Cytogenetics, FISH and Molecular Testing in Hematologic

Malignancies - 1st Edition

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© 2008 Informa UK Ltd

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Gorczyca-FM 11/12/07 3:14 PM Page v

Contents

Preface vii
Acknowledgments ix

Chapter 1. Introduction 1
Karyotype 2
Nomenclature 3
Conventional cytogenetics 4
Fluorescence in situ hybridization 9
Molecular pathology 19
Immunophenotyping by flow cytometry 23
Immunohistochemistry 26

Chapter 2. Applications of cytogenetics, FISH, and PCR in diagnosis, 31

prognosis, and disease monitoring
Most common chromosomal/molecular abnormalities 31
Application of chromosomal and molecular markers in diagnosis 97
Application of chromosomal and molecular markers in prognostification 106
Application of chromosomal and molecular markers in disease monitoring 108

Chapter 3. Hematologic malignancies: correlation between morphology, phenotype, 115

and chromosomal/genetic markers
B-cell lymphoproliferations 115
Peripheral (mature) T-cell lymphoproliferative disorders 166
Hodgkin lymphoma 182
Acute myeloid leukemia 183
Precursor lymphoblastic lymphoma/leukemia (acute lymphoblastic leukemia) 198
Chronic myeloproliferative disorders 205
Mastocytosis 224

v
Gorczyca-FM 11/12/07 3:14 PM Page vi

vi CONTENTS

Myeloproliferative/myelodysplastic diseases 225

Myelodysplastic syndromes 226
Abbreviations/terminology 233

References 235
Index 311
Gorczyca-FM 11/12/07 3:14 PM Page vii

Preface

Cytogenetics, fluorescence in situ hybridization additions of genetic material (e.g. monosomies and
(FISH) and molecular tests, especially polymerase trisomies). Nonrandom chromosomal aberrations
chain reaction (PCR), play an important role in the detected by G-banding, FISH, and/or PCR identify
management of patients with hematologic malignan- patients at different risk and help to determine the
cies by helping to establish the diagnosis, as well as most appropriate management (e.g. aggressive treat-
predict prognosis, response to treatment and disease ment in high-risk groups versus the wait-and-watch
progression.1–48 Chromosomal and molecular abnor- approach or less aggressive treatment for low-grade
malities provide the most reliable criteria for classifica- neoplasms). Cytogenetic and molecular analysis helps
tion of hematopoietic tumors and often comprise the to identify chromosomal/oncogenic abnormalities
basis for targeted therapy. For example, Philadelphia that can be targeted by specific therapies exemplifying
chromosome (Ph), rising from t(9;22),BCR/ABL con- the ever-expanding personalized approach to manage-
firms the diagnosis of chronic myeloid leukemia ment of cancer patients. Imatinib (Gleevec) and novel
(CML) while the presence of t(15;17)PML/RARa confirms tyrosine kinase inhibitors (Dasatinib, Nilotinib) suc-
the diagnosis of acute promyelocytic leukemia (APL). cessfully target BCR/ABL fusion in CML. Neoplastic
Others include: t(8;21) or inv(16) in a subset of acute cells in CEL or systemic mastocytosis with
myeloid leukemia (AML), chromosome 14 abnormali- eosinophilia with FIP1L1/PDGFRA also respond to
ties in T-prolymphocytic leukemia (T-PLL), iso(7q) in imatinib, albeit with higher sensitivity. All-trans-
hepatosplenic T-cell lymphoma, t(11;14)CCND1/IGH in retinoic acid (ATRA) therapy revolutionized treat-
mantle cell lymphoma, t(14;18)IGH/BCL2 in follicular ment of APL by targeting PML/RARα fusion.
lymphoma, t(2;5)NPM/ALK in anaplastic large cell lym- Myelodysplastic plastic (MDS) patients with del(5q)
phoma (ALCL), t(8;14)c-MYC/IGH in Burkitt respond to lenalidomide. A combination of molecular
lymphoma, JAK2V617F mutation in classic non-CML genetic techniques and G-banding allows for early
chronic myeloproliferative disorders, especially detection of recurrence, identification of low levels of
Polycythemia vera, and cryptic deletion on 4q12 asso- residual disease (minimal residual disease; MRD), and
ciated with the FIP1L1/PDGFRA fusion gene in evaluation of engraftment status after allogeneic stem
chronic eosinophilic leukemia (CEL) or systemic mas- cell transplantation.
tocytosis with eosinophilia. Lymphomas most often This book provides a review of chromosomal and
display balanced translocations, whereas myelodys- molecular changes in hematologic malignancies and
plastic syndromes are often marked by deletions or correlates the karyotypic and genetic abnormalities

vii
Gorczyca-FM 11/12/07 3:14 PM Page viii

viii PREFACE

with morphology, immunophenotype, and clinical Chapter 3 covers specific hematologic malignancies
data. It is divided into three chapters. The first and provides a short description of the clinical,
describes the karyotype and the basics of cytogenetics, pathologic, and cytogenetic/molecular characteristics.
FISH, and molecular testing. Chapter 2 lists the most
common chromosomal and molecular aberrancies and
their utility in diagnosis, prognosis, and monitoring. Wojciech Gorczyca
Gorczyca-FM 11/12/07 3:14 PM Page ix

Acknowledgments

I would like to express my great appreciation to encouragement, expertise and professionalism during
Dr Kelly Cornish (Commissioning Editor at work on this book as well as my prior publications.
Informa Healthcare, London, UK) for her help and This book would not possible without the help of
support during preparation of this book. I am also Michael Lorenzo, Joseph Tripodi, and Charles
grateful to Robert Peden and the whole staff of Barnabe from Genzyme’s laboratory in New York,
Informa Healthcare in the UK for years of support, whose contributions I want to acknowledge.

ix
Gorczyca-FM 11/12/07 3:14 PM Page x
Gorczyca-Ch01 11/7/07 3:42 PM Page 1

CHAPTER 1

Introduction

Recurring chromosomal abnormalities are involved the diagnosis or management of hematologic disor-
in the pathogenesis of hematologic malignancies and ders. Automation and high sensitivity have led to the
are important indicators for their diagnosis and recent increase in popularity of PCR technologies in
prognosis. The methods used to detect the genetic diagnosis and especially in disease monitoring.
changes in hematologic malignancies include: Many chromosomal and molecular changes define
specific hematologic entities and syndromes, and
(a) conventional cytogenetics (karyotyping on cells have important therapeutic and prognostic impact:
derived from direct preparations or short-term t(15;17)PML/RARa is characteristic for acute promyelo-
cultures using banding analysis; G-banding) cytic leukemia (APL), a unique variant of acute
(b) molecular cytogenetics, e.g. fluorescence in situ myeloid leukemia (AML) treated with ATRA and
hybridization (FISH), multicolor FISH, and arsenic dioxide; t(8;21) or inv(16) comprises the
spectral karyotyping (SKY) favorable risk group of AML, whereas a complex
(c) molecular techniques to analyze DNA, RNA, or karyotype in AML predicts a poor prognosis;
proteins directly, e.g. the polymerase chain reac- t(2;5)NPM/ALK defines a subset of anaplastic large cell
tion (PCR), reverse transcriptase PCR (RT-PCR), lymphoma associated with a good prognosis and
quantitative real-time RT-PCR (RQ-PCR; qRT- chemosensitivity; t(8;14)MYC/IGH is important in the
PCR), Southern blotting, and microarray analysis. diagnosis of Burkitt’s lymphoma and, when accom-
panied by BCL2 or BCL6 rearrangements, defines
Conventional cytogenetics using classical the aggressive subset of diffuse large B-cell lym-
karyotyping of chromosomes remains the most phoma; iso(7) is seen typically in hepatosplenic γδ
comprehensive method for assessing chromosome T-cell lymphoma and inv(14) in T-prolymphocytic
abnormalities, especially numeric and structural leukemia (T-PLL). The t(9;22)BCR/ABL is typical for
chromosome aberrations. Technical issues associated chronic myeloid leukemia (CML), although it may
with cytogenetics (e.g. requirement for fresh sample, be seen in a subset of precursor B-lymphoblastic
difficulties in identification of masked or cryptic leukemia (B–ALL) or AML, where it is associated
aberrations due to limited resolution by classic band- with a poor prognosis. Understanding the role of
ing techniques) have resulted in an increased use of BCR/ABL fusion in the pathogenesis of CML led to
molecular cytogenetic techniques, such as FISH, to the development of imatinib (Gleevec), a selective
identify specific abnormalities that are useful in either BCR/ABL kinase inhibitor that replaced interferon
1
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2 CYTOGENTICS, FISH AND MOLECULAR TESTING IN HEMATOLOGIC MALIGNANCIES

and/or allogeneic stem cell transplantation as the 23 pairs (Figure 1.1). Chromosomes are distinct
frontline therapy for CML.42, 49–54 bodies found in the nucleus of cells, best visible in
A significant subset of hematopoietic malignan- the phase of the cell cycle called metaphase.
cies shows lack of chromosomal rearrangements in Chromosomes are composed of protein and DNA
conventional cytogenetic studies and only molecular and hold the genetic information in the form of
tests are able to visualize the underlying genetic linear sequences of four bases (A,T,C,G). The DNA
defect(s). For example, FLT3 mutations belong to sequence for a single trait is called a gene. Each chro-
the most frequent mutations in AML (~23%) with mosome contains a few thousand genes, which range
the majority of patients (~70%) displaying a normal in size from a few thousand bases up to 2 million
karyotype.55–62 Similarly, MLL mutations occur bases. The first 22 pairs are labeled longest to short-
mainly in cytogenetically normal AML.63–68 Molecular est. The last pair are called the sex chromosomes,
technology, especially real-time quantitative RT-PCR which are labeled X or Y. Females have two X chro-
(qRT-PCR), plays an important role in monitoring mosomes (XX), and males have an X and a Y chro-
patients after treatment, best documented in CML mosome (XY). Each chromosome has a short or
patients on imatinib.41,42 p (petit) arm and long or q (next letter in the
alphabet) arm, which are separated by a region
known as the centromere. The centromere is a con-
KARYOTYPE
densed part of the chromosome which binds
A karyotype is a set of the chromosomes from together two sister chromatids and constitutes the
one cell. There are 46 chromosomes occurring in attachment site for spindle fibers during cell division.

1 2 3 4 5

Figure 1.1 Normal karyotype (male)

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y
Gorczyca-Ch01 11/7/07 3:42 PM Page 3

INTRODUCTION 3

Group Chromosomes Characteristics Example

A 1-3 Large metacentric chromosomes

B 4-5 Large submetacentric

chromosomes

C 6-12 and X Medium-sized submetacentric

chromosomes Figure 1.2 Types of chromo-
somes
G 13-15 Large acrocentric chromosomes

E 16-18 Medium-sized acrocentric

chromosomes

F 19-20 Short metacentric chromosomes

G 21-22 and Y Short acrocentric chromosomes

Types of chromosomes are presented in Figure 1.2. include −5, −7, −X, and −Y, and those most commonly
Each chromosome arm is defined further by num- duplicated (+; trisomy) include +4, +6, +8, +9, +10,
bering the bands (light and dark bands are visible +11, +12, +13, +14, +19, +20, and +21. Common
under the microscope after staining with various chromosomal deletions include del(5q), del(6q),
dyes); the higher the number, the further that area is del(7q), del(13q), and del(20q). Isochromosome
from the centromere. The band width and the order (i) is an abnormal chromosome with two chromosome
of bands are specific for each chromosome and allow arms positioned as mirror images of each other
their identification (Figures 1.3–1.25). (duplication of one of the arms, resulting in a meta-
centric chromosome with identical genes on both
arms). The most common isochromosomes include
NOMENCLATURE
i(1q), i(7q), i(9q), i(11q), i(17q), i(21q), and i(22q).
Chromosomal aberrations include numeric and A chromosomal inversion (inv) is a 180° rotation of
structural abnormalities (Figure 1.26). A cell with a chromosome segment (part of a chromosome is
46 chromosomes is called diploid, and a cell with an reversed in orientation). The common inversions
abnormal number of chromosomes is called aneu- include inv(3) and inv(16). A chromosomal transloca-
ploid (<46, hypodiploid; >46, hyperdiploid). An tion (t) is a relocation of material from one chromo-
insertion (ins) is a structural rearrangement in which some to a different chromosome. Translocations can
part of a chromosome is inserted to a new location be either reciprocal (mutual exchange of segments of
on a chromosome. Monosomy refers to a single chromosomes) or Robertsonian (centric fusion of the
chromosome and trisomy to three chromosomes. long arms of acrocentric chromosomes and loss of
Loss of a chromosome is designated by a minus sign their short arms). Most of the translocations are recip-
(−; monosomy) and loss of part of a chromosome by rocal and result in either synthesis of novel fusion pro-
del. Deletions can be either terminal or interstitial. tein or relocation of an oncogene to a locus that is
The chromosomes that are most commonly lost highly transcribed. The common translocations
Gorczyca-Ch01 11/7/07 3:42 PM Page 4

4 CYTOGENTICS, FISH AND MOLECULAR TESTING IN HEMATOLOGIC MALIGNANCIES

t(1;3) 36.3
MDS 36.2
PRDM16 t(1;3)
36.1
t(1;7)
35
ET 24.3 t(1;14)
MPL 24.2
CIMF 24.1
33
32 TAL1 T-ALL

31
p t(1;14)(p22;q32)
MALT
22 BCL10 lymphoma

AML mutation 21
Acute
MDS NRAS 13 OTT megakaryoblastic
CMML 12 leukemia (AML-M7)
t(1;22)
t(1;14)(q21;q32)
Figure 1.3 Chromosome 1 t(1;22) 12
ALL
B-cell Chronic
BCL9 21 PDE4DIP eosinophilic
Iymphoma leukemia
22 t(1;5)
B-ALL PBX1 23
24
t(1;19) [E2A] 25 TPM3 ALCL
q
31 t(1;2)

32

41

42
43
44
1

include t(9;22), t(15;17), t(14;18), t(11;14), t(8;14), (haploid number). Women have 22 pairs of autoso-
and t(8;21). A Robertsonian translocation product is mal chromosomes and two X chromosomes, while
considered a derivative chromosome and is described men have 22 pairs of autosomal chromosomes and
by der. Marker chromosomes (mar) are abnormal two sex chromosomes, X and Y (Figures 1.3–1.25).
chromosomal structures which cannot be subclassi- Based on the location of the centromere and
fied by cytogenetics. The presence of a marker chro- chromosome size, they can be divided into
mosome is depicted as a +mar. Two cells with metacentric, submetacentric, and acrocentric
identical structural aberrations are defined as a clone. and subclassified into seven groups (A to G)
Most common terms and abbreviations used in cyto- (Figure 1.2). The structures of all chromosomes and
genetics/FISH studies are presented in Table 1.1. the location of most common genes involved
in hematopoietic malignancies are presented in
Figures 1.3 through 1.25.
CONVENTIONAL CYTOGENETICS
Cytogenetic aberrations, both structural (transloca-
Cytogenetics is the study of chromosomes. Somatic tions and inversions) and numeric (deletion or gain of
cells in humans have 46 chromosomes (diploid whole or portion of chromosome) occur in the major-
number) and gametic cells have 23 chromosomes ity of hematopoietic tumors.5,8,10,13,15,22,28,69–109
Gorczyca-Ch01 11/7/07 3:42 PM Page 5

INTRODUCTION 5

25
24
23 ALK ALCL
22
21
t(2;5)
p 16 t(X;2)
t(2;14) 15 t(1;2)
B-CLL 14 t(2;3)
BCL11A
13 t(2;17)
12 t(2;22)
IGK
inv(2)
11.2

DLBCL t(2;3) [BCL6]

11.2
Burkitt 12
DLBCL t(2;8) [MYC] 13
B-ALL 14.1 Figure 1.4 Chromosome 2
14.2
14.3
21
22
23
q 24

31

32.1
32.2
32.3
33 inv(2)
34
35 ATIC ALCL
36
37
2

26
25
24
23
22 t(1;3)

21 TCTA T-ALL
p
DLBCL
14 FOXP1
MALT Iymphoma
13

12
t(2;3) Figure 1.5 Chromosome 3
11.2 TFG ALCL
12
13.1
13.2
t(3; 3) 13.3
t(3;12) 21
t(3;21) 22
inv(3) 23 t(3;5)
24 AML
q 25 MLF1 MDS
AML/tAML CML, blasts crisis
MDS/tMDS EVI1 26.1
CML 26.2
(MDS1)
26.3 Follicular Iymphoma
CML,blast crisis BCL6
27 DLBCL
28
29 t(3; 14)
3 t(3;2)
t(3;22)
Gorczyca-Ch01 11/7/07 3:42 PM Page 6

6 CYTOGENTICS, FISH AND MOLECULAR TESTING IN HEMATOLOGIC MALIGNANCIES

Multiple
16 FGFR3
myeloma
15.3 t(4;14)
15.2
p 15.1
del(14)(q12) Mastocytosis
14
13 CEL/HES
Mastocytosis mutations 12
AML (core binding)
CHIC2 t(4;12) AML
with t(8;21) KIT 12 PDGFRA
AML-M4eo
with inv(16) 13
B-ALL
21 AF4 Biphenotypic
(MLLT2) leukemia
Figure 1.6 Chromosome 4 22 AML
23 t(4;11)
24
25
q
26
27
28
31.1
31.2
31.3
32
33
34
35
4

15.3
15.2
15.1
p 14
13
12

11.2
12
13
t(1;5)
Figure 1.7 Chromosome 5 t(5;7) 14
t(5;12) 15 MDS
CMML ± eosinophilia t(5;14)
q
del(5q)
t(5;15) 21 AML
aCML
CEL/HES t(5;17) 22
SM-CEL/HES
23
AML ± eosinophilia
CMPD + eosinophilia PDGFRB t(2;5) [ALK ] ALCL
31 t(3;5) [MLF1] AML/MDS
B-ALL IL3 t(5;17) [RARA] APL
with 32
eosinophilia t(5;14) 33 ALCL
34 AML
T-ALL TLX3 35 NPM1 APL
CMML
t(5;14) [BCL11B] 5 CML, blasts crisis
[TCRD ]