Identification of fetal liver-type hepatocellular carcinoma based on a methylome analysis and its associations with genetic alterations

Aims: Hepatocellular carcinomas (HCCs) with expression-patterns similar to those of fetal livers reportedly showed aggressive behavior. As gene expressions are linked to epigenetic status during tissue development, we tried to identify and characterize fetal liver type-HCCs (F-HCCs) through methylome analyses. Methods: Using the Gene Expression Omnibus (GEO-NCBI), we found 854 genes with significant difference of methylation and expression levels between fetal and adult livers. We also analyzed 83 pairs of HCCs and their non-cancerous livers (NLs) for DNA methylation using HumanMethylation450 BeadChip and selected genes that showed difference of methylation between HCCs and NLs. Mutations of TP53, CTNNB1, and TERT promoter are determined for the 83 HCCs using deep sequencing and/or direct sequencing. Based on the data from GEO and our analyses of human HCCs, we selected top 14 target genes for further analyses. Results: The genes that were hyper- and hypo-methylated in fetal livers were enriched for metabolic pathways and pathways of differentiation processes, respectively. We classified HCCs into adult liver-type and F-HCCs based on the number of abnormally methylated genes and hierarchical clustering analysis using methylation levels. A subclass of F-HCCs is characterized by increased tumor size, presence of vascular invasion, hepatitis B, and TP53 mutation. Conclusion: Through the methylome analysis, we successfully identified F-HCCs and characterized the unique features with advanced tumor phenotype and TP53 mutation.