Molecular pathology library series Philip T. Cagle, MD, Series Editor For other titles published in this series, go to www.springer.com/series/7723 Molecular Pathology of Hematolymphoid Diseases Edited by Cherie H. Dunphy University of North Carolina, Chapel Hill, NC, USA Editor Cherie H. Dunphy Department of Pathology and Laboratory Medicine University of North Carolina Chapel Hill 27599-7525, NC USA cdunphy@unch.unc.edu Series Editor Philip T. Cagle, MD Pathology and Laboratory Medicine Weill Medical College of Cornell University New York, NY The Methodist Hospital Houston, TX USA ISBN 978-1-4419-5697-2 e-ISBN 978-1-4419-5698-9 DOI 10.1007/978-1-4419-5698-9 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010921203 © Springer Science+Business Media, LLC 2010 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 stor- age 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. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) The past two decades have seen an ever-accelerating growth in knowledge about molecular pathology of human diseases, which received a large boost with the sequencing of the human genome in 2003. Molecular diagnostics, molecular targeted therapy and genetic therapy, are now routine in many medical centers. The molecular field now impacts every field in medicine, whether clinical research or routine patient care. There is a great need for basic researchers to understand the potential clinical implications of their research whereas private practice clinicians of all types (general internal medicine and internal medicine specialists, medi- cal oncologists, radiation oncologists, surgeons, pediatricians, family practitioners), clinical investigators, pathologists and medical laboratory directors and radiologists require a basic understanding of the fundamen- tals of molecular pathogenesis, diagnosis, and treatment for their patients. Traditional textbooks in molecular biology deal with basic science and are not readily applicable to the medical setting. Most medical textbooks that include a mention of molecular pathology in the clinical set- ting are limited in scope and assume that the reader already has a working knowledge of the basic science of molecular biology. Other texts emphasize technology and testing procedures without integrating the clinical perspective. There is an urgent need for a text that fills the gap between basic science books and clinical practice. In the Molecular Pathology Library series, the basic science and the technology is integrated with the medical perspective and clinical application. Each book in the series is divided according to neoplastic and non-neoplastic diseases for each of the organ systems traditionally associated with medical subspecialties. Each book in the series is organized to provide specific application of molecular pathology to the patho- genesis, diagnosis, and treatment of neoplastic and non-neoplastic diseases specific to each organ system. These broad section topics are broken down into succinct chapters to cover a very specific disease entity. The chapters are written by established authorities on the specific topic from academic centers around the world. In one book, diverse subjects are included that the reader would have to pursue from multiple sources in order to have a clear understanding of the molecular pathogenesis, diagnosis, and treatment of specific diseases. Attempting to hunt for the full information from basic concept to specific applications for a disease from varied sources is time-consuming and frustrating. By providing this quick and user- friendly reference, understanding and application of this rapidly growing field is made more accessible to both expert and generalist alike. As books that bridge the gap between basic science and clinical understanding and practice, the Molecu- lar Pathology Series serves the basic scientist, the clinical researcher and the practicing physician or other health care provider who require more understanding of the application of basic research to patient care, from “bench to bedside.” This series is unique and an invaluable resource to those who need to know about molecular pathology from a clinical, disease-oriented perspective. These books will be indispensable to physicians and health care providers in multiple disciplines as noted above, to residents and fellows in these multiple disciplines as well as their teaching institutions and to researchers who increasingly must justify the clinical implications of their research. New York, NY Philip T. Cagle, MD Series Preface v Section I Molecular Pathology of Hematolymphoid Neoplasms: General Principles Chapter 1 Molecular Oncogenesis ................................................................................................... 3 Aniruddha J. Deshpande, Christian Buske, Leticia Quintanilla-Martinez, and Falko Fend Chapter 2 Genetic Predispositions for Hematologic and Lymphoid Disorders ............................... 21 Frederick G. Behm Chapter 3 Prognostic Markers.......................................................................................................... 65 David Bahler Chapter 4 Cancer Stem Cells: Potential Targets for Molecular Medicine ....................................... 73 Isabel G. Newton and Catriona H.M. Jamieson Chapter 5 Gene Therapy for Leukemia and Lymphoma.................................................................. 81 Xiaopei Huang and Yiping Yang Chapter 6 Chemical and Environmental Agents (Including Chemotherapeutic Agents and Immunosuppression) .................................................................................... 91 Richard J.Q. McNally Chapter 7 Viral Oncogenesis............................................................................................................ 107 Alexander A. Benders and Margaret L. Gulley Section II Specific Techniques and Their Applications in Molecular Hematopathology Chapter 8 Techniques to Determine Clonality in Hematolymphoid Malignancies ......................... 119 Daniel E. Sabath Chapter 9 Techniques to Detect Defining Chromosomal Translocations/Abnormalities ................ 129 Jennifer J.D. Morrissette, Karen Weck, and Cherie H. Dunphy Chapter 10 Molecular Techniques to Detect Disease and Response to Therapy: Minimal Residual Disease ............................................................................................... 153 Marie E. Beckner and Jeffrey A. Kant Chapter 11 Detection of Resistance to Therapy in Hematolymphoid Neoplasms ............................. 165 Karen Weck Chapter 12 Monitoring Engraftment of Bone Marrow Transplant by DNA Fingerprinting.............. 173 Jessica K. Booker Contents vii Contents Chapter 13 Gene Expression Profiling ............................................................................................... 177 Cherie H. Dunphy Chapter 14 Proteomics of Human Malignant Lymphoma ................................................................. 191 Megan S. Lim, Rodney R. Miles, and Kojo S.J. Elenitoba-Johnson Chapter 15 Mouse Models of Hematolymphoid Malignancies ......................................................... 203 Krista M.D. La Perle and Suzana S. Couto Section III Molecular Pathology of Hematolymphoid Neoplasms: Specific Subtypes Chapter 16 Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma ................................. 211 Patricia Aoun Chapter 17 Marginal Zone B-Cell Lymphoma .................................................................................. 221 Lynne V. Abruzzo and Rachel L. Sargent Chapter 18 Lymphoplasmacytic Lymphoma ..................................................................................... 233 Pei Lin Chapter 19 Molecular Pathology of Plasma Cell Neoplasms ............................................................ 241 James R. Cook Chapter 20 The Roles of Molecular Techniques in the Diagnosis and Management of Follicular Lymphoma .................................................................................................. 249 W. Richard Burack Chapter 21 Mantle Cell Lymphoma ................................................................................................... 257 Kai Fu and Qinglong Hu Chapter 22 Diffuse Large B-Cell Lymphomas .................................................................................. 267 Cherie H. Dunphy Chapter 23 The Molecular Pathology of Burkitt Lymphoma ............................................................ 277 Claudio Mosse and Karen Weck Chapter 24 Precursor B-Cell Acute Lymphoblastic Leukemia.......................................................... 287 Julie M. Gastier-Foster Chapter 25 Molecular Genetics of Mature T/NK Neoplasms............................................................ 309 John P. Greer, Utpal P. Davé, Nishitha Reddy, Christine M. Lovly, and Claudio A. Mosse Chapter 26 Precursor T-Cell Neoplasms ............................................................................................ 329 Kim De Keersmaecker and Adolfo Ferrando Chapter 27 Classical Hodgkin Lymphoma and Nodular Lymphocyte-Predominant Hodgkin Lymphoma ........................................................................................................ 347 Michele Roullet and Adam Bagg Chapter 28 Posttransplant Lymphoproliferative Disorder ................................................................. 359 Margaret L. Gulley Chapter 29 AIDS-Related Lymphomas ............................................................................................. 367 Amy Chadburn and Ethel Cesarman viii Contents Chapter 30 Chronic Myelogenous Leukemia .................................................................................... 387 Dan Jones Chapter 31 Molecular Pathogenesis of Nonchronic Myeloid Leukemia Myeloproliferative Neoplasms ........................................................................................ 395 Mike Perez and Chung-Che (Jeff) Chang Chapter 32 Molecular Pathology of Myelodysplastic/Myeloproliferative Neoplasms, Myeloid, and Lymphoid Neoplasms with Eosinophilia and Abnormalities of PDGFRA, PDGFRB, and FGFR1, and Mastocytosis ................................................... 405 Robert P. Hasserjian Chapter 33 Molecular Pathogenesis of Myelodysplastic Syndromes ................................................ 417 Jesalyn J. Taylor and Chung-Che “Jeff” Chang Chapter 34 Acute Myeloid Leukemias with Recurrent Cytogenetic Abnormalities ......................... 429 Sergej Konoplev and Carlos Bueso-Ramos Chapter 35 Acute Myeloid Leukemias with Normal Cytogenetics ................................................... 449 Sergej Konoplev and Carlos Bueso-Ramos Chapter 36 Acute Myeloid Leukemia with Myelodysplasia-Related Changes and Therapy-Related Acute Myeloid Leukemia ............................................................. 463 Sergej N. Konoplev and Carlos E. Bueso-Ramos Chapter 37 Molecular Pathology of Hemoglobin and Erythrocyte Membrane Disorders................. 473 Murat O. Arcasoy and Patrick G. Gallagher Chapter 38 White Blood Cell and Immunodeficiency Disorders ...................................................... 499 John F. Bastian and Michelle Hernandez Chapter 39 Molecular Basis of Disorders of Hemostasis and Thrombosis ....................................... 511 Alice Ma Chapter 40 Sarcoidosis: Are There Sarcoidosis Genes? .................................................................... 529 Helmut H. Popper Chapter 41 Castleman’s Disease ........................................................................................................ 541 Richard Flavin, Cara M. Martin, Orla Sheils, and John James O’Leary Chapter 42 Molecular Pathology of Histiocytic Disorders ................................................................ 545 Mihaela Onciu Chapter 43 Reactive Lymphadenopathies: Molecular Analysis ........................................................ 561 Dennis P. O’Malley Chapter 44 Molecular Pathology of Infectious Lymphadenitides ..................................................... 569 Kristin Fiebelkorn Chapter 45 Gene Therapy for Nonneoplastic Hematologic and Histiocytic Disorders ..................... 597 Kareem N. Washington, John F. Tisdale, and Matthew M. Hsieh Index ..................................................................................................................................................... 609 ix Lynne V. Abruzzo, MD, PhD Associate Professor of Hematopathology, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Patricia Aoun, MD, MPH Associate professor, Department of Pathology and Microbiology, University of Nebraska Medical Cen- ter, Omaha, NE, USA Murat O. Arcasoy, MD, FACP Associate Professor of Medicine, Division of Hematology, Department of Medicine, Duke University Medical Center, Durham, NC, USA Adam Bagg, MD Professor, Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA David Bahler, MD, PhD Associate Professor of Pathology, Department of Pathology, University of Utah, Salt Lake City, UT, USA John F. Bastian, MD Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA Marie E. Beckner, MD Fellow, Molecular Diagnostics, Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA Frederick G. Behm, MD Director of Clinical Pathology, Department of Pathology, University of Illinois at Chicago, Chicago, IL, USA Alexander A. Benders, MD Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands Jessica K. Booker, PhD Scientific and Assistant Director of Clinical Molecular Genetics Laboratory, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Carlos E. Bueso-Ramos, MD, PhD Professor, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Walter Richard Burack, MD, PhD Associate Professor, Director of Hematopathology Section, Department of Pathology and Laboratory Medicine, Strong Memorial Hospital, University of Rochester, Rochester, NY, USA Christian Buske, MD Professor, Institute for Experimental Tumor Resarch and Department of Internal Medicine III, University Hospital Ulm, Ulm, Germany Contributors xi Contributors Ethel Cesarman, MD, PhD Professor of Pathology and Laboratory Medicine, Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA Amy Chadburn, MD Professor, Department of Pathology, Northwestern University – Feinberg School of Medicine, Chicago, IL, USA Chung-Che (Jeff) Chang, MD, PhD Chief, Hematopathology Service and Director, Hematopathology Fellowship, The Methodist Hospital, Houston, TX, USA Professor, Department of Pathology, Weill Medical College of Cornell University, New York, NY, USA James R. Cook, MD, PhD Assistant Professor of Pathology, Department of Pathology, Cleveland Clinic Lerner College of Medi- cine, Cleveland, OH, USA Suzana S. Couto, DVM, DACVP Head, Laboratory of Comparative Pathology, Clinical Pathology Division, Research Animal Resource Center, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Utpal P. Davé, MD Assistant Professor of Medicine and Cancer Biology, Division of Hematology/Oncology, Vanderbilt University Medical Center, Nashville, TN, USA Kim De Keersmaecker, PhD Departments of Pediatrics and Pathology, Columbia University Medical Center, New York, NY, USA Department of Molecular and Developmental Genetics-VIB, Center for Human Genetics, K.U. Leuven Hospital, Leuven, Belgium Aniruddha J. Deshpande, PhD Department of Hematology/Oncology, Children’s Hospital Boston, Boston, MA, USA Cherie H. Dunphy, MD Professor and Director of Hematopathology and Hematopathology Fellowship, Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA Kojo S.J. Elenitoba-Johnson Professor, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA Falko Fend, MD Professor, Institute of Pathology, University Hospital Tuebingen, Eberhard-Karls University, Tuebingen, Germany Adolfo A. Ferrando, MD, PhD Assistant Professor of Pediatrics and Pathology, Institute for Cancer Genetics, Columbia University, New York, NY, USA Kristin R. Fiebelkorn, MD Assistant Professor, Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Richard Flavin, MB, FRCPath Department of Histopathology, Trinity College Dublin, Dublin, Ireland Kai Fu, MD, PhD Assistant Professor and Staff Hematopathologist, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA Patrick G. Gallagher, MD Associate Professor, Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA xii Contributors Julie M. Gastier-Foster, PhD Director, Cytogenetics/Molecular Genetics Laboratory, Department of Laboratory Medicine, Nationwide Children’s Hospital, OH,USA Department of Pathology, Ohio State University, Columbus, OH, USA John P. Greer, MD Professor of Medicine and Pediatrics, Department of Hematology/Stem Cell Transplantation, Vanderbilt University Medical Center, Nashville, TN, USA Margaret L. Gulley, MD Professor of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Robert P. Hasserjian, MD Assistant Professor, Department of Pathology, Harvard Medical School/Massachusetts General Hospital, Boston, MA, USA Michelle Hernandez, MD Department of Pediatrics, University of North Carolina, Chapel Hill, NC, USA Matthew M. Hsieh, MD Staff Clinician, NHLBI-NIDDK-MCHB, National Institutes of Health, Bethesda, MD, USA Qinglong Hu, MD, MSc Assistant Professor, Department of Pathology, Creighton University Medical Center/School of Medicine, Omaha, NE, USA Xiaopei Huang, PhD Senior Research Scientist, Department of Medicine and Immunology, Duke University Medical Center, Durham, NC, USA Catriona H.M. Jamieson, MD, PhD Assistant Professor, Division of Hematology-Oncology, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Dan Jones, MD, PhD Professor, MD Anderson Cancer Center, Houston, TX, USA, and Quest Diagnostics, Chantilly, VA, USA Jeffrey A. Kant, MD, PhD Director, Division of Molecular Diagnostics, Department of Pathology and Human Genetics, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Sergej N. Konoplev, MD, PhD Assistant Professor, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Krista M. D. La Perle, DVM, PhD, DACVP Director, Laboratory of Comparative Pathology, Research Animal Resource Center, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Megan S. Lim, MD, PhD Associate Professor, Department of Pathology, University of Michigan Medical Center, Ann Arbor, MI, USA Pei Lin, MD Associate Professor, Department of Hematopathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Christine M. Lovly, MD, PhD Clinical Fellow, Department of Hematology and Oncology, Vanderbilt University School of Medicine, Nashville, TN, USA xiii Contributors Alice D. Ma, MD Associate Professor of Medicine, Department of Hematology/Oncology, University of North Carolina, Chapel Hill, NC, USA Cara M. Martin, PhD, MSc, BSc Department of Histopathology, The Coombe Women and Infant’s University Hospital, University of Dublin, Trinity College, Dublin, Ireland Richard J.Q. McNally, BSc, MSc, DIC, PhD Department of Health and Society, Newcastle University, Newcastle upon Tyne, England, UK Rodney R. Miles, MD, PhD Assistant Professor, Department of Pathology, University of Utah, Salt Lake City, UT, USA Jennifer J.D. Morrissette, PhD, FACMG Director, Clinical Cytogenetics, Department of Pathology, St. Christopher’s Hospital for Children, Philadelphia, PA, USA Claudio A. Mosse, MD, PhD Assistant Professor, Department of Pathology, Vanderbilt University Medical Center and Nashville Veterans Administration Medical Center, Tennessee Valley Healthcare Systems, Nashville, TN, USA Isabel Gala Newton, MD, PhD Research Resident, Radiology Department, University of California San Diego Medical Center, San Diego, CA, USA John James O’Leary, MD, PhD, MSc, MA, FRCPath, HPath, RCPI, FTCD Professor, Department of Pathology, Trinity College Dublin, Dublin, Ireland Dennis P. O’Malley, MD Hematopathologist, Clarient Inc., Aliso Viejo, CA, USA Mihaela Onciu, MD Director, Anatomic pathology and Special Hematology Laboratories, Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA Mike Perez, MD Hematopathology Fellow, Department of Pathology, The Methodist Hospital and The Methodist Research Institute, Houston, TX, USA Helmut H. Popper, MD Professor of Pathology, Department of Pathology, Medical University of Graz, Graz, Austria Leticia Quintanilla-Martinez, MD Institute of Pathology, University Hospital Tuebingen, Eberhard-Karls University Tuebingen, Tuebingen, Germany Nishitha Reddy, MD Assistant Professor, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Michele Roullet, MD Assistant Professor, Department of Pathology and anatomy, Pathology Sciences Medical Group/Eastern Virginia Medical School, Norfolk, VA, USA Daniel E. Sabath, MD, PhD Associate Professor, Head of Hematology Division, Departments of Laboratory Medicine and Medicine, University of Washington School of Medicine, Seattle, WA, USA Rachel L. Sargent, MD Assistant Professor, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Orla Sheils, PhD, FAMLS, MA, MA (Med. Ethics and Law), FRCPath, FTCD Department of Histopathology and Morbid Anatomy, Trinity College Dublin, Dublin, Ireland xiv Contributors Jesalyn J. Taylor, MD Hematopathology Fellow, Department of Pathology, The Methodist Hospital and The Methodist Research Institute, Houston, TX, USA John F. Tisdale, MD Senior Investigator, NHLBI-NIDDK-MCHB, National Institutes of Health, Bethesda, MD, USA Kareem N. Washington, PhD Research Fellow, NHLBI-NIDDK-MCHB, National Institutes of Health, Bethesda, MD, USA Karen Weck, MD Associate Professor, Departments of Pathology and Laboratory Medicine and Genetics, University of North Carolina, Chapel Hill, NC, USA Yiping Yang, MD, PhD Associate Professor, Department of Medicine and Immunology, Duke University Medical Center, Durham, NC, USA xv Section I Molecular Pathology of Hematolymphoid Neoplasms: General Principles C.H. Dunphy (ed.), Molecular Pathology of Hematolymphoid Diseases , Molecular Pathology Library 4, DOI 10.1007/978-1-4419-5698-9_1, © Springer Science+Business Media, LLC 2010 Introduction The history of molecular pathology is inseparable from the advances in neoplastic hematopathology, since many advances, both in understanding mechanisms of disease development and progression, as well as of technical aspects of molecular pathology, are intimately linked with landmark findings in hematologic disorders. The detection of the Phil- adelphia chromosome in chronic myelogenous leukemia, which was subsequently shown to represent a translocation involving chromosomes 9 and 22 t(9;22)(q34;q11.2) result- ing in the BCR–ABL fusion gene (see Chap. 30), marks the beginning of an exciting journey, which in turn has led to the development of targeted therapies against this defining molecular aberration. The first clinical areas where molecular testing was incor- porated into routine diagnosis and clinical management of patients were hematology and hematopathology. Molecular studies are nowadays an integral part of state-of-the-art diag- nostics of hematologic neoplasms. Correct performance and interpretation of molecular studies in these disorders require an understanding of the underlying principles of oncogenesis. Therefore, this chapter tries to summarize the molecular changes that are important for the development and progression of hematolymphoid malignancies. The Initiation and Maintenance of Oncogenic Programs: Genetic and Epigenetic Changes Human tumors are often a result of the abnormal and limitless clonal expansion of one renegade cell. Like normal cells, tumor cells propagate by the transmission of their genetic and epigenetic information to daughter cells. The difference is that in tumor cells, this information is changed, usually in many ways, and the faithful propagation of this abnormal change is the key to the expansion of the tumor. These changes can occur at many levels, one of the most important being the change in genetic information due to changes in DNA sequence that is characteristic of most cancers. Recently, epigenetic changes or changes in genetic information without alterations in the sequence of DNA have been in the limelight because they have profound effects on gene expression and the maintenance of genome integrity. Genetic and epigenetic lesions are acquired by somatic cells, often progressively, and can work in tandem to induce tumor formation. Types of Genetic Changes in Hematolymphoid Neoplasms Recent studies involving genetic and molecular techniques have provided tremendous insights into the biology of hema- topoietic neoplasms. Genetic changes in hematopoietic and lymphoid malignancies are the result of either chromosomal alterations or epigenetic changes that induce deregulation of gene expression. Since the discovery of the Philadelphia chromosome, recurrent chromosomal abnormalities such as translocations, deletions, inversions and duplications associated with several types of leukemia, lymphoma, and certain types of epithelial tumors have been identified. 1–3 These chromosomal abnor- malities are often somatic mutations acquired by a clonally expanded malignant population. As is the case with CML, certain chromosomal abnormalities can be associated with specific types of disease, and the characterization of these abnormalities can be used for diagnosis, as well as for the determination of disease prognosis. Moreover, treatment regimens can be optimized to suit discrete subgroups divided according to these abnormalities. Chromosomal aberrations can be numerical (changes in chromosome numbers) or structural (changes in chromosome structure such as those arising from translocations, inversions, deletions, etc.). Even though several hundred different types of chromosomal alterations have been reported 4 , most of them occur at a very low frequency, with some recurrent translocations accounting for most of the cases. These translocations can, however, be 1 Molecular Oncogenesis Aniruddha J. Deshpande, Christian Buske, Leticia Quintanilla-Martinez, and Falko Fend 3 A.J. Deshpande et al. broadly classified into those that lead to the juxtaposition of oncogenes to strong regulatory elements, such as those of the immunoglobulins or chromosomal translocations that lead to oncogenic fusion gene formation. The former leads to the aberrant overexpression of structurally normal oncogenic gene products and are mostly observed in lymphoid malig- nancies. The latter types of gene rearrangements lead to the formation of aberrant fusion genes, many of which have been shown to be oncogenic in models of tumor formation. In contrast to the chromosomal translocations, other acquired somatic mutations such as point mutations, dele- tions, and insertions have been more difficult to detect. However, mutations in protein-coding genes constitute a significant proportion of genetic changes and may impact tumor progression. These mutations occur in a diverse set of genes, some of the most common being in genes governing signal transduction pathways or in lineage-specific transcrip- tion factors. While mutations in signaling pathway genes confer proliferative advantage to cells, abnormal changes in lineage-specific transcription factors impair differentiation of cells. These two types of mutations, as described below in the two-hit model of leukemogenesis, are often seen to be complementary and sequentially acquired steps. Although the assumption that signaling pathway alterations mostly affect proliferation, and transcription factor deregulation that mostly affects differentiation is simplistic and not entirely correct, for didactic purposes, this division is helpful and will be used to describe the two classes of mutations in more detail in the next subsections. Since the molecular mecha- nisms responsible for triggering leukemia and lymphoma are so different, the chapter is divided into two sections; one section deals with molecular mechanisms of leukemias and myeloid disorders and the second section deals with molecu- lar mechanisms of lymphoid neoplasms. Genetic Changes in Leukemia and Myeloid Disorders Multistep Pathogenesis and the Cooperativity of Genetic Alterations Cancer is now widely recognized as a multistep process involving progressive accumulation of multiple mutations involving the activation of oncogenes and the inactivation of tumor suppressor genes. Often, the deregulation of dis- tinct pathways and processes by these accumulating muta- tions is a necessary prerequisite for tumor formation. Several observations suggest that single mutations are insufficient for tumor development. Cells carrying certain leukemia- or lymphoma-specific lesions may be detected in normal indi- viduals, albeit at low frequencies. 5–7 A simplistic model for cooperative mutations in acute myeloid leukemias (AML) proposed by Gilliland and Griffin 8 postulates that these can be broadly classified into two major complementary subgroups: (1) mutations that confer proliferation or survival signals (usually involving aberrantly activated tyrosine kinases) and (2) mutations that impair differentiation (usually involving transcription factors) (Figure 1.1). 9 It is hypoth- esized that the combined action of these two classes of muta- tions is necessary for a full-blown AML to develop. This is supported by the fact that mutations in two genes belonging to the same sub-group are rarely seen in the same patient. In line with the finding that abnormal gene fusions can be found in normal individuals, the fusion genes AML1/ETO ( RUNX1- RUNX1T1 ) as a result of a t(8;21)(q22;q22) and TEL/AML1 ( ETV6-RUNX1 ) occurring as a result of t(12;21)(p13;q22) have been reported to occur at low frequencies without induc- ing disease. Accordingly, it was also shown that these fusion genes can rarely initiate complete leukemogenesis in murine models in the absence of cooperating mutations. 10,11 However, the introduction of appropriate “second hits,” which support the hypothesis of collaborative action, can induce a leuke- mic phenotype, resembling the corresponding human malig- nancy. For example, aggressive leukemias could be induced by the combined, but not separate, expression of the AML1/ ETO ( RUNX1-RUNX1T1 ) fusion protein and a mutated ver- sion of FLT3 ( FLT3 internal tandem duplication). 12 Similar evidence for a multistep pathogenesis exists for malignant lymphomas, both derived from experimental data, as well as clinical observations. For example, in monoclonal gammo- pathy of unknown significance (MGUS), clonal plasma cells carrying the pathognomonic immunoglobulin translocations characteristic for multiple myeloma may be detected in a significant percentage of normal elderly individuals. Trans- formation to overt multiple myeloma or lymphoma occurs at a rate of approximately 1% per year, again demonstrating the necessity to acquire additional genetic alterations for a fully malignant phenotype. In view of these findings, it is clear that full blown hematologic malignancies result from the deregulation of multiple different pathways and that under- standing them is the key to the establishment of treatment strategies. The most frequent recurrent translocations and mutations in acute myeloid leukemia are listed in Tables 1.1 and 1.2. These abnormalities are also discussed in Chaps. 34 and 35, respectively. Proliferation and/or Survival Signals The most frequently observed molecular abnormality in AML, are mutations in nucleophosmin (NPM), which usu- ally involve exon 12 of the NPM1 gene (Table 1.2). NPM is a ubiquitously expressed nucleolar phosphoprotein, which shuttles continuously between the nucleus and the cytoplasm. The prevalence of NPM1 in all de novo AML is roughly 35%. Furthermore, more than half of the AML patients with no cytogenetic abnormality bear this mutation 4 1. Molecular Oncogenesis (normal karyotype). This mutation appears to show a female predominance. 13 In AML, mutations in the NPM1 gene lead to increased nuclear export and aberrant accumulation of the NPM protein in the cytoplasm, which is thought to contribute to tumorigenesis by increasing proliferation and/ or inhibiting the programmed cell death. 14 A number of recent studies have increased our understanding of the role of NPM1 in leukemia, which are becoming very important for developing new therapeutical strategies to target this pathway. AML with mutated NPM1 and a normal karyotype, has in general a favorable prognosis and a good response to induction therapy. Malignant changes in signal transduction pathways confer survival and proliferative properties to leukemic cells. The alteration of these signal transduction pathways is often mediated by genetic changes in key signaling molecules such as the receptor tyrosine kinases (RTKs) or the RAS family of guanine nucleotide-binding proteins. An impressive body of evidence in the last decades has highlighted the role of aberrantly activated RTKs in leu- kemia. While some RTKs are involved in the formation of leukemia-specific fusion genes such as ABL, JAK2, PDG- FRs, SYK, and FGFRs, others such as JAK2, FLT3, and the KIT have been shown to be activated by gain of func- tion mutations in myeloproliferative disease and myeloid leukemia. One of the most common examples of a kinase acti- vated due to chromosomal translocation in leukemia is the BCR–ABL kinase, which is generated by the t(9;22) (q34;q11.2) translocation, which is present in all cases of CML and in a proportion of cases with ALL. The inhibi- tion of this kinase is seen to be crucial to the therapy of t(9;22) positive leukemias. 15 In AML, overexpression or aberrant constitutive activation of class III RTKs like FLT3 or KIT through point mutations, duplications etc., has been reported. 16–19 A class of tyrosine kinases termed Janus kinases (JAKs), which mediate cytokine/growth factor signaling are frequent targets of mutation in myeloproliferative disorders. The JAK2 V617F mutation in the pseudokinase domain of JAK2 is found in >95% polycythemia vera patients, essen- tial thrombocythemia (EM, 50% of patients) and primary myelofibrosis (PMF, 50% of patients). 20 In these disorders, hypersensitivity to growth factor signaling leads to uncon- trolled increase in mature hematopoietic elements with normal or near-to-normal function. At the molecular level, mutations in RTKs could affect dimerization, kinase func- tion, receptor conformation, or phosphorylation, leading to their constitutive activation. 21 The common pathological consequence of this constitutively active kinase signaling is uncontrolled proliferation, which is an important component in the pathogenesis of leukemia. Finally, mutations in p53 gene, which is probably the most frequently mutated gene in cancer, is observed at a much lower frequency in leukemia than in solid tumors; whereas RAS mutations, most of which involve the N-Ras gene, may be found in as much as 30% of the AML cases. 22,23 Normal BM Leukemia FLT3 KIT N-RAS/K-RAS AML1-ETO PML-RAR α CBF β /SMMHC Mutations Affecting Proliferation, Survival etc. Mutations Primarily Affecting Differentiation / Apoptosis Eg. FLT3 Inhibitors, Imatinib Eg. ATRA, HDAC Inhibitors Fig. 1.1. The two-hit model of leukemogenesis. This figure shows collaborating mutations between genetic alterations in factors that affect differentiation and activating mutations in genes causing proliferative/survival advantages. Potential therapeutic interventions are depicted below. Adapted and permission granted from Kuchenbauer et al. 9 5 A.J. Deshpande et al. Block of Differentiation Another important subset of genes that are frequently mutated in acute leukemias of both lymphoid and myeloid origin are transcription factors with essential functions in hematopoi- esis. Mutations in lineage-specific transcription factors are thought to lead to a block in differentiation and, therefore, contributing both to cellular transformation and the charac- teristic immature phenotype of acute leukemia. Deletions of the IKAROS gene occur in over 80% of patients with BCR– ABL positive B-ALL, but not in CML. These deletions result either in loss of expression or the expression of a dominant negative form of IKAROS in the tumor cells suggesting that the loss of function of this transcription factor is an important step in the development of Ph+ B-ALL. Moreover, the loss of IKAROS might explain the difference in maturation between Ph+ B-ALL and CML despite the common presence of the BCR-ABL. Point mutations in the granulocytic differentiation fac- tor CEBP a have been reported in over 10% of all AML patients, 24–26 ,whereas 7% of patients harbor mutations in the transcription factor PU.1. 27 The myeloid transcription factor RUNX1 (also known as AML1), which is recurrently involved in chromosomal translocations, is also mutated in Table 1.1. Examples of chromosomal translocations in patients with AML. Translocation Involved genes Protein function Translocations involving the “core binding factor” (CBF) family t(8;21)(q22;q22) t(3;21)(q26;q22) t(3;21)(q26;q22) t(3;21)(q26;q22) inv(16)(p13;q22) t(12;21)(p13;q22) AML1 Transcription factor and CBF complex subunit ETO Putative transcription factor AML1 Transcription factor and CBF complex subunit EVI1 Transcription factor AML1 Transcription factor and CBF complex subunit EAP Ribosomal Protein AML1 Transcription factor and CBF complex subunit MDS1 Unclear CBF b Heterodimeric Partner of AML1 MYH11 Smooth muscle myosin heavy chain TEL ETS related transcription factor AML1 Transcription factor and CBF complex subunit Translocations involving the retinoic acid receptor a (17q11) t(15;17)(q21;q11) PML1 Zinc finer protein t(11;17)(q23;q11) PLZF Transcriptional repressor t(5;17)(q31;q11) NPM Nuclear phosphoprotein t(11;17)(q13;q11) NUMA Mitotic spindle component Translocations involving the “mixed lineage leukemia” (MLL) gene (11q23) t(11;16)(q23;p13.3) CBP Histone acetylase t(11;22)(q23;q13) P300 Histone acetylase t(9;11)(p22;q23) AF9 Transcription factor? t(11;19)(q23;p13) ENL Transcription factor t(6;11)(q27;q23) AF6 Signal transduction protein? Translocations involving the nucleoporin family t(2;11)(q31;p15) t(7;11)(p15;p15) t(6;9)(q23;q34) Normal Karyotype NUP98 Component of the nuclear pore complex HOXD13