Study Of Multiple Gene Mutation All Sequence Detection And Prognostic Factors In Gastrointestinal Stromal Tumors

Objectives To investigate the status and significance of KIT, PDGFRAand DOG1gene mutation in gastrointestinal stromal tumors (GIST). To studythe prognostic factors of GIST.Methods100cases of GIST were diagnosed between May2002andMay2013at the First Affiliated Hospital of Guangxi Medical University.Medical history, patient follow-up, and pathological data were collected fromthe medical records. DNA from formalin-fixed paraffin-embedded tissues andfresh tumor tissues was isolated and amplified for the21exons of KIT,22exonsof PDGFRA and26exons of DOG1. Each PCR product was sequenced.Aminoacid sequences were inferred from DNA and aligned to Genbank referencesequences to determine the position and type of mutation. To investigate theprognostic factors of GIST. Kaplan-Meier,log-rank test and Cox regressionmodel were used to analyze statistics of GIST by SPSS17.0softwares.P＜0.05was considered statistically significant.Results Of the100GIST cases examined by molecular studies,75cases(75%) of KIT mutations were identified. The majority of KIT mutations wereobserved in exon11(59cases,59%) and in exon9(11cases,11%). Exon13 mutations were identified in3cases(3.0%). Exon17mutations were identifiedin2cases(2.0%). Across KIT, we observed51deletions,18simple substitu-tions and7insertion.The majority of these mutations involved the proximal partof KIT exon11between codons550and570, accounting for74.6%(44cases).This region should be considered as a mutational “hot spot” for GIST。Deletions of one or more codons in exon11KIT were the most commonmutations, accounting for51cases. Simple substitutions in KIT exon11was thenext most common type of mutation, occurring in8cases.1insertion in KITexon11was observed.6cases mutations in exon9had been identical with6-nucleotide duplications,encoding Ala502-Tyr503. Of the11KIT exon9mutations, we observed5simple substitutions. The mutations occuring in KITexon13and17were simple substitutions.16cases (16%) of PDGFRA mutations were observed. The majority ofPDGFRA mutations were observed in exon18(8cases,8%) and in exon12(5cases,5%). Exon8,11and14mutations were identified in3cases respectively.Of the8PDGFRA exon18mutations,5(50%) involved codon842, and4wereD842V substitutions. Across exon18, we observed5simple substitutions,2deletions, and1simple insertion. Of the5PDGFRA exon12mutations, weobserved4simple substitutions and1simple insertion. The mutation occuring inPDGFRA exon8leads to substitution of Tyr to Ser in codon392. The mutationoccuring in PDGFRA exon11leads to substitution of Leu to Pro in codon521.The mutation occuring in PDGFRA exon14leads to substitution of Thr to Lysin codon632.No DOG1mutations was found in100patients.9cases (9%) of GIST do notharbor either KIT or PDGFRA mutations and are known as wild-type GIST.The total5-year overall survival was58.8%. Log-rank univariate survival analysis showed that the primary location,the tumor size, metastasis, operationalmethod, type of tumor cells, invasion of the surrounding organs and tissues,invasion of the smooth muscle, mitotic counts of the tumor cells, deletions inexon11KIT and targeting therapy were significant prognostic factors (P<0.05).Cox regression model showed that the tumor size, metastasis,operationalmethod, invasion of the surrounding organs and tissues, mitotic counts of thetumor cells, deletions in exon11KIT and targeting therapy had independentprognostic value.Conclusions1. KIT and PDGFRA mutations play an important role in theformation and development of gastrointestinal stromal tumors.2. KIT and PDGFRA mutations are mutually exclusive, and they areinvolved in similar cellular signaling pathways that result in GIST oncogenesisbut act at a different receptor site.3.It was the first report that the whole sequence of the three genes weredetected. KIT gene mutation occurs only in the exon11,9,13and17. Mutationsin gastrointestinal stromal tumor can also occurred in8and11exon ofPDGFRA.4. The overexpression mechanism of DOG1is not related to DOG1genemutation. DOG1is possibly a fortuitous marker of the GIST,which is irrelevantto the kinase type III signal transduction pathways.5. The study shows that the tumor size, metastasis,operational method,invasion of the surrounding organs and tissues, mitotic counts of the tumor cells,deletions in exon11KIT and targeting therapy had independent prognosticvalue.6. The study suggests that early diagnosis and synthetic treatment consistingof operation and targeting therapy can significantly improve the survival of GIST.