Lymphoma (Methods in Molecular Medicine)

359 Pages·2010·5.253 MB·English

by  Tim Illidge, Peter W. M. Johnson

M E T H O D S I N M O L E C U L A R M E D I C I N ETM
LLyymmpphhoommaa
MMeetthhooddss aanndd PPrroottooccoollss
EEddiitteedd bbyy
TTiimm IIlllliiddggee
PPeetteerr WW.. MM.. JJoohhnnssoonn
Lymphoma
M E T H O D S I N M O L E C U L A R M E D I C I N E™
John M. Walker, SERIES EDITOR
123.Marijuana and Cannabinoid Research: 103.Pancreatic Cancer: Methods and Protocols,
Methods and Protocols, edited by Emmanuel edited by Gloria H. Su, 2004
S. Onaivi, 2006 102.Autoimmunity: Methods and Protocols,
122.Placenta Research Methods and Protocols: edited by Andras Perl, 2004
Volume 2, edited by Michael J. Soares and 101.Cartilage and Osteoarthritis: Volume 2,
Joan S. Hunt, 2006 Structure and In Vivo Analysis, edited by
121.Placenta Research Methods and Protocols: Frédéric De Ceuninck, Massimo Sabatini,
Volume 1, edited by Michael J. Soares and and Philippe Pastoureau, 2004
Joan S. Hunt, 2006 100.Cartilage and Osteoarthritis: Volume 1,
120.Breast Cancer Research Protocols, edited by Cellular and Molecular Tools, edited by
Susan A. Brooks and Adrian Harris, 2005 Massimo Sabatini, Philippe Pastoureau, and
119.Human Papilloma Viruses: Methods and Frédéric De Ceuninck, 2004
Protocols, edited by Clare Davy and John 99.Pain Research: Methods and Protocols,
Doorbar, 2005 edited by David Z. Luo, 2004
118.Antifungal Agents: Methods and Protocols, 98.Tumor Necrosis Factor: Methods and
edited by Erika J. Ernst and P. David Protocols, edited by Angelo Corti and Pietro
Rogers, 2005 Ghezzi, 2004
117.Fibrosis Research: Methods and Protocols, 97.Molecular Diagnosis of Cancer: Methods and
edited by John Varga, David A. Brenner, Protocols, Second Edition, edited by Joseph E.
and Sem H. Phan, 2005 Roulston and John M. S. Bartlett, 2004
116.Inteferon Methods and Protocols, edited by 96.Hepatitis B and D Protocols: Volume 2,
Daniel J. J. Carr, 2005 Immunology, Model Systems, and Clinical
115.Lymphoma: Methods and Protocols, edited Studies, edited by Robert K. Hamatake and
by Timothy Illidge and Peter W. M. Johnson, Johnson Y. N. Lau, 2004
2005 95.Hepatitis B and D Protocols: Volume 1,
114.Microarrays in Clinical Diagnostics, edited Detection, Genotypes, and Characterization,
by Thomas O. Joos and Paolo Fortina, 2005 edited by Robert K. Hamatake and Johnson
Y. N. Lau, 2004
113.Multiple Myeloma: Methods and Protocols,
edited by Ross D. Brown and P. Joy Ho, 94.Molecular Diagnosis of Infectious Diseases,
Second Edition, edited by Jochen Decker and
2005
Udo Reischl, 2004
112.Molecular Cardiology: Methods and Protocols,
93.Anticoagulants, Antiplatelets, and
edited by Zhongjie Sun, 2005
Thrombolytics, edited by Shaker A. Mousa,
111.Chemosensitivity: Volume 2, In Vivo Mod- 2004
els, Imaging, and Molecular Regulators, ed- 92.Molecular Diagnosis of Genetic Diseases,
ited by Rosalyn D. Blumethal, 2005 Second Edition, edited by Rob Elles and
110.Chemosensitivity: Volume 1, In Vitro As- Roger Mountford, 2004
says, edited by Rosalyn D. Blumethal, 2005 91.Pediatric Hematology: Methods and
109.Adoptive Immunotherapy: Methods and Protocols, edited by Nicholas J. Goulden
Protocols, edited by Burkhard Ludewig and and Colin G. Steward, 2003
Matthias W. Hoffman, 2005 90.Suicide Gene Therapy: Methods and
108.Hypertension: Methods and Protocols, Reviews, edited by Caroline J. Springer, 2004
edited by Jérôme P. Fennell and Andrew 89.The Blood–Brain Barrier: Biology and
H. Baker, 2005 Research Protocols, edited by Sukriti Nag, 2003
107.Human Cell Culture Protocols, Second 88.Cancer Cell Culture: Methods and Protocols,
Edition, edited by Joanna Picot, 2005 edited by Simon P. Langdon, 2003
106.Antisense Therapeutics, Second Edition, 87.Vaccine Protocols, Second Edition, edited by
edited by M. Ian Phillips, 2005 Andrew Robinson, Michael J. Hudson, and
105.Developmental Hematopoiesis: Methods Martin P. Cranage, 2003
and Protocols, edited by Margaret H. 86.Renal Disease: Techniques and Protocols,
Baron, 2005 edited by Michael S. Goligorsky, 2003
104. Stroke Genomics: Methods and Reviews, 85.Novel Anticancer Drug Protocols, edited by
edited by Simon J. Read and David Virley, 2004 John K. Buolamwini and Alex A. Adjei, 2003
M E T H O D S I N M O L E C U L A R M E D I C I N E™
Lymphoma
Methods and Protocols
Edited by
Tim Illidge
CRUK, Paterson Institute for Cancer Research,
University of Manchester, Manchester, UK
Peter W. M. Johnson
Cancer Sciences Division,
Southampton General Hospital, Southampton, UK
© 2005 Humana Press Inc.
999 Riverview Drive, Suite 208
Totowa, New Jersey 07512
www.humanapress.com
All rights reserved.
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permis-
sion from the Publisher. Methods in Molecular Medicine™ is a trademark of The Humana Press Inc.
All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not
necessarily reflect the views of the publisher.
This publication is printed on acid-free paper. ∞
ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.
Production Editor: Nicole E. Furia
Cover design by Patricia F. Cleary
Cover Illustration: Figure 1 from Chapter 14, “Antibody Techniques Used in the Study of Anaplastic Lym-
phoma Kinase-Positive ALCL,” by Karen Pulford, Helen M. Roberton, and Margaret Jones.
For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact
Humana at the above address or at any of the following numbers: Tel.: [phone]; Fax: [phone];
E-mail: [email protected]; or visit our Website: www.humanapress.com
Photocopy Authorization Policy:
Authorization to photocopy items for internal or personal use, or the internal or personal use of specific
clients, is granted by Humana Press Inc., provided that the base fee of US $30.00 per copy is paid directly
to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that
have been granted a photocopy license from the CCC, a separate system of payment has been arranged and
is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829-
159-6/05 $30.00].
Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
eISBN 1-59259-936-2
Library of Congress Cataloging-in-Publication Data
Lymphoma : methods and protocols / edited by Timothy Illidge, Peter W.M.
Johnson.
p. ; cm. — (Methods in molecular medicine, ISSN 1543-1894 ; 115)
Includes bibliographical references and index.
ISBN 1-58829-159-6 (alk. paper)
1. Lymphomas—Molecular aspects—Laboratory manuals.
[DNLM: 1. Lymphoma. 2. Clinical Laboratory Techniques. 3. Genetic
Techniques. WH 525 L9854 2005] I. Illidge, Timothy. II. Johnson, P. W. M.
III. Series.
RC280.L9L9527 2005
616.99’446—dc22
2004026632
Preface
The molecular investigation of hematological malignancies has always fore-
run that of other cancers. There are a number of potential reasons for this, some
of which are more logistical than theoretical, and include the ready access to
pathologic material with the presence of malignant cells generally in great
excess in comparison with the normal. These factors are combined with a set of
diseases for which potentially curative systemic therapies are available, making
the precise subclassification for prognostic and therapeutic purposes even more
important. In general, studies of the leukemias have first started with
immunophenotyping, classical cytogenetics, restriction digest analysis, poly-
merase chain reaction studies (PCR), fluorescent in situ hybridization (FISH),
and comparative genomic hybridization. Most recently, gene expression
arrays have been tested first in leukemia, closely followed by the application of
these techniques to lymphoma.
The different techniques described in Lymphoma: Methods and Protocols
have evolved in parallel over the last three decades. The first was classical
cytogenetics and the identification of consistent structural abnormalities in the
chromosomes of malignant cells, with the findings linked to morphologic and
immunophenotypic entities. From chromosomal rearrangements, reverse
genetics led to the characterization of key genes adjacent to breakpoints, whose
function was then related back to the abnormal cellular behavior. The finding
that an activating oncogene, c-Myc, was involved in the characteristic chromo-
some 8 breakpoint in Burkitt’s lymphoma was a key moment for this field.
Analysis of different breakpoints has yielded other, similarly important
insights, such as identification of a failure to undergo apoptosis as a central
mechanism in malignancy following the finding of the Bcl-2 gene at the
chromosome 18q21 breakpoint in follicular lymphoma. The field of cytogenetics
has been enhanced greatly by the development of FISH, in which insights into
more sophisticated patterns of chromosomal abnormality can be understood,
and there has subsequently been a steady rise in the number of regions for
study.
An increase in our understanding of lymphocyte biology, particularly B-cell
ontogeny and the germinal center reaction has led to the application of immu-
noglobulin gene rearrangements as markers of clonality and to patterns of
somatic mutation in the variable regions being used as indicators of the stage in
development at which transformation occurred. Study of these patterns of
v
vi Preface
mutation in low-grade lymphoma has yielded the fascinating information that
follicular types are preferentially mutated at glycosylation sites, suggestive of
a ligand-related selection pressure in the germinal center during pathogenesis.
In chronic lymphocytic leukemia (CLL) the more “immature” unmutated/
nongerminal center types show a considerably more aggressive course than the
post-germinal center mutated cases. There are interesting parallels in diffuse
large B-cell lymphoma, which is always mutated in the immunoglobulin
V genes, but in which a germinal center immunophenotype and pattern of
mRNA expression conveys a better prognosis. The application of gene arrays
to the question of biological heterogeneity in morphologically similar diseases
has been especially rewarding. Different subtypes of large B-cell lymphoma
have been clearly defined, and the grey zone between Hodgkin lymphoma and
sclerosing mediastinal lymphoma is being steadily illuminated.
Currently gene arrays are not a routine part of the diagnostic investigation
for lymphoma, although it seems highly likely that this will be the case for
subsequent editions of this book. However, it may be that the gene signatures
are sufficiently paralleled by the phenotype to allow immunohistochemistry to
be used instead, once a firm correlation has been established. This looks likely
to be the case for large B-cell lymphoma with the use of Bcl-6 and CD10 as
germinal center markers, and certainly broadens the applicability of the classi-
fication when fresh biopsy material is difficult to obtain.
The process of classifying and reclassifying lymphoma has been a source of
some frustration for clinicians and pathologists alike for many years, but the
advent of the Revised European American Lymphoma (REAL) classification
and subsequently its adoption into the new WHO classification have brought
unprecedented harmony into this area. The recognition of REAL disease enti-
ties has been critical to the assimilation of immunophenotypic and molecular
data into the classification, which has been a notable success. The process of
subdividing the different entities along biological lines is well underway, with
classes of CLL and diffuse large B-cell lymphoma already characterized as
described. Other entities such as MALT lymphoma are being split down
according to the precise translocation present, and in all these cases the sub-
divisions seem likely to have relevance to the type of therapy chosen. Thus,
gastric MALT lymphoma with a t(11;18)(q21;q21) is much more likely to
respond to the eradication of Helicobacter pylori than is one with
t(1;14)(p22;q32).
The use of such molecular information to guide therapy is slowly gaining
ground, although in other areas the benefits remain less clear. The detection of
minimal residual disease using PCR techniques has been extensively studied,
particularly in follicular lymphoma, with the general conclusion that absence
of the pathognomonic t(14;18)(q32;q21) translocation at the time of clinical
Preface vii
remission is preferable, but no firm data as to whether the pursuit of such
molecular remission actually conveys a survival benefit. Until the advent of
rituximab antibody therapy there were few patients in whom molecular remis-
sion was achieved with anything less than myeloablative treatment, but now
that there is an intervention that can achieve a high rate of conversion from
PCR-positive to PCR-negative it may be possible to test the validity of this
hypothesis. The use of quantitative PCR to follow progress sequentially has
added a further level of refinement to this approach and made it much easier to
consider therapy directed by molecular results.
As well as providing prognostic information, molecular analyses have been
used to produce novel therapeutics, especially in the area of immunotherapy.
Characterization of the lymphocyte cell surface molecules has been used to
identify antigens against which vaccines or antibodies may be deployed, and
the newer technique of serological identification by expression cloning
(SEREX) has provided a wealth of information on this. The idiotype represents
the truly unique antigen on a B-cell surface and here once again the application
of molecular techniques has been able to provide a highly specific immunogen,
in the form of a DNA vaccine with patient-specific sequences derived from the
lymphoma.
In conclusion, the aim of this book is to give some practical information on
the current molecular approaches being used in the understanding, classifica-
tion, and therapy of lymphoma. Though it cannot be exhaustive in providing
information for every chromosomal translocation and gene rearrangement, we
hope that it provides a useful background to the field and a means to get started
with the molecular diagnostics of a group of diseases that are both biologically
interesting and medically important.
Tim Illidge
Peter Johnson

Contents
Preface ..............................................................................................................v
List of Contributors...........................................................................................xi
1 Purification of Primary Malignant B-Cells and Immunoblot Analysis
of Bcl-2 Family Proteins
Claire Dallman and Graham Packham.................................................1
2 Molecular Diagnosis of Lymphoma: Outcome Prediction by Gene
Expression Profiling in Diffuse Large B-Cell Lymphoma
Kim Last, Silvana Debarnardi, and T. Andrew Lister..........................15
3 Demonstration of a Germinal Center Immunophenotype
in Lymphomas by Immunocytochemistry and Flow Cytometry
Andrew Jack, Sharon Barrans, David Blythe,
and Andrew Rawstron...................................................................65
4 Karyotyping Lymph Node Biopsies in Non-Hodgkin’s Lymphoma
Fiona M. Ross and Christine J. Harrison.............................................93
5 Identification of Lymphoma-Associated Antigens Using SEREX
Amanda P. Liggins, Barbara A. Guinn, and Alison H. Banham........109
6 Determining Mutational Status of Immunoglobulin V Genes
in Chronic Lymphocytic Leukemia: A Useful Prognostic Indicator
Surinder S. Sahota, Gavin Babbage, Niklas Zojer,
Christian H. Ottensmeier, and Freda K. Stevenson.....................129
7 Idiotype Gene Rescue in Follicular Lymphoma
Katy McCann, Surinder S. Sahota, Freda K. Stevenson,
and Christian H. Ottensmeier......................................................145
8 Immunoglobulin Gene Mutation Patterns and Heterogeneity
of Marginal Zone Lymphoma
Mariella Dono and Manlio Ferrarini................................................173
9 T-Cell Receptor Molecular Diagnosis of T-Cell Lymphoma
Elizabeth Hodges, Anthony P. Williams, Susan Harris,
and John L. Smith.........................................................................197
10 Cloning of Immunoglobulin Chromosomal Translocations
by Long-Distance Inverse Polymerase Chain Reaction
E. Loraine Karran, Takashi Sonoki, and Martin J. S. Dyer................217
ix