Bile cell‑free DNA as a novel and powerful liquid biopsy for detecting somatic variants in biliary tract cancer

Abstract

Tissue sampling of biliary tract carcinomas (BTCs) for molecular characterization is challenging. The aim of this study was to investigate the possibility of identifying individual actionable mutations derived from bile cell-free DNA (cfDNA) using targeted deep sequencing. Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in the present study. Using targeted deep sequencing with a panel of 150 tumor-related genes, paired bile cfDNA and tumor DNA were analyzed for mutational variants individually and then compared. The present study, to the best of our knowledge, is the first to reveal that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case for plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. When comparing bile cfDNA and tumor DNA for single nucleotide variation (SNV)/insertion and deletion (Indel), the results using targeted deep sequencing revealed high sensitivity (94.7%) and specificity (99.9%). Additionally, the sensitivity of detecting a copy number variation (CNV) was 75.0%, with a specificity of 98.9%. When comparing two bile extraction methods, including percutaneous transhepatic cholangial drainage and operation, no significant difference in SNV/Indel or CNV detection sensitivity was noted. Moreover, when examining the tumor stage and incidence site, AJCC stage II and the distal bile duct both had significantly decreased CNV detection sensitivities. The present study revealed that targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. These preliminary results may shed light on bile cfDNA as a promising liquid biopsy for BTC patients.

Bile-Based Cell-Free DNA Analysis Is a Reliable Diagnostic Tool in Pancreatobiliary Cancer

“…Brush cytology and forceps biopsies displayed an overall sensitivity of 77.1% for the diagnosis of suspected malignant biliary strictures when obtained by ERC(P) [ 29 ]. Our results are in line with the previous studies, which reported high sensitivity and specificity of bile cfDNA analysis in gallbladder cancer and cholangiocarcinoma [ 30 , 31 ] and showed its value as ancillary method in the diagnosis of malignant biliary strictures [ 32 ]. This is the first study reporting on the usefulness of bile as a source of liquid biopsy in PDAC.…”

Comparison of methods for the isolation of cell-free DNA from cell culture supernatant

“…Similarly, a recent study demonstrated the presence of larger cfDNA fragments (1000–6000 bp) in bile—a body fluid that does not likely possess the same DNA degradation qualities of plasma. 41 Alternatively, intact larger cfDNA fragments may be missed due to the use of DNA extraction methods that are biased toward the isolation of small cfDNA fragments. It is likely that the resolution of such an experimental problem may only be achievable through a method or combination of methods that allow the complete recovery and high resolution differentiation between all cfDNA populations (e.g.…”

Circulating Tumor DNA in Biliary Tract Cancer: Current Evidence and Future Perspectives

“…Lastly, the option to use the bile as source for DNA sequencing in BTC has been recently investigated and deserves to be mentioned, since bile is another component of liquid biopsy. A recent study by Shen et al from 10 BTC patients (including 4 cases of GBC) suggested that bile cfDNA could consist of long fragments, with a high correspondence between molecular features detected in bile and tissue sampling (129). Studies on larger cohorts of patients are needed to confirm the above results and to further assess the role of bile as source of cfDNA.…”