Cancer related gene alterations can be detected with next-generation sequencing analysis of bile in diffusely infiltrating type cholangiocarcinoma

• Bile samples have enough quality and quantity of DNA to secure NGS analysis.
• Genome-wide association study can be performed with bile samples especially in difficult condition of obtaining tumor tissue.
• Somatic mutations and damaging SNPs of diffusely infiltrating type CC were different from those of mass-forming type CC.
• Genomic analysis of bile in CC could be used for accurate diagnosis, pathogenesis study, and targeted therapy in the future.

Genome-wide association study in diffusely infiltrating type cholangiocarcinoma (CC) can be limited due to the difficulty of obtaining tumor tissue. We aimed to evaluate the genomic alterations of diffusely infiltrating type CC using next-generation sequencing (NGS) of bile and to compare the variations with those of mass-forming type CC. A total of 24 bile samples obtained during endoscopic retrograde cholangiopancreatography (ERCP) and 17 surgically obtained tumor tissue samples were evaluated. Buffy coat and normal tissue samples were used as controls for a somatic mutation analysis. After extraction of genomic DNA, NGS analysis was performed for 48 cancer related genes. There were 27 men and 14 women with a mean age of 65.0 ± 11.8 years. The amount of extracted genomic DNA from 3 cm3 of bile was 66.0 ± 84.7 μg and revealed a high depth of sequencing coverage. All of the patients had genomic variations, with an average number of 19.4 ± 2.8 and 22.3 ± 3.3 alterations per patient from the bile and tumor tissue, respectively. After filtering process, damaging SNPs (8 sites for each type of CC) were predicted by analyzing tools, and their target genes showed relevant differences between the diffusely infiltrating and mass-forming type CC. Finally, in somatic mutation analysis, tumor-normal paired 14 tissue and 6 bile samples were analyzed, genomic alterations of EGFR, FGFR1, ABL1, PIK3CA, and CDKN2A gene were seen in the diffusely infiltrating type CC, and TP53, KRAS, APC, GNA11, ERBB4, ATM, SMAD4, BRAF, and IDH1 were altered in the mass-forming type CC group. STK11, GNAQ, RB1, KDR, and SMO genes were revealed in both groups. The NGS analysis was feasible with bile sample and diffusely infiltrating type CC revealed genetic differences compared with mass-forming type CC. Genome-wide association study could be performed using bile sample in the patients with CC undergoing ERCP and a different genetic approach for accurate diagnosis, pathogenesis study, and targeted therapy will be needed in diffusely infiltrating type CC.