Table Of Contentc.
Wagener S. Neumann
(Eds.)
Molecular Diagnostics
of Cancer
With 50 Figures and 31 Tables
Springer-Verlag
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Budapest
Professor Dr. Christoph Wagener
Abteilung fUr Klinische Chemie, Medizinische Klinik
Universiti:its-Krankenhaus Eppendorf
MartinistraBe 52, W-2000 Hamburg 20, FRG
Dr. Siegfried Neumann
Diagnostische Forschung E. Merck
Frankfurter StraBe 250, W-6100 Darmstadt, FRG
ISBN-13: 978-3-540-55476-9 e-ISBN-13: 978-3-642-77521-5
DOl: 10.1007/978-3-642-77521-5
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© Springer-Verlag Berlin Heidelberg 1993
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Preface
Traditionally, in vitro diagnostics of cancer relies on the microscopic
examination of tissue sections or cells. Numerous studies based on
biochemical and immunochemical techniques focused on the meta
bolism, enzymes and structural components of tumor cells. Since
qualitative differences between normal and tumor cells could not be
detected, metabolic properties of human cells and surface-associated
or shed tumor markers did not have a significant effect on the clinical
differentiation between normal and malignant tissues.
Since the 1920s, a number of scientists have claimed that the cancer
ous cell is formed from normal cells by some kind of genetic mutation.
For half a century, no methods were available to fully define the
mutations on the molecular level. This changed dramatically, however,
during the past 15 years. New techniques were developed and are con
tinuing to be elaborated in the fields of molecular biology, molecular
genetics, cytogenetics and cell biology. Application of some of these
methods, such as transfer of human tumor DNA into cultured fibro
blasts and hybridization, as well as DNA- and protein sequencing
techniques, revealed homologies between retroviral oncogenes and
normal human genes and paved the way to the first identification of
specific genetic alterations in human tumors in 1982.
Since then many more genes altered in various malignancies have
been identified. Starting from the observation that cellular genes
homologous to retroviral oncogenes were located at breakpoints of
chromosomal translocations, many genes at chromosomal breakpoints
have been cloned which, because of homologies to structural motifs of
proteins with known functions, e.g., transcription factors or homeo
box genes, are thought to contribute to malignant transformation.
Furthermore, the detection of chromosomal deletions by cytogenetic
procedures or the use of polymorphic DNA markers led to the identi
fication of genes which contribute to malignancy when genes or gene
product are nonfunctional or lost.
VI Preface
A number of tumor-specific alterations in DNA structure, RNA syn
thesis and encoded proteins have been delineated. It can be antici
pated that the confirmation of such alterations by enlarged studies on
clinical specimens will gain significant diagnostic impact.
At present, established or putative areas of application have to be
defined. Furthermore, a number of technical problems have to be
solved before diagnostic procedures based on genetic alterations in
cancer cells will become routine. In those cases in which alterations
are of limited heterogeneity, the design of appropriate reagents and
methods is straightforward. Some genes, however, harbor multiple
mutations scattered over a significant portion of the gene. In these
cases, rational approaches to the sensitive detection of a mutation in a
given tumor are needed. Finally, the diagnostic significance of newly
described cancer genes has not yet been established.
In order to delineate present and putative future applications of
new diagnostic strategies based on the direct or indirect detection of
mutated cancer genes, a conference on the "Application of Methods
of Molecular and Cell Biology to the Diagnosis of Malignant Disease"
was held in Hamburg, FRG, in October 1991, by the German Society
for Clinical Chemistry. The canferene was sponsored by E. Merck,
Darmstadt, FRG.
Methodological aspects such as enzymatic target amplification by
the polymerase chain reaction, DNA fingerprinting and transfer of
putative cancer genes to appropriate recipient cells were covered, in
addition to recent aspects of the application of monoclonal antibodies
in immunohistochemistry and immunoscintigraphy.
The characterization of oncogenes and tumor suppressor genes was
correlated with growth control and dissemination of tumor cells on the
basis of in vitro or clinical findings. The contributions in this book pro
vide an update on established or newly described cancer genes and
may help in the translation of concepts from basic research into clini
cal practice.
The ultimate goal of new diagnostic strategies in cancer is to
improve diagnosis, prognosis and therapeutic regimens for patients.
We sincerely hope that the present volume will bring us closer to that
goal.
Hamburg/Darmstadt, C. Wagener
Autumn 1992 S. Neumann
Contents
The Polymerase Chain Reaction:
Methodology and Diagnostic Potential (A. Pingoud) . 1
Gene Loss as Marker in Tumor Development:
Considerations on the Origin of Specific Brain Tumors Due
to Combinant Loss of Function Mutations
of Tumor Suppressor Genes (K. D. Zang). . . . . . . . . . 17
p53: Oncogene, Tumor Suppressor, or Both? (W Deppert). 27
Tumor Genome Screening by Multilocus DNA Fingerprints
as Obtained by Simple Repetitive Oligonucleotide Probes
(J. T. Epplen, S. Bock, and P. Niirnberg) ............. 41
Activation of ras Oncogenes in Human Tumours
(N. R. Lemoine). . . . . . . . . . . . . . . . . . ..... 53
Laboratory Techniques in the Investigation
of Human Papillomavirus Infection (E.-M. de Villiers) .... 65
Detection of Minimal Residual Leukemia by Polymerase
Chain Reactions (c. R. Bartram) . . . . . . . . . . . .. .. 77
A Dominant Metastogene Confers Metastatic Potential
to Tumor Cells (H. Ponta, M. Zoller, K. -H. Heider, S. Seiter,
W Rudy, M. Hofmann, C. Tolg, and P. Herrlich). . . . . . 87
Progression in Human Melanoma Is Accompanied
by the Altered Expression of Cell Adhesion Molecules
(J. P. Johnson, B. G. Stade, U. Rothbacher, S. Stratil,
and G. Riethmiiller) . . . . . . . . . . . . . . . . . . . . 97
Structure, Function and Expression of the CEA Gene Family:
Diagnostic and Therapeutic Implications (W Zimmermann,
F. Grunert, G. Nagel, S. von Kleist, and J. Thompson) . ..... 107
VIII Contents
Membrane Proteins as Markers for Normal and Neoplastic
Endocrine Cells (G. Lahr and M. Gratzl). . . . . . . . . . . . . 117
Biological and Clinical Relevance of the Tumor-Associated
Serine Protease uPA (M. Schmitt, F. Janicke,
N. Chucholowski, L. Goretzki, N. Moniwa, E. Schiiren,
O. Wilhelm, and H. Graeff) . . . . . . . . . . . . .. . ... 129
Analysis of Growth Regulatory Pathways in Human
Neuro-Oncology (M. Westphal, W. Hamel, L. Anker,
and H.-D. Herrmann). . . . . . . . . . . . . . . .. . ... 151
HER-2/Neu/c-ERB-B2 Amplification: Clinical Relevance
in Mammary Carcinoma (w. Jonat, K. Friedrichs,
H. Eidtmann, J. Meybohm, and S. Singh) . . . . . . .. ..169
Cytokine-Mediated Regulation of Growth Factor Receptors
(EGF-R and erb-B2) in Pancreatic Tumors (H. Kalthoff,
C. Roeder, and W. Schmiegel) . . . . . . . . . . . . . . . .. . 175
Chimeric CEA-Specific Antibody Fragments and Anti-idiotypic
Antibodies for Immunoscintigraphy and New Therapeutic
Approaches to Colorectal Carcinomas (M. Neumaier) ...... 187
Tumor Localization by Immunoscintigraphy:
Potential and Limitations (S. Matzku and H. Bihl) . .207
Subject Index . . . . . . . . . . . . . . . . . . . . . 219
Contributors*
Anker, L. 1511 Meybohm, J. 169
Bartram, C. R. 77 Moniwa, N. 129
Bihl, H. 207 Nagel, G. 107
Bock, S. 41 Neumaier, M. 187
Chucholowski, N. 129 Niirnberg, P. 41
Deppert, W. 27 Pingoud, A. 1
Eidtmann, H. 169 Ponta, H. 87
Epplen, J. T. 41 Riethmiiller, G. 97
Friedrichs, K. 169 Roeder, C. 175
Goretzki, L. 129 Rothbacher, U. 97
Graeff, H. 129 Rudy, W. 87
Gratzl, M. 117 Schmiegel, W. 175
Grunert, F. 107 Schmitt, M. 129
Hamel, W. 151 Schiiren, E. 129
Heider, K.-H. 87 Seiter, S. 87
Herrlich, P. 87 Singh, S. 169
Herrmann, H.-D. 151 Stade, B. G. 97
Hofmann, M. 87 Stratil, S. 97
Janicke, F. 129 Thompson, J. 107
Johnson, J. P. 97 Tolg, C. 87
Jonat, W. 169 Villiers, E.-M. de 65
Kalthoff, H. 175 Westphal, M. 151
Kleist, S. von 107 Wilhelm, O. 129
Lahr, G. 117 Zang, K. D. 17
Lemoine, N.R. 53 Zimmermann, W. 107
Matzku, S. 207 Zoller, M. 87
* The addresses of the authors are given on the first page of each contribution.
1 Page on which contribution begins.
The Polymerase Chain Reaction:
Methodology and Diagnostic Potential
A. Pingoud
Institut fur Biophysikalische Chemie, Zentrum Biochemie, Medizinische Hochschule
Hannover, Konstanty-Gutschow-StraBe 8, W-3000 Hannover 61, FRG
Introduction
Since its discovery in 1983 (for an entertaining account of the discovery
see [1 D, the polymerase chain reaction (peR) has revolutionized the life
sciences. The remarkable success of this method is due to the fact that it is
simple, versatile, and robust. Most importantly, there is increasing interest
in the target of the peR, namely DNA (or RNA), the carrier of the genetic
information, whose precise composition one needs to know in order to
understand the physiology and pathophysiology of living organisms.
The peR involves the in vitro amplification of a specific DNA sequence,
starting from as little as a single DNA molecule and ending up with a billion
copies. The reaction is based on the precise annealing and faithful extension
of two oligodeoxynucleotide primers that flank and thereby define the target
region to be amplified in the double-stranded DNA template. A peR cycle
consists of denaturation of the double-stranded DNA, annealing of the
primers and their extension using a DNA polymerase. With denaturation
of the products a new cycle begins. Repeated cycles of denaturation,
annealing and polymerization result in the exponential amplification of
specific DNA fragments whose 5' ends are represented by the primers used
in the polymerization. The length of the DNA fragments produced in the
peR is determined by the length of the primers used and the distance
between the 3' ends of the primers when hybridized to the target DNA.
Thus, product identification can make use of the length of the peR pro
ducts. Sequence information characteristic for the amplified DNA between
the two primers, as well as "chemical tags" associated with the two primers
and incorporated into the DNA fragments, can also be used for this purpose.
The result of a peR analysis can be a qualitative one, as needed for the
detection of hereditary diseases and genetic polymorphisms (including
disease predisposition), the identification of somatic mutations (including
cancer) and the presence of viral or microbial pathogens. There is an
Wagener/Neumann (Eds.)
Molecular Diagnostics of Cancer
© Springer-Verlag Berlin Heidelberg 1993
2 A. Pingoud
increasing demand for quantitative PCR, for example to measure the "titer"
of a pathogen, the number of infected or mutated cells in a tissue, and the
concentration of certain mRNAs.
In the present contribution, I shall briefly describe the basic methodology
and review recent developments regarding PCR procedures, from sample
preparation to product identification. I shall also discuss improvements in
PCR machines, as well as the results of efforts to automatize PCR analysis.
I will briefly mention other amplification protocols as alternatives to
PCR, such as the ligation chain reaction (LCR) and self-sustained sequence
replication (3SR). Finally, some relevant recent applications of the PCR for
medical diagnostic purposes will be mentioned.
It must be pointed out that two excellent review articles, similar in scope
to this one, but with different emphasis, were published in 1991 [2,3]. For a
thorough introduction to the methodology and applications of the PCR, the
reader is referred to four review volumes published in the last two years
[4-7].
The Polymerase Chain Reaction
Template
The usual PCR template is double-stranded DNA. Single-stranded DNA or
RNA can be used, the latter requiring reverse transcription. Typically,
nanogram amounts of cloned DNA or microgram amounts of human
genomic DNA are employed, the equivalent of 104_105 target DNA
molecules. However, much lower amounts of DNA can be amplified, down
to the DNA of single cells. In general, a standard nucleic acid purification
involving a proteinase K treatment, phenol-chloroform extraction and
ethanol precipitation is sufficient for most purposes. For special purposes,
depending on the amount and complexity of the biological sample, more
simple (lysing cells by boiling in water) or more complicated (separation of
cells, removal of cell debris etc.) DNA extraction protocols have been
worked out. For example, DNA preparation from blood previously were
considered to be dependent on the separation of leukocytes from the vast
excess of erythrocytes by a Ficoll-Hypaque density gradient centrifugation;
now, protocols have been worked out in which erythrocytes are selectively
lysed, then the DNA is extracted from the leukocytes by boiling in the
presence of Chelex-100, which removes the iron ions [8]. Another example
is given by the PCR amplification of DNA contained in paraffin-embedded
fixed tissues for prospective studies. The success of the amplification
depends on the fixative used, the time of fixation and the length of the target
DNA. While formaldehyde fixation quickly leads to material that cannot be
amplified anymore, alcohol fixation allows amplification after long storage
times without affecting histological detail [9]. Other examples that demon-
