Flow Cytometry in Neoplastic Hematology Morphologic-immunophenotypic Correlation Includes Digital Download

465 Pages·2017·25.96 MB·English

by  Wojciech Gorczyca

Flow Cytometry in Neoplastic Hematology
Morphologic–Immunophenotypic Correlation
Third Edition

Flow Cytometry in Neoplastic Hematology
Morphologic–Immunophenotypic Correlation
Third Edition
Wojciech Gorczyca, MD, PhD
BioReference Laboratories
Elmwood Park, New Jersey, USA
CRC Press
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Library of Congress Cataloging-in-Publication Data
Names: Gorczyca, Wojciech, 1959- author.
Title: Flow cytometry in neoplastic hematology : morphologic-immunophenotypic correlation / Wojciech Gorczyca.
Description: Third edition. | Boca Raton, FL : CRC Press/Taylor & Francis Group, 2017. | Includes bibliographical references and index.
Identifiers: LCCN 2016045629| ISBN 9781498775021 (pack-book and ebook : alk. paper) | ISBN 9781498775038 (ebook) | ISBN 9781498775045 (ebook)
Subjects: | MESH: Hematologic Neoplasms—diagnosis | Flow Cytometry | Immunophenotyping | Leukemia, Lymphoid—diagnosis | Leukemia,
Myeloid, Acute—diagnosis
Classification: LCC RC280.H47 | NLM WH 525 | DDC 616.99/418—dc23
LC record available at https://lccn.loc.gov/2016045629
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Contents
Preface to the Third Edition vi
Abbreviations vii
1.  Introduction 1
2.  Gating Strategies 15
3.  Classification of Hematopoietic Tumors 25
4.  Identification of Clonal B-Cell Populations 29
5.  Identification of Abnormal T-Cell Populations 44
6.  Identification of Myeloblasts 60
7.  Identification of B-Lymphoblasts 84
8.  Identification of T-Lymphoblasts 94
9.  Antigen Expression during Myeloid and Lymphoid Maturation 99
10.  Identification of Myelodysplastic Syndrome by Flow Cytometry 105
11.  Phenotypic Markers 115
12.  Morphologic–Flow Cytometric Correlation 126
13.  Molecular-Flow Cytometric Correlation 145
14.  Flow Cytometry Limitations 150
15.  Phenotypic Classification of B-Cell Lymphoproliferations 156
16.  Mature B-Cell Lymphoproliferations 163
17.  Plasma Cell Neoplasms 227
18.  Mature T/NK-Cell Lymphoproliferations 240
19.  Hodgkin Lymphoma 302
20.  B-Cell Lymphoblastic Leukemia/Lymphoma 309
21.  T-Cell Lymphoblastic Leukemia/Lymphoma 322
22.  Myelodysplastic Syndromes 331
23.  Chronic Myelomonocytic Leukemia and Other Myelodysplastic/Myeloproliferative Neoplasms 341
24.  Myeloproliferative Neoplasms 353
2 5.  Acute Myeloid Leukemia: Introduction 378
26.  Acute Myeloid Leukemia with Recurrent Genetic Changes 392
27.  Acute Myeloid Leukemia, Not Otherwise Specified 412
28.  Other Tumors 434
Index 451
v
Preface to the Third Edition
This third edition is the result of the author’s 25 years of experi- MPN, chronic myelomonocytic leukemia (CMML), and other
ence with flow cytometry (1991–2016). It incorporates the revised chronic myeloid neoplasms is significantly expanded. This
2016 World Health Organization (WHO) classification of hema- third edition provides expanded criteria for identifying blasts,
topoietic tumors. The number of illustrations has increased from with emphasis on differential diagnosis between various types
372 in the second edition to 489 in this edition, the majority being of acute leukemias and between blasts and maturing elements
composites in color. The text is significantly expanded and bet- with or without dysmaturation, and detailed discussion of leu-
ter organized to help early as well as experienced flow cytometry kemia-associated phenotype (LAP). Acute myeloid leukemias
users dealing with both standard and difficult cases. Chapters (AMLs) and their differential diagnoses are now discussed in
1–14 present flow cytometry features helpful in identification of greater detail, utilizing the revised WHO classification, includ-
abnormal population(s), and Chapters 15–28 provide systematic ing distinct immunophenotypic patterns of acute promyelocytic
descriptions of hematopoietic neoplasms. Each chapter has its leukemia (APL) and AML with NPM1 mutations, pure erythroid
own up-to-date references. leukemia versus high-grade MDS, differential diagnosis between
Although the book covers the wide spectrum of hematopoi- hypogranular APL, acute monoblastic leukemia, and AML with
etic tumors, the focus remains on the most important clinical NPM1 and/or FLT3 mutations, and acute monoblastic leukemia
diagnoses, such as acute promyelocytic leukemia, identification versus blastic plasmacytoid dendritic cell neoplasm (BPDCN).
of blasts, identification of clonal B-cell population, differentiat- The statistical analysis of immunophenotypic features of T-cell
ing mature vs. immature T-cell proliferations, differential diag- disorders, CMML, and acute leukemias incorporates hundreds
nosis between hematogones and B-lymphoblastic leukemia/ of new cases. Additional detailed criteria are provided for iden-
lymphoma (B-ALL), or distinction between chronic and acute tification of abnormal B- and T- populations, with significantly
monocytic proliferations. expanded differential diagnosis. New tables and expanded tables
The number of chapters has increased from 13 to 28. New from the previous edition summarize phenotypic features, clas-
chapters include topics such as the correlation of immunophe- sification, prognosis, and differential diagnosis on most of the
notypic features with specific morphologic or laboratory find- entities discussed.
ings (e.g., cytopenia, hyperleukocytosis, circulating plasma cells,
etc.) and the correlation between flow cytometry findings and Wojciech Gorczyca MD, PhD
molecular (genetic) changes. Chapter 13 of the previous edition Chief for Hematopathology Services
(Myelodysplastic syndromes and chronic myeloproliferative Medical Director of Flow Cytometry
neoplasms) is now expanded to three chapters (myelodysplas- BioReference Laboratories, an OPKO Health Company
tic syndrome [MDS], myeloproliferative neoplasm [MPN], Elmwood Park, New Jersey
and mixed myeloproliferative/myelodysplastic neoplasms). The September 2016
description of flow cytometric features associated with MDS,
vi
Abbreviations
ABL1 Abelson leukemia homolog 1 gene ENKTL extranodal NK/T-cell lymphoma, nasal type
aCML atypical chronic myeloid leukemia (BCR-ABL1-) ET essential thrombocythemia
AITL angioimmunoblastic T-cell lymphoma ETP-ALL early T-cell precursor ALL
ALC absolute lymphocyte count FAB French-American-British [classification of acute
ALCL anaplastic large cell lymphoma leukemia]
ALIP abnormal localization of immature precursors FC flow cytometry
ALK anaplastic lymphoma kinase FGFR1 fibroblast growth factor receptor 1 gene
ALL acute lymphoblastic leukemia FISH fluorescence in situ hybridization
AML acute myeloid leukemia FITC fluorescein isothiocyanate
AMML acute myelomonocytic leukemia FL follicular lymphoma
ANC absolute neutrophil count FLT3 Fms-like tyrosine kinase 3 gene
AP accelerated phase FSC forward scatter
APL acute promyelocytic leukemia GCB germinal center B-cell
ATLL adult T-cell lymphoma/leukemia G-CSF granulocyte-colony stimulating factor
ATRA all-trans retinoic acid GPI glycosylphosphatidylinositol
B-ALL B-lymphoblastic leukemia/lymphoma HGBL high-grade B-cell lymphoma
BCL1 B-cell lymphoma 1 (cyclin D1) protein encoded by H&E hematoxylin and eosin
CCND1 gene HCL hairy cell leukemia
BCL2 B-cell lymphoma 2 apoptosis regulating protein HCL-v hairy cell leukemia variant
encoded by BCL2 gene HES hypereosinophilic syndrome
BCR breakpoint cluster region gene HHV-8 human herpesvirus-8
BL Burkitt lymphoma HL Hodgkin lymphoma
BM bone marrow HLA-DR human leukocyte antigen D-related
BP blast phase HPF high-power field
BPDCN blastic plasmacytoid dendritic cell neoplasm HSTL hepatosplenic T-cell lymphoma
B-PLL B-cell prolymphocytic leukemia ICUS idiopathic cytopenia of undetermined significance
BRAF v-raf murine sarcoma viral oncogene homolog B1 IDUS idiopathic dysplasia of undetermined significance
CALLA common acute lymphoblastic leukemia antigen IGVH immunoglobulin heavy-chain variable gene
CBF core-binding factor ISFN in situ follicular neoplasia
CD cluster designation ISMCN in situ mantle cell neoplasia
CEL chronic eosinophilic leukemia ITD internal tandem duplication
CGH comparative genomic hybridization IVLBCL intravascular large B-cell lymphoma
CHIP clonal hematopoiesis of indeterminate potential JMML juvenile myelomonocytic leukemia
CLL chronic lymphocytic leukemia LAP leukemia-associated phenotype
CML chronic myeloid leukemia (BCR-ABL1+) LBL lymphoblastic lymphoma
CMML chronic myelomonocytic leukemia LCH Langerhans cell histiocytosis
CMR complete molecular response LESA lymphoepithelial sialadenitis
CNL chronic neutrophilic leukemia LGL large granular lymphocyte
CP chronic phase L&H lymphocyte and histiocyte cell [popcorn cell;
CR complete response neoplastic cells in NLPHL]
CSF cerebrospinal fluid LP lymphocyte predominant (cell) [neoplastic cells in
DH double hit NLPHL]
DIC disseminated intravascular coagulopathy LPL lymphoplasmacytic lymphoma
DLBCL diffuse large B-cell lymphoma LYG lymphomatoid granulomatosis
EATL enteropathy-associated T-cell lymphoma LyP lymphomatoid papulosis
EBER Epstein–Barr virus early RNA MALT mucosa associated lymphoid tissue
EBV Epstein–Barr virus positivity by in situ hybridization MBL monoclonal B-cell lymphocytosis
EMA epithelial membrane antigen MBR major breakpoint region
EMT extramedullary myeloid tumor (granulocytic MCL mantle cell lymphoma
sarcoma) mcr minor cluster region
vii
abbreviations
MDS myelodysplastic syndrome PLL prolymphocytic leukemia
MDS-EB MDS with excess blasts PMBL primary mediastinal large B-cell lymphoma
MDS-f MDS with fibrosis PMF primary myelofibrosis
MDS-MLD MDS with multilineage dysplasia PML promyelocytic leukemia gene
MDS-RS MDS with ring sideroblasts PNH paroxysmal nocturnal hemoglobinuria
MDS-SLD MDS with single lineage dysplasia PV polycythemia vera
MDS/MPN myelodysplastic/myeloproliferative PTCL peripheral T-cell lymphoma, unspecified
neoplasms PTLD posttransplant lymphoproliferative disorder
MDS/MPN-RS-T MDS/MPN with ring sideroblasts and QR quantitative real-time
thrombocytosis RARA retinoic acid receptor α gene
MEITL monomorphic epitheliotropic intestinal T-cell R-CHOP Rituximab+Cyclophosphami de+Hydroxydauno
lymphoma rubicin+Oncovin+Prednisone
MF mycosis fungoides ROC receiver operating characteristics
MGUS monoclonal gammopathy of undetermined RQ-PCR real-time quantitative PCR
significance RT-PCR reverse transcriptase PCR
MPAL mixed phenotype acute leukemia R-S Reed–Sternberg [cell]
MPN myeloproliferative neoplasm SBP solitary bone plasmacytoma
MPO myeloperoxidase SLE systemic lupus erythematous
MRD minimal residual disease SLL small lymphocytic lymphoma
MRI magnetic resonance imaging SM systemic mastocytosis
MZL marginal zone B-cell lymphoma SMZL splenic marginal zone lymphoma
NHL non-Hodgkin lymphoma SPTL subcutaneous panniculitis-like T-cell lymphoma
NK natural killer SS Sézary syndrome
NLPHL nodular lymphocyte predominant Hodgkin SSB Sudan Black B
lymphoma SSC side scatter
NOS not otherwise specified t-AML therapy-related acute myeloid leukemia
NPM nucleophosmin gene TAM transient abnormal myelopoiesis
NSE nonspecific esterase T-ALL T-cell lymphoblastic leukemia/lymphoma
PAS periodic acid-Schiff stain T follicular helper T-cell
FH
PBL plasmablastic lymphoma TCR T-cell receptor
PCM plasma cell myeloma THRLBCL T-cell/histiocyte-rich large B-cell lymphoma
PCR polymerase chain reaction TdT terminal deoxynucleotidyl transferase
PDGFRA alpha-type platelet-derived growth factor T-LGL T-cell large granular lymphocyte [leukemia]
receptor gene t-MDS therapy-related myelodysplastic syndrome
PDGFRB beta-type platelet-derived growth factor t-MDS/MPN therapy-related myelodysplastic/
receptor gene myeloproliferative neoplasms
PE phycoerythrin T-PLL T-cell prolymphocytic leukemia
PEL primary effusion lymphoma TTF1 thyroid transcription factor 1
PerCP peridinium chlorophyll protein complex WBC white blood cell [count]
PFL pediatric follicular lymphoma WHO World Health Organization
Ph Philadelphia chromosome [result of t(9;22) WM Waldenström macroglobulinemia
translocation]
viii
1
Introduction
Flow cytometry (FC) plays a very important role in diagnosis, of thiazole orange (TOT-1) [38]. Data are displayed in single-
subclassification, and post-treatment monitoring of hematologic parameter histogram and 2-parameer dot plots [8,13,39,40].
neoplasms [1–26]. FC results allow one to choose in a timely Most of the antigens detected by FC are located on the cell sur-
manner the most appropriate further testing (such as FISH or face. In order to stain intracellular components, cells have to
PCR) to establish the definite diagnosis and to further character- be permeabilized (so that the antibody can enter cells through
ize the malignant process. Multiparameter FC measures simul- “holes” in their membrane). The optimal number of reagents
taneously several surface and/or intracytoplasmic markers on a required to evaluate hematolymphoid neoplasms depends on the
single cell, allowing for accurate phenotypic characterization of clinical diagnosis, relevant laboratory data, and differential diag-
analyzed population(s). While no single marker permits a defi- nosis based on cytomorphology [26,41]. Flow cytometry is much
nite lineage assignment, analysis with panels of antibodies allows faster than immunohistochemistry and can analyze thousands
for separation of hematologic tumors into very precise subtypes of cells within seconds. Another advantage of FC immunophe-
with different prognosis and treatment requirements, as defined notyping is that it allows correlation of several markers on a sin-
by current World Health Organization (WHO) classification of gle cell, and detects intensity of staining and aberrant expression
hematopoietic and lymphoid tumors [27]. FC analysis can pre- of antigens. FC has high sensitivity for B-cell lymphoprolifera-
cisely differentiate between B- and T-cell malignancies, between tive disorders and acute leukemia and high specificity for several
mature (peripheral) and precursor tumors, and among the latter, categories of those neoplasms. All these properties are used in
determine the myeloid or lymphoid lineage. In acute leukemias, diagnostic hematopathology for subclassification of neoplasms.
the role of FC is not limited to identification of blasts, but expands The major disadvantage of FC is a need for liquid cell suspension
to determine the lineage and specific phenotype, which often and therefore lack of c orrelation with histomorphologic features
prompts a dditional testing for final subclassification of leukemia. (tissue architecture). FC requires viable fresh (unfixed) mate-
For example, blasts (or blast equivalents) with high side scatter rial. In a subset of neoplasms, especially high-grade lymphomas,
(SSC), positive CD117, CD13, and CD33 and negative HLA-DR decreased viability often precludes accurate FC analysis. Flow
raise the possibility of acute promyelocytic leukemia (APL), cytometry analysis requires at least 10,000–20,000 cells (events)
which can be confirmed by testing for PML-RARA. Myeloblasts acquired by tube, which often limits its use in specimens from
with a berrant co-expression of CD19 and CD56 suggest acute CSF, fine needle aspirates, and paucicellular (or fibrotic) lesions.
myeloid leukemia (AML) with t(8;21) and B-lymphoblasts with Dropout of neoplastic cells due to low viability or sample bias
aberrant expression of CD13 or CD33 suggest either BCR-ABL1 due to focal (partial) tissue involvement may lead to false nega-
or ETV6-RUNX1 rearrangements. Availability of new markers tive flow results (see also Chapter 14).
(antibodies), new fluorochromes, and improvement in instru-
mentation increases accuracy of FC studies and allows for identi-
the major role of flow cytometry analysis
fication of a minute abnormal population among the majority of
Identification of Acute Promyelocytic Leukemia
benign cells, evaluates minimal residual disease (MRD), and also
Acute promyelocytic leukemia (APL) is characterized by high
expands FC applications into such disorders as myelodysplastic
SSC, positive CD117, negative CD11c, CD34, and HLA-DR, and
syndromes, paroxysmal nocturnal hemoglobinuria (PNH), and
positive myeloid markers (myeloperoxidase, CD13, and CD33)
myeloproliferative neoplasms [9,18,26,28–34].
(Figure 1.1). The less common, hypogranular variant of APL
FC analysis requires fresh (unfixed) material. Types of speci-
shows a similar phenotype to classic APL, except for low SSC
mens suitable for FC include blood, bone marrow (BM) aspirate,
and often positive expression of CD2 and CD34. For details see
fresh tissue samples, fine needle aspirates, effusions (pleural,
Chapters 6, 25, and 26.
peritoneal), and other body fluids (e.g., cerebrospinal fluid; CSF).
In FC protocol, the sample is incubated with antibodies, followed
by red blood cell lysis, washing, fixation in paraformaldehyde, Identification of Blasts, Their Lineage and Phenotype
and FC analysis. Whole blood lysis represents the most com- Myeloblasts are usually characterized by expression of CD117
monly used technique for sample preparation [35–37]. Routinely and/or CD34, HLA-DR, CD133, CD13, CD33, and CD38. The
5,000 to 10,000 cells are collected (in MRD protocols 500,000 expression of CD45 is moderate and SSC is low (Figure 1.2).
events). Monoclonal antibodies used in FC are conjugated with Subsets of myeloblasts may be positive for TdT, CD123, CD71,
fluorochromes, which are excited or stimulated by laser(s) in flow CD7, and CD11c. Occasional cases may be HLA-DR-negative.
cytometer. The most common fluorochromes excited at 488 nm AML is diagnosed with ≥20% blasts (or blasts equivalents) in
(argon laser) include fluorescein isothiocyanate (FITC), phyco- the BM, except for AML with t(15;17), inv(16), or t(8;21), which
erythrin (PE), propidium iodide (PI), 7-amino-actinomcyin D do not require a 20% threshold for the diagnosis. For details see
(7AAD), peridin-chloryophyl-A-protein (PerCP), and dimmers Chapters 6 and 25 through 27.
1