Hepatitis B virus pregenomic RNA is associated with malignant phenotypes and postoperative survival in HBV-related intrahepatic cholangiocarcinoma

Abstract Background Chronic hepatitis B virus (HBV) infection contributes to infection-associated liver carcinogenesis. However, the biological and clinical relevance of HBV pregenomic RNA (pgRNA), a marker of residual viral transcriptional activity, in HBV-related intrahepatic cholangiocarcinoma (iCCA) remains insufficiently defined. Methods This single-center retrospective exploratory translational study included 45 patients with HBV-related iCCA who underwent curative resection after long-term nucleos(t)ide analogue therapy and had suppressed preoperative serum HBV DNA. Tissue pgRNA expression was measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), serum pgRNA was quantified by simultaneous amplification and testing (SAT), and spatial localization was assessed by immunofluorescence–fluorescence in situ hybridization (IF-FISH). Stable pgRNA-overexpressing HuCCT1 and RBE models were used to evaluate cell viability, migration, invasion, colony formation and stemness-related protein expression. Associations between serum pgRNA status and postoperative outcomes were examined using Kaplan–Meier, Cox regression and restricted mean survival time analyses. Results Tumor tissues showed lower overall pgRNA expression than paired adjacent non-tumor liver tissues, whereas pgRNA signals were detectable in a subset of CK19-positive tumor epithelial cells. In HuCCT1 and RBE cells, pgRNA overexpression was associated with increased cell viability, migration, invasion, colony formation and upregulation of multiple stemness-related proteins. In the clinical analysis, the serum pgRNA HIGH group had poorer overall survival (adjusted HR = 5.22, 95% CI 1.82–14.95; P = 0.002) and recurrence-free survival (adjusted HR = 3.51, 95% CI 1.26–9.79; P = 0.017) than the BLQ group after adjustment for tumor size. Conclusions HBV pgRNA may be linked to malignant and stemness-related phenotypes in HBV-related iCCA. High preoperative serum pgRNA was associated with poorer postoperative overall and recurrence-free survival in this exploratory cohort. These findings support validation of pgRNA as a candidate infection-related biomarker in larger prospective multicenter studies.