A Prospective Multicenter Observational Study to Evaluate Genomic Profiling of Bile-Derived Circulating Tumor DNA (Bile ctDNA) and Its Concordance With Tumor Tissue DNA in Patients With Advanced Biliary Tract Cancer (BTC)

Biliary tract cancer (BTC) is a heterogeneous and aggressive malignancy with poor prognosis, especially in advanced or metastatic stages where surgical resection is not feasible. The current standard first-line therapy with gemcitabine and cisplatin, now combined with immune checkpoint inhibitors, still provides limited long-term survival. Targeted therapies - including FGFR inhibitors for FGFR2 fusions, IDH1 inhibitors, and HER2-directed agents - have improved outcomes in molecularly selected subsets of patients. However, tumor tissue acquisition remains challenging in BTC due to the anatomically inaccessible location of many tumors and frequently insufficient tumor content, limiting the ability to perform comprehensive genomic profiling in a substantial proportion of patients.

Circulating tumor DNA (ctDNA) has emerged as a promising, minimally invasive biomarker for molecular profiling in BTC. While plasma-based ctDNA has shown acceptable concordance with tissue-based next-generation sequencing (NGS) in prior studies, its sensitivity is reduced in patients with locally advanced disease due to lower circulating DNA concentrations. Because bile is in direct contact with biliary tumors, it may harbor tumor-derived DNA at substantially higher concentrations and variant allele frequencies than plasma, suggesting the potential for improved sensitivity and reliability in mutation detection. Preliminary data support higher bile DNA yield and higher concordance with tumor tissue NGS compared with plasma ctDNA, and bile ctDNA alterations have also shown associations with survival outcomes. However, evidence supporting the clinical utility of bile ctDNA remains limited, and no large-scale prospective study has systematically evaluated its concordance with tumor tissue genomic profiling.

This prospective, multicenter, observational study will enroll 100 Korean patients with advanced or metastatic BTC from 4 hospitals, using a competitive enrollment strategy with a target of approximately 50% intrahepatic cholangiocarcinoma. Eligible patients provide a single blood sample (approximately 20 mL) and a single bile sample (approximately 20 mL, collected as residual fluid during clinically indicated biliary drainage via ERCP or PTBD) prior to first-line systemic anti-cancer therapy; no additional invasive procedure is performed solely for research purposes. Collected samples are sent to an external laboratory for ctDNA extraction and NGS-based genomic analysis.

The primary objective is to evaluate the concordance of genetic variants between bile ctDNA and tumor tissue DNA in patients with advanced BTC. Secondary objectives include:

• Assessing the frequency of actionable genomic alterations (FGFR2 fusion, IDH1 mutation, HER2 amplification) detected in bile ctDNA
• Evaluating the concordance of genetic variants between bile ctDNA and plasma ctDNA
• Comparing mutation detection rates among bile ctDNA, plasma ctDNA, and tumor tissue DNA Exploratory analyses will examine the association between bile ctDNA maximum variant allele frequency (max VAF) and tumor burden/clinical prognosis, characterize additional genetic variants uniquely detected in bile ctDNA, and explore potential therapeutic targets identified through bile ctDNA-based genomic analysis.