96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

Conclusions. In conclusion, 454 parallel sequencing is a very useful and
economic approach for molecular pathology due to sample multiplexing
and simultaneous target analyses. Both, FFPE extracted DNA and DNA
from cell preparations may be applied to the approach. If the mutation
rate is higher than 10% in the FFPE sample, the mutation is easily detected.
DO-114
IMDA: a methodical approach enhancing molecular diagnostic of
microcarcinomas and small biopsies
F . Mairinger1 , K . Worm1 , W . Grüning2 , T . Mairinger3 , K .W . Schmid1 1University Hospital Essen, Department of Pathology und Neuropathology,
Essen, 2Helios Klinikum Emil von Behring, Department of Pneumology,
Berlin, 3Helios Klinikum Emil von Behring, Department of Pathology, Berlin
Aims. The isothermal multiple displacement amplification (IMDA)
would be a powerful tool in molecular routine diagnostics for preamplification
of extraordinary small tumor samples (biopsies containing
small amount of tumor, microcarcinomas) but is not banked in pathological
laboratories. We designed a study to check the feasibility and convenience
of these methods for routine diagnostics on LCM microdissected
FFPE samples.
Methods. For validation of the IMDA assay, different benign FFPE tissue
samples were microdissected using LCM technology (areas ca. 50×50 µm
up to 150×100 µm) and afterwards preamplified by an commercial IM-
DA-kit (Qiagen REPLI-g FFPE assay). Chromosomal representation was
tested using qPCR of genes spanning regions on different chromosomes.
Afterwards, patient samples from the Helios Klinikum Emil von Behring
with a too small amount of material for conventional molecular
analysis were pre-amplified by IMDA and further processed with the
“normal” routine samples for EGFR analysis.
Results. With an amplification time duration of 3h and a starting material
of 50×50 µm extension a yield of total 250 µg DNA (concentration
of 5 µg/µl) could be generated. Preliminary results show an acceptable
relative chromosomal representation, the presence of diagnosis relevant
genes in clinical samples could be proven. Mutational analysis of clinical
samples was accomplishable and shows concordance with earlier diagnostically
findings.
Conclusions. We could proof the diagnostic feasibility and convenience
of IMDA for routine diagnostics. Also small amount samples, until now
not analyzable with molecular methods, will be sufficient for all-embracing
molecular routine diagnostics.
DO-115
miRNA 26b stabilizes the pro-apoptotic DAP Kinase by inhibiting
its E3 ubiquitin ligase DIP1
S . Knaup1 , S . Wach2 , A . Agaimy1 , J . Schulze-Luehrmann 1 , M . Hugele1 ,
S . Chakilam1 , R . Atreya3 , R . Wirtz4 , T .T . Rau1 , R . Schneider-Stock 1
1University of Erlangen-Nuremberg, Institute of Pathology, Erlangen,
2University of Erlangen-Nuremberg, Institute of Urology, Erlangen,
3University of Erlangen-Nuremberg, Department of Medicine I, Erlangen,
4STRATIFYER Molecular Pathology GmbH, Cologne
Aims. Tumor necrosis factor α (TNF) is a pro-inflammatory cytokine
involved in the inflammatory reaction of the intestinal mucosa, but
also mediates tumor eliminating effect. We have shown recently that
treatment of HCT116 colorectal tumor cells with TNF led to a higher
expression of death-associated protein kinase (DAPK) and induced caspase-dependent
apoptosis. It is also known, that DAPK can be found
in a complex together with DAPK-inter-acting protein (DIP1) that antagonizes
the pro-apoptotic function of DAPK by ubiquitination. Upon
TNF treatment a decrease of the DIP1 protein level could be detected,
while there were no matching changes in the DIP1 mRNA levels. This
raised the question of a potential involvement of a miRNA binding to
the 3‘UTR of DIP1 regulating its degradation or translational inhibition.
A miRNA microarray analysis of TNF-treated HCT116 cells revealed a
significant up-regulation of miRNA 26b.
Methods. The potential of miRNA 26b to target DIP1 and thereby influencing
apoptosis through regulation of DAPK had to be verified. This
was achieved by a luciferase reporter assay and overexpression of miRNA
26b as well as knock-down of the miRNA and DIP1, followed by westernblot
and Real-Time PCR analysis. To assess the in vitro findings in vivo,
in situ hybridization was performed on tissue microarrays of normal, inflamed
(ulcerative colitis) and inflammation-associated colorectal tumor
samples to check the localization and abundance of miRNA 26b.
Results. In the luciferase reporter assay, miRNA 26b binds to DIP1,
confirming it as a miRNA 26b target. This was further verified by overexpression,
as well as a knock-down of miRNA 26b. More miRNA led
to a decrease of DIP1 protein levels and in return to a stabilization and
increase of DAPK protein levels. Vice versa, reduction of miRNA 26b
resulted in higher DIP1 protein levels and less DAPK protein. Knockdown
of DIP1 by siRNA showed an increase of apoptosis via caspase 3
cleavage. In human tissues of ulcerative colitis patients, in situ hybridization
verified a higher level of miRNA 26b in the inflamed colon crypts,
consistent with the grade of inflammation.
Conclusions. miRNA 26b promotes apoptosis in human colon cancer
cells by targeting the E3 ubiquitin ligase DIP1 and thereby stabilizing the
pro-apoptotic DAPK. miRNA 26b is strongly expressed in the inflamed
tissue of ulcerative colitis patients, suggesting a possible role in the regulation
of the inflammatory process.
DO-116
MRNA and microRNA stability in surgical tissue: an issue for biobanking
and biomarker identification
C . Schuster1 , W .E . Thasler2 , K .-F . Becker3 , T . Kirchner1 , F . Hlubek1 1Ludwig-Maximilians-University München, Institute of Pathology, München,
2Ludwig-Maximilians-University München, Department of Surgery,
Grosshadern Hospital, München, 3Technical University Munich, Institute of
Pathology
Aims. Human frozen tissues are one of the best sources for molecular
analyses such as microarrays, qPCR or Next-Generation-Sequencing. In
addition, frozen tissues are particularly valuable for biomarker identification.
Several biobanks comprising non-fixed frozen tissues have been
established alongside with corresponding clinical data repositories to
facilitate biomarker studies relevant for clinical diagnostics. Apart from
proteomic approaches, mRNA- and microRNA-expression profiles have
shown to be highly valuable for biomarker studies. Since RNA is generally
a fragile molecule, RNA integrity in tissue specimens has tremendous
impact on gene expression analyses, requiring a rigorous quality assessment
of biobank tissue samples.
Methods. To address this issue, we established a tissue quality test system
based on RNA integrity and differential gene expression. We used normal
and cancerous surgical tissue that was stored under various conditions
and for different time periods of ischemia prior to being snap frozen.
Results. The RNA was isolated and the quality was assessed in four steps:
First, the RNA was quantitated by spectrophotometry (NanoDrop) and
total RNA quality was determined by on-chip electrophoresis (Experion).
Second, the degree of RNA degradation and the maximum length of
RNA molecules available for downstream applications were determined
by amplicon length analysis of housekeeping genes using PCR amplification.
Third, the RNA expression level of selected genes were determined
and correlated to the time and condition of ischemia. The genes
analysed comprised highly regulated genes, signaling pathway genes and
genes induced by hypoxia or apoptosis. Fourth, the expression of selected
miRNAs representing different expression levels was determined by
quantitative PCR.
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