2008 Barcelona - European Society of Human Genetics

Cancer genetics
P04.127
FGFR3 mutations and 3p, 9p, 9q & p53 deletions in noninvasive
bladder cancer
A. Y. Babayan 1 , O. A. Kuznetsova 1 , S. V. Bashkatov 2 , O. B. Karyakin 2 , D. V.
Zaletaev 1,3 , M. V. Nemtsova 1,4 ;
1 Research Institute for Molecular Medicine, Moscow, Russian Federation,
2 Medical Radiology Reserch Centre RAMS, Obninsk, Russian Federation,
3 Research Centre for Medical Genetics, RAMS, Mosocw, Russian Federation,
4 Research Centre for Medical Genetics, RAMS, Moscow, Russian Federation.
The bladder cancer is one of the most severe oncological diseases .
Importance of the development of new diagnostic clinical markers is
dictated by its high incidence and aggressive tumor growth . Cancer
development is a complex, multistage process involving various genetic
and epigenetic alterations . In our study we have tried to establish
associations between several genetics alterations (loss of heterozygosity
(LOH) at 3p, 9p, 9q and p53 loci and S249C activating mutation
in FGFR3) and tumor clinical phenotype (tumor differentiation, type of
growth and a non-recurrent period) . During the last year and the half
we have studied 40 matched samples (blood and tissue) from patients
with primary bladder cancer and 35 samples from patients with recurrent
bladder cancer, of which 17 patients demonstrated recurrence
within one year . All samples were divided into groups by differentiation
rate: 53 samples classified as G1+G2 and 22 as G3. By growth type
53 samples were unifocal and 39 multifocal . LOH at 3p, 9p, 9q and
p53 loci were detected by microsatellite analysis, and SSCP and direct
sequencing sought for identification of FGFR3 activating mutations .
Statistical analysis of the results included comparison of the patients’
clinical groups by Fisher’s exact test, calculation of odds ratios and
corresponding 95% confidence intervals, with GraphPad InStat v.3.5
software. Our results demonstrate that 9p deletions are significantly
more frequent in tumors with high recurrence rate (within one year) .
FGFR3 mutations are prevalent in G1+G1 group of tumors . No significant
differences were found between tumors with various growth
types .
P04.128
Evidences for cancer cells showing stem features from human
bladder transitional cell carcinoma
A. Bentivegna1 , D. Conconi1 , E. Panzeri1 , E. Sala2 , G. Bovo2 , M. Casu2 , P.
Viganò2 , G. Strada2 , L. Dalprà1,2 ;
1 2 Università degli Studi di Milano-Bicocca, Monza, MI, Italy, S. Gerardo Hospital,
Monza, MI, Italy.
Transitional cell carcinoma (TCC) is the most common type of bladder
cancer accounting for more than 90-95% and it often recur (75% after
5 years) .
Emerging evidence has suggested that the capability of a tumor to
grow, propagate and recur is dependent on a small subset of cells
within it, termed cancer stem cells (CSCs) . Current failure of cancer
therapies may be due to their lower effect on CSCs that are resistant
and retain their full capacity to divide and restore the tumor cell mass .
Although data have been provided to support this theory in human
blood, brain, and breast cancers, the identity of bladder cancer stem
cells has not been determined. Here, we report the initial findings towards
the isolation and preliminary characterization of a putative CSCs
population from human bladder TCCs . These cells, isolated from fresh
surgical specimens, were induced to proliferate in vitro in serum-free
medium containing the mitogenic growth factors EGF and bFGF . The
proliferating cells generated within 48 h detached spheres that showed
clonal origin. Cells also resulted positive by immunofluorescence for
the stem cells’ markers CD133, Oct-4, Nestin and Cytokeratins . We
also conducted a cytogenetic study on fresh chromosome spreads
and, when possible, on chromosome spreads after different time of
culture and a parallel molecular cytogenetic study by FISH on paraffin
embedded tissue sections and on fresh and after culture nuclei.
We found important karyotype changes by culture selection, losing the
complexity present in fresh tumors and a marked molecular heterogeneity
between the tumors .
P04.129
Reliable methylation Analysis for Epigenetic Research - Novel
Technologies offering a complete and standardized workflow
G. Schock, T. Träger, C. Korfhage, R. Peist, N. Rudinger, N. Fang, D. Jansen,
D. Löffert;
QIAGEN GmbH, Hilden, Germany.
The analysis of changes in DNA methylation is challenging due to the
lack of standardized methods for providing reproducible data . Here we
present a complete and standardized workflow for methylation analysis
.
Bisulfite Conversion: QIAGEN’s EpiTect Bisulfite technology represents
a unique system for DNA protection against DNA degradation
which guarantees the highest conversion efficiency of ≥ 99% and highest
DNA quality .
Whole bisulfitome amplification: Since the quantity of converted
DNA is often limited, we here present a novel technology for the reliable
and representative amplification of the entire bisulfite converted
genomic DNA _ the bisulfitome _ to overcome limitations in methylation
analysis derived by limited DNA amounts .
Dedicated PcR technology for methylation analysis: Methylation
Specific PCR (MSP) reactions often require extensive optimization.
We present a mutant Taq DNA Polymerase that has been genetically
engineered to increase primer extension specificity through better discrimination
of 3’ single base mismatches of the primer .
For highly sensitive TaqMan probe based real-time PCR, we have developed
a novel reagent that yields accurate methylation analysis in
real-time. A selection of pre-developed MethyLight assays for a first set
of genes will also be made available .
Assay control reagents: Assay design and the success of methylation
analysis by PCR can be facilitated and assessed by the use of
standardized human control DNAs .
With its newly introduced EpiTect solutions, QIAGEN makes available
standardized, pre-analytical and analytical solutions from DNA sample
collection, stabilization and purification, to bisulfite conversion and
real-time or endpoint PCR methylation analysis or sequencing .
P04.130
Karyotype alterations of CD133(+) stem-like cells of glioblastoma
multiforme
W. Yang 1 , D. Yang 1 , C. C. Lin 2 , L. Hsieh 2 , D. Cho 3 , C. Hung 1 , T. Li 1 ;
1 Cell/Gene Therapy Research Lab., China Medical Univ Hospital, Taichung,
Taiwan, 2 Dept of Medical Research China Medical Univ Hospital, Taichung,
Taiwan, 3 Dept of Neurosurgery, China Medical Univ Hospital, Taichung, Taiwan.
Recent studies have demonstrated the existence of cancer stem-like
cells with CD133 surface marker in glioblastoma multiforme (GBM) .
It is believed that these minor CD133(+) tumor cells possess more
efficient DNA-repair function than the majority CD133(-) progeny cells
and thus can survive the radiation therapy and/or chemotherapy and
rapidly give rise to fatal tumor recurrence . To isolate CD133(+) GBM
cells, we administered ionizing radiation to short-term tumor cell cultures
from 8 new GBM patients (participants in a clinical trial of immunotherapy)
. In one particular case, the GBM cells that survived irradiation
contained 50- 60% highly clonogenic and neurosphere-forming
CD133(+) cells . We also used FACS to isolate the CD133(+) cells
from un-irradiated original GBM cell population of this patient . Karyotype
analysis showed that the majority CD133(-) cells of initial culture
were 46, XY, +7, +der(7;9)(p10;p10),-10, -18, der(19)(q13), with little
change in near diploid karyotype within 10 passages in vitro . In contrast,
majority of FACS-isolated un-irradiated CD133(+) cells showed
a hypo-tetraploid with variation of chromosome number among them .
The radiation-survived CD133(+) cells of this patient showed similar
hypo-tetraploid patterns with slightly higher variation in chromosome
number than the un-irradiated CD133(+) cells . Individual cell clones of
the two CD133(+) categories were consistently hypotetraploid but still
with variation of chromosome number, suggesting chromosomal instability
during cell proliferation . Using these hypotetraploid CD133(+)
GBM cells, we have obtained preliminary results that certain therapeutic
agents that down-regulated CD133(+) expression also could shift
the hypotetraploid to the near diploid karyotypes of CD133(-) GBM
cells .
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