Bile cell-free DNA as a powerful liquid biopsy of the somatic variants in biliary tract cancer

Purpose: Biliary tract cancers (BTCs) are less common, but rapidly lethal tumors. Despite recent improvements in multimodal therapy, the 5-year survival rate is less than 10% in patients with advanced or metastatic BTC. BTC patients lack personalized treatment options, partly because tissue samplings are often inadequate for molecular characterization. Thus, this study examined the use of bile cell-free DNA (cfDNA), which is released by tumor and normal cells, as a potential liquid biopsy able to provide genomic information for the whole tumor. Materials and Methods: Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in this study August 2017 to March 2018. To investigate the use of bile cfDNA as a liquid biopsy in BTC patients, targeted deep sequencing, with a panel of 150 tumor-related genes, was used to analyze paired bile cfDNA and tumor DNA for mutational variants individually and then compared. The sensitivites and specificities of detecting mutations including SNV/Indel and copy number variation (CNV) in bile cfDNA were analyzed. Results: This study is the first to show that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case in plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. The mutational profiles of bile cfDNA showed that of the SNV/Indel mutations, the highest variation was seen in TP53, followed by KRAS, NOTCH1, NOTCH2 and KMT2A. Among the CNV mutations, low recurrent amplified genes, such as CCNE1, ERBB2, CDKN1B, ZNF217, and CDK6, were identified; with CDKN2A shown to have a deep deletion. Among these genes, NOTCH1, NOTCH2, ERBB2 and CDK6 are of interest as potential drug targets. When comparing bile cfDNA and tumor SNV/Indel detection results using targeted deep sequencing, a high sensitivity (94.7%) and specificity (99.9%) were obtained. 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. Conclusions: This study shows that long DNA fragments are prevalent in bile cfDNA samples and targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. This is the first time that a mutational landscape, including SNVs, Indels and CNVs, has been obtained for bile cfDNA from BTC patients. These preliminary results maybe shed light on bile cfDNA as a promising liquid biopsy for BTC patients. Citation Format: Ningjia Shen, Dadong Zhang, Lei Yin, Yinghe Qiu, Jian Liu, Wenlong Yu, Xiaohui Fu, Bin Zhu, Xiaoya Xu, Anqi Duan, Zishuo Chen, Xiang Wang, Xinkai Cao, Teng Zhao, Zisong Zhou, Lianghe Yu, Hao Qin, Zheng Fang, Jing-Yu Li, Yuanjin Liu, Lei Xiong, Bo Yuan, Fugen Li, Yongjie Zhang. Bile cell-free DNA as a powerful liquid biopsy of the somatic variants in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2292.