POSTERS a c-met inhibition may decrease tumor recurrence and increase survival in RFA treated HCC patients. P0295 CD4+CD25+CD127LOW REGULATORY T CELLS PLAY PREDOMINANT ANTI-TUMOR SUPPRESSIVE ROLE IN HEPATITIS B VIRUS ASSOCIATED HEPATOCELLULAR CARCINOMA S. Sharma1 , R. Khosla1 , P. David1 , A. Rastogi2 , A.K. Vyas1 , A. Bhardwaj3 , A. Sahney3 , R. Maiwall3 , G. Ramakrishna1 , S.K. Sarin3 , N. Trehanpati1 . 1 Research, 2 Pathology, 3 Hepatology, Institute of liver and biliary sciences, New Delhi, India E-mail: trehanpati@gmail.com Background and Aims: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and hepatitis B is one of the common causes. T regulatory cells (Tregs) are strong immunomodulators and are likely to play major role in HCC development. We hypothesize that HBV infection expands Treg mediated immunosuppression creating ideal microenvironment for oncogenic transformations. Therefore, we investigated T regulatory cells CD4+CD25+CD127low and their regulation by TGF-b, IL-10 and PD1 in patients with HBV related HCC (HBV-HCC) vs. nonHBV-HCC. Methods: HBV-HCC (n = 17), non-HBV-HCC (n = 22; NASH =16, alcohol related=6) and Chronic hepatitis B infection (CHBV; n = 10) patients were recruited for study. Whole blood immunophenotyping was done by multicolor flow cytometry. Functionality of isolated CD4+CD25+CD127low Treg was assessed by in vitro suppression assay. Expression of FoxP3, IL-10, PD-1, TGF-b and Notch in Tregs and liver explants was analyzed by flow cytometry, immuno-histochemistry (IHC) and qRT-PCR. Results: Total lymphocyte count and CD8+Tcells were significantly lower in HBV-HCC compared to Non-HBV-HCC (p = 0.04 and p = 0.04) and CHBV (p = 0.003 and p = 0.05). Foxp3 expression in CD4+CD25+hi CD127low was significantly increased in HBV-HCC compared to Non-HBV-HCC and CHBV patients (P = 0.002, P = 0.006). In in vitro suppression assay Tregs isolated from HBV-HCC showed increased suppressive ability and secretion of immunosuppressive cytokines IL-10 and TGF-b compared to Non-HBV-HCC (P = 0.01 and P = 0.04) and CHBV (P = 0.02 and P=NS). PD1 expression in CD4+CD25+hi was significantly decreased in the HBV-HCC than nonHBV-HCC and CHBV (P = 0.04 and P=NS). In HBV-HCC, a fetoprotein (AFP) levels were significantly high (median 941, range 2–575736.6) than Non-HBV-HCC (median 13.5, range 2–18,900). In HBVHCC Foxp3 expression in CD4+ CD127low CD25+hi was significantly correlated with both, high (>1000 ng/ml, r = 0.914, P = 0.000) and low (<1000 ng/ml, r = 0.857, P = 0.014) AFP levels. Reduced PD1 expression in HBV-HCC also had negative correlation with FOXP3 in CD4+CD25+hi CD127low (r = −0.78, p = 0.04). Conclusions: Our results demonstrates that CD4+ CD25+hi Tregs from HBV-HCC patients have decreased expression of PD-1, resulting in higher IL-10 and TGF-b secretion. Increased suppressive ability of Tregs in HBV related HCC confers increased anti-tumor suppressive response than in non HBV-HCC. Modulation of T-regs and PD1 may serve as useful therapeutic targets. P0296 eNOS POLYMORPHISMS IN RELATION TO OUTCOME IN ADVANCED HCC PATIENTS RECEIVING SORAFENIB A. Caadei Gardini1 , M. Scartozzi2 , L. Faloppi3 , G. Marisi1 , P. Ulivi1 , E. Scarpi1 , G. Luca Frassineti1 . 1 IRST-IRCCS, Meldola, 2 Università Cagliari, Cagliari, 3 Università ancona, Ancona, Italy E-mail: casadeigardini@gmail.com Background and Aims: Cancer cells adapt to hypoxic microenvironment through the activation of many molecules, including endothelial nitric oxide synthase (eNOS). Sorafenib, by blocking the vascular endothelial growth factor receptors (VEGFRs), induces an inhibition of eNOS activitywith a consequent decrease of the production of nitric oxide (NO). NO is associated with an increase of tumor angiogenesis, tumor invasion and metastasis formation. In our study we analysed the role ofeNOS polymorphisms in relation to clinical outcome in patients with hepatocellular carcinoma treated with sorafenib. Methods: From a database of 257 patients diagnosed with hepatocellular carcinoma from 2004 to 2014, we selected 54 patients who received sorafenib. Peripheral blood samples or FFPE tumor tissues were available for DNA extraction and genotyping analysis. Three eNOS polymorphisms (eNOS +894 G/T, eNOS VNTR 27bp 4a/b, eNOS −786 C/T) were analyzed by direct sequencing or Real Time PCR method. We analyzed 21 patients for the VNTR 4a/b polymorphism and 32 patients for −786 C/T polymorphism. All the candidate genotypes were evaluated to identify a potential correlation with overall survival (OS) (log-rank test). Results: With regard to eNOS VNTR it was observed that patients carrying the b allele (5 repetitions of 27bp) both in homozygosity (4bb) and in heterozygosity (4ab) were associated with a better OS. The variants 4aa (4 repeats of 27bp in homozygosity), 4ab and 4bb, were associated with a median OS of 5.7, 13.9 and 23.6 months, respectively (p = 0.016). For eNOS −786 the presence of the T allele both in homozygosity (TT) and in heterozygosity (TC) was associated with a statistically significant longer OS with respect to patients with CC genotype (15.6 versus 13.9 months, respectively, p = 0.031). No correlations were observed in relation to PFS (p = 0.494). Conclusions: eNOS VNTR and eNOS −786 could represent prognostic markers in patients with advanced hepatocellular carcinoma treated with sorafenib. P0297 THE TRANSFORMING GROWTH FACTOR-BETA (TGF-b) GOVERNS HUMAN LIVER TUMOR CELL PLASTICITY A. Malfettone1 , J. Fernando1 , P. Koudelkova2 , J. Soukupova1 , E. Bertran1 , À. Fabra1 , M. Grubinger2 , B. Rani3 , G. Giannelli3 , W. Mikulits2 , I. Fabregat1,4 . 1 Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet, Barcelona, Spain; 2 Institute of Cancer Research, Medical University of Vienna, Vienna, Austria; 3 Department of Biomedical Sciences and Human Oncology, University of Bari, Medical School, Bari, Italy; 4 School of Medicine, University of Barcelona, Barcelona, Spain E-mail: amalfettone@idibell.cat Background and Aims: Transforming Growth Factor-beta (TGF-b) acts as a tumor suppressor in initial stages of hepatocarcinogenesis. However, tumour cells acquire mechanisms to overcome TGF-b suppressor effects and respond to it undergoing epithelial– Journal of Hepatology 2015 vol. 62 | S263–S864 S419 POSTERS mesenchymal transition (EMT). In some tumour cells a link exists between EMT and the expression of stem cell markers. Aim of this study was to analyze if autocrine activation of the TGF-b pathway in hepatocellular carcinoma (HCC) cells may control the expression of Cancer Stem Cell (CSC) markers concomitant with the acquisition of mesenchymal properties. Methods: Different HCC cell lines with different autocrine expression of TGF-b and epithelial–mesenchymal phenotypes were analysed. TGF-b Receptor I (TbRI) expression was stably silenced by shRNA. EMT and SC marker expression was analyzed by Flow Cytometry, Immunofluorescence and Real-Time PCR. Sphereforming and colony-formation assays were performed to explore the biological properties of liver CSCs. Results: Epithelial liver tumour cells expressed EpCAM and CD133, whereas mesenchymal-like cells expressed CD44. Cancellation of the TbRI in Hep3B (with a mixed epithelial–mesenchymal phenotype) prevented the TGF-b-induced EMT, but also induced a mesenchymal–epithelial transition (MET). Relevantly, TbRI knockdown decreased the basal expression of EpCAM and CD133 and reduced the formation of liver spheroids and number of clones. The TbRI knock-down in HLE and HLF (mesenchymal-like cells) decreased significantly the expression of the Snail family genes, but unexpectedly did not produce a full MET. Nevertheless, TbRI knock-down decreased expression of CD44, which correlated with a lower ability to form liver spheroids and clones. Interestingly, chronic treatment of Hep3B cells with TGF-b induced progressive down-regulation of EpCAM and CD133 and up-regulation of CD44, concomitant with the appearance of a mesenchymal phenotype. Furthermore, these cells formed liver spheres and colonies more efficiently. Conclusions: TGF-b not only modulates the EMT phenotype of HCC cells, but also the expression of CSC genes, although it is not clear yet the cross-talk among both processes. A mesenchymal phenotype and CD44 expression are associated with poor prognosis in HCC. Results of this study further support that activation of the TGF-b pathway may be considered a therapeutic target in HCC. Acknowledgements: People Programme (Marie Curie Actions) of the FP7–2013, under REA grant agreement #PITN-GA-2012-316549 (IT-LIVER). P0298 GLUCOSE TRANSPORTER ISOFORM 1 (GLUT1) EXPRESSION DETERMINES HEPATIC METASTASIS OF MELANOMA CELLS A. Koch1 , S.A. Lang1 , P. Wild2 , A. Bosserhoff3 , C. Hellerbrand1 . 1 University Hospital Regensburg, Regensburg, Germany; 2 University Hospital Zurich, Zurich, Switzerland; 3 University of Erlangen, Erlangen, Germany E-mail: claus.hellerbrand@ukr.de Background and Aims: The facilitative glucose transporter isoform 1 (GLUT1) is the key rate-limiting factor in glucose transport into cancer cells, and we have previously shown that GLUT1 is a tumorpromotor in hepatocellular carcinoma, while its expression is at the detection limit in normal hepatocytes. The aim of this study was to analyze whether GLUT1 expression and a high capacity for glucose uptake, respectively, are general pro-cancerogenic factors in the liver. Methods: We used malignant melanoma as a model-tumor, which is known to preferentially metastasize to the liver. Results: Similar as observed in HCC, GLUT1 expression was enhanced in melanoma cell lines compared to primary melanocytes, as well as in melanoma compared to naevi. Immunohistochemical analysis of a tissue micro array consisting of 140 human melanoma tissues showed that GLUT1 expression was significantly enhanced in metastasis compared to primary tumors. GLUT1 expression in primary tumors correlated with tumor staging, and most importantly, with progression- and overallS420 survival. To determine the role of GLUT1 in melanoma metastasis, GLUT1 expression was suppressed in the murine melanoma cell line B16 (i) by stable transfection with shRNA and (ii) by using the selective GLUT1-Inhibitor WZB117. GLUT1 suppression caused decreased anaerobic glycolysis and lactate secretion, and inhibited proliferation and migration of B16 cells. Moreover, GLUT1 suppression lowered apoptosis resistance of melanoma cells. Next, B16 cell clones with and without GLUT1 suppression were subjected to an established model of hepatic metastasis, in which tumor cells were injected into the spleen of syngeneic mice, from where they metastasize into the liver via the portal circulation. GLUT1 suppressed cells formed significantly less metastases and hepatic metastases derived from GLUT1 suppressed B16 cells revealed less immune-cell infiltration and more apoptosis as assessed by CD3immunohistochemistry and TUNEL staining. Conclusions: Our data promote the hypothesis that high glucose levels in the portal circulation and the liver, and the capacity to utilize those, respectively, promote hepatic metastasis. Our data indicate enhanced apoptosis resistance of tumor cells and known immunomodulatory effects of lactate as potential underlying mechanisms of this phenomenon. GLUT1, which is almost selectively expressed in malignant cells but not in healthy liver or other non-malignant tissues, appears as an attractive therapeutic target for hepatic metastasis. P0299 SORAFENIB EFFECT ON MITOCHONDRIAL FUNCTION PROVIDES A TARGET FOR INCREASING HCC THERAPY EFFICACY A. Tutusaus1 , M. Stefanovic1 , J.C. Fernandez-Checa1 , M. Marı́1 , A. Morales1 . 1 IIBB-CSIC/Hospital Clinic-IDIBAPS-CIBEREHD, Barcelona, Spain E-mail: amorales@clinic.ub.es Background and Aims: Multikinase inhibitor sorafenib has limited efficacy in the treatment of advanced hepatocellular carcinoma (HCC). Novel therapies, in combination with sorafenib or in monotherapy, are demanded to increase drug efficacy in HCC treatment. The lack of positive results from other drugs, underscores the importance of identifying weaknesses in HCC biology that current approaches have not recognized. A mitochondrial effect of sorafenib has been previously reported, although its participation in sorafenib toxicity and HCC therapy has drawn little attention. Methods: Hepatoma cell lines (HepG2 and Hep3B) were treated with sorafenib and Bcl2-inhibitors. Western blots in total, cytosolic and mitochondrial extracts and qPCRs were performed after sorafenib exposure. ROS production, mitochondrial membrane permeabilization, caspase activity and ATP measurements were analyzed in sorafenib-treated hepatoma cells. Tumor growth was determined after subcutaneous injection of HepG2 cells on the flanks of nude mice. Results: Sorafenib induces a rapid decline in mitochondrial membrane potential, with production of reactive oxygen species (ROS) and reduction in the levels of the antiapoptotic Bcl-2-family protein MCL-1. However, cytochrome c release from mitochondria and/or ATP depletion after sorafenib exposure were not observed during hours, suggesting modest mitochondrial contribution in cell death. Bcl-2 levels, that play an important role in mitochondrial dependent cell death, were not affected by sorafenib, so we decided to evaluate the effect of several Bcl-2 inhibitors (HA-14, ABT-263, ABT-767 and AT-101) on sorafenib toxicity in hepatoma cells. Among them, ABT-263 (Navitoclax) a potent inhibitor of Bcl-xL and Bcl-2 now in Phase II clinical trials for leukemia and solid tumors, was the drug that exhibited higher capacity to potentiate sorafenib effects on Hep3B and HepG2 cells. ABT-263 co-administration with sorafenib induced quick release of cytochrome c and enhanced caspase-3 activity. Moreover, in vivo administration of ABT-263 combined with sorafenib greatly potentiated sorafenib effects, Journal of Hepatology 2015 vol. 62 | S263–S864