Cell-free DNA methylation markers in the diagnosis of hepatitis B virus-related- hepatocellular carcinoma: a review

Hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) remains a major cause of cancer-related mortality worldwide, and its prognosis largely depends on detection at an early, potentially curable stage. However, current surveillance strategies based on ultrasonography and alpha-fetoprotein (AFP) have limited sensitivity, highlighting the need for more accurate and non-invasive biomarkers. This review examines the emerging role of cell-free DNA (cfDNA) methylation analysis as a liquid biopsy approach for the early detection and surveillance of HBV-HCC. Aberrant DNA methylation is a major epigenetic event in hepatocarcinogenesis, contributing to the silencing of tumor suppressor genes, while the HBV X protein can further promote methylation dysregulation through its interactions with DNA methyltransferases. Clinically, specific cfDNA methylation markers and multi-feature panels have demonstrated promising diagnostic performance for HCC detection. More complex multi-feature cfDNA models integrating methylation with additional fragmentomic features have achieved up to 95.42% sensitivity and 97.67% specificity (AUC = 0.997) for differentiating HCC from liver cirrhosis, while maintaining a high sensitivity of 90.0% for early-stage HCC detection. In specific cohorts, methylation-based approaches have complemented AFP, detecting nearly 75% of early malignancies missed by AFP alone and increasing the AUC of combined predictive model from 0.87 to 0.90. Furthermore, combined mutation-methylation assays that concurrently profile genetic mutations and hypermethylation patterns have achieved validation AUC values as high as 0.93. Overall, cfDNA methylation-based approaches exhibit promising diagnostic performance compared with conventional biomarkers in specific cohorts. However, prospective validation and direct head-to-head comparisons with existing surveillance strategies are still required before their clinical utility can be established.