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

Abstract

Objective: Despite 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).

Design: A 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.

Results: An 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.

Conclusion: Implementation 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.

Keywords: biliary strictures; cholangiocarcinoma; diagnostic and therapeutic endoscopy; mutation screening; pancreatic tumours.

Figure 1

Characteristics of the patients included in the study and their diagnosis. (A) Demographic description, location of biliary strictures and serum carbohydrate antigen 19–9 (CA19-9) levels of patients included in this study at the time of initial diagnosis. (B) Flow chart indicating the initial and final clinical diagnosis of the patients.

Figure 2

Analysis of bile and plasma cell-free DNA (cfDNA) and comparison of Pan-Cancer and Oncomine Comprehensive Assay (OCA) panels’ performance in bile cfDNA from patients with cholangiocarcinoma (CCA). (A). Representative electropherograms showing the size distribution of cfDNA obtained from human bile and plasma. (B) Representative images of agarose gels showing PCR-amplified fragments of TP53 gene of high and low molecular weight from four independent samples of bile or plasma cfDNA. (C) Heat maps show the mutated genes detected by both panels in the same bile samples, and the mutations detected by the Pan-Cancer panel in plasma cfDNA. Asterisks indicate specific mutations that are not included in the Pan-Cancer panel. ‘nt’: not tested. BP, base pairs; FU, fluorescence units.

Figure 3

Mutational profile of bile cell-free DNA (cfDNA) and paired tissue DNA samples from patients with malignant stenoses. The heatmap in upper panel shows the mutations detected with the Pan-Cancer panel in bile cfDNA, and the heatmap in the lower panel shows mutations identified with the Oncomine Comprehensive Assay (OCA) panel in the available paired tissues. Asterisks indicate specific mutations that are not included in the Pan-Cancer panel. Diagonal lines indicate the detection of two different mutations in the corresponding gene. The initial diagnosis (Dx), Bilemut diagnosis and final clinical diagnosis, as well as the type of tumour, are indicated. CCA, cholangiocarcinoma; PDAC, pancreatic ductal adenocarcinoma.

Figure 4

Diagnostic performance of the Bilemut assay in patients with an initial diagnosis of benign stenosis. (A) Schematic representation of the initial clinical diagnosis, the Bilemut assay diagnosis and the final clinical diagnosis of patients. The four Bilemut false-positive patients are encircled. (B) Heatmap showing the mutational profile of bile cell-free DNA (cfDNA), Bilemut assay, at the time of initial diagnosis. Diagonal lines indicate the detection of two different mutations in the corresponding gene. The initial diagnosis (Dx), Bilemut diagnosis, final diagnosis and extended follow-up diagnosis are indicated. The type of tumour diagnosed (pancreatic ductal adenocarcinoma (PDAC), cholangiocarcinoma (CCA) or gall bladder (GB)) is also indicated. (C). Chronology of malignancy detection during follow-up of patients with an initial diagnosis of benign stenosis. ERCP, endoscopic retrograde cholangiopancreatography.

Figure 5

Diagnostic performance of the Bilemut assay in patients with an initial diagnosis of indeterminate stenosis. (A) Schematic representation of the initial clinical diagnosis, the Bilemut assay diagnosis and the final clinical diagnosis of patients. (B) Heatmap showing the mutational profile of bile cell-free DNA, Bilemut assay, at the time of initial diagnosis. The initial diagnosis (Dx), Bilemut diagnosis, final diagnosis and extended follow-up diagnosis are indicated. The type of tumour diagnosed (pancreatic ductal adenocarcinoma (PDAC) or cholangiocarcinoma (CCA)) is also indicated. (C) Chronology of malignancy detection during follow-up of patients with an initial diagnosis of indeterminate stenosis. ERCP, endoscopic retrograde cholangiopancreatography.

Figure 6

(A) Implementation of the Bilemut assay in an algorithm for the management of patients with biliary stenosis. The four steps in which Bilemut could be applied are indicated by grey boxes. See text for details. This algorithm is based on the National Comprehensive Cancer Network guidelines. (B) Summary of the advantages and limitations of the Bilemut assay. ERCP, endoscopic retrograde cholangiopancreatography; NGS, next-generation sequencing.