Cell-free DNA from bile outperformed plasma as a potential alternative to tissue biopsy in biliary tract cancer

Abstract

Background: Biliary tract cancers (BTCs) are rare and highly heterogenous malignant neoplasms. Because obtaining BTC tissues is challenging, the purpose of this study was to explore the potential roles of bile as a liquid biopsy medium in patients with BTC.

Patients and methods: Sixty-nine consecutive patients with suspected BTC were prospectively enrolled in this study. Capture-based targeted sequencing was performed on tumor tissues, whole blood cells, plasma, and bile samples using a large panel consisting of 520 cancer-related genes.

Results: Of the 28 patients enrolled in this cohort, tumor tissues were available in eight patients, and plasma and bile were available in 28 patients. Somatic mutations were detected in 100% (8/8), 71.4% (20/28), and 53.6% (15/28) of samples comprising tumor tissue DNA, bile cell-free DNA (cfDNA), and plasma cfDNA, respectively. Bile cfDNA showed a significantly higher maximum allele frequency than plasma cfDNA (P = 0.0032). There were 56.2% of somatic single-nucleotide variant (SNVs)/insertions and deletions (indels) shared between bile and plasma cfDNA. When considering the genetic profiles of tumor tissues as the gold standard, the by-variant sensitivity and positive predictive value for SNVs/indels in bile cfDNA positive for somatic mutations were both 95.5%. The overall concordance for SNVs/indels in bile was significantly higher than that in plasma (99.1% versus 78.3%, P < 0.0001). Moreover, the sensitivity of CA 19-9 combined with bile cfDNA achieved 96.4% in BTC diagnosis.

Conclusion: We demonstrated that bile cfDNA was superior to plasma cfDNA in the detection of tumor-related genomic alterations. Bile cfDNA as a minimally invasive liquid biopsy medium might be a supplemental approach to confirm BTC diagnosis.

Keywords: DNA; bile; biliary tract neoplasms; cell-free nucleic acids; liquid biopsy; sequence analysis.

Figure 1

Distribution of somatic mutations in plasma and bile samples. (A) Heatmap indicating the mutations detected in bile and matched plasma samples. Dark green indicates mutations detected from both sources, maroon indicates mutations that were present only in the plasma samples, and dark blue indicates mutations present only in the bile samples. (B) Venn diagram showing the overlap of genetic mutations in plasma and bile samples obtained from 28 patients with BTC. BTC, biliary tract cancer; CAV, carcinoma of ampullar of Vater; dCCA, distal cholangiocarcinoma; GBC, gallbladder cancer; iCCA, intrahepatic cholangiocarcinoma; pCCA, perihilar cholangiocarcinoma; Pla, plasma.

Figure 1

Distribution of somatic mutations in plasma and bile samples. (A) Heatmap indicating the mutations detected in bile and matched plasma samples. Dark green indicates mutations detected from both sources, maroon indicates mutations that were present only in the plasma samples, and dark blue indicates mutations present only in the bile samples. (B) Venn diagram showing the overlap of genetic mutations in plasma and bile samples obtained from 28 patients with BTC. BTC, biliary tract cancer; CAV, carcinoma of ampullar of Vater; dCCA, distal cholangiocarcinoma; GBC, gallbladder cancer; iCCA, intrahepatic cholangiocarcinoma; pCCA, perihilar cholangiocarcinoma; Pla, plasma.

Figure 2

The difference in (A) somatic mutation detection rate, (B) maxAF, and (C) TMB between bile cfDNA and plasma cfDNA samples. cfDNA, cell-free DNA; maxAF, maximum allele fractions; Pla, plasma; Tis, tumor tissue; TMB, tumor mutation burden.

Figure 3

The distribution of somatic mutations detected in eight patients with BTC who had available tumor tissue, plasma, and bile samples. (A) The distribution of matched, bile-specific, and tissue-specific mutations in eight patients. (B) The distribution of matched, plasma-specific, and tissue-specific mutations in eight patients. BTC, biliary tract cancer; cfDNA, cell-free DNA; Pla, plasma; Tis, tumor tissue.

Figure 4

Somatic mutations identified in bile, plasma, and tissue samples. (A) Heatmap showing somatic mutations identified in bile and matched tissue samples. Dark green indicates mutations identified in both sources, olive green indicates mutations present only in the tissue samples, and dark blue indicates mutations present only in the bile samples. (B) The by-variant sensitivity and PPV of utilizing capture-based targeted sequencing to identify SNVs/indels in bile cfDNA were calculated, with tissue samples used as references. (C) Heatmap displaying somatic mutations identified in plasma and matched tissue samples. Dark green indicates mutations identified in both sources, olive green indicates mutations present only in the tissue samples, and maroon indicates mutations present only in the plasma samples. (D) The by-variant sensitivity and PPV of utilizing capture-based targeted sequencing to identify SNVs/indels in plasma cfDNA were calculated, with tissue samples used as references. CAV, carcinoma of ampullar of Vater; cfDNA, cell-free DNA; dCCA, distal cholangiocarcinoma; iCCA, intrahepatic cholangiocarcinoma; indels, insertions and deletions; pCCA, perihilar cholangiocarcinoma; Pla, plasma; PPV, positive predictive value; SNVs, single-nucleotide variants; Tis, tumor tissue.

Figure 4

Somatic mutations identified in bile, plasma, and tissue samples. (A) Heatmap showing somatic mutations identified in bile and matched tissue samples. Dark green indicates mutations identified in both sources, olive green indicates mutations present only in the tissue samples, and dark blue indicates mutations present only in the bile samples. (B) The by-variant sensitivity and PPV of utilizing capture-based targeted sequencing to identify SNVs/indels in bile cfDNA were calculated, with tissue samples used as references. (C) Heatmap displaying somatic mutations identified in plasma and matched tissue samples. Dark green indicates mutations identified in both sources, olive green indicates mutations present only in the tissue samples, and maroon indicates mutations present only in the plasma samples. (D) The by-variant sensitivity and PPV of utilizing capture-based targeted sequencing to identify SNVs/indels in plasma cfDNA were calculated, with tissue samples used as references. CAV, carcinoma of ampullar of Vater; cfDNA, cell-free DNA; dCCA, distal cholangiocarcinoma; iCCA, intrahepatic cholangiocarcinoma; indels, insertions and deletions; pCCA, perihilar cholangiocarcinoma; Pla, plasma; PPV, positive predictive value; SNVs, single-nucleotide variants; Tis, tumor tissue.

Figure 4

Somatic mutations identified in bile, plasma, and tissue samples. (A) Heatmap showing somatic mutations identified in bile and matched tissue samples. Dark green indicates mutations identified in both sources, olive green indicates mutations present only in the tissue samples, and dark blue indicates mutations present only in the bile samples. (B) The by-variant sensitivity and PPV of utilizing capture-based targeted sequencing to identify SNVs/indels in bile cfDNA were calculated, with tissue samples used as references. (C) Heatmap displaying somatic mutations identified in plasma and matched tissue samples. Dark green indicates mutations identified in both sources, olive green indicates mutations present only in the tissue samples, and maroon indicates mutations present only in the plasma samples. (D) The by-variant sensitivity and PPV of utilizing capture-based targeted sequencing to identify SNVs/indels in plasma cfDNA were calculated, with tissue samples used as references. CAV, carcinoma of ampullar of Vater; cfDNA, cell-free DNA; dCCA, distal cholangiocarcinoma; iCCA, intrahepatic cholangiocarcinoma; indels, insertions and deletions; pCCA, perihilar cholangiocarcinoma; Pla, plasma; PPV, positive predictive value; SNVs, single-nucleotide variants; Tis, tumor tissue.

Figure 5

The performance of bile cfDNA in BTC diagnosis. BTC, biliary tract cancer; CA 19-9, cancer antigen 19-9; CEA, carcinoembryonic antigen; cfDNA, cell-free DNA.