Dataset Information

Analysis of C-Kit Exon 9, Exon 11 and BRAFV600E Mutations Using Sangers Sequencing in Gastrointestinal Stromal Tumours.

ABSTRACT: Background Gastrointestinal stromal tumors (GIST) are the most common mesenchymal neoplasms in the gastrointestinal (GI) tract. The mutation of C-KIT is considered to be the crucial step in the tumorigenesis. Targeted therapies are being developed focusing these mutations. Various exon mutations of GIST responded in varied patterns to this targeted therapy. This study was carried out to evaluate the C-KIT exon 11, exon 9 and BRAF V600E mutations among GIST specimens. Methods This retrospective study was carried out among 20 DNA extracted specimens from paraffin blocks of GIST received in our tertiary teaching institution for a period of three years. DNA sequencing was carried out for mutational analyses on C-KIT exon 9, C-KIT exon 11 and BRAF V600E genes using Sanger sequencing. Results Histologically, majority of the tumors had spindle cell morphology. About 19 cases were positive for CD117. The analysis of type of mutations showed that three cases carried Exon 11 and three cases carried Exon 9 mutations. BRAF V600E mutation was seen in one case. Conclusion It is essential to conduct molecular studies on GISTs in order to get a clear understanding of the pathogenesis and behavior pattern. This will also help in designing targeted therapies and assessing recurrence. With the advent of rapidly evolving personalized therapy, the evaluation of genetic mutations is essential for diagnosis and prognostic value.

SUBMITTER: Madhala D

PROVIDER: S-EPMC7174865 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Analysis of C-Kit Exon 9, Exon 11 and BRAFV600E Mutations Using Sangers Sequencing in Gastrointestinal Stromal Tumours.

Madhala Divya D Sundaram Sandhya S Chinambedudandapani MohanaPriya M Balasubramanian Archana A

Cureus 20200322 3

Background Gastrointestinal stromal tumors (GIST) are the most common mesenchymal neoplasms in the gastrointestinal (GI) tract. The mutation of C-KIT is considered to be the crucial step in the tumorigenesis. Targeted therapies are being developed focusing these mutations. Various exon mutations of GIST responded in varied patterns to this targeted therapy. This study was carried out to evaluate the C-KIT exon 11, exon 9 and BRAF V600E mutations among GIST specimens. Methods This retrospective s ...[more]

PMID: 32328381

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Project description:Gastrointestinal stromal tumors (GISTs) are common mesenchymal neoplasms in the gastrointestinal tract of humans and dogs. Little is known about the pathogenesis of these tumors. This study evaluated the role of c-KIT in canine GISTs; specifically, we investigated activating mutations in exons 8, 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), all of which have been implicated in human GISTs.Seventeen canine GISTs all confirmed to be positive for KIT immunostaining were studied. Exons 8, 9, 11, 13 and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA, were amplified from DNA isolated from formalin-fixed paraffin-embedded samples.Of these seventeen cases, six amplicons of exon 11 of c-KIT showed aberrant bands on gel electrophoresis. Sequencing of these amplicons revealed heterozygous in-frame deletions in six cases. The mutations include two different but overlapping six base pair deletions. Exons 8, 9, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA had no abnormalities detected by electrophoresis and sequencing did not reveal any mutations, other than synonymous single nucleotide polymorphisms (SNPs) found in exon 11 of c-KIT and exons 12 and 14 of PDGFRA.The deletion mutations detected in canine GISTs are similar to those previously found in the juxtamembrane domain of c-KIT in canine cutaneous mast cell tumors in our laboratory as well as to those reported in human GISTs. Interestingly, none of the other c-KIT or PDGFRA exons showed any abnormalities in our cases. This finding underlines the critical importance of c-KIT in the pathophysiology of canine GISTs. The expression of KIT and the identification of these activating mutations in c-KIT implicate KIT in the pathogenesis of these tumors. Our results indicate that mutations in c-KIT may be of prognostic significance and that targeting KIT may be a rational approach to treatment of these malignant tumors. This study further demonstrates that spontaneously occurring canine GISTs share molecular features with human GISTs and are an appropriate model for human GISTs.

Project description:Background and aimsConsidering the indication of adjuvant therapy, the recurrence risk for primary gastrointestinal stromal tumor (GIST) after surgery needs to be accurately estimated. However, current risk stratification schemes may still have room for improvement. This study seeks to analyze prognostic factors for primary GISTs from 3 aspects, including clinicopathological parameters, immunohistochemical biomarkers, and gene mutational status, and attempts to find novel valuable factors predicting the malignancy potential of GISTs.MethodsRetrospective data from 114 primary GIST patients after R0 resection were collected. Clinicopathological data was obtained from medical records and re-evaluated. Immunohistochemical analysis was performed using the Tissue Microarray method for Ki67, p16, p27, p53, SKP2, CD133, and actin. KIT gene exons 9, 11, 13, and 17 and PDGFRα gene exons 12 and 18 were tested for mutations using PCR.ResultsUnivariate analysis revealed the following factors as poor prognostic indicators for relapse-free survival with a median follow-up of 50 months: male gender, gastrointestinal bleeding, mitotic index >5/50HPFs, tumor size >5 cm, non-gastric site, necrosis, epithelioid or mixed cell type, surrounding tissue invasion, Ki67>5%, p16>20%, p53 index >10, SKP2>10%, and KIT exon 11 deletion. Besides mitotic index, tumor size and site, SKP2 high expression (RR = 2.91, 95% CI: 1.41-5.99, P = 0.004) and KIT exon 11 deletion (RR = 2.73, 95% CI: 1.04-7.16, P = 0.041) were also independent risk factors in multivariate analysis, with gastrointestinal bleeding also showing a trend towards significance (RR = 1.88, 95% CI: 0.98-3.64, P = 0.059). In addition, gastrointestinal bleeding and SKP2 high expression showed a good ability to stratify high-risk patients further.ConclusionOur results show that gastrointestinal bleeding, SKP2 high expression, and KIT exon 11 deletions may be useful indicators of high recurrence risk for primary GIST patients.

Dataset Information

Analysis of C-Kit Exon 9, Exon 11 and BRAFV600E Mutations Using Sangers Sequencing in Gastrointestinal Stromal Tumours.

Publications

Analysis of C-Kit Exon 9, Exon 11 and BRAFV600E Mutations Using Sangers Sequencing in Gastrointestinal Stromal Tumours.

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