Studies Of Mutation And Expression Of C-kit And PDGFR-a Gene And Their Clinical Significance In Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the digestive tract and characterized by expression of CD117 protein. Its morbidity is gradually increased year by year. Most GISTs have oncogenic c-kit mutations that engender constitutive activation of this receptor tyrosine kinase, resulting in increased cell proliferation and survival, and such mutations appear to play a key role in the pathogenesis of the majority of GISTs. However, recent studies have reported in a small subgroup of CD117-negtive GISTs (5-7%), which no c-kit but PDGFR-α mutation was found. Except for PDGFR-α (platelet-derived growth factor receptor α) , DOG1 (discovered on GIST-1) and PKC9 (protein kinase Cθ) expression were detected in CD117-negtive GISTs. Thus, PDGFR-α, DOG1 and PKCθ can be considered as new useful markers in pathologic diagnosis of GISTs, especially in CD 117-negtive GISTs. In this study, to investigate the effect of the expression and mutation of c-kit and PDGFR-α gene and their relationship with clinical pathology and prognosis of GIST, 119 cases of GISTs were analysed for CD-I 17 and PDGFR-α, β expression by immunohistochemical method. The mutations of c-kit exons 9,11,13 ,17 and PDGFR-α exons 12,18 were evaluated in fifty cases of GISTs by PCR method as well as DHPLC and DNA sequencing. DOG1 transcriptional level, a protein ofunknown function, is detected in 25 cases of GISTs by RT-PCR technique, in order to investigating the role and clinical significance of DOG1 gene in GIST.Part 1: Detection of C-kit and PDGFR-α Genes Mutations in GISTsTo investigate the relationship between c-kit and PDGFR-α mutation, the effect of the mutation of c-kit and PDGFR-α gene and its association with clinical pathology and prognosis of GIST, c-kit exons 9,11 , 13 ,17 mutations and PDGFR-α exons 12,18 mutations were detected in 50 cases of GISTs by DHPLC and direct sequencing technique.Results showed : ① Of 50 cases of GISTs, c-kit mutations were identified in 21 GISTs which were all CD117-positive. All mutations were located in exon 11, and no mutations were identified in exon 9 ,13and 17. All were heterozygous mutations. These mutations included in-frame deletion in 7 GISTs, point mutations in 10 GISTs and frame shift mutation in 4 GISTs. All the mutations were clustered between codon 556 and 560,i.e a hotspot located in the proximal part of exon 11. ② PDGFR-α mutations were identified in 10 of all 50 cases, in which four cases of positive mutations did not express CD 117. Eight were exon 18 mutation and 2 were exon 12 mutation of PDGFR-α. Four were point mutation of D842V of exon 18, which is the commonest mutation type of PDGFR-α gene. ③ C-kit-mutated GISTs displayed histomorphologically a spindle cell phenotype and origination from the stomach and the small intestine. In contrast, the tumours with PDGFR-α mutations had an epithelioid or mixed phenotype and more likely arised outside gastrointestinal tract and presented an unfavorable clinical course. ④ in the study, simultaneous mutations of c-kit and PDGFR-α genes in same case of GISTs was not found, suggesting that c-kit and PDGFR-α mutations representing two differently pathogenetic mechanism, which were mutually exclusive.Part 2: Expression of CD117, PDGFR-α and PDGFR-β and Their Clinical Significance in GISTsIn order to investigate the expression level of CD117, PDGFR-α and PDGFR-β and potentially clinical significance in differrent biological aggressive risk of GISTs, immunohistochemical staining for CD117, PDGFR-α and PDGFR-β expression was performed in 119 cases of GISTs. At the same time, the relationship between mutations of c-kit and PDGFR-α genes and expressions of CD117 and PDGFR-α.was compared.Results showed : ①Of 119 cases of GISTs, positive percentages for PDGFR-α, PDGFR-β and CD117 were 65.5% (78/119), 10.9% (13/119) and 87.4% (104/119), respectively. In control group of non-GISTs, 17.9% (5/28) were positive for PDGFR-α and 7.2%(2/28) positive for PDGFR-β. PDGFR-α protein was more highly expressed in the majority of GISTs than that in non-GISTs. ② Expression of PDGFR-α and CD117 proteins could be mutually complemented in GISTs. ③ There was a close relationship between expressions of PDGFR-α and PDGFR-α gene mutation, described as that between CD117 expression and c-kit gene mutation. ④ There were not significantly statistically differences in PDGFR-α protein expressional level among VLR, LR, IR and HR subsets of GISTs, suggesting that PDGFR-α could not be an valuable prognostic index.Part 3: Expression of DOG1 mRNA in GISTsIn order to investigate clinical significance of DOG1 gene and provide a scientific data in finding a new molecular target in GIST, DOG1 mRNA expression level was detected in 25 cases of GISTs by RT-PCR technique. The relationship between DOG1 mRNA expression and mutation and expression of c-kit and PDGFR-α genes was also analyzed.Results showed : ①DOG1 mRNA expression was detected in 92% (23/25) of 25 GISTs,. In control group, 25% (3/12) were positive for DOG1 mRNA expression. ② To be another pathologically molecular mechanism, DOG1 maybe play a role in GISTs in the diagnosis of GISTs, including CD117(—) and/or without c-kit and PDGFR-αmutations.Conclusions1. In our study, c-kit mutations rate were 42% in 50 cases of GISTs. All mutations occured in exon 11 and no mutations were identified in exon 9,11, 13 and 17. All were heterozygous mutations. All the mutations were clustered between codon 556 and 560, the classic"hot spot" at the 5'end of exon 11.2. PDGFR-α mutations were identified in 20% of all the 50 cases. Eight were exon 18 mutation and 2 were exon 12 mutation. Four were the point mutation of D842V of exon 18, which was the commonest mutation type.3. Mutation site of PDGFR-α/c-kit gene was associated with location and histological morphology of GISTs. PDGFR-α mutations had an unfavorable clinical course.4. No case of GIST found in this study had simultaneous mutations of c-kit and PDGFR-α genes, suggesting that c-kit and PDGFR-α mutations were mutuallyexclusive.5. PDGFR-α protein was highly expressed in the majority of GISTs, indicating that PDGFR-α and CD117 proteins could be mutually complemented in GISTs, together application of these two markers could improve the diagnostic accuracy of GISTs.6. There was a close relationship between expressions of PDGFR-α and mutations of PDGFR-α genes.7. Compared to control group, DOG1 gene in mRNA level was highly expressed in the majority of GISTs , suggesting it may aid in the diagnosis of GISTs without the expression of CD117 protein and/or c-kit and PDGFR-α mutations.