Early diagnosis is crucial for all cancers, especially HCC, due to its resistance to systemic therapy [4]. Curative HCC treatments, such as hepatectomy and radiofrequency ablation (RFA), are only feasible in the early stages. However, even with curative treatment, 5-year recurrence rates remain high. Early diagnostic tools are essential not only for high-risk individuals to detect HCC early but also for post-treatment surveillance in all HCC patients, regardless of treatment methods, including curative treatment, systemic therapy, targeted therapy, or immunotherapy [19,20,21,22].

Beyond conventional serum protein markers, considerable research focuses on identifying more effective recurrence predictors. Several factors have been reported, including larger tumor size, multiple tumors, microvascular invasion, absence of a tumor capsule, and poor tumor differentiation [23]. Patient-related risks include liver cirrhosis, hepatic dysfunction (Child–Pugh, MELD score, or Albumin-Bilirubin scores), high bilirubin, and systemic inflammation markers like the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and neutrophil-to-eosinophil ratio (NER) [24]. The results of our study may imply the potential of methylation markers for predicting HCC treatment outcomes.

In recent decades, the application of advanced epigenomic technologies has been adopted for the discovery of novel biomarkers for HCC diagnosis [25]. DNA methylation, a key mechanism for regulating gene expression, plays a crucial role in various physiological events, including embryonic development, X-chromosome inactivation, imprinting, and suppression of parasitic DNA sequences [26]. Reasonably, aberrant DNA methylation can lead to various human diseases, including cancers. Recent evidence suggests that DNA methylation is associated with the repression of not only tumor suppressor genes but also tumor suppressor microRNAs (miRNAs) in many cancer cells. In the present study, we identified six genes and two miRNAs, including APC, COX2, RASSF1A, RGS10, ST8SIA6, VIM, miR-129-2, and miR-203, as methylation markers for HCC early diagnosis according to our previous work [16,17,18].

RASSF1A, a key cell cycle regulator, is often inactivated in various cancers, including HCC. This tumor suppressor plays a crucial role in maintaining genomic stability and regulating the cell cycle [27]. Hypermethylation of RASSF1A has been observed in the serum of 93% of HCC patients, 58% of HBV carriers, and 8% of healthy individuals [28]. APC also found to be hypermethylated in a significant percentage of HCC plasma samples, plays a vital role in cell cycle regulation, apoptosis, and cell migration [29]. It interacts with β-catenin [30], promoting its degradation and inhibiting the WNT signaling pathway, a crucial pathway in HCC development. Vimentin, a type III intermediate filament protein, has been associated with increased metastatic potential and poor survival rates in various cancers due to its expression in tumor tissues. It plays a significant role in managing the metastatic process, including epithelial-mesenchymal transition (EMT), invasion, and migration [31]. The methylation of VIM promoters occurs at a significantly higher rate in HCC with frequencies of 61.67%, which was considerably higher than those observed in 24.14% of liver cirrhosis, 13.64% of chronic hepatitis B patients, and 12% of healthy controls [32]. RGS10, a member of the Regulator of G-Protein Signaling (RGS) proteins, plays a significant role in cellular regulation. Changes in RGS10 expression have been linked to various diseases, including cancers [33]. ST8SIA6, a member of the six sialyltransferases family, is known to create ligands for Siglec-E, which modulates immune responses to tumors [34]. The upregulation of lncRNA ST8SIA6-AS1 has been linked to enhanced growth, movement, invasion, and apoptosis resistance in HCC [35,36,37]. However, the role of ST8SIA6 in the development of HCC remains unclear.

Our previous studies have shown that miR-203 and miR-129-2 have higher methylation levels in HCC compared to normal controls, suggesting their role as tumor suppressors [12, 17]. MiR-203 targets ABL1 and BCR-ABL1, an oncogenic fusion gene. When miR-203 is silenced, it activates the BCR-ABL1 gene, leading to increased tumor cell growth [38]. This silence is common in various cancers, including oral cancer and HCC [12, 39]. MiR-203 also targets Bmi-1, a component of a histone modifier complex, and its expression can trigger apoptosis and inhibit cell growth [40]. MiR-129-2 directly targets SOX4, a transcription factor involved in the Wnt pathway, which is crucial in HCC development. SOX4 can bind to TCF/LEF or β-catenin, stabilizing the β-catenin protein [41, 42]. These findings highlight the significant roles of miR-203 and miR-129-2 in cancer development and their potential as therapeutic targets.

In summary, this study investigated a novel, non-invasive liquid biopsy method using methylated genes and miRNAs in cell-free DNA for HCC diagnosis. The MPM-8G model exhibited superior diagnostic performance compared to AFP alone, with further improvement when combined with AFP, demonstrating increased AUC, sensitivity, and specificity. Importantly, the combined MPM-8G and AFP approach shows promise for early HCC diagnosis, which is a critical factor for improved patient outcomes.

Despite these strengths, this study has several limitations. First, while the MPM-8G model shows promising results, the sample size is limited. Larger, more diverse cohorts are needed to validate these results in a multi-center study design. Second, the study focuses on methylation markers and AFP. While the combination improves diagnostic accuracy, other potential biomarkers or clinical factors were not explored, which might further enhance diagnostic performance. Finally, the study used a retrospective design, which can introduce biases due to incomplete data or patient selection. A prospective study design would be more robust.