pressure may occur. In addition, they are not amenable to conventional therapies and thus represent residual disease after primary treatment and are the cause of disease recurrence. Surgical excision or high-dose radiotherapy cannot extend to include all these cells without extensive brain damage. Even with basic levels of histologic detection, 30% of all gliomas are bilateral when subjected to microscopic examination of both hemispheres (7). Radiotherapy and many chemotherapeutic agents act on rapidly dividing cells and thus are ineffective, as are drugs that do not cross the intact blood–brain barrier. Antiangiogenic therapy will not be effective unless these cells later proliferate into a tumor mass of critical size.

PATHOLOGIC AND PHYSIOLOGIC MECHANISMS OF INVASION Regardless of the anatomical or pathologic type of invasion, the same basic physiologic mechanisms are involved (12–14).

55. Invasion in Brain Tumors

n n n

n

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The tumor cell detaches from the main tumor mass with loss of cell-to-cell adhesion. The tumor cell adheres to the local extracellular matrix (ECM) via specific cell surface receptors. The anchored tumor cell produces proteases to degrade the local ECM and its attachments to it (or induces host cells to do so). Cell adhesion molecules and intracellular processes such as restructuring of the actin cytoskeleton and extension of cytoplasmic processes produce motility in a process of continuous adhesion-movement-detachment. Continuous invasion is a cyclic repetition of these steps. Environmental factors (such as local cytokines and anatomic structures) influence the invasive tumor behavior.

The original description of the mechanisms of invasion by Liotta was based on observation of metastatic melanoma (12) but applies to all types of tumor invasion. This early report also noted that the invasive and metastatic potential of cells within a tumor or cell line population was not uniform. Individual variations in cellular expression of receptors, proteolytic enzymes, and other factors determined the ability of particular cells to move on or through different substrates. This has subsequently been confirmed by subselection of cells with an enduring in vitro and in vivo invasive phenotype greater than that of the parent population from tumors and cell lines (15). The patterns of invasion described in the previous sections demonstrate that gliomas utilize multiple substrates to invade both on and through. This will involve many different cell–cell and cell–matrix interactions. It is important to note that each of these interactions is a normal physiologic process that has become abnormal in either degree or timing, as is the case with much uncontrolled growth of tumor cells. Thus, treatment aimed at control or cessation of tumor invasion will potentially also affect physiologic processes such as tissue repair and immune function, which are dependent on invasion, just as treatments aimed at halting uncontrolled cell division also affect normal hematopoiesis and regeneration of the gut lining.

MODELS OF INVASION In Vitro Models Although there has been some correlation shown between in vitro invasiveness and clinical outcome (16), translation of in vitro findings to effective therapies has been slow. Assays to study invasion in vitro may be two- or threedimensional. Two-dimensional assays test either adhesion or migration of cells on specific substrates in culture, with or without the presence of stimulatory or inhibitory factors. The radial migration assay is commonly used. The cells of interest are plated as either a monolayer or spheroid (Fig. 55-1) in the center of a well coated with an

FIGURE 55-1. Multicellular tumor spheroids have many applications for in vitro and in vivo invasion assays. They more closely resemble the three-dimensional structure of tumor tissue than cell monolayers or suspensions and produce their own extracellular matrix and cell-cell interactions.

ECM component. Thus, adhesion to the substrate and migration away from a central point can be assessed and compared under different conditions (17–20). Three-dimensional assays test the invasion of cells of interest through various matrix or tissue preparations. The Boyden chamber assay is a well-characterized method for studying the movement of cells through ECM (15,21,22). Cells are placed in culture in an upper chamber that is separated from a lower chamber by a membrane with 8-␮mdiameter pores. The membrane may be coated with an ECM preparation and the lower chamber is filled with culture medium with or without a chemoattractant. At a defined time, cells that have actively moved through the membrane pores are counted. Movement through an uncoated membrane measures cell migration; movement through an ECM-coated membrane tests invasion through that matrix. Many models have been developed to more closely model the brain environment for study of glioma invasion than those described above. In “confrontation” or coculture assays, the cells to be assessed for invasion are prepared as either spheroids (Fig. 55-1) or a monolayer and are “confronted⬘” with tissue or another spheroid in spatial proximity to assess invasion of the tumor cells into the tissue or spheroid. A piece of tissue such as embryonic chick heart is commonly used as an invasion substrate, as are fetal brain cell aggregates, which form spheroids with multiple elements of brain tissue, including the ECM and neuronal and glial elements (16,23–27). Brain slices in organ culture (28,29) and three-dimensional gels composed of ECM components of brain (30,31) have also been

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used to more closely mimic the normal environment of glioma invasion.

In Vivo Models Despite the utility, simplicity, and economy of in vitro models of invasion, they do not simulate the movement of tumor cells through intact brain in a host with an intact immune system. The brain is anatomically complex, with intermingled structures and regions of varied embryological origin and biochemical composition not mimicked by current in vitro models. Similarly, the host immune response is an important aspect of tumor biology that is absent in these models. Thus, in vivo brain tumor models are needed despite the additional complexity and expense involved. No model is perfect, but a thorough understanding of the features of each may allow an appropriate model to be chosen for each clinical question. Transplantable cell lines implanted into rodents are the most common animal models of glioma, particularly those using carcinogen-induced cell lines implanted into the syngeneic host (32–44) (Fig. 55-2A). These models are convenient and well characterized in terms of growth and survival, but the single-step process of tumorigenesis, using nitrosureas or transforming viruses, does not mimic

the multistep process of human tumorigenesis (36,37). In a few cases, the cell lines are derived from spontaneously arising rodent tumors (45–49) (Fig. 55-2B). The cytopathologic and histopathologic characteristics of the cell line may be similar to human gliomas but the cells are rarely as invasive (Fig. 55-2A, B) and the cell of origin is not always clear. Although the original tumors may have been very invasive, and even metastasized, the subsequent selection and serial culture of transplantable cell lines leads to changes in the tumor characteristics, including degree of invasiveness (41). These models have a poor history for predicting human response to therapy (18,41,50). They do, however, have the advantage of being syngeneic in their immunocompetent hosts and relatively cheap. Thus, they have provided the best, most affordable means of investigating glioma biology and testing new therapies for many years. Even though these models have been used to study astrocytoma invasion, the actual degree of invasive growth is variable and they often display a minimally invasive phenotype. They commonly grow as a discrete mass with little extension into the surrounding brain. C6 has been widely used but is not truly syngeneic and generally appears minimally invasive, although some reports have suggested that single cells invade the surrounding brain (Fig. 55-2A)

FIGURE 55-2. Many transplantable cell lines have some invasive features but do not closely mimic human gliomas. A. Tumor (T) formed by the C6 cell line in the brain of a Fisher rat is seen. The tumor stands out darkly due to staining for the LacZ reporter gene product, ␤- galactosidase. Some characteristics of human gliomas are seen, including poor invasion into dense white matter structures traveling perpendicular to the tumor [in this case, the corpus callosum (CC)], but the invasive pattern is largely perivascular rather than the single-cell invasion characteristic of human tumors. The arrows show blood vessels outlined by invading tumor. B. A subclone of the spontaneous murine astrocytoma cell line SMA-560 is seen to grow with a defined expansile border in normal nude mouse brain (white arrow) or to invade as defined finger-like tumor projections (black arrow), not as single cells.

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(32–34,36,37) as there are for BT4Cn (38,39,51) and RT-2 (36,43,44). RG-2, D74, and F98 are also sometimes said to be invasive, but the evidence is scant (36,41,42). The 9L/T9 rat gliosarcoma is immunogenic (52) and poorly invasive into brain, but exhibits florid perivascular spread (37,40,53). Similarly, the CNS-1 glioma shows mostly perivascular and leptomeningeal spread (36,40). There are several models in which rodent tumors have been modified in an effort to increase invasive growth with some success. Strategies include overexpression of the ECM component BEHAB/brevican (54,55), overexpression of growth factors such as scatter factor (SF) (56) or transforming growth factor ␤1 (TGF-␤1) (57,58), and irradiation of the cell line prior to implantation (19). Although it is advantageous to use an immunocompetent animal to study tumor biology, the immune response adds an additional layer of complexity to the interpretation of results. Also, to study human cells in vivo, an immunocompetent host is not appropriate. Therefore, many glioma models have been based on the growth of human cell lines or direct tumor explants in immunosuppressed hosts. These cells are likely to be genetically and biologically more similar to spontaneous human tumors than to carcinogen-induced rodent tumors. However, many pathologic processes, including tumor invasion, are influenced by host–tumor interactions; therefore, the use of an immunodeficient host, either a genetically immunodeficient host or one immunosuppressed with drugs, is also a disadvantage. Similar to the rodent cell lines described above, most cell lines derived from human tumors do not have an invasive growth pattern in immunosuppressed animals and grow mostly as a discrete mass. However, in some cases, human cell lines do seem to replicate human tumor invasion. The GL15 line derived from a human glioblastoma multiforme demonstrates single-cell brain invasion distant to the tumor mass and across the corpus callosum, as well as perivascular spread when grown in immunosuppressed rats (59). A similarly derived line, SNB19, also shows invasive growth in athymic mice (60,61). Many other human cell lines have been reported to show some degree of invasive growth (17,62), but the histopathologic evidence is not as strong. Direct human astrocytoma explants, prepared as either cell suspensions or as tumor spheroids, can also show an invasive growth pattern in immunodeficient animals (63,64). These models overcome the disadvantages of using cells that have been in culture for long periods (65) and their invasive phenotype can closely mimic human tumors, but they are less convenient than established cell lines and less reproducible due to the variability of the cells implanted. The implantation of tumor cells via an intracerebral injection is an artificial construct for tumorigenesis on which all the models described above is based. A large number of tumor cells are introduced at one time, the

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blood–brain barrier is breached and there is an intracerebral wound to which the brain responds. The disease course is short (weeks), with only a brief window of opportunity for therapy. This does not closely replicate human tumorigenesis. However, until recently there was no viable alternative, as spontaneously arising tumors in animals were rare. More recently, genetically engineered mouse models with both loss and gain of genetic material, including the epidermal growth factor receptor (EGFR) and its signaling pathway p21-ras and cell cycle–regulating genes such as the INK4a-ARF locus, have been developed that closely mimic human tumors in terms of genetic defects, cell of origin, histopathologic characteristics, and invasive phenotype. It has been suggested that these models will have a higher success of predicting efficacy of therapies (66–69). The current deficiencies of these models are their expense and the fact that the tumor dynamics are not as well defined or predictable as the implantation models. However, this technology is developing rapidly, and genetically engineered models are likely to soon become the gold standard for glioma biology.

Tumor Cell Markers To accurately assess invasion in an in vivo glioma model it is necessary to identify individual tumor cells. While many models have relied on standard histochemical stains and differences in morphologic characteristics to assess invasiveness, this is suboptimal, as tumor cells may be difficult to identify with certainty. In particular, single invading tumor cells can resemble normal or reactive astrocytes and are admixed with them (8,63). No definitive tumor-associated antigen, not expressed by surrounding brain cells, has been identified although many have been suggested including integrins, proteases, and ECM components associated with invasion (29,65,70–78). A number of methods have been developed to aid identification of tumor cells in experimental models. The introduction of foreign genes with products that can be easily and enduringly detected is an excellent method to identify cells of interest. In particular, introduction of the Escherichia coli LacZ reporter gene (Fig. 55-2A) or jellyfish green fluorescent protein has been done to identify intracerebral tumor cells (52,60,79–81). In vitro or in immunodeficient animals, this is an effective method. However, in immunocompetent animals, the xenoprotein may act as an additional tumor antigen and enhance immune-mediated killing of the tumor (52,82,83). Thus, the growth and invasion profile may be changed or the cell line may not produce tumors at all. In addition, subselection of cells expressing the xenoprotein is necessary and may change the cell characteristics. Fluorescent dyes, incorporated directly into the tumor cell membrane or cytoplasm by preimplantation incubation, have also been used to assess invasion. The carbocyanine

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vital dyes, DiI or DiO, PKH-67 and fast blue, are some of the more commonly used (23,29,32,62). The dye is evenly distributed to daughter cells with cell division. Thus, staining intensity is halved with each cell division and dilution of the dye with loss of visualization is possible; however, effective labeling up to 21 days after tumor implantation in vivo has been reported. The most important limitation of dyes for identification of single tumor cells is uptake by phagocytes resulting in false-positive cells. Where human tumors are implanted into rodents, various species-specific markers can be employed (59,84).

the invading edge of tumors in which hyaluronan expression is up-regulated (21,85, 92,93). If metastasis occurs receptors for vascular endothelial cells such as the selectins and vascular cell adhesion molecule (V-CAM) are more relevant. Clearly a finely balanced regulatory system is needed. Glioma cells express many different cell adhesion receptors, but expression varies considerably between cells both in vivo and in vitro and there are no clear, single mediators (13, 21). Adhesion molecules expressed by at least some glioma cells include the following: n

MECHANISMS AND SUBSTRATES OF INVASION Over the last three decades, much of the work on glioma invasion has focused on understanding the underlying processes, with the aim of finding mechanisms of control. The amount of information gleaned has been enormous and suggested many possibilities but has not led to tangible changes in management or prognosis. The following section outlines many of the important mechanisms and substrates of cellular invasion but is by no means complete. There have also been a number of excellent reviews on this topic (85–88). Ultimately, it is most important to understand that each tumor, and possibly each tumor cell, can utilize a number of anatomic pathways, matrix substrates, proteolytic enzymes, cell receptors, and other mechanisms and respond to different local conditions. Therapies aimed at only one aspect of the invasion process may be partly useful but are unlikely to completely halt invasion, just as antiproliferative therapies with multiple mechanisms of action or in combination are usually required.

Adhesion Molecules on the Tumor Cell Surface and Surrounding Tissue Cell adhesion molecules have a complex role in glioma invasion. Both attachment and detachment is necessary at different stages. Tumor cells must loosen their cell contacts to detach from the main tumor mass. They must then attach to an ECM substrate for motility and matrix dissolution. In addition, adhesion receptors mediate the effects of the ECM on cell morphology, proliferation, differentiation, and gene expression (89–91). Thus, both up-regulation and down-regulation of adhesion molecules for multiple ligands will be necessary. Invasion along the perivascular space requires interaction with BM components such as laminin, collagen IV, and proteoglycans. Invasion through brain tissue may require interaction with collagen I, myelin, hyaluronan, or tumorderived ECM components. CD44 (the hyaluronan receptor) mediates extensive tumor–ECM interactions at

n n n n n

Integrin family receptors Immunoglobulin superfamily receptors (N-CAM, I-CAM, V-CAM) Selectin superfamily receptors CD44 and other hyaluronan receptors Endothelial (E-) and neural (N-) cadherin Other nonintegrin receptors (e.g., elastin-binding protein)

There is considerable variability in expression of integrin receptors on glioma cells. The ␤1 subunit is fairly consistently expressed; however, other ␤ chains and multiple ␣ chains are also expressed on different lines (13). Expression of other adhesion molecules also varies. For instance, variation in expression of N-CAM on the BT4C glioma line is related to variation in invasiveness (38,51). This intra- and intertumor and cell line variability is also true of most other cancers and creates subpopulations of cells that adhere, migrate, and move differently (94). In vitro experiments aimed at blocking invasion using neutralizing antibodies to specific receptors are effective only in the subgroup of cells that express the relevant receptor (88). Integrin expression is also regulated by cytokines produced by the tumor such as TGF-␤) (95,96). Cytokines regulate integrin expression differentially, and as more then one integrin can subserve the same type of adhesion, blocking the action of that cytokine or integrin may not block adhesion or invasion (97). There is also significant redundancy among cell adhesion molecules, that is, more then one receptor for each ligand and vice versa. For example, there are a number of different integrins that can bind fibronectin, or other ECM molecules with the “RGD” binding motif. In addition to hyaluronan, CD44 binds laminin, fibronectin, vitronectin, and some collagen subtypes with varying degrees of affinity. Conversely, as there are multiple receptors for hyaluronan, inhibition of CD44 will only partially inhibit astrocytoma cell adhesion, migration, and invasion on this substrate (21,85,92). There are only a handful of integrins that are specific to a certain cell type or ligand and therefore a single function. Other nonintegrin receptors, such as elastin-binding protein, can mediate adhesion to integrin ligands in the presence of integrin inhibitors (98). Novel receptors and ligands are being also investigated, adding new layers of complexity. Thrombospondin-1 (TSP-1)

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and its receptor have been shown to enhance adhesion and motility in glioma cell lines and to activate latent TGF-␤. However, inhibition of TSP-1 binding reduces migration and invasion only in cells lines with high baseline TSP-1 production; those with low production are unaffected (99). Thus, glioma cells may use many substrates and adhesion molecules for invasion. Each cell could conceivably change the substrate used depending on the local environment and its own adhesion molecule expression profile.

correlated with invasiveness (108). uPA overexpression is associated with malignant progression in astrocytomas (88), and its high-affinity binding receptors are correlated with invasiveness (39,61). PAI-1 is overexpressed by glioblastomas and may be associated with areas of necrosis (39). It should be noted that protease expression may not correlate with activity (88) and is regulated by multiple factors including other ECM proteases and their inhibitors, TGF-␤ and other cytokines, the cytoskeleton, and growth factors (86,104,110,111).

Proteases and Protease Inhibitors Expressed by the Tumor and Surrounding Tissue

Cell Motility

The enzymes that degrade the ECM, and their inhibitors, form a complex, multilayered system, similar to that of the cell adhesion receptors. Proteolytic enzymes are part of the normal physiologic repertoire for cellular migration required for inflammation, tissue remodeling, and wound repair. These enzymes interact with other mechanisms of cellular invasion, including adhesion receptors and the ECM (100,101). Gliomas express a wide range of enzymes (and their inhibitors) that in theory could degrade the majority of ECM components, including vascular BM (31,39, 102–105). Again there is inter- and intratumor and cell line variability (85,98). Enzyme expression is not uniform throughout a tumor and tends to be highest at the tumor edge (106). ECM proteases may also be produced by cells other than the tumor cells; thus at the tumor edge, invading angiogenic endothelial cells also secrete proteases to breakdown the ECM and conceivably also influence tumor invasion (107). The proteases and inhibitors variably expressed by gliomas include the following: n n n n n

The matrix metalloproteinases (MMPs) (1–3 and 7–13) The “membrane-type” MMPs (MT-MMPs) (1–3) The serine proteases urokinase and tissue plasminogen activator (uPA and tPA) The tissue inhibitors of MMPs (TIMPs) (1–3) The plasminogen activator inhibitors (PAIs) (1–3) and proteonexin-1

MMP-2, 3, and 9, in particular, are overexpressed by astrocytomas and associated with in vitro and in vivo invasiveness and, in a few instances, with prognosis (19,88,108). The MT-MMPs are expressed on the surface of tumor cells. They can activate other MMPs (particularly MMP-2 and 9) and can function as receptors for them. This facilitates pericellular proteolysis. Expression of MT-MMP-1 in particular is associated with invasiveness (88). The TIMPs are protease inhibitors that regulate MMP function (109). Decreased expression of TIMP-1 and 2 has been

Multiple in vitro and in vivo studies have shown glioma cells to be constitutively motile on various substrates (13,20–22,98,112,113). Binding to the ECM leads to formation of filopodia and lamellipodia, essential membrane structures for migration and restructuring of the cytoskeleton. Motility factors and cytokines such as TGF-␤ modulate this process (111,114). Motility and proliferation seem to be temporally exclusive events (the “grow or go” theory) (115).

Mitogenic Growth Factors and Their Receptors Expressed by the Tumor Overexpression, amplification, or mutation of EGFR is a critical event in astrocytoma tumorigenesis. Production of EGF, or the alternative ligand TGF-␣, by the tumor completes an autocrine stimulatory loop. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, insulin-like growth factor, vascular endothelial growth factor, SF, and their receptors are also expressed by large subsets of glioma cells. There is in vitro and in vivo evidence that these factors not only cell affect growth and proliferation, but also increase cell scattering, migration, and invasion, depending on the cell type and experimental conditions (25,116–124).

Transforming Growth Factor ␤ and Its Receptors Expressed by the Tumor The three human TGF-␤ isoforms and its receptors are variably overexpressed by gliomas and cell lines (95,125,126), completing autocrine and paracrine loops. Overexpression has been related to malignancy and prognosis (125). As noted above, factors secreted by the tumor can modulate invasion. TGF-␤, in particular, is a potent modulator of the substrates of invasion, including ECM, integrin, protease, and growth factor expression (95,97,104,110,111,127). It has been strongly related to invasiveness in vitro and in vivo for both gliomas and other malignant cell types (57,58,86,97,110,128), and overexpression of TGF-␤, abnormalities of its signaling pathways, and interactions with other growth factors such as SF have been clearly linked

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to invasion in other cancers (56,104, 11,129–132). TGF-␤ also suppresses the host immune response by multiple mechanisms [reviewed in (133,134)]. Conceivably, inhibition of the immune response to invading glioma cells could also promote invasiveness.

Cytogenetic Abnormalities of the Tumor Cell A multitude of genetic abnormalities are now being described in relation to glioma tumorigenesis. Many may have as yet undetermined effects on invasiveness. For instance, phosphatase and tensin homologue deleted from chromosome ten (PTEN) is a tumor suppressor gene, frequently mutated or deleted in malignant astrocytoma, leading to uncontrolled proliferation. It is also down-regulated by TGF-␤. It has many functions, one of which is enhancement of focal adhesion kinase (FAK), mediating integrin function (135,136). The effect on invasion is unclear. A reduction in FAK activity and thus adhesion could promote invasion by mediating cell detachment from the tumor mass or reduce invasion by inhibiting ECM adhesion. In some studies strength of adhesion is related to migration (13). As noted above, the overexpression, amplification or mutation of EGFR may also promote invasion. Additionally, cellular abnormalities that favor resistance to apoptosis (such as changes in the BCL-2/BAX rheostat) seem to be related to the invasive phenotype (19,104,111). Clearly, further investigation of the effects of genetic abnormalities on invasiveness will be necessary.

Local ECM Environment As adhesion to the ECM is a critical step in tumor invasion, the composition of the ECM is of great interest. In vitro work has shown that the behavior of migrating and invading tumor cells is highly dependent on the type of ECM substrate (137). The ECM of the brain is poorly characterized but differs from that of other tissues and is regionally variable (85). In vitro, glioma cells adhere to the ECM of many brain structures, including blood vessels, arachnoid, choroid plexus, ventricular ependyma, and myelinated fiber tracts (85). The ability to adhere in vitro does not necessarily correspond to the pattern of invasion observed in vivo. The ECM of the brain parenchyma is an amorphous matrix of hyaluronan, a little collagen, and other fibrous proteins, except where mesodermally derived endothelial cells enter as vasculature. In these regions there is a welldefined BM: around vessels, at the ependymal ventricular lining, and at the glial limitans externa (85,138). It has long been thought that BM components act as a barrier to invasion (13,20,22,87,139,140) as gliomas rarely invade the leptomeninges or cross the vascular BM to metastasize to the periphery, although there are reports of both

(141–153). However, in vitro, many gliomas can invade BM components using a complex interplay of integrins and the ECM (154); thus, the relationship is not clear. Both in vitro and in vivo gliomas migrate along BM and also synthesize BM components, which could be a mechanism for migration along nonpermissive substrates or structures (155,156). White matter is more permissive to invasion than gray matter (20,157). Mathematical modeling of glioma invasion shows that tumor cells moving through white matter have a “diffusion coefficient” five times that of gray matter (157) and this may be related to differences in ECM composition as well as tissue density. Gliomas also express their own extracellular matrix, which differs from that of the normal brain. BEHAB/brevican, tenascin, and hyaluronan are all up-regulated in glioma and other tumor matrices and may be involved in providing a permissive ECM for invasion, even if the local ECM is incompatible (54,55,71,158–163). There is also evidence that the abnormal ECM found at the tumor edge is partly produced by host cells induced to do so by secreted tumor factors (62). Large, detailed investigations of dozens of cell lines, tumor explants, and many different substrates have looked at the ability of glioma cells to migrate on or invade ECM components that they might routinely encounter (13,20–22, 98,112,113). Overall, these studies show broad patterns consistent with what is seen in vivo but do not identify a single substrate or mechanism for all cells. There is significant variability for different cells, assays, ECM isoforms, and ECM combinations. Prediction of behavior is not possible from simplistic equations such as which integrins the cells express or which ECM components they can invade through or adhere to in vitro (13,88,98,137,164). Glioma-derived ECM is often an effective substrate for adhesion and invasion in many assays but is variable in composition. Thus the invasive behavior of glioma cells in vivo is most likely an interplay of tumor manipulation of the environment and local constitutive ligands associated with specific regions or structures of the brain (13,20–22,165,166).

Other Factors in the Local Environment While invasion is generally an active process, there seem to be some pathways of low resistance along which cells may move more with less effort, including the perivascular space and some white matter pathways. Injection of fluorescent microspheres into the brain will result in a few millimeters of “invasion” across the corpus callosum and more widespread distribution in the cerebrospinal fluid and perivascular spaces (62). This may be partly why edematous white matter is favored by invading glioma cells as opposed to densely cellular gray matter.

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Effects of Tumor Therapy Sublethal irradiation has been shown to induce MMP activity leading to enhanced migration and invasion of glioma cells in vitro and possibly in vivo. This was associated with up-regulation of TGF-␤ expression (19). Thus, treatment factors may also enhance the invasiveness of brain tumors.

DEVELOPMENT OF THERAPIES AGAINST INVASION The development of therapies against brain tumor invasion has become of great interest as our understanding of the mechanisms of invasion has increased. Unfortunately, understanding the pathogenesis has as not yet led to efficacious therapies. Unraveling the pieces of this complex process has not necessarily suggested simple, effective targets. Treatments targeting proteases and ECM components are commonly proposed, with limited success in some models. Strategies to halt invasion in gliomas and other cancers include inhibition of uPA receptor expression (61,102), forced overexpression of TIMP-3 (167), MMP inhibition and fragmentation (168,169), thromboxane synthase inhibition (170), inhibition of signal transduction (171), and administration of radiolabeled antibodies to ECM components (70,172,173). However, the target of each therapy is unlikely to be the only mechanism of invasion used by all cells in a tumor, and thus such therapies have shown limited efficacy. Targets with a broad role in glioma invasion, such as TGF-␤, are also being investigated (86,104,174–177). The limited nature of the models available to test therapies, as described above, also hampers translation into clinical studies. However, a question remains: will inhibition of cellular invasion in brain tumors improve prognosis? Immune effector cell invasion and subsequent immune-mediated killing of tumor cells is beneficial but inadequate in glioma and other cancers (86,120,126,133,134,178–181). Promotion of immune cell invasion could be an important advance in therapy, especially to “track down” tumor cells distant to the primary tumor mass, which are unaddressed by conventional therapy. Immune invasion may also be used to carry therapeutic agents to individual tumor cells—a potential vehicle for many novel treatments (82,182,183). The likelihood of differential control of tumor and immune cell invasion is yet to be extensively explored. There are also other considerations when proposing treatment of gliomas with “anti-invasion” therapies. First, it can be argued from studies using serial stereotactic biopsy, as well as from autopsy studies and case reports, that glioma cells will already have invaded widely at the time of diagnosis (2,8–10,32,184–186) and may even

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have metastasized (149,150,187–189). If invasion has already occurred to such an extent, anti-invasion therapy may not be useful. However, as use of MRI becomes routine, diagnosis of small tumors, with only minor radiologic abnormalities and few if any symptoms, is possible (190). Invasion of tumor away from the primary mass at such an early stage may be more limited and therefore amenable to therapy. Unfortunately, this is only speculative, as there is still no way to easily assess the extent of invasion in an individual patient at any one time. If the behavior of gliomas proves to be similar to that of other invasive tumors, such as breast cancer, extended survival may be related to early detection of limited disease. Perhaps a genuine role for a “screening brain scan” will be developed. Second, glioma recurrences generally occur locally, close to the main tumor mass and within the radiotherapy field (191–193). Although these recurrences probably arise from invading cells, they are from those that have invaded only a short distance and are likely to have already done so at the time of diagnosis. However, there is evidence that recent improvement in the control of the main tumor mass results in recurrences at sites distant to the primary site (194–196). A therapy that can limit ongoing invasion from primary and secondary tumor sites may limit the extent of more distant invasion and the volume of brain necessary for inclusion in therapies that are potentially harmful to normal brain. Third, there is emerging evidence that conventional therapy, particularly irradiation, may actually increase invasion in glioma cells that it does not kill (19) and control of the main tumor mass may encourage invading cells to proliferate and form secondary tumors (148,197). Thus, a treatment aimed against ongoing invasion, commencing at the time of diagnosis and continuing throughout other treatments, would be a significant addition to the treatment armamentarium.

CONCLUSIONS Invasion in brain tumors continues to be a significant clinical problem, precluding their effective management and resulting in a dismal prognosis that has not changed since the 1980s (198). Many advances have been made in our understanding of this complex and multifaceted process, but no simple, effective therapeutic strategies have yet come into common use. With further investigation of the mechanisms of invasion, using better tumor models, hopefully the future will bring efficacious anti-invasion therapies to add to the current treatment armamentarium for brain tumors.

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w Molecular Genetics of Brain Tumors Jianhong Zhu, Helen Li Zhang, and Eric T. Wong As rapid advances occur in brain tumor research, it is increasingly acceptable that brain tumor is a disease involving dynamic changes in the genome (1,2). However, current information about molecular genetics of brain tumors makes the complexity of this science almost beyond measure. So it is necessary to develop this sophisticated knowledge into a logical science, where the complexities of the disease will become understandable in terms of a small number of underlying principles. There are many distinct types of cancer and subtypes of tumors in brain. The vast catalog of cancer cell genotypes is a manifestation of seven essential alterations in cell physiology that collectively dictate malignant growth: deviation of cell development, perturbation of growth factor signaling pathway, unarrest of cell cycle, limitless replicative potential, evasion of apoptosis, sustained angiogenesis, and invasion. Under these principles, neuro-oncologists concentrate on applying those genetic and molecule findings into clinical practice, which includes the genetic reclassification of brain tumors (3), the assessment of prognosis, and the guidance for treatment (1,4). Furthermore, evidence about the existence of neural stem cells (NSCs) in adults brain and the resemble features between brain cancer cells and NSCs make it necessary to rethink the origin of brain tumors, some of which maybe arise from neural progenitor cells (5,6). To confirm this hypothesis, genetic models have been established, which provide unparalleled opportunities for exploring the etiology, maintenance, and treatment of brain tumors (7,8).

ESSENTIAL ALTERATIONS IN CELL BIOLOGY OF BRAIN TUMORS The barriers to development of cancer are embodied in teleology: cancer cells have defects in regulatory circuits that govern normal cell proliferation and homeostasis. In general, seven alterations, namely deviation of cell development, perturbation of growth factor signaling pathway, unarrest of cell cycle, limitless replicative potential, evasion of apoptosis, sustained angiogenesis, and invasion, are essential to progress normal cells into malignant cells.

Deviation of Cell Development In the last few years, striking parallels between cancer and normal development have emerged. Molecules originally discovered based on their role in cancer—oncogenes and tumor suppressor genes—have now been shown to function as fundamental regulators of cell growth and differentiation during development. Similarly, genes identified as regulators of pattern formation in invertebrates and vertebrates have been implicated in a variety of human cancers. In light of these findings, the field of developmental biology and tumor biology, which were distinct for many years, has begun to converge and inform each other in fascinating ways. In the context of the nervous system, the interface between development and cancer is the study of brain tumors. Brain tumors represent a kind of natural genetic screen that can provide valuable information about genes that regulate proliferation, differentiation, and death in the nervous system. On the other hand, exploring the mechanisms that control cell fate in the developing nervous system can yield important insights into the mechanisms of tumorigenesis and discover new targets for therapy. Wnt signaling and Sonic hedgehog (Shh) signaling pathways have important functions in brain development and tumor formation (9). Wnt is the homolog of the Drosophila segment polarity gene wingless, which initially was found as mouse mammary tumor virus proviral insertion site, collectively called int. Wnt proteins are a large family of signaling molecules that interact with Frizzled receptors to activate downstream pathways, which are important to regulate cell growth and cell fate determination in the nervous system (10). Many of the Wnt proteins activate gene transcription through a pathway controlled by -catenin. In the absence of pathway stimulation, -catenin protein is destabilized by a cytoplasmic complex containing the proteins Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase-3 (GSK-3). The action of this complexion is antagonized by Dishevelled. Wnt signaling thus stabilizes catenin, which acts as a transcriptional coactivator by associating with the TCF/LEF family of transcription 793

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FIGURE 56-1. A1. Wnt binding to Frizzled receptors activates Dishevelled, which in turn inhibits glycogen synthase kinase-3 (GSK-3) and results in the stabilization of -catenin. -Catenin forms nuclear complexes with TCF transcription factors, thus regulating the expression of numerous genes. A2. When Wnt is not active, -catenin is held in a complex with GSK-3, Axin, and the product of the adenomatous polyposis coli tumor suppressor gene (APC). This enables the phosphorylation of -catenin by GSK-3, targeting it for ubiquitination and degradation. In the absence of active -catenin, the TCF transcription factors associate with Groucho, a transcription inhibitor. Hence, through this signaling system, the presence or absence of Wnt can control the positive or negative influence, respectively, of TCF on gene expression. B1. In the unbound form, PTCH suppresses the activity of a second membrane associated protein, smoothened (Smo). In the absence of Smo activity, the intracellular components of the hedgehog pathway including fused (FUS), costal-2 (COS2), and Ci/Gli (Ci155), are held in a microtubule-associated complex. In this state, the full-length active form of Ci/Gli is cleaved, generating a truncated transcription repressor (Ci75). B2. Binding of sonic hedgehog (Shh) to PTCH releases suppression of Smo, permitting dissociation of the microtubule-associated complex and release of full length transcription activating Ci/Gli (Ci155) which in turn mediates the transcription of a number of genes, including PTCH itself and members of decapentaplegic and Wnt families. Loss of PTCH expression may similarly relieve Smo suppression. Signaling via this pathway is critical for normal cerebellar development, specifically cells of the external germinal cell layer.

factors (Fig. 56-1A). Activated TCF/LEF transcription factors induce transcription of c-myc and cyclin D1, which can promote cell cycle progression and thereby contribute to tumorigenesis. The Wnt signaling pathway was first implicated in medulloblastoma tumorigenesis after the identification of APC as the “target” gene mutated in a subgroup of patients with Turcot syndrome. This heterogeneous familial cancer syndrome is characterized by the co-occurrence of colonic carcinoma and a malignant brain tumor; individuals affected by the APC mutation frequently develop medulloblastoma. Although initial attempts to identify APC mutations or loss of heterozygosity (LOH) at the APC locus on chromosome 5q in sporadic tumors were unsuccessful, more recent analyses have identified Wnt

pathway alterations. Studies of -catenin mutation in medulloblastoma have focused on exon 3, the coding region containing the four potential GSK-3 phosphorylation sites. Codons 33 and 37, which encode serine residues, are potential hotspots for medulloblastoma mutation. Seven of 113 cases (6%) of sporadic medulloblastomas have been identified with these mutations, whereas APC mutations have been detected in 2 of 46 cases (4%). Moreover, APC and -catenin mutations appear to be mutually exclusive in sporadic medulloblastoma, generating an overall incidence of Wnt pathway alterations in this disease of around 13%. Interestingly, all were missense mutations in contrast to the truncating alterations normally seen in medulloblastoma patients with Turcot syndrome (11–15).

56. Molecular Genetics of Brain Tumors

Shh proteins are secreted glycoproteins that activate a membrane–receptor complex, which activates Gli zincfinger transcription factors. Shh signaling activates a receptor complex that is formed by Patched (Ptc) and Smoothened (Smo). Shh inhibits Ptc, which in turn normally inhibits Smo. Inhibition of Ptc by Shh activates the Shh pathway and results in Gli activity. Therefore, the function of Ptc is to silence the pathway in the absence of active Shh ligands (Fig. 56-1B). Shh controls the growth of the cerebellum and orchestrates the positioning and differentiation of several cell types (16–18). Shh is expressed by Purkinje cells. Components of the Shh signaling pathway—the membrane proteins Ptc1, Ptc2, and Smo and the transcription factors Gli1 and Gli2—were found to be produced in granule cell precursors in the external germinal layer. The Purkinje cell–derived signal, Shh, controls granule cell precursor proliferation, which was supported by some researches who found ablating Purkinje cells led to local failure of granule cell precursors proliferation and soluble Shh protein induces proliferation of granule cell precursors and inhibits their differentiation and migration into the internal granule layer. Normally, this signaling and patterning information is tightly regulated by endogenous factors, which are secreted by Bergman glia, and when it persists inappropriately, precursor cells might be instructed to proliferate abnormally, thereby initiating a tumor. The findings that Shh is normally expressed in Purkinje neurons of the cerebellar cortex open up a new avenue of inquiry on brain and tumor development (19). Of sporadic medulloblastomas, 10% to 20% contain mutations at the Ptch locus (20). And among mouse Ptch1 heterozygotes, 10% to 25% develop medulloblastomas (21–23). Furthermore, cyclopamine, an Shh pathway antagonist, can block proliferation of medulloblastoma cells and induce changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character (24). The Shh–Gli pathway might also be involved in gliomagenesis (25). In addition to regulating cerebellar cortical development, the Shh–Gli pathway also regulates the growth of the cerebral cortex. During late embryogenesis and postnatal development, Shh is expressed by cells in the cortical and tectal plates in a layer-specific manner. It seems to regulate the proliferation of laterdeveloping Gli1-expressing precursors in the ventricular and subventricular zones. The Shh–Gli pathway therefore regulates the growth of the dorsal brain, in contrast to its role in cell fate determination in the early ventral neural tube. Because all these neuronal and glial precursor populations in the brain seem to express the Gli genes, analyses of various primary central nervous system (CNS) tumor samples and tumor cell lines show that nearly all tumors tested—including astrocytoma,

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oligodendroglioma, glioblastoma multiforme, and primitive neuroectodermal tumors—consistently express the Gli1 and Ptc genes. Therefore, expression of Gli1 might not only be a marker of the origin of these varied brain tumors but is also likely to indicate that tumor cells harbor an active Shh–Gli pathway. Inappropriate expression of Gli in time or space rather than amplification of the gene is more important for initiating glioma formation. Experimental evidence for an involvement of Gli1 function in initiating brain tumorigenesis, possibly including glioma formation, derives from the ability of the transient somatic misexpression of human Gli1 in the developing tadpole CNS to initiate tumors (25). These tumors express platelet-derived growth factor receptor (PDGFR)—a marker of oligodendrocyte precursors and of human gliomas—and form well after the injected RNA and its protein product have been degraded, indicating that some endogenous components carry the “imprint” of the action of the injected materials. Further analyses show that endogenous Gli1 is consistently expressed in the tumors. Because inhibition of endogenous Gli1 protein synthesis by an antisense oligonucleotide led to the inhibition of tumor formation by the coinjected human Gli1 messenger RNA (mRNA), these results indicate that the maintenance of tumorigenesis, which is initiated by human Gli1, requires the continuous action of the endogenous pathway. Direct experimental evidence that Shh–Gli signaling activity contributes to the maintenance of gliomas and other brain tumors comes from the finding that cyclopamine—a plant alkaloid that selectively inhibits the activity of the Shh receptor complex—acting at the level of Smo (smoothed), inhibits the growth of several primary human gliomas as well as human medulloblastoma and glioma cell lines (26). Because NSCs and cancer cells share the ability to self-renew, it seems reasonable to propose that newly arising cancer cells appropriate the machinery for selfrenewing cell division that is normally expressed in NSCs. The researches in Wnt and Shh pathway definitely provide a new insight into a relationship between arising of cancer cells and development of NSCs.

Perturbation of Growth Factor Signaling Pathway Dependence on growth signaling is apparent when propagating normal cells in culture, which typically proliferate only when supplied with appropriate diffusible mitogenic factors and a proper substratum for their integrins. Such behavior contrasts sharply with that of tumor cells, which invariably show a greatly reduced dependence on exogenous growth stimulation. Tumor cells generate many of their own growth signals, thereby reducing their dependence on stimulation from their normal tissue microenvironment.

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At least three molecular strategies for achieving autonomy are adopted by brain tumors. The first strategy is autocrine stimulation of growth factor. Many cancer cells acquire the ability to synthesize growth factors to which they are responsive, creating a positive feedback–signaling loop. Second, growth factor receptor overexpression may enable the cancer cell to become hyperresponsive to ambient levels of growth factor that normally would not trigger proliferation. And finally, ligand-independent signaling can be achieved through overexpression and structural alteration of growth factor receptors. Many well-characterized growth factors and their receptors are actually involved in the formation of brain tumors in these three manners. These factors include PDGFs A and B, epidermal growth factors (EGFs), transforming growth factors (TGFs)-, acidic and basic fibroblast growth factors (FGFs), insulin-like growth factors (IGFs) I and II, and their cognate receptors (2,27). PDGFA and PDGFB binding to their receptors PDGFR- and PDGFR- induce the PDGFR dimers -, -, and -. The factors and the receptors are expressed widely in gliomas, meningiomas, ependymomas, and neuroblastomas (27). Reactivation of expression of the normal developmentally regulated genes appears to be the cause of overexpression in most cases because amplification and rearrangements occur in only 10% to 20% of gliomas (28); however, there may be cell type specificity because amplification of PDGFR- appears to be restricted to oligodendrogliomas with anaplastic features (29). The induction of PDGF expression appears to be a sufficient and early event in tumorigenesis; the injection of a retrovirus coding the PDGFB chain into mouse brains induced tumors with features similar to glioblastoma multiforme (GBM) or primitive neuroectodermal tumor (30). These tumor cells expressed not only the PDGFB chain but also increased PDGFR-. The resultant autocrine stimulation could be specifically inhibited in vitro (31). Such experiments show the significant contributions to tumorigenesis can arise from the change in the expression of a single growth factor. The acidic and basic FGFs (aFGF, bFGF) belong to a class of heparin-binding factors that bind to isoforms of their cognate receptors FGFR1 and FGFR2 (27). Increases in aFGF and bFGF or FGFRs have been reported in astrocytoma, meningioma, schwannoma, and neuroblastoma. Coexpression of FGFs and their receptors contributes to autocrine stimulation. The mitogenic effects may be the result of overexpression of normal proteins; however, studies on glioblastomas have shown that the -exon of FGFR1 was alternatively spliced out of the transcript, resulting in a high-affinity receptor believed to contribute to glial malignancy (32). Thus, changes in either the growth factor levels or changes to the receptor can have significant impact on tumor growth. IGF-I and IGF-II are endocrine factors that circulate in the bloodstream and bind to their cognate receptors

(IGF-IR), a tyrosine kinase receptor, and IGF-IIR, the mannose 6-phosphate receptor (27). The IGFs also bind to IGF binding proteins (IGFBP), thought to modulate IGF function. IGF-I, IGF-II, or IGFIR are increased in glioma, meningioma, schwannoma, neuroblastoma, ependymoma, and medulloblastoma. IGFBP1 and IGFBP2 are increased in gliomas and meningiomas, and expression of IGFBP2 is increased with gliomas grades (33–35). In medulloblastomas, studies suggested that the lack of Ptc promotes enhanced IGF-II expression and that this overexpression was indispensable for tumor formation (36). Ligand binding of a member of the EGF family of growth factors (EGF, TGF-, HB-EGF, and NRGs) to an EGF receptor (EGFR/c-ErbB1–4) leads to receptor dimerization (27). The pleiotropic effects of EGFR activation are due to the specificity and affinities of the various ligands for the various receptors (37). In brain tumors, the ligands TGF- and EGF contribute significantly to the autocrine stimulation of EGFR. TGF- is increased in gliomas, meningiomas, and neuroblastomas (28). EGF and EGFR are increased in meningiomas and gliomas; however, the EGF receptor is amplified with rearrangements or mutations in approximately 40% to 50% of all GBMs (presumably de novo GBMs) and in low-grade and high-grade oligodendrogliomas. In a study of 44 GBMs, 17 tumors had amplified EGFR with alterations (38). The most common mutation in deletion of exons 2 to 7 results in a truncated receptors lacking amino acids 6 through 273 (called EGFRvIII), which is constitutively tyrosine phosphorylated despite the lack of ligand binding (Fig. 56-2). The EGFRvIII proteins detected in 60% of GBMs and 20% of anaplastic astrocytomas. EGFR gene amplification has been associated with a poor prognoses for patients with GBM, and patients with EGFRvIII-expressing GBMs have shorter life expectancies. EGFRvIII confers enhanced tumorigenicity in vivo not only by increasing proliferation through constitutively activating Ras-MAPK pathway, but also by evading cell death through up-regulating expression of the antiapoptotic protein, Bcl-XL, which is activated by PLC-JAK pathway (39,40). Other EGFR mutations also occur in gliomas. Therefore, multiple, functional EGFR mutants may contribute to the ability of those gliomas to evade multimodal therapies and greatly increases the complexity in designing therapeutic targeting based on EGFR stimulation (38). In addition to growth factors and growth factor receptors alterations, signaling transducers are also involved in the formation of brain tumors. When the growth factors bind to their receptors, they induce receptor dimerization. This pairing activates the kinase domain of both receptors, inducing the trans-autophosphorylation of the receptors in several locations. This phosphorylation creates docking sites to recruit the signaling proteins Shc adapter protein or growth factor receptor binding protein-2 (Grb2). Grb2 binds to the son of sevenless (SOS) protein that engages and

56. Molecular Genetics of Brain Tumors

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FIGURE 56-2. Linear representation of the human epidermal growth factor receptor (EGFR) and the EGFR-vIII. The extracellular part consists of four subdomains (L1 to S2). Domains S1 and S2 are cysteine-rich domains involved in disulfide bonds, and domains L1 and L2 are involved in ligand binding. The intracellular part consists of a juxtamembrane region, protein kinase domain, and a carboxyterminal region.

phosphorylates the Ras protein, which in turn induces two major signaling pathways. In pathway 1, Ras activates Raf, followed by mitogen-activated protein kinase (MAPK) kinase kinase (MEK) 1/2, followed by MAPK/extracellular signal-regulated kinase (ERK) activation. Phosphorylated MAPK translocates into the nucleus, where it up-regulates the transcription of the proto-oncogenes c-myc and c-fos. The c-myc and c-fos regulate the transcription of proliferation-associated genes, which in turn promote cell division. In pathway 2, Ras activates phosphatidylinositol-3 kinase (PI-3K), which in turn induces a cascade of events: vAkt murine thyoma viral homolog (Akt)/PKB becomes phosphorylated and induces phosphorylation of the IKB kinase (IKK) complex. The complex induces the release of nuclear factor B (NF-B) by phosphorylation of its inhibitor I_B. Free NF-B translocates to the nucleus, where it regulates expression of antiapoptotic genes and also promotes cell division (Fig. 56-3) (1,41). Recently, a study showed that ErbB-4, a member of EGF tyrosine kinase receptor family, could activate gene expression in a more direct manner. The ErbB-4 receptor undergoes proteolysis within its plasma membrane domain—a process called regulated intramembrane

proteolysis—and its intracellular portion moves to the nucleus, where it affects the transcription of target genes (42). There are two steps to the proteolysis of ErbB-4. First, most of this receptor’s ectodomain is cleaved off by a membrane-associated metalloprotease called TACE. Next, the remaining part of the receptor is cleaved within its transmembrane domain by a second protease. This new signal transduction pathway might be involved in the progression of glioblastoma. Mutations in signaling molecules have not been documented for brain tumors. It appears that the increase in enzyme activation in response to increased growth factor activation is sufficient to induce proliferation in astrocytomas and meningiomas. Levels of activated Ras are increased in glioblastomas in comparison with levels in nonneoplastic brain. The importance of Ras in this signaling cascade has been shown by inhibition of proliferation by blocking Ras farnesylation in glioblastoma cells by the drug lovastatin (43) or by the inhibition of Ras function using adenovirus-mediated gene transfer of dominant negative H-Ras into meningioma cells (44). These antitumoral effects and the pivotal role that Ras plays in controlling several signal transduction pathways

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FIGURE 56-3. Biologic interactions involved in the acquisition of tumor phenotypes. The acquisition of seven essential alteration—deviation of cell development, perturbation of growth factor signaling pathway, unarrest of cell cycle, limitless replicative potential, evasion of apoptosis, sustained angiogenesis (not showed), and invasion—are sophisticated, interweaved, and regulated by intrinsic mechanisms in tumor itself and by extrinsic environment signals.

suggest that it may be an important therapeutic target; however, the activation of Ras may be mediated by growth factor-independent mechanisms because it is postulated that the loss of the guanosine triphosphatase activating protein, neurofibromin, in neurofibromatosis type 1 (NF-1) tumors leads to Ras activation (41). Other mitogen-stimulated signaling molecules elevated in brain tumors include the phospholipase C family of proteins and other members of the Ras signaling pathways, including Shc2, Grb2/SOS, Raf-MAPK, and PI3K/Akt (41,45). Recently, one study showed that JNK played a major role in promoting tumorigenesis and enhanced JNK activation was frequently found in primary brain tumors. And expression of a domain-negative form of JNK potently inhibited EGF mediated transformation and protection from apoptosis (46). These observations further emphasize the importance of these signaling pathways for CNS tumor proliferation and suggest that targeted inhibition of selected signaling pathway components could be exploited to inhibit tumor growth (41).

Cytokine receptors are devoid of intrinsic tyrosine kinase activity. When activated by specific ligands, they recruit one of the four members of the cytoplasmic protein tyrosine kinases, the Janus kinases (JAKs), including Tyk2 and JAK1–3. The downstream effectors are a family of proteins called STATs (signal transducer and activator of transcription). After JAK receptor tyrosine phosphorylation, the STAT proteins homodimerize or heterodimerize and translocate to the nucleus, where they bind to specific DNA elements that regulate cytokine-inducible genes (Fig. 56-3). Increased levels of JAK1, JAK2, and STATs have been found in gliomas, medulloblastomas, meningiomas, and ependymomas (47). Interleukin-13 receptor is highly expressed in all grades of gliomas, especially high-grade gliomas (48).

Unarrest of Cell Cycle Much of the circuitry that enables normal cells to respond to antigrowth signals is associated with the cell cycle clock,

56. Molecular Genetics of Brain Tumors

specifically the components governing the transit of the cell through the G1 phase of its growth cycle. Cells monitor their external environment during this period and, on the basis of sensed signals, decide whether to proliferate, to be quiescent, or to enter into a postmitotic state. The cell possesses an intricate series of checks and balances regulated by positive effectors and negative regulators that control proliferation. The positive effectors include the cyclin-dependent kinase (CDK)/cyclin complexes (CDK4, 6/cyclin D1, 2, 3) and the proto-oncogenes MDM2, possibly its relative MDM4, and PAX5. The negative effectors include the cyclin-dependent kinase inhibitors (p16INK4A, p15, p18INK4C, p19ARF, p21, and p27) and the pivotal tumor suppressor genes Rb and p53. Many of the mutations or genetic alterations observed in CNS tumors to date occur in the cell cycle control genes. These mutations or alterations promote unregulated cell proliferation, an essential biologic function for tumors of all grades. There are two major axes that are implicated in the deregulation of the cell cycle, and these are centered on the tumor suppressor genes p53 and Rb. pl6INK4A-CDK4, 6/CyclinD-Rbl Axis Loss of the wild-type allele with mutation of the remaining allele or loss by homozygous deletion has been well documented for all components of the pl6INK4A-CDK4, 6/cyclinD-Rbl axis (Fig. 56-3). Deletion or mutation of each of these genes is prevalent in astrocytomas and oligodendrogliomas, and they are associated with specific stages of glioma progression (28). Although it was thought that loss of p16INK4A was restricted to tumors of glial origin (49), p16INK4A mutations have been reported in malignant meningiomas (50) and malignant peripheral nerve sheath tumors (51). CDK4 and CDK6 amplification have been reported in gliomas. Although uncommon, amplification and overexpression of cyclin Dl and D3 also have been reported. The loss of Rb in gliomas due to mutations, deletions, and rearrangements is well documented (28). p21–p53-MDM2/MDM4/PAX5-p14ARFAxis The p21–p53-MDM2/MDM4/PAX5-pl4ARF axis involves the p53 gene (Fig. 56-3), which is mutated in approximately 60% to 65% of astrocytomas. Mutation in this gene is one of the earliest observed in astrocytomas, and the resultant lack of DNA damage monitoring most likely contributes to the short time to tumor recurrence and increased malignant progression observed in these tumors. p53 mutations also occur, although less frequently, in atypical meningiomas, high-grade ependymomas, and neurofibromas (28). Mutations to p53 affect p21, which regulates the same cyclinD/CDK4 complex of the pl6 pathway, indirectly regulating Rb phosphorylation.

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Although mutations or deletions of the p21 gene are rare in brain tumors, expression of p21 was decreased in seven malignant glioma cell lines; however, the decreased expression did not correlate with the mutational status of p53, suggesting that the regulation of p21 is complex and there are other unknown regulators of p21 (52). Recent research found that the downstream proteins of p53 signaling pathway, such as p53RDL1, p53AIP1, and IRF-5, was also involved in gliomagenesis through inhibiting apoptosis and promoting transcription of oncogenes (53,54). Transferring p53RDL1 gene into gliomas in vivo can inhibit proliferation of glioma cells and induce apoptosis of these tumor cells (55). Inhibitors of p53 include PAX5, MDM2, and possibly MDM4 (56,57). PAX5 represses the transcriptional activation of the p53 gene by binding to a 5 regulatory region that is necessary for p53 promoter activity. Increased expression of PAX5 has been reported only for astrocytomas. Overexpression was observed in areas exhibitor an increased expression of the EGFR, suggesting that these two genes may be connected intricately to the de novo glioblastoma pathway. MDM2 functions to inhibit p21 transcriptional activation by p53 by binding to and concealing the p53 DNA binding domain and inducing the rapid degradation of the p53 protein. MDM2 amplification and overexpression occur in a greater percentage of de novo glioblastomas as compared with progressionrelated glioblastomas, suggesting that MDM2 overexpression also may constitute an alternate molecular mechanism of escape from p53-regulated growth control. MDM2 is also amplified and overexpressed in ependymomas. MDM4 was amplified and overexpressed in some malignant gliomas that did not have p53 mutations or MDM2 mutations, suggesting that it plays a role in suppressing p53 function in a few tumors. Another important regulator of this pathway is pl4ARF (Fig. 56-3). This protein arises from the alternative splicing of the CDKN2A locus that also codes for the pl6INK4A protein; pl4ARF binds to the p53/MDM2 complex and inhibits MDM2-mediated degradation of p53. In a study of 190 astrocytic gliomas, 40% of GBMs and 5% of anaplastic astrocytomas were found to have mutant or deleted pl4ARF (56). FoxO Forkhead transcription factors act as signal transducers at the confluence of Smad, P13K, and FoxG1 pathways. Smad proteins activated by TGF- form a complex with FoxO proteins to turn on the growth inhibitory gene p21Cip1. This process is negatively controlled by the P13K pathway and by the telencephalic development factor FoxG1, which binds to FoxO-Smad complexes and blocks p21Cip1 expression. The activity of this network confers resistance to TGF--mediated cytostasis during the development of the neuroepithelium and in glioblastoma brain tumor cells. Evidence shows that mutation to any one of the members of these two axes is sufficient to promote cell divi-

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sion, supporting the existence of redundant pathways to malignant progression. That mutation of one of these genes is sufficient to subvert cell cycle control is supported by the reports showing that the reexpression of pl6INK4A, pl5, Rb, and p53 in glioma tumor cells inhibits their growth (58–60).

Evasion of Apoptosis The ability of tumor cell populations to expand in number is determined not only by the rate of cell proliferation, but also by the rate of cell attrition. Programmed cell death (apoptosis) represents a major source of this attrition. The evidence is mounting that acquired resistance toward apoptosis is a hallmark of most cancers. The apoptotic machinery can be broadly divided into two classes of components—sensors and effectors. The sensors are responsible for monitoring the extracellular and intracellular environment for conditions of normality or abnormality that influence whether a cell should live or die. These signals regulate the second class of components, which function as effectors of apoptotic death. The sentinels include cell surface receptors that bind survival or death factors. Intracellular sensors monitor the cell’s well-being and activate the death pathway in response to detecting abnormalities, including DNA damage, signaling imbalance provoked by oncogene action, and survival factor insufficiency. Further, the life of most cells is in part maintained by cell–matrix and cell–cell adherence based survival signals whose abrogation elicit apoptosis. Both soluble and immobilized apoptotic regulatory signals likely reflect the needs of tissues to maintain their constituent cells in appropriate architectural configurations (Fig. 56-3). Many of the signals that elicit apoptosis converge on the mitochondria, which respond to proapoptotic signals by releasing cytochrome c, a potent catalyst of apoptosis. Members of the Bcl-2 family of proteins, whose members have either proapoptotic (Bax, Bak, Bid, Bim) or antiapoptotic (Bcl-2, Bcl-XL, Bcl-W) function, act in part by governing mitochondrial death signaling through cytochrome c release. The p53 tumor suppressor protein can elicit apoptosis by up-regulating expression of proapoptotic Bax in response to sensing DNA damage; Bax in turn stimulates mitochondria to release cytochrome c. The ultimate effectors of apoptosis include an array of intracellular proteases termed caspases. Two “gatekeeper” caspases, -8 and -9, are activated by death receptors or by the cytochrome c released from mitochondria, respectively. These proximal caspases trigger the activation of a dozen or more effector caspases that execute the death program through selective destruction of subcellular organelles and the genome (61). Apoptosis is a major barrier to cancer that must be circumvented. Altering components of the apoptotic ma-

chinery can dramatically affect the dynamics of tumor progression, providing a rationale for the inactivation of this machinery during tumor development. Resistance to apoptosis can be acquired by cancer cells through a variety of strategies. Surely, the most commonly occurring loss of a proapoptotic regulator through mutation involves the p53 tumor suppressor gene. The resulting functional inactivation of p53 protein is seen in most of brain cancers and results in the removal of a key component of the DNA damage sensor that can induce the apoptotic effector cascade. Additionally, the PI3 kinase– Akt/PKB pathway, which transmits antiapoptotic survival signals, is likely involved in mitigating apoptosis in a substantial fraction of brain tumors. This survival signaling circuit can be activated by extracellular factors such as IGF-I/II or interleukin-13, by intracellular signals emanating from Ras, or by loss of the PTEN tumor suppressor. Moreover, Bcl-2 family are also involved in evade from apoptosis. Some studies showed that gliomas had a high level of Bcl-2 overexpression, which can be an early event in gliomas tumorigenesis (62). And the expression of Baxpsi (a new variant of the proapoptotic molecule Bax) is correlated with an increased survival of GBM patients (63). Other mechanisms, such as caspase family gene methylation (64) and overexpression of survivin (65), a novel antiapoptotic gene, are supposed to play important roles in tumorigenesis. Recently, a mechanism for abrogating the FAS death signal has been revealed in malignant gliomas: A nonsignaling decoy receptor for FAS ligand is up-regulated, titrating the death-inducing signal away from the FAS death receptor (Fig. 56-3) (66). When laid out a provisional apoptotic signaling circuit, it is evident that most regulatory and effector component are present in redundant form. This redundancy holds important implications for the development of novel of antitumor therapy, because tumor cells that have lost proapoptotic components are likely to retain other similar ones.

Limitless Replicative Potential Most living cells have a finite replicative potential, which are considered to be result of the existence of a molecular “clock” that can keep track of cell divisions. Telomeres, which represent the ends of linear chromosomes, are repetitive silent DNA segments exerting structural functions that are crucial for maintaining chromosome integrity. The cell is not able to ensure the replication of the distal tract of DNA of its lagging strand, which results in the progressive shortening, division after division, of telomeres. After a genetically defined number of cell cycles have been completed, telomeres reach a critically short extension, which triggers a proliferative crisis known as senescence. Telomere shortening, which typically occurs in normal somatic cells, can be regarded as a point in a biologic clock, which marks the progression of a cell

56. Molecular Genetics of Brain Tumors

toward the end of its replicative life span. Most cells stop dividing when they reach a “threshold to senescence.” Some cells, however, ignore warnings to stop dividing despite critically shortened telomeres. It is known that the wearing away of the telomeres with successive cell divisions eventually abrogates their protective functions and chromosomes become unstable, fused, or lost (Fig. 56-4A). Cells with such defects not only are unable to divide but also may not survive. Immortal cells solve the problem of the truncated telomeres by means of a complex of proteins and RNA called telomerase. Two essential components have been identified in human telomerase: an RNA subunit, called hTR (telomerase RNA), containing a template sequence on which the telomeric repeats are synthesized, and a protein, which shows structural and

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functional similarities with viral reverse transcriptases, called hTERT (telomerase reverse transcriptase). The RNA component is ubiquitously present, whereas hTERT expression correlates with telomerase activity and its expression is essential to ensure the enzymatic activity of the complex. Turning on this enzymatic complex allows the malignant clones to escape senescence and to divide indefinitely. In neuroepithelial tumors, the rate of detection of telomerase activity correlates with tumor malignancy (67). Some researches have demonstrated a difference in telomerase pattern between anaplastic astrocytomas and glioblastomas, with a high percentage of glioblastomas (ranging from 80% to 100%) showing telomerase activity, as opposed to anaplastic astrocytomas, only a minor percentage of samples of which (ranging from 20% to 30%)

FIGURE 56-4. A. Telomere attrition and perpetuation of the breakage-fusion-bridge (BFB) cycle. As cells divide in the absence of telomerase, telomeres erode, exposing the ends; DNA repair functions can then create chromosome fusions. Here, a fusion between two sister chromatids forms a dicentric chromosome, which results in anaphase bridging during segregation in mitosis. The dicentric chromosome is broken when pulled to opposite spindle poles, creating changes in gene dosage (amplifications [Amp] and deletions [Del]) for the resulting daughter cells. The broken chromosome must then be repaired again and can become fused to another chromosome, generating a second dicentric chromosome and perpetuating the BFB cycle. This cycle likely facilitates the accumulation of genetic changes that enable cells to emerge from crisis and proceed to malignancy. B. A model for crisis induced by telomere attrition. Shortened telomeres elicit replicative senescence in vitro, blocking further proliferation unless checkpoint responses are disrupted. Continued cell division will eventually cause telomere dysfunction and crisis. The early stages of crisis can be averted by loss of p53. Eventual emergence from late crisis requires not only p53 inactivation but also the acquisition of telomere maintenance mechanisms.

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Cancer of the Nervous System

expressed telomerase. It appears that the progression from anaplastic astrocytoma to glioblastoma implies the activation of telomerase and that secondary glioblastomas express telomerase in a higher percentage of cases than do primary glioblastomas. When the telomere length of glioblastoma samples was analyzed, telomeres were found to be of normal or elongated length, supporting the view that the telomerase machinery is active in all glioblastoma tumors. Moreover, there seems to be a relationship among the degree of hTERT expression, telomerase activity, and proliferative rate in glioblastoma tumors. A study showed that introducing hTERT into normal astrocytes was sufficient to initiate the tumorigenic process by circumventing cellular senescence in astrocytes regardless of the activation of p53 and Rb, which demonstrated that telomerase expression appears to be a perquisite in the formation of malignant glioma (68). It is well known that cells with critically shortened telomeres may die due to activation of the p53 checkpoint pathway (Fig. 56-4B). In cells lacking these checkpoints, telomere loss can result in chromosome instability, and this can promote some of the mutations required to achieve cellular transformation. Therefore, early p53 inactivation in gliomas may provide the means for the genetic instability required for creation and selection of telomerase-positive cells, which is manifested by a research that found the secondary glioblastomas with p53 gene mutations demonstrated significantly higher telomerase activity than those without p53 mutation (69). Using in situ hybridization methods, the existence of two different expression patterns of hTERT mRNA in glioblastoma was demonstrated. A diffuse pattern, with most of the tumor cells expressing hTERT, was found in 56% of the tumors. The remaining cases (44%) showed a focal pattern, with hTERT-positive neoplastic cells interspersed among hTERT-negative viable cells or necrotic tumor areas. Interestingly, tumors with the diffuse pattern of hTERT expression were smaller and exhibited a higher proliferative rate than tumors with the focal pattern. In addition, survival was significantly shorter in the latter group (70). This suggests that at an early stage of tumor progression, a small tumor with high proliferative rate and limited necrotic phenomena diffusely expresses hTERT. At a later stage, which might be related to a critical tumor size, glioblastoma becomes necrotic, the proliferative rate declines, groups of cells lose hTERT expression, and telomerase activity might disappear. From a clinical point of view, these data on hTERT mRNA expression seem to provide a prognostic parameter for glioblastoma cases, because a worse prognosis is associated with the focal hTERT expression. Neuroblastoma is the one in which the correlation between telomerase activity and outcome is best demonstrated. Telomerase activity was expressed in 94% of neuroblastomas but not in benign ganglioneuromas or adjacent adrenal tissues. Among untreated neuroblastomas, 75% of tumors with high telomerase activity had a

poor prognosis, 97% of tumors with low telomerase activity had a good prognosis, and 100% of tumors with no detectable telomerase activity regressed. Recently, researchers cast light on the dysfunction of spindle checkpoint in p53-deficient cancer cells, which is a critical factor in resistance to mitotic catastrophe, an important mechanism for the induction of cell death in cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphasepromoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint function by BubR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis (71).

Sustained Angiogenesis Tumor growth cannot be sustained without angiogenesis (the induction of new blood vessels from preexisting vessels). The formation of new vessels requires the coordination of angiogenic and antiangiogenic factors that regulate the induction of vascular discontinuity, endothelial cell proliferation, endothelial cell migration, and structural reorganization of the new vasculature. Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells. Hypoxia, which is the result of a lack of perfusion and vascular compression in tumor tissue, is one of the strongest inducers of VEGF expression. VEGF is considered to be the most important mediator of neovascularization in gliomas, and its expression increases during glioma progression. Overexpression of VEGF has been shown for meningiomas, ependymomas, hemangioblastomas, and neuroblastomas (72). The three VEGF receptors, VEGFR-1 (flt-1), VEGFR-2 (flk-1), and VEGFR-3 (flt-4), which belong to the family of tyrosine kinase receptors that initiate signaling events on dimerization, are selectively expressed on endothelial cells. Both receptors are increased on endothelial cells in malignant astrocytomas, anaplastic oligodendrogliomas, and ependymomas. The expression of flt-1 receptor occurs early in glioma tumorigenesis, whereas the flk-1 gene is up-regulated during glioma progression (73). In our own study, in vivo gene transfer of soluble flk1 (sflk1) by recombinant adenoviruses (Adsflk1-FC) (74) could inhibit glioblastoma growth in the mouse brain. U87MG human glioblastoma cells were injected intracerebrally in athymic nude mice. Seven days after tumor inoculation these mice were injected with Ad-sflk1-FC. Administration of Ad-sflk1-Fc produced a significant reduction in mean tumor size compared with controls (p < 0.01). Several other growth factors and their tyrosine kinase receptors, such as scatter

56. Molecular Genetics of Brain Tumors

factor and its receptor c-met, bFGF and its receptor (FGFR1 and FGFR2), and cytokine SDF1 and its receptor CXCR4, also play a role in angiogenesis in astrocytomas, glioblastomas, oligodendrogliomas, ependymomas, and schwannomas (75,76). Thrombospondin-1, angiostatin, and endostatin were potent angiostatic factors. We generated recombinant adeno-associated virus vectors encoding the endostatin gene (77). The intramuscular administration of adenoassociated virus-endostatin vectors yielded sustained secretion of endostatin into blood over 90 days and inhibited the growth of human U87MG glioblastoma in an intracerebral model using nude mice. Moreover, the combined use of intramuscular and intratumoral administration of the adenoassociated virus vector resulted in 91% suppression of brain tumor growth. This treatment also significantly prolonged survival of brain tumor-bearing mice (p < 0.01). Tumor vessel density demonstrated a marked reduction of tumor vascularization after treatment. Anti-angiogenic therapy could be a feasible strategy for glioma therapy. The inappropriate expression of several growth factors or secreted proteins tips the balance in favor of overall tumor neovascularization, suggesting that inhibition of angiogenesis may be an important therapeutic strategy. Currently, a new candidate tumor suppressor gene, ING4, is involved in regulating angiogenesis and brain tumor growth of ING4 is significantly reduced in gliomas as compared with normal human brain tissue, and the extent of reduction correlate progression from lower to higher grades of tumors. In mice, xenografts of human glioblastoma U87MG, which has a decrease of ING4, grow significantly faster and have higher vascular volume fractions than control tumors (77).

Tissue Invasion The invasive or infiltrative phenotype renders brain tumors most difficult to cure. Invasion as a biologic phenotype is complex, but the activation of extracellular proteases and the altered binding specificities of cadherins, cell adhesion molecules, and integrins are clearly central to the acquisition of invasive capability. For a tumor cell to invade brain tissue, the adjacent matrix must be degraded partially to make room for the tumor cell (Fig. 56-3). Many matrix-degrading proteinases are implicated in brain tumor invasion. Matrix metalloproteinases (MMPs) can degrade almost all known extracellular matrix (ECM) components (79). MMP-2 and MMP-9 are expressed in gliomas, and increased expression of MMP-9 is observed in higher grades. Moreover, the inhibition of MMP-9 using antisense gene transfer impairs glioblastoma invasion in vivo, and MMP inhibitors reduced glioma invasion in vitro (80). The cysteine proteinase cathepsin B functions as an endopeptidase and exopeptidase and has a broad range of substrates. Its expression is increased in human gliomas and is expressed in invading tumor cells,

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suggesting that it may contribute in glioma invasion. The tip-regulation of urokinase-type plasminogen activator (uPA) can initiate a cascade of events to promote pericellular matrix degradation and promote cell migration. uPA and uPA receptor (uPAR) are coexpressed in malignant astrocytomas, and uPA was found to be expressed predominantly at the leading edge of the tumors, suggesting that they may contribute to brain tumor invasion. When glioma cells were infected with adenovirus-mediated antisense uPAR, glioma invasion and tumor growth were suppressed (81). Integrins are transmembrane receptors composed of heterodimers of  and  subunits that mediate adhesion of cells to the surrounding ECM (Fig. 56-3). Changes to integrin and ECM components and their resultant interactions have profound effects on tumor cell adhesion and migration. To date, the integrins 2, 3, 4, 5, 6, 1, 4, v, v3, and v5 are found to be significantly expressed in gliomas and modulate adhesion and migration. It seems that the tumor cells themselves may modulate the ECM environment by secreting matrix molecules and provide a suitable matrix for adhesion and migration. The synthesis of vitronectin by GBM cells invading into adjacent brain has been reported, and glioma cell line, U87MG, U251MG, AN1/lacZ, and HF66 all expressed fibronectin, laminin, tenascin, collagen type IV, and chondroitin sulfate to varying degrees (82). The uPA/uPAR complex also plays a role in tumor cell migration. uPAR is an adhesion receptor for vitronectin, and the uPA/uPAR complex binds to 1 and 2 integrins (83). When human glioblastoma cells were transfected with an antisense uPAR construct, transfectants showed lower invasion and decreased migration, which suggest that the up-regulation of uPAR observed in gliomas could enhance invasion. Because uPAR binds to integrins, one possible mechanism is through the sequestration of integrins by enhanced uPAR so that cell–matrix interactions decrease and invasion is enhanced.

GENETIC APPROACHES IN BRAIN TUMOR CLASSIFICATION Currently, the 2000 World Health Organization classification in brain tumor is widely applied, which mainly reflects the histologic and location feature and, for the most part, is a practical and effective approach to brain tumor analysis. But classification schemes that are based on visual criteria alone are subjective and allow considerable interobserver variation. In addition, traditional groupings are only satisfactory for series of cases and not necessarily adequate predictors of behavior, response to therapy, or survival for individual tumors and patients. Therefore, a classification for brain tumor based on specific genetic events in brain tumorigenic mechanisms has a greater likelihood of achieving universal clinical relevance (84). The main genes implicated in CNS tumors are shown in Table 56-1.

w TABLE 56-1

Genes Implicated in Central Nervous System Tumors

Nervous System Neoplasia Gliomas Progression pathway (secondary pathway) Astrocytoma

Negative Effectors/Tumor Suppressor Genes

Positive Effectors/Oncogenes

LOH17p, TP53 mutation, LOH13q, LOH1p, LOH3p, LOH22q (not NF2), LOH11p, LOH16p (TSC2), p27
10, LOH9p (p16 and p15), LOH13q(Rb), LOH11p,LOH19q, LOH22q, LOH10q, LOH17p13 (not p53), p14ARF LOH10p, LOH10q23, PTEN mutation, LOH10q24–25 (MXI1?,LGI1?), LOH10q25.3–26.1, DMBT1 mutation, LOH18q (DCC loss of expression)

+7 (PDGFs/PDGF-Rs), IGFs/IGF-R, IGFBP2 EGF, a/bFGF/a/bFGFRs, TGF-/


10, LOH10p and 10q, PTEN mutation, LOH9p, P16 deletion, LOH13, Rb mutation

+7 (EGFR amplification, overexpression, truncation), 9P(PAX5), 12q (MDM2 amplification, overexpression), PDGFA overexpression

LOH1p, LOH19q(p16), LOH4q rare, LOH14q rare, LOH15, LOH18, LOH9q, LOH10 (10q25–26) LOH4q, 9p loss, LOH CDKN2A/p16, LOH CDKN2C/p18, LOH10p and 19q,
10, LOH15 rare, LOH18 rare, TP53 mutation, rare, 22q(NF2), 16p(TSC2), 6q

7 (EGFR)

Spinal: -22, LOH22q, NF22 mutation, LOH11q Intracranial: LOH11q, LOH22q, MEN1 mutation, LOH1q, LOH6q, LOH9q, LOH13q 9p loss, 13q loss

PDGFs/PDGFRs, SV40, IGF-II, MDM2, ShcA

Medulloblastoma

17p (not p53), 6q, 16q, 9q22 (PTCH), 10q25.3–26.1 (DMBT1) APC

7, 17q, 1, 4, 6, IGF-I/II,IGFR1,myc-N, myc-C

Atypical teratoid/rhabdoid tumor

monosomy 22, deletion 22q11(Hsnf5/INI1)

Anaplastic astrocytoma

Glioblastoma multiforme

De novo pathway (primary pathway) Glioblastoma

Oligodendroglioma Low grade

Anaplastic

Ependymoma Low grade

Anaplastic

Meningioma Benign

Atypical

Malignant

22q (NF2; subtype specificity), 18(DAL-1), 1p3, 3p, 5p, 5q, 11, 13, 17p 1p36(ALPL), 1p34–1p32, 1p22, 1p21.1–1p13, 2p, 6q, 10, 14q24.3–31, 13q32.1–32.2, 18q, p53 9p9(p16)

+7, +20, 12q13 Amplification (MDM2, CDK4), cyclin D1 and D3, PCNA, granulin PDGFR-, 1q32, CDK2, CDK6 (MDM4?), Ras, c-myc, myb, gli, VEGF

PEGF, EGFR, VEGF overexpression

1q gain, 11q23 gain, myc amplification, 2p23–24 amplification

EGF/c-Erb2, bFGF/bFGFR, IGFs/IGFRs, IGFBP1–3, PDGF-BB/PDGFR, TGH-/, interleukin13-R, ROS1, c-myc, c-fos, RAS 1q,9q,12q,15q,17q,20

Amp17q

Neurofibroma Benign Malignant(MPNST)

17q11.2(NF1),17p(p53), 13q14–q21, 9p, 3, 18p P16, P27

17q24-qter (not NF1) Ras, cyclin E

Choroid plexus tumor Choroid plexus papilloma Choroid plexus carcinoma

21q 5q, 9q, 11, 15q, 18q

5q, 6q, 7, 9q, 12, 15q, 18q 1, 4q,10, 14q, 20q, 21q

Schwannoma Sporadic NF-2 associated

22q (NF2), 1P? 22q (NF2)

bFGF, IGFs, CD44

Neuroblastoma

11q, 14q, 17p and q, 1p36(p73), 3p, activin A

N-myc, NM23H1, VEGF, bFGF, IGFs, PDGFA, TGF-

56. Molecular Genetics of Brain Tumors

Glioma Diffuse Fibrillary Astrocytoma Diffuse fibrillary astrocytomas are the most common type of primary brain tumor in adults. These tumors are divided histopathologically into three grades of malignancy: astrocytoma, anaplastic astrocytoma, and GBM. Astrocytomas are the most indolent of the diffuse astrocytoma spectrum. Nonetheless, astrocytomas have a marked potential for malignant progression. At least two different pathways are involved in the formation of GBM. Distinct genetic events occurred in each pathway (85). Progression Pathway Formation of Astrocytoma This pathway is a progressive

stepwise evolution from a low-grade diffuse astrocytoma, to anaplastic astrocytoma, to GBM. Diffuse astrocytoma may grow slowly, but it is not benign because of its invasive quality and location. Patients diagnosed with this progressive glioma generally are young (mean age, 39 years) and usually have a history of a less malignant glioma at the time of diagnosis (86). The most common finding is a mutation or allelic loss of chromosome arm 17p, the target gene being TP53 (17p13.1). The most common genetic lesion is a missense mutation that inactivates the TP53 gene. Approximately 65% of patients between the ages of 18 years and the mid-40s had a mutation in the TP53 gene compared with 9% of older patients with a mean age of 60. A similar observation has been made with immunohistochemical analysis of the p53 protein. These investigations have shown that approximately three-fourths of these gliomas have an abnormal accumulation of p53 protein. This increase in accumulation of p53 protein is expected because it can occur as a result of a mutation in TP53 and aberrations in other genes controlling the expression of the TP53 gene (87). The PDGF family of genes appears to be important to astrocytoma evaluation. In astrocytomas, the A chain and  receptor are predominantly overexpressed. This observation is significant because the most common chromosomal abnormality identified in astrocytomas involves aneuploidy of chromosome 7; the chromosomal location of the PDGFA chain and gain of chromosome 7 strongly correlates with shorter survival in astrocytomas (88). Several studies described regions of common deletion on 22q12.3–13.1, 22q13.2, and 22q13.3 were found in 11% of human astrocytomas (2) and associated with astrocytoma progression (89,90). A novel putative tumor suppressor gene, SCN6, localized to 22q13.2 and could be a candidate for the postulated tumor suppressor gene at the deleted locus in 22q13 in glioma. Progression to Anaplastic Astrocytoma Anaplastic astrocytomas can be identified in young and old patients, with the peak incidence occurring in the mid-50s. When young patients are separated from older patients, the

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mutation rate of the TP53 gene, overexpression of PDGF and PDGF receptors, and allelic loss on 22q are similar in frequency to that found in diffuse astrocytomas. This similarity supports the concept of the progressive nature of the diffuse astrocytoma to a more malignant stage defined as the anaplastic astrocytoma (91). Changes that mark this transition from diffuse astrocytoma to anaplastic astrocytoma include additional allelic loss on chromosome arms 9p, 13q, and 19q (92) and amplification of a chromosome region occurs to chromosome 12 (12q13–14) (93). Those genetic alterations converge on one critical cell-cycle regulatory complex, which includes the p16INK4A, CDK4, cyclin D1 and retinoblastoma (Rb) proteins. The simplest schema suggests that p16INK4A inhibits the CDK4/cyclin D1 complex, preventing CDK4 from phosphorylating pRb, and so ensuring that pRb maintains its brake on the cell cycle. Chromosome 9p loss occurs in approximately 50% of anaplastic astrocytomas and GBMs, with 9p deletions occurring primarily in the region of the CDKN2/p16INK4A (or MTS1) gene, which encodes the p16INK4A protein. The frequency of 9p loss increases not only at the transition from astrocytoma to anaplastic astrocytoma, but also at the transition from anaplastic astrocytoma to GBM, implying that the 9p tumor suppressor plays a role in different stages of astrocytoma progression (94). Loss of chromosome 13q occurs in one-third to one-half of high-grade astrocytomas, suggesting the presence of a progression-associated astrocytoma tumor suppressor gene on that chromosome. The 13q14 region containing the Rb gene is preferentially targeted by these losses, and inactivating mutations of the Rb gene occur in primary astrocytomas. Overall, analysis of chromosome 13q loss, Rb gene mutations, and Rb protein expression suggests that the Rb gene is inactivated in about 20% of anaplastic astrocytomas and 35% of GBM. Interestingly, Rb and CDKN2/p16INK4A alterations in primary gliomas are inversely correlated, rarely occurring together in the same tumor (95). An important site of allelic loss in leading to anaplastic astrocytomas occurs on chromosome 19q13.2–13.3. A number of candidate genes have been isolated from or mapped to this region, including the Bax gene, whose product negatively regulates apoptosis with Bcl-2, but the tumor suppressor gene has not been identified. This tumor suppressor gene may be unique to glial tumors and is limited primarily to the progressive type of glioma. When tissue was selected for secondary gliomas and primary gliomas, the allelic loss reported was 54% for secondary gliomas versus 6% for primary gliomas (96). A frequent amplification of a chromosome region occurs to chromosome 12 (12q13–14) (95). The genes MDM2 and CDK4 are mapped to this chromosome region. Because amplification of the CDK4 gene and overexpression of cyclin D1 may have similar effects as p16INK4A or pRb inactivation, these mechanisms may provide additional alternatives to subvert cell-cycle control and facilitate

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progression to GBM. CDK4 is amplified in 15% of malignant gliomas, although this frequency may be higher among cases without CDKN2/p16INK4A loss, perhaps reaching 50% of GBMs without CDKN2/p16INK4A loss. CDK4 amplification and CDKN2/p16INK4A deletions do not occur together in GBM cell lines, and some GBM cell lines overexpress cyclin D1. On the other hand, in some GBMs and GBM cell lines, CDK4 amplification and cyclin D1 overexpression appear to represent alternative events to CDKN2/p16INK4A deletions, because these genetic changes only rarely occur in the same tumors. In combination, it is likely that up to 50% of anaplastic astrocytomas and perhaps all GBM have alterations in at least one component of this critical cell-cycle regulatory pathway. Progression to Glioblastoma Multiforme (Secondary Glioblastoma)  GBM is considered the most malignant of the

gliomas. The genetic instability of this tumor results in numerous and varied genetic changes, which are reflected in the disparate number of cell types and chromosomal and molecular aberrations. Karyotypic changes can range from near-diploid to several hundred chromosomes per cell. Structural rearrangements are common and highly variable. These structural rearrangements often are complex, and the use of comparative genomic hybridization, fluorescent technologies, or spectral karyotyping has been helpful in determining the origin of these derivative or marker chromosomes (92). The frequency of TP53 gene mutations or allelic loss or both is approximately the same in GBMs as in anaplastic astrocytomas and diffuse astrocytomas. There is a substantial increase in the LOH reported for 10p, 10q23.3, and 10q25.3–26.1, however, in addition to the loss of an entire copy of chromosome 10. The tumorsuppressor gene, PTEN (phosphate-tensin), was identified on chromosome 10q23.3. The PTEN gene encodes a dual specificity phosphatase that has been shown to function in the regulation of cell growth, apoptosis, cell migration, and interactions with the ECM. Initially, it was thought that LOH of PTEN was a late event that initiated the progression of an anaplastic astrocytoma to become a GBM (97). But currently it was confirmed that only a few of the secondary glioma had a mutation in this gene, and allelic loss was confined primarily to 10q. A second gene, DMBT1 (deleted in malignant brain tumors 1) on chromosome arm 10q, may be important to progression of secondary glioma. Aberrations in this gene are thought to contribute to genetic instability. DMBT1 is located at 10q25.3–26 and is considered the candidate tumor suppressor gene found deleted in some 38% of the secondary GBMs (98). But some studies showed that DMBT1 polymorphisms are not likely primary targets of 10q loss in malignant gliomas and do not support a major role for DMBT1 in gliomagenesis (99,100). Other genes found to be aberrant in GMB that might contribute to the malignant and invasive phenotype have

been described. High-grade gliomas have abnormalities of chromosome 18, and several molecular studies have indicated that it is deleted in approximately 50% of GBMs. Allelic loss in chromosome 19, carrying a putative tumor suppressor gene, may be found to have a similar frequency to anaplastic astrocytomas when tissue is selected. De Novo Pathway (Primary Glioblastoma)

The second pathway for GBMs appears to arise de novo or rapidly from a preexisting tumor, although they are indistinguishable from secondary glioblastomas histopathologically. This type of GBM appears to have no evolutionary component. This tumor usually is associated with older patients (mean age, 55 years) who have not had a previous history of a lower grade of tumor. Although TP53 mutations or allelic loss is the most common genetic aberration of secondary GBMs, the amplification of EGFR is most common in the primary GBM, and more than half of these tumors have a rearrangement of this gene. This mutated form of the EGFR has a high level of tyrosine kinase activity in the absence of the EGF ligand, which essentially keeps this receptor in a turned on autocrine mode. The amplification of EGFR potentially overrides the normal negative regulation of the PTEN gene product. EGFR is mapped to chromosome 7p13–11, and although the DNA copy number may be normal, the gene also can be overexpressed, a feature identified in more than 60% of these tumors (91). Allelic loss on two regions of chromosome arms 10p and 10q is restricted almost entirely to the primary glioma (97). It has been suggested that the loss of whole chromosome 10 is a major factor in the evolution of the highly malignant GBM. In some tumors, two different grades of tumor can coexist side by side, and chromosome 10 loss is not a feature associated with the diffuse astrocytoma. It is postulated that it is the loss of chromosome 10 that permits the abrupt change from a low-grade to a high-grade mass. Differing from the secondary GBM, the primary GBM tends to show the loss of the entire chromosome 10 as opposed to the loss of 10q identified as the primary loss in the secondary GBM (101). Amplification of MDM2 gene, located at 12q14.3–15, also appears to be restricted to the primary de novo pathway. The amplification of MDM2 gene by virtue of binding to p53 essentially inactivates this protein. This is an alternative mechanism that allows a tumor cell to be removed from the control of normal TP53 gene expression. Tumors that overexpress MDM2 generally lack a mutation or allelic loss in TP53. Amplification of MDM2 as a functional inhibitor of p53 can compensate for a lack of allelic loss of p53 (2). Genes important in the cell cycle appear to be highly associated with the primary GBM. These genes include the CDKN2 locus that codes for p16INK4A and p14ARF, CDK amplification and allelic loss, and the mutation of

56. Molecular Genetics of Brain Tumors

Rb1. The aberrant expression of any of these genes contributes to the uncontrolled proliferation associated with the GBM, and as studies progress that select tumors on the basis of clinical criteria, these genes are restricted to the primary GBM (91). Oligodendroglioma Oligodendrogliomas are tumors that occur primarily in adults, with a peak incidence in the fourth and fifth decade. Histologically, oligodendroglial tumors comprise a continuous spectrum ranging from well-differentiated neoplasms to malignant invasive tumors. In contrast to astrocytoma, however, the histopathology does not always correlate with survival. Therefore, prognostic markers are needed. Molecular analysis of oligodendrogliomas has shown allelic loss on chromosome arms 1p and 19q and that this loss was preferential for oligodendrogliomas. The most frequent allelic loss occurs on chromosome arm 19q. It has been observed in 50% to 80% of the tumors analyzed despite there being little evidence of numerical or structural abnormalities of chromosome 19 2(2). Many genes known to be lost or mutated in tumors reside on chromosome arm 19q (e.g., DNA repair gene, ERCC1, ERCC2, XRCC, and DNA ligase; proapoptosis gene, Bax; and TGF-1 gene), but the importance of these genes is still needed to be elucidated (102). The location of a potential tumor suppressor gene is not well defined for chromosome arm 1p. CDKN2C (p18INK4C, a negative regulator of the cell cycle) and p73 (a homolog of p53), respectively located on chromosome 1p32 and 1p36, are not the gene crucial to this deletion (103). The candidate gene at this site awaits further investigation. Additional genetic lesions have been reported on chromosome arms 9p and 10q. These genetic changes have been associated with the transition of the welldifferentiated oligodendroglioma to anaplastic oligodendroglioma (104). A potential target gene on 9p21 is the cell cycle inhibitor, CDKN2A (p16INK4A). In studies that assessed the involvement of this gene, no allelic loss or mutations were observed for CDKN2A gene in well-differentiated oligodendrogliomas (104). This is in contrast to the findings of anaplastic oligodendrogliomas, in which 42% of the cases had an allelic loss or mutation (104). For chromosome 10 involvement, the well-differentiated oligodendrogliomas and the anaplastic oligodendrogliomas showed LOH on 10p, 10q23, and 10q25 in a few cases analyzed, although none of these tumors had a mutation of PTEN, suggesting this is not the targeted gene (105). Other chromosomes reported to have occasional LOH include 4q and chromosomes 14,15,11p,18, and 22q (104), but additional cases are needed more study to determine the relative importance of these findings.

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Occasionally, oligodendrogliomas have mutations in the TP53 gene but with far less frequency than that observed in astrocytic tumors (92,104); this is in contrast to the immunohistochemistry studies for p53 protein. A much higher percentage of oligodendrogliomas express the p53 protein, but only a few of these tumors have a mutation in the TP53 gene (104). This observation is similar to that of the astrocytomas and is explained in part by the aberrant expression of other genes that can affect directly the expression of TP53, such as the MDM2 gene. Growth factors and their receptors most likely play an important role in oligodendrogliomas as they do in the astrocytic tumors. PDGFA and PDGFB and their receptors PDGFR- and PDGFR- are expressed in oligodendrogliomas (106). Despite the aberrant expression of this growth factor and receptor, gene amplification was detected only in the anaplastic oligodendroglioma and the anaplastic oligoastrocytoma. The aberrant expression of EGFR has been identified in oligodendrogliomas and anaplastic oligodendrogliomas, and this does not appear to be the result of gene amplification, such as described in astrocytic tumors. The allelic loss on chromosome arm 1p is emerging rapidly as a marker of chemotherapeutic sensitivity and longer survival for oligodendrogliomas and for some anaplastic oligodendrogliomas (107,108). Generally, tumors carrying 1p deletions also carry 19q allelic loss, and these patients appear to have better outcomes than patients with LOH of 1p alone (108). The allelic loss of 1p and 19q is almost 100% predictive for drug sensitivity and survival for oligodendrogliomas. The results of the anaplastic astrocytoma were ambiguous, however, and a larger and more homogeneous set of patients was investigated to determine whether 1p was an important marker of drug sensitivity and survival in this group of tumors. The results of this investigation suggest that anaplastic oligodendrogliomas can be divided genetically into four therapeutically and prognostically relevant subgroups (109). Patients whose tumors have combined but isolated losses of 1p and 19q have a marked response to chemotherapy and associated long survival even if postoperative radiation therapy has not been given. The second group of tumors in which there is only a chromosome 1p alteration has a therapeutic response, but the duration of this response is shorter. The third group of tumors is characterized by no 1p loss, but they do have a TP53 mutation. These tumors have a therapeutic response, but it is short, and the tumor recurs quickly. The last group of tumors lacks a 1p loss and a TP53 mutation. These tumors have little to no response to chemotherapy. They are the most aggressive of the anaplastic oligodendrogliomas and behave clinically similar to a glioblastoma. The genetic aberrations possessed by this group of tumors reflect similar aberrations (i.e., PTEN mutations,

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10q loss, EGFR amplification, gain chromosome 7q34– 36, and CDKN2A deletion) to those identified in the primary glioblastoma. Recently, after examining gene expression profiles of oligodendroglioma by Genechip, the tumors with 1pLOH had similar expression profiles to the normal brain for those differentially expressed genes. This research provided baseline information to elucidate the mechanism of chemosensitivity in oligodendrogliomas (110). Oligoastrocytoma Oligoastrocytomas are tumors composed of mixtures of oligodendroglial and astrocytic cells. Molecular studies have not been able to identify a consistent distinct from either oligodendrogliomas or astrocytomas. In one study, microdissection was used to determine if the different cellular components carried the same or different genetic lesions. Three tumors analyzed by this method were found to carry the same genetic lesions in both components, suggesting that this type of tumor has a monoclonal origin rather than two components simultaneously. Other investigations have determined that approximately 30% of the oligoastrocytomas carry genetic lesions that frequently are found in astrocytic gliomas, especially TP53 mutations and LOH on 17p (111). Therefore, tumors with a 17p loss or TP53 mutation would not commonly have allelic loss on 1p and 19q, whereas oligoastrocytomas with a 1p, 19q deletion would not have a 17p deletion or TP53 mutation. An extensive study comparing allelic loss between astrocytic tumors and oligodendrogliomas for chromosomes 1p, 17p, and 19q suggested two genetic subsets in mixed tumors. One subset is related genetically to astrocytomas, and the other is related genetically to oligodendrogliomas. When these tumors acquire anaplastic features, they also are thought to acquire changes in 9p, 10, and 11p with occasional amplification of the PDGF genes and EGFR gene or changes similar to the progressive changes of anaplastic oligodendroglioma and diffuse astrocytoma (104,106). Ependymoma Ependymomas are tumors that develop from the ciliated epithelium that lines the ventricles of the brain and spinal canal. Ependymomas are common in the first two decades of life. The myxopapillary ependymoma is located exclusively in the spinal cord. The ependymoma may originate from the cerebral ventricles or from the spinal canal. These tumors are defined more clearly from surrounding brain than any other glioma, although these neoplasms are capable of showing a spectrum of anaplasia. The anaplastic ependymoma is a more malignant tumor associated with a higher mitotic index and microvascular proliferation.

The most frequent cytogenetic abnormality of ependymomas is the loss of whole chromosome 22 in about 30% of the cases. This chromosome is involved in many structural rearrangements, with the most breakpoints localizing to 22q12. Molecular studies indicate that allelic loss is the most common finding at 22q12, the location of the NF2 gene that encodes the protein merlin. More recently, several reports have indicated that the mutation of NF2 occurs primarily in spinal cord ependymomas and have no relation with intracranial ependymoma (112). Allelic loss on chromosome arm 11q was identified in addition to the chromosome 22q loss and a mutation in the NF2 gene. Unlike the allelic loss on 22q, the 11q loss was found in both the spinal cord and intracranial tumors, and LOH11 exhibited a highly significant inverse associated with LOH22q. The multiple endocrine neoplasia type 1 gene (MEN1) is localized to this region, and this group suggested that a mutation in MEN1 appeared to be associated with the progression of ependymoma to anaplastic ependymoma. Differences between intracranial and spinal cord tumors now appear to include the gain of chromosome 7 in addition to the loss of chromosome 22q. Some studies showed that the frequent chromosomal aberrations in intracranial tumors were gain of 1q and losses on 6q, 9, and 13. Gain of 1q and loss on 9 were preferentially associated with anaplastic ependymomas. The gain of chromosome arm 1q and loss of chromosome 9 and chromosome arm 13q are associated preferentially with adults, whereas children younger than age 3 had a loss of chromosome arms 9p and 22q (113). Genes localized to these particular chromosomes suggest that the cyclinD/CDK4 pathway is deregulated in the more malignant tumors because the Rb1 gene is localized to 13q and the INK4A gene to 9p. TP53 mutations are rare in this tumor; however, it is possible to alter the function of the TP53 gene product (p53 protein) by p14ARF gene product, which is derived from INK4A gene, or by the MDM2 gene, which is found amplified in a small subset of ependymomas (114). Additionally, losses of chromosome 22 and 6q were most frequent in childhood intracranial ependymomas. These two chromosome imbalances were never associated. This may suggest at least two different progressions for ependymoma, one involving chromosome 22 lose and one involving 6q loss. Despite recurrent losses of 6q occur in 20% to 25% of the patients with ependymoma, no candidate tumor suppressor gene has been identified. As more studies of this nature are reported, it is becoming clear that the genetic aberrations do differ from other glial tumors. Scheil et al. (115) identified that myxopapillary ependymomas and ependymomas had a loss of genetic material on 13q14–q31 and several gains of chromosomes arms 17p, 9q, and 20q. It is possible that subsets of ependymoma will be found in which genetic differences will have prognostic value; however, more

56. Molecular Genetics of Brain Tumors

cases are needed to confirm or clarify the genetic aberrations that occur in ependymomas.

Medulloblastoma Deletions involving the short arm of chromosome 17 represent the most frequent genetic abnormality in medulloblastoma and occur in 40% to 50% of primary tumors. Such deletions of 17p may occur in the absence of other gross abnormalities of 17 or, more frequently, as a component of an isochromosome of 17q (i (17q)) (116). Although a number of studies in the literature have reported a significantly worse prognosis for patients whose tumors harbor deletions of 17p, this has not been a universal finding. The molecular consequence of 17p loss in medulloblastoma has also to be established. High-resolution mapping studies have identified a common region of LOH at 17p13.3. This region excludes TP53, which appears to be rarely mutated in medulloblastoma, but includes the site of a number of other candidate tumor suppressor genes, including the hypermethylated in cancer-1 (HIC-1) gene, which resides in a CpG island and is hypermethylated in medulloblastomas (117). Recently, research in molecular basis of medulloblastoma focused on the Ptc gene, which encodes Patched protein, a membrane receptor of Shh (Fig. 56-1B). Studies detected mutations in the Ptc gene in approximately 10% to 20% of sporadic medulloblastoma and a potential, but not exclusive, relationship with the thermoplastic phenotype (21,118). Interestingly, a number of cases of human medulloblastoma have been reported in which LOH at the Ptc locus (9q22.3–q31) is associated with preservation of the remaining wild-type allele (119). Studies of the Ptc heterozygous mice indicated that Ptc haploinsufficiency predisposes to medulloblastoma, but additional genetic alterations are probably necessary to product a full-blown tumor (24), which was confirmed by a study that showed the mice heterozygous for Ptc that lack p53 increased in the incidence and accelerated development of medulloblastoma (120). Regions of gene amplification have also been identified in approximately 10% of medulloblastoma. Rather than identifying a common region of gain, these studies have detected a diverse array of amplified loci including the ErbB1 (7p12), myc (8q24), and n-myc (2P24.1) oncogenes and a variety of other amplified loci, including 11q22.3 and 5p15.3, which are also amplified in glioblastoma (121,122). Although the clinical significance of most of these abnormalities remains unclear, evidence suggests that myc amplification is associated with aggressive disease. In two of the largest series in the literature, no long-term survivors were observed among a total of 17 cases with evidence of myc amplification. Furthermore, Scheurlen and colleagues (123) reported a myc amplification rate of 17%, three times that observed by other

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authors (124), among a population of clinically high-risk patients. Other frequent chromosomal abnormalities detected in medulloblastoma include gains of 7, gains and losses of chromosome 1q, and loss of chromosome 22. Chromosome 1 is frequently involved in chromosomal abnormalities in medulloblastoma. Although no consistent breakpoint has been identified in its alteration, gross rearrangement often results in partial or complete gain of 1q (121, 122). Indeed, chromosome 1q31–32 is a known hot spot for deletions in breast cancer and may harbor a tumor suppressor gene. However, a recent microsatellite analysis failed to demonstrate the loss of 1q31 in primary medulloblastoma (123). Gain of chromosome 7 also occurs with significant frequency in medulloblastoma and may be associated with the presence of i (17q) (121). Recently, atypical teratoid/rhabdoid tumors, which show an extremely poor outcome, were distinguished from medulloblastoma. Most atypical teratoid/rhabdoid tumors demonstrate monosomy chromosome 22 or deletions of 22q11 with Hsnf5/INI1 gene mutation, but the molecular consequence of Hsnf5/INI1 loss of function is unclear (4,125).

Choroid Plexus Tumors Choroid plexus tumors are rare intraventricular papillary neoplasms derived from choroid plexus epithelium, which account for only 0.4% to 0.6% of all intracranial and 2% to 3% of pediatric neoplasms. Plexus papillomas outnumber choroid plexus carcinomas by a ratio of 5:1 and around 80% of choroid plexus carcinomas arise in children. Among choroid plexus tumors, gains of chromosome were more frequent than losses. Choroid plexus papillomas typically show hyperdiploidy with gains on chromosomes 7, 9, 12, 15, 17, and 18, whereas the occasional association of choroid plexus carcinoma with constitutional duplication of the short arm of chromosome 9 suggests a relationship between 9p abnormality and abnormal growth of the choroid plexus. The most frequent abnormalities were gain of chromosome 7 and 12, and these imbalances were always associated. Several chromosomal imbalance differences could be found that were characteristic for a tumor entity or age group. In choroid plexus papillomas, gains of 5q, 6q, 7q, 9q, 15q, and 18q and losses of 21q were significantly more common, whereas choroid plexus carcinomas were characterized by gains of 1, 4q, 10, 14q, 20q, and 21q and losses of 5q, 9p, 11, 15q, and 18q. Among choroid plexus papillomas, children more often showed gains of 8q, 14q, 12, and 20q; adults mainly presented with gains of 5q, 6q, 15q, and 18q and losses of 22q. Although the number of aberrations overall as well as of gains and losses on their own bore no significance on survival among choroid plexus tumors, a

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significantly longer survival among patients with choroid plexus carcinomas was associated with gains of 9p and losses of 10q. This shows that aberrations differ between choroid plexus papillomas and choroid plexus carcinomas as well as between pediatric and adult choroid plexus papillomas, supporting the notion of different genetic pathways (126,127).

Meningioma Meningiomas are common intracranial tumors that arise from the meninges and compress the underlying brain. Meningiomas are usually benign, but some “atypical” meningiomas may recur locally, and some meningiomas are frankly malignant and may invade the brain or metastasize. Monosomy 22 is common in meningiomas, pointing to a chromosome 22q meningioma suppressor gene. Indeed, the NF2 gene on chromosome 22q is frequently mutated in meningiomas, clearly implicating it in meningothelial tumorigenesis. In sporadic meningiomas, both chromosome 22q allelic loss and NF2 gene mutations are more common in fibroblastic and transitional subtypes than in meningothelial forms. As in schwannomas, NF2 gene alterations result predominantly in immediate truncation, splicing abnormalities, or altered reading frames, producing grossly truncated proteins. Interestingly, NF2 gene mutations in meningiomas cluster in the moesin-ezrin-radixin homology domain in the first half of the coding sequence. Approximately 40% of meningiomas have neither NF2 gene mutations nor allelic loss of chromosome 22q. For these tumors, it is likely that a second meningioma tumor suppressor gene is involved. This putative second gene is probably not on chromosome 22q, because NF2 gene mutations in meningiomas correlate fairly closely with chromosome 22q loss. Nonetheless, a few meningiomas have been described with loss of portions of chromosome 22q that do not include the NF2 gene, suggesting the possibility of a second meningioma locus on chromosome 22. One candidate gene from this second chromosome 22q region is BAM22, a member of the b-adaptin gene family, which may be inactivated in some sporadic meningiomas. Another candidate is the MN1 gene, which is disrupted by a translocation in a meningioma. Furthermore, a family with multiple meningiomas but without vestibular schwannomas does not show linkage to the NF2 locus on chromosome 22q, suggesting yet another meningioma predisposition gene. Allelic losses in meningiomas have been noted on a variety of other chromosomes, including 1p3, 3p, 5p, 5q, 11, 13, and 17p. Atypical and malignant meningiomas are rare. Genetic analyses of malignant meningiomas have revealed preferential losses of chromosomes 1p, 10, and 14q. In one anaplastic meningioma, molecular genetic analysis of three morphologically distinct regions revealed LOH for

chromosomes 1p and 22q in all regions but for chromosomes 17p and 9q only in the malignant region. Chromosome 10 loss, in particular, has been associated with those meningiomas with morphologic features of malignancy, rather than those designated as malignant on the basis of brain invasion alone. Losses of the long arm of chromosome 18 are frequent in atypical and anaplastic meningiomas but rare in benign meningiomas, but four tumor suppressor genes (MADH2, MADH4, APM-1, and DCC) located at 18q21 do not play a significant role in the pathogenesis of meningiomas (128). History suggests that molecular profiling will not replace histology entirely, at least not in the near future. Ultrastructural and immunohistochemical analyses provided valuable ancillary techniques for tumor classification but did not supplant standard light microscopic evaluation. Histologic examination is simple and efficient. As a result, it should be part of the diagnostic armamentarium for many years to come. Nonetheless, molecular approaches, such as mRNA expression profiling and eventually delineation of protein pathway activity, will no doubt refine tumor classification. Novel methods may also come from other disciplines, such as neuroradiology. A perfect classification system, based on both phenotypic and molecular features, should provide accurate prediction of response to effective therapies and lack of response to ineffective toxic therapies.

FIGURE 56-5. Microscopic appearance of human glioblastoma induced by combination of myc and EGFRvIII expression (magnification, 40). The glioblastoma lesions were characterized by increased cell density, nuclear pleomorphism, and prominent mitotic figures. Microvascular proliferation was present within the tumor. One of the most characteristic morphologic features of human glioblastomas is necrosis with pseudopalisading (the presence of serpiginous zones of tumor necrosis bordered by palisades of viable tumor cells); this architectural pattern was also seen in the stem cell-induced glioblastoma. The arrowhead shows necrosis within a densely cellular region of tumor. The black arrow shows necrosis with pseudopalisading. The white arrow shows giant cell formation and nuclear pleomorphism.

56. Molecular Genetics of Brain Tumors

ORIGIN OF CANCER CELLS: STEM CELLS AND CANCER STEM CELLS Although there are great advances in research on genetic and molecule mechanism of brain tumor formation, it is still unclear what is the origin of brain tumor, differential cells or progenitor cells. Moreover, provided that some brain tumors originate from progenitors, which stage in the development of progenitors those tumors initiate from is still unknown. Therefore, a better understanding of the relationship between brain tumor and stem cell might provide insight into brain tumor biology and potential therapeutic strategies for this disease.

Some Cancer Cells May Originate from Stem Cells NSCs have a proliferative potential, are highly migratory, and can pursue remarkably diverse pathways to differentiate—all features intrinsic to glioma cells and likely characteristics for neoplastic cells of origin. Moreover, many brain tumor cells resemble undifferentiated cells in their gene expression and phenotypic characteristics. Identification of the cell of origin is crucial for understanding gliomagenesis. Some oncogenes transfer into cell types of specific stages of differentiation and allow identification of candidate cell types capable of giving rise to gliomas. We use an example from our own work to illustrate the differentiation characteristics of cells responding to the oncogenic effect of myc and EGFR. We isolated and cloned multipotent NSCs from human embryonic forebrain. Two human NSC lines were established (129). These self-renewing clones give rise to neurons, astrocytes, and oligodendrocytes in culture and in the newborn mouse brain after transplantation. Two retroviruses carrying full-length v-myc cDNA (130) and the deletes form of the EGFR (EGFRvIII) cDNA (eliminating the sequence of exons 2 to 7) (131) were used to transfer human NSCs and astrocytes. In soft agar, we observed a low level of anchorageindependent growth after infection with retro-myc (5.2% and 2.1% colony-forming efficiencies in NSCs and astrocytes, respectively), which was not statistically different compared with uninfected control cels or EGFRvIII expressing cells. However, EGFRvIII cooperates with myc overexpression to promote anchorage-independent growth of NSCs in culture (27.8% colony-forming efficiency in NSC-myc/EGFRvIII cells, p < 0.01). The trend toward anchorage-independent growth after infection with retromyc/retro-EGFRvIII was observed only in NSCs, not astrocytes (3.8% colony-forming efficiency in astrocytemyc/EGFRvIII cells, p > 0.05). After subcutaneous inoculating of the NSC-myc/ EGFRvIII cells into nude mice of BALB/c background, NSC-myc/EGFRvIII caused palpable tumors in 8 weeks.

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The animals that received astrocyte-myc/EGFRvIII cells had no tumors. To determine whether this growth advantage had site specificity, the NSC-myc/EGFRvIII, astrocyte-myc/EGFRvIII, NSC-myc, NSC-EGFRvIII, and NSC cells were stereotactically implanted into the brains of nude mice. After 11 weeks, 10 of 15 animals that received NSC-myc/EGFRvIII cells had tumors (Fig. 56-5), whereas only 1 of 15 mice implanted with NSC-myc cells developed a tumor. The animals that received astrocytemyc/EGFRvIII or NSC-EGFRvIII cells had no tumors. These results support the hypothesis that NSCs or early progenitors could be the targets of transformation in gliomagenesis. Holland et al.’s study also demonstrated that transfer of Akt and Ras yield glioblastomas only after transfer into nestin-expressing CNS-progenitor cells (Ntv-) but not after transfer into GFAP-expressing astrocytes (Gtv-) (132). In contrast, the activated form of the EGFR yields lesions resembling gliomas in both Gtv- and Ntv- mice but does so more commonly in Ntv- mice, suggesting that gliomas maybe arise from a certain developmental stage of NSC (133). Moreover, in a study by transferring the overexpression of PDGFB into either nestin-expressing neural progenitors or GFAP-expressing astrocytes, PDGF gene transfer converted cultured astrocytes into cells with morphologic and gene expression characteristics of glial precursors and gene transfer of PDGF to neural progenitors induced the formation of oligodendrogliomas more efficiently than transfer to astrocytes. The highest percentage of PDGF-induced malignant gliomas arose from of INK4A-ARF null progenitor cells. These data suggest that chronic autocrine PDGF signaling can promote a proliferating population of glial precursors and is potentially sufficient to induce gliomagenesis. Loss of INK4A-ARF is not required for PDGF-induced glioma formation but promotes tumor progression toward a more malignant phenotype (134). From the view of cell development, stem cells are vulnerable targets of transformation in some brain cancers because the machinery for self-renewal in stem cells is already activated, maintaining this activation may be simpler than turning it on de novo in a more differentiated cell and by self-renewing, and stem cells often persist for long periods of time, which provide a much greater opportunity for mutations, such as PDGFR and EGFR, to accumulate in individual stem cells than in most mature cell types. PDGF is crucial during normal glial development, and EGF is vital to NSC proliferation and survival. During embryogenesis, PDGF is expressed by neurons and astrocytes, where glial progenitors and neurons express the PDGF receptor. Low-grade astrocytomas have been shown to exhibit PDGF ligand and receptor overexpression, in addition to TP53 mutation. Oligodendrogliomas also show expression of the PDGF_receptor and amplifications of the PDGF receptor. Similarly,

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EGF is required for survival and proliferation to stem cells in ventricular zone. Overexpression of EGFR via retroviral vector transfer results in proliferation of stem cells as well as premature astrocytic differentiation, and EGF-responsive stem cells in the ventricular and subventricular zones retain the capacity to generate all three major cell types in vitro (135). NSCs transplanted into the lateral ventricle remain undifferentiated with simultaneous infusion of EGF but differentiate into astrocytes without the simultaneous infusion of EGF (136). As mentioned previously, EGFR is often amplified and mutated in high-grade gliomas. About 40% of glioblastomas with amplification express an activated form of EGFR, called EGFRvIII, which lacks a portion of the extracellular ligand binding domain and is constitutively autophosphorylated. This mutant form of EGFR confers enhanced tumorigenicity. Moreover, metastasis has many parallels with migratory ontogenic states; genes involved in developmental processes such as neural crest formation are also involved in tumor invasion. These clues of glioma originated from NSCs provided a new insight into glioma biology, which may contribute to glioma classification and treatment. We could expect some adult gliomas to derive originally from adult neural progenitor cells, which reside in several locations, including the subventricular zone of the lateral ventricle, although such tumor stem cells could migrate and establish tumor in different areas. Because not all gliomas derive from stem cells, an alternative is that gliomas could arise from dedifferentiating glia cells, which is confirmed by a study that showed transduction of INK4A/ARF(
/
) NSCs or astrocytes with constitutively active EGFR induced a common high-grade glioma phenotype and demonstrated NSCs and astrocytes as equally permissive compartments for gliomagenesis in INK4A/ARF knockout mice (96).

Cancer Stem Cells Many observations suggest that analogies between normal stem cells and tumorigenic cells may be appropriate. Both normal stem cells and tumorigenic cells have extensive proliferative potential and the ability to give rise to new (normal and abnormal) tissues. Some tumors are composed of heterogeneous combinations of cells, with different phenotypic characteristics and different proliferative potentials. Because most tumors have a clonal origin, tumorigenic cancer cells can give rise to phenotypically diverse progeny, which include cancer cells with indefinite proliferative potential, as well as cancer cells with limited or no proliferative potential. This suggests that tumorigenic cancer cells undergo processes that are analogous to the self-renewal and differentiation of normal stem cells. Thus, tumorigenic cells can be thought of as cancer stem cells that undergo an aberrant and poorly

regulated process of organogenesis analogous to what normal stem cells behave. Pioneer studies in human leukemia and breast cancer cells have shown that a very small fraction of the original tumor is uniquely capable of recapitulating the tumor following transplant (136,137). In the case of brain tumors, the presence of NSC-like cells in brain tumors has suggested that these tumors contain “cancer stem cells” that are critical for their propagation (138–140). We have sorted out SP cells from cultured human primary tumor cells originated from 1 sample of ependymoma and 1 sample of glioblastoma. It demonstrated that SP cells expressed nestin and vimentin, which originally used as mark of neural stem cells. In the test of single cell cloning, 25% of SP cells developed clones, but only 2% in non SP cells. Brain tumors were found in 6 of 12 null mice 8 weeks after intracerebral implantation of SP cells, whereas 1 of 12 were found in non SP groups (p < 0.01). The SP cells were resistant to chemotherapeutic drugs Vm26, Vp and Cisplatin, but non SP cells were sensitive to VM26 and Vp (141). Sorting of SP cells is a simple approach for isolation of brain cancer stem cells. Cancer stem cells present higher capability to form colonies in cell culture and to develop tumors in the brain. It is critical to stress the distinction beween neural stem cells and cancer stem cells. Neural stem cells exhibit extensive self-renewal and have the capacity to differentiate into multiple cell types (e.g., neurons, astrocytes, and oligodendrocytes). Cancer stem cells may also exhibit extensive self-renewal and multipotency. But most importantly, cancer stem cells are defined by their capacity to maintain the long-term growth of a tumor in vivo. This capacity for tumor propagation is most compellingly demonstrated by transplanting putative cancer stem cells into a new host and showing that they can recapitulate the growth of the tumor. To develop more effective therapies for malignant brain tumors, we need a deeper understanding of their molecular and cellular basis. This understanding may come from an appreciation of the relationship between brain tumors and stem cells.

GENETICALLY ENGINEERED MOUSE MODELS OF GLIOMAS In addition to the histologic features that define gliomas, mutations and other alterations in gene expression and signal transduction are classically found in these tumors. Some of these alterations are likely to be the effects of neoplastic phenotype, whereas others may be causative agents essential to the etiologic origin of the disease. The determination of whether specific genetic alterations, either individually or in combination, can serve as the etiology of glioma requires modeling in animals with the fulfillment of Koch’s postulates. Animal modeling studies not only

56. Molecular Genetics of Brain Tumors

provide information on the potential causes of glioma formation and origin of glioma, they also identify novel candidate targets for therapy and provide tumor-bearing animals for preclinical trials. Recently, remarkable strides have been made in the generation of mouse models of the diffuse glioma that provide unparalleled opportunities for advancing our knowledge of the etiology, maintenance, and treatment of this lethal class of tumors.

Genetic-Modification Strategies Mainly, two genetic-modification strategies are established to develop the models of glioma formation. Germline Genetic Modification Strategy This strategy includes gain-of-function transgenic approaches and targeted-deletion approaches. Transgenic mice express the gene of interest in all cells that use the promoter that drives a construct’s expression, whereas knockout mice lose expression of the targeted gene in all cells that would normally express it. In both cases, large numbers of cells of a specific cell type are modified by these strategies. In germline-modification models, the tissue with the gene expression alteration is frequently developmentally normal. Tumors are often initiated and develop in this cell population from secondary, usually unknown, genetic events. These strategies are ideal for demonstrating components that contribute to the induction and progression of tumorigenesis. Furthermore, breeding can be used to combine both transgenes and targeted deletions. Most studies to model cancer in mice use these germlinemodification strategies at present. However, because extensive breeding is required to combine multiple mutations, the analysis of large numbers of permutations of genetic alterations is expensive and time consuming. Somatic-Cell Gene-Transfer Strategy In this strategy combinations of mutations are transferred by retroviral infection to specific cell types postnatally. The analysis of permutations of multiple mutations with such systems is faster and less labor intensive than with germline strategies. Because the number of cells that are infected initially is small, secondary events required for tumor initiation are unlikely to occur. However, if a tumor forms, the introduced alterations were probably sufficient to induce tumor formation. This point is striking in cases where abnormally elevated expression of a single gene, such as that encoding the polyoma virus middle T antigen, in a few hundred astrocytes induces the formation of gliomas at high frequency. Both germline and somatic cell gene-transfer strategies have advantages and disadvantages, and comparing

813

results from each can be informative. In germline strategies, mutations that contribute to, but are insufficient for, oncogenesis will give rise to tumors by the acquisition of other mutations, which can then be identified. In somatic-cell gene-transfer strategies, insufficient combinations are unlikely to score positively for tumor formation and therefore might be missed. Furthermore, causing retroviral infection by direct injection into the brain generates injury and inflammation that disrupts the local environment and alters gene expression in potential target cells. The contribution that these local effects have on tumor formation in such models is not clear. By contrast, germline strategies do not produce local injury. Additional mutations and alterations in gene expression are likely to occur during tumor progression regardless of the strategy for tumor induction. However, in some cases most tumor cells might evolve so as to continue to depend on the initiating mutations. There are reports of reversible oncogenesis in mice using tetracycline-induced H-Ras expression to produce melanomas and that of myc to generate lymphomas and skin tumors. Despite genomic instability in these tumors, they completely regress with the removal of the initiating genetic alteration. Whether any of the gliomas are initiated by a single event or a by summation of many contributing factors is unknown. However, identifying the pathways that have the potential to initiate glioma formation is an important step towards rational therapeutic approaches for this disease.

Glioma Mouse Models with Defined Genetic Alterations Recently, a number of animal models have been introduced that exquisitely replicate many of the quintessential morphologic features of the different classes of human gliomas. Models have been created that reproduce the morphologic features of virtually all of the specific subtypes of diffuse glioma with remarkable fidelity, including the full spectrum of diffuse astrocytoma, low-grade and anaplastic oligodendrogliomas, and mixed glioma. The modeling systems that have been used to generate these tumors in mice have also identified common biologic pathways that appear capable of contributing to or causing the formation of gliomas. A summary of the different mouse models, including defined genetic alterations, cell of origin, and resultant glioma histology, is provided in Table 56-2. Diffuse Astrocytoma Several transgenic mouse lines have been generated that develop low-grade diffuse astrocytomas. In one system, a transgene containing the GFAP promoter/enhancer and V12 H-Ras was introduced into the mirine germline (143).

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w TABLE 56-2

Mouse Models with Defined Genetic Backgrounds Cell of Origin/ Affected Cells

Tumor

GFAP/v-src

All GFAP-expressing cells (astrocytes)

Low-grade (early) and high-grade (late) astrocytoma

GFAP/V12H-Ras

All GFAP-expressing cells (astrocytes)

Heterozygous; predominantly single low-grade astrocytomas Hemozygous; predominantly multifocal high-grade astrocytomas

All cells in animal

Astrocytomas to predominantly GM Strain specific penetrance of astrocytoma phenotype

Murine retrovirus/ PDGF B-chain

Mixed cell population in brain

GBM; primitive neuroectodermal tumor

K-Ras plus Akt

Infected nestin-producing CNS progenitor cells (Nt-va)

GBM

K-Ras plus Akt

Infected astrocytes (Gt-va)

No tumors

(p16INK4A/P19ARF)+/+

PDGF-B

Infected nestin-producing CNS progenitor cells (Nt-va)

Low-grade oligo

(p16INK4A/P19ARF)
/


PDGF-B

Infected nestin-producing CNS progenitor cells (Nt-va)

High-grade oligo

(p16INK4A/P19ARF)+/+ (p16INK4A/P19ARF)
/


PDGF-B

Infected astrocytes(Gt-va)

Mixed oligoastrocytomas

(p16INK4A/P19ARF)+/+

Polyoma middle T

Infected astrocytes(Gt-va)

Mixed oligoastrocytomas

All cells in animal

Oligodendrogliomas

Gene Disruption

Transgene

Somatic Transfer

NF1
/+; P53
/+in cis

P19ARF
/


One line developed solitary tumors resembling low-grade astrocytomas in 80% of the animals and multiple tumors resembling anaplastic astrocytomas in 20% of animals when the transgene was in the heterozygous state. With the transgene in the homozygous state, the animals developed multifocal tumors resembling anaplastic astrocytomas. Consistent with the uniformly higher grade of the tumors in the homozygous mice, their survival time was shortened compared with the heterozygous mice; the median survival of heterozygous mice was 3 months compared with 4 weeks for the homozygous mice. In another model, transgenic mice were generated using the v-src kinase under the control of GAFP regulatory elements. In 14.4% of mice, small proliferative foci as well as overt astrocytomas developed in the brain and spinal cord. Early lesions were similar to low-grade astrocytomas; at late stages the tumors exhibited the histological characteristics of anaplastic astrocytoma and glioblastoma (144).

Glioblastoma The formation of glioblastoma has been observed in both GFAP/v-src and GFAP/v12H-Ras transgenic mice (143,144). Cell lines established from the high-grade astrocytomas of the GFAP/v12H-Ras transgenic mice show abnormal karyotypes, as well as expression of proteins known to be involved in apoptosis and cell cycle progression, including MDM2, p16INK4A, p19ARF, PTEN, and EGFR (143). From these experiments, it is unclear which of these genes in combination with v12H-Ras is sufficient for gliomagenesis. The GFAP/v-src induced glioblastomas have not yet been studied on the genetic or karyotypic level. A spectrum of low- to high-grade astrocytomas has also been reported in knockout mice heterozygous for NF1 and TP53 (145). Because neurofibromin, the gene product of NF1, is a negative regulator of Ras, this is further evidence for the central role of Ras in gliomagenesis.

56. Molecular Genetics of Brain Tumors

The introduction of combined activated G12D mutation form of K-Ras and Akt into Ntv- transgenic mice induces glioblastoma formation (132). The mice develop tumors within 9 weeks. Tumor formation is not observed with either oncogene alone or when the combination is injected into Gtv- mice. Thus, modeling of glioblastoma in mice requires precursor cells on the other. Transfer of K-Ras and Akt into Ntv- mice carrying an inactivated allele of INK4A-ARF accelerates the development of glioblastoma. Interestingly, transfer of K-Ras into Ntv- mice carrying the INK4A-ARF deletion yields gliosarcoma in 30% of offspring. This is not seen with the transfer of either K-Ras or Akt alone into Gtv- mice. However, the combination of K-Ras and Akt transferred together into Gtv- mice carrying an inactivated allele of INK4A-ARF produces spindle cell gliomas and gemistocytic astrocytomas. The production of tumors in Gtv- mice by the transfer of K-Ras/Akt requires the absence of p16INK4A/ p19ARF. Glioblastoma induced by somatic transfer of KRas and Akt into Ntv- transgenic mice do not show karyotypic abnormalities as confirmed by karyotype analysis. A separate series of experiments has demonstrated that the ectopic expression of V12H-Ras, hTERT, and the papilloma virus oncogenes E6/E7, which inhibit the p53/Rb pathway, converts human astrocytes into cells resembling those of anaplastic astrocytoma (146). With the further transfer of Akt, the cells acquire features of glioblastoma (147). These data support the observation that Ras can cooperate with Akt signaling to generate glial tumors of high malignancy. Oligodendroglioma Transfer of the gene encoding PDGFB into Ntv- mice carrying an inactivated allele of INK4A-ARF generates highgrade oligodendroglioma (134). Features characteristic of oligodendroglioma were also found among the tumors induced by the transfer of the gene encoding the PDGFB chain through the MoMULV into newborn mice. A second series of experiments using transgenic mice that express the viral oncogene v-ErbB, an active homologue of EGFR, from the S-100b promoter (which is active in astrocytes and glial precursors) generated similar oligodendrogliomas. Thus, ectopic expression of activated RTK growth factors may lead to oligodendroglioma formation. Mice carrying a deletion of p19ARF develop oligodendrogliomas with some frequency, in contrast to the lack of gliomas arising in p16INK4A/p19ARF deficient mice. The reasons for this discrepancy are unclear. Finally, infection of newborn mouse brains with replication-competent virus expressing the PDGFB chain results in a variety of high-grade gliomas, including glioblastoma and oligodendroglial tumors, in addition to tumors resembling primitive neuroectodermal tumor (31).

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Mixed Oligoastrocytoma Polyoma middle T antigen stimulates Shc (leading to Ras activation), PI3-kinase (leading to Akt activation), and Src. The transfer of polyoma middle T antigen into Gtv- mice via the RCAS vector induces mixed oligoastrocytomas (144). Tumors were seen at 9 weeks in 9 of 33 mice. The histologic features of these tumors were similar to those seen in human anaplastic astrocytomas, anaplastic oligodendrogliomas, and anaplastic mixed oligoastrocytomas. Features of other primary CNS neoplasms, such as primitive neuroectodermal tumors or ependymomas, were not seen in any of the lesions. Polyoma middle T antigen does not require additional genetic alterations, as also reported for the induction of gliomas by EGFR (133) and glioblastomas by K-Ras/Akt (132). This is consistent with the activation of multiple pathways required for tumor induction by polyoma middle T antigen alone. A second approach for the generation of mixed gliomas is the transfer of PDGF into Gtv- mice (134). These tumors have a very similar histologic appearance to those generated by polyoma middle T antigene. Unclassified Glioma The transfer of the gene encoding constitutively active EGFR into Ntv- or Gtv- mice leads to glioma production when the mice are also deficient at the INK4A-ARF locus (coding for p16INK4A and p19ARF); tumors are more efficiently generated in Ntv- mice than in Gtv-. The somatic transfer of active EGFR into mice carrying the wild-type allele of the INK4A-ARF locus does not result in glioma formation (133). Furthermore, mice lacking the INK4AARF locus develop lymphomas and sarcomas with a mean latency of 34 weeks. Thus, activated EGFR receptor requires the inactivation of the INK4A-ARF locus to yield glioma formation. Determination of which of the two gene products encoded by the INK4A-ARF locus, p16INK4A, p19ARF (the mouse homologue of human p14ARF), or both, needs to be inactivated to yield glioma formation in combination with EGFR requires further investigation. Introduced mouse models of the diffuse gliomas are likely to yield significant insight into the complex process of gliomagenesis. They provide an unparalleled approach for precise manipulation and study of the effects gene alterations that has heretofore been unavailable. The new mouse models may also provide an opportunity for improvement of glioma therapy through identifying the critical events involved in gliomagenesis.

SUMMARY As with most other tumors, there are seven essential alterations in brain tumor cell physiology that collectively dic-

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tate malignant growth: deviation of cell development, perturbation of growth factor signaling pathway, insensitivity to antigrowth signals, limitless replicative potential, evasion of apoptosis, sustained angiogenesis, and invasion. These intrinsic and extrinsic mechanisms result in heterogeneous brain tumor, which mainly present the morphologic difference from traditionally pathologic views. Therefore, it is necessary to explore a molecular genetic approach to refine current classification schemes, which can provide more valuable information for therapy and prognosis of this fatal disease. The research on multipotent progenitor cells/NSC makes it necessary to rethink the origin of brain tumors. Studies on biology of stem cells and brain tumor cells suggest at least some brain tumors may arise from neural progenitors. Moreover, transfer oncogenes to different progenitors allows identification of candidate cell types capable of giving rise to gliomas, which confirm this hypothesis. Compared with the common features between stem cells and cancer cells, it is possible that some cancer stem cells existed in tumors, which maybe result in some heterogeneity in tumors and refractory to therapy. Animal modeling studies not only provide information on the potential causes of glioma formation, they also identify novel candidate targets for therapy and provide tumorbearing animals for preclinical trials. Remarkable strides have been made in the generation of mouse models of the diffuse glioma that provide unparalleled opportunities for advancing our knowledge of the etiology, maintenance, and treatment of this lethal class of tumors.

ACKNOWLEDGMENT This work was partially supported by the Chinese Nature Science Foundation, Shanghai Science Foundation, and 973 Grant CB509907.

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C h a p t e r

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w Predicting Medulloblastoma Outcome Stephanie Greene and Scott L. Pomeroy Medulloblastomas, the most common malignant pediatric brain tumors, represent about 20% of pediatric brain tumors. They largely affect children under the age of 10, striking boys nearly twice as often as girls. The tumors most often arise in the vermis of the cerebellum, extending into and often filling the fourth ventricle. They arise in the cerebellar hemispheres in about 25% of cases, typically in older children. Medulloblastomas express neuronal markers and transcription factors specific to cerebellar granule cells, such as PAX6, EN2, NSCL1, and Zic (1,2), consistent with the conclusion that they arise by oncogenic transformation of cerebellar granule cell progenitors. Research is focusing on the molecular genetic events leading to medulloblastoma formation and progression in an attempt to elucidate markers of biologic aggressiveness. Modern therapeutic strategies for medulloblastoma combine surgery, external beam radiation, and multipledrug chemotherapy to promote long-term survival to rates approaching 80% (3,4). Despite these successes, treatments that have enabled substantial improvements in mortality are injurious to the central nervous system, leaving most survivors with life-altering neurologic and cognitive sequelae (5). As mortality from the disease has declined, a growing focus of new therapeutic protocols has been to decrease the morbidity of treatment methods (3,5,6). A major challenge, then, is to identify less aggressive disease at the time of diagnosis so that toxic therapy can be minimized while the efficacy of treatment is maintained and to target highrisk patients for more intensive therapy to maximize their chances of survival. A method of reliably stratifying patients according to the aggressiveness of their disease and the ability to predict their outcome with near certainty are prerequisites for tailored therapy.

CLINICAL PROGNOSTIC FACTORS Staging for risk stratification is based on clinical criteria that have been established over several decades. Initially proposed in 1969, the Chang system combines preoperative tumor size (T stage) and the presence of metastases

(M stage) to provide the most widely used criteria for judging extent of disease at the time of initial diagnosis (7). Recent studies have not found T stage to predict outcome (4,8–10). Positive cerebrospinal fluid cytology (stage M1) was historically linked to poor prognosis, although it has not been linked to decreased survival in recent clinical trials (4,11). Local or distant metastases (stages M2, M3, M4) have consistently been found to correlate with lower survival rates (6,8). Moreover, extent of surgical resection has been correlated with survival in several trials (4,12,13), leaving M stage and postoperative residual disease as the primary prognostic criteria for risk stratification at the time of initial treatment. The possible effect of age on outcome is confounded by differences in treatment. For the past two decades, infants have been treated with protocols that delay or exclude radiation, because the sequelae of craniospinal irradiation are inversely related to the patient’s age at the time of treatment (4,14). Radiation therapy is a mainstay of treatment of all other medulloblastoma patients, however, and has been shown to be highly effective in the treatment of this tumor (15). Younger children are more likely to have disseminated disease at the time of diagnosis (16), and large-cell/anaplastic tumors are more common in younger children (17,18). A better outcome for adult medulloblastoma has been reported (19). In studies of children older than ages 2 or 3 years, in whom radiotherapy was used, the prognostic effect of age is unclear (4,9,12,20). Previous studies of the effect of gender on clinical outcome in patients with medulloblastoma reached conflicting conclusions. In several studies, shorter survival times for boys were statistically significant (21–23). Other groups were able to demonstrate little or no association between gender and outcome (8–10,12,24–26). In summary, these clinical criteria provide the basis of current risk stratification criteria, which include Chang M stage and amount of residual disease. Children older than age 3 years with no evidence of metastatic disease and minimal postoperative residual disease (less than 1.5 cm3) are considered to be “standard risk.” Those with metastases or high residual tumor burden are considered 821

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to be at higher risk for recurrence. Infants are considered high risk, but the issue is confounded by treatment factors. About 80% of standard risk patients can be considered cured, whereas only about 50% of higher risk patients will achieve long-term survival (20). In practice, some patients respond poorly to therapy for unclear reasons, whereas others respond better than predicted by their clinical grade alone. Because of inaccuracies of clinical predictive criteria, biologic markers have been sought that may distinguish those patients who can be cured with reduced therapy from those requiring more extensive treatment.

TISSUE MARKERS Histopathologic grading has been documented to be of clinical significance, but no pathologic staging system is in use at present (23). Four subtypes of medulloblastoma are described in the neuropathology literature. Classic medulloblastoma is composed of densely packed small blue cells, with frequent mitoses and apoptosis. Desmoplastic medulloblastoma is characterized by abundant reticular stroma and reticulin-free nodules containing cells with uniform nuclei that undergo neuronal differentiation (23,27). A better prognosis has been historically associated with the desmoplastic variant, but many studies have found no correlation between the desmoplastic variant and survival (10,23,28). Allelic loss of 9q has been reported and has been associated with the desmoplastic variant of medulloblastoma (29). The sonic hedgehog (SHH) receptor PTCH is on chromosome 9q22 and is a likely tumor suppressor gene for desmoplastic medulloblastoma (2). The features of two other subtypes have been used to attempt prediction of outcome, given the dramatically different prognoses associated with them. Each of these subtypes makes up about 5% of medulloblastoma diagnoses and has distinct histologic findings. A variant of the desmoplastic subtype, known as “medulloblastoma with extensive nodularity,” has an excellent prognosis and usually occurs in patients younger than age 1 year (23). Large-cell/ anaplastic medulloblastoma, a recently described aggressive variant containing cells with large nuclei, abundant mitoses and apoptosis, and large areas of necrosis, results in rapid dissemination of disease and progression to death within months, despite otherwise favorable clinical features (17,18,23,30). Nodularity, apoptosis, proliferative potential, and anaplasia have been assessed as possible predictors of outcome. The evidence for nodularity is not suggestive of a link with outcome, and that for apoptotic activity is controversial (31,32). Although markers of proliferation and increasing degrees of anaplasia have been associated with worse outcomes, no histologic classification scheme has emerged (12,23).

Atypical teratoid/rhabdoid tumors have been recently distinguished from medulloblastoma and may on occasion have identical histology, but they have an extremely poor prognosis (33). They typically occur in very young children and can be distinguished from other malignant brain tumors by the presence of characteristic “rhabdoid” cells or mutations of the INI-1 gene, or both (34,35).

GENETIC MARKERS Early efforts focused on DNA ploidy and were very inconclusive. In some cases, aneuploidy was correlated with a poor prognosis, whereas others have found it to be associated with a better outcome or lower incidence of metastasis (12,25,36,37). Genetic studies have more recently focused on specific chromosomal deletions, amplifications, and mutations. Several common chromosomal anomalies, deletions of 17p and 9q and isochromosome 17q, have been identified with cytogenetic analysis but have inconsistently been associated with outcome. A deletion of the short arm of chromosome 17 (17p) is the most frequent genetic abnormality in medulloblastoma, occurring in 40% to 50% of tumors (20,29,38). Deletions of chromosome 17p have been linked to poor outcome (39,40), although a few groups have found no association with outcome (41,42). Isochromosome 17q (loss of chromosome 17p with gain of chromosome 17q), seen in about 30% of medulloblastoma samples, has been found to be variably associated with outcome (43,44). Several groups have reported that patients with isolated 17p loss had substantially higher 5-year mortality rates than those with isochromosome 17q (20,45). Although TP53, the most commonly mutated tumor suppressor gene in cancer, is located at 17p13.1, this location is proximal to the usual breakpoint of 17p in medulloblastoma (42,46). Moreover, TP53 mutations have been identified in only some medulloblastoma samples (47). TP53 is overexpressed in less than 5% of medulloblastomas, and this overexpression is not related to TP53 mutation (45,48). High levels of TP53 expression have been associated with both the Ki-67 proliferation index and poor outcome (45,47,48). A second candidate gene, the hypermethylated-in-cancer-1 (HIC-1) gene, is located at 17p13.3 (49). MYC (or c-MYC), a proto-oncogene, is a transcriptional regulator implicated in the regulation of genes involved in the cell cycle, apoptosis, and embryonal differentiation and is the most commonly amplified gene in medulloblastoma (50,51). MYC amplification has been found to be highly associated with poor outcome in medulloblastoma (50,51,52). It has been linked to large cell morphology and to early dissemination of disease through cerebrospinal fluid pathways (30,51). MYC messenger RNA expression

57. Predicting Medulloblastoma Outcome

has been shown to be a more robust predictor of outcome than clinical factors (50). Amplification of N-MYC has also been identified in a small subset of medulloblastoma samples (30,51,53). Amplification of N-MYC is well characterized as being linked to aggressive disease in neuroblastoma, and has been reported to be linked to aggressive disease in medulloblastoma as well (30,51). It has also been linked to the large cell phenotype (30). In summary, abnormalities of chromosome 17 have been inconsistently linked to outcome, whereas amplification of c-MYC and N-MYC, which have been more reliably associated with outcome, occur in only a small percentage of medulloblastoma samples. For these reasons, cytogenetic markers have not been used routinely for clinical staging.

GENE EXPRESSION- BASED MARKERS A great amount of attention has been given to the idea of expressed genes as outcome markers. Initially, glial fibrillary acidic protein expression was associated with poor outcome, but the association is weak. For example, an increased risk of relapse has been quantitatively associated with the extent of glial fibrillary acidic protein expression in some series, whereas others have shown glial fibrillary acidic protein expression to be associated with increased 5-year survival rates (24,28). High levels of expression of the neurotrophin-3 (NT3) receptor TRKC, a transmembrane glycoprotein receptor with tyrosine kinase activity, have been correlated with longer intervals of progression-free survival as well as with a more favorable outcome overall (1,54,55). Patients with high TRKC expression were less likely to have metastatic disease at the time of diagnosis, and their specimens were more likely to demonstrate apoptosis (1). Under normal conditions, mature (and thus more differentiated) granule cells respond to NT3 by promoting axonal maturation (56). TRKC might be a marker of a more differentiated, and therefore less aggressive, tumor; alternatively, it may promote differentiation and apoptosis, thereby slowing tumor growth, as is seen with TRKA and neuroblastoma (54). TRKC expression has been shown to be a better predictor of outcome than clinical factors (55). Dysregulated overexpression of ErbB2 and ErbB4, two type I tyrosine kinase receptors, has been associated with aggressive disease (20,45,57). The coexpression of the ErbB4 receptor, the main receptor for neuregulin 1-beta (NRG1B), with the ErbB2 receptor occurs in over 50% of tumors and has been associated with decreased survival in medulloblastoma patients (57–59). Coexpression of ErbB2, ErbB4, and NRG1B has been shown to be

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significantly related to the presence of metastases at the time of diagnosis (57). Combinations of these biologic markers have been used to predict outcome with better accuracy than either clinical factors or any individual marker. An analysis combining c-MYC and TRKC expression was a better predictor of outcome than clinical models (55). A classification of high- and low-risk tumors with high risk defined by a combination of high ErbB2 receptor expression and/or isolated 17p loss yielded an 83% 5-year mortality rate for the high-risk group, with a 32% mortality rate for the lowrisk group (20). These studies suggest that combining multiple markers may be more accurate than single markers for predicting outcome. The idea that combinations of markers have increased accuracy is supported by DNA array-based gene expression profiling. One recent study identified genes that were differentially expressed in 23 metastatic and nonmetastatic tumors, and a model predictive of metastatic risk was developed (60). Platelet-derived growth factor receptor ␣ (PDGFRA) was shown to be associated with metastatic disease, as were downstream members of the RAS/mitogenactivated protein kinase signal transduction pathway (60). Biologic activity of these markers is suggested by experiments demonstrating that antibodies to PDGFRA could block both PDGFA-induced cell migration in the Daoy medulloblastoma cell line and PDGFA-induced phosphorylation of downstream mitogen-activated protein kinases (60). Moreover, SPARC (secreted protein acidic and rich in cysteine) and other proangiogenic genes, growth factors, transcriptional regulators, and oncogenes were identified by array analysis as being present at elevated levels in metastatic medulloblastoma samples. SPARC expression also has been linked to metastatic prostate cancer. Expression-array profiling has demonstrated differential gene expression in classic and desmoplastic medulloblastoma (2). Interestingly, PTCH, GLI, insulin-like growth factor II, and N-MYC, all of which are transcriptionally regulated by the SHH pathway, were associated with desmoplastic histology (62). Earlier work showed mutations of PTCH to be the genetic lesion in patients with Gorlin syndrome (nevoid basal cell carcinoma syndrome), an autosomal dominant disorder with several manifestations, including an increased incidence of desmoplastic medulloblastoma (3% to 5%), and mutations in PTCH have also been reported to occur in a fraction of sporadic desmoplastic medulloblastoma (61). These results suggest that other molecules of the SHH pathway may be linked to desmoplastic medulloblastomas. Gene expression profiles were also highly correlated with response to therapy (2). A clinical outcome model based on gene expression profiles was superior to clinical staging criteria or single gene outcome predictors. Novel genes were identified that corresponded to outcome; those

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correlated with favorable outcome were related to cerebellar differentiation (NSCL1, TRKC) or encoded extracellular matrix proteins (collagen type V, elastin), whereas those correlated with poor prognosis involved cell proliferation and metabolism (MYBL2, enolase 1, LDH), including genes encoding ribosomal proteins (2). Further validation of array-based outcome predictors is needed before such models could be implemented clinically.

FUTURE DIRECTIONS High-risk patients at present are identified by clinical factors, including age less than 3 years, residual tumor burden after surgery, and the presence of metastatic disease. The presence of histopathologic markers of proliferation and anaplasia may indicate higher risk, although the risk assessment must remain clinically based until the validity of molecular markers is established. Several expressed markers have been able to identify poor-outcome patients with better accuracy than clinical staging. High levels of expression of TRKC have been correlated with a favorable outcome (1,54,55), whereas high ErbB2 receptor and c-MYC expression identify a subpopulation of high-risk patients (52,55,59). Combinations of molecular risk factors have been applied to better define high- and low-risk groups of patients. Gene expressionarray analysis has been used to identify genes that might be predictive of outcome. These biologic markers require further study to more reliably identify their roles in the pathogenesis of medulloblastoma and to use this knowledge to better classify patients. The ability to better stratify patients according to risk will enable therapy to be tailored to the individual patient to minimize the toxicity of therapy while maximizing the likelihood of cure.

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57. Predicting Medulloblastoma Outcome 30. Reardon DA, Jenkins JJ, Sublett JE, et al. Multiple genomic alterations including N-MYC amplification in a primary large cell medulloblastoma. Pediatr Neurosurg 2000;32:187–191. 31. Haslam RH, Lamborn KR, Becker LE et al. Tumor cell apoptosis present at diagnosis may predict treatment outcome for patients with medulloblastoma. J Pediatr Hematol Oncol 1998;20:520–527. 32. Schiffer D, Cavalla P, Chio A, et al. Tumor cell proliferation and apoptosis in medulloblastoma. Acta Neuropathol 1994;87: 362–370. 33. Rorke LB, Packer RJ, Biegel JA. Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood: definition of an entity. J Neurosurg 1996;85:56–65. 34. Biegel JA, Zhou JY, Rorke LB, et al. Germ-line and acquired mutations of INI1 in atypical teratoid and rhabdoid tumors. Cancer Res 1998;59:74–79. 35. Versteege I, Sevenet, Lange, et al. Truncating mutations of hSNF5/ INI1 in aggressive paediatric cancer. Nature 1998;394:203–206. 36. Latimer FR, AlSaadi AA, Robbins TO. Cytogenetic studies of human brain tumors and their clinical significance. J Neurooncol 1987;4:287–291. 37. Yasue M, Tadanori T, Engelhard H, et al. Prognostic significance of DNA ploidy in medulloblastoma of childhood. J Neurosurg 1989;70:385–391. 38. Bigner SH, Mark J, Friedman HS, et al. Structural chromosomal abnormalities in human medulloblastoma. Cancer Genet Cytogenet 1988;30:91–101. 39. Cogen PH. Prognostic significance of molecular genetic markers in childhood brain tumours. Pediatr Neurosurg 1991/1992; 17:245–250. 40. Batra SK, Mclendon RE, Koos JS, et al. Prognostic implications of chromosome 17p deletions in human medulloblastoma. J Neurooncol 1995;24:39–45. 41. Emadian JM, Mcdonald JD, Gerken SC, et al. Correlation of chromosome 17p loss with clinical outcome in medulloblastoma. Clin Cancer Res 1996;2:1559–1564. 42. Biegel JA, Janss AJ, Raffel C, et al. Prognostic significance of chromosome 17p deletions in childhood primitive neuroectodermal tumours (medulloblastomas) of the central nervous system. Clin Cancer Res 1997;3:473–478. 43. Biegel JA, Rorke LB, Packer RJ, et al. Isochromosome 17q in primitive neuroectodermal tumors of the central nervous system. Genes Chromosomes Cancer 1989;1:139–147. 44. Cogen PH, McDonald JD. Tumor suppressor genes and medulloblastoma. J Neurooncol 1996;29:103–112. 45. Nam DH, Wang KC, Kim YM, et al. The effect of isochromosome 17q presence, proliferative and apoptotic indices, expression of c-ErbB2, BCL2 and p53 proteins on the prognosis of medulloblastoma. J Korean Med Scie 2000;15:452–456. 46. Biegel JA, Burk CD, Barr FG, et al. Evidence for a 17p tumor-related locus distinct from P53 in pediatric primitive neuroectodermal tumors. Cancer Res 1992;52:3391–3395.

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47. Adesina AM, Nabantoglu J, Cavenee WK, et al. P53 gene mutations and MDM2 gene amplification are uncommon in medulloblastoma. Cancer Res 1994;54:5649–5651. 48. Jaros E, Lunec J, Perry RH, et al. P53 protein overexpression identifies a group of central primitive neuroectodermal tumours with poor prognosis. Br J Cancer 1993;68:801–807. 49. Rood BR, Zhang H, Weitman DM, et al. Hypermethylation of HIC-1 and 17p allelic loss in medulloblastoma. Cancer Res 2002;62:3794–3797. 50. Grotzer MA, Hogarty MD, Janss AJ, et al. MYC messenger RNA expression predicts survival outcome in childhood primitive neuroectodermal tumor/medulloblastoma. Clin Cancer Res 2001;7:2425–2433. 51. Aldosari N, Bigner SH, Burger PC, et al. MYCC and MYCN oncogene amplification in medulloblastoma. Arch Pathol Lab Med 2002;126:540–544. 52. Herms J, Neidt I, Luscher B, et al. C-MYC expression in medulloblastoma and its prognostic value. Int J Cancer 2000; 89:395–402. 53. Tomlinson FH, Jenkins RB, Scheithauer BW, et al. Aggressive medulloblastoma with high-level N-MYC amplification. Mayo Clin Proc 1994;69:359–365. 54. Segal RA, Goumnerova LC, Kwon YK, et al. Expression of the neurotrophin receptor TRKC is linked to a favorable outcome in medulloblastoma. Proc Natl Acad Sci USA 1994;91: 12867–12871. 55. Grotzer MA, Janss AJ, Fung K, et al. TRKC expression predicts good clinical outcome in primitive neuroectodermal brain tumors. J Clin Oncol 2000;18:1027–1035. 56. Segal RA, Pomeroy SL, Stiles CD. Axonal growth and fasciculation linked to differential expression of BDNF and NT3 receptors in developing cerebellar granule cells. J Neurosci 1995;15(7 Pt 1):4970–4981. 57. Gilbertson RJ, Clifford SC, MacMeekin W, et al. Expression of the ErbB-neuregulin signaling network during human cerebellar development: implications for the biology of medulloblastoma. Cancer Res 1998;58:3932–3941. 58. Gilbertson R, Hernan R, Pietsch T, et al. Novel ErbB4 juxtamembrane splice variants are frequently expressed in childhood medulloblastoma. Genes Chromosomes Cancer 2001;31:288–294. 59. Gilbertson RJ, Perry RH, Kelly PJ, et al. Prognostic significance of HER2 and HER4 coexpression in childhood medulloblastoma. Cancer Res 1997;57:3272–3280. 60. MacDonald RJ, Brown KM, LaFleur B, et al. Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease. Nat Genet 2001;1–10. 61. Kenney AM, Rowitch DH. Sonic hedgehog promotes G1 cyclin expression and sustained cell cycle progression in mammalian neuronal precursors. Mol Cell Biol 2000;20: 9055–9067. 62. Hahn H. Wojnowski L, Specht K, et al. Patched target IGF2 is indispensable for the formation of medulloblastoma and rhabdomyosarcoma. J Biol Chem 2000;275:28341–28344.

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w Predictive Markers in Human Gliomas Arnab Chakravarti The advent of powerful technologies in molecular biology has dramatically enhanced our understanding of the underlying molecular pathogenesis of gliomas. Recent studies have identified molecular pathways enhancing the proliferative, invasive, and angiogenic potential of gliomas. Further, important insight has been gained into the molecular mechanisms, contributing to treatment resistance of gliomas. Such a fundamental understanding is important two reasons. One is to identify specific subsets of patients who will respond most optimally to a particular type of therapy (e.g., radiation, chemotherapy and/or biologic therapy). The second is to identify molecular pathways that may serve as promising therapeutic targets. In this chapter we focus on prognostic markers in gliomas that have been found to be important in one or both of these regards.

COMMON CHROMOSOMAL ALTERATIONS IN GLIOMAS Gliomas exhibit a wide range of genomic alterations, including loss of heterozygosity for 1p, 9p, 10, 13q, 17p, 19q, and 22q loci and gain of chromosome 10 (1). Corresponding genes for these loci include CDKN2A (cyclindependent kinase inhibitor 2A, containing p16 and p14ARF) (1,2), PTEN/MMAC1 (phosphatase and tensin homolog/mutated in multiple advanced cancers 1) on 10q (3–7), RB (retinoblastoma gene) on 13q, TP53 (tumor protein 53) on 17p (8), and amplification of the following genes: EGFR (epidermal growth factor receptor) (9–12), PDGFRA (platelet-derived growth factor receptor alpha polypeptide) (13,14), CDK4 (15,16), and MDM2 (17), among others. In gliomas that undergo progressive change from low to high grade, there are characteristic genetic alterations for each stage of progression (Fig. 58-1). Low-grade astrocytomas, which carry median survivals of about 5 to 10 years, are characterized by p53 mutations and PDGFR overexpression. Anaplastic astrocytomas, which carry median survivals of about 3 years, are

characterized by RB gene mutations/loss of heterozygosity, CDK4 amplification, CDKN2A deletion, PTEN loss of heterozygosity, DMBT1/mxi loss, 19q loss, and/or 11p loss. Glioblastomas, which carry median survivals of about 9 to 12 months, are characterized by EGFR amplification, EGFR vIII mutation (deletion of extracellular binding domain), CDKN2A deletion, PTEN loss, and/or RB mutation (18). It is not surprising that many of these genes which are associated with glioma progression regulate key cellular functions such as survival, proliferation, invasion, and apoptosis.

PROGNOSTIC SIGNIFICANCE OF LOSS OF CHROMOSOMES 1p AND 10q IN ANAPLASTIC OLIGODENDROGLIOMAS Oligodendrogliomas tend to have specific genetic alterations that distinguish them from other types of gliomas, including astrocytomas. Allelic losses of chromosome 1p and 19q occur in approximately 50% to 70% of both low-grade and anaplastic oligodendrogliomas (16,19–23). Anaplastic oligodendrogliomas additionally demonstrate losses of chromosomes 9p and 10q, as well as p53 gene mutations (16,20,23,24). It was determined that loss of chromosome 1p alone or when combined with loss of 19q was associated with significantly improved chemotherapeutic response (as measured radiographically) and with improved overall survival (24). This effect was found to be quite dramatic, as 95% of these patients were alive at 5 years after diagnosis. The authors suggested that patients whose tumors demonstrate losses of chromosomes 1p and 19q may be candidates for postsurgical treatments where PCV-based chemotherapy precedes radiation. CDKN2A gene deletions were found to be associated with significantly reduced survival. The significant inverse relationship between CDKN2A gene deletions and losses of chromosomes 1p and 19q further suggests that the observed differences in clinical behaviors reflect distinct genetic pathways leading to development of these tumors.

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FIGURE 58-1. Common molecular and genetic anomalies at each step of gliomagenesis. Note that some glioblastomas arise from the linear pathway illustrated here, whereas others arise de novo. The net effect of these molecular/ genetic events is ultimately to generate a tumor phenotype characterized by increased proliferative, invasive, and angiogenic capabilities. Further, many of these molecular events have been demonstrated to increase resistance of glioblastomas to radiation and chemotherapy.

CHROMOSOMAL DETERMINANTS OF RADIATION RESPONSE IN GLIOMAS Cytogenetic aberrations associated with radiation outcome in gliomas was addressed in a recent study from the University of California at San Francisco (UCSF) (25). In this study, radiation response was defined radiographically. Radiation-sensitive tumors were those that demonstrated greater than 50% reduction in contrast-enhanced volume, whereas radiation-resistant tumors were defined as those tumors with greater than 50% increase in contrastenhanced volume. Cytogenetic analysis was performed by comparative genomic hybridization. It was determined that relative gain of chromosome 7 (locus of EGFR gene) occurred in 70% of the radiation-resistant cases versus 30% of the radiation-sensitive cases and was the most significant difference involving a single change between the two groups (p = 0.06). Also of borderline significance were 9p23-24 and 13q14 losses, which tended to be more frequent in radiation-resistant (6 of 20) compared with radiation-sensitive (0 of 10) cases.

PROGNOSTIC MARKERS IN GLIOMAS AS DETERMINED BY GENE EXPRESSION ANALYSIS The advent of DNA chips and membrane-based microarrays has the potential to make a major impact on cancer classification, diagnosis, and therapy. In gliomas, several studies have yielded informative findings to this end. A comparative analysis between low-grade astrocytomas versus nontumorous brain tissue revealed that exclusive

expression of six genes in the former (TIMP, c-myc, EGFR, DR-nm23, nm23-H4, and GDNPF) (26). A subset of 7 genes (AAD14, SPARC, LRP, PDGFR-alpha, 60S ribosomal protein L5, PTN, and hBAP) were found to be up-regulated more than twofold in 20% to 60% of cases, whereas 11 genes (IFI9-27, protein kinase CLK, TDGF1, BIN1, GAB1, TYRO3, LDH-A, adducin 3, GUK1, CDC10, and KRT8) were found to be down-regulated to less than 50% of normal levels in 64% to 100% of cases. A separate study compared the gene expression profiles of high-grade versus low-grade gliomas and found 167 genes significantly up-regulated and 193 genes significantly down-regulated in grade IV versus grade I tumors (27). The authors noted that many of the genes with increased expression in grade IV tumors encoded proteins that are involved in cellular proliferation (e.g., CDK4, CDC28 protein kinase 2, etc.), migration (e.g., VCAM1, CD24, myosin light chain kinase, etc.), DNA repair (e.g., thymidylate synthetase, topoisomerase II␣, etc.), and survival (e.g., Survivin). Curiously, Survivin, which is an antiapoptotic protein in the inhibitor of apoptosis family of proteins, was significantly associated with adverse outcome in a separate study using quantitative Western analysis (28). Interestingly, many of the down-regulated in genes in grade IV versus grade I tumors were those involved in cellular adhesion (e.g., NCAM1, EMP2, etc.), signal transduction (protein kinase C, phosphoinositide 3-kinase [regulatory subunit], g-protein coupled receptor 17, activated cdc42Hs kinase, etc.), and transcription (early growth response 3, smad3, zinc finger protein 145, etc.), among others. It should be noted that approximately 6,800 genes were analyzed in this study, which is only a subset of the approximately 30,000 different genes in the entire human

58. Predictive Markers in Human Gliomas

genome. Therefore, these differences likely reflect only a portion of the actual differences in gene expression between high- and low-grade gliomas. The question of which subsets of genes may be responsible for tumor recurrence after radiotherapy also has substantial therapeutic implications. A study comparing gene expression patterns in primary versus recurrent gliomas after radiotherapy revealed that messenger RNA (mRNA) levels of growth factors participating in paracrine loops such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor ␤ (PDGF-␤) were decreased in postradiation recurrent tumors as compared with primary tumors in 75% of patients (29). However, mRNA levels of growth factors involved in autocrine loops such as EGFR, PDGF␣, PDGFA, and basic fibroblast growth factor (bFGF) were increased in most postradiation recurrent tumors compared with the primary tumor. The results of this study shed some fascinating insights into the molecular mechanisms involved in tumor recurrence after radiation.

EPIGENETIC DETERMINANTS OF CHEMOSENSITIVITY The epigenetic determinants of chemosensitivity were examined in a recent study. It has been previously reported that the DNA repair enzyme, O(6))-methylguanine-DNA methyltransferase (MGMT), can reduce the efficacy of alkylating chemotherapeutic agents (30). The activity of MGMT is regulated through methylation of its promoter, where methylation prevents expression of MGMT. In this study, 47 patients diagnosed with high-grade gliomas whose treatment included alkylating agents were examined for MGMT promoter methylation from their corresponding tumor specimens. It was found that the MGMT promoter was methylated in 19 of 47 patients (40%). This finding of MGMT methylation (or MGMT inactivation) was associated with enhanced regression of tumor radiographically (p < 0.001) and prolonged overall and diseasefree survival rates (p < 0.001). In fact, MGMT methylation status was an independent and stronger prognostic factor than age, stage, tumor grade, or performance status. Given the findings of this study, the authors advocate consideration of agents that inhibit MGMT, such as O(6))benzylguanine for future clinical studies.

CELL CYCLE MARKERS It has been found that cell cycle abnormalities in gliomas are ubiquitous. The G1/S checkpoint, regulated by the retinoblastoma protein (pRB), has been found to be deregulated in virtually all gliomas in some manner. The RB pathway involves a complex of regulatory proteins including pRB, p16, cdk4, and E2F-1, all of which has been found to

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be deregulated in gliomas. The current paradigm suggests that pRB maintains the integrity of the G1/S checkpoint, whereas the cdk4/cyclin D complex serves to phosphorylate and thereby inactivate pRB. p16, a cyclin-dependent kinase inhibitor, serves to inhibit the cdk4/cyclin D complexes. It has been found that allelic loss of 13q, the locus of the RB gene, occurs in approximately one-third of glioblastoma multiformes (GBMs) and is associated with progression from low-grade to anaplastic astrocytoma (31–34). However, loss of pRB itself was not found to be of prognostic significance independent of histology. Homozygous deletions of the CDKN2A locus, containing both p16 and p14ARF genes, have been found in up to approximately 60% of GBMs (32). Further, it has been found that loss of CDKN2A is a more common event in GBM than lower grade gliomas. Because both loss of p16 and p14ARF have both been associated with enhanced malignant potential in gliomas, the loss of CDKN2A affects two critical regulatory pathways: the pRB and p53 pathways. Intriguingly, loss of p14ARF protein expression was found to be a more powerful negative predictor of survival than loss of p16 protein expression, indicating that corresponding protein levels of these genes may be influenced by mechanisms other than gene deletion alone (35). These results also suggest that understanding the functions of p16 and p14ARF in gliomagenesis is far from complete. Another critical component of the pRB-E2F axis is the G1 cyclin-dependent kinase family, in particular, cdk4. It has been found that the CDK4 gene is amplified 10- to 100-fold in approximately 15% to 20% of GBMs (15,16). Likewise, overexpression of CDK4 protein has been found to be more frequent in high-grade gliomas and has been found to carry negative prognostic value among this subset of patients.

PROGNOSTIC VALUE OF THE EPIDERMAL GROWTH FACTOR RECEPTOR The most frequent gene amplification event in high-grade gliomas involves EGFR. In fact, EGFR gene amplification is seen in approximately 50% of GBMs (11). EGFR is a 170-kDa RTK that is composed of an extracellular binding domain, a transmembrane lipophilic segment, and an intracellular domain that has protein kinase activity (36–38). The ligands for EGFR include EGF as well as transforming growth factor (TGF)-␣. After binding of ligand, EGFR dimerizes, which activates the intrinsic protein tyrosine kinase and triggers a cascade of downstream signaling events (Fig. 58-2). Of the four EGFR family members, erbB1 to -B4, erbB1 has been the best characterized to date. It has been observed that EGFR gene amplification (located on chromosome 7) is quite common in GBMs, occurring in up to 50% of cases

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FIGURE 58-2. Downstream signaling pathways regulated by epidermal growth factor receptor (EGFR). EGFR activity is enhanced in many glioblastomas either through gene amplification events or through overexpression of the constitutively active EGFRvIII mutant. The net effect of EGFR signaling in glioblastomas is to enhance the malignant phenotype through the mechanisms listed.

(11). In lower grade gliomas, EGFR gene amplification has been found to be far less common. Approximately 40% of the GBMs with EGFR amplification express a mutant form of EGFR, referred to as EGFR vIII. The EGFR vIII mutant lacks a portion of the extracellular ligandbinding domain as the result of genomic deletions of exons 2 through 7 in the EGFR mRNA (12,39). This results in constitutive phosphorylation, or activation, of the EGFR pathway. Introduction of EGFR vIII into glioma cells has been shown to enhance cell proliferation and invasion, while inhibiting apoptosis (15,40,41). Additionally, it has been demonstrated to confer resistance to chemotherapeutic agents via up-regulation of Bcl-XL expression (42).

ROLE OF EPIDERMAL GROWTH FACTOR RECEPTOR IN GLIAL DEVELOPMENT There is increasing evidence that EGFR plays an important role in normal astrocyte development and differentiation. Indeed, understanding fundamental mechanisms by which

EGFR enhances survival in progenitor cells has the potential to shed valuable insights into how EGFR enhances survival in glioma cells in response to radiation. It has been reported that highest levels of EGFR expression occur with the peak of gliogenesis in the embryonic and early perinatal period, suggesting an association with astrocyte and/or oligodendrocyte development (18,43). There are several lines of evidence directly associating EGFR with the glial differentiation process. First, targeted deletion of EGFR in mice results in embryonic or perinatal lethality with the affected mice demonstrating cortical dysgenesis and reduced numbers of astrocytes (44–46). Second, retroviral-mediated overexpression of EGFR in the early ventricular zone results in proliferation of stem cells as well as premature astrocyte differentiation (43). Third, although cultured neural stem cells upon transplantation into the adult brain preferentially differentiate into astrocytes, if EGF is simultaneously infused into the lateral ventricles, then the transplanted neural stem cells remain undifferentiated and continue to proliferate (47). These results, taken together, suggest that EGFR appears to have a role in normal astrocyte differentiation and survival

58. Predictive Markers in Human Gliomas

of the neural stem cell compartment. It is entirely possible that increased EGFR pathway activation may interfere with the normal differentiation process and serve to enhance malignant potential in gliomas. As the net effect of EGFR pathway activation, as illustrated by these models, is to enhance cellular survival, it is not surprising that there is increasing evidence that EGFR signaling is associated with resistance to conventional cytotoxic agents, as described below.

PRECLINICAL DATA ON THE EPIDERMAL GROWTH FACTOR RECEPTOR IN RADIATION RESISTANCE There is strong evidence suggesting EGFR plays a key role in contributing to radiation resistance of malignant gliomas. These results have emerged from EGFR inactivation studies using one of several approaches: monoclonal antibodies, tyrosine kinase inhibitors, antisense oligonucleotides, dominant-negative EGFR mutants, and so forth. These reports suggest that when EGFR antagonism is combined with radiation, a significant radiosensitization effect is observed (48–53). In one study, glioma cells were treated with an adenoviral construct containing dominant-negative EGFR (Ad-EGFR-CD533) (50,52). A dose-enhancement ratio of 1.85 was observed. The authors suggest that because Ad-EGFR-CD533 inhibits the activation of all ErbB receptors by preventing functional receptor heterodimerization and transphosphorylation, there may be at least a theoretical advantage in combining Ad-EGFR-CD533 with other types of EGFR inhibitors. However, another report suggested that not all EGFRexpressing GBMs are amenable to anti-EGFR radiosensitization and that absolute EGFR expression levels may not predict which tumors respond best to this treatment strategy (54). In this study, two primary GBM cell lines with equivalent EGFR expression levels were found to have very different sensitivities to the EGFR tyrosine kinase inhibitor, AG1478. This was apparent despite similar reductions in EGFR signaling in both cell lines, as measured by phosphoEGFR levels. It was found that the resistant GBM cell line demonstrated an up-regulation of insulin-like growth factor receptor 1 (IGFR1) levels upon AG1478 administration. This resulted in sustained signaling through the phosphatidylinositol 3-kinase (PI3-K)/AKT pathway and ultimately in resistance to AG1478. Cotargeting IGFR1 with EGFR greatly enhanced both spontaneous and radiation-induced apoptosis and the invasive potential of this resistant cell line. AKT and p70s6k appeared to be important downstream targets of IGFR1-mediated resistance to anti-EGFR targeting. These findings suggest that IGFR1 signaling through PI3-K may represent a novel and

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potentially important mechanism of resistance to antiEGFR therapy.

DOWNSTREAM EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING TARGETS: RAS/RAF/MAPK AND PI3-K/AKT EGFR has distinct downstream signaling pathways, which have been previously reported to play important roles in radiation resistance. The RAS/RAF/MAPK cascade is one of the best characterized EGFR-regulated pathways. RAS is a guanine nucleotide-binding protein, which in its GTP-bound form results in the downstream activation of numerous important signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway. RAS-GTP is inactivated to RAS-GDP through intrinsic GTPase activity, catalyzed by GTPase activating proteins (RAS-GAPs). Oncogenic RAS is resistant to RAS-GAPs and is therefore locked into its GTP-bound form, resulting in constitutive activation. Although oncogenic RAS has been found to be common in other tumor types, this has not been observed in human gliomas (55,56). However, it has been reported that high-grade gliomas commonly have elevated levels of RAS-GTP (57–63). As gliomas commonly have amplification/overexpression of RTKs such as EGFR, PDGFR, and IGFR1, this may represent an important mechanism through which RAS signaling is up-regulated in gliomas. It has been demonstrated that antagonizing RAS either by using dominant-negative constructs or farnesyltransferase inhibitors results in antiproliferative, antiangiogenic, and proapoptotic effects in glioma cell lines in vitro (58–62). Combining farnesyltransferase inhibitors with chemotherapy was found to result in approximately 69% growth inhibition in preclinical in vivo models (62). Further, it has been demonstrated that astrocyte-specific expression of activated RAS results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Therefore, given RAS is a major effector of EGFR signaling, this may represent one of the major mechanisms by which EGFR enhances the malignant potential of glioma cells. To further support this observation, a recent study reported that EGFR plays a critical role in mediating resistance to sequential administration of radiation and chemotherapy in primary human glioblastoma cells in a RAS-dependent manner (49). It is well known (and a point of frustration) that unlike other tumor types, combined modality therapy involving chemotherapy and radiation has failed to appreciably improve outcome for GBM patients compared with radiation alone. In three primary GBM cell lines, an actual antagonistic effect was observed between radiation and BCNU chemotherapy. These cell

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lines were coincidentally found to have strong expression of EGFR. Upon inhibition of EGFR with AG1478 (an EGFR tyrosine kinase inhibitor), it was found that this crossresistance between sequentially administered radiation and BCNU was effectively abrogated. It was found that BCNU inhibited radiation-induced apoptosis through EGFR-mediated activation of PI3-K/AKT via RAS. On the other hand, radiation was found to inhibit BCNU-induced apoptosis through EGFR-mediated activation of both PI3-K and MAPK (p44/p42) pathways, also via RAS. Inhibition of either EGFR or RAS activity appears to not only abrogate the observed antagonism between sequentially administered radiation and chemotherapy, but actually results in a greater enhancement of apoptosis in the setting of combined modality therapy than when administered with either radiation or chemotherapy as single agents. Therefore, these findings suggest that strategies to inactivate EGFR or RAS signaling may be critical to improving not only the efficacy of single agent therapy, but also of combined modality therapy in gliomas.

determined quantitatively through Western blotting and image analysis and were found to be significantly correlated with reduced survival (p < 0.0001) (35). Studies on EGFR gene amplification also appear to suggest an association with both adverse radiation response, as determined radiographically, and association with more malignant histologies (25,67). In a study from UCSF, comparative genomic hybridization was performed on 30 frozen GBM specimens (25). These cases were designated as either radioresistant or radiosensitive based on imaging criteria. Radiosensitive tumors were those that demonstrated greater than 50% reduction in contrastenhanced volume after radiation; radioresistant tumors were those that demonstrated greater than 50% increase in contrast-enhanced volume postradiation. It was observed that a simultaneous gain of chromosomes 7 (locus of EGFR gene) and 19 were found in 30% of radioresistant cases but were absent from radiosensitive cases (p = 0.05). These data provides compelling evidence that up-regulation of EGFR is directly associated with radioresistance in vivo as well as reduced patient survivals reported in prior studies.

PROGNOSTIC VALUE OF EPIDERMAL GROWTH FACTOR RECEPTOR EXPRESSION IN GLIOMAS

INVASION AND ANGIOGENESIS MARKERS

There is increasing evidence that the observed role of EGFR in radiation resistance in preclinical models translates into adverse clinical outcome. In a study from UCSF, it was reported that positive EGFR immunoreactivity was associated with poor radiographically assessed radiation response (p = 0.046) (64). Thirty-three percent of tumors with no EGFR immunoreactivity had good radiation responses (more than 50% reduction in tumor size by computed tomography or magnetic resonance imaging), compared with 18% of tumors with intermediate EGFR staining and 9% of tumors with strong staining. In other studies, it has been demonstrated that EGFR immunostaining is of independent prognostic value in gliomas. Another study performed a multivariate analysis on clinical and biologic prognostic factors predictive of glial tumor outcome. A multivariate analysis including age, histology, tumor resection, EGFR immunostaining, and labeling index revealed that EGFR, labeling index, and tumor resection were the only independent significant predictors of survival (65). In a separate study, multivariate analysis of EGFR immunostaining was performed with other variables including immunostaining for proliferating cell nuclear antigen, p53, bcl-2, and for apoptotic index (66). It was found that only EGFR positivity and apoptotic index were significant on multivariate analysis (p = 0.0053 and p = 0.0039, respectively). Quantitative measurements of EGFR expression in gliomas also appear to support previously reported immunohistochemical observations. EGFR levels in frozen glioma specimens were

It has been well established that glioblastoma are highly invasive tumors, which also have high angiogenic potential. The mechanisms underlying GBM invasion are becoming better understood. Matrix metalloproteinases (MMPs), which are cation-dependent endopeptidases, may play an important role in enhancing invasive potential of gliomas. It has been reported that consistently expressed with increasing tumor grade are interstitial collagenase (MMP-1), gelatinase A (MMP-2), matrilysin (MMP-7), membrane-type 1 MMP (MMP-14), and membrane type 2 MMP (MMP-15) (68–72). MMP-9 expression has also been found to be directly associated with tumor grade; however, some believe that in consideration of immunohistologic and in situ hybridization studies demonstrating localization of MMP-9 within and around vasculature that MMP-9 expression is most likely linked with angiogenesis. There have been intriguing reports of cooperative effects between these proteases and extracellular matrix receptors. For example, interaction of ␣v␤3 integrin with MMP-2 has been found to be an essential mechanism in tumor cell and endothelial cell motility (68). Likewise, the interaction of ␣v␤3 integrin with MT1MMP has been shown to greatly enhance the activation of MMP-2 (68). Given this compelling data, MMP inhibitors may prove to be of at least theoretical value in the management of high-grade glioma patients. Gliomas also demonstrate elevated production of a host of angiogenic factors, including VEGF, acidic (aFGF) and bFGF, TGF-␣ and -␤, angiogenin, EGF, hepatocyte

58. Predictive Markers in Human Gliomas

growth factor, interleukin-8, IGF, among others (73,74). One angiogenic marker of particular interest is VEGF. VEGF is a 34- to 45-kDa dimeric glycosylated protein with structural similarity to PDGF. It is known that VEGF acts specifically on endothelial cells as a potent mitogen and chemotactant. VEGF is known to enhance survival of endothelial cells as well. Other properties of VEGF relate to microvascular permeability, with effects over 1,000 times as great as histamine. In GBMs, VEGF mRNA is up to 50-fold overexpressed when compared with normal brain tissue (75–77). In situ hybridization has revealed that VEGF mRNA is expressed at relatively low levels in normal brain, somewhat up-regulated in low-grade gliomas, and highly expressed in GBMs, in which VEGF mRNA is spatially restricted to perinecrotic (palisading) cells (77). This association suggests that hypoxia may be able to induce VEGF expression in vivo and thereby regulate angiogenesis. A recent study also suggests that VEGF expression at the mRNA level is associated with recurrence after radiation therapy (29). There is also strong evidence that two of the first angiogenic factors isolated, FGF-1 and FGF-2, are important players in glioma angiogenesis. It has been demonstrated that 93% of gliomas have elevated levels of FGF-1 mRNA compared with normal brain tissue (78). Higher levels of FGF receptors (FGFR) have also been found in gliomas compared with normal brain (79,80). There also appears to be differential expression of FGFR isotypes in progression to GBM, where there are diminishing levels of FGFR-2 and increasing levels of FGFR-1 (79–81). It is thought that FGF-1 and FGF-2 participates in angiogenesis in two ways: by modulating endothelial cell proliferation and by regulating VEGF expression in tumor cells (73). Another angiogenic factor that has been found to be of prognostic value in gliomas is TGF-␤. TGF-␤ family members have been found to regulate cellular proliferation, differentiation, motility, apoptosis, and angiogenesis in a variety of tumor types (82). In many systems, TGF-␤ has been found to suppress proliferation. However, TGF-␤ has been demonstrated to actually enhance glioma proliferation in a number of studies (83–85). Perhaps most significantly, it appears that increasing levels of TGF-␤ are associated with adverse outcome in patients with malignant gliomas (84). The precise reasons why TGF-␤ appears to have differing effects in glioma cells compared with other tumor types remains speculative.

SUMMARY In the past decade, there have been a host of molecular/ genetic anomalies identified in gliomas. In this chapter we described those molecular/genetic markers that appear to be of prognostic and, in some cases, of therapeutic value in gliomas. Precisely how this information can be

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optimally used in the future is debatable. The possibilities are to use this information to select patients for appropriate adjuvant therapies (e.g., radiation, chemotherapy, biotherapies) that would produce the greatest benefits with the least amount of toxicity. The second possibility is to design targeted biotherapies such as small molecule inhibitors and gene therapy that when used alone or in combination with conventional cytotoxic therapy may improve outcome for glioma patients. Given the wide range of molecular/genetic abnormalities in gliomas, it is possible, if not likely, that the most effective therapeutic strategies may be those that target more than one pathway or more than one functional parameter (e.g., signal transduction + angiogenesis). Regardless of how this information is ultimately used in the future, understanding the consequences of these molecular/genetic aberrations is a critical first step.

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C h a p t e r

5 9

w Molecular Neuropathology Catherine L. Nutt and David N. Louis

In modern clinical neuro-oncology, no variable affects therapeutic decisions and prognostic estimation more than tumor classification. Unfortunately, there are many situations in which the most universal classification system—that of the 2000 World Health Organization (WHO) (1)—is problematic, primarily because pathologic diagnosis remains quite subjective (2). For example, some brain tumors are difficult to place neatly into one of the categories. For others, the histologic diagnosis and corresponding predicted clinical behavior do not concur with the actual clinical course. Finally, it is doubtful that the current histopathologic system alone will accurately predict patient response to targeted therapies once available. Inquiries into the genetic basis of gliomas have yielded much information on specific genetic events that underlie brain tumorigenesis. Significant advances in molecular pathology have begun to provide pertinent clinical information, aiding in both the classification and the management of brain tumors. In this chapter we review the current molecular pathology of primary neoplasms of the nervous system.

DIFFUSE GLIOMAS Gliomas are extremely heterogeneous, and from a practical point of view, the most important initial distinction is to separate diffuse from circumscribed gliomas and then to classify the variants of diffuse and circumscribed gliomas. The infiltrative growth pattern of diffuse gliomas essentially prevents surgical cure, and most of these tumors are resistant to standard radiotherapeutic and chemotherapeutic approaches. Nonetheless, some subtypes of diffuse glioma respond to therapy, highlighting the importance of proper classification and grading of glial tumors.

Diffuse Astrocytomas, Including Glioblastoma WHO grade II astrocytomas are best characterized by inactivating mutations of the TP53 tumor suppressor gene 837

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on chromosome 17p as well as overexpression of the platelet-derived growth factor (PDGF) A chain and the PDGF-␣ receptor (3). Allelic loss of chromosome 17p and TP53 mutations have been observed in at least one-third of adult astrocytomas, irrespective of tumor grade (4). An integral role for p53 in the early stages of astrocytoma tumorigenesis is further evidenced by so-called secondary glioblastomas; it has been demonstrated that grade IV lesions with homogeneous TP53 mutations evolve clonally from subpopulations of similarly mutated cells present in the initial grade II astrocytic tumors (5). Functional studies have recapitulated a role for p53 inactivation in the early stages of astrocytoma formation (6,7). The transition from WHO grade II astrocytoma to WHO grade III anaplastic astrocytoma is accompanied by a number of molecular abnormalities. Studies suggest that most of these alterations converge on one critical cell-cycle regulatory complex that includes the p16, retinoblastoma (Rb), cyclin-dependent kinase 4 (cdk4), cdk6, and cyclin D1 proteins. Individual members of this pathway are altered in up to 50% of anaplastic astrocytomas and in most, if not all, glioblastomas. Loss of chromosome 9p primarily affects the region of the CDKN2A gene and occurs in approximately 50% of anaplastic astrocytomas and glioblastomas (8). The CDKN2A gene encodes the p16 and p14ARF proteins, and expression of these proteins is most commonly altered by homozygous deletion of the CDKN2A gene, although point mutations and hypermethylation of CDKN2A have also been found to alter p16 and p14ARF expression (9–11). Chromosome 13q loss occurs in one-third to one-

half of high-grade astrocytomas, with the RB gene preferentially targeted by losses and inactivating mutations (12). Analyses of the loss of chromosome 13q, RB gene mutations, and Rb protein expression suggest that the RB gene is inactivated in approximately 20% of anaplastic astrocytomas and 35% of glioblastomas (8,9). CDK4 is amplified in about 15% of malignant gliomas (13) and alterations of cyclin D1 and CDK6 are sometimes encountered (14–16). Progression to glioblastoma is characterized by the loss of chromosome 10; although occurring far less commonly in anaplastic astrocytomas, this alteration can be found in 60% to 95% of glioblastomas (17,18). PTEN mutations occur in about 20% of glioblastomas (19–21). The DMBT1 gene, located on 10q25–26, has also been suggested as a candidate tumor suppressor gene in malignant gliomas, but some DMBT1 deletions simply reflect unmasking of constitutional deletion polymorphisms by 10q loss (22). Nonetheless, given the remarkably high frequency of chromosome 10 loss in malignant glioma, additional tumor suppressor genes likely reside on this chromosome. The epidermal growth factor receptor (EGFR) gene is the most frequently amplified oncogene in astrocytic tumors (23) and is characteristic of so-called de novo glioblastomas. EGFR gene amplification may be regionally heterogeneous in glioblastomas, which may present practical problems for molecular diagnostic approaches (24). Glioblastomas with EGFR gene amplification display overexpression of EGFR at both the messenger RNA and protein levels, and approximately one-third of glioblastomas with EGFR gene amplification also display

FIGURE 59-1. Molecular genetic subsets of glioblastoma.

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EGFR gene rearrangements, which produce truncated proteins similar to the v-erbB oncogene (25). EGFR amplification and TP53 mutations appear to be mutually exclusive genetic aberrations in glioblastomas. One-third of glioblastomas have TP53/chromosome 17p alterations, one-third display EGFR gene amplification, and one-third have neither change (26). Cells lacking functional p53 are not transformed when cultured in the presence of EGF but are transformed in the presence of other growth factors (7); glioblastomas with TP53 mutations may therefore not be expected to acquire EGFR gene amplification if activation of the EGF–EGFR pathway does not produce an increased growth advantage in such cells. In fact, it was demonstrated recently that isolated EGFR-amplified cells can be found in TP53 mutated glioblastomas (24). Thus, although EGFR amplification occurs frequently at the cellular level in TP53-mutant glioblastomas, TP53 mutation does not facilitate selection of EGFR-amplified cells. Less common genomic alterations associated with low-grade astrocytomas include loss of chromosome 22q (27) and gains of chromosome 7q (28). In anaplastic astrocytomas and glioblastomas, allelic loss on 19q is quite common, being observed in up to 40% of these tumors (29). Comparative genomic hybridization studies have identified numerous other amplifications and deletions in astrocytic gliomas (30–35). Moreover, the recent implementation of array-based comparative genomic hybridization has enabled the identification of amplicons with single gene resolution in these lesions (36). Further exploration with this technique should allow the development of a more comprehensive overview of the genetic aberrations found in astrocytic tumors. Molecular Diagnostics and Prognostics As discussed above, TP53 mutations and EGFR gene amplification appear often to be mutually exclusive genetic aberrations in glioblastomas. These two distinct alterations provide additional information for the classification of glioblastomas (Fig. 59-1). The genetic pathway involving TP53 mutations often involves malignant progression from a lower grade astrocytic tumor to a secondary glioblastoma (26,37,38). In contrast, those glioblastomas with EGFR gene amplification usually arise de novo, without a clinically evident preceding lower grade astrocytoma (26,37). Furthermore, glioblastomas with loss of chromosome 17p tend to occur in patients younger than those characterized by EGFR gene amplification (39,40). Interestingly, younger age at initial diagnosis has been an important prognostic parameter among patients with glioblastoma, with younger patients faring better than older patients. These genetic alterations may therefore reflect the age-based difference in prognosis, suggesting that genetic analysis may begin to explain the clinical observations concerning age differences in astrocytic lesions.

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Although convincing differences in prognosis have not been observed in earlier studies of either TP53 or EGFR alterations in astrocytic gliomas, recent studies suggest that the relationship between age, p53, EGFR, and survival may be more complex than originally anticipated in patients with glioblastoma. In one study, when glioblastoma patients were differentiated initially based on length of survival, nuclear expression of p53 was significantly more frequent in long-term survivors, and EGFR overexpression appeared slightly more frequent in shortterm survivors (41). However, when glioblastoma patients were differentiated initially based on age, EGFR overexpression indicated worse prognosis in younger glioblastoma patients but better prognosis in older patients (42,43). Furthermore, within the subgroup of glioblastoma patients younger than the median age, EGFR overexpression has been negatively associated with survival in p53 wild-type cases but not in tumors positive for p53 immunohistochemistry (42). The prognostic effects of TP53, 1p, and CDKN2A/p16 alterations also appear to be dependent on patient age (44). The complexity of these results suggests that prognostic estimation in patients with glioblastoma may require analysis of subgroups that incorporate information for both age and specific genetic alterations. In addition to the diagnostic and prognostic information provided by p53 and EGFR status, other genetic alterations have been implicated as prognostic indicators in astrocytic lesions. Gains of 7p and 7q have been associated with shorter patient survival in anaplastic astrocytomas, independent of age (34). Furthermore, 10q loss of heterozygosity (45–47) and PTEN mutations (43) have been significantly associated with shorter survival in astrocytic tumors. Recently, genetic alterations have also been correlated with therapeutic response in astrocytic tumors; relative radioresistance of some glioblastomas may be associated with EGFR overexpression (48). Moreover, the vast number of gene expression profiling studies being conducted on gliomas continues to provide additional molecular insight into the classification and prognosis of malignant gliomas (49–57). Taken together, these studies provide strong evidence for molecular genetic subgroups of astrocytic gliomas that vary in treatment response and prognostic outcome.

Oligodendroglioma The most common allelic losses in oligodendrogliomas occur on chromosomes 1p and 19q, occurring together and affecting 40% to 80% of the tumors (29,58,59). Anaplastic oligodendrogliomas may display allelic losses of chromosome 9, involving the CDKN2A gene, and chromosome 10, involving the PTEN gene (58,60,61). Disruption of the RB1/CDK4/p16INK4a/p15INK4b and the TP53/p14ARF/MDM2 pathways appears frequently in anaplastic oligodendrogliomas (62,63), with simultane-

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FIGURE 59-2. Molecular genetic subsets of oligodendrogliomas. “Other” is defined as tumors that display 1p loss but either do not have 19q loss or have other genetic alterations such as TP53 mutation, PTEN mutation, 10q loss, CDKN2A deletion, or EGFR amplification.

ous disruption of both pathways occurring in 45% of lesions in one study (63). In particular, hypermethylation may be one epigenetic mechanism by which oligodendroglial tumors may escape cell cycle control (62–64). Molecular Diagnostics and Prognostics The value of molecular genetic analysis in modern clinical neuro-oncology is best exemplified by anaplastic oligodendrogliomas. Anaplastic oligodendrogliomas that have 1p loss are sensitive to procarbazine, CCNU, and vincristine (PCV) chemotherapy, with approximately 50% of these tumors demonstrating a complete neuroradiologic response (60). In contrast, anaplastic oligodendrogliomas with an intact 1p are only PCV sensitive in about 25% of cases and rarely exhibit a complete neuroradiologic response. Furthermore, patients whose anaplastic oligodendrogliomas have 1p and 19q loss have a median survival of approximately 10 years compared with a median survival of about 2 years for patients whose tumors lack these genetic alterations (60). Subsequently, it has been shown that anaplastic oligodendrogliomas can be divided genetically into four therapeutically and prognostically relevant subgroups (Fig. 59-2) (65). Moreover, studies have demonstrated that the molecular subtypes of anaplastic oligodendroglioma may arise preferentially in particular lobes of the brain and have differential patterns of growth, providing additional information pertinent to clinical patient management (66). Because a diagnosis of oligodendroglioma affects both therapeutic decisions and prognostic estimation, the ability to recognize oligodendroglial tumors has become extremely important. Recently, in an attempt to not overlook patients with a better prognosis and who might benefit from chemotherapeutic treatment, an increase in oligodendroglioma diagnoses has been noted. To provide a more objective method of classifying oligodendrogliomas, a number of current studies investigated the correlation between tumor morphology and molecular genetic profile. In a study of glioblastomas with an oligodendroglial compo-

nent, evidence was provided for a subgroup of glioblastomas of oligodendroglial origin; these lesions displayed the genetic aberrations of a “standard” glioblastoma but differed by having a higher incidence of 1p and 19q loss (67). Similarly, one report distinguished a set of glioblastomas with 1p and 19q loss from oligodendrogliomas with 1p/19q loss by the presence of 10q loss; these results suggest the presence of a subgroup of glioblastomas of oligodendroglial origin where 10q loss may be characteristic of progression (68). Interestingly, one study of low-grade gliomas suggested that histologic appearance correctly predicted genotype in approximately 80% of these lesions and that neuropathologists can be “trained” to recognize the particular histopathologic features responsible for an accurate diagnosis (69). It should be noted, however, that even trained neuropathologists do not achieve 100% accuracy, and the incorporation of objective molecular genetic analyses is still warranted (69). Recently, it was demonstrated that classification based on gene expression profiles was better able to distinguish between anaplastic oligodendrogliomas and glioblastomas than standard pathologybased classification, especially among diagnostically challenging tumors with nonclassic histology; moreover, this microarray-based classification model provided a more accurate predictor of prognosis in these tumors (54).

Oligoastrocytoma Many oligoastrocytomas, like oligodendrogliomas, display allelic losses on chromosomes 1p and 19q (29,58,59) as well as losses on chromosomes 9 and 10 (58,60). However, other oligoastrocytomas display TP53 mutations and 17p loss, more akin to the genotype of astrocytoma (70). In one study, TP53 mutation was inversely related to 1p loss of heterozygosity in gliomas, providing further evidence of two distinct lineages (68). Interestingly, although oligoastrocytomas display the histologic features of both oligodendrogliomas and astrocytomas, microdissection of the oligodendroglial and astrocytic components has demonstrated that, despite histologic differences, the molecular

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alterations are identical in the two components (59). Moreover, as demonstrated for oligodendrogliomas (66), recent data suggest that molecular subtypes of oligoastrocytoma correlate with tumor location (71).

OTHER GLIOMAS “Circumscribed” Astrocytomas Inquiries into the molecular pathology of these tumors have been fewer and less detailed than studies of the diffuse gliomas. The clinical relevance of the detected changes has also not been evaluated in any detail, because these tumors are not as common as the diffuse gliomas and are low-grade neoplasms that may be cured by surgical resection. Pilocytic Astrocytoma Pilocytic astrocytomas do not display the same genomic alterations as diffuse fibrillary astrocytomas. Pilocytic astrocytomas preferentially affect patients with neurofibromatosis type 1 (NF-1). Accordingly, allelic loss of the NF1 gene on chromosome 17q has been found in 25% of these tumors (72), with loss of the NF1 allele being much more frequent in NF-1–associated pilocytic astrocytomas than in sporadic cases (73). Nonetheless, some data argue against a major role for NF1 as a tumor suppressor gene in sporadic pilocytic astrocytomas (73,74). Pleomorphic Xanthoastrocytoma The genetic events that underlie pleomorphic xanthoastrocytoma (PXA) formation and progression differ from those responsible for diffuse astrocytoma tumorigenesis (75,76). Although p53 mutations have been found in PXAs, the few documented cases have displayed mutations somewhat different from those usually found in diffuse fibrillary astrocytomas (75–77). EGFR gene amplification does not occur in PXAs, although glioblastomas that arise from PXAs may display EGFR amplification (75,76,78). One recent study used comparative genomic hybridization and demonstrated gain on chromosome 7 and loss on 8p in two of three PXAs (78). Subependymal Giant Cell Astrocytoma Because subependymal giant cell astrocytomas (SEGAs) are associated with tuberous sclerosis, it is reasonable to hypothesize that the tuberous sclerosis genes, TSC1 on chromosome 9q and TSC2 on chromosome 16p, are involved in SEGA formation. In fact, loss of heterozygosity studies have shown allelic loss of chromosome 9q and 16p loci in some SEGAs. Detailed mutation analysis for these genes has not been completed to date in SEGA le-

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sions, although one survey of TS hamartomas revealed a SEGA with a mutation in TSC2 and corresponding loss of heterozygosity (79). An immunohistochemical study of seven SEGAs found only one tumor that was focally positive for tuberin, the protein encoded by the TSC2 gene, providing evidence for TSC2 as a tumor suppressor gene in SEGAs (80). Interestingly, a comparative genomic hybridization study of eight SEGAs revealed no chromosomal imbalances, suggesting that such aberrations are absent or very rare events in SEGA (81).

Ependymoma Chromosome 22q loss is a common genetic alteration in ependymomas, with NF2 gene mutations occurring in spinal ependymomas (82,83). For cerebral ependymomas, the paucity of NF2 mutations suggests that another, as yet unidentified, chromosome 22q gene may be responsible in the genesis of these tumors. In addition to 22q alterations, comparative genomic hybridization analysis has demonstrated gain of 1q and losses on 6q, 9, 13, and X in intracranial ependymomas, whereas gain on chromosome 7 was found almost exclusively in spinal cord lesions (84). In a fine mapping study using 384 microsatellite markers, chromosomal losses were detected on 1p, 6q, 16p, 16q, 17q, 19q, 20q, and 22q as well as the loss of whole chromosomes 13, 16, 19, and 20 (85). Chromosome 11q loss may be significantly inversely correlated with 22q loss in ependymomas, and approximately half of the tumors displaying 11q loss had a mutation in the MEN1 (multiple endocrine neoplasia type 1) gene (86). Although mutations of the TP53 gene do not appear to play a role in ependymoma tumorigenesis (87), disruption of the p53 pathway may be affected through MDM2 (88) or p14ARF (89,90). Knowledge of these genetic alterations has not been shown to be of practical clinical importance.

CHOROID PLEXUS TUMORS Choroid plexus tumors have been reported occasionally in patients with von Hippel-Lindau (VHL) disease and Li-Fraumeni syndrome, but studies of human choroid plexus tumors have shown neither VHL nor TP53 mutations, and it is possible that the VHL tumors were actually the papillary middle ear tumors typical of VHL (91). Sequences similar to SV40 virus, an oncogenic virus with the ability to inactivate both the p53 and Rb proteins, have been identified in human choroid plexus papillomas and ependymomas (92), but the role of oncogenic viruses in these tumors remains controversial (93–99).

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Molecular Diagnostics and Prognostics Comparative genomic hybridization has provided recent insights into the molecular pathology of choroid plexus tumors; abnormalities include chromosomal gains of 5, 7, 9, 12, and 20 as well as losses of 10 and 22q (100,101). The hSNF5/INI1 gene, which encodes a member of the chromatin-remodeling SWI/SNF multiprotein complex and is localized to 22q, has recently been implicated as a possible tumor suppressor gene in choroid plexus tumors (102). Several chromosomal aberrations have been found to be characteristic for a tumor entity or age group; patterns of alterations in choroid plexus papillomas were significantly different from those in choroid plexus carcinomas, and children presented with changes distinct from those seen in adults (100). As well, chromosomal gains of chromosome 7 and 12q were shown to distinguish choroid plexus papillomas from ependymomas (101). Moreover, gain of 9p and loss of 10q appeared to correlate with a more favorable prognosis in choroid plexus carcinomas (100).

EMBRYONAL TUMORS Medulloblastoma One-third to one-half of medulloblastomas have isochromosome 17q with allelic loss of 17p, but TP53 mutations are rare. Other genetic alterations frequently noted in medulloblastomas include allelic losses of chromosomes 6q, 10q, 11, and 16q (103,104) as well as genomic losses on chromosomes 10q, 11, 16q, 17p, and 8p (105). Although oncogene amplification has not been a common finding in medulloblastomas, comparative genomic hybridization studies have demonstrated amplification of chromosome bands 5p15.3 and 11q22.3 and gains of chromosomes 17q and 7 (105). The oncogene c-myc is amplified in a significant number of cases; however, this alteration appears more frequently in medulloblastoma cell lines than in primary tumors (106). Two hereditary syndromes have provided insight into genetic pathways involved in medulloblastoma tumorigenesis. Turcot syndrome, characterized by colonic and brain tumors, has been linked to medulloblastomas; patients with adenomatous polyposis often display mutations of the APC gene on chromosome 5q and may develop medulloblastomas (107). Although loss of chromosome 5q and APC gene mutations are infrequent in sporadic medulloblastomas (108,109), rare mutations of ␤-catenin, a protein that functions in a common molecular pathway, have been noted (109,110). Mutations affect the phosphorylation sites of the degradation targeting box of ␤-catenin and result in nuclear ␤-catenin protein accumulation, suggesting that inappropriate activation of the WNT/wingless signaling pathway may contribute to the pathogenesis of

these lesions (109). Indeed, mutations and deletions in AXIN1, a component of the WNT/wingless pathway, have also been detected in sporadic medulloblastomas (111). Additional insight into medulloblastoma tumorigenesis has been gained through the study of Gorlin syndrome. This condition is characterized by multiple basal cell carcinomas, bone cysts, dysmorphic features, and medulloblastomas. Gorlin syndrome has been shown to arise from defects in the PTCH gene, a homolog of the Drosophila patched gene, located on chromosome 9q. Medulloblastomas, particularly the nodular desmoplastic forms characteristic of Gorlin disease, can display allelic loss of chromosome 9q and PTCH mutations (112–114). Sporadic medulloblastomas have also demonstrated rare mutations in the smoothened (SMO) gene (115), and both germline and somatic mutations have been detected in SUFU (suppressor of fused) (116); PTCH, SMO, and SUFU proteins function in a molecular pathway regulated by the Sonic hedgehog protein. Although likely, it remains to be determined whether additional genes involved in these two pathways may by implicated in medulloblastoma tumorigenesis.

Atypical Teratoid/Rhabdoid Tumors Abnormalities of chromosome 22, including deletions and mutations of the hSNF5/INI1 gene, distinguish atypical teratoid/rhabdoid tumors from other embryonal tumors, such as medulloblastomas and supratentorial PNETs, and may be of considerable diagnostic help in diagnostically difficult cases (117–121).

Molecular Diagnostics and Prognostics Medulloblastomas have benefited greatly from gene expression analysis. Original studies provided evidence that the level of expression of the neurotrophin-3 receptor (trkC) is related to prognosis, with high trkC expression correlating with a more favorable outcome (122). More recently, it has been suggested that expression levels of the ErbB2 receptor may also act as a prognostic factor (123). DNA microarray expression profiling has provided vast quantities of new information. This type of analysis has demonstrated that medulloblastomas are molecularly distinct from PNETs and atypical teratoid/rhabdoid tumors, that medulloblastomas may be derived from cerebellar granule cells through activation of the Sonic hedgehog pathway, and that the clinical outcome of this disease is highly predictable on the basis of gene expression profiles (124). Furthermore, a comparison between metastatic and nonmetastatic medulloblastomas has identified 85 genes that are differentially expressed between these two classes; notably, PDGF-␤ receptor and members of the RAS/mitogenactivated protein kinase signal transduction pathway are upregulated in metastatic lesions (125–127).

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MENINGIOMA Monosomy 22 and NF2 mutations are common in meningiomas, clearly implicating NF2 in meningioma tumorigenesis (128). In sporadic meningiomas, allelic loss of chromosome 22q and NF2 mutations are more common in fibroblastic and transitional forms than in meningothelial lesions (129). A few meningiomas have been described with loss of portions of chromosome 22q that do not include the NF2 gene, suggesting that a second meningioma locus might be found on chromosome 22q (130). One study demonstrated a potential mutation hotspot in exon 9 of the hSNF5/INI1 gene in a small number of meningiomas, raising the possibility that hSNF5/INI1 may be a second tumor suppressor gene on chromosome 22 in these lesions (131), but a more recent report suggests alterations in hSNF5/INI1 are relatively rare (132). Approximately 40% of meningiomas display neither NF2 gene mutations nor chromosome 22q allelic loss, and, as such, it is likely that additional meningioma tumor suppressor genes exist. One study has suggested the possibility of alternative meningioma genes on chromosomes 1p and 3p (133). Moreover, allelic losses in meningiomas have been demonstrated on a number of chromosomes, including 1p, 3p, 5p, 5q, 11, 13, and 17p (134,135). Atypical meningiomas often exhibit allelic losses of 1p, 6q, 9q, 10q, 14q, 17p, and 18q, implicating these loci in tumor progression (135–138). Furthermore, more frequent losses of 6q, 9p, 10, and 14q are seen in anaplastic meningiomas (135,139). It has been suggested that most anaplastic meningiomas display genetic alterations of CDKN2A, p14ARF, and CDKN2B, indicating that inactivation of cell cycle control is an important aberration in these lesions (140). In addition to chromosomal losses, higher grade meningiomas have also demonstrated chromosomal gains, with gains of 20q, 12q, 15q, 1q, 9q, and 17q being the most common (135,139). Overexpression of PDGF B chain and the PDGF-␤ receptor has also been demonstrated in meningiomas and appears to correlate with tumor grade (141).

Molecular Diagnostics and Prognostics Molecular genetic analysis has aided the diagnosis of meningioma by further defining the markers of malignancy. Although invasion into the surrounding brain was once an indicator of malignancy in meningioma, molecular studies have shown that histologically benign lesions that invade the brain do not display the genetic alterations indicative of a higher grade meningioma (135); in particular, chromosome 10 loss has been associated with lesions designated as malignant on the basis of morphologic features but not those meningiomas thought to be malignant due to the presence of brain invasion (137). In the search for prognostic indicators, both 1p and 14q deletions have been implicated in the ability to predict

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tumor recurrence (142,143) and chromosomal 10 loss may predict higher rates of recurrence as well as shorter survival (144).

PERIPHERAL NERVE SHEATH TUMORS Schwannomas, like meningiomas, occur frequently in NF-2 patients, have frequent loss of chromosome 22q, and harbor NF2 gene mutations in at least 50% of cases (145). Loss of merlin, the NF2 encoded protein, occurs in all schwannomas, consistent with an integral and universal role for NF2 inactivation in both inherited and sporadic forms of schwannoma (146). In addition to loss of 22q, comparative genomic hybridization has demonstrated gain of copy number on chromosome 9q34 as well as gain on 17q (147). Multiple neurofibromas are associated with NF-1 (148,149). The NF1 gene encodes neurofibromin, a RasGAP (guanosine triphosphatase activating protein); loss of neurofibromin expression in NF-1–associated tumors can result in elevated RAS activity. Neurofibromas, particularly the NF-1–associated plexiform variants, can undergo malignant progression to malignant peripheral nerve sheath tumors, a transition that can be accompanied by genetic alterations including inactivation of the NF1, TP53, and CDKN2A genes (150–152). However, although CDKN2A inactivation appears to occur almost equally in NF-1–associated and sporadic malignant peripheral nerve sheath tumors, alterations of p53 expression are more common in sporadic malignant peripheral nerve sheath tumors (153); these findings and other cytogenetic studies (154) suggest that NF-1–associated and sporadic malignant peripheral nerve sheath tumors may exploit different oncogenetic pathways.

HEMANGIOBLASTOMA Multiple hemangioblastomas are characteristic of VHL disease, an inherited tumor syndrome in which patients may develop a variety of lesions, including hemangioblastomas, retinal angiomas, renal cell carcinomas, and pheochromocytomas (155). Allelic loss has been demonstrated in hemangioblastomas in the region of the VHL gene on chromosome 3 (156), and the VHL gene is mutated in sporadic hemangioblastomas (157). These observations suggest that VHL acts as a classical tumor suppressor gene and is involved in both familial and sporadic hemangioblastomas. The biologic function of the VHL protein appears complex, with evidence suggesting it may stabilize messenger RNA of angiogenic compounds such as vascular endothelial growth factor (158) as well as extracellular matrix components such as fibronectin (159). Moreover, VHL complexes have been shown to target the ubiquitination of hypoxia-inducible

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factor-1␣ (160), and expression of hypoxia-inducible factor-1␣ has been demonstrated in stromal cells throughout hemangioblastomas (161). These actions of VHL provide a possible explanation for the highly vascular nature of hemangioblastomas. pVHL is a microtubule-binding protein with associated microtubule-stabilizing function; altered microtubule dynamics may contribute causally to the development of hemangioblastoma in the setting of type 2A VHL disease (162). Comparative genomic hybridization in hemangioblastomas has revealed common losses of chromosomes 3, 6, 9, and 18q and a gain of chromosome 19 (163–165).

SUMMARY In this chapter we reviewed the molecular pathology of primary neoplasms of the nervous system. Molecular genetic alterations have been discussed that are characteristic of specific tumor types and stages of progression, offering the potential to augment the current WHO classification system. In addition, the value of genetic profiling in clinical patient management, aiding in diagnostic and therapeutic decisions as well as prognostic estimation, has been illustrated. Of note, microarray expression profiling is now providing vast quantities of new information. Currently, a plethora of expression profiling studies are being conducted and initial results, especially in medulloblastomas and diffuse gliomas, suggest that microarray analysis will be diagnostically informative for many different classes of brain tumor. Most importantly, as more is understood about the molecular pathways involved in brain tumorigenesis, such knowledge will likely contribute to the development of more effective treatment for these tumors.

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w Molecular Markers of Metastatic Disease

George Perides and Julian K. Wu Over the last 40 years efforts have been made to develop molecular markers that could be used to identify the presence of metastatic tumors to the brain. Ideally, these markers would not only aid in the diagnosis and prognosis of the disease but also in assessing treatment efficacy. Such markers have been successfully used in a number of systemic cancers. An outstanding example is the detection of an elevated prostate-specific antigen level in blood for the diagnosis of prostate cancer. The usefulness of the prostate-specific antigen assay is furthered by our ability to use prostate-specific antigen levels to assess ongoing treatment efficacy. In this chapter we review and summarize our current knowledge of tumor markers for metastatic tumors to the brain. Because most published studies on tumor markers for metastatic tumors to the brain have been on tumors from a variety of different histologic types, we discuss these studies first. We then focus our discussion on the various markers according to the tissue of origin, in particular breast cancer, lung cancer, and melanoma. Finally, we present the markers related to carcinomatous meningitis.

STUDIES ON METASTATIC TUMORS WITH DIVERSE PRIMARIES Using comparative genomic hybridization, Petersen et al. (1) investigated quantitative changes in specific chromosomal regions. They found DNA gains at the chromosomal regions 1q23, 8q24, 17q24-25, 20q13, and 7p12 in 77% to 82.5% of the metastatic cases compared with the DNA from systemic cancers without metastases to the brain. Deletions were found at a slightly lower frequency at 4q26, 4q22, 5q21, 9q21, 17p12, 4q32-q34, 10q21, 10q23-24, and 18q21-22 in 67.5% to 77.5% of the cases. In a small study of 11 patients with tumors metastatic to the brain, 64% of the patients had increased expression of the proto-oncogene c-erbB-2 and 67% had abnormal DNA content (2). The Ki-67 proliferation index of 145 brain tumors was investigated by Niemiec (3). The Ki-67 proliferation index in metastatic tumors to the brain was

found to be significantly higher than all primary brain tumors tested, including high-grade gliomas. The density of the omega-3 peripheral type benzodiazepine binding sites has also been used as an indicator of tumor proliferation (4). 3H-PK 11195 binding was determined on tissues obtained in biopsies or autopsies to evaluate the omega-3 binding sites. Tumor tissues from patients with metastatic brain tumors secondary to lung or kidney cancer contained the highest densities in omega-3 binding sites (up to 20-fold higher density than control brain parenchyma). This increase was significantly higher than the one observed in high-grade gliomas (up to 12-fold) and low-grade gliomas (2- to 3fold higher than control brain) (4). It is remarkable that the increased density correlated extremely well with the histopathologic features of these specimens. Only a few studies compared paired primary tumors to their metastatic tumors to the brain. Arnold et al. (5) compared by immunohistochemistry the expression levels of several tumor-related products. They found that overexpression of MMP-9 and p53 correlated with primary tumors and matched brain metastases. Low or no expression of tissue inhibitor of metalloproteases-1 was also found in brain metastases. Their findings suggested that in metastatic tumors the balance was shifted toward degradation of the extracellular matrix. In the same study, changes in the expression of E-cadherin or bcl-2 levels did not correlate with any preferences to metastasize (5). In another study, the ganglioside GM3 was found in both the primary and tumors metastatic to the brain, whereas the ganglioside GT1b was found in all but one metastatic tumors to the brain but not in the primary tumors (6). The authors suggested that GT1b ganglioside may be an indicator of an end organ target of metastasis. Several studies have looked for markers in the cerebrospinal fluid (CSF) of patients with brain tumor metastases. The rationale for studying the CSF for markers is the hypothesis that any molecule released in the brain is likely to diffuse into the CSF and therefore can be detected. Unfortunately, some CSF markers have the drawback of being elevated not only in the CSF of patients 849

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with brain tumors but also in patients with inflammatory or other central nervous system (CNS) diseases. Lactic dehydrogenase-5 (LDH) isoenzyme was elevated in the CSF of patients with malignant melanoma, breast, or lung carcinoma to the brain but not in other types of CNS metastases. Elevated levels of LDH in CSF have been used as a marker for CNS metastasis but may be falsely elevated in cases of CNS infection (7). Matrix metalloproteinases (MMPs), enzymes associated with tumor progression and metastasis, have also been investigated as a marker for CNS metastases. Our laboratory has found that 64 of 66 CSF samples analyzed from patients with systemic tumors with CNS metastases contained MMP-9, whereas the CSF from patients with systemic tumors without CNS involvement did not have detectable levels of MMP-9 (8). Similarly, lipid-associated sialoprotein (LSP) was found to be elevated five- to sixfold in the CSF of patients with tumors metastatic to the brain compared with the levels found in healthy individuals, patients with non-neoplastic neurologic diseases, or patients with systemic tumors but without CNS metastasis. In addition, we found LSP levels in the CSF corresponded with patients’ response to treatment. Patients with progressive disease had LSP levels similar to the ones seen in patients with tumor metastasis at presentation, whereas the CSF from patients with stable disease, partial response, or complete response to treatment showed reduced levels similar to those seen in healthy individuals (9). Adrenocorticotropic hormone concentrations in the CSF of patients with lung cancer with CNS metastases were also determined to be significantly higher when compared with cancer patients without CNS metastasis (10). The CSF-to-serum ratio was even higher in patients with leptomeningeal disease. Using a telomeric repeat amplification protocol, it was found that 32 of 35 brain metastases was positive for telomerase in the CSF of patients with metastatic tumors but negative in all CSF tested from patients without brain malignancies (11).

STUDIES ON SPECIFIC CANCERS WITH BRAIN METASTASIS Breast Cancer Annually, approximately 180,000 patients are newly diagnosed with breast cancer in the United States. Depending on the study, 15% to 30% of patients with breast cancer patients will develop brain metastases. A number of laboratories focused their studies on the identification of molecular markers for breast cancer metastasis to the brain. Creatine kinase (CK)-BB activity has been evaluated as a diagnostic marker in the CSF of patients with breast cancer (12). It was found to be significantly higher in the CSF of patients with metastasis to the brain than those without. The assay

had 83% sensitivity and 87% specificity and a positive predictive value of 60% and negative predictive value of 96%. For patients with leptomeningeal disease, the assay had a 57% sensitivity and 100% specificity and 72% negative predictive value and 100% positive predictive value. The same laboratory examined whether the concentration of the nonspecific proliferation marker tissue polypeptide antigen in the CSF can distinguish patients with brain metastasis from those without. The sensitivity of the assay was 74% and the specificity, 100%. The negative predictive value was 86% and the positive predictive value, 100%. Comparison of CSF cytology, CK-BB activity, and tissue polypeptide antigen concentration in CSF samples from patients with intraventricular reservoir indicated that CK-BB and tissue polypeptide antigen are more sensitive in identifying leptomeningeal disease than the CSF cytology (12). In an attempt to determine whether specific markers characterize predilection for brain metastasis, Domagala et al. (13) reported that vimentin-positive breast tumors metastasize twice as often to the brain compared with vimentin-negative breast tumors, whereas the p53 status seemed to have no effect on visceral metastasis. Comparison of primary breast carcinomas and 22 corresponding brain, liver, lung, and bone metastases was performed by Hampl et al. (14). Their analysis of 19 microsatellite markers on seven breast cancer- and metastasis-related chromosomal regions showed that loss of heterozygosity correlated with distant metastasis, including the brain. Combination of the three breast cancer–related chromosomal regions alone (17q21-BRCA1, 13q12-13 BRCA2, and 11q22-23 ATM) or combination with allelic loss of the p53 region 17p13.1 correlated with aggressive breast cancer with metastases (14). Loss of heterozygosity at the 19p13.3 locus has been identified in 56.3% of the breast tumors metastatic to the brain and 58.3% of the lung tumors metastatic to the brain. Such changes have been found less frequently in primary brain tumors, including gliomas (17.3%), pituitary adenoma (18.8%), and meningiomas (5.3%) (15). Similarly, loss of heterozygosity in the PTEN/MMAC1 locus has been found in 67% of breast cancers and 64% of lung cancers metastatic to the brain. Thus far, there has been no metastasis associated specifically with the PTEN/MMAC1 gene, and an additional tumor suppressor gene on chromosome 10q has been suggested to explain this finding (16). A large difference in loss of heterozygosity in chromosome 15q14 was also seen in primary breast cancer without metastasis to the brain (11%) compared with ones with metastases to the brain (70%) (17,18). Immunohistochemical analysis of breast cancer revealed that c-erbB-2 protein overexpression correlated with a threefold higher rate of brain metastasis compared with cancers that do not express c-erbB-2 (18). Interestingly, these cancers with c-erB-2 overexpression metastasize three times less often to the bone (18). Immunohistochemical

60. Molecular Markers of Metastatic Disease

evaluation also showed that breast and lung metastases to the brain were positive for cytokeratin 7, with a 98% sensitivity and 78% specificity. In the same study it was found that cytokeratin 20 staining had a 94% sensitivity and specificity for gastrointestinal carcinomas (19).

Lung Flow cytometry has been used to analyze DNA content of tumor tissue from patients with lung carcinoma who developed brain metastases. The DNA analysis of tumor tissue from patients who underwent surgical resection and mediastinal nodal resection for stage I adenocarcinoma at the M.D. Anderson Cancer Center showed a significant correlation between high DNA index in the primary lung cancer and high frequency of brain metastasis. Eighty-six percent of patients developing brain metastases had at least 30% of abnormal DNA content in their primary site, whereas only 4% patients without brain metastasis had abnormal DNA content (p = 0.01) (20,21). D’Amico et al. (21) conducted an immunohistochemical study in an attempt to identify specific markers that would indicate lung tumors predisposed to metastasize to the brain (21). The authors tested lung tumor specimens from patients who underwent surgical resection for stage I non–small cell lung carcinoma and correlated the markers with the development of brain metastases. Of eight potential markers tested, two were associated with the development of brain metastases, the elevated expression of p53 (p = 0.02), and urokinase-type plasminogen activator (p = 0.002) (24). CK, its BB-isoenzyme (CK-BB), and ␤2-microglobulin were tested as markers for CNS metastases in the CSF of patients with small cell lung carcinoma (25). Fifteen of 17 patients (88%) with leptomeningeal involvement had levels of CK-BB above normal (more than 0.4 U/L), whereas 4 of 26 patients with parenchymal CNS metastasis had elevated CKBB levels. Interestingly, 6 of 12 patients with negative CSF cytology were found to have leptomeningeal disease by the CK-BB measurement (22). Similarly, 93% of patients with bombesin levels in the CSF greater than 20 fmol/mL had leptomeningeal disease (23). In a prospective randomized trial, LDH status was found to be the most important prognostic factor for survival of patients with lung carcinoma metastatic to the brain. The mean survival of patients with normal LDH status undergoing radiation therapy was 5.1 months, whereas the mean survival of patients with high LDH levels survived 2.9 months (24). In another study, histologic staining of brain metastases for fucose was shown to be stronger than in the primary site. The intensity of fucose staining and the percentage of the tumor stained for fucose was reported to correlate with survival (25). Thyroid transcription factor-1 is expressed only by lung adenocarcinomas and thyroid carcinomas and is being routinely used to distinguish primary lung cancer from

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cancer metastatic to the lung (26). Using a thyroid transcription factor-1 antibody, 31 of 40 lung carcinomas metastatic to the brain stained for thyroid transcription factor-1 and only 1 of 31 metastatic nonpulmonary carcinomas was positive. All lung adenocarcinomas, 86% of small cell lung carcinomas, and 79% of large cell lung carcinomas metastatic to the brain were positive for thyroid transcription factor-1, whereas none of the metastatic tumors from squamous cell carcinoma was positive (26). Based on these data, Srodon and Westra (26) suggested that thyroid transcription factor-1 might be useful in identifying tumors of unknown primary.

Melanoma Chromosomal rearrangements in melanomas metastatic to the brain mostly (75%) involve 11q and 17q aberrations (27). In separate study the same group reported that isochromosome 17q formation was identified in a case of melanoma metastatic to the brain (28). S-100 protein is a valuable marker for melanocytic tumors in cases of amelanotic metastasis from an unknown primary but is not specific for melanomas (29). The HMB45 antibody is associated with melanoma and used as a melanoma marker (30). HMB-45 has been identified as a specific marker for melanoma metastasis to the CNS, whereas other types of carcinomas were negative (31). The combination of negative cytokeratin staining and positive HMB-45 has been suggested to be diagnostic for malignant melanoma (32). A comparison study among three monoclonal antibodies the S1/61/69 raised against S100B, the HMB-45 raised against gp-100, and the SM5-1 that recognizes a 200- to 225-kDa protein indicated high specificity for SM5-1 and HMB-45 but a large number of false-positive results for the S1/61/69 anti-S100B antibody (29). In a comparison study, Hoon et al. (33) reported that reverse transcriptase-polymerase chain reaction analysis for MAGE-3, MART-1, and tyrosinase are more sensitive than cytology or immunohistochemistry using S-100 and HMB-45 antibodies. In a study involving primary and metastatic malignant melanomas, enolase isoenzymes were tested by immunohistochemistry but were found not to be reliable for the diagnosis of melanoma metastases to the brain (34). Recently, Hau et al. (35) reported that melanoma inhibiting activity (MIA/CD-RAP) in the CSF of patients with melanoma metastases to the brain was significantly higher than levels found in patients without metastasis. The CSF levels are also frequently higher than the levels in the blood (35). In a series of 99 patients with metastatic carcinoma to the brain, Giordana et al. (31) attempted to differentiate the metastases with various markers. Only HMB-45 was specific for skin melanomas, whereas the other antibodies tested against carcinoembryonic antigen (CEA), carbohydrate antigen CA19.9, CA125, BCA-225, and cytokeratin 20

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were not specific. Interestingly, CEA was found more frequently in early metastases of lung carcinomas than late metastases.

LEPTOMENINGEAL METASTASES Diagnosis of leptomeningeal metastasis is based on CSF cytology. However, there are frequently false-negative results despite evidence suggestive of leptomeningeal disease by radiologic imaging data (36). Several techniques have been explored to evaluate markers that can complement CSF cytology in making the diagnosis of leptomeningeal metastases. Glucosephosphate isomerase was studied in a series of 66 patients with malignancies. An elevated concentration of glucosephosphate isomerase (more than 20 U/L) was used as the cutoff value and provided a 53% sensitivity and 92.1% specificity for the presence of leptomeningeal metastases. Although the sensitivity was not high, an increased concentration of glucosephosphate isomerase suggests the presence of leptomeningeal disease (37). Our studies show that the levels of LSP are elevated in the CSF of patients of patients with neoplastic meningitis secondary to metastatic disease and correspond to tumor progression and are reduced to physiologic levels in cases of complete response to treatment (9). At least two laboratories have tested for the presence of vascular endothelial growth factor in CSF from patients with carcinomatous meningitis (38,39). Both groups reported that vascular endothelial growth factor was identified only in the CSF from patients with leptomeningeal disease and not in the CSF from patients with parenchymal tumors in the CNS. Not only was the vascular endothelial growth factor concentration in the CSF of all patients with carcinomatous meningitis elevated before treatment, it also returned to lower levels after antineoplastic treatment (39). In all neurologic controls without neoplastic involvement of the CNS, including infectious neurologic disease, vascular endothelial growth factor levels were below the level of detection (less than 25 pg/mL) (38,39). Fluorescence in situ hybridization with a chromosome 1q12 as a probe was performed on 15 CSF samples to determine whether chromosomal additions can be used to determine carcinomatous meningitis. Eighty-seven percent of the samples tested positive, suggesting that fluorescence in situ hybridization can be used at the single cell level for the diagnosis of leptomeningeal disease (40). Over the last 15 years, Twijnstra and collaborators investigated several biochemical markers for the diagnosis of leptomeningeal disease. In a study involving 57 breast cancer patients, a ␤-glucuronidase assay had 93% sensitivity and 93% specificity in identifying carcinomatous meningitis (41). After testing CSF samples from 350 patients, increased LDH activity had a 79% sensitivity and 83% specificity for

carcinomatous meningitis, indicating that LDH may be useful as a diagnostic test for leptomeningeal disease and in monitoring response to treatment (42). In a study evaluating ␤-glucuronidase, ␤2-microglobulin, CEA, and LDH, the authors found that the combination of ␤-glucuronidase and ␤2-microglobulin assays is highly sensitive and specific for leptomeningeal disease (43). Twijnstra et al. (44) also reported that the levels of ␤2-microglobulin and CEA before treatment were predictive for response to treatment and survival. These results have been reproduced by other laboratories. Weller et al. (45) reported that increases in ␤2-microglobulin corresponded to leptomeningeal disease secondary to malignant melanoma, whereas no increases were seen in patients with cerebral metastasis. CEA has been found to be significantly higher in the CSF of patients with leptomeningeal disease compared with other neurologic controls and patients with primary tumors or patients with parenchymal metastasis (46,47). Recently, similar results have been reported for the elevated levels of CEA and CA15.3 in CSF from patients with leptomeningeal carcinomatosis secondary to breast cancer (48). Several biochemical markers have been evaluated with variable success. Tumor necrosis factor-␣ was found associated with leptomeningeal disease secondary to melanoma (45,49), whereas differences in vasopressin, although statistically significant, were not deemed useful for clinical use (50). Although a large number of markers have been identified associated with tumors metastasis to the brain, their use has been rather limited, mostly to the identification of the tumor’s origin. The use of such markers to monitor disease progression except in the case of patients with leptomeningeal disease is virtually nonexistent. Current methods to identify CNS products in the bloodstream associated with tumor metastasis have not been successful. Further investigation for specific and sensitive markers that would allow early diagnosis as well as regular and frequent assessment of tumor response to treatment would be a valuable tool in the treatment of patients with metastases to the brain. Developments in new technologies such as DNA microarray analysis have recently shown that some primary tumors also had a metastatic gene expression pattern and a poorer prognosis (51). This study suggests, counter to current views of cancer progression, that the metastatic property of cancers may be preordained by early mutations in these tumors. Using this technique, one may be able to screen and predict at the time of primary tumor diagnosis which patients will likely develop metastases, leading to earlier preventative therapies.

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42. Twijnstra A, van Zanten AP, Hart AA, et al. Serial lumbar and ventricle cerebrospinal fluid lactate dehydrogenase activities in patients with leptomeningeal metastases from solid and haematological tumours. J Neurol Neurosurg Psychiatry 1987; 50:313–320. 43. van Zanten AP, Twijnstra A, Ongerboer de Visser BW, et al. Tumour markers in the cerebrospinal fluid of patients with central nervous system metastases from extracranial malignancies. Clin Chim Acta 1988;175:157–166. 44. Twijnstra A, Ongerboer de Visser BW, van Zanten AP, et al. Serial lumbar and ventricular cerebrospinal fluid biochemical marker measurements in patients with leptomeningeal metastases from solid and hematological tumors. J Neurooncol 1989;7:57–63. 45. Weller M, Stevens A, Sommer N, et al. Intrathecal IgM response in disseminated cerebrospinal metastasis from malignant melanoma. J Neurooncol 1993;16:55–59. 46. Rombos A, Evangelopoulu-Katsiri E, Mariatos P, et al. Cerebrospinal fluid carcinoembryonic antigen and alpha fetoprotein in patients with central nervous system neoplasia. Acta Neurol Scand 1988;77:440–444.

47. Weller M, Stevens A, Sommer N, et al. Tumor cell dissemination triggers an intrathecal immune response in neoplastic meningitis. Cancer 1992;69:1475–1480. 48. Machado AB, Machado AA, Barbuto JA, et al. Central nervous system metastases from breast carcinoma. A clinical and laboratorial study in 47 patients. Arq Neuropsiquiatr 1998;56: 188–192. 49. Nakamura S, Nagano I, Yoshioka M, et al. Immunocytochemical detection of tumor necrosis factor-alpha in infiltrating tumor cells in the cerebrospinal fluid from five patients with leptomeningeal carcinomatosis. Acta Neurol Scand 1995;91: 137–140. 50. Pedersen AG, Hammer M, Hansen M, et al. Cerebrospinal fluid vasopressin as a marker of central nervous system metastases from small-cell bronchogenic carcinoma. J Clin Oncol 1985; 3:48–53. 51. Ramaswamy S, Ross K, Lander E, et al. A molecular signature of metastasis in primary solid tumors. Nat Genet 2003; 33:49–54.

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w Stem Cell Biology and Brain Tumors Kadir Erkmen and Peter McL. Black In recent years, there has been great interest in the use of neural stem cells for the management of neurologic diseases. Different sources of cells for transplantation have been used, including neural progenitor cells, neural stem cells, or embryonic stem cells. This research has spurred many debates in the scientific community as well as in the lay press as to the feasibility, safety, and ethics of such work. Studies are currently underway to further define neural stem cell biology as well as their potential in the management of human neurologic diseases. Stem cells are defined as immature, uncommitted cells that can self-replicate in tissue culture and differentiate into most lineages of cells. Embryonic stem cells (ES cells) are derived from the inner cell mass of an embryo in the blastocyst stage and are totipotent, with the ability to form all cell types and tissues from all embryonic germ layers, including neural tissues (1,2). Multipotent neural stem cells (NSCs), which have the ability to differentiate into all neural and glial cell types, are derived from the subventricular zone or germinal matrix of a developing fetus brain and can be propagated in specific growth factor– enhanced media (3,4). These cells have the ability to form neurons, astrocytes, and oligodendrocytes given the appropriate developmental cues or culture conditions. Indeed, when transplanted into the developing nervous system, NSCs disseminate throughout the CNS and integrate within the developing neural networks. Both ES cells and NSCs have been investigated for their potential role in the management of neurologic disease. Previous research has looked at the transplantation of fetal brain tissue grafts for neurologic disorders, most notably for Parkinson disease (5–11). These studies have shown that cellular replacement therapy may be a promising methodologic approach to the management of neurodegenerative diseases. A drawback of this therapeutic approach is the ethical concerns of obtaining sufficient tissue grafts from embryonic brains to treat a large number of patients. Stem cells, on the other hand, can provide an unlimited source of donor cells for transplantation through in vitro expansion in an undifferentiated state. The use of stem cells has been proposed for disorders

ranging from stroke, to neurodegenerative diseases including Parkinson disease and Huntington disease, to spinal cord injury, to demyelinating disorders. In experimental models, neural stem cells have been shown to have the ability to engraft and integrate into diseased central nervous system (CNS), repopulate specific types of degenerating cells, and express therapeutic foreign genes (4). Postulated mechanisms of action that can be harnessed for therapeutic value include (a) replacement of degenerating or injured neural cells, (b) secretion of neurotrophic factors to support host neurons and glia, and (c) delivery of a deficient factor, such as a gene product or neurotransmitter. Recent experimental studies have introduced the concept of using neural stem cells as a potential therapy for malignant brain gliomas (12–18). Despite advances in neurosurgery, chemotherapy, and radiation therapy, there has been little progress in the treatment and overall survival of patients with glioblastoma. One feature that makes glioblastomas incurable is the ability of malignant cells to infiltrate away from the primary tumor mass, which has made conventional treatment modalities ineffective. Newer modalities of treatment, such as gene therapy, have also proved ineffective due to difficulties in efficient delivery of viral vectors. Recent investigations have looked at the possible use of neural progenitor cells (NPCs) and NSCs as a novel delivery mechanism for therapeutic molecules to tumor cells due to their unique ability to migrate through normal brain along similar white matter pathways as gliomas. A study by Benedetti et al. (12) used interleukin-4 (IL-4)–secreting NPCs as a therapeutic modality for intracranial gliomas. IL-4 cDNA was transferred to NPCs derived from newborn C57BL6 mice. The investigators demonstrated that GL261 glioma cells exposed to conditioned medium from NPC IL-4 cells showed significant attenuation of growth as measured by incorporation of 3 H-thymidine. In addition, the therapeutic potential of IL-4–secreting NPCs were tested in vivo using GL261 glioma cells injected into syngeneic C57BL6 mice; alone, coinjected at a 1:1 ratio with NPCs expressing IL-4, or 855

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coinjected at a 1:1 ratio with NPCs expressing galactocerebroside (GALC). Mice injected with IL-4 NPCs lived significantly longer than control mice injected with glioma cells alone. Interestingly, mice injected with GALC NPCs also live longer than animals injected with tumor alone, indicating that NPCs themselves may have an antitumor effect. These in vivo experiments were repeated in a model that more closely imitates human disease, in which tumors were allowed to grow for 5 days prior to injection of NPCs. Results in this model also showed significant survival prolongation in mice treated with IL-4–expressing NPCs. NPCs were prelabeled with BrdU prior to injection to allow detection of cells in situ. NPCs were seen up to 20 days after injection within the tumor and at the tumor edges. In vivo production of IL-4 was also demonstrated. With this study, the authors have provided evidence that NPCs expressing IL-4 have a strong antitumor effect when injected into an intracranial model of murine gliomas. Mice that received NPC IL-4 lived significantly longer than mice without NPC transplantation. Interestingly, mice injected with NPCs expressing GALC, which has no antitumor effect, also lived longer, indicating that NPCs may have an antitumor effect of their own. Another study by Aboody et al. (13) also demonstrated the ability of NSCs expressing a therapeutic gene to prolong survival of mice with intracerebral tumors. However, this study also demonstrated the ability of NSCs to migrate through the CNS to areas of tumor growth. NSCs expressing a therapeutic transgene were injected (a) into the tumor, (b) into a location distant to the tumor but in the same hemisphere, (c) into the contralateral hemisphere from the tumor, and (d) into the cerebral ventricle. Histologic evaluation showed NSCs within the tumor mass in all four paradigms, demonstrating the extensive migratory ability of NSCs. NSCs injected into the contralateral hemisphere were seen on histologic staining to be present in the corpus callosum, crossing hemispheres, as well as within the tumor mass. In addition, NSCs appeared to demonstrate a strong tropism for tumor cells. NSCs were shown juxtaposed to tumor cells infiltrating away from the main tumor mass, indicating the ability of NSCs to track infiltrating cells along the same migratory pathways. These studies provide evidence that therapeutic molecules can be delivered to infiltrating tumors using the unique migratory abilities of NSCs. The infiltrating quality of glioma cells is what makes other more conventional treatment modalities fail. NSCs appear to be capable of migrating to areas of tumor, as well as tracking infiltrating tumor cells away from the tumor mass. A study by Tang et al. (14) demonstrated this migratory ability in vivo using bioluminescence imaging of NSCs expressing the firefly luciferase gene. They also demonstrated NSC migration toward tumor from the contralateral hemisphere across the corpus callosum. Others have proposed

that these migratory NSCs can potentially help repair damaged areas within the brain, in contrast to other therapeutic modalities for gliomas such as chemotherapy and radiation (15,16). In an attempt to take advantage of the unique migratory capability of NSCs, many different treatment paradigms have been tried using NSCs as a delivery platform for therapeutic molecules. Some examples of therapeutic modalities delivered to tumor cells by NSCs include cytotoxic chemicals, apoptosis-inducing molecules, and immune cytokines. In one study (13), NSCs were transfected to secrete the enzyme cytosine deaminase (CD), which converts the prodrug 5-fluorocytosine (5-FC) into the cytotoxic chemical 5-fluorouracil (5-FU). Tumor-bearing mice injected with CD-secreting NSCs were treated systemically with the prodrug, allowing local conversion of 5-FU to 5-FC in areas of tumor growth. The advantage of this prodrug conversion paradigm is the possibility for the “bystander effect” with the ability to cause death of multiple tumor cells surrounding a single CD-secreting NSC. In other studies, NSCs secreting therapeutic cytokines IL-4 and IL-12 were delivered as therapeutic molecules (12,17) to induce an immune-mediated cytotoxicity. These studies improved on previous viral vector–mediated immune therapy of brain tumors, presumably due to the increased ability to deliver the cytokines to tumor cell rests with the migratory ability of NSCs. Finally, NSCs were used to deliver the proapoptotic protein TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) to tumor cells resulting in increased apoptosis within tumor rests and inhibition of tumor growth (18). These studies have demonstrated the ability of NSCs to migrate through the brain toward areas of tumor growth. In fact, NSCs appear to have a strong tropism for tumor cells and may be able to track infiltrating cells away from the primary tumor mass. It is these unique characteristics of NSCs that make them an attractive platform on which to deliver therapy to brain tumors. Many different treatment paradigms have been attempted using NSCs as a novel delivery mechanism for therapy. Further study of the biology of NSCs is required before these types of therapies can be made available for the management of human glioblastoma; however, such therapies offer a novel approach to the management of a disease for which conventional therapies have failed.

REFERENCES 1. Reubinoff BE, et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134–1140. 2. Zhang SC, Wernig M, Duncan ID, et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–1133. 3. Snyder EY, et al. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 1992;68:33–51.

61. Stem Cell Biology and Brain Tumors 4. Flax JD, et al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol 1998;16:1033–1039. 5. Piccini P, et al. Dopamine release from nigral transplants visualized in vivo in a Parkinson’s patient. Nat Neurosci 1999; 2:1137–1140. 6. Studer L, Tabar V, McKay RD. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1998;1:290–295. 7. Kordower JH, et al. Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease. N Engl J Med 1995;332:1118–1124. 8. Hauser RA, et al. Long-term evaluation of bilateral fetal nigral transplantation in Parkinson disease. Arch Neurol 1999; 56:179–187. 9. Piccini P, et al. Delayed recovery of movement-related cortical function in Parkinson’s disease after striatal dopaminergic grafts. Ann Neurol 2000;48:689–695. 10. Lee CS, Cenci MA, Schulzer M, Bjorklund A. Embryonic ventral mesencephalic grafts improve levodopa-induced dyskinesia in a rat model of Parkinson’s disease. Brain 2000;123: 1365–1379.

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11. Freed CR, et al. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Engl J Med 2001;344:710–719. 12. Benedetti S, et al. Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med 2000;6:447–450. 13. Aboody KS, et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci U S A 2000;97:12846–12851. 14. Tang Y, Shah K, Messerli SM, et al. In vivo tracking of neural progenitor cell migration to glioblastomas. Hum Gene Ther 2003;14(13):1247–1254. 15. Noble M. Can neural stem cells be used as therapeutic vehicles in the treatment of brain tumors? Nat Med 2000;6:369–370. 16. Noble M. Can neural stem cells be used to track down and destroy migratory brain tumor cells while also providing a means of repairing tumor-associated damage? Proc Natl Acad Sci U S A 2000;97:12393–12395. 17. Ehtesham M, Kabos P, Kabosova A, et al. The use of interleukin 12-secreting neural stem cells for the treatment of intracranial gliomas. Cancer Res 2002;62:5657–5663. 18. Ehtesham M, Kabos P, Gutierrez AR, et al. Induction of glioblastoma apoptosis using neural stem cell–mediated delivery of tumor necrosis factor–related apoptosis-inducing ligand. Cancer Res 2002;62:7170–7174.

C h a p t e r

6 2

w Issues Regarding Death and

Dying in Brain Tumor Patients

Malcolm P. Rogers, Bernardino Vaccaro, and Peter McL. Black GENERAL REVIEW OF THE ISSUES OF DEATH AND DYING Any textbook dealing with the treatment of patients with malignant brain tumors would be incomplete without a chapter on the issue of treating the dying patient and his or her family. The treatment for malignant brain tumors is improving rapidly, yet most patients will eventually die from this disease. The goals of treatment are to prolong life and ensure the best quality of life possible. Achieving these goals means providing care through the final stages of dying and, if needed, providing bereavement care to the surviving family and friends. Over the past few decades the medical profession has taken more seriously its role in providing care and comfort to the dying patient. Although curing brain tumors is the ultimate goal, an important mission of physicians will always remain the relief of suffering. Brain tumor patients illustrate some of the issues found in the care of all patients with terminal disease: discussing the prognosis with patient and family; providing satisfactory palliative care; evaluating medical therapies, including tube feedings; understanding and evaluating the expressed desire for suicide; minimizing the adverse impact on the family; and following through with support when it is needed. Brain tumor patients also have special features: the effect of the brain injury caused by the tumor and treatment on the patients’ behavioral difficulties and drug effects. Finally, they require an understanding of the role of the surgeon, oncologist, nurse, psychiatrist, and social worker in an integrated approach to the patient.

HOW BRAIN TUMOR PATIENTS DIE Many patients with brain tumors and their families want to know what will actually happen in the process of dying. They want to know what symptoms and signs will occur, how rapid the process will be, what they can expect, what

they will be required to do, and whether there will be pain and suffering. Relieving uncertainty by forecasting the future makes coping with the process much easier for patients and families. Although the specifics are hard to answer in any given situation, there are some common patterns to dying from a brain tumor. At the point at which there is no clearly effective therapy, many patients are aware they are facing death. However, at this point, most patients are debilitated to the point that they have a diminished investment in living and have less fear of death than they do at the beginning of their illness. Most patients become increasingly lethargic, have difficulty walking because of hemiparesis or ataxia, and may become incontinent. Many of these symptoms are related to an overall increase in pressure within the brain or diffuse involvement of the brain with tumor. If patients already have some weakness in an arm or leg, their mobility will be even more limited. In general, the pattern is an increase of a preexisting deficit, with increasing drowsiness superimposed. As lethargy progresses, patients may lapse into semi- or full coma. Because the level of consciousness diminishes, there is relatively little pain and suffering for the patient. However, the family may become increasingly distressed as issues of nutrition supervene. Patients are generally bedridden at this point in their illness. If the patient remains at home, most families will need the assistance of some nursing care at home, generally provided through a hospice program. At the very end of a patient’s life, breathing becomes more labored, irregular, and eventually ceases. However, because of the lack of consciousness, patients do not experience the fear associated with respiratory failure and suffocation. Intravenous hydration is to be avoided, and ice chips or wet mouth soaks may be used for comfort. Because of the lapsing consciousness, death is not usually painful. When there are no further therapeutic options, the process of dying occurs relatively rapidly, generally within days or weeks. 859

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Although this pattern of peaceful lapse into coma is the most common for patients with malignant hemisphere tumor, it is by no means the only one. Some patients are increasingly frustrated by difficulty speaking and understanding as the tumor progresses in the left hemisphere. Some have swallowing difficulties while they are still alert, making issues of gastrostomy or permanent nasogastric tube placement necessary to face. Some have headaches that can be very difficult to palliate, requiring large doses of narcotics. Some have vomiting, which makes care difficult for the family. It is important in these variations for the physician to be available and helpful in prescribing palliative medications and not brushing these problems aside or becoming distant. Sometimes sympathetic listening is all that can be done, but it should be the physician’s role to do this.

ESSENTIALS OF PALLIATIVE CARE FOR THE DYING PATIENT During the last century, the place of death generally has moved out of homes and into institutions. More than 50% of all deaths of Medicare patients occurred in hospitals often after days or weeks of care in critical care settings. Yet many reports have emphasized that medical students and house officers receive relatively little training in the care of the dying patient (1–3). Most agree that communication between doctors and dying patients and their families needs to be improved (4). Steinmetz and colleagues (5) studied the role of primary care and family physicians in the care of dying patients. The physicians studied listed the most important and problematic aspects of their care of dying patients. They agreed that effective communication was essential to the comfort of both the patient and the physician. They found symptom management most difficult, especially breathlessness, followed by depression, pain, and anxiety. Less emotionally laden symptoms such as bedsores, nausea, vomiting, and constipation were easier to manage. The Institute of Medicine report on approaching death defines a good death as “one that is free from avoidable distress and suffering for patients, families, and caregivers; in general accord with patients’ and families’ wishes; and reasonably consistent with clinical, cultural, and ethical standards” (6). Patients and their families have further identified six major components of a so-called good death: pain and symptom management, clear decision making, preparation for death, completion, contributing to others, and affirmation of the whole person (7). Recently, new training and related curriculum (www.epec.net) have been developed for many of the critical points in cancer care, including breaking bad news, truth telling, explaining treatment options, and giving information about clinical trials (8–11). From these and

other reports, the following recommendations can be made about managing the dying patient.

Truth Telling Delivering bad news is extremely difficult for all sides and needs to be done in a way that provides something positive for the patient and family to hang on to. Too often it is perceived as cold and becomes disruptive to the relationship. For physicians, dealing with their own intense emotions and those of the patient, who may direct anger at the messenger, can be very difficult. Buckman and Kason (12) recommended a six-step protocol for delivering bad news to patients and their families: 1. Arrange to meet in a private setting to avoid interruptions. 2. Establish what the patient (and/or family) already knows. 3. Identify how much the patient (and/or family) wants to know. 4. Share the diagnosis and prognosis with the patient (and/or family). Present the various treatment options available and provide a realistic appraisal of the benefits and burdens of each. 5. Respond to the patient’s (and/or family’s) feelings and identify and acknowledge their reactions. 6. Formulate a plan of care and establish a contract for the future. Buckman and colleagues (13) also developed some helpful training materials. Additional important elements to this process include making good eye contact; listening carefully to the language of the patient and sharing the same vocabulary; obtaining an invitation to provide more information and, if declined, an open invitation to provide it in the future; giving information in clear digestible segments while checking to see if the patient has understood, or denied; maintaining empathy throughout; and summarizing the conversation at the end to avoid misunderstanding. Buckman et al. recommended scheduling a follow-up visit, which assures patients they are not being abandoned. There should also be a discussion of advance directives (living wills, health care proxies, durable powers of attorney, etc.) relatively early on in the process, in part to make it routine and in part to ensure that patients make these judgments while they still retain clear comprehension and judgment. In practice, there may be numerous visits dealing with the above steps. Timing is everything. Patients with malignant brain tumors are generally more focused on aggressive treatment in the beginning of their care, whether on surgery or radiation. Often the discussion of chemotherapy conveys the reality that the tumor has not and cannot be entirely eliminated by these other methods and that the goal is to reduce the size or keep the growth in check.

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When there is growth of the tumor despite aggressive treatments, patients are more open to discussions of the realities of a grim prognosis and to discussions about hospice care. Occasionally, some families request that patients not be told the diagnosis or prognosis directly. This is potentially a difficult situation, but our experience has been that sooner or later the patient learns the truth and often has great distress at the previous attempt at hiding it. Although it is important to allow families latitude in their own management of this issue, we also encourage families to be truthful to the patient, especially when he or she is requesting more information. Deception is to be avoided.

Pain Management Pain in terminally ill patients should be treated aggressively (14). Medication should generally be administered on a fixed regular schedule rather than on an as-needed basis. The best initial regimen is a nonopiate pain medication, such as acetaminophen 650 mg every 4 hours or 975 mg every 6 hours, aspirin (in the same dosage), or ibuprofen 400 to 600 mg every 3 to 4 hours. If this does not provide sufficient relief, a weak opiate such as oxycodone (30 mg every 3 to 4 hours) or codeine with aspirin or acetaminophen (180 to 200 mg every 3 to 4 hours) should be tried next. OxyContin is an effective controlled-release preparation of oxycodone that can be given every 12 hours, at doses ranging from 10 to 80 mg, as relief of pain requires. If the relief of pain is still insufficient, stronger opiates are used, including fentanyl patches, which provide a steady rate of analgesia over a 72-hour period (absorption is hastened by heat, so heating pads or hot water bottles should not be placed over patches); morphine 30 to 60 mg orally every 3 to 4 hours around the clock or 10 mg parenterally every 3 to 4 hours around the clock; morphine-controlled release 90 to 120 mg orally every 12 hours; or hydromorphone 7.5 mg orally every 3 to 4 hours or 1.5 mg parenterally every 3 to 4 hours. Usual concerns about overdosage or addiction are not as important here as they are in patients with postoperative pain or other reversible short-lived causes of pain. If these measures fail, additional help from a pain service or anesthesiologist with in-dwelling catheters or patient-controlled morphine pump should be considered. For patients with spinal or bone pain, neurosurgical ablative procedures may be considered as well.

Shortness of Breath Shortness of breath is best managed with humidified oxygen. In general, a vigorous search for the cause is not appropriate unless the family is very committed to prolonging life. Suctioning may be helpful if there is visible

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evidence of secretions, and families can be taught to do this. The issue of antibiotics or diuretics to treat presumed pneumonia or congestive heart failure should be discussed with the family but are generally not recommended in a hospice care situation. Often the diagnosis of a cause of dyspnea requires a trip to the emergency ward that is unsatisfactory and time consuming and not consonant with terminal care.

Hydration and Feeding Generally, hydration with intravenous fluids and feeding tubes are not regarded as comfort care (15); from a legal and ethical perspective, they are seen as any other invasive medical intervention (16). They can therefore be avoided in the terminal setting. Studies have shown that patients at the end of life lose their appetite without undue suffering. Most patients die 3 to 14 days after artificial hydration and nutrition are stopped, generally without discomfort and often slipping into coma.

Comfort Measures Measures to maximize comfort should be used. Pressure sores should be minimized with frequent turning and repositioning. Families can be helpful in this aspect of caregiving. Mouth care should be given, including swabs, mouth washes, ice chips, and shallow suction if needed. Antiemetics, antianxiety, and antidepressant medications should be given as needed, the latter with psychiatric consultation if possible. Hospice nurses are usually helpful in arranging these interventions with the physician. A bowel program is important to reduce discomfort. Unnecessary blood tests or other procedures should be eliminated. Decisions regarding the treatment of infections should be thought out in terms of providing maximum comfort to a patient rather than mindlessly prolonging life. They should be discussed with the family and, if possible, with the patient before they are made.

SPECIAL ISSUES IN BRAIN TUMOR PATIENTS Diffuse Brain Injury One of the unique aspects of many patients with brain tumor is surviving with brain damage that has altered the person irrevocably. Destruction of brain tissue by tumor and its treatment clearly alter the very essence of a person, through effects on judgment, comprehension, humor, memory, enthusiasm, energy, capacity for empathy, and capacity for emotional excitement. Sometimes these changes are subtle. But in a few instances they are profound and can have the same devastating effects that

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Alzheimer’s disease may have. The person is physically there but in some significant way is already gone. This can pose special burdens and conflicts for the spouse or family. It is difficult for patients to acknowledge that prolongation of life under these circumstances is often not a desirable goal. Many spouses have already assumed the total burden for childcare and financial support. To add the burden of custodial care of a spouse who is irrevocably brain damaged can be overwhelming.

of radiation effect, further tumor growth, and edema. In the end, his rapid decline and death brought the family closer together, with the support of the brain tumor center staff. The medical team can help the family acknowledge that prolongation of life, when its quality is drastically reduced, is not necessarily a desirable goal.

Behavior and Personality Change Case Vignette 2

Clinical Vignette 1 A 38-year-old father of a 4-year-old daughter lived for 10 years after the diagnosis of a left frontal oligodendroglioma. This tumor had responded well to several surgical procedures, radiation, and chemotherapy. Moreover, the cumulative effects of his tumor growth and treatment left his personality and cognitive function permanently changed in what turned out to be his last year of life. He was unable to work or to provide care for his 4-year-old daughter. He remained at home all day watching television. For their financial support, his wife needed to continue to work full time. She was pained by his lack of emotional responsiveness to her but especially to their young daughter, who didn’t understand why her father was less affectionate. His short-term memory difficulties, slowness, and self-centered behavior sometimes elicited his wife’s anger, although she understood that the disease had caused these changes. She believed that his own parents and siblings did not fully understand the situation and did not provide much support for her and her daughter. They, at times, felt excluded from his care by her. His illness, therefore, became a source of division within the larger family. She felt ambivalent about further prolongation of his life and was able to verbalize this ambivalence in counseling. It led to considerable guilt, however. As he began to deteriorate more rapidly, meetings of the medical team with the patient, his wife, and his family allowed for a more cooperative effort as they cared for him at home during the last days of his life. It was helpful to have a coordinated approach to his care by his neurosurgeon, radiation oncologist, neurologist, and medical oncologist, and the guidance of the psychiatrist was invaluable. He died at home. After his death, his wife was able to resume a more normal life and to maintain a relationship with her in-laws. Comment: This case is a good illustration of the family’s burden of living and caring for a family member with cognitive, emotional, and personality change. To put it into perspective, the patient did have a significant prolongation of a life of reasonably good quality until the final year of life. This is typical of patients with a brain tumor, who usually function well until they approach a precipitous decline. In this case, the changes were a combination

A 39-year-old married father of an infant had a left frontal lobe glioblastoma. His tumor had grown aggressively through surgery, radiation, and chemotherapy. Increasing problems with edema required him to be on large doses of dexamethasone, up to 24 mg/day. He had a significant expressive aphasia. The patient and his parents and siblings strongly believed that an alternative treatment clinic in Mexico offered him the prospect of cure. His wife was more realistic and persuaded him to defer such a trip. Instead, in the last 2 months of his life, he and his wife went on a vacation to Hawaii. He was able to swim and soak up the sun in an environment similar to his native California. He felt rejuvenated. He became increasingly agitated as the vacation came to an end, feeling that a few more days there would help him to defeat the tumor. On the airplane flight back, he refused to sit in the back of the plane, complaining about the quality of air. His behavior embarrassed his wife, who was trying to manage the care of her infant in addition to helping him. It also troubled the pilot, who was not willing to fly until he heard an explanation of one of his passenger’s erratic behavior. After they returned to New England, it became clear to the doctors and his wife that there were no further aggressive treatment options. His behavior continued to be erratic. At one point, he threatened to jump from the passenger seat of the car his wife was driving and at another point cut himself superficially with a knife. He was seen by a psychiatrist, who believed the dexamethasone had triggered a manic episode, on top of the psychologic threatened loss of life and function. He was treated with antipsychotics and high doses of antianxiety medications, but his behavior remained very difficult for his wife to manage at home. His aphasia made communication more difficult. Eventually, he was hospitalized in an acute care institution both for management of his behavior and relief of pain and other palliative care. He died a few weeks later in the hospital. Comment: This is a somewhat dramatic example of the sudden changes in behavior that may occur toward the end of life. One needs to provide vigorous psychopharmacologic intervention, family support, and, if needed, as it was in this case, hospitalization. It is important to recognize that clinically abnormal behavior should be treated

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in this setting with the same treatments that would be used if there were no brain tumor, including antianxiety, antipsychotic, and antimanic medication if needed. Another difficult aspect of this case was the unrealistic search for a miraculous cure by the patient and extended family, which made his wife’s burden in managing his death significantly more difficult. On the one hand, she had come to the painful realization that he would soon die and hoped for a more peaceful and less chaotic death. On the other hand, her in-laws were telling her, in effect, that she was negligent in not striving for a cure and should elect instead a desperate trip far from home. She was put in a position of conflict, rather than being supported by his family in the final weeks of his life. The example underscores the importance of family cohesion in the care of the dying patient. His team of doctors supported her goals, reassuring her that her approach was appropriate, and supported her efforts to calm his agitation and provide care at home as long as possible. This case also illustrates the special difficulties at the end of life posed by aphasia. Aphasia can be enormously frustrating for patients and their families and can occasionally be associated with uncharacteristic aggressive outbursts. Antianxiety or antipsychotic medications, such as lorazepam and haloperidol, may be helpful in managing such aggressive behavior.

COPING WITH DYING Patients Fear of death is a natural reaction of most patients. It helps to know more precisely what a given patient most fears: whether it be separation from loved ones, loss of control, pain and suffering, abandonment, or punishment. Some patients, by virtue of their religious life and experience, have very specific expectations of what follows death. Others are less certain. The age of the patient influences many of these reactions. Older patients who have lived full and gratifying lives will accept it more easily than younger patients, especially those who have younger children. Case Vignette 3 A 38-year-old computer programmer was diagnosed as having a glioblastoma multiforme. He underwent surgery, followed by radiation and then chemotherapy. He lost his appetite during the chemotherapy and experienced marked fatigue. He was unable to work, and his stamina diminished. His marriage revolved around their mutual work and a shared passion for gourmet cooking. There were no children. They both believed that life as they had known it was over. The patient himself had little tolerance for any further medical intervention. He wanted to “hurry

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up and get it over with.” He and his wife had discussed suicide but dismissed it as an option because the life insurance policy would be nullified by suicide. Comment: This case represents an exceptional response. Both the patient and his wife were starkly realistic about the future. It was as if they had permitted themselves to be hopeful for the last time and now wanted no medical intervention, false hope, and anything that would prolong life. They had already grieved the loss of life as they knew it. There certainly was an element of depression in his response. On the other hand, this was also a rational wish for death. His fatigue, loss of interest, and pleasure in some of the activities that remained were evident. His internist prescribed fluoxetine, which was rejected after a few days because of nausea. He was also seen in psychiatric consultation. A stimulant, methylphenidate, was prescribed. It had a brief dramatic effect on his energy level and desire to do things. However, he found the stimulating effect to be too intense and simply stopped taking it after 2 to 3 days rather than titrating the dosage downward. The event confirmed their decision to avoid any further medical intervention. Stimulants such as methylphenidate or dopamine agonists, such as bromocriptine (17), may be helpful in the treatment of abulia or akinetic mutism associated with frontal lobe injuries.

Families The most important gift friends and family can give to those who are dying is to be with them. The last few months of life can be particularly meaningful. Fear of abandonment, fear of loss of control, or pain and suffering are generally the greatest worries of those who are dying. Case Vignette 4 A college teacher and his wife sought psychiatric help in dealing with the discovery of a left temporal glioblastoma in his 78-year-old mother-in-law. Their biggest concern was how to soften the impact on the patient’s husband, who had become withdrawn and refused to discuss the implications of his wife’s cancer. The son-in-law and daughter believed the father should be protected from further information about the prognosis. They opted for radiation therapy, which they presented to him in a very positive way. They planned a cross-country vacation, lasting several months, as a final period of shared time together. Even after the patient became totally aphasic as a result of her tumor, the entire family arranged to be together virtually all of their time over the months before her death. Comment: Even though in some cases verbal communication with the patient is no longer possible, families can experience great joy in being together during the last few months of life.

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Doctors Dealing with intimate end-of-life decision making with patients and families can be emotionally enriching, but it can also be psychologically draining. It is helpful for physicians to share their own uncertainties about treatment options and to reach out to other members of the health care team. Doctors do need to answer questions about prognosis and death with sensitivity as discussed above under truth telling. For most physicians and other health care providers, acknowledging death may raise uncomfortable feelings about their own mortality or the deaths of their own loved ones. Feelings of failure are also common. Opportunities to share experiences with their peers may be quite helpful (18). There are times when questions from the patient and family suggest a denial of the possibility of death, and the physician’s judgment in dealing with such denial is critically important. Case Vignette 5 A divorced 49-year-old mother of a 17-year-old daughter presented with a series of metastatic brain lesions from a primary breast cancer. She had a good response to radiosurgery with two of the lesions, allowing her to continue to live independently for over a year and even to continue her work as a therapist. After a year and a half, her condition took a marked turn for the worse, with lethargy, memory loss, and ataxia. She was no longer able to live independently, and her sister moved in with her. During a clinic visit to help sort out the possibilities for her condition, the sister asked what would happen if the reason for her deterioration turned out to be carcinomatous meningitis as suspected. “Death,” answered the neurosurgeon calmly but unflinchingly. “Oh,” responded the sister, who then quietly started crying. There was no surprise or sadness expressed by the patient. The tension in the room seemed to dissipate. Comment: There are moments when doctors need to be unflinchingly direct about what patients and families are facing. Knowing when that moment is and how to present it requires as much skill from the doctor as is needed in brain surgery in the operating room or in the administration of radiation therapy or chemotherapy.

Nurses Nurses play a pivotal role in all phases of the care of brain tumor patients, including terminal care and bereavement counseling. In hospital settings, nurses have the most frequent contact with patients, and, as such, often play a critical role in providing emotional support. As one patient wrote in an article about his cancer treatment, even a brief

empathic exchange made an enormous difference in his comfort level (19). This exchange might take the form of a comforting word, a gentle touch, a hand held, or a genuine expression of concern. Of course, such interactions should not be limited to nursing but should include physicians as well. Nurses may, however, play a particularly important role in identifying symptoms and problems, which the patient may not wish to discuss with the physician; in assisting the physician in prescribing appropriate palliative care; and in providing outreach care at home, through the Visiting Nurse Association (VNA) or hospice programs.

BEREAVEMENT The Bereavement Process Bereavement is the loss of a loved one through death. Grief is the affect or feelings associated with the loss (20). Mourning refers to the socially sanctioned rituals, actions, and expectations that are part of the bereavement process. There are many different models describing the bereavement process. Elisabeth Kubler-Ross (21) described five stages of death and dying experienced both by the individual and his system of supports from the time of identifying the actual or anticipated loss, through the resolution of the grieving process. These stages include denial of the illness or impending loss; anger concerning the individual’s plight; bargaining, a period of trying to forestall the inevitable until some goal can be achieved; depression, the acknowledgment of the reality of the impending loss; and acceptance, the process of coming to terms with the reality of the loss and incorporating this reality into the actions of one’s life. A second model describing the bereavement process is that of John Bowlby (22), who observed the impact on children of the loss of their parents. He noted that children went through an initial phase of protest, crying out, and calling for their parents, followed by efforts to regain the lost parents. When these efforts failed, the children went through a period of despair over losing their parents and then a period of disorganization. During the period of disorganization, the children were not attached to any adults in the hospital and often would refuse interactions with other caregivers or only minimally acknowledge their presence. Finally, they would begin to reorganize and reconnect to other individuals in their environment and form new relationships. The grieving or bereavement process occurs as a parallel process, affecting both the person diagnosed with cancer and the family system. The person diagnosed with cancer may lose a sense of personal identity and integrity. He or she is now a cancer patient. His or her life becomes reorganized around doctors’ visits, chemotherapy, radiation

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therapy, and monitoring for recurrence. If the brain tumor or its treatment alters brain structure and function and results in cognitive, physical, or personality changes, the loss can be devastating. The once independent intelligent individual may no longer be able to function independently and effectively as before. There are often concurrent losses of job, status, mobility, independence, and physical attractiveness. These losses are real and must be acknowledged and worked through. The family may no longer be able to rely on the patient for support, assistance with household duties, and child rearing. The patient may not be safe alone because of confusion, forgetfulness, and irritability. Other issues of grieving concern the stage of life at which the diagnosis is made. A younger person may have to grieve the loss of a career cut short or a relationship never developed. If there are children, a dying parent faces the loss of never seeing them grow up, concerns for their future support and well-being, and strong emotions around leaving or abandoning them. An older person, who has raised a family or had a career, may have a very different reaction to the threat or death. The bereavement process may last from 6 to 18 months. Anticipatory grief, a term coined by Lindemann (23) begins with the assumption that death will occur. Unlike the kind of grief that begins with peak intensity at the moment of an unexpected sudden death, anticipatory grief is a gradual process that often accelerates as death approaches. It is also a process that the family and the patient can experience together. In either case, the process of grieving involves acknowledging the reality of the loss of the loved one, coming to terms with the both practical and psychologic meanings of this loss, and reorganizing one’s life without the loved one. In the early stages, survivors are preoccupied with the loved one. They will experience strong emotions, such as anger, tearfulness, sadness, depressive affect, sleeplessness, decreased appetite, lack of energy, aimlessness, and, in more severe cases, despair. Funerals, wakes, and memorial services serve an important role in helping the survivors to acknowledge the reality of the loss, receive extra support, and begin the grieving process. Psychologically, the grieving process is about acknowledging the loss of the loved one and gradually withdrawing the psychologic energy and emotions connected to the deceased. As time goes on, this energy and emotion becomes available for daily living and new relationships. This is often accomplished by means of reminiscing about the deceased, either alone or with family and friends. Also, as time goes on, the strong emotions experienced early on in the grieving process will begin to subside. Sleep patterns will stabilize. Appetite, enjoyment, and energy will begin to return. To some extent, grieving is never completed. Individuals will often report feelings of sadness and grief for years

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after the death of a loved one. These will often occur around important anniversaries of the individual and other important family events such as holidays, birthdays, graduations, and so forth. The question arises, when does an individual need a referral to a psychiatrist or a counselor? This is often not an easy question to answer. Some signs of concern are when an individual demonstrates continued weight loss, sleep disturbance, or anhedonia that do not appear to be resolving over 2 to 4 weeks. Any signs that an individual is not able to attend to activities of daily living, personal hygiene, or self-care are also cause for immediate concern and consultation. Any statements suggesting suicidal ideation or planning are also cause for alarm. Careful questioning as to the meaning of such statements and prompt referral to mental health professionals are indicated. Helpful interventions may include the short term use of low-dose benzodiazepines for sleep and a referral to bereavement or support groups within the hospital, hospice, or their church or synagogue. The grieving process can often be aided if the family is supported or encouraged to talk with the dying member. Taking care of unfinished business around relationships, disagreements, and the future can help bring a sense of closure. This facilitates the bereavement process for all involved. Unexpressed feelings, thoughts, goodbyes, and relationships that were conflicted or ambivalent can lead to complicated bereavement reactions. Case Vignette 6 A 43-year-old woman had multiple brain tumors, including right parietal and left temporal lesions. She had been treated with surgery and radiation and had multiple admissions for management of her declining function. On one such admission, she was evaluated by a psychiatrist, who believed that she had an adjustment reaction to her illness in addition to her organic mental disorder. Brief psychotherapy was begun. The patient had the opportunity to review her life and come to terms with her disease and possible impending death. She had been a leader in early childhood education and who started her own school. She talked about hoping to return to teaching. Later she acknowledged that being able to do so was unlikely. She had been with her common-law husband for 15 years. During the last weeks of her life, they decided to marry. A service was held in the hospital. As her lesions grew, she became more aphasic at times but continued to look forward to her visits with her husband and the psychiatrist until shortly before her death. Comment: This case demonstrates that even though a patient may have significant neurologic and cognitive impairment, he or she may still be able to do a significant

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amount of psychologic work around mourning the loss of career and future. This patient was able to make significant decisions around important personal issues such as marriage. Even though her life was shortened, the final weeks were especially meaningful for this patient and her husband.

Atypical Grief Reactions Bereavement can also be complicated in situations in which an individual may have previously unresolved grief, excessive dependency, or anger toward the loved one. Other possible predictors of poor outcome include previous psychiatric or substance abuse history and poor social support. Pathologic bereavement is a term that describes the failure of the grieving individual to process and work through the death of a loved one and the failure to move on with one’s life. Signs of this may be continued idealization of the deceased disproportionate to their realistic qualities or continued disturbance of sleep, appetite (often with weight loss), and mood. Other signs include feelings of hopelessness and helplessness and the inability to attend to responsibilities of daily life. In some individuals, grief reactions may progress to major depression (20). Any of these developments may indicate the need for formal mental health consultation.

Health Consequences of Bereavement Bereavement can have important health consequences for the survivors (24). Studies have shown a slight increase in morbidity and mortality after the death of a loved one. Men who are living alone appear to be at particular risk. There may be more visits to the doctor, more days out of work, and more physical disease, particularly cardiac in nature (25). There is also some evidence for diminished immune function in men during bereavement (26). There also appears to be an increase in depression 1 year after bereavement, especially in those with a prior history of depression (27).

Bereavement Services Most hospice programs provide counseling and support for bereavement. There are other established self-help community programs, such as the widow-to-widow program. There are also numerous books written for the bereaved family, such as Edgar Jackson’s You and Your Grief (28) and Simos’s A Time to Grieve (29). Silverman (30) focused on helping children to deal with the death of a parent. Others who have written on bereavement more for health professionals include Parkes and Weiss (31), Schneider (32), and Pollock (33). There are multiple resources available through the Internet.

HOSPICE CARE The word “hospice” has been used for centuries to describe a place of shelter and rest for the weary or sick travelers on long journeys. It was first applied to the care of dying patients in 1967 in a residential suburb of London. The movement has grown rapidly (34). As of January 2001, there were 2,273 hospice programs recognized by Medicare, existing in all 50 states. They serve more than 400,000 terminally ill Medicare patients and their families every year. There are many more volunteer programs. Information about available programs exists in most hospital social service and continuing care departments and in community visiting nurse programs or on the Internet. The National Hospice and Palliative Care Organization can be contacted through a toll-free Hospice Helpline at 800-658-8898 or at www.nhpco.org. The Hospice Education Institute offers referrals and regional seminars on hospice care around the country (1-800-3311620 or at www.hospiceworld.org. There are many other national resources available on the Internet. More than 90% of hospice care is delivered at home rather than in specific residential treatment programs. Patients who are not expected to live for more than 6 months are accepted for hospice care. The goals of hospice programs are to provide palliative rather than curative care and to emphasize comfort and quality of life rather than prolongation. All patients are on a “do not resuscitate” status. Hospice programs treat the patient and family as a unit. They provide 24-hour coverage. Depending on the level of need, home health aides and homemaker services are provided in the home during the day. Nurses make periodic visits as needed and are available on a 24-hour basis. Hospice care programs have special expertise in bed care to avoid pressure sores, pain management, catheter care, mouth care, and other approaches described above as palliative care. The guiding principle is always to provide comfort and quality rather than simply prolonged life. Other hospice services include physical, occupational and speech–language therapists; medical supplies and appliances; social service; and spiritual, dietary, and other counseling, including bereavement. If needed, brief hospitalizations are coordinated. Each hospice program has a physician medical director, who coordinates the prescribing of medications with the primary physician. With respect to brain tumor patients, the primary physician may in some cases be a specialist in the care of brain tumors, such as a neurosurgeon, neurologist, medical, or radiation oncologist. Patients and families generally want to maintain a strong link, in part because of the special expertise of this group regarding anticonvulsants and dexamethasone to control cerebral edema and general explanations as to the meaning of various symptoms and signs. Psychiatrists who are knowledgeable about brain tumors

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can be very helpful in providing psychopharmacologic and psychotherapeutic support. In our brain tumor center, most patients make use of hospice programs, frequently providing a transition to them from visiting nurse associations or other comprehensive home health care programs. Dying at home, as opposed to the hospital, is generally preferable for the patient and the family.

ETHICAL ISSUES In one extensive survey of physicians treating dying patients in a hospital setting (35), most physicians believed that patients were overtreated with ventilation and resuscitation efforts. In contrast, they believed that patients were undertreated in terms of pain management. A recent study of 9,000 desperately ill hospitalized patients found, despite considerable efforts to the contrary, that these patients died prolonged and painful deaths (4). In almost half the cases, physicians were unaware of the wishes of their patients. As a result, even when patients clearly indicated that they wanted “heroic” treatments withheld, these measures were often given anyway. This study did not include significant numbers of brain tumor patients, but it underscores the need for clear communication with patients and their families about treatment at the end of life. It is often the failure to provide appropriate end-of-life care that raises the issue of patientrequested suicide (36). Dr. Jack Kevorkian made assisted suicide a highly public issue. Many disagree with his methods, but few doubt the need for renewed attention to this complicated issue. Other physicians, such as Timothy Quill, have approached and written about this issue in a more balanced and thoughtful fashion (37–40). He has pointed out that failure to provide appropriate information about prognosis and palliative care contributes to unnecessary pain and suffering. Meaningful end-of-life options and issues—such as health care proxies, do not resuscitate orders, other life-sustaining measures such as ventilation and feeding tubes, and palliative care—should be discussed sooner than they are. The level of public support for change in this area became evident with the 1994 Death with Dignity Act, passed by most voters in the state of Oregon and finally implemented in 1997. It remains in effect despite attempts to overturn it. Other states have not enacted similar laws, and several medical societies have opposed it (41). The Oregon law allows certain terminally ill patients with less than 6 months to live to commit suicide with the assistance of their attending physicians. It further requires that the patient be competent and not mentally ill. The Oregon experience suggests that as patients underlying concerns are addressed, the likelihood of a request for physician-assisted suicide decreases markedly.

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Approximately 1% of dying patients request it and approximately 10% of those persist in their request. For many the possession of a lethal prescription is sufficient to provide a needed sense of control over the time and manner of their death (42).

Rational Suicide There are differences of opinion about the prevalence of so-called rational suicide. Most studies that have actually investigated the issue have found that the desire for death in the terminally ill is closely associated with clinical depression in most cases. Most studies support the need for careful evaluation of depression. During a 2-week followup period, Brown and colleagues (43) studied the final days of 44 terminally ill patients. They found that most (77%) had never wished for death to come early. Of the remaining 10 patients, 3 had been suicidal and 7 had wished for an early death. All 10 patients were found to be suffering from clinical depression. When one looks carefully for psychiatric diagnosis among people who have committed suicide, one finds compelling evidence for the presence of significant mental illness. One group of investigators (44) found that in young men completing suicide, the combination of borderline personality disorder, affective disorder, and untreated alcoholism was particularly lethal. Well-known risk factors include male gender, increasing age, living alone, recent loss, early parental loss, and family history of suicide. Thus, there is compelling evidence that depression can be driving the seemingly rational wish to die and should be carefully evaluated in every terminally ill patient. Nevertheless, most physicians also agree that the decision of a terminally ill patient to shorten the period of suffering before death can be rational. However, when our care of dying patients is as thoughtful and attentive as our most aggressive therapies, patients do not commonly seek assisted suicides (36). We should strive to create an approach that allows death to be a natural and expected milestone of human existence rather than only as an unwanted outcome of disease. (45). What patients really want at the end of life are relief of suffering, help in minimizing the burden on families, closer relationships with their family, and a sense of control (46).

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