Genome-wide 5-hydroxymethylcytosine in circulating cell-free DNA reflects DNA methylation dynamics for non-invasive detection of early hepatocellular carcinoma.

BACKGROUND

Early hepatocellular carcinoma (HCC) remains difficult to diagnose because of its insidious and asymptomatic onset. Liquid biopsy provides a promising non-invasive approach for early tumor detection. 5-Hydroxymethylcytosine (5-hmC), the oxidation product of 5-methylcytosine (5-mC), has emerged as an informative epigenetic mark reflecting tumor-associated alterations during hepatocarcinogenesis. This study aimed to identify and validate a circulating cell-free DNA (cfDNA) 5-hmC-based signature for the early detection of HCC and to evaluate its diagnostic and prognostic potential.

METHODS

Using an XGBoost-based ensemble learning framework, we identified cfDNA 5-hmC markers that defined an early HCC 5-hmC signature. The signature was assessed in training and validation cohorts using receiver operating characteristic (ROC) analysis, with the area under the curve (AUC) as the primary performance metric.

RESULTS

We defined a 19-gene cfDNA 5-hmC signature (eHMS), which showed high diagnostic accuracy for early HCC across multiple precancerous states, with AUCs of 92.3% (healthy controls vs. early HCC), 82.5% [benign liver lesions (BLL) vs. early HCC], 78.8% [chronic hepatitis B (CHB) vs. early HCC], 81.3% (liver cirrhosis vs. early HCC), and 89.4% when pooling all non-HCC, further validated in an external cfDNA cohort (AUC =93.5%). When traced to tissue 5-mC, the 5-mC-related eHMS (MeHMS) score distinguished tumors from adjacent tissues with an AUC of 99.9% and showed improved discriminatory performance compared with alpha-fetoprotein (AFP, AUC =71.5%). Moreover, the signature may reflect early metabolic reprogramming and provide prognostic value by stratifying patients for survival and disease progression.

CONCLUSIONS

Our findings indicate that the eHMS represents a promising biomarker for liquid biopsy in early HCC surveillance.