The clinical stakes are considerable. Hepatocellular carcinoma is a leading cause of cancer-related mortality worldwide, and its prognosis depends heavily on whether the tumor is caught while it is still small and potentially curable. Current surveillance for people at high risk, particularly those with cirrhosis or chronic hepatitis B infection, relies on abdominal ultrasonography combined with the blood protein alpha-fetoprotein. Yet these tools have well-documented limitations. Meta-analyses cited in the review show that surveillance imaging and alpha-fetoprotein together miss a substantial fraction of early tumors, and even refined protein markers such as AFP-L3 have struggled to reliably distinguish early-stage disease from benign cirrhotic liver tissue. This diagnostic gap has pushed researchers toward liquid biopsy approaches that interrogate molecular signals released by tumors into the circulation.

Cell-free DNA refers to small fragments of genetic material that circulate in blood plasma, shed by dying cells throughout the body. In people with cancer, a portion of this DNA originates from tumor cells and is known as circulating tumor DNA. What makes methylation analysis particularly attractive is that DNA methylation, the addition of methyl groups to cytosine bases in CpG dinucleotides, is an epigenetic modification that does not alter the underlying genetic sequence but profoundly changes gene activity. Aberrant methylation is a hallmark of hepatocarcinogenesis: hypermethylation of promoter CpG islands silences tumor suppressor genes, while broader patterns of methylation dysregulation reshape the epigenetic landscape of the malignant cell. Because these methylation changes are cancer-specific and occur at defined genomic loci, they can be detected in the small quantity of tumor-derived DNA present in plasma.

The review places particular emphasis on the interplay between hepatitis B virus and the host epigenome. The viral X protein, a multifunctional regulator encoded by HBV, can promote methylation dysregulation through direct interactions with DNA methyltransferases, the enzymes that establish and maintain methylation patterns. Studies summarized in the review show that HBx upregulates DNA methyltransferases DNMT1, DNMT3A and DNMT3B, leading to hypermethylation and silencing of genes such as SOCS-1 and RASSF1A, and that the viral protein can induce epigenetic repression of E-cadherin through its interaction with DNMT3A. The virus genome itself also undergoes methylation during infection, and methylation of the HBV genome has been associated with the development of hepatocellular carcinoma and occult infection. This virus-driven epigenetic rewiring means that methylation signatures in HBV-related liver cancer carry information about both the tumor and the viral history that shaped it.

On the clinical performance side, the review assembles a striking set of results from recent studies. Multi-feature cfDNA models that integrate methylation data with additional fragmentomic features, such as fragment size and end-motif patterns, have achieved sensitivity of 95.42 percent and specificity of 97.67 percent, with an area under the receiver operating characteristic curve of 0.997, for differentiating hepatocellular carcinoma from liver cirrhosis. Critically, these models maintained a sensitivity of 90.0 percent for early-stage disease, the setting in which detection matters most. In specific cohorts, methylation-based approaches complemented alpha-fetoprotein by detecting nearly 75 percent of early malignancies that the protein marker alone missed, and adding methylation data to a combined predictive model raised its AUC from 0.87 to 0.90. Combined mutation-methylation assays, which simultaneously profile genetic mutations and hypermethylation patterns in circulating DNA, have reached validation AUC values as high as 0.93.

Several individual methylation markers have emerged as recurring candidates across studies. The RASSF1A promoter, frequently silenced in hepatocellular carcinoma, has been detected as methylated cell-free DNA in plasma, and a study of patients with hepatitis B-related cirrhosis found that cell-free methylation of RASSF1A and CDKN2AIP helped distinguish malignant from non-malignant disease. Genome-wide methylation profiling of plasma cell-free DNA across the stages of HBV-related hepatocarcinogenesis has identified potential biomarkers for early detection and risk monitoring, and more recent plasma cfDNA methylome profiling has pointed to GJA4 as a candidate biomarker specific to HBV-related disease. Whole-genome bisulfite sequencing approaches have further refined the search for blood-based methylation biomarkers, while digital droplet PCR has been applied to quantify circulating tumor DNA methylation with high sensitivity.

The technical foundations of these assays deserve attention because they explain both the promise and the difficulty of methylation-based liquid biopsy. Detecting a tumor-specific methylation signal requires distinguishing a small tumor-derived fraction from a large background of DNA released by normal cells, particularly in patients with cirrhosis, where inflammation and cell turnover elevate background cfDNA levels. Bisulfite conversion, which chemically converts unmethylated cytosines to uracil while leaving methylated cytosines intact, remains the workhorse of methylation analysis, but it degrades DNA and complicates quantification. Newer approaches, including methylation-aware sequencing and single-molecule assays, aim to preserve more information from each fragment. Computational methods for cell-type decomposition of circulating cell-free DNA, such as the CelFiE framework, help researchers account for the tissue origins of cfDNA and isolate the tumor-specific component of the methylation signal.

The review is careful to note that these impressive numbers come largely from retrospective, case-control studies in specific cohorts, and that the field has not yet demonstrated clinical utility in the way guidelines demand. Prospective validation and direct head-to-head comparisons with existing surveillance strategies, namely ultrasonography and alpha-fetoprotein, are still required before methylation-based liquid biopsy can be recommended for routine screening of high-risk populations. Issues of patient selection bias, standardization of assay protocols, and the cost and accessibility of sequencing-based platforms in the regions where hepatitis B is most prevalent all remain practical obstacles. The authors, writing from Burkina Faso, where chronic hepatitis B infection is endemic, underscore that any diagnostic advance must ultimately be deliverable in the health systems that carry the greatest burden of disease.

Even with those caveats, the trajectory of the field is clear. Methylation markers are stable, cancer-specific, and detectable in plasma at levels that modern assays can measure, and their performance in early-stage disease exceeds what protein biomarkers alone have achieved. As multi-feature models combine methylation with fragmentomics and mutation data, and as prospective trials test these panels against current standards of care, cell-free DNA methylation analysis may move from a research tool to a clinical mainstay in the surveillance of hepatitis B-related hepatocellular carcinoma, offering patients the possibility of catching a deadly cancer while it is still curable.

Subject of Research: Cell-free DNA methylation biomarkers for early diagnosis of hepatitis B virus-related hepatocellular carcinoma

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

Article References: Soubeiga, S. T., Ouattara, A. K., Ilboudo, C., Djigma, W. F., & Simpore, J. (2026). Cell-free DNA methylation markers in the diagnosis of hepatitis B virus-related- hepatocellular carcinoma: a review. Molecular Biology Reports, 53(1), Article 1722. https://doi.org/10.1007/s11033-026-12942-0

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12942-0

Keywords: hepatocellular carcinoma, hepatitis B virus, cell-free DNA, DNA methylation, liquid biopsy, biomarkers, epigenetics, cirrhosis, alpha-fetoprotein, early detection, tumor suppressor genes, HBx protein