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 ¼ 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.

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

“…Referring to this segment feature of cfDNA from plasma or serum, the commercial kits, including the QIAamp Circulating Nucleic Acid Kit (Qiagen) among others were originally designed for the purification of highly fragmented cfDNA from plasma or serum, and capturing DNA fragments longer than 75 bps according to the manufacturer ( 15 ). 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.…”

“…Thus, it may be a better source of tumor markers for detecting biliary tract and other gastrointestinal cancers ( 10 ). In patients with biliary tract cancers (BTCs), bile cfDNA exhibited better sensitivity and specificity in the detection of gene mutations (94.7% and 99.9%, respectively) than plasma cfDNA, indicating its potential advantage in BTC diagnosis ( 11 – 13 ). Furthermore, bile cfDNA fragments are much longer than the highly fragmented plasma cfDNA, which may be due to the extreme physiochemical condition of bile, indicating that bile cfDNA may carry more information about genome aberration ( 12 ).…”

Circulating tumour DNA — looking beyond the blood

“…Emerging sources of cfDNA Beyond the fluids already discussed, several other possible sources of cfDNA are available. cfDNA has been identified in bile from patients with biliary tract cancers and was shown to be superior to plasma ctDNA for the detection of cancer-related somatic mutations 4,139,140 . Uterine lavage fluid has been studied as a source of cfDNA in a cohort of 107 women undergoing hysteroscopy and curettage following post-menopausal bleeding.…”

“…To date, the majority of liquid biopsy research has focused on blood-based biomarkers, including circulating tumour DNA (ctDNA), circulating tumour cells (CTCs) and circulating microRNAs 3 . However, data published over the past decades indicate that these cancer-derived cells and molecules can also be found within non-blood biological fluids, including, among others, cerebrospinal fluid (CSF), urine, pleural fluid and peritoneal fluid [4][5][6][7][8][9][10] . This expanding interest in liquid biopsies from a wider range of sources is supported by the improved availability of ctDNA detection and analysis technologies.…”

Bile cell‑free DNA: a game changer for management of biliary tract cancer?

“… 1 Preliminary studies have suggested a high correspondence between mutational profile in BTC tissue and bile, and the detection of tumor-specific genetic aberrations with utilization of bile cell-free DNA (cfDNA) is under evaluation, although this strategy has not yet been implemented into clinical routine. 2 In ESMO Open , Gou and colleagues report the results of a prospective study carried out in a Chinese institution to assess the role of bile as a liquid biopsy medium in patients with BTC 3 ; interestingly, the authors highlighted that bile cfDNA was superior to plasma cfDNA in the detection of tumor-related alterations.…”