Cancer Associated Viruses (Current Cancer Research) 144199999X, 9781441999993

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Current Cancer Research

Series Editor Wafik El-Deiry

For further volumes: http://www.springer.com/series/7892

Erle S. Robertson Editor

Cancer Associated Viruses

Editor Erle S. Robertson Professor of Microbiology Director of the Tumor Virology Training Program Perelman School of Medicine University of Pennsylvania Philadelphia, PA, USA [email protected]

ISBN 978-1-4419-9999-3 e-ISBN 978-1-4614-0016-5 DOI 10.1007/978-1-4614-0016-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011940811 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

In Memoriam

Baruch S. Blumberg, M.D., D.Phil.

Baruch Samuel (Barry) Blumberg died suddenly on April 5, 2011 shortly after giving a presentation on “citizen science” at the NASA Lunar Science Institute in Ames, California. Barry’s talk concerned making spacecraft data available to the public so that ordinary people could contribute to its interpretation. That final talk reflected his deep belief that anyone who was willing to invest time and thought could have ideas that would lead to new understandings of research information. This volume is about viruses and cancer, but Barry was neither a virologist nor an oncologist. However, he contributed fundamentally to both. Barry began his research career as a medical student at Columbia University when he took an elective in v

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Tropical Medicine in Suriname. There, he observed that filariasis was rampant, but that only some of the many infected people showed signs of disease. Suriname had many different ethnic groups and some of the diversity in responses was associated with ethnicity. That led him to wonder for the rest of his life why humans living in the same environment responded so differently to infectious agents. After an internship and assistant residency in medicine at Bellevue Hospital in New York City and a fellowship in rheumatology, Barry went to Oxford University to study biochemistry with Alexander Ogston. He was in the lab at the same time as Oliver Smithies who also went on to win a Nobel Prize. In England, he was exposed to the history of scientific discovery and began formulating his scientific ideas. It was there that he found his scientific inspiration in the lives and work of the nineteenth-century naturalists Charles Darwin and Alfred Russell Wallace. He began to apply the principles of evolution to his research. After receiving a doctorate, Barry went to an obscure unit called Geographic Medicine and Genetics of the National Institute of Arthritis and Metabolic Disease (NIAMD) in Bethesda, Maryland. He chose this hidden corner because he thought it would allow him to pursue his own ideas and travel wherever he pleased. He began a lifelong pattern of collecting blood samples everywhere he went, making observations about the people from whom they were drawn and often collecting samples of vegetation from their environments. It was at the NIH that Barry honed his interest in genetic polymorphisms in human blood, inherited variants of proteins or blood groups, which he believed were likely to be associated with human diseases. As a believer in the central importance of natural selection, he thought all such variants had to be important. Otherwise, they would not have persisted in human populations. In 1964, the Director of the Institute for Cancer Research (ICR, precursor to the Fox Chase Cancer Center) recruited Barry to become the head of a new Division of Clinical Research. The lure was that Barry was promised he could do whatever he wanted as long as his research ultimately had consequences for disease in humans. Barry was intrigued and immediately began assembling a small group of physicians to staff this new enterprise. He had complete faith that his approach: identifying variants in human blood and then finding out what they meant, would be much more informative than starting with a disease and trying to identify its causes. It was this approach that first resulted in identifying an antigen on a lipoprotein in serum, and subsequently to a different antigen, the “Australia antigen.” Barry focused the efforts of his new division on understanding the biological significance of Australia antigen. In a series of studies of diseases associated with the antigen, the group found that Australia antigen was closely associated with one form of viral hepatitis, later called hepatitis B. At the same time, they observed that Australia antigen was a particle similar in appearance to a virus. That was enough information for Barry to begin to develop a unique vaccine, one that was prepared from antigenic particles in human blood. The patent for the vaccine was submitted in 1969 and granted in 1971. By 1975, before the vaccine had even been tried in humans, Barry predicted in print that the vaccine would not only prevent infection

In Memoriam

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with the hepatitis B virus but that it would also prevent liver cancer. Therefore, it would be the first cancer vaccine. In 1976, Barry was awarded the Nobel Prize in Physiology or Medicine for “discoveries concerning new mechanisms for the origin and dissemination of infectious diseases.” Very few people are privileged to win a Nobel Prize. Barry made his success a celebration for everyone in his group. He took as many of his colleagues and staff in the division to the ceremony as he was permitted, and least 15 and their spouses. In 1989, he returned to Oxford to become the Master of Balliol College serving until 1994. Balliol was founded in 1263 and Barry was its first American Master. From 1999 to 2002, he was director of the NASA Astrobiology Institute. He continued his affiliation with NASA and in 2008 became a Senior Scientist at the NASA Lunar Science Institute. In 2005, he became the President of the American Philosophical Society, founded by Benjamin Franklin, and the oldest learned society in the Americas. Barry was always a happy person. He celebrated his own life by living it to the fullest. He cycled, hiked, ran, rock-climbed, canoed, and kayaked until the end of his life. Barry had a long and happy marriage. He was always proud of the accomplishments of his wife, Jean, his four children and nine grandchildren. He left a legacy of accomplishments that saved an enormous number of lives and prevented hundreds of millions of people from becoming ill with the hepatitis B virus. On every continent, his many friends and colleagues mourn his loss. Fox Chase Cancer Center Philadelphia, PA, USA

W. Thomas London

Preface

For almost 30 years, there has been no comprehensive text covering the many viral agents and their contributions to cancers or cell proliferation. The goal was to provide a relatively up-to-date tome, which would be a wonderful resource for the many investigators in the field of viral oncology. The chapters are meant to be a thorough review of the literature, which covers specific viruses as well as provide some synthesis of what we now know about viruses, cell proliferation and the genes that target specific cellular pathways. The previous works are outdated, and as this book is put to press, we would still have additional works to be published that will certainly be missed. We have tried to be as comprehensive as possible within the guidelines of the text without sacrificing the science and we have allowed authors much flexibility that would only be fitting if one takes on a job to complete a chapter that is as comprehensive and current as we have attempted in this book. The primary goal here was to provide the most comprehensive version of chapters covering the majority of viruses and cancers, which will be a major resource for all trainees in the field of viral oncology from undergraduates and graduate students to post-doctoral fellows in basic science and translational or clinical studies, as well as investigators related to viral oncology. This approach was certainly limited as we will, without a doubt, be missing some of the detail and intricate nuances of each viral system. That said, we certainly tried to encourage a general theme throughout and so the experienced readers in the field may find some aspects of it less inviting. Nevertheless, I think that overall, we have attained a level of scientific sophistication for each of the chapters that it would be worth the time of experts in the field to read and it would be a solid contribution enjoyed by all including novices, as well as the experts. Thus, I take full responsibility for any omission that may have occurred, unintentionally. I also want to say that each contributor has done a fantastic job in completing his or her chapter, and that one would have to give them all a tremendous thank-you for their efforts in making this project happen even with the burdensome task of meeting deadlines and responding to my many emails in nudging them along. The book begins with a chapter that introduces the father of viral oncology Professor Peyton Rous with some of his many interesting findings and his training, ix

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as well as his international interactions with many scientists across the world. This year would be the 100th anniversary of the initial discovery of the Rous Sarcoma Virus. This is followed by a chapter contributed by Baruch Bloomberg, one of the many Nobel laureates whose contributions to the field has made a huge impact in saving lives throughout the world. He was passionate about pushing for the development of the hepatitis B vaccine and in doing so, led to the vaccination of millions throughout the world who would have been infected and would have a higher probability of developing hepatocellular carcinoma. He presents a historical perspective on viruses and cancer. The chapters by Drs. Jae Jung, Blossom Damania and Robin Weiss present a broad outline of how viruses can contribute or drive the oncogenic process and the potential for cancer transmission. Dr. Alwine wrote the introductory chapter for the DNA tumor viruses and suggests that while some large DNA viruses may not be able to directly transform a cell, they can certainly alter signaling and metabolism in ways that can certainly drive the transformation and possible immortalization of the infected cells. The chapter by Dr. Bala Chandran brings together the many contributions by the large DNA herpesviruses and their ability to induce the oncogenic process. Further on this theme, we also cover specific chapters on viruses in herpesviridae with my group looking at the Lymphocryptoviruses; Dr. Schultz on the Rhadinoviruses; Dr. Rose on the contribution of the retroperitoneal fibromatosis herpesvirus to retroperitoneal fibromatosis, a Kaposi’s sarcoma-like disease in macaques with simian AIDS; Dr. Wong exploring the viruses in nonhuman primates; Dr. Speck presenting the murine herpesvirus model of tumorigenesis; and Drs. Parcells and Morgan who describe the Marek’s disease virus and its contribution to T-cell lymphomas in chickens. These chapters provide an in-depth analysis of these viral agents and their similarities and differences in driving the oncogenic process. We have had a great deal of success in bringing in a number of talented investigators looking at the small DNA tumor viruses, in particular the Polyoma and Papilloma viruses as well as the Adenoviruses. Dr. Gjoerup did a fantastic job in describing the many facets of the Polyomas and their contribution to cancer and this was followed by chapters from Drs. Butel, Hirsch, Khalili and Becker who provided a thorough review of the SV40 as a model system, the BK virus, the JC virus and the new Merkel cell Polyoma virus, respectively. I should also mention at this stage, that more recently in July 2010, there was a report of another virus, which belongs to the Polyoma virus family now called Trichodysplasia Spinulosa-associated Polyoma virus (TSV), which was identified in a rare skin disease called Trichodysplasia Spinulosa, exclusively seen in immunocompromised patients. It is yet to be seen if this is a ubiquitous virus in the population, which becomes opportunistic in these group of patients. A review of the Papilloma viruses covering the HPV and BPV systems was completed by Drs Jianxin You and Suzannne Wells. Adenoviruses, another group of oncogenic DNA viruses, were also included, although to date there has been no direct association with Adenoviruses and human cancers. However, there has been a wealth of information over more than 30 years showing that Adenoviruses are fully capable of meeting the major criteria for driving the oncogenic process using in vitro studies and also inducing tumors in an animal model.

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The Hepadnaviruses have also been addressed in the compendium, where we take a closer look at the contributions of the hepatitis viruses B and C. Professor Tim Block has done a marvelous job of reviewing the many general attributes of the hepatitis viruses and the cancers they are associated with from a virological to a more molecular perspective. This is followed by chapters on hepatitis B virus by Dr. Mason from the Fox Chase Cancer Center, where Barry Bloomberg spent a great many of his years as a scientist working on the hepatitis virus. Dr. Mason did a fantastic job in getting us up to date on the causes of chronic liver disease, cirrhosis and many years later HBV-induced hepatocellular carcinoma, which takes at times as long as 40 years. Dr. Bret Lindenbach explores the contributions of hepatitis C virus to the development of hepatocellular carcinoma and summarizes the clinical and molecular virology links between the HCV virus and HCC. Dr. Kathleen Boris-Lawrie finds an interesting angle to explore further the role of HIV-1 as a risk factor for the development of malignancies in AIDS patients by describing why Kaposi’s sarcoma, non-Hodgkin’s lymphoma and cervical carcinomas can function as prognostic indicators of AIDS and begins to suggest the relationship of coinfection and how this may contribute to the oncogenic process. We also cover the HTLV-1 and HTLV-2 where their contributions to cell proliferation were well described. Dr. Chou-Zen Giam focuses on the role of HTLV-1 in causing adult T-cell leukemia paying attention to the role of two viral proteins Tax and HBZ in viral replication and leukemogenesis. Dr. Patrick Green focuses on the biology and pathogenesis of HTLV-2 and further dissects the various cellular processes utilized by the virus in contributing to cell proliferation. The chapter on avian and murine retroviruses was skillfully put together by Drs. Karen Beemon and Naomi Rosenberg. This chapter provides information on viral oncogenes and the cooperation between these viral oncogenes as a major step in the development of cancer. They also describe the potential role of these viruses as vector systems. Dr. Leslie Parent goes into further detail in contributing the chapter on the Rous Sarcoma Virus which takes the reader from the provirus concept and how the integrated provirus, which certainly has transforming activities, led to the identification of the a cellular gene highly homologous to the viral transforming gene. Dr. Susan Ross thoroughly presents the mouse mammary tumor viruses (MMTV), which can cause breast cancer in mice by causing insertional activation of mutation of cellular oncogenes and provides an extremely useful model for understanding human breast cancer. Another very interesting virus is the Jaagsiekte Sheep retrovirus, which is associated with lung cancer. This virus causes ovine pulmonary adenocarcinoma which is derived from the secretory lung epithelial cells. Dr. Hung Fan describes here how the pathology of the virus is directly linked to the envelope protein (Env) and that JSRV is mostly pathogenic in the lung as the viral LTR is transcriptionally active only in differentiated airway epithelial cells. Drs. Renne and Swaminathan now contributed a chapter on the small RNAs and their role in viral-mediated cancers. The final chapter was a wonderful contribution by Dr. Charles Wood, who explores the role of immunodeficiency and opportunistic infections and their cooperation in driving the oncogenic process.

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Finally, I wanted to personally thank all the authors for their patience and their hard work in getting their contributions to me as timely as can be expected. Even the folks who were somewhat tardy in their delivery have made it worthwhile, and I can say overall that I am personally happy with the final product. I hope this project provides a renewed vigor to our community of scientists to explore the contributions of viruses to cancer. In my many discussions with investigators in the cancer field it is still amazing and a bit puzzling to me that a great many are still hesitant to acknowledge that viruses or infectious agents on a whole have much to do with cancer, even though it is well known that about 20% of all known cancers are associated with infectious agents. I hope this renewed thrust will minimize these concerns and provide new support for the many investigators who have spent their entire lives working towards understanding how viruses contribute to the oncogenic process. Happy reading. Cheers Philadelphia, PA, USA

Erle S. Robertson

Contents

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Peyton Rous: A Centennial Tribute to the Founding Father of Cancer Virology...................................................................... Volker Wunderlich and Peter Kunze

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Viruses and Cancer: A Historical Perspective – HBV and Prevention of a Cancer .................................................................... Baruch S. Blumberg

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Virus-Mediated Cell Proliferation ......................................................... Sun-Hwa Lee, Stacy Lee, and Jae Ung Jung

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Viral-Encoded Genes and Cancer ......................................................... Blossom Damania

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5

Oncogenic Viruses and Cancer Transmission ...................................... 101 Robin A. Weiss

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DNA Viruses and Cancer: Taking a Broader Look ............................. 119 James C. Alwine

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Herpesviruses and Cancer ..................................................................... 133 David Everly, Neelam Sharma-Walia, Sathish Sadagopan, and Bala Chandran

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Lymphocryptoviruses: EBV and Its Role in Human Cancer ............. 169 Santosh Kumar Upadhyay, Hem Chandra Jha, Abhik Saha, and Erle S. Robertson

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Nonhuman Primate Gamma-herpesviruses and Their Role in Cancer .................................................................................................. 201 Ryan D. Estep and Scott W. Wong

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Rhadinoviruses: KSHV and Associated Malignancies ........................ 215 Susann Santag and Thomas F. Schulz

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Retroperitoneal Fibromatosis Herpesvirus and Kaposi’s Sarcoma-Like Tumors in Macaques ...................................................... 251 Laura K. DeMaster and Timothy M. Rose

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Murine Gammaherpesvirus-Associated Tumorigenesis...................... 267 Kathleen S. Gray and Samuel H. Speck

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Marek’s Disease Virus-Induced T-Cell Lymphomas ........................... 307 Mark S. Parcells, Joan Burnside, and Robin W. Morgan

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Polyomaviruses and Cancer ................................................................... 337 Ole Gjoerup

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Polyomavirus SV40: Model Infectious Agent of Cancer ..................... 377 Janet S. Butel

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BK Polyomavirus and Transformation ................................................. 419 Tina Dalianis and Hans H. Hirsch

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Polyomavirus JC and Human Cancer: Possible Role of Stem Cells in Pathogenesis ................................................................. 433 Kamel Khalili, Martyn K. White, Jennifer Gordon, and Barbara Krynska

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Merkel Cell Polyomavirus ...................................................................... 449 David Schrama and Jürgen C. Becker

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Human Papillomaviruses and Cancer................................................... 463 Jianxin You and Susanne Wells

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Tumorigenesis by Adenovirus Type 12 E1A ......................................... 489 Hancheng Guan and Robert P. Ricciardi

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Overview of Hepatitis Viruses and Cancer ........................................... 509 Timothy M. Block, Jinhong Chang, and Ju-Tao Guo

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Hepadnaviruses and Hepatocellular Carcinoma ................................. 531 William S. Mason

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Hepatitis C Virus and Hepatocellular Carcinoma ............................... 571 Brett Lindenbach

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Human and Animal Retroviruses: HIV-1 Infection Is a Risk Factor for Malignancy ............................................................ 585 Amy M. Hayes and Kathleen Boris-Lawrie

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HTLV-1 and Oncogenesis ....................................................................... 613 Chou-Zen Giam

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Human T-Cell Leukemia Virus Type 2 (HTLV-2) Biology and Pathogenesis ..................................................................................... 647 Rami Doueiri and Patrick L. Green

Contents

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Mechanisms of Oncogenesis by Avian and Murine Retroviruses ............................................................................................. 677 Karen Beemon and Naomi Rosenberg

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Rous Sarcoma Virus: Contributions of a Chicken Virus to Tumor Biology, Human Cancer Therapeutics, and Retrovirology.................................................................................... 705 Leslie J. Parent

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Mouse Mammary Tumor Virus and Cancer ........................................ 739 Susan R. Ross

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Jaagsiekte Sheep Retrovirus and Lung Cancer ................................... 755 Chassidy Johnson and Hung Fan

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Small RNAs and Their Role in Herpesvirus-Mediated Cancers ........ 793 Sankar Swaminathan and Rolf Renne

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Viral Malignancies in HIV-Associated Immune Deficiency ................ 819 Pankaj Kumar, Veenu Minhas, and Charles Wood

Index ................................................................................................................. 853

Contributors

James C. Alwine Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Jürgen C. Becker Director, Division of General Dermatology, Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, A-8036, Graz, Austria [email protected] Karen Beemon Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA [email protected] Timothy M. Block Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA Hepatitis B Foundation, Pennsylvania Biotechnology Center, Doylestown, PA, USA [email protected] Baruch S. Blumberg Fox Chase Cancer Center, Philadelphia, PA 19111, USA [email protected] Kathleen Boris-Lawrie Department of Veterinary Biosciences, Center for Retrovirus Research, Center for RNA Biology, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA [email protected] Joan Burnside Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA

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Contributors

Janet S. Butel Department of Molecular Virology and Microbiology, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA [email protected] Bala Chandran H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA [email protected] Jinhong Chang Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA [email protected] Tina Dalianis Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska R8:01, Karolinska University Hospital, 171 76, Stockholm, Sweden [email protected] Blossom Damania Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA [email protected] Laura K. DeMaster Department of Global Health, University of Washington, Seattle Children’s Research Institute, Seattle, WA, USA Rami Doueiri Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA Ryan D. Estep Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, USA David Everly H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Hung Fan Department of Molecular Biology and Biochemistry, Cancer Research Institute, University of California, Irvine, CA 9269, USA [email protected] Chou-Zen Giam Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA [email protected] Ole Gjoerup Cancer Virology Program, University of Pittsburgh Cancer Institute, Research Pavilion Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA [email protected]

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Jennifer Gordon Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA Kathleen S. Gray Department of Microbiology & Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA Patrick L. Green Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210, USA Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA [email protected] Hancheng Guan Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Ju-Tao Guo Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA Amy M. Hayes Department of Veterinary Biosciences, Center for Retrovirus Research, Center for RNA Biology, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA Hans H. Hirsch Department of Biomedicine, Clinical and Transplantation Virology, Institute for Medical Microbiology, University of Basel, Basel, Switzerland Infectious Disease and Hospital Epidemiology, University Hospital Basel, Basel, Switzerland [email protected] Hem Chandra Jha Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Chassidy Johnson Department of Molecular Biology and Biochemistry, Cancer Research Institute, University of California, Irvine, CA 9269, USA Jae Ung Jung Department of Molecular Microbiology & Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA [email protected]

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Contributors

Kamel Khalili Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA [email protected] Barbara Krynska Center of Neural Repair and Rehabilitation and Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA Department of Neurology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA Pankaj Kumar Nebraska Center for Virology and the School of Biological Sciences, Morrison Center, University of Nebraska-Lincoln, Lincoln, NE 68583, USA Peter Kunze Institute of Pathology “Georg Schmorl”, 01067, Dresden, Germany [email protected] Sun-Hwa Lee Department of Molecular Microbiology & Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA [email protected] Stacy Lee Department of Molecular Microbiology & Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA Brett Lindenbach Section of Microbial Pathogenesis, Yale University School of Medicine, 354C Boyer Center for Molecular Medicine, New Haven, CT 06536-0812, USA [email protected] William S. Mason Fox Chase Cancer Center, Philadelphia, PA 19111, USA [email protected] Veenu Minhas Nebraska Center for Virology and the School of Biological Sciences, Morrison Center, University of Nebraska-Lincoln, Lincoln, NE 68583, USA Robin W. Morgan Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA Mark S. Parcells Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA [email protected]

Contributors

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Leslie J. Parent Department of Medicine, Penn State College of Medicine, Hershey, PA 17033, USA Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, PA 17033, USA [email protected] Rolf Renne Department of Molecular Genetics and Microbiology, UF Shands Cancer Center, University of Florida, Gainesville, FL 32610-3633, USA [email protected] Robert P. Ricciardi Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Erle S. Robertson Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Timothy M. Rose Department of Pediatrics, University of Washington, Seattle Children’s Research Institute, Seattle, WA, USA [email protected] Naomi Rosenberg Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA [email protected] Susan R. Ross Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Sathish Sadagopan H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Abhik Saha Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

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Contributors

Susann Santag Institute of Virology, Hannover Medical School, Hannover, Germany David Schrama Director Division of General Dermatology, Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, A-8036, Graz, Austria Thomas F. Schulz Institute of Virology, Hannover Medical School, Hannover, Germany [email protected] Neelam Sharma-Walia H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology Facilities, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Samuel H. Speck Department of Microbiology & Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA [email protected] Sankar Swaminathan Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132, USA [email protected] Santosh Kumar Upadhyay Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Robin A. Weiss Division of Infection & Immunity, University College London, London, UK [email protected] Susanne Wells Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Division of Hematology/Oncology, Cincinnati Children’s Hospital, Cincinnati, OH, USA [email protected] Martyn K. White Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA

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Scott W. Wong Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th AvenueBeaverton, OR 97006, USA Division of Pathobiology and Immunology, Oregon National Primate Research Center, Beaverton, OR, USA Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, USA [email protected] Charles Wood Nebraska Center for Virology and the School of Biological Sciences, Morrison Center, University of Nebraska-Lincoln, Lincoln, NE 68583, USA [email protected] Volker Wunderlich Max Delbrück Center for Molecular Medicine, 13125, Berlin, Germany [email protected] Jianxin You Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected]

Chapter 1

Peyton Rous: A Centennial Tribute to the Founding Father of Cancer Virology Volker Wunderlich and Peter Kunze

Introduction On December 10, 1966, the American pathologist and cancer researcher Francis Peyton Rous (1879–1970) (Fig. 1.1), professor emeritus at the Rockefeller Institute for Medical Research, New York, was awarded the Nobel Prize for Physiology or Medicine “for his discovery of tumor-inducing viruses.” He received the award in Stockholm from the hands of the Swedish King Gustav VI Adolf (1882–1973). Fifty-five years after his discovery (Rous 1911a, b) and forty years after his first nomination by Karl Landsteiner (1868–1943) (Nomination Database 1901–1951), one of the great scientists of the twentieth century was awarded this long-deserved honor. His work launched a new era of medicine (Vogt 1996). Amazingly, however, up to now, science historians have not written a biography of Rous. Just a few months before the Nobel ceremony, Rous had received the prestigious Paul Ehrlich and Ludwig Darmstaedter Prize (Germany’s supreme medical accolade) in St. Paul’s Church in Frankfurt am Main on March 14, 1966. There, he began his award acceptance speech with the words: The joy that moves me on this festive occasion is particularly great because it brings to mind some personal memories. When I studied at The Johns Hopkins School of Medicine at the beginning of this century, all young physicians looked to Germany as a model. The dean of the school, Professor William Welch, an eminent pathologist, had many years earlier worked with Paul Ehrlich in Breslau (today Wroclaw) [both under the supervision of the pathologist Julius Cohnheim (1839–1884)], and upon his return to the United States had informed the American medical community of the major progress made in Germany during this time. (Rous 1966: 20) [German in original]

V. Wunderlich (*) Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany e-mail: [email protected] P. Kunze Institute of Pathology “Georg Schmorl”, 01067 Dresden, Germany E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_1, © Springer Science+Business Media, LLC 2012

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Fig. 1.1 Photograph of Peyton Rous, 1959 in Israel, courtesy of Dr. Inge Graffi, Berlin. Photo credit: Weizmann Institute of Science, Rehovot, Israel

In fact, during those years, German science (and in a broader sense European science) was a world leader in many fields within and outside of medicine, not only in the inner circle of Paul Ehrlich. As a result, it was extremely attractive for young scientists from other countries to come to Germany to work here temporarily. For a future career in the USA, proof of European experience could be quite helpful, not unlike as it is today with many appointments of professors in Germany, where a previous work stay in the USA is regarded a conditio sine qua non. If the young Rous, while still a student, dreamed of a sojourn in Germany, this dream was soon to become reality. In the official Nobel biography, which is based on Rous’s own autobiographical notes, an additional personal recollection of the laureate is recorded: [At The Johns Hopkins Medical School] he graduated in 1905 [Doctor of Medicine] and became an intern in its hospital. Then, finding himself unfit to be a “real doctor,” he turned to medical research instead, and for this purpose became an instructor in pathology at the University of Michigan on a beggarly salary. His work in the laboratory turned out to be mainly that of a technician because the University had small funds only, but with noble generosity Professor Alfred [sic] Warthin, head of the Department, came to his rescue, actually offering to “teach summer school” in his stead, and give Peyton the sum thus earned, if he would study German hard and use the money to go for the summer to a certain hospital in Dresden where morbid anatomy was taught. Dresden in 1907! Exquisite city in an exquisite land, with no hint of war in the air! (Anonymous 1966)

Rous, who in 1966 was well along in years, could still remember Dresden, although he seemed to have forgotten the names of the host institution (the specific hospital) and his Dresden mentor. Thus, these details remained largely unclear and

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were mentioned only very briefly in the Biographical Memoirs (Andrewes 1971; Dulbecco 1976) dedicated to Rous. However, his sojourn in Dresden was the only one abroad before Rous began his exceptionally successful career as independent research scientist at the Rockefeller Institute, and apparently – even in retrospect – the time spent in Dresden was very important to him. In this paper, we report on new research inquiries concerning Warthin, Schmorl, and Rous. But first, we shall briefly present some of the pathologists who influenced Rous before his stay in Dresden. In subsequent sections, we shall briefly present several aspects of Rous’ work in the years immediately following his Dresden stay that were crucial for tumor virology.

A Fresh Age of Medical Endeavor in America: William Henry Welch Welch and Warthin influenced the career of the young Rous in different ways. He studied at the School of Medicine at The Johns Hopkins University, America’s first research university, which opened in 1892. Apparently, William Henry Welch (1850–1934) (Fig. 1.2), the founding dean and professor of pathology at the medical school and at that time academic teacher, was able to spark Rous’ interest in experimental medicine in general and in pathology in particular. In Baltimore, Welch established the first pathology teaching laboratory in the USA. In general, Welch dedicated himself to an extraordinary degree to the modernization of American medicine in teaching and research [“a fresh age of medical endeavor came in – an

Fig. 1.2 Photograph of William H. Welch. Photo credit: Courtesy of the Rockefeller Archive Center

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age in which experiment largely took over from observation,” Rous wrote, in retrospect (Rous 1948: 611)], whereby the experience gained during his research stays in Europe helped him (1875–1878, 1884) (MacCallum 1936; Flexner and Flexner 1941; Flexner 1943; Brieger 1970). As first president of the Board of Scientific Directors at the Rockefeller Institute for Medical Research in New York (1901–1933), Welch took a keen interest in Rous’ subsequent rise to become a distinguished scientist. Just a few years after his death, Rous had the honor of presenting a William Henry Welch Lecture (Rous 1941). On another occasion, Rous noted: “It is not too much to say that modern scientific medicine reached America through William Henry Welch” (Rous 1949: 411). The Journal of Experimental Medicine was launched in 1896 by Welch as founding editor of a new type of medical publication, which was then developed into a highly prestigious journal by Rous during his extremely long tenure (1922–1970, until 1945 together with Simon Flexner).

Aldred Scott Warthin: A Consummate Pathologist As a young, freshly graduated medical doctor, Rous came to Aldred Scott Warthin (1866–1931) (Fig. 1.3) at the University of Michigan with the aim of doing experimental work and obtaining training in pathology. He could not have made a better choice. Warthin was initially trained as church musician before turning to the study of medicine. He received his MD in 1891 and his PhD in 1893. He worked temporarily in the Department of Internal Medicine at the University of Michigan before he

Fig. 1.3 Photograph of Aldred S. Warthin. Photo credit: A.S. Warthin Papers, Bentley Historical Library, University of Michigan

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started his academic career there as pathologist. Following various positions from 1896 on (among them as instructor, a position Rous later held under him), he was appointed professor and director of the Pathological Laboratories at the University of Michigan in Ann Arbor in 1903. Between 1893 and 1900, Warthin regularly traveled to Europe during the summer months in order to work at the institutes of pathology in Vienna, Freiburg, and Dresden and at the same time to pursue his multifaceted interests (music, art history, collection of old books, particularly of medical incunabula). While doing so, he in no way neglected his extensive pathological research. A number of eponyms are today associated with the name Warthin: Warthin’s sign (exaggeration of pulmonary sounds in acute pericarditis), Warthin’s tumor (benign salivary gland tumor with lymphoid tissue covered by epithelium), and Warthin–Finkeldey giant cells (multinucleated giant cells seen in the lymphoid tissues of patients with measles). Moreover, he translated Ernst Ziegler’s (1849–1905) Lehrbuch der allgemeinen und speziellen pathologischen Anatomie und Pathogenese [Text-Book of Pathological Anatomy and Pathogenesis] (two volumes; Jena, 1881–1882) from German into English. Warthin was, as one would say today, very well-networked and held important posts in numerous medical societies. In person and manner, Warthin was a model of virile fastidiousness. […] Warthin’s approach to pathology was based upon a familiarity with and keen interest in internal medicine. He had a full appreciation of the biological significance of pathology, but to him, study in this field represented a particular opportunity for advancement of medicine as a science. (Anonymous 1932: 134–35) [And Rous later noted]: Warthin was bright-eyed and fresh-colored, quick and strong. He was drastic yet kind, earnest yet cheerful, and most sensitive to beauty. He loved music, gardens, books, and friends. (Rous 1936: 494)

Warthin’s research on the familial incidence of cancer had a particularly lasting impact. In 1895, he initiated one of the most thoroughly documented and longest family histories ever recorded (Warthin 1913, 1925). Recently, a new update of this family, originally referred to as Warthin’s family G and subsequently described as Lynch syndrome family, has been published (Douglas et al. 2005). “He [Warthin] can properly be called the father of cancer genetics,” Henry T. Lynch affirmed, 90 years after the beginning of this study (Lynch 1985). In his later years, Rous drew attention to some forgotten, yet at their time very far-sighted, works of his teacher. In 1904 and again in 1906 Professor Aldred Warthin of the University of Michigan, a consummate pathologist whose abilities I came to know through serving under him as instructor, reported facts making plain that human leukemia is a neoplastic disease; and in 1907 he published a study showing that this held true of a leukemia he came upon in a chicken [Warthin 1907. At the end of this paper he stated: “The problem of leukemia, then, becomes identical with that of malignant neoplasms in general.” Not bad for 1907]. No causative agent was then perceptible within the neoplastic tissue, but in 1908 two Dutch [sic, correct would be Danish] workers, Ellermann and Bang, reported on a virus as causing a chicken leukemia [Ellermann and Bang 1908]. Soon after, they procured another agent from a leukemia of a differing sort, and by means of these agents they transferred the two diseases in fowl after fowl. Their findings were wholly convincing yet were written off because leukemia was not generally realized then to be a neoplastic disease. Indeed, this did not come about until the 1930s. Warthin’s papers had been completely overlooked. He and the [Danish] workers were more than 20 years ahead of their time. (Rous 1967a: 844)

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In the summer of 1898, Warthin was a guest of Schmorl at Dresden Friedrichstadt Municipal Hospital. During these months, he performed a number of dissections, the protocols of which are still preserved today in the journals of the institute (Fig. 1.4). Warthin must have enjoyed his stay so much that he – several years later – recommended his protégé Rous to go to Dresden, too, and work with Schmorl. From his own experience, he knew that for this purpose a sufficient command of the German language was required. As mentioned before, he proposed to Rous to finance his trip and stay in Dresden. Rous gladly accepted this suggestion and remained grateful to his mentor throughout his life. One can assume that Warthin also saw the famous Dresden Dance of Death while he was in Dresden, an over 12-m long sandstone relief with 27 figures dating from the year 1534 (Dresdner Totentanz). He must have pointed this out to Rous too. In any case, Rous titled his later lecture in Oxford, named after the British humanist and physician Thomas Linacre (1460–1524), “The Modern Dance of Death” with a picture of “The Physician” from Holbein’s “Dance of Death” from 1523 to 1526 on the cover of the printed version (Rous 1929). Warthin had been engaged with this subject for many years and published an in-depth and famous study in 1931 which traces the dance-of-death motif through six centuries (Warthin 1931).

Georg Schmorl: A Scientist with Most Infectious Enthusiasm The Pathological–Anatomical Institute of the Dresden Friedrichstadt Municipal Hospital had existed since 1849 and since 1894 was situated in a spacious new building (the present Institute of Pathology “Georg Schmorl”) (Kunze 1999: 22–25, 70–79) (Fig. 1.5). It was the domain of well-known pathologists, among them were Albert von Zenker (1825–1898), Felix Victor Birch-Hirschfeld (1842–1899), and Adolf Neelsen (1854–1894). Since 1894, Christian Georg Schmorl (1861–1932) (Fig. 1.6) had been head of the institute, which was already then very attractive, and helped give the place its special aura. Again and again, physicians from many countries came to Dresden to work with Schmorl. Their number reached hundreds, which is why it was not possible to mention the names of later prominent guest researchers in Schmorl’s obituaries. For instance, the British pathologist Hubert Maitland Turnbull (1875–1955) was a guest researcher at the institute from 1905 to 1906, and after him a long series of British scientists. Under the influence of Schmorl, Turnbull later performed a great service to British pathology (Russell 2004). Work in morbid anatomy at Dresden under the inspiring guidance of Professor Georg Schmorl revolutionized Turnbull’s ideas about his future. Schmorl was noted for his work on bone pathology, and Turnbull had gone to Dresden primarily to become versed in this as a prelude to entering orthopedic surgery. In the early years of this century morbid anatomy was regarded, at least in this country, as a subject which had nothing more to offer: it had been sucked dry by Virchow and his school, and bacteriology was in the ascendant. But at Dresden the lively teaching and work of Schmorl gave the lie to all this; so much so that Turnbull resolved to give his future to morbid anatomy and to redeem its status in England. (Russell 1956)

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Fig. 1.4 Excerpts from autopsy reports written by Warthin 1898 in Dresden. Reproduction with the permission of the Archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

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Fig. 1.5 View of the Pathologic–Anatomical Institute, Dresden Friedrichstadt Municipal Hospital, in the year 1907, seen from Friedrichstrasse. The institute’s auditorium is located on the left side under the cupola. On the right, St. Matthew’s Church can be seen, which was constructed according to the plans of Daniel Pöppelmann in 1732. Contemporary postcard. Reproduction with the permission of the archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

Fig. 1.6 Portrait of Christian Georg Schmorl. Oil painting (118 × 85 cm) by Robert Sterl (1867–1932), an important representative of German Impressionism, 1921. Reproduction with the permission of the Dresden Friedrichstadt Municipal Hospital, photo: Martin Würker

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The high standard of British pathology that is based on the schools of Schmorl and Turnbull (Storey 2008) certainly was a prerequisite for the remarkable performance that was achieved in this country in the field of chemical carcinogenesis since the 1920s (Lawley 1994). Like the famous German poet Gotthold Ephraim Lessing (1729–1781), Schmorl attended the renowned Fürstenschule (the Prince’s school) of St. Afra in Meißen. Later, he always emphasized the enduring influence of his schooling. After graduating, he studied mathematics and natural sciences for several semesters, before deciding to study medicine. After earning his doctorate in medicine in 1887, he became the assistant of Birch-Hirschfeld in Leipzig. There, he qualified to become lecturer in pathology in 1892. After Neelsen’s early death, he took on the position as prosector and director of the Pathological Institute of the Dresden Friedrichstadt Municipal Hospital in 1894, a position he held until 1932 (from 1903 on, as professor) (Turnbull 1932; Geipel 1934; Junghanns 1983; Scholz 2007). Schmorl was particularly interested in microscope technology. He was the author of the standard textbook “Pathological Histological Examination Methods” (1897), which within 37 years had seen 16 editions and throughout Germany became an indispensable reference as “Der kleine Schmorl” (the little Schmorl) for generations of young pathologists. Moreover, he had an affinity for photography, microphotography, and X-ray photography which he had developed for use in pathology and in which he achieved true mastery; preferably, he wanted to perform all the necessary work with his own hands. Noteworthy are his pioneering works on bone pathology, especially the work in which he for the first time describes “Nodulus intraspongiosus Schmorl.” Through his unique collection of pathological bone specimens (the “Georg Schmorl” Pathology Collection), he gained an international reputation. “Schmorl was possessed of tireless energy, a most infectious enthusiasm, great diligence, an astonishing memory and a lively imagination. To search with knife or microscope was obviously a joy to him” (Turnbull 1932: 982).

Peyton Rous in Dresden Whether Rous only stayed a summer (Anonymous 1966), a few months (Huggins 1970), or a whole year (Andrewes 1971; Dulbecco 1976) in Dresden cannot be concluded from the preserved documents. What can be verified is that he performed 23 dissections (Fig. 1.7) between the beginning of July until the end of August 1907 (Wunderlich and Kunze 2008). As the protocols show, he was proficient in German. Furthermore, a photograph is preserved showing Rous and other physicians together with Schmorl (Fig. 1.8) – further evidence for Rous’ stay in Dresden. The picture deserves a place in a future biography of Rous to be written by historians of science. Schmorl, the passionate photographer, was probably responsible for the carefully staged arrangement of people and objects in the photograph.

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Fig. 1.7 Excerpts from autopsy reports written by Rous 1907 in Dresden. Reproduction with the permission of the Archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

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Fig. 1.8 Christian Georg Schmorl (fourth from left, seated) surrounded by visiting researchers (among them, the almost 28-year-old Peyton Rous, third from left) and some assistants. Pathological– Anatomical Institute of Dresden Friedrichstadt Municipal Hospital, summer 1907. Gustav Molineus (1880–1954), later professor of medicine in Düsseldorf, is standing to the left of Rous. Second from the right is Curt Oehme (1883–1963), later professor of internal medicine in Heidelberg. Details regarding the other people depicted in the photograph are not preserved. The photograph is clearly posed, as can be seen in the symmetrical arrangement of the people, among other things. The pathologists’ most important equipment of that time (microscope, hand microtome, staining solutions, and solvents) round off the picture. Reproduction with the permission of the Archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

In view of the severe destruction through the devastating air raids in Dresden in February 1945 and the worst flood of the century in August 2002, which did not exclude Dresden Friedrichstadt Municipal Hospital, it is a stroke of good fortune that these documents could survive the past 100 years. “No hint of war in the air,” is how Rous in 1966 melancholically remembered the undamaged Dresden of 1907 (Anonymous 1966). On June 15, 1907, an article by Rous was published in the Journal of Infectious Diseases in which he suggested an improvement of Schmorl’s celloidin-plate method (Rous 1907). This enabled the simultaneous staining of many paraffin sections with additional dyes, making the overall staining procedure more efficient.

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This work, which apparently already originated in Ann Arbor, must have pleased Schmorl, the methodologist in quest of perfection. In a way, it was the admission ticket for Rous. From reports of other Schmorl students (Turnbull 1932; Geipel 1934; Junghanns 1983), we know a great deal about the work flow in the Schmorl Institute. Every coworker had to prepare his microscopic specimens himself, cut and stain them as well as obtain the necessary tools (staining solutions, microscope slides, and glass covers) himself. Although very many autopsies had to be performed, Schmorl found the time to discuss the results in the autopsy room every day and to give important information to the other autopsists. On Saturday mornings, the always well-attended demonstrations took place in the auditorium, which physicians from other institutions were allowed to attend as well. “Schmorl’s teaching was of the nature of personal coaching, and his pupils, who were all graduates, had the inestimable advantage of learning his method of working. […] He gave without stint from his store of knowledge, whether published or not, to any who wanted to learn” (Turnbull 1932: 984). Supposedly, Rous learned quite a lot about how to present results as well. Many of his later papers were “illustrated with excellent photographs and microphotographs […]. Descriptions were detailed, for Rous liked to have his observations fully documented, and the accounts were often full of vivid imagery” (Andrewes 1971: 648). Like Schmorl, he “photographed with meticulous attention to detail” (ibid: 652). Besides pathology, bacteriology was another key area in the Schmorl institute. A bacteriological research center was opened in 1897 in the Dresden Municipal Hospital, subordinate to the Pathological Institute and thus to Schmorl. By 1907, the number of examinations had increased to almost 7,000 per year. Among them were cases of important infectious diseases, such as diphtheria, typhoid fever, cholera, infection of wounds, anthrax, tetanus, influenza, pneumonia, tuberculosis, syphilis, and rabies. Therefore, Rous must have learned many new things about infectious diseases. This may have been unexpected for him, since for a long time his teacher Welch had not considered the etiological role of bacteria as having any particular significance (Temkin 1950) and Warthin had concentrated solely on research on syphilis and tuberculosis (Anonymous 1932; Rous 1936). It was also not clear at that time if the still young field of bacteriology was to be considered part of pathology or rather of hygiene. In any case, the broadening of Rous’ horizon very soon had consequences for his own research. Even in 1923, Rous commented on his own “inborn lack of aptitude for bacteriology” (Rous to Gye, quoted from Becsei-Kilborn 2010: 141). As Warthin had intended, the stay in Dresden superbly rounded off Rous’ training. Here, he experienced methodological perfection using state-of-the-art technology. After Welch and Warthin, Schmorl was another research personality who had a profound impact on his further career. Following the extraordinary achievements of bacteriology at the end of the nineteenth century and the associated perception that many diseases had a monocausal etiology, around 1900, many hopes were also placed on finding a rapid solution to the problem of cancer. In the following years, it was up to Rous to work out the first evidence of a multicausal explanation of carcinogenesis in order to later come to the confident realization that under natural conditions malignant tumors gradually develop in a multifactorial process (Rous 1967b).

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Simon Flexner and Medical Discovery During his studies, Rous was infected with tuberculosis bacteria while performing an autopsy, developed lymph-node TB, and was forced to interrupt his studies for a year. After his stay in Dresden, he was diagnosed with pulmonary tuberculosis, which forced him to pause again, but fortunately he recovered quickly. Afterward, it was Warthin, once again, who gave him a crucial bit of information: Dr. Warthin told Peyton Rous that the Rockefeller Institute for Medical Research [in New York] was casting a wide net of grants for beginners, and he asked him if Peyton would like him to apply for one that would free Peyton for experimental work. That grant enabled Rous to find out enough about lymphocytes to be deemed worth publishing in the Journal of Experimental Medicine [Rous published in 1908 three papers on lymphocytes in this journal], edited by Simon Flexner, who was also the director of the Institute; and after another few months Flexner asked Rous to take over the laboratory for cancer research [at the Department of Bacteriology and Pathology] which Flexner was quitting to learn more about poliomyelitis, then crippling many American children. (Anonymous 1966)

The Rockefeller Institute for Medical Research (from 1964 on, Rockefeller University) was founded in 1901 as a private institute by John D. Rockefeller (1839–1937) and opened in 1904. It was the first American institute that exclusively engaged in biomedical research. At first, research on infectious diseases and bacteriology was the focus of interest; however, the problem definition was very broad and comprised many branches of basic biological research (Corner 1964; Hollingsworth 2004). The founding director was the pathologist Simon Flexner (1863–1946) (Fig. 1.9), who held this position from 1901 to 1935 with great success. In his view,

Fig. 1.9 Photograph of Simon Flexner. Courtesy of the Rockefeller Archive Center

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the institute was “an attempt to add knowledge by discovery and to apply that knowledge to the prevention and alleviation of disease” (Rous 1949: 416). Flexner had studied pharmacy and medicine at the University of Louisville, and earned his PhD in 1889. As 27-year-old, he came to William Welch at The Johns Hopkins University in Baltimore and there experienced the stimulating founding phase of the School of Medicine. Welch became his teacher, promoter, and later his fatherly friend; Flexner soon became Welch’s closest colleague. The focus of Flexner’s research was on experimental pathology, bacteriology, and immunology. Because of his expertise in these areas, he was repeatedly appointed head of commissions to combat epidemics at home and abroad. In the years 1893 and 1909, he spent some time in Europe in order to work with Friedrich Daniel von Recklinghausen (1833–1910) in Strasbourg and later with Emil Fischer (1852–1919) and Ernst Leopold Salkowski (1844–1923) in Berlin, among others. Prior to becoming director of the Rockefeller Institute, he had been professor for Pathological Anatomy at The Johns Hopkins University since 1899 and since 1900 Head of the Department of Pathology at the University of Pennsylvania (Corner 1972). As researcher, Flexner became known especially for his work on epidemic cerebrospinal meningitis. At the Rockefeller Institute, he developed a serum treatment (Flexner’s serum), which proved successful first with monkeys and later, during an epidemic, with humans. A treatment with serum from immunized horses was introduced simultaneously and independently of Flexner by the German physician Georg Jochmann (1874–1915). Flexner’s research on poliomyelitis was of particular importance for Rous and the work that he had already begun at the Rockefeller Institute. At that time, outbreaks of polio were a major problem, not only in America. From 1908 to 1909, the team led by Flexner identified a filterable virus (!) as responsible agent of the disease [the RNA virus which was discovered independently in Vienna by Karl Landsteiner (1868–1943) and Erwin Popper (1879–1955) is today assigned to the Picornaviridae family]. Flexner and colleagues also identified the transmission path of the virus. They showed that it enters the body through the nose, attacking the olfactory nerve. They could also experimentally infect monkeys with poliomyelitis by administering the virus in the nasopharynx (Flexner and Lewis 1909). For his work on serum treatment of epidemic cerebrospinal meningitis and on transmission of poliomyelitis to monkeys, Flexner was nominated ten times for a Nobel Prize in Physiology or Medicine (Nomination Database 1901–1951), but did not get the Prize. However, contemporaries and historians regard Flexner’s greatest achievement to lie in the organization of medical research and especially in the unprecedented success of the Rockefeller Institute (Corner 1964; Hollingsworth 2004). “Perhaps no man save Welch has done so much for American medicine” (Rous [commenting on Flexner] 1948: 613). Although many other high-ranking researchers had worked at the Rockefeller Institute for a long time and could have been considered to pay tribute to Flexner in obituaries, this task fell on Peyton Rous (Rous 1948, 1949). He did it in a loving way. “He [Flexner] had proved that the experimental method can meet human needs if it be given its head, wide and free; and he had shown that discoverers can be discovered” (Rous 1948: 613).

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At the beginning, Flexner had also been head of a laboratory for cancer research at the Rockefeller Institute. Together with James W. Jobling (1876–1961), he discovered a transplantable tumor, a rat adenocarcinoma most likely of prostatic origin (Flexner–Jobling carcinoma). This particular tumor has served for many decades as a unique test material in cancer research (Triolo 1964: 12; Corner 1964: 59), for instance in the famous experiments of Otto Warburg on the energy metabolism of cancer cells. Because of urgent work in other areas, particularly in the research of poliomyelitis, Flexner gave up his position as head of the laboratory, after he had found an appropriate successor in the then 30-year-old Rous. However, a number of obstacles had to be overcome before Rous could take over. Rous later described that at this time youngsters were warned off from the Institute with the slurring phrase “Rockefeller money” (Rous 1949: 418). Since cancer research was considered a futile field among scientists, even Welch had implored him: “Whatever you do, don’t commit yourself to the cancer problem” (Andrewes 1971: 644). Luckily, Rous did not follow this advice. Presumably, it was again Warthin who, besides Flexner, encouraged Rous despite all the prophecies of doom to turn toward cancer research. Toward the end of his life, Rous was able to acknowledge that “[Cancer is] the most intellectually worthwhile of all diseases” (Rous to Heagensen 1957, quoted in Becsei-Kilborn 2010: 117).

A Discovery Greeted with Skepticism At the Rockefeller Institute, Rous soon had the luck of the diligent: still in 1909, a poultry farmer consulted him who had noticed a big tumor in the chest of one of his chickens (a Plymouth Rock breed). Worrying that this could be a threat for other chickens of his flock, the breeder consulted different pathologists without success. Only Rous realized that this was a spindle cell sarcoma. This malignant tumor served Rous in the following years as a model for basic research studies; it was, therefore, later termed the Rous sarcoma (classic chicken sarcoma). To begin with, Rous succeeded for the first time in the transmission of a chicken tumor by means of injection into healthy animals – however, only when the recipients were genetically compatible animals (Rous 1910). If a homogenate of such tumors was passed through a filter that was not permeable to cells or bacteria, this filtrate in turn generated spindle cell sarcomas in healthy chicks after inoculation, which Rous as an experienced pathologist could clearly identify (Rous 1911a, b). Although he initially did not use the term “virus” to explain his results (he spoke cautiously only of a “filterable agent”), the chosen experimental setup did not allow for any other interpretation: for the first time, it was shown that a “real” tumor was caused by an infectious agent, probably a virus. Leukemias which showed similar results, however less convincing, were reported by Vilhelm Ellermann (1871–1924) and Oluf Bang (1881–1937) (Ellermann and Bang 1908). However, at that time, leukemias were not considered to belong to the neoplastic diseases. In the 1890s, viruses as novel biological entities were recognized for their characteristic of passing through

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bacteria-proof filters while preserving their biological activity. In 1911, Rous published his findings. He soon succeeded in developing a detection method for the agent on the chorioallantoic membrane of the chicks. Shortly thereafter, he proved a virus etiology for two further distinct chicken sarcomas. Today, the term Rous sarcoma virus (RSV) not only includes Rous’ original isolates, but numerous other chicken viruses that have been isolated independently from each other and that induce sarcomas through a genetic mechanism similar to the original isolate. RSV was the first known tumor virus and the first representative of the retroviruses. The initial euphoria about Rous’ discovery was soon followed by disillusionment. Apparently, viruses were not generally responsible for the induction of tumors. Also, the virus theory could hardly be brought into conformity with contemporary beliefs about the causal genesis of cancer. Furthermore, with the outbreak of World War I, Rous had to devote himself to other subjects (among them, with great success, the conservation of blood). Recent historical research substantiates that Rous’ discovery triggered a longlasting scientific discussion (van Helvoort 1999, 2004; Becsei-Kilborn 2010). However, a detailed description of this discussion would go beyond the framework of this article. Rous found himself facing considerable skepticism, even resistance. Even James B. Murphy (1884–1950), temporarily his close colleague when carrying out these experiments and who later gained increasing influence in the US cancer research community, did not believe in the involvement of viruses, but interpreted the agent as a “transmissible mutagen.” Later, he thought to have proved it to be a ferment. Rous’ sharpest critic was James Ewing (1866–1943), who was very conscious of his power as pathologist and director of research at the Memorial Hospital for Cancer and Allied Diseases in New York City. Ewing largely rejected experimental pathology for the research of cancer etiology and believed the origin of cancer to be within the cell itself. Frequent points of criticism by other scientists about Rous’ experiments were: (1) when preparing the filtrate, a few tumor cells could have passed through the filter; (2) the effect might have been caused not by an infectious agent, but by products synthesized by tumor cells; (3) it was doubted that the induced sarcomas were real tumors, and it was suggested that they should rather be seen as “granulomas”; (4) since the agent could not infect most cells – with the exception of certain chicken cells – the high specificity of its effect remained enigmatic; and (5) whether tumor development could be caused by external factors at all or if tumors develop in an endogenous way was not decided at that time. However, the belief favoring purely endogenous reasons prevailed at that time. Although Rous could dispel some objections, he did not succeed in convincing his critics. All in all, the results were for a long time considered an oddity of chickens that were of no relevance to the situation in humans. But there were also advocates for Rous, in America most notably Flexner and the highly respected Leo Loeb (1869–1959). In Japan, Akira Fujinami (1870–1934) found a very similar agent in chicken sarcomas that is known today as Fujinami virus (Fujinami and Inamoto 1914). From the mid 1920s on, much-discussed studies by the British scientists Willam E. Gye (1884–1952) and Christopher H. Andrewes (1896–1988) kept Rous’ experiments from being forgotten. Rous himself did not take up work in this area until 1933, after Richard E. Shope (1901–1966) had

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succeeded with a cell-free transmission of papillomas of cottontail rabbits (Shope 1933). Shope left the alleged virus tumor to his colleague Rous for further experiments. The model gave Rous the opportunity to study many characteristics of the natural development of tumors. Under special conditions, real carcinoma developed from the papilloma. Rous found that tumors develop gradually. A phase of tumor initiation is followed by phases of promotion and progression up to the fully developed metastasizing tumor (Rous and Kidd 1941). This may cause a synergistic effect of viruses and chemical carcinogens. The terms “latent or dormant tumor cells” and “cancer as a multifactorial disease” were also introduced by Rous on the basis of these experiments (see Rous 1967b). The real breakthrough of the virus theory of cancer came in the 1950s. In 1951 and 1954, respectively, Ludwik Gross (1904–1999) in New York and Arnold Graffi (1910–2006) in Berlin were able to prove that viruses caused lymphatic (Gross 1951) and myeloid leukemia (Graffi et al. 1954) in mice. Numerous other isolations of DNA- or RNA-containing oncogenic viruses were made in a variety of animal species. Many of these became outstanding models of the emerging molecular biology. The golden age of tumor virology had begun. Rous had always believed in the viral nature of his agent. In a letter to his British colleague Stephen L. Baker (1888–1978), he confessed in 1930: “My own belief has always been that the agents causing these tumors are viruses.” But at that time, he had to continue carefully “[…] though the statement is confidential to you” (quoted from Becsei-Kilborn 2010: 132).

The Great Good Fortune of Rous I will always consider it good fortune that just when I finished my apprenticeship in physics, chemistry and medicine, the Kaiser Wilhelm Institute for Biology was founded in Berlin, and that upon its founding Emil Fischer, then Vice President of the Kaiser Wilhelm Society, forthwith appointed me as scientific member [upon the recommendation of Theodor Boveri (1862–1915)]. […] I have no doubt that my scientific achievements can mainly be attributed to the unusual degree of freedom and independence that I […] have received. [German in original]

This is how Otto Warburg (1883–1970) commented in retrospect on his career, according to his student and biographer Sir Hans Krebs (1900–1981) (Krebs 1978: 351; Krebs 1981). Warburg and Rous, who were almost the same age, were probably the most important cancer researchers of the early twentieth century (Fujimura 1996a, b). Like Warburg, Rous had completed a long period of apprenticeship served with Welch, Warthin, and Schmorl when he joined the new Rockefeller Institute, in which he then (from 1920, as member) was to experience an impressive career. Rous, too, always regarded these circumstances as good fortune: “Environment is everything for a scientific man,” he wrote (Rous 1949: 412). Both scientists benefited from developments that took place in these years. On the one hand, the character of cancer research changed: “Cancer research became an experimental science at the turn of the century, along with most of the biological

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sciences” (Fujimura 1996b: 247). On the other hand, novel institutions were founded for nonuniversity research: at first, the Rockefeller Institute for Medical Research in New York which later served as a model for the foundation of the German Kaiser Wilhelm Society with its various institutes. Therewith, a culture of excellence was institutionalized (Hollingsworth 2004) that was to lead to great achievements in the cases of Rous and Warburg: “Discovery [is] the most satisfying of human experiences” (Rous 1949: 423). However, the priorities which Flexner set from the beginning for the Rockefeller Institute were also a stroke of good fortune for Rous. Until the end of Flexner’s term of office, the role of pathology was sacrosanct: “Pathology is far more important for us than physiology and pharmacology, and the background of medicine than general science. Our pathologists are all moving on; pathology is the fundamental branch of medicine” (Flexner 1993, quoted from Corner 1964: 187). This was in the spirit of Rous, who told Jacob Furth (1896–1979) in 1942: “Experimental pathology has always been to me one of the most exciting of human activities” (quoted from Becsei-Kilborn 2010: 116). As mentioned before, Flexner was also a major proponent of virus research. At precisely the time when Rous began his research on chicken sarcoma, the studies of Flexner’s team reached a milestone with the evidence of a virus etiology of poliomyelitis. Rous’ decisive proposition – a cell-free transmission of chicken sarcoma – probably arose out of discussions within the Rockefeller Institute. Fortunately, Rous could count on Flexner’s continuing support during the yearlong controversies on the significance of his discovery. Rous himself was very confident early on. The identification of the chicken sarcoma had already been a considerable achievement in 1909 (very little research had been done on poultry tumors at that time), and he was now able to fend off his critics because of his versatility and expertise as a pathologist. He was aware that the nature of the tumors induced by cell-free transmission was a decisive argument. Evidence of their malignancy was in every respect unambiguous: cell-free filtrates of the same tumor induced reproducible, histologically identical neoplasms, each independently obtained agent induced tumors of various types, and all induced tumors had the ability for invasive growth and metastasizing, i.e., they were genuine malignant neoplasms. At the latest during these experiments, it became clear that Peyton Rous had been very fortunate in the choice of his teachers. He kept them in honorable memory throughout his life. In his obituaries for Simon Flexner, he also remembered William H. Welch, who had imparted a vision of an experimental pathology to the young Rous and who let him take part in the vibrant ambiance of scientific discovery (Rous 1948, 1949). Rous was indebted to Aldred S. Warthin for a thorough education as pathologist and for key professional guidance, such as providing the contact to Schmorl and Flexner, as well as encouraging the shift to cancer research. He commemorated him in a beautifully written obituary (Rous 1936) and as late as 1967 alluded to long-forgotten work of his teacher (Rous 1967a). In Dresden, he encountered European science and culture and was full of praise even 60 years later (Anonymous 1966). He studied the advances in bacteriology in Germany under Georg Schmorl. In the working method of Schmorl, he saw methodological perfection

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combined with the use of modern technical (especially photographic) equipment. Thus, he was well-prepared to seize the opportunities that were offered to him at the Rockefeller Institute. Among his teachers and promoters, it was Simon Flexner, who discovered the discoverer in Rous, whom he had known the longest and probably revered the most. He commemorated him in obituaries that are well worth reading even today (Rous 1948, 1949). Flexner was the guarantor of freedom of research at the Rockefeller Institute, and this proved to be of crucial importance for the seminal discovery of tumor-inducing viruses.

Epilogue Although Peyton Rous is first and foremost known as the “father of the tumor virus” (van Epps 2005; Moore and Chang 2010), his name is also associated with other important achievements. At the Rockefeller Institute, his well-rounded education bore rich fruit. During World War I, the preservation of living blood cells was an urgent medical need. Together with Joseph Richard Turner, Jr. (1889–?), Rous developed a method which enabled long-term storage of blood without clotting. “In a mixture of 3 parts of human blood […], 2 parts of isotonic citrate solution […] and 5 parts of isotonic dextrose solution […], the cells remain intact for about 4 weeks” (Rous and Turner 1916). This method enabled Oswald H. Robertson (1886–1966), the pioneer of transfusion medicine, to set up the world’s first blood bank behind the front line in Belgium in 1917. Rous cooperated closely with Robertson, as a number of joint publications demonstrate. The “Rous–Turner solution” is still in use for human blood transfusions, and in the 90 years since its first use it has saved countless lives. Rous was particularly proud of this achievement – and rightly so. A cell and tissue culture method which has been a laboratory standard up to the present day can also be traced back to Rous’ research. To obtain a suspension of individual living cells from the fixed tissue, he performed a digestion with trypsin (Rous and Jones 1916). The optimal trypsin concentration had to be determined separately for each kind of tissue. Once again, with this method, Rous proved his talent for finding solutions that could be carried out easily. In conjunction with his work on blood conservation, Rous also focused on the functions of the liver, gall bladder, and kidney as well as on the permeability of small vessels. The Rous test for hemosiderin in urine to detect hemosiderosis (Rous 1918) was one of the results of his research at that time. Any appraisal of Rous would be incomplete if it neglected to draw attention to Rous’ great merits as long-time editor (from 1922 to 1970) of the renowned Journal of Experimental Medicine. He invested a lot of time and energy in this task. According to contemporary witnesses, he continued working well into old age and was equally meticulous in revising manuscripts as he was in conducting experiments in the laboratory. Both activities – along with his modesty – contributed to Rous’ extraordinary reputation. As a young man, Cornelius Rhoads (1898–1959) experienced Rous as he revised his manuscript, and Rhoads described this later

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quite humorously: “Dr. Rous, gravely and patiently, reviewed my efforts with me, demolished my conclusions, refuted my claims and made clear the proper use of my native tongue. He then rebuilt on the ruins such a clear picture of the problem, and the procedure required to solve it, that my conceit was converted almost imperceptibly to inspiration, my enthusiasm to resolution. As I left the generous, patient, and kindly man, I was no longer the same individual” (Rhoads 1959). Time and again during the twentieth century, the RSV as a model for fundamental studies proved to be a serendipitous choice, and its scope extended far beyond oncology. After Rous, several scientists received the Nobel Prize in Medicine for studies in which this virus played a decisive role. In 1974, one of the prize winners was Albert Claude (1899–1983), who pioneered the fractionation of cells by differential centrifugation. This discovery was the prerequisite for making images of the structure of the RSV, among others, using an electron microscope – the first time for a tumor virus (Claude et al. 1947). Howard M. Temin (1934–1994) and David Baltimore (born 1938) discovered reverse transcriptase and were honored for their work in 1975. Their research was conducted with Rous sarcoma and Rauscher murine leukemia viruses (Temin and Mizutani 1970; Baltimore 1970). J. Michael Bishop (born 1936) and Harold E. Varmus (born 1939) received the Nobel Prize in 1989 for the discovery of cellular oncogenes. They were able to identify the cellular origin of retroviral oncogenes, among others, first for the src gene, the viral homolog of which is responsible for the transforming activity of the RSV (Stehelin et al. 1976). For their discovery of human immunodeficiency virus (Barré-Sinoussi et al. 1983), today besides the RSV the most well-known retrovirus, Françoise Barré-Sinoussi (born 1947) and Luc Montagnier (born 1932) were awarded the Nobel Prize in 2008 along with Harald zur Hausen. Other equally important discoveries in the field can also be mentioned, further substantiating George Klein’s appraisal: “Few fields in modern biology and certainly no other field in cancer research can be traced back to the work of one man in the same way that the foundation in the field of viral oncology can be traced back to the work of Peyton Rous in 1911” (Klein 1980). Acknowledgments We thank Carol Oberschmidt (Berlin) for translation and Professor Manfred F. Rajewsky (Essen) for critical reading of the manuscript.

References Andrewes CH (1971) Francis Peyton Rous 1879–1970. Biographical Memoirs of Fellows of The Royal Society 17:643–62 (with bibliography) Anonymous (1932) Aldred Scott Warthin. J Pathol Bacteriol 35:133–5 Anonymous (1966) Peyton Rous. Biography. The Nobel prize in physiology or medicine 1966. http://nobelprize.org/nobel_prizes/medicine/laureates/1966/rous-bio.html. Accessed 20 Jan 2011 Baltimore D (1970) RNA-dependent DNA-polymerase in virions of RNA tumour viruses. Nature 226:1209–11 Barré-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vézinet-Brun F, Rouzioux C, Rozenbaum W, Montagnier L (1983) Isolation of a

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T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868–71 Becsei-Kilborn E (2010) Scientific discovery and scientific reputation: the reception of Peyton Rous’ discovery of the chicken sarcoma virus. J Hist Biol 43:111–57 Brieger GH (1970) Welch, William Henry. Dictionary of Scientific Biography 14:248–50 Claude A, Porter KR, Pickels EG (1947) Electron microscope study of chicken tumor cells. Cancer Res 7:421–30 Corner GW (1964) A history of the Rockefeller Institute, 1901–1953. Origins and growth. New York, Rockefeller Institute Press Corner GW (1972) Flexner, Simon. Dictionary of Scientific Biography 5:39–41 Douglas JA, Gruber SB, Meister KA et al (2005) History and molecular genetics of Lynch syndrome in family G: a century later. J Am Med Assoc 294:2195–202 Dresdner Toten Tanz (the Dresden dance of death) [in German]. http://www.derevo.org/common/ de/actions/projects/totentanz/tt1.html. Accessed 20 Jan 2011 Dulbecco R (1976) Francis Peyton Rous. October 5, 1879–February 16, 1970. Biogr Mem Members Natl Acad Sci 48:275–306 (with bibliography) Ellermann V, Bang O (1908) Experimentelle Leukämie bei Hühnern. Zentralbl Bakteriol 46:595–609 [in German] Flexner S (1943) William Henry Welch 1850–1934. Biogr Mem Natl Acad Sci 22:215–31 Flexner S, Flexner JT (1941) William Henry Welch and the heroic age of American medicine. New York, Viking Press [Reprinted 1966] Flexner S, Lewis PA (1909) The nature of the virus of epidemic poliomyelitis. J Am Med Assoc 53:2095 Fujimura JH (1996a) Standardizing practices: a socio-history of experimental systems in classical and virological cancer research, ca. 1920–1978. Hist Philos Life Sci 18:3–54 Fujimura JH (1996b) Crafting science. A sociohistory of the quest for the genetics of cancer. Harvard University Press, Cambridge, MA; London, UK Fujinami A, Inamoto K (1914) Über Geschwülste bei japanischen Haushühnern insbesondere über einen transplantablen Tumor. Z Krebsforsch 14:94–119 [in German] Geipel P (1934) Georg Schmorl. Verh Dtsch Pathol Ges 27:326–39, (with bibliography) [in German] Graffi A, Bielka H, Fey F et al (1954) Gehäuftes Auftreten von Leukämien nach Injektion von Sarkom-Filtraten. Naturwissenschaften 41:503–4 [in German] Gross L (1951) “Spontaneous” leukemia developing in C3H mice following inoculation, in infancy, with Ak-leukemic extracts, or Ak-embryos. Proc Soc Exp Biol Med 76:27–32 van Helvoort T (1999) A century of research into the cause of cancer: is the new oncogene paradigm revolutionary? Hist Philos Life Sci 21:293–330 van Helvoort T (2004) The start of a cancer research tradition: Peyton Rous, James Ewing, and viruses as a cause of cancer. In: Stapleton DH (ed) Creating a tradition of biomedical research: contributions to the history of The Rockefeller University. The Rockefeller University Press, New York, pp 191–209 Hollingsworth JR (2004) Institutionalizing excellence in biomedical research: the case of The Rockefeller University. In: Stapleton DH (ed) Creating a tradition of biomedical research: contributions to the history of The Rockefeller University. The Rockefeller University Press, New York, pp 17–64 Huggins CB (1970) Peyton Rous and his voyages of discovery. J Exp Med 150:734–5 Junghanns H (1983) Georg Schmorl, der Forscher und Lehrer (2.5.1861–14.8.1932). Medizinhist J 18:324–37 [in German] Klein G (1980) Introduction. In: Klein G (ed) Viral oncology. Raven Press, New York Krebs H (1978) Otto Warburg: Biochemiker, Zellphysiologe, Mediziner. Naturwiss Rundsch 31:349–56 [in German] Krebs H (1981) Otto Warburg: cell physiologist, biochemist, and eccentric. Oxford University Press, Oxford, UK Kunze P (1999) Vom Adelspalais zum Städtischen Klinikum. Geschichte des Krankenhauses Dresden-Friedrichstadt. Krankenhaus Dresden-Friedrichstadt, Dresden [in German]

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Lawley PD (1994) Historical origins of current concepts of carcinogenesis. Adv Cancer Res 65:17–111 Lynch HT (1985) Classics in oncology. Aldred Scott Warthin, M.D., Ph.D. (1866–1931). CA: Cancer J Clin 35:345–7 MacCallum WG (1936) Welch, William Henry. Dictionary of American Biography 19:621–4 Moore PS, Chang Y (2010) Why do viruses cause cancer? Highlights of the first century of human tumour virology. Nat Rev Cancer 10:878–89 Nomination Database for the Nobel Prize in Physiology or Medicine, 1901–1951. http://nobelprize.org/nobel_prizes/medicine/nomination/database.html. Accessed 20 Jan 2011 Pathologie-Sammlung “Georg Schmorl”. Technische Universität Dresden [in German] http://publicus.culture.hu-berlin.de/sammlungen/detail.php?dsn=766&view=2. Accessed 20 Jan 2011 Rhoads CP (1959) Citation and presentation of the Academy Medal to F. Peyton Rous, M.D. Bull N Y Acad Med 35:216–9 Rous FP (1907) A method for the simultaneous passage of many paraffin sections through the more difficult stains. J Infect Dis 4:382–4 Rous FP (1910) A transmissible avian neoplasm (sarcoma of the common fowl). J Exp Med 12:696–705 [Reprinted: (1979); ibid 150:729–753] Rous P (1911a) Transmission of a malignant new growth by means of a cell-free filtrate. J Am Med Assoc 56:198 [Reprinted: (1983) ibid 250:1445–1449 and http://caonline.amcancersoc.org/ cgi/reprint/22/1/23. Accessed 20 Jan 2011] Rous P (1911b) A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med 13:397–411 [Reprinted at http://www.euchromatin.com/Rous11b.htm. Accessed 20 Jan 2011] Rous P (1918) Urinary siderosis. Hemosiderin granules in the urine as an aid in the diagnosis of pernicious anemia, hemochromatosis, and other diseases causing siderosis of the kidney. J Exp Med 28:645–58 Rous P (1929) The modern dance of death. Cambridge University Press, Cambridge, UK Rous P (1936) Warthin, Aldred Scott. Dictionary of American Biography 19:493–4 Rous P (1941) The William Henry Welch lecture. I. The conditions determining cancer. II. The known causes of cancer. J Mt Sinai Hosp 8:184–7 Rous P (1948) Simon Flexner and medical discovery. Science 107:611–3 Rous P (1949) Simon Flexner. 1863–1946. Obituary Notices of Fellows of The Royal Society 6, No. 18:408–445 (with bibliography) Rous P. (1966) The dualism of the discoverer. In: Heymann G (ed) Festschrift anläßlich der Verleihung des Paul-Ehrlich-und Ludwig-Darmstaedter-Preises 1966 an Prof. Dr. Peyton Rous. Gustav Fischer, Stuttgart, pp 20–30 [text in German, the English version is unpublished]. Andrewes (1971) and Dulbecco (1976) did not include this paper in the list of all works by Rous. Rous P (1967a) Symposium on RNA viruses and neoplasia: comment. Proc Natl Acad Sci USA 58:843–5, Andrewes (1971) and Dulbecco (1976) did not include this paper in the list of all works by Rous Rous P (1967b) The challenge to man of the neoplastic cell. Nobel Prize lecture. Cancer Res 27:1919–24 Rous P, Jones FS (1916) A method for obtaining suspensions of living cells from the fixed tissues, and for the plating out of individual cells. J Exp Med 23:549–55 Rous P, Turner JR (1916) The preservation of living red blood cells in vitro I. Methods of preservation. J Exp Med 23:219–37 Rous P, Kidd JG (1941) Conditional neoplasms and subthreshold neoplastic states. A study of the tar tumors of rabbits. J Exp Med 73:365–90 Russell DS (1956) Hubert Maitland Turnbull. J Clin Pathol 9:78–9 Russell DS (2004) Turnbull, Hubert Maitland (1875–1955). Oxford Dictionary of National Biography 55:590–1 Scholz A (2007) Schmorl, Christian Georg. Neue Dsch Biogr 23:263–4 [in German]

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Shope RE (1933) Infectious papillomatosis of rabbits. J Exp Med 58:607–24 Stehelin D, Varmus HE, Bishop JM, Vogt PK (1976) DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170–3 Storey GO (2008) Hubert Maitland Turnbull (1875–1955): pathologist at The London Hospital. J Med Biogr 16:227–31 Temin HM, Mizutani S (1970) RNA-dependent DNA-polymerase in virions of Rous sarcoma virus. Nature 226:1211–3 Temkin O (1950) The European background of the young Dr Welch. Bull Hist Med 24:308–18 Triolo VA (1964) Nineteenth century foundation of cancer research. Origins of experimental research. Cancer Res 24:4–27 Turnbull HM (1932) Christian Georg Schmorl. J Pathol Bacteriol 35:982–5 Van Epps HL (2005) Peyton Rous: father of the tumor virus. J Exp Med 201:320 Vogt PK (1996) Peyton Rous: homage and appraisal. FASEB J 10:1559–62 Warthin AS (1907) Leukemia of the common fowl. J Infect Dis 4:369–81 Warthin AS (1913) Heredity with reference to carcinoma. Arch Intern Med 12:546–55 Warthin AS (1925) The further study of a cancer family. J Cancer Res 9:279–86 Warthin AS (1931) The physician of the dance of death. A historical study of the evolution of the dance of death mythus in art. Hoeber, New York Wunderlich V, Kunze P (2008) Vor 101 Jahren: Peyton Rous, ein zukünftiger Nobelpreisträger, im Krankenhaus Dresden-Friedrichstadt. Ärzteblatt Sachsen 19:375–81 [in German]

Chapter 2

Viruses and Cancer: A Historical Perspective – HBV and Prevention of a Cancer Baruch S. Blumberg

Introduction Viruses are thought to be the causative agent of about 15–20% of cancers, including some of the world’s most common cancers. As is the case for most diseases, cancers have multiple causes or factors that contribute to etiology, pathogenesis, prognosis, and response to treatment. In addition to the cancers with identified viral causes, there are probably additional cancer disease entities in which viruses contribute to pathogenesis and in which prevention of infection may significantly alter the outcome. In recent years, there have been reports, primarily in the general press, on the disaffection with the progress of cancer control and treatment. These often refer to the so-called War on Cancer (although that term was not the official designation) initiated on December 23, 1971 and, as perceived by the public, its failure to have a dramatic effect on control and treatment. There have been improvements in treatments for many cancers; for several childhood and usually relatively rare adult cancers, therapies have been impressive. But for many cancers, treatment has resulted in only limited increased survival and the treatments themselves may diminish the quality of life. The search for better treatments is a major research program. That War on Cancer campaign started with many goal-directed research programs. Viral-caused cancer was one of the goal-directed projects based on the models from virus/cancer relations in experimental, domestic, and wild animals. However, human cancers did not appear to closely follow the animal models, and by the mid-1970s support for virus-caused cancers in humans had diminished. But, as in many research areas, interest appears to wax and wane over the years. As evidenced by this book and others on the same topic, the increase in the number of research groups studying cancer and infectious agents, interest is growing once again.

B.S. Blumberg (*) Fox Chase Cancer Center, Philadelphia, PA 19111, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_2, © Springer Science+Business Media, LLC 2012

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Prevention has had a major effect on decreasing the load of cancer in human populations. Primary prevention, for example smoking cessation programs, has resulted in major decreases in the incidence of cancer of the lung and other cancer as well as noncancer diseases. Dietary measures have probably decreased cancer of the colon and other organs. Cancer of the stomach has decreased dramatically, presumably as a consequence of changes in the environment, in diet, or some other factors that are not clearly identified. Secondary prevention, i.e., early detection and treatment, appears to have decreased the cancer load for cancers of the breast, colon, and others. The research on cancer-causing viruses (and other infectious agents) promises to facilitate even greater advances in prevention and treatment. The hepatitis B vaccine was invented in 1969 two years after the discovery and identification of hepatitis B virus (HBV). Product development began in the mid-1970s and it was approved for use in the early 1980s. It is now one of the most commonly used vaccines worldwide. The hepatitis B vaccination campaigns and other control measures have dramatically reduced the incidence of infections, the HBV carrier state, and acute and chronic liver disease. HBV, along with hepatitis C virus (HCV), is an etiological agent for over 80% of all primary cancers of the liver (hepatocellular carcinoma, HCC) and it is expected that in time there will be dramatic drops in the incidence of the cancer. Several studies have already shown significant decreases in the HCC incidence in countries with early and effective vaccination programs and a high incidence of the cancer. The CDC in its Hepatits B Vaccine Fact Sheet stated (Hepatitis B Vaccine: Fact Sheet First Anti-cancer Vaccine (2006) http:// www.cdc.gov/hepatitis, May 17, 2006): Hepatitis B vaccine prevents hepatitis B disease and its serious consequences like hepatocellular carcinoma (liver cancer). Therefore, this is the first anti-cancer vaccine.

The WHO has noted that, second to smoking cessation, HBV vaccination is the major cancer interventional prevention program. In 2007, about 25 years after the approval of HBV vaccine, a second cancer prevention vaccine was accepted by the FDA. Vaccines for several strains of papilloma virus have been shown to effectively prevent cancer of the cervix, other cancers, and common diseases of the reproductive system. Its use is now spreading widely as the appropriate populations for vaccination are being identified. As a consequence of these practical advances and a growing understanding of the viral-caused cancer process at the molecular level, we are now in a period of increased interest in cancer virology, as witnessed by this book and others on the subject, and the organization of new research groups. In this paper, I briefly review some of the established cancer virus relations and then discuss the research, public health, and clinical processes that led to the HBV vaccine and its consequences. The HBV story can serve as an example that can help to advance similar mass prevention programs. Most cancer therapies are dependent on either removing cancer by surgery or by the destruction of existing cancer cell with radiation, chemotherapy, or by altering the host immune system to reject cancer cells. For viral-induced cancers, these harsh methods may not be necessary. The cancer could be averted or delayed by the

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antiviral treatment of those already infected before symptoms appear and, possibly, also after clinical disease develops. This should be less toxic than many current therapies. The effectiveness of antiviral therapy would also be another direct demonstration of the etiological role of a virus. This is an exciting time for virus/cancer studies as the effectiveness of vaccines for at least two common cancers inspires a search for additional viral connections and applications.

Viral Origins of Human Cancers There are several excellent reviews of this subject, with an emphasis on molecular virology, for example Boccardo and Villa (2007) and DeVita et al. (2008). As Boccardo and Villa have noted, the strongest direct evidence for a meaningful etiologic relation is the prevention of the cancer by vaccination. This has been shown for HBV and HCC in large national studies (see, for example, Liang et al. 2009), and for papilloma virus [human papilloma virus (HPV)] and cancer of the cervix in field trials. As noted above, another direct demonstration of etiologic relation would be the prevention and/or treatment of a cancer with an antiviral. There are many other accepted virus/cancer etiologic relations. In many cases, they are believed to be indirect which appears to mean that they do not conform to molecular biological models of carcinogenesis. From a practical point of view, the question of a direct or secondary cause is not as important as an understanding of process that allows practical prevention or treatment. An important feature of cancer prevention by vaccination is the question of instituting universal vaccination to protect the relatively small portion of the population who develop cancer. However, vaccination is further justified if the program also protects against more common diseases. This is true for HBV vaccine, where protection is also offered against acute and chronic hepatitis, cirrhosis, and liver failure and probably some forms of kidney diseases, polyarteritis nodosa, and others. The same could be said for HCV if a vaccine is found for it. The HPV vaccine in addition to protecting against cancer of the cervix and other cancers protects against several common sexually transmitted and other diseases, including condyloma acuminate, warts, and recurrent respiratory papillomatosis. Vaccines are not currently widely used for other cancer-related viruses, but there is hope that they will be; other preventive methods against infectious agents can currently be used. The Epstein–Barr virus (EBV) is etiologically related to several cancers, some of them common in particular locations. They include nasopharyngeal carcinoma, Burkitt’s Lymphoma – one of the first virus cancer relations suspected on epidemiologic grounds – Hodgkin’s Disease, and several others. Intensive research on inventing a vaccine and determining its optimal use is in progress. Human T-cell leukemia virus (HTLV-1) is etiologically associated with adult T-cell leukemia as well as other noncancer diseases. These include HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), uveitis, and infective dermatitis.

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There are probably other causes of death associated with chronic HTLV-1 infection. In a historic prospective study in Zaire (now Democratic Republic of the Congo) where HTLV-1 is common (Lechat et al. 1997), after about 20 years, individuals who were HTLV-1 carriers in 1969 had a significantly lower survival compared to noncarriers. These studies confirmed a similar report from Japan. If further supported, this could justify the widespread use of an HTLV-1 vaccine when and if one is produced, in that vaccination could prevent many diseases in addition to the relatively rare leukemia and other identified associated diseases. HTLV-II has been isolated from a patient with hairy cell leukemia and there may be an etiologic relation of the virus with this disease. The EBV is associated with several cancers that are common in particular geographic regions. Denis Burkitt, a physician practicing and doing epidemiologic studies in equatorial Africa, described an unusual lymphoma, soon named after him. He showed that its distribution followed that of malaria and hypothesized that it had an infectious origin associated with mosquito transmission. Epstein and Barr, then, isolated a virus from the cells of a Burkitt’s lymphoma case and the virus was found to be extremely common in African cases. This was not true of sporadic cases found away from the tropics, suggesting multiple etiologies operating in different environment. EBV is also associated with nasopharyngeal carcinoma that is extremely common in China and elsewhere, and Hodgkin’s lymphoma. Human immunodeficiency virus types I and II (HIV-I, HIV-II) are associated with several cancers. These include other virus-related cancers: Kaposi’s sarcoma [associated with human herpes virus 8 (HHV-8) or Kaposi’s sarcoma herpes virus (KSHV)], Hodgkin’s lymphoma (associated with EBV), and cervical carcinoma (associated with HPV). The immune deficiency that characterizes AIDS may increase the susceptibility to these cancers, but there also appears to be an interaction of the genome of HIV in the cancer pathogenic process. These findings illustrate that the pathogenesis of viral-caused cancers may involve multiple viruses or other factors in pathogenesis. Humans have many more bacterial cells in their body than their “own” cells and even more viruses – and one or more of these may interact with the virus associated with the specific cancer. This may appear to make the problem incomprehensively complex, but the history of medicine has shown that effective measure can be found even though the entire process is not fully understood.

Discovery of the Hepatitis B Virus The discovery of the HBV did not start as a directed search for the virus but as a project in basic clinical research. It was a circuitous and convoluted process whose outcome would have been difficult to predict at its onset. (This and following sections are adapted in part from Blumberg 2002, 2010.) A striking feature of medicine is the great variation in host response to diseasecausing agents. Starting in 1957, we set out to find inherited polymorphic and acquired variation in the blood proteins that could be related to disease susceptibility

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(Blumberg 1961). This is analogous, at the phenotype (proteome) level, to the contemporary search for the relation between genomic polymorphic variation and disease using SNIPS and other databases. It was an interesting process as it required worldwide collections in different disease environments to compare the distribution of the polymorphic alleles and try to understand their relation to disease. Protein variation was determined using the recently introduced method of electrophoresis in gel. In 1961, another technique was deployed. Many serum proteins are polymorphic; hence, patients who had multiple transfusions would likely to be exposed to proteins they had not inherited. If some of the polymorphic proteins were antigenic, the patients might develop antibodies against them and their blood could be used as a “reagent” to discover and study antigenic protein polymorphisms. Using double diffusion in agar gel, we identified a complex, inherited antigenic system of the serum low-density lipoproteins (the “Ag System,” Allison and Blumberg 1961). Using these and other anti-lipoprotein antibodies identified by us (and to a greater measure by others), associations were found with cardiovascular disease, Alzheimer’s, and diabetes. We continued to test the sera of transfused patients against a panel of sera with the expectation that we would find additional antigen-antibody systems of interest. In 1963, a precipitin band was detected between the sera of a transfused hemophilia patient from New York and, among others, the sera of Australians (Blumberg et al. 1965). Much of the subsequent research was done on these sera; the reactant protein was called “Australia antigen” (Au). The next problem was to learn what it was. Au was rare in normal Americans but common in leukemia patients; this generated the hypothesis that people at high risk of leukemia might also have high frequencies of Au. Since patients with Down’s syndrome (DS, chromosome 21 trisomy associated with mental retardation) are at high risk for an unusual form of leukemia, we predicted that they would have a high frequency of Au; this was confirmed in a series of studies in large institutions for the mentally retarded (Blumberg et al. 1967) (Fig. 2.1). In the meanwhile, there were a series of observations that raised the possibility that Au was associated with hepatitis. It was found in transfused people, occasional patients with hepatitis, and in institutionalized patients (i.e., leprosy, mental retardation), where infectious spread would be expected. But, the most convincing observation was in a DS patient. He did not have Au when first seen but did so on subsequent testing; the appearance of Au coincided with the onset of hepatitis. We, then, formally tested the hypothesis that Au was associated with hepatitis by comparing the prevalence of Au in patients with and without clinical hepatitis; Au was much more common in the patients. The next series of tests were designed to determine if Au was a hepatitis virus or part of it. This was confirmed by clinical, electron microscope, transmission and other studies. The initial observations were soon confirmed by Okochi and Murakami (1968), Vierucci et al. (1968) and Prince (1968). Prince associated Au with the HBV that had been postulated by Krugman and other pioneers in the hepatitis field before our discovery of HBV. The identification of one virus facilitated the identification of others (HAV, HDV, HCV, HEV, etc.) in several other laboratories that greatly increased the probabilities of control and treatment of viral hepatitis (Fig. 2.2).

Fig. 2.1 Scan. ppt 112K. The first published image of the precipitin reaction in agar gel between “Australia antigen” (the surface antigen of HBV) in the top well, and the antibody against it in the bottom well. The top well contains serum form a patient with leukemia who is a carrier of HBV. The bottom well contains serum from a patent with hemophilia who has received many blood transfusions, some from blood donors who were HBV carriers, and developed antibodies against the surface antigen (Australia antigen)

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Fig. 2.2 Blumberg Surinam. ppt (299K). Surinam, South America, September 1950. The photograph was taken in a Djuka community near the mining town of Moengo on the Cottica River (Kotikaliba) during the course of an infectious disease survey and clinical care project. It was during these surveys that striking differences in response to infection were noted and reported

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There were immediate applications of the discovery of HBV. The “Au test,” as it was called, became widely used for the diagnosis of acute and chronic hepatitis, a further aid to control and treatment. It was a major step forward in viral diagnosis; a virus could be diagnosed within a few hours by direct detection. Previously, viral diagnosis often depended on comparing the titers of specific antibodies early in infection to titers during convalescence (Merigan 1997, personal communication). In 1969, we suggested testing blood donors to detect asymptomatic carriers of HBV (Blumberg et al. 1969). It was soon adapted at Philadelphia General Hospital (Senior et al. 1974), and later, after some controversy, at many other places. Soon, posttransfusion hepatitis due to HBV appeared to be under control. Subsequently, the discovery of HCV further reduced the frequency of posttransfusion hepatitis to the extent that it is no longer a major medical and surgical problem.

Invention of the Vaccine One of the major problems in vaccine invention is the identification of the specific antibody or cellular immune reaction that provides protection. The failure to do so has slowed the development of vaccines against HIV, HCV, tuberculosis, malaria, and other pathogens. The identification of a protective antibody for HBV was possible soon after the research began. By 1968, we recognized that antibodies (anti-HBs) against the surface antigen (HBsAg) of HBV were probably protective. We had rarely seen individuals who had both HBsAg (indicating infection) and anti-HBs in their blood; this is consistent with protection. Further, Okochi in Tokyo (Okochi et al. 1970) had shown that patients, who had anti-HBs before they were transfused with donor blood containing HBV, were much less likely to develop hepatitis than those who did not have anti-HBs. Later, in a multiyear study in a renal dialysis unit where HBV infection was endemic, Lustbader demonstrated a striking level of anti-HBs’ protection (Lustbader et al. 1976). A peculiar feature of HBV recognized after EM visualization of particles of the virus (Bayer et al. 1968; Dane et al. 1970) was that, in addition to the whole virus particles, there were very large numbers of spherical and rod-shaped particles in the peripheral blood of carriers and other infected individuals that contained only HBsAg. In some carriers, this amounted to more than 1% of their total serum protein. The vaccine was prepared from these particles. In 1969, the Institute for Cancer Research filed a patent in the USA and foreign countries for the process for extracting surface antigen particles free from whole virus as the source of the vaccine and for the product (Blumberg and Millman 1972; Blumberg 1972). To quote from the summary of the patent application: “… a vaccine against viral hepatitis is derived from blood containing Australia antigen, having particles resembling viruses which are substantially free of nucleic acid, of a size range of 180–210 A, substantially free of infectious particles. The vaccine where required is attenuated or altered. The preferred procedure of removing impurities including infectious components involves centrifugation, enzyme digestion, column

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gel filtration, differential density centrifugation in a solution of sucrose, dialysis, differential centrifugation in a solution of cesium chloride and dialysis.” At the time, this was a unique method for producing a vaccine. None had previously been produced from the blood of viral carriers and none has since then. Studies by Krugman and his colleagues increased the interest in the Blumberg/ Millman vaccine (Krugman et al. 1971). They inoculated children with a preparation of HBV-positive serum which had been boiled for 1 min. Subsequently, they evaluated whether these children had been protected against hepatitis by injecting them with untreated serum containing the virus. The heated serum provided substantial (but not complete) protection against HBV infection. This experiment had the effect of impressing potential manufacturers that the vaccine was feasible. In 1971, we received an assurance of interest from Merck Pharmaceuticals (Hilleman 1971, personal communication). By 1975, a sufficient amount of work had been done in laboratories in the USA and elsewhere to encourage us to recommend production and field testing of the vaccine; a licensing agreement was concluded with Merck. The noted vaccine expert Maurice Hilleman was given responsibility for the project and an experimental vaccine was produced and tested in animals (Buynak et al. 1976).

Vaccine Field Trials By the early 1980s, a series of vaccine field trials were completed, primarily by Szmuness and colleagues (Szmuness et al. 1980, 1981). The Szmuness trial has been described as “one of the best organized and executed trials of any human vaccine” and “a milestone in preventive medicine” (London and Blumberg 1985). It was primarily on the basis of this trial that the vaccine was approved by the FDA; it is described in some detail (summarized from London and Blumberg 1985). The first problem was to choose a population with a sufficiently high risk of infection to make a vaccine trial feasible. Szmuness believed that the trial should be carried out in a population which stood to benefit from an effective vaccine (Szmuness et al. 1980). Among the populations at high risk considered for the trial were residents of institutions for the mentally retarded (in whom we had earlier reported a high HBsAg frequency), patients undergoing maintenance hemodialysis, members of the medical staff of hemodialysis centers, American Indians, and homosexual men. By the late 1970s, very few new residents were being admitted to state institutions for the retarded, and the rate of new infections in long-term residents was quite low. Quarantine procedures had been instituted in Philadelphia (Snydman et al. 1976) and subsequently elsewhere; they had greatly reduced the incidence of hepatitis B infections. By 1975, Szmuness ascertained that the risk of infection among homosexual men in New York City was high and that they were cooperative, intelligent, and well-educated (Szmuness et al. 1975). The prevalence of hepatitis B markers was 68% among more than 10,000 men surveyed, and the annual incidence of infection was projected to be 19.2% (Szmuness et al. 1978), later estimated at 30% (Szmuness

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1980, personal communication). The study included 1,083 male subjects admitted between November 1978 and October 1979, of whom 549 received vaccine and 534 a placebo. The first two inoculations were given 1 month apart, and the third was given 6 months after the first. Follow-up was carried out for at least 6 months after the last dose of vaccine but for 12 months for most participants. Ninety-three percent of the subjects received all three inoculations, an outstanding compliance rate. The results were convincing. First, there was no difference in the frequency of severity of side effects between the vaccine and placebo groups. Second, the antibody response in the vaccinated groups was excellent; 77% of the vaccines had significant levels of anti-HBs within 2 months of the first inoculation, and 96% had antibody after the third dose, while only 2–5% of placebo recipients without evidence of active HBV infection developed anti-HBs. Third, there was a clear difference in the number of HBV infectious events between the vaccine and placebo recipients. Of 122 such events, 93 (76.2%) occurred in the placebo recipients and 29 (23.8%) in the vaccines (p < 0.0001). Fifty-two of the subjects had an event classified as “hepatitis B” (alanine aminotransferase levels ³90 IU plus the appearance of HBsAg in their serum). Only seven of these most serious events occurred in vaccinated men, and all but one of these occurred prior to the third dose of vaccine. There were 73 HBV events in the placebo group and 14 in the vaccines (p < 0.0001), and only 4 of the 14 events occurred after the third dose of vaccine. The efficacy ratio (incidence in placebo recipients over those in vaccines) reached 14.0 for the period from 5 to 18 months after vaccination. HBV infections which occurred in vaccine recipients of the full immunization schedule only happened in those who had not produced anti-HBs antibody. Finally, an unforeseen but clinically and biologically important result was that those vaccinated subjects who did not produce anti-HBs were not more likely to develop persistent infections than placebo recipients who became infected. Thus, Szmuness and his colleagues (Szmuness 1980, personal communication) were able to conclude that “this placebo-controlled, randomized, double-blind clinical trial, conducted in 1,083 subjects who had an unusually high risk of hepatitis B virus infection, proves the efficacy of the vaccine ….” Subsequent trials supported this conclusion (Maupas et al. 1981; Francis et al. 1982; Chan et al. 2004; Desmyter et al. 1983; Benahamon et al. 1984). The Szmuness trial was not only effective, but also efficient. Just over a 1,000 people were involved. Compare this to the more than one million children involved in the testing of polio vaccine and the thousands that have been involved in the so-far unsuccessful trials of an HIV vaccine. Millions of doses of the plasma-derived vaccine have been used. Reports of side effects have on occasion led to suspension of the vaccine programs, but they were subsequently reinstated (Marshall 1998). The effectiveness of HBsAg derived from plasma as a protection-inducing antibody validated its manufacture by recombinant methods, and recombinant vaccine is now the major source of the vaccine (McAleer et al. 1984). It was the first vaccine produced by the recombinant method and for many years the only one; it has helped to make the vaccine available worldwide as the cost of manufacture and distribution decreases.

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Global Vaccination Programs National vaccination programs began soon after the vaccine was available (see, for example, Gatcheva et al. 1995; Bonnanni 1995; Ginsberg and Shouval 1992; De la Torre and Esteban 1995). In February 1990, we convened The International Conference on Prospects for Eradication of Hepatitis B Virus that that included reports on the extent of the HBV endemic, the national resources available for vaccination programs, and the possibilities for control and, possibly, eradication (Blumberg 1990). In 1992, the WHO placed HBV vaccine on the Extended Program on Vaccination setting a target of 1997 for integration into national programs. By April of 2005, 158 of the 192 members of the World Health Organization had national vaccination programs (Kim et al. 2007). HBV is one of the most widely used vaccines worldwide. The results are impressive. Newborn and childhood vaccination was started in Taiwan in 1984 with excellent national participation and long-term reporting of the results (Chan et al. 2004). In 1999, 15 years later, the carrier prevalence had dropped from 9.8 to 0.7% (p < 0.001). The prevalence had also dropped significantly (but not as much) in children in a similar cohort who had not been vaccinated. There have been similar findings elsewhere (see, for example, Da Villa et al. 1992, 1995). This implies a type of “herd immunity” that could hasten the overall effect of the program and accelerate control and, possibly, eradication. There was also a striking drop in deadly fulminant hepatitis in young children (Kao et al. 2001). From 1975 to 1984, the average mortality from fulminant hepatitis was 5.36/100,000 infants; from 1985 to 1998 – after the vaccination program had started – it was 1.71/100,000. There have been reports from elsewhere of striking drops in the prevalence of HBV carriers and decrease in the incidence of clinical hepatitis. Several HBV carrier surveys before and after vaccination programs have been summarized (Blumberg 2004). In a regional study in the Peoples Republic of China, the prevalence of carriers decreased from 16.0 to 1.4%. The before and after percentages in other countries are similar: The Gambia 10.0 and 0.6%; Japan 2.7 and 0.9%; Saudi Arabia, 6.7 and 0.3%; Catalonia (Spain), 9.3 and 0.9%. In the USA, the rate of new HBV infections has declined significantly since 1991. It dropped from a peak of more than 70,000 cases in 1984 to less than 20,000 in 2006. The decline has been greatest among children born since 1991, when routine vaccination of children was recommended by the CDC (CDC Web site 2006). In Alaska, following an intensive vaccination campaign among Native Americans, the incidence dropped from 215 cases/100,000 before the vaccination programs to 7–14 cases/100,000 in 1993 after the program was in place. In 1995, no cases were reported (McMahon et al. 1996). A national hepatitis serological survey was conducted in China in 1992 (Liang et al. 2009). The authors estimated that there were 120 million HBV carriers, that 20 million patients suffered from chronic effects of HBV including chronic liver disease (cirrhosis, liver failure, etc.) and HCC, and that about 300,000 die annually from the consequences of late-stage HBV infection. Liver cancer and cirrhosis are

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both on the list of the ten most common causes of fatal diseases and HBV is the major cause of both of these. About one-third of all the HBV carriers in the world are in Peoples Republic of China. In 2009, a report was published on the effects of the vaccine program based on a national epidemiological study involving 160 counties, 369 township and village clusters, and 81,775 persons (Liang et al. 2009). The authors concluded that China had achieved the goal of reducing HBsAg prevalence to less than 1% in the vaccinated population (mostly children under 5 years) and 16–20 million HBV carriers had been prevented as a result of the infant vaccination program. Furthermore, in the vaccine-impacted population, i.e., young children, 2.8–3.5 million future deaths have been prevented. There are about 375–400 million carriers worldwide. A tentative projection to the whole world population can be attempted using the Chinese data. In many places worldwide, the vaccination programs began earlier and the compliance rates were somewhat higher. The Chinese estimates were based on the results in the childhood vaccinated population. In several studies, it has been shown that in countries with successful childhood vaccination program there is also a significant drop in HBV carrier incidence in the unvaccinated population, presumably as a consequence of herd immunity (see above). Taken these factors into account, about 15–25 million future deaths have been prevented worldwide. Plans for the continuation of the successful campaign in China are being discussed, including the possibility of eradication (So 2006).

HBV: The First Cancer Vaccine Perhaps, the most conceptually important outcome of the vaccination program is the decrease of HCC, primary cancer of the liver. HCC is one of the most deadly and common cancers worldwide. It is the third most common cause of death from cancer in males and the seventh most common in females. Most HCCs are caused by infection with HBV or HCV; HBV is said to account for 65–75% of the cases worldwide. In Taiwan, the yearly incidence of HCC in the vaccine-impacted population (age 6–14 years) declined from 0.7/100,000 (1981–1986) before vaccination programs were fully implemented to 0.36/100,000 between 1990 and 1994 after the vaccination program was in place (Huang and Lin 2000; Chan et al. 2004). A follow-up report found that the prevention of HCC has continued from “childhood to early adulthood” (Chang et al. 2009). The major cause of continued disease was inadequate vaccination and highly infective mothers. This is another indication for the treatment of HBV carrier mothers, particularly those with titers of HBV DNA. HBV vaccine is the first preventive cancer vaccine with a demonstrated impact on the world’s cancer load. In 2007, about 25 years after the introduction of the first, the second cancer prevention vaccine was launched. It provided effective protection against strains of papilloma virus preventing cancer of the cervix and other cancers (Chen and Berek 2007). There are also continuing studies on the

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possibility of vaccine protection against EB virus and the prevention of nasopharyngeal and other cancers (Hepeng 2008).

Secondary Antiviral Prevention: Prevention by Delay National vaccination programs are the most effective means of controlling HCC. But what of those already infected? There are about 400 million carriers of HBV and some 300 million carriers of HCV at risk for chronic liver disease and HCC. Antiviral treatment may delay or abort the risk of these diseases. We termed this “prevention by delay” (Blumberg and London 1981) as the antiviral treatment can prolong the symptom-free period until the carrier dies of other causes. Treatment of HBV carriers with antivirals (see, for example, Liaw et al. 2004) or by other means can greatly decrease the risk of HCC; this is discussed elsewhere in this book. A further advantage of treatment is that decreasing titers of virus decreases the infectivity of carriers and patients and, therefore, the risk of transmission to those who have not been vaccinated or were inadequately vaccinated. In particular, it could reduce the transmission of HBV from mothers to their children at the time of birth and soon afterward. This could hasten the control of HBV and increase the possibility of eradication. Fortunately, prevention of HCC, and probably of other viral-caused cancers, has the double arm of primary prevention and secondary prevention to aid in control.

The Genetics of Hepatitis B Virus The initial research on hepatitis B began as a study in the inheritance of susceptibility to HBV chronic infection (Blumberg et al. 1966), and more recent population and genomic studies have added rich detail (for review, see, Blumberg 2006a, b). There are multiple loci at which one allele increases susceptibility to chronic infection and an alternate allele increases the probability of developing protective antibody. An added interesting aspect of these observations is that these same alleles may affect susceptibility to other infectious agents. For example; the DRB1*1302 allele at the MHC Class II locus (chromosome 6) is related to susceptibility to HBV chronic infection, response to falciparum malaria, and response to papilloma virus. The VDR locus (chromosome 12) is related to responses to HBV, Mycobacterium tuberculosis, and Mycobacterium leprae; there are many other examples. We have categorized the organisms with affinities to the same genetic locus as Microorganism Gene Affinity Clusters (MIGAC). In some cases, an allele that confers an advantage to the host for one member of the cluster may be disadvantageous for another. These allelic variations constitute genetic polymorphisms and as such both advantages and disadvantages may be expected [for example, carriers of HBV bind larger quantities of iron than uninfected people. This may be an advantage in regions with low dietary

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iron intake (Sutnick et al. 1974; Felton et al. 1979)]. It is interesting to conjecture how members of the cluster are affected if one of the infectious agents in the cluster is greatly decreased as a result of a successful vaccination program.

Conjectures on the Future of Research on Viruses and Cancer 1. The Editor has asked me to comment on the possible directions for future research, including my preferences, in respect to cancer and viruses or, more broadly, infectious agents. Preventive measures have made an important contribution to the decrease of the load of human cancer and, obviously, research should be encouraged and increased. The apparent success of the HBV vaccine program in decreasing the incidence of one of the most common and deadly cancers was an encouragement to seek others. As already noted, it required about 25 years for the introduction of the second cancer prevention vaccine, against the papilloma virus, an indication of the lack of funding for this theater of research in the past. 2. There are multiple factors in the pathogenesis of cancers. Independent of what is considered the “real” cause, often expected to be some gene selection, introduction, or alteration, the most important “cause” may be distant from genetic effects but amenable to interventional change. For example, HBV and HCV may not be directly involved in genetic change consistent with theoretical expectations, but prevention or treatment of infection can considerably decrease risk. Control of other contributors to advancing pathogenesis, for example the removal of aflatoxins and/or other environmental enhancers of cancer, decreasing iron and iron storage levels (Weinberg 1984; Hann et al. 1989) controlling excessive alcohol intake, and probably many others, can contribute significantly to decreasing the risk of HCC and other untoward consequences of HBV infection (Blumberg 2002). 3. Viruses that are designated as the “main” cause of a cancer interact with other infectious agents in the host. As noted above, there are interactions of the genome of HIV with other cancer-associated virus. Humans are infected normally with vast numbers of bacteria and virus. It is unlikely that they would not interact with cancer-related viruses. Also, hosts are often coinfected if viruses have similar transmission routes. HBV infection is associated with infection with HCV, HIV, HTLV-1, and probably other blood-borne infectious agents. The host can respond differently to each of the following: acute disease, carrier state, development of chronic infection, production of protective and other antibodies, integration into the host genome, or genomes of the other organisms. The study of the interactions of viruses with each other and with the genome of the host can provide insight into the process of carcinogenesis, therapy, and prevention. 4. In their review, Boccardo and Villa (2007) cite many examples in which the same or similar viruses may cause several cancers (i.e., EBV and Burkitt’s Lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, etc.; HPV and genital carcinoma,

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carcinoma of the oropharynx, etc.). It would be fruitful to continue this search. There are several studies showing an association of HBV with cancer of the pancreas (see, for example, Iloeje et al. 2010). Duck HBV, a hepadnavirus (HBV-related viruses), localizes in the pancreas and HBV, although primarily localized to the liver, and is also found in the pancreas in humans (Coyne et al. 1970). These findings suggest that HBV has a role in cancer of the pancreas or that another virus that is cross antigenic with HBV may be involved. It is informative to determine if the HBV vaccination campaign has had an effect of the incidence of cancer of the pancreas. The first disease associations reported for HBV were with leukemias, but there has been little follow-up on these leads (Blumberg et al. 1967). HBV has recently been associated with lymphoma (Engels et al. 2010), and further studies on this connection are warranted. 5. Most cancer viruses are associated with more noncancer disease; in many cases, they are more common than the cancer. The association (noted above) of chronic HTLV-1 infection with decreased survival is an example of the potential public health importance of chronic infections with viral-caused cancers. The recent studies of zur Hausen and his colleagues (zur Hausen 2010) on the Torque Teno Virus (TTV) that is related to cancers, MS, and other disease characterized by inflammation is an important example of how this research is proceeding. 6. Prevention against and treatment of infectious agents has been one of the most successful accomplishments of scientific medicine and this can be extended to cancers caused or influenced by infectious agents. A future direction for cancer centers and research institutes, then, could or should be to seek viruses in patients with various cancers and, perhaps more importantly, those who are at the risk of cancer. A strategic approach would be to start with the null hypothesis that all cancers include one or more microorganisms (including viruses) in their etiological roster, and then use available methods (and methods to be developed) for detecting past or present infection. This could include infection in prior generations of the host that remain in the host’s genome. 7. Another fruitful area for research would be to continue the search for polymorphic genetic variation that increases the risk for and/or alters the pathogenesis and treatment of cancer. There have been important advances in this field since the early days of searching for polymorphic variants in serum proteins and elsewhere that influence susceptibility to disease and affect response to drugs (see, for example, Blumberg 1961). The use of whole genome sequences has facilitated the identification of many sites, but it has also made it more complicated and difficult to apply. As data accumulates, it is likely that general laws will emerge and the use of these variations to protect the most susceptible will increase. Identifying the genetic components of susceptibility makes it easier to find environmental agents in that a subpopulation can be identified in which the environmental influences are strong. Further investigation of “Magac” clusters (see above) can provide leads on disease connections and operations of many genes on the same outcome.

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8. The discovery and development of new and the improvement of current antivirals effective against specific or many viruses should be a major research program. The intensive research on treatments for HIV and AIDS has given strong support to pharmaceutical companies seeking useful and profitable products, and this can be extended to HBV and HCV. (There are worldwide about 700 million carriers of HBV and HCV.) It was recognized early in the HBV research programs that complete removal of the virus from its host may be difficult or impossible as the virus is often integrated into the host genome. However, decreased titers can considerably reduce the risk for the development of chronic liver disease and HCC. There are now several antivirals that are effective in doing this and others are in the process of development. In many cases, carriers of cancer-related viruses require long-term treatment using relatively low doses as reduction of titer rather than total elimination is the goal. This could also result in decreased side effects. Long-term treatments are attractive to pharmaceutical companies and could encourage research and development of the antivirals. The availability of effective and safe antivirals encourages the detection of carriers, of whom currently only a small percentage has been identified.

Conclusions Viruses cause many of the cancers that afflict humans and these same viruses often cause other noncancer diseases that may be more common than the cancers. This provides an additional justification for national and universal vaccination programs. Known cancer viruses are often implicated in the etiology or pathogenesis of other cancers and it is important to find these connections. If this is common, then the existing cancer vaccine programs and those added in the future may lead to decreases in the incidence of other cancers not now recognized as virally caused. There are now two vaccination programs – HBV and HPV – that are in place and appear to be successful; it is expected that others will be added in time as vaccines or other preventive measures are found for the existing virus-related cancers. It is likely that viruses have a role in the pathogenesis of many other cancers than those already identified and an important direction for cancer research would be to find these and develop countermeasures, including vaccination. There is also the possibility of developing new and better antvirals to treat those chronically infected with known cancer viruses. As experience with secondary prevention of cancer with antivirals increases, treatment may be a useful method to determine if a cancer is caused by a virus. The HBV vaccine cancer prevention program has been in place for nearly 30 years. It has served as a model for future programs and has been described in some detail in this chapter. HBV was discovered in a nongoal-directed project, as is often the case in the solution of medical and biological problems. The original vaccine was derived from a portion of the virus – the surface antigen particles that

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exist in large quantities in the blood of carriers of HBV; this was a unique method of making a vaccine that did not require cell culture. HBV vaccine was subsequently made by the recombinant method, the first vaccine so produced. It is now one of the most commonly used vaccines in the world. (However, there are still regions that are inadequately served.) The reduction in the prevalence of HBV carriers and the incidence of acute and chronic liver disease and HCC has been profound. Rigorous control and even eradication may be possible. We are in a period of increased awareness of the virus–cancer connection and the major effect it has and can have on the control of several and perhaps many cancers. Strong support for continued research and application moves these programs forward.

References Allison AC, Blumberg BS (1961) An isoprecipitation reaction distinguishing human serum protein types. Lancet 1:634–637 Bayer ME, Blumberg BS, Werner B (1968) Particles associated with Australia antigen in the sera of patients with leukemia, Down’s syndrome and hepatitis. Nature 218:1057–1059 Benahamon E, Courouce AM, Jungurs P et al (1984) Hepatitis B vaccine: randomized trial of immunogenicity in hemodialysis patients. Clin Nephrol 3:102–103 Blumberg BS, Millman I (1972) Vaccine against viral hepatitis and process. US Patent Office no. 3,636,191 Blumberg BS (1961) Inherited susceptibility to disease: Its relation to environment. Arch Environ Health 3:612–636 Blumberg BS (1990) Proceedings of International Conference on Prospects for Eradication of Hepatitis B Virus. In Vaccine 8 Introduction S5 Conclusion S139 Blumberg BS (1972) Viral hepatitis, Au antigen, and hope for a vaccine. Gastroenterology (Med. World News) 14–18 Blumberg BS, Alter HJ, Visnich S (1965) A “new” antigen in leukemia sera. JAMA 191:541–546 Blumberg BS, Gerstley BJS, Hungerford DA et al (1967) A serum antigen (Australia antigen) in Down’s syndrome leukemia and hepatitis. Ann Int Med 66:924–931 Blumberg BS, Melartin L, Guinto RA et al (1966) Family studies of a human serum isoantigen system (Australia antigen). Am J Human Genet 18:594–608 Blumberg BS, London WT, Sutnick AI (1969) Relation of Australia antigen to virus of hepatitis. Bull Path 10:164 Blumberg BS (2002) Hepatitis B. Princeton University Press, Princeton NJ, The Hunt for a Killer Virus Blumberg BS (2006a) Hepatitis B virus: conjectures on human interactions and the origin of life. In: Seckbach J (ed) Life as We Know It. Springer, New York, pp 213–235 Blumberg BS (2006b) The curiosities of hepatitis B virus: prevention, sex ratio, and demography. Proc Am Thorac Soc 3:14–20 Blumberg BS (2004) The impact of hepatitis B vaccine worldwide. In: Vierucci Alberto (ed) The Proceedings of the Società Italiana de Allergolgia ed Immunologia Pediatrica, 57–63 Firenza, 18–20 EDITEAM, Cento, (Florence) Blumberg BS (2010) Hepatitis B. In: Artenstein AW (ed) Vaccines: a biography. Springer, New York, pp 301–315 Blumberg BS, London WT (1981) Hepatitis B virus and the prevention of primary hepatocellular carcinoma. Editorial. N Engl J Med 304:782–784

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Boccardo e, Villa LL (2007) Viral origins of human cancers. Curr. Med. Chem. 14(24): 2526–2539 Bonnanni P (1995) Implementation in Italy of a universal vaccination program against Hepatitis B. Vaccine 13:68–71 Buynak EB, Roehm RR, Tytell AA et al (1976) Development and chimpanzee testing of a vaccine against human Hepatitis B. Proc Soc Exp Biol Med 151:694–700 Chan C-Y, Lee S-D, Lo K-J (2004) Legend of Hepatitis B vaccination: the Taiwan experience. J Gastrolenterol and Hepatol 19:121–126 Chang MH, You SL, Chen CJ, Liu CJ, Lee CM, Lin SM, Chu HC, Wu TC, Yang SS, Kuo HS, Chen DS, the Taiwan Hepatoma Study Group (2009) Decreased incidence of hepatocellular carcinoma in Hepatitis B vaccinees: a 20-year follow-up study. J Natl Cancer Inst 101(19):1348–1355 Chen JK, Berek JS (2007) Impact of the human papilloma vaccine on cervical cancer. J Clin Oncology 25:2975–2982 Coyne (Zavatone) V, Millman I, Cerda J, Gerstley BJS, London WT, Sutnick AI, Blumberg BS (1970) The localization of Australia antigen by immunofluorescence. J Exp Med 131:307–320 Da Villa G, Piazza M, Iorio R et al (1992) A pilot model of vaccination against hepatitis B virus suitable for mass vaccination campaigns in hyperendemic areas. J Med Virol 36:274–278 Da Villa G, Picciottoc L, Elia S et al (1995) Hepatitis B vaccination: universal vaccination of newborn babies and children at 12 years of age versus high risk groups. A comparison in the field. Vaccine 13:1240–1243 Dane DS, Cameron CH, Briggs M (1970) Virus-like particles in serum of patients with Australia antigen-associated hepatitis. Lancet 1:695 Devita VT, Chu E (2008) A history of cancer chemotherapy. Cancer Res. 68(21):8643–8653. De la Torre J, Esteban R (1995) Implementing universal vaccination programs: Spain. Vaccine 13:72–74 Desmyter J, Colaert J, DeGroote G et al (1983) Efficacy of heat-inactivated hepatitis B vaccine in hemodialysis patients and staff: double blind placebo-controlled trial. Lancet 2:1323–1328 Engels EA, Cho ER, Jee SH (2010) Hepatitis B virus infection and risk of non-Hodgkin lymphoma in South Korea: a cohort study. Lancet Oncol 11:827–834 Felton C, Lustbader ED, Merten C et al (1979) Serum iron levels and response to hepatitis B virus. Proc Natl Acad Sci USA 76:2438–2441 Francis DP, Hadler SC, Thompson SE (1982) The prevention of hepatitis B with vaccine: report of the centers for disease control multi-center efficacy trial among homosexual men. Ann Intern Med 97:362–366 Gatcheva N, Vladimirova N, Kourtchatova A (1995) Implementing universal vaccination programs: Bulgaria. Vaccine 13:82–83 Ginsberg GM, Shouval D (1992) Cost-benefit analysis of a nationwide neonatal inoculation program against hepatitis B in an area of intermediate endemicity. J Epidemiol Community Health 46:597–594 Hann HL, Kim CY, London WT, Blumberg BS (1989) Increased serum ferritin in chronic liver disease: A risk factor for primary hepatocellular carcinoma. Int J Cancer 43:376–379 Hepatitis B Vaccine: Fact Sheet First Anti-cancer Vaccine (2006) http://www.cdc.gov/hepatitis May 17, 2006 Hepeng J (2008) A controversial bid to thwart the “Cantonese Cancer”. Science 321:1154–1155 Huang K-Y, Lin S-R (2000) Nationwide vaccination: a success story in Taiwan. Vaccine 18:S35–S38 Kao JH, Hsy HM, Shau WY et al (2001) Universal hepatitis B vaccination and the decreased mortality from fulminant hepatitis in infants in Taiwan. J Pediatr 139:349–352 Kim S-Y, Salomon JA, Goldie SJ (2007) Economic evaluation of hepatitis B vaccination in lowincome countries: using cost-effectiveness affordability curves. Bull World Health Organ 85:821–900

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Krugman S, Giles JP, Hammond J (1971) Viral hepatitis, type B (MS-2 strain): studies on active immunization. JAMA 217:41–45 Lechat MF, Shrager DI, Declercq E, Bertrand F, Blattner WA, Blumberg BS (1997) Decreased survival of HTLV-1 carriers in leprosy patients from the Democratic Republic of the Congo: a historical prospective study. J Acquir Immune Defic Syndr Hum Retrovirol 15:387–390 Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, Zhang Y, Liu J, Gong X, Chen Y, Wang F, Zheng H, Wang F, Guo J, Jia Z, Ma J, Wang H, Luo H, Li L, Jin S et al (2009) Epidemiological serosurvey of Hepatitis B in China. Declining HBV prevalence due to Hepatitis B vaccination. Vaccine 27:6550–6557 Iloeje UH, Yang HI, Jen CL, Su J, Wang LY, You SL, Lu SN, Chen CJ (2010) Risk of pancreatic cancer in chronic hepatitis B virus infection: data from the REVEAL-HBV cohort study. Liver Int 30:423–429 Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, Tanwandee T, Tao QM, Shue K, Keene ON, Dixon JS, Gray DF, Sabbat J, Cirrhosis Asian Lamivudine Multicentre Study Group (2004) Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 351:1521–1531 London WT, Blumberg BS (1985) Comments on the role of epidemiology in the investigation of hepatitis B virus. Epidemiol Rev 7:59–79 Lustbader ED, London WT, Blumberg BS (1976) Study design for a hepatitis B vaccine trial. Proc Natl Acad Sci USA 73:955–959 McAleer WJ, Buynak EB, Maigetter RZ et al (1984) Human hepatitis B vaccine from recombinant yeast. Nature 307:178–180 Marshall E (1998) A shadow falls on hepatitis B vaccination effort. Science 281:630–631 Maupas P, Chiron VP, Barn F et al (1981) Efficacy of hepatitis B vaccine in prevention of early HBsAg carrier in children: controlled trial in an endemic area (Senegal). Lancet 1:289–292 McMahon B, Mandsager R, Wainwright K, et al. (1996) The Alaska native hepatitis B control program. Proc. IX Triennial International Symposium on Viral Hepatitis and Liver Disease, Rome, Italy, abstract 74 Okochi DJ, Murakami S (1968) Observations on Australia antigen in Japanese. Vox Sang 15:374–385 Okochi DJ, Murakami S, Nonomiya K et al (1970) Australia antigen, transfusion and hepatitis. Vox Sang 18:289–300 Prince AM (1968) An antigen detected in the blood during the incubation period of serum hepatitis. Proc Natl Acad Sci USA 60:814–821 Senior JR, Sutnick AI, Goeser E et al (1974) Reduction of post-transfusion hepatitis by exclusion of Australia antigen from donor blood in an urban public hospital. Amer J Med Sci 267:171–177 Snydman DR, Bryan JA, London WT et al (1976) Transmission of hepatitis B associated with hemodialysis: role of malfunction (blood leaks) in dialysis machines. J Infect Dis 134:562–570 So S (2006) A comprehensive national strategy to eliminate hepatitis B in China should include an expanded national immunization program to provide free catch-up vaccination for every child and adolescent in addition to universal newborn vaccination. Opening keynote address, China Hepatitis Prevention and Control Conference, Nov 16–18, 2006, Chengdu, China Sutnick AI, Blumberg BS, Lustbader ED (1974) Elevated serum iron levels and persistent Australia antigen (HBsAg). Ann Intern Med 81:855–856 Szmuness W, Harley EJ, Ikram H et al (1978) Socio-demographic aspects of the epidemiology of hepatitis B. In: Vyas G, Cohen SN, Schmid R (eds) Viral hepatitis. Franklin Institute Press, Philadelphia, pp 297–320 Szmuness W, Much MI, Prince AM et al (1975) On the role of sexual behavior in the spread of hepatitis B infection. Ann Intern Med 83:489–495 Szmuness W, Stevens CE, Harley EJ et al (1980) Hepatitis B vaccine: demonstration of efficacy in a controlled clinical trial in a high-risk population in the United States. N Engl J Med 303:833–841

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Szmuness W, Stevens CE, Harley EJ et al (1981) A controlled clinical trial of the efficacy of the hepatitis B vaccine (heptavax B); a final report. Hepatology 8:119–121 Vierucci A, Bianchini AM, Morgese G et al (1968) L’antigene Austrlia. I Rapporte con l’epatite infettiva e da siero. Una ricerca in pazienta pediatrici. Pediatria Int 18:3–11 Weinberg ED (1984) Iron withholding: a defense against infection and neoplasia. Physiol Rev 64:65–102 Zur Hausen H (2010) In New virus behind cancer, MS? – The scientist – Magazine of the Life Sciences http://www.the Scientist.com/news/display/57623/#ixzz0x1SJXy4g.

Chapter 3

Virus-Mediated Cell Proliferation Sun-Hwa Lee, Stacy Lee, and Jae Ung Jung

Introduction It is estimated that 15–20% of all human cancers are linked to human tumor viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), human T-cell lymphotropic virus (HTLV), Epstein–Barr virus (EBV), and Kaposi’s sarcoma-associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8). HTLV is an RNA tumor virus associated with adult T-cell leukemia, whereas HBV, HPV, EBV, and KSHV are DNA tumor viruses associated with liver cancer (HBV), cervical and other anogenital cancers (HPV), Burkitt’s lymphoma and nasopharyngeal carcinoma (EBV), and Kaposi’s sarcoma (KS) (KSHV) (Dayaram and Marriott 2008). Simply defined, cell proliferation is the increase in cell numbers as a result of cell growth and division. For normal cells, entry into an active proliferative state from a quiescent state (G0) depends on the presence of exogenous, mitogenic, growth stimulatory signals, such as diffusible/soluble growth factors, components of the extracellular matrix (ECM), and cell-to-cell adhesion/interaction molecules, since the binding of these stimulatory signals to their receptors induces a variety of intracellular signaling transduction pathways involved in cellular proliferation (Hanahan and Weinberg 2000). Tumor cells, however, depend less on exogenous, growth stimulatory signals in the initiation of proliferation. Thus, the ability to proliferate in the absence of external growth factors is suggested to be one of the hallmarks of tumor cells and is generally achieved by either overexpression of growth receptors and/or ligands, mutations in receptors, or downstream signaling molecules whose activities are independent of ligand binding or defects in specific components of the cell cycle machinery (Hanahan and Weinberg 2000).

S.-H. Lee (*) • S. Lee • J.U. Jung Department of Molecular Microbiology and Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_3, © Springer Science+Business Media, LLC 2012

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Likewise, tumor viruses appear to have evolved numerous strategies that promote the proliferation of infected host cells in the absence of external growth stimulatory signals for their replication and survival, thereby ultimately contributing to the transformation of infected host cells. To this end, several viral proteins encoded by tumor viruses have been shown to dysregulate normal cellular processes, such as cell cycle progression, apoptosis, immune surveillance, and antiviral responses, allowing viral replication and survival. Many excellent reviews on the impact of tumorigenic viruses on cellular processes, including cell cycle progression, have been published (Damania 2007; Dayaram and Marriott 2008; Gatza et al. 2005; Helt and Galloway 2003; Hoppe-Seyler and Butz 1995; McLaughlin-Drubin and Munger 2008; Sears and Nevins 2002). This review focuses on the biochemical and molecular strategies used by oncogenic HHVs, including EBV and KSHV, to enhance cell proliferation.

Oncogenic Human Herpesviruses The Herpesviridae family comprises large, double-stranded DNA viruses with a genome size of 100–200 kb. Members of this family are classified as three subfamilies based on their genomic organization and biological characteristics: alpha (a), beta (b), and gamma (g). Eight HHVs are known so far. Members of the a-HHV include herpes simplex viruses (HSV) 1 and 2 (HHV-1 and HHV-2) and varicellar zoster virus (VZV; HHV-3). Members of the b-HHV include cytomegalovirus (CMV; HHV-5), HHV-6 variants A and B, and HHV-7. Members of g-hepesviruses are further classified as g1-herpesviruses (lymphocryptoviruses) and g2-herpesviruses (rhadinoviruses). EBV (HHV-4) and KSHV (HHV-8) belong to g1- and g2HHV, respectively (Damania 2007). Among the members of the HHV family, only EBV and KSHV have been implicated in a variety of human cancers. Association of both EBV and KSHV with a number of human cancers derives from two distinct features of their life cycles, latency and lytic cycle. In a lytic cycle, viruses replicate extensively and express virtually all viral genes, ultimately leading to the production of progeny viruses and the death of infected host cells (Ganem 2007). This lytic infection probably occurs either during primary infection or periodically in certain physiologic conditions, causing viral spread between cells and hosts (Kalt et al. 2009). In latency, on the other hand, the viral genome is maintained as a circular episome in the nuclei of infected host cells. Only a handful of viral genes are expressed and progeny viruses are not produced. In addition to these two life cycles, both EBV and KSHV have other common features: (1) both can infect B lymphocytes, (2) their latency is associated with human cancers, and (3) it is difficult to model their lytic replication cycles in vitro (Hume and Kalejta 2009). EBV (HHV-4). EBV is the first human virus to be directly implicated in carcinogenesis and over 90% of the global adult population is infected with EBV. It is usually asymptomatic, but a proportion of EBV-infected individuals develop infectious mononucleosis (IM), a disease characterized by lymphadenopathy and fatigue, later in life. During acute infection, EBV primarily infects and replicates in the stratified

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squamous epithelium of the oropharynx. This is followed by latent infection of B lymphocytes. While lytic infection is known to be associated with oral hairy leukoplakia, a proliferative disorder in immunocompromised patients, most EBVassociated malignancies are caused by latent infection (Kutok and Wang 2006). EBV-associated malignancies include Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), nasopharyngeal carcinoma (NPC), T and natural killer (NK) cells lymphoma, and posttransplant lymphoma (Kutok and Wang 2006). BL is a malignancy principally found in children, especially those who live in regions of Africa with a high incidence of malaria, although it also occurs more sporadically in other areas of the world. BL tumor cells are highly linked with EBV as almost all African BL patients are EBV positive. NPC is a malignancy of the squamous epithelium situated in the nasopharynx. EBV-associated NPC frequently occurs in Southern China, Northern Africa, and among Eskimo populations and is thought to arise from clonal expansion of latently infected cells. HL is the most common EBV-associated malignancy occurring in the Western world (about 30–90% of all HL patients are EBV positive). EBV is also highly present in immunoblastic lymphomas in HIVinfected individuals (about 70%) as well as in immunosuppressed posttransplant patients (100%) (Damania et al. 2000). EBV can infect primary human B lymphocytes in vitro, converting them into continuously growing, semiactivated, immortalized, and transformed lymphoblastoid cell lines (LCLs). Within LCLs, EBV is latent. Among the >85 genes encoded by EBV, only 11 viral proteins are expressed in LCLs: six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and 5), three latent membrane proteins (LMPs 1, 2A, and 2B), and two EBV-encoded small nonpolyadenylated (noncoding) RNAs (EBERs 1 and 2). Among these, EBNA-2, -3A, -3C and LMP1 are required for the in vitro immortalization of B lymphocytes by EBV (Rickinson and Kieff 2007). KSHV (HHV-8). KSHV is the second HHV implicated in human malignancies. KSHV is uncommon in the general population (less than 7%, but some geographical areas have infection rates as high as 60%). KSHV is primarily transmitted through saliva, although other transmission routes, including vertical, sexual, blood, and transplantrelated transmission, have also been reported (Pica and Volpi 2007). KSHV can infect many different types of cells in vitro, including B cells, epithelial cells, endothelial cells, and cells of the monocyte/macrophage lineage (Brinkmann and Schulz 2006). In addition, KSHV has been shown to immortalize primary human endothelial cells to have long-term proliferation and survival and to establish latency in B cells and endothelial cells in vivo (Brinkmann and Schulz 2006; Damania et al. 2000). KSHV is the etiologic cause of Kaposi’s sarcoma, an endothelial neoplasm. Globally, KS is the fourth most common cancer caused by infection, after gastric cancer (Helicobacter pylori), cervical cancer (human papillomavirus), and liver cancer (hepatitis cirrhosis). KS remains a major cause of cancer-related deaths among immune-suppressed and organ-transplant patients (Kalt et al. 2009). KSHV infection is also highly associated with two rare atypical B-cell lymphoproliferative diseases: primary effusion lymphomas (PEL)/body cavity B-cell lymphomas (BCBL) and multicentric Castleman’s disease (MCD). These are principally or exclusively of B-cell origin. MCD is a polyclonal B-cell lymphoproliferative

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disease with dissemination to multiple lymph nodes and other lymphoid tissues. In AIDS patients, MCD is invariably associated with KSHV infection, whereas approximately half the cases of HIV-negative individuals are KSHV associated. PEL is a monoclonal B-cell lymphoproliferative disease marked by rapid proliferation of cells in the pleural, peritoneal, and pericardial cavities. It usually occurs in AIDS or other immunosuppressed individuals and is often associated with KSHV and EBV coinfection. Most of what we know about KSHV biology has been obtained from studying viral gene expression in cultured PEL cells, which grow readily in culture, stably maintain latent KSHV genome, and express latent transcripts and proteins in virtually all cultured cells (Ganem 2006). KSHV proteins expressed during latency are believed to contribute to the development of KSHV-associated diseases. Five major latency-associated viral proteins have been identified in latently infected lymphoma cells: the ORF73-71 locus encoding latency-associated nuclear antigen (LANA-1, ORF73), viral cyclin (v-Cyclin, ORF72), viral FLICE inhibitory protein (vFLIP, ORF71, K13), viral interferon regulatory factor (vIRF) 3 (LANA-2, K10.5), and vIL-6 (K2) (Ganem 2007). All, except for LANA-2 which is exclusively expressed in PEL and MCD, are also expressed in all KSHV-infected cells and have been shown to affect cellular proliferation and survival (Ganem 2006).

Viral Strategies for Self-Proliferation Oncogenic HHVs, including EBV and KSHV, are equipped with strategies that promote the proliferation of infected host cells for their survival and replication. Both lytic and latent EBV and KSHV viral proteins demonstrate the ability to activate growth signaling by functioning as (1) growth factor receptors, (2) growth factor receptor ligands, (3) signal transduction molecules, (4) cell cycle regulators, or (5) transcription factors. Interestingly, some viral proteins are of cellular origin.

Viral Proteins Mimicking Growth Receptor A common feature shared by EBV and KSHV is the presence of membrane-associated viral proteins located at the left and right ends of the coding regions of their viral genomes. These viral proteins are LMP1 (EBV), LMP2A (EBV), K1 (KSHV), and K15 (KSHV). Unlike LMP1 and LMP2A, both K1 and K15 are mainly expressed during the lytic replication cycle (Brinkmann and Schulz 2006). There is no sequence homology among the proteins, although there is a limited structural similarity (Fig. 3.1). Yet, they all function as constitutively active receptors capable of inducing an array of cellular signaling pathways in a ligand-independent manner. In addition, they all have distinct oncogenic/transforming potentials. Thus, it has been suggested that these viral proteins act by mimicking normal growth signals required for the

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EBV LMP1

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Lyn

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I T A M

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Vav

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TRAF6

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TRADD

COOH

MAPK

PI3K/Akt

MAPK

NH2

JAK

MAPK

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2+

Ca

Fig. 3.1 Membrane-associated viral proteins encoded by both ends of EBV and KSHV genomes

proliferation and survival of cells, which ultimately contributes to the transformation of infected cells (Brinkmann and Schulz 2006; Damania et al. 2000; Schulz 2006). This section briefly discusses EBV and KSHV viral proteins which enhance selfproliferation by acting as growth factor receptors and their ligands. EBV LMP1. LMP1, the first open reading frame (ORF) of EBV, is expressed in all EBV-related malignancies. It is essential for the immortalization of primary B lymphocytes to LCLs in vitro (Rickinson and Kieff 2007). It is an integral membrane protein containing a short cytoplasmic N-terminus (23 aa), six transmembrane (TM)-spanning domains, and a long cytoplasmic C-terminus (200 aa). A substantial amount of experimental evidence suggests that the six TM domains and the two cytoplasmic C-terminal domains of LMP1, termed transformation effector sites (TESs) 1 and 2 or C-terminal NF-kB-activating regions (CTARs)1 and 2, are critical for the conversion of primary B lymphocytes to LCLs by LMP1 (Fig. 3.1, Soni et al. 2007). It has been shown that LMP1 markedly mimics an important B-cell activation receptor CD40, which induces the activation of NF-kB, a key transcription factor involved in the regulation of cell growth, antiapoptosis, and expression of numerous cytokines upon CD40–ligand interaction (Glenn et al. 1999; Hatzivassiliou et al. 1998; Kilger et al. 1998). Like CD40, LMP1 when expressed in B cells recruits tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and the TNF receptor-associated death domain (TRADD) through its CTAR1 and CTAR2, respectively (Devergne et al. 1998; Eliopoulos et al. 1996; Eliopoulos and Young 1998; Izumi et al. 1997). Unlike CD40, however, LMP1 transduces signals in the absence of extracellular ligands or cross-linking by self-oligomerizing through its N-terminal and the TM domains on the plasma membrane. This self-oligomerization mimics the effects of receptor/ligand interactions, giving rise to the constitutive activation of a number of downstream signaling pathways (Clausse et al. 1997;

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Eliopoulos and Young 1998; Floettmann et al. 1996; Hatzivassiliou et al. 1998; Huen et al. 1995; Izumi et al. 1997; Izumi and Kieff 1997; Laherty et al. 1992). Thus, by mimicking the B-cell activation receptor CD40, LMP1 contributes to the progression of EBV-associated malignancies. More detailed molecular mechanisms by which LMP1 activates NF-kB signaling are discussed in later in this review. In addition to NF-kB signaling, activation of both MAPK and JAK/STAT is also implicated to be the function of LMP1 (Eliopoulos et al. 1999; Eliopoulos and Young 1998; Gires et al. 1999; Huen et al. 1995). The MAPK family, a group of serine/threonine kinases activated in response to extracellular environment signals such as growth factor, cytokines, and stress signals, is involved in a variety of key events, including proliferation, differentiation, apoptosis, and migration. The MAPK family consists of three parallel pathways, namely ERK, JNK, and p38. In epithelial cells, LMP1-mediated activation of JNK and p38 depends more on its CTAR2 domain (Eliopoulos et al. 1999; Eliopoulos and Young 1998). In lymphocytes, however, both CTAR1 and CTAR2 are necessary for the activation of JNK and p38 (Soni et al. 2007). LMP1-mediated activation of JNK requires TRAF6, TAK1, and TAB1, but not TRAF2, TRADD, IRAK4, MyD88, and RIP, indicating that LMP1 selectively utilizes cellular signaling molecules involved in TNFR or IL-1/TLR receptors that maximize growth and survival signals without inducing apoptosis (Soni et al. 2007). Meanwhile, JAK/STAT signaling mediated by LMP1 integrates with the AP-1 transcription factor pathway. The region between CTAR1 and CTAR2 contains proline-rich sequences and is involved in the interaction with members of the JAK family. Thus, this motif of LMP1 is believed to play a role in JAK3-mediated activation of STAT3 (Gires et al. 1999). Taken together, LMP1 appears to mediate constitutive activation of cellular signaling pathways important for controlling EBV-infected cell survival and proliferation by mimicking activated receptors. EBV LMP2A. LMP2A is another membrane protein expressed in EBV latently infected B cells. LMP2A contains 12 TM domains linked by loops, a long cytoplasmic N-terminal (119 aa), and a short C-terminal domain (27 aa). LMP2A aggregates in the plasma membrane. The N-terminal cytoplasmic domain of LMP2A contains three tyrosine-based SH2 binding motifs, two of which form a functional immunoreceptor tyrosine-based activation motif (ITAM) (Fig. 3.1, Fruehling and Longnecker 1997). ITAM motifs [(D/E)x7(D/E)x2YxxL/I/Vx6–8YxxL/I/V, where x is any amino acid] are composed of a stretch of negatively charged amino acids followed by two SH2 binding motifs (YxxL/I/V). Found in the cytoplasmic domains of T- or B-cell receptors, ITAM motifs are tyrosine phosphorylated by Src family protein tyrosine kinases (SF-PTKs) upon ligand engagement. Signaling molecules containing SH2 domains are then subsequently recruited, leading to the induction of an array of intracellular signaling pathways. In the case of LMP2A, its ITAM is tyrosine phosphorylated and is required for LMP2A association with the SH2 domain of the nonreceptor tyrosine kinases, such as Lyn, Fyn, Syk, and Csk (Beaufils et al. 1993; Burkhardt et al. 1992; Longnecker et al. 1991; Scholle et al. 1999), to induce intracellular calcium mobilization and cytokine production.

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LMP2A interaction with Lyn and Syk mimics BCR signaling, inducing the activation of the PI3K/Akt survival pathway in the absence of BCR signals (Caldwell et al. 1998). The PI3K/Akt signaling pathway plays an important role in mediating transformation, antiapoptotic effects, invasion, and adhesion. Akt is a serine/threonine kinase, which phosphorylates and regulates the activities of cell cycle regulatory proteins, such as GSK-3b and cyclin D. LMP2A is also involved in the activation of MAPK signaling in various EBV-infected cell lines in vitro (Chen et al. 2002; Panousis and Rowe 1997). LMP2A binds to and is phosphorylated by ERK, leading to the activation of MAPK signaling in B-cell lines (Panousis and Rowe 1997). LMP2A activates JNK, but not p38, in NPC cell lines (Chen et al. 2002). This in vitro observation is supported by a recent study, in which LMP2A expression in transgenic mice induced the constitutive activation of the ERK/MAPK and PI3K/Akt pathways, resulting in cell proliferation and survival (Anderson and Longnecker 2008). KSHV K1. KSHV K1 is the first ORF located at a position equivalent to that of the LMP1 of EBV (Lagunoff et al. 1999; Lee et al. 1998b; Zong et al. 1999). K1 is a 46-kDa transmembrane glycoprotein consisting of an N-terminal extracellular domain, a TM domain, and a short cytoplasmic C-terminal domain (Fig. 3.1). Its N-terminal extracellular domain contains several N-glycosylation sites and displays a high degree of genetic variability between different KSHV isolates. A survey of isolates led to the identification of five major subtypes of K1 (A–E), each containing several distinct variants (Brinkmann and Schulz 2006). In contrast, its C-terminal cytoplasmic (38 aa) is relatively well-conserved and contains a functional ITAM similar to the one found in LMP2A (Lee et al. 1998a). The K1 ITAM motif is phosphorylated and recruits Lyn, Syk, PLCg2, the p85 subunit of PI3K, Vav1, SHP1 and SHP2, RAS-GAP, and growth factor receptor-bound protein 2 (Lagunoff et al. 1999; Lee et al. 2005). Interaction of K1 with these cellular proteins results in the activation of several transcription factors, including AP-1, NF-AT, and Akt-driven forkhead box containing proteins, all of which are involved in preventing apoptosis (Tomlinson and Damania 2004). In addition, K1 interaction with Lyn leads to the activation of NF-kB in B cells (Prakash et al. 2005). Similar to EBV LMP1 and LMP2A, K1-mediated signaling occurs constitutively, independent of ligand binding by self-oligomerization through its extracellular domain (Lagunoff et al. 1999). In addition, K1 induces VEGF and matrix metalloproteinase-9 (MMP-9) in endothelial cells (Greene et al. 2007), suggesting that K1 may contribute to angiogenesis and cell division. KSHV K15. The K15 gene of KSHV is located at a position equivalent to that of EBV LMP2A (Choi et al. 2000; Glenn et al. 1999; Poole et al. 1999). Sequence analysis of the K15 gene from different KSHV isolates revealed that the K15 gene is highly variable, showing as much as 60–70% divergence at the amino acid level (Poole et al. 1999). K15 is a transmembrane protein consisting of 4–12 TM domains and a short cytoplasmic C-terminal domain (amino acid 335–489) (Fig. 3.1). Like LMP2A, the cytoplasmic domain of K15 contains multiple signaling motifs, which have been shown to be highly conserved in most isolates (Poole et al. 1999): a putative SH3 binding motif (P386PPLPP), a potential SH2 binding motif (Y481EEVL), a

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tyrosine-based signaling motif (Y431ASIL), and a putative TRAF binding motif (A473TQPTDD) (Glenn et al. 1999; Poole et al. 1999). The cytoplasmic domain of K15 binds to TRAF1, TRAF2, and TRAF3 in vitro (Glenn et al. 1999). The tyrosine residue within the Y481EEVL motif of K15 is constitutively tyrosine phosphorylated by cellular SF-PTKs, including Src, Hck, Lck, Fyn, and Yes (Burkhardt et al. 1992; Choi et al. 2000; Rajcani and Kudelova 2003). Once phosphorylated, K15 activates the ERK2 and JNK pathways, which together lead to AP-1 activation (Brinkmann et al. 2003). TRAF2 interaction with the region containing Y481EEVL is involved in K15mediated activation of the MAPK pathway. Tyrosine phosphorylation within the Y481EEVL motif is required for TRAF2 binding and NF-kB activation (Brinkmann et al. 2003), indicating the importance of the Y481 residue of K15 in its activation of diverse signaling pathways. It has been reported that K15 is expressed in KSHVlatently infected PEL and MCD cells (Sharp et al. 2002), leading to the suggestion that the interaction between K15 and SF-PTKs plays a role in the maintenance of latency (Cho et al. 2008; Pietrek et al. 2010). Taken together, these findings suggest that K15 seems to function similarly to LMP1 (e.g., the recruitment of TRAFs and the activation of NF-kB and JNK) as well as to LMP2A (e.g., the recruitment of SF-PTKs).

Modulation of Chemokine/Cytokine System The mammalian chemokine system is composed of chemokines and chemokine receptors. Members of the chemokine superfamily currently consist of at least 46 members that are structurally related small proteins around 8–10 kDa in size. Four subfamilies have been identified so far based on the relative position of their cysteine (C) residues, which form conserved disulfide bonds: CCL, CXCL, XCL, and CX3L. The majority are either CCL chemokines (with no intervening amino acids between two cysteine residues) or CXCL (with a single intervening amino acid (X) between two cysteine residues) (Mantovani et al. 2010). The main function of chemokines is to attract different cells. For example, the CCL family members are known to attract a variety of cells from immune system, whereas the CXCL family members mainly attract neutrophils and lymphocytes. However, chemokines also regulate other biological activities, such as cell proliferation and differentiation, survival, angiogenesis, and senescence (Mantovani et al. 2010). Thus, deregulation of chemokine expression has been implicated in tumor growth, angiogenesis, and metastasis (Mantovani et al. 2010). Biological effects of chemokines are mainly mediated by G protein-coupled receptors (GPCRs), a diverse family of membrane receptors containing seven TM domains. While the extracellular domains of GPCRs engage with a variety of ligands, its cytoplasmic domain couples to heterotrimeric G proteins made up of an a subunit and a bg heterodimer. Following ligand binding, its TM domains undergo a series of conformational changes, catalyzing GDP to GTP exchange on a Ga subunit and subsequently generating a free GTP-bound Ga subunit and a free Gbg heterodimer to activate downstream signaling effector proteins, including PLC and

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adenylate cyclase (AC), which function as secondary messenger molecules. Although mutant forms of GPCR or heterotrimeric G proteins are not frequently found in human cancer, malignant cells often subvert the normal physiological functions of GPCRs to promote autonomous proliferation, evade immune responses, increase blood supply, and invade surrounding tissues and disseminate to other organs (Dorsam and Gutkind 2007). Several members of the HHV family, including EBV, KSHV, HHV-6, -7, and hCMV-1, encode mimicries of cellular chemokines and chemokine receptors, indicating that these viruses might have evolved to hijack host chemokine system for their propagation and replicative advantage (Rosenkilde et al. 2008). KSHV Viral Chemokines. KSHV expresses at least three chemokine homologs of cellular macrophage-inhibitory proteins (MIPs), which are members of the CCL chemokine family (Moore et al. 1996). For this reason, they were previously named vMIPI (vCCL1), vMIPII (vCCL2), and vMIPIII (vCCL3) and are encoded by ORFs K6, K4, and K4.1, respectively. These KSHV-encoded chemokines act on cellular chemokine receptors expressed on Th2 cells, such as CCR8 (vCCL1 and vCCL2), CCR3 (vCCL2), and CCR4 (vCCL3), from which their immunomodulatory functions are derived (Boshoff et al. 1997; Dairaghi et al. 1999; Endres et al. 1999; Stine et al. 2000). Interestingly, vCCLs, unlike their cellular homologs, have been shown to promote angiogenesis in certain experimental systems (Boshoff et al. 1997; Simonart et al. 2001; Stine et al. 2000). Since angiogenesis is a key feature of KS, vCCLs were speculated to be possible angiogenic factors of KS. For instance, vCCL1 activates the induction of a potent angiogenic factor, VEGF, and its receptor KDR (Flt-1) in vCCL1-expressing cells. Thus, it has been suggested that increased signaling mediated by upregulated VEGF and its receptor by vCCL1 may enhance new blood vessel formation and proliferation of tumor cells within the microenvironment of angiogenic KS lesions (Liu et al. 2001). Since KS lesions are also characterized by the presence of infiltrating leukocytes and high levels of inflammatory cytokines, the chemotactic properties of vCCLs may further promote infiltration of monocytes/macrophages to KS lesions to facilitate the production of inflammatory cytokines and proangiogenic factors (Direkze and Laman 2004). KSHV vGPCR. The KSHV vGPCR, encoded by ORF74, is homologous to the human chemokine receptors CXCR1 and CXCR2, which are the receptors for the angiogenic chemokines IL-8 (also known as CXCL8) and growth-related oncogene a (Gro-a, also known as CXCL1). Similar to cellular GPCRs, KSHV vGPCR is a seven TM protein with conserved glycosylation sites in its N-terminal and cysteine residues in its extracellular loops (Fig. 3.2a, Arvanitakis et al. 1997; Bais et al. 1998). Unlike cellular GPCRs, however, vGPCR is ligand-independent and constitutively active due to the presence of several structural changes, including a mutation (Asp142Val) within its highly conserved DRY (Asp-Arg-Tyr) motif (Rosenkilde et al. 2008). Moreover, compared to other viral and cellular chemokine receptors, vGPCR shows a higher degree of promiscuity, capable of binding not only cellular CXCL1 and CXCL8 but also vCCL2, indicating that vGPCR can induce signaling in a ligand-dependent fashion as well (Geras-Raaka et al. 1998; Gershengorn et al. 1998).

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a

b

KSHV vGPCR

EBV BILF1

Cellular, Viral ligands

ligands ?

NH2

NH2

a

b

a

g

COOH

a

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GTP

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a GTP

GTP

MAPK

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Fig. 3.2 Signaling mediated by viral chemokine receptors

Thus, both ligand-dependent and -independent activation of vGPCR may contribute to tumorigenesis and/or viral replication uniquely in the microenvironment of KSHV-associated tumors. The broad signaling capability of vGPCR also encompasses signaling through several G-protein a subunit families (Gas, Gaq, and Gai) (Shepard et al. 2001; Smit et al. 2002) as well as Rac-1, a member of the Rho family of monomeric G-proteins (Montaner et al. 2004). Signaling pathways constitutively activated by KSHV vGPCR include MAPK, PLC, PI3K, and Akt. Expression of KSHV vGPCR in human umbilical vascular endothelial cells (HUVECs) results in the induction of PI3K and Akt activity, which in turn plays a central role in promoting cell survival (Bais et al. 2003; Montaner et al. 2001; Sodhi et al. 2004). Akt activity is tightly regulated by PI3K. When growth factor receptors bind to ligands, the catalytic subunit (p110) of PI3K is activated via recruitment of the regulatory subunit (p85) of PI3K or via Ras activation, both of which lead to the production of PIP3. Akt in the cytoplasm then binds to PIP3 and subsequently translocates to the plasma membrane, where its kinase activity is then fully induced by PI3K-dependent kinases (PDKs). The tumor-suppressor protein PTEN antagonizes PI3K by dephosphorylating PIP3, reducing Akt translocation to the cellular membrane, thereby downregulating Akt activity. It has been reported that treatment of an Akt inhibitor prevents the proliferation of vGPCR-expressing endothelial cells in vitro and inhibits the tumorigenic potential of vGPCR in mice (Sodhi et al. 2004). This suggests that the activation of Akt is a significant factor in the development of vGPCR-induced sarcoma and perhaps KS. Recent studies have further demonstrated that vGPCR-mediated Akt activity can activate the mTOR pathway, which also plays a central role in cell proliferation, metabolism, and angiogenesis (Sodhi et al. 2006). Constitutive activation of Akt by vGPCR-mediated PI3K

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activation results in phosphorylation and subsequent degradation of the tuberous sclerosis complex (TSC), a negative regulator of mTOR (Sodhi et al. 2006). As a result, mTOR activity is induced, giving rise to the activation of ribosomal p70 S6 kinase and 4EBP1, the key regulators of the translational machinery, and the promotion of cell proliferation. Treatment of rapamycin, a pharmacological inhibitor of mTOR, showed a dramatic suppression of vGPCR-expressing cell proliferation in vitro as well as tumor growth in vivo, indicating that vGPCR-mediated activation of the PI3k/Akt/mTOR cascade may contribute to vGPCR-mediated sarcomagenesis (Montaner 2007). Similar to vCCLs, vGPCR has also been reported to have angiogenic functions. Endothelial cells ectopically expressing vGPCR have constitutively active VEGF receptors and increased proliferation (Bais et al. 2003). In addition to VEGF, vGPCR also upregulates angiogenic chemokines IL-8 and Gro-a in a manner similar to human CXCR2, a chemokine receptor associated with angiogenesis in a number of tumors (Montaner et al. 2004). Another signaling pathway induced by vGPCR is the activation of p38 and ERK and the subsequent phosphorylation of hypoxia-induced factor 1a (HIF-1a). This, then, acts on the VEGF promoter, resulting in the induction of VEGF expression and secretion (Sodhi et al. 2000). vGPCR can also activate such transcription factors as NF-AT, AP-1, and NF-kB and promote the expression of a number of autocrine and paracrine proinflammatory cytokines and growth factors, such as IL-1b, IL-6, GM-CSF, TNFa, IL-8, and MIP-1, as well as adhesion molecules, such as VCAM-1, ICAM-1, and E-selectin (Cannon et al. 2003; Couty et al. 2001; Montaner et al. 2001; Pati et al. 2001, 2003; Schwarz and Murphy 2001; Shepard et al. 2001; Smit et al. 2002). As discussed above, vGPCR appears to exert broad effects on cell proliferation, angiogenesis, and inflammation for a mere single viral gene product. Thus, KSHV vGPCR, although expressed during the lytic phase of its viral life cycle, clearly stands out as one of the key factors involved in the proliferation of KSHV-associated tumor cells. EBV BILF1. EBV encodes a GPCR homolog called BILF1, which is expressed in the lytic phase of the viral replication cycle. Although it displays a limited homology to chemokine receptors, sequence analysis revealed that BILF1 has several features belonging to GPCRs. It contains seven TM domains, conserved cysteine residues in its N-terminus and in its extracellular loops, seven N-glycosylation sites, and four intracellular phosphorylation sites (Fig. 3.2b, Paulsen et al. 2005). BILF1 is known to constitutively activate signaling pathways involved in proliferation (NF-kB) through Gai (Paulsen et al. 2005). Similar to KSHV vGPCR, the DRY motif in BILF1 is replaced with an EKT (Glu-Lys-Thr) motif (Paulsen et al. 2005). Alternative DRY motifs found in KSHV vGPCR have been previously shown to be associated with constitutive activity as well as transforming activity (Rosenkilde et al. 2008). Unsurprisingly, a recent study demonstrated that BILF1, similar to KSHV vGPCR, has both of these abilities in vitro and in vivo (Lyngaa et al. 2010). This study shows that the EKT motif plays a key role in the constitutive activation of BILF1 and that constitutive signaling through Gai is associated with BILF1-mediated cell transformation, VEGF secretion, and tumor formation (Lyngaa et al. 2010). Although these

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studies imply that like KSHV vGPCR BILF1 may play an important role during EBV infection by mimicking a functional GPCR, additional roles of BILF1 in immune evasion by targeting PKR and MHC class I (Garcia et al. 2006; Zuo et al. 2009) suggest that BILF1 may function distinctly from KSHV vGPCR. Moreover, it still remains unknown whether chemokines and other ligands bind to BILF1. KSHV vIL-6 (ORF K2). Cellular interleukin (IL)-6 is a multifunctional cytokine involved in the regulation of immune responses, inflammation, oncogenesis, and angiogenesis (Nishimoto and Kishimoto 2006). Signaling induced by IL-6 requires the IL-6 receptor (IL-6R) complex composed of gp80 (the a subunit) and gp130 (the b subunit). gp130 is ubiquitously expressed and homodimerizes to transduce intracellular signaling, whereas gp80 binds to IL-6 but is limited in its availability. IL-6/IL-6R signaling induces signaling pathways, such as the Ras/MAPK and JAK/ STAT pathways, which are in turn involved in the activation of transcription of IL-6 responsive genes (c-Fos and c-Jun). Many B-cell tumor cell lines depend on IL-6 signaling for growth (Nishimoto and Kishimoto 2006). KSHV encodes a homolog of cellular IL-6, vIL-6 encoded in (ORF) K2 (Neipel et al. 1997). It has 24.6% amino acid sequence homology to cellular IL-6. Although expressed abundantly during lytic replication, various levels of vIL-6 expression have been detected in latently infected cells (MCD>PEL>>KS;