DNA methylation profiling of human hepatocarcinogenesis.

BACKGROUND Mutations in TERT promoter are established gatekeepers in early hepatocarcinogenesis, but little is known about other molecular alterations driving this process. Epigenetic deregulation is a critical event in early malignancies. Thus, we aimed: 1) to analyze DNA methylation changes during the transition from preneoplastic lesions to early HCC (eHCC) and to identify candidate epigenetic gatekeepers, and 2) to assess the prognostic potential of methylation changes in cirrhotic tissue. METHODS Methylome profiling was performed using Illumina HumanMethylation450 (485,000 CpG, 96% of known CpG islands), with data available for a total of 390 samples: 16 normal liver, 139 cirrhotic tissue, 8 dysplastic nodules and 227 HCC samples, including 40 eHCC below 2cm. RESULTS A phylo-epigenetic tree derived from the euclidean distances between differentially DNA methylated sites (n=421,997) revealed a gradient of methylation changes spanning normal liver, cirrhotic tissue, dysplastic nodules and HCC with closest proximity of dysplasia to HCC. Focusing on promoter regions, we identified epigenetic gatekeeper candidates with an increasing proportion of hypermethylated samples (beta value >0.5) from cirrhotic tissue ( =25%), to eHCC (>50%), and confirmed inverse correlation between DNA methylation and gene expression for TSPYL5, KCNA3, LDBH and SPINT2 (all p<0.001). Unsupervised clustering of genome-wide methylation profiles of cirrhotic tissue identified two clusters, M1 and M2 with 42% and 58% of patients, respectively, which correlate with survival (p<0.05), independent of etiology. CONCLUSIONS Genome-wide DNA methylation profiles accurately discriminate the different histological stages of human hepatocarcinogenesis. We report novel epigenetic gatekeepers in the transition between dysplastic nodules and eHCC. DNA methylation changes in cirrhotic tissue correlate with clinical outcomes.