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

SA-P-064
Evolution of molecular pathology at the Institute of Basel
M .P . Bihl 1 , S . Hoeller 1 , A . Foerster 1 , R . Chaffard 1 , S . Schneider 1 , A . Rufle 1 ,
L . Terracciano 1 , L . Tornillo 1
1 University of Basel, Institute of Pathology, Basel, Switzerland
Aims. Molecular genetics in pathology is a very young field. It began with
analysis of haematological diseases or inherited disorders, but in the last
decade, also many of solid tumors have been found to be related to specific
somatic mutations. These mutations can give a hint to drug sensitivity
in a given tumor and therefore are urgently required in modern
oncology. Here we show how this field has developed in the recent years
using the example of the activity of the Institute of Pathology during the
last 6 years.
Methods. The number of PCR based analysis increased from 206 (in
2006) to over 800 analyses (in 2011).
Results. In the beginning only CKIT/PDGFRA mutation and EGFR
mutation analysis and clonality analysis were performed. However, the
overall percentage of analysed sites remained stable with 50% from the
lung, 10–20% from the blood or lymph node and 10–25% from colorectal
or gastrointestinal sites. Recently, new mutations are also routinely
tested like (IDH 1 and 2, BRAF and CTNNB1) and therefore new organs
were included like brain (glioma), skin (melanoma) and liver (adenomas).
From three available assays in 2006 the spectrum of our analysis
expanded to 20 different assays today. The number of performed FISH
analysis stayed stable, while the number of HER2 hybridisations dropped,
but new assays were introduced into the routine panel like 1p19q and
ALK translocation analysis for glioma and adenocarcinoma of the lung,
respectively. Therefore, the dynamic changes in molecular pathology are
due to new tumor classification systems, the development of new tumor
specific drugs and the increased knowledge of drug sensitivity in cancer
patients harboring specific somatic mutations.
Conclusions. In the upcoming years molecular based prognostic markers
and mutation specific therapies will even more expand the spectrum of
molecular testing in pathology. Its role is crucial to improve and optimize
the diagnosis and therapy of tumors and is essential in modern oncology.
Adaptation of the increasing knowledge of pathogenetic pathways
will be a major issue for molecular laboratories in the future.
SA-P-065
The amyloid precursor protein (APP) is a novel biomarker for
transformed human pluripotent stem cells
V . Venkataramani1 , K . Thiele2 , C .-L . Behnes2 , G .G . Wulf1 , P . Thelen3 , L . Opitz4 ,
G . Salinas-Riester4 , O . Wirths5 , T .A . Bayer5 , H .-J . Radzun2 , S . Schweyer2 1University Medicine Göttingen, Department of Hematology and Oncology,
Göttingen, 2University Medicine Göttingen, Department of Pathology, Göttingen,
3University Medicine Göttingen, Department of Urology, Göttingen,
4 5 University Medicine Göttingen, DNA Microarray Facility, Göttingen, University
Medicine Göttingen, Division of Molecular Psychiatry, Göttingen
Aims. There is no doubt that the amyloid precursor protein (APP) and its
proteolytically derived Aβ species significantly contribute to the pathogenesis
of Alzheimer disease. However, the normal physiological role of
this ubiquitously expressed protein has remained largely unknown. In
the current study, we characterized APP expression in a panel of human
testicular germ cell tumors (TGCT) of different histological origin. Furthermore,
we analysed whether histone deacetylase (HDAC) inhibitors
effectively induce cell differentiation and impact stem cell signature and
APP protein levels in embryonal carcinoma (EC) cell lines. These analyses
were also performed in a physiologically relevant in vivo setting
using an established xenograft mouse model.
Methods. Paraffin-embedded tissue blocks from orchiectomy specimens
were used for tissue microarray construction consisting of 173 cases of
pure and mixed TGCTs as well as eight randomly selected normal testicular
tissues. Following TGCT cell lines were used: NCCIT, NTera-2 (EC
cell lines) and TCam-2 (seminoma cell line). Cellular differentiation was
analysed by cell proliferation and cytotoxicity assays, cell morphology
via fluorescence microscopy and expression analyses of stem cell genes
and lineage-specific differentiation markers were determined using microarray
analysis, qRT-PCR and Western blot analysis. APP expression
was selectively down-regulated using target-specific siRNA duplexes.
Xenografts inoculated with NTera-2 were orally treated with the HDAC
inhibitor VPA and tumor growth as well as APP protein levels were compared
to vehicle treated animals.
Results. APP is exclusively expressed in pluripotent germ cell cancer
subtypes (EC and seminoma). Differentiated TGCTs (e.g. teratoma) only
presented low or lack of APP expression. APP knock-down induced the
expression of lineage-specific differentiation markers. HDAC inhibitor
treatment induced cell differentiation, accompanied by down-regulated
APP protein levels and stem cell genes. Moreover, GRP78 could be
identified as a key factor that specifically triggers proteasomal degradation
of APP. Oral administration of VPA significantly suppressed tumor
growth and depleted APP protein levels in vivo.
Conclusions. Our results indicate that APP behaves as a reliable biomarker
for transformed human pluripotent stem cells and also shed light
on the significance of APP as a novel molecular target and furthermore
broaden the therapeutic potential of HDAC inhibitors in the clinical treatment
of TGCT.
SA-P-066
Thymoquinone lowers toxicity and increases efficacy of
5-fluorouracil
C . El-Baba1 , S . Morgenthal2 , M . Ocker3 , H . Gali-Muhtasib4 , R . Schneider-Stock 1
1 2 University of Erlangen-Nuremberg, Institute of Pathology, Erlangen, Christian-Albrechts-University,
Institute of Pathology, Köln, 3Phillips-University of Marburg, Institute of Surgical Research, Marburg, 4American University of
Beirut, Department of Biology, Beirut, Lebanon
Aims. Colorectal cancer is a non-negligible cause of mortality worldwide.
5-fluorouracyl (5-FU) has proved to be one of the most effective chemotherapeutics
for colorectal cancer but an inactivated p53 status leads to a
resistance to 5-FU. We have shown that thymoquinone (TQ), a bioactive
compound extracted from Nigella sativa (black seed) exerts promising
anti-apoptotic effects independent of the p53 status of tumor cells. Our
aim is to investigate if TQ is able to sensitize colorectal cancer cells to
5-FU to minimize its cytotoxic side-effects in the clinical setting.
Methods. Human colon cancer cells HT29 (mutant p53), HCT116 (p53+/+)
and normal intestinal cells HCEC were treated with TQ and/or 5-FU.
Cell viability was measured via crystal violet, mitochondrial activity and
colony formation assays. Cell cycle distribution and apoptosis were assessed
by flow cytometry via propidium iodide (PI), Annexin-V staining,
and Western blotting. A mouse xenograft study was conducted for a better
assessment of potential in vivo effects.
Results. HCT116wt cells were more sensitive to TQ than HT29 cells.
HCEC cells were highly resistant to TQ treatment, which indicates that
TQ has an effect mainly on cancerous cells. In HT29 cells, the combination
TQ/5-FU was equally efficient as the treatment with ten times
higher concentration of 5-FU alone. Annexin-V, propidium iodide-cell
cycle analysis, as well as Caspase 3 and Caspase 9 cleavage along with the
increase of Bax to Bcl2 ratio, revealed a higher apoptosis induction when
the cells are treated with both drugs in comparison to either TQ or 5-FU.
In vivo study showed that the relative tumor size of mice co-treated with
TQ and 5-FU was significantly reduced in comparison with the tumors
of control mice or mice treated with either drug alone.
Conclusions. TQ when combined with the antineoplastic agent 5-FU
was increasing apoptosis in colon cancer cells. The combination therapy
could overcome the resistance to 5-FU in the p53 mutant tumor cells without
showing dramatic cytotoxicity on normal colon cells. Combined
with further clinical studies this approach might be promising for the
improvement of colorectal cancer treatment.
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