Cholangiocarcinoma (CCA), a highly aggressive hepatobiliary malignancy, faces significant diagnostic challenges in early stages due to nonspecific clinical manifestations. Although plasma cell-free DNA (cfDNA) analysis via next-generation sequencing (NGS) is widely used for noninvasive cancer detection, its sensitivity in CCA remains suboptimal owing to the low tumor DNA abundance. This study aims to compare bile and plasma as sources for liquid biopsy in CCA genomic profiling, focusing on analytical accuracy and biomolecular stability.

Ten patients with resectable CCA were prospectively enrolled in this study. DNA extracted from matched plasma, bile, and tumor tissue underwent NGS for somatic mutation, microsatellite stability (MSI), and tumor mutation burden (TMB) analysis.

The concentration of cfDNA in bile was significantly higher than in plasma. Somatic mutations were detected in 100% (10/10) of tumor tissue DNA, 70% (7/10) of bile cfDNA, and 10% (1/10) of plasma cfDNA. Compared to plasma cfDNA, bile cfDNA exhibited higher TMB (median TMB 1.0 vs. 0; p < 0.05) and greater maximum allele fractions (median maxAF 0.057 vs. 0; p < 0.001). While tumor stage significantly influenced plasma cfDNA detection rates, bile cfDNA maintained high detection sensitivity independent of clinical variables. Among CCA patients, bile cfDNA mutation positivity demonstrated sensitivity comparable to CA19-9 serology.

Bile cfDNA outperforms plasma cfDNA in detecting somatic mutations and retains high sensitivity independent of tumor stage, positioning it as a promising tool for early CCA diagnosis and molecular characterization.