- Email: [email protected]
F-01 Baseline and early dynamic evaluation of HCV NS3-resistance
Abstracts of the 46th A.I.S.F. Annual Meeting 2013 / Digestive and Liver Disease 45S (2013), S1–S48 trioxide. Furthermore, HuCCT-1 cells silenced for CXCR7 blocked the ability of CXCL12 to induce cell migration. Conclusions: These data demonstrate that two CCA cell lines express CXCR7 and that this receptor has a key function during CXCL12-induced CCA migration. Both CXCR4 and CXCR7 contribute to CXCL12-induced signaling in CCA cells.
T-56 Oxidative genomic and mitochondrial damage in relation to telomeric dysfunction in liver carinogenesis M. Piciocchi 1 , R. Cardin 1 , U. Cillo 1 , M. Bortolami 1 , A. Kotsafti 2 , A. Vitale 1 , F. Farinati 1 1 Department of Surgical, Oncological and Gastroenterological Sciences, University of Padova, Padua, Italy; 2 Venetian Oncological Institute, Padua, Italy
Introduction: Oxidative stress by hepatitis C (HCV) and B (HBV) virus is a molecular event leading to hepatocellular carcinoma (HCC). Telomeres are prone to oxidative modifications, inducing progressive telomere shortening and chromosomal instability. Telomerase activity plays a role in telomeres maintenance and cell immortalization. TERT, the rate limiting factor for telomerase transcription, is regulated by epigenetic mechanism and under oxidative stress migrates to mitochondria, where it ameliorates the membrane stability and exerts an anti-apoptotic role. Aim: To investigate the molecular network underlying virus-related liver carcinogenesis. Materials, methods and results: One hundred sixty-two patients [21 with HCC (tumor/peritumoral tissue), 71 with HCV/35 with HBV and 15 controls] were investigated to evaluate: 8-hydroxydeoxyguanosine (8-OHdG), marker of oxidative DNA damage (HPLC-EC), telomerase activity and telomeres length (RealTime-PCR), TERT promoter methylation status (Quantitative Methylation Specific-PCR) and mitochondrial TERT translocation (Western Blot). Overall, 8-OHdG levels were significantly higher in tumor tissues than in controls (p=0.02), telomeres were significantly shorter in HCC than in less advanced disease stages (p=0.01), whereas telomerase activity was significantly higher in tumor tissues than in controls (p=0.01). 8-OHdG levels inversely correlated with telomere length in HCC. Overall, TERT promoter was hypermethylated in HCC and peritumoral tissue (p=0.0001). Sub-grouping the patients on the etiology basis, HBV-related liver damage progression was characterized by later 8-OHdG accumulation and a pronounced telomerase activation limited to HCC tissue. TERT was localized in mitochondria in all HCC investigated and, in mitochondria, DNA 8-OHdG levels were significantly lower than in genomic DNA (p= 0.0003). Conclusions: These data describe a network in which oxidative DNA damage is linked to changes in telomeres length, extent of telomerase activation and higher methylation of TERT promoter in HBV/HCV related carcinogenesis. The role of mitochondrial TERT in HCC is open to debate but an intriguing work hypothesis is that it could downregulate apoptosis.
Friday, February 22nd F-01 Baseline and early dynamic evaluation of HCV NS3-resistance D. Di Paolo 1 , V. Cento 2 , S. Cucchiarelli 1 , S. Francioso 1 , F. De Luca 2 , V.C. Di Maio 2 , F. Antenucci 1 , F. De Leonardis 1 , I. Lenci 1 , F.P. Antonucci 2 , A. Bertoli 2 , M. Mapfumo 3 , C. Sarrecchia 3 , L. Nosotti 4 , C.F. Perno 2 , F. Ceccherini Silberstein 2 , M. Angelico 1 1 Hepatology
Unit, University of Tor Vergata, Rome, Italy; 2 Virology Chair, University of Tor Vergata, Rome, Italy; 3 Infectious Disease Unit, University of Tor Vergata, Rome, Italy; 4 Hepatology Unit, National Institute of Health, Migration and Poverty, Rome, Italy Background: Telaprevir (TVR) and Boceprevir (BOC) are now first-choice therapeutic options for genotype 1 chronic hepatitis C, but their use is as-
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sociated with a significant risk of inducing viral resistances. A structured monitoring of the HCV genome by NS3-region sequencing has not yet been performed during treatment with TVR/BOC. Aim: To evaluate the presence of TVR/BOC-resistance mutations through the systematic genotypic characterization of viral quasispecies at the NS3-gene before and during the early and late phases of TVR/BOC-treatment. Methods: 21 patients (HCV-1a/1b = 9/12; advanced fibrosis/cirrhosis = 8/13) treated with TVR (N=19) or BOC (N=2) were enrolled. HCV-RNA (detectionlimit=12 IU/ml) and NS3-protease sequences were evaluated at baseline, after 48 hours and at week 4, 8, 12, 16 and 24 TVR/BOC-treatment, and during the follow-up. Results: This interim analysis includes data at month 6. One genotype 1a-infected cirrhotic receiving TVR experienced viral breakthrough at week 8 which was associated with evidence of major resistance mutations (V36M+R155K). Another TVR-treated, 1a-cirrhotic patient who was a partial virological responder (>2 log IU/ml drop of HCV-RNA at week 12) developed the resistant-mutations V36M+R155K at week 8. Other two cirrhotic patients developed the resistance mutations V36M and T54A after only 48 hours of TVR treatment: the first experienced a viral breakthrough later, at week 24, associated with the additional emergence of R155K mutation, while the other obtained RVR. One BOC-treated 1b-cirrhotic patient had the minor resistant-variant Q80S at baseline and reported an RVR (BOC is on-going). Conclusions: Major TVR/BOC-resistance mutations were undetectable at baseline. To evaluate the risk of selecting resistant variants, 48 hours after TVR/BOC start could be a crucial timing to study HCV-NS3 mutations. The emergence at 48 hours of V36M but not T54A was associated with TVR virological-failure. The combination of resistant-variants V36M+R155K was always associated with TVR-treatment failure.
F-02 Incidence and consequences of thrombocytopenia in 1523 HIV/HCV coinfected patients treated with PEGylated interferon and ribavirin A.M. Cattelan 1 , E. Angeli 2 , E. Pontali 3 , R. Gulminetti 4 , I. Maida 5 , P. Nasta 6 , G. Verucchi 8 , A. Caputo 9 , C. Iannacone 10 , M. Puoti 11 1 Ospedale
Santa Maria della Misericordia, Rovigo, Italy; 2 Azienda Ospedaliera Universitaria Luigi Sacco, Milano, Italy; 3 Galliera Hospital, Genova, Italy; 4 IRCCS Policlinico San Matteo, Pavia, Italy; 5 University of Sassari, Sassari, Italy; 6 Azienda Ospedaliera Spedali Civili, Brescia, Italy; 7 Azienda Ospedaliero, Universitaria Policlinico S. Orsola Malpighi, Bologna, Italy; 8 Università degli studi di Torino, Torino, Italy; 9 Roche Spa, Monza, Italy; 10 SPARC consulting Spa, Milano, Italy; 11 Ospedale Niguarda, Milano, Italy Introduction: Haematological side effects are a major limitation of HCV/HIV combination therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV). Thrombocytopenia can lead to PEG-IFN dose reduction or withdrawal thus potentially reducing the Sustained Virologic Response (SVR). Aim: To define the incidence and consequences of treatment-induced thrombocytopenia in HIV/HCV coinfected patients. Materials and methods: This was an observational study (OPERA) of HIV/HCV coinfected patients starting PEG IFN+RBV treatment in Italy between 2005 and 2009. Thrombocytopenia was defined as <100,000 platelets/mL (mild 80,001–100,000/mL, moderate 50,001–80,000/mL, severe 25,001–50,000 or life threatening ≤25,000/mL). Multivariate regression analysis, Chi-Square or Fisher’s Exact test and t-test for unpaired samples were used for statistical analysis. A p value <0.05 was considered to be significant. Results: Of 1523 patients showing a median platelet count of 180,000/mL at baseline (IQR 141,000–224,000/mL), 595 (39.1%) developed thrombocytopenia: mild, 221 (14.5%), moderate, 275 (18.1%) and severe, 87 patients (5.7%). Only 0.8% had platelets ≤25,000/mL; 2.7% discontinued because of thrombocytopenia and only 1 patient had bleeding during treatment. Among cirrhotic patients, 106/148 had thrombocytopenia. Multivariate analysis adjusted for baseline platelet count and cirrhosis diagnosis, showed that only undetectable HIV-RNA (AOR 1.65; 95% CI 1.16–2.34, p=0.003) was independently associated with thrombocytopenia during treatment. Thrombocytopenia occurrence
T-56 Oxidative genomic and mitochondrial damage in relation to telomeric dysfunction in liver carinogenesis M. Piciocchi 1 , R. Cardin 1 , U. Cillo 1 , M. Bortolami 1 , A. Kotsafti 2 , A. Vitale 1 , F. Farinati 1 1 Department of Surgical, Oncological and Gastroenterological Sciences, University of Padova, Padua, Italy; 2 Venetian Oncological Institute, Padua, Italy
Introduction: Oxidative stress by hepatitis C (HCV) and B (HBV) virus is a molecular event leading to hepatocellular carcinoma (HCC). Telomeres are prone to oxidative modifications, inducing progressive telomere shortening and chromosomal instability. Telomerase activity plays a role in telomeres maintenance and cell immortalization. TERT, the rate limiting factor for telomerase transcription, is regulated by epigenetic mechanism and under oxidative stress migrates to mitochondria, where it ameliorates the membrane stability and exerts an anti-apoptotic role. Aim: To investigate the molecular network underlying virus-related liver carcinogenesis. Materials, methods and results: One hundred sixty-two patients [21 with HCC (tumor/peritumoral tissue), 71 with HCV/35 with HBV and 15 controls] were investigated to evaluate: 8-hydroxydeoxyguanosine (8-OHdG), marker of oxidative DNA damage (HPLC-EC), telomerase activity and telomeres length (RealTime-PCR), TERT promoter methylation status (Quantitative Methylation Specific-PCR) and mitochondrial TERT translocation (Western Blot). Overall, 8-OHdG levels were significantly higher in tumor tissues than in controls (p=0.02), telomeres were significantly shorter in HCC than in less advanced disease stages (p=0.01), whereas telomerase activity was significantly higher in tumor tissues than in controls (p=0.01). 8-OHdG levels inversely correlated with telomere length in HCC. Overall, TERT promoter was hypermethylated in HCC and peritumoral tissue (p=0.0001). Sub-grouping the patients on the etiology basis, HBV-related liver damage progression was characterized by later 8-OHdG accumulation and a pronounced telomerase activation limited to HCC tissue. TERT was localized in mitochondria in all HCC investigated and, in mitochondria, DNA 8-OHdG levels were significantly lower than in genomic DNA (p= 0.0003). Conclusions: These data describe a network in which oxidative DNA damage is linked to changes in telomeres length, extent of telomerase activation and higher methylation of TERT promoter in HBV/HCV related carcinogenesis. The role of mitochondrial TERT in HCC is open to debate but an intriguing work hypothesis is that it could downregulate apoptosis.
Friday, February 22nd F-01 Baseline and early dynamic evaluation of HCV NS3-resistance D. Di Paolo 1 , V. Cento 2 , S. Cucchiarelli 1 , S. Francioso 1 , F. De Luca 2 , V.C. Di Maio 2 , F. Antenucci 1 , F. De Leonardis 1 , I. Lenci 1 , F.P. Antonucci 2 , A. Bertoli 2 , M. Mapfumo 3 , C. Sarrecchia 3 , L. Nosotti 4 , C.F. Perno 2 , F. Ceccherini Silberstein 2 , M. Angelico 1 1 Hepatology
Unit, University of Tor Vergata, Rome, Italy; 2 Virology Chair, University of Tor Vergata, Rome, Italy; 3 Infectious Disease Unit, University of Tor Vergata, Rome, Italy; 4 Hepatology Unit, National Institute of Health, Migration and Poverty, Rome, Italy Background: Telaprevir (TVR) and Boceprevir (BOC) are now first-choice therapeutic options for genotype 1 chronic hepatitis C, but their use is as-
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sociated with a significant risk of inducing viral resistances. A structured monitoring of the HCV genome by NS3-region sequencing has not yet been performed during treatment with TVR/BOC. Aim: To evaluate the presence of TVR/BOC-resistance mutations through the systematic genotypic characterization of viral quasispecies at the NS3-gene before and during the early and late phases of TVR/BOC-treatment. Methods: 21 patients (HCV-1a/1b = 9/12; advanced fibrosis/cirrhosis = 8/13) treated with TVR (N=19) or BOC (N=2) were enrolled. HCV-RNA (detectionlimit=12 IU/ml) and NS3-protease sequences were evaluated at baseline, after 48 hours and at week 4, 8, 12, 16 and 24 TVR/BOC-treatment, and during the follow-up. Results: This interim analysis includes data at month 6. One genotype 1a-infected cirrhotic receiving TVR experienced viral breakthrough at week 8 which was associated with evidence of major resistance mutations (V36M+R155K). Another TVR-treated, 1a-cirrhotic patient who was a partial virological responder (>2 log IU/ml drop of HCV-RNA at week 12) developed the resistant-mutations V36M+R155K at week 8. Other two cirrhotic patients developed the resistance mutations V36M and T54A after only 48 hours of TVR treatment: the first experienced a viral breakthrough later, at week 24, associated with the additional emergence of R155K mutation, while the other obtained RVR. One BOC-treated 1b-cirrhotic patient had the minor resistant-variant Q80S at baseline and reported an RVR (BOC is on-going). Conclusions: Major TVR/BOC-resistance mutations were undetectable at baseline. To evaluate the risk of selecting resistant variants, 48 hours after TVR/BOC start could be a crucial timing to study HCV-NS3 mutations. The emergence at 48 hours of V36M but not T54A was associated with TVR virological-failure. The combination of resistant-variants V36M+R155K was always associated with TVR-treatment failure.
F-02 Incidence and consequences of thrombocytopenia in 1523 HIV/HCV coinfected patients treated with PEGylated interferon and ribavirin A.M. Cattelan 1 , E. Angeli 2 , E. Pontali 3 , R. Gulminetti 4 , I. Maida 5 , P. Nasta 6 , G. Verucchi 8 , A. Caputo 9 , C. Iannacone 10 , M. Puoti 11 1 Ospedale
Santa Maria della Misericordia, Rovigo, Italy; 2 Azienda Ospedaliera Universitaria Luigi Sacco, Milano, Italy; 3 Galliera Hospital, Genova, Italy; 4 IRCCS Policlinico San Matteo, Pavia, Italy; 5 University of Sassari, Sassari, Italy; 6 Azienda Ospedaliera Spedali Civili, Brescia, Italy; 7 Azienda Ospedaliero, Universitaria Policlinico S. Orsola Malpighi, Bologna, Italy; 8 Università degli studi di Torino, Torino, Italy; 9 Roche Spa, Monza, Italy; 10 SPARC consulting Spa, Milano, Italy; 11 Ospedale Niguarda, Milano, Italy Introduction: Haematological side effects are a major limitation of HCV/HIV combination therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV). Thrombocytopenia can lead to PEG-IFN dose reduction or withdrawal thus potentially reducing the Sustained Virologic Response (SVR). Aim: To define the incidence and consequences of treatment-induced thrombocytopenia in HIV/HCV coinfected patients. Materials and methods: This was an observational study (OPERA) of HIV/HCV coinfected patients starting PEG IFN+RBV treatment in Italy between 2005 and 2009. Thrombocytopenia was defined as <100,000 platelets/mL (mild 80,001–100,000/mL, moderate 50,001–80,000/mL, severe 25,001–50,000 or life threatening ≤25,000/mL). Multivariate regression analysis, Chi-Square or Fisher’s Exact test and t-test for unpaired samples were used for statistical analysis. A p value <0.05 was considered to be significant. Results: Of 1523 patients showing a median platelet count of 180,000/mL at baseline (IQR 141,000–224,000/mL), 595 (39.1%) developed thrombocytopenia: mild, 221 (14.5%), moderate, 275 (18.1%) and severe, 87 patients (5.7%). Only 0.8% had platelets ≤25,000/mL; 2.7% discontinued because of thrombocytopenia and only 1 patient had bleeding during treatment. Among cirrhotic patients, 106/148 had thrombocytopenia. Multivariate analysis adjusted for baseline platelet count and cirrhosis diagnosis, showed that only undetectable HIV-RNA (AOR 1.65; 95% CI 1.16–2.34, p=0.003) was independently associated with thrombocytopenia during treatment. Thrombocytopenia occurrence