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LIVER CANCER PROGRESSION GENE SIGNATURE OF CELL LINES FROM OVAL-CELL DERIVED HEPATIC TUMORS
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Abstracts / Digestive and Liver Disease 41S (2009), S1–S167
jected to surgery treatment in our centre from the 1st of January 2000 to the 31st of July 2007. Primary endpoint was survival. 243 were subjected to Liver Transplantation (OLT) and 137 to liver resection (LR). 82% of the OLT were in stage 1, 66% of the LR in stage 2. Patients in stage 0 were subjected to percutaneous treatments. In stage 1 OLT was performed in patients with an age under 65. For patients in stage 1 with an age over 65, LR was proposed in absence of Ixp, other patients were subjected to percutaneous treatments. Results: The median follow-up was 37 months. The survival rate at 1,3 and 5 years was 91%, 81%, 71% in OLT group and 84%, 63%, 50% in LR group. The 5 years survival rate of patients in stage 1 subjected to OLT was 73% while 51% in the LR group. Patients in stage 2 subjected to LR (78 patients), had 5 years survival of 48%. The mortality rate was 1,5% among the OLT and 1,1% among the LR. Conclusions: We consider OLT the gold standard for patients in the initial stage. We prefer to process patients in premature stage to percutaneous treatments with results comparable to LR but can still be treated with transplant without further morbility. LR is reserved to patients in stage 0 and 1 who are not eligible for percutaneous treatment or OLT and to patients in stage 2 still obtaining satisfactory results with low mortality rate. # H. Biliary 2. Surgery
OC.03.3 LIVER CANCER PROGRESSION GENE SIGNATURE OF CELL LINES FROM OVAL-CELL DERIVED HEPATIC TUMORS A.C. Piscaglia ∗ ,1 , N. Saulnier 1 , V. Tesori 1 , M. Barba 1 , M. Campanale 1 , G. Ianiro 1 , F. Purchiaroni 1 , F. Barbaro 1 , T. Galeotti 1 , B. Petersen 2 , G. Pani 1 , A. Gasbarrini 1 1 Catholic University - Gemelli Hospital, Roma; 2 University of Florida, Gainesville, USA
Background and aim: Cancer cell lines (named LCSCs) were established from a new model of hepatocholangiocarcinoma in rats, derived from liver oval cells. Aim of this study was to compare the gene expression profile of two of these lines: LCSC-5 and LCSC-2. Material and methods: LCSC immunophenotype was assessed on cytospins and chamber-slides. Their clonogenic potential was evaluated by soft-agar colony formation assay, and their tumorigenicity was investigated upon transplantation into nude mice. The cellular response to HGF was measured by western blot analysis for c-Met, p-ERK, pS6, p-Akt, and by functional assays. The growth kinetics was assessed by MTT assay. Gene expression profiling was performed on Affymetrix platform. Finally, the effects of a novel and promising anticancer drug, Sorafenib, were investigated on both cell lines. Results: LCSC-2 had higher proliferative capacity and clonogenicity in vitro, and, upon transplantation into nude mice, induced macroscopic tumors in less than 30 days, while LCSC-5 required approximately 80 days. Transcriptional analysis revealed that genes overrepresented in LCSC-2 were associated with tumor progression/growth (S100A4, ALDH3A1, ARMCX2), drug resistance (ABCC1) and invasion/cytoskeleton-remodelling (TPM2, PDLIM1, CAPG). Conversely, several liver-associated transcripts (IL6ST, ZFP36, IGF2, CYB5, LISCH7) and genes often downregulated in invasive cancers (GRO1, CEACAM1, NFKBIA, MGP) were reduced in LCSC-2. The expression level of the HGF receptor c-Met and its downstream signaling cascade appeared downregulated in LCSC-2 cells, compared to LCSC-5. Sorafenib had cytotoxic effects and, at cytostatic concentrations, potentiated HGF responses in both cell lines, this effect being again less pronounced in LCSC-2, which was also less sensitive to the drug. Conclusions: LCSC-2 displayed a more aggressive, anaplastic, and drug-resistant phenotype when compared to LCSC-5. The HGF/c-Met pathway may play a causative role in this difference, likely by pro-
moting hepatic differentiation, and may also mediate, at least in part, biological response to Sorafenib. The identification of a panel of genes underlying liver cancer progression offers novel insights to study the effects of molecularly targeted agents against hepatic tumors. # K. Pancreatic and liver oncology 1. Basic science
OC.03.4 OXIDATIVE DAMAGE, CELL IMMORTALIZATION AND miRNA EXPRESSION IN HEPATOCELLULAR CARCINOMA (HCC) AND HEPATITIS R. Cardin, M. Bortolami, M. Piciocchi, L. Barzon, A. Sinigaglia, E. Lavezzo, U. Cillo, G. Zanus, F. Farinati ∗ Università di Padova, Padova Background and aim: The molecular mechanisms driving hepatocarcinogenesis are complex and involve viral factors, inflammation and the production of ROS, the host’s repair mechanisms and cell immortalization. Little information is available about miRNA expression in HCC in relation to the above factors. The aim was to assess the extent of DNA oxidative damage (8-hydroxydeoxyguanosine 8OHdG) in different phases of the carcinogenetic process, the impact of DNA repair gene polymorphisms on such accumulation and any effect of the two on telomerase activity (TA). The expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death, was also considered. Material and methods: Hepatic samples obtained either at surgery, [neoplastic (HCC TU) and adjacent non-cancerous tissues (HCC NTU)], and at percutaneous biopsy (HCV/HBV-positive hepatitis patients) were analyzed for 8OHdG (HPCL-ED), OGG1 (a DNA repair gene) polymorphism (PCR-RFLP), TA (RT-PCR) and for microarray analysis. Nineteen HCC patients and 26 with chronic hepatitis gave informed consent and entered the study. Results: 8OHdG levels were significantly higher in HCC TU and NTU than in chronic hepatitis. TA was activated in 18/19 HCC specimens (94%), it was less frequently detected in adjacent non-cancerous tissues (52%) while it was never identifiable in chronic hepatitis. The mean levels of TA in HCC were significantly higher than those detected in non-cancerous tissues and in chronic liver diseases (p=0.01). In HCC we observed significant positive correlations among 8OHdG levels, TA, stage of associated liver disease, OGG1 polymorphisms and ALT/GGT levels. Additionally, in HCC tissues, a significant positive correlation was found between microRNA-92 expression, TA (p=0.0052) and 8OHdG levels (p=0.05). mir-199a, mir-199b, mir-195 and mir-122a were strongly downregulated in the majority (55%-70%) of HCCs, while mir-92 and mir-145 showed a less marked downregulation. In contrast mir-222 was upregulated in the HCC. Conclusions: The above analysis shows an oxidative damage-mediated induction of TA, modulated by host factors and impacting on some miRNA regulation. In particular miR-92 expression has been already linked to hepadnavirus-associated carcinogenesis and c-myc, frequently activated in HCC. This correlation among DNA oxidative damage, TA and miRNA expression is a novel finding. # K. Pancreatic and liver oncology 1. Basic science
OC.03.5 CONTRAST-ENHANCED US (CEUS) IN EARLY EVALUATION OF NON-SURGICAL TREATMENT OF HCC CAN CHANGE THE PATIENT’S FOLLOW-UP AND SURVIVAL F. Giangregorio ∗ , M. Di Stasi, L. Fanigliulo, M.G. Marinone, G. Comparato, G. Aragona, G. Sbolli, P. Tansini, F. Fornari Ospedale “Gugliemo Da Saliceto”, Piacenza Background and aim: Ceus is used in the assessment of loco-regional
Abstracts / Digestive and Liver Disease 41S (2009), S1–S167
jected to surgery treatment in our centre from the 1st of January 2000 to the 31st of July 2007. Primary endpoint was survival. 243 were subjected to Liver Transplantation (OLT) and 137 to liver resection (LR). 82% of the OLT were in stage 1, 66% of the LR in stage 2. Patients in stage 0 were subjected to percutaneous treatments. In stage 1 OLT was performed in patients with an age under 65. For patients in stage 1 with an age over 65, LR was proposed in absence of Ixp, other patients were subjected to percutaneous treatments. Results: The median follow-up was 37 months. The survival rate at 1,3 and 5 years was 91%, 81%, 71% in OLT group and 84%, 63%, 50% in LR group. The 5 years survival rate of patients in stage 1 subjected to OLT was 73% while 51% in the LR group. Patients in stage 2 subjected to LR (78 patients), had 5 years survival of 48%. The mortality rate was 1,5% among the OLT and 1,1% among the LR. Conclusions: We consider OLT the gold standard for patients in the initial stage. We prefer to process patients in premature stage to percutaneous treatments with results comparable to LR but can still be treated with transplant without further morbility. LR is reserved to patients in stage 0 and 1 who are not eligible for percutaneous treatment or OLT and to patients in stage 2 still obtaining satisfactory results with low mortality rate. # H. Biliary 2. Surgery
OC.03.3 LIVER CANCER PROGRESSION GENE SIGNATURE OF CELL LINES FROM OVAL-CELL DERIVED HEPATIC TUMORS A.C. Piscaglia ∗ ,1 , N. Saulnier 1 , V. Tesori 1 , M. Barba 1 , M. Campanale 1 , G. Ianiro 1 , F. Purchiaroni 1 , F. Barbaro 1 , T. Galeotti 1 , B. Petersen 2 , G. Pani 1 , A. Gasbarrini 1 1 Catholic University - Gemelli Hospital, Roma; 2 University of Florida, Gainesville, USA
Background and aim: Cancer cell lines (named LCSCs) were established from a new model of hepatocholangiocarcinoma in rats, derived from liver oval cells. Aim of this study was to compare the gene expression profile of two of these lines: LCSC-5 and LCSC-2. Material and methods: LCSC immunophenotype was assessed on cytospins and chamber-slides. Their clonogenic potential was evaluated by soft-agar colony formation assay, and their tumorigenicity was investigated upon transplantation into nude mice. The cellular response to HGF was measured by western blot analysis for c-Met, p-ERK, pS6, p-Akt, and by functional assays. The growth kinetics was assessed by MTT assay. Gene expression profiling was performed on Affymetrix platform. Finally, the effects of a novel and promising anticancer drug, Sorafenib, were investigated on both cell lines. Results: LCSC-2 had higher proliferative capacity and clonogenicity in vitro, and, upon transplantation into nude mice, induced macroscopic tumors in less than 30 days, while LCSC-5 required approximately 80 days. Transcriptional analysis revealed that genes overrepresented in LCSC-2 were associated with tumor progression/growth (S100A4, ALDH3A1, ARMCX2), drug resistance (ABCC1) and invasion/cytoskeleton-remodelling (TPM2, PDLIM1, CAPG). Conversely, several liver-associated transcripts (IL6ST, ZFP36, IGF2, CYB5, LISCH7) and genes often downregulated in invasive cancers (GRO1, CEACAM1, NFKBIA, MGP) were reduced in LCSC-2. The expression level of the HGF receptor c-Met and its downstream signaling cascade appeared downregulated in LCSC-2 cells, compared to LCSC-5. Sorafenib had cytotoxic effects and, at cytostatic concentrations, potentiated HGF responses in both cell lines, this effect being again less pronounced in LCSC-2, which was also less sensitive to the drug. Conclusions: LCSC-2 displayed a more aggressive, anaplastic, and drug-resistant phenotype when compared to LCSC-5. The HGF/c-Met pathway may play a causative role in this difference, likely by pro-
moting hepatic differentiation, and may also mediate, at least in part, biological response to Sorafenib. The identification of a panel of genes underlying liver cancer progression offers novel insights to study the effects of molecularly targeted agents against hepatic tumors. # K. Pancreatic and liver oncology 1. Basic science
OC.03.4 OXIDATIVE DAMAGE, CELL IMMORTALIZATION AND miRNA EXPRESSION IN HEPATOCELLULAR CARCINOMA (HCC) AND HEPATITIS R. Cardin, M. Bortolami, M. Piciocchi, L. Barzon, A. Sinigaglia, E. Lavezzo, U. Cillo, G. Zanus, F. Farinati ∗ Università di Padova, Padova Background and aim: The molecular mechanisms driving hepatocarcinogenesis are complex and involve viral factors, inflammation and the production of ROS, the host’s repair mechanisms and cell immortalization. Little information is available about miRNA expression in HCC in relation to the above factors. The aim was to assess the extent of DNA oxidative damage (8-hydroxydeoxyguanosine 8OHdG) in different phases of the carcinogenetic process, the impact of DNA repair gene polymorphisms on such accumulation and any effect of the two on telomerase activity (TA). The expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death, was also considered. Material and methods: Hepatic samples obtained either at surgery, [neoplastic (HCC TU) and adjacent non-cancerous tissues (HCC NTU)], and at percutaneous biopsy (HCV/HBV-positive hepatitis patients) were analyzed for 8OHdG (HPCL-ED), OGG1 (a DNA repair gene) polymorphism (PCR-RFLP), TA (RT-PCR) and for microarray analysis. Nineteen HCC patients and 26 with chronic hepatitis gave informed consent and entered the study. Results: 8OHdG levels were significantly higher in HCC TU and NTU than in chronic hepatitis. TA was activated in 18/19 HCC specimens (94%), it was less frequently detected in adjacent non-cancerous tissues (52%) while it was never identifiable in chronic hepatitis. The mean levels of TA in HCC were significantly higher than those detected in non-cancerous tissues and in chronic liver diseases (p=0.01). In HCC we observed significant positive correlations among 8OHdG levels, TA, stage of associated liver disease, OGG1 polymorphisms and ALT/GGT levels. Additionally, in HCC tissues, a significant positive correlation was found between microRNA-92 expression, TA (p=0.0052) and 8OHdG levels (p=0.05). mir-199a, mir-199b, mir-195 and mir-122a were strongly downregulated in the majority (55%-70%) of HCCs, while mir-92 and mir-145 showed a less marked downregulation. In contrast mir-222 was upregulated in the HCC. Conclusions: The above analysis shows an oxidative damage-mediated induction of TA, modulated by host factors and impacting on some miRNA regulation. In particular miR-92 expression has been already linked to hepadnavirus-associated carcinogenesis and c-myc, frequently activated in HCC. This correlation among DNA oxidative damage, TA and miRNA expression is a novel finding. # K. Pancreatic and liver oncology 1. Basic science
OC.03.5 CONTRAST-ENHANCED US (CEUS) IN EARLY EVALUATION OF NON-SURGICAL TREATMENT OF HCC CAN CHANGE THE PATIENT’S FOLLOW-UP AND SURVIVAL F. Giangregorio ∗ , M. Di Stasi, L. Fanigliulo, M.G. Marinone, G. Comparato, G. Aragona, G. Sbolli, P. Tansini, F. Fornari Ospedale “Gugliemo Da Saliceto”, Piacenza Background and aim: Ceus is used in the assessment of loco-regional