Accurate quantification of cell-free Ceruloplasmin mRNA as a biomarker for early detection of hepatocellular carcinoma

Accurate and early detection of hepatocellular carcinoma (HCC) is critical for improving patient outcomes. Current biomarkers like AFP have limited sensitivity, necessitating novel diagnostic markers. A novel semi-nested RT-PCR assay was developed to quantify circulating Ceruloplasmin (CP) mRNA in peripheral blood. This method co-amplifies CP mRNA and an internal control (IC) gene, followed by DNA melting analysis to distinguish and quantify CP mRNA. CP mRNA levels were significantly higher in the HCC group (median: 3.37) compared to both the CLD group (0.24, p = 0.0066) and the HD group (0.17, p < 0.0001). Further analysis using ROC curves highlighted the diagnostic performance of the assay. For differentiating HCC from CLD, the area under the ROC curve (AUC) was 0.704, with 50.98% sensitivity and 95.24% specificity. In comparison to HD, the AUC was 0.812, with 74.51% sensitivity and 80.65% specificity. Against the combined control group (CLD and HD), the AUC was 0.768, with 50.98% sensitivity and 96.15% specificity. Additionally, in 59.1% of HCC cases with AFP levels below 20 ng/mL, CP mRNA levels were elevated, indicating that CP mRNA could help detect a substantial proportion of AFP-negative HCC cases. This study, the first comprehensive clinical investigation of cell-free CP mRNA for HCC diagnosis, demonstrates its potential as a sensitive and specific non-invasive biomarker. Further validation in larger cohorts is needed to confirm its clinical utility.