Whole-exome sequencing to identify somatic mutations in peritoneal metastatic gastric adenocarcinoma: A preliminary study

Abstract

Peritoneal metastasis occurs in more than half of patients with unresectable or recurrent gastric cancer and is associated with the worst prognosis. The associated genomic events and pathogenesis remain ambiguous. The aim of the present study was to characterize the mutation spectrum of gastric cancer with peritoneal metastasis and provide a basis for the identification of new biomarkers and treatment targets. Matched pairs of normal gastric mucosa and peritoneal tissue and matched pairs of primary tumor and peritoneal metastasis were collected from one patient for whole-exome sequencing (WES); Sanger sequencing was employed to confirm the somatic mutations. G>A and C>T mutations were the two most frequent transversions among the somatic mutations. We confirmed 48somatic mutations in the primary site and 49 in the peritoneal site. Additionally, 25 non-synonymous somatic variations (single-nucleotide variants, SNVs) and 2 somatic insertions/deletions (INDELs) were confirmed in the primary tumor, and 30 SNVs and 5 INDELs were verified in the peritoneal metastasis. Approximately 59% of the somatic mutations were shared between the primary and metastatic site. Five genes (TP53, BAI1, THSD1, ARID2, and KIAA2022) verified in our study were also mutated at a frequency greater than 5%in the COSMIC database. We also identified 9genes (ERBB4, ZNF721, NT5E, PDE10A, CA1, NUMB, NBN, ZFYVE16, and NCAM1) that were only mutated in metastasis and are expected to become treatment targets. In conclusion, we observed that the majority of the somatic mutations in the primary site persisted in metastasis, whereas several single-nucleotide polymorphisms occurred de novo at the second site.

Keywords: Sanger sequencing; gastric cancer; peritoneal metastasis; somatic mutation; whole-exome sequencing (WES).

Figure 1. Distribution of the lengths of all INDELs in the on-target regions

A length >0 indicates an insertion mutation; otherwise, the length represents a deletion mutation.

Figure 2. Mutation spectrum of non-synonymous variations in gastric cancer

The blue bar represents somatic mutations in primary gastric cancer, and the red bar indicates somatic non-synonymous mutations identified in peritoneal metastasis.

Figure 3. Non-synonymous somatic mutations discovered by WES

From outside to inside: the outer ring represents the chromosome number and section, the blue ring and letters indicate mutated genes with non-synonymous somatic variations and their chromosomal locations, and the gray ring shows genes with somatic INDEL mutations. Figure 3A and 3B summarize the confirmed non-synonymous somatic mutations in primary gastric adenocarcinoma and peritoneal metastasis, respectively.

Figure 4. Distribution of confirmed non-synonymous somatic mutations in primary gastric cancer and peritoneal metastasis

Figure 5. Mutation distribution of the five genes in the COSMIC database

A, B, C, D, and E represent the genesTP53, BAI1, ARID2, THSD1and KIAA2022, respectively. The arrow indicates the site of the mutation identified in gastric adenocarcinoma and matched peritoneal metastasis.

Figure 6. Pipeline of somatic mutation analysis from raw sequencing data

Low-quality reads were removed, and bam was performed for alignment using GATK INDEL and SNV realigner after eliminating possible duplicates. MuTectwas used to identify somatic SNVs, and all somatic SNVs were annotated by ANNOVAR. By filtering false positives, confident somatic SNVs were obtained.