Table Of ContentMolecular Diagnosis of Cancer
METHODS IN MOLECULAR MEDICINE
TM
John M. Walker, sei~leS ROTIDE
Molecular Diagnosis of Cancer, edited by Finbarr E. Cotter, 1996
Human Cell Culture Protocols, edited by Gareth E. Jones, 1996
AntisenTshee rapeutics, edited by Sudhir Agrawal, 1996
Vaccine Protocols, edited by Andrew Robinson, Graham .11 Farrar,
and Christopher N. Wiblin, 1996
Prion Diseases, edited by Harry .F Baker and Rosalind M. Ridley, 1996
Molecular Diagnosis ofG enetic Diseases, edited by Rob Elles, 1996
Herpes Simplex Virus Protocols, edited by Moira S. Brown
and Alasdair MacLean, 1996
Helicobacter pylori Protocols, edited by Christopher L. Clayton
and Harry .T Mobley, 1996
Lectins in MedicalR esearch, edited by Jonathan M. Rhodes
and Jeremy D. Milton, 1996
Gene Therapy Protocols, edited by Paul Robbins, 1996
Molecular Diagnosis
of Cancer
Edited by
Finbarr .E Cotter
ytisrevinU of ,nodnoL KU
anamuH Press ~E~
Totowa, weN Jersey
© 1996 Humana Press Inc.
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Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in molecular medicine,s.
Molecular diagnosis of cancer / edited by Finbarr E. Cotter.
p. cm. -- (Methods in molecular medicine)
Includes index.
ISBN 0-89603-341-4 (alk. paper)
1. Cancer--Molecular diagnosis. I. Cotter, Finbarr E.
II. Series.
[DNLM: .1 Neoplasms~iagnosis. 2. DNA--analysis. 3. Polymerase
Chain Reaction. 4. Genetic Techniques. QZ 241 M7173 1996]
RC270.M64 1996
616.99'4075~c20
DNLM/DLC
for Library of Congress 96-20789
CIP
Preface
The aims of Molecular Diagnosis of Cancer are to introduce scientists
and physicians working in the field of diagnostics to the area of cancer
molecular pathology and to highlight the possibilities of its application to the
cancer physician in the clinic. The degree of molecular biological expertise
required should be minimal,a lthough those with more experience may also be
able to benefit from the book. All of the authors have considerable practical
experience in the method they describe and are working predominantly in the
setting of the cancer clinic. As such, the book pulls together a number of tech-
niques that are already being applied to a wide range of malignancies. More-
over, this field will continue to expand exponentially as further research leads
to a greater understanding of the molecular basis of cancer.
Detection of the changes to the DNA or RNA code within a diseased cell
often provides pathological information for the diagnosis, prognosis, andm an-
agement of the disease. Such DNA-related analysis is primarily the role of
molecular pathology. Methods to detect these alterations are being refined
and are evolving from the research to the diagnostic laboratory. One of the
single most powerful techniques in this new branch of pathology has been the
polymerase chain reaction (PCR), to which much of this book is devoted. The
use of PCR in pathology not only provides new diagnostic possibilities, but
also requires the acquisition of new expertise in its performance and in inter-
pretation of the results if the full potential is to be achieved.
PCR is an in vitro technique, invented by Kary Mullis, that produces
multiple copies of selected sequences of DNA, provided the sequence is present
in the test DNA sample. Its sensitivity lies in the region of a million-fold
amplification from a template equivalent of as little as a single human cell
and, for this reason, rigorous methods to minimize and control for contamina-
tion are required. An important property of PCR when considering diagnostic
applications is the ability of the method to amplifyf rom target DNA sequences
not only those well prepared from freshly frozen tissue, but also those from
degraded DNA templates, such as those found in paraffin-embedded tissue.
Amplification by PCR is ideally suited to target sequences of DNA below
1000 bases in size, above which the efficiency of the reaction falls dramati-
cally. However, lengthening the extension time fort he reaction can yield longer
amplification products. Specificity of the reaction is influenced by several
V
vi Preface
factors. Excess of qaT polymerase, excessive extension times, and low
annealing temperatures will all increase the risk of spurious amplification;
however, the opposite approach leads to greater specificity, with the penalty
of a reduced quantity of amplification product. PCR initially leads to an expo-
nential accumulation of DNA, but a plateau of amplification product is even-
tually reached (ideally after 25 cycles). Further amplification within thep lateau
phase may lead to accumulation of detectable product from low levels of con-
taminant DNA. Essentially, less rather than more cycles may, ironically, be
optimal in PCR.
Often, the gene sequences that are required to be detected consist of a
number ofexon sequences separated by long intron regions. DNA PCR would
not be possible because the distance between the primers is too great. How-
ever, nature normally splices outt he intron sequence when producing mRNA,
bringing the exon sequences considerably closer. If the mRNA is extracted
and converted back into complementary DNA (reverse transcription [RT] to
cDNA), then this would permit PCR, as described above, to uset he exon prim-
ers, as they are much closer. This process (RT-PCR) is useful for the examina-
tion of quite large pieces of DNA, such as chromosomal translocation, in which
the DNA breakpoints are varied and over a large distance but the resultant
chimeric fusion gene remains constant. The use of PCR in molecular diagnos-
tics of cancer primarily uses the RT methodology and has been applied pre-
dominantly in the field of hematological malignancies, in which chromosomal
translocations leading to the presence of chimeric genes are observed in many
cases. The abundance of molecular diagnostic methods in blood malignancies
in part reflects the relative ease of examination, which results from the acces-
sibility of the "tumor" cell from the bone marrow and its presence in a cell
suspension.
Identification of cancers associated with mutations in gene sequences
requires detection of as little as a single base sequence difference. Single-
strand conformation polymorphism (SSCP) analysis is a quick and effective
technique, based on PCR, for the detection of single nucleotide base substitu-
tions. The method relies on the principle that single-stranded DNA takes on
sequenced-based secondary structures (Conformers) under nondenaturing con-
ditions. In real terms, this means that molecules varying by as little as a single
base substitution may form different conformers and migrate differently in a
nondenaturing polyacrylamide gel. The best results are obtained when the DNA
molecule being examined is 200 base pairs or less in size. SSCP analysis for a
base mutation is carried by PCR using flanking oligonucleotide primers (com-
monly to a gene sequence) and incorporating a ]S53[ or [32p] labeled dATP in
the reaction. The radioactive PCR product is made single stranded by boiling
and is then run on the denaturing gel. The polyacrylamide gel is then dried and
Preface vii
exposed to an autoradiograph to show the presence or absence of a base muta-
tion. Directly sequencing the PCR product by the dideoxy method (to confirm
correct amplification and the presence of mutations suggested by SSCP) can
be executed in the thermal cycler by a method known as cycle sequencing,
directly incorporating radioactivity. The application of S SCP is predominantly
in the area of solid tumors and is usually suggestive of a tumor suppressor
gene, illustrated well by the P53 and Wilm's tumor genes.
There are two major problems related to PCR, namely the false positive
and the false negative results. These drawbacks can be largely overcome pro-
vided adequate care and good positive and negative control reactions are car-
ried out at all times with this extremely sensitive technique.
False Positive. Those arising by this contamination constitute a very se-
rious problem. Minute numbers of cells containinegq uivalent amounts of DNA,
often transferred by contaminated syringes, pipet tubes, tubes, or gloves, may
be readily amplified and cause problems with the interpretation of results.
Only the most stringent precautions taken while collecting and manipulating
samples, together with positive and negative controls (reaction mixes with no
DNA, or DNA known to be negative or positive for the set of primers used), will
help avoid false positive results. Precautions include physically isolating PCR
preparations and PCR product, autoclaving of solutions, UV irradiating solu-
tions, aliquoting reagents, using disposable gloves, avoiding splashes and using
positive displacement pipets, premixing reagents, and adding the test DNA last.
False Negative. Positive and negativec ontrols remain as important when
determining the possibility of a false negative result. The true false negative
means that the target DNA was present in the test DNA, but was not detected
in the PCR reaction. This may be caused by sampling errors in which the
target sequence for PCR was present in very low numbers in the tissue DNA
extracted. The quantity of DNA taken for the reaction would not always be
adequate to contain a PCR target sequence. Poor quality of DNA may also be
a problem. These can be guarded against by using PCR with known primers
that readily amplify human DNA to judge the quality of the DNA for PCR,
whereas multiple testing will help exclude low levels of disease as a cause for
the negative result. RT-PCR raises another problem concerning the false nega-
tive result, which may be owing to a "resting" cell that is temporarily not
expressing the disease-related gene. Here, the failure to detect the positive
result does not exclude its subsequendte tection and should be consideredw hen
interpreting RT-PCR results. The last element to be considered is the negative
result with such consensus primers as those used in antigen receptor PCR, in
which clonal evolution by the disease may lead to a negative result, or the
consensus is inadequate to give good annealing for PCR. Essentially, if results
raise doubts, then repeat the PCR.
viii Preface
As a technique, PCR has now had eight years of development. Modifica-
tions of the basic method in the field of cancer have now provided a sensitive
and flexible approach to the provision number of diagnostic possibilities,
including sequences analysis, detection of base mutations, demonstration of
chimeric gene products, and the determination of the presence or absence of
altered DNA related to disease. In the next few years, there will be a further
expansion with enzymatic amplification becoming even more automated and
entering the routine pathology laboratories to improve our understanding and
management of cancer.
Recently, a number of less sensitive but just as powerful techniques,
including fluorescence in situ hybridization (FISH), a modificaticoanl led com-
parative genome hybridization (CGH), the detection of apoptosis (programmed
cell death), and in situ hybridization (ISH), have added to the molecular diag-
nostic repertoire. The methodology for these newer methods is given in the
final chapters of this book.
It is our hope that workers in the field of cancer molecular pathology
will be able to use the practical guides given to initiate and evaluate cancer
patients and will also be able to use the experience gained to further develop
their molecular diagnostic skills as further consistent DNA alterations are dis-
covered in tumor material.
Finbarr E. Cotter
Contents
Preface ............................................................................................................. v
Contributors ..................................................................................................... xi
List of Color Plates ........................................................................................ xiii
TRAP I. LACIGOLOTAMEH SEICNANGILAM
1 PCR of Gene Rearrangements for the Detection of Minimal Residual
Disease in Childhood ALL,
Nick Goulden, Kenneth Langlands, Colin Steward, Chris Knechtli,
Mike Potter, and Tony Oakhill .......................................................... 3
2 Detection of BCR-ABL in Hematological Malignancies by RT-PCR,
Nicholas C. P. Cross ............................................................................ 25
3 Molecular Diagnosis of Acute Myeloid Leukemia with Maturation,
FAB-Type M2,
Ewald J. B. M. Mensink and Louis .T F. van de Locht ..................... 37
4 Analysis of the PML/RAR-c~ Fusion Gene in Acute Promyelocytic
Leukemia by Reverse-Transcription Polymerase Chain Reaction:
Technical Recommendations, Advantages, and Pitfalls,
Daniela Diverio, Anna Luciano, Roberta Riccioni,
Francesco Lo Coco, and Andrea Biondi ....................................... 47
5 RT-PCR Analysis of Breakpoints Involving the MLL Gene Located
at 1 lq23 in Acute Leukemia,
Chris F. E. Pocock and Finbarr E. Cotter .......................................... 55
6 Polymerase Chain Reaction for Detection of the t(14;18) Transtocation
in Lymphomas,
Peter W. M. Johnson ............................................................................ 63
7 NPM-ALK Reverse Transcriptase-Polymerase Chain Reaction Analysis
for Detection of the t(2;5) Translocation of Non-Hodgkin's Lymphoma,
Sheila A. Shurtleff, James R. Downing, and Stephan .W Morris ........ 75
8 Molecular Diagnosis of the 5q Deletion in Malignant Myeloid Disorders,
Jackie Boultwood ................................................................................. 91
9 Polymerase Chain Reaction Based Methods for Assessing Chimerism
Following Allogeneic Bone Marrow Transplantation,
Mark Lawler and Shaun .tF McCann ................................................. 105
