Mutational Analysis of Bile Cell-Free DNA in Primary Sclerosing Cholangitis: A Pilot Study

Abstract

Background: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterised by inflammation and fibrosis of the bile ducts, conferring an increased risk of cholangiocarcinoma (CCA). However, detecting CCA early in PSC patients remains challenging due to the limited sensitivity of conventional diagnostic methods, including imaging or bile duct brush cytology during endoscopic retrograde cholangiopancreatography (ERCP). This study aims to evaluate the potential of bile cell-free DNA (cfDNA) mutational analysis, termed the Bilemut assay, as a tool for CCA detection in PSC patients.

Methods: Sixty-three PSC patients undergoing ERCP due to biliary strictures were prospectively recruited. Bile samples were collected, and cfDNA was extracted and analysed using the Oncomine Pan-Cancer Cell-Free assay. Twenty healthy liver donors were included for comparison. Samples with a mutant allele frequency (MAF) ≥ 0.1% were considered positive. Correlations between mutational status and clinical characteristics were assessed.

Results: cfDNA mutational analysis was successful in all bile samples. Mutations predominantly in KRAS, GNAS, and TP53 were detected in 36.5% (23/63) of PSC patients, compared to 10% (2/20) of healthy donors (p = 0.0269). The clinical characteristics of Bilemut-positive and -negative patients were comparable, though there was a trend towards a lower prevalence of inflammatory bowel disease in the Bilemut-positive group. Among PSC patients diagnosed with CCA during follow-up, 75% were Bilemut-positive, suggesting an association between mutational status and malignancy risk.

Conclusions: Mutational analysis of cfDNA obtained from bile collected from PSC patients undergoing ERCP is feasible. Implementing the Bilemut assay may help identify patients needing closer surveillance and further imaging studies.

Keywords: PSC; bile; cfDNA; cholangiocarcinoma; cholangitis; chronic liver disease; liquid biopsy; mutations.

FIGURE 1

Mutational profile of bile cfDNA from control and PSC patients. (A) The heatmap shows the mutations detected with the Pan‐Cancer panel in bile cfDNA from healthy living liver donors (n = 20) and PSC patients (n = 63). The colour scale represents the percentage of mutational allele frequency (MAF), ranging from 0% to > 2%. The bar at the bottom shows the sum of the MAFs for all the mutations detected in each patient, and patients are ordered from lowest to highest based on this cumulative MAF. # marks the patients diagnosed with CCA among those with detected mutations. (B) The graph represents the time (in months) between PSC diagnosis and bile sample collection, comparing patients in which mutations were detected (Bilemut‐positive) or not (Bilemut‐negative). No significant differences were found between both groups. (C) The histogram shows the number of patients where the Bilemut assay resulted negative or positive in both control and PSC groups. The positivity rate in PSC patients (23/63; 36.5%) was significantly higher than that observed in healthy donor samples (Fisher's exact test, p = 0.027).

FIGURE 2

Comparison of the clinical characteristics of Bilemut‐negative and Bilemut‐positive PSC patients. (A) Biochemical parameters in Bilemut‐negative versus Bilemut‐positive PSC patients, including carbohydrate antigen 19–9 (CA19‐9; U/mL), carcinoembryonic antigen (CEA, ng/mL), alpha‐fetoprotein (AFP, ng/mL), aspartate aminotransferase (AST, U/L), alanine aminotransferase (ALT, U/L), gamma‐glutamyl transferase (GGT, U/L), alkaline phosphatase (ALP, U/L), albumin (ALB, g/dL), bilirubin (Bil, mg/dL), international normalised ratio (INR), white blood cells (WBC, 10³/μL), red blood cells (RBC, 10⁶/μL), haemoglobin (HGB, g/dL), platelets (PLT, 10³/μL), creatinine (Crea, mg/dL), sodium (Na, mmol/L), and potassium (K, mmol/L). Each dot represents an individual patient, with bars indicating the mean ± standard deviation. (B‐D) Distribution of (B) cirrhosis, (C) inflammatory bowel disease (IBD) or (D) liver trasplantation in Bilemut‐negative and Bilemut‐positive PSC patients. Statistical significance was determined using the Mann‐Whitney test (A) or Fisher's exact test (B‐D), with no significant differences found in any of the analyses.

FIGURE 3

Bilemut assay performance for the diagnosis of CCA in PSC patients. (A) Bilemut results (negative or positive) in the four PSC patients with CCA. (B) Chronology of PSC diagnosis, Bilemut result, and cholangiocarcinoma (CCA) detection of the three Bilemut‐positive patients with CCA.