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

pressed moderate levels of the protein. Then, Kindlin-2 gene was overexpressed
by transfected into MCF-7 cells. In comparison, short hairpin
RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in
HS578T cells. Vector controls were also done in the same cell lines. Ki67
Li, FCM cell cycle, anchorage-independent colony formation (in vitro
tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed.
Apoptotic cells were labeled by fluorescent annexin V assay and
quantified by FACS. Array CGH analysis and spectral karyotyping were
performed to detect the genomic instability of these cells.
Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much
quicker than that of the controls. The proportion of G2-M phase cells,
clone formation and tumorigenicity were significantly higher than these
of the controls. The change of Kindlin-2-ShRNA transfected cells was
just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce
the rate of apoptosis induced by the chemotherapy drugs, and these cells
showed much more genomic instability compared with the controls.
Conclusions. These findings suggested that up-regulation of Kindlin-2
promotes the progression of human breast cancer cells by increasing
their proliferation, drug resistance, genomic instability, and tumorigenesis.
SG-P-129
DKK3 and ITIH5 gene methylation as novel biomarkers for bloodbased
breast cancer screening: Improving early detection of
breast cancer
V . Kloten1 , B . Becker1 , M .G . Schrauder2 , P .A . Fasching2 , A . Hartmann3 ,
J . Veeck1 , R . Knüchel1 , E . Dahl1 1University Hospital of the RWTH Aachen, Institute of Pathology, Aachen,
2 3 University Hospital Erlangen, University Breast Center, Erlangen, University
Hospital Erlangen, Erlangen
Aims. For early detection of breast cancer the development of robust
blood-based biomarkers that accurately reflect the host tumor is mandatory
and thus a growing field of research. The most common alterations
in human cancers including breast cancer are changes in the status of
DNA methylation, which are therefore quickly emerging as a new pool
of potential biomarkers. Thus, we investigated the feasibility of detecting
aberrant tumor suppressor gene methylation in cancer cell-derived free
circulating DNA in the bloodstream of patients.
Methods. Using qualitative MSP, we examined the methylation status of
six biologically significant putative tumor suppressor genes, i.e. ITIH5,
DKK3, WIF1, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum.
Clinical performance was determined in a large training study on 150
serum samples (120 breast cancers, 30 healthy controls). 20 benign disease
and 30 colon cancer serum samples were included for additional
specificity testing.
Results. Based on the training study we could evaluate the top candidate
biomarkers with the best values for sensitivity and specificity. A marker
panel of DKK3 and ITIH5 detected breast cancer with a sensitivity of 46%
(55/120). Specificity of the panel was sufficient with 83%, 100% and 93% in
colon cancer samples, benign and healthy control samples, respectively.
Control samples revealed unacceptable high methylation rates of SFRP1
and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2
and WIF1 showed a considerable methylation frequency in sera from
healthy controls.
Conclusions. The current study suggests that cancer-specific methylation
of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be
valuable biomarkers for the early detection of breast cancer. In the second
phase of this project we are currently validating ITIH5 and DKK3
as reliable methylation biodiagnostic markers in an independent test set
consisting of 160 breast cancer serum samples and 160 control samples.
SG-P-130
Forced expression of ITIH5 in a luminal-type breast cancer model
confers growth suppressive properties
S . Huth1 , R . Knüchel1 , E . Dahl1 1University Hospital of the RWTH Aachen, Institute of Pathology, Aachen
Aims. Inter-α-trypsin inhibitors are protease inhibitors which consist of
one light chain (bikunin) and different heavy chains (ITIHs). We recently
characterized ITIH5 as a novel member of the ITIH gene family and
showed that its loss in human breast cancer is associated with parameters
of tumour invasion and distant metastasis. Thus, we aimed to analyze
the biological function of ITIH5 in an in vitro model of breast cancer,
the luminal-type breast cancer cell line T47-D.
Methods. T47-D clones expressing ITIH5 and corresponding mock
(empty vector) clones were generated using standard methods and subsequently
validated on DNA, mRNA and protein level. Functional analyses
of ITIH5 clones were performed by standardized assays including
XTT, colony formation, cell-matrix-adhesion and Apo-ONE® Homogeneous
Caspase-3/7 Assay.
Results. Forced ITIH5 expression in T47-D transfectants was successfully
achieved validating integration and expression of ITIH5 on the DNA,
mRNA and protein level, respectively. Using functional assays we observed
significantly reduced proliferation (p=0.01) as well as significantly
increased cell-matrix-adhesion (p