Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures

Abstract

ObjectiveDespite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA).DesignA prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay.ResultsAn initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut.ConclusionImplementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.

Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients

“…QIAamp Circulating Nucleic Acid Kit (Qiagen) is widely used to isolate bile cfDNA ( 11 – 13 , 16 ), however, it was found in our previous study and that of Arechederra M et al. that long fragments of cfDNA (> 6,000 bp) were prevalent in bile ( 12 , 17 ), suggesting it was required to establish the extraction method of bile cfDNA considering its fragmentation feature.…”

Evaluating Pancreatic and Biliary Neoplasms with Small Biopsy-Based Next Generation Sequencing (NGS): Doing More with Less

“…They reported that TP53 mutations were found in just 2/23 cases, whereas they did not find any non-synonymous SMAD4 alterations, suggesting that both mutations arise mostly at invasion [ 128 ]. Apart from its value assessing equivocal pancreatic lesions, NGS could be used in the evaluation and management of suspicious biliary strictures, as it has shown higher sensitivity than cytology or elevated serum CA19-9 levels to detect malignancy and an enhanced performance compared with FISH [ 78 , 88 , 89 , 90 , 91 , 92 ].…”

“…NGS was performed on biliary tract brushings, forceps biopsies, and bile, whereas the authors utilized LBC samples or material directly collected for molecular evaluation [ 78 , 88 , 89 , 90 , 91 , 92 ]. Mutations in the KRAS, TP53, SMAD4, and CDKN2A genes were the most common ones detected [ 78 ].…”

“…Two research groups evaluated the potential of bile-based NGS in the evaluation of suspicious biliary strictures. NGS was more sensitive to detect malignancy, compared with the initial pathomorphological evaluation, performed either with FNA or FNB [ 88 ]. Notably, results were highly concordant with the molecular analysis performed in the matched tissue specimens, as 96.2% of the alterations present in the tissues were detected with bile-based NGS [ 89 ].…”

Clinical relevance of biomarkers in cholangiocarcinoma: critical revision and future directions

“…Bile is also emerging as a promising liquid biopsy matrix for CCA diagnosis; a recent study showed that detection of mutations in bile cell-free DNA has a strong sensitivity for early malignancy detection within the biliary tract 40. Identification of differentially methylated markers in bile cell-free DNA has also been recently demonstrated to provide high sensitivity for CCA detection 41 42.…”