Mutation and Expression Analysis of Gastrointestinal Stromal Tumor Associated Genes Spectrum in a Pakistani Male through Comprehensive Next-generation Sequencing

Abstract

Gastrointestinal stromal tumors (GISTs) are the most prevalent mesenchymal sarcomas of the gastroenteric tract. They account for about 3% of the total malignancies of the gastrointestinal tract. These tumors dominantly arise in men with age above 60 years. It has an annual incidence rate of about 10 to 15 cases per million. These sarcomas originate from the interstitial cells of Cajal (ICC) underlying the complete gastrointestinal tract. These tumors mostly emerge in the stomach followed by the small intestine. Spindle cell morphology is the most frequent cell morphology occurring in GISTs. These tumors are identified by immunohistochemical markers. The KIT (CD117) marker is exclusively overexpressed in these tumors. The patients usually present symptoms of abdominal pain, nausea, vomiting, and gastrointestinal bleeding. Endoscopic ultrasonography- Fine needle aspiration biopsy (EUS-FNAB) is at present considered to be the most useful technique for diagnosing GIST. In about 85% of GISTS, KIT, and PDGFRA genes have activating mutations. While in rest of the cases, SDH-deficiency, BRAF, KRAS, NF1, PIK3CA and other uncommon genes are mutated. This case study aimed to investigate the extended mutational status of a GIST patient. The patient was a Pakistani male who was diagnosed with a tumor at the gastroesophageal junction. To achieve this objective, his control and tumor tissue was subjected to whole exome and transcriptome sequencing. After bioinformatic analysis, the whole exome annotated data was filtered for pathogenic mutations in genes potentially transforming normal ICCs into transformed tumor cells. This analysis revealed a number of mutations in genes related to metabolism and cancer-related, RAS/RAF, PI3K/AKT, and mTOR pathways. In exon 19 of the PDGFRA gene, missense mutations were found in the tyrosine kinase domain of the protein. There was a missense mutation in the SDHA gene, altering the protein sequence and structure in the FAD binding domain. The missense mutations in the Neurofibromatosis type 1 (NF1) gene caused significant alterations in its protein domains. There were missense mutations in genes that are enriched in the RAS/RAF signaling pathway. These include PTPRF, MAP2K1, MAP3K1, RASA1, FGFR1 and JAK1. PI3K/AKT and mTOR pathway genes also carried mutations including PIK3CA, PIK3R1, PIK3R2, AKT1, Mutation and Expression Analysis of Gastrointestinal Stromal Tumor Associated Genes Spectrum in a Pakistani Male through Comprehensive Next-generation Sequencing viii Abstract AKT3, TSC1, TSC2, IRS1, IRS2, and PTEN. This study provided a wide spectrum of molecular characterization of GISTs. The transcriptome analysis identified 2441 differentially expressed genes (DEGs), among which 63 were upregulated and 137 were downregulated. These genes showed enrichment in biological processes such as cell adhesion matrix, metabolism, cellular senescence, positive regulation of cellular component proliferation, growth, developmental processes, purinergic signaling. They were enriched in cancer associated pathways such as PI3K/AKT, mTOR, RAS and RAP-1. The common pathway results of the NGS analysis identified significant deregulated genes including the upregulated GNB1 and CSFR3 while downregulated FOXD2, HES5, CDKN2C, FOXO6, TP73, RAP1GAP, RPS6KA1, PRKCZ and INNP5B. These genes showed enrichment in mTOR, MAPK and PI3K/AKT pathways. These genes showed enrichment in EMT, cell migration and invasion by increased activation of PI3K/AKT pathway. This NGS data analysis revealed that these pathways can be the driver molecular pathways for gastrointestinal stromal tumorigenesis. When dealing with the therapeutic and prevention strategies against Gastrointestinal stromal tumors, this broad molecular screening can be of significant use. Keywords: Gastrointestinal stromal tumor (GIST), KIT, PDGFRA, NF1, Whole exome sequencing (WES), Transcriptome sequencing, SDHA.