Background: Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide. Surgical resection remains the mainstay treatment for early localized HCC, but hepatic recurrence occurs in >70% of patients within 5-years post-surgery. We previously reported that recurrent HCC can originate from either the same (Progressive pHCC) or different clonal lineage (Multi-centric mHCC), whereby the latter is associated with better prognosis. Interestingly, tumours from de novo clones (mHCC) still shared similar aberrant methylation profiles, suggesting that common epigenetic alterations already existed in the early preneoplastic cirrhotic lesions and could predispose to liver carcinogenesis. In this study, we aim to elucidate the underlying methylation events from preneoplastic lesions that lead to either mHCC or pHCC.

Methods: Samples were obtained from patients who underwent partial hepatectomy at PWH & QMH, Hong Kong. Recurrence type of mHCC or pHCC was defined by WGS or targeted NGS as previously described. Methylation profiling and bulk RNA sequencing was performed on Normal liver (NNL, n=4), Nontumoral adjacent (PN), primary tumor (PT) and matching relapsed tumor (RT) (mHCC n=8 sets; pHCC n=10 sets). 1260 differentially methylated (DM) probes from the PN tissues of mHCC versus pHCC were selected for subsequent time series clustering using MFuzz. Clusters were grouped by hyper- or hypomethylated predisposing (change occurring from NNL to PN) or transforming (change only occurring from PN to PT/RT) trajectories. Probes harboring differentially methylated regions (DMRcate >3 CpG probes) with inverse transcriptional correlation were prioritized for subsequent validation.

Results & Conclusion: Time series clustering revealed that differentially methylated events predisposing to mHCC recurrence are governed by changes in the microenvironment such as Regulation of Immune response (GO:0050778) and Cell Adhesion (GO:0022407). E.g. mHCC-specific hypomethylation of CTLA4 DMR at the Transcription Start Site (TSS) shows significant inverse correlation at the transcript level. Underlying immune profile of mHCC as inferred by CIBERSORT also indicated higher levels of Tregs in mHCC versus pHCC primary tumors, which is consistent with reports highlighting CTLA4 as predominantly expressed by Tregs. Given that CTLA4 methylation status has been reported to correlate with ICB response in other cancer types, this could be of clinical importance in the management of recurrent HCC. On the other hand, pHCC-specific methylation changes involve cell projection (GO:0030030), vascularization (GO:1900748) & developmental growth (GO:0048589) (eg. DNAH7 hypomethylation, CYFIP2 hypermethylation). This may explain in part the previously reported larger tumor size and poorer overall survival of pHCC versus mHCC. Further validation is ongoing to draw firm conclusions.

Xiaofan Ding

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