Wintrobe's Clinical Hematology [13 ed.] 9781451172683, 2013023525

Citation preview

Wintrobe’s Clinical Hematology T h i r t e e n th

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E d i t i o n

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Wintrobe’s Clinical Hematology T h i r t e e n th

E d i t i o n

Editors: John P. Greer, MD

Alan F. List, MD

George M. Rodgers, MD, PhD

Professor Departments of Medicine and Pediatrics Divisions of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

Senior Member Department of Malignant Hematology President and CEO Moffitt Cancer Center Tampa, Florida

Professor of Medicine and Pathology University of Utah School of Medicine Health Sciences Center Medical Director, Coagulation Laboratory ARUP Laboratories Salt Lake City, Utah

Daniel A. Arber, MD

Robert T. Means, Jr., MD

Professor and Vice Chair Department of Pathology Stanford University Director of Anatomic and Clinical Pathology Services Stanford University Medical Center Stanford, California

Professor of Internal Medicine Executive Dean University of Kentucky College of Medicine Lexington, Kentucky

Bertil Glader, MD, PhD Professor Departments of Pediatrics and Pathology Stanford University Medical Center Stanford, California Lucile Packard Children’s Hospital Palo Alto, California

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Editor Emeritus: John Foerster, MD, FRCPC Professor and Physician Emeritus Winnipeg, Manitoba, Canada

Frixos Paraskevas, MD Professor of Internal Medicine and Immunology (Retired) University of Manitoba Medical School Associate Member Institute of Cell Biology—Cancer Care Manitoba Winnipeg, Manitoba, Canada

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Senior Executive Editor: Jonathan W. Pine, Jr. Senior Product Manager: Emilie Moyer Production Product Manager: David Saltzberg Manufacturing Manager: Beth Welsh Marketing Director: Lisa Zoks Senior Designer: Stephen Druding Production Service: Integra Software Services Pvt. Ltd. © 2014 by LIPPINCOTT WILLIAMS & WILKINS, a WOLTERS KLUWER business Two Commerce Square 2001 Market Street Philadelphia, PA 19103 USA LWW.com First Edition: Lea & Febiger, 1942. Second Edition: Lea & Febiger, 1946; Third Edition: Lea & Febiger, 1951. Fourth Edition: Lea & Febiger, 1956. Fifth Edition: Lea & Febiger, 1961. Sixth Edition: Lea & Febiger, 1967. Seventh Edition: Lea & Febiger, 1974. Eighth Edition: Lea & Febiger, 1981; Ninth Edition: Williams & Wilkins, 1993; Tenth Edition: Lippincott Williams & Wilkins, 1999; Eleventh Edition: Lippincott Williams & Wilkins, 2004; Twelfth Edition: Lippincott Williams & Wilkins, 2009. All rights reserved. This book is protected by copyright. No part of this book may be reproduced in any form 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. Printed in China Library of Congress Cataloging-in-Publication Data Wintrobe's clinical hematology / editors, John P. Greer, Daniel A. Arber, Bertil Glader, Alan F. List, Robert T. Means Jr., Frixos Paraskevas, George M. Rodgers ; editor emeritus, John Foerster. —13th edition. p. ; cm. Clinical hematology Includes bibliographical references and index. ISBN 978-1-4511-7268-3 (hardback) I. Greer, John P., editor of compilation. II. Title: Clinical hematology. [DNLM: 1. Blood. 2. Hematologic Diseases. 3. Hematology—methods. WH 100] RB145 616.1'5—dc23 2013023525 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 the 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 the 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 FDA status of each drug or device planned for use in their clinical practice. To purchase additional copies of this book, call our customer service department at (800) 638-3030 or fax orders to (301) 223-2320. International customers should call (301) 223-2300. Visit Lippincott Williams & Wilkins on the Internet at: LWW.com. Lippincott Williams & Wilkins customer service representatives are available from 8:30 am to 6 pm, EST. 10 9 8 7 6 5 4 3 2 1

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c o n t r i b u t o r s

Darryl J. Adamko, MD, FRCPC

Robert J. Arceci, MD, PhD

A. Dean Befus, PhD

Associate Professor Department of Pediatrics University of Saskatchewan Saskatoon, Saskatchewan Canada

Professor Department of Pediatric Oncology Johns Hopkins University Baltimore, Maryland

Professor Department of Medicine University of Alberta Edmonton, Alberta Canada

Donald M. Arnold, MD, MSc Archana M. Agarwal, MD Assistant Professor Department of Pathology University of Utah Medical Director Special Genetics ARUP Laboratories Salt Lake City, Utah

Blanche P. Alter, MD, MPH Senior Clinician Clinical Genetics Branch Division of Cancer Epidemiology and Genetics National Cancer Institute Rockville, Maryland

Claudio Anasetti, MD Professor Oncologic Sciences University of South Florida Senior Member Department Chair Department of Blood and Marrow Transplantation H. Lee Moffitt Cancer Center Tampa, Florida

Stephen M. Ansell, MD, PhD Professor of Medicine Mayo Clinic College of Medicine Consultant Division of Hematology Department of Internal Medicine Mayo Clinic Rochester, Minnesota

Daniel A. Arber, MD Professor and Vice Chair Department of Pathology Stanford University Director of Anatomic and Clinical Pathology Services Stanford University Medical Center Stanford, California

Associate Professor Department of Medicine McMaster University Associate Medical Director Medical Affairs Canadian Blood Services Hamilton, Ontario Canada

P. Leif Bergsagel, MD Professor College of Medicine Mayo Clinic Medical School Rochester, Minnesota Department of Hematology/Oncology Mayo Clinic Phoenix, Arizona

Maria R. Baer, MD Professor Department of Medicine University of Maryland School of Medicine Director Hematologic Malignancies University of Maryland Marlene and Stewart Greenebaum Cancer Center Baltimore, Maryland

Charles D. Bangs, BS Cytogenetics Laboratory Supervisor Clinical Laboratories Stanford Hospital and Clinics Palo Alto, California

Kirstin F. Barnhart, DVM, PhD, DACVP Principal Pathologist AbbVie, Inc. Preclinical Safety North Chicago, Illinois

James C. Barton, MD Clinical Professor of Medicine Department of Medicine University of Alabama at Birmingham Medical Director Southern Iron Disorders Center Brookwood Medical Center Birmingham, Alabama

Minoo Battiwalla, MD, MS Staff Clinician, Hematology Branch National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland

Nancy Berliner, MD Professor Department of Medicine Harvard Medical School Chief Division of Hematology Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts

Kristie A. Blum, MD Associate Professor Division of Hematology, Internal Medicine The Ohio State University The Ohio State University Wexner Medical Center Columbus, Ohio

Caterina Borgna-Pignatti, MD Full Professor of Pediatrics Clinical and Experimental Medicine University of Ferrara Chief Department of Pediatrics Arcispedale Sant’Anna Ferrara, Italy

Linette Bosques, MPhil PhD Candidate Departments of Cell Biology and Pathology Yale School of Medicine New Haven, Connecticut

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Contributors

Sylvia S. Bottomley, MD

William C. Chapman, MD

Professor Emeritus of Medicine Department of Medicine University of Oklahoma College of Medicine Staff Physician Medical Service VA Medical Center Oklahoma City, Oklahoma

Professor of Surgery Department of Surgery Washington University—St. Louis Attending/Chief of Surgery Department of Surgery Barnes Jewish Hospital St. Louis, Missouri

Robert A. Brodsky, MD Professor of Medicine and Oncology Division of Hematology Department of Medicine Director Division of Hematology Johns Hopkins University Baltimore, Maryland

Kathleen E. Brummel-Ziedins, PhD Associate Professor Department of Biochemistry University of Vermont Burlington, Vermont

Francis K. Buadi, MD, ChB Assistant Professor Consultant Hematologist Department of Internal Medicine Mayo Clinic Rochester, Minnesota

David C. Calverley, MD Associate Professor of Medicine Division of Hematology/Medical Oncology Oregon Health and Science University Portland, Oregon

Ralph Carmel, MD Professor of Medicine Department of Medicine Weill Cornell Medical College New York, New York Director of Research Department of Medicine New York Methodist Hospital Brooklyn, New York

Howard H. W. Chan, MBChB, FRCP(C), MSC Assistant Professor Department of Medicine McMaster University Hamilton, Ontario Canada

Karen L. Chang, MD Physician-in-Charge Histology/Immunohistochemistry Kaiser Permanente Southern California Regional Laboratory North Hollywood, California

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Judah A. Denburg, MD, FRCPC William J. Walsh Professor of Medicine Department of Medicine McMaster University Hamilton, Ontario Canada

Athena M. Cherry, PhD Professor Department of Pathology Stanford University School of Medicine Cytogenetics Laboratory Director Clinical Laboratories Stanford Hospital and Clinics Stanford, California

Andrew Chow, BA MD-PhD Candidate Stem Cell Institute Albert Einstein College of Medicine New York, New York

Robert D. Christensen, MD Director of Neonatology Research Women and Newborn Services Intermountain Healthcare Salt Lake City, Utah

Matthew Collin, BM, BCh, DPhil, FRCPath Professor of Hematology Institute of Cellular Medicine Newcastle University Consultant Hematologist Northern Centre for Cancer Care Newcastle upon Tyne Hospitals Newcastle upon Tyne, United Kingdom

Steven E. Coutre, MD Professor of Medicine (Hematology) Department of Medicine Stanford University School of Medicine Stanford Cancer Center Stanford, California

Utpal P. Davé, MD Assistant Professor Department of Medicine Division of Hematology/Oncology Vanderbilt University School of Medicine Attending Physician Department of Medicine/Division of Hematology/Oncology Tennessee Valley Health Systems VA Nashville, Tennessee

Robert J. Desnick, PhD, MD Dean for Genetic and Genomic Medicine Professor and Chairman Emeritus Genetic and Genomic Sciences Mount Sinai School Medicine Physician-in-Chief Genetics and Genomic Medicine Mount Sinai Hospital New York, New York

David Dingli, MD, PhD Professor of Medicine Hematology and Internal Medicine Mayo Clinic College of Medicine Consultant, Hematology Mayo Clinic Rochester, Minnesota

Angela Dispenzieri, MD Professor of Medicine and Laboratory Medicine Department of Medicine Mayo College of Medicine Mayo Clinic Rochester, Minnesota

Ahmet Dogan, MD, PhD Professor of Pathology Department of Laboratory Medicine and Pathology Mayo Medical School Consultant Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota

Michael W. N. Deininger, MD, PhD

M.B. Majella Doyle, MD

M.M. Wintrobe Professor of Medicine Chief, Division of Hematology and Hematologic Malignancies Department of Internal Medicine University of Utah/Huntsman Cancer Institute Salt Lake City, Utah

Assistant Professor of Surgery Department of Surgery Washington University—St. Louis Attending, Department of Surgery Barnes Jewish Hospital St. Louis, Missouri

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Contributors



Cynthia E. Dunbar, MD

Haydar Frangoul, MD

Renzo Galanello, MD

Senior Investigator and Section Head Hematology Branch The National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland

Professor Director Pediatric Blood and Marrow Transplant Program Division of Pediatric Hematology/Oncology Vanderbilt University School of Medicine Monroe Carell Jr. Children's Hospital at Vanderbilt Nashville, Tennessee

Professor of Pediatrics Department of Biomedical Sciences and Biotechnologies University of Cagliari Head, 2nd Pediatric Clinic Thalassemia Unit Cagliari, Italy

Anne F. Eder, MD, PhD Executive Medical Officer National Headquarters, Biomedical Services American Red Cross Rockville, Maryland Adjunct Assistant Professor Department of Pathology and Laboratory Medicine Georgetown University School of Medicine Washington, D.C.

Corwin Q. Edwards, MD Professor of Medicine Department of Medicine University of Utah School of Medicine Director of Graduate Medical Education Intermountain Medical Center and LDS Hospital Salt Lake City, Utah

Ashkan Emadi, MD, PhD Associate Professor Department of Medicine Division of Hematology/Oncology Leukemia and Hematologic Malignancies University of Maryland School of Medicine Marlene and Stewart Greenebaum Cancer Center Baltimore, Maryland

Paul S. Frenette, MD Chair and Director Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Albert Einstein College of Medicine New York, New York

Richard C. Friedberg, MD, PhD Professor and Deputy Chairman Department of Pathology Tufts University School of Medicine Western Campus Chair Department of Pathology Baystate Medical Center Springfield, Massachusetts

Debra L. Friedman, MD, MS Associate Professor Department of Pediatrics Vanderbilt University School of Medicine Director, Division of Pediatric Hematology/Oncology Monroe Carell Jr. Children’s Hospital at Vanderbilt Nashville, Tennessee

Rafael Fonseca, MD Professor of Medicine Hematology/Oncology Mayo Clinic Scottsdale, Arizona

Magali J. Fontaine, MD, PhD Assistant Professor Department of Pathology Stanford University Associate Medical Director Department of Pathology, Transfusion Service Stanford, California

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Associate Professor Department of Pathology Stanford University Stanford, California Director of Clinical Operations Stanford Blood Center Palo Alto, California

Patrick G. Gallagher, MD Professor Department of Pediatrics and Genetics Yale University School of Medicine Attending Physician Yale—New Haven Hospital New Haven, Connecticut

Jacob R. Garcia, MD Associate Hematologist/Oncologist Pediatric Hematology/Oncology Children’s Hospital and Research Center Oakland Oakland, California

Guillermo Garcia-Manero, MD Professor Department of Leukemia University of Texas MD Anderson Cancer Center Houston, Texas

Amy E. Geddis, MD, PhD

Stephen J. Everse, PhD Associate Professor Department of Biochemistry University of Vermont Burlington, Vermont

Susan A. Galel, MD

Michael M. Fry, DVM, MS, DACVP Associate Professor Department of Biomedical and Diagnostic Sciences College of Veterinary Medicine, University of Tennessee Knoxville, Tennessee

Vijayakrishna K. Gadi, MD, PhD Assistant Member Clinical Research Division Fred Hutchinson Cancer Research Center Assistant Professor Medical Oncology University of Washington Seattle, Washington

Associate Professor Pediatric Hematology/Oncology University of Southern California, San Diego Rady Children’s Hospital San Diego San Diego, California

Tracy I. George, MD Associate Professor of Pathology Chief, Hematopathology Division Director, Hematopathology Fellowship University of New Mexico Health Sciences Center Albuquerque, New Mexico

Morie A. Gertz, MD, MACP Professor and Chair Department of Medicine Mayo Clinic Rochester, Minnesota

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Contributors

Spencer B. Gibson, PhD

Michael R. Grever, MD

Caron A. Jacobson, MD

Professor Biochemistry and Medical Genetics Manitoba Institute of Cell Biology University of Manitoba Provincial Director of Research Manitoba Institute of Cell Biology CancerCare Manitoba Winnipeg, Manitoba Canada

Professor Department of Internal Medicine The Ohio State University Chairman Department of Internal Medicine Arthur G. James Cancer Hospital and Richard J. Solove Research Institute Columbus, Ohio

Instructor Department of Medicine Harvard Medical School Clinical Instructor Medical Oncology Division of Hematologic Malignancies Dana-Farber Cancer Institute Boston, Massachusetts

Thomas G. Gross, MD, PhD

Madan Jagasia, MBBS, MS

Bertil Glader, MD, PhD Professor Departments of Pediatrics and Pathology Stanford University Medical Center Stanford, California Lucile Packard Children’s Hospital Palo Alto, California

Christopher L. Gonzalez, MD Assistant Clinical Professor Department of Pathology Assistant Medical Director Stanford Blood Center Stanford University Palo Alto, California

Lawrence T. Goodnough, MD Professor Department of Pathology and Medicine Division of Hematology Stanford University Director Transfusion Service Stanford University Medical Center Stanford, California

Siamon Gordon, MB, ChB, PhD, FRS Glaxo Professor of Cellular Pathology Emeritus Sir William Dunn School of Pathology University of Oxford Oxford, United Kingdom

Jason Gotlib, MD, MS Associate Professor of Medicine (Hematology) Department of Medicine/Division of Hematology Stanford University School of Medicine Stanford, California

John P. Greer, MD Professor Departments of Medicine and Pediatrics Divisions of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

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Professor Department of Pediatrics The Ohio State University Medical Center Gordon Teter Chair for Pediatric Cancer Hematology/Oncology/BMT Nationwide Children’s Hospital Columbus, Ohio

Roy M. Gulick, MD Professor Department of Medicine Chief, Division of Infectious Disease Weill Cornell Medical College New York, New York

Kenneth R. Hande, MD Professor Departments of Medicine and Pharmacy Division of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

Jane S. Hankins, MD, MS Associate Member Hematology St. Jude Children’s Research Hospital Memphis, Tennessee

Associate Professor Department of Medicine Division of Hematology/Oncology Section Chief, Hematology and Stem Cell Transplant Vanderbilt University Medical Center Nashville, Tennessee

Vandita P. Johari, MD Assistant Professor Department of Pathology Tufts University School of Medicine Western Campus Chief, Clinical Pathology Department of Pathology Baystate Health Springfield, Massachusetts

James B. Johnston, MD Professor Department of Internal Medicine University of Manitoba Hematologist/Oncologist Department of Medical Oncology and Hematology CancerCare Manitoba Winnipeg, Manitoba Canada

Suzanne R. Hayman, MD Assistant Professor of Medicine Department of Internal Medicine/ Hematology Mayo Clinic College of Medicine Consultant Department of Internal Medicine/ Hematology Mayo Clinic Rochester, Minnesota

Nancy M. Heddle, MSc Professor Department of Medicine McMaster University Hamilton, Ontario Canada

Derralynn A. Hughes, MA, DPhil, FRCP, FRCPath Senior Lecturer Department of Haematology University College London London, United Kingdom

Dan Jones, PhD Medical Director Cancer Diagnostics Quest Diagnostics Nichols Institute Chantilly, Virginia

Adetola A. Kassim, MD Associate Professor Department of Medicine Division of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

John G. Kelton, MD Dean and Vice President Faculty of Health Sciences Professor Department of Pathology and Molecular Medicine and Department of Medicine McMaster University Hamilton, Ontario Canada

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Contributors



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Annette S. Kim, MD, PhD

Larry W. Kwak, MD, PhD

Jeffrey M. Lipton, MD, PhD

Assistant Professor Department of Pathology, Microbiology, and Immunology Vanderbilt University Medical Center Nashville, Tennessee

Professor and Chairman Department of Lymphoma/Myeloma The University of Texas MD Anderson Cancer Center Houston, Texas

Rami Komrokji, MD

Robert A. Kyle, MD

Professor Pediatrics and Molecular Medicine Hofstra North Shore—LIJ School of Medicine Hempstead, New York Chief Hematology/Oncology and Stem Cell Transplantation Steven and Alexandra Cohen Children’s Medical Center of New York New Hyde Park, New York

Clinical Director Associate Member Department of Malignant Hematology H. Lee Moffitt Cancer Center & Research Institute Tampa, Florida

Ashish Kumar, MD, PhD Assistant Professor of Pediatrics Division of Bone Marrow Transplantation and Immune Deficiency University of Cincinnati College of Medicine Staff Physician Division of Bone Marrow Transplantation and Immune Deficiency Cancer and Blood Diseases Institute Cincinnati Children’s Hospital Medical Center Cincinnati, Ohio

Shaji Kumar, MD Professor of Medicine Division of Hematology Mayo Clinic Rochester, Minnesota

Thomas J. Kunicki, PhD Senior Scientist II Department of Hematology Research CHOC Children’s Hospital University of California—Irvine Orange, California

Professor of Medicine Laboratory Medicine and Pathology Department of Internal Medicine Division of Hematology College of Medicine Mayo Clinic Rochester, Minnesota

Paige Lacy, PhD Associate Professor Department of Medicine University of Alberta Edmonton, Alberta Canada

Martha Q. Lacy, MD Department of Hematology Mayo Clinic Rochester, Minnesota

Jeffrey E. Lancet, MD Associate Professor University of South Florida Section Chief—Leukemia Associate Member of Malignant Hematology/Oncology H. Lee Moffitt Cancer Center & Research Institute Tampa, Florida

Paul J. Kurtin, MD Professor of Pathology Mayo Medical School Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota

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Assistant Professor Oncologic Sciences University of South Florida Assistant Member Blood and Marrow Transplantation H. Lee Moffitt Cancer Center Tampa, Florida

Mignon Lee-Chuen Loh, MD Professor of Clinical Pediatrics Department of Pediatrics University of California San Francisco Benioff Children’s Hospital San Francisco, California

John A. Lust, MD, PhD Associate Professor of Medicine Mayo Clinic College of Medicine Consultant Division of Hematology Department of Internal Medicine Mayo Clinic Rochester, Minnesota

Andre Larochelle, MD, PhD Principal Investigator, Hematology Branch The National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland

Gary M. Kupfer, MD Professor Department of Pediatrics Yale School of Medicine Chief, Pediatric Hematology/Oncology Department of Pediatrics Yale New Haven Children’s Hospital New Haven Connecticut

Frederick L. Locke, MD

Christopher M. Lehman, MD Professor (Clinical) Pathology Department University of Utah Medical Director of Hospital Laboratories Pathology Department University of Utah Health Care Salt Lake City, Utah

Meghan S. Liel, MD Fellow Department of Hematology and Medical Oncology Oregon Health and Science University Portland, Oregon

William R. Macon, MD Professor of Pathology Mayo Medical School Consultant Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota

Suman Malempati, MD Assistant Professor Department of Pediatrics Oregon Health and Science University Doernbecher Children’s Hospital Portland, Oregon

Kenneth G. Mann, PhD Professor Emeritus, Biochemistry University of Vermont Burlington, Vermont

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Contributors

Catherine A. Manno, MD

Dean D. Metcalfe, MD

Richard A. Nash, MD

Chair Department of Pediatrics Pat and John Rosenwald Professor of Pediatrics New York University Langone Medical Center New York, New York

Chief Laboratory of Allergic Diseases Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland

Peter Maslak, MD

Joseph R. Mikhael, MD

Affiliate Member Affiliate Professor Clinical Division/Medical Oncology Fred Hutchinson Cancer Research Center University of Washington Seattle, Washington Transplant Physician Colorado Blood Cancer Institute Presbyterian/St. Luke’s Denver, Colorado

Professor of Clinical Medicine Department of Internal Medicine Weill Cornell Medical College Chief Hematology Laboratory Service Laboratory Medicine Memorial Sloan-Kettering Cancer Center New York, New York

Thomas L. McCurley, MD Associate Clinical Professor Department of Pathology Vanderbilt University Medical Center Nashville, Tennessee

Laura Y. McGirt, MD Assistant Professor of Medicine/ Dermatology Division of Dermatology Department of Medicine Vanderbilt University School of Medicine Nashville, Tennessee

Margaret M. McGovern, MD, PhD Professor and Chair Department of Pediatrics Stony Brook University School of Medicine Physician in Chief Stony Brook Long Island Children’s Hospital Stony Brook, New York

Kelly M. McNagny Professor Medical Genetics The Biomedical Research Centre University of British Columbia Vancouver, British Columbia Canada

Robert T. Means, Jr., MD Professor of Internal Medicine Executive Dean University of Kentucky College of Medicine Lexington, Kentucky

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Associate Professor Department of Medicine Mayo Clinic Scottsdale, Arizona

Andrew J. Moore, MD Clinical Fellow Department of Medicine Division of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

Redwan Moqbel, PhD, FRCPath Professor and Head Department of Immunology Faculty of Medicine University of Manitoba Winnipeg, Manitoba, Canada

David S. Morgan, MD Associate Professor Department of Medicine Division of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

William G. Morice II, MD, PhD Associate Professor Laboratory Medicine and Pathology Mayo School of Graduate Medical Education Consultant Division of Hematopathology Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota

Claudio A. Mosse, MD, PhD Assistant Professor of Pathology Department of Pathology Vanderbilt University School of Medicine Chief of Pathology and Laboratory Medicine Department of Pathology and Laboratory Medicine Tennessee Valley Health Systems VA Nashville, Tennessee

Sattva S. Neelapu, MD Associate Professor Department of Lymphoma and Myeloma The University of Texas MD Anderson Cancer Center Houston, Texas

Elizabeta Nemeth, PhD Associate Professor Department of Medicine David Geffen School of Medicine University of California, Los Angeles Los Angeles, California

Huong (Marie) Nguyen, MD Hematology/Oncology Fellow Department of Medicine Stanford School of Medicine Stanford Hospital and Clinics Cancer Institute Stanford, California

H. Stacy Nicholson, MD, MPH Professor and Chair Department of Pediatrics Oregon Health and Science University Physician-in-Chief Doernbecher Children’s Hospital Portland, Oregon

Ariela Noy, MD Associate Member, Associate Attending Department of Medicine Memorial Sloan-Kettering Cancer Center and Cornell Weill Medical College New York, New York

Diane J. Nugent, MD Pediatric Hematology Chief of Hematology Department of Hematology CHOC Children’s Hospital University of California—Irvine Orange, California

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Contributors



Maureen M. O’Brien, MD

Charles J. Parker, MD

Jerry S. Powell, MD

Assistant Professor Department of Pediatrics University of Cincinnati Associate Director Leukemia/Lymphoma Program Department of Oncology Cincinnati Children’s Hospital Medical Center Cincinnati, Ohio

Professor Department of Medicine University of Utah School of Medicine Salt Lake City, Utah

Professor of Medicine Division of Hematology/Oncology University of California University of California Davis Medical Center, Davis Sacramento, California

Robin K. Ohls, MD Professor Department of Pediatrics University of New Mexico Director of Pediatric Integration Clinical Translational Science Center University of New Mexico Health Sciences Center Albuquerque, New Mexico

Mihaela Onciu, MD

Robert C. Pendleton, MD, FACP Associate Professor Department of Medicine University of Utah Director Hospitalist Program Medical Director Thrombosis Service Department of Medicine University of Utah Healthcare Salt Lake City, Utah

Sherrie L. Perkins, MD, PhD

Hematopathologist OncoMetrix Memphis, Tennessee

Professor of Hematology Department of Pathology University of Utah Salt Lake City, Utah

Attilio Orazi, MD, FRCPath (Engl.)

Joseph Pidala, MD, MS

Professor of Pathology and Laboratory Medicine Vice Chair for Hematopathology Department of Pathology and Laboratory Medicine Weill Cornell Medical College Director Division of Hematopathology Department of Pathology and Laboratory Medicine New York, New York

Thomas Orfeo, PhD Research Associate Department of Biochemistry University of Vermont Colchester, Vermont

Eric Padron, MD Assistant Member Department of Malignant Hematology H. Lee Moffitt Cancer Center & Research Institute Tampa, Florida

Frixos Paraskevas, MD Professor of Internal Medicine and Immunology (Retired) University of Manitoba Medical School Associate Member Institute of Cell Biology—Cancer Care Manitoba Winnipeg, Manitoba Canada

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Assistant Professor Oncologic Sciences College of Medicine—University of South Florida Assistant Member Blood and Marrow Transplantation H. Lee Moffitt Cancer Center Tampa, Florida

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John G. Quigley, MB, FRCPC Associate Professor Department of Medicine University of Illinois at Chicago Attending Physician Division of Hematology/Oncology Department of Medicine University of Illinois Hospital and Health Sciences System Chicago, Illinois

Charles T. Quinn, MD, MS Associate Professor Department of Pediatrics University of Cincinnati College of Medicine Director of Hematology Clinical and Translational Research Department of Hematology Cincinnati Children’s Hospital Medical Center Cincinnati, Ohio

Elizabeth A. Raetz, MD Associate Professor Department of Pediatrics Division of Hematology/Oncology New York University New York, New York

Annette Plüddemann, PhD Senior Researcher Department of Primary Care Health Sciences University of Oxford Oxford, United Kingdom

Michael R. Porembka, MD

S. Vincent Rajkumar, MD Professor of Medicine Division of Hematology Mayo Clinic Rochester, Minnesota

Michael Recht, MD, PhD

Department of Surgery Washington University School of Medicine St. Louis, Missouri

Associate Professor of Pediatrics and Medicine Pediatric Hematology-Oncology Oregon Health & Science University Portland, Oregon

Anna Porwit, MD, PhD

Nishitha M. Reddy, MD

Professor Department of Laboratory Medicine and Pathobiology University of Toronto Hematopathologist Department of Laboratory Hematology University Health Network Toronto General Hospital Toronto, Ontario Canada

Assistant Professor Department of Medicine Division of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

Matthew M. Rees, MD Rutherford Hospital Rutherford, North Carolina

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Contributors

George M. Rodgers, MD, PhD Professor of Medicine and Pathology University of Utah School of Medicine Health Sciences Center Medical Director Coagulation Laboratory ARUP Laboratories Salt Lake City, Utah

Stephen J. Russell, MD, PhD Dean for Discovery and Experimental Research Department of Molecular Medicine Richard O. Jacobson Professor of Molecular Medicine Mayo Clinic Rochester, Minnesota

John T. Sandlund, Jr., MD Member Department of Oncology St. Jude Children’s Research Hospital Professor Department of Pediatrics University of Tennessee College of Medicine Memphis, Tennessee

Bipin N. Savani, MBBS Associate Professor of Medicine Director Long Term Transplant Clinic, Hematology and Stem Cell Transplant Vanderbilt University Medical Center Nashville, Tennessee

Matthew Seftel, MD Assistant Professor Department of Internal Medicine University of Manitoba Hematologist Department of Medical Oncology and Hematology CancerCare Manitoba Winnipeg, Manitoba Canada

Paul J. Shami, MD Professor of Medicine Division of Hematology and Hematologic Malignancies Adjunct Professor Department of Pharmaceutics and Pharmaceutical Chemistry University of Utah Salt Lake City, Utah

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Luke R. Shier, MD

Steven L. Soignet, MD

Assistant Professor Department of Pathology University of Ottawa Hematopathologist Pathology and Laboratory Medicine The Ottawa Hospital Ottawa, Ontario Canada

Arcus Advisory New York, New York

Akiko Shimamura, MD, PhD Associate Member Clinical Research Division Fred Hutchinson Cancer Research Center Associate Professor of Pediatrics Pediatric Hematology/Oncology Seattle Children’s Hospital Seattle, Washington

Keith M. Skubitz, MD Professor Division of Hematology, Oncology and Transplantation Department of Medicine Musculoskeletal Tumor Program, Masonic Cancer Center University of Minnesota Attending Physician University of Minnesota Medical Center Minneapolis, Minnesota

James W. Smith, MT, BSc Assistant Professor Department of Medicine McMaster University Coordinator/Technical Director Platelet Immunology Laboratory Hamilton Health Sciences Hamilton, Ontario Canada

Kristi J. Smock, MD Assistant Professor Department of Pathology University of Utah Health Sciences Center Medical Director Hemostasis/Thrombosis Laboratory ARUP Laboratories Salt Lake City, Utah

Susan S. Smyth, MD, PhD Professor and Chief Division of Cardiovascular Medicine University of Kentucky Attending Physician Medical Services Lexington VA Medical Center Lexington, Kentucky

Nicole I. Stacy, DVM, DrMedVet Adjunct Clinical Assistant Professor Department of Large Animal Clinical Sciences University of Florida College of Veterinary Medicine Gainesville, Florida

Martin H. Steinberg, MD Professor of Medicine Department of Pediatrics, Pathology and Laboratory Medicine Boston University School of Medicine Director Center of Excellence in Sickle Cell Disease Boston Medical Center Boston, Massachusetts

A. Keith Stewart, MBChB Dean for Research Division of Hematology/Oncology Mayo Clinic Scottsdale, Arizona

Stephen A. Strickland, MD, MSCI Assistant Professor Department of Medicine Division of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

Mary Ann Thompson, MD, PhD Associate Professor Department of Pathology Vanderbilt University Medical Center Nashville, Tennessee

John Tisdale, MD Senior Investigator and Section Head Hematology Branch The National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland

Troy R. Torgerson, MD, PhD Assistant Professor Department of Pediatrics University of Washington School of Medicine Attending Physician Department of Immunology Seattle Children’s Hospital Seattle, Washington

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Contributors



Han-Mou Tsai, MD

Mark C. Walters, MD

Lawrence M. Weiss, MD

Professor Department of Medicine Pennsylvania State University College of Medicine Chief Section of Hemostasis and Thrombosis Milton S. Hershey Medical Center Hershey, Pennsylvania

Associate Adjunct Professor Department of Pediatrics University of California—San Francisco, San Francisco Director Blood Marrow Transplant Program Department of Hematology/Oncology Children’s Hospital and Research Center Oakland, California

Senior Consulting Pathologist Clarient Pathology Services, Inc. Aliso Viejo, California

Luc Van Kaer, MD Professor Department of Pathology, Microbiology and Immunology Vanderbilt University School of Medicine Nashville, Tennessee

Winfred C. Wang, MD Member Department of Hematology St. Jude Children’s Research Hospital Memphis, Tennessee

Srdan Verstovsek, MD, PhD Professor of Medicine Chief Section for Myeloproliferative Neoplasms (MPNs) Department of Leukemia Director Clinical Research Center for MPNs University of Texas MD Anderson Cancer Center Houston, Texas

Maurene K. Viele, MD Clinical Associate Professor Department of Pathology Stanford University Associate Medical Director Transfusion Service Stanford University Medical Center Stanford, California

Mary A. Vu, MD Clinical Fellow Department of Medicine Division of Hematology/Oncology Vanderbilt University Medical Center Nashville, Tennessee

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Russell E. Ware, MD, PhD Professor and Vice-Chair for Global Health Department of Pediatrics Baylor College of Medicine Director Texas Children’s Hematology Center Director Texas Children’s Center for Global Health Texas Children’s Hospital Houston, Texas

Kathryn E. Webert, MD, MSc, FRCPC Associate Professor Department of Medicine and Department of Molecular Medicine and Pathology McMaster University Director of Operations, Transfusion Medicine Hamilton Regional Laboratory Medicine Program Hamilton Health Sciences Hamilton, Ontario Canada

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James A. Whitlock, MD Professor Department of Pediatrics University of Toronto Division Head Division of Hematology/Oncology The Hospital for Sick Children Toronto, Ontario Canada

Michael E. Williams, MD Byrd S. Leavell Professor of Medicine Hematology/Oncology Division University of Virginia School of Medicine Department of Medicine University of Virginia Health System Charlottesville, Virginia

Steven R. Zeldenrust, MD, PhD Assistant Professor Hematology Mayo Clinic Rochester, Minnesota

John A. Zic, MD Associate Professor of Medicine/ Dermatology Division of Dermatology Department of Medicine Vanderbilt University School of Medicine Nashville, Tennessee

Jeff P. Zwerner, MD, PhD Assistant Professor of Medicine/ Dermatology Division of Dermatology Department of Medicine Vanderbilt University School of Medicine Nashville, Tennessee

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p r e f a c e

Welcome to the thirteenth edition of Wintrobe’s Clinical Hematology. This textbook strives to continue the Wintrobe tradition of being comprehensive yet accessible to all who seek to understand the history, science, and clinical practice of hematology. We have brought together clinicians and scientists who have given their time and expertise to produce a state-of-the-art resource which includes an online presence with expanded bibliographies, appendices, and updates.

The Wintrobe Legacy Few have appreciated the wealth of information to be gained by the study of blood more than Maxwell Myer Wintrobe (1906 to 1986). He cited poets, including John Donne’s “pure and eloquent blood” and Goethe’s “Blood is a juice of a very special kind”; but he added that “It is for the scientist that the blood has been especially eloquent.”1. It has been over 70 years since Wintrobe wrote the first edition of Clinical Hematology (1942); and at the time, he was uncertain if it would have much readership owing to the priorities of World War II. His objective was “to bring together the accumulated information in the field of hematology in a systematic and orderly form.” He felt the book should be “comprehensive, complete, and authoritative.” He emphasized the importance of “an accurate diagnosis as a prerequisite to efficacious treatment.” His goals were to link science to the clinical practice of hematology and to provide the best therapy possible for an individual patient. We have recruited an outstanding group of scientists and clinicians who have given their expertise and time to accomplish similar goals for the thirteenth edition of Wintrobe’s Clinical Hematology. Wintrobe’s career included medical school at the University of Manitoba (1921 to 1925) and academic appointments at Tulane University (1927 to 1930), The Johns Hopkins University (1930 to 1943), and the University of Utah (1943 to 1986). His interests were broad and his contributions to medicine and hematology were many.2 He had access to an abundance of clinical material at Charity Hospital (New Orleans, LA), where he invented the hematocrit glass tube, which came to bear his name and allowed him to collect information about the blood (Figure 1). The Wintrobe hematocrit tube not only allowed determination of the volume of packed red blood cells after centrifugation but also allowed measurement of the erythrocyte sedimentation rate, determination of the volume of packed white cells and platelets, and detection of changes in the appearance of the plasma. At Johns Hopkins University, Wintrobe made peripheral smears available to the clinic, reorganized the teaching of the third-year student laboratory course, and established himself as an investigator and leader in hematology.3 He and his colleagues showed that hypochromic anemia responded to iron,4 gave the first account of cryoglobulin in the blood,5 and provided the first evidence that thalassemias were inherited.6 He became chief of the Clinic for Nutritional, Gastrointestinal, and Hematologic Disorders in 1933 and was promoted to the position of Associate in Medicine in 1935. During World War II, he was assigned to study chemical warfare agents and his efforts led to a landmark paper with Louis Goodman et al. on the efficacy of nitrogen mustard as a chemotherapeutic agent.7

FIGURE 1. Original illustration of Wintrobe tubes depicting the appearance of centrifugal blood in various conditions, as published in the seventh edition.

In 1943, Wintrobe was offered the first Chair of Medicine at the University of Utah. He served as Chair for 24 years and in 1970 was named Distinguished Professor of Internal Medicine. He studied the role of nutritional factors, particularly the B vitamins, in hematopoiesis, and attempted to develop an animal model for pernicious anemia.3,8 His work with pig’s nutritional requirements resulted in discovering the effects of pyridoxine deficiency and the role of copper in iron metabolism.3 He studied the effects of the newly discovered adrenocorticosteroids on hematopoiesis,9 described the association of chloramphenicol with aplastic ­anemia,10 and became an advocate for reporting the adverse reactions to drugs.11 Wintrobe’s clinical interests extended beyond hematology, and he received the first research grant ever awarded by the National Institutes of Health. The grant was to study hereditary muscular dystrophy (a disorder that affected a number of Utah families) and was renewed annually for 23 years.2,3 He directed the Laboratory for the Study of Hereditary and Metabolic Disorder and Training Institute (1969 to 1973). Together with George Cartwright, he established a premier hematology training program at Utah. They trained 110 fellows, 85% of whom became associated with medical schools or research institutes.12

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Preface

Wintrobe was the sole author of the first six editions, and he recruited former fellows to assist him on the seventh and eight editions: Jack Athens, Tom Bithell, Dane Boggs, John Foerster, and Richard Lee with John Lukens joining them on the eighth edition (1981), the last one to involve Wintrobe. Lee, Bithell, Foerster, Athens, and Lukens were the editors for the ninth edition (1993). John Greer, Frixos Paraskevas, and George Rodgers joined Lee, Foerster, and Lukens for the tenth edition (1999) and Bert Glader was added for the eleventh edition (2004). Robert Means, Jr., and Daniel Arber joined Foerster, Glader, Greer, Paraskevas, and Rodgers for the twelfth edition (2009). We welcome Alan List as a new editor for this edition and honor John Foerster as the editor emeritus.

In Memory of John N. Lukens We remember our friend, John Nevius Lukens, Jr. (1932 to 2010), who was dedicated to the Wintrobe legacy and committed to ­the education of the next generation of health care providers. He was a graduate of Princeton University (1954) and Harvard Medical School (1958). After an internship in Medicine and Pediatrics at the University of North Carolina, he completed his residency at The Children’s Hospital in Cincinnati (1959 to 1961). He served 2 years in the U.S. Army Medical Corps at the Letterman General Hospital in San Francisco and became a research fellow at the University of Utah School of Medicine with Eugene Lahey and Wintrobe (1964 to 1967). His research contributed to the understanding of the anemia of chronic disease and iron deficiency. John was a founding member of the Children’s Cancer Group and was among the pioneers of pediatric hematology who contributed to a steady and marked increase in the curability of childhood acute lymphoblastic leukemia and other cancers. He held faculty appointments at the University of Missouri School of Medicine (1967 to 1971), Tufts Medical School (1971 to 1973), and the Charles R. Drew Postgraduate Medical School (1973 to 1975) before becoming Director of Pediatric Hematology/Oncology (1975 to 1997) at Vanderbilt University’s Children’s Hospital. He became an Emeritus in 2001 until his death in 2010. John is remembered as a role model as a physician, a loving husband to his wife Cauley of 51 years, a father devoted to their daughters, Ann, Rachel, and Betsy, and a grandfather to five.

there are numerous photomicrographs, which illustrate the role of hematopathology in diagnosis. The book is divided into eight parts: Laboratory Hematology; The Normal Hematologic System; Transfusion Medicine; Disorders of Red Cells, Hemostasis, and Coagulation; Benign Disorders of Leukocytes; The Spleen and/or Immunoglobulins; Hematologic Malignancies; and Transplantation. Throughout the chapters, there is an emphasis on the four components in hematology that contribute to diagnosis: the morphological exam of the peripheral blood smear, bone marrow, lymph nodes, and other tissues; flow cytometry, cytogenetics, and molecular markers. The expanding role of molecular genetics and flow cytometry is not only improving diagnosis but also providing targets for novel therapies. The role of tyrosine kinase inhibitors in chronic myeloid leukemia serves as a model for molecularly targeted therapy. The detection of minimal residual disease by either flow cytometry or polymerase chain reaction techniques is impacting therapeutic decisions. Chapters on gene therapy and immunotherapy are up-to-date reviews on these unique therapies for a variety of hematologic disorders. The role of stem cell transplantation is addressed in chapters on specific diseases and in an entirely new part, which reviews its application for both benign and malignant disorders, graft–versus-host disease, and the importance of longterm follow-up of transplantation survivors. For a textbook to meet its audience needs in the 21st century, there must be an online presence and a way to interact with and update its readers. The online text has a complete reference list for each chapter and two appendices, one reviewing the clusters of differentiation molecules by Dan Arber and Frixos Paraskevas and another by veterinarians Nicole Stacy, Kirstin Barnhart, and Michael Fry, who review lab values and photomicrographs of the blood of animals. We plan to issue updates online when there is either unique or sufficient information that influences the practice of hematology. We are indebted to the efforts of Jonathan Pine, who has kindly supported us as Senior Executive Editor at Lippincott Williams & Wilkins since the 10th edition; Emilie Moyer, Senior Product Manager; and Frannie Murphy, Development Editor.

References

Thirteenth Edition Our goal in the thirteenth edition is to continue Wintrobe’s commitment to link the past accomplishments in hematology to the present state of the art and to future developments. We are honored to have some of the best hematologists in the world contribute to this edition. They have continued the Wintrobe tradition of providing historical perspective and combining basic science with clinical practice. There are 74 new authors, and all of the chapters are new or have been completely revised. All of the authors are worth singling out, but space limits our ability to thank them individually. One of the new contributors is Michael Deininger, who is the Maxwell M. Wintrobe Professor of Medicine at the University of Utah Huntsman Cancer Institute. The audience for the book encompasses the entire spectrum of health care providers, including medical students, nurses, residents, clinicians, and scientists, who seek answers about hematology. The textbook reviews the science, the methods of diagnosis, and the evidence for the basis of therapeutic decisions. The artwork has been extensively redrawn for color and consistency and

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1. Wintrobe MM. Blood, pure and eloquent: a story of discovery, of people, and of ideas. New York: McGraw-Hill Book Company, 1980. 2. Herbert LF. Maxwell Myer Wintrobe: new history and a new appreciation. Tex Heart Inst J 2007;34:328–335. 3. Spivak JL. Maxwell Wintrobe, in his own words. Br J Haematol 2003;121:224–232. 4. Wintrobe MM, Beebe RT. Idiopathic hypochromic anemia. Medicine 1933;12:187–243. 5. Wintrobe MM, Buell MV. Hyperproteinemia associated with multiple myeloma. Bull Johns Hopkins Hosp 1933;52:156–165. 6. Wintrobe MM, Matthews E, Pollack R, et al. A familial hemopoietic disorder in Italian adolescents and adults; resembling Mediterranean disease (thalassemia). J Am Med Assoc 1940;114:1530–1538. 7. Goodman LS, Wintrobe MM, Dameshek W, et al. Nitrogen mustard therapy; use of methyl-bis (beta-chloroethyl) amine hydrochloride and tris (betachloroethyl) amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. J Am Med Assoc 1946;132:126–132. 8. Wintrobe MM. The search for an experimental counterpart of pernicious anemia. AMA Arch Intern Med 1957;100:862–869. 9. Wintrobe MM, Cartwright GE, Palmer JG, et al. Effect of corticotrophin and cortisone on the blood in various disorders in man. AMA Arch Intern Med 1951;88:310–336. 10. Smiley RK, Cartwright GE, Wintrobe MM. Fatal aplastic anemia following chloramphenicol (chloromycetin) administration. J Am Med Assoc 1952;149:914–918. 11. Wintrobe MM. The problems of drug toxicity in man—a view from the hematopoietic system. Ann N Y Acad Sci 1965;123:316–325. 12. Boggs DR. Maxwell M. Wintrobe. Blood 1973;41:1–5.

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a c k n o w l e d g m e n t s

Thanks to my wife, Gay, for her support and our grown children, Lesley, Adam, and Scott; Pamela Johnson, who diligently and kindly prepared manuscripts; Billi Bean, who worked on editions 9 through 12 and handed the reins to Pamela; Meera Kumar, P.A., the nurse practitioners and nurses who provide extraordinary care to the patients at Vanderbilt University Medical Center; and mentors and colleagues: Robert Collins, John Flexner, Stanley Graber, Marsha Kinney, Mark Koury, Sanford Krantz, Friedrich Schuening, Richard Stein, and Steven Wolff; and a special thanks to John Lukens, who brought me into the world of Maxwell M. Wintrobe, and my lifelong friend, Thomas McCurley.

I wish to thank my wife Stacey and our children, Casey, Robert, and Patrick, for their support and tolerance during the preparation of this book; the many teachers and colleagues who have guided me as mentors and examples in science and medicine, particularly Shu-Yung Chen, Joachim Pfitzner, James B. Walker, Robert D. Collins, Roger M. DesPrez, Richard Borreson, Richard Vilter, Herbert Flessa, John Flexner, and Sanford B. Krantz; and above all my late parents, Ann and Bob Means, who were my first and best teachers. Robert T. Means, Jr., MD

John P. Greer, MD

I wish to thank my wife, Carol Park, for her constant support. I also thank my current and past trainees, colleagues, and mentors, all of whom are continuous sources of knowledge.

I am deeply indebted to my pathologist wife, Dr. Maria Paraskevas, for her unwavering support, encouragement, and advice throughout the writing of my chapters. Frixos Paraskevas, MD

Daniel A. Arber, MD

I wish to acknowledge the many outstanding colleagues, both chapter authors and fellow editors, whom I have had the privilege to work with in the development of this new edition. I also want to acknowledge my students, residents, and fellows who continue to make the teaching of clinical hematology so meaningful. Lastly, but most of all, I want to recognize the understanding and support of my wonderful wife Lou Ann, my children, and their families.

I acknowledge Stephen Kling for expert word processing and my numerous contributors for their outstanding chapters. This is the fourth edition of this textbook I have been involved with; it has been a pleasure working with my coeditors and publishing colleagues on this edition. George Rodgers, MD, PhD

Bertil Glader, MD, PhD

As I am sure our readers understand, creating a state-of-the-art reference text is by no means a simple task. It begins with the authors who graciously give their time, often juggling deadlines with immediate demands from their own research, clinical duties, etc. I thank them for their diligence and perseverance to create an outstanding reference. Individuals at Lippincott Williams & Wilkins such as Emilie Moyer, Franny Murphy, and Jonathan Pine worked ever so patiently in providing the guidance and focus necessary to see this to completion. Finally, my sincere thanks to our senior editor and master medical coordinator of Wintrobe, Dr. John Greer, for his faith in the process, admirable leadership, and sensitivity to the mission.

I wish to thank my wife Gisela, who bore my commitments to this tome for seven editions with encouragement, patience, and grace. Deeply felt gratitude is extended to my mentors, Dr. L. G. Israels and Dr. M. M. Wintrobe, both now regrettably deceased, and Dr. B. Benacerraf, who nurtured my interests in Immunology. Dr. Israel’s enthusiasm for Hematology and his ability to combine effectively clinical excellence, teaching, and research drew me to this specialty as a medical student. Dr. Wintrobe taught me in his own unique way and gave me the opportunity to contribute as author and associate editor to this great textbook. Special thanks to my colleagues who have contributed valuable chapters to this text. John Foerster, MD

Alan F. List, MD

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c o n t e n t s

Contributors vii Preface xvii Acknowledgments xix Introduction xxv

Section 4:The Lymphocytes  227

11. Lymphocytes and Lymphatic Organs  227 Frixos Paraskevas

12. B Lymphocytes  251 Frixos Paraskevas

P a r T I Laboratory Hematology 1. Examination of the Blood and Bone Marrow  1 Kristi J. Smock, Sherrie L. Perkins

2. Clinical Flow Cytometry  19 Anna Porwit

3. Cytogenetics 46 Athena M. Cherry, Charles D. Bangs

4. Molecular Diagnosis in Hematology  58 Dan Jones

13. T Lymphocytes and Natural Killer Cells  279 Frixos Paraskevas

14. Effector Mechanisms in Immunity  313 Frixos Paraskevas Section 5: HEMOSTASIS   371

15. Megakaryocytes  371 Amy E. Geddis

16. Platelet Structure and Function in Hemostasis and Thrombosis  389 Susan S. Smyth

17. Platelet Function in Hemostasis and Thrombosis  411 David C. Calverley

18. Blood Coagulation and Fibrinolysis  428

P a r T II The Normal Hematologic System SECTION 1: Hematopoiesis  65

Kathleen E. Brummel-Ziedins, Thomas Orfeo, Stephen J. Everse, Kenneth G. Mann

19. Endothelium: Angiogenesis and the Regulation of Hemostasis  498 Paul J. Shami, George M. Rodgers

5. Origin and Development of Blood Cells  65 Andrew Chow, Paul S. Frenette SECTION 2: The Erythrocyte  83

6. The Birth, Life, and Death of Red Blood Cells: Erythropoiesis, The Mature Red Blood Cell, and Cell Destruction  83 John G. Quigley, Robert T. Means, Jr., Bertil Glader Section 3: Granulocytes and Monocytes  125

7. Neutrophilic Leukocytes  125 Keith M. Skubitz

PAR T III Transfusion Medicine 20. Red Cell, Platelet, and White Cell Antigens  509 Kathryn E. Webert, James W. Smith, Donald M. Arnold, Howard H. W. Chan, Nancy M. Heddle, John G. Kelton

21. Transfusion Medicine  547 Susan A. Galel, Magali J. Fontaine, Maurene K. Viele, Christopher L. Gonzalez, Lawrence T. Goodnough

8. The Human Eosinophil  160 Paige Lacy, Darryl J. Adamko, Redwan Moqbel

9. Mast Cells and Basophils: Ontogeny, Characteristics, and Functional Diversity  181

P a r T I V Disorders of Red Cells

A. Dean Befus, Kelly M. McNagny, Judah A. Denburg

10. Monocytes, Macrophages, and Dendritic Cells  193 Matthew Collin, Derralynn A. Hughes, Annette Plüddemann, Siamon Gordon

SECTION 1: Introduction  587

22. Anemia: General Considerations  587 Robert T. Means, Jr., Bertil Glader

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Contents

SECTION 2: Disorders of Iron Metabolism and Heme Synthesis  617

40. Congenital Dyserythropoietic Anemias  990

23. Iron Deficiency and Related Disorders  617

41. Anemias Secondary to Chronic Disease and Systemic Disorders  998

Lawrence T. Goodnough, Elizabeta Nemeth

24. Sideroblastic Anemias  643 Sylvia S. Bottomley

25. Hemochromatosis  662 Corwin Q. Edwards, James C. Barton

26. Porphyrias  682 Sylvia S. Bottomley

Gary Kupfer, Linette Bosques, Bertil Glader

Robert T. Means, Jr.

42. Anemias During Pregnancy and the Postpartum Period  1012 Robert T. Means, Jr.

43. Anemias Unique to the Fetus and Neonate  1018 Robert D. Christensen, Robin K. Ohls

44. Erythrocytosis  1032 Section 3: Hemolytic Anemia  707

27. Hereditary Spherocytosis, Hereditary Elliptocytosis, and Other Disorders Associated with Abnormalities of the Erythrocyte Membrane  707 Patrick G. Gallagher, Bertil Glader

28. Hereditary Hemolytic Anemias Due to Red Blood Cell Enzyme Disorders  728 Bertil Glader

29. Autoimmune Hemolytic Anemia  746 Richard C. Friedberg, Vandita P. Johari

30. Hemolytic Disease of the Fetus and Newborn  766 Charles T. Quinn, Anne F. Eder, Catherine S. Manno

31. Paroxysmal Nocturnal Hemoglobinuria  785 Charles J. Parker, Russell E. Ware

32. Acquired Nonimmune Hemolytic Disorders  809 Robert T. Means, Jr., Bertil Glader Section 4: Hereditary Disorders of Hemoglobin Structure and Synthesis  823

33. Sickle Cell Anemia and Other Sickling Syndromes  823 Jane S. Hankins, Winfred C. Wang

34. Thalassemias and Related Disorders: Quantitative Disorders of Hemoglobin Synthesis  862 Caterina Borgna-Pignatti, Renzo Galanello

35. Hemoglobins with Altered Oxygen Affinity, Unstable Hemoglobins, M-Hemoglobins, and Dyshemoglobinemias 914 Martin H. Steinberg Section 5: Other Red Cell Disorders  927

36. Megaloblastic Anemias: Disorders of Impaired DNA Synthesis  927 Ralph Carmel

37. Inherited Aplastic Anemia Syndromes  954 Akiko Shimamura, Blanche P. Alter

38. Acquired Aplastic Anemia  965 Robert A. Brodsky

39. Red Cell Aplasia: Acquired and Congenital Disorders  975 Jeffrey M. Lipton, Bertil Glader, Robert T. Means Jr.

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Robert T. Means, Jr.

PAR T V Disorders of Hemostasis and Coagulation SECTION 1: Introduction  1043

45. Diagnostic Approach to the Bleeding Disorders  1043 George M. Rodgers, Christopher M. Lehman SECTION 2: Thrombocytopenia:  1058

46. Thrombocytopenia: Pathophysiology and Classification  1058 George M. Rodgers

47. Thrombocytopenia Caused by Immunologic Platelet Destruction 1061 Meghan S. Liel, Michael Recht, David C. Calverley

48. Thrombotic Thrombocytopenic Purpura, Hemolytic-Uremic Syndrome, and Related Disorders  1077 Han-Mou Tsai

49. Miscellaneous Causes of Thrombocytopenia  1097 Archana M. Agarwal, George M. Rodgers

50. Bleeding Disorders Caused by Vascular Abnormalities  1106 George M. Rodgers, Matthew M. Rees

51. Thrombocytosis and Essential Thrombocythemia  1122 George M. Rodgers, Robert T. Means, Jr.

52. Qualitative Disorders of Platelet Function  1128 Thomas J. Kunicki, Diane J. Nugent Section 4: Coagulation Disorders  1143

53. Inherited Coagulation Disorders  1143 Jerry S. Powell, George M. Rodgers

54. Acquired Coagulation Disorders  1186 George M. Rodgers Section 5: Thrombosis  1218

55. Thrombosis and Antithrombotic Therapy  1218 Robert C. Pendleton, George M. Rodgers

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Contents



P a r T vI Benign Disorders of Leukocytes, the Spleen, and/or Immunoglobins 56. Diagnostic Approach to Malignant and Nonmalignant Disorders of the Phagocytic and Immune Systems  1259 Daniel A. Arber, Thomas L. McCurley, John P. Greer

57. Neutropenia  1279 Caron A. Jacobson, Nancy Berliner

58. Qualitative Disorders of Leukocytes  1290 Ashish Kumar, Keith M. Skubitz

59. Lysosomal Abnormalities of the Monocyte–Macrophage System: Gaucher and Niemann-Pick Diseases  1302 Margaret M. McGovern, Robert J. Desnick

60. Langerhans Cell Histiocytosis  1307 Suman Malempati, H. Stacy Nicholson

61. Pathology of Langerhans Cell Histiocytosis and Other Histiocytic Proliferations  1317 Karen L. Chang, Lawrence M. Weiss

62. Infectious Mononucleosis and Other Epstein-Barr Virus–Related Disorders  1324 Thomas G. Gross

63. Primary Immunodeficiency Syndromes  1342 Troy R. Torgerson

64. Acquired Immunodeficiency Syndrome  1358 Ariela Noy, Roy M. Gulick

65. Disorders of the Spleen  1369 Matthew R. Porembka, Majella Doyle, William C. Chapman

66. Tumors of the Spleen  1384 Daniel A. Arber

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Section 2: The Acute Leukemias  1523

72. Molecular Genetics of Acute Leukemia  1523 Mary Ann Thompson, Utpal P. Davé

73. Diagnosis and Classification of the Acute Leukemias and Myelodysplastic Syndromes  1543 Daniel A. Arber, Attilio Orazi

74. Acute Lymphoblastic Leukemia in Adults  1556 Steven E. Coutre

75. Acute Myeloid Leukemia in Adults  1577 Ashkan Emadi, Maria R. Baer

76. Acute Lymphoblastic Leukemia in Children  1616 Elizabeth A. Raetz, Mignon Lee-Chuen Loh, Maureen M. O’Brien, James A. Whitlock

77. Acute Myelogenous Leukemia in Children  1637 Robert J. Arceci

78. Acute Promyelocytic Leukemia  1656 Jeffrey E. Lancet, Peter Maslak, Steven L. Soignet

79. The Myelodysplastic Syndromes  1673 Guillermo Garcia-Manero SECTION 3: Myeloproliferative Disorders   1688

80. Pathology of the Myeloproliferative Neoplasms  1688 Luke R. Shier, Tracy I. George

81. Chronic Myeloid Leukemia  1705 Michael W.N. Deininger

82. Polycythemia Vera  1722 Robert T. Means, Jr.

83. Myelofibrosis  1734 Rami Komrokji, Eric Padron, Srdan Verstovsek

84. Eosinophilic Neoplasms and Hypereosinophilic Syndrome 1746 Huong (Marie) Nguyen, Jason Gotlib

PAR T V II Hematologic Malignancies SECTION 1: General Aspects  1391

67. Hematopoietic Neoplasms: Principles of Pathologic Diagnosis 1391 Daniel A. Arber

68. Principles and Pharmacology of Chemotherapy  1399 Kenneth R. Hande

69. Supportive Care in Hematologic Malignancies  1426 Andrew J. Moore, Mary A. Vu, Stephen A. Strickland

70. Immunotherapy  1467 Adetola A. Kassim, Sattva S. Neelapu, Larry W. Kwak, Luc Van Kaer

71. Gene Therapy for Hematologic Disorders  1492 Andre Larochelle, Cynthia E. Dunbar, John Tisdale

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85. Systemic Mastocytosis  1757 Dean D. Metcalfe SECTION 4: Lymphoproliferative Disorders 1770

86. Diagnosis and Classification of Lymphomas  1770 William R. Macon, Paul J. Kurtin, Ahmet Dogan

87. Molecular Genetic Aspects of Non-Hodgkin Lymphomas 1801 Annette S. Kim

88. Non-Hodgkin Lymphoma in Adults  1827 John P. Greer, Nishitha M. Reddy, Michael E. Williams

89. Non-Hodgkin Lymphoma in Children  1873 John T. Sandlund, Jr., Mihaela Onciu

90. Chronic Lymphocytic Leukemia  1888 James B. Johnston, Matthew Seftel, Spencer B. Gibson

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91. Hairy Cell Leukemia  1929 James B. Johnston, Michael R. Grever

92. Cutaneous T-Cell Lymphoma: Mycosis Fungoides and Sézary Syndrome 1951 John A. Zic, Jeff P. Zwerner, Laura Y. McGirt, Claudio A. Mosse, John P. Greer

93. Hodgkin Lymphoma in Adults  1984 David S. Morgan, Kristie A. Blum

94. Hodgkin Lymphoma in Children  2005 Debra L. Friedman SECTION 5: Plasma Cell Dyscrasias  2014

95. Practical Approach to Evaluation of Monoclonal Gammopathies 2014 Francis K. Buadi, Joseph R. Mikhael, William G. Morice II

96. Molecular Genetic Aspects of Plasma Cell Disorders  2022 P. Leif Bergsagel, A. Keith Stewart, Stephen J. Russell, Rafael Fonseca

97. Monoclonal Gammopathies of Undetermined Significance and Smoldering Multiple Myeloma  2029 S. Vincent Rajkumar, Robert A. Kyle, John A. Lust

98. Multiple Myeloma  2046

101. POEMS Syndrome, Cryoglobulinemia, and Heavy-Chain Disease  2141 Angela Dispenzieri, David Dingli, Morie A. Gertz

PAR T V i II Transplantation 102. Hematopoietic Cell Transplantation  2159 Richard A. Nash, Vijayakrishna K. Gadi

103. Hematopoietic Stem Cell Transplantation for Nonmalignant Disorders  2176 Jacob R. Garcia, Mark C. Walters

104. Allogeneic Hematopoietic Stem Cell Transplantation (HCT) for Hematologic Malignancies  2189 Bipin N. Savani, Nishitha M. Reddy, Madan Jagasia, Haydar Frangoul

105. Graft-Versus-Host Disease and Graft-Versus-Tumor Response 2206 Joseph Pidala, Frederick L. Locke, Claudio Anasetti

106. Late Effects After Transplantation  2221 Bipin N. Savani, Minoo Battiwalla

Angela Dispenzieri, Martha Q. Lacy, Shaji Kumar

99. Immunoglobulin Light-Chain Amyloidosis (Primary Amyloidosis)  2098 Morie A. Gertz, Martha Q. Lacy, Angela Dispenzieri, Steven R. Zeldenrust

1 00. Waldenström Macroglobulinemia  2124 Rafael Fonseca, Suzanne R. Hayman, Stephen M. Ansell

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Appendix A  Clusters of Differentiation  (eBook only) Frixos Paraskevas

Appendix B  Comparative Hematology  (eBook only) Michael M. Fry, Kirstin F. Barnhart, Nicole Stacy

Index 2235

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IN T RODU C T ION THE DIAGNOSTIC AND THERAPEUTIC APPROACH TO HEMATOLOGIC PROBLEMS Maxwell M. Wintrobe

The study of the blood has a long history. Humankind probably always has been interested in the blood because it is likely that even primitive peoples realized that loss of blood, if sufficiently great, was associated with death. In biblical references, to shed blood meant to kill.1

THE FOUNDATIONS OF DIAGNOSIS When and in what manner blood was first examined is unknown, but before the days of microscopy only the gross appearance of the blood could be studied. Blood allowed to clot in a glass vessel can be seen to form several distinct layers: at the bottom a dark red, almost black, jellylike material is seen; above this is a red layer; and still nearer the top of the clot is a pale green or whitish layer. Above these is the transparent, yellow serum. It has been suggested that perception of these layers in the blood after its removal from the body may have given rise to the doctrine of the four humors (black bile, sanguis, phlegm, and yellow bile), which were believed to constitute the substance of the human body. Health and disease were thought to be the result of the proper mixture or imbalance, respectively, of these four humors. This doctrine corresponding to the pervading concept of matter founded on the interrelationship of the four elements—earth, water, air, and fire—was set out clearly in the Hippocratic writings and was systematized into a complex metaphysical pattern by Galen in the 2nd century ad. It dominated medical thinking even into the 17th century. Microscopic examination of the blood by Leeuwenhoek and others in the 17th century, and subsequent improvements in their rudimentary equipment, provided the means whereby theory and dogma would gradually be replaced by scientific understanding. The advance of knowledge was slow; however; those who were willing to observe and to seek greater understanding were few compared with the multitudes who repeated the age-old formulations. In the 18th century, William Hewson (1739 to 1774) made many important observations, and over the next 150  years or more, others gradually left their mark, including Gabriel Andral (1797 to 1876), Alexander Donné (1801 to 1878), Georges Hayem (1841 to 1933), and Paul Ehrlich (1854 to 1915), as well as Virchow, Aschoff, Maximow, Pappenheim, and still others in more recent times.2 However, it was not until the 1920s, beginning with the investigations of Whipple, Minot, and Castle, that the modern era of hematology started. From that time on, the field of hematology has flourished, and knowledge and understanding have grown at an ever-accelerating pace. The story has been told elsewhere.2 The reader will find revealing the comparison of the first edition of this textbook published in 1942, with subsequent editions. At one time, hematology was a purely laboratory endeavor concerned with quantitation of the formed elements of the blood and the study of their morphology and that of the bone marrow,

spleen, and lymphoid tissues. In the past 70 years, however, hematology has become a broad-based science that, in seeking to understand the normal and pathologic physiology of the hematopoietic system, uses all the methods of diverse scientific disciplines such as biochemistry, cell biology, immunology, physical chemistry, molecular biology, genetics, and nuclear medicine. As a consequence, the study of a hematologic problem can involve the use of procedures of great complexity. Nevertheless, in the majority of patients whose illness may be regarded as being directly or indirectly related to the blood or blood-forming organs, examination requires only simple procedures. These begin with the steps fundamental and essential in the study of any clinical problem: a carefully taken history and a meticulous, discerning physical examination. It should be emphasized that the hematologist must be conversant with illness on a broad scale because, although certain diseases affect primarily the blood and blood-forming tissues, more often, disorders of other organ systems result in alterations in the hematopoietic system. A sound and thorough background is essential for the hematologist because modern oncologic therapy affects tissues other than those of the hematopoietic system and can be associated with diverse complications. The symptoms of hematologic disorders are so varied and nonspecific that in themselves they may not suggest a hematologic problem. Thus, unexplained fever, extreme fatigability, or recurrent infections may or may not be caused by a hematologic condition. Likewise, physical examination may or may not direct attention to the hematopoietic system. The physical signs may be mainly those of congestive heart failure when the primary condition is pernicious anemia in relapse, adenopathy and splenomegaly when the underlying condition is a self-limited childhood infection, or nothing other than pallor when leukemia or aplastic anemia is the underlying disorder. In other instances, sternal tenderness, or bone tenderness elsewhere, hemorrhages, or spoon nails may direct attention to the hematopoietic system. Certain details of history must receive special inquiry. These include exposure to physical or chemical agents, which may have caused injury, and to drugs, prescribed or self-medicated. Often overlooked are substances used at work and in the home, such as pesticides and solvents. Especially misleading is the fact that only the exceptional individual is harmed by most of these agents, thereby giving the patient and the physician a false sense of security. For example, in patients given chloramphenicol, only rarely did aplastic anemia develop. Also deserving special inquiry is the diet, the degree and frequency of menstrual blood loss, evidence of intestinal blood loss, and the presence or the absence of fever. In addition, family history is important in the differential diagnosis of hematologic disorders. Knowledge of ethnic origin or a history of jaundice, anemia, cholelithiasis, splenectomy, or bleeding in male rather than in female members of the family, for example, may offer useful clues. Furthermore, history alone may be insufficient. Although symptoms may be denied, a palpable

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Introduction

spleen on physical examination or morphologic changes seen in the blood smear, such as target cells and basophilic stippling, may direct attention to the hitherto unsuspected hereditary disorder. A family history is only as good as the thoroughness of the inquiry. In the physical examination, careful attention should be given to the color of the skin, the sclerae and the nails, the presence of lymphadenopathy, sternal or other bone tenderness, splenomegaly, and petechiae in the mouth, ocular fundi, or skin. Unless the patient has been examined while lying on his or her right side, with the abdomen relaxed, as well as in the usual manner in the supine position, a search for an enlarged spleen cannot be considered complete. One also should be careful not to miss a renal tumor or mistake it for an enlarged spleen. In addition to the history and physical examination, it is necessary to determine whether anemia is present, to calculate the red cell indices, and to measure the concentrations of the leukocytes and platelets. The erythrocyte sedimentation rate may be included as a useful, nonspecific indicator of acute or chronic conditions. Blood chemical examinations, especially those for blood urea nitrogen, creatinine, and uric acid, are also essential. Usually, in an automated laboratory, it is faster, equally accurate, and less costly in time and money to obtain a battery of such data as part of the initial evaluation. The stained blood smear must be examined not only to determine the differential leukocyte count but also to search for other signs of abnormality. The latter, at least, should be done by the hematologist. Even if one could count on perfect laboratory examinations and reports (which one cannot, even with the best laboratories), nothing can replace the careful scrutiny of the blood smear by a physician who knows the patient’s complaints and physical findings. Subtle abnormalities in red cell morphology may have been overlooked or a rare nucleated red cell or an immature or abnormal leukocyte may have been missed; a platelet count may have been reported as being low, but the blood smear may show otherwise. The indiscriminate selection of a battery of hematologically oriented tests, such as obtaining a Coombs test and levels of serum iron, B12, and folic acid, in every anemic patient is wasteful, unwise, and unnecessary. In particular, bone marrow examination, which too often is done almost automatically in conditions suspected to be hematopoietic, is unjustified as a routine part of the hematologic examination. Bone marrow examination is useful in certain situations but cannot be expected to be helpful in others. Again, the tendency to order procedures such as red cell survival studies, liver and spleen scans, or other costly or time-­ consuming examinations when the same information can be obtained or inferred by simpler means only taxes the financial resources of the patient and the health care system. Results of the initial investigation often direct further study in one of three or four areas, the approaches to which are described in later chapters. Thus, if anemia appears to be the outstanding feature, refer Chapter 22 for steps to follow in investigating its nature and cause. The investigation of patients with bleeding disorders is outlined in Chapter 45. Abnormalities in the numbers or morphologic characteristics or leukocytes, splenomegaly, lymphadenopathy, recurrent infection, or other signs of abnormal immunologic or phagocytic function are explored in Chapter 56. If all of the formed blood elements are deficient (pancytopenia), consult Chapters 37 and 38. The steps outlined in these chapters, along with the guidance of a provisional diagnosis on the basis of evidence disclosed to that point, make the diagnostic process a logical and orderly procedure. In such a stepwise fashion, an accurate diagnosis can be reached with a minimum of trouble to the patient and at minimal cost. Only in the most complex cases is an exhaustive investigation justified.

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PRINCIPLES OF MANAGEMENT The successful and intelligent management of disease depends on three elements: accuracy of diagnosis, understanding of the nature of the abnormalities discovered and their ultimate prognosis if unchecked, and appreciation of the character of the patient and his or her reaction to the illness. Other important factors that must be considered in this connection are the patient’s age, responsibilities, concerns, and fears. Accuracy of diagnosis obviously is fundamental and influences the treatment of the patient. If the diagnosis is uncertain, one must consider what additional steps must be undertaken in seeking the diagnosis; whether the suspected possibilities justify those steps; whether consultation might be helpful; whether one is justified in waiting to allow the disease process to progress without interference, thereby permitting it to declare itself more clearly; and whether a therapeutic trial based on the most likely diagnosis is justified. One must avoid diffuse testing and data gathering, which may be expensive and of limited value. Physicians should regard the discovery of anemia in a patient as a challenge. Anemia is a manifestation of disease, not a disease in itself. The anemia may be a subtle sign of chronic renal insufficiency, of malignancy, or of chronic infection that has not otherwise declared itself. In such cases, management depends on the nature of the underlying cause. If the anemia is of the irondeficient type, it is a signal to search for its cause and eliminate it if possible; moreover, the anemia can usually be relieved by appropriate iron therapy. In addition, it is essential that the physician understand the nature of the abnormality or the disease that has been discovered. It is as wrong to alarm the patient when this is not justified as it is to fail to discover some disorder that should have been recognized and treated. It is especially important to be cautious in the way one uses terms such as leukemia that have life-threatening implications. Some forms of chronic lymphocytic leukemia, for example, are so slow in their progress (even requiring no treatment for many years) that this term is misleading. Likewise, other terms that have serious implications and yet refer to diseases with wide ranges of prognostic implication must be used most cautiously. Accurate diagnosis and knowledge of the prognosis, both with and without various modes of therapy, should guide the physician in answering the three major questions of therapy: WHETHER to treat WHEN to treat with WHICH modality The therapeutic measures, particularly the chemotherapeutic agents available to hematologists, carry a substantial potential for harm. Potential gain must be weighed against potential risks. This is especially true when therapeutic expectation is palliation rather than cure of the disease. Although it is self-evident that the physician must be mindful of the patient’s fears and hopes and those of his or her family, it is too easy to overlook this need. The physician must take the time to give the patient and his or her family some understanding of the illness (if there is one) with sensitivity and sympathy. The physician must choose carefully each word he or she uses and consider how comments may be interpreted or misinterpreted. The nature and course of certain hematologic disorders are such that in some patients reassurance may be far more helpful than any other measure the physician can offer. Consultation with another physician, preferably an expert, may be of value to the emotional well-being of certain patients, even when the physician is certain of the diagnosis and the appropriate treatment. Undertaking meaningless therapy, treating only because of the magnitude of the white blood cell count, for example, without considering the psychologic effects of such attention to what may be a minor manifestation of the disease, and risking the possibility

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of injury by the therapeutic agents used without considering the normal course of the disease are common errors in judgment. Dealing with the terminally ill patient and his or her family requires compassion, wisdom, and tact. One must be truthful and also understanding; what is especially important is how the truth is told. Furthermore, it is rare that a patient wants to know the whole truth. It is wise to give the patient and his or her family an opportunity to ask questions. In that way, the facts come out more gradually, and a blunt announcement of the likely outcome is avoided. Patients and their families often ask how long the patient will live. No physician is able to predict this with any accuracy. On

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the one hand, the stamina of patients and their will to live vary greatly; on the other, it is difficult to guess what unexpected events may occur that will bring closer, or postpone, the ultimate end.

References

1. Wintrobe MM. Blood, pure and eloquent: a story of discovery, of people and of ideas. New York, NY: McGraw-Hill, 1980. 2. Wintrobe MM. The blossoming of a science, a story of inspiration and effort. Philadelphia, PA: Lea & Febiger, 1985.

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P a rT

I

Laboratory Hematology

Chapter 1

Examination of the Blood and Bone Marrow

Careful assessment of the blood is often the first step in assessment of hematologic function and diagnosis of related diseases, and many hematologic disorders are defined by specific blood tests. Examination of blood smears and hematologic parameters yields important diagnostic information about cellular morphology, quantification of the blood cellular components, and evaluation of cellular size and shape that allows formation of broad differential diagnostic impressions, directing additional testing. This chapter introduces the fundamental concepts and limitations that underlie laboratory evaluation of the blood and outlines additional testing that may aid in evaluating a hematologic disorder, including special stains and bone marrow examination. Blood elements include erythrocytes or red cells, leukocytes or white cells, and platelets. Red blood cells (RBCs) are the most numerous blood cells in the blood and are required for tissue respiration. RBCs lack nuclei and contain hemoglobin, an iron-containing protein that acts in the transport of oxygen and carbon dioxide. White blood cells (WBCs) serve an immune function and include a variety of cell types that have specific functions and characteristic morphologic appearances. In contrast to mature red cells, WBCs are nucleated and include neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Platelets are cytoplasmic fragments derived from marrow megakaryocytes that function in coagulation and hemostasis. Blood evaluation requires quantification of each of the cellular elements by either manual or automated methods. Automated methods, using properly calibrated equipment,1,2 are more precise than manual procedures. In addition, automated methods may provide additional data describing cellular characteristics such as cell volume. However, the automated measurements describe average cellular characteristics but do not adequately describe the scatter of individual values around the average. Hence, a bimodal population of small (microcytic) and large (macrocytic) RBCs might be reported as normal cell size. Therefore, a thorough blood examination also requires microscopic evaluation of a stained blood film to complement hematology analyzer data, especially when new findings are identified.3–5

the specimen is often helpful in ensuring proper handling and test performance. A number of factors may affect hematologic measurements, and specimens should be collected in a standardized manner to reduce data variability. Factor example, patient activity, level of hydration, medications, sex, age, race, smoking, and anxiety may significantly affect hematologic parameters.6,7,8 Similarly, the age of the specimen may affect the quality of the data collected.9,10 Thus, data such as patient age, sex, and time of specimen collection should be noted, as well as pertinent correlative clinical information. Most often, blood is collected by venipuncture into collection tubes containing anticoagulant.11 The three most commonly used anticoagulants are tripotassium or trisodium salts of ethylenediaminetetraacetic acid (EDTA), trisodium citrate, and heparin. EDTA and disodium citrate act to remove calcium, which is essential for the initiation of coagulation, from the blood.11 Heparin acts by forming a complex with antithrombin in the plasma to prevent thrombin formation. EDTA is the preferred anticoagulant for blood counts because it produces complete anticoagulation with minimal morphologic and physical effects on cells. Heparin causes a bluish coloration of the background when a blood smear is stained with Wright-Giemsa stains, but does not affect cell size or shape. Heparin is often used for red cell testing, osmotic fragility testing, and functional or immunologic analysis of leukocytes. Heparin does not completely inhibit white blood cell or platelet clumping. Trisodium citrate is the preferred anticoagulant for platelet and coagulation studies. The concentration of the anticoagulant used may affect cell concentration measures if it is inappropriate for the volume of blood collected and may also distort cellular morphology. Most often, blood is collected directly into commercially prepared negative-pressure vacuum tubes (Vacutainer tubes; Becton Dickinson, Franklin Lakes, NJ), which contain the correct concentration of anticoagulant when filled appropriately, thereby minimizing error.11 Anticoagulated blood may be stored at 4°C for a 24-hour period without significantly altering cell counts or cellular morphology.9 However, it is preferable to perform hematologic analysis as soon as possible after the blood is obtained.

Specimen Collection

Reliability of Tests

Proper specimen collection is required for acquisition of reliable and accurate laboratory data for any hematologic ­ specimen. Before a specimen is obtained, careful thought as to what studies are needed will aid in optimal collection of samples. Communication with laboratory personnel analyzing

In addition to proper acquisition of specimens, data reliability requires precise and reproducible testing methods. Both manual and automated testing of hematologic specimens must be interpreted in light of expected test precision, particularly when evaluating the significance of small changes. All laboratory tests

Laboratory Hematology

Kristi J. Smock, Sherrie L. Perkins

1

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2

Part i  Laboratory Hematology 

are evaluated with respect to both accuracy and reproducibility. Accuracy is the difference between the measured value and the true value, which implies that a true value is known. Clearly, this may present difficulties when dealing with biologic specimens. The National Committee for Clinical Laboratory Standards (NCCLS) and the Clinical and Laboratory Standards Institute (CLSI) have attempted to develop standards to assess the accuracy of blood smear examination11 and automated blood cell analyzers.2 Automated instrumentation requires regular quality assurance evaluations and careful calibration to reach expected performance goals and the ability to collect accurate and reproducible data.2,12,13 In addition, the International Consensus Group for Hematology Review has suggested criteria that should lead to manual review of a specimen after automated analysis and differential counting.3

Cell Counts Cell counts are important parameters in evaluating the blood. Cell counts may be determined either manually or by automated hematology analyzers. Whether performed by manual or automated methodologies, the accuracy and precision of the counts depend on proper dilution of the blood sample, even distribution of cells, and precise sample measurement. As blood contains large numbers of cells, sample dilution is usually required for accurate analysis. The type of diluent is dependent on the cell type to be enumerated. Thus, red cell counts require dilution with an isotonic medium, whereas in white cell or platelet counts, a diluent that lyses the more numerous red cells is often used to simplify counting. The extent of dilution also depends on the cell type. In general, red cell counts need more dilution than is required for the less abundant WBCs. Errors in cell counts are caused primarily by errors in sample measurement, dilution, or enumeration of cells. The highest degree of precision occurs when a large number of cells can be evaluated. Clearly, automated methods are superior to manual methods for counting large numbers of cells and minimizing statistical error. Table 1.1 lists the comparable values of reproducibility for automated and manual (hemocytometer) counting methods. Manual counts are done using a microscope after appropriate dilution of the sample in a hemocytometer, a specially constructed counting chamber that contains a specific volume. Red cells, leukocytes, and platelets may be counted. Due to the inherent imprecision of manual counting and the amount of technical time required, most cell counting is now performed by automated instruments that increase the accuracy and speed of analysis by the clinical laboratory, thereby minimizing levels of human

manipulation for test entry, sampling, sample dilution, and analysis.16 With increasing automation, some hematology analyzers can be coupled with instruments performing other laboratory tests using the same tube of blood.17 There is a variety of different automated hematology analyzers available, dependent on the volume of samples to be tested and the specific needs of the physician ordering testing. The analyzers range in price and workload capacity from those that would be appropriate for an individual physician’s office or point-of-care facility to those needed in a busy reference laboratory with capacity for over 100 samples to be analyzed per hour.16 Most automated hematology analyzers perform a variety of hematologic measurements, in addition to cell counting, such as hemoglobin concentration (Hb), red cell size, and leukocyte differentials. Many instruments also perform more specialized testing, such as reticulocyte counts.18 The ability of analyzers to perform accurate WBC differential counts, particularly those that can perform a five-part differential (enumerating neutrophils, lymphocytes, monocytes, eosinophils, and basophils), has been a significant technologic advance over the past 15 years. Automated methods for white cell counts and differentials use several distinct technical approaches, including measurement of electrical impedance, differential light scatter, optical properties, or surface antigen/cytochemical staining either alone or in combination.19,20 Most of the newer-generation hematology analyzers utilize optical flow cytometric technologies with or without additional cytochemical staining to detect specific cell types such as red cells, white cells, and platelets (Fig. 1.1).19,21,22 The newer analyzers have the additional ability to detect reticulocytes as part of the normal complete blood count (CBC) differential using a fluorescent RNA dye and many will also enumerate nucleated red blood cell numbers based on their optical properties.23 In addition, many of the current analyzers do auto sampling directly from tubes

Various Angles of Scattered Light

Sample Stream Focused Laser Beam

TA B L E 1.1

Reproducibility of Blood Counting Procedures Two Coefficients of Variation Cell Type Counted

Hemocytometera (%)

Automated Hematology Analyzer (%)

±11.0

±1.0

±16.0 ±22.0 ±33.9

±1.5 ±2.0 ±5.0

Red cells White cells Plateletsb Reticulocytes a Minimum error. Usual error. b Error may be greater with low

(450 × 109/L) platelet counts. Data derived from Bentley S, Johnson A, Bishop C. A parallel evaluation of four automated hematology analyzers. Am J Clin Pathol 1993;100:626–63214 and Wintrobe M. A simple and accurate hematocrit. J Lab Clin Med 1929;15:287–28915.

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Sample Feed Nozzle

Sheath Stream

Figure 1.1. Optical flow cytometric type of automated hematology analyzer.  A suspension of cells is passed through a flow chamber and focused into a single cell sample stream. The cells pass through a chamber and interact with a laser light beam. The scatter of the laser light beam at different angles is recorded, generating signals that are converted to electronic signals giving information about cell size, structure, internal structure, and granularity. (Adapted and redrawn from Cell-Dyn 3500 Operator’s Manual. Santa Clara, CA: Abbott Diagnostics, 1993.)

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Chapter 1  Examination of the Blood and Bone Marrow

and use a very small sample ranging from 35 to 150 ml for a full CBC ­analysis. Using flow cytometric technologies, some analyzers also have the ability to detect specific blood cell populations by specific antigen expression, such as detection of CD34 peripheral blood stem cells or leukemic blasts.24–26 Integration of data from cytochemical or antigenic staining and light scatter properties has improved the accuracy of the five-part differential and decreased the numbers of unidentifiable cells requiring technician review for identification. Instruments from Abbott Laboratories (CELL-DYN), 16,27 Horiba Medical (ABX Pentra series), and Sysmex (XE series, XT series, and XS series)16,28,29 primarily utilize fluorescent-based flow cytometry as the modality for analysis. Each system has slightly different fluorochrome staining combinations that aid in the identification of white cells, red cells, and platelets in combination with light scatter characteristics. All provide integrated reticulocyte counts and five-part differentials. Workload capacities range from 70 to 106 samples analyzed per hour. When reticulocytes are ordered as a part of the differential, the capacity falls to between 40 and 60 samples per hour (allowing for the staining and detection of the RNA dye fluorescence). Instruments by Siemens (Advia 120 and 2120 series) use a combination of flow cytometric techniques and a cytochemical peroxidase stain for the five-part differential. This instrument integrates electrical impedance data, flow cytometric light scatter, characteristic fluorescent staining, and cytochemical staining to generate an accurate white blood cell differential. Siemens technology also calculates hemoglobin levels, claiming that this causes less interference by high white blood cell counts or lipemia in the specimen.16,30,31 Instruments from Beckman/Coulter (Coulter DxH series, LH 500 series, LH 750 series, LH 780 series) also utilize electrical impedance or conductivity in combination with light scatter approaches, integrating these technologies to provide full analysis and five-part differentials (Fig.  1.2). The Beckman/ Coulter series includes nucleated RBCs and reticulocyte counts in every differential. Its capacity is 45 samples per hour when reticulocytes are included and 100 samples per hour for a CBC without reticulocyte counts.16

Red Blood Cell Analytic Parameters

WBC

3

RBCs are defined by three quantitative values: the volume of packed red cells or hematocrit (Hct), the amount of hemoglobin (Hb), and the red cell concentration per unit volume. Three additional indices describing average qualitative characteristics of the red cell population are also collected. These are mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). All of these values are routinely collected and calculated by automated hematology analyzers, largely replacing many of the previously used manual or semi-automated methods of RBC characterization, with certain exceptions as noted below. The use of hematology analyzers imparts a high degree of precision compared to manual measurements and calculations (Tables 1.1 and 1.2).

The hematocrit is the proportion of the volume of a blood sample that is occupied by red cells. Hct may be determined manually by centrifugation of blood at a given speed and time in a standardized glass tube with a uniform bore, as was originally described by Wintrobe.15 The height of the column of red cells after centrifugation compared with total blood sample volume yields the Hct. Macromethods (using 3-mm test tubes) with low-speed centrifugation or micromethods using capillary tubes and high-speed centrifugation may also be used. Manual methods of measuring Hct are simple and accurate means of assessing red cell status. They are easily performed with little specialized equipment, allowing adaptation for situations in which automated cell analysis is not readily available or for office use. However, several sources of error are inherent in the technique. The spun Hct measures the red cell concentration, not red cell mass. Therefore, patients in shock or with volume depletion may have normal or high Hct measurements due to hemoconcentration despite a decreased red cell mass. Technical sources of error in manual Hct determinations usually arise from inappropriate concentrations of anticoagulants,32 poor mixing of samples, or insufficient centrifugation.15 Another inherent error

Laboratory Hematology

Volume of Packed Red Cells (Hematocrit)

RBC REL#

V O L U M E

50 100

200

300 f

PLT

TA B L E 1 . 2

Reproducibility of Red Cell Indices

REL#

2

DF 1

ID# 1 ID# 2 Sequence # DATE: TIME: Cass/Pos

WBC

06/21/96 08:55:45 S

Normal WBC Pop Normal RBC Pop Normal PLT Pop

NE LY MO EO BA

6.7 % 59.4 31.6 7.7 0.7 0.6

# 4.1 2.1 0.5 0.0 0.0

10

20

30 f

RBC HGB HCT MCV MCH MCHC RDW

4.56 13.5 40.3 88.3 29.5 33.5 13.4

PLT

202

MPV

8.2

Figure 1.2. Histograms and printout generated by the Coulter automated hematology analyzer utilizing light scatter and electrical impedance.  BA, basophil; EO, eosinophil; HCT, hematocrit; HGB, hemoglobin; LY, lymphocyte; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MO, monocyte; MPV, mean platelet volume; NE, neutrophil; PLT, platelet; RBC, red blood cell; RDW, red cell distribution width; WBC, white blood cell.

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Index

Method Used

Hemoglobin concentration

Spectrophotometric Automated

Mean corpuscular volume

Hemocytometer Automated

Mean corpuscular ­hemoglobin Mean corpuscular ­hemoglobin concentration

Hemocytometer Automated Automated

% Error (±2 Coefficients of Variation) 1.0–2.0 Pr

Nonreactive P

IgG Panreactive Unclear > I> others

Warm Autoimmune Hemolytic Anemia

Drug-Induced Hemolytic Anemia

40–70% IgG ± C3; rarely C3 alone IgG, occasionally with IgA or IgM IgG Panagglutinin Rarely Rh

12–18% IgG or C3; occasionally IgG ± C3 IgG IgG or nonreactive Rh-related Drug-dependent

Anti-idiotypic antibodies and direct nucleotide sequencing of the rearranged immunoglobulin variable-region genes have revealed significant cross-reactivity and homologies among cold autoantibodies with similar specificity.48,49 For instance, the monoclonal anti-idiotypic antibody 9G4 recognizes an idiotypic determinant present on the heavy chains of both anti-I and anti-i cold agglutinins as well as the responsible neoplastic B-cells.50 Essentially all pathologic anti-I and anti-i cold agglutinins are derived from a distinct subset of heavy-chain variable-region genes called VH4 family genes, specifically VH4–21.51 In ∼40% of the patients, a circulating B-cell clone can be identified with a distinctive karyotypic marker (trisomy 3q11-q29; trisomy 12; or 48XX+3+12). The chromosomal abnormalities were associated with chronic idiopathic cold agglutinin syndrome as well as with monoclonal cold agglutinins secondary to a neoplasm.52,53,54 In addition, the cold agglutinins have the same serologic specificity and isoelectric focusing spectrotype and are therefore likely derived from a pre-neoplastic or neoplastic B-cell clone.7

I/i Blood Group System Specificity

INCREASING AFFINITY

More than 90% of cold-active antibodies have the I antigen as their target on the RBC, and the i antigen is the binding site for a significant portion of the remaining 10%.31 The closely related I/i antigens are high-frequency carbohydrates similar to the ABO

Range of Antibody Fixation

Range of Complement Action

Range of Hemolysis 0°

10°

15° 20° 25°

30°

40° C

FIGURE 29.4. Temperature ranges for cold agglutinin fixation and lytic complement action.  (From Schubothe H. The cold hemagglutinin disease. Semin Hematol 1966;3:27–47, with permission.)

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antigens. The RBC surface densities of I and i are inversely proportional, with neonatal RBCs exclusively expressing large amounts of i antigen, usually converting to exclusively I antigen by 18 months of age. Consequently, adult RBCs are used to detect anti-I agglutinins and cord RBCs are needed to detect anti-i agglutinins. Extremely rare adults have been described who never express I antigen on the RBCs. Other uncommon but reported antigen TA B L E 2 9 . 7

SECONDARY COLD AGGLUTININ DISEASE Neoplasms   Waldenstrom macroglobulinemia   Angioimmunoblastic lymphoma   Other lymphomas   Chronic lymphocytic leukemia   Kaposi sarcoma  Myeloma   Nonhematologic malignancy (rare) Infections   Mycoplasma pneumoniae   Mononucleosis (Epstein-Barr virus)  Adenovirus  Cytomegalovirus  Encephalitis   Influenza viruses  Rubella  Varicella   Human immunodeficiency virus  Mumps  Ornithosis   Legionnaires’ disease   Escherichia coli   Subacute bacterial endocarditis  Listeriosis  Syphilis  Trypanosomiasis  Malaria Other   Autoimmune diseases   Tropical eosinophilia

Disorders of Red Cells

RBC, red blood cell.

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Part iv  Disorders of Red Cells  •  SECTION 3  Hemolytic Anemia

targets include Pr. Anti-Pr cold agglutinins tend to be high-titer, with a wide thermal range, and cause symptomatic anemia.55,56 Other infrequent targets are Gd, Fl, Vo, Li, Sa, Lud, M, N, Me, Om, D, Sdx, and P.38,44,57 The fact that M. pneumoniae induces anti-I antibodies in the majority of patients is potentially related to the finding that sialylated I/i antigens serve as specific Mycoplasma receptors.58 Minor modification of this antigen may incite autoantibodies. Another theory suggests that an I-like antigen appears on the organism itself, and cross-reacting antibodies lead to RBC lysis.59 Despite the high rate of antibody production, clinically significant hemolysis occurs in very few patients.60 Infectious mononucleosis is also associated with CAD, but to a much lesser degree than Mycoplasma. Only 0.1% to 3.0% of mononucleosis patients have clinical hemolysis,61 although anti-i is present in 8% to 69% of sera post-infection.62–64 Therefore, the majority of patients with antibodies are asymptomatic. Anti-I activity is usually noted as well, but not to the same degree. Also, anti-Pr and anti-N have been reported.65 Both IgM and IgG antibodies as well as IgM rheumatoid-like factors reacting with IgG may act as cold agglutinins after infectious mononucleosis.66,67 See Table 29.7 for a list of other infectious diseases associated with cold agglutinins, most of which are anti-I, although anti-i has been seen in cytomegalovirus infections and in lymphomas.68

spleen may be enlarged or more frequently palpable in secondary cold agglutinins due to lymphoma or infectious mononucleosis.44 If hemolysis does occur after Mycoplasma infections, it typically begins during the post-pneumonia recovery period when cold autoantibody titers are at peaking. The process, even if severe, resolves spontaneously within 1 to 3 weeks.31 Hemolytic anemia after infectious mononucleosis may begin with the onset of illness or within the next 3 weeks.44 The self-limited, post-infectious CAD tends to affect younger patients, whereas the chronic idiopathic form is a disease of the elderly, with peak incidence at ∼age 70 years.43

Laboratory Features

Cold agglutinins attach to the RBC in the cooler peripheral circulation. As the blood returns to the warmer core circulation, the antibody dissociates from the RBC. Antibodies that attach, fix complement, and then dissociate are free to attack another erythrocyte and begin the process again.69 Complement fixation and activation, which are responsible for the destruction of the RBCs, are far more efficient at the warmer core temperatures. However, with a high antibody titer and a wide thermal amplitude, there may be sufficient temperature overlap to produce hemolysis at 22±10°C.43 See Figure 29.4. Because of this diversity of temperature requirements for optimal activity of the antibody and complement, RBC destruction is usually not particularly severe with cold autoantibodies. Quite impressive exceptions occur, and these are typically the antibodies with either high titers (>1:1,000) or activity up to 37°C even in the face of modest titers. Thermal amplitude is a better predictor of hemolysis than titer.46,70 High-titer cold agglutinins with a narrow thermal amplitude may produce a clinical picture with bursts of hemolysis associated with exposure to cold, often manifested as intermittent hemoglobinuria between quiescent periods.69 A frequent misconception about cold agglutinins is the assumption that they are cryoglobulins, whereas in fact they are two distinct disease processes. Both may cause cyanosis and Raynaud phenomenon in cooler temperatures. However, cryoglobulins do not fix complement on the RBCs or lead to hemolysis.

Mild chronic anemia is the rule, but the hemoglobin may fall to 5 to 6 g/dl, especially in the winter months in cold climates. The peripheral smear, if not obtained from a carefully collected prewarmed specimen properly maintained warm until spread on a warm slide, may show significant agglutination and RBC clumping under magnification. Occasionally, clumping is so extensive as to be grossly visible without magnification and may even preclude an adequate smear examination. Agglutinates are frequently visible in the specimen tube and can appear to be a large clot. Dissolution with warming demonstrates that the clumped and clotted appearance is a result of a cold agglutinin rather than Rouleaux formation or fibrin strands. Often, the first suspicion of a cold agglutinin comes from a failed attempt to obtain a valid RBC count and indices on an automated CBC. The initially reported RBC count is often artifactually low and the MCV artifactually high, producing a spuriously high MCHC. The reticulocyte count is modestly elevated except in rare cases of concomitant marrow failure, such as those due to parvovirus B19 infection.73 Spherocytosis is not pronounced as in warm AIHA. WBC and platelet counts are usually normal, but low levels of both have been reported, as has leukocytosis.44 Bilirubin is mildly elevated, rarely >3 mg/dl. LDH may be increased (reflecting RBC destruction), and complement and haptoglobin are often low or absent. During brisk hemolysis, hemoglobinuria and hemoglobinemia are manifest. The DAT is positive with polyspecific and anticomplement antisera. As above, IgM has dissociated and is not detectable. In extremely rare cases, the antibody involved is IgG or IgA, either alone or in addition to IgM.31,44 Mixed warm and cold autoantibodies are not rare (discussed later). Titers measured at 4°C may range from 1:1,000 to 1:1,000,000, although typical values are between 1:1,000 and 1:500,000. Much lower levels can be clinically significant if activity is measurable at 37°C. Post-infectious CAD titers are lower (80% of patients having a prompt reduction in hemolysis. In one series, only 7% took longer than 2 weeks, and even fewer more than 3 weeks. Therefore, if there has been no improvement after 3 weeks of therapy, the patient may be considered a steroid treatment failure. Recurrence of hemolysis after remission is usually gradual, especially if the steroids were weaned over a prolonged period.

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Chapter 29  Autoimmune Hemolytic Anemia

759

Relapse of warm AIHA occurs in the vast majority of patients. Approximately 40% to 50% of patients require maintenance doses of prednisone (5 to 20 mg/day).166 If the maintenance prednisone dose is >15 mg/day, other measures should be considered. Free autoantibody in the serum (positive IAT or antibody screen) may disappear, but the DAT remains positive in most patients, although perhaps weaker.44 Complete and lasting remission rates from steroids alone are reported as occurring in only 16% to 35% of patients,167,168 so the majority of patients with warm AIHA require additional therapy.

Second-Line Treatment

Second-line treatment is indicated in those surgical candidates who have not responded to prednisone, require prednisone doses >10 to 20 mg/day to maintain remission, or have suffered intolerable side effects. Splenectomy:  The rationale for splenectomy is twofold. The spleen is the major site of RBC sequestration and destruction in warm AIHA, resulting from the interaction of IgG antibodies with the macrophage Fc receptors. Splenectomy has little effect on the clearance of IgM-coated RBCs and therefore would not be indicated in the unusual patient with a warm-active IgM antibody. The spleen is also believed to be a major producer of IgG antibodies.31,32 Overall responses are probably ∼60% to 75%, but many of these patients relapse or remain on steroids, albeit at lower, more tolerable doses.31,169,170 The likelihood of response to splenectomy may be higher in idiopathic AIHA than in secondary AIHA.171 The complications of splenectomy are those inherent in major abdominal surgery and also include subdiaphragmatic abscess, pulmonary embolism, and increased susceptibility to infections, especially in children.44,172 Pneumococcal, meningococcal, and Haemophilus influenzae vaccinations should be given, preferably administered preoperatively.173 Subsequent to splenectomy, patients should be given antibiotics promptly with any febrile illness.

Disorders of Red Cells



Rituximab:  Rituximab may be used as a second-line treatment option in patients who refuse splenectomy or are at high surgical risk. Rituximab is a chimeric human/murine monoclonal anti-CD20 antibody approved for use in lymphoma. Successful rituximab use in refractory warm AIHA and Evans syndrome has been documented in several case reports and series.174,175-177,178 Regimens identical to the lymphoma treatment (375 mg/m2/week × 4 weeks) have produced remissions in some patients who were refractory to other therapeutic regimens. A response rate of 87% was reported for one series of refractory pediatric patients. Twenty-three percent of the responders relapsed, but subsequent courses of rituximab induced additional remissions.178 Thus far, few side effects have been reported, but rare reactions to the infusion have been documented.179 B-cell counts remain low for months after treatment, raising the risk of infections due to poor immune response.180 The efficacy and toxicity of rituximab monotherapy was tested in additional retrospective studies in a mixed population of refractory primary or secondary AIHA. These studies have been summarized in a review by Lechner et al.181 Overall response rate was 82%. Safety data available are limited and adverse events include two patients with severe infections and one patient with myocardial infarction.182 The most severe potential long-term complication of rituximab treatment was progressive multifocal leukoencephalopathy (PML), which, however, has been observed in only two patients with AIHA.181 If remissions remain durable and potential side effects are less harmful than other treatments for warm AIHA, such as prolonged steroid use or splenectomy, rituximab may become accepted second-line therapy.

Immunosuppressive Therapy

Immunosuppressive therapy is indicated for patients who have failed to respond to splenectomy and/or rituximab therapy.181

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Cyclophosphamide is an effective immunosuppressive agent with capability to suppress the immune response, even if administered after antigen presentation.183 This is particularly desirable for administration after the onset of immune hemolysis. Beneficial effects of cyclophosphamide therapy are reported in reviews.166,168,169 Blood counts should be followed closely. Other significant side effects from the antimetabolites and cyclophosphamide include hemorrhagic cystitis, bladder fibrosis, secondary malignancies, sterility, and alopecia.31,166,184,185 No properly controlled trial exists from which to draw conclusions, but the reviews and case reports suggest a response rate of approximately 40% to 60% in those patients who did not respond to steroids and splenectomy.168,169,170 A reasonable immunosuppressive regimen might include azathioprine (80 mg/m2/ day) or cyclophosphamide (60 mg/m2/day), concomitantly with prednisone (40 mg/m2/day). Prednisone may be tapered over 3 months or so, and the cytotoxic agent continued for 6 months before reducing the dose gradually.166 Bone marrow suppression may dictate minor dose adjustments. Rapid withdrawal has led to rebound immune response.186 Alternatively, high-dose cyclophosphamide (50 mg/kg/day ×4 days) has produced a complete remission in 66% of patients who were refractory to other therapies. Severe myelotoxicity and its attendant potential for complications are expected.173 Mycophenolate mofetil, an inhibitor of inosine 5′-­ monophosphate dehydrogenase, is an immunosuppressant initially employed to treat allograft rejection. It has been shown to induce complete or partial remission of hemolysis in case reports. Doses begin at 1 g/day and are then increased to 2 g/day. In cases of partial remission, reduction in doses of other immunosuppressives was possible without sacrificing efficacy. Long-term side effects are not fully established. Short-term side effects consist primarily of gastrointestinal intolerance and mild myelosuppression.176,177,187,188 Cyclosporine A has been used both successfully and unsuccessfully in refractory hemolytic anemia patients, as were many of the other immunosuppressive medications previously described. Doses of 3 mg/kg/day with target serum levels of 200 to 400 ng/ml produced remissions.178,189 It has also been used in combination with other remedies with some success, including danazol and prednisone.141,179,180,190,191

and cytokines.199 Other postulated mechanisms include modulating expression and function of Fc receptors, interfering with the activation of complement, modulating immune response through anti-idiotype antibodies, and effects on B- and T-cells.200 Plasmapheresis has been used with limited success in attempts to remove the antibody.31,201,202 However, in some patients fulminant hemolysis proceeds unchecked and plasmapheresis may serve as a temporizing measure until other immunosuppressive therapies can take effect. Selective removal of IgG with staphylococcal protein A columns has also been reported with some benefit.203 Stem cell transplantation (SCT) has been described for many severe, life-threatening autoimmune syndromes, including hemolytic anemia and Evans syndrome. Sources of the stem cells have been autologous, HLA-matched sibling, and cord blood.204,205 Relapses and the expected range of complications, including death, have occurred. For very severe and refractory cases of Evans syndrome, SCT offers the only chance of long-term cure. Available data suggest that allogeneic SCT may be superior to autologous SCT, but both carry risks of severe morbidity and of transplantrelated mortality. As more refractory patients are seen, stem cell reconstitution after high-dose immune suppressive regimens will no doubt expand. Cure of Evans syndrome following reducedintensity conditioning has been reported and should be considered for younger patients in the context of controlled clinical trials.206 Alemtuzumab is a humanized anti-CD52 monoclonal antibody that is an effective therapy for B-CLL, mycosis fungoides, and T-cell prolymphocytic leukemia. There have been recent case reports that suggest alemtuzumab may be useful for the treatment of AIHA in B-CLL patients who have failed other treatments.207,208

Transfusion

Drug-induced Immune Hemolytic Anemia

As with CAD, red cell transfusion support may be required in patients who are clinically symptomatic or severely anemic. Patients with WAIHA should be tested for the presence of coexisting alloantibodies, which may have developed following pregnancies or prior transfusions. Alloantibodies, rather than autoantibodies, may cause major transfusion reactions in such patients if not discovered. However, identifying an alloantibody in the presence of an autoantibody takes additional time. Thus, if the patient needs to be transfused emergently, transfusion should be given prior to the availability of the results. Most patients will tolerate even serologically incompatible blood. Blood bank personnel should be involved early in the decision to transfuse to minimize delays and possible confusion.192

Other Therapies

Intravenous immunoglobulin (IVIG) has not enjoyed the success in AIHA that it has in immune thrombocytopenia (ITP). Case reports of success193–195 and failure196,197 have appeared. Escalating the dose from the standard 0.4 to 1.0 g/kg/day (×5 days) may be helpful. In one refractory patient, weekly maintenance infusions of 800 mg/kg/week helped to control transfusion requirements.198 The mechanism of action of IVIG is not completely clear. Recent evidence suggests that IVIG exerts inhibitory effects on dendritic cells by down-regulating costimulatory molecules, blocking maturation, and modifying their interactions with lipopolysaccharide

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Complications Hoffmann suggests that venous thromboembolism is an underrecognized complication of AIHA and may in some instances be related to co-existent antiphospholipid antibodies.209 Although it is premature to recommend anticoagulant prophylaxis in general for patients with AIHA hemolytic episodes, consideration might be given to those at particularly high risk, such as those with evidence of co-existing antiphospholipid antibodies.

Drugs can produce hemolysis by both immune and nonimmune mechanisms. In the 1970s and 1980s, a-methyldopa and high-dose penicillin were responsible for the majority of cases of drug-induced immune hemolytic anemia (DI-IHA). In recent years there has been a significant decline in DI-IHA due to a-methyldopa and high-dose penicillin because of declining use of those medications. More recent estimates of the incidence of DI-IHA are approximately 1 per million.210 Second- and thirdgeneration cephalosporins, especially cefotetan and ceftriaxone, have been associated increasingly with cases of immune hemolytic anemia, accounting for ∼80% of the DI-IHA.211 Rarely, these cases of cephalosporin-induced immune hemolytic anemia are fatal. Other antibiotic agents associated with immune hemolytic anemia include b-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) found in combination with b-lactam antibiotics in Timentin (ticarcillin/clavulanate), Unasyn (ampicillin/sulbactam), Zosyn (piperacillin/tazobactam), and piperacillin.211 AIHA has been noted with increased incidence in patients receiving purine nucleoside analogs such as fludarabine, cladribine, and pentostatin for hematologic malignancies.209 The mechanism by which these drugs cause AIHA is unclear. See Figure 29.4 and Tables 29.6 and 29.10.

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Chapter 29  Autoimmune Hemolytic Anemia

TA B L E 29.1 0

DRUGS ASSOCIATED WITH IMMUNE HEMOLYSIS OR AUTOANTIBODIES Acetaminophen Aminopyrine Amphotericin B Ampicillin Antazoline Apazone (azapropazone) Buthiazide (butazide) Carbenicillin Carbimazole Carboplatin Catergen Cefotaxime Cefotetan Cefoxitin Ceftazidime Ceftriaxone Cephaloridine Cephalothin Chaparral Chlorambucil Chlorinated hydrocarbons 2-Chlorodeoxyadenosine Chlorpromazine Chlorpropamide Cianidanol Ciproflaxin Cisplatin Cladribine Cyclofenil Diclofenac Diethylstilbestrol Diglycoaldehyde Dipyrone

Doxepin “Ecstasy” Elliptinium acetate Erythromycin Etodolac Fenfluramine Fenoprofen Fludarabine Fluorescein 5-Fluorouracil Glafenine Hydralazine Hydrochlorothiazide Ibuprofen Insecticides Insulin Interferon-a Intravenous contrast media Isoniazid Latamoxef Levodopa Mefenamic acid Mefloquine Melphalan 6-Mercaptopurine Mephenytoin Methadone Methicillin Methotrexate Methyldopa Nafcillin Nalidixic acid Nomifensine

Omeprazole Oxaliplatin p-Aminosalicylic acid Penicillin G Phenacetin Podophyllotoxin Probenecid Procainamide Propyphenazone Pyramidon Quinidine Quinine Ranitidine Rifampin (rifampicin) Sodium pentothal Stibophen Streptomycin Sulfonamides Sulfonylurea derivative Sulindac Suprofen Suramin Teniposide Tetracycline Thiazides Thiopental Thioridazine Tolbutamide Tolmetin Triamterene Trimellitic anhydride Zomepirac

Modified from Arndt PA, Garratty G. The changing spectrum of drug-induced hemolytic anemia. Semin Hematol 2005;42:137–144.

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Some medications may produce hemolysis by more than one mechanism, and differentiating among them is not always possible. Nonimmunologic adsorption of proteins to the RBC membrane can also cause a positive DAT, but is not associated with increased RBC destruction.

Drug Adsorption Mechanism (Penicillin Type) Ina penicillin-type drug absorption mechanism, the drug binds tightly to the RBC membrane and the antibody attaches to the drug without direct interaction with the erythrocyte. Penicillin binds to the RBC membrane covalently and can be demonstrated on the RBC in most patients receiving high doses of the drug, even in the absence of antibody.213 Attachment of only the drug does not harm the erythrocyte. However, when the drug is given in large doses (>10 million units/day), it can induce production of IgG antibody, which attaches to the membrane-bound drug, thus producing a positive DAT with AHG sera.213 Eluates from these RBCs do not react with RBC panels, in stark contrast to the previously discussed true autoimmune antibodies, which display panagglutinin activity. The explanation lies in the fact that penicillin-induced antibodies are attached to the drug alone and not to membrane components of the erythrocyte. If reagent cell suspensions are first coated with penicillin, agglutination occurs with all RBCs, thus providing a diagnostic testing strategy when drug-induced hemolysis is suspected.31 The benzylpenicilloyl determinant is the primary antigenbinding site. High-titered IgG antibenzylpenicilloyl antibodies are responsible for the positive DAT and appear in ∼3% of patients receiving high doses of the drug.214 Only some of these patients develop hemolysis.31 Rare exceptions of complement fixation to the RBCs exist in patients on penicillin. These have been associated with IgG attachment, and the DAT is positive with both antiIgG and anti-C3 sera.215,216 RBC destruction in the drug adsorption mechanism of hemolysis is through sequestration by splenic macrophages of the IgGcoated RBCs.217,218 Rarely, when associated with complement fixation, the RBCs may be lysed (intravascular hemolysis).219 Anemia develops gradually over ∼7 to 10 days and can be lifethreatening if not recognized and the drug discontinued. Once the medication is stopped, the hemolysis resolves over the ensuing couple of weeks. However, the DAT may remain positive for several weeks. Of clinical importance is the observation that other signs of penicillin hypersensitivity, such as urticaria and airway reactivity, are usually absent.31 Other drugs can also cause hemolysis by this mechanism. Cephalosporins,220–224 tetracycline,225,226 tolbutamide,227 and semisynthetic penicillins228,229 can cause DI-IHA by this mechanism.

Disorders of Red Cells



Mechanisms Drug-induced antibodies can be divided into two main groupings based on the requirement for the drug in detection. Drug-dependent antibodies (penicillin type or immune complex type) require the presence of the drug in the test system, whereas drug-independent antibodies (autoantibodies) do not. See Table 29.11 and Figure 29.5. Drug-dependent antibodies may be true autoantibodies with serology that is identical to warm AIHA. There are three major mechanisms by which drugs can cause immune hemolysis in vivo212 (see Fig. 29.5): 1. The drug adsorption mechanism, in which the antibody reacts with a drug tightly bound to the RBC membrane 2. The neoantigen or immune complex mechanism, in which the drug combines loosely with the RBC membrane and the antibody reacts with new antigenic site(s) created by the combination of drug and membrane 3. The autoimmune mechanism, which is indistinguishable from true AIHA without drug exposure

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Neoantigen Mechanism (Quinidine/Stibophen Type) The neoantigen mechanism is also known as the immune complex mechanism. The antibody is specific for a combination antigen, or neoantigen, created jointly by the drug and RBCs. Investigations with rare antigen-negative RBCs revealed that 2 antibodies have specific sites on the RBC membrane to which they attach along with the drug.230,231 The neoantigen mechanism differs from the drug adsorption mechanism in a few key areas. Unlike the penicillin model, these drugs bind very loosely to the RBC membrane. Only a small dose of the medication is required for hemolysis to occur, as opposed to the very large doses of penicillin required. Hemolysis is usually sudden, severe, and accompanied by hemoglobinuria2 instead of the subacute anemia typically seen with the drug adsorption type. Renal failure is a frequent occurrence in the neoantigen mechanism.232,233 The effector phase is mediated predominantly by complement

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TA B L E 29.11

MECHANISMS OF DRUG-INDUCED HEMOLYSIS OR POSITIVE DIRECT ANTIGLOBULIN TEST Drug Adsorption

Neoantigen

Autoimmune

Nonimmune Adsorption

Prototype drug Role of drug

Penicillin Cell-bound hapten

Quinidine/stibophen Antibody binds drug + RBC

Typical direct antiglobulin test

IgG

Complement

a-Methyldopa Induces drug-independent RBC antibody IgG

First-generation cephalosporins Modifies RBC membrane; adsorbs proteins nonspecifically Non-Ig

Antibody reactions

Reacts only with drug-coated cells Subacute onset; mild to severe hemolysis

Reacts only with drug present

Drug-independent panagglutinin Insidious onset; chronic mild hemolysis

No antibody present

Typical clinical presentation

Acute onset; severe hemolysis

fixation and subsequent intravascular hemolysis. Some sequestration of RBCs occurs in splenic macrophages or the liver via complement receptors. The DAT is positive only for the presence of complement, and IgM or IgG are rarely still attached to the RBC.31 Therefore, eluates are nonreactive primarily because there is no immunoglobulin to elute. In vitro, serum reacts with RBCs only in the presence of the drug or a reactive metabolite.31 These drugs may also induce thrombocytopenia by similar mechanisms.234,235

RBC

Drug adsorption

No hemolysis

Autoimmune Mechanism (a-Methyldopa Type) Unlike the previous two mechanisms, which require the presence of the offending drug for antibody reaction with the RBC membrane, hemolysis induced by a-methyldopa is truly autoimmune in nature. Antibodies bind to erythrocyte membrane antigens in a manner indistinguishable from the sporadic AIHA discussed earlier. With declining use of a-methyldopa, these antibodies are now seen in association with cladribine, fludarabine, levodopa, mefenamic acid, and procainamide.211 The etiology of these antibodies is unknown, but the drugs likely directly stimulate the immune system to mimic an autoimmune disease. The DAT is positive with anti-IgG and is usually negative with anti-C3. The eluate shows a panreactive antibody. The characteristics of the IgG antibody eluted from the RBCs are strikingly similar to those in idiopathic warm AIHA. They are polyclonal236 and bind as a panagglutinin to reagent cells even in the absence of the drug. As in warm AIHA, these antibodies have a predilection for Rh antigens, with some specific anti-c and anti-e documented.236,237 Despite a high incidence of immunoglobulin coating of RBCs, only a minority of patients actually develop clinical hemolysis.2 Explanations of this phenomenon have been unsatisfactory. The amount of antibody on the RBC correlates poorly with in vivo hemolysis, and no threshold has been well established.61,238

Nonimmunologic Protein Adsorption Mechanism

RBC

Neoantigen

Drug RBC

Autoimmune

Antibody

FIGURE 29.5. A proposed theory of drug-induced antibody reactions.  A: The antibody attaches only to the drug, which is tightly bound to the red blood cell (RBC) membrane (penicillin type). B: The antibody attaches to a neoantigen created by components of both the drug and the RBC membrane (quinidine/stibophen type). C: The antibody attaches mainly to the membrane, not requiring the presence of the drug (a-methyldopa type). (Adapted from Habibi B. Drug induced red blood cell autoantibodies co-developed with drug specific antibodies causing haemolytic anaemias. Br J Haematol 1985;61:139–143.)

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Proteins other than immunoglobulins may attach nonspecifically to the RBC membrane and cause positive antiglobulin reactions. These do not cause increased RBC destruction and are of importance only because of the need to differentiate them from those of clinical significance. This is most commonly seen in patients on cephalosporins, which produce a positive DAT in ∼3% of patients.239,240 Other drugs that have been associated with this mechanism include cefotetan, cisplatin, diglycoaldehyde, oxaliplatin, suramin, and the b-lactamase inhibitors clavulanate, sulbactam, and tazobactam.211 Many different RBC-bound proteins have been detected within a few days of instituting the medication, including fibrinogen, albumin, complement, immunoglobulins, and a2-macroglobulin.240 Clinical distinction between this benign finding and other, potentially significant ones involves the demonstration of a nonreactive eluate with cephalosporin-treated RBCs and the absence or low titer of antidrug antibodies in the serum.31 In the absence of hemolysis, a positive DAT is not a cause for discontinuing the medication.

Multiple Mechanisms—Unifying Hypothesis Many medications have been implicated in producing hemolysis by more than one mechanism, sometimes simultaneously in the

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Chapter 29  Autoimmune Hemolytic Anemia

same patient. Arndt and Garratty have referred to a unifying hypothesis in their review.211 The drug or its metabolites interact with the constituents of the RBC membrane, resulting in production of different populations of antibodies. Of these antibodies, some react with drug epitopes alone (drug adsorption). Other antibodies may react with drug and membrane components (neoantigen mechanism), and others with RBC membrane components (autoantibody mimickers). See Figure 29.6.

Clinical Manifestations The clinical features of DI-IHA are similar to those found in idiopathic AIHA, including pallor, jaundice, and easy fatigability. Splenomegaly is not uncommon, but lymphadenopathy and hepatomegaly should not be attributed to drug-related hemolysis.241 The severity of these symptoms depends on the rate of hemolysis, which is, in part, dependent on the mechanism involved. Those patients with the neoantigen mechanism are at the greatest risk for plummeting hemoglobins, hemoglobinuria, and renal f­ailure.2,232,242 Cefotetan has been implicated in many severe hemolytic reactions.243–245 Fatal reactions are rare but may occur.224,246 Cefotetan, ceftriaxone, and fludarabine have been associated with fatalities.211,247 The drug adsorption and autoimmune varieties are typically characterized by insidious onset of hemolysis over days to weeks. A careful medication history is necessary to evaluate the possibility of a culprit drug in all patients with AIHA.

Laboratory Features Just as with idiopathic AIHA, anemia with reticulocytosis and a positive DAT are hallmarks of the condition. Elevated indirect bilirubin and LDH are common findings. Rampant RBC destruction leads to hemoglobinemia, hemoglobinuria, and elevated creatinine levels. Distinguishing the mechanisms involved can be accomplished by the serologic results. One can differentiate the neoantigen mechanism from cold autoantibodies by the absence of high-titer cold agglutinins or D–L antibodies in the druginduced cases. Drug-induced antibodies also do not react in the absence of the drug or a metabolite in IATs. Only a careful history and resolution of the hemolysis after discontinuation of the drug can separate the a-methyldopa variety from true autoimmune antibodies. Antibodies to cefotetan react to very high titers against drug-treated RBCs and at lower titers with untreated RBCs with or without drug present. Antibodies to ceftriaxone are the immune

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complex type.211 The positive DAT may persist for a few weeks to months after stopping the medication responsible, especially with the autoimmune mechanism.31

Management A careful drug history, including over-the-counter medications, nutritional or dietary supplements, and illicit drugs, is imperative. Discontinuing the implicated medication is usually all that is necessary in management of drug-induced hemolytic anemia. Problems may arise when a positive DAT occurs and uncertainty exists as to whether significant RBC destruction is occurring. As previously described, many medications may be associated with a positive DAT test and yet not cause hemolysis. The drug need not be stopped in these patients. In cases of brisk hemolysis associated with the neoantigen mechanism, stopping the offending agent can be life-saving. Although the helpfulness of prednisone therapy is questionable,248 it has been used with some success in fludarabine-associated DI-IHA.247 Transfusion can be accomplished, usually without difficulty in cross-matching, as the antibodies in the drug adsorption and neoantigen mechanisms are drug-dependent. However, patients with the autoimmune mechanism may encounter the same difficulties as previously discussed in the section on warm AIHA. It is important to keep in mind that transfused RBCs may be destroyed at the same rate as endogenous RBCs if drug or active metabolites are still circulating. Prognosis is typically excellent for these patients after discontinuation of the drug. With the variety of choices of pharmaceuticals available, alternative therapies are nearly always accessible to treat the underlying condition adequately.

Transplant-associated Immune Hemolytic Anemias

Disorders of Red Cells



Although it is not technically an autoimmune hemolytic process, the immune hemolysis associated with transplantation is analogous in many ways. In both cases, antibodies are generated against endogenous self-antigens; the key distinction lies in the source of the antibody-producing lymphocytes. That source can be either the lymphocytes and their precursors contained in a stem cell product being utilized for hematopoietic reconstitution, or they can be lymphocytes that are merely passengers contained in the vascular and perivascular regions of a solid organ being transplanted.249 See Table 29.12.

Hematopoietic Stem Cell Transplants Antibody to drug Antibody to (mainly) membrane components

Drug

Antibody to drug and membrane components

Red cell membrane FIGURE 29.6. Proposed unifying hypothesis for drug-induced antibody reactions.  The thicker darker lines represent antigen-binding sites on the Fab region of the drug-induced antibody. Drugs (haptens) bind loosely (or firmly) to cell membranes, and antibodies can be made to (a) the drug (producing in vitro reactions typical of a drug adsorption [penicillin-type] reaction); (b) membrane components, or mainly membrane components (producing in vitro reactions typical of autoantibody); or (c) part-drug, part-membrane components (producing an in vitro reaction typical of the so-called immune complex mechanism). (From Garratty G. Target antigens for red-cell bound autoantibodies. In: Nance SJ, ed. Clinical and basic science aspects of immunohematology. Arlington, VA: American Association of Blood Banks, 1991:33–72.)

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Hematopoietic stem cell transplants can be ABO-compatible or ABO-mismatched. The mismatch can include major, minor, or both major and minor ABO incompatibilities.250 ABO-compatible stem cell transplants have the same ABO type for the donor and recipient. A major ABO mismatch implies the introduction of a foreign ABO antigen, as would be seen with a group O recipient of a group A, B, or AB donor stem cell product. A minor ABO mismatch implies the introduction of a foreign ABO antibody(isohemagglutinin), as would be seen with a group A, B, or AB recipient of a group O donor stem cell product. Both major and minor incompatibilities would be seen with a group A recipient of a group B donor product or a group B recipient of a group A donor product. Appropriate selection of RBC- and plasma-containing blood products can help minimize the complications of passive antibody transfer.250 The acute impact of a minor incompatible stem cell transplant is ameliorated with a simple washing of the donor product to prevent passive transfer of pre-existing antibodies. However, when there is a minor incompatibility, the novel hematopoietic stem cells will eventually produce lymphocytes that generate antibodies against the recipient’s original remaining RBCs. This immune hemolysis

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TA B L E 29.12



TIMING OF POST-TRANSPLANT IMMUNE HEMOLYTIC ANEMIA Immediate

Days-Weeks-Months

Hemolysis of original RBCs due to ABO antibodies in the donor stem cell product (minor ABO group mismatch) Passive transfer of ABO antibodies: infusion of plasma, platelets, IVIG, intravenous anti-D, and antilymphocyte globulin Hemolysis of donor RBCs contained in stem cell product by original preexisting ABO antibodies (major ABO group mismatch)

Passenger lymphocyte syndrome

Autoimmune hemolytic anemia

Passive transfer of ABO antibodies: infusion of plasma, platelets, IVIG, intravenous anti-D, and antilymphocyte globulin Hemolysis of donor RBCs produced by the newly engrafted marrow caused by residual ABO antibodies (major ABO group mismatch) Alloantibodies produced by residual cells of the original immune system Alloantibodies produced by engrafted cells of the donor’s immune system (weeks-months) Autoimmune hemolytic anemia

IVIG, Intravenous immunoglobulin; RBC, red blood cell.

may begin 7 to 10 days post-transplant and can be abrupt and severe. As the patient’s original—but now incompatible—RBCs are destroyed, they are replaced by a combination of transfused cells and post-engraftment novel donor-type RBCs. Consequently, the immune-mediated RBC destruction is limited by the residual original-type RBCs remaining.

Solid Organ Transplants and the Passenger Lymphocyte Syndrome The immune hemolysis associated with solid organ transplantation is typically due to a passenger lymphocyte syndrome. This situation can be seen with just about any solid organ being transplanted, as long as the donor and recipient share an RBC incompatibility. The classic example involves a group O organ transplanted into a group A recipient. Donor lymphocytes that are merely passengers in the transplanted organ react to recipient (endogenous) RBCs and generate antirecipient RBC antibodies. The consequent hemolytic anemia is more frequent with transplanted organs containing significant lymphoid mass. The incidence is lowest in kidney transplant recipients (9% to 17%), intermediate among liver transplant recipients (29% to 40%), and highest in heart–lung transplant recipients (70%).250,251–253 Extensive perfusion of transplanted organs does not necessarily prevent this passenger lymphocyte syndrome, implying extravascular lymphocyte sequestration. Unlike the hemolysis associated with minor ABOincompatible hematopoietic stem cell transplants, the target RBCs are not replaced by transfusion and a novel engrafted marrow. Consequently, the immune-mediated RBC destruction is limited only by the survival of the passenger lymphocytes.

selected References The full reference list for this chapter can be found in the online version.

1. Sokol RG, Hewitt S, Stamps BK. Autoimmune haemolysis. Mixed warm and cold antibody type. Acta Haematol 1983;69:266–274. 2. Worlledge SM. Immune drug-induced hemolytic anemias. Semin Hematol 1969;6:181–200. 3. De Angelis, De Matteis MC, Cozzi MR, et al. Abnormalities of membrane protein composition in patients with autoimmune hemolytic anemia. Br J Haematol 1996;95:273–277.

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5. Shlomchik MJ. Mechanisms of immune self-tolerance and how they fail in autoimmune disease. In: Silberstein L, ed. Autoimmune disorders of blood. Bethesda, MD: American Association of Blood Banks, 1996:1–34. 7. Semple JW, Freedman J. Autoimmune pathogenesis and autoimmune hemolytic anemia. Semin Hematol 2005;42:122–130. 8. Hall AM, Ward FJ, Vickers MA, et al. Interleukin mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen. Blood 2002;100:4529–4536. 9. Worth GR, Jones BA, Schreiber AD. Fc receptor structure/function and role in immune complex-mediated autoimmune disease. Hematology (Am Soc Hematol Educ Prog) 2004;54–58. 10. Brown EJ. Complement receptors, adhesion, and phagocytosis. Infect Agents Dis 1992;2:63–70. 12. Ross GD, Medof ME. Membrane complement receptors specific for bound fragments of C3. Adv Immunol 1985;37:217–267. 14. Lachmann PJ, Voak D, Oldroyd RG, et al. Use of monoclonal anti-C3 antibodies to characterize the fragments of C3 that are found on erythrocytes. Vox Sang 1983;45:367–372. 15. Logue G, Rosse WF. Immunologic mechanisms in autoimmune hemolytic disease. Semin Hematol 1976;13:277–289. 16. Frank MM, Schreiber AD, Atkinson JP. NIH conference.Pathophysiology of immune hemolytic anemia. Ann Intern Med 1977;87:210–222. 17. Unkless JC. Function and heterogeneity of human Fc receptors for immunoglobulin G. J Clin Invest 1989;83:355–361. 19. Anderson DR, Kelton JG. Mechanisms of intravascular and extravascular cell destruction. In: Nance SJ, ed. Immune destruction of red cells. Arlington, VA: American Association of Blood Banks, 1989:1–52. 20. van Oss CJ, Absolom DR. Hemagglutination and the closest distance approach of normal, neuraminidase, and papain-treated erythrocytes. Vox Sang 1983;47:250–256. 21. Leikola J, Pasanen VJ. Influence of antigen receptor density on agglutination of red blood cells. Int Arch Allergy Appl Immunol 1970;39:352–360. 22. Fukuda M, Fukuda MV, Hakomori S-I. Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane. J Biol Chem 1979;254:3700–3703. 23. Economidou J, Hughes-Jones NC, Gardner B.The functional activities of IgG and IgM anti-A and anti-B. Immunology 1967;13:227–234. 25. Moreschi C. Neue Tatsachen über die Blut Körpenchenagglutination. Zentralbl Bakt 1908;46:49–51. 26. Coombs RRA, Mourant AE, Race RR. A new test for the detection of weak and “incomplete” Rh agglutinins. Br J Exp Pathol 1945;26:255–266. 27. Coombs RRA, Mourant AE, Race RR. In vivo isosensitization of red cells in babies with haemolytic disease. Lancet 1946;1:264–266. 31. Petz LD, Garratty G. Acquired immune hemolytic anemias. New York: Churchill Livingstone, 1980. 33. Garratty G. Novel mechanisms for immune destruction of circulating autologous cells. In: Silberstein L, ed. Autoimmune disorders of blood. Bethesda, MD: American Association of Blood Banks, 1996:79–114. 34. Burkhart P, Rosenfield RE, Hsu TC. Instrumental PVP-augmented antiglobulin tests I. Detection of allogeneic antibodies coating otherwise normal erythrocytes. Vox Sang 1974;26:289–304. 35. Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol 2005;42:156–164. 36. Issitt PD, Pavone BG, Goldfinger D, et al. Anti Wrb and other autoantibodies responsible for positive direct antiglobulin tests in 150 individuals. Br J Haematol 1976;34:5–18. 40. Sokol RJ, Hewitt S, Booker DJ, et al. Patients with red cell autoantibodies: selection of blood for transfusion. Clin Lab Haematol 1988;10:257–264. 41. Donath J, Landsteiner K. Ueber Paroxysmal Hämoglobinurie. Münch Med Wschr 1904;51:1590–1593. 42. Landsteiner K. Uber Besiehungen zwischen dem Blutserum and den Kürper Kürperzeller. Munch Med Wochenschr 1903;50:1812. 43. Schubothe H. The cold hemagglutinin disease. Semin Hematol 1966;3:27–47. 44. Dacie J. The auto-immune haemolytic anaemias, 3rd ed. Edinburgh: Churchill Livingstone, 1992. 46. Pruzanski W, Shumak KH. Biologic activity of cold reactive autoantibodies. N Engl J Med 1977;297:538–542. 48. Williams R, Kunkel H, Capra J. Antigenic specificities related to the cold agglutinin activity of gamma M globulins. Science 1968;161:379. 51. Pascual V, Victor K, Spellerberg M, et al. VH restriction among human cold agglutinins. The VH4–21 gene segment is required to encode anti-I and anti-i specificities. J Immunol 1992;149:2337–2344. 52. Michaux L, Dierlamm J, Wlodarska L, et al. Trisomy 3q11-q29 is recurrently observed in B-cell non-Hodgkin’s lymphomas associated with cold agglutinin syndrome. Ann Hematol 1998;76:201–204. 55. Roelcke D. Cold agglutination. Transfus Med Rev 1989;3:140–166. 61. Dacie JV, Worlledge SM. Autoimmune hemolytic anemias. In: Brown EB, Moore CV, eds. Progress in hematology VI. New York: Grune & Stratton, 1969:82–120. 67. Gronemeyer P, Chaplin H, Ghazarian V, et al. Hemolytic anemia complicating infectious mononucleosis due to the interaction of an IgG cold anti-i and an IgM cold rheumatoid factor. Transfusion 1981;21:715–718. 68. Horwitz CA, Skradski K, Reece E, et al. Haemolytic anaemia in previously healthy adult patients with CMV infections: report of two cases and an evaluation of subclinical haemolysis in CMV mononucleosis. Scand J Haematol 1984;33:35–42. 69. Evans RS, Turner E, Bingham M. Studies with radioiodinated cold agglutinins of ten patients. Am J Med 1965;38:378. 70. Rosse WF, Adams JP. The variability of hemolysis in the cold agglutinin ­syndrome. Blood 1980;56:409–416.

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74. Harboe M, Torvisk H. Protein abnormalities in the cold haemagglutinin ­syndrome. Scand J Haematol 1969;6:416–426. 78. Berentsen S, Ulvestad E, Gjertsen BT, et al. Rituximab for primary chronic cold agglutinin disease:a prospective study of 37 courses of therapy in 27 patients. Blood. 2004;103(8):2925–2928. 79. Schollkopf C, Kjeldsen L, Bjerrum OW, et al. Rituximab in chronic cold agglutinin disease: a prospective study of 20 patients. Leuk Lymphoma. 2006;47(2):253–260. 82. Rodenhuis S, Maas A, Hazenberg CA, et al. Inefficacy of plasma exchange in cold agglutinin hemolytic anemia—a case study. Vox Sang 1985;49:20–25. 84. Zoppi M, Oppliger R, Althaus U, et al. Reduction of plasma cold agglutinin titers by means of plasmapheresis to prepare a patient for coronary bypass surgery. Infusionstherapie Transfusionsmedizin 1993;20:19–22. 86. Agarwal SK, Ghosh PK, Gupta D. Cardiac surgery and cold-reactive proteins. Ann Thorac Surg 1995;60:1143–1150. 87. Siami FS, Siami GA.A last resort modality using cryofiltration apheresis for the treatment of cold hemagglutinin disease in a Veterans Administration hospital. Ther Apher Dial 2004;8:398–403. 89. Woll JE, Smith CM, Nusbacher J. Treatment of acute cold agglutinin hemolytic anemia with transfusion of adult i RBCs. JAMA 1974;229:1779–1780. 92. Schreiber AD, Herskovitz BS, Goldwein M. Low-titer cold-hemagglutinin ­disease. N Engl J Med 1977;296:1490–1494. 93. Hillmen P, Hall C, Marsh JC, et al. Effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med 2004;350:552–559. 95. Göttsche B, Salama A, Mueller-Eckhardt C. Donath-Landsteiner autoimmune hemolytic anemia in children. A study of 22 cases. Vox Sang 1990;58:281–286. 97. Hinz CB Jr, Picken ME, Lepow IH. Studies on immune human hemolysis. I. The kinetics of the Donath-Landsteiner reaction and the requirement for complement in the reaction. J Exp Med 1961;113:177–192. 98. Hinz CF Jr. Serologic and physicochemical characterization of DonathLandsteiner antibodies from six patients. Blood 1963;22:600–605. 100. Levine P, Celano MJ, Falkowski F. The specificity of the antibody in paroxysmal cold hemoglobinuria. Transfusion 1963;3:278–280. 102. van Loghem JJ, van der Hart M, Dorfmeier H. Serological studies in acquired hemolytic anemia. In: Sixth International Congress of the International Society of Hematology, 1958. New York: Grune & Stratton, 1958. 103. Sokol RJ, Hewitt S, Stamps BK. Autoimmune haemolysis associated with Donath-Landsteiner antibodies. Acta Haematol (Basel) 1982;68:268–277. 105. Hunt JH. The Raynaud phenomena: a critical review. Q J Med 1936;5:399–444. 107. Bunch DF, Schwarz DM, Bird GWG. Paroxysmal cold haemoglobinuria following measles immunization. Arch Dis Child 1972;47:299–300. 110. Sokol RJ, Hewitt S, Stamps BK. Autoimmune haemolysis in childhood and adolescence. Acta Haematol (Basel) 1984;72:245–257. 113. Rausen AR, LeVine R, Hsu TCS, et al. Compatible transfusion therapy for paroxysmal cold hemoglobinuria. Pediatrics 1975;55:275–278. 116. Shulman IA, Branch DR, Nelson JM, et al. Autoimmune hemolytic anemia with both cold and warm autoantibodies. JAMA 1985;253:1746–1748. 118. Sokol R, Hewitt S, Stamps BK. Autoimmune haemolysis: an 18 year study of 865 cases referred to a regional transfusion centre. Br Med J Clin Res Educ 1981;282:2023–2027. 124. Dacie J. Secondary or symptomatic haemolytic anaemias, 3rd ed. Edinburgh: Churchill Livingstone, 1995. 126. Ravetch J, Kinet J-P. Fc receptors. Annu Rev Immunol 1991;9:457–492. 129. Sokol RJ, Hewitt S, Stamps B. Erythrocyte autoantibodies, autoimmune haemolysis and pregnancy. Vox Sang 1982;43:169–176. 130. Chaplin H Jr, Cohen R, Bloomberg G, et al. Pregnancy and idiopathic autoimmune haemolytic anaemia: a prospective study during 6 months gestation and 3 months post-partum. Br J Haematol 1973;24:219–229. 131. Starksen NF, Bell WE, Kickler TS. Unexplained hemolytic anemia associated with pregnancy. Am J Obstet Gynecol 1983;146:617–622. 134. Downes KA, Domen RE, McCarron KF, et al. Acute autoimmune hemolytic anemia following DTP vaccination: report of a fatal case and review of the literature. Clin Pediatr 2001;40:355–358. 136. Evans RS, Duane RT. Acquired hemolytic anaemia. I. The relation of antibody activity to activity of the disease. II. The significance of thrombocytopenia and leukopenia. Blood 1949;4:1196–1213. 137. Evans RS, Takahashi K, Duane RT, et al. Primary thrombocytopenic purpura and acquired hemolytic anaemia. Evidence for a common etiology. Arch Intern Med 1951;87:48–65.

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140. Scaradavou A, Bussel J. Evans syndrome results of a pilot study utilizing a multiagent treatment protocol. J Pediatr Hematol Oncol 1995;17:290–295. 142. Liesveld JL, Rowe JM, Lichtman MA. Variability of the erythropoietic response in autoimmune hemolytic anemia: analysis of 189 cases. Blood 1987;69:820–826. 144. Hauke G, Fauser AA, Weber S, et al. Reticulocytopenia in severe autoimmune hemolytic anemia (AIHA) of the warm antibody type. Blut 1983;46:321–327. 150. Payne R, Spaet TH, Aggeler PM. An unusual antibody pattern in a case of idiopathic acquired hemolytic anemia. J Lab Clin Med 1955;46:245–254. 153. Chaplin H Jr. Clinical usefulness of specific antiglobulin reagents in autoimmune hemolytic anemias. Prog Hematol 1973;7:25–49. 154. Araguás C, Martí-n-Vega C, Massagué I, et al. “Complete” warm hemolysins producing an autoimmune hemolytic anemia. Vox Sang 1990;59:125–126. 158. Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia. Am J Hematol 2002;69:258–271. 159. Petz LD. Treatment of autoimmune hemolytic anemias. Curr Opin Hematol 2001;8:411–416. 167. Allgood JW, Chaplin H. Idiopathic acquired autoimmune hemolytic anemia: a review of forty-seven cases treated from 1955 through 1965. Am J Med 1967;43:254–273. 168. Zupanska B, Sylwestrowicz T, Pawelsi S. The results of prolonged treatment of autoimmune hemolytic anemia. Hematologia 1971;4:425–433. 174. Ahrens N, Kingreen D, Seltsam A, et al. Treatment of refractory autoimmune haemolytic anaemia with anti-CD20 (rituximab). Br J Haematol 2001;114:244–245. 178. Zecca M, Nobili B, Ramenghi U, et al. Rituximab for the treatment of refractory autoimmune hemolytic anemia in children. Blood 2003;101:3857–3861. 181. Lechner K, Jager U.How I treat autoimmune hemolytic anemias in adults Blood. 2010;116(11):1831–1838 182. Bussone G, Ribeiro E, Dechartres A, et al. Safety of rituximab in adults with warm antibody autoimmune haemolytic anemia: retrospective analysis of 27 cases. Am J Hematol 2009;84(3):153–157. 192. Garratty G, Petz LD. Approaches to selecting blood for transfusion to patients with autoimmune hemolytic anemia. Transfusion 2002;42(11):1390. 198. Vandenberghe P, Zachee P, Verstraete S, et al. Successful control of refractory and life-threatening autoimmune hemolytic anemia with intravenous immunoglobulins in a man with the primary antiphospholipid syndrome. Ann Hematol 1996;73:253–256. 199. Bayry J, Lacroix-Desmazes S, Carbonneil C, et al. Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin. Blood 2003;101:758–765. 203. Jaweed M, Nifong TP, Domen RE, et al. Durable response to combination therapy including staphylococcal protein A immunoadsorption in life threatening refractory autoimmune hemolysis. Transfusion 2002;42:1217–1220. 206. Norton A, Roberts I. Management of Evans syndrome. Br J Haematol 2006;132:125–137. 208. Cheung WW, Hwang GY Alemtuzumab induced complete remission of autoimmune hemolytic anemia refractory to corticosteroids, splenectomy and rituximab. Haematologica 2006 May;91(Suppl 5):ECR13. 209. Hoffmann P. Immune hemolytic anemia—selected topics. Hematology (Am Soc Hematol Educ Prog) 2006;13–18. 210. Petz LD, Garratty G. Immune hemolytic anemias, 2nd ed. Philadelphia, PA: Churchill Livingstone, 2004. 211. Arndt PA, Garratty G. The changing spectrum of drug-induced hemolytic anemia. Semin Hematol 2005;42:137–144. 213. Levine BB, Redmond A. Immunochemical mechanisms of penicillin induced Coombs positivity and hemolytic anemia in man. Int Arch Allergy Appl Immunol 1967;31:594–606. 217. Swanson MA, Chanmougan D, Schwartz RS.Immunohemolytic anemia due to anti-penicillin antibodies. N Engl J Med 1966;274:178–181. 236. Bakemeier RF, Leddy JP. Erythrocyte autoantibody associated with alpha methyldopa: heterogeneity of structure and specificity. Blood 1968;32:1–14. 238. Garratty G, Nance SJ. Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry. Transfusion 1990;30:617–631. 247. Borthakur G, O’Brien S, Wierda WG, et al. Immune anemias in patients with chronic lymphocytic leukaemia treated with fludarabine, cyclophosphamide and rituximab–incidence and predictors. Br J Haematol 2007;136(6): 800–805. 250. Friedberg RC. Transfusion therapy in the patient undergoing hematopoietic stem cell transplantation. Hematol Oncol Clin North Am 1994;8:1105–1116.

Disorders of Red Cells



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Chapter 30

Hemolytic Disease of the Fetus and Newborn Charles T. Quinn, Anne F. Eder, Catherine S. Manno

Hemolytic disease of the fetus and newborn (HDFN) results from the destruction of red blood cells by maternal immunoglobulin (Ig) G antibodies that gain access to the fetal circulation during pregnancy. These antibodies may be directed against Rhesus (Rh) or other blood group antigens on fetal red blood cells, inherited from the father and not present on the mother’s red blood cells. HDFN associated with blood group antibodies has a broad spectrum of effects, ranging from mild anemia and hyperbilirubinemia in an infant to life-threatening complications before birth.1 Clinical manifestations of severe hemolytic disease before birth include profound fetal anemia, hepatosplenomegaly, generalized edema, massive ascites, and congestive heart failure. The accelerated destruction of fetal red blood cells elicits extramedullary hematopoiesis and release of nucleated and other immature red blood cells into the peripheral circulation (erythroblastosis fetalis). Ongoing hemolysis of red blood cells after birth results in neonatal hyperbilirubinemia that may cause kernicterus and permanent neurologic injury or death.

Historical Background In the 20th century, HDFN was recognized as a distinct clinical entity, its pathogenesis was delineated, and an effective strategy for preventing its most common form was introduced. Case reports in the early 1900s (and before) described edematous (hydropic) stillborns and anemic infants with marked jaundice who died within days of birth. Not until 1932 were these phenomena realized to be the same hematologic disease process when Diamond et al. described the interrelationship of neonatal anemia, jaundice, and edema as symptoms that occur in varying degrees and combinations in erythroblastosis fetalis.2 The precise etiology of the disease remained obscure until seminal observations on blood group antigens and incompatibility in pregnancy were made in the 1940s. Landsteiner and Weiner first used immune sera raised in rabbits against red blood cells from Rhesus monkeys to agglutinate human red blood cells, thus discovering the Rhesus factor.3 This antigen, now known as the D antigen of the Rhesus blood group in humans, is present exclusively on red blood cells of Rh-positive individuals. In the year before this discovery, Levine and Stetson recognized that a woman could become immunized against paternally inherited red blood cell determinants of the fetus during pregnancy.4 Using Landsteiner’s anti-Rh antisera to investigate cases of erythroblastosis fetalis, Levine et al. subsequently demonstrated that 90% of the mothers were Rh negative, and all the fathers and infants were Rh positive.5 This statistical association as well as the presence of Rh agglutinins in the blood of mothers with affected infants supported their theory that alloimmunization and transplacental passage of these antibodies caused destruction of fetal red blood cells.5 The inciting stimulus for red blood cell alloimmunization in pregnancy, the passage of fetal red blood cells into the maternal circulation or fetal–maternal hemorrhage (FMH), was directly demonstrated by Chown in 1954.6 Methods to monitor alloimmunized pregnancies and to treat affected fetuses and infants were first introduced in the late 1940s. Neonatal exchange transfusion enabled simultaneous correction of the anemia and reduction of bilirubin concentration in affected infants.1,7 In 1961, Liley described the relationship between the concentration of bilirubin in amniotic fluid and the degree of destruction of fetal red blood cells, providing a tool to assess the severity of intrauterine hemolytic disease.8 Two years later, Liley

introduced the technique of intraperitoneal transfusion of anemic fetuses, which was used for more than 20 years before it was largely supplanted with intravascular transfusion techniques.9 Primary prevention of D alloimmunization became possible with the advent of anti-D immune prophylaxis. The ability of passively transferred antibodies to effectively block active immunization to foreign antigens was first demonstrated by Von Dungern in 1900.1 Experimental studies in the 1960s applied this approach to D alloimmunization, revealing that Rh-negative men could be protected if administered anti-D immune globulin (RhIG) before transfusion with Rh-positive red blood cells.10,11 Between 1963 and 1968, clinical trials involving Rh-negative pregnant women demonstrated that administration of RhIG within 72 hours of delivery was successful in reducing the incidence of D alloimmunization from 7% to 15% to 1% to 2%.12,13,14,15,16,17 Metaanalysis of clinical trials of postpartum RhIG administration confirms a >90% reduction in the alloimmunization rate among treated women compared to untreated women (Table 30.1).17 The recognition that FMH occurring primarily in the third trimester contributed to residual risk of alloimmunization during pregnancy led to the clinical observation that additional, antenatal RhIG prophylaxis could further reduce the risk of D alloimmunization to 0.5 mg/dl/hour), and the presence of comorbid factors such as hemolysis, asphyxia, significant lethargy, temperature instability, sepsis, or acidosis. For infants at 35 weeks’ or more gestation, exchange transfusion is recommended when the total serum bilirubin rises to threshold levels despite intensive phototherapy, based on the gestational age and the presence of risk factors (Fig. 30.8).126 It is also a clinical option to use the bilirubin-to-albumin ratio together with, but not in lieu of, the total serum bilirubin level as an additional factor in determining the need for exchange transfusion.126 Immediate exchange transfusion is recommended if infants show signs of acute bilirubin encephalopathy (e.g., hypertonia, arching, fever, high-pitched cry) or if total serum bilirubin is 25 mg/dl (428 mmol/L) or higher.126 If exchange transfusion is indicated, a transfusion volume approximately twice the infant’s total blood volume (85 ml/ kg × 2 for a term infant; 100 ml/kg × 2 for a pre-term infant) is administered incrementally while removing aliquots of the infant’s blood over a period of 1 to 2 hours. The procedure either involves a push–pull method with a single vascular access or an isovolemic method that requires two catheters to allow for simultaneous withdrawal and infusion.1,125 The umbilical artery is usually used for withdrawing blood, and the umbilical vein is usually used for infusion. The isovolemic method may be preferable because mean arterial pressure and cerebral blood volume may be more stable than during the single-catheter, push–pull method.125 An exchange of approximately two blood volumes removes approximately 85% of red blood cells but only approximately 45% of plasma bilirubin because the latter re-equilibrates between intravascular and extravascular spaces. Consequently,

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infants with aggressive hemolytic disease may require more than one exchange transfusion before an acceptable bilirubin concentration is achieved. Red blood cell units chosen for neonatal exchange transfusion should be O, Rh negative, or ABO/Rh type specific; should lack the blood group antigen implicated in hemolytic disease (e.g., ABO hemolytic disease requires the use of type O red blood cells); and should be compatible with maternal serum. Maternal serum or plasma is usually used in crossmatching red blood cells for infants because it contains the implicated antibody in high concentration and is available in large quantity. If a maternal sample is not available, the infant’s plasma or serum can be used for compatibility testing; however, the concentration of circulating antibody may be low if most is bound to the infant’s red blood cells. In this case, the bound antibody can be eluted from the surface of the infant’s red blood cells, and the resulting eluate can be used for crossmatching. Rarely, HDN is due to an antibody to a high-­incidence blood group antigen present in almost all persons, and no compatible units can be identified.41 In this case, maternal blood can be collected and washed to remove incompatible plasma for the infant. Any blood component collected from biologic relatives must be irradiated as a precaution against transfusion-associated graft-versus-host disease, as well as blood components transfused to infants who received intrauterine transfusion or who are otherwise immunocompromised due to premature birth or severe primary immune deficiency. Because of the difficulty in identifying all high-risk newborn infants, many transfusion services provide g-irradiated blood for all infants until the age of 4, 6, or 12 months.129 Another common standard of practice is the use of either leukocyte-reduced cellular components or units selected from CMV-seronegative donors to reduce the risk of CMV transmission to newborn infants.129 Most blood banks also transfuse only units that lack hemoglobin S to infants to avoid the potential for hypoxia-induced sickling in a critically ill infant and fresh (1% to 3% of the red cells can be identified by standard flow cytometry. Concern that recent red cell transfusion might result in a false-negative result seems unfounded. Because the assay is very sensitive and because the proportion of GPI-AP–deficient cells is greater in the reticulocyte population than in the peripheral blood as a

whole, massive transfusion that both replaces essentially all of the patient’s blood volume and also completely suppresses hematopoiesis would be required to produce a false-negative result. Transfusion will have an impact on the percentage of GPI-AP–­ deficient cells that are observed, but the possibility that the diagnosis would be obscured is remote. Conversely, it is unlikely that a recent hemolytic episode would result in a false-negative result because all the abnormal cells are destroyed. However, when documenting the proportion of affected cells and determining the precise erythrocyte phenotype, the analysis is best done when the patient has not been recently transfused, as well as when the patient is not experiencing a hemolytic crisis related to infection or some other cause. By careful gating and by using triple-antibody staining techniques, the sensitivity of flow cytometry can be enhanced by about three orders of magnitude, such that as few as 0.003% GPI-AP–deficient cells (RBCs and WBCs) can be consistently and reproducibly detected71 (Fig. 31.7). This high-resolution analysis is used to identify patients with PNH-sc (Fig. 31.7B). Patients with PNH-sc in the setting of aplastic anemia and the refractory anemia variant of MDS appear to have a more benign clinical course than patients without PNH-sc.71,105,106 In addition, some71,105,106 but

Figure 31.7. High sensitivity flow cytometry for diagnosis of PNH.  A: Two-color flow cytometry histogram of erythrocytes (upper panels) and neutrophils (lower panels) from a normal volunteer (left) and from a patient with classic PNH (right). Erythrocytes were stained with phycoerythrin (PE)-labeled anti-glycophorin A (vertical axis) and a combination of fluorescein isothiocyanate (FITC)-labeled anti-CD55 and anti-CD59 (horizontal axis). Neutrophils were stained with PE-labeled CD11b (vertical axis) and FITC-labeled anti-CD55 and anti-CD59 (horizontal axis). No GPI-AP–deficient erythrocytes or neutrophils were among ∼100,000 cells counted in this analysis for the normal control, whereas the patient had 72% GPI-AP–deficient erythrocytes and 96% GPI-AP–deficient neutrophils. B: Two-color flow cytometry histogram of erythrocytes and neutrophils from two patients with aplastic anemia but with no clinical evidence of PNH. In the example on the left, approximately 0.077% of the erythrocytes and 0.74% of the neutrophils failed to express CD55 and CD59. In the example on the right, 3% of the erythrocytes and 21% of the neutrophils failed to express CD55 and CD59.

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Chapter 31  Paroxysmal Nocturnal Hemoglobinuria

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Disorders of Red Cells



Figure 31.7. (continued)

not all107 studies suggest that patients with PNH-sc respond more favorably to immunosuppressive therapy. A diagnostic assay for PNH using fluorescent aerolysin (FLAER) that exploits the unique properties of the bacterial toxin aerolysin has been developed.108 This channel-forming protein binds directly to the GPI anchor. By fluorochrome labeling of a modified recombinant form of the protein that does not cause lysis, this reagent can be used to detect leukocytes with the PNH phenotype.108 The primary advantage of this assay is that because it detects all GPI-APs, it is specific for PNH. The primary disadvantage is that the FLAER reagent does not bind well to RBCs. Thus FLAER cannot be used to characterize GPI-AP expression on erythrocytes. Analysis of expression of GPI-AP on erythrocytes is a highly specific test for PNH. There is no other known disease in which the erythrocytes include a mosaic of both GPI-AP+ and GPI-AP− cells. Subjects with isolated deficiency of either DAF (the Inab phenotype) or MIRL (CD59) will be identified by this method (assuming that anti-DAF and anti-MIRL antibodies are used). Such patients are extremely rare, however, and their flow cytometry histograms are readily distinguishable from patients with PNH because 100% of the cells are abnormal and expression of only one GPI-AP is deficient. Patients with inherited abnormalities of GPI-AP synthesis would be distinguished from patients with PNH because in the former case, mosaicism would not be observed.

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Differential Diagnosis The diagnosis of PNH must be considered in any patient who has the following: (1) signs and symptoms of intravascular hemolysis (manifested by an abnormally high LDH) of undefined cause (i.e., Coombs-negative) with or without macroscopic hemoglobinuria often accompanied by iron deficiency; (2) pancytopenia in association with hemolysis; (3) venous thrombosis affecting unusual sites, especially intra-abdominal, cerebral, or dermal locations accompanied by evidence of hemolysis; (4) unexplained recurrent bouts of abdominal pain, low backache, or headache in the presence of chronic hemolysis; and (5) Budd-Chiari syndrome. It is important to document evidence of hemolysis before proceeding with tests more specific for clinical PNH. As discussed above, a history of gross hemoglobinuria (nocturnal or otherwise) is not part of the initial clinical presentation in approximately three-fourths of patients with PNH (Table 31.3). However, except for patients with PNH-sc, laboratory evidence of hemolysis is a relatively constant feature of the disease. Quantitation of serum LDH is particularly informative because intravascular hemolysis results in markedly elevated values. If LDH concentrations are difficult to interpret because of other comorbid conditions (e.g., liver disease), then alternative evidence for chronic intravascular hemolysis should be sought (e.g., low serum haptoglobin, urine hemosiderin).

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Part iv  Disorders of Red Cells  •  SECTION 3  Hemolytic Anemia

Without evidence of hemolysis, more specific tests for clinical PNH are generally unwarranted. PNH must be differentiated from antibody-mediated hemolytic anemias, especially paroxysmal cold hemoglobinuria and the cold agglutinin syndrome, and from HEMPAS (Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum Lysis Test, or Congenital Dyserythropoietic Anemia type II). The mechanism that underlies the abnormal susceptibility of HEMPAS erythrocytes to acidified serum lysis is different from that of PNH.109By using flow cytometry, there is no difficulty distinguishing PNH from other hemolytic diseases because deficiency of GPI-APs affecting a portion of the erythrocytes is diagnostic of PNH. By definition, patients with PNH-sc have no clinical or laboratory evidence of hemolysis. PNH-sc is diagnosed by using highresolution flow cytometry. Patients with aplastic anemia and the refractory anemia variant of MDS should undergo screening for PNH-sc at diagnosis and yearly thereafter. Finding PNH-sc appears to have important prognostic and therapeutic implications, as patients with PNH-sc/aplastic anemia or PNH-sc/MDS-RA may have a more benign clinical course and a higher rate of response to immunosuppressive therapy than those without PNH-sc.71,105,106

Treatment The size of the PNH clone and the type and severity of the bone marrow failure component of the disease are the main factors that determine the clinical course. Some patients have a relatively benign clinical course with only a moderate degree of anemia and minimal hemolysis, and in such patients, no PNH-specific treatment is required. Other patients have severe anemia punctuated by hemolytic crises and thromboembolic complications; in such patients, treatment of the complement-mediated hemolytic anemia is clearly warranted. In other patients, the disease is dominated by bone marrow failure rather than by hemolysis, and in those patients, the focus of treatment should be on the underlying marrow failure process. By taking into account the size of the PNH clone and the type and severity of the bone marrow failure component of the disease, a classification has been developed that provides a rational basis for management.

Clinical Classification The basic approach to classifying PNH is straightforward. Flow cytometric analysis of peripheral blood erythrocytes and granulocytes (± monocytes) is needed to determine the phenotype of the

red cells and the size of the PNH clone (based on the percentage of GPI-AP–deficient granulocytes (± monocytes). CBC, reticulocyte count, serum concentration of LDH, bilirubin (fractionated), haptoglobin, and iron stores are needed to assess the degree of marrow failure and hemolysis and whether iron deficiency is present. Bone marrow aspirate and biopsy and cytogenetic analysis are needed to characterize the status of the bone marrow. Once the basic evaluation is complete, patients should be classified based on the categories developed by the International PNH Interest Group.1 The three categories are as follows (Figure 31.8): Classic PNH. Patients with classic PNH have clinical evidence of intravascular hemolysis (reticulocytosis, abnormally high concentration of serum LDH and indirect bilirubin, and abnormally low concentration of serum haptoglobin), but have no evidence of another defined bone marrow abnormality. A cellular marrow with erythroid hyperplasia and normal or near-normal morphology, but without nonrandom karyotypic abnormalities, is consistent with classic PNH. The PNH clone is large (>50% and often >90%). Patients with a large population of PNH II erythrocytes, however, will have minimal hemolysis. PNH in the setting of another specified bone marrow disorder. This subcategory of patients has at least laboratory evidence of hemolysis but also has concomitantly a defined underlying marrow abnormality. Bone marrow analysis and cytogenetics are used to determine if PNH arose in association with aplastic anemia or MDS. Standard criteria are used for diagnosis of the bone marrow abnormality (e.g., aplastic anemia, low-risk MDS). Finding nonrandom karyotypic abnormalities that are associated with a specific bone marrow abnormality may contribute diagnostically (e.g., abnormalities of chromosomes 5q, 7, and 20q are associated with MDS). The large majority of patients with PNH/ AA and PNH/MDS have relatively small PNH clones (36 to 36.5 g/dl can suggest hereditary spherocytosis, particularly if accompanied by an MCV below the reference range for ­gestational age.35,36 Reticulocytosis and normoblastosis reflect the accelerated nature of fetal erythropoiesis. Reticulocyte counts at birth are approximately 5%, with a range of 4% to 7%.2 Counts in preterm infants are slightly higher, averaging 6% to 10%. Reticulocytes remain elevated for the first 1 to 2 days of life, then drop abruptly to 0% to 1%. Nucleated red blood cells (NRBC) are seen regularly on blood smears during the first day of life. The reference ranges for NRBC, according to gestational age at birth, are shown in Figure 43.5.37 Elevations in NRBC in preterm infants correlate with adverse outcomes of intraventricular hemorrhage (IVH) and periventricular leukomalacia.37,38 Elevations in NRBC in term infants correlate with hypoxic ischemic encephalopathy and with adverse neurodevelopmental outcomes.37,38

Disorders of Red Cells



Figure 43.1. Reference ranges for blood hemoglobin concentration at birth. Values are shown from 24,416 subjects after 22 to 42 weeks gestation. The solid line shows the mean value and the dashed lines show the 5% and the 95% reference range. (From Jopling et al. Reference ranges for hematocrit and blood hemoglobin concentration during the neonatal period; data from a multihospital healthcare system. Pediatrics 2009;123;e333–e337.)

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Figure 43.2. Reference ranges for blood ­hemoglobin concentration of term and late preterm neonates during the first month after birth. The solid line shows the mean value and the dashed lines show the 5% and the 95% reference range. (From Jopling et al. Reference ranges for hematocrit and blood hemoglobin concentration during the neonatal period; data from a multihospital healthcare system. Pediatrics 2009;123;e333–e337.)

Figure 43.3. Reference ranges for blood hemoglobin concentration of neonates 29 to 34 weeks gestation during the first month after birth. The solid line shows the mean value and the dashed lines show the 5% and the 95% reference range. (From Jopling et al. Reference ranges for hematocrit and blood hemoglobin concentration during the neonatal period; data from a multihospital healthcare system. Pediatrics 2009;123;e333–e337.)

Red cell morphology in the newly born preterm or term neonate is characterized by macrocytosis and poikilocytosis. Target cells and irregularly shaped cells are particularly prominent. A high proportion of stomatocytes is noted when viewed by phase contrast microscopy.39 Similarly a high proportion of siderocytes (3.16% vs. normal male adult mean of 0.09%) are seen.40 Differential staining of red cells for fetal hemoglobin (HbF) provides a demonstration of the switch in hemoglobin synthesis that precedes birth: the younger macrocytes contain a minimal amount of HbF, whereas the smaller older cells are rich in HbF.41 Variations in red cell size and shape are somewhat greater than those observed in term infants, and cytoplasmic vacuoles are evident in nearly one-half of all cells when viewed by using interference-contrast microscopy. Red cell survival is shorter in

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preterm than in term infants. For infants who undergo exchange transfusion or multiple transfusions, both erythropoietin concentrations and reticulocyte counts are lower at any given hemoglobin concentration.42 It had been assumed that oxygen delivery is decreased in newborns because of the presence of a high-affinity hemoglobin, but a leftward shift in the hemoglobin–oxygen dissociation curve due to high levels of HbF might actually better maintain oxygen delivery during episodes of severe hypoxemia.43 The capacity of a fetus or neonate to deliver oxygen to tissues is better estimated by the circulating red blood cell volume than by the hematocrit or hemoglobin concentration. However, measuring the circulating red blood cell volume in a fetus or neonate is particularly difficult. Therefore, either the hematocrit or the hemoglobin is often used in making transfusion decisions. Mock

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Chapter 43  Anemias Unique to the Fetus and Neonate

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Disorders of Red Cells

Figure 43.4. Reference ranges for mean corpuscular volume (MCV) upper panel, and mean corpuscular hemoglobin (MCH) on the day of birth.  Values are shown from subjects after 22 to 42 weeks gestation. The solid line shows the mean value and the dashed lines show the 5% and the 95% reference range. (From Christensen et al. The erythrocyte indices of neonates, defined using data from over 12,000 patients in a multihospital healthcare system. J Perinatol 2008;28:24–28.)

et al. used a nonradioactive method, based on in vivo dilution of biotinylated RBC enumerated by flow cytometry, to estimate the correlation between hematocrit and circulating RBC volume in infants below 1,300 grams, between 7 and 79 days of life. They found that venous hematocrit values correlated highly with the circulating erythrocyte volume (r = 0.907; p < 0.0001).44 Neonates have a shorter red cell survival than do children and adults.45 The life span of red cells from term infants is estimated to be 60 to 80 days with use of the 51Cr method and 45 to 70 days using methods involving 59Fe.45 Fetal studies using [14C] cyanatelabeled red cells in sheep revealed an average red cell life span of 63.6 ± 5.8 days.46 The mean red cell life span increased linearly from 35 to 107 days as the fetal age increased from 97 days (midgestation) to 136 days (term). Neonatal red cells transfused into adults have a similarly short survival,47 indicating that factors intrinsic to the newborn red cell are responsible. Also, adult red cells survive normally in newborn recipients.48 The life span is not parametrically distributed, in that most cells are destroyed before the mean survival is reached.

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Shortened red cell survival as well as demands imposed by an expanding red cell mass account for erythropoietic rates at birth that are three to five times higher than those of normal adults. The abrupt transition from the uterus to an oxygen-rich environment triggers responses that have a profound effect on erythropoiesis. During the first 2 months of life, the infant experiences both the highest and lowest hemoglobin concentrations occurring at any time in development. Although quite variable, erythropoietin levels at birth usually are well above the normal adult range. Erythropoietin levels fall in the immediate postnatal period, with a half-life of 2.6 ± 0.5 hours in infants with polycythemia and 3.7 ± 0.9 hours in infants born to mothers with preeclampsia.49 By 24 hours, the erythropoietin value is below the normal adult range, where it remains throughout the first month. The decrease in erythropoietin is followed by a decline in the number of bone marrow precursors50 and a fall in the reticulocyte count. The combination of shortened cell survival, decreased production, and growth-related expansion of the blood volume is responsible for a progressive fall of the hemoglobin concentration

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Figure 43.5. Reference ranges for blood concentrations of nucleated erythrocytes on the day of birth.  Values are shown from subjects after 23 to 42 weeks gestation. The solid line shows the mean value and the dashed lines show the 5% and the 95% referenc range. (From Christensen et al. Neonatal reference ranges for blood concentrations of nucleated red blood cells. Neonatology 2010;99:289–294.)

12,000 11,000 10,000 9,000 8,000 7,000 NRBC/µl

6,000 5,000 4,000 3,000 2,000 1,000 0 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Weeks gestation Average

to a mean of approximately 11 g/dl at 2 months of age.51 The lower range of normal for infants of this age is approximately 9 g/dl. This nadir is called physiologic anemia, because it is not associated with apparent distress and is not prevented with nutritional supplements. Stabilization of the hemoglobin concentration is heralded by an increase in reticulocytes at 4 to 8  weeks.50 Thereafter, the hemoglobin concentration rises to a mean level of 12.5 g/dl, where it remains throughout infancy and early childhood. At term, the placenta and umbilical cord contain 75 to 125 ml of blood, or approximately one fourth to one third of the fetal blood volume. The umbilical arteries constrict shortly after birth but the umbilical vein remains dilated, and blood flows in the direction of gravity. Infants held below the level of the placenta can receive half of the placental blood volume (30 to 50 ml) in 1 minute. Conversely, infants held above the placenta can lose 20 to 30 ml of blood back into the placenta per minute.52 The blood volume of infants with early cord clamping averages 72 ml/kg, whereas the volume of infants with delayed cord clamping averages 93 ml/kg. Linderkamp et al. compared postnatal alterations in blood viscosity, hematocrit, plasma viscosity, red cell aggregation, and red cell deformability in the first 5 days in full-term neonates with early (less than 10 seconds) and late (3 minutes) cord clamping.53 The residual placental blood volume decreased from 52 ± 8 ml/kg of neonatal body weight after early cord clamping to 15 ± 4 ml/kg after late cord clamping. The neonatal blood volume was 50% higher in the late cord-clamped infants than in the early cord-clamped infants. It is possible to promote placental transfer of blood to preterm infants by delaying the clamping of the umbilical cord for 30 seconds. In fact transfer of about 10 ml/k body wt can be expected using this method.54 In a randomized trial by Mercer et al., this maneuver of delayed cord clamping among infants 20%. Percutaneous umbilical blood sampling can determine if hemoglobin concentration differences of greater than 5 gm/dl exist between the two fetuses.113 After birth, the donor twin may require transfusions and can also experience neutropenia, hydrops from severe anemia, growth retardation, congestive heart failure, and hypoglycemia. The recipient twin is often the sicker of the two. Problems include hypertrophic cardiomyopathy, congestive heart failure, polycythemia, hyperviscosity, respiratory difficulties, hypocalcemia, and hypoglycemia. Neurologic evaluation and imaging are helpful because of the risk of neurologic cerebral lesions in 20% to 30% of both twins.114 Prenatal treatment for twin–twin transfusion consists of close monitoring and reduction amniocenteses to decrease uterine stretch and prolong the pregnancy. Treatment in utero using laser surgery to ablate bridging vessels has become more common in the past decade. Most recent reports conclude an improvement in fetal survival using selective laser ablation.115,116,117

Perinatal Hemorrhage Obstetric complications, such as placenta previa, abruption, incision or tearing of the placenta during cesarean section, and cord evulsion, can result in significant neonatal blood loss. Placental abruption involves premature separation of the placenta from the uterus and occurs in 3 to 6 per 1,000 live births.118 Risk factors for abruption include cigarette smoking, prolonged rupture of the membranes, chorioamnionitis, hypertension (before pregnancy and pregnancy-induced), and advanced maternal age.119–121 The incidence of abruption increases with lower gestational age, and abruption can be a cause of preterm delivery.120 Mortality ranges from 0.8 to 2.0 per thousand births, or 15% to 20% of the deliveries in which significant abruption occurs. Placenta previa involves part or all of the placenta overlying the cervical os. Maternal risk factors for developing a placenta previa are essentially the same as those for abruption.122,123 Vasa previa (anomalous vessels overlying the internal cervix os) can be made with transvaginal color Doppler, and should be suspected in any case of antepartum or intrapartum hemorrhage. Although vasa previa is uncommon (1 in 3,000 deliveries), the perinatal death rate is high, ranging from 33% to 100% when undetected before delivery.124 Infants born after placental abruption or previa can be hypovolemic due to prenatal hemorrhage. Although the majority of the blood loss is maternal, loss of fetal blood can also occur, thus in neonates born after abruption or previa it is important to monitor blood pressure, hemoglobin/hematocrit, and tissue perfusion. Cord rupture can occur during delivery due to traction on a shortened, weakened, or otherwise abnormal umbilical cord. Cord aneurysms, varices, and cysts can all lead to a weakened cord. Cord infections (funisitis) can also weaken the cord and increase the risk of rupture. Hematomas of the cord occur

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infrequently (1 in 5,000 to 6,000 deliveries) and can be a cause of fetal blood loss and fetal death. Hematomas of the cord can be diagnosed in utero by ultrasound and differentiated from other lesions of the placenta and cord.125 Velamentous insertion of the umbilical cord occurs when the cord enters the membranes distant from the placenta, and is present in approximately 0.5% to 2.0% of pregnancies. These vessels are more likely to tear, even in the absence of traction or trauma. The fetal mortality is high in this condition, because rapid detection is difficult.126

Postnatal Hemorrhage Loss of fetal blood during delivery can occur into the placenta. The fetal–placental–umbilical cord unit contains about 120 ml/kg of blood, at term. After delivery, but before the umbilical cord is severed, blood in this unit can flow predominantly toward or away from the neonate. A fetoplacental hemorrhage can occur when the neonate is held significantly higher than the placenta after birth, for instance, on the mother's abdomen. It has been suggested that neonates can lose 10% to 20% of their blood volume when born with a tight nuchal cord, which allows blood to be pumped through umbilical arteries toward the placenta, while constricting flow back from the placenta to the baby, through the umbilical vein, which is more easily constricted due to its thin wall structure. However, in a study of over 200,000 deliveries, those with a tight nuchal cord (6.7%) did not have outcomes different from those with loose nuchal cords or with no nuchal cords.127 On that basis, it is not clear that tight nuchal cords typically cause clinical problems. Hemorrhage into the subgaleal space can be a life-threatening neonatal complication.128 However, the spectrum of severity ranges widely, from a small asymptomatic hemorrhage to a massive one causing hypovolemic shock. Associations are well known between vacuum or forceps-assisted delivery and subgaleal hemorrhage, but some cases occur when neither vacuum nor forceps were applied. In 38 neonates recently reported with a subgaleal hemorrhage, 21 occurred after vacuum, two after forceps, four after vacuum followed by forceps, and 11 when neither vacuum nor forceps were used. Thirty-five were admitted to an intensive care unit. Transfusions were given to 13, but no transfusions were given in the group where neither vacuum nor forceps was used, suggesting their hemorrhages were less severe.128 Anemia appearing after the first 24 hours of life in a nonjaundiced infant can be a sign of hemorrhage. Visible hemorrhages, such as a cephalohematoma, as well as internal occult hemorrhages, can occur. Breech deliveries may be associated with renal, adrenal, or splenic hemorrhage into the retroperitoneal space. Delivery of macrosomic infants, such as infants born to diabetic mothers, can result in hemorrhage. Infants with overwhelming sepsis can bleed into soft tissue and organs, such as liver, adrenal glands, and lungs. The liver in a neonate is prone to iatrogenic rupture, resulting in a high morbidity and mortality.129 Infants may appear asymptomatic until the liver ruptures and hemoperitoneum occurs. This can occur in term and preterm infants130 and has been associated with chest compressions during cardiopulmonary resuscitation. Surgical intervention involving vascular tamponade has been reported to save some infants; however, the mortality remains high.131 Splenic rupture can result from birth trauma or as a result of distention caused by extramedullary hematopoiesis. Abdominal distension and discoloration, scrotal swelling, and pallor are clinical signs of splenic rupture; these signs may also be seen with adrenal hemorrhage or hepatic rupture.132 Other rare causes of postnatal hemorrhage include hemangiomas of the gastrointestinal tract,133 vascular malformations of the skin, and hemorrhage into soft tumors, such as giant sacrococcygeal teratomas or ­ovarian cysts.

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Fetal and Neonatal Anemia Due to Congenital Infection Neonatal bacterial sepsis can cause anemia on the basis of hemolysis, DIC, and/or hemorrhage. Neonates with sepsis are generally jaundiced and have hepatosplenomegaly. Some bacterial organisms responsible for neonatal sepsis produce hemolytic endotoxins that result in accelerated erythrocyte destruction.134 Congenital infections due to cytomegalovirus, toxoplasmosis, rubella, syphilis, and herpes simplex can also cause hemolytic anemia. Fetal and neonatal infection with parvovirus B19 can cause severe anemia, hydrops, and fetal demise.135 The fetus or neonate generally presents with a hypoplastic anemia, but hemolysis can occur as well. The virus replicates in erythroid progenitor cells and results in red cell aplasia. In utero transfusions for hydropic fetuses can be successful. Intrauterine fetal infusion of B19 IgG-rich high titer gammaglobulin has been reported to be successful.136 Other fetal infections associated with neonatal anemia include malaria and HIV. Congenital malaria is seen rarely in the United States, generally in large cities where imported cases of malaria are increasing. In certain African countries, congenital malaria has been reported in up to 20% of neonates.137 Congenital HIV infection in a neonate is generally asymptomatic. However, infants born to mothers on zidovudine can have a hypoplastic anemia due to suppressive effects of the drug on fetal erythropoiesis.138

Anemia of Prematurity and Other Hypoproliferative Disorders Impaired erythrocyte production can occur in a fetus or neonate for a variety of reasons. Lack of an appropriate or sufficient marrow environment (as seen in osteopetrosis), lack of specific substrates or their carriers (e.g., iron, folate, vitamin B12, or transcobalamin II deficiency), and lack of specific growth factors (e.g., decreased erythropoietin production or abnormalities in Epo receptors) can be causative.

Anemia of Prematurity Infants delivered before 32 completed weeks gestation typically develop a transient and unique anemia knows as the anemia of prematurity.139 During the first week or two after birth, while in an intensive care unit, anemia secondary to phlebotomy loss is common.140 However after this period has passed, a second anemia is sometimes seen; it is characterized as a normocytic, normochromic, hyporegenerative anemia, with serum erythropoietin concentrations significantly below those found in adults with similar degrees of anemia.141 This anemia is not responsive to the administration of iron, folate, or vitamin E. Some infants with the anemia of prematurity are asymptomatic, whereas others have clear signs of anemia that are alleviated by erythrocyte transfusion. These signs include tachycardia, rapid tiring with nipple feedings, poor weight gain, increased requirements for supplemental oxygen, episodes of apnea and bradycardia, and elevated serum lactate concentrations.139–141 The reason preterm infants do not significantly increase serum erythropoietin concentration during this anemia is not known.142 Indeed, it is unclear whether production of erythropoietin does in fact increase, yet the serum concentration does not. Certainly their erythroid progenitors are sensitive to erythropoietin,143,144 and concentrations of other erythropoietic growth factors appear to be normal.145 The molecular and cellular mechanisms responsible for the anemia of prematurity are multifactorial, and include the

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transition from fetal to adult hemoglobin, shortened erythrocyte survival, and hemodilution associated with a rapidly increasing body mass.139,140 It is unknown whether preterm infants rely on erythropoietin produced by the liver (the source of erythropoietin in utero), or that produced by the kidney, or a combination of the two. Regardless of the mechanism responsible for the anemia of prematurity, exogenous erythropoietin administered to preterm infants accelerates effective erythropoiesis.146,147 A meta-analysis of studies evaluating the use of “late erythropoietin administration to prevent and treat the anemia of prematurity reveals a positive effect on decreasing transfusion requirements in preterm infants.148 In addition, beneficial neurodevelopmental effects of recombinant erythropoietin administration have been reported in preterm infants.17,18,19,20,149,150 Pharmacokinetic studies of darbepoetin, the long-acting erythropoietic stimulator, have been conducted among neonates with the anemia of prematurity, with the speculation that less frequent dosing and cost savings might render darbepoetin a more attractive alternative than recombinant erythropoietin for treating the anemia of prematurity.151,152,153,154 Following subcutaneous and intravenous dosing, darbepoetin has a considerably shorter terminal half-life in neonates than in adults (Table 43.5). Intravenous dosing appears to be as effective as subcutaneous dosing.153 Newer, long-acting Epo mimetics, such as CERA (continuous erythropoiesis receptor agonist), have yet to be studied in neonates, but may hold promise as potential agents to decrease transfusions and provide neuroprotection. Although a minimal number of clinical studies evaluating darbepoetin administration to preterm infants have been published, numerous RCTs evaluating Epo administration to preterm infants have consistently shown evidence of increased erythropoiesis and a decrease in transfusions.141 A consistent finding in the largest RCTs has been an elevation in hematocrit among Epo-treated infants compared with placebo/controls, despite the implementation of strict transfusion guidelines aimed at maintaining hematocrits in a similar range. For those neonatal practitioners electing to maintain hematocrits at higher levels, the use of Epo can gain a “buffer” of 4% to 6% hematocrit points, decreasing the number of transfusions given. This may benefit preterm infants in a number of ways, given recent studies that have reported associations between RBC transfusions and necrotizing enterocolitis (NEC)155 and between RBC transfusions and IVH.156,157

Disorders of Red Cells



Other Hypoproliferative Anemias During the neonatal period, hypoproliferative anemias are rare, with the exception of the anemia of prematurity, which is common (Table 43.6). The hypoproliferative anemia Diamond-Blackfan syndrome can be diagnosed at birth, but characteristically is not recognized until after 2 to 3 months of age. In fact, 10% to 25% of infants with Diamond-Blackfan syndrome have at least a mild anemia at birth.158 Severe anemia with hydrops has been

TA B L E 4 3 . 5

Terminal T ½ of Darbepoetin Among Adults, Children, and Neonates after Subcutaneous or Intravenous Dosing After Subcutaneous Dosing (hours) Adults Children Neonates

49 43 22

After Intravenous Dosing (hours) 25 22 10

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TA B L E 43.6

Syndromes Associated with Fetal/Neonatal Anemia Genetic Syndrome

Phenotypic Features

Genotypic Features

Adenosine deaminase deficiency

Autoimmune hemolytic anemia, reduced erythrocyte adenosine deaminase activity Type I (rare): megaloblastoid erythroid hyperplasia and nuclear chromatin bridges between nuclei; type II (most common): “hereditary erythroblastic multinuclearity, positive acidified serum (HEMPAS) test, increased lysis to anti-i; type III: erythroblastic multinuclearity (“gigantoblasts ), macrocytosis Steroid-responsive hypoplastic anemia, often macrocytic after 5 mo of age Hypoproliferative anemia usually presenting between 5 and 15 yr of age Steroid-responsive hypoplastic anemia, reticulocytopenia, some macrocytic RBCs, shortened RBC lifespan. Cells are hypersensitive to DNA cross-linking agents Hemorrhagic anemia Hypoplastic anemia from marrow compression; extramedullary erythropoiesis Hypoplastic sideroblastic anemia, marrow cell vacuolization Iron-deficiency anemia from chronic blood loss ATR-X: hypochromic, microcytic anemia; mild form of hemoglobin H disease ATR-16: more significant hemoglobin H disease and anemia are present

AR, 20q13.11

Congenital dyserythropoietic anemias

Diamond-Blackfan syndrome Dyskeratosis congenita Fanconi pancytopenia

Osler hemorrhagic telangiectasia syndrome Osteopetrosis Pearson syndrome Peutz-Jeghers syndrome X-linked a-thalassemia/mental retardation (ATR-X and ATR-16) syndromes

Type I: 15q15.1-q15.3; type II: 20q11.2; type III: 15q21

AR; sporadic mutations and AD inheritance described; 19q13.2, 8p23.3-p22 X-linked recessive, locus on Xq28; some cases with AD inheritance AR, multiple genes: complementation; group A: 16q24.3; B:; C: 9q22.3; D2: 3p25.3; E: 6p22-p21; F: 11p15; G: 9p13 AD, 9q34.1 AR: 16p13, 11q13.4-q13.5; AD: 1p21; lethal: reduced osteoclasts Pleioplasmatic rearrangement of mitochondrial DNA; X-linked or AR AD, 19p13.3 ATR-X: X-linked recessive, Xq13.3; ATR-16: 16p13.3, deletions of a-globin locus

AD, autosomal dominant; AR, autosomal recessive; RBC, red blood cell.

reported in conjunction with this syndrome. Aase syndrome, another congenital hypoplastic anemia syndrome involving marrow and skeletal anomalies,159 is sometimes classified as a variant of Diamond-Blackfan syndrome. Congenital dyserythropoietic anemia is a rare disorder marked by ineffective erythropoiesis, megaloblastic anemia, and characteristic abnormalities of the nuclear membrane and cytoplasm seen on electron microscopy. Fanconi pancytopenia rarely is manifested during the neonatal period. This autosomal-recessive disorder is characterized by marrow failure and congenital anomalies, including abnormalities in skin pigmentation, gastrointestinal anomalies, renal anomalies, and upper limb anomalies.160 Approximately one third of patients have no obvious congenital anomalies, and anemia is less common than thrombocytopenia and leukopenia. Five genetic phenotypes of Fanconi pancytopenia have been reported, and two of the genes have been cloned.161 Cells are hypersensitive to DNA cross-linking agents such as diepoxybutane and mitomycin C. The diepoxybutane test represents a sensitive and specific diagnostic test. When Fanconi anemia is recognized in a neonate, it is generally on the basis of the congenital anomalies and not the hematologic abnormalities. However, congenital thrombocytopenia, manifested during the immediate newborn period, progressing to pancytopenia has rarely been reported.162 Erythropoietin concentrations are usually elevated, and HbF production is increased. A significant percentage of patients develop myelodysplastic syndrome or acute myelogenous leukemia later in life. Treatment of Fanconi pancytopenia includes androgen therapy, and, in many cases, bone marrow transplantation has been successful. Autosomal-recessive osteopetrosis is a rare disorder characterized by osteoclast dysfunction, resulting in a decreased bone marrow space.163 Developmental delay, ocular involvement, and neurodegenerative findings occur in association with hypoplastic anemia. Patients are generally treated with hematopoietic stem cell transplantation, but they are particularly susceptible

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to posttransplantation complications after myeloablation, and reduced-intensity conditioning programs may be helpful.164 Pearson syndrome is a congenital hypogenerative anemia that can progress to pancytopenia, and additionally affects the exocrine pancreas, liver, and kidneys.165 Patients with this syndrome can present during the neonatal period, but typically do so later in infancy, often in the first year of life. Presenting features include failure to thrive, with anemia, neutropenia, and/or thrombocytopenia. The marrow examination typically shows characteristic vacuoles within erythroid and myeloid precursors, hemosiderosis, and ringed sideroblasts. The syndrome is caused by a loss of large segments of mitochondrial DNA.166,167

Considerations Regarding Erythrocyte Transfusion in the Neonatal Period Transfusion of banked donor erythrocytes can be life saving for small and ill neonates with severe anemia or hemorrhage. However, risks of transfusions exist and must be weighed against potential benefits each time a transfusion is considered. Two transfusion risks are highly unique to VLBW( 0.60 or women with a hematocrit > 0.55, there is reported to be >99% likelihood that the red cell mass is elevated.39 In its 2007 criteria for the diagnosis of polycythemia vera, the World Health Organization (WHO) uses an Hb concentration of >18.5 g/dl in men or 16.5 g/dl in women to define an elevated red cell mass.40 In a comparative study, the hematocrit 0.60/0.55 standard was reported to identify elevated red cell mass more accurately than the Hb concentration 18.5/16.5 g/dl standard.41 It should be noted that in certain circumstances of severe hemoconcentration (e.g., in the systemic capillary leak syndrome42), Hb concentrations or hematocrits in this range may be observed in patients with a normal red cell mass. Such patients typically exhibit anasarca and other physical findings suggestive of severe intravascular volume depletion and redistribution of intravascular volume. The goal of the approach outlined in Figures 44.3 and 44.4 is initially to distinguish spurious (relative) polycythemia from actual polycythemia, then to distinguish polycythemia vera from secondary polycythemia and primary proliferative polycythemia, to rule out other primary polycythemia, and finally to identify the etiology of secondary polycythemia. The characteristics of polycythemia vera are outlined in Chapter 82; the other polycythemic syndromes are discussed below. A certain number of patients are not readily classified as having either polycythemia vera or secondary polycythemia. These patients fall into a category called (for want of a more physiologic term) idiopathic polycythemia or idiopathic erythrocytosis and appear to represent a heterogeneous group of disorders (see below). The diagnostic approach to polycythemia has been substantially altered by the observation that more than 95% of patients with polycythemia vera express a mutation in the JAK2 gene in which phenylalanine is substituted for valine at position 617.43–45 JAK2 V617F mutation-negative polycythemia vera patients have been reported to have mutations in other exons of JAK2.46

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Chapter 44  Erythrocytosis Increased Red Cell Mass

NO

1035

RELATIVE POLYCYTHEMIA

YES

NO JAK2 mutation YES

UNCHARACTERIZED MPD

Both NO

YES

Suspect Secondary Polycythemia

See Figure 44.4

NO

Low Epo or MPD Marrow

Either

Low Epo and MPD Marrow

Either YES

See Figure 44.4 NO

Both YES

From Figure 44.3

Disorders of Red Cells

POLYCYTHEMIA VERA FIGURE 44.3. Approach to patients with erythrocytosis.  EEC, endogenous erythroid colonies; EpoR, erythropoietin receptor; JAK2 mutation, testing for JAK2 V617F or other mutations associated with polycythemia vera; low Epo, serum or plasma erythropoietin concentration less than the lower limit of normal; MPD, myeloproliferative disorder; MPD marrow, bone marrow findings suggestive of a myeloproliferative disorder (see text); MPD, myeloproliferative disorder; VHL, von Hippel-Lindau protein.

O2 Saturation ≥ 92% NO

YES NO Elevated carboxyhemoglobin

Family history of erythrocytosis

NO

YES

Hemoglobin studies • Electrophoresis • P50 NORMAL YES ABNORMAL

YES

NO

Other studies if clinically appropriate: • Positional O2 saturation • Echocardiography • Endocrine evaluation

EpoR/VHL gene studies NORMAL

SECONDARY POLYCYTHEMIA

Renal or hepatic lesions

PRIMARY PROLIFERATIVE POLYCYTHEMIA

ABNORMAL

NORMAL

IDIOPATHIC POLYCYTHEMIA ? OBSERVATION ? EEC studies ? Periodically repeat JAK2 mutation

FIGURE 44.4. Approach to patients with erythrocytosis.   JAK2 V617F-negative erythrocytosis. CNS, central nervous system; EEC, endogenous erythroid colonies; EpoR, erythropoietin receptor; MPD, myeloproliferative disorder; VHL, von Hippel-Lindau protein.

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Part iv  Disorders of Red Cells  •  SECTION 5  Other Red Cell Disorders

T A B L E 44.3

Normal Values For Red Blood Cell, Plasma, And Total Blood Volume (ML/KG ± 1 Standard Deviationa)

Women Sea level Wennesland et al.34 Huff and Feller31 Men Sea level Wennesland et al.34 Huff and Feller31 Weil et al.30 1,600 mb Weil et al.30 3,100 mb Weil et al.30

No.

Red Blood Cells

Plasma

 97  20

25.4 ± 2.6 24.4 ± 2.6

36.8 ± 3.7 34.8 ± 3.2

—

199  42  16  19  39

28.3 ± 2.8 28.3 ± 4.1 27.1 ± 3.7 26.8 ± 3.2 31.8 ± 6.7

34.4 ± 4.0 33.5 ± 5.2 33.0 ± 5.3 31.9 ± 3.6 35.2 ± 5.3

—

Total Blood Volume

58.9 ± 4.9

61.5 ± 8.6 60.0 ± 8.6 58.7 ± 5.8 66.8 ± 8.5

a Red blood cell volume measured by 51Cr method. Other values calculated without correction for trapped plasma. b Only values of which we are aware at altitudes significantly above sea level. They may be somewhat low for unknown reasons; the packed cell volumes at 1,600 m were the same as at sea level, a finding that contradicts an earlier author’s own large experience.

It is tempting to regard the JAK2 mutation in polycythemia vera as conceptually analogous to the bcr/abl mutation in chronic myelogenous leukemia; however, the JAK2 V617F mutation is also found in other myeloproliferative disorders.44 Its implications are discussed in more detail in Chapter 82, but in terms of differential diagnosis, it should be regarded as a marker of a myeloproliferative state. Polymerase chain reaction–based assays for JAK2 V617F versions of this test are widely available through reference laboratories in the United States and Europe at costs in the $300 to $500 range. As the role of JAK2 assessment in the approach to myeloproliferative disorders continues to expand, increasing numbers of medical centers are developing in-house tests for JAK2 V617F. The WHO criteria for the diagnosis of polycythemia vera requires evidence of an increased red cell mass, and a JAK2 or other functionally similar mutation and one minor criterion; or evidence of an increased red cell mass and all three minor criteria.40 The minor criteria are a serum or plasma erythropoietin concentration below the range of normal for the laboratory; a bone marrow examination exhibiting the characteristic features of a myeloproliferative disorder (hypercellularity with trilineage hyperplasia, clustered pleomorphic megakaryocytes, and no features of inflammation) and the formation of erythroid colonies in vitro in the absence of added erythropoietin (“endogenous erythroid colonies”[EECs]).40 Although these tests can be evaluated in any order desired, ease of test access would suggest that erythropoietin concentration be ordered first, followed by marrow evaluation if necessary. In a JAK2 mutation-positive patient, either a characteristic erythropoietin concentration or a characteristic marrow would permit the diagnosis of polycythemia vera. In a JAK2 mutation-negative patient, absence of both a characteristic erythropoietin concentration and a characteristic marrow would rule out the diagnosis. EEC assays, a hallmark of myeloproliferative disorders in general,47,48 are not readily available outside of research laboratories, which limits their diagnostic practicality. The roles of erythropoietin levels and EEC assays in the differential diagnosis of erythrocytosis are most strongly supported by their association with the WHO diagnostic criteria; as stand-alone tests in individual cases, utility is less clear.49,50,51 Although mean

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serum erythropoietin concentration in the subset of patients with polycythemia vera is significantly lower than that observed in secondary polycythemia, there is considerable overlap, making it less useful for individual cases. This would be expected physiologically. A patient with secondary polycythemia due to tissue hypoxia would have an elevated serum erythropoietin level until the hematocrit was sufficiently high to oxygenate tissue adequately; then the erythropoietin concentration would be expected to decrease. The intermittent nature of detection of an elevated serum erythropoietin concentration in secondary polycythemia has been described,52 as has the failure of serum erythropoietin concentrations to predict clinical course in idiopathic erythrocytosis.53 Studies have been reported demonstrating that polycythemia vera and secondary polycythemia can be distinguished based on the serum erythropoietin response to phlebotomy: after phlebotomy, serum erythropoietin levels increase in secondary polycythemia but remain stable in polycythemia vera.49 EECs corresponding to erythroid burst-forming units were observed in 12 of 17 polycythemia vera patients, 3 of 11 secondary polycythemics, 1 of 6 relative polycythemics, and 1 of 11 normal individuals in one series.54 Soluble transferrin receptors are typically elevated in all forms of polycythemia and thus do not distinguish polycythemia vera and secondary polycythemia.55 As a general approach to the evaluation of erythrocytosis, all patients with presumed polycythemia should undergo JAK2 V617F testing. If the clinical features are suggestive of a secondary etiology of polycythemia, patients who do not show a JAK2 mutation should follow the process outlined in Figure 44.4. Individuals without a documented JAK2 mutation but in whom suspicion of a myeloproliferative disorder is high, should be investigated for minor WHO criteria (Fig. 44.3).

Relative Polycythemia Lowered fluid intake, marked loss of body fluids, or a combination of both causes a decrease in plasma volume and may produce a relative erythrocytosis. The decrease in plasma volume may result from any cause of intravascular fluid loss, insensible fluid loss, persistent vomiting, severe diarrhea, copious sweating, postoperative complications, or shift of fluid into the extravascular space (“third spacing”)3,37,42,56 or may be an effect of high altitude.53 In severe burns, plasma loss leads to hemoconcentration. Chronic relative polycythemia or erythrocytosis has been variously referred to as Gaisböck syndrome,57 “stress” erythrocytosis,3 benign polycythemia,52,56 benign erythrocytosis,58 spurious polycythemia,59,60 pseudopolycythemia,61 and apparent polycythemia.62 The last three terms are the most accurate: in the absence of an elevated red cell mass, there is no polycythemia. In one series of 215 patients referred with a diagnosis of polycythemia vera,63 18 (8.3%) were believed to have chronic relative erythrocytosis, possibly caused by “stress.”3 Patients with relative polycythemia or erythrocytosis are typically male; the mean age at diagnosis is less than is seen in patients with polycythemia vera.62 Obesity is typically described as an associated feature,3 although not all studies support this association.64 Other features reported to be strongly associated with relative polycythemia are hypertension and smoking;62,64,65 associations with alcohol abuse and renal disease are occasionally reported.62,63 It is probable that this syndrome is not a true clinical entity.60 The red cell mass values generally accepted as normal at sea level, or at any given altitude, represent the mean ±2 standard deviations. Thus, on the basis of the normal frequency distribution curve for this physiologic parameter, the values in 2.5% of the population are above this range. The individuals in this group should not be regarded as necessarily abnormal.66 The optimal management of relative polycythemia is unknown. As noted previously, phlebotomy increases cerebral blood flow even in patients with relative polycythemia; whether it is of

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symptomatic benefit is less clear.20,21 It should probably be avoided. Theoretic arguments can be made that contracting the blood volume further in these patients who already are normovolemic or slightly hypovolemic may impair tissue perfusion. Satisfactory control of hematocrit can be obtained in at least two thirds of patients by reduction of excess weight, improved hypertension control, avoidance of diuretics, and reduction if not cessation of smoking.64 Potentially leukemogenic cytoreductive therapy, such as radioactive phosphorus or oral chemotherapeutic agents, is probably never indicated. Some types of high-affinity Hbs (Heathrow,67 Pierre-Benite,68 Rahere69) may show relative polycythemia.

Polycythemia (Absolute Erythrocytosis) Primary Polycythemia Polycythemia Vera Polycythemia vera is discussed in Chapter 82.

Primary Familial Polycythemia (“Chuvash Polycythemia”)/Primary Proliferative Polycythemia familial erythrocytosis or polycythemia is a term used to describe instances in which two or more members of a family have polycythemia, do not have polycythemia vera, and have no identifiable “secondary” causes.70 This finding can result from a constellation of pathophysiologic mechanisms, including abnormalities of oxygen–Hb interaction, or idiopathic constitutive erythropoietin secretion. These syndromes are discussed under etiologies of secondary polycythemia, below. Primary familial polycythemia is a term used to describe a syndrome observed in families with abnormalities of the erythropoietin receptor, resulting in hypersensitivity to erythropoietin and consequent erythrocytosis.71,72,73 This particular autosomal dominant trait does not necessarily confer an adverse prognosis early in life: the propositus of the first such family described was an Olympic gold medalist in cross-country skiing.71 However, these individuals are at increased risk for thrombotic and vascular mortality later on. A variant of this syndrome occurs with high frequency among the people of the Chuvashia region of the former Soviet Union.74 These individuals appear to have a mutation in the oxygen-sensing pathway regulating erythropoietin production, typically involving von Hippel-Lindau protein, and also in the response of erythroid progenitors to erythropoietin.75–78 This effect on erythropoietin signaling appears to be mediated by loss of JAK2 regulation of erythropoiesis.79 As in other patients with familial erythrocytosis due to aberrant erythropoietin signaling, Chuvash polycythemia patients have increased risk for vascular disease.80 Isolated cases with similar mechanisms are referred to as primary proliferative polycythemia.

Secondary Polycythemia (Physiologically Appropriate [Hypoxic]) Insufficient oxygen supply to the tissues may result from any of the following, alone or in combination: (a) decreased ambient oxygen pressure (e.g., high altitude); (b) pulmonary diffusion or mixing abnormalities; (c) right-to-left cardiopulmonary shunts, as in cyanotic congenital heart disease; (d) hypoventilation; or (e) altered oxygen-carrying affinity of Hb. In all of these disorders, insufficient tissue oxygenation leads to increased erythropoietin production and a consequent increase in red cell mass (see Chapter 6).

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High-altitude Erythrocytosis In 1890, Viault showed that erythrocytosis develops during sojourn at high altitude.81 He found erythrocyte counts of 7.5 to 8.0 × 1012 cells/L not only in natives of the Peruvian Andes working in a mine at an altitude of 4,392 m above sea level, but also in himself and in a traveling companion, although his blood count in Lima (160 m above sea level) had been normal. On a Mt. Everest expedition, researchers demonstrated that red cell volume and values of total Hb rose progressively as higher altitudes were attained; at 19,000 feet (5,800 m), mean values were 49% above those at sea level. The increase in total blood volume was partially masked by reductions in plasma volume.82,83 A sharp increase in erythropoietin production occurs within the first week of high-altitude exposure and is associated with mobilization of iron stores and evidence of iron-deficient erythropoiesis.84 Mechanisms of adaptation to living at high altitude apparently are multiple and differ between ethnic groups.85,86 The rapid ascent to high altitude is accompanied by symptoms of fatigue, dizziness, pulsating headache, anorexia, nausea, vomiting, insomnia, and irritability, a syndrome well known to mountain climbers and residents of high altitudes and referred to as acute mountain sickness or acute altitude disease.87–90 The symptoms first appear some 4 to 6 hours after reaching a high altitude but may be delayed for as many as 96 hours, suggesting that the pathogenesis represents more than simple hypoxia. The incidence is greatest in younger persons, in those flying to high altitude, or in those who climb fast and spend few nights acclimatizing. Gender, the weight of the load carried, and recent respiratory infection do not appear to affect the incidence.88 Severity is greatest in the young and in less-experienced climbers and correlates with the speed of ascent and the altitude reached.91 Thus, all persons develop symptoms if they are suddenly transported from sea level to 15,000 feet (4,570 m) or higher, whereas a few develop symptoms at 8,000 to 10,000 feet (2,400 to 3,000 m).83 After 4 to 8 days, acclimatization usually occurs, and symptoms remit spontaneously.88,92 In some individuals, however, symptoms may progress to cerebral confusion, coma, and even death related to pulmonary edema unless the subject is returned to low altitude.87,93 The pathogenesis of acute mountain sickness may involve hypoxia and subsequent excessive secretion of antidiuretic hormone and adrenal steroids with resulting fluid retention, increased blood volume, and finally cerebral edema, pulmonary congestion, or both.94–97,98 The incidence and severity of symptoms can be considerably reduced or prevented by appropriate treatment.99 The events associated with acclimatization after arrival at high altitude are not understood completely but probably include the following:

Disorders of Red Cells



• An increase in erythrocyte 2,3-biphosphoglycerate (BPG) levels and a shift to the right in the oxygen–Hb dissociation curve, thus allowing better tissue delivery of oxygen despite decreased arterial oxygen saturation.86,100–102 The increase in 2,3-BPG appears to compensate for the left shift in the curve that results from the initial hypocapnia and increase in arterial pH.100,103 • Increased erythropoietin production with subsequent increase in iron mobilization, reticulocytosis, and increase in red cell mass and blood volume.104 • Correction of the initial excessive antidiuretic hormone and adrenal steroid secretion and return to the normal diurnal variation of plasma steroid levels.98 The final result is a new equilibrium at decreased oxygen saturation and carbon dioxide tension with increases in alveolar ventilation, respiratory frequency, and red cell mass.92 These manifestations of acclimatization are quickly lost on descent to sea level, even after many years of residence at high altitude. In some individuals, however, after a few or many years of good adaptation, excessive erythrocytosis develops, and arterial oxygen saturation may fall to as low as 60% (normal, 81%).

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Part iv  Disorders of Red Cells  •  SECTION 5  Other Red Cell Disorders

An incapacitating illness characterized by alveolar hypoventilation develops. This entity is known as chronic mountain or altitude sickness or Monge disease.105 Diminished mental acuity, headaches, dyspnea, fatigue, reduced physical fitness, nausea, vomiting, diminution of visual acuity, dizziness, tinnitus, vague or even excruciating pains in the extremities, paresthesias, and cough are characteristic symptoms. If the condition advances, symptoms include incessant dyspnea, aphonia, profound lethargy, and even coma. The face is bluish violet or almost black, the eyelids are edematous and bluish, the sclerae are intensely colored by distended capillaries, the tongue is thick, the hands are enlarged and turgid, the fingers are clubbed, and dependent edema may be observed. The thorax is more barrel-shaped than in healthy inhabitants of the same region and altitude. Hypotension is often present. The spleen and the liver are infrequently enlarged, unless cardiac failure ensues. Erythrocytosis is more marked than in normal residents of high altitudes, with hematocrits up to 0.84 L/L and Hb values as high as 28.0 g/dl. MCV is normal or slightly increased, and the mean corpuscular hemoglobin concentration (MCHC) is normal. Normal reticulocyte and leukocyte counts are usually observed. Hyperbilirubinemia owing to unconjugated bilirubin may be pronounced. Red cell turnover is greater in these individuals than in normal residents of high altitudes. Platelet counts usually are normal or high, yet epistaxis is common, and hemoptysis, bleeding of the gums, and purpura may occur. Red cell volume is greatly increased (88 to 95 ml/kg body weight). The results do not appear to be completely explainable by differences in erythropoietin production.105–110 Affected individuals usually are in the fourth to sixth decade of life. Remissions and relapses are described. Ascent to still higher altitudes aggravates symptoms, whereas descent to sea level relieves them. Cardiac impairment does not appear until late in the disease course, and death occurs more often from hemorrhage, pulmonary tuberculosis, or bronchopneumonia than from cardiac insufficiency. At first, the disease was considered a distinct entity. It has been suggested that the disease is an exaggeration of the process of acclimatization and aging, because patients with chronic mountain sickness had Hb concentrations within the normally distributed values for large groups of native residents. Support for this suggestion comes from the observation that chronic lung disease increases the likelihood of chronic mountain sickness.111 Chronic mountain sickness has not been reported to occur in natives of the Himalayas.112 This may reflect in part occupational differences, namely mining, and a consequently high incidence of chronic lung disease in the Andes as compared with the pastoral occupation of the Sherpas. Investigation of selective gene expression in populations in whom chronic mountain sickness is prevalent, may further enhance understanding of this syndrome.113,114,115 Differentiation of chronic mountain sickness from other causes of hypoxic polycythemia should not be difficult. Cases of congenital or acquired cyanotic heart disease can be distinguished by the cardiac findings. Polycythemia vera is not altered by increased ambient oxygen tension, whereas in Monge disease, descent to sea level produces complete relief of symptoms, together with a pronounced reduction in the blood volume and restoration of normal blood counts.116

Pulmonary Disease A variety of diseases, such as chronic obstructive pulmonary disease, diffuse pulmonary infiltrates (fibrous or granulomatous), kyphoscoliosis, and multiple pulmonary emboli, leads to erythrocytosis as the result of inadequate oxygenation of the blood circulating through the lungs. Not all patients with lung disease and decreased arterial oxygen saturation, however, have

72683_ch044_1032-1042.indd 1038

elevated Hb or hematocrit levels,117,118 and only in approximately 50% is an increase in red cell mass noted.119 The reason for this suboptimal response to hypoxia is not clear, but it does not appear to result from a decrease in erythropoietin production or the presence of chronic infection.117,118,120 When polycythemia occurs, it usually is associated with increased MCV, reduced MCHC,119 and normal MCH120 values. The red cell morphology changes have been attributed to increased water uptake by the cell, which in turn may result from carbon dioxide retention.117 If polycythemia is present, it is corrected by chronic oxygen administration.121 Vascular malformations in the lung may also be associated with erythrocytosis.122,123 Pulmonary arteriovenous fistulae should be suspected when a murmur is heard in a lung field in association with erythrocytosis.

Chronic Cor Pulmonale

The clinical picture of chronic cor pulmonale varies, but oxygen deficiency with arterial desaturation and elevated pulmonary artery pressure is of central importance.124,125 Polycythemia with its associated increase in blood viscosity and volume appears to be the physiologic price of a compensatory mechanism progressively extended to the point at which it is more injurious than beneficial.126 As in less severe pulmonary disease, the MCV of the red cells tends to be elevated, whereas the MCHC generally is decreased.127

Cyanotic Heart Disease Marked degrees of polycythemia may be seen in patients with a partial shunt of the blood from the pulmonary circuit. Hematocrit levels greater than 0.60 are not uncommon. The most frequent defects producing such polycythemia are pulmonary stenosis (usually with defective ventricular or atrial septum, patent foramen ovale, or patent ductus arteriosus), persistent truncus arteriosus, complete transposition of the great vessels, and the tetralogy of Fallot (pulmonary stenosis, defective ventricular septum, dextroposition of the aorta, right ventricular hypertrophy). Individuals with such defects exhibit evidence of disturbed cardiorespiratory function, marked cyanosis, clubbing of the fingers and toes, and sometimes stunted growth. The total plasma volume may be reduced to below normal levels, but the increase in the size of the red cell mass is so great that the total blood volume usually is higher than normal.128 Erythroid hyperplasia is observed in the marrow.129,130 The general consensus is that low oxygen tension resulting from shunting of unoxygenated blood through or around the lungs with consequent desaturation of the arterial blood stimulates erythropoietin production. With successful operative intervention, this value may be significantly corrected, with resolution of polycythemia.

Acquired Heart Disease In 1901, Abel Ayerza described a syndrome characterized clinically by slowly developing asthma, bronchitis, dyspnea, right-sided heart failure, and severe cyanosis with associated polycythemia.131 The striking degree of cyanosis led Ayerza to describe these patients as “black cardiacs.” It was subsequently demonstrated that the unifying feature of these patients was not pulmonary disease but rather pulmonary arterial hyperplasia, leading to pulmonary hypertension and consequent right-sided heart failure.132 In all forms of acquired heart disease, any erythrocytosis that may develop is correlated to some extent with the degree of cardiopulmonary decompensation. It is typically minimal. Polycythemia is reportedly accompanied by evidence of intensified erythropoiesis in the bone marrow, an increase in red cell mass, and some macrocytosis.131

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Hypoventilation Syndromes Polycythemia is found occasionally in patients who exhibit no evidence of pulmonary disease or cardiovascular shunts. The primary defect in at least some of these patients appears to be an inadequate ventilatory drive from the respiratory center in the brain.126,133 A similar defect has been reported in patients with the Pickwickian syndrome, so called because of the description of Joe, the hypersomnolent fat boy, in Dickens’s The Pickwick Papers.134 In the setting of extreme obesity, these patients exhibit somnolence, cyanosis, and hypercapnia and may develop periodic respiration, ultimately with right ventricular failure. Voluntary hyperventilation alleviates the hypercapnia, and in many patients, loss of weight restores normal alveolar ventilation and reverses the syndrome.135,136 Alveolar hypoventilation and erythrocytosis, however, do not develop in all obese individuals; it appears that only in the presence of an insensitive respiratory center does a massive panniculus limit respiratory function and result in alveolar hypoventilation, hypoxemia, and hypercapnia.137 In some patients, the decreased ventilatory drive is of unknown cause or is a result of idiopathic disease of the medullary respiratory center (Ondine curse)133,138; other etiologies include bulbar poliomyelitis, vascular thrombosis, or previous encephalitis.126,137 In any case, the consequent hypoxemia results in elevated levels of erythropoietin and erythrocytosis, with hematocrits reported as high as 0.758. Patients with polycythemia and positional arterial oxygen desaturation have also been reported.139 Whether this results from alveolar hypoventilation while supine or from shunting through an arteriovenous malformation while upright is unclear.140 Obstructive sleep apnea has been associated with polycythemia (presumably due to episodic erythropoietin secretion during apneic episodes) in some141 but not all142 reports.

Abnormal Hemoglobins Inherited Abnormalities of Hemoglobin

Certain mutant Hbs are characterized by increased oxygen, and patients who carry such Hbs tend to develop erythrocytosis.143–145,146 More than 200 high-affinity Hbs have been characterized, of which roughly half produce significant erythrocytosis.147 Oxygen–Hb dissociation curves are shifted dramatically to the left in individuals carrying these abnormal Hbs. The degree of left shift can be quantified by determining the P50 (i.e., the oxygen pressure at which Hb is half-saturated). The normal value in whole blood is 23 to 29 mm Hg at standard pH, temperature, CO2 content, and barometric pressure. The whole-blood P50 is almost invariably decreased in patients with a high-affinity Hb; most values fall between 9 and 21 mm Hg. In a few instances, the P50 has been normal, or nearly so, in whole blood (e.g., HbG Norfolk148), necessitating the measurement of the oxygen dissociation curve of the purified Hb to demonstrate the defect. The approach to the diagnosis of high-affinity Hb variants, the characteristics of patients with representative mutations, and the molecular pathology are discussed in Chapter 35. The most important physiologic consequence of increased oxygen affinity is that release of oxygen is impaired at partial pressure of oxygen values normally found in tissues. Uptake of oxygen in the lungs is enhanced, but this effect is relatively unimportant, because normal Hb is nearly completely saturated in the lungs under the usual physiologic circumstances. As previously noted, however, the increased affinity may confer some advantages when environmental oxygen is low, such as at high altitudes. Individuals with high-affinity Hbs are not at a disadvantage under hypoxic conditions. They tolerate ascent to high altitudes as well as or better than normal subjects and thus appear to be preadapted to hypoxic stresses. Under such conditions, the enhanced oxygen loading seems more important than the impaired delivery.

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Similarly, exercise tolerance appears unimpaired.149 There is no evidence that oxygen delivery to the heart is defective in patients with high-affinity Hbs. Although myocardial infarctions and other findings of atherosclerotic cardiovascular disease are reported in these patients, it is unclear whether this is an actual association or simply reflects the high frequency of atherosclerosis in the general population.150–152 High-affinity Hbs appear to exert no adverse effects on fetal development in utero. Theoretically, oxygen delivery to a developing noncarrier fetus might be impaired when the mother is a carrier, because the normal differential in oxygen affinity between fetal and adult Hb (which is in favor of the developing fetus) would be narrowed. However, only in the family with Hb Yakima was there a suggestion that spontaneous abortions occurred at an increased rate.153,154 In contrast, normal pregnancy outcomes were recorded for mothers carrying the severe high-affinity variants Hb Bethesda, Hb Osler, and Hb Little Rock.154 Evidently, maternal and fetal polycythemia and increased uterine and fetal blood flow compensate for the theoretic deficit in placental oxygen transport. There are no data addressing whether carrier fetuses have a developmental advantage over noncarriers born to these mothers; however, in dizygotic twins born to a mother with Hb Osler, the carrier twin developed more fully than the noncarrier as measured by the ponderal index (weight/length3).154 It has been suggested that the homozygous state for highaffinity Hbs would be incompatible with life because of insufficient oxygen release to tissue. This may be true; however, at least four patients with abnormal Hb levels approximating those that would be observed in homozygotes have been described with no apparent ill effects (Hb Abruzzo,155 Hb Crete,156 and Hb Headlington157). The unusually high proportion of abnormal Hb was clearly due to concurrent b-thalassemia in two cases157 and probably in the others as well. No treatment is indicated for most patients with high-affinity Hbs. Their erythrocytosis is a compensation for a physiologic state and should be regarded as “normal for them.” In the rare patient with erythrocytosis and associated symptoms, phlebotomy may be used, but caution must be used to avoid lowering the hematocrit to a point at which oxygen delivery is impaired.158 A reasonable approach is probably to phlebotomize the individual patient to the highest hematocrit at which he or she is no longer symptomatic rather than to a specific number.149,158,159 Certainly, reducing blood Hb concentrations to normal levels would be undesirable. Cytoreductive agents should not be used for treatment.

Disorders of Red Cells



Acquired Abnormalities of Hemoglobin

Moderate elevations of carboxyhemoglobin in erythrocytes shift the oxygen dissociation curve. In heavy smokers, carboxyhemoglobin concentration may reach sufficiently high levels (4.0% to 6.8%) to produce polycythemia.160,161 In the older literature, polycythemia in association with phosphorus poisoning has been described, although it may have been merely relative erythrocytosis resulting from acute liver damage. Although certain drugs and chemicals (e.g., nitrites, nitrates, aniline dyes, sulfonamides, and nitrobenzene) produce toxic levels of methemoglobin, sulfhemoglobin, or both in the blood of even normal persons,162–167 erythrocytosis apparently has not been described in patients with toxic methemoglobinemia.

Familial Polycythemia (Physiologically Appropriate) Familial defects in 2,3-BPG metabolism (e.g., BPG mutase deficiency168 or elevated erythrocyte adenosine triphosphate169), which would have the effect of shifting the oxygen dissociation curve to the left, provide other physiologically appropriate (tissue hypoxia) reasons for polycythemia. A specific pyruvate kinase

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mutation resulting in higher than normal enzyme activity and decreased 2,3-BPG causing erythrocytosis has been described as well in association with erythrocytosis.170

Secondary Polycythemia (Physiologically Inappropriate [Normoxic]) Unlike patients with physiologically appropriate secondary polycythemia, individuals with inappropriate polycythemia receive no benefit from the higher red cell concentrations. Treatment should be aimed at correcting the underlying disease; phlebotomy can be considered in the symptomatic.

Aberrant Erythropoietin Secretion Erythrocytosis has been described in association with a variety of neoplasms, cysts, vascular abnormalities, and endocrinologic disorders. In the syndromes discussed in the preceding section, erythrocytosis was secondary (i.e., driven by increased erythropoietin); however, this erythropoietin secretion and the consequent erythrocytosis were physiologic responses to tissue hypoxia. In this section, disorders in which erythropoietin-driven erythrocytosis bears no relation to physiologic requirements are reviewed (Table 44.4).

Renal Disorders

Renal cell carcinoma (hypernephroma) is one of the disorders most frequently associated with erythrocytosis. Erythrocytosis is observed in 0.9% to 1.6% of patients with renal cell carcinoma (approximately one fourth as frequent a finding as anemia).171 Elevated serum erythropoietin levels, however, are observed in more than 60% of patients.172 Erythrocytosis also has been reported in patients with renal sarcoma, hemangioma, adenoma,173 Wilms tumor,153,174 renal cysts, hydronephrosis,175–177 horseshoe kidney,175 and polycystic kidneys.173 Renal artery stenosis has also been reported in association with erythrocytosis.178 Erythrocytosis in renal cell carcinoma is attributed to constitutive erythropoietin production by the tumor. Erythropoietin messenger RNA can be demonstrated in renal carcinoma cells.179 It is assumed that this is also the mechanism by which other parenchymal renal diseases produce erythrocytosis. Hydronephrosis and anatomic abnormalities probably produce erythrocytosis by increasing pressure on erythropoietin-producing cells in the renal parenchyma.180 Significant and measurable concentrations of erythropoietin (sometimes >100 mU/ml) can be detected in fluid aspirated from renal cysts associated with polycythemia. Production of erythropoietin in renal cell carcinoma is said to predict a good response to therapy.181 Management of erythrocytosis in these patients should be directed at treatment of the responsible renal lesion with phlebotomy as an adjunct, when necessary. Erythrocytosis is also observed in patients after renal transplantation.182 This phenomenon is associated with elevated serum erythropoietin; the source of erythropoietin is presumed to be the transplant recipient’s native kidneys.183,184 Effective therapeutic modalities include phlebotomy, angiotensin-converting enzyme inhibitors, and theophylline.185–187

Liver Diseases

During fetal development, the liver contributes to erythropoietin production (Chapter 6); hepatic disease, like renal disease, may be associated with erythropoietin production and polycythemia. Erythrocytosis has been identified in persons with hepatocellular carcinoma with incidence 2.5% to 10.0%.188,189 When measured, red cell mass has been shown to be increased,190 and elevated serum erythropoietin levels have also been described.191 As with

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TA B L E 4 4 . 4

Disorders Associated With Normoxic Secondary Polycythemia Renal Disease Renal cell carcinoma Renal sarcomaa Renal adenomaa Renal hemangiomaa Wilms tumora Solitary renal cysts Polycystic kidney disease Hydronephrosis Horseshoe kidneya Renal artery stenosisa Postrenal transplantation Hepatic Disease Hepatocellular carcinoma Hepatic hamartomaa Hepatic metastasesa Hepatic angiosarcomaa Hepatic angiomaa Viral hepatitisa Vascular cerebellar tumors Other Neoplasms Uterine leiomyomata Uterine fibroid tumorsa Cutaneous leiomyomataa Meningiomaa Placental trophoblastic tumorsa Chronic lymphocytic leukemiaa Systemic amyloidosisa Atrial myxomaa Endocrine Disorders Cushing syndrome Primary aldosteronism Virilizing ovarian tumors Bartter syndromea Pheochromocytomaa Other Human immunodeficiency virus infectiona a Polycythemia

infrequently reported.

renal cell carcinoma, erythropoietin production by the tumor has been demonstrated.192,193,194 Remission of erythrocytosis may be observed after successful tumor treatment.193,194 Erythrocytosis has also been reported with hepatic hamartomas and tumors metastatic to liver,173 as well as hepatic angiomas195 and hemangiosarcomas.196 Polycythemia has been reported in the early stages of viral hepatitis.197,198 Cirrhosis is occasionally listed in texts as associated with erythrocytosis, but this apparently does not occur except in the setting of another disease, such as cirrhosis with hepatocellular carcinoma.

Cerebellar Vascular Tumors The association of erythrocytosis with vascular tumors of the cerebellum is well established.173,199 Elevated serum erythropoietin

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levels and tumor production of erythropoietin have been demonstrated,200–202 but abnormalities in Von Hippel-Lindau protein are observed as well.203–205 Correction of erythrocytosis may be observed after effective therapy and erythrocytosis may return with recurrence of the tumor.173

Leiomyoma and Fibroid Tumors of the Uterus

Several cases in which large leiomyomas and fibroid tumors of the uterus were associated with erythrocytosis have been reported.206,207 Erythrocytosis tends to subside after effective therapy and is also associated with production of erythropoietin by tumor.207,208 Cutaneous leiomyomata have also been associated with erythrocytosis.209

Other Neoplasms

Rare instances of erythrocytosis in association with a variety of other tumors have been reported, but some of these associations may be coincidental.173 However, erythropoietin synthesis by tumor cells has been clearly demonstrated in a patient with meningioma.210 Erythrocytosis has also been reported in rare patients with chronic lymphocytic leukemia,211 systemic amyloidosis,212 placental trophoblastic tumors,213 and atrial myxomas.214

Endocrinologic and Other Disorders

Erythrocytosis has been reported in association with a number of endocrinologic disorders, including Cushing syndrome, primary aldosteronism,215 virilizing ovarian tumors,216 Bartter syndrome,217 and pheochromocytoma.218,219 In the latter disorder, tumor erythropoietin production has been reported.220 There have been a number of reports describing small numbers of patients with human immunodeficiency virus infection and polycythemia.221,222–226 It is unclear if there is an actual pathophysiologic association or if this is coincidental.

Drug-induced Erythrocytosis Anabolic and androgenic steroids may be abused by both recreational and professional athletes for purposes of improving performance.227 A consequence of androgen administration, either medicinal or extralegal, may be erythrocytosis.228,229,230 In some cases, the degree of erythrocytosis may be severe. Recombinant human erythropoietin has also been abused by athletes (particularly those in endurance sports) to increase the red cell mass and thus oxygen-carrying capacity.231,232,233 As indicated earlier in Figure 44.2, this may backfire if the athlete becomes hypovolemic as a result of exertion. Cases of surreptitious erythropoietin self-administration resulting in accelerated hypertension and unstable angina have been reported.234 A perceived advantage of erythropoietin over androgens for this purpose is the inability to distinguish endogenous from exogenous erythropoietin as well as the lack of hepatic toxicity. Newer approaches that allow discrimination between exogenous recombinant erythropoietin and endogenous erythropoietin may make this practice less frequent.235,236,237,238

Familial Polycythemia (Physiologically Inappropriate) Kindreds that exhibit an autosomal recessive erythrocytosis associated with increased erythropoietin production have been described.239

Idiopathic Polycythemia The term idiopathic polycythemia (or erythrocytosis) refers to patients who have an elevated red cell mass of unknown etiology after appropriate investigation. It would include most of the patients formerly categorized as “benign erythrocytosis.” The existence of this group, which is estimated to contain 20% to 30%

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of patients evaluated for polycythemia,141 essentially represents a failure to categorize all polycythemic patients correctly. Of 25 patients reported in one series, 12 were found to have elevated erythropoietin levels and were therefore assumed to represent patients with secondary polycythemia; these patients tended to be younger than the patients with normal erythropoietin levels.139 Progenitor culture studies were not helpful in subcategorizing the group in this particular study.139 Some studies have reported endogenous colony-formation studies to be useful and serum erythropoietin levels not helpful,50 whereas others have reported the opposite.240 Kiladjian et al. treated 39 patients with idiopathic erythrocytosis with pipobroman and compared their clinical course to 140 concurrently treated polycythemia vera patients.241 The risk of leukemia, thrombosis, and myelofibrosis was the same in the two groups. This study confirms that the idiopathic erythrocytosis group contains a certain number of polycythemia vera patients; however, it does not provide a way to identify individuals who do not have a myeloproliferative disorder and therefore should not be exposed to leukemogenic agents.241 Because this category probably represents a mixed bag, including early polycythemia vera, mild secondary polycythemia, and normal individuals at the higher end of the bell-shaped curve for red cell mass,64 a cautious approach is warranted. Observation may be the most reasonable intervention; this may be the patient subset in which otherwise low-yield studies, such as erythroid progenitor studies, are likely to be useful. Periodic retesting for the JAK2 V617F mutation or other JAK2 mutations may also be useful, although a study that addressed this question found that less than 2% of idiopathic erythrocytosis patients studied exhibited the mutation.242

Disorders of Red Cells



Selected References The full reference list for this chapter can be found in the online version. 2. Schwarz TH, Hogan LA, Endean ED, Roitman IT, Kazmers A, Hyde GL. Thromboembolic complications of polycythemia: polycythemia vera versus smoker’s polycythemia. J Vasc Surg 1993;17:518–522. 7. Osler W. Chronic cyanosis with polycythemia and enlarged spleen; a new clinical entity. American Journal of the Medical Sciences 1903;126: 187–201. 16. Milligan DW, MacNamee R, Roberts BE, Davies JA. The influence of irondeficient indices on whole blood viscosity in polycythaemia. British Journal of Haematology 1982;50:467–473. 18. Thomas DJ, Marshall J, Ross Russell RW, et al. Effect of haematocrit on cerebral blood-flow in man. Lancet 1977;2:941–943. 21. Humphrey PRD, Michael J, Pearson TC. Management of relative polycythaemia:studies of cerebral blood flow and viscosity. British Journal of Haematology 1980;46:427–433. 38. Djulbegovic B, Hadley T, Joseph G. A new algorithim for the diagnosis of polycythemia. American Family Physician 1991;44:113–120. 39. Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood 2007;110:1092–1097. 42. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779–1790. 43. Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005;106(6):2162–2168. 44. Means RT. JAK2 V617F mutation testing in polycythemia vera: use and impact in an academic practice. Am J Med Sci 2008;336(4):327–329. 45. Schnittger S, Bacher U, Haferlach C, et al. Detection of JAK2 exon 12 mutations in 15 patients with JAK2V617F negative polycythemia vera. Haematologica. Mar 2009;94(3):414–418. 46. Reid CDL. The significance of endogenous erythroid colonies in haematological disorders. Blood Reviews 1987;1:133–140. 48. Birgegard G, Wide L. Serum erythropoietin in the diagnosis of polycythemia and after phlebotomy treatment. British Journal of Haematology 1992;81:603–606. 53. Westwood N, Dudley JM, Sawyer B, Messinezy M, Pearson TC. Primary polycythaemia: diagnosis by non-conventional positive criteria. European Journal of Haematology 1993;51:228–232. 61. Messinezy M, Pearson TC. Apparent polycythaemia: diagnosis, pathogenesis and management. European Journal of Haematology 1993;51:125–131. 63. Messinezy M, Pearson TC. A retrospective study of spparent and relative polycythaemia: associated factors and early outcome. Clinical and Laboratory Haematology 1990;12:121–129.

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71. de la Chapelle A, Sistonen P, Lehaslaiho H, Ikkala E, Juvomen E. Familial erythrocytosis genetically linked to erythropoietin receptor gene. Lancet 1993;1:82–84. 73. Sergeyeva A, Gordeuk VR, Tokarev YN, Sokol L, Prchal JF, Prchal JP. Congenital polycythemia in Chuvashia. Blood 1997;89:2148–2154. 79. Gordeuk VR, Prchal JT. Vascular complications in Chuvash polycythemia. Semin Thromb Hemost 2006;32(3):289–294. 83. Richalet JP, Souberbielle JC, Antezana AM, et al. Control of erythropoiesis in humans during prolonged exposure to the altitude of 6,542 m. Am J Physiol Mar 1994;266(3 Pt 2):R756– R764. 98. Wright A, Brearey S, Imray C. High hopes at high altitudes: pharmacotherapy for acute mountain sickness and high-altitude cerebral and pulmonary oedema. Expert opinion onpharmacotherapy Jan 2008;9(1):119–127. 115. Ge RL, Helun G. Current concept of chronic mountain sickness: pulmonary hypertension-related high-altitude heart disease. Wilderness&environmental medicine Fall 2001;12(3):190–194. 119. Murray JF. Classification of polycythemic disorders with comments on the diagnostic value of arterial blood oxygen analysis. Annals of Internal Medicine 1966;64(4):892–903. 138. Messinezy M, Sawyer B, Westwood NB, Pearson TC. Idiopathic erythrocytosis— additional new study techniques suggest a heterogenous group. European Journal of Haematology 1994;53:163–167. 140. Moore-Gillon JC, Treacher DF, Gaminara EJ, Pearson TC, Cameron IR. Intermittent hypoxia in patients with unexplained polycythemia. British Medical Journal 1986;293:588–590. 146. Wajcman H, Galacteros F. Hemoglobins with high oxygen affinity leading to erythrocytosis. New variants and new concepts. Hemoglobin 2005;29(2):91–106. 148. Charache S, Achuff S, Winslow R, Adamson J, Chervenick P. Variability of the homeostatic response to altered p50. Blood Dec 1978;52(6):1156–1162.

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153. Charache S, Catalano P, Burns S, et al. Pregnancy in carriers of high-affinity hemoglobins. Blood Mar 1985;65(3):713–718. 160. Smith JR, Landaw SA. Smokers’ polycythemia. N Engl J Med. Jan 5 1978;298(1):6–10. 179. Mitus WJ, Toyama K, Brauer MJ. Erythrocytosis, juxtaglomerular apparatus (JGA), and erythropoietin in the course of experimental unilateral hydronephrosis in rabbits. Ann N Y Acad Sci. Mar 29 1968;149(1):107–113. 183. Martino R, Oliver A, Ballarin JM, Remacha AF. Postrenal transplant erythrocytosis: further evidence implicating erythropoietin production by the native kidney. Annals of Hematology 1994;68:201–203. 193. Sakisaka S, Watanabe M, Tateishi H, et al. Erythropoietin production in hepatocellular carcinoma cells associated with polycythemia: immunohistochemical evidence. Hepatology Dec 1993;18(6):1357–1362. 198. So CC, Ho LC. Polycythemia secondary to cerebellar hemangioblastoma. Am J Hematol Dec 2002;71(4):346–347. 221. Battan R, Ottaviano P, Porcelli M, Distenfeld A. Polycythaemia in patient with AIDS. Lancet 1990;335:1342–1343. 227. Dickerman RD, Pertusi R, Zachariah NY, Schaller F. Androgen-induced erythrocytosis. Am J Hematol Nov 1998;59(3):263–264. 233. Brown KR, Carter, Jr., Lombardi GE. Recombinant erythropoietin overdose. American Journal of Emergency Medicine 1993;11:619–621. 237. Sharpe K, Ashenden MJ, Schumacher YO. A third generation approach to detect erythropoietin abuse in athletes. Haematologica Mar 2006;91(3):356–363. 239. Shih LY, Lee CT, See LC, et al. In vitro culture growth of erythroid progenitors and serum erythropoietin assay in the differential diagnosis of polycythaemia. European Journal of Clinical Investigation 1998;28:569–576. 241. Percy MJ, Jones FG, Green AR, Reilly JT, McMullin MF. The incidence of the JAK2 V617F mutation in patients with idiopathic erythrocytosis. Haematologica 2006;91(3):413–414.

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PART

V

Disorders of Hemostasis and Coagulation SECTION 1

I nt r o d u ction

C h a p te r 4 5

George M. Rodgers, Christopher M. Lehman

Except for that which occurs during menstruation, spontaneous bleeding is abnormal. Surprisingly, little blood is lost, even after large injuries, because of the efficiency with which vascular integrity is normally maintained and the rapidity with which it is restored after injury. In general, these phenomena reflect the functional effectiveness of normal hemostasis (see Chapters 17 through 19). It must be recognized, however, that the adequacy of hemostasis is only relative, and despite the presence of normal vessels, platelets, and coagulation factors, bleeding can occur as the result of localized pathologic processes. The 11 chapters in Part V deal with disorders that result from abnormalities of the hemostatic process. This chapter is a summary of the diagnostic approach to these disorders and includes a brief discussion of laboratory methods for their study. In subsequent chapters, individual disorders are considered in six categories: thrombocytopenia (Chapters 46 through 49), bleeding disorders caused by vascular abnormalities (Chapter 50), thrombocytosis (Chapter 51), disorders of platelet function (Chapter 52), inherited coagulation disorders (Chapter 53), and acquired coagulation disorders (Chapter 54). The pathophysiology of thrombosis and the principles of antithrombotic therapy are summarized in Chapter 55.

Clinical Evaluation of the Bleeding Patient A careful evaluation of the patient presenting with a bleeding disorder can often provide valuable clues as to whether the abnormality resides in the vessels, platelets, or the process of blood coagulation; a carefully obtained history can usually establish whether the disorder is inherited or acquired; and the physical examination may reveal findings such as the characteristic skin lesions of hereditary hemorrhagic telangiectasia, which alone may provide the diagnosis of a previously perplexing bleeding problem. Results of the clinical evaluation should lead to a rational and efficient laboratory investigation. It is important to ask specific questions about bleeding because people with normal hemostasis may believe they bleed excessively.1 Certain questions may discriminate between those with normal and abnormal hemostasis, including whether excessive bleeding occurs after tooth extraction or small cuts, whether spontaneous bruising or muscle bleeding occurs, or whether the patient has ever been transfused or treated with blood products.1

Manifestations of Disordered Hemostasis Certain signs and symptoms are virtually diagnostic of disordered hemostasis. They can be divided arbitrarily into two groups: those seen more often in disorders of blood coagulation and those most commonly noted in disorders of the vessels and platelets. The latter group is often called purpuric disorders because cutaneous and mucosal bleeding usually are prominent. The clinical findings that are most valuable in distinguishing between these two broad categories are summarized in Table 45.1. Although these criteria are relative, they provide valuable clues to the probable diagnosis if they are applied to the predominant clinical features in a given patient.

Disorders of Hemostasis and Coagulation

Diagnostic Approach to the Bleeding Disorders

TABLE 45.1

Clinical Distinction Between Disorders of Vessels or Platelets and Disorders of Blood Coagulation Finding Petechiae Deep dissecting hematomas Superficial ecchymoses Hemarthrosis Delayed bleeding Bleeding from superficial cuts and scratches Sex of patient

Positive family history

Disorders of Coagulation

Disorders of Platelets or Vessels

Rare Characteristic

Characteristic Rare

Common; usually large and solitary Characteristic Common Minimal

Characteristic; usually small and multiple Rare Rare Persistent; often profuse

80%–90% of inherited forms occur only in male patients Common

Relatively more common in females Rare (except von Willebrand disease and hereditary hemorrhagic telangiectasia)

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Part v  Disorders of Hemostasis and Coagulation  •  SECTION 1  Introduction

Bleeding into Skin and Soft Tissues Petechiae are characteristic of an abnormality of the vessels or the platelets, such as thrombocytopenia, and are exceedingly rare in the coagulation disorders. These lesions are small capillary hemorrhages ranging from the size of a pinhead to much larger (Fig.  45.1). They characteristically develop and regress in crops and are most conspicuous in areas of increased venous pressure, such as the dependent parts of the body and areas subjected to pressure or constriction from girdles or stockings. In patients with scurvy, petechiae may be distributed around hair follicles in the “saddle area” of the thighs and buttocks (see Fig. 50.5). Petechiae must be distinguished from small telangiectasias and angiomas. Vascular structures such as telangiectasias or angiomas blanch with pressure, whereas petechiae do not. In the purpuric disorders, petechiae commonly are associated with multiple superficial ecchymoses, which usually develop without perceptible trauma but seldom spread into deeper tissues. Small isolated ecchymoses are commonly noted in apparently normal women, especially on the legs, and in small children. Although large superficial ecchymoses may be seen in association with the coagulation disorders, the most characteristic lesion is the large spreading hematoma (Fig. 45.2). Such hematomas may arise spontaneously or after trivial trauma and often spread to involve an entire limb by dissecting within muscles and deep fascial spaces, often with minimal discoloration of the overlying skin.

Hemarthrosis Hemorrhage into synovial joints is virtually diagnostic of a severe inherited coagulation disorder, most commonly hemophilia A or hemophilia B, and is rare in disorders of the vessels and platelets or in acquired coagulation disorders. This disabling problem often develops with pain and swelling as chief symptoms but without discoloration or other external evidence of bleeding (see Fig.  53.3). Subperiosteal hemorrhages in children with scurvy

Figure 45.2. Large dissecting hematoma of thigh in a patient with hemophilia.  A. The lesion resulted from a slight bump to the inguinal area and spread to involve the entire thigh. (Courtesy of Dr. John Lukens.)

and swollen painful joints that may develop in some patients with allergic purpura occasionally may be confused with hemarthrosis.

Traumatic Bleeding

Figure 45.1. Diffuse petechial rash induced by a tourniquet in a patient with chronic idiopathic thrombocytopenic purpura (platelet count = 40 × 109/L).

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The unavoidable traumas of daily life and even minor surgical procedures are a greater challenge to hemostasis than any test yet developed in the laboratory. In contrast to “spontaneous” bleeding manifestations, bleeding after trauma in a person with a hemorrhagic diathesis differs in a quantitative way from that which would normally be expected in terms of the amount, duration, and magnitude of the inciting trauma. Such variables are extremely difficult to assess accurately by taking the patient’s history. The amount of blood lost may be exaggerated by the patient. The  need for transfusions and the number administered may serve as a rough guide. The patient’s statement concerning the duration of bleeding is more reliable. Detailed inquiry as to past injuries and operations must be made because the patient is likely to forget procedures or injuries that were uncomplicated and to dwell on those in which bleeding was a problem. Whether reoperation was required for prolonged bleeding after tooth extraction or other minor surgical procedures may be helpful in identifying a patient with abnormal hemostasis. In individuals with a coagulation disorder, the onset of bleeding after trauma often is delayed. For example, bleeding after a tooth extraction may stop completely, only to recur in a matter of hours and to persist despite the use of styptics, vasoconstrictors, and packing. The temporary hemostatic adequacy of the platelet plug despite defective blood coagulation may explain this phenomenon of delayed bleeding, as well as the fact that patients with coagulation disorders seldom bleed abnormally from small superficial cuts such as razor nicks. In contrast, posttraumatic or postoperative surgical bleeding in thrombocytopenic patients usually is immediate in onset, as a rule responds to local measures, and rarely is as rapid or voluminous as that encountered

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in patients with coagulation disorders. However, it may persist for hours or days after surprisingly small injuries. Valuable information often is obtained from a careful review of dental procedures, because most patients have had one or more teeth extracted at some time during their lives. The amount of bleeding normally encountered varies greatly, but as a rough guide, uncomplicated extraction of a single molar tooth may result in brisk bleeding for up to 1 hour and slight oozing for up to 2 days in normal persons.2 Typically, bleeding is more profuse from upper than from lower sockets and is more extensive after extraction of molar teeth, particularly impacted third molars, than after removal of other teeth. In patients with inherited coagulation disorders, the shedding of deciduous teeth often is uncomplicated. The response to trauma is an excellent screening test for the presence of an inherited hemorrhagic disorder, and a history of surgical procedures or significant injury without abnormal bleeding is equally good evidence against the presence of such a disorder. The removal of molar teeth is a major challenge to hemostasis, as is a tonsillectomy, and it is a rare hemophiliac, however mildly affected, who can withstand these procedures without excessive bleeding.

Miscellaneous Bleeding Manifestations Despite the fact that structural causes for bleeding (such as polyps, varices, and tumors) are commonly seen in patients with hematuria, hematemesis, and melena, bleeding from these sites may also be associated with both purpuric and coagulation disorders. Severe menorrhagia may be the sole symptom of women with von Willebrand disease (vWD), mild thrombocytopenia, or autosomally inherited coagulation disorders. Recurrent gastrointestinal bleeding or epistaxis in the absence of other bleeding manifestations is common in hereditary hemorrhagic telangiectasia. A coagulation disorder or a disorder of platelet function should be considered if protracted hematuria is the only symptom. Bleeding into serous cavities and internal fascial spaces often occurs in patients with inherited coagulation disorders and may create serious diagnostic problems. In hemophilia, retroperitoneal hemorrhage or bleeding into the psoas sheath may mimic appendicitis, and hemorrhage into the bowel wall may be confused with intestinal obstruction. Signs and symptoms simulating a variety of acute intra-abdominal disorders also may be seen in association with allergic purpura. Bleeding into the central nervous system may complicate thrombocytopenia and may occur after minor trauma in patients with coagulation disorders. Multiple small retinal hemorrhages are common in patients with thrombocytopenia and other purpuric disorders but are uncommon in those with inherited coagulation disorders; large hematomas of the orbit may be seen in the latter group. The coexistence of bleeding and thromboembolic phenomena or bleeding from previously intact venipuncture sites is suggestive of diffuse intravascular coagulation (DIC). Protracted wound healing, wound dehiscence, and abnormal scar formation have been described in inherited afibrinogenemia, the dysfibrinogenemias, and in factor XIII deficiency.3 Hemoptysis rarely is associated with hemorrhagic disorders.

Clinical Features of Inherited Bleeding Disorders An inherited bleeding disorder is suggested by the onset of bleeding symptoms in infancy and childhood, a positive family history (particularly if it reveals a consistent genetic pattern), and laboratory evidence of a single or isolated abnormality, most commonly the deficiency of a single coagulation factor.

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Age at Onset: Bleeding in the Neonate Birth and the neonatal period provide unique challenges to the hemostatic mechanism,4 and bleeding during the first month of life often is the first evidence of an inherited disorder of hemostasis. Small cephalohematomas and petechiae are common in the newborn as a result of the trauma of delivery. Large cephalohematomas that progressively increase in size may result from hemophilia but are more common in association with acquired bleeding disorders such as hemorrhagic disease of the newborn (see Chapter 54). Bleeding from the umbilical stump and after circumcision is common in the acquired coagulation disorders, and it also occurs in the inherited coagulation disorders,5 with the exception of hypofibrinogenemia, dysfibrinogenemia, and factor XIII deficiency. The onset of bleeding from the umbilical cord may be delayed in these latter disorders. In the evaluation of bleeding in the neonate, the clinician should remember that hematochezia and hematemesis may originate from swallowed blood of maternal origin. Simple tests to distinguish such maternal blood from fetal blood have been described.5 Many infants with inherited coagulation disorders do not bleed significantly in the neonatal period. Less than one-third of patients with hemophilia A and B and only 10% of those with other inherited coagulation disorders have hemorrhagic symptoms during the first week of life. In such patients, the disorder may become clinically silent for a time. Hematomas may first be seen only when the child becomes active. Hemarthrosis commonly does not develop until a child is 3 or 4 years of age. A mild inherited hemorrhagic disorder may be difficult to distinguish from the insidious onset of an acquired defect. Patients with mild inherited coagulation disorders may enter adult life before characteristic bleeding manifestations occur. These patients and those with some forms of inherited thrombocytopenia and disordered platelet function often describe a history of posttraumatic bruising and hematoma formation that they have come to accept as normal. In hereditary hemorrhagic telangiectasia, the lesions become more prominent with advancing age and may not be symptomatic until middle age. Similarly, in patients with Ehlers-Danlos syndrome, bleeding may not be a problem until adult life.

Disorders of Hemostasis and Coagulation



Family History The family history is of great importance in the evaluation of bleeding disorders. In disorders inherited as autosomal dominant traits with characteristic symptoms and high penetrance, such as hereditary hemorrhagic telangiectasia, an accurate pedigree spanning several generations can often be obtained. The presence of typical bleeding manifestations in male siblings and maternal uncles is virtually diagnostic of X-linked recessive inheritance, which characterizes hemophilia A and hemophilia B. In such X-linked traits, the family history also may be helpful in a negative sense—that is, it may clearly exclude the disorder in certain offspring, such as the sons of a known hemophiliac. Details of the various genetic patterns that may be encountered are discussed in the chapters that deal with these conditions. The limitations of the family history, however, are greater than is commonly realized. Hearsay history is difficult to evaluate, and it is often impossible to assess the significance of easy bruising or to differentiate between manifestations of a generalized bleeding disorder and more common localized lesions, such as peptic ulcer and uterine leiomyomas. A negative family history is of no value in excluding an inherited coagulation disorder in an individual patient. As many as 30% to 40% of patients with hemophilia A have a negative family history.6 The family history usually is negative in the autosomal recessive traits, and consanguinity, which is commonly present in these kindreds, is notoriously difficult to document or exclude.

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Part v  Disorders of Hemostasis and Coagulation  •  SECTION 1  Introduction

Clinical Features of Acquired Bleeding Disorders Generalized bleeding may be a prominent feature of a wide variety of acquired disorders that encompass virtually the entire field of medicine. Bleeding manifestations usually are less severe than in the inherited forms, and the clinical picture often is dominated by evidence of the underlying disorder rather than by bleeding alone. In the neonate, for example, DIC usually is associated with significant complications such as sepsis, hypoxia, acidosis, or problems related to prematurity. The physician should suspect sepsis or occult thrombosis in any sick neonate with unexplained thrombocytopenia.5 Multiple hemostatic defects commonly are present in patients with acquired hemorrhagic diseases, which often include thrombocytopenia and significant coagulation abnormalities. In contrast, a single abnormality usually is found in patients with inherited hemorrhagic disorders. In general, the emphasis of the study of the acquired bleeding disorders should be on the patient, not on the laboratory. A thorough history and the physical examination often reveal the cause of thrombocytopenia, such as a drug or acute leukemia. In most vascular disorders, including senile purpura, allergic purpura, scurvy, and amyloidosis, the history and physical examination are of primary diagnostic importance, and the laboratory has little to offer.

Drug History The importance of exhaustive interrogation regarding drug use and chemical exposure cannot be overemphasized. The list of drugs associated with thrombocytopenia (see Table 47.6) or vascular purpura grows longer each year. Less common but more serious is drug-induced aplastic anemia, which may present initially with bleeding. Many commonly used drugs, notably aspirin, impair platelet function and produce abnormal findings on laboratory tests that often lead to expensive and unnecessary additional laboratory studies. The same drugs may provoke bleeding when administered to patients with pre-existing hemostatic defects such as hemophilia

A. Drug ingestion also may produce coagulation abnormalities, and drugs that potentiate or antagonize the anticoagulant effects of coumarin derivatives may lead to bleeding or erratic laboratory control. The surreptitious ingestion of such agents is not uncommon. Results of various coagulation tests may be abnormal in a surprisingly large percentage of hospitalized patients because of heparin that is administered therapeutically or is used in small amounts to maintain the patency of indwelling venous catheters, venous pressure lines, arteriovenous shunts, and various pumps and infusion machines. The partial thromboplastin time (PTT), in particular, may be greatly prolonged in patients who have received even a minute amount of this anticoagulant. Such coagulation abnormalities often are confused with DIC, inhibitors of factor VIII, and other serious coagulation disorders, and they commonly lead to repeated, and usually useless, coagulation studies. A thorough bedside inventory often is required to find out that heparin is indeed responsible. Prolongation of the thrombin time associated with a normal reptilase time or direct assay of heparin provides laboratory evidence of heparin contamination.

Laboratory Methods for Study of Hemostasis and Blood Coagulation No single test is suitable for the laboratory evaluation of the overall process of hemostasis and blood coagulation, but methods of varying complexity and use are available for assessing various components and functions individually. The emphasis of the following discussion is on methods that are simple and widely available in most laboratories. The interpretation of the most commonly used tests and the range of values obtained in normal subjects with representative techniques are summarized in Table 45.2. Definitive coagulation methods usually require a specially equipped laboratory and trained personnel, and are discussed here from a general standpoint only. Additional comments concerning the use and limitations of the various methods are included in chapters dealing with individual disorders.

TA B L E 45.2

Interpretation of Common Tests of Hemostasis and Blood Coagulation Test Platelet count   Phase microscopy  Automated Partial thromboplastin time (activated)b Prothrombin timeb

Normal Rangea (±2 SD)

Common Causes of Abnormalities

140,000–440,000/ml 177,000–406,000/ml 26–36 sec55,c

Thrombocytopenia, thrombocytosis

12.0–15.5 sec66,c

Thrombin timeb

14.7–19.5 sec

Fibrinogen assayb

150–430 mg/dl70

Factor VIII assayb Fibrin degradation product assay

50–150 U/dl

D-dimer assay

0–0.4 mg/ml

0–5 mg/ml80

Deficiencies or inhibitors of prekallikrein; high-molecular-weight kininogen; factors XII, XI, IX, VIII, X, and V; prothrombin or fibrinogen; lupus inhibitors; heparin; warfarin Deficiencies or inhibitors of factors VII, X, and V; prothrombin or fibrinogen; dysfibrinogenemia; lupus inhibitors; heparin; warfarin Afibrinogenemia, dysfibrinogenemia, hypofibrinogenemia, and hyperfibrinogenemia; inhibitors of thrombin (heparin) or fibrin polymerization (fibrin degradation products, paraproteins) Afibrinogenemia, dysfibrinogenemia, and hypofibrinogenemia; inhibitors of thrombin or fibrin polymerization Hemophilia A and von Willebrand disease; acquired antibodies to factor VIII Disseminated intravascular coagulation; fibrinogenolysis; thrombolytic drugs, liver disease; ­dysfibrinogenemia Disseminated intravascular coagulation; recent surgery; pregnancy; thromboembolism

aNormal range in the University of Utah coagulation laboratory. bTests affected by heparin. cSignificant variations depending on reagents and technique.

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Bleeding Time Hemostasis in a small superficial wound, such as that produced when measuring the bleeding time, depends on the rate at which a stable platelet plug is formed and, thus, provides a measure of the efficiency of the vascular and platelet phases. However, it does not discriminate between vascular defects, thrombocytopenia, and platelet dysfunction. The bleeding time leaves much to be desired in terms of reproducibility, because no two skin areas are exactly the same and it is impossible to produce a truly standard wound.7 Older studies using the bleeding time test supported the view that this test might be helpful in predicting bleeding in individual patients.8 More recent studies suggest that a bleeding time result is determined not only by platelet number and function, but also by hematocrit,9 certain components of the coagulation mechanism,10,11 skin quality,12 and technique.13 A careful analysis of this literature indicates that there is no correlation between a skin template bleeding time and certain visceral bleeding times,13,14 and that no correlation exists between preoperative bleeding time results and surgical blood loss or transfusion requirements.15 A clinical outcomes study reported that discontinuation of the bleeding time in a major academic medical center had no detectable adverse clinical impact.16 A position paper of the College of American Pathologists and the American Society of Clinical Pathologists concluded that the bleeding time was not effective as a screening test, and that a normal bleeding time does not exclude a bleeding disorder.17 Patients thought to have a platelet-type bleeding disorder based on their personal or family history (or both) should be evaluated for vWD and the inherited qualitative platelet disorders, using assays discussed in the section Platelet Function Assays. Newer assays that may be useful in screening patients for platelet dysfunction are also discussed in the section New Assays of Platelet Function.

Platelet Enumeration Platelets are considerably more difficult to count than erythrocytes or leukocytes. This difficulty is to be expected in view of the small size of these cells and their tendency to adhere to foreign surfaces and to aggregate when activated. In general, techniques for platelet counting may be classified into two groups: hemacytometer or direct methods, in which whole blood is diluted and the platelets are counted in much the same way as leukocytes or erythrocytes, and fully automated electronic methods. Virtually identical values for the normal range of the platelet count have been obtained with modern methods, as summarized in Table 45.2. An estimate of platelet numbers in a well-prepared blood smear by an experienced observer is a valuable check on the platelet count as determined by any method. In general, when a blood smear is examined at 100 × power, each platelet counted/field represents approximately 10,000 platelets × 109/L. Consequently, a normal blood smear should demonstrate, on average, at least 14 platelets/high-power field. Instruments for totally automated platelet counting are widely used. Details of automated cell counters are discussed in Chapter 1. When automated methods are used, various nontechnical factors may produce falsely low platelet counts.18 These factors include platelet agglutinins,19 abnormal amounts of plasma proteins in various paraproteinemias, previous contact of platelets with foreign surfaces such as dialysis membranes,20 large or giant platelets, platelet satellitism,21 lipemia,22 and ethylenediaminetetra-acetic acid (EDTA)–induced platelet clumping,23 a phenomenon that may produce platelet clumps of sufficient size to artifactually increase the leukocyte count.24 Spuriously high platelet counts may result from the presence of microspherocytes,25 fragments of leukemic or red blood cells,26 and Pappenheimer

72683_ch045_1043-1057.indd 1047

bodies.27 Special technical modifications and the use of careful manual counting methods may be required to eliminate these artifacts and to obtain accurate platelet counts.

Platelet Volume Measurements The widespread availability of particle counters in the clinical laboratory permits the accurate measurement of platelet volume on a routine basis. Mean platelet volume (MPV) is increased in disorders associated with accelerated platelet turnover as the result of large numbers of megathrombocytes28 or in patients with Bernard-Soulier syndrome. Normal or decreased values for MPV usually are obtained in patients with disorders associated with deficient platelet production, in some patients with sepsis,29 and in people with certain big-spleen syndromes.30 Some authors suggest that increased MPV provides evidence of accelerated platelet production and may be interpreted in the same way as the reticulocyte count. The method is difficult to standardize, however, and when determined on routinely collected specimens by automated counters, it is affected by numerous variables pertaining to specimen collection, anticoagulant, temperature, and duration of storage.31 In view of these problems and the difficulty in interpreting platelet size heterogeneity under normal and abnormal conditions,32 these measurements should be interpreted with caution. The presence of microcytic platelets in patients with some inherited thrombocytopenias such as Wiskott-Aldrich syndrome is reliably reflected by MPV measurements. On the other hand, giant platelets associated with Bernard-Soulier syndrome may be counted as leukocytes or erythrocytes and may not be reflected in the MPV.

Platelet Function Assays Since the 1960s, platelet aggregation using platelet-rich plasma has been the standard method to assess platelet function. This method uses aggregometers, which are modified nephelometers that permit measurement of changes in optical density of a platelet suspension under conditions of constant temperature and continuous agitation (Fig. 45.3). Most instruments measure

Disorders of Hemostasis and Coagulation

Tests of Vascular and Platelet Phases

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Chapter 45  Diagnostic Approach to the Bleeding Disorders

Optical Density (OD) of Platelet-Rich Plasma



5

0.2

Addition of 1µM ADP

0.4 4 6 0.6 3 2 0.8 1 0

1

2

3 4 Time (minutes)

5

6

7

Figure 45.3. The interpretation of aggregometer tracings.  Tracing of platelet aggregation produced by a low concentration of adenosine diphosphate (ADP), illustrating normal changes in optical density (OD)—that is, (1) a slight decrease caused by dilution with aggregating agent; (2) a transient increase caused by initial platelet swelling or shape change; (3) a rapid progressive decrease as platelet aggregates form, the size of which is roughly proportional to the amplitude of the oscillations in the tracing (4). The OD then reaches a nadir (5) from which maximal aggregation as a percentage of the initial OD may be calculated as follows: maximal aggregation (%) = OD at T0—minimum OD/OD at T0. After this (6), a slow increase in OD caused by disaggregation occurs under some conditions.

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a combination of light scatter and absorption. Instruments have been developed that permit both nephelometric and photometric measurements and the simultaneous measurement of aggregation and nucleotide release.33 Platelet aggregation usually is studied in suspensions of citrated platelet-rich plasma, in which the size and dimensions of the stirring bar, variations in plasma citrate concentration attributable to variations in hematocrit, the pH, and the nature of the buffers are important variables. Platelet suspensions usually are prepared by differential centrifugation, but methods that use albumin density gradient centrifugation and gel filtration have also been described.34 Although harvesting platelets from the blood of thrombocytopenic patients is difficult, testing such platelets in the aggregometer is reproducible in suspensions containing as few as 50,000 platelets/ml. Methods for the study of platelet aggregation in whole blood35,36 also have been described. Interfaced computer systems have been developed for calculating and expressing platelet function data.37 Adenosine diphosphate (ADP) in concentrations of 5 mmol/L or higher produces platelet aggregation directly that is independent of the release of platelet-contained ADP.38 Various other aggregating agents act mainly by inducing the release reaction, such as a suspension of connective tissue particles (collagen), epinephrine and norepinephrine, and thrombin. With epinephrine (5 mmol/L), a weak primary aggregating effect usually can be clearly distinguished from the subsequent release reaction, which produces a secondary wave of aggregation. Such primary and secondary waves of aggregation also may be seen with carefully titrated amounts of ADP (0.2 to 1.5 mmol/L).38 Ristocetin is an antibiotic that induces platelet agglutination (platelet metabolic activity not required) in the presence of von Willebrand factor (vWF). Patients deficient in vWF (vWD) or in the receptor for vWF (Bernard-Soulier syndrome) have an abnormal ristocetin response. Ristocetin is tested in concentrations of 0.6 to 1.2 mg/ml; the lower concentrations are helpful in identifying specific variants of vWD, type 2B and platelet-type vWD (see Chapters 52 and 53). The release reaction is measured only indirectly by routine aggregometry—that is, the aggregation associated with the release of ADP from the platelets (release-induced aggregation or secondary aggregation). Methods for the quantitation of various substances released from platelets have been described. For example, the amounts of ADP or serotonin released/unit of time serve as indices of dense body release39; the amount of various hydrolytic enzymes or platelet factor 4 released is a measure of the extent of a-granule release.40 Suggested guidelines for standardization of platelet aggregation methods have been proposed.41,42 Sensitive methods have been developed for the determination of platelet-derived substances in plasma that may serve as markers of intravascular platelet activation,43 including platelet factor 4, b-thromboglobulin, stable prostaglandins (6-keto prostaglandin F1a and thromboxane A2), and leukotrienes.43 These measurements may have diagnostic value in thromboembolic disorders and syndromes characterized by intravascular platelet aggregation.

New Assays of Platelet Function

An appreciation of the limitations of the bleeding time test has led to the development of newer assays to evaluate platelet function.44 Some of these are point-of-care tests. The clinical use and predictive value of these tests to identify patients with hemostatic disorders remain to be established. One assay, the platelet function analyzer (PFA-100), has been investigated for several years, and many published reports using this assay are available. In this method, citrated blood samples are exposed to high shear rates in a capillary flowing through an aperture within a membrane coated with collagen and either ADP or epinephrine.45 The closure time to hemostatic plug formation within the aperture is the

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endpoint of the test. A large study using the PFA-100 found that prolonged closure times could be attributed to specific quantitative or qualitative abnormalities in platelet function or vWF (or both) in 93% of patients tested.46 However, the International Society on Thrombosis and Haemostasis has taken the position that the PFA100 is insufficiently sensitive and specific to be used as a screening device for platelet disorders.47 It has been suggested that optimal use of the PFA-100 in evaluation of hemostasis would use an algorithmic approach, evaluating not only PFA-100 closure times, but also a complete blood count, blood smear, and assays for vWD and platelet aggregation to further evaluate abnormal closure times. A  recent addition to the PFA repertoire is the INNOVANCE PFA P2Y test designed to assess P2Y12-receptor blockade. Several additional platelet function analyzers are available on the market, though they are not as well studied as the PFA100.48 The ICHOR II-Plateletworks system (Helena Laboratories, Beaumont, TX) compares impedance-derived platelet counts in samples with and without added platelet agonists to assess platelet function. This system has historically been used to evaluate cardiopulmonary bypass patients, but more recently has been applied to patients undergoing coronary stent placement. The Impact-R (Matis Medical, Beersel, Belgium) is an automated cone-and-plate research analyzer that assesses platelet adhesion and aggregation on a polystyrene surface under laminar flow conditions. The VerifyNow system analyzes platelet agonist-induced aggregation of fibrinogen-coated microparticles to assess platelet function. Agonist cartridges are designed to evaluate the effects of aspirin, clopidogrel, and GPIIb/IIIa platelet receptor inhibitor administration on platelet function. Platelet mapping, a modification of thromboelastography, measures the platelet contribution to clot strength in the presence of specific agonists.49 To date, no platelet function analyzer assay has been sufficiently studied or validated to warrant routine clinical use.48,50,51

Tests of Coagulation Phase In general, meticulous performance of coagulation tests is more important than the exact technique chosen. Blood samples obtained by traumatic venipunctures or from indwelling catheters often are inadequate for coagulation studies.52 A poorly collected blood sample is a far more common cause of inaccurate results than is technical error. With the exception of one assay for fibrin degradation products (FDPs), all coagulation tests are performed on citrated plasma, most commonly obtained using blue-top vacuum blood collection tubes that pull in nine parts of blood to one part citrate. The  International Society for Thrombosis and Haemostasis recommends the routine use of 3.2% sodium citrate. A pool of freshly frozen citrated plasma from several normal donors is a suitable control for screening procedures in most laboratories. Lyophilized control plasma and borderline abnormal control plasmas are available commercially to standardize coagulation assays and to provide reference standards. The citrate ion does not enter the erythrocyte. Consequently, the plasma citrate concentration is abnormally high when blood with a high hematocrit (>55%) is collected in usual concentrations of this anticoagulant. This may produce artifactual prolongation of one-stage screening tests of coagulation, such as the PTT.53 To obtain interpretable data on such samples, tubes containing citrate concentrations appropriate for the hematocrit must be prepared by removing an aliquot of the citrate anticoagulant contained in standard blue-top tubes.

Activated Partial Thromboplastin Time The activated partial thromboplastin time (PTT) is a simple test of the intrinsic and common pathways of coagulation. When a mixture of plasma and a phospholipid platelet substitute is recalcified,

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Chapter 45  Diagnostic Approach to the Bleeding Disorders Contact Activation

Tissue Factor

EXTRINSIC

INTRINSIC XII

XI

om hr as pl bo

HMWK

VII

IX

tin e

m Ti

PL VIII

COMMON

V Prothrombin Fibrinogen

Fibrin

Prothrombin Time Thrombin Time

X

Stypven Time Prothrombin Time

PL

Figure 45.4. The interpretation of common screening tests of blood coagulation.  Coagulation factors are indicated within arrow-shaped blocks, which represent the major pathways of coagulation. Screening tests are indicated at the side of these blocks in relation to pathways and coagulation factors measured by each. HMWK, high-molecular-weight kininogen; PL, phospholipid; Pre-K, prekallikrein.

fibrin forms at a normal rate only if the factors involved in the intrinsic pathway (prekallikrein, high-molecular-weight kininogen, and factors XII, XI, IX, and VIII) and in the common pathway (factors X and V, prothrombin, and fibrinogen) are present in normal amounts (Fig. 45.4). Platelet substitutes of various kinds may be used, such as chloroform extract of brain54 and other crude cephalin fractions as well as soybean phosphatides (inosithin). In the PTT, such platelet substitutes are provided in excess, and the test is unaffected by the number of platelets remaining in the plasma (unless the sample contains antiphospholipid antibodies). Platelet substitutes are only partial thromboplastins, however, and they are incapable of activating the extrinsic pathway, which requires complete tissue thromboplastin (tissue factor). Thus, the PTT bypasses the extrinsic pathway and is unaffected by a deficiency of factor VII. The PTT assay is used to detect factor deficiency, screen for the lupus anticoagulant, and monitor heparin anticoagulation. The PTT is somewhat more sensitive to deficiencies of factors VIII and IX than to deficiencies of factors XI and XII or factors involved in the common pathway,55,56 but with most techniques, the test usually yields abnormal results if the plasma level of any of the essential factors is 72 hours, which made this treatment approach less efficient for bleeding patients.249 More recent studies have shown a more rapid increase of the platelet count within 24 hours and a longer duration of the increase when using a higher dose of 75 mg/kg compared with 50 mg/kg.250 Anti-D therapy now carries a black box warning that requires observation of the patient for 8 hours after administration due to the risk of intravascular hemolysis and disseminated intravascular coagulation.251

Disorders of Hemostasis and Coagulation



Treatment of Chronic Primary Immune Thrombocytopenia Many adult patients with ITP, even if they initially respond to steroids, will develop chronic ITP. Chronic ITP is defined as ITP lasting longer than 12 months.1 Patients who are asymptomatic and have platelet counts between 30,000 and 50,000/ml may be managed with careful observation.164,184 Symptomatic patients with platelet counts 30,000/ml. If patients can be maintained on 10 mg every other day, additional treatment may not be

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indicated. Some patients observed for years with platelet counts of 10,000/ml have had no significant bleeding other than ecchymoses or petechiae, even without steroid therapy.252 Splenectomy has been the traditional therapy for refractory cases of ITP. There are more options now with accumulating evidence regarding the role of rituximab and thrombopoiesis-stimulating agents.

Splenectomy Patients with severe thrombocytopenia (50,000 and less bleeding was observed in the eltrombopag arm.

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Chapter 47  Thrombocytopenia Caused by Immunologic Platelet Destruction

Platelet counts dropped again within 2 weeks of discontinuing therapy. A phase III study (RAISE) in patients with chronic ITP published in 2011 showed similar results.296 After two weeks of therapy, median platelet counts in the eltrombopag group were 53,000 to 73,500 versus 17,500 to 23,000 in the placebo arm. Patients in the eltrombopag arm had less bleeding and were more successfully tapered off their other ITP treatments. Similar results have been seen with romiplostim. Romiplostim is an injectable thrombopoeitin mimetic that is given once per week. It is a thrombopoietin mimetic peptide (peptibody) which binds to the thrombopoeitin receptor. In a randomized study published in 2008, patients with ITP were randomized in a 2:1 fashion to romiplostim versus placebo.297 In splenectomized and nonsplenectomized patients, 79% and 88% of patients receiving romiplostim achieved a platelet count >50,000. Only a very low number of patients in the placebo arm achieved this threshold (14% of nonsplenectomized patients, 0% of splenectomized patients). This correlated with decreased use of other ITP therapies. The most common side effects are arthralgias, fatigue, and nausea.298 Serious adverse events are rare. Additional trials have shown that romiplostim is safe and effective for prolonged use299 and that decreased bleeding is observed in patients receiving romiplostim due to higher platelet counts.300 With prolonged use patients do still require regular platelet monitoring as the dose required may vary over time.299 As with eltrombopag, indefinite treatment is required as platelet counts fall quickly when treatment is discontinued. There has been some concern regarding increased marrow fibrosis in patients receiving eltrombopag or romiplostim. Preclinical data showed increased fibrosis in the marrow of rats treated with romiplostim.301 This appeared to be dose dependent and decreased after the treatment was discontinued. In the same paper, a retrospective review of clinical trials using romiplostim was performed.301 Reticulin deposition was noted in 10 of 271 patients; however, most patients did not have bone marrow biopsies performed. In 5 patients who had pretreatment bone marrow biopsies, reticulin deposition increased in 4 patients. In all patients, reticulin staining decreased when romiplostim was discontinued. In 10 patients followed prospectively with serial bone marrow biopsies, only 1 of 10 patients treated developed new reticulin fibrosis on treatment. Similar findings are seen with eltrombopag. The clinical significance of this finding is unclear. Although the thrombopoietin mimetics are effective in both splenectomized and nonsplenectomized patients, expert panels recommend that they be reserved for patients with refractory ITP after splenectomy and patients with contraindications to splenectomy.5 This recommendation is based on concerns regarding the requirement for indefinite treatment which is accompanied by significant cost and risk of long-term toxicities that have not yet been identified.

Immunosuppressive Drugs Immunosuppressive therapy for ITP has yet to be evaluated thoroughly; the overall effectiveness of these potent drugs is variable, and remissions achieved have been short-lived. Poor results were reported in children.302 Favorable results are nevertheless noteworthy, because they were obtained in refractory patients who had not responded to splenectomy or steroids.303 Preliminary reports of successful treatment in refractory patients have been published using high-dose methylprednisolone234,304 and cyclophosphamide-based combination chemotherapy.305 Cyclophosphamide alone, either daily oral or pulse intravenous therapy, induced remissions in 16% to 55% of patients.14,303,306–308 However, this drug must be administered for several weeks before the platelet count rises and often must be continued for an indefinite period to maintain the remission, and side effects such as leukopenia and alopecia often are significant. Azathioprine,

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cyclosporin A, mycophenolate mofetil, actinomycin, and other immunosuppressive agents, either alone or in combination with corticosteroids, have variable success.309–315 Vincristine and vinblastine, administered intravenously at weekly intervals, may be as effective as cyclophosphamide but act more rapidly, often increasing the platelet count within 7 days.316–320 In addition to their suppressive effects on cellular and humoral immune responses, these agents increase platelet production in both animals and normal human subjects.321,322 In ITP, their mechanism of action has been postulated to be inhibition of microtubuledependent events required for monocyte–macrophage function.323

Other Proposed Therapies A number of other therapies have been reported to be successful in single case reports or in small series of patients. Danazol, an attenuated androgen, has been effective in increasing platelet counts in patients with ITP in doses ranging from 50 mg/day324 to 800 mg/day.325–327 The mechanism of action is postulated to be a danazol-induced reduction of Fc receptors on phagocytic cells.328 Long-term results indicate a final response rate of >40% at a median follow-up of 10 years with a reasonable safety profile.329 Recombinant a-interferon can increase platelet counts in up to 50% of patients when injected subcutaneously three times a week, and some responses were durable after stopping treatment.330,331

Supportive Measures Physical activity should be restricted to minimize the hazards of trauma, particularly head injury. Drugs, such as nonsteroidal anti-inflammatory drugs, that impair platelet function should be avoided. Blood loss should be treated as indicated, and platelet concentrates should be administered in the presence of significant bleeding.332 However, transfusions typically produce only a slight and transient increase in the platelet count, no doubt because of the rapidity with which they are destroyed in vivo.333 Platelet transfusions, nevertheless, may produce some increase in platelet numbers in many patients,334 often diminish bleeding for a time, and can be effective in the management of serious complications such as subarachnoid hemorrhage. They should be reserved for such life-threatening emergencies or for the immediate preoperative treatment of patients with serious hemorrhage before splenectomy. A single large dose of IVIG followed by a platelet transfusion can be effective in arresting hemorrhage in some critically ill patients.235 In most patients with platelet counts >50,000/ml, preoperative platelet transfusions are not indicated. Platelet transfusions should be avoided in patients with chronic ITP because of subsequent development of alloantibodies. Exchange plasmapheresis may be valuable in critically ill patients and may be particularly effective in children.335–337 Anovulatory hormones are useful when menorrhagia is a major complaint. Because of the risk of septicemia, polyvalent pneumococcal vaccine, Haemophilus influenzae B vaccine, and quadrivalent meningococcal polysaccharide vaccine should be administered at least 2  weeks before elective splenectomy in both adults and children.338

Disorders of Hemostasis and Coagulation



Immune Thrombocytopenia in Pregnancy Both ITP and non-ITP may occur during pregnancy. 339,340 Thrombocytopenia was present in 7% of women when they were admitted to hospital for a full-term delivery in a prospective 7-year study of 15,741 mothers and 15,932 newborns.341–343 Most platelet counts were between 100,000 and 150,000/ml, and 1% of women had platelet counts 15 × 109/L; age, anemia, and platelet count >1,000 × 109/L, are reported risk factors for leukemic transformation.27 Studies have reported that JAK2V617F-negative ET patients have a lower arterial thrombotic risk than JAK2V617F-positive ET patients.76 In contrast, the JAK2V617F allele burden itself may or may not predict a greater thrombotic diathesis.76,77 Among JAK2V617Fnegative ET patients, the presence of other markers of clonality such as mutations in human androgen receptor (HUMARA), TET2, ASXL1, or MPL, predicts an increased thrombotic risk.78

Disorders of Hemostasis and Coagulation

TA B L E 51. 3

FIGURE 51.2. Characteristic megakaryocyte proliferation in a marrow specimen from an essential thrombocythemia (ET) patient.

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Part v  Disorders of Hemostasis and Coagulation  •  SECTION 2  Thrombocytopenia

Cumulative Proportion Surviving

1.0

Essential thrombocythemia patients Reference cohort

0.9

p = 0.30

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

0

5

10 15 20

No. at risk 435 328 200 80 28

25 30 35 40 45 50 Time (years) 2

55 60 65

FIGURE 51.3. Survival curves of 435 patients with essential thrombocythemia, compared with life expectancy of the general population. From Passamonti et al., Life expectancy and prognostic factors for survival in patients with polycythemia vera and essential thrombocythemia. Am J Med 2004;117:755–761, with permission.

The standard model for thrombotic risk in ET has two components: age >60 years and history of thrombosis. The absence of both denotes low risk, and the presence of either denotes a high risk of thrombosis.79 A model proposed more recently, the International Prognostic Score for ET-thrombosis (IPSET-thrombosis), incorporates the two elements of the standard model as well as the presence of cardiovascular risk factors and JAK2V617F mutation status.80 The IPSET-thrombosis model defines low, intermediate, and high risk categories. Approximately half the patients in the low risk category from the standard model are relocated to the intermediate category in the IPSET-thrombosis model, whereas the standard model high risk patients are distributed essentially evenly between the three IPSET-thrombosis categories.80 The two models are outlined in Table 51.5.

Treatment The goal of treatment in ET is the prevention of thrombotic events, the primary cause of morbidity and mortality.26 Therefore, treatment strategy is based on thrombotic risk stratification.79,81,82 At present, only the standard risk stratification model has been

TA B L E 51.5

Prognostic Models For Thrombotic Risk In Essential Thrombocythemia

1 point 1 point 2 points 2 points

risk factors include diabetes, hypertension, and/or tobacco use.

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Aspirin therapy, usually at 100 mg/day or less, is generally well tolerated in ET patients, with a low risk of bleeding.84 Antiplatelet agents such as aspirin reduce the risk of venous thrombosis in ET patients with the JAK2V617F mutation, and the risk of arterial thrombosis in patients with cardiovascular risk factors. Its benefits in other low risk patients are unclear.85 Aspirin and antiplatelet agents should be avoided in patients with a history of bleeding. Although markedly elevated platelet counts are not a risk factor per se for aspirin-induced bleeding,85 acquired von Willebrand syndrome may be unmasked by aspirin in patients with platelet counts greater than 1,000 × 109/L.73 Many physicians regard it as prudent to assess von Willebrand factor activity prior to initiating aspirin in such patients.83

Cytoreductive Therapy Hydroxyurea Hydroxyurea is considered the first-line cytoreductive agent in ET.81,82,86,87 Some of the beneficial effects of hydroxyurea in ET and other myeloproliferative disorders may reflect reduction of leukocytosis and leukocyte-platelet interaction.88 The extent to which hydroxyurea reduces JAK2V617F expression in ET patients is disputed.89,90,91,92

Anagrelide The platelet-reducing drug anagrelide is a reasonable second choice for patients intolerant of, or unresponsive to, hydroxyurea.93,94 As a first-line agent in ET, anagrelide has been reported to be non-inferior to hydroxyurea.95 Unlike hydroxyurea, it does not affect leukocytosis.

Administration of recombinant interferon can also be considered in patients refractory to hydroxyurea or to both hydroxyurea and anagrelide.96–98 The systemic side effects and need for subcutaneous injection limit its acceptability to some patients.99 High risk pregnant females with ET can be successfully managed with interferon.100

High risk: ≥ 3 points a Cardiovascular

Aspirin

Interferon

Standard Model79  Age > 60 years   History of thrombosis Low risk: Neither factor High risk: One or both factors IPSET-Thrombosis Model80  Age > 60 years   Cardiovascular risk factorsa   History of thrombosis   JAK2V617F mutation present Low risk: 0 or 1 point Intermediate risk: 2 points

validated as a guide to therapy. Low risk patients can be managed with low dose aspirin or, under some circumstances, observation only. Cytoreduction of the platelet count is recommended for high risk patients.81,82 The IPSET-thrombosis model has not been prospectively validated as a guide to therapy, but the developers of the model have suggested that cytoreductive therapy be considered in any patient with a history of thrombosis and in older patients with cardiovascular risk factors and/or JAK2V617F mutation. Aspirin therapy is recommended for younger patients without a thrombosis history but with either cardiovascular risk factors and/or JAK2V617F mutation.80 The bleeding associated with acquired von Willebrand syndrome in ET can be resolved by reduction of the platelet count to a value approximating normal.83

Other Agents/Modalities Busulphan is an agent of established efficacy in myeloproliferative disorders such as ET,82,101 however, its risk profile as an alkylating agent makes it less attractive than hydroxyurea, anagrelide, or interferon. Apheresis can utilized to obtain rapid reduction in the platelet count in emergencies associated with severe thrombocytosis such as bleeding with acquired von Willebrand syndrome or limb gangrene or in preparation for surgery, but should be

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Chapter 51  Thrombocytosis and Essential Thrombocythemia

accompanied by cytoreductive drugs.83 JAK2 inhibitors currently available appear to be of primary benefit in post-ET myelofibrosis rather than in ET itself.

Post-essential Thrombocythemia Myelofibrosis Myelofibrosis may be a late complication of ET, with an incidence of approximately 1.6 per thousand person-years of follow-up and is the cause of death in approximately 4% of ET patients.26 Patients may develop transfusion-dependent anemia and/or thrombocytopenia, and symptomatic splenomegaly. JAK2 inhibitors appear to have benefit in the management of cytopenias and splenomegaly in myelofibrosis.102–104 In general, management follows the pattern outlined for primary myelofibrosis in Chapter 83.

Selected References The full reference list for this chapter can be found in the online version.

2. Buss DH, Cashell AW, O’Connor ML, Richards F 2nd, Case LD. Occurrence, etiology, and clinical significance of extreme thrombocytosis: a study of 280 cases. Am J Med 1994;96:247–253. 4. Aydogan T, Kanbay M, Alici O, Kosar A. Incidence and etiology of thrombocytosis in an adult Turkish population. Platelets 2006;17(5):328–331. 5. Griesshammer M, Bangerter M, Sauer T, Wennauer R, Bergmann L, Heimpel H. Aetiology and clinical significance of thrombocytosis: analysis of 732 patients with an elevated platelet count. J Intern Med 1999;245(3):295–300. 6. Stone RL, Nick AM, McNeish IA, et al. Paraneoplastic thrombocytosis in ­ovarian cancer. N Engl J Med 2012;366(7):610–618. 7. Ceresa IF, Noris P, Ambaglio C, Pecci A, Balduini CL. Thrombopoietin is not uniquely responsible for thrombocytosis in inflammatory disorders. Platelets 2007;18(8):579–582. 10. Imashuku S, Kudo N, Kubo K, Takahashi N, Tohyama K. Persistent thrombocytosis in elderly patients with rare hyposplenias that mimic essential thrombocythemia. Int J Hematol 2012;95(6):702–705. 12. Zecchina G, Ghio P, Bosio S, Cravino M, Camaschella C, Scagliotti GV. Reactive thrombocytosis might contribute to chemotherapy-related thrombophilia in patients with lung cancer. Clin Lung Cancer 2007;8(4):264–267. 13. Teofili L, Larocca LM. Advances in understanding the pathogenesis of familial thrombocythemia. Br J Haematol 2011;152(6):701–712. 18. Brusamolino E, Orlandi E, Morra E, et al. Hematologic and clinical features of patients with chromosome 5 monosomy or deletion (5q). Med Pediatr Oncol 1988;16(2):88–94. 20. Cabello AI, Collado R, Ruiz MA, et al. A retrospective analysis of myelodysplastic syndromes with thrombocytosis: reclassification of the cases by WHO proposals. Leuk Res 2005;29(4):365–370. 21. Rice L, Popat U. Every case of essential thrombocythemia should be tested for the Philadelphia chromosome. Am J Hematol 2005;78(1):71–73. 26. Passamonti F, Rumi E, Pungolino E, et al. Life expectancy and prognostic factors for survival in patients with polycythemia vera and essential thrombocythemia. Am J Med 2004;117(10):755–761.

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27. Gangat N, Wolanskyj AP, McClure RF, et al. Risk stratification for survival and leukemic transformation in essential thrombocythemia: a single institutional study of 605 patients. Leukemia 2007;21(2):270–276. 34. Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood 2007;110:1092–1097. 43. Kondo T, Okuno N, Naruse H, et al. Validation of the revised 2008 WHO diagnostic criteria in 75 suspected cases of myeloproliferative neoplasm. Leuk Lymphoma 2008;49(9):1784–1791. 44. Kwon M, Osorio S, Munoz C, Sanchez JM, Buno I, Diez-Martin JL. Essential thrombocythemia in patients with platelet counts below 600 ×10(9)/L: applicability of the 2008 World Health Organization diagnostic criteria revision proposal. Am J Hematol 2009;84(7):452–454. 54. Brecqueville M, Rey J, Bertucci F, et al. Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms. Genes Chromosomes Cancer 2012;51(8):743–755. 58. Lippert E, Boissinot M, Kralovics R, et al. The JAK2-V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera. Blood 2006;108(6):1865–1867. 67. Smalberg JH, Arends LR, Valla DC, Kiladjian JJ, Janssen HL, Leebeek FW. Myeloproliferative neoplasms in Budd-Chiari syndrome and portal vein thrombosis: a meta-analysis. Blood 2012;120(25):4921–4928. 74. Cesar JM, de Miguel D, Garcia Avello A, Burgaleta C. Platelet dysfunction in primary thrombocythemia using the platelet function analyzer, PFA-100. Am J Clin Pathol 2005;123(5):772–777. 77. Zhang S, Qiu H, Fischer BS, et al. JAK2 V617F patients with essential thrombocythemia present with clinical features of polycythemia vera. Leuk Lymphoma 2008;49(4):696–699. 79. Passamonti F. Prognostic factors and models in polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Clin Lymphoma, Myeloma Leuk 2011;11(Suppl 1):S25–S27. 80. Barbui T, Finazzi G, Carobbio A, et al. Development and validation of an International Prognostic Score of thrombosis in World Health Organizationessential thrombocythemia (IPSET-thrombosis). Blood 2012;120(26):5128–5133. 82. Barbui T, Finazzi MC, Finazzi G. Front-line therapy in polycythemia vera and essential thrombocythemia. Blood Rev 2012;26:205–211. 83. Elliott MA, Tefferi A. Pathogenesis and management of bleeding in essential thrombocythemia and polycythemia vera. Curr Hematol Rep 2004;3(5):344–351. 84. Finazzi G, Carobbio A, Thiele J, et al. Incidence and risk factors for bleeding in 1104 patients with essential thrombocythemia or prefibrotic myelofibrosis diagnosed according to the 2008 WHO criteria. Leukemia 2012;26(4):716–719. 85. Alvarez-Larran A, Cervantes F, Pereira A, et al. Observation versus antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia. Blood 2010;116(8):1205–1210, quiz 1387. 87. Dingli D, Tefferi A. Hydroxyurea: the drug of choice for polycythemia vera and essential thrombocythemia. Curr Hematol Malignancy Rep 2006;1(2):69–74. 89. Latagliata R, Rago A, Spadea A, et al. Decisional flow with a scoring system to start platelet-lowering treatment in patients with essential thrombocythemia: long-term results. Int J Hematol 2009;90(4):486–491. 94. Finazzi G, Barbui T. Evidence and expertise in the management of polycythemia vera and essential thrombocythemia. Leukemia 2008;22(8):1494–1502. 95. Gisslinger H, Gotic M, Holowiecki J, et al. Anagreilde compared with hydroxyurea in WHO-classified essential thrombocythemia: the ANAHYDRET Study, a randomized controlled trial. Blood 2012;121:1720–1728. 100. Elliott MA, Tefferi A. Interferon-alpha therapy in polycythemia vera and essential thrombocythemia. Semin Thromb Hemost 1997;23:463–472.

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Chapter 52

Qualitative Disorders of Platelet Function Thomas J. Kunicki, Diane J. Nugent

Much of our current understanding of normal platelet structure– function relationships has been derived from the study of patients with congenital platelet disorders. These syndromes are the subject of excellent recent reviews.1–5

Normal Platelet Function: A Summary When a blood vessel is damaged, circulating platelets interact with components of the extracellular matrix, particularly collagen, and a complex series of receptor–ligand interactions ensue that ultimately lead to the formation of a stable platelet plug or thrombus (Fig. 52.1). This process is a continuum of at least three phases that we can describe as initiation, extension, and consolidation, each of which entails the cooperation of a different group of receptors. In the initiation phase (Fig. 52.2), plasma von Willebrand factor (VWF) binds to collagen via its A3 domain and becomes structurally altered such that its A1 domain then binds to the platelet membrane receptor glycoprotein Ib–IX–V complex (GPIb complex). This association is a requisite step in the adhesion of platelets to exposed thrombogenic surfaces at sites of vessel wall injury or in regions of atherosclerotic plaque rupture. Concurrently, a more stable platelet monolayer is formed on the collagen surface mediated predominantly by the platelet-specific receptor GP VI (GPVI) and platelet integrin a2b1. The engagement of these receptors enhances platelet activation leading to the extension phase (Fig. 52.3), mediated largely by the conversion of prothrombin to thrombin at the activated platelet surface and the secretion of active compounds from platelet granules (a-granules and d-granules) that can further stimulate platelets. One of these, adenosine diphosphate (ADP), plays a particularly important role in the platelet response, binding to its cognate platelet receptors to augment platelet activation. The activated platelet also produces and/or releases additional agonists, including the agonist thromboxane A2 (TXA2). Most of the receptors involved in the events of the extension phase are members of the G protein-coupled receptor family. In the consolidation phase (Fig. 52.4), platelet–platelet cohesion (aggregation), mediated by the binding of fibrinogen and/or VWF to the activated platelet integrin aIIbb3 (GPIIb-IIIa), together with the assembly of a fibrin network, results in the generation of platelet-rich aggregates or thrombi. Further complexity is inherent in this final phase of platelet plug formation, and current research indicates an essential role for outside–in signaling

Figure 52.1. Platelet thrombus formation.  The formation of a platelet thrombus can be envisaged as occurring in three distinct phases. The initiation phase entails the transient tethering of the platelet followed by a firmer adhesion to components of the matrix, leading to platelet activation. In the extension phase, the activated platelets synthesize and/or release from granule stores potent agonists, such as adenosine diphosphate (ADP) or thromboxane A2 (TXA2). These augment platelet activation. At the same time, the procoagulant platelet surface facilitates the formation of thrombin, which is itself a potent platelet agonist. In the consolidation phase, platelet–platelet cohesion (platelet aggregation) mediates the formation of the stable platelet plug.

through integrins and via receptor tyrosine kinases, including members of the Eph kinase family. Inherited or acquired defects that affect any phase of this complex process can result in excessive thrombosis or bleeding. Hereditary disorders can be divided into groups (Table 52.1), based on whether they predominantly influence the initiation, extension, or consolidation phases of platelet-dependent hemostasis. Platelet dysfunction can also be a manifestation of many acquired disorders, and these are also discussed in this chapter.

Diagnosis and Classification of Platelet Dysfunction: An Algorithm The preliminary diagnosis of platelet dysfunction must be made on the basis of patient history, and the diagnosis then confirmed by specific laboratory tests of platelet function. A practical algorithm for this purpose, depicted in Figure 52.5, is reproduced with permission from the excellent review by Bolton-Maggs et al.5

Bedside Exam and Patient History An accurate and detailed patient and family history is a key element in the assessment of platelet disorders. One should bear in mind that bleeding histories are subjective and that bleeding can be variable, often evolving or decreasing throughout a person’s lifetime. In particular, children may not have yet had enough hemostatic challenges to develop a strong clinical. The subjectivity of the oral history is reflected in a recent statistic that at least one quarter of persons who complain of serious bleeding do not have a bleeding disorder, whereas at least one third of persons who

Figure 52.2. Initiation of platelet adhesion by matrix components, particularly collagen.  Platelets employ a number of collagen receptors. These include VWF-mediated binding of collagen to the glycoprotein Ib (GPIb) complex (a heptamer composed of GPIba, GPIbb, GPV, and GPIX), the direct engagement of collagen by the integrin a2b1 and GPVI/ FcRg. Engagement and clustering of GPVI initiates tyrosine phosphorylation of FcRg by a Src family kinase (SFK). The tyrosine kinase Syk then binds and is activated, in turn activating phospholipase Cg, which then initiates phosphoinositide hydrolysis, secretion of ADP, and the production of TXA2. ADP and TXA2 augment platelet activation by binding to their respective platelet receptors.

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TA B L E 5 2 . 1

Disorders of Platelet Function Hereditary platelet dysfunction   Initiation phase    Bernard-Soulier syndrome    GPVI deficiency   Extension phase    Secretion disorders/granule deficiencies    a-Granule abnormalities (gray platelet syndrome)

have no bleeding can be shown to have von Willebrand disease (vWD) or a platelet disorder.6 To overcome the subjectivity of a history for superficial mucocutaneous bleeding, the International Society on Thrombosis and Hemostasis (ISTH) suggests that bleeding should be considered clinically significant when there are two or more distinct bleeding sites such as the skin, nose, gums, vagina, gastrointestinal tract, or genitourinary tract. This includes either spontaneous bleeding or provoked bleeding, such as that which might result from dental work, parturition, trauma, or surgery. In addition, a bleeding history involving only a single site should be considered significant when it is so severe as to warrant blood transfusions. Finally, a single bleeding symptom that recurs on three or more unrelated and separate occasions should also be considered significant.7,8

Figure 52.4. The consolidation phase of platelet thrombus formation occurs through the bridging of adjacent integrin aIIbb3 complexes by fibrinogen or VWF, as well as other adhesive proteins.

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   a/d-Granule deficiency    Defects of signal transduction and secretion     Impaired liberation of arachidonic acid    Cyclooxygenase deficiency    Thromboxane synthetase deficiency    Thromboxane A2 receptor abnormalities    Defects in calcium mobilization    Defects of platelet procoagulant activity   Consolidation phase    Glanzmann thrombasthenia   Miscellaneous     Hereditary macrothrombopathy/sensorineural hearing loss Acquired disorders of platelet function   Drug-induced platelet dysfunction    Analgesics    Antibiotics    Cardiovascular drugs    Psychotropic drugs   Secondary platelet dysfunction    Uremia    Paraproteinemia    Myeloproliferative disorders

Disorders of Hemostasis and Coagulation

Figure 52.3. The extension phase of platelet plug formation accelerates and augments the activation of the platelet and is mediated largely by G protein-coupled receptors, including: the purinogenic receptors P2Y1 and P2Y12, which are bound by ADP; the a and b isoforms of the thromboxane A2 (TXA2) receptor TP; the protease-activated receptor (PAR) family members PAR1 and PAR4, that are recognized by thrombin; and the a2A-adrenegic receptor that is specific for epinephrine.

   d-Granule (dense body) abnormalities    Hermansky-Pudlak syndrome    Chediak-Higashi syndrome    Wiskott-Aldrich syndrome

A number of quantitative approaches to assess the relative severity of patient bleeding have been proposed, some including a normalization based on patient age.9,10 However, such approaches work best for a comparison of cohorts of related individuals with comparable bleeding disorders, such as families of patients with vWD. The distinction between normal individuals and those with bleeding disorders is not always clear cut. Care should be taken in all patients to note the use of medicines, either “over the counter,” herbal, or prescription, that are known to influence platelet function. This is particularly true of medications such as aspirin, other nonsteroidal anti-inflammatory drugs (NSAIDs), ticlopidine or clopidogrel, integrin aIIbb3 antagonists (e.g., Abciximab, tirofiban, and eptifibatide), epoprostenol, statins, cilostazol, sildenafil, fluoxetine, and large doses of various b-lactam antibiotics (penicillins > cephalosporins).7 Bleeding manifestations typical of platelet dysfunction include: (1) Unexplained or extensive bruising; (2) epistaxis, particularly if lasting more than 30 minutes, causing anemia or admission to hospital; (3) menorrhagia, particularly if this has been present since the menarche; (4) oral cavity bleeding; (5) bleeding during

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Part v  Disorders of Hemostasis and Coagulation  •  SECTION 2  Thrombocytopenia

Suspected Heritable Platelet Disorder

Normal

NO NO

Platelet dysfunction ?

Thrombocytopenia ? YES

Other clinical conditions (e.g., albinism, hearing loss, Immunodeficiency, nephritis, Or mental retardation)?

YES Thrombocytopenia ?

NO

NO Platelet aggregation

YES

YES Large platelets

WAS

HPS

CHS

Quebec

WAS

MYHg

Blood smear Genetic analysis

BSS

lg levels Genetic analysis

GPS

Platelet factor V

Scott

EM Nucleotides / sec. lg Levels

P2Y12

YES

EM Nucleotides ATP Secretion

TXA2 pathyway

NO

Genetic analysis

NO

Absent RIPA Flow cytometry

Normal

EM α-granule proteins

Rev. ADP

Flow cytometry

Nucleotide secretion

GT

No AA

cAMP inhibition Receptor binding

To many agonists

NO

Large platelets

TXB2 RIA COX activity

RIPA only

Thrombocytopenia ?

YES

YES

Chapter 50

Signaling defect

δ-SPD (EM)

Flow cytometry Genetic analysis

NO YES

Figure 52.5. A scheme for the analysis of patients with suspected hereditary platelet dysfunction.  In order to establish a precise diagnosis, specific laboratory tests are needed to establish a defect in platelet number or function. Generally, an automated full blood count, blood film, platelet aggregometry, and quantification of platelet nucleotides are necessary. The purpose of this algorithm is to assist the investigator in the systematic interpretation of laboratory results. To complete a diagnosis, specialty analyses may be necessary, and these are included for the specific disorder. Such assays might include flow cytometry to measure expression of surface glycoproteins, such as aIIb or b3 (GT), GPIb or GPIX (BSS). In addition, quantitation of annexin V binding (Scott syndrome) and genetic analyses to identify specific gene mutations might be needed: aIIb or b3 (GT), WAS (WAS and XLA) and MYH9. Certain disorders can be caused by mutations in more than one gene (e.g., HPS and BSS), so genetic analysis has not been considered as a definitive test in all cases. The algorithm includes the most frequent or best characterized heritable platelet disorders. Symbols and abbreviations: ↓, reduced aggregation; AA, arachidonic acid; BSS, Bernard-Soulier Syndrome; CHS, Chediak-Higashi syndrome; COX, cyclooxygenase; d-SPD, dense-granule disorder; EM, electron microscopy; GPS, gray platelet syndrome; GT, Glanzmann thrombasthenia; HPS, HermanskyPudlak syndrome; MYH9, MYH-9–related disorder; P2Y12, deficiency of P2Y12 ADP receptor; QBS, Quebec platelet syndrome; Rev., reversible aggregation; RIA, radioimmunoassay; RIPA, ristocetin-induced platelet aggregation; Sec., secretion; TXA2, thromboxane A2; WAS, Wiskott-Aldrich syndrome. From Bolton-Maggs PH, Chalmers EA, Collins PW, et al. A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO. Br J Haematol 2006:603–633.

childbirth; (6) bleeding following invasive procedures; and (7) bleeding following dental extraction. Severe platelet dysfunction is present from early childhood onward. Following delivery of an affected infant one may find intracranial and/or subdural hemorrhage, excessive bleeding from the umbilical stump or after circumcision, or easy bruising after handling. As the infant becomes more mobile, easy or extensive bruising following relatively mild trauma can be indicative of platelet dysfunction. Prolonged epistaxis is a common finding throughout childhood and can even become life threatening. In adults, menorrhagia and bleeding during childbirth are common and potentially serious, whereas bleeding following any invasive procedure should be anticipated.5 Mild platelet dysfunction is more likely to manifest itself at any age most commonly following a definable hemostatic challenge, such as surgery or dental extractions. Easy bruising is a very nonspecific symptom, and many cases are difficult to distinguish from what would otherwise be considered a normal response. Consanguinity increases the likelihood of an autosomal recessive platelet disorder, and a family history is invaluable in establishing the diagnosis of inherited platelet dysfunction.

Laboratory Assessment A number of laboratory-based evaluations are critical for an accurate diagnosis of platelet dysfunction.

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Whole Blood Platelet Count A key element in assessing platelet dysfunction is an accurate whole blood platelet count. Automated counts should be viewed as provisional. Inasmuch as macrothrombocytes or platelet aggregates created inadvertently during blood processing will not be counted, the whole blood platelet count should be confirmed by an optical method.

Global Coagulation Tests To rule out abnormalities in clotting factors, all patients should have a prothrombin time, activated partial thromboplastin time, and thrombin time performed. Laboratories should determine their own age-related normal range. It is also critical to investigate all patients for VWD, which is far more common than platelet function disorders and creates a similar bleeding phenotype.

Bleeding Time The bleeding time is historically a common measure of platelet function. However, because the test is poorly reproducible, time consuming, and insensitive, the bleeding time has gradually fallen out of favor as a clinically useful test.11 In cases of mild platelet dysfunction, the bleeding time is often normal or minimally prolonged; 11 in severe cases, it will usually be prolonged.

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Unfortunately, the bleeding time does not correlate well with the in vivo bleeding tendency within individual patients, and an accurate bleeding history is considered by many to be a more valuable screening test. Nonetheless, a prolonged bleeding time should be considered sufficient grounds to perform additional tests of platelet function.

from adult values, whereas collagen-induced platelet nucleotide release has been shown to be reduced in neonates compared with children older than 1 year. Agonist-induced secretion of platelet granule contents has been shown to be reduced in both term and premature babies due to immature signal transduction pathways.18,19

Platelet Function Analyzer-100

Flow Cytometry

The platelet function analyzer-100 (PFA-100) measures the rate of thrombus formation under high shear in citrated whole blood that is perfused through a membrane aperture coated with collagen/epinephrine or collagen/ADP. The closure time (CT) will be significantly prolonged in Glanzmann thrombasthenia (GT) and Bernard-Soulier syndrome (BSS) using either ADP/collagen or epinephrine/collagen membrane cartridges.12,13 Consequently, the PFA-100 can be used to screen patients to exclude these diagnoses. The PFA-100 may be sensitive to platelet storage pool disease (SPD), primary secretion defects, the Hermansky-Pudlak syndrome (HPS), and the Quebec syndrome. However, because falsenegative results occur in patients with all of these disorders, there are those who question its usefulness as a screening tool.12,14 The PFA-100 is affected by platelet count and hematocrit, and is dependent on normal VWF levels and naturally occurring (genetic) differences in platelet membrane GPVI or a2b1 expression.13,14,15

Flow cytometry is routinely used to measure platelet surface receptor density, platelet activation, a-granule release, procoagulant phospholipid expression, and microvesicle production.20,21 A common application of flow cytometry is the assessment of GPIb complex and integrin aIIbb3 expression in the diagnosis of BSS and GT. Individuals who are heterozygous for these disorders are also readily distinguished. An important benefit of flow cytometry is the small quantities of blood required, an attractive feature in young children or thrombocytopenic individuals.

Platelet Aggregation Platelet aggregation in platelet-rich plasma remains an important test in the analysis of platelet function. The typical agonists that are used to induce platelet aggregation are ADP, epinephrine, collagen, arachidonic acid (AA), ristocetin, the TX receptor agonist U46619, thrombin or the thrombin receptor-activating peptide. Because the level and/or activity of each of the receptors for the agonists can vary among normal subjects, it is recommended that dose–response curves to each agonist be obtained from the patient under study and compared to a reference range obtained from multiple normal subjects.16 When thrombin is the agonist, an inhibitor of fibrin polymerization, such as glycine–proline– arginine–proline peptide, must be added to the PRP, or alternatively, plasma-depleted washed platelets must be used. Platelet aggregation is sensitive to platelet count, and at counts ≤120,000/ml, the response to some agonists will be impaired. In thrombocytopenic samples, the best options are to adjust a control sample to the same count as the patient or to perform studies on washed platelets where the platelet number can be normalized. Consideration should also be given to more specialized tests, such as a measure of P-selectin expression or integrin aIIbb3 activation by flow cytometry. The expected aggregation responses associated with specific diagnoses are covered in the appropriate sections.

Adenine Nucleotide Content and Release Measurement of platelet adenosine nucleotide (ADP and adenosine triphosphate [ATP]) content and release can be used in the diagnosis of storage pool and release defects.17 A finding of normal platelet aggregation does not exclude the diagnosis of SPD. It is recommended that patients suspected of having platelet dysfunction should have both platelet aggregation and adenine nucleotide release performed, unless it is certain that the laboratory performing the platelet aggregation assays can demonstrate that their assay conditions are sensitive to defects in platelet nucleotide amount or release. Platelet nucleotide content and release varies with age. Ideally, age-related normal ranges should be established for total and released levels of ATP and ADP and their ratios. Platelet aggregation, nucleotide content, and nucleotide release in children over 12 months of age do not differ significantly

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Electron Microscopy Transmission electron microscopy (TEM) of fixed/embedded platelet thin sections can be performed by a limited number of specialized personnel, but is critical in the assessment of platelet granule defects and changes in platelet ultrastructure (e.g., in the evaluation of patients with MYH-9 defects). Whole-mount EM can be used to quantitate d-granule content because this is readily identified in unstained preparations.22

Additional Assays Several additional assays are available in specialized laboratories that can provide further information relevant to the diagnosis of the particular platelet disorder, including analysis of receptor expression, specific molecular or genetic defects, protein phosphorylation, formation of signal transduction intermediates, or a characterization of the platelet proteome. The utility of these tests in clinical diagnoses is currently under intense investigation.

Disorders of Hemostasis and Coagulation



Overview of Treatment Options for Platelet Disorders Although the number of clearly identifiable platelet dysfunction syndromes is growing, our choice of treatment options remains limited. Minor membrane bleeding may be controlled with topical agents in the nasal or oral cavities with antifibrinolytic agents. Epsilon-aminocaproic or tranexamic acids will decrease blood loss associated with epistaxis or menorrhagia. Some patients may benefit from Stimate as documented by DiMichele and Hathaway,23 whereas others actually bleed more with this agent due to the fibrinolysis induced by this medication. For this reason many centers recommend the use of Stimate in conjunction with Amicar or Cyklokapron. In the case of menorrhagia, hormonal suppression is the mainstay for women who do not wish to undergo endometrial ablation for hysterectomies. In combination with antifibrinolytics and Stimate, even severe bleeding may be controlled; however, more aggressive therapy including platelet infusion may be required to control bleeding before these agents take effect. Platelet transfusion may pose a dilemma to those physicians wishing to avoid alloimmunization in patients who may require multiple transfusions throughout their lifetimes to control hemorrhage. This is particularly true for those patients who are lacking membrane GPs, such as aIIbb3 or GPIb. In this setting patients are at risk of developing isoimmunization, making antibodies against the “foreign” proteins that they lack and thus becoming refractory to all subsequent platelet transfusions.

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Although rare, this creates a significant challenge in patients who require frequent platelet infusions to control life-threatening bleeding. Patients with GT have not only developed antibodies to aIIb and/or b3 but also demonstrated anti-idiotypic antibodies that bind to fibrinogen, thus creating a hemorrhagic disorder far worse than the underlying platelet dysfunction. In general, physicians avoid platelet transfusion apart from cases of severe hemorrhage. Isoimmunization appears to be rare in Glanzmann but the risk of alloimmunization is still a major concern, therefore leukocyte depletion of transfused platelets is recommended to decrease the frequency of sensitization. Activated recombinant Factor VII (rFVIIa) has been used to slow or arrest bleeding associated with platelet dysfunction.24 Dosages have varied widely but many patients have responded to this regimen when others have failed. Used in combination with antifibrinolytics, minor bleeding can be controlled in certain patients. This treatment is often used prior to platelet transfusion in order to avoid blood product exposure and isoimmunization. For those patients who present with recurrent life-threatening bleeds, bone marrow transplant or stem cell infusion following immune ablation is recommended before the patients have extensive blood product exposure.25 Successful transplantation with normal stem cells represents long-term cure for these patients. Although complications related to stem cell transplantation cannot be overlooked, successful engraftment essentially eliminates the significant risk of mortality related to hemorrhage in patients with severe disorders. Patients with rare platelet dysfunction syndromes are now included in many of the rare bleeding disorder international and regional registries that will aid physicians in understanding the natural course, optimal therapy, and life expectancy for each of the syndromes listed below, as well as the numerous platelet disorders that have yet to be defined. Definitive molecular and biochemical diagnoses will dictate appropriate therapy in these patients. With improvement in platelet function measurement and proteomic approaches one should see significant improvement in early diagnosis and medical management.

Hereditary Disorders of Platelet Function Defects in the Initiation Phase: Bernard-Soulier Syndrome BSS is a rare disorder first described in 1948 as “dystrophie thrombocytaire hemorrhagipare congenitale” caused by abnormal expression or activity of the platelet GPIb complex.26

Etiology BSS platelets have a quantitative or qualitative abnormality of the membrane GPIb complex, a heptamer composed of four leucine-rich GP that are the products of distinct genes (Fig. 52.2). The prominent member of the complex, GPIb, is a heterodimer composed of disulfide-bonded GPIba and GPIbb subunits. GPIb then forms a noncovalent complex with GPIX, and two GPIb-IX trimers then associate noncovalently with one molecule of GPV. The amino-terminal type A domain of GPIba binds directly to VWF, mediating normal platelet adhesion during the initial phases of primary hemostasis, whereas BSS platelets do not adhere to the extracellular matrix when perfused at a high shear rate.27 The defect in the GPIb complex also explains the failure of affected platelets to agglutinate in the presence of ristocetin, even in the presence of normal plasma or VWF. Mutations in the gene for GPIba, GPIbb, or GPIX, but not GPV, have been shown to result in decreased expression of the GPIb

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complex and the BSS. Defects range from virtually absent GPIb to variant forms in which patients retain measurable amounts of apparently dysfunctional GPs.1 BSS is usually inherited as an autosomal-recessive disorder, and consanguinity is common in reported kindreds. Heterozygotes typically have “giant” platelets and reduced levels of the GPIb complex and may or may not be symptomatic.

Clinical Features Bleeding symptoms are usually evident shortly after birth or in early childhood. The clinical manifestations include purpura, epistaxis, gingival bleeding and menorrhagia, and more rarely gastrointestinal bleeding, major hematomas, or hematuria. Severe bleeding episodes can result from trauma and surgical procedures, such as tonsillectomy, appendectomy, splenectomy, oral surgery, and menses. However, individual bleeding can vary substantially in severity and frequency.

Laboratory Findings The typical laboratory findings include an increased bleeding time, mild thrombocytopenia, giant platelets on blood smear, and defective adhesion to collagens in vitro. Platelet morphologic abnormalities are a hallmark of BSS, featured by large platelets with a diameter as high as 10 mm. A defective platelet agglutination in response to ristocetin, as measured by aggregometry in vitro, is a unique characteristic of BSS that differentiates this syndrome from other rare inherited disorders that are also associated with macrothrombocytopenia, such as the MHY9-related disorders.1 A firm diagnosis requires the combined findings of increased bleeding times, macrothrombocytopenia, defective ristocetin-induced agglutination, and low or absent levels of platelet GPIb-V-IX (CD42a-d) by flow cytometry. In bone marrow aspirates, megakaryocytes are normal or increased in number, but they reveal no characteristic morphologic abnormalities when viewed by light microscopy. Electron microscopic studies have revealed abnormalities of the dense tubular system and vacuolization of the demarcation membrane system.28

Management Therapeutic approaches include both general and specific treatment of bleeding. Patients should be warned to avoid trauma and antiplatelet medication, such as aspirin, and to maintain proper dental hygiene. Females may benefit from contraceptive therapy once they reach puberty. Treatment of bleeding or prophylaxis during surgical procedures usually requires blood or platelet transfusion with the associated risk of developing antiplatelet alloantibodies. Desmopressin and rFVIIa administration have been shown to shorten the bleeding time in some patients. In rare cases of life-threatening bleeding, a bone marrow or umbilical cord hematopoietic stem cell transplantation may be considered.29 Responses to antifibrinolytic agents are more variable, and the administration of adrenal corticosteroids and splenectomy are usually ineffective. Platelet-reactive isoantibodies have been generated by BSS who have received multiple blood or platelet transfusions.30 These antibodies produce a particularly severe clinical complication because they will bind to and may neutralize the function of GPIb on transfused platelets. This results in impaired adhesion of the transfused platelets. The presence of such antibodies can be established by a finding of impaired in vitro aggregation of normal platelets induced by ristocetin and bovine VWF in the presence of patient plasma. Alloimmunization is also a common side effect of multiple platelet transfusions, and this may necessitate the subsequent use of HLA-matched platelets.

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Variation in the Initiation Phase: Collagen Receptor Polymorphisms A lifelong bleeding disorder and the unique absence of in vitro platelet aggregation to collagen have been associated with a deficiency of either of the collagen receptors, integrin a2b1 or GPVI,1 yet specific gene defects have not been identified. Careful evaluation is needed to establish a specific molecular defect in either receptor.

The Integrin a2b1 Among normal individuals, platelet a2b1 levels can vary up to tenfold and correlate with differences in adhesiveness to type-I or type-III collagens and genetic variants of the a2 subunit gene ITGA2. We identified several single nucleotide polymorphisms (SNPs) within the coding sequence of the a2 gene ITGA2 that correlate with platelet a2b1 density31 and define six major ITGA2 haplotypes.

Clinical Relevance of a2 Polymorphisms In VWD, platelet adhesive functions are impaired due to the decrease in functional VWF multimers in plasma and platelets. Inheritance of the ITGA2 haplotypes associated with lower a2b1 density will increase the risk of bleeding in patients with either type I and type2 VWD.9,10 The association of ITGA2 haplotypes with risk for arterial thrombosis has been studied in acute coronary syndromes, diabetic nephropathy, and stroke. Although several studies have found that the inheritance of high-density ITGA2 haplotypes correlates with increased thrombosis, this association is not a consistent observation.32

Platelet Glycoprotein VI GPVI is a major platelet GP (60 to 65 kDa) that has been confirmed as an important receptor for collagen since the initial identification of a patient with a mild bleeding disorder whose platelets lacked GPVI and exhibited defective collagen-induced responses.33 Autoantibodies against GPVI can cause substantial shedding of this receptor through metalloproteinase cleavage.34,35

Glycoprotein VI Polymorphism Aside from isolated cases of GPVI defects, it is important to note that there is variation in platelet GPVI content among normal healthy subjects,36 which is directly proportional to normal variation in mean platelet volume.37 This variation is manifested in a significant difference in prothrombinase activity induced by GPVIspecific agonists such as the snake venom protein convulxin or collagen-related peptide (CRP).36 The direct association between platelet a2b1 density and GPVI content is also attributable to variation in mean platelet volume, which has a proportional effect on levels of other receptors, including aIIbb3.37 Variation in GPVI content represents yet another genetically controlled risk factor predisposing individuals to hemorrhagic or thromboembolic disorders.

Defects in the Extension Phase Secretion Defects: Storage Pool Disease SPD is a heterogeneous group of congenital disorders that have in common a deficiency of granules or their constituents that results in a defect in ADP release from activated platelets and abnormal secretion-dependent platelet aggregation.1 Defective platelet secretion can result from the absence of or defects in one

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or both of the dominant types of platelet granules: The a-granule or the d-granule (dense body). Thus, three subgroups of SPD are now distinguished. The isolated deficiency of a-granules (a-SPD) has been studied for several years and is more commonly known as the Gray platelet syndrome (GPS)38; the exclusive abnormality of dense-granules (d-SPD) can be congenital or acquired, as in myeloproliferative syndromes or rheumatologic disorders; 17 and abnormalities of both a- and d-granules are classified as ad-SPD.17

a-Granule Storage Pool Disease: Gray Platelet Syndrome GPS is a very rare disorder with less than 100 cases reported worldwide and is characterized by a selective deficiency in the number and content of a-granules.17,38 The formation of a-granules in immature megakaryocytes proceeds normally, but the granule number then decreases during maturation, and the mature megakaryocytes are left with only small abnormal granules that are few in number. Some of the normal protein constituents of a-granule membranes, such as GPIV, integrin aIIb-b3, and P-Selectin remain in the abnormal granules and will be normally redistributed during platelet activation. The defect is limited to megakaryocytes and platelets. Recent evidence suggests that GPS is associated with mutations in NBEAL2 (neurobeachin-like 2 gene), which encodes a protein containing a BEACH domain that is predicted to be involved in vesicular trafficking and may be critical for the development of platelet a-granules.39–41 Several proteins are synthesized but not stored properly in the abnormal a-granules. These include platelet factor 4, b-thromboglobulin, fibrinogen, fibronectin, vWF, platelet-derived growth factor, and thrombospondin. Other organelles, such as lysosomal granules, mitochondria, and d-granules (dense bodies) are present in normal numbers, and d-granules contain normal amounts of adenine nucleotides and serotonin.

Clinical Features

GPS is inherited as an autosomal trait and is associated with mild to moderate thrombocytopenia (20,000 to 150,000/ml) and moderately enlarged platelets.17,38 The bleeding time is usually prolonged, and platelet survival may be shortened, but splenomegaly is not common. As in other platelet function defects, a history of mild bleeding is the norm, and treatment is seldom required. The bone marrow exhibits normal megakaryocyte numbers and increased reticulin, occasionally around clusters of megakaryocytes, but myelofibrosis is not a feature of this disorder. In Wrightstained blood smears, the platelets appear large, misshapen, agranular, and gray. In electron photomicrographs, an almost total lack of a-granules is evident in platelets and in megakaryocytes. When analyzed by EM, it is the only disorder that is characterized by the selective absence of platelet a-granules. Megakaryocytes show defective a-granule biogenesis, with impaired uptake and storage of endogenously synthesized proteins, such as platelet factor 4, b-thromboglobulin, or VWF, and defective sequestration of exogenous proteins, such as fibrinogen, albumin, or factor V. On the other hand, in the platelet, the a-granule marker P-selectin is retained and is redistributed to the surface upon activation. Both autosomal recessive and autosomal dominant inheritance have been reported, implicating more than one gene in the etiology of the disease. Myelofibrosis of bone marrow is an additional feature that is attributed to the spontaneous release of plateletderived growth factor and transforming growth factor-b1 from megakaryocytes.

Disorders of Hemostasis and Coagulation



Laboratory Findings

Gray platelets have a normal aggregation and release in response to AA or ionophore and generate TX normally. Platelet aggregation induced by ADP, epinephrine, thrombin, or collagen is variably affected. Release from dense bodies usually is subnormal,

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particularly when induced by thrombin. Abnormal calcium influx and mobilization from intracellular stores have also been reported. The platelet count is often reduced and can be as low as 50,000 per ml. By EM, the platelets have reduced numbers or a complete absence of a-granules.

Management

Because of the rare nature of a-granule disorders, there is not a very extensive clinical database upon which to make generalizations about effective treatment of the bleeding problem. However, these patients should be managed in the same manner as other patients with mild bleeding disorders. In the case of major bleeding or surgical challenge, they may require platelet transfusion.

Isolated d-Storage Pool Disease d-SPD is characterized by easy bruising, mucocutaneous bleeding, and excessive post-operative and post-partum hemorrhage.17

Clinical Features

The symptoms can become more severe if the patient ingests aspirin or other antiplatelet agents, and patients with SPD should be advised to avoid such drugs. Platelet counts are typically ­normal, and the bleeding time is usually prolonged. The platelets are morphologically normal on Wright-stained smears, but they are deficient in dense bodies by EM. The diagnosis of d-SPD must be made by the finding of a decrease in dense granule constituents and/or EM to demonstrate the absence of dense granule-limiting membranes and contents. The most consistent finding is that adenine nucleotides are reduced with an increased ratio of ATP to ADP and normal levels of lysosomal enzymes. The platelet content of serotonin, however, can be variably reduced. Whole-mount EM recognizes calcium, but it cannot differentiate the absence of a dense granule from the absence of its calcium. Dense granules can also be quantitated by fluorescent microscopy because, whether full and empty, d-granules take up the fluorescent dye quinacrine (mepacrine).42

Laboratory Findings

Because of the defect in d-granules and the decreased level of ADP, primary aggregation responses in vitro are normal, but secondary aggregation may be diminished. Typically, there is an impaired response to collagen and a deficient secondary wave with ADP, epinephrine, and low concentrations of thrombin, but ristocetin aggregation is normal. Although this functional defect is often encountered in this disorder, it cannot be relied upon as a diagnostic criterion. The platelets are also deficient in serotonin, calcium, and pyrophosphate, which are stored in the d-granules.17 The bleeding time is increased, with normal platelet counts and morphology. Subsets of patients have been identified who have a prolonged bleeding time, decreased dense bodies, and ADP release but normal platelet aggregation. The diagnosis of d-granule SPD can best be made following a combination of studies, including bleeding time, platelet aggregation, an assessment of storage pool adenine nucleotides, thrombin-induced radioactive serotonin uptake and release, and TEM. There is suggestive evidence that d-SPD is inherited as an autosomal dominant characteristic, but neither the causative gene nor the molecular nature of the basic defect have yet been determined.

Management

To treat the symptoms of SPD, desmopressin (DDAVP) can normalize the bleeding time in some patients, often within 1 hour after infusion.23 This treatment is particularly effective in improving hemostasis after procedures.

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Combined ad-Storage Pool Disease In ad-SPD, which is much less common than isolated d-SPD, d-granules and/or their contents are uniformly decreased, and the deficiency of a-granules and/or their constituents can vary.17 Platelets in these patients form significantly smaller thrombi in flowing blood than is seen with platelets from patients with d-SPD. The defects are also not uniformly among the platelet population, such that a-granules and d-granules may be completely absent in one portion of the population yet present in nearly normal levels in another.43 The mode of inheritance appears to be autosomal dominant, but a basic molecular defect and a genetic basis have not yet been determined.

Animal Models and Etiology

The comparative study of granule deficiencies in the mouse or rat can provide clues to understanding the etiology of SPD. For example, the gunmetal mouse44 and the fawn-hooded hypertensive rat45 are characterized by reduced platelet a- and d-granules, a disorganization of megakaryocyte internal membranes, and impaired a-granule protein retention. Mutations in RabggtA result in defective geranylgeranyl transferase, which catalyzes the attachment of lipid geranylgeranyl groups to Rab proteins.44 Rab proteins are small guanosine triphosphatases (GTPases), which split GTP to provide energy for membrane fusion events and for attachment to cytoskeletons. By analogy, it is possible that mutations in the human gene RABGGTA are also responsible for human ad-SPD, but this remains to be proven.

Clinical Features

Bleeding in this disorder is clinically similar to bleeding in d-SPD and GPS, and is manifested by a prolonged bleeding time with normal platelet counts.17 Except for their bleeding tendency, patients with ad-SPD appear otherwise healthy.

Laboratory Findings

Defects in secondary platelet aggregation are typical, and defects in primary aggregation are even more extreme than seen in d-SPD. Because of variability, in vitro platelet aggregation in ad-SPD is not an appropriate diagnostic criterion, and an accurate diagnosis requires measurement of d- and a-granule constituents and/or EM of platelet organelles. The lysosomal enzyme content of ad-SPD platelets is normal.

Management

Because of the few numbers of patients with combined ad-SPD, there is little clinical information upon which to develop a treatment scheme. However, this disorder can likely be managed in the same manner as the isolated d-SPD (see above).

Quebec Platelet Syndrome (Factor V Quebec) The Quebec platelet syndrome is an extremely rare, autosomal dominant disorder that was originally identified by the finding of low levels of platelet a-granule factor V but normal plasma factor V.1,17 It is characterized by protease-related degradation of many platelet a-granule proteins, including P-selectin. The a-granule ultrastructure is normal. Increased expression of a-granule urokinase-type plasminogen activator is thought to lead to spontaneous intracellular activation of the fibrinolytic pathway resulting in the generation of plasmin, which cleaves multimerin that would otherwise stabilize factor V.46

Clinical Features and Laboratory Findings

Many cases may go undiagnosed because there are no characteristic morphologic features or platelet aggregation abnormalities. The main laboratory finding is defective procoagulant activity,

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reflected by a failure to assemble the prothrombinase complex. A related defect, platelet Factor V New York, is also characterized by decreased levels of platelet Factor V, although there is no indication of Factor V proteolysis, as in Factor V Quebec.47

Management

The Quebec platelet syndrome is unresponsive to platelet transfusions. It is the current opinion that fibrinolytic inhibitors are the most effective means to control bleeding.48 Otherwise, treatment should be similar to that given to patients with bleeding disorders.

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associated with defective natural killer cell function.55 However, LYST mutations cannot be identified in all CHS patients, suggesting the existence of causative defects in other genes.56 Several proteins are known to interact with LYST, including the SNARE complex protein HRS, signaling protein 14-3-3, and casein kinase II.57 It has been proposed that LYST may be an adaptor protein that brings into close proximity proteins that mediate intracellular membrane fusion reactions. Along this vein, LYST contains several consensus motifs that play distinct roles in vesicle transport and fusion.

Clinical Features and Laboratory Findings

Hermansky-Pudlak Syndrome

Platelet counts are normal prior to development of the accelerated phase. The dense granule defect results in mucocutaneous bleeding and a prolonged bleeding time. The secondary wave of platelet aggregation is impaired, there is an increased ATP-toADP ratio, decreased platelet serotonin, and decreased platelet calcium. Some patients have a normal number and shape of dense bodies, but most often there is an absence of or marked reduction in dense bodies.

HPS is as an autosomal-recessive trait that has a worldwide distribution but is most prevalent in Puerto Rico.

Management

Hermansky-Pudlak and Chediak-Higashi Syndromes The HPS and Chediak-Higashi syndrome (CHS) are rare autosomal recessive disorders that have in common platelet dense granule deficiency, albinism, and lysosomal granule defects.49

Etiology

The genetic causes of HPS are diverse. Mutations in at least seven genes have been linked to this disease, and the absence of platelet dense bodies is thought to result from a defect in organelle development.50

Prior to the accelerated phase, hemostatic problems can be treated as in the case of other mild platelet disorders. With time, infections and a lymphoproliferative accelerated phase are serious developments that can result in death within the first decade.1,54 Although experience is still limited, hematopoietic cell transplantation is a potentially effective therapy for correcting and preventing hematologic and immunologic complications of CHS.58

Clinical Features and Laboratory Findings

A diagnosis of HPS is based on the presence of oculocutaneous albinism and absent platelet dense bodies on whole-mount EM.51 Typical findings are a lifelong history of easy bruising; minor bleeding episodes, such as mucus membrane bleeding; epistaxis, and metromenorrhagia. Many patients will require whole blood or platelet transfusions when bleeding symptoms become more severe. Pulmonary fibrosis and inflammatory bowel disease, associated with infiltration of ceroid-pigmented reticuloendothelial cells in the lung and colon, have been reported in several cases of this syndrome.52 In platelet aggregation assays, there is an absence of the secondary wave in response to ADP and epinephrine, and the response to collagen is also abnormal. Usually, the template bleeding time will also be longer than normal.

Management

Localized and limited treatment of the bleeding symptoms is the norm in HPS.49,53 The albinism necessitates skin and eye protection, and the bleeding can be controlled topically with thrombin and Gelfoam. Prophylactic use of intravenous 1-desamino-8-D-arginine vasopressin or Stimate is recommended for procedures such as tooth extractions or biopsies. Menstrual bleeding can be regulated by birth control pills. In the event of major surgeries or severe bleeding episodes, platelet or red blood cell transfusions may be required.

Chediak-Higashi Syndrome SPD also may be associated with the CHS.1,54 In addition to defective platelet dense granules and oculocutaneous albinism, CHS is also characterized by immune deficiency and progressive neurologic dysfunction. In CHS patients, the granule abnormalities are not restricted to the megakaryocyte lineage, and numerous cell types exhibit giant cytoplasmic inclusions that are enlarged vesicles.

Etiology

Numerous cases of CHS have been reported to result from mutations in the lysosomal trafficking regulator gene (LYST) located on chromosome 1q42.1-42.2. Mutations in the LYST protein can be

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Griscelli Syndrome Mutations in RAB27A, a gene for a small GTPase involved in vesicular transport and organelle dynamics, are considered to be the cause of GS.59 The ashen mouse with mutated RAB27A is a model of GS and exhibits a reduction in the number of platelet dense granules. However, the hemostatic phenotype of these mice is affected by the genetic background, and it is likely that additional modifier gene polymorphisms have an important influence on the hemostatic phenotype.

Disorders of Hemostasis and Coagulation



Clinical Features and Laboratory Findings

GS is a rare autosomal recessive disorder in which patients present with abnormal pigmentation, immunodeficiency, and development of the accelerated phase.60 GS patients have no obvious bleeding prior to the accelerated phase, and it has not been firmly established that there is a characteristic platelet dense granule defect. Hypopigmented hair with a silvery-gray sheen and pigment clumps is a characteristic of GS, but these are larger and less homogeneously distributed than one sees with CHS patients. The giant cytoplasmic granules typical of CHS cells are not seen in GS cells.

Management

Like CHS, the disease is usually fatal by the first decade of life. The same management as observed with CHS will likely be effective in patients with Griscelli syndrome.1,54

Paris-Trousseau (Jacobsen) Syndrome The Paris-Trousseau (Jacobsen) syndrome is an autosomal dominant disorder characterized by thrombocytopenia, a history of relatively mild bleeding, the presence of giant a-granules in a minor proportion of platelets, and two morphologically distinct populations of megakaryocytes in the bone marrow, some of which exhibit signs of abnormal maturation. Additional congenital abnormalities include mental retardation, cardiac abnormalities, and cranio-facial abnormalities. This is an extremely rare disorder, and only 10 childhood cases have been reported.61

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These patients have deletions of the long arm of chromosome 11q that include the gene FLI1, which encodes a transcription factor that plays a critical role in normal megakaryocytopoiesis. Thus, the hemizygous deletion of FLI1 generates a subpopulation of megakaryocyte progenitors that fail to differentiate normally, forming small immature megakaryocytes that undergo massive lysis.62 Those platelets with giant a-granules fail to release their contents normally upon stimulation.

Arthrogryposis-Renal Dysfunction-Cholestasis Syndrome The gene VPS33B encodes a protein that is involved in protein trafficking. A mutation of the gene is the cause of ARC syndrome.63 ARC is an autosomal recessive disorder with multiple systemic abnormalities, one of which is platelet dysfunction is caused by a deficiency in platelet a-granules.64 This disorder is diagnosed by severe distinguishing features other than the platelet dysfunction, and affected individuals usually do not survive their first year.

Inherited Disorders of Primary Membrane Receptors Congenital Defects of P2Y12 The adenine nucleotides ADP and ATP are both released from platelet d-granules during activation induced by a variety of agonists, and both can augment or modulate the platelet functional response through autocrine and paracrine mechanisms by binding to platelet purinergic receptors.65 Two platelet purinergic receptors are specific for ADP, the Gq-protein–coupled receptor P2Y1 and the Gi-protein–coupled receptor P2Y12. One receptor, the P2X1 ion channel, is bound by ATP. The P2Y and P2X receptors operate in a temporally distinct manner and selectively trigger distinct intracellular signaling pathways. Recent advances in our understanding of P2Y receptor physiology suggest that these receptors are a potentially relevant target of antithrombotic therapy. P2Y1 mediates ADP-induced intracellular calcium ion mobilization and shape change, and P2Y12 is coupled to adenylyl cyclase inhibition and is responsible for ADP-induced macroscopic platelet aggregation.66 At the same time, we have only a perfunctory understanding of the function of P2X1 in platelet activation. It is known, however, that P2X1 function is linked to calcium ion influx.66

Etiology

P2Y12 defects are inherited as autosomal recessive traits,67 and heterozygous individuals display a mild abnormality in platelet function similar to that observed in patients with SPD. In two cases, different homozygous frameshift mutations cause premature termination of translation. In another case, one allele presented a reading frameshift caused by the deletion of two nucleotides, whereas the other case had a normal coding sequence but a reduced expression, possibly resulting from another mutation in a regulatory region of the gene.68

Clinical Features and Laboratory Findings

A limited number of patients have been described with congenital abnormalities of P2Y12 resulting in bleeding diatheses and abnormalities of platelet function.4 The common clinical findings in these patients are a lifelong history of mucosal bleeding, easy bruising and/or excessive post-operative bleeding, and mildly to severely prolonged bleeding times. Laboratory findings were a weak and rapidly reversible primary wave of aggregation induced by ADP and abnormal aggregation induced by collagen, arachidonate, and TXA2 analogs. Aggregation induced by high concentrations of thrombin was normal. ADP-induced shape change is

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normal, but ADP fails to inhibit the expected normal rise in cAMP levels seen after stimulation with prostaglandin E1.

Management

Inasmuch as there are generally no serious side effects, DDAVP is recommended for prophylaxis and treatment in these patients.23

Thromboxane A2 Receptor Defective TXA2 receptor function is characterized by impaired platelet aggregation in response to the TXA2 analog U46619, a variable response to other agonists comprising a reduced primary wave and absent secondary wave, and defective secretion.69

Epinephrine Receptor A selective absence of an aggregation in response to adrenaline has been reported as a heritable trait in association with easy bruising and reduced expression of platelet a2-adrenoceptors.70,71 However, the aggregation response to adrenaline among normal individuals is itself quite variable, and a relative decrease in the aggregation response to adrenaline may represent the lower segment of a common asymptomatic population variant.72

Inherited Defects in Signal Transduction Pathways Cyclooxygenase Defects Defects in platelet cyclooxygenase (COX) or TX synthase73 give the same laboratory findings as seen in TXA2 receptor defects (see above), but can be distinguished from the latter by the preservation of platelet aggregation in response to prostaglandin-H2 or the TXA2 analog U46619.

Scott Syndrome Scott syndrome is a rare autosomal recessive disorder characterized by a defect in calcium-induced phospholipid scrambling and prothrombin conversion on platelets and other blood cells.74 When Scott platelets are activated, phosphatidylserine is not transported from the inner to the outer phospholipid leaflet of the membrane. The binding of factor Va–Xa and factor VIIIa–IXa complexes is impaired, resulting in decreased thrombin generation and impaired platelet-dependent fibrin formation on subendothelium. It has been reported that Scott syndrome may be explained, at least in part, by mutations in an ATP-binding cassette transporter A1 implicated in the exofacial translocation of phosphatidylserine.75

Additional Defects Patients have been described with: A lineage-specific Gaq subunit deficiency and impaired aIIbb3 activation; reduced Gai1 expression and signaling; defective calcium ion mobilization and an abnormal response to ionophore A23187; a reduced production of inositol 1,4,5-triphosphate and phosphatidic acid, diminished plekstrin phosphorylation and defective phospholipase C activation; and a specific defect in phospholipase Cb2 expression.76 Pseudohypoparathyroidism type 1b, which is associated with a mild bleeding disorder, is caused by a defect in the heterotrimeric G protein subunit Gsa, which regulates adenylyl cyclase.77

Defect in the Consolidation Phase: Glanzmann Thrombasthenia GT is an autosomal-recessive disorder characterized by defective in vitro platelet aggregation and a lifelong bleeding tendency due

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to quantitative or qualitative abnormalities of the platelet GPIIbIIIa,2 also known as the integrin aIIbb3.

Etiology A qualitative or quantitative defect of either GPIIb or GPIIIa is the basic biochemical basis for GT. GPIIb-IIIa is a calcium-dependent heterodimer that binds preferentially to fibrinogen or vWF, but also to fibronectin or vitronectin. The genes for GPIIb and GPIIIa are distinct but are physically located within 1 Mb of each other on the long arm of chromosome 17 (17q21-22). A genetic defect in either GPIIb or GPIIa can inhibit synthesis of that subunit and/or prevent normal assembly and processing of the functional receptor. This results in the lack of a fibrinogen receptor and defective fibrinogen binding after platelet activation. Platelet aggregation, which requires this protein, is therefore deficient or completely absent. A continually maintained database is accessible on the Internet (https://haemgen.haem.cam.ac.uk/thrombogenomics/) which contains a list of over 100 mutations that give rise to GT. The aIIb and b3 genes (ITGA2B and ITGB3, respectively) are both affected, and although post-translational defects predominate, reduced mRNA stability can also be a cause. Integrin synthesis occurs in the megakaryocytes with aIIbb3 complex formation in the endoplasmic reticulum. Noncomplexed or incorrectly folded gene products fail to undergo processing in the Golgi apparatus and are rapidly degraded intracellularly.78 In GT, isoantibodies specific for the deleted GPs have been demonstrated in the blood of multiply transfused patients,2 and these can reproduce the thrombasthenic defect in normal transfused platelets.

Clinical Features Thrombasthenia is inherited as an autosomal-recessive trait, and consanguinity is common within affected kindreds, resulting in geographic clusters of patients.2 Hemorrhagic symptoms occur only in patients homozygous for GT mutations, whereas the heterozygous condition is mostly asymptomatic, even though platelets from heterozygotes have only one half the normal level of aIIbb3. The sites of bleeding in GT are clearly defined: Purpura, epistaxis, gingival hemorrhage, and menorrhagia are nearly constant features; gastrointestinal bleeding and hematuria are less common but can cause serious complications. Bleeding at menarche is severe enough to require transfusions in most patients. Deep visceral hematomas, a characteristic of coagulation disorders such as hemophilia, are not usually seen in GT. Bleeding symptoms normally manifest rapidly after birth, even if most patients are diagnosed before the age of five. Epistaxis is a common cause of severe bleeding, and is typically more severe in childhood. In general, the bleeding tendency in GT decreases with age. Post-traumatic and post-operative hemorrhage can be serious, and pregnancy and delivery represent a severe hemorrhagic risk, because bleeding may not always be preventable by platelet transfusions. Although GT can be a severe hemorrhagic disease, the prognosis is excellent with careful supportive care. Death from hemorrhage in diagnosed patients is rare unless associated with trauma, other disease (e.g., cancer), or chronic isoimmunization.

Laboratory Findings A prolonged bleeding time, deficient clot retraction, and deficient platelet aggregation with ADP, collagen, epinephrine, or thrombin are typical findings. Ristocetin-induced aggregation, on the other hand, and coagulation tests are normal. Thrombasthenic platelets are present in normal numbers and are morphologically normal when viewed by light microscopy.

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Management With GT patients, it is important to anticipate bleeding risks before performing procedures, and because the risk for bleeding is so unpredictable, it is prudent to administer prophylactic platelet transfusions even in the absence of a prior history of bleeding. Localized bleeding can be treated by limited measures, such as fibrin sealants or topical thrombin and antifibrinolytic agents (â-aminocaproic acid, tranexamic acid). Epistaxis and gingival bleeding can be controlled by nasal packing or the application of gel foam soaked in topical thrombin. The risk of gingival bleeding can be minimized by regular, proper dental care. When tooth extractions must be performed or when hemorrhage accompanies the loss of deciduous teeth, bleeding can be significantly reduced by the application of individually prepared plastic splints that provide physical support for hemostasis. Severe menorrhagia is common and can be effectively treated with high doses of progesterone, followed by maintenance treatment with birth control pills. Severe gastrointestinal bleeding is an isolated but severe problem. Iron deficiency anemia can develop insidiously with gingival oozing or minor menorrhagia. Confronted with a severe bleeding episode or as prophylaxis for invasive procedures, most GT patients receive blood transfusions, regardless of bleeding history.79 Transfusions should be continued until wound healing is complete. However, a significant number of transfused patients can develop HLA-specific alloantibodies or GPIIb-IIIa-specific isoantibodies, which seriously complicate transfusion therapy and limit future treatment. Isoantibodies can block normal transfused platelet aggregation and/or lead to the rapid removal of transfused platelets by immune mechanisms. It is not yet possible to predict the propensity of individual GT patients to develop such antibodies. Antibodies may be successfully removed prior to surgery by immunoadsorption on Protein A Sepharose, but this is a complex procedure restricted to specialized centers.80 Pregnancy and delivery represent a severe hemorrhagic risk. Platelet transfusions are required not only prior to delivery, but afterward as well. Successful delivery by cesarean section, with platelet transfusions, has been reported. The use of rFVIIa (NovoSeven®; NovoNordisk A/S, Malov, Denmark) is an alternative approach for early cessation of bleeding in GT, especially in the case of patients who have developed antibodies and/or have a history of transfusion refractoriness.81 It seems that rFVIIa enhances deposition of aIIbb3-deficient platelets on the subendothelial matrix through an interaction with fibrin formed by the increased thrombin generation82 leading to increased clot stability.83 However, some question the efficacy of rFVIIa in children with GT.84 In a few GT patients, the bleeding symptoms have been considered sufficiently serious to warrant allogeneic bone-marrow transplantation.85

Disorders of Hemostasis and Coagulation



Additional Hereditary Defects of Platelet Function (with Thrombocytopenia) MYH-9-related Thrombocytopenia Syndromes MYH-9-related disorder encompasses autosomal dominant macrothrombocytopenia syndromes previously classified as MayHegglin anomaly, Sebastian, Fechtner, and Epstein syndromes and certain inherited thrombocytopenias that have in common mutations within MYH9, the gene for nonmuscle myosin heavy chain II-A (NMMHC-IIA).86

Etiology

NMMHC-IIA is part of the nonmuscle myosin IIA hexamer that is a component of the contractile cytoskeleton in megakaryocytes, platelets, and other many other cell types. Mutations of MYH9

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that give rise to MYH-9-related disorder disrupt the stability of the nonmuscle myosin IIA hexamer or its association with regulatory proteins. This results in defective megakaryocytopoiesis that leads to thrombocytopenia. Additional platelet function defects arise from abnormal shape change or impaired expression of the GPIb complex.87 As in many other inherited hematologic disorders, incomplete penetrance accounts for a poor correlation between the MYH9 genotype and clinical phenotype as well as variability in phenotype among individuals with identical mutations.86

Clinical Features

Patients with MYH-9-related disorder present with bleeding, sensorineural hearing loss, glomerulonephritis, and/or eye abnormalities. The severity of the bleeding symptoms is out of proportion to the moderately decreased platelet count, suggesting the concurrence of a platelet function defect. Life-threatening bleeding has been reported, but is the exception. The combination of macrothrombocytopenia and one or more of the recognized clinical features of MYH-9-related disorder is strong evidence for this diagnosis.

Laboratory Findings

Platelet counts are normally from 20,000 to 130,000/ml, and are associated with an increased mean platelet volume and a conspicuous population of very large platelets. The presence of Dohle-like bodies within neutrophils on stained peripheral blood smears is highly suggestive of MYH-9-related disorder. An abnormal staining of neutrophil inclusions with antibodies specific for NMMHC-IIA is a more sensitive index of the syndrome, but a definitive diagnosis must be based on the identification of a causative mutation within MYH9.

Management

The symptoms of MYH-9-related disorders are highly variable, and therefore treatment of individual patients should be guided as much as possible by personal and family history.5 In general, management would be the same as for other mild platelet disorders.

Wiskott-Aldrich Syndrome Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by microthrombocytopenia, eczema, and immunodeficiency.1 The WAS gene encodes the WAS protein (WASP), and mutations in WAS result in decreased or absent expression of WASP, a key regulator of actin polymerization in hematopoietic cells playing a role as an adapter protein. The reduction in or absence of WASP results in premature proplatelet formation in the bone marrow and the microthrombocytopenia characteristic of WAS.

Etiology

WAS arises from mutations in WAS, the gene for the protein WASP that is expressed in all hematopoietic cell lineages. Isolated X-linked thrombocytopenia (XLT) is also caused by mutations in WAS and is a variant of the same disease but generally lacks severe immune deficiency.88

Clinical Features

Infants with WAS often present with bruising and purpura in the neonatal period with an increased risk of intracranial hemorrhage. Gastrointestinal bleeding or prolonged bleeding after circumcision may be a presenting feature. Eczema develops during the first year of childhood and can varying in severity, from widespread and debilitating in children with “classic” WAS to mild or absent in those with the XLT. Infections begin in the first 6 months of life. Bacterial infections occur more often, particularly otitis media and respiratory tract infections, whereas severe viral infections and opportunistic infections occur less frequently. In the XLT

72683_ch052_1128-1142.indd 1138

phenotype, eczema and infection do not occur. With increasing age, hemolytic anemia and vasculitis become the most frequent manifestations of the autoimmune component of this disease.89 Another life-threatening complication of WAS is malignancy, usually but not exclusively lymphoreticular in origin.

Laboratory Findings

Thrombocytopenia (ranging from 5,000 to 50,000/ml) with small platelets is evident at birth. Despite normal bone marrow megakaryocyte numbers and morphology, there may be ineffective thrombocytopoiesis with reduced platelet survival.

Management

The management of WAS should address bleeding manifestations, recurrent infections, eczema, autoimmune disorders, and the risk of malignancy. When available, bone marrow or stem cell transplant is the treatment of choice as early as possible. Proper management is benefited by a multidisciplinary approach, which should include an immunologist. When transfusion is necessary, HLA-compatible and irradiated platelets should be used in order to avoid sensitization. Because of the immune deficiency, platelets to be transfused should always be irradiated and certified free of cytomegalovirus. Splenectomy can result in an increase both in platelet number and size, but the risk of sepsis is increased post splenectomy, and a risk–benefit analysis of this operation must be considered for individual patients. Splenectomy does not influence the development of malignancy or autoimmune disorders, and it may thus be more successful in the milder phenotype of XLT.

Acquired Disorders of Platelet Function Drug-induced Platelet Dysfunction An adult patient that presents with a normal platelet count, mucocutaneous bleeding, and a negative family history, defined as no bleeding among first degree relatives, should be evaluated for an acquired disorder. Although inherited platelet disorders are rare, acquired disorders of platelet function are encountered frequently. A diverse variety of medications, from ionophores to ADP analogs, certain systemic diseases, and various procedures can induce platelet dysfunction (Table 52.2). Even common dietary substances, such as garlic or fish oil, can impair platelet function.90 This chapter focuses on commonly used drugs that can impair platelet function at therapeutic doses. Other drugs that are used to treat thromboembolic disorders are discussed in detail in Chapter 62. The extent of platelet dysfunction produced by drugs in healthy individuals is usually not clinically significant. On the other hand, patients with coagulation disorders, uremia, thrombocytopenia, and patients receiving heparin or Coumadin as anticoagulant

TA B L E 5 2 . 2

Drugs Affecting Platelet Function Analgesics Aspirin Nonsteroidal anti-inflammatory drugs Acetaminophen Antibiotics b-Lactams: Penicillins, cephalosporins Cardiovascular drugs

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therapy, can exhibit serious bleeding when they receive a drug that attenuates platelet function. In addition, the inherent variation in platelet reactivity among normal indviduals91 can greatly influence the susceptibility of an individual to the antiplatelet effects of dietary supplements or drugs.

Aspirin Aspirin inhibits platelet function via irreversible acetylation of platelet COX-1 and the resulting inhibition of TXA2 synthesis. A single high dose of aspirin (e.g., 325 mg or more) or consecutive smaller doses (e.g., 81 mg) taken for 3 or more days will produce essentially complete inhibition of TXA2 production. The hemostatic risk following aspirin is predictable, with a 5% to 10% incidence of minor bleeding and a 1% to 2% incidence of major bleeding, i.e., requiring hospitalization or red cell transfusion.92 In ex vivo whole blood perfusion assays, aspirin-treated platelets will adhere normally to denuded arterial segments or collagen-coated surfaces, but they fail to form thrombi due to inhibition of aggregate formation. By EM, one notes an inhibition of organelle centralization that normally follows platelet stimulation by collagen. In platelet aggregation assays, aspirin-treated platelets exhibit decreased responses to collagen and an absence of the secondary wave of aggregation that is normally induced by epinephrine and low concentrations of ADP, which is reflected in a marked reduction in the release of d-granule ADP, ATP, and serotonin. Individual differences in platelet responsiveness to aspirin have been noted. In some normal individuals, aspirin ingestion can modestly elevate the bleeding time and have a small impact on platelet aggregation in vivo, more so in men than in women, but in certain patients with otherwise minor platelet dysfunction, the bleeding time can be markedly prolonged and platelet aggregation severely impaired after aspirin ingestion. The search for a genetic basis for this difference in aspirin sensitivity is a hotly contested area of investigation.

Other Nonsteroidal Anti-inflammatory Drugs NSAIDs reversibly inhibit platelet COX-1, and normal platelet function is gradually restored when these drugs are stopped. For example, ibuprofen (600 mg daily) will prolong PFA-100 CTs, but these will conform to baseline within 24 hours after drug intake is ceased.93 It is uncommon to see clinical bleeding in normal individuals as a result of an NSAID, but gastrointestinal bleeding due to gastric ulceration is not uncommon. Paradoxically, ibuprofen can also exhibit a prothrombotic effect if it is ingested within 2 hours of taking aspirin, inasmuch as it can transiently block acetylation of the COX-1 target site.

Cyclooxygenase-2 Inhibitors COX-2 inhibitors provide the anti-inflammatory effects of COX blockade without affecting platelet function. COX-2 is expressed in endothelial cells, fibroblasts, and monocytes and is up-regulated in response to growth factors, cytokines, endotoxin, and hormones. Even though COX-2 inhibitors have no direct effect on platelets, they increase the risk for thrombosis and cardiovascular disease because they inhibit vascular (endothelial) cell synthesis of prostacyclin, a natural inhibitor of platelet activation and thrombosis.94

P2Y12 Antagonists Platelet responses to natural agonists are normally enhanced by the binding of released ADP to platelet purinogenic receptors. The thienopyridines clopidogrel and ticlopidine bind irreversibly to the platelet purinogenic receptor P2Y12 and thereby inhibit platelet responses induced by both exogenous ADP and ADP released from

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Chapter 52  Qualitative Disorders of Platelet Function

1139

platelet d-granules. However, clopidogrel has replaced ticlopidine as the preferred medication because the latter is associated with potentially life-threatening hematologic side effects (thrombotic thrombocytopenic purpura, agranulocytosis, and aplastic anemia). Steady-state inhibition of platelet function occurs after 3 to 5 daily doses of 75 mg clopidogrel, but it can be achieved sooner with a 300-mg loading dose. Even though clopidogrel has a greater effect than aspirin on the bleeding time,95 there is no greater risk of bleeding in vivo. The combination of clopidogrel and aspirin is currently used to prevent or treat arterial thrombosis.96 The effect of clopidogrel on platelet function is irreversible, such that after cessation of clopidogrel, platelet function returns to roughly 50% of normal levels by 72 hours, and its effect is completely reversed by 7 days.97

Antibiotics Large doses of various b-lactam antibiotics (penicillins more often than cephalosporins) can cause clinical bleeding, abnormal platelet function in vitro, and increased bleeding times, because of a nonspecific effect on ligand–receptor interactions.98 The effect on platelet function can be manifested by a dose-dependent reduction in platelet aggregation in vitro to ADP, epinephrine, and collagen. The clinical effect is exaggerated in hypoalbuminemic patients because the level of free drug is increased and thus more interact with the platelet surface. b-Lactam compounds influence clinical bleeding when there is co-existing hemostatic defect, such as in uremia, thrombocytopenia, or vitamin K deficiency. b-Lactams appear to modify the platelet membrane and decrease agonist binding. The effect can become event after several days of treatment and will not resolve until 7 to 10 days after discontinuation. Moxalactam and cefotetan can produce clinical bleeding as a result of both platelet dysfunction and the N-methythiotetrazole side-chain effect on vitamin K–dependent clotting factor synthesis.99,100

Integrin aIIbb3 Antagonists

Disorders of Hemostasis and Coagulation



Specific inhibitors of integrin aIIbb3 are potent inhibitors of platelet function, and three are currently approved by the FDA: Abciximab is a chimeric human–mouse monoclonal Fab fragment; tirofiban is an arginine–glycine–aspartate-based peptidomimetic; and eptifibatide (integrelin) is a synthetic cyclic heptapeptide based on the lysine–glycine–aspartate motif of the snake venom disintegrin barbourin. These antagonists, which inhibit platelet aggregation effectively because they prevent fibrinogen and VWF binding to aIIbb3, are used commonly in patients undergoing percutaneous coronary intervention (PCI), concurrently with heparin and other antiplatelet therapy such as aspirin. Bleeding is a common side effect of these antagonists and occurs in ∙10% of recipients, but intracranial bleeding (35,000 U of heparin per 24-hour period, regardless of patient weight, to reflect this form of heparin resistance.398 With true heparin resistance, both a measurement of anticoagulant activity such as the aPTT and a measurement of antithrombotic activity such as the anti–factor Xa activity assay demonstrate inadequate degrees of heparin activity. True heparin resistance most likely results from the nonspecific binding of heparin to mononuclear white cells, vascular endothelial cells, and acute-phase proteins such as histidine-rich glycoprotein, vitronectin, and PF4, resulting in an inadequate quantity of free or AT-bound heparin.396 Another potential cause of heparin resistance is acquired AT deficiency such as can be seen in cancer patients. Patients can also manifest an “apparent” heparin resistance characterized by dissociation between the aPTT and heparin assays.398 In these patients, the aPTT may be normal or near normal, whereas the anti–factor Xa activity assay reveals an appropriate target heparin activity level between 0.3 and 0.7 U/ml. Simply escalating the dose of heparin to achieve the desired aPTT without checking a heparin assay may result in a pronounced bleeding risk. Dissociation between the aPTT and

Disorders of Hemostasis and Coagulation

FIGURE 55.5. Inhibition of thrombin activity by the heparin (H)–antithrombin (AT) mechanism.  Reaction 1 indicates that in the absence of heparin catalysis, AT can irreversibly inactivate thrombin (T), albeit in an inefficient manner. In the presence of H, a conformation change occurs in the AT molecule, and heparin also acts as a template to bind both AT and T, promoting rapid inactivation of coagulation (reaction 2 ). In reaction 3, heparin is released from the AT–T complex and is available to catalyze subsequent reactions. Inhibition of factor Xa by AT–H does not require binding of heparin to the factor Xa molecule.

TAB L E 5 5 . 7

Weight-Based Nomogram Of Unfractionated Heparin (Ufh) In Treatment Of Venous Thromboembolism Initial UFH Bolus

80 U/kg

Initial UFH infusion rate Check aPTT after 6 h of IV infusion; modify infusion rate as follows:

18 U/kg/h

 If aPTT3 × control, hold infusion for 1 h, then decrease infusion by

4 U/kg/h 2 U/kg/h — 2 U/kg/h 3 U/kg/h

aPTT, activated partial thromboplastin time. Data from Raschke RA, Reilly BM, Guidry JR, et al. The weight-based heparin dosing nomogram compared with a “standard care” nomogram. Ann Intern Med 1993; 119:874–881.

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heparin concentration likely reflects elevated levels of factor VIII that can shorten the in vitro aPTT without affecting the antithrombotic actions of the drug.

Low-molecular-weight Heparins LMWH is derived from the enzymatic or chemical cleavage of UFH to produce a mixture of low-molecular–weight glycosaminoglycan molecules with a mean molecular weight of ∼5,000 D (∼15 saccharide units).396 For example, enoxaparin sodium is produced by benzylation followed by alkaline hydrolysis, dalteparin sodium is produced by controlled nitrous acid depolymerization, and tinzaparin sodium is produced by enzymatic digestion with heparinase. LMWH binds AT via the same pentasaccharide sequence as UFH.396 However, because of the predominance of molecules 50 mg/day) to achieve therapeutic anticoagulation; the term warfarin resistance has been applied.440 Patients who are difficult to anticoagulate with warfarin, either because they exhibit warfarin resistance or because they are very sensitive to the drug and cannot be safely regulated, may be candidates for long-term parenteral anticoagulants, or a novel oral anticoagulant.

Disorders of Hemostasis and Coagulation



Laboratory Monitoring of Warfarin Therapy

The PT assay is useful to monitor warfarin therapy, because this assay measures three vitamin-K–dependent coagulation proteins: Factors VII, X, and prothrombin. The PT is particularly sensitive to factor VII deficiency; with a half-life of 4 to 6 hours, the factor VII level may drop rapidly after only 1 day of warfarin therapy and prolong the PT value. However, because the other vitamin-K– dependent proteins have longer half-lives, therapeutic anticoagulation takes 4 to 5 days. There is no advantage to giving larger loading doses of warfarin (e.g., >10 mg); this regimen only results in a more rapid drop in factor VII levels, delay in attainment of a stable PT, a precipitous fall in protein C levels, and predisposition to warfarin-induced skin necrosis.439,449,450 To understand current recommendations for monitoring ­warfarin therapy, it is important to appreciate the concept of the INR,

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Log PT Reference Reagent

TAB L E 55.10

Drugs And Medical Conditions Affecting Warfarin Potency Antagonists

Drugs Acetaminophen Anabolic steroids Broad-spectrum antibiotics Chloral hydrate Cimetidine Clofibrate Disulfiram Fluconazole Indomethacin Influenza vaccine Lovastatin Metronidazole Omeprazole Phenylbutazone Phenytoin Propranolol Protease inhibitors (except ritonavir) Quinine/quinidine Salicylates Tamoxifen Thyroid drugs Trimethoprim/sulfamethoxazole Medical Conditions Older age Liver disease Biliary disease Malabsorption Congestive heart failure Fever Hyperthyroidism Malnutrition Vitamin K deficiency Cancer

Drugs Adrenal corticosteroids Barbiturates Carbamazepine Chlordiazepoxide Cholestyramine Efavirenz Griseofulvin Nafcillin Rifampin Sucralfate Trazodone Medical Conditions Excess dietary vitamin K Inherited resistance to warfarin Hypothyroidism Nephrotic syndrome

Data from the 2003 Physicians’ Desk Reference. Montvale, NJ: Thomson Healthcare; Hirsh J, Dalen JE, Anderson DR, et al. Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest 2001;119(Suppl):108S–121S.

a method that standardizes PT assays.438,451 An international reference thromboplastin preparation has been adopted by the World Health Organization (WHO). Each new commercial thromboplastin is calibrated against the primary WHO reference preparation. These results are used to calculate the relative sensitivity of the unknown preparation compared with the WHO standard (international sensitivity index [ISI]). The method to determine the ISI for a particular thromboplastin is depicted in Figure 55.7. By adjusting for the ISI of a particular thromboplastin, an INR, defined as the PT ratio that would have been obtained if the WHO standard thromboplastin had been used, can be determined. The  INR is calculated using the following formula: INR = (PT ratio) ISI (Fig. 55.8). The American College of Chest Physicians’ consensus panel recommends low-intensity warfarin therapy (INR, 2.0 to 3.0) for all indications except prosthetic mechanical heart valves and prophylaxis of recurrent MI, for which higher-intensity warfarin therapy (INR, 2.5 to 3.5) is suggested.438

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Normal Log PT Laboratory Reagent FIGURE 55.7. Method for determination of an international sensitivity index (ISI) value for a laboratory’s thromboplastin preparation. Log prothrombin time (PT) values are determined using a reference thromboplastin reagent and the commercial laboratory thromboplastin reagent on patients receiving stable (2 weeks) oral anticoagulant therapy and a group of normal, untreated volunteers. The best-fit line is determined, and the slope of this line multiplied by the ISI of the reference thromboplastin reagent is the ISI value for the commercial thromboplastin reagent. From Rodgers GM. Laboratory monitoring of anticoagulant and fibrinolytic therapy. In: Kjeldsberg C, McKenna R, Perkins S, et al., eds. Practical diagnosis of hematologic disorders, 2nd ed. Chicago, IL: ASCP Press, 1995:745–755, with permission.

Adverse Effects of Warfarin Therapy Bleeding.  A direct relationship exists between the risk of bleeding and the intensity of anticoagulation, with patients receiving higher-intensity (INR > 3.0) therapy having a fivefold greater risk of bleeding.452 Other major factors contributing to bleeding include co-existing conditions such as structural gastrointestinal lesions, hypertension, renal disease, and cerebrovascular disease.453,454 Investigation of patients who experience visceral bleeding while on warfarin therapy often results in identification of structural disease.455 Highest bleeding rates occur early in the course of warfarin therapy and in patients with cerebrovascular disease.456,453 For patients given lowintensity warfarin therapy for prophylaxis of VTE, the risk of major bleeding is 12 mo and no other risk factors

a CHADS2

stroke score is calculated by adding one point for each of: congestive heart failure, hypertension, Age > 75 years old, diabetes and 2 points for prior stroke. Adapted from Douketis JD, Spyropoulos AC, Spencer FA, et al. Perioperative management of antithrombotic therapy: antithrombotic therapy and prevention of thrombosis, 9th ed: American college of chest physicians evidence-based clinical practice guidelines. Chest 2012;141(2):e326S–e350S.

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and liver. This complication may be difficult to distinguish from warfarin-induced skin necrosis, except that on biopsy, cholesterol emboli are present in the purple toe syndrome, and widespread vascular thromboses are seen in skin necrosis. Treatment involves prompt recognition of the syndrome and discontinuation of warfarin therapy.470 Novel Oral Anticoagulants. Warfarin has substantial limitations including inter- and intrapatient dosing variability, drug–drug and drug–diet interactions, and a narrow therapeutic index. These limitations necessitate routine laboratory monitoring and dose adjustment that can be cumbersome for some patients. An oral anticoagulant that is safe and effective, has predictable pharmacology, has few drug–drug and drug–dietary interactions, and does not require laboratory monitoring has the potential to revolutionize the chronic management of patients with atrial fibrillation, prosthetic heart valves, and recurrent venous thrombosis as well as facilitate better compliance with out-of-hospital thromboprophylaxis regimens. Over the past 5 years, there has been a rapid emergence of such novel oral anticoagulants (NOACs) into the clinical landscape. At the forefront are the oral DTI dabigatran etexilate and the oral factor Xa inhibitors (e.g., rivaroxaban, apixaban, and edoxaban). Tables 55.12 and 55.13 summarize the properties of the NOACs that are currently available for clinical use and the comparative results of large-scale studies in patients with atrial fibrillation, respectively.471 It is important to note that numerous additional agents are also currently under investigation.471

Dabigatran Etexilate

Dabigatran etexilate is an oral prodrug that is rapidly converted by serum esterases to dabigatran, a potent univalent DTI. Oral bioavailability is ∼6% to 7%, and uptake is reduced by proton pump inhibitors and delayed with food; however, these kinetic alterations are not thought to be clinically significant and do not require dose adjustments. Peak dabigatran serum levels occur ∼2 hours after an oral dose; dabigatran is primarily eliminated

TAB L E 55.12

Pharmacology Of Novel Oral Anticoagulants

Target

Dabigatran (Pradaxa®)

Rivaroxaban (Xarelto®)

Apixaban (Eliquis®)

Thrombin (Binds Reversibly) Capsule 6% 1–2 h Conjugation; No CYP involvement

Factor Xa (Binds Reversibly) Tablet 50–85% 1–3 h Oxidation (via CYP3A4) + conjugation

DrugInteractions

Strong p-gp inducers and inhibitors

Faxtor Xa (Binds Reversibly) Tablet 60–80% 2–4 h Oxidation (via CYP3A4 + CYP2J2) + hydrolysis Strong p-gp and CYP3A4 inducers and inhibitors

Renal Excretion Half-life Dosing Frequency, Major Trials

80%

66%

Stong p-gp and CYP3A4 inducers and inhibitors 25%

14–17 h BID

9–13 h QD

9–14 h BID

Dosage Form Bioavailability Time to Peak Metabolism

BID, twice daily; CYP, cytochrome p 450; p-gp, p-glycoprotein; QD, daily.

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by renal clearance, and has a half-life of ∼14 to 17 hours.471 Although a reduced dose has been approved for use in the United States by the FDA for patients with a creatinine clearance of less than 30 ml/minute, this recommendation is solely based upon drug kinetic modeling, and the drug is contraindicated in patients with a clearance 72.6% with hazard ratios of 0.67 (95% CI, 0.56 to 0.80) and 1.11 (95% CI, 0.91 to 1.35), respectively.476

Oral Factor Xa Inhibitors Because of the demonstrated efficacy and safety of factor Xa inhibition with fondaparinux, oral factor Xa inhibitors have entered the clinical landscape. Oral factor Xa inhibitors that are available for clinical use or are in development include rivaroxaban (Bayer Healthcare), apixaban (Bristol Myers Squibb/

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TAB L E 55.1 3

Trial

RE-LY

ROCKET-AF

ARISTOTLE

AVERROES

Drug and dose

Dabigatran etexilate 150 mg and 110 mg BIDa No

Rivaroxaban 20 mg QDa

Apixaban 5 mg BIDa

Apixaban 5 mg BIDa

Yes: 15 mg QD if CrCl 30–49 ml

Randomized open label (n = 18,113) 71.5 20% 2.1 50.4 Warfarin: INR 2–3 67% TTR

Randomized double blind (n = 14,000)

Yes: 2.5 mg BID if 2 of: age >80, weight 1.5 Randomized double blind (n = 18,201) 70 19% 2.1 43 Warfarin: INR 2–3 66% TTR

Yes: 2.5 mg BID if 2 of: age >80, weight 1.5 Randomized double blind (n = 5,599) 70 13.5% 2.1 60.5 Aspirin 81–324 mg

Adjusted dose? Design Mean age (y) Prior stroke/TIA Mean CHADS2 Warfarin naïve (%) Comparator

73 55% 3.5 37.5 Warfarin: INR 2–3 57.8% TTR

aBID,

twice daily; Cr, creatinine; CrCl, creatinine clearance; INR, international normalized ratio; QD, daily; TIA, transient ischemic attack; TTR, time in therapeutic range; wt, weight. Data from references 475, 479, 480, 481.

Pfizer), edoxaban (Daiichi), betrixaban (Portola), and TAK-442 (Takeda).471 Currently, rivaroxaban and apixaban are the most advanced in their clinical trials programs and are approved for use in the European Union, Canada, or the United States. Rivaroxaban is a reversible small molecule direct factor Xa inhibitor that binds to and inactivates both fluid-phase factor Xa as well as factor Xa associated with the prothrombinase complex. Rivaroxaban has an oral bioavailability of ∼60% to 80%, with peak levels achieved 2 to 3 hours after an oral dose. The drug is metabolized in part by both CYP3A4-dependent and CYP3A4independent pathways, and has an elimination half-life of ∼7 to 11 hours.471 The kidneys eliminate approximately 35% of active drug as well as inactive metabolites. Potent combined P-gp and CYP3A4 inhibitors (e.g., ketoconazole, itraconazole, lopinavir/ritonavir, ritonavir, indinavir/ritonavir, and conivaptan) and inducers (e.g., carbamezapine, phenytoin, rifampin, St John’s Wort) should be avoided in patients on rivaroxaban because they can significantly increase or decrease plasma drug concentrations, respectively.477 Rivaroxaban is currently approved for the prevention of VTE in patients undergoing elective joint replacement surgery, the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, and the treatment of VTE.477 In

the RECORD trials, rivaroxaban (10 mg daily) was compared to enoxaparin for thromboprophylaxis in patients undergoing elective joint replacement surgery. In a pooled analysis, the combined endpoint of symptomatic VTE and mortality occurred less often in patients receiving rivaroxaban (1.3% vs. 0.6%, p < 0.001), whereas clinically relevant bleeding was more frequent (2.6% vs. 3.3%, p = 0.026).478 The ROCKET-AF trial was a large (n = 14,000) double-blind, noninferiority, randomized trial comparing rivaroxaban (20 mg daily in patients with normal renal function and 15 mg daily in patients with a creatinine clearance of 30 to 49 ml/min) to adjusted-dose warfarin (target INR 2 to 3) in patients with atrial fibrillation who were at an increased risk of stroke or systemic embolism.479 Rivaroxaban met the prespecified endpoint of stroke/systemic embolism occurrence with a HR of 0.88 (95% CI 0.74 to 1.03, p < 0.001 for noninferiority), had similar rates of major bleeding (HR 1.04, 95% CI 0.9 to 1.2, p = 0.58), and less intracranial hemorrhage (HR 0.67, 95% CI 0.47 to 0.93, p = 0.02).479 Apixaban is a reversible, small molecule, direct factor Xa inhibitor that, as does rivaroxaban, binds to and inactivates both fluid-phase as well as clot-bound factor Xa. Apixaban has an oral bioavailability of >45%, achieves peak levels ∼3 hours after an oral dose, has an elimination half-life of ∼8 to 14 hours, and

Disorders of Hemostasis and Coagulation

Novel Oral Anticoagulant Atrial Fibrillation Clinical Trial Overview

TAB L E 5 5 . 1 4

Novel Anticoagulant Agents: Peri-Procedural Management

Preprocedure DABIGATRAN CrCl >50 CrCl 30–50 CrCl 50 CrCl 10 doses (>5 d)

8h 9–10 h

Skip 1 dose (1 d) Skip 2 doses (2 d)

Skip 2 doses (2 d) Skip 3–4 doses (3–4 d)

9h Skip 1 dose (1 d) Skip 2 doses (2 d) Delay re-initiation until hemostasis is certain (24–72 h) and no epidural catheter is present

CrCl, creatinine clearance (ml/min).

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is eliminated via multiple mechanisms. Similar to rivaroxaban, potent inhibitors of CYP3A4 (e.g., ketoconazole, itraconazole, lopinavir/ritonavir, ritonavir, indinavir/ritonavir, and conivaptan) and inducers (e.g., carbamezapine, phenytoin, rifampin, St John’s Wort) should be avoided.471 Apixaban has also been studied for thromboprophylaxis, stroke prevention in atrial fibrillation, and VTE treatment. Apixaban is currently approved for use in the European Union, Canada, and approved in the United States for the treatment of non-valvular atrial fibrillation. The AVERROES trial was a large (n = 5,599) double-blind, randomized trial comparing apixaban (5 mg twice daily) to aspirin (81 to 324 mg) in patients with atrial fibrillation who were unsuitable candidates for warfarin therapy. Compared to aspirin, apixaban was more effective and equally safe with a HR of 0.45 (95% CI 0.32 to 0.62, p < 0.001) for stroke/systemic embolism occurrence, HR 1.13 (95% CI 0.74 to 1.75, p = 0.57) for major bleeding, and a HR 0.85 (95% CI 0.38 to 1.9, p = 0.69) for intracranial bleeding.480 The ARISTOTLE trial was a large (n = 18,201) double-blind, double dummy, randomized trial comparing apixaban (5 mg twice daily) to adjusted dose warfarin (INR 2 to 3) in patients with atrial fibrillation. Compared to warfarin, apixaban was more effective and safer with a HR of 0.79 (95% CI 0.66 to 0.95, p = 0.01) for stroke/systemic embolism occurrence, HR 0.69 (95% CI 0.6 to 0.8, p < 0.001) for major bleeding, and a HR 0.42 (95% CI 0.3 to 0.58, p < 0.001) for intracranial bleeding.481 Because of the predictability in dose-response, laboratory monitoring of the oral factor-Xa inhibitors is unnecessary. If, however, lab assessment is required (e.g., need for urgent surgery or management of bleeding), a normal aPTT and a normal INR suggest little anticoagulant presence, although clinical judgment is required. 274a,482 Table 55.14 suggests a management approach to patients requiring temporary interruption of anticoagulation for an invasive procedure. Like all anticoagulants, the primary adverse effect of the factor Xa inhibitors is an increase in bleeding risk. Both rivaroxaban and apixaban have significant protein binding and so are not anticipated to be dialyzable. Management of bleeding in patients on these agents is problematic as there is no known reversal agent. Patients who have bleeding on rivaroxaban or apixaban should be managed with aggressive conservative measures including active control of bleeding site.474 In cases of life-threatening bleeding, the administration of activated prothrombin complex concentrates may be effective in reversing the effect.483

Thrombolytic Drugs The major reaction of the fibrinolytic (plasminogen) system involves the conversion by PAs of the inactive proenzyme, plasminogen, into the active enzyme, plasmin. Plasmin can degrade fibrinogen, fibrin monomers, and cross-linked fibrin (as found in thrombi) into FDPs. These plasmin-mediated reactions generate many species of FDPs including unique species of FDP such as fragment X from fibrinogenolysis and cross-linked FDPs such as (DD)E- and d-dimer from cross-linked fibrin.484,485 Knowledge of these reactions is necessary to appreciate the mechanisms of action and limitations of commercial PAs. A common feature of the management of all thromboembolic diseases is the desire to restore vascular patency in a timely fashion to prevent loss of tissue, organ, and limb function, as well as life. Acute arterial thrombosis warrants an attempt at immediate thrombolysis, whereas venous thrombosis only warrants such intervention in extreme cases. Recognition of the importance of the endogenous fibrinolytic system in limiting the size of hemostatic thrombi, clearing hemostatic thrombi after vascular repair, and preventing pathologic thrombosis has resulted in the development of pharmacologic fibrinolytic (thrombolytic) agents to facilitate rapid restoration of vascular patency. Most

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thrombolytic agents are recombinant forms of physiologic PAs. The commercially available PAs differ with regard to plasma half-life, fibrin selectivity, primary clinical usage, primary infusion strategy, and immunogenicity. Currently available and investigational PAs and their key characteristics are summarized in Table 55.15. Most thrombolytic agents are fashioned after endogenous t-PA or urokinase. Traditional thrombolytic drugs include bacteriaderived streptokinase (SK), anisoylated plasminogen SK activator complex, urokinase (two-chain u-PA), and recombinant t-PA (rt-PA). Newer molecules have been and are being developed in an attempt to improve on the traditional agents. Major goals of new thrombolytic agent development include increasing fibrin specificity, theoretically to reduce bleeding complications, prolonging initial plasma half-life to facilitate single- or double-bolus administration, reducing sensitivity to inactivation by PAI-1, and improving production efficiency. New thrombolytic agents include mutants of PAs, chimeric PAs, conjugates of PAs with monoclonal antibodies, and novel PAs from animal or bacterial origin. Non-PA thrombolytic agents that can degrade fibrin and/or fibrinogen directly (e.g., microplasmin, alfimeprase, and ancrod) are also under investigation. TAB L E 5 5 . 1 5

Properties Of Currently Available And Investigational Thrombolytic Agents Thrombolytic Agent

Molecular Weight (Daltons)

Plasma Half-life (Minutes)

Streptokinase

47,000

Anisoylated plasminogen streptokinase activator complex Urokinase

131,000

20 (drug); 90 (lytic effect) 40–90

Complexes with plasminogen to achieve activity Streptokinase and plasminogen complex

34,000/54,000

15

Recombinant urokinase

54,000

7

Recombinant prourokinase

49,000

7

Alteplase Reteplase (recombinant plasminogen activator) Tenecteplase

65,000 39,000

4–8 15

Direct plasminogen activator derived from fetal kidney cells Recombinant high-molecular– weight urokinase Active after conversion to urokinase A recombinant t-PA Truncated t-PA with an extended half-life

65,000

20 (initial); 90–130 (terminal)

Desmoteplase (rDSPAa1)

52,000

2.8 h

Plasmin

85,000

—

Key Properties

A modified t-PA with an extended half-life, enhanced ­plasminogen activator inhibitor-1 resistance, and greater fibrin specificity Highly fibrin specific and lacking in neurotoxic effects of alteplase Catheter-directed therapy

t-PA, tissue-type plasminogen activator.

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Streptokinase SK is obtained from cultures of b-hemolytic streptococci. By itself, SK has no PA activity, but after combining with plasminogen, a complex is formed that is capable of activating other plasminogen molecules to plasmin.486 Purified SK has a molecular weight of 47,000 D. SK was the first clinically used thrombolytic agent. It is not fibrin-selective in that its therapeutic use results in systemic fibrin(ogen)olysis and what is termed the lytic state from proteolysis of fibrinogen, factors V and VIII, and other plasma proteins.486 Platelet function may also be perturbed, because plasmin can proteolyze key platelet membrane receptors.487 Generation of FDPs also contributes to the significant hemostatic defect of thrombolytic therapy. Although the lytic state predisposes patients to bleeding, the benefit of decreased blood viscosity that results from the lytic state may be clinically important. The half-life of SK is ∼20 minutes. Because SK is a bacterial protein, it is antigenic, and allergic reactions occur in ∼6% of patients. Anaphylaxis during SK use occurs in ∼0.1% of patients.488 Patients previously exposed to SK or those with previous streptococcal infections may acquire antistreptococcal antibody levels sufficient to neutralize the activity of SK. Therefore, all patients receiving SK should be monitored to ensure attainment of the lytic state. This can be done with the thrombin time assay. SK has been used primarily to treat VTE and MI, as well as to treat central venous access-device–associated thrombosis.

Urokinase-type Plasminogen Activator In the past, u-PA was obtained from human fetal kidney cell cultures. It is currently produced using nonhuman mammalian tissue cultures. Its molecular weight is 34,000 D. u-PA is not fibrin-­selective, and this drug also produces a lytic state. The halflife of u-PA is ∼15 minutes. u-PA is used to treat VTE, MI, and thrombolysis of clotted catheters.

Tissue-type Plasminogen Activator Currently, t-PA is produced by recombinant technology as a twochain species, with a molecular weight of ∼65,000 D.489,490 In vitro, t-PA is fibrin-specific, because of its high affinity for fibrin with which it forms a ternary complex with plasminogen. However, with t-PA dosage regimens currently being used, the lytic state is produced in vivo.491 Consequently, bleeding complications with t-PA are similar to those with SK or u-PA.492 The half-life of t-PA is much shorter than that of SK or u-PA, ∼5 minutes. t-PA is used to treat VTE and acute MI492–496 and has also been approved for use in acute ischemic stroke (within 3 hours of stroke onset) and venous catheter withdrawal occlusion.497 Laboratory monitoring of t-PA therapy is usually not recommended.

Tissue-type Plasminogen Activator Variants Recombinant PA (r-PA; reteplase) is a nonglycosylated deletion mutant of wild-type human t-PA comprised of only the kringle 2 and the protease domains of the parent molecule. Lack of the finger domain imparts lower fibrin-binding affinity.498 Lack of glycosylation, a finger domain, and an epidermal growth factor domain impart an extended half-life (15 minutes vs. 5 minutes). The longer half-life allows for double-bolus administration. The longer half-lives of r-PA and TNK–rt-PA compared with t-PA facilitate bolus administration primarily for acute coronary thrombosis.

Other Plasminogen Activators Recombinant glycosylated prourokinase (single-chain u-PA) has a greater stability than recombinant nonglycosylated prourokinase and has been evaluated for catheter-directed, intra-arterial treatment of stroke.499 Staphylokinase is produced by Staphylococcus aureus.

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1247

It appears to have substantial thrombolytic activity, but it may also be immunogenic.500 Vampire bat (Desmodus rotundus) salivary PA (rDSPAa1, desmoteplase) possesses >72% primary structure homology to human t-PA but lacks a kringle 2 domain501 which may impart greater fibrin specificity. Finally, although not a PA, plasmin therapy has been investigated in arterial vascular disease.471

Thrombolytic Therapy–Associated Bleeding Bleeding is the most common complication associated with thrombolytic therapy, regardless of the agent. The bleeding stems from plasmin’s inability to distinguish between hemostatic and pathologic thrombi. This complication can range from minor bleeding at an intravenous infusion site to life-threatening ­hemorrhage.502 Intracranial hemorrhage is a relatively uncommon but serious complication of thrombolysis in patients being treated for acute MI. The factors that increase the risk for bleeding during thrombolytic therapy are not fully understood. However, Gurwitz and associates used the National Registry for MI to determine risk factors for this adverse event in individuals treated with t-PA.503 Their analysis of 673 patients with intracranial hemorrhage indicated that older age, female sex, black ethnicity, systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥100 mm Hg, history of stroke, t-PA dose ≥1.5 mg/kg, and lower body weight were all significantly associated with an increased risk for intracranial hemorrhage. It is also possible that the properties of the agent used for thrombolysis may contribute to the risk for bleeding complications.

Thrombolytic Fibrin Specificity and Hemorrhagic Risk Thrombolytic agents can be characterized along a variety of dimensions, but one that is often mentioned is fibrin specificity.504 The ability of a thrombolytic agent (PA) to distinguish between plasminogen in the general circulation and plasminogen bound to fibrin surfaces dictates its fibrin specificity. Activation of fibrin-bound plasminogen results in the generation of fibrin-bound plasmin that is protected from inactivation by a2-antiplasmin. Bound plasmin generates soluble fibrin degradation products; circulating plasmin degrades fibrinogen into FDPs. Fibrin specificity differs from fibrin affinity, which is a measure of how avidly a given agent binds to fibrin, but not its specificity for this molecule.504 At present, there is little evidence to support the view that differences in fibrin affinity among PAs are significantly correlated with either the efficacy or safety of these preparations.505 High fibrin specificity is thought to be associated with lower risk for hemorrhagic complications in patients undergoing thrombolytic therapy because of the belief that plasmin generated on the fibrin surface of a thrombus restricts its activity only to that surface. This view is not universally supported by available data from large-scale clinical trials. A relationship between high fibrin specificity and reduced bleeding risk has been demonstrated.506 The results of the Assessment of the Safety and Efficacy of a New Thrombolytic (ASSENT)-2 trial, which included 16,949 patients with acute MI, showed that the use of the highly fibrin-specific thrombolytic agent TNK-tPA, compared with rt-PA, was associated with a significantly lower risk for major noncerebral bleeding.506 This lower rate of bleeding complications was correlated with a significant reduction in the need for blood transfusions. The ASSENT-2 investigators also reported that TNK-tPA was associated with a significantly lower risk for noncerebral bleeding than the lessspecific agent alteplase.507 Intracranial bleeding rates were comparable with the two agents. Results from other large-scale studies support the opposing view that high fibrin specificity may actually be associated with increased risk for intracranial bleeding in patients undergoing

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thrombolytic therapy for acute MI. For example, the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries (GUSTO) trial showed that the risk of intracranial bleeding was slightly higher in 41,021 patients with MIs who received treatment with rt-PA as compared with SK.508 These findings are consistent with those from another very large-scale comparison of SK with rt-PA in 20,768 patients with MI (Gruppo Italiano per lo Studio della Streptochinasi nell’Infarto Miocardico 2 [GISSI-2]), which showed a significantly higher risk of stroke in patients who received the latter, more fibrin-specific agent.509 Similarly, the Third International Study of Infarct Survival (ISIS-3) showed that treatment of patients with anisoylated plasminogen SK activator complex was associated with increased risk for intracranial bleeding compared with SK in a large cohort of 41,299 patients who received thrombolytic therapy for suspected MI.510 There are a number of potential explanations for the association between high fibrin specificity and increased intracranial bleeding observed in the patients treated in the GUSTO, GISSI-2, and ISIS-3 trials. These include the inability of fibrin-specific agents to distinguish between pathologic thrombi and hemostatic thrombi, and the possibility that treatment with fibrin-specific agents resulted in greater degradation of hemostatic fibrinogen and other circulating coagulation factors than SK. Finally, it may be that fibrin-specific therapy resulted in increased production and accumulation of fragment X, and that this enhanced the bleeding risk.511

Venous Thromboembolic Disease VTE, encompassing both DVT and PE is a common disease that carries a substantial risk of morbidity and mortality, is associated with high healthcare costs, and for many patients can be prevented. It has been estimated that between 500,000 and 2 million VTE cases, including calf vein thrombosis, proximal DVT, and PE, occur annually in the United States alone. It is estimated that up to 50% of DVT and PE are asymptomatic or undetected.512 The major clinical consequences of extremity DVT include the PTS (chronic swelling, stasis dermatitis, stasis ulceration, and venous claudication: All secondary to venous insufficiency) and PE. The major clinical consequences of PE include acute lung infarction, chronic dyspnea, chronic pulmonary hypertension, and death. DVT restricted to the calf veins uncommonly results in clinically important PE and is rarely associated with a fatal outcome. In contrast, inadequately treated DVT involving the popliteal or more proximal leg veins is associated with a 20% to 50% risk of clinically relevant recurrence and is strongly associated with both symptomatic and fatal PE.513,514 In untreated patients, death from PE occurs most frequently within 24 to 48 hours of initial presentation. All-cause mortality rates in treated patients with PE is as high as 11% at 2 weeks and 17% at 3 months.512 Even small PE in patients with emphysema, cardiac disease, or lung involvement with malignancy may result in death. Any VTE in a patient with a contraindication to anticoagulation presents a therapeutic challenge and greater likelihood of adverse outcome.

Management of Venous Thromboembolism For a majority of patients with VTE, treatment is traditionally straightforward and includes an initial treatment phase (first 5 to 7 days) wherein there is an immediate initiation of a therapeutically dosed parenteral anticoagulant (e.g., heparin or low-­ molecular–weight heparin) and simultaneous initiation of long-term therapy (e.g., vitamin K antagonist), followed by a long-term treatment phase (from 5 to 7 days to 3 months) where the parenteral anticoagulant is discontinued, and VKA therapy is continued, and then an extended treatment phase (3 months

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to indefinite) where there is a decision to continue treatment or not.515,516,517 The emergence of the NOACs will greatly simplify this approach for a majority of patients.

Initial Treatment of Venous Thromboembolism The mainstay of pharmacologic therapy for VTE is anticoagulation. A delay in achieving therapeutic anticoagulant intensity may have a negative effect on a patient’s long-term VTE recurrence rate.518,519 VKA have a delayed onset of action and the necessity of instituting a rapid-onset parenteral anticoagulant (e.g., heparin) in patients with acute VTE during VKA initiation has been highlighted.520 As long as warfarin is dosed to a therapeutic level, overlap therapy with heparins can be minimized to 5 days.517,521,522 UFH remains the preferred initial parenteral anticoagulant for certain patients with acute VTE such as those with end-stage renal disease. When heparin is used, weight-based dosing (80-U/kg bolus followed by 18 U/kg/hour) with subsequent dose adjustments based on a standardized nomogram (Table 55.7) facilitates achieving a target aPTT.523 Use of any published nomogram helps facilitate the rapid achievement of target intensity anticoagulation. Adjusted-dose subcutaneous UFH, intermittent intravenous UFH boluses, and fixed dose subcutaneous heparin have also been used effectively in the treatment of VTE.513,524,525 The therapeutic intensity of UFH is monitored using the aPTT. An aPTT therapeutic range that correlates with an anti-FXa activity level of 0.3 to 0.7 U/ml is preferred.397 The aPTT should be checked every 4 to 6 hours until the aPTT surpasses the minimum of the therapeutic range. As compared to heparin, weight-based subcutaneous LMWH is a preferred treatment option for most patients with acute VTE.517 In 1992 two separate groups of investigators demonstrated that fixed-dose LMWH therapy was at least as safe and effective as adjusted-dose intravenous heparin in patients with proximal vein thrombosis.526,527 Subsequently, numerous other comparative trials have been performed, and in a meta-analysis of 13 comparative trials Dolovich et al.528 demonstrated the LMWHs to be comparable to heparin for the initial treatment of VTE with regard to recurrence, major bleeding, and total mortality.528 Similarly, in a meta-analysis of 2,110 patients who presented with acute symptomatic PE, LMWH were found to be at least as effective (OR for recurrence, 0.68, 95% CI, 0.42 to 1.09) and safe (OR for major bleeding, 0.67, 95% CI, 0.36 to 1.27) as heparin.529 Enoxaparin sodium is dosed at 1.0 mg/kg body weight every 12 hours or 1.5 mg/kg once daily.530 Tinzaparin is dosed at 175 U/kg once daily.517 Dalteparin is dosed at 200 U/kg (up to 18,000 U) once daily. The Matisse trials also support the use of fondaparinux in patients with VTE with efficacy and safety at least as good as LMWH.517,531 Efficient initial dosing of VKA therapy in patients with VTE minimizes excessive duration (i.e., beyond 5 to 7 days) of potentially costly parenteral anticoagulants and may allow for shorter hospitalization. The use of standardized warfarin initiation dosing nomograms has been shown to be efficient and safe.450,532,533 In two separate studies of predominantly inpatients, dosing nomograms using a 5-mg initial dose was compared to 10 mg and shown to be effective in achieving a therapeutic INR within 5 days with less risk of excessive anticoagulation.450,532 More recently a 10-mg initial VKA dose nomogram in outpatients being treated for VTE had a greater percentage of patients achieving a therapeutic INR by day 5 of therapy with no difference in excessive anticoagulation or adverse events as compared to a lower initial dose.533 Lower initial doses (e.g., 5 mg or less) may be most appropriate for those patients who are elderly, have low body weight, are on interacting medications, or have poor nutrition, whereas a higher dose (e.g., 10 mg) may be acceptable for others. The initial treatment of VTE used to be confined to the inpatient setting; however, two clinical trials demonstrated that

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outpatient LMWH treatment for carefully selected patients with acute DVT is safe and effective and can lead to substantial cost savings.534–536 Assuming an appropriate care-delivery system is in place, outpatient DVT treatment has become appropriate for a majority of DVT patients except perhaps, those with massive ileofemoral DVT, a high bleeding risk, or substantial co-morbid illness that otherwise necessitates hospitalization.537 Appropriate initial site-of-care for patients who present with acute PE is more controversial. Unlike patients with acute DVT, patients with acute PE represent a very heterogeneous risk-group with 3-month mortality rates ranging from 1.4% to 17.4%.512,538 Standardized clinical assessments, biomarkers, and right ventricular size on imaging can all help to identify patients with a favorable prognosis. For carefully selected, low-risk patients with acute PE, a predominantly outpatient management approach has been shown to be safe and effective.539

Anticoagulation Considerations in Special Populations with Venous Thromboembolism In occasional patients with thrombosis, the aPTT assay may not be reliable in monitoring UFH therapy, for example, patients with the lupus anticoagulant and a prolonged baseline aPTT. For these patients, one option would be to use UFH levels that can be obtained by an automated assay using an anti–factor Xa method. In this instance, the targeted therapeutic range would be 0.35 to 0.7 U/ml. Alternatively, these patients could be given LMWH or fondaparinux, with the dosage determined solely by body weight and no necessity for laboratory monitoring. Patients with marked elevation in factor VIII levels may also be more reliably anticoagulated using LMWH.400 Obese patients are underrepresented in VTE treatment trials, and that has led to uncertainty about optimal LMWH dosing in these patients.540,541 Studies evaluating drug (e.g., dalteparin, enoxaparin, tinzaparin) activity with the use of anti-Xa activity levels, suggest the pharmacodynamics of LMWHs in obese patients (weighing up to 190 kg) are similar to nonobese patients.542–544 Consequently, weight-based dosing (using total body weight) in obese patients without dose-capping and without anti-Xa monitoring appears appropriate.397,540 However, due to limited published experience, anti-Xa monitoring with subsequent dose adjustment should be considered in patients at extreme weights, i.e., those patients weighing over 190 kg in whom LMWH are used.540 The concomitant presence of renal insufficiency (creatinine clearance [CrCl] 60 years 1

• High Risk = >4% Symptomatic VTE Rate • Greater # RF = Greater RAM Sensitivity

Non-orthopedic Surgical Patients‡ 5 points each: Joint replacement surgery, hip/pelvic/leg fracture, stroke, multiple trauma, SCI 3 points each: Age >75, history of VTE, family history of thrombosis, HIT, thrombophilia 2 points each: Age 60–74, cancer, major surgery, laparoscopic or arthroscopic surgery, CVC, bed rest >72h, immobilizing cast 1 point each: Age 41–60, minor surgery, IBD, edema, BMI > 25, sepsis, serious lung disease, medical patient on bed rest, CHF, AMI, varicose veins, OCP/ERT, pregnant/postpartum High Risk = 8+ points Low Risk = 0–1+ points

FIGURE 55.9. Individualized venous thromboembolism risk assessment models (RAMs).  Individualized risk assessment for venous thromboembolism using validated risk assessment models may optimize patient selection and subsequent net benefit of thromboprophylaxis for acutely ill hospitalized patients or those undergoing major surgical intervention. In general, RAMs have a good positive predictive value in identifying high-risk patients, but may be relatively less useful in identifying lower-risk patients. AMI, acute myocardial infarction; BMI, body mass index; CHF, congestive heart failure; CVC, central venous catheter; HIT, heparininduced thrombocytopenia; IBD, inflammatory bowel disease; ICU/CCU, intensive care unit or critical care unit; LE, lower extremity; OCP/ERT, oral contraceptives/ estrogen replacement therapy; SCI, spical cord injury; VTE, venous thromboembolism. Adapted from *Kucher N, Koo S, Quiroz R, et al. Electronic alerts to prevent venous thromboembolism among hospitalized patients. N Engl J Med 2005;352:969–977. †Spyropoulos AC, Anderson FA, Fitzgerald G, et al. Predictive and associative models to identify hospitalized medical patients at risk for VTE. Chest 2011;140:706–714. ‡Bahl V, Hu HM, Henke PK, et al. A validation study of a retrospective venous thromboembolism scoring method. Ann Surg 2010;251:344–350.

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Chapter 55  Thrombosis and Antithrombotic Therapy

Strategy 1 (Opt In): INDIVIDUAL Risk Assessment

NO VTE Prophylaxis

FIGURE 55.10. General approach to venous thromboembolism (VTE) prophylaxis in hospitalized patients.  Some form of venous thromboembolism risk assessment should be performed in patients admitted to the hospital to identify those patients who are most likely to benefit from thromboprophylaxis. Risk assessment can be either individualized or considering at-risk patient groups (e.g., orthopedic surgery patients). Once risk has been identified, the choice of prophylactic strategy should be based upon the patient’s bleeding risk, history of heparin-induced thrombocytopenia, renal function, duration of therapy, and cost. CrCl, creatinine clearance; DTI, direct thrombin inhibitor; HIT, heparin-induced thrombocytopenia; LMWH, low-molecular–weight heparin; ml/min, milliliters per minute; NOACs, novel oral anticoagulants; VKA, vitamin-K–antagonists.

Regularly Reassess

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Strategy 2 (Opt Out): GROUP Risk Assessment

VTE Prophylaxis

Risk Develops

Bleeding Risk

Optimal Mechanical

History HIT

Fondaparinux, DTI

CrCl < 30 ml/min

Heparin, VKA

LMWH, Fondaparinux, VKA, NOACs

charged oligosaccharides, that can bind to positively charged PF4 tetramers, resulting in heparin-PF4 complex formation that is immunogenic under certain circumstances.608 Binding of IgG to the heparin-PF4 complex forms an immune complex that triggers cross-linking of the platelet FcgIIa receptor, an event that leads to platelet activation and formation of thrombogenic microparticles.609 The end result is development of thrombocytopenia in the setting of a profound hypercoagulable state. HIT typically develops between 5 and 14 days after the commencement of heparin therapy and produces a variable but often moderate degree of thrombocytopenia wherein nadir platelet counts are typically in the range of 60,000/ml and the development of severe thrombocytopenia (100°F) has been seen in two-thirds of patients, often beginning 5 to 7 days into therapy or during the period of neutropenia.99 Cladribine suppresses CD4+ lymphocytes. The CD4:CD8 ratio may remain depressed for up to 16 months after therapy, with associated opportunistic infections including Candida or Aspergillus.98 After high-dose cladribine therapy (5 to 10 times the recommended therapeutic dose), renal failure and progressive irreversible motor weakness with paraparesis have been reported. Betticher et al.100 have shown that

Hematologic Malignancies



Block DNA and RNA-polymerase plasma

F-ara AMP

deoxycytidine

into

F-ara A

F-ara A cells

F-ara ATP kinase

NAD

72683_ch068_1399-1425.indd 1415

DNA incorporation

Inhibits Ribonucleotide reductase Inhibits DNA primase and ligase Figure 68.14. Activation pathway and mechanism of action of fludarabine.

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reducing the dose of cladribine from 0.7 to 0.5 mg/kg per cycle decreases both the rate of grade 3 myelosuppression (33% to 8%) and the risk of infection (30% to 7%). No change in lymphoma response rate was noted with this dose reduction. The primary toxicity of clofarabine is myelosuppression leading to an increased risk of infection. Nausea and vomiting are seen but are generally mild. Severe reversible hepatic toxicity is noted in 15% to 40% of patients receiving clofarabine.96,101

Pentostatin Mechanism of Action

Pentostatin (deoxycoformycin, DCF) is a potent inhibitor of adenosine deaminase. It is active in the treatment of chronic lymphoid malignancies, particularly hairy cell leukemia.102 Inhibition of adenosine deaminase results in inability of the cell to catabolize adenosine and deoxyadenosine. Intracellular concentrations of deoxy-ATP increase and exert a negative feedback on ribonucleotide reductase, resulting in an imbalance in deoxynucleotide pools. This imbalance inhibits DNA synthesis and impairs replication, with arrest of cells in the G1 and S phases of the cell cycle. The malignant cells of hairy cell leukemia and CLL have low levels of adenosine deaminase, making them particularly sensitive to the effects of pentostatin.103

Clinical Pharmacology

Plasma pentostatin concentrations greatly exceed those needed to inhibit adenosine deaminase. Pentostatin’s terminal half-life is 3 to 15 hours in humans. Forty to 80% of the drug is excreted unchanged in urine within 24 hours.104 Plasma clearance correlates with creatinine clearance. Dosage reduction should be considered for patients with impairment in renal function. Pentostatin is not bioavailable by the oral route, because of its acid lability.

Toxicity

At doses used in hairy cell leukemia (4 mg/m2 biweekly), therapy is usually well tolerated.105 Toxicities include worsening of neutropenia, mild to moderate lethargy, anorexia, rash, and reactivation of herpes zoster late in therapy. Nausea, although usually mild, can occasionally be severe. Delayed emesis is seen.

Histone Deacetylase Inhibitors (Vorinostat, Romidepsin) and Hypomethylating Agents (5-Azacytidine, Decitabine) Eukaryotic DNA is organized in a macromolecular complex wrapped around histone proteins. Amino acid residues on the histone tail can be modified by acetylation and by methylation. These modifications change the structure of the histone-DNA complex. Histone acetyltransferases (HATs) are enzymes that acetylate histone molecules, resulting in opening of the chromatin, thereby allowing transcription factors access to DNA promoter regions. In contrast, HDACs remove acetyl groups from histone proteins, leading to condensation of DNA and inactivation of gene transcription.106 Increased HDAC activity has been associated with a decrease in tumor suppressor gene expression, alterations in intrinsic and extrinsic apoptotic pathways, and decreased apoptosis. Romidepsin (Istodax®) and vorinostat (Zolinza®) are HDAC inhibitors approved for treatment of cutaneous T cell lymphomas. Similarly, a large number of genes are silenced by methylation in some cancers. This methylation may block the gene promoter region of tumor suppressor genes.107 5-Azacytidine (Vidaza®) and decitabine (Dacogen®) are methyltransferase inhibitors which block the methylation of DNA with the hope of activating tumor suppressor genes. 5-Azacytidine and decitabine have been approved for treatment of MDS and are being used in AML.

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Mechanism of Action Vorinostat is a potent inhibitor of several HDACs (1, 2, 3, and 6), while romidepsin inhibits class 1 HDAC enzymes.106,108,109 It is uncertain whether specificity of any HDAD inhibition impacts antineoplastic activity. Inhibition of HDAC induces genes that cause apoptosis or differentiation. Clinical studies have demonstrated hyperacetylation in tumor cells following vorinostat administration. However, no correlation between tumor acetylation status and response has been seen, suggesting that other mechanisms of action may impact on antineoplastic activity.108 HDAC inhibitors also induce oxidative damage, have antiangiogenic effects, and disrupt cell cycle checkpoints. The specific mechanism of antineoplastic activity remains uncertain. 5-Azacytidine and decitabine (5-aza-deoxycitidine) are both nucleoside analogs. Like cytarabine, they undergo phosphorylation within the cell to a triphosphate form and then are incorporated into DNA. Once incorporated into DNA, these agents bind the enzyme DNA methyltransferase, inhibit methyltransferase activity, and result in DNA hypomethylation.110 Hypomethylation reverses silenced tumor suppressor gene loci. The silenced genes may play an important role in terminal differentiation, apoptosis, or senescence of leukemic cells.111

Clinical Pharmacology Vorinostat is available as an orally administered, 100-mg capsule that is generally given with food. Both vorinostat and romidepsin are primarily inactivated by metabolism (glucuronidation and hydrolysis) with metabolites excreted in the urine. Vorinostat has a short half-life (2 hours); despite the short plasma half-life, accumulation of acetylated histones continues for over 10 hours after administration.106 Use of vorinostat or romidepsin in patients with renal or hepatic insufficiency has not been studied. However, since drug clearance is primarily via metabolism, dose adjustment for renal insufficiency is not thought to be needed. Following IV or subcutaneous administration, decitabine and 5-azacytidine are rapidly inactivated by hepatic metabolism (primarily via the enzyme cytidine deaminase). There is little protein binding. The half-lives of 5-azacytidine and decitabine are 4 hours and 30 minutes, respectively. The drugs do not have oral bioavailability because of rapid decomposition in acidic solutions. Because of an association between the length of drug exposure with DNA methylation and the short half-life of these drugs, continuous treatment of azanucleosides over several days is recommended.111 Dose adjustments for hepatic or renal insufficiency have not been studied but are unlikely to be needed.

Toxicity The most common toxicities of HDAC inhibitors include nausea, vomiting, diarrhea, thrombocytopenia, anemia, and taste disorders. Dose-limiting side effects are generally thrombocytopenia and dehydration.112 Pulmonary emboli have been reported in 4% to 5% of cancer patients receiving vorinostat. QT prolongation is noted with both vorinostat and romidespin.106 The primary toxicity of decitabine and azacytidine is myelosuppression. Gastrointestinal toxicities, including nausea, vomiting, and constipation, are seen, usually transient in nature (1 to 4 days), and improve over time.113

Hormonal Therapies A comprehensive review of all hormonal therapies is beyond the space limitation of this chapter. Of the several types of hormonal agents used in cancer therapy, corticosteroids are the class of drugs used to treat hematologic malignancies. Corticosteroids are also useful in managing complications of cancer, including chemotherapy-induced emesis, hypercalcemia, and increased

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intracranial pressure. Prednisone and dexamethasone are synthetic glucocorticoids most commonly used in oncology. They are respectively 4- and 30-fold more potent than the naturally occurring glucocorticoid, cortisol. Prednisone and cortisol have roughly equivalent mineralocorticoid effects while dexamethasone lacks significant mineralocorticoid activity.

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Resistance is a result of the presence of p-glycoprotein or reduced tubulin binding resulting from mutations in tubulin.

Clinical Pharmacology

Several cancers depend on specific hormones for growth and cellular integrity. Altering the hormonal balance can cause tumor  regression in patients. The mechanism of corticosteroid cytotoxicity to lymphocytes is mediated via binding to the ­ glucocorticoid receptors, which induces apoptosis in these ­hormone-sensitive cells.114

All vincas are rapidly taken up into cells. However, vincas differ in cellular retention (vinblastine greater than vincristine). All vincas are extensively bound to tissues and to proteins with long terminal half-lives (20 to 60 hours).119 CNS penetration is poor. All of the vinca alkaloids are metabolized by the liver via hepatic cytochrome P450 3A4 (CYP 3A4) and excreted into the bile. Drugs that block CYP 3A4 may inhibit vinca clearance, causing increased toxicity.120 Doses of the vinca alkaloids should be reduced in patients with hepatic dysfunction, but not in patients with renal insufficiency. Specific dose reduction guidelines with impairment of hepatic function are not available.

Clinical Pharmacology

Toxicity

Mechanism of Action

Prednisone is an inactive prodrug that requires hepatic activation to produce prednisolone, the active moiety. Oral bioavailability of corticosteroids is excellent (>80%). The plasma half-life of prednisone is 1 hour for prednisolone and 4 hours for dexamethasone. In contrast to cortisol, synthetic glucocorticoids have little protein binding. Drugs activating the microsomal enzyme system (such as phenytoin) increase degradation, while cytochrome P450 inhibitors (such as aprepitant) increase glucocorticoid concentrations.115

Toxicity Prednisone and dexamethasone have little mineralocorticoid activity; therefore most side effects are related to suppression of the hypothalamic-pituitary-adrenal (H-P-A) axis and the development of iatrogenic Cushing’s syndrome. In general, suppression of the H-P-A axis is not seen in patients who have received glucocorticoids for less than 3 weeks. A gradual tapering of glucocorticoids is recommended for those on these agents for longer periods. Side effects are dose- and schedule-related.116 Commonly affected systems include: skin (purpura, thinning, acne), eye (cataracts), heart (hypertension, hyperlipidemia), endocrine (diabetes, adrenal insufficiency), GI (PUD, steatohepatitis), bone (osteoporosis), muscle (myopathy), psychiatric (euphoria, psychosis), and an increased risk of infections. Many of these toxicities are seen only with long-term therapy.

Microtubulin Agents Two general classes of microtubulin inhibitors are used in cancer therapy: the vincas and the taxanes. The vinca alkaloids block microtubulin formation, while the taxanes (docetaxel, ixabepilone, and paclitaxel) stabilize microtubulin bundles, leading to their dysfunction. Since taxanes are not generally used in therapy of leukemias and lymphomas, only the vinca alkaloids will be reviewed here.

Vinca Alkaloids (Vinblastine, Vincristine, and Vinorelbine) Mechanism of Action

The vinca alkaloids vinblastine, vincristine, and vinorelbine bind to tubulin and prevent the formation of microtubulin, a protein that is essential for maintenance of cellular shape and for formation of the mitotic spindle.117 Vinca alkaloids bind to a site on microtubulin distinct from the taxanes. Cells treated with vinca alkaloids are arrested in metaphase.118 Disruption in microtubular formation leads to initiation of apoptosis. Differences in activity and toxicity of the vinca alkaloids result from variation in their pharmacokinetics, their differential effects on various tubulin isoforms, and variations in tissue penetration and cellular retention.

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Vincristine causes little myelosuppression. Its dose-limiting and most frequent toxicity is neurotoxicity,118,121 manifested by a symmetric, distal, sensory-motor neuropathy. Loss of deep tendon reflexes in the lower extremities and paresthesias of the fingers and toes are common early findings. Continued use of the drug can result in further motor neuropathy, which may be only partially reversible when the drug is stopped. Neuropathies of the motor cranial nerves have also been reported, as have constipation, cramps, and paralytic ileus. Hair loss is commonly seen following vincristine administration. The dose-limiting toxicity of vinblastine is hematopoietic, with thrombocytopenia and leukopenia commonly occurring after administration of the drug.122 The onset of myelosuppression tends to occur earlier with this agent than with other antineoplastic agents, with the leukocyte nadir typically seen by day 4 to 7 and recovery by day 10 to 14. Severe neurotoxic symptoms are unusual with vinblastine, but use of the drug is associated with myalgias and an autonomic neuropathy manifested by orthostatic hypotension or paralytic ileus. The dose-limiting toxicity of vinorelbine is myelosuppression.123 Vinorelbine is less neurotoxic than vincristine. Injection-site reactions of erythema, pain, and vein discoloration occur in one-third of patients, with severe vein toxicity seen in 2% of patients treated with vinorelbine. Respiratory reactions have been reported. All vincas are potent vesicants, with severe local tissue damage associated with extravasation of these drugs into soft tissues.

Hematologic Malignancies



Monoclonal Antibodies Monoclonal antibodies have been used in cancer therapy over 30 years. Initial monoclonal antibodies were produced in mice. The development of techniques to convert portions of the murine antibody to a human subclass (chimerization), to convert all of the antibody, except the hypervariable region, to human amino acid sequences (humanization), or to make a totally human monoclonal antibody have advanced the field.124 The humanized proteins have a longer half-life and can better activate human complement stimulating complement-mediated cytotoxicity or activate antibody-dependent cell-mediated cytotoxicity (ADCC). Seven monoclonal antibodies (alemtuzumab, brentuximab vedotin, gemtuzumab, ibritumomab, ofatumumab, rituximab, and tositumomab) have been approved for treatment of hematologic malignancies and are briefly reviewed.

Rituximab (Rituxan®) and Ofatumumab (Arzerra™) Mechanism of Action

Both rituximab and ofatumumab bind to the protein CD20. CD20 is a nonglycosylated protein of 33 to 35 daltons that is expressed on the surface of human B lymphocytes. CD20 is expressed on all B cell malignancies except most ALL and myeloma. Rituximab is a genetically engineered mouse/human chimeric IgG monoclonal

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antibody with human constant regions and mouse variable regions. Several mechanisms of cytotoxicity have been demonstrated.124,125 Antibody-mediated cellular toxicity (ADCC), resulting from the ligation of the Fc portion of the monoclonal antibody to Fc receptors expressed by accessory cells, occurs. Rituximab is also capable of binding complement and triggering complementdependent cell lysis (CDC) of human B cells. Cross-linking of CD20 molecules can trigger apoptosis. Ofatumumab is a fully human monoclonal antibody which binds to a site on CD20 different from rituximab. Compared to rituximab, ofatumumab enhances in vitro CDC and ADCC due to greater reactivity with human complement and effecter cells.126,127 Head-to-head clinical trials comparing of rituximab to ofatumumab have not been performed.

Clinical Pharmacology

Rituximab and ofatumumab antibody concentrations in plasma following drug administration are proportional to the dose of drug administered, although there is marked inter-individual variation in maximal drug concentration (5- to 50-fold). Antibody half-life ranges from 30 to 400 hours for rituximab and 7 days for ofatumumab.128,129 With weekly administration schedules, there is progressive accumulation of rituximab from week 1 to week 4. Drug clearance is not affected by chemotherapy. Drug elimination probably occurs by protein binding and proteolytic degradation. Clearance may be related to variable amounts of tumor cells present in the body and the concentration of CD20 antigens to which the drug binds.129

Toxicity

Mild toxicity is seen in the majority of patients receiving rituximab or ofatumumab, but severe toxicity (NCI grade 3 or 4) is rare. Infusion reactions, resulting from release of cytokines occurring when antibody binds to CD20 cell, result in mild nausea, chills, rigors, tachycardia, fever, and skin rash within 1 to 3 hours following treatment in some patients. Slow drug administration is recommended for patients receiving their first cycle of drug to minimize such reactions. Serious infusion-related reactions (bronchospasm, hypotension, acute respiratory distress syndrome, and shock) have been reported during drug infusions in less than 1% of infusions. The incidence of infusion-related reactions decreases with subsequent infusions. Grade 3 to grade 4 neutropenia (2% to 6%), anemia (1% to 3%), and thrombocytopenia (1% to 2%) are uncommon. B cell lymphocyte counts are reduced for ∼6 months.128,129 Late onset neutropenia, occuring 40–350 days following therapy, has been noted in 3–7% of patients. Multifocal leukoencephalopathy has been reported with rituximab.130 Interstitial lung disease and reactivation of hepatitis B have been noted with rituximab therapy. A late o ­ ccurring neutropenia has been noted following rituximab therapy. 90Y-Ibritumomab

and

Tiuxetan (Zevalin®) (Bexxar®)

131I-Tositumomab

Mechanism of Action

90Y-ibritumomab and 131I-tositumomab are also monoclonal antibodies directed at the CD20 antigen but have an attached radioactive moiety designed to result in radiation-induced cytotoxicity to cells expressing the CD20 antigen.131,132 Ibritumomab is a monoclonal antibody combined with tiuxetan, which acts as a chelation site for Indium-111 and Yttrium-90 (Y-90). Indium-111 is a gamma-emitter used to assess biodistribution of ibritumomab while Y-90 emits beta particles. Beta-emission induces cellular damage through the formation of free radicals (in both target cells and surrounding cells). Roughly 85% of non-Hodgkin lymphomas express the CD20 antigen on their surface.133 A pretreatment therapy of unlabeled antibody to CD20 is given prior to administration of radioactive-tagged antibody to minimize targeting of radio labeled antibody to normal B cells in the circulatory system.

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131I-Tositumomab is a radio-iodinated derivative of a murine IgA lambda monoclonal antibody covalently liked to iodine 131.

Clinical Pharmacology 90Y-Ibitumomab

is a pure b-emitter with a half-life of 64 hours. has both b and a radiation. Clearance of 131I-tositumomab–labeled antibodies varies significantly among patients, and dosing for an individual patient is derived from quantitative whole-body imaging of a test dose. The half-life of elimination averages 67 hours. More rapid clearance is noted in patients with a more extensive tumor burden. 131I-Tositumomab

Toxicity

The primary toxicity of both 131I-tositumomab and 90Y-ibitumomab is myelosuppression occurring about 1 month after dosing. The median platelet and neutrophil nadirs are 43,000 and 690, respectively, with tositumomab. Using 90Y-ibitumomab, grade 4 neutropenia, thrombocytopenia, and anemia occur in 35%, 14%, and 8% of patients, respectively.131,133 Nadirs occur 7 to 9 weeks posttherapy. Nonhematologic toxicity includes asthenia, hypothyroidism, infusion reactions, and fevers. Infusion reactions including anaphylaxis are seen in 25% to 30% of patients (anaphylaxis less common). Myelodysplasia and mucocutaneous reactions (erythema multiforme) have been noted days to months following therapy.134

Alemtuzumab (Campath®) Alemtuzumab is a humanized monoclonal antibody directed against the CD52 antigen expressed not only by most lymphomas and lymphoid leukemias but also by normal B and T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, and neutrophils. The CD52 antigen is not internalized after antibody binding; antitumor effects are mediated by antibody-dependent cell-­mediated cytotoxicity.135 Alemtuzumab also alters signal transduction, which may contribute to cytotoxicity. Because it binds to normal B and T cells and normal neutrophils (triggering complement lysis), transfusion reactions are frequent. The drug is therefore administered by slow infusion or by subcutaneous injection. Patients receiving subcutaneous alemtuzumab have local skin reactions which may be severe. Treatment results in myelosuppression and long-term immunosuppression. There is a high risk of opportunistic infections.136

Brentuximab Vedotin—(AdcetrisTM) Mechanism of Action

Brentuximab vedotin is a conjugate of a chimeric IgG1 monoclonal antibody against CD 30 linked to a cytotoxic drug, monomethyl auristatin E. CD 30 is a member of the TNF family expressed on the tumor cells of Hodgkin’s lymphoma and anaplastic large cell lymphoma. Monomethyl auristatin E (MMEA) is a synthetic inhibitor of tubulin polymerization. After binding to cells expressing CD30, the antibody-drug conjugate is internalized in lysosomes. The peptide linking the antibody to the drug is then cleaved, releasing MMEA into cells where it binds to tubulin and causes cell cycle arrest and apoptosis.140 A small fraction of MMEA diffuses out of tumor cells where it results in cytotoxicity to cells in the microenvironment.141

Clinical Pharmacology

Brentuximab is administered intravenously every 3 weeks. The half-life of the antibody- drug conjugate is 4 to 6 days, while the half-life of MMEA is 3 to 4 days.137 Steady state plasma levels are achieved in 21 days. Most MMEA is cleared via the liver into the stool (72%) as intact drug. There is modest metabolism via CYP 3A4. Information regarding dose adjustments or renal or hepatic insufficiency is not yet available, but it is unlikely that renal impairment would affect drug clearance.

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Toxicity

As with many monoclonal antibodies, infusion reactions can be seen during drug infusion (10%) with occasional anaphylaxis.138,139 Peripheral neuropathy, which often improves with drug discontinuation, is noted in 20% to 50% of patients. Other toxicities seen with brentuximab vedotin include fatigue, fever, nausea and diarrhea, and neutropenia. Progressive multifocal leukoencephalopathy (PML) due to reactivation of JC virus has been seen. A high incidence of pulmonary toxicity was noted when brentuximab has been combined with bleomycin.

Gemtuzumab Ozogamicin (Mylotarg®) Gemtuzumab is a monoclonal antibody to the CD33 antigen, found on myeloid cells, linked to the antibiotic calicheamicin. The postulated mechanism of action is through binding of the antibody to myeloblast with internalization of calicheamicin. Calicheamicin binds to DNA, resulting in DNA strand breaks. Because of lack of clinical studies clearly demonstrating antineoplastic activity, gemtuzumab was withdrawn from clinical use in the United States in 2010.

Molecularly Targeted Therapies In the early years of cancer drug development, antineoplastic agents were often discovered by testing biologics or chemicals for ability to kill cancer cells in culture. If activity was identified, then the mechanism of action of that material was later determined. With advances in cancer biology and sequencing of the human genome, our understanding of how cancers develop and what processes are critical in cancer proliferation has grown. For the past two decades, most cancer drugs have been developed by identifying a critical pathway needed by a particular cancer and then identifying a drug which blocks that critical pathway. Molecular targeted therapies are drugs which selectively target specific molecular features of cancer cells such as aberrations in genes or proteins which regulate tumor cell growth. The best of these drugs target genes or proteins which are critically responsible for tumor growth or survival, such as the BCR-ABL protein in CML. Of the many target agents developed in the past 15 years, those used in treatment of hematologic malignancies will be reviewed here. The targets include: the IL-2 receptor (denileukin diftitox), the BCR-ABL oncoprotein (imatinib, dasatinib, nilotinib), the retinoic acid pathway (ATRA, arsenic trioxide), and the p ­ roteosome (bortezomib).

Denileukin Diftitox Mechanism of Action

Denileukin diftitox (Ontak®) is a fusion protein containing peptide sequences for human interleukin (IL-2) and diphtheria toxin.140 The IL-2 portion of this fusion protein binds to cells that express IL-2 receptors. Once bound to the IL-2 receptor, the diphtheria toxin protein is internalized, inhibits cellular protein synthesis, and results in cell death. Three types of IL-2 receptors exist: high-, intermediate-, and low-affinity. The fusion protein is internalized only with intermediate- or high-affinity IL-2 receptors. Expression of high-affinity IL-2 receptors is normally restricted to activated T lymphocytes and lymphomatous cells of T or B cell origin. Denileukin diftitox has been approved for use in cutaneous T cell lymphomas.141

Clinical Pharmacology

There is wide variation in serum concentrations of denileukin diftitox after intravenous administration (coefficient of variation >50%).141 A terminal half-life of ∼75 minutes is seen with initial therapy, but this decreases to 43 minutes with continued therapy, as antibodies to denileukin diftitox appear. Animal studies have shown that the drug is cleared via hepatic protein degradation.

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Toxicity

Hypersensitivity reactions, such as dyspnea, back pain, rash, hypertension, and chest tightness, occur during or within 24 hours of drug infusion in 60% of patients.141,142 These symptoms resolve within 48 hours and can usually be controlled with use of steroids, antihistamines, or slowing of drug infusion. Constitutional and gastrointestinal symptoms (nausea/vomiting, asthenia, myalgias, headache, diarrhea) are seen in 92% of patients (grade 3 or 4 in one-third). A vascular leak syndrome (hypotension, edema hypoalbuminemia) occurs up to 2 weeks postinfusion in 25% of treated patients. The drug should not be given if the serum albumin is less that 3.0 g/dL. Myelosuppression is uncommon.

Retinoids All-Trans Retinoic Acid or Tretinoin Mechanism of Action.  In acute promyelocytic leukemia, the retinoic acid receptor (RAR) is translocated next to a nuclear protein gene (PML). Fusion of RAR protein with the nuclear protein, PML, prevents normal differentiation of myeloid cell.143 Retinoids are vitamin A derivatives that are essential for normal controlled cellular growth and development. ATRA binds to one or more nuclear receptors to decrease proliferation and induces differentiation of APL cells.144 Pharmacologic concentrations of ATRA (1  mM) reverse the inhibition of differentiation of the promyelocytes and induce remission of APL by providing retinoic acid to activate repressed genes. Clinical Pharmacology. Tretinoin is well absorbed. Peak plasma ATRA concentrations of 350 ng/mL are achieved 1 to 2 hours following ATRA dosing and drop rapidly (t1/2 = 50 minutes). ATRA is metabolized by the hepatic P450 microsomal enzyme system. Use of ATRA stimulates this degradative pathway. ATRA plasma concentrations, therefore, decrease with prolonged drug use.145 Variation in hepatic microsomal metabolism accounts for significant patient-to-patient and day-to-day variation in ATRA clearance.146

Hematologic Malignancies



Toxicity.  Many patients tolerate tretinoin with minimal morbidity. However, 20% to 25% of patients may develop a syndrome characterized by unexplained fever, leukocytosis, dyspnea with interstitial pulmonary infiltrates, peripheral edema, pleuropericardial effusions, hypotension, and acute renal failure.147 Patients with 4 of these symptoms are classified as having a severe differentiation syndrome (also call retinoic acid syndrome), while those with 3 symptoms have intermediate syndrome. The differentiation syndrome occurs more frequently in patients with elevated WBC or renal insufficiency. Steroids are used to treat and prevent this syndrome. Pseudo-tumor cerebri, hyperlipidemia, and abnormal liver function tests have been observed with therapy. ATRA, like other retinoids, has significant teratogenic properties, particularly during the first trimester of pregnancy.148

Bexarotene Mechanism of Action.  Bexarotene (Targretin®) is a retinoid approved for therapy of cutaneous T cell lymphoma. Retinoid modulation of tumor growth is mediated though binding to nuclear receptors that function as transcription factors to regulate gene expression in T cells. Two receptor families, retinoic acid nuclear receptors (RARs) and retinoid X nuclear receptors (RXRs) have been identified.149 Bexarotene selectively binds and activates RXR receptors, inducing apoptosis in selected cell types through triggering of a variety of downstream events.150 Clinical Pharmacology. Bexarotene is available as a topical gel for application to skin lesions or as an oral capsule for systemic therapy. Peak plasma concentrations are seen 2 to 3 hours after oral administration. Drug half-life is 4 to 7 hours. Bexarotene, like

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other retinoids, undergoes metabolism by the hepatic cytochrome P450 enzyme system (CYP3A4). Drug interactions with other CYP 3A4–metabolized drugs may occur. Gemfibrozil increases ­bexarotene concentrations and toxicity. Toxicity. The most common bexarotene-associated toxicities are hyperlipidemia (82%), hypercholesterolemia (30%), central hypothyroidism (29%), headache (20%), asthenia (16%), pruritus (13%), and leukopenia (11%).151,152 Side effects are more common at higher drug doses. Pancreatitis may result from hyperlipidemia. Use of lipid-lowering agents and thyroid replacement are often required. Thyroid function tests must be monitored.

Signal Transduction Inhibitors Several antineoplastic agents have been FDA approved that inhibit a tyrosine kinase associated with signal transduction molecules activated in cancer cells. Inhibitors of the BCR-ABL (imatinib, dasatinib, nilotinib), PDGFR (sunitinib and sorafenib), EGFR (erlotinib, gefitinib), BRAF (vemurafenib), hedgehog (vismodegib), and VEGFR (sunitinib, pazopanib, and sorafenib) tyrosine kinases are clinically available. Imatinib mesylate, nilotinib, and dasatinib are currently approved for treatment of chronic myelogenous leukemia (CML). Other approved tyrosine kinase inhibitors are used primarily for treatment of solid tumors and will not be discussed in this chapter.

Imatinib Mesylate Mechanism of Action

CML is characterized by a reciprocal exchange of genetic material between chromosomes 9 and 22 (t9; 22). A new gene is formed, the BCR-ABL proto-oncogene, which encodes a signal transduction protein that is autonomous. Increased activity of the BCR-ABL proto-oncogene leads to cellular proliferation, decreased apoptosis, or both.153 The intracellular component of the BCR-ABL signal transduction protein contains a tyrosine kinase that activates subsequent signaling molecules by taking a phosphate from ATP and transferring it to a second signaling molecule. Imatinib mesylate (Gleevec®) binds to the ATP-binding site of the BCR-ABL oncoprotein and prevents transfer of phosphate from ATP to the second messenger (Fig. 68.15). Imatinib inhibits the tyrosine kinase of the BCR-ABL, c-kit, and PDGF oncogenes.154

Extracellular

Clinical Pharmacology

Imatinib mesylate is given orally at doses of 400 to 800 mg/day. Bioavailability is 98%, with a drug half-life of 18 hours. Imatinib is cleared by hepatic microsomal cytochrome P450 metabolism (CYP 3A4) to a metabolite that has similar potency.155 Biliary excretion of parent drug and metabolite account for 70% of drug clearance. However, no evidence of decreased clearance or increased drug toxicity has been seen in patients with hepatic or renal dysfunction.156,157 Drugs that alter the hepatic CYP 3A4 metabolism have the potential to alter clearance of all BRC-ABL TKIs.158

Toxicity

Mild nausea (70%), diarrhea (56%), and fluid retention are the most common toxicities associated with imatinib.154,159 Edema can usually be managed with diuretics or dose reductions. Hematologic toxicity is mild and associated with a more advanced stage (e.g., CML blast crisis). Severe toxicity (grade 3 or 4) is rare (15% of patients). Abnormal liver function tests have been reported but require discontinuation of therapy in 30%) associated with bortezomib treatment include fatigue/weakness, GI disturbances (nausea/ anorexia/diarrhea/constipation), myelosuppression (neutropenia/ thrombocytopenia), and peripheral neuropathy.168 Peripheral neuropathy may be seen in up to 35% of patients; however, many myeloma patients have an underlying neuropathy prior to starting bortezomib. The neuropathy is related to the duration of treatment. Dose reductions based on the presence of neuropathy are needed and can result in improvement of neuropathy in 65% to 70% of patients.169

Topoisomerase II Inhibitors (Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, Mitoxantrone, Etoposide, and Teniposide) Mechanism of Action Topoisomerases are nuclear enzymes that make the transient strand breaks in DNA to allow the cell to manipulate DNA topology by passing an intact helix through a transient break in the DNA backbone.170 This is a mechanism to relieve super coiling and tension on the DNA molecule. DNA topoisomerase I makes single-strand breaks in the DNA, whereas topoisomerase II makes double-strand breaks and passes double-stranded DNA through the break. Topoisomerase enzymes are needed for DNA replication, chromosome condensation, and chromosome segregation. Topoisomerase II inhibitors (doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, etoposide, and teniposide) act by poisoning this enzyme to prevent it from relegating cleaved DNA

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Mg 2+

Figure 68.16. The catalytic cycle of topoisomerase II.  Topoisomerase II binds to DNA. In the presence of magnesium and ATP, an intact DNA helix can pass through a temporary break in DNA with subsequent relegation. Topoisomerase II inhibitors block this cycle at the stage of DNA cleavage. (From Osheroff N, Zechiedrich EL, Gale KC. Catalytic functions of DNA topoisomerase II. Bioassays 1991;12:269–275.)

Hematologic Malignancies

Bortezomib targets and blocks the action of the proteosome. The proteosome is a large enzyme complex which breaks down proteins that have been selected for degradation.165 The degradation process requires the proteins to transverse the regulatory gate of the proteosome. Bortezomib is a modified boronic peptide. It inhibits the chymotryptic site of the 26S proteosome, an enzyme that regulates protein degradation.166 Bortezomib inhibits the degradation of proteins involved in regulation of cell proliferation and survival. It deregulates signaling molecules that are critical to the interaction of myeloma cells with the bone marrow microenvironment, leading to growth inhibition and apoptosis. Several intracellular molecules important in apoptosis, including NF-kB, JNK, Bcl-2, p53, and gp130 are modulated by proteosome degradation.

(Fig. 68.16).171 This converts topoisomerase II into a toxin, by introducing high levels of transient protein-associated breaks in the genome of treated cells. Failures to repair the DNA break by the cell results in apoptosis. Currently available topoisomerase I inhibitors are irinotecan (CPT-11) and topotecan. As these agents are not frequently used in treatment of hematologic neoplasms, they will not be further discussed in this section. Topoisomerase II inhibitors include the anthracyclines, epipodophyllotoxins and mitoxantrone.

Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin, and Idarubicin) Clinical Pharmacology

Clearance of all anthracyclines occurs through hepatic metabolism and biliary excretion. Urinary excretion accounts for only ∼10% of anthracycline clearance.172–174 Dose reduction is required for patients with jaundice, although specific dose reduction guidelines are not available. Use of doxorubicin in patients with hepatic dysfunction does not appear to increase cardiac toxicity, but an increase in mucositis and myelosuppression occurs. Liposomalencapsulated anthracyclines (liposomal doxorubicin [Doxil ®, MyocetTM] or liposomal daunomycin [DaunoXome®]) act as depot forms of drug.175,176 The delayed release of the a ­ nthracyclines from the liposome produces lower peak plasma concentrations and less cardiotoxicity.

Toxicity

The acute dose-limiting toxicity of the anthracyclines is myelosuppression, with a nadir in leukocytes expected around day 10 to 14, with recovery usually by day 21 to 28.171 Other acute systemic toxicities include nausea, vomiting, alopecia, and

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mucositis. Anthracyclines cause severe local tissue reactions if extravasation occurs during infusion. The most serious toxicity associated with anthracyclines is cardiotoxicity. Anthracyclines cause a dose-dependent congestive cardiomyopathy that often leads to congestive heart failure. Late-onset cardiomyopathy can appear months to years after treatment is completed.177 The mechanism underlying the cardiotoxic effects of anthracyclines remains uncertain but is thought to be via formation of free radicals, generated by iron-doxorubicin complexes that damage cardiac cellular membranes178; cardiotoxicity may also be related to anthracycline damage to cardiac stem cells. The cardiac damage caused by anthracyclines is cumulative. With total doses of doxorubicin 10,000 infants.226 Birth defects include absent or hypoplastic limbs, ear or eye deformities, and heart defects. Thalidomide administration to pregnant women is absolutely contraindicated. Women of childbearing age must have a negative pregnancy test before starting thalidomide, use two effective forms of birth control, and have a pregnancy test every 4 weeks. Breastfeeding is prohibited. Men must use a condom or refrain from intercourse. Common thalidomide toxicities include neuropathy, somnolence, and constipation. The incidence of neuropathy is 38% at 6 months and 73% at 12 months.227 Clinical features are tingling or painful distal paresthesias affecting primarily the feet but sometimes the hands. The duration of thalidomide therapy correlates directly with the development of neurotoxicity. Some degree of sedation is universal, dose-dependent, and usually appears 2 weeks after initiation of therapy. Some patients suffer from depression. Tremors and/ or headaches occur in 5% to 20% of patients. Venous thrombosis is seen in 2% to 23% of patients, with higher risk in patients >60 years of age. Sinus bradycardia is noted in 5% of patients. Constipation develops a few days after starting treatment in >50% of patients. Hypothyroidism occurs in 20% of patients occurring 1 to 6 months after starting therapy. A maculopapular skin rash or pruritus is seen in 20% to 50% of patients. Stevens-Johnson syndrome is occasionally seen (2 weeks) contribute to the risk of serious infections.3 Neutropenia is usually caused by decreased production. Localizing signs and symptoms are often absent in the setting of severe neutropenia because of a lack of inflammatory response from absent granulocytes. Fever remains the most common sign of infection associated with neutropenia.

Neutropenia is a common complication of acute leukemia (AL) and is often prolonged during induction therapy.4 In chronic myeloid leukemia (CML), neutropenia typically occurs with the development of blast crisis, with the evolution of myelofibrosis (MF), or with therapy. Mild neutropenia is observed in patients with MF and multiple myeloma (MM), but it is uncommon during untreated phases. Neutropenia occurring in patients with Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) is typically a result of marrow invasion with tumor or marrow fibrosis and occurs in conjunction with other cytopenias. Hairy cell leukemia patients may become neutropenic secondary to tumor cell invasion, splenomegaly, or both, but may also result from defects in cellmediated immunity, monocytopenia, and decreased T cells after nucleoside analog therapy.5 In patients with T cell large granular lymphocytic leukemia, neutropenia may be the primary problem. Functional defects in morphologically normal neutrophils have been described in hematologic malignancies, particularly myeloproliferative neoplasms (MPNs) and myelodysplastic syndrome (MDS).6 Such defects increase the susceptibility to infection.7 Neutrophils from untreated patients with CML may be mildly defective with respect to phagocytosis, oxygen consumption, and bactericidal capacity, and tend to have decreased concentrations of lactoferrin, elastase, collagenase, and peroxidase.8 Myeloblasts and lymphoblasts found in AL patients are of no benefit to the host against infection.

Deficient Immunoglobulin Production The humoral immune response is one of the two main arms of the immune system. In this response, the immune system triggers specific B cells to proliferate and secrete their specific antibodies. Impaired humoral immunity is a major cause of frequent and severe infection in patients with hematologic malignancies. A decrease in Th2 CD4 T-lymphocyte–B-lymphocyte interaction results in decreased antibody production, complement-mediated damage, and phagocytosis. Diminished immunoglobulin synthesis is a major contributor to infection in patients with CLL, MM, and some B cell types of NHL.9,10,11 Myeloma and other plasma cell dyscrasias are often functionally hypogammaglobulinemic despite elevated total immunoglobulin. Splenectomized patients have impaired antibody response, reduced levels of tuftsin (natural activator of phagocyte cells), and are at increased risk for infections similar to those of patients with hypogammaglobulinemia, particularly encapsulated organisms (Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis).12

Defects in Cellular Immunity Cellular immunity comprised of T lymphocytes, macrophages, and natural killer cells recognizes and combats pathogens that proliferate intracellularly. Cellular immune mechanisms are important in immunity to all classes of infectious agents, including most viruses and many bacteria (e.g., Mycoplasma, Chlamydophila, Listeria, Salmonella, and Mycobacterium), parasites (e.g., Trypanosoma, Toxoplasma, and Leishmania), and fungi (e.g., Histoplasma, Cryptococcus, and Coccidioides).13 T lymphocytes are activated by dendritic cells, macrophages, and B lymphocytes, which present foreign antigens in the context of the host’s own major histocompatibility complex antigen to the T cell receptor. Activated T cells then act in several ways to fight infection. Cytotoxic CD8+ T cells directly attack and lyse host cells that express foreign antigens. Helper CD4+ T cells stimulate the proliferation of B cells and the

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T a b l e 69.1

HOST DEFECTS PREDISPOSING TO COMPLICATIONS IN HEMATOLOGIC MALIGNANCIES Disease

Host Defect

Complications

Acute myeloid leukemia

Neutropenia

Bacterial infections, including perirectal abscess, typhlitis, sinusitis; superinfections when hospitalized; increasing problem of methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci; Clostridium difficile colitis; aspergillosis with prolonged neutropenia; viral infections (herpes simplex) Hemorrhage

Chronic myeloid leukemia

Chronic lymphocytic leukemia

Hodgkin lymphoma

Non-Hodgkin lymphoma Small B cell lymphoma PTCL, particularly angioimmunoblastic and subcutaneous panniculitis like PTCL Large B cell lymphoma T-lymphoblastic lymphoma Mantle cell lymphoma Burkitt lymphoma Multiple myeloma

Waldenström macroglobulinemia Hairy cell leukemia

T cell large granular lymphocyte leukemia

Adult T cell leukemia/lymphoma

Leukostasis, tumor lysis Bacterial infections (see Acute myeloid leukemia) Pneumocystis jirovecii, disseminated varicella

Cellular immunity Immune dysfunction Cellular immunity Cytokine production Splenectomized Immune dysfunction Mediastinal disease

Hemorrhage Tumor lysis, leukostasis No increased risk of infections except in blast crisis Increased risk of thrombosis and hemorrhage, similar to other myeloproliferative disorders Infections with encapsulated organisms (pneumococcus, Haemophilus influenzae, meningococcus) Mycobacteria, fungal, viral (herpetic), Salmonella AIHA, ITP, red cell aplasia Viral (herpes zoster, other), P. jiroveci, fungal, mycobacteria, listeriosis, Salmonella B symptoms, pruritus, eosinophilia Encapsulated organisms (above), increased risk of leukemia ITP, AIHA SVC syndrome

Decreased Igs Immune dysfunction Cytokine production Mediastinal disease Mediastinal disease Colonic polyposis Gastrointestinal primary Paraprotein Decreased Igs Osteoclast overactivity IgM paraprotein Neutropenia Cellular immunity Immune dysfunction Monocytopenia Neutropenia

Similar infections to CLL AIHA B symptoms, hemophagocytic syndrome, eosinophilia SVC syndrome, pericardial disease SVC syndrome, tumor lysis, CNS disease Gastrointestinal bleed Obstruction, perforation, tumor lysis, CNS disease with advanced-stage disease Hyperviscosity, hemorrhage Similar infections to CLL Hypercalcemia Hyperviscosity, hemorrhage Bacterial and fungal infections P. jirovecii, atypical mycobacteria Periarteritis nodosa, lymphocytic vasculitis — Bacterial infections

Immune dysregulation, positive rheumatoid factor, antinuclear antibody Cellular immunity Parathyroid hormone–related protein

ITP, AIHA, red cell aplasia

Decreased immunoglobulins

Hematologic Malignancies

Acute lymphoblastic leukemia

Thrombocytopenia, disseminated intravascular coagulation Hyperleukocytosis Neutropenia Cellular immunity while on maintenance therapy Thrombocytopenia Hyperleukocytosis Mild defects in neutrophil function Thrombocytosis, platelet dysfunction

Opportunistic infections (Strongyloides stercoralis, P. jirovecii ) Hypercalcemia

AIHA, autoimmune hemolytic anemia; CLL, chronic lymphocytic leukemia; CNS, central nervous system; Ig, immunoglobulin; ITP, immune thrombocytopenic purpura; PTCL, peripheral T cell lymphoma; SVC, superior vena cava.

production of immunoglobulins. Defects in cell-mediated immunity characterized by impaired Th1 CD4+ T lymphocytes and/or macrophage function results in increased risk of infections with intracellular bacteria, fungi, parasites, and viruses (Table 69.2). Multiple factors determine the severity and frequency of impaired cellular immunity. There may be differences in the stages of disease

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studied, the therapy used, and the sensitivity of the tests used to measure cellular immunity. Patients with HL often do not respond to new antigens and lose prior sensitivity as well.13,14 Patients with CLL usually show reduced or absent lymphocyte transformation with phytohemagglutinin but do not lose skin hypersensitivity to antigens such as old tuberculin.15 Patients with HL do not mount

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Part vii  Hematologic Malignancies  •  SECTION 1  General Aspects

T a b l e 69.2

OPPORTUNISTIC INFECTIONS ASSOCIATED WITH DEFECTS IN IMMUNITY IN HEMATOLOGIC NEOPLASIA Defect

Infections

Neutropenia

Gram-negative bacteremia (Escherichia coli, Pseudomonas, Klebsiella, Proteus) Gram-positive bacteremia (methicillin-resistant Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus viridans) Fungemia (Candida species, aspergillosis) Encapsulated organisms   Streptococcus pneumoniae   Haemophilus influenzae   Neisseria meningitidis Bacteria   Listeria monocytogenes  Mycobacteria  Legionella species  Nocardia species  Salmonella species Viruses Herpes simplex Varicella zoster Parainfluenza, respiratory syncytial virus, cytomegalovirus Fungi Cryptococcus neoformans Coccidioides immitis Histoplasma capsulatum Pneumocystis jirovecii Parasites Toxoplasma gondii Strongyloides stercoralis

Humoral immunity

Cellular immunity

either a primary or a secondary immune response, whereas those with CLL maintain secondary responses but cannot mount a primary response. Depressed cellular immunity is uncommon in AL16 except during maintenance therapy in ALL.17

Approach to Infection in the Immunocompromised Host The initial assessment of a febrile immunocompromised host is dependent on the underlying hematologic condition and other associated risk factors. It should focus on determining the potential sites and causative organisms and assessing the patient’s severity of illness. Although fever remains the most important clue to an infectious process, the characteristic signs and symptoms of infection may be absent in more than one-half of infected neutropenic patients, and routine cultures are often negative.3 It is estimated that 60% or more of neutropenic patients who become febrile have an established or occult infection.18 No known factors accurately predict which patients with fever and neutropenia are most likely to have bacteremia. As a result, a careful history and screening physical examination must be performed with special attention to the most common sites of infection: skin, oropharynx, nares, sinuses, lungs, GI tract (including perianal area), soft tissues, and indwelling catheter devices. Risk assessment should be performed as part of the initial evaluation as it helps stratify the severity and facilitate goal-directed therapies. Assessing risk may determine the type of empiric antibiotic therapy (oral vs. intravenous), venue of treatment (inpatient

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vs. outpatient) and duration of antibiotic therapy. The most commonly used index for the stratification of risk for complications in febrile neutropenic patients is the Multinational Association for Supportive Care in Cancer (MASCC) index (Table 69.3).19 The following independent factors were found to be predictive of lower risk for complications: (1) burden of illness characterized by low or moderate symptoms, (2) absence of hypotension, (3) absence of chronic obstructive pulmonary disease, (4) presence of solid tumor or absence of previous fungal infection in patients with hematologic malignancies, (5) outpatient status, (6) absence of dehydration, and (7) an age less than 60 years. These variables predicting low risk were assigned an integer weight, and a risk index score consisting of the sum of these integers was derived. A score of 21 or greater identified low-risk patients with a positive predictive value of 91%, specificity of 68%, and sensitivity of 71%; whereas those with scores less than 21 are at higher risk for complications.19 In general, most experts consider high-risk patients to be those with anticipated prolonged (>7 days duration) and profound neutropenia (ANC < 0.1 × 109/L) or significant medical comorbid conditions, including hypotension, pneumonia, new abdominal pain, or neurologic changes. High-risk patients warrant inpatient therapy with intravenous antibiotics. Lower-risk patients, including those with anticipated brief ( 0.5 × 10 /L 9

Reassess for infection site

Reassess

Stop if no disease and condition is stable

FIGURE 69.2. Common modifications of empiric antimicrobial therapy in the febrile neutropenic patient.

atypical fungi, and several clinically relevant Candida species (C. krusei, C. tropicalis, C. lusitaniae, and Torulopsis glabrata) due to the absent or poor activity of fluconazole against these organisms. Echocardiography is recommended for S. aureus bloodstream infections to determine the presence or absence of endocarditis, and thus clarify the need for prolonged antibiotic therapy. Transesophageal echocardiography is more sensitive and preferred when compared with a transthoracic approach.35

Myeloid Colony-stimulating Factors Prophylactic use of myeloid colony-stimulating factors (CSFs) is common in the setting of intensive chemotherapy regimens such as stem cell transplantation. Multiple randomized clinical trials of

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prophylactic recombinant granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have shown benefits in reducing the time to neutrophil recovery and the duration of fever and hospitalization in patients with hematologic neoplasms.36,37 Prophylactic G-CSF and GM-CSF in autologous and allogeneic hematopoietic stem cell transplantation (HSCT) recipients have been associated with a small reduction in the risk of documented infections but do not appear to affect infection-related or treatment-related mortality.38 Empiric use of CSFs in the management of neutropenic fever is not standard practice, as no consistent benefit has been demonstrated in terms of morbidity or mortality among randomized controlled trials of their use in patients with febrile neutropenia.39,40 Neither ASCO nor EORTC recommend the routine use of growth

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Part vii  Hematologic Malignancies  •  SECTION 1  General Aspects Step 1 Assess frequency of FN associated with the planned chemotherapy regimen

FN risk ≥ 20%

FN risk 10–20%

FN risk < 10%

Step 2 Assess factors that increase the frequency/risk of FN High risk

Age >65 years

Increased risk Advanced disease (level I and II History of prior FN evidence) No antibiotic prophylaxis, no G-CSF use Other factors: (level III and IV evidence)

Poor performance and/or nutritional status Female gender Hemoglobin 10 days) and profound (18 mm Hg. Older age and prior chemotherapy, particularly with anthracyclines, are contributing factors. Diagnostic confirmation strategy varies among institutions; suspected with clinical features, elevated D-dimer, and V/Q scan and confirmed by spiral CT pulmonary angiography. Sudden onset of respiratory distress after transfusion. The incidence of transfusion-related lung injury (TRALI) is 0.04%–0.1%; mortality is estimated at 5%–8%. Acute-onset, bilateral lung infiltrates on chest radiography, pulmonary artery wedge pressure ≤ 18 mm Hg, or the absence of clinical evidence of left atrial hypertension, and PaO2/Fi02 ≤ 300 (acute lung injury) or ≤ 200 (ARDS). Lung biopsy showing nonspecific inflammation and variable fibrosis with no evidence of lung infection, and without alternative clinical diagnosis. Data from imaging, cultures, serology, and FB inconclusive for a firm diagnosis.

ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage; CMV, cytomegalovirus; CT, computed tomography; DFA, direct immunofluorescent antibody; FB, fiberoptic bronchoscopy; PCR, polymerase chain reaction; RSV, respiratory syncytial virus; TBB, transbronchial biopsy.

rates in patients refractory or intolerant to triazoles or amphotericin B.141 Posaconazole has been used successfully as salvage therapy for a variety of invasive fungal infections refractory to standard therapy.142 Posaconazole is currently approved by the FDA for prophylaxis of invasive Aspergillus and Candida infections, and in the European Union is indicated for treatment of invasive aspergillosis and other invasive fungal infections refractory to standard antifungal agents.143

Endemic Fungi Commonly known endemic fungi include Histoplasma capsulatum, Coccidioides immitis, and Blastomyces dermatitidis.

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These dimorphic fungi exist in nature in the fruiting mycelial stage and then convert to the yeast stage at body temperature. Endemic mycoses in the central United States include histoplasmosis and blastomycosis. Immunocompetent hosts are typically asymptomatic following inhalation of Histoplasma microconidia but may manifest acute fever, pulmonary infiltrates, and hypoxia. Immunocompromised patients have a higher risk of disseminated histoplasmosis involving the liver, spleen, lymph nodes, bone marrow, adrenal glands, mucocutaneous tissues, gastrointestinal tract, and CNS. Chest radiographs may show a miliary reticulonodular appearance similar to that seen with tuberculosis. Blood cultures may be positive in disseminated histoplasmosis. Antigen detection in blood, urine, and BAL is both

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A

sensitive and specific.144 Antibody detection may also be useful, but false-negative results may occur in immunocompromised patients.145 Biopsy specimens showing small intracellular or narrow budding yeast are suggestive of the diagnosis and should be confirmed by culture. IDSA guidelines recommend amphotericin B for severe pulmonary or disseminated histoplasmosis.145 Prolonged therapy with itraconazole may be initiated after ­stabilization of disease, and should be continued for the duration of ­immunosuppression.145 Coccidioides immitis is endemic in the southwestern United States. C. immitis is more likely to be pathogenic in patients with compromised cell-mediated immunity. High rates of treatment failure and death have been reported in patients with hematologic malignancies.146 The diagnosis is most often established by finding the fungus in BAL, sputum, or biopsies. Serology is positive in only 55% of patients. Coccidioidomycosis can involve virtually any organ in disseminated disease but has trophism for bone and the CNS. Therapy for disseminated disease generally requires amphotericin B followed by maintenance fluconazole.147

Pneumocystis Jirovecii Pneumonia (PJP) Pneumocystis jirovecii (formerly P. carinii) is classified as a fungus rather than a protozoan based on gene sequence data, although it lacks ergosterol, the main fungal cell-wall component. Defective T cell immunity and steroid use are risk factors for PJP. Pneumocystis jirovecii can have a fulminant course with rapid progression to respiratory failure in immunocompromised patients.148 Patients with pneumonia from P. jirovecii usually present with rapid onset of dyspnea, nonproductive cough, hypoxemia, and fever. Radiologic studies generally show diffuse bilateral interstitial infiltrates but can show focal infiltrates. Pleural effusion is uncommon. Diagnosis of PJP relies on visualization of the organism microscopically, as it does not grow in culture. BAL is the standard diagnostic modality for PJP, but induced sputum has acceptable yield in some institutions.149 Immunofluorescent staining with monoclonal antibodies is more sensitive than silver staining or Wright-Giemsa staining.150 PJP frequently results in positive serum beta-d-glucan testing.151 Treatment should be started based on clinical suspicion, and TMP-SMX (5 mg/kg IV every 8 hours) remains the treatment of choice.3 Prednisone should be added to the empiric treatment

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FIGURE 69.5. A: Chest CT of a hematopoietic cell transplant recipient who developed pulmonary Aspergillus infection after prolonged immunosuppression.  B: Photomicrograph of the characteristic 45° angle branching of septate hyphal forms of Aspergillus. Gomori methenamine silver stain, × 400. (Courtesy of Margie Scott.)

regimen if the pO2 is 7 days.182

Viral Infections Cytomegalovirus CMV infection has been a major source of morbidity and mortality in transplant recipients prior to the era of proper prophylaxis and monitoring for reactivation. Transplant recipients are at risk for reactivation (if CMV seropositive pre-HSCT) and also primary infection (from stem cells or blood products from CMV seropositive donors). Incidence of primary infection has decreased with the increasing use of leukofiltered blood products. CMV infection is defined as the reactivation of the virus and the detection of the virus in the blood or other body fluids in the absence of organ-specific abnormalities (pneumonitis, hepatitis, colitis, and retinitis). CMV disease is defined as the isolation of the virus from body fluids or tissues in a symptomatic patient or the histopathologic evidence of CMV on tissue biopsy (see Fig. 69.6). Risk factors for the development of CMV disease include older recipient age, pretransplant seropositivity of the recipient or donor, or both, and severe acute GVHD.192,193 T cell depletion of the stem cell graft or treatment of the recipient with antithymocyte globulin for GVHD increases the likelihood of CMV reactivation.194 The incidence of CMV reactivation in allogeneic transplant recipients who are seropositive before transplant is 60% to 70%, as compared to a 10% to 30% incidence of primary infection in seronegative recipients.195–197 Prior to the use of ganciclovir, CMV interstitial pneumonitis occurred in 15% to 30% of HSCT allograft recipients and the ensuing mortality was as high as 85%; however, mortality remained high at 30% to 50% even with the combined use of ganciclovir and CMV-specific immune globulin (Ig).194 Administration of CMV-safe or leukofiltered blood products is recommended to all seronegative autologous or allogeneic HSCT recipients to prevent primary CMV infection.195,198 CMV surveillance starting at the time of engraftment is recommended in instances of recipient and/or donor CMV seropositivity. Tests used for CMV surveillance include CMV pp65 antigenemia assay, DNA PCR detection methods, or CMV blood cultures.199 Detection of CMV in the blood is the strongest predictor of CMV disease, but 12% to 20% of patients with negative surveillance cultures still develop CMV disease. While high-dose intravenous acyclovir significantly reduces the incidence of all forms of CMV disease or delays the onset of CMV infection, it does not prevent CMV viremia.200,201 There are two recommended strategies for prevention of CMV disease. One strategy is prophylaxis with ganciclovir, valganciclovir, or valacyclovir, starting from engraftment until day 100, or longer if the patient remains at risk for CMV reactivation (active GVHD, high-dose steroids, low CD4 count).199,202 The other strategy is close surveillance and preemptive therapy with ganciclovir or foscarnet when CMV reactivation is detected. Both strategies reduce the incidence of CMV disease in the first 100 days; however, the median onset of CMV disease has shifted from 50–60 days to 160–176 days post-HSCT in the preemptive era.197 Preemptive therapy with ganciclovir for patients with positive CMV surveillance cultures (blood, urine, throat, or bronchoalveolar lavage fluid) has demonstrated improved survival at 100 and 180 days post-HSCT, but has failed to provide an overall survival advantage.203 Protracted ganciclovir prophylaxis can lead to emergence of resistant strains and the failure of natural immunity against CMV to develop, thus resulting in late

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recrudescence.196,204,205 Optimal preemptive therapy appears to be 1 to 2 weeks of twice-a-day induction followed by maintenance until PCR or antigen negativity.182 Ganciclovir is the drug of choice for CMV infection and disease, but its myelosuppressive effects may preclude its use in patients with significant cytopenias. Foscarnet is an equally effective alternative and can be used to treat ganciclovir-refractory CMV infections or in patients with significant cytopenias. Although it is not myelosuppressive, foscarnet is associated with renal toxicity and electrolyte imbalances.160 Cidofovir is another nephrotoxic antiviral with efficacy against CMV, but there are few data on its use in the stem cell transplant patient population.206 Maribavir is the latest antiviral in the armamentarium of drugs available for management of CMV and has been found to decrease rates of CMV infection when used as prophylaxis in the stem cell transplant setting,207 but has failed to prevent CMV disease in a randomized phase III study.208

Herpes Simplex Virus HSV reactivation occurs in as many as 80% of seropositive allogeneic transplant recipients, causing mucocutaneous oral or genital lesions, esophagitis, and, occasionally, pneumonia or encephalitis. Testing all transplant recipients for herpes simplex virus exposure (HSV IgG) is recommended. Antiviral prophylaxis with acyclovir, valacyclovir, or famciclovir is recommended for all seropositive patients until the time of engraftment. Although its use is not recommended past 1 month after transplant, some patients with recurrent lesions might benefit from longer use of the ­prophylaxis.209,210

Varicella Zoster Virus Impaired cellular immunity is the principal risk factor for VZV disease. Current recommendations are to test every transplant patient for varicella zoster virus serostatus (IgG). VZV reactivation may occur at any time after engraftment in autologous and allogeneic transplant recipients. Disseminated VZV is seen in as many as 30% of cases and is associated with a high mortality. Many centers administer oral acyclovir or valacyclovir for ∼12 months after transplant to VZV-seropositive patients.210 Seronegative patients should be given varicella zoster immune globulin within 96 hours of exposure to a VZV vaccine or upon contact with active infection.

Fungal Infections Most fungal infections in the SCT population are due to Candida or Aspergillus. The etiology in the remaining 7 days

Levofloxacin 500 mg orally daily or TMP/SMX may be used for those allergic to or intolerant of fluoroquinolones

Start when ANC < 1.0 × 109/L and continue until resolution of neutropenia

Induction therapy for AML and MDS

Posaconazole 200 mg orally tid is drug of choice if high risk for aspergillosis. Alternatives include itraconazole, voriconazole, lipid formulation of amphotericin B, or an echinocandin (may choose to use fluconazole prophylaxis if incidence of aspergillosis is low.) Fluconazolea or an echinocandin Azoles are inhibitors of cytochrome P-450 isoenzymes, and are expected to interfere with metabolism of vinca alkaloids and other drugs; close monitoring is recommended Fluconazolea or echinocandin

Begin with initiation of chemotherapy and continue until resolution of neutropenia.

Acute lymphoblastic leukemia

Autologous HSCT recipient during neutropenia Allogeneic HSCT recipient during neutropenia

Allogeneic HSCT with significant GVHD receiving intensive immunosuppressive therapyb Pneumocystis jirovecii Prophylaxis

VIRUSES Prophylaxis for HSV

Prophylaxis for VZV Prophylaxis for CMV (preemptive therapy)

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Fluconazole,a itraconazole, voriconazole, and micafungin have each been evaluated in this setting Posaconazole 200 mg orally tid is drug of choice. Alternatives include voriconazole, itraconazole, lipid formulation of amphotericin B, or an echinocandin

Continue prophylaxis for duration of neutropenia

Continue prophylaxis for duration of neutropenia Continue prophylaxis for duration of neutropenia Continue prophylaxis for 16 wk and for at least the duration of intensive immunosuppressive therapy,b whichever occurs later

Acute lymphocytic leukemia, allogeneic HSCT recipients, alemtuzumab recipients, fludarabine recipients, or patients receiving corticosteroids (≥20 mg of prednisone equivalent) for ≥1 mo in the presence of other immunosuppression or myelotoxic chemotherapy.

TMP/SMX 1 DS (TMP 160 mg + SMX 800 mg) orally daily or 3 days per week OR dapsone 100 mg orally daily OR inhaled pentamidine 300 mg every 4 wk OR atovaquone 1,500 mg/d.

In allo-HSCT, continue prophylaxis for 2 mo after stopping immunosuppression. In patients treated with alemtuzumab, continue prophylaxis for 2 mo after the last dose or until the CD4 count is >200.

HSCT recipients (HSV-seropositive recipients) Induction chemotherapy for acute leukemia (HSV-seropositive) Patients treated with alemtuzumab, or in patients with recurrent HSV reactivation following chemotherapy Allogeneic HSCT recipients with a history of chicken pox or shingles Patients requiring CMV surveillance 1. Allogeneic HSCT recipients who are CMV+ or whose donor is CMV+ (standard of care) 2. Autologous SCT recipients receiving a CD34-enriched autograft 3. Patients treated with alemtuzumab

Acyclovir 400 mg orally bid or tid OR 800 mg orally bid OR 250 mg/m2/12 h or valacyclovir 500 mg orally once or twice (higher doses have been used up to 1,000 mg tid) or famciclovir 250 mg orally tid

Continue prophylaxis for HSV until resolution of neutropenia and mucositis. In patients treated with alemtuzumab, continue prophylaxis for 2 mo after the last dose or until the CD4 count is >200

Acyclovir 800 mg orally bid OR valacyclovir 500 mg orally daily Induction for 1 wk followed by maintenance for 1 wk as follows: Ganciclovir 5 mg/kg IV q12h for 7 days (induction), followed by Ganciclovir 5 mg/kg IV daily 5 times a week (maintenance) OR Foscarneta 60 mg/kg IV q12h times 7 days (induction) followed by Foscarnetc 60 mg/ kg IV daily (maintenance) Oral valganciclovir (900 mg bid) is an acceptable alternative to IV formulations in patients who do not have severe gut GVHD (see text)

CMV surveillance: 1. Allogeneic HSCT recipients: day 30 to at least 6 mo after allogeneic HSCT, during periods of GVHD, and until the CD4+ count is >100 mL. 2. Recipients of CD34-enriched autologous grafts: day 30 to day 100 and until the CD4+ count is >100 ml. 3. Alemtuzumab recipients: time of initiation until at least 2 mo after completion of therapy and until the CD4 count is >100 ml. The level of CMV reactivation that triggers preemptive therapy varies with the method. The CDC recommends any positive CMV antigenemia (pp65) or two consecutive qualitative PCR results within the first 100 days, and five cells per slide after the first 100 days

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T a b l e 69.6 ( c o ntinued )

PROPHYLAXIS OF INFECTIONS IN HEMATOPOIETIC STEM CELL TRANSPLANT RECIPIENTS AND HEMATOLOGIC CANCER PATIENTS Prophylaxis

Indication

Prophylaxis against CMV

Agent

Duration/Comments

Ganciclovir or foscarnet at the same dose as in preemptive regimen

Treatment is given for the first 100 days, then weekly or biweekly monitoring and preemptive management is initiated

ANC, absolute neutrophil count; AML, acute myelogenous leukemia; bid, twice daily; CDC, Centers for Disease Control and Prevention; CMV, cytomegalovirus; DS, double strength; GVHD, graftversus-host disease; HSCT, hematopoietic stem cell transplant; HSV, herpes simplex virus; IV, intravenous; MDS, myelodysplastic syndrome; tid, 3 times a day; PCR, polymerase chain reaction; SCT, stem cell transplant; TMP/SMX, trimethoprim/sulfamethoxazole; VZV, varicella zoster virus. aFluconazole

is effective as prophylaxis against candidal, but not mold, infections. If prophylactic fluconazole is used in patients with prolonged neutropenia, a strategy of empirical modification to a mold-active drug in patients with persistent neutropenic fever should be considered. Doses apply to adults with normal renal function. include acute grade II to IV GVHD, or extensive chronic GVHD, or treatment with intensive immunosuppressive therapy consisting of either high-dose corticosteroids (1 mg/kg of body weight per day for patients with acute GVHD or 0.8 mg/kg every other day for patients with chronic GVHD), antithymocyte globulin, or a combination of two or more immunosuppressive agents. cDoses apply to adults with normal renal function. bCriteria

prophylaxis with an oral penicillin or TMP/SMX is recommended for at least 6 months post-HSCT or until discontinuation of immunosuppressive therapy.243 Vaccination against S. pneumoniae is recommended for all HSCT recipients, preferably with pneumococcal 7-valent conjugate vaccine.243

Antifungal Prophylaxis Antifungal prophylaxis should be based on risk stratification and should target specific pathogens in different patient groups. Patients with hematologic malignancies or HSCT recipients have several risk factors for fungal infections: defects in cellular immunity, prolonged profound neutropenia, immunosuppressive therapy, use of broad-spectrum antibiotics, use of parenteral nutrition, and use of indwelling vascular devices. The Transplant Associated Infections Surveillance Network (TRANSNET) prospectively analyzed HSCT recipients with proven or probable invasive fungal disease. Aspergillosis (43%), candidiasis (28%), and zygomycosis (8%) were the most common.244 Preventive efforts should be directed toward the reduction of Candida and Aspergillus species, as they have traditionally been the most common causes of fungal disease in immunocompromised patients.196,244 Patients should be counseled to avoid dust or soil exposure when traveling to areas that are endemic for organisms such as Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum when possible. Most other pathogens such as Aspergillus species, C. neoformans, and the Mucorales order (Zygomycetes class) are ubiquitous in the environment. Hospital outbreaks of Aspergillus are more likely to occur during periods of construction or renovation.245,246 HEPA filtration, regular maintenance of ventilation systems, and floor-to-ceiling barriers around construction sites are important measures in reducing the risk of invasive aspergillosis.247 Prior to the development of the triazole class of drugs, few effective antifungal agents other than amphotericin B were available. Nystatin and clotrimazole may prevent oropharyngeal candidiasis but are not well tolerated in patients with severe mucositis. Oral polyenes (nystatin and oral amphotericin B) are not orally bioavailable and have failed to demonstrate a reduction of systemic fungal infections.248,249 Inhaled amphotericin B has shown promise in preventing colonization and infection with Aspergillus but tolerability remains a concern and

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direct comparison to systemic mold-active azoles has not been studied.250–252 Multiple randomized trials in patients with leukemia and patients undergoing allogeneic HSCT have demonstrated that fluconazole prophylaxis at a dosage of 400 mg/day reduces the incidence of superficial and invasive candidal infections, excluding C. krusei.211,253,254 Fluconazole prophylaxis has also been found to confer improved survival in HSCT recipients,189,255 but may increase the risk of colonization by azole-resistant Candida strains.189 Itraconazole prophylaxis for candidiasis has been shown to provide similar efficacy and may additionally reduce the risk of aspergillosis. However, itraconazole has also demonstrated higher toxicity (drug-drug interactions due to cytochrome P450 3A4 inhibition) and gastrointestinal intolerance. It is also contraindicated in patients with a decreased cardiac ejection fraction or a history of congestive heart failure.256 Micafungin appears to be as effective as fluconazole in prevention of candidiasis in HSCT recipients257 and may also reduce the risk of Aspergillus. Prevention of aspergillosis requires prophylaxis with broader spectrum antifungals. Comparison of standard prophylaxis (fluconazole or itraconazole) versus posaconazole in AML/MDS patients receiving myelosuppressive induction therapy revealed that posaconazole prophylaxis led to fewer invasive fungal infections, including aspergillosis, and to improved survival.142 A similar comparison in HSCT recipients with severe GVHD demonstrated that prophylactic posaconazole also led to fewer cases of invasive aspergillosis but failed to impact overall survival.142 Comparison of voriconazole versus fluconazole prophylaxis in allogeneic HSCT recipients showed a trend toward reduction of aspergillosis infections in voriconazole recipients, but no difference in overall or fungal infection–free survival.3 Fluconazole is a well-studied agent and remains the standard drug of choice to prevent invasive candidiasis, but it has no activity against molds.258 Mold-active agents should be considered when there is significant risk of aspergillosis. Posaconazole is an oral agent with antimold activity and requires administration with food or enteral preparations to enhance bioavailability. The mold-active azoles (i.e., itraconazole, voriconazole, posaconazole) are potent inhibitors of cytochrome P450 3A4 isoenzymes and may lead to reduced clearance of other drugs, such as calcineurin inhibitors and vinca alkaloids. Close monitoring of drug-drug interactions and appropriate dose modifications are required.109

Hematologic Malignancies

Adapted from the NCCN Clinical Practice Guidelines in Oncology. Prevention and Treatment of Cancer-Related Infection. NCCN. Version 1.2012, the clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America (IDSA), and the comprehensive guideline cosponsored by the Center for International Blood and Marrow Research, the National Marrow Donor program, the European Blood and Marrow Transplant Group, the American Society for Blood and Marrow Transplantation, the Canadian Blood and Marrow Transplant Group, the Infectious Diseases Society of America (IDSA), the Society for Healthcare Epidemiology of America, the Association of Medical Microbiology and Infectious Disease Canada, and the CDC makes evidence-based recommendations for HSCT that may be applicable to other cancer patients. Tomblyn M, Chiller T, Einsele H, et al. J Am Soc Blood Marrow Transplant 2009;15:1143–1238.

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Histamine blockers and proton pump inhibitors, such as omeprazole, should be avoided, and serum levels must be monitored during therapy.259

Antiviral Prophylaxis Opportunistic viral disease may occur by primary infection or by reactivation of latent infection. Herpesviruses, including CMV, VZV, and HSV-1 and -2, are by far the most common infection-causing viruses. Antiviral prophylaxis (acyclovir, valacyclovir, or famciclovir) against HSV is advised during the period of neutropenia in seropositive patients receiving chemotherapy for acute leukemia, and during neutropenia and at least 30 days after HSCT for both allogeneic and autologous transplant recipients.3,239 Prolonged prophylaxis should be considered in allogeneic HSCT recipients with GVHD or with frequent HSV reactivation before transplantation.210 Acyclovir is an effective prophylaxis against reactivated HSV infections (gingivostomatitis, esophagitis) in patients who are receiving intensive chemotherapy for acute leukemia or BMT.260–262 Acyclovir or valacyclovir are each commonly used as prophylaxis, treatment, and suppressive therapy against HSV and VZV in immunocompromised patients.263,264 Although acyclovir has proven to be highly effective as prophylaxis, there are reports of acyclovir-resistant HSV developing while on therapy.265,266 Treatment with foscarnet or cidofovir is recommended in the setting of acyclovir-resistant HSV. Foscarnet-resistant HSV strains have been reported in allogeneic stem cell transplant patients and such patients have been treated with cidofovir.267 Newer nucleoside analogs (such as BV-ara U, Brovavir) are being studied. Acyclovir-related prodrugs (valacyclovir, famciclovir) appear as effective as acyclovir for HSV and VZV prophylaxis.160,172,268 Avoidance of close contact with infected or exposed individuals is advisable for immunocompromised seronegative patients. Prophylaxis should also be considered in patients  receiving T  cell–depleting agents (e.g., fludarabine, calcineurin inhibitors, and proteasome inhibitors).

Prevention of Viral Hepatitis Reactivation of latent hepatitis B virus occurs in various settings (e.g., HSCT, cytotoxic chemotherapy, anti-CD20 monoclonal antibodies treatments). The immunosuppressive effect of the chemotherapy allows virus reactivation in the liver, and the subsequent immune reconstitution may result in hepatocellular damage.269 Patients with lymphoma, or who use steroids or receive anthracycline chemotherapy, seem to be at higher risk.270 Fulminant hepatitis and death may occur following HBV reactivation in immunocompromised patients. Evaluation of HBV surface antigen, core antibody, and surface antibody should be considered for those in whom intensive immunosuppressive therapy in planned.271 Lamivudine prophylaxis has been shown to be relatively effective in the prevention of hepatitis B reactivation, though randomized trials are still needed.204,270,272 Baseline quantitative PCR for HBV DNA should be obtained in HBsAg-positive individuals. Antiviral therapy should be strongly considered in patients with active HBV infection undergoing HSCT or other intensive immunosuppression therapies, but an optimal antiviral treatment regimen remains unclear.

Augmentation of Host Defense Optimization of patients’ immune status via prophylactic immunization is another consideration to assist in the reduction of infection risk. Table 69.7 summarizes the recommended vaccine schedule of patients with hematologic malignancies and HSCT recipients as per the Advisory Committee on Immunization Practices (ACIP), ASBMT, and EBMT.182,212,239,273,274

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Active Immunization Patients with hematologic neoplasms are at increased risk for infection with polysaccharide-encapsulated bacteria, viruses, and fungi due to impaired T cell, granulocyte, and reticuloendothelial cell function, as well as to defective antibody responses. Immunization as a means of preventing infectious morbidity has been best studied in children with ALL or solid tumors, patients with HL with or without splenectomy, and marrow transplant recipients. Although the optimal timing of immunizations in these patients and specific recommendations for each type of cancer are still unclear, general guidelines have been established. If indicated, the administration of inactivated vaccines should be completed more than 10 days before initiation of chemotherapy or 3 months after completion of chemotherapy in adults with hematologic malignancies, including acute leukemia and myeloproliferative diseases.239 CDC recommendations require all infants to be vaccinated for Haemophilus influenzae type B (Hib) and thus almost all adults in theory should possess adequate immunity against this organism. However, splenectomized Hodgkin’s disease patients who were receiving antineoplastic therapy were found to have a significantly greater decrease in Hib titer at a 6- to 12-month period after the primary vaccination (given before starting chemotherapy) as compared to normal controls.275 Most children who receive maintenance chemotherapy for leukemia or lymphoma are able to generate protective antibody responses to a single dose of conjugate polysaccharide Hib vaccine, although responses are less than those that are seen in healthy children.276 Recommendations for children are therefore to continue the primary series of Hib-conjugate vaccinations during chemotherapy treatment and to administer a booster immunization 1 year after completion of chemotherapy.277 For adults with hematologic malignancies, it is recommended that a dose of Hib vaccine be given before initiation of chemotherapy or before splenectomy, if one is planned.278 The need for a booster a year after chemotherapy in adults has yet to be determined, and further studies are warranted.275,278,279 Pneumococcal vaccines are available as either a conjugate vaccine (PCV-7, PCV-13) or a polysaccharide vaccine (pneumococcal polysaccharide vaccine 23-valent, PPSV23). Conjugated vaccines elicit improved immune responses in HSCT recipients compared with pure polysaccharide vaccines, and are therefore preferred.274,280–283 Conjugated vaccines induce a robust T cell– dependent immune response and generate long-term memory loss. It is recommended that HSCT recipients receive 3 sequential doses of PCV-13 starting 3 to 6 months after transplant. A fourth vaccination with PPSV-23 is given 8 weeks after the third dose of PCV-13 to broaden the immune response to include serotypes not included in PCV-13. Given that patients with active chronic GVHD are likely to have a poor response to PPSV-23, a fourth dose of PCV-13 should be considered in these patients.182 One-time revaccination with PPSV-23 at 5 years after the first dose is currently recommended for immunocompromised patients.278 The ACIP recommends that adults 19 years of age or older with immunocompromising conditions who have not previously received PCV13 or PPSV23 should receive a dose of PCV13 first, followed by a dose of PPSV23 at least 8 weeks later. Complete protection might not be achieved if the vaccine is given within 3 years after antineoplastic therapy.284,285 Immunization for N. meningitidis by using a polysaccharide vaccine is recommended in cancer patients requiring splenectomy.286 However, this vaccine offers no protection from serogroup B (which is responsible for one-third of cases). Ongoing studies are being conducted to test new meningococcal serogroup B vaccine.287 Children undergoing chemotherapy and who have not completed all of the diphtheria-tetanus (DT) (or diphtheria, pertussis, and tetanus; pertussis is included if the child is 10 d before initiation of chemotherapy, or 3 mo after completion of chemotherapy Not during immunosuppressive therapy >2 wk before initiation or between cycles of intermittent chemotherapy For leukemic patients in remission, >3 mo after completion of therapy; otherwise contraindicated in patients with leukemia, lymphoma, or those undergoing immunosuppressive therapy Before staging splenectomy

Annually each fall/winter

Measles, mumps, rubella

Nonimmune leukemic patients in remission and household contacts of all immuno-suppressed individuals

Meningococcal (Neisseria meningitides) Poliomyelitis (inactivated poliovirus vaccine only)c

Lymphoma patients

23-Valent polysaccharide pneumococcal (Streptococcus pneumoniae) PCV13 (Pneumococcal Conjugate) Vaccine

Tetanus and diphtheria toxoids combined

Varicella

aAdapted

Adults at increased risk of infection; all susceptible household contacts of cancer patients Any nonimmune cancer patient, especially lymphoma and multiple myeloma a patients 19 yr of age or older with immunocompromising conditions who have not previously received PCV13 or PPSV23; 19 y of age or older with immunocompromising conditions, who have previously received one or more doses of PPSV23 All cancer patients

Nonimmune household contacts of cancer patients

No data available; recommend each dose >10 d before initiating chemotherapy Before staging splenectomy, >10 d before initiation of chemotherapy, or 3 mo after completion of chemotherapy Give a dose of PCV13 first followed by a dose of PPSV23 at least 8 wk later. Subsequent doses of PPSV23 should follow current PPSV23 recommendation. Give a dose of PCV13 one or more years after the last PPSV23 dose was received No data available; recommend administration >10 d before initiating chemotherapy

Not applicable—contraindicated in patients with leukemia, lymphoma, or those undergoing immunosuppressive therapy

Once

2 doses; second dose 3–5 y after the first 3 doses; second dose 4–8 wk after the first, third dose 6–12 mo after the second 2 doses; second dose at least 5 y after the first

Primary 3-dose series if not previously immunized; second dose 4–8 wk after the first, third dose 6–12 mo after the second; booster doses at 10-y intervals throughout life or with dirty wound if >5 y since last dose For persons >13 y of age, 2 doses separated by 4–8 wk

Hematologic Malignancies

Influenza

3 doses: second dose 1–2 after the first, third dose 4–6 mo after the first

from the Centers for Disease Control and Prevention.

bMay

be used in conjunction with hepatitis B immunoglobulin prophylaxis.

cLive

oral polio vaccine is no longer available for general use in the United States and is contraindicated in immunosuppressed persons or their household contacts.

and polio immunization series should complete the boosters as scheduled, although aggressive and prolonged chemotherapy may blunt the response. Only the inactivated polio vaccine (IPV) should be given to immunocompromised patients because of the risk of acquiring polio from the live attenuated oral polio vaccine. Booster doses of DT or diphtheria, acellular pertussis, and tetanus and the IPV should be administered 1 year after chemotherapy completion. It is recommended that adult cancer patients receive DaPT immunization boosters in the same dose and schedule as for healthy individuals, as responses can be elicited in immunocompromised patients.239 The inactivated influenza vaccine is generally recommended in immunocompromised patients on a yearly basis.278 Several studies have evaluated the efficacy of this vaccine in cancer patients; there is definite efficacy in patients with solid tumors, but the response may be blunted in patients with hematologic malignancies.288 Despite a lack of a clear-cut benefit in immunocompromised patients, the likelihood of at least partial protection has urged physicians to vaccinate all cancer patients with the inactivated influenza vaccine.

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The live attenuated measles, mumps, and rubella (MMR) vaccine may be considered in previously unimmunized children with leukemia who are in remission, or who have been off or not received chemotherapy for at least 3 months; but it is otherwise contraindicated in immunocompromised individuals.239,277 The live attenuated varicella vaccine is not recommended for use in immunocompromised patients, except in seronegative children with HIV.239,289,290 Protective antibodies to common childhood diseases (polio, tetanus, diphtheria, mumps, measles) wane after ablative therapy and autologous or allogeneic transplantation and thus necessitate revaccination.291–293 All HSCT patients should be vaccinated with combined tetanus-diphtheria toxoids at 12, 14, and 24 months after transplant.243,292,294–296 Antibody levels against H. influenzae gradually decline after transplant, and revaccination using the Hib-conjugate vaccine is recommended at 12, 14, and 24 months after transplant to restore immunity.243 The oral polio vaccine is contraindicated in all immunocompromised individuals.278 IPV and hepatitis B vaccines are recommended at 12, 14, and 24 months after transplant, owing to the loss of immunity against

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the virus by 1 year after transplant in the majority of transplant recipients.297,298 Vaccination against Meningococci, rabies, and hepatitis A is not routinely recommended in this patient population.182 Annual seasonal administration of inactivated influenza vaccine is recommended following 6 months posttransplant.182 The MMR vaccine, a live attenuated vaccine, may be given to BMT patients about 2 years after transplant and may be administered to household contacts before then. Patients with GVHD who are continuing to receive immunosuppressive therapy should not be given the MMR vaccine.291 The varicella vaccine is a live attenuated vaccine and is contraindicated in transplant recipients. Use of the Bacille Calmette-Guérin vaccine is contraindicated during the first 2 years posttransplant.299,300

Passive Immunization Intravenous immunoglobulins (IVIG) therapy for prevention of infection has been evaluated primarily in patients with lymphoproliferative disorders and multiple myeloma and SCT recipients. Currently available IVIG preparations are safe, well tolerated, and consist primarily of IgG, although small amounts of IgA and IgM are present.301 IgA-poor preparations must be used to prevent anaphylaxis in patients with known or suspected congenital IgA deficiency and/ or those with high anti-IgA titers. IVIG (400 mg/kg given every 3 weeks) has been shown to reduce frequency of moderately severe bacterial infections in patients with CLL who have hypogammaglobulinemia or a history of recurrent infections.302–304 However, this practice has not been proven to be cost-effective, to prolong survival, or to demonstrably improve the quality of life of patients with CLL.194 Reduction in the number of symptomatic and life-threatening infections has also been reported in patients with multiple myeloma receiving monthly infusions of IVIG.201,305 A meta-analysis of multiple trials involving prophylactic IVIG in patients with CLL and MM illustrated a significant decrease in occurrence of major infections and reduction in clinically documented infections, but no survival benefit.306 Thus, prophylactic use of IVIG for patients with CLL or MM with hypogammaglobulinemia and/or recurrent infections should be considered on an individual basis. In allogeneic HSCT patients, prophylactic use of IVIG has been shown to prevent grades II–IV acute GVHD,307,308 decrease gramnegative septicemia, and decrease local infections; but it does not improve mortality.309,310 Decisions to use IVIG in allogeneic transplant patients should be made on an individual basis because of the cost of IVIG and the availability of suitable antimicrobial

alternatives. Autologous transplant recipients do not appear to benefit from prophylactic globulin therapy, and its use may actually lead to increased incidence of fatal hepatic venoocclusive disease in these patients.311 IVIG and CMV hyperimmune globulin can confer passive immunity against CMV infection, but other methods of CMV prophylaxis, such as leukofiltration of blood products, may be more cost-effective.312 Passive immunization with varicella zoster immune globulin is indicated in seronegative immunosuppressed patients who are exposed to an active case and should be given within 72 to 96 hours of the exposure.313 Patients who are exposed to measles, mumps, or rubella may benefit from passive Ig prophylaxis if it is given within 6 days of exposure.314,315 Intramuscular Ig is recommended for patients who travel to areas that are endemic for hepatitis A or for postexposure prophylaxis.316 Two doses of hepatitis B Ig, given 1 month apart, are recommended for postexposure prophylaxis.

MANAGEMENT OF CATHETERRELATED INFECTIONS Catheter-related bloodstream infections (CRBSI) are associated with prolonged hospitalizations, increased healthcare costs, and increased morbidity and mortality.317,318 The incidence of device-associated infection varies depending on the type of device inserted, its length of use, and the extent of the patient’s immunosuppression. Neutropenic patients, particularly those with a hematologic malignancy, have a greater risk for developing CRBSI and bacteremia.317,319,320 Overall, the incidence of port-associated infections is low at 0.1 per 1,000 port-days, and that of tunneled catheters is approximately 1 to 3 per 1,000 days of patient use. Cumulative incidence of PICC-related blood stream infections is 1.1 per 1,000 PICC-days (range 0.9 to 1.3), but higher in the inpatient setting (2.1 per 1,000 PICC-days).318 Number of lumens is an independent risk factor for CRBSI and multiple-lumen catheters have higher infection rates.142,321 Standardized infection control procedures as recommended by the CDC are increasingly being used to help reduce catheter-related bloodstream infections.322 Several types of catheter-related infections have been defined: exit site infection, tunnel infection (or pocket infection in the case of ports), septic phlebitis, and CRBSI (Table 69.8).55 The majority of these infections are caused by gram-positive pathogens, with coagulase-negative staphylococci recovered most frequently.

T a b l e 69.8

DEFINITION OF CATHETER-ASSOCIATED INFECTIONS Localized catheter colonization Exit site infection Tunnel infection Pocket infection Catheter-related bloodstream infection (CRBSI)

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• Significant growth of a microorganism (greater than 15 CFU) from the catheter tip, subcutaneous segment of the catheter, or catheter hub. • Erythema, or induration within 2 cm of the catheter exit site, in the absence of concomitant bloodstream infection (BSI) and without concomitant purulence. • Tenderness, erythema, or site induration of more than 2 cm from the catheter exit site along the subcutaneous tract of a tunneled catheter (i.e., Hickman or Broviac) in the absence of concomitant BSI. • Purulent fluid in the pocket of a totally implanted intravascular catheter that may or may not be associated with spontaneous rupture and drainage or necrosis of the overlaying skin in the absence of concomitant BSI. • Bacteremia or fungemia in a patient with an intravascular catheter with at least one positive blood culture obtained from a peripheral vein, clinical manifestations of infections (e.g., fever, chills, or hypotension) and no apparent source for the BSI except the catheter. • One of the following should be present: a positive semiquantitative (greater than 15 CFU/catheter segment) or quantitative (greater than 102 CFU/catheter segment) culture whereby the same organism (species and antibiogram) is isolated from the catheter segment and peripheral blood; simultaneous quantitative blood cultures with a 3:1 ratio for CVC versus peripheral; or differential period of CVC culture versus peripheral blood culture positivity of more than 2 hours.

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CHAPTER 69  Supportive Care in Hematologic Malignancies

Catheter-related infections with S. aureus, gram-negative bacteria, corynebacteria, bacillus species, and mycobacterial and fungal organisms have also been reported. Simultaneous cultures from peripheral sites and all catheter ports are helpful in distinguishing infection of the catheter itself from infection arising from another source. Successful treatment of CRBSIs caused by coagulase-negative staphylococcal or even gram-negative organisms does not always require catheter removal. Infections of the skin pocket of an implanted port also resolve in ∼70% of patients without removal of the device.55 Appropriate intravenous antibiotics should be administered for 1 to 3 weeks and should be rotated through all lumens. If clinical improvement is observed and surveillance cultures from each lumen remain negative after 3 days of antibiotic therapy, the catheter sterilization is likely.55 Uncomplicated exit site infections will also usually resolve with aggressive local care and systemic antibiotic therapy. However, some bacterial infections (e.g., S. aureus, some Bacillus species, Corynebacterium group, and Stenotrophomonas species) will require catheter removal in order to increase the likelihood of successful treatment. Other indications for immediate removal of the catheter include evidence of complicating endocarditis, osteomyelitis, septic thrombosis, septic pulmonary embolism, or sepsis with signs of shock/end-organ dysfunction. Certain organisms, such as Candida and fungi, are extremely difficult to eradicate and necessitate prompt catheter removal to avoid the complications of disseminated infection. Fungemia caused by Malassezia furfur (Pityrosporum orbiculare) tends to occur in patients who receive parenteral lipids and may be resistant to amphotericin B. This infection often manifests as fever, pulmonary infiltrates, and thrombocytopenia; discontinuation of the lipid, as well as removal of the catheter, is needed.323

Occlusion and Venous Thrombosis of Catheters Although a clot is the most common cause of occlusion, inability to aspirate blood from the port or catheter does not always suggest clot formation. Other causes of impaired catheter flow include a malpositioned Huber needle, catheter compression on the wall of the vein, catheter kinking, catheter pinch-off, precipitation of drug solutions in the catheter lumen, development of fibrin sheaths, and catheter migration resulting in a malpositioned tip.324 Fibrin blockage is common, and simple repositioning of the patient and/

or Valsalva maneuvers may allow blood to be withdrawn. If these strategies fail, the catheter position should be confirmed by chest radiograph after injection of contrast dye through the catheter. Patients in whom there is no contraindication to thrombolytic therapy may be given an infusion of urokinase at 200 U/kg/hour for as long as 12 hours to reopen the catheter. Otherwise, 5,000 U of urokinase (in 2 ml of sterile water) may be injected into the catheter, and a blood draw may be attempted again in 30 minutes. This procedure may be repeated twice in 24 hours if necessary. Recombinant tissue plasminogen activator (alteplase) at a dose of 2 mg/2 ml instilled for 30 minutes may also restore function in occluded venous catheters.325,326 Contrary to early reports, data from two randomized studies failed to demonstrate a benefit of low-dose warfarin327 or LMWH328 to reduce the incidence of symptomatic catheterassociated thrombotic events in patients with cancer. Prophylactic use of urokinase (5,000 IU/ml) every 1 to 2 weeks into long-term CVADs has shown reduced incidence of thrombosis and also of catheter-related infections.329 Thrombosis can occur in the catheter itself or in the superior vena cava or veins of the upper extremity. While asymptomatic thromboses have been reported in rates up to 60%, recent studies show a variable incidence of symptomatic catheter-related thrombosis of 4% to 5% up to 28% of adults and 12% of children with a CVC.325,330,331 Almost all central indwelling venous access devices become coated with a fibrin sheath within days of insertion and the majority of CVC-related thrombi arise within 30 days of initial placement.332 Pain, ipsilateral extremity edema, and superficial venous dilation require evaluation with venography or noninvasive contrast imaging techniques. The catheter should be removed if it is no longer needed or if treatment with systemic anticoagulation fails. If the catheter remains functional and needed for clinical use, then the recommendation from the 2012 American College of Chest Physicians is to keep the catheter in place and to continue systemic anticoagulation with warfarin or low molecular weight heparin (LMWH) as long as the catheter remains indwelling. If the catheter is removed, it is recommended to continue systemic anticoagulation for a total of 3 months regardless of diagnosis.333 Another recommendation has a variable length of anticoagulation, from 6 weeks to 6 months depending upon the size of the clot and whether the patient is considered prothrombotic331 (Fig. 69.8).

High risk of emboli

Heparin or LMWH for 3–5 days then remove line Warfarin or LMWH for 6 weeks to 6 months

CVC not needed

Suspected catheter-related thrombosis

Doppler ultrasonography +/-venography

1451

Hematologic Malignancies



Low risk of emboli

Remove CVC

Thrombosis

CVC needed

Heparin or LMWH for 5–7 days

Warfarin or LMWH for 3–12 months

Prophylactic LMWH until CVC removed

FIGURE 69.8. Algorithm for diagnosis and management of catheter-related thrombosis.  (CVC, central venous catheter; LMWH, low-molecular-weight heparin.) Ultrasonography (U/S) can be used as the initial screening test for thrombosis, whereas venography may be required if U/S is negative or inconclusive. The duration of anticoagulation depends upon clinical factors, including the need for the CVC, the size of the clot, and the presence of a prothrombotic state. In patients with cancer, LMWH is preferred over warfarin, and duration of anticoagulation should be 6 months or longer. (Adapted from Baskin JL, Pi C-H, Reiss U, et al. Management of occlusion and thrombosis associated with long-term indwelling central venous catheters. Lancet 2009;374:159–169.)

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Chemotherapy-Induced Nausea and Vomiting Chemotherapy-induced nausea and vomiting (CINV) is a common adverse effect of cancer therapies. Nausea and vomiting can be very distressing to both patients and their caregivers. Nausea can have a negative impact on quality of life, both physical and cognitive functions, and may lead to critical delays in the administration of potentially curative therapy.334,335 Nausea and emesis can be induced by a number of factors besides chemotherapy, and thus it is important to investigate the underlying etiology. Radiation therapy, especially in those undergoing total body irradiation (TBI) prior to HSCT, infections, metabolic derangements, electrolyte abnormalities, metastases (especially brain and liver), medications (antibiotics, antifungals, opiates), and other psychological factors can all lead to nausea and vomiting in patients with cancer.336 Based upon the emetogenic potential of the chemotherapy administered and specific patient characteristics, personalized therapeutic decisions can be made to try to prevent CINV.337 A breakdown of the relative emetogenicity of commonly used chemotherapeutic agents can be found in Chapter 68, Table  68.2. Patient characteristics associated with increased risk for CINV include: age < 50, female gender, history of motion sickness, history of low prior chronic alcohol intake, and emesis during pregnancy.335 Coadministration of chemotherapeutic agents and repeated cycles of chemotherapy can also increase the potential for CINV.338 CINV can be broken down into 5 distinct yet related syndromes: (1) acute CINV, (2) delayed CINV, (3) anticipatory CINV, (4) breakthrough CINV, and (5) refractory CINV.338 Acute CINV is typically defined as occurring within the first 24 hours of chemotherapy administration. Delayed CINV is typically defined as occurring 1 to 5 days after chemotherapy administration. Anticipatory CINV occurs prior to the chemotherapy administration, and has been associated with just one episode of nausea and vomiting with a prior regimen. Breakthrough CINV occurs despite the use of appropriate preventative strategies, and refractory CINV occurs due to failure of preventative and rescue therapies.338 To understand the rationale behind different strategies for the prevention and treatment of CINV, it is helpful to understand the physiology of the emesis response. The current model of the emesis response (Fig. 69.9) is primarily mediated through neurotransmitters in the gastrointestinal (GI) system and the central nervous system (CNS).338 Afferent signals from the chemotherapy trigger zone (CTZ), cerebral cortex, and GI tract converge on the vomiting center (VC) within the medulla oblongata. Ultimately,

the VC sends out efferent signals to the organs responsible for completing the motor events of emesis (esophagus, stomach, abdominal musculature).339 Serotonin and substance P are the main neurotransmitters implicated in CINV; however dopamine, histamine, endorphins, acetylcholine, and GABA have also been shown to be involved. Based upon the identification of active neurotransmitters and their receptors within the VC, control of CINV may depend ultimately upon blocking the neurotransmitter receptors in the VC from afferent inputs of the GI tract and CNS.338 Prevention is the key treatment of CINV. Development of effective preventative antiemetic regimens requires consideration of the emetic potential of the chemotherapy, dosing and duration of the chemotherapeutic regimen (1 day vs. multiday), single vs. multi-drug regimens, personal risk factors, and the mechanism of action of the prescribed antiemetic therapy. Maximum benefit from antiemetic therapy is achieved when it is initiated prior to chemotherapy and continued throughout the duration of the emetic response to each chemotherapeutic agent. Multiple organizations, including ASCO, NCCN, and MASCC, have developed guidelines for antiemetic prevention, and a summary of these recommendations is listed in Table 69.9.

Agents 5-HT3 Receptor Antagonists Perhaps the most significant advance in antiemetic therapy, as well as supportive therapy in general, has been the development of the 5-HT3 receptor antagonists. Agents in this class (e.g., granisetron, ondansetron, palonosetron, and dolasetron mesylate) have all shown efficacy in controlling acute nausea associated with chemotherapy.340–342 As well, both IV and PO routes of administration of these medications are effective when appropriate doses are given.343 While all 5-HT3 receptor antagonists have been shown to be effective in controlling acute nausea and vomiting caused by chemotherapy, palonosetron has recently been shown to be much more effective than other available 5-HT3 receptor antagonists in preventing delayed nausea and vomiting.344,345,346 A recent meta-analysis of randomized controlled trials comparing palonosetron with other 5-HT3 receptor antagonists revealed that it was significantly more effective in preventing acute and delayed nausea and vomiting for highly emetogenic and moderately emetogenic chemotherapy.347 Palonosetron’s half-life of 40 hours is much longer than that of the other 5-HT3 receptor antagonists and it has an approximately 100fold higher binding affinity to the 5-HT3 receptor. These qualities likely explain palonosetron’s superiority in preventing chemotherapy-induced nausea and vomiting, especially delayed onset nausea and vomiting.348 Palonosetron is currently FDA approved for use as a single infused dose on day 1 for the prevention of acute and delayed nausea and vomiting associated with highly and moderately emetogenic chemotherapy. While repeat dosing is considered safe based on NCCN guidelines, the additional benefit of repeated dosing for multiple-day chemotherapy regimens is unknown. Adverse effects of 5-HT3 receptor antagonists are usually mild and transient. Headache is the most common side effect, followed by gastrointestinal side effects of abdominal discomfort, diarrhea, and constipation, which occur less frequently.349 Association of IV dolasetron with increased risk for cardiac arrhythmias has been reported by the FDA, and thus IV dolasetron is not recommended.

Neurokinin 1 Receptor Antagonists

FIGURE 69.9. Model of the emesis response.  (With permission from Navari RM. Pathogenesisbased treatment of chemotherapy-induced nausea and vomiting–two new agents. J Support Oncol 2003;1:89–103.)

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Substance P is a mammalian tachykinin found in vagal afferents that send impulses to the vomiting center and thus are involved in the vomiting response. Substance P induces emesis through its binding to neurokinin 1 (NK-1) receptors in the brainstem nucleus tractus solitarius, the area postrema, and the abdominal vagus nerves.338,350 Two randomized, double-blind, multicenter

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Ta b l e 69.9

SUMMARY OF GUIDELINES FOR THE PREVENTION AND TREATMENT OF CINV High Acute CINVa

Group

Low

Minimal

Acute CINV

Delayed CINV

Acute/Delayed CINV

Acute/Delayed CINV

-5-HT3 (palonosetron preferred) + dexamethasone + (fos)aprepitant ± lorazepam ± H2d blocker or PPIe

± 5-HT3 RA (unless palonosetron given day 1) +Dexamethasone (8 mg po daily days 2–4 with aprepitant or fosaprepitant 150 mg IV day 1) (8 mg po day 2, then 8 mg po BID days 3 and 4 with fosaprepitant 150 mg IV day 1) + Aprepitant 80mg po days 2 and 3 if aprepitant given on Day 1 ± lorazepam day 2–4

-5-HT3 RA (palonosetron preferred) +Dexamethasone ± Aprepitant or Fosaprepitant 150 mg IV day 1 ± lorazepam ± H2 blocker or PPI

(repeat daily for multiple-day regimens) -Dexamethasone 12 mg po/ IV daily Or -Metoclopramide 10–40 mg PO/IV Or -Prochlorperazine 10 mg PO/IV ± lorazepam ± H2 blocker or PPI

No routine prophylaxis before or after chemotherapy is recommended

ASCOf 2

-5-HT3 RA + Dexamethasone +(fos)aprepitant ±Lorazepam ±diphenhydramine

-Dexamethasone Day 2–3, or day 2–4 + Aprepitant day 2–3 if Fosaprepitant is NOT used day 1.

-5-HT3 RA (palonosetron preferred) + Dexamethasone ±aprepitant ±lorazepam ±diphenhydramine

-5-HT3 RA on days 2 and 3. (unless palonosetron given day 1) Or Dexamethasone Daily day 2–3 Or Aprepitant 80 mg po day 2–3 (if aprepitant given day 1) ± dexamethasone ± Lorazepam ± H2 blocker or PPI -Dexamethasone daily day 2–3 ± aprepitant (if used on day 1)

-Dexamethasone 8 mg PO/IV prior to chemotherapy

No antiemetic should be administered routinely before or after chemotherapy

MASSC/ ESMOg 3

For HECh and ACi regimens: -5-HT3 RA + Dexamethasone + (fos)aprepitant

-Dexamethasone day 2–4 + aprepitant day 2–3 if aprepitant given day 1

Non-AC regimens: -Palonosetron + Dexamethasone

-Dexamethasone day 2–3

-Dexamethasone or 5-HT3 RA or Dopamine antagonist prior to chemo

-No routine prophylaxis is recommended

RAc

bNational

Induced Nausea and Vomiting;

Comprehensive Cancer Network;

Hydroxytryptamine 3 receptor antagonist;

d Histamine e Proton

2;

Pump Inhibitor;

fAmerican

Society of Clinical Oncology;

gMultinational hHighly

Association of Supportive Care in Cancer/European Society of Medical Oncology;

Emetogenic Chemotherapy;

iAdriamycin/Cyclophosphamide.

Data from Ettinger DS, Armstrong DK, Barbour S, et al. Antiemesis. Clinical practice guidelines in oncology. J Natl Comp Cancer Netw: JNCCN 2009;7:572–595; Basch E, Prestrud AA, Hesketh PJ, et al. Antiemetics: American Society of Clinical Oncology Clinical Practice Guideline update. J Clin Oncol 2011;29:4189–4198; Roila F, Herrstedt J, Aapro M, et al. Guideline update for MASCC and ESMO in the prevention of chemotherapy- and radiotherapy-induced nausea and vomiting: results of the Perugia Consensus Conference. Ann Oncol/ESMO 2010;21(Suppl 5):v232–v243.

CHAPTER 69  Supportive Care in Hematologic Malignancies

Delayed CINV

NCCNb1

aChemotherapy c5

Moderate

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controlled studies found the addition of aprepitant (125 mg po on day 1 and 80 mg po daily on days 2 and 3) to standard therapy (5-HT3 receptor antagonist on day 1) and dexamethasone (prechemotherapy on day 1 and daily on days 2 to 4) improved rates of acute N/V and delayed N/V when compared to standard therapy alone in patients receiving cisplatin chemotherapy.351,352 Thus, in 2003, aprepitant became the first neurokinin-1 receptor antagonist to be approved for the prevention of acute and delayed nausea and vomiting with highly-emetogenic chemotherapy. Fosaprepitant, a parenteral NK-1 receptor antagonist, has since become available for the prevention of CINV associated with highly emetogenic chemotherapy. Unlike aprepitant, fosaprepitant is given at a dose of 150 mg IV 30 minutes prior to chemotherapy on day 1 only.353 Important to note is that NK-1 receptor antagonists are moderate inhibitors of the enzyme CYP34A, the enzyme responsible for glucocorticoid metabolism, and thus in clinical trials the dosing of concurrent dexamethasone was reduced to 12 mg on day 1 and 8 mg on days 2 and 3.351,352

Olanzapine Olanzapine is an atypical antipsychotic with an affinity to multiple neurotransmitter receptors involved in the emetic response, including: dopaminergic, serotonergic, adrenergic, histaminergic, and muscarinic receptors, thus suggesting a potential benefit for the prevention and treatment of nausea and vomiting.354,355 In a pilot study, Passik et al. explored the antiemetic activity of olanzapine in patients with advanced cancer, with their results suggesting benefit. Subsequent trials, including a randomized phase III trial, confirmed these results.356–358 Navari et al. conducted a randomized phase III trial comparing the efficacy of olanzapine vs. aprepitant, both in combination with palonosetron and dexamethasone, for the prevention of CINV in patients receiving highly emetogenic chemotherapy. Complete response rates, no emesis, and no need for rescue medications were similar results for both groups during the acute and delayed period; however, nausea was better controlled in the olanzapine group.358 The total dose of dexamethasone was also less in the olanzapine group, as this group received only a one-time dose of dexamethasone on day 1 vs. 4 days of dosing in the aprepitant group. This finding led the authors to conclude that the use of olanzapine for the prevention of CINV with highly emetogenic chemotherapy may be more beneficial, since the exposure to steroids and potential adverse effects of their use with multiple rounds of chemotherapy would be much less. An increase in extrapyramidal symptoms (EPS) associated with olanzapine use has not been seen in studies evaluating olanzapine’s efficacy in the prevention of CINV, and this lack of increase in EPS as compared to other neuroleptic drugs used for the treatment of nausea (e.g., prochlorperazine and metoclopramide) is another reason to consider its use. These preliminary studies also found an improvement in mood and appetite in patients who were treated with olanzapine.354 At this time, expert guidelines recommend the use of aprepitant along with 5HT-3 receptor antagonists and steroids for the prevention of CINV associated with highly emetogenic chemotherapy; however, further studies evaluating olanzapine’s benefit in this group of patients are ongoing and the results of these studies may alter recommendations in the future.

Chemotherapy-induced Nausea and Vomiting with High-dose Chemotherapy/Hematopoietic Stem Cell Transplant Controlling nausea and vomiting in patients undergoing HDC with HSCT rescue is a challenge. Many factors, including HDC, total body irradiation (TBI), medications to prevent graft-versushost disease, antimicrobials, and opioids, can induce nausea and vomiting.359 Multiple studies have shown a benefit of 5-HT3

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receptor antagonists along with steroids in the prevention of CINV in patients undergoing HDC; however, complete responses with these regimens were seen in only 15% to 50% of patients.360–363 Given the suboptimal rate of control seen with 5HT-3 receptor antagonists and steroids, studies examining the addition of aprepitant, an NK-1 antagonist, to 5HT-3 receptor antagonists have been conducted. The role of newer agents, such as aprepitant, in the prevention and treatment of CINV in patients undergoing HDC with stem cell rescue is less clear. There is data to support the efficacy and safety of aprepitant along with 5HT-3 receptor antagonists and steroids, albeit from small, single institution studies. Despite the small sample sizes, these studies have shown efficacy for a number of preparative regimens, including: BEAM (BCNU [carmustine] + etoposide + ARA-C [cytarabine] + melphalan), T-ICE (paclitaxel + ifosfamide + carboplatin + etoposide), BuCy (busulfan + cyclophosphamide), Cy/TBI (cyclophosphamide + total body irradiation), Cy/TBI/VP16, and BCV (BCNU + cyclophosphamide + VP16). The data so far has looked at multiple different dosing regimens for aprepitant, including the traditional 3-day regimen (125 mg po on day 1 followed by 80 mg po on days 2 and 3) and a 12-day regimen (125 mg po on day -7 followed by 80 mg po daily thereafter until day +4) conducted in a pilot study by Bubalo et al.364 As aprepitant is a moderate inhibitor of CYP34A, busulfan doses need to be followed closely, and in pilot studies all patients required dose adjustments.365,366,367,368,369 These studies support the added benefit of aprepitant to standard antiemetic therapy without increasing toxicity. Because of the limited amount of data, ASCO guidelines currently recommend considering the use of aprepitant in patients undergoing HDC with HSCT until the results of larger phase III studies become available.

Miscellaneous Multiday chemotherapy regimens and highly emetogenic radiation regimens are both commonly used in the management of hematologic malignancies. ASCO guidelines recommend the use of antiemetics appropriate for the emetogenic risk class of the chemotherapy being administered for each day of chemotherapy and for 2 days after, if appropriate.370 Both MASCC and ASCO guidelines recommend the use of a 5HT-3 receptor antagonist along with steroids for the treatment of highly emetogenic radiation therapy, such as TBI, with ASCO guidelines recommending a minimum of 5 days of steroid therapy.343,370

CANCER-RELATED FATIGUE Fatigue is one of the most common symptoms reported by cancer patients, and has a reported incidence of 60% to 90% among patients with cancer. Cancer-related fatigue (CRF) is defined as an unusual sense of tiredness that is persistent and not improved with rest, that can occur with cancer or cancer treatment, and that may affect physical and/or mental functioning.371 Fatigue can negatively impact a patient’s quality of life by interfering with their ability to perform activities of daily living, keep employment, and maintain financial stability.372 CRF is more intense and more unpredictable than fatigue that was experienced before the cancer diagnosis.373 Multiple etiologies for CRF are often present in patients with cancer and include anemia, sleep disorders, depression, endocrinopathies, nutritional deficiencies, and uncontrolled pain. Anemia is likely the most common reversible cause of fatigue during active cancer treatment. Although CRF is often attributed to the initiation of cancer therapies, this fatigue may persist long after the completion of treatment with up to 30% of cancer survivors reporting loss of energy years after completion of treatment.372,374 Escalante and Manzullo recommend using the question “How

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would you rate your fatigue on a scale of 0 to 10 over the past week?” along with a simple visual analog scale from 0 to 10 (with 0 as no fatigue and 10 as the worst imaginable fatigue) to screen patients and to follow response to interventions. Specific causes for fatigue should be sought out, and once specific therapies for fatigue are initiated, then assessments for response should take place as the identified cause is being corrected.372 Multiple pharmacologic and nonpharmacologic interventions have been employed to treat CRF. Nonpharmacologic interventions include exercise programs, nutritional assessments, and sleep analysis. Exercise has been the most studied nonpharmacologic strategy for treating fatigue.372 Schwartz et al. evaluated the effect of a moderate-intensity home exercise regimen in women with newly diagnosed breast cancer receiving active treatment with chemotherapy. Low- to moderate-intensity exercise significantly decreased fatigue in these patients, and the intensity of fatigue declined as exercise tolerance increased.373 Initiation of an exercise program should begin at low intensity levels and shortened intervals with gradual increase in intensity and duration based on the patient’s physical conditioning. Patients with severe debilitation and extensive disease may require consultation with a physical therapist or a physical medicine and rehabilitation specialist to assist in developing a safe and effective exercise program.372 Pharmacologic strategies that have been used to combat CRF include stimulants, antidepressants, and low-dose steroids. Welldesigned clinical trials evaluating the efficacy of these pharmacologic interventions to guide treatment decisions are unfortunately lacking. Methylphenidate, a stimulant, has been studied in CRF and may have a beneficial effect. Sarhill et al. studied the efficacy of methylphenidate for the treatment of fatigue in patients with advanced cancer. In this open labeled pilot study, 9 of 11 patients studied achieved benefit regardless of whether anemia was present. Only 1 patient stopped treatment because of adverse effects.375,376 Therapy is typically initiated at 5 mg in the morning and 5 mg in the early afternoon. Methylphenidate should not be administered in the late afternoon as it may interfere with sleep patterns. Patients may require titration of dosing over time in order to maintain a clinical benefit because of the potential development of tolerance. Tachycardia and hypertension are known adverse effects of methylphenidate and it is therefore not recommended for use in patients with known coronary artery disease, uncontrolled hypertension, or tachyarrhythmias.372 Modafinil, a nonamphetamine psychostimulant, has been studied in a randomized phase III trial to evaluate its effectiveness in the treatment of cancer-related fatigue.377 Jean-Pierre et al. reported a benefit from modafinil 200 mg daily in patients with severe fatigue at baseline; however, there was no statistically significant improvement in fatigue in patients with mild to moderate fatigue at baseline. Thus, it is recommended that intervention with modafinil be limited to those with severe fatigue at baseline.377 The inability of current pharmacologic interventions to produce significant improvements in CRF for the majority of cancer patients underscores the importance of a multidisciplinary approach to CRF that incorporates assessments for fatigue, recognition of underlying etiologies, coping strategies, nonpharmacologic therapies, and intervention with pharmacologic therapy only when necessary.

Cancer Pain Pain is one of the most feared symptoms of cancer by patients and family members.378 Sixty to eighty percent of patients with advanced cancer experience moderate to severe pain on a monthly basis, and control of cancer-related pain is one of the most important goals of supportive care. Management of cancer pain can be quite complex because the etiologies are diverse and

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can adversely affect multiple domains of quality of life, including activities of daily living, psychological well-being, physical functioning, and social interactions.379 Coexisting cancer symptoms, such as weakness, fatigue, nausea, dyspnea, constipation, and impaired cognition, can magnify the perception of and exacerbate cancer pain. Modifying the source of the pain, altering the perception of pain, and blocking the transmission of pain to the central nervous system can effectively treat cancer pain in 85% to 95% of cases.380 A comprehensive patient-centered approach to pain management that assesses the patient’s pain, barriers to pain control, concurrent medical problems, response to pharmacologic interventions, and psychosocial status is key to achieving adequate pain control.381–384

Types of Cancer Pain To adequately address and control pain, it is imperative that the treating physician understand the pathophysiology of cancer pain. The majority of cancer-related pain can be classified as nociceptive, neuropathic, or both. The distinction between these 2 types of pain is important, as neuropathic pain is often refractory to opioid therapy. Nociceptive is the term used to describe pain that is propagated by continual tissue injury, and it is classified into somatic or visceral pain. Nociceptive pain is termed somatic when the continual tissue injury is relayed via primary afferent nerves in somatic tissues such as those of the musculoskeletal system. Somatic pain is classically described as being sharp or aching and localized to the area of tissue damage. Nociceptive pain is termed visceral when tissue injury is relayed via primary afferents of the viscera. Visceral pain tends to be poorly localized and intermittent, many times described as dull or aching.378,385,386 Neuropathic pain is a result of injury to peripheral or central nerves and typically causes paroxysmal burning, shooting, or aching sensations that may or may not be associated with paresthesias. In patients with malignancy, neuropathic pain can be caused by a number of different etiologies, such as direct nerve injury from the tumor itself, or the result of anticancer treatments such as surgery, radiation, and chemotherapy. Identification of neuropathic pain often indicates that nontraditional adjuvant therapies may be required to achieve adequate pain relief.385,387

Hematologic Malignancies



Barriers to Pain Control Up to 50% of patients with cancer have uncontrolled pain.388 Several reasons for inadequate treatment of pain exist and can be classified as physician barriers or patient barriers to pain control. Multiple studies have identified physician-related barriers to cancer pain management, and all have a recurring theme.389,390 Two large studies surveying US medical oncologists’ attitudes and practices in cancer pain management have been undertaken over the past 20 years.391,392 Despite the almost 20-year time between the 2 studies, both concluded that knowledge deficits, inadequate training in pain management, inadequate opioid prescribing as recommended by established guidelines, and poor pain assessment are the main physician-related barriers to adequate pain management. The potential for addiction, the development of tolerance, scrutiny by regulatory agencies, and inadequate side effect management are other potential reasons for nonadherence to established pain management guidelines. There are also many patient-related barriers that hinder patients’ use of appropriate analgesics for cancer pain. Reluctance of patients to report pain and take pain medications have been shown to be the most prominent patient-related barriers to cancer pain management.393,394 The Barriers Questionnaire, a tool designed to identify patient-related barriers to cancer pain management, has identified 9 areas of concern that patients may have in reporting and taking pain medications. (1) Fatalism, belief that cancer pain is inevitable; (2) fear of addiction; (3) “desire to be a

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good patient” and not complain; (4) fear of distracting one’s physician from treating the disease; (5) concern that increasing pain signifies disease progression; (6) concerns about drug tolerance; (7) concerns about side effects; (8) religious fatalism, the religious belief that pain is caused or given by a deity and that patients have to tolerate the pain in order to avoid carrying the pain into their next life; and (9) concerns that pain medications are better when given on an as needed basis.393 Patient education is one way to overcome patient barriers to cancer pain management. Multiple studies have proven the effectiveness of pain education programs in improving patient adherence to analgesic therapy and ultimately decreasing pain intensity.395–397 Educational interventions for both physicians and patients will likely lead to more appropriate guideline-based analgesic prescribing and patient adherence to prescribed therapies.

produced within the tumor microenvironment and are responsible for pain associated with local tumor growth and infiltration.406,407 The benefit and role of NSAIDs in the treatment of cancer-related pain has been shown in two large meta-analyses.407,408 There does seem to be a ceiling effect for analgesia with NSAIDs and doses beyond the daily recommended maximum dose are not advised. Doses of nonopioid analgesics should be titrated to maximal daily doses or until adverse effects emerge, as long as mild to moderate pain persists. NSAIDs do carry a risk for GI toxicity, and cancer patients may be particularly susceptible to these toxicities, given concurrent medications that can lead to mucositis, gastritis, and anorexia. Selective COX-2 inhibitors are available and have been associated with less GI toxicity than nonselective NSAIDs; however, there have been reports of adverse GI effects with their use in cancer patients.409 For acetaminophen, it is not recommended to dose more than 6 g per day; however, for those with underlying

Pain Assessment A comprehensive pain assessment is crucial to adequate pain control and includes a detailed pain history that evaluates pain intensity, location, and type, as well as an assessment of psychological stressors and the patient’s network of social support. A detailed physical exam paying close attention to impending oncologic emergencies is also important in determining the etiology of pain so that timely and appropriate interventions can be initiated. Pain is subjective, and thus patient report is the “gold standard” for measurement. In order to try and make pain measurement objective and reliable, a number of scales have been developed. Pain intensity has been shown to have an inverse correlation with quality of life and functioning. Pain intensity can be measured by a number of validated scales.398,399 Some scales are available to assess pain intensity for those with limited literacy as well as those who are cognitively impaired.378,400–402 Other scales, such as the Memorial Pain Assessment Card and the Wisconsin Brief Pain Inventory, have been developed to assess pain intensity as well as psychological distress. These two scales are not only multidimensional but also practical, given the efficiency in which they can be completed in a clinical setting.403,404 Numerical ratings of pain intensity obtained from the different scales can then be used to guide analgesic therapy.

Analgesic Management: World Health Organization Analgesic Ladder The World Health Organization (WHO) “three-step analgesic ladder” (Fig. 69.10) developed in the 1980s continues to provide the framework for the management of cancer-related pain. The analgesic ladder introduced a stepwise approach to pain management and selection of appropriate analgesia based on pain intensity. Based on WHO recommendations, mild pain, rating 1 to 4, is treated with nonsteroidal antiinflammatory drugs (NSAIDs) and acetaminophen plus adjuvant analgesics; moderate pain, rating 5 to 6, is treated with weak opioids such as codeine and tramadol; and for severe pain, rating 7 to 10, strong opioids such as hydromorphone, morphine, fentanyl, oxycodone, and methadone are recommended.405,406 Additional principles endorsed by the WHO for optimal pain management include: (1) oral administration of pain medications if possible; (2) around-the-clock dosing for chronic pain; (3) pain severity should determine drug choice; (4) individualized treatment plans because of variability of response to analgesic agents among patients; and (5) frequent reassessments of patient’s pain.406 NSAIDs and acetaminophen are the initial nonopioid step 1 analgesics recommended for use by the WHO guidelines for mild pain. NSAIDs block cyclooxygenase 1 and cyclooxygenase 2 enzymes (COX-1 and COX-2), thus decreasing prostaglandin synthesis. Prostaglandins and other inflammatory cytokines are

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Freedom from cancer pain

Opioid for moderate to severe pain ± non-opioid ± adjuvant therapy

3

Pain persisting or increasing

Opioid for mild to moderate pain ± non-opioid ± adjuvant therapy

Pain persisting or increasing

Non opioid ± adjuvant therapy

2

1

PAIN FIGURE 69.10. The WHO “three-step analgesic ladder.”

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hepatic dysfunction or those taking concomitant potentially hepatotoxic medications, 4 to 6 g per day may be more appropriate. If pain remains uncontrolled with appropriate step 1 interventions, then the introduction of opioid therapy into the management of cancer pain is recommended by step 2 of the WHO analgesic ladder. Opioids are the backbone of pain management in patients with cancer. Interindividual variation in opioid receptors (polymorphisms of the mu, kappa, and/or delta receptors), absorption, intensity of the painful stimulus, administration of concurrent medications, and enzymes responsible for opioid metabolism underscore the importance of an individualized approach to opioid prescribing for patients experiencing cancer pain.410,411,412 Step 2 opioids, previously referred to as “weak opioids,” include codeine, tramadol, hydrocodone, and oxycodone. These opioids are typically combined with nonopioid analgesics such as NSAIDs or acetaminophen which limit their total daily dose. Codeine has a weak affinity for mu opioid receptors and its analgesic activity requires metabolism to morphine by CYP2D6. The time-to-peak effect for codeine is typically 1 to 2 hours, and it has an effective half-life of approximately 3 hours. Like many other opioids that require CYP2D6 metabolism to produce active metabolites, medications that induce or inhibit the CYP2D6 enzyme can alter the analgesic effects of codeine. In addition, some patients are “fast” or “slow” metabolizers, further adding to the variability in effectiveness.413 Tramadol, a synthetic analog of codeine, is also considered a “weak opioid.” Tramadol is unique because it not only has analgesic properties that are mediated through mu receptor activation, but it also has serotonin and norepinephrine reuptake inhibitor activity. This dual mechanism is felt to be responsible for its effectiveness, especially in selected patients with chemotherapy-induced neuropathic pain.414,415 O-desmethyl tramadol, the active metabolite of tramadol, requires metabolism via the CYP2D6 enzyme and is thus subject to interactions to CYP2D6 inducers and inhibitors. Maximal daily dose is 400 mg per day, as doses above this can lead to neurotoxicity and seizures. Hydrocodone is an oral opiate that is indicated for the treatment of moderate pain and is available only as a combination product with an NSAID or acetaminophen. The metabolism of hydrocodone is through the CYP2D6 enzyme as well; however, its active metabolite, hydromorphone, is a very strong mu receptor agonist.416 Given hydrocodone’s prodrug properties, analgesic response is affected by different polymorphisms in the CYP2D6 gene, as well as inducers and inhibitors of this enzyme.410 If pain persists at a level of 5 or greater after achieving the maximal daily dose of the step 2 analgesic, then the addition of a step 3 analgesic is recommended. Morphine, fentanyl, oxycodone, and hydromorphone are the most commonly used step 3 analgesics used for the management of moderate to severe cancer pain. Morphine has classically been the opioid of choice for the treatment of moderate to severe cancer pain. Due to morphine’s multiple routes of administration (oral tablets, oral solutions, parenteral, and rectal), formulations are available to appropriately treat acute pain crisis, chronic stable pain, and those in a terminal condition. Morphine can cause adverse effects that are classic for all opioids; however, it can also produce adverse effects that are unique to itself. Morphine can cause a histamine release, which can lead to bronchospasm and rash. Morphine can also decrease sympathetic tone that leads to peripheral vasodilatation and resultant orthostatic hypotension.410,413 It is metabolized through glucuronidation, producing active metabolites morphine-6 glucuronide (M6G) and morphine-3 glucuronide (M3G). M3G is associated with neurohyperexcitability and hyperalgesia. Both metabolites are excreted in the urine; thus, renal insufficiency can lead to an accumulation of these metabolites and result in oversedation and increased adverse effects.417,418 Oxycodone is available in its pure form as well as in combination with acetaminophen or an NSAID. Combination products

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limit oxycodone’s titration due to maximal daily doses of the nonopioid analgesic partner medication, thus limiting its efficacy to that of other step 2 analgesics. In its pure form, oxycodone has no ceiling effect, the same as the other step 3 opioids. Oxycodone is metabolized by CYP2D6 to oxymorphone, thus making it susceptible to drug-drug interactions that induce or inhibit CYP2D6.410 Hydromorphone is a semisynthetic opioid that is approximately 5 times more potent than morphine once steady state oral and parenteral dosing is reached.419 Hydromorphone has multiple routes of administration: intravenous, intramuscular, subcutaneous, oral, and rectal, and is extensively metabolized by the liver; thus its use may be preferred in patients with renal failure.410 Fentanyl is another step 3 opioid that is a very strong opiod agonist, approximately 80 times more potent than morphine. Fentanyl is unique in that it not only has a parenteral form, but also has transdermal and transbuccal formulations. These preparations are particularly useful for patients with stable chronic pain who are unable to swallow pills, are noncompliant, or are in the terminally ill phase when swallowing may be difficult. When transitioning to transdermal fentanyl preparations it is important to note that there is a delay of 8 to 16 hours until the onset of analgesia, and thus initial overlap with other opioid medications may be required. Steady state of transdermal fentanyl is not reached until approximately 72 hours.410,420 Fentanyl undergoes extensive metabolism in the liver, thus making it an attractive option for use in those with renal failure. There is some controversy as to the appropriate relative potency ratio to be used when converting oral morphine to transdermal fentanyl. Donner et al. showed that an oral morphine to transdermal fentanyl ratio of 100:1 was safe and effective in converting patients with cancer pain from oral morphine to fentanyl; however, they noted that this ratio was too low and that the true ratio determined by their study was 70:1. Thus, if one uses 100:1 as a conversion ratio, then one should be aware that this will likely lead to insufficient dosing, and supplemental breakthrough medications will likely be necessary until appropriate doses can be achieved.421 Oxymorphone, a newer strong opioid multireceptor agonist, is available in oral preparations and is 10 times more potent than morphine. Oxymorphone is not metabolized by CYP2D6 and thus is not as susceptible as some of the other aforementioned opioids to drug-drug interactions.422 Methadone is another effective opioid receptor agonist that has been used to treat chronic cancer pain. Due to its long half-life, erratic metabolism, and propensity for drug-drug interactions, it is recommended that methadone only be managed by providers with knowledge of its pharmacology and experience in its prescribing.410 The oral route of administration is the most preferred as long as patients are able to swallow, and this route of administration is controlling the pain. Acute pain exacerbations should be treated with short-acting medications around the clock along with breakthrough dosing in order to ultimately come up with a total 24-hour opioid dose that will control the pain. As long as patients are experiencing severe unrelieved pain, the total opioid dose should be increased by 50% to 100% every 24 hours until the pain is controlled. If patients are experiencing unrelieved moderate pain, the total opioid dose should be increased by 25% to 50% every 24 hours until the pain is controlled. Once the patient’s pain is adequately controlled, then long-acting preparations of oral opioid therapy can be incorporated. Appropriate doses of longacting opioid medications can be calculated by taking the 24-hour total  oral opioid requirement to control the pain and converting this into a long-acting opioid dose. Because of the individualized nature of the metabolism of different formulations of opioid pain medications, as a general rule it is safe to start with 75% of the total dose of the previous opioid when transitioning between opioids, and titrate the dose up as necessary. Once scheduled long-acting opioids are instituted, short-acting breakthrough

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medications should be made available to the patient on an as needed basis with dosing intervals determined by the medications’ time to peak effect. Again, frequent reassessment of pain control is of paramount importance, especially during times of transition between different formulations of opioid medications.386 Despite aggressive and appropriate management with opioid and other adjuvant analgesic medications, a minority of patients will continue to have uncontrolled pain related to their malignancy. In these circumstances, a referral to an anesthesiologist or a neurosurgical pain specialist is warranted. Epidural nerve blocks, spinal cord stimulation, or implantable epidural catheters with opioid pain pumps may be needed to achieve adequate pain control.423,424

Adjuvant Therapies Opioid therapy alone may not be adequate to control all types of cancer-related pain, and thus throughout all steps of the WHO analgesic ladder adjuvant analgesic treatments are recommended when they are felt to benefit the patient (Table 69.10). Corticosteroids have long been used to treat pain due to spinal cord compression associated with metastatic disease; however, their benefit has been shown in the treatment of bone and neuropathic pain as well.425–427 Anticonvulsants such as gabapentin and lamotrigine, as well as antidepressants such as amitriptyline and nortriptyline, have been shown to improve neuropathic pain and can be used in combination.428,429,430 As pain can be exacerbated by depression, and depression can be influenced by pain, it is crucial that prompt recognition and treatment of depressive symptoms occur when identified.431 Pain that is caused by malignant bone involvement may respond to therapy with agents that limit osteoclast activity, such as bisphosphonates and Miacalcin, as well as agents such as NSAIDs and corticosteroids, which can decrease local inflammation caused by tumor invasion.432,433

TA BL E 69.10

ADJUVANT ANALGESICS Indication/Drug Class Multipurpose Analgesics   Tricyclic Antidepressants   Selective Serotonin Reuptake Inhibitors   Noradrenaline/serotonin Reuptake Inhibitors  Others   Corticosteroids   Neuroleptics For Neuropathic Pain  Anticonvulsants   N-methyl-d-aspartate receptor antagonists   Topical Drugs For Bone Pain  Corticosteroids  Calcitonin  Bisphosphonates For Musculoskeletal Pain   Muscle Relaxants  Baclofen  Tizanidine  Benzodiazepines

Examples Amitriptyline, Nortriptyline, Desipramine Paroxetine, Citalopram Venlafaxine Dexamethasone, Prednisone Olanzapine Gabapentin, Topiramate, Lamotrigine, Levetiracetam, Pregabalin Ketamine, Dextromethorphan Lidocaine/Prilocaine Dexamethasone, Prednisone Calcitonin Pamidronate, Zoledronic Acid Cyclobenzaprine, Orphenadrine, Carisoprodol, Metaxalone Diazepam, Lorazepam, Clonazepam

Adapted from Lussier D, Huskey AG, Portenoy RK. Adjuvant analgesics in cancer pain management. Oncologist 2004;9:571–591.

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There is some evidence to suggest that complementary therapies such as massage, acupuncture, and yoga are beneficial in the treatment of cancer-related pain; however, larger studies are needed to confirm these possible benefits.434–436

Adverse Effect Management Despite the efficacy of opioids in the treatment of pain, almost all patients who use opioids for analgesia will experience side effects. The most common adverse effects of opiates are listed in Table 69.11. Prevention is the key to the management of these adverse effects; however, once side effects emerge, aggressive measures to abate these undesired effects, along with opioid titration and possible rotation, are necessary. Constipation is one of the most psychologically distressing side effects from opioid therapy that patients may experience and can lead to anorexia and bowel obstruction.437 All patients taking opioids should be on a prophylactic bowel regimen to try and prevent constipation. Methylnaltrexone, a peripheral opioid antagonist, has been demonstrated in multiple studies to be effective in relieving opioidinduced constipation. Weight-based subcutaneous dosing can be given as needed, however not to exceed 1 dose per 24-hour period. Inclusion criteria for randomized clinical trial enrollment were either < 3 bowel movements within the preceding week or >2 days without a bowel movement.438–441 As methylnaltrexone is unable to cross the blood-brain barrier, it is able to reverse peripheral inhibition of opioid receptors of the GI tract without affecting analgesia.442 Respiratory depression is a potentially fatal yet uncommon adverse effect of opioid treatment. Naloxone, a nonselective opioid antagonist, is the only therapy currently available for the reversal of opioid-induced respiratory depression.443 Due to its low bioavailability, naloxone is given by intravenous infusion. The extent and duration of naloxone’s effect depends on the receptor affinity of the opioid being reversed. Due to its rapid elimination, naloxone many need to be dosed repeatedly or by continuous infusion to maintain reversal of respiratory depression. Since naloxone is a nonselective opioid antagonist, reversal of respiratory depression results in reversal of analgesia, and thus research into therapies that can reverse respiratory depression while preserving analgesia are currently underway.444 Urinary retention can be relieved with insertion of a Foley catheter into the bladder. Excessive sedation can be combated by dose reduction of the opioid or with addition of a psychostimulant such as caffeine or methylphenidate.445 There is preliminary data that opioid-induced neurotoxicity, specifically hyperexcitability, can be managed with medications such as lorazepam, baclofen, valproic acid, and midazolam. Small studies have shown improvement in opioid-induced nausea with centrally acting antiemetics such as

TA BL E 6 9 . 1 1

ADVERSE EFFECTS OF OPIOID TREATMENT Common Adverse Effects of Opioid Therapy Constipation Respiratory Depression Hyperexcitability Urinary Retention Nausea Pruritus Sedation

Hallucinations Impotence Fatigue Sweating Vomiting Myoclonus Xerostomia

Adapted from Cheung WY. Pharmacologic management of cancer-related pain, dyspnea, and nausea. Semin Oncol 2011;88:450–459.

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metoclopramide and phenothiazines, as well as antipsychotics such as olanzapine and chlorpromazine.354,446

Mucositis Mucositis is a common toxicity related to systemic chemotherapy, especially in those patients receiving HDC and HSCT, in which mucositis affects 80% to 100% of patients undergoing preparative regimens.447 Mucositis can make the host susceptible to infection, modulate the intensity of treatment able to be given, and hinder the ability for oral intake, all of which can have a negative impact on quality of life (QOL).448 The choice of chemotherapeutic agent (e.g., methotrexate, bleomycin, infusional 5-FU, cytarabine, etoposide), preexisting oral disease, and concurrent use of radiation therapy are all factors that can increase the likelihood of a patient developing treatment-related mucositis.449,450 As with nausea, prevention is key; unfortunately, the data for effective preventative interventions is sparse. Pretreatment prophylactic oral care, consisting of a comprehensive oral examination that includes caries treatment, endodontic therapy, and tooth extraction if necessary, has been shown to decrease the frequency of treatment-related oral complications.451 Other preventative therapies such as cryotherapy, palifermin, and low-level laser therapy have data to support their use in patients undergoing treatment for hematologic malignancies. The strongest data supporting oral cryotherapy (ice chips swished around the mouth for 30 minutes) for the prevention of treatment-related mucositis has been in patients receiving bolus 5-FU treatments.452 There is also data supporting the use of oral cryotherapy in patients receiving high-dose melphalan and stem cell transplantation, and thus MASCC guidelines recommend the use of oral cryotherapy for the prevention of oral mucositis in this patient population.453 Palifermin is a recombinant human keratinocyte growth factor that stimulates the growth and differentiation of epithelial cells. Spielberger et al. evaluated the effectiveness of palifermin in preventing the development of severe mucositis in patients undergoing HDC and TBI followed by autologous HSCT in a double-blind phase III randomized controlled trial.454 Palifermin (60 µg/kg of body weight per day intravenously for 3 days before the initiation of conditioning therapy and for 3 days after autologous HSCT) was shown to decrease the incidence of WHO grade 3 or 4 mucositis, duration of mucositis, use of opiod analgesics, and need for total parenteral nutrition (TPN). These results led to clinical practice guidelines recommending the use of prophylactic palifermin in patients with hematologic malignancies who are undergoing highdose chemotherapy with autologous HSCT.453,455 Palifermin’s utility in allogeneic HSCT is not as well defined. Retrospective data from Goldberg et al. evaluated the safety and efficacy of palifermin in patients who underwent T cell–depleted allogenic HSCT and found a benefit in those patients undergoing TBI-based conditioning regimens but not those undergoing chemotherapy-based conditioning regimens.456 Prospective studies to evaluate palifermin’s role in allogenic HSCT are currently underway and thus guidelinebased recommendations currently advocate for palifermin’s use in patients undergoing HDC with autologous HSCT.453,455,456 Low-level laser therapy (LLLT) has also been reported to be an effective treatment for the prevention of oral mucositis associated with HDC and TBI conditioning regimens prior to HSCT. This type of low energy radiation therapy has been shown to have analgesic, antiinflammatory, and wound healing properties.457 MASCC guidelines currently recommend the use of LLLT to reduce the incidence of oral mucositis associated with HDC or TBI conditioning regimens and HSCT. Unfortunately, LLLT requires expensive equipment and expert training, and thus few centers have the capability to support such therapy.453 Supersaturated calcium phosphate rinses (SCPRs) have been evaluated in the prevention of treatment-related oral mucositis. In

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a small randomized single-institution study of patients undergoing allogeneic HSCT comparing SCPR 4 times daily to a control group consisting of a solution made of saliva leaf extract, iodine-povidine, and fluconazole administered at the same frequency, SCPRs were found to decrease the incidence, severity, and duration of oral mucositis, as well as the need for analgesic and TPN interventions. Larger multicentered trials are needed to confirm these results.458 Once oral mucositis occurs, treatment is supportive and consists of appropriate oral care, mucosal protectants, and analgesia (topical and/or systemic); however, data to validate these interventions is limited. Routine oral care should focus on limiting trauma which could further damage the oral mucosa. Soft toothbrushes and gentle cleansing rinses such as salt and soda rinses (½ teaspoon of salt and 1 teaspoon of baking soda in a quart of water every 4 hours) should be used. In an open labeled pilot study conducted by Innocenti et al., Gelclair, a mucosal protectant, was found to be beneficial in reducing oral pain. Of note, only 3 of the 30 patients in this trial had chemotherapy-related mucositis.459 Studies of analgesic mouthwashes have documented pain relief with topical lidocaine and morphine preparations; however, if these preparations do not control the mucositis-related pain, patients should be treated with systemic analgesia by the oral route if possible, and if unable, through parenteral routes.460,461

Anorexia Cancer-related anorexia (CRA) leads to weight loss as a result of decreased appetite from the systemic effects of advanced cancer.462 Anorexia is one of the most frequent and troubling symptoms experienced by patients and their family members, occurring in more than half of patients with advanced cancer.463,464 CRA and cachexia are provoked by metabolic changes induced by advanced malignancies in the host, with a milieu of proinflammatory cytokines felt to be responsible.465 Dramatic changes in weight and body habitus can occur when the hypermetabolic state of malignancy is coupled with decreased caloric intake associated with CRA. The resultant changes in body habitus can be quite psychologically distressing to patients and their families, and thus patient education and attempts to improve anorexia are of utmost importance.466 There are multiple nonpharmacologic and pharmacologic interventions that are effective in the treatment of CRA; however, the first step in the management of CRA is to evaluate for any reversible causes. Reversible causes of anorexia in patients with advanced cancers and those receiving directed therapies for advanced cancers include constipation, uncontrolled pain, nausea, vomiting, gastroparesis, depression, stomatitis, mucositis, and delirium. Evaluation for a possible reversible cause and its treatment should be the first step in management of CRA.466 If no reversible cause can be identified, then interventions directed toward management of CRA should be initiated. Nonpharmacologic measures include nutritional counseling, increasing physical activity, and encouraging the intake of caloriedense foods and supplements. Corticosteroids, megestrol acetate (Megace), and cannabinoids have all been evaluated in the treatment of anorexia. Megestrol acetate at doses of 800 mg/day have been the most extensively studied and have been shown to be effective in treating anorexia associated with advanced malignancy and acquired immune deficiency syndrome (AIDS).467–472 Jatoi et al. performed a large randomized controlled trial of more than 400 patients with advanced cancer, comparing the effectiveness of megestrol acetate (800 mg/day) vs. dronabinol (2.5 mg twice a day) vs. a combination of both agents for the treatment of cancer-associated anorexia. Megestrol acetate was found to improve appetite and induce weight gain more than dronabinol. Combination therapy did not provide an additional benefit over megestrol acetate therapy

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alone.471 A study conducted by Navari et al. found that the addition of olanzapine (5 mg/day) to megestrol acetate (800 mg/day) led to improvements in weight gain, anorexia, and quality of life (QOL) when compared to megestrol acetate alone. No additional grade III or IV toxicities were observed in the group receiving megestrol acetate and olanzapine.473 Megestrol acetate is fairly well tolerated, with increased risk for thromboembolism being the most worrisome adverse effect. A prospective study of dexamethasone 4 to 16 mg daily has been shown to be effective in treating anorexia; however, given the multiple potential adverse effects of corticosteroids, the lowest effective dose should be used and treatment discontinued if no benefit is observed within 3 to 5 days of starting treatment.466,474 For patients unable to consume all of their caloric needs by mouth, enteral tube feedings are the treatment of choice, as this method of delivery can help prevent mucosal atrophy of the gastrointestinal tract.

Complications of Hematologic Malignancies There are numerous complications secondary to hematologic neoplasms, and selected topics on thrombocytopenia, anemia, and tumor burden are described in the subsequent section. Neutropenia was addressed in the preceding sections on neutrophil defects, febrile neutropenia, and myeloid colony-stimulating factors. Problems due to tumor burden include hyperleukocytosis, acute tumor lysis, hypercalcemia, and cord compression.

Thrombocytopenia Thrombocytopenia may develop as a result of direct marrow infiltration, chemotherapy, infection, DIC, immune-mediated platelet destruction, and hypersplenism, as well as other idiopathic etiologies, and is considered the most common cause of serious hemorrhagic events in patients with acute or chronic leukemia.475 The relationship between spontaneous bleeding events and decreased platelet counts in the setting of induction chemotherapy or HSCT has been well described.476,477 As a result, prophylactic and/or therapeutic platelet transfusion to reduce bleeding risk is a generally accepted practice in these settings.478–481 The optimal threshold for prophylactic platelet transfusion remains controversial, but levels in the 10 to 20 × 109/L range have commonly been utilized with no major differences in outcome demonstrated between these levels.476,477,482 However, the risk of bleeding events in thrombocytopenia has been estimated to be increased 8-fold when counts are < 5 × 109/L and 2-fold with counts from 5 to 15 × 109/L compared to platelets in the 20 to 29 × 109/L range.483 Therapeutic (in the presence of bleeding) versus prophylactic (platelet count < 10 × 109/L) platelet transfusion strategies have been compared in patients undergoing intensive chemotherapy for AML or autologous HSCT.484 The therapeutic platelet transfusion strategy led to a reduced number of platelet transfusions without an increased risk of major hemorrhage in autologous HSCT patients. However, nonfatal grade 4 (mostly CNS) bleeding was increased among AML patients, suggesting a continued role for prophylactic strategies in this population. Clinical practice guidelines set forth by ASCO currently recommend a threshold of 10 × 109/L for prophylactic platelet transfusion in adult patients receiving therapy for acute leukemia.485 Patients with either chronic or recurrent thrombocytopenia requiring multiple platelet transfusion exposures are at increased risk for experiencing platelet refractoriness due to alloimmunization. The presence of fever is also associated with increased refractoriness to platelet transfusions486 and further compounds the risk of bleeding in this setting. When administering platelet transfusions to this population, the use of ABO-compatible

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platelet products is important in reducing alloimmunization as well as improving the incremental response to a given platelet transfusion.487,488 Platelets are generally available in the form of either single-donor apheresed products or pooled from multiple random donors. Each of these products may also be modified by either leukoreduction or irradiation. Reduction of leukocytes from platelet products by filtration has been demonstrated to be equally as effective as irradiation in the prevention of alloimmunizationrelated platelet refractoriness.489 Similarly, there is no difference in alloimmunization risk to support empiric use of leukoreduced single-donor (apheresed) platelet products compared to randomdonor platelets in this setting.489 Leukoreduction provides additional benefits by preventing CMV transmission490 and by reducing the incidence of febrile transfusion reactions.491 The benefit of platelet irradiation for the prevention of transfusion-associated graft-versus-host disease (GVHD) appears limited. Use of irradiated platelet products should be restricted to patients receiving allogeneic HSCT, those receiving blood products from related donors, and those who are severely immunocompromised.492 Thrombocytopenia caused by accelerated platelet consumption can occur in patients with hematologic malignancies. ITP has been well described in the setting of CLL493–495 and has been reported in patients with HL, NHL, MM, and ALL.494–498 The mechanisms that contribute to the initiation of ITP in each of these situations are largely unknown, but it is presumed that pathogenic autoantibodies are produced and react to antigens present on the platelets. The prognostic significance of ITP in the setting of the various hematologic malignancies is unclear. Standard therapeutic interventions for ITP (Chapter 47) with steroids, intravenous immunoglobulin (IVIG), and/or splenectomy are typically employed in addition to definitive treatment for the underlying malignancy. Diffuse intravascular coagulation (DIC) is well recognized to cause a consumptive thrombocytopenia associated with hematologic malignancies, most notably APL (Chapter 78). DIC is characterized by widespread intravascular activation of coagulation, intravascular fibrin deposition, and simultaneous consumption of coagulation factors and platelets which increase bleeding risk.499 APL-associated DIC is characterized by marked hyperfibrinolysis with a clinical presentation of severe bleeding and laboratory parameters consistent with hypofibrinogenemia, elevated fibrin split products, elevated fibrinogen degradation, quantitative D-dimers, and consumption of plasminogen and á2-antiplasmin.500 It is suspected that the hyperfibrinolytic state in APL-associated DIC is superimposed on the prothrombotic characteristics known to occur in DIC as a result of coagulation activation and fibrin deposition, providing concurrent risk for both thrombotic and hemorrhagic complications. Chemotherapy administration to treat an underlying malignancy may transiently increase thrombotic risk by causing endothelial damage as well as release of procoagulant factors, cytokines, and proteases from damaged malignant cells.501,502,503,504 Therapeutic intervention for DIC remains largely supportive, with the focus on treatment of the underlying malignancy in these situations. Limited data exists to provide recommendations for the management of DICrelated complications. Transfusion support of platelets or plasma products is typically determined by active bleeding and/or the risk of hemorrhagic complications.505 The use of therapeutic heparin should be considered on an individual patient basis and may benefit those at risk for thrombotic complications.503,506 Thrombocytopenia as a direct result of the malignant condition, or the aggressive chemotherapy that may be required, poses significant challenges in the treatment of patients. Recombinant human IL-11 (Oprelvekin) has been approved for the treatment and prevention of chemotherapy-induced thrombocytopenia in nonmyeloid malignancies.507 Randomized placebo-controlled testing in breast cancer patients receiving chemotherapy demonstrated reduced platelet transfusion events.508 Use of rIL-11 has

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also been shown to improve platelet nadirs and shorten duration of thrombocytopenia in patients receiving chemotherapy for hematologic malignancies.509 It is not indicated for use in children or in patients receiving myeloablative chemotherapy. Despite its proven effectiveness, the use of rIL-11 has been limited in part because of the potential for hypersensitivity reactions, ventricular arrhythmias, and papilledema associated with visual field defects and possibly blindness. Genetically modified recombinant human IL-11 (mIL-11) is under active investigation, but early reports suggest that it is able to provide equivalent thrombopoietic activity with an improved safety profile.510 Romiplostim is a thrombopoietin (TPO) peptide mimetic which increases platelet counts by binding to and activating the TPO receptor (Chapter 47). Although approved for use in chronic ITP, off-label use has been reported for chemotherapy-induced thrombocytopenia511 and romiplostim’s role in this setting is under active investigation (NCT01676961; NCT01516619). Eltrombopag is a nonpeptide TPO agonist also approved for chronic ITP. Eltrombopag increases platelet production by binding to and activating the TPO receptor, but also increases proliferation and differentiation of marrow progenitor cells by activation of intracellular signal transduction pathways. Increased platelet numbers have been reported with its use in patients with radiationinduced thrombocytopenia,512 patients receiving chemotherapy for advanced solid tumors,513 and patients with refractory aplastic anemia.514 Eltrombopag use in patients undergoing chemotherapy for hematopoietic malignancies is also an area of active investigation (NCT01656252, NCT01488565).

Anemia Anemia commonly occurs as a result of the presence of a hematologic malignancy and/or the subsequent therapeutic interventions. Marrow infiltration and replacement with neoplastic cells is the primary cause, but other mechanisms must be considered. Patients with inflammatory and/or malignant conditions commonly present with normocytic, normochromic or microcytic, hypochromic anemia. Laboratory findings are typically suggestive of a hypoproliferative state characterized by low reticulocyte counts and the absence of appropriate erythroid hyperplasia in the marrow in response to the anemia. Serum erythropoietin (EPO) levels tend to be inappropriately low in response to the malignancy-associated anemia,515 which is suspected to result from proinflammatory cytokines that interfere with the regulation of EPO gene expression.516 Erythroid marrow response to EPO is blunted, and higher doses of EPO supplementation are often required to improve anemia associated with hematologic neoplasms.475,517 The use of EPO stimulating agents has been curtailed because of an inferior survival in randomized trials for cancer patients and an increased risk for thrombosis.518 Inflammatory cytokines have been demonstrated to increase hepcidin production,519,520 causing decreased intestinal iron absorption and impaired iron release from the reticuloendothelial system.521 Increased IL-6 levels have been implicated in stimulating hepcidin production in patients with HL, MM, ALL, and AML, and provides insight into the pathophysiology of anemia commonly found in patients with various malignancies.522–524 Targeting IL-6 in the treatment of cancer-related anemia has shown promise and remains an area of active investigation.525,526 Macrocytic anemia is often seen in association with MDS and AML and is also commonly caused by exposure to cytotoxic chemotherapy. Agents such as cytarabine, hydroxyurea, methotrexate, and 6-mercaptopurine inhibit DNA syntheses and are frequently associated with the development of macrocytic anemia in exposed patients. In the absence of exposure to these types of agents, abnormal nucleated red cells may occur in AML or as part of the terminal phase of myeloproliferative disorders. Certain

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morphologic features allow their differentiation from the megaloblasts resulting from vitamin deficiencies. The nuclear chromatin is typically not as fine as in true megaloblasts, and a greater proportion of the erythroid precursors are more immature in most patients with erythroleukemia than in those with vitamin B12 or folate deficiencies. In addition, the neutrophils characteristic of these vitamin deficiencies, such as hypersegmentation, giant metamyelocytes, and macropolycytes, are usually absent in the macrocytic anemia seen in AML/MDS. Hemolytic anemias have been observed in association with various hematologic malignancies and may precede diagnosis of the malignant condition. While their presentation may be dramatic, concomitant hemolytic anemias do not carry prognostic significance. Warm antibody–mediated hemolytic anemia has been classically described to occur in 10% to 20% of patients with CLL but has also been observed in association with various other hematologic cancers.527–530 Therapy is directed at decreasing the production of antibody and the splenic destruction of red blood cells (Chapter 29). Glucocorticoids, transfusion support, and/or splenectomy are used in autoimmune hemolytic anemia of warm antibody type, in addition to therapy directed toward the underlying neoplasm. Anemia is usually mild in cold agglutinin disease, and intervention with steroids and splenectomy is of little benefit. Avoiding cold exposure, plasmapheresis, and treatment of the underlying lymphoproliferative disorder may be effective in improving the anemia associated with cold agglutinin disease. Microangiopathic hemolytic anemia is seen most often as part of the DIC syndrome but has been reported in patients with hematologic neoplasms without evidence of DIC. Erythrocyte trapping and/or destruction in the spleen may contribute to anemia in hematologic cancers such as low-grade lymphomas and CLL. Splenectomy has been used for treatment of nonautoimmune hemolytic anemias in lymphoid malignancies when this hypersplenism occurs.531,532 Hemophagocytic syndrome is a rare but often dramatic cause of anemia in patients with a variety of hematologic malignancies. Its presence has been described in association with acute leukemias, lymphomas, and myeloma,533–537 as well as a result of infectious complications of both bacterial and viral origin.538,539 The mechanism by which this potentially devastating macrophage activation occurs is poorly understood, but is felt to result from the inability to turn off the inflammatory response of the immune system to the particular inciting factor (malignancy, infection, etc.).540 Prognosis in these patients is quite poor.

Hematologic Malignancies



Hyperleukocytosis and Leukostasis Hyperleukocytosis has been variably defined as a white blood cell (WBC) count greater than 50 × 109/L or 100 × 109/L (Table 69.12). TA BL E 6 9 . 1 2

MANAGEMENT OF HYPERLEUKOCYTOSIS 1. Risk of leukostasis depends upon disease and level of WBC (× 109/L). AML (monocyte variants) and CML, blast crisis (>50–100) > ALL (>150–300) > CML (>150–250) > CLL (300–500) 2. Target organs for leukostasis are: brain (stupor, blurred vision), lungs (dyspnea), kidneys(azotemia), penis (priapism), heart (arrhythmia) 3. Spurious lab may occur with hyperleukocytosis: elevated platelet count, pseudohypoxemia (“leukocyte larceny”), pseudohyperkalemia, pseudohypoglycemia, prolonged coagulation tests 4. Transfusion: limit red cells due to risk of increasing viscosity; transfuse platelets due to increased risk of bleeding and loss of platelets with leukapheresis 5. Therapy: hydration, allopurinol or rasburicase, hydroxyurea, consider leukapheresis if symptomatic, dexamethasone, institute chemotherapy, manage tumor lysis

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It is typically associated with increased morbidity and mortality in patients with leukemic diagnoses as a result of associated leukostasis and/or tumor lysis syndrome. The absolute count at which the hyperleukocytosis becomes clinically relevant is somewhat dependent on the specific underlying leukemic process. For instance, patients with AML may develop severe complications at a WBC count of 50 × 109/L, whereas patients with CLL may tolerate WBC counts above 400 × 109/L. Hyperleukocytosis has been estimated to occur in approximately 5% to 13% of AML and between 10% to 30% of ALL,541 and has historically been associated with a poor prognosis when occurring in patients with acute leukemia.542–544 Supportive care measures such as aggressive intravenous fluid support, allopurinol, and hydroxyurea are typically employed as temporizing measures until some form of cytoreductive therapy can be initiated. Leukostasis is a symptomatic manifestation of hyperleukocytosis which qualifies as a medical emergency caused by the “sludging” of the leukemic cells within the capillaries, leading to vascular obstruction and resultant tissue hypoxia (Fig. 69.11). Presenting symptoms of leukostasis are reflective of the location where this tissue hypoxia is occurring. CNS manifestations of confusion, headaches, visual disturbances, somnolence, delirium, or ataxia may occur. Respiratory findings may include bilateral infiltrates on chest imaging and associated dyspnea, tachypnea, hypoxia, and rhonci on physical exam. Leukostasis remains a diagnosis of clinical suspicion rather than one confirmed by a laboratory test. Given the potential for catastrophic consequences, reasonable

A

C

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clinical suspicion of diagnosis should prompt emergent intervention. Hydroxyurea is an oral antimetabolite with the ability to rapidly lower leukocyte counts and whole blood viscosity.545,546 Definitive antileukemic therapy should be initiated as soon as possible. Some centers prefer to initiate leukapheresis prior to the initiation of chemotherapy in order to reduce the peripheral leukemic burden, ameliorate the symptoms of leukostasis, and minimize the risk of tumor lysis syndrome upon initiation of the chemotherapy.547,548 While retrospective data has shown that leukapheresis may reduce early mortality, it has failed to provide improvements in remission or overall survival.549

Acute Tumor Lysis Syndrome ATLS occurs most frequently in patients with rapidly proliferating neoplasms such as high-grade lymphomas (e.g., Burkitt) and acute leukemias (Fig. 69.12). ATLS may occur spontaneously and be observed at the time of initial diagnosis or in response to the initiation of cytotoxic chemotherapy. Tumor lysis results from the rapid breakdown of malignant cells and the subsequent abrupt release of the cellular contents (intracellular ions, nucleic acids, proteins, etc.) into the extracellular space. Laboratory evidence of tumor lysis is more commonly seen than the clinical manifestations. Laboratory tumor lysis requires at least 2 of the following metabolic abnormalities occurring simultaneously within 3 days prior to or up to 7 days after the

B

FIGURE 69.11. Hyperleukocytosis and leukostasis.  A: An elevated leukocrit is present in this tube of centrifuged peripheral blood from a patient with T cell acute lymphoblastic leukemia who had a peripheral blood blast count of 250 × 109/L. B: Pulmonary alveolar capillaries are expanded by leukocyte aggregates indicative of leukostasis in patient with acute myeloid leukemia (hematoxylin and eosin stain). C: Ring-enhancing lesions on magnetic resonance imaging were attributed to hemorrhage in a patient with chronic granulocytic leukemia, hyperleukocytosis, and blurred vision (TI-weighted image).

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CHAPTER 69  Supportive Care in Hematologic Malignancies Measure: potassium, phosphorous, calcium, uric acid Assess kidney function: creatinine, urine output ≤1 Abnormal value No TLS

≥ 2 Abnormal values Laboratory TLS No symptoms/signs

Assess tumor burden: LDH, Disease type, size of mass, WBC, bone marrow Small or resected

Medium size LDH ≤ 5 x Nl

Clinical TLS Azotemia Hypocalcemia Hyperuricemia Dysrhythmia

FIGURE 69.12. Management of tumor lysis syndrome (TLS).  Serum chemistries and renal function are assessed when instituting therapy in any patient with a hematologic neoplasm. The risk of TLS is based on assessing laboratory values, disease type, and tumor burden. Patients at a high risk for TLS include the acute leukemias with hyperleukocytosis and the aggressive lymphomas with large masses or elevated LDH (lactate dehydrogenase). Volume repletion, agents to lower uric acid, and laboratory monitoring are warranted in all patients. As the risk of TLS increases, the interval of monitoring shortens and the level of care intensifies with cardiac monitoring and the possible requirement for dialysis. (Adapted from Howard SC, Jones DP, Pui C-H. The tumor lysis syndrome. N Eng J Med 2011;364:1844–1854.)

Large Mass LDH ≥ 5 x Nl Hyperleukocytosis Packed marrow

Assess Lysis Risk: Nephropathy Dehydration Acidosis Hypotension Nephrotoxins

Negligible

Low

Hydration Allopurinol Daily lab

Intermediate Hydration Allopurinol/Rasburicase Inpatient monitoring Lab every 8–12 hrs

High Hydration Rasburicase Cardiac monitoring Lab every 6–8 hrs

initiation of therapy: hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia.550,551,552 The clinical tumor lysis syndrome is defined as the above abnormal laboratory findings in combination with acute renal failure, seizures, cardiac dysrhythmias, or death. Hyperuricemia plays a major role in the development of the acute renal failure seen in ATLS. The etiology of the renal failure is presumably from crystal-induced tissue injury that occurs when calcium, uric acid, and xanthine precipitate in the renal tubules and lead to crystal-induced obstruction (Fig. 69.13). Allopurinol is commonly used in the prevention and treatment of

Established TLS Hydration Rasburicase Cardiac monitoring Intensive care Lab every 4–6 hrs Possible dialysis

ATLS. Allopurinol is a competitive inhibitor of xanthine oxidase and prevents the formation of uric acid. Allopurinol is available in both oral and IV preparations. If uric acid levels remain elevated despite allopurinol, rasburicase (recombinant uric acid oxidase) may be administered. Rasburicase converts uric acid to allantoin which is highly water soluble. Randomized data from high-risk pediatric patients receiving rasburicase versus allopurinol demonstrated reduction of uric acid levels by 86% within 4 hours of rasburicase administration versus only a 12% reduction in the allopurinol group (P < 0.0001).553 The FDA approved dosing schedule for rasburicase is 0.2 mg/kg intravenously over

Hematologic Malignancies

TLS Risk

B

A FIGURE 69.13. Tumor lysis:  A: Karyorrhectic lymphoblast nuclei are present throughout the kidney in a patient with acute tumor lysis syndrome in whom cytolysis was initiated by administration of corticosteroids. B: Renal tubules contain calcium phosphate precipitates that contributed to acute lysis-associated renal failure (hematoxylin and eosin stain). (Courtesy of Dr. William R. Macon, Department of Pathology, Mayo Medical Center, Rochester, MN.)

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30 minutes daily for up to 5 days. Multiple reports in the literature suggest that lower fixed dose strategies may be sufficient and more cost-effective.554,555–557 Patients with significant electrolyte imbalances and/or renal insufficiency may require therapeutic dialysis support until resolution of the ATLS. Prophylactic dialysis prior to initiation of chemotherapy should also be considered in high-risk patients such as those with bulky high-grade tumors.

Hypercalcemia Hypercalcemia can occur in association with many various hematologic malignancies but is more commonly seen in patients with either MM or human T cell leukemia virus type-1 (HTLV-1)– associated adult T cell leukemia/lymphoma (ATL). Hypercalcemia in patients with cancer is primarily due to increased bone resorption and release of calcium from bone. The 3 main mechanisms leading to hypercalcemia are increased osteolytic activity, tumor secretion of parathyroid hormone-related protein (PTHrP), and ectopic production of calcitriol (1, 25-dihydroxyvitamin D). The symptoms of hypercalcemia are generally nonspecific and include fatigue, anorexia, nausea, constipation, pain, frequent urination, and altered mental status. Correction of the calcium levels typically leads to rapid and effective palliation of these symptoms.558 MM has an affinity for diffuse bone involvement and releases osteoclast activating factors leading to osteoclast-induced bone resorption that present as lytic lesions which may occur throughout the skeleton.559 Myeloma has also been associated with osteoblast inhibition leading to decreased bone formation.560 This impaired bone formation in the setting of increased destruction disrupts calcium homeostasis and results in the hypercalcemia. Numerous molecules have been implicated in the development of lytic disease and hypercalcemia, including interleukin (IL)-6, the receptor activator of nuclear factor kappaB ligand (RANKL), macrophage inflammatory protein 1a, osteoprotegerin, and IL-3. Calcium homeostasis in the normal host involves conversion of 1-OH vitamin D to calcitriol in the kidney. Calcitriol leads to increased calcium absorption from the gastrointestinal tract as well as increased bone resorption and results in hypercalcemia. Ectopic production of calcitriol has been described in both HL and NHL.561,562 Normal calcium metabolism is mediated by parathyroid hormone. In cases of HTLV-1–associated ATL,563 as well as other hematologic malignancies (AML,564 CML,565,566 HL,567 NHL,568 and MM569), the secretion of PTHrP stimulates osteoclasts, causing increased calcium release from the bone and resulting in a process known as humeral hypercalcemia of malignancy (HHM). Endogenous PTH is typically suppressed secondary to the hypercalcemia, and serum PTH levels will be very low. Unlike PTH, PTHrP does not typically stimulate production of calcitriol570 and thus does not increase intestinal calcium absorption. The uncoupling of bone resorption and formation that occurs in HHM results in the influx of calcium into the circulation and resultant hypercalcemia. Regardless of the mechanism leading to the cancer-related hypercalcemia, successful treatment ultimately requires antineoplastic therapy directed toward the underlying malignancy. The severity of the patient’s symptoms and the prevention of catastrophic consequences will typically require interventions with more rapid impact. Patients with hypercalcemia often experience significant volume depletion as a result of an osmotic diuresis. Thus, aggressive volume repletion with isotonic intravenous fluids (e.g., normal saline) should be initiated upon discovery of the hypercalcemia. Once the patient is appropriately volume replete, loop diuretics may be added to increase calciuresis. Thiazide diuretics increase calcium resorption and should be avoided.

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The effectiveness and availability of bisphosphonates have made them a mainstay of hypercalcemic therapy. These agents inhibit calcium release by disrupting osteoclast-mediated bone resorption.571 Multiple intravenous bisphosphonate choices are available for the treatment of malignancy-associated hypercalcemia: pamidronate, zoledronic acid, and ibandronate. Intravenous administration of the bisphosphonates is generally well tolerated, but patients may experience flu-like symptoms (fever, arthralgias, myalgia, fatigue, and bone pain), uveitis, hypocalcemia, hypophosphatemia, impaired renal function, and osteonecrosis of the jaw.572 Calcitonin results in rapid lowering of serum calcium levels and may be combined with bisphosphonates,573–575 but calcitonin tachyphylaxis is common and limits the benefit of repeated dosing. Cases of renal insufficiency have been reported in association with bisphosphonates, and caution should be employed with their use in the treatment of hypercalcemia in patients with impaired renal function.576 Denosumab is a fully human monoclonal antibody that targets RANKL and inhibits osteoclast maturation, activation, and function, leading to reduced bone resorption. It is approved for the treatment of osteoporosis and prevention of skeletal-related events in at-risk patient populations. Its effectiveness has been reported in the treatment of malignancy-associated hypercalcemia refractory to bisphosphonates.577 Denosumab has also demonstrated less nephrotoxicity than bisphosphonates,578,579 and its use has been reported in the setting of renal insufficiency.580 Common side effects of denosumab include fatigue, headache, hypophosphatemia, hypocalcemia, nausea, weakness, dyspnea, and cough.

Cord Compression Spinal cord impingement is a medical emergency requiring rapid recognition and intervention in the hopes of minimizing complications. It has been frequently reported in patients with MM and is also known to occur in association with HL and NHL. Cord compression as a result of a leukemia diagnosis is rare but has been reported.581–583 Pain is the most common presenting symptom of patients with cord compression and may precede neurologic symptoms by several weeks. Radicular pain, pain not relieved with lying down, and proximal muscle weakness are of particular concern for the possibility of an underlying cord impingement. The development of autonomic symptoms such as urinary retention and fecal incontinence typically occurs late in the course and is predictive of poor outcomes. Patients with known hematologic malignancies that complain of severe back pain and/or any neurologic symptom warrant urgent imaging to rule out cord compression. Emergent referrals for the possibility of surgical intervention should be considered for patients in whom there is a reasonable index of suspicion. Empiric steroids may be appropriate in clinical contexts of concern, or if there are unavoidable delays in being able to obtain imaging. It should be noted, however, that the initiation of steroids may cause rapid necrosis of the tumor and impair the ability to make a tissue diagnosis. Magnetic resonance imaging (MRI) is the imaging modality of choice and should include the entire spine, as multiple areas of compression can occur simultaneously584 (Fig. 69.14). Intervention for cord compression is directed at pain control, avoidance of complications, and the preservation or improvement of neurologic function. Frontline therapy typically consists of urgent initiation of corticosteroids (dexamethasone). The optimal dose of steroids is not known. High-dose dexamethasone (up to 100 mg) has been shown to improve neurologic recovery and ambulatory status,426,585 but carries the risk of serious adverse events such as psychosis and gastrointestinal complications.586,587 In general, it is recommended that patients with suspected or confirmed cord compression be started on dexamethasone at an

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selected References The full reference list for this chapter can be found in the online version.

FIGURE 69.14. Spinal cord involvement shown by magnetic resonance imaging.  Epidural mass on sagittal view with evidence of cord compression and cord impingement at &7 level (T1-weighted image). The diagnosis by biopsy was multiple myeloma.

initial dose of 10 to 100 mg followed by at least 16 mg daily (in divided doses).588 Following the initiation of steroids and confirmation of cord impingement, definitive treatment with surgical intervention, radiation therapy, and/or chemotherapy should be initiated.588,589 Surgery should be considered in those who are medically and surgically appropriate and/or those in whom a tissue diagnosis has not yet been established. Radiation therapy should be administered to those who are not candidates for surgical intervention.

Summary This chapter has addressed selected aspects of supportive care in hematologic malignancies. The improved prognosis for leukemias, lymphomas, and myeloma has been attributed to better drugs, including targeted therapy and immunotherapy; however, progress could not have occurred without the concomitant advances in supportive care, particularly in the management of infections and in transfusions. The goals of supportive care are to prevent and reduce the toxicities of therapy and the complications of the diseases. There is an emphasis on survivorship, including rehabilitation after therapy and recognition of the long-term risks of therapy. When therapeutic options no longer offer survival benefit, access to palliative care and end-of-life issues take precedence over other components of supportive care.

Resources for Supportive Care • Multinational Association of Supportive Care in Cancer: www. mascc.org • National Institutes of Health: www.cancer.gov/cancertopics/ coping • ASCO Supportive Care and Quality of Life • NCCN Guidelines for Supportive Care

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Aapro MS, Bohlius J, Cameron DA, et al. 2010 update of EORTC guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapy-induced febrile neutropenia in adult patients with lymphoproliferative disorders and solid tumours. Eur J Cancer 2011;47:8–32. 50. Legrand M, Max A, Peigne V, et al. Survival in neutropenic patients with severe sepsis or septic shock. Crit Care Med 2012;40:43–49. 51. Hamalainen S, Kuittinen T, Matinlauri I, et al. Neutropenic fever and severe sepsis in adult acute myeloid leukemia (AML) patients receiving intensive chemotherapy: causes and consequences. Leuk Lymphoma 2008;49:495–501. 55. Mermel LA, Allon M, Bouza E, et al. Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 2009;49:1–45. 57. Chang FY, Peacock JE Jr, Musher DM, et al. Staphylococcus aureus bacteremia: recurrence and the impact of antibiotic treatment in a prospective multicenter study. Medicine (Baltimore) 2003;82:333–339. 60. Rybak M, Lomaestro B, Rotschafer JC, et al. Therapeutic monitoring of vancomycin in adult patients: a consensus review of the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, and the Society of Infectious Diseases Pharmacists. Am J Health Syst Pharm 2009;66:82–98. 70. Guidelines for the management of adults with hospital-acquired, ventilatorassociated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005;171:388–416. 84. Mera RM, Miller LA, Amrine-Madsen H, et al. Acinetobacter baumannii 2002–2008: increase of carbapenem-associated multiclass resistance in the United States. Microb Drug Resist 2010;16:209–215. 91. Kuse ER, Chetchotisakd P, da Cunha CA, et al. 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Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2010;50:291–322. 120. Carson KR, Evens AM, Richey EA, et al. Progressive multifocal leukoencephalopathy after rituximab therapy in HIV-negative patients: a report of 57 cases from the research on adverse drug events and reports project. Blood 2009;113:4834–4840. 131. Mandell LA, Wunderink RG, Anzueto A, et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 2007;44(Suppl 2):S27–S72. 142. Cornely OA, Maertens J, Winston DJ, et al. Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia. N Engl J Med 2007;356:348–359. 145. Wheat LJ, Freifeld AG, Kleiman MB, et al. Clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the Infectious Diseases Society of America. Clin Infect Dis 2007;45:807–825.

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146. Blair JE, Smilack JD, Caples SM. Coccidioidomycosis in patients with hematologic malignancies. Arch Intern Med 2005;165:113–117. 154. Small TN, Casson A, Malak SF, et al. Respiratory syncytial virus infection following hematopoietic stem cell transplantation. Bone Marrow Transplant 2002;29:321–327. 157. Harper SA, Bradley JS, Englund JA, et al. Seasonal influenza in adults and children--diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 2009;48:1003–1032. 165. Cohen SH, Gerding DN, Johnson S, et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol 2010;31:431–455. 166. Zar FA, Bakkanagari SR, Moorthi KM, et al. A comparison of vancomycin and metronidazole for the treatment of Clostridium difficile-associated diarrhea, stratified by disease severity. Clin Infect Dis 2007;45:302–307. 169. Louie TJ, Miller MA, Mullane KM, et al. Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med 2011;364:422–431. 171. Ison MG. Adenovirus infections in transplant recipients. Clin Infect Dis 2006;43:331–339. 172. Jancel T, Penzak SR. Antiviral therapy in patients with hematologic malignancies, transplantation, and aplastic anemia. Semin Hematol 2009;46:230–247. 182. Tomblyn M, Chiller T, Einsele H, et al. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol Blood Marrow Transplant 2009;15:1143–1238. 211. Robenshtok E, Gafter-Gvili A, Goldberg E, et al. Antifungal prophylaxis in cancer patients after chemotherapy or hematopoietic stem-cell transplantation: systematic review and meta-analysis. J Clin Oncol 2007;25:5471–5489. 213. De Castro N, Neuville S, Sarfati C, et al. Occurrence of Pneumocystis jiroveci pneumonia after allogeneic stem cell transplantation: a 6-year retrospective study. Bone Marrow Transplant 2005;36:879–883. 220. Gardner A, Mattiuzzi G, Faderl S, et al. Randomized comparison of cooked and noncooked diets in patients undergoing remission induction therapy for acute myeloid leukemia. J Clin Oncol 2008;26:5684–5688. 226. Perlroth J, Choi B, Spellberg B. Nosocomial fungal infections: epidemiology, diagnosis, and treatment. Med Mycol 2007;45:321–346. 237. Imran H, Tleyjeh IM, Arndt CA, et al. Fluoroquinolone prophylaxis in patients with neutropenia: a meta-analysis of randomized placebo-controlled trials. Eur J Clin Microbiol Infect Dis 2008;27:53–63. 244. Kontoyiannis DP, Marr KA, Park BJ, et al. 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Should immunoglobulin therapy be used in allogeneic stem-cell transplantation? A randomized, doubleblind, dose effect, placebo-controlled, multicenter trial. Ann Intern Med 2003;139:8–18. 322. O’Grady NP, Alexander M, Burns LA, et al. Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis 2011;52:e162–e193. 331. Baskin JL, Pui CH, Reiss U, et al. Management of occlusion and thrombosis associated with long-term indwelling central venous catheters. Lancet 2009;374:159–169. 333. Guyatt GH, Akl EA, Crowther M, et al. Executive summary: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141:7S–47S. 343. Roila F, Herrstedt J, Aapro M, et al. Guideline update for MASCC and ESMO in the prevention of chemotherapy- and radiotherapy-induced nausea and vomiting: results of the Perugia Consensus Conference. Ann Oncol 2010;21(Suppl 5):v232–v243. 344. Saito M, Aogi K, Sekine I, et al. Palonosetron plus dexamethasone versus granisetron plus dexamethasone for prevention of nausea and vomiting during chemotherapy: a double-blind, double-dummy, randomised, comparative phase III trial. Lancet Oncol 2009;10:115–124. 359. Navari RM. Prevention of emesis from multiple-day and high-dose chemotherapy regimens. J Natl Compr Canc Netw 2007;5:51–59. 367. Jordan K, Kinitz I, Voigt W, et al. Safety and efficacy of a triple antiemetic combination with the NK-1 antagonist aprepitant in highly and moderately emetogenic multiple-day chemotherapy. Eur J Cancer 2009;45:1184–1187. 370. Basch E, Hesketh PJ, Kris MG, et al. Antiemetics: American Society of Clinical Oncology clinical practice guideline update. J Oncol Pract 2011;7:395–398. 374. Escalante CP, Manzullo EF. Cancer-related fatigue: the approach and treatment. J Gen Intern Med 2009;24(Suppl 2):S412–S416.

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377. Jean-Pierre P, Morrow GR, Roscoe JA, et al. A phase 3 randomized, placebocontrolled, double-blind, clinical trial of the effect of modafinil on cancerrelated fatigue among 631 patients receiving chemotherapy: a University of Rochester Cancer Center Community Clinical Oncology Program Research Base Study. Cancer 2010;116:3513–3520. 389. Jacobsen R, Liubarskiene Z, Moldrup C, et al. Barriers to cancer pain management: a review of empirical research. Medicina (Kaunas) 2009;45:427–433. 398. Caraceni A. Evaluation and assessment of cancer pain and cancer pain treatment. Acta Anaesthesiol Scand 2001;45:1067–1075. 405. Jadad AR, Browman GP. The WHO analgesic ladder for cancer pain management. Stepping up the quality of its evaluation. JAMA 1995;274:1870–1873. 406. Cheung WY, Zimmermann C. Pharmacologic management of cancer-related pain, dyspnea, and nausea. Semin Oncol 2011;38:450–459. 410. Trescot AM. Review of the role of opioids in cancer pain. 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Chapter 70

Immunotherapy

History There probably is no field in medicine that has provided as much hope, or as much disappointment, as the field of tumor immunology. A major relationship between the immune system and the oversight of neoplasms was postulated in the early part of the last century by Paul Ehrlich.1 This theory of immunosurveillance envisioned that, in long-lived animals, inheritable genetic changes in somatic cells must be common, and some proportion of these changes must represent steps toward malignant transformation. It was considered an evolutionary necessity, therefore, that some mechanisms exist for eliminating or inactivating such potentially dangerous mutant cells. This mechanism was thought to be immunologic. The theory of immunosurveillance was restated in the 1950s by Lewis Thomas, then popularized and championed by Sir Macfarlane Burnet.2 Supported by these powerful figures in medicine, the theory of immunosurveillance was so inherently appealing that it often was accepted uncritically, and evidence to the contrary often overlooked.3 For instance, although patients or animals who are immunosuppressed tend to have an increased incidence of tumors, these tumors are disproportionately of lymphoid origin or associated with an oncogenic virus. The development of common epithelial neoplasms (with the exception of certain skin cancers) in these patients occurs with much less impressive frequency.4 The most obvious evolutionary necessity of the immune system was to survey a variety of infections, especially viral infections. Early evidence seemed to indicate that immunity played a significant role in eradicating virally induced tumors.4,5 On the other hand, it appeared to play a less significant, or less effective, role in prevention of tumors induced by physical or chemical carcinogens.6,7 Experimentation in the early part of the 20th century demonstrated that spontaneously arising tumors in outbred animals could occasionally be transplanted from one animal to another of the same species and propagated in that fashion. Attempts to immunize against transplantable tumors soon followed. Animals injected with a small number of tumor cells often were able to eliminate those tumor cells—that is, there appeared to be a threshold number of tumor cells required for tumor propagation. Animals that had eliminated a sublethal inoculum of tumor cells were often able to withstand inoculation with a large number of tumor cells that would have been lethal in a naive animal. Furthermore, preexposure to normal tissue of the donor often rendered the recipient resistant to challenge with a lethal number of tumor cells.8 These experiments brought into question the idea of tumor-specific antigens and ultimately led to the discovery of major histocompatibility complex (MHC) genes and their products.9,10 Modern tumor immunology finds its roots in the classic experiments of Prehn and Main.11 These investigators demonstrated, in genetically identical mice, that previous exposure to a chemically induced sarcoma rendered animals resistant to challenge with the same tumor, but that these animals would accept normal, nonneoplastic tissues transplanted from the tumor donor animal. Similarly, prior exposure to normal tissues from the donor animal did not render the recipient animal resistant to tumor challenge. These experiments revived the notion that tumorspecific (transplantation) antigens did exist. Subsequent experiments demonstrated that protection afforded by prior exposure to tumor cells was tumor specific.12 Thus, the host response to transplanted tumors behaved like an adaptive immune response, demonstrating memory and specificity.

Tumor immunity could be passively conveyed from one animal to another by transfer of lymphoid cells.13 The relevant cells for protection were shown to be T lymphocytes.14 Thus, it should have been clear to workers in the field that the relevant tumor antigens were those that could be recognized by T lymphocytes. However, as this work was beginning there was little understanding of how T lymphocytes recognized antigens or how those antigens were processed and presented to the T lymphocyte by antigen-presenting cells (APCs) or the tumor target cells. Much time and effort were expended in search of membrane structures or tumor cell products that would distinguish the tumor from all others. Particularly after the description of monoclonal antibody technology,15 a fervent search was undertaken to define structures on tumor cells that would be tumor specific and potential targets for therapeutic intervention. Although many cell surface structures were defined, and the contribution to the understanding of biology cannot be overstated, this adventure produced only a single truly specific tumor antigen, the idiotype (Id) of clonally distributed immunoglobulin present on certain lymphomas. Only recently has convincing evidence for an effect of immunosurveillance been produced.16,17,18 This new evidence relies, in great measure, on the availability of genetically manipulated animal systems. A variety of knockout mice with defects in components of immune activation or effector function develop, at high frequency, spontaneous tumors or tumors after carcinogen exposure. Interferon-g receptor–deficient mice are more likely to develop methylcholanthrene-induced sarcomas and are more susceptible to spontaneous development of sarcomas and lymphomas after loss of p53 alleles.19,20 One in two aged perforin-deficient mice develops disseminated lymphomas.21 These tumors are rejected by histocompatible wild-type mice through a mechanism dependent on CD8+ T lymphocytes. A high incidence of lymphoma is also seen in aged mice deficient in Fas/Fas ligand interactions.22 Aged mice doubly deficient in signal transducer and activation of transcription (Stat) 1 and recombination activating gene (Rag) 2 develop adenocarcinomas (colon, breast, and lung) with high frequency.23 The frequency and distribution of tumors are increased in the doubly deficient mice over the frequencies and distributions seen in singly deficient mice. Many of these observations have been interpreted as evidence that the primary (both first and predominant) mechanism underlying tumor immunosurveillance is the system of innate immunity.16,17,18

Hematologic Malignancies

Adetola A. Kassim, Sattva S. Neelapu, Larry W. Kwak, Luc Van Kaer

Innate Immunity Against Tumors The innate immune system is a widespread and evolutionarily ancient form of host defense against infection. In recent years, there has been an explosion of information regarding innate immunity, including its role in host defense and its regulation of inflammation and adaptive immunity.24,25,26 The innate immune system is made up of many cells. These include dendritic cells (DCs), macrophages, mast cells, neutrophils, eosinophils, natural killer (NK) cells, natural killer T (NKT) cells, and certain subsets of gd T cells. Each of these cell types has been implicated in immune responses against tumors.

Phagocytic Cells Many cells of the innate immune system, including neutrophils, macrophages, and DCs, bear receptors that detect “danger”27

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in the form of pathogen-associated molecular patterns (PAMPs). Examples of PAMPs include bacterial lipopolysaccharide, lipoprotein, peptidoglycan, and lipoteichoic acids; bacterial CpG DNA; and viral RNA and DNA. These PAMPs are recognized by a variety of pattern recognition receptors (PRRs) expressed by cells of the innate immune system.26,28 The innate immune system is said to distinguish “infectious nonself” from “noninfectious self.” The PRRs of the innate immune system are encoded in the germline. Unlike genes of the T cell antigen receptor and the immunoglobulins, these genes do not undergo rearrangement. They are fixed and detect critical microbial components. Engagement of PRRs with PAMPs can result in pathogen uptake and/or cellular activation.26,28 One important group of PRRs is the family of evolutionary conserved Toll-like receptors (TLRs), which are critically important for innate immune cell activation. Thirteen TLRs, each with specificity for a different PAMP, have been described in mammals. Other PRR families include the nucleotide-binding domain leucine-rich repeats (NLR) receptors and the caspase recruitment domain helicases. Neutrophils and macrophages typically exert little antitumor activity, unless these cells are activated by bacteria, their products, or cytokines produced by tumor-specific T cells.29,30 Recent studies have suggested that dying tumor cells or damaged tissues can release damage-associated molecular patterns (DAMPs) that can interact with PRRs and thus serve as danger signals.31 NLR receptors such as NLRP3 (NLR family pyrin domain-containing 3), which form large cytoplasmic signaling complexes called inflammasomes, are thought to play a critical role in detecting DAMPs and might regulate the development of tumors either positively or negatively.32–34 Macrophages can kill tumor cells using the same mechanisms utilized for killing of microorganisms. These mechanisms include phagocytosis and release of cytotoxic molecules such as reactive oxygen intermediates and nitric oxide.35 Activated macrophages also produce a variety of cytokines. Among these cytokines, tumor necrosis factor (TNF)-a plays a major role in the tumoricidal effects of macrophages in vitro.36 Another important role of phagocytes in tumor immunity is to present tumor antigens to T lymphocytes. DCs (and other APCs such as macrophages) can phagocytose tumor cells and present tumor antigens in the context of MHC molecules and costimulatory signals to T lymphocytes.37

Natural Killer Cells It has been recognized for a long time that NK cells kill MHC class I–deficient tumor cells in vivo and in vitro.38 However, the identity and characterization of receptors mediating NK activation proved elusive for many years. Activation of NK cells now is understood to be dependent on the balance of activating and inhibitory signals emanating from activating and inhibitory receptors on the NK cell surface.39,40 These receptors fall into two major structural classes, those of the immunoglobulin superfamily (KIRs and LIRs) and those of the C-type lectin-like family (NKG2D, CD94/NKG2A, and Ly49). Most inhibitory receptors (e.g., CD94/NKG2A, KIR, and Ly49) recognize classic or nonclassic MHC molecules. An activating receptor on NK cells, NKG2D (also called KLRK1), has now been shown to recognize a variety of stress-induced MHC class I–like molecules (e.g., Rae-1, H60, and MICA/B). Of note, activated CD8+ T cells and mucosal gd T cells also express NKG2D. Another activation receptor on NK cells is FcgRIII, which can target NK cells to immunoglobulin G (IgG) antibody-coated tumor cells and induce antibody-dependent cell-mediated cytotoxicity. NK cells can also discriminate between different allelic variants of MHC molecules.41 This phenomenon was originally identified in the context of the hybrid resistance transplant model in mice, where parental bone marrow grafts were rejected by a subset of

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host F1 NK cells. When faced with mismatched allogeneic targets, a subset of donor NK cells can sense the missing expression of self–human leukocyte antigen (HLA) class I alleles and mediate alloreactions. These alloreactive NK cells can improve engraftment and control the relapse of acute myeloid leukemia (AML) in mismatched hematopoietic transplants.41,42

Natural Killer T Cells NKT cells are a subset of T lymphocytes that share receptor structures and functions with the NK cell lineage.43 Prototypical NKT cells, often referred to as invariant (i)NKT cells, express a semi-invariant T cell receptor (TCR), which is specific for glycolipid antigens presented by the MHC class I–like protein CD1d. Although NKT cells express an antigen-specific receptor that is generated by somatic DNA rearrangement, these cells belong to the innate rather than the adaptive arm of the immune system.44 The invariant TCR expressed by NKT cells recognizes a limited set of self- and foreign antigens and, therefore, bears similarity to the PRRs expressed by cells of the innate immune system. Further, NKT cells have a natural, activated phenotype and are unable to generate classic memory responses against their cognate glycolipid antigens. Mice that are deficient in NKT cells have increased susceptibility to MCA-induced sarcomas, indicating that these cells contribute to natural immunity against tumors.45 NKT cells in mice and humans respond to the marine sponge–derived glycosphingolipid a-galactosylceramide (a-GalCer), which has potent antimetastatic activities in mice.45 a-GalCer and related NKT cell antigens are being explored as potential cancer immunotherapies.46

Mucosal gd T Cells T cells expressing the gd TCR are enriched in mucosal tissues such as the mucosa of the gut and skin.47 These mucosal gd T cells have a highly restricted TCR repertoire, suggesting specificity for a limited set of antigens selectively expressed in their respective epithelial compartments. Like NKT cells, mucosal gd T cells can be classified as being at the interface between innate and adaptive immunity.44 Epidermal and intestinal gd T cells express NKG2D and become activated when NKG2D binds to stress-induced MHC class I–related molecules. gd T cells play a crucial role in immune surveillance against malignant epidermal cells. The incidence of cutaneous malignancies after treatment with a combination of initiator and promoter carcinogens was substantially increased in mice lacking the TCR d-chain.48 Activation of gd T cells required both NKG2D and the gd TCR, suggesting that engagement of NKG2D with its ligand(s) synergizes with signals received through the autoreactive gd TCR.

Adaptive Immunity Against Tumors The adaptive immune system is composed of B and T cells that express diverse antigen-specific receptors, immunoglobulins, and TCRs, respectively. Diversity of these receptors is generated by somatic DNA rearrangement, in a process referred to as VDJ recombination.49,50 Currently, there is little evidence that adaptive immunity plays a major role in natural immunity against tumors (with the exception of tumors induced by viruses). Although mice lacking T lymphocytes have increased susceptibility to the development of MCA-induced sarcomas, this might be largely due to the lack of iNKT cells and/or gd T cells.18,51 Nevertheless, it is clear that tumors can induce adaptive immune responses (Fig. 70.1), which can be exploited for the development of cancer immunotherapies.

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FIGURE 70.1. Immune responses against tumors.  A variety of cells and soluble factors of innate and adaptive immunity can participate in immune responses against tumors. Examples of mechanisms that can suppress immune responses against tumors (i.e., production of TGF-b by tumor cells and induction of Treg cells) are also depicted. Ab, Antibody; FcR, Fc receptor; IFN, interferon; MDSC, myeloid-derived suppressor cell; MHC I, major histocompatibility complex class I; MHC II, major histocompatibility complex class II; NK, natural killer; NKT, natural killer T; PRR, pattern recognition receptor; TAA, tumor-associated antigen; TCR, T cell receptor; TGF, transforming growth factor; TNF, tumor necrosis factor; Treg, regulatory T cell.

Antibodies and B Cells The role of B cells in regulating tumor immunity remains poorly understood. In some tumor models, B cells appear to be important for priming of T cell responses and tumor resistance, whereas in other models, B cells have an inhibitory effect on the generation of cytotoxic T lymphocyte (CTL) responses and tumor rejection.52 Nevertheless, it is clear that tumor-bearing hosts can produce antibodies against a variety of tumor antigens.53 However, strong humoral responses rarely correlate with tumor resistance. Nevertheless, antibodies can be utilized for immunotherapy of cancer, in particular tumors of hematopoietic origin. Antibodies may kill tumor cells by activating complement and promoting phagocytosis by macrophages. Alternatively, antibody-coated tumor cells may be killed by antibody-dependent cell-mediated cytotoxicity, in which Fc receptor–bearing NK cells, macrophages, or neutrophils mediate the killing. In addition, in certain cases, antibodies may directly interfere with the growth of tumor cells, as illustrated by the beneficial effects of anti–HER-2/neu antibodies against breast cancer, which likely involves downregulation of the HER-2/neu growth factor receptor.54

T Lymphocytes Classic studies with transplantable tumors have demonstrated a critical role of T lymphocytes in tumor immunity.11 CTLs play a particularly important role in tumor rejection, as these cells can directly lyse malignant cells that display tumor antigens in association with MHC class I molecules.55,56 The importance of CD4+ T cells in tumor immunity is less clear. CD4+ T cells may secrete cytokines that promote the development of CD8+ T cell responses, increase the sensitivity of tumor targets to CTL lysis by inducing MHC class I expression, and activate macrophages. Because of their critical role for the development of tumor immunotherapies, we will briefly describe the mechanisms that lead to the induction of T cell responses to tumors.

Antigen Processing and Presentation T lymphocytes recognize peptide antigens in the context of MHC molecules. These peptides are derived from two distinct

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pathways.57 Peptides representing proteins sampled from the extracellular world are generally presented in the context of class II MHC proteins, whereas peptides resulting from intracellular synthesis of proteins are presented in the peptide groove of the class I MHC proteins (Fig. 70.2). The binding cleft of MHC molecules has a b-pleated sheet floor and a-helical sides. An immunogenic peptide must be capable of forming noncovalent attachments to key residues along the cleft and interacting with the T cell antigen receptor with other residues. The MHC contact residues of the peptide tend to be near the amino and carboxy terminal ends of the peptide. The cleft of the class I MHC molecule has closed ends and accommodates only a peptide of proper length, 9 to 11 amino acids. The cleft of the class II MHC molecule, on the other hand, is open-ended and can bind peptides of more diverse lengths, 10 to 30 amino acids, with most being 12 to 19.58 Peptides located in class II MHC molecules are derived from proteins that have been consumed by APCs59 (Fig. 70.2A). The proteins are taken up by phagocytosis, or receptor-mediated endocytosis in clathrin-coated pits, or engulfed by pinocytosis. Once internalized, the antigens are located in membrane-bound vesicles called endosomes. The endosomes then become continuous with lysosomes. The enzymology of the endolysosome has been described in some detail.59,60 There, in an acidic environment, disulfide bonds in proteins are first reduced by the enzyme GILT (gamma interferon-inducible lysosomal thiol reductase), and the resulting products are subsequently cleaved to peptides by proteases, predominantly cathepsins. The endosome fuses with an exocytic vesicle budding from the Golgi apparatus that contains newly made class II MHC molecules associated with invariant chain, which has been shown to play a critical role in the assembly, intracellular transport, and function of MHC class II molecules.61 In addition, a chaperone, HLA-DM, plays a critical role in the loading of peptides onto MHC class II molecules. HLA-DM is a peptide exchange factor that binds with empty and peptide-loaded class II molecules in endosomal and lysosomal compartments.61 In the fused vesicle, peptides are loaded into the class II MHC molecules. Fusion of the endosome with the plasma membrane ultimately displays the class II MHC molecule–peptide complexes on the cell surface.

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FIGURE 70.2. Presentation of peptides by major histocompatibility complex (MHC) molecules.  A: MHC class II–restricted antigen processing and p­ resentation to CD4 T cells. Exogenous protein antigens are taken up by antigen-presenting cells, disulfide bonds are reduced by the interferon (IFN)-g-inducible lysosomal thiol reductase (GILT), and the proteins are then degraded in endosomal/lysosomal compartments by cathepsins (Cat). MHC class II a and b are synthesized in the endoplasmic reticulum (ER) and associate there with the MHC class II–associated invariant chain (Ii). The class II/Ii complexes then egress to endosomal compartments, where Ii is degraded by cathepsins, until only its class II–associated invariant chain (CLIP) region remains bound by class II. CLIP is then removed from class II by the human leukocyte antigen (HLA)-DM peptide exchange factor. Finally, class II is loaded with peptide and delivered to the cell surface for presentation to class II–restricted CD4 T cells. B: MHC class I–restricted antigen processing and presentation to CD8 T cells. Cytosolic proteins, derived from endogenously synthesized proteins or from cross-presented antigens (as indicated by the arrow), are degraded by immunoproteasomes that contain the interferon IFN-g–inducible subunits LMP2 (2), LMP7 (7), and MECL-1 (M). Some of the resulting peptides are further processed in the cytoplasm by peptidases and then transported to the lumen of the endoplasmic reticulum (ER) by the transporter of antigen processing (TAP). Some of the peptides undergo further processing in the ER by ER-associated aminopeptidases (ERAP). Peptide-receptive MHC class I heavy chain (HC)/b2-microglobulin (b2m) heterodimers in the ER associate with a variety of chaperones, including calreticulin (crt), ERp57, and tapasin (tpn). After binding with peptides, class I molecules undergo a conformational change, permitting their egress to the cell surface for presentation to class I–restricted CD8 T cells.

Peptides are prepared for presentation on class I molecules in a different fashion (Fig. 70.2B). These peptides are derived from intracellular protein synthesis.62,63 After protein synthesis, proteins introduced into the cytoplasm become the target of the proteasome, a cytoplasmic organelle whose major function is the degradation of proteins tagged for turnover by the addition of ubiquitin.64,65 During conditions of interferon-g production such as infection, several proteasome subunits (LMP2, LMP7, and MECL-1) become upregulated and are incorporated into newly assembled proteasomes. Proteasomes that include these IFN-g–inducible subunits are referred to as immunoproteasomes, whereas those that lack these subunits are called constitutive proteasomes. The IFN-g–inducible proteasome subunits favor the generation of peptides that have increased affinity for MHC class I molecules.66–68 In addition to the proteasome, several cytoplasmic peptidases (e.g., TPPII, LAP, TOP) have been implicated in the generation of antigenic peptides, although they can cleave some epitopes as well.65,69 These peptides are then transported into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) proteins.62 Within the ER, peptides may be further trimmed by ER-resident aminopeptidases (i.e., ERAP1 and ERAP2).65,69,70 Assembly of class I MHC heavy chain molecules with b2-microglobulin requires the presence of peptides. Within the ER, empty MHC class I molecules are associated with a variety of chaperones, including calnexin, calreticulin, ERp57, and tapasin. Tapasin is a transmembrane protein that tethers empty class I molecules in the ER to TAP.71 Emerging evidence suggests that tapasin retains unstable MHC class I molecules within peptide-loading compartments until they bind with high-affinity peptides. The assembled MHC class I–peptide

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complex transits the Golgi apparatus, proceeds in a vesicle to the cell surface, and is displayed on the cell surface after fusion of the vesicle membrane with the plasma membrane. There has been a lot of interest in the mechanisms whereby tumor cells initiate CD8+ T cell responses. Few tumors are derived from professional APCs and, therefore, do not effectively prime naive CD8+ T cells. It has now been established that tumor cells can be processed and presented by host APCs, particularly DCs, in a process that is referred to as cross-presentation (Fig. 70.2).72,73 Tumor antigens are then processed inside the APC, and peptides derived from these antigens are displayed on MHC class I molecules for recognition by CD8+ T cells. These APCs also express MHC class II molecules and can prime naive CD4+ T cells, which may be important for the generation of effective CD8+ memory responses. Once tumor antigen-specific CTLs are generated, they can kill tumor cells without the requirement for costimulation. While the precise mechanisms of cross-presentation remain poorly understood,74 the concept of cross-presentation has important applications in the development of tumor vaccines.72,75

T lymphocyte Activation The goal of antigen processing and presentation is the activation of appropriate T lymphocytes to proliferate, produce cytokines, and promote an immunologic reaction or become cytotoxic cells. Although the interaction of the T cell antigen receptor with antigen-MHC provides specificity of response and initiates the crucial events of activation, the interactions are few and have low affinity.76 The interaction between T lymphocytes and APCs or target cells is initially stabilized by a number of nonspecific

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Chapter 70  Immunotherapy

activation motifs (ITAMs) that are targets for intracellular protein tyrosine kinases that catalyze the phosphorylation of tyrosine residues in various protein substrates. The tyrosine kinase lck interacts with the cytoplasmic domains of CD4 and CD8 and the tyrosine kinase fyn interacts with the TCR–CD3 complex. Binding of the TCR with peptide/MHC complexes results in receptor clustering, bringing CD4/CD8 and lck in close proximity of the ITAMs within the CD3 and z-chains. Lck and fyn subsequently phosphorylate tyrosine residues within the ITAMs, which become docking sites for the z-associated protein, ZAP-70, a member of the syk family of protein tyrosine kinases. The bound ZAP-70 then becomes a substrate for lck, and phosphorylation of ZAP-70 results in its activation. Activated ZAP-70 phosphorylates several scaffolding proteins, including LAT (linker of activated T cells) and SLP-76, which, when phosphorylated, serve as docking sites for other proteins that, in turn, activate multiple signaling pathways. One of these signaling pathways involves changes in inositol lipid metabolism. Phospholipase Cg1 (PLCg1) becomes tyrosine

Hematologic Malignancies

receptor–counterreceptor interactions, leading to development of an immunologic synapse with its central supramolecular activation cluster.77,78,79 Chief among these interactions is the coupling of CD2 on the T lymphocyte with lymphocyte function antigen-3 on the APC. Also involved is the interaction of the lymphocyte function antigen-1 molecule with intercellular adhesion molecule-1 and intercellular adhesion molecule-2. Once the cells have been apposed, the specific interaction of the T cell antigen receptor and the antigen-MHC can occur. It now appears that the T cell proceeds toward activation only if certain threshold numbers of TCR-MHC/antigen interactions occur.80 The signal transduction pathways that result in T cell activation have been extensively reviewed,81–83,84 and we will focus here on the most salient features (Fig. 70.3). Ligation of the TCR with an agonist peptide/MHC complex results in phosphorylation of the cytoplasmic portions of the CD3 and z components of the TCR. The cytoplasmic domains of CD3 and z contain several conserved peptide sequences called immunoreceptor tyrosine-based

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γ ζ

θ κ

κ κ

κ

κ

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FIGURE 70.3. Overview of signal transduction events involved in T lymphocyte activation.  Interaction of the T cell receptor (TCR) and coreceptors with major histocompatability complex (MHC)–peptide complexes on antigen-presenting cells (APCs) results in multiple signaling events that lead to the activation of several transcription factors that stimulate expression of numerous genes (e.g., the interleukin-2 [IL-2] gene). Note that the precise interactions between different adaptor proteins that participate in proximal TCR signaling events remain incompletely understood. AP, activated protein; DAG, diacylglycerol; Elk, Ets-like transcription factor; Erk, extracellular signal-regulated kinase; Grb, growth factor receptor-bound protein; IkB, inhibitor of kB; IP3, inositol triphosphate; Itk, interleukin-2-inducible tyrosine kinase; Jnk, Jun N-terminal kinase; Jnkk, Jnk kinase; LAT, linker of activated T cells; Lck, lymphocyte-specific protein tyrosine kinase; Mek, Mapk/Erk kinase; Mekk, Mapk/Erk kinase; NF, nuclear factor; NFAT, nuclear factor of activated T cells; PIP2, phosphatidylinositol biphosphate; PKC, protein kinase; PLC, phospholipase C; RasGRP, Ras guanyl nucleotide-releasing protein; SLP-76, SH2 domaincontaining leukocyte protein, 76-kD; Sos, son of sevenless; ZAP-70, z-associated protein kinase, 70-kD.

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phosphorylated and activated as it associates with LAT. Activation of PLCg1 leads to the hydrolysis of a minor membrane lipid, phosphatidylinositol biphosphate (PIP2), to yield inositol triphosphate (IP3) and diacylglycerol (DAG). Each of these products activates downstream events. IP3 induces a rapid increase of free Ca2+ by release from membrane-sequestered Ca2+ stores, whereas DAG and Ca2+ activate protein kinase C (PKC) u. T cell activation also results in the activation of the ras and rac signaling pathways. Adapter proteins that are activated by phosphorylated LAT and SLP-76 result in the activation of the guanine nucleotide exchange factors SOS (son of sevenless) and vav, which activate the ras and rac signaling pathways, respectively. These signaling events ultimately result in the activation of a number of transcription factors, including NFAT, NF-k B, and AP-1. Cytosolic Ca2+ binds with the Ca2+-dependent protein calmodulin, and Ca2+-calmodulin complexes subsequently activate several enzymes, including the serine/threonine phosphatase calcineurin. Calcineurin then dephosphorylates NFAT, which uncovers a nuclear localization signal that permits NFAT to translocate to the nucleus. Activation of NF-kB is dependent, at least in part, on activated PKCu. NF-kB is normally found in the cytoplasm in association with a protein called inhibitor of kB (IkB). TCR signals result in phosphorylation of IkB, which is then targeted for degradation by the proteasome. Release of IkB uncovers a nuclear translocation signal in NF-kB that permits its translocation to the nucleus. AP-1 is a transcription factor composed of the proteins Fos and Jun, which are activated by the ras and rac signaling pathways, respectively. The net result of this extremely complex activation system is the expression of new proteins, the acquisition of functional capacity, or the ability to proliferate. T lymphocyte activation is best understood as a culmination of events leading to IL-2 production.85 The constraints on production of this cytokine are more rigorous than those relevant for production of other gene products (such as IL-2 receptor a-chain and transcription factors). The promoter of the IL-2 gene is made up of a number of binding sites for transcription factors, including two NFAT sites, an NF-kB site, and an AP-1 site.86 The combination of production of IL-2 receptor a-chain and IL-2 provides an adequate stimulus for the T lymphocyte to successfully proliferate, giving rise to the antigen-specific clonal expansion of lymphocytes characteristic of immunologic responses. However, there are extraordinary controls against inappropriate activation of T lymphocytes.87 In addition to a first signal delivered via the T cell antigen receptor complex, full activation of T cells also requires a second signal.88 The best characterized origin of these second signals is the interaction of CD28 on the T lymphocyte surface with its cognate ligands CD80 (B7–1) and CD86 (B7–2) on the APC, the most potent of which is the DC. Failure to receive a second signal can lead the T lymphocyte to undergo anergy or apoptosis. Activation of T cells is also regulated by a variety of negative signals, including inhibitory receptors of the CD28 family such as CTLA-4 (cytotoxic T lymphocyte-associated protein 4), which interacts with CD80 and CD86, and PD-1 (programmed death-1), which interacts with PD-L1 and PD-L2.88

Tumor-Associated Antigens Tumor antigens are like all other antigens of adaptive immunity. That is, with few exceptions, they are peptides that are presented to T lymphocytes in the cleft of an MHC-encoded protein.55 The nature of peptide antigens responsible for immune responses to tumors has been described in a classic set of experiments.55 In essence, two approaches were used. Neither made assumptions regarding the nature of the antigenic peptides. In the first approach, tumor-derived cloned CTLs were established. Next, a library of tumor complementary DNA or genomic DNA was

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constructed and used to transfect cells expressing appropriate MHC molecules but lacking the tumor-specific epitope. Transfected cells were tested for their ability to activate the tumor-specific CTLs. The transfected DNA was then recovered and sequenced, thus identifying the gene of origin. In the second approach, MHC molecules were isolated from tumor cells. Subsequently, peptides were eluted from the MHC molecules and fractionated chromatographically. These peptide fractions were then used to load APCs and presented to tumor-specific CTLs. Peptide fractions that stimulated T cell responses were then sequenced using conventional Edman degradation or tandem mass spectrometry. These approaches have revealed some surprising characteristics of tumor-specific antigens. Most tumor-specific antigenic peptides discovered thus far have been derived from proteins not usually expressed in any normal adult tissues89 (with the exception of testis and ovary), such as P1A90,91 and MAGE-1,92,93 or they represent differentiation antigens characteristic of the cellular lineage of the tumor, such as tyrosinase,94–96 gp100,97–99 and MART1/ Aa100,101 in melanoma. Early definition of tumor antigens focused on MHC class I– restricted peptides.102 This seemed to be the obvious approach because most tumors express MHC class I structures, but few express MHC class II molecules. Also, the point of immunotherapy was to eliminate tumors—a job for cytolytic cells (i.e., for CD8+ cytotoxic lymphocytes that recognize antigen in the context of class I MHC molecules). Early clinical immunization trials103–105 demonstrated the feasibility and the potential efficacy of immunotherapy with peptides recognized by CD8+ T cells. However, immune responses were, in general, weak and short-lived. At the same time that the trials were being conducted, there was a growing realization of the importance of CD4+ T cells in the immune response against tumors.102,106,107 Techniques similar to those used to define antigens recognized by CTLs have been used to define antigens for CD4+ T cells. However, these techniques are slow and labor intensive. A genetic targeting expression system has been designed to expedite antigen screening.107 It is likely that incorporation of both MHC class I– and II–restricted epitopes in tumor vaccines will be required to generate potent antitumor responses.108 While this direct approach to tumor antigen recognition has proceeded, other investigators have asked whether certain appealing target proteins could be immunogenic. In particular, molecules involved in the process of malignant transformation provide attractive targets for therapeutic intervention.109 Because loss variants of tumor cells bearing these oncogenic proteins would presumably be nonmalignant,110 an immunologic assault on these proteins might be particularly effective. Evidence has been provided that immune responses to both mutated and overexpressed oncogenic proteins can occur in patients with malignancy or can be elicited in animals. Target oncogenic proteins include mutated Ras,111 HER-2/Neu,112,113 BCR-ABL,114,115 PMLRARa,116 and mutated p53.117 A newer approach to definition of tumor-specific antigen targets for humoral immunity, termed SEREX, has been introduced.118 In the SEREX approach, a complementary DNA library is prepared from a patient’s tumor specimen, packaged into phage vectors, and expressed in bacteria. Recombinant proteins from bacterial clones are transferred to nitrocellulose membranes and identified as relevant antigens by reactivity with IgG antibodies present in the patient’s serum. Early studies defined three classes of antigens: (a) known tumor antigens, such as MAGE-1, MAGE-4a, and tyrosinase; (b) products of known genes, such as restin; and (c) unknown gene products.119,120 Because the cellular and humoral arms of immunity work in concert, it is likely that targets of antibody production will also prove to be targets of cellular immunity. The SEREX method provides a direct approach to the definition of potentially relevant tumor antigens.

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While the immune system can protect against the development of tumors, interactions between developing tumors and the host are complex. Tumors often develop means to evade immune responses, and tumors that develop in immunocompetent hosts are often more immunogenic than those that develop in immunodeficient hosts. Finally, it is now also well-recognized that the immune system can play both tumor-suppressing and tumor-promoting roles.

Immune Evasion By Tumors Many tumors have devised ways to evade immune responses.18,56,121 First, tumors may lose expression of the antigens that were recognized by antibodies or CTLs. Second, many tumors downregulate expression of MHC class I molecules, rendering these cells resistant to lysis by CTLs.122,123 Third, tumors may fail to induce effective CTL responses because of the absence of costimulatory molecules and/or resistance to uptake by APCs and cross-presentation.124 Instead of inducing an effective immune response, some tumors may actively promote tolerance induction, by inducing anergy, exhaustion, or deletion of tumor antigen-specific T cells.125,126 This might involve the generation of tumor antigen-specific regulatory T cells (Tregs),127 induction of inhibitory costimulatory molecules such as CTLA-4 and PD-1 on tumor antigen-specific T cells,126 and/or expansion of myeloid-derived suppressor cells, a heterogeneous group of myeloid progenitor cells and immature myeloid cells that can inhibit lymphocyte function.128 Fourth, tumor cells may actively suppress immune responses by secretion of suppressive cytokines such as TGF-b or by expression of the Fas ligand, which may engage with Fas on lymphocytes to induce apoptosis.125 Fifth, the tumor microenvironment, most notably the tumor stroma, may be critical in preventing immunologic destruction of tumor cells by effectively generating an immune privileged site.

Immune Sculpting of Tumors In 2001, an important study showed that the immune system not only can protect against the development of tumors, but also can influence the quality of tumors—that is, the immune system of the host in which a tumor develops influences the immunogenicity of the tumor.23 These investigators showed that RAG2-deficient mice not only develop MCA-induced tumors at higher frequency, but that a substantial portion of these tumors was spontaneously rejected upon transplantation in syngeneic immunocompetent mice. In sharp contrast, tumors derived from immunocompetent mice usually grew progressively in immunodeficient mice. Thus, these findings demonstrated that the immune system not only protects the host from tumor formation but also sculpts the immunogenicity of the tumors, in a process that is now referred to as cancer immunoediting.17,18,129 Cancer immunoediting has been posited to proceed through three sequential stages: (a) an elimination phase where the immune system recognizes and destroys tumors before they become clinically apparent, (b) an equilibrium phase where tumor cells that escaped the elimination phase are continuously destroyed, with emergence of resistant tumor cell variants due to immune pressure, and (c) an escape phase where tumor cells that have successfully evaded immune responses progressively grow. The cancer immunoediting hypothesis represents an extension or modern version of the immunosurveillance hypothesis.

Tumor-promoting Immune Responses Discussion of tumor–host interactions would not be complete without at least a mention of the tumor-promoting role of the immune

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system.130 Many environmental factors, including chronic infections, tobacco smoke, and inhaled pollutants, as well as dietary factors and obesity, are associated with a low-level chronic inflammation and represent risk factors for cancer development. Chronic inflammation can contribute to tumor genesis at all stages, by generating nontoxic stress during the initiation of cancer, inducing cellular proliferation to promote cancer development, and enhancing angiogenesis and tumor invasion to promote cancer progression.130

Approaches to Immunotherapy Immunotherapy is the use of the immune system or its components to target and eradicate tumors. B cell lymphomas are considered to be the most immune responsive of all human cancers.131 They can undergo spontaneous regression,132 and partial responses have been elicited through the use of nonspecific immune activators, such as bacillus Calmette-Guérin and IL-2.133,134 Thus, follicular B cell lymphomas represent excellent candidates for immunotherapy.

Antibody Approaches The most common form of immunotherapy employed in the treatment of cancer is passive immunotherapy, which involves the administration of manufactured antibodies that target a particular antigen (Table 70.1). Monoclonal antibodies have emerged as a potent and effective molecularly targeted therapy for human cancer, usually in combination with chemotherapy.135,136 Therapeutic mAbs, such as rituximab and alemtuzumab, are examples of a passive approach. Currently, several B lymphocyte antigens, including CD20, CD22, and CD52, have been utilized as targets for immunotherapy. These targets, while found uniformly on lymphoma cells, are also expressed on normal immune system components, such as normal B lymphocytes.134,137,138 Personalized immunotherapy, also referred to as Id vaccine therapy, is a patient- and tumor-specific approach. This modality targets unique protein determinants of the immunoglobulin molecules produced by the malignant B cell clone and does not appear to result in depletion of normal lymphocytes or subsequent impairment of the immune system. This technique stimulates the patient’s immune system to attack the tumor through the use of both the humoral and cellular arms of the immune system. As a result, immunologic memory may be established which could translate into long-term remission. Some of these are being tested in phase II and III trials (Fig. 70.4).139

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Ta bl e 7 0 . 1

Personalized Active Immunotherapy Versus Passive Immunotherapy Personalized Active Immunotherapy Passive Immunotherapy Tumor-specific Stimulates host immune response Induces immunologic memory May produce long-term immunity Induces both the cellular and humoral arms of the immune system Requires patient tumor sample for production

Not tumor-specific Does not stimulate host immune response Temporary antitumor effect Requires retreatment Induces the humoral arm of the immune system only (ADCC, CDC) Does not require patient tumor sample

ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity.

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FIGURE 70.4. Idiotype as a tumor-specific antigen for B-lymphoma cells. Each B lymphocyte expresses an immunoglobulin molecule on its surface, the idiotype (Id) protein, which is capable of recognizing and binding to a unique antigen. When B lymphocytes undergo malignant transformation, the Id sequences are maintained by the malignant clones and can thus serve as tumor-specific antigen. MHC, major histocompatability complex; TCR, T cell receptor. (Adapted from Vose JM. Personalized immunotherapy for the treatment of non-Hodgkin’s lymphoma: a promising approach. Hematol Oncol 2006;24:47–55.)

Unconjugated Antibodies Unaltered antibodies have been used since the earliest trials of monoclonal antibody therapy in humans.140–142 In early trials, success was limited by the absence of suitable tumor cell surface targets, antigenicity of first-generation (murine) mAbs in humans, modulation of the target structure from the tumor cell surface, and poor recruitment of immune effector mechanisms.143,144 However, enthusiasm for this approach was rekindled by the enormous success of genetically engineered, chimeric, or fully humanized versions of mAbs, most notably rituximab for lymphoid malignancies and trastuzumab in solid tumors.144 Rituximab is a chimeric monoclonal antibody with humanized framework and Fc regions. It is directed against the CD20, pan B cell antigen. CD20 is present on pre-B cells and mature B cells, but not on precursor cells or terminally differentiated plasma cells. The function of CD20 remains poorly understood,145 although it has been implicated in B cell activation, regulation of B cell growth, and regulation of transmembrane calcium flux. The antibody fixes human complement and elicits antibody-dependent cellular cytotoxicity (ADCC). Rituximab was approved for use as monotherapy in patients with low-grade or follicular CD20+ non-Hodgkin lymphoma (NHL) in 1997. In the pivotal trial involving 166 patients, reported by McLaughlin et al.,146 patients had relapsed or chemotherapy-resistant disease. Patients received four weekly infusions of rituximab at 375 mg/m2. Rituximab produced a tumor response in one-half of the patients, with a median duration of response of 11.8 months, comparable to the patients’ response duration on the last chemotherapy treatment. Human antichimeric antibody responses were uncommon. The most common adverse experience associated with rituximab was a constellation of acute infusion-related events, including chills, fever, headache, rhinitis, pruritus, vasodilation, asthenia, and angioedema. This syndrome can progress to hypotension, urticaria, bronchospasm, and, rarely, death. The risk is particularly great in patients with high circulating white cell counts.147 Other

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toxicities were, in general, mild and infrequent.148 Neutropenia and thrombocytopenia were unusual. Circulating B cells were depleted and remained low until recovery at a median of 12 months. Immunoglobulin levels, however, remained normal. No increased incidence of infections was seen. Rituximab was evaluated again in the relapsed, refractory, low-grade, or follicular NHL patient population, using eight weekly infusions rather than four.149 This extended regimen produced a response rate of 57% and a time to progression (TTP) in responding patients of more than 19.4 months. Adverse event reporting was commensurate with the longer treatment period. Davis et al.150 reported a response rate of 43% and a TTP of 8.1 months in the patients with a significant poor prognostic factor, importantly in patients with low-grade or follicular NHL who had bulky disease, using the standard 4-infusion regimen. These investigators also showed an overall response rate (ORR) of 40% in patients who had progressed after an initial response to rituximab, with an estimated median TTP for responding patients of 17.8 months.151 Rituximab has been used in the first-line treatment of patients with indolent lymphoma, both as monotherapy and in combination with chemotherapy. Hainsworth150 reported the results of rituximab as monotherapy in 39 previously untreated patients. Patients received rituximab x 4 at the usual dose and schedule, with an ORR of 54%. Patients who had responded or who had stable disease were treated with an additional four weekly treatments of rituximab at 6 months intervals to a maximum of 4 treatment cycles. The ORR rose to 72% after the second course of treatment. Progression-free survival (PFS) at 1 year was 77%. The addition of rituximab to cyclophosphamide, hydroxyldaunomycin, oncovin (vincristine), and prednisone (CHOP) chemotherapy produced impressive results. Patients were treated with six infusions of rituximab, one associated with each of six cycles of CHOP. In 40 patients with newly diagnosed (n = 31) or relapsed/refractory (n = 9) low-grade or follicular NHL, rituximab plus CHOP produced an ORR of 95% and complete response (CR) rate of 55%. With a median follow-up of 29 months, median duration of response and median TTP had not been reached. Eight of 18 patients tested for the BCL-2 [t(14;18)] translocation by PCR testing were positive at initiation of therapy. Seven of these 8 patients, after therapy, became negative for the translocation. The authors concluded that the addition of rituximab to CHOP produced benefits in efficacy parameters without significant additional toxicity. Elimination of PCR positivity for the t(14;18) translocation had not been previously reported with CHOP alone. Rituximab also has been used with success in patients with more aggressive lymphomas. Vose et al.151 reported the results of rituximab plus CHOP chemotherapy (again using 6 infusions of rituximab in association with 6 cycles of CHOP) in 33 previously untreated patients with advanced aggressive B cell NHL. The combination produced an ORR of 94% and a CR rate of 61%. With a median observation time of 26 months, 29 of 31 patients achieving a remission were in continuing remission at the time of the report. Thirteen patients were BCL-2 positive at study entry. Eleven of these 13 patients became BCL-2 negative after treatment, and 10 of the 11 remained BCL-2 negative. The authors concluded that the results were achieved without significant added toxicities above those expected with CHOP. The Groupe d’Etude des Lymphomes de l’Adulte undertook a study to compare the utility of CHOP with that of rituximab plus CHOP in elderly patients with diffuse B cell lymphoma.152 Patients between the ages of 60 and 80 years with untreated, diffuse large B cell lymphoma were eligible for the study. Patients were randomly assigned to receive 8 cycles of CHOP chemotherapy (197 patients) or to receive 8 cycles of CHOP, each given after an infusion of rituximab (202 patients). Rituximab plus CHOP produced a superior rate of remission, 76% versus 63% (P = 0.005). With

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a median follow-up of 2 years, event-free and overall survivals (OSs) were significantly higher in the rituximab plus CHOP group (P < 0.001 and P = 0.007, respectively). These results were achieved without a significant incremental increase in toxicity. The addition of rituximab to CHOP reduced the risk of treatment failure (risk ratio, 0.54; 95% confidence interval [CI]: 0.44, 0.77) and the risk of death (risk ratio, 0.64; CI: 0.45, 0.89). The addition of immunotherapy to standard chemotherapy had accomplished what 25 years of chemotherapy manipulation had failed to do (i.e., improve on the results of CHOP chemotherapy).153,154 Maintenance rituximab has also been found to prolong event-free survival (EFS) and response duration in follicular lymphoma.155 In subsequent ongoing randomized phase III studies,156,157 rituximab maintenance regimen provided significant PFS and OS at 3-year156 and 4-year157 follow-up assessments in both previously treated156 and untreated157 patients with follicular NHL, compared with no further treatment, though no difference in OS has been reported by other investigators.158 The mechanism by which rituximab produces these impressive results is less clear. A number of possible mechanisms have been considered: initiation of complement-mediated cell lysis, induction of ADCC, and signaling via CD20 leading to programmed cell death and/or sensitization to cytotoxic drugs. Pretreatment lymphoma cells from 29 patients were examined by flow cytometry for expression of complement inhibitors CD46, CD55, and CD59.159 Expression of these cell surface inhibitors of complement activation was not predictive of outcome to rituximab therapy. Considerable evidence suggests that induction of ADCC plays an important role in rituximab’s antilymphoma effects. A rituximab-like antibody for which an IgG4g framework was substituted for the IgG1 framework of rituximab was incapable of producing B cell depletion in primates.160 Rituximab was relatively ineffective in eliminating Raji B cell implants in FcRg−/−/ nu/nu knockout mice compared to nu/nu mice.161 These mice lack the activating receptor for Fc portions of antibodies, a critical component of the antibody-dependent cell-mediated cytotoxicity mechanism. In patients, response to rituximab has been shown to be associated with homozygosity for the high-affinity allotype of the FcgRIIIa receptor.162 Evidence also exists that rituximab signaling or interference with normal signaling via CD20 may directly induce apoptosis or sensitize cells to the deleterious effects of chemotherapeutic agents.163 A direct, growth inhibitory effect of rituximab, with accompanying apoptosis, on cell lines cultured in the absence of complement was demonstrated.164 Anti-CD20–associated apoptosis has been associated with upregulation of the proapoptotic protein, Bax165 and downregulation of antiapoptotic protein BCL-2 through inactivation of STAT3.166 Downregulation of STAT3 appears to be a result of downregulation of an IL-10 autocrine pathway.167 These changes and/or others may be responsible for increased sensitivity to chemotherapeutic agents.168 In the wake of the success of rituximab, a number of other antilymphoma mAbs have entered the clinic.169 Alemtuzumab is a humanized IgG1k monoclonal antibody directed against the CD52 cell surface antigen.144 CD52 is expressed on normal and malignant lymphocytes of B- and T cell lineage, as well as NK cells, monocytes, and macrophages. Alemtuzumab is indicated for the treatment of B cell chronic lymphocytic leukemia (CLL) in patients who have been treated with alkylating agents and who have failed fludarabine therapy. The pivotal clinical trial was carried out in 93 patients with fludarabine-refractory CLL .170 Alemtuzumab produced a response rate of 33%. Virtually all of the responders were partial responders; the CR rate was 2%. Median duration of response was 7 months. Median TTP was 4.7 months for the group as a whole; 9.5 months for responders. The most common adverse events were infusion related—most were grade 1 or 2 in severity, including rigors in 90% of patients (grade 3 in 14%), fever in 85% of patients (grade 3 or 4 in 20%),

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nausea in 53% of patients, and vomiting in 38% of patients. Infusion-associated side effects declined with subsequent infusions. During the study, 28% of patients experienced dyspnea, 17% experienced hypotension, and 3% experienced hypoxia. Overall, 55% of patients developed an infection during the study. Approximately one-half of these infections were considered serious (grade 3 or 4). Septicemia occurred in 15% of patients, and two deaths resulted. Opportunistic infections occurred in 12% of patients. Ten percent of patients died during or within 30 days of treatment—one-third of these were attributed to progressive disease. Twenty-four percent of patients discontinued treatment because of a drug-related side effect. Most patients who discontinued had not responded to therapy. Serious infusionrelated events associated with alemtuzumab appear to result from ligation of CD16 on NK cells resulting in what has been termed cytokine storm—release of IL-6, TNF-a, and interferong.171 Prolonged immunosuppression after use of alemtuzumab can result in opportunistic infections.172 Treatment schemas now include the routine use of prophylaxis with both antibiotics and antivirals. To improve on the immunogenicity and efficacy of rituximab, the last few years have seen the development of new generations of anti-CD20 monoclonal antibodies (mAbs) with enhanced antitumor activity resulting from increased complement-dependent cytotoxicity (CDC) and/or ADCC and increased Fc binding affinity for the low-affinity variants of the FcgRIIIa receptor (CD16) on immune effector cells. These second-generation mAbs, such as ofatumumab, veltuzumab, and ocrelizumab, are in clinical development. They are humanized or fully human to reduce immunogenicity, but with an unmodified Fc region. Ofatumumab is a fully human anti-CD20 IgG1 mAb in clinical development for hematologic malignancies and autoimmune diseases. Ofatumumab specifically recognizes an epitope encompassing both the small and large extracellular loops of the CD20 molecule, and is more effective than rituximab at CDC induction and killing target cells. Veltuzumab (IMMU-106, hA20) is a humanized anti-CD20 mAb with complementarity-determining regions similar to rituximab. This antibody has enhanced binding avidities and a stronger effect on CDC compared to rituximab. Ocrelizumab is a humanized mAb with the potential for enhanced efficacy in lymphoid malignancies compared to rituximab because of increased binding affinity for the low-affinity variants of the FcgRIIIa receptor. Third-generation mAbs are also in clinical development. They are also humanized mAbs, but in addition they have an engineered Fc to increase their binding affinity for the FcgRIIIa receptor. Third-generation mAbs also in clinical development include AME-133v, PRO131921, and GA-101 (Table 70.2), with enhanced affinity for the FcgRIIIa receptor and an enhanced ADCC activity compared to rituximab.173 Two other mAbs with potential utility in the treatment of lymphoma are in early clinical development.169,174 Epratuzumab is a humanized IgG1 monoclonal antibody directed against the CD22 antigen. CD22 is a pan–B cell antigen with distribution similar to that of CD20. Epratuzumab has a favorable safety profile in early trials. Approximately 50% of follicular lymphoma patients and 25% of diffuse large-cell lymphoma patients responded in a small phase II trial. Some of the responses have been long-lived. A recent phase II trial testing the safety and efficacy of combining epratuzumab with R-CHOP (ER-CHOP) in untreated DLBCL showed that the addition to standard R-CHOP, E 360 mg/m2 intravenously, administered for 6 cycles in 107 patients, showed similar toxicity to standard R-CHOP. ORR in the 81 eligible patients was 96% (74% CR/ CR unconfirmed [Cru]) by computed tomography scan and 88% by positron emission tomography. By intention to treat analysis, at a median follow-up of 43 months, the EFS and OS at 3 years in all 107 patients were 70% and 80%, respectively. Comparison with a cohort of 215 patients who were treated with R-CHOP showed improved EFS in the ER-CHOP

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Ta bl e 70.2

Anti-CD20 Monoclonal Antibodies (mabs) Approved or Potentially Useful for Lymphoid Malignancies mAb

Company

Rituximab (Rituxan®, Mabthera®) Rituximab (Rituxan®, Mabthera®) Veltuzumab (IMMU-106, hA20) Ocrelizumab

Hoffman La Roche

PRO131921

Antibody characteristics

ADCC

CDC

Direct effects

Type I, first-generation mouse/human chimeric IgG1 Type I, first-generation, human IgG1 Type I, second-generation, humanized IgG1 Type I, second-generation, Humanized fusion IgG1

++

++

+

++

++++

+

++

++

+

+++

+/−

+

Genentech, Inc.

Type I, third-generation, humanized fusion IgG1

+++

+++

+

Improved binding to FcgRIIIa, better ADCC, superior antitumor efficacy

AME-133 v (LY2469298)

Lilly

Type I, third-generation, humanized fusion IgG1

+++

++

++

GA-101 (RO5072759)

Glycart Biotechnology AG, Genentech, F Hoffmann-La Roche Ltd Trubion Pharmaceuticals Inc., Pfizer Inc.

Type II, third-generation, humanized IgG1

++++

−

++++

Enhanced affinity for FcgRIIIa, superior ADCC Superior ADCC and direct cell killing

SMIP-derived humanized fusion protein

+++

+

?

TRU-015

GlaxoSmithKline plc/ Genmab A/S Immunomedics Inc. Genentech Inc./Biogen Idec Inc./Chugai Pharmaceutical Co. Ltd/Roche Holding Ag

Comparison with rituximab

Binding to different CD20 epitope; more effective at CDC Slower off-rate, enhanced binding avidity, a superior CDC Binding to different CD20 epitope, enhanced ADCC, reduced CDC, enhanced affinity for FcgRIIIa RIIIa

Single-chain polypeptide, enhanced ADCC, reduced CDC

ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; IgG, immunoglobulin G; SMIP, small modular immunopharmaceutical; + indicates low cytotoxicity; ++ indicates intermediate cytotoxicity; +++ indicates high cytotoxicity; ++++ indicates very high cytotoxicity; +/− indicates very low cytotoxicity; − indicates lack of cytotoxicity; ? indicates cytotoxicity unknown. Reproduced with permission from Robak et al. BioDrugs 2011;25:13–25.

patients. ER-CHOP was well tolerated and results appear promising as a combination therapy.175 Apolizumab is a humanized IgG1 monoclonal antibody that binds to a variant of the HLA-DR b-chain. The antibody induces complement-mediated lysis, ADCC, and tyrosine phosphorylation signaling events in cell lines in vitro. The antibody binds to approximately 70% of lymphoma specimens. Administration of the antibody to patients results in typical infusion-related side effects. Four of 8 patients with follicular lymphoma responded to apolizumab. A phase I/II dose-escalation study of thrice-weekly apolizumab (1.5, 3.0, 5.0 mg/kg/dose) for 4 weeks in relapsed CLL resulted in significant toxicity and lack of efficacy; thus, further clinical trials of apolizumab were discontinued, as were other trials in lymphoma and solid tumors.176 Milatuzumab (hLL1, IMMU115; Immunomedics) is a fully humanized mAb specific for CD74, a cell surface–expressed epitope of the HLA class II–associated invariant chain. CD74 plays an important role as an accessory signaling molecule and survival receptor in the maturation and proliferation of B cells by activating the PI3K/Akt and the NF-kB pathways.177 Milatuzumab demonstrated antiproliferative activity in transformed B cell lines, improved survival in preclinical models, and is presently being evaluated for the treatment of several hematologic malignancies. Inroads are being made in other hematologic cancers. In multiple myeloma, it was recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) was highly and universally expressed on myeloma cells while having restricted expression in normal tissues. Preclinical studies showed that elotuzumab (formerly known as HuLuc63), a humanized mAb targeting CS1, could induce patient-derived myeloma cell killing within the bone marrow microenvironment using a SCID-hu mouse model and that the CS1 gene and cell surface protein expression persisted on myeloma patient-derived plasma cells collected after bortezomib administration. In vitro bortezomib pretreatment of myeloma targets significantly

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enhanced elotuzumab-mediated ADCC, both for OPM2 myeloma cells using NK cells or peripheral blood mononuclear cells from healthy donors and for primary myeloma cells using autologous NK effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib exhibited significantly enhanced in vivo antitumor activity. Elotuzumab is currently in a phase I clinical trial in relapsed/refractory myeloma.178 In AML, one strategy for the development of mAbs targeting human AML stem cells involves first identifying cell surface antigens preferentially expressed on AML LSC (leukemia stem cell) compared with normal hematopoietic stem cells. In recent years, a number of such antigens have been identified, including CD123, CD44, CLL-1, CD96, CD47, CD32, and CD25. Moreover, mAbs targeting CD44, CD123, and CD47 have demonstrated efficacy against AML LSC in xenotransplantation models. Hopefully, these antibodies will ultimately prove to be effective in the treatment of human AML.179

Anti-idiotype Therapy Much time and energy were expended searching for tumor antigens, particularly after the development of mAb techniques.17 These brute-force immunization, hybridization, and screening procedures yielded little. They defined only a single truly tumorspecific antigen. That was the Id of clonally distributed antibody expressed on the surface of certain B cell lymphomas. Because it represents a unique protein structure within the combining site of the antibody, the Id can serve as an antigen for antibody production. This fact was demonstrated by Sirisinha and Eisen180 in the early 1970s. Furthermore, they demonstrated that an immunologic response to Id could lead to tumor protection.181 Levy and Miller182 have explored the utility of anti-Id strategies in indolent B cell lymphoma over many years. Initially, these investigators raised custom-made anti-Id mAbs183 for passive administration. A first patient with far advanced,

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chemotherapy-refractory disease received anti-Id mAbs. Gradual reductions in serum Id and tumor volume were noted. The patient then entered a long-term complete remission.184 In an initial series of patients treated with anti-Id therapy, 11 patients were reported.185 In this group, a second near-complete remission, four partial remissions, and five insignificant responses were seen. There was little toxicity associated with administration of the anti-Id antibodies. The most common side effects were chills and fever. Transient shortness of breath, headache, nausea, emesis, diarrhea, and myalgias were occasionally observed. Unusual toxicities included transient leukopenia or thrombocytopenia and transient elevations of hepatic enzymes. Several interesting problems of anti-Id therapy were noted in this early series: the interfering effect of circulating Id, the development of human antimouse antibody, and the emergence of Id-negative tumor cell variants.186–189 An attempt to reduce the incidence of emergence of Id-negative lymphoma variants with a short course of chlorambucil was unsuccessful.190 The cumulative experience suggests that anti-Id therapy can result in a 15% CR rate and a 50% partial response rate. The mechanism of tumor response in these trials remains unclear. However, response in these trials correlated with anti-Id–induced signal transduction events in the lymphoma cells, suggesting that activation of apoptotic pathways may lead to lymphoma cell death after interaction of the anti-Id antibody with the lymphoma surface-bound immunoglobulin receptor.191 This group has turned to active immunization strategies in indolent lymphoma (see section “Immunization Strategies”).

Radioimmunotherapy The use of immunoglobulin–radionuclide conjugates in cancer treatment represents appropriation of a classic guided-missile strategy. In theory, the antibody homes to its antigenic target and delivers a cytotoxic assault on the cell to which it attaches. Radionuclides offer certain advantages over other cytotoxic agents. They do not have to be internalized. Radioactive particles can deliver their effects over distances of 1 to 5 mm, thus limiting collateral damage to normal tissues while still potentially providing antitumor effects against antigen-negative variants in the vicinity in what has been termed a cross-fire effect. The principles of radiation physics underlying radioimmunotherapy (RIT) have been reviewed by Press and Rasey.192 Radiolabeled antibodies deliver continuous, exponentially decreasing, low-dose-rate radiation. Traditional external beam radiotherapy delivers intermittent, fractionated radiation at higher dose rates. The most commonly used isotopes for RIT have been iodine 131 (I-131) and yttrium 90 (Y-90). These radionuclides kill cells primarily through emission of b particles, resulting in DNA strand breaks. The b particles of Y-90 are more energetic than those of I-131. They affect cells in a radius of approximately 5 mm compared to approximately 1 mm for those of I-131. I-131 also emits g rays. This allows direct imaging of the distribution of the radioconjugate but raises issues regarding shielding and health care worker and family member safety. Several recent reviews attest to the evolution of this field.193,194 A number of theoretical and experimentally generated concerns with RIT appear to have been overcome in the successful clinical studies described below. There had been concern that effective delivery of radioimmunoconjugates would be impeded by heterogeneous tumor vasculature, slow diffusion of these large molecules in interstitial spaces, heterogeneous biodistribution in tumor nodules, and high intratumoral pressures. The two products in clinical use presently, Y-90 ibritumomab tiuxetan (Zevalin) and I-131 tositumomab (Bexxar), are both directed against the anti-CD20 antigen of B lymphocytes, the same structure targeted by rituximab. Both products are based on murine mAbs. Both are administered after infusion of unconjugated anti-CD20 antibodies—rituximab in the case of Zevalin

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and tositumomab in the case of Bexxar. Both have used nuclear medicine imaging as a preparatory step to administration, but it is no longer required for Y-90 ibritumomab. Simple dosimetry is accomplished for I-131 tositumomab by capturing whole-body gamma counts after infusion of a 5 mCi “dosimetric dose” of the agent. Imaging was carried out in Y-90 ibritumomab tiuxetan– treated patients to assure normal biodistribution. Whole-body dosimetry is carried out in I-131 tositumomab–treated patients to allow calculation of a patient-specific activity (mCi) to deliver a desired total-body dose of radiation (cGy). Both have been studied most extensively in indolent lymphoma and have been approved for treatment of patients with relapsed or refractory follicular, including rituximab refractory, or transformed B (CD20+) NHL. The approval for ibritumomab tiuxetan rested primarily on two clinical studies. The first was a randomized controlled comparison of the effectiveness of Y-90 ibritumomab tiuxetan to that of rituximab in patients with relapsed or refractory, follicular, or transformed B cell NHL.195 The study involved 143 patients; 73 randomized to Y-90 ibritumomab tiuxetan (single administration), and 70 randomized to rituximab (weekly × 4). The median number of prior therapies was 2. Approximately one-half of the patients failed to respond to or had a TTP of less than 6 months to their last chemotherapy regimen. Y-90 ibritumomab tiuxetan produced a statistically superior response rate (using the response definitions of the International Workshop), 80% versus 56% (P = 0.002). The number of durable responders at 6 months favored Y-90 ibritumomab tiuxetan–treated patients, but the significance of the observation was lost at 9 months and 12 months. Median TTP (estimated by Kaplan-Meier methods) was 11.2 months for patients treated with Y-90 ibritumomab tiuxetan and 10.1 months for patients treated with rituximab (P = 0.173). Grade 3 and 4 nonhematologic adverse events were unusual in both groups. Y-90 ibritumomab tiuxetan produced grade 3 or 4 neutropenia in 57% of patients, grade 3 or 4 thrombocytopenia in 60% of patients, and grade 3 or 4 anemia in 2% of patients. One patient in the Y-90 ibritumomab tiuxetan–treated group developed myelodysplasia. One patient in the rituximab-treated group developed pancreatic carcinoma. The second trial was a phase II experience in 57 patients who had failed to respond to rituximab or had a TTP of ≤6 months.196 These patients had a median of 4 prior therapies, and 74% had bulky tumors (greatest diameter ≥ 5 cm). In this patient population, Y-90 ibritumomab tiuxetan produced a response rate of 74% and CR rate of 15%. The median TTP was estimated at 6.8 months. Grade 4 neutropenia occurred in 35% of patients, grade 4 thrombocytopenia in 9% of patients, and grade 4 anemia in 4% of patients. The pivotal study for I-131 tositumomab enrolled 60 patients with refractory or transformed low-grade NHL who had been treated with at least two different qualifying chemotherapy regimens.197 Patients must also have failed to achieve an objective response or relapsed within 6 months after completion of their last qualifying chemotherapy (LQC) regimen. Median age was 60 years, and other poor prognostic features included: median of 4 prior therapies, bulky disease, bone marrow involvement, elevated serum lactate dehydrogenase, advanced stage, and transformation from an initial low-grade histology to a highergrade histology in 38% of the patients. A statistically significant improvement in the primary endpoint was achieved, with a longer duration of response (>30 days) after I-131 tositumomab therapy (n = 26) compared to patients after their LQC (n = 5; P < 0.001). Improvements in secondary efficacy endpoints after I-131 tositumomab compared to those after LQC were also achieved: overall response (47% vs. 12%; P < 0.001), duration of response (11.7 vs. 4.1 months; P < 0.001), and CR (22% vs. 2%; P = 0.002). Fifteen of 60 patients (25%) were classified as long-term responders (patients with a MIRROR Panel–assessed TTP of a year or more). Nine (15%) of the 60 patients remained in CR, with TTP ranging from 41+ to 57+ months.

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A second trial examined the incremental benefit of the radioconjugate (I-131 tositumomab) compared to the nonradioactive antibody (tositumomab).198 This study was a randomized, twoarm, open-label, multicenter study that enrolled patients with chemotherapy-relapsed or refractory low-grade or transformed low-grade NHL. Patients were randomized to receive either I-131 tositumomab therapy or unlabeled tositumomab alone. The primary endpoint was a comparison of the rates of CR. Secondary endpoints included ORR, duration of responses, and TTP. A total of 78 patients (18% with transformation) participated in the study. Patients had been previously treated with one to three chemotherapy regimens. One or more therapies must have included an anthracycline, anthracenedione, or alkylating agent. A significant difference was observed for the primary efficacy endpoint. The CR rate was 33% (14 of 42 patients) for the patients treated with I-131 tositumomab compared to 8% (3 of 36) for patients treated with unlabeled tositumomab (P = 0.012). In addition, the ORR was greater after treatment with I-131 tositumomab: 23 of 42 patients (55%) compared to 7 of 36 patients (19%; P = 0.002). Nineteen patients initially treated with the unlabeled antibody crossed over to receive I-131 tositumomab after disease progression. A CR then was observed in 42% (8 of 19 patients) and an ORR in 68% (13 of 19 patients) in the crossover patient population. A total of 20 patients (33%) from the I-131 tositumomab–treated populations, including patients in the crossover arm, were classified as having a long-term response, including ten patients continuing in CR, with TTP ranging from 23+ to 59+ months. The efficacy of I-131 tositumomab was also evaluated in patients who had progressed after rituximab.199,200 Patients must have had prior treatment with at least four doses of rituximab without an objective response, or to have progressed during or after treatment. Twenty-four patients did not respond to their last treatment with rituximab, and, of the 16 patients who did respond to rituximab, five patients had a duration of response exceeding 6 months. A response occurred in 27 of 40 patients (68%), with a median duration of response of 14.7 months (95% CI; 10.6 months no response). A CR occurred in 12 of 40 patients (30%); the median duration of CR had not been reached (95% CI; 11 months no response). Twenty-four patients had a longer (at least 30 days) duration of response after I-131 tositumomab than after rituximab; 5 patients had a longer duration of response after rituximab than after I-131 tositumomab; 9 patients had equivalent durations of response; and 2 patients were censored (P < 0.001). A total of 14 patients (35%) had a TTP of 12 months or longer. The median PFS was 10.4 months (95% CI, 5.7 to 8.6) for all patients and 24.5 months for confirmed responders (95% CI, 16.8 to not reached [NR]). PFS for 15 confirmed CR patients was NR with an estimated 3 years PFS of 73%. Prior response to rituximab did not significantly affect the confirmed OR rate, duration of response, or median PFS. Radioiodinated tositumomab and Y-90 ibritumomab tiuxetan have also been used at myeloablative doses, with stem cell rescue in patients with relapsed B cell lymphomas.201,202,203,204 Radioiodinated tositumomab was used initially as a single agent.202 Twenty-five patients were imaged after a tracer dose of radioiodinated tositumomab. Twenty-two of these 25 patients achieved favorable biodistributions (i.e., had tumor doses in excess of doses to normal organs). These 21 patients received therapeutic infusions of radioiodinated tositumomab (345 to 785 mCi) followed by reinfusion of autologous hematopoietic stem cells. All patients achieved bone marrow engraftment (19 with bone marrow stem cells, two with peripheral blood stem cells). However, two patients died before full neutrophil recovery; one of sepsis, one of progressive lymphoma. Nonhematologic toxicities included nausea in most patients, mild mucositis in five patients, and partial alopecia in four patients. One patient experienced reversible cardiomyopathy and interstitial pneumonitis. Eighteen patients responded to this therapy; 16 patients experienced a CR. With a median follow-up of

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2 years, 2 years PFS was estimated at 62%, with OS estimated at 93%. Press et al. then combined radioiodinated tositumomab with chemotherapy and autologous stem cell transfusion in a series of patients with relapsed B cell lymphomas.203 Fifty-two patients received the planned therapy. Patients were again given tracer doses of radioiodinated tositumomab and underwent sequential gamma camera imaging. Absorbed doses of radiation to tumor sites and normal organs were determined. Thereafter, patients received a therapeutic infusion of radioiodinated tositumomab calculated to deliver between 20 and 27 Gy to normal organs (e.g., liver, kidneys, and lungs). Patients then received etoposide, 60 mg/ kg, and cyclophosphamide, 100 mg/kg, followed by reinfusion of autologous hematopoietic stem cells. The maximal tolerated dose of radioiodinated tositumomab to be combined with chemotherapy was determined to be that dose that delivered 25 Gy to normal organs. Eight patients experienced 13 grade 3 or 4 toxic events. These included 3 patients with acute respiratory distress syndrome, 3 patients with severe mucositis or gastrointestinal toxicity, 1 patient with venoocclusive disease, and 4 patients with fatal infections. At 2 years, the Kaplan-Meier estimates of OS and PFS for all treated patients were 83% and 68%, respectively. These findings were considered superior to results previously observed in patients who had undergone conventional external beam total-body radiation with etoposide/cyclophosphamide preparation for transplantation in the same institution. Thirty-one patients with CD20+ NHL were treated with high-dose Y-90 ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide and were followed by autologous hematopoietic cell transplantation (HCT).204 Treatment also was well tolerated; there were 2 deaths and 5 relapses. At a median follow-up of 22 months, the 2 years estimated OS rates are 92% and 78%, respectively. Retreatment with tositumomab and I-131 tositumomab, has also been found possible in patients with progressive disease after treatment with I-131 tositumomab, who were able to receive subsequent therapy, including cytotoxic chemotherapy and stem cell transplantation.205 In patients with prior response, I-131 tositumomab can produce second responses that can be durable.206 Use of RIT is also being investigated in the allogeneic HCT setting.207,208,209 RIT with b-emitters has been successfully used for further dose intensification of myeloablative conditioning regimens for HCT. Using a canine model of nonmyeloablative HCT, Bethge et al. used pretransplant RIT with the a-emitter bismuth-213 coupled to anti-CD45 or anti-TCRab mAb together with postgrafting immunosuppression with mycophenolate mofetil and cyclosporine, which resulted in stable engraftment and long-term mixed chimerism.208 In 2 relapsed patients with NHL, Y-90 ibritumomab tiuxetan was given as part of the conditioning for an HLA-matched donor transplant. Rituximab 250 mg/m2 was given on days −21 and −14, 0.4 mCi/kg Y-90 ibritumomab tiuxetan on day -14 and fludarabine (30 mg/m2) plus cyclophosphamide (500 mg/m2) on days −7 and −3, resulting in fast and reliable engraftment, offering an attractive new therapeutic option for relapsed lymphoma patients.209 A method of dose-intensified RIT called “pretargeting” RIT (PRIT) dissociates the slow distribution phase of the antibody molecule from the delivery of the therapeutic radionuclide. This might achieve improved outcomes with less toxicity, and is being explored in preclinical studies of hematologic malignancies.210 In a preclinical study using an acute leukemia xenograft model, comparing conventional and pretargeted antihCD45 RIT, investigators showed clearly that anti-CD45 PRIT provided rapid tumor localization and improved biodistributions of radioactivity with significant improvements in efficacy compared with conventional anti-CD45 RIT.211 In allogeneic HCT, RIT is being explored to reduce toxicity associated with external g-beam radiation. RIT with an anti-CD45 mAb labeled with the a-emitter astatine-211 has been used as a conditioning regimen in dog leukocyte antigen-identical HCT, resulting in good engraftment and minimal toxicity.212

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Immunotoxins and Fusion Toxins This category of treatment reagents resembles those guided missiles of RIT. In this case, however, the warheads are chemical rather than nuclear, and the targeting is provided by antibodies or by lymphokines, growth factors, and so on, that specifically bind receptors on the surfaces of target tumor cells. Attached to the targeting moiety is the cytolytic moiety. This is usually a toxin, derived from plants or bacteria, which works by inhibiting protein synthesis. They kill either resting or dividing cells and require fewer than ten molecules in the cytosol to be effective.213,214 Toxins of this type tested in phase I trials include ricin A-chain, blocked ricin, saporin, pokeweed antiviral protein, Pseudomonas exotoxin A, and diphtheria toxin. Recently, calicheamicin, a highly potent antitumor antibiotic that cleaves double-stranded DNA at specific sequences,215 has been successfully targeted to leukemia cells.216 A number of factors influence the efficacy of immunotoxins. These include the binding affinity of the ligand for its target and the target density on the tumor cell surface.217 The epitope to which binding occurs can affect the potency of the immunotoxin.218 Membrane-proximal epitopes appear to confer greater efficacy. Immunotoxin binding must lead to internalization of the target structure and the attached immunotoxin.219 Once internalized, the toxin moiety must translocate to the cytoplasm to be effective. This process is aided by certain translocation sequences in the toxin. The need for translocation signals provides the rationale for using blocked ricin toxin; targeting via the binding subunit is eliminated but translocation signals are preserved.220 The site of translocation may vary for different toxins. Increasing lysosomal pH protects cells from Pseudomonas exotoxin and diphtheria toxin but increases sensitivity to ricin,221–223 suggesting that ricin may undergo translocation in the Golgi apparatus. Finally, these toxins affect protein synthesis by ADP-ribosylating elongation factor 2224 in the case of diphtheria toxin and Pseudomonas toxin, or by alteration of the 60S ribosomal subunit in the case of ricin.225 A number of phase I clinical trials using ricin-based, anti-pan B cell antibody immunotoxins have been reported in B cell lymphoma.226–233 These trials demonstrated that therapeutic doses of immunotoxins can be delivered with tolerable, reversible side effects. Toxicities include systemic symptoms of fever, nausea, vomiting, headache, and muscle aches; evidence of hepatocyte damage with transaminase elevations; and significant problems with capillary leak syndrome.234 Again recognized were the problems of human antimouse antibody formation and rapid clearance of immunotoxin in the presence of circulating antigenemia. Sporadic responses were seen in these trials, with response rates perhaps approaching 25% and CRs approaching 10%. There were hints that targeting via CD22 might be more useful than targeting via CD19.235 Some experts have suggested that these agents will not become useful clinical tools until problems of their immunogenicity have been solved.143,214 However, recent success has been reported for two agents of this class. A recombinant immunotoxin containing the antiCD22 variable domain (Fv) fused to a truncated Pseudomonas exotoxin has produced CRs in patients with hairy-cell leukemia (HCL).236 Sixteen patients whose disease was resistant to nucleoside analogs were treated by intravenous infusion every other day for three doses. Thirteen of 16 patients responded—11 had CRs. The treatment was generally well tolerated. Common side effects included transient elevations of liver enzymes and hypoalbuminemia. Median follow-up was 16 months, during which 3 of the 11 complete responders relapsed. These three patients were retreated with the immunotoxin. Two of the three developed hemolytic uremic syndrome. Moxetumomab pasudotox, is an improved, more active form of a predecessor recombinant immunotoxin, BL22 (also called CAT-3888), and is up to 50-fold more active on lymphoma cell lines and leukemic cells from

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Chapter 70  Immunotherapy

1479

patients with CLL and HCL. A phase I trial was recently completed in HCL patients, who achieved response rates similar to those obtained with BL22 but without dose-limiting toxicity. In addition to further testing in HCL, moxetumomab pasudotox is being evaluated in phase I trials in patients with CLL, B cell lymphomas, and childhood ALL. Moreover, protein engineering is being used to increase its activity, decrease nonspecific side effects, and remove B cell epitopes.237 Gemtuzumab ozogamicin (Mylotarg) is an immunotoxin composed of a recombinant human IgG4k monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin,238 and was previously approved for the treatment of elderly patients with CD33-positive AML in first relapse. The antibody is directed against the CD33 antigen found on the surface of leukemic blasts and normal immature cells of myelomonocytic lineage, but not on hematopoietic stem cells. CD33 is a sialic acid–dependent adhesion molecule. In a phase I dose-escalation trial, treatment with gemtuzumab ozogamicin resulted in elimination of leukemic cells from peripheral blood and bone marrow in 8 of 40 patients.239 The basis for approval of the drug was the experience in 142 patients participating in one of three similar trials designed to examine the efficacy and safety of gemtuzumab ozogamicin in patients in first relapse of AML.216 Across the three studies, 80 patients were 60 years of age or older. The median duration of first remission for the group was 11.1 months. All 142 patients received a first dose of drug, 109 patients received two doses (the recommended treatment course), and five patients received three doses. Roughly 40% of patients were treated as outpatients. Median duration of hospitalization was 24 days. The ORR was 30%. This included patients achieving a CR (defined as [a] leukemic blasts absent from peripheral blood, [b] bone marrow blasts 4 megabases).7,8 More focused analyses such as interphase fluorescence in situ hybridization (FISH) have improved the resolution of standard cytogenetics so that amplifications or deletions of 100 kilobases may be visualized.9 In the last 10 years, oligonucleotides covering the entire human genome have been printed onto arrays allowing hybridization of fluorescently labeled leukemic and control genomic DNA.10,11 This array comparative genomic hybridization (CGH) can identify copy number variation (CNV), and has been powerfully employed in identifying recurrent

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Chapter 72  Molecular Genetics of Acute Leukemia

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T ab le 72.3

Resolution Of Technologies Available To Identify Genetic Alterations In Acute Leukemia Technique

Resolution

Molecular Lesion

References

Whole genome sequencing

1 bp

13,325

Whole exome sequencing Transcriptome sequencing

1 bp 1 bp

Gene expression microarray Interphase FISH Array comparative genomic ­hybridization

– 100 kb 100 kb amplification 3–5 Mb deletion 100 kb 3–4 Mb

Point mutations, amplifications, insertions, ­deletions, translocations Point mutations, amplifications, deletions Gene expression, fusion transcript detection, point mutations Gene expression Focal amplifications or deletions Amplifications, deletions Amplifications, deletions Amplifications, deletions, translocations

11,12,331 8,332

SNP array Cytogenetics/karyotyping

326,327 328,329 244,246,330 9 10,11

deletions and amplifications in acute leukemias. Similar microarrays were designed to cover common single nucleotide polymorphisms (SNPs) for genotyping purposes, but they too have been used to identify CNVs, particularly copy number neutral loss of heterozygosity (also known as uniparental disomy) in leukemia.12 Array CGH and SNP arrays display the human genome as small probes and will not detect balanced translocations. Most excitingly, major advances in whole genome sequencing now provide single base pair resolution so that point mutations may be identified as well as chromosomal rearrangements.13 These powerful technologies are readily deployed in the diagnosis of acute leukemia and provide a complete genomic perspective of the disease; however, there are important caveats. Human genomes are full of rare and common structural variants and SNPs.14–18 Therefore, mutations can only be interpreted as being acquired in leukemogenesis if they are not present in normal somatic tissue. Thus, matched normal somatic tissue is frequently subjected to the same genomic analyses as leukemic blasts. The sequence of the human genome was completed in 2001 at 90% coverage after 10 years of collaborative and arduous work from multiple laboratories for a cost of 1 billion dollars; the draft genome sequence contained gaps that were filled in 2004, resulting in a highly accurate reference sequence currently at build 37(2012).19–21 At this writing, a sample human genome can be sequenced in 6 weeks for approximately 40 thousand dollars.22 This remarkably short processing time and reduction in cost were made possible by massively parallel sequencing technologies, collectively known as next generation (or second generation) sequencing.23 The original draft human genome sequence was generated by Sanger sequencing or dideoxynucleotide chain termination and capillary electrophoresis, first generation technologies. In second generation technologies, the addition of nucleotides occurs in parallel on multiple DNA strands and is multiplexed frequently on microchips or beads.24 This has resulted in an incredible information glut. As DNA sequencing technologies race toward the $1,000 genome, the burden has shifted toward efficient and timely informatics analysis. Some of the complexity of genome assembly can be avoided by targeted sequencing or whole exome sequencing. Here, capture technologies utilize DNA hybridization to purify all the exons of a given genome. These are then fully sequenced; of course, in this case the mutations discovered are limited only to the coding regions of the genome, and mutations within regulatory regions may also be present. Besides mutation analysis, next generation sequencing has expedited our ability to define whole genome chromatin marks,25 CpG methylation,25,26 microRNAs,27 and noncoding RNAs.28 For chromatin analysis,

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immunopurification can enrich for genomic DNA that is associated with specific histone marks that denote active or inactive gene expression. These tools will be important in analyzing the effects of mutant leukemia proteins that modify histone residues. In 2008 the entire genome sequence of a cytogenetically normal AML patient was completed for the first time.29 In 2009 further refined techniques were used to sequence a second AML genome to a higher level of coverage (98%),30 and in 2010 DNA from the first patient was subjected to deeper sequencing.31 In each patient multiple acquired somatic mutations were identified by comparison to normal skin from the same patient. Somatic mutations unique to the leukemia cells were found in coding sequences, as well as in conserved or regulatory portions of genes. All of the mutations were shown to be heterozygous and were present in the majority of the blasts. Screening of a large panel of AML samples for novel coding region mutations identified in the two sequenced genomes demonstrated two genes not previously thought to be involved in leukemogenesis that were recurrently mutated: IDH1, (mutated in 16% of cytogenetically normal AML samples),32 and DNMT3A (mutated in 22% of AML samples).31 A recent study greatly expanded this breakthrough work by deep sequencing the genomes of 12 patients with AML with a normal karyotype and 12 patients with acute promyelocytic leukemia (APL) with the t(15;17).33 Again, bone marrow and skin samples were compared, and all single nucleotide variations (SNVs) were validated by Illumina sequencing. An average of 440 SNVs per genome were identified. The number of SNVs per genome was not different between the cytogenetically normal leukemia cases and the t(15;17) leukemia cases, and the number was proportional to age. Interestingly, the number of mutations detected in HSCs from normal patients was similar and also varied with age, suggesting that mutations randomly accumulate in stem cells and that the hundreds of mutations in the AML genomes most likely preexisted before the initiating mutation that gave growth advantage to the AML clone.33 Thus only a few mutations may be relevant to the pathogenesis of the clone. An average of 11 mutations with translational consequences were present in cytogenetically normal AML genomes and 10 in APL (counting promyelocytic leukemia-retinoic acid receptor-a [PML-RARA] itself). Only a few were recurring in other AMLs, with an average of 3 recurring mutations in each cytogenetically normal AML genome and 2 recurring mutations, including PML-RARA, in the AML genomes with t(15;17).33 The complexity of mutations that contribute to the leukemic phenotype is visually appreciated in Figure 72.1, a summary of coexpression of mutations in a cohort of 398 patients with AML that were screened for mutations of eighteen gene loci.34

Hematologic Malignancies

FISH, fluorescence in situ hybridization; SNP, single nucleotide polymorphism.

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Part vii  Hematologic Malignancies  •  SECTION 2  The Acute Leukemias

Figure 72.1. Mutational complexity of acute myeloid leukemia (AML).  The Circos diagram depicts the relative frequency and pairwise co-occurrence of mutations in patients with newly diagnosed AML who were enrolled in the Eastern Cooperative Oncology Group E1900 clinical trial. The length of the arc along the outer circle corresponds to the frequency of mutations in the first gene, and the width of the ribbon corresponds to the percentage of patients who also had a mutation in the second gene listed on the opposite end of the ribbon. Pairwise co-occurrence of mutations is denoted only once, going in the clockwise direction. The frequency of occurrence in the test cohort of the 18 genes in the test panel is listed to the right of the Circos diagram. From Patel JP, Gonen M, Figueroa ME, et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012;366:1079–1089, Copyright 2012 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

Acute Myeloid Leukemia World Health Organization Classification A subset of the new WHO classification of acute myeloid leukemia is entitled “acute myeloid leukemia with recurrent genetic abnormalities”35 (see Table 72.4). These recurrent translocations occur most often in de novo acute leukemia, but are not restricted to

T ab le 72.4

World Health Organization Classification Of Acute Myeloid Leukemia AML with recurrent genetic abnormalities   AML with t(8;21)(q22;q22); RUNX1-RUNX1T1   AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11   Acute promyelocytic leukemia with t(15;17)(q24.1;q21.1); PML-RARA   AML with t(9;11)(p22;q23); MLLT3-MLL   AML with t(6;9)(p23;q34); DEK-NUP214   AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1   AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1   AML with mutated NPM1   AML with mutated CEBPA AML with myelodysplasia-related changes Therapy-related myeloid neoplasms AML, NOS Myeloid sarcoma Myeloid proliferations related to Down syndrome AML, acute myeloid leukemia; NOS, not otherwise specified. From Swerdlow, SH, Campo, E, Harris, NL, et al., Eds. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon, France: IARC Press, 2008.

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this category. Another major category of acute myeloid leukemia is AML with myelodysplasia-related changes,35 where genetic mutations and chromosomal deletions occur secondary to the mutator phenotype of the underlying myelodysplastic syndrome (MDS) in some cases. Whole genome sequencing has clarified our understanding of the clonal evolution from MDS to AML. Walter et al compared seven sets of samples from patients for whom there were paired samples of normal skin, preleukemic bone marrow diagnosed as MDS, and bone marrow involved by secondary AML.36 In each genome there were 304 to 872 somatic point mutants in coding regions or consensus splice site regions, and those point mutations with translational consequences comprised an average of 24 mutations per genome. These functional mutations occurred in a total of 168 genes over the 7 genomes. The strength of the study was that the number of mutations allowed study of clonal evolution from MDS to secondary AML. The clonal evolution described by the sequential acquisition of mutations (defined as five mutation clusters by unsupervised clustering analysis) is visually shown in Figure 72.2. A majority of the cells in the MDS sample contained the same cluster of mutations, meaning that before blasts were even detected morphologically, the marrow was involved by a clonal process. At the secondary AML stage, all the samples contained several clones, all of which had the original set of mutations, but which were defined by acquisition of new sets of mutations as well. Presumably most of these somatic mutations are “passenger” mutations, but the multiplicity of mutations tracked adds credence to the description of clonal evolution.36 The mutations that were in characterized genes ranged the gamut of genes involved in adhesion, cell death, cell cycle, differentiation, metabolism, motility, signaling, transcription, and transporters.36 In the subsequent sections, we will discuss how alterations of genes that fall in these functional classes contribute to leukemogenesis.

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Transcription Factors Promyelocytic Leukemia-Retinoic Acid Receptor-a One of the most elegant examples of the interaction between clinical and molecular advances in the treatment of acute leukemia is APL. The association between the t(15;17)(q24.1;q21.1) translocation and the characteristic morphology of APL (hypergranular blasts with frequent Auer rods or microgranular variant with dumbbell-shaped nuclei) has been known for a long time. The ability to treat APL with retinoic acid (RA) and the understanding of the molecular basis for this treatment is a compelling example of the power of molecular medicine. The initial report from China37 that all-trans retinoic acid (ATRA) could induce complete remission (CR) in APL patients actually preceded the discovery that the t(15;17) translocation involved the RARA gene on chromosome 17.38,39,40 Of four translocations associated with APL, the most common is t(15;17)(q24.1;q21.1), in which the 5′ portion of the fusion protein is encoded by the PML gene from 15q24.1 and the 3′ portion is encoded by the RARA gene from 17q21.1. The RARA gene is a ligand-dependent steroid receptor that mediates the effects of the ligand, RA, on the cell. The breakpoint is invariant in intron 2 of RARA, yielding the C-terminal portion of the fusion protein, which includes the DNA-binding, ligand-binding, dimerization, and repression domains of RARA. There are three major breakpoints in the PML gene. The most common generates PML(L)-RARA, which includes the first six exons of PML encoding 554 amino acids of PML.41 The wild-type RARA is a nuclear receptor that acts as a transcription factor and binds to retinoic acid response elements (RAREs) in the promoters of many genes, including genes important in myeloid differentiation. RARA binds as a heterodimer with retinoid X receptor protein (RXR) and acts as a transcriptional repressor until ligand (RA) binding occurs, changing the conformation of the protein and resulting in transcriptional activation.42 Target genes important for myeloid differentiation include colony-stimulating factors (granulocyte colony-stimulating factor [G-CSF]), colony-stimulating factor receptors (G-CSFRs), neutrophil granule proteins (leukocyte alkaline phosphatase, lactoferrin), cell-surface adhesion molecules (CD11b, CD18), regulators of the cell cycle, regulators of apoptosis (BCL2), and transcription factors (RARs, STATs, HOX genes) (reviewed in Ref. 43). Expression of a

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dominant negative RARA in either a murine hematopoietic cell line or primary murine bone marrow cells, followed by stimulation with granulocyte–macrophage colony-stimulating factor (GM-CSF), results in arrest of granulocytic differentiation at the promyelocyte stage.44 In the absence of RA, the wild-type RARA, present as a heterodimer with RXR on the RARE, binds to the corepressor proteins SMRT, N-CoR, mSin3, and histone deacetylases (HDACs). Deacetylation of the histones at the target gene promoter results in transcriptional repression. Ligand binding at physiologic concentrations of ATRA causes a conformational change that results in release of corepressors and recruitment of a coactivator complex (SRC-1), which associates with histone acetyltransferases (Fig. 72.3A).45 Acetylation of the histones at the target gene promoter is associated with transcriptional activation (reviewed in Ref. 43). Wild-type PML protein is normally localized in subnuclear PML oncogenic domains, also called nuclear bodies (NBs), in which other nuclear factors colocalize.46 PML may act as a tumor suppressor protein and is involved in growth suppression as well as in induction of apoptosis (reviewed in Ref. 43). Although it does not bind DNA directly, it influences transcription by interacting with both CREB-binding protein (CBP),47 a transcriptional activator, and HDACs, transcriptional repressors, possibly within the NBs. The protein encoded by the PML-RARA fusion transcript resulting from the t(15;17) is delocalized from the NBs to a microspeckled nuclear pattern.48 In APL, the PML-RARA protein binds to RAREs with similar affinity to the RARA protein and is able to heterodimerize with RXR. It acts in a dominant negative manner, competing with wild-type RARA for binding to the RAREs. It binds corepressor proteins in the absence of ligand (via the RARA portion of the protein). However, physiologic levels of ATRA (10−8 M) are not able to convert PML-RARA into a transcriptional activator; pharmacologic concentrations are required (10−6 M; Fig. 72.3B).45,49 This provides the mechanistic basis for the efficacy of treatment of APL patients with ATRA to include differentiation of the promyelocytes. Understanding of the mechanism of the response of APL to ATRA was furthered by studies of an alternative translocation, t(11;17)(q23;q21.1), which is rarely seen in patients with APL.50 Patients with this translocation are resistant to treatment with pharmacologic doses of ATRA. The fusion partner gene on chromosome 11q23 encodes ZBTB16 (previously known

Hematologic Malignancies

Figure 72.2. Clonal progression of MDS to secondary acute myeloid leukemia (AML).  This model summarizes the clonal evolution from MDS to secondary AML (sAML) in one patient. Cells in clone 1 (yellow ) contain cluster 1 mutations, 323 somatic single nucleotide variants (SNVs) present in approximately 74% of the bone marrow cells. Cells in clone 2 (orange) originated from a single cell in clone 1 and therefore contain all cluster 1 and 2 mutations. This clone became dominant in the sAML sample, in which three subsequent subclones (red, purple, and black ) evolved through serial acquisition of SNVs (clusters 3, 4, and 5). From Walter MJ, Shen D, Ding L, et al. Clonal architecture of secondary acute myeloid leukemia. NEJM 2012;366:1090–1098. Copyright 2012 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

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Part vii  Hematologic Malignancies  •  SECTION 2  The Acute Leukemias

A

Corepressor RXR RAR N-CoR

Sin3A HDAC-1

1528

Co-activator +ATRA (10-8 M)

SRC-1 RXR RAR

X

RARE

RARE

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

N-CoR

RXR RAR N-CoR

SRC-1 RXR RAR

X

RARE

Ac

Ac

N-CoR

SRC-1

?

PLZF-RARα RXR RAR N-CoR POZ

+ATRA (10-6 M)

RXR RAR

X

POZ

RARE

Core-Binding Factor Translocations The t(8;21) is present in approximately 15% of patients with acute myeloid leukemia,56,57 and the RUNX1 (runt-related transcription factor 1, formerly called AML1) gene, cloned from the t(8;21) (q22.3;q22) breakpoint,58,59 is mutated in another 3% of AML. The activity of the murine counterpart of RUNX1 was first described as part of the core-binding factor (CBF), which binds to a core enhancer sequence of the Moloney murine leukemia virus long terminal repeat.60 Another component of CBF, the non-DNA-binding CBFb was found to be associated with inversion 16 in AML.61

N-CoR

Sin3A HDAC-1

C

as promyelocytic zinc finger), a transcriptional repressor. The N-terminal portion of the fusion protein encoded by ZBTB16 includes the N-terminal POZ/BTB protein interaction domain, transcriptional activation and repression domains, and a variable number of zinc fingers important for protein and DNA interactions (reviewed in Refs. 43,45). ZBTB16 interacts with N-CoR, SMRT, mSin3A, and HDAC1 via the POZ/BTB domain,51,52 and therefore contributes a second binding site for corepressor proteins. Therefore, although pharmacologic doses of ATRA induce release of corepressors from the RARA portion of the fusion protein, the corepressors binding to ZBTB16 are unaffected (Fig. 72.3C).43,53 Significantly, concomitant treatment of cells with HDAC inhibitors such as trichostatin A (TSA) restores ATRA sensitivity, since TSA inhibits the deacetylase activity of the corepressors on the ZBTB16 moiety.49,52 Subsequent studies have demonstrate that PML-RARA recruits the polycomb-repressive complex 2 (PRC2) to the promoters of its gene targets. The PRC2 has a H3K27 methylase activity and can initiate gene repression through trimethylation of H3K27.54 ZBTB16-RARA additionally recruits polycomb-repressive complex 1 (PRC1); treatment with RA releases PRC2 from both PML-RARA and ZBTB16-RARA, but does not release PRC1 from ZBTB16-RARA.55

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+ATRA (10-6 M)

RARE

Sin3A HDAC-1

Figure 72.3. Model for the role of nuclear corepressors and retinoid acid receptor a (RARA) fusion proteins in the pathogenesis and treatment of acute promyelocytic leukemia.  A: In the absence of all-trans retinoic acid (ATRA), RARA, promyelocytic leukemia (PML)-RARA, and promyelocytic leukemia zinc finger (PLZF; now known as ZBTB16)-RARA associate with N-CoR/sin3A/HDAC1 corepressor complex, which deacetylates histone tails, resulting in a compressed chromatin and transcriptional repression. Binding of ATRA at a physiologic concentration induces a conformational change in RARA, causing release of the corepressor complex and binding of coactivator (SRC-1) with histone acetyltransferase activity. Acetylation (Ac) of histone tails opens up the chromatin, facilitating transcriptional activation. B: In the case of PML-RARA protein, pharmacologic doses of ATRA are required to achieve dissociation of the N-CoR repressor complex. C: Because of additional interactions of the PLZF (ZBTB16) moiety of PLZF-RARA fusion protein with corepressors, they do not dissociate even in the presence of pharmacologic doses of ATRA. Therefore, the chromatin still remains in the repressed state. From Guidez F, Ivins S, Jhu J, et al. Reduced retinoic acid-sensitivities of nuclear receptor corepressor binding to PML- and PLZF-RARa underlie molecular pathogenesis and treatment of acute promyelocytic leukemia. Blood 1998;91:2634–2642, with permission.

PML-RARα Sin3A HDAC-1

B

X

RARE N-CoR

Finally, the fusion partner of RUNX1 in t(8;21), named RUNX1T1, or ETO (eight-twenty-one), also encodes a transcriptional regulator.62 A gene related to RUNX1T1, CBFA2T3 (or MTG16), is involved in yet another translocation involving RUNX1, t(16;21).63 The structures of the fusion proteins resulting from these CBF translocations are shown in Figure 72.4. RUNX1 is located at chromosome 21q22.3 and is encoded by 12 exons over 260 kb of DNA. The N-terminal portion of the protein contains the runt homology domain (RHD), which is homologous to the Drosophila runt protein64 and is responsible for the official HUGO name, RUNX1. This is the DNA-binding domain and it is mutated in familial platelet disorder (FPD) and in AML associated with RUNX1 mutations.65,66 CBFB interacts via this domain and changes the conformation of RUNX1 to increase DNA-binding affinity.67 C-terminal to the RHD are potential MAP kinase phosphorylation sites, followed by three weak activation domains, a nuclear matrix target signal, a dimerization domain, and sequences that are recognized by corepressor proteins (reviewed in Ref. 68). The CBFs are essential for hematopoietic development. Gene deletion of either Runx169 or Cbfb70 in mice results in fetal death at E11.5 to 12.5. These embryos lack all fetal hematopoiesis. Further transgenic experiments have demonstrated that Runx1 is essential for development of HSCs in the aorta/gonadal/mesodermal (AGM) region, the source of definitive hematopoiesis.71 The essential role of RUNX1 in hematopoietic development appears to be through its function as a transcriptional activator. It regulates lymphoid genes such as B-cell tyrosine kinase, T-cell receptor a and b,72 cytokines (interleukin-3 [IL3],73 GM-CSF74), and granulocyte proteins (myeloperoxidase and neutrophil elastase),75 to name a few. In addition, RUNX1 acts as a transcriptional repressor of genes such as p21Waf1/Cip1 via interactions with the mSin3a corepressor76 and with SUV39H1, a histone methyltransferase.77 The ETO gene, now called RUNX1T1, was cloned from the t(8;21) fusion58 and is the mammalian homolog of the Drosophila

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N-CoR

Dimerization mSin3A mSin3A

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A RUNX1⁄ RUNX1T1

DNA binding

TAF110

HHR

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HDAC-2

HDAC-1 HDAC-3

ZF

HDAC-1, 2, 3

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DNA binding

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C ETV6/RUNX1

PNT

DNA binding

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Dimerization

D Coiled-coil

CBFβ ⁄ SMMHC

RUNX1

Figure 72.4. Schematic diagram of the t(8;21), t(16;21), t(12;21), and inv(16) fusion proteins with known corepressor contacts.  A: t(8;21) RUNX1/ RUNX1T1. The RUNX1 portion is shown in light pink, with the DNA-binding domain indicated. The RUNX1T1 portion is the dark pink box with domains conserved between RUNX1T1 and its Drosophila homolog in light gray boxes. Known contacts with corepressors and histone deacetylases are shown. B: t(16;21) RUNX1MTG16 (now known as CBFA2T3). RUNX1 is shown as a light pink box, and MTG16 is shown in a similar manner to RUNX1T1 in A. C: t(12;21) ETV6-RUNX1. ETV6 is the dark pink box, with the conserved pointed (PNT) domain indicated. The RUNX1 portion is the light pink box. Interactions with corepressors and HDACs are shown. D: Inv(16) CBFB-SMMHC (now known as MYH11). The CBFB portion, which interacts with RUNX1, is light pink, and the SMMHC is dark pink, with the coiled-coil domain indicated as well as the C-terminal portion, which is necessary for interaction with mSin3A and HDAC8.77 HHR, hydrophobic heptad repeat; ND, nervy domain; TAF110, a domain with homology to the TAF110 coactivator; ZF, zinc finger domain. From Hiebert SW, Lutterbach B, Amann J. Role of corepressors in transcriptional repression mediated by the t(8;21), t(16;21), t(12;21), and inv(16) fusion proteins. Curr Opin Hematol 2001;8:197–200, with permission.

nervy gene.78 The four homology domains shared with the Drosophila protein include a region of similarity to TAF110, a hydrophobic heptad repeat (HHR), an ND domain of undetermined function, and two zinc finger motifs that may be a protein– protein interaction domain (Fig. 72.4A).68 RUNX1T1 does not appear to bind DNA specifically on its own. However, it may act as a corepressor protein.79 It associates with N-CoR and mSin3A, and directly binds to the Class I HDACs, HDAC-1, HDAC-2, and HDAC-3 (Fig. 72.4A).80 In the t(8;21) translocation, the RUNX1 gene is fused to the RUNX1T1 gene on chromosome 8. The breakpoint in the RUNX1 locus is between exons 5 and 6,81 yielding a fusion protein with the N-terminal 177aa of RUNX1.58 In this fusion protein, the DNAbinding domain is present, but the C-terminal activation domains, corepressor interaction sites, and nuclear localization signals (NLSs) of the wild type are not present (Fig. 72.4A).68 The breakpoint in the RUNX1T1 gene occurs in the introns between the first two alternative exons of RUNX1T1, resulting in the inclusion of almost all of the coding region for RUNX1T1 in the fusion transcript.58 The RUNX1-RUNX1T1 protein specifically binds to the same DNA-binding site as RUNX1 and can heterodimerize with CBFB.82 Therefore, the RUNX1-RUNX1T1 protein can act as a dominant negative inhibitor of wild-type RUNX1. However, cotransfection experiments demonstrated that RUNX1-RUNX1T1 can also function as an active transcriptional repressor, not only inhibiting activation of a reporter gene containing the GM-CSF receptor

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promoter by cotransfected RUNX1, but also reducing the expression of the reporter gene below baseline.83 The ability of RUNX1RUNX1T1 to act as a transcriptional repressor depends on its association with HDACs (via RUNX1T1; Fig. 72.4A), since the HDAC inhibitor TSA can abrogate effects of RUNX1-RUNX1T1 on the cell cycle.80 In addition, examination of the M-CSFR gene in Kasumi-1 cells (high expressors of RUNX1-RUNX1T1) reveals an increase in histone H3Lys9 methylation.84 Targets of RUNX1RUNX1T1 repression are presumed to include genes important for granulocyte differentiation. In addition, RUNX1-RUNX1T1 represses the tumor suppressor genes P14ARF and NF1.85,86 P14ARF stabilizes TP53 by antagonizing MDM2, an inhibitor of TP53.87 Therefore, repression of P14ARF reduces the checkpoint control path of TP53, and may be a key event in t(8;21) leukemogenesis. The promoter of P14ARF has eight RUNX1 DNA-binding sites, and wild-type RUNX1 can activate P14ARF. However, transfection of RUNX1-RUNX1T1 into cells that have only low levels of RUNX1 and high endogenous levels of P14ARF results in repression of P14ARF. Samples of bone marrow from patients with t(8;21) leukemia have low levels of P14ARF transcript by quantitative real-time polymerase chain reaction analysis.85 Surprisingly, expression of RUNX1-RUNX1T1 in myeloid progenitor cells inhibits cell cycle progression. However, this may contribute to leukemogenesis by allowing time for accumulation of mutations in a cell immune from TP53-induced apoptosis due to inactivation of P14ARF.85

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Finally, inversion 16, present in about 8% of AML cases, involves the CBF complex member CBFB and is associated with a morphologically distinct subset of AML, previously considered M4Eo in the French-American-British Cooperative Group Classification. This disease is a myelomonocytic leukemia with abnormal eosinophils that have dark purple as well as orange granules.35 This cytogenetic abnormality in which the CBFB gene is fused to the smooth muscle myosin heavy-chain (SMMHC) gene, MYH11, results in fusion of the first 165aa of CBFB to the C-terminal coiled-coil region of SMMHC protein (Fig. 72.4D).61 A C-terminal region of SMMHC/MYH11 is necessary for the activity of CBFB/MYH11 as a transcriptional corepressor, and this region also associates with mSin3a and HDAC8. Presumably CBFB/MYH11, which cannot bind DNA on its own, interacts with RUNX1 to form a transcriptional repressor complex.88 A number of experiments demonstrate that the CBF translocations are necessary but not sufficient for induction of leukemia. In order to determine whether expression of RUNX1-RUNX1T1 is sufficient to produce leukemia, mice were generated with a conditional Runx1-Runx1t1 knock-in allele using the Lox-Cre system. This obviates the embryonic lethality that results when Runx1-Runx1t1 is introduced into transgenic mice (recapitulating the phenotype of the Runx1 null mouse). No leukemia developed in 20 Runx1-Runx1t1+ mice in 11 months, and no hematologic abnormality was detected except for a slight increase in the number of hematopoietic colony-forming cells. Interestingly, expression of Runx1-Runx1t1 did not cause a significant block in differentiation of hematopoietic precursors. When the mice were mutagenized with the DNA alkylating agent, ethylnitrosourea (ENU), 31% of the mice developed granulocytic sarcoma or AML.89 This supports the hypothesis that several genetic “hits” are necessary for the development of leukemia. Another study used retroviral transduction of CD34+ human hematopoietic progenitor cells to investigate the effect of RUNX1-RUNX1T1 on proliferation and differentiation.90 In mice reconstituted with RUNX1-RUNX1T1 expressing HSCs there was an expansion of the HSC population and immature myeloid cell populations, although the mice did not develop acute leukemia.91 Therefore, the expression of RUNX1-RUNX1T1 promotes accumulation of immature cells and prolongs the period of time during which progenitor cells may accumulate additional mutations. Further support for the hypothesis that genetic mutations besides a mutant RUNX1 locus are necessary for development of acute leukemia comes from the study of patients with FPD with a propensity to develop AML (FPD/AML). These patients have mutations in one allele of RUNX1.92 They have defective platelets and progressive pancytopenia, and develop myelodysplasia and a high incidence of AML with age. However, second mutations appear to be necessary before progression to AML occurs. This implies that acquisition of additional mutations is necessary for development of leukemia.

CCAAT/Enhancer Binding Protein-a CCAAT/enhancer binding protein-a (CEBPA) is a transcription factor that regulates granulocytic differentiation.93 Cytogenetically silent mutations of CEBPA have been identified in about 10% of AML cases.94 In addition, mutations in other oncogenes in leukemia often lead to CEBPA downregulation. For example, RUNX1RUNX1T1 represses the CEBPA promoter.95 FLT3-ITD activation of ERK leads to modification of CEBPA which reduces its activity.96 In addition, the CEBPA promoter is methylated in half of AML cases.97 The importance of CEBPA in granulocyte differentiation is demonstrated by the lack of mature granulocytes in Cebpa knockout mice,98 while its conditional expression triggers granulocyte differentiation in bipotential precursors.99 CEBPA

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transactivates the genes for G-CSF and GM-CSF receptors and several granulocyte-specific proteins. The gene produces two proteins using alternative start sites. The larger and predominant 42-kD protein consists of two N-terminal transactivating domains, with a C-terminal bZIP domain consisting of a basic (b) region that mediates DNA sequence binding and a leucine zipper (ZIP) domain that mediates dimerization.100 The shorter 30-kD protein is transcribed from an alternative internal start site, and retains its bZIP domain but lacks the first transactivation domain. Mutations in CEBPA are of two types: C-terminal bZIP domain mutations and N-terminal truncating mutations that lead to enhanced production of the 30-kD protein.101,102,103 The former type inhibits dimerization and DNA binding. The latter type dimerizes with the long form, but inhibits transactivation by the dimer, functioning in a dominant negative manner. In two-thirds of AML with CEBPA gene mutations, one allele has an N-terminal mutation and the other allele has a C-terminal variant. Several families with familial AML have been documented to have germline CEBPA N-terminal mutations, and progression to AML has been shown to correlate with a somatic mutation in the C-terminus.104 CEBPA mutations most often occur in intermediate-risk AML with normal cytogenetics, and these patients have a significantly improved outcome.94 Interestingly, mutation of CEBPA at both alleles is associated with a better overall survival than mutation of CEBPA at a single allele.105 Approximately onethird of AML with CEBPA mutations also have FLT3 mutations, and the CEBPA mutation confers a more favorable prognosis in this group of AML patients as well.34

GATA1 GATA1 is a zinc finger transcription factor that regulates erythroid and megakaryocytic differentiation. In the acute megakaryoblastic leukemia (AMkL) that occurs in children with Down syndrome (DS), mutations of GATA1 have been described in all tested cases.106,107,108 Familial missense mutations in GATA1 result in a syndrome of dyserythropoietic anemia and thrombocytopenia, while conditional knockout of Gata1 in megakaryocyte precursors in mice leads to thrombocytopenia and megakaryoblast proliferation. Approximately 10% of DS patients develop transient myeloproliferative disorder (TMD) in the neonatal period (usually in the first week, almost always within the first 2 months of life), and these patients have mutations in GATA1.106,109 About one-third of DS patients with TMD later develop AMkL within 5 years, and identical GATA1 mutations have been identified in the AMkL blasts as were present in the TMD. A large study demonstrated that there is no difference in the GATA1 mutations present in patients who just developed TMD compared to patients who went on to AMkL.110 The mutations in GATA1 result in transcription of a truncated form that lacks its N-terminal transactivation domain, GATA-1s. This shorter form has similar DNA-binding activity but reduced transactivation compared to wild-type, and it therefore can act in a dominant negative manner. By introducing the truncated GATA1 into GATA1-deficient fetal liver progenitor cells by retroviral transduction, Muntean and Crispino demonstrated that GATA-1s restored terminal differentiation but that abnormal proliferation occurred.111 Interestingly, analysis of a knock-in mouse model where Gata-1s replaces wild-type Gata-1 demonstrates that fetal liver megakaryocytes abnormally proliferate, but that megakaryocyte proliferation is normal in adult bone marrow.112 This may explain why TMD occurs in the neonatal period in DS patients. Research into the gene(s) on chromosome 21 that cooperate with mutant GATA1 to produce AMkL in DS children is ongoing; a recent mouse study reported evidence that Dyrk1a (dual-specificity tyrosine-phosphorylation-regulated kinase 1A) can cooperate with mutant Gata1 to promote megakaryoblast expansion.113

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AMkL in Down syndrome is sensitive to cytosine arabinoside/ anthracycline-based chemotherapy, with event-free survival rates of 80% to 100%.114 Interestingly, a putative target of GATA1 regulation is cytidine deaminase (CDA), which inactivates ara-C by deamination to the inactive uridine arabinoside. Presumably failure of GATA-1s to transactivate CDA increases the efficacy of ara-C treatment.111

Epigenetic Factors Modifying Chromatin and DNA IDH1/2 and TET2 Mutations In the whole genome sequencing of blasts from a patient with cytogenetically normal AML, mutations in isocitrate dehydrogenase 1 (IDH1) were detected and were found to be present in 16% of a panel of 80 cytogenetically normal AML samples.30 IDHI mutations had previously not been described in AML, though IDH1/2 mutations are common in gliomas.115 In a further screen of AML DNA, it was found that IDH1/2 mutations are mutually exclusive with mutations in TET2 (ten-eleven translocation 2) in de novo AML (Fig. 72.5). Additional findings suggesting a functional link between the products of these two genes are that AMLs with mutations in IDH1 have similar patterns of DNA hypermethylation as AMLs with mutations in TET2.116 In addition, these two mutational categories of AML share patterns of aberrant gene expression at the level of 93%.116 The link became clear upon further investigation of the enzymatic activity of IDH1/2 and TET1/2. Wild-type IDH1/2 catalyzes production of a-ketoglutarate (a-KG), whereas the neomorphic enzymatic activity of mutant IDH1/2 produces 2-hydroxyglutarate (2-HG). a-KG–dependent enzymes such as histone demethylases and TET1/2 are inhibited by 2-HG, which is structurally similar enough to a-KG that it can bind in place of a-KG and inhibit these enzymes.117 The TET proteins catalyze the conversion of 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC), which is thought to be a first step in demethylation of the cytosine.118 Experiments using a fluorescent-tagged antibody to

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5hmC demonstrate that cotransfection of plasmids encoding IDH1 and TET2 result in a global increase in 5hmC, whereas cotransfection of plasmids encoding mutant IDH1 and wild-type TET2 fail to demonstrate an increase in 5hmC.117 Therefore, mutations in IDH1 and TET2 both produce increased DNA methylation: mutant IDH1 by inhibiting TET2, and mutant TET2 by loss of its ability to convert 5mc to 5hmC, and thereby promote demethylation.116–119 The significance of these changes for pathogenesis of AML is demonstrated by experiments in which either stable expression of mutant IDH1 or shRNA-mediated knock-down of TET2 in primary mouse bone marrow cells resulted in increased c-kit expression and decreased expression of the mature myeloid markers Mac-1 and Gr-1 by flow cytometric analysis.116 Therefore, the hypermethylation and resultant silencing of genes as a result of IDH1 and TET2 mutations presumably inhibit myeloid differentiation and thereby promote development of AML. This mechanism suggests new avenues of molecular therapy in that drugs that mimic a-KG or that inhibit the mutant IDH1/2 may restore proper enzymatic function to histone demethylases and TET2, promoting normal histone and DNA methylation patterns.117 In studies performed to date, mutations in IDH1 and TET2 have not been shown to have a significant impact on survival.105

DNMT3A Deep sequencing of DNA from the first AML patient with a normal karyotype revealed additional mutations, including a mutation in DNA methyltransferase (DNMT3A). Sequencing of the DNMT3A gene in DNA from 281 AML patients revealed a mutation rate of 22.1%. The DNMT3A mutations were found in patients with intermediate-risk cytogenetics. As a group these patients had a mean overall survival significantly shorter than the patients with wild-type DNMT3A: 12.3 months compared to 41.1 months.31 The mutations detected in patients with AML map predominantly at the interface where they disrupt tetramerization of the DNMT3A molecules. Dimeric DNMT3A molecules still have methylase activity, but they dissociate from DNA more quickly than wild-type, so

Hematologic Malignancies



Figure 72.5. IDH1/2 mutations are mutually exclusive with mutations in TET2 in de novo AML.  A: Circos diagram revealing relative frequency and pairwise co-occurrences of mutations in IDH1 and IDH2 in a cohort of 385 patients with de novo AML. B: Circos diagram revealing relative frequency and pairwise co-occurrences of mutations in TET2 in a cohort of 385 patients with de novo AML. Reprinted from Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 2010;18:553–567, with ­permission from Elsevier.

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that fewer cytosines in a CpG island are methylated.120 The effect on tetramerization explains why the DNMT3A mutations are dominant negative, usually occurring in just one allele. Global methylation does not seem to be affected in DNA from AML with mutated DNMT3A, but analysis of DNA methylation by MeDIP-Chip (methylated DNA immunoprecipitation-Chip) analysis in a matched set of DNAs from 5 AML patients with mutated DNMT3A and 5 AML patients with wild-type DNMT3A demonstrated 182 genomic sites where the DNA from patients with mutated DNMT3A was hypomethylated.31 Studies of DNA methylation in Dnmt3a-null murine HSCs demonstrated a complex story with poor correlation between changes in methylation sites and changes in gene expression comparing wild-type to Dnmt3a-null HSCs. However, changes in gene expression patterns may be due to changes in methylation of regulatory regions of directly affected genes, whose expression then alters regulation of many other genes by mechanisms other than methylation. Transcriptional profiling of Dnmt3a-null HSCs did reveal that genes involved in the multipotency of normal HSCs were upregulated, whereas genes necessary for differentiation of the HSCs were downregulated. This suggests that the DNMT3A mutations may contribute to the block in differentiation that occurs in leukemic blasts. Interestingly the Dnmt3a-null mice have not yet developed leukemia, suggesting that DNMT3A mutation alone is not sufficient for leukemogenesis.121

Mixed Lineage Leukemia: 11q23 Translocations A transcriptional activator that is characteristically rearranged in infant leukemia, therapy-related leukemia, and mixed phenotype acute leukemia is the mixed lineage leukemia gene (MLL), which maps to chromosome 11q23 (reviewed in Ref. 122). The MLL gene consists of 34 exons over 100 kb encoding a 3,969 aa protein.122 MLL is the mammalian homolog of trithorax, a Drosophila transcriptional regulator that positively regulates homeobox genes.123 Homeobox genes are a large family of genes which are developmental regulators essential for growth and differentiation. They were first identified in Drosophila during the study of genes whose mutations led to developmental abnormalities involving misassignment of body segment identity.124 The mammalian homologs consist of 39 HOX genes, which are important in mammalian development and cell fate determination.125 Analysis of chimeric mice reconstituted with Mll-deficient embryonic stem cells demonstrates that MLL expression is required for definitive hematopoiesis and expansion of HSCs in the AGM region.126 Wildtype MLL appears to be responsible for the regulation of homeobox gene expression during development, including HOXA9, HOXC8, HOXA7, HOXA10, HOXC6, and HOXC9.126,127,128,129 Wild-type MLL regulates HOX gene expression by methylation of histone H3 lysine (H3K4), requiring the SET domain. The SET ALL

14.9% 8.5% 2.7%

domain is a protein domain shared by a number of transcriptional regulators that have histone methyltransferase activity.130 H3K4 methylation is associated with transcriptional activation. MLL rearrangements involve approximately 10% of chromosomal rearrangements overall in patients with ALL, AML, and MDS, and are associated with poor prognosis.131 More than 60 different partner loci have been identified132; the 10 most common translocation partners are listed in Figure 72.6, along with a pie chart demonstrating the frequency of the translocation in pediatric vs. adult leukemia and AML vs. ALL. In pediatric and adult ALL, the most common translocation partners are the AFF1 gene (previously known as AF4) at 4q21.3 in t(4;11), the MLLT3 gene (previously known as AF9) at 9p22 in t(9;11), and the MLLT1 (ENL) or ELL genes at 19p13.3 and 19p13.1, respectively, in t(11;19). Interestingly, the t(9;11) and t(11;19) are also associated with AML133; thus the name mixed lineage leukemia, since the gene is involved with leukemias of both myeloid and lymphoid origins. The breakpoints of 11q23 usually occur between exons 8 and 11 of MLL (Fig. 72.7),122 leaving approximately the N-terminal 1,400 amino acids of the MLL protein.134 The retained protein contains three AT-hook sequences thought to bind DNA at the minor groove,135 the CxxC domain that specifically binds unmethylated DNA, and a lysine-rich RD2 region. The conserved C-terminal SET domain is usually lost, though it is the domain that has the H3K4 histone methyltransferase activity that is the mechanism by which the wild-type protein activates HOX gene transcription (Fig. 72.7).122 There has been much investigation as to the role of the multiplicity of fusion partners of MLL. Two of the most common fusion partners, MLLT10 (AF10) and MLLT1, associate with DOT1L, which is a histone methyltransferase with a different activity than wild-type MLL, as it methylates histone H3 lysine 79 (H3K79me).136 This histone modification is also associated with transcriptional activation, however. DOT1L is one of many proteins in a Dot.com complex, which consists of DOT1L, AF10 (MLLT10), AF17 (MLLT6), and ENL (MLLT1) (Fig. 72.8).122,136 Thus the plethora of MLL fusion partners begins to make sense, as many of them normally associate together in complexes that regulate transcription. Therefore the fusion protein associates with its usual partners, but the H3K79me marking occurs at different sites than usual, as the N-terminal MLL protein has the DNA-binding sites that bring the activating histone methyltransferase activity to MLL target genes. Demonstration of an abnormal increase in the H3K79 methylation pattern at target HOXA genes confirms this hypothesis, and this increase was shown to be dependent on DOT1L function.137 Dependence on functional DOT1L not only for the H3K79 methylation pattern but also for the development of leukemia has been demonstrated with a conditional knockout mouse model for Dot1L expression.137 If cells AML 23.3%

1.5%

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6.2%

2.5%

66%

30.4%

10.9% 14.5%

10.1% MLL–AFF1 MLL–ELL

MLL–ENL MLL–AF17

MLL–AF9 MLL–SEPT

5.4%

MLL–AF10 Others

MLL–AF6

Figure 72.6. The distribution of the most common MLL chimaeras in acute lymphoid and myeloid leukemias.  In MLL-rearranged ALL, AFF1, AF9 (now known as MLLT3 ), ENL (now known as MLLT1 ), and AF10 (now known as MLLT10 ) account for 90% of all the translocations, and in AML 70% of MLL translocations involve AF9 (MLLT3 ), ENL (MLLT1 ), AF10 (MLLT10 ), ELL, and AF17 (now known as MLLT6 ). AFF1 is the most frequent translocation partner of MLL, which is typically associated with ALL. Reprinted by permission from Macmillan Publishers Ltd: Mohan M, Lin C, Guest E, Shilatifard A. Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer 10:721–728, copyright 2010.

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Figure 72.7. Structure of wild-type and leukemia-associated mixed lineage leukemia (MLL) proteins.  Top: Domain architecture of wild-type MLL. Cleavage of MLL (denoted by the blue arrow ) results in 320-kDa MLLN and 180 kDa MLLC fragments, which noncovalently associate. Domains within MLLN include three AT-hooks (red ), two subnuclear localization motifs (SNL1 and -2) (purple ), a DNMT1 homology region (CxxC) (green), four plant homeodomain (PHD) fingers (blue ), an atypical bromodomain (orange ), and a FYRN domain (open circle ). The breakpoint cluster region (BCR) spans an 8.3-kb region encompassing exons 7 to 13 (according to the new nomenclature) and is the site of chromosomal translocations involving MLL. Between the CxxC and the first PHD finger is repression domain 2 (RD2). A host cell factor-binding motif (HBM) (yellow ) is found between the bromodomain and PHD3. MLLC contains a transactivation domain (TAD) (filled oval ), a FYRC domain (open square), and a C-terminal SET domain (dark blue ). Middle: Chromosomal translocations involving MLL result in chimeric MLL-fusion proteins that include the N-terminal sequence of MLL up to the BCR (dotted vertical line), followed by one of several different fusion partners. Also shown are examples of fusion partner proteins. MLL-fusion proteins invariably retain AT-hooks, SNL and -2, and the CxxC domain of MLLN, while losing the downstream PHD fingers and further C-terminal domains, including the SET domain. Bottom: MLL is also prone to internal partial tandem duplications (MLL-PTD), leading to duplication of MLL sequences comprising the AT-hooks, SNL1 and -2, and the CxxC domain, that are inserted at the BCR. Republished with permission of Annual Reviews, Inc. Muntean AG, Hess JL. The pathogenesis of mixed lineage leukemia. Annu Rev Pathol Mech Dis 2012;7:283–301.

are transduced by a retrovirus expressing MLL-MLLT3, smaller colonies of blast-like cells grow if the Dot1L is inactivated by the introduction of Cre. In addition, the colonies then demonstrate morphologic signs of differentiation. If the MLL-MLLT3 cells are transplanted into irradiated mice, in vivo colonies are also reduced if Dot1L is inactivated, and these colonies have more differentiation and fewer proliferating cells. 137 In addition, methylation analysis of HOXA and MEIS1, target genes of MLL, demonstrate reduced H3K79me2 modification after disruption of DOT1L. Even more dramatic is the effect of a small molecule inhibitor of DOT1L, EPZ004777, which specifically inhibits DOT1L and prevents its H3K79 methylase activity.138 The growth of MLL-rearranged cell lines such as MV4-11 and MOLM-13 is inhibited by EPZ004777 after several days of exposure in cell culture, and levels of HOXA9 and MEIS1 expression are decreased. However, the growth of a leukemia cell line in which MLL is not rearranged (Jurkat) was not affected by EPZ004777. In addition, treatment of a mouse xenograft model of MLL with EPZ004777 resulted in a statistically significant increase in median survival. Therefore, inhibition of DOT1L holds promise for targeted treatment of MLL leukemia.138 Data also exists for participation of MLL fusion proteins with other transcriptional regulatory complexes. Purification of the proteins involved in the super elongation complex (SEC) revealed participation of several of the MLL translocation partners, including AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), and ELL (Fig. 72.8).139 The SEC complex also contains elongation factors such as ELL2, ELL3, P-TEFb, EAF1, and EAF2, and it functions to promote transcriptional elongation by RNA polymerase II.

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Therefore, interaction of this complex with the MLL fusion protein may provide another mechanism for increasing HOX target gene transcription.122,139 The end result of the recruitment of these transcriptional regulatory complexes by the MLL fusion protein is thought to be abnormally sustained homeobox (HOX) gene expression. Usually HOX genes are expressed highly during early development, but then are downregulated during hematopoiesis. However, the MLL fusion protein inappropriately activates the HOX genes.122 Microarray analysis of MLL leukemia cells supports this hypothesis, as high levels of HOXA9, HOXA5, HOXA4, and HOXA10, as well as MEIS1, characterize the gene expression profile of leukemias with MLL translocations.140,141 MLL rearrangements are associated with several unique types of leukemia. First, in infant acute leukemia (birth to 1 year), there is a 60% to 80% incidence of 11q23 rearrangement.142 Second, in acute leukemias related to treatment with DNA topoisomerase II inhibitors, there is a 70% to 90% incidence of MLL rearrangements, particularly t(4;11)(q21;q23) and t(9;11) (p21-22;q23).143,144 Topoisomerase II is an enzyme involved in unwinding of DNA during replication and transcription. It does so by producing double-stranded in the DNA, after which (ds) breaks the ends are rejoined by a ligase activity of topoisomerase II. Topoisomerase II inhibitors such as epipodophyllotoxins inhibit this ligase function so DNA double strand breaks accumulate, triggering apoptotic events. In MLL there are 11 sites similar to topoisomerase II consensus binding sites in the breakpoint cluster area.145 Therefore, if DNA ds breaks created by the topoisomerase II are incorrectly religated, translocations in MLL are likely to

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Menin

MLL • AF10 • ENL • AF9

LEDGF

Figure 72.8. Proposed mechanism by which mixed lineage leukemia (MLL) fusion proteins increase transcription of Hoxa9.  MLL fusion proteins lose a large carboxy-terminal portion that includes the H3K4me3 writing methyltransferase SET domain, retain the chromatin-targeting property, and also acquire aberrant transactivation mechanisms through MLL fusion partners. On the left, a subset of MLL fusions MLL-AF10 (MLLT10), MLL-ENL (MLLT1), and MLL-AF9 (MLLT3), directly interact with DOT1L through the MLL fusion partner and induce the methylation of H3K79 at Hoxa9. Some other MLL fusions, MLL-AF4 (AFF1), MLL–AF5q31 (AFF4), and MLL-ELL, interact with and recruit the P-TEFb transcription elongation complexes to HOXA9. On the right, DOT1L complexes (DOT1L–AF10-AF17-ENL [or AF9]) associate with P-TEFb complexes through the shared component ENL. Reprinted by permission from Macmillan Publishers Ltd: Chi P, Allis CD, Wang GG. Covalent histone modifications-miswritten, misinterpreted and mis-erased in human cancers. Nat Rev Cancer 2010;10:457–469, copyright 2010.

occur. Interestingly, infant leukemia with MLL translocations has a similar distribution of breakpoints, whereas sporadic cases of acute leukemia have more random breakpoints.146 This observation has triggered speculation that in utero exposure to environmental topoisomerase II inhibitors such as flavonoids may have a role in the etiology of infant leukemia.147 Recently a different type of MLL gene alteration involving an in-frame partial tandem duplication of exons 5 to 11 has been described in approximately 4% to 7% of patients with AML148 (Fig. 72.7). This occurs in the absence of visible chromosome abnormalities and is often associated with FLT3 mutations.148 This mutation retains the C-terminal SET domain, unlike all known MLL fusions resulting from balanced translocations. A mouse knock-in model replacing one copy of Mll with the MllPTD (MllPTD/WT mice) results in overexpression of Hoxa7, Hoxa9, and Hoxa10 in bone marrow, blood, and spleen.149 Inspection of the promoter of Hoxa7 and Hoxa9 by chromatin immunoprecipitation assay demonstrates an increase in H3K4 methylation, as would be predicted due to the retention of the SET domain (Fig. 72.7). The latency of development of leukemia appears to be shorter for MLL rearrangements than for other leukemogenic rearrangements. In studies of twins who develop infant leukemia, those bearing a shared MLL rearrangement have a concordance of nearly 100% in the first year of life, whereas in twins sharing another rearrangement, the concordance is 25% and the time to development of leukemia may be years instead of months.150,151 Similarly, therapy-related leukemias based on MLL rearrangement occur sooner after therapy than those occurring after alkylating agents or radiation, usually with 7q- or 5q-cytogenetics.144,152 This suggests that the oncogenic fusion protein produced by the MLL rearrangement can deregulate the cell without the accumulation of many secondary mutations. However, in genetic experiments in mice where the Mll-Mllt3 fusion gene is knocked in, there is still a latency of 6 months before development of acute leukemia, suggesting that some secondary mutations are necessary.153 An additional reflection of the potency of MLL rearrangements is that they are a poor prognostic indicator in infant leukemia, ALL, and most AML cases.142 This section demonstrates that mutations in genes involved in epigenetic regulation have emerged as a significant mechanism of leukemic transformation. In addition to the genes discussed above, mutations in ASXL1 (addition of sex combs-like 1), a member of the polycomb-repressive deubiquitylase complex, and mutations in EZH2 (enhancer of zeste homolog 2), an H3K27 methyltransferase in the PRC2, have been found in AML that is secondary to MDS or myeloproliferative diseases. This enlarging group of genes has led Shih et al. to propose two additional categories of genes involved in the pathogenesis of AML besides the Class I genes promoting proliferation and the Class II genes promoting block in differentiation. These two additional categories are: (1) Mutations in genes involved in the hydroxymethylation

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ENL and AF9

AF10 DOT1L complex

MLL • AF4 • AF5q31 • ELLs LEDGF

Menin AF5q31 and AF4 ELL1, ELL2 EAF and ELL3

DOT1L

AF17

P-TEFb transcription elongation complex

CDK9 and Cyclin T (p-TEFb)

?

Pol II CTD (Ser2-P)

Hoxa9 transcripts elongated

pathway (IDH1/2, TET2), and (2) Mutations in genes involved in epigenetic modification (DNMT3A, ASXL1, MLL).154

Kinases FLT3 Mutations FLT3 may be the single most commonly mutated gene in AML (reviewed in 155). Originally cloned from CD34+ HSCs, it encodes a type III receptor tyrosine kinase. FLT3 ligand (FL) is a type I transmembrane protein that is expressed on the surface of support and hematopoietic cells in the bone marrow. It normally stimulates growth of immature myeloid cells and stem cells.156 When FL ligand binds to the FLT3 receptor, FLT3 dimerizes and autophosphorylates intracytoplasmic tyrosine residues. The phosphorylated, activated FLT3 then activates downstream signal transduction pathways, including PI3K/AKT, MAPK/ERK, and STAT5.157,158 Several types of mutations in FLT3 have been cloned from leukemic cells. The most common are internal tandem repeat (ITD) mutations, in which head-to-tail duplications of various lengths and positions occur in the juxtamembrane (JM) portion of the molecule (Fig. 72.9).159 These elongation mutations may occur due to DNA replication errors as a result of a potential palindromic intermediate that may form at that site.160 The JM domain is an autoinhibitory domain whose inhibitory function is usually relieved by autophosphorylation after ligand binding.155 The in-frame insertions in the JM domain produce mutant proteins that are constitutively activated; they are able to dimerize

D835X Extracellular WT

TM JM

TK1

KI

TK2

AL

COOH

QFRYESQLQMVQVTGSSDNEYFYVDFREYEYDLKWEFRPRENLEF GLVQVTGSSDNEYFYVDFREYE GLYVDFREYEY REYEYDL YEYDLK CSSDNEYFYVDFREYEYDLKWEFRPRENL GYVDFREYEYDLKWEFRPRENLEF YDLKWEFRPRENLEF

Figure 72.9. Schematic of the internal tandem repeat (ITD) and activation loop FLT3 mutations in acute myelogenous leukemia (AML).  The structure of the FLT3 receptor tyrosine kinase is shown, with the position of the transmembrane domain (TM), the juxtamembrane (JM) domain, the kinase domains (TK1 and TK2), kinase insert (KI), and activation loop (AL). The amino acid sequence of the wild-type JM domain is listed, and underneath are the tandem duplication sequences found in individual patients with AML. These are always in-frame insertions. The position of the amino acid that is commonly substituted in activation loop mutations is indicated above the schematic of the protein domains. From Mizuki M, Fenski R, Halfter H, et al. FLT3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the RAS and STAT5 pathways. Blood 2000;96:3907–3914 and Kelly LM, Liu Q, Kutok JL, et al. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood 2002;99:310–318.

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and autophosphorylate in the absence of ligand.160 Other types of mutations include activation loop mutations, usually an Asp825Tyr substitution resulting from a point mutation in the second tyrosine kinase domain (TKD), producing constitutive activation of FLT3.155 The overall frequency of FLT3-ITD in adult AML is 24% of patients, while in pediatric AML the frequency is somewhat lower at 10% to 15% (reviewed in Ref. 155). The frequency is very low in MDS and ALL. In contrast, the FLT3 activation loop mutation is reported in 7% of AML, 3% of MDS, and 3% of ALL patients.161 FLT3-ITD is detected most frequently in APL, but has been detected in all AML subtypes.155 In addition, FLT3 is overexpressed at the mRNA and protein levels in many cases of AML and ALL.162 The role of the FLT3-ITD in leukemogenesis has been investigated by retroviral transduction of murine bone marrow stem cells followed by transplantation into mice. These mice develop a myeloproliferative disease with predominantly maturing myeloid elements, but they do not develop acute leukemia.163 Therefore, the FLT3 mutations may confer the proliferative signal in patients with acute leukemia, whereas a concomitant balanced translocation or other genetic defect confers the block in differentiation necessary for development of acute leukemia.155 Mutation of FLT3 is a significant independent prognostic factor for poor outcome in patients younger than 60 years. In a study of 91 pediatric AML patients on the Children’s Cancer Group (CCG) protocol, the remission induction rate was 40% in patients with FLT3-ITD compared to 74% with wild-type FLT3. The difference in event-free survival at 8 years was even more striking, at 7% for patients with FLT3-ITD compared to 44% for patients with wild-type FLT3.164 In a study of 398 patients younger than 60 with AML, FLT3-ITD mutations were the primary predictor of outcome in patients with intermediate-risk cytogenetics and were associated with reduced overall survival.34 As with BCR-ABL1 for CML, the implication of a mutant constitutively active tyrosine kinase receptor in the pathogenesis of AML opens up the possibility of identifying a selective kinase inhibitor as a specific treatment for AML patients with a mutant FLT3. Several kinase inhibitors have been identified by inhibition of IL3-independent growth of cell lines expressing FLT3-ITD in culture.165,166 Eight of these FLT3 inhibitors (FLT3-TKI) have been tested in phase I/II trials as single agents (reviewed in Ref. 167). In each study a majority of patients achieved >50% reduction in peripheral blast count, but these reductions were transient, lasting several weeks to months. The partial efficacy may be in part due to the refractory nature of the phase I/II patient population. However, a study performed by Piloto et al.168 on resistant human cell lines developed through prolonged coculture with FLT3 tyrosine kinase inhibitors demonstrated that although FLT3 phosphorylation was still inhibited, downstream signaling pathways were activated. In two cell lines, activating NRAS mutations were detected. No mutations in FLT3 were detected. However, in other studies, SMRT sequencing was used to demonstrate secondary FLT3 kinase domain mutations in 4 of 8 patients who relapsed after treatment with FLT3-TKI. The mutation frequency in the patients’ blasts was 20% to 50%, consistent with most of the leukemic blasts having a mutation on one allele. This degree of mutation in relapsed patients supports the idea that FLT3 mutations are “driver” mutations necessary for growth of the leukemic clone. From the crystal structure of FLT3 several of the mutations would force the molecule into an active kinase confirmation which is not recognized by the AC220 type II kinase inhibitor used in the study.169

Nuclear Pore Proteins Nucleophosmin Nucleophosmin (NPM) is a molecular chaperone that shuttles between cytoplasm and nucleus, with particular nucleolar

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concentration of protein.170 While NPM appears to function to transport preribosomal particles from the nucleolus to cytoplasm, other functions have also been described, including regulation of centrosome duplication, regulation of p53, and functional regulation and stabilization of P19ARF (reviewed in Ref. 171). Cytogenetically silent mutations of NPM1 have now been identified in 35% of adult AML with normal karyotype. The frequency is less (9% to 27%) in pediatric AML with normal karyotype.171,172,173 The NPM1 mutation is stably expressed, being consistently present in leukemic blasts at relapse.174 The wildtype NPM has two NLSs and two nuclear export signals (NES) that mediate the nuclear–cytoplasmic shuttling of wild-type NPM. Over 50 different mutations in NPM1 have been found in patients with AML; all of these mutations cause changes in the C-terminus of the NPM protein, including generation of a new NES motif, and loss of tryptophan residues 288 and 290, or 290 alone, causing unfolding of the C-terminal domain and disruption of binding to the nucleolus.174,175 The presence of a mutation correlates absolutely with abnormal subcellular localization of NPM, with relocation from its normal predominantly nucleolar location to the cytoplasm; this can be detected in tissue sections by immunohistochemistry. The mutation is always heterozygous, which may be related to the fact that the homozygous mutant is embryonic lethal.176 The mutant NPM appears to function in a dominant negative manner through heterodimerization with normal NPM, to cause relocation of some of the normal NPM, as well as the mutant NPM, to the cytoplasm.177 The mechanism by which the cytoplasmically located NPM promotes leukemia is under investigation. Conditional expression of mutated NPM in transgenic mice results in overexpression of HOX genes in Lin− marrow progenitor cells.178 Another mechanism by which mutant NPM1 may promote leukemogenesis is by destabilizing the tumor suppressor protein P14ARF, which regulates the TP53 response. P14ARF colocalizes with NPM to the nucleolus, and their interaction stabilizes P14ARF. Without this interaction and nuclear location, p19Arf (the mouse homolog) is more rapidly degraded by proteasomes. By this mechanism, mutant NPM may indirectly cause decreased amounts of the tumor suppressor protein P14ARF.174 NPM1 mutations frequently occur in conjunction with IDH1 and IDH2 mutations and with DNMT3A mutations (see Fig. 72.1).34,173 Several studies have shown that patients with NPM mutations in the absence of FLT3 mutations have a favorable response to chemotherapy.173,175 Patients with intermediate-risk cytogenetics and mutations in NPM1 and either IDH1 or IDH2 mutations have an improved rate of overall survival and are considered to have an overall favorable risk.34

Hematologic Malignancies



Risk Stratification of Acute Myeloid Leukemia A major purpose of molecular genetic testing performed on clinical AML samples in the molecular diagnostics laboratory is to have enough information to choose the optimal therapy and to assess the prognosis of the patient. Besides determining information about BCR-ABL1 or PML-RARA rearrangements that have an obvious impact on treatment decisions, the risk stratification of the patient helps the doctor determine whether it is best to treat with aggressive therapy, to adopt a gentler chemotherapy regimen, or to rush the patient to transplant. A recently published attempt to integrate the newly expanded knowledge of molecular defects, both mutations and translocations, that occur in AML reported the results of testing for mutations in a panel of 18 genes in 398 patients with AML at diagnosis. 97.3% of the patients had at least one somatic mutation. The frequency of each of the 18 mutations tested is demonstrated in Figure 72.1, as well as the Circos diagram depicting the interrelationships between the mutations.34 The Circos diagram visually conveys the multiplicity of mutations that occur in most patients. This integrated mutational analysis demonstrated patterns of co-occurring mutations and

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also mutually exclusive mutations. Some of the more important co-occurrences are KIT mutations and CBF alterations, co-occurrence of IDH1/2 mutations and NPM1 mutations, and DNMT3A mutations with NPM1, FLT3, and IDH1 mutations. As mentioned previously, IDH1/2 mutations do not occur in patients with TET2 mutations (Fig. 72.5). In addition, IDH1/2 and WT1 mutations are mutually exclusive, as well as DNMT3A mutations and MLL translocations.34 The effect of multiple mutations and the interplay with underlying cytogenetic changes on risk stratification are shown in Table 72.1.34,179 The most statistically significant predictors of overall survival are as follows: FLT3-ITD and MLL-PTD mutations are associated with reduced overall survival. CEBPA mutations and CBF translocations are associated with improved overall survival. PHF6 and ASXL1 mutations are associated with reduced survival. IDH2 mutations (R140Q) confer a favorable outcome. In intermediate-risk AML, FLT3-ITD mutations are the primary factor influencing outcome. In this cohort of patients the group with favorable risk had a 3-year overall survival of 64%, whereas the group with intermediate risk had a 3-year overall survival of 42% and the group with adverse risk had a 12% overall survival. Multivariate analysis demonstrated that outcomes predicted from the risk stratification are independent of age, WBC, induction dose, transplantation status, and post-remission therapy.34 Grossman et al.105 developed a similar prognostic model based entirely on molecular mutational analysis rather than karyotype, in which they constructed an algorithm with 5 prognostic subgroups based upon analysis of 1,000 patients for mutations in: PML-RARA, RUNX1-RUNX1T1, CBFB, MYH11, FLT3-ITD, MLL-PTD, NPM1, CEBPA, RUNX1, ASXL1, and TP53. The very favorable prognostic group (overall survival at 3 years of 82.9%) consisted of patients with PML-RARA rearrangement or CEPBA double mutations, and the very unfavorable group with an overall survival of 0% at 3 years consisted of patients with TP53 mutations. TP53, a well-characterized tumor suppressor gene, is mutated in many cancers, and mutations in TP53 are the basis of the cancer-prone Li-Fraumeni syndrome.180 However, this study is the first to characterize the frequency of TP53 mutations in AML (11.5%) and its prognostic significance.105 The particular combination of mutations and percentage survival will change with the discovery of additional mutations, new treatment trials, and additional patient cohorts, but the principle is important that risk stratification must take into account a multiplicity of genetic events, both translocations and mutations. The role of the molecular diagnostics lab will certainly increase; the difficult question will be what is the most prognostically significant and cost effective panel of molecular tests to perform on an AML patient at diagnosis.

B-Acute Lymphoblastic Leukemia Introduction Table 72.5 outlines the WHO classification for B-ALL, which lists several of the major translocations that have been repeatedly seen in B-ALL. Recent genome-wide analysis of DNA from multiple B-ALL cases has demonstrated, as in AML, that there are multiple deletions and mutations in many other genes besides the well-characterized translocations. A recent survey of 242 cases of pediatric ALL using Affymetrix SNP arrays identified an average of 6.46 somatic copy number alterations per case, predominantly deletions.181 Interestingly, alterations in genes regulating B-lymphocyte differentiation were noted in 40% of B-ALL cases. The B-cell differentiation genes most commonly translocated or mutated include EBF1, PAX5, and IKZF1. Copy number changes in PAX5 occurred in 29.7% of this series, with the most common alteration being mono-allelic deletion of PAX5. PAX5 is essential for B-cell development, and it controls B-cell–specific transcription of B-lineage–specific genes such as CD19, CD79a, BLNK, and

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Tab l e 7 2 . 5

World Health Organization Classification Of Precursor Lymphoid Neoplasms B lymphoblastic leukemia/lymphoma   B lymphoblastic leukemia/lymphoma, NOS   B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities    B-ALL with t(9;22)(q34;q11.2); BCR-ABL1    B-ALL with t(v;11q23); MLL rearranged    B-ALL with t(12;21)(p13;q22.3); ETV6-RUNX1    B-ALL with hyperdiploidy    B-ALL with hypodiploidy    B-ALL with t(5;14)(q31;q32); IL3-IGH@    B-ALL with t(1;19)(q23;p13.3); TCF3-PBX1 T-lymphoblastic leukemia/lymphoma B-ALL, B-cell acute lymphoblastic leukemia; NOS, not otherwise specified. From Swerdlow SH, Campo E, Harris NL, et al., Eds. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon, France: IARC Press, 2008.

CD72.181 In Pax5−/− mice, B-cell development is arrested prior to the B220+ pro–B-cell stage, and these pro-B cells are uncommitted lymphoid progenitors.182 Mutations in genes necessary for B-cell differentiation are a perfect example of Class II mutations that cause a block in cell differentiation in the classic model of acute leukemia pathogenesis.

Transcription Factors PAX5 In the above-mentioned study, the PAX5 gene was altered by deletion or mutation in 31.7% of cases in a series of 242 B-ALLs.181 It is rearranged in 2.6% of pediatric B-ALL cases, with 17 different fusion partners documented. PAX5 is a paired box domain (PRD) transcription factor, encoded by the PAX5 gene at chromosome 9p13, and consists of 10 exons that encode a 391 amino acid protein. In all the fusion proteins, the paired box domain DNA-binding region and nuclear localization region are retained, but the C-terminal transactivation domain is deleted.183 The C-terminal fusion proteins may act in contrast as transcriptional repressors, which would be brought to PAX5 target gene promoters by the retained PAX5 DNAbinding region. The PAX5 fusion proteins may act as dominant negative molecules in conjunction with the product of the wild-type PAX5 allele, and in this way may repress genes whose products are necessary for B-cell differentiation. The PAX5 translocations are correlated with a relatively normal karyotype, suggesting that they have a major role as a driver mutation; PAX5 deletions, however, are usually associated with a complex karyotype.184

Core Binding Factors RUNX1 is also involved in a translocation that is present in 25% of pediatric B-ALL, t(12;21)(p13;q22.3).185 This translocation is associated with a good prognosis, although it is often missed by standard karyotype analysis. In this translocation, the N-terminus of ETV6, formerly called TEL (translocation-ETS-leukemia), is fused to most of the coding region of RUNX1.186 ETV6 contains a DNA-binding ETS domain and a “pointed” domain homologous to the Drosophila development protein, pointed.187 ETV6 is a transcriptional repressor and as such it contains a DNA-binding ETS domain and domains that interact with mSin3A (pointed domain), N-CoR, and HDAC-3 (Fig. 72.4C).188 The functional significance of these interactions with HDACs was demonstrated by the ability of TSA to inhibit two properties of 3T3 fibroblast cells transformed with ETV6 and Ras: expression of the stromelysin-1 gene and aggregation.188

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Another chromosomal alteration involving the RUNX1 locus that occurs in approximately 2% of pediatric patients with B-ALL is the intrachromosomal amplification of chromosome 21 (iAMP21). The significance of recognizing this cytogenetic abnormality is that it is associated with a dismal prognosis if treated with standard chemotherapy. There is a 6.6 Mbp common region of amplification (CRA) on chromosome 21, and in the majority of cases an associated 3.3 Mbp common region of deletion at the telomere. The average copy number of this CRA is 4.8, and the amplification is usually detected by FISH using the RUNX1 probe. Although RUNX1 is in the CRA, gene expression studies do not reveal increased transcription of RUNX1. This cytogenetic abnormality is associated with a complex karyotype and multiple mutations; however, clonal analysis indicates that the precipitating genetic event is the iAMP21 amplification.189

TCF3 (E2A) Translocations A common translocation in childhood B-ALL, present in 5% of pre–B-ALL cases,190 is the t(1;19)(q23.3;p13.3) translocation, which fuses the TCF3 (E2A) gene on chromosome 19p13.3 with the PBX1 gene on chromosome 1q23.3 (Fig. 72.10).191,192 The TCF3 locus encodes three transcripts, E12, E47, and E2-5, which are generated by alternative splicing.193 They belong to class I of the basic helix-loop-helix (bHLH) family of transcription factors which bind to specific E-box (CANNTG) sequences in promoters and enhancers, the first of which were identified in the enhancer regions of the immunoglobulin heavy-chain and k-chain genes.193 Usually the ubiquitous E2A proteins heterodimerize through the HLH domain with members of the class II bHLH proteins, most of which are tissue specific in expression. These heterodimers are crucial in transcriptional regulation of tissue-specific genes during development. Although E2A proteins are ubiquitous, they are preferentially expressed in B lymphocytes,194 and E47 forms homodimers exclusively in B cells.195 The requirement for E2A proteins in B-cell development is demonstrated by Tcf3−/− null mice, which exhibit a complete block in B-cell differentiation at the pro–B-cell stage prior to immunoglobulin gene rearrangement, as well as defective thymocyte differentiation.196,197 These mice have an increased frequency of T-lymphoblastic lymphoma.196 PBX1 (pre–B-cell leukemic homeobox 1), identified as the fusion partner of TCF3 in t(1;19),198 encodes a member of the homeodomain family of transcription factors, encoded by homeobox (HOX) genes. The PBX1 gene is the mammalian homolog of the Drosophila gene Extradenticle, whose protein product Fusion Site

Dimerization/ DNA binding

Activation Domain

E2A AD1

AD2

b HLH

homeodomain

HLF

PBX1 b ZIP

E2A

E2A

HLF

E2A-HLF

PBX1

E2A-PBX1 AD1

AD2

b ZIP

AD1

AD2

H

Figure 72.10. Structural features of E2A (TCF3) fusion proteins.  The N-terminus of the E2A (TCF3 ) gene encodes a transcriptional activation domain that is translocated to hepatic leukemia factor (HLF ) or pre–B-cell leukemic homeobox1 (PBX1) by chromosome translocations in acute lymphoblastic leukemia. In the case of E2A (TCF3)-HLF, the DNA-binding and dimerization domains of E2A (TCF3) are replaced by similar domains in HLF. For E2A(TCF3)-PBX1, the same DNA-binding and dimerization domains of E2A (TCF3) are replaced with the DNA-binding homeodomain of PBX1. AD, activation domain; b HLH, basic helix-loop-helix; bZIP, basic leucine zipper domain; H, homeodomain.

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cooperates with other homeodomain proteins during development.199 Likewise, PBX1 forms heterodimers with other homeodomain proteins via the homeodomain and the C-terminal HOX cooperativity motif (HCM).200 Cotransfection experiments with reporter genes containing DNA-binding sites for PBX1 have demonstrated that PBX1 is not a strong transcriptional activator.201 The Pbx1−/− mice have late embryonic lethality associated with multiple organ abnormalities,202 supporting the hypothesis that PBX1 interactions regulate homeodomain protein function. PBX1 is not normally expressed in lymphocytes. In the t(1;19) translocation, the breakpoint on chromosome 19 occurs within the intron between exons 13 and 14 of TCF3, so that the N-terminal two-thirds of TCF3 (E2A), aa1-483, are included in the fusion protein.192 This includes both of the transcriptional activation domains (AD1 and AD2), but excludes the bHLH DNAbinding and dimerization domains (Fig. 72.10). Therefore, the TCF3-PBX1 fusion protein depends on the homeodomain of PBX1 for DNA-binding specificity. The most straightforward model for how expression of the TCF3-PBX1 fusion protein results in the development of leukemia is that fusion of the TCF3 activation domains onto the PBX1 sequence results in abnormally strong transactivation of target genes recognized by the PBX1 homeodomain.203 These target genes would be activated in lymphocytes, where PBX1 is usually not expressed. Mapping experiments demonstrate that the activation domains AD1 and AD2 of TCF3 are necessary for transactivation of reporter genes containing PBX1 binding sites, and they are also necessary for transformation of NIH 3T3 cells.204 Gene expression profiling of a pre–B-cell line inducibly expressing TCF3-PBX1 demonstrated upregulation of BMI1,205 a gene which is expressed in normal HSCs, but whose expression normally decreases during hematopoietic development.206 Bmi1−/− mice have a reduced number of HSCs, with defective adult self-renewal of HSCs.206 Cells from Bmi1−/− mice are resistant to transformation by Tcf3-Pbx1,205 suggesting that the mechanism by which TCF3-PBX1 transforms cells is through overexpression of BMI1. Interestingly, BMI1 is a transcriptional repressor of the CDKN2A locus,205 which encodes the two tumor suppressor genes P16INK4A and P14ARF.207 P16INK4A binds to CDK4 and 6, preventing association with cyclins and phosphorylation of Rb. Therefore, repression of P16INK4A results in increased phosphorylation of Rb, which loses its affinity for the transcription factor E2F, increasing expression of E2F target genes necessary for progression to the S phase and proliferation.208,209 The tumor suppressor P14ARF inhibits MDM2, a repressor of TP5387; therefore, inhibition of P14ARF results in increased MDM2, repression of TP53, and loss of the checkpoint functions of TP53 in preventing propagation of cells with DNA damage.87 Therefore, activation of BMI1 by TCF3-PBX1 results in downregulation of two powerful tumor suppressor pathways. Another translocation involving TCF3 occurs in approximately 1% of pediatric ALL, t(17;19)(q22;p13.3), which fuses TCF3 to HLF (hepatic leukemia factor).210,211 Clinically, these patients are adolescents; they may present with disseminated intravascular coagulation and hypercalcemia and usually have a poor prognosis. HLF encodes a transcription factor of the basic leucine zipper (bZIP) family, in which the basic region is the DNA-binding region and the leucine zipper refers to an amphipathic a-helical domain through which HLF can homodimerize or heterodimerize with other bZIP proteins. HLF is usually expressed in liver, kidneys, and central nervous system neurons, but not in hematopoietic cells.212 The TCF3-HLF fusion protein is homologous to the TCF3-PBX1 fusion protein, in that the N-terminal 483 amino acids of TCF3, contributing the activation domains AD1 and AD2, are fused to the C-terminal portion of HLF, which contains the bZIP DNAbinding and dimerization domains (Fig. 72.10).213 Unlike PBX1, wild-type HLF is a strong transactivator, but the alteration in cell type expression and alterations in DNA-binding affinity and protein interactions by virtue of fusion to TCF3 may contribute to the transforming properties of TCF3-HLF.214

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Recent experiments using BaF3 cells inducibly expressing TCF3-HLF have implicated LMO2 and BCL2 as transcriptional targets of the abnormal TCF3-HLF transcription factor.215 Microarray analysis was performed on RNA from the cell lines before and after induction of TCF3-HLF expression; subsequent ChIP experiments demonstrated that TCF3-HLF directly binds to the promoters of these two genes to transcriptionally activate them. Relevant to potential new therapies of this poor prognosis leukemia is the finding that knock-down of LMO2 expression or inhibition of BCL2 activity in the cells expressing TCF3-HLF reduces the proliferation of these immortalized cells.215 LMO2 is a transcription factor expressed in hematopoietic progenitors and is known to play a role in development of T-ALL216 BCL2 is an antiapoptotic protein with a well-known role in the pathogenesis of B-cell lymphoma (reviewed in Ref. 217).

IKAROS SNP array studies first demonstrated that IKZF1, encoding the transcription factor IKAROS, is deleted in 76.2% of pediatric Ph+ B-ALL and 90.9% of adult Ph+ B-ALL.218 A subsequent study of adult B-ALL cases demonstrated that 75% of adult Ph+ B-ALL cases had alterations of the IKZF1 gene, compared to 58% of adult Ph− B-ALL cases.219 IKAROS is a zinc finger–containing transcription factor that is required for lymphoid lineage commitment. It is expressed in multipotent, self-renewing HSCs and is necessary for induction of genes important for the lymphoid lineage, as well as repression of genes responsible for self-renewal and multipotency in the differentiating progeny of HSCs.220 Deletions are usually of one allele, and in most cases the deletion involves a subset of exons, most commonly exons 4 to 7.219 Sequencing of the deletion sites demonstrates the presence of heptamer recombination signal sequences that are recognized by RAG enzymes during immunoglobulin gene recombination.218 These deletions result in loss of the DNA-binding domain but preservation of the dimerization domain of the protein, creating a dominant negative molecule.219 Use of gene expression arrays to compare RNA from B-ALL cases with intact or mutated IKZF1 loci demonstrates that IKZF1 mutations are associated with downregulation of several genes in the B-cell differentiation pathway and upregulation of genes involved in cell cycle regulation, apoptosis regulation, DNA damage, and the JAK-STAT signaling pathway. In addition, in B-ALL with IKZF1 mutations, there is downregulation of RAG and EBF1, two genes whose products are involved in IgH VDJ recombination.219 Downregulation of genes involved in normal B-cell differentiation may cause the arrest of B-cell maturation that occurs in B-ALL. As the gene expression patterns in B-ALL with mutations in IKZF1 are similar to gene expression patterns in BCR-ABL1 positive ALL, these cases are referred to as Ph-like ALL.221 Though the prognosis of these cases is poor, discovery of frequent concomitant mutations in cytokine receptors such as CRLF2 and other signaling molecules such as JAK2 suggests that use of kinase inhibitors may play a role in treating this subset of B-ALL.222

Kinases BCR-ABL1 The Philadelphia chromosome is the result of the t(9;22) (q34;q11.2) translocation in which the 5′ domain of the BCR gene from chromosome 22 is fused with the 3’ TKD of the ABL1 gene from chromosome 9.223,224 The Philadelphia chromosome is the resultant shortened chromosome 22. It is the most frequent recurring translocation in adult ALL, occurring in 15% to 30% of patients,225 and also is present in 5% of pediatric B-ALL.226 Unfortunately, it is an adverse prognostic factor in children and adults.

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The BCR-ABL1 fusion gene is associated most commonly with CML. The pathogenesis of CML will be discussed in Chapter 81. A lymphoid blast crisis arising from CML may be difficult to distinguish from a Philadelphia chromosome–positive (Ph+) ALL. The size of the BCR-ABL1 fusion protein and whether it is restricted in expression to lymphoid cells may be helpful in making this distinction. The most common breakpoint region, the major breakpoint cluster region (M-bcr), spans almost 6 kb between exons 12 and 16 of BCR and results in a fusion protein of 210 kD, referred to as p210bcr-abl.227 A minor breakpoint, the m-bcr, is farther 5′, after exon 2 of BCR, resulting in a truncated fusion protein of 190 kD that contains only the first two exons of BCR (p190bcr-abl).2 Interestingly, p210bcr-abl is much more common in CML and CML with lymphoid blast crisis, whereas p190bcr-abl is much more commonly expressed in Ph+ ALL. p190bcr-abl is present in 80% to 90% of pediatric Ph+ ALL and 50% of adult Ph+ ALL.225 However, some cases of Ph+ ALL contain both p190bcr-abl and p210bcr-abl. Transgenic mice expressing p190bcr-abl develop an aggressive leukemia restricted to pre–B cells, whereas transgenic mice expressing p210bcr-abl develop a more chronic disease involving B and T cells and myeloid lineages.228 In some cases of Ph+ ALL the aberrant fusion protein is present in lymphoid and myeloid marrow cells, whereas in other cases the aberrant fusion protein, usually p190bcr-abl, is restricted to lymphoid cells. Those cases in which p210bcr-abl is present in both lymphoid and myeloid cells are most likely to represent a CML lymphoid blast crisis.229 Studies of BCR-ABL1 expression in CML have demonstrated the leukemogenic properties of BCR-ABL1 as a constitutive tyrosine kinase.230 This constitutive kinase activates by phosphorylation multiple downstream signal transduction intermediates, including RAS, PLCg, and PI3 kinase.231 Activation of these pathways results in proliferation and resistance to apoptosis.232 Presumably similar mechanisms are at work in Ph+ ALL. Restriction of expression of BCR-ABL1 to the lymphoid lineage would explain the development of ALL. However, in those cases of Ph+ ALL in which BCR-ABL1 is expressed in the stem cell compartment, it is unclear why ALL has resulted instead of CML. A high percentage of Ph+ ALL, as well as B lymphoid blast crisis of CML, have concomitant mutations in IKAROS, as described above.218,219,233 Treatment of Ph+ ALL remains problematic. Initial response to chemotherapy is similar in Ph+ ALL and Ph− ALL, but remissions tend to be short lived. Transplantation appears to be the best means of attaining a lasting remission. In a phase II trial of imatinib in relapsed or refractory Ph+ ALL, 60% of patients achieved a hematologic response, but it was usually short lived.234 However, trials using imatinib in conjunction with standard chemotherapy followed by bone marrow transplant in CR have proven more successful. The combination approach induces CR in 96% of patients with a 30% incidence of relapse and 65% overall survival rate at 18 months.235,236 Posttransplant BCR-ABL1 positivity has a poor prognosis; therefore in current protocols imatinib therapy is usually reinitiated 6 to 8 weeks after transplant.233 Secondary resistance to imatinib is common, however, and occurs in approximately 70% of relapse patients with CML blast crisis or Ph+ ALL. In 50% to 90% of relapse patients, point mutations have been identified in the adenosine triphosphate–binding pocket of BCR-ABL1 that is targeted by imatinib.237 New kinase inhibitors that can bind to the mutated as well as wild-type BCR-ABL1, such as dasatinib and nilotinib, are being utilized in patients who develop resistance to imatinib.233

T-Cell Acute Lymphoblastic Leukemia/Lymphoma T-ALL is an uncommon acute leukemia but its genetics have been studied in considerable depth. The driver mutations in T-ALL were first identified by cloning the breakpoints at chromosomal

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translocations.238,239,240,241 These experiments revealed oncogenes whose expression was deregulated by rearrangement with the promoters and enhancers of T-cell receptor genes. Subsequently, it was shown that these same oncogenes are overexpressed by mechanisms other than translocation.242,243,244,245 Indeed, gene expression clustering based on these oncogene signatures has been highly informative in classifying T-ALL into distinct subtypes that relate to cell of origin.244,246,247 Whole exome sequencing and targeted sequencing have revealed numerous important mutations in T-ALL. As shown in Table 72.6, these oncogenes and tumor suppressor genes are divided into major gene classes. The CDKN2A locus encodes cell cycle regulatory genes P14ARF and P16INK4A, and is the most commonly inactivated gene in human T-ALL.238 CDKN2A is located on 9p21 which shows homozygous deletion in more 72% of patients.182,241 CDKN2A is also inactivated by other mechanisms.248,249 Deletions of 13q14.2 including the RB1 gene occur in up to 12% of T-ALL patients182; these deletions are found in patients with intact CDKN2A, an expected finding since both of these tumor suppressors act in the same pathway.244,250 Mutations in TP53 and CDKN1B (p27) are uncommon, each occurring in less than 1% of T-ALL patients.

Notch The NOTCH pathway is mutated in the majority of T-ALL patients. NOTCH1 is a regulatory protein that is important in many cell fate decisions, including commitment to T-cell lineage and choice of ab lineage.251,252,253 It was first cloned from a t(7;9)(q34;q34) translocation occurring in a T-ALL patient that involved NOTCH1 on chromosome 9q34 and the T-cell receptor b-chain gene on chromosome 7q34.3.254 The t(7;9) translocation turned out to be rare in T-ALL but targeted sequencing revealed that over 60% of T-ALL patients have activating mutations in NOTCH1.255 NOTCH1 is synthesized as a single polypeptide protein that is cleaved in the Golgi at site S1 into two subunits, the ligand-binding NEC (extracellular) and NTM (transmembrane), which bind noncovalently at the heterodimerization domain. Upon ligand binding to NEC, NTM is cleaved at site S2 by a metalloprotease, and cleaved at S3 by regulated intramembrane proteolysis catalyzed by a multiprotein enzyme, the gamma (g) secretase.256 The remaining intracellular portion, ICN1, translocates to the nucleus, where it acts as a transcriptional regulator with the DNA-binding protein CSL and with coactivators of the Mastermind-like family.257 The majority of the activating mutations in NOTCH1 found in T-ALL occur in the heterodimerization domain or in the PEST domain.255 The PEST domain regulates the turnover of NOTCH1. Therefore, the heterodimerization domain mutants uncouple NOTCH1 activation from ligand binding and the PEST domain mutants increase NOTCH1 protein stability.255 The NOTCH1/CSL complex has

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numerous transcriptional targets that affect diverse pathways required for cell transformation; among these, MYC and HES1 appear to be important for T-cell leukemogenesis.258,259,260,261 The FBXW7 gene is mutated in T-ALL and fits into the NOTCH pathway since it encodes a component of a multiprotein E3 ubiquitin ligase that targets NOTCH1, MYC, and CCNE for degradation.262,263,264 Inactivating mutations in FBXW7 are present in 9% to 16% of T-ALL patients.265,266 The unique proteolytic pathway leading to activated NOTCH1 can be targeted by small molecule inhibitors of the gamma-secretase enzyme that is required for S3 cleavage.267,268,269 These inhibitors have been used in clinical testing; however, since NOTCH1 regulates cell fate decisions for intestinal epithelial cells, patients have experienced significant gut toxicity.267

Transcription Factors Tal1 and LMO Factors Class B bHLH transcription factors, TAL (SCL) (T-cell acute lymphoblastic leukemia 1/stem cell leukemia), TAL2, LYL1, and OLIG2 are frequently deregulated in T-ALL.241 The TAL1 gene is deregulated in 25% of T-ALL. TAL1 was originally cloned from a translocation, t(1;14)(p32;q11), present in 3% of patients with T-ALL.270 In the translocation, the breakpoint is 5′ to the coding region of TAL1 on chromosome 1, and the translocation places TAL1 under the regulation of the T-cell receptor a/b genes on chromosome 14.271,272 A second series of rearrangements that occurs in 26% of patients with T-ALL results in deletion of 90 to 100 kb of DNA from the 5′ upstream region of TAL1, placing the gene under the control of the upstream constitutively active SIL promoter.273 In both cases, the coding region of TAL1 is intact, unlike the fusion proteins that are usually expressed in other acute leukemias. Also, in some cases of T-ALL, overexpression of TAL1 occurs when there is no evident gene rearrangement by Southern blot analysis, suggesting a mutation in regulatory sequences.274 During development, TAL1 is expressed in early hematopoietic elements, in both the yolk sac blood islands and the definitive blood cells of the AGM region and fetal liver.275 Postnatally, it is expressed in erythroid, megakaryocyte, and mast cell lineages, but not in T cells. In nonerythroid cells, TAL1 is expressed in stem cells but is not expressed as the cells differentiate; however, in erythroid cells, TAL1 expression increases with early erythroid differentiation but decreases with terminal differentiation.276 The essential role of TAL1 in hematopoietic development is demonstrated by mice made null for Tal1; embryonic lethality occurs due to a total deficiency in hematopoietic progenitors.277,278 Conditional gene targeting experiments using the Lox-Cre system to delete Tal1 in adult mice demonstrate that continued expression

Hematologic Malignancies



T a ble 7 2 .6

Functional Classes Of Genes Mutated In T-Lymphoblastic Leukemia Gene Class

Frequency (%)

Oncogene or Tumor Suppressor

Cell cycle genes Notch and its targets bHLH and partners Homeobox genes Other transcription factors and chromatin modifiers Cytokine and signal transduction

85 70 35 35 5–15

CDKN2A, RB1, TP53, CDKN1B NOTCH1, FBW7, MYC TAL1, TAL2, LYL1, OLIG2, LMO1-3 TLX1, TLX3, HOXA cluster, MLL, CALM MYB, BCL11B, PHF6, EZH2

5–15

ABL1, FLT3, IL7R, JAK1, LCK, NRAS, IGF1R

The frequency shown is the estimated mean of all the frequencies reported for each individual gene mutation in a given class.

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of TAL1 is not necessary for maintenance of HSCs, but it is necessary for erythrocyte and megakaryocyte differentiation.279 TAL1, like LYL1, OLIG2, and TAL2, binds E-box sequences in DNA by heterodimerizing with class I bHLH transcription factors TCF3. Tandem E boxes or E-box GATA sequences can be bound by two TAL1/TCF3 heterodimers that are bridged by LIM-domain-only 1 or 2 (LMO1/2) proteins. Interestingly, LMO1-3 genes are also important drivers of T-ALL which can be deregulated by chromosomal rearrangements with T-cell receptor genes and other mechanisms.216,280,281 Other TAL1 protein partners include GATA1, LDB1, coactivators p300 and pCAF, and corepressors mSin3A and HDAC1.282,283 Interestingly, LMO2 can be coimmunoprecipitated with TAL1 from T-ALL cell lines,284 and mice overexpressing both Tal1 and Lmo2 develop T-ALL with shorter latency than transgenic mice overexpressing either gene alone, providing evidence for cooperativity.285–287 The transcriptional targets of activation or repression by TAL1 that are required for T-ALL have not been fully elucidated.288,289,290 Alternatively, TAL1 may act as a dominant negative inhibitor of the TCF3 transcription factors.291,292 Human and murine T-ALLs induced by bHLH or LMO gene overexpression show repression of TCF3 target genes.293 LYL1, OLIG2, and TAL2 may all behave similarly to TAL1 in that they cooperate with LMO proteins to regulate gene expression, although LYL1-overexpressing T-ALLs are more immature in the T-cell differentiation hierarchy than TAL1-overexpressing T-ALLs.294,295,296,297 The expression of these bHLH genes was quantified in a large panel of T-ALLs showing mutually exclusive expression patterns, which also argues for functional redundancy in leukemia pathogenesis. Interestingly, LYL1 and TAL1 are also functionally redundant in the maintenance of adult hematopoietic stem and progenitor cells.244,298

Homeodomain Proteins The homeobox genes are a major group of genes deregulated in T-ALL, mutually exclusive to the bHLH genes and LMO genes discussed above.244 TLX1 (HOX11) is overexpressed by chromosomal rearrangement or other mechanisms in 7% of T-ALL.242,299 TLX3 (HOX11L2) is deregulated most commonly by a t(5;14)(q35.1;q32) in 20% of adult T-ALL cases.300,301 Finally, the HOXA gene cluster is deregulated in 5% of T-ALL cases by inv(7)(p15.2q34) and other transcriptional mechanisms.302–304 TLX1-overexpressing T-ALLs have a distinctive block at the cortical stage of T-cell differentiation. Recent data using transgenic mice overexpressing Tlx1 show that the protein may repress mitotic checkpoint genes, leading to aneuploidy, and may disrupt the normal factors needed for T-cell receptor a rearrangement.305,306 MLL and its fusion partners PICALM and MLLT10 are rearranged and overexpressed in 5% to 10% of T-ALL cases and may function by deregulating the expression of the HOXA gene cluster.140,304,307,308 Other transcription factors mutated in T-ALL include the MYB oncogene, which is duplicated in 8.4% of T-ALL cases as analyzed by array CGH and fiber FISH studies.309,310 Array CGH, SNP arrays, and sequencing revealed deletion or missense mutation in the BCL11B gene in 9% of T-ALL patients.311 Bcl11b knockout mice show major defects in T-cell differentiation consistent with a role in T-cell progenitor transformation.312 Loss of function mutations were found in PHF6, a transcription factor gene encoded on Xq26. This finding was very intriguing in that the mutations were 10-fold more prevalent in males than females, which correlates with the higher prevalence of the disease in males; however, the PHF6 mutations were highly concordant with TLX gene deregulation and uncommon in TLX-negative cases.313 EZH2 and SUZ12 loss of function mutations occur in 25% of T-ALL cases. These two genes encode protein components of the PRC2. PRC2 is responsible for methylation of Lysine-27 on histone H3, a mark associated with silenced genes.314

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Mutations in Signaling Pathways T-ALL, like most acute leukemias, harbors mutations predominantly in transcription factors. These proteins are difficult therapeutic targets in comparison to cytokine receptors and signal transducing tyrosine kinases. The latter group of genes was very recently implicated in T-ALL with targeted sequencing approaches. For example, gain of function mutations in the IL7R gene have been described in 5% to 10% of T-ALL patients.295,315,316 Some of the missense substitutions in IL7R introduce a cysteine residue in place of the wild-type amino acid in the JM domain. This creates a disulfide bond linking two receptors, resulting in constitutive phosphorylation of STAT5 and other downstream substrates.316 The IL7R protein utilizes the nonreceptor tyrosine kinases, JAK1 and JAK3, for signal transduction, and these too are mutated in 5% to 10% of T-ALL cases.295,317,318,319 FLT3-ITD mutations similar to those found in AML are also found in T-ALL. NRAS mutations have also been described in ∼10% of patients.181,320,321 The ABL1 gene is amplified involving simple translocations and complex rearrangements in 5% to 10% of T-ALLs.322 Mutations in signaling pathways are more common in early T-cell precursor ALL, a form of T-ALL that is resistant to induction chemotherapy and prone to relapse.295,296 Some of the oncogenes and tumor suppressor genes involved in T-ALL pathogenesis are also targeted by a group of microRNAs, offering yet another pathway toward regulation of expression.323,324

Summary This chapter has reviewed the major translocations found in acute leukemia, with a focus on understanding the function of the fusion proteins encoded by the translocated genes. In addition, genes that are common sites of point mutations or deletions in acute leukemia are reviewed. A major theme has been alteration of transcriptional regulation. A major focus of future research will be identification of downstream targets of these aberrant transcription factors. Study of the aberrant transcription factors resulting from translocations and mutations has increased our understanding of the importance of histone acetylation, deacetylation, and methylation in transcriptional regulation. Study of mutated tyrosine kinases present in leukemia led to the first specifically engineered kinase inhibitor for the therapy of CML and Ph+ ALL. Kinase inhibitors have also been developed for the relatively common mutant FLT3 protein present in AML. This is a truly exciting time for hematologic oncology, where elucidation of the pathogenesis of acute leukemia is leading to development of new therapeutic agents. However, powerful genomic methods have revealed the incredible complexity of the mutational events necessary for the development of acute leukemia.

Selected References The full reference list for this chapter can be found in the online version.

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120. Holz-Schietinger C, Matje DM, Reich NO. Mutations in DNA m ­ ethyltransferase (DNMT3A) observed in acute myeloid leukemia patients disrupt processive methylation. JBC 2012;287:30941–30951. 122. Muntean AG, Hess JL. The pathogenesis of mixed-lineage leukemia. Annu Rev Pathol Mech Dis 2012;7:283–301. 127. Hess JL. MLL: a histone methyltransferase disrupted in leukemia. Trends Mol Med 2004;10:500–507. 132. Meyer C, Kowarz E, Hofmann J, et al. New insights to the MLL recombinome of acute leukemias. Leukemia 2009;23:1490–1499. 136. Chi P, Allis CD, Wang GG. Covalent histone modifications—miswritten, misinterpreted and mis-erased in human cancers. Nat Rev Cancer 2010;10: 457–469. 137. Bernt KM, Zhu N, Sinha AU, et al. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell 2011;20:66–78. 138. Daigle SR, Olhava EJ, Therkelsen CA, Majer CR, et al. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell 2011;20:53–65. 139. Mohan M, Lin C, Guest E, Shilatifard A. Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer 2010;10: 721–728. 148. Basecke J, Whelan JT, Griesinger F, Bertrand FE. The MLL partial tandem duplication in acute myeloid leukaemia. Br J Haematol 2006;135:438–449. 152. Gill Super H, McCabe NR, Thirman MJ, et al. Rearrangements of the MLL gene in therapy-related acute myeloid leukemia in patients previously treated with agents targeting DNA-topoisomerase II. Blood 1993;82:3705–3711. 154. Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer 2012;12:599–612. 155. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002;100:1532–1542. 160. Kiyoi H, Towatari M, Yokota S, et al. Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia 1998;12:1333–1337. 167. Kinder T, Lipka DB, Fischer T. FLT3 as a therapeutic target in AML: still ­challenging after all these years. Blood 2010;116:5089–5102. 171. Grisendi S, Pandolfi PP. NPM mutations in acute myelogenous leukemia. N Engl J Med 2005;352:291–292. 176. Bolli N, De Marco MF, Martelli MP, Bigerna B, et al. A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF. Leukemia 2009;23:501–509. 178. Vassilliou GS, Copper JL, Rad R, Li J, Rice S, Uren A. Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice. Nat Genet 2011;43:470–475. 179. Dohner H, Estey E, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European Leukemia Net. Blood 2010;115:453–474. 181. Mullighan CG, Goorha S, Radtke I, et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 2007;446:758–764. 183. Medvedovic J, Ebert A, Tagoh H, Busslinger M. Pax5: a master regulator of B cell development and leukemogenesis. Adv Immunol 2011;111:179–206. 184. Coyaud E, Struski S, Prade N, et al. Wide diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogenetique Hematologique study. Blood 2010;115:3089–3097. 186. Golub TR, Barker GF, Bohlander SK, et al. Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci U S A 1995;92:4917–4921. 188. Wang L, Hiebert SW. TEL contacts multiple co-repressors and specifically associates with histone deacetylase-3. Oncogene 2001;20:3716–3725. 189. Rand V, Parker H, Russell LJ, et al. Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia. Blood 2011;117:6848–6855. 196. Bain F, Engel I, Robanus EC, et al. E2A deficiency leads to abnormalities in ab T-cell development and to rapid development of T-cell lymphomas. Mol Cell Biol 1997;17:4782–4791. 205. Smith KS, Chandra SK, Lingbeek M, Ross DT, et al. Bmi-1 regulation of INK4AARF is a downstream requirement for transformation of ­hematopoietic progenitors by E2a-Pbx1. Mol Cell 2003;12:393–400. 207. Chin L, Pomerantz J, DePinho RA. The INK4a/ARF tumor suppressor: one gene-two products-two pathways. Trends Biochem Sci 1998;8:291–296. 208. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev 1998;12:2245–2262. 210. Raimondi SC, Privitera E, Williams DL, et al. New recurring ­chromosomal translocations in childhood acute lymphoblastic leukemia. Blood 1991; 77:2016–2022. 215. de Boer J, Yeung J, Ellu J, Ramanujachar R, et al. The E2A-HLF oncogenic fusion protein acts through Lmo2 and Bcl-2 to immortalize hematopoietic progenitors. Leukemia 2011;25:321–330. 218. Mullighan CG, Miller CB, Radtke F, Phillips LA. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 2008;453:110–114. 219. Iacobucci I, Iraci N, Messina M, Lonetti A, Chiaretti S, al e. IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia. PLOS One 2012;7:1–10. 222. Roberts KG, Morin RD, Zhang J, et al. Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia. Cancer Cell 2012;22:153–166. 225. Secker-Walker LM, Craig JM, Hawkins JM, et al. Philadelphia positive acute lymphoblastic leukemia in adults: age distribution, BCR breakpoint and ­prognostic significance. Leukemia 1991;5:196–199. 226. Uckun FM, Nachman JB, Sather HN, et al. Clinical significance of Philadelphia chromosome-positive pediatric acute lymphoblastic leukemia in the context of contemporary intensive therapies: a report from the Children’s Cancer Group. Cancer 1998;83:2030–2039.

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233. Ribera J-M. Advances in acute lymphoblastic leukemia in adults. Curr Opin Oncol 2011;23:692–699. 235. de Labarthe A, Rousselot P, Huguet-Rigal F, et al. Imatinib combined with induction or consolidation chemotherapy in patients with de novo Philadelphia chromosome-positive acute lymphoblastic leukemia—results of the GRAAPH-2003 study. Blood 2007;109:1408–1413. 238. Rabbitts TH, Axelson H, Forster A, et al. Chromosomal translocations and leukaemia: a role for LMO2 in T cell acute leukaemia, in transcription and in erythropoiesis. Leukemia 1997;11(Suppl 3):271–272. 241. De Keersmaecker K, Marynen P, Cools J. Genetic insights in the pathogenesis of T-cell acute lymphoblastic leukemia. Haematologica 2005;90:1116–1127. 244. Ferrando AA, Neuberg DS, Staunton J, et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell 2002;1:75–87. 246. Yeoh EJ, Ross ME, Shurtleff SA, et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002;1:133–143. 252. Sambandam A, Maillard I, Zediak VP, et al. Notch signaling controls the generation and differentiation of early T lineage progenitors. Nat Immunol 2005;6:663–670. 256. Aster JC, Pear WS, Blacklow SC. Notch signaling in leukemia. Annu Rev Pathol Mech Dis 2008;3:587–613. 257. Roy M, Pear WS, Aster JC. The multifaceted role of Notch in cancer. Curr Opin Genet Dev 2007;17:52–59. 260. Weng AP, Millholland JM, Yashiro-Ohtani Y, et al. c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev 2006;20:2096–2109. 264. O’Neil J, Grim J, Strack P, et al. FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors. J Exp Med 2007;204:1813–1824. 268. Weng AP, Nam Y, Wolfe MS, et al. Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol Cell Biol 2003;23:655–664.

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280. McCormack MP, Forster A, Drynan L, Pannell R, Rabbitts TH. The LMO2 T-cell oncogene is activated via chromosomal translocations or retroviral insertion during gene therapy but has no mandatory role in normal T-cell development. Mol Cell Biol 2003;23:9003–9013. 282. Grutz GG, Bucher K, Lvenir I, Larson T, Larson RA, Rabbitts TH. The ­oncogenic T cell LIM-protein Lmo2 forms part of a DNA-binding complex specifically in immature T cells. EMBO J 1998;17:4594–4605. 288. Ono Y, Fukuhara N, Yoshie O. Transcriptional activity of TAL1 in T  cell acute lymphoblastic leukemia (T-ALL) requires RBTN1 or -2 and induces TALLA1, a highly specific tumor marker of T-ALL. J Biol Chem 1997;272: 4576–4581. 290. Palomero T, Odom DT, O’Neil J, et al. Transcriptional regulatory networks downstream of TAL1/SCL in T-cell acute lymphoblastic leukemia. Blood 2006;108:986–992. 293. Dave UP, Akagi K, Tripathi R, et al. Murine leukemias with retroviral insertions at Lmo2 are predictive of the leukemias induced in SCID-X1 patients following retroviral gene therapy. PLoS Genet 2009;5:e1000491. 295. Zhang J, Ding L, Holmfeldt L, et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012;481:157–163. 305. Dadi S, Le Noir S, Payet-Bornet D, et al. TLX homeodomain oncogenes ­mediate T cell maturation arrest in T-ALL via interaction with ETS1 and ­suppression of TCRalpha gene expression. Cancer Cell 2012;21:563–576. 311. Gutierrez A, Kentsis A, Sanda T, et al. The BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia. Blood 2011;118:4169–4173. 317. Mullighan CG, Zhang J, Harvey RC, et al. JAK mutations in high-risk ­childhood acute lymphoblastic leukemia. Proc Natl Acad Sci U S A 2009;106: 9414–9418. 323. Mavrakis KJ, Van Der Meulen J, Wolfe AL, et al. A cooperative microRNAtumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL). Nat Genet 2011;43:673–678. 332. Grimwade D, Mrozek K. Diagnostic and prognostic value of cytogenetics in acute myeloid leukemia. Hematol Oncol Clin North Am 2011;25:1135–1161.

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Chapter 73

Diagnosis and Classification of the Acute Leukemias and Myelodysplastic Syndromes Daniel A. Arber, Attilio Orazi

The diagnosis and classification of acute leukemias and myelodysplastic syndromes (MDSs) has grown increasingly complex.1,2 Cases can no longer be fully classified by the use of only morphologic evaluation and cytochemical studies. Historic information, such as the presence of Down syndrome, prior therapy, or prior MDS all impact the final diagnosis. Additionally, immunophenotypic studies are needed for many cases; and cytogenetic, with possible molecular genetic, studies are required for essentially all cases. This increase in complexity in the evaluation of these neoplasms has resulted in more precise diagnostic categories and recognition that the broad categories of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and MDS actually represent heterogeneous groups of diseases. The newer disease categories are more predictive of outcome than older classification systems, in part because of their ability to separate disease groups within each category.3–6 Unfortunately, many physicians continue to use older terminology for these diseases, relying on terminology from the French-American-British Cooperative Group (FAB) classification7,8,9,10 of these neoplasms. While the FAB classification provided firm diagnostic criteria and useful terminology for communication of findings using the methods available at the time, its use is no longer appropriate. The third (2001) and fourth (2008) editions of the World Health Organization (WHO) classification of hematopoietic tumors have dramatically changed the approach to diagnosis of many of these neoplasms, and the WHO system should be considered the current standard of care. Modifications from the LeukemiaNet group and others will certainly continue to aid in the refinement of our classification systems.11,12,13 This evolution from the FAB to the WHO is reminiscent of changes in lymphoma classification with evolution from the Rappaport14 and Kiel15 classifications to the Working Formulation16 to the REAL17 classification and finally to the WHO classification.2,18 Perhaps because the changes in lymphoma classification were more stepwise with shorter time intervals between the changes than the leukemia classification changes, they have been more widely adopted. We no longer refer to diffuse large B-cell lymphoma as histiocytic lymphoma and should no longer refer to AML with t(8;21)q22;q22) as M2, or to B-lymphoblastic leukemia with BCRABL1 as L2. Several discoveries have impacted the classification of these neoplasms and many of them are covered in great detail in other chapters, but genetic discoveries associated with the acute leukemias and MDSs are probably the most significant. The finding of recurrent cytogenetic abnormalities with prognostic significance impacts all of these diseases.19,20,21–24 While balanced translocations are more common in the acute leukemias, the presence of single and complex abnormalities in the MDS has helped define disease prognosis as well as defining a specific disease category of MDS with isolated del(5q).5,6 The more recent discoveries of specific gene mutations have further impacted both acute leukemia and MDS diagnosis.13,25–31 While many of these mutations have their greatest frequency in AMLs with a normal karyotype, others offer prognostic significance that complements other morphologic and karyotypic features. The classification of AML and MDS has also been impacted greatly by the understanding of similarities between the two.

The so-called myelodysplasia-related AML and de novo AML described by Head32 helped lead to a new way of thinking about this disease; especially AML occurring in older patients. This chapter will highlight key classification issues in the acute leukemias and MDSs, which are discussed in detail in the chapters that follow.

Diagnostic Evaluation The diagnostic approach to acute leukemia and MDSs still begins with a morphologic evaluation, but requires careful integration of the morphologic findings with clinical information and relevant laboratory data, including cytogenetic and molecular results.33 Laboratory data, particularly results of a recent complete blood count (CBC) must be reviewed with the samples. Morphologic evaluation requires a well-stained (usually Wright-stained) peripheral blood (PB) smear prepared from a recent sample (less than 2 hours from procurement). A 200-cell manual differential count is required for PB smears. The bone marrow (BM) aspirate smears are best prepared at the bedside immediately after procurement and promptly stained. The review of BM aspirate smear includes a 500-cell differential count. In patients with inaspirable marrow, touch preparation of the BM biopsy can be used in lieu of an aspirate smear. The BM biopsy is usually stained with hematoxylin and eosin (H&E) or Giemsa and is useful in many settings.34 The morphologic assessment allows for the appropriate use of ancillary tests in these disorders. Details of the ancillary tests used for the workup of the various diseases are provided in Chapters 2 through 4 and will not be repeated here. Cytochemical studies are still performed at many institutions and can often provide quick general information about the cell type of an acute leukemia (myeloid versus myeloperoxidase negative), but because more detailed information can now be obtained by flow cytometry in the same time frame, the use of these less specific cytochemical studies has decreased. Immunophenotyping, usually by multicolor flow cytometry, is now standard for acute leukemias and is absolutely required to accurately diagnose the lymphoblastic leukemias and some AMLs.7,11,12,35,36 In cases of acute leukemia with marrow fibrosis, as may occur with some acute megakaryoblastic leukemias and with acute panmyelosis with myelofibrosis (APMF) (see Fig. 73.1), paraffin section immunohistochemistry performed on a BM trephine biopsy is essential.37 The use of flow cytometry immunophenotyping in the MDSs is the subject of much study, and many centers have incorporated this technique into the evaluation of patients with potential MDS not only to help quantitate blood and marrow blast cell percentages, but also to detect abnormal maturing cell populations.11,38,39 These methods, however, do not replace morphologic evaluation, including morphologic blast cell counts on smears. Cytogenetic studies should also be performed on all cases of suspected acute leukemia or MDS.21,40,41 While specific gene mutations and some structural abnormalities will be missed by this method, karyotype analysis currently provides the best overall assessment of chromosomal abnormalities and should not be supplanted by other studies. Fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) studies are often helpful to detect cryptic abnormalities that may be missed by karyotype analysis and are often added in panels for suspected

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A

B

C

D FIGURE 73.1 Acute panmyelosis with myelofibrosis illustrating the use of immunohistochemistry in diagnosis.  The marrow is inaspirable due to marked marrow fibrosis (A) with a mixed cellular population that includes immature mononuclear cells. The cells show a mix of granulocyte precursors marking with myeloperoxidase (B), erythroid precursors marking with glycophorin B (C), and immature megakaryocytes marking with von Willebrand’s factor (D).

acute leukemia or MDS.42 Finally, molecular studies for specific gene mutations are now routinely performed on samples from patients with these disorders, but when each mutation is studied individually the specific tests performed should be ordered based on the findings of other studies. However, with the rapid growth of next-generation sequencing technology,43 more cost-effective gene mutation panels will become available that may reduce the need for selective testing. Once this broad array of studies is complete, the results should be incorporated into a single report with a final diagnosis. Because they cannot always be completed in the time interval needed to begin therapy, this approach requires the use of preliminary and amended reports. Because the WHO classification relies on use of cytogenetic studies and some prognostic risk groups are defined by mutation analysis, the diagnosis often needs to be refined, and thus amended, when these studies are complete.

Myelodysplastic Syndromes Patients with MDS typically show persistent (>6 months) unexplained cytopenias. The majority of MDS patients present with anemia. Neutropenia and thrombocytopenia are less common presenting symptoms. The cytopenias are defined by a hemoglobin level of less than 10 g/dL, an absolute neutrophil count

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of less than 1.8 × 109/L, and a platelet count of less than 100 × 109/L.33,44 Of note, CBC values higher than those listed above are not exclusionary for a diagnosis of MDS if definitive morphologic and/or cytogenetic findings are consistent with such a diagnosis. Since the presence of cytopenia(s) is required for the diagnosis, the most current and preferably previous CBCs have to be reviewed at the time of BM exam. Other pertinent data include medications, chemical exposure history, and previous and current illnesses, since all of these can cause morphologic dysplasia indistinguishable from MDS. Comorbidities associated with morphologic dysplasia are frequent in elderly patients affected by MDS, and include liver and kidney failure, autoimmune disorders, neoplasms, and systemic infections. In particular, morphologic evaluation is best performed when the patient is off medication. Dysplastic features can be present in a single hematopoietic lineage (unilineage dysplasia) or involve all marrow populations (multilineage dysplasia) (Fig. 73.2). At least 10% of all cells in a given lineage (erythroid, myeloid, megakaryocytic) have to be dysplastic for establishing the presence of MDS-associated dysplasia. The majority of MDS subtypes show dyserythropoiesis. PB shows normocytic, normochromic, or macrocytic anemia with macroovalocytes. Microcytosis can be present in rare cases of MDS (e.g., cases associated with congenital or acquired alpha thalassemia).45 The most common dysplastic features seen in erythroid precursors include nuclear abnormalities such as nuclear

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B

C

D

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A

F E FIGURE 73.2 Dyspoietic changes in myelodysplastic syndrome and acute myeloid leukemia. Peripheral blood (A, B) and bone marrow (C, D) n­ eutrophils may show cytoplasmic hypogranulation, clumped nuclear chromatin, and hyposegmentation, including pseudo–Pelger-Huët anomaly as shown in image A. ­Dyserythropoiesis may include nuclear-cytoplasmic asynchrony and irregular nuclear shapes in the marrow erythroid precursors (C, D). Megakaryocyte changes include hypersegmented cells (E) and smaller, hypolobated cells (F).

budding, nuclear fragmentation, irregular nuclear outlines, karyorrhexis, internuclear bridging, multinucleation, and megaloblastoid, coarsely condensed chromatin. Cytoplasmic vacuoles with coalescing vacuoles, defective hemoglobinization and increased numbers of ring sideroblasts can also be encountered. Similarly,

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dysgranulopoiesis is often manifested by abnormal nuclear features including hypolobation (pseudo-Pelger-Huët or monolobated neutrophils), hypersegmented and/or enlarged nuclei, nuclear sticks or fragments, macropolycytes, and abnormally condensed chromatin, which often coexists with nuclear hypolobation.

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Cytoplasmic features of neutrophil dysplasia include hypogranulation, pseudo-Chediak-Higashi granules, and rarely Auer rods. Hypogranulation occurs frequently and is related to the defective formation of secondary granules. However, the evaluation of this feature is highly subjective and dependent on the staining quality. A well-stained segmented neutrophil or neutrophilic precursor with well-developed secondary granules present, preferably in the same microscopic field, can be used as an internal control. Megakaryocyte morphology can be evaluated using both aspirate smear and the histologic sections (biopsy or clot section) with careful examination of at least 30 megakaryocytes.33,44 Dysplastic features include monolobated, hypolobated, and hyperlobated nuclei, and multiple, widely separated nuclei including “pawn-ball” forms. Normal size or small megakaryocytes with a single (nonlobated) eccentrically placed nucleus are common in 5q- syndrome and in cases with abnormalities of chromosome 3. The latter also shows typically numerous bilobated forms. Megakaryocytic dysplasia is often associated with thrombocytopenia and platelets of variable size, including large forms occasionally showing hypogranulation. The biopsy allows for the evaluation of cellularity, architectural features, fibrosis, and the presence of previously undiagnosed focal lesions such as metastatic neoplasms or infections. In MDS marrows, the typical architectural organization is lost. Clusters of immature cells are frequently found in the center of the marrow space. This finding was originally termed abnormal localization of immature precursors and is more frequently seen in cases of high-grade MDS. The identification of blast clusters is facilitated by CD34 immunohistochemistry, which is also useful to determine the number of blasts in cases with inaspirable marrows. The presence of CD34 positive cell clusters is a prognostically significant finding that is predominantly seen in high-risk MDS.46 The BM biopsy is necessary for the assessment of BM fibrosis by the Gomori silver impregnation method. Significant fibrosis occurs predominantly in high-risk and therapy-related MDS; however, it may also occur in low-risk disease. Finally, BM biopsy is critical for the exclusion of other hematologic and also nonhematologic diseases associated with unexplained cytopenia(s) which can clinically mimic MDS. In addition to dysplasia, the evaluation of blasts constitutes a cornerstone of morphologic diagnosis of MDS. The optimal quality of BM aspirate and PB smears cannot be overemphasized.33 The blast percentage is derived from the 500-cell differential count of marrow aspirate smear and 200-cell PB manual differential count. Both differential counts are essential for the subclassification of individual cases. The agranular and granular (type II and type III blasts), and, if present, monoblasts, promonocytes, and megakaryoblasts are included in the blast category.47 In the absence of adequate BM aspirate smears due to causes such as an inaspirable marrow, careful inspection of the histologic material aided by a CD34 immunohistochemical stain can be used. The latter is particularly helpful in cases with significant marrow fibrosis or in hypoplastic marrows, which often yield hemodilute marrow aspirates.48,49,50 Of note, blasts in MDS can be negative for CD34, therefore careful correlation with the visual blast identification is required in all cases. It is also important to emphasize that morphologic dysplasia is not equivalent to the diagnosis of MDS. Megakaryocytic and erythroid dysplasia are commonly seen in individuals in a variety of conditions. Nutritional deficiencies, heavy metal exposure, medications, and systemic diseases can produce morphologic changes resembling those seen in MDS. Therefore, as discussed previously, the correlation with clinical history is critical. Cytogenetics is crucial and is also required for proper classification. The MDS-associated abnormalities are listed in Table 73.1. In addition, mutational analysis is becoming an important additional tool in the prognostic assessment of MDS30,51; but mutation studies are not routinely performed in the evaluation for MDS at this time.

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TA B L E 7 3 . 1

Recurrent Chromosomal Abnormalities and Their Frequency in Myelodysplastic Syndrome Abnormality Unbalanced  +8  −7 or del(7q)  −5 or del(5q)  del(20q)  −Y   i(17q) or t(17q)   −13 or del(13q)  del(11q)   del(12q) or t(12q)  del(9q)  idic(X)(q13) Balanced   t(1; 3)(p36.3;q21.2)   t(2; 11)(p21;q23)  inv(3)(q21;q26.2)  t(6;9)(p23;q34)

Frequency (%) 10 10 10 5–8 5 3–5 3 3 3 1–2 1–2 1 1 1 1

The 2008 WHO classification further refined the criteria for diagnosis and classification of MDS, and seven categories are now recognized (Table 73.2). Cases currently recognized as refractory cytopenia with unilineage dysplasia (RCUD) were considered as refractory anemia (RA) or MDS, unclassifiable in the 2001 WHO classification. Such cases encompass patients with one isolated cytopenia or bicytopenia associated with unilineage dysplasia. An advantage of the new category of RCUD is that one can further specify the lineage: refractory anemia, refractory neutropenia, or refractory thrombocytopenia. Regardless of the lineage involved in RCUD, blasts are absent or represent less than 1% of the PB differential count. Patients with 1% blasts in PB or patients with unilineage dysplasia associated with pancytopenia are now classified as having unclassifiable MDS (MDS-U), owing to the uncertain but presumably more severe clinical significance of these findings.2 Overall, the category of MDS-U is currently better defined and also includes cytopenic patients lacking significant dysplasia, but presenting with cytogenetic abnormalities considered presumptive evidence of MDS. The 2008 WHO classification acknowledged a subset of pediatric patients with specific MDS features different from those commonly seen in adults, and included a separate category of childhood MDS termed refractory cytopenia of childhood. This category encompasses children with MDS that have persistent cytopenia with less than 2% blasts in the PB and less than 5% blasts in the BM.52,53 Some subtypes or presentations of MDS are more challenging and will be discussed in more detail. MDS-U includes cases which do not fulfill criteria of other MDS subtypes. MDS-U can be diagnosed in patients fulfilling the following criteria: (1) patients who fit the criteria for a diagnosis of RCUD or refractory cytopenia with multilineage dysplasia (RCMD), but in whom 1% blasts in the blood are found on at least two consecutive occasions; (2) patients with MDS with pancytopenia and morphologic dysplasia limited to one hematopoietic lineage; (3) patients with persistent cytopenias, no increase in blasts, and lacking diagnostic morphologic features of MDS (less than 10% dysplastic cells in any lineage), but with clonal cytogenetic abnormalities considered as a presumptive

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TA B L E 73. 2

The WHO 2008 Classification Of Mds Subtype

Blood findings

Refractory cytopenias with unilineage dysplasia (RCUD)   Refractory anemia (RA)   Refractory neutropenia (RN)   Refractory thrombocytopenia (RT)

Unicytopenia or bicytopeniaa No or rare blasts ( 30%) appear to have clinically aggressive behavior.358,359 Median survivals for FL were static in the range of 8 to 12 years for several decades, but are now improving due, in part, to monoclonal antibody therapy and better supportive care.351,352,360,361 The microenvironment and gene expression profiling in FL are likely to add prognostic information and potential therapeutic targets. There has been conflicting data about the presence of tumor-associated CD68+ macrophages (TAM) and T-reg cells, with initial reports indicating a poor prognosis for the former and a favorable prognosis for the latter. Subsequent studies have not confirmed these findings.330,332,364 Gene profiling identified a favorable “immune response 1” signature encoding T cells (CD7, CD8B1, ITK, LEF1, and STAT4) and macrophages. (ACTN1 and TNFSF3B) and an unfavorable “immune response 2” signature of genes in macrophages and/or dendritic cells (TLR5, FCGR1A, SEPT10, CCR1, LGMN, and C3AR1). Future studies will incorporate genetic, molecular, and biologic data into prognostic models which will influence the selection of therapy.

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Small B lymphocytic lymphoma (SLL) comprises 3% to 10% of all NHL and is part of the spectrum of diffuse small B cell lymphomas which includes lymphoplasmacytoid lymphoma, marginal zone lymphomas, and mantle cell lymphoma (Chapter 86).365,366 There is extensive clinical overlap with chronic lymphocytic leukemia (CLL) (Chapter 90), although there is more prominent lymphadenopathy and less lymphocytosis in SLL than in CLL.367–369,370 The WHO considers SLL and CLL different clinical presentations of the same disease. An initial lymphocyte count greater than 5 × 109/L is considered diagnostic of CLL. Over time, 10% to 20% of patients develop a lymphocytosis consistent with CLL. SLL represents 4% to 6% of NHL in the West, is a disease of the elderly (median age, 55 to 65 years), and has a male:female ratio of approximately 2:1.367,368 Patients usually present with generalized adenopathy, and marrow involvement is found in most patients (70% to 80%). Median survivals have been variable from a low of 4 to 6 years to 10+ years, may depend upon prognostic factors and the criteria utilized to separate SLL from CLL, but are similarly improving in the rituximab era.371 Deletion at chromosome 6q is the most common cytogenetic abnormality in SLL, but has had no effect on prognosis,372 whereas del(17p) and del(11q) are associated with poorer prognosis.373 Both unmutated genes as opposed to hypermutated genes and expression of CD38 or ZAP 70 have been associated with a worse prognosis in CLL.374 Between 2% and 8% of SLL and CLL evolve into an aggressive large cell process known as Richter syndrome, which is characterized by bulky retroperitoneal adenopathy, rising LDH, and survival usually less than 1 year.375,376 Prognosis is better if transformation occurs in previously untreated patients.370 Lymphoplasmacytic lymphoma (LPL) largely overlaps with Waldenström macroglobulinemia (WM) (Chapter 100). LPL represents 1% to 2% of NHL, usually occurs in the elderly (median age 60 to 70 years), and presents with lymphadenopathy (15% to 20%), splenomegaly (10% to 20%), and marrow involvement in most patients.377,378 A paraprotein is found in 29% to 50% of patients, with IgM the most prevalent type, and can contribute to hyperviscosity (6% to 20%), neuropathy, and glomerular disease.377 A positive Coombs’ test, cold agglutinin disease, cryoglobulinemia, autoimmune diseases, and positive hepatitis C serology can be associated with LPL and WM.379,380 Mutation of the myeloid differentiation primary response gene88 (MYC88) is nearly universal in WM and can be useful in differentiating it from other disorders.381 Translocation (9;14) involving the paired box gene PAX5 can be detected in LPL, but its frequency and significance are a matter of debate.378 Deletion of 6q is the most common cytogenetic abnormality but it is not specific for LPL/ WM.378 Median survivals have been variable, usually in the 7- to 10-year range, and are worse with advanced age, cytopenias, organomegaly, elevated B2 M, and hypoalbuminemia.378,382,383 Marginal zone B cell lymphomas (MZL) usually account for between 5% and 7% of all NHL and include extranodal MALToma, nodal based disease, and SMZL.384 Extranodal MALTomas represent the most common MZL (50% to 70%) and are discussed in the section “Management of Extranodal Lymphomas”. Nodal MZL occurs in the elderly (median age, 59 to 65 years) and preferentially in women (up to 2:1 female:male ratio), may present with localized lymphadenopathy, and has less marrow involvement (28% to 45%) than other indolent lymphomas.371,384,385,386 Cytopenias or a paraprotein (10%) are rarely present. The most frequent cytogenetic abnormalities are gains in chromosomes 3 and 18q23.387 Median survival has been variable due to limited numbers of patients but has been recorded in the 9- to 12-year range.371 SMZL occurs in the elderly (median age, 61 to 70 years), has a slight female predominance, and usually presents because of symptoms of splenomegaly.384,388,389 Cytopenias are common (46% to 60%); peripheral adenopathy is rare (10% to 15%); and marrow involvement is usually detected (73% to 100%).390,391

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Chapter 88  Non-Hodgkin Lymphoma in Adults

A paraprotein may be present and is usually IgM. Despite disseminated disease, splenectomy can alleviate symptoms and improve cytopenias.389,390 Median survival has been recorded to be 10.5 years, but is shorter in the presence of a paraprotein, elevated b-2m, or lymphocytosis (>9 × 109/L).391,392 A simple prognostic scoring system is based on 3 factors: hemoglobin 90% of cases and is considered the most reliable immunophenotypic marker in the diagnosis of MCL,472 although variant MCL with cyclin D2 or cyclin D3 expression also occur.473,474 A minimally deleted segment of 11q22–q23 affecting the ATM gene in 50% of MCL suggests that this is an early genetic event along with the

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BCL-1/CCND1 (cyclin D1) translocation to chromosome 14.475 Loss of tumor suppressor genes, including TP53 and CDKN2/p16, appears to occur later and has been associated with an aggressive clinical course.476–479 Marked clinical and biologic heterogeneity of MCL is now well recognized, with about a quarter of patients having “indolent” disease that may not require immediate therapy. These patients often present with lymphocytosis and splenomegaly, and their symptoms may be confused with CLL if proper flow cytometry and FISH markers are not obtained at diagnosis. Others present with low tumor burden lymphadenopathy and lack systemic symptoms, and watchful waiting for the assessment of the pace of disease has been proposed as initial management in these cases.480 Biomarkers of clinically indolent cases include mutated immunoglobulin heavy chain variable genes, lack of nuclear SOX11 expression and TP53 mutation, few if any karyotypic changes aside from the t(11;14), and a characteristic molecular signature.481 Clinically aggressive MCL typically has high tumor burden, blastoid morphology, and complex cytogenetics in addition to the t(11;14) and/or high Ki-67 expression. Molecular markers that appear both pathogenically and prognostically relevant include the NOTCH1 mutation,482 dysregulation of the Hippo tumor suppressor pathway,483 and a unique microRNA signature and expression of miR-29.484,485 The percentage of tumor cells expressing the proliferation marker Ki-67 has been incorporated into the MIPI score to enhance the discrimination of clinical outcomes among low-, intermediate-, and high-risk MCL patients treated with CHOP or R-CHOP regimens (Fig. 88.16).306,486 Clinical outcomes and survival have improved in recent years as treatment options have expanded, although MCL remains incurable with standard approaches. Most studies show the absence of a plateau on survival curves. Response rates to initial therapy are typically above 80% with a number of chemoimmunotherapy regimens; CRs are usually 100 × 109/L in 75% of patients), splenomegaly (73%) and lymphadenopathy (53%); and approximately 20% of patients have Hematologic Malignancies

The differential diagnosis of CLL in Chapter 90 includes the mature T/NK leukemias. Large granular lymphocyte (LGL) leukemia was associated with chronic neutropenia in 1977, recognized as a clonal disorder in 1985, and classified into T (CD3+) and NK (CD3-) types in 1993(Fig. 88.21A).204,627,628 The median age is 55 to 60 years, and there is an association with rheumatoid arthritis (20%) and autoimmune features.204,629 Lymphocytosis is usually between 2 and 20 × 109/L and mild splenomegaly (25% to 50%) may be present, but the usual clinical presentation is neutropenia or anemia. Recurrent infections are usually respiratory or mucocutaneous and occur in 15% to 56% of patients.204 Pure red cell aplasia, autoimmune hemolytic anemia, aplastic anemia, ITP, and MDS may occur in LGL leukemia. LGL cells express high levels of both FAS and FAS ligand, but the cells are resistant to FASmediated death due to STAT 3 activation which upregulates an antiapoptotic protein.629,630 LGL leukemia is an indolent disease

1857

B

A

D

C FIGURE 88.21. Mature T/NK leukemias:  A: T cell large granular lymphocytic leukemia: The lymphocytes are enlarged with dense nuclear chromatin and abundant cytoplasm with fine azurophilic granules. B: Natural killer/T cell leukemia: Three leukemic cells and one myelocyte are present. The leukemia cells have blastic, fine nuclear chromatin with irregular nuclei and basophilic cytoplasm. Some cases may show cytoplasmic granules. C: T cell prolymphocytic leukemia: The lymphocyte has clumped nuclear chromatin with irregular nuclear contours, sometimes referred to as “knobby cells.” Nucleoli may be inconspicuous, in contrast to B cell prolymphocytic leukemia, which has prominent nucleoli. D: Abnormal T lymphocyte in the peripheral blood of a patient with adult T cell leukemia. The cell is enlarged with a multilobated nucleus. These “flower cells” are usually easily found with this disorder. Images provided by Dr. Dan Arber, Stanford University, Palo Alto, CA.

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skin infiltration.639,640,642 Cell morphology is variable; the nuclei are irregular, include a “knobby” variant, and usually have prominent nucleoli; cytoplasmic protrusions are characteristic. Sixty per cent of patients are CD4+/CD8-; 25% coexpress CD4+/CD8+; and 15% are CD4-/CD8+ 639,640,642. The most common cytogenetic abnormality occurring in 80% of patients is an inversion of chromosome 14 with break points in q11 and q32; 10% have a reciprocal translocation t(14;14) (q11;q32).639,644 These translocations juxtapose the locus of the TCR a/b gene with the TCL1 and TCL1b genes at 14q32.645 Deletions at 11q23, the locus for the ATM gene, and at 12p13, and abnormalities of chromosome 8 are other common cytogenetic findings in T-PLL.639,646 Response rates in T-PLL are 10% to 48% with few complete remissions utilizing chemotherapy, including CHOP or nucleoside analogs.642,643 Median survival is usually 1 year although up to one-third of patients can have an indolent phase.642,643,647Alemtuzumab (Campath 1H) is the most active single agent in T-PLL with a 74% and 91% response rate in previously treated and untreated patients, respectively.641,648 Preliminary data suggest the best survival occurs in patients treated with intravenous alemtuzumab followed by an allogeneic HCT. Nucleoside analogs, particularly pentostatin, can be effective in slow responders.641 The clinical course of ATL, which follows infection by HTLV1, is variable and takes 4 basic forms: 1) acute (leukemic) (60%), 2) lymphoma (20%), 3) chronic (20%), and 4) smoldering (5%). The male to female ratio is 1.3–2.2:1, and the median age is 47 to 65 years.59 The acute form is characterized by lymphadenopathy, organomegaly, skin lesions, elevated white count with multilobed lymphocytes, often referred to as “cloverleaf” or “flower” cells (Fig. 88.21D), hypercalcemia, elevated lactate dehydrogenase (LDH) level, and usually a rapidly fatal course. The cutaneous lesions have a diverse appearance, including papules, nodules, plaques, tumors, and ulcers. Histologically, dermal invasion predominates, although the lesions of ATL may resemble primary cutaneous T cell lymphoma with epidermotropism and Pautrier’s microabscesses. Anemia and thrombocytopenia are infrequent findings because of a low degree of marrow infiltration. Central nervous system involvement may develop in as many as 10% of patients with ATL.649 Lymphoma is distinguished by prominent adenopathy without significant peripheral blood involvement. Primary extranodal lymphoma occurs in approximately 5% of lymphomatous presentations and has involved skin, Waldeyer’s ring, gastrointestinal tract, sinuses, and pleura.650 The chronic type is associated with an increased white blood cell count and occasionally with slight adenopathy and organomegaly. Patients with smoldering ATL have few ATL cells (0.5% to 3%) in the peripheral blood, and may have skin lesions as well as slight adenopathy, organomegaly, and marrow infiltration. Chronic and/or smoldering ATL may evolve into an acute form after many years of indolent disease.651 No consistent cytogenetic abnormality has been identified in ATL. The most common abnormalities are gains at chromosomes 14q, 7q, and 3p and losses at 6q and 13q.652,653 Aneuploidy, multiple chromosomal breaks, and loss of tumor suppressor genes are associated with an aggressive course.653,654 Despite combination chemotherapy, which can yield brief and low responses, median survivals in the acute and lymphomatous forms of ATL are usually less than 1 year.59,655,656,657 Chemotherapy with a complex regimen, the LSG15 protocol, is marginally superior to CHOP and has higher response rates in the lymphoma form compared to acute.658 Overexpression of MDR (ABCB1) and TP53 contribute to chemoresistance.659,660 Poor performance status, high LDH, age above 40 years, tumor bulk, and hypercalcemia are adverse prognostic factors.655 The chronic and smoldering forms have a longer survival rate regardless of therapy. The major causes of death in ATL are opportunistic

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pulmonary infections and progressive disease, often in association with hypercalcemia.661,662 Because of its chemoresistance and its HTLV-1 viral leukemogenesis, ATL has been a unique disease for investigating therapy. Interferon a (IFN) plus zidovudine (AZT) has had a higher response rate (67% to 92%) than chemotherapy regimens in ATLL.663 The current recommendation is to first initiate antiviral therapy for the acute, chronic, and smoldering forms.657 Others advocate debulking with chemotherapy followed by antiviral therapy or concomitant treatment, particularly for the lymphoma form. NF-kB inhibition has been proposed as a therapeutic target in ATL. Arsenic trioxide synergizes with IFN, has been shown to shut down the NF-kB pathway, and has been effective in phase II trials.664 Conjugated and unconjugated monoclonal antibodies directed at the IL-2 receptor (CD25), CD52 (alemtuzumab), chemokine receptor 4(KW-0761), and CD2 (Siplizumab) have activity in ATL and are being evaluated in conjunction with other therapy. Allogeneic hematopoietic cell transplantation (alloHCT) was first reported as a curative option in ATL in 1996. Although the median survival with alloHCT (9.9 months in a review of 586 patients) does not appear superior to other therapy, the 3-year estimated overall survival of 36% suggests HCT may offer the best chance for long-term survival.665 Despite the new therapies under investigation in ATL, the ultimate goal is prevention of the disease. Avoiding breast-feeding in mothers infected with HTLV-1 can reduce infection in the newborn by 80%.59 Other proposed methods to prevent ATL are antiretroviral therapy and a TAX-targeted vaccine.

Therapy For Localized Large Cell Lymphoma Localized disease is defined by either stage I or II disease which is nonbulky (no tumor mass ≥ 10 cm; no mediastinal mass > onethird the chest diameter). Radiation therapy alone has resulted in 20% to 85% cure rates for limited stage large cell lymphomas, with the best results in patients who have stage I disease after undergoing pathologic staging.18,666 With clinical staging, cure rates with radiation alone have usually been less than 50%; therefore, most investigators are advocating chemotherapy, usually CHOP-type regimens, often followed by radiation, with cure rates of 70% to 90%.667 Using a stage-modified IPI for localized disease, patients with no adverse risk factors (stage I, age ≤60, 60 year vs. 93% (P = 0.03). No difference between BL and unclassified B cell lymphoma

BM, bone marrow involved; CALGB, Cancer and Leukemia Group B; CR, complete remission; CODOX, cyclophosphamide, vincristine, doxorubicin, high-dose methotrexate; D, liposomal cytarabine; DFS, disease-free survival; dm, dose-modified; EPOCH, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin; M, methotrexate; GMALL, German Multicenter ALL; Hyper CVAD, cyclophosphamide, vincristine, Adriamycin (doxorubicin), dexamethasone alternated with high doses of methotrexate and cytarabine; HIV, human immunodeficiency virus; IVAC, ifosfamide, VP-16 (etoposide), Ara-C (cytarabine); LMB, Lymphoma Malins de Burkitt; R, rituximab.

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genes, a and d at 14q11.2, b at 7q35, and g at 7p14-5, which produce high levels of transcription factor genes such as HOX11/ TLX1, TAL1/SCL, TAL2, and LYL. GEP can subdivide T-LBL into subtypes with differing prognoses.829–831 Patients with HOX11 have a pattern of gene expression associated with the early cortical thymocyte and have a better prognosis than patients with TAL1 or LYL1 expression, which correlate with late cortical and early pro-T lymphocytes, respectively.832 NOTCH1 is a transmembrane receptor and activating mutations occur in 50% to 60% of patients with childhood T-ALL.833,834 FBXW7 is a ubiquitin ligase that triggers ubiquitination and degradation by the proteasome. Inactivating mutations FBXW7 have been reported in 11% to 13% of patients. NOTCH1 and FBXW7 mutational status together identify a favorable group of lymphoblastic lymphoma.835 Rarely, T-LBL is associated with eosinophilia, myeloid malignancy, specific cytogenetic translocations t(8;13) (p11;q12), t(8;9)(p11;q32), or t(6;8)(q27;p11), and is known as 8p11 myeloproliferative disorder.836 Unusual cases of T cell LBL have been associated with prior epipodophyllotoxin chemotherapy and a cytogenetic translocation involving the MLL gene, t(11;19)(q23;p23).837 Because LBL is rare in adults, only a few series have addressed therapy and some patients have received early transplantation, which compromises the assessment of the impact of chemotherapy.458 Studies in the 1980s indicated a CR rate of 53% to 95% using CHOP-like induction regimens and high CNS relapse (∼40%) if CNS prophylaxis was not given.838–840Using predominantly ALL-like regimens with a maintenance phase lasting 12 to 36 months results in 3- to 5-year survivals of 40% to 72%.838–840 Poor prognostic features included age (> 30 years), bone marrow involvement, elevated white-cell count (>50 × 109/L), CNS disease, elevated LDH, and slow response.839,840 Coleman reported a 5-year survival of 94% in low-risk patients compared to 19% (P< 0.001) with high-risk features, defined by bone marrow or CNS disease or elevated LDH (>1.5 × normal).840 Short course, dose intensive regimens without maintenance have been tried in lymphoblastic lymphoma, but the relapses appear excessive and the numbers are too small to utilize this approach.841 Controversial management issues for LBL include the optimal type of CNS prophylaxis, the role of radiation to the mediastinum,

the type of maintenance therapy, and the role of transplantation. With present-day ALL regimens, the CR rates are 75% to 90%, with prolonged DFS of 40% to 70% of responders (Table 88.15).842–853 An option to consider for young adults is to utilize pediatric-based protocols which have had better EFS when compared to adult ALL protocols.854 A retrospective analysis comparing patients irradiated to those not irradiated showed less mediastinal relapse but no difference in FFP or OS.855 In the era of intensive ALL regimens, however, routine mediastinal radiation is no longer used in pediatrics and is probably not warranted in most adults. PET imaging may assess residual mediastinal disease, but it has not been well studied in LBL. Because of poor survival with chemotherapy in high-risk patients, there may be a role for early transplantation.851 The most significant predictor of survival for patients undergoing HCT is the status of the disease at the time of transplant.853,856–859 The European Bone Marrow Transplantation Group retrospectively reviewed their experience in 214 patients with LBL who underwent autologous transplantation with 6-year DFS varying according to disease status: 63% in first CR, 31% in second CR, and 15% with resistant disease.853 Trials that evaluate HCT often fail to include intent to treat analysis and result in a bias favoring the transplant arm. In a randomized trial comparing early autologous HCT to conventional chemotherapy,858 there was a trend favoring autoHCT (55% versus 24%, P = 0.065) but there was no improvement in OS.858 Only two-thirds of patients eligible for randomization were actually randomized. The role of allogeneic transplant in LBL in first CR is even more controversial. In an International Bone Marrow Transplant Registry retrospective review of transplants for LBL, there were fewer relapses with allogeneic HCT compared to autologous HCT, but there was no survival advantage due to higher treatment-related mortality.859 Recent randomized trials in adult ALL, some of which include LBL, however, are favoring the allogeneic arm.860,861 Novel agents are being studied in relapsed LBL. Nelarabine, an analog of AraG, had an ORR of 55% in pediatric patients with T-ALL/LBL in first relapse and 27% in second relapse.862 An ORR of 46% (36% CR) was reported for adults with relapsed/ refractory T-ALL/LBL.863 Clofarabine, a nucleoside analog that

Hematologic Malignancies



TA B L E 88.1 5

Chemotherapy In Adults With Lymphoblastic Lymphoma Survival by Risk Group Author, Year (Reference)

No. of Patients

Median Age (y)

Thomas, 2001842 Sweetenham, 2001858 Hoelzer, 2002848 Jabbour; 2006849 Song, 2007850 Hunault, 2007851

 24 119  45  27  34  45

Cortelazzo, 2012852

 30

Overall Survival (%) (at x years)

Low

Higha

Regimen

CR (%)

SCT in 1st CR (%)

28 26 25 31 26 27

Hyper CVAD LSA L2/Stanford GMALL-89/93 LMT-89 Hybrid ALL/NHL ALL, randomized to auto-SCT

96 57 93 74 66 89

0 36b 0 — 85c 44

80 (3) 46 (3) 51 (7) 63 (5) 72 (4) 64 (7)

— — 56 85 — 69

— — 48 30 — 60

27

ALL ± auto-SCT

62d

47

72 (5)

—

—

ALL, acute lymphoblastic leukemia; CR, complete response; GMALL, German Multicenter Acute Lymphoblastic Leukemia; HyperCVAD, cyclophosphamide, vincristine, doxorubicin, dexamethasone alternated with high-dose methotrexate and cytarabine followed by maintenance; LMT, Lymphoma Malignant T; NHL, non-Hodgkin lymphoma; SCT, stem cell transplant. a High

risk defined by marrow involvement.

b Transplant:

12 allo, 31 auto; auto had 55% vs. 24% RFS (3 year) for chemotherapy (n = 34) (P = 0.065).

c Transplant:

4 allo, 25 auto.

d CR

rate up to 93% with radiation to 6 pts.

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inhibits ribonucleotide reductase, had an ORR of 30% (24% CR) in relapsed pediatric ALL and 17% in adult ALL.864 Clofarabine combined with cyclophosphamide and/or etoposide had a 31% CR rate in adults with relapsed ALL/LBL, but had 23% treatmentrelated deaths.865 The prevalent NOTCH1 mutations in T-ALL are targets for inhibition, and mutations of NUP214-ABL1, an activated tyrosine kinase in episomal DNA, present in 6% of T-ALL, may respond to tyrosine kinase inhibitors.866

Management of Extranodal Lymphomas Approximately one-third of NHLs arise from sites other than nodes, spleen, or bone marrow, and have occurred in almost every organ.867 There is geographic and ethnic variation for sites and types of extranodal lymphomas.868 The gastrointestinal tract accounts for up to one-third of extranodal NHL in the Western hemisphere.867,869 If Waldeyer’s ring and tonsils are included, head and neck localizations are the second most common site, accounting for one-fifth of cases. SEER data reports stomach, skin, intestine, and brain to be the most common sites of extranodal disease.35 The sites of extranodal lymphoma

reflect the homing mechanisms of lymphocytes and may depend upon antigen-driven lymphoproliferation. Management of extranodal lymphomas does not necessarily follow that of their nodal counterparts.870 Site(s) of involvement and histologic type are both important factors in the management of extranodal lymphomas (Table 88.16). It is important to determine whether the disease is localized or disseminated. The Ann Arbor staging system has been used with stage IE, representing localized extranodal lymphoma, and IIE, representing extranodal lymphoma with involvement of adjacent lymph nodes; a subclassification of IIE, which separates involvement into contiguous nodes (II1E) and noncontiguous nodes (II2E), has been used in gastric lymphomas; stages III and IV in extranodal lymphomas represent disseminated disease with little advantage in distinguishing the 2 stages. Extranodal lymphomas of the gastrointestinal tract, nasopharynx, and testes are generally more aggressive than those of the lung, orbit, or salivary gland. Therapy, however, depends not only on the site, but also on the histologic characteristics, immunophenotype, epidemiologic factors, size, and stage of the lymphoma. The stomach accounts for approximately one-half of the gastrointestinal lymphomas; 60% of gastric lymphomas are aggressive, usually DLBCL, sometimes in conjunction with low grade histology, and 40% are indolent, usually of the MALT type. The

TA B L E 88.16

Management Issues For Extranodal Lymphomas Site

Usual Pathology

Clinical Associations

Suggested Therapya

Stomach

B cell MALToma

Helicobacter pylori

Intestine Ileum Immunoproliferative small intestinal disease Enteropathy-associated T cell lymphoma Waldeyer’s ring Paranasal (sinus) Nasal

Burkitt B cell MALToma Peripheral T cell lymphoma

Obstruction Malabsorption IgA heavy chain, Middle East Celiac disease, West Obstruction

Antibiotic trial, serial endoscopy; other therapy if t(11;18), t(1;14), transformation or progressive disease High-dose cyclophosphamide combination chemotherapy ±surgery Antibiotics, steroids, ± combination chemotherapy Combination chemotherapy ± surgery; nutrition

Large B cell Large B cell Natural killer/T cell lymphoma

Salivary gland Thyroid Lung Orbital Primary CNS

B cell MALToma Large B cell, B cell MALToma Small B cell, B cell MALToma Small B cell, B cell MALTtoma Large B cell

Other gastrointestinal disease CNS disease Epstein-Barr virus; angiocentric features, Asia Sjögren syndrome Hashimoto thyroiditis

Testis

Large B cell

Breast Ovary

Large B cell Burkitt Large B cell Large B cell Mycosis Fungoides B cell MALTomas, primary cutaneous follicular lymphoma, primary cutaneous large B cell lymphoma

Bone Cutaneous

Ocular involvement Leptomeningeal disease AIDS Contralateral testicular disease, CNS disease, retroperitoneal spread Pregnancy Bilateral disease

Combination chemotherapy or combined modality Combined modality, consider CNS prophylaxis Combined modality, consider CNS prophylaxis Single agent and/or radiation Radiation or combined modality, depending on histology and stage Single agent, surgery, or radiation Radiation Steroids, high-dose methotrexate ± radiation Orchiectomy, combination chemotherapy, radiation to contralateral testis, CNS prophylaxis Combination chemotherapy ± radiation Combination chemotherapy Combination chemotherapy ± radiation Skin-directed Radiation if stage IE Combined modality (see Chapter 92)

AIDS, acquired immunodeficiency syndrome; CNS, central nervous system; MALToma, lymphoma of mucosa-associated lymphoid tissue. aCombined

modality refers to combination chemotherapy and radiation. Combination chemotherapy refers to doxorubicin-based therapy. Rituximab is added to therapy of most B cell (CD20+) lymphomas.

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recognition of MALTomas has led to unique therapeutic options. MALTomas were previously called pseudolymphomas, but the latter term is no longer valid because MALTomas are monoclonal, can be associated with a higher grade histology, and have cytogenetic abnormalities.107–110,871 Clinically, MALTomas are different from other indolent lymphomas in that they tend to remain localized, rarely disseminate, and respond favorably to local therapy.871,872 Management of gastric MALToma changed when antibiotic therapy for H. pylori resulted in regression of lymphoma.873–877 Ultrasound endoscopy and biopsies assist in determining the extent of disease and depth of invasion, which correlate with spread to regional lymph nodes and response to antibiotics.878 CR rates to antibiotics are over 80% when disease is confined to the mucosa and submucosa.876 Antibiotics are also curative in over half the patients with H. pylori associated gastric large B cell lymphoma, with either a mixed marginal zone/DLBCL or pure DLBCL.101 Infiltration of the muscularis propria or nodal extension and the presence of t(11;18) or t(1:14) predict poor response to antibiotics.875,876,879 Serial endoscopies are warranted to ensure eradication of H. pylori and gradual disappearance of the lymphoma over many months.873–876 Follow-up endoscopy is recommended 2 months after antibiotic therapy and, subsequently, at least twice a year for 2 years.878 B cell clonality as detected by PCR may persist after histologic regression, but is of uncertain clinical significance.104,880 If there is H. pylori negative or progressive disease, alternative therapies are usually effective and include radiation, rituximab, and/or single agent or combination chemotherapy.872,881,882 With the recognition of MALToma and the effectiveness of other therapies, the role of surgery has diminished in the management of gastrointestinal lymphoma. While early studies advocated surgical resection in gastric and other intestinal lymphomas to cure localized disease, debulk, accurately stage, and/or prevent perforation,247,883,884 recent reports have achieved good results with either combination chemotherapy or combined modality without surgery, particularly for the aggressive ­ histologies.869 While a partial or subtotal gastrectomy may be justified in rare cases, more extensive surgery, such as total gastrectomy, should not be routinely performed because of the increased morbidity and the curability with alternative therapies. In a series of 398 patients with localized primary gastric lymphoma (38% MZL and 49% DLBCL) who received radiation and/or chemotherapy, there was no difference in survival at 42 months for those treated with surgery (n = 63; 86% OS) compared to those without surgery (n = 335; 91% OS).885 Similar surgical issues pertain to intestinal lymphomas as to gastric lymphomas, particularly if they present with obstruction; however, they usually are less amenable to total resection.886 Other B cell tumors which can have unique gastrointestinal presentations are Burkitt in the ileocecal region, which is common in the Middle East,887 mantle cell lymphoma, which can present with lymphomatous polyposis,250 and FL in the duodenum.888 Immunoproliferative small intestinal disease (IPSID), also called alpha heavy chain disease or Mediterranean lymphoma, can be considered a subtype of B cell MALToma distinguished by its occurrence in older children and young adults (age range, 10 to 35 years) from low socioeconomic groups in the Middle East and North Africa and its association with the synthesis of an IgA heavy chain and Campylobacter jejuni.114,889 Although the prognosis is usually considered poor in IPSID, a combination of antibiotics (tetracycline), steroids, and/or anthracycline-based chemotherapy, along with aggressive supportive care with hyperalimentation, can achieve complete remissions in two-thirds of patients, with survivals in over half the patients at 3.5 years.889,890 A unique T cell lymphoma of the small intestine is EATL, which can be associated with celiac disease (type I), may require emergent surgery due to obstruction or perforation, and responds poorly

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1867

to chemotherapy (see section on “Extranodal Peripheral T Cell Lymphoma”).612,891,892 Lymphomas involving Waldeyer’s ring account for up to one-third of extranodal lymphomas and are second to gastric lymphoma as the most common extranodal site.893 Prognosis depends upon histology, which is usually a DLBCL, size of the tonsillar mass, and stage.894 Up to one-half of patients have advanced disease, and simultaneous involvement of the gastrointestinal tract is detected in 10% to 15% of patients with Waldeyer’s ring lymphoma.895,896 Because of the usual DLBCL histology and the advanced stage of many patients, chemotherapy plus rituximab, often in combination with radiation, is usually considered the therapy of choice.897 With combined modality therapy, 5-year PFS is 70% to 90% for clinical stage IE and 40% to 60% for stage IIE.898–900 Lymphomas of the nasal cavity and paranasal sinuses often are evaluated in series that include Waldeyer’s ring lymphomas, despite differences in presentation, diagnosis, and therapy. The primary lymphoma is often advanced in nasal lymphomas with invasion of adjacent bones and an increased risk for CNS involvement. Although uncommon, cervical node involvement in nasal lymphomas is associated with a poor prognosis.898 Using the American Joint Committee TNM staging for carcinoma of the paranasal sinuses, patients with T 1 to 2 lesions had an 89% 5-year DFS compared to 25% for patients with T 3 to 4 lesions.898 DLBCL is the most common histology in the West; but in series from Asia, NK/T cell lymphomas predominate, present with nasal septal perforation or destruction, and are associated with EBV (see section on “Extranodal PTCL”).617,901 Concurrent chemoradiotherapy or novel chemotherapy using agents which bypass MDR is advocated for most nasal NK/T cell lymphoma.902 The best therapeutic results in nasal lymphomas have tended to be in those series with combined modality therapy.901,903 Adding CNS prophylaxis to combined modality therapy resulted in a 5-year OS of 47% and disease-specific survival of 62%.904 Although somewhat variable according to series, the majority of salivary gland lymphomas are indolent (MALToma) and localized, most often to the parotid, and have survivals following radiation therapy alone of 80% to 90% at 5 years and 50% to 70% at 10 years.188,905,906,907 Most patients (approximately 80%) with thyroid lymphomas have localized stage IE or IIE disease, and the majority are DLBCL, sometimes with a MALToma component.908,909 Approximately half of the patients have a history of HT.910 A worse prognosis occurs with penetration of the thyroid capsule, tumor bulk, advanced stage, and intermediate- to highgrade histologic types. Because of the frequency of these factors, many have advocated combined modality therapy for thyroid lymphoma except in the patient with stage I MALToma, who can receive radiation alone.909,910 Five-year OS for DLBCL of the thyroid ranges from 52% to 90%.906,911 Five-year DFS for MALToma of the thyroid following radiation is 88% to 100%.912 Most primary pulmonary lymphomas are small B lymphocytic with or without plasmacytic differentiation and are considered a part of the spectrum of MALTomas. Two previously diagnosed entities, pseudolymphoma and lymphocytic interstitial pneumonitis, likely represent MALTomas. Therapy options include rituximab;chemotherapy, either single agent or combination; surgical resection; and radiation. The prognosis is good with 94% OS at 5 years without reaching a median survival at 10 years; and there is no advantage for a specific type of therapy.274,913,914 Other pulmonary lymphomas are heterogeneous, and therapy depends upon pathology and extent of disease. A favorable prognosis has been reported for DLBCL without bulk disease and normal LDH.915 Lymphomatoid granulomatosis (LYG) is a rare type of EBV lymphoproliferation of B cells which most commonly presents with lung lesions varying from nodules to necrotic cavitary lesions, and which can have associated CNS and skin lesions.916 Therapy and prognosis depend upon grading with steroids and

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interferon used for grades 1/2 and chemotherapy plus rituximab for grade 3 lesions. Orbital lymphomas usually involve small B lymphocytes with or without plasmacytic differentiation and need to be distinguished from benign lymphoid hyperplasia. Some are associated with Chlamydia psittaci and can respond to antibiotics (see section on “Prelymphomatous Conditions”). They tend to be localized (80%+), and 10% to 20% are bilateral. They respond to radiation, with survival of 75% to 95% at 5 years and 70% to 80% at 10 years.269,917,918 Large B cell lymphomas are less common, tend to involve the lacrimal gland or retro-orbital area, and are treated with (10 to 200) combined modality therapy plus rituximab.269,919 CNS lymphomas are predominantly DLBCL and are responsive to steroids, radiation, intrathecal or Ommaya reservoir therapy, and systemic chemotherapy which crosses the blood brain b ­ arrier.920,921 Standard treatment historically for PCNSL was whole brain radiation therapy (WBRT) alone; and the prognosis was poor with survival less than 6 months in AIDS patients and 1 to 2 years in immunocompetent hosts.922,923 However, recent data utilizing early chemotherapy, particularly high-dose methotrexate or cytosine arabinoside, usually followed by WBRT, have improved results in PCNSL with median survivals from 30 to 60 months.920,924–926 Severe neurologic toxicity manifested as leukoencephalopathy secondary to radiation is common, particularly in the patient over 60 years of age, and trials with chemotherapy alone and deferred radiation are being conducted.266,927–929 In a randomized trial of 551 patients with PCNSL, there was no difference in OS when WBRT was omitted from primary chemotherapy.930 Rituximab has poor penetration into the CNS, but it is being used in protocols with PCNSL and its role is yet to be ­determined.920,931 Alkylating agents with good CNS penetration which are a part of chemotherapy combination include thiotepa, ifosfamide, nitrosoureas, procarbazine, and temozolomide.920 There may be a role for high-dose chemotherapy and autologous transplant for PCNSL in both the relapsed and upfront setting.932,933 Testicular lymphoma, representing approximately 5% of testicular neoplasms, is the most common testicular tumor beyond the age of 60 years and the most common bilateral testicular tumor.934,935,936 It is associated with involvement of the contralateral testis in 20% to 35% of patients, and with involvement of the skin, Waldeyer’s ring, and CNS, each in approximately 10% of patients. The predominant histologic type is DLBCL (80% to 90%). In early series, median survivals have been 1 to 2 years, and 5-year survivals have varied from 12% to 48%, depending in part upon the extent of disease.934,935 Because of the poor prognosis and early systemic spread, anthracycline-based chemotherapy and rituximab are warranted, usually with radiation to the involved testis if unresected, as well as to the contralateral testis. Because of the CNS relapse rate of 15% to 31%, CNS prophylaxis should be considered.934,937 In an international phase II trial of 53 patients treated with R-CHOP, usually with CNS prophylaxis and testicular radiation, the 5-year PFS and OS were 74% and 85%, respectively; CNS relapse was 6%.938 Breast lymphomas usually manifest as a rapidly enlarging mass, may be multiple, and involve the opposite breast in 10% to 20% of patients.273,939,940 The histologic type is variable, but the majority are DLBCL; low grade lesions with MALToma features and an association with lymphocytic lobulitis have been described.941 Massive bilateral breast involvement with BL has been described in pregnant or lactating women, predominantly from Africa, and is characterized by rapid dissemination with ovarian and CNS disease.273 After simple biopsy and staging, radiation therapy can be considered for local control of low grade lesions, but combination chemotherapy plus rituximab is warranted for aggressive histologies. Primary ovarian lymphomas are rare, except in countries where BL is endemic; however, the ovary is the most common site of female genital tract involvement by lymphoma.942 Ovarian lymphomas may be associated with bilateral involvement in 20%

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to 50% of patients and have had a poor prognosis, with 5-year survival of less than 25%. Prognosis depends on the extent of disease and the histologic type. DLBCL is the most common type, followed by FL, all grades, and Burkitt. Combination chemotherapy plus rituximab is warranted in most patients because of aggressive histologies. Primary bone lymphomas are usually DLBCL (85%+), and approximately one-third have more than one bone involved.271 Prognosis depends on the bones involved, with the femur the best and the spine the worst, histologic type, stage, and the presence of soft tissue involvement.271,943 Although combined modality has been recommended for aggressive histologies,944,945 chemotherapy alone has been successful. Because radiologic abnormalities persist after chemotherapy, local radiation is often given to the entire bone with a boost to the tumor bed.946 For localized bone lymphoma treated with combined modality, FFTF exceeds 80% at 5 years.947 Therapy of CTCL and the differential diagnosis of cutaneous lymphomas are addressed in Chapter 92. Local radiation therapy is often adequate for localized (IE) cutaneous MALToma or PCFCL, whereas multiple therapeutic options similar to other indolent lymphomas can be considered for advanced disease. Both the primary cutaneous MALToma, or marginal zone lymphoma (PCMZL), and PCFCL have a good prognosis with 5-year OS exceeding 90%.948,949 The majority of PCMZL have multifocal skin lesions involving the trunk and extremities, and up to 50% will relapse within 5 years of their initial therapy, but will still have a good prognosis (5-year OS>90%).948 PCFCLs tend to involve the head and neck and lack BCL-2 expression and the BCL-2 gene rearrangement. Alternatively, BCL-2 expression is often present in PCBCL of the leg as opposed to other sites, is primarily seen in the elderly, and has a poor prognosis (5-year OS 43% to 63%) despite the use of rituximab and combined modality therapy.517,949–951

Role of Hematopoietic Cell Transplantation Transplantation is addressed in Chapters 102 and 104 and has taken an expanded role in the therapy of NHL; its use has been noted in preceding sections on individual diseases. Outcomes depend upon disease state (type of lymphoma, remission status), patient factors (age, performance status), and source of stem cells, either autologous (auto) or allogeneic (allo). In 1978, Appelbaum reported long-term DFS in 3 of 9 patients with relapsed BL who underwent high-dose chemotherapy and autologous marrow transplantation.952 Subsequent series reported a 5-year OS rate of 20% to 50% using autoSCT for relapsed NHL.953,954,956 In the rituximab and RIT era, the OS has improved to 40% to 70%, but is variable depending upon type of lymphoma, prognostic factors, and chemosensitivity.957,958 Patient selection determines outcome, with the best results achieved in patients who are either in first CR or have minimal residual disease before the transplant and have a good performance status. Transplants in first CR or in patients with indolent histologies are controversial. Patients with predominantly DLBCL who are in a sensitive relapse—i.e., they are responding to additional chemotherapy—have a 30% to 70% salvage rate with autoHCT, compared to 0% to 15% patients who are in a resistant relapse. There is no superior preparative regimen, which may include various chemotherapy agents with or without total body irradiation. Rituximab and/or RIT are being incorporated into preparative regimens to try to decrease relapses.957,959 In a randomized trial comparing BEAM (carmustine, etoposide, cytarabine, and melphalan) plus rituximab vs. BEAM plus conventional tositumomab (Bexxar) followed by autoHCT, there was no difference in PFS, OS, or transplant relapsed mortality (TRM).960

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AlloHCT has been used less than autoHCT because of higher mortality rates, cost, and/or lack of a donor. It was initially reserved for selected young patients (usually less than 50 years of age) with marrow involvement, highly aggressive lymphomas, or after relapse from an autoHCT. Early (day 100) mortality rates previously were 5% to 25% for autoHCT and 15% to 45% for alloHCT, but are now 5 cm, but is not very effective for patients with loss of p53 function. 8. Prophylaxis against Pneumocystic jirovecii and herpes infections should be given to patients receiving nucleoside analogues, steroids, or alemtuzumab and continued for 6 months following therapy. The authors use trimethoprimsulfamethoxazole (co-trimoxazole) 1 double strength twice a day on Saturdays and Sundays with valacyclovir 500 mg/day (or an equivalent). For patients allergic to trimethoprim-sulfamethoxazole, dapsone 100 mg 3 times a week or pentamidine by aerosol once a month may be used.

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9. Steroids should primarily be reserved to treat immune cytopenias and should be avoided, if possible, when patients are receiving chemotherapy. There is no evidence that steroids increase the response rate obtained with alkylating agents or nucleoside analogues alone, and they increase the risk of infection. However, if used judiciously, prednisone may be combined with chlorambucil to enhance marrow clearing of tumor and to reduce organomegaly. 10. Patients with immune cytopenias or red cell aplasia should initially be treated with prednisone 1 mg/kg/day orally, but may also benefit from gammaglobulin, cyclosporine, cyclophosphamide, or rituximab. Patients who are going to receive a prolonged course of prednisone should be maintained on pamidronate 30 mg IV every 3 months, or an equivalent, to prevent osteoporosis and prophylactic antibiotics for Pneumocystic jirovecii and herpes infections. 11. Radiotherapy is reserved for local lesions that are particularly bulky and troublesome and is used only when chemotherapy is not required for control of more disseminated disease. The lowest dose of radiotherapy capable of shrinking the tumor mass should be used. Splenic irradiation may be helpful in patients who require a splenectomy but who are not surgical candidates. Radiotherapy is most useful in patients who have had little prior therapy. 12. Splenectomy may be useful in patients with painful splenomegaly or who have cytopenias that are unresponsive to other therapies. Minimally invasive surgical techniques should be considered. 13. Prophylactic gammaglobulin is useful in reducing the fre quency of infections in patients with hypogammaglobulinemia and frequent bacterial infections. G-CSF and erythropoietin may be useful to maintain neutrophil counts and hemoglobin. 14. Although not standard therapy, alloSCT may be considered for younger patients (30% of patients,83,93 and infections can occur with unusual organisms such as atypical mycobacteria, including Mycobacterium kansasii, which is a unique feature of HCL, in comparison to other lymphoproliferative disorders.83,94,97 Other organisms include Toxoplasma gondii, Legionella, Listeria monocytogenes, and Pneumocystis jirovecii, as well as various fungi and viruses.83,98–100 The high infection rate can be ascribed primarily to neutropenia, although several other immune defects have been described. Monocytopenia is often a prominent feature,101,102 and functional abnormalities of monocytes and granulocytes may occur.99,101,103–105 The defects in monocyte production and function may account for the unusual susceptibility of these patients to atypical mycobacterial and fungal infections.95,96 The T-cells are also highly abnormal in HCL, demonstrating an inversion in the CD4:CD8 ratio, and they have a poor antigen response which is likely related to absence of CD28.106,107 Moreover, there is clonogenic expansion of CD8+ cytotoxic lymphocytes but the target for these cells has not yet been identified.108 These abnormalities can resolve with IFN-a therapy, although it may take up to 2 years to see this effect.109 A depressed helper to suppressor T-cell ratio associated with a decrease in the number of T-helper cells and an increase in the number of T-suppressor cells also has been demonstrated.109 As expected, lymphocyte functional studies reveal impaired delayed-type hypersensitivity to recall antigens, as well as near-absent antibody-dependent cellular cytotoxicity.106 In contrast to CLL, the serum Ig levels are normal.110

Hematologic Malignancies

FIGURE 91.4. Immune suppression in hairy-cell leukemia (HCL).  Immune suppression is a result of T-cell dysfunction and impaired hematopoiesis with pancytopenia, bone marrow fibrosis, and hypersplenism. T-cell activation, decreased numbers of memory T-cells, restricted T-cell repertoire, and opportunistic infections are the result of inappropriate activation and suppression of T-cell responses directly by cytokines produced by the neoplastic B-cells. Cytopenia with severe monocytopenia is caused by the secretion of tumor necrosis factor (TNF)-a by the hairy cells. TNF-a also has autocrine prosurvival effects on the tumor clone. Treatment of HCL with IFN-a is able to restore the abnormal T-cell repertoire and hematopoiesis by inhibiting cytokine (including TNF-a) mediated effects on the T-cells and tumor cell. Fibrosis is caused by production of fibroblast growth factor-2 (FGF-2) and overexpression of its receptor FGFR1. The FGF2-FGFR1 interaction increases with CD44v3 co-receptor and syndecan family members. FGFR1 signals secretion of autocrine fibronectin and of transforming growth factor-b (TGFb) by hairy cells. TGFb stimulates adjacent fibroblasts to produce fibronectin and collagen type III. (From Forconi F. Hairy cell leukaemia: biological and clinical overview from immunogenetic insights. Haematol Oncol 2011;29:55–66, with permission.)

Weakness, easy fatigue Fever, sweats, weight loss, anorexia Infection Easy bruising, bleeding Left upper quadrant abdominal discomfort Autoimmune disorders Splenomegaly Hepatomegaly Ecchymoses, petechiae

Incidence (%)

Autoimmune Disorders Clinical manifestations secondary to various autoimmune disorders are being recognized with increasing frequency in patients

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with HCL.83,111,112,113–115 In one series of patients, these complications were second only to infection as a cause of morbidity.83,112 The onset may occur any time during the course of the disease and is not related to the tumor burden. Most frequently, patients present with arthritis, arthralgias, palpable purpura, or nodular skin lesions resulting from cutaneous vasculitis, and low-grade fever.83,112,114 Occasionally, patients may have involvement of the lung, liver, intestine, and kidney, with a clinical picture that resembles polyarteritis nodosa.83,112,113,115 These patients often have fever, malaise, and weight loss, and a co-existent infection must be ruled out.83,112 If skin lesions are present, the diagnosis can be confirmed by biopsy, which usually shows changes compatible with a diagnosis of polyarteritis nodosum or leukocytoclastic vasculitis; occasionally, a vasculitis related to the invasion of the vessel wall by hairy cells occurs; this may appear very similar to polyarteritis nodosa with the presence of aneurysms.114 In some organs, such as the lung, a granulomatous vasculitis may be found.112 Angiography may reveal peripheral aneurysms.112 Antinuclear antibodies, rheumatoid factor, immune complexes, and hepatitis B antigen are variably positive.111 Cryoglobulinemia has been detected in some patients.116,117 It has been postulated that the increased incidence of vasculitis in HCL may be related to infections with hepatitis B and other viruses, cross-reactivity of antibodies against hairy cells with epitopes on endothelial cells, and decreased clearance of immune complexes by the impaired immune system.114 These autoimmune manifestations may be self-limited, but if therapy is required, a short course of corticosteroids is usually effective.114 Remissions have also been observed after splenectomy, IFN-a, and pentostatin therapy.112,114,115

Unusual Manifestations Lytic Bone Lesions Although the immunophenotypic profile of hairy cells closely resembles that of B-cells at a developmental stage just before terminal differentiation to plasma cells, lytic bone lesions are distinctly unusual (Table 91.3).118–121 In some patients with osteolytic lesions, HCL and multiple myeloma were thought to coexist.122,123 However, several patients with classic HCL and without any evidence of plasma cell proliferation have been reported to develop osteolytic lesions.119–121 These lesions have a predilection for the proximal femur and usually are associated with extensive bone marrow infiltration by hairy cells.120,121 The lesions, as with those seen in association with multiple myeloma, respond well to radiotherapy. The administration of corticosteroids can produce prompt relief of bone pain.118

Skin Involvement Cutaneous lesions referable to thrombocytopenia (ecchymoses, petechiae), infection, or vasculitis are common during the course

of HCL, but lesions caused by infiltration of the skin by hairy cells are unusual.124,125 In a retrospective review of 600 cases, skin lesions thought to be due to infiltration by hairy cells were reported in 8.0% of cases, but histopathologic verification was present in only 1.3%.124 Infiltrative lesions usually are widely disseminated and consist of erythematous maculopapules. Biopsy shows the infiltrates to be perivascular, involving the dermis but not the epidermis.126

Splenic Rupture Surprisingly, even with massive splenomegaly, spontaneous splenic rupture is rare in HCL, occurring in ∼2% of cases.1,127

Other Organ Dysfunction Although hairy cell infiltration of multiple organs and tissues is a frequent finding at autopsy, clinically significant organ dysfunction is unusual.128 Infiltration of connective tissue and fat surrounding organs is common.128 Central nervous system involvement is unusual, and only a rare case of meningeal involvement has been documented.128,129 Infection is by far the most frequent cause for neurologic complications.129 Pleural effusions, ascites, proteinlosing enteropathy, and spinal cord compression may occur rarely in HCL and result from tissue infiltration by hairy cells.118

Laboratory Findings The relative incidence of the most characteristic laboratory findings is listed in Table 91.4. In a series of 725 cases studied by the Italian Cooperative Group, 80% of patients had pancytopenia at presentation, with one third of all patients having a hemoglobin level 100 × 109/L, was achieved in 40% of patients.179 An increase was noted in the hemoglobin in 92%, neutrophils in 84%, and platelets in 92% of patients.179 Jansen et al.179 also reported that patients with larger spleens responded better than those with smaller spleens, but this has been disputed.180 The duration of response after splenectomy is variable, but some patients remain asymptomatic for years. Approximately one third, however, achieve only a minimal response or relapse within a few months. In an analysis of prognostic variables after splenectomy in 194 patients, the most important were bone marrow cellularity and the platelet count.181 Failure-free survival, defined as time from splenectomy to death or the need for more therapy, was significantly worse if the postoperative bone marrow cellularity was ≥85% or the platelet count was 120 g/L, platelets >100 × 109/L, and neutrophils >1.5 ×109/L) for at least 1 month with no morphologic evidence of hairy cells in the marrow or peripheral blood and resolution of organomegaly. A partial remission (PR) was defined as the normalization of peripheral blood counts and the persistence of >5% hairy cells in the marrow; however, treatment must have produced a >50% fall in the hairy cell infiltration in marrow. The aggregate results show that the overall response rate with IFN-a is ∼80%, with 13% having a CR and 69% having a PR.189–191 Responding patients have a reduced incidence of infections, even if they remain neutropenic.187 The responses occur rapidly, regardless of whether the patients have previously had a splenectomy, with hairy cells disappearing from the peripheral blood within the first week; the platelet counts return to normal within 2 months, the hemoglobin level within 4 months, and the neutrophil counts within 4 to 6 months.182,187 The percentage of hairy cells in the bone marrow decreases, but they rarely disappear completely, and the reticulin fibrosis persists.193,194 Patients with CD5+ hairy cells195 and HCL-variant patients149 respond poorly to IFN-a. The optimal dose schedule for IFN-a- in HCL has yet to be established. In most series, the dose is 2 to 4 × 106 U/m2 subcutaneously (SC) three to seven times weekly for 12 months. Higher doses do not appear to increase the response rate and are associated with more toxicity.186,196 In addition, extending the treatment beyond 12 months does not improve the response rate, and the development of a chronic fatigue syndrome is more prevalent and severe.197 Doses 1 log lower (2 × 105 U/m2 three times weekly) show activity, but the response is inferior to that achieved with higher IFN-a doses.198 However, the relapse rate is high (33% to 77%) after the discontinuation of IFN-a, usually 6 to 31 months after cessation of therapy.188,199 It has been demonstrated that maintenance low-dose IFN-a (1 × 106 U, three times/ week, or 3 × 106 U, once per week) can prolong remissions with minimal toxicity.191,200,201 In one study, patients received either no maintenance therapy or 1 × 106 U IFN-a three times/week; 37 of the 56 patients who did not receive maintenance therapy relapsed at a median time of 19 months, whereas none of the 28 patients receiving maintenance therapy relapsed, with the median follow-up time being 30 months.200 When patients relapse after IFN-a therapy, a further remission can generally be obtained with IFN-a200,201 or the nucleoside analogs.136,175,176 In addition, patients who relapse after therapy with the nucleoside analogs may respond to IFN-a, confirming the lack of cross-reactivity between these agents.202 Neutralizing anti-IFN antibodies appear to develop in one third of patients treated with IFN-a2a,203 but this does not occur with IFN-a2b.204 The clinical significance of these neutralizing antibodies and their role in the induction of resistance to therapy are controversial. In 51 patients with HCL treated with INF-a2a, 31 (61%) developed antibodies after a median of 6 months of therapy, and in 16 of these, the antibodies neutralized the antiviral activity of recombinant INF-a2a in vitro but had no effect on natural IFN-a.203 It is interesting that six of the patients with antibodies were clinically resistant to IFN-a, whereas none of the patients without antibodies were resistant. However, in a follow-up study, no further patients were noted to have developed antibodies, and antibodies to IFN could no longer be detected in patients who previously had antibodies, although they continued to receive IFN-a.205 Virtually all patients experience toxicity with IFN-a therapy, and the frequency and severity are dose- and age-related. Flu-like symptoms occur in most patients with the initiation of treatment; these symptoms can usually be controlled with acetaminophen or by reducing the IFN-a dosage, and symptoms usually resolve

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within 2 to 4 weeks. Less common symptoms include: nausea and vomiting; diarrhea; central nervous system manifestations, such as somnolence and confusion; cardiovascular disorders, including hypotension and tachycardia; and skin changes, such as rash and pruritus.196 With high doses, leukopenia, thrombocytopenia, and anemia may occur, but this is rare with the doses of IFN-a used for HCL. A worsening of pre-existing autoimmune disorders, or the emergence of new autoimmune problems, has also been reported with IFN-a therapy; this appears to be related to the development of multiple autoantibodies.206 In most reports, patients developed thyroiditis, autoimmune thrombocytopenia, or anemia.207,208 Kampmeier et al.209 reported an increased incidence of second malignancies in HCL patients treated with IFN. Of 69 patients treated with IFN-a2b for 12 to 18 months, 13 (19%) developed second malignancies; this incidence was substantially higher than predicted. Six of the tumors were hematologic, and seven were adenocarcinomas. The tumors developed 17 to 105 months after the initiation of IFN-a. However, these results have not been confirmed, and no increase in the incidence of second malignancies was observed in 200 HCL patients in another study, of whom 147 had been treated with IFN-a.210

Nucleoside Analogs The nucleoside analogs pentostatin (pentostatin; Nipent, SuperGen, San Ramon, CA), cladribine (cladribine; Leustatin, Ortho Biotech, Raritan, NJ), and fludarabine (F-ara-A AMP; Fludara, Berlex Laboratories, Richmond, CA) have significant activity in the low-grade lymphoid malignancies (Fig. 91.8).136,175,176 Both pentostatin and cladribine have now replaced IFN-a as first-line therapy for HCL, and fludarabine is now one of the standard treatments for CLL. After therapy with pentostatin, deoxyadenosine and adenosine accumulate in the plasma; after uptake into cells, deoxyadenosine is phosphorylated to deoxyadenosine monophosphate, deoxyadenosine diphosphate, and deoxyadenosine triphosphate (dATP); this occurs preferentially in lymphocytes.211 The intracerebral accumulation of deoxyadenosine and adenosine likely causes the nausea and vomiting that are a major toxicity of this agent.212 Cladribine and F-ara-A are halogenated derivatives of deoxyadenosine that are resistant to degradation by adenosine deaminase. For clinical use, F-ara-A is administered as the more water-soluble monophosphate, F-ara-AMP (fludarabine), which is rapidly dephosphorylated in the plasma to F-ara-A.213,214 As with deoxyadenosine, cladribine and F-ara-A accumulate in lymphocytes as their phosphorylated derivatives and their mechanisms of action have been recently reviewed.215–217 These agents can kill lymphocytes in three ways (Figs 91.9 and 91.10).215–217 First, the triphosphate forms can trigger DNA breaks, which result in the release of cytochrome c from the mitochondria; the released cytochrome c interacts with Apaf-1 and dATP, causing the activation of caspase 9 and, subsequently, apoptosis. Second, the increased levels of triphosphates can enhance the effects of endogenous dATP on the apoptosome, inducing apoptosis. Finally, cladribine differs from deoxyadenosine and F-ara-A in that it is phosphorylated by deoxyguanosine kinase in the mitochondria to cdATP, which is directly toxic to the mitochondria. Pentostatin is highly effective in HCL and produces a much higher rate of durable CR than is observed with IFN-a (Table 91.7).189 Pentostatin has been administered in a variety of different doses and schedules for HCL, but regardless of the mode of administration, it produces responses in most patients. Of 960 patients in nine studies, the CR rate ranged from 44% to 89% (median, 76%), and the PR rate varied from 0% to 52% (median, 16%).189,216,217,218–223,224,225 Patients who relapse after splenectomy or who are resistant to IFN-a also respond to pentostatin, with a median CR rate of 42% and a PR rate of 45%.225,226,227 In two large studies, the response rates to pentostatin were

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Chapter 91  Hairy Cell Leukemia

H

NH2

OH

NH2 N

N

N

N

N

HN CL

N

HOCH2

F

N

N

N

HOCH2

HOCH2

O

O

O

N

N

HO

OH 2-Chlorodeoxyadenosine (CdA)

OH 2’-Deoxycoformycin (dCF)

OH F-ara-A

NH2 N

N

N

N

N

N

Hematologic Malignancies

NH2

N N

HOCH2

HOCH2

O

O

OH OH Adenosine

OH Deoxyadenosine

FIGURE 91.8. Structures of nucleoside analogs.

similar in untreated patients and in patients previously treated with IFN-a.189,224 Moreover, these studies identified young age, initial high hemoglobin, high white cell count, and little or no splenomegaly as favorable prognostic features.189,224 The most commonly used treatment regimen is pentostatin 4 mg/m2 intravenously (IV) every second week; the average number of treatments to CR is 8 (range, 4 to 15).189,220 The peripheral blood lymphocyte count falls rapidly after the initiation of treatment, with the hairy cell count decreasing by 50% to 95% in the first week.228 Concomitantly, there is a rapid increase in platelets followed by recovery of neutrophils and hemoglobin; the median time to documented peripheral and marrow CR is 4 months.228 In contrast to IFN-a, there is resolution of the marrow fibrosis after therapy with pentostatin.228,229 Using immunophenotyping of peripheral blood or bone marrow, immunohistochemistry of the bone biopsy, or gene rearrangement studies, one can detect MRD in HCL patients who are in morphologic CR after pentostatin, suggesting that pentostatin cannot entirely eliminate the hairy cell population.229,230 In addition, relapses are observed after discontinuation of treatment without evidence of a plateau, although the duration of remissions is considerably longer than for IFN-a.189,221,231,232,233

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Several studies have evaluated the long-term outcome of patients treated with pentostatin223,225,232–236 (Table 91.8). The longest follow-up is of 188 patients followed for a median of 14 years with the relapse rates at 5, 10, and 15 years being 24%, 42%, and 47%, respectively.233 The likelihood of relapse depended on response and pre-treatment parameters with the longest remission being in those who achieved a CR and had a pre-treatment hemoglobin of >100 g/L and platelets >100 × 109/L, and the worst prognosis was in those who achieved a PR and were anemic and/or thrombocytopenic pre-treatment (Fig. 91.11).233,234 For patients who remain in CR at 5 years the likelihood of remaining in CR by 15 years is 75%. For patients requiring another treatment, the likelihood of achieving a CR decreased but for those who did achieve a CR the prognosis was similar as for those with a first-time CR.233,234 In the Phase III intergroup study, patients were randomized to receive pentostatin, 4 mg/m2 IV every 2 weeks, or IFN-a, 3 × 106 U SC three times per week.189,236 Patients not responding to one treatment were switched to the other agent. There were 241 patients who received pentostatin and were followed for a median of 9.3 years; 154 received pentostatin as initial therapy, and 87 received pentostatin after failure with IFN-a. For all

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FIGURE 91.9. Metabolism of the nucleoside analogs.  ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; CdA, 2-chlorodeoxyadenosine; d, deoxy; dCF, 2′-deoxycoformycin.

patients, the estimated 5- and 10-year survivals were 90% and 81%, similar to those predicted for the general population. The survival was similar whether patients were treated initially with pentostatin or were crossed over to pentostatin after treatment with IFN-a (Fig.91.12A). Patients younger than 55 years of age did significantly better than patients 55 years of age or older, and the 10-year survivals for the two groups were 93% and 68%, respectively (Fig. 91.12B). Similarly, in a large multicenter retrospective study from France, the estimated survivals at 5 and 10 years in 230 evaluable patients treated with pentostatin were both 89%.224 In that study, a hemoglobin level 1 cm in diameter

T4 N Lymph Node Stage N0 N1 N2 N3 Nx M Visceral Stage M0 M1 B Blood Stage

Confluence of erythema (erythroderma) covering >80% BSA

B0

Sézary cell count ≤5% lymphocytes

B1

Sézary cell count >5% lymphocytes when B2 criteria are not met

B2

T-cell clone in blood + >1,000 Sézary cells/ml, OR 2 of 3:

Clinical Stage Early-Stage Disease IA IB IIA Late-Stage Disease IIB IIIA IIIB IVA1 IVA2 IVB Sézary Syndrome IVA1 or 2 or IVB

No clinically abnormal peripheral lymph nodes (pLNs) Clinically abnormal pLNs with NCI grade 1 or 2 pathology Clinically abnormal pLNs with NCI grade 3 pathology Clinically abnormal pLNs with NCI grade 4 pathology Clinically abnormal pLNs with no histologic confirmation No visceral involvement Visceral involvement

CD4/CD8 ratio >10, or CD4+CD7− >40% lymphocytes, or CD4+CD26− >30% lymphocytes AND lymphocytes or CD4 or CD3 count must be elevated. TNMB Stages T1 N0 M0 B0–1 T2 N0 M0 B0–1 T1–2 N1–2 M0 B0–1 T3 N0–2 M0 B0–1 T4 N0–2 M0 B0 T4 N0–2 M0 B1 T1–4 N0–2 M0 B2 T1–4 N3 M0 B0–2 T1–4 N0–3 M1 B0–2 T4 N0–3 M0–1 B2

BSA, body surface area; EORTC, European Organization for the Research and Treatment of Cancer; ISCL, International Society of Cutaneous Lymphomas; NCI, National Cancer Institute; pLNs, peripheral lymph nodes. Modified from Olsen E, Vonderheid E, Pimpinelli N, et al. Revisions to the staging and classification of mycosis fungoides and Sézary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the Cutaneous Lymphoma Task Force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 2007;110:1713–1722.

lymphadenopathy.182 Because invasive tests such as liver biopsy or staging laparotomy have added little diagnostic information for patients with early and advanced-stage disease,175,373,374 these staging studies are not recommended by the recent consensus conference.176 Table 92.6 outlines staging procedures for MF/SS adapted from the ISCL/EORTC guidelines.176 All patients should have a complete physical examination with special attention to skin and lymph nodes. Careful mapping of skin involvement and/ or total-body photographs are recommended to document the initial extent of disease at diagnosis and to assess response to treatment.375 Laboratory studies should include serum comprehensive

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metabolic panel, lactate dehydrogenase, and complete blood count with manual differential. In addition, peripheral blood should be examined for abnormal lymphocytes by either a Sézary cell count (number per microliter) and/or flow cytometry with attention to CD4+/CD8+ ratio and CD4+/CD7− or CD4+/CD26− gated populations if indicated. TCR gene rearrangement of peripheral blood is also recommended, including an attempt to correlate a positive finding with any clone in the skin. A recent study showed no significant benefit to performing bone marrow biopsies for staging purposes.181 They may be helpful in working up unexplained hematologic abnormalities.

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Chapter 92  Cutaneous T-Cell Lymphoma: Mycosis Fungoides and Sézary Syndrome

TA B L E 92.5

NCI VA GRADING SCHEME FOR LYMPH NODE HISTOLOGY IN CTCL Grade

Histopathologic Features

LN-1

DL with occasional CTC

LN-2 LN-3

DL with CTC singly or in small clusters (15 cells)

LN-4

Partial or complete effacement by MF/SS ± DL

CTC, cerebriform T-cells; CTCL, cutaneous T-cell lymphoma; DL, dermatopathic lymphadenopathy; LN, lymph node; MF, mycosis fungoides; NCI, National Cancer Institute; SS, Sézary syndrome; VA, Veterans Affairs. Modified from Sausville EA, Eddy JL, Makuch RW, et al. Histopathologic staging at initial diagnosis of mycosis fungoides and the Sézary syndrome. Definition of three distinctive prognostic groups. Ann Intern Med 1988;109:372–382.

Imaging The role of diagnostic imaging in the initial staging of CTCL has been examined by several authors.376–379 Kulin et al. studied the results of gallium citrate Ga-67 scintigraphy, liver–spleen

1969

scans, lymphangiography, and computed tomography (CT) used in the initial staging of 62 CTCL patients (85% with stage I or II disease) and found that none of the results added significantly to the information obtained from physical examination and routinely performed lymph node biopsy (73% of 62).377 In contrast, a study of 63 CTCL patients (78% with stage I or II disease) who had staging body CT scans found positive findings in 18 (29%) patients, one half of whom had clinically unsuspected advancedstage disease.376 Eight of these 18 patients had biopsies, with 5 of 8 confirming extracutaneous CTCL. Of the 38 patients with stage I disease, however, only 2 had positive findings on CT scan.376 Another retrospective study of 33 CTCL patients (70% with stage I or II disease) who had CT scans found that 3 of the 20 patients with initial clinical stage I disease were staged higher on the basis of CT findings as stage II.378 Subsequent lymph node biopsies confirmed extracutaneous disease in all three patients (stage IVA). In summary, pelvic, abdominal, and thoracic CT scans have a low yield in patients without palpable adenopathy (stage I) and are not necessary for staging these patients. The highest yield of CT scans appears to be in cases of non-MF/SS CTCL (nonepidermotropic, transformed CTCL, ALCL) and stage III disease (erythroderma), in contrast to stage II and IV patients, in whom CT findings often do not change treatment or stage.376 Therefore, staging CT scans of the chest, abdomen, and pelvis are recommended for patients with generalized plaques, erythroderma, tumors, palpable lymph nodes, or blood involvement.176

TA B L E 92.6

RECOMMENDED INITIAL STAGING EVALUATION OF MYCOSIS FUNGOIDES/SÉZARY SYNDROME Evaluation and Studies Skin Physical exam

Identify primary skin lesion (patches, plaques, tumors, erythroderma) and % BSA involved.

Skin biopsy Immunophenotyping Molecular genetics Lymph Nodes

Punch biopsy of thickest or oldest skin lesions (>1 skin biopsy) CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30. T-cell receptor rearrangement analysis if needed.

Physical exam Radiologic tests LN biopsy Immunophenotyping Molecular genetics Viscera Physical exam Radiologic tests Liver biopsy Spleen biopsy Bone marrow biopsy Other biopsies Immunophenotyping Molecular genetics Blood Chemistries Blood cells Immunophenotyping

Identify abnormal peripheral lymph nodes ≥1.5 cm or irregular. CT scans of chest, abdomen, pelvis except in stage IA or limited IB. PET scans may be helpful to identify which LN to biopsy. Complete LN biopsy preferred over core LN biopsy or FNA. CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30. T-cell receptor rearrangement analysis if needed.

Molecular genetics

Hematologic Malignancies



Identify abnormal liver, spleen, or other organs. CT scans of chest, abdomen, pelvis except in stage IA or limited IB. PET scans may be helpful to identify visceral abnormalities. Not indicated, unless involvement would change management. Not indicated, unless involvement would change management. Not indicated, unless abnormalities would change management. Not indicated, unless involvement would change management. CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30. T-cell receptor rearrangement analysis if needed. Comprehensive panel including liver enzymes, lactate dehydrogenase, HTLV-1 serology if indicated. Complete blood count and manual Sézary cell count/differential. Flow cytometry for absolute count and %: CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD26 (if available), CD30. Also include CD4/CD8 ratio, CD4+CD7−, CD4+ CD26− (if available). T-cell receptor rearrangement analysis.

BSA, body surface area; CT, computed tomography; FNA, fine-needle aspiration; HTLV-1, human T-lymphotropic virus-1; LN, lymph node; PET, positron emission tomography. Modified from Olsen E, Vonderheid E, Pimpinelli N, et al. Revisions to the staging and classification of mycosis fungoides and Sézary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the Cutaneous Lymphoma Task Force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 2007;110:1713–1722.

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Part vii  Hematologic Malignancies  •  SECTION 4  Lymphoproliferative Disorders

Positron emission tomography (PET) may provide an alternative staging and response-assessment tool for patients with CTCL. A recent study evaluated 13 patients with MF and SS at risk for secondary lymph node involvement using integrated PET/ CT followed by excisional biopsy of the lymph nodes.380 Two of seven patients with LN4 effaced lymph nodes had nodes 6 cells) that do not efface architecture (grade LN3). ISCL/EORTC stage N1 lymph nodes may show dermatopathic lymphadenopathy, or small clusters of atypical lymphocytes (3 to 6 cells) that do not distort nodal architecture (grade LN1–2). In general, there is good correlation between histologic and molecular evaluation of lymph nodes for involvement by MF/SS. Three studies have shown that most histologically negative lymph nodes (LN1–2) do not show T-cell–receptor gene rearrangements, whereas ∼90% or more histologically involved LNs (LN4) do show clonal T-cell–receptor gene rearrangements by Southern blot analysis.383–385 All three studies also showed a mixture of clonal and polyclonal populations in histologically borderline cases (LN3 or histologically equivalent to LN3). Although not statistically significant in all studies, histologically borderline cases that have a positive TCR gene rearrangement tend to have a poorer prognosis, similar to cases with histologically involved lymph nodes (LN4).384 Fine-needle aspiration of lymph nodes to assess for involvement by MF/SS has been evaluated in a limited number of patients, with good correlation between cytologic grade of the FNA specimen and histologic classification of the lymph node biopsy.382,386 However, as with other types of lymphomas, there is an inherent risk of sampling only low-grade involvement and missing a focal area of transformation to a large-cell lymphoma. Therefore, excisional biopsy of peripheral lymph nodes >1.5

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cm in diameter is recommended, with preference for the largest lymph node draining the involved skin area or, if available, the lymph node with the highest SUVfrom PET scan data.176

Peripheral Blood The 1979 Committee on Staging and Classification of CTCLs defined peripheral blood involvement by MF/SS as >5% peripheral blood lymphocytes with Sézary cell morphology (Fig. 92.7);372 however, this criterion is not uniformly agreed on. Because Sézary cells can be seen in the peripheral blood of patients with benign dermatitis or erythroderma, investigators have suggested that other criteria be used to define peripheral blood involvement.176,371 The 2007 ISCL/EORTC staging criteria of peripheral blood involvement includes B2 staging for high blood tumor burden and B1 blood stage for low blood tumor burden (>5% Sézary cells) when criteria for B2 are not met. More specifically, B2 is defined as clonal TCR rearrangement in the blood and one or more of the following: (a) absolute Sézary cell count of 1,000 cells/ ml or more, (b) CD4/CD8 ratio of 10 or more due to an increase in CD3+ or CD4+ cells by flow cytometry, (c) increase in CD4+ cells with an aberrant phenotype: ≥40% CD4+/CD7−, or ≥30% CD4+/ CD26− as suggested by Vonderheid and Bernengo.387 As discussed earlier, aberrant phenotypes include the absence of T-cell markers such as CD2, CD3, CD5, CD7, and CD26, which are usually expressed on normal T-cells, or co-expression or absence of both CD4 and CD8. A CD4:CD8 ratio of >10:1 or an aberrant phenotype constitutes an abnormal population. Because decreased CD7 expression on T-cells has been seen in patients with benign skin conditions, the ISCL recommends that 40% or more of the CD4+ T cells lack CD7 for it to be considered significant.176 PCR of peripheral blood has been shown to detect a clonal population of T-cells in approximately one third of patients with stage I to II disease and a majority of patients with stage III to IV disease.187,388 In addition, loss of CD26 expression on circulating CD4+ T-cells has been reported in most cases of MF and SS.199,200,389 Therefore, gating of CD4+CD26− T-cells may provide a more sensitive way to use flow cytometry to evaluate peripheral blood for involvement by MF/SS.29,38,235,390 A recent study of CD27 expression in peripheral blood T-cells showed a significantly higher expression of the CD4+CD27+CD45RAcentral memory T-cell subset in patients with SS as compared to patients with idiopathic erythroderma patients who showed increased CD4+CD27–CD45RA– effector memory T-cell levels.391

Bone Marrow Studies of bone marrow involvement in MF patients at initial staging found disease in 2% to 22% of patients.174 Histologic findings in involved marrows include clusters of CD3+ atypical lymphocytes with cerebriform nuclei and occasional large dysplastic cells.174 Bone marrow involvement by MF is more common in higher-stage disease and correlates with a poorer prognosis; however, when other factors such as skin stage and visceral involvement were considered, bone marrow involvement was not shown to be an adverse prognostic factor.392 These findings were supported by a recent study that evaluated the prognostic significance of histologic and molecular evidence of bone marrow involvement at the time of diagnosis. This study also showed a correlation between histologic or molecular bone marrow involvement and clinical stage of disease, but bone marrow involvement failed to be an independent prognostic indicator.181 Therefore, despite being considered in initial staging of non-MF/SS lymphoma patients, routine staging bone marrow biopsies in patients with MF are not currently recommended. The recent ISCL/EORTC consensus conference recommended performing bone marrow biopsies only in patients with B2 blood involvement or unexplained hematologic abnormalities.176

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Chapter 92  Cutaneous T-Cell Lymphoma: Mycosis Fungoides and Sézary Syndrome

Prognosis Prognosis correlates with the extent of skin disease and status of the lymph nodes, blood, and visceral involvement. MF behaves in a manner similar to other low-grade or indolent NHLs, with prolonged survival despite recurrent relapses (see Chapter 88). Zackheim et al. assessed relative (observed/expected) long-term survival among the four skin stages. Stage and survival data for 489 patients with CTCL was extracted from a University of California, San Francisco, CTCL registry dated 1957 to 1994 and compared to a control group matched for age, sex, race, and geographic variables. Using the control group to generate expected survival values, researchers found a relative survival at 10 years for each group as follows: 100% for T1, 67% for T2, 40% for T3, and 41% for T4.393 The TNM staging system has correlated well with prognosis, demonstrating the following 5-year survival rates: 95% for stage I, 76% for stage II, 45% for stage III, and 51% for stage IV.394 Utilizing the newest staging criteria from the ISCL/EORTC, the 5-year survival rates are similar: 94% for stage IA, 84% for stage IB, 78% for stage IIA, 47% for stage IIB, 47% for stage IIIA, 40% for stage IIIB, 37% for stage IVA1, 18% for stage IVA2, and 18% for stage IVB.189 Data from a retrospective cohort analysis suggest that the long-term (30-year) survival of patients with stage IA (limited patch/plaque) MF is similar to the expected survival of a matched control population.389,393 Therefore, it is unlikely that stage IA MF will affect the life expectancy of afflicted patients. Kim et al. performed a retrospective cohort analysis of 525 patients with MF/SS at Stanford University from 1958 through 1999.389 In the multivariate analysis, patient age, T classification, and the presence of extracutaneous disease were the most important independent factors. The risk for disease progression to a more advanced TNM or B classification, worse clinical stage, the development of extracutaneous disease, or death due to MF correlated with the severity of the initial T classification. None of the patients had T1 disease when their extracutaneous disease was detected.389 Using histology to differentiate between patch and plaque predominance within stage T2, one study found plaque predominance to influence relative survival negatively.395 A retrospective analysis by Sausville et al. of 152 consecutively staged patients at the NCI identified skin stage (T3, T4 vs. T1, T2) and visceral involvement as the most significant independent predictors of survival with palpable adenopathy and lymph node histopathology classification showing marginal significance using multivariate analysis.173 Three prognostic groups were identified, with the most favorable, low-risk group having limited skin disease without visceral or blood involvement (TNM stages IA, IB, IIA) and the least favorable, high-risk group demonstrating effaced lymph nodes (NCI VA LN4, TNM stage IVA) or visceral involvement (TNM stage IVB).173,396 The intermediate-risk group included all other patients (TNM stages IIB, III) and stage IVA patients with grade LN3 lymph node histopathology. Several reports suggest that patients with dermatopathic or early lymph node involvement with TCR gene rearrangement have a worse prognosis than similar patients without evidence of gene rearrangement.171,383,384,397 A study of 57 patients demonstrated serum LDH to reflect tumor burden in erythrodermic CTCL, with levels being inversely related to hematologic stage and survival.398 This same study used a univariate model, which found lymph node stage and hematologic stage to predict survival. Upon multivariate assessment, however, only lymph node stage served as an important prognostic indicator of survival. A more recent study that retrospectively analyzed 1,502 MF/ SS patients with the revised ISCL/EORTC staging for CTCL found a significant difference in survival between those with patch-only disease (T1a/T2a) versus patch and plaque disease (T1b/T2b).189

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Multivariate analysis revealed that advanced T stage, the presence of a peripheral blood tumor clone without Sézary cells (B0b), increased LDH, and folliculotropic MF were associated with both decreased survival and increased risk of disease progression.189 The same analysis showed that large-cell transformation was predictive only of increased risk of disease progression, and male sex, increasing age, and poikilodermatous MF were associated with decreased survival.189 In a retrospective analysis of 100 patients with transformed MF, Benner et al. identified decreased overall and diseasespecific survival in those with folliculotropic MF and those lacking CD30.166 They developed a “prognostic index” for those with transformed MF based upon the following four negative prognostic factors: CD30 negativity, generalized skin lesions, extracutaneous transformation, and folliculotropic MF. There was a significant decrease in survival in those with 2+ negative prognostic factors versus those with 0 to 1.166 Patients with SS have a relatively poor prognosis, with a median survival of ∼3 to 4 years.136,389,399 In a series of 29 patients with SS, features linked with a bad prognosis included fast evolution of the disease (from symptoms onset up to diagnosis) (p = 0.0274), raised levels of serum lactate dehydrogenase (p = 0.0379), and b2-microglobulin (p = 0.0151), the latter being the most important prognostic factor.399 Some of these findings were confirmed in another series of 28 patients with SS in whom the detrimental prognostic value of increasing age and LDH level, and the identification of the EBV genome in the skin, were reported.400 The prognosis for patients with extracutaneous disease is poor, with median survivals between 1 and 2.5 years.401 Virtually all patients with extracutaneous disease die of CTCL, compared to nearly one third of patients with generalized plaques and a majority of patients with tumors or erythroderma without visceral involvement.375,402 Very few patients with limited plaques (T1) actually die of MF, with most deaths due to cardiovascular events or other malignancies.375,389,393 Overall, illnesses attributed directly to CTCL or indirectly implicated via CTCL-related complications contribute up to a mere 19% of deaths in CTCL patients.394 More recently, Agar et al. found that 26% died due to their cutaneous lymphoma.189 Overall, the bulk of patients with CTCL do not die from their malignancy.401,403 Second malignancies other than skin cancers include NHL, HD, colon cancer, and lung cancer.404 Infection remains the most common cause of death in patients who succumb to CTCL, with Staphylococcus aureus and Pseudomonas aeruginosa being the most common pathogens infecting the skin, leading to bacteremia and sepsis.402,403 Visceral involvement with CTCL may lead to organ failure and ultimately death.

Hematologic Malignancies



Therapy The mainstay of treatment of CTCL has been control of the cutaneous manifestations of disease with topical therapies in the hope of preventing spread to extracutaneous sites. However, because of the risk of progression to extracutaneous sites and worsening cutaneous symptoms, systemic agents alone or in combination with topical therapies have been studied to control more advanced disease. Therapy in MF/SS is based on the extent of disease, age, performance status, potential for remission, availability of treatments, efficacy, and treatment toxicity.405,406 Because MF usually behaves as a low-grade or indolent lymphoma, controversial issues have involved the timing, selection, and intensity of systemic therapy. Unfortunately, there are few randomized clinical trials comparing the efficacy of the numerous therapeutic options available for patients with MF/SS. The following discussion summarizes the efficacy and toxicity of each therapy, and relates these parameters to disease stage (Fig. 92.16).

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Part vii  Hematologic Malignancies  •  SECTION 4  Lymphoproliferative Disorders

FIGURE 92.16. Cutaneous T-cell lymphoma (CTCL) treatment algorithm.  alloSCT, allogeneic stem cell transplant; Bex, bexarotene capsules; IFN, interferon; LN,lymph node; MF/ SS, mycosis fungoides/ Sézary syndrome; NBUVB, narrow beam ultraviolet B phototherapy; PUVA, psoralen + ultraviolet A phototherapy. Note that single-agent chemotherapeutic agents with impressive benefit/risk ratios include pegylated liposomal doxorubicin, gemcitabine, and ­pralatrexate.

Topical Chemotherapy Mechlorethamine hydrochloride, or nitrogen mustard (HN2), was introduced in 1947 as the first topical agent to demonstrate activity in treating MF407 and remains one of the major therapies of choice for early-stage MF. In 1942, HN2 became the first chemotherapy agent to be infused into a human being for the treatment of cancer.408 HN2 is an alkylating agent that undergoes rapid degradation to an active ethylenimonium ion that has high antimitotic activity and a brief half-life.401 Several studies have suggested that the mode of action of HN2 may involve induction of G2 arrest,409 gene-specific DNA cross-links,410,411 and blockage of transcription factor–binding sites.412 Some investigators have suggested that immunogenic properties of HN2 demonstrated by its propensity to induce delayed-type hypersensitivity contribute to its antineoplastic activity.413 Topical HN2 may be prepared in a variety of ways; initial studies involved dissolving HN2 in water to reach a concentration of 10 to 20 mg/dl,414 whereas more recently HN2 has been suspended in an emollient such as Aquaphor or a gel at concentrations of 0.01% and 0.02%.415,416 Topical HN2 is usually applied once daily to the entire skin surface with relative sparing of the eyelids, genitalia, rectum, lips, and intertriginous areas. The length of treatment is variable but usually involves daily applications until the patient achieves a complete or significant clearing of skin lesions, followed by a maintenance regimen of daily or every-other-day applications for a period of 6 months to 2 years. No author has advocated a maintenance regimen of indefinite HN2 applications. Recently, Kim et al. found that longer maintenance regimens had no impact on the relapse rate in patients treated with topical HN2.417 Treatment may be intensified for localized lesions by increasing either the concentration or frequency of application. The response rate of topical HN2 is related to the morphology (patch/plaque vs. tumor) and extent of disease (T1 vs. T2). Several large series with >100 patients have been reported.417,418,419 It is difficult to compare overall response rates among these studies because of differences in the clinical stages treated, adjunctive therapies, and staging systems. The percentage of patients with early-stage MF achieving an initial complete response (CR) ranged from 64%418 to 75%419 in stage I disease. Including more advanced stages, the complete response ranged from 37% in stage I/IIA disease to 50% in stage I to III disease.417 The median time to achieve CR is shorter for stage I patients than for those with

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more advanced stages.417,418 Vonderheid et al. found a durable CR to HN2 lasting more than 8 years occurring in 34 of 324 (10%) patients with early-stage MF,419 and Kim et al. demonstrated that freedom-from-progression rates at 10 years were 85% and 83% for T1 and T2 patients, respectively.417 Relapse is common, as Kim et al. observed relapses in 42% of 107 patients after an initial CR, all of which occurred within 5 years, and disease progression to a higher skin stage has been recently reported in 12% and 17% of T1 and T2 patients, respectively.417 However, among patients who had an initial response to HN2 and relapsed, 67% achieved a CR after receiving HN2 salvage therapy. In summary, HN2 is effective in achieving an initial response in early-stage disease. Relapse occurs frequently but can often be treated with a second course of therapy. The side effects of topical HN2 include local allergic reactions, xerosis, hyperpigmentation, and secondary cutaneous cancers.413 Immediate hypersensitivity reactions manifesting as urticaria are rare but do occur in ∼5% of patients using topical HN2 and necessitate discontinuing treatment to avoid a potentially lifethreatening anaphylactic reaction.413,420,421 Delayed-type hypersensitivity reactions (allergic contact dermatitis) manifesting as erythematous eczematous patches occur in up to 64% of patients treated with aqueous HN2418,419,422 but occur less frequently (8 months.468 Etretinate is no longer available and has largely been replaced with its metabolite, acitretin, because of its superior safety profile. Acitretin may reduce the thick palmoplantar keratoderma of advanced MF or SS.465 In 1999 the FDA approved the use of bexarotene capsules, a novel retinoid, for the treatment of CTCL. Unlike isotretinoin and acitretin, which bind to nuclear retinoic acid receptor (RAR), bexarotene binds to and activates the nuclear retinoid X receptor RXR and is therefore referred to as a “rexinoid.” RXRs are unique in that they form heterodimers with a vast array of nuclear receptors including the RARs, liver X receptors (LXR), and peroxisome proliferator activator receptors.469,470 The ultimate antiproliferative effect is mediated, in part, by the induction of apoptosis and expression of adhesion molecules.471,472 Two multicenter clinical trials established the optimal dose of 300 mg/m2/day, with an overall response rate of 45%.473,474 Higher response rates were seen in patients with higher initial doses (up to 650 mg/m2), but side effects of hypertriglyceridemia were dose-limiting. Subsequent pancreatitis occurred in 4 of the 152 patients enrolled in the two clinical trials.473,474 The advanced-stage CTCL trial showed a relapse rate of 36% but an impressive median duration of response of 299 days.473 Talpur et al. recently summarized the experience of treating 70 patients with CTCL using bexarotene capsules as monotherapy and combined with other modalities.475 Many of the patients participated in the two pivotal clinical trials. The overall response rate seen in the monotherapy group (n = 54) was 48%, in contrast to the 69% response rate in the combination-therapy group. Bexarotene was safely added to photopheresis (extracorporeal photochemotherapy [ECP]), ECP/IFN, IFN/PUVA, and ECP/IFN/ PUVA. Adverse effects were similar between the clinical trials and included hypertriglyceridemia (87%), central hypothyroidism requiring thyroid supplementation (80%),476 neutropenia (41%), skin peeling (43%), hypercholesterolemia (20%), and pancreatitis (3%). Of the monotherapy group, 78% (n = 54) and 100% of the combination-therapy group (n = 16) required at least

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one lipid-lowering agent (LLA). Of the 10 patients with diabetes, 3 had hypertriglyceridemia that could not be controlled with an LLA.475 In this series, atorvastatin and fenofibrate were the LLAs of choice. It is interesting that 9 of 10 (90%) patients on bexarotene monotherapy taking two LLAs responded, which was significantly higher than those groups on one or no LLA (p = 0.0001). The explanation for this finding is not clear, but using two LLAs may allow patients to maintain maximum doses of bexarotene.475 Vigilance should be exercised when combining atorvastatin and fenofibrate to monitor for rhabdomyolysis. Gemfibrozil and drugs that inhibit the CYP 3A4 enzyme are contraindicated with bexarotene, to avoid elevated drug levels and worsened side effects. Although patients taking bexarotene are at increased risk for elevated triglycerides, elevated LDL, and decreased HDL, there are no reports to date indicating an increased risk of cardiac events. Several animal studies have shown that bexarotene may have a favorable pharmacologic effect on atherosclerosis despite the induction of hypertriglyceridemia via LXR,477 due to a beneficial action on intestinal absorption and macrophage efflux,478 inhibition of the initial inflammatory response that precedes the atherogenic process by targeting different steps of the mononuclear recruitment cascade,469 and protective effects against H2O2-induced apoptosis in H9c2 rat ventricular cells through antioxidant and mitochondria-protective mechanisms.479 Bexarotene is also available as a 1% gel. The gel formulation is most helpful in early-stage patients (IA) without prior therapies. In a phase I/II multicenter trial, bexarotene 1% gel was applied to lesional skin with increasing frequency, once daily the first week and twice daily the second week, with a goal of four times daily if tolerated.480 Patients achieved an overall response rate of 63% and a clinical CR of 21%. Median projected time to onset of response was 20.1 weeks (range, 4.0 to 86.0 weeks), and the estimated median response duration from the start of therapy was 99 weeks.480 The most common side effect was irritation (retinoid “paint splatter” dermatitis) at the sites of application, which might make it difficult to assess response to the drug. The study was followed by a phase III study that included patients with stages IA/B and IIA disease. The overall response rate was 44% according to the Physicians Global Assessment. Similar to the previous study, the median time to response was 142 days (range, 28 to 505 days), and the relapse rate in responding patients was 26%. The most frequent adverse response was irritant dermatitis, which occurred more frequently in patients applying the drug more frequently.481 Overall, topical bexarotene is a moderately effective and well-tolerated treatment for early-stage MF most often used for refractory patches or thin plaques. The retinoid dermatitis may obscure evaluation of the patches of MF so patients should be instructed to avoid enlarging the original treatment area and to stop applying the gel 1 to 2 weeks before clinical evaluation.

Interferon IFNs are glycoproteins, naturally occurring or synthesized by recombinant DNA technology. These agents act as immunomodulators with both cytostatic and antiviral activity.482 Although three classes, IFN-a, IFN-b, and IFN-g, are described, the a-IFNs have been most extensively studied in MF and SS, and their efficacy was first reported by Bunn et al. in 1984.483 The exact mechanism of action of IFN in MF and SS is unknown. IFN may act to inhibit IL-4 and IL-5 production by normal and aberrant T-cells in patients with SS, induce myelomonocytic My7 antigen (CD13) in epidermal basal cells,484 induce the ds-RNA–dependent enzyme 2′5′-oligoadenylate synthetase leading to cleavage of cellular RNAs, and phosphorylate eIF-2, a peptide elongation initiation factor that blocks protein synthesis.482 The pharmacokinetics of IFN delivered via the intramuscular and subcutaneous routes are equivalent, allowing patients the opportunity to self-administer the drug subcutaneously.

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The phase II National Cancer Institute (NCI) trial reported by Bunn et al. in 1984 used high doses of recombinant IFN-a2a (50 × 106 U/m2 = 50 MU/m2 subcutaneously three times per week) in 20 heavily pretreated MF patients and demonstrated an objective response in 45%, including 3 patients (15%) who achieved a CR.485,486 Treatment of MF and SS with IFN-a has been reported in >200 patients and the results have been summarized.482 The overall response rate for IFN-a alone was 52%, with a 17% CR among 207 MF and SS patients, summarized by Bunn et al.487 Over three quarters of the patients received IFN-a2a, which is no longer available in the United States, although there were no apparent differences in clinical efficacy between IFN-a2a and IFN-a2b (Intron-A, Schering-Plough Research Institute).482,487 However, interpretation of pooled data is complicated by variations in initial dose, target dose, frequency, and length of therapy among study centers.482 A trial evaluating PUVA and IFN-a2a versus PUVA and IFN-a2b in CTCL patients demonstrated increased myelosuppression and liver toxicity, but also reduced constitutional effects, reduced study drop-out, and increased overall response (89% versus 50%) in the IFN-a2b arm.488 There are conflicting reports regarding the impact of clinical stage on the likelihood of response to systemic IFN-a.482,489,490 In their study of 51 MF and SS patients, Jumbou et al. found that patients with early disease (stages I and II) demonstrated a higher response rate to IFN-a than those with advanced disease.491 In an attempt to define the optimal dose of IFN-a in CTCL, a randomized study from Duke and Northwestern Universities was designed comparing low-dose (3 MU/day) to escalating doses (up to 36 MU/day); however, because of slow patient accrual, the proposed study was terminated.489 For the 22 patients evaluated, the objective response rate was 64% and was greater, although not statistically significant, for those receiving high doses (11 of 14) than for those receiving low doses (3 of 8), in part because of late responses in unresponsive patients who crossed over into the higher-dose arm.487,489 Of interest, two of the three CR patients were induced by the low-dose regimen, suggesting that patients could achieve a CR with 3 MU daily of IFN-a2a.482,489 An intermediate daily dose of 18 MU of IFN-a2a for 3 months followed by the same dose three times a week resulted in an impressive 80% objective response rate (27% CR, 53% PR) in 15 CTCL patients reported by Tura et al.492 Daily dosing of IFN-a2a for an induction period of several months was used by all of the larger studies of IFN-treated CTCL patients,490,493 whereas many fewer patients have been studied using an initial three times a week schedule.494–496 Nonetheless, some authorities487 recommend an optimal dose of 3 MU of IFN-a2a three times a week, based in part on randomized dose studies in B-cell indolent NHL showing no benefit of higher-dose IFN with respect to response rate or duration.497 Maximum daily dosing is dependent on several patient factors but, in general, should not exceed 15 M.498 Two to five months is generally necessary to obtain an objective response with IFN, but a complete or maximal response can take much longer.482,490 Treatment is generally continued for ∼1 year after a CR to prevent the high potential for relapse when treatment is discontinued before clearing.482 Three studies have reported a median duration of response to IFN-a as follows: 6 months,485 8 months,493 and 14 months.490 Olsen et al. noted a mean duration of PR while on therapy of 7.9 months (range, 2.1 to 26.5 months) and durations of CR off therapy ranging from 4 to 28 months.482 Two NCI phase II trials combined IFN-a2a with the adenosine analogs DCF and fludarabine to treat CTCL and demonstrated no clear advantage over either drug alone.499 The numerous side effects associated with IFN have led to dose reductions in 50% to 86% of patients in some studies.489,492 Besides producing anti-IFN antibodies, patients with MF can also exhibit decreased responsiveness to IFN through acquired resistance to IFN-a–induced gene expression. Specifically, a

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resistant CTCL cell line exhibits disrupted signal transduction in a pathway that is normally activated by IFN-a when therapeutic. In particular, STAT1 expression is reduced in these cells, which interrupts the JAK/STAT signaling pathway.500 Recombinant IFN-g and IFN-b have been studied in many fewer CTCL patients, as they appear to offer no major advantage over IFN-a.501–503 IFN-b has reportedly shown little effectiveness in treating CTCL,501 and IFN-g has been linked with more intense and frequent side effects than IFN-a.482 Interestingly, it has been demonstrated in vitro that the combination of IFN-g and a Tolllike receptor (TLR) 7/8 agonist increases natural-killer–cell cytotoxic activity against CTCL cell lines.504 This combination of the two therapies led to an increase in IL-12 and IFN-a levels which was regulated through the IRF8.504

Electron Beam Radiotherapy and Photon Beam Irradiation Radiation was one of the earliest effective treatments for MF, first reported in 1902.505 In 1953, Trump et al. were first to suggest using accelerated electrons for treatment.506 Modern total-skin electron beam radiotherapy (TSEBRT) is among the most effective and well-studied therapies for MF.200,507,508 The total skin surface can be treated by linear accelerator, generated electron beams that are scattered by a penetrable plate at the collimator site.509 The usual depth of penetration is 100%, and shielding all of these structures for part of TSEBRT is not uncommon.507 The long-term results of 561 MF patients treated at Stanford University and Hamilton Regional Cancer Centre from the mid1950s to 1993 with TSEBRT alone were summarized by Jones et al.507 Over 80% of stage IA patients can be expected to achieve a CR after TSEBRT, with 40% to 60% remaining relapse-free at 5 years.507 Two more recent studies showed similar CR rates, ranging from 80% to 90% for IA and IB disease.511,512 Less encouraging were the relapse-free rates, at 2.5 years for stages IB (35% to 40%), IIA (21% to 37%), IIB (7% to 26%), and III (10% to 23%).507 However, most patients who relapse with what is usually minimal disease will re-enter remission with other topical therapies.507 In these studies, a new diagnosis of MF, low stage, lack of blood involvement, and intensity of TSEB were independently associated with progression-free survival (PFS).507,511 TSEBRT was less effective in advanced-stage disease (II to IV), with a CR rate of 60%.511 Another report suggests that prognosis for tumorstage patients with 10% involvement.513 Acute cutaneous side effects, peaking 1 to 2 weeks after TSEBRT, include erythema, edema, dry or moist desquamation, tenderness, and rare blister formation that is most severe at sites of disease.507,514 Total-body alopecia and loss of nails will occur in all unshielded patients, but the skin appendages will normally regrow within 6 months.507 During the first year, heat intolerance may develop as a result of the suppression of sweat gland production, which may be permanent.403,515 Patients with erythroderma (T4) may experience more severe acute side effects

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from TSEBRT; however, radiation may be effective in stage III, especially with no blood involvement.516 When there is blood, lymph node, or visceral involvement in patients with erythroderma, combined-modality therapies, in particular photopheresis, should be explored.516,517 Chronic cutaneous side effects most commonly include xerosis, superficial atrophy, telangiectasia, and dyspigmentation.507,515 The role of TSEBRT in the development of secondary cutaneous malignancies has not been clearly established, as most patients have received a variety of therapies that could contribute to the development of skin cancer.507 Several studies have evaluated the response and toxicity of multiple courses of TSEBRT for CTCL, with similar results.512,518,519,520 Wilson et al.518 reported on 14 CTCL patients, 5 receiving three courses of TSEBRT and 9 patients who received two courses. The total median dose was 5,700 cGy (range, 4,500 to 8,200 cGy) with 86% (n = 12) achieving a CR after the second course of therapy (median relapse-free interval, 11.5 months) and 3 of 5 (60%) achieving a CR after a third course (limited follow-up).518 Because of the toxicities of TSEBRT and the effectiveness of topical therapies, most authorities recommend using TSEBRT for patients with progressive disease, those failing topical therapies, or patients with extensive, deeply infiltrated plaques and tumors.511,521 Because of the high relapse rates after TSEBRT, post-treatment with nitrogen mustard, PUVA, or photopheresis have been studied to maintain remissions.511,517,522 A recent retrospective study of patients with MF that received TSEBRT (n = 180, T2 and T3 skin stage) from Stanford University found no difference in outcomes between those that received adjunctive topical nitrogen mustard to prevent relapse and those patients that did not.520 Small-field megavoltage photon beam irradiation can be applied as palliation to either deep-seated cutaneous lesions in the tumor phase or for extracutaneous disease.523 Nearly all cutaneous lesions respond completely, with the risk of relapse (up to 45%) being inversely proportional to the dose, and with recurrence usually developing within 2 years of treatment.

Photopheresis or Extracorporeal Photochemotherapy Because of the development of resistance to conventional chemotherapy and radiation and the high potential for relapse in advanced-stage patients, new modalities to treat CTCL have been developed. Leukapheresis, which had been used in patients with high Sézary cell counts,524 was the forerunner for a new adaptation of PUVA called extracorporeal photochemotherapy (ECP) or photopheresis. In the original protocol, patients ingested 8-MOP prior to undergoing fractionation of their blood. A leukocyteenriched blood fraction was then isolated and exposed to UVA in an extracorporeal system, which photoactivated the psoralen.525 The photopheresis procedure currently performed uses liquid 8-MOP injected directly into the collection bag containing the enriched white blood cell fraction to achieve a concentration of 340 ng/ml within the collection bag. All treated and untreated blood products were then returned to the patient. In 1987, Edelson et al. were the first to report responses in 27 of 37 patients (64%) with resistant CTCL treated with ECP, including 8 of 10 patients with lymph node involvement and 24 of 29 patients with erythroderma. However, patients with extensive plaques or tumors did not respond as well (3 of 8 patients).525 The immunomodulatory mechanism underlying patient response to ECP is still under debate. However, evidence currently supports the following two simultaneous and synergistic processes occurring during ECP: induction of apoptosis in malignant T-cells, and a mass conversion of blood monocytes to DCs.434,526,527 Animal studies demonstrate that ECP induces a CD8+ T-cell response against expanded clones of pathogenic

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T-cells,528 as well as an increased synthesis of class I MHC molecules on murine T-cell lymphoma cells.529 Recent in vitro studies using family-specific monoclonal antibodies and magnetic bead technology demonstrated a tumor-specific cytolytic CD8+ T-cell response to distinctive Class I surface peptides on CTCL tumor cells of four patients with advanced disease. These results suggest that reduced Class I expression of relevant tumor antigen epitopes may limit the extent of CD8+ T-cell–mediated cytolysis.530 In support of this hypothesis, investigators have found a favorable response to correlate with the following two scenarios at the onset of ECP: normal or near-normal numbers of CD8+ peripheral blood T-cells531 and a lower CD4/CD8 ratio in the peripheral blood.532 Other investigators have found that ECP and in vitro PUVA induce apoptosis in peripheral blood lymphocytes but not in monocytes.434 The apoptosis induction mechanism remains unknown, but may be explained by the observation that a significant amount of tumor necrosis factor-a (TNF-a), which mediates various antitumor effects, is produced by macrophages following ECP.533 Berger et al. identified monocytes transitioning to immature DCs during overnight incubation in gas-permeable bags of ECP-treated WBCs from five patients with intractable CTCL.534 Both the initial leukapheresis step as well as the subsequent passage through the narrow photoactivation plate initiated and contributed to monocytes-to-DC differentiation.534,535 Edelson proposed that the innumerable encounters of monocytes with the plastic surface of the photoactivation plate activated the cells to begin differentiation to immature DCs.526 An immature DC can engulf an apoptotic T-cell and present tumor antigen via MHC Class I molecules, which stimulates a potent antitumor CD8 T-cell response.526,527,534–536 Treatment of CTCL with ECP has been reported in >400 patients and has been recently summarized.537 The majority of CTCL patients treated with ECP have exhibited generalized erythroderma (skin stage T4), a finding most likely due to the encouraging preliminary study results of Edelson et al.537 A combined analysis of >400 patients treated with ECP and adjunctive therapies showed an overall response rate for all stages of CTCL of 55.7% (244 of 438), with 17.6% (77 of 438) achieving a complete response537 Efficacy in treating certain clinical stages (IB, IIA, III, and IVA) and skin stages (T2 and T4) of MF and SS is favorable, although randomized trials comparing ECP to other standard therapies are needed. Combined analysis of five North American series525,538–542 of ECP-treated CTCL patients (N = 157) demonstrates an objective response (>25% improvement of skin lesions) in 67 of 111 stage T4 patients (60%), with ∼20% achieving a CR. A long-term follow-up study of the original 29 erythrodermic CTCL patients in the report of Edelson et al.525 demonstrated a median survival of 60 months, which compared favorably to historical controls.532 Many authorities recommend that ECP be considered the first line of treatment for erythrodermic-stage patients.543–545 However, other authorities differ in their opinions regarding the role of ECP in the treatment of CTCL, stating that the data have been inconsistent and the need for prospective randomized studies.546,547,548 Preliminary and long-term follow-up studies by Zic et al. on 20 refractory CTCL patients treated with ECP and adjunctive therapies demonstrated an objective response (>50% clearing of skin lesions) in 9 of 14 (64%) early-stage T2 patients, with CR in 4.538,549 Talpur and colleagues reported the results of a prospective study of 19 patients with MF stages IA, IB, and IIA who were treated with photopheresis administered 2 days every 4 weeks for 6 months.550 Patients with partial responses by skin weighted assessment continued for an additional 6 months and nonresponders added oral bexarotene and/or IFN-a. The overall response rate was 42% and the authors concluded that ECP is effective for patients with early-stage MF alone or in combination with biologic response modifiers with low toxicity and improved quality of life.550 Observations by other investigators question

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the use of ECP to treat stage IB patients when less expensive and more widely accessible therapies are available.551,552,553 ECP is well tolerated, with few complications or adverse effects.537,545 Uncommon adverse reactions are usually vascular-related and include the fluid-responsive hypotension and venipuncture-site hematomas.549 Rarely, adverse reactions have included exacerbation of congestive heart failure or arrhythmias,543 superficial thrombophlebitis,543 catheter-related sepsis,554 herpes infections,543 disseminated fungal infection,543 and a single episode of hemolysis.538 Several studies have focused on the effects of combining ECP with systemic chemotherapy, PUVA, IFN and other cytokines,555–558 radiation therapy, bexarotene with and without IFN,559,560 and nitrogen mustard. Recently, Raphael and colleagues summarized their experience treating 98 patients with SS with at least 3 months of photopheresis and one or more systemic immunostimulatory agents (IFN-a, oral retinoids, IFN-g, granulocyte-macrophage colony-stimulating factor).561 A total of 73 patients (75%) responded (30% CR, 45% PR) and a lower CD4/ CD8 ratio, a higher percentage of monocytes, and lower numbers of circulating abnormal T-cells at baseline were the strongest predictive factors for complete response compared with nonresponse.561 A much smaller cohort of patients with SS (n = 12) showed a lower response rate of 42% and the parameters that correlated best with response were number of Sézary cells, CD4/ CD8 ratio, and white blood cell count.562 McGirt and colleagues found increased eosinophils and decreased percentages of Sézary cells were associated with a favorable clinical response to ECP, but they were not able to identify the predictors of ECP response within the first 3 months of treatment.563

Systemic Chemotherapy Single agent and combination chemotherapy are reserved for patients who have advanced stage (IIb–IVb), relapsed or refractory disease or are part of a clinical trial.564 Treatments that maintain or augment the immune response, such as IFN, bexarotene, and ECP may be preferable over chemotherapy which is immunosuppressive. Sequential use of single agents should be considered over combinations inasmuch as CTCL is chronic and the clinical goals are control of the disease, amelioration of pruritus, and prevention of skin breakdown; however, combination chemotherapy may be warranted if the disease is progressive, extensive, or in stage IV.565,566 There is no consensus about the optimal combination of agents.564 Previously, single agents used for MF/SS were the same as those used in all types of NHL and included oral corticosteroids, alkylating agents, methotrexate, doxorubicin, bleomycin, vinka alkaloids, cisplatin, and etoposide, and resulted in an objective response in a majority of patients with CR rates ranging from 15% to 20%.559,567 However, median relapse-free intervals were usually short and lasted less than 6 months with a range from 3 to 22 months.567 Daily or pulse chlorambucil with a steroid has been used to successfully treat SS.568,569 In two retrospective series using weekly low-dose oral methotrexate, Zackheim et al. reported a 33% response rate (12% CR) in T2 skin disease and a 55% response rate (41% CR) in erythrodermic CTCL with time to progression and OS of 15 months and 31 months, respectively.570,571 Novel chemotherapy agents have been evaluated for therapy of CTCL and include nucleoside analogs, liposomal doxorubicin, histone deacetylase inhibitors, the antifolate pralatrexate, the proteasome inhibitor bortezomib, and a novel alkylating agent, temozolomide. In a phase II trial of gemcitabine, a pyrimidine antimetabolite, in refractory patients, Zinzani et al. reported a 70% ORR, including 10% CR and a median response duration of 8 months.572 Gemcitabine was given at a dose of 1,200 mg/m2 on days 1, 8, and 15 of a 28-day cycle for a total of three courses. One

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to two cycles were sufficient to induce tumor reduction in 83% of patients with stage IIB MF. Side effects with gemcitabine included neutropenia (34%), thrombocytopenia (25%), cutaneous hyperpigmentation (17%), and elevated liver enzymes (13%).572 Duvic et al. demonstrated a similar response rate (68%) and side-effect profile in another phase II trial, with dosing 1,000 mg/m2 on days 1, 8, and 15 for 6 cycles.573 Marchi et al. reported a 78% ORR, including 22% CR, in previously untreated patients.574 Isolated cases of cardiotoxicity, including atrial fibrillation, myocardial infarction, and congestive heart failure; and of pulmonary toxicity suggest that patients with CTCL may be prone to unusual, nonhematopoietic side effects of gemcitabine.575 The purine nucleoside analogs, 2-deoxycoformycin (DCF or pentostatin), 2 chlorodeoxyadenosine (2CdA or cladribine), and fludarabine monophosphate (FAMP) have activity in CTCL.576,577 DCF inhibits adenosine deaminase, preventing the transformation from adenosine to inosine, which is found in high concentrations of T lymphocytes. Accumulation of metabolites blocks DNA synthesis by inhibiting ribonucleotide reductase. Dosing of DCF has varied among studies and yields an ORR of 33% to 71% with approximately one third of the responses being CR.578 The median time to progression ranges from 1.3 to 8.3 months, and there appears to be a better response in SS than in MF.578–580 The most common side effects are granulocytopenia, renal insufficiency, and prolonged CD4 lymphopenia. A combination study of DCF with high-dose IFN resulted in an ORR of 41% with a median PFS of 13.1 months.581 2CdA is a chlorinated purine analog that is activated by phosphorylation and accumulates in lymphocytes with high deoxycytidine kinase, resulting in DNA strand breaks and cell death. 2-CdA was initially given by continuous infusion over 5 to 7 days and resulted in an ORR of 28% to 41% with a median duration of response of 4 to 5 months in pretreated patients.577,582,583 Toxicity included leukopenia (62%) with grade 3 or 4 neutropenia (24%), thrombocytopenia (33%), and infections (62%) of both bacterial and opportunistic types.582 FAMP is activated by phosphorylation and inhibits DNA repair to induce cell death in lymphocytes. The ORR of FAMP as a single agent in relapsed CTCL has been 19% to 29.5%;584,585 however, response rates improved in small series when FAMP was combined with IFN (51%), cyclophosphamide (42%), or ECP (63%).499,585,586 The median PFS for IFN and ECP were 5.9 months and 13 months, respectively.499,585 Grade 3 or 4 cytopenias occurred in 60% of FAMP+IFN but only 5% of FAMP+ECP. Forodesine is a selective purine nucleoside phosphorylase inhibitor and causes increased levels of deoxyguanosine and deoxyguanosine triphosphate, which inhibit T-cell proliferation.587,588 In a phase I intravenous trial of 13 patients with CTCL, there were 4 responses (3 CR, 1 PR) and 6 with stable disease (SD). In a phase I/II trial using an oral formulation at 80 mg/m2 once daily, the objective response rate was 53.6% (2 CR, 13 PR and 22 SD).589 Forodesine was well tolerated with primarily mild (grade 1 or 2) side effects, including nausea, dizziness, headache, peripheral edema, and fatigue. Altering a drug’s vehicle can improve its efficacy and lower its toxicity, as evidenced by the pegylated liposomal preparation of doxorubicin.590 In an open clinical trial, 10 patients with relapsing CTCL were treated with pegylated liposomal doxorubicin at a dosage of 20 mg/m² once a month, with an upper limit of 400 mg or 8 infusions.591 A total of 60% of patients experienced a CR, 10% PR, and 10% SD following this monotherapy. These results were supported by a retrospective review of 34 patients using doses of 20 to 40 mg/m2 every 2 to 4 week.592 They demonstrated an overall response rate of 88% (44% CR + CRu [patients who achieved a CR defined by clinical criteria only with no biopsy] and 44% PR), an OSof 17.8 months and event-free survival of 12 months. Adverse events were reported in 41% of patients with 17% grade 3/4, including only one with palmar-plantar erythrodysesthesias.

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Histone deacetylase (HDAC) inhibitors (HDI) were discovered serendipitously to have activity in T-cell lymphomas in phase I trials by Piekarz et al. at the NCI and by O’Connor et al. at Memorial Sloan Kettering Cancer Center (MSKCC).593,594 HDI cause histone hyperacetylation, alter chromatin structure, and modulate gene expression.595 They also acetylate nonhistone proteins such as p53 and down-modulate cytokines such as IL-10, both of which contribute to apoptosis of cancer cells.595–597 HDI can be divided into 6 groups based on their chemical structures, and it is unknown whether pan-HDAC inhibition (HDACi) or more selective HDACi is better in treating malignancies.598 Two HDI have been approved for therapy in relapsed CTCL, vorinostat (Zolinza, Merck & Co, Whitehouse Station, NJ, suberoylanilide hydroxamic acid [SAHA]), a relatively nonselective inhibitor of HDAC, and romidepsin (Istodax, formerly depsipeptide, Celgene Corp, Summit, NJ), a specific inhibitor of class I HDAC enzymes.596 Vorinostat was approved for the treatment of relapsed CTCL in October 2006. The maximum tolerated daily oral doses of vorinostat were determined to be 400 mg. In a phase II trial of 33 heavily treated patients with CTCL (median number of prior systemic therapy = 5), Duvic et al. reported an ORR of 24% (all PR) and an additional 33% had SD, pruritus relief, or both.599 In a multicenter phase II trial in 74 patients treated with at least two previous systemic therapies, the ORR was similar at 29.7%.600 The median time to progression was 4.9 months and 9.8 months for responders with stage IIB–IVB disease. The adverse effects for the two phase II trials were diarrhea (49% to 60%), fatigue (46% to 78%), nausea (43% to 60%), thrombocytopenia (22% to 54%), and dysgeusia (24% to 51%). Grade 3/4 adverse events were thrombocytopenia (5% to 19%), dehydration (1% to 8%), and pulmonary embolism (5%).599,600 Cardiac complications have been reported including QT prolongation.601 Vorinostat is being evaluated in combinations with bexarotene, bortezomib, and lenalidomide.595,602 Romidepsin achieved orphan drug status in 2007 and was approved for the treatment of relapsed CTCL in November 2009.603,604 It is a natural product and a stable prodrug that is converted into its active form by a glutathione-mediated step. Thus, a benefit of romidepsin lies in its ability to counteract glutathione-mediated drug resistance.593,605 In two phase II trials in relapsed CTCL patients (n = 71 and 96), the ORRs were 34% in both, including 6% CR in both and the median durations were 13.7 and 15.4 months.606,607 Both studies used the same dose and schedule derived from the NCI phase I study: 14 mg/m2 as a 4-hour infusion once weekly times 3 out of 4 weeks. Most of the adverse events with romidepsin were mild (grade 1 or 2) and were gastrointestinal symptoms or fatigue. Adverse events (all grades) were nausea (52% to 54%), fatigue (41% to 42%, emesis (19% to 26%), anorexia (20% to 21%), diarrhea (8% to 14%), and ageusia (13% to 19%). Grade 3/4 cytopenias were more pronounced in the NCI study: neutropenia (14%), lymphopenia (21%), thrombocytopenia (6%),608 and anemia (6%).606 There was concern that HDI, including romidepsin, would have significant prolongation of the QT interval and risk of arrhythmia, but the studies found minimal risk; however, antiemetics which prolong QT should be limited and the potassium and magnesium levels should be normalized before romidepsin infusion. The potential to alter the expression of a more focused, disease-related subset of genes and to limit adverse effects has prompted the development of isoform-specific HDAC inhibitors in various stages of study.597,609 Belinostat is an intravenous hydroxamic acid with an ORR of 14% in CTCL and 25% in PTCL.610–613 Panobinostat is a hydroxamic acid with both intravenous and oral formulations and had a 60% ORR in a study of 10 patients with CTCL;614 however, a larger study of 95 patients reported only 16% ORR.615 Clinically relevant QTc prolongation is not associated with current dose schedules of panobinostat.616

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Pralatrexate (Folotyn; Allos Therapeutics Inc.) is a novel antifolate with a high affinity for the reduced folate carrier-1 which is overexpressed in neoplastic cells. Pralatrexate inhibits dihydrofolate reductase, an enzyme involved in the synthesis of deoxythymidine and the purine DNA nucleotides, and promotes apoptosis. Pralatrexate received approval in September 2009 for relapsed or refractory PTCL at a dose of 30 mg/m2 weekly for 6 of 7 weeks.617 A de-escalation trial identified an effective dose of 15 mg/m2 weekly for 3 of 4 weeks in patients with relapsed CTCL.618 The ORR was 45% (13/29: 1 CR, 12 PR); 73% of responses were continuing at 6 months by Kaplan–Meier estimate. The main toxicities were mild except for mucositis (48% all grades, 17% grade 3). To reduce mucositis, vitamin B12 and folate supplementation are started before therapy with pralatrexate. Ongoing trials are investigating combination therapies that incorporate pralatrexate. Although in vitro studies suggested synergy with sequential gemcitabine, the toxicities, primarily marrow suppression, were excessive.619,620 Pralatrexate is synergistic with bortezomib in both in vitro and in vivo models of T-cell malignancies.621 Bortezomib (Velcade; Millennium Pharmaceuticals, Boston, MA); a proteasome inhibitor, down-regulates NF-kB activation and causes apoptosis of CTCL cell lines.622 In a phase II trial of 12 relapsed patients (10 MF, 2 PTCLU with skin involvement), the ORR was 67% (2CR, 6PR) and all responses were durable, lasting from 7 to >14 months.623 There were no grade 4 toxicities, and the grade 3 toxicities were neutropenia (n = 2), thrombocytopenia (n = 2), and sensory neuropathy (n = 2). Bortezomib is synergistic with other agents. A phase I trial of bortezomib, gemcitabine, and liposomal doxorubicin reported a PR in 6 of 7 relapsed CTCL patients.624 The combination of bortezomib with the HDAC inhibitor, vorinostat, induced apoptosis in CTCL and was associated with an up-regulation of cell cycle regulating proteins p21 and p27, an increased expression of phosphorylated p38, and a suppression of vascular endothelial growth factor by tumor cells.625 Temozolomide, an oral derivative of dacarbazine that causes DNA damage by methylating nucleotide bases, has demonstrated some efficacy in the treatment of CTCL. This alkylating agent produces O6-alkylguanine adducts, which are deactivated by O6-alkylguanine-DNA alkyltransferase (AGT), a DNA repair enzyme often found in tumor cells. Dolan et al. found that patients with MF demonstrated lower than expected levels of AGT in tumor cells, thus emphasizing temozolomide’s potential therapeutic efficacy in this specific malignancy.626 In a phase II trial, 9 patients were treated with 150 mg/m² of temozolomide orally for 5 days for the first 4-week cycle and then 200 mg/m2 of temozolomide for 5 days for the second and third 4-week cycles. A total response rate of 33% was observed with 2 patients developing grade 3 hematopoietic toxicities.627 In a larger trial of 26 relapsed patients using the 200 mg/m2 dosing schedule, the ORR was 27% (2CR, 5 PR); the median disease-free survival (DFS) and OS were 4 and 24 months, respectively.628 The response did not correlate with levels of AGT. The most common grade 1/2 toxicities were gastrointestinal and treatment was stopped in three patients due to grade 3 thrombocytopenia, lymphopenia, and skin reaction. Combination chemotherapy regimens tend to be reserved for relapsed, aggressive, and advanced-stage MF/SS. Responses occur in the 40% to 89% range but are brief with PFS in the 5 to 9 month range.629–631 In a phase II trial of EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) in 15 refractory patients with CTCL, including 4 ALCL, the ORR was 80% (4 CR, 8 PR); the median PFS was 8.0 months and the median OS was 13.5 months.631 Infectious complications, including staphylococcal bacteremia related to open skin lesions, are a significant problem for patients receiving combination chemotherapy.

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Combined-modality Therapy

Percent surviving

A

C

100 90 80 70 60 50 40 30 20 10 0

Percent surviving

100 90 80 70 60 50 40 30 20 10 0

No. of Total deaths

Combination 52 Conservative 51

21 19

P = 0.72 12 24 36 48 60 72 84 96 108 Survival (mo)

Percent surviving

Percent surviving

Because there is no single therapy for CTCL that can consistently induce long-lasting remissions, various combinations of therapeutic modalities have been studied. The results of several small studies suggested that better survival could be achieved by combining TSEBRT with chemotherapy.632,633 Hallahan et al. evaluated TSEBRT followed by combination chemotherapy for 21 patients with tumor-stage CTCL demonstrating an objective response in 19 (90%) with a median duration of remission of 12 months, but all patients relapsed within 25 months.632 Two nonrandomized studies suggested a benefit for early-stage CTCL patients who receive TSEBRT followed by chemotherapy.633,634 Following TSEBT, subsequent PUVA therapy appears to aid in maintaining remission status in patients with CTCL. A significant benefit in DFS but no statistically significant improvement in OS was observed. However, prospective, randomized data are needed to confirm these results. Incidentally, PUVA has also demonstrated effectiveness as a salvage therapy after TSEBT in early-stage patients with recurrence, with acceptable toxicity.635 The most significant study to evaluate the role of combinedmodality therapy was that of Kaye et al. at the NCI who compared TSEBRT and combination chemotherapy (cyclophosphamide, doxorubicin, etoposide, and vincristine) to conservative topical therapy (beginning with topical HN2 followed sequentially, if needed, by PUVA, TSEBRT, and combination chemotherapy) in a randomized trial of 103 patients with MF.636 Although the rate of CR was significantly increased in the combined-modality arm (38% vs. 10%, p = 0.032), toxicity was greater and no significant difference was noted between the groups in disease-free or OS (Fig. 9.17). Thus, this study indicated that, similar to other lowgrade lymphomas, early aggressive therapy in MF does not have a major impact on survival. A nonrandomized study from Yale University compared relapse-free survival (RFS) and OS between CTCL patients who achieved a CR following TSEBRT, with subsequent treatment consisting of either adjuvant chemotherapy (cyclophosphamide and doxorubicin; n = 77), photopheresis (n = 11), or no adjuvant therapy (n = 43).637 Adjunctive therapy was also offered to 32 patients who achieved a “good PR” to TSEBRT. The statistical analysis found no appreciable impact on RFS among the patients receiving adjuvant chemotherapy or photopheresis when compared to patients receiving no adjuvant therapy. However, a marginally significant (p < 0.06) improvement on OS was demonstrated

No. of Total deaths

Combination 28 Conservative 28

12 12 P = 0.72

12 24 36 48 60 72 Survival (mo)

84

96 108

100 90 80 70 60 50 40 30 20 10 0

100 90 80 70 60 50 40 30 20 10 0

Total

Combination 16 Conservative 15

No. of deaths

1 2

P = 0.36 12 24 36 48 60 72 84 96 108 Survival (mo)

B

No. of Total deaths

8 Combination 8 Conservative 8 5 P = 0.16

12 24 36 48 60 72 84 96 108 Survival (mo)

D

FIGURE 92.17. Survival curves from randomized study at the National Cancer Institute comparing intensive combined therapy.  A: Overall survival. B: Survival among low-risk patients (stage IA, IB, or IIB). C: Survival among intermediate-risk patients (stage IIB, III, or IVA). D: Survival among high-risk patients (stage IVB). (From Kaye FJ, et al. A randomized trial comparing combination electron beam radiation and chemotherapy with topical therapy in the initial treatment of mycosis fungoides. N Engl J Med 1989;321:1784, with permission.)

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1979

when stage T3/T4 patients treated with adjuvant photopheresis (n = 7) were compared to stage T3/T4 patients receiving no adjuvant therapy (n = 22).637 Retinoids and IFN-a have been combined to treat CTCL in several open studies.638–640 Combined analysis of the results of 102 reported patients treated with retinoids and IFN-a showed that approximately 60% of patients respond and 10% achieve a CR similar to the response for IFN-a alone.487 PUVA therapy has been combined with systemic retinoids to treat CTCL in two studies.641 demonstrating response rates similar to PUVA alone. Bexarotene has been combined with ECP, PUVA phototherapy, and IFN-a in several patients with potential beneficial effects, as well as no increased toxicity.475,642 The combination of PUVA and IFN-a is well tolerated and generates impressive complete response rates.643–645 In the combined analysis of the phase I and phase II trials of IFN-a and PUVA for CTCL at Northwestern University, 39 CTCL patients (Stage IB, n = 14; IIA, n = 5; IIB, n = 6; III, n = 8; IVA, n = 5; IVB, n = 1) received intramuscular or subcutaneous IFN-a2a three times a week at initial intermediate doses (6MU, n = 3; 12MU to 18MU, n = 13; 21MU to 30MU, n = 23) with subsequent dose reduction in 19 patients due to apparent toxicity.643 IFN-a was continued for the planned 2-year period in only 10 of 39 patients (26%) with 8 patients receiving 4 or less months of IFN-a due to tumor progression, toxic effects, or patient request. PUVA was initiated three times per week and tapered to 1 monthly treatment indefinitely for patients achieving a CR. The overall objective response rate was 90% with 24 patients (62%) achieving a pathologically confirmed CR, 15 of whom had early-stage disease (Stage IB/IIA). A total of 19 patients (54%) relapsed demonstrating a median duration of response of 28 months (range, 1 to 64 months). Median survival for the entire cohort was 62 months with mean survivals for stage I/II and stage III/IV patients of 55 and 35 months, respectively.643 Thus, although patients respond impressively, a majority of patients will relapse despite maintenance PUVA and experience nontrivial toxicities at higher doses of IFN. The overall impact on survival of combined PUVA and IFN-a has yet to be determined. A prospective phase II trial examined escalating doses of IFNa2a combined with PUVA in 63 symptomatic patients representing all stages of MF and SS.645 A total of 51 patients achieved a CR (74.6%) or PR (6%), with a median response duration of 32 months. The 5-year OS rate was 91% and included 17 patients with advanced disease.645 Rupoli et al. completed a multicenter prospective phase II clinical study on 89 patients with early-stage IA to IIA MF treated for 14 months with low-dose IFN-a2b (6 to 18 MU/wk) and PUVA.646 Complete remission (CR) was achieved in 84% and an overall response rate in 98% of cases. Long-term CR was associated with high epidermal CD1a+ dendritic-cell density (P = 0.030) and high CD8+ lymphoid T-cell density was associated with a lower relapse rate (P = 0.002).646 Stadler and colleagues completed a prospective randomized multicenter trial to compare IFN plus PUVA and IFN plus acitretin in stage I and II patients with CTCL (n = 82 evaluable patients).647 IFN-a2a was administered subcutaneously at 9 MU three times weekly and combined with either PUVA at an initial interval of five times weekly, or with acitretin up to 50 mg daily. IFN-a + PUVA (n = 40) was significantly superior to the IFN + acitretin (n = 42), as marked by a 70% complete remission rate in the former, versus a 38.1% complete remission rate in the ­latter.647 A recent report appeared to suggest that elevated levels of the cutaneous T-cell attracting chemokine CTACK/CCL27 in skin and sera after combined PUVA and IFN-a2b therapy might be correlated with risk of recurrence.26 Reports have emerged of the successful combination of low-dose bexarotene and phototherapy. Narrow-band UVB phototherapy has been combined with oral bexarotene with success in one recent case.648

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The successful combination of low-dose bexarotene with PUVA phototherapy has also been reported.475,642,649–654

Immunotherapy Over the past decade, researchers have developed immunotherapies to correct abnormalities in the immune response, and cellular growth and differentiation pathways in patients with CTCL. As described in previous sections, recombinant forms of natural cytokines such as IFNs and immunomodulation with photopheresis have shown promise in the treatment of CTCL, with tolerable toxicity profiles and reasonable efficacy. This section focuses on other cytokines and monoclonal antibody therapies for the treatment of CTCL. Fusion toxin therapy takes advantage of the preferential expression of specific receptors on the surface of malignant cells. Recombinant fusion of a plant or bacterial toxin gene to a specific receptor ligand can guide the toxin gene to the target cell, where it can be internalized via receptor-mediated endocytosis and translocated into a toxic moiety in the cytosol.655 The interleukin-2 receptor (IL-2R) is present in low-, medium-, and high-affinity forms. The high-affinity form of IL-2R is a three-subunit peptide, and one of the three subunits contains CD25. The high-affinity IL-2R has been a specific target that is commonly present on cells in ATLL,656 and on mature, activated T-cells. Approximately 60% of patients with CTCL will show expression of IL-2R on their mature T-cells, but there is both interpatient and intrapatient variability of expression.657 DAB486IL2 and DAB389IL2 were the first fusion toxins to be used in clinical trials and are composed of the nucleotide sequence of the enzymatically active and the membrane translocating domains of diphtheria toxin conjugated to the amino acid sequence of human IL-2.655,658,659 In a phase II trial of DAB486IL2 reported by Foss et al.,660 3 of 14 CTCL patients demonstrated an objective response (1 patient with a PR and 2 patients with slightly less than 50% improvement). The authors observed that IL-2R expression was necessary, but not sufficient, to predict response. DAB389IL2 (denileukin diftitox), a second-generation molecule, replaced DAB486IL2 because it showed a more favorable pharmacokinetic profile. A pivotal phase III trial evaluated the safety, efficacy, and pharmacokinetics of two dose levels of denileukin diftitox.661 This randomized, blinded, parallel-grouped study focused on the use of denileukin diftitox in the treatment of 71 patients with persistent or recurrent stage IB to IVA CTCL. For study inclusion, patients had to have detectable CD25 on ≥25% of their tissue biopsy lymphocytes via immunoperoxidase assay. Following therapy, 30% of patients experienced an objective response (20% PR and 10% CR), 32% experienced SD, and 3% demonstrated progressive disease. Both 9- and 18-mg/kg/day doses were compared, and showed similar tolerability and no evidence of cumulative toxicity. No statistically significant difference was found between the two dosing regimens with respect to response rate and duration of response (median 6.9 months, range 2.7 to 46.1 + months).661 Upon stratification with respect to disease stage, 18 mg/kg/day of denileukin diftitox proved more effective in treating stage IIB patients (36% OR) than the lower dose (23% OR).661 Denileukin diftitox was approved by the FDA in 1999 for the treatment of patients with persistent or recurrent CTCL whose malignant cells express the CD25 component of the IL-2 receptor. A phase III, placebo-controlled trial, again evaluating the two dosing regimens of 9- and 18-mg/kg/day doses was performed with 144, CD25 CTCL patients of any stage.662 The overall response rate was higher in the 18-mg/kg/day group as compared to the 9-mg/kg/ day group (49.1% vs. 37.8%) and both were higher than placebo (15.9%).662 There was also increased estimated PFS in the 18-mg/ kg/day group versus the 9mg/kg/day group (32.4 months vs. 26.5 months), but the adverse events were similar for both dosing

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arms.662 Not surprisingly, the placebo group had significantly fewer adverse events and reduced PFS. The effectiveness of denileukin diftitox in patients with 35

>2a

>3

50 or any B MMR >.33 or >10 cm >3

any

Figure 93.7. Progression-free survival (A) and survival (B) as related to number of risk factors.  (From Hasenclever D, Diehl V. A prognostic score for advanced Hodgkin’s disease. N Engl J Med 1998;339:1506–1514, with permission.)

patients with one risk factor (22% of all patients), FFP was 77%. For patients with two risk factors (29% of all patients), FFP was 67%. For patients with three risk factors (23% of all patients), FFP was 60%. For patients with four risk factors (12% of all patients), FFP was 51%. For patients with five or more risk factors (7% of all patients), FFP was 42%. Of note, B symptoms did not have independent prognostic value in this model. At present this model is validated only for advanced-stage patients. A number of prognostic classifications have been used for early-stage HL and have been used to tailor therapy further in clinical trials. These commonly employ some combination of age, erythrocyte sedimentation rate, mediastinal bulk, and number of involved nodal sites. See Table 93.7, “Unfavorable Prognostic Factors for Stage I–II HL, by Three Cooperative Groups.”

OVERVIEW AND HISTORICAL PERSPECTIVE OF RADIATION THERAPY Six years after the discovery of x-rays by Roentgen in 1895, Pusey reported that x-rays could shrink enlarged nodes in patients with HL.102 However, given the orthovoltage techniques of the day, therapy was only palliative. The modern radiotherapy era began with the Swiss radiotherapist Gilbert in 1925.103 Based on observed patterns of spread in patients with HL, Gilbert advocated treatment of both involved areas and adjacent, apparently uninvolved, areas. This approach was also adopted by Peters, who, in 1950, was the first to report that radiation therapy of HL could produce cures.104 Over the next two decades, the curability of HL was confirmed by Peters,105,106 Easson and Russell,107 and Kaplan,108,109 the latter investigator establishing the critical relationship between radiation dose and the risk of recurrence in the treatment field (Fig. 93.8).

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100 Recurrence rate, percent

B

As originally suggested by Gilbert, the principle of treating involved and adjacent apparently uninvolved areas (i.e., extended field therapy) became a standard radiotherapeutic approach to HL. With the advent of staging laparotomy and evidence that the retroperitoneum could represent a potentially involved adjacent area of disease, the concept of extended field therapy came to include the standard mantle, para-aortic/splenic, and pelvic radiation ports shown in Figure 93.9.110 Although the radiotherapy pioneers were the first to cure HL, and they did so by conducting elegant and groundbreaking clinical trials, the era of radiation as single modality for the initial treatment of HL has passed. However, HL is a very radiation-sensitive disease, and radiation remains a useful component of the treatment of certain patients. Currently the most well documented role for radiation is in the early stage setting, discussed below. Complications of radiation therapy depend on technique, dose, and the volume of irradiated tissue. Common complications include hair loss within the treatment fields, radiation pneumonitis, radiation pericarditis, mediastinal fibrosis, and pulmonary fibrosis. Symptoms of radiation pneumonitis generally occur within 1 to 3 months of completing therapy and are nonspecific because they include dyspnea, cough, and fever. Such

Hematologic Malignancies

GSHG, German Hodgkin Study Group (auses alternate definition of nodal sites); EORTC, European Organization for the Research and Treatment of Cancer; NCIC, National Cancer institute of Canada; MMR, mediastinal mass ratio: maximal width of mass/maximal intrathoracic width; MTR, mass to thoracic width at T5–T6 interspace on standing PA chest radiograph.

Recurrences total fields rate

80

76/97

78%

60

147/243

60%

58/121

48%

120/342 53/206

35% 26%

40 20

24/208 11.5% 4/91 4.4% 1000 2000 3000 4000 Dose, r Figure 93.8. Rate of recurrence in a given treatment field as a function of radiation dose delivered to that field.  (From Kaplan HS. Evidence for a tumoricidal dose level in the radiotherapy of Hodgkin disease. Cancer Res 1966;26:1221–1224, with permission.)

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Figure 93.9. Treatment fields used as extended field irradiation of Hodgkin disease.  A: Mantle field. B: Inverted Y field. C: Mantle and para-aortic field (extended mantle field). D: Total nodal field. The spleen is irradiated in conjunction with the fields in B, C, and D, unless it has been surgically removed.

A

complications are rarely seen at lung doses 10% in the preceding 6 mo

Hematologic Malignancies

  Unexplained recurrent fever >38°C   Drenching night sweats. X  Bulk disease (see C below)   E  Involvement of a single extranodal site that is contiguous or proximal to the known nodal site. C.  Bulk disease One or both of the following presentations are considered “bulk” disease: • Large mediastinal mass: tumor diameter >1/3 the thoracic diameter (measured transversely at the level of the dome of the diaphragm on a 6-foot upright PA CXR) In the presence of hilar nodal disease the maximal mediastinal tumor measurement may be taken at the level of the hilus. This should be measured as the maximum mediastinal width (at a level containing the tumor and any normal mediastinal structures at the level) over the maximum thoracic ratio. • Large extramediastinal nodal aggregate: A continuous aggregate of nodal tissue that measures >6 cma in the longest transverse diameter in any nodal area. aSome

studies use 10 cm for definition of extramediastinal bulk disease.

Initial Treatment for Pediatric Hodgkin Lymphoma To our knowledge, the biology and natural history of HL does not differ between children and adults. As a result, early therapeutic approaches for pediatric HL were similar to or modeled after those developed for adults with HL. This seemed particularly logical and HL is most commonly a disease of adolescents as opposed to adults. However, with high cure rates, contemporary standard approaches to adolescent and young adult HL include multiagent chemotherapy with or without low-dose involved field radiotherapy. The overriding principles of these treatment regimens are to balance efficacy with both acute and, perhaps, more important, long-term toxicities.81–83 A summary of common protocols for upfront therapy is found in Table 94.3. Figure 94.1 shows the timeline of evolution of therapy for pediatric HL. Challenges inherent in comparing data across pediatric HL trials are differing patient populations, with differing definitions of stratification. However, overall, results are quite good with 3- to 10-year EFS rates ranging from 67% to 94% for combined modality therapy and 60% to 91% for chemotherapy alone, as reviewed by Olson and Donaldson.84 Chemotherapy regimens have largely built upon the MOPP and ABVD backbones and have been quite successful overall. To decrease alkylator therapy and potential cardiotoxicity and to provide dose-intensive treatment, the Pediatric Oncology Group (POG) developed a series of studies built upon the ABVD backbone, substituting dacarbazine with etoposide. The use of vincristine instead of vinblastine allowed for escalation of doxorubicin and etoposide. POG 9226 piloted therapy with 4 cycles of DBVE followed by low-dose IFRT in patients with Stages I, IIA and IIIA. The 5-year EFS was 89%.85 This pilot was followed by a Phase III study using the same therapy (POG 9426) in a nonrandomized response-based manner. Those with a rapid response

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TAB L E 9 4 . 3

SOME COMMON STANDARD THERAPY PROTOCOLS FOR PEDIATRIC CLASSICAL HODGKIN LYMPHOMA Stage and Presentation

Commonly Accepted Chemotherapy Regimens

IA, IIA < 4 nodal regions without B symptoms, bulk disease, or extranodal extension

VAMP × 4 COPP/ABV hybrid × 4 ABVE × 4 OEPA or OPPA × 2 CHOP × 3–4 ± Rituximab ABVD × 2–4

IA, IIA with bulk disease, ≥3 nodal regions, or extranodal extension IIB*, IIIA, IVA

COPP/ABV × 6 ABVE-PC × 3–5 OPPA/OEPA × 2 + COPP × 2 OEPA-COPAC ABVD × 4–6

IIB*, IIIB, IVB

ABVE-PC × 3–5 BEACOPP × 8 (or BEACOPP × 4 + ABVD × 2 or + COPP/ABV × 4) OPPA/OEPA × 2 + COPP × 4 OEPA-COPAC Stanford V ABVD × 6–8

All regimens should consider LD-IFRT (low-dose involved field radiotherapy; 15 to 25 Gy), but there are variations in recommendations based on risk and early and complete response to chemotherapy.

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FIGURE 94.1. Timeline of Hodgkin lymphoma upfront therapy.

1970s MOPP Full-dose extended field RT

1950s – 1960s Radiation therapy (RT) Full-dose Extended fields

1990s COPP/ABVD BEACOPP ABVE-PC OEPA/OPPA VAMP Reduction in RT use Response-based therapy Gender-based therapy

(60% tumor reduction, gallium negative) after 2 cycles proceeded to consolidative IFRT without further chemotherapy; slow responders continued to receive 4 cycles plus IFRT. The overall EFS was 88.3%.86 In POG 9425 (for patients with advanced stage disease) prednisone and cyclophosphamide were added to the ABVE backbone, with more rapid delivery of therapy to intensify the weekly therapeutic intensity. Response was assessed after three cycles (9 weeks). Those with rapid response did not receive the additional two cycles of chemotherapy. All patients received consolidative regional low-dose (20 Gy) RT. Overall 3-year EFS was 87%.79 Both studies evaluated the efficacy of dexrazoxane in reducing cardiac and pulmonary toxicity. Although it is still too early to know whether there was a significant benefit, dexrazoxane was associated with an increased risk of acute (hematologic/ infectious) toxicities and development of second malignancies, mostly treatment-related leukemia.87 With the goal of balancing efficacy with toxicity, and specifically to decrease long-term risk for organ dysfunction and second malignancies, there have been a number of studies conducted to evaluate the avoidance of RT in the POG and Children’s Cancer Group (CCG), and more recently in the Children’s Oncology Group (COG), representing the merger of those groups. In the Children’s Cancer Group (CCG) 521 study, patients were randomized between 12 cycles of alternating MOPP/AVBD versus 6 cycles of AVBD + low-dose regional chemotherapy. Event-free survival was 77% for Stage III and IV patients and there was excess pulmonary toxicity with the ABVD arm.88 POG 8725 compared eight cycles of MOPP/ AVBD with and without IFRT in Stage IIB, III, and IV patients. There was no significant benefit for RT, but patients were exposed to significant doses of alkylating agents and anthracyclines, which are associated with significant long-term risk for gonadal toxicity, cardiac toxicity, and secondary leukemia.89 Response post three cycles was a strong prognostic factor, with a 94% EFS in the rapid early responders as opposed to a 78% EFS in those who did not respond quickly.89 In a randomized Phase III trial, CCG 5942 evaluated the avoidance of radiotherapy in patients who had a complete response following chemotherapy. Patients were treated with four cycles of COPP/ABV, six cycles of COPP/ABV, or two cycles each of COPP/ABV, cytarabine/etoposide, and CHOP, depending on the stage of disease and presence of bulk disease. Of the 829 eligible patients, complete response was achieved in 83% and 501 were randomized to receive IFRT or no further therapy. Event-free survival at 3 and 10 years was 87% and 83%, respectively. In all groups, the difference in EFS post randomization was highly significant and overall EFS was 91% at 3 and 10 years for the group who received IFRT and 86% at 3 years and 83% at 10 years for those who did not receive IFRT. Despite the differences

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1980s MOPP/ABVD Reduction of RT fields and dose

2000–2012 ABVE-PC OEPA/OPPA/COPAC VAMP CVP, CHOP, R-CHOP FOR NLPHL Further reduction in RT use Targeted RT fields Response-based therapy Gender-based therapy

in EFS, early evaluation of overall survival is not affected by the inclusion of IFRT.63,90 A pilot study of 99 patients in CCG 59704 utilized the German Hodgkin Disease Group’s BEACOPP backbone for patients with B symptoms or advanced stage disease. Overall the 5-year EFS was 94% with patients receiving six to eight cycles of chemotherapy with or without radiotherapy on the basis of a gender-stratified, response-based therapeutic algorithm.91 A series of studies in the COG have just completed accrual and have evaluated the principles of response-based riskadapted therapy, where therapy is decreased for those at lowest risk and with favorable early responses to chemotherapy and augmented for those with more advanced disease or a slow response to initial chemotherapy. These studies have utilized the ABVE-PC backbone of the POG 9425 and 9,426 studies. The Dana Farber-Stanford-St. Jude pediatric Hodgkin lymphoma consortium studies have focused efforts on reduction in alkylating agent, anthracycline, and radiotherapy doses. Low-risk patients have been treated with four cycles of an alkylator-free chemotherapy protocol, VAMP, with radiotherapy doses of 15 Gy or 25.5 Gy based upon response to the first two cycles of chemotherapy. This approach resulted in a 5-year EFS of 93% and 10-year EFS of 89%.92 For patients with higher risk disease, two protocols have been evaluated, VAMP/COP and VEPA, both with IFRT. Results have been less acceptable with 5-year EFS of 74% and 68%, respectively.93,94 The group is now evaluating the Stanford V regimen95 that has been successfully employed in adult patients. In the same era, the German cooperative groups have built upon COPP chemotherapy in both pediatric and adult HL. To decrease gonadotoxicity, which is more prevalent in males exposed to alkylating agents, some of these therapies have been gender-based. The DAL-HL-90 study used OEPA/COPP for males and OPPA/COPP for females, both followed by IFRT. EFS for Stages II, III, and IV was 92%, 86%, and 90%, respectively.96 The GPOH-95 study was designed to assess elimination of radiotherapy for those with a complete response. It built on the OPPA/ OEPA backbones by adding cycles of COPP for patients with more advanced disease. Radiotherapy dose was determined by the post-chemotherapy disease reduction. Complete response was defined as complete resolution of all disease (as opposed to a 70% reduction in CCG 5942 and 59704). Only 22% achieved a complete response. A total of 50% achieved between 75% and 95% disease reduction and were treated with 20 Gy radiotherapy, and 4% had less than a 75% reduction and were treated with 30 Gy. In addition, 20% of patients had residual masses treated with boost doses to 35 Gy. Overall the EFS was 92% for those receiving radiotherapy, compared with 88% in those treated with chemotherapy alone, but for patients with IA, IB, and IIA disease, EFS was not

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different among those with a CR to chemotherapy and no other therapy (97%) and those with a partial response, subsequently treated with radiotherapy (94%). However, for all other patients the EFS was 79% for those treated with chemotherapy alone compared with 91% for those treated with combined modality therapy.97 This was followed by the GPOH-HD-2002 study, where OEPA-COPDAC chemotherapy was tested in males compared with OPPA-COPP in females for intermediate and advanced HL. The 5-year EFS was 89.0% without significant difference between males and females.98 For low-risk patients with NLPHL, some patients may be treated with surgery alone. There are multiple small reports in both adult and pediatric HL of patients of successful treatment of completely resected Stage I disease. For those who did relapse, salvage rates were encouraging and death from disease very low.99–102 This has prompted a study in the COG to formally evaluate surgery only in patients with involvement of a single completely resected lymph node with NLPHL. Although adults with NLPHL may be treated with radiotherapy alone, the dose required without adjuvant chemotherapy exceeds that optimal in children and adolescents, and thus this strategy is not used in pediatric NLPHL. The optimal treatment of patients with advanced stage NLPHL remains undetermined. These patients are traditionally treated with regimens that are used for patients with advanced stage classical HL with outcomes equivalent to or better than those with CHL. However, the biology of NLPHL may be more similar to indolent CD20+ B-cell non-Hodgkin lymphoma (NHL) than classical HL and thus the question remains of whether regimens for NHL would be more effective with potentially less toxicity in these patients. A retrospective analysis at MD Anderson Cancer Center from 1995 to 2010 found that patients with NPLHL treated with R-CHOP had better 5-year progression-free and overall survival compared to those treated on the HL regimen.103 From 2005 to 2010, a study was conducted in France and the United Kingdom, testing CVP in 55 patients with early-stage NLPHL. The 40-month freedom from treatment failure was 75.4%, but with an overall survival of 100%, respectively.104 However, prospective multicenter randomized studies have not been conducted to answer such questions and it is unlikely, given the exceptional overall survival, that such studies will be successfully conducted.

Radiotherapy for Hodgkin Lymphoma It has been recognized since the 1950s that HL is an extremely radiosensitive disease. Currently, for children and adolescents with HL, radiotherapy is exclusively delivered in the context of multimodality therapy. Doses of 15 to 25 Gy are commonly used, and as noted above, clinical trials have been and continue to be conducted to identify groups of patients for whom the exclusion of radiotherapy is possible, without affecting disease-free survival. Below are some basic principles for radiotherapy in pediatric and adolescent HL. However, delivery of radiotherapy, particularly in children and adolescents, requires careful considerations of the age, tumor burden and location, response to chemotherapy, and an assessment of both short- and long-term potential toxicity. Megavoltage energies are now the treatment of choice for pediatric and adolescent HL. Although there are differences in treatment techniques among radiation oncologists and specific institutions, the general technical principles remain constant.105 Proton beam radiotherapy is currently under investigation at several centers across the United States, but there is insufficient data to know whether this modality will provide the best option for long-term cure, while decreasing adverse long-term effects of treatment.106,107 Radiotherapy fields must be designed with the goal to deliver the optimum volume of radiotherapy for disease control, while avoiding normal tissue damage. Customized shielding blocks

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should be utilized as appropriate to protect normal tissue. Blocking the genitalia is of specific importance when pelvic fields are included. In females, oophoropexy results in doses of 8% to 10% and 4% to 5%, respectively, of the pelvic dose,108 which will be compatible with the preservation of fertility. For males, a frog leg position and an individually fitted shield will provide the greatest shielding to the testes, reducing scatter to approximately 0.75% of the pelvic lymph node dose.109

Treatment for Relapsed Hodgkin Lymphoma HL may be cured even if it has recurred after initial treatment. Potentially curative options include conventionally dosed combined modality protocols: radiotherapy alone for those relapsing in a limited nodal pattern, re-induction chemotherapy followed by autologous hematopoietic stem cell transplantation (HSCT), full and reduced intensity allogeneic transplants, and evolving targeted antibodies.110,111 Some common retrieval regimens are shown in Table 94.4 and the choice of regimen is dependent on previous treatment and patterns of relapse. Two ifosfamide-based regimens have been used with good response rates, ICE and IV with EFS exceeding 75%.112,113 Gemcitabine has also been used in combination with vinorelbine with excellent overall response rates in recurrent HL with similar EFS.114,115,116,117 Autologous HSCT is most commonly used for recurrent or refractory HL, particularly when used following dose-intensive regimens or for high-risk disease.115,116–119,120–125,126,127,128 However, an allogeneic effect has been observed, and full and reduced intensity allogeneic approaches as well as immune modulation and induction of autologous graft versus host disease have been explored to enhance the allogeneic effect.129–131,132,133–136 The most promising new agent in the treatment for HL is brentuximab vedotin (SGN35), which is also being utilized in the earlier relapse period with several recently reported as well as ongoing trials, although pediatric experience is quite limited.137 In a recent Phase 2 study, the efficacy and safety of brentuximab vedotin was evaluated in 102 patients with relapsed or refractory HL after ASCT. Reductions in tumor size were observed in 94% of patients. The overall response rate was 75%, with complete remission achieved by 34% of patients and partial response in 40%.138 A retrospective analysis by the German Hodgkin Study Group with brentuximab vedotin as single agent was reported in 45 patients with refractory or relapsed CD30 (+) HL, with an objective response rate of 60%, including 22% complete remissions.139 A case series of 20 transplant-naive patients who either refused or were ineligible for transplant, enrolled in two Phase I multicenter studies, reported six responses.140 In a study of

Hematologic Malignancies



TAB L E 9 4 . 4

COMMON TREATMENT OPTIONS FOR RECURRENT HODGKIN LYMPHOMA ICE (Ifosfamide, carboplatin, and etoposide) DECA (Dexamethasone, etoposide, cisplatin, cytarabine) IV (Ifosfamide and vinorelbine) GV (Gemcitabine and vinorelbine) IEP–ABVD–COPP (Ifosfamide, etoposide, prednisone–doxorubicin, bleomycin, vinblastine, dacarbazine–cyclophosphamide, vincristine, procarbazine, prednisone) APE (Cytosine arabinoside, cisplatin, etoposide) SGN-35 (brentuximab vedotin)—antibody-drug conjugate targeting CD30 These therapies can be used as stand-alone therapy or as re-induction prior to planned stem cell transplant. Radiotherapy can be considered together with this therapy.

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25  patients with recurrent HL post-allogeneic transplant, who had received a median of 9 (5–19) prior regimens, overall and complete response rates were 50% and 38%, respectively, among 24 eligible patients, with a median progression-free survival time of 7.8 months.141 These data led to accelerated approval of brentuximab vedotin for the treatment of patients with HL after failure of autologous stem cell transplantation (ASCT) or after failure of at least two prior multiagent chemotherapy regimens in patients who are not ASCT candidates.142 Studies are now ongoing evaluating brentuximab vedotin as part of multiagent protocols in the upfront and recurrent setting.

Adverse Long-term Outcomes of Therapy In pediatrics, a significant consideration when deciding therapy is the risk of adverse long-term risks of therapy, which include organ dysfunction and second malignancies.143 The common potential long-term effects of radiotherapy and chemotherapy for HL are summarized together with general monitoring recommendations in Table 94.5. In a review from the Childhood Cancer Survivor Study (CCSS), among 2,633 HL 5-year survivors, initially treated between 1970 and 1986, there were 500 deaths with the following causes: HL, 35%; second malignancies, 23%; cardiovascular disease, 14%; pulmonary disease, 4%; external cause, 7%; other and unknown cause, 17%. Adjusting for demographics

and medical conditions, treatment with radiotherapy or alkylating agents were independent risk factors for overall mortality and those same exposures as well as anthracycline treatment for second malignancies. In this cohort, the 30-year cumulative incidence for second malignancy was 18.7%. Based on self-report, the 30-year cumulative incidence of grade 3 to 5 chronic health conditions in the cohort were as follows: cardiovascular,11%; pulmonary, 5%; and thyroid, 51%.144 Other studies of subsequent malignancy cite female gender, radiotherapy field and dose, and follow-up time since diagnosis as risk factors. The most significant risk is for secondary breast cancer in female survivors treated with thoracic radiotherapy, where even lower doses of radiotherapy, with ongoing follow-up now are associated with increased risk.145–149 Thyroid dysfunction and thyroid cancer are also of concern in this population. In the CCSS, self-report thyroid status was assessed in 1,791 HL survivors treated from 1970 to 1986. Of the entire cohort, 34% have been diagnosed with at least one thyroid abnormality. Hypothyroidism was the most common disturbance, with a relative risk of 17.1 compared to sibling controls. Hyperthyroidism was reported by 5% of survivors, which was 8-fold greater than the incidence reported by the controls. The risk of thyroid nodules was 27 times that in sibling controls. Female gender and higher radiation dose increased risk for thyroid abnormalities.150 In a pooled cohort of childhood cancer survivors including 16,757 patients, with 187 developing primary thyroid cancer, radiation-dose–related relative risks increased approximately linearly for 50 Gy. Dose-related excess relative risks increased with decreasing age at exposure but did not vary with attained age or time-since-exposure, remaining elevated 25+ years after exposure. Gender and number of treatments did not modify radiation effects. In another pooled analysis, three models of assessing absolute risk for secondary thyroid cancer were developed and validated. Model M1 included self-reported risk factors, model M2 added basic radiation and chemotherapy treatment information abstracted from medical records, and model M3 refined M2 by incorporating reconstructed radiation absorbed dose to the thyroid. All models were associated with elevated relative risks (M1 = 10.8; M2 = 6.8; M3 = 8.2) and had good discriminatory ability. Past thyroid nodule was consistently the strongest risk factor in the models.151,152 Cardiac toxicity following anthracycline therapy and mediastinal radiotherapy is well recognized among survivors.144,153 Among 1,132 survivors of HL treated between 1978 and 1995, treated with a relatively low doxorubicin dose (160 mg/  m2), at a median of 19.5 years, cardiac diseases had been diagnosed in 50 patients. Valvular defects were diagnosed most frequently, followed by coronary artery diseases, cardiomyopathies, conduction disorders, and pericardial abnormalities. Multivariate analysis showed the mediastinal radiotherapy dose to be the only significant variable associated with cardiac disease.154 In a cohort of 1,362 5-year survivors diagnosed between 1966 and 1996, with 50 cardiac events reported, cumulative incidence of anthracycline (dose), cardiac irradiation (dose), combination of these treatments, and congenital heart disease were significantly associated with developing a cardiac event.155 Reproductive outcomes have been examined for all survivors in the CCSS, but risk factors for impairment are generally those seen with HL therapy. Sklar and colleagues evaluated premature menopause and a multiple Poisson model showing risk factors to include attained age, exposure to radiation to the ovaries in a dose-dependent manner, number and cumulative dose of alkylating agents, and a diagnosis of HL.156 Green and colleagues found that male survivors were less likely to sire a pregnancy than siblings. Among survivors, the hazard ratio of siring a pregnancy was decreased by RT to the testes, higher cumulative alkylating agent dose, and treatment with cyclophosphamide or procarbazine.157 Studies of HL survivors treated in the more contemporary age of chemoradiotherapy are underway to see if reduction in anthracyclines, alkylating agents, and radiotherapy have resulted in fewer long-term adverse chronic health conditions. Various pediatric cooperative groups have developed longterm follow-up guidelines, which provide a foundation for the type of follow-up care that should be delivered. However, the recommendations are not completely consistent with one another and recommendations are consensus-based, and thus, the optimal follow-up strategy is yet to be fully elucidated. Efforts are underway for international harmonization among large childhood cancer groups.158–161,162–164,165–167

Conclusions Cure rates for HL are one of the highest in pediatric oncology, but the very therapies that have afforded such cure rates share the etiologic limelight for adverse long-term health outcomes. Ongoing clinical trials thus seek to balance efficacy with both short- and long-term toxicity. For a meaningful minority of patients with higher risk initial disease, refractory, and recurrent disease, the challenge remains increasing the cure rate and incorporating novel agents that may better target the underlying biology and pathophysiology. And last, additional study is required to understand the complex biology and epidemiology of HL better to understand better how they interface with one another, affect disease presentation, response to therapy, and long-term outcomes.

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2011

Key Summary Points For initial diagnosis: Work-up: History and exam; CT neck, chest, abdomen, pelvis, FDG-PET scan, Bone marrow; CBC/differential, metabolic panel, erythrocyte sedimentation rate, ferritin, bone marrow; excisional lymph node biopsy. Initial treatment: Risk-based (Table 94.3); Enroll on open clinical trial when available. For refractory or recurrent disease: Work-up: As per initial diagnosis. Treatment: Risk based (Table 94.4). If relapse is not low stage after low-density low-risk chemotherapy protocol without radiotherapy, consider stem cell transplant once response is evident (FDG-PET non-avid). For follow-up: Follow clinically with imaging studies for first year to18 months, then clinically follow for long-term toxicity using COG guidelines (www.survivorshipguidelines.org).

Useful Web-based References Children’s Oncology Group: http://www.childrensoncologygroup.org/index.php/hodgkindisease http://www.survivorshipguidelines.org National Cancer Institute PDQ: http://www.cancer.gov/cancertopics/ pdq/treatment/childhodgkins/HealthProfessional

selected References The full reference list for this chapter can be found in the online version. 10. DeVita VT Jr, Canellos GP, Moxley JHD. A decade of combination chemotherapy of advanced Hodgkin’s disease. Cancer 1972;30:1495–1504. 11. Santoro A, Bonadonna G, Valagussa P, et al. Long-term results of combined chemotherapy-radiotherapy approach in Hodgkin’s disease: superiority of ABVD plus radiotherapy versus MOPP plus radiotherapy. J Clin Oncol 1987;5:27–37. 12. Donaldson SS, Link MP. Combined modality treatment with low-dose radiation and MOPP chemotherapy for children with Hodgkin’s disease. J Clin Oncol 1987;5:742–749. 13. Percy CL, Smith MA, Linet M, et al. Lymphomas and reticuloendothelial neoplasms. Bethesda, MD: National Cancer Institute, SEER program, 1999. 20. Sleckman BG, Mauch PM, Ambinder RF, et al. Epstein-Barr virus in Hodgkin’s disease: correlation of risk factors and disease characteristics with molecular evidence of viral infection. Cancer Epidemiol Biomarkers Prev 1998;7:1117–1121. 21. Grufferman S, Delzell E. Epidemiology of Hodgkin’s disease. Epidemiol Rev 1984;6:76–106. 30. Karin M, Delhase M. The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatory signalling. Semin Immunol 2000;12:85–98. 34. Ravi R, Bedi GC, Engstrom LW, et al. Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB. Nat Cell Biol 2001;3:409–416. 35. Durkop H, Foss HD, Demel G, et al. Tumor necrosis factor receptor-associated factor 1 is overexpressed in Reed-Sternberg cells of Hodgkin’s disease and Epstein-Barr virus-transformed lymphoid cells. Blood 1999;93:617–623. 36. Wang CY, Mayo MW, Korneluk RG, et al. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281:1680–1683. 37. Mosialos G, Birkenbach M, Yalamanchili R, et al. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 1995;80:389–399. 42. Weiss LM, Movahed LA, Warnke RA, et al. Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin’s disease. N Engl J Med 1989;320:502–506. 48. Tzankov A, Zimpfer A, Pehrs AC, et al. Expression of B-cell markers in classical hodgkin lymphoma: a tissue microarray analysis of 330 cases. Mod Pathol 2003;16:1141–1147. 49. Anagnostopoulos I, Hansmann ML, Franssila K, et al. European Task Force on Lymphoma project on lymphocyte predominance Hodgkin disease: histologic and immunohistologic analysis of submitted cases reveals 2 types of Hodgkin disease with a nodular growth pattern and abundant lymphocytes. Blood 2000;96:1889–1899. 53. Lister TA, Crowther D, Sutcliffe SB, et al. Report of a committee convened to discuss the evaluation and staging of patients with Hodgkin’s disease: cotswolds meeting. J Clin Oncol 1989;7:1630–1636. 57. Friedberg JW, Fischman A, Neuberg D, et al. FDG-PET is superior to gallium scintigraphy in staging and more sensitive in the follow-up of patients

Hematologic Malignancies



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Part vii  Hematologic Malignancies  •  SECTION 4  Lymphoproliferative Disorders

with de novo Hodgkin lymphoma: a blinded comparison. Leuk Lymphoma 2004;45:85–92. 58. Hudson MM, Krasin MJ, Kaste SC. PET imaging in pediatric Hodgkin’s lymphoma. Pediatr Radiol 2004;34:190–198. 63. Nachman JB, Sposto R, Herzog P, et al. Randomized comparison of low-dose involved-field radiotherapy and no radiotherapy for children with Hodgkin’s disease who achieve a complete response to chemotherapy. J Clin Oncol 2002;20:3765–3771. 64. Ruhl U, Albrecht M, Dieckmann K, et al. Response-adapted radiotherapy in the treatment of pediatric Hodgkin’s disease: an interim report at 5 years of the German GPOH-HD 95 trial. Int J Radiat Oncol Biol Phys 2001;51: 1209–1218. 65. Krasin MJ, Rai SN, Kun LE, et al. Patterns of treatment failure in pediatric and young adult patients with Hodgkin’s disease: local disease control with combined-modality therapy. J Clin Oncol 2005;23:8406–8413. 66. Smith RS, Chen Q, Hudson MM, et al. Prognostic factors for children with Hodgkin’s disease treated with combined-modality therapy. J Clin Oncol 2003;21:2026–2033. 68. Landman-Parker J, Pacquement H, Leblanc T, et al. Localized childhood Hodgkin’s disease: response-adapted chemotherapy with etoposide, bleomycin, vinblastine, and prednisone before low-dose radiation therapy-results of the French Society of Pediatric Oncology Study MDH90. J Clin Oncol 2000;18:1500–1507. 69. Schwartz CL. Prognostic factors in pediatric Hodgkin disease. Curr Oncol Rep 2003;5:498–504. 71. Bohlen H, Kessler M, Sextro M, et al. Poor clinical outcome of patients with Hodgkin’s disease and elevated interleukin-10 serum levels. Clinical significance of interleukin-10 serum levels for Hodgkin’s disease. Ann Hematol 2000;79:110–113. 73. Chronowski GM, Wilder RB, Tucker SL, et al. An elevated serum beta2-microglobulin level is an adverse prognostic factor for overall survival in patients with early-stage Hodgkin disease. Cancer 2002;95:2534–2538. 74. Nadali G, Tavecchia L, Zanolin E, et al. Serum level of the soluble form of the CD30 molecule identifies patients with Hodgkin’s disease at high risk of unfavorable outcome. Blood 1998;91:3011–3016. 77. Dukers DF, Meijer CJ, ten Berge RL, et al. High numbers of active caspase 3-positive Reed-Sternberg cells in pretreatment biopsy specimens of patients with Hodgkin disease predict favorable clinical outcome. Blood 2002;100: 36–42. 78. Carde P, Koscielny S, Franklin J, et al. Early response to chemotherapy: a surrogate for final outcome of Hodgkin’s disease patients that should influence initial treatment length and intensity? Ann Oncol 2002;13(Suppl 1):86–91. 79. Schwartz C, Constine LS, Villaluna D, et al. Risk-adapted, response-based approach using ABVE-PC for children and adolescents with intermediate- and high-risk Hodgkin lymphoma: the results of P9425. Blood 2009;114:2051–2059. 80. Weiner MA, Leventhal BG, Marcus R, et al. Intensive chemotherapy and low-dose radiotherapy for the treatment of advanced-stage Hodgkin’s disease in pediatric patients: a pediatric oncology group study. J Clin Oncol 1991;9:1591–1598. 81. Donaldson SS. Pediatric Hodgkin’s disease—up, up, and beyond. Int J Radiat Oncol Biol Phys 2002;54:1–8. 82. Hudson MM. Pediatric Hodgkin’s therapy: time for a paradigm shift. J Clin Oncol 2002;20:3755–3757. 83. Schwartz CL. Special issues in pediatric Hodgkin’s disease. Eur J Haematol 2005;(Suppl):55–62. 84. Olson MR, Donaldson SS. Treatment of pediatric hodgkin lymphoma. Curr Treat Options Oncol 2008;9(1):81–94. 85. Tebbi CK, Mendenhall N, London WB, et al. Treatment of stage I, IIA, IIIA(1) pediatric Hodgkin disease with doxorubicin, bleomycin, vincristine and etoposide (DBVE) and radiation: a Pediatric Oncology Group (POG) study. Pediatr Blood Cancer 2006;46:198–202. 86. Tebbi CK, Mendenhall NP, London WB, et al. Response-dependent and reduced treatment in lower risk Hodgkin lymphoma in children and adolescents, results of P9426: a report from the children’s oncology group. Pediatr Blood Cancer 2012;59:1259–1265. 87. Tebbi CK, London WB, Friedman D, et al. Dexrazoxane-associated risk for acute myeloid leukemia/myelodysplastic syndrome and other secondary malignancies in pediatric Hodgkin’s disease. J Clin Oncol 2007;25:493–500. 88. Hutchinson RJ, Fryer CJ, Davis PC, et al. MOPP or radiation in addition to ABVD in the treatment of pathologically staged advanced Hodgkin’s disease in children: results of the children’s cancer group phase III trial. J Clin Oncol 1998;16:897–906. 89. Weiner MA, Leventhal B, Brecher ML, et al. Randomized study of intensive MOPP-ABVD with or without low-dose total-nodal radiation therapy in the treatment of stages IIB, IIIA2, IIIB, and IV Hodgkin’s disease in pediatric patients: a pediatric oncology group study. J Clin Oncol 1997;15:2769–2779. 90. Wolden SL, Chen L, Kelly KM, et al. Long-Term Results of CCG 5942: a randomized comparison of chemotherapy with and without radiotherapy for children with Hodgkin’s lymphoma—a report from the children’s oncology group. J Clin Oncol 2012;30:3174–3180. 91. Kelly KM, Sposto R, Hutchinson R et al. BEACOPP chemotherapy is a highly effective regimen in children and adolescents with high-risk Hodgkin lymphoma: a report from the Children’s Oncology Group. Blood 2011;117: 2596–2603. 92. Donaldson SS, Link MP, Weinstein HJ, et al. Final results of a prospective clinical trial with VAMP and low-dose involved-field radiation for children with low-risk Hodgkin’s disease. J Clin Oncol 2007;25:332–337. 93. Hudson MM, Krasin M, Link MP, et al. Risk-adapted, combined-modality therapy with VAMP/COP and response-based, involved-field radiation for unfavorable pediatric Hodgkin’s disease. J Clin Oncol 2004;22:4541–4550.

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94. Friedmann AM, Hudson MM, Weinstein HJ, et al. Treatment of unfavorable childhood Hodgkin’s disease with VEPA and low-dose, involved-field radiation. J Clin Oncol 2002;20:3088–3094. 95. Horning SJ, Hoppe RT, Breslin S, et al. Stanford V and radiotherapy for locally extensive and advanced Hodgkin’s disease: mature results of a prospective clinical trial. J Clin Oncol 2002;20:630–637. 96. Schellong G, Potter R, Bramswig J, et al. High cure rates and reduced longterm toxicity in pediatric Hodgkin’s disease: the German-Austrian multicenter trial DAL-HD-90. The German-Austrian pediatric hodgkin’s disease study group. J Clin Oncol 1999;17:3736–3744. 97. Dorffel W, Luders H, Ruhl U, et al. Preliminary results of the multicenter trial GPOH-HD 95 for the treatment of Hodgkin’s disease in children and adolescents: analysis and outlook. Klin Padiatr 2003;215:139–145. 98. Mauz-Korholz C, Hasenclever D, Dorffel W, et al. Procarbazine-free OEPACOPDAC chemotherapy in boys and standard OPPA-COPP in girls have comparable effectiveness in pediatric Hodgkin’s lymphoma: the GPOH-HD-2002 study. J Clin Oncol 2010;28:3680–3686. 99. Shankar A, Daw S. Nodular lymphocyte predominant Hodgkin lymphoma in children and adolescents—a comprehensive review of biology, clinical course and treatment options. British J Hematol 2012:1–11. doi:10.1111/bjh.12055.– 100. Sandoval C, Venkateswaran L, Billups C, et al. Lymphocyte-predominant Hodgkin disease in children. J Pediatr Hematol Oncol 2002;24:269–273. 101. Pellegrino B, Terrier-Lacombe MJ, Oberlin O, et al. Lymphocyte-predominant Hodgkin’s lymphoma in children: therapeutic abstention after initial lymph node resection—a study of the French Society of Pediatric Oncology. J Clin Oncol 2003;21:2948–2952. 102. Murphy SB, Morgan ER, Katzenstein HM, et al. Results of little or no treatment for lymphocyte-predominant Hodgkin disease in children and adolescents. J Pediatr Hematol Oncol 2003;25:684–687. 104. Shankar A, Hall GW, Gorde-Grosjean S, et al. Treatment outcome after low intensity chemotherapy [CVP] in children and adolescents with early stage nodular lymphocyte predominant Hodgkin’s lymphoma—an Anglo-French collaborative report. European J Cancer 2012;48:1700–1706. 105. Tenezakis SA, Hudson MM, Constine LS. Hodgkins’ disease, in Halperin EC, Constine LS, Tarbell NJ, et al., eds. Pediatric radiation oncology, 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins, 2010:137–165. 106. Li J, Dabaja B, Reed V, et al. Rationale for and preliminary results of proton beam therapy for mediastinal lymphoma. International J Radiat Oncol Biol Phys 2011;81:167–174. 110. Copeland A Younes A. Current treatment strategies in Hodgkin lymphomas. Current Opin Oncol 2012;24:466–474. 111. Daw S, Wynn R, Wallace H. Management of relapsed and refractory classical Hodgkin lymphoma in children and adolescents. Br J Haematol 2011;152:249–260. 112. Bonfante V, Viviani S, Devizzi L, et al. High-dose ifosfamide and vinorelbine as salvage therapy for relapsed or refractory Hodgkin’s disease. Eur J Haematol Supplementum 2001;64:51–55. 114. Cole PD, Schwartz CL, Drachtman RA, et al. Phase II study of weekly gemcitabine and vinorelbine for children with recurrent or refractory Hodgkin’s disease: a children’s oncology group report. J Clin Oncol 2009;27: 1456–1461. 115. Suyani E, Sucak GT, Aki SZ, et al. Gemcitabine and vinorelbine combination is effective in both as a salvage and mobilization regimen in relapsed or refractory Hodgkin lymphoma prior to ASCT. Ann Hematol 2011;90:685–691. 120. Colpo A, Hochberg E, Chen YB. Current status of autologous stem cell transplantation in relapsed and refractory Hodgkin’s lymphoma. Oncologist 2012;17:80–90. 121. Harris RE, Termuhlen AM, Smith LM, et al. Autologous peripheral blood stem cell transplantation in children with refractory or relapsed lymphoma: results of children’s oncology group study A5962. Biol Blood and Marrow Transpl 2011;17:249–258. 122. Smith SD, Moskowitz CH, Dean R, et al. Autologous stem cell transplant for early relapsed/refractory Hodgkin lymphoma: results from two transplant centres. British J Haematol 2011;153:358–363. 123. Lieskovsky YE, Donaldson SS, Torres MA, et al. High-dose therapy and autologous hematopoietic stem-cell transplantation for recurrent or refractory pediatric Hodgkin’s disease: results and prognostic indices. J Clin Oncol 2004;22:4532–4540. 124. Moskowitz CH, Yahalom J, Zelenetz AD, et al.High-dose chemo-radiotherapy for relapsed or refractory Hodgkin lymphoma and the significance of pretransplant functional imaging.British J Haematol 2010;148:890–897. 125. Josting A, Muller H, Borchmann P, et al. Dose intensity of chemotherapy in patients with relapsed Hodgkin’s lymphoma. J Clin Oncol 2010;28: 5074–5080. 127. Baker KS, Gordon BG, Gross TG, et al. Autologous hematopoietic stem-cell transplantation for relapsed or refractory Hodgkin’s disease in children and adolescents. J Clin Oncol 1999;17:825–831. 128. Gorde-Grosjean S, Oberlin O, Leblanc T, et al. Outcome of children and adolescents with recurrent/refractory classical Hodgkin lymphoma, a study from the Societe Francaise de Lutte contre le Cancer des Enfants et des Adolescents (SFCE). British J Haematol 2012;158:649–656. 129. Claviez A, Canals C, Dierickx D, et al. Allogeneic hematopoietic stem cell transplantation in children and adolescents with recurrent and refractory Hodgkin lymphoma: an analysis of the European group for blood and marrow transplantation. Blood 2009;114:2060–2067. 130 Sureda A, Canals C, Arranz R, et al. Allogeneic stem cell transplantation after reduced intensity conditioning in patients with relapsed or refractory Hodgkin’s lymphoma. Results of the HDR-ALLO study—a prospective clinical trial by the Grupo Espanol de Linfomas/Trasplante de Medula Osea (GEL/ TAMO) and the lymphoma working party of the European group for blood and marrow transplantation. Haematologica 2012;97:310–317.

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131. Holmberg L, Maloney DG. The role of autologous and allogeneic hematopoietic stem cell transplantation for Hodgkin lymphoma. J Natl Comp Canc Network 2011;9:1060–1071. 133. Chen R, Palmer JM, Popplewell L, et al. Reduced intensity allogeneic hematopoietic cell transplantation can induce durable remission in heavily pretreated relapsed Hodgkin lymphoma. Ann Hematol 2011;90:803–808. 134. Peggs KS, Hunter A, Chopra R, et al. Clinical evidence of a graft-versusHodgkin’s-lymphoma effect after reduced-intensity allogeneic transplantation. Lancet 2005;365:1934–1941. 135. Faulkner RD, Craddock C, Byrne JL, et al. BEAM-alemtuzumab reducedintensity allogeneic stem cell transplantation for lymphoproliferative diseases: GVHD, toxicity, and survival in 65 patients. Blood 2004;103:428–434. 136. Younes A. Novel treatment strategies for patients with relapsed classical Hodgkin lymphoma. Hematology 2009;2009:507–519. 137. Senter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nature Biotech 2012;30:631–637. 138. Younes A, Gopal AK, Smith SE, et al. Results of a pivotal phase II study of brentuximab vedotin for patients with relapsed or refractory Hodgkin’s lymphoma. J Clin Oncol 2012;30:2183–2189. 141. Gopal AK, Ramchandren R, O’Connor OA, et al. Safety and efficacy of brentuximab vedotin for Hodgkin lymphoma recurring after allogeneic stem cell transplantation. Blood 2012;120:560–568. 142. de Claro RA, McGinn KM, Kwitkowski VE, et al. U.S. Food and Drug Administration Approval Summary: brentuximab vedotin for the treatment of relapsed hodgkin lymphoma or relapsed systemic anaplastic large cell lymphoma. Clin Cancer Res 2012;16:5845–5849. 143. Friedman DL, Constine LS. Late effects of treatment for Hodgkin lymphoma. J Natl Comp Canc Network 2006;4:249–257. 144. Castellino SM, Geiger AM, Mertens AC, et al. Morbidity and mortality in longterm survivors of Hodgkin lymphoma: a report from the childhood cancer survivor study. Blood 2011;117:1806–1816. 145. Constine LS, Tarbell N, Hudson MM et al. Subsequent malignancies in children treated for Hodgkin’s disease: associations with gender and radiation dose. Intl J Radiat Oncol Biol Phys 2008;72:24–33. 146. O’Brien MM, Donaldson SS, Balise RR, Whittemore AS, Link MP, Second malignant neoplasms in survivors of pediatric Hodgkin’s lymphoma treated with low-dose radiation and chemotherapy. J Clin Oncol 2010;28:1232–1239. 147. Friedman DL, Whitton J, Leisenring W, et al. Subsequent neoplasms in 5-year survivors of childhood cancer: the childhood cancer survivor study. J Natl Canc Inst 2010;102:1083–1095. 148. Bhatia S, Yasui Y, Robison LL et al. High risk of subsequent neoplasms continues with extended follow-up of childhood Hodgkin’s disease: report from the late effects study group. J Clin Oncol 2003;21:4386–4394. 149. Inskip PD, Robison LL, Stovall M, et al. Radiation dose and breast cancer risk in the childhood cancer survivor study. J Clin Oncol 2009;27:3901–3907.

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2013

150. Sklar C, Whitton J, Mertens A, et al. Abnormalities of the thyroid in survivors of Hodgkin’s disease: data from the childhood cancer survivor study. J Clin Endocrinol and Metab 2000;85:3227–3232. 151. Kovalchik SA, Ronckers CM, Veiga LH, et al. Absolute risk prediction of second primary thyroid cancer among 5-year survivors of childhood cancer. J Clin Oncol 2013;31:119–127. 152. Veiga LH, Lubin JH, Anderson H, et al. A pooled analysis of thyroid cancer incidence following radiotherapy for childhood cancer. Radiat Res 2012;178:365–367. 153. Constine LS. Cured of Hodgkin lymphoma, but suffering a broken heart. Leuk Lymphoma 2008;49:1433–1435. 154. Schellong G, Riepenhausen M, Bruch C, et al. Late valvular and other cardiac diseases after different doses of mediastinal radiotherapy for Hodgkin disease in children and adolescents: report from the longitudinal GPOH followup project of the German-Austrian DAL-HD studies. Pediatr Blood Cancer 2010;55:1145–1152. 156. Sklar CA, Mertens AC, Mitby P, et al. Premature menopause in survivors of childhood cancer: a report from the childhood cancer survivor study. J Natl Canc Inst 2006;98:890–896. 157. Green DM, Kawashima T, Stovall M, et al. Fertility of male survivors of childhood cancer: a report from the childhood cancer survivor study. J Clin Oncol 2010;28:332–339. 158. Taylor A, Hawkins M, Griffiths A, et al. Long-term follow-up of survivors of childhood cancer in the UK. Pediatr Blood Cancer 2004;42:161–168. 159. Landier W, Bhatia S, Eshelman DA, et al. Development of risk-based guidelines for pediatric cancer survivors: the children’s oncology group long-term follow-up guidelines from the children’s oncology group late effects committee and nursing discipline. J Clin Oncol 2004;22:4979–4990. 160. Landier W, Wallace WH, Hudson MM. Long-term follow-up of pediatric cancer survivors: education, surveillance and screening. Pediatr Blood Cancer 2006;46:149–158. 161. Skinner R, Wallace WH, Levitt GA. Long-term follow-up of people who have survived cancer during childhood. Lancet Oncology 2008;7(6):489–498. 165. Landier W, Armenian SH, Lee J, et al. Yield of screening for long-term complications using the Children’s oncology group long-term follow-up guidelines. J Clin Oncol 2012;30:4401–4408. 166. Kenney LB, Bradeen H, Kadan-Lottick NS, Diller L, Homans A, Schwartz CL. The current status of follow-up services for childhood cancer survivors, are we meeting goals and expectations: a report from the consortium for New England childhood cancer survivors. Pediatr Blood Cancer 2011;56: 1062–1066. 167. Kremer LC, Mulder RL, Oeffinger KC, et al. A worldwide collaboration to harmonize guidelines for the long-term follow-up of childhood and young adult cancer survivors: a report from the international late effects of childhood cancer guideline harmonization group. Pediatr Blood Cancer 2012. doi:10.1002/pbc.24445.

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SECTION 5

P l a s m a C e l l D y sc r a si a s

C h a p te r 9 5

Practical Approach to Evaluation of Monoclonal Gammopathies Francis K. Buadi, Joseph R. Mikhael, William G. Morice II

The real incidence of monoclonal gammopathy is unknown; however, it increases with age, with most cases being identified in the 7th or 8th decade of life.1–3 The presence of a monoclonal protein is indicative of an underlying clonal plasma cell or B cell disorder. These disorders encompass a spectrum of disease entities ranging from clinically benign monoclonal gammopathy of undetermined significance (MGUS) to clinically significant diseases such as multiple myeloma, and also including Waldenström macroglobulinemia, chronic lymphocytic leukemia, and various neurologic and cutaneous diseases.4–12 Among 43,000 monoclonal gammopathies evaluated at Mayo Clinic from 1960 to 2010 most had MGUS (57.5%), multiple myeloma (18%), or primary (AL) amyloidosis (9%) (Fig. 95.1). However, 4% of these cases were associated with other conditions, such as POEMS syndrome, cryoglobulinemia, Castleman’s disease, and light chain deposition disease, which do require therapy and should not be missed during evaluation. A systematic approach to the patient with a monoclonal immunoglobulin (monoclonal protein) disorder is required, on the one hand to prevent unnecessary testing in the majority who will not need treatment for the underlying condition, and on the other hand to ensure that those with a clinically significant condition will be adequately diagnosed. This chapter addresses the initial approach to an individual with a monoclonal protein or suspected immunoglobulin disorder. We review the various conditions to consider in a patient with a monoclonal protein, so as to help guide the evaluation. The basic principles on the use of the various tests used, their

interpretation, and limitations are also reviewed. Subsequent chapters in this book will deal with the detailed evaluation of specific diseases associated with monoclonal gammopathy.

Classification Of Monoclonal Immunoglobulin Disorders A prior understanding of the various conditions associated with the production of monoclonal immunoglobulins (monoclonal protein) is essential in the evaluation of an individual with a monoclonal gammopathy. The clinical presentation may be due to the magnitude of the underlying tumor burden or the direct toxic effect of the monoclonal protein. However, a simple way of approaching the evaluation of these cases is to start with the type of monoclonal protein (Table 95.1). Although the various diseases can be broadly classified based on the type of monoclonal protein, there is overlap in the underlying conditions. For example, an IgM monoclonal protein is usually associated with lymphoproliferative diseases such as Waldenström macroglobulinemia and some lymphomas, but may also be associated with cases of IgM-associated multiple myeloma.13

Initial Evaluation The usual test that will detect a monoclonal protein is not part of the typical healthy adult physical evaluation.14 It is therefore usually detected during the evaluation of a clinical symptom or during further evaluation of an abnormal routine blood test. Abnormalities in routine clinical tests that suggest the possibility of a monoclonal protein disorder include rouleau formation in a peripheral blood smear, elevated total serum protein, proteinuria, anemia, renal dysfunction, or hypercalcemia. Clinical situations that may require evaluation for

TABLE 95.1

Classification Of Monoclonal Proteins Laboratory finding

FIGURE 95.1. Distribution of monoclonal gammopathies seen at Mayo Clinic between 1960 and 2010. MGUS, monoclonal gammopathy of undetermined significance; SMM, ­smoldering multiple myeloma.

Clinical implication

Non-IgM monoclonal proteins

IgM monoclonal proteins

Premalignant or undetermined

IgG, IgA, light chain, and other MGUS

IgM MGUS other lymphoproliferations

Intermediate Malignant

SMM Active MM Plasma cell leukemia

Smoldering macroglobulinemia Waldenström macroglobulinemia or other lymphoproliferative disorders

MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma; SMM, smoldering multiple myeloma.

2014

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2015

M-protein Immunofixation

Non-IgM

IgM

Bone marrow biopsy [type of clonal cell]

Bone marrow biopsy [type of clonal cell]

Symptomatic

Differential Diagnosis • CLL • LPL

Lymphocytes or Lymphoplasmacytic

Plasma cells

Amyloidosis • • • • •

Restrictive Cardiomyopathy Non-selective proteinuria Peripheral neuropathy Autonomic dysf unction Hepatomegaly

POEMS • • • •

Multiple Myeloma C-Hypercalcemia R -Renal failure A -Anemia B -Bone disease

Polyneuropathy

Asymptomatic

Symptomatic

MGUS M-spike < 3g/dl BM Clonal cell < 10% SMM/SWM M-spike > 3g/dl BM Clonal cell > 10%

Organomegaly

Waldenström Macrogobulinemia • Anemia • Thrombocytopenia • Bulky lymphadenopathy

• Splenomegaly • Hyperviscosity syndrome

• Bleeding (epistaxis)

Endocrinopathy

Differential Diagnosis

M-protein (Almost always Lambda light chain restricted)

• CLL • Other B-cell

lymphoproliferative disorders

• Skin lesions

LCDD MGUS-neuropathies Solitary plasmacytoma

a monoclonal protein include back pain, osteoporosis disproportionate to age, pathologic fracture, osteolytic or sclerotic bone lesions, recurrent sinopulmonary infections, progressive peripheral neuropathy, infiltrative or restrictive cardiomyopathy, and Raynaud’s phenomenon. It must, however, be noted that in these conditions a monoclonal protein may not always be detected by standard testing, and further evaluation may be needed to confirm or exclude an underlying plasma cell or lymphoproliferative disorder, particularly if the index of suspicion is high. Most patients with monoclonal protein are asymptomatic, especially those in whom a routine blood abnormality leads to further testing, resulting in the identification of a monoclonal protein. A simple algorithm taking into consideration the various tests available and clinical presentation is shown in Figure 95.2. Although there may be some overlap, following this algorithm should lead to the correct diagnosis in most cases.

FIGURE 95.2. Simple diagnostic algorithm for patients with a monoclonal gammopathy.  CLL, chronic lymphocytic leukemia; MGUS, monoclonal gammopathy of undetermined significance; SMM, smoldering multiple myeloma; SWM, Smoldering Waldenström macroglobulinemia; LCDD, light chain deposition disease; LDL, Lymphoplasmacytic lymphoma.

a spike in the gamma region (Fig. 95.4). This is referred to as the M-protein or M-spike with the “M” referring to monoclonal, not IgM. Further testing is then required to determine the type and quantity. All spikes should be evaluated, since in certain cases there may be two (biclonal gammopathy), or more, different monoclonal proteins in a single M-spike that should not be missed.20–23 It must also be stressed that the gamma region contains all immunoglobulin isotypes (IgM, IgA, IgD, and IgE) and not only IgG. This same test can be used to determine the amount of monoclonal protein present using the densitometer tracing or peak size by capillary zone electrophoresis (Fig. 95.4). The measured M-protein by densitometry tracing is usually lower than the total involved immunoglobulin measured by nephelometry.16 Protein electrophoresis occasionally

Normal Serum

Laboratory Evaluation The following are important tests that will help in the evaluation of a patient with or suspected to have a monoclonal protein.

Albumin = 38.6 g/L

α1 = 2.6 g/L α2 = 8.8 g/L β = 9.6 g/L γ = 11.3 g/L

Protein Electrophoresis Serum and urine should both be evaluated for the presence of a monoclonal protein. High-resolution agarose gel electrophoresis or capillary zone electrophoresis is the preferred method for screening for a monoclonal protein.15–19 These tests will separate serum or urine proteins into their various components in an electric field based primarily on their physical properties such as size and charge; the proteins are detected either by staining a solid matrix or by their electrical impedance as they exit the column. There are usually five components seen, albumin, a-1, and a-2, b-, and g-globulin (Fig. 95.3). Monoclonal proteins will usually migrate into the gamma regions; however, occasionally they may be seen in the b or a-2 region. Samples with a monoclonal protein usually will result in

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Hematologic Malignancies

Lymphocytes or Lymphoplasmacytic

TP = 71.0 g/L

PEL Alb α1

α2 β

γ

FIGURE 95.3. Images of a normal serum electrophoresis, showing the five protein components.

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Part vii  Hematologic Malignancies  •  SECTION 5  Plasma Cell Dyscrasias

Polyclonal Hypergammaglobulin

Monoclonal Gammopathy Serum

gamma fraction = 34 g/L

M-Spike = 7.2 g/L

PEL

PEL Alb α1

α2

β

Alb α1

γ

α2 β

γ PEL

PEL G

G A

A IFE

IFE M

M K

K L

L FIGURE 95.4. Images of a serum protein electrophoresis and immunofixation depicting a monoclonal protein.

may fail to identify the presence of a monoclonal protein, especially in cases where there is only minimal production of the monoclonal protein, or minimal amounts of immunoglobulin free light chains.24 Currently in such cases performing immunofixation despite a negative protein electrophoresis or using the serum free light chain analysis may be the only way to confirm the presence of an underlying clonal disorder, although tandem mass spectroscopy methods are currently being explored.21,25,26 A monoclonal protein spike should not be confused with a polyclonal increase in immunoglobulin, which usually will be seen as a broad-based band in the gamma region and is not associated with a clonal cell disorder27 (Fig. 95.5).

Identification Of The Type And Quantitation Of The Monoclonal Protein The heavy and light chain isotypes of a monoclonal protein is usually determined by immunoelectrophoresis or

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FIGURE 95.5. Images of a serum protein electrophoresis depicting a polyclonal gammopathy.

­immunofixation.15,16,21 Immunoelectrophoresis, although less expensive, is infrequently used, as it is relatively insensitive. Immunofixation involves the application of anti-heavy and antilight chain antibodies to the electrophoretic gel and has higher detection sensitivity. Immunofixation will identify both the heavy chain isotype (IgG, IgM, IgA, IgD, or IgE) and the light chain type (k or l) and should always be performed on all cases of protein electrophoresis with an M-spike. A case with an IgG heavy chain isotype and a k-light chain will be reported as an IgG-kmonoclonal protein (Fig. 95.6). Most laboratories will initially only perform immunofixation for IgG, IgA, and IgM. Immunofixation for IgD and IgE must be performed if an M-spike is present and either initial immunofixation studies are negative or detect only a monoclonal light chain (Fig. 95.7). The isotype and quantity of the monoclonal protein is important for classification and prognosis. For example in MGUS, the risk of progression is lower in patients with IgG isotype and an M-spike less than 0.5 g/dl. The type of protein and quantity is also important for monitoring patients during therapy. Immunofixation should still be performed in cases where there is a strong suspicion of the presence of a clonal plasma cell or

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Chapter 95  Practical Approach to Evaluation of Monoclonal Gammopathies

Large M-spike

2017

IgD Myeloma

M–spike = 44.9 g/L

PEL

M-spike = 8.9 g/L

PEL Alb α1

α2 β

γ

Alb α1

α2 β

γ

PEL PEL G Hematologic Malignancies

G A

IFE

A M

IFE M

K IgG = 64.9 g/L IgA = 0.7 g/L IgM = 0.7 g/L

K L

FIGURE 95.6. Protein electrophoresis and immunofixation studies characterize a monoclonal IgG-k protein.  The arrows indicate the lanes that show reactivity with the specific antibodies. The reactivity is consistent with the same migration shown in the extreme left column that shows the electrophoresis. Thus, in this assay the nature of the monoclonal protein seen in the electrophoresis is elucidated: isotype G and light chain-k.

lymphoproliferative disorder, even if the protein electrophoresis is negative for an M-spike. Capillary zone electrophoresis with immunosubtraction is an automated system that is slightly more sensitive than high-resolution agarose gel electrophoresis and can be used in the identification, subtyping, and measurement of the M-protein.17,19 This method is laborious and time consuming, however, making it less frequently used for typing of the monoclonal protein.

L

PEL

D

E

IFE

Quantitation Of Immunoglobulins

K

Immunoglobulin (IgG, IgA, IgM) levels should always be measured at diagnosis and monitored regularly during therapy, especially the involved immunoglobulin. If immunofixation showed an IgD or IgE M-protein, then their levels should also be determined. In most clonal plasma cell disorders the levels of the uninvolved immunoglobulins are suppressed or reduced. Rate nephelometry is a reliable and rapid method for immunoglobulin quantification, although this method does not differentiate between monoclonal and polyclonal immunoglobulin.28,29 Rate nephelometry also does not provide information on how much of the measure immunoglobulin is abnormal; therefore, in patients with normal total immunoglobulin it is not clinically helpful. This test does have

L

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FIGURE 95.7. Images of a positive protein electrophoresis but initial negative immunofixation for IgG, IgA, or IgM isotype with further testing for IgD and IgE showing an IgD isotype with l-light chain.

value in clinical care however, as knowing the level in severely hypogammaglobulinemic states will help determine who should receive immunoglobulin replacement, especially those with recurrent sinopulmonary infections.

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Part vii  Hematologic Malignancies  •  SECTION 5  Plasma Cell Dyscrasias

Free Light Chain Assay A significant population of patients with clonal plasma disorders produces excess amounts of light chain but not a full immunoglobulin molecule: Bence Jones proteinemia.30 Most of these patients will have a negative protein electrophoresis and immunofixation. In these patients the free light chain assay test may be the only way to detect the presence of a clonal plasma cell disorder.24–26 Immunoglobulin free kappa (k) and lambda (l) light chains concentration in the serum is usually dependent on the rate of production from plasma cells and renal clearance. This results in a defined serum concentration and ratio. In clonal plasma cell disorders there is an excess production of only one of the light chain types, resulting in higher levels, with suppression of the uninvolved light chain, leading to an abnormal k/l ratio. The levels and ratio, however, may be affected by renal failure, since the light chains are cleared by the kidneys. This test has become very important in the evaluation and monitoring of patients with amyloidosis, since this is the major protein involved in AL-type amyloid deposits.31

Urine Evaluation In all patients with a serum M-protein or suspected to have a clonal plasma cell or lymphoplasmacytic disorder, a 24-hour urine collection should be examined for the presence of a monoclonal protein and 24-hour urine protein and monoclonal protein excretion. The excretion of immunoglobulin free light chain in the urine is referred to as Bence Jones proteinuria.30 The pattern of protein excretion, whether the urine protein is solely albumin or Bence Jones protein, is of diagnostic importance. For example, nonselective proteinuria (albumin predominance) is associated with glomerular diseases such as AL amyloidosis caused by the presence of a monoclonal protein.12,31

Bone Marrow and Tissue Evaluation Bone marrow aspiration and biopsy should be performed in all patients except in a selected group of completely asymptomatic patients with a very small monoclonal protein if a diagnosis of MGUS is favored.32–34 Even in the latter setting, however, most hematologists will recommend this test, since apart from giving information on the type of clonal cell disorder, it also provides information on the extent of disease. For example, the presence of less than 10% marrow plasma cells distinguishes MGUS from smoldering multiple myeloma (SMM).10 Basic evaluation of the bone marrow sample should include extent of infiltration by the cells of interest, reported as a percentage of the total marrow nucleated cells and/ or cellularity. Flow cytometric or immunophenotyping can confirm both the cell type (lymphoid vs. plasma cell) and clonality 35,36 (Fig. 95.8). Flow cytometry is particularly useful when following patients on therapy, as it is more sensitive in assessing the depth of response. Plasma cell DNA content can also be measured by flow cytometry, allowing measurement of ploidy status and proliferation rate, which, along with the proportion of normal plasma cells provides important prognostic information. 37 Plasma cell proliferation can also be measured by the labeling index (PCLI) which uses a BrdU pulse label and immunofluorescent detection. 38 However, it is not generally accessible and is not necessary in the majority of patients. Metaphase cytogenetics and fluorescent in situ hybridization for specific gene targets should be obtained in a subset of cases, since these provide prognostic information in certain clonal cell disorders. For example, in multiple myeloma, cytogenetic abnormalities provide important prognostic information, and therefore this must be obtained in all suspected cases at the time of initial evaluation.39–42 Gene expression profile analysis of the malignant plasma cells in multiple myeloma is of prognostic value and should be obtained if available.43–45

FIGURE 95.8. Identification of abnormal and normal plasma cells (PCs) by flow cytometry. All plasma cells are CD38 and CD138 positive.  The ­abnormal plasma cells are CD19 and CD45 negative and l-immunoglobulin light chain restricted.

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Chapter 95  Practical Approach to Evaluation of Monoclonal Gammopathies

Congo Red Stain In certain cases, a Congo red stain of subcutaneous fat aspirate or a bone marrow biopsy will have to be performed, looking for amyloid deposition.12,46,47 In about 70% of cases of AL amyloidosis Congo red staining of bone marrow or subcutaneous fat aspirate will be sufficient to make the diagnosis. The remaining cases will require biopsy of the involved organ. All patients with peripheral neuropathy, significant albuminuria, or infiltrative cardiomyopathy in the setting of a monoclonal protein should have this test done. It should also be considered in patients in whom there is a high clinical suspicion of amyloidosis. If this is positive, then liquid chromatography tandem mass spectometry of peptide extracts from the congophilic material should then be done for subtyping of the amyloid deposits.48

Imaging The imaging required for the evaluation of a patient with monoclonal protein depends on the clinical syndrome and type of monoclonal protein. A metastatic skeletal survey should be performed in all patients with non-IgM monoclonal protein. In multiple myeloma this may show lytic bone lesions (Fig. 95.9). One should also look for vertebrae compression fractures, and osteoporosis or osteopenia. These radiologic studies should also be performed in IgM monoclonal gammopathies if there is a strong clinical suspicion of IgM myeloma. For all cases of

2019

suspect multiple myeloma, a skeletal survey is still considered the standard test for bone evaluation.49–51 More sensitive modalities such as magnetic resonance imaging or PET-CT may provide further information.52–55 Magnetic resonance imaging is particularly useful for the assessment of the extent and nature of soft tissue disease arising from bone lesions, especially those in the spine which may cause neurologic compromise. PET-CT is usually helpful in the evaluation of plasmacytomas and cases of nonsecretory multiple myeloma56–58 (Fig. 95.10). In our experience, this modality has been helpful in the evaluation of patients with POEMS syndrome, by helping identify hypermetabolic osteosclerotic bone lesions. Computer tomography (CT) scanning of the chest, abdomen, and pelvis should be performed in cases of IgM gammopathy, since they are usually associated with lymphoproliferative disorders which may involve the lymph nodes, spleen, and mucosaassociated lymphoid tissues.

History and Physical Evaluation A detailed history and physical examination should be obtained any time a monoclonal protein is identified. Certain signs and symptoms may help guide the evaluation and ultimately indicate the appropriate diagnosis. For instance, patients presenting with bone pain, pathologic fractures, weight loss, and symptoms of hypercalcemia or acute renal failure are more likely to have multiple myeloma.9 Alternatively, those with night sweats, epistaxis,

Hematologic Malignancies



FIGURE 95.9. Skeletal bone survey showing lytic lesions typically seen in multiple myeloma.

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2020

Part vii  Hematologic Malignancies  •  SECTION 5  Plasma Cell Dyscrasias type of monoclonal protein and underlying clonal cell disorder has been established, the physician then needs to look into the patients’ history, physical examination, and laboratory data to determine whether this clonal disorder is having any systematic effect. The typical issues are usually with the hematopoietic (anemia, thrombocytopenia, bulky lymphadenopathy), skeletal (lytic lesion, pathologic fracture, and sclerotic lesions), renal (cast nephropathy, immunoglobulin deposition disease, and nephrotic range proteinuria), nervous system (peripheral neuropathy, autonomic dysfunction), cardiovascular (restrictive cardiomyopathy), and/or the endocrine systems. There may be overlap of symptoms, but understanding the various syndromes is important in helping to arrive at the correct diagnosis.

FIGURE 95.10. PET-CT scan showing multiple soft tissue plasmacytomas in a patient with negative marrow biopsy.

and lymphadenopathy are more likely to have lymphoproliferative disorders such as Waldenström macroglobulinemia, marginal zone lymphoma, or chronic lymphocytic leukemia/small lymphocytic lymphoma.8,35,59–61 Other less common conditions may also be revealed by patient assessment. A diagnosis of amyloidosis may be indicated by the presence of edema, shortness of breath, hepatomegaly, peripheral neuropathy (e.g., carpal tunnel syndrome), autonomic dysfunction, periorbital purpura, and macroglossia.12,31,47 Skin rash, neuropathy, and Raynaud’s phenomenon should prompt evaluation for cryoglobulinemia.5,62 Those presenting with polyneuropathy, multiple endocrinopathy, skin lesions like glomeruloid hemangiomata, and organomegaly may have POEMS syndrome.11 Importantly, one should never overlook the initial reason that led to the identification of the monoclonal protein. Most patients in whom a monoclonal protein is identified are asymptomatic. For these patients, the key to a correct diagnosis may be the reason for the initial testing for the monoclonal protein. In a case where a mildly elevated serum creatinine was the key factor, a nephrology evaluation with possible kidney biopsy may be the only way to diagnose immunoglobulin deposition disease of the kidney.6,63,64 Monoclonal gammopathies are common in the elderly, who are also at risk for other pathologic conditions and are usually on multiple medications with their side effects. Therefore it is necessary to always confirm that the identified abnormality is due to the clonal cell disorder or monoclonal protein rather than to another unrelated pathologic condition. For example, mild hypercalcemia in association with a monoclonal protein may be due to parathyroid disease or medication in a patient with benign MGUS rather than myeloma. Renal failure or proteinuria in a hypertensive or diabetic patient with monoclonal protein should be investigated thoroughly before attributing it to the monoclonal protein. Such cases may require a kidney biopsy.

Diagnostic Algorithm (Fig. 95.2) In a patient suspected of having an underling gammopathy, one should first define the isotype and then quantity of the monoclonal protein. This will certainly help in identifying and defining the underlying clonal cell disorder. In the majority of cases, appropriate tissue biopsy and histopathologic evaluation should aid in providing a definitive diagnosis. Bone marrow biopsy is usually requisite. In certain situations, directed biopsy of a single bone lesion, lymph node, or soft tissue mass may also be needed. Tissue diagnosis can also be helpful in deciding therapeutic intervention; for example, the use of rituximab for the treatment of an IgM MGUS associated neuropathy can be indicated by finding an underlying CD20-positive lymphoproliferative disorder. Once the

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Differential Diagnosis of Monoclonal Gammopathy (Table 95.2) The majority of non-IgM monoclonal gammopathies are initially classified as MGUS.10 In most MGUS patients the M-protein is a complete immunoglobulin molecule; however, some may have only light chain and therefore are classified as light chain MGUS.2 MGUS patients do not have any symptoms or laboratory abnormalities attributable to the underlying clonal plasma cell disorder; specifically, they do not have anemia, hypercalcemia, renal insufficiency, or disease-associated bone disease. They should have less than 3 g/dl of monoclonal protein and less than 10% bone marrow plasma cell. Patients with similarly minimal clinical sequelae and an M-protein ≥ 3gm/dl or ≥ 10% clonal bone marrow plasma

TA BL E 9 5 . 2

Differential Dignosis Of Monoclonal Gammopathies IgM type IgM MGUS (may also be biclonal) Smoldering Waldenström macroglobulinemia Waldenström macroglobulinemia Other (including lymphoma and IgM MM) Non-IgM type Non-IgM MGUS (may also be biclonal) SMM MM Plasma cell leukemia Solitary plasmacytoma Amyloidosis complicating a B cell neoplasm (AL) Miscellaneous monoclonal gammopathy–associated conditions Osteosclerotic MM with peripheral neuropathy POEMS syndrome Cryoglobulinemia Peripheral neuropathy associated with MGUS SLONM Fanconi’s syndrome Light or heavy chain deposition disease Castleman’s disease Scleromyxedema Necrobiotic xanthogranuloma Systemic capillary leak syndrome Angioimmunoblastic lymphadenopathy with monoclonal protein Other MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma; SLONM, sporadic late onset nemaline myopathy SMM, smoldering multiple myeloma.

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Chapter 95  Practical Approach to Evaluation of Monoclonal Gammopathies

cell are classified as having SMM.65 Distinguishing between MGUS and SMM has prognostic significance and will determine frequency of follow-up. As bone marrow examination may be the only factor that will upgrade an MGUS to SMM, this must be strongly considered in the evaluation of all monoclonal gammopathies. Neither MGUS nor SMM require therapeutic intervention; patients with MGUS have a lower risk of progression to MM at about 1% per year, compared to 10% to 20% in patients with SMM. Closer follow-up is needed for SMM compared to MGUS.10 Patients with myeloma should meet the CRAB criteria, defined as hypercalcemia, renal insufficiency, anemia, or bone disease.66 It is necessary to confirm that these complications are due to the underlying clonal plasma cell disorder. These patients do require therapy and should be under the care of a specialist with detailed knowledge of the management of multiple myeloma and associated complications. Solitary plasmacytomas should also be recognized, since these require only limited therapy.56 These patients have a single bone or soft tissue mass, a negative bone marrow biopsy, and do not meet the CRAB criteria. Local therapy such as radiation, cryotherapy, or surgical excision is usually all that is required.67 They do have a better prognosis. A significant number of patients with monoclonal gammopathy develop complications as a result of the toxic effect of the monoclonal protein. In amyloidosis, misfolding of the monoclonal protein, usually the light chain component, results in the formation of insoluble amyloid deposits in major organs of the body.12,31,47 This deposition results in structural and physiologic dysfunction of the affected organ. An amyloid diagnosis is easy to make when presenting with macroglossia in association with periorbital purpura.68 Most, however, will present with symptoms secondary to the organ involvement, such as chest pain and shortness of breath due to restrictive cardiomyopathy in cardiac cases, and peripheral neuropathy and autonomic dysfunction in nervous system disease. Nonselective proteinuria with hypoalbuminemia is seen in kidney involvement. Hepatomegaly with elevated alkaline phosphates will be seen in those with liver disease, and those with gastrointestinal involvement will have constipation or diarrhea. Patients may present with multiple organ involvement, causing a protean constellation of findings; in such cases a strong suspicion of amyloidosis is required in order not to miss the diagnosis. Nonamyloidotic immunoglobulin deposition diseases such as light chain deposition diseases are a group of conditions that should be considered during the evaluation of monoclonal gammopathy.69 This involves the deposition of light chains in the kidney or heart. Renal involvement presenting with renal failure with some proteinuria is the most common.64 Heart involvement, although not common, does occur, presenting with restrictive cardiomyopathy, and should be differentiated from amyloidosis.70 IgM monoclonal gammopathy is usually associated with an underlying B cell lymphoproliferative disorder, and true IgM myeloma is exceedingly rare.13 A large percentage of these IgM monoclonal gammopathy patients will have an IgM MGUS or Waldenström macroglobulinemia.59,71 The remainder often has a variety of other B cell lymphoproliferative diseases such as chronic

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lymphocytic leukemia, marginal zone lymphoma, and large cell lymphoma.8 Those with Waldenström macroglobulinemia may present with cytopenias, hyperviscosity syndrome, epistaxis, lymphadenopathy, and splenomegaly.35,61,72 There are other rare monoclonal gammopathy associated conditions that should be considered during the evaluation of a monoclonal protein. The evaluation in these cases is usually dictated by the clinical presentation; for example, those with neuromuscular complications should be evaluated for conditions such as POEMS syndrome, the antimyelin associated glycoprotein–associated neuropathy, or sporadic late onset nemaline myopathy.11,73,74 POEMS patients, in addition to the neuropathy, do have organomegaly (lymphadenopathy, splenomegaly, cardiomegaly, and hepatomegaly), multiple endocrine dysfunction, and skin changes (glomeruloid hemangiomata, hyperpigmentation). Almost all POEMS patients do have a l-light chain restricted monoclonal protein, and it may be a non-IgM or IgM monoclonal protein. If a patient has a k-light chain monoclonal protein, they most probably do not have POEMS syndrome. Skin conditions such as scleromyxedema, necrobiotic xanthogranuloma, and cryoglobulinemia should be considered in the differential diagnosis when patients present with cutaneous lesions.7,62,75–78

Summary and Recommendations The diagnostic approach to monoclonal gammopathy should be thorough and involve a detailed history, physical examination, appropriate laboratory testing, and imaging. This should distinguish benign from clinically relevant conditions that need immediate intervention. A good understanding of conditions associated with monoclonal gammopathy is essential. Testing and evaluation should include the following considerations.

Hematologic Malignancies



• Serum and urine protein electrophoresis, followed by immunofixation will confirm the presence of the monoclonal protein, identify and classify the isotype, and provide an estimation of the amount. Immunoglobulin free light assay will help identify cases with only free light chain production, but negative protein electrophoresis. • Immunoglobulin isotype quantification is helpful for monitoring. • A bone marrow evaluation will determine the nature of the underlying B-lineage disorder (plasma cell or lymphoproliferative) and also the extent of marrow infiltration. • Complete blood count analysis will identify cytopenias which may be due to bone marrow replacement or immune mediated causes. • Comprehensive metabolic panel, looking at serum calcium, creatinine, bilirubin, lactate dehydrogenase, and liver transaminases is essential. • Bone and soft tissue imaging, looking for bony lesion and soft tissue masses such as lymphadenopathy or plasmacytoma is also needed. • A detailed history and physical evaluation should help direct further testing.

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Chapter 96

Molecular Genetic Aspects of Plasma Cell Disorders P. Leif Bergsagel, A. Keith Stewart, Stephen J. Russell, Rafael Fonseca

Introduction Multiple myeloma (MM) is an age-dependent monoclonal tumor of bone marrow (BM) plasma cells (PCs), often with significant end organ damage that can include lytic bone lesions, anemia, loss of kidney function, immunodeficiency, and amyloid deposits in various tissues.1 It has an estimated incidence of 21,700 in 2012, with 10,710 deaths in the United States.2 Despite recent therapeutic advances, MM continues to be a mostly incurable disease but with a 5-year survival rate reported in the SEER database that has increased from 28% (1987 to 1989) to 43% (2002 to 2008).3 In fact, a subset of younger patients initially treated in 1999 can be identified with a 10-year survival rate of 75%,4 with presumably even better results possible for patients starting treatment today. The incidence is higher in blacks than whites, and in men than women (Fig. 96.1),3 and it is evident from the SEER registry data that although the incidence of MM continues to rise with an annual percentage change of close to 1%, since 1995 the mortality rate has been decreasing at an even faster rate. MM cells are similar to postgerminal center (GC) long-lived PCs, characterized by strong BM dependence, extensive somatic hypermutation (SHM) of immunoglobulin (Ig) genes, and absence of IgM expression in all but 1% of tumors.5 However, MM cells differ from healthy PCs because they retain the potential for a low rate of proliferation (1% to 3% of cycling cells). MM has served as a useful model for understanding the pathogenesis of lymphoid tumors because it is characterized by

20

White Male

Black Male

the presence of a premalignant precursor tumor and defined stages, with researchers able to isolate purified tumor cell populations at all stages.

Multiple Myeloma Is A Plasma Cell Tumor Of Postgerminal Center B Cells Pre-GC B cells can generate short-lived PC that mostly remain in the primary lymphoid tissue (Fig. 96.2). Post-GC B cells can generate plasmablasts (PB) that have successfully completed multiple rounds of SHM and antigen selection, followed by IgH switch recombination, with both B cell–specific DNA modification processes having oncogenic potential.6 These PB typically migrate to the BM, where stromal cells facilitate terminal differentiation into long-lived PC.7 The surface immunophenotype of normal BM PCs is CD38+CD138+ CD19+CD45+CD56−. Although monoclonal gammopathy of undetermined significance (MGUS), SMM, and MM tumor cells also are CD38+CD138+, 90% are CD19−, 99% are CD45− or dim, and 70% are CD56+.8,9 The reason for this immunophenotypic difference is not known. Possibly there is a normal PC with an MM phenotype, but it is rare, and/or transient, and it has yet to be identified. Alternatively, the change in the phenotype may be a consequence of the transformation process.

White Female

Black Female

20

15

15

0.9 10

10

0.8 –1.4 0.5

5

–1.8

5

Incidence rate per 100,000

Mortality rate per 100,000

0.6

–0.9 –2.8 0 1975 1985 1995

2009 1985 1995

2009 1985 1995

2009 1985 1995

0 2009

Year of Diagnosis/Death FIGURE 96.1. Differences in US incidence and mortality rates over time, by race and gender.  Source is the SEER 9 areas and US mortality files. The most recent annual percentage change for each rate is indicated adjacent to the respective regression line.

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Chapter 96  Molecular Genetic Aspects of Plasma Cell Disorders

2023

BONE MARROW

GERMINAL CENTER

CD138

PB IgG IgH switch recombination IgD

PC

PB IgA

IgM

B t(4;14)

PB IgA

Ig somatic hypermutation t(11;14)

PB IgG

MM

CD45 CD19

CD138 CD56

Multiple Myeloma Is Almost Always Preceded By An Asymptomatic Monoclonal Gammopathy An age-dependent premalignant tumor called monoclonal gammopathy of undetermined significance (MGUS)10,11 is present in about 4% of individuals over the age of 5012,13 and precedes almost every case of MM. A PC tumor must contain at least a billion cells to produce enough monoclonal Ig (M-Ig) or M-IgL (in the 15% of tumors that produce only IgL) to be detected by serum or urine electrophoresis or a serum–free IgL assay. It can be subclassified as lymphoid (15%) or PC (85%) MGUS, which can progress sporadically at average rates of 1% per year to chronic lymphocytic leukemia/lymphoma/ lymphoplasmacytoma/Waldenstrom’s macroglobulinemia and MM, respectively.14 Lymphoid MGUS and PC MGUS can be distinguished by morphology, although clinically the distinction is primarily based on the type of M-Ig detected in serum or urine: mostly IgM for lymphoid MGUS and mostly non-IgM (including Ig light chain only) for PC MGUS. MGUS is distinguished from MM by having a M-Ig of 90% in HMCL.25,26 Limited studies indicate that IgL translocations are present in about 10% of MGUS/SMM tumors, and about 15% to 20% of intramedullary MM tumors and HMCL.18 Translocations involving an IgK locus are rare, occurring in only 1% to 2% of MM tumors and HMCL.18 There are three recurrent primary IgH translocation groups, with the chromosomal sites, target oncogenes, and approximate prevalence in MM (∼40% prevalence for all three groups) as follows: CYCLIN D (11q13-CYCLIN D1-15%; 12p13-CYCLIN D2- < 1%; 6p25-CYCLIN D3-2%) MAF (16q23-MAF-5%; 20q12MAFB-2%; 8q24.3-MAFA-< 1%; MMSET/(FGFR3)-4p16-(MMSET in all but also FGFR3 in 80% of these tumors)-15%.

Chromosome Content Is Associated with at Least Two Different Oncogenic Pathways There is a consensus that chromosome content reflects at least two pathways of pathogenesis. Nearly half of MGUS and MM tumors are hyperdiploid (HRD), with 48 to 75 (mostly 49 to 56) chromosomes, usually with extra copies of three or more specific chromosomes (3,5,7,9,11,15,19,21). Nonhyperdiploid (NHRD) tumors have 75 chromosomes. Strikingly, HRD tumors rarely (∼10%) have a primary IgH translocation, whereas NHRD tumors usually (∼70%) have an IgH translocation.27 Tumors with a t(11;14) may represent a distinct category of NHRD tumors as they often are diploid or pseudodiploid. Curiously, EMM tumors and HMCLs nearly always have a NHRD genotype, suggesting that HRD tumors are more stromal cell dependent than NHRD tumors. Although it has been proposed that NHRD and HRD tumors represent different pathways of pathogenesis, the timing, mechanism, and molecular consequences of hyperdiploidy is unknown. Interestingly, in patients with t(4;14) or t(14;16) or t(14;20) or del17p, the presence of one or more trisomies is associated with a substantially better prognosis than with the absence of trisomies. This suggests that the phenotype associated with trisomies may be dominant.28

Universal Cyclin D Dysregulation Almost all cases of PC neoplasms starting from the MGUS stage express 1 or more of the CYCLIN D genes in an aberrant fashion, despite a low proliferation index.29 Therefore, it has been proposed that dysregulation of a CYCLIN D gene provides a unifying, early oncogenic event in MGUS and MM. MGUS and MM appear closer to normal, nonproliferating PCs than to normal proliferating PB, for which 30% or more of the cells can be in S phase; yet the expression level of CYCLIN D1, CYCLIN D2, or CYCLIN D3 mRNA in MM and MGUS is distinctly higher than in normal PCs. This can be due to direct dysregulation in MM tumors with a CYCLIN D gene translocation or indirectly in tumors with a translocation of MAF, encoding a transcription factor that markedly upregulates CYCLIN D2. Although MMSET/FGFR3 tumors express moderately high levels of CYCLIN D2, the cause of increased CYCLIN D2 expression remains unknown. While normal BM PC express little or no detectable CYCLIN D1, the majority of HRD tumors express CYCLIN D1 biallelically, whereas most other tumors express increased levels of CYCLIN D2 compared to normal BM PC, both by unknown mechanisms. Only a few percent of MM tumors do not express increased levels of a CYCLIN D gene compared to normal PC, but many of these tumors appear to represent samples that are substantially contaminated by normal cells and another large fraction of these tumors often have inactivated RB1, the

72683_ch096_2022-2028.indd 2024

inhibitor downstream of CYCLIN D, eliminating the necessity of overexpressing a CYCLIN D gene.

Primary IgH Translocations It is thought that CYCLIN D translocations only dysregulate expression of a CYCLIN D gene. By contrast, MAF translocations dysregulate expression of a MAF transcription factor that causes increased expression of many genes, including CYCLIN D2 and adhesion molecules that are thought to enhance the ability of the tumor cell to interact with the BM microenvironment.29,30 The contributions of the two genes dysregulated by t(4;14) remain controversial. MMSET is a chromatin-remodeling factor that is overexpressed in all tumors with a t(4;14), whereas about 20% of tumors lack der(14) and FGFR3 expression. The rare acquisition of FGFR3 activating mutations during progression confirms a role for FGFR3 in MM pathogenesis. Although an activated mutant FGFR3 can be oncogenic, it recently was shown that wildtype FGFR3 (as is found in most t[4;14]) can contribute to B cell oncogenesis.31 It remains to be determined if FGFR3 is critical early in pathogenesis but becomes dispensable during progression of t(4;14) MM. Preclinical studies suggest that tyrosine kinase inhibitors are active only against t(4;14) HMCL with activating mutations of FGFR3, whereas anti-FGFR3 monoclonal antibodies that inhibit FGFR3 signaling but also elicit antibody-dependent cell-mediated cyotoxicity are active against HMCLs expressing wild-type FGFR3.32,33 Despite an apparently indispensable role in t(4;14) MM, it remains to be determined how MMSET, which sometimes has amino-terminal truncations caused by the translocation, contributes to MM pathogenesis. There are some clues. It is a histone methyltransferase for H3K36me2, and when overexpressed it results in a global increase in H3K36 methylation, and a decrease in H3K27 methylation, which might explain some of the many changes in gene expression associated with t(4;14) tumors.29,34,35,36 In addition, it recently has been determined that MMSET has a role in DNA repair (Fig. 96.3). Following DNA damage MMSET is phosphorylated on Ser102 by ATM and is recruited to sites of double strand breaks (DSBs), where it results in methylation of H4K20, which is required for recruitment of p53 binding protein (53BP1). 53BP1 is required for p53 accumulation, G2/M checkpoint arrest, and the intra-S–phase checkpoint in response to ionizing radiation. Approximately half of the translocation breakpoints in t(4;14) MM result in a truncated MMSET that lacks Ser102 and cannot be recruited to DSBs, resulting in a failure to recruit 53BP1 and a loss of the normal DNA damage response pathway. It is not known whether this biologic difference results in a different clinical outcome for t(4;14) MM patients with a truncated versus full-length MMSET.37 Importantly, loss of MMSET expression alters adhesion, suppresses growth, and results in apoptosis of HMCLs, suggesting that it is an attractive therapeutic target.35

Molecular Classification of Multiple Myeloma The patterns of spiked expression of genes deregulated by primary IGH@ translocations and the universal overexpression of CCNDs genes either by these translocations or other mechanisms led to the translocations and cyclin D (TC) classification that includes eight groups: those with primary translocations (designated 4p16, 11q13, 6p21, MAF), those that overexpressed CCND1 and CCND2 either alone or in combination (D1, D1 & D2, D2), and the rare cases that do not overexpress any CCND genes (“none”) (Table 96.1).29 Greater than 95% of the D1 group are HRD. In addition, most of the patients with HRD MM and trisomy 11 fall within the D1 and D1 & D2 groups, while those without trisomy 11 fall within the D2 group, although a majority of the D2 group are NHRD. This classification system therefore focuses on the different kinds of mechanisms that dysregulate a CCND gene as an early and unifying event in pathogenesis.

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Chapter 96  Molecular Genetic Aspects of Plasma Cell Disorders

2025

Breakpoints Ser102

PHD fingers

NLS PWWP

1 2

HMG

NLS

3 PWWP

SET

MMSET2

4

ATM

Ser102

MMSET Me Me K20 53BP1

PO4 MDC1 H2AX

FIGURE 96.3. MMSET is involved in DNA repair.  An ideogram of MMSET highlights the important functional domains of the protein, with arrows indicating the initiation of translation of the truncated forms, lacking Ser102, that result from translocation breakpoints in between the coding exons. Following DNA damage MMSET is phosphorylated on Ser102 by ATM and is recruited to sites of double strand breaks by MDC1, where it methylates H4K20. Dimethylation of H4K20 recruits p53-binding protein (53BP1), a key transducer of the DNA damage checkpoint signal.

An MM classification based on an unsupervised analysis of microarray gene expression profiling from the UAMS identified seven tumor groups characterized by the coexpression of unique gene clusters.38 This classification was partially replicated in an independent unsupervised analysis of a combined HOVON-GMMG dataset that identified 10 tumor groups with considerable overlap with the UAMS groups.39 Interestingly, these clusters also identify subgroups corresponding to the different primary translocations and hyperdiploidy. Importantly, however, they also highlight other important secondary events that can occur in each subtype of MM: proliferation (PR), expression of NFkB target genes (NFkB), cancer-testis antigens, and the phosphatase PTP4A3/PRL3 (PRL3). The CD-1 and CD-2 groups represent subgroups of patients with t(11;14) and t(6;14), with the former characterized by arginosuccinate synthetase 1 expression, and the latter by expression of B cell antigens (CD20, VPREB, CD79A). Interestingly, they identify patients with markedly different clinical outcomes. Of all the molecular subgroups, CD-1 has the quickest onset and highest frequency of CR (90%), whereas CD-2 has the slowest onset and lowest frequency of CR (45%), when treated with Total Therapy 3. However, after the MF, the CD-1 has the shortest CR duration

(77% at 2 years), whereas the CD-2 has the longest (100% at 2 years).40

Additional Oncogenic Events in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma MYC Dysregulation

Hematologic Malignancies

H4

There is increased expression of c-MYC in most newly diagnosed MM tumors compared to MGUS tumors.41 Recently, it was shown that sporadic activation of a MYC transgene in GC B cells in an MGUS-prone mouse strain led to the universal development of MM tumors.42,43 Hence, increased MYC expression seems to be responsible for progression from MGUS to MM. Complex translocations involving MYC (c-MYC>>N-MYC>L-MYC) appear to be secondary progression events that often do not involve Ig loci.44 They are rare or absent in MGUS, but occur in 15% of newly diagnosed tumors, 50% of advanced tumors, and 90% of HMCLs.18,45 A recent report suggests that a small molecule inhibitor of BRD4 can inhibit MYC RNA expression in MM, with therapeutic effect.46

TA B LE 96.1

Comparison Of Different Molecular Classifications In Multiple Myeloma Group

TC

Gene

%

CYCLIN D

UAMS

HOVON-GMMG

Cyclin D translocation

11q13 12p13 6p25 16q23 20q12 8q24 4p16 D1 D1 + D2

CCND1 CCND2 CCND3 MAF MAFB MAFA MMSET/FGFR3

15 NFkB activation BCMA-Fc TACI-Ig

B

Bortezomib IKKβ inhibition

FIGURE 96.4.  (continued)

Other Pathogenic Events Secondary Ig translocations, including most IgK and IgL translocations and IgH translocations not involving one of the seven primary partners, can occur at all stages of disease, and with a similar frequency in HRD and NHRD tumors, but apart from MYC, few partner loci have been identified.18 Other genomic rearrangements are frequent, but only a few specific target genes have been identified.63,66,67 Changes in DNA methylation are frequent, with one study suggesting that a marked increase in hypomethylation is associated with the MGUS to MM transition,68 whereas a second study suggests only a small increase in hypomethylation for MM compared to MGUS.69 Mutations in seven genes regulating RNA metabolism, protein translation, and homeostasis were identified in 16 of 38 patients.54 In addition to previous studies implicating roles for MMSET and KDM6A (UTX), genomic sequencing studies found that other histone-modifying enzymes are frequent targets of mutation, although the epigenetic consequences are unknown.54 Similarly, changes in microRNA expression at different stages have been identified, but more extensive studies are needed.62,70

Intraclonal Tumor Heterogeneity Associated with High-risk Multiple Myeloma Although the evidence is still emerging, it appears that many of the genetic events in MM are secondary and often present only in subclones of the tumor population.48,67,71 Recently, a high level of intraclonal tumor heterogeneity has been described in some patients with high-risk MM48,67,71 associated in one case with alternating clonal dominance under therapeutic selective pressure, observations with important clinical implications. The findings suggest a competition between subclones for limited resources and raise the possibility that early, suboptimal treatment may eradicate the “good” drug-sensitive clone, making room for the “bad” drug-resistant clone to expand. They support the use of aggressive multidrug combination approaches for highrisk disease with unstable genomes and clonal heterogeneity, and sequential one- or two-drug approaches for low-risk disease with stable genomes and lacking clonal heterogeneity.

72683_ch096_2022-2028.indd 2027

Conclusion Significant progress has been made in understanding the molecular pathogenesis and biology of MM (Fig. 96.5). Oncogenic pathways can be activated through cell intrinsic or extrinsic mechanisms. Similar to other cancers, MM is characterized by multistage accumulation of genetic abnormalities deregulating different pathways. Much of this knowledge is already being utilized for diagnosis, prognosis, and risk-stratification of patients. Importantly, from a clinical standpoint, this knowledge has led to development of novel therapeutic strategies, some of which are already in clinical use, and many others showing promise in preclinical and early clinical studies.

Hematologic Malignancies

with increased proliferation and a poor prognosis, whereas monoallelic deletion is not. Mutations of FAM46C—often with hemizygous deletion—were identified in 3.4% and 13% of MM tumors in two studies, and in 25% of 16 HMCL.54,64

Selected References The full reference list for this chapter can be found in the online version.



8. Perez-Persona E, Vidriales MB, Mateo G, et al. New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smoldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells. Blood 2007;110(7):2586–2592. 10. Landgren O, Kyle RA, Pfeiffer RM, et al. Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study. Blood 2009;113(22):5412–5417. 11. Weiss BM, Abadie J, Verma P, Howard RS, Kuehl WM. A monoclonal gammopathy precedes multiple myeloma in most patients. Blood 2009;113(22): 5418–5422. 13. Kyle RA, Therneau TM, Rajkumar SV, et al. Prevalence of monoclonal gammopathy of undetermined significance. N Engl J Med 2006;354(13): 1362–1369. 24. Bergsagel PL, Kuehl WM. Chromosome translocations in multiple myeloma. Oncogene 2001;20(40):5611–5622. 27. Fonseca R, Debes-Marun CS, Picken EB, et al. The recurrent IgH translocations are highly associated with nonhyperdiploid variant multiple myeloma. Blood 2003;102(7):2562–2567. 29. Bergsagel PL, Kuehl WM, Zhan F, Sawyer J, Barlogie B, Shaughnessy J Jr. Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood 2005;106(1):296–303. 35. Martinez-Garcia E, Popovic R, Min D-J, et al. The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells. Blood 2011;117(1):211–220. 37. Pei H, Zhang L, Luo K, et al. MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites. Nature 2011;470(7332):124–128. 38. Zhan F, Huang Y, Colla S, et al. The molecular classification of multiple myeloma. Blood 2006;108(6):2020–2028. 39. Broyl A, Hose D, Lokhorst H, et al. Gene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients. Blood 2010;116(14):2543–2553. 41. Chng WJ, Huang GF, Chung T-H, et al. Clinical and biological implications of MYC activation: a common difference between MGUS and newly diagnosed multiple myeloma. Leukemia 2011;25(6):1026–1035. 42. Chesi M, Robbiani DF, Sebag M, et al. AID-dependent activation of a MYC transgene induces multiple myeloma in a conditional mouse model of postgerminal center malignancies. Cancer Cell 2008;13(2):167–180.

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2028

Part vii  Hematologic Malignancies  •  SECTION 5  Plasma Cell Dyscrasias Rare de novo MM

Germinal center B cell

Intramedullary Myeloma

MGUS

Extramedullary Myeloma

NON-HYPERDIPLOID

Karyotypic abnormalities and epigenetic abnormalities

1ry IgH tx

11q13 6p21 16q23 20q11 4p16 Oth r Other

Secondary (Ig) TLC NFKB activating mutations PI3K/AKT dysregulation: DEPTOR, PTEN, PIK3CA

DEL13

HYPERDIPLOID

Protein translation/RNA Processing FAM46C, DIS3, XBP1, LRRK2 mutation N-RAS

K-RAS, FGFR3, BRAF

TRISOMY 3,5,7,9,11 15,19,21

MYC RNA ± MYC rearrangement ?BLIMP1, IRF4 mutation

MYC (Ig) rearrangement P18, RB inactivation p53 inactivation

Cyclin D dysregulation FIGURE 96.5. A model for the multistep molecular pathogenesis of multiple myeloma.  Two largely nonoverlapping pathways (immunoglobulin [Ig] translocations versus multiple trisomies) are primary events associated with dysregulated cyclin D expression. The most common secondary genetic events associated with tumor progression are shown, including early and late dysregulation of MYC, and late inactivating mutations of p53.

43. Chesi M, Matthews GM, Garbitt VM, et al. Drug response in a genetically engineered mouse model of multiple myeloma is predictive of clinical efficacy. Blood 2012;120(2):376–385. 46. Delmore JE, Issa GC, Lemieux ME, et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 2011;146(6):904–917. 54. Chapman MA, Lawrence MS, Keats JJ, et al. Initial genome sequencing and analysis of multiple myeloma. Nature 2011;471(7339):467–472. 56. Annunziata CM, Davis RE, Demchenko Y, et al. Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma. Cancer Cell 2007;12(2):115–130.

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57. Keats JJ, Fonseca R, Chesi M, et al. Promiscuous mutations activate the noncanonical NF-kappaB pathway in multiple myeloma. Cancer Cell 2007;12(2):131–144. 67. Keats JJ, Chesi M, Egan JB, et al. Clonal competition with alternating dominance in multiple myeloma. Blood 2012;120(5):1067–1076.

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Chapter 97

Monoclonal Gammopathies Of Undetermined Significance And Smoldering Multiple Myeloma S. Vincent Rajkumar, Robert A. Kyle, John A. Lust

Nomenclature Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic, pre-malignant clonal plasma cell proliferative disorder.1–4 It was initially referred to as essential hyperglobulinemia by Jan Waldenström, as well as several other terms such as benign, idiopathic, asymptomatic, nonmyelomatous, discrete, cryptogenic, and rudimentary monoclonal gammopathy; dysimmunoglobulinemia; lanthanic monoclonal gammopathy; idiopathic paraproteinemia; and asymptomatic paraimmunoglobulinemia.5,6 However, because there is an indefinite risk of progression to multiple myeloma (MM) or related disorder such as Waldenström macroglobulinemia (WM) or amyloidosis (AL), the term MGUS is now the accepted nomenclature.1,2,7,8 Smoldering multiple myeloma (SMM) is a clinically defined pre-malignant stage between MGUS and MM.9,10 MGUS and SMM must be differentiated from MM, and from a number of related plasma cell disorders using the criteria listed on Table 97.1.8,11

Detection of Monoclonal Proteins Immunoglobulins consist of two heavy polypeptide chains of the same class and subclass and two light polypeptide chains of the same type. The various types of immunoglobulins are designated by capital letters that correspond to the isotype of their heavy chains, which are designated by Greek letters: gamma (g) constitutes immunoglobulin G (IgG), alpha (a) is found in IgA, mu (m) is present in IgM, delta (d) occurs in IgD, and IgE is characterized by epsilon (â). IgG1, IgG2, IgG3, and IgG4 are the subclasses of IgG; the subclasses of IgA are IgA1 and IgA2. Kappa (k) and lambda (l) are the two types of light chains. An intact immunoglobulin consists of two heavy chains of the same class and two light chains of the same type. A monoclonal increase in immunoglobulins results from a clonal process such as MGUS or MM, and a polyclonal increase in immunoglobulins is caused by a reactive or inflammatory process. The monoclonal immunoglobulin secreted by clonal plasma cells in MGUS, SMM, MM, and related monoclonal gammopathies is referred to as a monoclonal protein or M protein.

Electrophoresis and Immunofixation Monoclonal proteins are detected using agarose gel or capillary electrophoresis of the serum and urine.12 An M-protein is usually visible as a localized band on protein electrophoresis, and as a tall narrow spike or peak in the b or g region or, rarely, in the a2globulin area of the densitometer tracing (Fig. 97.1A). A polyclonal increase in immunoglobulins produces a broad band or broadbased peak that migrates in the g region. A suspected M protein on electrophoresis must be confirmed on immunofixation, which also determines the immunoglobulin heavy-chain class and its light chain type.13 In addition, immunofixation is also done when MM, WM, AL (light chain) amyloidosis, or a related disorder is suspected, because small M proteins may not be detected with electrophoresis alone. Immunofixation is performed using commercial kits or systems such as Sebia, or Pentafix (Fig. 97.1B).

Quantitative Immunoglobulins In patients with detectable M proteins, another assay that aids in monitoring is quantitation of immunoglobulins performed with a rate nephelometer. It can accurately measure 7S IgM, polymers of IgA, and aggregates of IgG. However, levels of IgM obtained by nephelometry may be 1,000 to 2,000 mg/dl higher than those expected on the basis of the serum protein electrophoretic tracing. The quantitative IgG and IgA levels may be increased similarly.14

Serum Free Light Chain Assay The serum free light chain (FLC) assay (Freelite™, The Binding Site Limited, Birmingham, UK) is an automated nephelometric assay that measures free kappa (k) and lambda (l) light chains that are not bound to intact immunoglobulin.15,16 The normal serum free-k level is 3.3 to 19.4 mg/L and the normal free-l level is 5.7 to 26.3 mg/L.17 The normal ratio for FLC-k/l is 0.26 to 1.65. The normal reference range in the FLC assay reflects a higher serum level of free l light chains than would be expected given the usual k/l ratio of 2 for intact immunoglobulins. This occurs because the renal excretion of free k (which exists usually in a monomeric state) is faster than free l (which is usually in a dimeric state).15,16 Patients with a k/l FLC ratio 1.65 are defined as having a monoclonal k free light chain. If the FLC ratio is >1.65, k is referred to as the “involved” FLC and l the “uninvolved” FLC, and vice versa if the ratio is less than 0.26. The serum FLC assay can be used in place of urine protein electrophoresis and immunofixation in the initial screening algorithm for M proteins. In a study of 428 patients, Katzmann et al. found that urine studies can be eliminated by using the serum FLC assay in combination with the SPEP and immunofixation.18 However, if a monoclonal plasma cell disorder is identified on screening, a 24-hour urine collection followed by electrophoresis and immunofixation should always be done to aid in the assessment of disease progression and response to therapy over time. In addition to its role as a substitute for urine studies in the screening of plasma cell disorders, the FLC assay is used to predict prognosis in MGUS, SMM, AL, and solitary plasmacytoma.19–21 In addition, it is also used to monitor oligo-secretory MM, nonsecretory MM, light chain only form of MM, and AL amyloidosis.16,22,23,24 In order to use the FLC assay to monitor disease progression, the baseline FLC ratio must be abnormal and the involved FLC level ≥100 mg/L.24,25

Hematologic Malignancies

Introduction

Monoclonal Gammopathy Of Undetermined Significance Definition MGUS is defined by the presence of a serum M protein