Accurate detection of cholangiocarcinoma in primary sclerosing cholangitis using DNA methylation biomarkers in bile and plasma.

BACKGROUND & AIMS: Current methods for cholangiocarcinoma (CCA) detection in primary sclerosing cholangitis (PSC) lack accuracy, often leading to late, non-curative diagnoses. In this study, we aim at identifying DNA methylation biomarkers in liquid biopsies for earlier and more accurate CCA detection.

METHODS

Reduced representation bisulfite sequencing was used to analyze tissue samples from individuals with CCA (n = 10) and PSC (n = 9), yielding 12 candidate DNA methylation biomarkers. After droplet digital PCR assay development, quality control, and verification in tissue, we selected four biomarkers for evaluation in bile (n = 272) and plasma (n = 128) samples from individuals with PSC and/or CCA collected across three different hospitals.

RESULTS

Four biomarkers demonstrated perfect discrimination (AUC = 1.00) between PSC-associated CCA (PSC-CCA) and PSC alone in tissue samples. In bile, all four biomarkers achieved an AUC >0.9 for distinguishing PSC-CCA (n = 20) from PSC alone (n = 207). A two-biomarker panel (5-42952178 and PRKCB) achieved 95% sensitivity and 90% specificity; the addition of KCNA6 and ZFP82 increased the specificity to 97% while maintaining the sensitivity at 90%. Notably, the two-biomarker bile panel successfully detected all eight CCA cases (100% sensitivity) in samples collected 3-12 months before diagnosis using conventional methods. Performance was lower in plasma, where an alternative two-biomarker panel (KCNA6 and PRKCB) achieved 70% sensitivity and 90% specificity for distinguishing PSC-CCA (n = 17) from PSC alone (n = 101).

CONCLUSIONS

Four DNA methylation biomarkers accurately discriminate CCA from PSC in bile, with lower performance in plasma. Their ability to detect CCA months before clinical diagnosis supports further evaluation in prospective studies to enable earlier, potentially curative intervention in PSC.

IMPACT AND IMPLICATIONS: In this study, we demonstrate that liquid biopsy-based DNA methylation biomarkers offer high sensitivity and specificity for early detection of CCA in individuals with PSC, directly addressing a critical unmet need. Early and accurate detection may enable timely surgical intervention, with the potential to significantly improve survival in this high-risk group. Both bile and plasma samples are accessible via routine clinical care, and implementation of such biomarkers would not introduce additional procedural risk. If validated in prospective studies, these biomarkers could transform standard CCA surveillance in PSC by providing a minimally invasive, reliable tool for longitudinal monitoring and earlier detection and intervention.