Proteomic Analysis of Serum Extracellular Vesicles Revealed Fibulin‐3 as a Novel Predictive Biomarker for Hepatocellular Carcinoma in Patients With Chronic Hepatitis B

BACKGROUND AND AIM: Although nucleos(t)ide analog (NUC) therapy reduces hepatocellular carcinoma (HCC) incidence in chronic hepatitis B (CHB) patients, HCC can develop despite viral suppression. Identifying predictive biomarkers of HCC during NUC therapy remains clinically important. This study aimed to identify novel biomarkers by proteomic analysis of serum extracellular vesicle (EV) proteins in CHB patients receiving NUC therapy. METHODS: We enrolled 242 CHB patients treated with NUCs for over one year, with serum HBV DNA < 3.0 log IU/mL, no prior HCC, and available stored serum samples. Proteomic analysis of serum EV proteins was carried out to identify candidate biomarkers, followed by validation of their serum levels using enzyme-linked immunosorbent assay (ELISA). RESULTS: The median age was 55 years; 144 were male. During a median follow-up of 10.1 years, 44 patients (18.2%) developed HCC. Five candidate EV proteins were identified: fibulin-3, VCAM-1, fibronectin, vWF, and FGFBP2. In univariate Cox analysis of serum levels of candidate EV proteins measured by ELISA, fibulin-3 and VCAM-1 were identified as significant predictive factors. In multivariate analysis, male sex, GGT, AFP, fibulin-3, and the FIB-4 index were independent predictors, whereas VCAM-1 was not. A cutoff value for fibulin-3 was set at 10.8 μg/mL. HCC incidence was significantly higher in patients with fibulin-3 ≥ 10.8 μg/mL and in those with ≥ 3 of five identified risk factors. The 10-year cumulative HCC incidence was 0% versus 37.2% in patients with 0 versus ≥ 3 risk factors. CONCLUSION: Serum fibulin-3 is a novel biomarker predictive of HCC in CHB patients undergoing NUC therapy.