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

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.