Atlas of Hematologic Neoplasms

Atlas of Hematologic Neoplasms

Tsieh Sun, M.D. Editor

Atlas of Hematologic Neoplasms

123

Editor Tsieh Sun, M.D. Director of Hematopathology and Flow Cytometry Pathology and Laboratory Medicine Service Veterans Affairs Medical Center Eastern Colorado Health Care System Professor of Pathology Department of Pathology University of Colorado School of Medicine Denver, Colorado 80220 USA [email protected]

ISBN 978-0-387-89847-6 DOI 10.1007/978-0-387-89848-3 Springer Dordrecht Heidelberg London New York

e-ISBN 978-0-387-89848-3

Library of Congress Control Number: 2009920689 c Springer Science+Business Media, LLC 2009  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 identif ed 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)

Preface

Due to its rapid development in recent years, hematopathology has become a very complex discipline. The current development is mainly in two aspects: the new classificatio of lymphomas and leukemias, and new techniques. The Revised European – American Classificatio of Lymphoid Neoplasms (REAL classification and the World Health Organization (WHO) classificatio of hematologic neoplasms require not only morphologic criteria but also immunophenotyping and molecular genetics for the diagnosis of hematologic tumors. Immunophenotyping is performed by either fl w cytometry or immunohistochemistry. There are many new monoclonal antibodies and new equipment in recent years that make immunophenotyping more and more accurate and helpful. There are even more new techniques invented in recent years in the fiel of molecular genetics. In cytogenetics, the conventional karyotype has been supplemented and partly replaced by the fluorescenc in situ hybridization (FISH) technique. The current development of gene expression profilin is even more powerful in terms of subtyping the hematologic tumors, which may help to guide the treatment and predict the prognosis. In molecular biology, the tedious Southern blotting technique has been largely replaced by the polymerase chain reaction (PCR). The recent developments in reverse-transcriptase PCR and quantitative PCR make these techniques even more versatile. Because of these new developments, hematopathology has become too complex to be handled by a general pathologist. Many hospitals have to hire a newly trained hematopathologist to oversee peripheral blood, bone marrow, and lymph node examinations. These young hematopathologists are geared to the new techniques, but most of them are still inexperienced in morphology. No matter how well-trained a hematopathologist is, they still need to see enough cases so that they can recognize the morphology and use the new techniques to substantiate the diagnosis. In other words, morphology is still the basis for the diagnosis of lymphomas and leukemias. Therefore, a good color atlas is the most helpful tool for these young hematopathologists and for surgical pathologists who may encounter a few cases of hematologic tumors from time to time. In a busy daily practice, it is difficul to refer to a comprehensive hematologic textbook all the time. There are a few hematologic color atlases on the market to show the morphology of normal blood cells and hematologic tumor cells. These books are helpful but not enough, because tumor cell morphology is variable from case to case and different kinds of tumor cells may look alike and need to be differentiated by other parameters. The best way to learn morphology is through the format of clinical case study. This format is also consistent with the daily practice of hematopathologists and with the pattern in all the specialty board examinations. Therefore, it is a good learning tool for pathology residents and hematology fellows as well as medical students. This book presents 85 clinical cases with clinical history and morphology of the original specimens. This is followed by further studies with pictures to show the test results. The reader is expected to make a preliminary diagnosis on the basis of the material provided before turning to the answer. At the end, a concise discussion and a correct diagnosis are rendered. The list of references is not exhaustive, but it provides the most recent information, current up to 2008. In fact, the entire book is based on the 2008 WHO classification The major emphasis is the provision of more than 500 color photos of peripheral blood smears, bone marrow aspirates, core biopsies, lymph node biopsies, and biopsies of other solid organs that are involved with lymphomas and leukemias. Pictures of other diagnostic parameters, such as fl w cytometric histograms, immunohistochemical stains, cytogenetic karyotypes, fluorescenc in situ hybridization, and polymerase chain reaction, are also included. v

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Preface

A comprehensive approach with consideration of clinical, morphologic, immunophenotypic and molecular genetic aspects is the best way to achieve a correct diagnosis. After reading this book, the reader will learn to make a diagnosis not only based on the morphology alone but also in conjunction with other parameters. Denver, Colorado, USA

Tsieh Sun

Acknowledgments

I wish to thank my pathology colleagues in the Veterans Affairs Medical Center, Drs. Chitra Rajagopalan, Mark Brissette, Deniel Merick, Samia Nawaz, Mona Rizeg Passaro, and Gaza Bardor, for their support and encouragement. I particularly appreciate John Ryder, M.D. of University Hospital of Colorado Denver Health Sciences Center for providing Cases 3 and 51, and Xiayuan Liang, M.D. of the Children’s Hospital of Denver for Cases 67 and 76. I also wish to thank my clinical colleagues in the Oncology-Hematology Service, Drs. Madeleine Kane, Thomas, Braun, Catherine Klein, David Calverley, and Eduardo Pajon, for providing me with clinical cases and intellectual stimulation. Wonderful technical assistance has been provided by technologists in the Flow Cytometry, Hematology and Histology Laboratories. My thanks are also due to the staff of the publisher, Springer, and the compositor, Integra, for their helpful cooperation. I am also thankful for valuable technical assistance in photography from Lisa Litzenbarger. Finally, I am most appreciative to my wife, Sue, for her faithful support, patience, and understanding.

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Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1

Part I

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classificatio of Lymphoma and Leukemia . . . . . . . . . . . . Morphology of Hematopoietic Cells . . . . . . . . . . . . . . . . Comparison Between Flow Cytometry and Immunohistochemistry Monoclonal Antibodies Used for Immunophenotyping . . . . . . Cytogenetic Techniques for Hematologic Neoplasms . . . . . . . Molecular Biology Techniques for Hematologic Neoplasms . . . . Diagnostic Procedures for Hematologic Neoplasms . . . . . . . .

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3 3 8 22 22 24 26 27

Part II Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33

Hematologic Neoplasms . . . . . . . . . . . . . . . . . . . . Case 1 Chronic myelogenous leukemia, accelerated phase . . . Case 2 Chronic myelogenous leukemia, blast crisis . . . . . . Case 3 Chronic neutrophilic leukemia . . . . . . . . . . . . . Case 4 Primary myelofibrosi . . . . . . . . . . . . . . . . . . Case 5 Essential thrombocythemia . . . . . . . . . . . . . . . Case 6 Chronic myelomonocytic leukemia . . . . . . . . . . . Case 7 Atypical chronic myelogenous leukemia . . . . . . . . Case 8 Refractory anemia with ring sideroblasts . . . . . . . . Case 9 Refractory cytopenia with multilineage dysplasia . . . Case 10 5q– syndrome . . . . . . . . . . . . . . . . . . . . . Case 11 Acute myeloid leukemia (AML) with t(8;21)(q22;q22) Case 12 AML with inv(16) . . . . . . . . . . . . . . . . . . . Case 13 Acute promyelocytic leukemia . . . . . . . . . . . . Case 14 AML without maturation . . . . . . . . . . . . . . . Case 15 AML with maturation . . . . . . . . . . . . . . . . . Case 16 Acute myelomonocytic leukemia . . . . . . . . . . . Case 17 Acute monoblastic leukemia . . . . . . . . . . . . . . Case 18 Acute monoblastic leukemia with t(8;16) . . . . . . . Case 19 Acute erythroid leukemia . . . . . . . . . . . . . . . Case 20 Acute megakaryoblastic leukemia . . . . . . . . . . . Case 21 Myeloid sarcoma . . . . . . . . . . . . . . . . . . . . Case 22 Leukemia cutis . . . . . . . . . . . . . . . . . . . . . Case 23 B-lymphoblastic leukemia/lymphoma . . . . . . . . . Case 24 T-lymphoblastic leukemia/lymphoma . . . . . . . . .

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35 35 41 45 50 57 62 68 73 80 85 91 96 101 107 112 116 120 125 129 135 141 146 152 157 ix

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Case 25 Lymphoblastic lymphoma . . . . . . . . . . . . . . . . . . Case 26 Chronic lymphocytic leukemia . . . . . . . . . . . . . . . Case 27 Richter syndrome . . . . . . . . . . . . . . . . . . . . . . Case 28 Small lymphocytic lymphoma . . . . . . . . . . . . . . . . Case 29 Paraimmunoblastic variant of small lymphocytic lymphoma Case 30 Prolymphocytic leukemia . . . . . . . . . . . . . . . . . . Case 31 Lymphoplasmacytic lymphoma . . . . . . . . . . . . . . . Case 32 Lymphoplasmacytic lymphoma transformation . . . . . . . Case 33 Splenic B-Cell marginal zone lymphoma . . . . . . . . . . Case 34 Hairy cell leukemia . . . . . . . . . . . . . . . . . . . . . Case 35 Plasma cell myeloma . . . . . . . . . . . . . . . . . . . . . Case 36 Plasma cell leukemia . . . . . . . . . . . . . . . . . . . . . Case 37 Plasmacytoma . . . . . . . . . . . . . . . . . . . . . . . . Case 38 Extranodal marginal zone lymphoma of the stomach . . . . Case 39 Extranodal marginal zone lymphoma of the lung . . . . . . Case 40 Extranodal marginal zone lymphoma of the salivary gland . Case 41 Nodal marginal zone lymphoma . . . . . . . . . . . . . . . Case 42 Follicular lymphoma, low-grade . . . . . . . . . . . . . . . Case 43 Follicular lymphoma, high-grade . . . . . . . . . . . . . . Case 44 Mantle cell lymphoma, mantle zone variant . . . . . . . . . Case 45 Mantle cell lymphoma, blastoid variant . . . . . . . . . . . Case 46 Mantle cell lymphoma in polyposis . . . . . . . . . . . . . Case 47 Diffuse large B-cell lymphoma, immunoblastic type . . . . Case 48 Diffuse large B-cell lymphoma, anaplastic type . . . . . . . Case 49 T-cell/histiocyte-rich large B-cell lymphoma . . . . . . . . Case 50 Primary mediastinal (thymic) large B-cell lymphoma . . . . Case 51 Intravascular large B-cell lymphoma . . . . . . . . . . . . Case 52 Body cavity lymphoma . . . . . . . . . . . . . . . . . . . Case 53 Burkitt lymphoma, lymph node . . . . . . . . . . . . . . . Case 54 Burkitt lymphoma, intestinal . . . . . . . . . . . . . . . . . Case 55 Burkitt leukemia . . . . . . . . . . . . . . . . . . . . . . . Case 56 T-cell large granular lymphocytic leukemia . . . . . . . . . Case 57 Adult T-cell leukemia/lymphoma . . . . . . . . . . . . . . Case 58 Natural killer cell leukemia/lymphoma . . . . . . . . . . . Case 59 Hepatosplenic T-cell lymphoma . . . . . . . . . . . . . . . Case 60 Subcutaneous panniculitis-like T-cell lymphoma . . . . . . Case 61 Blastic plasmacytoid dendritic cell neoplasm . . . . . . . . Case 62 Mycosis fungoides/S´ezary syndrome . . . . . . . . . . . . Case 63 Primary cutaneous anaplastic large cell lymphoma . . . . . Case 64 Angioimmunoblastic T-cell lymphoma . . . . . . . . . . . Case 65 Lymphoepithelioid T-cell lymphoma . . . . . . . . . . . . Case 66 Anaplastic large cell lymphoma, common variant . . . . . . Case 67 Anaplastic large cell lymphoma, small cell variant . . . . . Case 68 Anaplastic large cell lymphoma, lymphohistiocytic variant . Case 69 Hodgkin lymphoma, nodular lymphocyte predominant . . . Case 70 Hodgkin lymphoma, nodular sclerosis . . . . . . . . . . . . Case 71 Hodgkin lymphoma, mixed cellularity . . . . . . . . . . . Case 72 Hodgkin lymphoma, lymphocyte-rich . . . . . . . . . . . . Case 73 Hodgkin lymphoma, lymphocyte-depleted . . . . . . . . . Case 74 Extranodal Hodgkin lymphoma . . . . . . . . . . . . . . . Case 75 Post-transplant lymphoproliferative disorder . . . . . . . . Case 76 Langerhans cell histiocytosis . . . . . . . . . . . . . . . .

Contents

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161 166 172 178 184 188 194 200 204 211 218 224 228 234 241 246 252 259 267 273 279 286 290 296 301 307 314 319 323 332 339 344 349 354 361 366 370 375 381 385 393 397 402 407 415 423 430 434 439 443 451 458

Contents

Diseases Mimicking Hematologic Neoplasms . . . . . Case 77 Thymoma . . . . . . . . . . . . . . . . . . . . Case 78 Growth factor effect . . . . . . . . . . . . . . . Case 79 Hematogones in postchemotherapy bone marrow Case 80 Castleman disease . . . . . . . . . . . . . . . . Case 81 Rosai – Dorfman disease . . . . . . . . . . . . . Case 82 Kikuchi – Fujimoto disease . . . . . . . . . . . Case 83 Niemann – Pick disease . . . . . . . . . . . . . Case 84 Gaucher disease . . . . . . . . . . . . . . . . . Case 85 Sarcoidosis . . . . . . . . . . . . . . . . . . . .

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465 466 471 475 480 488 493 497 501 507

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

513

Part I

Introduction

Classification of Lymphoma and Leukemia Hematologic neoplasms are tumors of blood cells. All the blood cells are derived from a pluripotent stem cell that can differentiate into various cell lineages, including erythrocyte, megakaryocyte, basophil, eosinophil, neutrophil, monocyte, and lymphocyte (Fig. 1). These cell lineages are grouped into lymphoid cells and nonlymphoid cells or myeloid cells. Therefore, leukemias can be divided into lymphoid leukemia and myeloid leukemia. Leukemic cells originate from the bone marrow and circulate in the peripheral blood, whereas lymphoma is lymphoid tumor confine to the lymphoid organs or extranodal tissues. However, with the advent of new technology, lymphoma cells can be detected in the blood and bone marrow even in a relatively early stage, and thus the demarcation between lymphoma and leukemia is sometimes blurred. Leukemia can be further divided into acute and chronic types. In acute leukemia, the clinical course is rapidly progressive and the leukemic cells are immature blasts. Chronic leukemia, on the other hand, has a slow and indolent clinical course and the tumor cells are mature-looking in lymphoid leukemia and intermediate forms (promyelocytes, myelocytes, and metamyelocytes) in myeloid leukemia. Lymphoma does not have acute or chronic types, but its clinical course is essentially determined by the maturity of the tumor cells. The mature tumor cells behave like those in chronic leukemia, whereas the immature form is similar to acute leukemia. The homogeneity of lymphoma cells in terms of their maturation stage prompted the theory of maturation arrest as the mechanism of tumorigenesis [1].

Fig. 1 Development of hematopoietic cells (hematopoietic tree) T. Sun, Atlas of Hematologic Neoplasms, c Springer Science+Business Media, LLC 2009 DOI 10.1007/978-0-387-89848-3 1, 

3

4

Introduction

Development of B and T Lymphocytes The development of B cells is confine to the bone marrow. There are several schemes to defin the developmental stages of B cells, but the current scheme divides B cells into pro-B, pre-B, immature B, mature B, germinal center B, memory (marginal zone) B, and plasma cell stages [2]. The development of T lymphocytes starts when the T cells migrate from the bone marrow to the thymus. The stage I thymocyte is called prothymocyte, stage II, cortical thymocyte, and stage III, medullary thymocyte [2]. When the mature thymocyte enters the peripheral circulation, it becomes a postthymic or peripheral T cell. The third lineage of lymphocyte is natural killer (NK) cell. NK cells share a common progenitor cell with T cells and they attain maturity in the thymus preceding ␣␤ T-cell differentiation [3]. However, their exact developmental stages are still unclear.

Intranodal B-Cell Differentiation Both T cells and B cells recirculate in the blood and home to various lymphoid organs, including lymph nodes, spleen, and mucosa-associated lymphoid tissue (MALT), due to the attraction of their surface homing receptors to the high endothelial venules at the hilum of the lymph nodes and spleen. In the lymph node, lymphocytes travel from one compartment to another, undergoing further morphologic changes (Fig. 2) [4]. The recirculating B cells firs come to the mantle zone, where small lymphocytes develop into intermediate lymphocytes (mantle cells). The mantle cells then move into the germinal center and evolve through the stages of centroblasts and centrocytes. These cells are collectively called follicular center cells. Some activated B cells transform into memory B cells and migrate to the marginal zone to become marginal zone cells. Under certain conditions, the marginal zone cells move to the parafollicular perisinusoidal area and become parafollicular B cells. These cells have ovoid nuclei and relatively abundant clear cytoplasm resembling monocytes and are thus called monocytoid B cells, which are now called marginal zone B cells. Some B cells transform into effector cells, which are plasma cells. The plasma cell is the terminal stage of the B cell, which moves to the medullary cord and finall migrates

Fig. 2 Intranodal B-cell differentiation (maturation). Recirculating B cells migrate through the high endothelial venule in the hilum of lymph node to mantle zone, germinal center, marginal zone, paracortex, and finall the sinus

Classification of Lymphoma and Leukemia

5

back to the bone marrow. The recirculating B cells also migrate directly without passing through the germinal center and the mantle and marginal zones to the paracortex and become B immunoblasts.

Pre-germinal Center, Germinal Center and Post-germinal Center Lymphomas Lymphoma may develop at each stage of intranodal differentiation [2]. The origin of these lymphomas can be determined by the status of the variable region of heavy chain gene (VH ) mutation. Lymphomas that show no VH gene mutation represent a tumor from the pre-germinal center. Lymphomas that express VH gene mutation and intraclonal diversity are derived from the germinal center; whereas those that have VH gene mutation but not intraclonal diversity originate from post-germinal center B cells. Pre-germinal center lymphoma is represented by mantle cell lymphoma. Germinal center lymphoma includes follicular lymphoma, Burkitt lymphoma, a subset of diffuse large B-cell lymphoma, and Hodgkin lymphoma. Post-germinal center lymphoma includes nodal marginal zone-B-cell lymphoma, extranodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, lymphoplasmacytic lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, and a subset of diffuse large B-cell lymphoma [2].

Classification of Acute Leukemias The French – American – British (FAB) classificatio has been used as the basis for the classificatio of acute leukemia for many years [5]. However, the 2008 World Health Organization (WHO) classificatio has made many changes to the FAB classificatio [6]. The FAB divides acute lymphoblastic leukemia (ALL) into L1, L2, and L3, but the WHO classificatio considers that the division of L1 and L2 does not serve any clinical purpose and merges them into B-cell and T-cell ALLs. L3 is morphologically associated with Burkitt leukemia, but the 2008 WHO classificatio discourages the inclusion of Burkitt leukemia in the category of acute lymphoblastic leukemia. In the new WHO classification all acute lymphoblastic leukemias and precursor B- and T-cell lymphomas are classifie under precursor lymphoid neoplasms (Table 1). In acute myelogenous leukemia (AML), the original FAB categories, M0, M1, M2, M3, M4, M5, M6, and M7, are now classifie in the category of AML not otherwise categorized (Table 2). Also included in the AML classificatio are acute basophilic leukemia, acute panmyelosis with myelofibrosis myeloid sarcoma, myeloid proliferations related to Down syndrome, and blastic plasmacytoid dendritic cell neoplasm. However, the major addition is the acute myeloid leukemia with recurrent cytogenetic abnormalities, which includes nine well-define entities.

Classification of Lymphoma Modern classificatio of non-Hodgkin lymphoma started with Rappaport, whose classificatio was based on the histologic pattern (nodular or diffuse), cytology (lymphocyte or histiocyte), and cell differentiation (well differentiated or poorly differTable 1 WHO classificatio for precursor lymphoid neoplasms B-lymphoblastic leukemia/lymphoma, not otherwise specified B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities B-lymphoblastic leukemia/lymphoma with t(9;22)(q34;q11.2); BCR-ABL1 B-lymphoblastic leukemia/lymphoma with t(v;11q23); MLL rearranged B-lymphoblastic leukemia/lymphoma with t(12;21)(p13;q22); TEL-AML 1 (ETV6-RUNX1) B-lymphoblastic leukemia/lymphoma with hyperdiploidy B-lymphoblastic leukemia/lymphoma with hypodiploidy (hypodiploid ALL) B-lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32); IL3-IGH B-lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1) T-lymphoblastic leukemia/lymphoma

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Introduction

Table 2 WHO classificatio of acute myeloid leukemia Acute myeloid leukemia with recurrent cytogenetic abnormalities AML with t(8;21)(q22;q22), RUNX1-RUNX1T1 AML with inv(16)(p13q22) or t(16;16)(p13.1;q22), (CBF␤/MYH11) Acute promyelocytic leukemia with t(15;17)(q22;q12), (PML/RAR␣) (AML-M3) AML with t(9;11)(p22;q23); MLLT3-MLL AMLwith t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EV11 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 AML with mutated NPM1 AML with mutated CEBPA AML with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukemia not otherwise categorized AML, minimally differentiated (AML-M0) AML without maturation (AML-M1) AML with maturation (AML-M2) Acute myelomonocytic leukemia (AML-M4) Acute monoblastic and monocytic leukemia (AML-M5) Acute erythroid leukemia (AML-M6) Acute megakaryoblastic leukemia (AML-M7) Acute basophilic leukemia Acute panmyelosis with myelofibrosi Myeloid sarcoma Myeloid proliferations related to Down syndrome Blastic plasmacytoid dendritic cell neoplasm Acute leukemia of ambiguous lineage Acute undifferentiated leukemia Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); BCR-ABL1 Mixed phenotpe acute leukemia with t(v;11q23), MLL rearranged Mixed phenotype actue leukemia, B/lymphoid, NOS Mixed phenotype acute leukemia, T/myeloid, NOS Natural killer (NK)-cell lymphoblastic leukemia/lymphoma FAB classificatio in parenthesis, provisional entity in italic type

entiated). In the 1970s, there were many classifications the better known ones included Lukes and Collins, Kiel, Dorfman, British National Lymphoma Investigation, and the U.N. World Health Organization classifications These different schemes inevitably caused some confusion among pathologists; thus the National Cancer Institute in the United States organized a team of experts to evaluate the available classification and establish a “compromise” new scheme. As a result, a working formulation of non-Hodgkin lymphomas for clinical use was proposed [7]. The Working Formulation is relatively simple and yet incorporates all the major components from other schemes. Its major advantage is dividing the lymphomas into three prognostic groups that make the Working Formulation clinically relevant. It was promptly accepted and has been widely used, especially in North America. However, in Europe the Kiel classificatio is more popular than the Working Formulation [8]. The Working Formulation, nevertheless, does not identify individual disease entities and does not include many new entities, especially in the T-cell lymphoma category, that have appeared in recent years. In addition, the new treatments used currently have changed the outlook of many diseases; thus the prognostic grouping may no longer be valid for some of the lymphomas. Therefore, some American hematologists and oncologists believed that the Working Formulation has outlived its usefulness. Because of this situation, a Revised European – American Classificatio of Lymphoid Neoplasms (REAL classification was proposed [9]. This new scheme encompasses many new entities, covers both Hodgkin lymphoma and non-Hodgkin lymphomas, and incorporates immunophenotypes and cytogenetics as an integral part of the diagnosis. The REAL classification however, contains a number of provisional entities that required additional studies for confi mation or elimination in future schemes. The WHO classificatio fulfill this function by verifying these provisional entities and has been accepted universally as the standard classificatio [5]. In 2008, a revised WHO scheme with many new changes was proposed (Table 3).

Classification of Lymphoma and Leukemia Table 3 WHO classificatio of lymphoid neoplasms B-cell neoplasms Precursor B-cell neoplasms B-lymphoblastic leukemia/lymphoma, NOS B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities∗ Mature (peripheral) B-cell neoplasms B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma B-cell prolymphocytic leukemia Splenic B-cell marginal zone lymphoma (± villous lymphocytes) Hairy cell leukemia Splenic B-cell lymphoma/leukemia, unclassifiabl Splenic diffuse red pulp small B-cell lymphoma Hairy cell leukemia variant Lymphoplasmacytic lymphoma Waldenstr¨om macroglobulinemia Heavy chain diseases Alpha heavy chain disease Gamma heavy chain disease Mu heavy chain disease Plasma cell myeloma Solitary plasmacytoma of bone Extraosseous plasmacytoma Extranodal marginal zone lymphoma of MALT type Nodal marginal zone lymphoma Pediatric nodal marginal zone lymphoma Follicular lymphoma Pediatric follicular lymphoma Primary cutaneous follicle center lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma (DLBCL), NOS T-cell/histiocyte-rich large B-cell lymphoma Primary DLBCL of the CNS Primary cutaneous DLBCL, leg type EBV positive DLBCL of the elderly DLBCL associated with chronic inflammatio Lymphomatoid granulomatosis Primary mediastinal (thymic) large B-cell lymphoma Intravascular large B-cell lymphoma ALK-positive large B-cell lymphoma Plasmablastic lymphoma Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease Primary effusion lymphpoma Burkitt lymphoma B-cell lymphoma, unclassifiable with features intermediate between DLBCL and Burkitt lymphoma B-cell lymphoma unclassifiable with feature intermediate between DLBCL and classical Hodgkin lymphoma T- and NK-cell neoplasms Precursor T-cell neoplasms T-lymphoblastic leukemia/lymphoma Mature T-cell and NK-cell neoplasms T-cell prolymphocytic leukemia T-cell large granular lymphocytic leukemia Chronic lymphoproliferative disorder of NK cells Aggressive NK-cell leukemia Systemic EBV positive T-cell lymphoproliferative disease of childhood Hydroa vacciniforme-like lymphoma Adult T-cell leukemia/lymphoma Extranodal NK/T-cell lymphoma, nasal type Enteropathy-associated T-cell lymphoma

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Introduction

Table 3 (continued) Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Mycosis fungoides S´ezary syndrome Primary cutaneous CD30 positive T-cell lymphoproliferative disorders Lymphomatoid papulosis Primary cutaneous anaplastic large cell lymphoma Primary cutaneous gamma-delta T-cell lymphoma Primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma Primary cutaneous CD4-positive small/medium T-cell lymphoma Peripheral T-cell lymphoma, NOS Angioimmunoblastic T-cell lymphoma Anaplastic large cell lymphoma, ALK-positive Anaplastic large cell lymphoma, ALK-negative Hodgkin lymphoma Nodular lymphocyte predominant Hodgkin lymphoma Classic Hodgkin lymphoma Nodular sclerosis classical Hodgkin lymphoma Lymphocyte-rich classical Hodgkin lymphoma Mixed cellularity classical Hodgkin lymphoma Lymphocyte-depleted classical Hodgkin lymphoma MALT, mucosa-associated lymphoid-tissue; provisional entities in italic type ∗ See Table 2.

Morphology of Hematopoietic Cells The most important tool for the diagnosis of hematologic neoplasms is morphologic examination of the peripheral blood smears and bone marrow biopsies and aspirates [10, 11]. Automated instruments are now generally used in clinical laboratories that help to relieve the burden of hematology technologists/technicians from doing the time-consuming differential counts on peripheral blood smears. These instruments serve as a screening tool to distinguish the normal and abnormal smears so that further studies can be performed in patients with abnormalities. However, when qualitative abnormalities are present, manual differential counts are frequently required. There is no automated instrument for differential counts in bone marrow. This task is performed by technical staff in many hematology laboratories. However, in cases of leukemia and lymphoma, the morphology of blood cells frequently deviates from normal, such as the presence of micromyeloblasts, type III myeloblasts, and dysplastic myeloid cells and monocytes. These unusual morphologic variations may cause misidentificatio for other cell types and lead to misdiagnosis. A hematopathologist should be able to do a reliable manual differential count in the peripheral blood and bone marrow so that he or she is able to double-check if the counts done by the technicians are correct.

Myeloid Cells Myeloblast: The myeloblasts are the most immature cells in the myeloid series (Fig. 3). The enumeration of myeloblasts is most important in the differential diagnoses between acute and chronic myeloid leukemias, and myelodysplastic syndrome with excess blasts. In a normal bone marrow, the blast count should be less than 3%. The cut-off point is 5% for pathologic conditions, which can be seen in refractory anemia with excess blasts, chronic myeloid leukemia, chronic myelomonocytic leukemia, and residual or relapsed acute myeloid leukemia. When the blast count is over 20%, it is diagnostic of acute myeloid leukemia. The only exceptions are acute leukemias with special cytogenetic karyotypes, such as t(8:22) and inv(16); in those cases, the number of blasts can be less than 20%. Myeloblasts range from 15 to 20 ␮m in diameter. In acute leukemia, micromyeloblasts may appear and these cells can be as small as myelocytes. However, the major characteristic features of myeloblasts are their immature (finel reticular or dispersed) chromatic pattern, prominent nucleoli, and high nuclear/cytoplasmic ratio (7:1 to 4:1). The cytoplasm of a myeloblast is usually slightly basophilic and contains no granules, which is called type I myeloblast (Fig. 3A & D). Type

Morphology of Hematopoietic Cells

9

Fig. 3 A & D type I myeloblasts, B & E type II myeloblasts, C & F type III myeloblasts

II myeloblast contains less than 20 granules per cells (Fig. 3B & E). Type III myeloblast contains more than 20 granules (Fig. 3C & F) and is frequently present in acute myeloid leukemia with t(8:22) translocation. The presence of Auer rods (bodies) in the cytoplasm of myeloblasts is most helpful for the diagnosis of acute myeloid leukemia. Auer rods can be also seen in promyelocytes in acute promyelocytic leukemia and occasionally in more mature forms of myeloid cells in acute leukemias. Promyelocyte: Promyelocytes are the largest myeloid cells in the bone marrow, measuring 14–24 ␮m in diameter (Fig. 4A & B). They constitute 2–5% of nucleated cells in the bone marrow. Their nuclear chromatin is slightly more mature than that of the myeloblasts and the nucleoli are less prominent. Their major distinction from myeloblasts is the presence of primary (azurophilic) granules in the cytoplasm and the lower nuclear/cytoplasmic ratio (5:1 to 3:1). A paranuclear halo or hof, corresponding to the Golgi apparatus, is frequently present in normal promyelocytes but not in leukemic promyelocytes. The presence of multiple Auer rods in the cytoplasm is characteristic of acute promyelocytic leukemia. Prominent hypergranularity or hypogranularity is also characteristic of leukemic promyelocytes. However, the changes in cytoplasmic granules can also be seen in myelodysplastic syndrome. Myelocyte: The transition from promyelocyte to myelocyte is marked by the appearance of secondary granules in the cytoplasm (Fig. 4C). These secondary granules are also called specifi granules, as the color of the granules distinguishes neutrophils (lilac) from eosinophils (eosinophilic) and basophils (basophilic). However, in pathologic conditions, such as acute leukemia, large numbers of primary granules may persist in myelocytes. Therefore, it is important to look at other parameters to distinguish these two cell types. Myelocytes are smaller than promyelocytes, measuring 10–18 ␮m in diameter. They have more mature chromatin, showing a clumping or condensed pattern, and the nucleolus is no longer present. The nuclear/cytoplasmic ratio ranges from 2:1 to 1:1. A paranuclear hof is present in some myelocytes. The color of the cytoplasm varies from light basophilic to amphophilic. Myelocytes constitute 5–19% of the nucleated cells in normal bone marrow. Metamyelocyte: Metamyelocytes are characterized by the kidney-shaped nucleus (Fig. 4D). The indentation of the nucleus should be less than half the diameter of a hypothetical round nucleus. In comparison with myelocytes, metamyelocytes are smaller (10–15 ␮m), with lower nuclear/cytoplasmic ratio (1.5:1 to 1:1) and more mature chromatin pattern. No nucleolus is visible. The cytoplasm is pale blue to pink, containing fewer primary granules than myelocytes. The color of the secondary granules distinguishes neutrophils from eosinophils and basophils. They constitute 13–22% of nucleated cells in the bone marrow.

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Introduction

Fig. 4 A & B promyelocyte, C myelocyte, D metamyelocyte, E band, F segmented neutrophil

Band: Bands are similar to metamyelocytes in size (10–15 ␮m) but the nucleus is C or S shape, showing a deeper indentation (more than half the distance from the farthest nuclear margin) than that of the metamyelocytes (Fig. 4E). The constricted region contains chromatin that is different from the threadlike f lament seen in the segmented neutrophils. The chromatin is clumped and a nucleolus is not visible. The nuclear/cytoplasmic ratio varies from 1:1.5 to 1:2. The cytoplasmic color and the predominance of secondary granules in bands are also similar to metamyelocytes. Bands constitute 10–15% of the nucleated cells in the bone marrow and 5–10% of the nucleated cells in the peripheral blood. Segmented Neutrophil: Segmented neutrophils are characterized by their lobulated nucleus connected by thin filament without visible chromatin (Fig. 4F). The number of lobes varies from 2 to 5, but most cells have 3–4 lobes. In the blood, when the nucleus of segmented neutrophils contain more than f ve lobes, this condition is described as hypersegmentation (Fig. 5A). It is a manifestation seen in megaloblastic anemia, secondary to vitamin B12 or folate deficien y, chronic myeloproliferative disorder, and myelodysplastic syndromes. In the bone marrow, if there are more than 5% segmented neutrophil showing f ve lobes, it is sufficien to be considered hypersegmentation. When there are many cells with 1–2 lobed nuclei present, it is called hyposegmentation or hypolobation (Fig. 5B & C). In Pelger – Hu¨et anomaly, the nucleus of neutrophils shows a pince-nez or eyeglasses appearance (two round lobes connected by a single thin f lament) (Fig. 5C). A monolobated form can also be seen (Fig. 5B). The same forms of nucleus can be seen in myelodysplastic syndromes; these cells are then termed pseudo-Pelger – Hu¨et cells. Dysplastic myeloid cells can also show hypergranularity (Fig. 5A) or hypogranularity (Fig. 5F). In infections, the cytoplasmic granules become larger and darkly stained; these granules are commonly called toxic granules (Fig. 5D). Under the same condition, a pale blue inclusion of variable size and shape is also frequently present in the cytoplasm of segmented neutrophils, and is called a D¨ohle body (Fig. 5E). Toxic changes in neutrophils consist of toxic granulation, toxic vacuolation and D¨ohle bodies. Segmented neutrophils range from 10 to 15 ␮m in diameter. The nuclear/cytoplasmic ratio is 1:3. The chromatin is clumped and nucleolus is not visible. They constitute 3–11% of nucleated cells in the bone marrow and 50–70% of nucleated cells in the peripheral blood. Eosinophil: Eosinophils are similar to neutrophils in size (slightly larger), nuclear morphology, chromatin pattern, and nuclear/cytoplasmic ratio (Fig. 6A & D). The major difference between them is the presence of coarse, uniform, and orangered (eosinophilic) granules in the cytoplasm of eosinophils. These granules are also refractile due to their crystalline structure.

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11

Fig. 5 A hypersegmented neutrophil, B monolobated neutrophil, C pseudo-Pelger – Hu¨et cell, D toxic granulation, E D¨ohle body (arrow), F hypogranular neutrophil

Most eosinophils are bilobed and a minority of them may show 3–5 lobes. The immature eosinophils include eosinophilic myelocytes and metamyelocytes, which are similar to the corresponding stages of neutrophils except for the cytoplasmic granules. In the immature forms, a few purplish or basophilic granules can be visible in addition to the eosinophilic granules. They constitute 0–3% of nucleated cells in bone marrow and 0–5% in the peripheral blood. Basophil and Mast Cell: Basophils are similar to neutrophils and eosinophils in size, nuclear morphology, chromatin pattern, and nuclear/cytoplasmic ratio (Fig. 6B & E). The characteristic of basophils is the presence of basophilic (deep purple to black) granules, which are irregular in shape and larger than neutrophilic granules. These granules may be abundant and obscure the nucleus. They are also called metachromatic granules as they can be demonstrated by metachromatic stains, such as toluidine blue or Giemsa. The nuclei of basophils usually have 2–3 lobes. The mast cell is the tissue counterpart of basophil. It has only a single unsegmented nucleus but the granules are the same as the basophil, except that they are more abundant and more evenly distributed than those in the basophils (Fig. 6C & F). There are 0–1% basophils in the peripheral blood and less than 1% in the bone marrow. Mast cells are not seen in the peripheral blood, and they constitute less than 1% of nucleated cells in the bone marrow.

Monocytic Cells Monocyte: Monocytes are the largest leukocyte in the peripheral blood, measuring 12–20 ␮m in diameter. The nucleus is usually kidney-shaped with convolutions or folding, but many cells show an irregular configuratio (Fig. 7A & D). Monocytes have abundant cytoplasm with light blue color and vacuoles or ingested particles are frequently present. The active monocytes have irregular cell borders or pseudopod-like cytoplasmic projections, but inactive monocytes are round with smooth edges. Cytoplasmic granules are not commonly seen but small numbers of fine azurophilic granules may be present. Promonocyte: There is a spectrum of monocytes with morphology between a monoblast and a mature monocyte. These cells are designated promonocytes. Promonocytes are smaller than monoblasts with more mature chromatin pattern and less prominent nucleoli (Fig. 7B & E). The nuclear convolution in promonocytes is less prominent than that seen in mature monocytes but more striking than that in monoblasts. The demarcation between monoblasts and promonocytes is not clear-cut in

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Introduction

Fig. 6 A & D eosinophils, B & E basophils, C & F mast cells

Fig. 7 A & D monocytes, B & E promonocytes, C & F monoblasts. Note picture in C may represent transitional forms of promonocytes to monoblasts. Cells in C & F are at lower magnificatio than A, B, D, E, therefore they are disproportionately smaller

Morphology of Hematopoietic Cells

13

Fig. 8 Histiocyte/macrophage. A. A histiocyte with multiple cytoplasmic processes. B. A sea-blue histiocyte from a case of chronic myeloid leukemia. C. A macrophage with cytoplasmic vacuolation and erythrophagocytosis. D. A Gaucher cell

many cases (Fig. 7C). However, promonocytes are included in the blast count to defin the diagnosis of acute monoblastic/monocytic or myelomonocytic leukemia, and thus the distinction between these two developmental stages is not strictly required. Monoblasts: Monoblasts are not seen in normal bone marrow and are difficul to distinguish from myeloblasts. They are usually larger (15–25 ␮m) with lower nuclear/cytoplasmic ratio than myeloblasts (Fig. 7C & F). In acute leukemia, the monoblasts can be as large as 40–50 ␮m in diameter. Although the nucleus is round, it is usually slightly irregular and convoluted. Nucleoli are present and cytoplasmic granules are infrequently seen. The identificatio of monoblasts often requires the help of special cytochemical (non-specifi esterase) or immunohistochemical (CD68) stains. Macrophage and Histiocyte: Monocytes can transform into macrophages or histiocytes in tissue and both can be seen in the bone marrow. A macrophage is a large cell, measuring 15–80 ␮m in diameter (Fig. 8A & C). It has one or more round nuclei with 1–2 small nucleoli. The chromatin pattern is spongy or reticular. This cell is easily recognizable in the bone marrow because of its large size, low nuclear/cytoplasmic ratio, abundant cytoplasm, and irregular or poorly define cell border. Its phagocytic activity is usually demonstrated by the presence of ingested small particles, hemosiderin, blood cells, and vacuoles in the cytoplasm (Fig. 8C). Histiocytes are considered inactive macrophages. In tissue sections, they are elongated, generally smaller than the macrophages, with a single eccentric nucleus and inconspicuous nucleoli. They usually show very few inclusions in the cytoplasm. However, there are several storage histiocyte disorders that show various substances in the histiocytes. The seablue histiocyte contains blue granules, which are an insoluble lipid pigment called ceroid, in the cytoplasm (Fig. 8B). The Gaucher cell contains glycocerebroside and the cytoplasm shows the wrinkle tissue pattern appearance (Fig. 8D). The Niemann – Pick cell is a foamy cell with a mulberry-like appearance due to the accumulation of sphingomyelin in the cytoplasm.

Lymphoid Cells Lymphoblast: Lymphoblasts are not present in normal bone marrow. They are similar to myeloblasts and monoblasts in morphology, but are generally smaller (10–20 ␮m) with a higher nuclear/cytoplasmic ratio (7:1 to 4:1) and less prominent

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Introduction

Fig. 9 A L1 lymphoblast, D L2 lymphoblasts, B & E prolymphocytes, C small mature lymphocyte, F large granular lymphocyte

nucleoli. The cytoplasm varies from light blue to deep blue and contains no cytoplasmic granules. However, lymphoblasts show a great variation in morphology and can be divided into L1, L2, and L3 forms according to the FAB classification The L1 lymphoblasts are uniformly small with scanty cytoplasm (Fig. 9A). Their nuclei are regular in shape, with inconspicuous nucleoli. This form is usually seen in pediatric cases. The L2 lymphoblasts are generally large, but their size is variable, as is the cytoplasm (Fig. 9D). Their nuclei also vary in shape, with prominent nucleoli. This form is more frequently seen in adults than in children. The L3 lymphoblasts are uniformly large, with moderate amounts of deep basophilic cytoplasm, which contains many vacuoles. The nuclei are round and regular with immature chromatin and prominent nucleoli. This form is rare in comparison with L1 and L2, and is more frequently seen in adults. These cases usually represent Burkitt leukemia. In the WHO scheme, L1 and L2 are combined (L1/L2), as these two types are frequently indistinguishable and are similar clinically. Prolymphocyte: Prolymphocytes are slightly smaller than lymphoblasts (10–18 ␮m in diameter) with lower nuclear/cytoplasmic ratio (5:1 to 3:1) (Fig. 9B & E). The nucleus has a chromatin density between that of a small lymphocyte and that of a lymphoblast. A single prominent nucleolus is the hallmark of a prolymphocyte, but it may occasionally show more than one nucleolus. The cytoplasm is moderately abundant and is usually pale blue in color. No cytoplasmic granules are present in prolymphocytes. Lymphocyte: Lymphocytes are the smallest leukocytes in the peripheral blood and the bone marrow, measuring 7–15 ␮m in diameter (Fig. 9C). The nuclear chromatin is dense and clumped without the presence of a nucleolus. However, some lymphocytes may show a chromocenter that may mimic a nucleolus. Most lymphocytes have scanty cytoplasm so that the nuclear/cytoplasmic ratio is high (5:1 to 2:1). Usually, no cytoplasmic granules are present. When a larger lymphocyte shows moderate transparent cytoplasm with delicate or coarse azurophilic granules, it is called a large granular lymphocyte (Fig. 9F). The large granular lymphocytes represent natural killer (NK) cells or NK-like cytotoxic T cells. Some lymphocytes may show a clear zone surrounding the nucleus or a paranuclear hof; these are considered normal features of lymphocytes. Under pathologic conditions, most frequently viral infections, lymphocytes may show some morphologic changes. These lymphocytes are called reactive or activated lymphocytes. The basic changes are that the nuclear chromatin may become more immature, the volume of the cytoplasm is increased and frequently becomes more basophilic. Inconspicuous nucleoli are often present. When a lymphocyte shows an eccentric nucleus, a prominent paranuclear hof and basophilic cytoplasm, it is called a plasmacytoid lymphocyte.

Morphology of Hematopoietic Cells

15

Fig. 10 A Downey type II cell, D Downey type III cell, B plasma cells in bone marrow aspirate, C plasma cells in bone marrow biopsy: the cartwheel/clock-face chromatin pattern is readily appreciable. E immature plasma cells with a prominent nucleolus. F plasmablast with high nuclear to cytoplasmic ratio, and without a hof

Other types of reactive lymphocytes are classifie by Downey into three types. Downey type I cells are small lymphocytes that contain an indented or kidney-shaped nucleus, which is sometimes lobated (monocytoid nucleus). The cytoplasm is basophilic with azurophilic granules and frequently vacuoles. Downey type II cells are larger cells with abundant agranular, pale cytoplasm that are frequently indented by the surrounding red blood cells, producing the so-called ballerina skirt appearance (Fig. 10A). The edges of the cytoplasm usually stain darker (peripheral basophilia) and there is sometimes lineal blue staining radiating from the center to the periphery of the cytoplasm (radial bluing). Downey type III cells are the same as immunoblasts or the so-called nonleukemic lymphoblasts that show blastoid chromatin with one or more nucleoli (Fig. 10D). The cytoplasm is moderate to abundant with deeply basophilic color. Plasma cell: Plasma cells are the terminal stage of lymphocytes and are slightly larger than the latter (10–20 ␮m) (Fig 10B & E). Plasma cells are oval with eccentric nucleus. The clumped chromatin characteristically shows a cartwheellike or clock-face pattern (Fig. 10E). Mature plasma cells have no nucleoli. The cytoplasm is typically deeply basophilic with a prominent paranuclear hof. However, in a few myeloma cases, particularly IgA myeloma, the cytoplasm can be pink-red, and these cells are referred to as flam cells. In actively secreting plasma cells, the cytoplasm contains immunoglobulin inclusions (Russell bodies), and these cells are called Mott cells. When the inclusion is present in the nucleus, it is termed a Dutcher body. Dutcher bodies are only seen in pathologic conditions, such as myeloma and macroglobulinemia. Plasmablast: Plasmablasts are usually seen in the terminal stage of myeloma or plasma cell leukemia. These cells are larger than plasma cells with immature chromatin pattern and high nuclear/cytoplasmic ratio (Fig. 10F). The nucleus is no longer present eccentrically, and the paranuclear hof is no longer present, as most plasmablasts do not produce immunoglobulin. As the cytoplasm of plasmablasts is not necessarily basophilic, these cells are hardly recognized as plasma cells in origin. In immature plasma cells between plasmablasts and plasma cells, however, the characteristic nuclear eccentricity and paranuclear hof are still visible (Fig. 10C).

Erythroid Cells Pronormoblast (Proerythroblast): This is the earliest stage of erythroid cells and is the largest, measuring 17–24 ␮m in diameter (Fig. 11A). In Giemsa stain preparations, nuclei of all nucleated red cells are characterized by their dark staining

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Introduction

Fig. 11 A pronormoblast, B basophilic normoblast, C polychromatophilic normoblast, D orthochromatic normoblast

with very well define and perfectly round configuration The chromatin pattern is characterized by the so-called sievelike pattern with multiple chromatin gaps (fenestration or parachromatin) in the nucleus. The differences between various stages are the gradual maturation of the chromatin pattern and increasing hemoglobinization of the cytoplasm. Therefore, the pronormoblasts show the most immature (lacy) chromatin pattern with visible nucleoli and dark-blue cytoplasm (no hemoglobinization). The nuclear/cytoplasmic ratio is about 8:1. A paranuclear hof (Golgi zone) is frequently present. Pronormoblasts comprise 1–3% of the nucleated erythroid cells in the bone marrow. Basophilic Normoblast (Erythroblast): Basophilic normoblasts measure 10–17 ␮m in diameter with a nuclear/cytoplasmic ratio of 6:1 (Fig. 11B). The chromatin pattern is open with slight clumping and the nucleoli can be visible in the early stage but nonvisible in the mature stage. The cytoplasm is dark blue with the presence of a paranuclear hof in some cells. Basophilic normoblasts constitute 6–8% of the nucleated erythroid cells in the bone marrow. Polychromatophilic Normoblast (Erythroblast): Polychromatophilic normoblasts measure 10–15 ␮m in diameter with a nuclear/cytoplasmic ratio of 1:4 (Fig. 11C). The chromatin is clumped with no visible nucleolus. The cytoplasm starts to show hemoglobinization and is blue-gray to pink-gray in color, depending on its maturation. Paranuclear hof is not as prominent as in the earlier stages of normoblasts. Polychromatophilic normoblasts comprise 10–20% of nucleated erythroid cells in the bone marrow. Orthochromic Normoblast (Erythroblast): Orthochromic normoblasts measure 8–12 ␮m in diameter with a nuclear/cytoplasmic ratio of 1:2 (Fig. 11D). The nucleus is pyknotic without visible nucleolus. The cytoplasm has nearly full hemoglobinization, showing pink or salmon color. No paranuclear hof is seen. They comprise 40–60% of nucleated erythroid cells in the bone marrow. Abnormal Red Cell Morphology: In a normal red cell population, all cells are uniform in size and shape. When there is an obvious variation in the red cell size, it is called anisocytosis (Fig. 12A), which is usually indicated by a high red cell distribution width. Some cases may show a dimorphic population (Fig. 12B), which is frequently seen after blood transfusion, but can also be seen in patients with vitamin B12 , folate or iron deficiencies When the shape of the red cells is variable, it is termed poikilocytosis (Fig. 12C). The changes in the configuratio of the erythrocytes are collectively called speculated red cells. This group includes schistocyte (Fig. 12D), helmet cell (Fig. 12E), horn cell (keratocyte) (Fig. 12F), teardrop cell (dacrocyte) (Fig. 13A), sickle cell (drepanocyte) (Fig. 13B), acanthocyte (spur cell) (Fig. 13C), and echinocyte (burr cell) (Fig. 13D). Schistocytes, helmet cells, and horn cells are different morphologic manifestations of fragmented red cells and

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17

Fig. 12 A anisocytosis, B dimorphic population, C poikilocytosis, D schistocytes (arrows), E helmet cell (arrow), F horn cell (keratocyte) (arrow)

their presence usually indicates the existence of hemolytic anemia, particularly microangiopathic hemolytic anemia, such as in disseminated intravascular coagulation or thrombotic thrombocytopenic purpura. Teardrop cells are seen mainly in myelofibrosis Sickle cells are present in sickle cell diseases. Acanthocytes show 2–20 unevenly distributed spicules and the absence of central pallor and are usually seen in abetalipoproteinemia, severe liver disease, splenectomy and malabsorption. Echinocytes are characterized by 1–30 evenly spaced spicules with normal centrol pallor and are present in uremia, pyruvate kinase deficien y, and microangiopathic hemolytic anemia. Other abnormal morphology of red cells includes polychromatophilic red cell (Fig. 13E), spherocyte (Fig. 13F), ovalocyte (elliptocyte) (Fig. 14A), stomatocyte (Fig. 14B), and target cell (Fig. 14C). Polychromatophilic red cells (polychromatia) are in the maturation stage immediately after reticulocytes. Therefore, their presence signifie red cell regeneration, either due to blood loss or hemolysis. The presence of spherocytes, ovalocytes and stomatocytes may indicate a hereditary disorder. However, if these cells are not predominant, it usually represents an acquired disease or a nonspecifi findin in anemias. Spherocytosis are seen in hemolytic anemia, post-splenectomy, and after transfusion. Ovalocytosis is present in thalassemia major, iron deficien y anemia, and megaloblastic anemia. Stomatocytosis is detected in alcoholism or liver disease. Target cells can be seen in many conditions, but thalassemia, hemoglobinopathy, and iron deficien y anemia should be excluded when target cells are detected. Several inclusions can be demonstrated in erythrocytes by Wright – Giemsa stain, which include basophilic stippling (Fig. 14D), Pappenheimer body (Fig. 14E) and Howell – Jolly body (Fig. 14F). Basophilic stippling is the presence of multiple fin blue granules over the entire red cell and it is seen in lead poisoning, thalassemias, myelodysplasias, and sideroblastic anemias. Pappenheimer body is represented by a small cluster of small blue granules on the red cells and it can be seen in many types of anemias. Howell – Jolly body is usually a single round blue granule about 1 ␮m in diameter and is mainly present in patients after splenectomy or with hyposplenism. Cabot ring is a rare finding but it can be seen in myelodyplastic syndrome and megaloblastic anemia. Under pathological conditions, such as vitamin B12 or folate deficiencie and megaloblastic anemia, the nuclear maturation lags behind the cytoplasmic maturation (nuclear-cytoplasmic dyssynchrony), the cells become larger with varying degrees of hemoglobinization and yet the nucleus remains immature-looking. This phenomenon is called megaloblastic change. If the cells and nuclei are larger than their normoblastic counterparts but there is only minimal nuclear-cytoplasmic dyssynchrony, these cells are considered megaloblastoid (Fig. 15).

18

Introduction

Fig. 13 A teardrop cell (dacrocyte) (arrow), B sickle cell (drepanocyte) (arrow), C spur cell (acanthocyte) (arrow), D burr cell (echinocyte) (arrow), E polychromatophilic erythrocyte (arrow), F spherocyte (arrow)

Fig. 14 A elliptocyte (ovalocyte) (arrow), B stomatocyte (arrow), C target cell (arrow), D basophilic stippling (arrow), E Pappenheimer body, F Howell – Jolly body (arrow)

Morphology of Hematopoietic Cells

19

Fig. 15 A bone marrow smear shows several megaloblastoid normoblasts at various stages

Megakaryocyte: Megakaryocytes are the largest cells in the bone marrow. This cell lineage is unique in that when the cell becomes mature, it grows larger than its precursor; whereas in other cell lineages, the immature cells are usually larger than the mature cells. This phenomenon is due to the fact that the cell proliferation is through a sequence of nuclear duplication without cell division, which is called endomitosis or endoreduplication. As a result, the megakaryocytes may have 4, 8, 16, 32, or 64 sets of chromosomes. At certain point, these cells stop doubling the DNA content and start the maturation process. In the mature cells, the nucleus becomes lobated. The number of nuclear lobes and the volume of cytoplasm are proportional to the DNA content, leading to the pleomorphic morphology of megakaryocytes. Megakaryocytes can be divided into three stages. Stage I cell is called megakaryoblast, which is mononucleated with immature chromatin and visible nucleoli (Fig. 16A). There is a small amount of basophilic cytoplasm. The size is at least 15 ␮m but in acute megakaryoblastic leukemia the cell size shows great variation, which is characteristic of leukemia. Stage II cell is called promegakaryocyte or basophilic megakaryocyte (Fig. 16B). This cell is at least 20 ␮m in size and has a lobated or horseshoe-shaped nucleus. The chromatin pattern is more mature with chromatin clumping and no nucleolus is visible. The basophilic cytoplasm is moderate in amount and may show blebs on the surface. Endomitosis takes place at this stage. Stage III cell is termed mature or granular megakaryocyte (Fig. 16C). This cell is at least 25–50 ␮m in size with multilobated nucleus and coarse, clumped chromatin pattern initially, processing to pyknotic in full maturation. The cytoplasm is granular and pink, containing azurophilic granules. This stage produces platelets by cytoplasmic compartmentalization. In a normal bone marrow, only mature megakaryocytes are seen. Megakaryocytes are not included in the differential counts of the bone marrow; they are estimated as adequate, increased, or decreased in the report. Platelet: The normal range of platelet count is between 150,000 and 400, 000/␮l. Above this threshold is called thrombocytosis (Fig. 17), and below it, thrombocytopenia (Fig. 18). Thrombocytosis is usually reactive in nature. However, if the platelet count is above 450, 000/␮l, essential thrombocythemia or other myeloproliferative neoplasms should be considered. Thrombocytopenia is usually seen in myelodysplastic syndromes, or other causes of bone marrow failure, such as post-chemotherapy, or bone marrow infiltratio by lymphoma, leukemia, or metastatic carcinoma. Pseudothrombopenia is seen in patients with EDTA antibody, so that platelet clumping (Fig. 19) occurs, leading to falsely low platelet count by the instrument. Normal platelets are even in size and shape. In pathologic conditions, the size of platelets can be markedly variable (anisocytosis). When the size of a platelet is larger than an erythrocyte, it is designated a giant platelet (Fig. 17). Hypergranular or hypogranular platelets can be seen in various platelet disorders.

20

Fig. 16 A megakaryoblast, B promegakaryocyte, C mature megakaryocyte

Fig. 17 A peripheral blood smear shows thrombocytosis. A giant platelet is at the right margin of the picture

Introduction

Morphology of Hematopoietic Cells

Fig. 18 A peripheral blood smear shows features of thrombocytopenia, leukopenia and hypochromacia of erythrocytes

Fig. 19 A peripheral blood smear shows platelet clumping due to the presence of EDTA antibody in the patient

21

22

Introduction

Comparison Between Flow Cytometry and Immunohistochemistry For subclassificatio of lymphomas and leukemias, morphologic examination alone is frequently insufficien and immunophenotyping is often required [12]. Immunohistochemistry (IH) is the most popular technique for this purpose, because it provides direct morphologic correlation with the markers so that a pathologist feels more confiden to make the diagnosis. In addition, immunohistochemical staining can be performed manually, and expensive equipment is usually not required. The ability to perform IH retrospectively on archived material is also a great advantage. At this stage, the application of IH is limited by the availability of monoclonal antibodies that can be used for histologic staining. After fixatio and embedding, many antigenic epitopes are altered so that they can no longer react to most antibodies that are available for fl w cytometry. The major drawback of IH is its inability to demonstrate surface immunoglobulins in small lymphoid cells and thus to demonstrate the clonality of the B-cell population in most lymphomas except for those tumor cells with abundant cytoplasm, such as in plasma cell and immunoblastic neoplasms. IH is also unable to distinguish surface from cytoplasmic antigens. For instance, cytoplasmic CD3 is present in early T-cell stage (thymocytes) and surface CD3 is detected in mature T-cell stage (peripheral T cells). When a tumor stains positive for CD3 by IH, the developmental stage of the tumor cells cannot be pinpointed. The same is true for the distinction between NK cells (cytoplasmic CD3-positive) and NK-like T cells (surface CD3-positive). In addition, multiple staining cannot be performed on the same cells by IH and accurate quantitation of antigens for therapeutic monitoring is not possible. Even with the imaging technique, only semiquantitative results can be obtained. Finally, IH is usually ordered after examination of the hematoxylin and eosin stained sections, so that a conclusion cannot be made until the third day after the receipt of the specimen. Because of these limitations of IH, f ow cytometry (FC) is frequently required. With FC, multiple specimens can be promptly processed with a panel of 10 or more monoclonal antibodies. In a good FC laboratory, the turn-around time is within 3 hours. When there is an adequate specimen, fl w cytometers count 3,000–5,000 cells for the study of each antigen. The examination of large numbers of cells enhances the sensitivity and accuracy of FC and makes it possible to detect small numbers of neoplastic cells. FC is able to stain multiple antigens on the same tumor cells and can distinguish surface from cytoplasmic staining. The availability of six-color FC further enhances its function for characterization of tumor cells. The percentages of various cell groups obtained by FC are highly reproducible and are thus comparable between different laboratories. The major drawback of FC is the lack of morphologic correlation with the markers. In other word, the markers that are present may represent normal cells or tumor cells. Therefore, the recognition of the tumor cell population depends on the gating technique in FC to separate the tumor cells from the normal cells. The tumor cells can then be further identifie by a group of monoclonal antibodies.

Monoclonal Antibodies Used for Immunophenotyping Since the early 1980s, thousands of monoclonal antibodies specifi for leukocyte differentiation antigens have been developed. These antibodies are categorized into different functional groups, and those that react to the same epitope are assigned the same cluster designation (CD). At the 8th International Workshop on Leukocyte Differenitation Antigens held in Adelaide, Australia in December 2004, the last cluster designation was CD339 [13]. These antibodies have been used mainly on fresh and appropriately frozen cells and can be used for FC studies (Table 4). However, there are an increasing number of newly developed antibodies that are reactive with antigens in fi ed paraffin-embedde tissue that can be used for IH studies (Table 5). Monoclonal antibodies can be divided into six categories depending on the antigens they react with [12]. 1. Lineage-associated antigens: There are many lineage-associated antigens. The most common B-cell-associated antigens include CD10, CD19, CD20, CD22, CD23, CD24, CD38, CD79, CD138, and PCA-1. The common T-cell-associated antigens encompass CD1, CD2, CD3, CD4, CD5, CD7, CD8, T-cell receptor ␣␤, and T-cell receptor ␥␦. The NK-cellassociated antigens are CD16, CD56, and CD57. The myelomonocytic antigens are CD11b, CD11c, CD13, CD14, CD15, CD33, CD64, CD68, and CD117. 2. Immature cell antigens: This category includes CD10, CD34, CD117, and terminal deoxynucleotidyl transferrase (TdT).

Monoclonal Antibodies Used for Immunophenotyping

23

3. Activation antigens: This category is composed of CD25, CD26, CD30, CD38, CD54, CD71, and HLA-DR. 4. Histocompatibility antigens: Histocompatibility antigens are important in directing cell-to-cell interaction. For instance, the CD4 cells react with cells carrying HLA-II antigen, whereas the CD8 cells react with those bearing HLA-1 antigen. The HLA-II antigens include HLA-DP, HLA-DR, and HLA-DQ. 5. Adhesion molecules: This category includes CD11a/CD18 (lymphocyte function antigen type 1), CD44, CD56 (neural cell adhesion molecule), CD54 (intercellular adhesion molecule type 1), CD102 (intercellular adhesion molecule type 2), CD106 (VCAM-1), and CD31 (platelet-endothelial cell adhesion molecule type 1). 6. Proliferation-associated antigens: The commonly known proliferation-associated antigens include Ki-67 and PCNA. The former is frequently used in immunohistochemical staining to evaluate the proliferative activities of tumor cells.

Table 4 Cell specificit and clinical application of common monoclonal antibodies Cluster designation Monoclonal antibodies Cell specificit CD 1a

Leu6, OKT6, T6

Thymocyte, Langerhans cells

CD2 CD3 CD4 CD5 CD7 CD8 CD10 CD11b

Leu5, OKT11, T11 Leu4, OKT3, T3 Leu3, OKT4, T4 Leu1, OKT1, T1 Leu9, OKT16, 3A1 OKT8, T8 CALLA, OKBcALLa, J5 Leu15, OKM1, Mo1

CD11c CD13 CD14 CD15

LeuM5, ␣S-HCL3, LeuM7, OKM13, My7 LeuM3, OKM14, MY4, Mo2 LeuM1, My1

CD16 CD19 CD20 CD21 CD22 CD23 CD25 CD30

Leu11 Leu12, OKpanB, B4 Leu16, B1 CR2, OKB7, B2 Leu14, OKB22, B3, ␣S-HCL1 B6, Leu20 IL-2, OKT26a, Tac Ki-1, BerH2

CD33 CD34 CD38 CD41 CD42a,b CD43 CD45 CD45RA CD45RO CD56 CD57 CD61 CD64 CD68 CD71 CD74 CDw75

LeuM9, My9 HPCA-1, My10 Leu17, OKT10, T10 J15 HPL14, AN51, 10P42 MT-1, Leu22, L60 HLE-a, LCA MT-2 UCHL1 Leu19, NKH-1 Leu7, HNK-1 10P61, VI-PL2 Fc␥P1, gp75 KP1 Tr receptor, OKT9, T9 LN2 LN1

E-rosette receptor T-cell receptor complex Helper/inducer T cell T cell, B cell subset T-cell receptor for IgM-Fc Cytotoxic/suppressor T cell Immature B cell and T cell Monocyte, granulocyte, NK cell, T-suppressor cell Monocyte, B cell from HCL Monocyte, granulocyte Monocyte, granulocyte Monocyte, granulocyte, Reed – Sternberg cell NK cell, granulocyte, macrophage B cell B cell Follicular dendritic cell, B cell, C3d B cell B cell IL-2 receptor on T cell (Tac antigen) Reed – Sternberg cell, activated T or B cell Monocyte, granulocyte Hematopoietic progenitor cell Plasma cell, activated T or B cell Platelet GPIIb/IIIa Platelet GPIX and GPIb T cell, B cell subset All leukocytes T cell, B cell subset T cell, B cell, monocyte, granulocyte NK cell NK cell, T cell subset Platelet GPIIIa Monocyte Monocyte, histiocyte Activated T/B cell, macrophage B cell, monocyte B cell, T cell subset

Clinical application T-ALL, T lymphoma, histiocytosis T-ALL, T lymphoma T-ALL, T lymphoma Identificatio of T subset T-ALL, T/B lymphoma, CLL T-ALL, T lymphoma Identificatio of T subset ALL, B lymphoma AML AML, HCL AML AML Hodgkin lymphoma NK-cell disorder B-ALL, B lymphoma, CLL B-ALL, B lymphoma, CLL B lymphoma B lymphoma, HCL B lymphoma, CLL HCL, adult T-cell leukemia Hodgkin lymphoma, anaplastic large cell lymphoma AML Acute leukemia Myeloma Megakaryoblastic leukemia Megakaryoblastic leukemia T- or B-cell lymphomas Lymphomas, leukemias Follicular lymphoma T lymphoma NK-cell disorder NK-cell disorder Megakaryoblastic leukemia Monocytic disorder Monocytic/histiocytic tumors Acute leukemias, lymphomas B lymphoma B lymphoma

24 Table 4 (continued) CD79a CD79b CD103 CD117 CD123

Introduction

HM47, HM56, JAB117 SN8, B29/123, CH3-1 HML-1, B-ly7 C-kit, stem cell factor receptor IL-3 receptor

B cell B cell B cell Hematopoietic stem cell Mast cell/basophil, DC2 cells, B cell subset

B cell, plasma cell B cell B cell, activated T cell, myeloblast, monoblast PCA-1 Plasma cell, monocyte, granulocyte Glycophorin A Erythroid series TCR-1, ␤F-1, WT31 T cell TCR-␦1, TCS1, anti␦ T cell ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; interleukin 2; NK, natural killer; PLL, prolymphocytic leukemia; Tr, transferrin CD138

B-B4, 1D4, F59-2E9, M115 FMC-7 HLA-DR

B lymphoma B lymphoma HCL AML Mast cell disease, hairy cell leukemia, Plasmacytoid dendritic cell tumor B lymphoma, myeloma PLL, HCL, B lymphoma B-cell neoplasms Myeloma Erythroleukemia T lymphoma/leukemia T lymphoma/leukemia HCL, hairy cell leukemia; IL-2,

Since FC does not allow the users to have a direct vision of the cell examined, a set of criteria is established to distinguish hematologic neoplasms from normal leukocytes. 1. Immunoglobulin light chain restriction: The surface immunoglobulin light chain ratio is the most commonly used diagnostic criterion, because it define the B-cell lineage and clonality at the same time. When one light chain is dominant over the other, it is referred to as light chain restriction and is indicative of monoclonality. Monoclonality is frequently associated with lymphoid tumors. 2. Loss of surface immunoglobulin in a B-cell population: Normal B cells express both B-cell antigens and surface immunoglobulin. In certain lymphoid tumors, such as primary mediastinal B-cell lymphoma and precursor B-cell neoplasms, surface immunoglobulin is not detected. 3. Coexistence of two different cell lineage markers on the same cell population: Dual-cell lineage markers have become the hallmark of several lymphoid tumors. The most common example is dual staining of a B-cell marker (CD19 or CD20) and a T-cell marker (CD5) in chronic lymphocytic leukemia, small lymphocytic lymphoma, and mantle cell lymphoma. 4. Expression of immature cell markers in a large number of cells: For lymphoid neoplasms, the immature cell markers include terminal deoxynucleotidyl transferase (TdT), CD10, and CD34. For myeloid tumors, CD34 and CD117 are frequently expressed. 5. Selective loss of one or more cell lineage antigens: This criterion is particularly useful for diagnosis of T-cell lymphomas, because there are no clonal markers for T cells analogous to light chain restriction for B cells. Most laboratories use three pan-T-cell markers (CD3, CD5, CD7) for comparison. 6. Determination of T-cell clonality by TCR-V␤ antibodies: A set of TCR-V␤ antibodies can be used to identify the clonality of the T cells. If most T cells express the antigens of the same family, it may represent a monoclonal T cell population.

Cytogenetic Techniques for Hematologic Neoplasms Chromosomal aberrations are classifie into numerical and structural abnormalities. Structural abnormalities include translocations, deletions, inversions, duplications, and isochromosomes [14, 15]. Among these, reciprocal translocation is most common in hematologic neoplasms. The numerical abnormalities are subdivided into polyploid and aneuploid. The term polypoid refers to multiplication of the normal haploid number of 23: triploidy, 69, and tetraploidy, 92. Aneuploid, on the other hand, refers to multiplication of chromosomes in irregular numbers; for instance, monosomy and trisomy. Cytogenetics plays multiple roles in relation to hematologic neoplasms [14, 15].

Cytogenetic Techniques for Hematologic Neoplasms

25

Table 5 Monoclonal antibodies used in immunohistochemistry CD/Antigen Cell specificit

Clinical application

ALK Bcl-2 Bcl-6 CD1a CD3 CD4 CD5 CD8 CD10 CD15 CD20 CD21 CD23 CD30

ALCL B lymphoma B lymphoma Precursor T-cell lymphoma/leukemia T lymphoma/leukemia T lymphoma/leukemia T lymphoma/leukemia T lymphoma/leukemia ALL, follicular lymphoma Hodgkin lymphoma B lymphoma FDC tumor and follicle identificatio B lymphoma Hodgkin lymphoma

CD34 CD42b CD43 CD45 CD45RA CD45RO CD56 CD57 CD61 CD68 CD79a CD79b CD117 Cyclin D1 DBA-44 Ki-67 PAX/BSAP TdT

ALCL cell B cell B cell Thymocyte, Langerhans cell T cell T-helper cell T cell T-suppressor cell Immature B cell Reed–Sternberg and myeloid cells B cell Follicular dendritic cell (FDC) B cell, FDC Reed – Sternberg and activated T/B cells Hematopoietic stem cell Platelet/megakaryocyte T cell, B cell subset All leukocytes T cell, B cell subset T cell, B cell subset NK cell NK cell Platelet/megakaryocyte Monocyte/histiocyte B cell B cell Hematopoietic stem cell B cell B cell Proliferation fraction B cell Precursor T/B cells

Acute lymphoid/myeloid leukemia Acute megakaryoblastic leukemia T/B-cell lymphoma, myeloid sarcoma Lymphomas, leukemias T/B-cell lymphoma T/B-cell lymphoma NK/T-cell lymphoma/leukemia NK/T-cell lymphoma/leukemia Acute megakaryoblastic leukemia Monocyte/histiocyte tumors B lymphoma B lymphoma Acute myeloid leukemia Mantle cell lymphoma Hairy cell leukemia High-grade lymphoma Hodgkin and non-Hodgkin lymphoma Precursor T/B-cell lymphoma/leukemia TRAcp Lymphoid cells Hairy cell leukemia ALCL, anaplastic large cell lymphoma; CD, cluster designation; NK, natural killer; TdT, terminal deoxynucleotidyl transferase; TRAcp, tartrateresistant acid phosphatase

1. Diagnosis of lymphoma and leukemia: There are increasing numbers of karyotypes that are diagnostic for a specifi tumor. The most commonly encountered abnormal karyotypes in lymphomas are listed in Table 6 [16]. In some hematologic neoplasms, such as chronic myeloid leukemia and Burkitt lymphoma, abnormal karyotype is the only criterion for a definit ve diagnosis. 2. Determination of the malignant nature of a lesion: Some cytogenetic abnormalities may not provide a definit ve diagnosis, but their presence indicates a clonal nature of the lesion. This is particularly useful in cases of myelodysplastic syndrome and myeloproliferative neoplasms. 3. Prediction of prognosis and detection of minimal residual diseases: Cases of anaplastic large cell lymphoma that express t(2;5) have a more favorable prognosis than those without this karyotype. In childhood acute lymphoblastic leukemia, cases with hyperdiploidy carry a better prognosis than diploidy cases. The prediction of prognosis by karyotyping is sometimes associated with the histologic pattern. For instance, a favorable prognosis with t(14;18) is due to its association with follicular lymphoma, whereas the poor prognosis predicted by t(8;14) is due to its association with Burkitt lymphoma. Some numerical chromosomal abnormalities, such as +5, +6, or +8, are related to shorter survival in patients with non-Hodgkin lymphoma. Since the karyotype is very specifi for a particular lymphoma or leukemia, it is helpful to use cytogenetic markers to detect minimal residual disease.

26 Table 6 Common chromosomal translocations in lymphomas Neoplasm Translocation Anaplastic large cell Burkitt

t(2;5)(p23;q35) t(8;14)(q24:q32) t(2;8)(p12;q24) t(8;22)(q24;q11) Burkitt-like t(14;18)(q32;q21) Cutaneous T cell t(10;14)(q24;q32) Diffuse large B cell t(3;14)(q27;q32) t(14;15)(q32;q11–13) Follicular t(14;18)(q32;q21) Lymphoplasmacytic t(9;14)(p13;q32) Mantle cell t(11;14)(q13;q32) Marginal zone/MALT t(11;18)(q21;q21) t(1;14)(p22;q32) Plasma cell myeloma t(4;14)(p16;q32) t(14;16)(q32;q23) t(16;22)(q23;q11) Small lymphocytic/CLL t(14;19)(q32;q13) CLL, chronic lymphocytic leukemia; MALT, mucosa-associated lymphoid tissue

Introduction

Genes involved ALK; NPM c-MYC; IgH Igκ; c-MYC c-MYC; Igλ IgH; BCL-2 NFKβ2(LYT-10); IgH BCL-6; IgH IgH; BCL-8 IgH; BCL-2 PAX5 (BSAP); IgH BCL-1 (CCND1); IgH API2; MLT BCL-10; IgH FGFR3; IgH IgH;c-MAF c-MAF;IgλI IgH; BCL-3

4. Distinction of relapsed from secondary neoplasms: When a lymphoma patient shows a new tumor after treatment, it is hard for immunophenotyping to distinguish whether it is a different tumor or a relapse of the same tumor. However, if the original lymphoma has a specifi karyotype, cytogenetic study can reliably identify if it is a relapsed or a newly developed tumor. Cytogenetic abnormalities can be detected by conventional karyotyping, fluorescenc in situ hybridization, or molecular biological techniques. Karyotyping should be used for the screening or for initial diagnosis of a new case. If there is a possibility of cytogenetic evolution (e.g. changes of clinical presentation), karyotyping should be repeated. Karyotyping is performed at the metaphase, so cell culture is required. In low grade malignancy, mitosis is frequently not obtained. In addition, when tumor cells are in the minority, they will be overgrown by the normal population. For these reasons, karyotyping is not a sensitive technique and false-negative results are not infrequently encountered. Therefore, for cases with a known karyotype, FISH is the technique of choice, as it is more sensitive and specifi than the conventional karyotyping. Furthermore, it takes only 2–3 days to obtain the fina result from FISH, thus it provides a rapid diagnosis. However, for therapeutic monitoring, such as after treatment of chronic myelogenous leukemia, molecular biology techniques, such as the real-time polymerase chain reaction, are more sensitive in detecting the minimal residual disease (MRD).

Molecular Biology Techniques for Hematologic Neoplasms FISH can be considered a hybrid of molecular biology and cytogenetics [14, 15]. In addition to FISH, the most commonly used molecular biology techniques in clinical laboratories are Southern blotting and polymerase chain reaction (PCR). The variants of PCR include reverse transcriptase PCR (RT-PCR) and quantitative PCR (real-time PCR). In hematology, molecular biology techniques were firs used for antigen receptor gene rearrangement [17]. The rearrangement of heavy chain gene indicates the presence of a monoclonal B-cell population, while the rearrangement of T-cell receptor gene suggests the existence of a monoclonal T-cell population. The original technique used is Southern blotting, which has gradually been replaced by the PCR technique. As mentioned in “Classificatio of Lymphoma and Leukemia”, somatic mutation of immunoglobulin heavy chain gene is a hallmark to distinguish lymphoma cells from pre-germinal center, germinal center and post-germinal center. PCR and FISH can also detect the rearrangement of oncogenes, such as BCR oncogene in chronic myelogenous leukemia and c-MYC oncogene in Burkitt lymphoma. However, the detection of translocation of oncogene with an antigen receptor gene (or between two oncogenes) is even more specifi for the diagnosis of a particular neoplasm. For instance, c-MYC oncogene can be detected in most cases of Burkitt lymphoma, but can also be detected in a small percentage of diffuse large

Diagnostic Procedures for Hematologic Neoplasms

27

B-cell lymphomas. However, when translocation of c-MYC and a heavy or light chain gene is detected, it is very specifi for the diagnosis of Burkitt lymphoma. The detection of chromosomal translocation by FISH or PCR is frequently more sensitive than the conventional karyotyping technique. The condition in which translocation is detected by molecular technique but not karyotyping is frequently referred to as cryptic abnormality. The detection of oncogene translocation helps to elucidate the mechanism of tumorigenesis. There are two major mechanism of oncogene activation [18]: 1. Fusion transcript: The classic example is chronic myelogenous leukemia, but the same pattern is encountered in cases of acute lymphoblastic leukemia. In these cases, the cytogenetic abnormality is t(9;22)(q34;q11), or the so-called Philadelphia chromosome. The translocation results in the fusion of c-ABL, a proto-oncogene, on chromosome 9q34, and a restriction region on chromosome 22q11, called the breakpoint cluster region (BCR), leading to transcription to an aberrant hybrid c-abl-bcr RNA. The bcr domain activates the tyrosine kinase activity of the c-abl protein (47). The abnormal activity of the tyrosine kinase may disturb the normal process of transduction in the cell and cause malignant transformation. In follicular lymphoma, the BCL-2 oncogene forms a fusion transcript with the immunoglobulin heavy chain gene that encodes the inner mitochondrial membrane protein, leading to the blocking of programmed cell death (anti-apoptotic activity). 2. Transcriptional deregulation: The well-known example is Burkitt lymphoma, in which the c-MYC proto-oncogene is translocated from chromosome 8 to chromosome 14 and juxtaposed with the heavy chain gene. As a result of the translocation, c-MYC submits to the control of the transcriptional enhancer of the immunoglobulin gene and is thus activated or deregulated. Constitutive MYC expression may prevent cells from entering the resting state (G0 phase) and differentiating, leading to continuing proliferation of undifferentiated cells. The overexpression of the BCL-1 gene in mantle cell lymphoma belongs to the same category. Recently, a new molecular biology technique, gene expression profilin (GEP), has emerged as the most promising technique for the study of hematologic neoplasms [19, 20]. This technique tethers hundreds or thousands of gene-specifi probes in arrays on a solid face, such as glass. RNA is extracted from tissues of interest, and labeled with a detectable marker, usually fluorochromes The samples containing this messenger RNA are then hybridized with the gene-specifi probes on the array. Images are generated by the use of confocal laser scanning and the relative fluorescenc intensity of each gene-specifi probe represents the level of expression of the particular gene After data analysis with computer manipulations, gene expression signatures can be recognized. A gene expression signature is define as a group of genes that are characteristically expressed in a particular group of cells belonging to a certain cell lineage, disease entity, or subtype of leukemia/lymphoma. This technique has been used for the diagnosis and subclassificatio of lymphomas and leukemias, prediction of prognosis, and guidance of treatment for hematologic neoplasms. The usefulness of GEP is exemplifie by the studies of diffuse large B-cell lymphoma and chronic lymphocytic leukemia. Diffuse large B-cell lymphoma can be stratifie with GEP into two groups. The group with the germinal center B-cell-like signature shows a more favorable prognosis than the group with the activated B-cell-like signature. Some cases of diffuse large B-cell lymphoma can be very similar to Burkitt lymphoma in terms of morphology and cytogenetics with positive c-MYC gene rearrangement [21, 22]. However, GEP reveals distinctive patterns in these two neoplasms that help make a definit ve diagnosis. Chronic lymphocytic leukemia also shows two distinctive signatures that can separate the patients into two prognostic groups. Patients with unmutated VH require aggressive treatment, while those with VH somatic mutation should follow the policy of watch and wait.

Diagnostic Procedures for Hematologic Neoplasms The clinical presentation of hematologic neoplasms is similar and they are usually the result of bone marrow failure. When there is thrombocytopenia, the patient may have petechiae, ecchymosis, or bleeding from various organs. Anemia causes fatigue, weakness and pallor. The manifestation of leukopenia is often secondary bacterial infections. Some symptoms may be due to compression by enlarged lymph nodes or leukemic infiltration The characteristic “B symptoms” in lymphomas include fever, night sweats, and weight loss. Therefore, the diagnosis of lymphoma and leukemia should follow certain routine procedures due to the nonspecifi clinical presentation in most patients.

28

Introduction

The screening procedure in these patients is examination of the peripheral blood for a complete blood cell count. This step is most fruitful as it may reveal if the patient has anemia, leukopenia, leukocytosis, thrombopenia, or thrombocytosis. A differential count will further demonstrate the abnormal cell lineage, be it granulocyte, monocyte, or lymphocyte. An extremely high leukocyte count usually suggests acute or chronic leukemia including myeloid or lymphoid cell lineage or chronic myeloproliferative neoplasms. However, aleukemic leukemia is increasingly common recently, probably due to the existence of large numbers of secondary leukemias. Cytopenia is frequently due to myelodysplastic syndromes, but the etiology can be any hematologic or nonhematologic neoplasms involving the bone marrow, aplastic anemia, and myelofibrosis Therefore, the second diagnostic step for hematologic neoplasms is bone marrow examination [23–26]. A hypercellular bone marrow can be seen in leukemia, myelodysplastic syndromes, myeloproliferative neoplasms, or infections. A hypocellular bone marrow can be seen in aplastic anemia, myelodysplastic syndrome, and other causes of bone marrow suppression (e.g. drugs or chemicals). Bone marrow biopsy is also used for staging purposes. When lymphoma cells are demonstrated in the bone marrow, it indicates a stage IV disease. Thus bone marrow examination may give the clue to the diagnosis of lymphoma. However, lymph node biopsy is needed under most circumstances for the diagnosis of Hodgkin and non-Hodgkin lymphoma, particularly the latter. The subtyping of these lymphomas is mainly based on their histologic patterns. Most lymphomas are considered predominantly nodal lymphomas, including follicular lymphoma, mantle cell lymphoma, nodal marginal zone B-cell lymphoma, peripheral T-cell lymphomas, and anaplastic large cell lymphoma. However, extranodal marginal zone B-cell lymphoma and extranodal NK/T-cell lymphoma are always present in extranodal sites. Cutaneous lymphomas can be primary, such as mycosis fungoides, or secondary, such as in anaplastic large cell lymphoma. Other lymphomas are predominantly disseminated, such as chronic lymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenstr¨om macroglobulinemia, plasma cell myeloma, S´ezary syndrome, and hairy cell leukemia. Finally, splenic involvement is the major clinical presentation of splenic marginal zone lymphoma, hepatosplenic T-cell lymphoma and hairy cell leukemia. In those extranodal lymphoma cases, the spleen, liver, gastrointestinal tract, skin or brain and other tissues are needed for examination. In addition to morphologic examination, fl w cytometry, immunohistochemistry, cytogenetics, and molecular biology have played an increasingly important role in the diagnosis of hematologic tumors. Therefore, additional specimens for ancillary studies should be always considered. 1. Examination of bone marrow: This is the most important step for the diagnosis of leukemia because the diagnostic criteria of acute myeloid leukemia is based on the blast count in the bone marrow. There are many entities for which bone marrow examination is indispensable [23–26]. These include myelodysplastic syndrome, myeloproliferative neoplasms, myelodysplastic/myeloproliferative diseases, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, plasma cell myeloma, and lymphoplasmacytic lymphoma. For nodal-based lymphomas, bone marrow examination is frequently needed for staging. Bone marrow aspirate is important for a cytologic examination, particularly the identificatio of blasts and dysplastic cells. A 500-cell differential count is needed as the basis for the diagnosis in most cases. Aspirate also provides the information of myeloid to erythroid ratio (M:E ratio), which helps in differential diagnosis. In case of a dry tap, an imprint (touch preparation) of the core biopsy is necessary for differential counts. However, touch preparations have several drawbacks. The major deficien y is due to the drying effect, which makes the cytoplasm coiled-up or damaged, leading to a falsely high nuclear/cytoplasmic ratio and the false impression of absence of cytoplasmic granules. As a result, promyelocytes or myelocytes may be misidentifie as blasts. In addition, mast cells, megakaryocytes, and, to a lesser degree, monocytes are often concentrated in bone marrow particles (spicules), and thus these cells may be underestimated in the non-spicular imprints. Cytochemical stains can be done on bone marrow aspirates or imprints. Myeloperoxidase and specifi and nonspecifi esterases are useful to defin the cell lineage. Acid phosphatase and Oil Red O are helpful in substantiating the diagnosis of hairy cell leukemia and Burkitt lymphoma, respectively. However, immunophenotyping by fl w cytometry and immunohistochemistry has gradually replaced cytochemistry. The bone marrow core biopsy (trephine biopsy) is important for the recognition of the histologic pattern and the severity of the neoplastic process (tumor load). The general cellularity of bone marrow as well as the estimated percentage of tumor cells should be performed in the core biopsy. Myelofibrosis granulomatosis, and metastatic carcinoma can be readily identifie in core biopsy, and recognition of the distribution of the immature cells is also an important feature. The immature myeloid cells are usually distributed along the bony trabeculae and immature erythrocytes are normally present in the intertrabecular area. When clusters of immature myeloid cells are present in the intertrabecular area, it is considered an abnormal localization of immature precursors (ALIP) and is pathognomonic for myelodysplastic syndromes. Because of the random distribution of

Diagnostic Procedures for Hematologic Neoplasms

29

lymphoma and chronic lymphocytic leukemia cells in the bone marrow, aspirates may not contain the tumor cells. Therefore, the diagnosis of these cases frequently depends on the core biopsy, which examines a much larger sample size than the aspirate. Beside hematoxylin and eosin stain for the core biopsy, Prussian blue stain for iron is an indispensable part of bone marrow study. It is essential for evaluation of anemia and for the detection of ringed sideroblasts in myelodysplastic syndromes. Giemsa and periodic acid – Schiff (PAS) stains are helpful for the differential count in the core biopsy. Eosinophils, basophils, mast cells, plasma cells, and nucleated red blood cells are easier to identify in Giemsa-stained preparations. Mature myeloid cells, megakaryocytes, and fungus are PAS-positive. In lymphoblasts and leukemic normoblasts, a block pattern of PAS staining is characteristic, while megakaryoblasts show a peripheral PAS staining pattern. In some laboratories, reticulin stain is also routinely performed for evaluation of myelofibrosis One of the difficul tasks in bone marrow examination is the distinction between a benign lymphoid aggregate, which is frequently seen in the elderly population, and a low grade lymphoma, which may not show obvious atypia in the tumor cells. Flow cytometry and immunohistochemistry may help. Occasionally, immunoglobulin heavy chain gene rearrangement has to be performed to identify the clonality of the lymphoid cells. However, some morphologic criteria may help to avoid overuse of the ancillary tests. The malignant lymphoid aggregates are usually high in number, large in size, with irregular margin or infiltratin margin, absence of a germinal center and frequently paratrabecular in distribution. The distinctions between a benign and malignant lymphoid aggregate are listed in Table 7. 2. Examination of lymph nodes: The diagnosis of lymphoma can be made in extranodal tissues, but the subclassificatio of lymphoma has to depend on the histologic patterns in the lymph node, such as follicular, sinusoidal, mantle zone, marginal zone, and diffuse. Therefore, lymph node biopsy is indispensable for the diagnosis of lymphoma [27, 28]. Accordingly, a well fi ed specimen is of utmost importance for morphologic recognition of different zones in the lymph node. Since ancillary studies, such as fl w cytometry and cytogenetics, are frequently required for lymphoma studies, lymph nodes should be transported in saline or RPMI medium and not in formalin. The specimen should be promptly processed after receipt. A large lymph node should be sliced at 2–3 mm intervals and selected slices are f xed in formalin. B5 is frequently advocated as the desirable fixat ve for lymph nodes. However, this fixat ve contains mercury and over-fixatio in B5 may cause nonspecifi immunochemical staining. Some modifie formalin fixat ve, such as IBF (isopropanol buffered formalin) or B plus, is a good substitute for B5. A large lymph node should be f xed for several hours or overnight before it is processed. Before the specimen is f xed 5–8 touch imprints should be made from the cut surface of a bisected lymph node. The imprints should be air-dried and stained with Wright – Giemsa, Diff-Quick (rapid Wright method) or hematoxylin and eosin. The imprint can demonstrate the size and configuratio of the tumor cells, the cytoplasmic granules, the cellular composition of the lymph node, and sometimes the histologic pattern (such as a starry sky pattern). If the lymph node imprint shows a monotonous population, many atypical cells, or a starry sky pattern, lymphoma should be considered. If Reed – Sternberglike cells or Hodgkin-like cells are present on an eosinophilic background, Hodgkin lymphoma should be considered. If cytoplasmic granules are demonstrated, natural killer cell lymphoma is suspected. Accordingly, cytochemical stains can be done on the remaining imprints. Oil Red O should be done if Burkitt lymphoma is suspected. Esterase stain should be performed when monocytic or histiocytic tumor is considered. Myeloperoxidase is helpful to exclude myeloid sarcoma.

Table 7 The distinguishing features between benign and malignant lymphoid aggregates Benign Malignant Age Configuratio Number Size Germinal center Distribution Cytology Cell population Bone marrow aspirate Flow cytometry Immunohistochemistry

Elderly Well circumscribed Usually 1–3 < 3 mm 5% of cases Never paratrabecular Small mature lymphocytes Mixed population with other leukocytes No lymphoma cells T cells or polyclonal B cells T cells or mixed T and B cells

Wider age range Irregular with infiltratin margin Frequently more than 3 May be > 3 mm No May be paratrabecular Small to medium-sized lymphocytes with or without atypia Pure lymphoid population Lymphoma cells may be present Monoclonal B-cell population Predominantly B cells

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Introduction

When hematologic neoplasm is in the differential diagnosis, a piece of lymph tissue should be sent to the f ow cytometry laboratory immediately for immunophenotyping and another piece sent to the cytogenetic laboratory for karyotyping or molecular biologic studies. For highly aggressive tumors, such as Burkitt lymphoma, fluorescenc in situ hybridization should be initiated instead of waiting for the result of karyotyping. 3. Examination of splenectomy specimen: The tumor cells, particularly large cell lymphoma, are rapidly autolyzed in the spleen; supposedly the spleen contains a high content of autolytic enzymes in the cells of the red pulp. Therefore, every effort should be made to assure the timely delivery of the splenectomy specimen to the histology laboratory, ideally within one hour. Under special occasions, laboratory personnel should be waiting outside the operating room to avoid any delay in the transportation of the specimen. Once the specimen is obtained, the spleen should be cut through and small pieces of specimen should be obtained from the areas where tumor involvement is suspected (e.g. a nodule) and put into the RPMI tubes immediately. The importance of obtaining a fresh specimen of the spleen is due to the fact that immunophenotyping by fl w cytometry is indispensable for the diagnosis of lymphoma in the spleen. Once the cellular viability is below 60%, a diagnosis is no longer reliable. Most of the lymphomas in the spleen involve the white pulp. Therefore, the expansion of the follicles is the major clue to the diagnosis of lymphoma. As the normal follicular cells are of B-cell origin, the positive staining of CD20 cannot distinguish a B-cell lymphoma from follicular proliferation. Other stains, such as bcl-2 and CD43, are usually negative in splenic lymphoma. Kappa and lambda stains are usually not helpful in identify the clonality of lymphocytes in the spleen. Therefore, an immunophenotyping by fl w cytometry is usually the only means for a definit ve diagnosis of B-cell lymphoma in the spleen, unless the tumor cells show marked atypia. Immunoglobulin heavy chain gene or T-cell receptor gene rearrangement done on the paraffin-embedde tissue may demonstrate a monoclonal pattern, but it is not always positive. When a fresh, unfi ed splenectomy specimen is obtained, its processing is the same as the lymph node. The specimen should be sliced at 3-mm intervals. Touch preparations should be made to visualize the morphology and special stains can be done accordingly. If a hematologic tumor is suspected clinically, f ow cytometry should be routinely performed regardless of the morphologic presentation in the touch preparations, as it is difficul to identify the tumor cells in imprints. Since it is critical to recognize and distinguish the white pulp, the red pulp core and sinus for differential diagnosis (hairy cell leukemia and hepatosplenic T-cell lymphoma are in the red pulp and most other lymphomas are confine to the white pulp), one cannot emphasize enough the importance of a well f xed splenectomy specimen. Therefore, several small-sized specimens from representative areas should be f xed for several hours or overnight before processing in the machine. 4. Examination of specimens from other organs: Large specimens from other solid organs or a large tumor mass from hollow organs should be treated the same as lymph node and splenectomy specimens. In essence, they should be promptly processed, making touch preparations for morphologic examination, and obtaining specimens for f ow cytometry and cytogenetics, when indicated. Large specimens should be sliced at 2–3-mm intervals, and fi ed for long enough to assure good morphology. Brain biopsies are usually small, but touch preparations should also be made and ancillary tests (e.g. fl w cytometry and cytogenetics) should be sent, if enough specimen can be garnered. Skin and gastrointestinal biopsies are usually fi ed before delivery to the pathology laboratory; thus immunohistochemical stain is the only ancillary test used under most circumstances. If immunostain fails to draw a conclusion, fluorescenc in situ hybridization or gene rearrangement on paraffi sections may help. The fina resort is to obtain a new unfi ed specimen for fl w cytometry and karyotyping.

References 1. Salmon SE. B-cell neoplasia in man. Lancet 1974;2:1230–1233. 2. Jaffe ES, Harris NL, Stein H, et al. Introduction and overview of the classificatio of the lymphoid neoplasms. In Swerdlow SH, Campo E, Harris NL, et al. eds., WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 158–155. 3. Carlyle JR, Michie AM, Cho SK, et al. Natural killer cell development and function precede ␣␤ T cell differentiation in mouse fetal thymic ontogeny. J Immunol 1998;160:744–753. 4. Harris NL. Mature B-cell neoplasms: Introduction. In Jaffe ES, Harris NL, Stein H, et al., eds., Tumours of Haematopoietic and Lymphoid Tissues, 3rd ed., Lyon, France, IARC Press, 2001, 121–126. 5. Bennett JM, Catovsky D, Daniel MT, et al. French-American-British (FAB) Cooperative Group. Proposals for the classificatio of acute leukemias. Br. J Haematol 1976;33:451–458.

References

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6. Swerdlow SH, Campo E, Harris NL, et al. eds., WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed., Lyon, France, IARC Press, 2008. 7. The Non-Hodgkin’s Lymphoma Pathologic Classificatio project. National Cancer Institute sponsored study of classificatio of non-Hodgkin’s lymphomas. Cancer 1982;49:2112–2135. 8. Lennert K, Feller AC. Histopathology of non-Hodgkin’s Lymphomas (Based on the updated Kiel classification) Springer-Verlag, Berlin, 1992. 9. Harris NL, Jaffe ES, Stein H, et al. A revised European-American classificatio of lymphoid neoplasms. A proposal from the International Lymphoma Study Group. Blood 1994;84:1361–1392. 10. Glassy EF (ed.). Color Atlas of Hematology, College of American Pathologists, Northfield Illinois, 1998. 11. Carr JH, Rodak BF. Clinical Hematology Atlas, 2nd ed., Elsevier Saunders, St. Louis, 2004. 12. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008; 45–51. 13. Zola H, Swart B, Nicholson I, et al. CD molecules 2005: Human cell differentiation molecules. Blood 2005;106:3123–3126. 14. LeBeau MM. Role of cytogenetics in the diagnosis and classificatio of hematopoietic neoplasms, In: Knowles DM (ed). Neoplastic Hematopathology, Lippincott Williams & Wilkins, Philadelphia, 2001;391–418. 15. Dewald GW, Ketterling RP, Wyatt WA, et al. Cytogenetic studies in neoplastic hematologic disorders. In: McClatchey KD (ed). Clinical Laboratory Medicine, 2nd ed., Philadelphia, Lippincott Williams & Wilkins, 2002;658–685. 16. McKeithan TW. Molecular biology of non-Hodgkin’s lymphoma. Semin Oncol 1990;17:30–42. 17. Delves PJ. Roitt IM. The immune system: First of two parts. N Engl J Med 2000;343:37–49. 18. Tam W, Dall-Fall-Favera R. Protooncogenes and tumor suppressor genes in hematopoietic malignancies. In: Knowles DM (ed). Neoplastic Hematopathology, Lippincott Williams & Wilkins, Philadelphia, 2001;329–364. 19. Quackenbush J. Microarray analysis and tumor classification N Engl J Med 2006;354:2463–2472. 20. Davis RE, Staudt LM. Molecular diagnosis of lymphoid malignancies by gene expression profiling Curr Opin Hematol 2002;9:333–338. 21. Hummel M, Bentink S, Berger H, et al. A biologic definitio of Burkitt’s lymphoma from transcriptional and genomic profiling N Engl J Med 2006;354:2419–2430. 22. Dave SS, Fu K, Wright GW, et al. Molecular diagnosis of Burkitt lymphoma. N Engl J Med 2006;354:2431–2442.. 23. Brown DC, Gatter KC. The bone marrow trephine biopsy: A review of normal histology. Histopathology 1991;22:411–422. 24. Cotelingam JD. Bone marrow interpretation: Interpretive guidelines for the surgical pathologist. Adv Anat Pathol 2003;10:8–26. 25. Naresh KN, Lampert I, Hasserjion R, et al. Optimal processing of bone marrow trephine biopsy: The Hammersmith protocol. J Clin Pathol 2006;59:903–911. 26. Foucar K. Bone Marrow Pathology, ASCP Press, Chicago, 2001. 27. Warnke RA, Weiss LM, Chan JKC, et al. Atlas of Tumor Pathology: Tumor of the Lymph Nodes and Spleen. Armed Forces Institute of Pathology, Washington DC, 1995, 15–42. 28. Ioachim HL, Medeiros LJ. Ioachim’s Lymph Node Pathology, 4th ed., Philadelphia, Lippincott Williams & Wilkins, 2009, 2–20.

Part II

Case Studies

Hematologic Neoplasms

Case 1 A 60-year-old man was admitted to hospital because of fatigue, weight loss, and abdominal discomfort for a period two months. Physical examination showed mild splenomegaly but no lymphadenopathy and hepatomegaly. Peripheral examination revealed a total leukocyte count of 51,000/␮l with 52% segmented neutrophils, 9% bands, 2% metamyelocytes, 7% myelocytes, 2% promyelocytes, 1% blasts, 5% lymphocytes, 2% monocytes, 5% eosinophils, and 15% basophils (Fig. 1.1). His hemoglobin was 11 g/dL, hematocrit 34%, and platelets 950,000/␮l. A bone marrow aspirate demonstrated 11% myeloblasts, 6% promyelocytes, 19% myelocytes, 5% metamyelocytes, 6% bands, 23% segmented neutrophils, 1% monocytes, 2% lymphocytes, 5% eosinophils, 11% basophils, and 11% normoblasts (Fig. 1.2). The myeloid to erythroid (M:E) ratio was 8:1. No myelodysplastic changes were detected.

Fig. 1.1 Peripheral blood smear shows a wide spectrum of myeloid cells with an increase of basophils (arrow). Wright – Giemsa, × 60 T. Sun, Atlas of Hematologic Neoplasms, c Springer Science+Business Media, LLC 2009 DOI 10.1007/978-0-387-89848-3 2, 

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Fig. 1.2 Bone marrow aspirate reveals predominantly myelocytes and promyelocytes with a few myeloblasts. Wright – Giemsa, × 60

A core biopsy showed 85% cellularity with widening of the paratrabecular cuff of immature myeloid cells (Fig. 1.3). The cellular component was predominantly myeloid cells with increased megakaryocytes (Fig. 1.4). Differential diagnoses: acute versus chronic myeloid leukemia

Case 1

Fig. 1.3 Bone marrow biopsy demonstrates widening of the paratrabecular cuff of immature myeloid cells. H&E, ×40

Fig. 1.4 Bone marrow biopsy shows megakaryocytic proliferation with many hypolobated micromegakaryocytes. H&E, ×60

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Hematologic Neoplasms

Fig. 1.5 Karyotyping of bone marrow reveal t(9;22)(q34;q11)

Further Studies Karyotype of bone marrow: t(9;22)(q34;q11) (Fig. 1.5) Fluorescence in situ hybridization analysis of bone marrow: BCR-ABL 1 fusion signal was demonstrated in 85% of bone marrow cells.

Case 1

39

Discussion Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that originates from a pluripotent hematopoietic stem cell. Therefore, it involves not only the myeloid cells but also monocytes, erythrocytes, megakaryocytes and lymphocytes. Clinically, CML is divided into three phases: chronic, accelerated, and blast [1–4]. In the chronic phase, the peripheral blood shows leukocytosis, usually over 50,000/␮l and in most cases exceeding 100,000/␮l. The leukocytes are mainly composed of granulocytes of various stages, from myeloblasts to segmented neutrophils, but the major population is composed of myelocytes and segmented neutrophils. This phenomenon is sometimes referred to as myelocyte bulge and is characteristic of CML. Peripheral basophilia is probably the most important findin for a morphologic diagnosis of CML, because it helps to distinguish reactive granulocytosis. However, the absence of basophilia does not exclude CML. Eosinophilia is also a common feature in CML, but it can also be seen in allergy and many other reactive conditions, so its presence is not specific The blast count in the chronic phase is usually 1,000/␮l or >10%. As the absolute monocyte count can be more than 1,000/␮l in other myeloproliferative neoplasms, such as chronic myeloid leukemia, the 10% cutoff is more specifi for differential diagnosis. There is no requirement for a certain cutoff of monocyte count in the bone marrow, probably because monocytes are hard to recognize in the bone marrow, especially when there is prominent granulocytic hyperplasia. However, in the so-called “marrow predominant CMML”, the peripheral blood may show 7.5 ␮m) Grade 3: partial effacement of NL architecture; many atypical cerebriform mononuclear cells Grade 4: complete effacement

LN1 : occasional and isolated atypical lymphocytes LN2 : many atypical lymphocytes or in 3–6 cell clusters LN3 : aggregates of atypical lymphocytes; nodal architecture preserved LN4 : partial/complete effacement of nodal architecture by atypical lymphocytes or frankly neoplastic cells

Immunophenotyping is not very specifi for distinguishing MF/SS from other CTCLs, but is a sensitive technique to exclude non-neoplastic entities. MF/SS cells are generally positive for CD2, CD3, CD4, and CD5, but are negative for CD7, CD8, CD1, and terminal deoxynucleotidyl transferase (TdT) [5]. In immunohistochemical staining, CD45RO is usually

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positive, which makes S´ezary cells the memory helper T cells (CD4 + CD45RO+). However, in the early stage, the percentage of CD7 may be normal or only partly reduced. Recently, it has been found that the loss of CD26 is a highly specifi phenotype (CD4 + CD26−) for neoplastic lymphocytes in MF/SS [5]. Molecular biological techniques are the most sensitive means used to identify MF/SS cells and to distinguish them from reactive lymphoid cells in the skin, peripheral blood, lymph nodes, and visceral organs [6]. The identificatio of clonal T-cell receptor gene (TCR) rearrangement by polymerase chain reaction (PCR) is more sensitive than Southern blotting. However, patients in the early stage of MF/SS have a lower positive rate and occasional benign inflammator skin lesions may show clonal TCR gene rearrangement. Therefore, clonal identificatio is probably more useful for therapeutic monitoring and prediction of prognosis than for initial diagnosis. Flow cytometry, using TCR V␤ 14 antibodies, has also been successfully applied to therapeutic monitoring. There have been no recurrent specifi cytogenetic aberrations identifie in MF/SS cases. The clinical course of MF/SS is usually indolent and is frequently preceded by a premalignant phase for several years. Many cases have an orderly progression from limited patches to generalized patches, plaques, and tumors. Nodal and visceral involvement is usually seen in a later stage. Transformation into high-grade lymphomas and coexistence with second malignancies (colon and lung cancers) have been reported. The standard staging system for MF/SS is the tumor, node, metastasis, blood (TNMB) system firs proposed by the National Cancer Institute, which is currently modifie by ISCL/EORTC (Table 62.2) [1] Table 62.2 ISCL/EORTC revision to the classificatio of MF/SS Skin T1 Limited patches, papules, and/or plaques covering < 10% of the skin surface T2 Patches, papules or plaques covering ≥ 10% of the skin surface T3 One or more tumors (≥ 1 cm diameter) T4 Confluenc of erythema covering ≥ 80% body surface Node N0 No clinically abnormal peripheral lymph nodes, biopsy not required N1 ∗ Clinically abnormal peripheral lymph nodes; histopathology Dutch grade 1 or NCI LN0–2 N2 ∗ Clinically abnormal peripheral lymph nodes; histopathology Dutch grade 2 or NCI LN3 N3 Clinically abnormal peripheral lymph nodes; histopathology Dutch grade 3–4 or NCI LN4 Nx Clinically abnormal peripheral lymph nodes; no histologic confirmatio Visceral M0 No visceral organ involvement M1 Visceral involvement (pathology confirmed) involved organ specifie Blood B0 ∗ Absence of significan blood involvement (≤ 5% atypical lymphocytes) B1 ∗ Low blood tumor burden; > 5% atypical lymphocytes but < B2 criteria B2 High blood tumor burdern: ≥ 1000/␮l S´ezary cells with positive clone ∗ These stages are further divided into a and b subtypes: a, clonal negative; b, clonal positive.

References 1. Olsen E, Vonderheid E, Pimpinelli N, et tal. Revisions to the staging and classificatio of mycosis fungoides and S´ezary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization for Research and Treatment of Cancer (EORTC). Blood 2007;110:1713–1722. 2. Willemze R, Jaffe ES, Burg G, et al. WHO-EORTC classificatio for cutaneous lymphomas. Blood 2005;105:3768–3785. 3. Glusac EJ. Criterion by criterion, mycosis fungoides. Am J Dermatopathol 2003;25:264–269. 4. Vonderheid EC, Bernengo MG, Burg G, et al. Update on erythrodermic cutaneous T-cell lymphoma: Report of the International Society for Cutaneous Lymphoma. J Am Acad Dermatol 2002;46:95–106. 5. Bemengo MG, Novelli M, Quaglino P, et al. The relevance of the CD4 + CD26− subset in the identificatio of circulating S´ezary cells. Br J Dermatol 2001;144:125–135. 6. Fraser-Andrews EA, Mitchell T, Ferreira S, et al. Molecular staging of lymph nodes from 60 patients with mycosis fungoides and S´ezary syndrome: correlation with histopathology and outcome suggests prognostic relevance in mycosis fungoides. Br J Dermatol 2006;155: 756–762.

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Case 63 A 78-year-old man presented with a skin nodule on the forehead. The lesion grew rapidly over a period of two months. Physical examination showed a 3 × 3 cm f rm exophytic erythematous papule with very shallow overlying excoriation on the left forehead. A full skin check did not reveal any other lesion. There was neither palpable lymphadenopathy nor hepatosplenomegaly. A CT scan of chest and abdomen also showed no lymphadenopathy. A 4-mm punch skin biopsy was performed (Fig. 63.1). In the follow-up visit one month later, the patient claimed that the skin nodule was enlarged and two new small nodules appeared.

Fig. 63.1 Skin biopsy shows a large sheet of highly pleomorphic and anaplastic large lymphoid cells in the upper dermis. The epidermis is not involved. H&E, × 40

Differential diagnoses: carcinoma versus lymphoma.

Further Studies Immunohistochemistry of skin biopsy: Cytokeratin stain: negative CD20 stain: negative (Fig. 63.2) CD3 stain: positive (Fig. 63.3) CD30 stain: positive (Fig. 63.4) ALK1 stain: negative

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Fig. 63.2 CD20 stain is negative for tumor cells. Immunoperoxidase, × 60

Fig. 63.3 CD3 stain is positive for tumor cells. Immunoperoxidase, × 60

Hematologic Neoplasms

Case 63

Fig. 63.4 CD30 stain is positive for tumor cells. Immunoperoxidase, × 60

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Discussion Primary cutaneous anaplastic large-cell lymphoma (C-ALCL) is define by the World Health Organization – European Organization for Research and Treatment of Cancer (WHO-EORTC) classificatio as a neoplasm composed of large cells with an anaplastic, pleomorphic, or immunoblastic cytomorphology and expression of the CD30 antigen by >75% of the tumor cells [1]. The WHO-EORTC include lymphomatoid papulosis (LyP) and C-ALCL in a single category, designated primary cutaneous CD30+ lymphoproliferative disorders, which account for approximately 30% of cutaneous T-cell lymphomas (CTCLs). The 2008 WHO classificatio adopts this terminology [2]. The skin lesion in C-ALCL is usually solitary or localized nodules and sometimes papules. Ulceration is frequently present. Similar to LyP, the skin lesions may show partial or complete spontaneous regression. However, LyP shows generalized and recurrent multiple papules [2]. C-ALCL has an indolent clinical course without systemic symptoms, which is in contrast to systemic ALCL (S-ALCL) that is clinically aggressive and frequently presents with stage III or IV disease. The EORTC system considers a skin lesion without systemic disease for six months as a cutoff to distinguish C-ALCL from S-ALCL [3]. However, borderline cases occur from time to time, and 10% of C-ALCL cases may have extramedullary dissemination, mainly involving the regional lymph node [1]. Histologically, sheets of tumor cells may infiltrat collagen fibers skin appendages, and subcutateous fatty tissue without the involvement of epidermis. Mitoses and apoptosis are frequent features. The demonstration of vascular invasion may help to distinguish it from LyP. The tumor cells are frequently anaplastic large cells similar to those seen in S-ALCL, but in 20% to 25% of cases the tumor cells appear to be pleomorphic or immunoblastic [1]. The presence of ulcerative lesions with inflammator infiltrates including reactive lymphocytes, histiocytes, eosinophils, and/or neutrophils may mimic LyP [1, 2]. Under most circumstances, it is difficul to distinguish C-ALCL from LyP and from S-ALCL morphologically. Immunophenotyping, however, can help distinguish between C-ALCL and S-ALCL [2,4]. S-ALCL is positive for anaplastic lymphoma kinase (ALK), clusterin, epithelial membrane antigen (EMA), and BNH.9 (blood group antigen H and Y), but these markers are negative for C-ALCL. On the other hand, C-ALCL expresses cutaneous lymphocyte antigen (CLA) and CC-chemokine receptor 4 (CCR4), which are not shown in S-ALCL. T-cell markers, CD30, and cytotoxic proteins are present in both C-ALCL and S-ALCL cases, except for the null cell type, which does not show T-cell markers. Immunophenotyping is generally considered not helpful in distinguishing C-ALCL from LyP; only close clinical follow-up may help to differentiate these two entities. t(2;5)(p23;q35) or its variants is usually not demonstrated in C-ALCL and LyP, but all three entities (C-ALCL, S-ALCL, and LyP) may have monoclonal T-cell receptor gene rearrangement. In comparison, LyP usually has the most favorable prognosis, S-ALCL has the worst outcome, and C-ALCL is in between. Because of this clinical implication, it is important to distinguish these three entities.

References 1. Willemze R, Jaffe ES, Burg G, et al. WHO-EORTC classificatio for cutaneous lymphomas. Blood 2005;105:3768–3785. 2. Ralfkiaer E, Willemze R, Paulli M, et al. Primary cutaneous CD30-positive T-cell lymphoproliferative disorders. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France, IARC Press, 2008, 300–301. 3. Kato N, Mizuno O, Ito K, et al. Neutrophil-rich anaplastic large cell lymphoma presenting in the skin. Am J Dermatopathol 2003;25:142–147. 4. Kadin ME, Pinkus JL, Pinkus GS, et al. Primary cutaneous ALCL with phosphorylated/activated cytoplasmic ALK and novel phenotype: EMA/MUC1+. cutaneous lymphocyte antigen negative. Am J Surg Pathol 2008; 32:1421–1426.

Case 64

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Case 64 A 64-year-old man presented with fever, chills, night sweats, and weight loss for one week. Physical examination showed generalized peripheral lymphadenopathy and splenomegaly. Laboratory studies revealed neutrophilia and lymphopenia, elevated lactate dehydrogenase and polyclonal hypergammaglobulinemia. A biopsy of the right cervical lymph node was performed (Figs. 64.1, 64.2, 64.3, and 64.4).

Fig. 64.1 Lymph node biopsy shows extensive infiltratio of lymphoma cells with clear cytoplasm. Vascular arborization is present. H&E, × 20

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Fig. 64.2 Lymph node biopsy demonstrates prominent vascular arborization. H&E, × 20

Fig. 64.3 Area of predominantly clear large tumor cells. H&E, × 40

Hematologic Neoplasms

Case 64

Fig. 64.4 Area of mixed cellularity. H&E, × 40

Differential diagnoses: Reactive lymphadenopathy versus lymphomas.

Further Studies Immunohistochemical stains: CD3 stain: positive (Fig. 64.5) CD4 stain: positive (Fig. 64.6) CD8 stain: negative (Fig. 64.7) CD21 stain: positive (Fig. 64.8) CD10 stain: positive (Fig. 64.9) Bcl-6 stain: positive (Fig. 64.10)

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Fig. 64.5 CD3 stain demonstrates parafollicular T-cell infiltration Immunoperoxidase, × 10

Fig. 64.6 CD4 stain reveals perivascular T-cell infiltration Immunoperoxidase, × 20

Hematologic Neoplasms

Case 64

Fig. 64.7 CD8 stain shows scattered cytotoxic T cells. Immunoperoxidase, × 10

Fig. 64.8 CD21 stain demonstrates parafollicular follicular dendritic cell meshwork. Immunoperoxidase, × 20

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Fig. 64.9 CD10 stain demonstrated positive staining of all lymphoma cells. Immunoperoxidase, × 20

Fig. 64.10 bcl-6 stain demonstrates positive staining in a fraction of the lymphoma cells. Immunoperoxidase, × 60

Hematologic Neoplasms

Case 64

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Discussion Angioimmunoblastic T-cell lymphoma (AITCL) is one of the common specifi subtypes of peripheral T-cell lymphomas, accounting for 15–20% of cases [1]. It constitutes approximately 1–2% of all non-Hodgkin lymphomas [1]. However, this lymphoma was probably underdiagnosed because of the protean clinical presentation, partial preservation of lymph node architecture, and a mixed cellular infiltration particularly in cases where the characteristic large lymphoma cells are in the minority. Therefore, the lymphoma was originally considered a premalignant condition, referred to as immunoblastic lymphadenopathy, angioimmunoblastic lymphadenopathy with dysproteinemia, and lymphogranulomatosis X, until the recent advances in immunophenotyping and molecular genetic techniques [2]. Clinically, patients frequently have systemic symptoms (fever, chills, night sweats, malaise, and weight loss) accompanied with generalized peripheral lymphadenopathy and hepatosplenomegaly. Skin rash is also a common presentation. There are multiple laboratory abnormalities, but polyclonal or occasional monoclonal gammopathy is one of the distinguished features. In association with gammopathy, patients may have circulating immune complexes, cold agglutinins, cryoglobulinemia, rheumatoid factor, anti-smooth muscle antibodies, as well as elevated erythrocyte sedimentation rate and lactate dehydrogenase [2, 3]. AITCL is associated with several autoimmune disorders, including autoimmune hemolytic anemia, rheumatoid arthritis, and autoimmune thyroiditis [2]. Furthermore, patients may have pleural effusion, ascites, and peripheral edema. Most patients succumb to opportunistic infections due to T-cell dysfunction. Histologically, there is frequently a mixed cellular infiltratio in the partially effaced lymph node. Attygalle et al described three histologic patterns, which may represent the different developmental stages in this disease [4]. The major distinctions of these three stages are the gradual effacement of the normal lymph node architecture and the steady increase of follicular dendritic cells (FDC). In pattern I, hyperplastic follicles with poorly developed mantles and ill-define borders are present without an extrafollicular FDC proliferation. Pattern II shows lymphocyte-depleted follicles with proliferation of FDC extending beyond the follicles. Pattern III reveals total effacement of normal architecture with a prominent extrafollicular proliferation of FDC. In each pattern, there is an expansion of the paracortex by a mixed cellular population. The typical lymphoma cells are of medium to large size with characteristic clear cytoplasm and usually show an angiocentric distribution [1–4]. The background cells are composed of various proportions of small lymphocytes, histiocytes, eosinophils, plasma cells and immunoblasts. A few multinucleated giant cells or Reed – Sternberg-like cells can be seen in some cases. Another characteristic feature of AITCL is vascular arborization, which is manifested as many branching and anastomosing blood vessels arranged in a haphazard pattern. Most of the blood vessels are high-endothelial venules. The peripheral cortical sinuses are usually patent and distended. The tumor cells often infiltrat the perinodal tissues. In the cutaneous lesion, AITCL usually shows dense perivascular dermal lymphoid infiltrat [3]. The lymphoma cells appear atypical in most cases, but occasional cases may show only vasculitis without atypia of lymphoid cells. The bone marrow is frequently involved with features similar to those seen in the lymph node, i.e. increased vascularity and a patchy polymorphic infiltratio [5]. The cell components include small to medium-sized lymphocytes, plasma cells, histiocytes, eosinophils, and large transformed blasts. However, large tumor cells with clear cytoplasm are rare. Immunohistochemical studies are instrumental for a definit ve diagnosis. The lymphoma cells usually express all panT-cell antigens without “antigen loss” [3]. CD4 is often predominant. Recent studies show that the follicular center cell markers, CD10 and bcl-6, are frequently positive [3, 4, 6]. In addition, CXCL13, a chemokine that is produced by normal follicular helper T cells (TFH ) has been demonstrated in 100% of AITCL cases studied [6]. The TFH origin of AITCL is further supported by gene expression profilin studies [6]. Another helpful immunostaining technique is the demonstration of FDC with CD21, CD23, or CD35 staining. The dendritic meshworks characteristically arise from the extrafollicular highendothelial venules. Proliferation of immunoblasts in the paracortex may be present. Most of the immunoblasts and Reed – Sternberg-like cells stain for B-cell markers (CD20 and CD79a). Epstein – Barr virus (EBV) can be detected in the large, transformed B cells and Reed – Sternberg-like cells, using EBV latent membrane protein stain or in situ hybridization for EBV-encoded RNA (EBER). It is hypothesized that the EBV+ B cells activate the TFH cells, which, through upregulating CXCL13, recruite more B cells to the lymph node and induce expansion of follicular dendritic cells [1]. These B cells may become clonal in a few cases and transform occasionally into diffuse large B-cell lymphoma. In gene rearrangement studies, most cases show a T-cell receptor gene rearrangement, but immunoglobulin heavy chain gene rearrangement has also been reported in 25–30% cases, probably associated with the expanded EBV+ B cells [1]. The common cytogenetic changes in AITCL include trisomy 3, trisomy 5, and gain of an X chromosome. The prognosis for AITCL is poor, with a median survival of less than three years [3].

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References 1. Dogan A, Gaulard P, Jaffe ES, et al. Angioimmunoblastic T-cell lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classifi cation of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed., Lyon, France, IARC Press, 2008; 309–311. 2. Dogan A, Attygalle AD, Kyriakou C. Angioimmunoblastic T-cell lymphoma. Br J Haematol 2003;121:681–691. 3. Ferry JA. Angioimmunoblastic T-cell lymphoma. Adv Anat Pathol 2002;9:273–279. 4. Attygalle A, Al-Jehani R, Diss T, et al. Neoplastic T cells in angioimmunoblastic T-cell lymphoma express CD10. Blood 2002;99:627–633. 5. Dogan A, Morice WG.. Bone marrow histopathology in peripheral T-cell lymphomas. Br J Haematol 2004;127:140–154. 6. Dunleavy K, Wilson WH, Jaffe ES. Angioimmunoblastic T cell lymphoma: pathobiological insights and clinical implications. Curr Opin Hematol 2007;14:348–353.

Case 65

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Case 65 A 63-year-old man presented with fever, night sweats, and weight loss for three weeks. Physical examination revealed bilateral cervical and axillary lymphadenopathy. There was also splenomegaly but no hepatomegaly. CT scan further identifie enlarged lymph nodes in the mediastinum and retroperitoneum. A biopsy of left cervical lymph node was performed (Figs. 65.1, 65.2, and 65.3).

Fig. 65.1 Lymph node biopsy shows a mixed population of small lymphocytes, epithelioid histiocytes and eosinophils. H&E, × 40

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Fig. 65.2 A higher magnificatio to demonstrate the cytologic details. H&E, × 60

Fig. 65.3 In some areas, the epithelioid histiocytes appear atypical with several multinucleated giant cells. H&E, × 60

Differential diagnoses: lymphomas of T-cell or B-cell lineage.

Hematologic Neoplasms

Case 65

Further Studies Immunohistochemical stains: CD3 stain: positive for most lymphoid cells CD20 stain: positive for scattered lymphocytes CD68 stain: positive for histiocytes (Fig. 65.4) Flow cytometry: positive for CD2, CD3, CD4, and CD5, but weakly positive for CD7 and CD8

Fig. 65.4 The epithelioid histiocytes are highlighted by CD68 staining. Immunoperoxidase, × 40

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Discussion Lymphoepithelioid lymphoma (LEL) or Lennert lymphoma is a rare entity, accounting for only 1.4% of non-Hodgkin lymphomas in the Kiel collection [1]. This tumor was considered a variant of Hodgkin lymphoma by Lennert in 1952 and it was not until early 1980s that LEL was proved to be a T-cell lymphoma by immunophenotyping and T-cell receptor gene rearrangement. The earlier reported cases were composed of a heterogeneous group of lymphomas, including Hodgkin lymphoma, angioimmunoblastic T-cell lymphoma, immunocytoma and other B-cell lymphomas. Retrospective review of the old cases concluded that most were actually not LEL. The characteristic feature of LEL is the presence of small to medium-sized lymphoma cells intermixed with a large number of epithelioid histiocytes [1, 2]. The difficult in the diagnosis of LEL is that the lymphoma cells show minimal nuclear irregularity, the chromatin is clumped and nucleoli are not present. On the other hand, the histiocytes are more pleomorphic and frequently form small to large clusters that may be mistaken for tumor cells. The infiltratio is usually diffuse, but an interfollicular infiltratio pattern is demonstrated in a minority of cases. Small numbers of multinucleated giant cells and Reed – Sternberg-like cells can be seen in some cases. In the background, there are frequently eosinophils and plasma cells. The presence of Reed – Sternberg-like cells and the background cells invariably lead to the misdiagnosis of Hodgkin lymphoma, which can be distinguished from LEL only by immunohistochemistry. By immunophenotyping, the tumor cells usually express all T-cell markers, including CD2, CD3, CD5, and CD7. When LEL transforms into a high-grade malignancy, partial loss of pan-T-cell antigens may occur [1]. Most LEL cases express CD4 and are considered helper T-cell lymphomas, but a predominant CD8-positive cytotoxic T-cell variant has been reported [3]. Those CD8-positive cases may also express a cytotoxic protein, TIA1. Dual immunohistochemical staining with Ki-67/CD4 and Ki-67/CD8 may help to distinguish the lymphoma cells from the reactive T-cells, as the latter do not carry the Ki-67 antigen [4]. CD68 stain may facilitate the identificatio of the pleomorphic histiocytes. The Reed – Sternberg-like cells may be occasionally reactive to both CD15 and CD30 and lead to a misdiagnosis of Hodgkin lymphoma. However, in most cases, both markers are negative, particularly CD15. Another major differential is angioimmunoblastic T-cell lymphoma (see Case 64). This tumor usually shows more abundant large clear tumor cells with vascular proliferation and arborization. Extrafollicular proliferation of follicular dendritic cells is also characteristic for angioimmunoblastic T-cell lymphoma, which can be demonstrated by immunohistochemical staining with CD21, CD23, or CD35. When CD10 and/or Bcl-6 are shown on the tumor cells, the diagnosis of angioimmunoblastic T-cell lymphoma is confirmed As the LEL cells can be bland-looking and immunophenotyping is not always conclusive, T-cell-receptor gene rearrangement is needed for a definit ve diagnosis. Cytogenetic studies may also help; the most common aberrant karyotype is trisomy 3.

References 1. Feller AC, Diebold J. Histopathology of Nodal and Extranodal Non-Hodgkin Lymphoma. 3rd ed., Berlin, Springer, 2004, 154–160. 2. Pileri SA, Weisenburger DD, Sng I, et al. Peripheral T-cell lymphoma, not otherwise specified In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 306–308. 3. Yamashita Y, Nakamura S, Kagami Y, et al. Lennert’s lymphoma: A variant of cytotoxic T-cell lymphoma. Am J Surg Pathol 2000;24: 1627–1633. 4. Takagi N, Nakamura S, Ueda R, et al. A phenotypic and genotypic study of three node-based, low grade peripheral T-cell lymphomas: angioimmunoblastic lymphoma, T-zone lymphoma and lymphoepithelioid lymphoma. Cancer 1992;69:2571–2582.

Case 66

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Case 66 A 49-year-old man f rst noticed an engorged blood vessel on his right chest wall going into his right armpit. It was originally considered a blood clot. A few weeks later, he found a lump under his right arm with marked swelling. A skin biopsy of his chest wall showed nonspecifi inflammation An excisional biopsy of the lump turned out to be a lymph node with atypical lymphoid cells (Fig. 66.1).

Fig. 66.1 Lymph node biopsy shows many anaplastic large cells. A few cells show the kidney-shaped nuclei (arrow), representing the hallmark cells. H&E, × 100

Differential diagnoses: Hodgkin and non-Hodgkin lymphomas.

Further Studies Immunohistochemistry CD3 stain: positive CD20 stain: negative CD30 stain: positive (Fig. 66.2) CD15 stain: negative CD45 stain: partial positive (Fig. 66.3) Epithelial membrane antigen (EMA): positive (Fig. 66.4) Anaplastic lymphoma kinase 1 (ALK1) stain: positive (Fig. 66.5)

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Fig. 66.2 CD30 stain shows the membranous and Golgi patterns in various numbers of tumor cells. Immunoperoxidase, × 60

Fig. 66.3 CD45 stain is demonstrated on most of the tumor cells. Immunoperoxidase, × 60

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Fig. 66.4 Epithelial membrane antigen (EMA) stain is seen on some tumor cells. Immunoperoxidase, × 60

Fig. 66.5 There are cytoplasmic (broad arrow) and nuclear (narrow arrow) staining patterns demonstrated by anaplastic lymphoma kinase (ALK) stain. Immunoperoxidase, × 60

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Discussion Anaplastic large cell lymphoma (ALCL) is mainly seen in pediatric patients, accounting for 40% of non-Hodgkin lymphomas in this group, but it is rare in adults, so that the general incidence is