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

Abstracts
ted genes will in the near future allow for providing helpful information
on individual cancer-genomes for individualized tumor therapy. Also,
NGS will become applicable to rather small specimen, including biopsies,
circulating tumor cells and circulating free DNA in plasma. Even so
NGS is already used in the clinical setting, for instance for the detection
of disease gene mutations of familial breast and ovarian cancer and for
genetic diseases, data analysis and interpretation using biostatistics, bioinformatics,
systems biological and mathematical approaches including
mathematical modeling is still rather complex. Robust strategies for data
analysis are being needed and are being developed.
Solving the key problems in cancer biology will therefore only be accomplished
by orchestrating a manifold collaboration between different
disciplines (life sciences, mathematics, and informatics). Platform comparison
will be provided for expression data comparing RNA seq and
array based methods. We will glimpse into the future and discuss third
generation sequencing and single-molecule sequencing technologies.
Important goals are to improve our knowledge on the regulation and
function of genes and proteins in single cells, tissue and organs.
While NGS is just on its way to be established as a routine diagnostics
tool, the next next-gen sequencing techniques are already emerging.
Whereas current NGS techniques depend on optics and thus require
elaborate signal detection, the third and fourth generation techniques
simply measure changes of electric current, either when DNA passes
through nanopores or bends nanowires in a sequence dependent manner.
Being electronic devices like computer processors, the third and
fourth generation sequencing machines will be very fast, very small and
rather cheap.
FR-023
Prognostoc biomarkers of gastric cancer
V . Warneke 1 , H .-M . Behrens 1 , C . Röcken1 , J . Haag1 , K . Balschun1 , C . Böger1 ,
C . Böger1 , T . Becker2 , J . Hartmann3 , M . Ebert4 , C . Röcken5 1 2 Christian-Albrechts-University, Department of Pathology, Kiel, Christian-
Albrechts-University, Department of Surgery, Kiel, 3Christian-Albrechts-Uni versity, Department of Internal Medicine II, Kiel, 4University of Mannheim,
Dept . of Medicine II, 5University Hospital Schleswig-Holstein, Campus Kiel,
Department of Pathology, Kiel
Aims. Chemotherapy for the treatment of gastric cancer (GC) is evolving
rapidly and continues to improve patient survival. We studied phenotypic
and genotypic biomarkers for GC, in order to test whether these
biomarkers are independent prognosticators of patient survival and
whether any of these biomarkers should be considered to tailor patient
treatment in the future.
Methods. 485 patients (299 men, 186 women; median age 68 years) with
GC had undergone either total or partial gastrectomy for adenocarcinomas
of the stomach or oesophagogastric junction. Survival data and
date of death were available for 469 patients. The pTNM-stage was based
on surgical pathological examination. The Laurén and mucin phenotype
was assessed. H. pylori- and Epstein-Barr virus infections were documented.
The following markers were studied: BRAF-, KRAS-, NRAS-
and PIK3CA (exon 9 and 20)-genotype, microsatellite instability, Her2/
neu-status, E-cadherin, β-catenin and EpCAM-expression.
Results. An intestinal type GC was found in 184 patients, a diffuse type
in 224. A persistent H. pylori-infection was found in 64 (15.5%) patients,
an EBV-infection in 15 (4.0%). Seventeen (3.6%) GCs showed a KRAS-,
12 (2.5%) a PIK3CA (exon 9)- and 9 (1.8%) a PIK3CA (exon 20)-mutation.
No NRAS- and BRAF-mutation was found in our series. 424 (95.1%)
GCs were EpCAM- and 46 (10.2%) Her2/neu-positive. 33 (7.3%) GCs
were highly microsatellite unstable, 31 (93.9%) of which showed loss of
expression of MLH1 and PMS2. Patient survival correlated with Laurén
phenotype, MSI-H and BerEP4-expression. Patient age, stage grouping
according to the Kiel-proposal, lymph node ratio and Mucin 2 were independent
prognosticators of patient survival.
54 | Der Pathologe · Supplement 1 · 2012
Conclusions. A thorough staging and surgical pathological examination
is the most important tool to assess patient prognosis. KRAS, PIK3CA
(exon 9 and 20), BerEP4-, Her2/neu- and MSI-status may be considered
to tailor patient treatment in the future.
FR-024
Deep-sequencing: Speed-up diagnostics of colorectal carcinoma
M . Rechsteiner1 , A . Bohnert 1 , A . von Teichmann 1 , S . Schmid-Brun1 ,
P . Schraml1 , H . Moch1 1University Hospital Zurich, Surgical Pathology, Zürich, Switzerland
Aims. In colorectal carcinoma, KRAS mutations have emerged as a major
predictor of resistance to anti-EGFR antibody treatment. Although the
role of BRAF mutations, in predicting the response to anti-EGFR drugs
still remains controversial, patients with a mutated BRAF gene exhibit
a significant shorter survival than patients without a mutation. In this
project we aimed to establish a high-troughput ultra-deep sequencing
platform to cope with the increased demand for sequence information
at medical institutions. With this platform we intend to unravel low frequency
mutations below the detection limit of Sanger sequencing and to
elucidate throughput power for diagnostics.
Methods. A cohort of 120 patients, diagnosed with colorectal carcinoma,
was established. The cohort consisted of 45 patients with KRAS mutations
in exons 2 or 3 and 75 patients without a KRAS mutation. This was
assessed by Sanger sequencing. The 75 patients with a wild-type KRAS
gene were further analysed for BRAF mutations in exon 15 by Sanger
sequencing.
Results. Fifty ng of genomic DNA isolated from FFPE tissue blocks were
found to give reproducible results as input material of the PCR to generate
amplicons used for deep-sequencing. The target amplicons were
KRAS exons 2 and 3 and BRAF exon 15. The exons 5 to 8 of the p53 gene
were also analysed due to high mutation rates in colorectal carcinoma.
The amplicons of each patient were labelled with multiplex identifiers
(MIDs), and these were shown to be highly specific in the data analysis
after deep-sequencing. Seven amplicons of 9 patients were pooled in
one single 454 Junior Sequencing run. On average, each amplicon was
covered 1000-fold which allowed us to identify mutations at a 4% frequency.
Integrated computational down-stream analyses enabled us to
speed up the detection and classification of mutations. Results from the
first 17 patients yielded 14 mutations located in the p53 gene (82%) and 1
in the BRAF gene (6%). The BRAF mutation was identified as the well
known activating mutation V600E. We also included a patient with a
known KRAS mutation as control which was successfully verified by
deep-sequencing.
Conclusions. This newly established method allowed us to analyse 7 amplicons
of 9 patients (63 amplicons in total) in one deep-sequencing run
within 1 week. The capacity limit of a Junior 454 is 4 runs per week which
would allow us to analyse the mutation status for the KRAS, BRAF, and
p53 genes of 36 patients with colorectal carcinoma
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FR-026
Colocalization algorithms for conversion of traditional immunohistochemistry
into virtual multicolor stains
A .-S .K . Meyer1 , P . Möller1 , J .K . Lennerz1 1University Ulm, Institute of Pathology, Ulm
Aims. Diagnostic immunophenotyping is performed via “mentally”
combining single immunohistochemistry (IHC) stains. The discrepancy
to research settings, were co-visualization predominates, is due to technical-
(i.e. species identity, detection systems) and/or practical hurdles