Atlas of differential diagnosis in neoplastic hematopathology [Third edition] 9781482212259, 1482212250, 9781482212211, 1482212218

Table of contents :
Front Cover......Page 1
Dedication......Page 6
Contents......Page 8
Preface......Page 12
Abbreviations......Page 14
Chapter 1: Lymph Node......Page 18
Chapter 2: Bone Marrow......Page 38
Chapter 3: Flow Cytometry......Page 66
Chapter 4: Immunohistochemistry......Page 122
Chapter 5: Immunophenotypic Markers: Differential Diagnosis......Page 136
Chapter 6: Cytogenetics......Page 172
Chapter 7: Fluorescence In Situ Hybridization and Polymerase Chain Reaction......Page 206
Chapter 8: Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma and B-Cell Prolymphocytic Leukemia......Page 228
Chapter 9: Mantle Cell Lymphoma......Page 252
Chapter 10: Marginal Zone Lymphoma......Page 268
Chapter 11: Follicular Lymphoma......Page 284
Chapter 12: Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia......Page 314
Chapter 13: Diffuse Large B-Cell Lymphoma and its Variants......Page 324
Chapter 14: Hairy Cell Leukemia......Page 352
Chapter 15: Burkitt Lymphoma and B-Cell Lymphoma, Unclassifiable (with Features Intermediate between Burkitt Lymphoma and DLBCL)......Page 362
Chapter 16: Plasma Cell Neoplasms......Page 376
Chapter 17: B-Cell Lymphomas with Plasmablastic Morphology and/or EBV Expression......Page 400
Chapter 18: Adult T-Cell Leukemia/Lymphoma......Page 412
Chapter 19: T-Cell Prolymphocytic Leukemia and T-Cell Large Granular Lymphocyte Leukemia......Page 418
Chapter 20: Peripheral T-Cell Lymphoma......Page 434
Chapter 21: Angioimmunoblastic T-Cell Lymphoma......Page 450
Chapter 22: Anaplastic Large Cell Lymphoma......Page 464
Chapter 23: Extranodal Natural Killer/T-Cell Lymphoma, Nasal Type......Page 482
Chapter 24: Classical Hodgkin Lymphoma......Page 490
Chapter 25: Nodular Lymphocyte Predominant Hodgkin Lymphoma......Page 508
Chapter 26: Introduction to Myeloproliferative Neoplasms......Page 520
Chapter 27: Chronic Myeloid Leukemia......Page 534
Chapter 28: Polycythemia Vera......Page 550
Chapter 29: Essential Thrombocythemia......Page 556
Chapter 30: Primary Myelofibrosis......Page 562
Chapter 31: Other Myeloproliferative Neoplasms......Page 568
Chapter 32: Myelodysplastic Syndromes......Page 582
Chapter 33: Mixed Myelodysplastic–Myeloproliferative Neoplasms......Page 614
Chapter 34: Acute Myeloid Leukemia: An Introduction......Page 628
Chapter 35: Acute Myeloid Leukemia without Specific Genetic Changes......Page 662
Chapter 36: Acute Myeloid Leukemia with Recurrent Genetic Abnormalities......Page 692
Chapter 37: B-Lymphoblastic Leukemia/Lymphoma......Page 712
Chapter 38: T-Lymphoblastic Leukemia/Lymphoma......Page 732
Chapter 39: Other Tumors......Page 746
Chapter 40: Body Cavities......Page 768
Chapter 41: Liver......Page 776
Chapter 42: Lung and Mediastinum......Page 788
Chapter 43: Salivary Glands......Page 800
Chapter 44: Skin......Page 806
Chapter 45: Spleen......Page 848
Chapter 46: Stomach and Gastrointestinal Tract......Page 870
Chapter 47: Testis......Page 898

Citation preview

Gorczyca

C li ni ca l Me di c i ne

Atlas of Differential Diagnosis in Neoplastic Hematopathology Management of tumor patients now relies on new individualized approaches to treatment, requiring extensive knowledge of the molecular makeup of tumors. Updated and expanded, the third edition of Atlas of Differential Diagnosis in Neoplastic Hematopathology examines not only the differential diagnosis but also the detailed morphologic, immunophenotypic, and especially genetic characteristics of the majority of hematolymphoid malignancies. Featuring a new structure and including new chapters, the third edition updates all content and presents considerable expansion on many topics, including: • • • • • • • •

Metaphase cytogenetic and FISH Flow cytometry (overview and detailed analysis of specific tumors) Acute myeloid leukemia and new classification schemes MDS, AML, and B- and T-cell lymphoproliferations Abnormal patterns in the lymph node and bone marrow with detail differential diagnosis based on histologic features and cellular composition Detailed differential diagnosis based on the expression of broad list of antigenic markers (flow cytometry and immunohistochemistry) Extranodal lymphomas Diagnosis of MDS and myeloproliferative neoplasm and their differential diagnosis based on the morphologic, flow cytometric, and chromosomal features

The book also provides expanded differential diagnosis of the most common as well as most difficult and rare entities, including morphologic, immunophenotypic, and karyotypic/molecular features. This edition includes updated algorithms for most common diagnoses as well as several new algorithms. The majority of figures have been revised and are in full color.

About the Author:

Dr. Wojciech Gorczyca is a noted authority in clinical flow cytometry and hematopathology. He earned his medical degree and doctorate in pathology from Pomeranian Medical University in Szczecin, Poland. A well-respected lecturer and author, he has published numerous peer-reviewed journal articles and six textbooks.

K21710

Atlas of Differential Diagnosis in Neoplastic Hematopathology

Third Edition

Third Edition

Atlas of

Differential Diagnosis in Neoplastic Hematopathology Third Edition

Wojciech Gorczyca

Atlas of

Differential Diagnosis in Neoplastic Hematopathology Third Edition

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Atlas of

Differential Diagnosis in Neoplastic Hematopathology Third Edition Wojciech Gorczyca

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2014 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20131217 International Standard Book Number-13: 978-1-4822-1225-9 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical, scientific or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verified. The reader is strongly urged to consult the drug companies’ printed instructions, and their websites, before administering any of the drugs recommended in this book. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately. The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Dedication To my wife, Elżbieta, and my daughters, Marta, Ewa, and Małgorzata.

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Contents Preface xi Abbreviations xiii

SECTION I

General Histologic and Cytologic Features

Chapter 1

Lymph Node  1

Chapter 2

Bone Marrow 21

SECTION II

Immunophenotyping

Chapter 3

Flow Cytometry  49

Chapter 4

Immunohistochemistry  105

Chapter 5

Immunophenotypic Markers: Differential Diagnosis 119

SECTION III Introduction to Cytogenetics, FISH, and Molecular Testing Chapter 6

Cytogenetics  155

Chapter 7

Fluorescence In Situ Hybridization and Polymerase Chain Reaction 189

SECTION IV

Mature B-Cell Neoplasms

Chapter 8

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma and B-Cell Prolymphocytic Leukemia 211

Chapter 9

Mantle Cell Lymphoma 235

Chapter 10 Marginal Zone Lymphoma  251 Chapter 11 Follicular Lymphoma  267 Chapter 12 Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia  297 Chapter 13 Diffuse Large B-Cell Lymphoma and its Variants  307 Chapter 14 Hairy Cell Leukemia  335

vii

viii

Contents

Chapter 15 Burkitt Lymphoma and B-Cell Lymphoma, Unclassifiable (with Features Intermediate between Burkitt Lymphoma and DLBCL)  345 Chapter 16 Plasma Cell Neoplasms  359 Chapter 17 B-Cell Lymphomas with Plasmablastic Morphology and/or EBV Expression 383

SECTION V

Mature T-Cell Neoplasms

Chapter 18 Adult T-Cell Leukemia/Lymphoma  395 Chapter 19 T-Cell Prolymphocytic Leukemia and T-Cell Large Granular Lymphocyte Leukemia 401 Chapter 20 Peripheral T-Cell Lymphoma 417 Chapter 21 Angioimmunoblastic T-Cell Lymphoma 433 Chapter 22 Anaplastic Large Cell Lymphoma  447 Chapter 23 Extranodal Natural Killer/T- Cell Lymphoma, Nasal Type  465

SECTION VI

Hodgkin Lymphoma

Chapter 24 Classical Hodgkin Lymphoma  473 Chapter 25 Nodular Lymphocyte Predominant Hodgkin Lymphoma  491

SECTION VII Myeloproliferative Neoplasms Chapter 26 Introduction to Myeloproliferative Neoplasms  503 Chapter 27 Chronic Myeloid Leukemia517 Chapter 28 Polycythemia Vera 533 Chapter 29 Essential Thrombocythemia  539 Chapter 30 Primary Myelofibrosis  545 Chapter 31 Other Myeloproliferative Neoplasms 551

ix

Contents

SECTION VIII Myelodysplastic Syndromes and Mixed Myelodysplastic/ Myeloproliferative Neoplasms Chapter 32 Myelodysplastic Syndromes  565 Chapter 33 Mixed Myelodysplastic–Myeloproliferative Neoplasms  597

SECTION IX Acute Myeloid Leukemia: An Introduction Chapter 34 Acute Myeloid Leukemia: An Introduction 611 Chapter 35 Acute Myeloid Leukemia without Specific Genetic Changes  645 Chapter 36 Acute Myeloid Leukemia with Recurrent Genetic Abnormalities 675

SECTION X Acute Lymphoblastic Leukemia and Other Hematopoietic Tumors Chapter 37 B-Lymphoblastic Leukemia/Lymphoma  695 Chapter 38 T-Lymphoblastic Leukemia/Lymphoma  715 Chapter 39 Other Tumors 729

SECTION XI

Extranodal Lymphomas and Their Differential Diagnosis

Chapter 40 Body Cavities 751 Chapter 41 Liver  759 Chapter 42 Lung and Mediastinum  771 Chapter 43 Salivary Glands  783 Chapter 44 Skin  789 Chapter 45 Spleen  831 Chapter 46 Stomach and Gastrointestinal Tract  853 Chapter 47 Testis  881 Index  891

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Preface The field of neoplastic hematology is changing rapidly and the management of patients relies more than ever on morphologic, immunophenotypic, karyotypic, and genetic characteristics We are witnessing the emergence of truly individualized approaches to treatment; therefore, morphologic and even extensive immunophenotypic analyses of tumors are not sufficient anymore, as diagnosis, prognosis, and treatment strategies depend heavily on the molecular makeup of tumors Acute myeloid leukemias (AMLs) are now classified based on specific chromosomal changes and mutational status of an expanding list of genes The prognosis of patients with AML cannot be established by a single methodology such as metaphase cytogenetics or even evaluation of the mutation of one gene [eg, concomitant KIT mutations occurring in the context of core-binding factor-positive AML confer a negative prognosis and NPM1+ AML has a good prognosis only if associated with wild-type fms-related tyrosine kinase 3 gene (FLT3)] The list of mature B- and T-cell lymphoproliferations, both nodal and extranodal, continues to expand and includes (among others) “gray zone” lymphomas (double-hit lymphomas), T-cell lymphomas with a follicular T-helper phenotype, and numerous morphologic and immunophenotypic variants of diffuse large B-cell lymphoma Myeloproliferative neoplasms are classified based on the status of JAK2, BCR– ABL1, KIT, and PDGFRA, with morphologic analysis of the bone marrow still playing a crucial role Flow cytometric analysis with 6-, 8-, or even 10-color methodologies helps in the diagnosis and subclassification of acute leukemias as well

as B- and T-cell lymphomas, but is also expanding its role in patients with myelodysplastic syndrome (MDS) or myeloproliferative neoplasms The role of flow cytometry in the analysis of blood, effusions, cerebrospinal fluid (CSF), and limited samples cannot be overemphasized This updated and expanded third edition focuses, as before, on the differential diagnosis, but in addition describes the detailed morphologic, immunophenotypic, and especially genetic characteristics of the majority of hematolymphoid malignancies To ease the navigation through the text and almost 900 figures, the atlas is divided into 11 sections and 47 chapters Each chapter ends with the most relevant and updated references As with the prior editions, this book would not be possible without help of my colleagues and co-workers from BioReference Laboratories (Elmwood Park, New Jersey) and CSI Laboratories (Alpharetta, Georgia) Special thanks for the whole flow cytometry and FISH departments from CSI Laboratories I would like to thank Claire Bonnett and the whole editorial crew at CRC Press/Taylor & Francis Group for their invaluable help and support Wojciech Gorczyca Bioreference Laboratories, Elmwood Park, New Jersey Regional Cancer Care Associates (RCCA), Hackensack, New Jersey

xi

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Abbreviations ABL1 aCML AEL AITL ALC ALCL ALIP ALK ALL AML AMML ANC AP APL ATLL ATRA BCL1 BCL2 B-CLL BCLU BCR BL BM BP BPDCN B-PLL BRAF CBF CCyR CD CEL cen CGH CLL CML CMML CMR CNL CP CyR del DH DLBCL EATL EBER EBV EMS EMT ENKTL ET

Abelson leukemia homolog 1 gene atypical chronic myeloid leukemia (BCR–ABL1−) acute erythroid leukemia angioimmunoblastic T-cell lymphoma absolute lymphocyte count anaplastic large cell lymphoma abnormal localization of immature precursors anaplastic lymphoma kinase acute lymphoblastic leukemia acute myeloid leukemia acute myelomonocytic leukemia absolute neutrophil count accelerated phase acute promyelocytic leukemia adult T-cell lymphoma/leukemia all-trans retinoic acid B-cell lymphoma 1 (cyclin D1) protein encoded by CCND1 gene B-cell lymphoma 2 apoptosis regulating protein encoded by BCL2 gene B-chronic lymphocytic leukemia large B-cell lymphoma, unclassifiable with features intermediate between DLBCL and BL breakpoint cluster region gene Burkitt lymphoma bone marrow blast phase blastic plasmacytoid dendritic cell neoplasm B-cell prolymphocytic leukemia v-raf murine sarcoma viral oncogene homolog B1 core-binding factor complete cytogenetic response cluster designation chronic eosinophilic leukemia centromere comparative genomic hybridization chronic lymphocytic leukemia chronic myeloid leukemia (BCR–ABL1+) chronic myelomonocytic leukemia complete molecular response chronic neutrophilic leukemia chronic phase cytogenetic response deletion (loss of part of chromosome) double hit diffuse large B-cell lymphoma enteropathy-associated T-cell lymphoma Epstein–Barr virus early RNA Epstein–Barr virus 8p11 myeloproliferative syndrome (8p11 stem cell leukemia/lymphoma) extramedullary myeloid tumor (granulocytic sarcoma) extranodal NK/T-cell lymphoma, nasal type essential thrombocythemia

xiii

xiv

ETP-ALL FAB FC FGFR1 FISH FITC FL FLT3 FSC GPI H&E HCL HCL-V Hg HL IGVH ins inv iso IVLBCL L&H LBL LGL LP LPL LYG LYP MALT MBL MCL MCyR MDS MF MGUS MMR MPAL MPN MPO MRD MZL NHL NK NLPHL NPM NSE p PCFCCL PCM PCR PCSM-TCL PD-1 PDGFRA PDGFRB PE PEL PerCP PFL

Abbreviations

early T-cell precursor ALL French–American–British (classification of acute leukemia) flow cytometry fibroblast growth factor receptor 1 gene fluorescence in situ hybridization fluorescein isothiocyanate follicular lymphoma fms-like tyrosine kinase 3 gene forward scatter glycosylphosphatidylinositol hematoxylin and eosin hairy cell leukemia hairy cell leukemia variant hemoglobin Hodgkin lymphoma immunoglobulin heavy-chain variable gene insertion inversion isochromosome intravascular large B-cell lymphoma lymphocyte and histiocyte cell (popcorn cell), neoplastic cells in NLPHL lymphoblastic lymphoma large granular lymphocyte lymphocyte predominant (cell), neoplastic cells in NLPHL lymphoplasmacytic lymphoma lymphomatoid granulomatosis lymphomatoid papulosis mucosa-associated lymphoid tissue monoclonal B-cell lymphocytosis mantle cell lymphoma major cytogenetic response myelodysplastic syndrome mycosis fungoides monoclonal gammopathy of undetermined significance major molecular response mixed phenotype acute leukemia myeloproliferative neoplasm myeloperoxidase minimal residual disease marginal zone B-cell lymphoma non-Hodgkin lymphoma natural killer nodular lymphocyte predominant Hodgkin lymphoma nucleophosmin gene nonspecific esterase short arm of chromosome primary cutaneous follicle center cell lymphoma plasma cell myeloma polymerase chain reaction primary cutaneous CD4+ small/medium-sized T-cell lymphoma programmed death-1 alpha-type platelet-derived growth factor receptor gene beta-type platelet-derived growth factor receptor gene phycoerythrin primary effusion lymphoma peridinium chlorophyll protein complex pediatric follicular lymphoma

Abbreviations

Ph PLL PMBL PMF PML PTCL PTLD PV q RA RAEB RARA RARS RARS-T R-CHOP RCMD RCUD RQ-PCR RT-PCR R–S SDRPL SLL SM SMZL SPTL SS SSC t TFH TCR TdT THRLBCL T-LGL TMD T-PLL WBC WM

Philadelphia chromosome [result of t(9;22) translocation] prolymphocytic leukemia primary mediastinal large B-cell lymphoma primary myelofibrosis promyelocytic leukemia gene peripheral T-cell lymphoma, unspecified posttransplant lymphoproliferative disorder polycythemia vera long arm of chromosome refractory anemia refractory anemia with excess blasts retinoic acid receptor α gene refractory anemia with ringed sideroblasts refractory anemia with ringed sideroblasts and thrombocytosis rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone refractory cytopenia with multilineage dysplasia refractory cytopenia with unilineage dysplasia real-time quantitative PCR reverse transcriptase PCR Reed–Sternberg (cell) splenic diffuse red pulp B-cell lymphoma small lymphocytic lymphoma systemic mastocytosis splenic marginal zone lymphoma subcutaneous panniculitis-like T-cell lymphoma Sézary’s syndrome side scatter translocation follicular helper T cells T-cell receptor terminal deoxynucleotidyl transferase T-cell/histiocyte-rich large B-cell lymphoma T-cell large granular lymphocyte (leukemia) transient myeloproliferative disorder T-cell prolymphocytic leukemia white blood cell (count) Waldenström macroglobulinemia

xv

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1

Lymph Node

I

coNteNtS Normal Structure........................................................................................................................................................................... 1 abnormal Patterns ........................................................................................................................................................................ 1 Diffuse Pattern.......................................................................................................................................................................... 4 Large/Intermediate Cells ..................................................................................................................................................... 4 Small Cells .......................................................................................................................................................................... 4 Mixed (Pleomorphic) Cells ................................................................................................................................................. 4 Nodular Pattern ...................................................................................................................................................................... 10 Paracortical (Interfollicular; T-Zone) Pattern ......................................................................................................................... 10 Intrasinusoidal Pattern ............................................................................................................................................................ 14 Clear Cell Infiltrate................................................................................................................................................................. 15 anaplastic Infiltrate ................................................................................................................................................................ 15 Histiocyte-Rich Infiltrate ........................................................................................................................................................ 16 Plasma Cell-Rich Infiltrate ..................................................................................................................................................... 17 Eosinophil-Rich Infiltrate ....................................................................................................................................................... 19 References ................................................................................................................................................................................... 19

Normal Structure Lymph nodes act as a scaffolding system and home for lymphocytes, monocytes, and histiocytes in the lymphatic system. They are ovoid encapsulated structures composed of the cortex (with primary and secondary follicles), the paracortex (area between the superficial cortex and the medulla), and the medulla (medullary cords with vessels and sinuses) (Figure 1.1). The capsule and its extension within the lymph node parenchyma (trabeculae) together with a reticular meshwork form the supportive elements of the lymph node. The reticular meshwork is composed of reticular cells, dendritic cells, macrophages, and follicular dendritic cells (FDCs). Primary and secondary follicles are distributed within the cortex (at the periphery of the lymph node). Primary follicles are composed of small and relatively monotonous B cells, which are negative for CD10 and BCL6, and positive for BCL2. Secondary (or reactive) follicles are composed of two zones: central pale-staining germinal centers and a darker staining mantle zone composed of mostly small lymphocytes. The mantle zone in the lymph nodes is usually homogeneous without an overt marginal zone (Figure 1.2a), typical for follicles in the spleen. Certain reactive conditions in the lymph node may lead to the formation of an easily identifiable marginal zone composed of so-called monocytoid B cells (Figure 1.2B and C). The germinal center B cells express CD10 and BCL6 and are negative for BCL2. They contain numerous larger lymphocytes with nucleoli (centroblasts), centrocytes (lymphocytes with irregular nuclei), small lymphocytes, tingible body macrophages, and dendritic reticulum cells. Few scattered T cells expressing CD10 and programmed death-1 (PD-1) are also present within reactive follicles [1]. The secondary follicles often show polarization of their architecture

(Figures 1.3 and 1.4), with one pole composed of centrocytes (lighter zone) and the other with increased numbers of centroblasts and macrophages (darker zone). Polarization helps to differentiate reactive follicles from follicular lymphoma (FL). The polarization is easy to appreciate with Ki-67 (Figure 1.3) or PD-1 staining. FDCs support the B cells in the follicles, which are best visualized by staining with CD21 and CD23. Intact (compact) distributions of FDC meshwork favor a reactive process, whereas an expanded or disrupted meshwork is seen in lymphomas. The lymphoid cells between follicles (paracortex or interfollicular region) are composed predominantly of small T cells with rare centroblasts (depending on the degree of activation), scattered interdigitating reticulum cells, and high endothelial venules. Sinuses contain macrophages and patency is  best  evaluated by the examination of the subcapsular region.  Immunohistochemical stainings of the lymph node with selected antigens, including B-cell markers (CD19, CD20, CD22, CD79a, and PaX5), pan-T-cell markers (CD2, CD3, CD5, and CD7), CD10, PD-1, CD21, and BCL2, help to identify normal architectures and differentiate reactive processes from lymphomas. Figure 1.4 shows immunohistochemical staining with selected relevant markers in the benign lymph node.

abNormal PatterNS Evaluation of the lymph node requires correlating architectural characteristics (low power) with cytologic features (high power; Figure 1.5). The size of lymphoid cells is categorized by comparing the nuclei of histiocytes or endothelial cells. Small lymphocytes (Figure 1.5a) are roughly the size of the nuclei of histiocytes or endothelial cells and have scanty blue cytoplasm and small nuclei with dense (compact) chromatin. 1

2

Abnormal Patterns

I

B-follicle

Capsule

T-zone . . . .. .. . . . . Blood vessels

Septa

Sinus

Mantle zone

Blood vessels

Germinal center

Cortex

FIGure 1.1

Paracortex

Medulla

Lymph node—Normal histology. CD3 + BCL6

A

C

B

FIGure 1.2 (a) Benign lymph node with prominent mantle zone (arrow). (B) Benign lymph node with prominent monocytoid B cells forming distinct marginal zone. (C) Dual immunostaining for BCL-6 (brown) and CD3 (red) shows mantle (marginal) zone as negative population with blue nuclei (hematoxylin counterstaining). BCL2 + PAX5

Ki-67

A

B

FIGure 1.3 (a) Benign germinal center. The immunostaining with Ki-67 shows distinct polarization (positive cells predominate on the left). (B) Dual staining with PaX5 (brown) and BCL2 (red) shows lack of BCL2 expression by reactive germinal center cells.

Prolymphocytes are characterized by the increased cell size and nucleoli (Figure  1.5B). Small lymphocytes with prominent nuclear irregularities in the form of indentation of the nuclear membrane (cleaved nuclei) are called centrocytes (Figure 1.5C). Large B lymphocytes are generally divided into

centroblasts and immunoblasts. Centroblasts have large vesicular nuclei and several nucleoli often seen adjacent to the nuclear membrane (Figure 1.5D). Immunoblasts are characterized by a prominent central eosinophilic macronucleolus (Figure 1.5E). Prominent intracytoplasmic vacuoles may push the nucleus

3

Lymph Node

CD21

C

B

A CD20

D

BCL2

CD3

E

F

FIGure 1.4 Benign lymph node—Histology and immunohistochemistry. (a) Low power shows a preserved architecture with follicles composed of germinal centers (arrow) and mantle zone and vague marginal zone. (B) Higher magnification shows germinal center with one pole composed of larger lymphocytes with macrophages (arrow) and the other composed mostly of small lymphocytes (such polarization is typical for reactive follicles and helps to differentiate it from follicular lymphoma). (C) CD21 staining shows a preserved follicular dendritic cell meshwork. (D) B cells are visualized by staining with CD20. (E) T cells are restricted to perifollicular area (CD3 staining). (F) Germinal center cells do not coexpress BCL2, a useful parameter in distinguishing between follicular hyperplasia and follicular lymphoma.

A

B

C “Signet-ring” cells

Large cells, immunoblasts

E

F

Reed–Sternberg cells

M

FIGure 1.5

Plasma cells/Russell bodies

H Plasmacytoid dendritic cells

Myeloblasts

L

K Langerhans cells

Popcorn cells

N

D

G

J

Large cells, centroblasts

Monocytoid B cells

Lymphoblasts

Plasma cells, anaplastic

I

Centrocytes

Prolymphocytes

Small lymphocytes

O

(a–P) Cytologic features in histologic section of lymph node (see text for details).

Anaplastic cells

P

I

4

I

to the periphery by forming “signet ring cells” (Figure 1.5F). This is usually a secondary phenomenon seen in a number of tumors, which are both hematolymphoid and nonhematopoietic, including FL, peripheral T-cell lymphoma not otherwise specified (PTCL), and adenocarcinoma. Monocytoid B cells are small- to medium-sized lymphocytes with relatively abundant pale or clear cytoplasm (Figure 1.5G). They may be seen in reactive conditions and are best appreciated in the follicles of splenic white pulp. B cells in marginal zone lymphoma (MZL) often display monocytoid features. Plasma cells have an abundant dense cytoplasm with a paler perinuclear area (“hof” or Golgi zone) and an eccentric nucleus (Figure 1.5H). The chromatin is coarsely granular with the formation of dark clumps at the periphery (“cartwheel” or “clock-face” appearance). In a subset of plasma cell neoplasms, plasma cells may show significant anisopoikilocytosis with prominent nucleoli and immature, multinucleated, or blastic forms (anaplastic plasma cells; Figure 1.5I). Blastic features include a prominent nucleolus and a fine, evenly distributed chromatin (Figure 1.5j–L). Reed–Sternberg (R–S) cells are large multinucleated (Figure  1.5M), multilobated, or bilobed cells with prominent eosinophilic nucleoli. Lymphocyte predominant (LP) cells [also popcorn cells or lymphocyte and histiocyte (L&H) cells for lymphocytes and histiocytes; Figure 1.5N] are characteristic for nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). They have multilobated (multinucleated) nuclei, scanty cytoplasms, highly irregular nuclear contours, pale vesicular chromatin, and prominent nucleoli. LP cells differ morphologically from classic R–S cells by their smaller size, less prominent nucleoli, and paler chromatin with a thin delicate nuclear membrane. Langerhans cells are usually large with pale eosinophilic cytoplasm and nuclei with characteristic grooves (clefts) responsible for their “coffee-bean” appearance (Figure 1.5o). The chromatin is dispersed and the nucleoli are inconspicuous, which give Langerhans cells bland cytologic features. anaplastic cells are large cells with irregular (horseshoe- or kidney-shaped) hyperchromatic nuclei, prominent nucleoli, and abundant cytoplasm (Figure 1.5P). Evaluation of the lymph node at low power provides important information and is very often crucial to subclassify the tumor and establish a final diagnosis, for example, FL versus diffuse lymphoma or NLPHL versus T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL). Several major histologic patterns can be recognized in the lymph node: nodular (follicular), diffuse, interfollicular (T zone; paracortical), sinusoidal, and mixed.

Diffuse Pattern large/Intermediate cells Diagnostic considerations of a diffuse infiltrate composed of large- and/or intermediate-sized cells (Figure 1.6) include diffuse large B-cell lymphoma (DLBCL), PTCL, blastoid variant of mantle cell lymphoma (MCL), extramedullary myeloid tumor (EMT; granulocytic sarcoma, monoblastic sarcoma), anaplastic large cell lymphoma (aLCL), plasmablastic lymphoma, Burkitt lymphoma (BL), large B-cell lymphoma,

Abnormal Patterns

unclassifiable with features intermediate between BL and DLBCL (BCLU; “gray zone” lymphoma, “double-hit” lymphoma), plasma cell neoplasm, lymphoblastic lymphoma, histiocytic sarcoma, dendritic cell tumors, blastic plasmacytoid dendritic cell neoplasm (BPDCN), and nonhematopoietic tumors (e.g., carcinoma, melanoma, Ewing’s sarcoma). a diffuse infiltrate of intermediate or large cells with a blastic (blastoid) appearance is typically seen in precursor and high-grade neoplasms characterized by prominent nucleoli, increased nuclear/cytoplasmic ratio, and often evenly distributed fine chromatin. Some tumors may have hyperchromatic nuclei. Diagnostic considerations include B-cell lymphoblastic lymphoma (B-LBL; Figure 1.7a), T-cell lymphoblastic lymphoma (T-LBL; Figure 1.7B), blastoid variant of MCL (Figure  1.7C), immunoblastic variant of DLBCL (Figure 1.7D), monoblastic sarcoma (Figure 1.7E), plasmablastic lymphoma (Figure 1.7F), BPDCN (Figure 1.7G), and granulocytic sarcoma (Figure  1.7H). High-grade tumors with blastic features often show admixtures of histiocytes, which engulf cellular debris (apoptotic bodies) and give rise to a so-called starry-sky pattern when the lymph node is examined at low power. This pattern is characteristic for BL (Figure  1.7I), high-grade DLBCL, BCLU, other highgrade lymphomas (aLCL, some PTCLs), as well as precursor (lymphoblastic) tumors. Small cells a diffuse infiltrate of predominantly small lymphocytes (Figure 1.8) is seen in small lymphocytic lymphoma/B- cell chronic lymphocytic leukemia (SLL/B-CLL), MCL, nodal MZL, nodal involvement by hairy cell leukemia (HCL), diffuse follicle center cell lymphoma (grade 1), FL with prominent sclerosis, lymphoplasmacytic lymphoma, T-cell prolymphocytic leukemia (T-PLL), small cell variant of PTCL, small cell variant of aLCL, and lymphomatous or acute variants of adult T-cell lymphoma/leukemia (aTLL). mixed (Pleomorphic) cells a diffuse, pleomorphic infiltrate (Figure 1.9) with small, intermediate, and large cells can be seen in Kikuchi lymphadenitis, Epstein–Barr virus (EBV)-associated atypical (reactive) hyperplasia, pleomorphic variant of MCL, nodal MZL with increased large cells, diffuse follicle center cell lymphoma (grade 2), angioimmunoblastic T-cell lymphoma (aITL), PTCL, mixed cellularity classical Hodgkin lymphoma (HL), THRLBCL, EBV-associated DLBCL, large B-cell lymphoma with features intermediate between HL and DLBCL (“gray zone” lymphoma), Langerhans cell histiocytosis, and SLL/ CLL with large cell transformation (Richter’s syndrome). Pleomorphic infiltrate with increased vascularity is seen in late HIV infection and aITL. Kikuchi lymphadenopathy (Kikuchi–Fujimoto disease) is a benign, self-limited disease often confused with B- and especially T-cell lymphomas due to the effacement of the lymph node architecture (at least partial) and scattered atypical lymphocytes with immunoblastic features. The lymph node shows reactive germinal centers and focal areas of necrosis (necrosis

5

Lymph Node

Diffuse large B-cell lymphoma

Peripheral T-cell lymphoma

CD20

CD3

B

A Mantle cell lymphoma, blastoid variant

Granulocytic sarcoma (extramedullary myeloid tumor)

BCL1

CD34

D

C Anaplastic large cell lymphoma (T cell)

Plasmablastic lymphoma

ALK

EBER

E

F Burkitt lymphoma with starry-sky pattern

Blastic plasmacytoid dendritic cell neoplasm

Ki-67

CD56

G

H

FIGure 1.6 Lymph node with diffuse infiltrate of intermediate to large cells. Differential diagnosis includes (a) DLBCL; (B) PTCL, large cell type; (C) MCL, blastoid variant; (D) EMT; (E) aLCL; (F) plasmablastic lymphoma; (G) BL; and (H) BPDCN.

may be subtle in early stages, when only occasional apoptotic cells are present within histiocytic aggregates) [2–4]. The histiocytic cells with engulfed cellular debris have abundant cytoplasm and crescentic nuclei (C-shaped forms), a characteristic feature of Kikuchi lymphadenopathy. The infiltrate is composed of a pleomorphic population of small, intermediate, and large lymphocytes (predominantly T cells, especially in areas of necrosis; CD8+ T cells are more abundant than CD4+),

histiocytes (CD68+, CD163+, and CD4+), and plasmacytoid dendritic cells (CD123+, CD68+, and CD303+). Scattered weakly CD30+ immunoblasts may be present, which are of T-cell lineage, usually CD8+. Characteristically, there are no neutrophils and only rare plasma cells may be seen. Karyorrhectic debris is present in the necrotic areas and may be prominent. Based on the cellular composition, some authors distinguish several variants of Kikuchi lymphadenopathy:  lymphohistiocytic,

I

6

Abnormal Patterns

T-LBL

CD10

B-LBL

I

CD3

CD34

TdT

CD20

B

A

Monoblastic sarcoma

Immunoblastic lymphoma

Mantle cell lymphoma, blastoid

CD68

BCL1

D

C Plasmablastic lymphoma

E

EBER

F

Granulocytic sarcoma

Blastic plasmacytoid dendritic neoplasm

MPO

CD56

G Burkitt lymphoma

H BCL2

BCL6

FISH for MYC break apart probe

I

FIGure 1.7 Lymph node with blastic/blastoid infiltrate. Differential diagnosis includes (a) precursor B-LBL; (B) precursor T-LBL; (C)  MCL, blastoid variant; (D) DLBCL, immunoblastic variant; (E) monoblastic sarcoma (EMT with monocytic differentiation); (F) plasmablastic lymphoma; (G) BPDCN; (H) granulocytic sarcoma (EMT with myeloid/neutrophilic differentiation); and (I) BL.

phagocytic, necrotic, and foamy cell type [5]. Lymphadenopathy in Kikuchi–Fujimoto disease may be accompanied by similar changes in the skin. a pattern similar to Kikuchi lymphadenopathy may be seen in systemic lupus erythematosus (SLE). SLE adenopathy differs by the presence of numerous plasma

cells, hematoxylin bodies, and degenerated nuclear material in the blood walls (azzopardi phenomenon). Infiltrates with scattered large cells with irregular (binucleated, multilobed, or multinucleated) nuclei and prominent nucleoli are seen in classical HL, PTCL, aITL, THRLBCL,

7

Lymph Node

Mantle cell lymphoma

SLL/CLL

B

A Hairy cell leukemia

Nodal marginal zone B-cell lymphoma

C Lymphoplasmacytic lymphoma

Diffuse follicle center cell lymphoma

E Follicular lymphoma with fibrosis

D T-cell prolymphocytic leukemia

H

G

F Peripheral T-cell lymphoma

I

Adult T-cell leukemia/lymphoma

J

FIGure 1.8 Lymph node with diffuse infiltrate of predominantly small cells. Differential diagnosis includes (a) SLL/CLL, (B) MCL, (C) MZL, (D) HCL, (E) diffuse FL, (F) FL with fibrosis, (G) lymphoplasmacytic lymphoma, (H) T-PLL, (I) PTCL, and (j) aTLL.

EBV-associated large B-cell lymphoma, NLPHL, EBVassociated lymphadenitis (infectious mononucleosis), Kimura disease, and cytomegalovirus (CMV)-associated lymphadenitis (Figure  1.10). Classical HL is characterized by the presence of R–S cells, whereas NLPHL is characterized by the presence of LP cells. R–S cells are large binucleated or multinucleated (multilobated) cells with prominent eosinophilic nucleoli (often with a clear halo around them), a thick nuclear membrane, and an amphophilic or eosinophilic cytoplasm (Figure 1.11). LP cells have multilobated nuclei, scanty cytoplasm, highly irregular nuclear contours, pale vesicular chromatin, and prominent nucleoli.  LP cells are surrounded by a rim of small T cells (CD3+/CD57+ rosettes) and are most often located within nodules composed predominantly of

small B cells, in contrast to R–S cells, which are usually present within the T-cell-rich background. EBV lymphadenitis (infectious mononucleosis; Figure 1.12) often shows enough cytologic atypia to raise the possibility of B-cell lymphoma (DLBCL), T-cell lymphoma (PTCL), and especially classical HL. The lymph node is enlarged with mixed follicular and paracortical hyperplasia, small foci of necrosis, and a pleomorphic infiltrate composed of lymphocytes, histiocytes, and plasma cells. Interfollicular (paracortical) areas show increased numbers of larger atypical lymphocytes with immunoblastic, centroblastic, or plasmablastic cytomorphology, which are of either B- or T-cell lineages. The activated cells often express CD30. Scattered EBV-infected cells (either small or medium sized) are always present. Plasma cells are

I

8

Abnormal Patterns

Kikuchi lymphadenitis

I

A Angioimmunoblastic T-cell lymphomas

T-cell-rich diffuse large B-cell lymphoma

E Follicle center cell lymphoma, diffuse

G

Mixed cellularity Hodgkin lymphoma

F Nodal MZL with increased large cells

EBV-associated DLBCL

I

H Gray zone lymphoma (HL & DLBCL)

J

C

B

D

Peripheral T-cell lymphoma

EBV-associated lymphadenopathy

Langerhans cell histiocytosis

K

CLL/SLL with large cell transformation

L

FIGure 1.9 Lymph node with diffuse and pleomorphic infiltrates. Differential diagnosis includes (a) Kikuchi lymphadenitis, (B) EBVassociated atypical (reactive) hyperplasia, (C) PTCL, (D) aITL, (E) THRLBCL, (F) classical HL (mixed cellularity), (G) diffuse follicle center cell lymphoma (grade 2), (H) nodal MZL with increased large cells, (I) EBV-associated DLBCL, (j) large B-cell lymphoma with features intermediate between HL and DLBCL (“gray zone” lymphoma), (K) Langerhans cell histiocytosis, and (L) SLL/CLL with large cell transformation (Richter’s syndrome).

9

Lymph Node

DLBCL, “anaplastic” variant

HL, classical

CD30

CD20

CD15

A

PTCL

B

CD3

C

ALCL

THRLBCL

EBV-associated DLBCL

CD20

D

F

E NLPHL

EBV-associated lymphadenitis

CD20

G

EBV

H CMV-associated lymphadenitis

Kimura disease

CMV

I

J

FIGure 1.10 Scattered large multinucleated/multilobated cells. Differential diagnosis includes (a) classical HL; (B) DLBCL, anaplastic variant; (C) PTCL; (D) aLCL; (E) THRLBCL; (F) EBV-associated DLBCL; (G) NLPHL; (H) EBV-associated lymphadenitis; (I) Kimura disease with characteristic eosinophilic abscesses and large multinucleated cells; and (j) CMV-associated lymphadenitis with intranuclear inclusions.

I

10

Abnormal Patterns

I

A

B

C

D

FIGure 1.11 R–S cells in cytologic and histologic preparations: (a,B) touch smear [Wright–Giemsa (W–G) staining] and (C,D) tissue section (H&E staining).

polytypic. T cells often show increased numbers of CD8+ cells, some of which display cytologic atypia.

noDular Pattern The nodular (follicular pattern) is typical for several reactive conditions such as follicular hyperplasia (Figure 1.13a), toxoplasma lymphadenitis, Kimura disease, follicular hyperplasia with progressive transformation of germinal centers (PTGC; Figure  1.13B), reactive process with mantle zone hyperplasia (Figure  1.13C), Castleman’s disease (Figure  1.13D), rheumatoid lymphadenopathy, IgG4-related lymphadenopathy, early HIV lymphadenopathy (Figure  1.13E), and atypical follicular hyperplasia. The main differential diagnosis in a lymph node with a nodular pattern is between follicular hyperplasia and FL (Figure  1.13F). Nodularity (often vague) can be seen in B-small lymphocytic lymphoma/chronic lymphocytic leukemia (B-SLL/CLL) with paler staining proliferation centers composed of prolymphocytes (“pseudofollicular” pattern; Figure  1.12G), B-SLL/CLL with unusual nodular pattern (Figure 1.13H), MCL (Figure 1.13I), and nodal MZL (Figure  1.13j). Fibrous bands divide the lymph nodes into prominent nodules with scattered large multilobated cells, which is characteristic of the nodular sclerosis type of classical HL (Figure 1.13K), whereas a vague nodular pattern with scattered large cells in the background of small lymphocytes is

typical for lymphocyte-rich classical HL  (Figure  1.13L) and NLPHL (Figure 1.13M). The distinct nodularity of NLPHL at low magnification helps to distinguish it from THRLBCL. Expansion of the marginal zone with prominent monocytoid B cells is seen in MZL and reactive conditions. a  prominent mantle zone can be seen in reactive disorders and MCL. MCL can also be present with diffuse and nodular patterns. a concentric arrangement of small lymphocytes (“onion skin”) with follicles composed of more than one germinal center is typical for Castleman’s disease  (hyaline-vascular type). Large, expanded follicles composed of small lymphocytes mostly from the mantle zone, which disrupt the germinal center are termed progressively transformed germinal centers (PTGCs) [6,7]. PTGC only partially replaces the lymph node, which otherwise shows follicular hyperplasia. The transformed germinal centers are a few times larger than reactive follicles and at later stages show only a few clusters of centroblasts and centrocytes, predominance of small lymphocytes, disrupted follicular dendritic cell meshwork without tingible body macrophages, plasma cells, or eosinophils in most cases. In contrast to NLPHL, the large atypical cells with nucleoli (LP cells) with characteristic T-cell resetting are absent.

Paracortical (interfollicular; t-Zone) Pattern a paracortical (interfollicular) infiltrate (Figure 1.14) can be seen in aITL and occasionally in PTCL (so-called  T-zone

11

Lymph Node

×40

×40

A

B ×400

C

D CD3

PAX5

E

F

CD20

G EBER

CD30

K/L dual

H

×600

I

J

FIGure 1.12 EBV-associated lymphadenitis shows paracortical (a) and follicular (B) hyperplasia with pleomorphic infiltrate (C,D), which include immunoblasts, centroblasts, plasma cells, and occasional Hodgkin-like cells or R–S-like cells. PaX5 staining at low power (E) shows mostly preserved architecture in areas with follicular hyperplasia. Interfollicular (paracortical) areas are composed of many T cells (F), with increased number of CD8+ cells, scattered B cells (G), polytypic plasma cells (H), and many activated cells (B or T cells) with cytologic atypia, many of which express CD30 (I). EBV-infected cells are visualized by EBV early RNa in situ hybridization (EBER-ISH) staining (j).

variant; Figure 1.14a). It is also often seen in aLCL (Figure 1.14B). other lesions with a prominent paracortical pattern include DLBCL (variant with interfollicular lymph node involvement; Figure 1.14C), EMT (Figure  1.14D), BPDCN (Figure  1.14E), Castleman’s disease (Figure  1.14F), plasma cell neoplasms involving lymph nodes (Figure 1.14G), classical HL (Figure 1.14H), metastatic tumors, HCL (Figure 1.14I),

and Langerhans cell histiocytosis (Figure  1.14j). of these, EMT may be difficult to identify by hematoxylin and eosin (H&E) examination alone [careful comparison of CD3 and CD43 staining helps to identify the population of CD3−/ CD43+ cells, which can then lead to the expansion of phenotypic panel to include myeloperoxidase, CD33, CD34, CD117, and terminal deoxynucleotide transferase (TdT), or

I

12

Abnormal Patterns

Reactive follicular hyperplasia

I

Reactive lymph node with PTGC

A

B

D

Follicular lymphoma

E

SLL/CLL, proliferation centers

F Mantle cell lymphoma

SLL/CLL

H

HL, classical, lymphocyte-rich type

L

Nodal MZL

J

I

HL, classical, nodular sclerosis

K

C HIV-associated adenopathy

Castleman’s disease

G

Mantle zone hyperplasia

NLPHL

M

FIGure 1.13 Lymph node with nodular pattern. Differential diagnosis includes (a) reactive follicular hyperplasia; (B) reactive follicular hyperplasia with PTGC; (C) mantle zone hyperplasia; (D) Castleman’s disease; (E) HIV-related florid follicular hyperplasia; (F) FL; (G) B-SLL/CLL with proliferation centers (pseudofollicular pattern); (H) B-SLL/CLL with unusual nodular pattern; (I) MCL; (j) nodal MZL with follicular colonization; (K) HL, nodular sclerosis variant; (L) lymphocyte-rich classical HL; and (M) NLPHL.

13

Lymph Node

PTCL

ALCL

CD5

ALK1

B

A DLBCL

Granulocytic sarcoma

Plasmacytoid dendritic cell neoplasm

CD20

Myeloperoxidase

CD56

D

C Castleman's disease, plasma cell type

E Plasma cell neoplasm, lymph node

Hodgkin lymphoma, classical

Lambda

CD30

CD138

G

F Hairy cell leukemia, lymph node

H CD25

Langerhans cell histiocytosis

CD1a

DBA44

I

J

FIGure 1.14 Paracortical (interfollicular) infiltrate in the lymph node. Differential diagnosis includes (a) PTCL (T-zone lymphoma); (B) aLCL; (C) DLBCL with interfollicular pattern; (D) EMT (granulocytic sarcoma); (E) BPDCN; (F) Castleman’s disease, plasma cell type; (G) plasma cell neoplasm; (H) classical HL; (I) HCL; and (j) Langerhans cell histiocytosis.

I

14

I

Abnormal Patterns

monocytic markers]. The interfollicular pattern is also seen in some reactive conditions, such as dermatopathic lymphadenitis, postvaccination lymphadenitis, occasional cases of IgG4related lymphadenopathy, viral lymphadenitis, and in some drug reactions. Paracortical hyperplasia is often seen in reactive conditions, especially in viral infections, including infectious mononucleosis (EBV lymphadenitis) and toxoplasma lymphadenitis. In reactive paracortical hyperplasia, low magnification may show a “moth-eaten” appearance and higher magnification shows scattered immunoblasts dispersed among small lymphocytes (mostly T cells). In some cases, especially drug-induced lymphadenopathy, eosinophils may be abundant. In patients with skin conditions, some histiocytes contain melanin pigment (dermatopathic lymphadenopathy). Immunoblasts (which are of either B- or T-cell lineage and express CD30 and CD45) may be occasionally abundant or form clusters, prompting differential diagnosis with large cell lymphomas. Infectious mononucleosis shows paracortical hyperplasia, focal follicular hyperplasia, and sinus histiocytosis with heterogeneous lymphohistiocytic infiltrate including larger lymphocytes with nucleoli (immunoblasts), plasma cells,  eosinophils, and histiocytes (tingible body macrophages). Some follicles may show features of apoptosis and necrosis. Increased numbers of immunoblasts or the presence of large, R–S like cells may resemble DLBCL and HL, respectively, especially in minute core biopsy. The

intrasinusoiDal Pattern an intrasinusoidal pattern (Figure  1.15) is typical for aLCL (Figure  1.15a), metastatic nonhematopoietic tumors, occasional cases of DLBCL (Figure 1.15B), intravascular large B-cell lymphoma (IVLBCL; Figure 1.15C), hepatosplenic T-cell lymphoma (HSTL), and variants of PTCL. Rare cases of classical HL may show intrasinusoidal distribution (Figure  1.15D). Cutaneous CD30+ T-cell lymphoproliferations, such as lymphomatoid papulosis (LYP) and cutaneous aLCL, can involve local lymph nodes

A′

A

DLBCL

ALK1

ALCL

B Hodgkin lymphoma

Intravascular B-cell lymphoma

C

presence of a preserved (albeit distorted) architecture and a heterogeneous B- and T-cell lymphoid population with a spectrum of small, medium, and large cells; reactive follicles; and EBV positivity helps to differentiate infectious mononucleosis from lymphoma. The staining with CD30 by immunoblasts is often weak in contrast to strong membranous and Golgi staining in R–S cells. In addition, reactive immunoblasts are negative for CD15 and positive for CD45. Clinical follow-up with repeated biopsy and/or molecular testing for B-cell clonality may help in difficult cases (e.g., to exclude EBV-associated large B-cell lymphoma). CMV lymphadenitis usually shows changes similar to those of EBV lymphadenitis. Clusters of B cells with a monocytoid appearance may be present. Typical CMVinfected cells have an “owl’s eye” appearance due to viral inclusion within enlarged nuclei.

D

CD30

D′

FIGure 1.15 Intrasinusoidal/intravascular pattern. Differential diagnosis includes (a) aLCL [aLK1-positive tumor cells are seen within sinusoids (a′)]; (B) DLBCL with unusual intrasinusoidal distribution; (C) intravascular lymphoma; and (D) classical HL [intrasinusoidal tumor cells express CD30 (D′)].

15

Lymph Node

with sinusoidal infiltration mimicking classical HL or systemic aLCL.

clear cell infiltrate Hematopoietic tumors composed of clear cells include a signetring cell variant of PTCL (Figure 1.16a), aITL (Figure 1.16B), nodal MZL (Figure  1.16C), a signet-ring cell variant of FL (Figure  1.16D), Langerhans cell histiocytosis (Figure  1.16E), mast cell disease (Figure  1.16F), Rosai–Dorfman disease (Figure  1.16G), histiocytic sarcoma (Figure  1.16H), DLBCL composed of clear cells (Figure 1.16I), and HCL (Figure 1.16j).

anaPlastic infiltrate an anaplastic infiltrate is characterized by highly atypical, pleomorphic, often bizarre cells with hyperchromatic nuclei PTCL, signet-ring cell variant

A

AITL

BCL6

D

C

Langerhans histiocytosis

CD1a

E

H

Mast cell disease, lymph node

F

Histiocytic sarcoma

Rosai–Dorfman disease

Marginal zone B-cell lymphoma

PD-1

B

FL, signet-ring cell variant

G

and prominent nucleoli. anaplastic features can be seen in anaplastic variants of DLBCL (Figure  1.17a), anaplastic lymphoma kinase (aLK)-positive DLBCL (Figure  1.17B), aLK-positive aLCL (Figure  1.17C), aLK-negative aLCL (Figure 1.17D), anaplastic (poorly differentiated) plasma cell myeloma (PCM; Figure 1.17E), classical HL, nodular sclerosis (syncytial variant; Figure 1.17F), classical HL, lymphocyte-depleted type (Figure 1.17G), Langerhans cell sarcoma (Figure  1.17H), anaplastic carcinoma (Figure  1.17I), and rare cases of EMT (granulocytic sarcoma). Neoplastic cells in aLCL are large with abundant amphophilic or basophilic cytoplasm; round, lobulated, irregular, often hyperchromatic nuclei; and prominent nucleolus [8–13]. Characteristic hallmark cells have a “horseshoe”-shaped nucleus with a pale-staining perinuclear hof. an anaplastic variant of DLBCL shows pleomorphic, often bizarre nuclei, which

DLBCL

I

HCL

CD20

J

FIGure 1.16 Clear cell infiltrate in the lymph node. Differential diagnosis includes (a) PTCL, signet-ring cell variant; (B) aITL; (C) MZL; (D) FL, signet-ring cell variant; (E) Langerhans cell histiocytosis; (F) mast cell disease; (G) Rosai–Dorfman disease; (H)  histiocytic sarcoma; (I) DLBCL composed of clear cells; and (j) HCL.

I

16

Abnormal Patterns

I

Diffuse large B-cell lymphoma

ALK+ large B-cell lymphoma

Anaplastic large cell lymphoma, ALK1+

CD20

ALK1

ALK1

A

C

B Anaplastic large cell lymphoma ALK1−

Anaplastic plasma cell myeloma

CD30

CD138

D

F

E HL, lymphocyte depleted

Langerhans cell sarcoma

CD30

CD1a

G

Classical HL, syncytial NS

H

Anaplastic carcinoma

I

FIGure 1.17 anaplastic infiltrate in the lymph node. Differential diagnosis includes (a) DLBCL with anaplastic features; (B) aLKpositive DLBCL; (C) aLK-positive aLCL; (D) aLK-negative aLCL; (E) PCM, anaplastic variant; (F) HL, nodular sclerosis, syncytial variant; (G) classical HL, lymphocyte-depleted type; (H) Langerhans cell sarcoma; and (I) anaplastic carcinoma.

can be multilobated and may resemble R–S cells [14,15]. They express B-cell markers, may be positive for CD30, and do not express T-cell markers or aLK. Diagnostic considerations also include HL, especially syncytial and lymphocyte-depleted variants [16–19]. Tumor cells in HL express CD30, PaX5 (weak), MUM1, and often CD15, and are negative for aLK.

Histiocyte-ricH infiltrate Numerous histiocytes are typically seen in reactive conditions, including sinus histiocytosis, infections, cat scratch disease, tuberculosis, fungal lymphadenitis, toxoplasma lymphadenitis

(Figure  1.18a), sarcoidosis (Figure  1.18B), dermatopathic lymphadenitis (Figure  1.18C), Kikuchi lymphadenitis, and Rosai–Dorfman disease (Figure 1.18D). Toxoplasmosis is characterized by a prominent follicular hyperplasia with increased large cells and tingible body macrophages, aggregates of monocytoid B cells, plasma cells and activated lymphocytes (immunoblasts) in medullary cords, and small granulomas, which are present in interfollicular and paracortical areas and often infiltrate reactive follicles. Sarcoidosis is characterized by granulomas in which some epithelioid cells contain crystalloid structures forming so-called asteroid body. Histiocytes with large vesicular nuclei; prominent nucleoli; abundant, pale, “wispy,”

17

Lymph Node

Sarcoidosis, “asteroid body”

Toxoplasmosis

A

C

B THRLBCL

E

Dermatopathic changes

Lennert’s lymphoma

CD20

F

D DLBCL with granulomas

CD3

CD20

G

HL, classical

NLPHL with granulomas

H

Rosai–Dorfman disease

CD30

I

FIGure 1.18 Lymph node with prominent histiocytic infiltrate and/or granulomas. Differential diagnosis includes (a) toxoplasma lymphadenitis; (B) sarcoidosis; (C) dermatopathic lymphadenopathy; (D) Rosai–Dorfman disease; (E) THRLBCL; (F) Lennert’s lymphoma, variant of PTCL; (G) DLBCL with granulomas; (H) NLPHL with granulomas; and (I) classical HL.

or frothy cytoplasm; and engulfed lymphoid cells (emperipolesis or lymphocytophagocytosis) are typical for Rosai–Dorfman disease (sinus histiocytosis with massive lymphadenopathy) [20]. Histiocytes in Rosai–Dorfman disease are positive for S100, CD4, CD11c, CD68, and CD163, and do not express CD1a or Langerin. Differential diagnosis of Rosai–Dorfman disease includes Langerhans cell histiocytosis that is characterized by S100-, CD1a-, and Langerin (CD207)-positive cells. occasional hematolymphoid neoplasms have a histiocyte-rich background or numerous histiocytic aggregates (granulomalike). These include histiocyte-rich large B-cell lymphoma (Figure  1.18E), Lennert’s lymphoma (a variant of PTCL;

Figure 18F), DLBCL with granulomas (Figure 1.18G), NLPHL (Figure 1.18H), and classical HL (Figure 1.18I).

Plasma cell-ricH infiltrate Plasma cells often accompany both reactive and neoplastic infiltrates in the lymph node, including reactive (nonspecific) lymph node, SLE lymphadenopathy, Castleman’s disease, plasma cell type (Figure 1.19a) or its variant with monoclonal (lambda+) plasma cells (Figure  1.19B), rheumatoid arthritisassociated lymphadenopathy, lymphoplasmacytic lymphoma (Figure  1.19C), HL, B-SLL with plasmacytic differentiation

I

18

Abnormal Patterns

I

Castleman’s disease, plasma cell type

Kappa

Castleman’s disease, plasma cell type with monoclonal (lambda) plasma cells

Kappa Lambda

A

Lambda

B B-SLL with plasmacytic differentiation

IgM

CD19

Lymphoplasmacytic lymphoma

D

C Plasma cell myeloma

CD138

E

MZL with plasmacytic differentiation

F FL with plasmacytic differentiation

G

CD5

Kappa

AITL

Lambda

Dual κ & λ

H

FIGure 1.19 Lymph node with plasma cell-rich infiltrate. Differential diagnosis includes (a) Castleman’s disease, plasma cell type; (B) Castleman’s disease, plasma cell type with monoclonal (lambda+) plasma cells; (C) lymphoplasmacytic lymphoma; (D) B-SLL/CLL with plasmacytic differentiation; (E) PCM; (F) nodal MZL with plasmacytic differentiation; (G) FL with plasmacytic differentiation; and (H) aITL (kappa, brown; lambda, red).

19

Lymph Node

Hodgkin lymphoma, classical

Kimura disease

B

A Angioimmunoblastic T-cell lymphoma

C

Peripheral T-cell lymphoma, unspecified

D

FIGure 1.20 Lymph node with eosinophil-rich infiltrate. Differential diagnosis includes (a) Kimura disease; (B) classical HL; (C) aITL; and (D) PTCL.

(Figure  1.19D), plasmacytoma or PCM (Figure  1.19E), nodal MZL with plasmacytic differentiation (Figure  1.19F), FL with plasmacytic differentiation (Figure  1.19G), and aITL (Figure  1.19H). Plasma cells in B-cell lymphoma with plasmacytic differentiation, especially in MZL, tend to occur in clusters in contrast to often dispersed plasma cells in a reactive process.

eosinoPHil-ricH infiltrate Eosinophils are typically abundant in Kimura lymphadenopathy (Figure 1.20a), parasitic infections, drug reactions (hypersensitivity), Langerhans cell histiocytosis, classical HL (Figure  1.20B), and peripheral T-cell lymphoproliferations including aITL (Figure 1.20C) and PTCL (Figure 1.20D).

reFereNceS 1. Dorfman DM, et al. Programmed death-1 (PD-1) is a marker of germinal center-associated T cells and angioimmunoblastic T-cell lymphoma. am j Surg Pathol, 2006. 30(7):802–10. 2. Pileri S, et al. Histiocytic necrotizing lymphadenitis without granulocytic infiltration. Virchows arch a Pathol anat Histol, 1982. 395(3):257–71. 3. Felgar RE, et  al. Histiocytic necrotizing lymphadenitis (Kikuchi’s disease): in situ end-labeling, immunohistochemical, and serologic evidence supporting cytotoxic lymphocytemediated apoptotic cell death. Mod Pathol, 1997. 10(3):231–41. 4. Menasce LP, et  al. Histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto disease): continuing diagnostic difficulties. Histopathology, 1998. 33(3):248–54.

5. Tsang WY, Chan jK, Ng CS. Kikuchi’s lymphadenitis. a morphologic analysis of 75 cases with special reference to unusual features. am j Surg Pathol, 1994. 18(3):219–31. 6. Ferry ja, Zukerberg LR, Harris NL. Florid progressive transformation of germinal centers. a syndrome affecting young men, without early progression to nodular lymphocyte predominance Hodgkin’s disease. am j Surg Pathol, 1992. 16(3):252–8. 7. Nguyen PL, Ferry ja, Harris NL. Progressive transformation of germinal centers and nodular lymphocyte predominance Hodgkin’s disease: a comparative immunohistochemical study. am j Surg Pathol, 1999. 23(1):27–33. 8. Chan jK. anaplastic large cell lymphoma: redefining its morphologic spectrum and importance of recognition of the aLKpositive subset. adv anat Pathol, 1998. 5(5):281–313. 9. Chan jK, et al. anaplastic large cell Ki-1 lymphoma. Delineation of two morphological types. Histopathology, 1989. 15(1):11–34. 10. Falini B. anaplastic large cell lymphoma: pathological, molecular and clinical features. Br j Haematol, 2001. 114(4):741–60. 11. jaffe ES. anaplastic large cell lymphoma: the shifting sands of diagnostic hematopathology. Mod Pathol, 2001. 14(3):219–28. 12. Pileri S, et al. Lymphohistiocytic T-cell lymphoma (anaplastic large cell lymphoma CD30+/Ki-1+ with a high content of reactive histiocytes). Histopathology, 1990. 16(4):383–91. 13. Pileri Sa, et  al. anaplastic large cell lymphoma: a concept reviewed. adv Clin Path, 1998. 2(4):285–96. 14. Haralambieva E, et  al. anaplastic large-cell lymphomas of B-cell phenotype are anaplastic lymphoma kinase (aLK) negative and belong to the spectrum of diffuse large B-cell lymphomas. Br j Haematol, 2000. 109(3):584–91. 15. Maes B, et al. among diffuse large B-cell lymphomas, T-cellrich/histiocyte-rich BCL and CD30+ anaplastic B-cell subtypes exhibit distinct clinical features. ann oncol, 2001. 12(6):853–8.

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16. Slack GW, et al. Lymphocyte depleted Hodgkin lymphoma: an evaluation with immunophenotyping and genetic analysis. Leuk Lymphoma, 2009. 50(6):937–43. 17. Drakos E, et  al. Nodular lymphocyte predominant Hodgkin lymphoma with clusters of LP Cells, acute inflammation, and fibrosis: a syncytial variant. am j Surg Pathol, 2009. 33(11):1725–31.

References 18. Harris NL, Differential diagnosis between Hodgkin’s disease and non-Hodgkin’s lymphoma. Int Rev Exp Pathol, 1992. 33:1–25. 19. Harris NL, Hodgkin’s disease: classification and differential diagnosis. Mod Pathol, 1999. 12(2):159–75. 20. Rosai j, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy. a newly recognized benign clinicopathological entity. arch Pathol, 1969. 87(1):63–70.

2

Bone Marrow

I

coNteNtS Normal Structure and Hematopoiesis ......................................................................................................................................... 21 Cytomorphology (Abnormal Features) ....................................................................................................................................... 24 Histology (Abnormal Patterns) ................................................................................................................................................... 28 Increased Blasts...................................................................................................................................................................... 28 Diffuse Small Cell Infiltrate ................................................................................................................................................... 28 Diffuse Intermediate/Large Cell Infiltrate .............................................................................................................................. 30 Lymphoid Aggregates (Nodular Lymphoid Infiltrate) ........................................................................................................... 31 Paratrabecular Lymphoid Infiltrate ........................................................................................................................................ 32 Discrepancy between the Size of Lymphocytes in Primary Lymphoma and BM Involvement ............................................. 33 Intrasinusoidal Lymphoid Infiltrate ........................................................................................................................................ 35 Patterns of Involvement by Mature B-Cell Lymphoproliferative Disorders .......................................................................... 35 Patterns of Involvement by Mature T-Cell Lymphoproliferative Disorders........................................................................... 35 Scattered Atypical Large Cells (Other Than Megakaryocytes).............................................................................................. 38 Megakaryocytosis................................................................................................................................................................... 38 Myeloid Hyperplasia (Increased M:E Ratio) ......................................................................................................................... 38 Eosinophilia............................................................................................................................................................................ 39 Erythroid Hyperplasia (Decreased M:E Ratio) ...................................................................................................................... 40 Erythroid Hypoplasia ............................................................................................................................................................. 41 Marrow Infiltrate with Fibrosis .............................................................................................................................................. 42 Plasmacytosis ......................................................................................................................................................................... 43 Nonhematopoietic Tumors ..................................................................................................................................................... 43 Blood—Cytomorphologic Features ............................................................................................................................................ 43 References ................................................................................................................................................................................... 47

Normal Structure aNd HematopoieSiS The bone marrow (BM) in adults occupies the medullary spaces of large bones such as the femur, the hip, the sternum, and the humerus. The marrow cellularity changes with age and can be roughly estimated as (100  −  age)% (100% at birth, 80% in childhood, 50% in a 50-year-old person, and 20% in an 80-year-old person). The primary function of the BM is to produce blood cells (hematopoiesis), in which early progenitor cells progressively differentiate into intermediate and mature elements. The BM (Figures 2.1 and 2.2) is composed of a matrix requisite for hematopoiesis and hemopoietic cells including rare pluripotent hemopoietic stem cells, granulocytic precursors [myeloperoxidase (MPO+), including CD34 + blasts, CD117+ blasts, and promyelocytes], erythroid precursors [glycophorin A (GPHA+), CD71+], megakaryocytes (platelet precursors; e.g., CD61+), scattered monocytes  (CD68+, CD163+), plasmacytoid dendritic cells (CD123+), lymphocytes (positive for B- and T-cell markers), plasma cells (CD138+, MUM1+), mast cells (CD117+, mast cell tryptase +), adipocytes, blood vessels, and other stromal elements (e.g., osteoblasts, osteoclasts, fibroblasts, etc.). The area close to the bone (the paratrabecular

zone) is composed mostly of myeloid precursors, and the intertrabecular area shows myeloid and erythroid precursors, sinusoids, and scattered megakaryocytes. The BM contains progenitor cells called stem cells. The stem cells have the pluripotent capacity for both self-renewal and differentiation. The most undifferentiated cells (pluripotent stem cells) give rise to stem cells committed to particular cell lineage (unipotent stem cells including common myeloid and common lymphoid progenitors). The stem cells express CD34, CD133, and CD59, and are usually negative for CD38, CD117, and  CD33 (at  least until they start to display a lineagespecific potential). These cells give rise to all types of lymphocytes and myeloid cells. The myeloid lineage comprises all nonlymphoid white cells (neutrophils, monocytes, eosinophils, and basophils), mast cells, red cells, and megakaryocytes (platelets). The common myeloid progenitors differentiate into bipotent cells, either megakaryocyte–erythroid progenitors or granulocyte–macrophage progenitors. The sequence of maturation stages on a BM aspirate smear stained with Wright–Giemsa for different cell types is presented in Figure 2.3. In healthy adults, the granulocytic series predominates over the erythroid 21

22

Normal Structure and Hematopoiesis

I

Megakaryocyte Adipocytes

Erythroid precursors

Blood vessels

Endothelial cells

Bone trabeculae

Sinusoids Myeloid cells

Hematopoietic cords B

A

FiGure 2.1

BM—Normal histology: (A) low magnification; (B) higher magnification.

CD34

FiGure 2.2

CD61

MPO

GPHA

BM—Immunohistochemistry.

series (the ratio is roughly 2:1–4:1). The recognized morphologic maturation stages in granulocytic lineage are myeloblasts, promyelocytes, myelocytes, metamyelocytes, bands, and polymorphonuclear leukocytes (neutrophils). Myeloblasts are round cells with a high nuclear/ cytoplasmic ratio, a fine (immature) chromatin, a scanty pale basophilic cytoplasm, and two or more prominent nucleoli. In a normal marrow core biopsy, blasts (CD34+) comprise up to 1%–2% of marrow cells. Promyelocytes are larger than blasts and have prominent nucleoli but show (primary) azurophilic cytoplasmic granules and coarser chromatin features. Myelocytes are smaller than promyelocytes, have abundant cytoplasm with numerous granules, irregular nuclear contour and coarse chromatin. The neutrophil metamyelocytes are smaller than the promyelocytes and have coarser (more mature) chromatin, and its acidophilic cytoplasm shows azurophilic granules and many fine neutrophilic granules. They usually do not have nucleoli. Metamyelocytes are smaller than myelocytes and

have a more condensed chromatin, a C-shaped nucleus, and neutrophilic granules in the cytoplasm. The nuclei of bands are elongated (band-like) and narrow, and neutrophils show a segmented nucleus with two to five clumps joined by delicate strands of chromatin. Megakaryocytic lineage includes megakaryoblasts, promegakaryocytes, (granular) megakaryocytes, and “naked” nuclei. Megakaryoblasts have single nucleus (often irregular), few prominent nucleoli, and basophilic and agranular cytoplasm (very early megakaryoblasts are morphologically indistinguishable from myeloblasts). Promegakaryocytes are larger than megakaryoblasts, have less basophilic cytoplasm and decreased nuclear/cytoplasmic ratio, and start to show nuclear lobes and cytoplasmic granules. Megakaryocytes are scattered individually and usually do not exceed four to five cells per high-power field (×400). They have irregular nuclei and a pale, granular cytoplasm. Cytoplasmic fragments of megakaryocyte cells (platelets) are instrumental in the primary hemostasis. Adults produce 1011 platelets daily (in normal conditions).

23

Bone Marrow

Pluripotent stem cell

Unipotent stem cell

Unipotent stem cell

Proerythroblast (pronormoblast)

Lymphoblast

Early normoblast (basophilic erythroblast)

Unipotent stem cell

Granulocyte–monocyte colony-forming unit

I Unipotent stem cell

Monoblast

Myeloblast

Eosinophilic

Promyelocyte Neutrophilic

Basophilic

Eosinophilic

Myelocyte Neutrophilic

Basophilic

Unipotent stem cell

Megakaryoblast

Promonocyte

Prolymphocyte

Intermediate normoblast (polychromatophilic erythroblast)

Megakaryocyte

Eosinophilic Late normoblast (orthochromatic erythroblast)

Segmented eosinophil

Metamyelocyte Neutrophilic Basophilic

Segmented neutrophil

Segmented basophil

Reticulocyte

Erythrocyte

FiGure 2.3

Lymphocyte

Eosinophil

Hematopoiesis (see text for details).

Neutrophil

Basophil

Monocyte

Platelets

24

I

Cytomorphology (Abnormal Features)

The erythroid lineage matures from stem cells to red cells (which carry oxygen to the peripheral tissues) through proerythroblast (pronormoblast), basophilic normoblast (basophilic erythroblast), polychromatic normoblast (early polychromatic erythroblast), orthochromatic normoblast (late polychromatic erythroblasts), and reticulocyte. Pronormoblasts are large cells with a prominent nucleolus, a fine chromatin pattern, and a deeply basophilic cytoplasm, which may be vacuolated. Basophilic normoblast is smaller, has more basophilic cytoplasm and coarse (granular) chromatin, and lacks nucleolus. Polychromatic normoblast is characterized by chromatin clumps and polychromatic cytoplasm. Orthochromatic normoblast is smaller and has an eccentric nucleus with a condensed chromatin that becomes pyknotic at later stages of maturation. The lymphoid lineage also matures from the stem cell to the mature lymphocyte. A lymphocyte becomes either a B cell or a T cell. T cells are further subdivided into helper/ inducer cells, suppressor/cytotoxic cells, and natural killer (NK) cells. The T cells regulate the B cells and kill the infected cells in the body. The B cells are programmed at birth to react against a specific glycoprotein sequence or antigen. Each B cell has a surface immunoglobulin (antibody) that contains a specific kappa or lambda light-chain configuration. If the B cell encounters its antigen match, it undergoes clonal expansion, making millions of its copies and eventually differentiating into a plasma cell. Plasma cells pour out their immunoglobulin antibodies from their cytoplasm into the serum, thereby enabling the infection or  intruder proteins to be eliminated. Programmed cell death (apoptosis) as well as suppressor T cells prevents the clonal cells from becoming autonomous, and hence neoplastic.

cytomorpHoloGy (abNormal FeatureS) The normal BM (Figure  2.4A) shows myeloid and erythroid cells at different stages of maturation as well as scattered megakaryocytes, lymphocytes, and rare eosinophils (Figure  2.3). The quality of the aspirate with adequate

spicules and optimal Wright–Giemsa staining is crucial for proper analysis of the maturation sequence, cytomorphology, differential counts, assessment of the myeloid-toerythroid ratio (M:E), and identification of any abnormalities such as dysplasia or the presence of extrinsic cells. The BM aspirates are also used for evaluating the BM iron profile (amount and distribution of sideroblastic iron, including ring sideroblasts; Figure 2.4B) and for cytochemical stains such as MPO (Figure 2.4C) and nonspecific esterase (NSE; Figure 2.4D). Ring sideroblasts are defined by at least five iron granules per cell encircling one-third or more of the nuclear rim (normally less than four cytoplasmic ferritin particles can be seen in a subset of erythroid precursors) [1,2]. BM fibrosis [e.g., primary myelofibrosis (PMF), myelodysplastic syndrome (MDS) with fibrosis, metastatic tumor, or acute megakaryoblastic leukemia] may result in a nonrepresentative, hemodilute aspirate or a “dry tap.” In such a situation, a touch smear can be a valuable substitute. Lymphoid cells are normally present in the BM and account for approximately 10%–20% of marrow cells in adults. The number of lymphocytes generally rises with age and/or the degree of chronic immune stimulation. The presence of moderate to marked lymphocytosis of small lymphocytes is suggestive of a low-grade lymphoproliferative disorder, such as B-cell chronic lymphocytic leukemia (B-CLL; Figure 2.5A). Most of the lymphocytes of B-CLL have a scanty cytoplasm, round (or rarely irregular) nuclei, and a condensed, dark chromatin. Prolymphocytes are larger than small lymphocytes of B-CLL and have prominent nucleoli (Figure 2.5B). Lymphoplasmacytic lymphoma (LPL) is characterized by small lymphocytes, lymphocytes with more abundant cytoplasm (plasmacytoid lymphocytes), and plasma cells (Figure  2.5C). Mantle cell  lymphoma (MCL; Figure 2.5D) and follicular lymphoma (FL) display irregular, often indented (cleaved) nuclei. Lymphoid cells of Burkitt lymphoma (BL; Figure 2.5E) are of medium size and have a scanty, vacuolated cytoplasm; blastoid nuclei with finely dispersed chromatin; and prominent nucleoli. Large cell lymphomas, such as diffuse large B-cell lymphoma

C

A

B

D

FiGure 2.4 BM aspirate smear (A) with a normal (benign) pattern showing maturing myeloid and erythroid precursors (Wright–Giemsa staining, ×1000). Iron staining (B) showing an abnormal pattern in the form of ringed sideroblasts (Prussian blue, ×1000). MPO (C) and NSE (D) stains help to differentiate between granulocytic and monocytic cells.

25

Bone Marrow

B

A

C Burkitt lymphoma

Mantle cell lymphoma

FiGure 2.5

(A–F) Cytologic features in BM aspirate—Lymphoid cells (see text for details).

(DLBCL; Figure 2.5F), have large, often irregular nuclei; an abundant cytoplasm; and one to several nucleoli. The nuclear and cytoplasmic characteristics vary, depending on the type of large cell lymphoma. High-grade lymphomas may display cytologic features similar to BL. Because of the reticulin fibrosis that often accompanies the neoplastic lymphoid infiltrate in the BM and/or the tendency of some lymphomas (especially FL) for a paratrabecular distribution, the BM aspirate smear may not show lymphocytosis or atypical lymphoid cells, despite histologically proved involvement. The BM is often involved also in patients with monoclonal B-cell

Myeloblasts, type I

Erythroblasts

FiGure 2.6

lymphocytosis [3], benign condition defined by 2%–≤5%, 5%–10%, and >10% [6]. CMML with 1500/μL). Increased numbers of eosinophils in the BM (Figure 2.21) are often seen in both reactive and neoplastic processes. The differential diagnosis includes reactive eosinophilia in parasitic infections (strongyloidiasis, hookworm infection, filariasis, scabies, isosporiasis), bacterial

infections (chronic tuberculosis, resolving scarlet fever), HIV, allergic disorders (asthma, atopic dermatitis), drug hypersensitivity, Loeffler’s syndrome, skin diseases (such as angiolymphoid  hyperplasia), granulomatous disorders (sarcoidosis), neoplasms (leukemia, lymphoma, adenocarcinoma), abnormal T cells with aberrant phenotype (may or may not be clonal), vasculitis, collagen vascular disorders, inflammatory bowel disease, hypoadrenalism, interleukin (IL)-2 therapy, radiation exposure, cholesterol embolization, and Kimura’s disease. Chronic eosinophilic leukemia (CEL) is an MPN characterized by clonal proliferation of eosinophil precursors leading to persistent eosinophilia in the blood, BM, and peripheral tissues [2,23–26]. CEL, not otherwise specified (CEL, NOS), excludes patients with a Philadelphia chromosome (BCR–ABL fusion) or rearrangement of PDGFRA, PDGFRB, or FGFR1. The eosinophil count

I

40

Histology (Abnormal Patterns)

Essential thrombocythemia

CML

I

A

B Reactive myeloid hyperplasia

D

PMF (early phase)

C Parvovirus

E

E′

FiGure 2.20 BM with myeloid hyperplasia (increased M:E ratio): (A) CML; (B) ET; (C) PMF (early cellular phase); (D) reactive myeloid hyperplasia; (E and E′) increased M:E ratio due to marked erythroid hypoplasia (parvovirus infection; high magnification shows typical viral inclusion).

is >1.5  ×  10 9/L in the blood and the blasts are 1.5 × 109/L) persisting for at least 6 months resulting in tissue damage, without underlying reactive etiology and malignancy associated with eosinophilia. Cases fulfilling the criteria for HES but without tissue damage are classified as idiopathic hypereosinophilia. Eosinophilia associated with rearrangement of PDGFRA, PDGFRB, or FGFR1 is excluded from CEL and HES, and constitutes separate myeloid neoplasms [2,26]. Rearrangement of PDGFRA may lead

to eosinophilia with T-ALL, B-ALL, or myeloid leukemia  [37]. Figure  2.22 shows an algorithmic approach to patients with eosinophilia.

erythroId hyperplasIa (decreased M:E ratIo) BM with erythroid hyperplasia is typically seen in patients with anemia (most pronounced in pernicious anemia). Erythroid hyperplasia can be seen in hemorrhagic and hemolytic states, thalassemia, and secondary polycythemia, and after treatment with erythropoietin. Erythroid hyperplasia with cytomorphologic features of dyserythropoiesis is often seen in MDS and AEL [38–40]. The distinction between myeloid neoplasms with erythroid predominance, such as MDS, (especially MDS with erythroid hyperplasia), AML with myelodysplasia-related changes, therapy-related myeloid neoplasm, and AEL can be very difficult. Blasts > F Lymph node, liver, spleen, bone marrow Diffuse

M >> F Isolated peripheral adenopathy

F>M Mediastinum

M>F Mediastinum

Low-power pattern

Adolescent, young adults M>F Mediastinum, supradiaphragmatic sites > other Nodular > diffuse

Nodular

Sclerosis

+ (in NS subtype)

−

−

Variable (in the same tumor) +/− (may vary from area to area)

Cytology of tumor cells

Hodgkin cells, R–S cells, lacunar cells

Large cells resembling LP cells, centroblasts, or immunoblasts

CD19 CD20 CD79a PAX5 OCT2/BOB1 CD45 CD30

− −/+ − (rarely +) + (dim) − (may be weakly +) − +++

+ ++ ++ ++ ++ + + (weak)/− (rarely)

+ + + + + + +++

CD15 CD138 BCL6 MUM1 EMA EBV/EBER

+ (may be negative) − − (may be positive) +++ − −/+

+ ++ ++ ++ ++ + − (in rare cases weakly +) − − + (rarely −) +/− +/− − (rare cases +)

Large, multilobated cells with large folded or multilobated nucleolus and many nucleoli (LP; popcorn cells) + ++ ++ ++ ++ + −

Diffuse to vaguely nodular + (delicate compartmentalization sclerosis; broad collagenous bands) Large cells with pale cytoplasm, may resemble anaplastic cells or R–S cells

− (rarely +)

+ (may be −)

+/− +/− − −

+/− +/− − −

Sex Location

thrlBcl

nlPhl

gray Zone (hl/dlBcl)

feature

− − + − +/− −

PMBl

Sheets of tumor cells resembling PMBL

LP, lymphocyte predominant cells; NS, nodular sclerosis; R–S, Reed–Sternberg cells.

typically expresses CD10, MYC, CD43, BCL6, and Ki-67 (~100%), and is negative for BCL2. Apart from BL, MYC is positive in BCLU (“double-hit” lymphoma) and in a minor subset of DLBCLs. Figures 4.5 through 4.12 show the typical phenotypic profile of major mature B-cell tumors.

peripheral (mature/post-thymiC) t-Cell lymphoproliferations T-cell lymphomas express CD4 or CD8, pan-T-cell markers (CD2, CD3, CD5, and CD7), CD10 [angioimmunoblastic T-cell lymphoma (AITL)], CD15 [rare cases of ALCL and peripheral T-cell lymphoma (PTCL)], CD16 [natural killer (NK) cells or T-cell large granular lymphocyte (T-LGL) leukemia], CD25 [adult T-cell leukemia/lymphoma (ATLL), other], CD30 (ALCL, subset of PTCLs, pagetoid

reticulosis, scattered cells in other lymphomas), CD43, CD56 (NK cells), CD57 (T-LGL leukemia), CD103 [enteropathy-associated T-cell lymphoma (EATL), ALK (ALCL, ALK+], BCL6 (subset of AITLs), granzyme B, EBV (extranodal NK cell lymphoma, nasal type), T-cell receptor αβ (TCRαβ), and TCRδγ. Figures 4.13 through 4.17 show the typical phenotypic profile of some of the mature T-cell lymphomas.

hoDgkin lymphoma NLPHL (Figure 4.18) is characterized by expression of CD45, B-cell markers, EMA (subset), IgD (subset), and BCL6;  it is negative for MUM1 and CD30 (with rare exceptions). Neoplastic cells in classical HL express CD30 (strong membranous and Golgi staining), CD15 (majority of cases), CD20

111

Immunohistochemistry

B-small lymphocytic lymphoma/chronic lymphocytic leukemia (B-SLL/CLL) CD5+, CD11c+ (weak)/−, CD19+, CD20+ (dim), CD22+, CD23+, CD43+, CD79a+, CD79b−, BCL1−, BCL2+, BCL6−, FMC7−, LEF1+, SOX11−, Ki-67 low H&E; ×200

fIgure 4.5

CD20

II CD5

CD23

The most typical IHC profile of B-CLL/SLL.

Mantle cell lymphoma (MCL) CD5+, CD10−/+, CD11c−, CD19+, CD20+, CD22+, CD23− (rare cases weakly positive), CD43+ (rare cases negative), CD79a+, BCL1 (cyclin D1)+, BCL6− (rare cases positive), FMC7+, LEF1−, SOX11+, Ki-67 variable H&E; ×200

fIgure 4.6

CD20

CD5

BCL1

CD43

Ki-67

The most typical IHC profile of MCL. Marginal zone lymphoma (MZL)

CD5− (rare cases positive), CD10−, CD11c+/−, CD19+, CD20+, CD22+, CD23−, CD43−/+, CD79a+, CD103− (rare cases positive), annexin A1−, BCL1 (cyclin D1)−, BCL2+, BCL6−, Ki-67 low H&E; ×400

fIgure 4.7

The IHC profile of MZL.

CD20

112

Phenotypic Profiles of Major Hematopoietic Malignancies

Follicular lymphoma (FL)

II

CD5−, CD10+ (rare cases negative, but BCL6+), CD11c−, CD19+, CD20+, CD22+, CD23−/+, CD43−, CD79a+, BCL1 (cyclin D1)−, BCL2+ (rare cases negative), BCL6+, MUM1−, Ki-67 variable (correlates with grade) H&E; ×100

fIgure 4.8

CD20

BCL2

CD10

The IHC profile of FL.

Lymphoplasmacytic lymphoma (LPL) CD5−, CD10−, CD11c−, CD19+, CD20+, CD22+, CD23−, CD25+/−, CD38+ (may be negative), CD43+/−, CD79a+, BCL1 (cyclin D1)−, BCL2+, BCL6−, monoclonal IgM+ plasma cells (kappa or lambda), Ki-67 low H&E; ×200

CD20

Kappa-ISH

Lambda-ISH

IgM

CD20 CD2 CD C D2 D 20

fIgure 4.9

The IHC profile of LPL.

(subset), PAX5 (dim expression), MUM1, and EBV/EBER (subset), and are negative for CD45, CD22, CD79a, and pan-T-cell antigens (Figure 4.19).

aCute leukemias AML (with or without maturation) is most often positive for pan-myeloid markers (CD13, CD33, MPO), CD11c, HLA-DR, CD34, and/or CD117, and may be positive for CD4, CD7, CD19 [cases associated with t(8;21)], CD56, CD64, and TdT. Acute promyelocytic leukemia (APL) is positive for pan-myeloid markers, CD64 (dim expression), and CD117, and may be positive for CD34 and CD2 in hypogranular variant (HLA-DR is negative in APL). Acute

monoblastic leukemia is positive for monocytic markers (muramidase, CD68) and CD11c, and is often positive for CD2, CD4, CD56, and HLA-DR. Lymphoblasts express TdT, CD34, and lineage-specific markers depending on the type of leukemia [B-ALL is positive for CD19, CD22, and CD79a, and T-cell acute lymphoblastic lymphoma (T-ALL) is positive for pan-T-cell antigens and CD4/CD8 and may be positive for CD1a]. Mast cell disease is characterized by the expression of CD117, mast cell tryptase, and CD2. Langerhans cell lesions are typically positive for CD1a and S100. Blastic plasmacytoid dendritic cell neoplasm is positive for CD4, CD56, and CD123 (among other markers). Figures 4.20 through 4.25 shows the typical phenotypic profile of major types of acute leukemia.

113

Immunohistochemistry

Diffuse large B-cell lymphoma (DLBCL) CD5− (rare cases positive), CD10−/+, CD11c−, CD19+, CD20+, CD22+, CD23−/+, CD25+/−, CD30−/+, CD43− (rare cases positive), CD45+ (rare cases negative), CD79a+, BCL1 (cyclin D1)−, BCL2−/+, BCL6+/−, MUM1+/−, MYC−/+, PAX5+, Ki-67 moderate to high (30%−95%)

II

DLBCL with germinal center B-cell-like phenotype H&E; ×400

CD20

BCL6

CD10

CD20

MUM1

CD10

DLBCL with activated B-cell-like phenotype H&E; ×400

fIgure 4.10

The IHC profile of DLBCL.

Burkitt lymphoma (BL) CD5−, CD10+, CD19+, CD20+, CD22+, CD23−, CD38+, CD43+ (rare cases negative), CD79a+, BCL1−, BCL6+, BCL2− (rare cases may be weakly positive), MUM1− (rare cases positive), MYC+, EBV−/+, Ki-67 high (∼100%), TdT− H&E; ×400

fIgure 4.11

The typical IHC profile of BL.

CD20

CD10

BCL2

Ki-67

114

Phenotypic Profiles of Major Hematopoietic Malignancies

Plasma cell myeloma (PCM)

II

CD5−, CD10−, CD19−, CD20 (rare cases positive), CD22−, CD30+/−, CD38+, CD43+/−, CD45− (rare cases positive), CD56+/−, CD79a+/−, CD117+/−, CD138+, BCL1−/+, BCL2+, BCL6−, MUM1+ H&E; ×400

fIgure 4.12

MUM1

CD138

CD45

The typical IHC profile of PCM. Peripheral T-cell lymphoma, not otherwise specified (PTCL)

T–cell antigens variable (CD5 and CD7 often negative), CD4+ > CD8+ (rare cases double negative or double positive, CD30−/+, TIA1− (rare cases positive), CD10−, CD56−/+, TIA1−/+, BCL2+, BCL6−, ALK1−, CD25+/−, CD15− (rare cases positive), CD43+/−; PD1, CXCL13, CD10, and BCL6 are negative except for follicular variant of PTCL H&E; ×400

fIgure 4.13

CD4

CD3

CD8

The typical IHC profile of PTCL. Anaplastic large cell lymphoma (ALCL)

T-cell antigens (CD2, CD3, CD5, CD7) variable (may be negative), CD4+ > CD8+ (subset cases double negative), CD30+, TIA1+ (some cases negative, mostly ALK−), granzyme B+ (some cases negative, mostly ALK1−), CD10−, BCL2+/−, BCL6− (may be positive in ALK+ tumors), PD1− (rare cases positive), ALK1+/−, CD25+, CD15− (rare cases positive), CD43+/−, EMA+/− (often positive in ALK+ tumors)

H&E; ×400

fIgure 4.14

The typical IHC profile of ALCL.

ALK1

CD30

CD43

EMA

115

Immunohistochemistry

Angioimmunoblastic T-cell lymphoma (AITL) T-cell antigens (CD2, CD3, CD5, CD7) variable (often positive), CD4+, CD8−, CD10+ (may be negative), BCL6+/−, PD1+, CXCL13+, TIA1−, granzyme B−, scattered EBV+ cells and CD30+ cells (immunoblasts), expanded CD21+/CD23+ FDC meshwork H&E; ×200

fIgure 4.15

CD30

PD1

II EBER-ISH

The typical IHC profile of AITL.

Hepatosplenic T-cell lymphoma (HSTL) CD2+/−, CD3+, CD5−, CD7+/−, CD4−, CD8−/+, γδ+ (rare cases αβ+), TIA1+, granzyme B−/+, CD56+/− H&E; ×200

fIgure 4.16

CD2

CD3

CD5

CD7

CD56

TIA1

The typical IHC profile of hepatosplenic T-cell lymphoma (HSTL).

Enteropathy-associated T-cell lymphoma (EATL) CD2+/−, CD3+, CD5−, CD7+, CD4−, CD8−/+, CD25+/−, CD30+/−, CD56+, TIA1+, granzyme B+ H&E; ×200

fIgure 4.17

The typical IHC profile of EATL.

CD3

CD5

116

Phenotypic Profiles of Major Hematopoietic Malignancies

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)

II

CD19+, CD20+, CD22+, CD30−, CD45+, EMA+/−, BCL2−, BCL6+, CD10−, IgD+/−, EBV−, CD15−, PAX5+, MUM1−, T-cell rosettes (CD57+/PD1+/CD3+) around tumor cells H&E; ×40

fIgure 4.18

CD20

CD57

The typical IHC profile of NLPHL. Classical Hodgkin lymphoma (HL)

CD19−, CD15+/−, CD20−/+, CD30+, CD79a−, CD45−, CD138−, BCL2+/−, BCL6−/+, BOB1−/weakly positive, OCT2−/weakly positive, PAX5+ (dim), MUM1+, EBV/EBER−/+ H&E; ×400

fIgure 4.19

CD30

PAX5

CD15

EBER–ISH

The typical IHC profile of classical HL.

Acute myeloid leukemia with/without maturation (AML) CD2−, CD7−/+, CD11b−, CD14−, CD25+/−, CD33+, CD34+/−, CD38+, CD45+, CD64−/+, CD117+, MPO+ (may be on subset), TdT+/−, CD56−/+, HLA-DR+ (rare cases negative) H&E; ×400

fIgure 4.20

The typical IHC profile of AML.

CD34

HLA-DR

MPO

117

Immunohistochemistry

Acute promyelocytic leukemia (APL) CD2−/+ (hypogranular variant often positive), CD11b−, CD14−, CD33+, CD34− (hypogranular variant often positive), CD38+, CD45+, CD64+ (dim), CD117+, MPO+, CD56−/+, HLA-DR− H&E; ×400

fIgure 4.21

CD34

CD117

II MPO

HLA-DR

The typical IHC profile of APL. Acute monoblastic leukemia

CD2−/+, CD4+/−, CD11b+ (may be variable), CD14−/+ (may be partial), CD33+, CD34− (rare cases positive), CD38+, CD45+, CD64+, CD68+, CD117− (rare cases positive), MPO− (rare cases positive), CD56+/−, HLA-DR+ (rare cases negative), muramidase (lysozyme)+ H&E; ×400

fIgure 4.22

Muramidase

MPO

CD68

CD34

CD56

The typical IHC profile of acute monoblastic leukemia. Precursor B-lymphoblastic leukemia/lymphoma (B-ALL/LBL)

CD10+ (rare cases negative), CD19+, CD20− (rare cases positive), CD22+, CD33− (rare cases positive), CD34+ (rare cases negative), CD45+/−, PAX5+, TdT+ (rare cases negative) H&E; ×400

fIgure 4.23

The typical IHC profile of B-ALL/LBL.

TdT

CD10

CD34

118

Phenotypic Profiles of Major Hematopoietic Malignancies

Precursor T-lymphoblastic leukemia/lymphoma (T-ALL/LBL)

II

CD1a−/+, pan-T-cell antigens variable (usually CD3+ and CD7 strongly positive), CD4/CD8+ > CD4/CD8−, CD10+/−, CD33− (rare cases positive), CD34+/−, CD38+, CD45+, CD117− (rare cases positive), MPO−, TdT+/− H&E; ×400

fIgure 4.24

CD4

CD1a

CD8

TdT

The typical IHC profile of T-ALL/LBL. Blastic plasmacytoid dendritic cell neoplasm

CD2−/+, CD4+, CD3−, CD5−, CD7+/−, CD8−, CD33+/−, CD34−, CD38+, CD45+, CD56+, CD117−, CD123+,

MPO−, TdT−/+

H&E; ×200

fIgure 4.25

CD56

The typical IHC profile of blastic plasmacytoid dendritic cell neoplasm.

CD4

5 Differential Diagnosis

Immunophenotypic Markers II

Contents ALK ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 120 Annexin-1 ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 120 BCL1 (Cyclin D1)��������������������������������������������������������������������������������������������������������������������������������������������������������������������� 121 BCL2 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 121 BCL6 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 123 CD2 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 124 CD3 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 124 CD4 and CD8���������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 125 CD5 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 125 CD7 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 125 CD10 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 125 CD11b ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD11c ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD13 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD14 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD15 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD16 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD19 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD20 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 128 CD20− B-Cell Neoplasms �����������������������������������������������������������������������������������������������������������������������������������������������������131 CD21 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������131 CD22 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������131 CD23 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 132 CD25 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 132 CD30 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 135 CD33 and CD13������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 135 CD34 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 137 CD38 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 137 CD43 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 137 CD45 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 138 CD52 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 138 CD56 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 138 CD57 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 139 CD64 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 142 CD68 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 142 CD71 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 142 CD79 and CD81������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 142 CD103 ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 142 CD117 ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 142 CD123 ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 142 CD138 ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 144 CD163 ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 144 CD235a (GPHA)����������������������������������������������������������������������������������������������������������������������������������������������������������������������� 144 Cyclin D1 ���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 144 Cytotoxic Proteins (T-Cell-Restricted Intracellular Antigen-1, Granzyme B, Perforin) ���������������������������������������������������������� 144 CXCL13 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 145 EBV/EBER ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 145 EMA������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 145 119

120

II

Annexin-1

HLA-DR ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������147 HHV-8 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������147 Ki-67 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������147 LEF1 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 147 MUM1����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������147 MYC������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 148 PAX5 ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 149 PD-1 ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 149 SOX11 ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 150 TCR γ/δ ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 150 TdT�������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 151 References ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 151 In this Chapter we present the list of commonly used immunophenotypic markers and differential diagnosis of their expression in hematopoietic tumors�

ALK • Anaplastic large cell lymphoma (ALCL; ALK+) • Diffuse large B-cell lymphoma (DLBCL) with cytoplasmic anaplastic lymphoma kinase (ALK) expression ALCLs are divided into two categories: ALK+ ALCL and ALK− ALCL [1]� The t(2;5) disrupts the nucleophosmin (NPM) gene on 5q35 and the anaplastic lymphoma kinase (ALK) gene on 2p23, generating a novel NPM–ALK gene� NPM– ALK fusion leads to a chimeric mRNA molecule and a unique 80-kDa NPM–ALK fusion protein referred to as p80 [2]� The NPM–ALK fusion induces constitutive, ligand-independent activation of the ALK tyrosine kinase leading to an aberrant activation of cellular signaling pathways� The MYC may be a downstream target of ALK signaling and its expression is a defining characteristic of ALK+ ALCL [3]� The NPM–ALK fusion is present in ALCL, a T-cell malignancy characterized by CD30 expression� Patients with ALK− ALCL have worse prognosis and poor response to chemotherapy compared to patients with ALK+ tumors [but the prognosis is slightly better than in peripheral T-cell lymphoma (PTCL)]� Several cytogenetic and molecular studies have demonstrated that chromosomal aberrations other than the t(2;5) (p23;q35) may give rise to ALK fusion genes in ALCL� These alternative partners to NPM gene include TPM3 (nonmuscle ALCL

A

ALK

tropomyosin) associated with t(1;2)(q21;p23), TFG (TRK-fused gene) associated with t(2;3)(p23;q21), CLTC (clathrin heavychain gene) associated with t(2;17)(p23;q23), and MSN (moesin) [4–6]� The MSN gene at Xq11–12 acts as an alternative fusion partner for activation of ALK in ALCL and the lymphomas exhibit a distinctive membrane-restricted pattern of ALK labeling [7]� Primary cutaneous ALCLs are ALK− in majority of cases� Only sporadic ALK+ cutaneous ALK+ ALCLs have been reported� Most of them show a cytoplasmic ALK expression associated with a variant ALK rearrangement [different than t(2;5)], but cases with cytoplasmic and nuclear staining are also reported, most often in younger patients [8,9]� Fluorescence in situ hybridization (FISH) studies for ALK break-apart probe can identify ALK rearrangement in cases with both cytoplasmic ALK expression and cytoplasmic and nuclear staining� ALK+ DLBCLs are characterized by plasmablastic cytomorphology, aggressive clinical behavior, granular cytoplasmic expression of ALK by immunostaining, and presence of ALK–CLTC fusion as a result of t(2;17) or rarely, cryptic ALK insertions to chromosome 4q22–24 [10–13]� A few cases of ALK+ DLBCL with t(2;5) have also been reported [14,15]� Figure 5�1 shows ALK+ hematopoietic tumors�

Annexin-1 • Hairy cell leukemia (HCL) • T-cell acute lymphoblastic leukemia (T-ALL) • Granulocytes/myeloid precursors (benign and malignant) ALCL

B

ALK

DLBCL

ALK

C

FiGURe 5.1 ALK expression—Differential diagnosis: (A) ALCL with nuclear/cytoplasmic ALK expression; (B) ALCL with cytoplasmic ALK expression; (C) DLBCL with ALK expression (cytoplasmic)�

121

Immunophenotypic Markers

CD20

Annexin-1

II

FiGURe 5.2

HCL displays strong CD20 and annexin-1 expression�

Annexin-1 is rather a specific marker for HCL [16,17]� It is negative in majority of other B-cell lymphomas� In a series reported by Sherman et al� [16], annexin-1 was also positive in all three cases of precursor T-ALL and in two cases of lymphoplasmacytic lymphoma (LPL) (in addition, annexin-1 positivity was seen in one case of metastatic adenocarcinoma and one case of metastatic melanoma)� Marginal zone lymphomas (MZLs), including splenic MZL and HCL variant, are negative for annexin-1 [16,17]� Annexin-1 positivity is typically seen as a cytoplasmic and nuclear staining pattern (Figure 5�2)� Due to positive expression of annexin-1 by myeloid cells (at different stages of maturation), immunostaining of the bone marrow (BM) sample for annexin-1 is not useful in monitoring patients with HCL after treatment [for minimal residual disease (MRD)]�

BCL1 (CYCLin D1) • Mantle cell lymphoma (MCL) • Plasma cell myeloma (PCM) • HCL The BCL1 gene (also known as CCND1 and PRAD1) is located on chromosome 11q13 and codes for cyclin D1� It is activated by its juxtaposition near the enhancer region of the immunoglobulin heavy-chain locus on chromosome 14� This phenomenon is caused by t(11;14)(q13;q32) translocation and defines MCL� CCND1 transcript or its encoded protein (cyclin D1/BCL1) can be detected by polymerase chain reaction (PCR), cytogenetic/FISH, or immunohistochemical staining� BCL1 (cyclin D1) shows a nuclear pattern of immunostaining (Figure 5�3)� Almost all cases of MCL are positive for BCL1� Very rare cases of MCL which are negative for BCL1 by immunohistochemistry often show a blastoid morphology and are positive for t(11;14)/CCND1 rearrangement and SOX11 expression [18]� In addition to MCL, BCL1 expression is seen in the subset of PCM and HCL� In PCM, the expression of BCL1 is associated with either t(11;14)(q13;q32) or extra copies of chromosome 11� PCM patients with the t(11;14) or trisomy 11

significantly overexpress BCL1 in comparison with patients without 11q abnormalities, who have cyclin D1 mRNA levels similar to healthy donors� In HCL, the intensity of expression of BCL1 is lower than in MCL and the immunohistochemical positivity is not associated with t(11;14)/CCND1 rearrangements or CCND1 gene amplification� In a series reported by Sherman et al� [16], the majority of HCL cases (96%) were positive for BCL1, approaching the sensitivity of annexin-1� Similar proportion (100%) of HCLs staining with BCL1 was also reported by Miranda et al� [19]� Rare cases of DLBCL have been reported to express BCL1 by immunohistochemistry [20,21]� It is likely that these lymphomas represent an aggressive variant of MCL (pleomorphic or blastoid) rather than DLBCL� In the published reports, BCL1+ DLBCLs are usually CD5+ and display nongerminal center phenotype (CD10 is not expressed; BCL6 and MUM1 are often positive)� There is no CCND1 rearrangement, but occasional cases show both MYC and BCL6 rearrangements (double hit) [20]� The staining with SOX11 and FISH studies may be helpful in these difficult and unusual cases�

BCL2 • Benign B and T cells (CD10+ benign germinal center B cells are negative) • Follicular lymphoma (FL; majority) • B-cell lymphomas [except for Burkitt lymphoma (BL)] • T-cell lymphoma (subset; not in ALK+ ALCL) • Other hematopoietic tumors The B-cell lymphoma/leukemia 2 (BCL2) gene on chromosome 18q21 encodes a 26-kDa protein that inhibits apoptosis through the mitochondrial pathway� The BCL2 gene was originally discovered in FLs with t(14;18)(q32;q21) translocation [22]� The t(14;18) places BCL2 under the control of the immunoglobulin heavy-chain (IGH) Eμ enhancer that induces production of high levels of BCL2 protein� Apart from t(14;18), variant translocations leading to juxtaposition

122

BCL2

Mantle cell lymphoma

BCL1

Mantle cell lymphoma, blastoid

BCL1

Plasmablastic lymphoma

BCL1

II

A

B Plasma cell myeloma

BCL1

C

D Hairy cell leukemia

Hairy cell leukemia, lymph node

BCL1

E

BCL1

F

FiGURe 5.3 BCL1 (cyclin D1) expression—Differential diagnosis: (A) MCL; (B) MCL, blastoid variant; (C) PCM; (D) plasmablastic lymphoma; (E) HCL (BM); (F) HCL (lymph node)�

of the BCL2 with either IGK (on 2p11) or IGL (on 22q11) have been recognized in B-cell lymphomas� Clonal BCL2–IGH rearrangements by PCR can be observed in healthy individuals without evidence of FL [23,24]� BCL2 protein can easily be detected by routine immunohistochemistry� BCL2 is widely expressed in normal lymphoid tissues, but is absent in benign CD10+ germinal center B cells� BCL2 positivity (Figure 5�4) is very helpful in

differentiating FLs (BCL2+) from reactive follicular hyperplasia (BCL2−)� Some FLs do not express BCL2, suggesting the inhibition of apoptosis due to other factors (e�g�, Bcl-xL) rather than BCL2 overexpression [25]� Benign B cells from mantle and marginal zones are BCL2+, and therefore, immunostaining with BCL2 is not helpful in the diagnosis of MCL and MZL [including mucosa-associated lymphoid tissue (MALT) lymphoma]�

123

Immunophenotypic Markers

Follicular lymphoma

CD10

Follicular hyperplasia

CD10

II BCL2

BCL2

A

FiGURe 5.4

B

Positive BCL2 expression helps to differentiate FL (A; BCL2+) from reactive follicular hyperplasia (B; BCL2−)�

BCL2 expression has been detected frequently in aggressive non-Hodgkin lymphomas, regardless of t(14;18), and is associated with unfavorable prognosis [26–29]� The BCL2 gene amplification is another important mechanism for BCL2 protein overexpression in DLBCL [30–32]� High BCL2 protein expression is more frequent in B-cell lymphomas (51%) than in T-cell non-HL (NHL; 17%) and is heterogeneously distributed among the different histological subtypes [27]� DLBCL displays BCL2 expression in 30%–60% of  cases, more frequently in nodal than in extranodal tumors [30,33– 35]� BLs do not express BCL2, which helps to differentiate them from aggressive DLBCL and “gray zone” (double-hit) lymphomas� BCL2+ DLBCL with t(14;18) may represent a progression from FL� In DLBCL, BCL2 protein-positive cases significantly outnumbered the cases with t(14;18), suggesting that mechanisms other than translocation are operative in DLBCL [28]� Alternative mechanisms for BCL2 protein expression include, among others, increased BCL2 copy number (e�g�, 18q+) or transcriptional deregulation by nuclear factor (NF)-κB [36]� Cases with t(14;18) and additional chromosomal aberrations have worse prognosis than DLBCL with t(14;18) only� Hermine et al� [27] reported the independent effect of BCL2 protein expression to be predictive of poor disease-free survival, in agreement with the role of BCL2 in chemotherapy-induced apoptosis� Multivariate analysis confirmed the significant benefit for survival and event-free survival when rituximab is added to standard chemotherapy regimen in BCL2+ DLBCL, suggesting that rituximab is able to prevent chemotherapy failure in patients with BCL2 protein overexpression [37]� Maartense et al� [38] reported the difference in the impact of BCL2 overexpression on prognosis between elderly patients (>65 years) and younger patients (80%) [45–48]� BCL6 is positive in the subset of classical HLs� AITL displays aberrant CD10 and less often BCL6 expression by neoplastic T cells� CD10 and/or BCL6 expression has been reported in the subset of MCLs

124

CD3

Follicular lymphoma

BCL6

DLBCL

BCL6

AITL

BCL6

II

A

B NLPHL

BCL6

C

FiGURe 5.5

D

BCL6 expression—Differential diagnosis: (A) FL; (B) DLBCL; (C) NLPHL; (D) AITL�

(10%–12%) [49,50]� BCL6 is expressed by PCSM-TCL and rare cutaneous T-cell lymphomas with T-follicular helper cells (TFH) phenotype [51,52]�

CD2 • Peripheral (mature) natural killer (NK) and T-cell lymphoproliferations • T-ALL/lymphoblastic lymphoma (LBL) • Blastic plasmacytoid dendritic cell neoplasm (BPDCN; subset) • AML (especially acute monoblastic leukemia) • Hypogranular variant of acute promyelocytic leukemia (APL) • Mast cell proliferations CD2 is a pan-T-cell antigen expressed by immature and mature T-cell disorders, including precursor T-ALL/LBL; PTCL, not otherwise specified (PTCL, NOS); T-cell prolymphocytic leukemia (T-PLL); mycosis fungoides/Sézary syndrome (MF/ SS); adult T-cell leukemia/lymphoma (ATLL); T-cell large granular lymphocyte (T-LGL) leukemia; NK cell lymphoma/ leukemia; extranodal NK/T-cell lymphoma, nasal type (ENKTL); aggressive NK cell leukemia; AITL; ALCL; and cutaneous T-cell lymphoma� The subset of AMLs, especially acute monoblastic leukemia, mixed phenotype acute leukemia

(MPAL), and BPDCN, may show an aberrant expression of CD2� Neoplastic mast cell proliferations show a coexpression of CD2 and CD25� Hypogranular variant of APL often shows an aberrant CD2 expression (typical, hypergranular forms are most often negative)�

CD3 • • • •

Peripheral (mature) T-cell lymphoproliferations T-ALL/LBL Primary effusion lymphoma (PEL; rare cases) Bilineal lymphoma in 8p11 myeloproliferative syndrome

CD3 is the most specific marker for T-cell lymphoproliferations, being positive in both mature (peripheral) and subset of immature neoplasms� Immature T cells that lack T-cell receptor gene rearrangements are negative for surface CD3� Precursor T-lymphoblastic neoplasms (T-ALL/LBL) usually do not express surface CD3 when analyzed by flow cytometry (cytoplasmic CD3 staining by flow cytometric and immunohistochemistry is often positive)� CD3 may be occasionally positive in PEL, a high-grade neoplasm of B-cell lineage� Bilineal lymphoma that is characterized by an infiltrate composed of numerous eosinophils, T lymphoblasts, and myeloblasts, and occurs in the setting of 8p11 myeloproliferative

125

Immunophenotypic Markers

syndrome (EMS) often shows the expression of CD3 by both lymphoid and myeloid components�

CD4 AnD CD8 CD4 antigen is expressed by T-helper cells, monocytes, macrophages, and dendritic cells� The examples of hematolymphoid neoplasms expressing CD4 or CD8 are presented in Figure 5�6� CD4 is positive in AITL, MF/SS, ALCL, T-PLL, ATLL, BPDCN, majority of PTCL, and subset of myeloid proliferations including AML, chronic myelomonocytic leukemia (CMML), acute monoblastic leukemia, and APL, and histiocytic tumors� Only rare cases of precursor T-ALL/LBL have restricted CD4 expression; the majority of cases are either dual CD4/CD8+ or dual CD4/CD8−� Thymocytes from thymus or thymoma are dual CD4/CD8+� CD8 stains cytotoxic/suppressor T cells and subset of NK cells� CD8 is expressed by a significant subset of T-LGL leukemias and enteropathy-associated T-cell lymphomas (EATLs), and peripheral (mature) T-cell lymphoproliferations including PTCL, T-PLL, ALCL, and subcutaneous panniculitis-like T-cell lymphoma (SPTL)� In the latter, CD8+ T cells surround the adipocytes forming a characteristic low-power pattern� Only rare cases of T-ALL/LBL have restricted CD8 expression� Hepatosplenic T-cell lymphoma (HSTL), T-LGL leukemia with NK cell phenotype, and aggressive NK cell leukemia are CD4−/CD8−� Very rare cases of mature B-cell lymphoproliferations may display aberrant expression of CD8 [most often chronic lymphocytic leukemia (CLL)]�

CD5 • Peripheral (mature) T-cell lymphoproliferations • T-ALL/LBL • B-cell CLL (B-CLL)/small lymphocytic lymphoma (SLL) • MCL • De novo CD5+ DLBCL • MZL (subset) • Intravascular large B-cell lymphoma (subset) CD5, a 67-kDa pan-T-cell antigen, is expressed by mature and immature T-cell lymphoproliferations� The expression is often dimmer than in benign T cells� Benign CD5+ B cells may be present in blood and lymphoid tissues, more often in children and younger patients� The expression of CD5 is often aberrantly missing in neoplastic T-cell processes� HSTL and EATL are most often CD5−� NK cells and NK cell tumors are CD5−� Myeloblasts from AML more often show an aberrant expression of CD2 and CD7 than CD5, but MPAL may be CD5+� Among B-cell disorders, CD5 is typically expressed in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) and MCL� B-CLL/SLL differs from MCL by CD23 positivity, although some cases of B-CLL may be CD23− and rare cases of MCL may be CD23+� Therefore, definite differentiation between MCL and CLL should be based on the status of BCL1 (cyclin D1) by

FISH or immunohistochemistry� Only a small proportion of DLBCL expresses CD5� Lack of BCL1 (cyclin D1) and SOX11 expression and lack of history of B-CLL/SLL distinguish de novo CD5+ DLBCL from MCL and Richter’s syndrome, respectively� The prognosis of de novo CD5+ DLBCL is markedly worse than that for CD5− DLBCL� The subset of intravascular lymphomas, MZL (MALT lymphoma), and rare cases of LPL shows CD5 expression� CD5+ splenic MZLs do not differ clinically from typical CD5− cases, but may show CD13 expression [53]� Only a very few cases of CD5+ FL have been reported� B-cell ALL (B-ALL), HCL, and BL are CD5−�

CD7 • • • • •

Peripheral (mature) NK and T-cell lymphoproliferations T-ALL/ LBL AML (subset) MPAL BPDCN (subset)

CD7 is a membrane-bound glycoprotein that is expressed very early in T-cell development� CD7 is a pan-T-cell antigen expressed by peripheral (mature) T-cell lymphoproliferations (e�g�, PTCL, T-PLL, ATLL, MF, SS, T-LGL leukemia, NK cell leukemia/lymphoma), and T-ALL� CD7 is very often aberrantly expressed (either negative or dim) in peripheral T-cell disorders� In precursor T-cell neoplasms, CD7 is most often positive and shows a bright expression when analyzed by flow cytometry (only ~2% cases are CD7−)� Other hematopoietic tumors that may express CD7 include BPDCN, MPAL, and AML� APL and B-cell lymphoproliferations are typically CD7−�

CD10 • FL • BL • Large B-cell lymphoma, unclassifiable with features intermediate between BL and DLBCL (BCLU; “double-hit” lymphoma) • DLBCL (subset) • B-ALL/LBL [common ALL antigen (CALLA+)] • T-ALL/LBL (subset) • MPAL (B-ALL/AML) • AITL (majority) • Granulocytes (neutrophils) • HCL (subset) • MCL (subset) • PCM (rare cases) • Plasmablastic lymphoma (rare cases) • Thymocytes CD10 (Figure 5�7) is a 100-kDa cell surface metalloendopeptidase, known as CALLA� Among hematopoietic cells, CD10 is expressed by immature B and T cells, germinal center B cells, granulocytes (neutrophils), majority of FLs, subset of DLBCLs, BL, BCLU, AITL, and subset of MCLs and HCLs�

II

126

CD10

CD8 expression

Forward scatter

II

Forward scatter

CD4 expression

CD8

CD4 Peripheral T-cell lymphoma

T-cell lymphoma, NOS

Monocytes/monoblasts

CD8 Forward scatter

T-LGL leukemia

NSE T-PLL

CD16 T-PLL

Blastik NK-cell leukemia

CD4

CD8

CD8 Sézary’s syndrome

Mycosis fungoides

Anaplastic large cell lymphoma CD8

ALCL

T-ALL

CD30

Precursor T-lymphoblastic leukemia CD8

CD4

B-CLL/SLL with aberrant CD8

APL

CD8

AML

CD19

FiGURe 5.6

Expression of CD4 and CD8�

γδ T-cell lymphoma

CD8

ALK1

CD57

127

Immunophenotypic Markers

Follicular lymphoma

CD10

CD10

DLBCL

Burkitt lymphoma

CD10

II

A BCLU (BM clot)

BCLU

CD10

D

CD10

CD10

AITL

B-ALL

CD10

CD10

Cutaneous CD4+ T-cell lymphoma

CD10

I

H

G

MCL

F

E Hairy cell leukemia

J

C

B

PCM

CD10

MUM1

CD10

K

FiGURe 5.7 CD10 expression—Differential diagnosis: (A) FL; (B) DLBCL; (C) BL; (D) “double-hit” lymphoma; (E) large B-cell lymphoma with features intermediate between BL and DLBCL; (F) MCL; (G) HCL; (H) AITL; (I) cutaneous CD4+ small/intermediate T-cell lymphoma; (j) precursor B-ALL/LBL; (K) PCM�

Among precursor lymphoid neoplasms, CD10 is more often expressed by B-ALL than by T-ALL� The expression of CD10 in B-ALL is very bright when analyzed by flow cytometry (immunohistochemistry shows a strong membranous staining)� Benign B-cell precursors (hematogones) are

CD10+� CD10 is not expressed by erythroid and myeloid precursors� Rare cases of PCM and acute monoblastic leukemia may be CD10+� Among T-cell lymphomas, CD10 is positive in AITL� Similarly to AITL, PCSM-TCLs express BCL6, programmed

128

II

death-1 (PD-1), and CXCL13, but are CD10− [52]� CD10 is expressed by rare cutaneous T-cell lymphomas with TFH phenotype [51]� Lack of CD10 on mature granulocytes, although not specific, is often associated with myelodysplasia� Occasional FLs may be CD10− (some cases may show discrepant flow cytometric and immunohistochemistry results with CD10 staining)� FL without t(14;18) and with BCL6 rearrangement defines a subtype of FL which is often CD10 negative� Stromal cells (uterus), certain sarcomas, and malignant melanoma may express CD10�

CD11b CD11b is expressed by monocytes (macrophages), granulocytes, dendritic cells, and NK cells� It is negative in mature B and T cells, but activated CD8+ T cells (e�g�, in viral infections) and some T-LGL leukemia cells are often CD11b+ (at least partially)� AMLs with monocytic differentiation are usually CD11b+, whereas myeloblasts from AML with or without maturation only sporadically display aberrant expression of CD11b� The CD11b+ AML is seen more often in cases with unfavorable cytogenetics, and the expression of CD11b correlates with monosomal karyotype (defined as two or more autosomal monosomies or one monosomy with other structural aberrations) and predicts an extremely poor prognosis [54]� APLs are CD11b negative� Lack of CD11b and CD13 expression in transient myeloproliferative disorder helps to differentiate it from AML in infants with Down syndrome, which are usually CD11b+/CD13+ [55]�

CD11c CD11c is a member of the superfamily of glycoproteins that mediate cell–cell and cell–matrix interaction and is expressed strongly on monocytes and tissue macrophages, and less strongly on granulocytes and some lymphocyte subsets� Bright expression of CD11c is typical for HCL and most cases of acute monoblastic leukemia� Other AMLs may express CD11c (dim to moderate or partial), but APL is CD11c−� The CD11c may be dimly to moderately positive in other hematologic malignancies including B-CLL, MZL, and HCL variant (HCL-V)� CLL may be CD11c positive (expression has been reported in 4%–89% of cases) [56–58]� In contrast to CLL, MCLs are CD11c− (or only sporadically positive) [58]� Kraus et al� found CD11c expression in 27% of CLL and only in 2 of 44 cases of MCL� Other published data reported CD11c positivity in MCL in 5�4% [56,59–62]� NK cells and activated benign T cells (usually CD8+) are often CD11c� BPDCN is CD11c− [63]�

CD13 See “CD33 and CD13” section�

CD14 CD14 is brightly expressed by benign monocytes and majority of neoplastic monocytes from CMML and monocytic component of acute myelomonocytic leukemia (Figure 5�8)�

CD20

Monoblasts and promonocytes from acute monoblastic leukemia are either CD14 – or display variable expression of CD14, ranging from negative to moderate/bright by flow cytometric analysis� Often, only a small subset of neoplastic monocytes from acute monoblastic leukemia expresses CD14� Benign granulocytes are CD14 –, but granulocytes/ maturing myeloid precursors from the subset of chronic myeloproliferative neoplasms [e�g�, polycythemia vera (PV) or chronic myeloid leukemia (CML)] may be CD14+ (mild upregulation of CD14 expression)� Similarly, aberrant expression of CD14 may be seen in transient myeloproliferative disorders in children with trisomy 21�

CD15 CD15 is expressed by granulocytes, monocytes, neoplastic cells in CML, subset of AMLs, and majority of neoplastic cells in classical HL (Figure 5�9)� Only very rare cases of PTCLs (including ALCL and PTCL) and extremely rare cases of NLPHL display aberrant CD15 expression [64]� CD15 is often present in nonhematopoietic malignancies�

CD16 CD16 is often expressed by neutrophils, T-LGL leukemia, NK cell proliferations, HSTL, and subset of neoplasms with monocytic differentiation (Figure 5�10)� Benign monocytes usually display dim expression of CD16 on the subset� Myeloblasts and atypical promyelocytes of APL are CD16−� Dysplastic granulocytes often display aberrant downregulation of CD16 (lack of expression of CD16 suggests dysgranulopoiesis and leftward shift)�

CD19 • • • • • •

B-cell lymphomas B-ALL/LBL NLPHL Benign plasma cells and rare plasma cell neoplasms MPAL (AML/B-ALL) AML (subset)

The CD19 molecule is a 95-kDa glycosylated type I integral membrane protein whose expression is limited to B cells and follicular dendritic cells (FDCs)� CD19 is expressed by mature and immature B-cell neoplasms, NLPHL, benign plasma cells, very rare plasma cell neoplasms, MPAL, and subset of AMLs, usually associated with t(8;21)/RUNX1– RUNX1T1 (which often coexpresses CD19 and CD56)�

CD20 • • • • •

B-cell lymphomas B-ALL/LBL (rare cases) PCM (rare cases) NLPHL Classical HL (subset)

129

Immunophenotypic Markers

Granulocytes

Polycythemia vera

Negative staining

II

A

B CD14

Forward scatter

Acute myelomonocytic leukemia

Acute monoblastic leukemia

D

C

CD14 CMML

CML

F

E CD14 Transient myeloproliferative disorder

Blasts (CD14−)

Granulocytes with dim CD14

Blasts (CD34+)

G CD14

CD34

FiGURe 5.8 CD14 expression—Differential diagnosis� Benign granulocytes (maturing myeloid cells) are negative for CD14 (A)� Aberrant (dim) expression of CD14 (upregulation) may be observed in the subset of myeloproliferative disorders [e�g�, PV (B); CML (F)] and transient myeloproliferative disorder (G)� Benign monocytes and neoplastic monocytes in the majority of cases of CMML (E) and the monocytic component of acute myelomonocytic leukemia (C) display bright expression of CD14� In contrast, immature monocytic population from acute monoblastic leukemia is either CD14 – or displays variable expression (D; arrows)�

CD20 is present on late pre-B cells and mature B cells, but not on precursor cells or terminally differentiated plasma cells (CD20 appears on the B-cell surface between the immunoglobulin light-chain gene rearrangement and the expression of surface immunoglobulin: It is expressed after CD19

and CD10 but before cytoplasmic μ chain expression)� The function of CD20 remains poorly understood, although it has been implicated in B-cell activation, regulation of B-cell growth, and regulation of transmembrane calcium flux� Apart from B-cell lymphoproliferations, CD20 is expressed

130

CD20

Hodgkin lymphoma (classical)

CMML

CD15

CD15

II

A

B Acute myeloid leukemia

NLPHL (rare cases)

CD15

CD15

D

C ALCL (rare cases)

CD15

E

PTCL (rare cases)

CD15

F

FiGURe 5.9 CD15 expression—Differential diagnosis: (A) classical HL; (B) CMML; (C) AML; (D) NLPHL (rare cases fixed in B5); (E) ALCL (rare cases); (F) PTCL, NOS (rare cases)�

on neoplastic cells in NLPHL, subset of classical HLs, and occasional plasma cell neoplasms� Rituximab (Rituxan), the monoclonal antibody directed against the CD20 antigen, is widely used for the treatment of B-cell non-HLs� B-cell lymphomas with expression of CD20 may lose the CD20 positivity after anti-CD20 immunotherapy� Rituximab was shown to mediate antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity, and to induce nonclassic (caspase-independent) apoptosis of lymphoma cells in vitro� In B-CLL, B-cell prolymphocytic

leukemia (B-PLL), and MCL, the levels of CD20 measured by the standard immunofluorescence or using calibrated beads, correlated linearly with the lytic response to rituximab regardless of diagnostic group� Patients most likely to respond to rituximab can be predicated by overexpression of BCL2, Ki-67 expression, polymorphism for Fc receptor (FcR) γIII (binding site for rituximab), and DNA microarray� Favorable results were observed in the treatment of B-ALL with rituximab in combination with routine chemotherapy, especially those with mature phenotype� In multiple myeloma  (MM), CD20

131

Immunophenotypic Markers

Benign granulocytes

AML

II

A

B CD16 CMML

Forward scatter

MDS

C

D CD16 T-LGL leukemia

Acute monoblastic leukemia

F

E CD16

FiGURe 5.10 CD16 expression� CD16 is expressed by benign granulocytes (A; neutrophils), but myeloblasts (B; arrow) and promyelocytes are negative� Dysplastic granulocytes from the subset of MDS may display aberrant negative CD16 [C; arrow, downregulation)� Aberrant positive CD16 may be seen in neoplastic monocytes from CMML (D) and acute monoblastic leukemia (E)� CD16, separately or along with CD56 and/or CD57, may be expressed in T-LGL proliferations (F)�

expression was reported to be associated with small mature plasma cell morphology and t(11;14)� MM cases expressing CD20 showed the tendency toward a poorer prognosis with lower survival�

CD20− B-Cell Neoplasms B-cell neoplasms with negative CD20 expression include DLBCL (rare cases), plasmablastic lymphoma, PEL, majority of B-ALL/LBL, DLBCL with ALK expression, majority of classical HL, majority of PCM, and B-cell lymphomas after rituximab treatment (Figure 5�11)�

CD21 CD21 is expressed by mature B cells, FDCs and FDC-derived neoplasms, subset of T cells, and normal thymocytes� CD21 can

be expressed by mature B-cell lymphoproliferative disorders, subset of BLs, and subset of DLBCLs� CD21 is negative in precursor lymphoblastic lymphoma, HCL, and PCM� Immunostaining with CD21, showing the pattern of FDC meshwork, helps in differential diagnosis between MZL versus reactive hyperplasia, NLPHL versus THRLBCL, AITL versus reactive hyperplasia or PTCL, and FL versus DLBCL (Figure 5�12)�

CD22 CD22 is expressed by B-cell lymphoproliferations (both mature and precursor)� CD22 expression is bright in HCL and splenic MZL, and dim in other B-cell lymphoproliferations [65–68]� Plasma cell neoplasms are CD22−� Huang et al� [68] reported the usefulness of aberrant expression of CD22 for flow cytometric detection of clonal B cells admixed with numerous benign polyclonal B cells� Basophils show non-specific staining with CD22�

132

CD25

CD20

Diffuse large B-cell lymphoma

PAX5

Primary effusion lymphoma

CD20

II

A

B DLBCL with ALK expression

CD20

ALK

Plasmablastic lymphoma

CD20

EBER

C

D B cells (CD22+)

B cells (CD20−)

Forward scatter

B cells (CD19+)

E

T cells (CD19−)

F

CD19

G

T cells (CD22−)

CD20

CD22

FiGURe 5.11 B-cell lymphomas with negative CD20 expression—Differential diagnosis: (A) DLBCL (rare cases); (B) PEL; (C) DLBCL with ALK expression; (D) plasmablastic lymphoma; (E–G) after Rituxan treatment [lymphomatous cells are positive for CD19 (E) and CD22 (G), but display negative staining with CD20 (F; arrow)]�

CD23 • FDCs/FDC tumors • B-small lymphocytic lymphoma/chronic lymphocytic leukemia (B-SLL/CLL) • FL (subset) • PCM (subset) • Acute monoblastic leukemia (rare cases) CD23 may be expressed by benign B cells and is strongly expressed by the majority of B-CLL/SLL (Figure 5�13)� Coexpression of CD5 and CD23 is typical for B-CLL/SLL� In contrast, most MCL cases are CD23− (only rare cases of MCL may be dimly CD23+, and this aberrant expression of CD23 may also be associated with p53 expression and atypical cytology)� Subset of FLs and DLBCLs may be CD23+� Other hematopoietic tumors that may display aberrant CD23 expression include acute monoblastic leukemia and

some CMML� Analysis of CD23 helps to assess FDC meshwork when evaluating nodal lymphomas (see “CD21” section)� Subset of benign plasma cells and plasma cell tumors express CD23�

CD25 • • • • • •

HCL B-cell lymphomas (subset) PTCL (subset) ATLL ALCL AML (subset)

The CD25 [interleukin-2 receptor (IL-2R)] is expressed by ATLL, HCL, and subset of other T- and B-cell lymphoproliferative disorders (Figure 5�14)� The expression of CD25 in cutaneous T-cell lymphoma may identify a subset of patients

133

Immunophenotypic Markers

Follicular lymphoma

Benign lymph node

II

A

B Marginal zone B-cell lymphoma

Nodular lymphocyte predominant Hodgkin lymphoma

D

C Angioimmunoblastic T-cell lymphoma

E

Follicular dendritic cell sarcoma

F

FiGURe 5.12 CD21 expression—Differential diagnosis: (A) Benign lymph node—intact meshwork of FDCs visualized by CD21 immunohistochemical staining; (B) FL—neoplastic follicle with expanded, delicate follicular dendritic meshwork; (C) MZL—partially preserved residual follicles with focal distortion of dendritic cell meshwork; (D) NLPHL—prominent expansion of FDC meshwork; (E) AITL— expanded and distorted pattern of FDC meshwork� Neoplastic T cells are negative for CD21 and FDC sarcoma (F)�

at risk undergoing large cell transformation� The expression of CD25 in T-cell lymphoproliferations may be variable depending on the anatomical site: Decreased CD25 expression was noted in the lymph nodes compared to the blood or skin, implicating difference in response to IL-2R-targeted

immunotherapy� CD25 is often expressed by ALCL� The subset of PTCLs, NOS, may be CD25+, whereas AITL and HSTL are usually CD25−� Markedly elevated serum soluble IL-2R levels are a particularly prominent feature of certain hematologic malignancies, such as human T-lymphotropic

134

CD25

CD23 staining in reactive follicle

B-SLL

CD23

B-CLL (BM)

CD23

II

CD5

B

A Mantle cell lymphoma (spleen)

C AITL

CD23

BCL1

D

CD23

E Acute monoblastic leukemia Forward scatter

Forward scatter

CMML

G F

CD23

CD23 Plasma cells

Follicular lymphoma

CD23

Hodgkin lymphoma

CD23

CD15

CD23

H

I

J

FiGURe 5.13 CD23 expression—Differential diagnosis: (A) B cells and dendritic cells in reactive lymph node (note prominent polarization typical for benign follicle); (B) B-SLL; (C) B-CLL; (D) MCL with unusual CD23 expression; (E) expanded FDC meshwork in AITL; (F) acute monoblastic leukemia; (G) CMML; (H) plasma cells; (I) FL; (j) classical HL with unusual CD23 expression by Hodgkin cells and Reed–Sternberg cells�

retrovirus type I-associated ATLL and HCL, reflecting tumor burden and response to therapy� CD25 is not expressed by HCL-V, a rare chronic B-cell lymphoproliferative disorder clinically and morphologically distinct from classical HCL� HCL-V is thought to represent a hybrid between

prolymphocytic leukemia, MZL, and HCL which does not response to typical HCL treatment and has shorter survival� The subset of AMLs may be positive for CD25� The expression of CD25 correlates positively with FLT3-internal tandem duplication (ITD), DNMT3A, and NPM1 mutations [69]� The

135

Immunophenotypic Markers

Adult T-cell leukemia/lymphoma

CD25 A

B

CD103 Anaplastic large cell lymphoma

CD25 expression—Differential diagnosis: (A) HCL; (B) ATLL; (C) ALCL (BM); (D) PTCL�

adverse prognostic impact of FLT3-ITD+ AML was restricted to CD25+ patients and CD25 expression improved AML prognostication independent of integrated, cytogenetic, and mutational data, such that it reallocated 11% of patients with integrated intermediate-risk disease based on cytogenetic/ mutational profiling to the unfavorable risk group with high risk of relapse [69]�

CD30

• • • • • • • • • • • • •

CD25

D

C

• • • • • •

CD3

Peripheral T-cell lymphoma, unspecified CD25

FiGURe 5.14

II

CD25

Hairy cell leukemia

Classical HL ALCL DLBCL (subset) PEL FL (rare cases) Primary mediastinal large B-cell lymphoma (PMBL) PTCL, NOS (subset) Primary cutaneous ALCL Pagetoid reticulosis Lymphomatoid papulosis (LYP) Lymphomatoid granulomatosis (LYG) Benign (activated) cells (B and T lymphocytes/ immunoblasts, plasma cells) Atypical cells (immunoblasts) in Epstein–Barr virus (EBV)-lymphadenitis or Kikuchi lymphadenopathy Large cells in ATLL Large cells in AITL Large cells in EATL Large cells in MF Aggressive systemic mastocytosis AML (subset)

CD30 (Figure 5�15) is a transmembrane glycoprotein and a member of the tumor necrosis factor superfamily� CD30

is a promising target for antibody-based therapy in HL and ALCL, as well as in CD30+ DLBCL� CD30 cluster of antibodies (e�g�, Ki-1, BerH2) recognizes an activationassociated protein that is expressed in activated B and T lymphocytes (with immunoblastic cytomorphology), classical HL, ALCL (systemic and primary cutaneous), subset of DLBCLs, neoplastic cells in LYG, PEL (most cases), PMBL, large cells in EATL, LYP, pagetoid reticulosis (a  variant of MF), MF with large cell transformation, and occasional cases of FL and PCM� Benign mast cells are CD30 −, but CD30 expression has been reported in aggressive systemic mastocytosis and occasional mast cell leukemias� In classical HL and ALCL, CD30 shows a strong membranous staining and Golgi area staining� CD30 is expressed in 14%–21% of DLBCL and is associated with unique gene expression profiling, nongerminal center origin, and favorable prognosis [70,71]� In AML, CD30 expression was reported to correlate with the presence of FLT3-ITD mutation and leukocytosis [72] or high-risk disease [73]�

CD33 AnD CD13 The pan-myeloid antigens, CD13 and CD33, are expressed by AML and 10%–20% of ALL� CD13 is expressed by the majority of AML� The CD33 molecule is a cell surface differentiation protein that is expressed on normal progenitor and myeloid cells, as well as on >80% of AML blasts� It belongs to the family of the sialic acid-binding immunoglobulin-like lectin (Siglec)� With the availability of immunotherapy targeted for CD33, the analysis of CD33 expression is becoming very important for patients with acute leukemias� jilani et al� [74] showed that CD33 intensity in the total BM CD33+ cells differed significantly with the type of disease: The median number of CD33 molecules per cell was highest in AML,

136

CD33 and CD13

Activated cells in benign lymph node

CD30

DLBCL

HL, classical

CD30

CD30

II

A PMBL

ALCL

CD30

D

CD30

Pagetoid reticulosis

H Plasma cell myeloma

MF, large cell transformation

CD30

F

CD30

G

NK lymphoma, nasal type

CD30

E EATL

J

C

B

K

Cutaneous ALCL

CD30

I EBV+ lymphoma in HIV+ patient

CD30

CD30

L

FiGURe 5.15 CD30 expression—Differential diagnosis: (A) activated cells in reactive lymph node; (B) DLBCL; (C) classical HL; (D) PMBL; (E) ALCL; (F) ENKTL; (G) EATL; (H) pagetoid reticulosis (variant of MF); (I) primary cutaneous ALCL; (j) MF with large cell transformation; (K) PCM; (L) EBV-associated high-grade lymphoma in an HIV+ patient�

followed by myelodysplastic syndrome (MDS), CML, and control subjects, and lowest in chronic myeloproliferative neoplasms� CD33 antigen is rapidly internalized upon binding to Gemtuzumab ozogamicin (Mylotarg), delivering the drug (calicheamycin) into the cell, with subsequent double-stranded DNA breaks� Mylotarg is currently approved for relapsed AML in patients older than 65 years, but is being evaluated in combination with chemotherapy� Mylotarg is highly effective

as a single treatment for patients with molecularly relapsed APL including those with very advanced disease� The subset of precursor B- and T-ALL/LBLs may express CD13 and/or CD33� This phenotype is often associated with BCR–ABL rearrangement (Philadelphia chromosome) and poor prognosis; although with contemporary intensive chemotherapy and targeted therapy with tyrosine kinase (TK) inhibitors (Imatinib), the outcome of majority of patients has

137

Immunophenotypic Markers

changed� Among AML, the CD13 and/or CD33 are more often negative or dim in AML with t(8;21) compared to AML without this translocation� In pediatric population, increased CD33 expression by myeloblasts is directly associated with adverse disease features and inversely associated with low-risk disease [75]� Lower CD33 expression is associated with superior response to Mylotarg� In a series reported by Pollard, there was a higher prevalence of low-risk disease features (e�g�, CBF+ AML) in patients with low CD33 expression, whereas patients with high CD33 expression were more likely to have high-risk disease (e�g�, FLT3-ITD+ AML) [75]� In low-risk patients, high CD33 expression is associated with significantly inferior outcome� High expression of CD33 on blasts may be a predictor of poor outcome� Rare cases of mature low-grade B-cell lymphomas (e�g�, CLL, MZL) may display aberrant expression of CD13� The expression of CD13 is seen more often in splenic MZL with positive CD5 expression [53]�

CD34 The CD34 is a small peptide attached to the cell membrane of a hematopoietic cell� It is a marker of myeloid immaturity expressed by developmentally early hematopoietic stem cells (erythroid, myeloid, and megakaryocytic precursors) and terminal deoxynucleotidyl transferase (TdT)+ immature lymphoid cells� Apart from blasts, CD34 also stains blood vessels and sinuses� The CD34 can be used by flow cytometry and immunohistochemistry� CD34 is positive in both AML and ALL� Among AMLs, CD34 is not expressed by classic (hypergranular) APL and majority of acute monocytic leukemias� Hypogranular variant of APL, however, is often CD34+� Apart from AML, CD34 may be positive in precursor B- and T-ALL/LBLs (more often in the former than in the latter) and in MPAL� BPDCN is CD34−� AMLs with mutation of NPM1 are often CD34− and those without NPM1 mutation but with FLT3-ITD are often CD34+ and TdT+ [76]� The immunophenotypic analysis of the CD34 staining in the BM by immunohistochemistry (number and distribution of blasts) and flow cytometry (number of phenotype of CD34+ blasts) is also very useful in the evaluation of the BM for MDS [77]� The number of CD34+ blasts in normal BM is usually 2% blasts has prognostic significance for patients with MDS� By definition, blasts exceeding 5% are diagnostic for refractory anemia with excess blasts (RAEB), and blasts exceeding 20% is indicative of AML� Increased number of CD34+ precursors is also observed in myeloproliferative neoplasm, especially in accelerated phase or incipient blast crisis� Aberrant phenotype of CD34+ blasts by flow cytometry, including overexpression of CD117 or CD123; downregulation of CD13, CD33, or CD45; and aberrant expression of CD7 or CD56 as well as increased number of blasts often indicate MDS�

Correlation of the expression of CD34, CD117, PAX5, MUM1, CD71, and mast cell tryptase helps to differentiate between myeloblasts (CD34+/CD117+), hematogones (CD34+/ PAX5+), mast cells (CD117+/tryptase+), immature basophils (tryptase+/CD34−/CD117−), immature erythroid cells (CD117+/−/CD71+/CD34−), and plasma cells (CD117+/MUM1+)� Among nonhematopoietic tumors, CD34 is expressed by vascular tumors, Kaposi’s sarcoma, dermatofibrosarcoma protuberans, gastrointestinal stromal tumor (GIST) and some other soft tissue tumors�

CD38 CD38 is a transmembrane glycoprotein with a widespread cellular expression and functional activity� The CD38 expression is high in B-cell precursors and in terminally differentiated plasma cells, but low to absent in mature B cells, where it can be induced by activatory signals� Apart from plasma cells, which have bright CD38 expression, CD38 is often positive (dim to moderate) in AML� Among mature neoplasms, CD38 may be positive in the subset of B-CLL, FL, DLBCL, and occasionally other B- and T-cell lymphoproliferations� Based on the percentage of clonal cells expressing CD38, B-CLL could be divided into two categories: one with 95%) and rare cases of FL and DLBCL� MCL is negative for LEF1�

mUm1 • • • • • •

HHV-8 • PEL • Multicentric Castleman’s disease-associated plasmablastic lymphoma HHV-8 expression is seen in PEL, Kaposi’s sarcoma, occasional cases of Castleman’s disease, and plasmablastic lymphoma associated with multicentric Castleman’s disease (Figure 5�22)� Typical plasmablastic lymphomas are negative for HHV-8�

Primary effusion lymphoma

A

MUM1 (Figure 5�24) is expressed by PCM, DLBCL with activated B-cell-like phenotype (ABC) and subset of DLBCLs with BCL6 expression, classical HL, and occasional T-cell lymphoma (e�g�, subset of ALCLs)� NLPHL is MUM1− and a

Kaposi’s sarcoma

HHV-8

PCM DLBCL (subset) Plasmablastic lymphoma Classical HL ATLL Other T-cell lymphoproliferations (rare cases)

HHV-8

B Plasmablastic lymphoma associated with multicentric Castleman’s disease

Castleman’s disease

HHV-8

C CD20

HHV-8

Lambda

Kappa

D

FiGURe 5.22 HHV-8 expression—Differential diagnosis: (A) PEL; (B) Kaposi’s sarcoma; (C) Castleman’s disease; (D) plasmablastic lymphoma associated with multicentric Castleman’s disease�

II

148

MYC

Reactive lymph node

Ki-67

Ki-67

Follicular lymphoma

II

A

B DLBCL

Ki-67

C

D SLL/CLL

E

Ki-67

Burkitt lymphoma

Plasma cell myeloma: Ki-67+ CD138

Ki-67

G

F

FiGURe 5.23 Ki-67 (MIB-1) expression (pattern)—Differential diagnosis: (A) reactive lymph node (Ki-67 showing prominent polarization); (B) FL (Ki-67 not showing polarization); (C) DLBCL (Ki-67 40% of cells) is reported in ~30% of DLBCL [99,100]� The positive MYC protein expression was seen in 69% in the MYC

149

Immunophenotypic Markers

DLBCL

Anaplastic large cell lymphoma

MUM1

MUM1

II

B

A Classical Hodgkin lymphoma

MUM1

C

Plasma cell myeloma

MUM1

D

FiGURe 5.24 MUM1 expression—Differential diagnosis: (A) DLBCL (activated B-cell-like and subset of DLBCLs with BCL6 expression); (B) ALCL (subset); (C) classical HL; and (D) PCM�

translocation-positive group and 28% in the translocationnegative group [99]� Presence of MYC translocation (rearrangement) by FISH, strong expression of MYC and BCL2 by immunohistochemistry, and dim expression of BCL6 are associated with inferior survival [99]� The poor prognostic effect of both MYC and BCL2 protein expression remained significant in a cohort of 140 patients reported by johnson et al� [100] after adjusting for the presence of other high-risk features (activated B-cell molecular subtype and presence of concurrent MYC and BCL2 translocations)� In a series reported by Green et al� [101], ≥70% MYC+ lymphoma cells correlated with positive MYC rearrangement status�

PAx5 • • • • • • • • •

B-cell lymphomas CLL HCL HL (dim expression) NLPHL B-ALL/LBL MPAL (AML/B-ALL) Hematogones PCM (subset)

PAX5 is expressed by B-cell lymphomas, B-CLL/SLL, HCL, classical HL (usually dimmer expression than in normal B cells), NLPHL, precursor B-ALL/LBL, MPAL with

myeloid/B-lymphoid differentiation, rare cases of poorly differentiated carcinoma, and subset of PCMs (Figure 5�25)� T-cell lymphomas including ALCL, precursor T-ALL/LBL, AML, carcinoid tumors, and carcinomas are negative [102]�

PD-1 • AITL • Primary cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma (PCSM-TCL) • Atypical cutaneous T-cell hyperplasia (cutaneous pseudo-T-cell lymphoma) • PTCL, NOS (rare cases) • SS (majority) • MF (rare cases) PD-1 is located on chromosome 2q37 and is a member of the CD28/CTLA-4 receptor family that regulates cellular immune responses� PD-1 is a marker of TFH’s, which also express CXCL13 and BCL6, and are involved in germinal center formation and plasma cell development� It is positive in the majority of AITL and PCSM-PTCL� Rare cases of other T-cell lymphoproliferations, such as PTCL, may be positive� The expression of PD-1 in PTCL was reported, but it may vary from 0% to 71% [103–105]� Cutaneous pseudo-T-cell lymphoma (atypical T-cell hyperplasia) is often positive for PD-1 [52]� PD-1 expression by more than 50% of the neoplastic T cells is observed in 89% patients with SS [106]� In contrast, PD-1 expression by more

TCR γ/δ

150

DLBCL

PAX5

Gastric DLBCL

Burkitt lymphoma

CD20

PAX5

II PAX5

A

B Plasmablastic lymphoma

PAX5

D

Small cell carcinoma

Keratin

CD138

B-ALL

PAX5

PAX5

F

E Plasma cell myeloma

G

C

Hodgkin lymphoma, classical

PAX5

NLPHL

PAX5

PAX5

I

H

FiGURe 5.25 PAX5 expression—Differential diagnosis: (A) DLBCL; (B) gastric DLBCL (CD20−); (C) BL; (D) plasmablastic lymphoma; (E) small cell carcinoma; (F) B-ALL; (G) PCM (subset); (H) classical HL (subset); (I) NLPHL�

than 50% of the neoplastic T cells is found in 13% patients with MF (in PD-1+ cases, serial skin sections showed that CXCL13 and BCL6 generally stained 25%–50% of the PD-1+ cells, while expression of CD10 was uncommon) [106]� Similarly to CD57, PD-1+ T cells are increased in the majority of NLPHL� In a series reported by Churchill et al� [107], PD-1 positivity was found in 87% of NLPHL (compared with 50% of CD57 staining)� NLPHL with diffuse areas showed a gradual decrease in PD-1 reactivity, although a significant proportion of cases show PD-1+ T cells within diffuse areas [107]� Since T-cell/histiocyterich large B-cell lymphoma and classical HL may also show an increased number of PD-1+ T cells, PD-1 expression is not helpful in differential diagnosis between NLPHL and those entities�

sox11 SOX11 expression is present in the majority of MCL (>90%) [18,20,108,109]� It is negative in B-CLL/SLL� The subset of T-ALL/LBL, BL, and T-PLL may also express SOX11�

tCR γ/δ • • • • • •

Cutaneous γ/δ T-cell lymphoma T-LGL leukemia (γ/δ subtype) HSTL PTCL, NOS (small subset) T-ALL/LBL (small subset) Aggressive cytotoxic T-cell lymphomas in the skin

151

Immunophenotypic Markers

γδ T-cell lymphoma

Enteropathy-type T-cell lymphoma CD3

TCR alpha/beta

II A B

TCRγ/δ Subcutaneous panniculitis-like T-cell lymphoma

TCRγ/δ

Forward scatter

CD8

CD3

TCR alpha/beta

C

D

CD8

TCR alpha/beta

γδ T-LGL leukemia

TCRγ/δ

CD57

FiGURe 5.26 TCRγ/δ expression—Differential diagnosis: (A) γ/δ T-cell lymphoma (blood/BM); (B) EATL; (C) SPTL; (D) γ/δ T-LGL leukemia�

The majority of mature T-cell lymphoproliferations are positive for TCRαβ� Only the small subset of tumors expresses TCRγδ (Figure 5�26), while the subset of tumors is negative for both� The TCRγδ phenotype can be analyzed by flow cytometry and TCRγ by immunohistochemistry (γ 3�20; Thermo Fisher Scientific, Inc�, Waltham, MA)� Immunohistochemical analysis confirmed the expression of TCRγ in cutaneous cytotoxic T-cell lymphomas [110]�

tdt • • • • •

B-ALL/lymphoma T-ALL/lymphoma Thymocytes (thymic hyperplasia; thymoma) AML (subset) BPDCN

TdT is a unique intranuclear DNA polymerase� TdT is positive in ALL/LBL of both T- and B-cell lineages, subset of AMLs (most commonly minimally differentiated), very rare

cases of acute monoblastic leukemia, and immature T cells from thymic tissue (thymoma and thymic hyperplasia)� In the series reported by Thalhammer-Scherrer et al� [111], TdT was positive in 9�3% of AML (47% of AML with minimal differentiation), 86% of B-ALL, and 69% of T-ALL�

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152

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153 63� Garnache-Ottou F, et  al� Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia� Br j Haematol, 2009� 145(5):624–36� 64� Gorczyca W, et  al� CD30-positive T-cell lymphomas coexpressing CD15: an immunohistochemical analysis� Int j Oncol, 2003� 22(2):319–24� 65� Robbins BA, et al� Diagnostic application of two-color flow cytometry in 161 cases of hairy cell leukemia� Blood, 1993� 82(4):1277–87� 66� Sanchez ML, et al� Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation� Leukemia, 2002� 16(8):1460–9� 67� Rossmann ED, et  al� Variability in B-cell antigen expression: implications for the treatment of B-cell lymphomas and leukemias with monoclonal antibodies� Hematol j, 2001� 2(5):300–6� 68� Huang j, et  al� Diagnostic usefulness of aberrant CD22 expression in differentiating neoplastic cells of B-cell chronic lymphoproliferative disorders from admixed benign B cells in four-volor multiparameter flow cytometry� Am j Clin Pathol, 2005� 123(6):826–32� 69� Gonen M, et  al� CD25 expression status improves prognostic risk classification in AML independent of established biomarkers: ECOG phase III trial, E1900� Blood, 2012� 120(11):2297–306� 70� Campuzano-Zuluaga G, et al� Frequency and extent of CD30 expression in diffuse large B-cell lymphoma and its relation to clinical and biologic factors: a retrospective study of 167 cases� Leuk Lymphoma, 2013� 71� Hu S, et al� CD30 expression defines a novel subset of diffuse large B-cell lymphoma with favorable prognosis and distinct gene expression signature: a report from The International DLBCL Rituximab-CHOP Consortium Program Study� Blood, 2013� 121(14):2715–24� 72� Fathi AT, et  al� CD30 expression in acute myeloid leukemia is associated with FLT3-internal tandem duplication mutation  and leukocytosis� Leuk Lymphoma, 2013� 54(4):860–3� 73� Zheng W, et al� CD30 Expression in high-risk acute myeloid leukemia and myelodysplastic syndromes� Clin Lymphoma Myeloma Leuk, 2013� 13(3):307–14� 74� jilani I, et al� Differences in CD33 intensity between various myeloid neoplasms� Am j Clin Pathol, 2002� 118:560–6� 75� Pollard jA, et al� Correlation of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin containing chemotherapy in childhood AML� Blood, 2012� 119(16):3705–11� 76� Dalal BI, et al� Detection of CD34, TdT, CD56, CD2, CD4, and CD14 by flow cytometry is associated with NPM1 and FLT3 mutation status in cytogenetically normal acute myeloid leukemia� Clin Lymphoma Myeloma Leuk, 2012� 12(4):274–9� 77� De Smet D, et al� Diagnostic potential of CD34+ cell antigen expression in myelodysplastic syndromes� Am j Clin Pathol, 2012� 138(5):732–43� 78� Lee PS, et al� Coexpression of CD43 by benign B cells in the terminal ileum� Appl Immunohistochem Mol Morphol, 2005� 13(2):138–41� 79� Ngo NT, Lampert IA, Naresh KN� Bone marrow trephine morphology and immunohistochemical findings in chronic myelomonocytic leukaemia� Br j Haematol, 2008� 141(6):771–81�

II

154

II

80� Ponka P, Lok CN� The transferrin receptor: role in health and disease� Int j Biochem Cell Biol, 1999� 31(10):1111–37� 81� Nakahata T, Okumura N� Cell surface antigen expression in human erythroid progenitors: erythroid and megakaryocytic markers� Leuk Lymphoma, 1994� 13(5–6):401–9� 82� Marsee DK, Pinkus GS, Yu H� CD71 (transferrin receptor): an effective marker for erythroid precursors in bone marrow biopsy specimens� Am j Clin Pathol, 2010� 134(3):429–35� 83� Davis BH, et  al� US-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: medical indications� Cytometry, 1997� 30(5):249–63� 84� Malcovati L, et  al� Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome� Leukemia, 2005� 19(5):776–83� 85� Della Porta MG, et  al� Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome� Leukemia, 2006� 20(4):549–55� 86� Dong HY, Wilkes S, Yang H� CD71 is selectively and ubiquitously expressed at high levels in erythroid precursors of all maturation stages: a comparative immunochemical study with glycophorin A and hemoglobin A� Am j Surg Pathol, 2011� 35(5):723–32� 87� Morice WG, et al� Predictive value of blood and bone marrow flow cytometry in B-cell lymphoma classification: comparative analysis of flow cytometry and tissue biopsy in 252 patients� Mayo Clin Proc, 2008� 83(7):776–85� 88� Del Giudice I, et al� The diagnostic value of CD123 in B-cell disorders with hairy or villous lymphocytes� Haematologica, 2004� 89(3):303–8� 89� Bain Bj� Bone marrow trephine biopsy� j Clin Pathol, 2001� 54(10):737–42� 90� Bain Bj� Bone marrow aspiration� j Clin Pathol, 2001� 54(9):657–63� 91� Asano N, et  al� Clinicopathologic and prognostic significance of cytotoxic molecule expression in nodal peripheral T-cell lymphoma, unspecified� Am j Surg Pathol, 2005� 29(10):1284–93� 92� Falini B, et  al� A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells� Blood, 2000� 95(6):2084–92� 93� Tsuboi K, et al� MUM1/IRF4 expression as a frequent event in mature lymphoid malignancies� Leukemia, 2000� 14(3):449–56� 94� Natkunam Y, et al� Analysis of MUM1/IRF4 protein expression using tissue microarrays and immunohistochemistry� Mod Pathol, 2001� 14(7):686–94� 95� Colomo L, et al� Clinical impact of the differentiation profile assessed by immunophenotyping in patients with diffuse large B-cell lymphoma� Blood, 2003� 101(1):78–84� 96� Hallermann C, et al� New prognostic relevant factors in primary cutaneous diffuse large B-cell lymphomas� j Am Acad Dermatol, 2007� 56(4):588–97�

References 97� Sundram U, et al� Expression of the bcl-6 and MUM1/IRF4 proteins correlate with overall and disease-specific survival in patients with primary cutaneous large B-cell lymphoma: a tissue microarray study� j Cutan Pathol, 2005� 32(3):227–34� 98� Hans CP, et  al� Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray� Blood, 2004� 103(1):275–82� 99� Horn H, et al� MYC status in concert with BCL2 and BCL6 expression predicts outcome in diffuse large B-cell lymphoma� Blood, 2013� 100� johnson NA, et al� Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone� j Clin Oncol, 2012� 30(28):3452–9� 101� Green TM, et al� High levels of nuclear MYC protein predict the presence of MYC rearrangement in diffuse large B-cell lymphoma� Am j Surg Pathol, 2012� 36(4):612–9� 102� Desouki MM, et al� PAX-5: a valuable immunohistochemical marker in the differential diagnosis of lymphoid neoplasms� Clin Med Res, 2010� 8(2):84–8� 103� Matsumoto Y, et al� Expression of master regulators of helper T-cell differentiation in peripheral T-cell lymphoma, not otherwise specified, by immunohistochemical analysis� Am j Clin Pathol, 2010� 133(2):281–90� 104� Krishnan C, et al� PD-1 expression in T-cell lymphomas and reactive lymphoid entities: potential overlap in staining patterns between lymphoma and viral lymphadenitis� Am j Surg Pathol, 2010� 34(2):178–89� 105� Yu H, Shahsafaei A, Dorfman DM� Germinal-center T-helpercell markers PD-1 and CXCL13 are both expressed by neoplastic cells in angioimmunoblastic T-cell lymphoma� Am j Clin Pathol, 2009� 131(1):33–41� 106� Cetinozman F, et  al� Differential expression of programmed death-1 (PD-1) in sezary syndrome and mycosis fungoides� Arch Dermatol, 2012� 148(12):1379–85� 107� Churchill HR, et al� Programmed death 1 expression in variant immunoarchitectural patterns of nodular lymphocyte predominant Hodgkin lymphoma: comparison with CD57 and lymphomas in the differential diagnosis� Hum Pathol, 2010� 41(12):1726–34� 108� Mozos A, et al� SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative subtype� Haematologica, 2009� 94(11):1555–62� 109� Xu W, Li jY� SOX11 expression in mantle cell lymphoma� Leuk Lymphoma, 2010� 51(11):1962–7� 110� Rodriguez-Pinilla SM, et  al� TCR-γ expression in primary cutaneous T-cell lymphomas� Am j Surg Pathol, 2013� 37(3):375–84� 111� Thalhammer-Scherrer R, et  al� The immunophenotype of 325 adult acute leukemias: relationship to morphologic and molecular classification and proposal for a minimal screening program highly predictive for lineage discrimination� Am j Clin Pathol, 2002� 117(3):380–9�

6

Cytogenetics

Contents Introduction ............................................................................................................................................................................... 157 Karyotype.................................................................................................................................................................................. 157 Diagnosis................................................................................................................................................................................... 157 Prognosis ................................................................................................................................................................................... 158 Common Chromosomal Changes in Hematopoietic Tumors ................................................................................................... 160 Deletions and Monosomies .................................................................................................................................................. 160 del(5q)/monosomy 5 ....................................................................................................................................................... 160 del(6q) ..............................................................................................................................................................................161 del(7q)/monosomy 7 ........................................................................................................................................................161 del(9p) ............................................................................................................................................................................. 162 del(9q) ............................................................................................................................................................................. 163 del(11q) ........................................................................................................................................................................... 163 del(12p) ........................................................................................................................................................................... 163 del(13q)/monosomy 13 ................................................................................................................................................... 163 del(17p)/monosomy 17 ................................................................................................................................................... 165 del(20q) ........................................................................................................................................................................... 166 Inversions ............................................................................................................................................................................. 166 inv(3)/t(3;3) ..................................................................................................................................................................... 166 inv(7)(p15q34)/t(7;7)(p15;q34) ....................................................................................................................................... 166 inv(11)(p15q22) ............................................................................................................................................................... 167 inv(12) ............................................................................................................................................................................. 167 inv(14) ............................................................................................................................................................................. 167 inv(16) or t(16;16) ........................................................................................................................................................... 167 Isochromosomes................................................................................................................................................................... 168 i(3) ................................................................................................................................................................................... 168 i(7q) ................................................................................................................................................................................. 168 i(17) ................................................................................................................................................................................. 168 Translocations ...................................................................................................................................................................... 169 t(1;3)(p36.3;q21.2) .......................................................................................................................................................... 169 t(1;4) ................................................................................................................................................................................ 169 t(1;5)(q23;q33) ................................................................................................................................................................ 169 t(1;6) ................................................................................................................................................................................ 169 t(1;14)(p22;q32) .............................................................................................................................................................. 169 t(1;14)(p32;q11) ...............................................................................................................................................................170 t(1;19) ...............................................................................................................................................................................170 t(1;22)(p13;q13) ...............................................................................................................................................................170 t(2;3) .................................................................................................................................................................................170 t(2;5)(p23;q35) .................................................................................................................................................................170 t(2;8) .................................................................................................................................................................................170 t(2;14) ...............................................................................................................................................................................170 t(2;17) ...............................................................................................................................................................................170 t(2;19) ...............................................................................................................................................................................170 t(3;3)(q26;q21)/inv(3).......................................................................................................................................................170 t(3;5) .................................................................................................................................................................................171 t(3;8)(q26;q24) .................................................................................................................................................................171 t(3;8)(q27;q24.1) ..............................................................................................................................................................171 t(3;14) ...............................................................................................................................................................................171 t(3;21) ...............................................................................................................................................................................171

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t(4;11) .............................................................................................................................................................................. 172 t(4;14) ...............................................................................................................................................................................174 t(5;10)(q22;q24) ...............................................................................................................................................................174 t(5;12) ...............................................................................................................................................................................174 t(5;17) ...............................................................................................................................................................................175 t(6;7)(p24;q21) .................................................................................................................................................................175 t(6;9) .................................................................................................................................................................................175 t(6;11) ...............................................................................................................................................................................175 t(6;14) ...............................................................................................................................................................................175 t(7;7) .................................................................................................................................................................................175 t(7;11) ...............................................................................................................................................................................175 t(7;12) ...............................................................................................................................................................................175 t(8;9)(p11;q33) .................................................................................................................................................................175 t(8;9)(q24;p13) .................................................................................................................................................................175 t(8;9)(p22;p24) .................................................................................................................................................................175 t(8;13)(p11;q12) ...............................................................................................................................................................175 t(8;14)(q24;q32) ...............................................................................................................................................................175 t(8;16) ...............................................................................................................................................................................176 t(8;21)(q22;q22.3) ............................................................................................................................................................176 t(8;22) ...............................................................................................................................................................................176 t(9;11)(p21;q23) ...............................................................................................................................................................176 t(9;12) ...............................................................................................................................................................................176 t(9;14)(p13;q32) ...............................................................................................................................................................176 t(9;22) ...............................................................................................................................................................................176 t(10;11) ............................................................................................................................................................................ 177 t(10;14) ............................................................................................................................................................................ 177 t(10;17)(p15;q21) ............................................................................................................................................................ 177 t(10;22) ............................................................................................................................................................................ 177 t(11;14)(q13;q32) ............................................................................................................................................................ 177 t(11;14)(p13;q11) and t(11;14)(p15;q11) ........................................................................................................................ 177 t(11;17)(q23;q21) ............................................................................................................................................................ 177 t(11;18)(q21;q21) ............................................................................................................................................................ 177 t(11;19)(q23;p13.1) and t(1;19)(q23;p13.3) .....................................................................................................................178 t(12;12) .............................................................................................................................................................................178 t(12;17) .............................................................................................................................................................................178 t(12;21) .............................................................................................................................................................................178 t(12;22)(p13;q11) .............................................................................................................................................................178 t(13;17) .............................................................................................................................................................................178 t(14;16) .............................................................................................................................................................................178 t(14;17) .............................................................................................................................................................................178 t(14;18)(q32;q21) .............................................................................................................................................................178 t(14;18)(q32;q21) ............................................................................................................................................................ 179 t(14;19)(q32;q13) ............................................................................................................................................................ 179 t(15;17)(q24;q21) ............................................................................................................................................................ 179 t(17;17)(qq11.2;q21) ....................................................................................................................................................... 180 t(17;20)(q21;q12) ............................................................................................................................................................ 180 Trisomies .............................................................................................................................................................................. 180 Trisomy 2/duplication 2p ................................................................................................................................................ 180 Trisomy 3/duplication 3p ................................................................................................................................................ 180 Trisomy 5......................................................................................................................................................................... 180 Trisomy 8......................................................................................................................................................................... 180 Trisomy 9......................................................................................................................................................................... 180 Trisomy 11....................................................................................................................................................................... 180 Trisomy 12....................................................................................................................................................................... 180 Trisomy 15........................................................................................................................................................................181 Trisomy 17........................................................................................................................................................................181

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Trisomy 18........................................................................................................................................................................181 Trisomy 21........................................................................................................................................................................181 Trisomy 22........................................................................................................................................................................181 References ..................................................................................................................................................................................181

IntroduCtIon Conventional cytogenetic (G-banding), fluorescence in situ hybridization (FISH), and molecular methods are commonly applied in the diagnosis of hematologic malignancies and, in addition, are of increasing practical importance to accurately evaluate the prognosis and minimal residual disease [1–25]. Cytogenetic analysis has become routine in the evaluation of patients with myeloid and many lymphoid diseases, in conjunction with morphology and immunophenotyping helps to establish definite diagnosis and recognize disease subsets, and is essential in evaluating disease progression. FISH probes are now available for many common chromosomal abnormalities and often provide advantage over classic cytogenetics by detecting chromosomal abnormalities that can be masked or cryptic in conventional G-banding technologies. Polymerase chain reaction (PCR) methods for the determination of clonality by the analysis of IGH and TCRγ gene rearrangements belong to the most widely used, and involve different IGH V framework regions for B-cell analysis and TCRγ V and J genes for T-cell analysis.

Karyotype A karyotype is a set of chromosomes from one cell. There are 46 chromosomes occurring in 23 pairs (Figure 6.1). Chromosomes are distinct bodies found in the nucleus of cells, best visible in the phase of the cell cycle called metaphase. Chromosomes are composed of protein and DNA, and hold the genetic information in the form of linear sequences of four bases (A, T, C, and G). The DNA sequence for a single trait is called a gene. Each chromosome contains a few thousand genes, which range in size from a few thousand bases up to 2 million bases. The first 22 pairs are labeled longest to shortest. The last pair are called the sex chromosomes, which are labeled X or Y. Females have two X chromosomes (XX), and males have an X and a Y chromosome (XY). Each chromosome has a short or p (petit) arm and long or q (next letter in the alphabet) arm, which are separated by a region known as the centromere. The centromere is a condensed part of chromosome that binds together two sister chromatids and constitutes the attachment site for spindle fiber during cell division. Types of chromosomes are presented in Figure 6.2. Each chromosome arm is further defined by numbering the bands (light and dark bands visible under the microscope after staining with various dyes); the higher the number, the further the area is from the centromere. The band width and the order of bands are specific for each chromosome and allow their identification. Chromosomal aberrations include numerical and structural abnormalities (Figure 6.3). A cell with 46 chromosomes

is called diploid, and a cell with abnormal number of chromosomes is called aneuploid (46, hyperdiploid). An insertion (ins) is a structural rearrangement in which part of a chromosome is inserted into a new location on a chromosome. Monosomy refers to a single chromosome and trisomy to three chromosomes. Loss of a chromosome is designated by a minus sign (−; monosomy) and loss of part of a chromosome is designated by del. Deletions can be either terminal or interstitial. The chromosomes that are most commonly lost include −5, −7, −X, and −Y, and that are most commonly duplicated (+; trisomy) include +4, +6, +8, +9, +10, +11, +12, +13, +14, +19, +20, and +21. The common chromosomal deletions include del(5q), del(6q), del(7q), del(13q), and del(20q). Isochromosome (i) is an abnormal chromosome with two chromosome arms positioned as mirror images of each other (duplication of one of the arm resulting in a metacentric chromosome with identical genes on both arms). The most common isochromosomes include i(1q), i(7q), i(9q), i(11q), i(17q), i(21q), and i(22q). A chromosomal inversion (inv) is a 180° rotation of a chromosome segment (part of a chromosome is reversed in orientation). The common inversions include inv(3) and inv(16). A chromosomal translocation (t) is a relocation of material from one chromosome to a different chromosome. Translocations can be either reciprocal (mutual exchange of segments of chromosomes) or Robertsonian (centric fusion of the long arms of acrocentric chromosomes and loss of their short arms). Most of the translocations are reciprocal and result in either synthesis of novel fusion protein or relocation of an oncogene to a locus that is highly transcribed. The common translocations include t(9;22), t(15;17), t(14;18), t(11;14), t(8;14), and t(8;21). A Robertsonian translocation product is considered a derivative chromosome and is described by der. Marker chromosomes (mar) are abnormal chromosomal structures that cannot be subclassified by cytogenetics. The presence of a marker chromosome is depicted as a +mar. Two cells with identical structural aberrations are defined as clone.

dIagnosIs Cytogenetic aberrations, both structural (translocations and inversions) and numerical (deletion or gain of whole or part of chromosome), occur in the majority of hematopoietic tumors. Chromosomal translocations result in altered gene function, due to either deregulated gene expression or abnormal activity of a novel fusion protein, resulting from the juxtaposition of coding sequences from two different genes, which under normal circumstances are separated. Chromosomal deletions result in the loss of genetic material crucial in maintaining proper functions of the cells, such as proliferation, cell cycle control, and programmed cell death (apoptosis).

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158

Prognosis

III

1

6

2

7

3

8

4

9

5

10

11

12

13

14

15

16

17

18

19

20

21

22

X

Y

FIgure 6.1

Normal karyotype (male).

choice, since the results with other agents [e.g., ethylenediaminetetraacetic acid (EDTA)] are less optimal. Many chromosomal and molecular changes define specific hematologic entities and syndromes, and have important therapeutic and prognostic impact: the t(15;17)/PML–RARA (retinoic acid receptor α) is characteristic for acute promyelocytic leukemia (APL), a unique variant of acute myeloid leukemia (AML) treated with all-trans retinoic acid (ATRA) and arsenic trioxide (among other medications); AML with t(8;21) or inv(16) comprises the favorable risk group, whereas complex karyotype in AML predicts a poor prognosis; the t(2;5)/NPM–ALK defines a subset of anaplastic large cell lymphomas (ALCLs) associated with a good prognosis and chemosensitivity; the t(8;14)/ MYC–IGH is important in the diagnosis of Burkitt lymphoma (BL) and when accompanied by BCL2 or BCL6 rearrangement defines the aggressive subset of B-cell lymphomas; and i(7) is seen typically in hepatosplenic T-cell lymphoma (HSTL) and inv(14) in T-cell prolymphocytic leukemia (T-PLL). The t(9;22)/BCR–ABL1 confirms the diagnosis of chronic myelogenous leukemia (CML), although it may also be seen in a subset of B-cell acute lymphoblastic leukemia (ALL) or AML.

Group

Chromosomes

Characteristics

A

1−3

Large metacentric chromosomes

B

4−5

Large submetacentric chromosomes

C

6−12 and X

Medium-sized submetacentric chromosomes

Example

1

4 7

G

13−15

Large acrocentric chromosomes 13

E

16−18

Medium-sized acrocentric chromosomes

F

19−20

Short metacentric chromosomes

G

21−22 and Y

Short acrocentric chromosomes

17 20

22

FIgure 6.2

Types of chromosomes.

Conventional cytogenetics studies are performed on fresh (unfixed) specimen containing viable cells. The specimen is either prepared immediately (direct preparation) or more often cultured for 1–3 days, with or without mitogenic stimulants (in the cases of acute leukemias, cells are cultured usually without mitogens). For hematologic malignancies, suitable specimens include bone marrow aspirate (preferable source), fresh tissue (lymph node, extranodal tumorous infiltrate), effusion (pleural, abdominal, and pericardial), cerebrospinal fluid (CSF), and blood. Specimen should be collected under sterile conditions. Sodium heparin (green top tube) is the anticoagulant of

prognosIs Cytogenetics and molecular testing play an important role in personalized approach to manage patients with hematologic malignancies. Acquired genomic aberrations have been shown to significantly impact survival in several hematologic malignancies. Chromosomal and molecular aberrations used in the prognostic stratification of the most common hematopoietic tumors are presented in Table 3.2. Chromosomal aberrations as revealed by conventional cytogenetic (G-banding) and/or FISH are widely recognized as the most important

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Cytogenetics

Diploid

Tetraploid

4

5

del(6q)

6

4

5

−7

+8

inv(14)

7

8

14

t(8;14)

7

FIgure 6.3

8

14

15

Examples of chromosomal aberrations.

prognostic determinants in AML. Although the prognosis for patients with AML is generally poor, the presence of certain chromosomal abnormalities is associated with better response to therapy and improved survival. AML patients with t(15;17), t(8;21), or inv(16) comprise the favorable risk group, whereas complex karyotype predicts an extremely poor prognosis (normal karyotype and other noncomplex abnormalities comprised the intermediate AML group). Myelodysplastic syndromes (MDSs) encompass the heterogeneous group of disorders with variable prognosis and risk of progression into acute leukemia. There is no single clinical or biologic parameter that can accurately predict prognosis, and it can often be challenging for clinicians to choose the most appropriate treatment for an individual patient. The treatment options depend on whether patient has low, intermediate, or high risk, and include supportive care measures, including blood transfusions (red cells or platelets), recombinant growth factors, allogeneic stem cell transplantation, DNA methyltransferase inhibitors (azacitidine and decitabine), cyclosporin, TLK199/valproic acid, and immunomodulatory agents (lenalidomide, thalidomide). The presence of specific cytogenetic abnormalities can predict disease manifestations and provide a basis for prognosis and direct treatment. Based on the prior International Prognostic Scoring System (IPSS) for MDS, normal karyotype, −Y, del(5q), and del(20q) were associated with good prognosis, and the presence of −7 or complex karyotype (three or more abnormalities) was associated with a poor prognosis (all other karyotypic changes and any double abnormalities had an intermediate prognosis) [25,26]. In the new five-group classification [Revised IPSS (R-IPSS)], “good” and “intermediate” groups largely overlap with IPSS scheme, and the IPSS group “poor” is split into “intermediate,” “poor,” and “very poor” [27] as follows: • Very good • −Y; del(11q)

• Good • Normal; del(5q); del(20q); del(12p) • Double abnormalities including del(5q) • Intermediate • del(7q); +8; i(17q); +19; any other • Any other double abnormalities • Poor • −7; inv(3)/t(3q)/del(3q) • Double abnormalities including −7/del(7q) • Complex (three abnormalities) • Very poor • Complex (>3 abnormalities) Patients with 5q− abnormality display marked erythroid and cytogenetic responses when treated with thalidomide derivative (lenalidomide), often resulting in transfusion independence and cytogenetic remission. Similarly, del(20q) is known to be a favorable prognostic factor in MDS when it is the sole change. The late appearance of del(20q) at any phase, however, is linked to a significantly unfavorable prognosis, thus indicating the clinical and biological heterogeneity of del(20q) in MDS. Independent adverse prognostic factors in MDS include complex karyotype, chromosome 5 and/or 7 abnormalities, older age, and prior MDS therapy. The use of DNA microarrays has defined two molecular subgroups of diffuse large B-cell lymphoma (DLBCL): germinal center B-cell-like (GCB) and activated-B-cell like (ABC) with significantly different mortality rates and responses to conventional therapy. The presence of t(2;5) in ALCL is associated with a good prognosis and chemosensitivity. The molecular or cytogenetics factors associated with a prognosis B-cell chronic lymphocytic leukemia (B-CLL) include the mutational status of the variable region of the immunoglobulin heavy-chain gene (IGVH), the abnormalities of chromosomes 11, 12, 13, and 17, and the status of the p53/TP53 gene. Deletion of chromosomes 11q and 17p, unmutated IGVH, and mutation of p53/TP53 are associated with an aggressive

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disease and poor prognosis, whereas the presence of del(13q) or mutated IGVH predicts indolent form of B-CLL. In plasma cell myeloma (PCM), the genomic aberrations t(4;14) and del(17p) are important independent predictors of survival.

III

Common Chromosomal Changes In hematopoIetIC tumors The common chromosomal and molecular aberrations found in hematological malignancies are shown in Table 6.1 and Figure 6.4. The structures of all chromosomes and the location of most common genes involved in hematopoietic malignancies are presented in Figures 6.5 through 6.27.

Deletions anD MonosoMies del(5q)/monosomy 5 • MDSs • Acute myeloid leukemia (AML)

• Therapy-related myeloid neoplasm • Blastic plasmacytoid dendritic cell neoplasm (BPDCN) • ALL (rare cases) Deletion of 5q occurs in MDSs and AMLs [14,28–35]. Patients with MDS and del(5q) can be grouped into those with isolated del(5q) (good prognosis), del(5q) with additional abnormality [prognosis worse than for isolated del(5q)], and del(5q) with complex karyotype (poor prognosis). The 5q− syndrome is a distinct category of MDS  defined  by less  than 5% blasts, isolated deletion of the long arm of chromosome 5, and low probability of  transformation to AML [29,30,32–37]. MDS cases with 5q− plus additional abnormalities,  including del(12p), del(7q), del(14q), i(11q), and i(17q), show a neoplastic evolution in a short period of time [38]. Patients  with  MDS  associated with del(5q)  respond well to lenalidomide treatment, although the target for the drug is unknown.

taBle 6.1 most Common Chromosomal and molecular markers in the diagnosis of hematopoietic malignancies marker t(2;5)/ALK1–NPM BCL6 rearrangements [t(3;n)] t(9;22)/BCR–ABL1

t(14;18)/BCL2–IGH BRAF mutation del(5q) del(5)/del(7)

del(13) del(17p) i(7q) inv(14) (TCL1) t(16;16)/inv(16) JAK2 mutation

t(8;14)/MYC–IGH t(15;17)/PML–RARA t(8;21)/RUNX1–RUNX1T1 t(9;14)/PAX5–IGH t(11;14) t(11;18)/API2–MALT1

neoplasm

Comments

ALCL (ALK1+) Some DLBCL CML CML in blast crisis AML ALL FL Some DLBCL Hairy cell leukemia

Good prognosis Does not occur in BL Confirms the diagnosis Poor prognosis Poor prognosis

5(q−) syndrome (MDS) MDS or AML AML MDS Therapy-related myeloid neoplasm CLL PCM Various neoplasms HSTL T-PLL AML (acute myelomonocytic leukemia with eosinophilia) PV Essential thrombocythemia Primary myelofibrosis

Sole abnormality, good prognosis del(5q) as part of complex aberrations, poor prognosis

BL Subset of DLBCL Acute promyelocytic leukemia AML LPL Mantle cell lymphoma Subset of PCM Extranodal marginal zone B-cell lymphoma (MALT type)

Does not occur in BL

Poor prognosis

Good prognosis Intermediate risk Poor prognosis (high-risk disease) Characteristic abnormality Characteristic abnormality Favorable prognosis

BCL2−/BCL6− BCL2+ and/or BCL6+ Very good prognosis Good response to ATRA Favorable prognosis Confirms the diagnosis of MCL Poor response to Helicobacter pylori eradication therapy in gastric lymphoma

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Cytogenetics

Lymphoid

Myeloid

III

del(5q)/-5 del7 del(9q) del(20q) dic(X)(q13) i(17q) inv(3) inv(16) t(1;7) t(2;3) t(3;3) t(3;5)

FIgure 6.4

t(4;12) t(5;12) t(5;14) t(6;9) t(8;9) t(8;21) t(9;11) t(15;17) Trisomy 11 Trisomy 19 Trisomy 21

del(7q) del(11q) del(12p) del(13q) del(17p) inv(12) t(4;20) t(9;22) Trisomy 8 Trisomy 9 Trisomy 20

del(6q) dic(9;12) dic(9;20) dup(21q) inv(14) t(2;5) t(2;8) t(2;17) t(2;19) t(3;14)

t(8;14) t(8;22) t(9;14) t(11;14) t(12;21) t(14;14) t(14;18) t(14;19) t(19;22) Trisomy 3 Trisomy 18

Chromosomal changes seen in myeloid vs. lymphoid proliferations.

Monosomy 5/del(5q) may be seen in all types of AML, but is more common in acute erythroid leukemia (AML-M6). Monosomy 5/del(5q) and monosomy 7/del(7q) represent the most common cytogenetic abnormalities in the therapyrelated MDS (t-MDS) and AML (t-AML) and are strongly associated with prior exposure to alkylating agents [39]. AML with del(5q) or monosomy 5 (−5) is associated with a very poor prognosis, similarly to AML with complex chromosomal abnormalities, monosomy 7 (−7), and t(9;22) [40–44]. Del(5q) is observed in other hematolymphoid malignancies, including plasmacytoid dendritic cell leukemia/lymphoma (blastic NK cell lymphoma/leukemia; DC2 acute leukemia) [45–47], adult T-cell leukemia/lymphoma [48,49], and rare cases of ALL [50–52]. Santana-Davila et al. [53] showed that del(5q), although most prevalent in MDS, can be seen across the spectrum of myeloid disorders including myeloproliferative neoplasms and its occurrence in lymphoid disorders might signify, for the most part, an occult myeloid clone. del(6q) • Follicular lymphoma (FL) • B-CLL • DLBCL • Marginal zone lymphoma [MZL; mucosa-associated lymphoid tissue (MALT) lymphoma] • Peripheral T-cell lymphomas • Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) • NK/T-cell leukemia/lymphoma • Extranodal NK/T-cell lymphoma, nasal type • ALL/lymphoblastic lymphoma (LBL)

Aberrations of the long arm of chromosome 6 are among the most common chromosomal abnormalities in lymphoid neoplasms [54–59]. The del(6q) has been reported in FL [54,55,60,61], WM [62–64], B-CLL [54,65–67], MZL [54], ALL [54,56,68], multiple myeloma [65,69], DLBCL [7,54,60,70,71], and T-cell lymphoproliferations [72,73]. In a series reported by Taborelli et al. [54], conventional cytogenetics revealed a del(6q) in 46% of lymphomas, including two cases that showed del(6q) as the sole chromosome anomaly. Deletion of the long arm of chromosome 6 is found in about one-half of patients with WM [62–64]. The area of minimal deletion falls between 6q23 and 6q24.3, but the deletion usually encompassed a large fragment of the 6q arm. The presence of del(6q) can help to distinguish WM from IgM+ monoclonal gammopathy of undetermined significance (MGUS) since the latter is negative for del(6q) [64]. The del(6q) (in conjunction with other abnormalities, such as +3p and +1p) plays a role in MGUS progression to multiple myeloma [74]. The del(6)(q21q25) is a recurrent chromosomal abnormality in NK cell lymphoma/leukemia, a highly aggressive malignancy [73,75]. del(7q)/monosomy 7 • AML • MDSs • Therapy-related myeloid neoplasm • ALL/LBL • CML (disease progression) • Chronic myeloproliferative neoplasms [polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)]

162

Common Chromosomal Changes in Hematopoietic Tumors

t(1;3) MDS ET PMF

III

PRDM16

36.3 36.2 36.1

MPL

35 34.3 34.2 34.1 33

Mutation

32

t(1;3) t(1;7) t(1;14)

TAL1

T-ALL

31 p

t(1;14)(p22;q32) 22

AML MDS CMML

Mutation

21

NRAS

13 12

t(1;14)(q21;q32) t(1;22) ALL B-cell lymphoma B-ALL

BCL10

OTT t(1;22)

MALT lymphoma Acute megakaryoblastic leukemia

12

BCL9 PBX1 t(1;19) [E2A] q

21

PDE4DIP

22 23

t(1;5)

24 25

TPM3

31

t(1;2)

CEL

ALCL

32 41 42 43 44 1

FIgure 6.5

Chromosome 1.

• Splenic MZL (SMZL) • Low-grade B-cell lymphomas including LPL/WM and B-CLL/small lymphocytic lymphoma (SLL) Loss of chromosomal material due to deletion of the long arm of chromosome 7 [del(7q)] or loss of one homolog (−7) is a common finding in all types of myeloid disorders, especially in patients with AML, MDSs, and therapy-related myeloid neoplasms, and is invariably associated with worse response to treatment and a poor prognosis [76–79]. Deletion of 7q in MDS is associated with an intermediate prognosis. Monosomy 7/del(7q), similarly to monosomy 5/del(5q), represents the most common cytogenetic abnormalities in t-MDS and t-AML and is strongly associated with exposure to alkylating agents. Monosomy 7 is also reported in myeloid proliferations in children [80], which is the most common cytogenetic abnormality in pediatric MDS [81]. In a series reported by Heerema et al. [82], among 1880 children with ALL, 75 (4%) had losses involving chromosome 7, 16 (21%) had monosomy 7, 41 (55%) had losses of 7p, 16 (21%) had

losses of 7q, and 2 (3%) had losses involving both arms. Deletion of chromosome 7 or del(7q) occurs frequently in both CML and non-CML chronic myeloproliferative neoplasms (PV, ET, and PMF) [80,83,84]. Rearrangements of 7q, especially del(7q), are seen in SMZLs (up to 40%), where they are associated with a poor prognosis and risk of large cell transformation [85–88]. Lymphomas with del(7q) often show plasmacytoid features (plasmacytic differentiation) [89,90]. SMZL patients with genetic losses [del(7q) or del(17p)] had a shorter survival than the remaining patients, including those with chromosomal gains [91]. Imbalances on 7q are associated with a shorter survival in patients with BL [92]. del(9p) • ALL/LBL • CML, myeloid blast crisis • Mantle cell lymphoma (MCL) • DLBCL/posttransplant DLBCL • Peripheral T-cell lymphoma (rare cases)

163

Cytogenetics

B-CLL BL

t(2;14)

25 24 23 22 21

p

16 15 14 13 12

BCL11A

IGK

11.2 DLBCL BL DLBCL B-ALL

t(2;3) [BCL6]

ALK

ALCL

t(2;5) t(X;2) t(1;2) t(2;3) t(2;17) t(2;22) inv(2)

11.2 12 13 14.1 14.2 14.3 21

t(2;8) [MYC]

22 23 24

q

31 32.1 32.2 32.3 33 34 35 36 37

SF3B1

MDS CLL

ATIC

ALCL

inv(2)

2

FIgure 6.6

Chromosome 2.

In pediatric ALL, del(9p) belongs to the most common structural rearrangements (17%), followed by t(12;21) (15%), del(6q) (8%), and MLL rearrangements (4%) [93]. Adult ALL patients with del(9p) have a significantly improved outcome, in contrast to those with Philadelphia (Ph) chromosome, t(4;11)(q21;q23), t(8;14)(q24.1;q32), or complex karyotype (five or more chromosomal abnormalities) [18]. Although deletions of 9p in ALL show considerable variability in both the extent and the location, all include the CDKN2A locus [94]. Deletion of 9p and other structural changes [+3q, +12q, del(6q), del(1p), del(13q), del(10q), del(11q), and del(17p)] may be observed in MCL, including blastoid variant and mantle cell leukemia [95–97]. Posttransplant DLBCL shows frequently losses of 6q, 17p, 1p, and 9p [98]. Both de novo CD5+ and typical (CD5−) DLBCL may have del(9p) [99,100]. The 9p abnormalities have been reported in enteropathy-associated T-cell lymphoma [101]. del(9q) • AML del(11q) • B-CLL • MCL • DLBCL • AML • MDSs • ALL/LBL

• Chronic myeloproliferative neoplasms (PV, ET, PMF) • T-PLL Structural aberrations involving 11q are among the most common aberrations in a number of hematological malignancies [102,103]. The 11q contains three genes: BCL1 (11q13), ATM (11q22–23), and MLL (11q23). Translocations or deletions involving the 11q23 region have been observed in ALL, AML, MDS, T-PLL, and B-cell lymphoproliferations including B-CLL and MCL. In B-CLL, deletions have been detected in 20%–30% of the cases and deletions of 11q identify a new clinical subset characterized by younger age; extensive peripheral, abdominal, and mediastinal lymphadenopathy; more advanced clinical stages; more rapid disease progression; and a shorter treatment-free interval [104]. Apart from B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL), del(11q) can be observed in other non-Hodgkin lymphomas, mostly MCL [95,105] and DLBCL [103], but also occasionally in MZL [106]. Deletion of 11q, similarly to +8, del(7q), and del(20q) are seen often in non-CML chronic myeloproliferative neoplasms (PV, ET, PMF). The del(11q) in MDS is often associated with sideroblastosis (ringed sideroblasts are also seen in Xq13 abnormalities). del(12p) • Chronic myeloproliferative neoplasms (PV, ET, PMF) • ALL • MDSs • AML • Therapy-related myeloid neoplasm • DLBCL, posttransplant • Non-Hodgkin lymphomas del(13q)/monosomy 13 • B-CLL • MCL (subset) • B-cell lymphomas, high grade • PCM/MGUS • Peripheral T-cell lymphomas • Chronic myeloproliferative neoplasms (PV, ET, PMF) Deletion of 13q14 is the most frequent genetic change in CLL, occurring in 50%–65% of the cases. The presence of either cryptic deletion of the single locus (q14) or deletion of larger portion of the chromosome is associated with a favorable prognosis. Patients with a deletion of 13q14 as single aberration have the longest estimated median treatment-free interval and survival time [107,108]. The most common cytogenetic aberrations in MCL include del(13q14), followed by del(17p) and +12. In a series reported by Parry-Jones et al. [109], 81% cases had at least one abnormality in addition to t(11;14). Trisomy 12 and deletions of 6q21 and 13q14 were associated with poorer prognosis [109,110]. Deletion of chromosome 13 by metaphase cytogenetics in PCM (Figure 6.28) is associated with a poor prognosis when treated with standard chemotherapy.

III

164

Common Chromosomal Changes in Hematopoietic Tumors

26 25 24 23 22

p

III

t(1;3)

21

TCTA

T-ALL

14

FOXP1

DLBCL MZL (MALT)

13 12 t(2;3) 11.2 12 13.1 13.2 13.3 21 22 23 24 25

t(3;3) t(3;12) t(3;21) inv(3) AML/tAML MDS/tMDS CML CML (blast crisis)

q EVI1 (MDS1)

26.1 26.2 26.3 27 28 29 3

FIgure 6.7

ALCL

t(3;5) MLF1

AML MDS CML, blast crisis

BCL6

FL DLBCL MZL

t(3;14) t(3;2) t(3;22)

Chromosome 3.

p Mastocytosis AML with t(8;21) AML with inv(16)

Mutations KIT

16

FGFR3

15.3 15.2 15.1 14 13 12

t(4;14)

12

22 23 24 25 26

q

27 28 31.1 31.2 31.3 32 33 34 35 4

Chromosome 4.

del(4)(q12) CHIC2 PDGFRA

PCM

Mastocytosis CEL/HES t(4;12)

13 21

FIgure 6.8

TFG

AF4 (MLLT2) t(4;11)

B-ALL MPAL AML

AML

165

Cytogenetics

15.3 15.2 15.1 14

p

13 12

III

11.2 12 13

CMML ± eosinophilia aCML CEL/HES SM-CEL/HES AML ± eosinophilia MPN + eosinophilia

t(1;5) t(5;7) t(5;12) t(5;14) t(5;15) t(5;17)

15 q

del(5q)

MDS AML

22 23 31

IL3

32 33 34

t(5;14) TLX3

35 5

t(5;14) [BCL11B] [TCRD]

FIgure 6.9

21

PDGFRB

B-ALL with eosinophilia T-ALL

14

t(2;5) [ALK] ALCL t(3;5) [MLF1] AML/MDS t(5;17) {RARA] APL ALCL AML APL NPM1 CMML CML (blasts crisis)

Chromosome 5.

AML MDS

25 24 23 22

DEK t(6;9)

MUM1 (IRF4)

PCM DLBCL Other lymphomas

21.3 21.2 21.1 12

p

12 13 14 15 16 q

21 22

FOXO3A

tAML (monocytic)

t(6;11)

23 24 25 26 27

t(6;11) MLLT4

AML (AMML)

6

FIgure 6.10

Chromosome 6.

del(17p)/monosomy 17 • Non-Hodgkin lymphomas • B-CLL • PCM • MDSs

• AML • CML (disease progression/clonal evolution) Deletion of 17p (Figure 6.29) or monosomy 17 occurs in a wide range of hematopoietic tumors and is associated with

166

III

Common Chromosomal Changes in Hematopoietic Tumors

a poor prognosis and resistance to standard chemotherapy [111–116]. CLL with del(17p) or mutations of the TP53 gene has a very poor outcome. The abnormalities, del(17p), t(4;14), and del(13q), have been established as predictors of poor outcome in patients with PCM treated with conventional chemotherapy or stem cell transplant. del(20q) • AML • MDSs • Chronic myeloproliferative neoplasms (PV, ET, PMF) • Angioimmunoblastic T-cell lymphoma (AITL; rare cases)

• • • •

LPL/WM (rare cases) MZL (rare cases) ALL/LBL (rare cases) CML (rare cases)

Deletion of 20q is associated most often with myeloid neoplasms [MDS, myeloproliferative neoplasm (MPN)], but rare B-cell lymphomas, including MZL and LPL/WM with del(20q), have been reported (follow-up in these patients did not reveal myeloid malignancies, including therapy-related tumors). Deletion of  20q alone (similarly to –Y and trisomy 8) is not diagnostic of MDS in the absence of morphological criteria [25].

inversions AML

22

t(7;11)

t(7;11)

CML

inv(3)/t(3;3) • MDSs • AML • Therapy-related myeloid neoplasms

21 HOXA9

HOXA11

15 14 13 12 11.2

p

inv(7)

T-ALL

The 3q21q26 inversion (Figure 6.30) is associated with both MDS and AML, often in association with monosomy 7. The AML with inv(3)(q21q26)/t(3;3)(q21;q26) has a poor prognosis [12].

11.2 TCRG 21

inv(7)(p15q34)/t(7;7)(p15;q34) • T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) • AML • Therapy-related myeloid neoplasm

22

q

31 32 33 34 35

TCRB

BRAF

HCL

Monosomy 7 in AML is rare and occurs often together with aberrations of 3q. The inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (7q34) and the HOXA gene locus (7p15) occurs in 5% of T-ALL patients leading to a transcriptional activation of HOXA10.

36

7

FIgure 6.11

Chromosome 7.

23 22 21

p AMML AML, monocytic

12 11.2

MYST3

11.2 12

t(2;8) t(8;16) [CREBBP] t(8;22) [EP300] inv(8)

13 21.1 21.2 21.3

q

22

t(2;8) [IGK] t(8;14) [IGH] t(8;22) [IGL]

BL DLBCL BCLU (double hit)

Chromosome 8.

t(6;8) t(8;9) t(8;11) t(8;12) t(8;13) t(8;17) t(8;19)

BAALC

AML CML, blast crisis

23

MYC

8

FIgure 6.12

ALL MPN ±eosinophilia (often progress to AML)

FGFR1

24.1 24.2 24.3

ETO (RUNX1T1) t(8;21)

AML

167

Cytogenetics

Mutations t(9;11) [MLL]

24

AF9 (MLLT3)

AML B-ALL B-NHL HL PCM

JAK2

23 22 21 13

p

PAX5

12

t(8;9) PCM1-JAK2

P.vera PMF ET AML MDS CEL aCML

12 13 21

t(7;9) [TCRB] T-ALL

TAL2

22 q

31 32 33

t(9;11) [MLL] AML AML MDS

FBP17

34

ABL1

9

t(9;12) ABL1-ETV6

ALL

Chromosome 9.

• Chronic myeloproliferative neoplasms • Non-Hodgkin lymphomas • CML, blast crisis

15

p

14 13 12

MLLT10

11.2

t(10;11)

AML T-ALL

11.2

22

q

23 24

MYST4

AML/tAML tMDS

t(10;16) NFKB2

25 26

t(10;14)

B-cell lymphomas T-ALL B-CLL PCM

10

Chromosome 10.

inv(11)(p15q22) • MDSs • AML • Therapy-related myeloid neoplasm • CML, disease progression/clonal evolution inv(12) • MDSs • AML • ALL/LBL

inv(14) • T-PLL The inversion of chromosome 14 [inv(14)] occurs in T-PLL (Figure 6.31).

21

FIgure 6.14

CML AML ALL

NUP214 t(6;9;11) [DEK]

FIgure 6.13

t(9;22) ABL1-BCR

inv(16) or t(16;16) • AML, especially acute myelomonocytic leukemia with eosinophilia • CML Abnormalities of chromosome 16 in AML have been associated with high complete remission and survival rates, and a favorable prognosis [117,118]. The inv(16)(p13.1q22), t(16;16)(p13.1;q22), and del(16)(q22) are nonrandom abnormalities associated with acute myelomonocytic leukemia with eosinophilia, and rarely with other subset of AMLs. The t(16;16) or inv(16) results in the fusion of MYH11 at 16p13 (SMMHC) with a part of CBFβ gene at 16q22. The prognosis of AML with del(16q) differs from that in AML with inv(16)/t(16;16) [119]. Additional karyotypic abnormalities may be present in up to 50% of AML with t(16;16)/ inv(16) and most often include trisomies 8, 21, and 22. The presence of additional changes, particularly +22, predicts a better outcome [12], although some other reports did not find the difference in the overall outcome compared to the patients with sole chromosome 16 aberrations [118,119].

III

168

Common Chromosomal Changes in Hematopoietic Tumors

tMDS AML/tAML

NUP98

15

t(11;20) t(7;11) inv(11) T-ALL

III

14

p

LMO2

Mutations WT1

13 12 11.2

t(11;14) [TCRD-A]

AML ALL MPAL

12 BCL1 (CCND1)

13

t(11;14)

t(11;18)

14

q

API2 (BIRC3)

ATM

22

API1 (BIRC2)

23

PLZF

24 25

11

FIgure 6.15

B-CLL T-PLL MCL

21

MZL

APL

MCL PCM

AML/tAML ALL MPAL MDS t(4;11) [AFF1] t(6;11) [EF6] t(9;11) [EF9] t(10;11) [EF10] t(11;19) [ELL] Partial tandem duplications MLL

Chromosome 11.

AML

ETV6 (TEL)

13

CDKN1B

12

p Interstitial deletions Monoallelic deletions

11.2 12 13 14 15 q

21

MDS ALL AML Lymphoma

t(1;12) [ARNT] t(1;12) [ABL2] t(1;12) [MDS2] t(3;12) [EVI1] t(4;12) [FGFR3] t(4;12) [CHIC2] t(5;12) [PDGFRB] t(7;12) [HLXB9] t(9;12) [JAK2] t(9;12) [SYK] t(12;13) [CDX2]

22 23 24.1 24.2

t(12;21) [RUNX1] t(12;22) [MN1]

AML APL MDS CML T-NHL AML CMML AML Leukemia MDS CML, blast crisis MDS AML ALL ALL MDS

24.3 12

FIgure 6.16

Chromosome 12.

The coexistence of t(9;22) and inv(16) in CML appears to correlate with more rapid transformation into blast phase (BP) [120].

i(7q) • HSTL

isochroMosoMes

i(17) • CML/CML in blast crisis • MDS • Other hematologic malignancies

i(3) • Persistent polyclonal B-cell lymphocytosis

169

Cytogenetics

AML MDS B-ALL APL (AML-M3)

D835 point mutations; internal tandem p duplications

13 12 11.2

FLT3

t(12;13) [ETV6]

12

CDX2

AML

13 FL BL

LCP1

AML ALL DLBCL Mutations; ALCL MDS deletions HL PCM RB1

14

t(3;13) [BCL6]

21

q

22 31 32 33 34 13

FIgure 6.17

Chromosome 13. 13 12 11.2

p T-ALL T-PLL ATLL T-cell NHL

TRD TRA t(1;14) [TAL1] t(8;14) [MYC] t(10;14) [HOX11] t(11;14) [RBTN2] t(14;14) [TCL1] t(14;21) [OLIG2]

11.2 12 13 21

t(1;14) [BCL10] t(3;14) [BCL6] t(4;14) [FGFR3] t(5;14) [IL3] t(8;14) [MYC] t(9;14) [PAX5] t(11;14) [CCND1/BCL1] t(14;18) [BCL2] t(14;19) [BCL3]

22 23

q

24

IGH

31 T-ALL

BCL11B t(5;14) [TLX3]

FIgure 6.18

MZL DLBCL PCM B-ALL BL DLBCL B-ALL LPL/WM MCL PCM FL DLBCL B-CLL

32

TCL1A t(14;14) t(7;14) inv(14)

14

B-cell lymphomas PCM T-PLL T-ALL T-cell NHL

Chromosome 14.

translocations t(1;3)(p36.3;q21.2) • Therapy-related myeloid neoplasm (rare) • ALL/LBL • MDSs t(1;4) • HSTL t(1;5)(q23;q33) • MDS with eosinophilia • MPN with eosinophilia

t(1;6) • B-CLL Reciprocal translocations in CLL are very rare and may include t(1;6) (Figure 6.32). t(1;14)(p22;q32) • MZL (MALT lymphoma) A few recurrent translocations have been associated with extranodal MZL of MALT type, including t(11;18) (q21;q21), t(14;18)(q32;q21), and t(1;14)(p22;q32). The t(1;14)(p22;q32) leads to an overexpression of BCL10

III

170

Common Chromosomal Changes in Hematopoietic Tumors

t(1;14)(p32;q11) • T-ALL/LBL

13 12 11.2

p

t(1;19) • B-ALL/LBL

11.2 12 13 14

III

The t(1;19)(q23;q13) translocation, associated with fusion of the E2A (TCF3) gene on chromosome 19 and PBX1 gene on chromosome 1, is present in 20%–30% of precursor B (pre-B) ALL [126].

15 21 q

PML

22 23

APL

t(15;17)(q22−24;q12−21) [PML/RARA]

24 25 26

Chromosome 15.

13.3 13.2 13.1

AMML with inv16 MDS

MYH11 CREBBP

12

p

11.2 t(16;16)(p13;q22) inv(16)(p13;q22) del(16)(q22)

11.2 12.1 12.2 13 21 q

CBFb

22

AML with inv16 MDS

23 CBFA2T3

24 16

FIgure 6.20

t(2;3) • AML t(2;5)(p23;q35) • ALCL

15

FIgure 6.19

t(1;22)(p13;q13) • Acute megakaryoblastic leukemia

tAML

t(16;21) [RUNX1]

Chromosome 16.

[121,122]. Through t(1;14)(p22;q32), the BCL10 gene is entirely transferred to the IGH gene, resulting in its overexpression. Wild-type BCL10 is implicated in activation of nuclear factor (NF)-κB. The t(1;14) translocation is seen most often in pulmonary (~4%) and gastric (~8%) MALT lymphoma, and only sporadically occurs in other locations [123,124]. In contrast to t(11;18), MALT lymphomas with t(1;14) often show other chromosomal aberrations, such as trisomies 3, 12, and 18 [125]. The t(1;14)-positive cases, similarly to t(11;18) cases, are resistant to Helicobacter pylori eradication therapy. The t(1;14) has not been identified in other lymphoma types.

The t(2;5)(p23;q35) is the specific aberration occurring in ALCL and is associated with NPM–ALK fusion, which can be detected at a molecular (FISH, PCR) and an immunohistochemical level. FISH appears to be more sensitive with higher rate of detection compared to reverse transcriptase PCR (RT-PCR) [127]. While t(2;5)/NPM–ALK represents the majority of ALK rearrangements (75%), the subset of cases has a variant translocation, which includes t(1;2) (TPM3 gene on 1q25), t(2;3) (TFG gene on 3q11–12), t(2;2) (ATIC gene on 2q35), and t(2;17) (CLTC gene on 17q23) [128–131]. The rare variant of DLBCL may express ALK by immunophenotyping (cytoplasmic localization) without t(2;5) translocation. This protein appears to be a full-length ALK receptor (and not a chimeric molecule characteristic for ALCL) [132,133]. The majority of ALK+ DLBCLs display t(2;17)(p23;q23) involving the clathrin gene (CLTC) [133]. t(2;8) • BL • B-ALL/LBL t(2;14) • CLL/SLL The t(2;14)(p16;q32) juxtaposing the BCL11A with IGH is very rare but recurrent translocation, and is associated with balky adenopathy, unmutated IGVH, and atypical morphology and immunophenotype. t(2;17) • DLBCL t(2;19) • ALCL t(3;3)(q26;q21)/inv(3) • AML • MDSs

171

Cytogenetics

13 t(5;17) [PDGFRB] CMML juvenile

12

p

HCMOGT-1

APL T-LGL leukemia

11.2

STAT5b

mutations del(17p) i(17q)

11.2 RARA

APL

t(11;17) [AF17] MLLT6

AML

22

t(15;17) [PML] t(11;17) [PLZF] t(5;17) [NPM] t(11;17) [NuMa]

MSI2

23

CLTC

t(7;17)

24

21

t(2;17) [E2A] B-ALL CML accelerated

ALCL

HLF

q

ALO17

25

t(2;17)(p23;q25) [ALK]

FIgure 6.21

B-CLL B-ALL HL CML, blasts crisis MDS AML PCM B-NHL/BL ATLL HIV-related lymphoma

p53 (TP53)

17

ALCL DLBCL with ALK1

t(2;17)(p23;q23) [ALK] MSF

AML

t(11;17) [MLL]

Chromosome 17.

t(3;8)(q27;q24.1) • DLBCL

The proto-oncogene B-cell lymphoma 6 (BCL6; LAZ3) on chromosome 3q27 encodes a transcription protein, with a POZ/zinc finger motif that is involved in cell cycle control, proliferation, lymphocyte differentiation, immunologic response, and repression of genes involved in lymphocyte activation and differentiation and inflammation [134–136]. The BCL6 gene was initially identified through its involvement in the DLBCL translocation between 3q27 and the immunoglobulin gene at 14q32 [137,138]. The t(3;14)(q27;q32) occurs in 7%–14% of DLBCL cases [139–142]. Au et al. [143] reported three patients (one was HIV+) with nonclonal t(3;14) or t(3;22); their lymphadenopathy resolved spontaneously, and none progressed to lymphoma at 4–6 years of follow-up. The BCL6 rearrangements are diverse and include translocations, microdeletions, point mutations, and hypermutation. It can be translocated to a number of translocation partners, most commonly immunoglobulin heavy (IGH/14q32) and light chain (IGK/2p12; IGL/22q11) genes. The other partners include 1q21, 2q21, 4p11, 5q31, 6p21, 7p12, 8q24, 9p13, 11q13, 11q23, 12q11, 13q14–21, 14q11, 15q21, and 16p11. Because of the large number of translocation partners, FISH analysis of BCL6 is best achieved by using break-apart probe. BCL6 translocations are found in ~40% of DLBCL and 5%–15% of FL [144–147]. Rearrangement of BCL6 can be identified in other types of lymphoma, including MZL and MCL [106,148,149]. The t(3;14)(p14;q32)/FOXP1–IGH has been recently identified in rare cases of MZL of MALT type (thyroid, ocular adnexae, skin), most often accompanied by additional genetic abnormalities [150].

t(3;14) • DLBCL (3q27; 3p14) • MZL (MALT type; 3p14)

t(3;21) • AML • Therapy-related myeloid neoplasm

11.3 p

11.2 11.2 12

q MZL

21

MALT1 t(11;18) [API2-MALT1] t(14;18) [IGH-MALT1]

22 23 18

FIgure 6.22

Chromosome 18.

t(3;5) • MDSs • AML t(3;8)(q26;q24) • AML • MDSs • Therapy-related myeloid neoplasm • CML, blast crisis

BCL2 t(14;18) [IGH-BCL2]

FL DLBCL

III

172

Common Chromosomal Changes in Hematopoietic Tumors

t(7;19) [TCRB] t(1;19) t(9;19) t(10;19) t(11;19) t(15;19) T-ALL AML

III

t(1;19) t(17;19) 13.3

LYL1

p

B-ALL

TCF3 (E2A)

13.2

t(11;19) [MLL]

13.1 12

ELL

AML

12 AML

CEBPA

Mutations

13.1 13.2

q Rarely

13.3 19

FIgure 6.23

FLT3

13.4

B-ALL t(14;19) [IGH]

PV ET, PMF

PRV1

AML B-ALL MDS

Internal tandem duplication (ITD) mutations

Chromosome 19.

13 p MDS AML AML

12 11.2

t(X;20) 11.2 TOP1

12

q

t(11;20)

CBFA2T2

13.1 13.2 13.3

del(20q)

20

FIgure 6.24

AML MDS CML B-ALL

AML MDS PV ET; PMF Other (rare)

Chromosome 20.

t(8;21) t(X;21) t(19;21) t(3;21) t(12;21) t(1;21)

13 12 11.2

p

11.2 21 ERG

q

22

RUNX1

OLIG2 21

FIgure 6.25

AML

ALL AML T cell NHL

t(14;21)(q11.1;122)

Chromosome 21.

• CML, BP • Precursor ALL/LBL Two recurring chromosomal translocations involving RUNX1 gene associated with myeloid leukemias are t(8;21)(q22;q22) and t(3;21)(q26;q22) (Figure 6.33). The latter occurs in chemotherapy-related myeloid neoplasm (primarily following

treatment with topoisomerase II inhibitors) and occasionally in the BP (blast crisis) of CML [151,152]. De novo AMLs with t(3;21) are rare. The t(3;21) involves RUNX1 on 21q and EVI1 on 3q26 [153]. RUNX1 corresponds to CBFA2 and encodes one of the DNA-binding subunits of the enhancer core-binding factor (CBF) [151]. t(4;11) • ALL/LBL The translocations involving MLL gene (also known as ALL1 gene) on 11q23 are common in leukemia and include several different partners: 1p32, 4q21, and 19p13.3 in ALL and 1q21, 2q21, 6q27, 9p22, 10p11, 17q25, 19p13.3, and 19p13.1 in AML [t(4;11) being the most common] [154–157]. Those leukemias are characterized by higher white blood cell counts, but only those with t(4;11) show a female predominance. The t(4;11) occurs usually in early-pre-B-ALL (pro-B-ALL), whereas other 11q23 translocations may occur in both B- and T-ALL. The t(4;11) translocation leads to the fusion of MLL

173

Cytogenetics

DLBCL

t(3;22) BCL6-IGL

13 12 11.2

p

BL DLBCL B-ALL

t(8;22) MYC-IGL

ALCL

q

FIgure 6.26

12

t(9;22) ABL1-BCR

13

MKL1

MYH9

III

Acute megakaryoblastic leukemia

t(1;22)

22

t(2;22)

CML AML ALL

BCR

11.2

IGL

Chromosome 22. 22.3 22.2 22.1 Down syndrome + acute megakaryocytic leukemia or transient myeloproliferative syndrome

21 p

11.4 11.3 11.2

Mutations GATA1

12

p

t(X;11)

11.2 q

21 q

11.2

AML T-ALL

MLLT7

13

11.3

22

12

23

Y

24 25 t(X;14) t(X;7)

26 27 28

MTCP1

T -PLL

X

FIgure 6.27

Chromosomes X and Y.

1

6

A

FIgure 6.28

B

2

7

3

8

4

9

5

10

13

14

15

16

19

20

21

22

11

17

X

12

18

Y

PCM with complex karyotype including monosomies 13 and 17: (A) cytomorphology; (B) metaphase cytogenetics.

174

Common Chromosomal Changes in Hematopoietic Tumors

15

16

17

18

21

22

X

Y

and AF4 genes. B-ALL with t(4;11) is associated with a poor prognosis [18,154,158,159]. Similarly to patients with Ph chromosome+ ALL, the presence of t(4;11) is associated with an increased percentage of minimal residual disease (MRD), and therefore an increased risk of relapse.

III

t(4;14) • PCM

FIgure 6.29 Deletion 17p(11.1): Metaphase cytogenetics (partial karyotype).

1

FIgure 6.30

13

2

3

4

The t(4;14)(p16;q32) translocation involves the subtelomeric regions of chromosome arms 4p and 14q, is karyotypically silent, and causes an overexpression of FGFR3 and MMSET [160]. The t(4;14)/FGFR3–IGH is detected most often by interphase FISH, but can also be detected by metaphase FISH, RT-PCR with capillary electrophoresis, gene expression profiling (microarray assay), and immunohistochemistry. The t(4;14)/FGFR3–IGH is found in ~15% of patients with multiple myeloma [160–162] and is much less common (~3%) in patients with MGUS [163–166]. Patients with t(4;14) have a poor prognosis with significantly shorter progression-free (median 9.9 vs. 25.8 months) and overall survival (median 18.3 vs. 48.1 months) than those without this translocation despite intensive chemotherapy and autologous stem cell transplant [162,167–170].

5

Inversion 3: Cytogenetics (partial karyotype).

14

FIgure 6.31 karyotype).

15

16

17

Inversion 14: Metaphase cytogenetics (partial

1

6

t(5;10)(q22;q24) • ALL/LBL t(5;12) • Chronic myelomonocytic leukemia (CMML) • Chronic myeloproliferative neoplasms (non-CML) with eosinophilia • Chronic eosinophilic leukemia (CEL)

2

7

Chromosome 5 is a site for platelet-derived growth factor receptor β (PDGFRB). The t(5;12) leads to the disruption of PDGFRB in which the 5′ end of ETV6 (earlier known as TEL) is juxtaposed to the 3′ end of PDGFRB (Figure 6.34). PDGFRB is disrupted by other translocations, and to date, four additional partner genes have been reported (H4, HIP1,

8

FIgure 6.32 Translocation t(1;6): Metaphase cytogenetics (partial karyotype).

FIgure 6.33

1

2

3

6

7

8

13

14

15

19

20

Translocation t(3;21): Metaphase cytogenetics.

9

21

4

5

10

11

12

16

17

18

22

X

Y

175

Cytogenetics

t(7;7) See “inv(7)” section. 3

9

4

10

t(7;11) • AML • CML, disease progression/clonal evolution (blast crisis)

5

11

12

FIgure 6.34 Translocation t(5;12): Metaphase cytogenetics (partial karyotype).

CEV14, and Rab5). Chronic myeloproliferative disorders (CMPDs) associated with t(5;12) are infrequent and represent a minority of patients within BCR–ABL-negative group. Clinically, most patients present with either myeloproliferative disorder with eosinophilia, CEL, or CMML [171]. Most patients are male with the 2-year survival of 55% [171,172]. t(5;17) • APL • MDSs (rare cases) • Acute myelomonocytic leukemia with eosinophilia (rare cases) The t(5;17)(q35;q21) is a rare translocation, in which RARA gene recombines with the NPM1 (Nucleophosmin) gene on chromosome 5q35 (NPM1–RARA). This translocation occurs in occasional cases of (hypergranular) APL. t(6;7)(p24;q21) • CML t(6;9) • AML • MDSs t(6;11) • T-PLL t(6;14) • PCM The MUM1 (multiple myeloma 1 or interferon regulatory factor 4) is a proto-oncogene that is deregulated as a result of (6;14) (p25;q32) chromosomal translocation in PCM, and is also expressed in various malignant lymphomas. MUM1, which can be identified by immunohistochemistry, may provide a marker for the identification of transition from BCL6 positivity (germinal center B cells) to CD138 expression (immunoblasts and plasma cells). In normal B cells, MUM1 expression is thought to denote the final step of intragerminal center B-cell differentiation and subsequent maturation toward the plasma cells. In DLBCL, MUM1 is detected in 50%–75% of cases, and is seen both with and without BCL6 expression [173–175].

t(7;12) • AML (pediatric) • Precursor ALL/LBL (pediatric) t(8;9)(p11;q33) • 8p11 myeloproliferative syndrome (EMS) t(8;9)(q24;p13) • B-ALL/LBL t(8;9)(p22;p24) • CML • CEL • AML • ALL/LBL • MDSs t(8;13)(p11;q12) • EMS Several recurrent translocations that involve chromosome band 8p11 have been described in myeloid malignancies. These translocations target FGFR1, a receptor tyrosine kinase for fibroblast growth factors, and MYST3. The 8p11 with disruption and activation of FGFR1 is associated with rare, aggressive hematological malignancy termed 8p11 myeloproliferative syndrome (EMS) or 8p11 stem cell leukemia/lymphoma, which frequently presents with hypereosinophilia and associated T-cell lymphoblastic lymphoma [176–178]. The disease rapidly transforms to acute leukemia, usually of myeloid phenotype. The most commonly observed translocation of this syndrome is t(8;13)(p11;q12), which results in the fusion of ZNF198–FGFR1 genes. Other gene fusions associated with distinct translocations described in EMS include t(8;9)(p11;q33), t(6;8)(q27;p11), t(7;8)(q34;p11), and t(8;22)(p11;q22), which fuse CEP110, FOP, TIF1, and BCR, respectively, to FGFR1 [176–179]. The t(8;17)(p11;q23) has also been reported. t(8;14)(q24;q32) • BL • B-ALL/LBL • DLBCL (subset) • B-cell lymphoma, unclassifiable with features intermediate between BL and LBCL (BCLU; “gray zone” lymphoma) • Other B-cell lymphomas (including FL, B-PLL, and MCL), often undergoing transformation to aggressive lymphoma

III

176

• PCM (rare cases) • Plasmablastic lymphoma

III

The MYC gene is translocated to the IG loci in all BLs [180,181]. The t(8;14)(q24;q32) translocation that involves MYC (on chromosome 8q24) and IGH (on chromosome 14q32) is the most frequent translocation (80% of BL). The remaining (variant) translocations include t(2;8) (p11.2;q24) involving IGK (on chromosome 2p11.2) and t(8;22)(q24;q11) involving IGL (on chromosome 22q11). The MYC translocations are characteristic of BL, but they have also been reported in a subset of DLBCLs (6%) [182] and other hematolymphoid tumors including BCLU, FL, a blastoid variant of MCL, PCM, prolymphocytoid transformation of B-CLL/SLL, Burkitt-type B-AML, and ALK+ ALCL [183–194]. Overexpression of MYC has been shown to be a consistent finding in ALK+ ALCL, but not ALK− ALCL, and the MYC gene is considered a downstream target of deregulated ALK signaling [193–195]. In addition to the characteristic t(2;5)(p23;q35) translocation, Monaco et al. [193] reported a t(3;8)(q26.2;q24) translocation and MYC gene rearrangement (confirmed by FISH analysis) in pediatric ALCL. MCL is genetically characterized by the t(11;14)(q13;q32) that deregulates cyclin D1. Small subsets of cases have been identified with variant CCND1 translocations with the immunoglobulin light-chain genes or with alternative translocations involving CCND2 and CCND3. Additionally, double-hit MCLs with MYC rearrangements with a highly aggressive clinical course have been reported. Plasmablastic lymphoma is often associated with MYC–IGH rearrangement [196,197]. t(8;16) • AML [most acute myelomonocytic leukemia (AMML)] • Therapy-related myeloid neoplasm • EMS AML with translocation t(8;16)(p11;p13) is an infrequent leukemia subtype with characteristic clinicobiological features. This translocation leads to fusion of MYST3 (MOZ; monocytic leukemia zinc finger protein) gene on chromosome 8p11 and CREBBP (CBP) gene on chromosome 16. The t(8;16) (p11;p13) is associated with de novo or therapy-related AML, often displaying monocytic differentiation, erythrophagocytosis, extramedullary infiltration, and disseminated intravascular coagulation [198]. t(8;21)(q22;q22.3) • AML The t(8;21)(q22;q22.3) translocation is seen in AML and is characteristically associated with AML with maturation subtype and frequent coexpression of CD34, CD19, and CD56 [36,199–202]. This translocation is one of the most frequent structural chromosomal abnormalities seen in AML and are reported in 6%–20% of AML cases. The t(8;21) results

Common Chromosomal Changes in Hematopoietic Tumors

in fusion between RUNX1 (Runt-related gene family; formerly termed AML1 gene) and RUNX1T1 genes (formerly ETO for eight twenty-one), producing a chimeric protein, RUNX1–RUNX1T1 [203]. AML with t(8;21) belongs to a group of CBF leukemias. CBF is also rearranged in AML with t(3;21) and t(16;16)/inv(16). AML with t(8;21) is found more frequently in children and young adults and displays predisposition for extramedullary localization [204,205]. This subtype of AML is associated with a favorable prognosis [12,118,204,205]. Additional cytogenetic abnormalities in AML with t(8;21) are common (75%) and may include −X, −Y, del(9)(q22), +8, and +4 [206,207]. Approximately 28% of patients show more than one chromosomal abnormality [206]. Additional abnormalities did not have a significant adverse effect in t(8;21) AML [12]. t(8;22) • BL • B-ALL/LBL t(9;11)(p21;q23) • AML The translocations of 11q23 involve MLL gene (also known as ALL1 or HRX); they are common in leukemias and include several different partners: 1p32, 4q21, and 19p13.3 in ALL, and 1q21, 2q21, 6q27, 9p21, 10p11, 17q25, 19p13.3, and 19p13.1 in AML. The t(9;11)(p21;q23) is associated with an intermediate prognosis in AML [36,118]. Other translocations involving 11q23, except t(11;19), usually have a poor prognosis [12]. The t(9;11), similarly to t(11;19), is often associated with monoblastic differentiation, but other variants of AML (e.g., AML with differentiation) have been reported as well. In childhood AML, the t(9;11) is a favorable genetic factor [208]. t(9;12) • AML • ALL/LBL • CML, disease progression t(9;14)(p13;q32) • LPL/WM The t(9;14)(p13;q32) is a translocation that occurs in B-cell lymphomas with plasmacytic differentiation with indolent presentation followed by a gradual clinical progression of disease [209]. In a more recent series reported by Cook et al. [210], the incidence of this translocation in LPL/WM was less common than previously reported. The t(9;14) results in the juxtaposition of the PAX5 (paired homeobox-5) gene with the IGH on chromosome 14. t(9;22) • CML • AML • ALL/LBL

177

Cytogenetics

The t(9;22)(q34;q11) translocation occurs in chronic myeloid leukemia (CML), subset of ALLs, and rare cases of AML. Ph chromosome, a shortened chromosome 22, results from a reciprocal translocation between the BCR gene on chromosome 22 and the ABL1 gene on chromosome 9. Acute leukemia with t(9;22) can be either de novo or results from transformation from CML (BP, myeloid, or lymphoblastic). The t(9;22) is frequent and prognostically unfavorable translocation in pre-B-ALL despite intensified chemotherapy [211,212]. Progression of CML, including accelerated phase (AP) or BP (blast crisis), is often associated with chromosomal evolution, that is, the appearance of chromosomal abnormalities in addition to Ph chromosome. These may include additional copies of Ph chromosome, isochromosome 17 [i(17)(q10)], a gain of chromosome 8 or 19, and less often −7, −17, +17, +21, −Y, and t(3;21)(q26;q22). t(10;11) • T-ALL/LBL • Peripheral T-cell lymphoproliferative disorders • AML The t(10;11) translocation is associated with a poor prognosis in AML, similarly to the majority of aberrations involving MLL gene (11q23), except for t(9;11) and t(11;19) [12]. t(10;14) • T-ALL/LBL • Peripheral T-cell lymphoproliferative disorders t(10;17)(p15;q21) • AML • B-ALL/LBL t(10;22) • Acute megakaryoblastic leukemia (AML-M7) t(11;14)(q13;q32) • MCL • PCM The t(11;14)(q13;q32) is the hallmark of MCL, where it can be detected virtually in all cases [139,213–216]. In this translocation, gene encoding cyclin D1 protein at 11q13 (CCND1 or BCL1 gene) is relocated to an IGH gene on 14q32 resulting in upregulation of cyclin D1 expression. Apart from MCL, the t(11;14)(q13;q32) is also observed in the subset of multiple myelomas (3%–20%) [217,218]. In multiple myeloma, cyclin D1/BCL1 upregulation is detected more often in the t(11;14) (q13;q32), suggesting that other chromosome 11 abnormalities, as well as additional mechanisms, must be responsible for the cyclin D1 overexpression. Panani et al. [219] showed that t(11;14) had a worse impact on disease outcome compared to t(14q32) with an unidentified partner chromosome. t(11;14)(p13;q11) and t(11;14)(p15;q11) • T-ALL/LBL

t(11;17)(q23;q21) • APL • Mixed phenotype acute leukemia (MPAL; rare cases) • MDS (rare cases) The t(11;17)(q23;q12) is present in a subset of patients with APL. This translocation results in the fusion of RARA gene (17q12–21) with the PLZF (promyelocytic leukemia zinc finger) gene (11q23). The t(11;17)/PLZF–RARA+ APL differs from classic APL with t(15;17) by its poor response to chemotherapy, ATRA resistance, and poorer prognosis, when treated with ATRA alone [220,221]. In the t(11;17(q13;q21), RARA gene partners with NuMA (nuclear mitotic apparatus) gene. Another translocation of RARA gene involves STAT5b gene on chromosome 17(q11). APLs with either t(11;17)(q13;q21)/NuMa–RARA or t(17;17) (q11;q21)/STAT5b–RARA present with morphologic and clinical features similar to classic type with t(15;17)/ PML–RARA. The t(11;17) translocation has also been identified in patients with MPAL and rare cases of primary MDS [222]. t(11;18)(q21;q21) • MZL (MALT lymphoma) There are two translocations involving MALT1 gene at 18q21: t(11;18)(q21;q21) involving API2 and MALT1 genes, and t(14;18)(q32;q21) involving IGH and MALT1. The t(11;18)(q21;q21) resulting in the API2–MALT1 fusion transcript is an exclusive finding in extranodal MZL of MALT type [223–225]. Within gastrointestinal tract, extranodal MALT lymphoma occurs most often in stomach and rarely in small and large intestines and esophagus. Gastric MALT lymphoma is associated with H. pylori infection. Apart from t(11;18)(q21;q21), other chromosomal aberrations in MZL lymphoma include t(1;14)(p22;q32); t(14;18)(q32;q21); t(1;14)(p22;q32); trisomies 3, 12, and 18; and p53 loss of heterozygosity (LOH)/mutation and fas gene mutation. The cases with t(11;18) usually do not show other genetic aberrations [226], such as trisomies 3 and 18, frequently seen in t(11;18)-negative tumors [227–230]. The t(11;18) has been reported in 40%–50% of MZLs arising in the stomach (MALT lymphoma) [231,232]. Gastric MALT lymphomas positive for t(11;18) are more often associated with the involvement of the lymph node and distal sites than t(11;18)negative cases [224]. The presence of t(11;18) translocation is a negative predictor for response to H. pylori eradication therapy [225]. The t(11;18)-positive MALT lymphomas are distinct from other MALT lymphomas, including those with t(1;14) or t(14;18). The t(11;18)-positive MALT lymphoma rarely undergoes high-grade transformation [227,233] despite more advanced stages and lack of response to H.  pylori eradication therapy. Gastric MALT with the t(11;18)(q21;q21), however, does not adversely affect the response of gastric MALT lymphoma to chemotherapy with cladribine (2CdA) [234]. This makes the cladribine

III

178

Common Chromosomal Changes in Hematopoietic Tumors

an attractive agent for the treatment of gastric MALT lymphoma unresponsive to H. pylori eradication.

III

t(11;19)(q23;p13.1) and t(1;19)(q23;p13.3) • AML • B-ALL/LBL • MPAL The 11q23 abnormalities occur in acute leukemias (Figure  6.35). Patients with t(9;11), t(6;11), or other 11q23 balanced translocations present at a younger age and with higher percentage of bone marrow blasts [235]. Unbalanced 11q23 abnormalities are commonly associated with deletions of chromosomes 5q and 7q, and/or complex karyotypes. Among the 11q23 translocations, t(9;11) has an independent intermediate prognostic significance, while t(11;19) and others have a poor prognostic impact [12]. The t(11;19) (q23;p13.1) occurs mainly in adult patients with AML, while t(11;19)(q23;p13.3) occurs in infants with either AML or ALL [236]. t(12;12) • ALL/LBL

The t(12;22)(p13;q11) translocation involving TEL and MN1 genes is a rare translocation seen in AML patients (Figure 6.36). t(13;17) • AML The t(13;17) translocation in AML is associated with a poor prognosis. It may also occur as a three-way translocation [t(11;13;17)].

t(14;17) • AML

t(12;21) • ALL/LBL Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. The t(12;21)(p12;q22) is commonly found in pediatric AML [93]. This translocation generates the ETV6–RUNX1

2

3

8

4

9

t(14;18)(q32;q21) • FL • DLBCL (subset • ALL/LBL • CLL/SLL (rare cases) The t(14;18)(q32;q21) juxtaposes the BCL2 gene at 18q21 with the immunoglobulin heavy-chain locus (IGH gene) at 14q32. It is characteristic of FL in which the breakpoints of t(14;18) occur in the major breakpoint region (mbr) or in the minor cluster region (mcr) at the 3′ end of BCL2. The frequency of

1

5

6

7

13

14

15

16

17

18

19

20

21

22

X

Y

FIgure 6.35

t(12;22)(p13;q11) • AML (rare cases)

t(14;16) • PCM

t(12;17) • ALL/LBL

1

chimeric gene. ETV6–RUNX1+ ALLs often express CD10, occur between age 1 and 10 years, and have a favorable prognosis. The ETV6 gene is also involved in t(5;12), t(9;12), and t(12;22), which occur in other hematopoietic tumors. Raimondi et al. [237] analyzed 815 children with newly diagnosed ALL and found 94 cases (11.5%) with 12p abnormalities involving ETV6, which was associated with a favorable treatment outcome.

10

11

12

Translocation t(11;19): Metaphase cytogenetics.

2

3

6

7

13

14

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FIgure 6.36 cytogenetics.

8

4

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AML with

5q−

5

10

11

12

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17

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22

X

Y

and t(12;22): Metaphase

179

Cytogenetics

FISH (IGH–BCL2 rearrangement) or PCR (IGH rearrangements for clonality). FISH may also be useful to differentiate FL from other lymphomas with a nodular growth pattern, although morphologic and immunophenotypic features are sufficient in the majority of cases. FISH evaluation of IGH– BCL2 and IGH–MALT1 is also useful to distinguish between FL and MZL with t(14;18) [conventional cytogenetics cannot distinguish the t(14;18)(q32;q21) involving IGH and BCL2 from t(14;18)(q32;q21) involving IGH and MALT1].

t(14;18)(q32;q21) in nodal FL by metaphase cytogenetics and/ or interphase FISH has been shown to vary between reports, most often ranging between 80% and 100% [139,147,216,238– 240]. The t(14;18) is also present in a subset of DLBCLs, being detected in 12%–35% of cases [139,241,242]. The incidence of t(14;18) is higher in lower grade FL (grade 1 or 2), compared to grade 3 [243,244]. Cytogenetic analysis is not always successful due to the low proliferative activity of lymphomatous cells. Although FISH methodology is more sensitive compared to conventional cytogenetics, standard cytogenetic analysis is helpful in identifying accompanying chromosomal abnormalities or unusual changes, such as the three-way translocation involving two major lymphoma-specific abnormalities, 3q27 and t(14;18)(q32;q21) [245]. The t(14;18) translocation results in overexpression of BCL2 protein, which functions to inhibit apoptosis (programmed cell death). The overexpression of BCL2 is also observed in other lymphomas, including B-CLL and MALT lymphoma, and is not always associated with t(14;18). The rare cases of de novo B-ALL display t(14;18) [246–250]. B-ALL with t(14;18) may arise as a blastic transformation of a preceding lymphoma [247,250,251] or as de novo ALL [246]. The t(14;18)/BCL2–IGH can be detected by cytogenetic, FISH, and PCR studies; FISH using either a single-fusion or a dual-fusion strategy and PCR using a consensus primer targeting the IGH joining region in combination of one of three primers flanking the major, minor, and intermediate breakpoint cluster regions on chromosome 18q21. Break-apart FISH probes to detect BCL2 gene rearrangements are also available [252]. Quantification of t(14;18) can be achieved by RT-PCR, especially using a multiplex RT-PCR protocol that allows amplification of control and target genes in the same reaction and precise size determination of BCL2–JH fusion sequences by capillary electrophoresis to evaluate treatment efficacy and minimal residual disease [253]. The analysis of t(14;18) [IGH–BCL2] by FISH is most useful in distinguishing between FL and atypical follicular hyperplasia. Although most cases can be easily diagnosed by morphology combined with immunohistochemistry, in the subset of cases the histomorphologic and immunophenotypic features are equivocal, and therefore require additional testing by either

1

FIgure 6.37

2

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t(14;18)(q32;q21) • MZL (MALT lymphoma) • Extranodal DLBCL (breast, testis) The breakpoint on chromosome 18, involving MALT1 gene, is seen in MZLs of MALT type. The BCL2 and MALT1 genes lie in very close proximity at the 18q21 locus. FISH studies for BCL2 and MALT1 can help to differentiate between MZL and FL, since both may be positive for t(14;18). In MALT lymphomas with t(14;18), there appears to be interaction between MALT1 and BCL10 genes. Both genes and their products can be detected by FISH and/or immunohistochemistry. The t(14;18)/IGH–MALT1 cases typically involve ocular adnexae and lung, and are not reported in gastric location. Another translocation involving the MALT1 gene at 18q21 of MZL is associated with t(11;18) and involves the API2 gene. The t(14;18) with breakpoints in IGH and MALT1 can also be found in DLBCL of breast and testis [254]. t(14;19)(q32;q13) • B-CLL t(15;17)(q24;q21) • APL The reciprocal translocation t(15;17)(q24;q12–21) is the diagnostic hallmark of APL [220]. The t(15;17) translocation creates two chimeric genes: the PML–RARA gene is formed on the derivative 15, whereas the reciprocal RARA–PML fusion is located on the derivative 17 (Figure 6.37). PML (promyelocytic leukemia) gene possesses growth suppressor and

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APL with t(15;17) (PML–RARA): (A) cytogenetics; (B) FISH.

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proapoptotic activity [255,256]. RARA is a transcription factor that mediates the effect of retinoic acid. The PML–RARA plays an important role in leukemogenesis by impairing the growth suppressor and proapoptotic activities of PML. It is also important in mediating the differentiation response to the ATRA treatment [41,220,257]. The introduction of novel targeted therapies in the form of ATRA and arsenic trioxide changed the clinical course of APL over the past 25 years from one that was fatal for the majority of patients to the most curable subtype of AML. Reciprocal translocation of 17q21 is found in >95% of APL. The remaining APL cases show complex or variant translocations involving chromosome 15 or 17 and other chromosomes as well as masked (cryptic) insertions. To date, five different fusion partners of RARA have been identified. The vast majority of cases are characterized by the presence of the t(15;17)(q22;q12–21), which involves the PML gene. The other chromosomal aberrations seen in APL include t(11;17)(q23;q21)/PZL–RARA, t(5;17)(q35;q12– 21)/NPM–RARA, t(11;17)(q13;q21)/NuMa–RARA, t(17;17) (q11;q21)/STAT5b–RARA, and der(17). APL associated with t(15;17), t(5;17), and t(11;17)(q13;q21) appears to be sensitive to ATRA. In contrast, APL associated with t(11;17)(q23;q21) leading to PZL–RARA rearrangement is typified by a lack of differentiation response to retinoids, and patients treated with ATRA alone have a poor prognosis [221]. t(17;17)(qq11.2;q21) • APL t(17;20)(q21;q12) • APL

trisoMies trisomy 2/duplication 2p • DLBCL • Other non-Hodgkin lymphomas trisomy 3/duplication 3p • MZL • SMZL • Other non-Hodgkin lymphomas • Persistent polyclonal B-cell lymphocytosis Trisomy 3 represents the most common recurring abnormality in MZL (MALT lymphoma) [88,240,258–260]. In nonHodgkin lymphomas, trisomy 3 or duplications of 3p predict a favorable clinical outcome [17]. Based on the most frequent recurrent abnormalities in SMZL, Sole et al. [88] divided it into two groups: one with gain of 3q and the other with deletion of 7q. trisomy 5 • AML • ALL/LBL

Common Chromosomal Changes in Hematopoietic Tumors

trisomy 8 • AML • APL • MDSs • Peripheral T-cell lymphoma • CML (disease progression; blast crisis) • PV • B-CLL • HSTL • ALL (rare cases) Trisomy 8 is the most common trisomy in de novo AML. The impact of trisomy 8 on AML patients is best predicted by the presence and nature of other chromosomal abnormalities [118,243]. Patients with sole +8 and +8 with additional abnormality other than t(8;21), inv(16)/t(16;16), and t(9;11) have significantly inferior overall survival, whereas patients with +8 and a complex karyotype with three or more abnormalities have significantly inferior complete remission rate and overall survival [118]. As a sole abnormality, the frequency of +8 varies among different AML subtypes, which is most frequent in acute monoblastic leukemia, followed by acute megakaryoblastic leukemia, AML without maturation, and AML with maturation types. Trisomy 8 is the most common additional chromosomal abnormality in APL (46% of APL with secondary changes) [261]. Outcome is similar among patients with t(15;17) alone and patients with t(15;17) and other clonal abnormalities [261]. Trisomy 8 occurs in CMML and refractory anemia with ringed sideroblasts (RARS). Gain of 8q in cutaneous T-cell lymphoma is associated with significantly shorter survival [72]. Trisomy 8, alone or in combination with a gain of chromosome 19 and additional copies of Ph chromosome, is often observed during clonal evolution of CML (AP or BP). Gain of chromosome 8 is often observed (15%) in chronic phase of PV. Lau et al. [262] described trisomy 8 as a sole cytogenetic abnormality in B-CLL patients. Apart from i(7q), HSTL often displays trisomy 8 [263,264]. Trisomy 8 alone [similarly to −Y and del(20q)] is not diagnostic of MDS in the absence of morphological criteria [25]. trisomy 9 • PV trisomy 11 • AML • MDSs trisomy 12 • B-CLL • FL • MZL (MALT lymphoma) • MCL (rare cases) In MCL, trisomy 12 is the only single cytogenetic parameter predictive of a poor prognosis [265]. Trisomy 12 is a

Cytogenetics

common abnormality in B-CLL, followed by 14q+, 13q, and 11q. Patients with trisomy 12 have advanced clinical stage, atypical morphology and immunophenotype, and shorter survival [266,267]. In a series reported by Geisler et al. [268], however, an additional copy of chromosome 12 without other chromosomal abnormalities was compatible with classical CLL and had no prognostic influence. Trisomy 12 is more often observed in patients with Richter’s syndrome than in overall population of patients with B-CLL [269]. FLs with del(17p) and +12 have an adverse clinical outcome [270]. Trisomy 12 occurs frequently in MZLs [240]. trisomy 15 • AML • MDSs • Non-Hodgkin lymphomas trisomy 17 • CML, disease progression/clonal evolution trisomy 18 • DLBCL • PCM • Other B-cell lymphomas, for example, MZL trisomy 21 • AML • Precursor ALL/LBL • CML, disease progression/clonal evolution Trisomy 21 is frequently observed in myeloid malignancies, but can also be seen in AML. Children with Down syndrome have a 150-fold increased risk of myeloid leukemia in the first 5 years of life. Leukemias in patients with Down syndrome are associated with +8 or −7. In addition, increased white blood cell count and circulating myeloblasts (almost invariably megakaryoblast by phenotypic studies) may be seen in up to 10% of newborns with Down syndrome [transient myeloproliferative disorder (TMD)]. Trisomy 21 in AML is associated with intermediate risk factor for induction therapy success and overall survival [118]. trisomy 22 • AML

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References 267. Foon KA, Rai KR, Gale RP. Chronic lymphocytic leukemia: new insights into biology and therapy. Ann Intern Med, 1990. 113(7):525–39. 268. Geisler CH, et al. In B-cell chronic lymphocytic leukaemia chromosome 17 abnormalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: a cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients. Leuk Res, 1997. 21(11–12):1011–23. 269. Tsimberidou AM, Keating MJ. Richter syndrome: biology, incidence, and therapeutic strategies. Cancer, 2005. 103(2):216–28. 270. Hoglund M, et al. Identification of cytogenetic subgroups and  karyotypic pathways of clonal evolution in follicular lymphomas. Genes Chromosomes Cancer, 2004. 39(3):195–204.

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Contents Introduction ............................................................................................................................................................................... 190 Fluorescence In Situ Hybridization ........................................................................................................................................... 190 Polymerase Chain Reaction ...................................................................................................................................................... 190 Reverse Transcriptase PCR .................................................................................................................................................. 192 Real-Time Quantitative PCR................................................................................................................................................ 192 Immunoglobulin Gene Rearrangement ................................................................................................................................ 193 T-Cell Receptor Gene Rearrangement ................................................................................................................................. 194 Genetic Markers—Differential Diagnosis ................................................................................................................................ 196 ALK ...................................................................................................................................................................................... 196 BCL1 .................................................................................................................................................................................... 196 BCL2 Rearrangement ........................................................................................................................................................... 196 BCL6 Rearrangement ........................................................................................................................................................... 196 BCR–ABL1 ........................................................................................................................................................................... 197 BCR–ABL1 Fusion and JAK2 Mutations ............................................................................................................................. 197 BRAF Mutations ................................................................................................................................................................... 197 Core-Binding Factor............................................................................................................................................................. 197 CBFB .................................................................................................................................................................................... 197 CEBPA Mutations ................................................................................................................................................................ 198 CCND1 ................................................................................................................................................................................. 199 CDKN2A .............................................................................................................................................................................. 199 ETV6 Rearrangement ........................................................................................................................................................... 199 FLT3 Mutations .................................................................................................................................................................... 199 IDH Mutations ..................................................................................................................................................................... 200 IGH Rearrangement ............................................................................................................................................................. 200 IRF4 (MUM1) Rearrangements ........................................................................................................................................... 200 JAK2 V617F Mutation ......................................................................................................................................................... 200 JAK2 Exon 12 Mutation ....................................................................................................................................................... 200 KIT Mutations ...................................................................................................................................................................... 200 MAF Rearrangement ............................................................................................................................................................ 201 MALT1 Rearrangement ........................................................................................................................................................ 201 MLL Rearrangement............................................................................................................................................................. 201 MPL Mutations..................................................................................................................................................................... 202 MYC Rearrangement ............................................................................................................................................................ 202 MYD88 L265P Mutations .................................................................................................................................................... 202 NPM1 Mutations .................................................................................................................................................................. 202 PAX5 Rearrangement ........................................................................................................................................................... 203 PDGFRA, PDGFRB, and FGFR1........................................................................................................................................ 203 PML–RARA .......................................................................................................................................................................... 203 RUNX1–RUNX1T1 ............................................................................................................................................................... 204 RUNX1 Mutations ................................................................................................................................................................ 204 Spliceosome Gene Mutations (SF3B1, SRSF2, ZRSR2, U2AF35) ...................................................................................... 204 STAT5b ................................................................................................................................................................................. 204 TCL1 Rearrangement ........................................................................................................................................................... 204 TET2 Mutations.................................................................................................................................................................... 204 References ................................................................................................................................................................................. 204

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Conventional cytogenetics using classical karyotyping of chromosomes remains the most comprehensive method for assessing chromosome abnormalities, especially numerical and structural chromosomal aberrations. Technical issues associated with cytogenetics (e.g., requirement for fresh sample, difficulties in identification of masked or cryptic aberrations due to a limited resolution by classic banding techniques) have resulted in an increased use of molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH), to identify specific abnormalities that are useful in either the diagnosis or the management of these disorders. Automation and high sensitivity lead recently to increased popularity of polymerase chain reaction (PCR) technologies in diagnosis and especially disease monitoring.

FluoresCenCe In SItu HybrIdIzatIon FISH uses test probes against the target DNA in the nucleus of interphase cells or metaphase chromosomes [1–27]. FISH allows the analysis of specific DNA changes in tissues (cells) or intact chromosomes and does not require metaphase chromosomes. Many of the clinical applications of FISH include chromosome enumeration using α-satellite probes (e.g., gain or loss of a chromosome), marker identification, gene mapping, deletion, amplification, or translocation as well as whole chromosome “painting.” In hematologic malignancies, the most common chromosomal abnormalities targeted by FISH include t(9;22)/BCR–ABL1 in chronic myeloid leukemia [CML; and subset of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)]; t(15;17)/PML–RARA in acute promyelocytic leukemia (APL); t(14;18)/IGH–BCL2 in follicular lymphoma (FL); t(11;14)/CCND1–IGH in mantle cell lymphoma (MCL); del(13q), del(11q), and del(17p)/TP53 in B-cell chronic lymphoblastic leukemia (B-CLL); del(5q) in myelodysplastic syndrome (MDS) and AML; del(13q) RB1, t(11;14)/CCND1–IGH, and del(17p)TP53 in plasma cell myeloma (PCM); t(8;14)/MYC–IGH in Burkitt lymphoma (BL); MYC–IGH, BCL2, and BCL6 rearrangements in B-cell lymphoma, unclassified with features intermediate between BL and diffuse large B-cell lymphoma (DLBCL) (BCLU; double-hit lymphoma; gray zone lymphoma); t(11;18)/API2– MALT1 and t(14;18)/IGH–MALT1 in marginal zone lymphoma [MZL; mucosa-associated lymphoid tissue (MALT) lymphoma]; t(8;21)/RUNX1–RUNX1T1 in AML; inv(16) in AML; and t(12;21)/ETV6–RUNX1 in ALL. The test can be performed indirectly or directly. FISH protocol includes the following steps: denaturation, in which the probe and the target DNA are denatured by incubation at high temperature; hybridization, in which the probe is hybridized to the chromosomal target; and washing to remove the unbound probe and analysis under fluorescence microscope. In contrast to Southern blot hybridization, in FISH protocol DNA is analyzed in the cell or on the chromosome and is not being extracted and run in a gel. In the indirect FISH method, biotin- or digoxigenin-labeled nucleotides are

Polymerase Chain Reaction

visualized in the second step by fluorescein isothiocyanate (FITC)-conjugated CY3-conjugated avidin- or rhodamineconjugated antidigoxigenin antibodies, whereas in the direct method, the probe(s) are already labeled with fluorochrome(s). With the application of several filters, two or three probes can now be detected simultaneously (Figure 7.1). The FISH probes can be generally subclassified into the following categories: centromere-specific probes, whole chromosome (painting) probes, single-copy (locus-specific) genomic probes, and spectral karyotyping (SKY; multiplex metaphase FISH, multicolor FISH). FISH probes for chromosomal translocations are most widely used in the evaluation of hematopoietic tumors. In the detection of translocation, two probes are labeled with a different fluorochrome: normal cell displays four signals (two of each color), whereas cell with translocation shows two adjacent signals leading to a different color of fluorescence signal. Dual-fusion probe consists of a pair of probes labeled with two different colors (fluorochromes): green (e.g., FITC) and red (e.g., rhodamine) directed against translocation breakpoint regions in the two different genes involved in a reciprocal translocation. In normal cell, there are two green and two red signals corresponding to two separate loci that are not in close proximity (no translocation). In cells with translocation between the targeted loci, there is one green and one red signal (normal chromosome) and one yellow signal indicating the fusion between two loci (yellow fluorescence being the result of overlap between green and red signals). Variant and complex pattern may also be identified and provide an additional clinical information on the underlying chromosomal changes. The third color may be used to label specific chromosome (e.g., chromosome 9 in BCR–ABL1 analysis; Figure 7.2). In the case of break-apart (BA) probe, the target DNA is labeled with two different probes directed at two opposite areas of the gene (3′ and 5′). The interpretation of results is opposite to that with fusion probes: fusion (yellow) signal is normal, and separate green and red signals indicate translocation (BA). The fusion of the signal seen as different colors (e.g., yellow) would indicate the normal allele, whereas the two different signals (e.g., red and green) would indicate the presence of translocation (Figure 7.1). The counterstain using either propidium iodide (PI) or 4′,6′-diamidino-2phenylindole (DAPI) can be used to identify chromosomes. The commonly used BA probes in hematologic tumors include MLL-BA (AML/ALL), CBFB-BA (AML), RARA-BA (APL), MYC-BA (BL and BCLU), MALT1-BA (MALT lymphoma), ALK-BA (ALCL), and IGH-BA (NHL/PCM).

Polymerase CHaIn reaCtIon PCR developed by Mullis and Faloona is a core technique for most tests used in molecular diagnostics [28–30]. It targets a segment of DNA [or RNA in reverse transcriptase PCR (RT-PCR)] and produces multiple copies (usually between 107 and 1011) of a DNA region of interest. PCR enables the detection of malignant cells below the threshold of karyotyping or morphology, even when combined with immunophenotyping. Fresh

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Abnormal pattern

One-probe FISH

Normal pattern

Two copies of analyzed locus are detected

Two copies each of analyzed two loci are detected and are well separated

One (top) and three (bottom) copies of analyzed locus are detected (in the case of centromeric probes this would imply monosomy and trisomy, respectively; in the case of probe for a specific gene, one signal would indicate the deletion of that gene, while three signals would indicate additional copy).

One signal with probe for retinoblastoma gene, indicating deletion of Rb gene

Three signals with CEP8 probe indicating trisomy 8

Two copies of one locus (green) and three copies of another locus (red) are present.

Acute lymphoblastic leukemia stained with TEL/AML1 probe: three copies of AML1 gene (three red signals are present)

Two copies of one locus (“red”) and one copy of another locus (“green”) are present. The deletion of “green” locus is implied.

Acute lymphoblastic leukemia stained with MLL probe: deletion of MLL (only one green signal is present)

Two-probe FISH

Two “red” and two “green” loci are present, but one “red” and one “green” locus are juxtaposed on one chromosomes (fusion results in different color due to overlap of two fluorochromes). Two loci are adjacent to each other

FIGure 7.1

Example

One locus is adjacent to another locus like in a normal cell, but second pair is separated. This implies some type of rearrangement, which separated two loci that are usually found together.

CML with BCR–ABL fusion (proximity of two probes give rise to “yellow” fluorescence fusion signal)

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“Break apart” probe (MYCBA); (it implies that c-Myc is broken and translocated; does not have to be 8;14, may be 8;22 and others)

Interpretation of FISH results.

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FIGure 7.2 FISH analysis for BCR–ABL using three-color probes: aqua for argininosuccinate synthetase 1 (ASS1) gene on chromosome 9q34, green for BCR gene on chromosome 22, and red for ABL gene on chromosome 22. Several positive signals (yellow) are seen in cases of CML (A); control sample (B) shows two green signals for normal chromosome 22 and two red and blue signals close to each other for normal chromosome 9.

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tissue is the best source of tissue for PCR analysis, but fixed tissue (formalin, ethanol) may also be used. Carnoy’s, Zanker’s, B5, and Bouin’s fixatives are suboptimal for PCR. Tissues exposed to decalcifying solutions (e.g., bone marrow trephine core biopsy) are not suitable for PCR analysis. Three major steps in PCR include denaturation of double-stranded DNA into single-stranded DNA (ssDNA), hybridization (annealing) of oligonucleotide primers to both ends of a target sequence, and a synthesis achieved by addition of four nucleotide bases and a Taq polymerase. A PCR reaction usually involves 30–40 cycles. The denaturation is achieved by high temperature (95°C), which by breaking the hydrogen bonds between complementary bases creates ssDNA. In the next step, the two primers join (hybridize) the single-stranded template DNA. In the final step (synthesis), Taq polymerase synthesizes new DNA strands, using the oligonucleotide primers as starting points.

ReveRse TRanscRipTase pcR RT-PCR methodology amplifies RNA and consists of two major steps: in the first step, it employs an enzyme reverse transcriptase, which converts RNA into the single-stranded complementary DNA (reverse transcription), which in the second step serves as a template for conventional PCR (second-strand reaction). The original RNA is degraded by RNase. RT-PCR can be performed on fresh or fixed tissue. RT-PCR is widely used in the diagnosis of genetic diseases, since it makes it easier to detect the presence of specific aberration by detecting directly the product (mRNA), for example, fusion transcripts encoded by the translocations. Additionally, it is very useful in monitoring some hematologic malignancies during treatment, since it allows for quantification of the analyzed products (see below).

Real-Time QuanTiTaTive pcR Qualitative PCR (e.g., RT-PCR) fails to detect diseasespecific changes in the subset of patients, especially in

patients monitored for minimal residual disease (MRD). This is often associated with poor quality of RNA. Real-time quantitative PCR (qRT-PCR, also RQ-PCR) became more powerful tool for monitoring patients, giving the clinicians the best chance of detecting the earliest stages of molecular relapse. qRT-PCR allows simultaneous amplification and detection of PCR product in a single reaction tube. The reaction is monitored in real time after each cycle of amplification by computer software permitting both detection and quantification of PCR products. After each amplification cycle, a dye is released and its amount is measured in real time to ensure that quantitation of DNA occurs in the exponential phase. The results are presented in the form of graph that plots the dye (corresponding to the amount of starting template) versus the number of PCR cycles (Figure 7.3). The quantitation is based on the number of cycles required to reach a designated threshold (the lower the number of cycles to reach the threshold, the higher the expression of mRNA or DNA copy number), using the fluorescence-based quenching methods [fluorescence resonance energy transfer (FRET)]. The most commonly used systems for qRT-PCR include TaqMan system, Molecular beacons, Scorpions, and SYBR green. TaqMan probes depend on the 5′-nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. The fluorochromes used in TaqMan (reporters) include FAM, HEX/ JOE/VIC, and TET. The fluorescence signal is quenched by either TAMRA or DABCYL (quenchers). Figure 7.4 illustrates the chemistry of TaqMan methodology. In the unhybridized state, the fluorochrome (reporter) and the quencher are close to each other, allowing for FRET (there is no fluorescent signal from the probe because the fluorescence of the reporter dye is being quenched). During PCR, when the Taq polymerase replicates a template and the 5′-exonuclease activity of the polymerase degrades the probe, the reporter and the quencher become unlinked and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage.

Amplification—BCR/ABL-positive sample

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The chemistry of TaqMan methodology in qRT-PCR.

immunoglobulin gene ReaRRangemenT The majority of lymphomas are of B-cell lineage, and consequently analysis of immunoglobulin gene rearrangements belongs to the most frequently used molecular test. Diagnosis of B-cell lymphomas is based most often on histomorphology and immunophenotyping (flow cytometry and/or immunohistochemistry), although many cases require addition of conventional cytogenetics, FISH, and/or PCR. Assessment of clonality by immunoglobulin gene rearrangement is crucial in establishing the definite diagnosis in cases with atypical (ambiguous) histology and phenotype, very early (incipient) lymphoproliferations, pleomorphic infiltrate with paucity of neoplastic cells, low-grade B-cell lymphoproliferations that may be difficult to diagnose based on the morphology or cases with an insufficient amount of tissue for morphologic/phenotypic analysis. Molecular detection of clonality also plays an important role in the diagnosis of posttransplant lymphoproliferative disorders. Among three genes

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that rearrange (kappa heavy-chain gene and lambda lightchain gene), IGH gene rearranges before the light-chain gene and is most frequently analyzed. The immunoglobulin molecule is made up of two heavy chains and two light chains, joined by disulfide bonds. Both heavy and light chains have variable and constant regions corresponding to the V and Cμ genes. The genes encoding heavy chains are located on chromosome 14, and the genes encoding light chains are located on chromosomes 2 (kappa) and 22  (lambda). During the maturation of lymphocytes, B cells rearrange their genes producing fusion gene composed of variable (V), diversity (D), joining (J), and constant (C) segments, which encode an antigen receptor that is expressed on the surface of B cells and become secreted when B cells differentiate into plasma cells. All maturing B cells rearrange their genes differently by splicing out and deleting a portion of the IGH gene, in which 1 of 30 D regions is juxtaposed first with 1 of 6 J regions, followed by joining of 1 of ~200 V regions. Antibody type (IgA, IgM, IgD, IgE, and IgG) depends on which C region (Cα, Cμ, Cδ, Cε, or Cγ) joins the rearranged VDJ genes. The heavy-chain protein (IGH) joins either kappa (κ) or lambda (λ) light-chain proteins (which are encoded by genes rearranged in a similar manner) to produce antibody. The unique coding sequence for both heavy- and light-chain genes ensures the diversity of antibody production by the plasma cells. Normal B-cell population, therefore, consists of polyclonal IG gene rearrangements. In B-cell neoplasms, the coding sequence characteristic of the B cells that gave rise to a malignant clone becomes inherited by all malignant cells. This clonal immunoglobulin gene rearrangement can be visualized by Southern blot analysis or by PCR amplification. Although there are no established criteria for identification of clonality, the results are usually easy to interpret as shown in Figure 7.5. The positive results

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FIGure 7.5 PCR analysis for IGH rearrangement: (A and B) polyclonal sample; (C) monoclonal peak in the polytypic background; (D) monoclonal peak; (E) indeterminate results (“oligoclonal” peaks and polytypic background).

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show clonal peaks well above the polyclonal background. Consensus primers targeting three families of IGH variable segments (frameworks 1, 2, and 3) are commonly used for PCR identification of B-cell clonality (FR1-PCR, FR2-PCR, FR3-PCR). Approximately 70% of lymphomas have clonal rearrangements detectable with framework 3 primers, 20% with framework 2, and additional few cases with framework 1. The combined use of primers yields the best results with detection of clonality approaching 90%. In a multicenter comparison of PCR sensitivity in detecting clonality, a significant difference was observed between FR3 alone compared with FR3 + FR2 (~57% vs. ~74%). Analysis of only a single FR region has several limitations, mostly associated with somatic hypermutations. Somatic hypermutations increase the diversity of the VH segments encoded in the germline genome and may lead to primer mismatching and consequently falsely negative results limiting the use of single FR region-PCR protocols. The main limitation of IGH PCR is false negative results, which vary depending on the methodology used, the subtype of lymphoma, and the degree of somatic hypermutation of IGH. The falsely negative results due to somatic hypermutation may be seen in B-CLL, but are more typical for lymphomas of germinal center and postgerminal center origin (i.e., FL, subset of DLBCL, and multiple myeloma) since they display higher rate of somatic mutation. Using Southern blot analysis or PCR targeting, incomplete DJH rearrangements and BCL2–IGH fusion may overcome problems associated with somatic hypermutations, and therefore be more sensitive in confirming clonality [31–36]. The detection rate can be further improved when multiplex IGH (DH−JH) and IGK PCR-based strategies are combined [37–39]. The European BIOMED-2 Concerted Action study group described a very sensitive method with primers targeting different FRs and incomplete DH−JH rearrangements (VH−JH and DH−JH sets of primers), which can detect virtually all monoclonal B-cell proliferations, regardless of high levels of somatic hypermutation [14,31,37,40]. The products of PCR analysis using fluorochromelabeled primers can be separated by capillary electrophoresis. Polyclonal (random) rearrangement shows a Gaussian distribution with multiple, heterogeneous fragments (peaks) (Figure 7.6A and B). A monoclonal B-cell population is distinguished from polyclonal cells based on the presence of one dominant peak (the amplified fragment of one size) (Figure 3.8C and D). When the ratio of the major peak to the second largest peak is low (e.g., between two and three), the results are indeterminate (Figure 3.8F). A number of benign (reactive) conditions may yield “clonal” PCR results, and therefore, molecular results should be correlated with clinical and laboratory data, especially morphology and phenotype, before treatment. Table 3.3 lists nonmalignant disorders that may show “clonal” rearrangements by PCR. The type of tissues acceptable for PCR analysis of B-cell clonality include fresh (unfixed) solid tissue, fresh blood, fresh bone marrow aspirate, formalin-fixed paraffin-embedded

Polymerase Chain Reaction

tissue, and frozen tissue. Ethylenediaminetetraacetic acid (EDTA) is the preferred anticoagulant for blood cell counts, due to interference of heparin with DNA amplification.

T-cell RecepToR gene ReaRRangemenT Similarly to B cells, T cells also rearrange the genes, but instead of producing different antibodies, the (protein) products of T-cell receptor genes are fixed to the surface of lymphocytes creating complex receptor molecules, allowing proper interaction with other components of the immune system. The T-cell receptors contain four major protein chains, α, β, γ, and δ, encoded by their corresponding T-cell receptor (TCR) genes (TCRA, TCRB, TCRG, and TCRD). Gene loci encoding the α chain (TCRA) and the δ chain (TCRD) are clustered on chromosome 14q11, and those encoding the β chain (TCRB) and the γ chain (TCRG) are located on chromosomes 7q34 and 7p15, respectively. Similarly to immunoglobulin genes, TCR genes are composed of V and C genes. The V region has three segments (V, J, and D) in the β and δ genes, and only two segments (V and J) in the α and γ genes. The earliest thymocytes express HLA-DR, CD34, and CD7; have germline configuration of the TCR genes; and are pluripotent. TCRδ gene rearranges as soon as the progenitor cell commits to the T lineage. The earliest definite T cells (early prothymocytes; pro-T cells) evolve in the subcapsular region of the thymic cortex and are referred as triple-negative (TN) cells, due to lack of expression of CD4, CD8, and surface CD3 (sCD3−/CD4−/CD8−). They are positive for CD2, CD7 (strong expression), CD34, CD44, terminal deoxynucleotidyl transferase (TdT; nucleus), and cytoplasmic CD3 (cCD3), and are characterized by a germline configuration of TCRβ chain. In the process of differentiation, TN cells start to express CD1 and CD5, and progressively lose the expression of CD34 and the intensity of CD7 expression. Further differentiation occurs in the thymic cortex through several stages. In the thymic cortex, double-negative (DN) T cells (CD4−/ CD8−) start to differentiate into double-positive (DP) T cells (CD4+/CD8+) through several stages. DN stage is characterized by the continuous rearrangement of TCRB, TCRD, and then TCRG, while TCRA rearrangement starts to occur during the DP stage. During the transition from pro-T cells to DN T cells, the cells rearrange TCRB gene, a process that is similar to IGH gene rearrangement (Dβ segment joins the Jβ segment, and then Vβ segment joins the DβJβ complex). At this point, cells start the commitment to either TCRγδ or TCRαβ lineages. If the TCRGD rearrangement is successful, cells become Tγδ+. In the subset of cells that undergo the rearrangements of β, α, and γ genes, β chain forms a heterodimer with the pre-Tα chain (surrogate α chain), termed pre-TCR complex, and starts differentiation into αβ lineage, entering the DP phenotypic stage. The pre-TCR forms a complex with CD3 at the thymocyte surface; those cells are characterized by surface expression of pre-TCR and low CD3. The preTCR complex blocks further γδ differentiation (TCRG gene rearrangements are dysfunctional) and plays a major role in T-cell commitment to αβ lineage (with subsequent production

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FIGure 7.6 Monoclonal T-cell receptor (TCR) gamma gene rearrangement (multiplexed PCR). Amplification products are analyzed by capillary electrophoresis: (A) polyclonal sample; (B) monoclonal peak in the polytypic background; (C) monoclonal peak; (D) monoclonal peaks.

of CD4+/CD8+ cells). T cells committed to TCRαβ lineage mature into CD4+/CD8+ cells that express sCD3 and become negative for TdT. In the thymic medulla, DP cells finish the differentiation process, becoming either CD4+ or CD8+, and then enter the blood as mature T-helper (CD4+) or T-suppressor/cytotoxic (CD8+) cells. Mature T cells, either αβ or γδ, demonstrate nongermline patterns of TCRG or TCRD genes. Mature T cells are characterized by the membrane expression of TCR–CD3 complex. T-cell lymphoproliferative disorders may display rearrangements of one to four TCR genes. PCR analysis of TCRB and TCRG genes is most often used to confirm clonality (Figure 7.6), whereas TCRD is rarely used. TCRA gene is usually not targeted. Most of the T cells rearrange 1 of the 11 variable segments of TCRG, 8 of which are homologous to one another and can be targeted by a single consensus primer. Using several consensus primers targeting variable segments and joining region of TCRG, for example, Vγ1–8, Vγ9, Vγ10, Vγ11, and Jγ1/Jγ2 allow for detection of T-cell monoclonality in up to 95% of cases with sensitivity between 1% and 5% [41]. Multiplex PCR with four TCR loci [TCRG, TCRD, and TCRB including complete (Vβ–Jβ) and incomplete (Dβ–Jβ) rearrangements] offers a sensitive approach to determine the clonality early in the diagnostic work-up of T-cell disorders, with TCRG being the single most informative locus (clonal rearrangement in 89%), followed by TCRB (79%) and TCRD (39%). Multiple primer set PCR methods should obviate a need for the more expensive and timeconsuming Southern blot technique and are the preferred

diagnostic molecular test for assessing T-cell clonality [42,43]. The results can be visualized using capillary or sequencing gel electrophoresis [44,45]. Although capillary gel electrophoresis is superior in assessing T-cell clonality, caution must be exercised when interpreting results, because pseudospikes appear to be common in benign tissues with lymphoid populations and are not necessarily indicative of clonal malignant T-cell population. Using capillary electrophoresis, Luo et  al. [45] proposed the threshold for identification of a predominant monoclonal population within a polyclonal background—the peak height ratio (Rn) of the peak of interest and the average of the two immediate flanking peaks. After the evaluation of monoclonal, reactive, and normal T-cell populations, an Rn of ≥3.0 was determined to be consistent with a monoclonal population, whereas an Rn between 1.9 and 3.0 was considered an intermediate range. Lee et al. [46] defined relative peak heights as h1/h2, where h1 represents the peak height of the largest peak above the normally distributed population and h2 represents the peak height of the normally distributed curve. Pseudospikes were found in almost 20% of histologically benign lymph nodes with relative peak heights being >0.5 and up to 1.5 [46]. Peaks with relative height of at least 3 represent a true clonal population in diagnostic samples, peaks with relative heights of 50% in patients over 50 years old) and 5% of children with ALL, more often in B-ALL than in T-ALL. In CML, the most common breakpoints involve the major breakpoint cluster region close to exon 13 (b2) or 14 (b3) in BCR and the upstream of exon 2 in ABL1, resulting in the creation of fusion transcripts e13a2 (b2a2) and e14a2 (b3a2). Both transcripts are associated with p210 BCR–ABL1 protein. Only rare cases (~1%) have e1a2 rearrangement (leading to p190 fusion protein) or e19a2 associated with p230. Alternative splicing may occur in CML leading to mixed transcripts (e.g., e1a2 in cases with e13a2 or e14a2 in cases with e13a2). The e1a2 transcript (p190) is the most common fusion detected in Philadelphia (Ph+) B-ALL.

BCR–ABL1 Fusion and JAK2 muTaTions • Myeloproliferative neoplasms (CML and non-CML myeloproliferative neoplasms) Concurrent presence of BCR–ABL1 and JAK2 in myeloproliferative neoplasms is rare, and only a few cases have been reported [75–77]. The majority of these patients present with typical CML (BCR–ABL1+), who also have low levels of JAK2+ clone. The latter usually becomes more prominent after patients achieve molecular remission of CML with imatinib treatment.

BRAF muTaTions • Hairy cell leukemia (HCL) BRAF mutations, reported typically in thyroid cancer and melanoma, have also been associated with HCL. The presence of BRAF mutations helps to distinguish HCL (BRAF+) from HCL variant (HCL-V) and other B-cell lymphomas including splenic MZL and splenic diffuse red pulp small B-cell lymphoma (BRAF−) [78–85]. BRAF mutations have been reported only in sporadic cases of other B-cell lymphoproliferations, including ~3% of B-CLL/CLL [86]. HCL with BRAF mutations was reported to respond to vemurafenib [87].

coRe-binding FacToR • AML (subset) The core-binding factor (CBF)-positive AML includes AML with inv(16)/t(16;16) (CBFB–MYH11) and t(8;21) (RUNX1– RUNX1T1) is associated with a favorable prognosis, except for patients with KIT mutation, especially D816 mutation [88–90].

CBFB • AML (subset) AML with inv(16)(p13q22) or t(16;16)(p13;q22) is associated with a relatively favorable prognosis [90–94]. On the molecular level, inv(16)/t(16;16) results in the juxtaposition of the myosin heavy chain 11 gene (MYH11) at 6p13 and the CBF, β subunit gene (CBFB) at 16q22 and the creation  of the CBFB–MYH11 fusion gene. More than 10 differently sized CBFB–MYH11 fusion transcript variants have been reported, with 85% of type A and 5%–10% each of types D and E. The CBFB–MYH11 fusion is seen typically in AML with eosinophilia [former French–American–British (FAB) classification: acute myelomonocytic leukemia with eosinophilia (AML-M4eo)]. Patients with non-type A variants lack KIT mutations, whereas 27% of patients with type A transcript carry additional KIT mutations [91].

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Genetic Markers—Differential Diagnosis

CEBPA muTaTions

but appears to be more pronounced in patients with biallelic (double) mutation [97,98]. AMLs with double-mutated CEBPA have other mutations, including TET2 (34%), GATA2 (21%), WT1 (13.7%), DNMT3A (9.6%), ASXL1 (9.5%), NRAS (8.4%), KRAS (3.2%), IDH1/2 (6.3%), FLT3internal tandem duplication (ITD) (6.3%), FLT3-tyrosine kinase domain (TKD) (2.1%), NPM1 (2.1%), and RUNX1 (1  of 94 cases) [99]. Figure  7.10 shows single and double mutations of CEBPA in AML.

• AML

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FIGure 7.10

PCR analysis for CEBPA mutation in AML (A) negative (control); (B) single mutations; (C) double mutations.

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Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. Mutation or methylation is rare, whereas genomic deletion occurs in 21% of B-cell precursor ALL and 50% of T-ALL patients [100–102].

• MCL • PCM (subset) Cyclin D1 (CCND1, BCL1) is rearranged in MCL (Figure 7.11) and the subset of patients with PCM.

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ETV6 ReaRRangemenT • B-ALL • T-ALL ETV6 (TEL) is an important hematopoietic regulatory factor and ETV6 gene rearrangement is involved in a wide variety of hematological malignancies. The partner genes include RUNX1, JAK2, ABL2, NCOA2, SYK, and PAX5 [103]. ETV6 rearrangements are generally associated with a favorable outcome in pediatric ALL, although ETV6–ABL1 predicts poor prognosis. ETV6 rearrangement are very rare (90% small lymphocytes with weak expression of surface immunoglobulin and CD20, and coexpression of CD5 and CD23. Atypical CLL shows larger lymphocytes with abundant cytoplasm, prolymphocytes-like or cleaved cells, and aberrant phenotype. The clinical course of CLL ranges from a very indolent disorder with a normal lifespan to a progressive disease with poor prognosis. Patients having aggressive disease require therapy within relatively short time after diagnosis, whereas patients with asymptomatic disease are not likely to benefit from palliative chemotherapy. The median survival of patients with CLL is 10 years. Patients with poor prognostic factors have median survival of ~3 years. The course of the disease depends on a number of factors including age, gender, Binet/Rai stage, performance status, laboratory parameters [lymphocyte count, thymidine kinase, soluble CD23, β2-microglobulin, lactate dehydrogenase (LDH)], atypical cytologic features, pattern and extent of BM infiltration, 17p deletion, TP53 mutation/loss, deletion of chromosome 11q23, ATM status, IGVH mutational status, BIRC3 abnormalities, NOTCH1 mutational status, SF3B1 mutational status, and CD38 and ZAP-70 expression [9–13]. The significant improvements in remission rates have been achieved with newer therapeutic approaches including purine analogs (e.g., fludarabine) in combination with monoclonal antibodies and stem cell transplantation [14–16]. The combination of rituximab and fludarabine or fludarabine-containing regimens has yielded overall response rates of 95%, with complete response rates up to 66% in previously untreated CLL patients [17]. CLL cases refractory to treatment have a very poor prognosis, despite various salvage therapy strategies [18–20].

rIsk categorIes In cLL Several risk categories can be distinguished in CLL patients based on the response to treatment, and cytogenetic and molecular changes. In the scheme proposed by Zenz et al. [12], the patients with CLL are divided into highest risk, high risk, and low risk: • Highest-risk CLL category includes patients with TP53 loss/mutations, lack of response to purine analogs, or very short response (55% prolymphocytes favors the diagnosis of B-cell prolymphocytic leukemia (B-PLL). In the absence of lymphadenopathy or organomegaly, cytopenias, or disease-related symptoms, the presence of ≤5 × 109/L B lymphocytes in blood is defined as “monoclonal B lymphocytosis.” The cytopenia caused by a typical marrow infiltrate defines the diagnosis of CLL regardless of the number of blood B lymphocytes. Immunophenotype. CLL cells express CD5, CD23, and B-cell antigens (CD19, CD20, CD22, CD79a). The expression of CD20 and surface immunoglobulins is weak. Staging. The two main staging methods that are used in CLL are Rai and Binet systems. Other tests performed at diagnosis. With the exception of fluorescence in situ hybridization (FISH), the application of additional tests at diagnosis should not be used in routine practice to influence therapy and is generally not recommended. However, certain parameters, such as immunoglobulin mutational status, are useful for predicting the clinical course in individual cases. The following tests should be recommended for patients who want a better prediction of the rate at which their disease might progress: • FISH • Mutational status of IGVH, expression of ZAP-70 or CD38

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma and B-Cell Prolymphocytic Leukemia

• Serum markers (CD23, thymidine kinase, β2-microglobulin) • Marrow examination (generally not required for the diagnosis of CLL, but can help in evaluating the factors that might contribute to cytopenias and that may or may not be directly related to leukemia cell infiltration of the marrow)

MonocLonaL B-ceLL LyMphocytosIs The presence of low numbers (3:1 or 20% in persons aged >60 years and being more frequent among the relatives [22,25] of patients with CLL [26–29]. CLL-like MBL can further be subdivided into clinical MBL and population-screening MBL. The former is diagnosed in a clinical setting, is associated with lymphocytosis, and has a concentration of clonal B cells ≥1500/µL. Populationscreening MBL is only detected during screening studies of healthy persons in the general population and is characterized by ≤50 clonal B cells/µL (low-count MBL). Virtually, all patients with CLL had a preceding MBL. The potential risk of progression of clinical MBL (previously termed MBL with lymphocytosis) into clinically overt CLL is ~1.1% per year. Rawstron et al. [25] reported minute monoclonal B-cell populations (30% of clonal cells) or ZAP-70 is associated with worse prognosis and often correlates with unmutated IGVH status. Among B cells, CD38 is expressed at high levels by B-lineage progenitors in BM and by B-lymphocytes in germinal center, in activated tonsils, and by terminally differentiated plasma cells. In CLL, CD38 expression identifies two subgroups of patients with different clinical outcomes, including OS, time to first treatment, number of leukemic cells with abnormal morphology, extent of adenopathy, ALC, and LDH and β2-microglobulin levels. In the majority of studies, the threshold is considered as ≥30% CD38+ clonal cells [9,32–36]. At the molecular level, the CD38+ and CD38− clones differ in the level of expression of activation markers CD69 and HLA-DR, Ki-67 fraction, ZAP-70 expression,

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FiGure 8.1 SLL/CLL in the lymph node (A; H&E staining). Lymphomatous cells are positive for CD20 (B), CD5 (C; compare with CD3 staining, F), and CD23 (D). The BCL6 staining is often very dimly positive and is considered nonspecific. Rare cells may express Ki-67 and/ or p53 (G–H; usually prolymphocytes and paraimmunoblasts). Subset of SLL/CLL cases is ZAP-70+ (I).

IGVH mutational status, telomere lengths, and telomerase levels, and in high-risk genomic abnormalities [32,37–39]. Although CD38 expression is associated with mutated CLL, recent reports suggest that combination of CD38 and IGVH mutational status had greater prognostic power than either marker alone [40,41].

MorphoLogy Blood and Bm aspirate In the BM aspirate and blood smears, CLL cells are small with scanty pale cytoplasm, round nuclei, and clumped (coarse) chromatin (see Figure 8.3). Occasional smudge cells and larger lymphocytes including prolymphocytes and paraimmunoblasts are usually also present. Prolymphocytes are distinguished from small lymphocytes by their relatively larger size, increasingly abundant pale cytoplasm, and prominent central nucleolus (Figure 8.4). Paraimmunoblasts have oval nuclei with large central nucleolus and grayish-blue cytoplasm. CLL with >15% and 55% of prolymphocytes.

Bm Biopsy Patterns of BM involvement by CLL include nodular, interstitial, and diffuse (Figure 8.5). The infiltrate is composed of mostly small lymphocytes with scattered prolymphocytes and paraimmunoblasts. In cases with prominent BM involvement, proliferation centers are often present. The presence of large atypical cells should raise the possibility of Richter’s transformation. Pattern and extent of BM involvement (diffuse vs. nondiffuse) have prognostic implications [42], although it is not independent of the staging and with novel phenotypic, chromosomal, and molecular markers, histologic pattern became less relevant [43]. Nodular and interstitial patterns are associated with early disease and better prognosis, while a diffuse marrow infiltrate is associated with a worse prognosis and  advanced disease, although not all studies support this [42,44–46]. Patients with >70% marrow involvement before therapy have a significantly shorter time to progression [47]. A significant correlation between poor survival and grade of BM reticulin fibrosis was recently reported [48]. Advanced reticulin fibrosis (grades 2–3 in 0–3 grading system) was associated with thrombocytopenia, anemia, elevated β2-microglobulin, and the presence of 11q deletion and poor survival [48].

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FiGure 8.2 CLL—Typical immunophenotypic profile (BM). BM shows prominent lymphocytosis (A; aspirate smear). FC shows dim expression of CD20 (B and C), moderate CD19 and CD5 (D), dim CD22 (E), positive CD23 (E), and dim expression of CD38 (F). BM biopsy from the same patient shows diffuse BM involvement (G) and positive expression of CD20 (H), CD5 (I), and CD23 (J) by immunohistochemistry.

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FiGure 8.3 B-CLL—Cytology. BM aspirate with marked lymphocytosis of small lymphocytes with scanty cytoplasm and small nuclei with dense, clumped nuclear chromatin.

FiGure 8.4 cytes (P).

B-CLL: small lymphocytes (L) and prolympho-

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FiGure 8.5 B-CLL—Pattern of BM involvement: (A) nodular involvement of the BM; (B) interstitial BM infiltrate; and (C and D) diffuse lymphoid infiltrate (with proliferation centers, D).

The extent of BM involvement after chemotherapy does not correlate with the interval between the treatment and relapse [47]. A comparison between the infiltration pattern and IGVH mutational status revealed that the samples with a diffuse pattern were IGVH-unmutated and the nondiffuse CLL samples were IGVH-mutated [49]. The same report showed that the expression of ZAP-70 was related to the infiltration type: in all samples with a diffuse infiltration pattern, the leukemic cells showed ZAP-70 staining, whereas leukemic cells in a nodular infiltration pattern were negative (the mixed-pattern type showed a variable ZAP-70 expression) [49]. Lymph node SLL in the lymph node shows effacement of the architecture with a characteristic pseudofollicular pattern caused by clusters of prolymphocytes and paraimmunoblasts (proliferation centers) in the background of diffuse small lymphocytic infiltrate (Figure 8.6). A minority of cases show only minimal amount of larger cells creating diffuse pattern without proliferation centers. The majority of lymphocytes in CLL/SLL are small and round with scanty cytoplasm, dense and clumped

chromatin, and round nuclei without prominent nucleoli or irregular nuclear borders. Prolymphocytes are medium-sized cells with prominent central nucleoli. Occasional cases of SLL show an interfollicular pattern or only partial involvement of the lymph node (Figure 8.7). other organs SLL/CLL frequently involves extranodal sites, including the tonsil, liver, spleen, skin, among others. The histomorphologic and immunophenotypic features are similar to those seen in the lymph nodes (Figures 8.8). Similar to the diagnosis of follicular lymphoma (FL) in situ or mantle cell lymphoma (MCL) in situ, immunohistochemical stains are very helpful in identifying subtle or partial involvement by SLL/ CLL (Figure 8.9).

dIsease progressIon/transforMatIon Transformation of CLL to more aggressive disease occurs in 5%–10% of patients. Clinical and laboratory features observed in patients with transformation include generalized adenopathy, systemic symptoms (fever, weight loss,

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma and B-Cell Prolymphocytic Leukemia

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FiGure 8.6 B-SLL: (A–D) Histology; (E) cytology. Low-power examination (A and B, two different cases) reveals diffuse lymphoid infiltrate with typical paler areas (proliferation centers) giving rise to a pseudofollicular pattern. Intermediate magnification (C) shows predominance of small lymphocytes with dark chromatin and scanty cytoplasm. Proliferation centers (D; high magnification) are composed of prolymphocytes with conspicuous nucleoli. Touch smear (E) shows small round lymphocytes with regular nuclear outlines and compact, darkly stained chromatin.

and worsened performance status), cytopenia (anemia and/ or thrombocytopenia), increased LDH level, hypercalcemia, and monoclonal gammopathy [50–54]. Morphological progression of CLL/SLL is represented by a predominance of prolymphocytes [55,56] or paraimmunoblasts [57], Hodgkin lymphoma (HL) [58–62], diffuse large B-cell lymphoma (DLBCL) [51,53,63–66], and rarely B-lymphoblastic lymphoma/leukemia (B-ALL) [67]. Hodgkin variant of Richter’s syndrome morphologically and immunophenotypically resembles classical HL (Figure  8.10) [53,54,68,69]. In a series reported by Tsimberidou et al. [68], 0.4% patients with CLL developed Hodgkin transformation with the median time from CLL diagnosis to transformation of 4.6 years. Large neoplastic cells express PAX5, CD15, and CD30, similar to de novo HL. Epstein–Barr virus (EBV) plays an important role in the pathogenesis of Hodgkin transformation of CLL [68,70]. Two types of Hodgkin transformation of CLL/SLL have been described, one with large tumor cells (Reed–Sternberg cells and their variants) in the background of CLL cells (type 1) and the other with polymorphous lymphohistiocytic infiltrate separate from CLL cells (type 2) [59–61,71–73]. It is likely that type-1 transformation represents histologic progression of the underlying CLL, and type-2 transformation represents two different, albeit related diseases.

A clonal relationship between CLL cells and Hodgkin/ Reed–Sternberg cells was demonstrated in three out of four patients who had Hodgkin transformation of CLL by using single-cell polymerase chain reaction (PCR) analysis and DNA sequencing [58]. In contrast to the generally favorable outcome of patients with de novo HL, only 34%–47% of patients with Hodgkin variant of Richter’s syndrome respond to multiagent chemotherapy and the median OS is 8 months [54,68]. Even patients responding to multiagent chemotherapy, such as adriamycin, bleomycin, vinblastine, dacarbazine (ABVD), eventually develop recurrent disease after a short period of time. Stem cell transplantation may be considered for patients who respond to chemotherapy, as the remission duration is short. It is suggested that patients with CLL and EBV infection may benefit from antiviral therapy, and such therapy may decrease the probability of Hodgkin transformation [69]. Neoplastic cells in prolymphocytic/paraimmunoblastic transformation (Figure 8.11) often express p53 and Ki-67. Progression of CLL/SLL to DLBCL can present as a localized enlargement of a single lymph node histologically showing DLBCL (Figure 8.12) or increasing generalized adenopathy with rapid deterioration of patient’s performance. Figure 8.13 shows flow cytometric features for CLL undergoing large cell transformation.

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FiGure 8.7 B-SLL—Partial involvement of the lymph node at the periphery. (A) Histology (H&E staining). (B–D) Immunohistochemistry. The central, not involved part of the lymph node (*), shows lack of CD20 staining (B), positive CD5 (C, reactive T cells are slightly darker than neoplastic B cells), and positive CD3 (D). Neoplastic B cells (arrow) at the periphery shows CD20 (B) and CD5 (C) expression and lack of CD3 (D).

The chances of development of Richter’s syndrome in CLL patients with multiple chromosome changes is higher than in those with either simple trisomy 12 or a normal karyotype [74]. Richter’s syndrome can occur in both mutated and unmutated variants of CLL. Progression of CLL may be associated with del(11q), overexpression of the MYC gene, deletions of the RB1 gene, and mutations of the p53 gene [51,53,75,76]. Apart from genetic defects, Richter’s syndrome may be triggered by viral infections [53].

cytogenetIcs/fIsh and MoLecuLar features Genomic aberrations in CLL are important independent predictors of disease progression and survival (Table 8.1). Chromosomal abnormalities are detected in the majority of CLL patients using molecular cytogenetic technologies, including FISH. Genetic aberrations are found more often by FISH studies when compared to conventional cytogenetics (68%–80% vs. 37%) [77,78]. The most frequent chromosomal abnormalities in CLL include del(13q), trisomy 12 (Figure 8.14), del(11q)/ATM, del(14q), del(6q), and del(17p)/TP53 [51,79–81]. Complex karyotype is more often seen as disease progresses (Figure 8.15). Patients with normal karyotype or deletion of 13q14 as the sole genetic abnormality have better prognosis than those with a complex karyotype or deletion of 11q23 or 17p13. Response rate to chemotherapy is significantly higher in patients with normal karyotypes than in those with abnormal karyotypes, especially with complex changes. In a series by

Dohner et al. [78], the median survival times for patients with 17p deletion, 11q deletion, 12q trisomy, normal karyotype, and 13q deletion as the sole abnormality were 32, 79, 114, 111, and 133 months, respectively. Patients with 17p deletions had the shortest median treatment-free interval (9 months), and those with 13q deletions had the longest (92 months) [78]. The response to rituximab was noted to vary by cytogenetic group: del(17)(p13.1), 0%; del(11)(q22.3), 66%; del(13)(q14.3), 86%; and +12, 25% [82]. Alemtuzumab (Campath, Genzyme, Boston, MA) may be an effective initial therapy for patients with TP53 mutations or del(17q)(p13.1) or both, as opposed to fludarabine, chlorambucil, or rituximab [83]. Reciprocal translocations are uncommon in CLL. The translocations involving IGH gene have been reported in 5%–15% of cases, and based on most reports are associated with shorter OS [84]. The most frequent is t(14;18). Whole genome sequencing offered new insights into the mutational status of CLL, and showed poor survival in patients with mutations of NOTCH1 or SF3B1 [85,86]. del(13q) The del(13q14) is the most frequent genetic change in CLL (Figure 8.16), occurring in 40%–66% [87–90]. The presence of either cryptic deletion of the single locus (q14) or deletion of larger portion of the chromosome is associated with a favorable prognosis. Patients with a del(13q14) as single aberration have the longest estimated median treatmentfree interval and survival time [78,91]. Deletions of 13q are

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FiGure 8.8 SLL/CLL involving tonsil. H&E sections (A–C) show typical pseudonodular pattern with proliferation centers. SLL cells are positive for CD23 (D), CD20 (E), and CD5 (F) by immunohistochemistry. FC analysis (G–L) shows typical immunophenotypic profile with positive CD5 (G), CD19, CD20, and CD23 (J). The expression of CD20 and lambda is dim (H and I, K, and L).

associated with typical morphologic and phenotypic features [92–95]. Although CLL with 13q deletion as the sole abnormality usually has a good prognosis, more aggressive clinical courses are documented for del(13)-only CLL carrying higher percentages of 13q deleted nuclei or the presence of

larger deletions involving the RB1 locus [96,97]. CLL carrying 100 × 109/L. The median OS time is 5 years, and the event-free survival time is 37 months [150,151]. The probability of OS for 3, 5, and 10 years is 63%, 56%, and 35%, respectively [151]. As detected

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FiGure 8.21 CLL with prominent atypia mimicking T-cell process by cytomorphology. The immunophenotype by FC is typical for CLL (dim lambda, dim CD20, and positive CD5 and CD23 expression).

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FiGure 8.22 B-PLL, FC. Leukemic cells show moderate expression of both CD20 and kappa (A–D), as well as positive CD23 (E), and CD11c (G). CD 5 is present on subset (E) and CD10 is negative (F).

by univariate and multivariate analyses, the advanced age, lymphocytosis (>100 × 109/L) and anemia (20 per 10 high-power fields (hpf), >50/mm2] [2,32]. A high proportion of Ki-67-positive cells (>40%–60%) is also an adverse prognostic indicator [32,33]. The three groups with different Ki-67 index of 30 × 109/L) and less likely to have adenopathy than those without deletion, and cases with deletions at 11q23 and 6q21 are associated with extranodal disease [15]. Complex karyotype

(Figure 9.19) is associated with poor prognosis [56]. Although both leukemic and nodal MCL show similar genomic patterns of losses (involving 6q, 11q22–23, 13q14, and 17p13) and gains (affecting 3q and 8q), genomic loss of chromosome 8p occurs more frequently in patients with leukemic disease (79% vs. 11%) [57]. This may indicate the presence of a novel tumor suppressor gene locus on 8p, whose deletion may be associated with leukemic dissemination. In a series reported by Au et al. [58], the common aneuploidies in MCL included −Y, −13, −9, −18, +3, and +12, and the common structural changes included +3q, +12q, del(6q), del(1p), del(13q), del(10q), del(11q), del(9p), and del(17p). The commonest breakpoint clusters were 1p21–22, 1p31–32, 1q21,

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6q11–15, 6q23–25, 8q24, 9p21–24, 11q13–23, 13q12–14, and 17p12–13. When analyzed separately as lymph node-based versus blood-based disease, deletions and chromosomal losses were more common in the lymph node group, while gains of chromosome segments 3q and 12q were similar [58]. Among 60 patients with MCL in leukemic phase reported by ParryJones et al. [15], the most common chromosomal abnormalities included −17p13 (46%), −13q14 (43%), −11q23 (25%), −6q21 (12%), and +12 (8%).

dIsease progressIon/transForMatIon Morphological progression of MCL is associated with an increase in large blastoid cells, resulting in the so-called blastoid variant [2,59]. The blastoid variant of MCL is a very aggressive subtype of lymphoma with a median overall survival of 14.5 months compared to 53 months for the patients with a common form of MCL [2,60]. A subset of blastoid variants of MCL may have p53 gene mutations [61]. Additionally, blastoid MCL subtypes are characterized by distinctly elevated mitotic counts, proliferation indices

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FIgure 9.10 MCL—BM (paratrabecular pattern). (A) Histology of BM core biopsy shows a dense paratrabecular lymphoid infiltrate. (B) Higher magnification displays nuclear contour irregularities. (C) Neoplastic lymphocytes are positive for BCL1. CD79a

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FIgure 9.12 MCL—Flow cytometry (blood). Lymphomatous cells display moderate CD20 (A and D) and kappa (B) expression, positive CD5 (A and B), negative CD23 (C), and positive CD38 (E). FISH studies performed on blood sample confirm the diagnosis of MCL by positive rearrangement of CCND1 (F; yellow fusion signal).

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FIgure 9.13 CD5-negative MCL. (A) Histology. Neoplastic cells are positive for CD20 (B) and BCL1 (D); they lack CD5 (C). (E–g) Flow cytometric analysis shows positive CD19, CD20, and lambda, and negative CD5. CD5-negative MCL comprises ~11% of all MCL cases. CD10

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FIgure 9.14 CD10+ MCL. (A) Histology; (B and C) immunohistochemistry; (D and E) flow cytometry. MCL cells express BCL1 (B) and coexpress CD5 and CD10 (C–E).

(53% vs. 27% in common MCL), frequent BCL1 rearrangements at the major translocation cluster locus (59% vs. 40%), and overexpression of p53 (21% vs. 6%) [62]. Blastoid MCL subtypes display a tendency for tetraploid number of chromosomes (36% of blastoid and 80% of pleomorphic types vs. 8% of common MCL), a feature clearly separating these neoplasms from other types of B-cell non-Hodgkin’s

lymphoma and possibly being related to cyclin D1 (BCL11) overexpression  [62]. CgH analysis showed an increased number of chromosome imbalances associated with blastoid variants of MCL (e.g., gains of 3q, 7p, and 12q, and losses of 17p), which may have a prognostic significance [63]. CgH losses of 17p correlated with p53 gene deletions and mutations. Leukocytosis, an elevated LDH level, and a high

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FIgure 9.15 MCL with aberrant CD23 expression and lack of CD43. Spleen with atypical lymphoid infiltrate (A). Lymphomatous cells express CD20 (B), BCL1 (C), and CD23 (D), and are negative for CD43 (E).

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FIgure 9.16 Aggressive (blastoid) variant of MCL—Flow cytometry and FISH studies. Lymphomatous cells are positive for CD20 (A), partially CD71 (B), CD5 (C and D), partially CD10 (D), and lambda (E and F), and are negative for CD23 (g). FISH studies (H) showed the rearrangement of CCND1 (red) and IGH (green) giving yellow fusion signal.

proliferative activity at diagnosis as assessed by the mitotic count and Ki-67 index are associated with an increased risk of blastoid transformation, and an elevated serum LDH and blood leukocytosis with a shorter time interval

to transformation [64]. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes (cyclin-dependent kinase inhibitors suggested as candidates for tumor-suppressor genes) are detected in aggressive MCL, but not in the typical

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FIgure 9.17

FISH: CCND1

MCL (blastoid variant) with unusual, strong BCL6 expression. FISH studies confirmed the diagnosis of MCL.

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Normal 14

der 11

der 14 Normal 11

FIgure 9.18

MCL showing CCND1–IGH fusion: FISH analysis.

variants. The p21Waf1 and p16INK4a alterations occur in a subset of tumors with a wild-type p53 gene [65]. Deletions of INK4a/ARF gene locus are found in up to 30% of MCL and are associated with poor prognosis [65,66].

dIFFerentIal dIagnosIs Differential diagnosis of MCL includes B- and T-cell lymphoproliferative disorders and other hematopoietic tumors. Occasional cases of MCL involve the lymph node

only focally and may be misinterpreted as a reactive process without immunostaining for CD20, CD3, CD5, and BCL1 (cyclin D1) (Figure 9.20). Based on cytologic and/ or histologic features, differential diagnosis includes the following: • Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) • DLBCL • FL

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Differential Diagnosis

• B- and T-cell prolymphocytic leukemia (B- and T-PLL) • Peripheral T-cell lymphoma (PTCL), not otherwise specified • B- and T-cell lymphoblastic leukemia/lymphoma (B- and T-ALL/LBL) • Acute myeloid leukemia (AML)/extramedullary myeloid tumor (EMT) • Blastic plasmacytoid dendritic cell neoplasm (BPDCN) • Reactive follicular hyperplasia • Acute and chronic monocytic leukemias

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FIgure 9.19 MCL with complex chromosomal abnormalities [near tetraploid cells with t(11;14), +21, XXY]: Metaphase cytogenetics.

Based on the BCL1 (cyclin D1) expression, the differential diagnosis includes the following: • Plasma cell myeloma (PCM) • HCL Based on CD5 or CD5/CD10 expression, the differential diagnosis includes the following: • • • •

SLL/CLL (CD5+, CD10−, CD23+) FL (very rare cases are dual CD5/CD10+) De novo CD5+ DLBCL or Richter’s syndrome Marginal zone lymphoma (MZL; subset)

Based on the cytologic features, the differential diagnosis of MCL (including blastoid and pleomorphic variants) includes FL (CD5−, CD10+, BCL6+), mature T-cell lymphoproliferations (CD2±, CD3±, CD7±, CD4±, CD8±), DLBCL (usually CD5−, CD10±, BCL6±, MUM1±, BCL1−), AML/ EMT (CD34+/CD117+/CD33+/MPO+, CD68+/muramidase+/ CD56±), monocytic proliferations (CD163+, CD68+, muramidase+), BPDCN (CD4+, CD56+, CD123+), precursor B-ALL/ LBL [terminal deoxynucleotidyl transferase (TdT)+, CD34+, CD10±], and precursor T-ALL/LBL (CD34±, TdT±, CD3+, CD1a±). Both FL and MCL may present with leukemic blood involvement (Figure 9.21). Flow cytometry and FISH studies (CCND1, BCL2) are helpful in these cases. Histologic differential diagnosis of MCL depends on cellular composition (small, medium, or large cells) and BCL1

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FIgure 9.20 MCL—Minimal lymph node involvement mimicking reactive process. (A and B) Low magnification shows follicular hyperplasia with well-demarcated mantle zone. (C and D) Higher magnification shows germinal center surrounded by mantle cells expressing BCL1.

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Cytologic features of (A) MCL and (B) FL.

whether the infiltrate is diffuse or nodular. In MCL with mostly a diffuse pattern, the diagnostic considerations include diffuse follicle center cell lymphoma, nodal MZL, B-small lymphocytic lymphoma/chronic lymphocytic leukemia (B-SLL/CLL), some PTCLs, DLBCL, AML/EMT, BPDCN, and lymphoblastic lymphomas. In MCL with a nodular pattern, the differential diagnosis includes FL, reactive lymph node with follicular hyperplasia, B-chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) with prominent proliferation centers, and nodal MZL. Based on the expression of CD5, MCL has to be distinguished from other CD5+ B-cell proliferations, including B-CLL/SLL (especially atypical variants of B-CLL/SLL, which may lack CD23 expression and show a moderate expression of CD20 and surface immunoglobulins, and cytologic atypia with irregular nuclei), de novo CD5+ DLBCL, and other B-cell lymphoproliferations, which may occasionally show aberrant expression of CD5 (MZL). As mentioned above, a subset of MCLs may display an aberrant phenotype, including lack of CD5, positive CD10, and positive CD23. Broader immunophenotypic panel that includes BCL1 (cyclin D1) may be necessary, when evaluating the tissue section suspicious for lymphoma. When tissue section or cell block is not available for immunohistochemical staining for BCL1 [e.g., blood sample, effusion, fine needle aspiration sample, BM aspirate smear, colony-stimulating factor (CSF), etc.], correlation with FISH studies for t(11;14)/CCND1 is often indicated to establish the correct diagnosis. BCL1 expression is not specific for MCL. PCM, subset of HCLs, and epithelial tumors may be BCL1+ by immunohistochemistry (HCL and epithelial cells expressing BCL1 do not carry CCND1–IGH rearrangement typical for MCL or PCM).

reFerences 1. Williams ME, Swerdlow SH, Meeker TC. Chromosome t(11;14)(q13;q32) breakpoints in centrocytic lymphoma are highly localized at the bcl-1 major translocation cluster. Leukemia, 1993. 7(9):1437–40. 2. Argatoff LH, et al. Mantle cell lymphoma: a clinicopathologic study of 80 cases. Blood, 1997. 89(6):2067–78.

3. Campo E, Raffeld M, Jaffe ES. Mantle-cell lymphoma. Semin Hematol, 1999. 36(2):115–27. 4. de Boer CJ, et al. Cyclin D1 protein analysis in the diagnosis of mantle cell lymphoma. Blood, 1995. 86(7):2715–23. 5. Harris NL. Mantle cell lymphoma. J Clin Oncol, 1994. 12(4):876–7. 6. Lenz g, Dreyling M, Hiddemann W. Mantle cell lymphoma: established therapeutic options and future directions. Ann Hematol, 2004. 83(2):71–7. 7. Pittaluga S, et al. Mantle cell lymphoma: a clinicopathological study of 55 cases. Histopathology, 1995. 26(1):17–24. 8. Campo E. genetic and molecular genetic studies in the diagnosis of B-cell lymphomas I: mantle cell lymphoma, follicular lymphoma, and Burkitt’s lymphoma. Hum Pathol, 2003. 34(4):330–5. 9. Thieblemont C, et al. Small lymphocytic lymphoma, marginal zone B-cell lymphoma, and mantle cell lymphoma exhibit distinct gene-expression profiles allowing molecular diagnosis. Blood, 2004. 103(7):2727–37. 10. Harris AW, et al. Cyclin D1 as the putative bcl-1 oncogene. Curr Top Microbiol Immunol, 1995. 194:347–53. 11. Leitch HA, et al. Limited-stage mantle-cell lymphoma. Ann Oncol, 2003. 14(10):1555–61. 12. Majlis A, et al. Mantle cell lymphoma: correlation of clinical outcome and biologic features with three histologic variants. J Clin Oncol, 1997. 15(4):1664–71. 13. Swerdlow SH, et al. The morphologic spectrum of nonHodgkin’s lymphomas with BCL1/cyclin D1 gene rearrangements. Am J Surg Pathol, 1996. 20(5):627–40. 14. Weisenburger DD, et al. Mantle cell lymphoma. A clinicopathologic study of 68 cases from the Nebraska Lymphoma Study group. Am J Hematol, 2000. 64(3):190–6. 15. Parry-Jones N, et al. Cytogenetic abnormalities additional to t(11;14) correlate with clinical features in leukaemic presentation of mantle cell lymphoma, and may influence prognosis: a study of 60 cases by FISH. Br J Haematol, 2007. 137(2):117–24. 16. Jares P, Campo E. Advances in the understanding of mantle cell lymphoma. Br J Haematol, 2008. 142(2):149–65. 17. Williams ME, et al. Mantle cell lymphoma: report of the 2009 Mantle Cell Lymphoma Consortium Workshop. Leuk Lymphoma, 2010. 51(3):390–8. 18. Swerdlow SH, Williams ME. From centrocytic to mantle cell lymphoma: a clinicopathologic and molecular review of 3 decades. Hum Pathol, 2002. 33(1):7–20. 19. Swerdlow SH, et al., eds. WHO classification of tumors of haematopoietic and lymphoid tissues. Lyon: IARC; 2008.

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20. Meusers P, et al. Multicentre randomized therapeutic trial for advanced centrocytic lymphoma: anthracycline does not improve the prognosis. Hematol Oncol, 1989. 7(5):365–80. 21. Feller AC, Diebold J. Histopathology of nodal and extranodal non-Hodgkin’s lymphomas. 3rd ed. Berlin/Heidelberg: Springer-Verlag; 2004. 22. Bosch F, et al. Mantle cell lymphoma: presenting features, response to therapy, and prognostic factors. Cancer, 1998. 82(3):567–75. 23. Fu K, et al. Cyclin D1-negative mantle cell lymphoma: a clinicopathologic study based on gene expression profiling. Blood, 2005. 106(13):4315–21. 24. Hiddemann W, Dreyling M. Mantle cell lymphoma: therapeutic strategies are different from CLL. Curr Treat Options Oncol, 2003. 4(3):219–26. 25. Berinstein NL, Mangel J. Integrating monoclonal antibodies into the management of mantle cell lymphoma. Semin Oncol, 2004. 31(1 Suppl 2):2–6. 26. Brugger W, et al. Rituximab consolidation after high-dose chemotherapy and autologous blood stem cell transplantation in follicular and mantle cell lymphoma: a prospective, multicenter phase II study. Ann Oncol, 2004. 15(11):1691–8. 27. Jacquy C, et al. Peripheral blood stem cell contamination in mantle cell non-Hodgkin lymphoma: the case for purging? Bone Marrow Transplant, 1999. 23(7):681–6. 28. Andersen NS, et al. Failure of immunologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection of minimal residual disease. Blood, 1997. 90(10):4212–21. 29. Howard OM, et al. Rituximab and CHOP induction therapy for newly diagnosed mantle-cell lymphoma: molecular complete responses are not predictive of progression-free survival. J Clin Oncol, 2002. 20(5):1288–94. 30. Raty R, et al. Ki-67 expression level, histological subtype, and the International Prognostic Index as outcome predictors in mantle cell lymphoma. Eur J Haematol, 2002. 69(1):11–20. 31. Hoster E, et al. A new prognostic index (MIPI) for patients with advanced-stage mantle cell lymphoma. Blood, 2008. 111(2):558–65. 32. Tiemann M, 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(1):29–38. 33. Katzenberger T, et al. The Ki67 proliferation index is a quantitative indicator of clinical risk in mantle cell lymphoma. Blood, 2006. 107(8):3407. 34. Determann O, et al. Ki-67 predicts outcome in advancedstage mantle cell lymphoma patients treated with anti-CD20 immunochemotherapy: results from randomized trials of the European MCL Network and the german Low grade Lymphoma Study group. Blood, 2008. 111(4):2385–7. 35. Klapper W, et al. Ki-67 as a prognostic marker in mantle cell lymphoma-consensus guidelines of the pathology panel of the European MCL Network. J Hematop, 2009. 2(2): 103–11. 36. Espinet B, et al. Clonal proliferation of cyclin D1-positive mantle lymphocytes in an asymptomatic patient: an earlystage event in the development or an indolent form of a mantle cell lymphoma? Hum Pathol, 2005. 36(11):1232–7. 37. Nodit L, et al. Indolent mantle cell lymphoma with nodal involvement and mutated immunoglobulin heavy chain genes. Hum Pathol, 2003. 34(10):1030–4. 38. Adam P, et al. Incidence of preclinical manifestations of mantle cell lymphoma and mantle cell lymphoma in situ in reactive lymphoid tissues. Mod Pathol, 2012. 25(12):1629–36.

References 39. Aqel N, et al. In-situ mantle cell lymphoma—a report of two cases. Histopathology, 2008. 52(2):256–60. 40. Carbone A, Santoro A. How I treat: diagnosing and managing “in situ” lymphoma. Blood, 2011. 117(15):3954–60. 41. Edlefsen KL, et al. Early lymph node involvement by mantle cell lymphoma limited to the germinal center: report of a case with a novel “follicular in situ” growth pattern. Am J Clin Pathol, 2011. 136(2):276–81. 42. Neto Ag, et al. Colonic in situ mantle cell lymphoma. Ann Diagn Pathol, 2012. 16(6):508–14. 43. Wilcox RA. Cutaneous T-cell lymphoma: 2011 update on diagnosis, risk-stratification, and management. Am J Hematol, 2011. 86(11):928–48. 44. Kelemen K, et al. CD23+ mantle cell lymphoma: a clinical pathologic entity associated with superior outcome compared with CD23– disease. Am J Clin Pathol, 2008. 130(2):166–77. 45. Zeng W, et al. Cyclin D1-negative blastoid mantle cell lymphoma identified by SOX11 expression. Am J Surg Pathol, 2012. 36(2):214–9. 46. Hsiao SC, et al. SOX11 is useful in differentiating cyclin D1-positive diffuse large B-cell lymphoma from mantle cell lymphoma. Histopathology, 2012. 61(4):685–93. 47. Xu W, Li JY. SOX11 expression in mantle cell lymphoma. Leuk Lymphoma, 2010. 51(11):1962–7. 48. Dong HY, et al. B-cell lymphomas with coexpression of CD5 and CD10. Am J Clin Pathol, 2003. 119(2):218–30. 49. gualco g, et al. BCL6, MUM1, and CD10 expression in mantle cell lymphoma. Appl Immunohistochem Mol Morphol, 2010. 18(2):103–8. 50. Espinet B, et al. Translocation (11;14)(q13;q32) and preferential involvement of chromosomes 1, 2, 9, 13, and 17 in mantle cell lymphoma. Cancer genet Cytogenet, 1999. 111(1):92–8. 51. Wlodarska I, et al. Secondary chromosome changes in mantle cell lymphoma. Haematologica, 1999. 84(7):594–9. 52. Bentz M, et al. t(11;14)-positive mantle cell lymphomas exhibit complex karyotypes and share similarities with B-cell chronic lymphocytic leukemia. genes Chromosomes Cancer, 2000. 27(3):285–94. 53. Allen JE, et al. Identification of novel regions of amplification and deletion within mantle cell lymphoma DNA by comparative genomic hybridization. Br J Haematol, 2002. 116(2):291–8. 54. Schraders M, et al. Novel chromosomal imbalances in mantle cell lymphoma detected by genome-wide array-based comparative genomic hybridization. Blood, 2005. 105(4):1686–93. 55. Cuneo A, et al. 13q14 deletion in non-Hodgkin’s lymphoma: correlation with clinicopathologic features. Haematologica, 1999. 84(7):589–93. 56. Cuneo A, et al. Cytogenetic profile of lymphoma of follicle mantle lineage: correlation with clinicobiologic features. Blood, 1999. 93(4):1372–80. 57. Martinez-Climent JA, et al. Loss of a novel tumor suppressor gene locus at chromosome 8p is associated with leukemic mantle cell lymphoma. Blood, 2001. 98(12):3479–82. 58. Au WY, et al. Cytogenetic analysis in mantle cell lymphoma: a review of 214 cases. Leuk Lymphoma, 2002. 43(4):783–91. 59. Norton AJ, et al. Mantle cell lymphoma: natural history defined in a serially biopsied population over a 20-year period. Ann Oncol, 1995. 6(3):249–56. 60. Bernard M, et al. Blastic variant of mantle cell lymphoma: a rare but highly aggressive subtype. Leukemia, 2001. 15(11):1785–91.

Mantle Cell Lymphoma 61. Hernandez L, et al. p53 gene mutations and protein overexpression are associated with aggressive variants of mantle cell lymphomas. Blood, 1996. 87(8):3351–9. 62. Ott g, et al. Blastoid variants of mantle cell lymphoma: frequent bcl-1 rearrangements at the major translocation cluster region and tetraploid chromosome clones. Blood, 1997. 89(4):1421–9. 63. Bea S, et al. Increased number of chromosomal imbalances and high-level DNA amplifications in mantle cell lymphoma are associated with blastoid variants. Blood, 1999. 93(12):4365–74.

249 64. Raty R, et al. Predictive factors for blastoid transformation in the common variant of mantle cell lymphoma. Eur J Cancer, 2003. 39(3):321–9. 65. Pinyol M, et al. Deletions and loss of expression of p16INK4a and p21Waf1 genes are associated with aggressive variants of mantle cell lymphomas. Blood, 1997. 89(1):272–80. 66. Pinyol M, et al. p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin’s lymphomas. Blood, 1998. 91(8):2977–84.

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10

Marginal Zone Lymphoma

CoNteNts Nodal Marginal Zone Lymphoma ............................................................................................................................................. 251 Introduction .......................................................................................................................................................................... 251 Morphology .......................................................................................................................................................................... 251 Lymph Nodes .................................................................................................................................................................. 251 Extranodal Sites............................................................................................................................................................... 252 BM and Blood ................................................................................................................................................................. 252 Immunophenotype................................................................................................................................................................ 254 Cytogenetics/Fluorescence In Situ Hybridization and Molecular Features ......................................................................... 256 Differential Diagnosis .......................................................................................................................................................... 256 Reactive Lymphoid Hyperplasia ..................................................................................................................................... 258 Diffuse Large B-Cell Lymphoma .................................................................................................................................... 258 Low-Grade B-Cell Lymphomas ...................................................................................................................................... 258 Plasma Cell Neoplasm .................................................................................................................................................... 259 PTCL and AITL .............................................................................................................................................................. 260 Secondary Lymph Node Involvement by MZL ............................................................................................................... 260 Extranodal Marginal Zone Lymphoma ..................................................................................................................................... 260 Introduction .......................................................................................................................................................................... 260 Morphology .......................................................................................................................................................................... 260 Immunophenotype................................................................................................................................................................ 261 Large Cell Transformation ................................................................................................................................................... 261 Cytogenetics/FISH and Molecular Features ........................................................................................................................ 262 Differential Diagnosis .......................................................................................................................................................... 263 References ................................................................................................................................................................................. 263

Nodal MargiNal ZoNe lyMphoMa IntroductIon Marginal zone lymphoma (MZL) can be subdivided into three major categories: splenic MZL, extranodal MZL [mucosa-associated lymphoid tissue (MALT) lymphoma], and nodal MZL. Nodal MZL is a low-grade lymphoma with the morphologic and immunophenotypic features similar to extranodal MZL (MALT type) but without splenic or extranodal disease. It occurs in the peripheral lymph nodes (most commonly in the head and neck area) and the abdomen, often presents in the advanced clinical stage, and has more aggressive clinical course than extranodal MALT lymphoma [1–7]. Patients with nodal MZL have lower 5-year overall survival and failure-free survival than those with MALT lymphoma [7]. Traverse-Glehen et al. [3] found peripheral blood involvement in 23%, anemia in 24%, thrombocytopenia in 10%, and presence of serum M component in 33%. In a series of 47 patients with primary nodal MZL reported by Arcaini et  al. [1], 45% had stage IV disease and 24% had positive hepatitis C virus serology. Based on the Follicular Lymphoma International Prognostic Index (FLIPI), 33% of nodal MZLs were classified

as low risk, 34% as intermediate risk, and 33% as high risk with the 5-year overall survival of 69%. In univariate analysis, worse overall survival was associated with high-risk FLIPI, age >60 years, and raised LDH, but in multivariate analysis, only FLIPI predicted a worse overall survival [1]. MZLs with both bone marrow (BM) and nodal involvement are associated with shorter overall survival [2].

Morphology lymph Nodes Nodal MZL was earlier divided into two morphologic variants: one resembling nodal involvement by extranodal MZL (MALT type) and the other resembling splenic MZL  [8]. More recently, four architectural patterns can be identified: diffuse, nodular, interfollicular, and perifollicular [9]. The diffuse pattern is characterized by sheets of neoplastic cells with effacement of nodal architecture (Figure 10.1). The nodular (marginal zone) pattern is characterized by wellformed nodules surrounding and colonizing reactive germinal centers (Figure 10.2). The nodules are rather sharply demarcated from the uninvolved interfollicular areas. The interfollicular pattern shows the neoplastic B cells limited 251

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FigUre 10.1 Nodal MZL with mostly a diffuse pattern without any residual follicles with reactive germinal centers. Neoplastic B cells show monocytoid appearance, strong expression of CD20 and PAX5, and only scattered Ki-67+ cells.

to the interfollicular areas with sparing of normal secondary follicles and often prominent perivascular/perisinusoidal involvement. The least common, perifollicular pattern is characterized by an annular distribution of the neoplastic cells around uninvolved normal secondary follicles (colonization of germinal centers is minimal or absent). Interfollicular and perifollicular patterns show an increased number of large cells, compared to other types. The lymphoid cells are mostly small to occasionally medium in size with variable admixture of large B cells with prominent nucleoli (immunoblast-like). Cellular pleomorphism (variable shape and size of neoplastic cells) is seen in the majority of cases, and only a subset of lymphomas is composed of predominantly small cells with occasional, scattered intermediate-sized cells. Large cell component, usually 50% large cells or sheets of large cells [9]. MZL with an increased number of large cells (20%–50%) does not behave more aggressively [3]. In contrast to follicular lymphoma (FL), there are no defined criteria for grading of MZL. Transformation to diffuse large B-cell lymphoma (DLBCL) is currently diagnosed only in the presence of sheets of large cells. In many cases, B cells have a monocytoid appearance with an abundant pale or eosinophilic cytoplasm (Figure  10.3). Occasional cases of MZL show an extensive plasmacytoid or plasmacytic differentiation and an increased number of large

cells with high mitotic rate [3]. Figures 10.4 and 10.5 show MZL with prominent plasma cell component. Residual germinal centers are usually preserved but are disrupted. Some cases show a prominent colonization of reactive follicles (either complete or partial) with characteristic expansion and disruption of follicular dendritic cell (FDC) meshwork  [6]. Residual follicle center cells express CD20, CD10, and BCL6, and are negative for BCL2, whereas neoplastic B cells infiltrating follicles express CD20, BCL2, and often MUM1. extranodal sites By definition, there is no evidence of involvement of the spleen or other extranodal sites, such as gastrointestinal (GI) tract, skin, lung, salivary gland, or ocular adnexa. BM and Blood BM and blood may be involved in nodal MZL more frequently than in extranodal MALT lymphomas. The circulating cells are small to intermediate in size and may display a monocytoid appearance in the blood smear. BM shows usually a nodular pattern of involvement (Figure 10.6) and less often paratrabecular distribution [10]. Lymphomatous cells often display a monocytoid appearance with an admixture of small lymphocytes, plasma cells, and occasional lymphocytes with irregular nuclei (centrocyte-like). B cells predominate, but T cells are also present. CD21+/CD23+ FDCs are intermixed between lymphoid cells.

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FigUre 10.2 Nodal MZL with a characteristic marginal zone pattern (A–C; nodular pattern). Lymphomatous cell are strongly positive for CD20 (D) and PAX5 (E). FDC meshwork is rather well preserved (F). Scattered residual CD10+ germinal center cells are present (G), but follicles show predominance of TFH cells (H–I; CD4+/PD1+).

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Nodal MZL with a typical monocytoid appearance of neoplastic B cells (A–C).

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FigUre 10.4 MZL with prominent plasmacytic differentiation: Two cases. Case 1 (A–H) shows a diffuse lymphoplasmacytic infiltrate (A  and B) of mostly small lymphocytes without a monocytoid appearance and focal prominent clusters of plasma cells with numerous Dutcher bodies (C). Lymphomatous cells are positive for CD20 (D), negative for CD5 (E), and positive for CD21 (F). Plasma cells are positive for MUM1 (G) and lambda (H; dual kappa and lambda staining; brown indicates kappa and red indicates lambda). Case 2 (I and J) shows prominent clusters of mature plasma cells with eccentric nuclei and intracytoplasmic inclusions (Russel bodies; original magnification shown; pictures have been digitally resized to fit the composite illustration).

IMMunophenotype Phenotypically, MZL is positive for B-cell markers (CD19, CD20, CD22, CD79a, and PAX5) without the expression of BCL1 (cyclin D1), BCL6, CD10, CD23, and CD103. CD5 is usually negative (Figure 10.7), but a minor subset of MZLs may be CD5+. The expression of CD20 and surface immunoglobulins is moderate when analyzed by flow cytometry (Figures 10.8 and 10.9). Often, there is dim to moderate expression of CD11c and/or CD43 (21%–50%) [9,11]. Some MZLs may show an aberrant expression of CD13 (Figure 10.10). The

CD43 expression is especially useful in evaluating small biopsy specimens, which help to confirm neoplastic process (by comparing CD20 vs. CD3 and CD43). BCL2 is most often dimly positive. Immunohistochemical evaluation of the lymph node shows disruption of the FDC meshwork (CD21 and CD23 staining) caused by follicle colonization, predominance of B cells (CD20 staining), and lack of CD10 expression by neoplastic cells. Plasma cells are often monotypic (~30%)—another useful parameter in confirming nodal MZL. Other cases may show an increased number of polytypic plasma cells.

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FigUre 10.5 Nodal MZL with extensive plasmacytic differentiation and only focal clusters of monocytoid B cells (arrows). Low magnification (A) shows mostly a preserved architecture of the lymph node with secondary follicles, aggregates of monocytoid B cells (arrow), and expanded interfollicular (paracortical). Monocytoid B cells (B) express CD20 and PAX5 (D–F; different magnifications) and are negative for MUM1 (G). Plasma cells (C) strongly express MUM1 (G) and kappa (H). Kappa and lambda (H and I) were analyzed by in situ hybridization (ISH) methodology.

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FigUre 10.6 BM involvement by MZL. Note atypical interstitial lymphoid aggregate with a monocytoid appearance.

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FigUre 10.7 Nodal marginal zone B-cell lymphoma—Immunohistochemistry. (A) Typical histologic pattern of MZL. (B and C) Expanded and disrupted FDC meshwork visualized by staining with CD23 (B) and CD21 (C). Neoplastic and non-neoplastic B cells are positive for CD20 (D), and negative for CD10 (E), and show plasmacytic differentiation with kappa restriction (F).

cytogenetIcs/Fluorescence In SItu hybrIdIzatIon and Molecular Features Abnormalities identified in >15% of patients with non-MALT MZLs included +3/+3q (37%), 7q deletions (31%), +18/+18q (28%), 6q deletions (19%), +12/+12q (15%), and 8p deletions (15%) [12]. Trisomy 3/3q, 7q deletions, +18, and +12 were seen in different combinations in >30% of patients in comparison with 2% in B-small lymphocytic lymphoma/chronic lymphocytic leukemia (B-SLL/CLL), 1% in mantle cell lymphoma (MCL), and 7% in FL [12].

dIFFerentIal dIagnosIs • Reactive process (e.g., follicular hyperplasia, monocytoid B-cell hyperplasia) • FL • MCL • DLBCL • Lymphoplasmacytic lymphoma (LPL) • Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) • Plasma cell neoplasm

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FigUre 10.8 Nodal marginal zone B-cell lymphoma—Flow cytometry. (A) Histology of the lymph node shows a residual germinal center surrounded by small lymphocytic infiltrate with plasma cells. Neoplastic B cells are positive for CD11c (B), kappa immunoglobulins (C and D), and are negative for CD103 (E) and CD25 (F).

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FigUre 10.9 Nodal MZL—Flow cytometry. Flow cytometric analysis shows partial lymph node involvement with CD20 + (A) monoclonal B cells (kappa+; B, arrow), reactive polytypic B cells (kappa+ and lambda+; B and C), and reactive T cells (CD20 −/CD5+; A and E). Monoclonal B cells are negative for CD71 (D) and show nonspecific phenotype without expression of CD5 (E) or CD10 (F).

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FigUre 10.10 MZL (blood) with aberrant expression of CD13—Flow cytometry (A–D, all events; E and F, lymphocytes only). Monoclonal B cells express CD20 (A) and kappa (B) and display an aberrant expression of CD13 (on subset of monoclonal B cells with brighter CD20; D and F). CD5 and CD10 are not expressed (E).

• Secondary lymph node involvement by MZL • Hodgkin lymphoma with clusters of monocytoid B cells • Peripheral T-cell lymphoma (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) The differential diagnosis of nodal MZL lymphoma (Figure 10.11) includes reactive lymph node (with monocytoid B-cell hyperplasia, mantle zone hyperplasia, or follicular hyperplasia), FL (especially with a reversed pattern or monocytoid differentiation), LPL, SLL/CLL, and other lymphoproliferations with an increased number of monocytoid B cells. MZL with extensive plasmacytic differentiation needs to be differentiated from plasmacytoma. reactive lymphoid hyperplasia Nodal MZL needs to be differentiated from reactive process, especially with follicular hyperplasia and clusters of monocytoid B cells, such as seen in toxoplasmosis, HIV infections, or Epstein–Barr virus (EBV) infections. Lack of monoclonal B-cell population by flow cytometric analysis is very helpful to exclude MZL. On immunohistochemical analysis, lack of aberrant phenotype of B cells (e.g., CD43 expression), monoclonal B cells, and/or plasma cells; disrupted FDC meshwork; and/or prominent colonization of follicles by neoplastic B cells (BCL2+) indicate reactive process. Reactive monocytoid B cells occur in rather well-demarcated clusters and are often BCL2−.

diffuse large B-Cell lymphoma An important morphologic consideration in the diagnosis of nodal MZL is its relationship with DLBCL, which is particularly problematic in cases with increased transformed large B cells. Nathwani et al. [5,13] described progression to DLBCL with >20% large cells. Recent data suggest that the presence of increased numbers of large cells is very common and may approach up to 50% (without sheet formation) [3,9]. Increased numbers of scattered large cells should not be confused with sheets of large cells, particularly if the latter is associated with an increase in proliferation (Ki-67 index). Presence of sheets of large cells and/or >50% large cells should raise the possibility of progression into DLBCL (DLBCL and nodal MZL). Kojima et al. [14] recently reported a series of 65 cases of nodal MZL, of which 20 cases had >50% large cells or sheets of large cells and were classified as nodal MZL and DLBCL. These cases had significantly worse outcome. The overall criteria for progression of nodal MZL into DLBCL are not well established, and there is no cutoff for proliferation (Ki-67) to aid in this distinction. low-grade B-Cell lymphomas FL may be difficult to differentiate from MZL, especially in cases of MZL with prominent follicle colonization, or FL with marginal zone differentiation or reversed pattern. The coexpression of CD10, BCL2, and BCL6 by neoplastic cells favors FL, but the subset of FL cases may be CD10 − or BCL2−. In difficult cases, correlation of fluorescence in situ

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B Hodgkin lymphoma with unusual monocytoid B-cell hyperplasia

FL, reversed pattern

CD30

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FigUre 10.11 Differential diagnosis of nodal marginal zone B-cell lymphoma. (A) Reactive lymph node with prominent germinal centers and foci of monocytoid B cells. (B) Reactive lymph node with mantle zone hyperplasia. (C) FL with a reversed pattern. (D) Classical Hodgkin lymphoma with unusual large foci of monocytoid B cells mimicking MZL (inset: higher magnification shows large cells that are positive for CD30).

hybridization (FISH) with polymerase chain reaction (PCR) testing for BCL2 rearrangement [t(14;18)] may be needed for final subclassification. Morphologic analysis of the BM core biopsy may also be helpful, as FL often shows prominent paratrabecular lymphoid infiltrate. MCL differs from MZL by positive CD5, SOX11, and BCL1 (cyclin D1) expression. It most often shows more monomorphic lymphoid infiltrate with scattered histiocytes and without large cell component typical for nodal MZL. SLL/CLL can be differentiated by the presence of proliferation centers [on low-power examination of hematoxylin and eosin (H&E) sections] and the coexpression of CD5 and CD23. Ki-67 index is usually much lower in SLL/CLL than in MZL, except for SLL/CLL cases with an increased number of prolymphocytes and/or paraimmunoblasts. Also plasmacytic differentiation is less common in SLL than in nodal MZL. LPL may be difficult to separate from MZL. High level of serum IgM protein favors LPL. Adenopathy is reported in ~15% of LPLs

and the histologic examination shows a mixed population of small lymphocytes, plasmacytoid B cells, and plasma cells. In contrast, MZL often shows a monocytoid appearance of neoplastic B cells. Analysis of the BM may also be helpful in the differential diagnosis of LPL. The lack of BM involvement favors LPL. LPL/Waldenström macroglobulinemia (WM) usually shows an interstitial BM involvement, whereas MZL (nodal and extranodal) often displays a nodular pattern and less often a paratrabecular pattern. Analysis of MYD88 L265P mutation is also helpful in the differential diagnosis, as it occurs in LPL and not MZL. Figure 10.12 compares the FDC meshwork in a normal lymph node, MCL, MZL, and FL. Note the prominent, asymmetrical disruption of the FDC in MZL. plasma Cell Neoplasm Extensive plasmacytic differentiation is often seen in MZL. Lack or very minimal number of B cells and/or aberrant

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CD21 in mantle cell lymphoma

CD21 in reactive lymph node

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B

CD21 in marginal zone B-cell lymphoma

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CD21 in follicular lymphoma

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FigUre 10.12 The pattern of CD21 immunohistochemical staining in reactive lymph node (A), mantle cell lymphoma (B), MZL (C), and FL (D). Colonization of follicles by neoplastic B cells in MZL causes disruption of FDCs (C).

phenotype of plasma cells (such as expression of BCL1, CD56, and/or CD117) would favor plasma cell neoplasm. Flow cytometric analysis may also be helpful. Identification of monoclonal B-cell population and monoclonal plasma cell population would favor MZL. Correlation of BM morphology, radiologic imaging data, and serum electrophoresis/ immunofixation may also be helpful. ptCl and aitl PTCL may show a prominent interfollicular (T-zone) pattern of involvement, mimicking MZL. Pleomorphic infiltrate with histiocytes and eosinophils and expression of pan-Tcell antigens (CD2, CD3, CD5, and/or CD7) point toward PTCL. AITL which mimicks MZL by its clear cell appearance, differs from MZL by immunophenotype (negative B-cell markers and positive PD-1, CD10, CD4 and pan-T-cell markers). secondary lymph Node involvement by MZl The differential diagnosis is based on the clinical data (e.g., presence of splenomegaly or history of MALT lymphoma would indicate secondary lymph node involvement by MZL).

other locations [15–25]. MALT lymphomas involve organs that acquire the lymphoid tissue after chronic inflammatory events, such as Helicobacter pylori-associated chronic gastritis, Campylobacter jejuni-associated immunoproliferative small intestine disease (IPSID), Chlamydia psittaci-induced inflammation (ocular area), or autoimmune disorders such as Hashimoto’s thyroiditis and lymphoepithelial sialadenitis (LESA)/Sjögren’s syndrome [26–28]. H. pylori is critical in the development of gastric MALT lymphoma, and eradication of H. pylori by antibiotics leads to regression of lymphoma in majority of cases. Patients with MALT lymphoma often have multiple sites of disease at initial diagnosis or during the clinical course and are usually not clonally related but arise independently, likely due to chronic antigenic stimulation, inducing oligoclonal B-cell proliferations and eventually a dominant B-cell clone [29]. MALT lymphoma is the most common type of lymphoma occurring in the ocular adnexae [24,30–32]. Infection with C. psittaci may predispose to the development of MALT lymphoma in ocular adnexae in some geographical areas, and antibiotic therapy induces regression of lymphoma [33–37].

Morphology extraNodal MargiNal ZoNe lyMphoMa IntroductIon Extranodal MZL (MALT lymphoma) is one of the most common lymphoma types. It occurs frequently in the stomach and other parts of the GI tract (Chapter 46), followed by the salivary gland (Chapter 43), lung (Chapter 42), head and neck, thyroid, orbit and ocular adnexae, skin (Chapter 44), breast, and

MALT lymphomas, regardless of location, share the same morphology and immunophenotype (Figure 10.13). MALT lymphoma is composed of morphologically heterogeneous lymphoid cells including small lymphocytes, lymphocytes with irregular nuclei resembling centrocytes, lymphocytes with abundant clear cytoplasm (monocytoid features), scattered immunoblasts, and centroblast-like cells. The lymphomatous cells arise in the marginal zone and extend

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Lambda

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Colon

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CD19

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FigUre 10.13 Extranodal MZL—Flow cytometry.

into follicles (follicle colonization) and between lymphoid follicles. Many cases display plasmacytic differentiation. Plasma cells may display Dutcher bodies. Apart from the presence of scattered large cells (at different proportions), most cases also show lymphoid follicles, which are infiltrated (colonized) by lymphomatous cells. In some occasions, only positive staining with FDC markers (CD21, CD23) demonstrates the site of the residual follicle. Some cases with prominent follicle colonization may mimic FL. The accompanying epithelial structures are often infiltrated by lymphocytes to form lymphoepithelial lesions. The lymphoepithelial lesions are most prominent in MZL of the salivary gland, thyroid, and lung, followed by the thymus and stomach [38]. The lymphoepithelial lesions are absent or sparse in MZL of the skin, breast, and ocular adnexae [38].

IMMunophenotype MALT lymphoma has non-specific phenotype (CD5–, CD10 –). CD43 expression is seen most often in MZL of the salivary gland, stomach, and upper aerodigestive tract, and monoclonal plasma cell infiltrate most often accompanies MZL of the breast, upper aerodigestive tract, skin,

and salivary glands [38]. Benign B cells in terminal ileum mucosa are often CD43+ [39].

large cell transForMatIon Transformed centroblast- or immunoblast-like cells may be present in variable numbers in MALT lymphoma, but when solid or sheet-like proliferations of transformed cells are present, the tumor should be diagnosed as DLBCL [11]. Transformation of MALT lymphoma to DLBCL is heralded by the emergence of increased numbers of large cells that form clusters (>20 cells) or sheet-like proliferations [15, 40–42]. Progression to high-grade lymphoma (DLBCL) occurs in ~8% of MALT lymphomas [43]. When diagnosing DLBCL in the stomach, the presence of lowgrade component (MALT lymphoma) should be noted in the report. Morphologic transformation of MZL to DLBCL occurs much more frequently in extranodal lymphomas of MALT type than in nodal and splenic MZLs [5,13,44–47]. Figure 10.14 shows an MZL in the lung, which 1 year later transformed into DLBCL (PCR tests revealed a clonal peak at the same location, which confirmed clonal evolution rather than secondary malignancy).

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60

Lung: marginal zone B-cell lymphoma 3200

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FigUre 10.14 Extranodal marginal zone B-cell lymphoma involving lung (A and B) with transformation into large B-cell lymphoma 1 year later (E and F). Molecular analysis confirmed a clonal IGH gene rearrangement, which is identical in both the primary pulmonary low-grade lymphoma and the transformed nodal large cell lymphoma (C and D).

cytogenetIcs/Fish and Molecular Features Four types of chromosomal translocation are commonly present in MZL (MALT type): t(11;18)(q21;q21)/API2– MALT1, t(1;14)(p22;q32)/BCL10–IGH, t(14;18)(q32;q21)/ IGH–MALT1, and t(3;14)(p14.1;q32)/FOXP1–IGH, which involve the activation of nuclear factor (NF)-κB. Other common genetic alterations include +3, +7, +12, +18, p53/ TP53 mutations, and p16 deletions with trisomy 3 [48–58]. Both chromosomal gains and losses are far more frequent in t(11;18)-negative cases than in t(11;18)-positive cases [59]. Recurrent chromosomal gains involving whole or major parts of a chromosome are seen for chromosomes 3, 12, 18, and 22 (23%, 19%, 19%, and 27%, respectively) [59]. Among the recurrent changes, gains at chromosomes 18q and 9q34 may be linked to pathogenesis of lymphoma. MALT lymphomas without translocations, but carrying trisomies of chromosomes 3, 7, 12, or 18, respond to H. pylori eradication, but they have higher risk of large cell transformation. The chromosome translocations occur at markedly variable incidences in MALT lymphoma of different sites, but are always mutually exclusive [20,60]. For chromosomal gain at 9q34, Zhou et al. [59] suggested that TRAF2 and CARD9 may be the target genes. Both genes have been shown to interact with BCL10 and activate NF-κB. Partial inactivation of the p53 gene may play a role in the development of low-grade MALT lymphoma [61], whereas complete inactivation may be associated with high-grade

transformation (similar to other hematologic malignancies) [49]. Homozygous deletions of p16 also play a role in large cell transformation [49]. Transformation of MALT lymphoma to DLBCL occurs in ~8% (more frequently in extranodal lymphomas of MALT type than in nodal and splenic MZLs) and is proceeded by the emergence of increased numbers of transformed blasts that form sheets or clusters on histologic examination [15,40,41,43]. Gastric MALT lymphoma usually remains localized for long periods within the tissue of origin. BM involvement at presentation is uncommon [19,23]. Disseminated disease appears to be more common in non-GI MALT lymphomas [43,62]. The t(11;18) leads to fusion of API2 gene at 11q21 and MALT1 gene at 18q21 [63–65]. The functional API2–MALT1 fusion product activates NF-κB. API2 is a member of inhibitor of apoptosis (IAP) gene family and is essential for suppression of caspase-dependent apoptosis [66]. In contrast to trisomy 3, which is nearly always accompanied by other numerical and structural chromosomal alterations, t(11;18) (q21;q21) usually occurs as a single abnormality [52,67]. Although amplifications of MALT1 are rare, gain of an extra copy of MALT1 gene is a frequent event in MALT lymphoma [57,59,68]. The t(11;18) occurs most often in lymphomas from the lung (~40%) and stomach (24%–40%), followed by the ocular adnexae (~15%) and orbit (~20%), and is rarely reported in the salivary gland, thyroid, and skin [69,70]. The API2– MALT1 fusion gene is characteristic for MALT lymphomas

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and is very rare in splenic MZL or DLBCL. In gastric MALT, t(11;18)(q21;q21) is strongly associated with failure to respond to treatment targeted at eradication of H. pylori [18,71,72]. This translocation, however, seems to be associated with a low risk of both the onset of additional genetic changes and the transformation into DLBCL [15,22,67,73,74], but exceptions may occur. Based on the response to H. pylori eradication therapy and presence of API2–MALT1 fusion gene, Inagaki et al. divided gastric MALT lymphomas into three groups: eradication-responsive and fusion-negative (group A), eradication-nonresponsive and fusion-negative (group B), and eradication-nonresponsive and fusion-positive (group C). The most common group A tumors are characterized by low clinical stage and superficial gastric involvement, and group C tumors by low H. pylori infection rate, low-grade histology, advanced clinical stage, and nuclear BCL10 expression. Group B tumors have frequent nodal involvement, deep gastric wall involvement, advance clinical stage, and sometimes an increased large cell component. The t(1;14) juxtaposes the BCL10 gene located on 1p22 to the immunoglobulin gene locus on 14q32 leading to deregulated expression of the oncogene and activation of NF-κB [55,75]. The t(1;14)(p22;q32) and its variant t(1;2)(p22;p12) occur in ~3% of MALT lymphomas. MALT lymphomas with t(1;14) tend to be at more advanced stage. The t(14;18)(q32;q21) that involves IGH gene on 14q32 and MALT1 gene on 18q21 (not BCL2 gene located on the same chromosome seen in a majority of FLs) can be identified by IGH–MALT1 FISH probe. It occurs in 5%–18% of MALT lymphomas, most often involving the lung, liver, skin, ocular adnexae, and salivary gland, but not the spleen, stomach, or GI tract [60,75]. All of the MALT lymphomas featuring the t(14;18)(q32;q21) are negative for t(11;18)(q21;q21) by reverse transcriptase PCR (RT-PCR), but trisomies 3 and/or 18 are identified in one-third of cases [75]. The t(3;14)(p14;q32) fuses FOXP1 gene at 3p14 with IGH gene [58]. In a series reported by Streubel et al. [58], 10% of MALT lymphomas harbored t(3;14)(p14.1;q32) comprising tumors of the thyroid (3/6), ocular adnexae (4/20), and skin (2/20), whereas the tumors of the stomach, spleen, and lung were negative. Most of the t(3;14)(p14.1;q32)+ MALT lymphomas harbored additional genetic abnormalities, such as trisomy 3 [58].

dIFFerentIal dIagnosIs Identification of clonal B-cells and/or plasma cells by immunohistochemistry, PCR, and/or flow cytometry helps to diagnose extranodal MALT lymphoma. Other features, which favor lymphoma over reactive process, include prominent lymphoepithelial lesions, plasma cells with Dutcher bodies, lymphoid cells with cytologic atypia, prominent monocytoid appearance of B cells, prominent follicle colonization by neoplastic B cells, aberrant expression of CD43 by B cells, and marked predominance of B cells over T cells [25,42,76]. Another helpful feature suggestive of MALT lymphoma is the presence of small B cells infiltrating into the upper parts

of the mucosa away from lymphoid follicles [25]. Infiltration or splitting of the muscularis mucosae by lymphoid nodules is often seen in MALT lymphoma but is not specific. Extranodal MZLs (MALT type) and their differential diagnosis are described in more detail in Chapters 42 (pulmonary MALT), 43 (salivary gland MALT), 44 (cutaneous MZL), 45 (splenic MZL), and 46 (gastric MALT).

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265 68. Streubel B, et al. Translocation t(11;18)(q21;q21) is not predictive of response to chemotherapy with 2CdA in patients with gastric MALT lymphoma. Oncology, 2004. 66(6):476–80. 69. Ye H, et al. MALT lymphoma with t(14;18)(q32;q21)/IGHMALT1 is characterized by strong cytoplasmic MALT1 and BCL10 expression. J Pathol, 2005. 205(3):293–301. 70. Streubel B, et al. Variable frequencies of MALT lymphomaassociated genetic aberrations in MALT lymphomas of different sites. Leukemia, 2004. 18(10):1722–6. 71. Liu H, et al. Resistance of t(11;18) positive gastric mucosaassociated lymphoid tissue lymphoma to Helicobacter pylori eradication therapy. Lancet, 2001. 357(9249):39–40. 72. Liu H, et al. T(11;18) is a marker for all stage gastric MALT lymphomas that will not respond to H. pylori eradication. Gastroenterology, 2002. 122(5):1286–94. 73. Rosenwald A, et al. Exclusive detection of the t(11;18)(q21;q21) in extranodal marginal zone B cell lymphomas (MZBL) of MALT type in contrast to other MZBL and extranodal large B cell lymphomas. Am J Pathol, 1999. 155(6):1817–21. 74. Dierlamm J, et al. Detection of t(11;18)(q21;q21) by interphase fluorescence in situ hybridization using API2 and MLT specific probes. Blood, 2000. 96(6):2215–8. 75. Streubel B, et al. T(14;18)(q32;q21) involving IGH and MALT1 is a frequent chromosomal aberration in MALT lymphoma. Blood, 2003. 101(6):2335–9. 76. Zukerberg LR, et al. Lymphoid infiltrates of the stomach. Evaluation of histologic criteria for the diagnosis of lowgrade gastric lymphoma on endoscopic biopsy specimens. Am J Surg Pathol, 1990. 14(12):1087–99.

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11

Follicular Lymphoma

contents Introduction ............................................................................................................................................................................... 267 Grading ..................................................................................................................................................................................... 268 Morphology............................................................................................................................................................................... 269 Lymph Nodes ....................................................................................................................................................................... 269 Bone Marrow and Blood ...................................................................................................................................................... 272 FL in Children and Young Adults ............................................................................................................................................. 273 Differential Diagnosis of PFL .............................................................................................................................................. 275 FL In Situ .................................................................................................................................................................................. 276 Immunophenotype .................................................................................................................................................................... 278 Extranodal FL ........................................................................................................................................................................... 280 Primary FL of the GI Tract................................................................................................................................................... 280 Primary FL of the Testis ....................................................................................................................................................... 280 Primary FL of the Thyroid Gland ........................................................................................................................................ 281 FL of Spleen ......................................................................................................................................................................... 281 Primary Cutaneous Follicle Center Cell Lymphoma ........................................................................................................... 282 Cytogenetics/FISH and Molecular Features ............................................................................................................................. 282 Transformation of FL ................................................................................................................................................................ 282 Disease Monitoring ................................................................................................................................................................... 283 Differential Diagnosis ............................................................................................................................................................... 285 Follicular Hyperplasia .......................................................................................................................................................... 285 Other Reactive Processes ..................................................................................................................................................... 285 Other B-Cell Lymphomas .................................................................................................................................................... 287 Nodular Lymphocyte Predominant HL ................................................................................................................................ 287 Mast Cell Disease................................................................................................................................................................. 287 References ................................................................................................................................................................................. 292

IntroductIon Follicular lymphoma (FL) is one of the most common type of malignant lymphomas in adults and is defined as a B-cell neoplasm of follicle center cell origin that usually displays a nodular pattern of growth (Figure 11.1), t(14;18) (q32;q21) by cytogenetic [or fluorescence in situ hybridization (FISH)], and coexpression of CD10, BCL2, and BCL6 by flow cytometry and/or immunohistochemistry [1–14]. FL is composed of a variable proportion of small and large centrocytes (cleaved cells) and centroblasts (large noncleaved cells). If diffuse areas of any size composed predominantly of blastic cells are present in case of FL, a diagnosis of concurrent diffuse large B-cell lymphoma (DLBCL) is also made. FL is a heterogeneous group of tumors with variable course, but the majority of cases have an indolent and slowly progressive clinical course with relatively long median survival, good response to initial treatment, and a continuous pattern of relapses, sometimes followed by histologic transformation into high-grade lymphoma [7,8,15–19]. Prognosis

is closely related to the extent of the disease at the time of diagnosis. The FL International Prognostic Index (FLIPI) is a strong predictor of outcome [7]. Approximately 65% of patients are in stage III or IV at the time of diagnosis [20]. Bone marrow (BM) involvement, which often shows a characteristic paratrabecular distribution, is found in ~40% of patients and B symptoms in 17% of patients. Long-term survival is relatively high when the disease is diagnosed in stage I or II [21]. With the current therapy, the expected median survival is ~8–10 years [15]. Adverse prognostic factors in FL include age >60 years, Ann Arbor stages III and IV, hemoglobin level 4, high number of extranodal involvement sites, high serum beta-2-microglobulin level, poor performance status, high erythrocyte sedimentation rate, and high serum LDH level [7,22–26]. The peripheral blood absolute lymphocyte count (ALC) at the time of diagnosis of FL was identified as a predictor for overall survival: ALC ≥1.0 × 109/L predicted a longer overall survival versus ALC 267

IV

268

Grading

Prominent nodular pattern

Vague nodular pattern

IV A

B Nodules of different sizes

C

Irregular nodules

D

FIGure 11.1 FL—Lymph node. (A) FL with a prominent nodular pattern; (B) FL with vague nodularity; (C) FL with nodules of different sizes; (D) FL with irregular nodules.

25% (now recognized as  areas of DLBCL) have a worse prognosis than purely follicular cases [29,30]. The presence of more than six chromosomal breaks and a complex karyotype has been shown to be associated with a poor outcome; in addition, del(6q23–26), del(17p) and mutations in TP53 as well as −1p, −12, +18p, and +Xp confer a worse prognosis and a shorter time to transformation [31,32]. In selected stage I and II follicular non-Hodgkin lymphoma (NHL) patients, deferred therapy is an acceptable approach; more than half of patients in the series reported by Advani et al. [16] remained untreated at a median of six or more years, and survival was comparable to that seen in reports with immediate treatment. Allogeneic BM transplant results in a long-term disease-free survival for ~50% of patients with advanced FL [33]. The influence of BM biopsy histology on prognosis and management of FL remains controversial. Extensive BM involvement, however, is a significant predictor of poor survival of patients with grade 1 and 2 FLs [34].

The survival rate is higher in extranodal FL than in nodal FL. Patients with primary cutaneous FL have a more favorable long-term prognosis than those with equivalent nodal disease [35]. FLs in noncutaneous extranodal sites have a similar favorable outcome [36]. The majority of extranodal FLs do not harbor t(14;18) [35,36]. In FL of the gastrointestinal tract (GI), the estimated 5-year disease-free survival was 62%, and the median disease-free survival is 69 months [37].

GradInG FL is graded based on the proportion of centroblasts (small and large) in 10 neoplastic follicles (Figure 11.2; Table 11.1), expressed per 40× high-power microscopic field (hpf). Grade 1 and 2 cases have a marked predominance of centrocytes and only a few centroblasts (grade 1 = 0–5 centroblasts/hpf; grade 2 = 6–15 centroblasts/hpf). Since grades 1 and 2 represent a continuum and are both clinically indolent, the current WHO classification does not encourage the distinction between grades 1 and 2, and suggests reporting “grade 1-2 of 3” [11]. Grade 3 cases have >15 centroblasts/hpf. Grade 3 FL is a heterogeneous group, which may be further subdivided based on the proportion of centrocytes into 3A (with centrocytes) and 3B (without centrocytes) [6,11,38,39]. If distinct areas of grade 3 FL are present in a biopsy of an otherwise grade 1–2 FL, a separate diagnosis of grade 3 FL should also be made, and the approximate amount of each grade reported [11].

269

Follicular Lymphoma

Grade 2

Grade 1

IV B

A Grade 3A

C

Grade 3B

D

FIGure 11.2 FL—Grading. (A) Low-grade FL (grade 1) is composed predominantly of small cells with irregular nuclei; (B) intermediate grade FL (grade 2) has a mixture of small (centrocytes) and larger (centroblasts) lymphocytes; (C and D) high-grade FL (grade 3) has predominantly large lymphocytes. Depending on the presence of few scattered small lymphocytes, grade 3 FL can be subdivided into 3A (small cells present) and 3B (small cells absent).

taBle 11.1 Fl Grading Grading 1–2 (low grade) 1 2 3 3A 3B reporting of pattern Follicular Follicular and diffuse Focally follicular Diffuse

definition 0–15 centroblasts per hpf 0–5 centroblasts per hpf 6–15 centroblasts per hpf >15 centroblasts per hpf Centrocytes present Solid sheets of centroblasts proportion of Follicles (%) >75 25–75