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Hepatocellular carcinoma and metabolic risk factors in a main reference center in Italy
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Abstracts / Digestive and Liver Disease 49S (2017) e19–e42
T-38
T-39
Absolute quantification of intrahepatic HBV covalently-closed-circular DNA by droplet digital PCR
Hepatocellular carcinoma and metabolic risk factors in a main reference center in Italy
G.P. Caviglia 1 , M.L. Abate 1 , A. Olivero 1 , C. Rosso 1 , A. Ciancio 1 , E. Bugianesi 1 , G.M. Saracco 2 , A. Smedile 1 1
Department of Medical Sciences, University of Turin, Turin, Italy 2 Department of Oncology, University of Turin, Turin, Italy Introduction: Novel antiviral strategies for chronic hepatitis B virus (HBV) treatment are under development in order to achieve not only viral suppression but covalently-closed-circular DNA (cccDNA) elimination. Therefore, a sensitive quantification of intrahepatic cccDNA is becoming crucial. Aim: To develop a new assay for cccDNA absolute quantification using droplet digital PCR (ddPCR) technology. Materials and methods: Plasmid AM-12 containing whole HBV genome and DNA extracts from HBsAg-positive and -negative liver specimens were used for assay characterization. Intrahepatic total DNA was isolated by phenol/chloroform method. DNA concentration and quality were assessed by NanoDrop ND1000 (NanoDrop Technologies). For cccDNA quantification, plasmidsafe ATP-dependent DNase (Epicentre) was added to digest linear single- and double-strand DNA. Digested samples were further purified by phenol/chloroform. Specific primers and FAM-labeled probe flanking HBV DNA gap region were used for cccDNA quantification. Intrahepatic cccDNA values were normalized for cellular DNA content using RPP30 probe assay (BioRad) and reported as cccDNA copies/cell. Results: A 10-fold dilution of AM12 plasmid was used to test linearity (105 –100 copies/ddPCR reaction, R2 = 0.9998, p < 0.0001) and concordance correlation coefficient between expected and observed values (rc = 0.9953, 95%CI 0.9927–0.9970). Reproducibility was assessed by intra- and inter-run tests using 2 different cccDNA-positive DNA extracts. Average coefficient of variation (CV) was 8.29% and 7.92% for intra- and inter-run test, respectively. To test cccDNA recovery, quantification of cccDNA was performed on digested and undigested paired samples; average cccDNA copies/ddPCR reaction were 153 ± 23 vs. 434 ± 36, respectively (recovery = 35%; p < 0.0001). Probit analysis was performed to determine lower limit of quantification (LLoQ) and detection (LLoD) using 10 replicates of a serially diluted cccDNA-positive sample. LLoQ was 1.1 × 10−4 copies/cell equal to 2.4 copies/ddPCR reaction whereas LLoD was 3.4 × 10−5 copies/cell equal to 0.8 copies/ddPCR reaction. Conclusions: Our ddPCR-based cccDNA quantitation system is sensitive and could be suitable for monitoring patients with chronic HBV infection during antiviral treatment. http://dx.doi.org/10.1016/j.dld.2017.01.076
R. Ibrahim Kamal Jouness, C. Rosso, M. Marietti, G.P. Caviglia, A. Nascè, D. Campion, A. Cantamessa, P. Carucci, E. Bugianesi Division of Gastroenterology and Hepatology, Department of Medical Sciences, University of Torino, Turin, Italy Background and aims: Hepatocellular carcinoma (HCC) is increasingly reported to be related with metabolic risk factors, particularly in patients with nonalcoholic fatty liver disease (NAFLD) and Chronic Hepatitis C (CHC). Methods: Following the introduction of a centralized specialist team to manage patients with HCC, we characterized the demographics of patients referred to the GI Division at the University Hospital of Torino and sought the relationship between the severity of HCC and their metabolic comorbidities. Results: In total 1039 patients with HCC were consecutively referred since 2011. The large majority were HCV (n = 640, 61.6%), followed by HBV (n = 126, 12.1%), NAFLD (n = 86, 8.3%) and alcohol (n = 157, 15.1%). Across the 5-years period (2011–2016), there was a trend in increase of HCV- and NAFLD-HCC, while alcoholHCC decreased and HBV-HCC was stable. In the same time lag, the age of patients with HCV-HCC progressively decreased (p < 0.001), while it was unchanged in the other etiologies. Diabetes mellitus (DM) was diagnosed in 30% of the whole HCC cohort (23% in HCV-, 28.3% in HBV-, 51% in NAFLD- and 47% in alcohol-HCC). Barcelona Clinic Liver Cancer (BCLC) stage was inversely related with BMI (OR 0.64, 95% CI 0.42–0.98; p = 0.0384) in the whole cohort and in the NAFLD-HCC cohort it was directly related to the presence of DM (OR 11.3, 95% CI 2.1–62.1; p = 0.0052). However, the association between DM and BCLC score was not maintained when the analysis was restricted to Child-Pugh A. Diabetic patients were less likely to be treated by surgery (OLT or liver resection) in the whole HCC cohort. Accordingly, DM was associated with multiple loco-regional treatments (p = 0.041). Conclusions: DM is associated with HCC severity according to BCLC score in the NAFLD-HCC cohort and it is also associated with multiple loco-regional therapy and less likelihood of surgical treatment in the whole HCC cohort. http://dx.doi.org/10.1016/j.dld.2017.01.077
Abstracts / Digestive and Liver Disease 49S (2017) e19–e42
T-38
T-39
Absolute quantification of intrahepatic HBV covalently-closed-circular DNA by droplet digital PCR
Hepatocellular carcinoma and metabolic risk factors in a main reference center in Italy
G.P. Caviglia 1 , M.L. Abate 1 , A. Olivero 1 , C. Rosso 1 , A. Ciancio 1 , E. Bugianesi 1 , G.M. Saracco 2 , A. Smedile 1 1
Department of Medical Sciences, University of Turin, Turin, Italy 2 Department of Oncology, University of Turin, Turin, Italy Introduction: Novel antiviral strategies for chronic hepatitis B virus (HBV) treatment are under development in order to achieve not only viral suppression but covalently-closed-circular DNA (cccDNA) elimination. Therefore, a sensitive quantification of intrahepatic cccDNA is becoming crucial. Aim: To develop a new assay for cccDNA absolute quantification using droplet digital PCR (ddPCR) technology. Materials and methods: Plasmid AM-12 containing whole HBV genome and DNA extracts from HBsAg-positive and -negative liver specimens were used for assay characterization. Intrahepatic total DNA was isolated by phenol/chloroform method. DNA concentration and quality were assessed by NanoDrop ND1000 (NanoDrop Technologies). For cccDNA quantification, plasmidsafe ATP-dependent DNase (Epicentre) was added to digest linear single- and double-strand DNA. Digested samples were further purified by phenol/chloroform. Specific primers and FAM-labeled probe flanking HBV DNA gap region were used for cccDNA quantification. Intrahepatic cccDNA values were normalized for cellular DNA content using RPP30 probe assay (BioRad) and reported as cccDNA copies/cell. Results: A 10-fold dilution of AM12 plasmid was used to test linearity (105 –100 copies/ddPCR reaction, R2 = 0.9998, p < 0.0001) and concordance correlation coefficient between expected and observed values (rc = 0.9953, 95%CI 0.9927–0.9970). Reproducibility was assessed by intra- and inter-run tests using 2 different cccDNA-positive DNA extracts. Average coefficient of variation (CV) was 8.29% and 7.92% for intra- and inter-run test, respectively. To test cccDNA recovery, quantification of cccDNA was performed on digested and undigested paired samples; average cccDNA copies/ddPCR reaction were 153 ± 23 vs. 434 ± 36, respectively (recovery = 35%; p < 0.0001). Probit analysis was performed to determine lower limit of quantification (LLoQ) and detection (LLoD) using 10 replicates of a serially diluted cccDNA-positive sample. LLoQ was 1.1 × 10−4 copies/cell equal to 2.4 copies/ddPCR reaction whereas LLoD was 3.4 × 10−5 copies/cell equal to 0.8 copies/ddPCR reaction. Conclusions: Our ddPCR-based cccDNA quantitation system is sensitive and could be suitable for monitoring patients with chronic HBV infection during antiviral treatment. http://dx.doi.org/10.1016/j.dld.2017.01.076
R. Ibrahim Kamal Jouness, C. Rosso, M. Marietti, G.P. Caviglia, A. Nascè, D. Campion, A. Cantamessa, P. Carucci, E. Bugianesi Division of Gastroenterology and Hepatology, Department of Medical Sciences, University of Torino, Turin, Italy Background and aims: Hepatocellular carcinoma (HCC) is increasingly reported to be related with metabolic risk factors, particularly in patients with nonalcoholic fatty liver disease (NAFLD) and Chronic Hepatitis C (CHC). Methods: Following the introduction of a centralized specialist team to manage patients with HCC, we characterized the demographics of patients referred to the GI Division at the University Hospital of Torino and sought the relationship between the severity of HCC and their metabolic comorbidities. Results: In total 1039 patients with HCC were consecutively referred since 2011. The large majority were HCV (n = 640, 61.6%), followed by HBV (n = 126, 12.1%), NAFLD (n = 86, 8.3%) and alcohol (n = 157, 15.1%). Across the 5-years period (2011–2016), there was a trend in increase of HCV- and NAFLD-HCC, while alcoholHCC decreased and HBV-HCC was stable. In the same time lag, the age of patients with HCV-HCC progressively decreased (p < 0.001), while it was unchanged in the other etiologies. Diabetes mellitus (DM) was diagnosed in 30% of the whole HCC cohort (23% in HCV-, 28.3% in HBV-, 51% in NAFLD- and 47% in alcohol-HCC). Barcelona Clinic Liver Cancer (BCLC) stage was inversely related with BMI (OR 0.64, 95% CI 0.42–0.98; p = 0.0384) in the whole cohort and in the NAFLD-HCC cohort it was directly related to the presence of DM (OR 11.3, 95% CI 2.1–62.1; p = 0.0052). However, the association between DM and BCLC score was not maintained when the analysis was restricted to Child-Pugh A. Diabetic patients were less likely to be treated by surgery (OLT or liver resection) in the whole HCC cohort. Accordingly, DM was associated with multiple loco-regional treatments (p = 0.041). Conclusions: DM is associated with HCC severity according to BCLC score in the NAFLD-HCC cohort and it is also associated with multiple loco-regional therapy and less likelihood of surgical treatment in the whole HCC cohort. http://dx.doi.org/10.1016/j.dld.2017.01.077