Circulating Cell-Free DNA Fragment Size as a Potential Biomarker for Hepatocellular Carcinoma: An Exploratory Proof-of-Concept Study

Background: Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancers worldwide. Alpha-fetoprotein (AFP), a widely used and accessible tumoral marker, has limited performance in the early detection of HCC among high-risk populations. This study aims to evaluate the potential added value of ccfDNA (circulating cell-free DNA), alone or in combination with AFP, using accessible, feasible ccfDNA analysis. Methods: A prospective cohort of 125 patients with chronic liver disease was analyzed. Patients with incomplete clinical or laboratory data and patients without cirrhosis were excluded from the final analysis. Nonparametric tests, logistic regression and ROC curve analysis were performed. ccfDNA concentration was assessed by fluorimetry and fragment size was measured using on-chip electrophoresis. Results: ccfDNA fragment size was significantly lower in the cirrhosis-HCC subgroup compared to the cirrhosis-only subgroup (p< 0.001). While AFP remains an independent predictor of HCC among cirrhosis patients, ccfDNA fragment size did not prove to be an independent predictor in this cohort. However, AUROC (Area Under the Receiver Operating Characteristic Curve) analysis revealed that a combined model of AFP and ccfDNA fragment size showed modest additional discriminatory value between the two groups, compared to either ccfDNA peak size or AFP alone. Conclusions: ccfDNA fragment size may provide modest complementary value within multimarker panels. However, the marker needs further validation in a larger cohort, and adequate assessment of potential confounders such as severity of liver dysfunction and age.