PMC:6440663 JSONTXT 7 Projects

Somatic Mutations from Whole Exome Sequencing Analysis of the Patients with Biliary Tract Cancer Abstract Biliary tract cancer (BTC) is a rare cancer and is associated with a poor prognosis. To understand the genetic characteristics of BTC, we analyzed whole-exome sequencing data and identified somatic mutations in patients with BTC. Tumors and matched blood or normal samples were obtained from seven patients with cholangiocarcinoma who underwent surgical resection. We discovered inactivating mutations of tumor suppressor genes, including APC, TP53, and ARID1A, in three patients. Activating mutations of KRAS and NRAS were also identified. Our analyses identified somatic mutations in Korean patients with BTC. Introduction Biliary tract cancer (BTC) is a heterogeneous group of cancers, including gallbladder cancer and extrahepatic and intrahepatic cholangiocarcinoma. BTC has been known as a disease with a poor prognosis, with few treatment options, regardless of its low incidence. In Korea, in 2014, the crude incidence rate of BTC was 11.2 per 100,000 men, and the crude mortality rate was 7.7 per 100,000 in both men and women [1]. Most patients have been diagnosed at an advanced stage. Although surgical resection is a curative treatment, recurrence of disease has been a clinical issue for patients after surgery [2]. Combination treatment with cisplatin plus gemcitabine has been reported as an appropriate treatment for patients with advanced BTC [3]. The genomic characteristics of BTC have been revealed through several studies using high-throughput next-generation sequencing technologies [4–6]. Jiao et al. [5] reported exome sequencing results of 32 patients with intrahepatic cholangiocarcinoma. Inactivating mutations in chromatin remodeling genes, including BAP1, ARID1A, and PBRM1, were identified with frequent mutations in IDH1 and IDH2. The oncogenes KRAS, PIK3CA, NRAS, GNAS, and ERBB2 were also significantly mutated in BTC [6]. Genomic analyses need to be performed in more BTCs to understand their biology and develop therapeutic strategies. Here, we reported the whole-exome sequencing (WES) data of seven patients with BTC and analyzed their somatic mutations to compare the genetic features between patients. Methods Patients and ppub of clinical data All patients had been enrolled in a phase II clinical trial at the National Cancer Center of Korea, as approved by the institutional review board (IRB No. NCCCTC-09-411). Enrolled patients had undergone surgical resection for BTC and were then treated with gemcitabine (1,000 mg/m2) as an adjuvant chemotherapy. The participants voluntarily agreed to donate the materials by signing an informed consent form. Tumor samples were obtained from surgical specimens and blood from seven patients with BTC. Whole-exome sequencing and data analysis We extracted genomic DNA from tissue specimens and blood samples using the QIAamp DNA mini kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). The concentration and quality of DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). WES was performed using the SureSelect Human All Exon V5 Target Enrichment kit (Agilent Technologies, Santa Clara, CA, USA) and processed on the HiSeq 2500 platform (Illumina, San Diego, CA, USA). The size and quantity of the libraries were checked using a 2100 Bioanalyzer (Agilent Technologies). Sequence reads were aligned to the human reference genome hg19 using the Burrows-Wheeler Aligner–MEM algorithm [7]. The alignments were refined using the Genome Analysis Tool Kit (Broad Institute), and duplicate or low-quality reads were excluded [8]. Somatic mutations were called using Mutect and Strelka with default settings by comparing the sequences of tumor samples with those of matched normal samples [9, 10]. Only mutations showing a total read depth of greater than 30 in normal samples were considered for further analysis. Results and Discussion The demographic and clinical characteristics of the patients with BTC, including age, sex, cancer stage, recurrence, and survival, are summarized in Table 1. The patients included three females and four males with an average age of 60 years. Among the seven patients, five patients were defined as experiencing recurrence of disease after surgery. We analyzed whole-exome sequencing data of matched tumor and normal DNA from the patients. The mean coverage depth for all samples is demonstrated in Supplementary Table 1. In total, 274 somatic mutations in protein-coding regions were called by both callers, Strelka and Mutect. The average number of somatic mutations per case was 34, showing a range of 25 to 59 (Fig. 1). We found two patients harboring activating mutations in KRAS, such as G12V and G13D (Table 2). Missense mutations in codon 61 of the NRAS gene and codon 766 of the NTRK1 gene were also detected in two patients. A nonsense mutation in TP53 was found in a patient, and APC was inactivated by frameshift mutations in two patients. A patient harbored a frameshift mutation that was caused by a deletion of G in the ARID1A gene. Detailed information on the somatic mutations is provided in Supplementary Table 2. This study analyzed somatic mutations using WES in a limited number of patients with BTC. To understand the genetic features of BTC, more data need to be analyzed from more patients. SUPPLEMENTARY INFORMATION Supplementary data including two tables can be found with this article online at https://doi.org/10.5808/GI.2018.16.4.e35. Supplementary Table 1. Coverage Information Supplementary Table 2 Detailed information on total somatic mutations Fig. 1 Somatic mutation count for patients with biliary tract cancer. Table 1 Demographic information for patients with BTC BTC, biliary tract cancer; RFS, recurrence-free survival; OS, overall survival. Table 2 Somatic mutations that were annotated with cancer gene census