Landscape of (epi-)genetic alterations during malignant transformation in liver cancer

Background and aims:

Development of primary liver cancer is a multi-stage process. Pre-neoplastic dysplastic lesions emerge on the basis of chronic liver damage and evolve into early hepatocellular carcinoma (eHCC) and, subsequently, progressed HCC (pHCC). Detailed molecular characterization and prediction of pre-neoplastic lesions at high risk for malignant transformation would significantly advance our diagnostic and therapeutic approaches. We here utilized integrative molecular analyses to characterize the sequential evolution of liver cancer and aimed to define key epigenetic drivers and biomarkers of HCC development and progression.

Methods:

Methylation 450k-beadchip analyses were performed on cirrhotic liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, eHCC (n = 5) and pHCC (n = 3) from 8 HCC patients with chronic hepatitis B infection. Differentially methylated gene regions (DMGR) were identified in comparison to non-cirrhotic and non-infected liver (n = 9). Potential epi-drivers and biomarkers were identified by integrative analyses of transcriptomic changes and validated in an independent cohort from the TCGA database.

Results:

The proportion of hypermethylated DMGR progressively increased from cirrhosis over dysplastic- to HCC and peaked in eHCC lesions. Early epigenetic alterations involved signaling pathways related to cell death, apoptosis and immune regulation, while late changes centered on cell survival, growth and migration. A common regulation of stem cell-associated pathways including Wnt/b-catenin signaling was revealed in dysplastic as well as eHCC potentially predisposing tumor progression. Moreover, we identified 101 genes with significant methylom changes in dysplastic and cancerous lesions with concomitant progressive gene expression alterations in cancer tissue. We further defined an epi-panel of early epigenetic marks in dysplastic lesions including selected CpG-sites with confirmed differential methylation in cancer tissue and consequential transcriptional alterations of the target genes using an independent cohort of 362 HCC and 49 surrounding liver samples. Unsupervised hierarchical clustering confirmed a robust classification in malignant and non-malignant lesions.

Conclusion:

Our results confirm that epigenetic changes occur early during hepatocarcinogenesis. Epigenetic modifications, therefore, might be of high diagnostic/predictive utility for the identification of dysplastic lesions at risk for cancer progression. The identified epi-panel of oncogenic epigenetic marks might be useful to complement phenotypic classifications and facilitate selection of lesions amenable to early therapeutic interventions.