Ultra-low-pass whole-genome sequencing of bile cfDNA in patients with biliary strictures.

Background: Accurate discrimination of malignant versus benign biliary strictures remains clinically challenging. We previously showed that targeted bile cell-free DNA (cfDNA) mutation analysis (Bilemut assay) enables sensitive detection of malignancy, but it does not distinguish cholangiocarcinoma (CCA) from pancreatic ductal adenocarcinoma (PDAC), and its prognostic value remains unclear. In this study, we evaluated ultra-low-pass whole-genome sequencing (ULP-WGS) of bile cfDNA as a potentially cost-effective, technically simple and low-DNA input demanding genomic approach for early tumour detection, tumour-origin discrimination and prognostic stratification. Methods: Bile samples from 95 patients with biliary strictures under radiological suspicion of malignancy, classified as malignant (n=26) or indeterminate (n=69) based on the result of cytology or biopsy at the first endoscopic retrograde cholangiopancreatography (ERCP), were analysed by ULP-WGS. Tumour fraction (TF) and copy-number alterations (CNAs) were inferred using ichorCNA. Tumour mutational burden (TMB) was assessed with the Bilemut panel. Overall survival was analysed by Kaplan-Meier analysis and log-rank test. A supervised classifier based on discriminant analysis of principal components was used to distinguish CCA from PDAC. Results: In matched samples, bile LP-WGS showed higher sensitivity than plasma for tumour detection. Among patients with an initial malignant diagnosis, bile ULP-WGS detected circulating tumour DNA (ctDNA) in 73% of cases. In patients initially classified as indeterminate but later confirmed as malignant, tumour DNA was detected in 51%, anticipating final diagnosis by a median of 38 days; notably, in seven patients, ctDNA was detectable more than 100 days before routine diagnosis, highlighting its potential for very early tumour detection. Specificity in strictures with a final benign outcome was 96%. CNA profiling revealed recurrent chromosomal alterations consistent with tissue-based data, and a 50-region CNA classifier discriminated CCA from PDAC with an accuracy of 0.864. Although neither TF nor TMB alone was associated with overall survival, their combination identified a subgroup with poorer prognosis. Conclusions: Our exploratory study shows that bile ULP-WGS performed at the first ERCP could represent a complementary, scalable and potentially cost-effective genomic strategy for early malignancy detection in biliary strictures, with additional value for tumour-origin classification and, combined with TMB analysis, for identifying patients with poorer prognosis.