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Molecular and Translational Medicine

Series Editors
William B. Coleman
Gregory J. Tsongalis

For further volumes:

http://www.springer.com/series/8176
Domnita Crisan
Editor

Hematopathology

Genomic Mechanisms of Neoplastic Diseases

Editor
Domnita Crisan
William Beaumont Hospital
Department of Clinical Pathology
Molecular Pathology Lab
3601 West 13 Mile Road
48073-6769 Royal Oak, Michigan
USA
[email protected]

ISBN 978-1-60761-261-2 e-ISBN 978-1-60761-262-9

DOI 10.1007/978-1-60761-262-9
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2010931434

© Springer Science+Business Media, LLC 2010

All rights reserved. This work may not be translated or copied in whole or in part without the written
permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring
Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly
analysis. Use in connection with any form of information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed is
forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are
not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject
to proprietary rights.
While the advice and information in this book are believed to be true and accurate at the date of going
to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any
errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect
to the material contained herein.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

I dedicate this book to my mother who gave me
her gene for optimism, to my brave father who
lost his life fighting communists, and to my
husband, Dan, who offered his loving support,
with patience and enthusiasm during work on this
book and my entire professional career
Preface

Hematopathology: Genomic Mechanisms of Neoplastic Diseases in the book

series Molecular and Translational Medicine addresses our current knowledge of
genomics as applied to the pathogenesis, diagnosis, prognosis, monitoring, and
targeted therapy of hematologic malignancies. Hematology has been at the van-
guard of the application of molecular technologies in diagnosis, classification, risk
stratification, and use of molecularly defined therapeutic targets. These advances in
molecular technologies, diagnostics, and gene-related therapy have seen an extraor-
dinary rapid pace since the completion of the Human Genome Project. Hematology
has integrated the discoveries of genomic lesions underlying hematologic malignan-
cies and applied the tools of molecular pathology, making them essential in clinical
practice.
The scope of this book is to keep pathologists and clinicians abreast of the
rapid and complex changes in genomic medicine, as exemplified by the molecu-
lar pathology of leukemias and lymphomas. This is a timely opportunity to not only
update physicians on the complexity of genomic abnormalities but also offer an
integrated framework encompassing molecular diagnostics, the new WHO (World
Health Organization) classification of hematologic neoplasms with focus on molec-
ular pathology, prognostic value of molecular tests, and molecular monitoring of
response to gene-targeted therapy.
The rapid pace of discovery, the explosion in genomic information, and the ever
changing molecular technologies make it necessary to constantly update our knowl-
edge and I hope that the readers will use this book as a practical resource and place
it next to their microscope, in their laboratories or clinical offices.
The first two chapters should be helpful for practicing pathologists and for clin-
icians, providing overviews of molecular techniques and cytogenetics, both well
established and new, as used in molecular hematology. Chapter 3 is a concise review
of the new 2008 WHO classification, which integrates molecular abnormalities
in the diagnosis of hematologic neoplasms. The following chapters offer compre-
hensive discussions of the molecular pathology of lymphoid and myeloid acute
leukemias, the mature B-cell and T-cell lymphomas, the myeloproliferation neo-
plasms, chronic lymphocytic leukemia, overall representing the major diagnostic
entities in neoplastic hematology.

vii
viii Preface

The new fields of targeted therapy in hematologic malignancies and microRNAs

as applied in hematologic malignancies are reviewed in the last two chapters and
offer comprehensive discussions of the current state of these novel approaches.
I am extremely grateful to all the authors for their excellent contributions to this
book; each chapter is an in-depth and thought-provoking update, as well as easily
readable and practical.
In a specialty as exciting and rapidly evolving as Molecular Hematology, it is
my hope that this will be just the first of many editions of this book. It will be
interesting and challenging to see the progress in genomics in the next years and ask
the question, Quo Vadis Hematology?

Royal Oak, Michigan Domnita Crisan

Contents

1 Molecular Techniques in Hematopathology . . . . . . . . . . . . . 1

Bobby L. Boyanton Jr. and Jennifer R. Rushton
2 Classical and Molecular Cytogenetic Analysis
of Hematolymphoid Disorders . . . . . . . . . . . . . . . . . . . . 39
Mark A. Micale
3 Using Cytogenetic and Molecular Tests in Diagnostic
Workups with the WHO Classification – 2008 . . . . . . . . . . . 79
Clarence C. Whitcomb
4 Update on the Molecular Pathology
of Precursor Lymphoid Leukemias . . . . . . . . . . . . . . . . . 103
Robert B. Lorsbach
5 Molecular Pathology of Acute Myeloid Leukemias . . . . . . . . . 127
Karen P. Mann and Debra F. Saxe
6 Molecular Pathology of Mature B-Cell and T-Cell Lymphomas . . 157
Sophia L. Yohe, David W. Bahler, and Marsha C. Kinney
7 Molecular Pathology of Myeloproliferative Neoplasms . . . . . . 215
David S. Bosler
8 Molecular Pathology of Chronic Lymphocytic Leukemia . . . . . 255
Daniela Hoehn, L. Jeffrey Medeiros, and Sergej Konoplev
9 Targeted Therapy in Hematologic Malignancies . . . . . . . . . . 293
Barbara Zehnbauer and Mona Nasser
10 Micro-RNAs in Hematologic Malignancies . . . . . . . . . . . . . 325
Muller Fabbri and George A. Calin
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

ix
Contributors

David W. Bahler, MD, PhD Department of Pathology, University of Utah, Salt

Lake City, UT, USA
David S. Bosler, MD Department of Clinical Pathology, Cleveland Clinic,
Cleveland, OH, USA
Bobby L. Boyanton Jr, MD Department of Clinical Pathology, Beaumont
Hospitals, Royal Oak, MI, USA
George A. Calin, MD, PhD Departments of Experimental Therapeutics and
Cancer Genetics, University of Texas, M.D. Anderson Cancer Center, Houston,
TX, USA
Muller Fabbri, MD Department of Molecular Virology, Immunology and
Medical Genetics, The Ohio State University Comprehensive Cancer Center,
Columbus, OH, USA
Daniela Hoehn, MD Department of Hematopathology, M.D. Anderson Cancer
Center, Houston, TX, USA
Marsha C. Kinney, MD Division of Hematopathology, University of Texas
Health Sciences Center, San Antonio, TX, USA
Sergej Konoplev, MD, PhD Department of Hematopathology, M.D. Anderson
Cancer Center, Houston, TX, USA
Robert B. Lorsbach, MD, PhD Department of Pathology, University of Arkansas
for Medical Sciences, Little Rock, AR, USA
Karen P. Mann, MD, PhD Department of Pathology and Laboratory Medicine,
Emory University, Atlanta, GA, USA
L. Jeffrey Medeiros, MD Department of Hematopathology, M.D. Anderson
Cancer Center, Houston, TX, USA
Mark A. Micale, PhD Beaumont Laboratory, Department of Anatomic
Pathology, Beaumont Hospitals, Royal Oak, MI, USA

xi
xii Contributors

Mona Nasser, MD Department of Clinical Chemistry, School of Medicine, Beni

Suef University, Beni Suef, Egypt
Jennifer R. Rushton, MD Department of Pathology, Baylor College of
Medicine – BCM 315, Houston, TX, USA
Debra F. Saxe, PhD Department of Pathology and Laboratory Medicine, Emory
University, Atlanta, GA, USA
Clarence C. Whitcomb, MD Department of Pathology, Miller School of
Medicine, University of Miami, Miami, FL, USA
Sophia L. Yohe, MD Department of Laboratory Medicine and Pathology,
University of Minnesota Medical Center, Minneapolis, MN, USA
Barbara Zehnbauer, PhD Division of Laboratory Systems, Laboratory Practice
Evaluation and Genomics Branch, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Chapter 1
Molecular Techniques in Hematopathology

Bobby L. Boyanton Jr. and Jennifer R. Rushton

Keywords DNA · RNA · Specimen collection · Specimen handling · Speci-

men processing · Cell enrichment · Nucleic acid · Stability · Storage · Spectro-
photometric · Fluorometric · Absorbance · Asymmetric PCR · Clonality ·
Immunoglobulin · T-cell receptor · Antigen receptor · Gene · Hematology ·
Hematolymphoid · Hematopathology · Paraffin · Formalin · Fixative ·
Extraction · Purification · Phenol–chloroform · Chaotropic salt · Silica col-
umn · Ethidium bromide · SYBR green · Gel electrophoresis · Capillary
electrophoresis · Agarose · Polyacrylamide · Restriction enzyme · Sanger
sequencing · Chain termination · Pyrosequencing · Sequencing by synthesis · Next-
generation sequencing · High-throughput sequencing · Automation · Polymerase
chain reaction · PCR · Reverse transcriptase PCR · Allele-specific
PCR · Nested PCR · Real-time PCR · Quantitative PCR · Methylation
PCR · FRET · TaqMan · Probe · Hydrolysis · Hybridization

Introduction

The discipline of hematopathology traditionally relies upon morphologic evaluation,

cytochemical stains, immunohistochemistry, flow cytometry, and karyotypic analy-
sis to classify hematolymphoid neoplasms. Although these time-honored methods
still comprise the primary diagnostic arsenal of the pathologist, the last few decades
have borne witness to the widespread acceptance of molecular techniques to clas-
sify these neoplasms. No longer considered ancillary, molecular analyses have led
to a greater understanding of the biological and clinical heterogeneity of hema-
tolymphoid neoplasms, and now form the primary diagnostic criteria for many
diagnoses as set forth by the World Health Organization [1]. They also provide
extremely sensitive and specific methods for prognostic marker detection and mini-
mal residual disease monitoring. These techniques have evolved rapidly over the last

B.L. Boyanton Jr. (B)

Department of Clinical Pathology, Beaumont Hospitals, 3601 W. Thirteen Mile Rd, Royal Oak,
MI 48073, USA
e-mail: [email protected]

D. Crisan (ed.), Hematopathology, Molecular and Translational Medicine, 1

DOI 10.1007/978-1-60761-262-9_1,  C Springer Science+Business Media, LLC 2010
2 B.L. Boyanton Jr. and J.R. Rushton

decade from Southern blot and hybridization assays to polymerase chain reaction
and its variants to gene expression profiling and single-nucleotide polymorphism
analysis, and more recently to microarray technology and whole-genome analysis.
Despite technological advancements, molecular techniques are critically dependent
upon the nature of nucleic acids retrieved from the specimen. Results cannot be
correctly interpreted if the quantity and/or the integrity of nucleic acids are not opti-
mal for the desired molecular application. As such, the purpose of this chapter is
twofold. First, issues pertaining to specimen collection, handling and processing,
and nucleic acid extraction, stability, and storage are reviewed. Second, molecular
techniques commonly utilized in hematopathology are reviewed. Cytogenetics, flu-
orescent in situ hybridization (FISH), and microarray techniques are discussed in
Chapter 2.

Part I: Specimen Collection and Processing

Standard Precautions and Safety

The collection, processing, and storage of biological samples pose risks to the han-
dler for the acquisition of a variety of infectious agents. All personnel handling
biological samples should follow “standard precautions”; guidelines are avail-
able from the US Centers for Disease Control and Prevention (www.cdc.gov).
Additionally, the Clinical and Laboratory Standards Institute (www.clsi.org) pub-
lishes literature pertaining to laboratory safety [2] and the protection of laboratory
workers from occupationally acquired infections [3].

Patient Identification and Labeling

Specimen labeling and tracking throughout the entire testing process is paramount
to ensure valid test results. Unique patient identifiers should be utilized (i.e.,
full name, date of birth, medical record number). In addition, the test requisi-
tion should also include (1) date and time of specimen collection, (2) specimen
type and source, (3) ordering physician and contact information, (4) billing infor-
mation, and (5) pertinent clinical and laboratory information. A copy of the
pathology report should be included with tissue specimens to ensure accurate spec-
imen identification and the ability to correlate molecular-based test results with
the histopathologic diagnosis. Every attempt should be made to obtain stained
slides for review. This will ensure that the correct tissue is submitted and that
the tissue is representative of the intended test, and will allow for the qualitative
assessment of cellularity. Compliance with regulations protecting personal health
information as set forth by the US Department of Health & Human Services
(www.hhs.gov/ocr/hippa) is of paramount importance and must be adhered to at all
times.
1 Molecular Techniques in Hematopathology 3

Cell Enrichment and Selection Techniques

The ability to selectively obtain desired cell populations increases the sensitivity and
specificity of molecular-based testing and facilitates the removal of potential con-
taminating substances that may be inhibitory to amplification-based methods, such
as polymerase chain reaction (PCR). This becomes vitally important with minimal
residual disease (MRD) testing, where it is not uncommon for residual malignant
cells to represent a minor fraction of the cellular milieu. Basic techniques of cell
selection and enrichment are discussed in this section.
The isolation of DNA or RNA from select cell populations within liquid spec-
imens (i.e., whole blood, marrow aspirates, body fluids) can be accomplished
by several techniques. Perhaps the most basic approach to obtain leukocytes
involves the preparation of a leukocyte-rich layer [4]. Following centrifugation
(3,300–3,500×g for 10–15 min), the specimen will partition into three distinct
layers: an upper aqueous layer, a middle leukocyte-rich layer (buffy coat), and a
lower layer (erythrocytes). The “buffy coat” is easily recovered following removal
of the upper layer. Another approach is selective erythrocyte lysis with hypotonic
buffer (e.g., ammonium chloride) [5]. After centrifugation, the released hemoglobin
will partition into the upper aqueous layer, while the nucleic acid of interest is
retained within the leukocyte pellet at the bottom of the tube. Following decant-
ing of the aqueous layer, the leukocyte pellet is washed several times with isotonic
buffer to remove any residual aqueous layer. Alternatively, density-gradient cen-
trifugation facilitates the selective recovery of lymphocytes and monocytes from
other cellular constituents [6]. Several commercial products incorporating Ficoll-
Hypaque or other density-gradient media into evacuated collection tubes specifically
for molecular-based testing are available – Vacutainer CPT Mononuclear Cell
Preparation Tube (Becton–Dickinson, Franklin Lakes, NJ). A more recent approach
uses antibody-coated magnetic beads to obtain desired cell populations from liq-
uid specimens. After incubation of the liquid specimen with the magnetic beads,
a magnetic field is applied, allowing unwanted cellular constituents to be removed
by decanting. Magnetic bead-bound cells of interest are then washed with isotonic
buffer. Cells of interest are released from the magnetic beads by either enzymatic
cleavage or competitive displacement using high-affinity monoclonal antibodies [7].
Alternatively, fluorescent antibody cell sorting (FACS) is also useful for captur-
ing selected cell populations. Fluorescent, differentially labeled antibodies bind to
desired cellular constituents. Using a modified flow cytometer, cells of interest are
routed to separate collection chambers based upon their fluorescence profile. The
cells of interest are thusly obtained and ready for nucleic acid extraction.
Laser capture microdissection is a common technique that facilitates the selec-
tion of desired cell populations from tissue sections. In short, tissue is mounted on a
glass slide and covered with a translucent coating. Using a microscope, cells of inter-
est are located, followed by user-defined infrared or UV laser activation that melts
the translucent coating containing the cell(s) of interest from the slide. The selected
regions of dissected film are removed, followed by routine nucleic acid extraction
protocols [7]. Commercial systems are available from Arcturus/MDS Analytical
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