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

one patient revealed more than 9 million cmV/10E5 cells. Histological
tissue assessment of the latter patient also revealed the existence of inclusion
bodies which are typical for a severe cmV infection. Interestingly,
samples with viral loads higher than 8000 cmV/10E5 cells were also
positive for cmV detection using immunohistochemistry. By analysing
different tissue sections of individual patients major differences in viral
loads were notable indicating that cmV infection in ulcerative colitis
could be locally quite heterogeneous. From three patients consecutive
samples were analysed after initiation of antiviral therapy. In every case
a significant reduction in cmV viral load could be detected indicating a
positive response to the therapy.
Conclusions. Quantitative real-time PCR is useful for detection of cmV
infection in tissue samples of patients suffering from ulcerative colitis.
Moreover, the assay seems to be capable for follow up monitoring of therapy
response during antiviral treatment.
SA-P-051
Ion semiconductor sequencing: a novel technology for analysis
of somatic mutations in formalin-fixed and paraffin-embedded
tumor biopsies
K . König1 , U . Koitzsch1 , J . Altmüller2 , S . Merkelbach-Bruse1 , J . Fassunke1 ,
C . Vollbrecht1 , L . Heukamp1 , P . Nürnberg2 , R . Büttner1 , M . Odenthal1 1 2 University Hospital Cologne, Institute for Pathology, Köln, Unversity
Cologne, Cologne Center of Genomics
Aims. Somatic mutations in a panel of genes encoding signal transducers
that are involved in cell growth, proliferation and differentiation take
center stage in molecular pathology due to their impact on tumor prognosis
and therapy response. Recently, an ion semiconductor sequencing
system, based on the semiconductor determination of proton release
after each nucleotide coupling to the DNA strand, was described (Rothberg
et al.; Nature 2011). In the present study, we aimed to evaluate this
novel sequencing technology for mutation detection in formalin-fixed
paraffin-embedded (FFPE) tumor biopsies.
Methods. DNA was purified from FFPE biopsies or from macrodissected
tumor materials by the M48 platform (Qiagen). PCR amplification
of a wide panel of target genes including EGFR exon 18, 19, 20, 21, Kras
codon 12, 13 locus, the Braf codon 600 locus, and Pik3Ca was performed
according to the routine processing in molecular pathology. Up to
109 molecules of the target amplicons of each patient were pooled, sheared,
and adapter and multiple identifier were ligated following the Xpress
protocol (ABI life Technologies). Multiplexed libraries were then used
for clonal amplification by means of emulsion PCR and applied to ion
semiconductor sequencing using the Ion Torrent platform.
Results. Amplicons from EGFR exon 18, 19, 20, 21, Kras codon 12, 13 locus,
the Braf codon 600 locus, and PikCa exons were successfully sequenced
and somatic mutations were detected after bioinformatic analyses.
Therefore, the ion semiconductor sequencing technology combined with
the Xpress adapter ligation approach revealed that amplicons of well established
PCR assays such as assays that are already established in the
routine diagnostics, are suitable templates for parallel sequencing.
Conclusions. In conclusion, parallel ion semiconductor sequencing is a
promising and efficient technology for multiplexed mutation analyses
that may be easily linked to existing PCR approaches of molecular routine
diagnostics.
SA-P-052
Inactivation of JNK as pathogenic factor in colorectal carcinogenesis
upon inflammation
K . Reissig1 , T . Guenther1 , A . Silver2 , R . Hartig3 , P . Steinberg4 , A . Roessner1 ,
A . Poehlmann1 1Otto-von-Guericke University Magdeburg, Department of Pathology,
Magdeburg, 2Queen Mary University London, Colorectal Cancer Genetics,
London, United Kingdom, 3Otto-von-Guericke University Magdeburg,
Department of Molecular and Clinical Immunology, Magdeburg, 4Institut of
Food Toxicology and Analytical Chemistry, Hannover
Aims. We have recently developed a cell culture model using human colon
epithelial cells (HCEC) and H2O2 to study tumor-initiating molecular
events in inflammation-associated colorectal cancer. As we have supposed
epigenetic changes that may account for HCEC transformation,
we aim to find epigenetic signaling events. As DNA damage checkpoints
are important in cancer cell proliferation, their role in HCEC transformation
was analyzed.
Methods. We simulated acute inflammation by treating HCEC with a pathophysiologic
concentration of H2O2 as ROS (reactive oxygen species).
Furthermore, we developed transformed HCEC by repeated treatment
cycles to mimic chronic inflammation. In order to analyze DNA damage
checkpoints, we performed cell cycle analysis using FACS. The function
of JNK was investigated using the JNK inhibitor SP600125. The cells were
further analyzed by immunoblotting.
Results. To prove whether single H2O2 treatment leads to activation of
DNA damage checkpoints, cell cycle analysis was performed. Acute ROS
activated the G1/S and G2/M DNA damage checkpoints. At the same
time, there was prominent activation of JNK signaling. This coincided
with periods when cell cycle arrest was observed. As the JNK inhibitor
was able to rescue S and G2/M arrest, the observed cell cycle arrest is
JNK-dependent. Immunoblotting analysis after JNK inhibition identified
p21, an inhibitor of cell cycle progression, as cellular JNK target. Importantly,
in line with uncontrolled proliferation of transformed HCEC,
we observed altered JNK activation. This down-regulation of activated
JNK caused down-regulation of p21 in transformed HCEC.
Conclusions. JNK inactivation plays an important role in HCEC transformation
and seems to be a pathogenic factor. Subsequent down-stream
p21 down-regulation occurs early, seems to be as efficient as p53 mutation,
and fits very well with the suggestive role of p21 as a potential tumor
suppressor in the colon. We speculate that both inactivation of JNK and
p21 down-regulation might cause the cell to turn on a one way street to
uncontrolled proliferation as one defining feature of the initiation of the
inflammation-carcinoma pathway in the colon.
SA-P-053
Validation of primary antibodies for immunohistochemistry
H .J . Grote1 1Merck KGaA, Histopathology – Biomarker Technologies, Darmstadt
Aims. Tissue biomarkers are gaining increasing importance in establishing
targeted therapies. In oncology tissue biomarkers are used for target
validation and indication seeking, for patient stratification and for pharmacodynamic
assays. Immunohistochemistry on formalin-fixed paraffin-embedded
(FFPE) tissue (IHC-P) is a key technology in biomarker
discovery and development. However, there is increasing evidence that
IHC-P is frequently impacted by limited specificity of primary antibodies.
This report presents strategies, technologies and examples for improved
primary antibody validation.
Methods. Antibody validation is a multi-step process. It starts with gathering
information on target expression and identification of positive
and negative expression controls. Standard laboratory procedures like
Western blot, FACS or qRT-PCR may be useful to corroborate or complement
published expression data in cancer cell lines. A Western blot
may also provide first information about selectivity of a candidate pri-
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