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