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Bone Marrow Biopsy Pathology: Christine Beham-Schmid Annette Schmitt-Graeff

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Essentials of Diagnostic Pathology

Series Editor: Farid Moinfar

Christine Beham-Schmid
Annette Schmitt-Graeff

Bone Marrow Biopsy

Pathology
A Practical Guide
Essentials of Diagnostic Pathology
More information about this series at: http://www.springer.com/series/8171
Christine Beham-Schmid
Annette Schmitt-Graeff

Bone Marrow Biopsy

Pathology
A Practical Guide
Christine Beham-Schmid Annette Schmitt-Graeff
Institute of Pathology Albert-Ludwigs-University of Freiburg
Medical University Graz Freiburg
Graz Germany
Austria

Series Editor
Farid Moinfar
Department of Pathology, Ordensklinikum Linz/Hospital of the Sisters of Charity
Department of Pathology, Medical University of Graz
Graz, Austria

ISSN 2194-6256 ISSN 2194-6264 (electronic)

Essentials of Diagnostic Pathology
ISBN 978-3-662-60307-9 ISBN 978-3-662-60309-3 (eBook)
https://doi.org/10.1007/978-3-662-60309-3

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed
to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer-Verlag GmbH, DE part of Springer Nature.
The registered company address is: Heidelberger Platz 3, 14197 Berlin, Germany
Preface

When I (C.B-S) was asked by Prof. F. Moinfar to write a book on bone marrow pathology, I
was hesitant in view of the abundance of excellent textbooks on this topic. However, the fact
that this book should be intended as a practical guide dominated rather by illustrations than by
text, and the promise of Prof. Annette Schmitt-Graeff to act as a coauthor, at least encouraged
me to start this project.
As a young internship doctor in the small public hospital in Oberndorf/Salzburg, I (C.B-S)
was stimulated by Primarius Dr. K. Mittermayer to have a look on bone marrow aspirates. This
resulted in my interest in hematology, which was deepened when I consecutively started a
training in pathology with focus on hematopathology at the Institute of Pathology, Faculty of
Medicine, University of Graz. During this time, I received intensive training in bone marrow
biopsy pathology in Munich (Prof. Burkhardt) and Tel Aviv (Bertha Frisch) and in lymphoma
diagnostics in London (P.G. Isaacson). Up to now, I have seen ten thousands of bone marrow
biopsies and gained a lot of experience. Since the knowledge of clinical data is essential for
exact pathohistological diagnosis of bone marrow biopsies, a close cooperation with the clini-
cal colleagues is mandatory.
My (A.S-G) enthusiasm for bone marrow (BM) diagnostics dates back to my residency in
hematology/oncology at the Departments of Internal Medicine, University Hospitals of
Cologne and Essen, Germany. I was keen on evaluating blood and BM cytology. I also realized
the impact of BM and lymph node biopsies on therapeutic strategies for our patients suffering
from myeloid or lymphoproliferative neoplasms. This appreciation was the rationale to pursue
a residency and a fellowship in anatomical pathology at the University Hospital Düsseldorf,
Germany, where I established histologic BM assessment. My research activities were on the
cytoskeleton including BM stroma with Prof. Dr. Dr. h.c. Giulio Gabbiani, University Hospital
Geneva, Switzerland, where I assumed the position as consultant. There and at the Berlin
Reference Center for Lymph Node Pathology of Prof. Dr. Dr. h.c. Harald Stein I was princi-
pally in charge of BM pathology including supervising residents in this field.
Later, I joined the faculty at the Medical Center, University Freiburg, Germany, where I was
appointed Professor of Hematopathology and GI Pathology. My activities were focused on
integrating morphological, immunophenotypic, and molecular aspects of hematological neo-
plasms and primary immunodeficiency disorders with the late Prof. Dr. Paul Fisch in close
clinico-pathologic cooperation.
As previous president of the German Division of the IAP (GDIAP) and actual member of
the General IAP and GDIAP Education committees, I have taught and directed numerous
hematopathology courses of the IAP nationally and internationally. In the context of my active
involvement in education and teaching, I am delighted to contribute to this textbook with a mix
of basic information and challenging diagnostic cases.
This book is intended as a practical guide in the complex field of bone marrow pathology.
We hope it will turn out helpful in the daily diagnostic work.

June 2020
Graz, Austria Christine Beham-Schmid
Freiburg, Germany Annette Schmitt-Graeff

v
Acknowledgments

I would like to thank my coauthor and friend Annette Schmitt-Graeff, without her support, my
own contributions for this book would not have been possible.
Moreover, I am deeply indebted to Prof. Farid Moinfar for giving me the opportunity to
write this book with my coauthor.
My special acknowledgment is also attributed to my teachers in hematology and hematopa-
thology Prim. Dr. K. Mittermayer, Oberndorf/Salzburg, Prof. Dr. R. Burkhardt, Munich/
Germany, Prof. Dr. B. Frisch, Tel Aviv/Israel, Prof. P.G. Isaacson, London/G.B., and Prof. Dr.
J. Thiele, Cologne/Germany.
In addition, I would like to recognize the excellent work of the team of the laboratory of
hematopathology and the photographers, Institute of Pathology, Medical University of Graz,
Austria.
Last but not least, I am most grateful to my husband Alfred and my son Clemens for their
never-ending patience and support during this project.

Christine Beham-Schmid

First, I would like to acknowledge the great dedication of my coauthor Prof. Dr. Christine
Beham-Schmid, Graz, and the series Editor Prof. Dr. Farid Moinfar, Linz, Austria, to the proj-
ect of our book.
I express my deep appreciation to many experts who inspired me with their devotion to
hematopathology and hematology. In particular, I want to mention Prof. Dr. Dr.hc Harald
Stein, Berlin, and Prof. Dr. Juergen Thiele, Cologne/Hannover, Germany, and Prof. Dr. Kristin
Henry, London, G.B. Special thanks go to Prof. Dr. Rüdiger Hehlmann who founded the
European LeukemiaNet (ELN). I would also like to acknowledge Prof. Dr. Attilio Orazi, El
Paso, and Prof. Dr. Daniel A. Arber, Chicago, USA, for their exciting investigations and teach-
ing in hematopathology.
I am especially indebted to Prof. Dr. Robert Zeiser, Dr. Dietmar Pfeifer, Dr. Milena Pantic,
and Dr. Justina Rawluk, Department of Hematology and Oncology, Medical Center, University
Freiburg, Germany, for their support and helpful contribution to molecular analyses, cytoge-
netics, and cytology of myeloid neoplasms.
Lastly, I would like to thank my mother Margret Gräff, my husband Eberhard Schmitt, our
son Rafael J. P. Schmitt, and my daughter-in-law Zelda Othenin for their patience and love.

Annette Schmitt-Graeff

vii
Contents

1 Normal Bone Marrow�� 1

Christine Beham-Schmid
1.1 Introduction�� 1
1.1.1 Bone Components�� 2
1.2 Cellularity of the Marrow �� 6
1.2.1 Topography �� 8
1.3 Cellular Components of the Bone Marrow �� 10
1.3.1 Hematopoiesis�� 10
1.3.2 Erythrocytopoiesis�� 10
1.3.3 Granulocytopoiesis �� 12
1.3.4 Monocytopoiesis and Macrophages�� 14
1.3.5 Megakaryocytopoiesis�� 16
1.3.6 Lymphocytes �� 19
1.3.7 Plasma Cells�� 22
1.3.8 Mast Cells �� 22
1.3.9 Bone Marrow Stroma�� 24
References�� 26
2 Non-neoplastic Conditions with Quantitative and Qualitative
Changes in Hematopoiesis�� 27
Christine Beham-Schmid
2.1 Common Non-neoplastic Erythroid Lineage Disorders �� 27
2.1.1 Common Examples of Anemia �� 27
2.2 Acquired Anemias�� 28
2.2.1 Anemia of Chronic Disease (ACD)�� 28
2.2.1.1 Caution�� 28
2.2.2 Iron Deficiency Anemias (IDA)�� 30
2.2.2.1 Caution�� 30
2.2.3 Megaloblastic Anemias�� 32
2.2.3.1 Caution�� 32
2.2.4 Hemolytic Anemia�� 34
2.2.4.1 Caution�� 34
2.2.5 Aplastic Anemia�� 36
2.2.5.1 Caution�� 38
2.3 Bone Marrow Biopsy in Collagen Vascular Diseases (CVD),
Especially in Systemic Lupus Erythematosus (SLE)�� 40
2.3.1 Caution�� 42
2.4 Bone Marrow Involvement in Castleman’s Disease �� 44
2.4.1 Caution�� 44
2.5 Non-neoplastic Conditions with Quantitative and Qualitative
Changes in Granulopoiesis �� 49
2.5.1 Severe (Acquired) Neutropenia�� 49
2.5.1.1 Caution�� 49

ix
x Contents

2.6 Kostmann Disease (Infantile Genetic Agranulocytosis,

Severe Congenital Neutropenia) �� 51
2.6.1 Caution�� 51
2.7 Leukemoid Reaction (LR)�� 53
2.7.1 Caution�� 53
2.8 Granulopoiesis with Increased Eosinophils�� 54
2.8.1 Caution�� 54
2.9 Quantitative and Qualitative Changes in Megakaryocytopoiesis�� 57
2.9.1 Morphologic Abnormalities of Megakaryocytes�� 57
2.9.2 Megakaryocytic Emperipolesis�� 57
2.10 Micromegakaryocytes and Hypolobulated/Nonlobulated
Megakaryocytes�� 58
2.11 Enlarged Megakaryocytes with Hyperlobulated Nuclei �� 61
2.12 Megakaryocytes with Naked Nuclei �� 62
2.13 Clustering of Megakaryocytes�� 63
2.13.1 Caution�� 63
2.14 Intrasinusoidal Megakaryocytes �� 66
2.15 Paratrabecular Location of Megakaryocytes�� 68
2.16 Gray Platelet Syndrome�� 70
2.17 Bone Marrow Alterations After Chemotherapy/Stem
Cell Transplantation�� 72
2.17.1 Caution�� 74
2.18 Monoclonal Antibody Treatment (Rituximab)�� 76
2.18.1 Caution�� 76
2.19 Bone Marrow Alterations after Bone Marrow Transplantation�� 77
2.19.1 Caution�� 77
References�� 79
3 Bone Changes Found in Bone Marrow Biopsies �� 81
Christine Beham-Schmid
3.1 Osteodystrophy �� 81
3.1.1 Renal Osteodystrophy�� 81
3.1.1.1 Caution�� 81
3.1.2 Hyperparathyroid Osteodystrophy�� 83
3.1.2.1 Caution�� 83
3.1.3 Osteopenia�� 85
3.1.3.1 Caution�� 85
3.1.4 Osteomalacia�� 87
3.1.4.1 Caution�� 87
3.1.5 Osteopetrosis (Marble Bone Disease)�� 88
3.1.5.1 Caution�� 88
3.1.6 Paget’s Disease of Bone�� 89
3.1.6.1 Caution�� 89
References�� 90
4 Non-hematological Malignancies in the Bone Marrow�� 91
Christine Beham-Schmid
4.1 Caution�� 92
References�� 102
5 Increase of Plasma Cells, Reactive and Neoplastic �� 103
Christine Beham-Schmid
5.1 Reactive Plasmacytosis �� 103
5.1.1 Caution�� 103
Contents xi

5.2 Monoclonal Gammopathy of Undetermined Significance (MGUS)�� 105

5.2.1 Plasma Cell Myeloma (MM)�� 105
5.2.2 Epidemiology�� 105
5.2.3 Morphology�� 105
5.2.4 Immunohistochemistry �� 110
5.2.5 Genetic Abnormalities�� 110
5.2.6 Caution�� 110
5.3 Plasma Cell Myeloma with Amyloid Deposition �� 111
5.3.1 Caution�� 111
References�� 113
6 Inflammatory and Infectious Diseases �� 115
Christine Beham-Schmid
6.1 Infectious Diseases�� 120
6.1.1 Hemophagocytic Syndrome�� 120
6.1.1.1 Caution�� 123
6.2 Visceral Leishmaniasis�� 124
6.2.1 Caution�� 125
6.3 Parvovirus B19 Infection�� 126
6.3.1 Caution�� 127
6.4 Infection with Mycobacterium Tuberculosis�� 128
References�� 130
7 Malignant Lymphomas�� 131
Christine Beham-Schmid
7.1 Introduction�� 131
7.2 Chronic Lymphocytic Leukemia (CLL) �� 134
7.2.1 Epidemiology�� 134
7.2.2 Morphology�� 134
7.2.3 Transformation of CLL�� 134
7.2.4 Immunohistochemistry �� 134
7.2.5 Cytogenetic Abnormalities and Molecular Characteristics�� 134
7.2.6 Caution�� 143
7.3 B-Cell Prolymphocytic Leukemia (B-PLL)�� 144
7.3.1 Epidemiology�� 144
7.3.2 Morphology�� 144
7.3.3 Immunohistochemistry �� 144
7.3.4 Cytogenetic Abnormalities and Molecular Characteristics�� 144
7.3.5 Caution�� 147
7.4 Lymphoplasmacytic Lymphoma (LPL)�� 148
7.4.1 Epidemiology�� 148
7.4.2 Morphology�� 148
7.4.3 Immunohistochemistry �� 148
7.4.4 Cytogenetic Abnormalities and Molecular Characteristics�� 148
7.4.5 Caution�� 155
7.5 Mantle Cell Lymphoma (MCL)�� 156
7.5.1 Epidemiology�� 156
7.5.2 Morphology�� 156
7.5.3 Immunohistochemistry �� 156
7.5.4 Cytogenetic Abnormalities and Molecular Characteristics�� 156
7.5.5 Caution�� 159
7.6 Hairy Cell Leukemia (HCL) �� 162
7.6.1 Epidemiology�� 162
7.6.2 Morphology�� 162
xii Contents

7.6.3 Immunohistochemistry �� 162

7.6.4 Cytogenetic Abnormalities and Molecular Characteristics�� 162
7.6.5 Caution�� 168
7.7 Follicular Lymphoma (FL) �� 169
7.7.1 Epidemiology�� 169
7.7.2 Morphology�� 169
7.7.3 Immunohistochemistry �� 170
7.7.4 Cytogenetic Abnormalities and Molecular Characteristics�� 170
7.7.5 Caution�� 172
7.8 Splenic Marginal Zone Lymphoma (SMZL)�� 173
7.8.1 Epidemiology�� 173
7.8.2 Morphology�� 173
7.8.3 Immunohistochemistry �� 173
7.8.4 Cytogenetic Abnormalities and Molecular Characteristics�� 173
7.8.5 Caution�� 177
7.9 Extranodal Marginal Zone Lymphoma of Mucosa-Associated
Lymphoid Tissue (MALT Lymphoma) �� 178
7.9.1 Epidemiology�� 178
7.9.2 Morphology�� 178
7.9.3 Immunohistochemistry �� 178
7.9.4 Cytogenetic Abnormalities and Molecular Characteristics�� 178
7.9.5 Caution�� 182
7.10 Diffuse Large B-Cell Lymphoma (DLBCL)�� 183
7.10.1 Epidemiology�� 183
7.10.2 Morphology�� 183
7.10.3 Immunohistochemistry �� 183
7.10.4 Cytogenetic Abnormalities and Molecular Characteristics�� 183
7.10.5 Caution�� 187
7.11 Intravascular Large B-Cell Lymphoma�� 188
7.11.1 Caution�� 188
7.12 Burkitt Lymphoma (BL) �� 190
7.12.1 Epidemiology�� 190
7.12.2 Morphology�� 190
7.12.3 Immunohistochemistry �� 190
7.12.4 Cytogenetic Abnormalities and Molecular Characteristics�� 190
7.12.5 Caution�� 192
7.13 T-Cell Chronic Lymphocytic Leukemia (T-CLL)/T-Cell
Prolymphocytic Leukemia (T-PLL)�� 193
7.13.1 Epidemiology�� 193
7.13.2 Morphology�� 193
7.13.3 Immunohistochemistry �� 193
7.13.4 Cytogenetic Abnormalities and Molecular Characteristics�� 193
7.13.5 Caution�� 197
7.14 T-Cell Large Granular Lymphocytic Leukemia (T-LGLL)�� 198
7.14.1 Epidemiology�� 198
7.14.2 Morphology�� 198
7.14.3 Immunohistochemistry �� 198
7.14.4 Caution�� 202
7.15 Hepatosplenic T-Cell Lymphoma (HSTL)�� 203
7.15.1 Epidemiology�� 203
7.15.2 Morphology�� 203
7.15.3 Immunohistochemistry �� 203
Contents xiii

7.15.4 Cytogenetic Abnormalities and Molecular Characteristics�� 203

7.15.5 Caution�� 206
7.16 Mycosis Fungoides (MF)/Sezary Syndrome (SS)�� 207
7.16.1 Epidemiology�� 207
7.16.2 Morphology�� 207
7.16.3 Immunohistochemistry �� 207
7.16.4 Cytogenetic Abnormalities and Molecular Characteristics�� 207
7.16.5 Caution�� 209
7.17 Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL, NOS) �� 210
7.17.1 Epidemiology�� 210
7.17.2 Morphology�� 210
7.17.3 Immunohistochemistry �� 210
7.17.4 Cytogenetic Abnormalities and Molecular Characteristics�� 210
7.17.5 Caution�� 212
7.18 Angioimmunoblastic T-Cell Lymphoma (AITL)�� 213
7.18.1 Epidemiology�� 213
7.18.2 Morphology�� 213
7.18.3 Immunohistochemistry �� 213
7.18.4 Cytogenetic Abnormalities and Molecular Characteristics�� 213
7.18.5 Caution�� 215
7.19 Anaplastic Large Cell Lymphoma (ALCL)�� 216
7.19.1 Epidemiology�� 216
7.19.2 Morphology�� 216
7.19.3 Immunohistochemistry �� 216
7.19.4 Cytogenetic Abnormalities and Molecular Characteristics�� 216
7.19.5 Caution�� 219
7.20 Adult T-Cell Leukemia/Lymphoma (ATLL)�� 220
7.20.1 Epidemiology�� 220
7.20.2 Morphology�� 220
7.20.3 Immunohistochemistry �� 220
7.20.4 Cytogenetic Abnormalities and Molecular Characteristics�� 220
7.21 Hodgkin Lymphoma �� 223
7.21.1 Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHD)�� 223
7.21.1.1 Epidemiology�� 223
7.21.1.2 Morphology�� 223
7.21.1.3 Immunohistochemistry�� 223
7.21.1.4 Cytogenetic Abnormalities and Molecular
Characteristics�� 223
7.21.1.5 Caution�� 225
7.22 Classical Hodgkin Lymphoma (cHL) �� 226
7.22.1 Epidemiology�� 226
7.22.2 Morphology�� 226
7.22.3 Immunohistochemistry �� 226
7.22.4 Cytogenetic Abnormalities and Molecular Characteristics�� 226
7.22.5 Caution�� 231
References�� 231
8 Mastocytosis�� 235
Annette Schmitt-Graeff
8.1 Epidemiology and Clinical Features�� 236
8.2 Morphologic and Immunohistochemical Features of Bone
Marrow Involvement by SM �� 236
xiv Contents

8.3 Genetics�� 237

8.4 Caution�� 237
References�� 251
9 Myeloproliferative Neoplasm (MPN) �� 253
Christine Beham-Schmid
9.1 Introduction�� 253
9.2 Chronic Myeloid Leukemia (CML), BCR-ABL1-Positive�� 253
9.2.1 Epidemiology�� 253
9.2.2 Morphology�� 253
9.2.3 Chronic Phase (CP)�� 253
9.2.3.1 Morphologic Findings with TKI Therapy�� 254
9.2.3.2 Disease Progression of CML �� 254
9.2.4 Accelerated Phase (AP)�� 254
9.2.5 Blast Crisis (BC) �� 254
9.2.6 Immunohistochemistry �� 255
9.2.7 Genetics�� 255
9.2.8 Caution�� 267
9.3 Chronic Neutrophilic Leukemia (CNL)�� 269
9.3.1 Epidemiology�� 269
9.3.2 Morphology�� 269
9.3.3 Immunohistochemistry �� 269
9.3.4 Genetics�� 269
9.3.5 Caution�� 271
9.4 Polycythemia Vera (PV)�� 272
9.4.1 Epidemiology�� 272
9.4.2 Morphology�� 272
9.4.3 “Spent Phase” of PV, Post-polycythemic Myelofibrosis,
and Myeloid Metaplasia�� 272
9.4.4 Caution�� 274
9.5 Primary Myelofibrosis (PMF)�� 275
9.5.1 Epidemiology�� 275
9.5.2 Morphology�� 275
9.5.3 PMF, Prefibrotic/Early Stage�� 275
9.5.4 PMF, Overt Fibrotic Stage�� 278
9.5.5 Immunohistochemistry �� 278
9.5.6 Caution�� 280
9.6 Essential Thrombocythemia (ET) �� 281
9.6.1 Epidemiology�� 281
9.6.2 Morphology�� 281
9.6.3 Immunohistochemistry �� 281
9.6.4 Caution�� 284
9.7 Myeloproliferative Neoplasm, Unclassifiable (MPN-U)�� 287
9.7.1 Immunohistochemistry �� 287
9.7.2 Caution�� 289
9.8 Monocytoid Progression of MPN and Monocytosis in MPN �� 290
9.9 Chronic Eosinophilic Leukemia, NOS (CEL-NOS)�� 292
9.9.1 Epidemiology�� 292
9.9.2 Morphology�� 292
9.9.3 Immunohistochemistry and Genetics�� 292
9.9.4 Caution�� 294
References�� 295
Contents xv

10 Myeloid/Lymphoid Neoplasms with Eosinophilia

and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2�� 297
Annette Schmitt-Graeff
10.1 Epidemiology and Clinical Features�� 297
10.2 Morphology�� 298
10.3 Genetics�� 298
10.4 Caution�� 299
References�� 309
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)�� 311
Annette Schmitt-Graeff
11.1 Introduction�� 311
11.2 Chronic Myelomonocytic Leukemia (CMML)�� 311
11.2.1 Epidemiology�� 311
11.2.2 Clinical Features�� 312
11.2.3 Bone Marrow and Blood Findings�� 312
11.2.4 Immunohistochemistry Applied to BM Trephine Biopsies�� 312
11.2.5 Molecular Pathogenesis�� 312
11.2.6 Prognosis �� 312
11.2.7 Caution�� 313
11.2.7.1 Transition to AML�� 313
11.2.7.2 Monocytosis in Classical MPN and Other
Myeloid Neoplasms�� 313
11.2.7.3 Secondary Acquisition of MPN-Typical Genetic
and Hematological Features in CMML�� 313
11.2.7.4 Benign Conditions�� 313
11.3 Juvenile Myelomonocytic Leukemia (JMML) �� 325
11.3.1 Epidemiology�� 325
11.3.2 Clinical and Laboratory Features�� 325
11.3.3 Morphology�� 326
11.3.4 Immunohistochemistry�� 329
11.3.5 Genetics�� 329
11.3.6 Caution�� 329
11.3.6.1 Viral infections�� 329
11.3.6.2 Osteopetrosis �� 329
11.3.6.3 Noonan Syndrome�� 330
11.4 Atypical Chronic Myeloid Leukemia, BCR-ABL1-Negative�� 331
11.4.1 Epidemiology�� 331
11.4.2 Clinical and Laboratory Features�� 331
11.4.3 Morphology�� 331
11.4.4 Immunohistochemistry�� 331
11.4.5 Genetics�� 331
11.4.6 Caution�� 331
11.5 Myelodysplastic/Myeloproliferative Neoplasm,
Unclassifiable (MDS/MPN-U)�� 334
11.5.1 Epidemiology�� 334
11.5.2 Clinical and Laboratory Features�� 334
11.5.3 Morphology�� 334
11.5.4 Genetics�� 334
11.5.5 Caution�� 334
xvi Contents

11.6 Myelodysplastic/Myeloproliferative Neoplasm with Ring

Sideroblasts and Thrombocytosis (MDS/MPN-RS-T) �� 337
11.6.1 Epidemiology�� 337
11.6.2 Morphology�� 337
11.6.3 Genetics�� 337
11.6.4 Caution�� 337
References�� 340
12 Myelodysplastic Syndromes (MDS) �� 343
Annette Schmitt-Graeff
12.1 Introduction�� 343
12.2 Epidemiology�� 344
12.3 Clinical Features �� 344
12.4 Morphology and Immunohistochemistry: Dysplasia
Assessment in MDS�� 344
12.4.1 Special Stains in MDS�� 345
12.4.2 Dysplastic Features Observed in Peripheral Blood and
Bone Marrow Specimens�� 345
12.4.2.1 The Peripheral Blood in MDS�� 345
12.4.2.2 The Bone Marrow in MDS: Dysplasia Involving the
Hematopoietic Lineages�� 345
12.4.2.3 The Bone Marrow Core Biopsy in MDS: Bone Marrow
Architecture and Stromal Components �� 347
12.5 Genetics in MDS�� 347
12.5.1 Chromosomal Abnormalities�� 347
12.5.2 Somatic Mutations�� 348
12.5.3 Prognostic Relevance of Genetic Alterations in MDS�� 348
12.5.4 Selected Genetic Profiles Associated
with Prognostic Relevance in MDS�� 348
12.6 Caution�� 349
References�� 380
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage
and Related Malignancies �� 383
Annette Schmitt-Graeff
13.1 Introduction�� 383
13.1.1 Diagnostic Approach to Patients with Suspected AL�� 383
13.2 Acute Myeloid Leukemia; General Aspects �� 383
13.2.1 Classification �� 383
13.2.2 Epidemiology�� 385
13.2.3 Morphology and Cytochemistry of Blood and Bone
Marrow Smears �� 385
13.2.4 Immunophenotype�� 385
13.2.5 Genetics�� 386
13.3 AML with Recurrent Genetic Abnormalities�� 388
13.3.1 Acute Promyelocytic Leukemia with PML-RARA�� 388
13.3.1.1 Genetics�� 388
13.3.1.2 Immunophenotype�� 388
13.3.1.3 Morphology�� 388
13.3.1.4 Caution�� 389
Contents xvii

13.3.2 Acute Myelogenous Leukemia (AML) with

inv(3)(q21.3;q26.2) or t(3;3)(q21.3;q26.2); GATA2; MECOM�� 394
13.3.2.1 Genetics�� 394
13.3.2.2 Immunophenotype�� 394
13.3.2.3 Morphology�� 394
13.3.2.4 Caution�� 394
13.3.3 AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1�� 397
13.3.3.1 Genetics�� 397
13.3.3.2 Immunophenotype�� 397
13.3.3.3 Morphology�� 397
13.3.3.4 Caution�� 397
13.3.4 Acute Myeloid Leukemia with inv(16)(p13.1q22) or
t(16;16)(p13.1q22); CBFB-MYH11�� 402
13.3.4.1 Epidemiology�� 402
13.3.4.2 Morphology�� 402
13.3.4.3 Immunophenotype�� 402
13.3.4.4 Genetics�� 402
13.3.4.5 Caution�� 402
13.3.5 Acute Myeloid Leukemia with Translocation
t(6;9)(p23;q34.1); DEK-NUP214�� 408
13.3.5.1 Epidemiology�� 408
13.3.5.2 Morphology�� 408
13.3.5.3 Immunophenotype�� 408
13.3.5.4 Genetics�� 408
13.3.6 AML with Translocation t(9;11)(p21.3;q23.3);
KMT2A-MLLT3�� 411
13.3.6.1 Epidemiology and Clinical Presentation�� 411
13.3.6.2 Morphology�� 411
13.3.6.3 Immunophenotype�� 411
13.3.6.4 Genetics�� 411
13.3.6.5 Caution�� 411
13.3.7 AML (Megakaryoblastic) with Translocation
t(1;22)(p13.3;q13.1); RBM15-MKL1�� 416
13.3.7.1 Epidemiology and Clinical Features �� 416
13.3.7.2 Morphology�� 416
13.3.7.3 Immunophenotype�� 416
13.3.7.4 Genetics�� 416
13.3.7.5 Caution�� 416
13.3.8 AML with Translocation t(9;22);BCR-ABL1�� 419
13.3.8.1 Epidemiology and Clinical Features �� 419
13.3.8.2 Morphology�� 419
13.3.8.3 Immunophenotype�� 419
13.3.8.4 Genetics�� 419
13.3.8.5 Caution�� 419
13.4 AML with Gene Mutations�� 422
13.4.1 AML with Mutated NPM1�� 422
13.4.1.1 Epidemiology and Clinical features�� 422
13.4.1.2 Morphology�� 422
13.4.1.3 Immunophenotype�� 422
13.4.1.4 Genetics�� 422
13.4.1.5 Caution�� 422
xviii Contents

13.4.2 AML with Biallelic Mutation of CEBPA�� 429

13.4.2.1 Epidemiology�� 429
13.4.2.2 Morphology�� 429
13.4.2.3 Immunophenotype�� 429
13.4.2.4 Genetics�� 429
13.4.2.5 Caution�� 429
13.4.3 AML with Mutated RUNX1�� 434
13.4.3.1 Epidemiology�� 434
13.4.3.2 Morphology�� 434
13.4.3.3 Immunophenotype�� 434
13.4.3.4 Genetics�� 434
13.5 AML with Myelodysplasia-Related Changes (AML-MRC)�� 437
13.5.1 Epidemiology�� 437
13.5.2 Morphology�� 437
13.5.3 Immunophenotype�� 437
13.5.4 Genetics�� 438
13.5.5 Caution�� 438
13.6 Therapy-Related Myeloid Neoplasms�� 442
13.6.1 Epidemiology and Etiology�� 442
13.6.2 Morphology�� 442
13.6.3 Immunophenotype�� 442
13.6.4 Genetics�� 442
13.7 AML, Not Otherwise Specified�� 447
13.7.1 AML with Minimal Differentiation�� 447
13.7.2 AML Without Maturation �� 450
13.7.3 AML with Maturation �� 452
13.7.4 Acute Myelomonocytic Leukemia�� 454
13.7.5 Acute Monoblastic and Monocytic Leukemia�� 458
13.7.6 Pure Erythroleukemia�� 461
13.7.6.1 Epidemiology�� 461
13.7.6.2 Morphology�� 461
13.7.6.3 Immunophenotype�� 461
13.7.6.4 Genetics�� 461
13.7.6.5 Caution�� 461
13.7.7 Acute Megakaryoblastic Leukemia�� 467
13.7.7.1 Epidemiology�� 467
13.7.7.2 Morphology�� 467
13.7.7.3 Immunophenotype�� 467
13.7.7.4 Genetics�� 467
13.7.7.5 Caution�� 467
13.7.8 Acute Basophilic Leukemia�� 470
13.7.8.1 Morphology�� 470
13.7.8.2 Immunophenotype�� 470
13.7.8.3 Genetics�� 470
13.7.8.4 Caution�� 470
13.7.9 Acute Panmyelosis with Myelofibrosis�� 473
13.7.9.1 Morphology and Immunophenotype�� 473
13.7.9.2 Genetics�� 473
13.7.9.3 Caution�� 473
Contents xix

13.8 Myeloid Sarcoma�� 476

13.8.1 Epidemiology and Clinical Features �� 476
13.8.2 Morphology and Immunophenotype�� 476
13.8.3 Genetics�� 476
13.8.4 Caution�� 476
13.9 Myeloid Neoplasms with Germline Predisposition�� 479
13.9.1 Epidemiology�� 479
13.9.2 Morphology and Immunophenotype�� 479
13.9.3 Genetics�� 479
13.9.4 Caution�� 479
13.10 Myeloid Proliferations Associated with Down Syndrome�� 485
13.10.1 Epidemiology and Clinical Findings�� 485
13.10.2 Morphology and Immunophenotype�� 485
13.10.3 Genetics�� 485
13.11 Blastic Plasmacytoid Dendritic Cell Neoplasm�� 490
13.11.1 Epidemiology and Clinical Features �� 490
13.11.2 Morphology and Immunophenotype�� 490
13.11.3 Genetics�� 490
13.11.4 Caution�� 490
13.12 Acute Leukemias of Ambiguous Lineage�� 494
13.12.1 Epidemiology and Clinical Features �� 494
13.12.2 Morphologic and Immunophenotypic Features�� 494
13.12.3 Genetics�� 495
13.12.4 Caution�� 495
13.13 Precursor Lymphoid Neoplasms: T-Lymphoblastic Leukemia/Lymphoma �� 499
13.13.1 Epidemiology and Clinical Features �� 499
13.13.2 Morphology and Immunophenotype�� 499
13.13.3 Genetics�� 500
13.13.4 Caution�� 500
13.14 Precursor Lymphoid Neoplasms: B-Lymphoblastic Leukemia/Lymphoma�� 513
13.14.1 Epidemiology and Clinical Findings�� 513
13.14.2 Morphology and Immunophenotype�� 513
13.14.3 Genetics�� 514
13.14.4 Caution�� 514
References�� 527
Normal Bone Marrow
1

Abbreviations hematopoiesis, which is present in skull, sternum, clavicle,

scapula, vertebrae, ribs, pelvic bone, and proximal sec-
BBL Berlin blue iron stain tions of hollow bone [3]. The term “marrow cellularity,”
BCR-ABL Breakpoint cluster region and Abelson tyro- used by hematologists and hematopathologists, refers to
sine kinase the actively producing compartment and does not include
BMB Bone marrow biopsy fatty tissue. Thus, aplastic marrow or hypoplastic marrow
CML Chronic myeloid leukemia shows complete or partial replacement by fat cells. Usually,
H&E Hematoxylin and eosin stain bone marrow biopsies are taken from the posterior iliac
HPF High power field crest [2]. Preferably, bone marrow examination should be
IHC Immunohistochemistry done in conjunction with clinical data (including question
ITP Idiopathic thrombocytopenia and/or indication for examination, previous illnesses,
MDS Myelodysplasia drugs) and complete blood count data and, ideally with
MGG May–Grünwald Giemsa stain bone marrow aspirate imprints or smears (in our experi-
MPO Myeloperoxidase ence, only in rare cases cytology is available for the inves-
PCR Polymerase chain reaction tigating hematopathologist; thus, this book focuses on
B-PLL B-cell prolymphocytic leukemia histology, immunohistochemistry and, if necessary, molec-
ular pathological methods of bone marrow biopsies). A
bone marrow biopsy containing at least five intertrabecular
spaces (excluding subcortical spaces containing mainly
1.1 Introduction fatty tissue) is considered adequate for diagnosis. All
biopsy specimens should have sections routinely stained
Histological investigation of bone marrow biopsy is an inte- with hematoxylin and eosin (H&E), May–Grünwald
gral examination method for diagnosis of various hemato- Giemsa (MGG) or Giemsa, BBL and reticulin stain (e.g.,
logical and also non-hematological diseases [1]. Since Gomori’s stain). Metachromatic stains (e.g., MGG) give
sampling of bone marrow biopsies (BMB) from newborns as additional information, which are not available in rou-
well as geriatric patients can easily be performed, the indica- tinely H&E stained sections. For example, identification of
tions for retrieving BMB have increased, especially in hema- eosinophils, plasma cells, and mast cells is facilitated; fur-
tology, internal medicine, oncology, but also osteology [2]. thermore, differentiation of proerythroblasts and myelo-
Various fixatives and different methods are described for blasts is easily possible. Iron stains (e.g., Prussian blue)
preparation of bone marrow biopsy sections; however, each should be done from each bone marrow biopsy; however,
hematopathologist prefers his own technique used in his lab- one has to be aware that decalcification might remove iron.
oratory. Therefore, it is not our intention to persuade the Therefore, the report should state “iron stain is negative”
investigating pathologists to use a special method, but the and not “there is absence of storage iron.”
need for well-fixed and well-stained thin sections cannot be The most important and indispensable information for
emphasized enough. diagnosis of bone marrow biopsies is the knowledge of
As the name “bone marrow” implies, histological the patient’s age (without knowledge of the patient’s age,
examination includes bone structure as well as marrow. In bone marrow diagnosis is not accurate and always incom-
adults, normal bone marrow consists of fatty tissue and plete). This knowledge helps to avoid pitfalls in histological

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 1

C. Beham-Schmid, A. Schmitt-Graeff, Bone Marrow Biopsy Pathology,
Essentials of Diagnostic Pathology, https://doi.org/10.1007/978-3-662-60309-3_1
2 1 Normal Bone Marrow

diagnosis, for example, recognition of normally present 1.1.1 Bone Components (Fig. 1.1)
subcortical hypoplasia or non-representative tangentially
retrieved BMB. It is also important to recognize and Representative bone marrow biopsies show cortical bone
report changes in hematopoietic cells due to inadequate (cortex, compact bone) and trabecular bone (trabeculae, can-
fixation, decalcification, or staining procedures. cellous bone, ossicles) (Fig. 1.1a, b) with appendant bone

a b

c d

e f

Fig. 1.1 (a, b) Low magnification of representative BMBs with corti- female with rarefication of trabecular bone (Gomori’s stain). (f) A
cal bone (left side) and trabecular bone (left biopsy: Gomori’s stain, 48-year-old female showing rarefication of trabecular bone (Gomori’s
right biopsy: Ladewig). (c) Higher magnification of (b) showing tra- stain). (g) Severely thickened cancellous bone of a 60-year-old male
becular bone constituting the honeycomb of bone. (d) Normal bone (Gomori’s stain). (h) BMB of a 73-year-old male with marked osteo-
structure (MGG) of a 50-year-old female. (e) BMB of a 52-year-old sclerosis (Gomori’s stain)
1 Normal Bone Marrow 3

g h

Fig. 1.1 (continued)

cells, which are flat endothelial cells, lining trabecular and The cytoplasmic processes of osteocytes connect with the
subcortical surface, osteoblasts, osteoclasts, and osteocytes. processes of other osteocytes and also with osteoblasts on
The cortex is a solid layer of compact bone of various the surface of the bone (Fig. 1.2d–f). Thus, a circulatory net-
thickness, on the outside periosteum, on the inside the endos- work within the osseous system, within the bone and to its
teum, a single layer of cells, are attached. The cortex mainly surface, is built, providing the subsistence of the osteocytes
consists of lamellar bone, but some woven bone is also pres- and enables the transfer between bone, bloodstream, and
ent. There is constant remodeling of the bone. In adults, interstitial fluid.
remodeling of the bone mainly takes place in the subcortical Osteoclasts are the bone-resorbing cells on or near the
regions. A new layer of bone is added by osteoblasts, while surface of the bone (Fig. 1.2g), often interposed between
the osteoclasts resorb other areas of the bone. Up to 20–25% endothelial and endosteal cells and also between subcortical
of the bone surface may be covered by osteoid. bone surface and endothelium on the trabecular surface.
Trabecular bone constitutes the honeycomb of bone Osteoclasts, with their villous extensions bind to matrix
(Fig. 1.1c, d) and is enclosed by the cortical bone. A rarefi- adhesion proteins and produce resorption pits, shallow con-
cation of trabeculae (osteopenia) results in enlargement of cavities, called Howship lacunae (Fig. 1.2h inset). The
marrow cavities (Fig. 1.1e, f). A thickening of cancellous appearance of osteoclasts is either uni- or multinuclear.
bone, osteosclerosis, causes a decreased size of marrow Multinuclear osteoclasts may even become larger than mega-
spaces (Fig. 1.1g, h). Semi-automatic and/or computerized karyocytes (Fig. 1.2h). The relatively flat uni-nucleated
quantification systems for bone and bone marrow struc- osteoclasts equally do resorb bone as do the multinucleated
tures are not generally required for the diagnosis and inter- forms. Bone marrow biopsies of children and young adults
pretation of bone marrow biopsy sections. For routine normally show marked bone remodeling with the presence
diagnosis, subjective assessment by naked eye of the com- of many osteoclasts, whereas in adults and older people the
ponents of the biopsy sections in a number of fields in the presence of many osteoclasts indicates a metabolic or a neo-
light microscope, or, if requested by the clinicians, assess- plastic disease.
ment by use of a graticule in the ocular is recommended [3] Mean values from healthy adults for osteoblastic index
(Fig. 1.2). (percentage of trabecular surface covered by cuboidal osteo-
Osteoblasts with a diameter from 20 to 50 μm produce the blasts) is 5%. The osteoclastic index (number of osteo-
bone matrix, osteoid, which gets subsequently ossified [4]. clasts/100 mm trabecular circumference) is 3–4/100 mm [4].
Osteoblasts line the surface of the cancellous bone and if Although osteoclasts and osteoblasts share the surface of the
these cells are activated, they become cuboidal (Fig. 1.2a, b). trabeculae (Fig. 1.2g), these cells originate from different
After mineralization of bone, some osteoblasts are trapped stem cells, osteoclasts from hematopoietic, and osteoblasts
within the bone and transformed into osteocytes (Fig. 1.2c). from mesenchymal stem cells.
4 1 Normal Bone Marrow

a b

c d

e f

Fig. 1.2 (a) Osteoblasts lining the surface of bone (MGG). (b) of osteocyte (MGG). (g) Multinucleated osteoclast on bone surface
Cuboidal activated osteoblasts (MGG). (c) Trabecular bone with osteo- (MGG). (h) Huge multinucleated osteoclasts. Inset reveals Howship
cyte (MGG). (d–f) Cytoplasmic processes of osteocytes connecting lacunae (MGG)
with processes of adjacent osteocytes. Inset shows high magnification
1 Normal Bone Marrow 5

g h

Fig. 1.2 (continued)

6 1 Normal Bone Marrow

1.2 Cellularity of the Marrow sue, or, similar to an aspirate, to a marrow cavity excluding
bone tissue [2]. The latter procedure is comparable to mea-
While BM aspirates (BMA) and BM trephine biopsies surements of the cellularity of aspirated fragments. Slight
(BMTB) are excellent for assessment of lineage percentages differences in marrow cellularity have been described in dif-
and morphology, only BMTB can provide accurate informa- ferent sites like sternum, lumbal vertebrae, and iliac crest [4].
tion about the overall degree of cellularity and organization The bone marrow of newborns mainly consists of cells
of all hematopoietic lineages. Also, if lymphocytic cells or with a negligent amount of fat cells (0–<5%). With increasing
plasma cells are increased, only BMTB will reveal the pat- age, cellularity decreases with an accelerated rate of reduc-
tern of their distribution. tion beyond the age of 70. The decrease in hematopoietic
However, it is important that not only the width of the tissue with advanced age is due to a loss of bone substance
BMTB is adequate but also the length which ideally should and also caused by a true loss of the amount of hematopoietic
measure at least 2 cm. Length is particularly important not tissue. The marrow cavities consequently are filled with fat
only for the “staging” of neoplasms as to whether there is cells. At the age of 20, the average cellularity is 70–80%, at
BM involvement but also in many infectious disease affect- the age of 50 cellularity is 50–60%, and at 70 years and
ing BM (Fig. 1.3). beyond, cellularity is reduced to 20–30% [2, 6–8] (Fig. 1.3a).
The term “cellularity” refers to hematopoietic as well as The spatial arrangement of hematopoiesis is a potential
fat cells and indicates the relative amount of these compo- pitfall with regard to the normally present subcortical
nents. Normal values are age-dependent (Fig. 1.3a) with hypoplasia (subcortical fatty tissue) (Fig. 1.3b–d). To
individual deviations [5, 6]. For routine diagnosis, the cellu- avoid the error of estimating cellularity in subcortical
larity is assessed subjectively; otherwise, there is the possi- regions, it is necessary to pay attention to bone structures
bility of computerized image analysis or histomorphometry. in the biopsy (e.g., a tangentially taken sample with plenty
Bone marrow cellularity is demonstrated as the percentage of cortical bone Fig. 1.3e). A representative biopsy (as
of a section occupied by hematopoietic tissue. This percent- mentioned before) should show at least three intertrabecu-
age can either refer to the entire biopsy including bone tis- lar spaces [9, 10].
1 Normal Bone Marrow 7

b c

d e

Fig. 1.3 (a) Schematic representation of cellularity in normal bone tical fatty tissue) (MGG). (c, d) Physiological subcortical hypoplasia: a
marrow biopsies showing decrease of hematopoiesis with advanced common diagnostic pitfall (MGG). (e) The whole length of the biopsy
age. (b) BMB of a patient with myeloproliferative neoplasm (left side is shows cortical bone (tangentially taken sample) (MGG)
representative for diagnosis, the biopsy on the right side reveals subcor-
8 1 Normal Bone Marrow

1.2.1 Topography (Fig. 1.4) one hand, “myeloid” implies the complete hematopoiesis, on
the other hand this term describes the granulopoietic and
In the marrow cavities, hematopoietic tissue is distributed monocytopoietic lineage only, as demonstrated by the
interstitial in the extravascular compartment (between the myeloid: erythroid (M:E) ratio. Usually, there is no interpre-
sinusoids) (Fig. 1.4a, b). In some pathological conditions, tation problem, but ambiguousness in using the term
hematopoiesis can be found intrasinusoidal also. Myeloid “myeloid” should be avoided.
precursor cells are closely attached to the endosteal surface Groups of erythropoietic cells and megakaryocytes show
and arterioles (Fig. 1.4c–f), more mature granulopoietic cells a close association to the marrow sinusoids [3, 5, 8]
are seen in central intertrabecular areas. Attention should be (Fig. 1.4g, h). In normal bone marrow, considerable varia-
paid to the term “myeloid,” which, in the German- and tions in the qualitative as well as quantitative distribution of
English-speaking parts, has two different meanings. On the cellular components can be observed.

a b

CD15 MPO

c d

CD15 MPO

Fig. 1.7 (a–d) Normal bone marrow: Comparison of staining reactivity in myeloid cells using antibodies to CD15 (figures on the left side) and
MPO (figures on the right side)
14 1 Normal Bone Marrow

1.3.4 Monocytopoiesis and Macrophages cells, or lipid. Occasionally, especially when there is an
(Fig. 1.8) increased turnover of hematopoiesis, sea-blue histiocytes are
found. These cells are macrophages with huge sea-blue cyto-
Even in optimal prepared histological sections, monocytes can plasm corresponding to granules containing lipofuscin or
hardly be detected. These cells are easily mistaken for granulo- ceroid (Fig. 1.8g, h). Quite often aggregates of sea-blue histio-
poietic precursor cells with their slightly kidney-shaped vesicu- cytes are seen in myeloproliferative neoplasms and myelodys-
lar nuclei and eosinophilic variably granulated cytoplasm. plastic syndromes [21, 22].
Small numbers of monocytes are located in the midst of hema- Interstitial histiocytosis can occur due to an increased
topoietic islands or periarteriolar. Immunohistochemically, turnover (sea-blue histiocytes) in the course of storage disor-
monocytes can be uncovered using antibodies to CD11c or ders (congenital, kappa-positive crystal storage).
CD14 [20, 21] (Fig. 1.8a, b). In the bone marrow, monocytes Increased histiocytes can be found in infectious disease
mature and become macrophages, which are found in great (e.g., Mb. Whipple, atypical mycobacteriosis).
numbers diffusely dispersed (Fig. 1.8c–e). Macrophages often A marked increase in histiocytes can be seen during resto-
contain hemosiderin (Fig. 1.8f), nuclear debris, hematopoietic ration after myeloablative therapy.

a b

CD14 CD14

c d

CD163

Fig. 1.8 (a, b) Monocytes in normal bone marrow highlighted immu- CD68. (f) Macrophages containing hemosiderin (Berlin blue iron
nohistochemically with an antibody to CD14. (c, d) Many macrophages stain). (g, h) Aggregates of sea-blue histiocytes (in a patient with MDS)
(stained with an antibody to CD163) in normal bone marrow. (e) stained with MGG
Macrophages in normal bone marrow stained with an antibody to
1 Normal Bone Marrow 15

e f

CD68

g h

Fig. 1.8 (continued)

16 1 Normal Bone Marrow

1.3.5 Megakaryocytopoiesis (Figs. 1.9 20–80 μm, their cytoplasm is less basophilic and often peri-
and 1.10) nuclear granules can be detected. (c) Mature megakaryo-
cytes with a diameter up to 150 μm exhibit an eosinophilic
Megakaryocytes with a diameter of 12 to 150 μm are the cytoplasm and show a variable granularity [4]. Especially
largest of bone marrow cells with an abundant cytoplasm when megakaryocytes are increased, emperipolesis
and lobulated nuclei (Fig. 1.9a–c). For detection of small (pseudo-phagocytosis), the presence of other cells (lym-
forms, immunohistochemical stains are required. The pref- phocytes, erythroblasts, erythrocytes, or granulocytes)
erential localization is near sinusoids (Fig. 1.9c, d). within megakaryocytes, is obvious [23] (Fig. 1.9g, h). This
However, since the sinusoids are often collapsed and thus emperipolesis however, is without pathological signifi-
hardly apparent, megakaryocytes often seem to be diffusely cance and can be seen in megakaryocytes of any size.
distributed within the marrow spaces. In normal hemato- Occasionally, denuded megakaryocyte nuclei are seen in
poiesis, usually megakaryocytes do not form clusters, but normal bone marrows. Normal routinely stained bone mar-
are either seated singly or form loose cell groups counting row sections reveal 8–15 megakaryocytes per mm2 corre-
up to 3–5 cells (Fig. 1.9e, f). During maturation, three sponding to 2–5 megakaryocytes per HPF [9]. Using
stages can be recognized. (a) Megakaryoblasts with a size immunohistochemistry (e.g., CD41, CD42b, CD61) con-
of 12–20 μm show a kidney shaped or an oval nucleus with siderably more megakaryocytes, especially small ones, are
a basophilic cytoplasm. (b) Pro-megakaryocytes measure visible (up to 25/mm2) [5] (Fig. 1.10a–d).

a b

c d

Fig. 1.9 (a, b) Megakaryocytes with lobulated nuclei, the largest bone ocytes in a patient with ITP. MGG. (g, h) Megakaryocytes with
marrow cells, are easily detected. MGG. (c, d) Normal perisinusoidal emperipolesis. MGG
localization of megakaryocyte. MGG. (e, f) Loose groups of megakary-
1 Normal Bone Marrow 17

e f

g h

Fig. 1.9 (continued)

18 1 Normal Bone Marrow

a b

CD41

c d

CD42b CD61

Fig. 1.10 (a) BMP of patient with untreated CML: In H&E stained biopsy, few small megakaryocytes are detectable. (b–d) Same biopsy as (a):
Immunohistochemically, using antibodies for megakaryocytes, much more megakaryocytes are visible: (b): CD41; (c): CD42b; (d): CD61
1 Normal Bone Marrow 19

1.3.6 Lymphocytes (Figs. 1.11 and 1.12) c arcinoma, cirrhosis, diabetes mellitus, systemic Castleman’s
disease and drug therapy [24–26]. Nodular B- and T-cell aggre-
Lymphocytes are a component of the normal bone marrow pop- gates increase with age and after medication (Fig. 1.12a, b).
ulation and appear either diffusely dispersed in the interstitium, Whereas in the normal bone marrow of children the num-
or in the form of small aggregates or lymphoid nodules ber of lymphocytes is rather high with 30–60% (B-cells
(Fig. 1.11a–c). Lymphoid nodules or aggregates, especially if comprise about 65% of lymphocytic cells in the first 4 years
they are rather small, are easily identified in sections stained for of life), in adults the number of lymphocytes is much lower
reticulin fibers (e.g., Gomori’s stain), since these lymphoid with 10–20%. Normally in adulthood, T-lymphocytes
aggregates show more fibers than the surrounding hematopoie- (CD3+) outnumber B-lymphocytes (CD20+) (Fig. 1.12c, d).
sis. With increasing age lymphoid nodules occur more frequent The rate of T-lymphocytes: B-lymphocytes is 4:1 to 6:1 [27].
and may cause differential diagnostic problems whether these The ratio of CD 4 to CD8 cells of approximately 1:2 is the
lymphoid nodules are benign or malignant. In most cases, the reverse of the ratio of CD4 to CD8 of 2:1 present in lymph
combination of adequate, representative, and good quality bone nodes (Fig.1.12 CD4, CD8). Furthermore, CD8+ cells are
marrow t rephine biopsy, careful analysis of the cytomorphology more frequent in the bone marrow than in peripheral blood.
of the lymphoid cells, number and pattern of their distribution, An increase of CD8 is skewed in virus infection and autoim-
immunophenotype based on immunohistochemistry munity, CD4 is skewed in drug reactions. NK cells constitute
(Fig. 1.11d–h), and clinical and laboratory data allow the correct about 2–4%.
interpretation to be made. In a few cases, cytogenetic and A reactive increase of T-cells with formation of lymphoid
molecular studies are needed for a correct diagnosis. aggregates is common (up to 40%) in elderly patients. An
It should also be stressed that neoplastic BM involvement increase of reactive T-cells is quite common in patients with
by hematolymphoid neoplasms may incite reactive inflam- MPN and MDS. In younger people, an increase of T-cells is
matory changes such as benign lymphoid aggregates, a dif- found in viral infections and autoimmune disorders. Clonality
fuse increase in reactive mainly T-lymphocytes, plasma cells, analyses can be misleading because of the presence of benign
histiocytes, and granuloma formation—the result of cytokine reactive T-cell clones.
production by the lymphoma cells. Furthermore, a reactive lymphocytosis can occur in the
Another result of BM involvement by small cell lymphomas form of hematogones after chemotherapy or post infectious.
is to cause reactive dysplastic features in the erythroid, granulo- Characteristics of benign lymphoid aggregates of small
cytic and megakaryocytic lineages (reactive dyshematopoiesis). size (<1 mm) with well defined borders are:
This secondary reactive myelodysplasia may mimic a primary 1. Random distribution; often perivascular and distant from bone
myelodysplastic syndrome (see Chapter - Myelodysplasia). trabeculae (lymphoid aggregates located paratrabecular are
Lymphoid nodules are either well demarcated or with irreg- always suspicious of infiltration of malignant lymphoma).
ular borders and may comprise germinal centers. Reactive lym- 2. Sometimes lymphoid follicles with germinal centers.
phoid infiltrates occur in a wide variety of diseases. These are 3. No cellular atypia.
infectious diseases such as hepatitis and HIV infection; con- 4. Predominance of B-lymphocytes, except in HIV
nective tissue disorders, particularly rheumatoid arthritis; infection.
hematological neoplasia including lymphoma, MDS, and 5. If plasma cells are found within lymphoid nodules, these
MPNs as well as idiopathic thrombocytopenic purpura, and a are polyclonal.
variety of anemias especially aplastic anemia, metastatic 6. Few reticulin fibers, except in HIV infection.

a b

Fig. 1.11 (a, b) Low and high magnification of lymphoid nodule: H&E. (c–h) Reactive lymphoid nodule with germinal center: (c): H&E; (d):
CD10; (e): CD20; (f): CD23; (g): CD3; (h): Ki67
20 1 Normal Bone Marrow

c d

CD10

e f

CD20 CD23

g h

CD3 MIB1

Fig. 1.11 (continued)

1 Normal Bone Marrow 21

a b

CD20

c d

CD3 CD3
e f

CD4 CD8

Fig. 1.12 (a) Normal bone marrow: H&E. (b) Small number of B-lymphocytes in normal bone marrow (CD20). (c, d) Higher number of
T-lymphocytes in normal bone marrow (CD3). (e, f) Low number of CD4+ lymphocytes. Much higher number of CD8+ lymphocytes
22 1 Normal Bone Marrow

1.3.7 Plasma Cells (Fig. 1.13) 5. Plasma cells are polyclonal.

6. No cytological atypia.
Plasma cells rarely amount to more than 5% in normal
bone marrow. Studies, using high-resolution flow cytom-
etry show averagely 0.25% plasma cells of all nucleated 1.3.8 Mast Cells (Fig. 1.13e)
bone marrow cells [28]. Usually, plasma cells are located
along the adventitia of blood vessels (Fig. 1.13a, b), but A few mast cells are a component of normal bone. The
single plasma cells or small groups of these cells are dif- preferential localization is adjacent to sinusoidal endothe-
fusely distributed around macrophages, but seldom lial cells (Fig. 1.13c, d), at the endosteal surface of trabecu-
around fat. Normal plasma cells reveal a cartwheel lae, in the wall of small arterioles, and at the periphery of
nuclear chromatin pattern and in metachromatic stains a lymphoid nodules [4]. Usually, mast cells show oval to
pale blue cytoplasm (mature plasma cells). Increased round nuclei and contain densely packed red granules in the
numbers of PCs are a common response to many dis- cytoplasm (Fig.1.13e). The best stain to identify mast cells
eases (see above in chapter Lymphocytes). The plasma is a metachromatic stain (e.g., Giemsa) or immunohisto-
cells on IHC analysis with antibodies to light chains chemical staining with an antibody to mast cell tryptase
kappa and lambda are polyclonal and do not usually (Fig. 1.13f). In neoplastic conditions, mast cells may be
exceed 10–20%. In some diseases, notably HIV infec- spindle-shaped and fibroblastic-like and may contain only
tion and multicentric Castleman’s disease (MCD) (see single granules; occasionally, neoplastic mast cells reveal
figures in relevant chapters) plasma cells may number up an abundant clear cytoplasm. In such circumstances, these
to 50% [29]. In the context of HIV infection, there cells are best detected using immunohistochemistry with
should be awareness that PC neoplasms do occur and the antibodies to tryptase, CD117c and CD25 [30, 31]. A reac-
BM may be involved [30]. tive increase of mast cells (mast cell hyperplasia) usually is
The main problems encountered in the correct interpreta- part of a nonspecific chronic inflammatory response, espe-
tion of bone marrow plasma cells are in separating various cially after toxic exposures, such as chemotherapy.
reactive plasmacytoses from multiple myeloma and its vari- Furthermore, mast cell hyperplasia in the bone marrow can
ants or MGUS. Morphologically, the reactive plasma cells be found in conditions like uremia, osteoporosis, and hema-
are mainly mature and nucleocytoplasmic asynchrony and tologic conditions like lymphomas, MDS, and leukemias
nucleoli are infrequent. Mott cells, Russell-bodies, Dutcher– [31, 32].
Fahy bodies, intracytoplasmic crystals, and bi-nucleated Characteristics of reactive mast cell increase:
forms may all be identified. 1. No mast cell granulomas (these are often associated with
Characteristics of reactive plasmacytosis: fibrosis and osteosclerosis)
1. Interstitial; scattered singly or in small clusters. 2. No spindle-shaped mast cells
2. Plasma cell number 10–20%, rarely may be 50%. 3. No cytological atypia
3. Present around capillaries or macrophages; rarely around 4. No occurrence of mast cells with abundant amounts of
fat spaces. clear cytoplasm
4. Rarely form large clusters and do not form nodules. 5. No increase of CD25-positive mast cells
1 Normal Bone Marrow 23

a b

c d

e f

try

Fig. 1.13 (a, b) Plasma cells with typical cartwheel cytoplasm around red granules in the cytoplasm. MGG. (f) Mast cells in normal bone mar-
a vessel. MGG. (c, d) Mast cells located close to sinusoidal endothelial row. (IH: tryptase)
cells. MGG. (e) High magnification of mast cells with densely packed
24 1 Normal Bone Marrow

1.3.9 Bone Marrow Stroma (Fig. 1.14) 2. Bone structure (architecture, osteopenia, osteosclerosis,
increased remodeling)
Hematopoietic cells are embedded in a framework con- 3. Cellularity (with consideration of the patients age: nor-
sisting of fat cells, fibroblasts, reticular cells, and the net- mocellular, or mild, moderate, high hypocellularity or
work of blood vessels and sinusoids, which are often hypercellularity. Ideally, percentage of cellular compo-
collapsed and difficult to identify. Rarely nerves are nents and fatty tissue should be specified)
detected in marrow spaces (Fig. 1.14a, b). In special stains 4. Hematopoietic cells (erythro-, granulo-, megakaryocyto-
(e.g., Gomori’s stain), few fibers are seen in the normal poiesis: description of quantity, quality, and topography)
marrow, mostly around blood vessels and endosteum 5. Stroma: fibroblasts, reticulin and collagen fibers, fat,
(Fig. 1.14c, d). This microenvironment supports the extra- iron, blood vessels, stromal cells
vascular hematopoietic compartment [3, 8]. The content 6. Other cellular components: B- and T-lymphocytes,
of iron, either within macrophages or in the bone marrow plasma cells, mast cells, macrophages, neoplastic infiltra-
stroma, can be graduated from 0 (no iron detectable) to 6 tion, neoplastic hematological disease, (special reactive
(masses of clumped iron and extracellular iron) [33] changes).
(Fig. 1.14e–h).
“Check list” for diagnosis of bone marrow biopsies: Diagnosis (ideally with consideration of clinical data): either
1. Assessment of length and quality (e.g., excellent, suffi- descriptive (with statement of differential diagnoses) or
cient, not adequate) definitive (according to WHO criteria).

a b

c d

Fig. 1.14 (a, b) Nerve accompanying blood vessel (a: MGG, b: detectable (grade 0). (f–h) Increase of iron (BBL): according to Gale:
Gomori’s stain). (c, d) Fibers around vessels in normal bone marrow (f): grade 4, (g): grade 5. (h): grade 6 with very large deposits of intra-
(Gomori’s stain). (e) Using an iron stain (BBL) no iron deposits are and extracellular iron
1 Normal Bone Marrow 25

e f

g h

Fig. 1.14 (continued)

26 1 Normal Bone Marrow

References 18. Naeim F. Topobiology in hematopoiesis. Hematol Pathol.

MGG MGG

e f

GOM GlycC

g h

MPO CD42b

Fig. 2.3 (continued)

34 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.2.4 Hemolytic Anemia gene mutation [11]. The paroxysmal nocturnal hemoglobin-
uria-phenotype granulocytes can be assessed by using mul-
In hemolytic anemias, the red blood cells are destroyed before tiparameter flow cytometry immunophenotypic analysis
their life span is over. In cases of extrinsic hemolytic anemia, with antibodies specific for glycosylphosphatidylinositol-
known as autoimmune hemolytic anemia, a bone marrow anchored proteins (CD55, CD59, CD16, CD66b). For the
biopsy is performed to exclude an underlying hematologic investigating hematopathologist, the knowledge of existing
neoplasm. There are several diseases and even medications PNH clones is essential for making a correct diagnosis.
that can cause such a condition. Some underlying causes of
extrinsic hemolytic anemia are splenomegaly, drugs, lupus, Case History
hepatitis, infection with EBV, but also leukemias, lymphomas, A 63-year-old female with hemolytic anemia and verified
myeloproliferative neoplasms [9], and solid tumors. PNH underwent a bone marrow biopsy (Fig. 2.4a–h) for
Among the acquired hemolytic anemias, paroxysmal exclusion of a concomitant disease. The information, given
nocturnal hemoglobinuria (PNH) is of special interest. PNH by the clinicians was reticulocytosis, high concentration of
can be associated with aplasia and pancytopenia, imitating LDH and indirect bilirubin, and abnormally low concentra-
aplastic anemia, but usually the bone marrow shows a tion of serum haptoglobin. The bone marrow was hypercel-
marked increase of nucleated erythroid precursors without lular mainly caused by an increased erythropoiesis. No
dysplastic features and a reversed myeloid to erythroid ratio dysplastic changes could be detected. A descriptive diagno-
(Fig. 2.4, case history) [10]. PNH is a clonal hematopoietic sis consistent with hemolysis and PNH was performed.
stem cell disease, caused by mutations in the PIG-A gene
[11]. An increased number of cells with deficiency of glyco- 2.2.4.1 Caution
sylphosphatidylinositol (GPI)-anchored membrane proteins Based upon morphology and also immunohistochemistry, a
are a result of hematopoietic progenitor cells with a phos- diagnosis of PNH cannot be made. Clinical information
phatidylinositol glycan complementation group A (PIG-A) about the presence of a PNH clone is mandatory.

a b

Fig. 2.4 PNH. (a, b) Routinely stained H&E section shows marked enlarged erythropoietic cell nests with a slight left shift. No dysplastic
hypercellular bone marrow in this 63-year-old female due to an increase changes are evident. (g) Megakaryocytes, stained with an antibody to
of erythropoietic cells. Erythropoiesis is stained with antibodies to CD42b are slightly decreased and small. (h) Myelopoiesis, stained with
CD71 (c, d), Glycophorin C (e) and Glycophorin A (f). There are rather an antibody to MPO appears normal
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 35

c d

CD71 CD71

e f

GlycC GlycA

g h

CD42b MPO

Fig. 2.4 continued)

36 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.2.5 Aplastic Anemia 2. Severe Aplastic Anemia (SAA).

• BM cellularity <25%, or 25–50% with <30% residual
Aplastic anemia, the paradigm of human bone marrow fail- hemopoietic cells.
ure syndrome, is defined by pancytopenia with bone marrow • Two out of three of the following: neutrophils
hypocellularity (Fig. 2.5, case history). Most cases are <0.5 × 109/L, platelets <20 × 109/L, reticulocyte
acquired and immune-mediated, with activated type 1 cyto- <20 × 109/L.
toxic cells implicated [12]. Rarely, there are inherited forms 3. Non-severe Aplastic Anemia (NSAA).
such as the genetic diseases Fanconi anemia and dyskerato- • Patients not fulfilling the criteria for severe or very
sis congenita. Usually, acquired aplastic anemia presents severe aplastic anemia.
with an abrupt onset of low blood cell counts in previously
healthy persons. Aplastic anemia has been shown to be asso- Cytogenetic abnormalities have been reported infre-
ciated with a multitude of autoimmune diseases, like, most quently with acquired aplastic anemia [14].
frequently, psoriasis, systemic lupus erythematosus, rheuma-
toid arthritis, celiac disease, colitis ulcerosa, multiple sclero- Case History (Post-hepatitic SAA)
sis, vasculitis, Sjögren’s syndrome, and ITP. Aplastic anemia A 7-year-old icteric girl was diagnosed with hepatitis A. Two
can be caused by environmental triggers including toxins, months later she presented with fever, headache, and patho-
chemicals, drugs, and viruses, especially hepatitis. A bone logical blood picture with Leuko 1.79 G/L, Ery 2.92 T/L, Hb
marrow biopsy is performed to assess the overall cellularity, 9.2 g/dL, Hkt 25.6%, MCV 87.7 fL, MCH 31.5 pg, MCHC
the topography of hematopoietic cells, and to estimate atypi- 35.9 g/dL, Thrombo 118 G/L. Hepatitis-associated aplastic
cal and/or dysplastic features within hematopoiesis and to anemia was suspected and a bone marrow biopsy was done
exclude abnormal or neoplastic infiltrates. during clinical workup. The bone marrow was highly hypo-
Cases of aplastic anemia are grouped as very severe, severe, cellular for age with more than 80% fat cells (Fig. 2.5a–f).
and non-severe according to the criteria as shown below [13]. Occasionally, in the marrow a few erythropoietic islands (hot
1. Very Severe Aplastic Anemia (VSAA). spots) were present. A diagnosis of subtotal bone marrow
• As for severe aplastic anemia but neutrophils aplasia, consistent with the clinical diagnosis of hepatitis-
<0.2 × 109/L. associated aplastic anemia, was made.

a b

Fig. 2.5 Post-hepatitic SAA. (a) The bone marrow is highly hypocel- an antibody to CD71. (d) Only a few diffusely distributed myeloid cells
lular for age (MGG). (b) Occasional erythropoietic islands, the so- can be seen (MPO). (e) No fibrosis is seen (Gomori’s stain). (f) Slight
called hot spots, are seen in higher magnification (MGG). No increase of iron deposits in the bone marrow (BBL)
megakaryocytes are visible. (c) Erythropoietic cells are visualized with
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 37

c d

CD71 MPO

e f

GOM BBL

Fig. 2.5 continued)

a b

c d

e f

CD20 CD23

Fig. 2.9 Castleman’s disease. (a, b) In H&E stained section, the over- cal for a reactive lymphoid follicle. (o) Most of the blasts are CD20-
view shows highly hypercellular bone marrow with multiple nodular positive. (p) Many blasts show reactivity for CD30 (EBV done with
lymphoid infiltrates, most of which are closely attached to the bony EBER, [not shown], is negative). (q, r) Using a plasma cell marker
trabeculae (b). (c, d) Some of the lymphoid infiltrates contain diffusely (Vs38c) in the periphery of the lymphoid aggregates many plasma cells
dispersed blasts. Most lymphoid aggregates resemble reactive follicles are evident. (s) With antibodies to light chains kappa (left) and lambda
with many CD20-positive B-cells (e), a network of follicular dendritic (right) the plasma cells are polyclonal. Within the lymphoid aggregates
cells (f, CD23), many CD3-positive T-cells (g) and BCL2-negative fol- the B lymphocytes do not show clonality. (t) There is no increase of
licle center (h). Lymphoid nodules stained with various T-cells markers IgG4-positive plasma cells. (u) Granulopoiesis (MPO) is increased. (v)
show a distribution like in reactive follicles (i: CD4; j: CD5; k: CD7; l: Erythropoietic cells (CD71) are increased. (w, x) There is an increase of
CD8). (m) Lymphoid nodule with many diffusely dispersed blasts rather small not atypical megakaryocytes (CD42b)
(H&E). (n) The proliferation fraction (Ki67) is quite low and not typi-
46 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

g h

CD3 BCL2

i j

CD4 CD5

k l

CD7 CD8

Fig. 2.9 (continued)

2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 47

m n

Ki67

o p

CD20 CD30

q r

Vs38c Vs38c

Fig. 2.9 (continued)

48 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

s t

kappa lambda IgG4

u v

MPO CD71

w x

CD42b CD42b

Fig. 2.9 (continued)

2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 49

2.5 Non-neoplastic Conditions with the presence of immature precursors and absence of
with Quantitative and Qualitative mature granulopoiesis, the bone marrow may resemble pro-
Changes in Granulopoiesis myelocytic leukemias.

2.5.1 Severe (Acquired) Neutropenia Case History (Agranulocytosis)

We show the bone marrow of a 61-year-old woman treated
The normal neutrophil levels vary with age and race roughly for colitis ulcerosa for several years (Fig. 2.10a–f). Due to
between 1.8–7.0 × 109/L. Patients with severe neutropenia increasing pain, the patient took pain relievers with a combi-
with less than 0.5 × 109/L. have a high risk of developing nation of acetylsalicylic acid, paracetamol, and caffeine.
fatal life-threatening infections. Acquired non-malignant After 14 days of this drug administration, the patient became
neutropenia is more common than chronic neutropenia. One severely ill with agranulocytosis, fever, and sore throat as
of the commonest causes of severe neutropenia or agranulo- well as urinary tract infection. Her leucocyte count was
cytosis in addition to treatment with chemotherapy is drug- 0.75 G/L, neutrophils abs. 0.14 G/L. To exclude other dis-
induced neutropenia [21]. The two basis mechanisms of eases, a bone marrow biopsy was performed.
drug-dependent neutropenia are either immune-mediated
destruction of circulating neutrophils by drug-dependent or 2.5.1.1 Caution
drug-induced antibodies or a direct toxic effect on marrow Severe neutropenia can also occur in autoimmune states like
granulocytic precursors. The onset in many cases is delayed rheumatoid arthritis and SLE. For exact diagnosis, the
and can arise as much as a month after the drug has been knowledge of the patient’s history is recommendable.
stopped, but usually presents within the first 6 months after Cyclic neutropenia is characterized by recurrent epi-
beginning the offending drug. Various drugs can cause neu- sodes of abnormally low levels of neutrophils. In most
tropenia and agranulocytosis, the most frequent agents are cases, episodes of neutropenia recur cyclic every 21 days
antimicrobial followed by antithyroid agents [22] and pain and may last for 3–6 days. The cycling period usually
relievers. In addition, there are reports of severe neutropenia remains constant and consistent among affected individu-
and agranulocytosis after the use of cocaine contaminated als. Cyclic n eutropenia may be inherited or acquired. Most
with levamisole [23]. cases of cyclic neutropenia are thought to be congenital;
During the acute phase of severe neutropenia/agranulocy- however, in some cases, the symptoms may not become
tosis, the bone marrow histology is variable. This variability obvious until childhood, adolescence, or early adulthood.
ranges from hypocellularity to edematous stroma often with There are, as shown above, many different causes of neu-
extravasation of erythrocytes from disrupted sinusoids and tropenia. Neutropenia may also result from viral infection.
marked reduction of myeloid precursors and/or maturation In addition, rarely the patient may produce antibodies
inhibition to hypercellularity, while erythropoietic and mega- against neutrophils (autoimmune neutropenia) causing an
karyocytic cells are not affected. There may also be just little abnormal decrease in white blood cells. The bone marrow
reduction of myeloid precursors or even an increase of during an episode of neutropenia shows a marked decrease
myeloid precursors with no mature forms. In such cases, of neutrophil precursors.
50 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

a b

c d

MPO MPO

e f

CD42b CD71

Fig. 2.10 Agranulocytosis. The bone marrow is normo- to slightly myeloid precursors (MPO). (e) Megakaryocytopoiesis is normal
hypercellular for age (a, H&E). In some areas, marked edematous appearing (CD42b). (f) Erythropoietic cells are slightly increased
stroma with inflammatory cells is evident (b, H&E). (c, d) (CD71)
Granulocytopoiesis is highly decreased with only some dispersed
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 51

2.6 ostmann Disease (Infantile Genetic

K reduced bone density, which can lead to osteopenia or
Agranulocytosis, Severe Congenital osteoporosis.
Neutropenia) All patients with severe congenital neutropenia, regard-
less of their treatment or response, are at risk to develop
Kostmann disease, first described in 1956, is an autosomal MDS or leukemia.
recessive disorder characterized by severe neutropenia below
0.50 × 109/L and onset of severe bacterial infections early in Case History (Kostmann Disease)
life [24]. Kostmann reported that the neutropenia was accom- The bone marrow of a 2-year-old girl with congenital agranu-
panied by “a primary insufficiency of the bone marrow” and locytosis is shown (Fig. 2.11a–h). The girl suffers from recur-
that the disease is determined by a “single recessive gene rent bacterial infections and is treated permanently with GCSF.
difference.” More than 50 years later, homozygous muta-
tions in the gene encoding the mitochondrial protein HCLS1-
associated X1 (HAX1) were found in affected descendants of 2.6.1 Caution
the original Kostmann family. The bone marrow of patients
suffering from Kostmann disease shows a characteristic mat- The differential diagnosis of CN includes a number of congeni-
uration arrest at the promyelocyte/myelocyte stage of myelo- tal and acquired diseases [26]. The most common difficulty is
poiesis (Fig. 2.11, case history) [25]. Besides neutropenia, determining whether the patient has cyclic neutropenia, auto-
patients with congenital neutropenia (CN) often have a immune neutropenia of infancy, or idiopathic neutropenia.

a b

c d

MPO CD71

Fig. 2.11 Kostmann disease. (a–d) These figures show the first bone (d). A bone marrow biopsy was taken 6 months after GCSF therapy: In
marrow biopsy of the girl. In routinely stained H&E sections, the bone routinely stained H&E sections (e, f), normocellular bone marrow is
marrow is normocellular for age but no mature granulopoiesis is seen seen. Using an antibody to MPO immature granulopoietic cells are con-
(a, b). Using an antibody to MPO (c) the granulopoiesis is immature centrated around vessels and trabeculae (g). Erythropoietic cells
with a marked maturation arrest. The erythropoiesis (CD71) is normal (CD71) are not affected (h)
52 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

e f

g h

MPO CD71

Fig. 2.11 (continued)

2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 53

2.7 Leukemoid Reaction (LR) karyocytopoiesis appeared rather normal. A descriptive diag-
nosis was made. Two weeks later, the WBC count decreased
In LR, a marked elevation in the WBC count resembling leuke- to normal levels without intensive therapy. The reason for her
mia is found. The WBC count can exceed 50.00 × 109/L and is increased WBC count could not be evaluated.
composed mainly of segmented neutrophils which may or may
not show toxic changes. The leukocytosis is associated with a
cause outside the bone marrow: this can be some sort of infec- 2.7.1 Caution
tious diseases, paraneoplastic reactions, injuries or hemor-
rhages and hemolytic events [27]. The bone marrow in LR is LR is an important, probably the most important, differential
mildly to marked hypercellular caused by an increase of neu- diagnosis in hematological malignancies since misinterpretation
trophilic myeloid hyperplasia and occasionally additional of secondary leukemia or relapse of a leukemia is possible.
megakaryocytic hyperplasia (see Fig. 2.12, case history below). Chronic neutrophilic leukemia, an extremely rare neo-
plasm, has to be taken into consideration, if a patient has
Case History prolonged neutrophilia. Clonality of neutrophils thus has to
An 81-year-old woman, who has been treated for diffuse be demonstrated or excluded for differential diagnosis.
large B-cell lymphoma 6 years previously, was admitted with BCR-ABL-positive CML morphologically can resemble
a WBC of 62.50 × 109/L. Clinical workup including cytoge- LR. However, in LR the megakaryocytes can be helpful for
netics did not reveal any evidence of malignant disease. A differential diagnosis since in LR the megakaryocytes are
bone marrow biopsy was performed (Fig. 2.12a–d). This larger (normal looking) than in CML. Furthermore, in LR
biopsy was markedly hypercellular for age due to an increased usually no increase of basophils is found.
granulopoiesis with maturation. Erythropoiesis and mega- LR can also occur as a rebound effect after neutropenia [28].

a b

MGG

c d

MPO

Fig. 2.12 Leukemoid reaction. In routinely stained H&E (a) and MGG (b) sections, the bone marrow is highly hypercellular for age. No neoplas-
tic infiltrate is seen. (c) The maturing granulopoiesis is highly increased (MPO). (d) Note the rather polymorphic megakaryocytopoiesis (MGG)
54 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.8 Granulopoiesis with Increased dysplastic changes in multiple cell lines, it is more likely to
Eosinophils deal with a hematological malignancy.

Increased eosinophils in the blood (>0.5 × 109/L) are a fre- Case History (Reactive Eosinophilia)
quent finding, and is in most cases connected with eosino- A 68-year-old male was admitted with pruritus and dyspnea.
philia (>10%) in the bone marrow. Patients with mild The patient is under observation for treated diffuse large
eosinophilia usually are asymptomatic, in cases with moder- B-cell lymphoma 5 years previously. At admission, the patient
ate to marked eosinophilia there is a risk of severe organ showed leucocytosis with eosinophilia and anemia. His blood
damage, irrespective of the cause of eosinophilia. To avert count was:: Leuko 39.78 G/L, Ery 3.58 T/L, Hb 11.0 g/dL,
organ damage and fibrosis, which is caused by the release of Hkt 31.2%, normal values of MCV, MCH, and MCHC,
special proteins from the eosinophilic granules, it is essential Thrombo 271 G/L, MPV 9.5 fL; segmented 12% (normal val-
to identify the cause of eosinophilia as soon as possible. ues 50–75), eosinophils 82% (normal: 5%), monocytes 3%
An absolute eosinophilic count of more than 500/μL is (normal), and lymphocytes 3% (normal 20–40). According to
defined as eosinophilia. An eosinophilia may occur either the patient, he suffered from an influenza infection 2 weeks
as (1) reactive (secondary) eosinophilia, (2) clonal (neo- ago. Cytogenetically and by molecular techniques, no abnor-
plastic) eosinophilia, and as (3) idiopathic eosinophilia, if malities could be detected. The clinical workup included a
no underlying reactive cause is found and there is no evi- bone marrow biopsy (Fig. 2.13a–h).
dence that the eosinophils are derived from a clonal myeloid The tumor-free bone marrow biopsy was moderately
neoplasm. hypercellular for age. Granulocytopoiesis, which showed
Various causal connections for reactive eosinophilia are maturation, was increased and marked eosinophilia with
known [29]. Frequent causes for eosinophilia are infections more than 80% eosinophils was detected in routinely stained
(especially helminths, Borrelia Burgdorferi, fungi), allergic sections and immunohistochemically. The diagnosis was
reactions and asthma, respiratory diseases like Loeffler syn- descriptive with a comment that a differential diagnosis
drome and hypersensitivity pneumonitis, cytokine infusions, between primary and secondary eosinophilia is not possible
vasculitis, non-hematological malignant diseases, drug reac- based on morphology.
tions, and connective tissue diseases. Furthermore, eosino-
philia can occur in combination with neoplastic disease,
especially Hodgkin’s disease, Langerhans cell histiocytosis, 2.8.1 Caution
and T-cell lymphomas, but also in combination with solid
tumors, especially in stomach and lung. If an unexplained In many cases with eosinophilia, it may be impossible to dif-
eosinophilia of more than 1.500/μL persists for a longer ferentiate primary from secondary eosinophilia. Thus, cyto-
period than 6 months, this eosinophilia is termed “hypereo- genetic and molecular studies are essential.
sinophilia” and may be more likely primary than secondary. In bone marrow biopsies with marked eosinophilia, an
The morphology of eosinophils does not differentiate infiltration by malignant lymphoma (T-cell lymphoma,
between primary and secondary eosinophilia (Fig. 2.13, case Hodgkin’s disease) has to be excluded. Since lymphoma
history). For an exact diagnosis, complex cytogenetic and infiltration can be subtle, at least some immunohistochemi-
molecular abnormalities have to be investigated. If the bone cal stains with B- and T-cell markers and CD30 should be
marrow with an increase of eosinophils shows atypia and performed.
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 55

a b

MGG

c d

EPX MPO

Fig. 2.13 Reactive eosinophilia. (a) The bone marrow is moderately cells are stained. To exclude an infiltration by mast cells, which can
hypercellular. Even at low power magnification, an increase of eosino- resemble eosinophils, mast cell markers CD117c, (e) and tryptase (f)
phils is seen (H&E). (b) Increased granulopoiesis with marked eosino- were used. Only a few mast cells are evident. (g) Erythropoiesis is
philia (>80%) is verified using a metachromatic stain (MGG). (c) The slightly decreased and left shifted, but not dysplastic (CD71). (h)
eosinophils are immunohistochemically stained with an antibody to Megakaryocytes are rather small and slightly decreased, but character-
EPX. (d) Using an antibody to MPO, the more immature granulopoietic istic atypias within megakaryocytes could not be detected (CD42b)
56 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

e f

CD117c Trypt

g h

CD71 CD42b

Fig. 2.13 (continued)

2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 57

2.9 uantitative and Qualitative Changes

Q paraneoplastic conditions. After application of growth fac-
in Megakaryocytopoiesis tors, the number of megakaryocytes can markedly increase.

Megakaryocytes are the largest cells normally seen in the bone

marrow with a size ranging from 12 to 150 μm. In many diseases, 2.9.1 Morphologic Abnormalities
megakaryocytic morphology is the clue for a c orrect diagnosis. of Megakaryocytes
Small megakaryocytes might be missed in routinely stained sec-
tions, but these small forms can be detected by immunohisto- Qualitative abnormalities of megakaryocytes offer morpho-
chemistry (e.g., antibodies to CD36, CD41, CD42b, CD61). logic indications to a number of non-neoplastic and neoplas-
A decrease in megakaryocytes can be congenital or tic disorders.
acquired, for example in aplastic anemia or megaloblastic ane-
mia. A hypoplasia of megakaryocytes can be seen as a para-
neoplastic phenomenon in malignant lymphomas (especially 2.9.2 Megakaryocytic Emperipolesis
T-LGLL) or carcinomas, but also as a toxic reaction by various (Fig. 2.14a–d)
drugs and radiation. Furthermore, hypoplasia of megakaryo-
cytes is often seen parainfectious (e.g. CMV infection). Quite often megakaryocytes show blood cells, mainly neu-
An increased number of megakaryocytes can be detected trophils, within the cytoplasm. This phenomenon is com-
in ITP, hemorrhage, hemolysis, hypersplenism, after sple- monly found in patients with thrombocytosis [30], either
nectomy and in many malignant disorders, like carcinomas neoplastic or reactive. Especially, neutrophils pass through
and lymphomas. Even in bone marrow biopsies, which are the megakaryocyte tubular system to enter the circulation. In
densely infiltrated by malignant tumor tissue, an increased our experience, emperipolesis is a common finding in
number of pleomorphic megakaryocytes is often seen, while myeloproliferative neoplasms, and myelodysplasia, in the
erythro- and granulocytopoiesis are highly decreased. rare gray platelet syndrome but also in reactive thrombocyto-
Hyperplasia of megakaryocytes is common in septic and sis, for example, in patients after splenectomy.

a b

MGG MGG

c d

MGG MGG

Fig. 2.14 Megakaryocytes (MGG) with emperipolesis in PMF (a) and karyocyte within another megakaryocyte could be detected (MGG). (d)
ET (b). (c) In the bone marrow of a patient with MDS/MPN with ring A bone marrow biopsy with dense infiltration by AML-blasts shows an
sideroblasts and thrombocytosis an unusual emperipolesis of a mega- emperipolesis by a neoplastic blast (MGG)
58 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.10 Micromegakaryocytes A left shifted megakaryocytopoiesis without atypia is

and Hypolobulated/Nonlobulated typical in patients with platelet survival defects, especially
Megakaryocytes (Fig. 2.15a–l) ITP [32].
Small megakaryocytes with hypolobulated nuclei, often
High numbers of nonlobulated micromegakaryocytes with a arranged in sheets, are frequently seen in patients after
rather dense chromatin is a characteristic finding in BCR- chemotherapy.
ABL-positive CML (“dwarf megakaryocytes”) [31] and in Hypolobulated megakaryocytes of normal size can occur
patients with myelodysplasia. These micromegakaryocytes in elderly patients without hematological disorders.
are often the hallmark cells for the diagnosis.

a b

MGG

c d

CD42b

Fig. 2.15 Increase of small megakaryocytes with often condensed cytes (H&E). (f): immunohistochemical stain with CD61. (g, h)
chromatin are a characteristic finding in BCR-ABL-positive CML (a: Increase of megakaryocytes with a left shift in a patient with ITP. (g:
H&E). Staining with MGG (b) reveals also an increase in basophils. (c) H&E; h: MGG). (i, j) This bone marrow was taken after chemotherapy
In a patient with MDS with multilineage dysplasia small atypical mega- for acute myeloid leukemia. In this bone marrow, a marked increase of
karyocytes with hypolobulated nuclei are seen (H&E). (d) rather small left shifted megakaryocytes is evident (i: H&E; j: immuno-
Immunohistochemically, much more small atypical megakaryocytes histochemical stain with CD42b). (k, l) This bone marrow biopsy of an
can be found in the bone marrow of the same patient (see c) with MDS old patient (staging was done for prostatic carcinoma) shows mega-
with multilineage dysplasia (CD42b). (e) Bone marrow biopsy with karyocytes of normal size, but with hypolobulated nuclei (H&E)
MDS with isolated del(5q) reveals masses of small atypical megakaryo-
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 59

e f

CD61

g h

MGG

i j

CD42b

Fig. 2.15 (continued)

60 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

k l

Fig. 2.15 (continued)

2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 61

2.11 Enlarged Megakaryocytes

with Hyperlobulated Nuclei
(Fig. 2.16a, b)

Such forms are seen in MPNs, mostly in ET [33]. Usually,

such giant forms of megakaryocytes are not found in reactive
conditions.

a b

MGG MGG

Fig. 2.16 (a, b) Bone marrow biopsy of a patient with ET showing huge megakaryocytes with hyperlobulated nuclei (MGG)
62 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.12 egakaryocytes with Naked Nuclei

M
(Fig. 2.17a–d)

Naked or “denuded” megakaryocytes can be seen in MPNs,

especially in PMF [33], and in HIV-infected patients. These
megakaryocytes reveal dark nuclei with not visible (absent)
cytoplasm.

a b

c d

CD42b

Fig. 2.17 Bone marrow biopsy in a patient with PMF. Even at low In routinely stained H&E section within an edematous stroma, mega-
magnification in routinely stained H&E sections (a), the naked nuclei karyocytes with naked nuclei are seen (c). Staining with an antibody to
of megakaryocytes are visible due to their dense dark chromatin. In CD42b (d) highlights the megakaryocytes, some of which are denuded
higher magnification, the naked forms do not show a visible cytoplasm
(b). This bone marrow biopsy was taken from an HIV-positive patient.
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 63

2.13 Clustering of Megakaryocytes 2.13.1 Caution

(Fig. 2.18a–p)
Clustering of megakaryocytes is not seen in “normal”
Small clusters of usually small to normal-sized non-atypical bone marrow biopsies. Clustering can be seen in reactive
megakaryocytes are visible in regenerative bone marrow biopsies, conditions, like regenerating bone marrow, but clustering
especially after chemotherapy. Clustering of small atypical mega- of megakaryocytes is more typical in neoplastic condi-
karyocytes with hypolobulated nuclei is common in MDS. Clusters tions. A “cluster of megakaryocytes” is defined as a group
of highly atypical, often large, megakaryocytes are seen in neo- of three or more megakaryocytes closely attached to one
plastic myeloid disorders, especially in MDS/MPN with ring sid- another.
eroblasts and thrombocytosis and in MPN, PMF [34].

a b

CD42b

c d

CD42b

Fig. 2.18 (a) Bone marrow biopsy of a 55-year-old male after chemo- increase of small atypical megakaryocytes forming clusters and sheets
therapy of AML. The bone marrow is hypercellular with increased left (CD42b). (i) Highly hypercellular bone marrow of a 63-year-old female
shifted hematopoiesis (H&E). (b) Clusters of rather small megakaryo- diagnosed with MDS/MPN with ring sideroblasts and thrombocytosis
cytes after chemotherapy are visualized immunohistochemically (MDS/MPN-RS-T) (H&E). (j) Increased highly atypical megakaryo-
(CD42b). (c) Highly hypercellular bone marrow of a 46-year-old male cytes in this patient are forming clusters (CD42b). (k) Highly hypercel-
diagnosed with MDS with blast excess-2 (H&E). (d) Clusters of small lular bone marrow of a 74-year-old female with MPN, early phase of
atypical megakaryocytes with hypolobulated nuclei are seen by immu- PMF (pre-PMF) (H&E). (l) Clusters of large atypical megakaryocytes
nohistochemistry in this 46-year-old male with MDS with blast excess-2 in the patient with pre-PMF (CD61). (m) Highly hypercellular bone
(CD42b). (e) Normocellular marrow in a young 18-year-old male diag- marrow of a 50-year-old male with MPN pre-PMF (H&E). (n) The
nosed with MDS with multilineage dysplasia (H&E). (f) Clusters and increased atypical rather large megakaryocytes in the 50-year-old
even sheets of atypical small megakaryocytes are highlightened immu- patient with pre-PMF are occasionally arranged in clusters (CD61). (o)
nohistochemically (CD42b). (g) Hypercellular bone marrow biopsy of Hypercellular bone marrow in a 70-year-old male with MPN PMF
a 87-year-old male diagnosed with MDS with isolated del(5q) (H&E). (fibrotic phase) (H&E). (p) Clusters of rather large atypical megakaryo-
(h) The hallmark of the bone marrow in this patient is the markedly cytes are found in this 70-year-old male with PMF (CD61)
64 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

e f

CD42b

g h

CD42b

i j

CD42b

Fig. 2.18 (continued)

2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 65

k l

CD61

m n

CD61

o p

CD61

Fig. 2.18 (continued)

66 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.14 Intrasinusoidal Megakaryocytes Especially, if the sinuses are dilated, megakaryocytes within
(Fig. 2.19) these vessels easily can be recognized. Intrasinusoidal local-
ization of megakaryocytes is not a feature of “normal” bone
Intrasinusoidal megakaryocytes usually are a feature of marrows.
MPNs and of the rare autoimmune caused myelofibrosis [35].

a b

MGG MGG

c d

Fig. 2.19 (a, b) Intrasinusoidal megakaryocyte with hyperlobulated (g) Intrasinusoidal megakaryocyte in a patient with PMF (MGG). (h)
nuclei typically for MPN, ET (MGG). (c, d) Low and high power mag- Intrasinusoidal megakaryocytes in a patient with MPN. This bone mar-
nification of a case with MPN, PV. Even at low power magnification, row morphologically resembles in one way MPN PV and in another
intrasinusoidal megakaryocytes are seen (H&E). (e) Huge megakaryo- way PMF. Clinically, the patient has no signs of polyglobulia, periph-
cytes located within a sinus in MPN, ET (MGG). (f) Intrasinusoidal eral blood shows slight thrombocythemia and leukocytosis. (MGG)
megakaryocytes in a patient with MPN, post-PV MF (H&E).
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 67

e f

MGG

g h

MGG MGG

Fig. 2.19 (continued)

68 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.15 Paratrabecular Location Rarely, paratrabecular megakaryocytes can be seen in reac-

of Megakaryocytes (Fig. 2.20a–h) tive bone marrows, mostly in regenerative marrows or in
patients with Lupus.
Megakaryocytes, closely attached to the bony trabeculae are
a feature of myeloid neoplasms, like MDS or MPN [36].

a b

MGG GOM
c
d

GOM

Fig. 2.20 (a, b) Paratrabecular megakaryocytes in a 87-year-old male lular bone marrow of a 67-year-old male with MDS/MPN with ring
patient with MDS with isolated del(5q). (left: MGG; right Gomori’s sideroblasts and thrombocytosis (MDS/MPN-RS-T). Many megakary-
stain). (c, d) A 47-year-old patient with treatment-associated MDS with ocytes are found closely attached to the bony trabeculae (e: H&E; f:
fibrosis (left: H&E, right: Gomori’s stain). (e, f, g, h) Highly hypercel- CD42b; g, h: CD61)
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 69

e f

CD42b

g h

CD61 CD61

Fig. 2.20 (continued)

70 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.16 Gray Platelet Syndrome brother also had low platelet counts and died aged 33 due to
spontaneous cerebral hemorrhage. Her parents, husband, and
Gray platelet syndrome (GPS) is a rare inherited thrombocy- the children of the deceased brother were clinically
topenia characterized by macrothrombocytopenia and the unaffected. Further studies showed that the respective family
absence of platelet α-granules resulting in typical gray plate- members are affected by gray platelet syndrome and a novel
lets on peripheral smears [37]. The disease is present at birth, homozygous single nucleotide insertion in GFI1B
but occasionally the disease is discovered in adulthood. (NM_004188.5; c.551insG) could be detected in the affected
Among the 60 well-documented cases, no female-male gen- mother.
der inequality and no ethnic or geographic predisposition is The bone marrow biopsy of the 36-year-old woman
found. Patients with gray platelet syndrome are often diag- was slightly hypercellular with a slight increase of reticu-
nosed as immune thrombocytopenic purpura (ITP). Long- lin fibers. The most conspicuous finding was that of a
term follow-up data show the progressive nature of moderately increased megakaryocytopoiesis. The mega-
thrombocytopenia and myelofibrosis of GPS resulting in karyocytes were rather small with either normally lobu-
fatal hemorrhages in many patients. lated or hypolobulated nuclei with condensed chromatin.
All megakaryocytes showed strong positivity with the
Case History of Gray Platelet Syndrome usual megakaryocytic markers. Additionally, the mega-
The bone marrow biopsy of a 36-year-old woman is shown karyocytes were strongly CD34-positive but alpha-gran-
who underwent a bone marrow biopsy (Fig. 2.21a–h) with ule marker (GF11B) was reduced (this marker was
the clinical supposed diagnosis of ITP. She and her children provided by the clinicians who excessively studied the
had episodes of life-threatening bleedings and the woman patients findings and reported this family). Erythropoiesis
and her children suffered from recurrent hematoma and pete- was slightly increased and left shifted, the granulopoiesis
chiae since childhood with platelets less than 45/nL. The appeared normal.

a b

GOM

Fig. 2.21 Gray platelet syndrome. (a) Routinely stained H&E section megakaryocytes (CD42b). (f) Higher magnification of CD42b-positive
shows slightly hypercellular bone marrow. In this figure, rather small megakaryocytes with hypolobulated nuclei. (g and h) All megakaryo-
megakaryocytes with hypolobulated nuclei can be seen. (b) Gomori’s cytes are CD34-positive. Inset shows no or rather weak staining of
stain reveals a slight increase of diffusely dispersed reticulin fibers. (c) megakaryocytes with an antibody to GF11B, whereas the erythropoi-
Erythropoietic cells are slightly increased and left shifted (CD71). (d) etic cells are rather strongly positive
Normal appearing granulopoiesis (MPO). (e) Increase of rather small
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 71

c d

CD71 MPO

e f

CD42b CD42b

g h

CD34

Fig. 2.21 (continued)

72 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.17 one Marrow Alterations After

B rather response to therapy, to estimate regeneration of the
Chemotherapy/Stem Cell hematopoiesis, and to evaluate the emergence of therapy-
Transplantation associated hematopoietic neoplasms. Consequently, it is of
great importance for the investigator to know the original
A dazzling array of bone marrow morphologic changes can be disease and also to know the therapy-regimen.
seen after and during chemotherapy for the treatment of vari- Myeloablative therapy (Fig. 2.22a–h) causes early
ous malignant neoplasms. Such potent therapies also affect changes after 1 week therapy. These can be visualized as
normal bone marrow cells, a phenomenon which must be bone marrow aplasia, marked edema, disappearance or
appreciated by the investigating pathologists. In addition to reduction of fatty tissue, fibrinoid necrosis (without tumor
chemotherapeutic regimens, marked bone marrow changes are necrosis), and increase of lymphocytes, plasma cells, and
also occurring after antibody therapy and treatment with granu- histiocytes, and dilatation of the sinusoids (Fig. 2.22a, b).
locyte colony-stimulating factors. In addition, therapy-related After 2–3 weeks, intermediate changes such as reappearance
myeloid neoplasms, such as therapy-related myelodysplastic of fatty tissue, mild reticulin fibrosis, foci of left shifted
syndromes (t-MDS) and therapy-related acute myeloid leuke- erythro- and granulopoietic cells, and an increase of
mia (t-AML), have to be considered in consequence of conven- B-precursor cells can be observed (Fig. 2.22c–f). Finally, the
tional anticancer chemotherapy and radiotherapy for solid late changes after 3 to 4 weeks therapy occur in the form of
tumors, various sarcomas, and hematologic malignancies [38]. the appearance of megakaryocytic clusters, resolution of
A bone marrow biopsy after various therapeutic regimens reticulin fibers, and recurrence of normocellularity or even
is performed to determine the degree of tumor reduction or slight hypercellularity (Fig. 2.22g, h).

a b
early changes

MGG MGG

Fig. 2.22 Bone marrow biopsy of an 18-year-old male treated for hematopoietic cells reveals diffusely dispersed large lymphoid imma-
acute myelomonocytic leukemia at day 7. The bone marrow is aplastic ture appearing cells (d, H&E). Immunohistochemical investigation
showing edema and fibrinoid necrosis (a, MGG). Markedly dilated shows CD10-positivity (e) and strong CD20-positivity (f) in these
sinusoids are evident (b, MGG). Bone marrow biopsy of a 24-year-old immature looking lymphoid cells uncovering these as hematogones. (g,
male treated for acute T-cell leukemia at day 21. The bone marrow h) Bone marrow biopsy of a 14-year-old girl treated for c-ALL at day
shows areas with increased fatty tissue and areas with confluent hema- 21 showing normocellularity for this age (g, H&E) and the occurrence
topoietic cells (c, H&E). High magnification of bone marrow field with of small groups of megakaryocytes (h, CD42b)
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 73

c d
intermediate changes

e f
intermediate changes

CD10 CD20

g h
late changes

CD42b

Fig. 2.22 (continued)

74 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.17.1 Caution very problematic in patients treated for acute B-cell leuke-
mias. Sheets of promyelocytes might be confused with
In some patients, uncommon findings in regenerating bone acute promyelocytic leukemia. And finally, an increase of
marrows can be seen (Fig. 2.23). Such findings, which can megakaryocytes (Fig. 2.23e, f), often combined with a dif-
be confused with a neoplastic process, comprise the appear- fuse reticulin fibrosis can cause difficulties, especially in
ance of large sheets of promyelocytes (Fig. 2.23a–d), a patients with underlying myeloproliferative neoplasms.
marked increase in hematogones and a rather florid increase Rarely, patients can show a marked delay in the regenera-
of megakaryocytes. An increase of hematogones can be tion of hematopoiesis.

a b

c d

Fig. 2.23 Uncommon findings in regeneration. This is the bone mar- age (a, b: MGG). In the hypercellular areas nearly exclusively promy-
row of a 76-year-old woman treated for acute myelomonocytic leuke- elocytic cells could be seen (c, d: H&E) The bone marrow biopsy of a
mia. The patient was pancytopenic after 3 weeks of treatment and 55-year-old male treated for AML (FAB M0) after 3 weeks therapy
received treatment with granuolcyte-stimulating factor. The bone mar- shows a marked increase of megakaryocytes (e: H&E, f: CD42b)
row biopsy was partly highly hypocellular and partly hypercellular for
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 75

e f

CD42b

Fig. 2.23 (continued)

76 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

2.18 Monoclonal Antibody Treatment tration, especially paratrabecular. After RTX therapy, an
(Rituximab) antigen loss of CD20 is found, and the infiltrates are mainly
or completely composed of reactive T-cells (Fig. 2.24a–d). In
The chimeric murine-human anti-CD20 monoclonal anti- addition to an immunohistochemical stain with an antibody
body Rituximab (RTX) is in worldwide use for the treatment to CD20, some more B-cell markers have to be used to avoid
of B-cell lymphomas, either alone or in combination with the mistake of missing a residual neoplastic infiltrate.
standard regimens. There is a high response rate with a rather Furthermore, areas of former lymphoma infiltration in para-
low toxic effect. The pathologists, investigating marrow trabecular areas may reveal a reticulin fibrosis.
biopsies after RTX treatment, have to be familiar with the
morphologic and immunophenotypic sequelae this treatment
causes. The pitfalls for pathologists after RTX treatment 2.18.1 Caution
were first described by Douglas et al. in 1999 [39]. An
evaluation of different small B-cell lymphomas treated with Since there is an antigen loss of CD20 in B-cells in patients
RTX emphasizes the use of a panel of different antibodies to treated with RTX, bone marrows have to be studied immuno-
avoid false-positive and false-negative interpretations [40]. histochemically with additional B-cell markers, such as
Especially, problematic are bone marrow biopsies of patients CD19, CD22, and CD79a. If CD20 is the only B-cell marker
with bone marrow infiltration by follicular lymphoma treated used, neoplastic residual lymphoid cells might be missed. In
with RTX. In these bone marrows, lymphocytic infiltrates addition, also PCR studies for evaluation of clonality might
can be detected in areas of former neoplastic lymphoid infil- be necessary.

a b

c d

CD20 CD3

Fig. 2.24 RTX treatment. (a–d) Bone marrow biopsy of a 54-year-old stained sections a slight fibrosis is recognizable. A nodular lymphocytic
male treated with RTX for follicular lymphoma. (a) (H&E) shows an infiltrate (b, H&E) shows no reactivity with an antibody to CD20 (c),
area of former paratrabecular lymphoma infiltration. Even in H&E the cells within this infiltrate are exclusively T-cells (d, CD3)
2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 77

2.19 one Marrow Alterations after Bone

B cytes and plasma cells (Fig. 2.25c–f) [42]. In addition, great
Marrow Transplantation (Fig. 2.25a–h) attention for persisting tumor cells is obligatory.
After 3–4 weeks of transplantation, erythroid and myeloid
The aim of a bone marrow or stem cell transplantation is to cell nests are present; dyserythropoiesis and dysmyelopoie-
cure the patient either from a neoplastic or non-neoplastic sis can still persist (up to 50% of hematopoietic cells may
disorder. Depending on the patient’s disease, the preparatory show dysplastic morphology). The regeneration of mega-
and conditioning regimens for transplantation are variable. karyocytes can be quite patchy. Characteristically, after
However, to eradicate tumor cells in patients with solid 4 weeks (day 28) trilineage engraftment should be seen. The
tumors or hematologic neoplasias, radiation and multiagent biopsy at day 28 is important to diagnose engraftment failure
chemotherapy are necessary. A bone marrow biopsy, com- or delayed engraftment. In approximately 15% of cases,
bined with aspirate smear preparations, is required in the marrow failure without recurrent disease at 3–6 months after
evaluation after bone marrow transplantation for eradication transplant is detected by engraftment study [42].
of underlying disease and engraftment [41]. Since chemo- Rarely, usually 12 months after transplantation, avascular
therapy is essential for preparative regimens of transplanta- bone necrosis can be found. Also, bone marrow aplasia, due
tion, the morphologic alterations in the bone marrow are to absent or delayed engraftment, persistent dysplastic hema-
similar to those described after myeloablative therapy. topoiesis, or monotonous immature groups of erythroid or
In addition, there are some particular morphologic myeloid cells, can be found.
changes after transplantation. Within the first week after
transplantation marked bone marrow destruction with apla-
sia, edema, and fat necrosis (Fig. 2.25a, b) as well as mild 2.19.1 Caution
reticulin abnormalities due to cell death are seen. After
1–2 weeks, regeneration effects can be observed. Such It is of great importance to look for residual disease.
changes may occur as dysplastic erythro- and granulopoietic Especially, concerning patients with transplantation because
cells, myeloid hyperplasia, and atypical localization of of AML, it is helpful or even necessary to know the pheno-
immature myeloid precursors, megaloblastic erythropoiesis, type of AML-blasts. In some instances, highly sensitive flow
scattered atypical small megakaryocytes, irregular calibra- cytometric and/or molecular studies for residual or recurrent
tion or vacuolization of fat cells, and an increase of lympho- disease might be necessary.

a b
early 1 week

Fig. 2.25 BM after TX. Bone marrow biopsy of a 47-year-old male positive T-lymphocytes (e). Plasma cells (stained with an antibody to
1 week after transplantation. Underlying disease was follicular lym- Vs38c) are increased (f). Further studies with antibodies to light chains
phoma. In H&E stained section, the bone marrow is highly hypocellular kappa and lambda revealed polytypic plasma cells. Bone marrow
(aplastic) (a). Higher magnification (b) reveals fat necrosis. (c–f) Bone biopsy of a 12-year-old boy 11 months after bone marrow transplanta-
marrow biopsy of a 29-year-old female 2 weeks after stem cell trans- tion. Underlying disease was precursor T-cell neoplasia (T-lymphoblastic
plantation. Underlying disease was AML. High magnification shows leukemia). Avascular bone necrosis with empty osteocytic lacunae in
dysplastic erythropoiesis and abnormal localization of immature bone trabeculae is detected. Furthermore, a diffuse necrosis of marrow
myeloid precursors (c, H&E). Immunohistochemically scattered small cells can be seen (H&E, g, low magnification, h, high magnification)
megakaryocytes (CD42b) are seen (d). There is an increase of CD3-
78 2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis

c d
1-2 weeks

CD42b
e f

CD3 Vs38c
g h
12 month

Fig. 2.25 (continued)

2 Non-neoplastic Conditions with Quantitative and Qualitative Changes in Hematopoiesis 79

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Bone Changes Found in Bone Marrow
Biopsies 3

The skeleton function is diversified. It is the instrument for Since it is impossible to show all these complex aspects,
direct locomotion and movement and protects vulnerable tis- selected important entities will be discussed.
sues. It is important for the homeostasis of minerals, and it
shelters the bone marrow which provides the blood elements.
The metabolites of Vitamin D, parathyroid hormones, and 3.1 Osteodystrophy
calcitonin are among the most important regulators in the
complex mechanism for the control of the calcium mecha- 3.1.1 Renal Osteodystrophy
nism and the sound condition of bone. These complex mech-
anisms explain the vulnerability of trabecular bone which is Renal osteodystrophy, a disease spectrum and not a uni-
variably affected in many primary or secondary diseases. A form progressive bone disease [2], is caused by derange-
bone marrow biopsy can be helpful in diagnosis since mor- ments in homeostatic and metabolic control of minerals.
phological changes can be detected before abnormalities are This progressive bone disease histologically reveals a mix-
evident on X-rays. Bone remodeling and bone production ture of osteopenia, osteosclerosis, osteomalacia, and oste-
are typical features in pediatric marrows. However, even in itis fibrosis cystica (Fig. 3.1a–d). Often reduction in
adults bone remodeling is continuing for mechanical support hematopoiesis as well as fibrosis and edema give rise to
and to provide adequate calcium levels. This bone remodel- the anemia of chronic renal disease. In any individual
ing in adults is morphologically not noticeable in the figura- patient, different morphologic and clinic changes may pre-
tive sense of peculiar osteoblasts or osteoclasts [1]. The dominate. Concerning the bone changes, these are affected
evaluation of trabecular bone on bone marrow biopsies com- mainly by the manifestations of secondary hyperparathy-
prises an estimation of the thickness, size, and shape and a roidism. Secondary hyperparathyroidism, in contrast to
general survey of the matrix and cellular components of the primary form, shows various degrees of a malacic com-
bony trabeculae. Normally, a structural support for medul- ponent. Distinctive forms of secondary hyperparathyroid-
lary cavities is evident by anastomosing bone spicules. ism reveal fibro osteoclastic lesions. According to Delling
Continuously connected anastomosing bony trabeculae are a [3], at least a rough estimation of renal osteodystrophy
pathologic finding. Careful evaluation of the mesenchymally should be made. He suggests Type I: the main histologic
developed osteoblasts and osteocytes, and the osteoclasts, finding is a fibro-osteoclastic morphology (Fig. 3.1a, b);
which are of hematopoietic origin, is essential. The mono- Type II: marked osteomalacia is present (Fig. 3.1c, d);
nuclear osteoblasts are located in the periphery of trabeculae. Type III: combination of Type I and II with low (a), moder-
In young patients, these cells often form a row along bony ate (b), and marked (c) bone remodeling and “+” increased
trabeculae. Osteoclasts, mostly multinucleated cells, occupy bone volume density and “−” decreased bone volume
small depressions on the bone’s surface, called Howship density.
lacunae. A patchy distribution of osteoclasts along bony tra-
beculae is normal in young patients. Mineralized bone pre- 3.1.1.1 Caution
dominates and comprises osteocytes. Unmineralized newly In patients with renal diseases, especially in dialysis patients,
formed bone matrix, the osteoid, is prominent in young chil- a special stain for osteoid (non-mineralized bone) has to be
dren, but minimal in adults. performed.

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 81

a b

c d

Fig. 3.1 Renal osteodystrophy. Bone marrow biopsy of a 59-year-old 46-year-old dialysis patient with marked osteomalacia (Ladewig’s
female dialysis patient with marked anemia. In the bone marrow biopsy, stain). The blue bone is mineralized; the red color is indicative of oste-
marked fibro-osteoclastic lesions without a malacian component were oid (non-mineralized bone)
seen (a: H&E; b: Ladewig’s stain). (c, d) Bone marrow biopsy of a
3 Bone Changes Found in Bone Marrow Biopsies 83

3.1.2 Hyperparathyroid Osteodystrophy formations and rarefication with osteomalacia is found.

Histomorphometry on iliac crest biopsies in PHPT has
In primary hyperparathyroidism (PHPT), an elevated PTH shown increased bone remodeling mainly in the trabecular
stimulates the kidney and bone causing a condition with bone and to a lesser degree in the cortical bone where the
high bone turnover, elevated plasma calcium, and metabolism is at a much lower level [4].
increased fracture risk. In PHPT as well as secondary
hyperparathyroidism osseous remodeling is excessive 3.1.2.1 Caution
(Fig. 3.2) without orderly arrangement and balance between Among others, especially metastatic tumors and hematopoi-
resorption and formation. Paratrabecular fibrosis is evident etic neoplasms with increased bone turnover, or a giant cell
in affected areas resulting in reduction of hematopoiesis. In tumor of bone must be considered in the differential diagno-
severe conditions, hematopoiesis can be completely replaced sis of findings caused by hyperparathyroidism (also called
by fibrosis. Especially in PHPT marked osteoclastic remod- “brown tumor”). Giant cell tumors mostly occur in long
eling with excavation of the bony trabeculae forming cystic bones, but these tumors can occur in the pelvis [5].

a b

c d

Fig. 3.2 Hyperparathyroidism. (a–f) Bone marrow biopsy of a seen. Excavated trabeculae with cystic formation (b, MGG), and marked
35-year-old woman diagnosed with primary hyperparathyroidism. osteoclastic remodeling (c, d, MGG) is evident. (e, f) Paratrabecular
(a–d) In overview (a, MGG) distortion of trabecular bone structure is coarse fibrosis extending into the marrow cavities (Gomori’s stain)
84 3 Bone Changes Found in Bone Marrow Biopsies

e f

Fig. 3.2 (continued)

3 Bone Changes Found in Bone Marrow Biopsies 85

3.1.3 Osteopenia resorption and formation throughout life. The aim is the
adaption to stimuli and the preservation of structure and
Osteopenia is characterized by thinned bony trabeculae strength. The bone volume is regulated by balanced-coupled
(Fig. 3.3). As a generalized change, osteopenia is noted as osseous remodeling in space within the skeleton and in time
osteoporosis; patchy osteopenia may occur in patients with throughout life. The cortex is a low remodeling area, and the
renal disorders or in patients with neoplastic infiltrates, espe- trabeculae are a high remodeling area. A complex interaction
cially myeloma patients. Osteoporosis is known to occur due of pathologic stimulation of osteoclasts and inhibition of
to hormonal changes (menopause and andropause), meta- osteoblasts induces an imbalance in bone remodeling [6].
bolic dysfunctions (insulin deficiency), and due to adverse
effects of various chronic diseases affecting the skeleton: 3.1.3.1 Caution
these include cardiovascular, gastro-intestinal, musculo- Osteopenic trabeculae are more common in females with an
skeletal, hematological, infectious, nephrological, oncologi- increasing incidence in menopause. The age of the patient is
cal, rheumatic, and hepatic diseases. The skeletal integrity is important in diagnosing osteopenia. If you find osteopenia,
maintained at remodeling sites by continuous cycles of check for comorbidities.

a b

c d

Fig. 3.3 Osteopenia. (a) bone marrow biopsy with marked osteopenia trabeculae are markedly thinned (H&E). (e) Bone marrow biopsy of a
in a 41-year-old woman after bone marrow transplantation due to fol- 25-year-old male after bone marrow transplantation because of acute
licular lymphoma. Note the filigree disconnected bony trabeculae myeloid leukemia. The trabeculae appear osteopenic; they are sur-
(H&E). (b) Bone marrow of a 51-year-old female with liver cirrhosis. rounded by adipose tissue. (H&E). (f) Bone marrow biopsy of a
Trabeculae are disconnected and markedly thinned (H&E). (c) Bone 22-year-old male who underwent transplantation because of acute
marrow biopsy of a 42-year-old male patient who had acute myeloid myeloid leukemia. Bone marrow is normocellular, but the trabeculae
leukemia and chemotherapy (H&E). (d) Bone marrow biopsy of a are thinned for this age (H&E)
31-year-old female with pure red cell anemia and thymoma. The bony
86 3 Bone Changes Found in Bone Marrow Biopsies

e f

Fig. 3.3 (continued)

3 Bone Changes Found in Bone Marrow Biopsies 87

3.1.4 Osteomalacia deficiencies caused by renal, intestinal, or hepatic diseases as

well as from dietary insufficiencies, aluminum toxicity, and
Osteomalacia may be a component of disorders already men- several drugs.
tioned above. Osteomalacia occurs when the newly formed
bone matrix is inadequately mineralized (Fig. 3.4). The 3.1.4.1 Caution
upper limit of trabecular bone surface covered by osteoid is The histological picture of osteomalacia seen in various dis-
24%; higher values of osteoid are suggestive of osteomalacia eases cannot be distinguished from osteomalacia seen in
[7]. The cause of osteomalacia may evolve from vitamin renal osteodystrophy.

a b

Fig. 3.4 (a, b) Bone marrow biopsy of a 73-year-old male with chronic intestinal disease (colitis ulcerosa). Ladewig’s stain highlights the
increased osteoid (red)
88 3 Bone Changes Found in Bone Marrow Biopsies

3.1.5 Osteopetrosis (Marble Bone Disease) encoding the a3 subunit of the vascular proton pump, which
mediates acidification of the bone osteoclast interface. This
In osteopetrosis (OP), a heterogeneous disease group with defect could be located to the osteoclasts [8]. This disorder is
genetic disorders, excessive unbalanced bone formation is evi- often lethal within the first years of life and stem cell trans-
dent leading to trabecular sclerosis (Fig. 3.5). The result is a plantation remains the only curative therapy option. An adult
diminution of the marrow spaces required for an effective form of OP (called benign osteopetrosis) is diagnosed in late
hematopoiesis. The disease can cause pancytopenia, extra- adolescence or adulthood and presents as anemia.
medullary hematopoiesis resulting to hepatosplenomegaly,
cranial nerves compression, and severe growth failure. The 3.1.5.1 Caution
underlying pathophysiological defect in all types of OP is the Keep in mind, that the cause of hepatosplenomegaly in child-
failure of the osteoclasts to reabsorb bone, leading to thick- hood could be osteopetrosis. Also remember that OP can
ened sclerotic bone with poor mechanical properties. Recently, occur in young adults who undergo a bone marrow biopsy
mutations have been identified in the ATP 6 (ICIR GI gene) because of anemia.

a b

GOM GOM

c d

Lad Lad

Fig. 3.5 (a–d) Bone marrow biopsy of a 4-year-old girl diagnosed with osteopetrosis. There is a jumbled bone formation causing obliteration of
the marrow cavities (a and b Gomori’s stain, c and d are stained according to Ladewig)
3 Bone Changes Found in Bone Marrow Biopsies 89

3.1.6 Paget’s Disease of Bone involvement of pelvis, femur, scull, clavicles, ribs, and tibia
is found. The osteoclasts are enlarged and show a variable
Paget’s disease of bone is the second most common meta- number of nuclei, some of the osteoclasts revealing more
bolic bone disease in the Western world [9]. Paget’s disease than 100 nuclei. Due to the rapid bone formation in many
is a localized disorder of uncontrolled bone remodeling. cases, a typical mosaic pattern of the trabecular bone is
Osteoclastic hyperactivity stimulates osteoblasts resulting seen.
in an overgrowth of bone formation (Fig. 3.6). This osteo-
clastic hyperactivity followed by compensatory osteoblas- 3.1.6.1 Caution
tic activity leads to a structurally disorganized mosaic of In the early stage of Paget’s disease, the morphology may
woven bone, which is less compact and more susceptible to mimic the changes seen in primary hyperparathyroidism.
deformities and fracture than normal adult lamellar bone. Osteoclasts in Paget’s disease are markedly increased in
The main symptom is bone pain. In most cases, a focal number and size and can contain up to 100 nuclei.

a b

Fig. 3.6 Paget disease. (a, b) The trabecular bone shows the characteristic mosaic pattern. Huge osteoclasts cover the trabecular surface (a:
H&E; b: MGG). (c) Typical giant osteoclast with multiple nuclei (H&E)
90 3 Bone Changes Found in Bone Marrow Biopsies

References 6. Colucci S, Brunetti G, Rizzi R, Zonno A, Mori G, Colaianni G, Del

Prete D, Faccio R, Liso A, Capalbo S, Liso V, Zallone A, Grano M,
et al. T-cells support osteoclastogenesis in an in vitro model derived
1. Foucar K. Miscellaneous disorders of bone marrow, including stro-
from human multiple myeloma bone disease: the role of the OPG/
mal and bone abnormalities. In: Bone marrow pathology. 2nd ed.
TRAIL interaction. Blood. 2004;104(127):344–7.
Chicago: ASCP Press; 2001. p. 546–84.
7. Bartl R, Frisch B. Principles of bone biopsy pathology. In: Biopsy
2. Al Badr W, Martin KJ. Role of bone biopsy in renal osteodystrophy.
of bone in internal medicine: an atlas and sourcebook, vol. 21.
Saudi J Kidney Dis Transpl. 2009;20(1):12–9.
New York: Springer; 2012. p. 65–74.
3. Delling G. Generalisierte Osteopathien. In: Remmele W, editor.
8. Sreehari S, Naik DR, Malini Eapen M. Osteopetrosis: a rare cause
Pathologie, vol. 3. Berlin: Springer; 1985. p. 689.
of anemia. Hematol Rep. 2011;3(1):e1.
4. Rolighed L, Rejnmark L, Christiansen P. Bone involvement in pri-
9. Abelson A. A review of Paget’s disease of bone with a focus on the
mary hyperparathyroidism and changes after parathyroidectomy.
efficacy and safety of zoledronic acid 5 mg. Curr Med Res Opin.
US Endocrinol. 2013;9(2):181–4.
2008;24(3):695–705.
5. Zheng K, Wang Z, Wu SJ, Ye ZM, Xu SF, Xu M, Hu YC, Yu
XC. Giant cell tumor of the pelvis: a systematic review. Orthop
Surg. 2015;7(2):102–7.
4
Non-hematological Malignancies
in the Bone Marrow

In patients with known or suspected non-hematological mens from different areas of a single iliac crest are recom-
malignancies, the indications for a bone marrow biopsy mended [1]. In adults, the most common primary tumors
include the following: metastasizing into the bone marrow are breast-, lung-, prostate-,
gastrointestinal tract-, renal cell, and genitourinary carcino-
1. Integral part of the initial staging mas, but also metastases from malignant melanomas and
2. Metastases are suspected clinically rarely sarcomas can be detected.
3. For monitoring the therapy and follow-up In children, the most common tumors, metastasizing
4. For unexplained fever into the bone marrow are neuroblastomas, Ewing sarco-
5. For hypercalcemia mas, rhabdomyosarcomas, osteosarcomas, and rarely
6. For high LDH-levels medulloblastomas.
7. For suspicious X-ray or scan Marrow infiltration by metastatic tumor in most cases is
focal, but diffuse infiltration can occur. In the bone marrow,
A bone marrow biopsy is useful in staging and determin- areas with metastases commonly reveal reticulin and/or col-
ing of bone marrow metastases of solid tumors. The diagno- lagen fibrosis and are most pronounced in marrows with
sis of bone marrow metastases is important because of greater degrees of marrow infiltration. Other stromal reac-
treatment methods and also survival. Anemia, thrombocyto- tions may include edema, increased blood vessel formation,
penia, leukopenia, bi-cytopenia, and pancytopenia, but also an inflammatory infiltrate at the margin of the neoplasm and
leukemoid reactions and leucoerythroblastic reactions can be lytic, as well as osteoblastic trabecular changes. Lytic
found after the metastatic infiltration of the bone marrow. changes show an overall loss of bony trabeculae with often
Occasionally, bone marrow metastases are verified without an increased number of osteoclasts. Osteoblastic changes
any changes in hematologic parameters. Metastases in the are combined with new bone formation and an increased
bone marrow can be seen in patients with widely metastatic number of osteoblasts, occasionally revealing a pagetoid
solid tumors, but also in tumors firstly diagnosed by bone appearance.
marrow biopsies. In many, or even most cases, a classification of the metas-
The incidence of bone marrow metastases by various tases in the bone marrow is possible with immunohistochem-
malignant tumors has changed over the years due to the ear- ical stains (Figs. 4.1, 4.2, 4.3, and 4.4). Overall, the
lier diagnosis of some tumors and due to the improved detec- immunohistochemical characterization of metastatic carci-
tion of micrometastases in the bone marrow. Since metastatic nomas to the bone marrow shows a good correlation with the
infiltrates are usually focal, the length of the bone marrow established staining pattern of the primary tumors [2].
biopsy material is important for the detection of metastases, Some examples of neoplastic bone marrow infiltrations
and bilateral bone marrow biopsies or double biopsy speci- (Figs. 4.1–4.9) are shown.

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 91

C. Beham-Schmid, A. Schmitt-Graeff, Bone Marrow Biopsy Pathology,
Essentials of Diagnostic Pathology, https://doi.org/10.1007/978-3-662-60309-3_4
92 4 Non-hematological Malignancies in the Bone Marrow

4.1 Caution tion might be missed without immunohistochemical stains.

Cells of Langerhans cell histiocytosis are detected by posi-
Micrometastases in a patient with known carcinoma might tivity for antibodies to S-100-protein, CD1a, and langerin.
be missed without immunohistochemical stains. Thus, at Follicular dendritic cell sarcoma is a rare neoplasm and
least immunohistochemistry with an antibody to cytokeratin extremely rarely the bone marrow is infiltrated (see Case 6).
should be done in patients with known carcinoma. It accounts for only 0.4% of soft-tissue sarcomas [5]. This
In children, metastases from neuroblastoma can be easily tumor usually occurs in lymph nodes, but it can occur in
missed in cases with only small neoplastic foci. It is important extranodal sites such as in the liver, lungs, tonsils, or spleen
to investigate biopsies with immunohistochemical methods. and even in the bone marrow.
Antibody to CD71 (transferrin receptor) does not only stain Bone marrow infiltration by neuroblastoma (see Cases 7
cells of erythroid lineage, but also carcinoma cells (see Fig. 4.1f and 8) is seen in about 50% of patients [6]. The morphology
in Case 1). In breast carcinomas, CD71 appears to be a candi- of the tumor cells is variably resembling ganglion cells
date marker of a subgroup of ER+/luminal-like breast cancer (Case 7) or small blue round cell tumor (Case 8).
characterized by poor outcome and resistance to tamoxifen [3]. Metastases of angiosarcoma to the bone marrow are
Langerhans cell histiocytosis (see Case 5) is a reactive extremely rare (case 9) and more often found in Kaposi’s sar-
proliferative disease of unknown etiology, occurring more coma. However, angiosarcoma arising in an irradiated breast is
frequent in children than in adults [4]. Bone marrow infiltra- being reported in the literature with increasing frequency [7].
4 Non-hematological Malignancies in the Bone Marrow 93

Case 1 (Fig. 4.1)

a b

c d

CKAE1 CK7

e f

GATA3 CD71

g h

estrogen progesteron

Fig. 4.1 (a–h) Case 1 (breast carcinoma): Bone marrow biopsy of a The tumor cells are stained with an antibody to CKAE1/3 (c) and with
69-year-old female with suspected carcinoma. The patient presented an antibody to CK7 (d). GATA3-positivity (e) gives the link to a metas-
with bone pain, anemia, and increased LDH. In H&E stained sections tasis from a breast carcinoma. A strong positivity for all tumor cells is
(a, b), an infiltration by carcinoma is seen. Hematopoiesis is replaced seen with an antibody to CD71 (Transferrin receptor), (f). The tumor
by an extensive neoplastic epithelial tumor with new bone formation. cells are estrogen-positive (g) and progesterone-negative (h)
94 4 Non-hematological Malignancies in the Bone Marrow

Case 2 (Fig. 4.2)

a b

c d

CKAE1 CK7

e f

CK20 TTF1

Fig. 4.2 (a–f) Case 2 (lung carcinoma): A 34-year-old female devel- H&E stained sections, the bone marrow shows extensive replacement
oped dyspnea after giving birth by Caesarean section. She developed by metastatic adenocarcinoma with marked new bone formation (a, b).
pleural effusion. Pneumonia was suspected, but despite treatment, her The tumor cells are strongly cytokeratin-positive (c, CKAE1/3) and
condition worsened. MR-tomography revealed pathologic signals in the show positivity for CK7 (d) and also CK20 (e). TTF-1-positivity of the
whole spinal column, and a bone marrow biopsy was performed. In tumor cells (f) is suggestive of a primary in the lung
4 Non-hematological Malignancies in the Bone Marrow 95

Case 3 (Fig. 4.3)

a b

c d

CKAE1 CD56

e f

synaptophysin TTF1

Fig. 4.3 (a–h) Case 3 (small cell carcinoma of lung): A 55-year-old patchy infiltration by rather small tumor cells (a, b: H&E). Strong reac-
female presented with leucocytosis and polyglobulia. Medical exami- tivity of the tumor cells is seen with antibodies to CKAE1/3 and CD56
nations were susceptive of a centrally located carcinoma of the bron- (c, d). Few tumor cells are weakly synaptophysin-positive (e). Strong
chus. A bone marrow biopsy was performed revealing a dense reactivity of the tumor cells with an antibody to TTF1 confirming the
infiltration by small carcinoma cells with neuroendocrine differentia- primary tumor in the lung (f)
tion, confirming small cell carcinoma of the bronchus. Diffuse and
96 4 Non-hematological Malignancies in the Bone Marrow

Case 4 (Fig. 4.4)

a b

c d

CK5/6

Fig. 4.4 (a–d) Case 4 (squamous cell carcinoma): A 59-year-old male The bone marrow was infiltrated by metastases of squamous cell carci-
with squamous cell carcinoma of the hypopharynx, diagnosed noma (a–c: H&E). Note the new bone formation (a, b). Squamous cell
12 months previously, developed increasing pancytopenia. A bone mar- carcinoma was immunohistochemically verified by staining with an
row biopsy was done with the suspected diagnosis of myelodysplasia. antibody to CK5/6 (d)
4 Non-hematological Malignancies in the Bone Marrow 97

a b

c d

NSE CD56

Fig. 4.8 (a–d) Case 8 (neuroblastoma): In this case of a 2-year-old tumor (a, H&E). The infiltrating cells are immunohistochemically reac-
boy, the rather fragmented bone marrow biopsy is infiltrated by tumor tive with antibodies to a neuroblastoma marker (b), NSE (c), and CD56
cells of neuroblastoma with the morphology of a small blue round cell (d)
4 Non-hematological Malignancies in the Bone Marrow 101

Case 9 (Fig. 4.9)

a b

c d

FVIII-rag

Fig. 4.9 (a–d): Case 9 (angiosarcoma): A 75-year-old woman devel- area). Higher magnification reveals irregular dissecting vascular chan-
oped angiosarcoma of the breast 32 months after lumpectomy and nels lined by pleomorphic cells with prominent nucleoli (b, c, H&E).
radiotherapy for a ductal breast carcinoma. The angiosarcoma infil- Reactivity with an antibody to FVIII-rag (d) in the tumor cells and in
trated the chest wall, liver, and bone marrow. In the bone marrow, a megakaryocytes (top right)
patchy infiltrate absorbing a whole marrow space is seen (a, H&E, right
102 4 Non-hematological Malignancies in the Bone Marrow

References 4. Kumar M, Sachdeva MUS, Naseem S, Ahluwalia J, Das R,

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Aleskandarany M, Paish EC, Douglas Macmillan R, Nicholson Indian J Hematol Blood Transfus. 2015;31(1):57–60.
RI, Ellis IO, Gee JM. Transferrin receptor (CD71) is a marker of 7. Ashour O, Fasih T. Radiation induced angiosarcoma of the breast:
poor prognosis in breast cancer and can predict response to tamoxi- case series—review at a single breast screening institution and
fen. Breast Cancer Res Treat. 2010;119(2):283–93. https://doi. review of the literature. Arch Can Res. 2016;4:2.
org/10.1007/s10549-009-0345-x.. Epub 2009 Feb 24.
Increase of Plasma Cells,
Reactive and Neoplastic 5

5.1 Reactive Plasmacytosis Case History 1

A 50-year-old male with a diagnosis of acute myeloid leuke-
Reactive plasmacytosis is common in patients with infec- mia 7 years previously, was admitted with high fever, pneu-
tious diseases (especially HIV), cirrhosis, and rheumatic dis- monia, and slight anemia. To exclude a hematologic
orders as well as autoimmune diseases, especially in patients neoplasm, especially a relapse from AML, a bone marrow
with lupus. biopsy was performed. In this hypercellular bone marrow, a
A reactive increase of plasma cells can be differentiated marked increase of reactive plasma cells was evident
from monoclonal plasma cell neoplasms by immunohisto- (Fig. 5.1a–d). Further evaluation of the patient revealed
chemical investigation with antibodies to light chains kappa Legionnaires’ disease.
and lambda. In contrast to neoplastic plasma cells, which
often show CD56-positivity and reactivity for Cyclin D1,
reactive plasma cells usually do not express these markers. 5.1.1 Caution
Also, the pattern of plasma cell infiltration is quite helpful,
since plasma cells in reactive conditions tend to surround In some instances, a marked increase of reactive plasma cells
blood vessels and occur in small clusters rather than in is seen in the bone marrow (up to 30%, rarely up to 50%,
large sheets or aggregates away from blood vessels [1]. especially in HIV-infected patients). This increase of plasma
Secretory plasma cells with Russell- or Dutcher-bodies can cells might simulate a bone marrow infiltration by plasma
be found. However, even if the plasma cells look overtly cell myeloma. It is essential to investigate the plasma cells
malignant, it is recommended that plasma cell clonality is for clonality using antibodies to light chains.
proven by light chain immunohistochemistry and/or flow It is advisable to get an accurate plasma cell count using sen-
cytometry. sitive plasma cell markers such as Vs38c, CD38, and CD138.

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 103

C. Beham-Schmid, A. Schmitt-Graeff, Bone Marrow Biopsy Pathology,
Essentials of Diagnostic Pathology, https://doi.org/10.1007/978-3-662-60309-3_5
104 5 Increase of Plasma Cells, Reactive and Neoplastic

a b

MGG Vs38c

c d

kappa lambda

Fig. 5.1 Reactive plasmacytosis. (a) In the hypercellular bone marrow, d) With antibodies to light chains (c: kappa; d: lambda) the plasma cells
a marked increase of mature plasma cells is seen (MGG). (b) The are polyclonal
plasma cells are highlighted with a plasma cell marker (Vs38c). (c,
5 Increase of Plasma Cells, Reactive and Neoplastic 105

5.2 Monoclonal Gammopathy plasma cell infiltration without myeloma-defining symptoms

of Undetermined Significance (MGUS) [4]. Clinically, the distinction between MGUS and SMM is
important since the risk of progression to MM is 10% in
MGUS is a clinically asymptomatic premalignant clonal SMM and only 1% in MGUS [5].
plasma cell or lymphoplasmacytic proliferative disorder. It is
defined by the presence of a serum monoclonal protein
(M-protein) at a concentration <3 g/dL, a bone marrow with 5.2.2 Epidemiology
<10% monoclonal plasma cells, and, most importantly,
absence of organ damage like lytic bone lesions, anemia, MM comprise about 15% of hematologic neoplasms. The
hypercalcemia, renal insufficiency, and hyperviscosity. incidence has been increasing by 0.7 % each year for the last
MGUS is mostly detected as an incidental finding when 10 years, but mortality has come down by 1.7 each year over
patients undergo a protein electrophoresis as part of an eval- the same period [6]. Children are not affected by this disease,
uation for various clinical symptoms. The detection of and rarely MM-patients are under the age of 30.
MGUS requires evaluation for a possible plasma cell neo-
plasm. The risk of progression to plasma cell myeloma in
patients with MGUS is 1% per year [2]. In patients with 5.2.3 Morphology
MGUS, the plasma cells are characteristically, but not
always, monoclonal by immunohistochemical light chain The infiltration pattern of plasma cell myeloma in the bone
investigation. A bone marrow aspirate and biopsy must dem- marrow is variable. There are cases with dense infiltration, and
onstrate fewer than 10% clonal plasma cells, or in the case of cases with diffuse or interstitial clusters or nodules, or cases
IgM MGUS, fewer than 10% infiltration by clonal lympho- with solitary patchy infiltrates. Commonly, when 30% or more
plasmacytic cells. of the bone marrow volume is infiltrated by plasma cells, a
MGUS may occur as non-IgM MGUS, IgM MGUS, or diagnosis of plasma cell myeloma is probable, but (see above)
light chain MGUS. In the bone marrow, MGUS show fewer reactive plasmacytosis has to be excluded. If a patchy infiltra-
than 10% clonal lymphoplasmacytic/plasma cells. tion is seen, a diagnosis of plasma cell myeloma is likely, even
Furthermore, patients with non-IgM MGUS and light chain when the percentage of plasma cells is less than 30%.
MGUS do not reveal lytic bone lesions, anemia, hypercalce- In most marrows infiltrated by plasma cell myeloma,
mia, and renal insufficiency related to the plasma cell increased osteoclastic activity is evident.
proliferation. In the following series of figures, various examples of
morphological presentations of plasma cell myeloma are
shown.
5.2.1 Plasma Cell Myeloma (MM) Not only the infiltration pattern of MM shows great vari-
ability, but also the morphology of the infiltrating plasma
The diagnosis of MM requires special diagnostic criteria cells is variable (see Fig. 5.2). Mature plasma cells, which
including the clonal plasma cell percentage, the quantity of can be seen in well-differentiated plasma cell myeloma (see
monoclonal protein, and the presence of CRAB symptoms, Fig. 5.3, Case 1), show a cartwheel nuclear chromatin pat-
which are anemia, bone lesions, hypercalcemia, and renal tern, a pale blue cytoplasm, and occasionally a perinuclear
insufficiency. According to the revised criteria of the inter- “hof.” In MM, the plasma cells may show prominent nucleoli
national myeloma working group (IMWG), the percentage (according to plasmablasts), there can be multinuclearity,
of bone marrow plasma cells is discriminating between polymorphism with cleaved nuclei, crystalline inclusions,
MGUS and MM. Thus, MM requires >10% clonal plasma and asynchronies of nucleocytoplasmic relation. There may
cells [3]. be neoplastic plasma cells with anaplastic appearance (see
Smoldering plasma cell myeloma (SMM) is an asymp- Fig. 5.4, Case 2) and spindle-shaped plasma cells. Some
tomatic clonal plasma cell disorder defined by the presence cases of plasma cell myelomas show marked sclerosis of the
of ≥3 g/dL serum M-protein and/or 10–60% bone marrow surrounding stroma (see Fig. 5.5, Case 3).
106 5 Increase of Plasma Cells, Reactive and Neoplastic

a b

c d

e f

CD56 κ λ

Fig. 5.2 This shows morphologic variants of plasma cells in plasma roglobulinemia. (c) The bone marrow of this 75-year-old woman is
cell myelomas. (a, b) Bone marrow infiltration by plasma cell myeloma infiltrated by plasma cell myeloma with foamy appearance of the
in a 53-year-old woman: on the one hand (a left side), the patchy infil- plasma cells. (d) In higher magnification, some multinucleated cells
trating plasma cells show a characteristic plasmacytic appearance, on with prominent eosinophilic nucleoli are visible. (e) The neoplastic
the other hand (right side) the tumor cells reveal an abundant eosino- plasma cells are reactive with an antibody to CD56. (f) Clonality is
philic morphology (H&E). (b) Higher magnification of the plasma cells evident by expression of the light chain kappa (left), whereas the plasma
with eosinophilic cytoplasm. These plasma cells are called “flame cells are lambda-negative (right)
cells.” Flame cells can occasionally be observed in Waldenström’s mac-
5 Increase of Plasma Cells, Reactive and Neoplastic 107

a b

c d

Vs38c Vs38c

e f

κ λ

Fig. 5.3 Case 1. This shows dense bone marrow infiltration of plasma remodeling is also highlighted with the antibody Vs38c, which is
cell myeloma in a 39-year-old woman with plasmacytic appearance and known to stain osteoblasts. (e) The plasma cells are not reactive with an
abnormal expression of CD23. (a, b) Dense infiltration of the bone mar- antibody to kappa light chain. (f) Monoclonality of the plasma cell
row by plasmacytic type of plasma cell myeloma (H&E). (c) Strong population is shown by strong lambda-positivity. (g) The monoclonal
staining of plasma cells with an antibody to Vs38c (antigen p63: this plasma cells are reactive with an antibody to CD23. (h) Only single
protein is an intracellular type II transmembrane protein which is local- plasma cells are Cyclin D1-positive
ized in the rough endoplasmic reticulum) (d) The increased osseous
108 5 Increase of Plasma Cells, Reactive and Neoplastic

g h

CD23 Cyclin D1

Fig. 5.3 (continued)

a b

c d

CD138 λ

Fig. 5.4 Case 2. This shows dense bone marrow infiltration by an ana- cells shows polymorphic blasts with clear irregularly formed nuclei and
plastic variant of plasma cell myeloma. In H&E stained sections, the multiple nucleoli (H&E). (c) Strong reactivity of the tumor cells with a
morphology of the infiltrating cells does not reveal the typical plasma- plasma cell marker (CD138). (d) The neoplastic plasma cells are
cytic differentiation. (a) Highly hypercellular bone marrow with dense lambda-positive (not shown is kappa-negativity)
infiltration of tumor cells (H&E). (b) High magnification of the tumor
5 Increase of Plasma Cells, Reactive and Neoplastic 109

a b

c d

CD56 CD56

e f

GOM Vs38c

Fig. 5.5 Case 3. Bone marrow biopsy of a 63-year-old female with CD56-positivity of plasma cells, also paratrabecular endothelial cells
dense sclerosis (sclerosing type of plasma cell myeloma). (a) Even in show reactivity with CD56. (e) Gomori’s stain reveals collagen fibrosis.
H&E stained section a dense sclerosis can be seen. In addition, there is (f) Positivity of the plasma cell with Vs38c. (g) The infiltrating plasma
marked osseous remodeling with irregularly formed bone. (b) Higher cells are monoclonal expressing the light chain kappa. (h) No reactivity
magnification shows plasma cell infiltration within the sclerotic stroma of the plasma cells with an antibody to light chain lambda
(H&E). (c) The plasma cells are CD56-positive. (d) In addition to the
110 5 Increase of Plasma Cells, Reactive and Neoplastic

g h

κ λ

Fig. 5.5 (continued)

5.2.4 Immunohistochemistry undermined significance. Without clinical information, an

increase of monoclonal plasma cells in the bone marrow can
Immunohistochemistry with plasma cell markers (e.g., be diagnosed as “plasma cell neoplasm.”
Vs38c, CD38, CD138) is helpful in estimating the exact Awareness of rare anaplastic or sarcomatoid variants of
number of plasma cells. Monoclonality is confirmed by plasma cell myelomas is important. Such plasma cell myelo-
applying antibodies to light and heavy chains. The tumor mas can be confused with high grade sarcoma, anaplastic
cells characteristically have monotypic cytoplasmic Ig, but carcinoma, or lymphoma. Immunohistochemical investiga-
lack surface Ig. In addition to the plasma cell markers, cells tions and thorough clinical and pathological correlation is
of MM are CD79a-positive. Up to 80% of MM aberrantly essential. Plasma cell myeloma might behave like a chame-
express CD56, furthermore in some cases aberrant expres- leon with its anaplastic appearance.
sion of CD20, CD23 (see Fig. 5.3, Case 1), CD117c (Fig. Reactive plasma cells can show variations in cell size.
5.6a, b), CD10, and CD52 can be observed. Many cases of Some cases of MGUS, plasma cell myelomas, or even
plasma cell myelomas are Cyclin D1-positive, correlating cases with reactive plasmacytosis may show immunoglobu-
with the presence of t(11;14)(q13;q32). lin inclusion bodies resembling a bunch of grapes. These
plasma cells are referred to as Mott cells or morula.
Reactive and neoplastic plasma cells may contain a single
5.2.5 Genetic Abnormalities inclusion in the cytoplasm (the so-called Russell-body) or
within the nucleus (the so-called Dutcher-body).
Many chromosomal rearrangements are found in the tumor Problems might be caused by the rare forms of non-
plasma cells at the time of diagnosis. The most frequent ones secretory plasma cell myeloma without detectable M com-
are monosomy 13 (45% of the patients), duplication of the ponent in the serum or urine. These rare variants are mostly
long arm of chromosome 1 (1q gains, 30% to 35% of the combined with an atypical morphology and aberrant immu-
patients), and different deletions involving the 1p, 6q, 8p, nophenotype often expressing CD23 [8].
12p, 14q, 16q, 17p, or 20p chromosomal regions [7]. Plasma cell leukemia is a rare variant of plasma cell
myeloma. It presents either as a progression of a previously
diagnosed plasma cell myeloma (secondary form) or as an
5.2.6 Caution initial manifestation of disease (primary form). The findings
on bone marrow aspiration and biopsy are similar to those
New or untreated cases with less than 10% bone marrow found in plasma cell myeloma without plasma cell leukemia;
plasma cells, even if they are shown to be monotypic, must however, the leukemic form usually lacks expression of
be placed into the category of monoclonal gammopathy of CD56.
5 Increase of Plasma Cells, Reactive and Neoplastic 111

5.3 lasma Cell Myeloma with Amyloid

P 5.3.1 Caution
Deposition (Fig. 5.6)
Be careful, when staining for amyloid P is used. Amyloid P
Amyloidosis in the bone marrow in most cases is associated component is present in all types of amyloid, but it is also
with clonal disorders, like plasma cell myeloma or lympho- found in normal elastic tissue and basement membranes.
plasmacytic lymphoma/Waldenström macroglobulinemia. Thus, a positive amyloid P is only relevant in the context of
Amyloid, a fibrillar material, is deposited extracellularly and a positive Congo red stain.
is found in the wall of small vessels and/or interstitial [9]. Amyloid A component is only found in secondary
Amyloid is easily detectable in Giemsa stained sections, amyloidosis and Mediterranean type amyloidosis.
Congo red stain or immunohistochemical stains with antibod- Amyloid P-positive and amyloid A-negative cases include
ies to amyloid A or P are applied for verification. Amyloid primary amyloidosis, dialysis-associated amyloid (beta2
deposits demonstrate apple-green birefringence when stained microglobulin amyloid), and also hereditary amyloidosis
with Congo red and viewed under polarizing microscopy [10]. [11].
In Giemsa stained sections, amyloid appears as bluish deposits Differential diagnosis to amyloid deposits includes gelati-
within the vessel walls or as cloud-like amorphous material in nous transformation of the bone marrow, fibrinoid necrosis,
the interstitium. In routinely H&E stained sections, amyloid and also edema.
appears as amorphous smudgy eosinophilic deposit.

a b

CD117c

Fig. 5.6 Plasma cell myeloma with expression of CD117c (a H&E, b vessel wall and small cloud-like amyloid deposits in the interstitium
CD117c). (c–h) Plasma cell myeloma with amyloidosis. In a 75-year- (left from vessel wall). (f) Amyloid deposits within a vessel wall in a
old male patient with plasma cell myeloma, amyloid deposits are easily rather large vessel in the subcortical area. (g, h) In the bone marrow of
visible in Giemsa stained sections. (c) Even at low magnification, thick- a 67-year-old woman with treated plasma cell myeloma extensive inter-
ened vessel walls with bluish deposits can be seen. (d) Plasma cells are stitial amyloid deposits are evident (g H&E) (h) immunohistochemical
surrounding a small vessel with amyloid deposits. (e) Amyloid within a stain with an antibody to amyloid P
112 5 Increase of Plasma Cells, Reactive and Neoplastic

c d

e f

g h

Amyloid P

Fig. 5.6 (continued)

5 Increase of Plasma Cells, Reactive and Neoplastic 113

5. Rajkumar SV, Landgren O, Mateos M-V. Smoldering multiple

References myeloma. Blood. 2015;125(20):3069–75.
6. Jagannath S, Richardson PG, Munshi NC. Multiple myeloma and
1. Hasserjian RP. Reactive versus neoplastic bone marrow. Problems other plasma cell dyscrasias. Cancer management; 2016.
and pitfalls. Arch Pathol Lab Med. 2008;132:587–94. 7. Corre J, Munshi N, Avet-Loiseau H. Genetics of multiple myeloma:
2. Kyle RA, Therneau TM, Rajkumar SV, Offord JR, Larson DR, another heterogeneity level? Blood. 2015;125(12):1870–6.
Plevak MF, Melton LJ. A long-term study of prognosis in mono- 8. Gupta R, Saxena P, Garg R. A rare case of non secretory plasma
clonal gammopathy of undetermined significance. N Engl J Med. cell leukemia with unusual morphology and aberrant expression of
2002;346(8):564–9. CD23 and CD56. J Hematol Transf. 2017;5(1):1058.
3. Lee N, Moon SY, Lee J-H, Park H-K, Kong S-Y, Bang S-M, Lee 9. Leung KF, Ma ES. Amyloid deposits in the bone marrow. Br J
JH, Yoon S-S, Lee DS. Discrepancies between the percentage of Haematol. 2003;121:679.
plasma cells in bone marrow aspiration and BM biopsy: impact on 10. Sanchorawala V. Light-chain (AL) amyloidosis: diagnosis and
the revised IMWG diagnostic criteria of multiple myeloma. Blood treatment. Clin J Am Soc Nephrol. 2006;1(6):1331–41.
Cancer J. 2017;7:e530. 11. Jang BG, Koh Y, Seo J-W. Immunohistochemical classification
4. Mateos MV, Landgren O. MGUS and smoldering multiple myeloma: of amyloid deposits in surgical pathology. Basic Appl Pathol.
diagnosis and epidemiology. Cancer Treat Res. 2016;169:3–12. 2009;2(1):1–8.
Inflammatory and Infectious Diseases
6

The investigation of the bone marrow by bone marrow 4. Chronic inflammation, “fibrotic” type: There is an
biopsy is one of the most valuable diagnostic tools for evalu- increase in reticulin and/or collagen fibers with reduction
ating hematologic disorders. Furthermore, bone marrow of fat cells and hematopoiesis (“sclerosing myelitis”). In
analysis has great importance in the evaluation of non- most cases, reactive T-lymphocytes and plasma cells are
hematologic conditions. Various chemical toxins, including increased, edema might be present and even new bone
minerals, as well as radiation may severely affect the bone formation can be evident. This condition is often found in
marrow cells and stroma. In patients with fever of unknown infections (especially HIV) and lupus as well as other
origin (FUO) and in patients with autoimmune deficiency autoimmune diseases [2] (see Fig. 6.3a, b).
syndrome (AIDS), the marrow may reveal various microor- 5. Chronic inflammation “proliferative” type: The
ganisms, such as leishmaniasis and tuberculosis, and fungal bone marrow is either normocellular or hypercellular
as well as viral infections. with an increase of reactive T-lymphocytes, plasma
Roughly, nonspecific inflammatory changes in the bone cells, mast cells, and/or eosinophils. This condition is
marrow can be subdivided into several types; however, these mainly seen in patients with rheumatoid arthritis [3]
inflammatory changes may occur in combined forms. (see Fig. 6.3c, d).
6. Chronic inflammation, “leukemoid” type: The bone
1. Acute inflammation, “exudative” or “necrotic” type: marrow is slightly or marked hypercellular with increased
Necrosis of hematopoietic cells and capillaries as well as neutrophils or eosinophils and often increased megakary-
edemas may be present (see Fig. 6.1a, b). Some hemato- ocytes. There is an increase in the estimated M:E ratio,
poiesis may be left. In addition, bone necrosis, especially but maturation synchrony is often unaffected. Due to
caused by chemotherapy, might be present. increases in granulopoietic cells, quantitative bone mar-
2. Acute inflammation, “hemorrhagic” type: Extensive row differential cell counts reveal an increased M:E ratio
hemorrhage is associated with hypoplasia of hematopoi- [4] (see Fig. 6.3e, f).
etic cells. This condition is seen, for example, in hepatitis 7. Chronic inflammation “granulomatous” type (see
B infection or in patients with severe histiocytic activa- Fig. 6.4a–f): In this condition, giant cell granulomas,
tion syndrome (see Fig. 6.1c, d) [1]. lipoid granulomas, or epithelioid cell granulomas are
3. Chronic inflammation, “atrophic” type: In addition to seen. Granulomas are nodular aggregates of inflamma-
edema, an increase of lymphocytes (mainly reactive tory cells, mainly consisting of epithelioid cells (Fig. 6.4a,
T-cells), plasma cells, and mast cells is often seen. b) and/or giant cells with various numbers of lympho-
Hematopoiesis is markedly reduced. Synonymously to cytes, plasma cells, mast cells, and fibroblasts in and
“atrophic” type, this condition is known as gelatinous around the granulomas. Occasionally, inclusion bodies
transformation, serous atrophy, or exudative myelitis. within giant cells can be found. Granulomas can be seen
Such changes may occupy all marrow spaces or circum- in hypo-, normo-, or hypercellular bone marrow as a
scribed marrow areas. This condition is seen in various response to various processes including sarcoidosis,
diseases, especially in patients with poor nutrition or mal- tuberculosis, collagen diseases, chemotherapy, fungi,
nutrition, chronic infections, and malignancies. The bone drugs, and various malignant diseases (paraneoplastic),
marrow is markedly hypocellular; also the fat cells are especially lymphomas [5]. Granulomas can also be found
reduced (see Fig. 6.2a–h). without any manifest diseases [6].

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 115

a b

c d

Fig. 6.1 Acute inflammation. (a, b) Bone marrow of a 16-year-old edematous stroma (H&E). (c, d) Hemorrhagic inflammation of the
pancytopenic male after chemotherapy because of a Ewing sarcoma. bone marrow in a pancytopenic 55-year-old female with hepatitis B
There is exudative inflammation with reduced hematopoiesis and (H&E)

a b

MGG MGG

Fig. 6.2 Chronic inflammation, atrophic type. (a, b) Gelatinous trans- bone marrow with gelatinous transformation. (f) Another bone marrow
formation of all marrow spaces in an 18-year-old pancytopenic female space of the same patient stained with PAS. In this marrow space, a few
with anorexia nervosa. Hematopoiesis is highly reduced; marked exu- hematopoietic cells are evident. (g) Immunohistochemical stain with an
dative myelitis is found (MGG). (c, d) In H&E stained section, the mar- antibody to CD71 shows a few erythropoietic cells. (h) A single mega-
row spaces in this young woman reveal stromal gelatinous karyocyte is detected using an antibody to CD42b
transformation without hematopoietic cells. (e) Gomori’s stain of the
6 Inflammatory and Infectious Diseases 117

c d

e f

GOM

g h

CD71 CD42b

Fig. 6.2 (continued)

118 6 Inflammatory and Infectious Diseases

a b

c d

CD3

e f

MPO

Fig. 6.3 Chronic inflammation. (a, b) Fibrotic type of inflammation in lar (c, MGG). Immunohistochemical stain with an antibody to CD3
a 26-year-old female patient with systemic lupus. The bone marrow shows increased T-lymphocytes (d). (e, f) Leukemoid type of chronic
biopsy was taken because of marked anemia and slight leukopenia as inflammation in a 67-year-old male patient with colonic carcinoma.
well as thrombocytopenia. In H&E stained section, the marrow is hypo- Overview of the MGG stained bone marrow shows highly hypercellular
cellular for age (a), Gomori’s stain reveals irregular fibrosis (b). (c, d) marrow spaces (e), mainly caused by an increased granulocytopoiesis
Proliferative type of inflammation in a 56-year-old woman with a long (f), visualized by immunohistochemical stain with an antibody to MPO
history of rheumatoid arthritis. The bone marrow is highly hypercellu-
6 Inflammatory and Infectious Diseases 119

a b

c d

e f

Fig. 6.4 Chronic inflammation (a, b) Chronic inflammation, granulo- thelioid cell granulomas. This patient suffered of night sweats and dys-
matous type. H&E stained bone marrow in a young 28-year-old woman pnea as well as back pain. PCR studies of the bone marrow with this
with fever of unknown origin. An epithelioid cell granuloma is closely marked granulomatous infiltrates for germ analysis, including myco-
attached to the bone. Clinical workup revealed sarcoidosis. (c–f) The bacteria were negative. Clinical work-up finally yielded sarcoidosis
bone marrow biopsy of this 52-year-old woman reveals masses of epi- (H&E)
120 6 Inflammatory and Infectious Diseases

6.1 Infectious Diseases lation. Patients with XLP are usually young male children
with mutations of the SAP/SH2D1A gene. If these patients
For most systemic infections, the bone marrow reveals non- survive the EBV infection (infectious mononucleosis), they
specific changes which, however, are compatible with a reac- might develop lymphomas or dysgammaglobulinemia.
tive, non-neoplastic process. Such reactive changes might be
the occurrence of granulomas or hemophagocytosis (see Case History 1
Figs. 6.5 and 6.6). In the following chapter, various charac- A 68-year old female presented to Intensive Care Unit with
teristic morphological features are described. high fever, weight loss, and rash as well as pancytopenia.
Furthermore, evidence of hemophagocytosis with hyperferri-
tinemia was found. Pneumonia and hepatitis B were diagnosed.
6.1.1 Hemophagocytic Syndrome She had a past history of multidrug-resistant tuberculosis.
In the hypercellular bone marrow biopsy marked hemo-
Hemophagocytic syndromes (HPS) are either primary/ phagocytosis was found, suggestive of secondary infection-
familiar diseases or secondary reactive disorders caused by associated hemophagocytic syndrome (Fig. 6.5a, b) [9].
various infections, neoplasms, or vascular collagen diseases Despite intensive treatment, the patient died of multiple
[7]. The bone marrow is hypercellular with myeloid hyper- organ failure.
plasia in many cases (Fig. 6.5), but also hypocellularity can
be found. The bone marrow (see Fig. 6.5) shows an increase Case History 2
of histiocytes with phagocytosis of erythropoietic and gran- A 87-year-old male patient was admitted with massive epi-
ulopoietic cells and occasionally also platelets. While hemo- staxis and high fever. Past history revealed cardiac problems
phagocytosis is easily detected, the underlying disease and carcinoma of the urinary bladder 12 years previously. At
causing hemophagocytosis often remains obscure. The admission, the patient was pancytopenic: Leuko 0.93·109/L,
infection-associated HPS can be caused by parasitic or bac- Ery 4.24·1012/L, Hb 11.8 g/dL, Hkt 36.9%, MCV 87.0 fL,
terial infections, but in most cases HPS is caused by viral MCH 27.8 pg, MCHC 32.0 g/dL, Thrombo 51·109/L, Ferritin
infections, especially EBV. EBV-associated HPS compro- was increased with 888.63 ng/mL. Bone marrow cytology
mises two distinct entities: (1) patients with sporadic hemo- revealed hypocellularity with dysplastic erythropoietic cells
phagocytic lymphohistiocytosis and (2) patients with and hemophagocytosis (Fig. 6.5c, d) A bone marrow biopsy
X-linked lymphoproliferative disorder (XLP) (Fig. 6.6). showed highly hypocellular bone marrow with increased
Both diseases originate from an abnormal T-cell immune macrophages, many of which showed hemophagocytosis,
response to the cells infected by EBV. XLP is a rare primary especially erythrophagocytosis (Fig. 6.5e–h). The patient
immunodeficiency first described in 1975 by Purtilo et al. was treated symptomatically and died 2 days after
[8]. The disease is accompanied by severe immune dysregu- admission.

a b

CD68

Fig. 6.5 (a) (Case history 1) Hypercellular hemorrhagic bone marrow sive erythrophagocytosis. Highly hypocellular bone marrow (H&E e)
(H&E) (b) (Case history 1) Immunohistochemically, using an antibody with an increase of macrophages with erythrophagocytosis (H&E f). (g,
to CD68, many macrophages with hemophagocytosis are present. (c–h) h) Immunohistochemical staining with an antibody to CD163 high-
(Case history 2) Bone marrow smear with dysplastic erythropoietic cell lighting the macrophages showing erythrophagocytosis
and macrophage with erythrophagocytosis. (d) Macrophage with mas-
6 Inflammatory and Infectious Diseases 121

c d

e f

g h

CD163 CD163

Fig. 6.5 (continued)

122 6 Inflammatory and Infectious Diseases

Case History 3: X-linked Lymphoproliferative tions of the SAP/SH2D1A gene. A bone marrow biopsy was
Syndrome (XLP) (Fig. 6.6) performed (Fig. 6.6). The bone marrow was hemorrhagic
A 3-year-old male child was admitted from another hospital with decreased hematopoiesis, increased T-lymphocytes, and
with sustained high fever non-responsive to antibiotic ther- occurrence of B-blasts. Hemophagocytosis was evident and
apy. The child was healthy until now, family history was unre- EBV infection could be visualized (Fig. 6.6a–h). Despite
markable. The child was transferred with the supposed immediate intensive treatment, the child died soon after
diagnosis of EBV infection. Clinical workup revealed muta- admission.

a b

CD71

c d

MPO CD61

Fig. 6.6 Infectious hemophagocytosis. (a) H&E stained bone marrow CD61. (e) T-lymphocytes are markedly increased (CD3). (f) There are
of the 3-year-old boy shows hypocellular hemorrhagic marrow spaces. many activated looking B-blasts (CD20). (g) Staining with a macro-
(b) Decreased erythropoietic cells stained with an antibody phage marker (CD163) shows many macrophages with hemophagocy-
to CD71. (c) Granulopoietic cells are highly decreased (MPO). tosis. (h) EBV infected cells visualized by EBER
(d) There are still many megakaryocytes stained with an antibody to
6 Inflammatory and Infectious Diseases 123

e f

CD3 CD20

g h

CD163

Fig. 6.6 (continued)

6.1.1.1 Caution 2. T-NK-cell lymphoma or leukemia (there are obvious

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 131

d e

Fig. 7.1 Schematic representation of infiltration patterns of malignant (e) Diffuse infiltration pattern. (f) Patchy infiltration. (g) Dense infiltra-
lymphomas. (a) Nodular infiltration pattern. (b) Paratrabecular infiltration (packed marrow)
tion pattern. (c) Intrasinusoidal infiltration. (d) Interstitial infiltration.
7 Malignant Lymphomas 133

f g

Fig. 7.1 (continued)

a schematic representation of the growth patterns of malig- obscure the often subtle lymphoma infiltration. Therefore, it
nant lymphomas. However, a mixture of various growth pat- is advisable to perform immunohistochemical stains using
terns in one biopsy of a patient can be seen. at least one B- and one T-cell marker (preferably CD20 and
Differential diagnosis concerning reactive (benign) and CD3).
neoplastic infiltrates must be carefully made especially in In this chapter, characteristic and atypical examples of
cases with plasmacytosis (reactive or plasma cell myeloma), BMBs with lymphoma infiltration are presented including
diffuse lymphocytosis or lymphoid nodules (reactive versus clinical presentation, morphology, and immunohisto
malignant lymphoma) and granulomatous lesions (e.g., infec- chemistry and, if necessary by PCR-techniques. Essential
tious disease or rheumatic diseases versus classical (c) HL). antibodies currently used for correct classification are men-
One has to be aware that lymphoma infiltration in the tioned. We try to show the most important and rather com-
bone marrow can induce severe changes and alterations of mon malignant lymphomas and the pitfalls; however, this
hematopoietic cells and the stroma. Such changes might chapter cannot completely cover this issue.
134 7 Malignant Lymphomas

7.2 Chronic Lymphocytic Leukemia (CLL) 7.2.3 Transformation of CLL

7.2.1 Epidemiology Transformation to a large cell lymphoma is seen during the

course of CLL in about 5% of the patients. This transfor-
CLL is the most frequent form of adult leukemia in Western mation is known as Richter’s syndrome. A much less fre-
countries. Typically, CLL is a disease of the elderly (mean age quent transformation to classical Hodgkin lymphoma may
is 65 years), but this leukemia is also diagnosed in younger occur [11].
adults and even, extremely rarely, in children [6]. If infiltration
by mature B-lymphocytes with the immunophenotype of CLL
is found in tissues (e.g., lymph node, tonsil) and the patient is 7.2.4 Immunohistochemistry
neither leukemic nor shows bone marrow infiltration, the diag-
nosis is “small lymphocytic lymphoma” (SLL). Tumor cells of CLL are CD19+, CD20+, CD22+, CD23+,
Monoclonal B-cell lymphocytosis (MBL) is a clonal CD79a+, CD43+, CD11c+ (weak) and CD5+, but nega-
B-cell disorder characterized by less than 5 × 109/L tive for CD10 and BCL6. Cyclin D1 can be detected in
B-lymphocytes in the peripheral blood, with a specific proliferation centers. CD38 and ZAP-70 have been sug-
immunophenotype lacking lymphadenopathy or organomeg- gested as surrogate markers to establish the mutational
aly. Most MBL cases reveal the immunophenotype of CLL, status [12, 13].
but non-CLL MBL also exists. MBL is divided into low-
count MBL (median B-cell count: 0.001 × 109/L) and high-
count MBL (clinical MBL, median B-cell count: 2.9 × 109/L).
Low-count MBL has a small risk of progression to CLL. In 7.2.5 Cytogenetic Abnormalities
contrast, patients with high-count MBL have a 1–2% per and Molecular Characteristics
year risk of progression to CLL and also a higher risk of
infectious complications and secondary malignancies [7]. CLL has the highest genetic predisposition of all hemato-
logical neoplasias. About 50% of CLL patients show del
13q14.3, about 20% have trisomy 12, rarely del 11q22-23,
7.2.2 Morphology 17p13, and 6q21 are found.
The discovery that IGVH genes in CLL can be either
CLL cells are small lymphocytes with dense chromatin and mutated or unmutated was a huge progress in the under-
scanty cytoplasm. A various number of prolymphocytes, standing of CLL and its prognosis: patients with mutated
slightly larger cells with prominent nucleoli, are intermingled IGVH genes (mutated CLL) have much better outcomes than
singularly or in a pseudofollicular arrangement, called prolif- those with unmutated IGVH genes (unmutated CLL) [14].
eration centers. Usually, the number of prolymphocytes is less Genetic prognostic markers such as immunoglobulin
than 2%. With increasing number of prolymphocytes, the dis- heavy chain gene mutational status and FISH studies for
ease course is said to be more aggressive. If prolymphocytes specific chromosomal abnormalities have now been devel-
exceed 50%, a diagnosis of prolymphocytic leukemia (B-PLL) oped to refine the risk of progressive disease. CD38 and
is favored. In cases of atypical CLL, the nuclear chromatin can ZAP-70 have been identified as surrogate markers for
be less condensated; marked nuclear irregularities can be seen mutation status and can be evaluated by flow cytometric
in peripheral blood (PB) lymphocytes. Such patients often immunophenotyping.
show trisomy 12 and other chromosomal abnormalities [8]. ZAP-70 (70-kDa zeta-associated protein) is an intracel-
The growth pattern of CLL in the bone marrow is either lular tyrosine kinase discovered initially because of its role
interstitial, nodular (see Fig. 7.2), diffuse, dense (packed mar- in T-cell signaling. It has also been found to be associated
row), or mixed. In cases of nodular infiltration, occasionally with the B-cell receptor in CLL. The expression of ZAP-70
paratrabecular infiltrates can be found. The infiltration pattern (≥ or =20% of B-cells) has been associated with an increased
is correlated with the patients’ prognosis. Thus, nodular infil- risk for an adverse outcome in B-cell CLL and is considered
tration pattern is associated with a better outcome [9, 10]. an important risk factor in these patients.
7 Malignant Lymphomas 135

Case History 1 (CLL) lymphocytes: 52.8%. Spleen and liver showed normal size,
A 70-year-old male was admitted to the hospital because of no enlarged lymph nodes were found. FACS-analysis was
recurrent cardiac problems. During clinical examination, a suspicious of transformed lymphocytic lymphoma; there-
pathological blood count was noticed with leukocytosis and fore, a bone marrow biopsy was performed (see Figs. 7.2
lymphocytosis: leukocytes: 12.2 G/L, neutrophils: 36.6%, and 7.3).

Fig. 7.2 CLL (to case history 1). (a) Moderately hypercellular bone are composed of small lymphocytes (H&E) (c–e) Infiltrating lympho-
marrow with multifocal nodular and patchy infiltration by lymphocytic cytes show strong immunohistochemical reactivity for CD19 (c), CD20
lymphoma. The tumor burden is 30% (MGG). (b) Neoplastic infiltrates (d), CD23 (e)
136 7 Malignant Lymphomas

CD19

b c

C D2 0

CD23

d e

Fig. 7.2 (continued)

7 Malignant Lymphomas 137

CD5

MIB1
CD3

b c

Fig. 7.3 CLL (to case history 1). (a) Infiltrating lymphocytes show lymphoma characteristically reveals a low proliferation fraction (Ki67).
strong immunohistochemical reactivity for CD5. (b) Reactive CD3+ (d, e) Lymphoma cells in this case are CD38− (d) and ZAP-70- (e)
lymphocytes are found within the neoplastic infiltrate. (c) Lymphocytic
138 7 Malignant Lymphomas

CD38
ZAP-70

d e

Fig. 7.3 (continued)

7 Malignant Lymphomas 139

Case History 2 (CLL/SLL) lymphocytic lymphoma and associated with peripheral lym-
Tonsillar involvement by small lymphocytic lymphoma phocytosis, the diagnosis is CLL.
(SLL) was incidentally detected in a 31-year-old male A bone marrow biopsy was performed 10 days after the
patient. Bilateral tonsillectomy was performed because of diagnosis of tonsillar infiltration. Hematological work-up
chronic tonsillitis and tonsillar abscess. Both tonsils were showed absolute B-lymphocytosis. Although the biopsy was
diffusely involved by SLL (see Fig. 7.4) with foci of resid- markedly fragmented, nodular infiltration by lymphocytic
ual uninvolved tonsillar lymphoid tissue. Typical morpho- lymphoma, could be diagnosed (see Fig. 7.5a–f). However, a
logic features, such as small round lymphocytes and reliable statement concerning the tumor burden was not pos-
proliferation centers, were present and the neoplastic cells sible due to fragmentation.
exhibited the characteristic CLL/SLL immunoprofile Diagnosis: fragmented bone marrow biopsy with nodular
(Fig. 7.4c–f): CD5+, CD20+, CD23+, negative for CD10 infiltration by CLL.
and Cyclin D1. Paraffin-embedded tissue from the bone marrow biopsy
Diagnosis: Multifocal infiltration of both tonsils by was used for genetic analysis using NGS. Deletion of 17p13
SLL. Comment: if the bone marrow biopsy is infiltrated by was detected suggestive for an unfavorable diagnosis.

a b

c CD23 d CD23

Fig. 7.4 CLL (to case history 2 tonsil). (a, b) Infiltration of tonsil by (MIB-1) is less than 5% within the neoplastic lymphocytic infiltrate. In
small lymphocytic lymphoma (H&E). (c, d) Tumor cells are positive contrast, high proliferation fraction is seen in residual germinal centers
for CD23. (e) Strong positivity for CD5. (f) The proliferation fraction
140 7 Malignant Lymphomas

e CD5 f M IB 1

Fig. 7.4 (continued)

a b

c CD20 d CD5

Fig. 7.5 CLL (Case 2 bm). (a, b) Despite distinct fragmentation para- to fragmentation). (c–e) Strong reactivity of infiltrating cells by CD20
trabecular and nodular infiltration can be seen by lymphocytic lym- (c), CD5 (d), and CD23 (e). (f) Low proliferation fraction within neo-
phoma (MGG, H&E). Details about tumor burden are not possible (due plastic cells in contrast to surrounding hematopoiesis (MIB-1)
7 Malignant Lymphomas 141

e CD23 f M IB 1

Fig. 7.5 (continued)

142 7 Malignant Lymphomas

Case History 3 (CLL/Richter’s syndrome) Immunohistochemical investigations revealed an identi-

A 76-year-old male was admitted to the hospital because of cal immunohistochemical reactivity of small lympho-
increasing pancytopenia and fever attacks. The patient was cytes and blasts (CD5+, CD20+, CD23+) according to
known to have CLL for 11 years. multifocal transformation into diffuse large B-cell lym-
In the bone marrow biopsy multiple, nodular and phoma (Richter’s syndrome).
patchy infiltrates by small lymphocytes with the immun- Diagnosis: Hypercellular bone marrow with multifocal
ophenotype of CLL were seen. In addition, multiple nodular and patchy infiltration by B-cell lymphoma with a
patchy infiltrates, consisting of blasts with prominent tumor burden of 60%. The majority of the lymphoma (80%)
nucleoli and broad clear cytoplasm were found (see corresponds to the known lymphocytic lymphoma (B-CLL),
Fig. 7.6). These blastoid infiltrates were often located in the minority (20%) shows transformation into high grade
the neighborhood of small lymphocytic infiltrates. B-cell lymphoma (Richter transformation).

a b

c d

Fig. 7.6 CLL—Richter’s syndrome (to case history 3; transformation The infiltrates of CLL and the tumor cells of transformed lymphoma
to Richter’s syndrome). (a–d) Hypercellular bone marrow with nodular reveal an identical phenotype with positivity for CD19 (e), CD20 (f),
infiltrates of typical CLL and nodular to patchy infiltration by diffuse CD23 (g), and CD5 (h)
large B-cell lymphoma according to Richter’s syndrome (H&E). (e–h)
7 Malignant Lymphomas 143

CD19 e CD20 f

CD23 g CD5 h

Fig. 7.6 (continued)

7.2.6 Caution Paratrabecular infiltration does not exclude infiltration

by CLL.
Some cases of CLL may show an atypical phenotype with Bone marrow infiltration by monoclonal
CD5 and/or CD23 being negative. B-lymphocytosis is indistinguishable from that of
ZAP-70 expression is considered to be a risk factor for CLL. Therefore, a final diagnosis of lymphoma in the bone
disease progression in patients with CLL, and ZAP-70 marrow should not be made if the hematological data are
expression assessed by immunohistochemistry can be used in favor of monoclonal B-lymphocytosis with a low periph-
as a reliable surrogate marker of the somatic mutation status. eral lymphocyte count [16]. However, there are studies
However, many commercially available antibodies give demonstrating that CLL is always preceeded by monoclo-
unreliable results [15]. nal B-lymphocytosis [17].
CLL in childhood is extremely rare but can occur [18].
144 7 Malignant Lymphomas

7.3 -Cell Prolymphocytic Leukemia

B In bone marrow biopsies, the distinction from other lym-
(B-PLL) phomas can be difficult and even impossible and requires
immunophenotypic as well as genetic studies. There are no
7.3.1 Epidemiology or very few mitoses in contrast to large cell lymphomas.

De novo B-PLL is rare, accounting for only 1% of lymphocytic

leukemias. The patients are usually older than 60, men and 7.3.3 Immunohistochemistry
women are equally affected. The patients present with spleno-
megaly and extreme leukocytosis, often exceeding 100,000/μL. B-prolymphocytes strongly express surface IgM as well as
B-cell antigens (CD19, CD20, CD22, CD79a). CD5-
positivity is found in up to 30% and CD23 in up to 20% of
7.3.2 Morphology cases. Often CD38 and ZAP-70 are expressed, however
without relation to the mutational status [20].
The majority of tumor cells (>55%, mostly >90%) are pro-
lymphocytes. The original description of clinical and patho-
logical features of B-PLL was done by Galton et al. [19]. 7.3.4 Cytogenetic Abnormalities
The tumor cells are larger than normal lymphocytes (up to and Molecular Characteristics
twice the size of a normal lymphocyte) and reveal a moder-
ately condensed chromatin in a round nucleus with central Complex karyotypes are common. Fifty percent of B-PLL
nucleolus and a basophilic cytoplasm. In contrast to bone patients show del(17p), a finding which is associated with
marrow infiltration by CLL, B-PLL may show fibrosis. TP53 gene mutations. This is probably the cause of progres-
Prolymphocytes infiltrate the bone marrow in an interstitial, sive course and treatment resistance. In several cases, aberra-
nodular, or mixed interstitial/nodular or even dense pattern. tions of MYC has been observed [21, 22].
7 Malignant Lymphomas 145

Case History (PLL) Diagnosis: Highly hypercellular bone marrow with diffuse
A 76-year-old male was admitted to the hospital because infiltration by B-cell prolymphocytic leukemia. The percent-
of splenomegaly. Laboratory investigation revealed age of tumor infiltration compared to hematopoiesis is 80%.
marked leukocytosis (85.809/L) and lymphocytosis At the same time as the bone marrow biopsy was per-
(95%). Furthermore, cervical lymphadenopathy (1 cm in formed, a cervical lymph node was removed for histological
diameter) was observed. Blood biochemistry was normal investigation (Fig. 7.8)
except for elevated lactate dehydrogenase level with The lymph node showed a diffuse infiltration by lympho-
546 U/L (normal: 120–140 U/L). A bone marrow biopsy cytes identical to those in the bone marrow, (Fig. 7.8a–c).
(see Fig. 7.7) and lymph node extirpation (see Fig. 7.8) Similar to the bone marrow, the tumor cells were strongly
were performed. CD20+ (Fig. 7.8d). Many reactive CD3+ T-cells were intermin-
gled (Fig. 7.8e). Using CD5 antibody more cells were stained
Bone marrow biopsy revealed highly hypercellular than with CD3, implicating that both T-cells and tumor cells
bone marrow with diffuse infiltration by medium-sized expressed CD5 (Fig. 7.8f). In the lymph node, most of the infil-
lymphocytes with round nuclei and moderately dense trating cells were CD23+ (Fig. 7.8g). The proliferation fraction
chromatin as well as centrally located nucleoli (Fig. 7.7a– using MIB1 in the lymph node was about 20% (Fig. 7.8h).
c). Immunohistochemical investigations showed strong Diagnosis: Lymph node infiltration by B-cell prolympho-
expression of CD19, CD20 (Fig. 7.7d), CD22, and cytic leukemia.
CD79a. About 30% of these tumor cells were positive for Genetic analyses on paraffin-embedded lymph node tis-
CD23 (Fig. 7.7e), but there was no reactivity for CD5. sue were performed using next-generation sequencing
The proliferation fraction (Fig. 7.7f) using MIB1 ranged (NGS) detecting trisomy 12, a rather uncommon cytogenetic
from 10 to 15%. abnormality in B-PLL.

a b

Fig. 7.7 PLL (bone marrow). (a, b) (B-PLL). Highly hypercellular for CD20. (e) A small proportion of tumor cells is CD23+. (f)
bone marrow with diffuse infiltration by B-PLL (MGG). (c) Most of the Proliferation fraction using MIB1 is 10–15%
tumor cells show nucleoli (H&E). (d) Strong reactivity of tumor cells
146 7 Malignant Lymphomas

c CD20 d

CD23 e MIB1 f

Fig. 7.7 (continued)

a b

Fig. 7.8 PLL (lymph node). (a–c) Dense lymph node infiltration by tumor cells show CD5-positivity. (g) In the lymph node, more tumor
B-PLL (H&E). (d) Strong reactivity of tumor cells using antibody to cells are CD23+ than in the bone marrow. (h) Proliferation fraction is
CD20. (e) Many reactive T-lymphocytes are interspersed (CD3). (f) about 20% (MIB1)
Compared to CD3, in addition to reactive T-lymphocytes, also some
7 Malignant Lymphomas 147

c CD20 d

CD3 e CD5 f

CD23 g MIB1 h

Fig. 7.8 (continued)

7.3.5 Caution The appearance of B-PLL in bone marrow biopsies may

be undistinguishable from CLL and large cell lymphomas,
Most B-PLL cases show an immunohistochemical profile especially if fixation and decalcification is not optimal. In
identical to CLL. Look for the morphologic details, espe- addition to immunohistochemistry, genetic analyses might
cially nucleoli. be useful and necessary.
148 7 Malignant Lymphomas

7.4 Lymphoplasmacytic Lymphoma (LPL) 7.4.3 Immunohistochemistry

7.4.1 Epidemiology Characteristically, LPLs show a monoclonal B-cell pheno-

type expressing CD19, CD20, CD22, CD79a, and PAX5.
Lymphoplasmacytic lymphoma (LPL) has a reported incidence Usually, the cells lack CD5, CD10, and CD23 although cases
of 3.4–7.3 for males and 1.7–4.2 for females/one million people with aberrant expression of these markers can be seen.
[23]. There is an increase of incidence with age with a median Expression of CD5, however in a variable number of tumor
age about 70 years [24]; however, also young patients and even cells is described in more than 40% of cases [28]. Many
teens have been reported with this disease [25]. Familiar predis- studies show positivity for CD23, but coexpression of CD5
position has been recorded in about 20% of patients [26]. If a and CD23 is rare. Rarely, CD10 is expressed in LPL-cells
patient with bone marrow involvement has IgM monoclonal [28]; expression of Cyclin D1 is not described in LPL. The
gammopathy of any concentration, the disease is Waldenstrom’s majority of tumor cells express surface Ig, the plasma cells
macroglobulinemia (WM). A typical precursor disease of WM show cytoplasmic Ig, usually of IgM-type, in some cases IgG
is IgM MGUS. The average risk for progression of IgM MGUS and rarely IgA. Normally, IgD is negative. The plasma cell
to WM is approximately 1.5% per year [27]. population can be highlighted with plasma cell markers,
such as CD38, CD138 or Vs38c.

7.4.2 Morphology
7.4.4 Cytogenetic Abnormalities
LPL shows a mixture of small lymphocytes, lymphocytes with and Molecular Characteristics
plasmacytoid differentiation and plasma cells. Russell bodies
corresponding to cytoplasmic or extracellular i mmunoglobulin NGS studies have detected somatic mutations of MYD88, a
deposits, as well as intranuclear inclusions, referred to as key component of the Toll-like receptor signaling machin-
Dutcher bodies can be present. Some blasts can be intermin- ery, in most cases of LPL/WM. Deregulated MYD88 signal-
gled. The number of these cells is variable. Occasionally, ing promoted by mutations support tumor cell survival in
plasma cells are the predominant cells, especially in treated LPL/WM, demonstrating that they are gain-of-function
LPLs. Distinction from plasma cell myeloma can be difficult driver events in this lymphoma [29]. Otherwise, no specific
morphologically and may require immunohistochemical and chromosomal or oncogenic abnormalities are found in
molecular investigations. Usually, many mast cells can be seen LPL. Frequently, deletion 6q is reported in LPL; however,
between the lymphoma cells or surrounding infiltrates. LPL this finding is not specific. Translocations involving the
involves the bone marrow in a nodular, diffuse, and/or intersti- immunoglobulin heavy chain locus occasionally are
tial pattern and paratrabecular infiltrates are frequently found. reported [30].
7 Malignant Lymphomas 149

Case History 1 (LPL) lymph node involvement by LPL, a bone marrow biopsy was
A 65-year-old woman was admitted to the hospital for strip- performed showing lymphoma infiltration (Fig. 7.9a–h).
ping operation of right vena saphena magna. During this DNA extracted from paraffin-embedded material of the bone
operation, a small (diameter 9 mm) inguinal lymph node was marrow biopsy revealed a MYD88 L265P mutation.
removed. Clinical records of the patient, who was treated for
osteoporosis since 13 years, revealed monoclonal gammopa- Diagnosis: Highly hypercellular bone marrow with dense
thy of IgM lambda type since 9 years. Complete blood count infiltration by LPL/Waldenström macroglobulinemia with
and differential blood count were normal; however, serum expression of IgM kappa. Tumor burden, in relation to hema-
protein kappa was highly elevated. After the diagnosis of topoiesis was more than 90%.

a b

c d

Fig. 7.9 Case history 1 (LPL). (a–c) Highly hypercellular bone mar- Russell bodies are lambda-negative. (g) The lymphocytes are CD20-
row with dense infiltrate of lymphocytes, plasma cells, and Russell bod- positive. (h) Strong reactivity of lymphoplasmacytic infiltrate and
ies (H&E). (d) Delicate fibrosis is detected by Gomori’s stain. (e) Russell bodies with an antibody to IgM
Expression of immunoglobulin kappa. (f) The infiltrating cells and
150 7 Malignant Lymphomas

e f

kappa lambda

g h

CD20 IgM

Fig. 7.9 (continued)

7 Malignant Lymphomas 151

Case History 2 (LPL/Waldenström Figs. 7.10, 7.11, tion of Codon 265 (exon 5) of the MYD88 gene was detected
and 7.12) in the DNA extracted from the bone marrow biopsy.
A 74-year-old woman was admitted to the hospital to undergo
a thorough diagnostic work up because of anemia and slight Diagnosis: Highly hypercellular bone marrow with multifo-
splenomegaly as well as monoclonal gammopathy of IgM cal nodular and patchy infiltration by LPL with expression of
lambda type. Furthermore, slightly enlarged axillary lymph IgM lambda, consistent with Waldenström macroglobulinemia.
nodes (Fig. 7.10) were found. A bone marrow biopsy The tumor burden was estimated as 40% of nucleated cells.
revealed multifocal infiltration by LPL (Fig. 7.11). A muta-

a b

c d

CD20

Fig. 7.10 Lymph node (case history 2). (a–c) H&E stains of the ingui- nodules, suggestive of residual follicles, are detected. (e) Plasma cells
nal lymph node with blurred architecture and focal sinus histiocytosis are negative for light chain kappa. (f, g) Lambda-positive plasma cell
(a, b). High magnification shows increased number of plasma cells and population. (h) The plasma cells express IgM heavy chain
occasional Dutcher bodies (c). (d) With an antibody to CD20 B-cell
152 7 Malignant Lymphomas

e f

kappa lambda

g h

lambda IgM

Fig. 7.10 (continued)

a b

Fig. 7.11 Bone marrow (case history 2). (a–d) Highly hypercellular Infiltrate stained with H&E showing some Dutcher bodies. (g) Dutcher
bone marrow with multifocal nodular to patchy neoplastic lymphoplas- body (center) highlighted by PAS-stain. (h) Within the neoplastic infil-
macytic infiltrates with many interspersed mast cells (MGG). (e, f) trate a delicate fibrosis is found (Gomori’s stain)
7 Malignant Lymphomas 153

c d

e f

g h

Fig. 7.11 (continued)

154 7 Malignant Lymphomas

a b

kappa kappa

c d

lambda lambda

e f

CD20 CD23

Fig. 7.12 (a, b) Lymphocytes and plasma cells are kappa-negative. (c, an antibody to CD23. (g) The many interspersed mast cells are stained
d) Strong lambda-positivity of infiltrating cells. (e) Majority of infiltrat- with an antibody to CD117c. (h) Low proliferation fraction of the infil-
ing cells show CD20-positivity. (f) Many tumor cells are reactive with trating cells (MIB-1)
7 Malignant Lymphomas 155

g h

CD117c MIB1

Fig. 7.12 (continued)

7.4.5 Caution myeloma. Lack of bone remodeling, lack of CD56-staining

as well as Cyclin D1-negativity is in favor of LPL. In the
Studies, antedating the actual WHO classification, show bone marrow of patients treated for LPL often monoclonal
poor reproducibility of LPL. There is no specific immuno- plasma cells without lymphocytes remain. In such cases, the
histochemical marker for this lymphoma, thus the diagnosis clinical history and mutational analysis are essential for cor-
is often made by exclusion. rect diagnosis. In addition, plasma cell myelomas rarely
Helpful for diagnosing LPL, in our opinion is a very express IgM.
strong immunohistochemical light chain restriction (Fig. In cases containing mainly plasma cells, the hemato-
7.12a–d). Furthermore, LPL usually has an increase in mast pathologist has to be extremely cautious. If these plasma
cells, which are easily detected in special stains (MGG) or cells show strong positivity for IgM, LPL is more likely than
immunohistochemically using antibodies against CD117c plasma cell myeloma. Furthermore, plasma cell myelomas,
(Fig. 7.12g) or mast cell tryptase. in contrast to LPL, usually reveal a more pronounced bone
One has to be aware that plasmacytic differentiation is a remodeling.
feature of many other small B-cell lymphomas. Testing for One has to be aware, that abnormal phenotype (e.g.,
MYD88 mutation, found in about 90% of LPL cases, can be CD5+ or CD23+) does not exclude the diagnosis of LPL (see
diagnostically helpful. immunhistochemical stains on bone marrow biopsy, Case
Cases of LPL with predominant plasmacytic morphology History 2, Fig. 7.12f).
can cause problems in differential diagnosis to plasma cell
156 7 Malignant Lymphomas

7.5 Mantle Cell Lymphoma (MCL) morphology closely resembles other small B-cell lympho
proliferative diseases, such as chronic lymphocytic leukemia,
7.5.1 Epidemiology prolymphocytic leukemia, follicular lymphoma, and mar-
ginal zone lymphoma. Correct diagnosis is only possible with
The reported incidence of MCL is 3–10 % of all NHLs with immunophenotyping. In the blastoid variant of MCL, the
a marked male predominance (2:1 or even greater) [1–3]. lymphoma cells mimic large cell lymphoma or lymphoblastic
Rarely, patients under the age of 50 are affected; the median leukemia revealing tumor cells with dispersed chromatin,
age at first diagnosis is about 65 years [31]. Although there small nucleoli, and scant cytoplasm. In blastoid variants of
are reports detecting skewed immunoglobulin variable MCL, the infiltration pattern is either diffuse or interstitial.
gene segment usage stimulating lymphomagenesis, a con-
clusive link to autoimmune disorders or pathogens could
not be identified until now [31]. MCL may be an aggressive 7.5.3 Immunohistochemistry
disorder with a fast-growing tumor mass, but it can also be
rather indolent in some patients, especially in those, show- In contrast to B-CLL, cells of MCL are strongly CD20+ and
ing exclusively blood and bone marrow involvement. The show a rather strong surface immunoglobulin light chain
disease is called “mantle cell lymphoma” because the restriction. Usually, cells of MCL are CD23-negative; how-
tumor cells originate from the mantle zone of the lymph ever, weak expression of CD23 is possible. Characteristically,
node. MCL is usually diagnosed as a late-stage disease the tumor cells are CD5-positive and CD10 as well as BCL6-
with multiple organ involvement. MCL confined to the gas- negative. Almost all cases are Cyclin D1+ and all cases are
tro-intestinal tract is referred to as lymphomatoid polyposis BCL2+. Classical cases of MCL are SOX11 (a transcription
(Fig. 7.13). factor) positive (nuclear staining), blastoid variants of MCL
are frequently SOX11-negative [34].

7.5.2 Morphology
7.5.4 Cytogenetic Abnormalities
The pattern of bone marrow involvement is rather variable and Molecular Characteristics
from nodular, interstitial, and paratrabecular to diffuse [32].
Occasionally, prominent intrasinusoidal infiltration has been The translocation t(11;14) can be detected in more than 90%
observed in MCL [33]. of MCLs.
Mantle cell lymphoma has a high frequency for marrow Alterations of the TP53 locus at chromosome 17p13 are
involvement (up to 95%) with small lymphoid cells, some frequently found in MCL. In about 20% of patients, muta-
with round and others with indented or angulated nuclei. tions of TP53 have been reported and these patients are
Reticulin fibers are mostly increased in infiltrated areas. The known to be associated with a rather poor outcome [35].
7 Malignant Lymphomas 157

Case History 1; MCL in colonic biopsies (Fig. 7.13) and Colonic biopsies showed infiltration by mantle cell lym-
bone marrow biopsy (Fig. 7.14) phoma (Fig. 7.13a–h). A bone marrow biopsy was per-
A 75-year-old male was admitted to the hospital because of formed. This biopsy was infiltrated by mantle cell lymphoma
obscure abdominal pain. The diagnostic work-up revealed in a paratrabecular and interstitial pattern (see Fig. 7.14). The
slight splenomegaly, slight anemia, and thrombocytopenia. tumor burden in relation to hematopoiesis was 30%.

a b

c Cyclin D1 d

CD21 e CD21 f

Fig. 7.13 MCL Colon. (a–b) Colonic biopsy with nodular and patchy (e, f) Within the infiltrate, dense irregular meshwork of FDCs is pres-
infiltration by MCL. (c) High magnification of the infiltrate shows ent. (g) Reactive CD3-positive lymphocytes are intermingled. (h)
the typical cleaved tumor cells. (d) Strong nuclear Cyclin D1-positivity. Lymphoma cells are CD5-positive
158 7 Malignant Lymphomas

CD3 g CD5 h

Fig. 7.13 (continued)

a b

c d

Fig. 7.14 MCL bone marrow. (a–d) Accentuated paratrabecular lym- Coexpression of CD5 in the lymphoma cells. (h) In most tumor cells,
phoma infiltration (H&E). (e, f) CD20-positive tumor cells are infiltrat- nuclear staining for Cyclin D1 is seen (inset shows another area with
ing the bone marrow in a paratrabecular and interstitial pattern. (g) Cyclin D1-positive cells)
7 Malignant Lymphomas 159

CD20 e C D20 f

CD5 g Cyclin D1 h

Fig. 7.14 (continued)

7.5.5 Caution proliferation centers of B-CLL), and also in plasma cell

myelomas. Usually, endothelial cells show expression of
SOX11 staining has been reported in about 90% of MCL Cyclin D1.
cases. However, SOX11 has also been detected in hairy cell Aberrant phenotypes, mostly in blastoid/pleomorphic
leukemia, Burkitt lymphoma, lymphoblastic lymphomas, variants, have been reported. Such cases show expression of
and T-cell PLL. BCL6 and CD10, whereas CD5 may be absent.
The rare Cyclin D1-negative cases of MCL show expres- In situ MCL is rare but well known in lymph nodes. But
sion of Cyclin D2 and/or Cyclin D3. A diagnosis of Cyclin an in situ component of MCL can also be found in bone mar-
D1-negative MCL must be made with great care, considering row biopsies (see Fig. 7.15 MCL in situ). In such cases, a
all immunohistochemical aspects (e.g., meshwork of FDCs) “normal” looking follicle is present; however, immunohisto-
and in some instances even FISH or other cytogenetic studies chemistry reveals Cyclin D1-positive cells restricted to the
might be necessary for correct diagnosis. mantle zone. Thus, immunohistochemical investigations
Immunohistochemically, Cyclin D1 can also be detected should be performed, even if there is a normal looking
in other B-cell lymphomas (e.g., in hairy cell leukemia, follicle.
160 7 Malignant Lymphomas

Case History 2; MCL In Situ

In the demonstrated case (Fig. 7.15), a 53-year-old male
patient underwent a bone marrow biopsy for staging reasons
since MCL was diagnosed in a lymph node.

a b

c d

CD20 CD23

e f

Cyclin D1 Cyclin D1

Fig. 7.15 In situ MCL. (a, b) Closely attached to the bony trabeculae, reactive BCL6 (left side) positive and CD10 (right side) positive germi-
a lymphoid follicle is found (H&E). (c) Staining with an antibody to nal center cells. (h) High proliferation fraction in the reactive germinal
CD20 shows a rather “normal” looking follicle. (d) A concentric net- center and rather low proliferation fraction in the neoplastic mantle
work of CD23-positive follicular dendritic cells is seen in this follicle. zone (MIB1)
(e, f) Cyclin D1-positive mantle zone cells. (g) The follicle contains
7 Malignant Lymphomas 161

g h

BCL6 CD10 MIB1

Fig. 7.15 (continued)

162 7 Malignant Lymphomas

7.6 Hairy Cell Leukemia (HCL) mast cells, plasma cells, small lymphocytes, a variable number
of hematopoietic precursors, and typically extravasated eryth-
7.6.1 Epidemiology rocytes [36]. Hematopoiesis, especially erythrocytopoiesis, in
most instances shows dysplastic features. In most cases, the
HCL is a rare mature B-cell neoplasm accounting for 2% of bone marrow biopsy is hypercellular, however, hypocellular
lymphoid leukemias. The median age is 52 years with a biopsies, resembling aplastic anemia, may occur [37].
male: female ratio of 5:1.

7.6.3 Immunohistochemistry
7.6.2 Morphology
The characteristic immunophenotype of HCL shows expres-
Hairy cells are medium-sized lymphocytes, about twice the sion of CD19, CD20, CD22, CD79a, and CD11c and addi-
size of normal small lymphocytes. The nuclei are round, oval, tional expression of CD103, CD25, CD123, Annexin A1,
bean-shaped, or convoluted with a rather homogenous chro- BRAF, TRAP (tartrate-resistant acid phosphatase), DBA44,
matin. Rarely, the nuclei are bilobulated. If nucleoli are pres- FMC-7, and variably Cyclin D1 [38].
ent, these are inconspicuous. The cytoplasm is broad and
irregularly shaped with lateral interdigitating extensions
(“hairy” projections are only seen in smears). Due to these 7.6.4 Cytogenetic Abnormalities
projections, the infiltration consists of widely separated cells; and Molecular Characteristics
the cells almost never show overlapping in normal thickness
sections. The bone marrow infiltration is highly variable, rang- There is no specific cytogenetic abnormality for
ing from a minimal interstitial infiltrate to obvious diffuse to HCL. Numerical abnormalities of chromosomes 5 and 7
even solid infiltration. The hairy cells are usually dispersed have been described [39]. The majority of cases of HCL
within a reticular fiber network. The clinician thus often gets a show VH genes with somatic hypermutation [39]. Usually,
dry tap and the bone marrow biopsy is the only reliable tech- BRAF-V600E mutation as the disease-defining genetic event
nique for confirming the diagnosis. The infiltrate also contains in HCL can be detected [40].
7 Malignant Lymphomas 163

Case History 1; HCL The bone marrow was slightly hypercellular for age. This
An 81-year-old male was admitted to the hospital because of was due to a diffuse infiltration of atypical lymphocytes
progredient pancytopenia, monocytopenia (Leuko 2.70 G/l, which exhibited the immunophenotype of HCL. The tumor
Ery 3.8 T/L, Thrombo 55 G/l, Mono 1%), and splenomegaly burden in relation to hematopoiesis was 50%.
(18 cm). No enlarged lymph nodes were found. In the periph- The diagnosis of diffuse bone marrow infiltration by HCL
eral blood, hairy cells were seen, FACS-analysis was suspi- was straightforward (see Figs. 7.16 and 7.17) with a tumor
cious of clonal B-cell proliferation, consistent with HCL. A burden of 50%.
bone marrow biopsy was performed (Figs. 7.16 and 7.17).

a b

c d

e f

CD19 CD19

Fig. 7.16 Case 1 (HCL) Slightly hypercellular bone marrow for age investigations reveal diffuse bone marrow infiltration by typical HCL.
(a–c). In higher magnification (d), the atypical lymphocytic cells are (e, f) CD19-positive cells; (g, h) strong CD20-positivity
surrounded by extravasated erythrocytes. (e–h) Immunohistochemical
164 7 Malignant Lymphomas

g h

CD20 CD20

Fig. 7.16 (continued)

a b

CD11c CD11c

c d

CD25 CD103

Fig. 7.17 Case 1 (HCL) (a–d) Immunohistochemical investigations detectable. (f) Megakaryocytes, stained with an antibody to CD61, are
reveal diffuse bone marrow infiltration by typical HCL. (a, b) Specific seen diffusely dispersed. (g, h) Diffuse reticulin fiber fibrosis (Gomori’s
staining for CD11c; (c, d) Tumor cell are CD25 and CD103-positive. stain) is typically seen between the HCL cells
(e) Between tumor cells erythropoietic islands (stained with CD71) are
7 Malignant Lymphomas 165

e f

CD71 CD61

g h

GOM GOM

Fig. 7.17 (continued)

166 7 Malignant Lymphomas

g h

BCL2 BCL6

Fig. 7.21 (continued)

7.7.5 Caution DLBCL, classical Hodgkin lymphoma, peripheral T-cell

lymphoma).
1. Follow-up biopsies after treatment with anti-CD20 ther- 3. Rarely, a follicular infiltration pattern is seen in FL. This
apy may still show lymphatic infiltrates, which on mor- infiltration pattern can be seen also in marginal zone lym-
phological grounds, resemble the characteristic FL phoma and mantle cell lymphoma [46].
infiltrates with a similar localization and similar cyto- 4. Occasionally, bone marrow involvement is scarcely vis-
morphology. However, these lymphocytic infiltrates ible. Especially in cases which show a small layer of
may either be composed of reactive T-lymphocytes or of neoplastic cells round the trabeculae, infiltrates easily
residual neoplastic B-cells that have lost CD20 expres- can be missed. Immunohistochemistry must be applied,
sion secondary to antibody treatment, but show positiv- especially when patients were treated with targeted
ity for other B-cell markers (CD19, CD79a). therapy.
Immunohistochemistry is absolutely necessary to differ- 5. Keep in mind that signet ring cell morphology can occur
entiate reactive from neoplastic infiltrates. in lymphomas.
2. Paratrabecular infiltrations are quite common for FL, but 6. Some cases of FL are CD23-positive [47]
paratrabecular infiltration can also be seen in other types 7. In the bone marrow FL tumor cells are mostly
of lymphomas (e.g., lymphocytic lymphoma, MCL, CD10-negative.
7 Malignant Lymphomas 173

7.8 plenic Marginal Zone Lymphoma

e f

CD21 CD23

g h

CD20 kappa lambda

Fig. 7.25 (continued)

7 Malignant Lymphomas 181

Case History 2 (MALT) Fig. 7.26 lesions in both lungs. Histologic examination of the lesions
A 35-year-old woman was diagnosed with Helicobacter in stomach, liver, and lung confirmed the diagnosis of gen-
pylori-positive MALT lymphoma. HP eradication therapy eralized MALT lymphoma. Furthermore, infiltration by
was performed without any staging procedures. Since con- this lymphoma was seen in the bone marrow biopsy.
trol endoscopy after 6 months still revealed lymphoma, the Diagnosis: Normocellular bone marrow with nodular
patient was referred to the hospital. Computed tomography infiltration by “low grade” B-cell lymphoma, consistent with
scans revealed a diffuse gastric infiltration of the antrum an infiltration of the already verified extranodal MALT lym-
and corpus, enlargement of epigastric lymph nodes, as phoma. The percentage of the infiltration is 10% of nucleated
well as an 8 × 7 cm hypodense liver lesion and multiple cells.

a b

c d

Fig. 7.26 Case history 2. MALT (a, b) show the infiltration in the liver infiltrating cells in the bone marrow biopsy. (f) The proliferation frac-
by small centrocyte-like cells with occasional intermingled blasts. (c, d) tion (MIB 1) is less than 5% in the lymphoma population. (g, h)
Nodular infiltration of the bone marrow by an identical lymphoma pop- Lymphoma cells reveal positivity for kappa light chain, whereas the
ulation as shown in the liver (H&E). (e) Strong CD20-positivity of the cells are negative using an antibody to lambda light chain
182 7 Malignant Lymphomas

e f

CD20 MIB1

g h

kappa lambda

Fig. 7.26 (continued)

7.9.5 Caution In some cases of MALT lymphomas involving the bone

marrow, a correct diagnosis is difficult or even impossible to
Overlapping patterns of infiltration in MALT lymphomas achieve since specific immunohistochemical markers are
may be seen [55]. still not available.
7 Malignant Lymphomas 183

7.10 iffuse Large B-Cell Lymphoma

D Expression of CD10, BCL6, and IRF4/MUM1 as well as
(DLBCL) GCET1 and FOXP1 varies. These markers are important for
identifying the two subgroups of DLBCL [59], termed ger-
7.10.1 Epidemiology minal center B-cell like or activated B-cell like (short GCB-
and ABC-like). In addition to defining the two subgroups of
At diagnosis, the incidence of a bone marrow infiltration DLBCL’s immunohistochemical staining with antibodies to
by DLBCL is low with 10–25% [56]. In rare instances, MYC and BCL2 is recommended in the new WHO classifi-
the bone marrow is the primary localization at initial cation for prognostic indications: the so-called double-
diagnosis [57]. expressor DLBCL with expression of MYC and BCL2 [60]
have a worse outcome than other DLBCLs. However, dou-
ble-expressor DLBCL is not a separate category in the
7.10.2 Morphology updated WHO classification.
In most cases, the proliferation fraction (Ki67) is > 40%
In most instances, infiltrates of DLBCL are easily identified
in H&E stained sections. There is either a diffuse or nodular
to patchy, often paratrabecular or mixed infiltration pattern 7.10.4 Cytogenetic Abnormalities
of the lymphoma cells. These cells are large blasts with one and Molecular Characteristics
or more prominent nucleoli. Occasionally, a dense infiltra-
tion is seen resembling an infiltration of acute leukemia. In Clonal rearrangement of immunoglobulin light chain genes
addition, a discordant morphology of the lymphoma in the and immunoglobulin heavy chain genes are usually found.
bone marrow compared to the initial site of diagnosis (most About one-third of DLBCLs reveal abnormalities of the
often a lymph node) can be observed. In most of such cases, 3q27 region involving the BCL6 gene. Translocation of the
the infiltrate in the bone marrow looks less aggressive than in BCL2 gene is found in 20–30% of cases and a MYC rear-
the primary site. Often there are many small B lymphocytes rangement can be observed in 10% of DLBCL. FISH analy-
with intermingled B-blasts [58]. sis to demonstrate c-MYC, BCL2, and BCL6 rearrangement
might be necessary to diagnose high grade B-cell lym-
phoma, with MYC and BCL2 and/or BCL6 translocations
7.10.3 Immunohistochemistry (formerly called “double-/triple-hit” lymphomas).
The two subgroups (GCB/ABC) are associated with dif-
The tumor cells express B-cell markers like CD19, CD20, ferent chromosomal aberrations. The GCB subgroup often
CD22, and CD79a. CD30 may be found and about 10% of show gains at 12q12, the ABC subgroup reveals gains of 3q,
cases show positivity for CD5. 8q21-q22 and losses of 6q21-q22 [61].
184 7 Malignant Lymphomas

Case History 1 (DLBCL) Diagnosis: Highly hypercellular bone marrow with dense
A 58-year-old previously healthy woman presented with infiltration by a “high grade” B-cell lymphoma. The mor-
dyspnea, retrosternal pain, and increased tendency to phology and immunohistochemical results are consistent
bleed. Examination revealed splenomegaly (diameter with infiltration by DLBCL; immunohistochemical algo-
15 cm) and pancytopenia with marked anemia (Hb 6.9 g/ rithm: activated B-cell like, immunohistochemical double hit
dL, Hkt17.8%). A bone marrow biopsy was performed. score according to Green: 2. The percentage of infiltration in
This biopsy revealed a dense infiltration by DLBCL of relation to hematopoiesis is >90%.
ABC subtype (Fig. 7.27).

a b

c d

CD20 BCL2

Fig. 7.27 (a, b) The highly hypercellular bone marrow shows dense the blasts are c-MYC-positive; strong positivity is seen with an anti-
infiltration by lymphoid blasts (H&E). (c, d) The blasts are strongly body to BCL6. (g, h) The blasts show intranuclear FOXP1-positivity
reactive with antibodies to CD20 and BCL2. (e, f) More than 30 % of and a high proliferation fraction (Ki67)
7 Malignant Lymphomas 185

e f

cMYC BCL6

g h

FOXP1 Ki 67

Fig. 7.27 (continued)

186 7 Malignant Lymphomas

Case History 2 (DLBCL) Diagnosis: Highly hypercellular bone marrow with

A 79-year-old man presented with an acute abdomen caused nodular and patchy infiltration by DLBCL, immunohisto-
by rupture of the spleen. Splenic specimens were diffusely chemical algorithm: activated B-cell like. The percentage
infiltrated by a DLBCL, activated B-cell type. Staging proce- of the infiltration in relation to the increased hematopoie-
dure showed nodular and patchy infiltration of the bone mar- sis is 30%.
row (Fig. 7.28).

a b

CD20

c d

CD3 GCET1

Fig. 7.28 (a) Highly hypercellular bone marrow with multiple nodular the blasts with an antibody to GCET1. (e) Using an antibody to CD10
and patchy, occasionally paratrabecular located infiltrates by blasts only dendritic cells are stained, the tumor cells are negative. (f) Only
(inset shows the blasts with one or more nucleoli). (b) The infiltrates are some blasts are weakly BCL6-positive. (g) Strong FOXP1-positivity of
highlighted by staining with an antibody to CD20. (c) Reactive CD3- the blasts. (h) The proliferation fraction (MIB1) is higher than 80%
positive T-lymphocytes are intermingled. (d) There is no reactivity of
7 Malignant Lymphomas 187

e f

CD10 BCL6

g h

FOXP1 MIB1

Fig. 7.28 (continued)

7.10.5 Caution and even molecular pathothologic investigations (PCR)

might be necessary to establish B- or T-cell lineage.
Bone marrow infiltration by DLBCL may mimic T-cell lym- In DLBCL with CD5 expression, the differential diagno-
phoma or Hodgkin lymphoma. Especially, T-cell/histiocytic- sis includes the blastoid variant of mantle cell lymphoma.
rich large B-cell lymphoma may be challenging. To diagnose Expression of Cyclin D1 and SOX11 by MCL, but not by
these lymphomas, a large panel of immunohistochemistry DLBCL might be helpful.
188 7 Malignant Lymphomas

7.11 Intravascular Large B-Cell Lymphoma Case History (intravascular large B-cell lymphoma, see
Fig. 7.29)
This is an aggressive lymphoma in which the tumor cells An 82-year-old woman was admitted to the hospital because of
grow within vascular lumina of various organs including marked anemia. The patient had no enlarged lymph nodes and a
the bone marrow (see Fig. 7.29), which can be the initial normal size of spleen and liver. Since the anemia could not be
site of diagnosis [62, 63]. Because this type of lymphoma is explained, a bone marrow biopsy was performed. In a moderate
extremely rare, no detailed descriptions concerning epide- to highly hypercellular bone marrow, the sinusoids and some
miology, morphology, and cytogenetic abnormalities are small vessels were filled with huge B-blasts corresponding to an
available. infiltration by intravascular large B-cell lymphoma (Fig. 7.29).
In bone marrow sections, aggregates of blasts are seen in Diagnosis: Highly hypercellular bone marrow with infil-
vessels, especially sinusoids. These blasts can be highlighted tration by intravascular large B-cell lymphoma.
by immunohistochemical stains with B-cell markers. More
than 30% of such lymphomas show expression of CD5,
about 10% reveal expression of CD10. In cases with CD10- 7.11.1 Caution
negativity, the blasts, like in our case shown below, are
MUM1-positive. Minimal extravascular infiltration may be seen in some cases
of intravascular large B-cell lymphoma.

a b

c d

CD20 CD20

Fig. 7.29 (a) The bone marrow is highly hypercellular with only a few antibody to CD34 labels endothelial cells thus highlighting the intravas-
fat cells. (b) Sinusoids and small vessels are filled with huge blasts. (c, cular blast infiltration. (f) Most blasts express CD5. (g, h) The blasts are
d) The intravascular blasts are positive for CD20. (e) Staining with an CD10-negative (g) and MUM1-positive (h)
7 Malignant Lymphomas 189

e f

CD34 CD5

g h

CD10 MUM1

Fig. 7.29 (continued)

190 7 Malignant Lymphomas

7.12 Burkitt Lymphoma (BL) mingled macrophages give rise to the typical “starry sky”
appearance.
7.12.1 Epidemiology

Three variants of BL are known. The endemic BL occurs in 7.12.3 Immunohistochemistry

African countries, the immunodeficiency-associated BL is
mainly seen in HIV-infected patients and the sporadic BL is The blasts of Burkitt lymphoma express B-cell-associated anti-
seen throughout the world, mainly in young adults and chil- gens, such as CD19, CD20, CD22 and typically show expres-
dren. The incidence of sporadic BL is 1–2% of all lympho- sion of IgM, CD10, BCL6, and CD38, while there is no or only
mas in Western countries [64]. weak BCL2-positivity. The tumor cells are negative for anti-
In about 60% of patients, the bone marrow is infiltrated in bodies to CD34, TdT, and CD30. Characteristically, the prolif-
either a diffuse or interstitial pattern. eration fraction (MIB1) is 100% (see Fig. 7.30d). Only a few
reactive T-lymphocytes are intermingled.

7.12.2 Morphology
7.12.4 Cytogenetic Abnormalities
The bone marrow, which is infiltrated in more than half of the and Molecular Characteristics
patients, shows an interstitial or diffuse infiltration pattern
(see Fig. 7.30e, f). The tumor cells are of medium size with The majority of Burkitt lymphomas reveal a MYC transloca-
multiple small nucleoli. The characteristic intracytoplasmic tion at band 8q24 to the IG heavy chain region 14q32. A few
vacuoles are better visualized in smears or touch prepara- cases have MYC translocation at the lambda 22q11 or kappa,
tions. Many mitoses and apoptosis are seen. Numerous inter- 2p12 light chain loci [65].
7 Malignant Lymphomas 191

Case History (Burkitt Lymphoma) lymphoma (Fig. 7.30a–d). The monomorphic blasts, infil-
A 29-year-old patient initially presented with acute abdo- trating the small bowel, revealed high nuclear to cytoplasmic
men, leucocytosis 15.8 × 103/μL, thrombocytopenia 72 × 103/ ratio and two to three nucleoli. The proliferation fraction was
μL, and mild anemia. Emergency operation revealed perfora- 100%. Bone marrow biopsy showed diffuse infiltration by
tion of the small bowel due to a large solid tumor mass. In c-MYC-positive Burkitt lymphoma (Fig. 7.30e, f).
H&E sections, the tumor masses showed blasts of Burkitt

a b

c d

MIB1

Fig. 7.30 (a) Dense infiltration of the small bowel by Burkitt lym- Routinely stained H&E section shows diffuse blastic infiltration of the
phoma. (b) Characteristic “starry sky” pattern of this lymphoma. (c) At bone marrow. (f) Immunohistochemical staining reveals c-MYC-
higher magnification, monomorphic blasts with several nucleoli are positivity in most blasts. (g) MYC translocation at band 8q24 to the IG
seen. (d) Proliferation fraction using MIB1-antibody is 100%. (e) heavy chain region could be demonstrated by FISH analysis
192 7 Malignant Lymphomas

e f

c-myc

Fig. 7.30 (continued)

7.12.5 Caution lymphoma entity called Burkitt-like lymphoma with 11q

aberration [66].
A minority of cases with Burkitt lymphoma (about 10%) One has to be aware that cases of DLBCL may show
may lack MYC translocation by FISH. According to the new MYC translocation.
WHO classification, there is a new provisional B-cell
7 Malignant Lymphomas 193

7.13 -Cell Chronic Lymphocytic Leukemia

T 7.13.3 Immunohistochemistry
(T-CLL)/T-Cell Prolymphocytic
Leukemia (T-PLL) T-prolymphocytes are CD1a-negative and TdT-negative
peripheral T-cells of post-thymic origin with positivity for
7.13.1 Epidemiology CD2, CD3, and CD7 (see Fig. 7.31). Expression of membra-
nous CD3 can be weak. A rather strong reactivity is seen for
T-CLL/T-PLL is a rare neoplasm, representing about 2% of CD52 (see Fig. 7.31), which can be used as a target therapy
mature lymphocytic leukemias in adults. The median age is [68]. In most patients (60%), the tumor cells are CD4-
65 years with a range from 30 to 94 years [67]. The disease is positive and CD8-negative, in about 25% of cases the tumor
characterized by marked lymphocytosis and splenomegaly. cells coexpress CD4 and CD8. This coexpression is nearly
unrivaled for T-PLL. A minority of cases show CD4− and
CD8+ tumor cells.
7.13.2 Morphology (Fig. 7.31)

The expression “T-CLL” is used as a synonym for small cell 7.13.4 Cytogenetic Abnormalities
variants of T-PLL. The leukemia is characterized by the and Molecular Characteristics
proliferation of small- to medium-sized lymphoid cells with
non-granular basophilic cytoplasm and small visible nucle- Clonal rearrangement for T-cell receptor genes is found.
oli. The lymphocytes are of post-thymic origin. The small Common chromosome abnormalities in 80 % of T-PLL
cell variant (T-CLL) may lack visible nucleoli. In some patients are inversion of chromosome 14 with breakpoints in
cases, the nuclei of this leukemia can be very irregularly the long arm at q11 and q32. Frequent findings are abnor-
formed and reveal cerebriform features. Usually, the periph- malities of chromosome 8, abnormalities of chromosome 6
eral blood, bone marrow and lymph nodes are infiltrated, and 17 have also been described [69]. Some cases show dele-
involvement of liver, spleen, and skin may occur. The bone tion of the TP53 gene.
marrow shows an interstitial or diffuse infiltration.
194 7 Malignant Lymphomas

Case History (T-cell chronic lymphocytic leukemia The bone marrow biopsy showed diffuse fibrosis and
[T-CLL]/T-cell prolymphocytic leukemia [T-PLL]) slight to moderate hypercellularity. An interstitial and diffuse
A 65-year-old woman was treated with cortisone for acute hearing infiltration by small lymphocytes, with an identical immuno-
loss and iridocyclitis. The patient developed leucocytosis, general- phenotype to that described in the lymph node, was seen (see
ized lymphadenopathy and splenomegaly. A cervical lymph node Fig. 7.33). The amount of the T-cell infiltration, in relation to
extirpation and bone marrow biopsy were performed. the still normoquantitative hematopoiesis was 25%.
Diagnosis was infiltration of lymph node and bone mar-
The lymph node was diffusely infiltrated with some unaf- row by T-PLL.
fected follicles still present (see Figs. 7.31 and 7.32). The Because of the rarity of this case, in addition to T-cell
infiltrating cells were small lymphoid cells with round nuclei receptor gene analysis, which showed clonally rearranged
without visible nucleoli. These cells were positive with anti- TCR beta and TCR gamma chain genes, low density whole
bodies to CD2, CD3, CD4, CD5, CD7, CD52 (very strong genome copy number variation (CNV) analysis using Ion
positivity), and BCL2 and negative for antibodies to CD8, Torrent NGS (Next-Generation Sequencing) was performed
CD16, CD57, TIA1, and granzyme B. The proliferation frac- (see Fig. 7.32c).
tion (MIB1) was very low with less than 5%

a b

c d

CD3 CD4

Fig. 7.31 T-PLL (lymph node). (a, b) Diffuse infiltration of the lymph and CD7-positive (right side). (g) No CD8 reactivity is seen in the infil-
node by small lymphocytes sparing follicles (H&E). (c) Infiltrating trating lymphocytes. (h) Lymphocytes are strongly stained with an anti-
lymphocytes are CD3-positive. (d) Strong CD4-positivity of infiltrating body to CD52
lymphocytes. (e, f) Infiltrating lymphocytes are CD5-positive (left side)
7 Malignant Lymphomas 195

e f

CD5 CD7

g h

CD8 CD52

Fig. 7.31 (continued)

a b

CD20

Fig. 7.32 T-PLL lymph node. (a) Preserved follicles are highlighted (c) CNV-analysis using next-generation sequencing shows deletion 9p,
with an antibody to CD20. (b) Low proliferation fraction within deletion 9q12-22, deletion 11q14-24
the neoplastic cell population as shown with an antibody to MIB1.
196 7 Malignant Lymphomas

Fig. 7.32 (continued)

a b

c d

CD3 CD4

Fig. 7.33 T-PLL (bone marrow). (a, b) Hypercellular bone marrow and CD8-negative (right side). (g) Strong reactivity of neoplastic lym-
diffusely infiltrated by small lymphocytes (H&E). (c, d) Diffuse infil- phocytes with an antibody to CD52. (h) Slight fibrosis can be seen
tration of the bone marrow by CD3 (left side) and CD4 (right side)- between the infiltrating lymphocytes (Gomori’s stain)
positive lymphocytes. (e, f) Infiltrating cells are CD7-positive (left side)
7 Malignant Lymphomas 197

e f

CD7 CD8

g h

CD52

Fig. 7.33 (continued)

7.13.5 Caution weak or negative [70]. Especially, if you find an exception-

ally strong reactivity for CD52, you should think of infiltra-
The strong CD7 and CD52 intensity is in contrast to other tion by T-PLL/T-CLL
mature T-cell malignancies, where these markers may be
198 7 Malignant Lymphomas

7.14 -Cell Large Granular Lymphocytic

T in about 50% slightly hypercellular. A left shifted granulocy-
Leukemia (T-LGLL) topoiesis is frequently seen. In most cases, there is a slight to
moderate fibrosis. The percentage of bone marrow infiltration
7.14.1 Epidemiology is variable; the infiltration pattern is interstitial and/or intrasinu-
soidal. In routinely stained H&E sections, the tumor cells are
About 2–3% of cases with mature lymphocytic leukemias are difficult to be seen. In many cases, reactive nodular lympho-
T-LGLLs. There is no age peak, but T-LGLL is rather rare at cytic aggregates, containing mainly B-cells are present.
the age under 25. Males and females are equally affected.
T-LGLL is a chronic, indolent lymphoproliferative disorder
with distinctive clinical and laboratory manifestations [71]. 7.14.3 Immunohistochemistry
Patients usually present with severe neutropenia, some-
times combined with anemia, even pure red cell aplasia. T-LGLL-cells are mature CD3, CD8, and T-cell receptor
Thrombocytopenia is rather rare. Often the patients show a (TCR)αβ-positive cytotoxic T-cells. Rare variants show
moderate splenomegaly CD4+/TCRαβ-positivity or TCRγδ-positivity. TCRγδ-
positive cases are either CD8-positive or CD4/CD8-
negative. Commonly, there is a loss of CD5 and CD7. Most
7.14.2 Morphology (Figs. 7.34 and 7.35) cases are reactive for CD16 and CD57. T-LGLL expresses
cytotoxic proteins like granzyme B, granzyme M, and TIA1
The histology of the bone marrow pathology of T-cell granular [72]. Often aberrant NK cell antigens are found (CD16,
lymphocytic leukemia (T-LGLL) is subtle and ill-defined. In CD57).
peripheral blood and bone marrow aspirates the neoplastic Cases with T-LGLL are clonally rearranged by TCR gene
lymphocytes are large granular lymphocytes with a broad cyto- studies. The TRG gene is rearranged in all cases irrespective
plasm and fine azurophilic granules. Although there is no con- of the type of receptors expressed. No unique karyotypic
sent on the level of lymphocytosis for diagnosis of T-LGLL, a abnormality is known, numeric chromosomal abnormalities
LGL count of >2 × 109/L is associated with a clonality in most are described [73].
cases. Although the patients with T-LGLL present with severe Up to 40% of T-LGLL show STAT3 mutations in the Src-
neutropenia with or without anemia, the bone marrow in half like homology domain 2, mainly in three hot spots. About
of the cases is normocellular or only slightly hypocellular, and 2% of T-LGLL harbor STAT5b mutations.
7 Malignant Lymphomas 199

Case Histories (T-LGLL) lular granulocytopoiesis. There was an increase of atypical

Case History 1 (Fig. 7.34) T-lymphocytes expressing CD3, CD8, CD57, and granzyme B
A male 86-year-old patient underwent an examination with a loss of CD7 (Fig. 7.34) A slight reticulin fibrosis was
including bone marrow biopsy with a tentative diagnosis of observed. PCR showed TCR rearrangement. The diagnosis, in
myelodysplasia. The absolute neutrophil count was accordance with the clinical findings was: slight hypercellular
1.95 × 109/L, platelet count was normal and slight anemia with bone marrow with interstitial and intrasinusoidal infiltration by
Hb 11.8 g/dL (normal value 13.0–17.5) was found. The bone T-LGLL. The amount of the infiltration was 25% compared to
marrow was slightly hypercellular with left shifted normocel- hematopoiesis.

a b

c d

CD3

Fig. 7.34 Case 1. (a, b) Slightly hypercellular bone marrow (H&E) (f) CD8-positive lymphocytes are markedly increased. (g) There is an
with a slight increase of lymphocytes and a small lymphocytic aggre- increase of CD57- positive lymphocytes which are predominantly
gate. (c) Slight reticulin fibrosis is revealed by Gomori’s stain. (d) located intrasinusoidal. (h) Cytotoxic phenotype is revealed by using
Interstitial increase of small- to medium-sized CD3-positive granzyme B
T-lymphocytes. (e) Compared to CD3, there is a loss of CD7-positivity.
200 7 Malignant Lymphomas

e f

CD7 CD8

g h

CD57 granzymeB

Fig. 7.34 (continued)

7 Malignant Lymphomas 201

Case History 2 (Fig. 7.35) lymphocytes were phenotypically of T-LGLL-type.

A 76-year-old patient presented with anemia (Hb 7.4 g/ Furthermore, multilineage dysplastic hematopoiesis with an
dL) and neutropenia with an absolute neutrophil count of increase of CD34-positive blasts (>5%) was found. Identical
1.36 × 109/L. A clonal T-cell population in the peripheral clonal TCR-rearrangements were found in peripheral blood
blood was found. Myelodysplastic syndrome was and bone marrow samples.
suspected. The bone marrow diagnosis was myelodysplasia with
The bone marrow biopsy revealed moderate hypercellu- excess of blasts-1 in association with an interstitial infiltra-
larity with an atypical interstitial T-lymphocytic infiltrate tion by T-LGLL (percentage of infiltration in relation to dys-
and a reactive lymphoid B-cell aggregate (Fig. 7.35). These plastic hematopoiesis: 20%).

a b

c d

CD20 CD3

Fig. 7.35 Case 2. (a, b) In H&E stained sections, hypercellular bone ing CD3 (d) and CD8 (e). (f) Only a few CD5-positive lymphocytes can
marrow is seen. The higher magnification (b) reveals dysplastic hema- be seen corresponding to a loss of T-cell marker. (g) Typically, the
topoiesis with prominent megaloblastic erythropoiesis. (c) A reactive T-cell population shows reactivity for granzyme B. (h) Increase of
lymphoid aggregate is mainly composed of CD20-positive CD34-positive blasts according to myelodysplasia with excess of
B-lymphocytes. (d, e) Interstitial infiltration of T-lymphocytes express- blasts-1
202 7 Malignant Lymphomas

e f

CD8 CD5

g h

granzymeB CD34

Fig. 7.35 (continued)

7.14.4 Caution organ-transplantation. It may also develop as a form of post-

transplant lymphoproliferative disease [74].
A diagnosis of T-LGLL should not be made without PCR Some investigators suggest that T-LGLL may be consid-
for confirmation of clonality since an increase of T-LGLL- ered as indolent clonal disorder of uncertain significance and
cells is not uncommon in infectious diseases, like EBV- or not as a leukemia.
CMV-infection and in patients with rheumatoid arthritis, Concerning STAT3 mutations, one has to be aware, that
Felty syndrome, autoimmune disorders, aplastic anemia, this mutation can be observed in other T-cell lymphomas
myelodysplasia, or PNH. and in some cases of aplastic anemia and hypocellular
T-LGLL can also occur in association with low grade MDS.
B-cell lymphomas. Moreover, a clonal increase of T-LGLL STAT5b mutations can be found in T-ALLs and hepato-
cells can occur after allogeneic bone marrow- and solid splenic gamma-delta T-cell lymphomas.
7 Malignant Lymphomas 203

7.15 Hepatosplenic T-Cell Lymphoma which is mostly involved, reveals a marked intrasinusoidal
(HSTL) infiltration pattern (Fig. 7.37). Spleen and liver are enlarged;
splenic infiltration occurs in the red pulp (Fig. 7.36), the liver
7.15.1 Epidemiology shows intrasinusoidal infiltration.

HSTL is a rare lymphoma originating from cytotoxic T-cells,

with either gamma-delta or alpha-beta T-cell receptor expres- 7.15.3 Immunohistochemistry
sion. Only about 5% of peripheral T-cell lymphomas are
HSTL. This lymphoma mainly occurs in young adults and The neoplastic cells are CD2+ and CD3+. Most cases are
adolescent males and shows an aggressive clinical course. TCRδ1+, TCRαβ−, CD56+/−, CD4−, CD8−, CD5−,
About 20% of HSTL develop in the context of chronic TIA1+, granzyme M+, granzyme B−. NK-related antigens
immune suppression, often following immunosuppressive CD16 and CD56 are frequently expressed.
therapy for organ transplantation. In addition, HSTL is
observed in patients treated by azathioprine and infliximab
for Crohn’s disease [75]. An association with hemophago- 7.15.4 Cytogenetic Abnormalities
cytic lymphohistiocytosis is frequently observed. and Molecular Characteristics

HSTL reveals rearrangement of TRG genes. In most cases,

7.15.2 Morphology isochromosome 7q is found.

Cells of HSTL show medium-sized nuclei and a rim of pale

cytoplasm. Small nucleoli can be seen. The bone marrow,
204 7 Malignant Lymphomas

Case History (HSTL) The massively enlarged spleen showed multiple infarc-
A 35-year-old male with Crohn’s disease had been treated tions and an infiltration of the red pulp by neoplastic
with Imurek® for more than 6 years. He presented with lymphocytes with the immunophenotype of HSTL
weight loss, fatigue, pancytopenia, and massive hepato- (Fig. 7.36). The bone marrow was infiltrated by the same
splenomegaly as well as purpura. Splenectomy, because tumor cell population interstitial and intrasinusoidal
of infarction and a bone marrow biopsy were performed. (Fig. 7.37).

a b

c d

CD3

e f

CD4 CD5

Fig. 7.36 HSTL. (a) Overview shows marked expansion of splenic red CD3-positivity of the infiltrating cells with sparing of focally visible
pulp and atrophy of follicles. (b, c) Higher magnification reveals lym- white pulp. (e–g) The neoplastic cells do not react with antibodies to CD4
phatic infiltration of the red pulp by monomorphic medium-sized lym- (e), CD5 (f), and CD7 (g). (h) Only a few lymphocytes are stained with
phocytes with small nucleoli and a rather pale cytoplasm. (d) Strong an antibody to CD8. Note the sinusoidal lining by CD8-positive cells
7 Malignant Lymphomas 205

g h

CD7 CD8

Fig. 7.36 (continued)

a b

c d

Fig. 7.37 HSTL in the bone marrow. (a–d) Hypercellular bone mar- of intrasinusoidal lymphocytes by CD2. (f, g) CD3-positive neoplastic
row biopsy with interstitial and intrasinusoidal infiltration by atypical cells infiltrate the bone marrow in an interstitial and intrasinusoidal pat-
lymphatic cells. Especially in the highest magnification (d) sinusoidal tern. (h) Tumor cells are TIA1-positive
infiltration by monomorphic lymphocytes is visible. (e) Weak staining
206 7 Malignant Lymphomas

e f

CD2 CD3

g h

CD3 TIA1

Fig. 7.37 (continued)

7.15.5 Caution Recognition of this lymphoma entity, despite the contro-

versial expression of markers, is possible by considering the
Difficulties regarding the diagnosis of HSTL may arise from primary involvement of the spleen and the liver with a typi-
not considering this rare entity as a differential diagnosis cal sinusoidal infiltration pattern, and other rather common
[76]. The diagnosis of HSTL is not always straightforward clinical features as presentation with hepatosplenomegaly
especially because of the rarity of the disease. In addition, and peripheral cytopenia.
there are varieties to the “common phenotype”; expression Differential diagnoses include T-lymphoblastic lympho-
of CD5, CD7, CD8, CD16, and CD56 is quite variable. mas/leukaemias, peripheral cytotoxic T-cell lymphomas, and
The data concerning expression of cytotoxic granule- unusual forms of T-chronic lymphocytic/promyelocytic leu-
associated proteins are contradictory. kemias with expression of CD8.
7 Malignant Lymphomas 207

7.16 ycosis Fungoides (MF)/Sezary

M tial infiltration by atypical lymphoid cells with often cerebri-
Syndrome (SS) form nuclei can be found. In transformed cases, defined by
>25% large blastoid cells in the cutaneous infiltrate, in the
7.16.1 Epidemiology bone marrow infiltration with T-blasts can be seen.

MF is the most frequent type of cutaneous T-cell lymphoma

(CTCL) and usually limited to the skin. Only in advanced 7.16.3 Immunohistochemistry
stages extracutaneous infiltration in lymph nodes, spleen, and
liver and rarely in the bone marrow is observed [77]. SS is In MF as well as SS the infiltrating cells typically are CD2+,
defined by generalized lymphadenopathy, erythroderma, and CD3+, TCRβ+, CD5+, and CD4+. Rare cases with CD8-
clonal neoplastic T-cells with cerebriform nuclei (Sezary positivity are described.
cells) in skin, lymph nodes, and peripheral blood. Furthermore,
according to WHO classification, one or more criteria, like
Sezary cell count of at least 1000 cells/mm3 and an increased 7.16.4 Cytogenetic Abnormalities
T-cell population with a CD4/CD8 ratio of more than 10 and/ and Molecular Characteristics
or loss of one or more T-cell antigens are required.
T-cell receptor genes show clonal rearrangement. In MF
complex karyotypes with structural and numerical altera-
7.16.2 Morphology tions are common, recurrent chromosomal abnormalities in
SS have not been found [78].
The histology in SS is similar to that of MF in cutaneous
lesions. If the bone marrow is involved, rather sparse intersti-
208 7 Malignant Lymphomas

Case History (MF/SS) (see Fig. 7.38) biopsy showed a diffuse and focally nodular infiltration by
A 59-year-old female patient suffering from generalized neoplastic T-lymphocytes with many intermingled CD30-
skin disease with erythroderma was treated for MF for 2 positive blasts (Fig. 7.38). A transformation of SS could be
years. Because the patient did not respond to treatment, a diagnosed with a percentage of 70% in relation to
bone marrow biopsy was performed. This bone marrow hematopoiesis.

a b

c d

CD3 CD4

Fig. 7.38 Case history MF/SS. (a, b) The bone marrow is hypercellu- SS-cells showing positivity for CD3 (c), CD4 (d), and CD5 (e). (f) Loss
lar due to a neoplastic lymphocytic infiltration (a) with many intermin- of the T-cell marker CD7. (g) Only a few reactive small CD8-positive
gled blasts (b). (c–e) The infiltrating cells reveal the phenotype of lymphocytes can be detected. (h) Many blasts with CD30-positivity
7 Malignant Lymphomas 209

e f

CD5 CD7

g h

CD8 CD30

Fig. 7.38 (continued)

7.16.5 Caution or SS is known, and the bone marrow shows an interstitial

T-cell population, careful immunohistochemical investiga-
The infiltration of the bone marrow in non-transformed cases tions with T-cell markers and PCR for clonality should be
of MF and SS might be sparse and thus can be missed. If MF done.
210 7 Malignant Lymphomas

7.17 eripheral T-Cell Lymphoma, Not

P in PTCL, NOS is intrasinusoidal, diffuse, interstitial, nod-
Otherwise Specified (PTCL, NOS) ular, or focal patchy [81].

7.17.1 Epidemiology
7.17.3 Immunohistochemistry
PTCLs, NOS account for 10–15% of lymphoproliferative
disorders in Western countries [79]. Mainly, this lymphoma PTCL, NOS in most cases show an aberrant T-cell phenotype
occurs in adults and is extremely rare in children. There is a with frequent downregulation or loss of CD5 and CD7. In nodal
male: female ratio of 2.1. cases, CD4+ and CD8− cases predominate, but double positiv-
ity or double negativity can be detected in some cases.
Expression of CD30, CD56, and cytotoxic markers is variable.
7.17.2 Morphology More than half of the cases are CD52-negative. Aberrant expres-
sion of one or more B-cell markers (mainly CD20 and CD79a)
The morphological appearance of PTCL, NOS is quite is seen in a few cases. The proliferation fraction, detected with
variable from monomorphous to highly polymorphous MIB1 is usually high. EBV-positivity is not uncommon.
infiltration. In most cases, medium-sized and large lym-
phocytes with irregular formed nuclei, prominent nucle-
oli, and many mitoses can be seen. Cells with a rather 7.17.4 Cytogenetic Abnormalities
clear cytoplasm and Reed-Sternberg (RS-)-like cells may and Molecular Characteristics
be present. But there are also cases with mainly small
lymphoid cells with irregular nuclei. The frequency of In most cases, clonal rearrangement of TCR is found. PTCLs,
bone marrow infiltration varies greatly from study to NOS often show complex karyotypes [82]. The genetic
study; on average one-third of PTCL, NOS show bone imbalances detected in PTCL, NOS vary from those of other
marrow infiltration [80]. The infiltration pattern described T-cell lymphomas.
7 Malignant Lymphomas 211

Case History (PTCL, NOS) (see Fig. 7.39) was moderately hypercellular with increase of all hematopoietic
A 39-year-old female presented with hemolytic anemia and cells. In addition, a diffuse and multifocal patchy infiltration by
generalized lymphadenopathy. In a cervical lymph node infiltra- PTCL, NOS was seen (Fig. 7.39). The percentage of the infiltra-
tion by PTCL, NOS was diagnosed. The bone marrow biopsy tion in relation to hyperplastic hematopoiesis was 20%.

a b

c d

GlycC MPO

Fig. 7.39 PTCL, NOS. (a, b) H&E stained section shows hypercellu- (d) Slightly increased myelopoiesis is revealed by an antibody to
lar bone marrow with hyperplastic erythropoietic cells and increase of myeloperoxidase. (e, f) Diffuse infiltration and several small patchy
lymphatic cells. (c) Erythropoiesis is markedly increased (consistent aggregates of CD3-positive lymphocytic cells. (g, h) The lymphoma
with hemolytic anemia) as shown with an antibody to glycophorin C. cells show double positivity for CD4 (g) and CD8 (h)
212 7 Malignant Lymphomas

e f

CD3 CD3

g h

CD4 CD8

Fig. 7.39 (continued)

7.17.5 Caution The morphologic patterns of bone marrow infiltration

by PTCL, NOS are variable, however, in most cases the
The neoplastic infiltrate of PTCL, NOS may be obscured by cytol ogy is similar to that of the diagnostic specimen.
other changes in the marrow, like erythroid hyperplasia in Occasionally, the diagnosis of PTCL, NOS is based on
the context of hemolytic anemia. findings in the bone marrow biopsy, when no other biopsies
are available. PCR-studies in such cases are advisable.
7 Malignant Lymphomas 213

7.18 Angioimmunoblastic T-Cell 7.18.3 Immunohistochemistry

Lymphoma (AITL)
The tumor cells express pan-T-cell antigens, such as
7.18.1 Epidemiology CD2, CD3, and CD5. The majority of cases are CD4-
positive, but many reactive polymorphic CD8-positive
AITL is a disease of the middle-aged and elderly persons. T-cells are intermingled. The neoplastic cells show the
Males and females are equally affected. With about 20% this immunophenotype of follicular helper T-cells (TFH) with
disease is one of the common specific subtypes of peripheral expression of CD10, CXCL13, BCL6, and PD-1 in more
T-cell lymphomas. Up to 2% of all non-Hodgkin lymphomas than half of the cases [83]. Follicular dendritic cells,
are this type of lymphoma. stained with antibodies to CD21 and/or CD23 may be
expanded. Often, many EBV-positive B-immunoblasts
are present. The B-blasts and the plasma cells are
7.18.2 Morphology polyclonal.

In most cases, the diagnosis of AITL results from a lymph

node biopsy. An infiltration of the bone marrow is found in 7.18.4 Cytogenetic Abnormalities
most cases, with histologic features similar to the affected and Molecular Characteristics
lymph nodes [79]. In most cases, there are nodular to patchy
infiltrates (see Fig. 7.40), mostly located paratrabecular, but Clonal rearrangement of the T-cell receptor gene is found in
also intertrabecular associated with an increased vascularity. up to 90% of cases. In 25–30% of AITL clonal immuno-
The infiltrates are polymorphic and composed of aggregates globulin gene rearrangements are detected resulting from the
of small- to medium-sized lymphocytes, intermingled blasts, presence of EBV-positive B-blasts.
histiocytes (often epithelioid), eosinophils, and plasma cells. Cytogenetically, trisomy 3, trisomy 5, and an additional
Occasionally, groups of clear cells are present. In some X-chromosome are frequent findings [84].
cases, the amount of plasma cells is quite prominent mis-
leading to a diagnosis of plasma cell myeloma. An increase
of reticulin fibers is always seen in the infiltrate.
214 7 Malignant Lymphomas

Case History (AITL) (Fig. 7.40) diagnosed as infiltration by AITL. The hypercellular bone
A 74-year-old woman was admitted to the hospital with gen- marrow biopsy showed multiple nodular to patchy mostly
eralized lymphadenopathy and weight loss of 10 kg within intertrabecular infiltrates consistent with an infiltration by
the last 4 months. The biopsy of a cervical lymph node was AITL with an amount of neoplastic infiltration of 30%.

a b

c d

CD3 CD4

Fig. 7.40 AITL. (a) In routinely H&E stained sections, the bone mar- positivity with an antibody to CD3. (d) There is a high amount of
row is markedly hypercellular due to multiple atypical nodular to CD4-positive tumor cells. (e) CXCL13 is expressed by most neoplas-
patchy intertrabecular infiltrates. (b) Higher magnification reveals an tic cells. (f) Intermingled, a few reactive CD8-positive T-cells are pres-
infiltrate composed of medium-sized lymphocytes, plasma cells, and ent. (g) CD30-positive blasts can be detected within the infiltrates.
blasts (H&E). (c) Most cells within the infiltrate are T-cells showing (h) Some EBV-positive cells are visualized by EBER in situ hybridization
7 Malignant Lymphomas 215

e f

CXCL13 CD8

g h

CD30 EBER

Fig. 7.40 (continued)

7.18.5 Caution classical Hodgkin lymphoma and even diffuse large B-cell
lymphoma, especially if CD20 and CD30-positive EBV-
In contrast to AITL in the lymph node, CD10 is rarely positive cells are found. In such cases, a descriptive diagno-
expressed by the tumor cells in the bone marrow. sis should be made (e.g., bone marrow infiltration by
A proliferation of follicular dendritic cells (FDC), a com- malignant lymphoma with many T-cells and intermingled
mon feature in the lymph node, is scarcely seen in infiltrates B-blasts: differential diagnosis comprises AITL, classical
of the bone marrow. Hodgkin lymphoma and DLBCL). PCR-studies for clonality
If the bone marrow biopsy is taken without a previous are essential in such cases to classify the lymphoma
lymph node, infiltrates of AITL may resemble infiltrates of correctly.
216 7 Malignant Lymphomas

7.19 naplastic Large Cell Lymphoma

A tions, especially when hallmark cells are absent and the
(ALCL) infiltrate is composed of the so-called small cell variant of
ALCL [86].
7.19.1 Epidemiology

The WHO classification differentiates between systemic 7.19.3 Immunohistochemistry

nodal ALCL and a cutaneous form. While the cutaneous
form is clinically rather indolent and mostly limited to the The cells of ALCL as a rule are CD30-positive (Figs. 7.41d
skin without bone marrow infiltration, the nodal form of and 7.42d) and express cytotoxic T-cell markers, such as
ALCL is a systemic disease. In the majority of cases of TIA1, perforin, or granzyme B. The expression of T-cell
ALCL translocations involving the ALK-1 gene are seen. markers, EMA and CD45 is variable [87]. Due to loss of
These lymphomas are termed ALCL, ALK-positive. several pan T-cell antigens, cases with a “null cell pheno-
However, there are lymphomas with identical histology and type” are described. In more than two-thirds of cases, the
identical immunophenotype lacking ALK-1 translocation. most widely used pan T-cell marker CD3 is negative. CD2,
These lymphomas are diagnosed as ALCL, ALK-negative. CD4, and CD5 are positive in 70% of cases. Rarely, cases
ALCL, ALK-positive comprises 3% of adult non-Hodgkin with CD8-positivity have been detected. In most cases, the
lymphomas and up to 20% of childhood lymphomas. There neoplastic cells are CD25-positive. ALK-1 protein expres-
is a male predominance of 1.5:1. ALK-positive ALCL is sion, either nucleocytoplasmic or cytoplasmic, is detected in
most frequent in the first three life decades. the majority of cases. The neoplastic cells of ALCL are virtu-
ALCL, ALK-negative shows a peak incidence in adults ally always negative for EBV.
older than 40 years. There is also a male predominance.

7.19.4 Cytogenetic Abnormalities

7.19.2 Morphology and Molecular Characteristics

In the lymph node, ALCL shows a variable morphologic About 90% of ALCL show clonal rearrangement of the
spectrum. The hallmark cells are large lymphoid cells T-cell receptor genes. The most frequent genetic abnormal-
with an often horseshoe-shaped nucleus. The cytoplasm ity is a translocation t(2;5)(p23;q35) between the ALK
of the tumor cells is mostly abundant. An infiltration of gene on chromosome 2 and the nucleophosmin gene on
the bone marrow is rather rare with about 25% [85]. The chromosome 5. There are variant translocations between
infiltration pattern is either interstitial and/or vaguely ALK and other genes on chromosomes 1, 2, 3, 17, 19, 22,
nodular or intrasinusoidal. If hallmark cells are present, and X. Using RT-PCR, the t(2;5) can be detected, but cases
these cells can easily be detected. In many cases, the infil- with variant translocations are negative by standard RT-PCR
trate is subtle and is missed in routinely stained H&E sec- method.
7 Malignant Lymphomas 217

Case History (ALCL) (Figs. 7.41 and 7.42) with an unknown primary tumor. An abdominal lymph
A 73-year-old male presented with weight loss, enlarged node (Fig. 7.42) and bone biopsy of lumbar vertebra
abdominal lymph nodes and bone pain in the shoulder as revealed an infiltration by ALCL, ALK-negative. A bone
well as the vertebral column and lower extremities. marrow biopsy was performed showing a vaguely nodular
Skeletal scintigraphy revealed numerous osteodestructive to patchy paratrabecular infiltrate by ALCL, ALK-negative
lesions within the bone, suggestive of metastatic process (Fig. 7.41).

a b

c d

CD30 CD30

Fig. 7.41 ALCL in the bone marrow. (a, b) In routinely stained H&E nized in the overview. (e) Whereas the tumor cells are strongly CD2-
sections, a normocellular bone marrow is seen. There is a single patchy positive (f), no reactivity is seen using CD3 (f). (g, h) Rather weak
neoplastic infiltrate consisting of large lymphoid cells. (c, d) The tumor positivity with an antibody to CD4 (g) and strong positivity with a an
cells in the infiltrate are strongly CD30-positive, a staining even recog- antibody to CD25 (h)
218 7 Malignant Lymphomas

e f

CD2 CD3

g h

CD4 CD25

Fig. 7.41 (continued)

a b

Fig. 7.42 ALCL in the lymph node. (a, b) To a large extent, the lymph (f) Weak CD4-positivity is found in a few blasts. (g) Typically, the
node shows an intact architecture (a). In higher magnification (b), an tumor cells of ALCL are BCL2-negative. (h) High proliferation fraction
accentuated intrasinusoidal infiltration by huge blasts is obvious. (c, d) of the neoplastic cells (MIB1)
The intrasinusoidal blasts are strongly positive for CD30 and (e) EMA.
7 Malignant Lymphomas 219

c d

CD30 CD30

e f

EMA CD4

g h

BCL2 MIB1

Fig. 7.42 (continued)

7.19.5 Caution cases of known ALCL always immunohistochemical stains

should be done not to miss interstitial infiltration.
Be aware, that infiltration of the bone marrow by ALCL can
be subtle and hard to see in routinely stained sections. In
220 7 Malignant Lymphomas

7.20 Adult T-Cell Leukemia/Lymphoma coarsely granular with distinct, occasionally prominent
(ATLL) nucleoli. A variable amount of blast-like cells is usually pres-
ent. The infiltration pattern of the bone marrow is usually
7.20.1 Epidemiology patchy or diffuse to dense.

This disease occurs mostly in endemic regions linked to the

prevalence of HTLV-1. The areas mostly affected are Central 7.20.3 Immunohistochemistry
Africa, Southwestern parts of Japan, and the Caribbean
countries [88]. ATLL is occurring in adults with a male to The tumor cells express T-cell markers (Fig. 7.43), such as
female ratio of 1.5:1. Some sporadic cases do arise; however, CD2, CD3, and CD5, but usually the neoplastic cells are neg-
the patients mostly originate from endemic regions. Four ative for CD7. Most cases are CD4-positive and CD8-
distinct clinical variants are described, and the prognosis and negative, rarely the immunohistochemical profile is the other
clinical course range from highly aggressive to a more pro- way round. In most cases, the tumor cells are CD25-positive
tracted course depending on the subtype [89]. and frequent expression of CXCR4 and FOXP3, chemokine
receptors, is seen. The large blasts can express CD30 (inset
Fig. 7.43f), but are negative for ALK1 and cytotoxic
7.20.2 Morphology molecules.

The affected organs, usually lymph nodes, spleen, peripheral

blood, and bone marrow, but also extranodal sites, like skin, 7.20.4 Cytogenetic Abnormalities
liver, gastro-intestinal tract, and central nervous system, and Molecular Characteristics
show a proliferation of highly pleomorphic lymphatic cells.
There is a mixture of medium-sized and large lymphatic There is a clonal rearrangement of the T-cell receptor genes.
cells with highly pleomorphic features (Fig. 7.43). Often, Most ATLL cases show clonal chromosome numerical and
especially in the peripheral blood, neoplastic cells with lobu- structural abnormalities, but no specific abnormalities for
lated nuclei (flower cells) can be found. The chromatin is this disease are known.
7 Malignant Lymphomas 221

Case History (ATLL) (Fig. 7.43) cells, which were medium sized with a variable amount of
A 42-year-old HIV-negative African male presented with intermingled larger blasts. A few eosinophils were intermin-
bone pain and dysphagia due to enlarged tonsils. CT scan gled, occasionally hematopoietic cells could be detected.
revealed masses of osteolytic lesions in the whole skeletal The immunohistochemistry revealed positivity of the tumor
system. Notable laboratory values at presentation revealed a cells with antibodies to CD2, CD3, CD4, CD5, CD99, and
white blood cell count of 32,000/L, hemoglobin of 11.8 g/ FOXP3. The blasts showed variable reactivity with an anti-
dL, thrombocytopenia with 82,000/L, and a high LDH with body to CD30. Cytotoxic markers and CD7 were negative.
780 U/L. A bone marrow biopsy from an osteolytic lesion in HTLV-1 antibody testing was positive by enzyme-linked
the iliac crest was performed (Fig. 7.43). The bone marrow immunosorbent assay. The diagnosis of ATLL with more
was densely infiltrated by highly pleomorphic lymphatic than 80% infiltration of the bone marrow was confirmed.

a b

c d

CD2

Fig. 7.43 Case history ATLL. (a–c) In routinely stained H&E sections, infiltrating tumor cells with an antibody to CD7, only a few small reac-
there is a dense bone marrow infiltration by highly pleomorphic lym- tive T-lymphocytes are stained. The inset highlights a huge CD30-
phoid cells. Many mitoses are seen, occasional larger blasts with promi- positive blast. (g) Strong reactivity of the neoplastic cells is seen with an
nent nucleoli are visible. The infiltrating pleomorphic cells are strongly antibody to CD25. (h) Intranuclear reactivity is seen in most tumor cells
CD2-positive (d) and CD4-positive (e). (f) There is no reactivity of the using an antibody to FOXP3
222 7 Malignant Lymphomas

e f

CD30

CD4 CD7

g h

CD25 FOXP3

Fig. 7.43 (continued)

7 Malignant Lymphomas 223

7.21 Hodgkin Lymphoma cells [91]. Within the bone marrow infiltrates small lympho-
cytes, scattered LP-cells and plasma cells as well as histio-
7.21.1 N
odular Lymphocyte Predominant cytes are present. Some cases with NLPHD show a follicular
Hodgkin Lymphoma (NLPHD) pattern of infiltration [92].

7.21.1.1 Epidemiology 7.21.1.3 Immunohistochemistry

NLPHD is rare comprising about 5% of all Hodgkin lym- The diagnostic LP-cells are positive for CD20, CD79a,
phomas. The affected patients are usually young male adults. BCL6, CD45, and CD75. In most instances, LP-cells are
More than 80% of cases present with stage I or II disease. positive for J-chain (see Fig. 7.44e), EMA, BOB1, OCT2,
Bone marrow involvement is uncommon and accounts for IgD, and kappa. LP-cells are usually negative for CD30 and
5–9% of cases. Patients with bone marrow infiltration tend to CD15, but weak CD30-positivity does not exclude the
show a more aggressive course of an otherwise rather benign diagnosis of NLPHD. The tumor cells are ringed by CD3-
course with a 10-year overall survival of more than 80% of positive and to a less extent CD57-positive T-cells. Whereas
patients with stage I and II [90]. in the lymph node a dense network of CD21-positive or
CD23-positive FDC-network is seen in the nodules (see
7.21.1.2 Morphology (Fig. 7.44) Fig. 7.44d), such is usually absent in the bone marrow.
The lymph node is totally or partially infiltrated by a typi- EBV is negative.
cally nodular or a nodular/diffuse infiltrate of small lympho-
cytes, histiocytes and intermingled tumor cells, called 7.21.1.4 Cytogenetic Abnormalities
LP-cells (Fig. 7.44) LP-cells are large blasts with an irregu- and Molecular Characteristics
larly formed nucleus and prominent nucleolus. Reports about LP cells have a clonal immunoglobulin rearrangement.
morphology of bone marrow infiltration in cases of NLPHD However, clonality is not detected in the whole tissue DNA,
are rare. The infiltration pattern is either diffuse, nodular to but only in DNA of isolated single LP-cells. A rearrange-
patchy (see Fig. 7.44f), or subtle with few scattered tumor ment of BCL6 is a frequent finding.
224 7 Malignant Lymphomas

Case History (NLPHD) healthy. Lymph node extirpation revealed infiltration by

A 28-year-old man presented with an enlarged 3.5 cm in NLPHD (Fig. 7.44). The bone marrow biopsy, which was
diameter measuring axillary lymph node. According to the rather fragmented, was infiltrated revealing a slightly nodu-
patient, the lymph node was growing slowly, and the enlarge- lar and patchy infiltrate consisting of small lymphocytes, his-
ment was noticed for more than 8 months. No other lymph tiocytes, and LP-cells (Fig. 7.44f–h). The amount of the
nodes were enlarged. Apart from this node, the patient felt neoplastic infiltrate was 20% compared to hematopoiesis.

a b

c d

CD20 CD21

Fig. 7.44 NLPHD. (a, b) Routinely stained H&E sections of the The LP-cells are characteristically J-chain-positive. (f) Despite
lymph node show a vaguely nodular neoplastic infiltrate (a) consisting fragmentation, a nodular and patchy neoplastic infiltrate is visible
of small lymphocytes with scattered LP-cells (b). (c) The LP-cells are in the bone marrow biopsy. (g, h) MGG-stain and H&E stain reveal
highlighted with an antibody to CD20; also, small lymphocytes are an infiltrate with small lymphocytes, histiocytes, and huge blasts
CD20-positive B-cells. (d) Within the nodules an irregular network consistent with LP-cells
of follicular dendritic cells is stained with an antibody to CD21. (e)
7 Malignant Lymphomas 225

e f

J-chain MGG

g h

MGG

Fig. 7.44 (continued)

7.21.1.5 Caution Compared with LP-cells in the corresponding lymph

Infiltration of the bone marrow by NLPHD may be subtle node, these cells often appear smaller in the bone marrow
in the interstitium. Thus, at least staining with an anti- biopsy [91].
body to CD20 is recommended for detection of an The differential diagnosis to T-cell and histiocyte-rich
involvement. large B-cell lymphoma can be challenging.
226 7 Malignant Lymphomas

7.22 Classical Hodgkin Lymphoma (cHL) focal bone marrow infiltration with nodular or patchy infiltrates
surrounded by normal bone marrow. A minority of patients
7.22.1 Epidemiology with cHL reveal a paratrabecular infiltration. The infiltrates are
polymorphous with small lymphocytes, histiocytes, and plasma
High variability is reported on the incidence of BM infiltra- cells as well as eosinophils. The infiltrates are accompanied by
tion by cHL. In children, the incidence is very low with a reticulin or collagen fibrosis confined to the lesions.
reported 1.8% in a large series [93], thus the significance of
a bone marrow biopsy is questionable in pediatric patients.
In adults, the reported incidence of bone marrow involve- 7.22.3 Immunohistochemistry
ment is 2–32%. In mixed cellularity Hodgkin lymphoma
bone marrow involvement is more frequent than in nodular HRS show a typical immunohistochemical profile, facilitat-
sclerosis type [94]. ing the detection in bone marrow biopsies. The tumor cells
Infection by EBV may play a role in the pathogenesis of are always CD30-positive (Fig. 7.45e), often reactive with
cHL. While in endemic areas nearly 100% of Hodgkin lym- antibodies to CD15, CD20, CD23, PAX5, BCL6, and MUM1.
phomas are EBV-positive, in Western countries EBV- Usually, the tumor cells do not express LCA and BCL2.
positivity is found only in a proportion of cases [95]. The tumor cells, especially of mixed cellularity type often
express EBV-associated markers, such as LMP1 and are pos-
itive for Epstein–Barr encoding region (EBER) in situ
7.22.2 Morphology (Figs. 7.45 and 7.46) hybridization (Fig. 7.45h) [96].

The following histological criteria have been recommended

to establish the diagnosis of bone marrow involvement by 7.22.4 Cytogenetic Abnormalities
cHL: characteristic Reed-Sternberg cells or mononuclear and Molecular Characteristics
Hodgkin cells (HRS) in a typical cellular environment (see
Fig. 7.45b). In addition, atypical cells in an eosinophil-rich HRS contain clonal immunoglobulin (Ig) gene rearrange-
fibrotic environment even in the absence of HRS-cells are ments in more than 98% of the cases and clonal T-cell recep-
strongly suggestive of bone marrow involvement in patients tor gene rearrangements in a very few cases. The clonal
diagnosed with cHL. HRS are never detected in areas of rearrangements are found only in the DNA of isolated tumor
inconspicuously appearing marrow since HRS-cells are cells and not in the whole tissue specimen.
always surrounded by an inflammatory microenvironment. The tumor cells, which originate from germinal center
The infiltration pattern is often diffuse, affecting entire mar- B-cells have lost most of the specific B-cell expression pro-
row spaces between the bone trabeculae. Some cases show a gram and have acquired other gene products [97].
7 Malignant Lymphomas 227

Case History (classical Hodgkin Lymphoma)

Case History 1 (Fig. 7.45)
A 64-year-old male was diagnosed with cHL, nodular scle-
rosing subtype, in a cervical lymph node. The bone marrow
biopsy revealed multiple patchy infiltrates consistent with cHL.

a b

c d

GOM CD20

Fig. 7.45 cHL case 1. (a, b) Routinely stained H&E section shows The tumor cells are strongly CD20-positive. (e) Specific CD30 staining
hypercellular bone marrow with multiple patchy infiltrates (a), which, of the tumor cells. (f) Some tumor cells are CD15-positive; further-
in higher magnification (b) reveal HRS-cells in a background of poly- more, the intermingled eosinophils are CD15-positive. (g) The small
morphic lymphocytes, histiocytes, and granulocytes. (c) Within the polymorphic lymphocytes are T-cells (CD3). (h) Tumor cells are EBV-
neoplastic infiltrates, a dense fibrosis is evident (Gomori’s stain). (d) positive (EBER)
228 7 Malignant Lymphomas

e f

CD30 CD15

g h

CD3 EBER

Fig. 7.45 (continued)

7 Malignant Lymphomas 229

Case History 2 (Fig. 7.46)

A 59-year-old male patient was diagnosed with cHL,
mixed cellularity, in an axillary lymph node. This patient
showed a dense infiltration by this cHL in the bone marrow
biopsy

a b

c d

Fig. 7.46 cHL, case 2. (a–b) Bone marrow biopsy is densely infil- cytes are strongly CD20-positive. (f) Granulocytes and some of the
trated by a neoplastic infiltrate (a). In higher magnification (c–d) atypi- mononuclear blasts are CD15-positive. (g) Strong CD30-positivity of
cal, mainly mononuclear blasts can be identified in an inflammatory the neoplastic cells (h) Typically, HRS-cells are stained for the
background. (e) The mononuclear blasts as well as some small lympho- transferrin-receptor CD71, an activation antigen
230 7 Malignant Lymphomas

e f

CD20 CD15

g h

CD30 CD71

Fig. 7.46 (continued)

7 Malignant Lymphomas 231

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Mastocytosis
8

Mastocytosis comprises a group of clonal mast cell (MC) Table 8.1 Updated WHO classification of mastocytosis 2016
disorders whose clinical course ranges from indolent to 1. Cutaneous mastocytosis (CM):
aggressive. In the revised 2016 WHO classification, masto- Urticaria pigmentosa (UP)/Maculopapular cutaneous mastocytosis
cytosis is included as a separate category and not any more (MPCM)
Diffuse cutaneous mastocytosis
listed among myeloproliferative neoplasms [1, 2].
Solitary mastocytoma of skin
Non-neoplastic MCs are present in many human organs 2. Indolent systemic mastocytosis (ISM)
including the lymphatic system and particularly abundant Meets criteria for systemic mastocytosis (SM). No “C” findings.
at major body interfaces with the external environment No evidence of associated hematological neoplasm
such as the skin, the lung, and the gastrointestinal tract Isolated bone marrow mastocytosis
[3]. MCs have pleiotropic functions including the multi- As above (ISM), but with bone marrow involvement and no skin
involvement, generally low burden of MC
valent capacity to recognize and to react to internal and
3. Smouldering systemic mastocytosis (SSM)
external dangers and form a bridge between innate and As above (ISM), but with 2 or more “B” findings, and no “C”
adaptive immunity. MCs store preformed mediators findings, 1 generally high burden of MC
within granules that are rapidly released thus promoting 4. Systemic mastocytosis with an associated hematological neoplasm
inflammation and local recruitment of other innate immu- (SM-AHN)
Meets criteria for SM and criteria for AHN as a distinct entity per
nity cells [3].
the WHO classification
The spectrum of mastocytosis is heterogeneous and sub- 5. Aggressive systemic mastocytosis (ASM)
divided into cutaneous mastocytosis, systemic mastocytosis Meets criteria for SM. One or more “C” findings. No evidence of
(SM), and localized mast cell tumors (Table 8.1). SM is mast cell leukemia
characterized by the accumulation of atypical MCs in one or 6. Mast cell leukemia (MCL)
more organ systems. The SM category includes indolent Meets criteria for SM. Bone marrow biopsy shows diffuse
infiltration, usually dense, by atypical, immature mast cells. BM
SM, smouldering SM and SM with an associated hemato- aspirate smears show ≥20% mast cells. In classic cases, mast cells
logical neoplasm (SM-AHN), aggressive SM (ASM), and account for ≥10% of peripheral blood white cells. Aleukemic
mast cell leukemia (MCL) [4, 5]. The term advanced sys- MCL variant (<10% circulating mast cells)
temic mastocytosis is used for aggressive SM, SM-AHN, 7. Mast cell sarcoma (MCS)
and mast cell leukemia. For the diagnosis of SM, one major No evidence of SM. Generally localized destructive growth
pattern. High-grade cytology
and several minor criteria have been defined. The major cri-
Adopted from Horny et al. [2], Pardanani [5]
terion is the presence of multifocal clusters of abnormal The diagnosis of SM can be made when the major criterion and one
≥15 MCs in the bone marrow or other organs. Minor diag- minor criterion or at least three minor criteria are present (see Table 8.2)
nostic criteria include spindle-shaped or morphologically

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 235

C. Beham-Schmid, A. Schmitt-Graeff, Bone Marrow Biopsy Pathology,
Essentials of Diagnostic Pathology, https://doi.org/10.1007/978-3-662-60309-3_8
236 8 Mastocytosis

Table 8.2 Major criterion and minor criteria, B and C findings that are 8.1 Epidemiology and Clinical Features
relevant for the classification of SM subtypes
Major criterion A prevalence of mastocytosis of 1 in 10,000 inhabitants has
Multifocal, dense infiltrates of mast cells (≥15 mast cells in been reported, but underdiagnosis is assumed [7]. CM pre-
aggregates) detected in sections of bone marrow and/or other
extracutaneous organ(s)
dominantly affects children and may disappear during
Minor criteria puberty [8]. SM mastocytosis generally develops in adults
(a) In biopsy sections of bone marrow or other extracutaneous with a light male predominance [2].
organs, >25% of the mast cells in the infiltrate are spindle- The clinical features are variable and related to the sub-
shaped or have atypical morphology or, of all mast cells in type of mastocytosis. CM may present as urticaria pigmen-
bone marrow aspirate smears, >25% are immature or atypical
(b) Detection of an activating point mutation at codon 816 of KIT
tosa, diffuse CM, or mastocytoma of skin.
in bone marrow, blood, or other extracutaneous organ SM may be associated with four categories of
(c) Mast cells in bone marrow, blood, or other extracutaneous symptoms:
organ express CD25 with/without CD2 in addition to normal
mast cell markers. (Mast cell CD25 is the more sensitive
(a) Constitutional symptoms (e.g., fatigue)
marker, by both flow cytometry and immunohistochemistry)
(d) Serum total tryptase persistently exceeds 20 ng/mL (unless (b) Skin manifestations (e.g., urticarial)
there is an associated myeloid neoplasm, in which case this (c) Mediator-related symptoms (e.g., abdominal pain, gas-
parameter is not valid) trointestinal problems)
“B” findings (d) Musculo-skeletal symptoms (osteoporosis)
1. High mast cell burden shown on BM biopsy: >30% infiltration
of cellularity by mast cells (focal, dense aggregates) and serum
total tryptase level > 200 ng/mL Patients with advanced SM such as aggressive SM and
2. Signs of dysplasia or myeloproliferation in non-mast cell mast cell leukemia may develop hepato-splenomegaly and
lineage(s), but insufficient criteria for definitive diagnosis of an organ impairment for example liver failure secondary to
associated hematological neoplasm (AHN), with normal or only mast cell infiltrates. Bone marrow involvement may be asso-
slightly abnormal blood counts.
ciated with anemia, secondary eosinophilia, neutropenia,
3. Hepatomegaly without impairment of liver function, palpable
splenomegaly without hypersplenism, and/or lymphadenopathy and thrombocytopenia or even bone marrow failure. In
on palpation or imaging. patients who develop SM-AHN, the outcome principally
“C” findings depends on both disorders. However, the non-mast cell
1. Bone marrow dysfunction caused by neoplastic mast cell hematologic neoplasm such as chronic myelomonocytic leu-
infiltration, manifested by ≥1 cytopenia(s) (ANC <1.0 × 109/L,
Hgb < 10 g/dL, and/or platelet count <100 × 109/L)
kemia, myelodysplastic/myeloproliferative neoplasm,
2. Palpable hepatomegaly with impairment of liver function, unclassifiable, or acute myeloid leukemia often determines
ascites, and/or portal hypertension patient prognosis.
3. Skeletal involvement with large osteolytic lesions with/without Serum tryptase released from MCs is important for the
pathological fractures (pathological fractures caused by diagnosis and monitoring of SM. Total serum tryptase levels
osteoporosis do not qualify as a “C” finding)
≥20 ng/mL are suggestive of SM. However, serum tryptase
4. Palpable splenomegaly with hypersplenism
5. Malabsorption with weight loss due to gastrointestinal mast cell may be normal in patients with low MC burden, for example,
infiltrates with indolent SM or isolated bone marrow mastocytosis [5].
Adopted from Horny et al. [2], Pardanani [5] It is worth noting that tryptase levels may also be increased
in non-MC neoplasms such as chronic myeloid leukemia.

atypical MCs, elevated serum tryptase level, abnormal 8.2 Morphologic

CD25 expression by MCs, and KITD816V mutation and Immunohistochemical Features
(Table 8.2). There is evidence that adequate diagnosis and of Bone Marrow Involvement by SM
subclassification of SM requires an in-depth evaluation of
the BM by experienced hematopathologists in combination Bone marrow involvement by SM is characterized by the
with molecular genetics, serum tryptase level, and clinical presence of multifocal clusters of abnormal MCs. Such clus-
parameters [6]. ters should be composed of aggregates comprising 15≥ MCs.
8 Mastocytosis 237

They are predominantly localized in the perivascular and/or mastocytosis by the revised 2016 WHO classification. In
paratrabecular bone marrow compartments and frequently cases with minimal bone marrow involvement molecular
associated with focal reticulin fibrosis. MC aggregates may analysis of laser microdissected MCS may be used to detect
be accompanied by reactive eosinophilia and/or lymphoid the KIT D816V mutation. Mast cell leukemia may harbor
infiltrates. However, 20% of patients with indolent SM atypical mutations in the KIT gene such as D816H/Y or
patients lack MC clusters in the bone marrow [5]. BM neo- F522C [10]. Rare SM cases with normal morphologic and
plastic MCs are generally spindle-shaped with oval nuclei immunophenotypic features may also harbor non-D816V
and elongated cytoplasmic processes. They may also have KIT mutations such as germline F522C or somatic I817V
round nuclei and abundant clear cytoplasm. In high grade SM mutations [5].
such as aggressive SM or MC leukemia, MCs may have bilo- Additional mutations may be found especially in
bated or multilobated nuclei or even resemble to metachro- advanced SM and include mutations in TET2, SRSF2,
matic blasts. MCs may easily be detected on Giemsa and/or ASXL1, RUNX1, CBL, JAK2, and/or RAS genes [9]. There is
Naphthol-ASD-chloroacetate esterase stained bone marrow evidence that mutations in SRSF2, ASXL1, RUNX1, and ele-
sections. However, since neoplastic MCs may contain rare vated alkaline phosphatase are predictive adverse prognostic
metachromatic granules or may even be degranulated, they markers for overall survival in SM [11]. Models for SM
may be missed on conventionally stained sections. Therefore, based on clinical and integrated clinical-genetics informa-
immunohistochemical staining for tryptase and CD117 (c-kit) tion have demonstrated the independent prognostic contri-
is mandatory when SM is suspected. However, these markers bution of mutations [12].
do not discriminate between neoplastic and non-neoplastic
MCs. By contrast, the aberrant expression of CD25 by neo-
plastic MCs is considered as a hallmark of mastocytosis. CD2 8.4 Caution
immunostain is less helpful. Aberrant CD30 expression is
observed in subsets of neoplastic MCs especially in aggres- • Mast cell infiltrates may easily be missed on H&E
sive SM and mast cell leukemia. stained sections. The application of immunohistochemi-
Mast cell leukemia is diagnosed when bone marrow aspi- cal markers for assessment of mast cell infiltration is
rate smears contain ≥20% MCs that are generally immature, mandatory.
atypical, and rather round than spindle-shaped. Mast cell • Atypical mast cells especially in mast cell leukemia are
leukemia may be accompanied by a leukemic blood picture frequently devoid of prominent polychromatic cytoplas-
with ≥10% peripheral blood MCs or be aleukemic. mic granules, may show lobulated nuclei, and may mimic
A retrospective study of bone marrow biopsies based on a monocytes/monoblasts.
cohort from the German Registry of Disorders on Eosinophils • Expression of tryptase is not restricted to mast cells but
and Mast Cells has shown that SM may be overlooked by may also be observed in basophilic leukemia that, how-
pathologists especially when immunohistochemical markers ever, does not express CD117.
were not applied [6]. • Eosinophils may be abundant in mastocytosis so that
myeloid/lymphoid neoplasms with eosinophilia and
gene rearrangement may be considered as a differential
8.3 Genetics diagnosis especially as these disorders often harbor a
spindle-shaped CD25+ mast cell population. However,
Mutations in KIT, most frequently KIT D816V, are present in mast cells in this category are generally loosely scattered
over 80% of all systemic mastocytosis patients [9]. The KIT between eosinophils and rarely arranged in clusters ≥15
D816V mutation is listed as minor diagnostic criterion for mast cells.
238 8 Mastocytosis

Case History 1. Bone Marrow Mastocytosis (Fig. 8.1) mast cell infiltrates accounting for about 15% of bone mar-
Routine laboratory examination of this 74-year-old man row cells. Aggregates were formed by >15 mast cells. The
revealed a mild chronic anemia. The CBC was as following: immunophenotype was characterized by a co-expression by
WBC 5.1 × 109/L, HB 11.3 g/dL, platelets 152 × 109/L. The CD25, CD117, and tryptase. CD2 was only expressed by
differential count was normal. No hepato-splenomegaly, rare lymphocytes. The mast cell infiltrates were associated
lymphodenopathy, or skin lesions were present. The serum with focal fibrosis. A focal moderate lacunar bone resorp-
tryptase level was 17 ng/mL. A bone marrow aspirate smear tion was observed but no large osteolytic lesions. A KIT
contained rare spindle-shaped mast cells containing abun- D816V mutation was present. The diagnosis was bone mar-
dant metachromatic granules. A bone marrow core biopsy row mastocytosis, a subtype of indolent systemic
was performed and showed paratrabecular and perivesicular mastocytosis.

a b

MGC Giemsa

c d

CD25 CD25

Fig. 8.1 Bone marrow mastocytosis. (a) Bone marrow aspirate smear CD25; e, f, CD117). Note focal moderate lacunar bone resorption (b).
with spindle-shaped mast cells showing polychromatic cytoplasmic (g, h) Perivascular and paratrabecular fibrosis associated with the mast
granulation (MGG). (b–f) Bone marrow core biopsy with paratrabecu- cells (Gomori)
lar (b, e) and perivascular (c, d, f) mast cell infiltrates (b; Giemsa; c, d,
8 Mastocytosis 239

e f

CD117 CD117

g h

Gomori Gomori

a b

H&E H&E

Fig. 8.7 Acute mast cell leukemia. (a, b) Extensive infiltration of the esterase (c) and myeloperoxidase (d) while both reactions stained resid-
bone marrow core biopsy by atypical mast cells showing oval or slightly ual myeloid cells. (e–h) The atypical mast cells expressed CD117 (e),
lobulated nuclei and a wide faintly granulated eosinophilic cytoplasm tryptase (f), partially CD30 (g) and CD25 (h)
(H&E). (c, d) Mast cells were negative for naphthol-ASD-chloroacetate-
250 8 Mastocytosis

c d

NASDCL MPO

e f

CD117 Tryptase

g h

CD30 CD25

Fig. 8.7 (continued)

8 Mastocytosis 251

References A. Impact of centralized evaluation of bone marrow histology in

e f

2D7 CD15
g h

CD34 CD42b

Fig. 9.3 (continued)

262 9 Myeloproliferative Neoplasm (MPN)

Case History 4 (CML, BC-AML) (Figs. 9.4 and 9.5) 33.2 g/dL, and Thrombo 20 G/L. A bone marrow biopsy
A 40-year-old male with treated CML for 8 years presented showed irregular cellularity with marked fibrosis and
with pancytopenia: leukocytes: 3.41 G/L, Ery 3.22 T/L, Hb increase of more than 30% myeloid blasts, thus myeloid
12.9 g/dL, Hkt 38.8%, MCV 120.5 fL, MCH 40.1 pg, MCHC blast crisis was diagnosed.

a b

c d

Fig. 9.4 Case 4 (CML, BC-AML). (a) Variable cellularity of the bone of small megakaryocytes. (e–h) The highly hypercellular, as well as the
marrow spaces (H&E). (b–d) In the highly hypercellular marrow hypocellular marrow spaces reveal a dense fibrosis (reticulin: grade 3,
spaces, a diffuse infiltration of blasts is seen, in addition to an increase collagen: grade 3) as shown with Gomori’s stain
9 Myeloproliferative Neoplasm (MPN) 263

e f

GOM GOM

g h

GOM GOM

Fig. 9.4 (continued)

a b

MPO CD33

Fig. 9.5 Case 4 (CML, BC-AML). (a) Most blasts are reactive with an ity of blasts are reactive with an antibody to CD34. (f) Strong specific
antibody to MPO. (b) Using an antibody to CD33, most blasts show CD7-positivity of the myeloid blasts. (g, h) High number of small
specific reactivity. (c) Most blasts are CD15-positive. (d, e) The major- megakaryocytes (dwarf forms) stained with an antibody to CD61
264 9 Myeloproliferative Neoplasm (MPN)

c d

CD15 CD34

e f

CD34 CD7

g h

CD61 CD61

Fig. 9.5 (continued)

9 Myeloproliferative Neoplasm (MPN) 265

Case History 5 (CML, BC-ALL) (Fig. 9.6) with Leuko 6.89 G/L, Ery 1.84 T/L, Hb 5.8 g/dL, Hkt 16.3%,
A 60-year-old female with Alzheimer’s disease and treated Thrombo 43 G/L, and LDH 340 U/L. The bone marrow
BCR-ABL1+ CML for 2 years was admitted to the hospital biopsy showed lymphoid BC.
because of a fainting fit. The blood picture was pathologic

a b

c d

CD10 CD20

Fig. 9.6 Case 5 (CML, BC-ALL). Lymphatic BC CML. H&E stained for CD10 (c), CD20 (d), CD34 (e) and TdT (f). (g, h) Morphologically,
section shows a highly hypercellular bone marrow (a) with dense infil- and also by immunohistochemistry, no CD42b-positive megakaryo-
tration of blast (b) with round nuclei and several nucleoli (inset). cytes and no MPO-positive granulopoietic cells could be identified
Immunohistochemistry revealed infiltration by lymphoid blasts positive
266 9 Myeloproliferative Neoplasm (MPN)

e f

CD34 TdT

g h

Fig. 9.9 (continued)

9.4.4 Caution Some patients have “masked PV.” This term was re-
introduced for JAK2-mutated patients with latent (ini-
The presence of reticulin fibrosis does not exclude the diag- tial, occult pre-polycythemic) disease manifestations,
nosis of PV. but who present with a BM morphology consistent with
If iron deposits are found, the patient might have a bleeding PV [18].
history. Otherwise, the diagnosis of PV has to be reassessed.
9 Myeloproliferative Neoplasm (MPN) 275

9.5 Primary Myelofibrosis (PMF) correct diagnosis of prefibrotic PMF is the atypical mega-
karyocytopoiesis. There are clusters of megakaryocytes,
9.5.1 Epidemiology often located paratrabecular or perisinusoidal, and most
megakaryocytes are enlarged, but there are also small forms.
The incidence rate of PMF ranges from 0.1 per 100,000 per The nuclei often show clumped chromatin, and the nuclei
year to 1 per 100,000 per year [19]. The median age at diag- appear “cloud-like” with hypolobulation. Naked megakaryo-
nosis is 65 years with a preponderance for males. cytic nuclei are often seen. Lymphoid nodules are not uncom-
mon and usually the bone marrow shows vascular
proliferation. Most patients with prefibrotic PMF gradually
transform into overt PMF. The diagnostic criteria for diagno-
9.5.2 Morphology sis of pre PMF are listed in the following table.

In addition to morphology of the bone marrow biopsy, driver WHO prePMF criteria [1, 15]
Major criteria
mutations are integrated into WHO diagnostic criteria. The
1. Megakaryocytic proliferation and atypia, without reticulin fibrosis
mutational frequency in PMF is 58% JAK2, 25% CALR, and >grade 1, accompanied by increased age-adjusted BM cellularity,
7% MPL [15]. granulocytic proliferation, and often decreased erythropoiesis
2. Not meeting the WHO criteria for BCR-ABL1+ CML, PV, ET,
myelodysplastic syndromes, or other myeloid neoplasms
3. Presence of JAK2, CALR, or MPL mutation or in the absence of
9.5.3 PMF, Prefibrotic/Early Stage these mutations, presence of another clonal marker, or absence of
minor reactive BM reticulin fibrosis
About 30–40% of patients with PMF are diagnosed in the Minor criteria
early stage without showing a significant reticulin or colla- Presence of at least one of the following, confirmed in two
gen fibrosis (grade 0 or 1). The bone marrow biopsy is consecutive determinations:
a. Anemia not attributed to a comorbid condition
hypercellular with hyperplastic granulopoiesis and increase
b. Leukocytosis ≥11 × 109/L
of atypical megakaryocytes. There may be a slight left shift c. Palpable splenomegaly
of granulopoietic cells without an increase of myeloblasts in d. LDH increased to above upper normal limit of institutional
percentage. There is no increase of CD34+ blasts. Usually, reference range
erythropoiesis is decreased, but some patients show rather Diagnosis of prePMF requires meeting all three major criteria and at
prominent erythroid precursor cells. The key feature for a least one minor criterion
276 9 Myeloproliferative Neoplasm (MPN)

Case History 1 (Pre-PMF) 24.2/pg, MCHC 33.3 g/dL, Thrombo 810 G/l, LDH
A 74-year-old female was admitted to the hospital because 288 U/l.
of cardiac insufficiency. During the medical work-up, a These findings were clinically suspicious for PV, addi-
pathologic blood picture, slightly increased LDH and tionally JAK2-positivity was found. A bone marrow
slight splenomegaly were found: Leuko 12.0 G/l, Ery biopsy was performed and diagnosed as MPN, pre-PMF
5.99 T/l, Hb 14.5 g/dL, Hkt 43.6%, MCV 72.8/L fL, MCH (Fig. 9.10).

a b

c d

GOM

Fig. 9.10 (a) The bone marrow is highly hypercellular with only a few (d) or show a slight circumscribed fibrosis (e). (f) Immunohistochemically
irregularly distributed fat cells (H&E). The most striking feature is the stained megakaryocytes (CD42b) are markedly increased with cluster
increased, highly polymorphous and atypical megakaryocytopoiesis formation. (g) Large atypical megakaryocytes with hypolobulated
with cluster formation (b) and hypolobulated nuclei with dense chro- nuclei forming a cluster (CD61). (h) The marrow reveals rather promi-
matin (b, c). Most marrow spaces are either devoid of reticulin fibers nent erythroid precursor cells (CD71)
9 Myeloproliferative Neoplasm (MPN) 277

e f

GOM CD42b

g h

CD61 CD71

Fig. 9.10 (continued)

278 9 Myeloproliferative Neoplasm (MPN)

9.5.4 PMF, Overt Fibrotic Stage 3. Presence of JAK2, CALR, or MPL mutation or in the absence of
these mutations, presence of another clonal marker, or absence of
reactive myelofibrosis
In this stage the bone marrow shows marked reticulin fibro-
Minor criteria
sis (grades 2 and 3) and frequently also collagen fibrosis of Presence of at least one of the following, confirmed in two
various extents. Cellularity is quite variable. In one and the consecutive determinations:
same biopsy, there can be areas with hypercellularity and a. Anemia not attributed to a comorbid condition
marked hypocellularity. In most cases the bone marrow is b. Leukocytosis ≥11 × 109/L
hypocellular for age with foci of hematopoietic cells alter- c. Palpable splenomegaly
d. LDH increased to above upper normal limit of institutional
nating with fatty tissue or fibers. Megakaryocytes are highly
reference range
atypical and can be arranged in dense sheets. Often mega- e. Leukoerythroblastosis
karyocytes are present within dilated sinusoids. Some Diagnosis of overt PMF requires meeting all three major criteria and
patients show dense fibrosis and nearly no hematopoietic at least one minor criterion
cells or a few immature hematopoietic islands within ves-
sels. Secondary bone formation of various degrees can be
detected [1, 20].
Disease acceleration is diagnosed, if patients with previ- 9.5.5 Immunohistochemistry
ously assured PMF show 10–19% blasts in the peripheral blood
or bone marrow, or if there is an increase of CD34+ blasts There are no documented abnormal phenotypic features.
detected immunohistochemically. Acceleration of overt PMF However, in PMF (early stage and fibrotic stage) immuno-
may additionally be indicated by an increase of monocytoid histochemistry is helpful for detecting the atypical mega-
differentiated cells [21]. Transformation to acute leukaemia is karyocytes, which are highlighted by using markers for
indicated by the finding of ≥20% blasts. The diagnostic criteria megakaryocytes, such as CD41, CD42b, or CD61.
for PMF, fibrotic stage are listed in the following table. Furthermore, the exact number of erythropoietic cells can
be estimated with antibodies to CD71, glycophorin A or
gylycophorin C. Also the amount of granulopoietic cells
WHO overt PMF criteria [1, 15] and an increase of blasts might need immunohistochemical
Major criteria
investigation (CD15, MPO, CD33, CD34, CD117c).
1. Presence of megakaryocytic proliferation and atypia, accompanied
by either reticulin and/or collagen fibrosis grades 2 or 3 Recently, an antibody to calreticulin has been developed.
2. Not meeting WHO criteria for ET, PV, BCR-ABL1+ CML, The use of this antibody allows a specific and rapid detec-
myelodysplastic syndromes, or other myeloid neoplasms tion of calreticulin mutations [22].
9 Myeloproliferative Neoplasm (MPN) 279

Case History (PMF Overt Stage) Hb 10.3 g/dL, Hkt 31.3%, MCV 92.3 fL, MCH 30.4 pg,
A 52-year-old male was admitted to the hospital because of MCHC 32.9 g/dL, Thrombo 98 G/L. LDH was increased
back pain, anemia, and thrombocytopenia. Furthermore the with LDH 486 U/l. JAK2 was detected.
patient complained about night sweats since 6 weeks without A bone marrow biopsy was performed revealing MPN,
infectious disease. The spleen was markedly enlarged. The overt PMF (reticulin fibrosis grade 2, collagen fibrosis grade
blood count was as following: Leuko 7.73 G/L, Ery 3.39 T/L, 2 and osteosclerosis grade 2) (Fig. 9.11).

a b

c d

GOM

Fig. 9.11 Overt PMF. (a–c) In H&E stained sections, the bone marrow lin fibrosis grade 2 and focal collagen fibrosis (gold-colored fibers vis-
is markedly hypercellular. The bone trabeculae focally are irregularly ible in (e). (f) Immunohistochemically, with an antibody to CD61, there
formed with newly formed bone (osteosclerosis grade 2). This second- is a marked increase of atypical megakaryocytes with focal cluster for-
ary bone formation is seen as focal budding, hooks, spikes, and focal mation. (g) Erythropoietic cells are decreased and irregularly distrib-
paratrabecular apposition of new bone. Striking increase of atypical uted (CD71). (h) Granulopoietic cells (stained immunohistochemically
megakaryocytes is evident. (d, e) Gomori’s stain reveals diffuse reticu- with MPO) are still prominent
280 9 Myeloproliferative Neoplasm (MPN)

e f

GOM CD61

g h

CD71 MPO

Fig. 9.11 (continued)

9.5.6 Caution In addition to the grading of reticulin fibers (according

to WHO grade 0–3) grading of collagen fibrosis and sec-
Careful bone marrow examination is essential for differenti- ondary bone formation might be useful, especially for
ating pre-fibrotic PMF with thrombocytosis from ET. The estimating these parameters in follow-up biopsies after
key features for correct diagnosis are the highly atypical therapy [20].
megakaryocytes with often hypolobulated nuclei with cloud-
like chromatin.
9 Myeloproliferative Neoplasm (MPN) 281

9.6 Essential Thrombocythemia (ET) WHO ET criteria [1]

Major criteria
9.6.1 Epidemiology 1. Platelet count ≥450 × 109/L
2. BM biopsy showing proliferation mainly of the megakaryocyte
lineage with increased numbers of enlarged, mature
The estimated incidence of ET ranges from 0.38 per 100,000 megakaryocytes with hyperlobulated nuclei. No significant
per year and 1.7 per 100,000 per year [19]. Most patients increase or left shift in neutrophil granulopoiesis or erythropoiesis
with ET are between 50 and 60 years old without sex and very rarely minor (grade 1) increase in reticulin fibers
predilection. However, there is a peak in women at the age of 3. Not meeting WHO criteria for BCR-ABL1+ CML, PV, PMF,
myelodysplastic syndromes, or other myeloid neoplasms
around 30 years [23].
4. Presence of JAK2, CALR, or MPL mutation
Minor criterion
Presence of a clonal marker or absence of evidence for reactive
9.6.2 Morphology thrombocytosis
Diagnosis of ET requires meeting all four major criteria or the first
The bone marrow biopsy is in most cases normocellular or three major criteria and the minor criterion
only slightly hypercellular without pathological increase of
reticulin or collagen fibers. The key feature of ET is the marked
increase of megakaryocytes. Large megakaryocytes or even 9.6.3 Immunohistochemistry
giant forms predominate. These forms often show hyperlobu-
lated (stag-horn like) nuclei. These megakaryocytes are dif- In ET cases, no aberrant phenotypic features are described.
fusely dispersed and may form loose clusters. Some patients, However, immunohistochemical stains are helpful in estimat-
especially after bleeding episodes, may reveal a proliferation ing the exact quantity of hematopoietic cells. Usually ET cases
of erythroid precursors, but usually the erythropoietic cell line show normal erythro- and granulopoiesis. The increased
is normal. Granulopoiesis is normal or only slightly increased. megakaryocytes with hyperlobulated nuclei can be highlighted
Due to the increase of megakaryocytes, many of these cells with antibodies to megakaryocytes (e.g. CD41, CD42b,
may show an emperipolesis. Iron can be found. Diagnostic CD61). In addition, calreticulin antibody can be used to estab-
criteria for ET are listed in the following table. lish calreticulin mutation.
282 9 Myeloproliferative Neoplasm (MPN)

Case History (ET) The bone marrow biopsy was normocellular with normal
A 54-year-old male was admitted to the hospital after an appearing erythro- and granulocytopoiesis. There was a mod-
accident with rib fracture. During work-up, marked throm- erate increase of normal sized and enlarged megakaryocytes
bocytosis (937 G/L) was found. All remaining blood test with often markedly hyperlobulated nuclei. Occasionally,
results were normal, and the patient had no history of throm- loose clusters of megakaryocytes were found. No fibrosis was
bosis or other complaints. JAK2 mutation was found and a seen. The diagnosis was MPN, ET (Fig. 9.12).
bone marrow biopsy was performed.

a b

c d

Fig. 9.12 ET (a) Normocellular bone marrow. (b) No pathological seen (MGG). (f–h) Rather large megakaryocytes with hyperlobulated
fibrosis is seen in Gomori’s stain. (c) Megakaryocytes are increased (“stag-horn” like) nuclei (MGG)
(MGG). (d, e) Loose cluster of pleomorphic megakaryocytes can be
9 Myeloproliferative Neoplasm (MPN) 283

e f

g h

Fig. 9.12 (continued)

284 9 Myeloproliferative Neoplasm (MPN)

9.6.4 Caution If the patients with suggested ET show highly atypical

megakaryocytes, pre-PMF has to be excluded.
Reactive thrombocytosis must be discriminated from A few bone marrow biopsies reveal morphologic features
ET. Frequent causes for reactive thrombocytosis include typical for ET (large to giant megakaryocytes with hyper-
infectious diseases, inflammation, hemolysis, iron defi- lobulated nuclei) and also features resembling PV
ciency anemia, drugs, splenectomy, and malignant disor- (proliferation of granulopoiesis and erythropoiesis).

ders as well as the rare congenital thrombocytoses, which According to Thiele, such peculiar cases exhibiting a histo-
are associated with mutations of thrombopoietin or its pathologic chimeric appearance are diagnosed as ET/PV. The
receptor. course of the disease with follow-up biopsies will uncover
If patients suggestive of suffering of ET show an increase the underlying disease [24] (Fig. 9.13).
in erythropoietic and granulopoietic cells, masked PV has to
be taken into consideration.

a b

c d

Fig. 9.13 Addendum to MPN: PV, PMF, and ET. Since megakaryocytes like nuclei as well as cluster-formation (e, f: H&E; g, h: MGG). (i–l)
are the hallmark cells in MPNs, figures of megakaryocytes in the bone mar- Megakaryocytes in ET: Most megakaryocytes are enlarged with abundant
row of these diseases are shown: Megakaryocytes in PV are highly pleo- cytoplasm and hyperlobulated, occasionally “stag-horn”-like (l) nuclei.
morphic and are of different size. Marked maturation defects concerning These megakaryocytes are often arranged in loose clusters (i, j: MGG).
the nuclear-cytoplasmic differentiation are not seen (a, b: H&E; c, d: Megakaryocytes in post-PV-MF (m, n, H&E) and post-ET-MF (o, p,
MGG). Megakaryocytes in PMF are highly atypical with abnormal nuclear- H&E) are highly pleomorphic and atypical with hyper- or hypolobulated
cytoplasmic ratio, abnormal clumping of chromatin and occasional cloud- nuclei and often clumped chromatin
9 Myeloproliferative Neoplasm (MPN) 285

e f

g h

i j

Fig. 9.13 (continued)

286 9 Myeloproliferative Neoplasm (MPN)

k l

m n

o p

Fig. 9.13 (continued)

9 Myeloproliferative Neoplasm (MPN) 287

9.7 Myeloproliferative Neoplasm, 2. Advanced stages of MPN: reticulin or collagen fibrosis,

Unclassifiable (MPN-U) secondary bone formation or transformation to an aggres-
sive stage (i.e., increased blasts and/or dysplasia) obscures
According to WHO criteria, the term myeloproliferative neo- the underlying MPN [25].
plasm, unclassifiable (MPN-U) should be applied only to 3. Patients with compelling evidence for MPN who have a
cases that have clinical, laboratory, and morphological fea- coexisting neoplastic or reactive disease which masks the
tures of MPN but do not meet the criteria for specific MPN underlying disease.
entities or that present with features that overlap two or more
of the MPN entities.
Most cases of MPN-U will belong into one of three 9.7.1 Immunohistochemistry
groups:
There is no known abnormal phenotype. However, immuno-
1. Early stages of PV, PMF, or ET: the characteristic features histochemistry is quite helpful for establishing the exact
are not yet completely developed. quantity of hematopoietic cells and for the detection of blasts.
288 9 Myeloproliferative Neoplasm (MPN)

Case History megakaryocytes. No fibrosis was detected, iron deposits were

A 49-year-old man presented with slight splenomegaly, decreased, but iron could be detected. Using antibodies to
thrombocytosis (856 G/L), leukocytosis (26.84 G/L), and CD34 and CD117c, no increase of blasts was detected.
normal Hb and normal erythropoietic count. LDH was The diagnosis was descriptive: Highly hypercellular bone
increased with 410 U/l. Further evaluation revealed negativ- marrow with increase of maturing granulocytopoiesis, increase
ity for BCR-ABL1 and positivity for JAK2. A bone marrow of polymorphic megakaryocytes, and increase of a slightly
biopsy was performed. atypical erythropoiesis. The morphologic features did not fit
The bone marrow was highly hypercellular with an increase “classical” MPN, thus a diagnosis of MPN-U was made.
of all hematopoietic components. The granulocytopoiesis The differential diagnosis included pre-PMF; however,
showed maturation, erythropoiesis was slightly left shifted only a few megakaryocytes showed hyperchromatic
with a few intermingled megaloblastic appearing cells. The hypolobulated nuclei. Especially the blood count with nor-
megakaryocytes were slightly increased and polymorphic; no mal erythropoiesis did not fit PV. However, within 8 months,
cluster formation was seen. There were a few enlarged mega- the patient developed erythrocythemia and an increased Hb
karyocytes, some small forms and many normal looking level, thus the diagnosis of PV was possible (Fig. 9.14).

a b

c d

GOM BBL

Fig. 9.14 (a) Representative bone marrow biopsy with highly hyper- topoiesis can be seen (MPO). (f) Erythropoiesis (CD71) is slightly to
cellular bone marrow (MGG). (b) Increase of all hematopoietic cells moderately increased with a few enlarged (megaloblastic appearing)
can be seen even in H&E stained section. (c) No increase of reticulin cells. The megakaryocytes are increased and polymorphic; some mega-
fibers is evident. (d) Iron stain (Berlin Blue) detects a few iron granules. karyocytes show hypolobulated nuclei (g: MGG, h: CD61)
(e) Immunohistochemically a marked increase of maturing granulocy-
9 Myeloproliferative Neoplasm (MPN) 289

e f

MPO CD71

g h

CD61

Fig. 9.14 (continued)

9.7.2 Caution If the diagnosis MPN-U is made, the pathologist should

describe all the features leading to this diagnosis.
The diagnosis of MPN-U should not be made, if the bone You should give consideration, despite the clinical
marrow biopsy is inadequate concerning the size or the assumption of MPN that the bone marrow might be reactive
quality [26]. (if no relevant mutations are known).
290 9 Myeloproliferative Neoplasm (MPN)

9.8 onocytoid Progression of MPN

M ential count, no monocytosis was found. Two years later, in the
and Monocytosis in MPN meantime the patient had only blood-letting therapy, the patient
presented with increasing thrombocytopenia. The blood count
Among progression to acute leukaemia and myelofibrosis some revealed: Leu 58.66 G/L, Ery 5.12 T/L, Hb 12.0 g/dL, Hkt
cases of Ph-negative MPNs show a striking monocytoid pro- 38.1%, Thrombo 71.0 G/L. In differential count a monocytosis
gression, the morphology resembling MDS/MPN of 18% was found. LDH was increased with 311 U/l. A FACS
CMML. These cases, histologically resembling CMML, should analysis of the peripheral blood showed 18% CD14+, CD33+,
be diagnosed as “monocytoid progression” of the preexisting CD13 DIM, DR+, CD4+ monocytes with aberrant expression
MPN, and not as CMML. During the course of disease, such of CD56 consistent with CMML. The patient was still JAK2-
cases can develop the original MPN-type again. In addition, positive. A bone marrow biopsy was performed. The highly
monocytosis (absolute monocyte count, AMC ≥ 1 × 109/L) hypercellular bone marrow showed moderately to highly
might accompany PV [27]. increased granulopoiesis with 15–20% monocytoid differenti-
An example of a monocytoid progression in a patient with ated cells. Erythropoiesis was moderately decreased and left
PV is shown. shifted with occasional megaloblastic forms. The megakaryocy-
topoiesis was slightly increased. Interestingly, many megakary-
Case History ocytes showed the typical morphology of PV; however, there
A 79-year-old man was diagnosed with JAK2-positive PV. At were small atypical forms as one can see in CMML. A slight
presentation a typical blood count was seen: Leu 18.92 G/L, Ery increase of CD34-positive blasts of more than 5% was found.
7.85 T/L, Hb 18.4 g/dL, Hkt 58.3%, Thrombo 626 G/L. LDH The diagnosis was “monocytoid progression” of PV. Since this
was normal, no splenomegaly was seen. The bone marrow is a very recent case, the outcome of the patient is not known
biopsy revealed the typical morphology of MPN, PV. In differ- (Fig. 9.15).

a b

CD61

Fig. 9.15 Monocytoid progression of PV. (a) The first biopsy shows Erythrocytopoiesis is decreased and dysplastic. A few small atypical
highly hypercellular bone marrow with the characteristic morphology megakaryocytes are seen in this marrow space (H&E). (e, f) Increase of
of PV with increase of all hematopoietic cell lines (H&E). (b) First monocytoid cells stained with an antibody to CD14. (g) Slight increase
biopsy with increased polymorphic megakaryocytes (CD61). (c) (>5%) of CD34-positive blasts. (h) Left side: In some areas large mega-
Second biopsy 2 years later: There is highly increased cellularity with- karyocytes, typical for PV can be seen, other areas (right side) show
out fat cells (H&E). (d) Second biopsy: Higher magnification with quite small atypical megakaryocytes which usually are seen in cases of
highly increased granulocytopoiesis with increased monocytoid forms. CMML (MGG)
9 Myeloproliferative Neoplasm (MPN) 291

c d

e f

CD14 CD14

g h

CD34

Fig. 9.15 (continued)

292 9 Myeloproliferative Neoplasm (MPN)

9.9 hronic Eosinophilic Leukemia, NOS

C nostic criteria imply that the diagnosis cannot be made with-
(CEL-NOS) out exact knowledge of clinical and cytogenetic data.
In the peripheral blood the hallmark of this disease is
9.9.1 Epidemiology eosinophilia. Tissue infiltration by eosinophils, especially in
the heart, skin, nervous system, and lungs, can cause serious
CEL-NOS is an extremely rare entity with clonal prolifera- complications.
tion of eosinophilic precursors and variable prognosis [28]. The bone marrow is always hypercellular because of a
The true incidence is yet not known. marked eosinophilic proliferation. In most patients with
CEL-NOS the maturation is normal. Charcot Leyden crys-
tals are a common finding. Megakaryo- and erythrocytopoi-
9.9.2 Morphology esis are usually normal in number but may show dysplastic
and atypical features. If there is an increase in myeloblasts
Difficulties in diagnosis of CEL arise, since this neoplasm (5–19%), CEL-NOS should be considered. Often the bone
often is, or has been merged with idiopathic HES and neo- marrow is fibrotic.
plasms with FIP1L1-PDGFRA (F/P) fusion transcript [29].
The diagnosis of CEL-NOS requires persistent eosino-
philia ≥1.5 × 109/L, exclusion of other myeloid neoplasms 9.9.3 Immunohistochemistry and Genetics
including CML, ET, PMF, PV, and MDS/MPN overlap syn-
drome, negativity for t(5;12)(q31–35;p13) or other rear- Since there are no immunophenotypic abnormalities
rangement of PDGFRB, negativity for FIP1L1/PDGFRA or described, immunohistochemistry is only helpful to exclude
other rearrangement of PDGFRA, negativity for FGFR1 an increase of blasts (CD34, CD117c).
rearrangement, <20% blasts in PB and BM, negativity for There are no specific cytogenetic or molecular genetic
cytogenetic and other features associated with AML and abnormalities. However, many chromosomal abnormalities
>2% blasts in PB or >5% in BM or positivity for a clonal were detected in eosinophilic leukemias, the most frequent
cytogenetic or molecular genetic abnormality. These diag- being trisomy of chromosome 8 [30].
9 Myeloproliferative Neoplasm (MPN) 293

Case History A descriptive diagnosis was made: moderately hypercel-

A 52-year-old man, a heavy smoker, presented with dyspnea, lular bone marrow with an increase of maturing granulocyto-
slight splenomegaly, and leukocytosis. The blood count was poiesis with marked eosinophilia. In a comment differential
Leuko 41.3 G/L, normal Ery, normal Thrombocytes, and marked diagnoses were listed, including reactive conditions, HES,
Eosinophilia with 54.2%. No genetic abnormalities were found. CEL-NOS and also eosinophilia with abnormalities of
A bone marrow biopsy was performed. The bone marrow PDGFRA, PDGFRB, and FGFR1.
was hypercellular with marked eosinophilia and normal Further clinical evaluation with eosinophilia in lung and
appearing erythro- and megakaryocytopoiesis. Blasts were heart and NGS from the bone marrow biopsy with a TET2
not increased, no fibrosis was detected. mutation, led to the diagnosis of CEL-NOS (Fig. 9.16).

a b

MGG MGG

c d

MGG MPO

Fig. 9.16 CEL-NOS. Highly hypercellular bone marrow with marked appearing erythropoiesis (CD71). (f) Megakaryocytes are not increased,
eosinophilia with maturation (a, b, c: MGG). (d) Granulopoietic cells they are rather small but not atypical (CD61)
immunohistochemically stained with an antibody to MPO. (e) Normal
294 9 Myeloproliferative Neoplasm (MPN)

e f

CD71 CD61

Fig. 9.16 (continued)

9.9.4 Caution have to suggest CEL-NOS, but the diagnosis is only possible
with a complete knowledge of clinical and genetic results.
If the bone marrow is hypercellular with marked eosinophilia In cases with eosinophilia also consider reactive eosino-
and normal appearing erythro- and megakaryocytopoiesis, you philia (e.g., allergic) and idiopathic HES.
9 Myeloproliferative Neoplasm (MPN) 295

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Myeloid/Lymphoid Neoplasms
with Eosinophilia and Rearrangement 10
of PDGFRA, PDGFRB, FGFR1,
or with PCM1-JAK2

Myeloid/lymphoid neoplasms (M/LN) with eosinophilia and Table 10.1 Myeloid/lymphoid neoplasms with eosinophilia and rear-
gene rearrangement are listed in a distinct WHO category. rangement of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2a
This category includes three specific disease groups involv- Diagnostic criteria of a MPN, AML, or ALL/LBL with eosinophilia
associated with FIP1L1-PDGFRA
ing rearrangement of platelet-derived growth factor receptor
A myeloid or lymphoid neoplasm, usually with prominent
α (PDGFRA), platelet-derived growth factor receptor β eosinophilia and a FIP1L1-PDGFRA fusion gene resulting from a
(PDGFRB), fibroblast growth factor receptor 1 (FGFR1) or cryptic del(4)(q12) or a variant fusion gene with rearrangement of
is associated with PCM1-JAK2, a provisional WHO entity PDGFRA or clinic-pathologic surrogate markers
[1–3] (Table 10.1). The presentation of the disorders is het- Diagnostic criteria for myeloid/lymphoid neoplasms associated with
ETV6-PDGFRB fusion gene or other rearrangement of PDGFRB
erogeneous: Phenotypic features may vary between those of
A M/LN myeloid or lymphoid neoplasm, often with prominent
a myeloproliferative neoplasm (MPN), especially chronic eosinophilia and sometimes with neutrophilia or monocytosis and
eosinophilic leukemia (CEL), myelodysplastic syndrome/ presence of t(5;12)(q32;p13.2) or a variant translocation or
MPN (MDS/MPN), or occasionally of an acute myeloid leu- demonstration of an ETV6-PDGFRB fusion gene or rearrangement
kemia (AML), a myeloid sarcoma (MS), or an acute lympho- of PDGFRB. Molecular confirmation is highly desirable. Cases with
fusion genes typically associated only with BCR-ABL1-like
blastic leukemia/lymphoma (ALL/LBL). It has been B-lineage ALL are excluded
strengthened that although the classification of this category Diagnostic criteria of MPN or acute leukemia associated with
of neoplasms is predominantly based on genetic aberrations, FGFR1 rearrangement (also referred to as 8p11myeloproliferative
diagnosis should still be anchored to a combination of histo- syndrome)
A MPN or MDS/MPN neoplasm with prominent eosinophilia, and
morphology and clinical and laboratory criteria [3].
sometimes with neutrophilia or monocytosis or acute myeloid
leukemia or precursor T-ALL/LBL or precursor B-ALL/LBL or
mixed phenotype acute leukemia (usually associated with peripheral
10.1 Epidemiology and Clinical Features blood or BM eosinophilia) and presence of t(8;13)(p11.2;q12.1) or a
variant translocation leading to FGFR1 rearrangement demonstrated
in myeloid cells, lymphoblasts, or both
M/LN with eosinophilia and gene rearrangement represent Diagnostic criteria for myeloid/lymphoid neoplasms with
rare disorders. A striking male predominance is reported in PCM1-JAK2
neoplasms with PDGFRA, PDGFRB rearrangements, and A myeloid or lymphoid neoplasm, often with prominent eosinophilia
with PCM1-JAK2 while the M/LN with FGFR1 rearrange- and presence of t(8;9)(p22;p24.1) or a variant translocation leading
to JAK2 rearrangement
ment has a male-to-female ratio of 1.5:1 [2]. The age range
a
Adapted and modified from [3]
of the patients is wide ranging from the first to eighth decade
in all types.
Patients with M/LN with PDGFRA rearrangement gener-
ally present as a chronic MPN with eosinophilia. Peripheral M/LN associated with ETV6-PDGFRB fusion gene or
blood and bone marrow eosinophilia are frequently associ- other rearrangement of PDGFRB include several variants.
ated eosinophilia-related secondary effects such as pruritus, Patients often present with eosinophilia, spleno ± hepato-
splenomegaly, and/or organ fibrosis. Serum tryptase is often megaly, occasionally skin involvement and/or cardiac fail-
mildly elevated (>12 ng/mL). The disease is responsive to ure. Serum tryptase may be mildly or moderately elevated.
tyrosine kinase inhibition, especially imatinib. Rare patients However, eosinophilia is not always present.
develop AML or T-ALL/LBL [4].

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 297

C. Beham-Schmid, A. Schmitt-Graeff, Bone Marrow Biopsy Pathology,
Essentials of Diagnostic Pathology, https://doi.org/10.1007/978-3-662-60309-3_10
298 10 Myeloid/Lymphoid Neoplasms with Eosinophilia and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2

In M/LN associated with FGFR1 rearrangement the ini- 10.3 Genetics

tial presentation is variable. Cases with MPN, splenomegaly,
MS, AML, B-/or T-ALL/LBL, mixed phenotype acute leuke- More than 70 fusion genes have been detected in M/LN with
mia are reported. All these manifestations are generally asso- eosinophilia and rearrangement of PDGFRA, PDGFRB,
ciated with eosinophilia. FGFR1, or with PCM1-JAK2 [3]. Essential hematologic and
M/LN with PCM1-JAK2 is generally associated with genetic features of M/LN associated with eosinophilia and
eosinophilia, bone marrow and blood involvement, and hep- gene rearrangement are listed in Table 10.2 [1].
atosplenomegaly. The morphologic and immunophenotypic A cytogenetically invisible interstitial deletion of the
features include MPN/CEL, MDS/MPN, AML, B-/T-ALL/ CHIC2 gene on chromosome 4q12 which can be detected by
LBL. The clinical course of JAK2-rearranged neoplasms is RT-PCR (FP fusion) or FISH (CHIC2 deletion) is the molecu-
aggressive, with rapid progression from chronic phase dis- lar basis for the M/LN with PDGFRA rearrangement. The
ease to myeloid or lymphoid blast phase. The disorder may resulting constitutively activated fusion tyrosine kinase is the
also initially present as acute leukemia/blast phase M/LN. rationale for successful treatment with imatinib [7, 8].
Additional fusion partners of PDGFRA are for example BCR,
ETV6, KIF5B, CDK5RAP2, STRN, TNKS2, and FOXP1 [3].
10.2 Morphology In addition to ETV6-PDGFRB, more than 30 additional
fusion partners of PDGFRB have been characterized [3].
Blood and bone marrow eosinophilia that may be associated FISH or RT-PCR is necessary to prove involvement of the
with tissue eosinophilia is observed in the majority of PDGFRB gene. A high responsiveness to Imatinib has been
patients. However, there are cases without significant reported [3].
eosinophilic. Eosinophils are predominantly mature but In M/LN associated with FGFR1 rearrangement, at least
eosinophilic myelocytes or promyelocytes may be detected. 14 different fusion genes have been detected. Reciprocal
As mentioned above, the morphologic features range from translocations include t(8;13)(p11;q12), t(8;9)(p11;q33),
those of CEL to MDS/MPN, AML, MS, T-/or B-ALL/ and t(6;8)(q27;p11), resulting in fusions of ZMYM2,
LBL. Therefore, blood and bone marrow may show dysplas- CNTRL, and FGFR1OP, respectively, to FGFR1. FGFR1
tic features of all three hematopoietic lineages, blast excess, fusion genes are associated with a disease entity initially
and basophilia. Moreover, lymphoblasts of T- or B-lineage referred to as the 8p11 myeloproliferative syndrome or
may be observed. stem cell leukemia/lymphoma [3]. In a large cohort of
A hallmark of the bone marrow core biopsy involved by
M/LN with PDGFRA rearrangement is the presence of
spindle-shaped mast cells, exhibiting an aberrant tryptase+/ Table 10.2 Summary of hematologic and genetic features of myeloid/
CD117+/CD25+ phenotype and often co-expressing CD2. lymphoid neoplasms associated with eosinophiliaa
Mast cells in this category are generally loosely scattered PDGFRAb Eosinophilia Cryptic deletion at
between eosinophils and rarely arranged in clusters ≥15 4q12
mast cells that represent the major criterion for the diagnosis ↑Serum tryptase FIP1L1-PDGFRA, at
↑Marrow mast cells least 66 other partners
of systemic mastocytosis. However, these mast cells may
PDGFRBb Eosinophilia t(5;12)(q32;p13.2)
occasionally even harbor a PDGFRA rearrangement as ETV6-PDGFRB, at
Monocytosis
shown by fluorescence in situ hybridization (FISH) com- Mimicking CMML least 25 other partners
bined with immunohistochemistry (FICTION) [5]. FGFR1c Eosinophilia FGFR1 various
The bone marrow of cases of M/LN with PCM1-JAK2 Often presents with T-ALL or partners
may show a characteristic striking sheet-like proliferation of AML
erythroid precursors in addition to a proliferation of myeloid PCM1- Eosinophilia t(8;9)(p22;p24.1)
JAK2d Rarely presents with T-LBL PCM1-JAK2
cells including eosinophils. or B-ALL
Morphologic features may be considered as surrogate Bone marrow shows
markers for different M/LN subsets. However, the final left-shifted erythroid
evaluation of BM core biopsies in the diagnostic work-up of predominance, lymphoid
persistent eosinophilia should include comprehensive mor- aggregates
phologic (stains for myeloid blast cells, mast cells, and
a
Adapted and modified from [1]
b
Respond to tyrosine kinase inhibitor (TKI)
fibers) and genetic analyses before a final diagnosis is estab- c
Do not respond to TKI
lished [6]. d
May respond to JAK2 inhibitors
10 Myeloid/Lymphoid Neoplasms with Eosinophilia and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2 299

patients, the two most frequently observed cytogenetic MPN such as atypical BCR-ABL1-negative chronic myeloid
abnormalities were t(8;13)(p11.2;q12)(partner gene leukemia.
ZMYM2) and t(8;22)(p11.2; q11.2)(BCR). The large major-
ity of patients had a RUNX1 mutation, and all developed an
acute leukemia [9]. 10.4 Caution
PCM1-JAK2 as a consequence of t(8;9)(p22;p24.1) is
the most frequent JAK2 fusion gene in this extremely rare • Blood and bone marrow eosinophilia may be inconspicu-
provisional WHO entity [3, 10]. Since both myeloid and ous or even absent in some cases harboring the typical
lymphoid lineages may be involved in PCM1-JAK2-positive mutational spectrum of the category M/LN with eosino-
neoplasms, it has been suggested that the disorder origi- philia and gene rearrangement.
nates from an early pluripotent hematopoietic progenitor or • Patients may initially present with T-/ or rarely B-ALL/
stem cell [3]. Only a few patients with t(9p24;v)/JAK2 at LBL mimicking precursor lymphoid neoplasms NOS.
diagnosis have been reported. In a common study from • The presence of atypical CD25+ spindle-shaped mast
large medical centers, most patients with 9p24/JAK2 pre- cells and the increased serum tryptase levels may hint to
sented with MPN-associated features characterized by vari- systemic mastocytosis associated with eosinophilia.
able degrees of eosinophilia, myelofibrosis, frequent • M/LN with eosinophilia and rearrangement of PDGFRA
proliferations of early erythroblasts in bone marrow and are extremely rare in female patients. Therefore, this
extramedullary sites, and infrequent/absent somatic muta- diagnosis assigned to a woman should be further
tions [10]. They may also present with features of a MDS/ discussed.
300 10 Myeloid/Lymphoid Neoplasms with Eosinophilia and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2

Case History 1. Myeloid/Lymphoid Neoplasm with showed a marked proliferation of left-shifted eosinophils and
Eosinophilia and Rearrangement of PDGFRA (Fig. 10.1) rare neutrophil precursors. Spindle-shaped mast cells coex-
A 57-year-old male patient consulted a dermatologist with pressing tryptase, CD117, and CD25 were scattered among
pruritus and fatigue. The CBC was as follows: WBC eosinophils. No mast cell aggregates were present.
25 × 109/L, Hb 7.3 g/dL, RBC 1.9 × 1012/L, platelets Immunohistochemical stain showed nuclear positivity for
98 × 109/L. The peripheral blood smears showed 58% pre- phospho(p)-SAT5. Reverse transcriptase polymerase chain
dominantly mature but partially degranulated eosinophils, 9% reaction (RT-PCR) revealed a FIP1L1-PDGFRA fusion gene.
neutrophils, 3% myelocytes, 4% band forms, and 17% lym- The diagnosis was myeloid/lymphoid neoplasm with
phocytes. The bone marrow core biopsy was hypercellular and PDGFRA rearrangement.

a b

Giemsa Giemsa

c d

NASDCL MPO

Fig. 10.1 Myeloid/lymphoid neoplasm with eosinophilia and rear- scattered through the marrow spaces. (f) Slight increase in reticulin
rangement of PDGFRA. (a, b) The bone marrow core biopsy is strik- (Gomori stain). (g) Presence of atypical spindle-shaped mast cells is
ingly hypercellular and contains about 80% eosinophils of all stages of highlighted by tryptase immunolabeling. (h) Nuclear expression pat-
maturation (Giemsa). (c) Naphthol-ASD-chloroacetate-esterase reac- tern of pSTAT5. The diagnosis was myeloid/lymphoid neoplasm with
tion and (d) myeloperoxidase immunolabeling performed on the FFPE eosinophilia and rearrangement of PDGFRA presenting with features
marrow sections. (e) Erythropoiesis is not arranged in a typical island of chronic eosinophilic leukemia
10 Myeloid/Lymphoid Neoplasms with Eosinophilia and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2 301

e f

HB Gomori

Fig. 10.4 (continued)

10 Myeloid/Lymphoid Neoplasms with Eosinophilia and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2 307

Case History 4. Myeloid/Lymphoid Neoplasm with CD34 labeled endothelial cells but did not provide evidence
Eosinophilia and Rearrangement of FGFR1 (Fig. 10.5) of increased blasts. Numerous spindle-shaped atypical mast
This 39-year-old male patient complained about pruritus and cells were highlighted by tryptase, CD117, and CD25 immu-
fatigue. Clinical examination revealed splenomegaly. The nolabeling. They were loosely arranged but did not form
CBC was as following: WBC 27 × 109/L, Hb 9.3 g/dL, MCV dense clusters. Moreover, nodular lymphoid infiltrates com-
89 fL, platelets 94 × 109/L. The peripheral blood smears posed of small inconspicuous B- and T-cells were present.
showed 35% partially hypogranulated eosinophils, 28% neu- No clonal T-cell receptor γ or β and immunoglobulin heavy
trophils, 3% myelocytes, 2% band forms, 9% monocytes, chain rearrangements were present. FISH analysis for
and 23% lymphocytes. Eosinophilia was also a prominent PDGFRA and PDGFRB rearrangements were negative.
feature observed in the bone marrow core biopsy. Neutrophil However, FGFR1 rearrangement was detected by further
precursors were also increased while erythropoiesis was screening by FISH. The diagnosis was myeloid/lymphoid
diminished. Megakaryocytes showed dysplastic features. neoplasm with eosinophilia and rearrangement of FGFR1.

a b

c d

MGG CD20

Fig. 10.5 Myeloid/lymphoid neoplasm with eosinophilia and rear- Lymphoid nodules composed of CD20+ B-cells (d) and of CD3+
rangement of FGFR1. (a) Hypercellular bone marrow core biopsy T-cells (e). (f) Evidence of numerous loosely arranged CD117+ spindle-
nearly devoid of fat cells showing two nodular lymphoid infiltrates shaped atypical mast cells. (g) CD34 decorates predominantly vascular
adjacent to trabecular bone (H&E). (b) Increased neutrophil precursors endothelial cells. No excess of blasts. (h) CD15 expression by the left-
and numerous dysplastic megakaryocytes (H&E). (c) Markedly shifted myeloid lineage
increased eosinophils at different stages of maturation (MGG).
308 10 Myeloid/Lymphoid Neoplasms with Eosinophilia and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2

e f

CD3 CD117

g h

CD34 CD15

Fig. 10.5 (continued)

10 Myeloid/Lymphoid Neoplasms with Eosinophilia and Rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2 309

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Myelodysplastic/Myeloproliferative
Neoplasms (MDS/MPN) 11

Table 11.1 Diagnostic criteria for CMML (modified from [7])

11.1 Introduction
• Persistent PB monocytosis ≥1 × 109/L, with monocytes accounting
for ≥10% of the WBC count
The five different clonal myeloid neoplasms that exhibit
• Not meeting WHO criteria for BCR-ABL1+ CML, PMF, PV, or ET
simultaneously both myelodysplastic and myeloproliferative • No evidence of PDGFRA, PDGFRB, or FGFR1 rearrangement or
features at initial presentation are listed in the MDS/MPN PCM1-JAK2 (should be specifically excluded in cases with
category: chronic myelomonocytic leukemia (CMML), eosinophilia)
atypical chronic myeloid leukemia, BCR-ABL1-negative • <20% blasts in the blood and BM
• Dysplasia in one or more myeloid lineages. If myelodysplasia is
(aCML), juvenile myelomonocytic leukemia (JMML),
absent or minimal, the diagnosis of CMML may still be made if
myelodysplastic/myeloproliferative neoplasms with ring sid- the other requirements are met
eroblasts and thrombocytosis (MDS/MPN-RS-T), and • An acquired clonal cytogenetic or molecular genetic abnormality
myelodysplastic/myeloproliferative neoplasms, unclassifi- is present in hemopoietic cells
able (MDS/MPN-U) [1]. TET2, ASXL1, and SFRS2 are the Or
most commonly mutated genes in adult MDS/MPN but • The monocytosis (as previously defined) has persisted for at least
3 months
numerous molecular pathogenetic lesions have been • All other causes of monocytosis have been excluded
detected. Most of them are not absolutely specific for any
adult MDS/MPN subtype [2].

≥3 months was excluded, or cytogenetic and molecular aber-

11.2 hronic Myelomonocytic Leukemia
C rations are present. A proliferative type with a WBC
(CMML) ≥13 × 109/L is separated from a dysplastic type with a
WBC <13 × 109/L. The diagnostic criteria according to the
CMML is a clonal stem cell neoplasm with heterogeneous 2018 WHO classification are listed in Table 11.1 [6]. Refined
features and risk of progression into acute myeloid leukemia criteria for classical (CMML), CMML variants and pre-
(AML) [3, 4]. Patients who are diagnosed with CMML must CMML conditions have been proposed [4].
fulfill the following criteria:
Persistent circulating monocytes ≥1 × 109/L with <20%
myeloblasts and promonocytes in the peripheral blood (PB) 11.2.1 Epidemiology
and/or bone marrow (BM), evidence of dysplastic features
involving at least one myeloid lineage, and absence of gene CMML is generally diagnosed in elderly patients with a
rangements such as BCR-ABL1, PDGFRA, or PDGFRB. median age ranging between 65 and 75 years, a peak over
According to the percentage of blasts and promonocytes, 70 years and a higher frequency in men than in woman (ratio
a subdivision into CMML-0 (<2% in PB, <5% in the BM), about 2:1). Information about the incidence rate is rarely
CMML-1 (2–5% in the peripheral blood, <10% in the BM), available. In a CMML cohort from the Netherlands, the age-
or CMML-2 (5–19% in PB, 10–19% in the BM or presence standardized incidence rate per 100,000 was 0.4 when bone
of Auer rods) should be performed [4]. Cases showing no marrow (BM) examination was included in the diagnostic
dysplastic features can be classified as CMML when a reac- work-up but may be underreported in cancer registries with-
tive condition associated with a monocytosis persisting for out BM confirmation [7].

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 311

C. Beham-Schmid, A. Schmitt-Graeff, Bone Marrow Biopsy Pathology,
Essentials of Diagnostic Pathology, https://doi.org/10.1007/978-3-662-60309-3_11
312 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

11.2.2 Clinical Features 11.2.5 Molecular Pathogenesis

Clinical symptoms include fatigue, night sweats, weight Cytogenetic aberrations involving many different chromo-
loss, and hepatosplenomegaly, especially in cases with somes have been reported in 30–40% patients. Examples are
higher monocyte counts. Extramedullary disease involving trisomy 8, trisomy 10, del(5q), monosomy 7 and complex
the skin for example may be present [3, 4]. karyotypes [2, 3].
Molecular alterations are more frequent and detected in
about 90% of patients. The molecular profile is complex and
11.2.3 Bone Marrow and Blood Findings includes mutations in genes involved in methylation (TET2,
50–60%), RNA splicing (SFSR2, 36–46%; U2AF35,
The BM is generally hypercellular with a focally accentuated 5–15%), histone modification (ASXL1, 43–44%; EZH2,
expansion of myelomonocytic cells. They show slightly 6–10%), transcription (RUNX1, 15–20%; CEBPA, 4–20%),
folded nuclei and a rather broad pale cytoplasm. signaling pathways (CBL, 10–20%; KRAS, 7–11%; NRAS,
Promonocytes are characterized by a fine stippled chromatin 4–16%; SETBP1, 6–16%; JAK2, 10%) [2].
structure and distinct nucleoli and abundant light gray or
slightly basophilic cytoplasm. These typical features may
better be detected in BM smears than in sections [6]. 11.2.6 Prognosis
Dysplasia may involve one or more myeloid lineages.
Micromegakaryocytes are often the hallmark of dysmega- Several prognostic scoring systems have been proposed
karyopoiesis. Reticulin fibrosis is reported in about one third to evaluate the overall survival with special consideration
of the cases and associated with a shorter median time to for the risk of transformation into acute myeloid leuke-
disease progression [8–10]. Since fibrosis grade was shown mia. These scores consider different variables such as
to be an important indicator of prognosis, with high-grade age, monocytosis, circulating blasts, anemia, thrombocy-
fibrosis predicting inferior survival, it has been proposed thattopenia, fibrosis, and genetic findings [8, 10]. A moderate
fibrosis grading should be incorporated into prognostic to severe myelofibrosis was reported in a 3.1% of CMML
assessment and therapeutic decision-making [9]. cohort and associated with worse overall survival [9, 10].
Fifty percent of these patients had JAK2 p.V617F muta-
tion [10].
11.2.4 Immunohistochemistry Applied to BM It is well accepted that somatic mutations are involved in
Trephine Biopsies the heterogeneous prognosis of CMML 10 [11]. In a large
CMML cohort, mutations in ASXL1, RUNX1, NRAS, and
Myelomonocytic cells of CMML are positive for CD33, SETBP1 were important molecular prognostic factors. In a
myeloperoxidase (MPO), often for naphthol-ASD-multivariable regression model, a CMML-specific
chloroacetate esterase and show variable expressions of Prognostic Scoring System-Mol was defined which inte-
CD14, CD68, CD163, overexpression of CD56, and aberrant grated the genetic score with RBC transfusion requirement,
expression of CD2 and CD65. Small nodules of plasmacy- WBC and BM blast counts, and was able to identify four
toid dendritic cells within the marrow are highlighted by risk groups with significantly different survival and risk of
CD123 staining and are considered as a part of the malignant leukemic evolution [12]. In a cohort of CMML patients
clone. treated with hypomethylating agents TET2mut/ASXL1wt gen-
Special point: An increase in CD34+ may be a hallmark otype predicted higher complete response rate and pro-
of disease progression. However, the evaluation of CD34+ longed survival while RUNX1mut and CBLmut genotypes were
BM cells is no reliable tool to detect the percentage of blasts associated with poorer outcome, independently of higher
and promonocytes since they often do not express CD34. leukocyte count [11].
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 313

11.2.7 Caution monocytic leukemia, and PDGFRB-rearranged neoplasm.

These cases may simulate CMML [6].
11.2.7.1 Transition to AML
The diagnosis of a transition to AML also designated as 11.2.7.3 econdary Acquisition of MPN-Typical
S
CMML blast phase depends on the percentage of blasts Genetic and Hematological Features
and promonocytes in the peripheral blood and especially in CMML
bone marrow (≥20%). The exact quantification of pro- A JAK2 V617F mutation may initially be present or occur
monocytes remains challenging since no characteristic as a second genetic event in some CMML cases. This
immunocytological marker is actually available to dis- additional mutation may be associated with a modulation
criminate between mature monocytes and promonocytes. of the disease with a more proliferative phenotype charac-
Promonocytes are better detected on cytologic blood and terized by the presence of MPN-like megakaryocytes in
bone marrow smears than on sections of paraffin-embed- the bone marrow and increased peripheral platelet counts
ded BM biopsies. [4, 10].

11.2.7.2 onocytosis in Classical MPN

M 11.2.7.4 Benign Conditions
and Other Myeloid Neoplasms • Chronic bacterial infections: tuberculosis, sarcoidosis,
Cases of MPN can be associated with monocytosis or they rheumatologic disorders
can develop it during the course of the disease. Other • Viral infections: varicella zoster
examples include chronic myeloid leukemia, juvenile myelo- • Bone marrow regeneration post chemotherapy
314 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

Case History 1 (CMML-1) (Figs. 11.1 and 11.2) decreased but showed dysplastic features including micro-
A 66-year-old male patient presented with reduced physical forms. The erythropoiesis was quantitatively decreased
performance state, fatigue, and enlarged spleen size (Fig. 11.1). with mild dyserythropoiesis. The granulopoiesis was left
Laboratory findings: Hb 9.5 g/dL, platelets 110 × 109/L, shifted. CD34-positive precursor/blast cells and promono-
WBC 25 × 109/L. The differential count showed 38% monocytes represented about 8% of nucleated bone marrow
cytes, 1% promonocytes, 45% neutrophils, 1% eosinophils, cells.
and 15% lymphocytes. The karyotype was normal. Next-generation sequencing
The BM smears and the trephine BM biopsy were revealed a TET2 mutation. The diagnosis was CMML-1 pre-
hypercellular with rare fat cells. The myelomonocytic lin- senting with a proliferative phenotype.
eage expressing myeloperoxidase and CD33 accounted for Case 1, Follow-up (CMML, Splenectomy)
more than 90% of all nucleated cells. Monocytes express- Because of abdominal discomfort and increasing symp-
ing CD14 and partially CD68 and CD56 were increased to tomatic thrombocytopenia splenectomy was performed
about 20%. Promonocytes and blast cells accounted for showing a dense infiltration of the red pulp by myelomono-
about 8% of BM cells. The megakaryopoiesis was not cytic cells (Fig. 11.2).

a b

HE HE
c d

CD14 CD34

Fig. 11.1 Bone marrow core biopsy (a–e), peripheral blood smear (f), of small hypolobated CD61-positive dysplastic megakaryocytes. (f)
and bone marrow aspirate smears (g, h) from the 66-year-old patient Peripheral blood smear showing mature monocytes and monocyte pre-
diagnosed with chronic myelomonocytic leukemia-1 (CMML-1). (a, b) cursors as well as dysplastic granulocytes and (g) bone marrow aspirate
Hypercellular marrow spaces showing proliferation of myeloid and characterized by a proliferation of myeloid and monocytic lineages and
monocytic cells (H&E). (c) The monocytic lineage is immunolabeled rare erythroid cells (MGG). (h) Alpha-naphthyl acetate esterase stain-
by CD14 antibody. (d) Rare CD34-positive precursor cells. (e) Presence ing highlighting the monocytic lineage
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 315

e f

CD61 MGG

g h

MGG ANAE

Fig. 11.1 (continued)

a b

HE HE

Fig. 11.2 Splenectomy specimen from the patient with chronic myelomonocytic leukemia (CMML) showing predominant infiltration of the red
pulp by myelomonocytic cells (a–c H&E) that are partially positive for NASDCL (d, e), CD33 (f), and CD14 (g, h)
316 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

c d

HE NASDCL

e f

NASDCL CD33

g h

CD
CD1
14
CD144 CD1
CD
CD14
14
4

Fig. 11.2 (continued)

11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 317

Case History 2 (CMML-2) (Figs. 11.3 and 11.4) monocytic lineages and 8% were eosinophils. Erythropoiesis
A 63-year-old lady was admitted with anemia (Hb 8.2 g/dL), and megakaryopoiesis were significantly decreased. By CD61
leukocytosis (WBC 52 × 109/L), and thrombocytopenia immunohistochemistry, micromegakaryocytes and rare mega-
(platelets 67 × 109/L). The differential count showed aniso- karyoblasts were detected. Promonocytes, monoblasts, and
cytosis of red blood cells, 32% monocytes, 7% promono- CD34-positive blasts accounted for about 17% of nucleated
cytes, 4% myelocytes, 3% metamyelocytes, 14% band bone marrow cells consistent with CMML-2.
forms, 39% neutrophils, 1% blasts, 8% eosinophils, and 13% Case 2, Follow-up (CMML, Progression to AML)
lymphocytes. (Fig. 11.4).
Immunophenotyping of peripheral blood revealed neutro- Seven months later, the condition of the patient deterio-
phils with an aberrant expression pattern of CD13-CD16 and rated. Promonocytes, monoblasts, and myeloblasts repre-
CD11b-CD13, monocytes with a reduced expression of sented 60% of peripheral blood and 85% of bone marrow
CD14, CD45, HLA-DR, and a coexpression of CD56. cells consistent with a progression into an acute myeloid leu-
Molecular analysis by next-generation sequencing reported kemia (AML) that was classified as AML with myelodyspla-
negativity for BCR-ABL but mutations in ASXL1, EZH2, and sia related changes according to 2016 WHO and fulfilled
TET2 genes. morphologic criteria of acute myelomonocytic leukemia
Sections of the hypercellular BM core biopsy (Fig. 11.3) according to the previous French-American British classifi-
showed that 85% of nucleated cells belonged to the myeloid and cation [7].

a b

H&E
H&
&E H&E
c d

Giiem
G emssa
a NASDCL
NA
NASSD
DCL
C

Fig. 11.3 Chronic myelomonocytic leukemia-2 (CMML-2). NASDCL). (e) Strikingly diminished megakaryopoiesis with rare
Proliferation of the myeloid and monocytic lineage with an increase in CD61-positive micromegakaryocytes and megakaryoblasts. (f)
immature precursors including promonocytes and monoblasts associ- Scattered CD71-positive predominantly immature erythroid cells. (g,
ated with an expansion of eosinophils (a, b H&E, c Giemsa, d h) Focal increase of CD34-positive myeloblasts
318 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

e f

CD61 CD71

g h

CD34
C
CD
D334
4 CD34
CD
CD3
34
4

Fig. 11.3 (continued)

a b

MGG ANAE

Fig. 11.4 Transformation of CMML-2 in overt acute myeloid leuke- is densely infiltrated by basophilic blast cells (d, e) that are partially
mia (AML). Peripheral blood smears show myelomonocytic blast cells NASDCL-positive (f) and frequently express CD34 (g, h). Note that a
with (a) slightly indented immature nuclei and a basophilic cytoplasm small dysplastic megakaryocyte is also labeled by the CD34 antibody
(MGG) staining for ANAE (b) and partially MPO (c). The core biopsy (h)
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 319

c d

MP
M PO Giiems
G ems
em sa
a

e f

Giie
G emm
msa
sa
sa NA
N AS
SDDC
CLL
g h

CD
C D
D334
34 CD
C D34
34

Fig. 11.4 (continued)

320 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

Case History 3 (Myelodysplastic Syndrome Evolving gene (ex3 and ex7), and in the JAK2 gene (ex14
into CMML, JAK2 V617F Mutated with Fibrosis, c.1849G > T p.V617F). A mixture of abundant small dys-
Further Progressing to AML) (Figs. 11.5, 11.6, and 11.7) plastic and large polymorphic megakaryocytes reminis-
Initially, a hematologic work-up of this 56-year-old male cent of a myeloproliferative neoplasm was a hallmark of a
patient was done because of thrombocytopenia. CBC: WBC newly performed bone marrow biopsy. Abundant imma-
4.2 × 109/L, hemoglobin 10.2 g/dL, MCV 88 fL, platelet ture myeloid and monocytic precursors with an increase in
count 45 × 109/L. The peripheral blood smear showed mild promonocytes and monoblasts to 12% and dysplastic ery-
granulocytic dysplasia and monocytosis (16%). The bone throid islands were observed. Reticulin fibrosis grade 1
marrow aspirate and core biopsy were hypercellular with a had developed (Fig. 11.6). The diagnosis was chronic
proliferation of myeloid and monocytic precursors and more myelomonocytic leukemia-2 with fibrosis grade 1 associ-
mature forms and mild erythroid dysplasia. Megakaryocytes ated with a JAK2 pV16F mutation and myeloproliferative
were clearly dysplastic with a predominance of small dys- features.
plastic forms. No fibrosis was observed (Fig. 11.5). The Case 3, Transformation into AML (Fig. 11.7):
karyotype was normal. No BCR-ABL1 or JAK2 pV16F muta- The patient’s clinical condition and hematologic parame-
tion was found. The diagnosis was myelodysplastic syndrome ters rapidly deteriorated and acute myeloid leukemia devel-
with multilineage dysplasia, evolution to chronic myelo- oped. Three months later, the CPC was: WBC 95 × 109/L
monocytic leukemia possible. The patient refused treatment. (86% blasts) hemoglobin 8.4 g/dL, platelet count
Case 3, Follow-up (Fig. 11.6): Progression to CMML 21 × 109/L. Immunophenotype of CD34+ blasts: positive for
During the following 5 years, progressive leukocytosis with CD13, CD200; partially positive for CD117, HLA-DR,
monocytosis and splenomegaly developed. The CPC was: CD38, CD11c, MPO. FISH analysis of bone marrow cells
WBC 30.64 × 109/L (19% monocytes, blast cells 1%), hemo- revealed a monosomy 7 (Fig. 11.7). The core biopsy showed
globin 9.8 g/dL, platelet count 68 × 109/L, LDH 970 U/L. a diffuse infiltration by CD33- and CD34-positive blasts
Molecular testing by NGS (targeted sequencing) associated with a dense reticulin meshwork consistent with
revealed mutations in the SRSF2-gene (ex1), the TET2 fibrosis grade 2.

a b

MGG
MGG
MG MGG

Fig. 11.5 CMML initially presenting as MDS. (a) Peripheral blood and myeloid dysplasia including the presence of pseudo-Pelger cells
smear with anisocytosis of red blood cells and relative monocytosis (d) (MGG). (e, f) Hypercellular core biopsy showing dysplasia of
insufficient for the diagnosis of CMML (MGG). (b–d) Bone marrow megakaryocytes, diminished erythropoiesis, left-shifted granulopoiesis,
aspirate demonstrating megakaryocytic dysplasia including the pres- and monocytosis (Giemsa). (g, h) Focal increase in monocyte precur-
ence of micromegakaryocytes (c), monocyte precursors and monocytes, sors that are predominantly negative for NASDCL
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 321

c d

MGG MGG
MG
GG

e f

Giemsa Giemsa

g h

NA
NAS
SD
DC
CL
L
NASDCL NASDCL
NA
NAS
SD
DCCL
L

Fig. 11.5 (continued)

322 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

a b

Giiem
G ms
saa Giemsa

c d

Giie
G em
msa
msa
sa Giemsa
Giie
G emm
msa
sa
sa

e f

CD61 CD14

Fig. 11.6 Overt CMML, JAK2 V617F positive. (a–d) The Giemsa- lineage is immunolabeled by CD14 antibody. (d, g) Note intertrabecu-
stained bone marrow core biopsy is completely devoid of fat cells as a lar nodules composed of mononuclear cells that correspond to (g)
consequence of myelomonocytic proliferation. (d, e) Abundant mega- CD123-positive cells derived from the monocytic lineage. (h)
karyocytes focally form loose clusters of large CD61-positive polymor- Predominant grade 1 reticulin fibrosis with focal increase to grade 2
phous cells with features of MPN rather than MDS. (f) The monocytic present in less than 30% of bone marrow spaces
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 323

g h

CD123 GOMORI

Fig. 11.6 (continued)

a b

Giemsa Giemsa

c d

CD34 CD33

Fig. 11.7 Transformation of the CMML in AML MRC. (a, b) The cells are rarely observed. (f) Megakaryopoiesis is strikingly diminished
Giemsa-stained core biopsy reveals abundant blasts showing a pale with the exception of scattered CD61-positive micromegakaryocytes
basophilic cytoplasm, nuclei with round or slightly folded nuclei and and megakaryoblasts. (g, h) The reticulin fiber network is increased to
expression of (c) CD34 and (d) CD33. (e) CD14-positive monocytic grade 2 with small foci of grade 3 fibrosis
324 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

e f

CD14 CD61

g h

GOMORI GOMORI

Fig. 11.7 (continued)

11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 325

11.3 J uvenile Myelomonocytic Leukemia 11.3.1 Epidemiology

(JMML)
JMML is rare neoplasm with a male predominance and
Juvenile myelomonocytic leukemia (JMML) is a pediatric an incidence of about 1.2 per million children per year
type of MDS/MPN type occurring in infants and young chil- [15]. The median age at presentation is 2 years (range
dren. The hematologic hallmark of this aggressive neoplasm is 0.1–11.4) [16]. Rarely, the affected patients are older
an expansion of clonal myelomonocytic cells in bone marrow, than 5 years. Hematopoietic stem cell transplantation
blood, spleen, and often multiple extramedullary sites [13, (HSCT) is the only curative therapy for most patients;
14]. The diagnostic criteria are summarized in Table 11.2. however, non-transplant options are actually studied as
pre-transplant therapy or therapy for relapse after trans-
plantation [17].
Table 11.2 Diagnostic criteria of JMMLa
I. Clinical and hematologic features (all four features mandatory)
Peripheral blood monocyte count ≥1 × 109/L 11.3.2 Clinical and Laboratory Features
Blast percentage in peripheral blood and bone marrow <20%
Splenomegaly (not always apparent at diagnosis)
The children typically present with fever, pallor, skin
No Philadelphia chromosome (BCR/ABL rearrangement)
II. Genetic criteria (one finding is sufficient)
bleeding and/or rash, hepato-splenomegaly, and lymphade-
Somatic mutation in PTPN11, KRAS, or NRAS nopathy. Laboratory features include anemia, thrombocyto-
Clinical diagnosis of NF1 or germline NF1 mutation penia, and elevated WBC count that rarely exceeds
Germline CBL mutation and loss of heterozygosity of CBL 100 × 109/L. Diagnostic criteria for JMML include an
III. Other criteria (for cases with the clinical and hematologic absolute monocyte count ≥1 × 109/L. Fetal hemoglobin

features under (I) but without a genetic criterion(II)) (HbF) is increased in many JMML patients with normal
Monosomy 7 or any other chromosomal abnormality; or ≥ of the
karyotype but not in those with monosomy 7 [14, 15]. An
following criteria must be fulfilled:
HbF increased for age additional finding is hypersensitivity of hematopoietic pro-
Myeloid or erythroid precursors in peripheral blood genitors to granulocyte-macrophage colony-stimulating fac-
Granulocyte-macrophage colony-stimulating factor tor (GM-CSF) [16, 17, 18].
hypersensitivity in colony assay
Hyperphosphorylation of STAT5
Modified from [6, 14, 18]
a
326 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

11.3.3 Morphology than in the peripheral blood. Erythropoiesis that is fre-

11.4 typical Chronic Myeloid Leukemia,

A clumped nuclear chromatin, abnormal hyper- or hypogranula-
BCR-ABL1-Negative tion) may also be observed [20, 26]. The bone marrow is hyper-
cellular resulting from a proliferation of dysplastic neutrophil
Atypical chronic myeloid leukemia, BCR-ABL1-negative precursors and often multilineage dysplasia.
(aCML) is a MDS/MPN entity characterized by neutrophilia with
dysplastic neutrophils and their precursors in the peripheral blood
11.4.4 Immunohistochemistry
[20–23]. The diagnostic criteria according to the 2016 WHO clas-
sification that are helpful to separate aCML from chronic neutro-
No characteristic immunophenotypic features are reported.
philic leukemia (CNL) are summarized in Table 11.3. The most
important diagnostic criteria are BCR-ABL1-negativity and
peripheral blood leukocytosis ≥13 × 109/L with the presence of 11.4.5 Genetics
dysplastic neutrophils and ≥10% neutrophil precursors in the
absence of significant basophilia and monocytosis. No disease-specific cytogenetics or mutation has been
described until now. Multiple karyotypic abnormalities have
been described and include most often trisomy 8 and del(20q)
11.4.1 Epidemiology [20]. A normal karyotype is rarely present. The diagnosis
requires absence of BCR-ABL1, PDGFRA, PDGFRB, FGFR1,
This myeloid neoplasm is diagnosed in elderly adults pre- and PCM1-JAK2 rearrangements. JAK2, CALR, and MPL
dominantly in their seventh or eighth decade. Since aCML is mutations are generally not detected in aCML. SETBP1 muta-
extremely rare, the exact incidence is unknown. A study tions represent one of the mostly frequently mutated genes in
from Italy reported 55 aCML cases, a multicenter study from aCML [21, 22, 25]. SETBP1 and/or ETNK1 mutations have
USA included only 65 aCML patients [24]. been reported in about a third and CSF3R mutations in <10%
of cases. However, it has been suggested that cases showing
CSF3R-T618 l mutation and prominent granulocytic dysplasia
11.4.2 Clinical and Laboratory Features should be clinically and biologically defined separately from
CNL and aCML [23]. SETBP1 and CSF3R might help to dis-
The clinical symptoms are not specific for the disease. The tinguish aCML from CNL [2]. SETBP1 mutations are not spe-
affected patients develop leukocytosis generally associated with cific for aCML but have been identified in up to 32% of aCML,
anemia, thrombocytopenia, and splenomegaly. The prognosis is 24% of JMML, 18% of CMML, and 10% of MDS/MPN-U
dismal with reported overall survival times from 12.4 to patients [27]. Additional mutations observed in aCML but also
25 months and a high risk of transformation into AML [20–23]. in other MDS/MPNs involve N/KRAS, CBL, RUNX1, and KIT
[23]. CBL mutations have been reported in approximately 10%
and activating mutations or internal tandem duplications in
11.4.3 Morphology FLT3 gene in 5% of aCML cases [2].

The detection of dysplastic neutrophils, promyelocytes, myelo-

cytes, metamyelocytes in the peripheral blood without signifi- 11.4.6 Caution
cant basophilia, and monocytosis is mandatory for the diagnosis
of aCML. The percentage of blasts is generally low and <20%. In patients with neutrophilia several differential diagnoses
Pseudo-Pelger-Huet forms showing bilobulated nuclei and should be excluded:
other abnormalities (hypersegmentation, nuclear projections,
• Reactive conditions with so-called leukemic reaction—
neutrophils are left shifted but show no dysplasia.
Table 11.3 Diagnostic criteria for aCML, BCR-ABL1-negativea
• Chronic myeloid leukemia (CML)—Philadelphia
Peripheral blood leukocytosis (WBC count ≥13 × 109/L) because of chromosome/BCR-ABL1 mutation is mandatory.
increased numbers of neutrophils and their precursors with
prominent dysgranulopoiesis Prominent basophilia may be observed.
Promyelocytes, myelocytes, metamyelocytes ≥10% of leukocytes • Chronic neutrophilic leukemia (CNL)—CNL neutrophils
No Ph chromosome or BCR-ABL1 fusion gene, no criteria for PV, generally show no or minimal dysplastic features and har-
ET, or PMF bor the characteristic CSF3R mutations.
No or minimal basophilia (<usually 2%), no or minimal absolute • Chronic myelomonocytic leukemia—persistent monocy-
monocytosis (<10%) of leukocytes
tosis ≥1 × 109/L is required.
Blasts <20% in blood and bone marrow
Hypercellular bone marrow with granulocytic proliferation and • Classical BCR-ABL1-negative myeloproliferative neo-
dysplasia, ± dysplasia in the erythroid and megakaryocytic plasms—characteristic driver mutations are present in the
lineages majority of cases. Dysplastic changes should not be
Modified from [20]
a
prominent at initial presentation.
332 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

Case History pseudo-Pelger-Huet cells (Fig. 11.11). The bone marrow core
A 71-year-old lady was admitted with weakness, malaise, and biopsy was hypercellular with a predominance of the granulo-
splenomegaly. CBC at presentation: WBC 98 × 109/L, hemo- cytic lineage exhibiting features of dysgranulopoiesis. The
globin 7.9 g/dL, MCV 94 fL, platelet count 68 × 109/L. The erythroid lineage was diminished. Megakaryocytes were gen-
differential count was as follows: blasts 2%, promyelocytes erally small and dysplastic. CD34+ blasts accounted for 9% of
10%, myelocytes 8%, metamyelocytes 13%, band forms 27%, hematopoietic cells. By FISH, no karyotypic abnormalities
neutrophils 25%, eosinophils 4%, basophils 1%, monocytes were found. Molecular analyses revealed SETBP1, AXL1,
3%, lymphocytes 7%. Cytologic examination of blood smears FLT3 ITD, and CBL mutations. No BCR-ABL1 or JAK2, MPL,
showed abundant neutrophilic precursors and abnormalities of or calreticulin mutations were found. These findings were
neutrophils including hypolobulation, hypergranularity, and consistent with a diagnosis of aCML.

a b

MGG MGG

c d

MGG MGG
MG
MGG

Fig. 11.11 Atypical chronic myeloid leukemia, BCR-ABL1-negative. (a– The hypercellular bone marrow core biopsy exhibits granulocytic prolifera-
d, MGG) Peripheral blood smears showing neutrophilic precursors and tion with maturation but left shift and dysplasia (e, f, Giemsa; g, NASDCL).
dysplastic hypolobulated neutrophils. Note the presence of rare blasts in d. (h) CD34+ blasts are increased but represent <10% of hematopoietic cells
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 333

e f

Giemsa Giemsa

g h

NASDCL CD34

Fig. 11.11 (continued)

334 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

11.5 Myelodysplastic/Myeloproliferative 11.5.3 Morphology

Neoplasm, Unclassifiable ( MDS/MPN-U)
There must be evidence of peripheral blood leukocytosis
The definition of MDS/MPN-U has been updated in the 2016 (≥13 × 109/L) and/or thrombocytosis (≥450 × 109/L).
version of the WHO classification [28]. This “by exclusion” However, dysplastic features of neutrophils are not as promi-
category includes cases that present with overlapping myelo- nent as in aCML. Blasts account for <20% in blood and bone
dysplastic and myeloproliferative features at initial diagnosis marrow cells. The bone marrow is hypercellular and shows
and do not fulfil the WHO-defined criteria of any other well- both proliferative and dysplastic features. Dysplasia affect-
defined MPN or MDS/MPN entity [2, 28]. Especially, BCR- ing ≥10% should involve at least one cell line. No specific
ABL1, PDGFRA, PDGFRB, FGFR1, PCM1-JAK2, or immunophenotypic features are observed.
CSF3R mutations should be excluded. Patients who develop
a MDS/MPN-U-like disease secondary to chemo- and/or
radiotherapy are diagnosed as therapy-related myeloid neo- 11.5.4 Genetics
plasm [28]. The diagnostic criteria are summarized in
Table 11.4. Until now, there are no specific molecular biomarkers for
MDS/MPN-U. Mutations of a variety of genes such as
SETBP1, TET2, ASXL1, NRAS, RUNX1, CBL, DNMT3A, and
11.5.1 Epidemiology IDH1/2 occur in subsets of MDS/MPN-U [2]. SETBP1 muta-
tions were reported in 9.3–10% of MDS/MPN-U patients who
No representative epidemiologic data are actually available. presented with higher WBC counts and lower platelet counts
Rare patients can be assigned to this entity. In a cohort of and hemoglobin patients with wild-type SETBP1 [21, 27, 30].
MDS/MPN patients, cases diagnosed as MDS/MPN-U Dysplastic morphology was more common in mSETBP1
represented only about 2% [29]. cases as compared with wild-type cases. An association of
SETBP1 with isochromosome i(17)(q10) and ASXL1 mutation
has also been described [29, 30]. However, SETBP1 mutations
11.5.2 Clinical and Laboratory Features occur in a large variety of myeloid neoplasms including other
MDS/MPN entities such as 32% of aCML, 24% of JMML,
Clinical symptoms at presentation are similar to those 18% of CMML, and 10% of MDS/MPN-U patients.
observed in other MDS/MPN categories. The prognosis is BCR-ABL1, PDGFRA, PDGFRB, FGFR1, and PCM1-
unfavorable. A median survival of 21 months has been JAK2 rearrangements must be excluded for the diagnosis of
reported [28]. MDS/MPN-U. The presence of classical MPN-associated
driver mutations such as JAK2, MPL, or CALR in cases with
morphologic features of a MDS/MPN-U suggests a disease
progression of a classical MPN. Rare cases such as cases
Table 11.4 Diagnostic criteria for MDS/MPN-U without a previously known chronic phase may nevertheless
Mixed myeloproliferative and myelodysplastic features, not meeting
be designated as MDS/MPN-U [28].
criteria of any other MDS/MPN category
No criteria for any MDS or MPN category
Clinical and morphologic MPN features (platelet count 11.5.5 Caution
≥450 × 109/L associated with bone marrow megakaryocyte
proliferation and/or WBC count ≥13 × 109/L)
No history of cytotoxic or growth factor therapy
• The patients should report no previous history of cyto-
Blasts <20% in blood and bone marrow toxic or growth factor therapy.
No BCR-ABL1 fusion gene, no PDGFRA, PDGFRB, FGFR1, • Reactive conditions associated with anemia, leukocytosis,
PCM1-JAK2 rearrangements and thrombocytosis should be excluded but generally
Modified from [28] show no frank dysplastic features.
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 335

Case History MPN-like cells, and formed loose clusters. The erythropoi-
A 74-year-old male patient was diagnosed with anemia, leu- esis was decreased and showed rare islands with disturbed
kocytosis, thrombocytosis, and mild splenomegaly. CBC at maturation. Granulopoiesis was increased and left shifted.
presentation: 16.1 × 109/L, hemoglobin 8.9 g/dL, MCV Blasts accounted for 4% of nucleated bone marrow cells.
96 fL, platelet count 470 × 109/L. The differential count was Karyotyping showed a monosomy 7. Genetic studies by
as follows: blasts 0%, band forms 4%, neutrophils 48%, NGS revealed TET2 and AXL1 mutations but no BCR-ABL1
eosinophils 1%, monocytes 4%, lymphocytes 41%. fusion gene and no JAK2, PDGFRA, PDGFRB, FGFR1,
The bone marrow was hypercellular (Fig. 11.12). PCM1-JAK2 rearrangements. Since no criteria of any other
Megakaryocytes were increased, showed MDS- and MPN- MDS/MPN were present, the diagnosis was MDS/
like features with a mixture of small dysplastic and large MPN-U.

a b

MGG
MG MGG

c d

MGG
MG Giemsa
Gi
G ie
em
ms
sa
a

Fig. 11.12 Myelodysplastic/myeloproliferative neoplasm, unclassifi- hypercellularity (d, Giemsa). Erythroid islands are rare but enlarged
able (MDS/MPN-U). a–c (MGG) bone marrow aspirate smears show- with decreased maturation (e, Giemsa, f, CD71). Both small hypolobu-
ing clusters of small dysplastic and large megakaryocytes, left-shifted lated and enlarged megakaryocytes are present (g, CD61). No reticulin
myeloid cells with hypogranulated and hypolobated forms and dimin- fibrosis (h, Gomori)
ished erythropoiesis. Examination of bone marrow core biopsy reveals
336 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

e f

Giemsa CD71

g h

CD6
CD61
CD611 Gomori
G
Goomo
mori
r

Fig. 11.12 (continued)

11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 337

11.6 Myelodysplastic/Myeloproliferative mutation). Ring sideroblasts are detected on iron-stained

Neoplasm with Ring Sideroblasts bone marrow aspirate smears but generally not on paraffin-
and Thrombocytosis (MDS/MPN-RS-T) embedded decalcified core biopsy specimens.
Erythropoiesis is increased and shows megaloblastoid and
The updated 2016 WHO classification has designated the pre- dysplastic features. Both large hyperlobulated and small
viously provisional category RARS-T as myelodysplastic/ dysplastic megakaryocytes are observed. Dense clus-
myeloproliferative neoplasm with ring sideroblasts and throm- ters and cloud-like megakaryocyte nuclei are reminis-
bocytosis (MDS/MPN-RS-T). This myeloid neoplasm actu- cent of primary myelofibrosis, but some biopsies may
ally represents a distinct MDS/MPN category [31, 32]. The contain more essential thrombocythemia- like mega-
hallmark of this myeloid neoplasm are the overlapping fea- karyocytes [36]. The reticulin network may be normal
tures between a MDS with ring sideroblasts and an MPN with or increased. Even collagen fibrosis and osteosclerosis
thrombocytosis [33]. The essential diagnostic features are a may develop.
platelet count ≥450 × 109/L and ≥15% ring sideroblasts (≥5%
in cases harboring a SF3B1 mutation; 34). Ring sideroblasts
are erythroblasts showing a perinuclear ring of ≥5 ferritin- 11.6.3 Genetics
loaded mitochondria that are highlighted by iron stain per-
formed on bone marrow aspirate smears. This phenomenon is Common mutations observed in MDS/MPN-RS-T are
linked to ineffective erythropoiesis and to SF3B1 haploinsuf- spliceosome gene mutations in the SF3B1 gene in >80%,
ficiency in >80% of cases [34, 35]. The iron in the mitochon- JAK2V617F mutation in up to 75% [32]. CALR or MPL
dria of ring sideroblasts corresponds a ferritin isoform with mutations are present in a small percentage of patients
ferroxidase activity that is likely to sequester potentially harm- [32, 33].
ful free iron [32]. The diagnostic criteria of MDS/MPN-RS-T Apparently, the mutant SF3B1 protein retains struc-
are summarized in Table 11.5. Some patients initially present tural integrity, albeit with altered function. It has been
MDS-RS and subsequently develop thrombocytosis consis- suggested that MDS/MPN-R-ST may result from a com-
tent with a transformation into MDS/MPN-RS-T [32, 33]. bination of SF3B1, responsible for myelodysplastic fea-
tures (i.e., ring sideroblasts), and subclonal JAK2, MPL,
or CALR mutations, conferring the myeloproliferative
11.6.1 Epidemiology phenotype (i.e., thrombocytosis) [37]. Numerous other
mutations have been reported including TET2 in about
This myeloid neoplasm is generally diagnosed in elderly adults 20% ASXL1 in about 20%, DNMT3A in about 15%, and
aged >70 years with a female preponderance. No data reporting SETBP1 in about 10% of patients [34]. The karyotype is
the incidence of this rare myeloid neoplasm are available. normal in most patients [31]. However, in a Mayo Clinic
study, anemia and abnormal karyotype were indepen-
dently prognostic for inferior survival. Univariate analy-
11.6.2 Morphology sis showed association between poor survival and ASXL1
or SETBP1 mutations [34].
The hallmark of this MDS/MPN entity is overlapping
MPN and MDS features in association with the presence
of 15% ring sideroblasts (≥5% in cases harboring a SF3B1 11.6.4 Caution

• Ring sideroblasts may be observed in clonal sideroblastic

Table 11.5 Diagnostic criteria for MDS/MPN-RS-T
anemias especially MDS-RS that, however, do not fulfil
Anemia associated with erythroid-lineage dysplasia (±multilineage
the criteria of MDS/MPN-RS-T.
dysplasia), ≥15% ring sideroblasts (≥5% in cases harboring a SF3B1
mutation) • Moreover, ring sideroblasts may be present in a variety non-
SF3B1 mutation or in the absence of SF3B1 no recent cytotoxic or clonal acquired or congenital sideroblastic anemias that
growth factor therapy may eventually be associated with reactive thrombocytosis.
Concomitant JAK2 V617F or CALR or MPL mutations strongly
support the diagnosis
Examples of non-clonal sideroblastic anemias include excess
Persistent thrombocytosis (platelet count ≥450 × 109/L)
No history of another myeloid neoplasm with the exception of alcohol use, copper deficiency, lead and zinc toxicity, and
MDS-RS drugs. Congenital sideroblastic anemias can result from
Blasts <1% in peripheral blood, <5% bone marrow mutations in mitochondrial pathways [32].
No BCR-ABL1 fusion gene, no PDGFRA, PDGFRB, FGFR1,
PCM1-JAK2 rearrangements
• Therefore, genetic studies especially JAK2 and SF3B1
No t(3;3)(q21.3;q26.2), inv(3)(q21.3;q26.2), or deletion (5q)
mutation analysis are mandatory in difficult cases.
Modified from [31]
338 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

Case History abundant both large MPN-type polymorphous and small

The patient was a 69-year-old female who presented with dysplastic MDS-type megakaryocytes often arranged in
persistent thrombocytosis, anemia, and splenomegaly. dense clusters. Erythropoiesis was increased, left shifted,
Laboratory studies showed the following: CBC and megaloblastoid. Iron staining performed on aspirate
12.4 × 109/L, hemoglobin 9.1 g/dL, RBC 2.6 × 1012/L, smears provided evidence of multiple ring sideroblasts
MCV 106 fL, platelet count 980 × 109/L. The differential (≥30%). Granulopoiesis was slightly increased. There was
count was: blasts 0%; band forms 1%, neutrophils 72%, no significant fibrosis. The karyotype was normal. Molecular
eosinophils 2%, monocytes 3%, basophils 1%, lympho- analyses were positive for JAK2V617F and SF3B1 Exon 15
cytes 22%. She underwent a bone marrow aspiration and mutations. The findings were consistent with MDS/
biopsy (Fig. 11.13). The hypercellular marrow showed MPN-RS-T.

MDS/MPN-RS-T

a b

Giiem
Giemsa
ems
em sa
a Giemsa

c d

Iron IIron
Irro
onn

Fig. 11.13 (a, b; MGG) Aspirate smears showing cluster of large megakaryocytes with a predominance of large hyperlobulated cells but
MPN-type and small dysplastic more MDS-type megakaryocytes. (c, d; also small dysplastic forms. The erythroid series represents about 60%
Prussian blue stain) The presence of abundant ring sideroblasts is of nucleated cells and shows megaloblastoid features and a decreased
detected by iron stain performed on bone marrow aspirate smears. The maturation (e, f, Giemsa; g, CD61; h, CD71)
hypercellular bone marrow core biopsy contains markedly atypical
11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN) 339

e f

HE HE

g h

CD
CD71
71
CD71 CD61
C
CD
D6
61
1

Fig. 11.13 (continued)

340 11 Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

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Myelodysplastic Syndromes (MDS)
12

Table 12.1 Terminology of and diagnostic features of MDS entities

12.1 Introduction according to the revised 2016 WHO Classificationa
• MDS with single lineage dysplasia MDS-SLD
Myelodysplastic syndromes (MDS) comprise a heteroge- – blasts BM < 5%, PB < 1%, no Auer rods
neous spectrum of clonal stem cell disorders. • MDS with multilineage dysplasia MDS-MLD
Six distinct MDS entities and the provisional entity – blasts BM < 5%, PB < 1%, no Auer rods
refractory cytopenia of childhood (RCC) are listed in the • MDS with ring sideroblasts MDS-RS
revised 2016 WHO classification of tumors of the hemato- – blasts BM < 5%, PB < 1%, no Auer rods
(If SF3B1 mutation status negative or unknown, ≥15% RS will
poietic and lymphoid tissues (Table 12.1, [1–4]).
be required, if SF3B1 mutation is present only ≥5% RS)
Several criteria are mandatory for the diagnosis of MDS: – single lineage dysplasia MDS-RS-SLD
the presence of at least single lineage cytopenia (anemia and/ – multilineage dysplasia MDS-RS-MLD
or neutropenia, and/or thrombocytopenia) associated with • MDS, with isolated del(5q)
one or several additional features: morphologic evidence of – blasts BM < 5%, PB < 1%, no Auer rods
dysplasia in ≥10% of cells involving at least one hematopoi- – one additional cytogenetic abnormality allowed
etic lineage, increased blasts, and/or a MDS-defining cytoge- – but no −7, or del(7q)
– no impact of SF3B1 mutation or RS
netic abnormality [2, 4]. Ineffective hematopoiesis is
• MDS unclassifiable MDS-U
considered to be related to enhanced apoptosis, stem cell – with single lineage dysplasia and pancytopenia
senescence, and immunologic dysregulation. blasts BM < 5%, PB < 1%, no Auer rods
For the assignment of a myeloid neoplasm to the MDS – with exactly 1% peripheral blood blasts
category, the blast must represent <20% of peripheral blood blasts BM < 5%, PB = 1%, no Auer rods
and/or bone marrow cells. This threshold distinguishes MDS – MDS without excess blasts or dysplasia, but with an
from acute myeloid leukemia (AML) [2]. The evolution of MDS-defining cytogenetic abnormality
blasts BM < 5%, PB < 1%, no Auer rods
an AML secondary to a MDS is classified as AML with
• MDS with excess blasts MDS-EB
myelodysplasia-related changes (AML-MRC) and defined – blasts BM 5–9% or PB 2–4%; BM <10% MDS-EB-1
by the boundary of ≥20% bone marrow blasts according to and PB < 5% no Auer rods
the WHO criteria. However, the presence of certain recurrent – blasts BM 10–19% or PB 5–19% MDS-EB-2
genetic abnormalities such as inv(16)(p13.1q22) qualifies a or Auer rods, blasts BM and PB < 20%
myeloid neoplasm for the diagnosis of AML without regard Note: ≥50% erythroids and 5–19% blasts: RAEB, no longer
to the percentage of blasts ([5]; see Chap. 13). Therapy- AML. These cases share features with MDS more than with AML;
≥20% blasts and ≥50% erythroids: AML (predominantly AML-MRC);
related myeloid neoplasm even those presenting with fea- BM bone marrow, PB peripheral blood
tures of MDS are not included in this chapter but are classified a
Modified according to [2]
as therapy-related myeloid neoplasms (see Table 12.2).
MDS subtypes have initially been classified according to
the French-American (FAB) classification that predominantly eters for the assignment of an MDS to a specific subtype [1,
considered cytologic features including the percentage of 2]. Diagnostics in MDS relies on complementary consider-
blasts [6]. With the availability of immunophenotyping, cyto- ation of hematological, morphological, and cytogenetic/
genetics, and molecular genetics, this approach has changed molecular parameters. Methods include marrow and periph-
significantly. In the revised 2016 WHO classification, cytoge- eral blood cytology, cytogenetics, fluorescence in situ hybrid-
netic and molecular features became the most relevant param- ization (FISH), bone marrow core biopsy examination,

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 343

Table 12.2 Myelodysplastic syndromes: the spectrum of common and reticulocyte counts. Patients with an absolute neutrophil
heterogeneous featuresa count <1.8 × 109/L are considered to be neutropenic.
Common features Thrombocytopenia is diagnosed when thrombocytes are
– Ineffective hematopoiesis with one or more peripheral cytopenias <100 × 109/La [2]. Anemia is the most frequent symptom of
– Morphologic dysplasia of maturing hematopoietic elements
MDS, while isolated neutropenia or thrombocytopenia are
Heterogeneous features
– Clinical course: indolent with a nearly normal life expectancy to rare [4]. Death may result from the consequences of refrac-
rapidly progressing to AML tory cytopenias or from transformation into AML with MDS-
– Different numbers of cytopenias related changes (AML MRC). The clinical course is highly
– Variable expansion of myeloblasts (<20%) variable ranging from a rather indolent disease in low-risk
– Different cytogenetic and molecular abnormalities MDS to a rapidly unfavorable outcome in high-risk disease.
– Variable risk for shortened overall and leukemia-free survival
Predicting the individual risk category is important for
(see prognostic scoring systems)
Exceptions patient counseling and treatment recommendations.
• AML- defining cytogenetic features without consideration of Prognostic factors in MDS can be divided into patient-related
blasts cell percentage or disease-related factors. Patient-related factors can include
Cytogenetic abnormalities typical for de novo AML age at diagnosis, performance status, and comorbidities.
t(8;21)(q22;q22)RUNX1-RUNX1T1, inv(16)(p13.1q22), t(16;16) Disease-related factors include pathological, laboratory, and
(p13.1;q22)CBFB-MYH11, t(15;17)(q24.1;q21.2); PML-RARA
biological features [12]. Different prognostic scoring systems
• MDS secondary to chemotherapy or irradiation: Therapy-
related myeloid neoplasms such as the International Prognostic scoring system (IPSS),
Modified according to [2, 7]
a the revised IPSS (IPSS-R), the World Health Organization
(WHO) Prognostic Scoring System (WPSS), and others are
currently used to risk-stratify MDS patients. These systems
immunophenotyping, and the evaluation of the genetic and are principally based on the karyotype, the bone marrow
epigenetic profile by established and new technologies. blast percentage, and/or cytopenias, transfusion dependency,
and/or features of the WHO classification but have different
cutoffs for various parameters [12].
12.2 Epidemiology

MDS is predominantly diagnosed in elderly patients with a 12.4 Morphology

median age of 70 years and with a male predominance [2, 3]. and Immunohistochemistry:
About five new MDS cases are diagnosed a year per 100,000 Dysplasia Assessment in MDS
inhabitants [3]. However, the incidence is probably even
higher since not every MDS patient will be adequately regis- Morphologic evaluation of dysplastic features present in
tered. The annual incidence in adult patients >70 years is peripheral blood, bone marrow aspirate smears, and bone mar-
about 20 cases per 100,000 individuals [2]. In Asia, MDS row core biopsy specimens is still an important component for
develops more frequently at a younger age [3]. In adults, the the diagnostic work-up of MDS cases even if scoring systems
different MDS subtypes are considered to be collectively the focus on genetic and hematological parameters. However,
most common acquired adult bone marrow failure syn- morphologic changes suggesting uni- or multilineage dyspla-
dromes [8]. Pediatric MDS cases have an annual incidence sia may also be observed in other myeloid neoplasms and in
of 1–4 cases per million and represent <5% of childhood non-neoplastic conditions, for example, autoimmune cytope-
hematologic malignancies [9, 10]. Most pediatric MDS cases nias [3, 4, 7, 13]. For the diagnosis of MDS, minimal diagnos-
correspond to refractory cytopenia of childhood (RCC). tic criteria should be fulfilled ([7]; see Table 12.3).
These cases are challenging since congenital bone marrow
failure syndromes and myeloid neoplasms with germline Table 12.3 Morphologic criteria for defining bone marrow dysplasiaa, b
mutations have to be considered [11]. • Cytology to evaluate abnormalities of hematopoietic cells:
– 200 cells in blood smears
– 500 cells in bone marrow smears
12.3 Clinical Features – At least 100 erythroid precursors, 100 granulocytic cells,
30 megakaryocytes
• Marrow dysplasia (≥10% of cells of the individual lineage)
The clinical presentation is frequently characterized by
• Bone marrow biopsy to assess cellularity, topography, and
unexplained isolated or multiple peripheral blood cytopenias fibrosis (grade 2–3)
or increased susceptibility to infections. Anemia is defined as Combination of overt dysplasia and clonal cytogenetic
hemoglobin <10 g/dL [<13 g/dL (males) or <12 g/dL abnormalities: conclusive diagnosis of MDS
(females)]a and is often macrocytic (MCV > 97 fL) with low higher threshold [2] bModified from [7]
a
12 Myelodysplastic Syndromes (MDS) 345

12.4.1 Special Stains in MDS ated by morphologic analysis of smears and biopsies are pro-
vided in Table 12.4.
The microscopic analyses of smears prepared from peripheral
blood and bone marrow aspirates as well as histologic slides 12.4.2.1 The Peripheral Blood in MDS
obtained from bone marrow core biopsies have still an impor- In peripheral blood smears, red blood cells frequently show
tant impact on the work-up of MDS particularly as a rapid anisocytosis, poikilocytosis and macrocytosis. Dual hyper-
initial diagnostic procedure [14]. A high technical quality of and normochromic cell populations can be observed. In the
specimens is mandatory [15]. Basic staining methods for the rare cases of acquired α-thalassemia-MDS (ATMDS), hypo-
evaluation of aspirates include the panoptic Pappenheim’s chromasia and microcytosis are present. Granulocytes of
(May Grünwald-Giemsa, MGG) or Wright’s stain, Pearl’s (or MDS patients are generally scarce reflecting granulocytope-
Berlin blue) iron stain and, as required, the cytochemical nia. Cytoplasmic hypogranularity or agranularity are often
determinations of myeloperoxidase (MPO), and non-specific noted. Hyposegmentation of nuclei may be similar to the
esterase, e.g., α-naphthyl acetate esterase. Bone marrow core inherited Pelger-Huet anomaly (so-called pseudo-Pelger
biopsies should preferentially be fixed in buffered 4% forma- cells). In addition, hypersegmented or giant forms of nuclear
lin, decalcified in EDTA and paraffin-embedded. In addition rings may be present. Abnormalities of platelets include
to panoptic stains (hematoxylin & eosin and/or Giemsa), spe- anisocytosis, hypo- or hypergranularity, and giant forms.
cial stains such as naphthol-ASD- chloroacetate esterase Platelet counts are often decreased with the exceptions of
(NASDCL) for granulopoiesis, Pearl’s for hemosiderin, MDS associated with isolated del(5q) or 3q26 abnormalities.
Gomori’s silver impregnation for reticulin fibers, and tri- Patients with these subsets of MDS have often normal or
chrome stains in order to demonstrate a dense collagen mesh- increased platelets. In MDS-EB-1 or -2, the peripheral blasts
work should be applied to sections cut from core biopsies. A represent 2–4% or 5–19%, respectively, and may contain
wide panel of antibodies is now available for further evalua- Auer rods in MDS-EB-2. The blast cell count should include
tion of the different myeloid cell lineages, highlighting the myeloblasts but also monoblasts, promonocytes, and mega-
presence of blasts, detecting lymphoid infiltrates, cancer karyoblasts but no proerythroblasts [14].
cells, and characterizing stromal components in bone marrow
core biopsies. Immunophenotyping of the neoplastic popula- 12.4.2.2 he Bone Marrow in MDS: Dysplasia
T
tion by flow cytometry (FC) is essential for the diagnostic Involving the Hematopoietic Lineages
work-up and monitoring of AML and is increasingly recog- According to the 2008 WHO criteria, unequivocal dysplastic
nized as part of an integrated diagnosis in MDS [16]. However, features in ≥10% of cells of at least one of the myeloid lin-
conventional stains and immunolabeling techniques applied eages are required to establish the diagnosis of MDS [2].
to cytologic and histologic specimens can still significantly Exceptions are cases with MDS-U (see Table 12.1).
contribute to the characterization of these MNs. Currently, dysplastic features of ≥10% of precursors of at
Immunohistochemistry of core biopsies remains most useful least two myeloid lineages are required for the diagnosis of
in cases presenting with hypoplastic and/or fibrotic variants MDS-MLD.
of MDS and AML in which sufficient material for immuno- Erythroid lineage: Erythropoiesis is often, but not in
phenotyping by FC is not available. Moreover, FISH to detect every MDS case, the preponderant lineage. Enlarged ery-
rearrangements and losses or gains of genetic material can be throid islands containing increased numbers of immature
routinely performed on smears, as well as on sections cut precursors may be dislocated from the center of the marrow
from paraffin-embedded biopsies [14]. spaces to the niches adjacent to the trabecular bone. Erythroid
precursors tend to show abnormalities such as megaloblas-
toid nuclear changes, nuclear lobulation, multinuclearity,
12.4.2 D
ysplastic Features Observed internuclear chromatin bridges, cytoplasmic vacuolization,
in Peripheral Blood and Bone Marrow or in rare cases PAS-positivity. While the latter features may
Specimens be detected on cytologic bone marrow aspirate smears, as
well as on sections from core biopsies, the presence of ring
The generally hyper-, rarely hypocellular, and occasionally sideroblasts is revealed only on Prussian blue stained bone
fibrotic bone marrow shows dysplastic features in ≥10% of marrow smears. Ring sideroblasts are erythroid precursors
cells of at least one of the hematopoietic lineages. Rare containing ≥5 iron granules which encircle the nucleus and
exceptions are cases with dysplastic features in <10% of correspond to mitochondrial iron deposits. Erythropoiesis
myeloid cells, but harboring MDS-type cytogenetic altera- can be immunolabeled by antibodies to hemoglobin, gly-
tions. These cases are assigned to as MDS-U (see Table 12.1). cophorin C, or CD71. Abnormal erythroid precursors may
Features that are suggestive of an MDS and may be evalu- also express CD117.
346 12 Myelodysplastic Syndromes (MDS)

Table 12.4 Morphologic and immunohistochemical features of dysplasia in peripheral blood and bone marrowa
Hematopoietic Cytology of bone marrow aspirate Cytology of peripheral Histology and immunohistochemistry of bone marrow
lineage smears blood smears Bone marrow core biopsies
Erythropoiesis/red Megaloblastic changes Anisopoikilocytosis Maturation defects
blood cells Maturation defects Hyperchromasia Makroblastic hyperplasia
Nuclear lobulation, budding Dual RBC populations Enlarged erythroid island
Bi-/multinuclearity Siderocytes Abnormal paratrabecular localization
Internuclear chromatin strands Basophilic stippling of erythropoiesis
Abnormal mitoses IH: glycophorin C, hemoglobin, CD71
Ring sideroblasts
PAS+ cytoplasmic inclusions
Cytoplasmic vacuolization
Granulopoiesis/ Maturation defects Pseudo-Pelger cells Maturation defects
neutrophils Hypo-/hypergranularity Hypersegmentation Increased number of immature precursors
Abnormal granules Hypogranularity Abnormal localization of immature precursors
Myeloperoxidase deficiency Giant forms (ALIP) in the central marrow cavity
Ring nuclei IH: myeloperoxidase, CD13, elastase, CD34
Megakaryopoiesis/ Micromegakaryocytes Anisocytosis Micromegakaryocytes
platelets Large cells Giant platelets Nuclear abnormalities (mono-/multilobation)
Monolobated or multilobated forms Vacuolization Megakaryocyte clustering
Hypersegmentation IH: CD61, CD42b, CD41 (CD34 in some cases)
Nuclear-cytoplasmic asynchrony
Blast cells Percentage of blasts Percentage of blasts Percentage of blasts
Auer rods Auer rods Spatial distribution of blasts (disseminated vs.
Hypergranularity Agranularity/hyper-granularity A-/hypergranularity clusters)
Myeloperoxidase Cytochemical features Immunohistochemical characterization:
(myeloperoxidase-/ CD34, CD117, CD33
esterase positivity) (CD14, myeloperoxidase, CD61,CD42b)
Additional features Percentage of dysplastic cells in the Bone marrow architecture
different myeloid lineages Cellularity
Lymphocytes, plasma cells, Fibrosis
Monocytic proliferation Vascularity
Hemosiderin in macrophages Lymphocytes, plasma cells
Monocytic proliferation
Hemosiderin in macrophages
IH: p53expression
IH immunohistochemistry, RBC red blood cells
a
Modified from [14]

Granulocytic lineage: Granulopoiesis is often hypo- from micromegakaryocytes to small mononuclear, hypo-
blastic and left-shifted and shows maturation defects. or bilobed megakaryocytes, cells with completely and
Abnormal hypo-or hypergranularity including pseudo-Che- widely separated nuclei or large hyperlobated forms.
diak-Higashi granules may be present. Similar to blood Megakaryocytes of smaller than normal size showing non-
smears, hyposegmented pseudo-Pelger-Huet cells may be lobated round nuclei are typical for MDS del(5q). Myeloid
seen. Granulation defects are evident especially on aspirate neoplasm harboring inv(3)/t(3;3)(q21.3q26.2) or t(3;3)
smears, while the bone marrow core biopsy may reveal the (q21.3q26.2) GATA2, MECOM that may present as MDS
atypical localization of immature CD34+ precursors or AML according to the blast cell count megakaryocytes
(ALIP) in the central parts of the marrow spaces. are small and typically mono- or bilobed. The immuno-
Immunohistochemistry for the detection of granulopoietic phenotype of megakaryocytes is characterized by the
cells include antibodies to myeloperoxidase, elastase, expression of CD42b, CD41 or CD61 and positive for
CD13, and CD33. linker of activated T lymphocyte and factor VIII–related
Megakaryocytic lineage: Megakaryopoiesis may be antigen. In MDS, megakaryocyte precursors may also be
hyperplastic, particularly in MDS with isolated del(5q) or immunolabeled by CD34 antibodies.
inv(3)(q21q26)/t(3;3)(q21;q26) but may also be dimin- Blasts: On bone marrow aspirate smears, blast cells
ished. On slides obtained from core biopsies, loose clus- show cytological features similar to those observed in the
ters of polymorphic cells may be observed and atypically peripheral blood. Megakaryoblasts, monoblasts, and pro-
localized in the periphery of the bone marrow spaces. monocytes have to be included when the percentage of
Megakaryocyte size and shape are quite variable ranging blasts is evaluated. The presence of myeloid blasts and
12 Myelodysplastic Syndromes (MDS) 347

immature myeloid precursors may be highlighted by CD34 moderate to marked fibrosis grade 2 and 3 characterized by a
and CD117 immunohistochemistry. diffuse dense increase in reticulin and collagen fibers which
may even be associated with osteosclerosis is an important
12.4.2.3 he Bone Marrow Core Biopsy in MDS:
T issue in MDS and has to be mentioned in each pathology
Bone Marrow Architecture report. Grading of fibrosis according to the content of so-
and Stromal Components called reticulin and collagen fibers should be performed [18].
The histologic examination of BMTBs is included in the cur- A hyperfibrotic MDS (MDS-F) is reported in about 10–20%
rent diagnostic approach of MDS to assess marrow cellular- of all MDS cases. It has been shown that a relevant moderate
ity, fibrosis, and topography. In addition to dysplastic features to marked marrow fibrosis was associated with multilineage
of the myeloid lineages, morphologic alterations in MDS dysplasia, high transfusion requirement, and poor-risk cyto-
pertain to topographical organization, cellularity, microves- genetics [19].
sel density, fiber content, and immune and stromal cells. The Hemosiderin: MDS patients frequently receive transfu-
microenvironment comprises among others macrophages, sions that can lead to iron overload. Iron-induced toxicity has
endothelial cells, fibroblasts/myofibroblasts, osteoblasts, a negative effect on hematopoietic stem cells and may con-
osteoclasts, adipocytes, extracellular matrix components, tribute to MDS progression [20]. The amount of iron storage
adhesion molecules, soluble factors, and other components by bone marrow macrophages can be evaluated both on aspi-
of the niche. The mesenchymal niche is actually considered rate smears and on Pearl’s stained sections of core biopsies.
as a critical contributor to disease initiation and evolution Hemosiderin granules in erythroblasts, especially in ring
[15]. In the normal bone marrow, precursor cells of the three sideroblasts, are detected in bone marrow aspirate smears
myeloid lineages reside in specialized bone marrow com- and not in sections cut from paraffin-embedded bone marrow
partments where they survive, differentiate, and give rise to core biopsies.
mature blood cells. Microvessels: MDS is often accompanied by an increase
Topography: In MDS spatial disturbances are frequently in microvascular structures that may be visualized by CD34
observed. Precursors of the three myeloid lineages are often immunohistochemistry.
dispersed throughout the marrow spaces. Collections of Immune cells: An immune-mediated impairment of
erythropoietic cells and megakaryocytes are no longer con- hematopoietic stem and progenitor cells, increased apopto-
fined to the central areas close to marrow sinuses but may be sis, high cytokine levels, and changes in the hematopoiesis-
dislocated to the paratrabecular zones. By contrast, granulo- supporting microenvironment may contribute to the
poietic precursors which are normally localized in the para- pathogenesis especially of hypoplastic MDS. Decreased
trabecular niche may be found in the central parts of marrow CD8 + T cells, less IFN-γ secretion by CD8 + T cells,
spaces. An abnormal localization of clusters (3–5 cells) or increased exhausted TIM3 + CD8 + T cells with lower
aggregates (>5 cells) of myeloblasts and promyelocytes perforin and granzyme B expression and higher CD95
(ALIP) observed in the central marrow cavity of BMTB sec- expression have been detected in MDS [21]. It has been sug-
tions may be observed. The presence of several ALIPs as well gested that the expansion of mutated clones may be driven
as CD34+ cell aggregates has been shown to be associated by inflammation in some patients with lower-risk MDS.
with an increased risk of leukemic transformation in MDS.
Bone marrow cellularity: Since the cellularity may vary
between different marrow areas, the overall cellularity may 12.5 Genetics in MDS
more accurately be assessed on BMTBs than on smears. In
MDS, cellularity is often increased but may also be normal 12.5.1 Chromosomal Abnormalities
or even decreased. The hypoplastic variant of MDS which is
observed in about 10% of all MDS cases may be diagnosti- Numerical and structural chromosomal abnormalities are
cally challenging. Depending on the age of the patient, an present in about 50% of MDS cases. Cytogenetic aberrations
MDS should be considered as hypoplastic MDS when fat can be detected by conventional chromosome banding analy-
cells occupy about 70–80% of the bone marrow spaces. The sis of bone marrow metaphases or by fluorescence in situ
differential diagnosis includes idiopathic cytopenia of uncer- hybridization (FISH) of bone marrow cells and circulating
tain clinical significance (ICUS), aplastic anemia, and toxic CD34+ cells [22]. In contrast to AML, balanced chromo-
or immunologic bone marrow damage. A detailed search for somal translocations are rare in MDS while unbalanced
dysplastic features is warranted and may allow a discrimina- translocations are more frequent. Losses or gains of large
tion between aplastic anemia and hypoplastic MDS or refrac- segments of chromosomes include −7/del(7q), −5/del(5q),
tory cytopenia of childhood [17]. +8, dup(1q), del(20q), del(11q), del(12p)/t(12p), del(17p)/
Fibrosis: A mild increase in bone marrow reticulin fiber iso(17q), del(18q), +21q gains, del(13q), der(1;7)(q10;p10).
content grade 1 is of no prognostic relevance in MDS. However, Complex karyotypes harboring ≥3 chromosomal aberrations
348 12 Myelodysplastic Syndromes (MDS)

are frequently associated with TP53 mutation and a dismal Table 12.5 Selected driver genes involved in MDSa
prognosis. Function/pathway Mutated genes, impact on outcome
Cytogenetic aberrations are associated with different RNA splicing SF3B1 (favorableb), SRSF2 (adverseb), U2AF1
prognoses and represent important components of the prog- (adverseb), U2AF2, SF1
DNA methylation TET2 (neutral), DNMT3A (neutral), IDH1/
nostic scoring systems. For example, cytogenetic subgroups
IDH2 (neutral)
defined according to the IPSS-R are: very good: −Y, del(11q); Chromatin ASXL1 (adverseb), EZH2 (adverse), MLL2
good: normal, del(5q), del(12p), del(20q), double including modification
del(5q); intermediate: del(7q), +8, +19, i(17q), any other Transcription RUNX1 (adverse), ETV6, GATA2, CEBPA,
single or double independent clones; poor: −7, inv(3)/t(3q)/ regulation CUX1, BCOR
Checkpoint/cell TP53 (adverse), CDKN2A
del(3q), double including −7/del(7q); complex: three abnor-
cycle
malities; very poor: complex: >3 abnormalities [23]. DNA repair ATM, BRCC3, FANCL
In addition, copy-neutral loss-of-heterozygosity RAS signaling PTPN11, NF1, NRAS, KRAS, CBL
(CN-LOH) or uniparental disomy (UPD), which are com- Cohesin complex STAG2, RAD21
monly seen in chromosomes 1p, 4q, 7q, 11q, 13q, 14q, 17p Other signaling JAK2, KIT, MPL, FLT3
may be detected in MDS. Chromosomal abnormalities are molecules
no stable features but may increase or even disappear during Modified from [12, 23]
a

blasts <5%
b
the disease course.

Table 12.6 MDS Cytogenetic Scoring Systema

12.5.2 Somatic Mutations Prognostic
subgroups Cytogenetic abnormalities
It is now well recognized that MDS are mostly diseases of Very good −Y, del(11q)
Good Normal, del(5q), del(12p), del(20q), double
epigenetic dysregulation and disordered spliceosome func- including del(5q)
tion [8]. Relevant gene mutations are listed in Table 12.6. Intermediate del(7q), +8, +19, i(17q), any other single or
Mutations in >40 genes and extensive subclonal diversifica- double independent clones
tion are a hallmark of MDS [24]. In many patients, several Poor −7, inv(3)/t(3q)/del(3q), double including −7/
mutations are present simultaneously. The neoplastic clone del(7q), complex: 3 abnormalities
Very poor Complex: >3 abnormalities
may remain stable for many years. In other patients, highly
dynamic shifts in clonal composition even without therapy Modified according to [23]
a

have been observed [25]. The total number of oncogenic

370 12 Myelodysplastic Syndromes (MDS)

Case History 10. Hypoplastic Myelodysplastic etic precursors including 7% blasts. The core biopsy was
Syndrome with Excess Blasts 1 (hMDS-EB-1) markedly hypocellular with focal presence of active hemato-
(Fig. 12.12) poiesis with reduced granulopoiesis, some erythroid islands,
A 67-year-old male patient was diagnosed with pancytope- and small dysplastic megakaryocytes. In addition, scattered
nia. The CBC was as follows: WBC 2.1 × 109/L, Hb 6.1 g/ CD34+ blasts were seen representing <10% of nucleated
dL, MCV 102 fL, platelets 45 × 109/L. The peripheral blood cells. Cytogenetic analysis revealed del5(q) and monosomy
showed 34% dysplastic neutrophils, 1% eosinophil, 4% 7. In addition, a SRSF2 mutation was detected. The diagnosis
monocytes, and 61% lymphocytes. The bone marrow aspi- was hypoplastic myelodysplastic syndrome with excess
rate smear contained abundant fat cells and rare hematopoi- blasts 1 (MDS-EB-1).

a b

MG
M
MGG
GG MGG
MG
GG
c d

MPO MPO

Fig. 12.12 Hypoplastic myelodysplastic syndrome with excess (myeloperoxidase). (e, f) Hypocellular bone marrow core biopsy show-
blasts 1 (MDS-EB-1). (a, b) Nuclear hypolobulation and decreased ing focal hematopoiesis with small dysplastic megakaryocytes
cytoplasmic granules of neutrophils in the peripheral blood. Presence of (Giemsa). (g) Rare cells of granulopoiesis are naphthol-ASD-
some pseudo-Pelger forms. (c, d) Bone marrow aspirate smear showing chloroacetate-esterase positive while small erythropoietic islands are
abundant fat cells and rare predominantly immature myeloid cells negative. (h) Scattered blasts are visualized by CD34 immunostaining
12 Myelodysplastic Syndromes (MDS) 371

e f

H&E H&E

g h

NASDCL CD34

Fig. 12.12 (continued)

372 12 Myelodysplastic Syndromes (MDS)

Case History 11. Hypoplastic Myelodysplastic The bone marrow core biopsy was hypocellular with focal
Syndrome with Excess Blasts 2 and Erythroid “hot spots” of predominantly composed of erythroid cells.
Predominance (hMDS-EB-2) (Fig. 12.13) Erythropoiesis represented 65% of bone marrow cells. The
A 69-year-old male patient was diagnosed with bicytopenia. percentage of CD34+ blasts was 14% calculated on all nucle-
The CBC was as follows: WBC 1.7 × 109/L, Hb 9.3 g/dL, ated cells. The karyotype was normal 46,XY. Molecular
MCV 98 fL, platelets 52 × 109/L. The peripheral blood con- analysis revealed a SRSF2 mutation. The diagnosis was
tained 19% neutrophils, 4% monocytes, 1% blasts, and 77% hypoplastic myelodysplastic syndrome with excess blasts 2
lymphocytes. The aspirate smears could not be evaluated. and erythroid predominance (MDS-EB-2).

a b

H&E
H& H&E
H&
H&E

c d

H&E
H& H&E
H&
&E

Fig. 12.13 Hypoplastic myelodysplastic syndrome with excess bone marrow cells. (f) Small dysplastic hypolobated CD61+ megakary-
blasts 2 and erythroid predominance. (a–d) H&E stained sections cut ocytes in the vicinity of a lymphoid aggregate. (g) Rare left-shifted
from the hypocellular bone marrow core biopsy showing predominance myeloperoxidase+ myloid precursors. (h) Clusters of CD34+ blasts in
of erythroid cells and small clusters of immature precursors/blasts in d. the central bone marrow area
(e) Increased CD71+ erythroid precursors accounting for about 65% of
12 Myelodysplastic Syndromes (MDS) 373

e f

CD71 CD61

g h

MPO
MP
MPOO CD34
CD
CD34

Fig. 12.13 (continued)

374 12 Myelodysplastic Syndromes (MDS)

Case History 12. Overlap Between Aplastic Anemia and composed of immature erythroid precursors. Myeloid cells
Hypoplastic MDS (Fig. 12.14) and megakaryocytes were rare. Focal lymphoid infiltrates
A 28-year-old woman was admitted to the hospital with a and an excessive storage of hemosiderin were present. No
history of pancytopenia diagnosed several years previously. excess blasts was observed. The karyotype was normal.
She had received multiple red blood cell transfusions. The Molecular analysis revealed a somatic DNMT3A mutation
CBC was as follows: WBC 1.3 × 109/L, Hb 5.6 g/dL, MCV (Exon 23: c.2645 G->A, p.R882H). No germline mutations
86.4 fL, platelets 24 × 109/L. The differential count showed were detected. The differential diagnosis included very
2% band forms, 38% neutrophils, 6% monocytes, and 52% severe aplastic anemia and hypoplastic MDS. Both condi-
lymphocytes. Some dysplastic neutrophils including pseudo- tions may harbor a DNMT3A mutation. The final diagnosis
Pelger forms were observed. The bone marrow aspirate favored very severe aplastic anemia associated with an
resulted in a dry tap. The bone marrow core biopsy was increased risk of developing a MDS as a consequence of the
markedly hypocellular with several hot spots predominantly DNMT3A mutation.

a b

Gie
em
mssa
Giemsaa
Gie
Giemmsa
sa

c d

Giem
Giemsa
em sa Giemsa
Gi
G iem
emsa
s

Fig. 12.14 Overlap between aplastic anemia and hypoplastic MDS. karyocytes by CD61 immunolabeling. (g) Rare predominantly immature
(a–d) Markedly hypocellular bone marrow core biopsy showing some cells of the granulocytic series (naphthol-ASD-chloroacetate-esterase).
hot spots composed of immature erythroid precursors (Giemsa). (e) (h) Rare CD34+ precursor/blast cells
CD71 immunoreactivity of erythroid cells. (f) Detection of rare mega-
12 Myelodysplastic Syndromes (MDS) 375

e f

CD71 CD61

g h

NASDCL
NAS
NA SD
DCL
C CD34
CD
CD34
D34
34

Fig. 12.14 (continued)

376 12 Myelodysplastic Syndromes (MDS)

Case History 13: Hypoplastic MDS with Multilineage myeloid cells were observed. Megakaryopoiesis was highly
Dysplasia and Fibrosis Grade 2 (Fig. 12.15) dysplastic with the presence of small hypolobated forms
A 62-year-old woman was diagnosed with severe pancyto- and some megakaryoblasts. CD34 immunostain revealed
penia. The CBC was as follows: WBC 0.8 × 109/L, Hb 5.1 g/ rare blast cells accounting for about 4% of nucleated cells.
dL, MCV 101 fL, platelets 18 × 109/L. The differential Furthermore, interstitial CD3+ lymphocytes were slightly
count showed 12% neutrophils, 1% eosinophils, 3% mono- increased. The karyotype was complex and included a sub-
cytes, and 84% lymphocytes. A bone marrow core biopsy clone carrying a del(5q). NGS provided evidence of a TP53
was markedly hypocellular and showed an edematous mar- mutation. The diagnosis was hypoplastic myelodysplastic
row with an increased reticulin fiber network consistent syndrome with multilineage dysplasia and fibrosis grade 2
with reticulin fibrosis grade 2. Hot spots with dysplastic ery- (hMDS-MLD-F) associated with an unfavorable prognostic
throid colonies and scattered predominantly immature score.

a b

H&E H&E

c d

H&E CD61

Fig. 12.15 Hypoplastic MDS with multilineage dysplasia and fibrosis CD61 immunostain. (e) Expression of CD71 by atypically localized
grade 2. (a–c) H&E stained section of the bone marrow core biopsy erythroid colonies. (f) Rare immature myeloid cells are positive for
showing hypocellular edematous marrow spaced containing a strikingly myeloperoxidase. (g) CD34 immunolabeling of rare myeloid blasts and
diminished hematopoiesis. Note the presence of a focally enlarged dys- some micromegakaryocytes. (h) Gomori stain consistent with fibrosis
plastic erythroid colony and of small megakaryocytes with hypolobated grade 2
nuclei in c. (d) Presence of micromegakaryocytes is highlighted by
12 Myelodysplastic Syndromes (MDS) 377

e f

CD71 MPO

g h

CD34 Gomori

Fig. 12.15 (continued)

378 12 Myelodysplastic Syndromes (MDS)

Case History 14: Myelodysplastic Syndrome Excess Blasts 2 showed markedly dysplastic megakaryocytes with com-
(MDS-EB-2) Progressing to Early AML with pletely separated nuclei, macroblastic erythroid precursors,
Myelodysplasia-Related Changes (AML MRC) (Fig. 12.16) rare myeloid precursors, and 22% blasts highlighted by
A 84-year-old man was diagnosed with pancytopenia. The CD34 immunolabeling. Cytologic examination of aspirate
CBC was as follows: WBC 1.2 × 109/L, Hb 5.2 g/dL, MCV smears revealed blasts with coarse myeloperoxidase+ gran-
98 fL, platelets 65 × 109/L. The peripheral blood smears ules and rare small Auer rods. The karyotype was complex.
showed 43% hypogranulated neutrophils, 11% monocytes, The diagnosis was myelodysplastic syndrome excess blasts 2
1% eosinophils, 1% basophils, and 44% lymphocytes. Bone transforming into early AML with myelodysplasia-related
marrow aspirate smears and trephine bone marrow biopsy changes (AML-MRC).

a b

H&E
H&E

c d

H
H&
H&E
&E H&E

Fig. 12.16 Myelodysplastic syndrome excess blasts 2 (MDS-EB-2) Enlarged macroblastic erythroid colony (hemoglobin). (f) Atypically
progressing to early AML with myelodysplasia-related changes (AML- localized clusters of CD34+ blast cells in the vicinity to CD34+ vascu-
MRC). (a–d) Age-related slightly hypercellular bone marrow showing lar endothelium. (g, h) Bone marrow aspirate smears showing blasts
enlarged macroblastic erythroid colonies close to atrophic trabecular containing myeloperoxidase+ granules and rare small Auer rods (MPO)
bone and small megakaryocytes with bilobated nuclei (H&E). (e)
12 Myelodysplastic Syndromes (MDS) 379

e f

HB
HB CD34

g h

M
MPPO
MPOO MPO
MPO

Fig. 12.16 (continued)

380 12 Myelodysplastic Syndromes (MDS)

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Acute Leukemia of Myeloid, Lymphoid,
and Ambiguous Lineage and Related 13
Malignancies

13.1 Introduction 13.1.1 D

iagnostic Approach to Patients
with Suspected AL
The descriptive term “Leukämie/leukemia” was intro-
duced by Rudolf Virchow who noticed the “white” color of A comprehensive approach including clinical and hemato-
blood samples obtained from patients presenting with high logical data, morphologic assessment of blood and bone
peripheral white blood cell counts and a reversed white marrow, immunophenotyping, cytogenetics, and molecular
and red blood cell balance [1]. The designation “acute leu- genetic testing is mandatory for the adequate diagnostic
kemia” (AL) is actually applied to a heterogeneous group work-up of AL. A detailed diagnostic evaluation should pro-
of immature clonal hematopoietic neoplasms occurring in vide important data required for adequate classification, risk
all age groups. The large majority of AL is derived from stratification, and decision for therapeutic strategies.
either the myeloid or the lymphoid lineage, allowing a Moreover, immunophenotyping by multiparameter flow
definite classification as acute myeloid leukemia (AML) or cytometry and molecular techniques detecting disease-
acute lymphoblastic leukemia (ALL). The rare category specific genetic lesions are useful to monitor therapy
“AL of ambiguous lineage” includes AL with no lineage- response, minimal residual disease, and relapse [6–8].
specific features and AL of mixed lymphoid/myeloid phe-
notype (MPAL) [2, 3].
The onset of AL may be insidious (de novo AL) or 13.2 cute Myeloid Leukemia; General
A
may be preceded by hematologic disorders such as Aspects
myelodysplastic syndromes (MDS) and/or an exposure
to radiation and/or toxic substances including chemo- 13.2.1 Classification
therapy. Moreover, there is now increasing evidence that
AL is not always an acquired malignancy but may The diagnosis of AML was traditionally based on the French-
develop in patients with germline mutations in genes American (FAB) classification. The FAB categories mainly
that are essential for normal hematopoiesis. Examples considered the percentage of blast cells and cytological and
are GATA factor mutations [4]. These mutations may ini- cytochemical features of the neoplastic population for the
tially present as bone marrow failure syndromes and diagnosis of AML [9]. With the availability of immunophe-
later progress to AL. Therefore, screening for a genetic notyping, cytogenetics, and molecular genetics, this approach
predisposition syndrome in newly diagnosed pediatric has significantly changed and is reflected by the WHO 2008
and adult patients with AML or MDS may have an and Revised 4th edition of the WHO classification of tumors
important impact on clinical care and management of the of hematopoietic and lymphoid tissue ([2, 3, 10]; see
patient and the family [5]. Table 13.1). The 2016 WHO classification defines a broad
The initial manifestation of AL is generally characterized category of AML with recurrent genetic abnormalities [11]
by peripheral blood and bone marrow involvement. However, that includes AML with balanced translocations/inversions
AL may also present with concurrent or even isolated extra- or cryptic translocations and AML with gene mutations.
medullary infiltrates. Tumor-forming tissue infiltrates with Other major categories are AML with myelodysplasia-
myeloid differentiation features are referred to as myeloid related changes [12], therapy-related myeloid neoplasms
sarcoma (MS). [13], AML, not otherwise specified (NOS; [14]), myeloid

C. Beham-Schmid, A. Schmitt-Graeff, Bone Marrow Biopsy Pathology,
Essentials of Diagnostic Pathology, https://doi.org/10.1007/978-3-662-60309-3_13
384 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Table 13.1 Important clinical and laboratory features and diagnostic Table 13.2 AML and related disorders according to the Revised 2016
procedures in patients with suspected AL WHO Classification (adopted from Arber et al. [2])
Important aspects Features, techniques AML with recurrent genetic abnormalities
Clinical presentation Symptoms: weakness, bleeding, AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1
infections, skin or mucosal infiltrates, AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);
hepatosplenomegaly, lymphadenopathy, CBFB-MYH11
age-related performance status Acute promyelocytic leukemia with PML-RARA
Patient history Medical history: antecedent cytopenias, AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A
recurrent infections, prior cytotoxic AML with t(6;9)(p23;q34.1); DEK-NUP214
therapy, exposure to toxins, irradiation, AML with inv(3)(q21.3;q26.2) or t(3;3)(q21.3;q26.2);
demographics GATA2,MECOM(EVI1)
Family history Hematologic disorders, AML (megakaryoblastic) with t(1;22)(p13.3;q13.1);
immunodeficiency states RBM15-MKL1
Peripheral blood Complete blood count including Provisional entity: AML with BCR-ABL1
differential count, cytologic evaluation
AML with gene mutations
of ≥200 white blood cells on blood
AML with mutated NPM1
smears, immunocytochemistry,
immunophenotyping, molecular AML with biallelic mutations of CEBPA
analyses (see below) Provisional entity: AML with mutated RUNX1
Bone marrow Aspirate: microscopic evaluation of AML with myelodysplasia-related changes
≥500 nucleated cells with special regard Therapy-related myeloid neoplasms
to blast cells on smears; trephine bone AML, NOS
marrow biopsy: overall cellularity, AML with minimal differentiation
hematopoietic compartment, atypical AML without maturation
cells, stroma including fibrosis AML with maturation
Additional techniques (see below) Acute myelomonocytic leukemia
Additional laboratory data Lactate dehydrogenase (LDH), Acute monoblastic/monocytic leukemia
coagulation tests, liver function tests Pure erythroid leukemia
Immunophenotype Cell surface and cytoplasmic markers Acute megakaryoblastic leukemia
by flow cytometry (liquid samples),
Acute basophilic leukemia
immunocytochemistry and/or
immunohistochemistry (smears, biopsy Acute panmyelosis with myelofibrosis
sections) using adequate antibody Myeloid sarcoma
panels Myeloid proliferations related to Down syndrome
Gross chromosomal Karyotyping (metaphase chromosomes Transient abnormal myelopoiesis
alterations from viable bone marrow or blood Myeloid leukemia associated with Down syndrome
cells), fluorescence or chromogen in situ Blastic plasmacytoid dendritic cell neoplasm
hybridization (interphase nuclei of bone Acute leukemias of ambiguous lineage
marrow aspirate or blood smears, Acute undifferentiated leukemia
trephine bone marrow biopsy sections) MPAL with t(9;22)(q34.1;q11.2); BCR-ABL1
Recurrent genetic Sequencing technologies including MPAL with t(v;11q23.3); KMT2A rearranged
mutations, epigenetics Sanger sequencing, next-generation MPAL, B/myeloid, NOS
targeted or whole genome sequencing,
MPAL, T/myeloid, NOS
epigenomics (liquid samples or
formalin-fixed paraffin-embedded In addition, various myeloid neoplasms with germline predisposition
material) with or without a preexisting disorder or organ dysfunction are listed in
the WHO classification [2, 16]

sarcoma [15], and myeloid proliferations associated with In AML patients, the genetic and mutational profiles that
Down syndrome [16]. are involved in disease pathogenesis are important for dis-
At initial presentation, AML is generally a bone ease classification and prognostic stratification [2, 17–19].
marrow-based neoplasm associated with the expansion of The European Leukemia Net (ELN) has defined four catego-
≥20% blast cells but may also involve extramedullary ries of AML risk based on six fusion genes, three genes with
sites. Exceptions are cases with low blast counts present- point mutations and cytogenetic abnormalities [20]. The
ing with AML-type recurrent cytogenetic abnormalities ELN recommendations have actually been refined [6]. In the
that are straightforwardly classified as AML (Table 13.2). future, individual patient outcome may be predicted from
AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1, t(15;17) genomic and clinical variables that may be important tools
(q22;q11-12); PML-RARA, and AML with inv(16) for precision oncology [21]. However, the phenotype of the
(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 are predominant blast cell population still remains pivotal for
considered as AML regardless of the percentage of blasts AML cases lacking specific molecular and/or cytogenetic
in the bone marrow. features. They are included in the “NOS” category of AML.
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 385

13.2.2 Epidemiology should be examined with special regard to the presence of

blast cells. The cytologic features of myeloblasts are variable
AML incidence rates in world populations are reported to (see case reports below). Myeloblasts may present as small
range from 3.0 to 4.0 cases per 100,000 person-years in adult cells with agranular cytoplasm, immature chromatin, and
populations with a male predominance of 1.25 [22]. The small nucleoli or as larger atypical cells with prominent
incidence of AML varies in different countries ranging from nucleoli and a broader rim of vacuolated or granular cyto-
0.9 per100,000 person-years in Algeria to 5.4 per 100,000 plasm. The presence of Auer rods that are frequently observed
person-years in Denmark. The significantly lower frequen- in classic acute promyleocytic leukemia (APL) but may also
cies reported for developing countries are generally attrib- occur in other entities is a hallmark of a myeloid differentia-
uted to the younger age of the population [22]. In Canada, tion of blast cells. Auer rods may rarely be observed in
the mean age of diagnosis is rising along with the AML inci- mature neutrophils especially in APL, in AML with t(8;21),
dence. The higher risk of AML in the elderly is also reflected or in AL of ambiguous lineage [31].
by the increase of AML by 73% in the UK in the last 40 years Monoblasts, promonocytes, and megakaryoblasts are
[22, 23]. The age at diagnosis of AML shows two peaks: the included in the blast count. In addition, non-blast cells of all
first small peak in early childhood and a second higher peak lineages should be carefully evaluated for quantitative and
in later adulthood [24]. AML is a rare childhood cancer with dysplastic features especially with regard to AML with
an occurrence of about seven cases per one million children myelodysplasia-related changes (AML-MRC).
annually [25] and accounts for about 25% of pediatric leuke- Myeloperoxidase (MPO) is the most important cyto-
mia [26]. The majority of AL occurring in the adult popula- chemical stain. A positive MPO reaction in at least 3% of
tion falls in the AML category. Cases of AML account for the blasts indicates a derivation from the myeloid lineage.
largest number of leukemia-related annual deaths in the However, blasts at early stages of myeloid differentiation
United States [27]. may be MPO negative [20]. Sudan black is also used as a
marker of myeloid differentiation but considered to be less
sensitive. Monoblasts may be negative for MPO but stain for
13.2.3 Morphology and Cytochemistry nonspecific esterase. Erythroblasts are not included in the
of Blood and Bone Marrow Smears blast cell count with the exception of pure erythroid leuke-
mia (PEL), an entity that does not harbor an increase in
Cytologic examination of peripheral blood and bone marrow myeloid blasts. In acute erythroid leukemia, periodic acid-
smears still remains helpful for the rapid classification of Schiff (PAS) may highlight positive vacuoles and globules in
AML subtypes (Table 13.3). Some entities even show an the cytoplasm of erythroblasts.
excellent phenotypic/genotypic correlation such as the core Iron stain allows the detection of sideroblasts, ring sidero-
binding factor leukemia (CBF) with inv16(p13;1q22) or blasts, and iron storage in bone marrow macrophages.
t(16;16)(p13.1;q22) and eosinophilia, AML with t(8;21)
(q22;q22.1); RUNX1/RUNX1T1 or AML carrying an NPM1
and FLT3 ITD mutation presenting with cup-like nuclei [20, 13.2.4 Immunophenotype
28–30].
May-Grünwald-Giemsa/Wright-Giemsa stained smears The immunophenotyping of leukemic cells by immunocyto-
prepared from peripheral blood and bone marrow aspirate chemistry/immunohistochemistry applied to cytological and/

Table 13.3 Cytochemical stains in AML

Cytochemistry Blast lineage determination Comments
a
Myeloperoxidase (MPO), Positive in blasts of granulocytic lineage AML without maturation and AMML ≥3% of blast MPO
Sudan Black B Highlight Auer rods positive
Weak or negative reaction in promonocytes. Strong positivity in APL with PML-RARA including the
Negative in monoblasts microgranular (hypogranular) variant
b
Naphthol AS-D Granulocytic precursors from the Strong positivity in hypergranular (typical) APL, negative in the
chloroacetate-esterase promyelocyte stage on. Atypical/immature microgranular (hypogranular) variant. Double positivity for CAE
(NASDCL or CAE) mast cells in mast cell leukemia and NSE or MPO in some dual-positive cells of AMML
a
Nonspecific esterase (NSE) Strong, weak, or absent reaction in Negativity does not exclude a monocytic differentiation of AML
monoblasts/promonocytes
Variable reactivity of megakaryoblasts
Evaluation of cytochemical stains rapidly allows blast delineation and enumeration but is not sufficient for diagnostic work-up of AML
a
MPO and NSE cytochemistry can be performed on cytologic blood and bone marrow smears
b
CAE works well on formalin-fixed paraffin-embedded trephine BM biopsies
386 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Table 13.4 Routine immunohistochemical stains on BM biopsies in Table 13.5 Routine genetic testing by FISH in AML
AML
FISH probes to detect chromosomal translocations, rearrangements,
Antigen Cell type Comments and deletions
Myeloperoxidase Myeloblasts±, Translocation t(15;17)/PML-RARA
(MPO) promyelocytes+, mature Translocation t(8;21)/RUNX1-RUNX1T1(AML1-ETO)
neutrophils+, monocytes/ Translocation t(6;9)/DEK-NUP214
promonocytes(±),monoblasts− CBFβ-Rearrangement/inv(16), t(16;16)
CD34 Myeloid precursors+, Low or absent KMT2a/MLL (11q23)-Rearrangement
myeloblasts+, endothelial expression in MECOM (3q26)-Rearrangement
cells+ APL
Deletion 5q31/5q33 (EGR1)/RPS14
CD117 Mast cells+,
Monosomy 7/Deletion 7q22, 7q31,7q36
myeloblasts±, monoblasts
(−/+) Deletion 17p13 (TP53)
CD33, CD13, CD15 Myeloblasts+, CD33: strong Deletion 20q12
promyelocytes+ expression in Analysis of 100 interphase nuclei is mandatory
AML with
mutated NPM1
HLA-DR Myeloblasts+, Low or absent can be applied to aspirate smears as well as to formalin-fixed
promyelocytes+, expression in paraffin embedded (FFPE) tissue sections. According to the
monoblasts+, APL 2017 ELN recommendations, FISH should be performed to
promonocytes+
detect gene rearrangements, such as RUNX1-RUNX1T1,
CD14, CD64, CD68 Monoblasts+,
CD11c, CD11c, promonocytes+ CBFB-MYH11, KMT2A (MLL), and MECOM (EVI1) gene
CD36, lysozyme fusions, or loss of chromosome 5q, 7q, or 17p material if con-
CD71, glycophorin CErythroblasts ventionally cytogenetic testing is not successful [6].
(pronormoblasts) Translocations/inversions are detected in ~50% and 30%
CD41, CD61, CD42b, Megakaryoblasts of AML arising in children and younger adults but are less
CD31
CD7 T-cells May aberrantly
frequent in older patients [18]. In about 45% adult AML
be expressed in cases, no cytogenetic abnormalities can be detected. These
AML cases are referred to as the category cytogenetically normal
(CN) AML. Rare children with normal karyotype by conven-
tional cytogenetic evaluation harbor socalled cryptic rear-
or histological specimens as well as by multiparameter flow rangements. In addition, some cases of PML-RARA+APL
cytometry of fluid samples is mandatory for the evaluation of lack the classic t(15;17).
blast cells and the delineation of cell lineages ([7], Table 13.4). In the actual WHO classification, eight balanced transloca-
Blast/precursor cells are highlighted by the expression of tions and inversions and their variants are listed [11].
markers such as CD34, CD117, CD33, HLA-DR. Note that Reciprocal translocations result in chimeric fusion genes
blasts of monocytic lineage often lack CD34 expression. involving for example RARA, RUNX1, the CBFβ subunits of
Myeloid lineage antigens are cytoplasmic MPO, CD13, the core binding factor complex, epigenetic regulators such as
CD33, CD15, and CD65. Monocytic lineage markers are KMT2A formerly designated as MLL, or components of the
CD14, CD36, CD64, frequently also CD4 and CD56. nuclear pore complex and other genes [18]. The detection of
Megakaryocytic markers are CD41, CD42b and CD61. certain recurrent genetic abnormalities in blood and/or mar-
Erythroid markers are CD235a (glycophorin A), CD36, CD71, row samples allows a diagnosis of AML without regard to the
and often weak CD117 [6, 32]. Flow cytometry is also helpful blast cell count. The AML-defining recurrent cytogenetic
for the evaluation of minimal residual disease in AML [33]. abnormalities actually include t(8;21)(q22;q22.1); RUNX1-
RUNX1T1; inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-
MYH11; t(15; 17)(q24.1;q21.2); PML-RARA or variant
13.2.5 Genetics translocations and t(1;22)(p13.3;q13.3); RBM15-MKL1.
The detection of nine balanced rearrangements and multiple
In 1973, cytogenetic studies performed by Janet Rowley unbalanced abnormalities allows the classification of a myeloid
revealed that subgroups of AML are associated with the bal- neoplasm with ≥20% blood or marrow blasts as “AML with
anced chromosomal rearrangements t[8;21] or t[15;17] first myelodysplasia-related changes (AML-MRC)” [6].
indicating that AML is a genetic disease [34–36]. Based on cytogenetic findings, a risk stratification has been
Karyotype analyses can be performed either by conven- established including favorable groups [t(8;21)(q22;q22.1);
tional cytogenetic methods applied to metaphase chromosomes inv(16)(p13.1q22) or t(16;16)(p13.1;q22); t(15; 17)
or by molecular cytogenetic methods performed on non-divid- (q24.1;q21.2)], intermediate groups [normal karyotype, all
ing cells (Table 13.5). A widely used method is the fluorescence prognostically not favorable or adverse types], and adverse
in situ hybridization (FISH) technique using a large panel of groups [numerous karyotypes including inv(3)/t(3;3); mono-
commercially available fluorescently labeled DNA probes that somal karyotype (loss of ≥2 unrelated abnormalities); complex
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 387

karyotype (≥3 unrelated abnormalities) and many other cyto- Table 13.6 Some important cytogenetic/molecular genetic alterations
genetic aberrations]. The cytogenetic risk stratification is vari- routinely tested in AML
able according to the age of patients. Genetic alteration Cytogenetics Relevance for outcome
Multiple techniques including Sanger sequencing of can- PML/RARA t(15; 17); Cryptic and Early complications due
variant translocations to coagulopathy, good
didate genes and high-throughput sequencing techniques may occur response to all-trans
have contributed to the understanding of the complex retinoic acid and arsenic
genomic alterations in AML with normal cytogenetics and trioxide
those with chromosomal losses or gains that were previously RUNX1/ t(8;21) Favorable, but adverse
RUNX1T1 effect of concomitant
poorly understood [18].
KIT mutation
A model of AML leukemogenesis has proposed two CBFB/MYH11 inv(16) or t(16;16) Favorable
classes of cooperating mutations conferring a proliferative MECOM (EVI1) inv3, t(3;3) but also Unfavorable
advantage (class I mutations) and inducing a block in overexpression with various normal
myeloid differentiation (class II mutations) has been pro- and abnormal
karyotypes
posed [37]. Class I comprises mutations leading to activation
BCR-ABL1 t(9;22) Rare in de novo AML,
of the receptor tyrosine kinase FLT3 or the RAS signaling poor response to
pathway and includes also JAK2 and SHP2. Class II com- tyrosine kinase inhibitor
prises mutations in CEBPA, MLL, NPM1, and gene fusions (TKI) therapy
resulting from t(8;21), inv(16)/t(16;16), or t(15;17). KIT t(8;21) Unfavorable in patients
inv(16)/t(16;16) with concomitant t(8;21)
In addition to somatically acquired driver mutations, sub- FLT3-ITD Normal karyotype Unfavorable, adverse
clones with differing mutational composition often develop outcome concomitant
during disease evolution. Whole-genome sequencing studies with NPM1
have provided evidence that AML is not a stable but a com- FLT3-TKD Various karyotypes
plex and dynamic disease with multiple competing clones (tyrosine kinase
domain)
[17, 38]. Mutations in genes encoding epigenetic modifiers, NPM1 Normal karyotype Favorable in FLT3-ITD
such as DNMT3A, ASXL1, TET2, IDH1, and IDH2, are com- wild-type
monly acquired early and are present in the founding clone. NPM1 and Normal karyotype
Mutations involving NPM1 or signaling molecules (e.g., FLT3-ITD
FLT3, RAS) are typically secondary events that occur later CEBPA Various karyotypes Biallelic mutation of
CEBPA: WHO entity
during leukemogenesis [38]. with longer disease-free
Genetic profiling including cytogenetic analyses and and favorable overall
sequencing of 111 genes resulted in a genomic and prognostic survival
classification of AML [17]. In the large AML cohort analyzed RUNX1 Normal or abnormal Unfavorable
karyotypes
in this study, NPM1-mutated AML was the largest class
DNMT3A Normal karyotype Unfavorable
(accounting for 27% of the cohort), with 73% of patients also IDH1/2 Predominantly normal Unfavorable, dependent
carrying mutations in DNA methylation or hydroxymethyl- karyotypes on mutation type
ation genes (DNMT3A, IDH1, IDH2R140, and TET2). In the ASXL1 Normal and abnormal Unfavorable
chromatin–spliceosome group (18% of the cohort), mutations karyotypes
in genes regulating RNA splicing (SRSF2, SF3B1, U2AF1, TET2 Normal and abnormal Dependent on mutation
karyotypes type and cytogenetics
and ZRSR2), chromatin remodelling (ASXL1, STAG2, BCOR, WT1 Normal and abnormal Unfavorable
KMT2A-PTD, EZH2, and PHF6), or transcription (RUNX1) karyotypes
were found. A third subgroup harbored mutations in TP53, TP53 Complex karyotypes, Unfavorable
complex karyotype alterations, cytogenetically visible copy- abnormalities of
chromosomes 5, 7, or 17
number alterations (aneuploidies), or a combination. A fourth
small subgroup (about 1%) carried IDH2R172 mutations [17].
Conventional karyotyping, FISH, and molecular testing classification includes AML with mutated NPM1 and AML
of large cohorts of AML patients have provided important with biallelic mutations of CEBPA as distinct entities that
insights into the pathogenesis, genetic complexity, and het- have in the absence of FLT3-ITD, a relatively favorable
erogeneous prognosis of the different AML types [6, 39]. prognosis [37]. CEBPA mutation may be inherited through
According to the 2017 ELN recommendations, screening for the germline, with the development of AML associated with
gene rearrangements such as PML-RARA, CBFB-MYH11, acquisition of additional mutations [18].
RUNX1-RUNX1T1, BCR-ABL1, and other fusion genes (if Taken together, genetic testing of all AML cases is man-
available) should be performed [40, 41]. Current interna- datory for risk stratification and decision-making whether
tional guidelines recommend molecular testing for a limited individual patients are eligible for conventional therapeutic
number of mutations including NPM1, CEBPA, RUNX1, regimens, targeted therapies, and/or candidates for alloge-
FLT3, TP53, and ASXL1 ([6], Table 13.6). The actual WHO neic hematopoietic cell transplantation.
388 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.3 ML with Recurrent Genetic

A promyelocytes to mature neutrophilic granulocytes.
Abnormalities Actually, the vast majority of APL patients treated with
ATRA in combination with arsenic trioxide (ATO) with or
13.3.1 A
cute Promyelocytic Leukemia without additional chemotherapy achieves complete remis-
with PML-RARA sion or is even cured [44]. International studies have pro-
vided evidence that molecular remission (defined as
Acute promyelocytic leukemia (APL) with PML-RARA is a negativization of the PCR test for PML/RARA) correlates
distinct subtype of the WHO category AML with recurrent with a significantly decreased risk of relapse and should be
genetic abnormalities that has previously been designated the therapeutic objective in APL [45].
as AML M3 according to the FAB classification [11]. APL In about 5% of PML-RARA+APL the classic t(15;17) is
occurs in both children and adults and accounts for up to not present but more complex RARA insertions or transloca-
12% of AML cases. A rapid diagnosis is mandatory since tions may occur such as t(11;17)(q23;q21.1) ZBTB16-
the risk of life-threatening hemorrhage and also of abnor- RARA. In some APL cases RARA translocations with NUMA1
mal coagulation is high due to the activation of fibrinolytic at 11q13.4, NPM1 at 5q35.1, or STAT5B at 17q11.2 are
and coagulation pathways by granule release from neoplas- reported. These patients may require different treatment
tic cells [11, 42]. Patients may initially present with dis- strategies.
seminated intravascular coagulation [42]. The platelet and Current methods to detect t(15;17) are conventional
WBC counts are often low, so that a detection of neoplastic karyotyping or FISH analyses. Cryptic t(15;17) transloca-
cells on peripheral blood cells may be difficult. However, tions that are present in less than 10% of patients are not
some cases especially variant forms may have high WBC detected by karyotyping or FISH but by RT-PCR or
counts. NGS. These techniques detect classical and rare RARA
The APL category includes several types of variant forms. translocations.
The term APL variant is used both for morphologic subtypes Primary or relapse APL can harbor additional mutations
such as the microgranular or hyperbasophilic APL or for in Fms-like tyrosine kinase 3 (FLT3), especially FLT3 inter-
molecular subtypes with variant RARA translocations. nal tandem duplication (FLT3-ITD), Wilms tumor 1 (WT1),
or N-RAS, but rarely in other genes. FLT3-ITD mutation that
13.3.1.1 Genetics is predominantly present in microgranular APL has appar-
In classical APL cases, a balanced translocation between ently no negative effect on long-term survival especially in
the long arms of chromosomes 15 and 17 results in the cases receiving ATRA and ATO.
PML-RARA fusion gene. The classical chromosomal break-
points are t(15; 17)(q24.1;q21.2). There are three break- 13.3.1.2 Immunophenotype
point regions on PML at 15q24.1. The bcr1 and bcr2 Classical APL cases are characterized by a myeloperoxi-
breakpoints are associated with in long transcripts and pres- dase+ CD33+ CD13+ myeloid phenotype in the absence of
ent in classical APL. The microgranular APL is generally HLA-DR, CD117, CD15, CD11b, and CD34 expression.
associated with a breakpoint at bcr3 that leads to a short CD2 and/or CD56 may be co-expressed. Microgranular APL
transcript. is more often CD2+ and may show weak or partial expres-
The abnormal PML-RARA transcription factor disrupts sion of HLA-DR and CD34. A moderate to high side-scatter
normal myeloid differentiation programs [43]. RARA binds appearance upon flow cytometric evaluation, depending
to retinoic acid responsive elements in a heterodimeric com- upon the degree of granularity of the leukemic cells, may be
plex with retinoid-X receptors (RXRA). In normal myeloid present.
cells, the effect of this complex is regulated by retinoic acid,
thus allowing gene transcription and cell differentiation. In 13.3.1.3 Morphology
APL, RARA-RXRA represses gene transcription. Targeted The classical APL is characterized by strong myeloperoxi-
treatment with all-trans retinoic acid (ATRA) achieves dase+ heavily granulated atypical promyelocytes. Auer rods
return of normal gene transcription and differentiation of of various shapes may be seen in both cells with granulated
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 389

and agranulated cytoplasm. Promyelocytes containing bun- immunophenotype (CD15− CD34− HLA-DR−) but are
dles of Auer rods are referred to as Fagot cells. negative for CD2.
On peripheral blood smears, the microgranular APL sub- • Atypical promyelocytes as well as the PML-RARA
type is characterized by bilobed nuclei surrounded by a baso- fusion transcript may persist for some weeks after suc-
philic cytoplasm devoid of distinct coarse granules. These cessful induction treatment without indicating treatment
cells are negative for naphthol-ASD-chloroacetate-esterase failure.
stain but strongly positive for myeloperoxidase cytochemical • Rapid diagnosis and treatment of APL is mandatory to
stains. The bone marrow, however, contains at least some prevent fatal hemorrhagic and/or thrombotic complica-
granulated cells and/or cells with Auer rods. In the hyperba- tions that may result from activation of both coagulation
sophilic APL variant, dark coarse granules are observed in a and fibrinolytic pathways by granule release from
basophilic cytoplasm. promyelocytes.
Other APL cases may show non-pathognomonic blasts on • APL should be suspected in cases with very low WBC
peripheral blood smears while neoplastic promyelocytes are counts and marked thrombocytopenia [46].
present in the bone marrow. • High leukocyte counts are often present in the hypogranu-
lar APL variant that, however, is also strongly
13.3.1.4 Caution myeloperoxidase-positive.
• APL represents a diagnostic emergency since coagulopa- • APL entity includes numerous morphologic, immuno-
thy and bleeding, particularly within the brain and lungs, phenotypic, and molecular variant forms.
are the most common causes of early death. • Differential diagnosis includes promyelocytic maturation
• Some cases of acute myeloid leukemia (AML) with inhibition by certain drugs, growth factor therapy, and
nucleophosmin (NPM) mutations have an APL-like AML with t(8;21) with densely granulated blasts.
390 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 1: APL dase, CD33, and CD45 (low). CD34 and HLADR were not
A 48-year-old woman was referred for the evaluation of expressed.
pancytopenia and skin hemorrhage. No significant past FISH studies provided evidence of a translocation
medical history was reported. Fibrinogen was decreased. t(15;17)/PML-RARA in >90% of cells and a trisomy 8 in a sub-
The CBC was as follows: Hb 9.5 g/dL, RBC 2.92 × 1012/L, clone. Molecular tests positive for a PML-RARA fusion transcript
platelets 18 × 109/L, WBC 1.2 × 109/L. The differential (isoform BCR1) confirmed the diagnosis of acute promyelocytic
count showed 48% atypical promyelocytes and blasts, 9% leukemia. The bone marrow core biopsy was infiltrated by atypi-
neutrophils, 43% lymphocytes. The bone marrow aspirate cal promyelocytes positive for myeloperoxidase and CD33.
contained 91% promyelocytes and blast cells with coarse Positivity for naphthol-ASD-chloroacetate-esterase was weak in
granules and Auer rods. Some cells were packed with abun- the neoplastic population but strongly positive in the cytoplasm
dant Auer rods (faggots) (Fig. 13.1). The neoplastic cells of macrophages that engulfed cellular debris. Erythropoiesis and
were positive for CD117 and co-expressed myeloperoxi- megakaryocytes were strikingly diminished (Fig. 13.1).

RUNX1-RUNX1T1 The neoplastic population is positive for myeloperoxi-
dase, CD34, CD13, CD33, and often CD56. Some blast
AML with t(8;21)(q22;q22.1) RUNX1-RUNX1T1 is a sub- cells may be stained by the naphthol-ASD-chloroacetate-
type of AML with core binding factor mutations and esterase cytochemical stain. B-lineage-associated anti-
accounts for about 8–15% of AML but is more rarely gens such as CD19 (weak), CD79a, and PAX5 may be
observed in the elderly population [11, 53]. This AML sub- detected [58]. CD117 is strongly expressed especially in
type has previously been included in the AML FAB M2 those cases carrying an additional KIT mutation. In cases
category. AML t(8;21)(q22;q22.1) is commonly associated lacking CD13 or CD33, other myeloid markers are
with a favorable prognosis that can however be changed as expressed.
a consequence of additional genetic alterations (see below).
13.3.3.3 Morphology
13.3.3.1 Genetics A typical feature of peripheral blood and bone marrow
RUNX1 is a member of the core-binding factor family of specimens is the presence of distinct myeloid blasts con-
transcription factors and acts as a key hematopoietic tran- taining pink granules, suggesting a tendency to matura-
scription factor. The translocation t(8;21) results in the fusion tion into the promyelocyte stage. These granules have
between the RUNX1 gene on chromosome 21 and the also been designated as “salmon-colored.” In addition,
RUNX1T1 locus on chromosome 8 and places RUNX1T1 Auer rods, cytoplasmic globules, and Chediak-Higashi-
(also designated as ETO) under control of the RUNX1 pro- like granules may be observed. Blast cells may show peri-
moter, resulting in high upregulation of the RUNX1T1 tran- nuclear clearing or “hof.” Eosinophils lacking the
scripts in t(8;21) AML. There is evidence this translocation abnormal granules of AML with inv(16) or t(16;16) may
leads to a deregulation of the myeloid gene regulatory pro- be increased.
gram, suppresses endogenous DNA repair in myeloid cells,
and favors mutagenesis. More than 50% of t(8;21) AML 13.3.3.4 Caution
patients have been shown to have a mutation in KIT, FLT3, • The differential diagnosis includes APL that lack however
N-RAS, or K-RAS, indicating a large genetic heterogeneity. expression of CD34 and B-lineage antigens.
Activating mutations in codon D816 of the KIT genes that • Other differential diagnosis are growth factor-induced
are present in about 20–25% of cases have an unfavorable changes and drug-associated maturation arrest of the
prognostic impact in t(8;21)-positive AML [54, 55]. myeloid series. In both conditions, no phenotypic abnor-
Moreover, frequent ASXL2 mutations in AML with malities are present. The promyelocytes are devoid of
t(8;21)/RUNX1-RUNX1T1 translocations have also been abnormal granules and do not express CD19, CD79a, or
reported to be associated with a worse prognosis [56, 57]. PAX5.
398 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 1 (Fig. 13.4) pink granules and a perinuclear clearing (“hof”). Blast cells
A 45-year-old male patient was admitted with an outside expressed myeloperoxidase, CD13, HLA-DR, CD117, and
diagnosis of AML. The CBC showed marked leukocytosis partially CD15 and PAX5. Cytogenetic studies showed t(8;21)
(WBC 30 × 109/L), anemia (Hb 7.5 g/dL), and thrombocyto- (q22;q22.1) RUNX1-RUNX1T1. Molecular analyses provided
penia (47 × 109/L). Differential count: 87% blasts, 2% neutro- evidence of additional KIT D816V and ASXL2 mutations.
phils, 11% lymphocytes. The basophilic cytoplasm of The diagnosis was AML with t(8;21)(q22;q22.1)
peripheral blood and bone marrow blasts frequently contained RUNX1-RUNX1T1.

a b

MGG
MG MGG

c d

MGG
MG MGG
MGG
MG
e f

Giemsa Giemsa

Fig. 13.4 AML with t(8;21)(q22;q22.1) RUNX1-RUNX1T1. (a, MGG) granules (d). (e–h) Bone marrow spaces were infiltrated by blasts with
Peripheral blood and (b–d, MGG) bone marrow smears showing blasts basophilic cytoplasm (e, f, Giemsa) that were positive for (g) naphthol-
with perinuclear clearing. The cytoplasm of some blasts contains pink ASD-chloroacetate-esterase and (h) expressed CD117
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 399

g
h

NASDCL CD117

Fig. 13.4 (continued)

400 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 2: AML with t(8;21)(q22;21q22) RUNX1- immunohistochemistry performed on the bone marrow core
RUNX1T1 and FLT3-ITD Mutation (Fig. 13.15) biopsy. The neoplastic population was negative for CD14,
A 62-year-old woman presented with anemia and thrombo- CD64, CD3, CD19, CD20, CD22, and CD79a. Despite the
cytopenia, suggesting a myelodysplastic syndrome (MDS). aberrant expression of CD7 and PAX5, the criteria for
The CBC was: WBC 2.8 × 109/L, Hb 8.68 g/dL, thrombo- ambiguous lineage acute leukemia specially of mixed pheno-
cytes 140 × 109/L. The blood smears showed 15% blasts type acute leukemia (MPAL) were not fulfilled since neither
while the bone marrow aspirate smear and the core biopsy CD3 nor CD19, CD79a, or CD22 was expressed.
contained about 90% blasts. The majority of blasts expressed Genetic studies revealed t(8;21)(q22;q22.1) RUNX1-
CD34, TdT, HLA-DR, CD13, CD33, and CD7 while small RUNX1T1 and cooperative t(8;21)(q22;q22.1) RUNX1-
subpopulations were positive for naphthol-ASD-RUNX1T1 and FLT3-ITD mutation. The diagnosis was AML
chloroacetate-esterase and myeloperoxidase. A strong aber- with t(8;21)(q22;21q22) RUNX1-RUNX1T1 and a coopera-
rant co-expression of PAX5 and CD7 was detected by tive FLT3-ITD mutation (Fig. 13.5).

a b

Gie
Giemms
sa
a Giemsa
c d

Giiiem
G em
e ms
sa
a NASDCL
N
NA
ASDC
SD
DC
CL
L

Fig. 13.5 AML with t(8;21)(q22;q22.1) RUNX1-RUNX1T1 and FLT3- Giemsa). A small subpopulation of blasts was positive for NASDCL (d)
ITD mutation. The bone marrow biopsy was infiltrated by medium- and myeloperoxidase (e). The large majority expressed CD34 (f), TdT
sized blast cells admixed with prominent eosinophil precursors (a–c, (g), and PAX5 (h)
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 401

e f

MPO CD34
g h

TdT PAX5

Fig. 13.5 (continued)

402 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.3.4 A
cute Myeloid Leukemia with inv(16) ers of blasts with high CD34 and CD117 may be expressed.
(p13.1q22) or t(16;16)(p13.1q22); An aberrant CD2 expression may be detected in some cases.
CBFB-MYH11
13.3.4.4 Genetics
Acute myeloid leukemias carrying inv(16)(p13.1q22) or In patients with this AML subtype, inv(16)(p13.1q22) is
t(16;16)(p13.1q22) chromosomal abnormalities belong to much more frequently detected than t(16;16) (p13.1q22).
the core binding factor (CBF)-AMLs. They are morpho- Both inversion (16)(p13.1q22) and translocation t(16;16)
logically related to the myelomonocytic subtype with (p13.1q22) fuse the beta subunit of core binding factor
abnormal eosinophils (AML-M4eo) of the French- (CBFB) gene located in 16q22 to the MYH11 gene located in
American-British classification [11]. The eosinophil com- 16p13, resulting in a chimeric protein product. The CBFB-
ponent also harbors the specific inv(16)(p13q22) MYH11 fusion protein blocks the differentiation process of
rearrangement and thus derive from the leukemic clone leukemic cells. CBFs are transcriptional regulators contain-
[28]. High rates of complete remission and favorable out- ing a common CBFβ (CBFB) subunit associated with one of
come in comparison to other AML subtypes can be achieved the three CBFα (CBFA) members. The MYH11 codes for a
by appropriate treatment strategies [59, 60]. smooth muscle heavy chain. The genetic alterations may be
missed by conventional karyotyping but are detected by
13.3.4.1 Epidemiology FISH and RT-PCR analyses. Additional genetic alterations
AML with inv(16)(p13.1q22) or t(16;16)(p13.1q22) is more are frequent and include gains of chromosome 22 and 8 or
frequent in children and young adults than in older patients. deletion (7q). Furthermore, mutations may be present, such
This AML subtype represents up to 10% in younger AML as KIT, NRAS, KRAS, or FLT3 genes [11, 60]. Various
patients but only up to 3% in older adult patients. genetic alterations have an impact on the prognosis. For
example, the coexistence of BCR-ABL1 and CBFB rear-
13.3.4.2 Morphology rangement is associated with poor outcome and a clinical
The characteristic morphologic feature of this AML subtype course similar to that of CML-BP [61]. Myeloid neoplasms
is the presence of blasts with monocytic and myelomono- with isolated del(16q) with the deletion of CBFB but lacking
cytic features both in the peripheral blood and the bone mar- CBFB-MYH11 rearrangement occur in older patients and are
row. In addition, the bone marrow of most, but not all cases associated with a shorter overall survival than AML with
shows abnormal eosinophils with coarse basophilic granules inv(16). Therefore, these cases should not be considered a
that may also be positive for naphthol-ASD-chloroacetate- variant of the AML-defining inv(16) [62].
esterase reaction. The abnormal granulation of eosinophils is
evident on bone marrow aspirate smears but usually cannot 13.3.4.5 Caution
be observed in section cut from paraffin-embedded biopsies. • The typical coarse basophilic granules of eosinophils may
By contrast, rare abnormal eosinophils may already be be missed in sections of paraffin-embedded bone marrow
detected in some cases with a low percentage of blasts <20% core biopsies.
that already carry a chromosome 16 abnormality and should • Eosinophils with abnormal basophilic granules are not
be diagnosed as overt AML (see case history). present in all cases of AML with inv(16)(p13.1q22) or
t(16;16)(p13.1q22).
13.3.4.3 Immunophenotype • Eosinophils may be increased in other AML subtypes,
Myeloid antigens (CD13, CD33, MPO), monocyte- for example, AML with t(8;21)(q22;q22.1) RUNX1-
associated markers (CD4, CD11b, CD14, CD64), and mark- RUNX1T1.
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 403

Case History 1: AML with inv(16)(p13.1q22) (CBFB/ However, molecular studies revealed a CBFB/MYH11
MYH11) (Figs. 13.6 and 13.7) translocation. Therefore, the diagnosis was revised to AML
A 29-year-old male was referred with pancytopenia and the with inv(16) despite the low percent of blasts. The patient
suspicion of a myelodysplastic syndrome. The CBC was: refused AML-type therapy. Five months later, peripheral leu-
WBC 2.5 × 109/L, Hb 9.1 g/dL, platelets 89 × 109/L. Differential kocytosis with monocytosis and the presence of 24% blasts
count: 8% monocytes, 11% neutrophils including some and promonocytes developed. The bone marrow aspirate
pseudo-Pelger forms, 1% blast, 3% promonocytes, 4% smears and core biopsy contained blasts and precursors of
eosinophils, and 67% lymphocytes. The bone marrow aspi- myelopoiesis and monocytopoiesis. Numerous abnormal
rate smear revealed a hypocellular marrow with 16% blasts eosinophils, some of which contained abnormal purple-vio-
and scattered atypical hypergranulated eosinophils let granules, were observed (Fig. 13.7). The bone marrow
(Fig. 13.6). By immunophenotyping, 12% CD117-positive biopsy was moderately hypercellular and showed an increase
cells were present that co-expressed (>90%) CD13, CD38, in eosinophils, but the abnormal granulation was only evi-
CD45 lo, and MPO and were partially positive for CD34 dent on the aspirate smears. The neoplastic population was
(35%), HLA-DR (35%), and CD33 (25%). The core biopsy positive for myeloperoxidase and partially expressed CD14,
was hypocellular and showed reduction of all three hemato- CD68, and CD34. Karyotyping, FISH, and RT-PCR analysis
poietic lineages with mild erythropoietic dysplasia. confirmed a CBFβ-rearrangement / inv(16)(p13.1q22) con-
Maturation of granulopoiesis was rarely observed. Blast sistent with the diagnosis of AML with inv(16)(p13.1q22).
cells accounted for about 15–16% of nucleated cells In addition, NGS using a myeloid panel revealed KRAS- and
(Fig. 13.6). The diagnosis was MDS-excess blasts 2. TET2 mutations.

a b

MG
M GG MGG
c d

MGG
MGG
MG MGG
MGG
MG

Fig. 13.6 AML with inv(16)(p13.1q22) and low blast count, case 1: precursors with coarse granules are appreciated (a–d, MGG). (e–h) The
(a) Eosinophils with normal granulation and (b) some monocytes with bone marrow biopsy is hypocellular and shows a relative predominance
abnormal nuclear lobation were detected in the peripheral blood smears. of erythropoietic cells, diminished granulopoietic maturation, and rare
(c, d) Hypocellular bone marrow aspirate smears with (c) rare blasts dysplastic megakaryocytes (e, f, Giemsa; g, h, NASDCL)
and groups of erythropoietic cells. (d) Scattered atypical eosinophil
404 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

Giemsa Giemsa

g h

N
NA
AS
SDDCL
C
NASDCL NASDCL
N
NA
AS
ASD
SDDCL
CL

Fig. 13.6 (continued)

a b

MGG MGG

Fig. 13.7 AML with inv(16)(p13.1q22), case 1: (a, b) Sequential bone stained sections. (d) Eosinophils show an aberrant Naphthol-ASD-
marrow smear contains among immature myelomonocytic precursors chloroacetate-esterase positivity. The neoplastic myelomonocytic cells
and blasts numerous abnormal eosinophils with pleomorphic coarse express myeloperoxidase (e). Subpopulations express CD34 (f), CD14
often purple-violet granules (MGG). (c–h) Bone marrow core biopsy. (g), and CD68 (h)
(c) The abnormal granulation of eosinophils is not evident on Giemsa
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 405

c d

Giemsa NASDCL

e f

MPO CD34

g h

CD14
CD1
CD 14
4 CD68
C
CD
D68
68

Fig. 13.7 (continued)

406 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 2: AML with CBFB/MYH11 inv(16) to a diffuse infiltration by blasts and immature myelomono-
(p13.1q22) CBFB/MYH11 (Fig. 13.8) cytic cells (Fig. 13.8). Eosinophils were increased; however,
A 43-year-old woman presented to the hospital with shortness no abnormal purple granules were observed on the sections. A
of breath, weakness, and enlarged cervical lymph nodes. CBC bone marrow aspirate was not available due to a dry tap result-
at presentation: WBC 58 × 109/L, Hb 8.6 g/dL, platelets ing from packed marrow. The karyotype was 46,XX,inv(16)
39 × 109/L. Differential count: 46% monocytes, 10% neutro- (p13.1 q22) [11]. FISH showed a CBFβ-rearrangement. A
phils, 24% promonocytes and monoblasts, 1% eosinophils, CBFB-MYH11 fusion transcript was detected by
and 19% lymphocytes (Fig. 13.8). The blasts were positive RT-PCR. Further molecular analysis using a next-generation
for sCD11c, sCD14, sCD64, sCD11b, sCD33, sCD13, myeloid panel revealed an additional NRAS mutation.
sCD15, and HLA-DR. The core biopsy was hypercellular due The diagnosis was AML with inv(16)(p13.1q22).

a b

MG
MG
GG
G MPO

c d

Giemsa Giem
Giemsa
sa
Giemsa

Fig. 13.8 AML with inv(16)(p13.1q22), case 2: (a, b) Blood smears blastic and monoblastic features. Eosinophil precursors are present (d);
with (a, MGG) atypical monocytes, granulated myelomonocytic cells, however, abnormal granulation is not prominent on the sections (c, d,
and (b) myeloperoxidase-positive blasts. (c–h) The bone marrow core Giemsa). The neoplastic population expresses (e) myeloperoxidase, (f)
biopsy is hypercellular and diffusely infiltrated by blasts with myelo- CD33, (g) partially CD68, and (h) strongly lysozyme
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 407

e f

MPO CD33

g h

CD68
CD
CD68
68 Lysozyme

Fig. 13.8 (continued)

408 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.3.5 A
cute Myeloid Leukemia with Translocation cation [63, 66]. Basophilia and multilineage dysplasia
t(6;9)(p23;q34.1); DEK-NUP214 especially of the megakaryocytic lineage may be seen.

Myeloid neoplasms with translocation t(6;9)/DEK-NUP214 13.3.5.3 Immunophenotype

are rare and may present as MDS or AML in children and Blasts express myeloperoxidase, CD33, CD123, HLA-DR,
adults. AML harboring this translocation that is listed as a and generally CD34 and CD117 [11].
distinct entity in the 2016 WHO classification is associated
with a dismal prognosis [11]. Patients relapse with a very 13.3.5.4 Genetics
high frequency [63, 64]. The t(6;9) fuses the 5′ part of the DEK gene at 6p22.3 and the
3′ part of the NUP214 gene, resulting in the chimeric DEK-
13.3.5.1 Epidemiology NUP214 gene. The DEK-NUP214 protein has leukemogenic
This neoplasm is diagnosed in both pediatric and adult potential in a small subpopulation of hematopoietic stem cells.
population and account for about 0.7–1.8% of all AML In a minority of patients, additional genetic alterations mainly
cases. n the pediatric population AML with translocation gains of chromosomes 8 and 13 are present. FLT3-ITD muta-
t(6;9)/DEK-NUP214 represents <1% of all childhood tions are much more frequent than in other AML subtypes and
AML [65]. have been reported in up to 78% of adult patients [11]. In a
multicenter study of pediatric cases, FLT3-ITD mutations
13.3.5.2 Morphology were reported in 42% [65]. This subtype of leukemia has a
Myeloid blasts of this AML subtype have no characteristic poor outcome in children regardless of FLT3-ITD status [67].
features. They may contain granules and Auer rods reminis- However, adult FLT3-ITD-positive patients relapse signifi-
cent of the M2 type of the French-American-British classifi- cantly faster than FLT3-ITD-negative ones [64].
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 409

Case History: AML t(6;9);DEK-NUP214 (Fig. 13.9) marrow biopsy was infiltrated by myeloid blasts (about 60%)
A 72-year-old male patient was admitted with leukocytosis and showed increased highly dysplastic megakaryocytes,
(WBC 20 × 109/L), anemia (Hb 7.9 g/dL), and thrombocyto- dyserythropoiesis, and some residual granulocytic precur-
sis (534 × 109/L). Peripheral blood contained about 80% sors. Karyotyping revealed t(6;9)(p23;q34.1).
blasts that were positive for CD13, CD33, CD38, HLA-DR, The diagnosis was AML with translocation
CD117, and partially myeloperoxidase (55%). The bone t(6;9);DEK-NUP214.

a b

HE
HE HE

c d

HE NASDCL

Fig. 13.9 Acute myeloid leukemia with translocation t(6;9);DEK- present. Blast represented about 60% of nucleated cells and expressed
NUP214. (a–c, H&E) The bone marrow core biopsy was hypercellular (e) myeloperoxidase and (f) partially CD68. (g) CD71 immunolabeling
and showed prominent megakaryocytic dysplasia with numerous highlighted the presence of dysplastic erythropoietic precursors. (h)
micromegakaryocytes and megakaryocytes with mono- or bilobated CD34+ blast were rare
nuclei. (d) Some NASDCL+ granulocytes and myeloid precursors were
410 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

MPO CD68

g h

CD71 CD34

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 419

13.3.8 AML with Translocation reminiscent of CML such as small megakaryocytes and/or
t(9;22);BCR-ABL1 pseudo-Gaucher cells.

In the revised 2016 WHO classification, de novo BCR-ABL1- 13.3.8.3 Immunophenotype

positive AML is listed as a provisional entity that is distinct The neoplastic population expresses various myeloid mark-
from chronic myeloid leukemia (CML) myeloid blast phase ers such as CD13 and CD33 and may be positive for MPO
(BP) [11]. In addition, mixed-phenotype acute leukemia and/or CD34. In some cases, co-expression of TdT, CD19, or
(MPAL), therapy-related myeloid neoplasms, and AML with CD7 has been reported. However, BCR-ABL1+ cases fulfill-
other recurrent genetic abnormalities do not fit into this ing criteria of MPAL are not included in this category [66].
category.
13.3.8.4 Genetics
13.3.8.1 Epidemiology and Clinical Features In de novo AML positive for t(9;22)(q34.1;q11.2), the BCR-
De novo AML cases with BCR-ABL1 is a rare subtype of ABL transcript p210 is more frequently detected than p190
AML accounting for <1% of all cases. It occurs predomi- [11, 80]. Other studies have suggested a nearly equal distri-
nantly in adults with a male predominance [11]. The distinc- bution in BCR-ABL1-positive AML [81]. Patients often show
tion between de novo AML with BCR-ABL1 and myeloid additional molecular and cytogenetic abnormalities includ-
blast phase of CML is challenging. Evaluation of patient ing monsomy 7, trisomy 8, or complex karyotypes. FLT3
clinical history is mandatory. Patients generally develop leu- mutation may be observed in de novo BCR-ABL1-positive
kocytosis with the presence of blast cells in the peripheral AML but not in CML BP. Differences between deletion of
blood, anemia, and thrombocytopenia. Generally, no spleno- antigen receptor genes (IGH, TCR), IKZF1, and/or CDKN2A
megaly develops. may support a diagnosis of de novo vs. CML BP but also
Without adequate treatment, patients with BCR-ABL1- occur in BCR-ABL-positive ALL [11]. Patients carrying
positive AML have a high relapse rate, and dismal prog- BCR-ABL1 in addition to recurrent genetic abnormalities
nosis [79]. However, outcome of patients <50 years of that define distinct entities such as CBF leukemias for exam-
age with BCR-ABL1-positive AML receiving allogeneic ple inv(16)(p13.1q22) are not included in this category.
stem cell transplantation (SCT) in complete remission is During TKI treatment, a clonal selection of a BCR-ABL1(−)
relatively favorable, possibly reflecting the beneficial subclone may be a cause of refractory disease [82].
effect of the additional use of tyrosin kinase inhibitors
(TKI) [80]. 13.3.8.5 Caution
• The exclusion of a CML blast phase may be challenging.
13.3.8.2 Morphology A detailed evaluation of the medical history and the
In contrast to CML BP, peripheral blood basophilia is rarely “background” features of the bone marrow biopsy may be
observed in BCR-ABL1-positive AML. The neoplastic helpful.
myeloid population may show different degrees of matura- • AML cases with a translocation t(9;22);BCR-ABL1-
tion including cases with minimal differentiation. positive that carry additional recurrent genetic abnormali-
Morphology may be quite similar to AML, NOS or AML ties that define specific entities are not included in this
with myelodysplasia-related changes (AML-MRC). The provisional category.
bone marrow cellularity is generally not as much increased • Therapy-related myeloid neoplasms positive for
as in CML-BP that, in addition, may show some features t(9;22);BCR-ABL1 are also excluded.
420 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History (Fig. 13.13) 92% of peripheral blood cells. An identical population was
A 59-year-old male patient was initially admitted to the hospi- present in bone marrow aspirate smears and in the trephine
tal with a suspected diagnosed of AML, NOS without matura- bone marrow biopsy (Fig. 13.13). Erythro- and granulocyto-
tion. No previous history of a hematologic or non-hematologic poiesis were significantly reduced. The marrow spaces were
neoplasm was reported. Clinical examination revealed no hypercellular. However, some fat cells were still present.
splenomegaly. CBC at presentation: WBC 33 × 109/L, Hb Karyotyping revealed a translocation t(9;22)(q34.1;q11.2).
6.8 g/dL, platelets 74 × 109/L. Myeloid blasts expressing Molecular analysis provided evidence of a p210 fusion tran-
CD13, CD33, CD34, CD7, and partially MPO represented script. The diagnosis was AML t(9;22);BCR-ABL1-positive.

a b

Giemsa Giemsa
c d

Giemsa NASDCL

Fig. 13.13 AML with translocation t(9;22)/BCR-ABL1-positive. (a– addition to residual granulopoietic cells, a minority of blasts expressed
h) The bone marrow biopsy was hypercellular, but in contrast to most MPO. (g, h). CD34 was strongly expressed in the total leukemic popu-
cases with CML BP still contained some fat cells. Blasts were medium- lation. No residual dwarf megakaryocytes or macrophages with fea-
sized without significant maturation (a–c; Giemsa). (d) Scattered tures of pseudo-Gaucher cells that may be present in CML BP were
NASDCL+ myeloid cells represented residual granulopoiesis. (e, f) In detected
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 421

e f

MPO MPO

g h

CD34 CD34

Fig. 13.13 (continued)

422 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.4 AML with Gene Mutations used to assess residual blasts after chemotherapy. Blasts gen-
erally express strongly CD33 and often monocytic markers
This category of the revised 2016 WHO classification such as CD14, CD36, and CD64 but are mostly negative for
includes the frequently diagnosed subtype AML with CD34. However, CD34 positivity may occur. A subpopula-
mutated NPM1, AML with biallelic mutation of CEBPA, and tion expressing stem cell markers may be detected by flow.
the provisional entity AML with mutated RUNX1. The expression of CD34, CD25, CD123, and CD99 has been
associated with FLT3-ITD co-mutations [11].

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 429

13.4.2 AML with Biallelic Mutation of CEBPA 13.4.2.4 Genetics

CEBPA-double-mutated cases usually bear biallelic N- and
AML with biallelic mutations in the transcription factor C-terminal mutations [98]. A variety of CEBPA mutations exist.
CCAAT/enhancer binding protein α (CEBPA) is actually rec- However, two classes of mutations predominate. N-terminal
ognized as a distinct entity by the revised 2016 WHO clas- mutations are located between the major translational start
sification [11]. This type is associated with a relatively codon and a second ATG in the same open reading frame.
favorable outcome [98, 99]. Mutations in the C-terminal basic leucine zipper (bZIP) region
are in-frame and may impair DNA binding and/or homodimer-
13.4.2.1 Epidemiology ization and heterodimerization. Numerous other CEBPA muta-
CEBPA-double-mutated cases are more frequent in pediatric tions are rare. Because of the variety of mutations, the
than in adult AML cases [11]. They represent about 4–9% of identification of CEBPA mutants has been considered to be
AML in children and young adults [11]. challenging [100]. Additional mutations may be present and
involve FLT3-ITD in up to 9% and GAT2 zinc finger 1 muta-
13.4.2.2 Morphology tions in about 39% [66]. The large majority of patients has a
This AML entity can present with various morphologies normal karyotype (about 70%), but abnormalities may be
including features of AML with or without maturation. detected for example del(5q) or del(9q) [66]. The additional
About a quarter of cases show multilineage background dys- GATA2 mutation has apparently no negative prognostic effect
plasia. Similar to NPM1-mutated cases, CEBPA-double- on the survival of biallelic CEBPA mutated cases [101]. It has
mutated cases with multilineage dysplasia should not be been suggested that GATA2 ZF1 mutations may collaborate
classified as AML with myelodysplasia-related changes with biallelic CEPBA mutations to deregulate target genes dur-
(AML-MRC). ing malignant transformation. These AML cases may represent
a genetically distinct subgroup of normal karyotype AML [101].
13.4.2.3 Immunophenotype
CEBPA-mutated AML expresses myeloid antigens CD13, 13.4.2.5 Caution
CD33, CD65, CD11b, and CD34. The double-mutated cases Cases of AML with biallelic mutation of CEBPA and mor-
are more frequently positive for HLA-DR, CD7, and CD15. phologic features of multilineage dysplasia fit in this new
Monocytic markers are rarely seen. Immunophenotypic WHO category (Fig. 13.17). However, cases with additional
analysis by flow has shown that CEBPA-double-mutated myelodysplasia-related cytogenetic abnormalities should
acute myeloid leukemia displays a unique phenotypic pro- rather be included in the category AML with myelodysplasia-
file [100]. related changes (AML-MRC).
430 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 1 (Fig. 13.17) The bone marrow biopsy was hypercellular and infiltrated by
The initial symptom of this 37-year-old male patient was the CD33-positive blasts partially positive for NASDCL and
development of cutaneous and mucosal hemorrhage. The rarely for CD14. No background myelodysplastic changes
CBC was: WBC 18 × 109/L, Hb 10.6 g/dL, platelets were observed. The karyotype was normal. Mutational anal-
21 × 109/L. Peripheral blood and bone marrow showed 85% ysis revealed a biallelic CEBPA mutation. Therefore, the
blasts mostly positive for myeloperoxidase and negative for case was assigned to the category AML with biallelic muta-
nonspecific esterase suggestive of an AML with maturation. tion of CEBPA.

a b

Giemsa Giemsa

c d

Giemsa NASDCL

Fig. 13.17 AML with biallelic mutation of CEBPA. The trephine bone cells and showed features of AML with maturation and were partially
marrow biopsy was hypercellular with some residual fat cells (a–c, positive for NASDCL (d). In addition, CD33 was expressed in the
Giemsa). Blasts accounted for about 90% of nucleated bone marrow majority of blasts (e, f) and CD34 in a subpopulation (g, h)
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 431

e f

CD33 CD33

g h

CD34
CD3
CD344 CD34
CD
CD3
34
4

Fig. 13.17 (continued)

432 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 2 (Fig. 13.18) The karyotype was normal. Molecular analysis revealed a
A 47-year-old woman was admitted with widespread cutane- biallelic CEBPA mutation in addition to a GATA2 zinc finger
ous hemorrhage. The CBC was: WBC 80 ×109/L, Hb 7.2 g/ (ZF)1 mutation. The diagnosis was normal karyotype AML
dL, platelets 13 × 109/L. The bone marrow aspirate contained with biallelic CEBPA-mutation with concomitant GATA2
85% blasts expressing myeloperoxidase, CD7, CD38, mutation.
CD117, and HLA-DR and partially positive for CD33, CD13
(50%), CD15 (45%), and CD86 (40%).

a b

MGG MGG

c d

Giemsa Giemsa

e f

NASDCL MPO

Fig. 13.18 AML with biallelic mutation of CEBPA and of GATA2. (a, marrow spaces diffusely infiltrated ba blasts that were largely negative
b) Peripheral blood (a) and bone marrow aspirate smears (b) containing for naphthol-ASD-chloroacetate-esterase (e) but strongly expressed
medium-sized blasts accounting for about 80% of nucleated bone mar- myeloperoxidase (f), CD7 (g), and CD117 (h)
row cells. (c, d) Trephine bone marrow biopsy showing hypercellular
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 433

g h

CD7 CD117

Fig. 13.18 (continued)

434 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.4.3 AML with Mutated RUNX1 13.4.3.3 Immunophenotype

The immunophenotype includes expression of CD34, CD33,
AML with mutated RUNX1 has been included as a and HLA-DR with variable presence of other antigens such
provisional entity in the revised 2016 WHO classifica- as CD33, MPO, and/or monocytic markers [11].
tion [11]. This type of AML has been associated with
distinct clinicopathologic features and inferior outcome 13.4.3.4 Genetics
[11, 102]. It has been proposed that RUNX1 mutational RUNX1 encodes a key transcriptional regulator of hematopoie-
status should be integrated into a diagnostic work-up of sis. RUNX1 has been implicated in mechanisms controlling
AML, particularly for AML, NOS or an intermediate- apoptosis, cell-cycle control, ribosome biogenesis, and decisions
risk group [103]. RUNX1 mutations predict for resis- involving self-renewal and differentiation. Moreover, it has been
tance to chemotherapy [104]. Improved survival may be shown that RUNX1 is a key determinant of megakaryocyte cell-
achieved by allogeneic h ematopoietic cell transplanta- fate decisions in multipotent progenitors. RUNX1 downregulates
tion [11, 104]. cell-adhesion factors that promote residency of stem cells and
megakaryocytes in their bone marrow niche [105]. RUNX1
13.4.3.1 Epidemiology mutations occur in a variety of hematologic neoplasm such as
The incidence of RUNX1 mutation in de novo is low myelodysplastic syndromes, de novo AML, therapy-related
(5–16%) [11, 103]. RUNX1 mutations have been associated myeloid neoplasms, and lymphoid neoplasms. Many different
with older age and male gender [102]. Cases developing RUNX1 mutations have been identified predominantly in exons
RUNX1-mutated AML after radiation or alkylating chemo- 3, 4, and 8 [104]. In this large patient cohort, RUNX1 mutations
therapy should be classified as therapy-related myeloid were almost mutually exclusive of AML with recurrent genetic
neoplasms. abnormalities [102]. However, karyotypic abnormalities such as
trisomies 8 and 13 may be found [11]. RUNX1 mutations co-
13.4.3.2 Morphology occur with a complex pattern of gene mutations, frequently
Blasts often show an immature morphology with minimal involving mutations in epigenetic modifiers (ASXL1, IDH2,
differentiation. However, various morphologic features have KMT2A, EZH2), components of the spliceosome complex
been reported including more mature myeloid or monocytic (SRSF2, SF3B1) and STAG2, PHF6, and BCOR [102].
differentiation. (See also case history 13.2.10.1 AML with minimal
differentiation.)
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 435

Case History positive for CD34, CD13, CD38, HLA DR, and CD117 and
A 68-year-old man consulted his physician because of a partially positive for CD7 (68%)and CD56. Morphology
perianal abscess pancytopenia. The CBC was: 1.2 × 109/L, and immunophenotype were suggestive of an AML with
Hb 6.8 g/dL, platelets 102 × 109/L. The differential count minimal differentiation. The karyotype was normal.
revealed 26% MPO-negative nongranulated medium-sized Molecular analysis revealed a monoallelic RUNX1 muta-
MPO-negative blasts, 2% metamyelocytes, 14% neutro- tion and a KMT2A partial tandem duplication (KMT2A-
phils, 1% eosinophils, 46% lymphocytes, and 11% smudged PTD). The diagnosis was AML with mutated RUNX1
cells. The bone marrow contained 70% blasts that were (Fig. 13.19).

a b

HE HE
c d

CD34 CD56

Fig. 13.19 AML with mutated RUNX1. The bone marrow biopsy was for MPO but strongly expressed CD34 (c). A majority of blasts was
hypercellular but still contained fat cells (a, b, HE). Immature appear- positive for CD56 (d) and CD7 (e, f). NASDCL reaction (g) and MPO
ing blasts represented about 70% of nucleated cells and were negative (h) immunohistochemistry marked residual granulopoietic cells
436 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD7 CD7
g h

NASDCL MPO

Fig. 13.19 (continued)

c d

MGG MGG

Fig. 13.22 Therapy-related myeloid neoplasm, case 1. (a–d, MGG) cytoplasm that were also observed in the bone marrow core biopsy (e,
Bone marrow aspirate smears were hypocellular with some more cel- f, Giemsa). Irregular reticulin fibrosis was assigned to a grade 2 (g, h,
lular areas showing a prominent dysplastic erythropoiesis with some Gomori)
vacuolated proerythroblasts and nests of myeloblasts with a basophilic
444 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

Giemsa
Giemsa

g h

Gomori Gomori

Fig. 13.22 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 445

Case History: Case 2 (Fig. 13.23) was: WBC 23 × 109/L, HB 9.3 g/dL, platelets 18 × 109/L. The
A 53-year old woman AML t(9;11)(p21.3;q23.3); KMT2A- peripheral blood contained 43% blasts with basophilic cyto-
MLLT3, therapy related plasm, prominent nucleoli. Blasts were positive for CD33
A 52-year old woman was admitted with leukocytosis and with co-expression of MPO, CD13 and partial co-expression
thrombocytopenia. Fifteen months earlier, she was diagnosed of CD117 and CD14. Blasts represented 80% of nucleated
with breast cancer, NOS, grade 3, and underwent neoadju- cells in the bone marrow aspirate and core biopsy. Genetic
vant chemotherapy with an etoposide containing chemother- studies revealed a t(9;11)(p22;q23); KMT2A-MLLTT3 trans-
apy. The suspected diagnosis of her physician was bone location. This translocation had apparently occurred second-
marrow involvement by metastatic breast cancer complicated ary to cytotoxic chemotherapy. Therefore, the AML was
by a reactive leukocytosis and thrombocytopenia. The CBC assigned to the t-MN category and diagnosed as t-AML.

a b

HE HE

c d

NASDCL CD33

Fig. 13.23 Therapy-related myeloid neoplasm with t(9;11) partially NASDCL-positive (c), express CD33 (d), myeloperoxidase
(p21.3;q23.3); KMT2A-MLLT3. The bone marrow core biopsy shows a (e), partially CD14 (f), and rarely CD34 (g). Some dysplastic mega-
dense infiltration by blasts with prominent nucleoli (a, b, H&E) that are karyocytes were observed (h, CD61)
446 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

MPO CD14

g h

CD34
C
CD
D34
34 CD61
CD
CD6
61
1

Fig. 13.23 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 447

13.7 AML, Not Otherwise Specified mutated genes per cytomorphological subtype were
RUNX1 in M0 (43%), NPM1 in M1 (42%), DNMT3A in M2
Myeloid neoplasms can only be designated as AML, NOS (26%), NPM1 in M4 (57%), M5a (49%), and M5b (70%),
when cytogenetic and molecular aberrations defining other and TP53 in M6 (36%). FLT3, NPM1, and WT1 mutations
AML categories have been excluded [14]. Especially, recur- were associated with an immature phenotype in myeloblastic
rent cytogenetic abnormalities, AML-defining mutations, AML, whereas other combinations involving ASXL1,
multilineage dysplasia, a prior MDS or MDS-defining cyto- RUNX1, MLL-PTD, CEBPA, or KRAS were more frequent in
genetic abnormalities, a history of cytotoxic or radiation myeloblastic AML with maturation. Within the NPM1-
therapy, and familial predisposition have to be excluded. mutated subcohort, ASXL1 mutations were significantly
With the exception of pure erythroid leukemia (PEL), blasts associated with a monoblastic differentiation and DNMT3A
must represent ≥20% of blood or bone marrow cells. Within mutations with a monocytic phenotype [123]. It is worth not-
this NOS category, subtypes are classified according to the ing that some AML cases that are assigned to the NOS cate-
morphologic and phenotypic features of the predominant gory at initial presentation are switched to a WHO-defined
myeloid lineage, the degree of maturation or even the clini- cytogenetic or molecular subtype when the results of further
cal course and the association with the bone marrow archi- analyses are available (see case 13.2.10.1).
tecture such as in acute panmyelosis with fibrosis (APMF)
[120]. Principally, the diagnostic criteria proposed by the
French-American-British (FAB) classification for AML are 13.7.1 AML with Minimal Differentiation
applied [121]. In most of the different subgroups, the AML,
NOS categories are not linked to patient prognosis. However, In AML with minimal differentiation (AML M0 according to
the FAB subtypes still retain a current prognostic role in pre- the FAB classification), the bone marrow comprises ≥20%
dicting the outcome of AML patients undergoing allogeneic blasts with a small rim of cytoplasm devoid of granules or
stem cell transplantation. In a large retrospective analysis, Auer rods [124]. Cytological or cytochemical features of a
M6/M7 patients had decreased leukemia-free survival and myeloid differentiation such as MPO or ANAE positivity are
increased nonrelapse mortality rates [122]. The AML absent or may be detected in <3% of blasts. However, a
phenotype- genotype associations were studied in a large myeloid differentiation is detected by flow cytometry and/or
cohort of patients diagnosed according to FAB/WHO defini- IH. CD34, CD117, CD13, and CD33 are often expressed.
tions by molecular profiling [123]. The most frequently Moreover, TdT, CD7, and CD19 may be expressed.
448 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History (Figs. 13.24 and 13.25) MPO and lymphocytic markers. The karyotype was normal.
AML with morphologic and immunophenotypic features Initially, mutational studies were performed by a limited
suggestive of minimal differentiation. A 74-year-old man panel. No NPM1, FLT3-ITD or TKD, CEBPA, DNMT3A,
presented with pancytopenia. The CBC was: WBC IDH1, or IDH2 mutations were detected. The initial diagno-
3.2 × 109/L, HB 7.6 g/dL, platelets 34 × 109/L. The periph- sis was AML with minimal differentiation. However, retro-
eral blood contained 26% blasts with small rim of agranular spective analysis of frozen material revealed a RUNX1
cytoplasm resembling lymphoblasts. The bone marrow aspi- mutation and a co-operating mutation in the SRSF2 gene.
rate smears and the core biopsy were hypocellular and RUNX mutations are frequently detected in AML with mini-
showed the same small blast population accounting for about mal differentiation [123]. Therefore, the case was moved to
85% of nucleated cells. Cytochemical and immunohisto- the provisional category AML with mutated RUNX1 (see
chemical stains revealed that only <3% of blasts contained also Sect. 13.3.3). This example highlights that in some cases
rare MPO-positive granules. By flow cytometry, the blasts the morphologic and immunophenotypic classification as a
were positive for CD34, HLA-DR, CD13, CD117, and TdT subtype of AML, NOS has to be changed later on to a more
but were negative for CD11b, CD14, CD64, CD15, and specifically defined AML category.

a b

MG
GG MGG
c d

MG
M GG MGG

Fig. 13.24 AML with phenotypic features of minimal differentiation. Small-sized blasts represented about 85% of nucleated cells. (c)
(a, MGG) Blood smears showed rare blasts resembling lymphoblasts. Scattered proerythroblasts were binucleated. (e–h) Rare blasts repre-
(b–d, MGG) The bone marrow aspirate smears were hypocellular. senting <3% contained some MPO-positive cytoplasmic granules
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 449

e f

MPO MPO

g h

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 461

13.7.6 Pure Erythroleukemia cells with ≥30% proerythroblasts. The chromatin is dark.
The cytoplasm of proerythroblasts is basophilic and fre-
The actual revised 2016 WHO classification restricts the quently contains vacuoles and PAS-positive inclusions.
diagnosis of erythroleukemia (AEL) to cases characterized There may be some megakaryocyte dysplasia in the back-
by a proliferation of immature cells of the erythroid lineage ground [14].
constituting >80% of bone marrow cells with ≥30% pro-
erythroblasts. This rare AML subtype has been designated as 13.7.6.3 Immunophenotype
pure erythroid leukemia (PEL; [14, 125]). The former 2008 There are no immunophenotypic markers to discriminate
WHO classification listed another type of AEL: the mixed neoplastic from non-neoplastic erythroid proliferations. The
erythroid/myeloid subtype defined by erythroid precursors erythroid lineage expresses glycophorin C, hemoglobin,
constituting ≥50% of bone marrow cells and the presence of CD71, and E-cadherin and may also be positive for CD117
blasts ≥20% in the non-erythroid compartment. However, but is CD34-, MPO, and HLA-DR-negative. In poorly
studies of large patient cohorts suggested that AEL, ery- differentiated cases, glycophorin C may be absent [14].
throid/myeloid subtype with <20% blasts may be more CD41 and CD61 expressions have been described in some
appropriately included in the spectrum of myelodysplastic cases leading to the differential diagnosis of acute mega-
syndromes with excess blasts rather than in AML, NOS karyoblastic leukemia.
[126, 127]. Therefore, the mixed AEL is now split into (a) The immunohistochemical detection of nuclear p53 over-
the categories MDS with erythroid predominance when expression correlated to TP53 aberrations is helpful to distin-
blasts are <20% or (b) in AML with myelodysplasia-related guish PEL from non-neoplastic erythroid proliferations and
changes (AML-MRC) when blasts are ≥20% of bone mar- should be included in the diagnostic work-up of suspected
row cells. The negative prognostic impact of erythroid pre- cases [130].
dominance, defined as 50% or more bone marrow erythroid
cells, in MDS is still challenging [125]. A retrospective eval- 13.7.6.4 Genetics
uation of cases previously diagnosed as AEL reclassified Until now, no specific mutational spectrum or chromosome
slightly over half of them as MDS EB-1 or -2 according to abnormalities have been described for PEL.
the 2017 WHO criteria. The overall survival was not differ- The cases generally have a complex, often highly com-
ent from other patients with MDS-EB [128]. Results of plex, karyotype [130]. Loss of chromosomes 5 and 7 or
another study suggested that cytogenetic, rather than MDS/ del(5q) or del(7q) has been reported [14].
AML subtypes, may have the most important impact on
prognosis for previously diagnosed AEL patients [129]. 13.7.6.5 Caution
• The differential diagnosis between PEL, MDS with ery-
13.7.6.1 Epidemiology throid predominance, and non-neoplastic hyperplasia of
Pure erythroid leukemia is an extremely rare AML category the erythroid lineage is challenging. Morphologic and
predominantly reported in elderly patients with a male pre- immunophenotypic features are overlapping.
dominance. The prognosis is dismal [130]. • Non-neoplastic erythroid-predominant conditions include
for example megaloblastic anemia or severe hemolysis.
13.7.6.2 Morphology Therefore, a significant degree of clinical, laboratory,
The erythroid lineage shows striking proliferation of immunophenotypic, and genetic investigation are recom-
immature cells accounting for >80% of bone marrow mended [130].
462 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 1: Pure Erythroid Leukemia (PEL) series was strikingly reduced. About 6% of nucleated bone
(Fig. 13.31) marrow cells expressed CD34. Nearly normal numbers of
A 69-year-old male presented with anemia and leukopenia. megakaryocytes were present. The p53 protein was strongly
CBC: WBC 2.1 × 109/L, hemoglobin 7.5 g/dL, platelet count overexpressed in the majority of bone marrow cells sugges-
of 173 × 109/L. Differential count: neutrophils 61%, lympho- tive of a TP53 mutation. Genetic studies demonstrated a
cytes 27%, monocytes 5%, eosinophils 2%, erythroblasts mixed lineage leukemia gene-partial tandem duplication
5%. The bone marrow biopsy was highly hypercellular with (MLL-PTD). No NPM1 or FLT3-ITD or -PTD mutations
a predominance of immature CD71-positive and glycopho- were detected. The karyotype was complex.
rin C-positive erythroblasts accounting for >85% of The diagnosis was acute erythroid leukemia (AEL) or pure
nucleated bone marrow cells. The MPO-positive myeloid erythroid leukemia (PEL) according to 2016 WHO criteria.

a b

HE HE

c d

HE CD71

Fig. 13.31 Pure erythoid leukemia (case 1). (a–c) Dense infiltration of MPO+ myeloid cells accounting for <10% of nucleated cells. (g) High
hypercellular bone marrow spaces by blast cells (H&E). (c) Immature KI67-positive proliferation fraction. (h) Striking nuclear overexpres-
erythroid precursors forming dense sheets (PAS). (d, e) More than 85% sion of the p53 protein by the large majority of blasts (immunostain
of blasts belong to the erythroid lineage expressing CD71. (f) Rare with anti-p53)
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 463

e f

CD71 MPO

g h

Ki67 p53

Fig. 13.31 (continued)

464 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 2: Pure Erythoid Leukemia (PEL) bone marrow core biopsy revealed large confluent sheets of
(Figs. 13.32 and 13.33) proerythroblasts without further maturation representing >90%
A 59-year-old man was admitted because of rapidly developing of nucleated cells. A striking feature was a strong nuclear over-
pancytopenia. The CBC was: WBC 2.2 × 109/L, HB 6.4 g/dL, expression of the p53 protein in nearly all erythroid precursors.
platelets 24 × 109/L. Examination of bone marrow aspirate The erythroid lineage was positive for glycophorin C, CD71,
smears revealed a striking expansion of the erythroid lineage E-cadherin, partially CD117, and negative for CD34. Rare
accounting for >90% of cells. The cytoplasm of proerythro- MPO-positive residual granulopoietic cells and some small
blasts was often vacuolated with PAS-positive inclusions. The CD61-positive megakaryocytes were detected.

a b

MGG MGG

c d

MGG PAS

Fig. 13.32 Pure erythroid leukemia (case 2). (a–d) Cytology of bone inclusions. (e–h) Trephine bone marrow biopsy showing a striking pre-
marrow aspirate smears. Abundant proerythroblasts with basophilic dominance of the erythroid lineage (e, f, PAS) highlighted by (g, h)
often vacuolated cytoplasm (a–c, MGG) and (d) coarse PAS-positive glycophorin C expression
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 465

e f

PAS PAS

g h

476 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.8 Myeloid Sarcoma 13.8.2 Morphology and Immunophenotype

Myeloid sarcomas (MS) formerly also referred to as chlo- MS are composed of neoplastic myeloid cells with or with-
roma are defined as extramedullary tumor-forming extra- out maturation lacking specific morphologic or immunophe-
medullary masses of myeloid blasts with or without notypic features [15]. They may express MPO, CD33, CD34,
maturation [15, 132, 133]. Even if these tumors occur by and CD117 and show reactivity for naphthol-ASD-
definition outside the bone marrow, they are briefly men- chloroacetate-esterase. However, a monoblastic or myelo-
tioned here, because their diagnosis has the same impact as monocytic phenotype is frequently reported and characterized
the diagnosis of AML. Diffuse infiltrates of extramedullary by the expression of CD14, CD11c, and CD163. However,
sites by myeloid blasts for example in the oral mucosa or the morphologic and immunophenotypic equivalents to other
skin do not fulfill criteria of an MS [15]. AML subtypes such as CD61+CD41+ acute megakaryoblas-
tic leukemia or CD71+ glycophorin C+ hemoglobin+ ery-
throid leukemia may also occur.
13.8.1 Epidemiology and Clinical Features

MS are rare neoplasms predominantly described as case 13.8.3 Genetics

reports and only a few studies of larger cohorts [15, 132, 134,
135]. They have a slightly male predominance and generally Until now, heterogeneous genetic profiles have been reported
occur in adult patients but may also be detected in children in MS including monosomy 7, trisomy 8, KMT2A rearrange-
and adolescents. MS may develop as the initial manifestation ments, NPM1 mutation, FLT3-ITD mutations, and others
of a myeloid neoplasm or simultaneously or subsequently to [15, 135]. Recently, an enrichment in RTK-RAS pathway
an AML, an MDS, or a MPN involving the bone marrow. mutation has been detected in myeloid sarcoma [136].
They may also develop as initial extramedullary AML
relapse after allogeneic hematopoietic transplantation.
A minority of patients with MS will never develop bone 13.8.4 Caution
marrow involvement, and the myeloid neoplasm will be
restricted to extramedullary sites. In many cases, immunohistochemistry using a large panel of dif-
MS are encountered at any localization but predominantly ferent antibodies and FISH/CISH probes is necessary to exclude
develop in the gastrointestinal tract, lymph nodes, and skin.
Multifocal involvement may occur. The prognosis is unfa- • malignant lymphoma,
vorable with the exception of patients offered allogeneic • undifferentiated carcinoma, or
hematopoietic transplantation. • various types of non-myeloid sarcoma.
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 477

Case History: Myeloid Sarcoma (Fig. 13.37) performed and showed a dense infiltration by medium-
A 23-year-old male patient developed a mediastinal to large-sized polymorphic blasts with prominent nucle-
mass with a maximal diameter of 12 cm. The CBC was: oli and a basophilic cytoplasm strongly co-expressing
WBC 15 × 10 9/L, HB 14.2 g/dL, platelets 280 × 10 9/L. The CD33 and CD43 and a high Ki67 proliferation fraction
peripheral blood smears contained 3% band forms, 68% >75%. About 65% of blasts expressed CD71 partially in
neutrophils, 3% eosinophils, 4% monocytes, and 22% co-expression with CD117. Smaller subpopulations were
lymphocytes. No dysplastic cells or blasts were observed. positive for CD68, CD4, lysozyme, and myeloperoxi-
The bone marrow was normocellular and showed a regu- dase (Fig. 13.37). No genetic testing was performed. The
lar maturation of all hematopoietic lineages. A mediasti- diagnosis was myeloid sarcoma with an isolated
noscopic wedge biopsy of the mediastinal mass was extramedullary manifestation.

a b

MGG

c d

Giemsa Gomori

Fig. 13.40 AML associated with germline CBL mutation. (a, b) marrow core biopsy specimen (Giemsa) showing (d) reticulin fibrosis
Peripheral blood films containing blasts, dysplastic monocytes, neutro- grade 1 (Gomori) and (e, f) a dense infiltration by a predominantly
phils, and rare erythroblasts (MGG). (c) Strikingly hypercellular bone naphthol-ASD-chloroacetate-esterase-positive myeloid population
484 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

NASDCL NASDCL

Fig. 13.40 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 485

13.10 M
yeloid Proliferations Associated ally >10%. In addition, the diagnosis requires the detection
with Down Syndrome of a GATA1 mutation (see below). Typically, the blast cells in
TAM have a megakaryoblastic morphology and phenotype.
Individuals with constitutional trisomy 21 (Down syn- They show cytoplasmic blebbing and express CD42 and
drome, DS) have a significantly increased risk to develop CD61 in co-expression with CD34, CD117, CD33, and other
different types of hematopoietic neoplasms especially myeloid markers but are negative for myeloperoxidase
myeloid proliferations in infancy, childhood, adolescence, CD15, CD14, or glycophorin A. An aberrant expression of
and adulthood [16]. CD56 and CD7 may be present. In addition, basophilia may
be observed.
In cases designated as silent TAM, no or very low num-
13.10.1 Epidemiology and Clinical Findings bers of blasts are present despite a GATA1 mutation. These
infants are also at risk to develop ML-DS.
Trisomy of chromosome 21 has been reported to be present ML-DS may present with pancytopenia and MDS-like
in one in 700–1000 live births worldwide [150]. In children features associated with low percentages of peripheral
with DS myeloid leukemia (ML-DS), and acute lymphoblas- blasts and macrocytic erythroid cells. The bone marrow
tic leukemia are increased by 150- and ∼30-fold, respec- shows marked dysplastic features. Overt AML is fre-
tively [151, 152]. About 10% of neonates and also some quently associated with bone marrow fibrosis, resulting in
fetuses with DS present with neonatal hematopoietic disor- a dry tap. Dysplastic CD42+CD61+ megakaryocytes and
der that is referred to as transient abnormal myelopoiesis megakaryoblasts are increased, often associated with a
(TAM). TAM may be the first symptom of infants with megaloblastic erythropoiesis and dysgranulopoiesis. The
mosaicism for trisomy 21 without the characteristic pheno- immunophenotype of blasts in ML-DS is similar to that
type of DS. TAM may be detected as an incidental finding on seen in TAM.
review of a blood film in asymptomatic infants or as a conse- AML in patients >5 years are not different from AML in
quence of clinical symptoms such as coagulopathy, multior- non-DS patients.
gan failure and extramedullary infiltrates associated with
hepatosplenomegaly and effusions [152]. In the majority of
infants, spontaneous remission of TAM occurs. However, 13.10.3 Genetics
about 20–30% of infants develop a myeloid leukemia [152]
especially acute megakaryoblastic leukemia 1–3 years later. Trisomy 21 is associated with a perturbation of fetal hematopoi-
AML may develop secondary to a myelodysplastic-syndrome esis preceding the acquisition of GATA1 mutations [152, 153].
(MDS)-like phase. Both MDS and AML in children with The fetal liver of DS-fetuses harbors increased dysplastic mega-
Down syndrome are designated as acute leukemia associated karyocytes while platelet counts are reduced. Next-generation
with DS (ML-DS). AML in children with Down syndrome sequencing-based methodology has recently shown that GATA1
younger than 5 years is 150-fold increased compared to mutations have been detected in up to 30% of infants with DS
healthy children. Moreover, the risk to develop a myeloid and in all patients with TAM or ML-DS [154]. GATA1 is the key
neoplasm is also significantly higher in adult patients with hematopoietic transcription factor. TAM has been attributed to
DS than in the general population. However, a globally the cooperation between constitutional trisomy 21 and acquired
decreased incidence of solid tumors as compared to age- somatic N-terminal truncating mutations in GATA1. Additional
adjusted non-DS controls has been reported [150]. genetic alterations including those in epigenetic regulators and
signaling molecules are involved in the progression from TAM
to ML-DS [155]. Examples are mutations of CTCF, EZH2,
13.10.2 Morphology and Immunophenotype KANSL1, JAK2, JAK3, MPL, SH3B3, and RAS pathway genes
[155]. Myeloid neoplasms developing in children with DS are
It is important to realize that blasts may be seen in the periph- similar to non-DS MDS or AML, may not harbor GATA1 muta-
eral blood smears of neonates with DS that do not develop tions but may show trisomy 8 in nearly half of the cases while
TAM. In infants with TAM, the blast cell counts are gener- monosomy 7 is very rare [16].
486 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 1: Intrauterine Transient Abnormal nodes and thymus were observed in addition to congenital
Myelopoiesis (TAM) (Fig. 13.41) heart disease. Microscopic evaluation revealed abundant
This preterm born male infant of 35 weeks of gestational age intravascular blasts associated with extravascular infiltrates in
was diagnosed with trisomy 21, a WBC of 120 × 109/L, multiple organs. Liver and spleen showed a striking intrasinu-
thrombocytopenia, liver failure, ascites, and coagulopathy. soidal expansion of CD34+ hematopoietic precursors and
He passed away from multiorgan failure 7 h after birth. Blood blasts predominantly of the megakaryocytic lineage express-
films showed 72% blasts, rare hypogranulated neutrophils, ing CD61. In addition, rare MPO+ granulocytic precursors
some circulating micromegakaryocytes, megakaryoblasts, and dysplastic erythroid cells were seen. Molecular analysis
and macrocytic red blood cells. At autopsy, hepatospleno- revealed a mutation in exon 2 of the GATA1 gene. The diag-
megaly and enlargement of multiple organs including lymph nosis was TAM in Down syndrome.

a b

H&E H&E

c d

H&E HE

Fig. 13.41 Down syndrome, intrauterine TAM. (a, b) Meningeal blood some larger abnormal megakaryocytes (c, d, H&E) that predominantly
vessels stuffed with abundant blast cells (H&E). (c–g) Hepatic involve- co-express CD61 (e, f). (g) Rare intrahepatic foci of MPO+ granulocytic
ment by TAM: The hepatic sinusoids are dilated due to immature hema- precursors. (h) Involvement of the thymus by the myeloid proliferation
topoietic cells including megakaryoblasts, micromegakaryocytes, and associated with a destruction of Hassal bodies (H&E)
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 487

e f

CD61 CD61

g h

MPO H&E

Fig. 13.41 (continued)

488 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History 2: Myeloid Leukemia Associated with aspirate resulted in a dry tap. The bone marrow core biopsy
Down Syndrome (Fig. 13.42) revealed reticulin fibrosis grade 2 with small foci of collag-
A 2-year-old boy had a history of TAM in the neonatal enous fibrosis and increased content of CD3+ lymphocytes.
period that spontaneously resolved within 3 months. He There was a diffuse infiltration by CD34+ blasts including
subsequently developed anemia and thrombocytopenia. At >50% CD61+ megakaryoblasts and small atypical mega-
admission to the Pediatric Hematology-Oncology karyocytes. By genetic testing, a JAK2 mutation in addition
Department, the CBC was: WBC 2.9 × 109/L, HB 5.8 g/dL, to GATA2 mutation, a trisomy 21 and a monosomy 7 were
platelets 25 × 109/L. The differential count showed 12% detected.
blasts with cytoplasmic blebbing, some dysplastic neutro- The diagnosis was myeloid leukemia associated with
phils including pseudo-Pelger cells, lymphocytes, and occa- Down syndrome (ML-DS) consistent with acute megakaryo-
sionally teardrop-shaped red blood cells. Bone marrow blastic leukemia.

a b

Gomori Gomori

c d

Giemsa Giemsa

Fig. 13.42 Myeloid leukemia associated with Down syndrome. (a–h) showing features of abnormal megakaryocytes (Giemsa). (e) Interstitial
Bone marrow core biopsy. (a, b) Reticulin fibrosis grade 2 with small and focal increased inconspicuous CD3+ lymphocytes. (f) High content
foci of collagenous fibrosis (Gomori reticulin stain). (c, d) Infiltration of CD34+ precursor/blast cells with (g, h) a predominance of CD61+
of bone marrow spaces by small- to medium-sized atypical cells often megakaryoblasts and small atypical megakaryocytes
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 489

e f

CD3 CD34

g h

CD61 CD61

Fig. 13.42 (continued)

490 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.11 B
lastic Plasmacytoid Dendritic Cell menting the diagnostic specificity alongside CD4, CD56,
Neoplasm CD123, and TCL1 [160]. Numerous other antigens such as
CD7, CD33, CD38, CD71, TdT, and BCL2 may be pres-
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an ent. A dot-like reactivity for CD68 is frequently seen.
aggressive myeloid malignancy that involves skin, bone mar- Some cases may lack CD4 or CD56 immunoreactivity.
row, peripheral blood, lymph nodes, and spleen. This rare BPDCN cases are negative for CD34, MPO, CD117,
WHO-defined entity is actually considered to be histogeneti- CD14, CD163, and lysozyme.
cally related to the precursors of interferon type-1 producing
plasmacytoid dendritic cells. BPDCN was previously referred
to as CD4+CD56+ hematodermic neoplasm [156, 157]. 13.11.3 Genetics

An abnormal karyotype that is frequently complex is present

13.11.1 Epidemiology and Clinical Features in more than half of the patients. Recurrent chromosomal
abnormalities predominantly involve chromosomes 5q, 8p,
BPDCN is rare and contributes to <1% of all hematologic 13q, 6q, 15q, and 9 [159]. Many genetic alterations including
neoplasms with a male predominance. BPDCN commonly loss of RB1 tumor suppressor gene loci and biallelic loss of
occurs in elderly men but may also affect infants and chil- 9p21.3 and the CDKN2A/CDKN2B genes encoding p16 and
dren. Most patients initially develop skin lesions and subse- cyclin-dependent kinase inhibitors have been detected.
quently bone marrow involvement and a leukemic blood Whole-exome sequencing revealed the epigenetic program
picture followed by a spread to spleen and lymph nodes. dysregulation as an important feature of BPDCN including
Different types of skin involvement may be observed: iso- mutations of ASXL1, TET2, SUZ12, ARID1A, PHF2, and
lated purplish nodules, isolated papules, or disseminated CHD8 [161]. BPDCN may harbor mutations of TP53,
cutaneous lesions [156]. There are also BPDCN cases with- NPM1, NRAS, FLT3, RUNX2, MYB, MYC and IKZF1 genes.
out cutaneous involvement. Compared to non-cutaneous Genomic aberrations involving 12p/ETV6 are considered to
BPDCN, cutaneous BPDCN was associated with an older be highly prevalent [162].
median age at onset (72 years versus 45 years, P < 0.05;
[158]). An association with other types of myeloid disor-
ders such as myelodysplastic syndrome (MDS), chronic 13.11.4 Caution
myelomonocytic leukemia (CMML), or acute myeloid leu-
kemia (AML) may occur in as much as 20% of BPDCN • Rare BPDCN cases may lack either CD4 or CD56.
cases. BPDCN may transform into AML. The prognosis of • The immunophenotype overlaps with subtypes of AML
BPDCN is dismal with a median survival of 12–14 months. cases especially with monocytic differentiation that are
However, new approaches to therapy have shown promis- negative for CD34 but also express CD4, CD56, and
ing results [159]. CD123. However, BPDCN does not express lysozyme
and is esterase-negative.
• CD71 may be positive in cytoplasmic vacuoles of BPDCN
13.11.2 Morphology and Immunophenotype thus mimicking erythroleukemia.
• The expression of CD4 and CD56 warrants the exclusion
The cutaneous lesions show dermal infiltration by blast of a NK/T-cell lymphoma.
cells sparing the epidermis but often involving the subcu- • In bone marrow and lymph biopsies from patients with
taneous fat [156]. The pattern of bone marrow involvement other myeloid disorders such as CMML or acute mono-
may be interstitial, nodular, or diffuse. Blasts are of cytic/monoblastic leukemia nodules of plasmacytoid den-
medium size and typically have a gray-blue elongated dritic cells expressing CD123 may be present. However,
cytoplasm resembling pseudopodia. They commonly they have a more mature phenotype and lack CD56
express CD4, CD56, CD123, CD43, BDCA-2/CD303, expression.
TCL1, and CTLA. Moreover, expression of the transcrip- • The cutaneous infiltrates of BPDCN may be confused
tion factor TCF4 is a sensitive marker for BPDCN, aug- with reactive nonmalignant lesions.
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 491

Case History (Figs. 13.43 and 13.44) CD303, TCL1, TCL4, CD7, and BCL2 but negativity for
A 76-year-old male patient developed multiple reddish- CD34, CD14, and myeloperoxidase. The bone marrow tre-
brown maculo-papular skin lesions. Clinical examination phine biopsy showed a focally accentuated infiltration by the
revealed anemia, thrombocytopenia, and leukocytosis. The blast cells. In addition to the above-mentioned antigens, a
peripheral blood, bone marrow smears, and skin biopsy weak expression of CD71 and a dot-like positivity for CD68
showed medium-sized blasts with irregular nuclear contours, were observed. The residual hematopoiesis showed dysplas-
small nuclei, and an agranular cytoplasm. Some blasts had a tic features. The genetic profile was characterized by muta-
hand-mirror-shaped cytoplasm. Immunophenotyping tions in TET2 and NRAS. The diagnosis was BPDCN
revealed expression of CD4, CD56, CD123, CD43, BDCA-2/ involving skin, bone marrow, and blood.

a b

MGG Giemsa

c d

Giemsa CD4

Fig. 13.43 Blastic plasmacytoid dendritic cell neoplasm (BPDCN), granulocytic precursors. (b, c) Focally accentuated infiltration of the
bone marrow. (a) Bone marrow smear showing numerous medium- bone marrow core biopsy by blasts with irregular nuclear contours,
sized partially hand-mirror-shaped agranular blast cells with an imma- small nucleoli, and a grayish-blue cytoplasm. (d–h) Blast cells express
ture chromatin structure. Note the presence of some dysplastic CD4 (d), CD123 (e, f), TCL1 (g), and CD7
492 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD123 CD123

g h

TCL1 CD7

Fig. 13.43 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 493

a b

H&E H&E

c d

CD123 CD123

e f

CD56 CD4

Fig. 13.44 41. Blastic plasmacytoid dendritic cell neoplasm (BPDCN), mis. (c–f) Immunohistochemical staining of the skin biopsy revealing
skin biopsy (a, b), H&E-stained sections of the skin biopsy showing expression of CD123 (c, d), CD56 (e), and CD4 (f) by blast cells
dermal infiltration by medium-sized neoplastic cells sparing the epider-
494 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.12 A
cute Leukemias of Ambiguous cells of AUL are generally small- to intermediate-sized with
Lineage round or irregularly shaped nuclei, stippled chromatin, and
agranular cytoplasm. They may resemble blasts from AML
Ambiguous lineage acute leukemias (ALAL) represent a with minimal differentiation or acute lymphoblastic leuke-
heterogeneous group of hematopoietic neoplasms that can- mia. They do not express markers that are lineage-specific,
not clearly be assigned either to the myeloid or lymphoid especially not myeloperoxidase.
lineage through immunophenotyping [163]. The category of MPAL may either comprise a single uniform population
ALA includes undifferentiated leukemias that lack lineage- of blasts showing a differentiation toward both lymphoid
specific antigens and mixed-phenotype acute leukemias and myeloid lineages or a hybrid morphology characterized
(MPALs; [163]). In some MPALs, blasts may simultane- by two distinct lymphoid and myeloid populations [165,
ously express markers of both the myeloid and B/T lymphoid 166]. Myeloid blasts may exhibit features of AML without
lineages, thus exhibiting a biphenotypic pattern. Other cases maturation or correspond to monoblasts. The diagnostic
of MPAL may harbor two distinct blast populations that each work-up of MPAL must include flow cytometry immuno-
fulfill criteria for B-cell, T-cell, or myeloid differentiation phenotyping and immunohistochemistry using a large
and may be designated as bilineal. panel of antibodies. In addition, cytochemistry studies for
myeloperoxidase and nonspecific esterase are essential
[167].
13.12.1 Epidemiology and Clinical Features The criteria for the assignment to lineage commitment
have been defined as follows (see Table 13.9): According to
ALALs are diagnosed predominantly in adults but may also Béné, myeloid engagement is characterized by the expres-
develop in children. These hematopoietic malignancies are sion of myeloperoxidase or, for cells of the monocytic lin-
rare and account for <4% of acute leukemias, but the exact eage, by the expression of at least two of the
incidence is unknown [163]. The so-called biphenotypic monocyte-associated antigens CD11c, CD14, CD64, lyso-
subtype of MPAL is considered to be more frequent than the zyme, and/or nonspecific esterase activity demonstrated in
bilineal subtype. ALALs are poor prognosis leukemias [164]. cytochemistry. T-lineage commitment is characterized by
cytoplasmic or surface (rare) expression of the epsilon chain
of CD3. B-lineage engagement is characterized by strong
13.12.2 Morphologic CD19 expression associated with at least one of the follow-
and Immunophenotypic Features ing: cytoplasmic or membrane expression of CD79a, CD22,
and/or CD10 surface labeling. If CD19 is weakly expressed,
According to the revised 2016 WHO classification, six dif- two of the latter markers must be present to confirm B-lineage
ferent entities are designated as ALALs (Table 13.8). Blast features [2, 165].

Table 13.8 Subtypes of acute leukemias of ambiguous lineage

ALAL entity Genetic features Morphology Immunophenotype
Acute Limited data available Undifferentiated blast cells, no Absence of myeloid, T- or B-lineage markers.
undifferentiated features of myeloid differentiation MPO negativity
leukemia (AUL)
MPAL with t(9;22) Philadelphia chromosome/ Dimorphic blast population: B- or rarely T-lineage markers. Separate
(q34.1;q11.2) BCR-ABL lymphoblasts and myeloblasts population positive for myeloid markers
MPAL with KMT2A/MLL rearrangement, Dimorphic blast population, Lymphoblast population: B-cell precursor, B1
t(v;11q23.3) various partner genes monoblasts, and lymphoblasts phenotype: CD19+, CD10−
Separate myeloid (monoblastic) population
expressing myeloid antigens
MPAL, B/myeloid, Clonal chromosomal Blasts may resemble lymphoblasts Blasts meet criteria for both B-cell and
NOS abnormalities; del(6p), 12p11.2; or may be dimorphic with myeloid lineage assignment
del(5q), abnormalities chr 7; lymphoblasts and myeloblasts
genetic signature between that of
ALL and AML
MPAL, T/myeloid, Clonal chromosomal Blasts may resemble lymphoblasts Blasts meet criteria for both T-cell and
NOS abnormalities without specificity or may be dimorphic with myeloid lineage assignment
lymphoblasts and myeloblasts
MPAL, NOS: rare Clonal chromosomal Nonspecific morphology No unique immunophenotype
types abnormalities without specificity
MPAL mixed phenotype acute leukemia, NOS not-otherwise-specified
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 495

Table 13.9 Criteria for lineage assignment for a diagnosis of MPAL the regulation of either epigenome or transcription such as
Lineage assignment criteria DNMT3A, RUNX1, TP53, and KMT2D (MLL2) and in
Myeloid lineage genes involved in DNA repair pathway [168]. In addition,
Myeloperoxidase (flow cytometry, immunohistochemistry, or numerous nonspecific other mutations have been detected
cytochemistry)
in ALAS.
Monocytic differentiation (at least two of the following:
nonspecific esterase cytochemistry, CD11c, CD14, CD64,
lysozyme)
T-lineage 13.12.4 Caution
Stronga cytoplasmic CD3 (with antibodies to CD3 ε chain)
Surface CD3 • Acute leukemias with genetic features of other well-
B-lineage
defined WHO entities are excluded from the ALAL
Stronga CD19 with at least one of the following strongly
expressed: CD79a, cytoplasmic CD22, or CD10 category.
Or • Examples include leukemias with t(8;21), inv16, or
Weak CD19 with at least two of the following strongly expressed: t(15;17) or myeloid neoplasms with FGFR1 rear-
CD79a, cytoplasmic CD22, or CD10 rangement even if they exhibit a bilineage
Adopted from Arber et al. [2] differentiation.
a
Strong defined as equal or brighter than the normal B or T cells in the • Chronic myeloid leukemia in blast crisis, AML with
sample
myelodysplasia-related changes, and therapy-related
AML also do not fit into this category.
13.12.3 Genetics • Various AML cases may harbor aberrant expression of
lymphoid-lineage-associated antigens such as CD7 but do
The genetic findings in ALALs are heterogeneous (see not fulfill the criteria listed in Table 13.9 that are required
Table 13.9). By whole-exome sequencing and transcrip- for the diagnosis of ALAL.
tome sequencing, common gene mutations seen in hema- • Acute B- or T-lymphoblastic leukemia may aberrantly
topoietic neoplasms were found in the majority of ALAL express myeloid-associated antigens such as CD13,
cases [168]. Most mutations occurred in genes involved in CD33, and/or CD117.
496 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

Case History. Bilineal MPAL B/Myeloid, NOS cytometry confirmed the presence of two distinct population:
(Figs. 13.45 and 13.46) The lymphoblastic population was CD19, CD10, partially
A 74-year-old male patient was admitted with the diagnosis CD20, and TdT positive while the myeloid population
of acute leukemia. The CBC was: WBC 35 × 109/L, HB expressed myeloperoxidase, CD33, and partially CD14 and
9.3 g/dL, platelets 25 × 109/L. Physical examination revealed CD15. The bone marrow core biopsy was diffusely infil-
a mild hepatosplenomegaly. On cytological smears, the trated by blasts that also exhibited a dimorphic phenotype.
peripheral blood and bone marrow aspirate showed a dimor- The majority of blasts expressed CD19, CD10, PAX5, and
phic blast cell population: about 65% of blasts were small TdT while a minority of immature cells were positive for
and resembled lymphoblasts and were myeloperoxidase neg- myeloperoxidase, CD33, and partially Cd15 and CD14. No
ative while about 35% of blasts were of larger size with more further genetic analyses were performed. The diagnosis was
abundant cytoplasm and expressed myeloperoxidase. Flow bilineal MPAL B/myeloid, NOS.

a b

MGG MGG

c d

MGG MGG

Fig. 13.45 Bilineal MPAL B/myeloid, NOS. (a, b) Peripheral blood biopsy is densely infiltrated by blasts (Giemsa) that are positive for
smears and (c, d) bone marrow aspirate smears showing a dimorphic CD34 (f). About 35% of blasts show features of immature myeloid cells
blast cell population comprising small lymphoblasts and larger that express myeloperoxidase (g, h)
myeloblast-like cells (MGG). (e) The hypercellular bone marrow core
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 497

e f

Giemsa CD34

g h

MPO MPO

Fig. 13. 45 (continued)

a b

CD15 CD14

Fig. 13.46 Bilineal MPAL B/myeloid, NOS. (a, b) Myeloid blasts are partially positive for CD15 (a) and CD14 (b) and express CD33 (c). (d–h)
The majority of blasts belongs to the B-cell lineage expressing CD19 (d, e), partially CD20 (f), strongly PAX5 (g), and TdT (h)
498 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

c d

CD33 CD19

e f

CD19 CD20

g h

PAX5 TdT

Fig. 13.46 (continued)

Fig. 13.52 Early T-cell precursor lymphoblastic leukemia. (a, b) (MGG). (c–h) Threphine core bone marrow biopsy shows a dense infil-
Cytologic aspect of peripheral blood (a) and bone marrow smears (b) tration by blasts with irregularly shaped nuclei (c, d, Giemsa).
showing abundant small- to medium-sized lymphoblasts with incon- Lymphoblasts strongly express CD7 (e), CD34 (f), CD117 (g), and par-
spicuous nucleoli. No cytoplasmic granulation or Auer rods are present tially CD33 (h). Note the presence of some residual megakaryocytes
512 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD7 CD34

g h

CD117 CD33

Fig. 13.52 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 513

13.14 P
recursor Lymphoid Neoplasms: Table 13.11 Subtypes of B-lymphoblastic leukemia/lymphoma
B-Lymphoblastic Leukemia/ B-lymphoblastic leukemia/lymphoma, NOS
Lymphoma B-lymphoblastic leukemia/lymphoma with recurrent genetic
abnormalities
B-lymphoblastic leukemia/lymphoma with t(9;22)(q34.1;q11.2);
B-lymphoblastic leukemia/lymphoma B-ALL/LBL is a malig- BCR-ABL1
nancy of precursor cells of the B-cell lineage generally present- B-lymphoblastic leukemia/lymphoma with t(v;11q23.3); KMT2A
ing with bone marrow and blood involvement. Primary rearranged
extramedullary tumor forming manifestation as B-lymphoblastic B-lymphoblastic leukemia/lymphoma with t(12;21)(p13.2;q22.1);
lymphoma is rare. B-ALL/LBL is subdivided into multiple enti- ETV6-RUNX1
B-lymphoblastic leukemia/lymphoma with hyperdiploidy
ties according to the genetic background (see Table 13.11) [178].
B-lymphoblastic leukemia/lymphoma with hypodiploidy
B-lymphoblastic leukemia/lymphoma with t(5;14)(q31.1;q32.3)
IL3-IGH
13.14.1 Epidemiology and Clinical Findings B-lymphoblastic leukemia/lymphoma with t(1;19)
(q23;p13.3);TCF3-PBX1
The estimated annual incidence of ALL is about 1–4.75 cases Provisional entity: B-lymphoblastic leukemia/lymphoma,
BCR-ABL1-like
per 100,000 population [178]. About 75% of cases are diag- Provisional entity: B-lymphoblastic leukemia/lymphoma with
nosed in children <6 years of age. B-All is much more frequent iAMP21
than T-ALL and accounts for up to 85% of ALL cases. By con- From Swerdlow et al. [177]
trast, lymphoblastic lymphoma is more frequently composed
of precursor T-cells than precursor B-cells [178]. The incidence
of B-ALL in infants, children, adolescents, and adults is vari- Table 13.12 Features of B-ALL of different maturation stages
able according to the different subtypes (see Table 13.11). Immunophenotype
The clinical symptoms are generally related to bone mar- Always CD19+ and/or
Subtype CD79a+ and/or CD22+ Cytogenetics
row involvement and replacement of the hematopoietic
Early precursor CD10− KMT2A (MLL)
lineages by malignant cells resulting in anemia, granulocyto- B-ALL (Pro-B) rearrangement
penia, and thrombopenia. Patients may complain about Common type CD10+ All types
bone pain and arthralgia. Lymphadenopathy and hepato- B-ALL
splenomegaly may develop. B-LBL may be asymptomatic. Pre-B-ALL Cytoplasmic μ+
By definition, bone marrow infiltration in B-LBL is <25%. Transitional Surface μ+; light chain−
pre-B-ALL
Mature precursor Cytoplasmic μ heavy chain+, t(1;19)
B-ALL cytoplasmic or surface kappa
13.14.2 Morphology and Immunophenotype or lambda; often CD34−

Lymphoblasts in B-ALL are generally small- to medium-

sized with a round to oval nucleus, a coarse reticular chroma-
tin and small nucleoli and a small rim of blue-gray cytoplasm. The typical immunophenotype is characterized by the
However, larger blasts and variant forms showing cytoplas- expression of B-lineage-associated antigens including CD19,
mic granulation or vacuoles or cytoplasmic hand-mirror-like CD22, CD10, and CD79a. In addition, non-lineage typical
cytoplasmic projection may be observed. The bone marrow is antigens are expressed such as TdT, CD34, HLA-DR, CD38,
generally dense packed with blast cells. Some cases present and CD45.
with a hypoblastic bone marrow with loosely arranged blasts According to the immunophenotype reflecting the matu-
associated with pancytopenia. Occasionally, extensive spon- ration stage, B-ALL is classified into different subtypes
taneous necrosis of neoplastic bone marrow cells is present. (Table 13.12) recognized by the EGIL classification [171].
514 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

13.14.3 Genetics represented in Hispanics and adult male patients with Down
syndrome and associated with inherited genetic variants in
Cytogenetic and molecular findings are essential for the risk GATA3 [179]. In Ph-like ALL/LBL, multiple genomic altera-
stratification of B-lineage ALL patients (see Table 13.13). tions may be present that activate kinase and cytokine recep-
Therefore, adequate genetic analysis by karyotyping, FISH, tor signaling. The genomic landscape varies across the age,
and gene expression profiling is important to determine prog- clinical features, outcomes, and genetic risk factors [180].
nosis and predictive factors in individual patients [19, 176].
BCR-ABL-like ALL/LBL is characterized by a gene expres-
sion profile similar to that of Ph-positive ALL but lacks the 13.14.4 Caution
BCR-ABL1 translocation [178, 179]. This subtype of
B-precursor neoplasms occurs in all age groups from child- • Burkitt lymphoma/leukemia is not included into this
hood to old age with prevalence in young adults. It is over- category.
• CD79a is not restricted to B-cells but may also be
expressed in specific for a B-lineage derivation but may
Table 13.13 Prognostic groups of B-ALL/LBL according to some also be expressed in T-ALL/LBL and in some cases of
genetic alterations AML.
Favorable Adverse • The blastoid variant of mantle cell lymphoma may resem-
High hyperdiploidy (51–65 Hypodiploidy (≤45 ble B-ALL but is TdT negative and generally express
chromosomes) chromosomes) cyclin D1 or SOX11.
t(12;21)(p13;q22); ETV6-RUNX1 BCR-ABL1 • Hematogones are B-precursors that may be detected espe-
BCR-ABL1-like
cially in bone marrow specimens from infants especially
t(1;19)
iAmp 21
after chemotherapy. They show a continuum of expres-
t(v;11q23.3); KMT2A sion of markers of B-cell differentiation. They are com-
rearranged posed of both immature TdT and CD34+cells as well as
Modified from Taylor et al. [19] more mature CD20+ B-cells negative for CD34.
13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 515

Case History 1: Pro B-ALL with KMT2A (v;11q23)- core biopsy was packed with the same small-sized blasts
Rearrangement (Fig. 13.53) expressing CD19, PAX5, and partially CD34 and TdT but
A 70-year-old woman was admitted with hyperleukocytosis, negative for CD10 and myeloperoxidase. FISH analysis
severe anemia, and thrombocytopenia. The CBC was: WBC revealed a KMT2A (v;11q23)-rearrangement. The diagnosis
212 × 109/L, HB 7.5 g/dL, platelets 22 × 109/L. Peripheral was ALL with KMT2A (v;11q23)-rearrangement according
blood and bone marrow smears showed small blast cells to the 2016 WHO classification. The immunophenotypic
positive for CD19, CD38, CD79a, HLA-DR, CD15 and par- subtype according to the maturation stage was pro B-ALL
tially for TdT and CD34. Blasts were negative for CD10, or early precursor B-ALL according to the
MPO, CD7, CD20, CD33, and CD117. The bone marrow EGIL-classification.

a b

MGG MGG

c d

Giemsa Giemsa

Fig. 13.53 Pro B-ALL with KMT2A (v;11q23)-rearrangement. (a, b) tration of the bone marrow core biopsy by lymphoblasts (Giemsa).
High number of small lymphoblasts in the peripheral blood (a) and Blasts are partially positive for CD34 (e) and TdT (f) and express CD19
bone marrow (b) smears (May-Grünwald-Giemsa). (c, d) Dense infil- (g) and PAX5 (h)
516 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD34 TdT

g h

CD19 PAX5

Fig. 13.53 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 517

Case History 2: Common B Lymphoblastic Leukemia in expressed cy79a, cyCD34, sCD34, sCD33, HLA-DR,
Down Syndrome (Fig. 13.54) sCD19, sCD1O, sCD22, and cyTdT and were negative for
A 24-year-old woman with Down syndrome (DS) was cyCD3 and myeloperoxidase. The karyotype was 47,XX,+21
admitted to the hospital because of pancytopenia. Myeloid indicating a constitutional trisomy 21. No other abnormality
leukemia associated with DS was suspected. The CBC was: was present. No translocation t(9;22)/BCR-ABL or MLL
WBC 2.8 × 109/L, HB 6.9 g/dL, platelets 29 × 109/L. The (11q23)-rearrangement was detected. The bone marrow
differential count showed 8% small-sized myeloperoxidase- core biopsy was diffusely infiltrated by small-sized blast
negative agranular blasts with scanty cytoplasma, 3% cells that were negative for myeloperoxidase but strongly
myelocytes, 2% band forms, 3% band forms, 4% mono- expressed CD34, CD10, CD19, TdT, and PAX5. In addition,
cytes, 23% neutrophils, 5% eosinophils, and 51% lympho- a weak co-expression of CD33 was observed. The diagnosis
cytes. By multiparametric flow cytometry, the CD45+ blasts was common B-ALL in DS.

a b

MGG Giemsa

c d

Giemsa MPO

Fig. 13.54 Common acute lymphoblastic leukemia in Down syn- presence of some eosinophils (Giemsa). (d) Myeloperoxidase is
drome. (a) Blood smear illustrates the presence of rare small-sized lym- expressed in rare residual granulopoietic precursors while the blast
phoblasts with fine open chromatin, small nucleoli, and a small rim of population is completely negative. (e–h) The blasts strongly express
cytoplasm (MGG). (b, c) The bone marrow core biopsy is densely infil- CD10 (e), CD34 (f), CD19 (g), and TdT (h)
trated by blast cells that account for >90% of nucleated cells. Note the
518 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD10 CD34

g h

CD19 TdT

Fig. 13.54 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 519

Case History 3: B-Lymphoblastic Leukemia with t(9;22) cCD3, CD11c, CD15, CD33, cIgM, and myeloperoxidase.
(q34.1;q11.2);BCR-ABL1 (Fig. 13.55) Bone marrow aspirate smears and the core biopsy showed a
A 37-year-old woman developed bone pain and fever. The CBC dense infiltration by lymphoblasts. Karyotyping and FISH anal-
was: WBC 56 × 109/L, HB 6.3 g/dL, platelets 48 × 109/L. The ysis revealed a t(9;22)(q34.1;q11.2); BCR-ABL1. By multiplex
peripheral blood contained 85% medium-sized blasts that were PCR, a BCR-ABL minor fusion transcript was detected. The
positive for CD19, cCD22, cCD79a, CD10, HLA DR, and TdT diagnosis was B-lymphoblastic leukemia with t(9;22)
and partially positive for CD20 and CD13 but negative for (q34.1;q11.2); BCR-ABL1.

a b

MGG Giemsa

c d

Giemsa CD19

Fig. 13.55 B-lymphoblastic leukemia with t(9;22)(q34.1;q11.2); and a pale basophilic cytoplasm. Note the presence of scattered pro-
BCR-ABL1. (a) A peripheral blood smear showing abundant medium- erythroblasts in b (Giemsa). (d) The strongly CD19+ lymphoblasts sur-
sized lymphoblasts with slightly irregular nuclei and evenly dispersed round a residual CD19-negative megakaryocyte. (e–h) The neoplastic
chromatin (May-Grünwald-Giemsa). (b, c) The trephine bone marrow cells also express CD10 (e), PAX5 (f), CD34 (g), and TdT (h)
core biopsy is densely infiltrated by blast cells with prominent nucleoli
520 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD10 PAX5

g h

CD34 TdT

Fig. 13.55 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 521

Case History 4: Mature B-Lymphoblastic Leukemia CD19, CD20, CD79a, surface Kappa-light chain, and
with TP53 Mutation (Fig. 13.56) IgM. They were partially positive for TdT and CD34 but
A 54-year-old woman developed hepatosplenomegaly, rap- were negative for CD10, myeloperoxidase, CD33, and
idly progressing lymphadenopathy, and fever. The CBC was: CD117. Genetic analysis showed a TP53 mutation indicating
WBC 48 × 109/L, HB 10.2 g/dL, platelets 19 × 109/L. The a high-risk group. In contrast to Burkitt lymphoma/leuke-
peripheral blood and bone marrow smears showed about mia, no MYC translocation was detected.
90% small- to medium-sized blasts that were positive for The diagnosis was mature B-ALL with TP53 mutation.

a b

Giemsa Giemsa

c d

CD79a CD10

Fig. 13.56 Mature B-lymphoblastic leukemia with TP53 mutation. for CD10 (d). Blasts expressed CD20 (e), IgM (f), and were partially
(a–h) Sections cut from bone marrow core biopsy showing a dense positive for p53 (g). The Ki67+ proliferation fraction was about 98%
infiltration by blasts (a, b, Giemsa) positive for CD79a (c) but negative (h)
522 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD20 IgM

g h

p53 Ki67

Fig. 13.56 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 523

Case History 5: Pre-B-ALL with Subtle Bone Marrow aspirate and core biopsy were performed and contained about
Infiltration at Relapse (Fig. 13.57) 20% blasts that were positive for CD34, CD19, CD22, TdT,
A 44-year-old woman was diagnosed with pre-B- surface μ chain, and partially for CD10 but negative for CD20,
ALL. Treatment by standard chemotherapy achieved complete CD3, CD33, CD117, and myeloperoxidase. In addition, an
remission. When she presented a year later for hematological aberrant expression of CD7 was present.
control, the CBC was: WBC 4.3 × 109/L, HB 12.6 g/dL, plate- Hematogones were excluded because of the homogeneous
lets 204 × 109/L. However, cytologic evaluation of peripheral immunophenotype and the aberrant expression of CD7 by
blood smears revealed rare myeloperoxidase-negative blasts B-lineage blasts. The diagnosis was relapse of a pre-B-ALL
accounting for about 4% of nucleated cells. A bone marrow with subtle bone marrow and peripheral blood involvement.

a b

MGG MGG

c d

Giemsa Giemsa

Fig. 13.57 Pre-B-ALL with subtle bone marrow infiltration at relapse. showing scattered blasts between hematopoietic precursors (Giemsa).
Rare lymphoblasts in a peripheral blood (a) and bone marrow aspirate (b) Immunohistochemical staining for CD19 (e), CD10 (f), CD34 (g), and TdT
smears (May-Grünwald-Giemsa). Note discrete signs of dyshematopoiesis (h) highlights interstitial bone marrow infiltration by B-lymphoblastic leu-
after chemotherapy. (c, d) Slightly hypercellular bone marrow core biopsy kemia without significant replacement of hematopoiesis
524 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

CD19 CD10

g h

CD34 TdT

Fig. 13.57 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 525

Case History 6: Pro-B-ALL with Partial Spontaneous aspirate smear was hypocellular and showed fat cells and
Bone Marrow Necrosis in a Patient with Down cellular debris. A bone marrow core biopsy was performed
Syndrome (Fig. 13.58) and revealed extensive bone marrow necrosis. Adjacent
A 43-year-old man with Down syndrome was admitted areas were infiltrated by small-sized blasts strongly express-
with pneumonia, bone pain, and tricytopenia. The CBC ing CD79a and TdT but negative for CD10. The necrotic
was: WBC 2.1 × 109/L, HB 6.1 g/dL, platelets areas were weakly immunolabeled by CD79a antibody. The
12 × 109/L. The differential count showed 30% neutrophils, diagnosis was pro-B-ALL (early B-ALL) with partial spon-
5% band forms, 2% metamyelocytes, 1% monocytes, 59% taneous bone marrow necrosis in a patient with Down
lymphocytes, and 3% small-sized blasts. Bone marrow syndrome.

a b

MGG MGG

c d

Giemsa Giemsa

Fig. 13.58 Pro-B-ALL with partial bone marrow necrosis in a patient of necrosis (c, d, Giemsa) and areas densely infiltrated by blasts (c, e,
with Down syndrome. (a) A rare peripheral blood lymphoblast with a f). (g) CD79a is strongly expressed by blasts that weakly stain the
high nuclear:cytoplasmic ratio, condensed chromatin, and inconspicu- necrotic areas. (h) Strong nuclear immunolabeling of non-necrotic blast
ous nucleoli (MGG). (b) Bone marrow aspirate showing fat cells and cells by TdT antibody
nuclear debris (MGG). (c–h) Bone marrow core biopsy showing areas
526 13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies

e f

Giemsa Giemsa

g h

CD79a TdT

Fig. 13.58 (continued)

13 Acute Leukemia of Myeloid, Lymphoid, and Ambiguous Lineage and Related Malignancies 527

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Bone Marrow Biopsy Pathology: Christine Beham-Schmid Annette Schmitt-Graeff

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Bone Marrow Biopsy Pathology: Christine Beham-Schmid Annette Schmitt-Graeff

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Essentials of Diagnostic Pathology

Series Editor: Farid Moinfar

Bone Marrow Biopsy

Bone Marrow Biopsy

ISSN 2194-6256 ISSN 2194-6264 (electronic)

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

2.6 Kostmann Disease (Infantile Genetic Agranulocytosis,

5.2 Monoclonal Gammopathy of Undetermined Significance (MGUS)������������������� 105

7.6.3 Immunohistochemistry ��������������������������������������������������������������������������� 162

7.15.4 Cytogenetic Abnormalities and Molecular Characteristics��������������������� 203

10 Myeloid/Lymphoid Neoplasms with Eosinophilia

11.6 Myelodysplastic/Myeloproliferative Neoplasm with Ring

13.3.2 Acute Myelogenous Leukemia (AML) with

13.4.2 AML with Biallelic Mutation of CEBPA��������������������������������������������� 429

Abbreviations hematopoiesis, which is present in skull, sternum, clavicle,

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 1

Fig. 1.1 (continued)

Fig. 1.2 (continued)

Fig. 1.4 (continued)

1.3 Cellular Components These erythroblastic islands, called erythrones, usually

Fig. 1.5 (continued)

Fig. 1.6 (continued)

Fig. 1.8 (continued)

Fig. 1.9 (continued)

Fig. 1.11 (continued)

1.3.7 Plasma Cells (Fig. 1.13) 5. Plasma cells are polyclonal.

Fig. 1.14 (continued)

References 18. Naeim F. Topobiology in hematopoiesis. Hematol Pathol.

2.1  ommon Non-neoplastic Erythroid

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 27

Fig. 2.1 (continued)

Fig. 2.2 (continued)

Fig. 2.3 (continued)

Fig. 2.4 continued)

2.2.5 Aplastic Anemia 2. Severe Aplastic Anemia (SAA).

Fig. 2.5 continued)

Fig. 2.6 (continued)

2.3  one Marrow Biopsy in Collagen

Fig. 2.7 (continued)

Case 2 SLE. Thus, be careful with a diagnosis of myelodysplasia;

CD3 CD20 kappa lambda

Fig. 2.8 (continued)

2.4  one Marrow Involvement

Fig. 2.9 (continued)

Fig. 2.9 (continued)

kappa lambda IgG4

Fig. 2.9 (continued)

2.5.1 Severe (Acquired) Neutropenia Case History (Agranulocytosis)

2.6  ostmann Disease (Infantile Genetic

Fig. 2.11 (continued)

Fig. 2.13 (continued)

2.9  uantitative and Qualitative Changes

Megakaryocytes are the largest cells normally seen in the bone

2.10 Micromegakaryocytes A left shifted megakaryocytopoiesis without atypia is

Fig. 2.15 (continued)

Fig. 2.15 (continued)

2.11 Enlarged Megakaryocytes

Such forms are seen in MPNs, mostly in ET [33]. Usually,

2.12  egakaryocytes with Naked Nuclei

Naked or “denuded” megakaryocytes can be seen in MPNs,

2.13 Clustering of Megakaryocytes 2.13.1 Caution

Fig. 2.18 (continued)

Fig. 2.18 (continued)

Fig. 2.19 (continued)

2.15 Paratrabecular Location Rarely, paratrabecular megakaryocytes can be seen in reac-

Fig. 2.20 (continued)

Fig. 2.21 (continued)

2.17  one Marrow Alterations After

Fig. 2.22 (continued)

Fig. 2.23 (continued)

2.19  one Marrow Alterations after Bone

Fig. 2.25 (continued)

References 20. Sato Y, Kojima M, Takata K, Morito T, Mizobuchi K, Tanaka T,

© Springer-Verlag GmbH Germany, part of Springer Nature 2020 81

3.1.2 Hyperparathyroid Osteodystrophy formations and rarefication with osteomalacia is found.

Fig. 3.2 (continued)

2.6 Kostmann Disease (Infantile Genetic Agranulocytosis,

5.2 Monoclonal Gammopathy of Undetermined Significance (MGUS)�� 105

7.6.3 Immunohistochemistry �� 162

7.15.4 Cytogenetic Abnormalities and Molecular Characteristics�� 203

10 Myeloid/Lymphoid Neoplasms with Eosinophilia

11.6 Myelodysplastic/Myeloproliferative Neoplasm with Ring

13.3.2 Acute Myelogenous Leukemia (AML) with

13.4.2 AML with Biallelic Mutation of CEBPA�� 429

1.3 Cellular Components These erythroblastic islands, called erythrones, usually

1.3.7 Plasma Cells (Fig. 1.13) 5. Plasma cells are polyclonal.

2.1 ommon Non-neoplastic Erythroid

2.2.5 Aplastic Anemia 2. Severe Aplastic Anemia (SAA).

2.3 one Marrow Biopsy in Collagen

2.4 one Marrow Involvement

2.5.1 Severe (Acquired) Neutropenia Case History (Agranulocytosis)

2.6 ostmann Disease (Infantile Genetic

2.9 uantitative and Qualitative Changes

2.10 Micromegakaryocytes A left shifted megakaryocytopoiesis without atypia is

2.11 Enlarged Megakaryocytes

2.12 egakaryocytes with Naked Nuclei

2.13 Clustering of Megakaryocytes 2.13.1 Caution

2.15 Paratrabecular Location Rarely, paratrabecular megakaryocytes can be seen in reac-

2.17 one Marrow Alterations After

2.19 one Marrow Alterations after Bone

3.1.2 Hyperparathyroid Osteodystrophy formations and rarefication with osteomalacia is found.

3.1.4 Osteomalacia deficiencies caused by renal, intestinal, or hepatic diseases as

4.1 Caution tion might be missed without immunohistochemical stains.

5.1 Reactive Plasmacytosis Case History 1

5.2 Monoclonal Gammopathy plasma cell infiltration without myeloma-defining symptoms

5.2.4 Immunohistochemistry undermined significance. Without clinical information, an

5.3 lasma Cell Myeloma with Amyloid

6.1.1.1 Caution 2. T-NK-cell lymphoma or leukemia (there are obvious

Case History 4 6.2.1 Caution

6.3 Parvovirus B19 Infection (Fig. 6.8) Case History

tochemically, no megakaryocytes were found. Granulopoiesis 6.3.1 Caution

6.4 Infection with Mycobacterium Case History

7.2 Chronic Lymphocytic Leukemia (CLL) 7.2.3 Transformation of CLL

7.2.1 Epidemiology Transformation to a large cell lymphoma is seen during the

7.2.6 Caution Paratrabecular infiltration does not exclude infiltration

7.3 -Cell Prolymphocytic Leukemia

7.3.5 Caution The appearance of B-PLL in bone marrow biopsies may

7.4 Lymphoplasmacytic Lymphoma (LPL) 7.4.3 Immunohistochemistry

7.4.1 Epidemiology Characteristically, LPLs show a monoclonal B-cell pheno-

7.4.5 Caution myeloma. Lack of bone remodeling, lack of CD56-staining

7.5.5 Caution proliferation centers of B-CLL), and also in plasma cell