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

well as MET activation were examined by Western blot analysis, flow
cytometry and immunofluorescence staining. Cells were treated with
varying concentrations of cetuximab and cisplatin and 5-fluorouracil in
tumor relevant concentrations. The biological end point was cell viability,
which was measured by XTT cell proliferation assay. Response to
treatment was evaluated using statistical methods.
Results. We assessed the activity of cetuximab in five gastric cancer cell
lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two
cell lines, MKN1 and MKN28, was significantly reduced by cetuximab
treatment. High EGFR expression and low levels of receptor activation
were associated with cetuximab responsiveness. MET activation as well
as mutations of KRAS and E-cadherin were associated with cetuximab
resistance.
Conclusions. These data indicate that our examinations may be clinically
relevant and the candidate markers should therefore be tested in clinical
studies.
DO-018
Notch2 expression and chemoresistance in neoadjuvant treated
gastric cancer
L . Bauer1 , R . Langer1 , M . Mandl1 , K . Becker1 , J . Slotta-Huspenina1 , A . Novotny2 ,
A . Hapfelmeier3 , H . Höfler1 , G . Keller1 1Technische Universität München, Department of Pathology, München,
2Technische Universität München, Department of Surgery, München,
3Technische Universität München, Department of Medical Statistics and
Epidemiology, München
Aims. In a recent study analyzing the prognostic significance of the expression
of cancer stem cell (CSC) related genes in residual gastric tumor
cells after neoadjuvant chemotherapy, Wnt and Notch signaling genes,
among others, showed a prominent association with survival. The aim of
this study was to assess selected genes for differential expression between
pretherapeutic biopsies and resected specimens. In vitro we investigated
the impact of Notch activity on chemosensitivity in gastric cancer cell
lines.
Methods. Expression of 12 genes was compared between corresponding
biopsies and resected specimens from patients treated with neoadjuvant
chemotherapy (CTx) demonstrating partial (n=22) or minimal/
no tumor regression (n=22). mRNA was isolated from macrodissected
FFPE tissues and gene expression was quantified by real time PCR using
TaqMan® low density arrays. Immunohistochemical staining (IHC) for
Notch2 was performed on biopsy/resected-specimen pairs from patients
with sub-total, partial and minimal/no tumor regression (n=22, each)
and from patients not treated by CTx. (n=16) and evaluated by a semiquantitative
scoring system. Chemosensitivity of three gastric cancer
cell lines to the gamma-secretase inhibitor DAPT alone or in combination
with cisplatin was determind by XTT or colony formation assays.
Results. Differential expression analysis revealed an increase of Notch2
and POU5F1 from biopsies to resected tumors in tumors with partial
response (p=0.002 and 0.028) and minimal/non responding tumors
(p=0.062 and 0.002). In contrast a decrease in expression was observed
in both tumor groups for Notch1 (p=0.072 and 0.001). Immunhistochemical
analysis of Notch2 revealed that cytoplasmic staining intensities of
tumor cells decreased significantly in all groups of CTx-treated patients
(p=0.016, .001 and 0.017) but not in non-CTx patients. IHC analysis of
Notch1 is in progress. Treatment of gastric cancer cell lines with 10 µM
DAPT and 2 µM cisplatin led to a synergistic reduction of metabolic activity
in comparison to the single drugs.
Conclusions. The comparison of mRNA expression between corresponding
biopsies and resected specimens revealed alterations consistent with
an enrichment of chemotherapy resistant residual tumor cells. Results of
Notch2 IHC are in line with the mRNA data. The synergistic effect of
cisplatin and the gamma-secretase inhibitor DAPT in vitro suggests that
Notch signaling might be involved in chemoresistance of gastric cancers.
AG Gastroenteropathologie VI – Unterer GI-Trakt
DO-020
Aurora-A protein expression is associated with multipolar mitoses
independent of molecular class of colorectal carcinomas*
D . Batarello 1 , A . Schoepflin1 , D . Hauschke2 , M . Werner3 , S . Lassmann1 1Institute of Pathology, University Medical Center Freiburg, Freiburg,
2Institute of Medical Biometry and Medical Informatics, University Medical
Center Freiburg, Freiburg, 3Institute of Pathology, University Medical Center
Freiburg
Aims. Aurora-A overexpression may induce supernumerary centrosomes,
respective multipolar mitoses, and aneuploidy in model systems.
Here, we examined the occurrence of Aurora-A positive multipolar mitoses
in aneuploid (microsatellite-stable, CIN-type) versus near-diploid
(microsatellite-instable, MIN-type) colorectal carcinomas (CRC).
Methods. Three-dimensional immunofluorescence (3D-IF) of Aurora-A
was performed on 8µm thick FFPE tissue sections of 18 previously characterized
colorectal carcinomas. Stained sections were screened by a
x63/1.3 oil objective at 0.7µm image stacks (one x63 field = high power
field/HPF; total of 374 HPFs, range 10–46 HPF per case). Total numbers
of mitoses (n=476, range 11–57 per case) and numbers of bipolar (2 Aurora-A
positive centrosomes/spindle poles) and aberrant multipolar (>2
Aurora-A positive centrosomes/spindle poles) mitoses were counted.
For differences of mitotic counts and frequencies between CIN-type and
MIN-type CRCs, the Wilcoxon Test (exact, two-sided; with p