2009 Vienna - European Society of Human Genetics

Cancer cytogenetics
mutations in codon 12 and 13 of the KRAS gene. The test is based
on PCR in the presence of a wild-type KRAS suppressor (mutant-enriched
PCR), followed by hybridization of PCR products to teststrips
presenting a parallel array of allele-specific oligonucleotide probes.
The performance of the StripAssay was evaluated on DNA obtained
from cultured cell lines, from formalin-fixed paraffin-embedded (FFPE)
tissue and from stool. Using serial dilutions of DNA from various KRASmutated
tumor cell lines into normal DNA, each of the 10 mutations
was shown to be detectable at levels as low as 1%.
DNA samples containing various proportions of mutated KRAS were
analyzed by the StripAssay in direct comparison to real-time PCR, dideoxy
sequencing and pyrosequencing. While all methods correctly
identified samples containing 25% mutated DNA, dideoxy sequencing
and pyrosequencing failed to detect levels of 12.5% or lower. Both the
StripAssay, as well as real-time PCR, unambiguously identified 10%,
5% and 1% of KRAS-mutated DNA in the presence of excess wild-type
DNA.
The simultaneous detectability of 10 different mutations with excellent
sensitivity will make the StripAssay a very useful tool for the assessment
of the KRAS mutation status in cancer patients. (oberkanins@
viennalab.co.at)
P06.208
A detailed analysis of K-ras point mutations in Russian patients
with sporadic adenomatous polyps and adenocarcinomas of the
colorectum
F. A. Amosenko 1 , E. L. Korchagina 2 , T. I. Matveeva 1 , N. V. Poltavets 1 , R. F.
Garkavtseva 2 , A. V. Polyakov 1 ;
1 Research centre for medical genetics, Moscow, Russian Federation, 2 NN
Blokchin Cancer Research Centre, Russian Academy of Medical Sciences,
Moscow, Russian Federation.
To investigate spectrum, frequency and clinical significance of K-ras
point mutations in colorectal (CR) adenocarcinomas and polyps of
Russian patients we examined alterations at codons 12 and 13 in primary
sporadic adenocarcinomas at various stages and differentiation
from 58 patients, in adenomas with different histology and displasia
from 33 patients, and in malignant polyps from 13 individuals. The average
age of patients with cancer was 63.2, with adenomas - 58.8, and
with malignant polyps - 62.1. DNA was extracted from surgical material.
The mutations were studied using PCR, SSCP, RFLP and automatic
sequencing. 25 (43.1%) of carcinomas, 16 (48.5%) of adenomas
and 9 (69.2%) of malignant polyps examined harbored
K-ras mutations. The mutation pattern of K-ras of CR carcinomas was
GGT->GaT (32%), GtT (16%), GcT (12%), aGT (8%), tGT (8%), GGC-
>GaC (20%), cGC (4%); in adenomas - GaT (37.5%), GtT (31.3%),
aGT (6.2%), cGT (6.2%), GaC (18.8%); and in malignant polyps - GaT
(33.3%), GtT (11.1%), tGT (22.3%), GaC (33.3%). Thus the mutation
profiles of K-ras at codons 12 and 13 in Russian patients are not different
from spectrum found in other parts of the world. The relationship
between the presence of K-ras mutation in samples and clinicopathological
data of the investigated individuals (age at diagnosis, sex,
staging of cancer, histology of adenomas, the location of tumours or
polyps) were analysed.
P07. cancer cytogenetics
P07.01
High-resolution mapping of chromosomal rearrangements at
common fragile sites on chromosome 14 in tumor cells
D. Ibragimova, A. Blumrich, L. Brückner, M. Schwab, L. Savelyeva;
DKFZ, Heidelberg, Germany.
Common fragile sites (cFS) are regions of genomic instability that are
particularly prone to breakage under conditions partially inhibiting DNA
synthesis. These regions are found in all individuals and appear to be
the hotspots of chromosomal rearrangements in cancer and neurological
diseases. Approximately 90 cFS regions have been cytogenetically
identified, but only few of these have been determined at DNA sequences
level and completely characterized. Two aphidicolin-inducible
cFS on chromosome 14, FRA14B and FRA14C, are listed in Genome
Database and located at 14q23 and 14q24.1, respectively. To identify
the precise genomic position of these cFS, we have performed sixcolour
FISH-mapping with BAC-probes on metaphase chromosomes
of lymphocytes treated with aphidicolin to activate breakage at cFS.
We have determined that FRA14B and FRA14C span large genomic
regions of 600 kb and 800 kb, respectively. To assess the possible role
of these two cFSs in cancer chromosome rearrangements we used
fine-tiling oligonucleotide array CGH with subsequent validation of obtained
results by PCR and FISH. We have detected multiple breakpoints
within FRA14B and FRA14C occurring in cancer samples from
breast, colon and neuroblastoma. Non-random distribution of breaks
along chromosome 14 with preferential involvement of cFS regions
was demonstrated in different cancer cell lines and primary breast tumors.
P07.02
cytogenetic Effect in Liquidators the Accident at chernobyl NPP
E. A. Domina;
Institute of experimental pathology, oncology and radiobiology, Kiev, Ukraine.
Objectives: To examine correlative relationship between radiation effect
rate cytogenetic and clinical effects in those who participated in
liquidation of the consequences of the accident at Chernobyl NPP during
early and remote terms.
Methods: cytogenetic (analysis of chromosomes aberrations in culture
of human lymphocytes) and statistic (model of spline regression).
Group of researched was 12 100 liquidators.
Results: The work is aimed at the improvement of biologic (cytogenetic)
dosimetry and indication of radiation affection rate in victims of
radiation. Approximation of dose-effect dependence has been elaborated
on the basis of chromosomes affection values in culture of human
lymphocytes and model of spline regression. The proposed model
differs from the other, based on traditionally used in biologic dosimetry
linear and linear-quadratic models, in more accuracy of approximation
and possibility to predict the effect of transition of calibring curve on
the plateau.
Inverse relationship of frequency of malignancies in liquidators of the
consequences of the accident: the highest values are noted at low
doses (10-50 mGy). Frequency of malignancies decreases with increase
of radiation doses. The obtained data gave grounds for the
hypothesis that low doses of absorbed radiation are statistically significant
factor of carcinogenic risk. A conclusion was made that it can
be connected with insufficient switching on anticarcinogenic defense
of human organism at a range of low doses. Cytogenetic criteria for
formation of groups of for formation of groups of increased cancer risk
are determined.
Conclusion: Low doses of absorbed radiation are statistically a significant
factor of carcinogenic risk.
P07.03
Array-based comparative genomic hybridization profiling
of diffuse astrocytomas for genomic aberrations linked to
prognosis
A. Brockschmidt 1 , E. Külshammer 1 , B. Klink 2 , C. Landwehr 1 , B. Radlwimmer 3 ,
M. Sabel 4 , J. Schramm 5 , M. Westphal 6 , G. Schackert 7 , J. Tonn 8 , T. Pietsch 9 , H.
Berger 10 , M. Löffler 10 , M. Weller 11 , G. Reifenberger 2 , R. G. Weber 1 ;
1 Institute of Human Genetics, Rheinische Friedrich-Wilhelms-University, Bonn,
Germany, 2 Department of Neuropathology, Heinrich-Heine-University, Düsseldorf,
Germany, 3 German Cancer Research Center, Heidelberg, Germany,
4 Department of Neurosurgery, Heinrich-Heine-University, Düsseldorf, Germany,
5 Department of Neurosurgery, Rheinische Friedrich-Wilhelms-University, Bonn,
Germany, 6 Department of Neurosurgery, University of Hamburg, Hamburg, Germany,
7 Department of Neurosurgery, Technical University, Dresden, Germany,
8 Department of Neurosurgery, Ludwig-Maximilians-University, Munich, Germany,
9 Department of Neuropathology, Rheinische Friedrich-Wilhelms-University,
Bonn, Germany, 10 Institute for Medical Informatics, Statistics and Epidemiology,
University of Leipzig, Leipzig, Germany, 11 Department of Neurology, University
Hospital Zürich, Zürich, Switzerland.
The clinical course of patients with diffuse astrocytomas of WHO grade
II (AII) is highly variable. To identify genomic alterations possibly linked
to prognosis, we screened 11 AII from patients with a long recurrence
free interval (RFI) of 60-144 months (AII-long), 9 AII from patients with
a short RFI of 2-25 months (AII-short), and 12 anaplastic astrocytomas
(WHO grade III, AAIII) for genomic imbalances by genome-wide
array-based comparative genomic hybridization. The number of genomic
imbalances was higher in AII-short as compared to AII-long, with
an average of 9.7±1.6 vs. 7.5±2.4 (mean±SEM) alterations per tumor