Intra-tumoral epigenetic heterogeneity and aberrant molecular clocks in hepatocellular carcinoma.

There is limited understanding of the epigenetic drivers of tumor evolution in hepatocellular carcinoma (HCC). Here we characterize the epigenetic contribution of methylation to intra-tumoral heterogeneity (mITH) using regional enhanced reduced-representation bisulfite sequencing DNA methylation data from 47 early stage, treatment-naive HCC biopsies across 9 patients by quantifying regional differential methylation across promoters and CpG islands, while overlapping with methylation age markers. Furthermore, we integrate these data with matching RNA-sequencing, targeted DNA sequencing, tumor-infiltrating lymphocyte (TIL), and hepatitis-B viral expression data. We found substantial mITH signatures in promoter and enhancer sites across 44% of patients in our cohort that highlight a novel axis of ITH that is not otherwise detectable from RNA analysis alone. Additionally, we identify an epigenetic tumoral aging measure that reflects a complex tumor fitness phenotype as a potential proxy for tumor clonality. Associating clinical outcomes with epigenetic tumoral age using 450k array data from 377 patients with HCC in the TCGA-LIHC single-biopsy cohort we found evidence implying that epigenetically old tumors have lower fitness yet higher TIL burden. Our data reveal a novel, unique epigenetic axis of ITH in HCC that merits further exploration.