Expression Of Nestin And Mutation Of PDGFR α In Human Gastrointestinal Stromal Tumor

PurposeTo observe the expression of nestin in gastrointestinal stromal tumors and analyze its value in diagnosis and origination of GISTs. To detect the mutations of PDGFR a gene in GISTs and investigate the correlation between PDGFR a mutation and C-kit mutation and the role of PDGFR a in the pathogenesis of GISTs.Methods116 cases of paraffin tissues of GISTs, among which 22 cases of fresh tissues were also collected at the same time , were studied for the expression of nestin by immunohistochemistry, RT-PCR and Western-Blot These GISTs occurred in the GI tract, omentum and mesentery. Three cases of leiomyomas and normal GI tract tissues were collected as controls. The C-kit gene mutations in exonll and the PDGFR a gene mutations in exonl2, exonl8 were detected by PCR-SSCP technique and DNA sequencing. Statistical analysis was performed using the SPSS software.Results1. Immunohistochemical analysis revealed strong and diffuse nestin expressions in 104 out of the 116 cases of GISTs (89.7%) . The positive signals were localized in cytoplasm and membrane. 90.5 percent of malignant GISTs and 88.7 percent of benign GISTs were nestin positive. There was no significance difference in the expression of nestin between benign and malignant GISTs ( x2 = 0.1, P > 0.05), as well as between differentage ( x2=1.43, P>0.05), sex ( x2 = 0.173, P>0.05), tumorsize ( x2 = 1.645, P > 0.05 ), location of tumors( x2 = 0.346, P > 0.05) and histological type( x 2 = 0.577, P > 0.05 ) . The rate of C-kit ,CD34 expression was 94.8%(110/116) ,75% (87/116), respectively. Six cases of GISTs in which C-kit negativewere nestin positive. There was significance difference between the expression ofCD34 and nestin ( x2 = 4.45, P < 0.05), while no significance difference between theexpression of C-kit and nestin ( x2 = 1.084, P > 0.05) .Leiomyomas were negative forC-kit and nestin. ICCs in the normal GI tract which C-kit positive were also nestinpositive.2. The results of RT-PCR showed nestin and C-kit expression in all of the GISTs and normal GI tract tissues. Each fragment of the nestin, C-kit was 337bp and 225bp, respectively. There was no significance difference in the expression of nestin and C-kit between benign and malignant GISTs. The expressions of nestin and C-kit in normal GI tract tissues were less than those in GISTs tissues. Neither nestin nor C-kit was expressed in leiomyomas.3. The results of Western-Blot revealed nestin expressions as 220KD proteins in all of the 22 cases of GISTs. C-kit were expressed in all of the GISTs as 145KD proteins. The expressions of nestin and C-kit in leiomyomas were negative. Both the two proteins were expressed faintly in normal GI tract tissues.4. Total DNA were extracted from 22 cases of GISTs, normal GI tract tissues, 3 leiomyomas and 1 schwannoma and amplified by PCR with primers of C-kit exon 11, PDGFR a exon 12 and PDGFR a exon 18. All PCR products were visualized by gel electrophoresis in 1.5g/L agarose and each product gave a single band of 225bp, 260bp, 250bp long, respectively. Analysis of PCR-SSCP showed that the rate of C-kit gene mutation was 31.8% (7/22). The PDGFR a gene mutations of exonl8 were detected in 5 out of the 22 GISTs (22.7%). And the mutation of PDGFR a gene in exonl2 was observed in 1 out of the 22 GISTs (4.5%). No mutant bands were found in normal GItract tissues, leiomyomas and schwannomas. Six cases of GISTs with PDGFR a mutations were sequenced and the results showed the three point mutations (codon2834T-C, codon2866C-T, codon2910T-C, codon2954A-G) and a 1 -bp insertion (G) at the codon 2891 in PDGFR a exonl 8.The sequencing results of one GIST with PDGFR a exonl2 mutation detected by PCR-SSCP is normal. 5. The rates of C-kit mutations were no significance difference between the malignant and thebenign GIST ( xa = 0.187, P>0.05), as well as between different age ( x2 = 0.646, P>0.05), sex( x2 = 1.18, P>0.05), tumor size ( x2 = 0.05, J?0.05), location of tumors ( x2 = 0.873, P > 0.05) and histological type ( x 2 = 1.4...