Fig. 5

Confirmation in an independent cohort showed a limited contribution of tissue-derived DMMs in detecting early-stage HCC in cfDNA (validation cohort). A qMSP-based methylation levels relative to the ACTB reference gene were illustrated in 89 cfDNA samples (24 late-stage HCC, 37 early-stage HCC, and 28 cirrhosis). All 5 DMMs exhibited hypermethylation in cfDNA of late-stage HCC compared to cirrhosis, and TSPYL5, NRIP3, and SPAG6 also showed statistical significance between early-stage HCC and cirrhosis (Mann–Whitney U test; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). B Using the model from the training cohort, the performance of the DMMs, ASAP/GAAD, and the combination of DMMs and ASAP/GAAD was evaluated for HCC detection. The p-value for the ROC curve was compared between the combined model and the ASAP/GAAD score. C, D Performance of the 5 DMMs in early-stage patients with small tumor size (< 3 cm) compared to cirrhosis, only TSPYL5 and SPAG6 showed statistical significance between early-stage HCC with small tumor size and cirrhosis (Mann–Whitney U test; ns, not significant; * p < 0.05)