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). We quantify epigenetic intra-tumoral heterogeneity (ITH) using regional enhanced reduced-representation bisulfite sequencing (eRRBS) DNA methylation data from 47 early stage, treatment-naive HCC biopsies across 9 patients. Integrating these data with matching RNAseq, targeted DNA sequencing, tumor-infiltrating lymphocyte (TIL) and hepatitis-B viral (HBV) expression, we computed regional differential methylation (DM) ITH signatures across 19,327 promoter regions, and 654,133 CpG islands, while overlapping with known methylation age marker genes (240/354). We found substantial ITH signatures in promoter and enhancer sites across 4/9 patients highlighting novel molecular pathways of tumor progression 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 evolution. In order to compute clinical associations with epigenetic tumoral age, we use 450k array data from 377 HCC patients in the TCGA-LIHC single-biopsy cohort to calculate tumoral age and find evidence implying that epigenetically old tumors have lower fitness yet higher TIL burden. Our data reveal a novel, unique epigenetic ITH axis in HCC tumors that furthers our understanding of tumor evolution and may serve as a potential avenue for enhancing patient stratification and treatment.