Metastases in the later stages of hepatocellular carcinoma (HCC) cause the majority of deaths associated with the disease, making early detection crucial to patient survival. Risk models assessing HCC risk in the general population can be used for risk stratification for further HCC surveillance, however, none have been validated externally. Methylation of circulating DNA shows potential for non-invasive diagnosis of HCC. We conducted a prospective case–control study nested within a community-based cohort. We measured methylation levels in six genes (CDKN2A, RASSF1A, STEAP4, TBX2, VIM and ZNF154) which were identified in our previous work, using pre-diagnostic plasma DNA from 237 HCC cases and 257 matched controls. We found TBX2 hypermethylation was associated with increased HCC risk, with ORs (95% CI) of 3.2 (1.8–6.0). The associations were mainly among high-risk subjects; among subjects infected with HBV/HCV, the OR (95% CI) of TBX2 methylation was 5.3 (2.2–12.7). Among subjects with high risk scores, the ORs (95% CIs) were 7.8 (1.5–38.6) for Wen-HCC model ≥16, 5.8 (2.2–15.5) for Hung-HCC ≥15 and 7.5 (2.2–26.0) for Michikawa-HCC ≥8. Adding TBX2 methylation improved the accuracy of risk models for a high-risk population, with the area under the curve (AUC) of 76% for Wen-HCC score with TBX2 methylation compared with 69% with Wen-HCC alone. The AUCs were 63% for Hung-HCC score plus TBX2 methylation, and 53% for Hung-HCC alone, 65% for Michikawa-HCC score plus TBX2 methylation and 58% for Michikawa-HCC alone. Our findings suggest the potential increase in risk assessment discrimination and accuracy from incorporation of DNA methylation.

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.

Background and Aims: Despite the substantial impact of environmental factors, individuals with a family history of liver cancer have an increased risk for HCC. However, genetic factors have not been studied systematically by genome-wide approaches in large numbers of individuals from European descent populations (EDP). Approach and Results: We conducted a 2-stage genome-wide association study (GWAS) on HCC not affected by HBV infections. A total of 1872 HCC cases and 2907 controls were included in the discovery stage, and 1200 HCC cases and 1832 controls in the validation. We analyzed the discovery and validation samples separately and then conducted a meta-analysis. All analyses were conducted in the presence and absence of HCV. The liability-scale heritability was 24.4% for overall HCC. Five regions with significant ORs (95% CI) were identified for nonviral HCC: 3p22.1, MOBP, rs9842969, (0.51, [0.40–0.65]); 5p15.33, TERT, rs2242652, (0.70, (0.62–0.79]); 19q13.11, TM6SF2, rs58542926, (1.49, [1.29–1.72]); 19p13.11 MAU2, rs58489806, (1.53, (1.33–1.75]); and 22q13.31, PNPLA3, rs738409, (1.66, [1.51–1.83]). One region was identified for HCV-induced HCC: 6p21.31, human leukocyte antigen DQ beta 1, rs9275224, (0.79, [0.74–0.84]). A combination of homozygous variants of PNPLA3 and TERT showing a 6.5-fold higher risk for nonviral-related HCC compared to individuals lacking these genotypes. This observation suggests that gene-gene interactions may identify individuals at elevated risk for developing HCC. Conclusions: Our GWAS highlights novel genetic susceptibility of nonviral HCC among European descent populations from North America with substantial heritability. Selected genetic influences were observed for HCV-positive HCC. Our findings indicate the importance of genetic susceptibility to HCC development.