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This study aimed to investigate the association of fasting insulin and glucose levels with hepatocellular carcinoma (HCC) risk in a case-cohort study within a cohort (1989-2006) of 2903 male government employees chronically infected with hepatitis B virus (HBV) in Taiwan. Insulin, glucose and HBV-related factors were assayed in baseline plasma among 124 HCC cases and a random subcohort of 1084 of the total cohort. After adjustment for demographics and HBV-related factors, including viral load and genotype, the HCC risk was higher for the highest [>6.10 μU/ml, hazard ratio (HR) = 2.36, 95% confidence interval (CI): 1.43-3.90] and lowest (<2.75 μU/ml, HR = 1.57, 95% CI: 0.96-2.58) categories of insulin, compared with insulin of 2.75-4.10 μU/ml. The dose-response relationship between insulin and HCC varied by follow-up time, with stronger association for the HCC cases that occurred ≥8 years after baseline (P for trend <0.0001). The effect of higher insulin on HCC risk remained after adjustment for other metabolic factors, and was fairly consistent across strata of age, body mass index, and HBV genotypic variants. However, it was more profound among those with viral load <4.39 log(10) copies/ml at recruitment (>6.10 μU/ml, HR = 6.15, 95% CI: 2.48-15.22). Higher insulin was also associated with an increased risk for cirrhosis diagnosed by ultrasonography and elevated alanine aminotransferase. No association with either cirrhosis or HCC was noted for glucose or diabetes after adjusting for insulin. In conclusion, elevated insulin levels are an independent risk factor for HCC among HBV carriers, especially for those with lower viral load.

ObjectiveThe hepatitis B virus (HBV)-polymerase region overlaps pre-S/S genes with high epitope density and plays an essential role in viral replication. We investigated whether genetic variation in the polymerase region determined long-term dynamics of viral load and the risk of hepatitis B progression in a population-based cohort study.MethodsWe sequenced the HBV-polymerase region using baseline plasma from treatment-naïve individuals with HBV-DNA levels≥1000 copies/mL in a longitudinal viral-load study of participants with chronic HBV infection followed-up for 17 years, and obtained sequences from 575 participants (80% with HBV genotype Ba and 17% with Ce).ResultsPatterns of viral sequence diversity across phases (i.e., immune-tolerant, immune-clearance, non/low replicative, and hepatitis B e antigen (HBeAg)-negative hepatitis phases) of HBV-infection, which were associated with viral and clinical features at baseline and during follow-up, were similar between HBV genotypes, despite greater diversity for genotype Ce vs. Ba. Irrespective of genotypes, however, HBeAg-negative participants had 1.5-to-2-fold higher levels of sequence diversity than HBeAg-positive participants (P<0.0001). Furthermore, levels of viral genetic divergence from the population consensus sequence, estimated by numbers of nucleotide substitutions, were inversely associated with long-term viral load even in HBeAg-negative participants. A mixed model developed through analysis of the entire HBV-polymerase region identified 153 viral load-associated single nucleotide polymorphisms in overall and 136 in HBeAg-negative participants, with distinct profiles between HBV genotypes. These polymorphisms were most evident at sites within or flanking T-cell epitopes. Seven polymorphisms revealed associations with both enhanced viral load and a more than 4-fold increased risk of hepatocellular carcinoma and/or liver cirrhosis.ConclusionsThe data highlight a role of viral genetic divergence in the natural course of HBV-infection. Interindividual differences in the long-term dynamics of viral load is not only associated with accumulation of mutations in HBV-polymerase region, but differences in specific viral polymorphisms which differ between genotypes.

Background: Coexistence of hepatitis B and nonalcoholic fatty liver disease is common; however, little is known about the impact of hepatic steatosis and its major genetic determinants on the natural history of HBV infection. We aimed to study the effects of hepatic steatosis and PNPLA3 variant p.I148M on the risk of hepatocellular carcinoma (HCC) and the lifetime probability of HBsAg seroclearance, which is associated with functional remission and improved long-term outcome of HBV infection. Methods: We conducted a cohort study of 2385 male, HBsAg-positive Taiwanese civil servants recruited in 1989-1992, and followed up until 2019. Cox regression with competing-risk models was used to estimate sub-distribution hazard ratios (sHRs) and 95% confidence intervals (CIs). Results: Of 2385 participants, 628 experienced HBsAg seroclearance and 217 developed HCC. Hepatic steatosis, excess body-mass index, and the PNPLA3-148M variant were significantly associated with higher HBsAg seroclearance rate. However, multivariate analyses accounting for HBsAg seroclearance and various HCC risk factors showed that, while steatosis was associated with decreased HCC risk (sHR [95% CI]: 0.49 [0.36-0.66]), carriage of the PNPLA3-148M variant allele for MM homozygotes) and obesity (1.51 [1.07-2.13]) were associated with increased risk. The inverse hepatic steatosis-HCC association persisted after additional adjustment for other viral factors or using different follow-up time cut-offs to account for reverse causality. Moreover, the PNPLA3 MM genotype was positively associated with elevations of ALT and AST and liver cirrhosis, while hepatic steatosis was positively associated with ALT but inversely associated with AST and liver cirrhosis. Conclusion: Hepatic steatosis and PNPLA3-148M variant appeared to have distinct impacts on the development of HBV-related progressive liver disease and HCC. PNPLA3 p.I148M, but not a diagnosis of hepatic steatosis, can help to identify HBV carriers with high-risk fatty liver disease in the progression to HCC.

To evaluate how hepatitis B virus (HBV) genetic variation affected progression from chronic carrier state to hepatocellular carcinoma (HCC), we analyzed HBV full-length sequences in blood obtained <1-20 years before diagnosis from 117 HCC cases and 118 controls nested in a cohort of 4,841 HBV carriers, for whom HBV genotypes B and C are predominant. The relationship between each viral single-nucleotide polymorphism (SNP) and HCC development was assessed using ordinal logistic models according to five periods of time to diagnosis (TTD). Thirty-one HBV-SNPs showed significant association with TTD after adjustment for HBV genotype, 24 of which could also be analyzed with an extended analysis on the full-length data in conjunction with 512 partial sequences (nucleotides 2,436-1,623) from the cohort. The obtained 10 robust candidate HBV-SNPs (P £ 0.0304), which showed odds ratios ranging from 1.89 to 8.68, were further confirmed in 163 GenBank HBV-HCC sequences from nine Asia regions, assayed after HCC diagnosis, representing the end stage of progressive hepatic diseases. The prevalence of these HBV-SNPs and their cumulative number, presented in terms of mutation score, increased with time approaching HCC diagnosis, with an odds ratio of 2.17, 4.21, 8.15, and 19.15, respectively, for the mutation score of 1, 2, 3, and 4 versus 0. The mutation score for predicting short-term HCC risk outperformed other factors, including HBV-DNA levels, viral genotype, and various combinations of risk factors, and revealed increasing accuracy with shorter TTD (<4.5 years before diagnosis: area under the curve 5 0.83-0.89; sensitivity 5 72.7%-94.1%; specificity 5 58.3%-70.5%; conditioned on optimized cutoff for genotype B and C, respectively). Conclusions: Identifying and tracking viral mutations is important for monitoring hepatitis B progression and early detection of HCC. (HEPATOLOGY 2016;64:720-731) T he pathogenesis of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is still unclear, but believed to involve complicated viralhost interactions.(1-4) Several host and viral factors have been associated with progression from chronic HBV carrier state to HCC.(3-8) HBV-related factors include viral load and viral genotypes and mutations. (3,4,(6)(7)(8) HBV replicates by reverse transcription with a viral polymerase lacking proofreading ability, allowing mutations to occur during chronic infection. Detection of naturally occurring HBV mutations has important implications for antiviral therapy and hepatic disease progression. (9,10) The size of the HBV genome is approximately 3.2 kilobases, with four overlapping open-reading