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

doi:10.21203/rs.3.rs-3316557/v1

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

https://doi.org/10.21203/rs.3.rs-3316557/v1

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Purpose

This case study covers a Pakistani man with GIST at the gastroesophageal junction, which is rare and confirmed by CT scan and immunophenotyping (Desmin+, CD117+, SMA+) and we aimed to probe the mutational landscape through NGS techniques to elucidate the key genes mutations involved in incidence and progression of GIST.

Methodology

Mutational status is examined through whole exome sequencing and mRNA sequencing using tumor tissue against adjacent control tissue.

Results

These analyses revealed a number of deleterious mutations in genes associated with cancer, metabolism and RAS/RAF pathways. These include PDGFRA, NF1, SDHA, and others. The mRNA transcripts analysis revealed 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, and positive regulation of cellular component proliferation along with cancer-associated pathways such as PI3K/AKT, mTOR, and RAS. The common pathway results of the NGS analysis identified significantly deregulated genes including the upregulated GNB1 and CSFR3 while downregulated FOXD2, HES5, CDKN2C, FOXO6, TP73, RAP1GAP, RPS6KA1, PRKCZ, INNP5B. These genes showed enrichment in EMT, cell migration, and invasion by increased activation of the PI3K/AKT pathway.

Conclusion

NGS data analysis confirmed that these genes could play a key role in driving molecular pathways for gastrointestinal stromal tumorigenesis. This study provided a wide spectrum of molecular characterization of GISTs in Pakistani man and its first time reported and could be a pilot study for driver mutations and therapeutic aspects.

Gastrointestinal stromal tumor (GIST)

Expression analysis

Tumor associated genes

Next Generation Sequencing (NGS)

Pathogenic mutations.

Gastrointestinal stromal tumors (GISTs) are the most prevalent mesenchymal neoplasms that arise in any region of the gastroenteric tract. Manzur et al. forged this term to relate an anatomically extensive continuum of tumors of the gut (Mavroeidis et al., 2018). They are considered rare tumors of the gastrointestinal tract and account for 0.1–3% of digestive tract malignancies (Al-Share et al., 2021; Qian et al., 2022). The pacemaker cells of the viscera, the interstitial cells of Cajal (ICC), involved in gastrointestinal tract vermiculation, are believed to be the cells of origin for these tumors. Primarily, GISTs typically appear in adults > 40 years of age, with an average age of 60–65 years (Al-Share et al., 2021). There’s no sex specification. As per the topographical disparities and due to lack of proper diagnosis, the factual incidence rate of GIST varies from as low as 4.3–6.8 cases per million to as high as 19–22 cases per million, with almost 5000 new cases being diagnosed each year in the USA (Mei et al., 2018). The incidence of familial autosomal dominant GIST is only about 5%, while most of the cases are sporadic (Al-Share et al., 2021; Wu et al., 2019).

These submucosal tumors appear anywhere in the digestive tract and are classified into two groups, the GISTs, and Extra-gastrointestinal GISTs. GISTs include typically occurring sarcomas, the most frequent primary sites are the stomach (60–65%) and small intestine (20–30%), followed by less common locations; duodenum (5%), colon, and rectum (< 6%), esophagus and appendix (< 1%). Whereas very rarely (5.5%), tumors develop in the mesentery, omentum, intestinal membrane, pelvis, and retroperitoneum and so are labeled as extra-gastrointestinal GISTs as shown in Fig. 1 (Al-Share et al., 2021; Gupta & Rateria, 2021; Ismail et al., 2022).

Upon hematoxylin and eosin-staining, these sub-epithelial tumors emanating from the gastrointestinal tract are classified into three cellular morphological classes, namely, the spindle type (70%), epithelioid type (20%), and mixed type (a mix of both, epithelioid and spindle type 10%)(Gupta & Rateria, 2021).

The clinical presentation of GISTs depend largely on the tumor size and location. In 18% of cases, the tumor is asymptomatic, particularly when the tumor is very small in size (< 2cm). Meanwhile, patients with larger tumors show symptoms such as gastrointestinal bleeding (~ 50% cases), abdominal pain (20–50% patients), ventral dilation, satiety, nausea, melena, hematemesis, and from mucosal ulceration. 10–30% of cases manifest obstruction of the gastrointestinal cavity resulting in dysphagia, jaundice, and pauperized urinary system (Gupta & Rateria, 2021).

GIST confirmation by immunohistochemistry (IHC) is crucial for tumor recognition. KIT (CD117) is a primary IHC marker (95% cases). CD117 is also expressed in small-cell lung carcinoma, metastatic melanoma, angiosarcoma, etc., so along with CD 117, other auxiliary biomarkers such as DOG1 (in ~ 95% GISTs), CD34 (70% cases), smooth muscle actin (SMA; in 25% of tumors), Desmin (< 5%), protein kinase C theta (PKC-θ) and S100 (~ 5% instances) are also used (Al-Share et al., 2021). Imaging techniques are usually used to diagnose GISTs. CT enterography is used to locate the tumor and chances of metastasis. MRI is the best approach to discern liver metastasis, hemorrhea, and rectal GISTs preoperative data (Al-Share et al., 2021; Blay et al., 2021; Gupta & Rateria, 2021; Parab et al., 2019). Endoscopic ultrasound with fine needle aspiration biopsy (EU-FNAB) is endorsed for pathological scanning by National Comprehensive Cancer Network (NCCN) (Qian et al., 2022).

GISTs is broadly divided into two types, the Succinate dehydrogenase competent and the Succinate dehydrogenase deficient. The following are included in the former category: i) GISTs with mutated KIT and PDGFRA, ii) BRAF, NF1, NRAS, and HRAS mutations leading to these tumors, iii) rarely reported mutated genes such as FGFR1, ATR, LTK, SUFU, PARK2, ZNF217, KRAS, and PIK3CA. Furthermore, some cases hold changes in chromosome structure in specific genes including FGFR1-HOOK3, KIT-PDGFRA, FGFR1-TACC1, ETV6–NTRK3, and (Boikos et al., 2016b; Brčić et al., 2021; Brenca et al., 2016; Charo et al., 2018; Hostein et al., 2010; Pantaleo et al., 2009).

Classification of GISTs.

About 80% of GISTs have gain-of-function mutations in the oncogenes located on chromosome 4 (4q12) encoding for the type III receptor tyrosine kinases KIT and Platelet-derived growth factor receptor α. These genes influence cell division, cell death, survival, and differentiation by controlling the RAS and PI3K signaling pathways. These are the disease's primary pathogens and the therapeutic basis for the effectiveness of Tyrosine Kinase inhibitors currently in use (Brčić et al., 2021; Qian et al., 2022). Deletions, insertions, and missense mutations arise in these genes. Exon 17, 13, 8, 10 (1% each), and 11 (70%) of KIT gene are all reported. The PDGFRA-affected mutations, which impact the exons 12, 14, and 18 of the oncogene, are identical to those seen in about 10% of GIST, with the exon 18 D842V mutation accounting for 70% of those.

The remaining 10 to 15% of the tumors without any of the above gene alterations constitute the Wild-type GISTs namely the SDH deficient (Bombac et al., 2020; Mavroeidis et al., 2018). The supposed "young adult," sporadic pediatric or CSS (Carney–Stratakis Syndrome), and wild type-GISTs in accordance with Carney Triad (CT) are included in this tumor group (Fig. 2) (Boikos et al., 2016a; Brčić et al., 2021).

27-year-old male was instituted at the Gastroenterology Department of Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan. The patient mentioned a 2-month history of vomiting, nausea, abdominal pain, weight loss, and constipation. He had a history of diabetes mellitus and hypertension. On investigations, EU-FNAB specified large amount of biliary secretions refluxing into the esophagus, heterogenous lobulated mainly hypoechoic mass with some septations measuring 7.1 cm was seen at the Gastroesophageal junction. Abdominal CT showed circumferential mural thickening of the esophagus noted at the gastroesophageal junction for a length of segment 4.7cm with an exophytic component predominantly at its posterior wall measuring 2cm in thickness. These findings favored the presence of a low-risk category gastrointestinal stromal tumor (GIST) at gastroesophageal junction. Immunophenotyping for Desmin, SMA, and CD117 expression confirmed the diagnosis of GIST. A tumor at this anatomical location is very rare to occur. Upon literature and clinical scrutinization, there was no alike case reported previously in Pakistan.

In Pakistan, very scarce data is reported on Gastrointestinal stromal tumors (GISTs). There exist published records of only clinicopathological details and immunohistochemical markers of GIST patients. The tumor cells have attained resistance to the available drug therapies. To come up with new therapeutic strategies, it is necessary to screen the molecular mutational spectrum of these tumors. The whole exome and transcriptome sequencing techniques of Next-generation sequencing employed in this study provide knowledge of the molecular characterization of the patient’s tumor. This data is helpful in assessing the mutational burden status along with expression profile of GIST case, first time in Pakistan.

Table 1

Clinicopathological characteristics of the patient included in the case study.
Case ID	J01
Gender	M
Age	27
Site of tumor	Gastro-Esophageal junction
Size (cm)	7.1
Mitotic Count (HPF)	< 1–2
Histological subtype	Spindle cell Morphology
Risk stratification	Low-risk category
Tumor status	Localized

Study design and sample collection

Tumor and normal tissue samples were collected from the patient receiving surgical treatment at the Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan, and were subjected to Next-Generation sequencing analyses. Written informed consent was acquired from the patient. Ethical approval for the conduction of this research was sought from the FMTI Ethics Research Review Board PIMS, Islamabad. The clinicopathological details of the patient are given in Table 1.

Whole Exome sequencing and Bioinformatic analysis

The tissue was processed for DNA extraction. Phenol Chloroform protocol was used to extract DNA from the samples. The extracted DNA was subjected to quantification via Thermo Scientific Multi Skan Go Instrument. The extracted DNA was subjected to whole exome sequencing. Illumina HiSeq platform X was used for sequencing. The resulting raw sequencing reads were processed via the GATK pipeline to obtain annotated variants files. Briefly, base calling provided raw sequence data of short reads in fastQ format. After quality checking by FastQC and adaptor’s removal via the FastP tool, the reads were mapped to Hg38 human reference genome using Burrows-Wheeler Aligner (BWA) tool. MarkDuplicate (Picard) tool was used to mark PCR duplicates after alignment. MuTect2 tool was used for somatic variants annotation. ANNOVAR was used for the functional annotation of the variants.

Mutation analyses of GIST-associated genes

Mutational screening focused on the genes already reported to be associated with GISTs. These genes were further screened for carrying the potential of being associated with the tumor on the following criteria: they must be non-synonymous alterations, variants classified as disease-causing or pathogenic by the tools used in the bioinformatic pipeline, variants residing in the exonic or splice site regions.

PPI and pathway enrichment analysis

Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway databases were used for pathway enrichment analyses of the target genes. STRING software was used to construct the protein-protein interaction network.

Transcriptome sequencing and Bioinformatic analysis

RNA was extracted using the TRIzol method. The extracted RNA was subjected to quantification via Thermo Scientific Multi Skan Go Instrument. cDNA was synthesized by viva cDNA synthesis kit (scientific RevertAid Reverse Transcriptase). This cDNA was subjected to transcriptome sequencing. Illumina was used for sequencing purposes. The transcriptome sequencing data was analyzed via the R software.

DEGs identification

For differential expression analysis of count data, DESeq2 was used. The log-fold change (FC) in expression and adjusted p-values (adj. P) were identified. DEGs were defined as genes that satisfied the specified cutoff criterion of adj. p > 0.05 and logFC > 2.0. Principal component analysis (PCA), Volcano plot, and heat map were constructed by the R software.

PPI and Pathway enrichment analysis

PPI networks were constructed using STRING software. The Differentially expressed genes were then subjected to pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway databases.

WES analysis of the Tumor sample

This case study presents a tumor at the gastroesophageal junction that was subjected to WES analysis. The analysis identified 98.6% exonic variants in the data. The remaining variants were classified into non-coding RNA exonic variants, splicing variants, and non-coding RNA splicing variants. Upon analyzing exonic variants, it was revealed that there were 14.5% missense variants in our data. While 68% of the variants were synonymous. The overall SNVs distribution in the sample is shown in Fig. 4 (A, B).

Novel mutations of the PDGFRA driver gene

WES analyses revealed novel mutations in the PDGFRA. There were three missense mutations at c.G2597C, c.T2624A, c.A2642T, and one deletion at c.CA2646-2647TG. All of these mutations existed in exon 19 of the gene which codes for the Tyrosine Kinase domain of the protein (Supplementary Fig. 1). There was no KIT mutation in our study.

Mutational screening of potential GIST-related gene

In addition to KIT and PDGFRA, mutations in genes such as SDHA, NF1, PIK3CA, and BRAF have also been reported to be associated with GISTs. To further screen the mutational spectrum of GIST-related genes, the WES data was investigated for mutations in genes reported in rare GISTs cases. Surprisingly, there was a novel mutation in SDHA (c.G379A). There were mutations in genes such as PTEN, IGF1R, PIK3CA, PIK3R1, PIK3R2, AKT1, AKT2, TSC1, TSC2, IRS1, and IRS. Interestingly our data also identified mutations in genes such as MAP3K1, MAP2K1, RASA1, FGFR1, PTPRF, and NF. These mutations are summarized in Table 1. There were 9 mutations in the NF1 gene. Among them, all were missense mutations except for one which was a stop-gain mutation. Our data showed diverse mutations in very important functional genes. Along with identifying a PDGFRA mutation, we also discovered mutations in genes that are classified into wild-type GISTs. Supplementry Table 1 summrizes these mutations.

Enrichment analysis of mutant genes

All 20 genes were analyzed using KEGG and GO pathway enrichment analysis. This showed that these genes engaged in highly important cancer or metabolism-associated pathways including PI3K/AKT, RAS signaling, PDGF signaling, SHC signaling, MAPK1/MAPK3 signaling, tyrosine kinase binding, and others as shown in Fig. 6 (A). The GO biological process showed that these genes participated in the Insulin-like growth factor receptor signaling pathway, PI3K signaling, PKB signaling, Regulation of Kinase activity, MAPK cascade, protein phosphorylation, and others shown in Fig. 6 (B). The GO cellular component analysis showed the existence of these genes in the protein kinase complex, succinate dehydrogenase complex, phosphatidylinositol 3-kinase complex IA, and others as shown in Fig. 6 (C). These genes were found to be involved in molecular functions such as kinase activity, phosphatase binding, and other processes as shown in Fig. 6 (D). To better comprehend the potential function relativity of these genes, a PPI network of these genes was constructed. It depicted that these genes have functional associations as shown in Fig. 7 (A). To investigate the pathway function association of these genes, a network of their pathway enrichment was constructed which suggested their close association. This network is shown in Fig. 7 (B). This network reflects that these genes are involved in the pathways such as RAS signaling, mTOR signaling, PI3K/AKT, and Insulin signaling pathways which are central to the regulation of biological processes such as growth, cell division, and proliferation.

Identification of differentially expressed genes (DEGs)

To investigate the mutational spectrum of GIST in our sample, we resorted to transcriptome sequencing. We identified the differentially expressed genes in our sample. There were 2641 DEGs identified with 63 upregulated and 137 downregulated genes. A volcano map of these DEGs is shown in Fig. 8 (A). Principal component analysis was performed for these DEGs. It is shown in Fig. 8 (B). The genes with significant differential expression were selected and a heatmap was generated for them. Figure 9 shows the heatmap for the most significant DEGs. In this heatmap, genes such as CSF3R, PER3, GNB1, KDM4A, and others in red have been upregulated. While the genes such as LRP8, FOXO6, PGD, and all in green have been downregulated.

Enrichment analysis

To analyze the role of the DEGs, they were subjected to KEGG and GO analysis. Figure 10 (A) shows the contribution of these DEGs to normal body functioning processes. They were found to be regulating critical biological processes as shown in Fig. 10 (A and C). The results revealed that these genes are involved in metabolism and cancer-related pathways, biological processes such as Mannose-type O-glycan biosynthesis, N-glycan synthesis, and others. They were also identified to be a part of RNA transport. Their association with other diseases was also seen including breast cancer, Cushing syndrome, Basal cell carcinoma, renal cell carcinoma, hepatocellular carcinoma, and gastric cancer. The association of these DEGs with different processes of the human body is summarized in Supplementary Table 3.

PPI network

To evaluate the association of the DEGs with each other, a PPI network was constructed. Figure 11 shows the network of these genes. They are highly associated to each other in terms of gene function.

Gastrointestinal stromal tumor is the most common malignancy of the gastrointestinal tract. It is a rare sarcoma that accounts for about 1 to 3% of total types. It has a yearly incidence of about ten to 15 cases per million. The tumor originates from the lineage of interstitial cells of Cajal (ICC). The stomach is the most frequently affected organ, followed by the small intestine, whereas, esophagus, the large intestine, the peritoneum, and omentum were rarely reported to have tumors. The primary tumor metastasizes to the liver. It is carried sporadically in ages between 60 to 65 having a slender male predominance. About 2% of GI stromal tumors are pediatric cases, with girls dominantly being affected. Moreover, other syndromes such as Carney Triad and Carney Stratakis syndrome occur simultaneously with GIST. About 85% of cases present KIT or PDGFRA gene mutations. These tyrosine kinase receptors are under activation in the absence of ligands, following the activation of molecular pathways including MAPK, PI3K, and STAT. These pathways code for unrestricted growth, survival, and proliferation of cells. The rest 15% of the cases may have SDH deficiency, RAS gene family aberrations, NTRK3-FGFR1 gene fusion, and other rare gene mutations. PCR-dependent mutation screening, which provides sequence data in hot-spot sites is currently being employed in clinical applications. High rates of drug resistance and recurrence, particularly in individuals with risk factors, were seen in GIST patients directing that other genes and pathways may be connected to the occurrence, progression, and chemoresistance of GIST. By administering mutation analysis at the genomic level with extensive sequencing depth and little input DNA volume required, NGS-based approaches bring up new possibilities. In this study, we used WES to analyze the genomic changes in GIST thoroughly. We found a number of unique variants in the PDGFRA or other tumor-related genes, and these genes were reinforced in a number of pathways involved in proliferation or metabolism. In the present study, a Pakistani patient with a tumor at his gastroesophageal junction was declared to be a Gastrointestinal stromal tumor (GIST). His DNA and RNA were subjected to Whole exome and Transcriptome sequencing, respectively, to discover the mutation spectrum of the tumor. Upon bioinformatic analysis, a number of variations were ascertained to be the driver and potential genes for the tumor under study. Four novel mutations were discovered in the PDGFRA gene. Three of them were missense while one was non-frameshift substitution. All four transitions existed in exon 19 and were of pathogenic nature.

These substitutions resided in the Tyrosine Kinase domain. This domain is incredibly important for the functioning of the receptor. On chromosome 4, PDGFRA is the driver gene for the GI stromal tumor to develop. Its mutations lead to the hyperactivation of kinase function via the MAPK and PI3K pathways to induce proliferation, development, and cell division (Kalfusova et al., 2019). Nine missense mutations were found in the wild-type class gene, NF1 on chromosome 17. NF1 gene constitutes a part of the remaining 10 to 15 percent of cases of wild-type genes. This tumor suppressor gene codes for Neurofibromatosis Type 1 disease and about 7% of these patients develop GI stromal cancer in their life (Mei et al., 2018). Mostly, NF1-associated GI stromal tumors develop in the duodenum and have spindle cell morphology (Andersson et al., 2005). There were 4 missense mutations in the PIK3CA gene. PIK3CA is a key mediator of PI3K pathway controlling the progression and transfiguration of GI stromal tumors (Lasota et al., 2016a). Our data also showed a novel mutation in the SDHA gene. Alteration of any SDH subunit (SDHA-D) leads to deficiency of these proteins, leading to accumulation of succinic acid stabilizing HIF1-α. This protein controls oncogenic transcription. The deficiency of these subunits has been related to the GI stromal tumor in many studies (Astolfi et al., 2020; Gupta & Rateria, 2021). This case was referred to as PDGFRA as well as wild-type mutant GIST.

Regulation of the PI3K/AKT, mTOR and RAS/RAF pathways is very crucial to the normal cell cycle and development. Our data exclusively showed mutations in genes that are central to these pathways. This includes genes such as PTEN, IGF1R, PIK3CA, PIK3R1, PIK3R2, AKT1, AKT2, TSC1, TSC2, IRS1, and IRS that are the key modulators of the PI3K/AKT and mTOR pathways. Interestingly our data showed mutations in genes such as MAP3K1, MAP2K1, RASA1, FGFR1, PTPRF and NF1 that are related to the RAS/RAF pathway. In some cancer types, PIK3CA mutations are linked to tumor progression and poor prognosis. The genotyping of PIK3CA can assist in identifying primary and secondary tumors that have the potential to become resistant to tyrosine kinase inhibitors and so can direct with therapy against this resistance (Lasota et al., 2016b). IGF1R has been expressed to be associated with the progression and development of GISTs (Li et al., 2015). Besides its alliance in diabetes and insulin resistance IRS1 gene also regulates the ERBb-PI3K-AKT pathway cascade and promotes tumorigenesis (Choi et al., 2019). Some variants of this gene have been reported to be associated with elevated risk of cancers (Maglio et al., 2013). PTPRF gene is associate with tumorigenesis and progression of tumors by regulating the RAS/ERK pathway (Davis et al., 2018). Many PTP proteins have been exercised to become therapeutic targets for many human disorders especially cancers (Feng et al., 2021). In this study, the genes such as TSC1, TSC2, and IRS1 that are common between both the signaling pathways (PI3K and mTOR) strongly indicate that they can be fruitful in presenting a promising treatment and prevention for GISTs. The GO and KEGG enrichment analysis located these genes in the pathways necessarily altered in cancers. The genes mutated in our data are enhanced in those biological processes, cellular components and molecular functions that are associated with tumor development, progression and poor prognosis.

The RNA sequencing analysis Identified about 63 upregulated and 137 downregulated genes in our sample. Upon GO and KEGG functional annotation, significant 100 genes were found to be enriched in processes like regulation of cell-matrix adhesion, anatomical structure maturation, positive regulation of cellular component biogenesis, cell population proliferation. They were also enriched in pathways involved in cancer such as PI3K/AKT, mTOR, Wnt signaling, HIF-1, p53, JAK/STAT, cAMP, TGF-β and others. Then a functional and biological enrichment of more significant genes was constructed. These genes were GNB1, FOXD2, HES5, RAP1GAP, RPS6KA1, INPP5B, CSF3R, FOXO6, CDKN2C, TP73, and PRKZC. These genes were involved in processes such as growth, cellular process, and developmental process. Among these, the upregulation of GNB1 and CSFR3 induces activation of PI3K/AKT, RAS, mTOR, signaling pathways that cause proliferation, invasion, metastasis, angiogenesis and cell growth. The downstream regulation of FOXD2, HES5, FOXO6, CDKN2C, TP73, PRKZC, RPS6KA1 and INPP5B genes inhibit apoptosis and induces cell survival and proliferation. The phosphatidylinositol PI3K/AKT/mTOR pathway is very crucial to the tumorigenesis, apoptosis, cell proliferation, translation and autophagy in neoplasms (Wang et al., 2018). Our WES and RNA-sequencing data both constituted of genes that are dominantly enriched in this pathway. Most of the deregulated genes in our data activate this pathway through their downstream signaling mediators. It can be hypothesized that this pathway is crucial to the development of Gastrointestinal stromal tumors. In many preclinical and early-stage clinical trials PI3K/AKT and mTOR signaling pathways inhibition has been declared as a promising targeted therapy approach for GISTs (Duan et al., 2020).

While undergoing metastasis, cancerous cells are supposed to travel through the extracellular matrix (ECM) for intravasation into lymphatic system and blood. The initial process of metastasis is a modification in the morphology of the cell, which is caused by the synchronization of intracellular signals and the microenvironment to enable cell separation from the primary site. This causes dense packing of cells leaving lesser room for protrusions. For stability of cells, cell matrix adhesion is a very crucial barrier to stop them from crossing the extracellular matrix (ECM) and hence, preventing their metastasis to surrounding environment (Maziveyi & Alahari, 2017). Cell adhesion also regulates proliferation, cell differentiation and apoptosis (Hynes, 2009). Aberrant extracellular cell matrix (ECM) results in abnormal proliferation of cells, irresponsiveness towards cell death, and failure towards cell differentiation. All these features are hallmarks of congenital abnormalities and malignancies such as cancers and tissue fibrosis. Due to enhanced significance of cell adhesion phenomena towards tumor progression and metastasis, treatment strategies are more focusing on targeting specific components of the ECM to reduce the metastatic event (Girigoswami et al., 2021; Paolillo & Schinelli, 2019; Walker et al., 2018). Our DEGs were dominantly enriched in genes that allow the cells to cross the matrix and migrate to neighboring environment. This migration is the crucial first step of metastasis. The deregulation of the genes in our data might have been the cause behind metastasis of the tumor from the primary gastrointestinal junction region to the neighboring nodes.

Cellular senescence is a persistent, terminal cell cycle arrest condition accompanied by multiple macromolecular alterations and a pro-inflammatory, hypersecretory phenotype. Senescent cell entry may serve as a barrier to carcinogenesis, making it possible for any anticancer therapy to aim towards this outcome (Muñoz-Espín et al., 2013). Senescent cells are widely identified in cancer patients with premalignant lesions (Schmitt et al., 2022). mTOR pathway regulates the events of senescence. The transcriptional products of this pathway controls the process of senescence (Casella et al., 2019). As the mediator genes of this pathway have been deregulated in our data, there are chances that the process of senescence might have been compromised leading to the onset of tumorigenesis.

The Nucleotide-binding protein beta 1 (GNB1) participates in the pathways of mTOR, AKT and ERK by encoding the heterotrimeric G protein subunit beta (β). Upregulation of GNB1 is involved in the increased activation of these pathways and has been reported in many cancers such as head and neck cancer, hepatocellular carcinoma and others (Usman & Hameed, 2022; Yoda et al., 2014). This gene was upregulated in our data and hence can be related to the occurrence of tumor due to the increased activation of its downstream mediators and pathways.

The first step of migration of cells is Epithelial-Mesenchymal transition (EMT). This transition is induced via the loss of epithelial cell-cell adhesion and achievement of mesenchymal morphology. It has been reported that tumor suppressor gene FOXO6, member of Forkhead transcription factor family (FOXO), suppresses cell proliferation, cell migration and invasion, and induces apoptosis hence suppressing EMT. Many members of this family have been regarded as tumor suppressors, as they inhibit cell proliferation and migration and foster apoptosis in many cancers including gastric cancers, hepatocellular carcinoma, breast cancer and lung cancer (Vandenberg et al., 2016; Ye & Duan, 2018). In our study, the expression of this gene was downregulated, so it can be conferred that the progression, proliferation and migration of the stromal tumor cells might have been due to the ectopic regulation of this gene.

Normal development requires controlled response to external stimuli and growth signals. Cellular and biological processes like transcription, DNA repair, DNA replication, purinergic signaling (regulating cell proliferation, differentiation and death), intracellular signaling, metabolism, synaptic signaling, and active transport all are the key to controlled developmental processes such as cell determination, division, growth and morphogenesis. Aberration of any of these pathways directs the cells towards abnormal development and growth, causing tumorigenesis (Collins et al., 1997; Motofei, 2022). The impact of the DEGs on the pathways associated with cancer lead to the abnormal regulation of the biological processes such as tissue invasion and metastasis, proliferation, evasion of apoptosis, focal adhesion, sustained angiogenesis, genomic instability, genomic damage, blockage of differentiation, and resistance to chemotherapy. The influence on above mentioned cellular components, molecular functions, gene-disease associations, and biological processes in our transcriptome analysis provide an insight on the previously reported and novel potential signaling pathways and mediators that can be associated with gastrointestinal stromal tumorigenesis.

It can be concluded that the whole exome sequencing analysis of our GIST patient revealed mutations in many genes having the potential to transform normal interstitial cells of Cajal into cancerous cells. This study elaborates new horizons in respect of prognostic and diagnostic evaluation. Further studies must be conducted to validate these findings. Real-time PCR and Reverse Transcriptase-PCR analysis of these novel genes will validate the mutational outcomes of our study. However, these novel discoveries can be considered while developing new therapeutic strategies.

Funding

This research is self-funded.

Authors Information

Authors and Affiliation

Cancer Genetics Laboratory, Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan

Naeem Maryam, Asad Laiba, Jahan Marium, Mahmood Hassan Asim, Anees Mariam, Murtaza Iram, Sultan Aneesa.

Alpha Genomics Lab (R&D), Islamabad, Pakistan

Ahmed Ibrar, Amin Humaira

Contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Maryam Naeem, Laiba Asad, Marium Jahan, Humaira Amin, Ibrar Ahmed, Asim Hassan Masood , Mariam Anees and Iram Murtaza. The first draft of the manuscript was written by Laiba Asad, while Maryam Naeem And Aneesa Sultan prepared the final manuscript. All authors read and approved the final manuscript

Corresponding author

Correspondence to Aneesa Sultan ([email protected])

Ethics Declaration

Conflict of interest

The authors declare they have no financial and non-financial interest to disclose.

Ethical Approval

The study was conducted according to the guidelines of institution review board of Quaid-I-Azam University, Islamabad, Pakistan.

Consent to participate

Written consent letter from patient was obtained.

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No competing interests reported.

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

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Abstract

Figures

Introduction

Case Study

Methodology

Study design and sample collection

Whole Exome sequencing and Bioinformatic analysis

Mutation analyses of GIST-associated genes

PPI and pathway enrichment analysis

Transcriptome sequencing and Bioinformatic analysis

DEGs identification

PPI and Pathway enrichment analysis

Results

WES analysis of the Tumor sample

Novel mutations of the PDGFRA driver gene

Mutational screening of potential GIST-related gene

Enrichment analysis of mutant genes

Identification of differentially expressed genes (DEGs)

Enrichment analysis

PPI network

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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