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Annals of Oncology
expression, but not in Gli3-undetectable HCT116 or DLD-1 cells. Silencing of
endogenous Gli3 down-regulated colony formation and proliferation in HT29 and
SW480 cells. However, truncated Gli3 (Gli3-R; repressor isoform) transduction had
no effect on these phenotypes although Gli1 expression was inhibited. After
implantation of Gli3- or mock-transfected DLD-1 cells into immunedeficient SCID
mice, tumor formation was observed in only Gli3-transfectant DLD-1 group but not
in mock control. In surgically resected colorectal cancer specimen, Gli3 expression
was heterogeneously detected and Shh expression was highly observed.
Conclusions: Gli3-FL and Shh signals induce tumorigenicity in Gli1 independent
manner, and Gli3-FL may be molecular targets for refractory colorectal cancer.
Disclosure: All authors have declared no conflicts of interest.
1695P FIBROBLAST INDUCED EPITHELIAL TO MESENCHYMAL
TRANSITION (EMT) IN A NOVEL NON-SMALL CELL LUNG
CANCER (NSCLC) MODEL
A. Amann 1 , M. Zwierzina 2 , M. Bitsche 2 , G. Gamerith 3 , J. Huber 1 , S. Koeck 1 ,
J. Kelm 4 , W. Hilbe 3 and H. Zwierzina 1
1 Internal Medicine 1, Medical University Innsbruck, Innsbruck, AUSTRIA,
2 Anatomy, Histology and Embryology, Medical University Innsbruck, Innsbruck,
AUSTRIA, 3 Internal Medicine 5, Medical University Innsbruck, Innsbruck,
AUSTRIA, 4 Insphero AG, Zürich, SWITZERLAND
Introduction: Different molecular processes lead to metastatic spread and the
occurrence of tumour cell resistance to therapeutic interventions. Among them, the
epithelial to mesenchymal transition (EMT) process plays a key role. During EMT,
epithelial tumour cells lose the expression of specific proteins and adopt the
phenotype of mesenchymal cells. These structural conversions are substantially
dependent on the tumour microenvironment. EMT of tumour cells can induce drug
resistance and metastasis. Thus, EMT inhibition may offer a new strategy for
overcoming tumour progression.
Methods: To evaluate EMT in a non-small cell lung cancer (NSCLC) model a 2D
and a 3D- cell culture system was applied using both the human lung cancer cell line
(A549) and the human lung fibroblast cell line (SV-80). For generating 3D cell
spheroids, a novel system was established consisting of 96-well hanging drop
microtiter plates (InSphero AG, Zürich, Switzerland). 2D co-culture assays were
performed in transwell filter inserts (Costar). EMT was induced with transforming
growth factor-β (TGF-β) or co-cultivation with fibroblasts in 2D/3D. The switch
from epithelial to mesenchymal cells was monitored by Western Blot (WB) analyses
of e-cadherin, vimentin and n-cadherin. Furthermore, immunohistochemical
analyses of e-cadherin, vimentin, α-smooth muscle actin, fibronectin, KI-67 and
CA-IX were done on paraffin embedded spheroids.
Results: EMT could be induced in the 2D not only by incubating tumour cells with
TGF-β but also by co-culturing them with fibroblasts in transwell filter inserts. In
A549 cells a change in morphology as well as in protein expression defined by WB
analysis (down regulation: E-cadherin; up regulation; Vimentin, n-cadherin) could be
detected. When cultivated in the 3D system, A549 cells showed an up regulation of
the mesenchymal protein vimentin without TGF-ß stimulation. Furthermore, a
significant up regulation of vimentin, KI-67, CA-IX, and a slight down regulation of
e-cadherin could be measured compared to monocultures.
Conclusion: 3D culture represents a model to study EMT in tumour cell lines
without addition of growth factors and thus reflects in vivo conditions closer than
2D culture.
Disclosure: All authors have declared no conflicts of interest.
1696P CANCER STEM CELL MARKERS IN CLINICAL PANCREATIC
CANCER: IMPACT OF CD44 + /CD24 + /EPCAM+
EXPRESSION ON HISTOLOGY AND PROGNOSIS
Y. Ohara, T. Oda, Y. Akashi, R. Miyamoto, K. Yamada, S. Hashimoto and
N. Ohkohchi
Surgery, University of Tsukuba, Tsukuba, JAPAN
Purpose: Emerging evidence suggests that the capability of a tumor to grow and
propagate is dependent on a small subset of cells within it, termed cancer stem cells
(CSCs). In pancreatic cancer, CD44 + /CD24 + /EpCAM+ cells have been reported to
be CSCs; however, the histological and clinical importance of these cells has not yet
been investigated. Here we clarified the characteristics of CD44 + /CD24 + /EpCAM+
cells in clinical specimens of pancreatic cancer using immunohistochemical assay.
Materials and methods: We used surgical specimens of pancreatic ductal
adenocarcinoma from 30 patients. In view of tumor heterogeneity, we randomly
selected 10 high-power fields per case, and triple-positive CD44 + /CD24 + /EpCAM+
expression was identified using our scoring system. The distribution, histological
characteristics, and prognostic importance of CD44 + /CD24 + /EpCAM+ cells were
then analyzed.
Results: Among a total of 300 assessed fields, 41 (14%) were evaluated as
triple-positive. The distribution of CD44 + /CD24 + /EpCAM+ cells varied widely
among the 30 cases examined, and CD44 + /CD24 + /EpCAM+ expression was
correlated with poor glandular differentiation and high proliferation (high Ki-67
labeling). Analysis of the three markers individually showed that CD44 and CD24
were also correlated with poor differentiation and high proliferation, while EpCAM
was not. Survival analysis showed that CD44 + /CD24 + /EpCAM+ expression was
not correlated with patient outcome; however, CD44 and CD24 each appeared to be
correlated with poor prognosis.
Conclusion: In pancreatic cancer, CD44 + /CD24 + /EpCAM+ cells overlapped with
poorly differentiated cells and possess high proliferative potential. In particular,
double-positive CD44 + /CD24+ cells seem to have relevance when considering
clinical aspects.
Disclosure: All authors have declared no conflicts of interest.
1697P SEMULOPARIN EFFICIENTLY INHIBITS THROMBIN
GENERATION TRIGGERED BY PANCREAS
ADENOCARCINOMA CELLS BXPC3. DISTINCT ROLES OF
ANTI-XA AND ANTI-IIA ACTIVITY
G. Gerotziafas 1 , M.P. Roman 1 , E. Mdemba 1 , M. Hatmi 1 , J. Fareed 2 ,J.
F. Bernaudin 1 and I. Elalamy 1
1 ER2UPMC, Faculty of Medicin, Université Pierre et Marie Curie (Paris VI), Paris,
FRANCE, 2 Pathology, Loyola University Medical Center, Maywood, IL, UNITED
STATES OF AMERICA
Background: Venous thromboembolism (VTE) is a common complication in
patients with cancer receiving chemotherapy. Currently no anticoagulant is approved
for VTE prophylaxis in this setting. Semuloparin is an ultra-LMWH generated
through a highly selective depolymerization of heparin which protects the
antithrombin (AT) binding site in order to improve the benefit/risk ratio compared
to existing anitcoagulants.
Aims: We studied in vitro the mechanism of action of semuloparin on the inhibition
of thrombin generation (TG) of human platelet poor plasma (PPP) triggered by
human pancreatic cancer cells BXPC3. We compared the antithrombotic efficiency of
semuloparin to that of enoxaparin and the specific AT-dependent factor Xa inhibitor
fondaparinux.
Materials and methods: BXPC3 cells were suspended in PPP spiked with clinically
relevant concentrations of semuloparin, enoxaparin and fondaparinux. The
endogenous thrombin potential (ETP) and the mean rate index (MRI) of the
propagation phase of TG were monitored with the CAT assay (Stago France) as
described previously (Gerotziafas et al Thromb Res 2011). Anti-Xa and anti-IIa
specific activities were assessed assays obtained from Diagnostica Stago.
Results: Both semuloparin and enoxaparin, at the concentration of 0.4 anti-Xa IU/
ml, completely abrogated TG. Total inhibition of TG occured in the presence of
0.002 anti-IIa IU/ml of semuloparin and 0.05 anti-IIa IU/ml of enoxaparin.
Fondaparinux, even at concentrations higher than 2 µg/ml, reduced the MRI but did
not significantly affect the ETP and did not completely inhibited TG,. The IC50 for
the ETP of the anti-IIa activity of enoxaparin was 37-fold higher as compared to that
of semuloparin.
Conclusion: In a cancer cell model of hypercoagulability, semuloparin reduced
efficiently thrombin generation. The unique anticoagulant profile of semuloparin
based on its high AT-affinity differentiates it from enoxaparin and fondaparinux. The
residual anti-IIa activity of semuloparin amplifies its antithrombotic efficacy. This
profile is expected to translate into an improved benefit/risk ratio.
Disclosure: All authors have declared no conflicts of interest.
1698P DO THE WELL-KNOWN RISK FACTORS OF BREAST CANCER
HAVE THE SAME IMPACT ON DEVELOPMENT OF
MOLECULAR SUBTYPES?
F. Paksoy Türköz 1 , M. Solak 1 , O. Keskin 2 ,Z.Arık 1 , F.S. Sarıcı 1 ,I_.H. Petekkaya 1 ,
T. Babacan 2 and K.M. Altundag 2
1 Medical Oncology, Abdurrahman Yurtaslan Ankara Oncology Research and
Education Hospital, Ankara, TURKEY, 2 Medical Oncology, Hacettepe University
Oncology Institute, Ankara, TURKEY
Background: Altough clinical differences between breast cancer (BC) subtypes have
been well-described, etiologic heterogeneity have not been fully studied. The aim of
this study was to assess the associations between risk factors and molecular subtypes
of BC.
Methods: 1884 invasive BC cases were retrospectively analyzed. The odds ratios (OR)
and 95% confidence intervals (CI) were estimated using multiple logistic regression
analysis.
Results: 1249 patients had luminal A, 234 had luminal B, 169 had HER-2
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