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

30

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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Hematologic Neoplasms

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

37

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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 <3%. The World Health Organization (WHO) classificatio designates 10% as the cut-off between chronic and accelerated phases [1], but most clinical studies still use 15% as the cut-off point [2]. The percentage of monocytes is usually <3%. If a high monocyte count is encountered, chronic myelomonocytic leukemia should be considered. Patients may have anemia or normal hemoglobin and hematocrit. Platelet count can be normal or markedly increased. The bone marrow shows prominent myelosis with a blast count less than 10%. Megakaryocytic hyperplasia is a frequent finding but the megakaryocytes are usually smaller and hypolobated, which is in contrast to the large, hyperlobated megakaryocytes frequently seen in essential thrombocythemia. The degree of myelofibrosi is usually proportional to megakaryocytosis. Erythrocyte precursors are generally decreased, resulting in an increased M:E ratio as high as 10:1. Seablue histiocytes and pseudo-Gaucher cells are frequently present because of an increase of cell turnover. The bone marrow biopsy shows marked hypercellularity with 5–10 layers of immature myeloid cells along the paratrabecular area. The progress from chronic phase to accelerated phase is indicated by one or more of the following criteria as define by the WHO classificatio [1]: (i) a blast count in the peripheral blood and/or bone marrow between 10 and 19%; (ii) peripheral blood basophils 20% or above; (iii) persistent thrombocytopenia (<100, 000/␮l) unresponsive to therapy; (iv) persistent thrombocytosis (>1, 000, 000/␮l) unresponsive to therapy; (v) increasing spleen size and increasing leukocyte count unresponsive to therapy; and (vi) cytogenetic evidence of clonal evolution. In addition, nucleated erythrocytes are more frequently seen in the peripheral blood, and reticulin or collagen fibrosi is more prominent in the bone marrow than in the chronic phase. The current case is, therefore, considered to be in the accelerated phase. The blast phase is define by the presence of >20% blasts in the peripheral blood or bone marrow by the WHO classification but many clinical studies still use 30% as cut-off [2]. The blast phase will be discussed under Case 2. Immunophenotyping does not play an important role in the initial diagnosis and therapeutic monitoring of CML. Flow cytometry cannot help to distinguish CML from reactive leukocytosis, but it is helpful in identifying the cell lineage in the blast phase [4]. Terminal deoxynucleotidyl transferase (TdT) and CD10 are used to identify lymphoblasts. Myeloblasts are positive for CD13 and CD33. Monoblasts are usually immunoreactive to CD14 and CD64. Erythroblasts are glycophorin A positive. Megakaryocytes are positive for CD41 and CD61. The stem cell markers, CD34 and CD117 are useful to separate three clinical phases. For instance, CD34-positive cells is in the range of 0–26% in the chronic phase, 6–64% in the accelerated phase, and 27–97% in the blast phase [4]. CD34 is positive for both myeloblasts and lymphoblasts, but CD117 is only positive for myeloblasts. CML is characterized by the presence of t(9;22)(q34;q11) as detected by conventional karyotyping in 95% of patients. The shortened chromosome 22 is called the Philadelphia (Ph ) chromosome. This genotype has now been verifie by molecular biology as the translocation of a proto-oncogene, Abelson murine leukemia virus (ABL 1) gene, on chromosome 9 to juxtapose the breakpoint cluster region (BCR) gene in chromosome 22. As a result, a BCR/ABL 1 fusion transcript is formed, and its product (a fusion bcr/abl 1 protein) acts as a constitutively active cytoplasmic tyrosine kinase. The abnormal activity of the tyrosine kinase may disturb the normal process of transduction in the cell and cause malignant transformation. For a detailed mechanism of pathogenesis in CML, the reader is referred to references [2] and [3]. Because the breakpoint in the BCR gene can be at the site of minor bcr (m-bcr), major bcr (M-bcr), or micro-bcr (␮-bcr), the fusion proteins are sized at 190 kd, 210 kd and 230 kd, respectively. All typical CML cases express a 210 kd bcr/abl proein. A subgroup of CML expressed a large 230 kd bcr/abl fusion protein and showed clinically a lower white cell count, prominent neutrophilic maturation, thrombocytosis and slower progression than the typical CML. This subgroup is called neutrophilic CML to distinguish from chronic neutrophilic leukemia (see Case 3), which is Ph -negative. Ph -positive acute

40

Hematologic Neoplasms

lymphoblastic leukemia (ALL) cases express either a 210 kd or a 190 kd bcr/abl protein. In childhood ALL, 80% of patients carry the 190 kd bcr/abl protein. Adult CML cases with 190 kd bcr/abl is associated with monocytosis, mimicking chronic myelomonocytic leukemia. Clinically, the chronic phase is manifested by an indolent clinical course. The symptoms are usually nonspecific including fatigue, malaise, headache, weight loss, and anorexia. About 50% of patients have splenomegaly caused by extrameduallary hematopoiesis. If the patient has a high basophil count, he or she may have flushin secondary to hyperhistaminemia. The chronic phase may persist for 3–5 years and progress to accelerated and blast phases. The accelerated phase may last for 1 year. When the patient reaches the blast phase, the median survival is about 18 weeks. The life expectancy of CML patient was about 4 years before the use of bcr/abl tyrosine kinase inhibitor for treatment. The use of imatinib mesylate (Gleevec or Glivec) has revolutionized the treatment of CML: many patients may now achieve cytogenetic or even molecular response, thus preventing the progression to the accelerated or blast phase. The diagnosis of CML depends on the identificatio of the abnormal karyotype, t(9;22), by conventional cytogenetic techniques. However, fluorescenc in situ hybridization (FISH) is the method of choice because it is 100% sensitive for the detection of BCR-ABL 1 fusion product at the initial diagnosis when tumor cell burden is high. For therapeutic monitoring, quantitative reverse transcriptase polymerase chain reaction (RT-PCR) should be used as it is more sensitive than FISH in detecting low levels of tumor cells. Recently, testing of phosphorylated Crkl (avian sarcoma virus CT10 regulator of kinaselike) protein is advocated for a rapid screening of ABL dysregulation and imatinib responsiveness [2].

References 1. Vardiman J, Melo JV, Baccarani M, et al. Chronic myelogenous leukemia, BCR-ABL 1 positive. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 32–37. 2. Ross DM, Hughes TP. Current and emerging tests for the laboratory monitoring of chronic myeloid leukaemia and related disorders. Pathology 2008;40:231–246. 3. Goldman JM, Melo JV. Chronic myeloid leukemia – Advances in biology and new approaches to treatment. N Engl J Med 2003;349:1451– 1464. 4. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 52–57.

Case 2

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Case 2 A 59-year-old man presented with malaise, intermittent nosebleed, petechiae and abdominal discomfort for several weeks. Physical examination on admission revealed marked hepatosplenomegaly but no lymphadenopathy. Peripheral blood examination showed a total leukocyte count of 53,000/␮l with 40% blasts, 35% immature myeloid cells and 21% neutrophils (Fig. 2.1). His hemoglobin was 9 g/dl, hematocrit 28%, and platelets 15,000/␮l.

Fig. 2.1 Peripheral blood smear shows an increase of blasts with a few basophils. Wright – Giemsa, × 60

The bone marrow aspirate revealed 95% cellularity with 90% myeloblasts, 4% other myeloid cells, 1% monocytes and 5% erythroid series (Fig. 2.2). The myeloid to erythroid (M:E) ratio was 19:1. There were mild myelodysplastic changes demonstrated in the myeloid series. The core biopsy demonstrated almost total replacement of normal hematopoietic cells by immature myeloid cells, mainly myeloblasts (Fig. 2.3). Megakaryocytes were markedly decreased. Differential diagnoses: acute and chronic myeloid leukemia.

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Fig. 2.2 Bone marrow aspirate reveals a marked increase of blasts with a high M:E ratio. Wright – Giemsa, × 60

Fig. 2.3 Bone marrow biopsy demonstrates total replacement of normal hematopoietic elements with blast cells. H&E, × 60

Case 2

43

Further Studies Karyotype of bone marrow: t(9;22)(q34;q11) and trisomy 8. Fluorescence in situ hybridization: BCR-ABL 1 fusion signals were identifie in 90% of cells (Fig. 2.4).

Fig. 2.4 Fluorescence in situ hybridization of bone marrow smear shows one green, one orange (red) and one fusion signal in two cells, representing BCR-ABL 1 fusion product

44

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Discussion The terminal phase of chronic myelogenous leukemia (CML) is blast crisis. The World Health Organization (WHO) classificatio system define the blast phase as follows: (i) the presence of 20% or more blasts in the peripheral blood or bone marrow, (ii) the existence of extramedullary proliferation of blasts, and/or (iii) detection of large aggregates and clusters of blasts in the bone marrow biopsy, even when it is focal in distribution [1]. In some clinical studies, however, 30% or more blasts in the blood or bone marrow are required to defin blast phase for therapeutic protocols [2]. The blast phase is the consequence of progression of the chronic and accelerated phases, so that the clinical symptoms is more prominent due to severe anemia, thromobocytopenia and marked splenomegaly. As CML originates from the pleuripotent stem cells, the blasts can be in the myeloid or lymphoid series. Myeloid series, including neutrophil, eosinophil, basophil, monocyte, erythroid or megakaryocyte or a combination of various cell lineages, accounts for about 70% of cases [1]. Approximately 20–30% of cases show lymphoblast crisis [1]. In rare occasions, simultaneous presence of myeloid and lymphoid populations may occur. The identificatio of the blast lineage frequently requires the help of immunophenotyping. The monoclonal antibody panel for the differentiation of various myeloid cell lineages includes myeloperoxidase, lysozyme, glycophorin A, CD11b, CD13, CD14, CD33, CD34, CD41, CD61, and CD117 [3]. For the identificatio of lymphoblasts, CD10, CD34, terminal nucleotidyl transferase (TdT) and a T-cell (CD7) as well as a B-cell marker (CD19 or CD20) are required [3]. Myeloblast crisis accounts for two-thirds of cases in blast phase. Most cases of lymphoblasts in blast phase are of B-cell lineage; T-lymphoblasts are rarely seen in CML blast crisis [2]. The distinction between CML in blast phase and acute leukemia with myeloid or lymphoid lineage is sometimes difficult The karyotype and molecular characteristics are most useful for the differential diagnosis. A history of chronic phase or accelerated phase of CML is most helpful. Acute myeloid leukemia (AML), particularly those with eosinophilia or basophilia, may mimic CML morphologically. Therefore, the demonstration of the karyotype of t(9;22)(q34;q11) or the BCR-ABL 1 fusion product by fluorescenc in situ hybridization is of utmost importance to confir the diagnosis of CML. AML with eosinophilia may show inv(16) and those with basophilia may have t(6;9) translocation. Rare cases of AML may be Philadelphia-chromosome positive, but such AML cases express e1a2 or e13a2/e14a2 bcr/abl transcript, whereas the blast phase of CML usually shows e13a2/e14a2 bcr/abl transcript [2]. The distinction between lymphoblastic blast crisis in CML and Philadelphia-chromosome-positive acute lymphoblastic leukemia (Ph + ALL) is difficult but the breakpoint cluster region differs in these two entities. Ph + ALL cases express either a 210 kd or 190 kd bcr/abl protein, and 80% of childhood ALL cases carry the 190 kd bac/abl protein. In contrast, all typical CML cases show 210 kd bcr/abl protein. The distinction between myeloblast and lymphoblast crisis has important therapeutic and prognostic implications. Before the imatinib era, cases with lymphoblast crisis had a better prognosis than those with myeloblast crisis. With imatinib or dasatinib treatment, myeloid cases have a much better progression-free survival than that of lymphoid cases [2]. In fact, with proper early treatment, most CML cases do not progress into the blast phase.

References 1. Vardiman JW, Melo JV, Bccarani M, et al. Chronic myelogenous leukemia, BCR-ABL 1 positive. In Swerdlow SH, Campo E, Harris NL, et al. WHO classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 32–37. 2. Ross DM, Hughes TP. Current and emerging tests for the laboratory monitoring of chronic myeloid leukaemia and related disorders. Pathology 2008,40:231–246. 3. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 52–59.

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Case 3 A 50 year-old man presented with leukocytosis. His total leukocyte count was 26,500/␮l with 2% myelocytes, 2% metamyelocytes, 3% bands and 81% segmented neutrophils (Figs. 3.1 and 3.2). His hemoglobin, hematocrit and platelets were within normal limits. A bone marrow biopsy showed a cellularity of 100% with a myeloid to erythroid (M:E) ratio of 18:1. There was a marked increase of mature neutrophils, but no basophilia or eosinophilia was demonstrated (Figs. 3.3, 3.4 and 3.5). No dysplastic changes were seen in three cell lines. The neutrophil alkaline phosphatase score (NAP) was high. Physical examination revealed splenomegaly.

Fig. 3.1 Peripheral blood smear shows neutrophilia. Wright – Giemsa, × 40

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Fig. 3.2 Peripheral blood smear shows toxic granulation in three neutrophils. Wright – Giemsa × 100

Fig. 3.3 Bone marrow aspirate reveals myelocytosis with hypergranular myeloid cells. × 100

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Case 3

Fig. 3.4 Bone marrow biopsy shows hypercellularity with increased granulocytes and megakaryocytes. H&E, × 60

Fig. 3.5 Bone marrow biopsy shows hypercellularity with increased granulocytes. H&E, × 60

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Differential diagnoses: Leukemoid reaction, chronic myelogenous leukemia, and chronic neutrophilic leukemia.

Further Testing Fluorescence in situ hybridization for BCR-ABL fusion gene product: negative. Reticulin stain: mild reticulin fibrosi (Fig. 3.6).

Fig. 3.6 Bone marrow biopsy reveals mild reticulin fibrosis Reticulin stain, × 60

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Discussion Peripheral leukocytosis is usually due to acute infection. Marked reactive leukocytosis can be seen in chronic inflammator reactions or occult malignancy. In those conditions, it is called leukemoid reaction. Chronic neutrophilic leukemia (CNL) is very similar to leukemoid reaction, therefore every effort should be made to rule out the latter, including detailed clinical history and physical examination, before making the diagnosis of CNL. CNL is a diagnosis by exclusion, as there are many conditions that may mimic CNL. The World Health Organization (WHO) classificatio has define a set of criteria for the diagnosis of CNL [1]. In the peripheral blood, the leukocyte count should be more than 25,000/␮l with more than 80% of segmented neutrophils and bands. Significan dysplasia should not be identifie in any of the cell lineage. Myeloblasts are < 1% and other immature myeloid cells are fewer than 10%. In the bone marrow, neutrophils should be markedly increased and myeloblasts should be less than 5%. Megakaryocytes and erythroid proliferation may also occur. No Philadelphia chromosome or BCR-ABL fusion gene should be identified There should be no rearrangement of PDGFRA, PDGFRB or FGFR1. Hepatosplenomegaly is frequently demonstrated in these patients. In addition, physiologic neutrophilia, another myeloproliferative disease, myelodysplastic syndrome and myelodysplastic/myeloproliferative disease should be excluded. Based on the above criteria, our case is consistent with CNL. However, similar manifestation may also be encountered in leukemoid reaction with the exception of splenomegaly [2]. Other differences are relative, such as the leukocyte count, the M:E ratio and the cellularity in the bone marrow, which are usually lower in leukemoid reaction than those presented in this case. The determination of C-reactive protein and erythrocyte sedimentation rate may be helpful to distinguish a reactive from a neoplastic process. The distinction between chronic myelogenous leukemia (CML) and CNL is mainly based on the presence or absence of Philadelphia chromosome and BCR-ABL 1 fusion gene. In addition, CML may show more than 10% immature myeloid cells in the peripheral blood, more than 5% of myeloblasts in the peripheral blood and bone marrow, and basophilia and eosinophilia are commonly seen in CML. The NAP score is usually decreased in CML. Toxic granulation in the granulocytes, as demonstrated in this case, is not seen in CML. There have been a few reported cases of CNL that showed BCR-ABL 1 fusion gene [2]. However, this fusion gene differs from those seen in CML or Philadelphilia chromosome-positive acute lymphoblastic leukemia (Ph’ AML) in terms of the breakpoint cluster region. The breakpoint in the M-bcr is typical of CML with a p210 protein product, while the m-bcr with a p190 protein product is seen in Ph’ ALL. The reported CNL cases had ␮bcr with a p230 protein product. It is recommended that these cases should be termed neutrophilic-chronic myeloid leukemia [2]. A high percentage of reported CNL cases was associated with plasma cell dyscrasia, either myeloma or monoclonal gammopathy with undetermined significanc (MGUS). However, in one such patient, a high level of G-CSF supposedly produced by the myeloma cells was demonstrated, indicating that the neutrophilia is reactive [2]. The polyclonal nature of the neutrophils is documented by X-linked polymorphism studies [3]. Therefore, this condition is designated plasma cell dyscrasia-associated neutrophilia instead of CNL. CNL can evolve from other myeloproliferative disorders, such as polycythemia vera and idiopathic myelofibrosi [2]. If significan myelodysplasia is identifie in a case suspected to be CNL, myelodysplastic/myeloproliferative diseases, such as atypical chronic myeloid leukemia, should be considered [1]. The clonal nature of CNL was supported by a study with the human androgen receptor gene assay and by the methylation pattern of the X-linked hypoxanthine phosphoribosyl transferase (HPRT) gene [3]. Cytogenetic abnormalities have also been found in 37% of cases studied, but at least some of these abnormalities may represent secondary events in the pathogenesis of CNL [2]. Most recently, JAK2 V617F tyrosine kinase mutation has been identifie in three cases of CNL [4].

References 1. Bain B, Brunning RD, Vardiman JW, et al. Chronic neutrophilic leukaemia. In: Swerdlow SH, Campo E, Harris HL, et al. eds: WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008,38–39. 2. Reilly JT. Chronic neutrophilic leukaemia: A distinct clinical entity? Br J Haematol 2002;116:10–18. 3. B¨ohm J, Kock S, Schaefer HE, et al. Evidence of clonality in chronic neutrophilic leukaemia. J Clin Pathol 2003;56:292–295. 4. Lea NC, Lim Z, Westwood NB, et al. Presence of JAK2 V617F tyrosine kinase mutation as a myeloid-lineage-specifi mutation in chronic neutrophilic leukaemia. Leukemia 2006;20:1324–1326.

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Case 4 A 60-year-old man was found to have leukocytosis and thrombocytopenia during a routine physical examination. He also noticed a new bruise on his trunk and blood-tinged secretion when he blew his nose in the past few weeks. Physical examination revealed prominent splenomegaly. His hematocrit was 38.5% and hemoglobin 12.4 g/dL. His total leukocyte count was 54, 000/␮l with features of leukoerythroblastosis (Fig. 4.1) and dacrocytosis (Fig. 4.2). The bone marrow aspirate revealed left-shifted myeloid series with a myeloid to erythroid (M:E) ratio of 13:1 (Fig. 4.3). The bone marrow biopsy showed myelofibrosi (Fig. 4.4) with a marked increase of reticulin fiber (Fig. 4.5). The patient received hydroxyurea, which reduced the leukocyte count, but his platelet count continued to drop. He finall had a splenectomy (Figs. 4.6, 4.7 and 4.8).

Fig. 4.1 Peripheral blood smear shows a myelocyte and a few nucleated red blood cells, consistent with leukoerythroblastosis. Wright – Giemsa, × 100

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Fig. 4.2 Peripheral blood smear shows features of anisocytosis with a few dacrocytes (arrows). Wright – Giemsa, × 100

Fig. 4.3 Bone marrow biopsy reveals myelofibrosi with a dilated sinus. Note all megakaryocytes appear to “stream“ through the marrow due to fibrosis H&E, × 20

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Fig. 4.4 Reticulin stain of the bone marrow shows marked increase of reticulin fibers × 60

Fig. 4.5 Bone marrow aspirate reveals left-shifted myeloid series with a high M:E ratio. Wright – Giemsa, × 60

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Fig. 4.6 Splenectomy specimen demonstrates many megakaryocytes and clusters of nucleated red blood cells inside and outside the sinuses. H&E, × 40

Fig. 4.7 Factor VIII stain of the splenectomy specimen shows many megakaryocytes of various sizes. × 60

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Fig. 4.8 Myeloperoxidase stain of the splenectomy specimen reveals extensive myeloid cell infiltration × 20

Differential diagnoses: Chronic myelogenous leukemia versus primary myelofibrosi

Further Studies Cytogenetic karyotype: Normal male karyotype – 46,XY Fluorescence in situ hybridization: no BCR-ABL 1 fusion product detected.

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Discussion The current case shows peripheral leukoerythroblastosis and dacrocytosis, bone marrow fibrosi with atypical megakaryocytic hyperplasia and splenomegaly with extramedullary hematopoiesis, fulfillin the major diagnostic triad of chronic idiopathic myelofibrosis However, due to the current discovery of the association of JAK2 mutation in about 50% of these patients, chronic idiopathic myelofibrosi is now considered a myeloproliferative neoplasm and has been renamed primary myelofibrosi (PMF) [1–3]. There are additional synonyms and some of them are still being used in the current literature, including agnogenic myeloid metaplasia, myelofibrosi with myeloid metaplasia, myelosclerosis with myeloid metaplasia and chronic granulocytic-megakaryocytic myelosis. There are a few sets of diagnostic criteria for PMF. The early diagnostic criteria adopted by the Italian consensus conference include two necessary criteria and six optional criteria [4]. The necessary criteria are (i) diffuse bone marrow fibrosis and (ii) absence of Philadelphia chromosome or BCR-ABL rearrangement in peripheral blood cells. The optional criteria include (i) splenomegaly of any grade, (ii) anisopoikilocytosis with tear-drop erythrocytes, (iii) presence of circulating immature myeloid cells, (iv) presence of circulating erythroblasts, (v) presence of cluster of megakaryoblasts and anomalous megakaryocytes in bone marrow sections, and (vi) myeloid metaplasia. The original World Health Organization (WHO) criteria include clinical finding and morphological finding [1]. The clinical presentations are splenomegaly and hepatomegaly, moderate to marked anemia, and normal, decreased or increased leukocytes and platelets. Morphological manifestations in the peripheral blood include leukoerythroblastosis, prominent red blood cell poikilocytosis and prominent dacrocytosis. The bone marrow criteria are composed of reticulin and/or collagen fibrosis decreased cellularity, dilated marrow sinuses, intraluminal hematopoiesis, neutrophilic proliferation, prominent megakaryocytic proliferation, megakaryocytic atypia and new bone formation (osteosclerosis). For prefibroti primary myelofibrosis the bone marrow fibrosi may be mild or absent with marked myelopoiesis (cellular phase) (Fig. 4.9). Compared to the fibroti stage, the other clinical or morphologic changes are milder or absent.

Fig. 4.9 Bone marrow biopsy of a case of PMF in cellular phase shows a few bizarrely shaped and hyperchromatic megakaryocytes. H&E, × 60

The revised WHO criteria proposed in 2007 simplifie the diagnostic items into three major and four minor criteria [1, 3]. The major criteria include: (i) Presence of megakaryocyte proliferation and atypia, usually accompanied by either reticulin and/or collagen fibrosis or, in the absence of significan reticulin fibrosis the megakaryocyte changes must be accompanied by granulocytic proliferation and often decreased erythropoiesis (i.e. prefibroti cellular phase disease). (ii). Not meeting

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WHO criteria for polycythemia vera, chronic myelogenous leukemia, myelodysplastic syndrome or other myeloid neoplasms. (iii) Demonstration of JAK2 V617F or other clonal marker (e.g. MPL W515K/L), or, in the absence of a clonal marker, no evidence of bone marrow fibrosi due to underlying inflammator or other neoplastic diseases. The minor critera include (i) leukoerythroblastosis, (ii) increase in serum lactate dehydrogenase level, (iii) anemia, and (iv) palpablesplenomegaly. A diagnosis of PMF requires meeting all three major criteria and two minor criteria. In addition to these basic morphological changes, the patient with PMF usually has intrasinusoidal hematopoiesis, angiogenesis and osteosclerosis in the bone marrow [5,6]. The pathogenesis of myelofibrosi and other changes is considered to be the release of cytokines from the megakaryocytes and monocytes. The cytokines frequently mentioned include transforming growth factor beta, platelet derived growth factor, basic fibroblas growth factor, vascular endothelial growth factor and tissue inhibitors of matrix metalloproteinases [6]. Extramedullary hematopoiesis may occur in the liver or other organs in addition to the spleen. Clinically, patients may have anemia, hypercatabolic symptoms (profound fatigue, fever, night sweat and weight loss), portal hypertension, and splenic infarction [5]. The causes of death include leukemic transformation, infections and thrombohemorrhagic events. PMF should be distinguished from polycythemia vera (PV) and essential thrombocythemia (ET) and the latter two entities may also transform into postpolycythemic myeloid metaplasia and post-thrombocythemic myeloid metaplasia, respectively [3, 5]. The megakaryocytes in PMF show a great variation in size with an aberrant nuclear/cytoplasmic ratio and hyperchromatic, bulbous, or irregularly folded nuclei [3]. These bizarre megakaryocytes are not seen in the other two entities. In ET, there is usually no leukoerythroblastosis in the peripheral blood and no granulocyte proliferation with left-shifted forms in the bone marrow. For the polycythemic stage of PV, the increases of red cell mass, hematocrit and hemoglobin may help to distinguish it from PMF. The bone marrow also shows prominent erythroid hyperplasia, in contrast to the high M:E ratio seen in PMF. However, in the spent phase of PV, patients may have leukoerythroblastosis and dacrocytosis in the peripheral blood, hypercellular bone marrow with myelofibrosis and splenomegaly with extramedullary hematopoiesis. At this stage, it is essentially indistinguishable from PMF [5]. PMF should also be differentiated from acute panmyelosis with myelofibrosis In contrast to PMF, this entity shows pancytopenia in the peripheral blood and trilineage hyperplasia in the bone marrow. Various degrees of myelodysplasia may be present but organomegaly is not seen. Myelodysplastic syndrome with myelofibrosi should be distinguished from PMF by the presence of prominent dysplasia in one or more cell lineages. Organomegaly is usually not present. In addition to JAK2 mutation, these patients may have other karyotypic aberrations. The most common ones are del(20)(q11;q13) and del(13)(q12;q22) [6]

References 1. Thiele J, Kvasnicka HM, Tefferi A, et al. Primary myelofibrosis In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues, Lyon, France, IARC Press, 2008, 44–47. 2. Tefferi A, Vardiman JW. Classificatio and diagnosis of myeloproliferative neoplasms: The 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia 2008;22:14–22. 3. Tefferi A, Thiele J, Oraz A, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis recommendations from an ad hoc international expert panel. Blood 2007;110:1092–1097. 4. Barosi G, Ambrosetti A, Finelli C, et al. The Italian consensus conference on diagnostic criteria for myelofibrosi with myeloid metaplasia. Br J Haematol 1999;104:730–737. 5. Tefferi A. Myelofibrosi with myeloid metaplasia. N Engl J Med 2000;347:1255–1265. 6. Tefferi A. Pathogenesis of myelofibrosi with myeloid metaplasia. J Clin Oncol 2005;23:8520–8530.

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Case 5 A 62-year-old man presented with transient ischemic accident with resultant speech defects and left-sided weakness lasting for less than 24 h. Blood examination showed a platelet count of 781,000/␮l with normal leukocyte count (Fig. 5.1). Hemoglobin and hematocrit were also within normal limits. The patient was started on aspirin immediately. Bone marrow biopsy was performed on the next day (Figs. 5.2, 5.3 and 5.4). Because the platelet count continued to increase to 1,158,000/␮l, hydroxyurea was added to the therapeutic regimen. The platelet count finall dropped to 257,000/␮l after two months.

Fig. 5.1 Peripheral blood smear shows thrombocytosis with platelet anisocytosis. Note two giant platelets and many small platelets with varying sizes. Wright – Giemsa, × 100

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Fig. 5.2 Bone marrow aspirate reveals a cluster of large, mature, hyperlobated megakaryocytes. Wright – Giemsa, × 40

Fig. 5.3 Bone marrow core biopsy shows a loose cluster of megakaryocytes. Two megakaryocytes have hyperlobated and deeply lobated nuclei. H&E, × 40

Case 5

Fig. 5.4 Bone marrow core biopsy shows two megakaryocytes with phagocytized lymphocytes (emperipolesis) (arrow). H&E, × 60

Differential diagnoses: reactive thrombocytosis versus essential thrombocythemia

Further Studies Peripheral blood mutation screening for JAK2V617F: positive.

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Discussion Essential thrombocythemia (ET) is a clonal myeloproliferative disorder involving primarily the megakaryocytic lineage. It was originally classifie in the category of chronic myeloproliferative disorders, but the recent discovery of the association of ET with JAK2 mutation leads to its new classificatio as a myeloproliferative neoplasm. The diagnostic criteria as formulated by the Polycythemia Vera Study Group are primarily based on the exclusion of other causes of thrombocytosis and neglect the bone marrow histology. However, the World Health Organization (WHO) scheme considers the bone marrow morphology as an integral part for the diagnosis [1–3]. The positive diagnostic criteria include marked proliferation of large, mature hyperlobated and deeply lobated megakaryocytes, forming loose clusters in the bone marrow. If dwarf megakaryocytes or highly bizarre megakaryocytes are present, chronic myelogenous leukemia and primary myelofibrosis respectively, should be considered. The peripheral blood shows marked thrombocytosis with varying sizes, but atypical forms are seldom seen. There should not be proliferation of other cell lineages without the coexistence of other complications, such as anemia. Phagocytosis of erythrocytes or leukocytes by megakaryocytes (emperipolesis) is frequently present in ET, but it is not a requirement for the diagnosis. Mild reticulin fibrosi can be seen in ET, but marked myelofibrosi or collagen fibrosi indicates the possible diagnosis of primary myelofibrosi or conditions secondary to other diseases rather than ET. Thiele et al. pointed out several distinguished features in primary myelofibrosis including collagen fibrosis splenomegaly, and leukoerythroblastosis in the peripheral blood [4]. In the bone marrow, there are prominent clusters of atypical or dysplastic megakaryocytes with marked nuclear-cytoplasmic deviation. It is also important to exclude polycythemia vera (PV), especially in cases with JAK2 mutation. PV may have marked thrombocytosis but graulocytic proliferation may also be present. The major distinguished features in PV are the increases of red cell mass, hemoglobin and hematocrit levels as well as the absence or decrease of iron stores in the bone marrow. Chronic myelogenous leukemia should be routinely excluded by testing BCR-ABL 1 fusion gene with fluorescenc in situ hybridization or karyotyping. Myelodysplastic syndrome should be distinguished from ET by the presence of dysplastic changes in one or more cell lineages. Finally, reactive thrombocytosis should be f rst ruled out by searching for inflammator reactions, neoplasms and history of splenectomy. In ET patients, there is increased sensitivity of megakaryocytes to thrombopoietin (Tpo) [5]. The plasma Tpo is either elevated or inappropriately normal in the face of thrombocytosis. The increased plasma level of Tpo is due to a clonal defect in platelet/megakaryocyte expression or down-regulation of Tpo receptor with resultant impaired binding and clearance of Tpo. In spite of the binding defect, the megakaryocyte progenitor cells are still markedly hypersensitive to the action of Tpo, causing proliferation of megakaryocytes and excess production of platelets. In approximately 50% of ET patients, the Janus kinase 2 (JAK2) gene has a somatic point mutation in exon 12, resulting in a valine-to-phenylalanine amino acid substitution at codon 617 (JAK2V617F). Normally, the Tpo binds to the Tpo receptor on the cell surface to initiate the intracellular sequence of phosphorylation and activation events leading to transcriptional activation of growth factor-responsive target genes. In patients with JAK2V617F mutation, the signal transducers and activators of the transcription (STAT) pathway are constitutively activated in the absence of binding of Epo to its receptor, thus accelerating the proliferative process [5]. Patients with JAK2 mutation have higher red blood cell counts, higher leukocyte counts, lower platelet counts and lower erythropoietin levels than those without the mutation. JAK2V617F is also associated with abdominal vein thrombosis. However, there is no difference in overall survival, myelofibroti and leukemic transformations between these two groups of patients [6]. In 1% of JAK2V617F-negative ET cases, a somatic mutation of a myeloid cytokine receptor, MPL (MPLW515L), is identifie [6]. The identificatio of these molecular markers, in combination with bone marrow histology, has made it possible to lower the platelet count threshold for ET diagnosis from 600 to 450 × 109 /L [3]. The major clinical manifestions in ET are thrombosis and bleeding. Hemorrhages characteristically involve the skin (ecchymoses and purpura) and mucous membranes (gastrointestinal and genitourinary tract bleeding, epistaxis and hemoptysis) [5]. The demonstration of altered neutrophil activation parameters and circulating platelet-leukocyte aggregates in ET cases implies a thrombogenic role for neutrophils and provides an explanation for the antithrombotic effects of hydroxyurea [7]. Thrombosis in ET occurs in arterial, venous or microcirculatory locations, but microcirculatory manifestations, such as headache, paraesthesia, erythromelalgia and transient neurologic and visual disturbances, are most common [8]. On the other hand, the bleeding diathesis in ET cases is considered to involve an acquired von Willebrand syndrome secondary to extreme thrombocytosis [7]. This syndrome is due to the enhanced proteolysis of von Willebrand factor multimer by the ADAMTS13 cleaving protease as a result of extreme thrombocytosis.

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The commonly used drugs for treatment of ET include aspirin, hydroxyurea and anagrelide (a platelet-specifi agent) or the combination of aspirin with hydroxyurea or anagrelide.

References 1. Thiele J, Kvasnicka HM, Orazi A, et al. Essential thrombocythaemia. In Swerdlow SH, Campo E, Harris NL, eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 48–50. 2. Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis Recommendations from an ad hoc international expert panel. Blood 2007;110:1092–1097. 3. Terreri A, Vardiman JW. Classificatio and diagnosis of myeloproliferative neoplasms: The 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia 2008;22:14–22. 4. Thiele J, Kvasnicka HM, Zankovich R, et al. Relevance of bone marrow features in the differential diagnosis between essential thrombocythemia and early stage idiopathic myelofibrosis Haematologica 2000;85:1126–1134. 5. Schafer AI. Molecular basis of the diagnosis and treatment of polycythemia vera and essential thrombocythemia. Blood 2006;107:4214–4222. 6. Pikman Y, Levine RL. Advances in the molecular characterization of Philadelphia-negative chronic myeloproliferative disorders. Curr Opin Oncol 2007;19:628–634. 7. Tefferi A. Essential thrombocythemia: Scientifi advances and current practice. Curr Opin Hematol 2006;13:93–98. 8. Elliott MA, Tefferi A. Thrombosis and haemorrhage in polycythaemia vera and essential thrombocythaemia. Br J Haematol 2004; 128:275–290.

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Case 6 A 61-year-old man presented with weakness, weight loss, low-grade fever and night sweats for 10 months. He was found to have a total leukocyte count of 20,400/␮l with an absolute neutrophil count of 14,400/␮l initially. His leukocyte count rose steadily. On admission, his total leukocyte count was 52,500/␮l with 36% neutrophils, 7% bands, 8% lymphocytes, 34% monocytes, 6% monoblasts, 3% myelocytes, 2% eosinophils and 4% basophils (Fig. 6.1). His hematocrit was 41.4 g/dL, hemoglobin 13.3 g/dL, mean corpuscle volume (MCV) 80 fl and platelets 511,000/␮l. Physical examination showed no lymphadenopathy and hepatosplenomegaly. A bone marrow biopsy revealed 95% cellularity with a myeloid to erythroid (M:E) ratio of 8:1 (Fig. 6.2). The aspirate showed 20% monocytes, and 14.6% monoblasts/promonocytes (Fig. 6.3). Trilineage dysplasia was demonstrated.

Fig. 6.1 Peripheral blood smear shows a few monocytes and a blast. Wright – Giemsa, × 100

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Fig. 6.2 Bone marrow biopsy reveals hypercellularity with increased monocytic cells. H&E, × 100

Fig. 6.3 Bone marrow aspirate shows mature and immature monocytes with a dysplastic monocyte (arrow). A few segmented granulocytes are also present. Wright – Giemsa, × 100

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Differential diagnoses: acute versus chronic myelomonocytic leukemia

Further Studies Cytochemical stain of bone marrow: alpha naphthyl butyrate esterase: positive for monocytes; chloroacetate esterase: positive for myeloid cells (Fig. 6.4) Immunohistochemical stain of bone marrow: CD68 (PGM-1)-positive (Fig. 6.5) Cytogenetic karyotype: 45,XY,-7[9]/46,XY[11] Fluorescence in situ hybridization for BCR-ABL fusion product: negative

Fig. 6.4 Cytospin of bone marrow aspirate reveals increased monocytes stained with alpha-naphthyl butyrate esterase (orange color) and a smaller number of myeloid cells stained with chloroacetate esterase (blue color). Combined esterase stain, × 60

Case 6

Fig. 6.5 Bone marrow biopsy shows a large number of monocytes as demonstrated by CD68 (PGM-1) stain. Immunoperoxidase, × 100

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Discussion In the 2008 World Health Organization (WHO) classification myelodysplastic/myeloproliferative diseases (MDS/MPD) are renamed myelodysplastic/myeloproliferative neoplasms (MDS/MPN), because this group of diseases represents clonal proliferation of malignant cells [1–3]. As the name implies, these diseases present with both myelodysplastic and myeloproliferative features. Some cases may be predominantly myelodysplastic while others are predominantly myeloproliferative. The new classificatio retains four subtypes of MDS/MPN: chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML) and MDS/MPN, unclassifiable CMML: This is the most common subtype in the MDS/MPN category, accounting for 3/100,000 persons annually. It is characterized by a substantial monocytosis in both the peripheral blood and the bone marrow with myelodysplastic changes in one or more myeloid lineages in the bone marrow. In the peripheral blood, the monocyte count should be >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 <1, 000/␮l of monocytes and the bone marrow must have at least 10% monocytes to be considered CMML [2]. Immature myeloid cells can be present in the peripheral blood, but they are usually <10% to be distinguished from aCML. If myelodysplasia is absent or minimal, the diagnosis of CMML can still be made when peripheral monocytosis has lasted for >3 months, other causes of monocytosis have been excluded, or clonal cytogenetic abnormality is demonstrated. CMML patients should be negative for t(9;22) or BCR-ABL 1 translocation. If eosinophilia is present, PDGFRA and PDGFRB rearrangements should be absent. If <5% of blasts (monoblasts and promonocytes) are demonstrated in the blood and <10% in the bone marrow, it is classifie as CMML-1, while demonstration of 5–19% blasts in the blood and 10–19% in the bone marrow is designated CMML-2. The M:E ratio in CMML is usually lower than that in chronic myeloid leukemia or aCML, as there are >15% of erythroid cells in the bone marrow. If eosinophils are >1,500/␮l, it is designated CMML with eosinophilia, which carries a specifi clinical syndrome with a specifi karyotype of t(5;12) in some cases. In the bone marrow, spleen, and lymph node, myelomonocytic clusters or nodules are frequently demonstrated. When the immature cell clusters are demonstrated in the intertrabecular area of bone marrow, it is called abnormal localization of immature precursors (ALIP). One unusual but characteristic findin is the demonstration in the above organs of aggregates of plasmacytoid dendritic cells, which have round nuclei, finel dispersed chromatin, inconspicuous nucleoli, and eosinophilic cytoplasm [1, 2]. The current case is characteristic of CMML-2. aCML: This subtype will be discussed under Case 7. JMML: This subtype is similar to CMML but the patients are children <14 years of age [3,4]. It has the lowest incidence in the MDS/MPN category (1.3 per million children per year). Similar to CMML, JMML has monocytosis in the blood and bone marrow and immature granulocytes in the blood. Philadelphia chromosome and BCR-ABL fusion product are absent. Unlike CMML, myelodysplastic changes are usually not prominent, and the total leukocyte count is usually higher. Blasts, including promonocytes, are <20% in the peripheral blood and bone marrow. However, myelomonocytic cells may infiltrat the skin, lung, liver, and spleen, mimicking acute leukemia. In nearly 70% of patients with JMML, the hemoglobin F level is >10% and the hemoglobin A2 level is low. MDS/MPN, unclassifiable: This subtype includes cases which show overlap features of MDS and MPN but do not meet the criteria of the above entities [5]. Flow cytometry may help to identify the monocyte with monocytic markers, including CD11b, CD11c, CD14 and CD64. The coexpression of CD56 or CD2 with the monocytic markers is characteristic of CMML [1, 2]. The demonstration of increases in CD34 and CD117 positive population may help to distinguish CMML-2 from CMML-1. Immunohistochemically, the monocyte can be demonstrated by CD68 (PGM-1) and lysozyme and the immature cells are identifie by CD34 and CD117 staining [6]. The plasmacytoid dendrictic cells are positive for CD123, CD4, CD14, CD33 (weakly), CD43, CD45RA, CD68, CD68R, and granzyme B [1,2]. Dysplastic megakaryocytes can be highlighted by CD42b and CD61 stains. For identificatio of monocytes, cytochemical stain for alpha-naphthyl butyrate esterase is still the most reliable technique. Approximately 20–40% of CMML patients show cytogenetic abnormalities, but none of them are specific The most common ones include +8, −7/del(7q) and structure abnormalities of 12p [1,2]. In JMML cases, the most common aberration are demonstrated in chromosome 7 [3, 4]. Monosomy 7, deletion of 7q and other chromosome 7 abnormalities occur in

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approximately 25–30% of cases. Point mutations of RAS gene are demonstrated in both CMML and JMML cases, and probably play an important role in the pathogenesis in both diseases.

References 1. Orazi A, Bennett JM, Germing U, et al. Chronic myelomonocytic leukemia. In Swerdlow SH, Campo E, Harris NL, et al. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed., Lyon, France, IARC Press, 2008, 76–79. 2. Orazi A, Germing U. The myelodysplastic/myeloproliferative neoplasms: myeloproliferative deseases with dysplastic features. Leukemia 2008;22:1308–1319. 3. Emanuel PD. Juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia. Leukemia 2008;22:1335–1342. 4. Baumann I, Bennett JM, Niemeyer CM, et al. Juvenile myelomonocytic leukemia, In Swerdlow SH, Campo E, Harris NL, et al. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed., Lyon, France, IARC Press, 2008, 82–84. 5. Vardiman JW, Bennett JM, Bain BJ, et al. Myelodysplastic/myeloproliferative neoplasms, unclassifiable In Swerdlow SH, Campo E, Harris NL, et al. WHO Classificatio of Tumours of Haematopoietic and Lymphoid tissues, 4th ed., Lyon, France, IARC Press, 2008, 85–86. 6. Ngo NT, Lampert IA, Naresh KN. Bone marrow trephine morphology and immunohistochemical finding in chronic myelomonocytic leukaemia. Br J Haematol 2008;141:771–781.

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Case 7 A 59-year-old man was found to have a high leukocyte count when he had a tooth extraction. He also noticed a new bruise on his left knee and an occasional nosebleed in the past month. Peripheral blood examination revealed a total leukocyte count of 118,000/␮l with 63% neutrophils, 11% bands, 6% lymphocytes, 4% monocytes, 1% eosinophils, 1% basophils, 5% metamyelocytes, 6% myelocytes, 1% promyelocytes, and 2% blasts (Fig. 7.1). His hematocrit was 40.3%, hemoglobin 13.0 g/dL, mean corpuscular volume 79.9 f , mean corpuscular hemoglobin 25.8 pg, and platelet count 45,300/␮l. Physical examination demonstrated an enlarged spleen, 4 cm below the left costal margin. There was no lymphadenopathy or hepatomegaly. A bone marrow aspirate showed a myeloid to erythroid (M:E) ratio of 9:1 with 1.7% blasts, and 3% monocytes (Fig. 7.2). The core biopsy revealed 90% cellularity with predominantly mature and immature granulocytes (Fig. 7.3). Extramedullary hematopoiesis was demonstrated in the splenectomy specimen (Fig. 7.4).

Fig. 7.1 Peripheral blood smear shows leukocytosis with several immature myeloid cells. Wright – Giemsa, × 40

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Fig. 7.2 Bone marrow aspirate reveals atypical myeloid cells (arrow). Wright – Giemsa, × 100

Fig. 7.3 Bone marrow biopsy demonstrate hypercellularity witho pure population of myelomonocytic cells without the presence of erythroid cells in this field H&E, × 60

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Fig. 7.4 Splenectomy specimen shows extramedullary hematopoiesis. A dilated sinus contains a large number of nucleated erythrocytes. A few megakaryocytes are seen in the red pulp cord (arrow). H&E, × 40

Differential diagnoses: chronic myelogenous leukemia versus atypical chronic myeloid leukemia

Further Studies Cytogenetic karyotype: 46,XY Fluorescence in situ hybridization for BCR-ABL 1 fusion product: negative Reticulin stain of bone marrow: increased reticulin fiber (Fig. 7.5).

Case 7

Fig. 7.5 Reticulin stain of bone marrow biopsy reveals marked increased of reticulin fibers × 60

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Discussion Atypical chronic myeloid leukemia (aCML) is a rare subtype of myelodysplastic/myeloproliferative neoplasms (MDS/MPN), affecting elderly patients in the sixth and seventh decades [1, 2]. aCML is similar to chronic myelogenous leukemia (CML) clinically and morphologically, but it is negative for Philadelphia chromosome and BCR-ABL 1 fusion product by definition It should also be negative for PDGFRA or PDGFRB gene rearrangement. Morphologically, the blast count is below 20% in both the peripheral blood and bone marrow. The severe dysplastic change in the granulocyte series is another hallmark to distinguish it from CML. In the peripheral blood, there is marked leukocytosis with mainly mature and immature granulocytes [1, 2]. The immature neutrophils are usually in the range of 10% to 20%, and the blasts are often below 5% but should not be higher than 20% by definition Dysgranulopoiesis is prominent and mainly manifests as hypogranular and hypolobated cells or the pseudo-Pelger – Hu¨et cells, but bizarre lobulation can also be seen. In some cases, the neutrophils and precursors may show exaggerated clumping of the nuclear chromatin. Those cases are described as having a syndrome of abnormal chromatin clumping. Besides the characteristic chromatin, there are no distinct clinical and pathologic differences between aCML and this syndrome, and the latter can be considered a variant of aCML. The absolute monocyte count can be elevated but the percentage should be below 10% to distinguish from chronic myelomonocytic leukemia (CMML). CMML cases may also have dysgranulopoiesis and immature granulocytes in the peripheral blood, but the immature cells are usually less than 10% of the total leukocytes. In contrast to CML, basophils and eosinophils are not elevated in the peripheral blood. Mild to moderate anemia is frequently present, and anisopoikilocytosis may be demonstrated. Thrombocytopenia is common but the platelet count can be normal or elevated in some cases. The bone marrow is usually hypercellular due to proliferation of granulocytes and their precursors [1, 2]. Monocytosis should be ruled out by cytochemical or immunohistochemical stains. Myeloblasts should be less than 20% and can be verifie with the help of CD34 and CD117 staining. The M:E ratio is often markedly elevated, but is not as high as that seen in CML cases. Dysgranulocytosis is prominent and the changes are similar to those observed in the peripheral blood. The number of megakaryocytes is variable, but dysplastic changes, such as micromegakaryocytes and hypolobated megakaryocytes, are frequently demonstrated. Erythrodysplasia may or may not be present, but some reports demonstrated this findin in more than one half of the cases studied. Reticulin fibrosi is often seen in the later stage of the disease. Immunophenotyping is usually not very helpful except for the exclusion of monocytosis and enumeration of blasts with fl w cytometry or immunohistochemistry [1, 2]. Aberrant karyotypes are reported in up to 80% of cases [1, 2]. The most common ones are +8, and del(20q). Other common finding are abnormalities in chromosomes 12, 13, 14, 17, and 19. About 30% of cases have acquired mutations of NRAS or KRAS. Although JAK2 mutation has been reported in rare cases of aCML, a recent report define aCML as a JAK2 v617F-negative neoplasm [3].

References 1. Vardiman JW, Bennett JM, Bain BJ, et al. Atypical chronic myeloid leukaemia, BCR-ABL 1 negative. In Swerdlow SH, Campo E, Harris NL, et al. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 80–81. 2. Orazi A, Germing U. The myelodysplastic/myeloproliferative neoplasms: myeloproliferative diseases with dysplastic features. Leuk Res 2008;22:1308–1319. 3. Fend F, Horn T, Koch I, et al. Atypical chronic myeloid leukemia as define in the WHO classificatio is a JAK2 V617F negative neoplasm. Leuk Res 2008;32:1931–1935.

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Case 8 A 56-year-old man presented with dyspnea, pallor and weakness for several months. Peripheral blood examination showed a total leukocyte count of 5,200/␮l with 74% neutrophils, 21% lymphocytes, 2% monocytes, 1% eosinophils, and 0% basophils (Fig. 8.1). His hematocrit was 28%, hemoglobin 9%, mean corpuscular volume (MCV) 82 f , mean corpuscular hemoglobin (MHC) 30 pg, and platelets 152,000/␮l. He was treated with iron pills and vitamin B12 for a few months to no avail and was admitted to the hospital for further investigation. Physical examination revealed no organomegaly. A bone marrow biopsy was performed (Figs. 8.2, 8.3 and 8.4).

Fig. 8.1 Peripheral blood smear shows anisopoikilocytosis. Wright – Giemsa, × 60

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Fig. 8.2 Bone marrow aspirate reveals erythroid predominance with several dysplastic nucleated red cells (arrow). Wright – Giemsa, × 60

Fig. 8.3 Bone marrow biopsy shows erythroid hyperplasia. H&E, × 60

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Fig. 8.4 Erythroid hyperplasia is readily appreciable in a Giemsa-stained bone marrow biopsy. × 100

Differential diagnoses: anemia of different etiology

Further Studies Flow cytometric analysis of bone marrow: CD7 0%, CD13 86%, CD14 6%, CD33 88%, CD34 35%, CD64 7%, CD117 22%, HLA-DR 65%, myeloperoxidase 52%. Iron stain of bone marrow: markedly increased iron stores with > 15% ringed sideroblasts (Fig. 8.5). Immunohistochemistry of bone marrow: CD34 stain: positive for a small number of scattered cells with no clustering

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Fig. 8.5 Bone marrow aspirate shows multiple ring sideroblasts (arrow). Iron stain, × 100

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Discussion Myelodysplastic syndromes (MDS) are a heterogenous group of disorders with ineffective hematopoiesis and myeloid dysplastic changes in the bone marrow and peripheral blood [1]. As a result, the bone marrow is usually hypercellular and the peripheral blood is cytopenic in one or more cell lineages. Hypocellular MDS can also be seen occasionally. The clinical symptoms in MDS depend on the cell lineage involved, i.e. whether the patient has anemia, neutropenia or thorombocytopenia. MDS are often seen in elderly persons, who may have normal hematopoiesis under normal conditions but may have latent age-associated defects that may lead to the development of MDS when under stress. The pathogenesis of MDS may be multifactorial, but apoptosis of myeloid cells before their maturation and release from the bone marrow may play an important role. The criteria for classificatio are based on both quantitative and qualitative changes [1, 2]. Quantitatively, the major parameters are the percentage of blasts and monocytes in the bone marrow and the peripheral blood and the percentage of ring sideroblasts among the erythrocyte precursors. Qualitatively, the criteria are the dysplastic changes seen in different cell lineages. Dysplasia mainly manifests as changes in the configuratio and lobulation of the nuclei, the size of the nuclei and of the entire cell, and cytoplasmic granularity. In the erythroid series, the most common finding are megaloblastoid changes and the presence of ring sideroblasts. The nuclear configuratio can be in a bizarre shape (e.g., budding, internuclear bridging), multilobated, fragmented, or karyorrhetic. The normoblasts may become periodic acid – Schiff (PAS)-positive. Anisocytosis and poikilocytosis are commonly seen in the peripheral blood. In the granulocytic series, the most common finding are hypolobation and hypogranularity. When a bilobed nucleus is present, these cells are referred to as pseudo-Pelger – Hu¨et cells. Hypersegmentation, hypergranularity, giant nuclei, or huge cell size are also features of myeloid dysplasia. Bizarre nuclear configuration ringed granulocytic nucleus, nuclear fragmentation, and separated nuclear lobes may also occur. In the megakaryocytic series, the most common finding are micromegakaryocytes, hypolobulation, mononucleation, and the presence of naked nuclei. The nuclei may be arranged in a bizarre pattern or in widely separated lobes. Myelodysplastic changes should be detected in more than 10% cells of the same lineage to be considered significant Conditions that may induce myelodysplastic changes, such as vitamin B12 or folate deficien y, heavy metal exposure, paroxysmal nocturnal hemoglobinuria, treatment with granulocyte growth factors, and congenital hematologic disorders, should be excluded before MDS is diagnosed. Histologic examination of core biopsy is not as helpful as aspirate in providing positive identificatio of MDS. The most distinguishing feature of MDS in tissue sections is the so-called abnormal localization of immature precursors (ALIP) [1, 2]. In normal hematopoiesis, the immature myeloid cells firs appear along the paratrabecular zone and gradually move to the intertrabecular area as they become mature. The definitio of ALIP is the presence of at least three aggregates of three or more myeloblasts and promyelocytes in the intertrabecular area (Fig. 8.6). In the World Health Organization (WHO) classificatio MDS is divided into 7 categories [1]: Refractory cytopenia with unilineage dysplasia (RCUD) [2,3]: RCUD is further divided into refractory anemia, refractory neutropenia and refractory thrombocytopenia. In refractory anemia, the anemia is usually normochromic and macrocytic, but may be normocytic. The granulocytes and platelets are generally normal but neutropenia and thrombocytopenia may occur in some patients. Blasts are seen in <1% in the peripheral blood and <5% in the bone marrow. The bone marrow is usually hypercellular with predominant erythroid precursors. Dyserythropoiesis is inevitably present. Megaloblastoid changes are frequently seen. Ring sideroblasts, if present, are <15%. Dysplastic changes in granulocytes and megakaryocytes are seldom demonstrated. In refractory neutropenia, there should be ≥10% dysplastic neutrophils present in the peripheral blood or bone marrow. Secondary neutropenia must be excluded. The erythroid and megakaryocytic lineage should show no dysplasia or <10% dysplastic cells. Refractory thrombocytopenia is characterized by the presence of ≥10% dysplastic megakaryocytes over evaluation of more than 30 megakaryocytes. The megakaryocytes may be increased or decreased. The erythroid and neutrophilic series should show <10% dysplastic cells. Refractory anemia with ring sideroblasts (RARS) [2, 4]: RARS is associated with anemia with dimorphic features; hypochromic and normochromic populations are present in the peripheral blood. The erythrocytes can be macrocytic or normocytic. Basophilic stipplings, Pappenheimer bodies, and nucleated erythrocytes are more frequently demonstrated in the peripheral blood in RARS than in other forms of MDS. The numbers of granulocytes and platelets are normal in most cases, but they may be decreased in some cases. Blasts, if present in peripheral blood, are <1%. The bone marrow is usually hypercellular with predominance of erythroid series. Dyserythropoiesis with megaloblastoid change is present in variable degrees. The major distinction between RARS and RCUD is the presence of >15% ring sideroblasts in the bone marrow.

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Fig. 8.6 Bone marrow biopsy from a case of refractory anemia with excess blasts reveals a cluster of immature myeloid cells in the intertrabecular area, representing abnormal localization of immature precursors (ALIP). Note the immature cells are large with a delicate, dispersed chromatin pattern (white arrow). H&E, × 100

Dysplastic changes are absent or mild in myeloid and megakaryocytic series. The number of blasts in the bone marrow is <5%. The current case belongs to this category. Refractory anemia with excess blasts (RAEB) [2, 5]: RAEB is define by the presence of 5–19% myeloblasts in the bone marrow and 2–19% blasts in the peripheral blood. Because the percentages of blasts in the bone marrow and peripheral blood affects the prognosis of patients, RAEB is further classifie as RAEB-1 when there are 5% to 10% blasts in the bone marrow and <10% blasts in the blood, and RAEB-2 when the bone marrow and blood show 11% to 19% blasts or when Auer rods are seen in the blasts even when the percentage is <11%. RAEB patients are inevitably anemic. The anemia is normocytic or macrocytic. Anisopoikilocytosis is frequently present together with nucleated erythrocytes in the peripheral blood. Most patients are pancytopenic with granulocytopenia and thrombocytopenia. The bone marrow is hypercellular with panmyeloid hyperplasia. Dysplasia can be demonstrated in all three cell lineages. ALIP is frequently present and erythroid and megakaryocytes, on the other hand, dislocated toward the paratrabecular area. Ring sideroblasts may also be detected. Refractory cytopenia with multilineage dysplasia (RCMD): Please refer to Case 9. MDS associated with 5q – syndrome: Please refer to Case 10. Myelodysplastic syndrome, unclassifiable (MDS-U) [6]: MDS-U is used for cases that do not satisfy the definitio of the above categories. The new WHO classificatio specifie three conditions that can be included in this category. First, patients with persistent cytopenia(s), ≤1% blasts in the blood and <5% blasts in the bone marrow, unequivocal dysplasia in <10% of cells in one or more myeloid lineages and abnormal cytogenetic karyotypes associated with MDS should be classifie as MDS-U. Secondly, patients with features of RCUD or RCMD, showing 1% blasts in the peripheral blood, should also be included in this category. The third group includes patients with unilineage dysplasia associated with pancytopenia. For pediatric patients, a separate classificatio has been recently proposed [7]. The f rst subtype is refractory cytopenia, which includes cases with anemia, neutropenia, or thrombocytopenia but with a blast count <5%. The second subtype is refractory anemia with excess blasts, which is identical to that seen in the adult population. Refractory anemia with excess blasts in transformation is retained as the third subtype of MDS in pediatric patients. The other WHO subtypes are so rare in pediatric patients that they are not included in this classification

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Immunophenotyping is not helpful for the diagnosis of MDS. An increase of CD34- and/or CD117-positive populations can be used to substantiate the morphologic diagnosis of MDS [2]. Cytogenetic abnormalities are demonstrated in 30–50% in primary MDS cases and in >80% of therapeutic-related MDS cases [1, 2]. Most of the abnormalities are numerical and more than half of the chromosomal abnormalities comprise deletions of chromosomes 5, 7, 11, 12, 13 and 20. Structural aberrations, such as inversion of chromosome 3 and translocations, also occur. Besides the 5q– syndrome, there are a few more chromosomal abnormalities that are associated with well-define morphologic clinical syndromes, such as monosomy 7 syndrome of childhood, del(17p) and inv(3)(q21–26).

References 1. Brunning RD, Orazi A, Germing U, et al. Myelodysplastic syndromes/neoplasms, overview. 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, 88–93. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 60–68. 3. Brunning RD, Hasserjian RP, Porwit A, et al. Refractory cytopenia with unilineage dysplasia. 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, 94–95. 4. Hasserjian RP, Gattermann N, Bennett JM, et al. Refractory anaemia with ring sideroblasts. 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, 96–97. 5. Orazi A, Brunning RD, Hassejian RP, et al. Refractory anaemia with excess blasts. 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, 100–101. 6. Orazi A, Brunning RD, Baumann I, et al. Myelodysplastic syndrome, unclassifiable 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, 103. 7. Hasle H. Myelodysplastic and myeloproliferative disorders in children. Curr Opin Pediatr 2007;19:1–8.

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Case 9 An 80-year-old man presented with feeling lightheaded on minimal exertion, generalized fatigue and palpitations for several months. Peripheral blood examination revealed a total leukocyte count of 2,900/␮l with 19.3% neutrophils, 72.5% lymphocytes, 5.6% monocytes, 1.6% eosinophils, and 1.0% basophils (Fig. 9.1). His hematocrit was 10.9%, hemoglobin 10.7 g/dL, mean corpuscular volume (MCV) 102 fl and platelets 60,000/␮l. Physical examination showed no lymphadenopathy and hepatosplenomegaly. A bone marrow biopsy was performed (Figs. 9.2, 9.3, 9.4 and 9.5).

Fig. 9.1 Peripheral blood smear shows pancytopenia with anisopoikilocytosis. Wright – Giemsa, × 60

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Fig. 9.2 Bone marrow aspirate reveals dysplastic erythoid cells (arrow) and three megaloblastoid normoblasts at the left upper corner. Wright – Giemsa, × 100

Fig. 9.3 Dysplastic granulocytes. a monolobated nucleus, b pseudo-Pelger – Hu¨et cell, c ringed nucleus, d hypersegmented nucleus, e hypogranularity, f hypergranularity. Wright-Giemsa, × 300

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Fig. 9.4 Bone marrow aspirate reveals two dysplastic megakaryocytes with hypolobulation and separated nucleus. Wright – Giemsa, × 100

Fig. 9.5 Bone marrow core biopsy shows hypercellularity with many hypolobated granulocytes barely recognizable. H&E, × 60

Case 9

Differential diagnosis: Myelodysplastic syndrome versus aplastic anemia

Further Studies Cytogenetic karyotyping: 45,XY,inv(2;?)(q21;?),t(3;5)(p12;q31),-6,del(7)(q22q32),add(15)(p11.2),del(22)(q12)[14]/46,XY[6]

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Discussion The current case belongs to the category of refractory cytopenia with multilineage dysplasia (RCMD). RCMD is characterized by the presence of one or more cytopenias in the peripheral blood and dysplastic changes in two or more myeloid lineages in the bone marrow. The blast count should be <1% in the peripheral blood and <5% in the bone marrow. Auer rods are not present in these cases and monocytes are not increased (<1000/␮l in the peripheral blood). The World Health Organization (WHO) recommends the following cut-offs to defin cytopenia: hemoglobin <10g/dL, absolute neutrophil count <1,800/␮l, and platelets <100,000/␮l [1]. However, these thresholds are f exible, depending on cytogenetic and morphologic findings To designate a condition as myelodysplastic syndrome, >10% of dysplastic cells in a specifi cell lineage should be demonstrated and certain numbers of cells should be counted. The minimal requirement is that 200 neutrophils and precursors, 200 erythroid precursors and 30 megakaryocytes should be evaluated. The new WHO classificatio also define a few unusual conditions that should not be considered RCMD. For instance, a case showing multilineage dysplasia with 2–4% blasts in the peripheral blood should be designated refractory anemia with excess blasts (RAEB)-1. A case showing features of multilineage dysplasia and Auer rods should be called RAEB-2. A case consistent with multilineage dysplasia but showing 1% blasts in the peripheral blood should be considered myelodysplastic syndrome, unclassifiable The bone marrow is usually hypercellular with dysplastic changes demonstrated in two or more cell lineages. The type and degree of dysplastic changes may vary from patient to patient, and no unifying morphologic feature has been established for this entity [2]. The new WHO classificatio describes the feature of cytoplasmic vacuoles in the normoblasts that are periodic acid – Schiff (PAS)-positive. It also emphasizes the specificit of micromegakaryocytes for the establishment of megakaryocytic dysplasia. A micromegakaryocyte is define as a megakaryocyte approximately the size of a promyelocyte or smaller with a nonlobated or bilobed nucleus. For neutrophils, the most common finding are hypogranulation and hypolobation, particularly the presence of the bilobed nucleus (pseudo-Pelger – Hu¨et cells). The additional morphologic features in the three cell lineage are described under Case 8 [2]. In RCMD, ring sideroblasts may be present and may be above 15%. However, in the new WHO classification the subtype of RCMD and ringed sideroblasts (RCMD-RS) is eliminated. There is no specifi immunophenotype for RCMD. The percentage of CD34- and CD117-positive cells may be increased in certain cases [2]. Approximately 50% of patients show cytogenetic abnormalities, including trisomy 8, monosomy 7, del(7q), monosomy 5, del(5q), and del(20q) as well as complex karyotype, as seen in the current case [1]. Clinically, the major feature is bone marrow failure with cytopenia of two or more myeloid cell lines.

References 1. Brunning RD, Bennett JM, Matutes E, et al. Refractory cytopenia with multilineage dysplasia. 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, 98–99. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Lippincott Williams & Wilkins, 2008.

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Case 10 A 69-year-old man presented with a perirectal lesion and was found to have severe anemia in the course of evaluation. His total leukocyte count was 4,200/␮l with normal differential. His hematocrit was 32%, hemoglobin 10.2 g/dL, MCV 103.4 f , and platelets 164,000/␮l. The peripheral blood smear showed prominent anisopoikilocytosis with many macrocytes (Figs. 10.1 and 10.2). The bone marrow aspirate revealed megaloblastoid and dysplastic changes in the erythroid series (Fig. 10.3) and hypolobated megakaryocytes (Fig. 10.4). The core biopsy demonstrated hypercellularity with clusters of hypolobated and monolobated megakaryocytes and bare pyknotic megakaryocytic nuclei (Figs. 10.5, 10.6 and 10.7).

Fig. 10.1 Peripheral blood smear shows anisopoikilocytosis with many macrocytes and schistocytes. Note a giant platelet (arrow). Wright – Giemsa, × 100

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Fig. 10.2 Periperhal blood smear shows a monolobated megakaryocyte on a background of anisopoikilocytosis. Wright – Giemsa, × 100

Fig. 10.3 Bone marrow aspirate shows predominantly erythroid cells with megaloblastoid and dysplastic changes. Wright – Giemsa, × 100

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Fig. 10.4 Bone marrow aspirate shows one monolobated and one bilobed megakaryocytes. Wright – Giemsa, × 60

Fig. 10.5 Bone marrow biopsy reveals a cluster of megakaryocytes with large eccentric, monolobated nuclei or nuclei with separate lobes. H&E, × 40

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Fig. 10.6 Bone marrow biopsy reveals a few hypolobated megakaryocytes with a bare pyknotic nucleus at the left lower corner. Giemsa, × 60

Fig. 10.7 Bone marrow biopsy shows a few hypolobated megakaryocytes and a single naked pyknotic nucleus. Periodic acid – Schiff (PAS), × 60

Case 10

Differential diagnoses: various subtypes of myelodysplastic syndromes

Further Studies Cytogenetic karyotype: deletion of chromosome 5q (Fig. 10.8).

Fig. 10.8 A karyotype shows deletion of chromosome 5q (arrow)

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Discussion Although karyotypic aberrations are found in about 50% of patients with myelodysplastic syndrome (MDS) at diagnosis, chromosome 5q31 interstitial deletions are far more common than other cytogenetic abnormalities [1]. Deletion of 5q may occur as an isolated abnormality or accompanying additional chromosome aberrations. Altogether 5q– accounts for approximately 15% abnormal karyotype in patients with MDS. 5q– can also be demonstrated in acute myeloid leukemia and chronic myeloproliferative disorders. Occasionally, 5q– may occur in acute lymphoblastic leukemia [2]. However, the World Health Organization (WHO) define a 5q– syndrome as those without accompanying cytogenetic abnormalities, and it also excludes cases of treatment-related MDS and those with more than 5% blasts in the bone marrow [3]. With this restricted definition 5q– syndrome represents a homogenous group of patients who have a median survival of 107–146 months, as compared to 45–47 months in those with additional chromosomal abnormalities [4, 5]. Due to the presence of relatively normal levels of leukocytes and platelets, patients with 5q– have a low incidence of infectious or bleeding complications [5]. Transformation to acute myeloid leukemia is rare with a frequency of about 10% [6]. The major clinical presentation in these patients is severe anemia requiring multiple blood transfusions. As a result, many patients have hemochromatosis, which can be fatal [6]. Splenomegaly has been reported in up to 20% of patients with 5q–, but its true incidence is probably lower [5]. In the peripheral blood, the most prominent feature is macrocytic anemia [3]. Anisopoikilocytosis is frequently present. Patients may also have neutropenia with normal morphology. The platelet count is usually normal or increased; platelet anisocytosis and giant platelets are often seen [7]. The bone marrow is usually hypercellular, but erythroid precursors are decreased with moderate dysplasia [7]. The most striking feature is the presence of small clusters of atypical megakaryocytes, which are large cells with hypolobated and most characteristically, monolobated nuclei [3, 6]. In 5q– syndrome, the chromosomal deletion is interstitial, of variable size, but with a predominance for large 5q13–33 deletion [6]. Most reported cases show that the deleted region is in band 5q31–32, which suggests that the inactivation of the genes in that region may play an important role in the development of the 5q– syndrome [6]. Lenalidomide has been found to be the drug of choice for the treatment of 5q– syndrome. Lenalidomid is an immunomodulatory drug with many biological properties, including the suppression of pro-inflammator cytokine production by monocytes, enhancement of T- and natural killer cell activation and inhibition of angiogenesis [1, 6]. Lenalidomide has a dramatic effect on the anemia of patients with 5q– syndrome. It can also normalize platelet counts and induce hematological and cytogenetic response. The mechanism of its effects is still unclear, but it appears to specificall target the del 5q clone [6].

References 1. Melchert M, Kale V, List A. The role of lenalidomide in the treatment of patients with chromosome 5q deletion and other myelodysplastic syndromes. Curr Opin Hematol 2007;14:123–129. 2. van den Berghe H, Michaux. 5q–, twenty-f ve years later: A synopsis. Cancer Genet Cytogenet 1997;94:1–7. 3. Hasserjian RP, Le Beau MM, List AF, et al. Myelodysplastic syndrome with isolated del(5q), In Swerdlow SH, Campo E, Harris NL, et al. eds., WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 102. 4. Giagoundidis AAN, Germing U, Aul C. Biological and prognostic significanc of chromosome 5q deletions in myeloid malignancies. Clin Cancer Res 2006;12:5–10. 5. Nimer SD. Clinical management of myelodysplastic syndromes with interstitial deletion of chromosome 5q. J Clin Oncol 2006;24:2576–2582. 6. Kelaidi C, Eclache V, Fenaux P. The role of lenalidomide in the management of myelodysplasia with del 5q. Br J Haematol 2008;140:267–278. 7. Nimer SD, Golde DW. The 5q– abnormality. Blood 1987;70:1705–1712.

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Case 11 A 15-year-old girl presented with cervical lymphadenopathy and petechiae in the upper extremities. She was treated with antibiotics to no avail. Physical examination showed mild splenomegaly. Peripheral blood examination revealed a total leukocyte count of 110,000/␮l with 30% blasts (Fig. 11.1). The hematocrit was 32% and platelet count 21,000/␮l. A bone marrow biopsy was performed (Figs. 11.2, 11.3, and 11.4).

Fig. 11.1 Peripheral blood smear shows two myeloblasts with multiple Auer rods. Wright – Giemsa, × 200

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Fig. 11.2 Bone marrow aspirate reveals a cluster of type 3 blasts with more than 20 cytoplasmic granules. Wright – Giemsa, × 100

Fig. 11.3 A composite picture of four myeloblasts containing lysosome inclusions (arrow), a condition designated pseudo-Chediak – Higashi anomaly. Wright – Giemsa, × 400

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Fig. 11.4 Bone marrow aspirate shows several myeloblasts with a prominent Golgi zone. Wright – Giemsa, × 100

Differential diagnoses: Acute lymphoblastic leukemia versus acute myeloid leukemia

Further Studies Flow cytometry: Presence of a low side-scatter, dim CD45 population showing positive CD13, CD33, HLA-DR, CD34, and CD117, but negative CD7, CD19, and terminal deoxynucleotidyl transferase. Cytogenetic karyotype: t(8;21)(q22;q22) (Fig. 11.5).

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Fig. 11.5 Cytogenetic karyotype: t(8;21)(q22;q22)

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Discussion The presence of a karyotype of t(8;21)(q22;q22) define a distinct clinicopathologic subtype of acute myeloid leukemia (AML) in the World Health Organization (WHO) classificatio [1]. This karyotype is so specifi that a diagnosis of AML can be established even when the blast count is below 20% in the bone marrow. The importance of identifying this karyotype is its association with a favorable prognosis and higher frequency of myeloid sarcoma. While AML in general is more frequently seen in elderly patients, this subgroup is mainly seen in children. Translocation (8;21) is one of the most common AML cytogenetic abnormalities, occurring in 7–8% of adult cases and 11.7% of pediatric cases. Molecular characterization has demonstrated that t(8;21) represents the fusion of the AML1 gene on chromosome 21q22 with ETO gene on chromosome 8q22 [1–4]. The AML1 gene is also called core binding factor protein alpha (CBF␣), RUNX1, and FEBP2. ETO is also called MTG8. AML1/ETO encodes a fusion transcript with a primary inhibitory role in normal hematopoietic differentiation. It regulates the expression of both AML1 target and nonAML1 target genes via its interaction with various transcription regulators. However, t(8;21) alone cannot induce leukemia; additional mutations are necessary for the development of AML. This subgroup of AML is usually associated with AML-M2 (acute myeloblastic leukemia with maturation) morphology. There are many characteristic morphologic and immunologic features that may help predict this particular karyotype [3, 4]. The most striking morphology in the blasts is the presence of the type 3 myeloblasts (with >20 cytoplasmic granules) and multiple Auer rods (although not as abundant as seen in cases of acute promyelocytic leukemia). Other features include pseudo-Chediak – Higashi anomaly (blasts with lysosome inclusions), large blasts with prominent Golgi area, salmoncolored granules and a rim of basophilic cytoplasm in myeloid cells, cells with pink, waxy cytoplasmic globules, cytoplasmic vacuoles in leukemic cells, and bone marrow eosinophilia. The immunophenotype is characterized by the presence of all myeloid markers (CD13, CD15, CD33 and myeoperoxidase) with coexistence of B-cell markers. CD19 can be demonstrated by fl w cytometry but CD79a and PAX5 protein (B-cell-specifi activator protein [BSAP]) by immunohistochemistry [1, 5]. CD56 can be demonstrated in a subset of cases; it confers an unfavorable prognosis and is associated with myeloid sarcoma [1]. CD34 is often present and helps to identify its malignant nature. C-KIT gene mutation and over-expression are found in this special subtype of AML, as well as the expression of its protein product, CD117, on leukemic cells [6].

References 1. Arder DA, Brunning RD, Le Beau MM, et al. Acute myeloid leukemia with recurrent genetic abnormalities. In Swerdlow SH, Harris NL, Campo E, eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissue. Lyon, France, IARC Press, 2008, 110–111. 2. Peterson LF, Zhang DE. The 8;21 translocation in leukemogenesis. Oncogene 2004;23:4255–4262. 3. Nucifora G, Dickstein JI, Torbenson V, et al. Correlation between cell morphology and expression of the AML1/ETO chimeric transcript in patients with acute myeloid leukemia without the t(8;21). 1994;8:1533–1538. 4. Andrieu V, Radford-Weiss I, Troussard X, et al. Molecular detection of t(8;21)/AML1-ETO in AML M1/M2: correlation with cytogenetics, morphology and immunophenotype. Br J Heamatol 1996;92:855–865. 5. Tiacci E, Pileri S, Orieth A, et al. PAX5 expression in acute leukemias: higher B-lineage specificit than CD79a and selective association with t(8;21)-acute myelogenous leukemia. Cancer Res 2004;64:7399–7404. 6. Wang XY, Zhou GB, Yin T, et al. AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: Implication in stepwise leukemogenesis and response to Gleevec. Proc Natl Acad Sci USA. 2005;102:1104–1109.

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Case 12 A 52-year-old man presented with shortness of breath, fatique, hypersomnolence and dry cough for 5 months. Physical examination revealed splenomegaly but no hepatomegaly and lymphadenopathy. Peripheral blood examination showed a total leukocyte count of 38,400/␮l with 3% neutrophils, 8% lymphocytes, 32% monocytes, 0.1% eosinophils, and 57% blasts (Fig. 12.1). Hemoglobin was 9.1 g/dL, hematocrit 28.4%, and platelets 11,400/␮l. The bone marrow aspirate showed 75% myeloblasts/monobasts, 2.75% promyelocytes, 8.0% monocytes, and 6.75% eosinophils (Fig. 12.2). The bone marrow biopsy revealed 90% cellularity with extensive infiltratio of immature myelomonocytic cells (Fig. 12.3). Eosinophils were also increased.

Fig. 12.1 Peripheral blood smear shows mature and immature myelomonocytic cells. Wright – Giemsa, × 60

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Fig. 12.2 Bone marrow aspirate reveals many immature myelomonocytic cells with eosinophilia. Note the eosinophils contain both eosinophilic and basophilic granules (arrow). Wright – Giemsa, × 100

Fig. 12.3 Bone marrow biopsy shows myelomonocytic cells with eosinophilia (arrow). H&E, × 100

Differential diagnoses: Different subtypes of acute myeloid leukemias.

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Further Studies Combined esterase staining: positive for alpha-naphthyl butyrate esterase and chloroacetate esterase in monoblasts and myeloblasts, respectively. Cytogenetic karyotyping: inversion of chromosome 16 (Fig. 12.4).

Fig. 12.4 Karyotype of bone marrow demonstrates inv(16) (arrows)

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Discussion The current case shows immature myelomonocytic cells in the peripheral blood and bone marrow, as well as >5% eosinophils in the bone marrow that fulfil the criteria of acute myelomonocytic leukemia with eosinophilia or M4Eo, as designated by the French – American – British (FAB) system [1]. The diagnostic criteria for M4Eo is similar to M4 except that more than 5% of eosinophils should be detected in bone marrow. In other words, there should be at least 20% of blasts, including myeloblasts, monoblasts and promonocytes, in the bone marrow [2]. Among the myeloid and monocytic components, the minority population should be more than 20% in the bone marrow. This subtype of acute myeloid leukemia (AML) is frequently associated with aberrations of chromosome 16, including inv(16)(p13q22), t(16;16)(p13;q22) and del(16)(q22), in order of frequency [2, 3]. M4Eo accounts for approximately 5% of all patients with AML and 20% of patients with M4 cases. However, these abnormal karyotypes can also been seen in other subtypes of AML, chronic myeloid leukemia and myelodysplastic syndromes. In approximately 10% of M4 cases with chromosome 16 abnormality may not show eosinophilia [3]. Cases with inv(16) or t(16;16) abnormality are classifie as a distinct subtype of AML in the World Health Organization (WHO) classificatio [2]. Therefore, even if there are less than 20% of blasts in those cases, a diagnosis of AML is established. Whether the eosinophils are leukemic cells in M4Eo is controversial. However, these eosinophils are definitel abnormal. The eosinophils include all maturation stages, but the abnormal granules with basophilic or purple-violet color are seen only in myelocyte and promyelocyte stages [2]. Ultrastructurally, these granules show no central crystalloids, which are the characterstic of normal eosinophils [4]. In cytochemical stains, these eosinophils react with chloroacetate esterase and periodic acid – Schiff [4]. The basophilic granules are positive for myeloperoxidase and negative for toluidine blue; these reactions are the opposite of those seen in normal basophils [4]. The immunophenotype of M4Eo is similar to other M4 cases, except that CD2, a T-cell marker, is coexpressed with myeloid markers [2, 3]. All myeloid markers, including CD13, CD33, myeloperoxidase, and HLA-DR are positive. One or more monocytic markers (CD4, CD11b, CD11c, CD14, CD36, CD64 and lysozyme) are expressed. The stem cell markers, CD34 and CD117, are often present. Immunohistochemical stains may demonstrate myeloperoxidase, lysozyme, CD68, CD34, and CD117 [3]. As mentioned before, there are three abnormal karyotypes demonstrated in the subtype of AML. Molecular genetic studies identify the core binding factor (CBF) ␤ at 16q22, and smooth muscle myosin heavy chain (MYH) 11 gene at 16p13 [2]. Under normal condition, CBF ␤ forms a heterodemeric complex with CBF ␣, thus stabilizing its interaction with DNA, while the pathologic fusion gene alters the transcriptional regulation of normal hematopoiesis. The fusion gene can be detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and by fluorescenc in situ hybridization (FISH), but the conventional karyotyping is sometimes not sensitive enough to detect these abnormalities. RT-PCR studies demonstrated the existence of marked molecular heterogeneity in terms of breakpoint location, and eight types of fusion transcripts have been reported, with the A type being most common (88%) [5]. In comparison with the A type, the rare fusion transcripts are associated with more frequent therapy-related M4Eo, additional chromosomal rearrangements and lower white blood cell count [5]. These patients usually show less than 5% or total absence of eosinophils, so that they may be misdiagnosed as other subtypes of AML [5]. Immunophenotypically, these patients show weaker expression of CD2, CD13, CD33 and CD90 [5]. Clinical symptoms of M4Eo are similar to those seen in other M4 cases. Specifi features of AML with inv(16) include a high leukocyte count, hepatosplenomegaly, and high incidence of central nervous system involvement [3]. Myeloid sarcoma may be present at initial diagnosis or at relapse. AML with inv(16) or t(16;16) is usually associated with a good prognosis [2]. There is no clinical difference between patients with inv(16) and those with t(16;16). However the outcome of del(16q) is no better than that of other M4 cases [6]. In addition, del(16q) cases lack relapse in the central nervous system and have lower incidence of eosinophilia and M4 subtype [6].

References 1. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classificatio of acute myeloid leukemia: A report of the FrenchAmerican-British Cooperative Group. Ann Intern Med 1985;103:620–624. 2. Arber DA, Brunning RD, Le Beau MM, et al. Acute myeloid leukaemia with recurrent genetic abnormalities. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumors of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 111–112.

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3. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 81–86. 4. Brunning RD. Acute myeloid leukemia. In Knowles DM, ed. Neoplastic Hematopathology, 2nd ed., Philadelphia, Lippincott Williams & Wilkins, 2001;1667–1715. 5. Schnittger S, Bacher U, Haderlach C, et al. Rare CBFB-MYH11 fusion transcripts in AML with inv(16)?t(16;16) are associated with therapyrelated AML M4Eo, atypical cytomorphology, atypical immunophenotype, atypical additional chromosomal rearrangements and low white blood cell count: a study on 162 patients. Leukemia 2007;21:725–731. 6. Marlton P, Keating M, Kantarjian H, et al. Cytogenetic and clinical correlates in AML patients with abnormalities of chromosome 16. Leukemia 1995;9:965–971.

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Case 13 A 48-year-old man presented with a two-month history of progressive shortness of breath and fatigue, which was signifi cantly worse over the last week. Physical examination showed petechiae over his extremities. Laboratory data revealed total leukocyte count of 1,200/␮l, with neutropenia (absolute neutrophic count was below 100/␮l). His hematocrit was 20% and platelet count 15,000/␮l. Coagulation studies revealed hypofibrinogenemia elevated levels of D-dimer, and prolonged prothrombin and thrombin times. The peripheral blood smear demonstrated abnormal leukocytes (Fig. 13.1), and a bone marrow biopsy (Figs. 13.2, 13.3 and 13.4) was performed.

Fig. 13.1 Peripheral blood smear demonstrates bilobed and monocytoid leukemic cells. Wright – Giemsa, × 100

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Fig. 13.2 Bone marrow aspirate shows several bilobed promyelocytes (arrow) and other leukemic cells are monocytoid. Wright – Giemsa, × 100

Fig. 13.3 A leukemic promyelocyte contains multiple Auer rods (arrow). Wright – Giemsa, × 500

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Fig. 13.4 Bone marrow biopsy shows many promyelocytes with eosinophilic cytoplasm and a few cells reveal bilobed nuclei (arrow). H&E, × 100

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Differential diagnoses: Acute leukemias.

Further Studies Combined esterase stain of the bone marrow aspirate (Fig. 13.5). Fluorescence in situ hybridization: Presence of PML-RAR␣ fusion product in 20% of leukocytes.

Fig. 13.5 Multiple Auer rods in one leukemic cell stain positive for chloroacetate esterase (arrow). Combined esterase stain, × 100

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Discussion Acute promyelocytic leukemia (APL) accounts for only 5–13% of all cases of acute myeloid leukemia (AML), but it is the most well-define subtype of AML. It was initially considered the most malignant form of acute leukemia, because, without treatment, patients die promptly due to hemorrhages [1]. However, it has now become a curable disease, almost comparable to that of childhood acute lymphoblastic leukemia. APL is classifie as AML-M3 in the French – American – British (FAB) classification This is the only subgroup of AML in which the leukemic cells are at the promyelocytic stage instead of blastic stage as seen in other AML cases. Most cases of APL show hypergranular promyelocytes. These cells are generally larger (14–25 ␮m) than normal promyelocytes and are devoid of a prominent paranuclear Golgi region, as is seen in normal promyelocytes [2]. The most characteristic feature is the abundance of cytoplasmic granules that cover the entire cytoplasm and mask the nucleus of the leukemic cells (Fig. 13.6). The cytoplasmic granules are believed to contain myeloperoxidase, procoagulant substances, and bactericidal enzymes.

Fig. 13.6 Bone marrow aspirate from another case reveals many hypergranular promyelocytes. Wright – Giemsa, × 100

However, the above features are not diagnostic for APL unless multiple Auer rods in bundles are demonstrated in the cytoplasm. The detection of the Auer rods can be facilitated with the chloroacetate esterase stain and sometimes with the myeloperoxidase stain, but they are negative with non-specifi esterase stain [2]. In about 15–20% of APL cases, the leukemic cells contain only a few cytoplasmic granules or the granules are so small that they can only be demonstrated by electron microscopy [3]. These cases are usually referred to as hypogranular or microgranular APL, respectively, and are designated AML-M3v in the FAB classification In M3v cases, the nuclei of the leukemic cells are usually folded or bilobed, mimicking those of monocytes. The current case belongs to this category. The diagnosis of APL can be substantiated by immunophenotyping. The leukemic cells are positive for panmyeloid markers, such as CD13, CD15, CD33, and cytoplasmic myeloperoxidase [2]. For stem cell markers, CD34 is frequently negative or weakly positive, but CD117 is consistently positive although the staining can be dim in some cases [2]. The low percentage or absence of HLA-DR is most helpful in distinguishing APL from other AML subgroups because all blasts are positive for HLA-DR while promyelocytes are negative for this marker. Promyelocytosis in the recovery phase of agranulocytosis is similar to APL in morphology (Fig. 13.6) and in the presence of myeloid markers but absence of HLA-DR. However, the former usually shows negative CD117 and positive CD11b and the latter is just the opposite [4]. CD2, a T-cell marker, is frequently demonstrated in the M3v cases, but rarely in the hypergranular M3 cases [2].

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Immunohistochemical stain with the promyelocytic leukemia (PML) gene product is specifi for the diagnosis of APL [5]. PML protein is present in the nucleus of normal cells and is characterized by a speckled pattern, which is the presence of 5–20 nuclear particles (nuclear bodies) per nucleus. The APL cells, in contrast, show a microspeckled or microgranular pattern, which is composed of >50 granules. This phenomenon is the result of disruption of the nuclear bodies and redistribution of the protein in the APL cells. It is a useful technique, particularly for therapeutic monitoring. However, it is seldom used in developed countries because molecular genetic techniques, particularly the fluorescenc in situ hybridization (FISH) technique and reverse-transcription polymerase chain reaction (RT-PCR), are easily accessible. Molecular cytogenetic techniques should be used routinely, not only to confir the diagnosis but to determine the therapeutic strategy [6]. Approximately 99% of APL cases including M3 and M3v show t(15;17)(q22;q21) translocation, which produces a promyelocytic leukemia (PML)-retinoic acid receptor ␣ (RAR␣) or, to a lesser extent, RAR␣-PML fusion transcript. The remaining APL cases involve four partner genes translocating with RAR␣: ZBTB16 (previously known as PLZF) in t(11;17)(q23;q21), nucleophosmin (NPM) gene in t(5;17)(q23;q12), nuclear matrix-associated gene (NuMA) in t(11;17)(q13;q21) and STAT5B at.t(17;17)(q11.2;q21). Leukemic cells with the ZBTB16-RAR␣ or STAT5B-RAR␣ fusion product are resistant to all-trans-retinoic acid (ATRA) therapy [7]. A study of gene expression profilin identifie two major clusters in APL cases [8]. The f rst cluster was represented by cases with M3v morphology, high leukocyte count, bcr3 PML-RAR␣ isoform, and Flt3-ITDs. The second cluster was composed of cases with typical M3 morphology, bcr1 PML-RAR␣ isoform, leukopenia, and Flt3-WT. The Flt3 mutation plays an important role in the leukemogenesis of APL [8]. Clinically, patients usually present with leukopenia in typical M3, but leukocytosis in M3v cases. A high leukocyte count usually predicts an unfavorable prognosis. Morbidity and mortality are mainly associated with coagulopathy. Many patients die of early fatal hemorrhage, especially intracranial or intrapulmonary hemorrhage. The mechanism in hemorrhages is due to the release of procoagulant substances from the leukemic cells that activate the coagulation cascade and generate thrombin, and deplete fibrinogen clotting factors, and platelets, so that patients with APL may have disseminated intravascular coagulation, fibrinolysi and proteolysis [2]. Chemotherapy alone is usually not sufficien in the treatment of APL. Since 1985, ATRA has been used effectively for the treatment of APL [1]. The mechanism of ATRA is to induce differentiation of the leukemic cells by reversing the transcriptional repression of PML/RAR␣. Since the mid-1990s, arsenic trioxide (ATO) has been used to treat patients with refractory or relapsed APL [1]. The therapeutic mechanism of ATO is to induce apoptosis of leukemic cells in addition to the induction of their differentiation. The combination of these two agents further enhances the therapeutic effect on APL cases. Since many therapeutic regimens have been developed in Shanghai, the Chinese scientists credit the success of their therapeutic approach to the combination of Confucian philosophy and Western biomedical science [1].

References 1. Wang ZY, Chen Z. Acute promyelocytic leukemia: From highly fatal to highly curable. Blood 2008;111:2505–2515. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 86–92. 3. Golomb HM, Rowley JD, Vardiman JW, et al. “Microgranular” acute promyelocytic leukemia: A distinct clinical, ultrastructural and cytogenetic entity. Blood 1980;55:253–259. 4. Rizzatti EG, Garcia AB, Pothan H, et al. Expression of CD117 and CD11b in bone marrow can differentiate acute promyelocytic leukemia from recovering myeloid proliferations. Am J Clin Pathol 2002;118:31–37. 5. Falini B, Flenghi L, Fagioli M, et al. Immunocytochemical diagnosis of acute promyelocytic leukemia (M3) with the monoclonal antibody PG-M3 (anti-PML). Blood 1997;90:4046–4053. 6. Lo Coco F, Diverio D, Falini B, et al. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood 1999;94:417–428. 7. Arber DA, Brunning RD, Le Beau MM, et al. Acute myeloid leukemia with recurrent genetic abnormalities. In Swerdlow SH, Campo E, Harris NL, et al. eds., WHO Classificatio of Tumours of Heamatopoietic and Lymphoid Tissues. 4th ed. Lyon, France, 2008, 112–114. 8. Marasca R, Maffei R, Zucchini P, et al. Gene expression profilin of acute promyelocytic leukemia identifie two subtypes mainly associated with Flt3 mutational status. Leukemia 2006;20:103–114.

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Case 14 A 68-year-old man presented with fatigue, malaise and frequent urinary tract infections. He was diagnosed with myelodysplastic syndrome in another hospital two years previously. Chemotherapy helped improve his condition until recently, when he felt extreme weakness and had repeated episodes of epistaxis. Peripheral blood examination revealed a total leukocyte count of 35,000/␮l with 21% blasts, 62% neutrophils, 2% monocytes, 12% lymphocytes, 2% eosinophils, and 1% basophils (Fig. 14.1). His hematocrit was 21.5%, hemoglobin 7 g/dl, and platelets 10,500/␮l. Physical examination showed mild splenomegaly but the liver was not palpable and no enlarged lymph nodes were detected. Petechiae were found on his trunk and extremities. The bone marrow aspirate demonstrated 95% of myeloblasts (Fig. 14.2). The core biopsy showed 90% cellularity with almost total replacement of normal hematopoietic cells by immature myeloid cells (Fig. 14.3).

Fig. 14.1 Peripheral blood shows many myeloblasts and a few of them reveal a single Auer rod in the cytoplasm (arrows). Wright – Giemsa, × 100

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Fig. 14.2 Bone marrow aspirate reveals many myeloblasts with very few mature cells and no erythroid cells in this field Wright – Giemsa, × 100

Fig. 14.3 Bone marrow biopsy shows high cellularity with an exclusively blastic population. H&E, × 60

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Differential diagnoses: acute myeloid leukemia of various subtypes.

Further Studies Flow cytometric analysis of bone marrow: CD13 89%, CD33 92% CD14 2%, HLA-DR 85%, CD34 75%, CD117 68%, myeloperoxidase 52%. Cytochemical stains of bone marrow: Myeloperoxidase stain: positive in 6% of blasts. Combined specifi and nonspecifi esterase stain: 97% of myeloid cells stained for chloroacetate esterase and 3% for alphanaphthyl butyrate esterase (Fig. 14.4).

Fig. 14.4 Bone marrow aspirate demonstrates chloroacetate esterase staining of the myeloblasts (arrows). Combined esterase, × 40

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Discussion Acute myeloid leukemia (AML) is originally define in the French – American – British (FAB) classificatio as acute leukemia with more than 30% of myeloblasts in the bone marrow [1]. The myeloid nature should be verifie by cytochemical stains including myeloperoxidase (MPO), Sudan black B, specifi and nonspecifi esterase or by the presence of Auer rods. The new classificatio of the World Health Organization (WHO) lowers the blast count to 20%, because recent studies have indicated that patients with 20–30% blasts have a prognosis similar to that of patients with >30% [2]. When the blast count is more than 90% as in the current case, it is designated AML-M1 in the FAB classificatio and AML without maturation in the WHO classification The WHO classificatio emphasizes the importance of clinical correlation of the AML entities, particularly the correlation of prognosis. Because cytogenetic abnormalities, multilineage dysplasia, and chemotherapy and/or radiation therapy have proved to be intimately related to prognosis in AML patients, they are established as new categories in the WHO classificatio (see Table 2 in “Introduction”). In the FAB classification more than 3% of blasts should be MPO or Sudan black B positive. If the blasts are negative for MPO in an otherwise typical AML case, it is classifie as AML-M0 or AML with minimal differentiation. The peroxidase in megakaryocytes can be demonstrated only by electron microscopy, and is called platelet peroxidase. The eosinophilic peroxidase is characterized by its resistance to cyanide. The basophils are negative for peroxidase. The esterases further divide the myeloid cells into granulocytic and monocytic series. The former is positive for specifi esterase (e.g. chloroacetate esterase) and the latter for nonspecifi esterase (e.g. alpha-naphthyl butyrate esterase). As f ow cytometry has become the preferred method for cell lineage identification cytochemistry and electron microscopy are seldom used for the diagnosis of AML. However, nonspecifi esterase stain on the bone marrow or blood smears is still the best technique to identify monocytic series because of its direct morphologic correlation. The major differential diagnosis for AML is acute lymphoblastic leukemia. Myeloblasts are usually larger, with more cytoplasm, more delicate chromatin and more prominent nucleoli than lymphoblasts. Lymphoblasts have no cytoplasmic granules, but myeloblasts may or may not have granules. The agranular myeloblasts are called type 1 blasts. Myeloblasts with less than 20 cytoplasmic granules are designated type 2 blasts, and those with more than 20 granules type 3 blasts. The immunophenotype of AML can be identifie by the presence of a MPO-positive, CD13- and CD33-positive population with a high percentage of either CD34- or CD117-positive cells [2, 3]. The percentages of monocytic markers, such as CD14, CD64, CD11b, and CD11c, should be low or absent. The coexpression of CD7 with a myeloid marker is helpful in supporting the malignant nature of the myeloid population. The expression of CD2 is associated with M4Eo/inv(16), and CD19 is associated with M2/t(8;21) [3]. Terminal deoxylnucleotidyl transferase (TdT) is demonstrated in about 18% of AML cases [3]. The percentage of TdT positive cells is usually lower than that seen in acute lymphoblastic leukemia. The TdT positive AML cases are usually associated with the more immature subtypes of AML, such as AML-M0 and AML-M1. CD56 is present in various subtypes of AML and is reported to be associated with unfavorable prognosis. A recent study considered that relapse of AML is due to the outgrowth of a population of CD34 + CD38− progenitor cells, which persist after chemotherapy, while the CD34 + CD38+ cells represent a more differentiated myeloid population [4]. Clonal chromosome abnormalities can be detected in 55–78% of cases of adult AML and in 79–85% of childhood AML. Table 14.1 lists the important karyotypic aberrations and their association with the FAB classificatio [3]. According to recent studies, at least two genetic alterations (class I and class II mutations) are required for the clinical manifestation of acute leukemia [5]. The class I mutations, including FLT3, KIT, RAS family members, and loss of function of neurofibromi 1, play a role in aberrant activation of signal transduction pathways. The class II mutations lead to a halt in differentiation via interference with transcription factors or co-activators, such as CBF ␣/ETO, CBF ␤/MYH11 , PML/RAR␣, and MLL gene rearrangements. The clinical manifestation is usually due to the failure of the leukemic cells to mature and to the inhibition of normal hematopoiesis. Most patients may have anemia and/or thrombocytopenia. As a result, these patients have symptoms of fatigue, malaise, weakness, or hemorrhages. When the mature granulocytes are markedly decreased, superimposed infections are a common phenomenon.

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Table 14.1 Correlation of cytogenetic and molecular abnormalities with FAB classificatio Cytogenetic abnormalities Genes involved Inv(3)(q21q26) t(3;3)(q21;q26)

Gene activation Ribophorin1/EVI 1

FAB type M0, M1, M2, M4, M5, M6, M7 M1, M2, M4, M6

Ronphorin 1/EVI 1 Gene fusion t(1;22)(p13;q13) N-RAS/C-SIS M7 (infantile) t(6;9)(p23;q13) DEK/CAN M1, M2, M4 t(7;11)(p15;p15) HOXA9/NUP98 M2, M4 t(8;16)(p11;q13) MOZ/CBP M5b/M4 t(8;21)(q22;q22) ETO/AML1 M2 t(9;11)(p22;q23) AF9/MLL M4, M5 t(10;11)(p12;q23) AF10/MLL M4, M5 +11 ALL1/MLL M1, M2 t(11;17)(q23;q21) MLL1/AF17 M5 t(11;19)(q23;q13.1) MLL1/ELL M4, M5 t(11;19)(q23;p13.3) MLL1/ENL M4, M5 t(15;17)(q22;q11–12) PML/RAR␣ M3 inv(16)(p13q22) MYH11/CBF␤ M4Eo t(16;16)(p13;q22) MYH11/CBF␤ M4Eo t(16;21)(p11;q22) FUS/ERG M1, M2, M4, M5 EVI 1, ecotrophic viral integration site 1; N-RAS, an oncogene derived from rat sarcoma virus; C-SIS, simian sarcoma oncogene; HOXA, homeobox A; MOZ, monocytic leukemia zinc finger CBP, CREB-binding protein; ETO, eight-twenty-one; AML, acute myeloid leukemia; MLL, mixed lineage leukemia; ALL, acute lymphoblastic leukemia; PML, promyelocytic leukemia; RAR, retinoic acid receptor; MYH, smooth muscle myosin heavy chain; CBF, core binding factor.

References 1. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classificatio of acute myeloid leukemia: a report of the FrenchAmerican-British Cooperative Group. Ann Intern Med 1985;103:620–629. 2. Arber DA, Brunning RD, Orazi A, et al. Acute myeloid leukaemia, not otherwise specified In Swerdlow SH, Campo E, Harris NL, et al. JW, eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 131. 3. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippicott Williams & Wilkins, 2008. 4. Buzzai M, Licht JD. New molecular concepts and targets in acute myeloid leukemia. Curr Opin Hematol 2008;15:82–87. 5. Frankfurt O, Licht JD, Tallman MS. Molecular characterization of acute myeloid leukemia and its impact on treatment. Curr Opin Oncol 2007;19:635–649.

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Case 15 A 60-year-old man presented with fatigue, malaise and shortness of breath. A hematology workup revealed hematocrit 36%, hemoglobin 12 g/dl, and platelets 90,000/␮l. His total leukocyte count was 34,500/␮l with 15% myeloblasts, 2% promyelocytes, 1% metamyelocytes, 10% bands, 50% neutrophils, and 22% lymphocytes (Fig. 15.1). A bone marrow biopsy showed 64% myeloblasts and 2% monocytes (Figs. 15.2 and 15.3). Physical examination revealed no hepatosplenomegaly and lymphadenopathy.

Fig. 15.1 Peripheral blood smear shows many blasts with two granulocytes but without intermediate forms demonstrated (leukemic hiatus). Note one blast contains an Auer rod (arrow). Wright–Giemsa, × 100

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Fig. 15.2 Bone marrow aspirate shows several blasts and many mature myelocytic cells. Wright–Giemsa, × 100

Fig. 15.3 Bone marrow biopsy reveals high cellularity with many immature mononucleated cells. Only a few nucleated red blood cells are present. H&E, × 60

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Differential diagnoses: acute leukemias.

Further Studies Cytochemistry: Combined esterase stain: the blasts were positive for chloroacetate esterase but negative for alpha-naphthyl butyrate esterase (nonspecifi esterase) (Fig. 15.4). Flow cytometry: A low side-scatter, dim CD45 population was gated that showed high percentages of CD13, CD33, CD34, CD117, and HLA-DR, but low percentages of CD14 and CD64.

Fig. 15.4 Bone marrow aspirate shows many blasts with chloroacetate esterase stain (blue). Combined esterase stain, × 60

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Discussion This is a case of acute myeloblastic leukemia (AML) with maturation, which is the most common subtype of AML, accounting for approximately 25–45% of AML cases [1]. The French – American – British (FAB) classificatio designates this subtype as AML-M2 and requires ≥30% of myeloblasts in the bone marrow or peripheral blood [2, 3]. The World Health Organization (WHO) scheme changes the threshold to 20% myeloblasts [1]. In addition, the mature myeloid population from the segmented neutrophils to promyelocytes should be >10% in the bone marrow to distinguish it from AML without maturation (AML-M1). The monocytic components should be <20% in the bone marrow and < 5,000/␮l in the peripheral blood to exclude acute myelomonocytic leukemia (AML-M4). The blast count should include myeloblast type 1 (no cytoplasmic granules), myeloblast type 2 (<20 cytoplasmic granules), and myeloblast type 3 (>20 cytoplasmic granules). If myeloblast type 3 is more than 10%, the case should be diagnosed as AML-M2 even though the mature granulocytes are <10% [2]. Myeloblasts types 2 and 3 can be distinguished from promyelocytes by the centrally located nucleus, absence of a prominent Golgi zone, and presence of a fin chromatin pattern with more prominent nucleoli. The FAB system requires cytochemical stains to verify the cell lineage of the blasts. The myeloperoxidase stain should be positive for >3% of blasts [2, 3]. The specifi esterase stain (chloroacetate esterase) should be positive in most blasts and nonspecifi esterase stain (alpha-naphthyl butyrate esterase) should be negative. However, immunophenotyping by fl w cytometry has essentially replaced cytochemistry for cell lineage identification The major myeloid markers used for the identificatio of myeloid lineage are CD13, CD33, and myeloperoxidase, but the mature granulocyte markers CD15 and CD65 are also positive [1]. The monocyte markers commonly used are CD14 and CD64, which should be negative. HLA-DR is also included in the panel to exclude AML-M3, which shows a very low percentage or absence of HLA-DR. Two stem cell markers (CD34 and CD117) are routinely used to defin the malignant nature of the myeloid cells. CD34 is a hematopoietic progenitor antigen; therefore, a high percentage of CD34-positive cells is suggestive of leukemia or myelodysplasia. CD117 is a stem cell factor receptor, also known as c-kit. CD117 is positive in all subtypes of AML and it is negative for lymphoblasts, thus helping to distinguish AML from acute lymphoblastic leukemia [4]. Lymphoblasts, however, may express CD34. The coexpression of CD7 with a myeloid marker is also an indication of leukemia [5]. Immunohistochemistry may demonostrate myeloid markers, such as myeloperoxidase, lysozyme, and CAE (Leder stain). CD34 and CD117 may also be demonstrated by immunohistochemical stains [1], which, however, are less sensitive than fl w cytometry. The most frequent cytogenetic abnormality seen in about 46% of AML-M2 cases is t(8;21)(q22;q22), which is a separate entity in the WHO classificatio [1]. Another group of AML-M2 is associated with basophilia in the bone marrow. At least two abnormal karyotypes have been found in AML-M2: t/del(12)(p11–13) and t(6;9)(p23;q34) [3]. In these cases, the blasts are agranular, but other cells show evidence of maturation toward basophils. There are no specifi clinical manifestations for AML-M2: symptoms depend on the degree of anemia, leukopenia, leukocytosis and thrombocytopenia.

References 1. Arber DA, Brunning RD, Orazi A, et al. Acute myeloid leukaemia, 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, 131–132. 2. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classificatio of acute myeloid leukemia: A report of the FrenchAmerican-British Cooperative Group. Ann Intern Med 1985;103:620–624. 3. Second MIC Cooperative Study Group. Morphologic immunologic and cytogenetic (MIC) working classificatio of the acute myeloid leukemias. Br J Haematol 1988;68:487–494. 4. Hans CP, Finn WG, Singleton TP, et al. Usefulness of anti-CD117 in the f ow cytometric analysis of acute leukemia. Am J Clin Pathol 2002;117:301–305. 5. Kita K, Miwa H, Nakase K, et al. Clinical importance of CD7 expression in acute myelocytic leukemia. Blood 1993;81:2399–2405.

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Case 16 A 55-year-old man presented with worsening fatigue and easy bruisability for f ve weeks. Two weeks prior to admission, he complained of severe gingival pain, treated by a dentist for gingivitis with amoxicillin and clindamycin, but his symptoms did not resolve. The laboratory workup showed that he had a total leukocyte count of 186,000/␮l, hematocrit 17.4% and platelets 62,000/␮l. Microscopic examination of the peripheral blood smear revealed 30% blasts of myelomonocytic lineage (Fig. 16.1). A bone marrow biopsy was performed (Figs. 16.2 and 16.3).

Fig. 16.1 Peripheral blood smear shows immature myelomonocytic cells. Wright – Giemsa, × 100

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Fig. 16.2 Bone marrow aspirate reveals many vacuolated monoblasts and myeloblasts. Wright – Giemsa, × 100

Fig. 16.3 Bone marrow biopsy demonstrates many immature cells, including monoblasts and promonocytes with folded nuclei (arrow). H&E, × 60

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Differential diagnoses: acute myeloid leukemias.

Further Studies Flow cytometry: CD13 92%, CD33 89%, CD33/CD7 65%, CD14 20%, CD64 42%, CD34 80%, CD117 90%, HLA-DR 85%. Cytochemical stain: Combined esterase stain: positive for both chloroacetate esterase and alpha-naphthyl butyrate esterase (Fig. 16.4).

Fig. 16.4 Bone marrow aspirate shows many myeloblasts and monoblasts stain with chloroacetate esterase (blue) and alpha-naphthyl butyrate esterase (brown), respectively. Combined esterase stain, × 60

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Discussion Acute myelomonocytic leukemia is classifie as acute myeloid leukemia (AML)-M4 by the French – American – British (FAB) scheme. It accounts for 20–30% of AML cases. The FAB diagnostic criterion is the presence of 30% of blasts in the bone marrow, including type I and type II myeloblasts, monoblasts and promonocytes [1]. The World Health Organization (WHO) classificatio lowers the cutoff point of blasts in the bone marrow to 20% [2]. In the differential count, both the granulocytic and monocytic components should exceed 20%; below this threshold, the leukemia is classifie as M5 or M2, respectively. Frequently, cytochemical stains are needed to help estimate the ratio of myelocytes versus monocytes. The combined esterase stain is most useful; the monocytes are identifie by the nonspecifi esterase, the myelocytes by specifi esterase. Myeloid cells are positive for myeloperoxidase and Sudan black, whereas monocytoid cells are weakly positive or negative for the above stains. When the percentage of monocytoid cells in bone marrow is <20%, the peripheral blood should have >5, 000/␮l monocytes to meet the diagnostic criteria [1]. If the blood monocyte count is below that level, the lysozyme concentration should exceed three times the normal value in serum or urine to support the diagnosis [1]. When eosinophilia is prominent, the lysozyme level can also be elevated. In those cases, lysozyme levels should be interpreted with caution. The leukemic cells may include type II myeloblasts, which has more than 20 granules in the cytoplasm that may mimic promyelocytes. The monocytes can be distinguished from myeloid cells by their folded or lobulated nuclei, larger cell size and abundant cytoplasm. The distinction between monoblasts and promonocytes depends on the nuclear configuration the chromatin pattern and the prominence of nucleoli. Promonocytes usually have more obvious lobulation of the nuclei, more mature chromatin pattern and less conspicuous nucleoli than the monoblasts have. Immunophenotyping by fl w cytometry usually demonstrates CD13, CD33, cytoplasmic myeloperoxidase, and monocytic markers [2]. CD64 is positive for all mature and immature monocytes, My4 is present in mature monocytes and promonocytes, and Mo2 is only expressed by mature monocytes [3]. When side-scatter versus CD45 gating is used, two clusters can be demonstrated: an immature myeloid cell population and an immature monocyte population. Sometimes, a third population, mature monocytes, can also be seen. In the myeloid cluster, both stem cells markers, CD34 and CD117, are frequently present, but the stem cell markers are usually expressed in lower percentages in monocyte clusters. Immunohistochemical stains may demonstrate myeloperoxidase, lysozyme, chloroacetate esterase, CD15, and CD68 [2]. There are two clones of CD68: KP-1 is present in both myeloid and monocytoid cells, whereas PG-M1 is specifi for monocytes and/or histiocytes. The most common cytogenetic aberration in M4 cases is the translocation of 11q23 with other partner chromosomes that is seen in about 20% of M4 and M5 cases [4]. Molecular studies have identifie a human homolog of the Drosophila trithorax gene at 11q23, designated mixed-lineage leukemia (MLL) gene, since it can be demonstrated in both acute myeloid and lymphoid leukemias [4]. As in other acute myeloid leukemia, patients usually have leukocytosis, anemia and thrombocytopenia. Similar to M5, M4 cases often have gingival hyperplasia and bleeding tendency. M4 can transform from chronic myelomonocytic leukemia.

References 1. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classificatio of acute myeloid leukemia. A report of the FrenchAmerican-British Cooperative Group. Ann Intern Med 1985;103:626–629. 2. Arber DA, Brunning RD, Orazi A, et al. Acute myeloid leukemia, 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, 132–133. 3. Yang DT, Greenwood JH, Hartung L, et al. Flow cytometric analysis of different CD14 epitopes can help identify immature monocytic populations. Am J Clin Pathol 2005;124:930–936. 4. Arber DA, Brunning RD, Le Beau MM, et al. Acute myeloid leukaemia with recurrent genetic abnormalities. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 114–115.

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Case 17 A 62-year-old man presented with epistaxis and gum bleeding. Peripheral blood examination showed a total leukocyte count of 11,400/␮l with 30% monocytes, 2% promonocytes, 2% monoblasts, 49% neutrophils, 3% metamyelocytes, and 10% lymphocytes (Fig. 17.1). The bone marrow aspirate showed 3.6% monocytes, 8.4% promonocytes, 77.8% monoblasts, 2.6% myeloblasts, 0.8% promyelocytes, 0.4% myelocytes, 0% metamyelocytes, 0.2% neutrophils, 0% eosinophils, 0% basophils, 0% lymphocytes, and 2% erythroid series (Fig. 17.2). The core biopsy revealed total replacement of normal hematopoietic cells by immature monocytes (Fig. 17.3).

Fig. 17.1 Peripheral blood smear shows high percentages of mature and immature monocytoid cells. Wright – Giemsa, ×60

Case 17

Fig. 17.2 Bone marrow aspirate reveals almost exclusively immature monocytes. Wright – Giemsa, ×60

Fig. 17.3 Bone marrow core biopsy shows replacement of normal hematopoietic cells by immature monocytes. H&E, ×60

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Differential diagnoses: Acute monoblastic leukemia, acute myelomonocytic leukemia, chronic myelomonocytic leukemia.

Further Studies Flow cytometry of bone marrow: CD7 4%, CD11b 74%, CD13 82%, CD14 (Mo2) 2%, CD14 (My4) 55%, CD33 92%, CD34 11%, CD64 98%, CD117 20%, HLA-DR 70%, myeloperoxidase (MPO) 99% (Fig. 17.4). Cytochemistry of bone marrow: Myeloperoxidase stain: negative. and chloroacetate esterase were negative but Combined esterase stain: positive for alphanaphthyl butyrate esterase and negative for chloroacetate esterase (Fig. 17.5). Immunohistochemistry: CD64 (PGM-1) stain positive (Fig. 17.6) Cytogenetic karyotype: 46 XY

Fig. 17.4 Flow cytometric histograms show strongly positive reactions to CD33, CD64, and myeloperoxidase, weakly positive for CD13 and My4, but negative for Mo2 and CD34

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Fig. 17.5 Combined esterase stain reveals predominantly alpha-naphthyl butyrate esterase reaction (brown) but only a few cells show chloroacetate esterase stain. ×40

Fig. 17.6 CD64 (PGM-1) stain highlights many monocytic cells in the core biopsy. Immunoperoxidase, ×40

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Discussion Acute monoblastic/monocytic leukemia (M5) is define by the French – American – British (FAB) classificatio as more than 80% of the myelomonocytic cells in the bone marrow are of monocytic lineage and the immature monocytes (monoblasts and promonocytes) are above 30%. The World Health Organization (WHO) scheme lowers the requirement of the immature cell count to 20% [1]. When the predominant cells are monoblasts, the diagnosis is M5a, while M5b define the entity with predominant promonocytes. The FAB system depends on cytochemistry to identify monocytes, which are myeloperoxidase-positive, alpha-naphthyl butyrate esterase (nonspecifi esterase)-positive, but chloroacetate esterase (specifi esterase)-negative. However, myeloperoxidase is frequently negative and even nonspecifi esterase can be false negative. Since morphologic identificatio of monoblasts and the distinction between monoblasts and myeloblasts in the bone marrow is difficult immunophenotyping is essential to make the diagnosis. There are many monoclonal antibodies applicable to fl w cytometry, which is the preferred technique for the diagnosis. Monocytic markers that are frequently used include CD4, CD11b, CD11c, CD14 (My4, LeukM3 and Mo2), CD64, CD68, and lysozyme. The panmyeloid markers, CD13, CD33, and myeloperoxidase, are usually coexpressed with the monocyte markers. The immature monocytes can be identifie by CD117 and CD34, but both markers can be negative. Tallman et al suggested that an immunophenotype of CD14- CD11b + CD117+ identifie M5a because CD14 is usually present in mature monocytes and CD11b are present in both mature and immature monocytes [2]. Yang et al. indicated that CD64 covers the entire spectrum of monocytes, My4 identifie mature monocytes as well as promonocytes, and M02 is expressed only on mature monocytes [3]. In the current case, CD64 was 98%, My4 55%, and Mo2 2%, indicating that the bone marrow contained mainly monoblasts, with small percentage of promonocytes and essentially no mature monocytes. This pattern is consistent with AML-M5a. When bone marrow aspirate is not available, immunohistochemical staining should be performed. CD68 of the PGM-1 clone is specifi for monocytes and histiocytes, but CD68 of the KP-1 clone stains for both monocytes and myelocytes so that the latter is not helpful for differential diagnosis. Lysozyme is also positive for both myeloid and monocytic cells. The distinction between M5 and acute myelomonocytic leukemia (M4) depends on the ratio of these two populations in the bone marrow. When the monocyte:myelocyte ratio is more than 4:1, it is M5; less than that is M4. Chronic myelomonocytic leukemia (CMML) is similar to M5 in that mature and immature monocytes are increased in the peripheral blood and bone marrow. However, the percentage of immature monocytes in the bone marrow is less than 20%. In addition, dysplasia in one or more myeloid lineage should be demonstrated in CMML. Disseminated intravascular coagulation is frequently seen in M5 cases, second only to those with acute promyelocytic leukemia (M3). The serum lysozyme level is elevated in most M5 cases. Since lysozyme is nephrotoxic, many M5 patients end up with renal failure, which is one of the reasons why M5 carries a poor prognosis.

References 1. Arber DA, Brunning RD, Orazi A, et al. Acute myeloid leukaemia not otherwise categorized. 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, 133–134. 2. Tallman MS, Kim HT, Paietta E, et al. Acute monocytic leukemia (French-American-British classificatio M5) does not have a worse prognosis than other subtypes of acute myeloid leukemia: A report from the Eastern Cooperative Oncology Group. J Clin Oncol 2004;22:1276–1286. 3. Yang DT, Greenwood JH, Hartung L, et al. Flow cytometric analysis of different CD14 epitopes can help identify immature monocytic populations. Am J Clin Pathol 2005;124:930–936.

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Case 18 A 71-year-old-man had a history of lung cancer metastasized to the brain. In a follow-up visit to the clinic, the patient was found to have a leukocyte count of 20,500/␮l with 15% blasts. Two weeks later, when he was admitted to the hospital, his leukocyte count rose to 62,700/␮l with 81% blasts (Fig. 18.1). A bone marrow aspirate showed 85% monoblasts, 7% promonocytes, and 3% monocytes (Fig. 18.2). Only 5% of normal hematopoietic cells in the myeloid and erythroid cell lines were present. The core biopsy revealed 95% cellularity with most of the normal hematopoietic components replaced by immature mononucleated cells (Fig. 18.3).

Fig. 18.1 Peripheral blood smear shows a few granular monoblasts. Wright – Giemsa, × 100

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Fig. 18.2 Bone marrow aspirate reveals erythrophagocytosis in two blasts. A nucleated red cell is in the left frame and the non-nucleated red cell in the right frame (arrows). Wright – Giemsa, × 100

Fig. 18.3 Bone marrow biopsy shows hypercellularity with predominance of large monoblasts. Erythrophagocytosis is seen in one blast (arrow). H&E, × 60

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Differential diagnoses: acute myeloid leukemias.

Further Studies Flow cytometry of bone marrow: Myeloperoxidase 30%, CD13-CD33 92%, CD14 48%, CD11c 29%, HLA-DR 87%, CD34 8%, CD7 0%. Cytochemistry: Combined esterase stain: blasts stained positive for alpha-naphthyl butyrate esterase, but negative for chloroacetate esterase (Fig. 18.4) Myeloperoxidase stain: positive for blasts Cytogenetic karyotype: t(8;16)(p11.2;p13.3)

Fig. 18.4 Combined esterase stain demonstrates nonspecifi esterase-positive monoblasts in a bone marrow aspirate. Cytochemical stain, × 100

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Discussion In the current case, the patient’s bone marrow contained 95% monocytic cells with 85% monoblasts, which were verifie by the nonspecifi esterase stain. Therefore, a diagnosis of acute monoblastic leukemia was established. In addition, the monoblasts in the peripheral blood showed prominent cytoplasmic granules and the blasts in the bone marrow revealed erythrophagocytosis. These cytologic features are characteristic of a subtype of acute myeloid leukemia (AML) with the karyotype of t(8;16)(p11;p13) [1]. This subtype of AML is very rare, with only about 50 de novo AML and treatment-related AML cases reported [2]. Most cases were monocytic (M5a or M5b) or myelomonocytic (M4) leukemia. The clinical features are characterized by the frequent presence of coagulopathy, either disseminated intravascular coagulation (DIC) or primary fibrinolysis The second clinical characteristics is extramedullary involvement with an increased risk for central nervous system disease. In a series of 29 patients, 41% had hepatomegaly, 33% splenomegaly, and 37% lymphadenopathy [3]. Other organ involvement includes skin, gum, bone, central nervous system, and testicles. The prognosis of this entity is ominous with a median survival of only two months [2]. Laboratory studies of this subtype include cytochemical staining and immunophenotyping by fl w cytometry. Although most monocytic leukemia cases show weak myeloperoxidase staining, M5 cases with t(8;16) reveal strong myeloperoxidase or Sudan black staining [1]. In M5 cases, the nonspecifi esterase (alpha-naphthyl butyrate esterase) stain is positive, and in M4 cases, both nonspecifi and specifi esterase (chloroacetate esterase) are positive. In M4 cases, dual esterase staining is frequently demonstrated in the same leukemic cells, a phenomenon described as transitional myelomonocytic variant [1]. Immunophenotypically, this subtype expresses all the myelomonocytic markers, including CD4, CD11b, CD13, CD14, CD15, and CD33 [1, 4]. The presence of HLA-DR is particularly significan for exclusion of acute promyelocytic leukemia (M3). The immature cell markers, such as CD34 and CD117, are usually absent or in a low percentage. The natural killer cell-associated marker, CD56, is frequently demonstrated [4]. Molecular studies identify the gene located at 8p11 as MOZ (monocytic leukemia zinc finger and that at 16q13 as CBP (CREB-binding protein) [1, 2]. As a result of the translocation, the fusion product (protein) of these two genes may lead to leukemogenesis through three possible mechanisms: (1) the fusion product mistargets the wrong gene instead of the genes these two individual proteins are supposed to target; (2) the fusion product misregulates the targeted gene(s); and (3) the fusion product loses the normal function in directing DNA transcription [5]. Other proposed mechanisms of leukemogenesis include aberrant chromatin acetylation by the mistargeting of specifi histone acetyltransferase (HAT) activity and an inhibition of RUNX1-mediated transcription [2]. The importance of recognizing this rare subtype of AML is because of its poor prognosis and its similarity to other malignant processes. Because presence of granular monocytoid cells is seen in the hypogranular M3 variant, t(8;16) AML may be mistaken as acute promyelocytic leukemia, particularly both may show features of coagulopathy. The presence of HLA-DR may help to exclude M3, but molecular genetic studies are critical for their distinction. The presence of erythrophagocytosis in the tumor cells may also lead to the misdiagnosis of malignant histiocytosis or T-cell neoplasms.

References 1. Sun T, Wu E. Acute monoblastic leukemia with t(8;16): A distinct clinicopathologic entity; Report of a case and review of the literature. Am J Hematol 2001;66;207–212. 2. Rozman M, Camos M, Colomer D, et al. Type 1 MOZ/CBP (MYST3/CREBBP) is the most common chimeric transcript in acute myeloid leukemia with t(8;16)(p11;p13) translocation. Genes Chromosomes Cancer 2004;40:140–145. 3. Hanslip JL, Swansbury JGJ, Pinkerton R, et al. The translocation t(8;16)(p11;p13) define an AML subtype with distinct cytology and clinical features. Leuk Lymphoma 1992;6:479–486. 4. Stark B, Resnitzky P, Jeison M, et al. A distinct subtype of M4/M5 acute myeloblastic leukemia (AML) associated with t(8;16)(p11;p13): Case report and review of the literature. Leuk Res 1995;19;367–379. 5. Jacobson S, Pillus L. Modifying chromatin and concepts of cancer. Curr Opin Genet Dev 1999;9:175–184.

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Case 19 A 69-year-old man presented with low-grade fever, nausea, and malaise for 1–2 weeks. A hematology workup showed a total leukocyte count of 8,800/␮l with 86% neutrophils, 4.7% lymphocytes, 8.4% monocytes, 0.1% eosinophils, and 0.2% basophils from an automated count. However, a manual differential revealed a few immature myeloid cells and nucleated red blood cells (Fig. 19.1). His hematocrit was 19.2%, hemoglobin 6.5 g/dl, MCV 87.4 f , and platelets 34,000/␮l. Physical examination showed no hepatosplenomegaly or lymphadenopathy. A bone marrow biopsy was performed (Figs. 19.2, 19.3 and 19.4)

Fig. 19.1 Peripheral blood shows three normoblasts of different stages. Wright – Giemsa, ×100

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Fig. 19.2 Bone marrow aspirate shows predominantly erythroid components of different developmental stages. Most of the pronormoblasts reveal a vacuolated cytoplasm. A binucleated pronormoblast and a few dysplastic nuclei (arrow) are demonstrated. Wright – Giemsa, ×100

Fig. 19.3 Bone marrow biopsy shows various developmental stages of erythroid cells. H&E, ×100

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Fig. 19.4 Bone marrow biopsy with Giemsa stain facilitates the recognition of erythroid elements. A pronormoblast is indicated by a large arrow and an orthochromatophilic normoblast is indicated by a small arrow. The remaining cells are basophilic and polychromatophilic normoblasts distinguished by their size and staining intensity. Giemsa, ×100

Differential diagnoses: Acute leukemias.

Further Studies Cytochemical stains: Immunoperoxidase: positive (Fig. 19.5) Periodic acid – Schiff (PAS): positive (Fig. 19.6) Immunohistochemical stains: Glycophorin A: positive (Fig. 19.7)

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Fig. 19.5 Bone marrow aspirate with myeloperoxidase stain demonstrates the myeloid elements. The erythroid cells are negative for myeloperoxidase. ×60

Fig. 19.6 Bone marrow aspirate with PAS stain shows coarse granules in the cytoplasm of nucleated red blood cells of various stages. PAS, ×100

Case 19

Fig. 19.7 Bone marrow biopsy shows immature erythroid cells stained for glycophorin. Immunoperoxidase, ×40

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Discussion Acute erythroid leukemia (AML-M6) was originally define in the French – American – British (FAB) classificatio as the presence of at least 50% normoblasts among the total number of nucleated cells and 30% type I and type II blasts among the nonerythroid population in the bone marrow. Subsequently, it was found that the presence of a significan number of immature erythroid components with a high pronormoblast to myeloblast ratio had even worse prognosis [1]. This subset of leukemia has been accepted as AML-M6b and the original subset is thus designated AML-M6a. AML-M6b is now define as the presence of more than 80% of normoblasts with no evidence of a singnifican myeloblast component [2]. In the World Health Organization (WHO) scheme, AML-M6a is designated erythroleukemia (erythroid/myeloid) and AML-M6b is called pure erythroid leukemia [2]. The requirement for myeloblast count in erythroid/myeloid erythroleukemia is lower at 20%. In M6a, the erythroid precursors are mainly pronormoblasts and basophilic normoblasts, while M6b usually shows undifferentiated blasts requiring cytochemical or immunochemical identificatio [1, 2]. The leukemic pronormoblasts and basophilic normoblasts are highly pleomorphic, varying in size and shape. The nuclei can be polylobated, multinucleated, fragmented, or extraordinarily large. One or a few prominent nucleoli are usually present. The cytoplasm is characterized by multiple vacuolation and lack of hemoglobinization. Erythrodysplasia is an integral component in erythroleukemia, which includes megaloblastoid/megaloblastic changes, nuclear budding, nuclear bridging, and other irregular configuration of the nucleus [1, 2]. The frequent presence of erythrodysplasia in M6 is because most cases of M6 evolve through a myelodysplastic phase. M6 is frequently associated with a history of myelodysplastic syndrome, chemotherapy, or exposure to toxin or alcohol. Occasionally, it may be the result of erythroblastic transformation from chronic myeloid leukemia [3]. In those cases, Philadelphia chromosome should be present and this karyotype is more frequently seen in M6b than in M6a [4]. Before a diagnosis of erythroleukemia is made, one should rule out vitamin B12 and folate deficien y, and erythropoietin therapy. The distinction between M6 from refractory anemia with excess blasts and AML with multilineage dysplasia is sometimes difficul and should be carefully excluded. Cytochemical stain is helpful in substantiating the diagnosis of M6. Normoblasts are normally negative for PAS stain, but it may show the characteristic block-like pattern with coarse cytoplasmic granules in the leukemic normoblasts [1, 2, 5]. Myeloperoxidase, Sudan black B and chloroacetate esterase are usually negative, but weak focal staining of alpha-naphthyl butyrate esterase can be demonstrated in early normoblasts. The specifi antigens for erythroblasts are glycophorin and hemoglobin A, which can be demonstrated by immunohistochemical staining [1, 2, 5]. Only glycophorin is available for fl w cytometry. The myeloid markers (CD13, CD33, and myeloperoxidase) and the stem cell markers (CD34 and CD117) can be demonstrated in bone marrow aspirate from erythroleukemia cases, but these markers represent the myeloblasts rather than the normoblasts. There are no specifi chromosome aberrations for erythroleukemia, but clonal abnormalities were found in 76% of cases in a large study series [4]. About one half of the abnormalities had hypoploidy. Therefore, most cases showed monosomy, which frequently involved chromosomes 5 and 7. However, a complex karyotype is often demonstrated, particularly in M6b cases. One study found that 4 of 16 M6a cases and 10 of 11 M6b cases had three or more cytogenetic abnormalities [1]. The new WHO classificatio suggests that if −5/del(5q), −7/del(7q), and/or complex chromosomal abnormalities are identified those cases should be considered AML with myelodysplasia if other requirements for that category are satisfie [2].

References 1. Mazzella FM, Kowal-Vern A, Shrit A, et al. Acute erythroleukemia: Evaluation of 48 cases with reference to classification cell proliferation, cytogenetics, and prognosis. Am J Clin Pathol 1998;110:590–598. 2. Arber DA, Brunning RD, Orazi A, et al. Acute myeloid leukaemia, 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, 134–136. 3. McFarlane R, Sun T. Detection of BCR/ABL fusion product in normoblasts in a case of chronic myelogenous leukemia. Am J Surg Pathol 2004;28:1240–1244. 4. Lessard M, Strucki S, Leymarie V, et al. Cytogenetic study of 75 erythroleukemias. Cancer Genet Cytogenet 2005, 113–122. 5. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Phildelphia, Lippincott Williams & Wilkins, 2008, 116–122.

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Case 20 A 50-year-old man was evaluated for pallor and easy bruisability. Laboratory studies revealed hemoglobin 7.2 g/dl, hematocrit 21%, platelets 15,000/␮l, and leukocyte count 7,100/␮l with 62% lymphocytes and rare (<1%) blasts. Physical examination revealed petechiae, and no organomegaly or lymphadenopathy. The bone marrow aspirate was a dry tap but the bone marrow imprint showed a few blasts (Fig. 20.1). The bone marrow biopsy was suspicious for acute leukemia (Figs. 20.2 and 20.3). A second attempt finall obtained a diagnostic aspirate (Fig. 20.4).

Fig. 20.1 Bone marrow imprint shows (left) a megakaryoblasts with high nuclear/cytoplasmic ratio and immature chromatin pattern. Note multiple blebs on the surface. The right panel is an immature megakaryocyte with high nuclear/cytoplasmic ratio, but the chromatin is clumped and more mature than that of the megakaryoblast. Wright – Giemsa, × 200

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Fig. 20.2 Bone marrow aspirate reveals a large cluster of megakaryoblasts with two cell types. The small blasts show scanty cytoplasm and dense chromatin, resembling lymphoblasts. The large blasts reveal moderate amount of cytoplasm and a f ne chromatin pattern with prominent nucleoli. Cytoplasmic blebs (arrows) are demonstrated on the surface of several blasts. Wright – Giemsa, × 100

Fig. 20.3 Bone marrow biopsy shows exclusively megakaryoblasts and megakaryocytes of various developmental stages. H&E, × 60

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Fig. 20.4 Bone marrow biopsy shows a few megakaryoblasts on a myelofibroti background. H&E, × 40

Differential diagnoses: acute myeloid leukemia versus acute lymphoblastic leukemia.

Further Studies Cytochemical stains for bone marrow aspirate: Myelperoxidase staining: negative Specifi and nonspecifi esterases: negative. Periodic acid-Schiff (PAS) staining: positive (Fig. 20.5) Immunohistochemical stains for bone marrow biopsy: CD42b staining: Positive (Fig. 20.6) Terminal deoxylnucleotidyl transferase staining: negative Flow cytometry of bone marrow aspirate: CD3 10%, CD19 21%, CD10 1%, CD33 67%, CD33/CD7 64%, CD33/CD61 66%, and HLA-DR 48%. Cytogenetic karyotype: 46,XY

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Fig. 20.5 Bone marrow aspirate with PAS stain shows a peripheral staining pattern with accentuation in cytoplasmic blebs (arrow). × 100 (From Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms, Philadelphia, Lippincott Williams & Wilkins, 2008)

Fig. 20.6 Bone marrow biopsy with CD42b stain highlights several mature megakaryocytes and a few smaller megakaryoblasts. Immunoperoxidase, × 40

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Discussion Acute megakaryoblastic leukemia (AMKL) is classifie as acute myeloid leukemia-M7 (AML-M7) by the French– American–British (FAB) system, which required the presence of more than 30% of megakaryoblasts in bone marrow for the diagnosis. The World Health Organization (WHO) system lowers the minimal blast count to 20%, of which at least 50% are of megakaryocytic lineage [1]. However, bone marrow fibrosi is frequently demonstrated in AMKL cases, so that a dry tap is usually obtained and an accurate blast count is not always possible. In those cases, the diagnosis of AMKL is allowed on the estimation of the number of blasts in the bone marrow biopsy, provided that the cell lineage of megakaryocytes is unequivocally identifie by immunohistochemistry, fl w cytometry or ultrastructural cytochemistry [1]. The morphology of megakaryoblasts is highly pleomorphic. They may appear as small round cells with scanty cytoplasm and dense chromatin, resembling lymphoblasts, or as larger cells with a fin chromatin pattern and prominent nucleoli [2]. The large-cell type usually has a moderate amount of basophilic cytoplasm with or without azurophilic granules. The most specifi morphologic feature is the presence of cytoplasmic blebs (budding), which recapitulates the process of platelet shedding from the cell surface. In the bone marrow biopsy, megakaryoblasts appear to be immature mononucleated cells with marked variation in size and shape. Their presence is usually suggested by the accompanying large numbers of pleomorphic megakaryocytes and varying degrees of myelofibrosi [1, 2]. In equivocal cases, electron microscopy may help distinguish megakaryoblasts from myeloblasts by cytochemical staining for platelet peroxidase, which is localized on the nuclear membrane and the endoplasmic reticulum. The peroxidase reaction in myeloblasts is localized in the Golgi area and cytoplasmic granules. Megakaryocytes can also be identifie by Factor VIII stain. However, it is more specifi to identify by CD36, CD41, CD42 and CD61 using f ow cytometry. Since CD41 and CD61 are present on mature and immature megakaryocytes but CD42 is only present on mature ones, the demonstration by f ow cytometry of a relative low percentage of CD42 in comparison with CD41 and CD61 is indicative of the presence of mostly immature megakaryoblasts, consistent with AMKL [2]. CD42b and CD61 antibodies are also available for immunohistochemistry. Since platelets may adhere to myeloblasts or monoblasts, causing false-positive results for platelet surface markers, the demonstration of cytoplasmic rather than surface CD41, CD42, and CD61 is more reliable for the diagnosis. Megakaryoblasts also express CD13 and D33, but are negative for other myeloid markers. CD34 and CD117 may or may not be positive. Glycophorin, an erythroid maker, is also demonstrated in some cases of AMKL, causing some confusion for the diagnosis [1, 2]. Lymphoid markers are usually negative except for CD7, which is often coexpressed with a myeloid marker. CD56 is also frequently expressed on megakaryoblasts. Cytochemical stains are also helpful in differentiate AMKL from other AML subtypes. AMKL is negative for myeloperoxidase by light microscopy, and for Sudan black B. PAS stain shows a characteristic peripheral staining pattern for AMKL cells, and the staining accentuated on the cytoplasmic blebs [2]. Megakaryoblasts may show acid phosphatase and a punctuate nonspecifi esterase staining. AMKL is a common form of childhood AML accounting for 7–10% of cases, as compared to an incidence of 1% among adult AML patients [2]. The incidence of AMKL in children with Down syndrome (DS) is estimated to be approximately 500 times greater than that in children without this syndrome. Approximately 10% of DS neonates and in normal neonates with trisomy 21 mosaicism develop transient leukemia [3]. About 20–30% of patients with transient leukemia transform into overt AMKL within three years. Recently, it has been found that nearly all cases of transient leukemia or AMKL in DS patients acquire mutation in the hematopoietic transcription factor gene GATA1, which results in the exclusive production of a short GATA1 isoform named GATA1s [3, 4]. It is hypothesized that there are at least three distinct steps in DS AMKL. First, the existence of trisomy 21 in DS patients or trisomy mosaicism in normal neonates. The second step is the acquisition of a GATA1 mutation. Finally, unidentifie genetic or epigenetic events are required for progression to AMKL [4]. The possible additional genetic anomalies include p53 mutation, reactivation of telomerase, and development of trisomy 8 [3]. In the new WHO classification myeloid leukemia associated with Down syndrome is considered a separate entity from AMKL [1]. In the infantile AMKL, the most common aberration is t(1;22)(p13;q13), occurring in >65% of cases [2]. It is now known that the OTT (RBM15) and MAL (MLK1) genes are located in 1p13 and 22q13, respectively [3, 4]. The OTT-MAL transcript can be identifie with molecular biology techniques. Another oncogene, c-cis, is also located at chromosome 22q13 and encodes platelet-derived growth factor B (PDGF-B), which plays an important role in the occurrence of myelofibrosis AML with t(1;22)(p13;q13), inv(3)(q21q26.2) and t(3;3)(q21;q26.2) are all associated with AMKL, but are now considered separate entities under the new WHO classificatio [1].

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In adults, AMKL is frequently secondary to chemotherapy, to leukemic transformation of either myelofibrosi or myelodysplastic syndrome, or as megakaryoblastic crisis of chronic myeloid leukemia [2, 5]. In contrast, AMKL in chidren generally appears de novo. Hematologically, AMKL patients are usually anemic and thrombocytopenic. The leukocyte count may be low at the beginning of the disease, but an abrupt and rapid increase in the number of peripheral blasts is frequently seen in the terminal stage [2]. AMKL cases usually have a rapidly progressive clinical course; the overall survival rate varies from 4.5 to 10.4 months in different studies.

References 1. Arber DA, Brunning RD, Orazi A, et al. Acute myeloid leukaemia, not otherwise specified In Sewerdlow SH Campo E, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 136–137. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 122–129. 3. Brink DS. Transient leukemia (transient myeloproliferative disorder, transient abnormal myelopoiesis) of Down syndrome. Adv Anat Pathol 2006;13:256–262. 4. Vyas P, Crispino JD. Molecular insights into Down syndrome-associated leukemia. Curr Opin Pediatr 2007;19:9–14. 5. Dastugue N, Lafage-Pochitaloff M, Pages MP, et al. Cytogenetic profil of childhood and adult megakaryoblastic leukemia (M7): A study of the Groupe Francais de Cytog´en´etique H´ematologique (GFCH). Blood 2002;100:618–626.

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Case 21 A 20-year-old man presented with low back pain and was found to have a paraspinal mass by radiology. The diagnosis of the biopsy was not conclusive due to prominent crush artifact. The peripheral blood showed no evidence of leukemia or dysplasia, but bone marrow biopsy revealed acute myeloid leukemia. The patient received several cycles of chemotherapy for acute leukemia. Two years later, he developed inguinal lymphadenopathy. A biopsy is depicted in Figs. 21.1 and 21.2.

Fig. 21.1 Lymph node biopsy reveals tumor cells showing large nuclei with dispersed chromatin and a single prominent nucleolus. Note two eosinophilic myelocytes are in the center (arrow). H&E, × 60

Differential diagnoses: Myeloid sarcoma versus treatment-related lymphoma.

Further Studies CD45 staining: positive (Fig. 21.3) CD20 staining: negative CD3 staining: negative Lysozyme staining: positive (Fig. 21.4) Myeloperoxidase staining: positive CD43 staining: positive (Fig. 21.5) Flow cytometry of lymph node: CD45 100%, CD33 100%, CD13–CD33/CD7 95%, CD14 21%, CD34 30%, HLA-DR 95% Cytogenetic karyotype: t(8:21)(q22;q22)

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Fig. 21.2 Lymph node biopsy reveals infiltratio of perinodal tissue with a cording pattern. H&E, × 40

Fig. 21.3 CD45 stain is demonstrated in all tumor cells. Immunoperoxidase, × 20

Hematologic Neoplasms

Case 21

Fig. 21.4 Lysozyme stain is positive for tumor cells. Immunoperoxidase, × 40

Fig. 21.5 CD43 stain shows positive reaction in all tumor cells. Immunoperoxidase, × 20

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Discussion Myeloid sarcoma (MS) is a solid tumor of extramedullary myeloid cells localized in soft tissues and in bones [1]. Extramedulary myeloid leukemic infiltratio can be seen in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML) at autopsy, but if a tumor mass is not formed, it should not be considered as MS. Besides AML and CML, MS is also seen in other chronic myeloproliferative disorders and in myelodysplastic syndrome (MDS). CML has a higher incidence of MS than AML (4.5% versus 2.5%) [2]. Less than 20 MS cases have been reported with MDS. Among the AML subtypes, some reports have claimed that acute myeloid leukemia with maturation (AML-M2) and acute myelomonocytic leuekemia with eosinophilia (AML-M4eo) have the highest incidence, as these two subtypes are associated with t(8;21) and inv(16), two of the most common cytogenetic aberrations in MS [2]. Other reports have stated that monocytic and myelomonocytic leukemias are most common [2]. These associated conditions can be present before, during or after the occurrence of MS. Although t(8:21) is more commonly seen in children, the incidence of MS is higher in adults with a mean age of 43–48 [2]. Skin involvement is most common and carries an ominous prognosis [3, 4]. In pediatric patients, orbital lesion is particularly common [4]. Female patients have a predilection for the involvement of ovaries and breasts [2]. In general, soft tissues, bone and lymph nodes are the frequent sites. Morphologically, MS can be divided into four cell lineages: granulocytic, monocytic, erythroid and megakaryocytic [1]. Granulocytic sarcoma can be further divided into blastic, immature and differentiated forms. The blastic form is composed primarily of myeloblasts, the immature form, myeloblasts and promyelocytes, and the differentiated form, promyelocytes and more mature neutrophils. The presence of eosinophilic myelocytes in granulocytic sarcoma cases is frequently the only clue to the diagnosis of MS [2]. Monocytic sarcoma is secondary to granulocytic sarcoma in frequency, and is composed of monoblasts, promonocytes and monocytes in various proportions. Erythroid and megakaryocytic sarcomas are extremely rare. MS usually presents in sheets of leukemic infiltrate frequently involving adjacent tissues. In the periphery of the tumor mass, tumor cells may form strands and cords, and sometimes a targetoid pattern, vaguely reminiscent of invasive lobular breast carcinoma [2]. The tumor infiltrate by expansion, so that normal tissues, such as the glandular and tubular structures, may be separated but the overall architecture is preserved. MS may show a starry-sky pattern with a high mitotic rate, mimicking Burkitt lymphoma and lymphoblastic lymphoma [2], but most frequently it is misdiagnosed as diffuse large B-cell lymphoma. The basic immunophenotype of MS is CD45 + CD3 − CD20−. This screening panel is important because most MS cases were misdiagnosed as lymphomas. On this basis, a panel of myelomonocytic markers should be used [1–5]. If a cell lineage is not conclusive, markers of erythroid cells (phycophorin or hemoglobin A) and megakaryocytes (CD41, CD42b, CD61, Factor VIII) should be added to exclude the rare cases of erythroid or megakaryocytic MS. Many studies have found that lysozyme and CD43 are the most sensitive markers for the diagnosis of MS [5]. Myeloperoxidase and CD117 are specifi for the identificatio of MS of myeloid lineage, while CD68 (PG-M1) and CD163 are specifi for monocytoid lineage. Historically, the Leder stain for chloroacetate esterase is the firs stain used for the diagnosis of MS but it has been found to be less sensitive than the above-mentioned stains [2]. CD99 is frequently mentioned in the literature but it does not make an additional contribution to the identificatio of MS cells. CD56 is seldom positive or only partially positive for MS cells, but its presence gives an unfavorable prognosis. For immunophenotyping, most studies were based on immunohistochemistry, but f ow cytometry is far superior, because some markers can be negative or weakly positive in immunohistichemical stains, and yet strongly positive by fl w cytometry [2]. This is particularly true for immunoperoxidase-negative cases (AML-M0), as exemplifie by several comparative studies. In the case report by Amin et al. [6] immunohistochemistry showed negative staining for MPO, lysozyme, Sudan black B, specifi and nonspecifi esterase, and terminal deoxynucleotidyl transferase (TdT) with weak CD34. However, fl w cytometry revealed strong HLA-DR, CD11c, CD13, CD15, CD34 and terminal deoxynucleotidyl transferase (TdT) staining. In two large study series, cytogenetic abnormalities have been detected in 54% of adult MS cases [4] and 87% pediatric MS cases [5]. The most frequently detected aberrations in the earlier literature include t(8;21)(q22;q22), characteristic of M2, inv(16)(p13;q22) or t(16;16)(p13;q22), characteristic of M4 with eosinophilia; and t(9;11)(p21;q23), characteristic of M5. While t(8;21) is still the most common findin in pediatric cases [5], monosomy 7, trisomy 8, and mixed lineage leukemia-splitting have been found to be more common among adult patients in recent studies [4, 5].

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References 1. Pileri SA, Orazi A, Falini B. Myeloid sarcoma. In Swerdlow SH, Campo E, Harris NL, et al. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008; 140–141. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 129–135. 3. Pileri SA, Ascani S, Cox MC, et al. Myeloid sarcoma: clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients. Leukemia 2007;21:340–350. 4. Dusenbery KE, Howells WB, Arthur DC, et al. Extramedullary leukemia in children with newly diagnosed acute myeloid leukemia. A report from the children’s cancer group. J Pediatr Hematol Oncol 2003;25:760–768. 5. Alexiev BA, Wang W, Ning Y, et al. Myeloid sarcomas: a histologic, immunohistochemical, and cytogenetic study. Diagn Pathol 2007;2: 42–49. 6. Amin KS, Ehsan A, McGuff HS, et al. Minimally differentiated acute myelogenous leukemia (AML-M0) granulocytic sarcoma presenting in the oral cavity. Oral Oncol 2002;38:516–519.

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Case 22 A 54-year-old man presented with a nodular lesion on the left neck at the site of a previous arterial line placement. It was originally considered a keloid. Upon consultation, the dermatologist suspected malignancy and a skin biopsy was performed (Figs. 22.1 and 22.2). Because of the histologic pattern, peripheral blood was studied and showed a total leukocyte count of 11,600/␮l with 69% neutrophils, 10% lymphocytes and 19% monocytes including about 5% immature forms. His hemoglobin was 11.9 g/dl, hematocrit 36.9% and platelets 270,000/␮l. This raised the suspicion of leukemia and a bone marrow biopsy was performed (Figs. 22.3 and 22.4).

Fig. 22.1 Skin biopsy shows that the normal architecture is replaced by extensive tumor cell infiltration Note the lineal or cording pattern of the infiltrate H&E, × 10

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Fig. 22.2 Higher magnificatio shows the monocytoid nuclei of the tumor cells and the lineal infiltratio pattern. H&E, × 60

Fig. 22.3 Bone marrow aspiration shows a cluster of blasts with high nuclear/cytoplasmic ratio, deep blue cytoplasm with vacuolation. Intermixed with the blasts are some normoblasts of various stages. Wright – Giemsa, × 100

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Fig. 22.4 Bone marrow biopsy reveals hypercellular marrow with predominantly immature cells with irregular nuclei. H&E, × 40

Differential diagnoses: leukemia cutis, cutaneous lymphoma, and dermatitis.

Further Studies Immunohistochemistry: CD68 stain: positive (Fig. 22.5) Lysozyme stain: positive (Fig. 22.6) Myeloperoxidase (MPO): negative (Fig. 22.7) Flow cytometry of bone marrow: CD13 90%, CD33 90%, MPO 18%, CD14 (Mo2) 35%, CD14 (My4) 49%, CD64 98%, HLA-DR 77%, CD34 1% and CD117 15% Cytochemistry: Alpha-naphthyl butyrate esterase: positive for blasts

Case 22

Fig. 22.5 Skin biopsy stained for CD68 shows positive staining for all tumor cells. Immunoperoxidase, × 10

Fig. 22.6 Lysozyme stain is also positive for tumor cells. Immunoperoxidase, × 10

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Fig. 22.7 Myeloperoxidase stain is negative for tumor cells. Immunoperoxidase, × 10

Hematologic Neoplasms

Case 22

151

Discussion In the current case, the diagnosis of a malignancy was unexpected and the patient had no clinical symptoms to suggest acute leukemia. However, the lineal infiltratio pattern and the monocytic lineage of the tumor cells raised the suspicion of leukemia. The peripheral blood did not show a full-blown feature of leukemia, but because of the presence of promonocytes, a bone marrow biopsy was performed. The bone marrow showed 85% blasts, which were identifie as monocytic origin by both f ow cytometry and immunohistochemistry. On the basis of the above studies, a diagnosis of leukemia cutis of monocytic lineage was established. Clinically and pathologically, the malignant cells resemble those of cutaneous lymphoma and many cases of leukemia cutis were originally misdiagnosed as lymphomas [1]. The diagnosis is particularly difficul in cases without leukemic presentation in the peripheral blood; those cases are designated aleukemic leukemia cutis (ALC). ALC is rare but it has been reported from time to time and is not an unusual presentation. However, many cases may not be truly aleukemic. Some reports of ALC cases were based on the absence of leukemic cells in the peripheral blood without examining the bone marrow [1]. In the current case, if the bone marrow had not been examined, we should have considered it a case of ALC. Under most circumstances, leukemia cutis carries a poor prognosis. In ALC cases, acute leukemia may finall emerge in a few months and patients die promptly. Most cases of myeloid leukemia cutis develop from acute myeloid leukemia, particularly acute monoblastic leukemia or acute myelomonocytic leukemia [2]. Patients with acute monoblastic or monocytic leukemia have an incidence of 10–30% for leukemic skin infiltrat [3]. A minority of cases develop from myelodysplastic syndrome and rare cases from chronic myelomonocytic leukemia [4, 5]. Immunophenotyping and cytochemistry are most important in definin the diagnosis of leukemia cutis [1–5]. It is probably easier to perform cytochemical stains on the touch preparations or frozen sections than to do immunohistochemical stains on the paraffi sections of the skin, because myeloperoxidase, esterases and lysozyme are highly specifi for the diagnosis of leukemic infiltratio of the skin. If enough cells are obtained, fl w cytometry can be useful. However, most studies are based on immunohistochemistry because the diagnosis of leukemia cutis is often unexpected and the specimen is usually fi ed in formalin for morphologic diagnosis. As mentioned before, lymphoma should be excluded before considering leukemia cutis. The screening markers are CD45, CD20, and CD3 to rule out either B- or T-cell lymphoma. Leukemia cutis should have an immunophenotype of CD45 + CD3 − CD20−. To confir the diagnosis, the most sensitive markers are CD68 (KP1 or PG-M1), CD43, and lysozyme. Leukemia cutis of myeloid origin is also positive for myeloperoxidase, which is frequently negative for those of monocytic origin. CD34 and CD117 are usually positive for immature myeloid sarcoma but are negative for well differentiated myeloid sarcoma [6]. Monoblastic sarcoma usually shows negative CD34 [5]. CD56 expression in acute myeloid leukemia has been associated with an increased incidence of leukemia cutis [7]. The difference between leukemia cutis and cutaneous myeloid or monobalstic sarcoma is that the latter forms a tumor mass.

References 1. Gil-Mateo MP, Miquel FJ, Piris MA, et al. Aleukemic “leukemia cutis” of monocytic lineage. J Am Acad Dermatol 1997;36:837–840. 2. Cibull TL, Thomas AB, O’Malley DP, et al. Myeloid leukemia cutis: a histologic and immunohistochemical review. J Cutan Pathol 2008;35:180–185. 3. Chen L, Rodgers TR, Chaff ns ML, et al. Acute monocytic leukemia with cutaneous manifestation. Arch Pathol Lab Med 2005;129:425–426. 4. Yavorkovsky LL, Zain J, Wu CD, et al. Monocytic leukemia cutis diagnosed simultaneously with refractory anemia with monocytosis: A case report. Am J Hematol 2001;66:120–122. 5. Sepp N, Radaszkiewicz T, Meijer CJLM, et al. Specifi skin manifestations in acute leukemia with monocytic differentiation. Cancer 1993;71:124–132. 6. Alexiev BA, Wang W, Ning Y, et al. Myeloid sarcomas: a histologic, immunohistochemical, and cytogenetic study. Diagn Pathol 2007;2:1–8. 7. Kuwabara H, Nagai M, Yamaoka G, et al. Specifi skin manifestations in CD56 positive acute myeloid leukemia. J Cutan Pathol 1999;26:1–5.

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Case 23 A 67-year-old man presented with increasing weakness and shortness of breath for approximately two months. Physical examination revealed no lymphadenopathy or hepatosplenomegaly. His skin showed no petechiae or ecchymoses. Peripheral blood examination demonstrated a total leukocyte count of 16,900/␮l with 18% segmented neutrophils, 4% bands, 47% lymphocytes, 1% monocytes, and 30% blasts (Fig. 23.1). The hemoglobin was 12.2 g/dl, hematocrit 39%, and platelets 21,000/␮l. His blood chemistry panel was unremarkable except for a high level of lactate dehydrogenase (889 IU/l). A bone marrow biopsy was performed and showed almost complete replacement of the normal hematopoietic elements by blastic cells (Figs. 23.2 and 23.3).

Fig. 23.1 Peripheral blood smear shows many large and medium-sized lymphoblasts, which have a high nuclear/cytoplasmic ratio and inconspicuous nucleoli. These features are consistent with the L2 morphology as define by the FAB classification Wright-Giemsa, × 100

Case 23

Fig. 23.2 Bone marrow biopsy reveals exclusively lymphoblasts with immature chromatin pattern. H&E, × 100

Fig. 23.3 Bone marrow aspirate shows many large and medium-sized lymphoblasts. Wright-Giemsa, × 100

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Differential diagnoses: acute myelogenous leukemia versus acute lymphoblastic leukemia.

Further Study Flow cytometric analysis of bone marrow: CD7 0%, CD10 2%, CD13 4%, CD19 96%, CD33 9%, CD34 100%, CD79a 99%, HLA-DR 93%, kappa 0%, lambda 0%, cytoplasmic ␮ chain 0%, terminal deoxynucleotidyl transferase (TdT) 100%. (Fig. 23.4) Cytochemical stains: Myeloperoxidase stain: negative Nonspecifi esterase stain: negative Specifi esterase stain: negative

Fig. 23.4 Flow cytometric histograms demonstrate positive reactions to CD19, CD79a, TdT, CD34, and HLA-DR. There is no reaction to CD33, kappa, lambda, CD117, and CD7. This immunophenotype is consistent with pro-B-lymphoblastic leukemia

Case 23

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Discussion In the World Health Organization (WHO) classification B-lymphoblastic leukemia/lymphoma includes acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL); the former is considered the leukemic phase and the latter the tissue phase of the same disease [1]. However, LBL of B-cell lineage is rare. The definitio of B-ALL is that there should be more than 25% of lymphoblasts in the bone marrow. In the French – American – British (FAB) system, ALL can be divided into three subtypes, L1, L2, and L3 [2]. The leukemic cells in L1 are uniformly small with scanty cytoplasm. Their nuclei are regular in shape, with inconspicuous nucleoli. This subtype is usually seen in pediatric cases. The leukemic cells in L2 are generally large, but their size is variable, as is the cytoplasm. Their nuclei also vary in shape, with prominent nucleoli. This subtype is more frequently seen in adults than in children. The neoplastic cells in L3 are uniformly large, with moderate amounts of deep basophilic cytoplasm, which contains many vacuoles. The nuclei are round and regular with prominent nucleoli. This subtype is rare in comparison with L1 and L2 and is more frequently seen in adults. It can also be the leukemic phase of Burkitt lymphoma. In the World Health Organization (WHO) classification this subclassificatio is no longer used. Instead, B-lymphoblastic leukemia/lymphoma is classifie by a selected group of karyotypes, which are “associated with distinctive clinical or phenotypic properties, have important prognostic implications, demonstrate other evidence that they are biologically distinct and are generally mutually exclusive with other entities” [1]. The major differential diagnosis for ALL is acute myelogenous leukemia (AML). The differences between lymphoblasts and myeloblasts are listed in Table 23.1 [3]. Hematogones should also be distinguished from lymphoblasts because they may assume the L1 morphology. However, they usually have a homogeneous nuclear chromatin pattern and contain no nucleoli. Hematogones are usually seen in pediatric bone marrow. In adult patients, hematogones are seen after chemotherapy or bone marrow transplantation. Immunologically, B-cell ALL can be divided into early precursor B-ALL or pro-B-ALL, common ALL and pre-B-ALL [1]. The blasts in pro-B-ALL express CD19, cytoplasmic CD79a, cytoplasmic CD22, and nuclear TdT. The blasts in common ALL show CD10. In the pre-B ALL, the blasts express cytoplasmic ␮ chain. The WHO scheme does not include the mature B-ALL (which should be positive for surface immunoglobulin and CD10 but negative for TdT) in the immunophenotypic classification Burkitt leukemia usually shows such an immunophenotype, but the WHO system excludes Burkitt lymphoma from the B-ALL classification Hematogones also express immature markers including CD10, CD34 and TdT, but they show a spectrum of mature and immature stages and never express aberrant phenotypes [3]. Based on the immunophenotype, the current case should be classifie as pro-B-ALL. On the basis of cytogenetic findings childhood precursor B-ALL can be divided into three distinct subgroups [3]. The low risk group includes ALL cases with hyperdiploidy (> 50 chromosomes), t(12;21), and dic(9;12). The high risk group includes those cases with 11q23 translocations, t(9;22) and hypodiploidy (< 46 chromosomes). The remaining cases, including those with t(1;19), are classifie in the intermediate subgroup. There are more than 30 structural abnormalities, including translocation, deletion, inversion, isochromosome, and dicentric chromosome, known to be present in ALL; the more important Table 23.1 Differential features between acute lymphoblastic and acute myeloblastic leukemias Lymphoblastic

Myeloblastic

Size of blasts Cytoplasm Cytoplasmic granules Auer rods Nuclear chromatin Nucleoli Myelodysplastic changes Myeloperoxidase Specifi esterase Nonspecifi esterase Periodic acid – Schiff TdT CD10 Myeloid antigens Gene rearrangement

Usually large and uniform Moderate amount Frequently present Seen in about 20% of cases Delicate and dispersed 1–4, often prominent May be present Often positive Positive in myeloid leukemia Positive in monocytic leukemia Positive in about 10–15% of cases Positive in occasional cases Negative Positive Occasionally positive

Variable, depending on subtype Scant Absent Absent Coarse to f ne 0–2, less prominent Absent Negative Negative Negative Often positive Frequently positive Frequently positive Negative Frequently positive

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Table 23.2 Important chromosomal abnormalities and genes involved in B-cell acute lymphoblastic leukemia Karyotype Genes involved Approximate incidence t(9;22)(q34;q11) t(8;14)(q24;q32) t(2;8)(p12;q24) t(8;22)(q24;q11) t(1;19)(q23;p13) t(17;19)(q22;p13) t(5;14)(q31;q32) t(1;11)(p32;q23) t(4;11)(q21;q23) t(9;11)(p22;q23) t(12;21)(p13;q22)

BCR, ABL1 c-MYC, IgH c-MYC, IgK c-MYC, IgL E2A, PBX1 E2A, HLF IL3, IgH MLL, AFIP MLL, AF4 MLL, AF9 TEL, AML1

Adult 30%; children 3% 1% < 1% < 1% 5% < 1% < 1% < 1% Infants 60%; adult 5% < 1% Adults < 1%; children 20%

ones are listed in Table 23.2 [4]. In the WHO classification the following karyotypes are selected as distinct ALL entities: t(9;22)(q34;q11.2), t(v;11q23), t(12;21)(p13;q22), t(5;14)(q31;q32), and t(1;19)(q23;p13.3) [1]. In addition, hyperploid and hypoploid ALL are also considered separate entities. ALL is mainly a pediatric neoplasm with 75% of cases occur in children under six years of age [1]. The incidence of ALL in adults is about one-third that in children. The clinical symptoms of ALL are due to suppression of hematopoiesis in the bone marrow and occasionally extramedullary leukemic infiltration The current cure rate is > 95% in children and 60–85% in adults [1]. This discrepancy is partly due to the higher frequency of adverse genetic aberrations and partly due to the usually higher leukocyte count in the adult ALL population.

References 1. Borowitz MJ, Chan JKC. B lymphoblastic leukaemia/lymphoblastic lymphoma, not otherwise specified and B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoieitc and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008;168–175. 2. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classificatio of acute leukemias. Br J Haematol 1976;33:451–458. 3. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippicott Williams & Wilkins, 2008, 136–143. 4. Thandla S, Aplan PD. Molecular biology of acute lymphocytic leukemia. Semin Oncol 1997;24:45–56.

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Case 24 A 5-year-old boy presented with nose bleeding, fever and fatigue for one week. Physical examination showed petechiae over his trunk and upper extremities. His cervical and axillary lymph nodes were palpable, but his liver and spleen were not enlarged. Chest X-ray examination shows no mediastinal mass. His peripheral blood showed a total leukocyte count of 21,500/␮l with 30% blasts (Fig. 24.1). His hematocrit was 32%, hemoglobin 10 g/dl, and platelets 10,000/dl. The bone marrow biopsy demonstrated 62% blasts (Figs. 24.2 and 24.3).

Fig. 24.1 Peripheral blood smear shows several small lymphoblasts with a high nuclear/cytoplasmic ratio and inconspicuous nucleoli. These features are consistent with the L1 morphology as define by the FAB system. Wright – Giemsa, × 60

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Fig. 24.2 Bone marrow biopsy reveals total replacement of normal hematopoietic elements by small lymphoblasts. H&E, × 100

Fig. 24.3 Bone marrow aspirate demonstrates small lymphoblasts. Wright – Giemsa, × 100

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159

Differential diagnoses: Acute lymphoblastic leukemia versus acute myeloid leukemia.

Further Studies Flow cytometric analysis of bone marrow: CD2 85%, CD3 60%, CD4 88%, CD5 88%, CD7 92%, CD8 86%, CD10 0%, terminal deoxynucleotidyl transferase (TdT) 85% Cytochemical studies: Myeloperoxidase stain: negative. Nonspecifi esterase stain: negative Specifi esterase stain: negative

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Discussion T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (LBL) are considered the leukemic and tissue phases of precursor T lymphoblastic leukemia/lymphoblastic lymphoma by the World Health Organization (WHO) system [1]. T-ALL mainly involves blood and bone marrow, but it may also present with a mediastinal mass or lymphadenopthy. On the other hand, LBL usually presents with a mediastinal mass or lymphadenopathy, but it may also involve blood and bone marrow. Therefore, the arbitrary distinction of these two entities is the percentage of lymphoblasts in the bone marrow. If the blast count is over 25%, it is designated T-ALL, while blast count lower than 25% is LBL. Patients with T-ALL are usually adolescents and children with predominance in males. The major clinical presentation is leukocytosis with a high percentage of lymphoblasts. There is no cutoff of blast count in the blood and bone marrow for the diagnosis. Morphologically, there can be uniformly small lymphoblasts with scanty cytoplasm and inconspicuous nucleoli (FAB-ALL-L1 morphology), or mixed large and small lymphoblasts with convoluted nuclei and distinct nucleoli (FAB ALL-L2 morphology). ALL-L3 subtype is equivalent to Burkitt leukemia, which is exclusively of B-cell type. Immunophenotyping is most important for the diagnosis of T-ALL as it helps to defin the cell lineage, the degree of maturation and characteristic of an aberrant phenotype for the detection of minimal residual disease. The WHO scheme divides T-ALL into pro-T, pre-T, cortical T, and medullary T stages [1]. The pro-T cells are cytoplasmic CD3 (cCD3)+, CD7+, CD2−, CD1a−, CD34±, CD4−, CD8−, and TdT+. The pre-T cells are cCD3+, CD7+, CD2+, CD1a−, CD34±, CD4−, CD8−, and TdT+. The cortical T cells are cCD3+, CD7+, CD2+, CD1a+, CD34−, CD4 + . CD8+, and TdT+. The medullary T cells are cCD3+, CD7+, CD2+, CD1a−, CD34−, surface CD3+, either CD4+ or CD8+, and TdT+. In general, the more immature the stage, the worse the prognosis for a particular immunophenotype. Cytochemical stains for myeloperoxidase, specifi and nonspecifi esterases are negative for the lymphoblasts. There are many cytogenetic aberrations discovered in T-ALL and LBL, and these two entities share the same karyotypes (Table 24.1) [2]. The most important molecular abnormality is activating mutations of NOTCH1, which is encountered in more than 50% of T-ALL cases [3]. NOTCH signaling in progenitor cells drives T-cell development at the expense of B-cell development. It is hypothesized that aberrant NOTCH signaling plays an important role in the pathogenesis of precursor T-lymphoblastic leukemia/lymphoma and NOTCH inhibitors may prove to be effective for the treatment of this entity. Table 24.1 Structural changes of chromosomes associated with T-ALL Abnormality Gene involved Approximate frequency (%) t(1;14)(p32;q11) t(8:14)(q24;q11) t(10;14)(q24;q11) t(11;14)(p15;q11) t(11;14)(p13;q11) inv(14)(q11q32) t(1;7)(p33;q35) t(7;9)(q35;q34) t(7;19)(q35;p13) t(7;11)(q35;p13) t(11;19)(q23;p13)

TAL1, TCR␣␦ c-MYC, TCR␣␦ HOX11, TCR␣␦ LMO1, TCR␣␦ LMO2, TCR␣␦ TCL1, TCR␣␦ SCL, TCR␤ TAL2, TCR␤ LYL1, TCR␤ TTG2, TCR␤ MLL, ENL

1–3 2 5–10 1 5–10 <1 <1 <1 <1 <1 <1

References 1. Borowitz M, Chan JKC. T lymphoblastic leukaemia/lymphoma. 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, 178–178. 2. Thandla S, Aplan PD. Molecular biology of acute lymphocytic leukemia. Semin Oncol 1997;24:45–56. 3. Pui CH, Robison LL, Look AT. Acute lymphoblastic leukemia. Lancet 2008;371:1030–1043.

Case 25

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Case 25 A 7-year-old boy presented with dyspnea, cough and wheezing for two weeks. He was treated for asthma, with no effect. Chest X-ray examination demonstrated a large anterior mediastinal mass. Physical examination revealed facial edema and palpable cervical and axillary lymph nodes. There was no hepatosplenomegaly. Examination of the peripheral blood showed mild anemia with normal leukocyte and platelet counts. A mediastinal biopsy was obtained through mediastinoscopy (Figs. 25.1 and 25.2).

Fig. 25.1 Lymph node biopsy shows a starry sky pattern. H&E, × 40

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Fig. 25.2 Lymph node biopsy reveals tumor cells with immature chromatin, multiple tangible-body macrophages and one mitotic f gure in the center. H&E, × 100

Differential diagnoses: mediastinal Hodgkin and non-Hodgkin lymphomas.

Further Studies Immunohistochemical stain of lymph node: Terminal deoxynucleotidyl transferase (TdT): positive (Fig. 25.3) CD43 stain: positive (Fig. 25.4) CD20 stain: negative Flow cytometry analysis of lymph node: CD2 96%, CD3 98%, CD4 95%, CD5 94%, CD7 98%, CD8 95%, CD10 0%, CD19 0%, TdT 92% (Fig. 25.5)

Case 25

Fig. 25.3 Lymph node biopsy shows positive terminal deoxynucleotidyl transferase stain in most tumor cells. Immunoperoxidase, × 100

Fig. 25.4 Lymph node biopsy shows CD43-positive stain in all tumor cells. Immunoperoxidase, × 40

163

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Fig. 25.5 Flow Cytometric histograms show positive reactions to terminal deoxynucleotidyl transferase, CD79a, CD10, CD7, and CD45, but negative reactions to CD19, CD13, CD14, and CD34.

Case 25

165

Discussion Lymphoblastic lymphoma (LBL) is commonly seen in the mediastinum with characteristic histological patterns and immunophenotypes. Most of the tumor is of T-cell origin but it may be of pre-B-cell or B-cell lineage in rare cases. When it is the T-cell type, LBL should be distinguished from T-acute lymphoblastic leukemia (T-ALL). As T-ALL may also present as a mediastinal mass, and LBL may have bone marrow and blood involvement, the somewhat arbitrary distinction is based on the percentage of lymphoblasts in the bone marrow. When there are more than 25% of lymphoblasts in the bone marrow, it is designated T-ALL; when the blasts are less than 25% with the presence of a mediastinal mass or lymphadenopathy, the diagnosis becomes LBL. However, the World Health Organization (WHO) groups the two entities together and designates it T-lymphoblastic leukemia/lymphoma [1]. In other words, they are considered the leukemic and tissue phases of the same disease entity. Morphologically, T-ALL and LBL are indistinguishable. Immunologically, they can be divided into pro-T-cell, pre-T-cell, cortical T-cell, and medullary T-cell stages, as discussed under Case 24. T-ALL often presents with a more immature T-cell phenotype and LBL shows a cortical and mature T-cell phenotype in most cases. Based on these findings it is suggested that the T-ALL cells are derived from the bone marrow, while the LBL cells are from the thymus [2]. Cytogenetically, the aberrations demonstrated in the LBL cases do not differ from those seen in the T-ALL cases [2, 3]. LBL cases often express T-cell receptor (TCR) ␣␤ chain gene rearrangement, while T-ALL shows more frequently TCR␥␦ gene rearrangement [4]. However, gene expression profilin studies demonstrated clear distinction between LBL and TALL [2, 4]. The differences are probably due to the variations in the microenvironment of the bone marrow and the lymph nodes, as gene expression profilin reflect an imprint of the affected tissue and its stromal bystander cells as well as a signature due to the host immune response to the tumor [2]. For other cytogenetic aberrations, the reader is referred to Case 24. Morphologically, T-ALL and LBL are indistinguishable. In a lymph node biopsy, the major differential diagnosis is Burkitt lymphoma. The typical morphologic feature of LBL is the presence of a “starry sky” histologic pattern due to the presence of numerous tangible-body macrophages as a result of accelerated apoptosis. Mitosis is also prominent. LBL cells are intermediate in size with scanty cytoplasm. Their nuclei are usually convoluted, containing dusky chromatin and inconspicuous nucleoli. Cells from Burkitt lymphoma are of medium size with round or ovoid nuclei that contain clumped chromatin and multiple nucleoli. Tissue imprints may help to distinguish LBL and Burkitt lymphoma. LBL cells have L1/L2 morphology, while Burkitt cells show the L3 morphology [5]. Burkitt cells have deep blue cytoplasm with multiple cytoplasmic vacuoles. Their chromatin pattern is very immature and multiple nucleoli can be seen. LBL cells show pale blue cytoplasm, which usually has no vacuoles or very few vacuoles. The chromatin pattern is more mature and the nucleoli are more inconspicuous in comparison with the Burkitt cells. LBL is most frequently seen in male adolescents and accounts for one-third of childhood non-Hodgkin lymphoma [5]. Pediatric patients with a typical T-LBL usually present with a mediatinal mass, which is frequently associated with supradiaphragmatic (cervical, supraclavicular, or axillary) lymphadenopathy. The mediastinal mass may cause airway obstruction, superior vena cava syndrome, pericardial effusions, and pleural effusions. In adult patients, the presentation is frequently extramediastinal, mainly abdominal or subcutaneous lesion. The extramediastinal tumors are often associated with the preB-cell or B-cell phenotype [5]. LBL is a highly aggressive tumor; patients usually die within 1.5 years.

References 1. Borowitz M, Chan JKC. T lymphoblastic leukaemia/lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Hematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 176–178. 2. Uyttebroeck A, Vanhentenrijk V, Hagemeijer A, et al. Is there a difference in childhood T-cell acute lymphoblastic leukaemia and T-cell lymphoblastic lymphoma? Leuk Lymphoma 2007;48:1745–1754. 3. Ellison DA, Parham DM, Sawyer JR. Cytogenetic f ndings in pediatric T-lymphoblastic lymphomas: One institution’s experience and a review of the literature. Pediatr Devel Pathol 2005;8:550–556. 4. Raetz EA, Perkins SL, Bhojwani D, et al. Gene expression profilin reveals intrinsic differences between T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. Pediatr Blood Cancer 2006;47:130–140. 5. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 143–151.

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Case 26 A 56-year-old man was found to have lymphocytosis on a preemployment examination. He denied having fever, weight loss and night sweats. Physical examination showed no lymphadenopathy or hepatosplenomegaly. Peripheral blood examination revealed a total leukocyte count of 22, 100/␮l with 71% lymphocytes, 23% neutrophils, 4% monocytes, 2% eosinophils, and 0% basophils (Figs. 26.1 and 26.2). His hemoglobin was 14 g/dl, hematocrit 43%, and platelets 190, 000/␮l. A bone marrow examination revealed lymphocytic infiltratio (Figs. 26.3 and 26.4).

Fig. 26.1 Peripheral blood smear shows numerous small lymphocytes. Wright – Giemsa, ×40

Case 26

167

Fig. 26.2 Higher magnificatio of the blood smear shows the characteristic chromatin clumping (snickerdoodle-like) pattern, as compared to the snickerdoodle cookies. Wright – Giemsa, ×300

Fig. 26.3 Bone marrow aspirate reveals closely packed tumor cells. Wright – Giemsa, ×100

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Fig. 26.4 Bone marrow biopsy shows two nonparatrabecular lymphoid aggregates. H&E, ×20

Differential diagnoses: chronic lymphoid leukemias and leukemic phase of lymphoma.

Further Studies Flow cytometry of peripheral blood: CD5 98%, CD10 4%, CD19 95%, CD19/CD5 93%, CD20 93%, CD20/CD38 9%, CD23 83%, CD19/kappa 94%, CD19/lambda 6%, FMC-7 5% (Fig. 26.5) Immunohistochemistry of bone marrow: CD20 stain: positive for the lymphoid aggregates (Fig. 26.6) CD3 stain: negative for the lymphoid aggregates

Case 26

169

Fig. 26.5 Flow cytometric histograms of peripheral blood demonstrate positive reactions to CD5, CD19, CD23, and a monoclonal kappa pattern. The gated population is negative for CD10, FMC-7 and lambda

Fig. 26.6 The lymphoid aggregate in the bone marrow is reactive to CD20 staining. Immunoperoxidase, ×20

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Discussion Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are the leukemic phase and the tissue phase, respectively, of the same disease [1, 2]. When both peripheral blood/bone marrow and lymph nodes are involved, it is designated CLL/SLL, which is the most common clinical presentation. CLL is the most common leukemia in Western countries, accounting for 40% of all leukemias in adults [3]. In contrast, the incidence of CLL in Asian countries, especially India, Japan, and China, is very low. The original requirement for the diagnosis of CLL was a lymphocyte count of over 125,000/␮l in the peripheral blood. However, with the advent of immunophenotyping, the new cutoff is at the 5,000/␮l level [2]. The International Workshop on Chronic Lymphocytic Leukemia (IWCLL) also requires that the lymphocytosis should be present for at least three months and a typical immunophenotype is identifie by fl w cytometry before a diagnosis of CLL is made. When patients have less than 5,000/␮l B lymphocytes in the peripheral blood, lymphadenopathy, organomegaly, cytopenias, or disease-related symptoms should be demonstrated to substantiate the diagnosis. Otherwise, the condition should be define as monoclonal B-lymphocytosis [2]. The morphology of CLL cells is variable. In most cases, the cytology of CLL is indistinguishable from that of normal mature small lymphocytes, but CLL can also be presented as a large cell type or mixed large and small cell type. A small percentage of prolymphocytes is frequently present in CLL cases. If the percentage of prolymphocytes is between 10% and 55%, the case is designated CLL/prolymphocytic leukemia (PLL) [2, 4]. If the prolymphocytes are more than 55%, it is designated PLL. About 2% to 8% CLL cases may transform into diffuse large B-cell lymphoma; those cases are called Richter transformation [1]. In rare occasions, CLL can also transform into acute lymphoblastic leukemia. Bone marrow involvement by CLL is define at the 30% level [3]. The infiltratio pattern can be nodular, interstitial, or diffuse. A paratrabecular pattern is seldom seen in CLL. Immunophenotyping by fl w cytometry is mandatory for the diagnosis of CLL. The minimal requirements in the immunophenotype are the coexpression of CD5 with a B-cell marker (CD19 or CD20) and positive CD23 in a monoclonal B-cell population, to distinguish CLL from mantle cell lymphoma, which also shows coexpression of CD5. There is a scoring system including f ve parameters (CD5+, CD23+, weak surface immunoglobulin, FMC7−, CD79b−) for differential diagnosis [4]. Each parameter scores one point. If a case scores 3–4 points, it is considered compatible with CLL diagnosis. A 5-point score is indicative a typical case of CLL. In addition, there are two markers that are used as prognostic indicators [1–4]. The firs one is CD38: the interaction of CD38 and its natural ligand CD31 may rescue CLL cell from apoptosis [5]; therefore, if CD38 is coexpressed with a B-cell marker on more than 30% of cells, it gives an unfavorable prognosis. Another marker is zeta-associated protein 70 (ZAP70): ZAP70 directly enhances the B-cell receptor transduction and IgM signaling in CLL cases, leading to increased tyrosine phosphorylation of key signal transduction proteins [5]. As a result, the unmutated CLL cells are activated, receiving continued stimulation for division and proliferation. In contrast, the mutated CLL cells are in the anergic state with down-regulated B-cell receptor and reduced signal reception, resulting in less aggressive behavior. Therefore, the most important prognostic predictor appears to be the mutational status of the variable region of the immunoglobulin heavy-chain gene (I gVH gene). About 40–50% of CLL cases may show abnormal karyotypes [1–4]. The most common one is trisomy 12, which is associated with Richter transformation. Other relatively common aberrations include deletion of 11q (site of the ataxia – telangectasia [ATM] gene) and deletion of 17p (site of the tumor suppressor gene p53). Both carry an unfavorable prognosis. On the other hand, 13q14 deletion is associated with a favorable prognosis. At the early stage, most patients are asymptomatic or have minimal symptoms. As the disease progresses, patients start to show lymphadenopathy, hepatomegaly, splenomegaly, anemia, and thrombocytopenia. On the basis of these clinical presentations, two staging systems have been developed (Tables 26.1 and 26.2). Treatment is usually not required until the patient is at Rai stage III or IV or Binet stage C. The therapeutic guideline for CLL recommended by the National Cancer Institute Working Group includes development of B symptoms, worsening anemia and/or thrombocytopenia, autoimmune cytopenias, progressive splenomegaly, progressive lymphadenopathy, and lymphocyte doubling time of six months [2, 3]. As mentioned before, unmutated I gVH gene and elevated CD38 and ZAP70 (>30%) are also indications for therapeutic intervention. Some serum markers, including lactate dehydrogenase, thymidine kinase, beta-2 microglobulin, and soluble CD23 level, have also been used to help the therapeutic decision [1, 2].

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Table 26.1 Rai staging system for chronic lymphocytic leukemia Stage 0 Stage I Peripheral and bone marrow lymphocytosis Lymphadenopathy Hepatomegaly or splenomegaly Anemia (Hb < 11 g/dl) Thrombocytopenia (platelets < 100, 000/␮l)

Stage II

Stage III

Stage IV

+

+

+

+

+

− −

+ −

± +

± ±

± ±

− −

− −

− −

+ −

± +

Table 26.2 Binet staging system for chronic lymphocytic leukemia Stage Area involved∗ Hemoglobin (g/dl) Platelets (×109 /l) A <3 ≥ 10 ≥ 100 B ≥3 ≥ 10 ≥ 100 C Variable < 10 < 100 ∗ The areas include cervical, axillary, and inguinal lymph nodes as well as spleen and liver.

References 1. M¨uller-Hermelink HK, Catovsky D, Montserrat E, et al. Chronic lymphocytic leukemia/small lymphocytic lymphoma. In Jaffe ES, Harris NL, Stein H, Vardiman JW, eds., Tumours of Haematopoietic and Lymphoid Tissues, Lyon, France, IARC Press, 2001, 127–130. 2. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: A report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institue-Working Group 1996 guidelines. Blood 2008;111:5446–5456. 3. Abbott BL. Chronic lymphocytic leukemia: Recent advances in diagnosis and treatment. Oncologist 2006;11:21–30. 4. Gentile M, Mauro FR, Guarini A, et al. New developments in the diagnosis, prognosis and treatment of chronic lymphocytic leukemia. Curr Opin Oncol 2005;17:597–504. 5. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 152–159.

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Case 27 A 59-year-old man presented with a low grade fever for two weeks with abdominal pain, increasing fatigue, and drenching night sweats. Physical examination revealed no lymphadenopathy or hepatosplenomegaly. However, a computed tomography scan showed marked splenomegaly and diffuse abdominal adenopathy. Laboratory testing demonstrated pancytopenia and a highly elevated lactate dehydrogenase (1076 IU/l). Due to his critical condition, lymph node biopsy was not attempted and a bone marrow biopsy was performed instead. The biopsy showed multiple intertrabecular lymphoid aggregates, suspicious of lymphoma (Fig. 27.1). Subsequently, a right groin lymph node was biopsied (Fig. 27.2).

Fig. 27.1 Bone marrow biopsy shows an intertrabecular lymphoid aggregate, consisting of small lymphocytes, representing the residual CLL cells. Wright – Giemsa, × 20

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173

Fig. 27.2 Lymph node biopsy reveals a confluen sheet of large tumor cells with vesicular nuclei and multiple membrane-bound nucleoli, consistent with the centroblastic variant of diffuse large B-cell lymphoma. H&E, × 100

Differential diagnoses: different subgroups of lymphomas.

Further Studies Immunohistochemistry of bone marrow: CD20 stain: positive CD3 stain: negative Bcl-2 stain: positive (Fig. 27.3) Ki-67 stain: > 90% proliferation fraction (Fig. 27.4) Flow cytometry: Two clusters of cells in the lymphoid region were identified Both clusters expressed CD19, CD20, CD23, and lambda, but were negative for kappa and CD10. One cluster in addition showed coexpression of CD19 and CD5 (Fig. 27.5). Cytogenetic karyotype: Markedly complex karyotype including add(14)(q32) and 32 other aberrations. These finding were consistent with a high-grade B-cell lymphoma transformation. Immunoglobulin heavy chain gene rearrangement by capillary electrophoresis: One monoclonal band was identifie in the bone marrow specimen and two monoclonal bands were present in the lymph node specimen. One of the two bands was identical to the bone marrow band (Fig. 27.6).

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Fig. 27.3 Bone marrow biopsy shows bcl-2 staining in most of the tumor cells. Immunoperoxidase, × 40

Fig. 27.4 Bone marrow biopsy shows Ki-67 staining in more than 90% of tumor cells. Note Ki-67 highlights the nucleoli in some cells (arrow). Immunoperoxidase, × 60

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Fig. 27.5 Flow cytometric histograms demonstrate three cell populations. The red cluster is consistent with CLL cells showing dual CD19/CD5 and CD23 staining in a monoclonal lambda population. The green cluster is consistent with the diffuse large B-cell lymphoma cells showing similar immunophenotype as the CLL population except for the absence of CD5 staining. The blue cluster represents the normal T-cell population

Fig. 27.6 Immunoglobulin heavy chain gene analysis shows a monoclonal spike (113 bp) in framework 3 of the bone marrow specimen. The lymph node specimen shows one monoclonal spike in framework 3 that is identical to the monoclonal spike in the bone marrow, representing the CLL component. Another one in framework 1 with a larger size (336 bp) represents the diffuse large B-cell lymphoma component

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Discussion Richter syndrome (RS) was initially described in a case of chronic lymphocytic leukemia (CLL) transformed into “reticular cell sarcoma”, which corresponds to diffuse large B-cell lymphoma in the modern terminology. The definitio of RS or Richter transformation has since been broadened to include many small cell lymphomas (small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma, and lymphoma of mucosa-associated lymphoid tissue) transforming into highgrade non-Hodgkin lymphoma as well as Hodgkin lymphoma [1, 2]. RS develops in about 3–10% of patients with CLL. It is probably not related to chemotherapy or radiation therapy because it happens also in untreated patients. Patients with CLL are known to have an increased risk of developing second neoplasms, but the incidence of transformation to large cell lymphoma is disproportionally high. The diagnosis of RS is based on the history of CLL or other small cell lymphomas and the subsequent development of a large cell lymphoma in the lymph node or extranodal sites. With this transformation, the patient’s clinical condition usually changes abruptly, with marked elevation of peripheral lymphocyte counts, lymphadenopathy, fever, and the involvement of the central nervous system or other extranodal sites. There are also other constitutional symptoms and elevation of serum lactate dehydrogenase or alpha-2 microglobulin [3]. Once RS develops, the patient runs a rapidly downhill clinical course and dies within 6 months. There is no significan improvement in survival with the current treatment modalities [3]. In a comparison of two groups of CLL patients, the group of age 55 years or younger appeared to have a higher incidence of RS than the group older than 55 [1, 3]. However, one possibility is that lymph node biopsy may be infrequently performed in the older age group even when there is a clinical progression [3]. The most common type of RS is CLL transforming to diffuse large B-cell lymphoma, mostly of the immunoblastic or centroblastic variant. The major histologic pattern in the lymph node or bone marrow is the presence of large lymphoma cells intermingled with small lymphocytes, which represent CLL or small lymphoma cells. In the peripheral blood, CLL cells are frequently detectable, which helps to establish the diagnosis. In the later stage, large tumor cells may become so predominant that the small cell component may be no longer recognizable. However, two cell populations may still be identifie by immunophenotyping or molecular biological techniques, as exemplifie by the current case. In cases of RS, the CLL and the large cell lymphoma cells may have the same immunophenotype, i.e. they share the same monoclonal immunoglobulin light chain, CD19, CD20, CD5, and CD23. In some cases, CD5 and IgD are downregulated in the large cell component [1]. Even if the immunophenotypes of the large cell lymphoma and CLL are identical, f ow cytometry may still demonstrate two separate populations thus providing evidence of Richter transformation, as seen in this case. When CLL cells are present in the peripheral blood or bone marrow and large cells are demonstrated in the lymph node, molecular genetic studies of these two specimens may determine their clonal relationship. Aproximately 60% of RS cases show that the large lymphoma cells share the same clonal origin with the CLL or small lymphoma cells, but the remaining cases may be a de novo diffuse large B-cell lymphoma developed after the small cell tumor [3]. One controversial issue in RS is whether CLL can transform into Hodgkin lymphoma [1]. When small cell lymphoma is coexistent with Hodgkin lymphoma, should it be called composite lymphoma or Richter transformation? Ohno et al demonstrated the same clonal origin of the tumor cells in Hodgkin lymphoma and CLL by single cell polymerase chain reaction for immunoglobulin heavy chain gene rearrangement [4]. It appears that most Hodgkin lymphomas in CLL cases are the result of clonal evolution of the CLL cells, but the remaining cases may represent de novo Hodgkin lymphoma or composite tumor. Some of these cases have a better prognosis than those RS cases with diffuse large B-cell lymphoma. Those cases are designated Hodgkin lymphoma variant of RS [5]. However, in one study series, the Hodgkin lymphoma variant showed generalized symptoms with progressive lymphadenopathy and four out of seven patients died within one year [6]. The development of RS is independent of disease stage, duration of disease, type of therapy or response to therapy. However, cases with high percentage of ZAP-70 or with unmutated VH gene have higher frequency of RS. CLL cases that transform into RS usually have a complex karyotype, but no specifi nonrandom aberration has been identified There are extensive studies of various oncogenes and tumor suppressor genes, but no conclusive evidence to incriminate any genes as responsible for transformation [1, 3]. However, p53 mutation or overexpression is more frequently demonstrated in RS than in CLL cases. The general concept is that transformation is associated with abnormalities in cell-cycle regulation and DNA repair [3].

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References 1. Nakamura N, Abe M. Richter syndrome in B-cell chronic lymphocytic leukemia. Pathol Int 2003;53:195–203. 2. Matolcsy A. High-grade transformation of low-grade non-Hodgkin lymphomas: mechanism of tumor progression. Leuk Lymphoma 1999;34:251–259. 3. Yee KWL, O’Brien SM, Giles FJ. Richter’s syndrome: Biology and therapy. Cancer J 2005;11:161–174. 4. Ohno T, Smir BN, Weisenburger DD, et al. Origin of the Hodgkin/Reed-Sternberg cells in chronic lymphocytic leukemia with “Hodgkin’s transformation”. Blood 1998;91:1757–1761. 5. Brecher M, Banks PM. Hodgkin’s disease variant of Richter’s syndrome. Report of eight ases. Am J Clin Pathol 1990;90:333–339. 6. Fayad L, Robertson LE, O’Brien S, et al. Hodgkin’s disease variant of Richter’s syndrome: Experience at a single institution. Leuk Lymphoma 1996;23:333–337.

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Case 28 A 72-year-old man was found to have an enlarged cervical lymph node in a routine physical checkup. He denied having fever, night sweats and weight loss. Physical examination showed no hepatosplenomegaly. CT scan revealed no deep-seated lymphadenopathy. Peripheral blood examination demonstrated a total leukocyte count of 4,500/␮l with 72% neutrophils, 23% lymphocytes, 4% monocytes, and 1% eosinophils. His hematocrit was 40%, hemoglobin 13 g/dl, and platelets 152,000/␮l. A bone marrow aspirate showed 9% lymphocytes and the core biopsy revealed no lymphoid infiltrate A lymph node biopsy was performed (Figs. 28.1 and 28.2).

Fig. 28.1 Lymph node biopsy shows multiple poorly defined pale-stained areas, representing proliferation centers (arrows). H&E, × 10

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Fig. 28.2 Higher magnificatio of the proliferation center reveals a mixed population of small lymphocytes (darkedly stained), prolymphocytes (lighter stained, black arrow) and a few paraimmunoblasts (white arrow). H&E, × 100

Differential diagnoses: low-grade lymphomas.

Further Studies Flow cytometric analysis of lymph node: CD5 92%, CD19 83%, CD19/CD5 83%, CD20 62%, CD10 3%, CD23 81%, FMC7 11%, CD19/kappa 81%, CD19/lambda 2%. Immunohistochemical stains of lymph node: Cyclin D1 stain: negative Ki-67 stain: positive in less than 10% of cells (Fig. 28.3)

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Fig. 28.3 Lymph node biopsy shows a few Ki-67 positive (arrow) lymphoid cells. Immunoperoxidase, × 100

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Discussion Small lymphocytic lymphoma (SLL) is a small B-cell lymphoma and the tissue phase of chronic lymphocytic leukemia (CLL). The term SLL is reserved for those cases with lymph node involvement and without a leukemic phase, as seen in the current case. In other words, the peripheral B lymphocyte count should be less than 5,000/␮l and no cytopenia caused by a clonal marrow infiltrat [1, 2]. However, even in those cases, the bone marrow and peripheral blood may eventually be involved. In patients showing both tissue and leukemic phases, the appropriate term should be CLL/SLL. SLL and CLL have identical immunophenotypes and are considered the same disease in the World Health Organization (WHO) classificatio [1]. This classificatio is distinguished from the Revised European – American Classificatio of Lymphoid Neoplasms (REAL classification [3] by separating prolymphocytic leukemia from CLL/SLL. CLL/SLL accounts for 6.7% of non-Hodgkin lymphomas [1]. Similar to other small cell lymphomas, the normal lymph node architecture is usually totally effaced by extensive infiltratio of small round cells with regular nuclei and a clumped chromatin pattern. Nucleoli are usually not present or inconspicuous. The cytoplasm is scant. The major distinction between SLL and other small cell lymphomas is the presence of proliferation centers or pseudofollicles, which are present in 90% of SLL cases. This structure is best detected under low-power magnificatio as a poorly define pale-stained area. It is composed of prolymphocytes and paraimmunoblasts on a small lymphocyte background [4]. Prolymphocytes are slightly larger than the small lymphocytes, with a dispersed chromatin pattern and a distinct or inconspicuous nucleolus. Paraimmunoblasts are large than prolymphocytes with more immature chromatin and prominent nucleoli [4]. A pseudofollicle should be distinguished from a residual germinal center, which can be present in mantle cell lymphoma or nodal marginal zone B-cell lymphoma. A germinal center is well-circumscribed and is composed of centrocytes, centroblasts, and macrophages, forming a pattern of polarity (Fig. 28.4). In some instances, a cluster of parafollicular monocytoid B-cells may also mimic the proliferation center.

Fig. 28.4 A well-circumscribed residual germinal center in a mantle cell lymphoma case. Note the polarity of the germinal center showing a dark zone with multiple tangible-body macrophages at the upper half and the light zone at the bottom half. × 20

In the spleen, most non-Hodgkin lymphomas are confine to the white pulp, but SLL usually shows both white pulp and red pulp involvement (Figs. 28.5 and 28.6). Pseudofollicles may be seen in the spleen, but they are not as conspicuous as they are in the lymph nodes. Bone marrow is eventually involved in SLL cases; the infiltratio pattern is identical to that seen in CLL. Extranodal involvement is mainly seen in the orbit and lungs.

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Fig. 28.5 A splenectomy specimen shows prominent expansion of the white pulp, representing lymphomatous infiltration H&E, × 10

Fig. 28.6 A splenectomy specimen reveals marked lymphomatous infiltratio in the red pulp cord. The red pulp sinuses are dilated. H&E, × 40

When the cells of the pseudofollicles predominate and proliferate diffusely with resultant nodal replacement, the subtype is designated as a paraimmunoblastic variant of SLL/CLL (see Case 29) [3]. When SLL transforms into diffuse large

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B-cell lymphoma, it is traditionally called Richter transformation (see Case 27). The paraimmunoblastic variant should be distinguished from prolymphocytoid transformation of CLL, which usually shows a large number of prolymphocytes in the peripheral blood and is much more common than the former, with a frequency of 15% of CLL cases. All these transformation variants should be distinguished from the blastoid variant of mantle cell lymphoma (see Case 45) by immunophenotyping. SLL has an immunophenotype identifcal to CLL [1, 2]. In other words, it is positive for all B-cell markers (CD19, CD20, CD21, CD23, CD24, CD79a, and HLA-DR). CD22 is the exception, as it can be negative or weakly positive. The characteristic feature in SLL is the coexpression of CD5 with a B-cell marker and the presence of a high percentage of CD23, which is negative in mantle cell lymphoma cases. The percentage of FMC-7 is high in mantle cell lymphoma and low in SLL/CLL. However, the percentages of CD23 and FMC-7 are not always reliable in distinguishing these two entities; the most specifi marker to differentiate mantle cell lymphoma from SLL is the presence of cyclin D1 in the former. Theoretically, the major phenotypic difference between SLL and CLL is the presence of adhesion molecules (CD11a/CD18) in SLL, which stay in the tissue. CLL is just the opposite. On the other hand, the chemokine receptors CXCR4 and CCR7 are expressed at higher levels in peripheral CLL cells than SLL cells in the lymph node [5]. SLL usually shows no abnormal karyotypes unless it is evolving into a high-grade lymphoma. However, with the fluores cence in situ hybridization technique, about 80% of CLL/SLL may show genetic aberrations [1]. For instance, about 50% show del13q13.3, and 20% trisomy 12. The less common karyotypes include deletions of 11q22.23, 17p13, and 6q21. Clinically, most patients are asymptomatic or mildly symptomatic with an indolent clinical course. If there is no transformation to high-grade lymphoma or superimposed infection, patients may have a normal lifespan. However, some patients may have autoimmune hemolytic anemia, infections, splenomegaly, hepatomegaly, or extranodal infiltration When anemia and thrombocytopenia develop, those patients are usually in the late stage of the disease (Rai stage 3–4 or Binet stage C).

References 1. M¨uller-Hermelink HK, Montserrat E, Catovsky D, et al. Chronic lymphocytic leukaemia/small lymphocytic lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 180–182. 2. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: A report from the International Workshop on Chronic Lymphocytic Leukemia updating the the National Cancer Institute-Working Group 1996 guidelines. Blood 2008111:5446–5455. 3. 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. 4. Feller AC, Diebold J. Histopathology of Nodal and Extranodal Non-Hodgkin Lymphoma. 3rd ed., Berlin, Springer, 2004, 23–29. 5. Ghobrial IM, Bone ND, Stenson MJ, et al Expression of the chemokine receptors CXCR4 and CCR7 and disease progression in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma. Mayo Clin Proc 2004;79:318–325.

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Case 29 A 53-year-old man presented with severe back pain, abdominal distention and weight loss for several days. He had a history of chronic lymphocytic leukemia/small lymphocytic lymphoma proven by peripheral blood examination and lymph node biopsy f ve years ago and was started with chemotherapy three years ago. On admission, the total leukocyte count was 18,000/␮l with 77.6% lymphocytes, 15.3% neutrophils, and 6.6% monocytes. A further manual differential count confirme moderate lymphocytosis with a predominant population of prolymphocytes (20%) and paraimmunoblasts (30%) (Fig. 29.1). His hematocrit was 37.8%, hemoglobin 13.5 g/dl, and platelet count 103,000/␮l. Serum lactate dehydrogenase was 343 IU/l.

Fig. 29.1 Peripheral blood smear shows three prolymphocytes and one paraimmunoblast (inset). Wright – Giemsa, ×200

Physical examination showed gross abdominal distention, with palpable splenomegaly and diffuse lymphadenopathy. Imaging revealed prominent intrathoracic, periaortic and pelvic lymphadenopathy. Bone marrow (Fig. 29.2) and lymph node (Figs. 29.3 and 29.4) biopsies were performed.

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Fig. 29.2 Bone marrow aspirate reveals large numbers of prolymphocytes (hollow arrow) and paraimmunoblasts (arrow). Wright – Giemsa, × 100

Fig. 29.3 Lymph node touch imprint shows many prolymphocytes (hollow arrow) with clear cytoplasm and several paraimmunoblasts (arrow). Wright – Giemsa, × 100

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Fig. 29.4 Lymph node biopsy demonstrates an increased number of paraimmunoblasts (arrow) among the prolymphocytes (hollow arrow) and small lymphocytes. H&E, × 60

Differential diagnoses: Small lymphocytic lymphoma with transformation to large cell lymphoma, prolymphocytoid transformation, or paraimmunoblastic transformation.

Further Studies Flow cytometric studies: Peripheral blood: CD19/CD5 33%, CD23 26%, FMC-7 16%, kappa 6%, lambda 29% Bone marrow: CD19/CD5 87%, CD23 70%, FMC-7 24%, kappa 2%, lambda 83% Lymph node: CD19/CD5 99%, CD23 65%, FMC-7 27%, kappa 0%, lambda 79% Cytogenetic studies of bone marrow and lymph node showed the same karyotype: 46,X,−Y,+12[2]/46,sl, del(11)(q22q23)[17]/46,XY[1]. This pattern indicates the presence of trisomy 12 and deletion of ATM gene at chromosome 11.

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Discussion The term paraimmunoblastic variant of small lymphocytic lymphoma/leukemia was f rst coined by Pugh et al, who reported 16 cases [1]. Since then there have only been a few cases reported. The paucity of this entity in the literature is probably due to the fact that the morphology of paraimmunoblasts is poorly define in various studies and the required number or percentage of paraimmunoblasts in this entity has never been clarified Lennert define a paraimmunoblast as a smaller immunoblast with weaker staining in the cytoplasm with Giemsa stain [2]. Pugh et al described paraimmunoblasts as “intermediate sized to large cells with partially vesicular nuclei, prominent central nucleoli, and a moderate amount of weakly staining eosinophilic (H&E) or gray-blue (Giemsa) cytoplasm” [1]. Brunning and McKenna simply described immunoblasts as morphologically in-between prolymphocytes and large cells in diffuse large B-cell lymphoma [3]. Muller-Hermelink et al stated that large cells in Richter transformation resemble paraimmunoblasts [4]. It appears that the demarcation between prolymphocytes, paraimmunoblasts, and diffuse large B-cell lymphoma cells is blurred. Therefore, it is most appropriate to consider prolymphocytoid transformation, paraimmunoblastic variant, and Richter transformation as a continuum of small lymphocytic lymphoma/leukemia, as suggested by Brunning and McKenna [3]. Accordingly, they used the term prolymphocytoid paraimmunoblastic transformation to replace that of paraimmunoblastic variant [3]. The increase of prolymphocytes is usually reflecte in the elevation of the percentage of FMC-7 determined by fl w cytometry. However, there are no immunologic markers for identificatio of paraimmunoblasts. In this variant, the immunophenotype is the same as that of small lymphocytic lymphoma/leukemia with increase of FMC-7. The mechanism that triggers small lymphocytic lymphoma/leukemia transformation is not entirely clear at this time, but multiple molecular cytogenetic aberrations have been proposed. These include trisomy 12, deletions, and translocations in chromosomes 11, 13, and 14, mutations of p53, p16INK4A and p21 genes, and microsatellite instability [5]. More recently, deletions of the ataxia – telangiectasia mutated (ATM) locus was found to be preferentially associated with chronic lymphocytic leukemia/prolymphocytic leukemia and prolymphocytic leukemia [6]. This aberration is usually late appearing or a secondary chromosome lesion. Reiniger et al found that a high frequency of a recently identifie form of genetic instability, termed aberrant somatic hypermutation, was detected in cases of prolymphocytoid transformation and Richter transformation [7]. These molecular genetic aberrations may become useful markers for the identificatio of transformation of small lymphocytic lymphoma/leukemia.

References 1. Pugh WC, Manning JT, Butler JJ. Paraimmunoblastic variant of small lymphocytic lymphoma/leukemia. Am J Surg Pathol 1998;12:907–917. 2. Feller AC, Diebold J. Histopathology of Nodal and Extranodal Non-Hodgkin’s Lymphomas (Based on the WHO classification) 3rd ed., New York, Springer, 2004, 22–29. 3. Brunning RD, McKenna RW. AFIP Atlas of Tumor Pathology: Tumors of the Bone Marrow, Washington DC, American Registry of Pathology, 1994, 266–270. 4. Muller-Hermelink HK, Catovsky D, Montserrat E, et al. Chronic lymphocytic leukemia/small lymphocytic lymphoma. In Jaffe ES, Harris NL, Stein H, Vardiman JW. eds., Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2001, 127–130. 5. Tsimberidou AM, Keating MJ. Richter syndrome: Biology, incidence, and therapeutic strategies. Cancer 2005;103:216–228. 6. Cuneo A, Bigoni R, Rigolin GM, et al. Late appearance of the 11q22.2–23.1 deletion involving the ATM locus in B-cell chronic lymphocytic leukemia and related disorders. Clinico-biological significance Haematologica 2002;87:44–51. 7. Reiniger L, Bodor C, Bognar A, et al. Richter’s and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation. Leukemia 2006;20:1089–1095.

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Case 30 A 59-year-old man presented with abdominal discomfort, early satiety, and easy fatigability for several months. Physical examination revealed splenomegaly and cervical lymphadenopathy, but the liver was not palpable. Hematology workup showed a total leukocyte count of 117,000/␮l with 80.5% lymphocytes, 14.3% neutrophils, and 2.8% monocytes. A manual differential count demonstrated 60% prolymphocytes in the lymphoid population (Fig. 30.1). His hematocrit was 41%, hemoglobin 12.5 g/dl, and platelets 140,000/␮l. After admission, bone marrow (Figs. 30.2 and 30.3) and lymph node (Figs. 30.4 and 30.5) biopsies were performed.

Fig. 30.1 Peripheral blood smear shows a cluster of prolymphocytes with medium size, moderate amount of cytoplasm, immature chromatin and a single prominent nucleolus (arrow). Wright – Giemsa, × 100

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Fig. 30.2 Bone marrow aspirate reveals predominance of prolymphocytes with a few nucleated red blood cells. Note the prolymphocytes in the bone marrow usually show only a small rim of cytoplasm and inconspicuous nucleoli. Wright – Giemsa, × 100

Fig. 30.3 Bone marrow biopsy demonstrates a predominant prolymphocyte population intermingled with small lymphocytes. H&E × 100

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Fig. 30.4 Lymph node biopsy shows total effacement of normal architecture by immature prolymphocytes. Note that no mitotic figure are present. H&E, × 40

Fig. 30.5 Lymph node biopsy shows a large sheet of prolymphocytes with vesicular nuclei and prominent nucleoli. Two paraimmunoblasts are present (arrows). H&E, × 100

Case 30

Differential diagnosis: chronic lymphocytic leukemia, prolymphocytic leukemia, leukemic phase of lymphoma.

Further Studies Flow cytometry: CD19 89%, CD5 15%, CD19/CD5 5%, CD20 80%, CD23 10%, FMC-7 80%, kappa 85%, lambda 5%

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Discussion Prolymphocytic leukemia (PLL) can be de novo or secondary to chronic lymphocytic leukemia (CLL), which is designated prolymphocytoid transformation [1]. However, the new World Health Organization (WHO) classificatio excludes transformed CLL from the category of B-cell PLL [2]. In cases of transformation, the peripheral blood may still show residual CLL cells intermixed with prolymphocytes, peripheral lymphadenopathy is frequently present, and the leukocyte count is not as high as in the de novo cases. On the other hand, primary PLL tends to have extremely high leukocyte counts (nearly or more than 100,000/␮l) with frequent splenomegaly. The definitio of PLL is that >55% of the lymphoid cells in the peripheral blood are prolympocytes [2,3]. If the prolymphocyte counts are between 10 and 55%, it is called CLL/PLL. When less than 10% of prolymphocytes are present in a CLL case, the diagnosis is not changed. A characteristic prolymphocyte of B-cell lineage is about 10–15 ␮m in diameter with a moderate amount of light basophilic cytoplasm. The nucleus has a chromatin density between that of a small lymphocyte and that of a lymphoblast. In PLL of T-cell lineage, there are two morphologic variants [3]. The small-cell variant is seen in about 25% of cases. The nucleoli are inconspicuous under light microscopy and require electron microscopy for identification In 5% of T-cell PLL cases, cerebriform nuclei are demonstrated (S´ezary-cell-like variant). Patients with PLL usually have extremely high leukocyte count and extensive infiltratio in internal organs [2]. In the spleen, both the red pulp and white pulp are involved in B-PLL, but only red pulp is involved in T-PLL [3]. A proliferative nodule with a bizonal pattern (darker at the center and lighter at the periphery) in the white pulp is characteristic of B-PLL (Fig. 30.6) [4]. The cells in the center are mature lymphocytes, which are encircled by a rim of prolymphocytes that stain lighter because of their dispersed chromatin.

Fig. 30.6 Splenectomy specimen reveals a lymphoid follicle with a bizonal pattern. H&E, × 10

In the lymph node, the infiltratio is diffuse with a pseudofollicular pattern in some cases [4]. In PLL cases secondary to CLL, the infiltratio may be patchy. When the normal architecture is completely replaced by prolymphocytes and paraimmunoblasts, it is frequently referred to as a paraimmunoblastic variant of small lymphocytic lymphoma/leukemia (see Case 29) [4]. The infiltrat ve pattern in the bone marrow is interstitial or diffuse, similar to that seen in CLL, or a mixed interstitial – nodular pattern [4]. In the skin, the characteristic of T-PLL is perivascular and periappendiceal infiltratio by atypical lymphoid cells in the dermis without epidermotropism [5].

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The prolymphocytes in tissue sections appear medium to large in size with a round or oval nucleus with a dispersed chromatin and a single nucleolus [4]. Despite the immature appearance of the tumor cells, the mitotic rate is usually low; this combination is considered characteristic of PLL. Flow cytometry may demonstrate positive reactions to CD19, CD20, CD22, CD23, CD79a, and FMC-7 in B-PLL cases [1, 2]. This immunophenotype is in contrast to that of most CLL cases, which show low percentages of CD22 and FMC-7 but high percentages of CD5 and CD23. The negative reactions to terminal deoxynucleotidyl transferase (TdT) and CD10 help distinguish PLL from lymphoblastic leukemia/lymphoma. In T-PLL cases, the immunophenotype is TdT − CD1a − CD2 + CD3 + CD5 + CD7+ [3]. 60% of T-PLL cases are CD4 + CD8−. About 25% cases show coexpression of CD4 and CD8, which distinguishes T-PLL from other peripheral T-cell lymphoma/leukemia. Only 15% cases are CD4 − CD8+. CD52 is also present in T-PLL cases and those cases can be treated with alemtuzumab (anti-CD52) [6]. In earlier studies, the most common cytogenetic aberration was t(11;14)(q13;q32). However, these cases are now considered to be the leukemic variant of mantle cell lymphoma which is excluded from the category of PLL [1, 2]. The most common cytogenetic aberrations in B-PLL are complex karyotype and del(17p). The latter is associated with the TP53 gene mutation [2]. In T-PLL cases, chromosome 14 inversion or translocation with breakpoints at bands q11 and q32 are the most frequent finding [3]. Chromosome 8 aberrations are also often encountered [3]. Deletion of TP53 gene is seen in 26% cases and is manifested with overexpression of p53 protein [3]. The median survival in B-PLL is 30–50 months. Patients with T-PLL have a median survival of less than one year and rarely survive more than two years after diagnosis.

References 1. Schlette E, Bueso-Ramos C, Giles F, et al. Mature B-cell leukemia with more than 55% prolymphocytes: A heterogeneous group that includes an unusual variant of mantle cell lymphoma. Am J Clin Pathol 2001;115:571–581. 2. Campo E, Catovsky D, Montserrat E, et al. B-cell prolymphocytic leukaemia. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumors of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 183–184. 3. Catovsky D, M¨uller-Hermelink HK, Ralfkiaer E. T-cell prolymphocytic leukemia. 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, 270–271. 4. Brunning RD, McKenna RW. Tumor of the Bone Marrow, Washington, DC, Armed Forces Institute of Pathology, 1994, 266–301. 5. Valbuena JR, Herling M, Admirand JH, et al. T-cell prolymphocytic leukemia involving extramedullary sites. Am J Clin Pathol 2005;123: 456–464. 6. Darden CD. T-cell prolymphocytic leukemia. Med Oncol 2006;23:17–22.

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Case 31 A 65-year-old man was admitted for investigation of syncope and blurred vision. On admission, his peripheral blood showed mild rouleaux formation. Serum protein electrophoresis demonstrated a monoclonal spike and immunofixatio confirme a monoclonal IgM-kappa pattern. Immunoglobulin quantitation revealed 3.2 g/dl of IgM with decreased IgG and IgA. His axillary lymph node was enlarged and a biopsy was done (Fig. 31.1). Subsequently, a bone marrow biopsy was performed (Figs. 31.2 and 31.3).

Fig. 31.1 Lymph node biopsy shows a mixed lymphoid and plasmacytic population. Note that many plasma cells contain Russell bodies (arrow). H&E, × 100

Case 31

Fig. 31.2 Bone marrow biopsy reveals a mixed lymphoid and plasmacytic population. H&E, × 100

Fig. 31.3 Bone marrow aspirate shows a mixed lymphoid and plasmacytic population. Wright – Giemsa, × 100

Differential diagnoses: Plasma cell myeloma, Waldenstr¨om macroglobulinemia, and lymphoma.

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Further Studies Immunohistochemistry: kappa/lambda light chain stains: positive for kappa and negative for lambda (Fig. 31.4) CD20 stain: positive for lymphoid cells (Fig. 31.5) Flow cytometry: Lymph node biopsy: positive for CD19, cytoplasmic and surface kappa light chain, and FMC-7, but negative for CD5, CD10, and cytoplasmic and surface lambda light chain (Fig. 31.6).

Fig. 31.4 Lymph node biopsy demonstrates many plasma cells with Russell bodies, as highlighted by kappa antibody staining. Immunoperoxidase, × 100

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Fig. 31.5 Lymph node biopsy shows CD20 staining of the lymphoma cells. Immunoalkaline phosphatase, × 100

Fig. 31.6 Flow cytometric histograms show positive reactions to CD19, FMC-7, and kappa light chain, but negative reactions to CD5, CD10, and lambda light chain. Note the coexistence of monoclonal surface and cytoplasmic kappa patterns

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Discussion In the World Health Organization (WHO) classification lymphoplasmacytic lymphoma (LPL) is define as “a neoplasm of small B lymphocytes, plasmacytoid lymphocytes and plasma cells, usually involving bone marrow, and sometimes lymph nodes and spleen, which does not fulfil the criteria for any of the other small B-cell lymphoid neoplasms that may also have plasmacytic differentiation” [1]. The Second and Third International Workshops on Waldenstr¨om Macroglobulinemia (WM) defin WM specificall as a bone-marrow-based LPL associated with serum monoclonal IgM [2]. Nevertheless, WM is only associated with a subtype of LPL and cases with t(9;14) are seldom, if ever, associated with WM [3]. Whereas WM is regarded as a primary bone marrow disorder, LPL frequently presents as a node-based lymphoma. There is no specifi histologic pattern to distinguish LPL from other small cell lymphomas, but LPL is characterized by a morphologic spectrum of small lymphocytes, plasmacytoid lymphocytes, and plasma cells, even though one cell type may be predominant. The presence of Dutcher body containing plasma cells, mast cells, and hemosiderin is characteristic of LPL but these features are not required for a diagnosis [1]. Because the lack of pathognomonic features in LPL, the WHO system suggests that LPL should be diagnosed by excluding other lymphomas that may contain considerable number of lymphoplasmacytic cells [1]. In some unclear cases, the diagnosis should be small B-cell lymphoma with plasmacytic differentiation, and a differential diagnosis provided [1]. Among the differential diagnoses, the marginal zone lymphomas are most difficul to distinguish from LPL, as they may have prominent plasma cell differentiation, monoclonal IgM gammopathy, and similar immunophenotype to LPL [1–3]. Owen et al considered both LPL and marginal zone lymphomas are part of a spectrum designated systemic marginal zone lymphoma, and their distinction clinically unnecessary [3]. However, the marginal zone pattern and the monocytoid morphology in marginal zone lymphomas are usually not present in LPL. Small lymphocytic lymphoma with plasma cell differentiation can be distinguished from LPL by the presence of proliferation centers and paraimmunoblasts. Plasma cell myeloma shows exclusively plasma cell infiltratio in the bone marrow without lymphadenopathy [4]. When monoclonal IgM is detected with osteolytic lesion in plasma cell dyscrasia, it should be designated IgM myeloma. In LPL/WM cases, the peripheral blood may show lymphoplasmacytic cells. However, the major abnormality can be seen in red blood cells, such as rouleaux formation or hemagglutination, when paraprotein level is high or IgM functions as cold agglutinin. In terms of immunophenotype, the tumor cells express all B-cell antigens, including CD19, CD20, CD22, CD24, CD79a, PAX5, and HLA-DR [1,3,4]. The absence of CD5, CD10, CD23, and bcl-1 is important for the exclusion of other lymphomas mentioned above. However, the most characteristic findin is the demonstration of the coexistence of monoclonal B-cells and plasma cells [5]. Flow cytometry may show both monoclonal surface and cytoplasmic immunoglobulin patterns. Most specificall , the percentages of these two populations overlap (for instance, 70% of the former and 50% of the latter). This overlapped pattern may be explained by the existence of some lymphoplasmacytic cells that can express both surface and cytoplasmic immunoglobulins. Immunohistochemical stains are also able to distinguish two cell populations: the lymphoid cells stains for CD20 and the plasma cells stains for monoclonal cytoplasmic immunoglobulin. Cytogenetically, t(9;14)(p13;q32) has been detected in 50% of LPL cases without monoclonal IgM gammopathy [6]. This translocation is characterized molecularly as the translocation of the PAX-5 gene in chromosome 9 to the IgH gene in chromosome 14. PAX-5 encodes the B-cell-specifi activator protein (BSAP) that is important in early B-cell development. The PAX-5 product abrogates the production of the J-peptid and downregulates IgH transcription. Therefore, no monoclonal IgM gammopathy is present in LBL cases with this translocation. The most common genetic aberration in LPL with IgM gammopathy is deletion of the long arm of chromosome 6, with 6q21 deletion observed in 42% of patients by fluorescenc in situ hybridization techniques. The most common clinical presentation is the hyperviscosity syndrome [6]. This often occurs in patients with a serum viscosity above 4 units (4 times as viscous as water) or an IgM level exceeding 3 g/dl. The symptoms of this syndrome are bleeding diathesis, retinopathy, hypervolemia, congestive heart failure, and various neurologic symptoms that may progress from headache and confusion to coma. The blurred vision seen in WM patients is thought to reflec rouleaux formation or “sludging” in occular vessels. Some monoclonal macroglobulins may function as antibodies against red cell antigens thus causing hemagglutination in the cold and leading to hemolysis [6]. Other cases may show precipitation of macroglobulin in the cold (cryoglobulin). Patients with cryoglobulinemia may show the Raynaud phenomenon, cold hypersensitivity, cold urticaria, cold purpura, or glomerulonephritis [6]. Anti-neural antibodies and amyloid light chain deposition may cause polyneuropathy [6].

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References 1. Swerdlow SH, Berger F, Pileri SA, et al. Lymphoplasmacytic lymphoma. 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, 194–195. 2. Berger F, Traverse-Glehen A, Felman P, et al. Clinicopathologic features of Waldenstr¨om’s macroglobulinemia and marginal zone lymphoma: are they distinct or the same entity? Clin Lymphoma 2005;5:220–224. 3. Lin P, Medeiros LJ. Lymphoplasmacytic lymphoma/Waldenstr¨om macroglobulinemia: An evolving concept. Adv Anat Pathol 2005;12: 246–255. 4. Vijay A, Gertz MA. Waldenstr¨om macroglobulinemia. Blood 2007:109:5096–5103. 5. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 178–185. 6. Foneca R, Hayman S. Waldenstr¨om macroglobulinemia. Br J Haematol 2007;138:700–720.

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Case 32 A 67-year-old man was initially diagnosed for idiopathic thrombocytopenic purpura because of persistent thrombocytopenia. Due to his failure in response to prednisone and intravenous immunoglobulin treatment, the patient finall had a splenectomy. Pathological examination of the spleen showed extramedullary hematopoiesis and his platelet counts stayed at a low level. In the current admission, the patient presented with fever and hypoxemia and was suspected to have either pneumonia or pulmonary embolism (PE). Chest X-ray did not demonstrate pulmonary infiltrate CT scan did not show any evidence of PE, but revealed generalized lymphadenopathy. Lymphadenopathy became increasingly prominent during the hospital course and a lymph node biopsy was performed (Figs. 32.1 and 32.2). The patient recalled that his finger and lower extremities sporadically became white and icy cold with blue fingernails Serum protein electrophoresis showed monoclonal gammopathy. Immunoglobulin quantitation revealed IgG 4690 mg/dl, IgA 614 mg/dl, IgM 339 mg/dl, kappa 3160 mg/dl, and lambda 1022 mg/dl.

Fig. 32.1 Lymph node biopsy shows lymphoplasmacytic cells with scattered immunoblasts. H&E, × 60

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Fig. 32.2 Higher magnificatio shows many immunoblasts and a few mitotic figures H&E, × 100

Differential diagnoses: different kinds of lymphomas.

Further Studies Immunohistochemical stains: Ki-67 (Mib-1): positive in 85% tumor cells (Fig. 32.3) CD68: positive for histiocytes (Fig. 32.4) Flow cytometry: CD19 81%, CD19/CD5 0%, CD20 3%, CD23 7%, CD56 3%, CD38/CD138 75%, CD10 0%, surface kappa 5%, surface lambda 52%, cytoplasmic kappa 1%, cytoplasmic lambda 64%

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Fig. 32.3 Lymph node biopsy demonstrates a high percentage of Ki-67 positive immunoblasts. Immunoperoxidase, × 60

Fig. 32.4 Lymph node biopsy reveals CD68-positive histiocytes. Immunoperoxidase, × 60

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Discussion In the current case, the lymph node showed total effacement of normal architecture by lymphoplasmacytic cells. Immunophenotyping by fl w cytometry revealed both monoclonal surface and cytoplasmic immunoglobulin patterns that is characteristic of lymphoplasmacytic lymphoma (LPL) [1]. The presence of a large plasma cell population was confirme by a high percentage of CD38/CD138-positive cells. The negative reactions to CD5, CD10 and CD23 helped rule out small lymphocytic lymphoma, mantle cell lymphoma, and follicular lymphoma. However, immunophenotyping is not useful in distsinguishing LPL from nodal marginal zone B-cell lymphoma. In the latter entity, the presence of typical marginal zone distribution of lymphoma cells and the monocytoid B-cell morphology may help differentiate it from LPL. Nevertheless, some authors consider that these two tumors actually belong to a spectrum, designated systemic marginal zone lymphoma [2]. This patient had a monoclonal IgG gammopathy, which is accepted by the World Health Organization system as a variant of Waldenstr¨om macroglobulinemia (WM) [3]. IgG is not a macroglobulin, but the presence of high levels of serum IgG may also cause hyperviscosity syndrome, particularly when the IgG molecule is asymmetric in configuratio [1]. In the current case, the paraprotein might function as cryoglobulin, leading to the Raynaud phenomenon, as recalled by the patient. Small numbers of immunoblasts are frequently seen in the lymph node of LPL [3], but the presence of a high percentage of immunoblasts should alert the pathologist to consider the possibility of high-grade lymphoma transformation. Ki-67 staining in this case demonstrated a proliferation fraction of 85%, which supported the diagnosis of immunoblastic transformation. Once the tumor transforms into a high-grade lymphoma, the clinical course becomes aggressive and the prognosis is grim. This patient died one month after the diagnosis. The incidence of immunoblastic transformation varies from 5 to 13% in two study series [4, 5]. Transformation usually occurs subsequently to LPL, but LPL and diffuse large B-cell lymphoma may also develop concurrently [2]. The initial signs of transformation include new onset or increasing size of lymphadenopathy, organomegaly, worsening constitutional symptoms, profound cytopenia, extramedullary disease, and rarely hypercalcemia [2, 4]. The current case showed new onset of lymphadenopathy that progressed rapidly during hospitalization. He also had constitutional symptoms, organomegaly, and extramedullary disease. Lin et al. found that clinicopathologic features, such as prognostic index, presence or absence of lymphadenopathy or splenomegaly at diagnosis of LPL/WM did not predict the risk of transformation. In addition to transformation to large cell lymphoma, a few cases of LPL have been reported to transform to classical Hodgkin lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myelogenous leukemia [1].

References 1. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 178–185. 2. Lin P, Medeiros LJ. Lymphoplasmacytic lymphoma/Waldenstr¨om macroglobulinemia: An evolving concept. Adv Anat Pathol 2005;12: 246–255. 3. Berger F, Isaacson PG, Piris MA, et al. Lymphoplasmacytic lymphoma/Waldenstr¨om macroglobulinemia. In Jaffe ES, Harris NL, Stein H, Vardiman JW. Tumours of Haematopoietic and Lymphoid Tissues, Lyon, France, IARC Press, 2001, 132–134. 4. Lin P, Mansoor A, Bueso-Ramos C, et al. Diffuse large B-cell lymphoma occurring in patients with lymphoplasmacytic lymphoma/Waldenstr¨om macroglobulinemia: Clinicopathologic features of 12 cases. Am J Clin Pathol 2003;120:246–253. 5. Kyrtsonis, MC, Vassilakopoulos TP, Angelopoulou MK, et al. Waldenstr¨om’s macroglobulinemia: Clinical course and prognostic factors in 60 patients. Experience from a single hematology unit. Ann Hematol 2001;80:722–727.

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Case 33 A 60-year-old man presented with hemolytic anemia and thrombocytopenia. The direct Coombs test was positive, and IgG antibody was identified Physical examination showed splenomegaly, but no peripheral lymphadenopathy was detected. Flow cytometric analysis of the peripheral blood revealed a monoclonal population. A bone marrow biopsy showed intrasinusoidal B-cell infiltratio (Fig. 33.1). His anemia and thrombocytopenia were progressive even with multiple blood transfusions. A splenectomy was finall performed. The histiology of the splenectomy specimen is illustrated in Figs. 33.2 and 33.3. One week after splenectomy, his hemoglobin, hematocrit, and platelet count returned to normal levels, and no transfusion was required.

Fig. 33.1 Bone marrow biopsy shows sinusoidal infiltratio of lymphoma cells. H&E, × 60

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Fig. 33.2 Splenectomy specimen shows several enlarged lymphoid follicles with mild sinusoidal infiltratio in the red pulp. H&E, × 10

Fig. 33.3 Splenectomy specimen reveals that the enlarged follicle extends to the periarterial sheath. H&E, × 20

Differential diagnoses: Splenic marginal zone lymphoma versus other small cell lymphomas.

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Further Testing Splenectomy specimen: CD20: positive for most lymphocytes in the nodules and periarterial lymphatic sheath (Fig. 33.4). Flow cytometry: Positive for CD19, CD20, FMC-7. and kappa, but negative for CD5, CD10, CD23, and lambda (Fig. 33.5). Immunoglobulin heavy chain gene rearrangement shows a monoclonal peak in each of three frameworks (Fig. 33.6). Cytogenetic karyotype: Gain of chromosome 3 and isochromosome 18 (Fig. 33.7).

Fig. 33.4 CD20 stain demonstrates B-cells in both follicles and the periarterial sheath area, supporting the diagnosis of a B-cell neoplasm. Immunoperoxidase, × 4

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Fig. 33.5 Flow cytometry shows positive reactions to CD19, CD20, FMC-7, and kappa but negative reactions to CD5, CD10, CD23, and lambda

Fig. 33.6 Polymerase chain reaction with capillary electrophoresis technique reveals a monoclonal peak in each of the three frameworks, representing immunoglobulin heavy chain gene rearrangement

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Fig. 33.7 Karyotyping of splenectomy specimen demonstrates 46,XY,i(18)(q10)[1]/47,sl,+(3)(q10)[2]/46,XY[15]. Gain of chromosome 3 is a recurrent findin in splenic marginal zone lymphoma

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Discussion Splenic marginal zone lymphoma (SMZL) is difficul to diagnose, because several lymphomas may involve the spleen and yet there is no special marker to identify SMZL [1–4]. In addition, there are inherent difficultie in diagnosing a lymphoma in the spleen. Besides hairy cell leukemia, hepatosplenic T-cell lymphoma, and natural killer cell lymphoma, all lymphomas primarily involve the white pulp showing expansion of the follicles, which can also be seen in reactive hyperplasia of lymphoid tissue. Immunohistochemistry does not help to establish a definit ve diagnosis of SMZL, because kappa and lambda light chain staining usually does not show a conclusive result for clonality. The identificatio of a B cell population in the follicles also cannot help to distinguish malignancy from reactive condition, as follicles are normally composed of B cells. However, when the periarterial lymphatic sheath reveals predominantly B lymphocytes, it denotes an abnormal or potentially malignant condition, as this area is the T-cell zone. A positive staining of bcl-2 is helpful to confir the malignant nature of the lymphoid cells, but follicular lymphoma should be excluded by demonstrating the absence of CD10 and bcl-6. A colonized follicle can also be recognized by the presence of a follicular dendritic cell meshwork with CD21 or CD35 staining in the absence of CD10 and bcl-6. Other markers that are positive in immunohistochemical staining include DBA-44, CD79a, CD79b, PAX/BSAP, and CD45 [1–4]. CD27, a marker of activated (memory) B cells, has been identifie in most cases of SMZL in recent studies [2, 3]. In addition to negative reactions to CD10 and bcl-6, SMZL is also negative for CD5, CD23, CD43, cyclin D1, and annexin A1, which help to exclude mantle cell lymphoma, small lymphocytic lymphoma, and hairy cell leukemia. CD43 is frequently positive in extranodal marginal zone B-cell lymphoma, therefore the negative reaction to CD43 helps to exclude splenic involvement with this lymphoma. Flow cytometry may play an important role in the diagnosis of splenic lymphomas, but splenectomy specimens autolyse rapidly. If fl w cytometric analysis starts more than one hour after the removal of the spleen, the results are not reliable. Flow cytometry may identify the clonality of the lymphoma by an immunoglobulin light chain restriction. It can also demonstrate an immunophenotype of CD5 − CD10 − CD11c ± CD19 + CD20 + CD22 + CD23 − CD25 ± FMC-7 + bcl-2+ [1–4]. When SMZL shows a CD11c+, CD25+, DBA-44 or tartrate-resistant acid phosphatase immunophenotype, it should be distinguished from hairy cell leukemia by demonstrating a negative reaction to either CD103, annexin A1, or HC2, which is positive in the latter entity. CD103, however, can be present in occasional cases. Morphologically, it is most useful to demonstrate the biphasic or bizonal pattern in the spleen, but this pattern is only seen in a fraction of cases [1–4]. This pattern shows that the germinal center of the white pulp is surrounded and finall replaced by small round lymphocytes (Fig. 33.8). The small lymphocytes are, in turn, surrounded by medium-sized lymphocytes with dispersed chromatin and abundant pale cytoplasm resembling the marginal zone cells. Scattered transformed large lymphoid cells are also seen in the outer zone. The mantle zone is effaced. Both cell types have been proven to be lymphoma cells. In the red pulp, besides sinusoidal infiltration small nodules are sometimes present with the composition of tumor cells similar to that in the periphery of the white pulp follicles. In SMZL, peripheral lymph nodes are seldom involved, but splenic hilar lymph nodes may demonstrate a micronodular pattern without the presence of marginal differentiation. The bone marrow is frequently involved in SMZL. The infiltratio can be nodular, interstitial, diffuse, or paratrabecular. However, the most characteristic pattern is intrasinusoidal, which can be demonstrated by immunohistochemical stains with CD20 or DBA-44 [1–4]. The demonstration of immunoglobulin heavy chain gene rearrangement or cytogenetic abnormality in the spleen, as seen in this case, offers strong support for the diagnosis of a lymphoma, but is not specifi for SMZL unless a special karyotype is identified The most common aberrant karyotype in SMZL is allelic loss at 7q21–32, gain of 3q, gain of 12q and t(11:14)(q13;q32) [2]. The t(11;14) may be different from that seen in mantle cell lymphoma in the molecular breakpoint in chromosome 11. SMZL is a low-grade B-cell lymphoma. Most patients are mildly symptomatic, associated with splenomegaly. A subset of SMZL cases may show villous lymphocytes in the peripheral blood. The current case presented with a monoclonal lymphoid population in the peripheral blood, but villous lymphocytes were not identified Autoimmune phenomena are present in 10% of patients, such as seen in our patient with autoimmune hemolytic anemia. Because of the presence of plasma cells among the tumor cells, one-third of patients may show monoclonal gammopathy, which can help in the differential diagnosis.

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Fig. 33.8 A characteristic bizonal pattern in the white pulp and a few satellite nodules in the red pulp are demonstrated in another case. H&E, × 10

References 1. Isaacson PG, Piris MA, Berger F, et al. Splenic B-cell marginal zone lymphoma. 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, 185–187. 2. Matutes E, Oscier D, Montalban C, et al. Splenic marginal zone lymphoma proposals for a revision of diagnostic, staging and therapeutic criteria. Leukemia 2008;22:487–495. 3. Papadaki T, Stamatopoulos K, Belessi C, et al. Splenic marginal-zone lymphoma: one or more entitites? A histologic, immunohistochemical, and molecular study of 42 cases. Am J Surg Pathol 2007;31:438–446. 4. Arcaini L, Paulli M, Boveri E, et al. Marginal zone-related neoplasms of splenic and nodal origin. Haematologica 2003;88:80–93.

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Case 34 A 62-year-old man presented with cellulitis. He was found to have pancytopenia with 0% monocytes in the peripheral blood. Physical examination revealed splenomegaly, but no hepatomegaly or lymphadenopathy. The peripheral blood smear showed a few atypical lymphoid cells (Fig. 34.1). A bone marrow aspirate was obtained after two dry taps (Fig. 34.2). The core biopsy revealed patchy distribution of lymphoid aggregates (Fig. 34.3).

Fig. 34.1 A characteristic hairy cell with delicate hairy projections and reticulated cytoplasm is present in the peripheral blood smear. Wright – Giemsa, × 100

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Fig. 34.2 Several clusters of hairy cells in the bone marrow aspirate. Note lymphoid nuclei and reticulated cytoplasm of the tumor cells. Wright – Giemsa, × 60

Fig. 34.3 Bone marrow biopsy shows a large sheet of hairy cells, forming a honeycomb pattern. H&E, × 60

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Differential diagnoses: Splenic marginal zone lymphoma versus hairy cell leukemia.

Further Testing Tartrate-resistant acid phosphatase stain of bone marrow aspirate: positive (Fig. 34.4) Immunohistochemistry of bone marrow biopsy: Tartrate-resistant acid phosphatase: positive for lymphoid cells (Fig. 34.5) DBA-44: positive for lymphoid cells (Fig. 34.6) Reticulin stain of bone marrow biopsy (Fig. 34.7) Flow cytometry: positive for CD19, CD11c, CD20, CD22, CD25, FMC-7, CD103, and lambda, but negative for CD23 and kappa (Fig. 34.8)

Fig. 34.4 A cytospin preparation of bone marrow aspirate shows strong, diffuse staining of tartrate-resistant acid phosphatase on many hairy cells. Cytochemical stain, × 60

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Fig. 34.5 A bone marrow biopsy shows positive tartrate-resistant acid phosphatase staining of the hairy cells. Immunoperoxidase, × 60

Fig. 34.6 A bone marrow biopsy shows DBA-44 staining of the hairy cells. Immunoperoxidase, × 40

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Fig. 34.7 A bone marrow biopsy shows moderate increase of reticulin fibers Reticulin stain, × 60

Fig. 34.8 Flow cytometric histograms demonstrate positive reactions to CD19, CD20, CD11c, CD22, CD23, FMC-7, CD103, and lambda, but negative reactions to CD23 and kappa

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Discussion Hairy cell leukemia (HCL) is a low-grade B-cell leukemia with unique morphologic and immunophenotypic features [1, 2]. Low-grade lymphoid neoplasm usually is incurable, but HCL is exceptional. With purine analogue treatment, most patients may achieve a durable complete remission. The common clinical features of this disease are pancytopenia or cytopenia in one cell line (neutropenia, anemia, or thrombocytopenia) together with splenomegaly. Monocytopenia is particularly striking and is one of the characteristics of HCL. Clinically, the patient may have hepatic involvement, but peripheral lymphadenopathy is highly unusual. If it occurs, transformation to high-grade lymphoma should be suspected. Patients without any complication may remain essentially asymptomatic for a long time. However, due to neutropenia and monocytopenia, superimposed bacterial and parasitic infections may occur. The leukemic hairy cells are of medium size (10–15 ␮m) with a moderate amount of cytoplasm, which usually shows a loose architecture [1, 2]. The nucleus is usually round or kidney-shaped with dispersed chromatin pattern and one or more nucleoli may or may not be present. The pathognomonic feature is the presence of multiple delicate (hairy) cytoplasmic projections, which are in contrast to the focal, short cytoplasmic projections seen on the villous lymphocytes in splenic marginal zone lymphoma. Ultrastructurally, the presence of a ribosome lamellar complex in the cytoplasm is highly specifi for hairy cells. However, because of cytopenia, hairy cells are difficul to fin in the peripheral blood except for the HCL variant. In the bone marrow, due to damage to the cytoplasm during the aspirate procedure, hairy cells are also hard to distinguish from other lymphocytes. However, the histologic patterns of HCL are highly characteristic. In the bone marrow, the individual hairy cells look like fried eggs as their nuclei are surrounded by a clear zone of unstained cytoplasm with a clear cell border. The leukemic cells are usually of patchy distribution forming a honeycomb pattern. The tumor cells of the nodal marginal zone B-cell lymphoma can also show a fried egg pattern in the lymph node, but they are seldom demonstrated in the bone marrow. As HCL cells may produce fibronectin reticulin fibrosi is a common phenomenon leading to dry tap in bone marrow aspirate [2]. In the spleen, the normal architecture is usually totally destroyed by the leukemic cells, so that the white pulp is atrophic and the red pulp sinuses and cords are hardly recognizable. The red blood cells are surrounded by the hairy cells, forming pseudosinuses or blood lakes (Fig. 34.9). Since most of the lymphomas, including splenic marginal zone lymphoma, show primarily white pulp involvement by tumor cells, HCL is readily distinguishable from other lymphomas on the basis of their histologic patterns. HCL also has a highly characteristic immunophenotype, which is most helpful in its differential diagnosis. The timehonored marker for HCL is tartrate-resistant acid phosphatase (TRAP) staining, either by cytochemical or immunohistochemical techniques. Although TRAP can be seen in other lymphoid tumors, it is still specifi for HCL if a high percentage of tumor cells shows a strong staining before and after tartrate treatment. Besides the presence of the B-cell antigens, CD19, CD20, CD22, and CD79a, HCL is characterized by the presence of CD11c, FMC-7, CD25, and CD103. However, these markers can also be demonstrated in splenic marginal zone lymphoma, except for CD103 and a rarely used marker HC2. In immunohistochemistry, DBA-44 and TRAP can be used to demonstrate the HCL cells [3]. DBA-44, however, can be demonstrated in other lymphomas, particularly splenic marginal zone lymphoma. There have been three new antibodies reported with promising results. Annexin A1 and T-bet can be demonstrated by immunohistochemistry [1,4] and CD123 can be demonstrated by fl w cytometry [5]. Recently, there have been increasing numbers of HCL variant reported [6, 7]. In contrast to typical HCL, the variant usually shows high leukocyte count with no monocytopenia, absence of reticulin fibrosi in the bone marrow and a different but characteristic immunophenotype (such as absence of CD25, CD123, annexin A1, and HC-2, as well as the infrequent presence of TRAP) [6]. It is important to recognize the HCL variant, because it does not respond to conventional HCL treatments (such as interferon alpha and 2-chlorodeoxyadenosine) and its prognosis is much worse than that of HCL. Most recently, a new entity designated “splenic red pulp lymphoma with numerous basophilic villous lymphocytes” has been reported [8]. The disease is similar to splenic marginal lymphoma in the presence of splenomegaly and moderate lymphocytosis instead of pancytopenia, as seen in HCL. On the other hand, the spleen shows atrophic white pulp and a diffuse lymphoid infiltratio of the red pulp, mimicking HCL. In terms of HCL markers, CD11c, CD103, and CD123 are positive, while annexin A1 and CD25 are negative. Thirty-seven cases reported in one series needed no treatment [8].

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Fig. 34.9 A splenectomy specimen reveals the effacement of the normal architecture with hairy cells surrounding the red blood cells, forming pseudosinuses. H&E, × 40

References 1. Foucar K, Falini B, Catovsky D, et al. Hairy cell leukemia. 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, 188–190. 2. Pettitt AR, Zusel M, Cawley JC. Hairy cell leukemia: Biology and management. Br J Haematol 1999;106:2–8. 3. Went PT, Zimpler A, Pehrs AC, et al. High specificit of combined TRAP and DBA-44 expression for hairy cell leukemia. Am J Surg Pathol 2005;29:474–478. 4. Filini B, Tiacci E, Liso A, et al. Simple diagnostic assay for hairy cell leukaemia by immunocytochemical detection of annexin A1 (ANXAI). Lancet 2004;363:1869–1870 5. Del Giudice I, Matutes E, Morilla R, et al. The diagnostic value of CD123 in B-cell disorders with hairy cell or villous lymphocytes. Haematologica 2004;89:303–308. 6. Cessna MH, Hartung L, Tripp S, et al. Hairy cell leukemia variant: fact or fiction Am J Clin Pathol 2005;123:132–138. 7. Piris M, Foucar K, Mollejo M, et al. Splenic B-cell lymphoma/leukemia, unclassifiable 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, 191–193. 8. Traverse-Giehen A, Baseggio L, Callet-Bauchu E, et al. Splenic red pulp lymphoma with numerous basophilic villous lymphocytes: A distinct clinicopathologic and molecular entity? Blood 2008;111:2253–2260.

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Case 35 A 67-year-old man presented with acute low-back pain after moving heavy furniture. Radiography and CT scan demonstrated a collapsed vertebral body at T9 level. Serum electrophoresis demonstrated a monoclonal spike in the gamma region. Immunofixatio identifie a monoclonal IgG-kappa gammopathy. Quantitation of serum immunoglobulins showed IgG 3.0 g/dl, IgA 80 mg/dl, and IgM 40 mg/dl. Bence Jones protein was present in the urine. His serum calcium level was 12 mg/dl and beta-2 microglobulin level was 11 mg/l. The peripheral blood smear showed rouleaux formation, but no plasma cells were seen (Fig. 35.1). The morphologic features of bone marrow aspirate and biopsy are illustrated in Figs. 35.2 and 35.3, respectively.

Fig. 35.1 A peripheral blood smear shows rouleaux formation. Wright – Giemsa, × 40

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Fig. 35.2 A bone marrow aspirate reveals many atypical plasma cells. Note two Mott cells contain Russell bodies (arrow). Wright – Giemsa, × 60

Fig. 35.3 A bone marrow aspirate shows many Russell bodies inside or outside the myeloma cells (arrow). H&E, × 60

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Differential diagnoses: Different clinical subtypes of plasma cell myeloma.

Further Testing Immunohistochemical stains Lambda light chain: positive (Fig. 35.4) Kappa light chain: negative (Fig. 35.5)

Fig. 35.4 Lambda stain highlights many myeloma cells in a bone marrow biopsy. Immunoperoxidase, × 40

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Fig. 35.5 Kappa stain is negative for tumor cells in the same bone marrow biopsy as Fig. 35.4. Immunoperoxidase, × 40

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Discussion Plasma cell myeloma or multiple myeloma (MM) accounts for 10% of all malignant hematologic neoplasms. It is a monoclonal plasma cell neoplasm that produces monoclonal gammopathy in most cases except for the subtype of nonsecretory myeloma and some cases of solitary plasmacytoma [1–3]. The World Health Organization (WHO) classificatio define three major diagnostic criteria for MM: paraprotein or M-protein in serum or urine, bone marrow clonal plasma cells or plasmacytoma, and related organ or tissue impairment (hypercalcemia, renal insufficien y, anemia, and bone lesion). The 2008 WHO classificatio emphasizes the clinical symptoms as the core requirement for the diagnosis of myeloma and does not require a minimum level of serum M-protein or minimum percentage of plasma cells in the bone marrow. When an asymptomatic patient shows less than 10% of plasma cells in the bone marrow and less than 30 g/l of M-protein in serum, and no evidence of other B-cell proliferative disorders, the condition is called monoclonal gammopathy of undetermined significanc (MGUS) [1]. If the patient is asymptomatic, but the serum shows >30 g/l of M-protein or ≥ 10% of monoclonal plasma cells in the bone marrow, the diagnosis should be asymptomatic or smoldering myeloma. As mentioned above, the major morphologic diagnostic criterion is the presence of increasing numbers of monoclonal plasma cells in the bone marrow [1–3]. In a borderline case, it is the clustering of plasma cells that is more reliable than the absolute count in determining the diagnosis. The plasma cells in most cases show a mature morphology with slight variation of size and shapes. Only high-grade myeloma, extramedullary myeloma involving the skin or the central nervous system, or plasma cell leukemia may show plasmablasts in the lesions. A plasmablast is a large plasma cell with large nucleus (>10 ␮m) with a high nuclear/cytoplasmic ratio, immature chromatin pattern and absence of a perinuclear hof region. A myeloma cell may show multiple cytoplasmic immunoglobulin inclusions, which are called Russell bodies. The inclusion body that is seen in the nucleus of myeloma cells is called the Dutcher body (Figs. 35.6 and 35.7). The presence of Russell bodies represents active production of immunoglobulins and can be seen in normal active plasma cells. Dutcher bodies, on the other hand, are only seen in pathologic conditions, such as MM, lymphoplasmacytic lymphoma, and some marginal zone B-cell lymphomas.

Fig. 35.6 A bone marrow aspirate demonstrates Dutcher bodies in two plasma cells. Wright – Giemsa, × 100

MM is usually confine to the bone marrow [1–3]. Extramedullary involvement is rare and is usually associated with unfavorable prognosis, particularly for those infiltratin the skin or the central nervous system. Plasma cells are seldom seen

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Fig. 35.7 A bone marrow biopsy reveals many Dutcher bodies in the nuclei of myeloma cells (arrow). H&E, × 100

in the blood, except for cases of plasma cell leukemia. However, rouleaux formation is frequently present in the peripheral smears. To determine the nature of plasma cell infiltratio in the bone marrow, immunophenotyping should be performed by immunohistochemistry or f ow cytometry [1–3]. Immunohistochemical demonstration of a predominant kappa or lambda light chain by immunohistochemical stain or RNA in situ hybridization is usually sufficien to identify the monoclonal nature of plasma cells and establish the diagnosis of MM. CD138 and CD38 are useful to highlight the plasma cells, in case the light chain staining does not work. The demonstration of CD56 on the plasma cells denotes their malignant nature, as normal plasma cells are negative for CD56. Myeloma cells are usually negative for mature B-cell antigens, such as CD19 and CD20, but are reactive to CD79a. The above immunophenotype can also be demonstrated by fl w cytometry. Clinically, MM patients usually have low back pain or pathologic fracture. The clinical signs for end-organ damage include hypercalcemia, renal insufficien y, anemia, and bone lesions, which is referred to as CRAB and is used as clinical diagnostic criteria for MM [2,3]. The demonstration of osteolytic bone lesion by imaging techniques and the demonstration of monoclonal gammopathy in serum and urine samples by protein electrophoresis and immunofixatio may further substantiate the diagnosis. However, morphologic diagnosis is still required to evaluate the tumor burden for therapeutic monitoring. In MM, there appear to be two pathways of pathogenesis [4]. Nonhyperdiploid tumors have a very high incidence of IgH translocations, involving f ve recurrent partners (11q13, 6p21, 4p16, 16q23, and 20q11) and a relative high incidence of chromosome 13/13q14 loss. Hyperdiploid tumors are more frequently associated with multiple trisomies involving chromosomes 3, 5, 7, 11, 15, 19, and 21.

References 1. McKenna RW, Kyle RA, Kuehl WM, et al. Plasma cell neoplasms. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, Framce. IARC Press, 2008, 200–213. 2. The International Myeloma Working Group. Criteria for the classificatio of monoclonal gammopathies, multiple myeloma and related disorders: A report of the International Myeloma Working Group. Br J Haematol 2003;121:749–757. 3. Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J Med 2004;351:1860–1873. 4. Hideshima T, Bergsagel PL, Kuehl WM, et al. Advances in biology of multiple myeloma: Clinical applications. Blood 2004;104:607–618.

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Case 36 A 76-year-old man presented with weakness and dyspnea for a week. The patient had been treated for plasma cell myeloma for the past three years. Laboratory examination revealed anemia, thrombocytopenia, and leukocytosis. The total leukocyte count was 13, 500/␮l with 74% lymphocytes by automatic cell count. However, manual examination revealed that many of the “lymphocytes” were immature plasma cells. Further examination showed that his ␤2 -microglobulin was 2.8 mg/l and lactate dehydrogenase 411 mg/dl. His immunoglobulin study demonstrated decreases in all immunoglobulins as compared with the levels six months ago. The morphology of the tumor cells in the peripheral blood and bone marrow is depicted in Figs. 36.1, 36.2 and 36.3.

Fig. 36.1 A peripheral blood smear shows three plasmablasts, which have a large nucleus with immature chromatin pattern and a high nuclear/cytoplasmic ratio. Wright – Giemsa, × 100

Case 36

Fig. 36.2 Bone marrow aspirate reveals a large cluster of plamablasts. One of them shows four nuclei. Wright – Giemsa, × 100

Fig. 36.3 Bone marrow biopsy shows many immature plasma cells with prominent nucleoli. A few of them are binucleated. H&E, × 100

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Differential diagnoses: Plasma cell leukemia versus lymphoblastic leukemia.

Further Testing Flow cytometry identifie a population of CD38- and CD138-positive population with negative CD10, CD19, CD56, and terminal deoxynucleotidyl transferase. A dim monoclonal cytoplasmic lambda light chain is demonstrated.

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Discussion Plasma cell leukemia (PCL) accounts for approximately 2% of cases of plasma cell dysplasia. It is define by the presence of more than 2, 000/␮l or 20% of plasma cells in the peripheral blood of the patient. About 60% of PCL is considered primary or de novo and 40% are secondary, usually developing at the terminal stage of plasma cell myeloma. Patients with primary PCL are usually younger, and have a higher incidence of hepatosplenomegaly and lymphadenopathy, a higher platelet count, fewer lytic bone lesions, a lower serum paraprotein level, and longer survival than patients with secondary PCL (7 months versus 2 months) [1–3]. Interestingly, a cell marker, CD28, may help to distinguish primary from secondary PCL, as it is seldom demonstrated in the former [4]. When the secondary PCL case is composed of plasmablasts, as seen in the current case, the immunoglobulin levels drop because plasmablasts are unable to produce paraproteins. PCL is considered a distinct clinicopathologic entity and it is important to distinguish PCL from the non-leukemic form of myeloma because the prognosis and treatment are markedly different between these two entities. Clinically, PCL patients display a higher prevalence of Durie – Salmon stage III disease, extramedullary involvement, more prominent anemia, thrombocytopenia, hypercalcemia, renal function impairment, and higher serum levels of lactate dehydrogenase and ␤2 microglobulin than those with myeloma [3, 5]. In addition, PCL cases have higher incidence of light chain disease, IgD, and IgE gammopathy but lower incidence of IgA gammopathy than those of myeloma [1, 5]. In terms of immunophenotype, PCL is similar to myeloma in the expression of CD38 and CD138, but differs in the expression of CD20, CD56, CD117, and HLA-DR [3, 5]. Myeloma shows frequent expression of CD56, CD117, and HLADR, while PCL has a higher frequency of CD20 expression. As CD56 plays an important role in anchoring plasma cells to bone marrow stroma, the lack of CD56 may partly explain why the plasma cells in PCL appear in the peripheral blood. CD56 is also associated with a good prognosis, whereas CD20 is associated with shorter survival [5]. In DNA analysis, myeloma cases have a high frequency of hyperdiploidy, which is associated with a good prognosis, while PCL cases show a high prevalence of diploidy or hypodiploidy that usually confers an unfavorable prognosis [4, 5]. PCL also differs from myeloma in cytogenetics. PCL has a higher incidence of chromosome 13 monosomy than myeloma (85 versus 26%). This aberrant karyotype is associated with a short survival [5]. On the other hand, many chromosomal abnormalities found in myeloma, such as trisomies 3, 7, 11, 15, and 17, are usually absent in PCL [5]. Finally, PCL patients show a poor response to melphalan and prednisone compared with polychemotherapy, while such difference has not been observed in myeloma patients [5]. Since PCL is a highly deadly disease, aggressive therapy, including bone marrow transplant, should be started in the initial treatment.

References 1. McKenna RW, Kyle RA, Kuehl WM. Plasma cell 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, 200–213. 2. The International Myeloma Working Group: Criteria for the classificatio of monoclonal gammopathies, multiple myeloma and related disorders: A report of the International Myeloma Working Group. Br J Hematol 2003;121:749–757. 3. Jim´enez-Zepeda VH, Dominguez VJ. Plasma cell leukemia: A rare condition. Ann Hematol 2006;85:263–267. 4. Blad´e J, Kyle RA. Nonsecretory myeloma, immunoglobulin D myeloma and plasma cell leukemia. Hemat/Oncol Clin North Am 1999;13:1259–1272. 5. Garcia-Sanz R, Orf˜ao A, Gonz´alez M, et al. Primary plasma cell leukemia: Clinical, immunophenotypic, DNA ploidy, and cytogenetic characteristics. Blood 1999;93:1032–1037.

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Case 37 A 64-year-old man presented with a rapidly growing cutaneous mass on the right leg. He was diagnosed plasma cell myeloma six years ago with multiple osteolytic bone lesions in the frontal parietal bones, right distal ulnar, left femur, and L4 vertebral body. Immunoglobulin studies confirme the diagnosis of lambda light chain disease. He subsequently developed a metastatic lesion in the nasopharynx. The histologic features of the skin biopsy are illustrated in Figs. 37.1 and 37.2.

Fig. 37.1 Cutaneous plasmacytoma shows a highly pleomorphic population consisting of atypical plasma cells and many multinucleated giant cells. H&E × 20

Case 37

Fig. 37.2 Higher magnificatio of Fig. 37.1 demonstrates atypical plasma cells and several multinucleated giant cells. H&E, × 60

Differential diagnoses: Cutaneous myeloma, lymphomas or carcinoma.

Further Testing Immunohistochemical stains: Lambda light chain: positive (Fig. 37.3). Kappa light chain: negative (Fig. 37.4).

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Fig. 37.3 The tumor cells are positive for lambda stain. Immunoperoxidase, × 40

Fig. 37.4 The tumor cells are negative for kappa stain. Immunoperoxidase, × 40

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Discussion Plasmacytoma is a well-circumscribed tumor mass consisting of a monoclonal plasma cell population. They may occur in bone or soft tissues, and are called solitary bone and extramedullary (extraosseous) plasmacytoma, respectively. Before a diagnosis is made, it is important to exclude a systemic disease for the absence of hypercalcemia, anemia, suppression of uninvolved immunoglobulins, and renal insufficien y that are attributable to myeloma [1]. Osseous plasmacytoma is a localized bone tumor consisting of a monoclonal plasma cell population and appearing as a solitary lytic lesion on radiological examination [2]. Strict staging criteria, including normal CT scan and magnetic resonance imaging (MRI) studies of the axial skeleton and the long bones, are required for a definit ve diagnosis [3]. In rare occasions, “solitary” plasmacytoma may present in multiple sites [4]. It is important to distinguish solitary plasmacytoma from extramedullary and extraosseous multiple myeloma, in which the patient has coexistent plasma cell myeloma and which may confer a very unfavorable prognosis [5]. For instance, involvement of the central nervous system is associated with plasmablastic morphology, poor cytogenetic abnormalities, high tumor burden and high lactate dehydrogenase levels [5]. Cutaneous involvement usually occurs in the late stages of myeloma as a reflectio of increased tumor cell burden and is often composed of immature plasmablasts even when the bone marrow contains predominantly mature plasma cells [6]. On the other hand, solitary plasmacytoma can be treated with radiation therapy with resultant long-term disease-free survival in approximately 30% of solitary bone plasmacytomas and 65% of extraosseous plasmacytomas [1]. However, 55% of osseous plasmacytomas may progress to myeloma in 10 years and 15% of extraosseous plasmacytomas develop myeloma [2]. Osseous plasmacytoma often involves the vertebrae (Fig. 37.5), ribs, skull, pelvis, femur, clavicle, and scapula [2]. Patients usually present with bone pain, spinal cord or nerve root compression or a pathological fracture from a single bone lesion [2, 3]. Monoclonal gammopathy is usually present at low levels in 24–72% of patients and can be used for therapeutic monitoring [1]. Otherwise MRI should be used to follow the patient. Extraosseous plasmacytoma typically occurs in the head and neck region, particularly the oropharynx, nasopharynx (Figs. 37.6 and 37.7), sinuses, and larynx [2]. These patients may present with symptoms such as nasal discharge, epistaxis, nasal obstruction, sore throat, hoarseness, or hemoptysis [3]. When the gastrointestinal tract is involved, extranodal

Fig. 37.5 A solitary plasmacytoma from the cell block of a vertebral mass shows a pure plasma cell population. H&E, × 40

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Fig. 37.6 A solitary plasmacytoma from the nasopharynx reveals many atypical plasma cells. H&E, × 20

marginal zone B-cell lymphoma with extreme plasmacytic differentiation should be excluded [2]. Reactive plasma cell granuloma should be ruled out by immunohistochemical stains for kappa and lambda light chains [2]. If the result is inconclusive, mRNA hybridization should be used to clarify the clonality. About 15–20% of patients may have monoclonal gammopathy. The immunophenotype and karyotype of solitary extramedullary plasmacytoma do not seem to differ from those of plasma cell myeloma.

Fig. 37.7 Higher magnificatio of Fig. 37.6 shows the atypical plasma cells with frequent binucleation. H&E, × 60

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References 1. Weber DM. Solitary bone and extramedullary plasmacytoma. Hematology, Am Soc Hematol Educ Program 2005; 373–376. 2. McKenna RW, Kyle RA, Kuehl WM, et al. Plasma cell 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; 200–213. 3. Dimophoulos MA, Kiamouris C, Moulopoulos LA. Solitary plasmacytoma of bone and extramedullary plasmacytoma. Hematol Oncol Clin North Am 1999;13:1249–1257. 4. The International Myeloma Working Group. Criteria for the classificatio of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 2003;121:749–757. 5. Damaj G, Mohty M, Vey N, et al. Features of extramedullary and extraosseous multiple myeloma: a report of 19 patients from a single center. Eur J Haematol 2004;73:402–406. 6. Requena L, Kutzner H, Palmedo G, et al. Cutaneous involvement in multiple myeloma. Arch Dermatol 2003;139:475–486.

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Case 38 A 56-year-old man presented with a long history of heartburn and abdominal pain. A stool guaiac test was positive. Serological test demonstrated positive result for Helicobacter pylori. The patient received antibiotics for the treatment of chronic gastritis without obvious improvement. A gastric biopsy was performed (Figs. 38.1, 38.2, 38.3, 38.4, 38.5, and 38.6).

Fig. 38.1 Gastric biopsy shows extensive lymphoid infiltratio replacing normal mucosal structures. H&E, × 10

Case 38

Fig. 38.2 Gastric biopsy reveals a reactive follicle with expansion of the marginal zone and destruction of the peripheral tissues. H&E, × 10

Fig. 38.3 Lymphoepitheial lesion demonstrated in another case of MALT lymphoma. H&E, × 20

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Fig. 38.4 Three lymphoid follicles in the mucosa: the upper one shows complete follicular colonization. H&E, × 10

Fig. 38.5 Plasma cell infiltratio is demonstrated in the gastric mucosa. H&E, × 60

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Fig. 38.6 A few Dutcher bodies (arrow) are present among a cluster of plasma cells. H&E, × 100

Differential diagnoses: chronic gastritis versus gastric lymphoma.

Further Studies CD20 stain: positive for most lymphoid cells (Fig. 38.7) Cytokeratin stain: positive for gastric glands (Fig. 38.8) Cytogenetic karyotyping: t(11;18)(q21;q21)

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Fig. 38.7 CD20 staining reveals predominantly B cells in the lymphoid infiltrate Immunoperoxidase, × 20

Fig. 38.8 Cytokeratin staining demonstrates a few fragmented acini, representing lymphoepithelial lesion. Immunoperoxidase, × 20

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Discussion Extranodal marginal zone B-cell lymphoma of the stomach (gastric MALT lymphoma) is a well-define clinicopathologic entity. It is one of the rare lymphomas in which its association with a infectious agent, H. pylori, is confirme and its eradication becomes an effective treatment of the early lesion [1, 2]. The normal gastric mucosa is devoid of lymphoid follicles, and therefore the mucosal associated lymphoid tissue (MALT) is induced firs by chronic inflammatio secondary to H. pylori or other infection. Proliferation of B cells is secondary to specifi activation of reactive T cells by H. pylori and cytokines. It is the presence of a continuous antigenic stimulation in combination with other microenvironmental factors that finall leads to the malignant transformation. The requirement for mucosal T-cell mediation of this pathologic process may explain why most MALT lymphomas remain localized and why about two-thirds of cases regress after H. pylori eradication [3, 4]. On the basis of this pathologic mechanism, it is understandable that the gastric lesion usually shows reactive lymphoid follicles and frequent plasma cell differentiation. The characteristic histologic pattern is the great expansion of the marginal zone of the reactive follicles, infiltratin and destroying the surrounding gastric acini, a phenomenon referred to as lymphoepithelial lesion [1–3]. This is probably the most important feature for the diagnosis of MALT lymphoma. Plasma cell infiltratio can be seen in many conditions and it does not distinguish lymphoma from chronic gastritis. However, the presence of the Dutcher body, an intranuclear inclusion, is relatively specific The lymphoma cells may also infiltrat back to the germinal center and replace it, which is another diagnostic feature, usually referred to as follicular colonization. The lymphoma cells are usually composed of small to medium-sized lymphocytes with cleaved nuclei reminiscent of the centrocytes in the germinal center, frequently referred to as centrocyte-like cells. In some cases, the tumor cells may assume the morphology of monocytoid B cells that have pale cytoplasm and well-define cell border. In the remaining cases, the tumor cells can be similar to small lymphocytes. A mixed population of these three cell types in different proportion can also be seen. As mentioned before, plasma cell differentiation is common. In addition, scattered transformed blasts (large lymphoid cells) are frequently present. The definitio of high-grade transformation is controversial. The suggested cutoff points include more than 10% large cells among the tumor cells or the presence of clusters of more than 20 large cells [3]. The WHO does not divide MALT lymphoma into low- and high-grade. If substantial large cells are present, it should be considered diffuse large B-cell lymphoma [1]. There are no specifi markers for MALT lymphoma. The role of immunophenotyping is to demonstrate a monoclonal Bcell population (CD19+, CD20+, CD22+, CD79a+, bcl-2+) with the absence of specifi markers for other non-Hodgkin lymphomas [1–3]. For instance, the absence of CD5 and CD23 excludes the diagnosis of small lymphocytic lymphoma and chronic lymphocytic leukemia. The absence of CD5 and bcl-1 rules out mantle cell lymphoma. Negative reactions to CD10 and bcl-6 in the germinal centers contradict the diagnosis of follicular lymphoma. Other markers that may facilitate the diagnosis are CD21, CD23, and CD35 for the demonstration of the follicular dendritic cell meshwork, so that follicular colonization can be readily recognized. Cytokeratin stain may highlight the lymphoepithelial lesions. The most common chromosome abnormality demonstrated in gastric MALT lymphoma is t(11;18)(q21;q21) with a frequency varying from 13.5 to 35% [1–4]. Molecular characterization has demonstated the inhibitor of apoptosis 2 (API2) gene on 11q21 and the MALT lymphoma-associated translocation (MALT1) gene on chromosome 18q21. The API2-MALT1 fusion gene and protein lead to increased inhibition of apoptosis with a resultant survival advantage of the tumor cells, independent of antigen. Cases with t(11;18) are unresponsive to H. pylori eradication therapy, partly because of the autonomous growth of the tumor cells and partly because most such cases are H. pylori-negative. Another characteristic feature of t(11;18) is the association of nuclear bcl-10 protein expression, in contrast to the normal expression of cytoplasmic bcl-10 protein in the germinal center cells [5]. Both the API2-MALT1 fusion protein and Bcl-10 protein may activate the nuclear factor-␬B pathway, which drives antigen-independent growth of the lymphoma cells. A second nonrandom translocation is t(1;14), which occurs in 1% to 2% of MALT lymphoma involving the lung, intestine and salivary gland [1]. The t(3;14)(.p14.1;q32) translocation is more frequently seen in ocular adnexa, skin, and thyroid, and t(14;18)(q32;q21) is often seen in stomach, ocular adnexa, lung, and thyroid [1]. Clinically, the most common presentations are nonspecifi dyspepsia and epigastic pain. The patient may have a long history of gastric symptoms, but those symptoms may be due to the precedent chronic gastritis.

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References 1. Isaacson PG, Chott A, Nakamura S, et al. Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classificatio of Tumours of Haematolpoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 214–217. 2. Isaacson PG. Recent developments in our understanding of gastric lymphomas. Am J Surg Pathol 1996;20 (Suppl 1):S1–S7. 3. Zucca E, Bertoni F, Roggero E, et al. The gastric marginal zone B-cell lymphoma of MALT type. Blood 2000;96:410–419. 4. Kahl BS. Update: gastric MALT lymphoma. Curr Opin Oncol 2003;15:347–352. 5. Ye H, Dogan A, Karran L, et al. BCL-10 expression in normal and neoplastic lymphoid tissue. Nuclear localization in MALT lymphoma. Am J Pathol 2000;157:1147–1154.

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Case 39 A 58-year-old man presented with a three months’ history of dyspnea on exertion, shortness of breath and pleuritic chest pain. The patient had no history of chronic lung disease and denied any smoking history. Chest X-ray showed bilateral lung masses with hilar involvement. The patient had undergone transbronchial biopsy and brushings, but both yielded no diagnostic features. A lung biopsy was then obtained through open thoractomy. The histologic features of the lung biopsy are illustrated in Figs. 39.1, 39.2, 39.3 and 39.4.

Fig. 39.1 A lung biopsy shows lymphoma cells in the alveolar wall (lymphangitic tracking). H&E, × 60

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Fig. 39.2 A lung biopsy reveals the marginal zone distribution of the lymphoma cells around the reactive follicles. The tumor cells expand to the adjacent bronchiolar wall and destroy part of the epithelium. H&E, × 10

Fig. 39.3 The germinal center and the expanded marginal zone are illustrated in two tumor nodules. H&E, × 40

Case 39

Fig. 39.4 The tumor cells infiltrat and partly destroy the bronchiolar epithelium (lymphoepithelial lesion). H&E, × 20

Differential diagnoses: pulmonary carcinoma versus lymphoma.

Further Studies Immunohistochemical stains: CD20 stain: positive (Fig. 39.5) CD3 stain: negative (Fig. 39.6) Flow cytometry: Positive for CD19, CD20, and kappa, but negative for CD5, CD10, CD23, and lambda.

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Fig. 39.5 CD20 stains for most of the tumor cells. Immuno-alkaline phosphatase, × 20

Fig. 39.6 CD3 highlights scattered T-lymphocytes. Immuno-alkaline phosphatase, × 20

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Discussion Extranodal marginal zone B-cell lymphoma or mucosa-associated lymphoid tissue (MALT) lymphoma is the most common primary pulmonary lymphoma, although it accounts for only 3.6% of extranodal lymphomas [1]. However, it is important to diagnose this rare lymphoma because it is markedly different from carcinomas of the lung in terms of clinical course, prognosis and treatment. In fact, the pulmonary MALT lymphoma has the most favorable behavior among MALT lymphomas of all other organs [2]. Clinically, the patient may be asymptomatic or may have nonspecifi symptoms, such as cough, dyspnea, chest pain, wheezing, and hemoptysis [1, 3]. Although a large lung mass or bilateral lesions may be found by imaging techniques, aggressive treatment is not required for MALT lymphoma [1]. Frequently, a complete resection is considered curative. A definit ve diagnosis of pulmonary MALT lymphoma can be only achieved by pathologic examination. Specimens may be obtained through open thoracotomy, thoracoscopy or CT-guided biopsy, but the positive rate of the CT-guided biopsy is only 25% [1]. As in other MALT lymphomas, chronic inflammatio is a predisposing factor. The characteristic feature is a marginal zone distribution of the lymphoma cells around individual reactive or “acquired” follicles with expansion to and infiltratio of the adjacent “dome” of bronchiolar epithelium (lymphoepithelial lesion). This is described as the lymphoma unit by Isaason and Norton [2]. As the lymphoma units coalesce, a nodular pattern is formed. In between the nodules, there are many obliterated bronchiolar lumens. The reactive lymphoid follicles may be replaced by the lymphoma cells, a process described as follicular colonization. A specifi feature is the spreading of the lymphoma in the lymphatics along the alveolar wall, interlobular septa, visceral pleura, and bronchovascular bundles, which is termed lymphangitic tracking [3]. Cytologically, the tumor cells may appear as small lymphocytes, centrocyte-like cells or monocytoid B cells, but most cases of the pulmonary MALT lymphoma are composed of small lymphocytes [2, 3]. Scattered transformed blasts are frequently present. Plasma cells or plasmacytoid cells with Dutcher bodies are also a constant feature. When large clusters or sheets of blasts are seen, this should be classifie as transformation to diffuse large B-cell lymphoma and not high-grade MALT lymphoma [2, 3]. Immunohistochemistry may demonstrate CD20 and CD79a on the tumor cells. The negative reactions to CD3, CD5, CD10, CD23, CD45RO, bcl-6, and cyclin D1 may help the exclusion of small lymphocytic lymphoma, mantle cell lymphoma and follicular lymphoma [4]. Cytogenetically, the most common aberrant karyotype is t(11;18)(q21;q21). Other two karyotypes, t(14;18)(q32;q21) and t(1;14)(q32;q21), are also specifi for MALT lymphomas but are seen in only a few cases of pulmonary MALT lymphoma [5].

References 1. Graham BB, Mathisen DJ, Mark EJ, et al. Primary pulmonary lymphoma. Ann Thorac Surg 2005;80:1248–1253. 2. Isaacson PG, Norton AJ. Extranodal Lymphomas, Edinburgh, Churchill Livingstone, 1994; 85–102. 3. Burke JS. Are there site-specifi differences among the MALT lymphomas – morphologic, clinical? Am J Clin Pathol 1999;111(Suppl 1):S133–139. 4. Xu HY, Jin T, Li RY, et al. Diagnosis and treatment of pulmonary mucosa-associated lymphoid tissue lymphoma. Chin Med J 2007;120: 648–651. 5. Chuang SS, Liu H, Ye H, et al. Pulmonary mucosa-associated lymphoid tissue lymphoma with strong nuclear B-cell CLL/lymphoma 10 (BCL-10) expression and novel translation t(1:2)(p22;p12)/immunoglobulin ␬ chain-BCL 10. J Clin Pathol 2007; 60:727–728.

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Case 40 A 56-year-old woman presented with a swollen neck at the left side for a few weeks. She had had a history of dry mouth and dry eyes for three years. Physical examination discovered an enlarged parotid gland and an excisional biopsy was performed (Figs. 40.1, 40.2, 40.3, 40.4, and 40.5).

Fig. 40.1 The salivary gland shows extensive small lymphoid cell infiltration leaving only a small rim of normal salivary tissue at the left side of this field Note a residual germinal center (short arrow) and an epimyoepithelial island (arrow) present at the right side. H&E, × 10

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Fig. 40.2 Another f eld shows three epimyoepithelial islands and a normal salivary duct present in a sea of small centrocyte-like cells. H&E, × 20

Fig. 40.3 Higher magnificatio of Fig. 41.2 shows an epimyoepithelial island with lymphoepithelial lesion. H&E, × 40

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Fig. 40.4 Another area reveals clusters of pale-staining monocytoid B cells. H&E, × 10

Fig. 40.5 Higher magnificatio of Fig. 40.4 shows an epimyoepithelial island being surrounded and infiltrate by monocytoid B cells. H&E, × 40

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Differential diagnoses: Myoepithelial sialadenitis versus parotid lymphomas.

Further Studies Immunohistochemistry: The lymphoid cells are positive for CD20, CD79a, and bcl-2, but are negative for CD3, CD5, CD10, and cyclin D1. Immunoglobulin heavy chain gene rearrangement: monoclonal pattern.

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Discussion Extranodal marginal zone B-cell lymphoma or mucosa-associated lymphoid tissue (MALT) lymphoma of the salivary gland is difficul to distinguish from benign lymphoepithelial lesion or myoepithelial sialadenitis (MESA) until a full-blown picture of lymphoma emerges [1]. The clinical presentation of MESA is frequently Sj¨ogren syndrome or other autoimmune disorders, which is also the predisposing condition in salivary gland MALT lymphoma, as in the current case. Morphologically, both are characterized by the presence of the epimyoepithelial islands, which is the result of the condensation of the ductal epithelium in the salivary gland with gradual narrowing of the lumen [1]. Lymphoid infiltratio of the ductal epithelium (lymphoepithelial lesion) is also present in both conditions. Furthermore, immunoglobulin heavy chain gene rearrangement can also be present in some cases of MESA [2]. Clinically, both MALT and MESA may have an indolent clinical course. As the disease progresses, MALT lymphoma shows a broad collar or halo of lymphoma cells around the epimyoepithelial islands or dilated ducts, growing to large sheets of tumor cells between the residual lymphoid follicles and finall replacing the germinal centers of the follicles [1–6]. The replacement of the germinal center cells with the lymphoma cells is called follicular colonization and is one of the characteristics of MALT lymphoma. Cytologically, the tumor cells may assume the morphology of small lymphocytes, centrocyte-like cells or monocytoid B cells. MALT lymphoma in salivary gland is characterized by the presence of predominantly monocytoid B cells [1–6]. Plasma cell differentiation is a frequent feature of this lymphoma, mimicking lymphoplasmacytic lymphoma. However, lymphoplasmacytic lymphoma is usually associated with macroglobulinemia and is a bone-marrow-based lymphoma, seldom involving the salivary gland [4]. In addition, scattered transformed blast cells or centroblasts are frequently present. When a large number of blasts are present in a MALT lymphoma, it is considered transformation to a diffuse large B-cell lymphoma and not a high-grade MALT lymphoma [2]. A small lymph node may be embedded in the parotid gland and may contain salivary ducts and acini in the medullary region (Figs. 40.6 and 40.7) [6]. However, the lymph node is usually separated from the residual parotid tissue by a capsulelike structure. Lymphoma can arise in the intraparotid lymph node and should be distinguished from primary salivary gland lymphoma. MALT lymphoma is the most common type of primary salivary gland lymphoma, but follicular lymphoma, small lymphocytic lymphoma, and others may be seen in the intraparotid lymph nodes [1].

Fig. 40.6 An intraparotid lymph node is separated from the salivary gland tissue by a fibrou capsule. Note a germinal center at the right upper corner of this field H&E, × 10

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Fig. 40.7 The intraparotid lymph node contains many salivary ducts. H&E, × 20

Immunohistochemistry may help to distinguish MALT lymphoma from MESA by demonstrating light chain restriction in both the B lymphocytes and plasma cells [4–6]. However, immunoglobulin stains may not be conclusive. In those cases, bcl2 positivity define lymphoma. MALT lymphoma is also positive for other B-cell antigens, such as CD19, CD20, CD22, and CD79a. The negative reactions to CD5, CD10, CD23, bcl-1, and bcl-6 are useful to exclude small lymphocytic lymphoma, mantle cell lymphoma, and follicular lymphoma.

References 1. Isaacson PG, Norton AJ. Extranodal Lymphomas, Edinburgh, Churchill Livingstone, 1994; 67–83. 2. Isaacson PG. Mucosa-associated lymphoid tissue lymphoma. Semin Hematol 1999;36:139–147. 3. Burke JS. Are there site-specifi differences among the MALT lymphomas – morphologic, clinical? Am J Clin Pathol 1999;111(Suppl 1):S133–139. 4. Harris NL, Isaacson PG. What are the criteria for distinguishing MALT from non-MALT lymphoma at extranodal sites? Am J Clin Pathol 1999;111(Suppl 1): S126–132. 5. Bacon CM Du MQ, Dogan A. Mucosa-associated lymphoid tissue (MALT) lymphoma: a practical guide for pathologists. J Clin Pathol 2007;60:361–372. 6. Kojima M, Shimizu K, Nishkawa M, et al. Primary salivary gland lymphoma among Japanese: A clinicopathological study of 30 cases. Leuk Lymphoma 2007;48:1793–1798.

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Case 41 An 81-year-old man presented with abdominal bloating for one month. His family physician treated him for diverticulitis but a 7-day course of antibiotics did not improve his condition. Physical examination showed no peripheral lymphadenopathy and no hepatosplenomegaly. A CT scan revealed that there were masses in the abdomen and chest. An exploratory laparotomy found multiple enlarged mesenteric lymph nodes. A lymph node biopsy was done and is depicted in Figs. 41.1 and 41.2.

Fig. 41.1 Lymph node biopsy reveals a multiple nodular pattern. H&E, × 10

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Fig. 41.2 Lymph node biopsy shows a residual germinal center surrounded by a greatly expanded area of small lymphoma cells. No mantle zone is recognizable. H&E, × 20

Differential diagnoses: mantle cell lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma and small lymphocytic lymphoma.

Further Testing Flow cytometry of the lymph node showed positive reactions to CD19, CD20 and lambda, but negative reactions to CD5, CD10, CD23, kappa and partial positive FMC-7. Immunohistochemical stains: CD3: negative for tumor cells (Fig. 41.3) CD20: positive for tumor cells (Fig. 41.4) CD79a: positive for tumor cells (Fig. 41.5) CD5: negative for tumor cells (Fig. 41.6) CD10: negative for tumor cells (Fig. 41.7) bcl-2: negative for tumor cells (Fig. 41.8)

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Fig. 41.3 The lymphoma nodule is negative for CD3 stain. Immunoperoxidase, × 20

Fig. 41.4 The lymphoma nodule is positive for CD20 stain. Immunoperoxidase, × 20

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Fig. 41.5 The lymphoma nodule is positive for CD79a stain. Immunoperoxidase, × 20

Fig. 41.6 The lymphoma nodule is negative for CD5 stain. Immunoperoxidase, × 20

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Fig. 41.7 The lymphoma nodule is negative for CD10 stain. The widely spaced centrocyte-like cell pattern is well illustrated in this field Immunoperoxidase, × 20

Fig. 41.8 The lymphoma nodule is negative for bcl-2 stain. Immunoperoxidase, × 20

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Discussion The marginal zone is an anatomically distinct B-cell compartment mainly seen in the spleen white pulp, Peyer’s patches of the small intestine, and tonsils, but it is inconspicuous in lymph nodes except for the mesenteric lymph node. Therefore, nodal marginal zone B-cell lymphoma (NMZBL) is rare, accounting for only 1.8% of lymphoid neoplasms [1]. The World Health Organization (WHO) classificatio includes three types of margin zone B-cell lymphoma, the extranodal, nodal and splenic. The relationship of these three tumors is still not conclusive, but they are three distinct clinicopathologic entities. It is particularly important to distinguish NMZBL from the extranodal form (lymphoma of mucosa-associated lymphoid tissue or MALT type) because the therapeutic response and prognosis are more favorable in the latter. Since MALT lymphoma may spread to lymph nodes, it is mandatory to exclude the coexistence of extranodal involvement before diagnosing NMZBL. Cytogenetically, the extranodal form is frequently associated with t(11;18)(q21;q21) and trisomy 3, but aberrant karyotypes are seldom seen in NMZBL, except for a few cases of trisomies 3, 7, and 18 [1]. Histologically, the infiltratio patterns include marginal zone-like/perifollicular, sinusoidal, nodular, diffuse, and inverse follicular [2, 3]. A combination of different patterns may be present in the same cases. In fact, the different patterns may represent various developmental stages. In the early stage, there is perifollicular proliferation of neoplastic cells, producing the marginal pattern (Fig. 41.9). The progressive expansion of interfollicular areas results in a nodular pattern (Fig. 41.10). In advanced stage, the lymph node architecture is entirely effaced with the resultant diffuse pattern. Frequently, the residual germinal center is infiltrate by the tumor cells, forming a follicular colonization pattern.

Fig. 41.9 Lymph node biopsy shows a residual follicle with a germinal center surrounded by a thin rim of mantle zone. The perifollicular area is infiltrate by pale-staining monocytoid B cells. H&E, × 20

The histologic patterns can also be divided into the splenic type and the MALT type [4]. The splenic type is characterized by a nodular proliferation of large and small lymphoid cells surrounding and infiltratin residual germinal centers. The mantle corona is absent in most cases. The nodal architecture is totally effaced. The MALT type is characterized by a predominantly perivascular, perisinusoidal, and parafollicular infiltration The mantle zone is preserved between the germinal centers and the tumor cells. Reactive follicles are always present. Cytologically, the tumor cells can be monocytoid B cell, centrocyte-like B cell or small B lymphocytes [1]. The monocytoid B cells are characterized by their medium-sized, abundant clear cytoplasm, centrally located bean-shaped or round nuclei, and fin chromatin pattern. The centrocyte-like cells are small cells with irregular nuclei and are widely spaced

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Fig. 41.10 Lymph node biopsy reveals a tumor aggregate composed of pale-staining monocytoid B cells. H&E, × 20

between cells. When monocytoid B cells are predominant, the perifollicular or nodular infiltratio is readily visualized as pale-staining areas. Large blasts, plasma cells and neutrophils are frequently intermixed with the monocytoid B cells, but are seldom seen when the centrocyte-like cells are prevalent. When the mantle zone pattern is predominant, NMZBL should be distinguished from mantle cell lymphoma. The nodular pattern in NMZBL may mimic follicular lymphoma. Occasionally, a perifollicular monocytoid B-cell proliferation may post a problem in differential diagnosis [5]. However, monocytoid B-cell proliferation secondary to infections (such as in toxoplasmosis) usually occurs in subcapsular sinuses [2]. The differential diagnoses between NMZBL and other lymphomas depend on immunophenotyping. It is a diagnosis by exclusion of similar lymphomas. Essentially, NMZBL is positive for B-cell markers, such as CD19, CD20, CD22, and CD79a. However, it is consistently negative for markers that are specifi for other lymphomas, such as CD5, CD10, CD23, bcl-6, and bcl-1/cyclin D1. The absence of these markers helps exclude mantle cell lymphoma, small lymphocytic lymphoma and follicular lymphoma. The colonized follicles are characterized by the demonstration of the follicular dendrictic cell meshwork with CD21, CD23, or CD35 in the absence of the normal germinal center markers, CD10 and bcl-6. When immunophenotyping fails to exclude other lymphomas, molecular genetic identificatio of abnormal karyotypes such as t(11;14)(q13;q32) or t(14;18)(q32;q21) can be helpful to rule out NMZBL.

References 1. Campo E, Pileri SA, Jaffe ES, et al. Nodal marginal zone B-cell lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. eds., WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2008, 218–219. 2. Arcaini L, Paulli M, Boveri E, et al. Marginal zone-related neoplasms of splenic and nodal origin. Haematologica 2003;88:80–93. 3. Traverse-Glehen A, Felman P, Callet-Bauchu E, et al. A clinicopathological study of nodal marginal zone B-cell lymphoma. A report on 21 cases. Histopathology 2006;48:162–173. 4. Campo E, Miquet R, Krenacs L, et al. Primary nodal marginal zone lymphomas of splenic and MALT type. Am J Surg Pathol 1999;23:59–68. 5. Kojima M, Motoori T, Iijima M, et al. Florid monocytoid B-cell hyperplasia resembling nodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue type. A histological and immunohistochemical study of four cases. Pathol Res Prac 2006;202:877–882.

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Case 42 A 60-year-old man presented with a history of a right groin mass for about three years. A CT scan of the pelvis showed numerous enlarged nodes. Physical examination revealed generalized lymphadenopathy with prominent bilateral axillary and inguinal adenopathy. An inguinal lymph node biopsy was performed (Fig. 42.1). Subsequently, a peripheral blood smear (Fig. 42.2) and a bone marrow biopsy (Fig. 42.3) were also examined.

Fig. 42.1 A lymph node biopsy shows the malignant follicles inside and outside the node. H&E, × 10

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Fig. 42.2 Peripheral blood smear reveals small lymphocytes with cleaved nuclei (arrows). Wright – Giemsa, × 300

Fig. 42.3 Bone marrow biopsy shows paratrabecular lymphoid infiltration characteristic for follicular lymphoma. H&E, × 40

Differential diagnoses: Hodgkin and non-Hodgkin lymphomas.

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Further Studies CD20 stain: positive (Fig. 42.4) CD79a stain: positive (Fig. 42.5) CD10 stain: positive (Fig. 42.6) Bcl-2 stain: positive (Fig. 42.7) Bcl-6 stain: positive (Fig. 42.8) CD21 stain: positive (Fig. 42.9) Ki-67 stain: positive in a high percentage of tumor cells (Fig. 42.10)

Fig. 42.4 Lymph node biopsy with CD20 stain shows selective staining of the malignant follicles. Note the follicles are uniform in size and in shape. Immunoperoxidase, × 10

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Fig. 42.5 CD79a stain of the same area shows positive staining of the malignant follicles. Immunoperoxidase, × 10

Fig. 42.6 CD10 stain of the same area shows weaker staining than other stains. Immunoperoxidase, × 10

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Fig. 42.7 Bcl-2 stain of the same area shows positive staining of malignant follicles. Immunoperoxidase, × 10

Fig. 42.8 Bcl-6 stain shows positive staining of a malignant follicle. Immunoperoxidase, × 20

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Fig. 42.9 CD21 stain demonstrates a follicular dendritic meshwork in the malignant follicles. Immunoperoxidase, × 20

Fig. 42.10 Ki-67 stain demonstrates a high proliferation fraction in the malignant follicles. This represents a subgroup of follicular lymphoma with low histologic grade and high proliferation index carrying an unfavorable prognosis similar to grade 3 follicular lymphoma. Immunoperoxidase, × 20

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Discussion Follicular lymphoma (FL) is one of the most common types of non-Hodgkin lymphomas, accounting for 20–30% of all and 40–50% of adult non-Hodgkin lymphomas [1]. FL is characterized by the presence of multiple nodules mimicking normal lymphoid follicles. In follicular hyperplasia, proliferation takes place in an extended period of time, so that the follicles are variable in size and shape and distributed irregularly [2]. The malignant follicles in FL formed in a relatively short period of time, and therefore the nodules are relatively uniform in size and shape and even in distribution. Unless there is a diffuse infiltratio area in between, the nodules usually show a “back-to-back” pattern. The tumor cells are derived from the germinal center, therefore the mantle zone is attenuated or totally absent. A normal germinal center has a light zone and a dark zone, composed of centrocytes (small and large cleaved cells) and centroblasts (small and large noncleaved cells), respectively. This polarization is no longer present in FL. In comparison to a normal follicle, the FL nodules show much fewer tangible-body macrophage and mitotic figures The differences between follicular hyperplasia and FL are summarized in Table 42.1. Table 42.1 Differentiation between follicular hyperplasia and follicular lymphoma Follicular hyperplasia

Follicular lymphoma

Lymph node architecture

Well preserved

Size and shape of germinal center Distribution of follicles Margin of follicles Mantle zone Polarity in germinal center Mitotic rate Tangible-body macrophages Cells in interfollicular area Surface immunoglobulin CD10 Bcl-2 Bcl-6 t(14;18) translocation

Variable

Completely or partially effaced Uniform

Irregular, well separated Sharp Intact Present High Prominent Normal lymphocytes Polyclonal Present on scattered cells Absent Present in scattered cells Absent

Even, back-to-back pattern Poorly define Absent or attenuated Absent Low Rare Atypical lymphoid cells Monoclonal Present Present Present Present

The World Health Organization (WHO) system adopts the Mann – Berard cell-counting method for grading [3]. The calculation is based on the average number of centroblasts in 10 neoplastic follicles examined under a 40× high-power fiel (hpf). Grade 1 FL is define by the presence of 0–5 centroblasts/hpf, grade 2, 6–15, and grade 3, >15. Grade 3 is further divided into grade 3a and 3b. In the former, centrocytes are still present, whereas the latter shows solid sheets of centroblasts. There are only minor differences in natural history and response to treatment between grades 1 and 2, but grade 3 is clinically and biologically distinct from the other two grades with worse prognosis and is closer to a diffuse large cell lymphoma. A centrocyte is characterized by its nuclei with clefts, indentation, or linear infoldings. The nuclear chromatin is condensed, and the small nucleoli are inconspicuous. The cytoplasm is scant. The centroblasts are two to three times as large as normal lymphocytes. Their nuclei are usually round but occasionally irregular in configuration with vesicular chromatin and one to three membrane-bound nucleoli. Their cytoplasm is scant and is basophilic on Giemsa stain. Centroblasts should be distinguished from follicular dendritic cells, because both cell types have vesicular nuclei of similar size. However, follicular dendritic cells have a distinct central eosinophilic nucleolus and are usually present in pairs with a back-to-back pattern. Diffuse areas are frequently present in various proportions with the follicles. A follicular pattern is define by the presence of 75% of follicular area [3]. A follicular and diffuse pattern should be reported when the follicular area is between 25 and 75%. When the follicular area is <25%, it is designated as minimally follicular. When in doubt, a diffuse area should be verifie by CD21 and CD23 stains, which should be negative [3]. The grade and percentages of follicular and diffuse areas should be included in the diagnosis. In the bone marrow, FL is characterized by the presence of a well-define paratrabecular lymphoid aggregate. FL cells are frequently demonstrated in the peripheral blood as small cleaved cells. In the spleen, the FL cells are confine to the white pulp showing irregular shaped large follicles. If cytologic atypia is not present, it is difficul to distinguish FL from follicular

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hyperplasia in the spleen. In the liver, FL cells usually infiltrat the portal area and may spread beyond the limiting plate of the lobules. The immunophenotype of FL is that of a monoclonal B-cell lymphoma with expression of all the common B-cellassociated antigens (CD19, CD20, CD22, CD24, CD79a) [1, 3]. The specifi markers include CD10, bcl-2, and bcl-6, which are antigens of the follicular center cells. However, CD10 and bcl-6 can also be present in the germinal centers of normal follicles and are not useful in distinguishing between FL and follicular hyperplasia by immunohistochemical stains. Bcl-2 is specifi in identifying the malignant follicles but it also cross-reacts with T lymphocytes and mantle zone B-cells. Therefore, only when it is detected in the germinal center should the diagnosis of FL be considered. Bcl-2 can be also positive in other lymphomas, but in other lymphomas there is usually absence of bcl-2 gene rearrangement. The identificatio of the follicular dendritic cells by the complement receptors (CD21 and CD35) and an immunoglobulin receptor (CD23) is also helpful in denoting the follicular origin of the tumor. However, follicular dendritic cells are not part of the tumor population. Ki-67 can be used to help grading and sometimes it is more reliable than morphologic grading for prediction of the clinical behavior of the tumor [4]. The characteristic karyotype of FL is t(14;18)(q32;q21) translocation [1, 3, 5]. Rare cases may have t(2:18)(p12;q21) or t(18;22)(q21;q11), representing a translocation with the kappa or lambda light chain gene, respectively [5]. The heavy chain gene is located at 14q32, whereas the proto-oncogene, bcl-2, is located at 18;q21. When bcl-2 moves into the proximity of the immunoglobulin (Ig) heavy chain gene enhancer region, it becomes deregulated or activated, and the function bcl-2-Ig fusion protein is overexpressed. The bcl-2 gene encodes for an inner mitochondrial membrane protein that plays the role of blocking programmed cell death (apoptosis). Therefore, cells with abnormal expression of this protein remain in stage G0 in the cell cycle and become immortalized.

References 1. Harris N, Ferry JA. Follicular lymphoma. In Knowles DM, ed. Neoplastic Hematopathology, 2nd ed., Philadelphia, Lippincott Williams & Wilkins, 2001, 805–822. 2. Schnitzer B. The reactive lymphadenopathies. In Knowles DM, ed. Neoplastic Hematopathology, 2nd ed., Philadelphia, Lippincott Williams & Wilkins, 2001;537–568. 3. Harris NL, Swerdlow SH, Jaffe ES, et al. Follicular lymphoma. 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, 220–226. 4. Wang SA, Wang L, Hochberg EP, et al. Low histologic grade follicular lymphoma with high proliferation index: morphologic and clinical features. Am J Surg Pathol 2005;29:1490–1496. 5. de Jong D. Molecular pathogenesis of follicular lymphoma: A cross talk of genetic and immunologic factors. J Clin Oncol 2005;23:6358–6363.

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Case 43 A 56-year-old man presented with a four-month history of a growing throat mass. The patient also had a history of right tonsillectomy a few years ago for the treatment of an infection. He felt fatigue and malaise. Physical examination found a large right tonsillar mass with regular smooth mucosa, measuring roughly 3 × 4 × 4 cm, which was limited to oropharynx. The mass was fir to palpation. No cervical lymphadenopathy was detected. A biopsy of the mass was performed (Figs. 43.1, 43.2, and 43.3).

Fig. 43.1 Tonsillar biopsy shows multiple malignant follicles of relatively uniform size and shape with a back-to-back pattern. H&E, × 10

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Fig. 43.2 Higher magnificatio shows a mixed population of centrocytes and centroblasts (arrow). H&E, × 100

Fig. 43.3 A pair of follicular dendritic cells (arrow) show f attening of the adjacent nuclear membranes and a centrally located prominent nucleolus. H&E, × 100

Case 43

Differential diagnoses: Lymphoma versus carcinoma.

Further Studies Immunohistochemical stains: CD20 stain: positive (Fig. 43.4) CD79a stain: positive (Fig. 43.5) CD10 stain: positive (Fig. 43.6) Bcl-2 stain: positive (Fig. 43.7) Bcl-6 stain: positive Bcl-1 stain: negative (Fig. 43.8) CD3 stain: negative Cytokeratin stain: negative Flow cytometry: A monoclonal kappa B-cell population with positive CD10, CD19, CD20, and bcl-2.

Fig. 43.4 CD20 stain show strong immunoreaction with tumor cells in the nodules. No diffuse area is present. Immunoperoxidase, × 20

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Fig. 43.5 CD79a stain shows similar features. Immunoperoxidase, × 10

Fig. 43.6 CD10 stain shows weaker reaction than other stains. Immunoperoxidase, × 10

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Fig. 43.7 Bcl-2 stain shows strong immunoreaction. Immunoperoxidase, × 20

Fig. 43.8 Bcl-1 stain shows negative reaction with tumor cells. × 20

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Discussion Follicular lymphoma, grade 3 (FL3) is a clinicopathologically distinct entity with marked difference in therapeutic response and prognosis from grades 1 and 2 follicular lymphomas (FL1 and FL2). It accounts for 29% of follicular lymphoma [1]. The clinical course of FL3 is significantl more aggressive than the other two types. While FL1 and FL2 are incurable, FL3 is potentially curable with aggressive treatments similar to that of diffuse large B-cell lymphoma [3]. Morphologically, FL3 shows an average of more than 15 centroblasts in a malignant follicle and is more likely to have diffuse areas than other types. FL3 can be further divided into FL3a and FL3b; the latter is composed of 100% centroblasts. In a review by Bierman, FL3 usually shows lower percentages or absence of expression for the follicular center cell proteins, including CD10, bcl-2, and bcl-6 [1]. The CD10 fluorescenc intensity as measured by fl w cytometry in FL3 was about one half of that of FL1 and FL2. In an immunohistochemical study, CD10 positivity was observed in 91% of FL1 and FL2, 48% of FL3a, and 57% of FL3b. The expression of bcl-2 was 97% in FL1, 96% in FL2, 48% in FL3a, and 57% in FL3b. Lower level of bcl-6 expression in FL3 was reported by a Norwegian group. On the other hand, the proliferation index as demonstrated by Ki-67 staining was reported to be higher in FL3 (>20%) than in FL1 and FL2 by several study groups. The multiple myeloma oncogene 1 (MUM1) is expressed on 50% to 75% of diffuse large B-cell lymphoma and is seldom seen in FL1 and FL2 (7.4%), but it is present in 78.9% of FL3 [1]. Cases that express MUM1 usually show lower percentages of CD10 and bcl-6. A subset of CD10 − /MUM1+ follicular lymphoma was reported from Japan, in which the tumor usually shows a FL3 morphology and is less likely to express bcl-2 protein or bcl-2 translocations [1]. Cytogenetically, FL3b is less likely to carry the t(14;18) translocation and more likely to show the 3q27/bcl-6 aberration that is usually associated with diffuse large B-cell lymphoma [3]. A recent study of gene expression profilin claimed that it could distinguish low-grade from high-grade follicular lymphoma with 100% accuracy and was more reliable for predicting the clinical behavior than morphologic grading [4]. Although this study and others reported that the genes involved in cellcycle regulation and DNA synthesis, such as CXCL12, NEK2, and MAPK1, helped to differentiate indolent and aggressive cases, the study by the National Cancer Institute found that nontumor cells played an important role in the prognosis [5]. This study identifie two discrete gene expression profiles The immune response 1 signature encodes genes expressed by T cells and macrophages and confers a favorable prognosis. The immune response 2 signature includes genes expressed predominantly by monocytes and follicular dendritic cells (FDCs) and confers an unfavorable prognosis. It concluded that the clinical course is mainly influence by the infiltratin nontumor cells and not the genes of the lymphoma cells. This assumption is further supported by other immunologic studies [6]. In the group with a favorable prognosis, the FDC population shows complete presence of CD21/CD23/CD35 and supports a dense, but relatively inactive T-cell infiltrate In the group with poor prognosis, the FDC population shows loss of CD21 and CD23; this represents an activated FDC network that induces highly activated specifi T cells.

References 1. Bierman PJ. Natural history of follicular grade 3 non-Hodgkin’s lymphoma. Curr Opin Oncol 2007;19:433–437. 2. Harris NL, Swerdlow SH, Jaffe ES, et al. Follicular lymphoma. 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, 220–226. 3. Bosga-Bouwer AG, van Imhoff GW, Boonstra R, et al. Follicular lymphoma grade 3B includes 3 cytogenetically define subgroups with primary t(14;18). 3q27, or other translocations: t(14;18) and 3q27 are mutually exclusive. Blood 2003;101:1149–1154. 4. Glas AM, Kersten MJ, Delahaye LJMJ, et al. Gene expression profilin in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment. Blood 2005;105:301–307. 5. Dave SS, Wright G, Tan B, et al. Prediction of survival in follicular lymphoma based on molecular features of tumor infiltratin immune cells. N Engl J Med 2004;351:2159–2169. 6. de Jong D. Molecular pathogenesis of follicular lymphoma: A cross talk of genetic and immunologic factors. J Clin Oncol 2005;23:6358–6363.

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Case 44 A 76-year-old man presented to the ENT clinic with a new onset of a left neck mass. He had no associated pain of the area and denied any weight loss or night sweats. Because of the acute nature of the neck mass, a fine-needl aspiration (FNA) was performed, which showed atypical cells, but was otherwise nondiagnostic. A neck dissection showed a lymph node with lymphoma (Fig. 44.1). A bone marrow biopsy also revealed lymphomatous involvement (Figs. 44.2 and 44.3). After chemotherapy, the patient was in remission for six months, but he subsequently developed splenomegaly and pancytopenia. A splenectomy was performed (Fig. 44.4).

Fig. 44.1 Lymph node biopsy shows three residual germinal centers surrounded by the expanded marginal zone. The 4th nodule at the right lower corner of this fiel may represent follicular colonization by the tumor cells. H&E, × 10

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Fig. 44.2 Bone marrow biopsy reveals an interstitial infiltratio pattern of the tumor cells. H&E, × 40

Fig. 44.3 Bone marrow aspirate demonstrates many atypical lymphoid cells with a few residual normal hematopoietic elements. Wright – Giemsa, × 60

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Fig. 44.4 Splenectomy specimen shows multiple lymphoid aggregates representing the expanded white pulp. H&E, × 10

Differential diagnoses: non-Hodgkin lymphomas.

Further Studies Flow cytometry of lymph node: CD5 94%. CD10 3%, CD19/CD5 36%, CD20 52%, CD23 3%, FMC-7 43%, kappa 43%, lambda 2%. Immunohistochemical stains: Cyclin D1 stain of spleen: positive (Fig. 44.5) CD20 stain of bone marrow: positive (Fig. 44.6) CD5 stain of bone marrow: positive CD3 stain of bone marrow: negative

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Fig. 44.5 Splenectomy specimen reveals positive nuclear staining of cyclin D1 for the tumor cells. Immunoperoxidase, × 60

Fig. 44.6 Bone marrow biopsy shows two lymphoid aggregates with CD20 staining. Immunoperoxidase, × 20

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Discussion Mantle cell lymphoma (MCL) originates from the mantle zone of the lymph nodes. Cytologically, the tumor cells are identical to the centrocytes in the lymphoid follicle and thus it was designated centrocytic lymphoma in the Kiel classification In addition to centrocytes, this tumor may also shows three morphologic variants as define by the World Health Organization (WHO) classificatio [1]. The small cell variant shows slight or moderate nuclear irregularity or cleft, with clumped chromatin pattern, no nucleoli, and scant cytoplasm. The marginal zone or monocytoid B-cell variant shows abundant pale cytoplasm resembling the tumor cells of marginal zone B-cell lymphoma. The agressive variant is divided into blastoid and pleomorphic subtypes. Tumor cells in the blastoid subtype resemble lymphoblasts with dispersed chromatin and a high mitotic rate. Tumor cells in the pleomorphic subtype are heterogeneous with large cleaved to oval nuclei and pale cytoplasm on Giemsa or methylene green pyronin stain. Nucleoli may be prominent in this subtype. The European MCL Network studied 304 MCL patients and divided MCL into the following subtypes: classical (87.5%), small cell (3.6%), pleomorphic (5.9%), and blastic (2.6%) [2]. The classical subtype is equivalent to the centrocytic subtype in WHO classification No monocytoid subtype was included in this study. Histologically, there are three patterns recognized: diffuse, mantle zone and nodular [1]. The diffuse type is most frequently encountered and is usually difficul to diagnose without immunophenotyping. However, when large numbers of pink histiocytes are intermixed with the tumor cells, MCL should be suspected (Fig. 44.7). The mantle zone type shows a residual or naked germinal center surrounded by an expanded mantle zone, as seen in this case. The nodular type may represent colonization of the germinal center or may arise from the primary lymphoid follicle (Fig. 44.8).

Fig. 44.7 Lymph node biopsy shows diffuse lymphoid infiltratio intermingled with many pink histiocytes. H&E, × 40

Bone marrow is frequently involved with nodular, interstitial, paratrabecular, or diffuse pattern, in that order. In the spleen, the white pulp is markedly expanded by the tumor cells, but reactive follicles with prominent germinal centers may also coexist. Lymphomatous polyposis of the gastrointestinal tract is most frequently due to MCL. This clinical presentation will be discussed in Case 46. Immunophenotypically, MCL is characterized by the coexpression of CD5 with a B-cell marker; CD19, CD20, and CD22 are all positive in MCL cases [3]. This immunophenotype is also present in small lymphocytic lymphoma/chronic lymphocytic leukemia, but the later expresses CD23 and not FMC-7, whereas MCL shows just the opposite. However, positive CD23 may occasionally occur in MCL, and negative CD23 may also be rarely seen in small lymphocytic lymphoma.

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Fig. 44.8 Lymph node biopsy reveals multiple lymphoid nodules. H&E, × 10

Immunohistochemical stain is most helpful in demonstrating the nuclear cyclin D1/bcl-1 stain. It may also demonstrate the follicular dendritic cell meshwork among the tumor cells by CD21, CD23 or CD35 stains. In contrast to most lymphomas, two thirds of MCL cases express surface lambda rather than kappa light chain. Two prognostic predictors, Ki-67 and Survivin, can be demonstrated by immunohistochemistry [3]. The proliferation fraction as demonstrated by these markers may predict the prognosis better than cytology and histologic pattern. MCL is characterized by the presence of t(11;14)(q13;q32) that represents the translocation of the proto-oncogene BCL-1 (11q13) juxtaposed to the heavy chain gene (14q32) [3]. A PRAD1 (parathyroid adenoma 1) or CCND1 gene is linked to the BCL-1 gene at its telomeric border. This gene encodes for cyclin D1, a cell cycle regulatory protein. As a result of the translocation, the CCND1/Cyclin D1 genes are deregulated. Therefore, the t(11;14)-carrying cells cannot exit from the cell cycle, resulting in an expanded B-cell department with developmental arrest. However, additional oncogenes may be required for a malignant transformation into MCL tumor. Gene expression profilin (GEP) is able to stratify MCL cases into various subtypes in terms of IgH gene mutation status, proliferation rate, and blastoid morphology [3]. GEP may also help to distinguish MCL from small lymphocytic lymphoma, diffuse large B-cell lymphoma, and splenic marginal zone lymphoma. MCL has the worst prognosis among all B-cell lymphomas, because it assumes an aggressive clinical course like other high-grade lymphomas and yet is incurable like other small cell lymphomas [4].

References 1. Swerdlow SH, Campo E, Seto M, et al. Mantle cell lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. eds., WHO Classificatio of Tumours of Hematopoietic and Lymphoid Tissues, Lyon, France, IARC Press, 2008, 229–232. 2. Tiemann M, Schrader C, Klapper W, et al. Histopathology, cell proliferation indices and clinical outcome in 304 patients with mantle cell lymphoma (MCL): a clinicopathological study from the European MCL network. Br J Haematol 2005;131:29–38. 3. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 232–240. 4. Smith MR. Mantle cell lymphoma: advances in biology and therapy. Curr Opin Hematol 2008;15:415–421.

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Case 45 A 64-year-old man presented with lymphadenopathy and splenomegaly. Based on a limited immunophenotyping and his clinical condition, the patient was initially diagnosed with chronic lymphocytic leukemia/small lymphocytic lymphoma. The patient had a relatively stable clinical course for nine years. In the ninth year after the initial diagnosis, the patient’s leukocyte count went up to 120,000/␮l with many blastoid cells (Fig. 45.1). A bone marrow biopsy confirme blastoid transformation (Figs. 45.2 and 45.3). Subsequently, the patient developed neurologic and pulmonary symptoms. Blasts were found in the cerebrospinal flui and bronchoalveolar lavage. The patient died nine months after the detection of blastoid cells. Autopsy showed systemic dissemination of lymphoma in practically all internal organs (Figs. 45.4 and 45.5) and lymph nodes (Fig. 45.6).

Fig. 45.1 Periperhal blood smear shows many pleomorphic lymphoid blastoid cells. Wright – Giemsa, × 60

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Fig. 45.2 Bone marrow biopsy reveals interstitial lymphoid infiltration H&E, × 60

Fig. 45.3 Bone marrow aspirate demonstrates many blastoid tumor cells with a few erythroid elements. Wright – Giemsa, × 100

Case 45

Fig. 45.4 Liver specimen from autopsy shows many large pleomorphic lymphoma cells. H&E, × 60

Fig. 45.5 Kidney specimen from autopsy reveals interstitial lymphoma cell infiltration H&E, × 20

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Fig. 45.6 Lymph node from autopsy shows many large pleomorphic tumor cells with immature chromatin pattern and prominent nucleoli. H&E, × 100

Differential diagnosis: Richter syndrome, blastoid transformation of mantle cell lymphoma.

Further Studies Flow cytometric studies: Peripheral blood: CD10 2%, CD19/CD5 92%, CD20 99%, CD23 30%, FMC-7 98%, kappa 98%, lambda 0% Bronchoalveolar lavage: CD10 4%, CD19/CD5 91%, CD20 94%, CD23 3%, FMC-7 94%, kappa 77%, lambda 1% Immunohistochemistry: CD20 stain: positive (Fig. 45.7) CD79a stain: positive Cyclin D1 stain: positive (Fig. 45.8) Ki-67 stain: positive (Fig 45.9) CD3 stain: negative

Case 45

Fig. 45.7 Positive CD20 staining of the tumor cells. Immunoperoxidase, × 60

Fig. 45.8 Positive nuclear staining of cyclin D1 for the tumor cells. Immunoperoxidase, × 100

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Fig. 45.9 Ki-67 stain shows a high proliferation fraction. Immunoperoxidase, × 100

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Discussion Mantle cell lymphoma (MCL) is characterized by the centrocyte-like tumor cells, but it also may show rare variants, including blastoid (blastic), pleomorphic, small cell and monocytoid B cells [1, 2]. The blastoid subtype has been reported to have a more aggressive clinical course by several studies, but other studies show that there is no statistical difference in prognosis between various cytologic subtypes [2]. Similarly, the histologic patterns in MCL, including the nodular, mantle zone, and diffuse pattern, show no differences in relation to prognosis by some studies, while other studies show that the mantle zone and/or nodular patterns have a better prognosis that that of the diffuse pattern. It is interesting to note that while MCL represents a lymphoma with aggressive clinical course and incurable outcome, there is a subgroup of MCL patients with an indolent course and relatively long survival [3]. Another unusual aspect of MCL is that it does not transform into diffuse large B-cell lymphoma, but transformation of other cytology subtypes to blastoid subtype does occur [1]. The indication of transformation is the loss of a mantle zone growth pattern, an increase in nuclear size, pleomorphism and chromatin dispersal [1]. The present case exemplifie both aspects: the initial clinical course of nine years represents the indolent form of MCL, but when it transformed to the blastoid form, the patient promptly went on a downhill clinical course and died within nine months. There are several risk factors that predict a poor prognosis in MCL cases, including high mitotic rate, leukemic phase, high proliferation fraction (> 40%) determined by Ki-67 stain, and more than 20% nuclear Survivin expression [1–4]. All these risk factors are common features of the blastoid subtype of MCL. In addition, the blastoid subtype also has frequent expression of CKD4, p53 mutation, and p16 deletion that all carry an unfavorable prognosis [4]. High levels of lactate dehydrogenase, (> 450 U/l), leukocytosis (> 10, 000/␮l), high percentage of Ki-67, and high mitotic rate at presentation are predictors for blastoid transformation [4].

References 1. Swerdlow SH, Campo E, Seto M, et al. Mantle cell lymphoma. 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, 229–232. 2. Tiemann M, Schrader C, Klapper W, et al. Histopathology, cell proliferation indices and clinical outcome in 304 patients with mantle cell lymphoma (MCL): a clinicopathological study from the European MCL network. Br J Haematol 2005;131:29–38. 3. Smith MR. Mantle cell lymphoma: advances in biology and therapy. Curr Opin Hematol 2008;15:415–421. 4. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 232–240.

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Case 46 A 65-year-old man was seen in the outpatient clinic for complaints of abdominal bloating and flatulence He had no history of diarrhea, constipation, melena, or bright red blood per rectum. Flex sigmoidoscopy found a 0.2 cm sessile polyp at 40 cm, but no biopsy was taken. Colonoscopy performed subsequently showed approximately 100 sessile polyps in the colon. Some polyps had central depressions and superficia erosion. Multiple biopsies were taken (Fig. 46.1).

Fig. 46.1 A colonic polyp shows dense lymphoid infiltratio in the submucosa. H&E, × 20

Differential diagnoses: familial polyposis versus different lymphomas.

Further Studies Immunohistochemistry of the polyps: CD3 stain: negative CD20 stain: positive CD79a stain: positive (Fig. 46.2) CD5 stain: positive (Fig. 46.3) CD23 stain: negative (Fig. 46.4) Cyclin D1 stain: positive (Fig. 46.5)

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Fig. 46.2 CD79a stains all lymphoma cells. Immunoperoxidase, × 10

Fig. 46.3 The tumor cells are also positive for CD5. Immunoperoxidase, × 10

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Fig. 46.4 CD23 stain is negative for tumor cells. Immunoperoxidase, × 10

Fig. 46.5 Cyclin D1 stain is positive for tumor cells. Immunoperoxidase, × 10

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Discussion Primary gastrointestinal lymphoma accounts for 13.7% of all malignant lymphoma cases [1]. Among these cases, 41% are diffuse large B-cell lymphoma, 35% are extranodal marginal zone B-cell lymphoma and 2% are in the form of multiple lymphomatous polyposis (MLP). It has been generally believed that MLP represents exclusively mantle cell lymphoma (MCL) of the gastrointestinal (GI) tract and that MCL presents in the GI tract mainly as MLP. In the light of current studies, this concept is not entirely true. In a study of 35 patients with MLP, 12 were found to be MCL, 14 follicular lymphoma (FL) and 9 extranodal marginal zone B-cell lymphoma (EMZBCL) [2]. Morphologically, MCL is characterized by centrocytic morphology, EMZBCL by lymphoepithelial lesions and FL by a nodular pattern. However, nodular pattern can be seen in other two lymphomas and lymphoepithelial lesions may be absent in some cases of EMZBCL [2]. In a small endoscopic biopsy, these three tumors are frequently indistinguishable. Therefore, the distinction between these three lymphomas depends on immunohistochemistry and molecular techniques. The minimal immunostaining includes CD5, CD10, and bcl-1/cyclin D1 in addition to the B-cell staining. The immunophenotype of MCL is CD5 + CD10 − bcl-1+, and that of FL is CD5 − CD10 + bcl-1−. EMZBCL shows negative reaction to all three markers. The simultaneous study of karyotypes can further confir the diagnosis. MCL shows t(11;14)(q13;q32), FL is characterized by t(14;18)(q32;q21), and EMZBCL does not show either karyotype. Since conventional karyotyping is not sensitive enough to demonstrate the abnormalities, most studies use molecular techniques to identify the BCL-1 and BCL-2 oncogenes. Because there is a large number of 11q13 breakpoints scattered over a region of more than 120 kb, the primers used in polymerase chain reaction (PCR) cannot cover the entire area, while the locus-specifi 11q13 probe used by the fluorescenc in situ hybridization technique (FISH) covers the entire region, so that the latter is more sensitive than the former technique. The distinction of these three lymphomas has important clinical implications, as the prognosis of the three is clearly different. The MCL has the worst prognosis, the EMZBCL is associated with a favorable outcome, and FL shows a prognosis between these two. MLP may involved any part of the GI tract, from the esophagus to the rectum, but colonic polyposis appears to be most prevalent [2]. Patients with MLP may show only nonspecifi symptoms, such as weight loss, fatigue and anemia, but severe cases may have abdominal pain, diarrhea, hematochezia, nausea, and vomiting [1, 2]. MCL has been reported to involve the GI tract in 15–30% of patients. However, two recent studies with thorough endoscopy and routine biopsies have demonstrated that the incidence of GI involvement in MCL is much higher [3, 4]. These two studies found endoscopic abnormalitites of 46 versus 49% in the lower GI tract and 38 versus 62% in the upper GI tract, respectively [3, 4]. Microscopic abnormalities were 77 versus 88% in the lower GI tract, and 77 versus 43% in the upper GI tract, respectively, in these two studies [3, 4]. Most strikingly, one study showed that 63% of patients with normal endoscopy of the upper GI tract and 71% with normal endoscopy of the lower GI tract demonstrated microscopic lymphomatous infiltratio [4]. However, as almost all cases with GI lesion also had bone marrow infiltration the findin of GI lesions does not change the Ann Arbor staging and the patient management. Therefore, both studies do not recommend a routine endoscopic studies in MCL patients without prominent GI symptoms.

References 1. Tamura S, Ohkawauchi K, Yokoyama Y, et al. Non-multiple lymphomatous polyposis form of mantle cell lymphoma in the gastrointestinal tract. J Gastroenterol 2004;39:995–1000. 2. Kodama T, Ohshima K, Nomura K, et al. Lymphomatous polyposis of the gastrointestinal tract, including mantle cell lymphoma, follicular lymphoma and mucosa-associated lymphoid tissue lymphoma. Histopathology 2005;47:467–478. 3. Romaguera JE, Medeiros LJ, Hagemeister FB, et al. Frequency of gastrointestinal involvement and its clinical significanc in mantle cell lymphoma. Cancer 2003;97:586–591. 4. Salar A, Juanpere N, Bellosillo B, et al. Gastrointestinal involvement in mantle cell lymphoma: A prospective clinic, endoscopic, and pathologic study. Am J Surg Pathol 2006;30:1274–1280.

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Case 47 A 82-year-old man presented with a rapidly growing mass on the left upper cheek for approximately three weeks. The mass measured 3 × 3 cm and was freely movable. The dentist removed it from the posterior buccal mucosa (Fig. 47.1) with a preliminary diagnosis of mixed salivary gland tumor. The patient denied chills and fever, but he claimed to have 12-pound weight loss in four months. A bone marrow biopsy was subsequently performed (Figs. 47.2 and 47.3).

Fig. 47.1 Lymph node biopsy shows predominantly immunoblasts with vesicular nucleus, prominent single nucleolus, and moderate amount of cytoplasm. H&E, × 100

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Fig. 47.2 Bone marrow biopsy shows many immunoblasts with the same characteristic as that shown in the lymph node. H&E, × 100

Fig. 47.3 Bone marrow aspirate reveals a cluster of large immunoblasts with a high nuclear/cytoplasmic ratio, immature chromatin, prominent nucleoli and basophilic cytoplasm. Wright – Giemsa, × 100

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Differential diagnoses: lymphoma versus salivary gland tumor.

Further Studies Immunohistochemical stains of the mass: CD20 stain: positive (Fig. 47.4) Bcl-2 stain: positive (Fig. 47.5) CD3 stain: negative (Fig. 47.6)

Fig. 47.4 Lymph node biopsy shows positive CD20 staining of tumor cells. Immunoperoxidase, × 100

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Fig. 47.5 Lymph node biopsy shows positive bcl-2 staining of tumor cells. Immunoperoxidase, × 100

Fig. 47.6 Lymph node biopsy shows negative CD3 staining of tumor cells. Immunoperoxidase, × 100

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Discussion Diffuse large B-cell lymphoma (DLBCL) is one of the most common lymphomas, accounting for 30–40% of adult nonHodgkin lymphoma in Western countries [1]. It consists of a group of large B-cell tumors with heterogeneous clinical, morphologic, immunologic, and molecular genetic features. A large cell is define by the World Health Organization (WHO) system and others as having a nucleus equal to or exceeding normal macrophage nuclei or more than twice the size of a normal lymphocyte [1]. In the 2008 WHO classification DLBCL is divided into three common morphologic variants, four distinct subtypes and eight other lymphomas of large B cells, including some lymphomas previously classifie as variants and subtypes in the 2001 classificatio [1]. The three common variants are centroblastic, immunoblastic, and anaplastic. DLBCL can be de novo or secondary, being transformed from low-grade lymphomas, including small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, nodal and extranodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma. The histologic pattern is characterized by a diffuse large cell infiltratio with effacement of the normal architecture, but it may show an interfollicular or intrasinusoidal infiltratio in some cases. Mitosis and apoptosis are prominent in most cases [2]. The most common variant is the centroblastic variant. The tumor cells are similar to the centroblasts seen in reactive germinal centers. These cells vary from medium to large size and are characterized by large round or oval nuclei with a vesicular chromatin pattern and two to four membrane-bound nucleoli (Fig. 47.7). The cytoplasm is scanty and amphophilic to basophilic. It can be further divided into monomorphic, pleomorphic, and multilobated subvariants.

Fig. 47.7 Lymph node biopsy from a case of centroblastic variant shows many centroblasts with multiple membrane-bound nucleoli (arrow) and scant cytoplasm. H&E, × 100

The second common variant is immunoblastic variant. This variant encompasses >90% of immunoblasts. The immunoblasts are large cells with large vesicular nuclei and a single prominent, centrally located nucleolus, as seen in the current case. There is a moderate amount of basophilic cytoplasm. The anaplastic variant will be discussed in Case 48. DLBCL expresses all B-cell markers, including CD19, CD20, CD22, CD79a, PAX5/BSAP, HLA-DR, and surface immunoglobulin [1–3]. Cytoplasmic immunoglobulin is expressed in cases when plasmacytic differentiation is present. In a subset of DLBCL, CD10, bcl-2, and bcl-6 are present and make it difficul to distinguish from follicular lymphoma. In fact, this subset is derived from the germinal center cells or it may well be transformed from follicular lymphoma. Another subset

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of DLBCL expressing MUM1/IRF4, VS38c, and CD138 is considered to originate from post-germinal center stage. CD30 is often expressed in the anaplastic variant. CD5 is present in 10% of DLBCL cases, but these cases are considered de novo DLBCL rather than transforming from small cell lymphomas. Another T-cell marker, CD43, can be expressed in 15–30% DLBCL cases [3]. Using DNA microarrays, two gene expression profile were demonstrated: the germinal center B-cell-like subgroup and the activated B-cell-like subgroup [2, 3]. The former was associated with a good prognosis and the latter with a poor outcome. A subsequent study identifie a third group, which did not express either set of genes from the above two groups [2, 3]. The germinal center B-cell-like subgroup expresses bcl-6+/CD10±/MUM1−/CD138− or bcl-6−/CD10+/ MUM1−/CD138− [4]. The nongerminal center B-cell-like subgroup expresses bcl-6±/CD10−/MUM1+/CD138− [4] In terms of oncogenes, BCL-6 is the most common cytogenetic aberration in DLBCL [1–3]. BCL-6 may be translocated with IgH, forming a karyotype of t(3;14)(q27;q32) or with other partner genes, forming t(3;6)(q29;q15) or t(3;22)(q27;q11). Another oncogene, BCL-2, is also commonly present in DLBCL with a frequency of 20–30% in the form of t(14;18)(q32;q21) (2,3). C-MYC rearrangement is uncommon. BCL-1 aberration is absent in DLBCL [1–3].

References 1. Stein H, Warnke RA, Chan WC, et al. Diffuse large B-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;1233–237. 2. Pileri SA, Dirnhofer S, Went PH, et al. Diffuse large B-cell lymphoma: one or more entities? Present controversies and possible tools for its subclassification Histopathology 2002;41:482–509. 3. de Leval L, Harris NL. Variability in immunophenotype in diffuse large B-cell lymphoma and its clinical relevance. Histopathology 2003;43:509–528. 4. Bai M, Skyrlas A, Agnantis NJ, et al. Diffuse large B-cell lymphomas with germinal center B-cell-like differentiation immunophenotypic profil are associated with high apoptotic index, high expression of the proapoptotic proteins bax, bak and bid and low expression of the antiapoptotic protein bcl-xl. Mod Pathol 2004;17:847–856.

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Case 48 A 58-year-old man was admitted because of the findin of a large left inguinal mass. The patient had night sweats for several weeks before he discovered the inguinal mass. There were no other superficia lymph nodes palpable. The CT scan showed no enlargement of hilar, mediastinal or axillary lymph node, but he had bulky retroperitoneal and pelvic adenopathy. The inguinal lymph node was biopsied (Fig. 48.1). A bone marrow biopsy was performed subsequently (Fig. 48.2).

Fig. 48.1 Lymph node biopsy shows anaplastic and pleomorphic tumor cells with bizarre, vesicular nuclei and prominent nucleoli. H&E, × 100

Case 48

Fig. 48.2 Bone marrow biopsy shows anaplastic tumor cells. H&E, × 100

Differential diagnosis: various lymphomas.

Further Studies Immunohistochemical stains of the bone marrow biopsy: CD20 stain: positive (Fig. 48.3) CD43 stain: positive (Fig. 48.4) CD30 stain: positive (Fig. 48.5)

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Fig. 48.3 Bone marrow biopsy shows positive CD20 staining of tumor cells. Immunoalkaline phosphatase, × 100

Fig. 48.4 Bone marrow biopsy shows positive CD43 staining of tumor cells. Immunoalkaline phosphatase, × 100

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Fig. 48.5 Bone marrow biopsy shows positive CD30 staining of tumor cells. Immunoalkaline phosphatase, × 100

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Discussion The anaplastic variant (AV) of diffuse large B-cell lymphoma (DLBCL) is similar to other DLBCLs in terms of clinical presentation, immunophenotype, molecular genetics, and prognosis [1]. The only distinguishing feature is its morphology. As its name implies, the tumor cells are highly anaplastic and pleomorphic with many bizarre neoplastic cells. The nuclei are vesicular with one or more prominent nucleoli. Many large cells are present and mitosis is striking. This variant may show a cohesive growth or a sinusoidal infiltratio pattern that may mimic metastatic carcinoma, which can be distinguished by its cytokeratin staining. These morphologic features are indistinguishable from the true anaplastic large cell lymphoma of T-cell or null-cell type [1, 2]. The latter tumor may also show cohesive growth and sinusoidal infiltratio pattern [3]. Furthermore, both tumors express CD30. However, their distinction is important because they are biologically different, so that their clinical manifestation and prognosis are not the same. Under most circumstances, these two tumors can be differentiated by their immunophenotypes; the AV expresses all B-cell markers (CD19, CD20, CD22, CD79a, and PAX5/BSAP) and the germinal center cell markers (CD10, bcl-2 and bcl-6) in some cases [2, 4]. However, AV may also express CD43, a T-cell marker, as seen in the current case [4]. Furthermore, in some cases, PAX5 is the only B-cell marker expressed in AV [2]. Therefore, molecular genetic studies are required for their distinction in difficul cases. AV usually shows monoclonal immunoglobulin heavy chain gene rearrangement, whereas T/null-cell anaplastic large cell lymphoma reveals T-cell receptor gene rearrangement. If T-cell receptor gene rearrangement is not demonstrated, cytogenetic study by karyotyping or fluorescenc in situ hybridization will be needed [1,2,4]. The former will show t(2:5)(p23;q35) and the latter, NPM-ALK fusion protein in anaplastic large cell lymphoma. The ALK gene product can also be demonstrated by immunohistochemcal stain. On the other hand, AV may show aberrations of BCL-6 or BCL-2 gene with corresponding abnormal karyotypes. BCL-6 abnormality is associated with t(3;14)(q27;q32), t(3;6)(q29;p15) or t(3;22)(q27;q11). BCL-2 abnormality is associated with t(14;18)(q32;q21).

References 1. Stein H, Warnke RA, Chan WC, et al. Diffuse large B-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, 233–237. 2. Pileri SA, Dirnhofer S, Went PH, et al. Diffuse large B-cell lymphoma: one or more entities? Present controversies and possible tools for its subclassification Histopathology 2003;41:482–509. 3. Delsol G, Falini B, M¨uller-Hermelink HK, et al. Anaplastic large cell lymphoma (ALCL), ALK-positive. 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, 312–316. 4. de Leval L, Harris NL. Variability in immunophenotype in diffuse large B-cell lymphoma and its clinical relevance. Histopathology 2003;43:509–528.

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Case 49 A 66-year-old man presented with chest pain, low-grade fever and night sweats for two weeks. Chest X-ray demonstrated a mediastinal mass, which was confirme by CT scan. A mediastinoscopic biopsy is depicted in Figs. 49.1, 49.2, and 49.3.

Fig. 49.1 Lymph node biopsy shows a few tumor cells in a lymphohistocytic background. H&E, × 20

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Fig. 49.2 Higher magnificatio of Fig. 49.1 demonstrates a Reed – Sternberg-like cell. H&E, × 100

Fig. 49.3 Another f eld reveals a large tumor cell surrounded by several atypical histiocytes. H&E, × 60

Differential diagnoses: Hodgkin lymphoma versus non-Hodgkin lymphoma.

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Further Testing Flow cytometry showed a predominant T-cell population with a normal CD4/CD8 ratio. The B-cell population revealed no monoclonality. Immunohistochemical stains: CD3: positive on background lymphocytes (Fig. 49.4) CD20: positive on large tumor cells (Fig. 49.5) CD79a: positive on large tumor cells CD15: negative (Fig. 49.6) CD30: negative (Fig. 49.7)

Fig. 49.4 CD3 stain highlights many T cells intermingled with unstained histiocytes. Immunoperoxidase, × 10

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Fig. 49.5 CD20 stain demonstrates two large lymphoma cells. Immunoperoxidase, × 60

Fig. 49.6 CD15 stain reveals no staining of tumor cells. Immunoperoxidase, × 60

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Fig. 49.7 CD30 stain reveals no staining of tumor cells. Immunoperoxidase, × 60

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Discussion T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is one of the subtypes of diffuse large B-cell lymphoma. It is characterized by less than 10% of large neoplastic B cells amid a majority of non-neoplastic T cells with or without histiocytes in the background [1–5]. Morphologically, the normal architecture of the lymph node is completely effaced by extensive lymphocytic infiltratio with scattered large tumor cells. A nodular pattern is usually not discernable, which is helpful to distinguish THRLBCL from nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). The tumor cells may resemble those in the centroblastic variant, immunoblastic variant, lymphocytic and histiocytic (L&H) cells, or Reed – Sternberg cells. In approximately one-third of cases, a mixed tumor cell morphology may be apparent. The paucity of other inflammator cells, including eosinophils, neutrophils, and plasma cells, is an important feature to separate THRLBCL from Hodgkin lymphomas. The scarcity of tumor cells and the occasional presence of Reed – Sternberg-like cells make THRLBCL difficul to distinguish from Hodgkin lymphoma, and immunohistochemistry is indispensable for the differential diagnosis [1–5]. Because the tumor cells are less than 10% of the total population, fl w cytometry usually is not helpful in identifying the monoclonality of B-cell population, as is seen in the current case. Immunohistochemical stains, on the other hand, may demonstrate CD45, CD20, CD79a, and the B-cell transcription factors PAX5/BSAP, OCT2, and BOB1 on the tumor cells [2–4]. The consistent negative reactions to CD15 and CD30 in THRLBCL help rule out classical Hodgkin lymphoma. The immunohistochemical phenotype is very similar between THRLBCL and NLPHL. The tumor cells in NLPHL are also positive for CD45, CD20, CD79a, PAX5/BSAP, OCT2, and BOB1, but are negative for CD15 and CD30. However, another B-cell transcription factor, PU.1, is frequently positive in NLPHL, but rarely in THRLBCL [2–4]. The most important distinction between these two entities depends on the immunohistochemical staining of the background cells. NLPHL usually shows large numbers of polyclonal B cells, and CD57-positive T cells are present, forming rosettes surrounding the L&H cells. A follicular dendritic cell network as demonstrated by CD21 staining is also present in NLPHL. On the other hand, CD20, CD21, and CD57 are negative in the background staining of THRLBCL. In addition, bcl-2 is positive in up to 50% of THRLBCL cases but is negative in NLPHL cases [2, 4]. The expression of CD10 and bcl-6 in THRLBCL denotes its germinal center B-cell origin [5]. Nevertheless, NLPHL and THRLBCL can be present in the same patient, and the latter is considered to be transforming from the former in some studies [2, 3]. There are also cases with a gray zone tumor between these two entities. THRLBCL was once considered the early stage of diffuse large B-cell lymphoma. However, the clinical manifestation indicates that THRLBCL is an aggressive lymphoma, with frequent extranodal involvement (liver, spleen, and bone marrow) or late stage at presentation. B symptoms (fever, night sweats, and weight loss) are more frequently seen than other diffuse large B-cell lymphomas. Without aggressive treatment, the prognosis of THRLBCL is usually worse than other diffuse large B-cell lymphomas.

References 1. De Wolf-Peeters C, Delabie J, Campo E, et al. T cell/histiocyte-rich large B-cell lymphoma. 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, 238–239. 2. Abramson JS. T-cell/histiocyte-rich B-cell lymphoma: Biology, diagnosis, and management. Oncologist 2006;11:384–392. 3. Boudov´a L, Torlakovic E, Delabie J, et al. Nodular lymphocyte-predominant Hodgkin lymphoma with nodules resembling T-cell/histiocyterich B-cell lymphoma: differential diagnosis between nodular lymphocyte-predominant Hodgkin lymphoma and T-cell/histiocyte-rich B-cell lymphoma. Blood 2003;102:3753–3758. 4. R¨udiger T, Gascoyne RD, Jaffe ES, et al. Workshop on the relationship between nodular lymphocyte predominant Hodgkin’s lymphoma and T cell/histiocyte-rich B cell lymphoma. Ann Oncol 2002;13 (Suppl 1):44–51. 5. Achten R, Verhoef G, Vanuytsel L, et al. T-cell/histiocyte-rich large B-cell lymphoma: A distinct clinicopathologic entity. J Clin Oncol 2002;20:1269–1277.

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Case 50 A 30-year-old man presented with respiratory distress syndrome and low-grade fever for a week. Physical examination found that he had facial edema and neck vein distention that were consistent with superior vena cava syndrome. Chest radiography demonstrated a large mediastinal mass and right pleural effusion. A mediastinal biopsy was obtained through mediastinoscopy (Figs. 50.1, 50.2, and 50.3). Test for serum alpha-fetoprotein was negative.

Fig. 50.1 Biopsy of a mediastinal mass shows tumor cells with clear cytoplasm that are separated by f brous septa (compartmentalization). H&E, × 20

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Fig. 50.2 Higher magnificatio shows large tumor cells with clear cytoplasm. H&E, × 60

Fig. 50.3 A thymic B-cell tumor from another case shows a Hassell’s corpuscle. H&E, × 60

Differential diagnoses: Hodgkin and non-Hodgkin lymphomas, and malignant germ-cell tumors.

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Case 50

Further Studies Immunohistochemical stains: CD20 stain: positive (Fig. 50.4) CD3 stain: negative (Fig. 50.5) Placental alkaline phosphastase stain: negative (Fig. 50.6) Cytokeratin stain: negative (Fig. 50.7) CD30 stain: negative CD15 stain: negative

Fig. 50.4 All tumor cells are positive for CD20. Immunoperoxidase, × 40

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Fig. 50.5 A few T lymphocytes stain for CD3, but the tumor cells are negative. Immuno-alkaline phosphatase, × 60

Fig. 50.6 The tumor cells are negative for placental alkaline phosphatase stain. Immuno-alkaline phosphatase, × 40

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Fig. 50.7 The tumor cells are negative for cytokeratin stain. Immuno-alkaline phosphatase, × 40

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Discussion Primary mediastinal (thymic) large B-cell lymphoma (PMLBCL) is classifie as one of the other lymphomas of large B cells in the 2008 World Health Organization (WHO) scheme [1]. PMLBCL is distinctive from other diffuse large B-cell lymphomas in clinical presentation, immunophenotype and cytogenetics. It is designated “primary” lymphoma to distinguish from secondary lymphomas that occur in the mediastinum. The Nebraska Lymphoma Study Group specificall emphasizes that PMLBCL is “a mediastinal mass of at least 5 cm in maximum dimensions, with no extramediastinal mass large than that in the mediastinum” [2]. In fact, the tumor is greater than 10 cm in diameter in three-quarters of patients. This tumor is characteristically seen in women in their third to fift decade of life. The major presentation is pulmonary symptoms, such as cough, chest pain, and dyspnea. Approximately one-half of patients manifest superior vena cava syndrome and one-third have pleural and pericardial effusion [3]. However, the tumor usually spreads locally without metastasis to distant lymph nodes. When metastasis occurs, it goes to internal organs and the kidney is most often involved. This tumor was called sclerosing large clear cell mediastinal lymphoma in the early literature because large lymphoma cell with clear cytoplasm is the most common morphology. The presence of fibrou bundles that separate the tumor cells in clusters (compartmentalization) is also characteristic of this tumor [1]. However, the tumor cells can be pleomorphic or they may be predominantly cleaved, noncleaved, centroblastic, or immunoblastic. When Reed – Sternberg-like cells are present, PMLBCL may mimic Hodgkin lymphoma. In fact, it is difficul to distinguish PMLBCL from the nodular sclerosing subtype of Hodgkin lymphoma in some patients, and these cases are designated mediastinal gray zone lymphoma [3]. Some thymic remnants, including Hassall’s corpuscles, can be demonstrated in some cases of PMLBCL and the similar mutation patterns of immunoglobulin heavy chain variable region and bcl-6 gene between PMLBCL and thymic B cells suggest that this tumor is derived from the thymus [3]. Immunophenotypically, the absence of surface immunoglobulin on the neoplastic B cells is most characteristic. The tumor cells express B-cell markers, including CD19, CD20, CD22, and CD23, but are negative for CD10 and CD21 [1–4]. This immunophenotype is also characteristic for thymic B cells [5]. On normal B cells, surface immunoglobulins and CD79 form a B-cell receptor complex, but on PMLBCL cells, there is a discordant expression of CD79a without that of surface immunoglobulins [6]. Bcl-2, bcl-6, and PAX5/BSAP (B-cell specifi activator protein) have also been reported positive in some PMLBCL cases [6]. Although the tumor cells do not show surface immunoglobulins, immunoglobulin gene rearrangements are demonstrated in them [1]. The absence of surface immunoglobulin expression is not due to a lack of functional Ig gene rearrangements nor to a defect in the transcription factors, but rather to a downregulation of the intronic heavy chain enhancer or posttranscriptional blockage [1]. The common cytogenetic abnormalities include gains in chromosome 9p and 2p, which correspond with Janus Kinase (JAK)-2 and REL, respectively [1, 3]. The expression of MAL gene is also specifi for PMLBCL and it can be identifie at the RNA or protein levels. MAL gene is present in a minor subpopulation of thymic medullary B cells but is not seen in other diffuse large B-cell lymphomas [3]. Rearrangements of bcl-2, bcl-6 and MYC genes are typically absent in PMLBCL [1]. Gene expression profilin demonstrates that PMLBCL is closer to classic Hodgkin lymphoma than to other diffuse large B-cell lymphomas [1, 3]. Nodular sclerosing Hodgkin lymphoma is top of the list for differential diagnosis, because this is frequently seen in the mediastinum and shows features of sclerosis. Phenotypically, Hodgkin lymphoma also shows no surface immunoglobulin and PMLBCL may express CD30. However, with a few exceptions, PMLBCL is negative for CD15 and positive for CD45, which helps to distinguish it from Hodgkin lymphoma. Cytogenetically, both show REL and JAK2 amplificatio [3]. The fina resort is to look at the gene expression profiling which may demonstrate distinct signatures between these two entities [1,3]. Another lymphoma commonly present in the mediastinum is lymphoblastic lymphoma, which is predominantly of Tcell origin and easily identifiabl by the demonstration of terminal deoxynucleotidyl transferase (TdT) in the nuclei of the tumor cells. Seminoma may also be present in the mediastinum; because this tumor shows a lobular pattern and large clear tumor cells, it may be mistaken for PMLBCL. However, seminoma is positive for placental alkaline phosphatase (PLAP) and CD117 and is negative for cytokeratin [7]. Other germ cell tumors should show coexistence of cytokeratin with alphafetoprotein or epithelial membrane antigen (EMA) [7].

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References 1. Gaulard P, Harris NL, Pileri SA, et al. Primary mediastinal (thymic) large B-cell lymphoma. 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;250–251. 2. Abou-Elella AA, Weisenburger DD, Vose JM, et al. Primary mediastinal large B-cell lymphoma: a clinicopathologic study of 43 patients from the Nebraska Lymphoma Study Group. J Clin Oncol 1999;17:784–790. 3. Savage KJ. Primary mediastinal large B-cell lymphoma. Oncologist 2006;11:488–495. 4. von Besien K, Kelta M, Bahaguna P. Primary mediastinal B-cell lymphoma: A review of pathology and management. J Clin Oncol 2001;19:1855–1864. 5. Kanavaros P, Gaulard P, Charlotte F, et al. Discordant expression of immunoglobulin and its associated molecule mb-1/CD79a is frequently found in mediastinal large B-cell lymphomas. Am J Pathol 1995;146:735–741. 6. Peleri SA, Zinzani PL, Gaidano G, et al. Pathobiology of primary mediastinal B-cell lymphoma. Leuk Lymphoma 2003;44 (Suppl 3):S21–S26. 7. Wick MR. Immunohistology of the mediastinum. In Dabbs D, ed., Diagnostic Immunohistochemistry, 2nd ed., Phildadelphia, Churchill Livingstone, 2006, 301–328.

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Case 51 A 70-year-old man presented with dementia. Cerebral magnetic resonance imaging showed patchy white matter inclusions. The histologic features of the brain biopsy are illustrated in Figs. 51.1, 51.2, and 51.3.

Fig. 51.1 Brain biopsy shows several blood vessels containing lymphoma cells. H&E, × 20

Case 51

Fig. 51.2 Brain biopsy shows intravascular lymphoma. H&E, × 40

Fig. 51.3 Brain biopsy shows intravascular lymphoma. H&E, × 40

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Differential diagnoses: Metastatic carcinoma and intravascular lymphoma.

Further Testing Immunohistochemistry: Cytokeratin: negative CD45: positive CD20: positive (Fig. 51.4) CD3: negative (Fig. 51.5) Epstein – Barr virus encoded RNA hybridization (EBER): negative

Fig. 51.4 The tumor cells are reactive to CD20 staining. Immunoperoxidase, × 40

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Fig. 51.5 CD3 stains perivascular T lymphocytes but not the intravascular tumor cells. Immunoperoxidase, × 40

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Discussion This brain biopsy shows several blood vessels that contain large tumor cells (Figs. 52.1, 52.2, and 52.3). These tumor cells have immature chromatin pattern and inconspicuous nucleoli. A small rim of cytoplasm is present. There is no extravascular neoplastic infiltration Mild perivascular petechial hemorrhages are seen in small areas. The tumor cells are positive for CD45 and CD20 (Fig. 51.4), but are not reactive to CD3 (Fig. 51.5) and cytokeratin. EBER is negative. The detection of intravascular tumor cells are most frequently seen in metastatic carcinoma, but the absence of cytokeratin staining excludes this diagnosis. The expression of CD45 and CD20 is indicative of a lymphoma of B-cell lineage. Thus a diagnosis of intravascular large B-cell lymphoma (IVBCL) is established. IVBCL is the term adopted by the World Health Organization (WHO) classificatio [1], but this entity has many synonyms, such as malignant angioendotheliomatosis, intravascular lymphomatosis, angio-endotheliotropic lymphoma, and angiotropic large cell lymphoma. IVBCL is an extremely rare disease, and it is estimated that less than 300 cases have been reported [2]. Although most of intravascular lymphomas are of diffuse large B-cell lymphoma, it can be seen in T-cell lymphomas including anaplastic large cell lymphoma and natural killer cell lymphoma. However, those cases are excluded from the category of IVBCL in the WHO classificatio [1]. Immunophenotyping in 82 cases of intravascular lymphoma revealed that 81% were of B-cell lineage and 15% of T-cell lineage [2]. Immunohistochemistry with CD45, CD3, and CD20 staining is usually sufficien to identify the cell lineage, but immunoglobulin heavy chain gene rearrangement and T-cell receptor gene rearrangement have also been used for the diagnosis of this entity [3]. CD5 and CD10 coexpression is detected in 38% and 13% of IVBCL cases, respectively [1]. The staining of endothelial cells with factor VIII or CD34 may help to delineate the intravascular nature of the lymphoma. The clinical presentation is mainly neurologic and dermatologic in Western countries; however, intravascular lymphoma have been reported in a wide range of other organs. In cases reported in the West, the involvement of hematopoietic tissues, including bone marrow, lymph node, spleen, and liver, is infrequent, and blood is seldom involved [2–5]. However, the Asian variant may have the opposite clinical manifestations, namely, frequent hepatospleomegaly and bone marrow involvement but infrequent skin and neurologic presentation [5]. In addition, these cases are often associated with pancytopenia and hemophagocytic syndrome. Except for cases with only skin involvement, intravascular lymphoma usually carries an ominous prognosis, probably due to its protean clinical presentation that makes an accurate early diagnosis difficul to achieve. This tumor usually shows clonal rearrangement of immunoglobulin genes, but nonrandom karyotypic aberrations have not been reported [1].

References 1. Nakamura S, Ponzoni M, Campo E. Intravascular large B-cell lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classifica tion of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 252–253. 2. Ko YH, Han JH, Go JH, et al. Intravascular lymphomatosis: a clinicopathological study of two cases presenting as an interstitial lung disease. Histopathology 1997;31:555–562. 3. Khoury H, Lestou VS, Gascoyne RD, et al. Muticolor karyotyping and clinicopathological analysis of three intravascular lymphoma cases. Mod Pathol 2003;16:716–724. 4. Ferreri ANM, Campo E, Seymour JF, et al. Intravascular lymphoma: clinical presentation, natural history, management and prognostic factors in a series of 38 cases, with special emphasis on the “cutaneous variant”. Br J Haematol 2004;127:173–183. 5. Murase T, Nakamura S, Kawauchi K, et al. An Asian variant of intravascular large B-cell lymphoma: clinical, pathological and cytogenetic approaches to diffuse large B-cell lymphoma associated with haemophagocytic syndrome. Br J Haematol 2000;111:826–834.

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Case 52 A 79-year-old man presented with pleural effusion with dyspnea on exertion for three months. These symptoms were attributed to congestive heart failure after myocardial infarction. Pleural flui showed WBC 6700/␮l with 62% lymphocytes. Physical examination revealed no peripheral lymphadenopathy and no hepatosplenomegaly. CT and MRI showed no lymphadenopathy and organomegaly in the thoracic and abdominal cavities. Bilateral bone marrow biopsy was negative for lymphoma. The patient had no risk factors for acquired immunodeficien y syndrome (AIDS). Cytospin smear and cell block of the pleural flui demonstrated large pleomorphic tumor cells (Figs. 52.1, 52.2, 52.3, and 52.4).

Fig. 52.1 Cytospin smear of pleural f uid shows a pleomorphic population of large anaplastic lymphoid cells. Note many cells show cytoplasmic vacuolation. Wright – Giemsa, × 100

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Fig. 52.2 Cell block section shows tightly packed pleomorphic lymphoid population. H&E, × 40

Fig. 52.3 Higher power view of the cell block reveals giant tumor cells. H&E, × 100

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Case 52

Fig. 52.4 Another view of the cell block shows atypical tumor cells. H&E, × 100

Differential diagnoses: Body cavity lymphoma, pyothorax associated lymphoma and primary effusion lymphoma.

Further Testing Serum human immunodeficien y virus (HIV) testing: negative Human herpesvirus type 8 (HHV-8) testing: negative In situ hybridization for Epstein – Barr virus (EBV): negative Flow cytometry: CD5 3%, CD19 95%, CD20 96%, CD30 5%, CD38 94%, CD10 96%, CD34 0%, CD45 100% Cytogenetic karyotype: questionable t(8;14)

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Discussion This patient has pleural effusion and the pleural flui contains large pleomorphic neoplasm with a monoclonal lymphoma phenotype and there is no evidence of lymphoma anywhere in the body. This is, by definition body cavity lymphoma. There are several kinds of body cavity lymphoma [1–3]. The case associated with HHV-8 infection is designated primary effusion lymphoma (PEL) and is accepted as a distinct clinicopathologic entity by the World Health Organization (WHO) classification PEL patients are frequently seen in AIDS patients, and many of them also have coexistent EBV infection. Morphologically, the tumor cells are large lymphoid cells resembling immunoblasts, plasmoblasts or anaplastic lymphoma cells. Immunophenotype is characterized by the expression of CD45 but not surface or cytoplasmic immunoglobulin as well as lack of B-cell and T-cell markers. In addition, activation and plasma cell-related antigens, such as CD30, CD36, CD38, Vs38c, and CD138, are usually demonstrated. Paradoxically, heavy chain gene rearrangement is always positive; therefore it is classifie under the category of diffuse large B-cell lymphoma. The current case does not have HHV-8 infection, which excludes the diagnosis of PEL. The immunophenotype by fl w cytometry is also not consistent with PEL. In HHV-8-negative body cavity lymphoma cases, several possibilities can be considered [1–3]. The pyrothorax-associated lymphoma (PAL) is EBV-associated HHV8-negative pleural B-cell lymphoma, usually after a longstanding chronic clinical condition. This tumor was originally identifie in patients with pyrothorax resulting from artificia pneumothorax for the treatment of pulmonary tuberculosis or tuberculous pleuritis. This disease is predominantly seen in Japanese patients. In addition to pleural effusion, a solid tumor of the pleura is also present. The current case has no clinical history of chronic infection and pyrothorax and there is no evidence of EBV infection, therefore it is not considered to be PAL. Other lymphomas, such as Burkitt lymphoma, immunoblastic lymphoma, and anaplastic large cell lymphoma, can present occasionally as primary lymphomatous effusion [1–3]. In our case, the immunophenotype is consistent with Burkitt lymphoma, and the karyotype is suspicious for t(8;14) translocation; therefore, Burkitt-like lymphoma is the most likely diagnosis. Further study of the c-MYC oncogene rearrangement by fluorescenc in situ hybridization on the paraffi section of the cell block is required for confirmatio of this diagnosis.

References 1. Said J, Cesarman E. Primary effusion lymphoma. 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, 260–261. 2. Ascoli V, Lo-Coco F. Body cavity lymphoma. Curr Opin Pulm Med 2002;8:317–320. 3. Carbone A. HHV-8-positive body-cavity-based lymphoma: A novel lymphoma entity. Br J Haematol 1997;97:515–522.

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Case 53 A 43-year-old man presented with a two-year history of HIV infection, complicated with cytomegaloviral retinitis and oral thrush. He initially noticed a swelling in his left axilla. CT scan identifie it as a large axillary lymph node. A lymph node biopsy was performed (Figs. 53.1 and 53.2). Peripheral blood smear showed no blasts. Physical examination revealed no hepatosplenomegaly. CT scan of abdomen demonstrated no mass. There were no tumor cells found in the cerebrospinal fluid The patient was started with two doses of intrathecal cytarabine, two doses of intravenous vincristine, one dose of intravenous doxorubicin, and f ve doses of intravenous cyclophosphamide.

Fig. 53.1 Lymph node biopsy shows a starry sky pattern due to the presence of numerous tangible-body macrophages. H&E, × 20

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Fig. 53.2 Touch preparation of a lymph node shows medium sized tumor cells with immature chromatin pattern, multiple inconspicuous nucleoli and a thin rim of deep blue cytoplasm containing multiple vacuoles. Wright – Giemsa, × 60

Differential diagnoses: Non-Hodgkin lymphomas.

Further Studies Ki-67 staining: positive (Fig. 53.3) CD20 staining: positive (Fig. 53.4) CD3 staining: negative CD10 staining: positive (Fig. 53.5) Bcl-2 staining: negative Bcl-6 staining: positive (Fig. 53.6) c-myc protein staining: positive (Fig. 53.7) Oil Red O staining of lymph node imprint: positive (Fig. 53.8)

Case 53

Fig. 53.3 Ki-67 staining shows positive immunoreaction in all tumor cells. Myeloperoxidase, × 20

Fig. 53.4 CD20 staining shows positive reaction on all tumor cells. Myeloperoxidase, × 20

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Fig. 53.5 CD10 staining shows positive reaction on all tumor cells. Myeloperoxidase, × 40

Fig. 53.6 Bcl-6 staining shows positive reaction in tumor cells. Myeloperoxidase, × 20

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Fig. 53.7 C-myc protein staining reveals numerous positive tumor cells. Myeloperoxidase, × 20

Fig. 53.8 Oil Red O stain demonstrates positive staining in the cytoplasmic vacuoles. Cytochemical stain, × 100

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Discussion Burkitt lymphoma/leukemia (BL) was f rst discovered in 1958 by Denis Burkitt, who described this tumor as a sarcoma involving the jaws of African children. Subsequently, many cases of “African lymphoma” were reported outside the African continent and these patients have a bimodal distribution (children and elderly) with the initial presentation of an abdominal mass, often involving the ileocecal region [1]. It accounts for 40% of lymphoma in children and 1–2% in adults in the United States and western Europe [2]. These two geographically different forms of tumor are now designated endemic and sporadic subtypes of BL, respectively (Table 53.1). Since the outbreak of the acquired immunodeficien y syndrome (AIDS), the incidence of BL has increased dramatically. It is estimated that BL is 1,000 times more common in AIDS patients than the general population [3]. This subtype involves mainly lymph nodes and bone marrow and is designated immunodeficien yassociated BL. Epstein – Barr virus (EBV) infection and malaria are probably the predisposing factors in the development of endemic BL, but evidence of EBV infection is much less common in sporadic and immunodeficien y-associated subtypes, and malaria is also not associated with these two subtypes [4]. Table 53.1 Comparison between endemic and sporadic Burkitt lymphoma Annual incidence per 100,000 population High risk age group Male/female ratio Initial presentation Bone marrow involvement Central nervous system involvement Leukemic phase Positive serologic test for EBV EBV receptor on tumor cells EBV genome in tumor cells EBV, Epstein – Barr virus.

Endemic subtype

Sporadic subtype

2.3–3.8 4–8 years 2:1 Jaw lesion About 8% About 30% Absent 88–97% Common 100%

0.1–0.3 Bimodal 2 or 3:1 Abdominal lesion 16–20% 5–20% Present About 20% Rare 11–20%

The morphology of this tumor is indistinguishable among the three subtypes. In tissue sections, the tumor cells are of medium size, with dispersed chromatin pattern, two to four small nucleoli, and a thin rim of cytoplasm. The histologic features are characterized by monotonous cohesive sheets of tumor cells with multiple mitotic figure and apoptotic bodies. The apoptotic bodies are frequently engulfed by phagocytic cells, which are commonly referred to as tangible-body macrophages. The scattered tangible-body macrophages impart the so-called “starry sky” pattern, which is characteristic of but not pathognomonic for BL, because it can also be seen in lymphoblastic lymphoma, immunoblastic lymphoma and other lymphomas with prominent apoptosis. Therefore, a touch preparation or bone marrow aspirate is important to identify the typical cytology for a preliminary diagnosis of BL. On the smears, the tumor cells are characterized by the moderate amount of deep blue cytoplasm containing multiple cytoplasmic vacuoles. The cytoplasm stains for methyl green pyronin due to its high content of polyribosome, whereas the lipid contents of cytoplasmic vacuoles are positive for Oil Red O. Cytoplasmic vacuoles can be seen in other lymphomas, but they are usually due to degeneration or high contents of glycoprotein or glycogen and not lipids. The chromatin pattern of the nucleus is more dispersed and immature than that perceived in tissue sections. Lymphomas with morphology between BL and diffuse large B-cell lymphoma were previously called Burkitt-like lymphoma (BLL) variant, but are now categorized as B-cell lymphoma, unclassifiabl with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma in the World Health Organization (WHO) classificatio [5]. Unlike BL, which shows a uniform round configuratio with multiple inconspicuous nucleoli, the BLL cells are pleomorphic with one or two prominent nucleoli, frequently eosinophilic. BLL is mainly seen in adults in the sporadic subtype. Extranodal presentation in internal organs is less frequently seen in BLL than BL (Figs. 53.9, 53.10, and 53.11).

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Fig. 53.9 Skin biopsy shows a pleomorphic tumor cell population and most cells reveal a single prominent nucleolus, characteristic of a Burkittlike lymphoma. H&E, × 100

Fig. 53.10 Liver biopsy of a BL case shows tumor cells in the portal area surrounding a bile duct and a small venule. H&E, × 40

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Fig. 53.11 Testicular biopsy of a BL case shows numerous tumor cells surrounding four seminiferous tubules. H&E, × 10

The second variant recognized in the 2001 WHO system is BL with plasmacytoid differentiation but it is no longer listed as a distinct variant in the 2008 classificatio [1]. The tumor cells show eccentric nucleus with often a single central nucleolus and basophilic cytoplasm. A monotypic cytoplasmic immunoglobulin pattern can be demonstrated in these cells. This variant is frequently seen in the immunodeficien y-associated subtype [3]. The BL cells usually show a monoclonal B-cell population derived from the follicular center cells. The B-cell markers that are expressed by BL include CD19, CD20, CD22, and CD79a [1–3]. The follicular center cell markers include CD10, bcl-6, and the newly discovered germinal center-associated lymphoma protein [1–3, 6]. However, one of the follicular center cell proteins, bcl-2, is characteristically absent or weakly positive in BL. C-myc protein is also demonstrated in most cases of BL. The negative markers that are helpful in differential diagnosis include CD5, CD23, CD138, bcl-1, and terminal deoxynucleotidyl transferase (TdT). However, the only marker that is accepted by the WHO system for a preliminary diagnosis is Ki-67 (mib-1), when more than 99% of the tumor cells are immunoreactive to this antigen [1]. A definit ve diagnosis depends on the demonstration of c-myc translocation with an immunoglobulin heavy chain or light chain gene, i.e., t(8;14)(q24;q32), t(2;8)(p11;q24) or t(8;22)(q24;q11). Although all subtypes share the same chromosomal aberrations, their breakpoints differ [1]. In endemic BL, the breakpoint in c-myc is more than 100 kb upstream from the f rst coding exon, and the breakpoint in the IgH gene is in the joining segment. In sporadic and HIV-associated BLs, the breakpoint in c-myc is between exons 1 and 2, and the break point in IgH is in the switch region. Gene expression profilin studies show that a subset of diffuse large B-cell lymphoma may have c-myc rearrangement but the partner genes are different [7, 8]. A few cases of BL may have bcl-2 rearrangement, and a small subset of BL has double translocation of c-myc and bcl-2, which carries the worst prognosis [3]. The distinction between BL and diffuse large B-cell lymphoma is clinically significant because BL has one of the highest cell proliferation rates of any human tumors with a doubling time of 24 to 48 hours. Therefore, BL requires brief-duration, high-intensity chemotherapy regimens containing aggressive central nervous system prophylaxis, whereas the treatment of diffuse large B-cell lymphoma is less intensive [2]. As mentioned above, the distinction between these two neoplasms is impossible in some cases and is categorized as B-cell lymphoma, unclassifiabl [5].

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References 1. Leoncini L, Raphael M, Stein H, et al. Burkitt lymphoma. 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, 262–264. 2. Blum KA, Lozanski G, Byrd JC. Adult Burkitt leukemia and lymphoma. Blood 2004;104:3009–3020. 3. Ferry JA. Burkitt’s lymphoma: clinicopathologic features and differential diagnosis. Oncologist 2006;1:375–383. 4. Brady G, MacAuthur GJ, Farrell PJ. Epstein-Barr virus and Burkitt lymphoma. J Clin Pathol 2007;60:1397–1402. 5. Kluin PM, Harris NL, Stein H, et al. B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma. 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, 265–266. 6. Natkunam Y, Lossas IS, Taidi B, et al. Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation. Blood 2005;105:3979–3986. 7. 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. 8. Dave SS, Fu K, Wright GW, et al. Molecular diagnosis of Burkitt lymphoma. N Engl J Med 2006;354:2431–2442.

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Case 54 A 80-year-old man presented with abdominal pain for one day. Radiologic examination of abdomen showed dilated loops of small intestine with air-flui levels. Exploratory laparotomy found an ileal mass and mesenteric lymphadenopathy. Biopsy of the ileum is illustrated in Figs. 54.1, 54.2, and 54.3.

Fig. 54.1 Ileal biopsy shows extensive lymphoid infiltratio of the mucosa and submucosa of the ileum with a starry sky pattern. H&E, × 10

Case 54

Fig. 54.2 Ileal biopsy reveals the medium-sized tumor cells with high mitotic f gures and apoptosis. H&E, × 40

Fig. 54.3 Another part of the ileal biopsy shows tumor cells confine to the submucosa. The mucosa is not involved. H&E × 20

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Differential diagnoses: Burkitt lymphoma, diffuse large B-cell lymphoma, extranodal marginal zone B-cell lymphoma.

Further Studies Immunohistochemistry of ileum specimen: CD3 stain: negative (Fig. 54.4) CD20 stain: positive (Fig. 54.5) CD10 stain: positive (Fig. 54.6) Bcl-2 stain: negative C-myc stain: positive (Fig. 54.7) Ki-67 stain: positive in >99% of cells (Fig. 54.8) Cytogenetics: t(8;14)(q24;q32) (Fig. 54.9) Molecular biology: Fluorescence in situ hybridization showed c-myc rearrangement.

Fig. 54.4 Ileal biopsy shows scattered CD3 staining of the T lymphocytes. Immunoperoxidase, × 10

Case 54

Fig. 54.5 Ileal biopsy shows strong, extensive CD20 staining of the tumor cells. Immunoperoxidase, × 10

Fig. 54.6 Ileal biopsy shows positive CD10 staining. Immunoperoxidase, × 10

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Fig. 54.7 Ileal biopsy shows positive c-myc staining. Immunoperoxidase, × 10

Fig. 54.8 Ileal biopsy shows positive Ki-67 staining in >99% of tumor cells. Immunoperoxidase, × 10

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Case 54

Fig. 54.9 Cytogenetic karyotype shows t(8;14)(q24;q32)

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Discussion In endemic areas, Burkitt lymphoma (BL) usually presents in the jaws of young children. Sporadic BL, on the other hand, is frequently located in the ileocecal region [1]. This case shows extensive lymphoid infiltratio in the ileum with involvement of the mesenteric lymph node, so that BL should be top of the list for differential diagnosis. The presence of a starry sky histologic pattern as seen in Fig. 54.1 further supports this assumption. Cytologically, the tumor cells are of medium size with relative uniform shape and multiple inconspicuous nucleoli. However, diffuse large B-cell lymphoma (DLBCL) can mimic BL and needs to be distinguished. BL and a subset of DLBCL are of follicular center cell origin, so that they have similar immunophenotype (i.e. positive for CD10, CD19, CD20, CD79a, bcl-6). However, BL is frequently negative for bcl2, whereas DLBCL is positive for this marker. Nevertheless, immunophenotyping alone is not acceptable by the World Health Organization (WHO) classificatio for a fina diagnosis of BL. The only acceptable immunohistochemical staining is Ki-67 (mib-1), which should be positive in more than 99% of tumor cells. Even if the fina diagnosis is not BL, the treatment will be as aggressive as BL if the proliferation fraction of the tumor is more than 99%. However, a definit ve diagnosis depends on the identificatio of c-myc oncogene rearrangement. Two recent studies [2, 3] with gene expression profilin show that a small percentage of DLBCL can also be positive for c-myc oncogene, but if the partner gene is the immunoglobulin heavy chain or light chain gene (e.g., t[8;14], t[2;8] or t[8;22]), it is still specifi for BL [4]. If the mucosa is the primary site of the lymphoma, extranodal marginal zone B-cell lymphoma (EMZBCL) should be considered. In this case, the primary site is submucosa with extension to mucosa since mucosa is intact in some areas whereas submucosa shows heavy infiltratio (Fig. 54.3). In addition, the immunophenotype is not consistent with EMZBCL, which is usually negative for all but the B-cell markers (e.g., negative for CD5, CD10, CD23, bcl-6). On the other hand, bcl-2 can be positive for EMZBCL, while it is negative for BL.

References 1. Leoncini L, Raphael M, Stein H, et al. Burkitt lymphoma, In: Swerdlow SH, Campo E, Harris HL, et al., eds: WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008: 262–266. 2. Dave SS, Fu K, Wright GW, et al. Molecular diagnosis of Burkitt lymphoma. N Engl J Med 2006;354:2431–2442. 3. Hummel M, Bertink 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. 4. Harris NL, Horning SJ. Burkitt’s lymphoma – The message from microarrays. N Engl J Med 2006;354:2495–2498.

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Case 55 A 64-year-old man presented with a 4-week history of increasing fatigue and malaise. The patient had an episode of coughing up blood and bleeding from his gum. At the emergency department, he was found to have severe thrombocytopenia and received platelet transfusion immediately. On admission, the laboratory studies showed a total leukocyte count of 5,900/␮l, hematocrit 32.5%, platelets 51,000/␮l and lactate dehydrogenase 926 IU/dl. His coagulation studies, including prothrombin time (PT) and activated partial thromboplastin time (aPTT), were within normal limits. Physical examination showed petechiae in the palate and the lower extremities, but no hepatosplenomegaly and lymphadenopathy. Radiologic examination showed no lymphadenopathy in the abdomen and no mass in the ileocecal area. The peripheral blood smear revealed rare blasts (Fig. 55.1). Bone marrow aspirate demonstrated 80% blasts (Fig. 55.2). The core biopsy showed 90% cellularity with extensive blastic infiltration replacing the normal hematopoietic cells (Fig. 55.3). There were many mitotic figures but apoptosis was not prominent.

Fig. 55.1 Peripheral blood smear shows a single blast. Wright – Giemsa, × 100

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Fig. 55.2 Bone marrow aspirate reveals many blasts with a high nuclear/cytoplasmic ratio and deep-basophilic cytoplasm with vacuolation. Wright – Giemsa, × 100

Fig. 55.3 Bone marrow biopsy shows hypercellularity with effacement of normal hematopoietic cells by monotonous immature cells. H&E, × 40

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Differential diagnoses: acute lymphoid or myeloid leukemia versus lymphoma.

Further Studies Ki-67 stain of bone marrow biopsy: high proliferation fraction (Fig. 55.4). Flow cytometry: Positive for CD10, CD19, CD45 and bcl-2 but negative for CD5, kappa, lambda and terminal deoxynucleotidyl transferase (TdT) (Fig. 55.5). Cytogenetic karyotype: t(8;22)(q24;q11.2) (Fig. 55.6).

Fig. 55.4 Ki-67 stain of bone marrow biopsy demonstrates a high proliferative fraction. Immunoperoxidase, × 40

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Fig. 55.5 Flow cytometry histograms demonstrate positive reactions to CD10, CD19, CD45 and bcl-2, but negative reactions to CD5, kappa, lambda and terminal deoxynucleotidyl transferase

Fig. 55.6 Cytogenetic karyotype shows 46,XY,t(8;22)(q24;q11.2),del(13)(q12q22),add(14)(q32)[18]/46,XY[2]

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Discussion The major clinical presentation in this patient was severe thrombocytopenia with hemoptysis and gum bleeding. The presence of a few blasts in the peripheral blood indicated acute leukemia. The bone marrow examination confirme this impression and the extensive blastic infiltratio explained the cause of thrombocytopenia. The f ow cytometry identifie the tumor cells as being of B-cell origin, and the absence of surface immunoglobulins together with positive reaction to CD10 is characteristic of acute lymphoblastic leukemia (ALL). The French – American – British (FAB) classificatio divides ALL into L1, L2 and L3 [1]. The morphology of L3 lymphoblasts is consistent with the Burkitt lymphoma cells. However, the World Health Organization (WHO) classificatio does not recognize the division of L1 and L2, because the morphologic difference based on the FAB scheme provides no correlation with clinical, immunophenotypic and cytogenetic finding [2]. L1 and L2 are now classifie as L1/L2 under the B-cell or T-cell acute lymphoblastic leukemia/lymphoma category in the WHO scheme. Burkitt lymphoma has three clinical subtypes with different tumor locations [3]. The endemic subtype usually shows the jaw lesion, the sporadic subtype, abdominal tumors and the immunodeficien y-associated subtype, nodal involvement. A leukemic presentation of Burkitt lymphoma is specifi for the sporadic subtype and it can also be seen in the immunodeficien y-associated subtype, but not in the endemic subtype [4]. Blood and bone marrow involvement is considered the leukemic phase of Burkitt lymphoma. Pure Burkitt leukemia without the involvement of lymph nodes or solid organs is rare. Burkitt leukemia has a strong tendency to involve the central nervous system and is very sensitive to chemotherapy, frequently leading to an acute tumor lysis syndrome [3]. Immunologically, in the 2008 WHO classificatio B-cell ALL is divided into pro-B-ALL, common ALL and pre-BALL [3]; Burkitt leukemia belongs to the mature B-ALL category that is no longer recognized by the WHO scheme, which emphasizes that B-ALL should not be used to indicate Burkitt leukemia [3]. In Burkitt leukemia, the tumor cells are positive for CD10, CD19, CD20, CD22 (cytoplasmic and surface), CD24, CD79a, bcl-6, HLA DR, and surface immunoglobulins, but are negative for CD34, cytoplasmic ␮ chain and TdT [4]. The major distinction between Burkitt leukemia and B-ALL (L1/L2) is that the former shows bright CD45 and is positive for surface immunoglobulins, but negative for TdT and CD34 [3]. The immunophenotype of the L1/L2 B-ALL is just the opposite. The current case is atypical in the absence of surface immunoglobulin and it could be mistaken as B-ALL. However, surface-immunoglobulin-negative Burkitt lymphoma has been reported in occasional cases. The bcl-2 positivity is due to the over-sensitivity of fl w cytometry for the detection of this protein. Again, weakly positive bcl-2 staining has been found in about 20% cases of Burkitt lymphoma [3]. Nevertheless, the identificatio of t(8;22) translocation in this case confirm unequivocally the diagnosis of Burkitt leukemia. The characteristic cytogenetic feature of Burkitt lymphoma/leukemia is the c-MYC translocation with the immunoglobulin heavy chain or light chain genes. In other word, it is t(8;14)(q24;q32), t(2;8)(p11;q24) or t(8;22)(q24,q11) translocation [3].

References 1. Bennett JM, Catovsky D, Danell MT, et al. French-American-British (FAB) Cooperative Group. Proposals for the classificatio of acute leukemias. Br J Haematol 1976;33:451–458. 2. Harris NL, Jaffe ES, Diebold J, et al. The World Health Organization Classificatio of Hematological Malignancies Report of the Clinical Advisory Committee Meeting, Airlie House, Virginia, November 1997. Mod Pathol 2000;13:193–207. 3. Leoncini L, Raphael M, Stein H, et al. Burkitt lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. eds., Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 262–264. 4. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lipincott Williams & Wilkins, 2008, 136–143, 253–261.

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Case 56 A 65-year-old-man was found to have neutropenia in a routine physical examination. He had been in relatively good health except for occasional sinusitis and for chronic arthritis. Physical examination demonstrated a large spleen about 3 cm below the left costal margin. No hepatomegaly or lymphadenopathy was detected. Peripheral blood examination revealed a total leukocyte count of 2, 200/␮l with 3% neutrophils and 88% lymphocytes (Fig. 56.1). His hematocrit was 34%, hemoglobin 11 g/dl, and platelets 110, 000/␮l. A bone marrow examination showed a decrease in myeloid to erythroid (M:E) ratio (0.8:1) (Figs. 56.2 and 56.3). A differential count revealed maturation arrest at the myelocyte stage (16% myelocytes, 2% metamyelocytes, 1% bands, and 1% segmented neutrophils) and an increase in lymphocytes (37%). The patient was treated with methotrexate, cyclophosphamide and cyclosporine with gradual improvement of neutropenia and anemia.

Fig. 56.1 Peripheral blood smear shows two large granular lymphocytes with transparent cytoplasm and cytoplasmic granules. Wright – Giemsa, × 300

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Fig. 56.2 Bone marrow aspirate reveals a cluster of large granular lymphocytes with a few small lymphocytes. Wright – Giemsa, × 100

Fig. 56.3 Bone marrow biopsy shows a lymphoid aggregate with a germinal center, representing reactive lymphocytes. The large granular lymphocytes are scattered in the periphery. H&E, × 20

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Differential diagnoses: aplastic anemia versus chronic lymphoid leukemias.

Further Studies Immunohistochemical stains of bone marrow: CD3 stain: positive for atypical lymphocytes CD20 stain: negative for atypical lymphocytes CD56 stain: negative for atypical lymphocytes CD57 stain: positive for atypical lymphocytes (Fig. 56.4) T-cell-receptor (TCR) gene rearrangement: clonal TCR beta chain gene rearrangement Cytogenetic karyotyping: normal male karyotype of 46,XY

Fig. 56.4 Bone marrow biopsy reveals CD57-positive large granular lymphocytes. Immunoperoxidase, × 60

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Discussion T-cell large granular lymphocyte (T-LGL) leukemia is define by the World Health Organization (WHO) as “a heterogeneous disorder characterized by a persistent (> 6 months) increase in the number of peripheral blood large granular lymphocytes, usually between 2 and 20 × 109 /L without a clearly identifie cause” [1]. This disease is similar to reactive large granular lymphocytosis in morphology and immunophenotype; the major distinctions are clinical manifestation and T-cell receptor (TCR) gene rearrangement. When LGL lymphocytosis persists for less than six months, it is designated transient LGL lymphocytosis, while cases showing lymphocytosis for more than six months are designated chronic LGL lymphocytosis [2]. Large granular lymphocytes (LGLs) vary from 15 to 18 ␮m in diameter [2,3]. The cytoplasm is usually pale or transparent, containing three or more azurophilic granules, but some LGLs may not contain any visible granules. The normal range of LGLs is 200–400/␮l or 10–15% among the mononuclear cells in the peripheral blood. When a patient has > 2, 000/␮l of LGLs or LGLs are > 40% of the lymphocyte fraction for more than six months, T-LGL leukemia should be suspected. Under most circumstance, a slight or moderate increase of LGLs is due to viral infections or autoimmune disorders. The bone marrow is frequently involved showing interstitial or nodular lymphocytic infiltration However, a linear accumulation of cytotoxic T cells within the bone marrow sinusoids is considered most specifi for this entity, while large lymphoid aggregates usually represent reactive mixed T- and B-cell population [4]. Nevertheless, LGL infiltratio may be inconspicuous and requires immunohistochemical stains to identify the tumor cells. Bone marrow aspirate may show increase of LGLs as well as small lymphocytes. A differential count may demonstrate maturation arrest of myeloid series at the myelocyte stage with resultant neutropenia in the peripheral blood [4]. The spleen usually shows leukemic infiltratio of the red pulp and prominent reactive germinal follicles consisting of polyclonal B cells [4]. In the liver, lymphoid infiltratio is mainly seen in hepatic sinusoids, but infiltratio of the portal areas may also be seen in severe cases [4]. The typical immunophenotype is CD3+ CD16+ CD56− CD57+ CD4− CD8+ [1–4]. Most LGL leukemic cells also contain cytotoxic granule proteins, such as T-cell intracellular antigen 1 (TIA1), granzyme B and perforin, in the cytoplasm. For T-cell receptor proteins, most cases express TCR␣␤. However, those cases with a CD3+ CD4− CD8− immunophennotype may express TCR␥␦ protein. Among other T-cell markers, CD2 is consistently expressed, but the expression of CD5 and CD7 is variable. The myeloid markers, CD11b and CD11c, are also frequently expressed in T-LGL leukemia. In addition, leukemic LGLs also express high levels of both Fas (CD95) and Fas ligand (CD95L) [3]. However, leukemic LGLs are resistant to Fas-mediated apoptosis, which can be one of the mechanisms of tumorigenesis in T-LGL leukemia. The recent discovery of the NK receptor (NKR) system in LGLs has a great potential in helping the diagnosis [5]. The NKR system includes two groups: killer cell immunoglobulin-like receptor and C-type-lectin-like receptor that consist of heterodimers of CD94 and NKG2 molecules. TCR gene rearrangement is the most important criterion for the identificatio of monoclonality in T-LGL leukemia [1–4]. Most cases show T C Rβ gene rearrangement, but T C Rγ gene rearrangement can be demonstrated in the minority of cases. Gene rearrangement can be performed with Southern blotting or polymerase chain reaction. Flow cytometry can also be used to analyze the T C Rβ gene variable region (V␤ ) protein expression to determine the clonality of T cells, but it is not as sensitive as the above techniques. Aberrant karyotypes have been reported in less than 10% of patients, including inversion of 12p and 14q, deletion of 5q, and trisomy of 3, 8, and 14 chromosomes [2]. The major clinical manifestations in T-LGL leukemia are chronic large granular lymphocytosis, neutropenia, splenomegaly, and rheumatoid arthritis; therefore it is difficul to distinguish T-LGL leukemia from Felty syndrome. Thus some authors considered these two entities to represent a spectrum of the same disorder [3]. Recurrent bacteria infections are secondary to neutropenia. These patients may also have B symptoms (fever, night sweats, and weight loss). Hepatomegaly is seen in 20% of patients. Lymphadenopathy is rare. Autoimmune antibodies, such as rheumatoid factor, antinuclear antibody, antineutrophil antibody, and antiplatelet antibody, are frequently found in these patients. Pure red cell aplasia and adult-onset cyclic neutropenia are also associated with T-LGL leukemia.

References 1. Chan WC. Foucar K, Morice WG, et al. T-cell large granular lymphocytic leukaemia. 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, 272–273. 2. Sokol L, Loughran TP Jr. Large granular lymphocyte leukemia. Oncologist 2006;11:263–273.

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3. Rose MG, Berliner N. T-cell large granular lymphocyte leukemia and related disorders. Oncologist 2004:9:247–258. 4. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 261– 266. 5. Lundell R, Hartung L, Hill S, et al. T-cell large granular lymphocyte leukemias have multiple phenotypic abnormalities involving pan-T-cell antigens and receptors for the molecules. Am J Clin Pathol 2005;124:937–946.

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Case 57 A 53-year-old man presented with pruritic skin rash over his forearms and trunk with low-grade fever for two weeks. The patient was born in Japan and migrated to the United States when he was 20 years old. Physical examination revealed generalized lymphadenopathy and splenomegaly. Peripheral blood examination showed a total leukocyte count of 13,000/␮l with 85% lymphocytes, 13% neutrophils, and 2% monocytes. His hematocrit was 45%, hemoglobin 14.5 g/dl, and platelets 195,000/␮l. Further examination of the peripheral blood smear found that about 20% of the lymphocytes showed atypical morphology (Fig. 57.1). A skin biopsy was performed (Figs. 57.2 and 57.3).

Fig. 57.1 A composite picture of four polylobated tumor cells in the peripheral blood. Wright – Giemsa, × 300

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Fig. 57.2 Skin biopsy shows extensive upper dermal infiltratio of tumor cells with epidermal involvement. H&E, × 20

Fig. 57.3 Higher magnificatio of skin biopsy reveals a cluster of large pleomorphic tumor cells invading the epidermis. H&E, × 40

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Differential diagnoses: cutaneous T-cell lymphoma, mycosis fungoides/S´ezary syndrome and adult T-cell leukemia/lymphoma.

Further Studies Viral serologic tests: positive for human T-cell leukemia virus-1 (HTLV-1) but negative for cytomegalovirus, Epstein – Barr virus and hepatitis B virus Blood chemistry profile lactate dehydrogenase 390 U/l, alkaline phosphatase 420 U/l and calcium 14 mg/dl. Other electrolytes and enzymes were within normal limits Flow cytometry of peripheral blood specimen: CD2 97%, CD3 98%, CD4 95%, CD5 93%, CD7 12%, CD8 4%, CD19 4%, CD20 3%, CD25 85%

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Discussion The current case showed peripheral lymphocytosis with 20% atypical lymphoid cells showing polylobated nuclei. The presence of skin lesion, lymphadenopathy and splenomegaly are also suggestive of lymphoid malignancy. The differential diagnoses should include mycosis fungoides/S´ezary syndrome and other cutaneous T-cell lymphomas. However, the presence of serologic evidence of HTLV-1 infection, the characteristic phenotype of CD4+ CD25+ for the tumor cells and marked hypercalcemia confir the diagnosis of HTLV-1-associated T-cell leukemia or adult T-cell leukemia/lymphoma (ATCL). The geographic distribution of this disease is also helpful to the diagnosis; most patients are from Japan, the Caribbean basin, and certain regions of South America, Africa and Middle Eastern Asia [1–3]. ATCL is the f rst leukemia linked to retroviral infection. HTLV-1 can be transmitted by blood transfusion, sexual contact and breastfeeding. In addition to ATCL, it may also cause HTLV-associated myelopathy/tropical spastic paraparesis, arthropathy and uveitis [3]. However, most ATCL patients in endemic areas contract the disease through breast milk, and the disease has a long latent period of several decades after the initial infection. Transmission requires transfer of HTLV-1infected cells and not free virus. The pathognomonic feature of ATCL is the presence of polylobated nuclei in the tumor cells (fl wer cells) [1–3]. The nuclear chromatin is moderately condensed, and nucleoli are inconspicuous. The cytoplasm is slightly to moderately basophilic. The percentage of such atypical leukemic cells present in the peripheral blood depends on the clinical form. The acute form accounts for 55–65% of patients [2, 4]. This form usually shows systemic symptoms, lymphadenopathy, organomegaly, and leukemia. The atypical lymphoid cells are usually numerous and they are highly pleomorphic [5]. Hypercalcemia and elevated lactate dehydrogenase (LDH) are the common laboratory findings Skin lesion is often the initial clinical presentation. The chronic form represents 20% of patients with moderate lymphocytosis that include the f ower cells, which are usually more uniform in size and shape [1, 3, 4]. Serum calcium and LDH are often normal. The patient may also have skin lesion and involvement of lymph node, lung or liver. The smoldering form is seen in 5% of patients with no systemic symptoms and no lymphocytosis [2, 4, 5]. However, less than 3% atypical lymphocytes may be present in the peripheral blood and skin and/or lung lesions may be seen. Serum calcium and LDH are usually normal in these patients. The lymphomatous form has a frequency similar to that of the chronic form (20%). The major clinical presentation is lymphadenopathy with or without hepatosplenomegaly [4,5]. Skin lesion and elevated LDH may be present, but peripheral lymphocytosis and hypercalcemia are characteristically absent. The skin lesion usually shows dermal infiltratio by small or large tumor cells, including CD30+ large cells mimicking anaplastic large cell lymphoma [6]. Pautrier microabscesses are often present which makes it difficul to distinguish from mycosis fungoides. In the lymph nodes, the normal architecture can be entirely effaced or the infiltrat is mainly seen in the paracortex (Fig. 57.4) [2, 6]. The tumor cells can be small cell, medium-sized cell, large cell, mixed type, or pleomorphic type. In patients with superimposed Epstein – Barr virus infection, Reed – Sternberg-like cells may be present [1, 2]. Immunophenotypically, ATCL is characterized by its helper T-cell phenotype (CD4+ CD8−), consistent presence of CD25 (Tac antigen or interleukin 2 receptor), and frequent loss of CD7 (a pan-T-cell antigen) [2, 3, 6]. Other positive T-cell markers include CD2, CD3, and CD5. A fina diagnosis of ATCL depends on the identificatio of HTLV-1 antibody by serologic techniques or the viral genome by molecular techniques. A monoclonal or oligoclonal pattern of HTLV-1 integration into cellular DNA can be demonstrated by the Southern blotting technique [6]. However, polymerase chain reaction (PCR) or reverse transcriptase PCR technique using a probe specifi for the HTLV-1 pol sequences or the pX gene is more specifi and sensitive than Southern blotting. The Tax protein in the virus is widely regarded as the key factor in the tumorigenesis of ATCL. The action of Tax protein is through transcriptional factors to affect a large number of human genes [7].

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Fig. 57.4 Lymph node biopsy shows the predominance of small tumor cells with irregular configuration intermingled with residual lymphocytes. H&E, × 100

References 1. Ohshima K, Jaffe ES, Kikuchi M. Adutl T-cell leukemia/lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. WHO Classificatio of Tumours of Haematolopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 281–284. 2. Matutes E. Adult T-cell leukemia/lymphoma. J Clin Pathol 2007;60:1373–1377. 3. Ratner L. Human T cell lymphotropic virus-associated leukemia/lymphoma. Curr Opin Oncol 2005;17:469–473. 4. Shimoyama M. Diagnostic criteria and classificatio of clinical subtypes of adult T-cell leukemia-lymphoma: a report from the lymphoma study group (1984–87). Br J Haematol 1991;79:428–437. 5. Brunning RD, McKenna RW. Tumor of the Bone Marrow. Washington, DC, Armed Forces Institute of Pathlogy, 1994, 301–308. 6. Watanabe S. Adult T-cell leukemia/lymphoma. In Knowles DM, ed. Neoplastic Hematopathology, 2nd ed. Philadelphia, Lippincott Williams & Wilkins, 2001;1603–1616. 7. Taylor G. Molecular aspects of HTLV-1 infection and adult T-cell leukaemia/lymphoma. J Clin Pathol 2007;60:1392–1396.

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Case 58 A 32-year-old man presented with a nontender right testicular mass. A testicular sonogram revealed a solid mass but alphafetoprotein and beta-human chorionic gonadotropin were negative. A right radical orchiectomy was performed (Fig. 58.1). Four cycles of chemotherapy were administered in conjunction with intrathecal methotrexate with resultant pancytopenia. The patient subsequently developed B symptoms (fever, night sweats, and weight loss) and CT scan demonstrated splenomegaly. A splenectomy (Fig. 58.2) and liver biopsy (Fig. 58.3) were performed. After splenectomy, pancytopenia resolved, but he suddenly developed leukocytosis (40, 000/␮l). Neoplastic lymphoid cells were found in the peripheral blood (Fig. 58.4) and the bone marrow (Figs.58.5 and 58.6). The patient died of massive gastrointestinal hemorrhage six months after the initial diagnosis.

Fig. 58.1 Testis shows extensive lymphoid infiltratio surrounding a seminiferous tubule. H&E, × 20

Case 58

Fig. 58.2 Splenectomy specimen reveals atypical lymphocytes infiltratin the red pulp core and sinuses. H&E, × 40

Fig. 58.3 Liver biopsy shows atypical lymphoid infiltratio in the portal area. H&E, × 40

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Fig. 58.4 Peripheral blood smear shows neoplastic large granular lymphocytes. Wright – Giemsa, × 100

Fig. 58.5 Bone marrow biopsy reveals interstitial and sinusoidal infiltratio of atypical lymphocytes. Giemsa, × 40

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Fig. 58.6 Bone marrow aspirate shows many neoplastic large granular lymphocytes with cytoplasmic granules (arrow). Wright – Giemsa, × 100

Differential diagnoses: lymphomas versus leukemias.

Further Studies Flow cytometric analysis of spleen: CD3 3%, CD4 2%, CD5 2%, CD7 11%, CD8 2%, CD10 5%, CD11c 96%, CD16 6%, CD19 3%, CD20 2%, CD56 89%, CD57 2%, HLA-DR 82% Flow cytometric analysis of blood: CD2 99%, CD3 5%, CD4 2%, CD5 5%, CD7 17%, CD8 3%, CD10 2%, CD11c 83%, CD16 12%, CD19 0%, CD20 0%, CD56 95%, CD57 1%, HLA-DR 54% Gene rearrangement studies: Germline configuratio for immunoglobulin heavy chain gene and for T-cell-receptor alpha-beta chain (TCR␣␤) and TCR␥␦ chain gene rearrangements.

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Discussion Natural killer (NK) cell lymphoma/leukemia (NKL) is a heterogeneous group of lymphomas/leukemias with various morphologic and clinical presentations. The fina classificatio depends on the immunophenotype, genotype and the status of Epstein – Barr virus (EBV) infection. In the 2008 World Health Organization (WHO) classification three mature NK-cell neoplasms are recognized: chronic lymphoproliferative disorder of NK cells, aggressive NK-cell leukemia and extranodal NK/T-cell lymphoma, nasal type [1, 2]. The blastic NK-cell lymphoma in the 2001 WHO scheme is now classifie as blastic plasmacytoid dendritic cell neoplasm (see Case 61). The myeloid/NK-cell acute leukemia and myeloid/NK-cell precursor acute leukemia reported in the earlier literature are probably acute myeloid leukemias with expression of the CD56 marker. Chronic lymphoproliferative disorder of NK cells is distinguished from T-cell large granular lymphocyte leukemia (Case 56) only by immunophenotype and genotype [3]. The current case exemplifie the entity of aggressive NK-cell leukemia, which has a fulminating clinical course with systemic dissemination of NK cells. NK cells assume the morphology of large granular lymphocytes (LGL). The morphology and normal ranges of LGL are described under Case 56. The original criteria for NK/T-LGL leukemia was the persistent presence of 2,000 LGL/␮l or > 40% of the lymphocyte fraction for at least six months and that all possible causes for reactive lymphocytosis are excluded. The new cutoff for NK-LGL leukemia is 600/␮l [4]. The tumor cells of the aggressive NK-cell leukemia are usually highly pleomorphic and often contain irregular cytoplasmic azurophilic granules, which are more prominent than those seen in normal LGLs [1–4]. Angioinvasion and angiodestruction are frequently observed in this tumor. Hemophagocytosis is also a common feature. In the spleen, this leukemia shows predominantly red pulp involvement. Extranodal NK/T cell lymphoma, nasal type, includes all extranodal tumors not limited to the nasal cavity and nasal pharynx; the gastrointestinal tract and skin are also frequently involved. Recently, NKL with primary nodal involvement is also included under this entity, designated nodal T/NK-cell lymphoma of nasal type [5]. Extranodal NK/T-cell lymphomas have a broad cytologic spectrum with variable cell size. The only morphologic evidence of NK cells is the demonstration of cytoplasmic granules in the touch preparation. In the early stage, the lymphoma cells are intermixed with reactive inflamma tory cells, including small lymphocytes, histiocytes, neutrophils, eosinophils, and plasma cells (Fig. 58.7). In the late stage, a full-blown feature of pleomorphic tumor cells with irregular nuclei and granular cytoplasm may finall prevail.

Fig. 58.7 Natural killer cell lymphoma in the duodenum shows extensive perivascular infiltratio of a mixed leukocyte population intermingled with tumor cells. H&E, × 10

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Fig. 58.8 Natural killer cell lymphoma in the duodenum shows perivascular tumor cell infiltratio with angioinvasive and angiodestructive lesion. The blood vessel wall is barely recognizable. H&E, × 40

The histologic pattern is more helpful than cytology in the diagnosis of this type of NKL. Regardless of the location, the pathognomonic features are angioinvasion, angiodestruction, mucosal ulceration and zonal necrosis in the majority of cases (Fig. 58.8). The major diagnostic criteria for NKL are the expression of NK cell marker, CD56 and cytotoxic granular protein (T-cell intracellular antigen-1 [TIA-1], granzyme B and perforin), presence of EBV infection (evidenced by EBV small-encoded RNA [EBER] or other serological tests), and the absence of TCR gene rearrangement [1–4]. However, the lack of one of these criteria does not entirely exclude the diagnosis, if other features are typical [1, 6]. In terms of T-cell markers, the most characteristic feature is the expression of cytoplasmic CD3 (CD3␧ ) but absence of surface CD3. The distinction between surface and cytoplasmic CD3 can be made only by f ow cytometry and not by immunohistochemistry. CD2 is persistently present on NK cells. Other T-cell markers, including CD4, CD5, CD7, CD8, CD43, and CD45RO, are usually negative. Two other NK cell markers, CD16 and CD57, are negative in NKL. Fas (CD95) and Fas ligand (CD95L) are commonly expressed and the interaction of these two molecules may facilitate local tissue invasion, distant metastasis, systemic tissue damage, and immune evasion. NKLs are characterized by the absence of clonal rearrangements of TCR genes and immunoglobulin heavy chain genes. The clonality of NK cells can be determined by X-linked DNA analysis in female patients, a method seldom used. There are two new receptor systems identifie in the NK cells [4]. The chemokine receptors have the function of controlling lymphocyte trafficking The NK receptors help to distinguish the autologous cells from the exogenous cells, thus protecting the former from NK-mediated cytotoxicity. Methods are being developed to identify the restriction of receptor repertoire as a means to establish the clonality of NK cells. The most common genetic abnormalitities in NKL are loss or gain of genetic materials. Deletion of 6q has the highest frequency in both aggressive NK-cell leukemia and extranodal NK/T-cell lymphoma. The latter also shows other abnormalities, such as del(11q), del(3q), del(17q), i(1q), i(6q) and 3 + xp (4). However, the numerical genetic abnormalities may represent secondary changes rather than the initial events. While it was suspected that aggressive NK-cell leukemia was merely the leukemic phase of extranodal NK/T-cell lymphoma, an array-based comparative genomic hybridization study has shown significan genetic differences between these two disorders [1].

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References 1. Chan JKC, Jaffe ES, Ralfkiaer E, et al. Aggressive NK-cell leukemia. 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, 276–277. 2. Chan JKC, Quintanilla-Martinez L, Ferry JA, et al. Extranodal NK/T-cell lymphoma, nasal type. 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, 285–288. 3. Sokol L, Loughran TP Jr. Large granular lymphocyte leukemia. Oncologist 2006;11:263–273. 4. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 267– 277. 5. Takahashi E, Asano N, Li C, et al. Nodal T/NK-cell lymphoma of nasal type: a clinicopathological study of six cases. Histopathology 2008;52:585–596. 6. Hasserjian RP, Harris NL. NK-cell lymphomas and leukemias. A spectrum of tumors with variable manifestations and immunophenotype. Am J Clin Pathol 2007;127:860–868.

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Case 59 A 30-year-old man presented with a one-week history of fever, chills, and abdominal pain. Peripheral blood examination showed pancytopenia with marked thrombocytopenia. Physical examination revealed hepatosplenomegaly, but no lymphadenopathy was detected. The patient was refractory to multiple transfusions. Finally, a splenectomy was performed (Figs. 59.1 and 59.2). A liver biopsy (Figs. 59.3 and 59.4) and a splenic hilar lymph node biopsy (Fig. 59.5) were taken at the same time.

Fig. 59.1 A splenectomy specimen shows marked distention of red pulp sinuses with lymphoid cells. H&E, × 20

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Fig. 59.2 Higher magnificatio of the dilated sinuses to show the atypical lymphoid cells. Note endothelial lining. H&E, × 60

Fig. 59.3 Liver biopsy reveals sinusoidal lymphoid infiltratio in contrast to the uninvolved portal areas. H&E, × 10

Case 59

Fig. 59.4 A close look at the sinusoidal infiltratio in a liver biopsy. H&E, × 20

Fig. 59.5 Splenic hilar lymph node biopsy shows prominent sinusoidal dilation without lymphoma cells. H&E, × 20

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Differential diagnoses: Hepatosplenic T-cell lymphoma versus splenic marginal zone lymphoma.

Further Studies Immunohistochemical stains: CD3 stain: positive (Fig. 59.6) CD20 stain: negative Flow cytometry of splenectomy specimen: positive for CD3 and negative for CD4, CD5, CD7, and CD8. All B-cell markers (CD19, CD20, kappa and lambda) were negative

Fig. 59.6 A liver biopsy reveals positive staining of the tumor cells with CD3 antibody. Immunoperoxidase, × 40 (From Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms, Philadelphia, Lippincott Williams & Wilkins, 2008)

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Discussion Hepatosplenic T-cell lymphoma (HSTCL) is a rare lymphoma, accounting for less than 5% of all peripheral T-cell lymphomas that are recognized by the World Health Organization (WHO) classification It is designated hepatosplenic ␥␦ T-cell lymphoma in the Reversed European American Classificatio of Lymphoid Neoplasms (REAL classification because most of the early cases showed an immunophenotype consistent with ␥␦ T cells. However, a few cases of ␣␤ T-cell lymphoma were reported subsequently, so that ␥␦ T cell has been changed into T cell in the WHO classificatio [1]. This lymphoma has an aggressive clinical course and patients have a median survival of 12–18 months [1,2]. Most patients are young (15–32 years at diagnosis) and predominantly male. The characteristic clinical presentation is hepatosplenomegaly without lymphadenopathy [1–5]. Peripheral blood usually shows pancytopenia, especially thrombocytopenia. Bone marrow is often involved at diagnosis. Most patients also have systemic symptoms, such as fever, chills, night sweats, weight loss and abdominal pain. In rare occasions, ␥␦ T-cell lymphomas may also involve skin, intestine and the nasal region, but they do not seem to belong to the same entity. The characteristic histologic pattern is sinusoidal infiltratio in the spleen, liver and bone marrow [1–5]. Most lymphomas involve the white pulp of the spleen, but HSTCL is one of the few lymphomas/leukemias infiltratin the red pulp exclusively. The white pulp, on the other hand, is atrophic. The lymphoma cells are present mainly in the sinuses but the red pulp cord of Billroth is also involved. The infiltratio in the liver is also confine to the sinuses without the involvement of the portal areas. The sinusoidal infiltratio in the bone marrow is so characteristic for this lymphoma that some authors consider it diagnostic if the patient has the right clinical setting (hepatosplenomegaly, no lymphadenopathy, pancytopenia, and B symptoms) [4]. The tumor cells are small to medium in size with a small amount of cytoplasm and a clumpy chromatin pattern. Nucleoli are usually inconspicuous. Immunohistochemistry is frequently required to highlight the lymphoma cells, particularly in the bone marrow [1–5]. Immunohistochemical positive stains include CD3, CD43, and CD45RO. On the other hand, the double negative staining of CD4 and CD8 is most characteristic and distinguishes HSTCL from other T-cell lymphomas. Flow cytometry may demonstrate positive CD3 reaction and negative CD4, CD5, CD7, and CD8 reactions. The identificatio of natural cell markers, including CD16, CD56, and CD11c, may further substantiate the diagnosis [4]. However, CD57 is consistently negative in HSTCL cases. Among the cytotoxic proteins, T-cell intracellular antigen 1 (TIA-1) is expressed by the tumor cells, but perforin and granzyme B are not. Therefore, HSTCL cells are considered to be inactivated cytotoxic T cells [1–3]. HSTCL cases usually show T-cell receptor ␥- and ␦-chain gene rearrangement, but ␤-chain gene rearrangement has also been demonstrated in some cases. Therefore, genotyping is not specific However, isochromosome 7q [i(7)(q10)] is a specifi primary aberration in HSTCL [1, 3, 5]. It may be present alone or coexistent with other abnormalities, such as trisomy 8. Recent studies also demonstrated aberrant coincidental expression of multiple killer immunoglobulin-like receptors (KIR) isoforms along with dim or absent CD94 [1].

References 1. Gaulard P, Jaffe ES, Krenacs L, et al. Hepatosplenic T-cell lymphoma. In Swerdlow SH, Campo E, Harris NL, et al. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed., Lyon, France, IARC Press, 2008, 292–293. 2. Gaulard P, Belhadj K, Reyes F. ␥␦ T-cell lymphoma. Semin Hematol 2003;40:233–243. 3. de Wolf-Peters C, Achten R. ␥␦ T-cell lymphomas: a homogeneous entity? Histopathology 2000;36:294–305. 4. Belhadj K, Reyes F, Farcet JP, et al. Hepatosplenic ␥␦ T-cell lymphoma is a rare clinicopathologic entity with poor outcome: report on a series of 21 patients. Blood 2003;102:4260–4269. 5. Salhany KE, Feldman M, Kahn MJ, et al. Hepatosplenic ␥␦ T-cell lymphoma: ultrastructural, immunophenotypic, and functional evidence for cytotoxic T lymphocyte differentiation. Hum Pathol 1997;28:674–685.

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Case 60 A 58-year-old man presented with a one-month history of multiple tender, erythematous subcutaneous nodules, involving bilateral lower extremities. Before onset of the nodules, the patient had fever, chills, malaise and myalgias for a few days. Despite treatment with antibiotics and local steroid preparation, there was no improvement of clinical symptoms. His peripheral blood showed no cytopenia. Physical examination revealed no hepatosplenomegaly and lymphadenopathy. A skin biopsy was performed (Figs. 60.1, 60.2, 60.3, and 60.4).

Fig. 60.1 Skin biopsy shows no tumor cell involvement of the epidermis and dermis. A small cluster of perivascular infiltratio of normal lymphocytes is present in the lower dermis. H&E, × 10

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Fig. 60.2 The subcutaneous tissue reveals tumor cell rimming of the adipocytes, forming a lace-like pattern. H&E, × 20

Fig. 60.3 A densely infiltrate area shows a large number of hyperchromatic, atypical lymphoma cells with numerous apoptotic bodies. H&E, × 40

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Fig. 60.4 A higher magnificatio demonstrates the hyperchromatic tumor cells in contrast with a few pale-stained histiocytes. A few mitotic figure are present. H&E, × 60

Differential diagnoses: Cutaneous T-cell or B-cell lymphoma versus benign panniculitis.

Further Studies Immunohistochemical stains: CD3 stain: positive CD4 stain: negative CD8 stain: positive CD20 stain: negative T-cell-receptor (TCR) gene rearrangement analysis: clonal rearrangement of TCR ␥ chain gene

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Discussion Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare entity accounting for less than 1% of all non-Hodgkin lymphomas [1]. It is seen predominantly in the elderly but a few pediatric cases have been reported [2]. The prognosis of this disease was considered to be poor because many cases of primary cutaneous ␥␦ T-cell lymphomas were classifie under this category [2, 3]. After exclusion of these cases, SPTCL generally has a much better prognosis [4]. One important prognostic factor is the presence of hemophagocytic syndrome (HPS). Once the HPS is present, treatment does not change the outcome [3]. The major clinical manifestation is the presence of multiple erythematous nodules, most commonly seen in the lower extremities, followed by the trunk, arms and face. Constitutional symptoms, such as fever, chills, fatigue, weight loss, joint and muscle pain, are present in about 40% cases [3]. When HPS develops, the patient may have hepatosplenomegaly and coagulopathy [3]. Histologically, SPTCL is characterized by a lobular panniculitis-like morphology. The lymphoma cells are of varying sizes with hyperchromatic, atypical nuclei. They tend to rim the adipocytes and form a lace-like pattern [4]. Karyorrhexis or apoptosis is one of the characteristic features and is considered to be mediated by the release of cytotoxic granular proteins by the tumor cells [5]. The presence of benign histiocytes with or without phagocytosis is a consistent feature. The dermis and epidermis are usually not involved by the lymphoma cells. There is no regional lymphadenopathy in the drained area. Immunophenotypically, the tumor cells usually express multiple T-cell markers, including CD2, CD3, CD43, and CD45RO [3]. Early reports with studies done on frozen sections showed many cases with predominantly CD4 phenotype, which was probably due to the staining of histiocytes [2, 5]. Current studies confir that the tumor cells belong to cytotoxic T cells because they express predominantly CD8, T-cell intracellular antigen-1 (TIA-1), granzyme B, and perforin [2, 5]. CD56 is always negative in the tumor cells [4]. Epstein – Barr virus is also negative in SPTCL. SPTCL cases are more frequently derived from ␣␤ T cells than ␥␦ T cells, as proven by TCR gene rearrangement studies [2, 5]. The “␥␦ SPTCL” is now classifie as primary cutaneous ␥␦ T-cell lymphoma [4], which is commonly associated with aggressive disease, systemic HPS, cytopenia and CD56 expression [2]. In contrast to SPTCL, cutaneous ␥␦ T-cell lymphoma also involves the dermis and epidermis. SPTCL needs to be differentiated from benign panniculitis and other lymphomas that involve the subcutaneous tissue. Benign panniculitis shows no atypia in the infiltratin lymphocytes, which are composed of aggregates of B cells [5]. There are also polyclonal plasma cells and dispersed T lymphocytes, which comprise an admixture of CD4+ and CD8+ cells [5]. In cutaneous T/NK-cell lymphoma, the neoplastic infiltrat is more confluen and tends to overrun adipose cells rather than to rim them [5]. The tumor cells also infiltrat the dermis and even the epidermis. Karyorrhexis is seldom seen. T/NK-cell lymphoma also shows a prominent angioinvasive and angiodestructive pattern and consistent expression of CD56. Anaplastic large cell lymphoma also shows a more confluen infiltratio pattern, invading both the dermis and epidermis. The demonostration of CD30 may help to distinguish it from SPTCL.

References 1. Gonzalez CL, Medeiros LJ, Braziel RM, et al. T-cell lymphoma involving subcutaneous tissue: a clinicopathologic entity commonly associated with hemophagocytic syndrome. Am J Surg Pathol 1991;15:17–27. 2. Salhany KE, Macon WR, Choi JK. Subcutaneous panniculitis-like T-cell lymphoma: Clinicopathologic, immunophenotypic, and genotypic analysis of alpha/beta and gamma/delta subtypes. Am J Surg Pathol 1998;22:881–893. 3. Weening RH, Ng CS, Perniciaro C. Subcutaneous panniculitis-like T-cell lymphoma. Am J Dermatopathol 2001;23:206–215. 4. Jaffe ES, Gaulard P, Ralfkiaer E, et al. Subcutaneous panniculitis-like T-cell lymphoma. In Swerdlow SH, Campo E, Harris NL, et al., WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France, IARC Press, 2008, 294–295. 5. Kumar S, Krenacs L, Medeiros J, et al. Subcutaneous panniculitic T-cell lymphoma is a tumor of cytotoxic T lymphocytes. Hum Pathol 1998;29:397–403.

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Case 61 A 39-year-old man presented with progressive skin lesions on his trunk associated with constitutional symptoms for six months. He had arthralgias, bone pain in the ankles and wrists, fatigue, fever, and a 20-pound weight loss over a period of six months. He was treated with a short course of antibiotics without benefit Physical examination revealed diffused subcutaneous nodules of varying sizes over his trunk with the largest nodule measuring 4 cm. Lymphadenopathy and hepatosplenomegaly were not detected. Laboratory test results, including liver function tests and hematology workup, were within normal ranges. A skin biopsy showed tumor cell infiltratio (Figs. 61.1 and 61.2). Subsequently, a bone marrow biopsy was performed (Figs. 61.3 and 61.4).

Fig. 61.1 A skin biopsy shows extensive leukemic infiltratio in the dermis. The epidermis is uninvolved and is separated from the dermal lesion by a clear zone (Grenz zone). H&E, × 10

Case 61

Fig. 61.2 Higher magnificatio of the skin biopsy shows the leukemic cells surrounding a hair follicle. H&E, × 60

Fig. 61.3 Bone marrow aspirate reveals a cluster of leukemic cells and many of them are bilobed (arrow). Wright – Giemsa, × 100

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Fig. 61.4 Bone marrow biopsy shows total replacement of normal hematopoietic cells by leukemic components. Note a few bilobed cells (arrow). H&E, × 100

Differential diagnoses: T-cell and B-cell lymphomas.

Further Studies Immunohistochemistry: Skin biopsy: positive for CD4, CD56, and CD68 but negative for CD3, CD5, CD8, CD20, myeloperoxidase, and lysozyme Flow cytometry: Skin biopsy: positive for CD2, cytoplasmic CD3, CD4, CD7, CD45, CD56, and HLA-DR, but negative for surface CD3, CD8, CD10, CD13, CD19, CD33, CD34, CD117, terminal deoxynucleotidyl transferase (TdT), and myeloperoxidase Bone marrow biopsy: positive for CD2, CD4, CD7, CD13, CD33, and CD56, but negative for CD3, CD8, CD11c, CD14, CD19, CD20, CD57, kappa, lambda, and TdT T-cell receptor gene rearrangement: negative

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Discussion In 1995, Brody et al reported a case of acute agranular CD4-positive natural killer cell leukemia [1]. This entity was later define by the World Health Organization (WHO) as blastic NK-cell lymphoma. However, because of the uncertainty of the cell lineage of this tumor, Petrella et al coined the term “agranular CD4/CD56 hematodermic neoplasm” [2]. Finally, the World Health Organization – European Organization for Research and Treatment of Cancer (WHO-EORTC) classificatio for cutaneous lymphomas modifie the term and designated it as CD4+/CD56+ hematodermic neoplasm [3]. Subsequent studies, including immunophenotyping, chemokine receptors, in vitro functional assays, gene expression profiling and the tumour-derived cell line CAL-1, all support the plamacytoid dendritic cell origin of this tumor [4]. Therefore, the 2008 WHO scheme classifie this tumor as blastic plasmcytoid dendritic cell neoplasm (BPDC) [4] Because of the great variations in the immunophenotype, there are probably many subtypes of dendritic cells. However, two subtypes are well established: dendritic cell type 1 (DC1), also called myeloid dendritic cells and dendritic cell type 2 (DC2), also known as plasmacytoid or lymphoid dendrictic cells (pDCs). DC1 expresses blood dendritic cell antigen 3 (BDCA3), while DC2 carries BDCA2 (CD303) and BDCA4 (CD304) [5]. Other commonly used pDCs-associated antigens include T-cell leukemia 1 (TCL1), cutaneous lymphocyte-associated antigen (CLA), and CD123 (IL-3␣ receptor) [6–8]. A recent study reported the adaptor protein CD2AP as the most specifi marker for pDCs [8]. The pDCs also express many signaling molecules, transcription factors, and Toll-like receptors, but many of these molecules are also expressed in other cell types, particularly B cells, and are thus of no practical signifi cance [8]. For routine immunophenotyping, the expression of CD4 and CD56 in the absence of lineage-specifi markers of T cells, B cells, or myelomonocytic cells is sufficien to diagnose BPDC when patients show a cutaneous presentation with bone marrow involvement [6, 7]. However, a fina diagnosis should be confirme by the presence of CD123, CLA, and/or TCL1. Other common positive markers include CD43, HLA-DR, and CD45RA [7]. The presence of EBV antigens, myeloperoxidase, lysozyme, PAX-5, CD20, CD22, and T-cell receptor protein should exclude a diagnosis of pDC leukemia/lymphoma [7]. Nevertheless, BPDC may be derived from myeloid dendritic cells. In those cases of acute myeloid dendritic cell leukemia, myeloid markers, such as CD13, CD14, CD15, CD33, myeloperoxidase, lysozyme, and BDCA3, can be identifie [5]. The current case belongs to this category. Cytogenetic anomalies have been identifie in two-thirds of pDC tumors [6]. These are usually complex karyotypes, characteristic of lymphoid and myeloid leukemias, but no specifi karyotype of pDC neoplasms has ever been identifie [6,7]. T-cell receptor gene and immunoglobulin heavy chain gene rearrangements should not be demonstrated in these tumors. In the earlier literature, pDCs were also called T-plasmacytoid cells, T-associated plasma cells, plasmacytoid monocytes, or plasmacytoid T cells [7,8]. These cells can be present in non-malignant conditions, such as Kikuchi disease and Castleman disease [8]. The clinical features of BPDC are highly characteristic. The initial presentation is usually in the skin with subsequent involvement of blood, bone marrow, or lymph nodes [6, 7]. Spleen, liver, central nervous system, lungs, kidneys, tonsils and muscles may also be involved [6]. The cutaneous lesions can be solitary or multifocal. The dermis is densely infiltrate but the epidermis is characteristically spared and is separated from the dermal lesion by a clear zone (Grenz zone) [5]. The cytology of the tumor cells is variable, but most cases show blastoid features, resembling lymphoblasts or myeloblasts. Cytoplasmic vacuoles and pseudopod-like extensions are frequently described [6, 7]. BPDC has been reported to be associated with precedent, concurrent, or subsequent myelomonocytic leukemias [7].

References 1. Brody JP, Allen S, Schulman P, Sun T, et al. Acute agranular CD4-positive natural killer cell leukemia. Comprehensive clinicopathologic studies including virologic and in vitro culture with inducing agents. Cancer 1995;75:2474–2483. 2. Petrella T, Meijer CJLM, Dalac S, et al. TCL1 and CLA expression in agranular CD4/CD56 hematodermic neoplasms (blastic NK-cell lymphomas) and leukemia cutis. Am J Clin Pathol 2004;122:307–313. 3. Willemze R, Jaffe ES, Burg G, et al. WHO-EORTC classificatio for cutaneous lymphomas. Blood 2005;105:3768–3785. 4. Facchetti F, Jones DM, Petrella T. Blastic plasmacytoid dendritic cell neoplasm. 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, 145–147. 5. Ferran M, Gallardo F, Ferrer AM, et al. Acute myeloid dendritic cell leukemia with specifi cutaneous involvement: a diagnostic challenge. Br J Dermatol 2008;158:1129–1133.

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6. Garnache-Ottou F, Feuillard J, Saas P. Plasmacytoid dendritic cell leukemia/lymphoma: toward a well define entity? Br J Haematol 2007;136:539–548. 7. Herling M, Jones D. CD4+/CD56+ hematodermic tumor: The features of an evolving entity and its relationship to dendritic cells. Am J Clin Pathol 2007;127:687–700. 8. Marafiot T, Paterson JC, Ballabio E, et al. Novel markers of normal and neoplastic human plasmacytoid dendritic cells. Blood 2008;111:3778– 3792.

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Case 62 A 51-year-old man presented with pruritic rash over his arms, abdomen, and joints for two years. He was treated for psoriatic dermatitis with no effect. His skin lesion progressed steadily and a skin biopsy was performed (Figs. 62.1 and 62.2). Peripheral blood (Fig. 62.3) and bone marrow (Fig. 62.4) examinations showed atypical lymphoid cells. After the correct diagnosis, his treatment produced temporary improvement of the skin lesions, but the disease progressed rapidly afterwards with involvement of 90% of his body surface area. He was noted to have axillary and groin lymphadenopathy and a lymph node biopsy was performed (Fig. 62.5).

Fig. 62.1 Skin biopsy shows intraepidermal lesion, and bandlike lymphoid infiltratio in the upper dermis that spares the dermal epidermal junction (Grenz zone). H&E, × 10

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Fig. 62.2 Skin biopsy reveals Pautrier microabscesses in the epidermis, consisting of single-haloed lymphocytes. H&E, × 40

Fig. 62.3 Peripheral blood shows four lymphoid cells with cerebriform nuclei. Wright – Giemsa, × 100

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Fig. 62.4 Bone marrow biopsy reveals diffuse infiltratio of atypical lymphocytes with irregular nuclei. H&E, × 100

Fig. 62.5 Lymph node biopsy shows large clusters of tumor cells with folded or convoluted nuclei, intermixed with residual small lymphocytes. H&E, × 60

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Differential diagnoses: cutaneous T- or B-cell lymphoma and mycosis fungoides.

Further Studies Flow cytometry: Peripheral blood: CD3 98%, CD3/CD4 95%, CD3/CD8 2%, CD5 70%, CD7 15%, CD19 2%, CD25 12% Bone marrow: CD3 95%, CD3/CD4 90%, CD3/CD8 8%, CD5 65%, CD7 10%, CD19 5%, CD25 0% Lymph node: CD3 92%, CD3/CD4 90% CD3/CD8 3%, CD5 68%, CD7 12%, CD19 4%, CD25 5% Immunohistochemistry: Skin biopsy: The lymphoid infiltrate in the skin showed positive staining for CD3 and CD4 but negative staining for CD8 and CD20

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Discussion Mycosis fungoides (MF) is a primary cutaneous T-cell lymphoma (CTCL) with a clinical presentation of patch, plaque, and tumor stages, and an epidermal and dermal skin infiltratio by small to medium-sized lymphoid cells with cerebriform nuclei. It is the most common CTCL, accounting for 65% of all primary cutaneous lymphomas [1]. In the earlier literature, S´ezary syndrome (SS) was considered an erythrodermic leukemia variant of MF. However, the current International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization for Research and Treatment of Cancer (EORTC) consider MF and SS as two separate diseases, partly because their prognoses are markedly different [1]. If a case meets the criteria of SS but is preceded by clinically typical MF, it is designated SS preceded by MF or secondary SS. The histologic features of MF/SS overlap with other cutaneous lymphomas and even some benign inflammator conditions. Therefore, clinical correlation is of utmost importance in making a morphologic diagnosis. The histologic pattern varies in different clinical stages of MF and in SS [2]. A patch lesion may reveal scanty to patchy lymphocytic infiltratio in the upper dermis. It is usually perivascular rather than bandlike. Intraepidermal lymphoid infiltratio (Pautrier microabscesses) is rare or absent. Cytologic atypia, if present, is minimal. The plaque lesion is characterized by a dense, bandlike infiltrat of atypical lymphoid cells in the upper dermis. Pautrier microabscesses are often present. The atypical lymphoid cells show hyperchromatic and convoluted nuclei and scant cytoplasm. The deep dermis and subcutis are seldom involved. The tumor lesion, in contrast, shows a dense infiltrat of atypical lymphoid cells that may extend into the lower dermis and subcutis. At this stage, the tumor cells increase in number and in size, so that a mixed population of small, medium-sized, and large cerebriform cells as well as blasts with prominent nucleoli is present. Epidermal infiltration in contrast, gradually disappears. The features that are considered most specifi for MF include Pautrier microabscesses and the presence of lymphocytes within the epidermis that are larger than those within the dermis [3]. The presence of convoluted lymphocytes 7 to 9 microns in diameter, epidermal infiltratio with single-haloed lymphocytes (lymphocytes surrounded with vacuoles), lining up of lymphocytes along the basal layer, and a lichenoid infiltrat that spares the dermal epidermal junction are also considered specifi diagnostic features for MF [3]. In the ISCL classification SS is define by the following features: 1. an absolute S´ezary cell count of ≥ 1000/␮l; 2. demonstration of immunophenotypic abnormalities: CD4/CD8 ratio of ≥ 10 or aberrant loss of pan-T-cell markers by fl w cytometry; and 3. demonstration of a T-cell clone in the peripheral blood by molecular or cytogenetic techniques [4]. The histologic features in the lymph node of MF/SS cases are most commonly those of dermatopathic lymphadenopathy. Characteristically, the lymph node contains sinus histiocytosis, an abundance of pigment-laden macrophages, and various numbers of atypical lymphocytes depending on the stage of the disease. The three major histopathologic grading systems for lymph nodes in MF/SS are the ISCL/EORTC, Dutch and NCI/VA classification on the basis of tumor cell numbers (Table 62.1) [1]. Table 62.1 Histopathologic grading systems of lymph node in MF/SS [1] ISCL/EORTC system Dutch system

NCI/VA system

N1

LN0 : no atypical lymphocytes

N2 N3

Grade 1: dermatopathic lymphadenopathy (DL)

Grade 2: DL; early involvement by MF (presence of cerebriform nuclei > 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

382

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

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

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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.

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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

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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

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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

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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.

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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.

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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.

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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 <5% of all lymphomas [1]. ALCL is characterized by its large, bizarre tumor cells, which, however, may not be predominant in its variants. ALCL was f rst discovered in 1982 and in the early era there were many variants, including pleomorphic, monomorphic, small cell, lymphohistocytic, Hodgkinlike, mixed, sarcomatoid, neutrophil-rich, eosinophil-rich, and signet-ring variant [2, 3]. However, in the 2001 World Health Organization (WHO) classification there are only three variants: common, small cell and lymphohistiocytic [3]. The common variant consists of a wide spectrum of large tumor cells with different morphologic features [1–4]. The most common cell type has vesicular horseshoe- or kidney-shaped nuclei with prominent nucleoli. A prominent eosinophilic Golgi region is frequently present. The cytoplasm is moderately abundant and mildly basophilic. Cells with these features have been called hallmark cells, because they are present in all ALCL variants in various proportions and their presence is the major diagnostic criterion. The small cell variant is characterized by the presence of predominantly small to medium-sized tumor cells. Nevertheless, the hallmark cells are invariably identifiabl around the blood vessels, particularly with the help of the CD30 stain. The lymphohistiocytic variant is define by the presence of a large number of histiocytes, which may occasionally show signs of erythrophagocytosis. As in the small cell variant, a diagnosis depends on the findin of perivascular infiltratio of the large tumor cells. In the 2008 WHO classification these variants are mentioned as patterns and a Hodgkin-like pattern is included [4]. The Hodgkin-like pattern was included in the Revised European – American Classificatio of Lymphoid Neoplasms (REAL classification as a provisional entity, which shows features mimicking nodular sclerosis classical Hodgkin lymphoma. Besides the angiocentric distribution of the tumor cells, the histologic features are characterized by a sheet-like cohesive growth pattern with tendency to sinusoidal or paracortical infiltratio [1–4]. These features frequently mislead to the diagnosis of carcinoma. The diagnosis of ALCL depends on immunophenotyping. This tumor is of predominantly T-cell type with occasional cases of null-cell type. When the tumor cells express B-cell markers, it is classifie as diffuse large B-cell lymphoma, because this type differs from ALCL in biologic, cytogenetic, and clinical aspects. However, the important diagnostic marker is CD30. Immunohistochemical staining can also demonstrate epithelial membrane antigen (EMA), clusterin, and anaplastic lymphoma kinase (ALK) [1, 2]. Cases with morphologic and immunophenotypic features of ALCL without the presence of ALK are classifie separately as ALCL, ALK-negative to distinguish from the classical ALCL, which is now designated ALCL, ALK-positive [4]. Cytotoxic granular proteins are often present. CD56 is present in a subset of ALCL and carries an unfavorable prognosis [1]. A high percentage of Survivin-positive cells also predicts a poor clinical outcome. Because one-third of ALCL cases may not express CD45, as well as the positive EMA reaction and the sinusoidal histologic features, ALCL is frequently confused with the diagnosis of carcinoma. The positive CD30 and the presence of Reed – Sternberg-like cells in some cases may mislead the diagnosis to Hodgkin lymphoma. The distinction between systemic ALCL and Hodgkin lymphoma is listed in Table 66.1. Table 66.1 Distinction between systemic ALCL and Hodgkin lymphoma Markers Systemic ALCL

Hodgkin lymphoma

Cytotoxic granular proteins + − ALK + − CD15 − + Clusterin + − EMA + − BSAP (PAX5) − + BNH.9 + − EBV − + ALCL, anaplastic large cell lymphoma; ALK, anaplastic lymphoma kinase; BNH.9, blood group antigen H and Y; BSAP, B-cell-specifi activation protein; EBV, Epstein – Barr virus; EMA, epithelial membrane antigen

A recurrent t(2:5)(p23;q35) translocation has been demonstrated in 40% to 60% of ALCL cases [1–5]. This translocation leads to the fusion of the anaplastic lymphoma kinase (ALK) gene at 2q23 and nucleophosmin (NPM) at 5q35. As a result, a chimeric NPM-ALK protein is produced. ALK is a novel tyrosine kinase, whereas NPM carries the newly synthesized

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protein from the cytoplasm to the nucleolus. Therefore, when ALK translocates with other partner genes, there is no nuclear staining of ALK. Other partner genes include TPM3, TFG, ATIC, CLTCL, MSN, TPM4, MYH9, and ALO17 and the translocation with ALK produces various fusion proteins (Table 66.2). The oncogenes can be demonstrated by Southern blotting, polymerase chain reaction and fluorescenc in situ hybridization (FISH), while the fusion proteins can be demonstrated by immunohistochemistry. As immunohistochemistry is comparable to FISH in sensitivity and specificit for the demonstration of ALK, it can be used to make a prompt diagnosis without resorting to molecular genetic techniques. Table 66.2 Characteristics of various ALK fusion proteins Frequency Genetic abnormality

Fusion proteins

Staining patttern

72.5% t(2:5)(p23;q35) NPM-ALK Cytoplasmic and nuclear 17.5% t(1;2)(q25;p23) TPM3-ALK Cytoplasmic and membrane 2.5% t(2;3)(p23;q21) TFG-ALK Cytoplasmic 2.5% inv(2)(p23;q35) ATIC-ALK Cytoplasmic 2.5% t(2;17)(p23;q11) CLTCL-ALK Granular cytoplasmic <1.0% t(2:X)(p23;q11–12) MSN-ALK Membrane <1.0% t(2;19)(p23;p13.1) TPM4-ALK Cytoplasmic <1.0% t(2;22)(p23;q11.2) MYH9-ALK Cytoplasmic <1.0% t(2;17)(p23;q25) ALO17-ALK Cytoplasmic ALK, anaplastic lymphoma kinase; NPM, nucleophosmin; TPM, tropomyosin; TFG, tropomyosin receptor kinase-fusion gene; ATIC, 5-aminoimidazole-4-carboxamide-1-beta-p-ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase; CLTCL, clathrin heavy polypeptide-like gene; MSN, moesin

ALK-positive systemic ALCL is usually seen in children and populations in their second and third decades with male predominance [1–5]. Systemic B symptoms (fever, night sweats, and/or weight loss) are present in about 75% of patients. About 70% of ALK-positive cases present with advanced stage disease or extranodal involvement, which is commonly present in the skin, bone, soft tissues, lung, and liver. Bone marrow involvement is detected in 30% of patients with immunostaining. The f ve-year survival rate is between 71% to 79% in various studies. ALK-negative systemic ALCL occurs in older persons between the ages of 40 and 60 with no gender preference. It is more frequently seen in secondary ALCL (transformed from T-cell lymphoma/leukemia) or HIV-related ALCL. The prognosis in ALK-negative cases are usually poorer than that of ALK-positive cases with a f ve-year survival rate between 11% and 46%. As mentioned before, this subtype is now separated from the ALK-positive ALCL as a distinct entity [6].

References 1. Jacobsen E. Anaplastic large-cell lymphoma, T-/null-cell type. Oncologist 2006;11:831–840. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 304–312. 3. Jaffe ES, Harris NL, Stein H, Vardiman JW. Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC, 2001, 230–235. 4. DelsolG, Falini B, M¨uller-Hermelink HK, et al. Anaplastic large cell lymphoma (ALCL), ALK-positive. 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, 312–316. 5. Kutok JL, Aster JC. Molecular biology of anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma. J Clin Oncol 2002;20:3691– 3702. 6. Mason DY, Harris NL, Delsol G, et al. Anaplastic large cell lymphoma, ALK-negative. 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, 317–319.

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Case 67 A 12-year-old girl presented with fever, sore throat, and right cervical lymphadenopathy. Peripheral blood examination revealed mild lymphocytosis. Serologic tests for antibodies against toxoplasma, cytomegalovirus and herpes simplex were all negative. A monospot test was also negative. The patient was treated with antibiotics without improvement of clinical symptoms. A lymph node biopsy was performed (Figs. 67.1 and 67.2).

Fig. 67.1 Lymph node biopsy shows effacement of normal architecture by diffuse small lymphocytic infiltratio with vascular proliferation. H&E, × 20

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Fig. 67.2 The same lymph node as in Fig. 67.1 demonstrates the presence of large lymphoma cells around the blood vessel. H&E, × 40

Differential diagnoses: Reactive lymphadenopathy versus lymphoma.

Further Studies Immunohistochemical stains: CD3 stain: positive CD20 stain: negative CD30 stain: positive (Fig. 67.3) Anaplastic lymphoma kinase 1 (ALK1) stain: positive (Fig. 67.4) Epithelial membrane antigen (EMA) stain: positive (Fig. 67.5) Flow cytometry: Strong positive reactions for CD3, CD8. and CD30, positive reactions for CD4 and CD5, but negative reaction for CD7 and CD20

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Fig. 67.3 CD30 stain highlights the angiocentric distribution of the large tumor cells. Immunoperoxidase, × 40 (From Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms, Philadelphia, Lippincott Williams & Wilkins, 2008)

Fig. 67.4 ALK1 stain shows cytoplasmic and nuclear staining in large lymphoma cells but only weak nuclear staining in small lymphoma cells. Immunoperoxidase, × 100 (From the same source as Fig. 67.3)

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Fig. 67.5 Epithelial membrane antigen (EMA) stain shows positive staining in most of the tumor cells. Immunoperoxidase, × 60 (From the same source as Fig. 67.3)

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Discussion The diagnosis of the small cell variant of anaplastic large cell lymphoma (ALCL) is frequently a challenge, because the patients are usually young, with a median age of 14, and because features of an inflammator process are frequently present clinically and morphologically [1]. It is particularly problematic when, in some cases, the lymph node architecture is only partially effaced. If a complete immunophenotyping is not performed, it can be misdiagnosed as peripheral T-cell lymphoma, not otherwise specifie [2, 3]. However, the prognoses of these two lymphomas are quite different. Although the small cell variant is rare, only accounting for 5–10% ALCL cases, it is important to recognize this entity, particularly in the pediatric population, because it is easily misdiagnosed as benign lesions. Clinically, this lymphoma predominantly involves the skin or the lymph node. B symptoms (fever, weigh loss, and night sweats) were demonstrated in 56% of patients in one series [1]. The small cell variant usually does not have the same favorable prognosis as other variants of ALCL, because these patients often present with disseminated disease at diagnosis [3]. Histologically, the tumor is composed predominantly of small to medium-sized lymphoid cells with a small number of large lymphoid cells [1–4]. The small lymphoid cells usually show irregular nuclei. In addition, inflammator cells are seen in varying numbers in the small cell variant. These include neutrophils, eosinophils, and plasma cells. Macrophages are present in most cases, with erythrophagocytosis in a few cases. Therefore, it is important to look for the small numbers of large tumor cells, which show the similar cytologic features of the common type and which are usually seen around the blood vessels or occasionally in the sinuses of the lymph node [1]. The detection of the large tumor cells is usually facilitated by immunohistochemical staining, mainly the CD30 staining. Immunophenotypically, almost all cases of the small cell variant are of T-cell origin. All the T-cell antigens, CD2, CD3, CD4, CD5, CD7, CD8, CD43, and CD45RO, can be positive but not all antigens are present in the same case [1, 4]. T-cell receptor gene rearrangement is also demonstrated in most cases studied. The most important markers for the diagnosis are CD30 and ALK. Both stainings are strongly positive for the large lymphoma cells and weakly positive in the small cell population. Some small tumor cells can be negative for both stainings. ALK stains both the cytoplasm and the nuclei of the large cells but only the nuclei of the small cells. The EMA stain, although not diagnostic, is highly characteristic for this lymphoma, as it is a specifi antigen of carcinomas. CD25 can be demonstrated in some cases, which should be distinguished from adult T-cell lymphoma/leukemia [1]. Whether the small cell component is malignant or reactive can be resolved by the following observations. The small cells stain for ALK, which is the most important evidence that it is the tumor cells. The transformation of the small cell variant to the ALCL common type has been demonstrated [1, 4]. Finally, most cases of the small cell variant show t(2;5) translocation that is characteristic for ALCL.

References 1. Kinney MC, Collins RD, Greer JP, et al. A small-cell-predominant variant of primary Ki-1 (CD30)+ T-cell lymphoma. Am J Surg Pathol 1993;17:859–868. 2. Falini B. Anaplastic large cell lymphoma: Pathological, molecular and clinical features. Br J Haematol 2001;114:741–760. 3. Delsol G, Falini B, M¨uller-Hermelink HK, et al. Anaplastic large cell lymphoma (ALCL), ALK-positive. 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, 312–316. 4. Benharroch D, Meguerian-Bedoyan Z, Lamant L, et al. ALK-positive lymphoma: A single disease with a broad spectrum of morphology. Blood 1998;91:2076–2084.

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Case 68 A 57-year-old man presented with left neck swelling for three weeks. He was treated with antibiotics to no avail and was admitted for a lymph node biopsy. Physical examination showed bilateral nontender lymphadenopathy over the cervical, axillary, and inguinal regions. CT scan also revealed diffuse adenopathy throughout the neck, chest, and abdomen. A left cervical lymph node biopsy is depicted in Figs. 68.1, 68.2, and 68.3.

Fig. 68.1 Lymph node biopsy shows several large lymphoma cells on a background of lymphohistiocytic infiltration H&E, × 40

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Fig. 68.2 Higher magnificatio reveals a few typical kidney-shaped lymphoma cells (arrow). H&E, × 100

Fig. 68.3 A Reed – Sternberg-like cell (arrow) is present. H&E, × 100

Differential diagnoses: Hodgkin lymphoma versus non-Hodgkin lymphoma.

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Case 68

Further Testing Immunohistochemistry: CD3: negative for tumor cells (Fig. 68.4) CD20: negative for tumor cells (Fig. 68.5) CD30: positive for tumor cells (Fig. 68.6) CD15: negative for tumor cells (Fig. 68.7) CD45: positive for tumor cells (Fig. 68.8) Epithelial membrane antigen (EMA): positive for tumor cells (Fig. 68.9) Anaplastic lymphoma kinase (ALK): positive for tumor cells (Fig. 68.10) CD68: positive for histiocytes (Fig. 68.11) Pancytokeratin: negative for tumor cells (Fig. 68.12)

Fig. 68.4 CD3 stain demonstrates many small T lymphocytes. Immunoperoxidase, × 20

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Fig. 68.5 The lymphoma cells are negative for CD20 stain. Immunoperoxidase, × 20

Fig. 68.6 CD30 stain demonstrates surface and Golgi staining patterns of the lymphoma cells. Immunoperoxidase, × 60

Case 68

Fig. 68.7 The lymphoma cells are negative for CD15 stain. Immunoperoxidase, × 40

Fig. 68.8 CD45 stain demonstrates surface and Golgi staining patterns of lymphoma cells. Immunoperoxidase, × 100

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Fig. 68.9 EMA stain highlights the lymphoma cells. Immunoperoxidase, × 20

Fig. 68.10 ALK1 stain reveals cytoplasmic and nuclear staining patterns of lymphoma cells. Immunoperoxidase, × 20

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Fig. 68.11 CD68 stain highlights the histiocytes. Immunoperoxidase, × 40

Fig. 68.12 The lymphoma cells are negative for cytokeratin stain. Immunoperoxidase, × 60

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Discussion The lymphohistiocytic variant of anaplastic large cell lymphoma (LH-ALCL) is characterized by intermixing of scattered large anaplastic tumor cells with large numbers of histiocytes that may obscure the tumor cells [1–5]. The anaplastic tumor cells are similar to those of other variants of ALCL, except that they may be smaller than those seen in the common type [1]. In other words, the tumor cells are large, anaplastic with eccentrically placed horseshoe- or kidney-shaped nuclei, dispersed chromatin pattern, and one or more nucleoli. The tumor cells are frequently present as perivascular cuff, easily demonstrated by immunohistochemical staining. The histiocytes are characterized by abundant acidophilic cytoplasm with an eccentric dense nucleus. Occasionally, erythrophagocytosis may be demonstrated in the histiocytes. The morphologic features of LH-ALCL are similar to lymphoepithelial lymphoma (Lennert lymphoma), T-cell/histiocyterich B-cell lymphoma, or Hodgkin lymphoma, which can be distinguished only by immunohistochemistry [1–5]. The tumor cells are characterized by the expression of CD30, EMA, and in most cases ALK. Most patients have T-cell tumor with positive CD3, CD43, CD45RO, and cytotoxic granular protein, but null-cell cases have been reported. The tumor cells also have a high proliferation fraction (Ki-67 >90%). On the other hand, B-cell markers and histiocyte markers are consistently negative. CD15 is negative in most cases. The histiocytes are positive for CD68 (KP1 and PG-M1), lysozyme, and CD45 but negative for CD30, ALK, EMA, and Ki-67, and are therefore not neoplastic [2]. A karyotype of t(2;5)(p23;q35) and T-cell receptor gene rearrangement have been demonstrated in a few cases studied [1, 2]. Lennert lymphoma is most similar to LH-ALCL morphologically, but the small lymphocytes are neoplastic in this tumor and the histiocytes are characteristically epithelioid. T-cell/histiocyte-rich B-cell lymphoma can be distinguished by its B-cell nature. In contrast to LH-ALCL, Hodgkin lymphoma is positive for CD15 and BSAP (PAX5) and negative for ALK. LH-ALCL accounts about 10% of all ALCL cases [1]. The patients are usually in the f rst two decades of life, younger than the common type of ALCL. The clinical presentation is superficia lymphadenopathy with systemic symptoms. Hepatosplenomegaly and mediastinal and retroperitoneal masses are also seen in some patients. Patients with this tumor have a more favorable response to chemotherapy than the common variant of ALCL.

References 1. Delso G, Falini B, M¨uller-Hermelink HK, et al. Anaplastic large cell lymphoma (ALCL), ALK-positive. 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, 312–316. 2. Pileri SA, Pulford K, Mori S, et al. Frequent expression of the NPM-ALK chimeric fusion protein in anaplastic large-cell lymphoma, lymphohistiocytic type. Am J Pathol 1997;150:1207–1211. 3. Pileri S, Falini B, Delsol G, et al. Lympho-histiocytic T cell lymphoma (anaplastic large cell lymphoma CD30+/Ki-1+ with a high content of reactive histiocytes). Histopathology 1990;73:806–813. 4. Yang CS, Chou G, Jan YJ, et al. Primary lymphohistiocytic variant of anaplastic large cell lymphoma of the stomach. J Chin Med Assoc 2007;70:71–75. 5. Paulli M, Vallisa D, Viglio A, et al. ALK positive lymphohistiocytic variant of anaplastic large cell lymphoma in an adult. Haematologica 2001;86:260–265.

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Case 69 A 44-year-old woman had a small nodule in the right armpit for a few weeks. The patient denied the presence of fever, weight loss, and night sweats. Physical examination found that the nodule was a lymph node, measuring 2 cm in diameter. It was movable and nontender. There were no additional superficia lymph nodes identified The liver and spleen were not palpable. Peripheral blood examination revealed mild anemia with normal leukocyte differential and platelet count. The pathology of the right axillary lymph node biopsy is depicted in Figs. 69.1, 69.2, and 69.3.

Fig. 69.1 Lymph node biopsy shows a nodular pattern. The tumor cells are represented by many small holes. H&E, × 5

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Fig. 69.2 Lymph node biopsy reveals many popcorn cells. H&E, × 40

Fig. 69.3 Higher power view of the lymph node demonstrates the multilobated or folded nuclei and scant cytoplasm of the popcorn cells. H&E, × 60

Case 69

Differential diagnoses: Hodgkin versus non-Hodgkin lymphomas.

Further Studies Immunohistochemistry: CD30 stain: negative for tumor cells (Fig. 69.4) CD15 stain: negative for tumor cells (Fig. 69.5) CD45 stain: positive for tumor cells (Fig. 69.6) CD20 stain: positive for tumor cells (Fig. 69.7) PAX5 stain: positive for tumor cells (Fig. 69.8) CD3 stain: negative for tumor cells (Fig. 69.9) CD57 stain: negative for tumor cells (Fig. 69.10) CD21 stain: negative for tumor cells (Fig. 69.11) Fascin stain: positive for tumor cells (Fig. 69.12)

Fig. 69.4 The popcorn cells are negative for CD30 stain. Immunoperoxidase, × 40

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Fig. 69.5 The popcorn cells are negative for CD15 stain. Immunoperoxidase, × 40

Fig. 69.6 The popcorn cells are positive for CD45 stain. Immumoperoxidase, × 100

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Fig. 69.7 CD20 stain highlights the popcorn cells and B lymphocytes. Immunoperoxidase, × 100

Fig. 69.8 PAX5 stain demonstrates the popcorn cells in a B lymphocyte nodule. The tumor cells characteristically show lighter staining than the B lymphocytes. Immunoperoxidase, × 40

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Fig. 69.9 CD3 stain reveals the T lymphocytes forming a rosetting or rimming pattern around the popcorn cells. Immunoperoxidase, × 40

Fig. 69.10 The CD57-positive T lymphocytes show the same rosetting around the popcorn cells. Immunoperoxidase, × 40

Case 69

Fig. 69.11 CD21 stain demonstrates that the popcorn cells are embedded in a follicular dendritic cell meshwork. Immunoperoxidase, × 40

Fig. 69.12 Fascin stain is positive for the popcorn cells. Immunoperoxidase, × 40

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Discussion In the World Health Organization (WHO) classification Hodgkin lymphoma (HL) is divided into nodular lymphocyte predominant HL (NLPHL) and four subtypes of classical HL [1, 2]. The current case belongs to the NLPHL, which has many distinct features that are different from the classical HLs. The hallmark cell for HLs is the Reed – Sternberg (RS) cell, which is characterized by its large size, bilobated or polylobated nuclei, with eosinophilic inclusion-like nucleoli and abundant amphophilic cytoplasm [1, 3]. There are several variants of RS cells. The presence of the variants is not diagnostic, but it should give a clue to the possible diagnosis of HL. The monoclear variant is a large cell with a single nucleus that contains a prominent eosinophilic nucleolus. This cell is called a mononucleated Hodgkin cell. A cell with a similar features, but multinuclear, is called a multinucleated Hodgkin cell. The degenerated form of a tumor cell is a dark smudged cell, which is called a “mummifie cell”. The other two variants are also not diagnostic of HL, but are characteristic of certain subtypes of HL [1, 3]. The lacunar cell is most frequently seen in the nodular sclerosing subtype of HL and will be discussed under this entity (Case 70). The lymphocytic and/or histiocytic (L&H) cell variant or popcorn cells are now designated lymphocyte predominant cells (LP cells) [1]. They are large cells with folded, convoluted, or multilobated nuclei; thin nuclear membrane, vesicular chromatin pattern; and inconspicuous nucleoli. The cytoplasm of these cells is pale-staining and varies from small to medium amount. This cell is typically seen in NLPHL. The characterisitic histologic feature for NLPHL is a vague or partially nodular pattern with diffuse areas [1, 3]. Reticulin stain may accentuate the nodular configuration The background is composed of small lymphocytes, histiocytes, and epithelioid histiocytes. Eosinophils, neutrophils, plasma cells, and necrosis are rarely seen. As mentioned before, the tumor cells are mainly LP cells, with accompanied mononuclear Hodgkin cells, mummifie cells, and abnormal mitosis. However, classical RS cells are hard to find In rare cases, follicular hyperplasia with progressive transformation of germinal centers (PTGC) may be present. In NLPHL, the LP and RS cells have an immunophenotype similar to that of non-Hodgkin lymphoma, such as negative for CD30 and CD15 but positive for CD45 and other B-cell associated antigens (e.g. CD20, CD79a, and PAX5/BSAP) [1–4]. The LP cells in NLPHL also express immunoglobulin (Ig) light chains, J chain, and clonal light chain messenger RNA. Furthermore, bcl-6 protein and two activation-associated molecules, CD40 and CD86, are also expressed by the LP cells. OCT2 is a transcription factor that activates the promoter of the Ig genes in conjunction with its coactivator BOB1. These two markers are positive in NLPHL cases but negative in classical HLs. The background lymphocytes are polyclonal B cells and CD4+/CD57+ T cells. The demonstration of CD57+ cells rosetting around the neoplastic cells is one of the diagnostic features of this subtype. On the other hand, the lymphocytes in T-cell/histiocyte-rich large B-cell lymphoma show absence of small B cells and CD4+/CD57+ T cells, but presence of CD4+ and TIA1+ T cells [1]. A follicular dendritic cell meshwork can be demonstrated in the background lymphocyte nodules by CD21, CD23, or CD35 staining. Unlike the classical HLs, NLPHL does not contain deleterious mutations that would prevent the transcription and translation of the Ig genes into mRNA and immunoglobulin, respectively [3, 4]. NLPHL also differs from the classical HLs in the consistent absence of Epstein – Barr virus [1, 4]. NLPHL cases show somatic mutations and ongoing mutations in the variable region of the immunoglobulin heavy chain genes [1]. BCL6 rearrangements are frequently found in NLPHL [1, 4]. NLPHL accounts approximately 5% of HL cases. It is characteristically seen in young (<35 years of age) males [3]. Most patients are asymptomatic. About 80% of patients are in stage I or II at presentation. The most frequently involved lymph nodes are in the cervical and axillary regions, followed by the inguinal region. The prognosis of NLPHL is very favorable, but it may transform into mixed cellularity or lymphocyte depleted HL with subsequent widespread extranodal disease. NLPHL also frequently transforms into non-Hodgkin lymphoma.

References 1. Poppema S, Delsol G, Pileri S, et al. Nodular lymphocyte predominant Hodgkin lymphoma. 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, 323–325. 2. Yung L, Linch D. Hodgkin lymphoma. Lancet 2003;361:943–951. 3. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 312–321. 4. Re D, K¨uppers R, Diehl V. Molecular pathogenesis of Hodgkin’s lymphoma. J Clin Oncol 2005;23:6379–6386.

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Case 70 A 53-year-old woman presented with a bulky neck mass. She had a history of night sweats, but denied fever and weight loss. Physical examination revealed left cervical lymphadenopathy, measuring about 3 × 5 cm. The spleen tip was palpable but the liver was not enlarged. Laboratory studies were unremarkable except for an elevated erythrocyte sedimentation rate (ESR) of 50 mm/h, but the lactate dehydrogenase was within normal limits (145 U/l). CT scan showed a cluster of matted lymph nodes at the base of the left neck, and slightly enlarged left axillary lymph node. No lymphadenopathy was identifie in the mediastinum and abdomen. Mild hepatosplenomegaly was demonstrated by abdominal CT scan. A biopsy was performed on the left cervical lymph node, which showed diagnostic lesions (Figs. 70.1 and 70.2). A subsequent bone marrow biopsy revealed no pathological findings

Fig. 70.1 Lymph node biopsy shows a large sheet of lacunar cells. H&E, × 60

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Fig. 70.2 Another f eld shows a Reed – Sternberg cell (broad arrow), a few mummifie cells (narrow arrow), and scattered mononuclear and multinuclear Hodgkin cells. Right upper corner inset: a typical Reed – Sternberg cell. H&E, × 100

Differential diagnoses: Hodgkin and non-Hodgkin lymphomas.

Further Studies Immunohistochemical stains of lymph node: CD30 stain: positive (Fig. 70.3) CD15 stain: positive (Fig. 70.4) CD45 stain: negative for tumor cells (Fig. 70.5)

Case 70

Fig. 70.3 CD30 stain highlights several tumor cells with membranous and Golgi staining patterns. Immunoperoxidase, × 60

Fig. 70.4 CD15 stain highlights several tumor cells with membranous and Golgi staining patterns. Immunoperoxidase, × 60

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Fig. 70.5 CD45 stain shows negative reaction to tumor cells. An unstained Reed – Sternberg cell is indicated by an arrow. Immunoperoxidase, × 100

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Discussion The classical Hodgkin lymphoma (HL) is divided into four types: nodular sclerosis (NS), mixed cellularity (MC), lymphocyte-rich (LR), and lymphocyte-depleted (LD) [1–3]. NS is most common subtype, accounting for 60% of HL. The characteristic histologic pattern of NS is the presence of interconnecting bands of collagen fiber separating lymphoid tissue into cellular nodules (Fig. 70.6) [1, 3]. The collagen bands can be identifie by their birefringent character when examined under polarized light. The lymph node capsule may also become thickened. Focal necrosis is commonly seen in NS.

Fig. 70.6 A partly necrotic cellular nodule is surrounded by a thick collagen f brous band. H&E, × 10

The characteristic tumor cells are the lacunar cells, which are large cells located in a lacuna-like space (Fig. 70.7) [1, 3]. This phenomenon is considered to be an artifact due to formalin fixation because the lacuna-like space is not seen in other forms of fixation The lacunar cells have monolobated or polylobated nuclei, delicate nuclear chromatin, small nucleoli, and an abundant water-clear or pale eosinophilic cytoplasm. The number of classical Reed – Sternberg cells varies in different subtypes of NS. The background cellular components may be predominantly lymphocytes or mixed populations of lymphocytes, eosinophils, and neutrophils. Plasma cells and histiocytes may also be present. On the basis of the cellular background of the nodules, NS can be further divided into subtypes of lymphocytic predominance, mixed cellularity, and lymphocyte depletion. The syncytial variant of NS is a special subtype that shows large aggregates or sheets of lacunar cells or other forms of tumor cells, as seen in the current case. In NS, the Hodgkin and Reed – Sternberg cells (HRS cells) are positive for CD30 and CD15 but negative for CD45 [1–3]. CD20 can be expressed in rare cases, but CD79a is seldom positive. The B-cell-specifi activation protein (BSAP), a product of the PAX5 gene, is present in approximately 90% of cases. HRS cells also express CD25, HLA-DR, ICAM-1, CD95, CD40, CD86, vimentin, and fascin [3]. The T cells surrounding the HRS cells are positive for CD40 and CD86 ligands [3]. In routine immunohistochemical staining, most background lymphocytes are CD4 with some CD57+ T lymphocytes. Microdissection of single HRS cells has demonstrated rearrangement of immunoglobulin genes, indicating that the tumor cell in HL is of B-cell origin [1–4]. The lack of the expression of B-cell immunophenotype in all classical HL types is due to crippling mutations in the immunoglobulin (Ig) genes of the HRS cells, leading to the absence of kappa and lambda gene transcripts. The absence of Ig transcripts is caused by the inactivation of the Ig promoter by the absence of octamer-dependent transcription factor (OCT2) and/or its coactivator, BOB.1. However, B cells acquiring crippling mutations are usually

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Fig. 70.7 Higher magnificatio of the sclerotic nodule in Fig. 70.6 shows multiple lacunar cells. H&E, × 100

efficientl eliminated within the germinal center by apoptosis, therefore HRS cells must be rescued by some transforming events so that they can survive. There are probably many antiapoptotic mechanisms that are involved in this rescue mission [2–4]. The most important one is constitutive expression of some transcription factors, such as nuclear factor-␬B (NF-␬B), Stat3, Notch1, and highly expressed cFlip molecules. Most of the classical HL cases express tumor necrosis factor receptor (TNFR) family members (CD30, CD40) and their ligands, leading to the activation of NF-␬B. Another possibility is that Epstein – Barr virus (EBV) infection induces the expression of latent membrane protein 1 (LMP1), which possesses antiapoptotic potential. Although clonal cytogenetic abnormalities are frequently found in classical HLs, they are not recurrent or specific NS is the only type of classical HL that is female-predominant [1, 3]. Patients are usually <50 years of age and 60% are stage I or II at presentation. The clinical manifestation is often cervical or supraclavicular lymphadenopathy or a mediastinal mass. NS has a greater histologic stability than other type of HL. The prognosis is usually good, except for the lymphocyte depletion subtype, which is more frequently seen in men with symptoms and with advanced stage of disease. The Ann Arbor system is generally used for staging, which shows a good correlation with the prognosis of HL (Table 70.1) [2]. Table 70.1 Ann Arbor staging system of Hodgkin lymphoma Stage Degree of involvement I

Involvement of one lymph node region or lymphoid structure (e.g. spleen, thymus, Waldeyer’s ring) II Two or more lymph node regions on the same side of the diaphragm III Lymph nodes on both sides of the diaphragm III1 : with or without splenic hilar, celiac, or portal nodes III2 : with para-aortic, iliac, or mesenteric nodes IV Involvement of extranodal site(s) beyond these designated E Symbols of additional features: A, No symptoms; B, Fever, drenching night sweats, weight loss greater than 10% in 6 months. X, Bulky disease: greater than a third widening of mediastinum or greater than 10 cm maximum diameter of nodal mass. E, Involvement of single, contiguous, or proximal extranodal site.

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References 1. Stein H, von Wasielewski R, Poppema S, et al. Nodular sclerosis classical Hodgkin lymphoma. 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, 330. 2. Yung L, Linch D. Hodgkin lymphoma. Lancet 2003;361:943–951. 3. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 312–321. 4. Re D, K¨upper R, Diehl V. Molecular pathogenesis of Hodgkin’s lymphoma. J Clin Oncol 2005;23:6379–6386.

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Case 71 A 30-year-old man presented with a left axillary mass for a week. He was diagnosed with a lymphoid tumor with bone marrow involvement three years ago and was treated with chemotherapy and autologous bone marrow transplantation. He had had multiple episodes of relapse and was refractory to multicycle chemotherapy. Physical examination revealed axillary lymphadenopathy and hepatomegaly. Abdominal ultrasound showed numerous lesions in the liver. CT scan demonstrated multiple pulmonary nodules with pleural effusion. MRI revealed paraspinal disease involving the upper thoracic epidural space (T2–T5 level). A biopsy of the left axillary lymph node was performed (Figs. 71.1 and 71.2). The patient died two months after the current admission.

Fig. 71.1 Lymph node biopsy shows a histiocytic granuloma in the upper f eld. The lower fiel reveals lymphohistiocytic infiltratio with scattered lacunar cells (arrows). H&E, × 20

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Fig. 71.2 Lymph node biopsy reveals a mixed population of histiocytes, neutrophils and eosinophils. A lacunar cell (broad arrow) and a popcorn cell (narrow arrow) are present. H&E, × 60

Differential diagnoses: Hodgkin and non-Hodgkin lymphomas.

Further Studies Immunohistochemistry of lymph node biopsy: CD30 stain: positive (Fig. 71.3) CD15 stain: positive (Fig. 71.4) CD45 stain: negative

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Fig. 71.3 CD30 stain highlights a few tumor cells. Note membranous and Golgi staining pattern. Immunoperoxidase, × 40

Fig. 71.4 CD15 stain highlights a few tumor cells. Note membranous and Golgi staining pattern. Immunoperoxidase, × 100

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Discussion Mixed cellularity Hodgkin lymphoma (MCHL) is the second most common Hodgkin lymphoma (HL), accounting for approximately 30% of the total cases [1, 2]. This type was considered a wastebasket of unclassifiabl cases in the Rye classification but is now recognized as a true type of HL. MCHL is characterized by diffuse mixed cellular infiltratio with easily identifiabl Hodgkin and Reed – Sternberg (HRS) cells. In some cases, an interfollicular growth pattern may be present. Broad collagen fibrou bands and thickened lymph node capsule should not be seen. The number of classical HRS cells and mature lymphocytes in MCHL falls between that of lymphocyte-rich HL (LRHL) and lymphocyte-depleted LH (LDHL). The background cells include lymphocytes, eosinophils, neutrophils, histiocytes, and plasma cells, but one of the cell types may be predominant. When the epithelioid histiocytes are predominant, granuloma-like clusters may be present. The immunophenotype and molecular genetic characteristics of MCHL are similar to other types of classical HL [1–4]. Clinically, MCHL is considered to be an intermediate form between lymphocyte predominance (including nodular lymphocyte predominance HL and LRHL) and LDHL. Therefore, the age range of patients, the clinical course, and the prognosis are all between these two groups of HL [2]. The stage at presentation is usually II or III. MCHL frequently transforms into LDHL and these two types of HL are often seen in HIV-infected patients. In comparison to nodular sclerosing HL, the abdominal lymph node and spleen are more frequently involved, but the mediastinum is less often affected in MCHL. The Ann Arbor staging system is a useful predictor for the prognosis in MCHL cases.

References 1. Weiss LM, von Wasielewski R, Delsol G, et al. Mixed cellularity classical Hodgkin lymphoma. In Swerdlow SH, Campo E, Harris NL, eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France, IARC Press, 2008, 331. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 312–321. 3. Yung L, Linch D. Hodgkin lymphoma. Lancet 2003;361:943–951. 4. Re D, K¨uppers R, Diehl V. Molecular pathogenesis of Hodgkin’s lymphoma. J Clin Oncol 2005;23:6379–6386.

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Case 72 A 72-year-old man was admitted for a lymph node biopsy. The patient had right neck swelling for a few months and a fin needle aspirate showed suspicious lymphoid cells but was not diagnostic. He had no fever, night sweats, or weight loss. Physical examination showed right cervical lymphadenopathy about 3 cm in diameter. The liver and spleen were not palpable. CT scan revealed that the subcarinal and mediastinal lymph nodes were also enlarged. No lymphadenopathy was detected in the abdomen and pelvis. A lymph node biopsy is depicted in Figs. 72.1, 72.2 and 72.3.

Fig. 72.1 Lymph node biopsy shows nodular pattern. H&E × 10

Case 72

Fig. 72.2 A Reed – Sternberg cell is present on a lymphocytic background. H&E, × 100

Fig. 72.3 A few popcorn cells (arrow) are present on a lymphocytic background. H&E, × 100

Differential diagnoses: Hodgkin and non-Hodgkin lymphomas.

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Further Studies Immunohistochemistry of lymph node biopsy: CD30 stain: positive (Fig. 72.4) CD15 stain: negative (Fig. 72.5) CD45 stain: negative for tumor cells CD20 stain: negative for tumor cells (Fig. 72.6) CD3 stain: negative for tumor cells

Fig. 72.4 CD30 stain highlights two Reed – Sternberg cells, showing both membranous and Golgi staining patterns. Immunoperoxidase, × 100

Case 72

Fig. 72.5 CD15 stain highlights two tumor cells. Immunoperoxidase, × 100

Fig. 72.6 CD20 stain demonstrates sheets of B lymphocytes in the background. Immuno-alkaline phosphatase, × 10

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Discussion Lymphocyte-rich classical Hodgkin lymphoma (LR) comprises 6% of Hodgkin lymphomas (HL). Its growth pattern can be nodular or diffuse [1, 2]. In the nodular subtype, the nodule is composed mainly of small lymphocytes and may contain regressed germinal centers. Eosinophils and neutrophils are seldom seen. Hodgkin and Reed – Sternberg cells (HRS cells) are usually present in the expanded mantle zone. They are mostly lymphocyte predominant (LP) cells/popcorn cells or mononuclear lacunar cells. In the diffuse subtype, histiocytes and epithelioid histiocytes may be abundant. Because of the presence of a nodular pattern as well as the LP cells, LR may mimic nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and can only be distinguished from the latter by immunophenotyping. In LR cases, the HRS cells show the typical CD30+, CD15+, and CD45− immunophneotype [1–3]. B-cell antigens may be expressed on the HRS cells in 3–5% of LR cases. Weak T-cell antigen expression may also be seen in rare cases. Similar to NLPHL but distinguished from other classical LHs, the HRS cells in LR cases also express bcl-6. The background lymphocytes may be CD57+ and form rosettes with the HRS cells. The cellular nodules in LR cases may show extensive CD20 staining and the follicular dendritic cell meshwork, as demonstrated by CD21, CD23, or CD35 staining. The dendritic meshwork highlights the inconspicuous germinal centers, which are frequently eccentrically located in the nodules of mantle cells (IgM+, IgD+) [1]. The molecular genetic features of LR are similar to other classical HLs [1–4]. The immunoglobulin (Ig) gene undergoes crippling mutations. There are no Ig transcripts and no B-cell antigen expression. However, Ig gene rearrangement is demonstrated in the tumor cells. The mechanism of the absence of Ig transcripts is due to the inactivation of a transcription factor OCT2 and its coactivator, BOB.1. Cytogenetic abnormalities are frequently demonstrated but specific recurrent karyotypes have not been identified LR is seen predominantly in men with an average age higher than that of patients with NLPHL [1, 2]. Most patients are in stage I or II at presentation. Mediastinal mass is more frequently encountered in LR than in NLPHL, but not as frequently as seen in the nodular sclerosis type. Although relapses are frequent, the prognosis is generally good in this group of patients.

References 1. Anagnostopoulos I, Isaacson PG, Stein H. Lymphocyte-rich classical Hodgkin lymphoma. 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, 332–333. 2. Sun T. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 312–321. 3. Yung L, Linch D. Hodgkin lymphoma. Lancet 2003;361:943–951. 4. Re D, K¨uppers R, Diehl V. Molecular pathogenesis of Hodgkin’s lymphoma. J Clin Oncol 2005;23:6379–6386.

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Case 73 A 62-year-old man presented with a left neck mass for a few weeks. The patient admitted that he had low-grade fever, night sweats, and weight loss in the last few months. Physical examination revealed left cervical lymphadenopathy measuring 5 cm in diameter. His supraclavicular and axillary lymph nodes on both sides were also enlarged. The liver and spleen were palpable. Laboratory examination showed elevated erythrocyte sedimentation rate and lactate dehydrogenase as well as lymphocytopenia. A lymph node biopsy was performed (Figs. 73.1 and 73.2). CT scan showed mediastinal and abdominal lymph node involvement. Subsequently, a bone marrow biopsy showed involvement with the neoplastic process.

Fig. 73.1 Lymph node biopsy shows a cluster of pleomorphic tumor cells with scanty lymphocytes in the background. H&E, × 100

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Fig. 73.2 Another f eld of the lymph node biopsy reveals mild fibrosi with a few tumor cells (arrow). H&E, × 100

Differenital diagnoses: Hodgkin and non-Hodgkin lymphomas.

Further Studies Immunohistochemistry of lymph node: CD30 stain: positive (Fig. 73.3) CD15 stain: positive (Fig. 73.4) CD45 stain: negative for tumor cells

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Case 73

Fig. 73.3 CD30 stain highlights several tumor cells with membranous and Golgi staining patterns. Immunoperoxidase, × 100

Fig. 73.4 CD15 stain hightlights several tumor cells with membranous and Golgi staining patterns. Immunoperoxidase, × 100

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Discussion Lymphocyte-depleted Hodgkin lymphoma (LDHL) accounts for only 1–5% of total cases of Hodgkin lymphoma (HL) [1–3]. It is considered to be the last histologic phase of HL, in which tumor cells predominate and reactive lymphocytes diminish. As a result, the classical Hodgkin and Reed – Sternberg (HRS) cells become abundant and lymphocytes are depleted along with fibrosis On the basis of variations in HRS cells and fibroti pattern, LDHL is subdivided into the reticular and diffuse fibrou subtypes. The reticular subtype is characterized by the presence of abundant classical HRS cells or of bizarre multinucleated RS cells, referred to as sarcomatous RS cells [1,2]. In this variant, there are occasional mononucleated Hodgkin cells, mummifie cells, and scattered aberrant mitoses. In the background, lymphocytes are scarce, and plasma cells, eosinophils, histiocytes, and neutrophils are also rare or absent. The fibrosi is diffuse, patchy, irregular, and fibrilla in nature. The fibrou tissue is not birefringent and thus not collagenous. Focal necrosis is common in this subtype. Many cases previously diagnosed as this subtype may well be non-Hodgkin lymphoma, particularly anaplastic large cell lymphoma. In the subtype of diffuse fibrosis lymphocytes are depleted, fibrosi is diffuse and disorderly, the number of HRS cells is variable, and focal necrosis is frequently present [1, 2]. Many of these cases are now classifie as nodular sclerosis type. Because many cases previously considered LDHL are now diagnosed as either anaplastic large cell lymphoma, or nodular sclerosis HL, LDHL is rarely diagnosed now [3]. The immunophenotype and molecular genetics in LDHL are similar to other classical HL lymphoma [1–4], and are not further discussed under this case. LDHL is usually seen in elderly patients, predominantly male, with a median age of 50–57 years in various reports [2]. Most patients have constitutional symptoms, namely, night sweats in 30% of patients, fever in 60%, and weight loss in 67%. Patients usually have abdominal and, rarely, peripheral lymphadenopathy. In addition, they may have hepatosplenomegaly, bone marrow involvement, lymphopenia, or subdiaphragmatic disease. Vascular invasion and extranodal spread are common autopsy finding in these cases. About 80% of the patients are found to have stage IIIB or IVB at presentation. The prognosis is very poor, especially for patients with the reticular subtype. In human immunodeficien y virus-infected patients, the clinical and pathologic presentations differ from those of nonacquired immunodeficien y syndrome (non-AIDS) patients [2]. In AIDS patients, 41–100% have either mixed cellularity HL or LDHL. Systemic B symptoms are seen in 70–100%, stage III or IV disease in 75–90%, and bone marrow involvement in 45–70% of AIDS patients. The median survival in this group of patients is approximately 18 months.

References 1. Benharroch D, Stein H, Peh SC. Lymphocyte-depleted classical Hodgkin lymphoma. 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, 334. 2. Sun T. Flow cytometry and Immunohistochemistry for Hematologic Neoplasms. Philadelphia, Lippincott Williams & Wilkins, 2008, 312–321. 3. Yung L, Linch D. Hodgkin lymphoma. Lancet 2003;361:943–951. 4. Re D, K¨uppers R, Diehl V. Molecular pathogenesis of Hodgkin’s lymphoma. J Clin Oncol 2005;23:6379–6386.

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Case 74 A 25-year-old man presented with fatigue, malaise, and frequent bruising after minor injury. Peripheral blood examination showed a total leukocyte count of 2,500/␮l, hemoglobin 11 g/dl and platelets 50,000/␮l. Physical examination revealed hepatosplenomegaly, but no lymphadenopathy. As the patient was refractory to multiple blood transfusions, splenectomy was performed (Fig. 74.1). A liver biopsy (Figs. 74.2 and 74.3) was done at the same time. Subsequently, the patient had a bone marrow biopsy (Figs. 74.4 and 74.5) and an intestinal biopsy (Figs. 74.6 and 74.7), due to the presence of severe gastrointestinal symptoms. The patient had a lymph node biopsy several years ago due to a lymphoid neoplasm and had been in remission after chemotherapy.

Fig. 74.1 Splenectomy specimen shows marked expansion of red pulp components, but no tumor cells are identified H&E, × 20

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Fig. 74.2 Liver biopsy shows marked cellular infiltratio and replacement of the liver parenchymal cells by eosinophils, neutrophils and scattered tumor cells. H&E, × 20

Fig. 74.3 Higher power view of the liver biopsy reveals many large tumor cells among numerous eosinophils. H&E, × 60

Case 74

Fig. 74.4 Bone marrow biopsy shows a restricted hypercellular area compared to the adjacent normocellular bone marrow. H&E, × 20

Fig. 74.5 Higher magnificatio of the bone marrow reveals many tumor cells with one Reed – Sternberg cell (arrow). H&E, × 100

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Fig. 74.6 Intestinal biopsy shows scattered tumor cells on an eosinophilic background. H&E, × 40

Fig. 74.7 Higher magnificatio of the intestinal biopsy reveals a few popcorn cells (arrow). H&E, × 100

Differential diagnoses: Hodgkin and non-Hodgkin lymphomas.

Hematologic Neoplasms

Case 74

Further Studies Immunohistochemical stains of bone marrow: CD15 stain: positive for tumor cells (Fig. 74.8) CD30 stain: positive for tumor cells (Fig. 74.9) CD45 stain: negative for tumor cells

Fig. 74.8 Bone marrow biopsy shows CD30 positive tumor cells. Immunoperoxidase, × 60

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Fig. 74.9 Bone marrow biopsy shows CD15 positive tumor cells. Immunoperoxidase, × 60

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Discussion Hodgkin lymphoma (HL) is a nodal-based disease; approximately 90% of patients show nodal involvement in the initial clinical presentation [1]. Extranodal involvement is usually secondary. The most common extranodal sites are the spleen (20%) and bone marrow (5%), which are the important sites for staging purposes [2]. However, because of the advances in imaging technique, splenectomy for staging is no longer necessary. The use of bone marrow biopsy for routine staging is controversial. The argument is that patients with prebioptic stage IA and IIA seldom have bone marrow involvement and that the impact of bone marrow infiltratio on prognosis is uncertain [3]. However, in some cases, the initial diagnosis of HL is made on bone marrow biopsy for evaluation of cytopenia, fever of unknown origin or organomegaly. In pediatric patients, the overall incidence of bone marrow disease is very low, so that bone marrow biopsy is usually not indicated [3]. The incidence of extranodal HL differs in various types of HL. It is most common in lymphocyte depleted HL (LDHL), which has an incidence of 50–75%. The incidence in mixed cellularity HL (MCHL) is 20–25%; nodular sclerosis HL (NSHL), 5–10%; and lymphocyte predominance, rare [3]. In MCHL, the spleen is involved in 30%, bone marrow in 10%, liver in 3%, and other organs in 1–3% [2]. In NSHL, spleen and/or lung involvement is seen in 10% of cases, and bone marrow involvement in 3% [2]. While most of the extranodal HL is secondary, there are about 0.25% HL cases that are primary medullary HL or primary involvement of other organs without evidence of lymph node lesions. In human immunodeficien y virus (HIV) infected patients, secondary extranodal involvement is more common than that of immunocompetent patients. Histologic diagnosis of extranodal HL is somewhat different from that of nodal HL. First of all, classical Reed – Sternberg (RS) cells are seldom detected in extranodal tissue. If a few RS-like cells are seen without the characteristic cellular background, it should be distinguished from anaplastic large cell lymphoma [3]. Secondly, the effacement of normal architecture in the lymph node is helpful to distinguish a neoplastic from a reactive process, while the overwhelming inflammator background with rare tumor cells in extranodal tissue frequently misleads the diagnosis to infection rather than lymphoma [1]. The identificatio of typical Hodgkin and Reed – Sternberg (HRS) cells is not required for the diagnosis of extranodal HL if the diagnosis has already been proven by previous lymph node biopsy [2]. In the bone marrow, the characteristic histologic pattern is a restricted area of either hypercellularity or fibrosis The infiltratio pattern is frequently diffuse but it can also be focal [3]. Focal infiltratio may show a nodular or a paratrabecular pattern. The cellular milieu is usually polymorphous, consisting of lymphocytes, eosinophils, neutrophils, plasma cells, and/or histocytes, but the presence of eosinophilia is most characteristic. Reticular or collagen fibrosi is invariably present. In the older lesions, fibrosi may become the predominant feature. The tumor cells are usually scanty. Classical RS cells are hard to find but mononuclear or multinuclear Hodgkin cells with prominent eosinophilic nucleolus and lacunar cells may be detected. The 1971 Ann Arbor symposium divided the diagnostic extranodal pattern into three categories [3]. The presence of typical RS cells with appropriate cellular environment is considered “certain”. The presence of atypical “histiocytes” without features of RS cells is considered “strongly suggestive”. The existence of fibrosi or necrosis alone is considered “suspicious”. However, the use of immunohistochemistry greatly facilitates the diagnosis of extranodal HL and a certain diagnosis can be achieved in almost all cases. The standard monoclonal antibody panel includes CD15, CD30 and CD45. CD30 is most reliable and should be positive in every case. CD15 may or may not be positive and its staining pattern is sometimes difficul to interpret in a focal lesion surrounded by many normal hematopoietic cells, because CD15 is a myeloid marker. CD45 should be negative on the tumor cells; a positive reaction indicates non-Hodgkin lymphoma. In equivocal cases, additional markers can be used for differential diagnosis [4]. The classical HLs are B-cell-specifi activator protein (BSAP)+, octamerbinding transcription factor 2 (OCT2)−, B-cell Oct-binding protein 1 (BOB1)−, while nodular lymphocyte predominant HL (NLPHL) is BSAP + Oct2 + BOB1+. The diffuse large B-cell lymphoma is immunophenotypically identical to NLPHL and anaplastic large cell lymphoma is negative for all three markers. The morphology and immunophenotype in extranodal HL of other organs are similar to those found in the bone marrow biopsy, as exemplifie by the current case. In this case, the bone marrow, liver and intestine were all involved, but the spleen was free of HL lesion.

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References 1. Citow JS, Rini B, Wollmann R, et al. Isolated, primary extranodal Hodgkin’s disease of the spine: case report. Neurosurgery 2001;49:453–457. 2. Stein H. Hodgkin lymphoma. In Swerdlow SH, Campo E, Harris NL, eds. WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France, IARC Press, 2008, 322–334. 3. Franco V, Tripodo C, Rizzo A, et al. Bone marrow biopsy in Hodgkin’s lymphoma. Eur J Haematol 2004;73:149–155. 4. Browne P, Petrosyan K, Hernandez A, et al. The B-cell transcription factors BSAP, Oct-2, and BOB.1 and the pan-B-cell markers CD20, CD22, and CD79a are useful in the differential diagnosis of classic Hodgkin lymphoma. Am J Clin Pathol 2003;120:767–777.

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Case 75 A 75-year-old man presented with multiple episodes of loss of consciousness, right-sided headaches, and retro-orbital pain over the previous year. The patient had received three cadaveric kidney transplants; the most recent one was six years prior to presentation. Some episodes were accompanied with facial drooping. Physical examination revealed a mild left facial paresis. Magnetic resonance imaging demonstrated a single 3 cm right parietal mass with surrounding edema. Laboratory studies showed a mild normocytic normochromic anemia without other hematologic abnormalities. An excisional biopsy of the right parietal mass was performed (Figs. 75.1 and 75.2).

Fig. 75.1 Brain biopsy shows an area with features mimicking Hodgkin lymphoma. Note Reed – Sternberg-like cell in the center of the field surrounded with a few Hodgkin-like cells with prominent eosinophilic nucleoli. H&E, × 100

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Fig. 75.2 Brain biopsy shows perivascular large lymphoma cell infiltratio with a concentric, laminar pattern, characteristic of cerebral diffuse large B-cell lymphoma. H&E, × 40

Differential diagnoses: post-transplant lymphoproliferative disorder versus primary brain tumor.

Further Studies Immunohistochemical stains Cytokeratin stain: negative CD45 stain: partial positive (Fig. 75.3) CD3 stain: negative (Fig. 75.4) CD20 stain: positive (Fig. 75.5) CD30 stain: positive (Fig. 75.6) CD15 stain: negative (Fig. 75.7) Epstein – Barr virus encoded RNA in situ hybridization (EBER): positive (Fig. 75.8) ALK1 stain: negative (Fig. 75.9) Immunoglobulin heavy chain gene rearrangement analysis: monoclonal pattern (Fig. 75.10)

Case 75

Fig. 75.3 The large lymphoma cells show positive CD45 stain. Immunoperoxidase, × 40

Fig. 75.4 The large lymphoma cells are negative for CD3 stain. Immunoperoxidase, × 60

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Fig. 75.5 CD20-positive stain is demonstrated on the large lymphoma cells. Immunoperoxidase, × 40

Fig. 75.6 The tumor cells are also CD30-positive. Immunoperoxidase, × 40

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Fig. 75.7 CD15 is negative for large lymphoma cells. Note that a few multinucleated tumor cells are present. Immunoperoxidase, × 60

Fig. 75.8 ALK1 stain is negative for tumor cells. × 40

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Fig. 75.9 Epstein – Barr virus encoded RNA in situ hybridization (EBER) shows positive staining on almost all tumor cells. × 40

Fig. 75.10 Capillary electrophoretogram after polymerase chain reaction (PCR) demonstrates monoclonal spikes in frameworks 2 and 3

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Discussion Post-transplant lymphoproliferative disorder (PTLD) is a complicated subject covering a broad spectrum of reactive and neoplastic lymphoid diseases. It has to be distinguished from allograft rejection and primary lymphoid neoplasms. The development of PTLD depends on the degree and duration of immunosuppression, the immunosuppressive drugs used, the transplanted organ and the original immune status of the patient, including the presence or absence of antibodies against the Epstein – Barr virus (EBV). The incidence of PTLD in kidney and liver transplant ranges from 1 to 3%, cardiac transplants, 1–6%, combined heart – lung transplants, 4–6%, lung transplants, 4–10%, and small intestinal transplants, up to 20% [1]. An azathioprine-based regimen tends to develop PTLDs involving extranodal sites, including allograft and the central nervous system [2]. Cyclosporine and tacrolimus-based regimens, on the other hand, tend to involve lymph nodes and the gastrointestinal tract. Cyclosporine does not confer added risk for the development of PTLD compared with azathioprine/steroid treatment, but tacrolimus increases the risk approximately two-fold [3]. The pathogenesis of PTLD is considered to be associated with the suppression of both tumor immune surveillance and antiviral activity [4]. EBV infection as transmitted from the graft is of particular importance, as EBV may induce B-cell transformation, resulting in uncontrolled lymphoproliferation. This partly explains the high incidence of PTLD in pediatric patients because most children are EBV-na¨ıve recipients. It is advocated that patients with EBV-negative serology should receive prophylactic antiviral treatment to prevent the development of PTLD [1]. EBV-positive PTLD cases tend to occur earlier than EBV-negative cases; the former have a median interval of 6–10 months compared with 4–5 years for the latter. The World Health Organization (WHO) classificatio divides PTLD into four categories: early lesions, polymorphic PTLD, monomorphic PTLD, and classical Hodgkin lymphoma type PTLD [2, 5]. The major histologic features in early lesions are plasmacytic hyperplasia and infectious mononucleosis-like PTLD. The diagnosis is based on the findin of lymphoproliferation in an allograft patient that is reversible after reduction of immunosuppressive drugs. Polymorphic PTLD is define as “polymorphic lesions composed of immunoblasts, plasma cells, and small and intermediate-sized lymphoid cells that efface the architecture of lymph nodes or form destructive extranodal masses” [2]. Immunophenotypically, the lymphocytes and plasma cells may be either polytypic or monotypic. However, monoclonality can be identifie in most cases by heavy chain gene rearrangement or episomal EBV genomes [2]. EBV can also be identifie by EBV-latent membrane protein 1 (EBV-LMP1) and Epstein – Barr nuclear antigen 2 (EBNA2), but EBER is a more sensitive technique for the detection of EBV [5]. A variable number of polymorphic PTLD cases are still reversible by reduction of immunosuppression. Monomorphic PTLD is morphologically identical to de novo B-cell or T-cell lymphomas. Most cases are of B-cell lineage, with the majority being diffuse large B-cell lymphoma or Burkitt lymphoma. Plasma cell myeloma is rare. Indolent B-cell lymphomas, such as follicular lymphoma and extranodal marginal zone B-cell lymphoma, are not included in this category even if the patient is in post-transplant status [2]. Monoclonality can be demonstrated by immunophenotyping or heavy chain gene rearrangement. EBV is frequently detected. T-cell lymphoma accounts for 4 to 14% of all lymphoma cases [2]. It covers a wide spectrum of neoplasms, including peripheral T-cell lymphoma, not otherwise specified hepatosplenic Tcell lymphoma, mycosis fungoides/S´ezary syndrome, anaplastic large cell lymphoma, NK/T-cell lymphoma, nasal type, T-cell large granular lymphocyte leukemia, and adult T-cell leukemia/lymphoma [2]. Most cases show clonal T-cell receptor rearrangement and about 25% of cases have clonal episomal EBV genomes [2]. Hodgkin lymphoma is rare, but Reed – Sternberg-like cells may be seen in polymorphic PTLD and occasionally monomorphic PTLD, as in the current case of diffuse large B-cell lymphoma. Therefore, it should be excluded by immunohistochemical stains before making the diagnosis of non-Hodgkin lymphoma. The identificatio of EBV in the tumor cells is helpful to distinguish de novo B-cell lymphoma from monomorphic PTLD cases and to distinguish polymorphic PTLD from rejection of allograft. Heavy chain gene analysis is useful in establishing the diagnosis of PTLD when a large population of polyclonal B-cells is present by immunophenotyping.

References 1. LaCasce AS. Post-transplant lymphoproliferative disorders. Oncologist 2006;11:674–680. 2. Swerdlow SH, Webber SA, Chadburn A, et al. Post-transplant 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, 343–349. 3. Opeiz G, Dohler B. Lymphomas after solid organ transplantation: a collaborative transplant study report. Am J Transplant 2004;4:222–230. 4. Buell JF, Gross TG, Woodle ES. Malignancy after transplantation. Transplantation 2005;80:S254–S264. 5. Wallace WAH, Bellamy COC, Rassl DM, et al. Transplant histopathology for the general histopathologist. Histopathology 2003;43:313–322.

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Case 76 A 2-year-old boy presented with anemia, thrombocytopenia, and pyelonephritis. CT scan showed splenomegaly and a retroperitoneal mass. An excisional biopsy was performed on the retroperitoneal mass. The histologic features are illustrated in Figs. 76.1, 76.2, 76.3 and 76.4.

Fig. 76.1 A lymph node biopsy shows a large sheet of Langerhans cells intermixing with small lymphocytes. H&E, × 40

Case 76

Fig. 76.2 Another f eld of the lymph node reveals a multinucleated giant cell (arrowhead) and a mummifie cell (arrow). H&E, × 40

Fig. 76.3 A Reed – Sternberg-like cell is present (arrow). H&E, × 100

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Fig. 76.4 Higher magnificatio shows the variable shapes of the Langerhans cell nuclei. A central groove is demonstrated in a few nuclei (arrows). H&E, × 100

Differential diagnoses: Hodgkin lymphoma, Langerhans cell histiocytosis, and histiocytic lymphoma.

Further Studies Immunohistochemistry: CD1a stain: positive (Fig. 76.5) S-100 stain: positive (Fig. 76.6) CD68 stain: positive for normal histiocytes (Fig. 76.7)

Case 76

Fig. 76.5 The Langerhans cells are positive for CD1a. Immunoperoxidase, × 60

Fig. 76.6 The Langerhans cells are positive for S-100. Immunoperoxidase, × 40

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Fig. 76.7 CD68 staining is only demonstrated in normal histiocytes. Immunoperoxidase, × 40

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Discussion In the current case, the normal architecture of the lymph node is largely replaced by sheets of histiocytoid cells with residual lymphoid tissue demonstrated in small areas (Figs. 76.1, 76.2, 76.3, and 76.4). The histiocytoid cells are of medium size with moderate amount of eosinophilic cytoplasm. The nuclei are mostly eccentrically placed and their shape varies from reniform, lobated, indented to clefted. A few cells show a groove or line in the middle of the nucleus. The chromatin pattern is delicate but no conspicuous nucleolus is present. A few multinucleated giant cells, mummifie cells, and Reed – Sternberg-like cells are also present. The mitotic rate is low. Immunohistochemical study demonstrates positive CD1a (Fig. 76.5) and S-100 (Fig. 76.6) staining, but CD68 is only seen in normal histiocytes (Fig. 76.7). The characteristic morphology of Langerhans cells in combination with strongly positive CD1a and S-100 is diagnostic of Langerhans cell histiocytosis (LCH). The presence of Reed – Sternberg-like cells, multinucleated giant cells, and mummifie cells raises the possibility of Hodgkin lymphoma. However, the background cellular components in Hodgkin lymphoma should be lymphocytes, plasma cells, and histiocytes and not typical Langerhans cells. Histiocytic lymphoma is a rare disease and its diagnosis is mainly by exclusion of other types of lymphomas and histiocytic disorders. The immunophenotype in this case excludes histiocytic lymphoma. In the early stage of LCH, Langerhans cells are usually intermixed with eosinophils and neutrophils [1]. In the later stage, fibrosi and foamy macrophages gradually become prominent. The characteristic features of Langerhans cells are described in the f rst paragraph. When there are marked cytologic atypia, anaplasia, and a high mitotic rate, Langerhans cell sarcoma should be suspected [2]. In the lymph node, LCH may be confine to the sinuses without disruption of the nodal architecture [2]. In severe cases, the entire lymph node architecture may be effaced with focal necrosis [2]. These cases are often seen in the multisystem disease. In the spleen, a nodular red pulp involvement is characteristic [1]. Although CD1a and S-100 are consistently present in Langerhans cells, the later stain is nonspecific Recent studies have found that the Langerhans cell-specifi lectin, langerin (CD207), is just as sensitive and specifi for the diagnosis of LCH [2, 3]. However, a definit ve identificatio of Langerhans cells depends on the findin of a membrane-demarcated intracytoplasmic organelle, the Birbeck granule, by electron microscopy. LCH cells do not express T- and B-cell associated antigens [2]. According to the Histiocyte Society, Langerhans cells are classifie in the dendritic cell family, which also includes interdigitating dendritic cells, dermal dendrocytes and follicular dendritic cells [4]. Another classificatio divides dendritic cells into CD34+ myeloid lineage, which includes Langerhans dendritic cells and interstitial dendritic cells, and CD34+ lymphoid lineage that is solely represented by plasmacytoid dendritic cells [5]. Clinically, LCH can be classifie into single-system disease and multisystem disease; the former is further divided into single site and multiple site (Table 76.1) [1, 6]. The single-system single site disease usually involves the bone and, less commonly, the lymph node, skin, or lung. The single-system multiple site disease almost always involves the bone. The multisystem disease may involve the bones, skin, liver, spleen, and lymph nodes. Cervical lymph nodes are most often affected. A full-blown hemophagocytic syndrome is usually seen in the multisystem disease. LCH is mainly a pediatric disease, but the single-system single site disease can be seen in both old children and adults. LCH is a clonal disease as proven by X-linked androgen receptor gene assay (HUMARA) [1, 2].

Table 76.1 Classificatio of Langerhans cell histiocytosis Organ involvement Single-system disease Single site (unifocal) Multiple site (multifocal) Multisystem disease

Bone, lymph node, skin, lung Bone Bone, skin, liver, spleen, lymph node

Age group

Synonym

Older children and adults Young chidren Infants

Solitary eosinophilic granuloma Hand – Sch¨uller – Christian dieease Letterer – Siwe disease

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References 1. Jaffe R, Weiss LM, Facchetti F. Langerhans cell histiocytosis. In: Swerdlow SH, Campo E, Harris HL, et al. eds: WHO Classificatio of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed., Lyon, France, IARC Press, 2008, 358–360 2. Ioachim HL, Medeiros LJ. Ioachim’s Lymph Node Pathology, 4th ed., Philadelphia, Lippincott Williams & Wilkins, 2009, 537–542. 3. Sholl LM, Hornick JL, Pinkus JL, et al. Immunohistochemical analysis of Langerin in Langerhans cell histiocytosis and pulmonary inflam matory and infectious diseases. Am J Surg Pathol 2007;31:947–952. 4. Favara BE, Feller AC, Pauli M et al. Contemporary classificatio of histiocytic disorders. Med Ped Oncol 1997;29:157–166. 5. Lipscomb MF, Masten BJ. Dendritic cells: immunologic regulators in health and disease. Physiol Rev 2002;82:97–130. 6. Stockschlaeder M, Sucker C. Adult Langerhans cell histiocytosis. Eur J Haematol 2006;76:363–368.

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T. Sun, Atlas of Hematologic Neoplasms, c Springer Science+Business Media, LLC 2009 DOI 10.1007/978-0-387-89848-3 3, 

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Case 77 A 60-year-old man was found to have an anterior mediastinal mass by routine chest X-ray. A fin needle core biopsy showed a mixed population of small lymphocytes and epithelial cells (Fig. 77.1). A thoracotomy biopsy revealed a lobulated tumor (Figs. 77.2 and 77.3).

Fig. 77.1 A f ne needle biopsy of the mediastinal mass shows a mixed population of small lymphocytes and polygonal epithelial cells. H&E, × 60

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Fig. 77.2 A mediastinal biopsy reveals that beneath a broad f brous septum is a densely populated lymphoid area with a sparsely populated center, recapitulating the cortex and medulla of the thymus. H&E, × 10

Fig. 77.3 A higher magnificatio of Fig. 76.2 demonstrates a few Hassall corpuscles. H&E, × 40

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Differential diagnoses: Thymoma versus lymphoma.

Further Studies Flow cytometry: The major population showed dual staining of CD4 and CD8. Terminal deoxynucleotidyl transferase (TdT) was positive, but CD10 was negative. No B-cell markers were identified Immunohistochemical stain: Cytokeratin stain was positive for epithelial cells (Fig. 77.4). CD3 was positive but CD20 was negative for background lymphocytes

Fig. 77.4 Cytokeratin stain highlights many immunoreactive epithelial cells and Hassall corpuscles (arrow). Immunoperoxidase, × 40

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Discussion Thymoma is an epithelial tumor of the thymus [1–3]. However, various proportions of immature T lymphocytes or thymocytes are always present. Although the thymocytes are not tumor cells, their immunophenotype can be used as surrogate markers for a preliminary diagnosis of thymoma. A fina diagnosis depends on the identificatio of the epithelial component by cytokeratin staining. Morphologically, thymoma is characterized by the presence of a lobular pattern separated by broad fibrou bands. Each lobule is composed of a mixed population of epithelial cells and thymocytes. The normal division of cortex and medulla is usually not discernable, except for the predominantly cortical thymoma (World Health Organization [WHO] classificatio type B1). Hassall corpuscles are only seen in predominantly cortical thymoma and occasionally in cortical thymoma (WHO classificatio type B2). The current case shows predominantly lymphocytes without any epithelial atypia, belonging to the WHO type B1. The epithelial cells are either polygonal (round or oval) or spindle-shaped. The nuclei of the tumor cells are usually vesicular with a small inconspicuous nucleolus. The cytoplasm is of variable amount and pale acidophilic. Most cases contain lymphocytes, but <4% of thymomas may show no lymphocytes at all. There are many classification of thymoma and there is no consensus about the nomenclasture of thymic epithelial tumors [1–3]. Therefore, the WHO committee chose a noncommittal alphabetic system for classificatio (Table 77.1). The Committee claimed that the new schema was not intended to replace any of the existing classifications but rather to serve as a means for translating the different terms used by the already existing classification [1, 2]. Table 77.1 WHO classificatio of thymoma WHO type Histogenetic type A AB B1 B2

Medullary thymoma Mixed thymoma Predominantly cortical Cortical

B3

Well-differentiated thymic carcinoma

C

Nonorganoid carcinoma

Morphologic description Predominantly spindle cells Mixed spindle and epithelial cells Lymphocyte-rich, no atypical epithelial cells Higher epithelial cell/lymphocyte ratio, more atypical tumor cells Atypical epithelial cells and some immature lymphocytes Various types of carcinoma

In the WHO classification type A is for tumor cells with a spindle or oval shape, whereas type B is for tumor cells with a dendritic or epithelioid appearance. Tumors with both features are called type AB. Type B thymomas are further divided into B1, B2, and B3 on the basis of an increasing epithelial cell/lymphocyte ratio and emergence of atypical neoplastic epithelial cells. Type C is designated for nonorganoid thymic carcinomas. Type C thymoma is composed of various kinds of carcinomas that are similar to nonthymic carcinomas, except that the thymic carcinoma is accompanied by mature lymphocytes and are located in the thymic region. These tumors include keratinizing and nonkeratinizing epidermoid (squamous) carcinomas, lymphoepithelioma-like carcinoma, basaloid carcinoma, mucoepidermoid carcinoma, and undifferentiated carcinoma. The modifie WHO classificatio excludes type C, segregating it into a separate category of thymic carcinoma [1]. The fina diagnosis depends on the identificatio of cytokeratin in the epithelial component. In case of an epithelial carcinoma, the demonstration of CD1-positive cells in a mediastinal tumor denotes thymic carcinoma and distinguishes it from metastatic carcinoma to the thymus. Based on the lymphoid markers, thymomas can be divided into early cortex, late cortex, and medulla stages (Table 77.2). Essentially, the early cortex stage is represented by the absence of both CD4 and CD8 markers, the cortex stage shows both CD4 and CD8 and the medullar stage expresses either CD4 or CD8. The immunophenotype of thymoma is sometimes difficul to distinguish from precursor T-lymphoblastic leukemia/lymphoma. In fl w cytometry, the demonstration of 3-dot clusters and the smear pattern of the T-cell markers is characteristic of thymoma [4]. Clinically, about one-third of the patients is asymptomatic, one-third has local symptoms, and one-third shows autoimmune disease at the time of diagnosis [2]. The most common autoimmune phenomena include myasthenia gravis, pure red cell aplasia, and hypogammaglobulinemia.

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Table 77.2 Immunophenotype of thymoma at various stages Stage Early cortex TdT CD1 CD2 CD3 CD4 CD5 CD7 CD8

+ − + − − − + −

Late cortex

Medulla

+ + + ± + + + +

+ − + + ± + + ±

References 1. Suster S. Diagnosis of thymoma. J Clin Pathol 2006;59:1238–1244. 2. M¨uller-Hermelink HK, Marx A. Thymoma. Curr Opin Oncol 2000;12:426–433. 3. Duwe BV, Sterman DH, Musani AI. Tumors of the mediastinum. Chest 2005;128:2893–2909. 4. Li S, Luco J, Mann KP, et al. Flow cytometry in the differential diagnosis of lymphocyte-rich thymoma from precursor T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma. Am J Clin Pathol 2004;121:268–274.

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Case 78 A 60-year-old woman was diagnosed with breast cancer. She received chemotherapy as well as prophylactic granulocyte colony-stimulating factor (G-CSF). Five days later, her peripheral blood showed a total leukocyte count of 30, 000/␮l with 1% blasts and 20% immature myelomonocytic cells (Figs. 78.1, 78.2, and 78.3). Dysplastic granulocytes were also noted.

Fig. 78.1 Peripheral blood smear shows leukocytosis with predominantly myeloid cells and greater than 10% immature forms including blasts. Wright – Giemsa, × 20

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Fig. 78.2 Higher magnificatio of peripheral blood smear reveals blasts and promyelocytes. Wright – Giemsa, × 100

Fig. 78.3 Bone marrow aspirate demonstrates mostly immature myeloid cells with 2% blasts. Wright – Giemsa, × 60

Case 78

Differential diagnoses: Chronic myeloproliferative disorder, leukemoid reaction and growth factor effect.

Further Studies A bone marrow biopsy showed 80% cellularity with 3% blasts and 5% dysplastic myeloid cells. Flow cytometry revealed no increase in the percentages of CD34 and CD117 in the myeloid population.

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Discussion The use of myeloid growth factors including G-CSF and granulocyte-macrophage CSF (GM-CSF) has become increasingly popular in recent years because of the broader application in various clinical situations [1, 2]. The most common usage is for the treatment or prophylaxis of febrile neutropenia in patients receiving myelosuppressive chemotherapy for various kinds of malignancy. G-CSF is also used to treat patients with congenital neutropenia, cyclic neutropenia, and aplastic anemia with great success. In addition, healthy stem cell donors are treated with G-CSF to mobilize stem cells from the bone marrow to the peripheral blood. However, the administration of myeloid growth factors may induce prominent morphologic changes in the peripheral blood and bone marrow, leading to confusion or misdiagnosis as leukemia, myeloproliferative disorders, or myelodysplastic syndrome. The presence of severe leukocytosis with immature cells including a high blast count may mimic acute or chronic leukemia [3]. A few patients even present with splenomegaly and extramedullary hematopoiesis, simulating a myeloid infil trate [4]. On the other hand, hypogranulation and abnormal nuclear lobulation of the neutrophils may be present that resembles myelodysplastic syndrome. The current case showed both myeloproliferation and myelodysplastic changes. In contrast to leukemia, the peripheral blood features in G-CSF effect also include those seen in leukemoid reactions, such as toxic granulation, D¨ohle bodies and cytoplasmic vacuolation in neutrophils [3]. Fortunately, these changes are transient. Once the myeloid growth factor is discontinued, the peripheral blood and bone marrow differential count may return to normal in a few weeks. In the current case, two months after discontinuation of G-CSF, the peripheral blood and bone marrow no longer showed myeloblasts and dysplastic changes. Nevertheless, true leukemia and myelodysplastic syndrome can be induced by myeloid growth factors in a small percentage of patients. In a study of 5510 breast cancer patients who received both chemotherapy and G-CSF and GM-CSF, 64 were subsequently diagnosed with either acute myeloid leukemia or myelodysplastic syndrome [5]. However, the benefit of growth factors may still outweigh the risks. It is possible that chemotherapy induces lethal mutation in a myeloid stem cell or progenitor cell, but the antiapoptotic effect of the myeloid growth factors save the mutant cells from destruction, leading to the subsequent development into myeloid leukemia [1].

References 1. Kaushansky K. Lineage-specifi hematopoietic growth factors. N Engl J Med 2006;354:2034–2045. 2. Lyman GH, Shayne M. Granulocyte colony-stimulating factors: findin the right indication. Curr Opin Oncol 2007;19:299–307. 3. Meyerson H, Farhi DC, Rosenthal N. Transient increase in blasts mimicking acute leukemia and progressing myelodysplasia in patients receiving growth factor. Am J Clin Pathol 1998;109:675–681. 4. Vaset MA, Neiman RS, Meletiou SD, et al. Marked granulocytic proliferation induced by granulocyte colony-stimulating factor in the spleen simulating a myeloid leukemic infiltrate Mod Pathol 1998;11:1138–1141. 5. Hershman D, Neugut AI, Jacobson JS, et al. Acute myeloid leukemia or myelodysplastic syndrome following use of granulocyte colonystimulating factors during breast cancer adjuvant chemotherapy. J Natl Cancer Inst 2007;99:196–205.

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Case 79 A 60-year-old man presented with a dome-shaped, red skin nodule in the left cervical region. The lesion grew rapidly and reached 30 mm in a few weeks. A skin biopsy showed clusters of tumor cells extending from the upper dermis to the subcutaneous tissue (Figs. 79.1 and 79.2). The epidermis was not involved. Physical examination revealed no superficia lymphadenopathy.

Fig. 79.1 Skin biopsy shows multiple clusters of tumor cells in the dermis. H&E, × 10

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Fig. 79.2 The tumor cells reveal hyperchromatic, dispersed chromatin pattern with imperceptible cytoplasm. H&E, × 100

Differential diagnoses: Non-Hodgkin’s lymphoma, small cell carcinoma, melanoma and Merkel cell carcinoma.

Further Studies Leukocyte common antigen (CD45) stain: negative S100 stain: negative Cytokeratin stains were positive with CK20 (Fig. 79.3) but negative with CK7 (Fig. 79.4)

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Fig. 79.3 CK20 staining reveals characteristic paranuclear dots in the tumor cells. Immunoperoxidase, × 60

Fig. 79.4 CK7 staining shows negative staining. Mitoses are abundant as demonstrated by the background staining. Immunoperoxidase, × 60

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Discussion Merkel cell carcinoma (MCC) is believed to be derived from the nondendritic, nonkeratinocytic cells in the basal layer of the epidermis, originally described by Merkel as tactile cells [1–3]. This tumor is now considered to be neuroendocrine in nature. Therefore, MCC is also called neuroendocrine cancer of the skin or small cell carcinoma of the skin. Morphologically, MCC mimics small cell carcinoma of lung, so that metastatic carcinoma should be always ruled out by imaging techniques. Histiologically, it is also similar to small cell lymphoma and melanoma. Grossly, this tumor varies from 20 to 200 mm in diameter with red or bluish color. It may present as a painless, nontender, dome-shaped nodule or plaque-like lesion [1–3]. Nonetheless, it is a rapid growing lesion, frequently with ulceration. This tumor affects primarily the areas of the skin exposed to the sun, with the head and neck region most frequently involved (about 50%). The extremities are involved in 40% of cases and the trunk and genitals account for 10% of patients. The statistics vary greatly in different reports [1, 2], but as many as 90% of patients may have regional lymph node involvement and up to 50% with distant metastases. When the tumor is localized, it is designated stage I, with lymph node involvement, stage II and with distant metastases, stage III. Because of its aggressive behavior and its frequent occurrence in elderly people, this tumor is usually fatal due to distant metastases. In spite of the rarity of this tumor, it is important to recognize it and to distinguish it from metastatic carcinoma, lymphoma, and melanoma. Histologically, this tumor can be divided into three morphological forms [1, 2]. The predominant type is the intermediate variant. The tumor cells are of medium size with scanty cytoplasm. The nuclei have a hyperchromatic, dispersed chromatin pattern with inconspicuous nucleoli. The tumor cells form nodular infiltratio in the dermis and frequently extend to the subcutaneous fat. The epidermis is rarely involved. The current case belongs to this variant. The small-cell variant is morphologically similar to other small cell carcinoma, especially those from the lung (Figs. 79.5 and 79.6). The trabecular variant shows delicate ribbons of small tumor cells with characteristic nuclear molding. Spindle cells are also seen in this variant. All variants show frequent mitoses and apoptosis. However, there are no clinically significan differences among these three variants and a mixed cell type is present in most cases.

Fig. 79.5 Skin biopsy shows the small cell variant in the dermis. H&E, × 10

The differential diagnoses between MCC, small cell carcinoma, lymphoma, and melanoma depend on immunohistochemical staining [1–3]. The negative staining of CD45 and S100 help to exclude the diagnoses of lymphoma and melanoma. The tumor cells are cytokeratin-positive, but the positive staining for CK20 and negative CK7 enable pathologists to distinguish

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Fig. 79.6 The tumor cells are variable in size with hyperchromatic and dispersed chromatin and imperceptible cytoplasm. H&E, × 100

MCC from bronchial small cell carcinoma. The paranuclear dot-like staining of cytokeratin is characteristic of MCC. In addition, MCC is also positive for neurofilamen protein, which is negative in bronchial small cell carcinoma. Other positive markers for MCC include CAM 5.2, epithelial membrane antigen, BEP-EP4, and CD117 [2, 3]. Some cases may show positive staining of bcl-2 [3], and those cases should be distinguished from lymphomas. Patients with local disease are usually treated with conservative surgery and postoperative radiation therapy (RT) [2]. When there is lymph node involvement, regional lymph node should be resected followed by postoperative RT [2]. Patients with unresectable nodal metastases should be treated with pre-operative chemotherapy and postoperative RT [2].

References 1. Goessling W, McKee PH, Mayer RJ. Merkel cell carcinoma. J Clin Oncol 2002;20:588–598. 2. Pectasides D, Pectasides M, Economopoulos T. Merkel cell cancer of the skin. Ann Oncol 2006;17:1489–1495. 3. Mendenhall WM, Mendenhall CM, Mendenhall NP. Merkel cell carcinoma. Laryngoscope 2004;114:906–910.

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Case 80 A 32-year-old man presented with hemolytic anemia and bulky periaortic pelvic lymphadenopathy. Core needle biopsy of the pelvic mass and excisional biopsy of the right inguinal lymph node were nondiagnostic. A transabdominal periaortic pelvic lymph node biopsy finall revealed diagnostic features, as shown in Figs. 80.1, 80.2, 80.3, 80.4, 80.5, 80.6, and 80.7.

Fig. 80.1 Lymph node biopsy shows follicular hyperplasia with atrophic germinal centers. H&E, × 10

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Fig. 80.2 The interfollicular area reveals proliferation of hyalinized blood vessels. H&E, × 20

Fig. 80.3 A hyperplastic follicle shows multiple concentrical layers of lymphocytes in the mantle zone. The germinal center is composed of eosinophilic cells representing follicular dendritic cells and histiocytes. H&E, × 60

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Fig. 80.4 A lymphoid follicle reveals a penetrating hyalinized blood vessel. H&E, × 40

Fig. 80.5 A large number of plasma cells are demonstrated in the interfollicular area. H&E, × 100

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Fig. 80.6 Numerous large pleomorphic tumor cells are present in an interfollicular area. H&E, × 100

Fig. 80.7 A Reed – Sternberg cell is shown in the center of this f eld. H&E, × 100

His leukocyte and platelet counts were normal, but hemolytic anemia was demonstrated by the presence of low hematocrit (28.7%) and hemoglobin (9.0 g/dl), with a high reticulocyte count (7.5%).

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Differential diagnoses: Reactive lymphadenopathy and lymphomas.

Further Studies Immunohistochemistry: Kappa stain: positive (Fig. 80.8) Lambda stain: positive (Fig. 80.9) CD30: positive (Fig. 80.10) CD15: positive (Fig. 80.11) HHV-8: negative (Fig. 80.12)

Fig. 80.8 Kappa stain demonstrates many plasma cells. Immunoperoxidase, × 10

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Case 80

Fig. 80.9 Lambda stain demonstrates many plasma cells. Immunoperoxidase, × 10

Fig. 80.10 CD30 stain shows the large Hodgkin and Reed – Sternberg cells. Immunoperoxidase, × 100

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Fig. 80.11 CD15 stain shows the large Hodgkin and Reed – Sternberg cells. Immunoperoxidase, × 100

Fig. 80.12 HHV-8 stain reveals no immunoreactivity. Immunoperoxidase, × 10

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Discussion The lymph node biopsy shows follicular hyperplasia (Fig. 80.1) and interfollicular vascular proliferation (Fig. 80.2). At higher magnification the follicles are composed of multiple concentric layers of lymphocytes (onion skin pattern) in the mantle zone with an atrophic germinal center, containing pale eosinophilic cells (Fig. 80.3). Penetration of hyalinized blood vessels into the germinal centers (lollipop pattern) is demonstrated in a few follicles (Fig. 80.4). Prominent plasma cell infil tration is present in the interfollicular area (Fig. 80.5). After careful search, large tumor cells are also demonstrated in the interfollicular areas in a few tissue sections (Fig. 80.6). A few Reed – Sternberg cells are also found (Fig. 80.7). The plasma cells are polyclonal, as demonostrated by the kappa and lambda staining (Figs. 80.8 and 80.9). The large tumor cells are positive for CD30 and CD15 staining (Figs. 80.10 and 80.11). Human herpes virus-8 (HHV-8) staining is negative (Fig. 80.12). These manifestations are consistent with the mixed type of hyaline vascular and plasma cell variants of Castleman disease (CD) or angiofollicular lymph node hyperplasia. In addition, Hodgkin lymphoma is present. The original report from Dr. Benjamin Castleman included patients with asymptomatic lymphadenopathy in the mediastinum [1–4]. The lymph node biopsies revealed marked follicular hyperplasia with a marginal zone of concentrical lymphocytes. The germinal centers are usually atrophic or regressively transformed, and contain penetrating hyalinized vessels and pale eosinophilic cells that represent follicular dendritic cells and histiocytes. Vascular proliferation is prominent in the interfollicular area, which is mainly composed of small lymphocytes and not plasma cells. In addition, sinuses are absent in the affected lymph nodes. This histologic pattern is designated the hyaline vascular variant (HV-CD), representing about 70% of CD. The proportion of the follicular and interfollicular abnormalities can be variable, and therefore it can be further divided into follicular HV-CD, classical HV-CD (equivalent degree of changes in the follicles and interfollicular area), and stroma-rich HV-CD [4]. In subsequent years, another pattern was recognized [1–4]. The characteristic of this pattern is that the germinal centers are hyperplastic instead of atrophic. Most strikingly, there is a prominent plasma cell infiltratio in the mantle zone as well as in the interfollicular areas. The normal architecture of the lymph node is usually not altered and the sinuses are patent. This histologic pattern is designated plasma cell variant (PC-CD), representing about 20% of CD. Unless HHV-8 is identified a diagnosis of CD is by exclusion, because this pattern can be seen in other hyperplastic reactive lymph nodes, such as rheumatoid arthritis. If the above two patterns involve a single node or chain of lymph nodes, they are classifie as unicentric CD. When there is multiple lymph node or extranodal involvement, it is designated multicentric CD, which represents 10% of the patient population [1–4]. This entity has a histologic pattern similar to the PC-CD or a mixed histologic pattern. These patients are usually symptomatic. The etiology of CD is still not entirely clear, but the plasma cell variant and the multicentric CD are frequently associated with HHV-8 infection [1–4]. Among HHV-8 positive patients, many have coexistent HIV infection. A plasmablastic variant of CD, as characterized by the presence of large plasmablasts (immunoblasts) in the mantle zone that express the HHV-8 latent nuclear antigen, is frequently present in HIV-infected patients [2–4]. The pathogenesis of CD is considered to be related to the release of interleukin-6 (IL-6) from the germinal center cells and from the HHV-8. Therefore, the clinicopathologic complex in CD is sometimes referred to as interleukin-6 syndrome [1]. Castleman disease is associated with several other diseases [2–4]. In the current case, Hodgkin lymphoma is identified This lesion is usually so scarce that a careful search of multiple sections is needed to make such a diagnosis. Some authors consider the manifestation of CD as secondary to Hodgkin lymphoma, and thus call it Castleman-like change instead of coexistent CD. Non-Hodgkin lymphomas, mostly immunoblastic type, are seen more frequently than Hodgkin lymphoma in CD cases. Other associated conditions include Kaposi sarcoma, which has the same causative agent as CD, the HHV-8; and the POEMS syndrome, which includes polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes.

References 1. Frizzera G. Atypical lymphoproliferative disorders. In: Knowles DM (ed), Neoplastic Hematopathology, Philadelphia, Lippincott, Williams & Wilkins, 2001: 569–622. 2. Casper C. The aetiology and management of Castleman disease at 50 years: translating pathophysiology to patient care. Br J Haematol 2005;129:3–17. 3. Dham A, Peterson BA. Castleman disease. Curr Opin Hematol 2007;14:354–369. 4. Ioachim HL, Medeiros LJ. Ioachim’s Lymph Node Pathology, 4th ed., Philadelphia, Lippincott Williams & Wilkins, 2009, 227–237.

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Case 81 A 14-year-old boy presented with bilateral cervical lymphadenopathy for a month. The patient had a brief period of low-grade fever and pharyngitis. A TORCH (toxoplasma, rubella, cytomegalovirus, and herpes simplex) serologic test was negative. A biopsy of the mass shows features of an abnormal lymph node, as illustrated in Figs. 81.1, 81.2, 81.3, 81.4, and 81.5.

Fig. 81.1 Lymph node biopsy shows marked sinusoidal dilatation and partial effacement of normal architecture with two residual follicles in this field H&E, × 10

Case 81

Fig. 81.2 A dilated sinus contains many lymphocytes, neutrophils, histiocytes, and static lymph. H&E, × 20

Fig. 81.3 Higher magnificatio demonstrates many multinucleated histiocytes with emperipolesis. H&E, × 40

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Fig. 81.4 The engulfed lymphocytes are seen inside intracytoplasmic vacuoles. H&E, × 100

Fig. 81.5 The medullary pulp is infiltrate by large numbers of plasma cells and lymphocytes. H&E, × 60

Differential diagnoses: Reactive lymphadenopathy, non-Hodgkin lymphoma and Rosai – Dorfman disease.

Case 81

Further Studies Serum protein electrophoresis: Polyclonal gammopathy Flow cytometry: No monoclonal B-cell population is identifie Immunohistochemistry: Positive reactions to S100 and CD68, and negative reaction to CD1a

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Discussion Sinus histiocytosis with massive lymphadenopathy (SHML) or Rosai – Dorfman disease is a disorder of benign histiocytosis, usually seen in children or young adults. Its major manifestation is lymphadenopathy, mostly seen in the cervical region, but axillary, para-aortic, inguinal, and mediastinal lymph nodes are also often affected [1, 2]. Involvement of extranodal sites is seen in a minority of patients. Patients are generally in good health with mild clinical symptoms, such as fevers, pharyngitis, and occasionally malaise, night sweats, or weight loss. The enlarged lymph node is usually painless, but pain and tenderness occur in some cases. Because of the presence of a prominent mass, which is unresponsive to antibiotics or other conventional treatment, malignancy is frequently suspected. However, spontaneous regression of the lesion may finall occur, although it may persist for a long duration. The salient histologic features of SHML are dilatation of sinuses and partial effacement of follicles. Proliferation of histiocytes is prominent in the sinuses and medulla and, characteristically, these histiocytes engulf lymphocytes. In fact, the lymphocytes actively penetrate the cytoplasm of the histiocytes, hence the name emperipolesis. The engulfed lymphocytes are inside an intracytoplasmic vacuole, protected from cytolytic enzymes. Erythrocytes and neutrophils can also be phagocytized by the histiocytes. The histiocytes in SHML are large with abundant eosinophilic cytoplasm. They can be variable in shape but atypia is not seen. The nucleus usually shows one or more nucleoli, while mitosis is seldom present. In the sinuses, there are also plasma cells, neutrophils, and static lymph. The medullary pulp is infiltrate by large numbers of plasma cells and lymphocytes, and occasionally lipid-laden histiocytes. As a result, many patients have polyclonal hypergammaglobulinemia. The rarity of eosinophils may help to distinguish eosinophilic granuloma or Langerhans cell histiocytosis. Necrosis is not seen in this lesion. Similar to the Langerhans cells, the histiocytes in SHML are positive for S100, but unlike the former they are negative for CD1a. The absence of expression of CD21, CD23, and CD35 distinguishes SHML histiocytes from dendritic histiocytes. However, these histiocytes express many other phagocytic and lysosomal markers, including CD11b, CD11c, CD14, CD15, CD64, CD68, HAM 56, CD163, EBM11, lysozyme, ␣1 -antitrypsin, ␣1 -antichymotrypsin, cathepsin D, and cathepsin E [1]. Although the clinical condition indicates that SHML is most likely to be infectious in nature, many studies failed to prove its etiologic correlation with bacteria and viruses. Current evidence suggests that it may be associated with the autoimmune lymphoproliferative syndrome [3]. Based on the clinical presentation, morphology and marker studies, the current case is consistent with SHML. Nonspecifi sinus histiocytosis does not show emperipolesis and the histiocytes are negative for S100. The absence of marked atypia and mitosis exclude malignant histiocytosis or histiocytic lymphoma.

References 1. Ioachim HL, Medeiros LJ. Ioachim’s Lymph Node Pathology. 4th ed., Philadelphia, Lippincott Williams & Wilkins, 2009, 193–197.. 2. Gaitonde S. Multifocal, extranodal sinus histiocytosis with massive lymphadenopathy: An overview. Arch Pathol Lab Med 2007;131: 1117–1121. 3. Maric I, Pittaluga S, Dale JK, et al. Histologic features of sinus histiocytosis with massive lymphadenopathy in patients with autoimmune lymphoproliferative syndrome. Am J Surg Pathol 2005;29:903–911.

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Case 82 A 24-year-old Japanese woman presented with left cervical lymphadenopathy for one week. The node was nontender and painless. She had a low-grade fever but was otherwise asymptomatic. The lymph node biopsy showed extensive necrosis (Figs. 82.1, 82.2, 82.3, and 82.4)

Fig. 82.1 Lymph node biopsy shows a necrotic area with fibrinoi necrosis and marked karyorrhexis. H&E, × 20

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Fig. 82.2 Higher magnificatio of Fig. 81.1 reveals f brinoid necrosis, karyorrhexis, and apoptosis. Note the absence of neutrophils, eosinophils, and plasma cells. H&E, × 40

Fig. 82.3 Higher magnificatio of Fig. 81.2 shows many plasmacytoid monocytes (arrow) and a few crescentic histiocytes (arrowhead). H&E, × 100

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Fig. 82.4 The uninvolved area of the lymph node reveals scattered immunoblasts and nonphagocytic histiocytes on a lymphocytic background. H&E, × 40

Differential diagnoses: Reactive lymphadenopathy versus lymphomas.

Further Studies Immunohistochemical stainings: Lymphocytes: CD20−, CD4−, CD8+ Histiocytes: CD68 (KP1)+, lysozyme+ Plasmacytoid monocytes: CD4+, CD68+

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Discussion Kikuchi – Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis, was described by two Japanese pathologists, Kikuchi and Fujimoto, independently in 1972. It has a benign self-limited clinical course, but morphologically it mimics malignant lymphoma and thus it is important to recognize this entity to avoid aggressive treatment in these patients. The early reported cases were mainly women under 40 years of age and the majority of cases involved the cervical lymph nodes [1–4]. Most patients were from Southeast Asian countries. However, later studies have shown that KFD may affect patients of any age, gender, and ethnic background. In addition, virtually any lymph node region may be involved. Clinically, most patients are asymptomatic, but a subset of patients may have fever, chills, myalgia, sore throat, leukopenia, and atypical lymphocytes in the peripheral blood [1–4]. Approximately 16–40% of patients have skin eruptions, including erythematous or papular rash, plaques or a variolliform eruption [5]. Most patients recover spontaneously, but multi-organ involvement has been reported in a few patients with possible underlying immunodeficien y, such as post-transplant patients. Other unusual manifestations include fatal myocarditis, pulmonary hemorrhage, hemophagocytic syndrome, bilateral panuveitis, and thyroiditis [1–4]. The characteristic pathology is patchy fibrinoi necrosis with prominent karyorrhectic nuclear fragments and eosinophilic apoptotic debris, involving mainly the paracortex of the lymph node [1–4]. Surrounding the necrotic areas is a mixed population of lymphocytes, histiocytes, and immunoblasts. There are two special cell types that are pathognomonic for this disease. The histiocytes with eccentric crescentic nuclei with karyorrhectic debris in the cytoplasm are designated crescentic histiocytes. Another cell type is called plasmacytoid monocytes (recently called plasmacytoid dendritic cells). These cells are of medium size with eccentric round nucleus similar to plasma cells and yet they are monocytes as identifie by immunostaining. Another diagnostic criterion is the almost complete absence of neutrophils, eosinophils, and plasma cells in the necrotic areas. In the uninvolved areas, there is paracortical expansion with scattered immunoblasts and histiocytes on a lymphocytic background, imparting the starry-sky or a “mottled” appearance. According to the predominant histologic features, KFD can be divided into four histologic subtypes: lymphohistiocytic, phagocytic, necrotic, and foamy cell types [1].The skin lesion may show necrotic keratinocytes, dense dermal lymphohistiocytic perivascular infiltration abundant karyorrhectic debris, and absence of neutrophils [1]. Since there is a marked proliferation of histiocytes and immunoblasts and some of these cells can be atypical, it is therefore important to distinguish KFD from malignant lymphoma, for which immunohistochemistry is most important. The immunoblasts are negative for B-cell markers, such as CD20, but are positive for T-cell markers, such as CD3 and CD5, as well as positive for CD30 and CD45 [1, 2, 4]. The histiocytes are positive for CD11b, CD11c, CD14, CD68 (KP1), Mac387, Ki-MP1, and lysozyme [1, 2]. The plasmacytoid monocytes are positive for all histiocytic markers as well as CD10 and CD74 [1]. Flow cytometric study should demonstrate a predominant T-cell population without the presence of a monoclonal B-cell population. Another major differential diagnosis is lupus erythematosus (LE), because LE also shows fibroinoi necrosis with nuclear debris in the lymph node [1, 2]. It is particularly important to exclude LE in KFD patients with skin eruptions. LE, however, may show hematoxylin bodies, lesions of vasculitis, and the presence of neutrophils. In a few cases, LE is considered coexistent with KFD. Several infectious diseases, such as tuberculosis, histoplasmosis, cat-scratch disease, and syphilis, should also be excluded before a diagnosis of KFD is made [1]. KFD has been considered infectious in nature, particularly viral, but so far there is no solid proof. Another possible mechanism is autoimmunity.

References 1. Ioachim HL, Medeiros LJ. Ioachim’s Lymph Node Pathology, 4th ed., Philadelphia, Lippincott Williams & Wilkins,2009, 199–202. 2. Onciu M, Medeiros, LJ. Kikuchi-Fujimoto lymphadenitis. Adv Anat Pathol 2003;10:204–211. 3. Tsang WYW, Chan JKC, Ng CS. Kikuchi’s lymphadenitis. A morphologic analysis of 75 cases with special reference to unusual features. Am J Surg Pathol 1994;18:219–231. 4. Kuo TT. Kikuchi’s disease (histiocytic necrotizing lymphadenitis). A clinicopathologic study of 79 cases with an analysis of histologic subtypes, immunohistology, and DNA ploidy. Am J Surg Pathol 1995;19:798–890. 5. Yasukawa K, Matsumura T, Sato-Matsumura KC, et al. Kikuchi’s disease and the skin: case report and review of the literature. Br J Dermatol 2001;144:885–889.

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Case 83 A 59-year-old man presented with splenomegaly and lymphadenopathy in the inguinal region. Hypersplenism was manifested as anemia and thrombocytopenia. The splenectomy specimen showed many foam histiocytes (Fig. 83.1). The lymph node biopsy also showed accumulation of similar histiocytes (Fig. 83.2).

Fig. 83.1 Splenectomy specimen shows the expansion of the cord of Billroth by foam histiocytes (arrow). The red pulp sinuses are not involved. H&E, × 60

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Fig. 83.2 Lymph node biopsy shows focal collection of foam histiocytes (arrow). H&E, × 60

Differential diagnoses: Storage histiocyte disorders.

Further Studies Periodic acid – Schiff (PAS) stain of splenectomy specimen: positive for histiocytes (Fig. 83.3) Sudan black B stain of splenectomy specimen: positive for histiocytes Electron microscopic examination of splenectomy specimen: concentric lamellar bodies PAS stain of lymph node: positive for histiocytes (Fig. 83.4) Culture skin fibroblasts markedly decreased sphingomyelinase activity

Case 83

Fig. 83.3 Periodic acid – Schiff (PAS) stain of the spleen reveals positive reaction in the Niemann – Pick cells. PAS, × 60

Fig. 83.4 PAS stain of lymph node shows PAS-positive cytoplasmic granules in the Niemann – Pick cells. PAS, × 100

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Discussion Niemann – Pick disease is an autosomal recessive lysosomal storage histiocytic disease [1–3]. It can be further divided into two categories [1]: those with a primary deficien y in acid sphingomyelinase activity are classifie as types A and B, and those with defective intracellular processing and transport of low-density lipoprotein-derived cholesterol are classifie as types C and D. Type A is caused by mutations in the acid sphingomyelinase gene on chromosome 11p15, leading to absence of acid sphingomyelinase activity and subsequent lysosomal accumulation of sphingomyelin [2]. Type B is associated with milder mutation of the acid sphingomyelinase gene, so that there is residual activity of the acid sphingomyelinase in those patients. Type C shows mutations in two genes: the NPC1 gene is located on chromosome 18q11 and the NPC2 gene on chromosome 14q24 [2]. Type D has a point mutation within the NPC1 gene. The NPC1 protein plays a role in the intracellular traffickin and distribution of low-density lipoprotein cholesterol. The disruption of this traffickin leads to accumulation of LDL cholesterol and neuronal degeneration. Clinically, type A is the infantile neurologic form, accounting for approximately three-quarters of all cases [1–3]. These patients may have central nervous system involvement, hepatosplenomegaly, mental retardation, generalized lymphadenopathy, and skin xanthomas. Death occurs in early childhood due to central nervous system involvement. Type B is a milder clinical variant, with hepatosplenomegaly but no neurological involvement. Type C disease may be manifested as severe neonatal jaundice, hepatosplenomegaly, dementia, and dystonic posturing in childhood. However, the onset of neurologic symptoms is extremely variable, and patients may not have neurologic changes until the f fth decade. Type D patients usually have only moderate degrees of hepatosplenomegaly. This variant has been reported only in descendants of an Acadian couple who immigrated to Nova Scotia from France in the 1600s. Based on the clinicopathological manifestations, the current case belongs to type B disease. Morphologically, Niemann – Pick cells range from 20 to 100 ␮m in diameter and contain numerous sharply define small vacuoles in the cytoplasm, imparting a foamy appearance. The vacuoles are positive for fat stains, PAS and Pearce’s phospholipid staining. The ultrastructure is characterized by membrane-bound concentric lamellated bodies resembling myelin figure or in the form of parallel lamellae designated “zebra bodies” [3]. These foam histiocytes can be found in the spleen, liver, lymph nodes, bone marrow, tonsils, gastrointestinal tract, and lungs. Sea-blue histiocytes may also be present in these organs. Unlike Gaucher disease, the neurons in Niemann – Pick disease are involved, showing vacuolation and ballooning. The accumulation of lipid substance in neurons finall leads to cell death and loss of brain substance. A definit ve diagnosis of types A and B depends on the demonstration of decreased sphingomyelinase activity in the spleen, liver, bone marrow biopsy, leukocyte extracts, or cultured skin fibroblasts In types C and D, cultured skin fibroblast may show reduction in intracellular cholesterol esterificatio and a f lipin stain may demonstrate free cholesterol in fibroblast [1]. Alternatively, cytogenetic studies may demonstrate mutations of acid sphingomyelinase gene in types A and B, and mutations of NPC genes in types C and D [1, 2].

References 1. Kolodny ED. Niemann-Pick disease. Curr Opin Hematol 2007;7:48–52. 2. Cruse RP. Overview of Niemann-Pick disease. UpToDate online15.3, 2007. 3. Nieman RS, Orazi A. Disorders of the spleen. 2nd ed., Philadelphia, W.B. Saunders, 1999, 171.

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Case 84 A 4-year-old boy presented with anemia and thrombocytopenia. Physical examination revealed moderate splenomegaly and mild hepatomegaly. His liver enzymes were slightly elevated. A thorough neurologic examination did not show any abnormality. The splenectomy specimen showed marked expansion of the red pulp cords by histiocytes with fibrillar cytoplasm (Figs. 84.1, 84.2, and 84.3). A bone marrow biopsy revealed infiltratio with the same histiocytes (Figs. 84.4 and 84.5).

Fig. 84.1 Splenectomy specimen shows a cluster of Gaucher cells with fibrillar cytoplasm. H&E, × 60

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Diseases Mimicking Hematologic Neoplasms

Fig. 84.2 Splenectomy specimen shows a cluster of Gaucher cells in the cord of Billroth. Note the dilated sinus next to the cluster. H&E, × 60

Fig. 84.3 A plastic section of splenectomy specimen reveals the detailed striation in the cytoplasm of Gaucher cells. Toludine blue O, × 100

Case 84

503

Fig. 84.4 Bone marrow biopsy reveals Gaucher cell infiltratio with residual hematopoietic cells in the right upper corner. H&E, × 40

Fig. 84.5 Bone marrow aspirate shows f ve multinucleated Gaucher cells, two mononucleated Gaucher cells (red arrows) and a single megakaryocyte (black arrow). Wright – Giemsa, × 60

504

Diseases Mimicking Hematologic Neoplasms

Differential diagnoses: Lipid storage diseases.

Further Testing Periodic acid – Schiff (PAS) stain in the spleen: positive and diastase-resistant in histiocytes (Fig. 84.6) PAS stain in the bone marrow: positive and diastase-resistant in histiocytes (Fig. 84.7) Blood leukocytes and cultured skin fibroblast showed a marked decrease in acid ␤-glucosidase activity

Fig. 84.6 Splenectomy section reveals PAS-positive Gaucher cells. PAS, × 60

Case 84

Fig. 84.7 Bone marrow biopsy shows PAS-positive Gaucher cells. PAS, × 40

505

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Diseases Mimicking Hematologic Neoplasms

Discussion Gaucher disease is characterized by the accumulation of typical Gaucher cells in various organs, frequently the spleen, liver, lymph node and bone marrow [1–4]. The Gaucher cells range from 20 to 100 ␮m in diameter with one or more nuclei and characteristic fibrillar or striated cytoplasm. They are PAS-positive and diastase-resistant. However, pseudo-Gaucher cells can be seen in the bone marrow of chronic myeloid leukemia, lymphoma, thalassemia, and plasma cell myeloma [2, 3]. In chronic myeloid leukemia, the excess catabolism of phagocytosed granulocytes and secondary depletion of glucocerebrosidase are probably the mechanism for the formation of the pseudo-Gaucher cells Gaucher disease is an autosomal recessive lysosomal storage disease with a deficien y in the enzyme acid ␤-glucosidase (glucocerebrosidase) [1–4]. As a result, there is an accumulation of its main substrate, glucosylceramide (glucocerebroside) in mononuclear phagocytes. The enzyme defici is due to the mutations at the glucocerebrosidase locus on chromosome 1q21. There have been over 200 mutations reported, but only three mutations (c.1226A>G, c.1448T>C and 84insG) have a high frequency [2]. In the spleen, the Gaucher cells accumulate in the cords of Billroth, because the cordal macrophages have a greater ability to ingest lipid than sinus lining cells [1]. Extramedullary hematopoiesis may occur in the spleen when the bone marrow is overwhelmed by the Gaucher cells. In the liver, K¨upffer cells are the site for glycosphingolipid accumulation [2]. In the brain, Gaucher cells are present around blood vessels. The neurons usually do not show features of lipid storage. In children, skeletal lesions are common, varying from osteopenia to osteolytic lesions. Hematologic changes are frequent finding in Gaucher disease, including anemia, thrombocytopenia, or pancytopenia. The mechanism is complicated, including bone marrow infiltration hypersplenism, or depletion of iron due to expansion of the macrophage pool [1]. Clinically, there are three types of Gaucher disease [1–4]. The most common form is type 1, which is usually referred to as adult form because many patients live to adulthood. Most patients are asymptomatic in spite of the fact that it is a multisystem storage disease. Symptomatic patients frequently have hepatosplenomegaly, anemia, thrombocytopenia or skeletal changes. Pediatric patients may have growth and pubertal retardation [4]. However, the central nervous system is characteristically uninvolved [2]. The current case belongs to this category. Type 2 disease is usually referred to as acute infantile neuronopathic form because it has an early onset with rapidly progressive neurological symptoms. These patients also have hepatosplenomegaly and frequently pulmonary infiltration Children with this form of disease usually die within two years [2]. Type 3 disease is sometimes called subacute neuropathic form and has a later onset in adolescence with slowly progressive neurologic impairment. However, it is clinically severe, with dementia and ataxia. These patients also have moderate multiorgan involvement. A definit ve diagnosis of Gaucher disease is the identificatio of acid ␤-glucosidase deficien y in leukocytes or in cultured skin fibroblasts Molecular genetic testing can also be helpful.

References 1. Neiman RS, Orazi A. Disorders of the spleen. 2nd ed., Philadelphia, W.B. Saunders Co., 1999, 169–171. 2. Germain DP. Gaucher’s disease: a paradigm for interventional genetics. Clin Genet 2004;65:77–86. 3. Jmoudiak M, Futerman AH. Gaucher disease: pathological mechanisms and modern management. Br J Haematol 2005;129:178–188. 4. Grabowski GA. Recent clinical progress in Gaucher disease. Curr Opin Pedatr 2005;17:519–594.

Case 85

507

Case 85 A 82-year-old man presented with pancytopenia and hypercalcemia. His thyroid function test was normal and serum electrophoresis showed no monoclonal gammopathy. A bone marrow biopsy revealed noncaseating granulomas (Figs. 85.1, 85.2, and 85.3). Computerized tomography of thorax showed extensive adenopathy throughout the mediastinum, as well as probable right hilar adenopathy. A transbronchial biopsy (Fig. 85.4) and a right axillary lymph node biopsy (Fig. 85.5) also demonstrated granulomatous lesions. A skin tuberculin test was negative. Serum coccidioidomycosis titers and urine antigen for histoplasmosis were also negative.

Fig. 85.1 Bone marrow biopsy shows noncaseating granulomas intermixing with focal lymphoid infiltration H&E, × 10

508

Diseases Mimicking Hematologic Neoplasms

Fig. 85.2 Bone marrow biopsy shows a spider-like asteroid body in a multinucleated giant cell (arrow). H&E, × 60

Fig. 85.3 Bone marrow biopsy shows an asteroid body in a multinucleated giant cell (arrow). H&E × 100

Case 85

Fig. 85.4 Transbronchial biopsy reveals a noncaseating granuloma along the bronchial wall. H&E, × 40

Fig. 85.5 Right axillary lymph node shows multiple noncaseating granulomas. H&E, × 5

Differential diagnoses: infectious diseases versus sarcoidosis.

509

510

Diseases Mimicking Hematologic Neoplasms

Further studies Special staining in bone marrow biopsy: Acid fast bacilli (AFB) stain: negative (Fig. 85.6) Gomeri methanamine silver (GMS) stain for fungi: negative (Fig. 85.7) Reticulin stain: increased reticular fiber (Fig. 85.8). Bone marrow biopsy culture for bacteria, acid fast bacilli and fungi were negative

Fig. 85.6 Acid-fast bacilli (AFB) stain of bone marrow biopsy shows no AFB organism in the granulomas. AFB, × 20

Case 85

Fig. 85.7 Gomeri methanamine silver stain of bone marrow biopsy shows no fungi. GMS, × 20

Fig. 85.8 Reticulin stain of bone marrow reveals reticulin fibrosis Reticulin × 10

511

512

Diseases Mimicking Hematologic Neoplasms

Discussion The diagnostic process in this case is somewhat unusual, by discovering non-caseating granulomas in the bone marrow f rst, followed by the identificatio of the same pathologic features in the lung and a lymph node. This clinical manifestation represents a systemic granulomatous disease, which is characteristic of sarcoidosis. However, because there are no pathognomonic features for sarcoidosis, infectious etiology has to be excluded before a fina diagnosis can be considered. This patient had undergone many studies and no infectious agents were identified Finally, he responded to corticosterioid therapy with prompt remission. There are no unique histologic features in sarcoidal granulomas to differentiate them from other granulomas [1–4]. These granulomas are composed of multinucleated giant cells, epithelioid histiocytes, lymphocytes, and plasma cells in various proportions for different individuals. No caseation or necrosis is present in sarcoidal granulomas. In later stages, fibrosi may occur gradually and finall replaces the granulomas, forming scar tissues. On the other hand, granulomas may resolve spontaneously with little consequence. Several cytoplasmic inclusions are described in the literature; their presence is supportive for the diagnosis but they are not diagnostic for this entity and their absence does not exclude the diagnosis of sarcoidosis [4]. The asteroid body is a spider-like structure inside the cytoplasm of the multinucleated giant cells, and is usually seen within a vacuole. The Schaumann bodies are basophilic oval structures composed of concentric layers, appearing like a raspberry. Hamazaki – Wesenberg bodies are giant lysosomes usually present extracellularly at or near the peripheral sinus of the lymph node. They are almost always apart from the granulomas. With Gomori silver stain, they appear like budding yeast cells. Clinically, most patients are asymptomatic or with nonspecifi systemic symptoms, such as fatigue, night sweats, weight loss, and malaise. Frequently, the diagnosis is made incidentally by routine chest radiographs. Sarcoidosis primarily affects the lungs and the lymphatic system and in 90% of patients involves the lungs. Pulmonary fibrosi is the most frequent severe manifestation and is responsible for the main morbidity and most of the deaths caused by sarcoidosis [2]. Ocular and cutaneous lesions are second most common. However, many other organs also may be involved. The relatively specifi clinical findin is L¨ofgren syndrome, which consists of bilateral hilar lymphadenopathy, ankle arthritis, erythema nodosum, fever, myalgia, and weight loss [1,3]. Approximately two-thirds of patients with sarcoidosis have a remission within 10 years after diagnosis. One-third of them progress to chronic phase with significan organ involvement. Less than 5% of patients die from sarcoidosis [1]. Cardiac sarcoidosis is the most common cause of death in sarcoidosis due to fibrosi in the heart, causing sudden death in some cases [4]. Corticosteroids remain the mainstay of treatment, but most patients require no treatment. The diagnosis of sarcoidosis is generally based on characteristic bilateral hilar adenopathy and supported by the detection of noncaseating granulomas by lymph node or lung biopsy. There are also a few tests that can help substantiate the diagnosis. Bronchoalveolar lavage flui with a CD4 to CD8 ratio greater than 3.5 is suggestive of sarcoidosis [3]. Sarcoidal granulomas produce angiotensin-converting enzyme (ACE), and the serum level of ACE is elevated in 60% of patients. However, its positive and negative predictive values were only 84 and 74%, respectively, in one series [1]. The Kveim – Siltzback skin test has been used for many years for the diagnosis of sarcoidosis. The test is performed by injecting human sarcoid tissue extract intradermally to the patient and a biopsy from the injected site is examined four weeks later [1]. However, the human sarcoid antigen is hard to obtain and to standardize; therefore this test is seldom used nowadays. Hypercalcemia is a common findin in sarcoidosis; although it is not specific its presence can be a clue to the diagnosis.

References 1. Iannuzzi MC, Rybicki BA, Teirstein AS. Sarcoidosis. N Engl J Med 2007;357:2153–2165. 2. Nunes H, Soler P, Valeyre D. Pulmonary sarcoidosis. Allegy 2005;60:565–582. 3. Wu JJ, Schiff KR. Sarcoidosis. Am Fam Physician 2004;70:312–322. 4. Ioachim HL, Medeiros LJ. Ioachim’s Lymph Node Pathology, 4th ed, Philadelphia, Lippincott Williams & Wilkins, 2009, 203–212.

Index

Aberrant somatic hypermutation, 187 Abetalipoproteinemia, 17 Abnormal localization of immature precursors (ALIP), 66, 77, 78 Abnormal red cell morphology, 16–17 Acanthocytes, 16, 17 Acid ␤-glucosidase, 507 Acquired immunodeficien y syndrome, see AIDS/HIV Activation antigens, 23 Acute basophilic leukemia, 5 Acute erythroid leukemia (AML-M6), 129–134 Acute leukemia, 3, 5 Acute lymphoblastic leukemia (ALL), 27, 39–40, 49 B-cell, 152–156, 343 chronic lymphocytic leukemia and, 170 classificatio of, 5 in differential diagnosis, 93, 110, 115, 137 lymphoplasmacytic lymphoma and, 203 T-cell, 157–160, 165 Acute lymphoid leukemia, 341 Acute megakaryoblastic lymphoma (AMKL), 135–140 Acute monoblastic/monocytic leukemia, 13 case histories, 120–124, 125–128 in differential diagnosis, 122 leukemia cutis and, 151 Acute myeloblastic leukemia with maturation (AML-M2), 95, 112–115 Acute myeloblastic leukemia without maturation (AML-M1), 115 Acute myelogenous leukemia (AML), 5, 154, 155 Acute myeloid leukemia (AML), 8, 9 case histories, 91–95, 107–111 in differential diagnosis, 36, 41, 97, 118, 127, 137, 159, 341 leukemia cutis and, 151 lymphoplasmacytic lymphoma and, 203 myeloid sarcoma and, 141, 144 WHO classification 6t Acute myeloid leukemia with maturation (AML-M2), 144 Acute myelomonocytic leukemia (AML-M4), 13

case history, 116–119 in differential diagnosis, 64, 115, 122, 124 leukemia cutis and, 151 Acute myelomonocytic leukemia with eosinophilia (AML-M4eo), 96–100, 144 Acute panmyelosis with myelofibrosis 5, 56 Acute promyelocytic leukemia (APL), 124 case history, 101–106 in differential diagnosis, 128 Adhesion molecules, 23 Adult T-cell leukemia/lymphoma, 349–353 AIDS/HIV Burkitt-like lymphoma and, 319, 321, 322 Burkitt lymphoma/leukemia and, 323, 328, 330 extranodal Hodgkin lymphoma and, 449 lymphocyte-depleted Hodgkin lymphoma and, 442 mixed cellularity Hodgkin lymphoma and, 433 Aleukemic leukemia cutis (ALC), 151 ALK gene, 300, 400 All-trans-retinoic acid (ATRA) therapy, 106 AML1 gene, 95 Anagrelide, 61 Anaplastic large cell lymphoma (ALCL), 28 case studies, 397–401, 402–406 cutaneous, 381–384 lymphohistiocytic variant, 407–414 systemic, 384 Anaplastic variant of diffuse large B-cell lymphoma (DLBCL), 296–300 Anemia aplastic, 83, 346 of different etiology, 75 hemolytic, 17, 204 iron deficien y, 17 megaloblastic, 10, 17 microangiopathic hemolytic, 17 refractory, with excess blasts, 78–79, 84, 134 refractory, with ring sideroblasts, 77–78 Aneuploidy, 24 Angioimmunoblastic T-cell lymphoma (AITCL), 385–392 513

514

Angiotensin-converting enzyme (ACE), 513 Anisocytosis, 16, 17f, 19 Ann Arbor staging system, 428, 449 API2-MALT1 fusion protein, 239 Aplastic anemia, 83, 346 Arsenic trioxide (ATO), 106 ATM gene, 170, 187 Atypical chronic myeloid leukemia (aCML), 66, 68–72 Auer rods, 9, 91f, 102f, 104f, 105, 107f, 110, 112f Azathioprine, 457 B Ballerina skirt appearance, 15 Bands, 10 Basophilic leukemia, acute, 5 Basophilic megakaryocytes, see Promegakaryocytes Basophilic normoblasts (erythroblasts), 16 Basophilic stippling, 17, 18f Basophils, 11, 12f B-cell acute lymphoblastic leukemia (ALL), 152–156, 343 B-cell lymphoma, 5, 7, 27–28, 30, 144, 175f, 183, 209–210, 232, 277, 368, 452, 457, See also Cutaneous B-cell lymphoma; Diffuse large B-cell lymphoma (DLBCL); Intravascular large B-cell lymphoma (IVBCL); Marginal zone B-cell lymphoma; Primary mediastinal large B-cell lymphoma (PMLBCL); T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) B-cell Oct-binding protein 1 (BOB1), 422, 427, 438, 449 B-cell prolymphocytic leukemia (PLL), 192, 193 B cells intranodal differentiation, 4–5 loss of surface immunoglobulin in a population, 24 B-cell-specifi activator protein (BSAP), 198, 427, 449 BCL-1 gene, 27, 289 BCL-2 gene, 27, 289, 295, 300, 330 BCL-6 gene, 295, 300, 312 BCR-ABL 1 fusion, 38, 39, 40, 43, 44, 48, 49 atypical chronic myeloid leukemia and, 70, 72 essential thrombocythemia and, 60 myelodysplastic/myeloproliferative neoplasms and, 64, 66 primary myelofibrosi and, 54, 55 BCR gene, 26, 27 Benign lymphoepithelial lesion, 250 Benign lymphoid aggregates, 29 B immunoblasts, 5 Binet staging system, 170, 171t Blastic plasmacytoid dendritic cell neoplasm (BPDC), 5, 370–374 Blastoid subtype mantle cell lymphoma (MCL), 279–285 B lymphocytes, development of, 4

Index

BOB1, see B-cell Oct-binding protein 1 (BOB1) Body cavity lymphoma, 321, 322 Bone marrow aspirate, 28 acute erythroid leukemia, 130f, 132f acute megakaryoblastic leukemia, 135, 136f, 138f acute monoblastic/monocytic leukemia, 121f, 125, 126f acute myeloblastic leukemia with maturation, 113f, 114f acute myeloid leukemia, 92f, 93f, 107, 108f, 109f acute myelomonocytic leukemia, 117f, 118f acute myelomonocytic leukemia with eosinophilia, 96, 97f acute promyelocytic leukemia, 102f atypical chronic myeloid leukemia, 68, 69f B-cell acute lymphoblastic leukemia, 153f blastic plasmacytoid dendritic cell neoplasm, 370f Burkitt lymphoma/leukemia, 338, 339f chronic lymphocytic leukemia, 166f chronic myelogenous leukemia, 35, 36f, 41, 42f chronic neutrophilic leukemia, 46f diffuse large B-cell lymphoma, 290f essential thrombocythemia, 58f Gaucher disease, 503f growth factor effect, 472f hairy cell leukemia, 211f, 213 leukemia cutis, 147f lymphoplasmacytic lymphoma, 194f mantle cell lymphoma, 273f mantle cell lymphoma, blastoid subtype, 279f multiple myeloma, 218f, 222 myelodysplastic/myeloproliferative neoplasms, 62, 63f, 64f myelodysplastic syndrome, 74f, 76f natural killer cell lymphoma/leukemia, 356f plasma cell leukemia, 224f primary myelofibrosis 50, 52f prolymphocytic leukemia, 188f 5q-syndrome, 85, 86f, 87f refractory cytopenia with multilineage dysplasia, 81f, 82f small lymphocytic lymphoma, 181 small lymphocytic lymphoma, paraimmunoblastic variant, 184f T-cell acute lymphoblastic leukemia, 158f T-cell large granular lymphocyte leukemia, 344f Bone marrow biopsy, 28–29 acute erythroid leukemia, 129, 130f, 131f, 133f acute megakaryoblastic leukemia, 135, 136f, 137f, 138f acute monoblastic/monocytic leukemia, 120, 121f, 125, 126f acute myeloblastic leukemia with maturation, 112, 113f acute myeloid leukemia, 91, 107, 108f acute myelomonocytic leukemia, 116, 117f acute myelomonocytic leukemia with eosinophilia, 96, 97f

Index

acute promyelocytic leukemia, 101, 103f atypical chronic myeloid leukemia, 68, 69f B-cell acute lymphoblastic leukemia, 152, 153f blastic plasmacytoid dendritic cell neoplasm, 372 Burkitt lymphoma/leukemia, 338, 339f, 340f chronic lymphocytic leukemia, 167f chronic myelogenous leukemia, 37f, 42f chronic neutrophilic leukemia, 45, 47f, 48f diffuse large B-cell lymphoma, 290f diffuse large B-cell lymphoma, anaplastic variant, 296f, 297f, 298f essential thrombocythemia, 58f, 59f extranodal Hodgkin lymphoma, 444f, 446f, 447f follicular lymphoma, 259f Gaucher disease, 503f, 505f hairy cell leukemia, 210, 211f, 212, 213f leukemia cutis, 148 lymphoplasmacytic lymphoma, 194f mantle cell lymphoma, 273f, 275f mantle cell lymphoma, blastoid subtype, 279f multiple myeloma, 219f, 220f mycosis fungoides/S´ezary syndrome, 376f, 378 myelodysplastic/myeloproliferative neoplasms, 63f, 65f myelodysplastic syndrome, 73, 74f natural killer cell lymphoma/leukemia, 355f plasma cell leukemia, 224f primary myelofibrosis 50, 51f prolymphocytic leukemia, 188f 5q-syndrome, 85, 87f, 88f refractory cytopenia with multilineage dysplasia, 80, 82f Richter syndrome, 171, 173f sarcoidosis, 507f, 508f small lymphocytic lymphoma, 178 small lymphocytic lymphoma, paraimmunoblastic variant, 186 splenic marginal zone lymphoma, 204 T-cell acute lymphoblastic leukemia, 157, 158f T-cell large granular lymphocyte leukemia, 344f Brain biopsy intravascular large B-cell lymphoma, 313f, 314f post-transplant lymphoproliferative disorder, 450f, 452 Breast cancer, 472, 475 BSAP, see B-cell-specifi activator protein (BSAP) Burkitt-like lymphoma, 318–321, 327, 328f Burkitt lymphoma/leukemia, 5, 14, 25, 26–27, 144, 155, 160 case studies, 323–331, 332–338, 339–343 in differential diagnosis, 165 endemic, 328, 330, 338 sporadic, 328, 338 Burr cells, 18f

515

C C-ABL gene, 27 Cabot ring, 17 Castleman disease (CD), 373, 481–488 CBP gene, 128 C-cis gene, 139 CCND1 gene, 278 Cell lineage antigens, selective loss of, 24 2-Chlorodeoxyadenosine, 216 Chronic leukemia, 3 Chronic lymphocytic leukemia (CLL), 27, 28, 176, 187 case study, 166–171 in differential diagnosis, 191, 192, 239 Chronic lymphocytic leukemia/prolymphocytic leukemia (CLL/PLL), 187, 192 Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 5, 170, 181–183, 184, 277 Chronic lymphoid leukemia, 346 Chronic myelogenous leukemia (CML), 26, 27 case studies, 35–40, 41–44 in differential diagnosis, 48, 49, 54, 70, 72 lymphoplasmacytic lymphoma and, 203 neutrophilic, 39 Chronic myeloid leukemia (CML), 8, 13f, 25, 134, 140 atypical, 66, 68–72 myeloid sarcoma and, 144 Chronic myelomonocytic leukemia (CMML), 8, 64, 66, 67, 119 in differential diagnosis, 64, 72, 122, 124 leukemia cutis and, 151 Chronic myeloproliferative disorder, 10, 474 Chronic neutrophilic leukemia (CNL), 39, 45–49 CKD4 gene, 285 C-KIT gene, 95 C-MYC gene, 26–27, 295, 322, 330, 338, 343 Cording pattern, 142f, 146f Cortical thymocytes, 4 CRAB, 223 Cryptic abnormality, 27 Cutaneous anaplastic large cell lymphoma (C-ALCL), 381–384 Cutaneous B-cell lymphoma, 368, 378 Cutaneous carcinoma, 229 Cutaneous lymphoma, 148, 229 Cutaneous myeloma, 229 Cutaneous T-cell lymphoma, 351, 368, 378 CXCL12 gene, 272 Cyclosporine, 457 Cytochemistry acute erythroid leukemia, 131, 134 acute megakaryoblastic leukemia, 137, 139 acute monoblastic/monocytic leukemia, 122, 124

516

acute myeloblastic leukemia with maturation, 114 acute myeloid leukemia, 109 acute myelomonocytic leukemia, 118 B-cell acute lymphoblastic leukemia, 154 leukemia cutis, 148, 151 T-cell acute lymphoblastic leukemia, 159 Cytogenetic karyotyping acute erythroid leukemia, 134 acute megakaryoblastic leukemia, 137 acute monoblastic/monocytic leukemia, 122, 127 acute myeloid leukemia, 93, 94f acute myelomonocytic leukemia with eosinophilia, 98 atypical chronic myeloid leukemia, 70 Burkitt-like lymphoma, 321 Burkitt lymphoma/leukemia, 333, 336f, 340, 341f chronic myelogenous leukemia, 38, 43 follicular lymphoma, 266 gastric MALT, 237 myelodysplastic/myeloproliferative neoplasms, 64 myeloid sarcoma, 141 primary myelofibrosis 54 pulmonary MALT, 245 5q-syndrome, 89 refractory cytopenia with multilineage dysplasia, 83 Richter syndrome, 173 small lymphocytic lymphoma, paraimmunoblastic variant, 186 splenic marginal zone lymphoma, 205, 207f T-cell large granular lymphocyte leukemia, 346 techniques, 24–26 Cytopenia, see Refractory cytopenia with multilineage dysplasia (RCMD); Refractory cytopenia with unilineage dysplasia (RCUD) Cytoplasmic blebs, 135f, 136f, 138f, 139 D Dasatinib, 44 Diagnostic procedures, 27–30 Diffuse large B-cell lymphoma (DLBCL), 5, 26–27, 144, 289, 400 anaplastic variant, 296–300 case study, 290–295 chronic lymphocytic leukemia and, 170, 176 in differential diagnosis, 278, 334, 338 lymphoplasmacytic lymphoma and, 203 small lymphocytic lymphoma and, 182–183 Dimorphic population, 16, 17f D¨ohle bodies, 10, 11f Downey type cells, 15 Down syndrome, 5, 139 Drosophila trithorax gene, 119 Dual-cell lineage markers, 24

Index

Durie-Salmon staging system, 227 Dutcher bodies, 15, 197, 221, 222f, 236f, 239 E Echinocytes, 16, 17 EDTA antibodies, 19, 21f Endemic Burkitt lymphoma/leukemia, 328, 330, 338 Endomitosis/endoreduplication, 19 Eosinophils, 10–11, 12f Epstein-Barr virus (EBV) adult T-cell leukemia/lymphoma and, 352 angioimmunoblastic T-cell lymphoma and, 391 blastic plasmacytoid dendritic cell neoplasm and, 373 Burkitt-like lymphoma and, 321, 322 Burkitt lymphoma/leukemia and, 328 intravascular large B-cell lymphoma and, 316 natural killer cell lymphoma/leukemia and, 358, 359 nodular lymphocyte predominant Hodgkin lymphoma and, 422 nodular sclerosis Hodgkin lymphoma and, 428 post-transplant lymphoproliferative disorder and, 451, 455f, 457 subcutaneous panniculitis-like T-cell lymphoma and, 369 Erythrodysplasia, 134 Erythroid cells, 15–21 Erythroid leukemia, acute, 129–134 Erythroid sarcoma, 144 Essential thrombocythemia (ET), 56, 57–61 ETO gene, 95 Extramedullary (extraosseous) plasmacytoma, 231–232 Extranodal Hodgkin lymphoma, 443–450 Extranodal marginal zone B-cell lymphoma, 5, 28, 257 in differential diagnosis, 231–232, 334, 338 of the lung (pulmonary MALT), 241–245 multiple lymphomatous polyposis and, 289 of the salivary gland (salivary gland MALT), 246–251 of the stomach (gastric MALT), 234–240 F FAB system, see French-American-British (FAB) system Felty syndrome, 347 Flow cytometry acute megakaryoblastic leukemia, 137, 139 acute monoblastic/monocytic leukemia, 122, 127, 128 acute myeloblastic leukemia with maturation, 114 acute myeloid leukemia, 93, 109 acute myelomonocytic leukemia, 118, 119 anaplastic large cell lymphoma, 403 B-cell acute lymphoblastic leukemia, 154 Burkitt-like lymphoma, 321 Burkitt lymphoma/leukemia, 340, 341f

Index

chronic lymphocytic leukemia, 167f, 168f, 170 follicular lymphoma grade 3, 269 hairy cell leukemia, 212, 214f hepatosplenic T-cell lymphoma, 364 immunohistochemistry compared with, 22 leukemia cutis, 148 lymphoblastic lymphoma, 162 lymphoepithelioid lymphoma, 395 lymphoplasmacytic lymphoma, 195, 196f, 198, 201 mantle cell lymphoma, 275 mantle cell lymphoma, blastoid subtype, 282 multiple myeloma, 223 mycosis fungoides/S´ezary syndrome, 378 myelodysplastic syndrome, 75 myeloid sarcoma, 141, 144 natural killer cell lymphoma/leukemia, 357 nodal marginal zone B-cell lymphoma, 253 plasma cell leukemia, 226 prolymphocytic leukemia, 191, 193 pulmonary MALT, 243 refractory cytopenia with multilineage dysplasia, 83 Richter syndrome, 172, 174f Rosai-Dorfman disease, 492 small lymphocytic lymphoma, 179 small lymphocytic lymphoma, paraimmunoblastic variant, 186, 187 splenic marginal zone lymphoma, 205, 206f, 209 T-cell acute lymphoblastic leukemia, 159 thyoma, 469 Fluorescence in situ hybridization (FISH), 26, 27 acute promyelocytic leukemia, 104, 105 anaplastic large cell lymphoma, 401 atypical chronic myeloid leukemia, 70 chronic myelogenous leukemia, 38, 40, 43, 44 chronic neutrophilic leukemia, 48 multiple lymphomatous polyposis, 289 myelodysplastic/myeloproliferative neoplasms, 64 primary myelofibrosis 54 small lymphocytic lymphoma, 183 Follicular center cells, 4 Follicular hyperplasia, 265–266 Follicular lymphoma (FL), 5, 28, 176 case study, 259–266 in differential diagnosis, 203, 253 multiple lymphomatous polyposis and, 289 Follicular lymphoma (FL) grade 1, 272 Follicular lymphoma (FL) grade 2, 272 Follicular lymphoma (FL) grade 3, 267–272 French-American-British (FAB) system, 5, 14 acute erythroid leukemia, 134 acute megakaryoblastic leukemia, 139 acute monoblastic/monocytic leukemia, 124 acute myeloblastic leukemia with maturation, 115

517

acute myeloid leukemia, 110, 111t acute myelomonocytic leukemia, 119 acute myelomonocytic leukemia with eosinophila, 99 acute promyelocytic leukemia, 105 B-cell acute lymphoblastic leukemia, 155, 343 Fried egg pattern, 216 Fusion transcript, 27 G Gastric biopsy, 233f, 234f Gastric mucosa-associated lymphoid tissue (MALT) lymphoma, 234–240 GATA1 gene, 139 Gaucher cells, 13 Gaucher disease, 502–507 Gene expression profilin (GEP), 27, 278 Germinal center B cells, 4 Germinal center lymphoma, 5 Glucocerebrosidase, 507 Granular megakaryocytes, see Mature megakaryocytes Granulocyte colony-stimulating factor (G-CSF), 472–475 Granulocytic sarcoma, 144 Growth factor effect, 472–475 H Hairy cell leukemia, 28 case study, 211–217 in differential diagnosis, 209 Hassall corpuscles, 307f, 311, 467f, 468f, 470 Helicobacter pylori, 234, 239 Helmet cells, 16, 17f Hematopoietic cells, morphology of, 8–21 Hematopoietic tree, 3f Hemoglobinopathy, 17 Hemolytic anemia, 17, 204 Hemophagocytic syndrome (HPS), 369 Hepatosplenic T-cell lymphoma (HSTCL), 28, 361–365 HHV-8, see Human herpes virus type 8 (HHV-8) Histiocytes, 13 Histiocytic lymphoma, 460 Histiocytic necrotizing lymphadenitis, see Kikuchi-Fujimoto disease (KFD) Histiocytosis, see Langerhans cell histiocytosis (LCH); Rosai-Dorfman disease Histocompatibility antigens, 23 HIV, see AIDS/HIV Hodgkin and Reed-Sternberg (HRS) cells Castleman disease and, 485f, 486f extranodal Hodgkin lymphoma and, 449 lymphocyte-depleted Hodgkin lymphoma and, 442 lymphocyte-rich Hodgkin lymphoma and, 438

518

mixed cellularity Hodgkin lymphoma and, 433 nodular sclerosis Hodgkin lymphoma and, 427, 428 Hodgkin lymphoma, 5, 488 chronic lymphocytic leukemia and, 176 classificatio of, 6 diagnostic procedures for, 28 in differential diagnosis, 162, 260, 302, 306, 308, 312, 396, 397, 400, 408, 414, 460 extranodal, 443–450 lymphocyte-depleted, 427, 428, 433, 439–442, 449 lymphocyte-rich, 427, 433, 434–438 lymphoplasmacytic lymphoma and, 203 mixed cellularity, 427, 430–433, 449 nodular lymphocyte predominant (see Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)) nodular sclerosis, 423–429, 449 Honeycomb pattern, 211f Horn cells, 16, 17f Howell-Jolly bodies, 17, 18f HPRT gene, 49 Human herpes virus type 8 (HHV-8), 320, 321, 484, 486f, 488 Human immunodeficien y virus, see AIDS/HIV Human T-cell leukemia (HTLV-1), 351, 352 Hydroxyurea, 61 Hypergranular neutrophils, 10 Hypersegmented neutrophils, 10, 11f Hyperviscosity syndrome, 198 Hypogranular neutrophils, 11f Hypolobation, 10 Hyposegmentation, 10 I IgA gammopathy, 226 IgD gammopathy, 226, 227 IgE gammopathy, 226 IgG gammopathy, 200, 203, 218 IgH gene, 198, 223, 278, 295, 330 IgM gammopathy, 198 IgM myeloma, 198 Ileal biopsy, 331f, 332f, 333f, 334f, 335f Imatinib, 40, 44 Immature B cells, 4 Immature cell antigens, 22 Immature cell markers, 24 Immunoglobulin heavy chain (VH ) gene, 5, 26, 27 Burkitt lymphoma/leukemia and, 330, 338, 343 chronic lymphocytic leukemia and, 170 post-transplant lymphoproliferative disorder and, 452 Richter syndrome and, 173, 176 salivary gland MALT and, 249

Index

splenic marginal zone lymphoma and, 206 Immunoglobulin light chain gene, 24, 209, 330, 338, 343, 422 Immunohistochemistry acute erythroid leukemia, 131 acute megakaryoblastic leukemia, 137 acute monoblastic/monocytic leukemia, 122, 124, 127 acute promyelocytic leukemia, 106 anaplastic large cell lymphoma, 397, 403 anaplastic large cell lymphoma, lymphohistiocytic variant, 409 angioimmunoblastic T-cell lymphoma, 387 blastic plasmacytoid dendritic cell neoplasm, 372 Castleman disease, 485 chronic lymphocytic leukemia, 168 cutaneous anaplastic large-cell lymphoma, 381 diffuse large B-cell lymphoma, 292 diffuse large B-cell lymphoma, anaplastic variant, 297 extranodal Hodgkin lymphoma, 447 fl w cytometry compared with, 22 follicular lymphoma grade 3, 269 hairy cell leukemia, 213 hepatosplenic T-cell lymphoma, 364 intravascular large B-cell lymphoma, 316 Kikuchi-Fujimoto disease, 496 Langerhans cell histiocytosis, 460 leukemia cutis, 148 lymphoblastic lymphoma, 162 lymphocyte-depleted Hodgkin lymphoma, 440 lymphocyte-rich Hodgkin lymphoma, 436 lymphoepithelioid lymphoma, 395 lymphoplasmacytic lymphoma, 196, 201 mantle cell lymphoma, 275, 278 mantle cell lymphoma, blastoid subtype, 282 mixed cellularity Hodgkin lymphoma, 431 multiple lymphomatous polyposis, 286 multiple myeloma, 220, 223 mycosis fungoides/S´ezary syndrome, 378 myelodysplastic/myeloproliferative neoplasms, 64 myelodysplastic syndrome, 75 nodal marginal zone B-cell lymphoma, 253 nodular lymphocyte predominant Hodgkin lymphoma, 417 nodular sclerosis Hodgkin lymphoma, 424 plasmacytoma, 229 post-transplant lymphoproliferative disorder, 452 pulmonary MALT, 243, 245 Richter syndrome, 173 Rosai-Dorfman disease, 492 salivary gland MALT, 249, 251 small lymphocytic lymphoma, 179 subcutaneous panniculitis-like T-cell lymphoma, 368 T-cell/histiocyte-rich large B-cell lymphoma, 303

Index

T-cell large granular lymphocyte leukemia, 346 thyoma, 469 Interferon alpha, 216 Interleukin-6 syndrome, 488 Intravascular large B-cell lymphoma (IVBCL), 314–318 Iron deficien y anemia, 17 J JAK-2 gene, 312 JAK2 V617F mutation, 49, 55, 56, 59, 60, 72 Juvenile myelomonocytic leukemia (JMML), 66, 67 K Karyotyping, see Cytogenetic karyotyping Kidney specimens, 280f Kiel classification 6, 277 Kikuchi-Fujimoto disease (KFD), 373, 494–497 L Langerhans cell histiocytosis (LCH), 458–464 Large cell lymphoma, 8, 25, 28, 176, 186, 300, 318, 369, 400, 432, 449, 457 see Anaplastic large cell lympoma; Diffuse large B-cell lymphoma (DLBCL); Intravascular large B-cell lymphoma; Primary mediastinal large B-cell lymphoma (PMLBCL); T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) Large granular lymphocytes, 14 Lead poisoning, 17 Lenalidomide, 90 Lennert lymphoma, see Lymphoepithelioid lymphoma (LEL) Leukemia, 3–8 acute, 3, 5 acute basophilic, 5 acute erythroid, 129–134 acute monoblastic/monocytic (see Acute monoblastic/monocytic leukemia) acute myeloblastic (see Acute myeloblastic leukemia with maturation (AML-M2); Acute myeloblastic leukemia without maturation (AML-M1)) acute promyelocytic (see Acute promyelocytic leukemia (APL)) chronic, 3 chronic lymphocytic (see Chronic lymphocytic leukemia (CLL)) chronic neutrophilic, 39, 45–49 diagnosis of, 25 hairy cell (see Hairy cell leukemia) human T-cell, 351, 352

519

lymphoblastic (see Lymphoblastic leukemia) lymphoid (see Lymphoid leukemia) myelogenous (see Acute myelogenous leukemia (AML); Chronic myelogenous leukemia (CML)) myeloid (see Myeloid leukemia) myelomonocytic (see Acute myelomonocytic leukemia (AML-M4); Chronic myelomonocytic leukemia (CMML); Juvenile myelomonocytic leukemia (JMML)) natural killer cell, 354–360 plasma cell, 224–227 prolymphocytic (see Prolymphocytic leukemia (PLL)) T-cell large granular lymphocyte, 344–348 Leukemia cutis, 146–151 Leukemoid reaction, 48, 49, 474 Lineage-associated antigens, 22 Lipid storage diseases, 505 Liver biopsy Burkitt lymphoma/leukemia, 328f extranodal Hodgkin lymphoma, 443f hepatosplenic T-cell lymphoma, 361f, 363f mantle cell lymphoma, blastoic subtype, 280f natural killer cell lymphoma/leukemia, 354f Low-density lipoprotein (LDL) cholesterol, 501 Lung biopsy, 240f, 241f Lung cancer, 125 Lupus erythematosus (LE), 497 Lymph node biopsy, 28, 29–30 adult T-cell leukemia/lymphoma, 352f anaplastic large cell lymphoma, 401f, 402f anaplastic large cell lymphoma, lymphohistiocytic variant, 406f angioimmunoblastic T-cell lymphoma, 384f, 385f Burkitt lymphoma/leukemia, 322f, 323f Castleman disease, 480f diffuse large B-cell lymphoma, 289f, 291f, 293f diffuse large B-cell lymphoma, anaplastic variant, 295f follicular lymphoma, 258f, 261 hepatosplenic T-cell lymphoma, 362f Kikuchi-Fujimoto disease, 493f, 494f, 495f Langerhans cell histiocytosis, 457f lymphoblastic lymphoma, 161f, 162f, 163f lymphocyte-depleted Hodgkin lymphoma, 438f, 439f lymphocyte-rich Hodgkin lymphoma, 433f lymphoepithelioid lymphoma, 392f lymphoplasmacytic lymphoma, 193f, 195, 196f, 199f, 201f mantle cell lymphoma, 272f mantle cell lymphoma, blastoid subtype, 281f mixed cellularity Hodgkin lymphoma, 429f, 430f mycosis fungoides/S´ezary syndrome, 376f, 378 myeloid sarcoma, 141f, 142f Niemann-Pick disease, 497, 498f

520

nodal marginal zone B-cell lymphoma, 251f, 252f nodular lymphocyte predominant Hodgkin lymphoma, 414f, 415f nodular sclerosis Hodgkin lymphoma, 422f prolymphocytic leukemia, 189f Richter syndrome, 172f Rosai-Dorfman disease, 488f small lymphocytic lymphoma, 179f, 180 small lymphocytic lymphoma, paraimmunoblastic variant, 186 T-cell/histiocyte-rich large B-cell lymphoma, 300f Lymphoblastic leukemia, 226 See also Acute lymphoblastic leukemia (ALL) Lymphoblastic lymphoma (LBL), 144, 155, 161–165 Lymphoblasts, 13–14 Lymphocyte-depleted Hodgkin lymphoma, 427, 428, 433, 439–442, 449 Lymphocyte-rich Hodgkin lymphoma, 427, 433, 434–438 Lymphocytes, 14–15 Lymphocytic leukemia, chronic, see Chronic lymphocytic leukemia (CLL) Lymphoepithelioid lymphoma (LEL), 393–396, 414 Lymphohistiocytic variant of anaplastic large cell lymphoma (LH-ALCL), 407–414 Lymphoid cells, 3, 13–15 Lymphoid leukemia, 3 acute, 341 chronic, 346 Lymphoid neoplasms, WHO classification 7–8t Lymphoma, 3–8 acute megakaryoblastic, 135–140 B-cell (see B-cell lymphoma) body cavity, 321, 322 Burkitt (see Burkitt lymphoma/leukemia) classificatio of, 5–8 common chromosomal translocations in, 26t cutaneous (see Cutaneous B-cell lymphoma; Cutaneous lymphoma; Cutaneous T-cell lymphoma) diagnosis of, 25 follicular (see Follicular lymphoma (FL)) germinal center, 5 histiocytic, 460 Hodgkin (see Hodgkin lymphoma) large-cell (see Large cell lymphoma) lymphoblastic, 144, 155, 161–165 lymphoepithelioid, 393–396, 414 lymphoplasmacytic (see Lymphoplasmacytic lymphoma (LPL)) mantle cell (see Mantle cell lymphoma (MCL)) marginal zone (see Marginal zone B-cell lymphoma; Marginal zone lymphoma) mediastinal gray zone, 312

Index

mucosa-associated lymphoid tissue (see Mucosa-associated lymphoid tissue (MALT) lymphoma) natural killer cell, 354–360 non-Hodgkin (see Non-Hodgkin lymphoma) parotid, 249 post-germinal center, 5 pre-germinal center, 5 primary effusion, 321, 322 pyrothorax-associated, 321, 322 small lymphocytic (see Small lymphocytic lymphoma (SLL)) splenic red pulp, 216 T-cell (see T-cell lymphoma) treatment-related, 141 Lymphomatoid papulosis (LyP), 384 Lymphoplasmacytic lymphoma (LPL), 28, 222 case studies, 194–199, 200–203 classificatio of, 5 M Macrophages, 13 MAL gene, 312 Malignant lymphoid aggregates, 29 MALT1 gene, 239 MALT lymphoma, see Mucosa-associated lymphoid tissue (MALT) lymphoma Mann-Berard cell-counting method, 265 Mantle cell lymphoma (MCL), 5, 28, 176, 183, 193 blastoid subtype, 279–285 case study, 273–278 in differential diagnosis, 203, 209, 239, 253, 258 multiple lymphomatous polyposis and, 289 Mantle cells, 4 MAPK1 gene, 272 Marginal zone B-cell lymphoma, 222 See also Extranodal marginal zone B-cell lymphoma; Nodal marginal zone B-cell lymphoma (NMZBL) Marginal zone B cells, 4 Marginal zone lymphoma, 198 See also Splenic marginal zone lymphoma (SMZL) Mast cells, 11, 12f Mature B cells, 4 Mature megakaryocytes, 19, 20f Mediastinal gray zone lymphoma, 312 Medullary thymocytes, 4 Megakaryoblastic lymphoma, acute, 135–140 Megakaryoblasts, 19, 20f Megakaryocytes, 19 Megakaryocyticsarcoma, 144 Megaloblastic anemia, 10, 17 Megaloblastic change, 17

Index

Megaloblastoid normoblasts, 17, 19f Melanoma, 479 Melphalan, 227 Memory B cells, 4 Merkel cell carcinoma (MCC), 476–480 Metamyelocytes, 9, 10f Microangiopathic hemolytic anemia, 17 Minimal residual disease (MRD), detection of, 25, 26 Mixed cellularity Hodgkin lymphoma (MCHL), 427, 430–433, 449 MLL gene, 119 Molecular biology techniques, 26–27 Monoblastic leukemia, acute, see Acute monoblastic/monocytic leukemia Monoblastic sarcoma, 151 Monoblasts, 11, 12f, 13 Monoclonal antibodies, immunophenotyping with, 22–24, 25t Monoclonal B-lymphocytosis, 170 Monoclonal gammopathy, 231, 232 Monoclonal gammopathy of undetermined significanc (MGUS), 222 Monocytes, 11, 12f Monocytic cells, 11–13 Monocytic leukemia, acute, see Acute monoblastic/monocytic leukemia Monocytic sarcoma, 144 Monocytoid B cells, 4 Monolobated neutrophils, 10, 11f Monosomy, 24 Mott cells, 15 MOZ gene, 128 Mucosa-associated lymphoid tissue (MALT) lymphoma, 4, 257 gastric, 234–240 pulmonary, 241–245 salivary gland, 246–251 Multiple lymphomatous polyposis (MLP), 286–289 Multiple myeloma, 218–223 MUM1 gene, 272, 295 MYC gene, 312 Mycosis fungoides/S´ezary syndrome, 28, 457 case study, 375–380 in differential diagnosis, 351, 352 Myeloblastic leukemia, acute, see Acute myeloblastic leukemia with maturation (AML-M2); Acute myeloblastic leukemia without maturation (AML-M1) Myeloblasts, 8–9 Myelocytes, 9, 10f Myelodysplastic/myeloproliferative neoplasms (MDS/MPN), 25, 62–67, 72 Myelodysplastic syndrome (MDS), 8, 10, 17, 19, 25, 140

521

case history, 73–79 in differential diagnosis, 83, 89 leukemia cutis and, 151 myeloid sarcoma and, 144 Myelodysplastic syndrome, unclassifiabl (MDS-U), 78 Myelofibrosis 17, 140 Myelogenous leukemia, see Acute myelogenous leukemia (AML); Chronic myelogenous leukemia (CML) Myeloid cells, 3, 8–11 Myeloid leukemia, 3 See also Acute myeloid leukemia (AML); Chronic myeloid leukemia (CML) Myeloid proliferations related to Down syndrome, 5 Myeloid sarcoma (MS), 5, 141–145, 151 Myeloid to erythroid (M:E) ratio, 28 atypical chronic myeloid leukemia, 68 chronic myelogenous leukemia, 35, 39, 41 chronic neutrophilic leukemia, 45 myelodysplastic/myeloproliferative neoplasms, 62 primary myelofibrosis 50 T-cell large granular lymphocyte leukemia, 344 Myeloma, 227 cutaneous, 229 IgM, 198 multiple, 218–223 plasma cell, 5, 28, 195, 220, 222, 224, 228 Myelomonocytic leukemia, see Acute myelomonocytic leukemia (AML-M4); Chronic myelomonocytic leukemia (CMML); Juvenile myelomonocytic leukemia (JMML) Myeloproliferative neoplasms, see Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) Myoepithelial sialadenitis (MESA), 249, 250, 251 N Natural killer (NK) cell lymphoma/leukemia (NKL), 354–360 Natural killer (NK) cells, 4 NEK2 gene, 272 Neoplasms blastic plasmacytoid dendritic cell, 5, 370–374 lymphoid, 7–8t myelodysplastic/myeloproliferative, 25, 62–67, 72 precursor lymphoid, 5t secondary, 26 Neutrophilic chronic myelogenous leukemia (CML), 39 Neutrophilic leukemia, chronic, see Chronic neutrophilic leukemia (CNL) Niemann-Pick cells, 13 Niemann-Pick disease, 498–501

522

Nodal marginal zone B-cell lymphoma (NMZBL), 5, 28, 216 case study, 252–258 in differential diagnosis, 203 Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), 306, 449 case study, 415–422 in differential diagnosis, 438 Nodular sclerosis Hodgkin lymphoma, 423–429, 449 Non-Hodgkin lymphoma, 181 classificatio of, 5–6 diagnostic procedures for, 28 in differential diagnosis, 162, 260, 265, 275, 302, 308, 324, 397, 408, 417, 424, 431, 440, 446, 477, 491 NOTCH signaling, 160 Notch 1 transcription factor, 428 NPC1 gene, 501 NPC2 gene, 501 NPM-ALK fusion protein, 300, 400–401 Nuclear-cytoplasmic dyssynchrony, 17 Nuclear factor-KB, 428 O Octamer-binding transcription factor 2 (OCT2), 422, 427, 438, 449 Orthochromic normoblasts (erythroblasts), 16 OTT-MAL transcript, 139 Ovalocytes (elliptocytes), 17, 18f P Pappenheimer bodies, 17, 18f Parafollicular B cells, 4 Paraimmunoblastic variant of small lymphocytic lymphoma (SLL), 184–187 Parotid lymphoma, 249 PAX-5 gene, 198 PDGFRA gene rearrangement, 66, 72 PDGFRB gene rearrangement, 66, 72 Pelger-Hu¨et anomaly, 10 Peripheral blood examination, 28 acute erythroid leukemia, 129 acute monoblastic/monocytic leukemia, 120f, 125 acute myeloblastic leukemia with maturation, 112f acute myeloid leukemia, 91f, 107f acute myelomonocytic leukemia, 116f acute myelomonocytic leukemia with eosinophilia, 96f acute promyelocytic leukemia, 101f adult T-cell leukemia/lymphoma, 348f atypical chronic myeloid leukemia, 68f B-cell acute lymphoblastic leukemia, 152f Burkitt lymphoma, 338f

Index

chronic lymphocytic leukemia, 166f, 167f chronic myelogenous leukemia, 35f, 41f chronic neutrophilic leukemia, 45f, 46f essential thrombocythemia, 57f extranodal Hodgkin lymphoma, 443 follicular lymphoma, 259f growth factor effect, 471f, 472f hairy cell leukemia, 211 mantle cell lymphoma, blastoid subtype, 278f, 282 multiple myeloma, 218 mycosis fungoides/S´ezary syndrome, 375f, 378 myelodysplastic/myeloproliferative neoplasms, 62f myelodysplastic syndrome, 73 natural killer cell lymphoma/leukemia, 355f plasma cell leukemia, 223f primary myelofibrosis 50f, 51f prolymphocytic leukemia, 187f 5q-syndrome, 85f, 86f refractory cytopenia with multilineage dysplasia, 80f small lymphocytic lymphoma, paraimmunoblastic variant, 184, 186 T-cell acute lymphoblastic leukemia, 157f T-cell large granular lymphocyte leukemia, 343f Peripheral T-cell lymphoma, 28 P21 gene, 187 P53 gene, 139, 170, 176, 187, 285 Philadelphia chromosome, 27, 39, 44, 49, 55, 66, 72, 134 P16INK4A gene, 187, 285 Plasmablasts, 15 Plasma cell leukemia (PLC), 224–227 Plasma cell myeloma, 5, 28, 195, 220, 222, 224, 228 Plasma cells, 4–5, 15 Plasmacytoid lymphocytes, 14 Plasmacytoma, 228–233 Platelet-derived growth factor B (PDGF-B), 139 Platelets, 19 PML-RARα fusion product, 104, 105 POEMS syndrome, 488 Poikilocytosis, 16, 17f Polychromatophilic normoblasts (erythroblasts), 16 Polychromatophilic red cells, 17, 18f Polycythemia vera (PV), 56, 60 Polymerase chain reaction (PCR), 26, 27 adult T-cell leukemia/lymphoma, 352 anaplastic large cell lymphoma, 401 multiple lymphomatous polyposis, 289 mycosis fungoides/S´ezary syndrome, 380 Polyploidy, 24 Popcorn cells, 415f, 416f, 417f, 418f, 419f, 420f, 421, 434f, 445f Post-germinal center lymphoma, 5 Post-transplant lymphoproliferative disorder (PTLD), 451–457

Index

PRAD1 gene, 278 Pre-B cells, 4 Precursor lymphoid neoplasms, WHO classification 5t Prednisone, 227 Pre-germinal center lymphoma, 5 Primary effusion lymphoma, 321, 322 Primary mediastinal large B-cell lymphoma (PMLBCL), 307–313 Primary myelofibrosi (PMF), 50–56 Pro-B cells, 4 Prognosis, prediction of, 25 Proliferation-associated antigens, 23 Prolymphocytes, 14 Prolymphocytic leukemia (PLL), 170, 187, 188–193 Prolymphocytoid transformation, 186, 192 Promegakaryocytes, 19, 20f Promonocytes, 11, 12f, 13 Promyelocytes, 9, 10f Promyelocytic leukemia, acute, see Acute promyelocytic leukemia (APL) Pronormoblasts (proerythroblasts), 15–16 Prothymocytes, 4 Pseudo-Chediak-Higashi anomaly, 92f, 95 Pseudo-Pelger-Hu¨et cells, 10, 11f, 81f, 84 Pseudothrombopenia, 19 Pulmonary carcinoma, 243, 245 Pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma, 241–245 Purine analogues, 216 Pyrothorax-associated lymphoma (PAL), 321, 322 Pyruvate kinase deficien y, 17 Q 5q-syndrome, 78, 79, 85–90 Quantitative (real-time) polymerase chain reaction (PCR), 26 R Rai staging system, 170, 171t Raynaud phenomenon, 198, 203 Reactive/activated lymphocytes, 14 Reactive lymphadenopathy, 387, 403, 485, 491, 496 Reactive lymphocytosis, 59 REAL, see Revised European-American Classificatio of Lymphoid Neoplasms (REAL) Red cell morphology, abnormal, 16–17 Reed-Sternberg cells adult T-cell leukemia/lymphoma and, 352 anaplastic large cell lymphoma, lymphohistiocytic variant and, 407f angioimmunoblastic T-cell lymphoma and, 391

523

Castleman disease and, 483f, 488 extranodal Hodgkin lymphoma and, 444f, 449 Langerhans cell histiocytosis and, 458f, 463 lymphocyte-rich Hodgkin lymphoma and, 434f, 435f lymphoepithelioid lymphoma and, 396 nodular lymphocyte predominant Hodgkin lymphoma and, 422 nodular sclerosis Hodgkin lymphoma and, 423f, 427 post-transplant lymphoproliferative disorder and, 457 primary mediastinal large B-cell lymphoma and, 312 T-cell/histiocyte-rich large B-cell lymphoma and, 301f, 306 See also Hodgkin and Reed-Sternberg (HRS) cells Refractory anemia with excess blasts (RAEB), 78–79, 84, 134 Refractory anemia with ring sideroblasts (RARS), 77–78 Refractory cytopenia with multilineage dysplasia (RCMD), 78, 80–84 Refractory cytopenia with unilineage dysplasia (RCUD), 77, 78 Relapsed neoplasms, 26 REL gene, 312 Reticulin fibrosi atypical chronic myeloid leukemia and, 70, 71f, 72 chronic neutrophilic leukemia and, 48 hairy cell leukemia and, 214f, 216 primary myelofibrosi and, 52f sarcoidosis and, 511f Reverse transcriptase polymerase chain reaction (RT-PCR), 26, 40, 99, 352 Revised European-American Classificatio of Lymphoid Neoplasms (REAL), 6, 181, 365, 400 Richter syndrome/transformation, 170, 183, 187 case study, 172–177 in differential diagnosis, 282 Ring sideroblasts, 74, 76f, 77–78, 84 Rosai-Dorfman disease, 489–493 Rouleaux formation, 217f, 223 RUNX1-mediated transcription, 128 Russell bodies, 15, 193f, 195, 218f, 222 S Salivary gland mucosa-associated lymphoid tissue (MALT) lymphoma, 246–251 Salivary gland tumors, 292 Sarcoidosis, 508–513 Sarcoma erythroid, 144 granulocytic, 144 megakaryocytic, 144 monoblastic, 151 monocytic, 144

524

myeloid, 5, 141–145, 151 Schistocytes, 16, 17f Secondary neoplasms, 26 Segmented neutrophils, 10 S´ezary syndrome, see Mycosis fungoides/S´ezary syndrome Sickle cells, 16, 17, 18f Sinus histiocytosis with massive lymphadenopathy (SHML), see Rosai-Dorfman disease Sj¨ogren syndrome, 250 Skin biopsy adult T-cell leukemia/lymphoma, 349f blastic plasmacytoid dendritic cell neoplasm, 369f, 372 Burkitt-like lymphoma, 328f cutaneous anaplastic large-cell lymphoma, 381 leukemia cutis, 146f, 149f Merkel cell carcinoma, 475f mycosis fungoides/S´ezary syndrome, 374f, 375f, 378 plasmacytoma, 227f, 228f subcutaneous panniculitis-like T-cell lymphoma, 365f Small cell carcinoma, 477, 479–480 Small lymphocytic lymphoma (SLL), 170, 176 case study, 178–183 in differential diagnosis, 203, 205, 209, 239, 253, 278 paraimmunoblastic variant, 184–187 See also Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Solitary bone plasmacytoma, 231 Southern blotting, 26, 347, 352, 380, 401 Spherocytes, 17, 18f Splenectomy specimens, 30 atypical chronic myeloid leukemia, 70f extranodal Hodgkin lymphoma, 442f Gaucher disease, 501f, 502f, 504f hairy cell leukemia, 216f hepatosplenic T-cell lymphoma, 360f mantle cell lymphoma, 274f, 275f natural killer cell lymphoma/leukemia, 354f primary myelofibrosis 53f prolymphocytic leukemia, 191f small lymphocytic lymphoma, 181f splenic marginal zone lymphoma, 204f, 206, 209 Splenic marginal zone lymphoma (SMZL), 5, 28, 216 case study, 204–210 in differential diagnosis, 213, 278, 364 Splenic red pulp lymphoma with numerous basophilic villous lymphocytes, 216 Sporadic Burkitt lymphoma/leukemia, 328, 338 Spur cells, 18f Starry sky histologic pattern, 144, 161f, 165, 322f, 328, 332, 338, 497 Stat3 transcription factor, 428

Index

Stomatocytes, 17, 18f Subcutaneous panniculitis-like T-cell lymphoma (SPTCL), 366–369 Surface immunoglobulin, loss of in a B-cell population, 24 Systemic anaplastic large-cell lymphoma, 384 T Tacrolimus, 457 Target cells, 17, 18f T-cell acute lymphoblastic leukemia (T-ALL), 157–160, 165 T-cell clonality, determination by TCR-V␤ antibodies, 24 T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL), 301–306, 414 T-cell large granular lymphocyte (T-LGL) leukemia, 344–348 T-cell lymphoma adult, 349–353 angioimmunoblastic, 385–392 cutaneous, 351, 368, 378 hepatosplenic, 28, 361–365 peripheral, 28 subcutaneous panniculitis-like, 366–369 T-cell prolymphocytic leukemia (PLL), 192, 193 TCR α␤ gene rearrangement, 165, 357 TCR ␥␦ gene rearrangement, 165, 357, 368, 369 TCR gene rearrangement, 346, 347, 359, 380 TCR-V␤ antibodies, T-cell clonality determined by, 24 Teardrop cells, 16, 17, 18f Testicular biopsy, 329f Tetraploidy, 24 Thalassemia major, 17 Thrombocytopenia, 19, 21f Thrombocytosis, 19, 20f Thymocytes, 4 Thyoma, 467–471 T lymphocytes, development of, 4 Tonsillar biopsy, 266f Toxic granulation, 10, 11f Toxic vacuolation, 10 TP53 gene, 193 Transcriptional deregulation, 27 Treatment-related lymphoma, 141 Triploidy, 24 Trisomy, 24 Trisomy 3, 227, 257, 396 Trisomy 7, 227, 257 Trisomy 8, 43, 139 Trisomy 11, 227 Trisomy 12, 170, 183, 187 Trisomy 15, 227 Trisomy 17, 227 Trisomy 18, 257

Index

525

cutaneous anaplastic large-cell lymphoma, 384 diffuse large B-cell lymphoma, 294 essential thrombocytopenia, 60 follicular lymphoma, 265 hepatosplenic T-cell lymphoma, 365 IgG gammopathy, 203 lymphoblastic lymphoma, 165 lymphoplasmacytic lymphoma, 198 mantle cell lymphoma, 277 mucosa-associated lymphoid tissue lymphoma, 239 multiple myeloma, 222 myelodysplastic/myeloproliferative neoplasms, 66 natural killer cell lymphoma/leukemia, 358 nodular lymphocyte predominant Hodgkin lymphoma, 422 post-transplant lymphoproliferative disorder, 457 primary effusion lymphoma, 322 primary myelofibrosis 55, 56 5q-syndrome, 90 refractory cytopenia with multilineage dysplasia, 78 small lymphocytic lymphoma, 181 T-cell acute lymphoblastic leukemia, 160 T-cell large granular lymphocyte leukemia, 347 thyoma, 470

Trisomy 21, 139 Tumor necrosis factor receptor (TNFR) family, 428 U Uremia, 17 V Vascular arborization, 384f, 391 VH gene, see Immunoglobulin heavy chain (VH ) gene W Waldenstr¨om macroglobulinemia, 28, 195, 198, 203 Working Formulation, 6 World Health Organization (WHO) system, 5t, 6t, 7–8t, 14 acute erythroid leukemia, 134 acute megakaryoblastic leukemia, 139 acute monoblastic/monocytic leukemia, 124 acute myeloblastic leukemia with maturation, 115 acute myeloid leukemia, 95 acute myelomonocytic leukemia, 119 anaplastic large cell lymphoma, 400 B-cell acute lymphoblastic leukemia, 155, 156, 343 blastic plasmacytoid dendritic cell neoplasm, 373 Burkitt lymphoma/leukemia, 328, 330, 338 chronic myelogenous leukemia, 39, 44 chronic neutrophilic leukemia, 49

Z ZAP-70, 170, 176 Zebra bodies, 501