Study On Prognostic Value Of FGFR3/P53and H-ras/CD9Gene Mutation In Bladder Cancer

Objective:Investigation on intrinsic molecular mechanism of urothelial carcinoma of the bladder in its divergent pathways of tumorigenesis. In order to provide theoretical basis for early diagnosis, prospective assessment of risk for individual patients and personalized medicine.Methods:The expressive mutations detection of FGFGR3Ⅲb, P53and H-ras gene were identified by PCR or COLD-PCR and direct sequencing in88tissue samples of human bladder cancer and5normal bladder mucosas, and the relative expressive level of CD9mRNA was detected by semiquantitative RT-PCR methods in the same tissues. The results were associated with the clinicopathologic parameters. The39cases in which CD9mRNA level decreased obviously were selected to detect mutations by PCR and direct sequencing. Following-up the patients, the association was compared between bladder cancer recurrence and each gene detected. The Logistic Regression and Correlation Analysis were used separately to compare each gene with bladder cancer recurrence and the correlations among genes detected.Results:1. The mutation rates of P53enhanced with increasing pathological grades in88cases, and were significantly lower in low grade (13.7%) than in high grade (64.3%) and low-high grade (60.9%); The mutation rates of P53increased with increasing pathological stages in88cases, and were significantly lower in pTa stage (9.8%) than in pT1(51.4%) and≥pT2stage (66.7%). Rate of recurrence in mutant-P53(46.7%) was significantly higher than that in wild-P53(22.4%).2. The mutation rates of FGFR3Ⅲb decreased with increasing pathological grades in88cases, and were significantly higher in low grade (60.8%) than in high grade (14.3%) and low-high grade (26.1%); The mutation rates of FGFR3Ⅲb decreased with increasing pathological stages in88cases, and were significantly higher in pTa (61.0%) than in≥pT2stage (16.7%). Rate of recurrence in wild-FGFR3Ⅲb (40.8%) was significantly higher than that in mutant-FGFR3Ⅲb (17.9%). In low grade and stage tumors, FGFR3mut/TP53wt was the most prevalent genotype, but in high grade and stage tumors FGFR3wt/P53mt was the most prevalent genotype. No significant correlation was found between P53and FGFR3Ⅲb mutations; We identified the FGFR3transcript variant2, an alternatively spliced isoform of FGFR3lacking exons8,9and10, and this splicing event leads to the generation of an mRNA encoding a FGFR3in which the COOH-terminal portion of the Ig-like-Ⅲ domain and transmembrane domain are deleted. FGFR3Ⅲb and FGFR3transcript variant2were found simultaneously expressing in88cases.3. Using COLD-PCR and direct sequencing, ten missense mutations (11.4%) and twenty-six silent mutations (29.5%) were detected, yielding a41.7%improvement in mutation detection compared with conventional PCR and direct sequencing; The activating mutations detected mainly presented in low grade and low stage tumors, and no significant correlation was found between activating mutations and tumor grade and/or stage; The recurrence rates were significantly higher in silent mutant H-ras than that in wild H-ras in bladder cancer; No significant correlation was found among H-ras,P53and FGFR3mutation.4. The relative CD9mRNA levels decreased with increasing pathological grades in88cases, and were significantly higher in low grade(0.81±0.22) than in high (0.38±0.11) and low-high grade(0.49±0.17); The relative CD9mRNA levels decreased with increasing pathological stages in88cases, and were significantly higher in pTa stage (0.81±0.22) than in≥pT2(0.37±0.14) and pT1stage (0.58±0.22), higher in pTl stage than≥pT2.15of39cases were detected CD9gene mutations, which were located at protein functional domain; The relative CD9mRNA levels were significantly negatively related to the P53mutations detected, and significantly positively related to the FGFR3Ⅲb mutations detected. The Logistic Regression analysis showed patients suffering from bladder cancer with wild FGFR3III b had a relative risk of3.88(P=0.022;95%confidence interval [95%CI],0.081-0.826) for recurrence compared with patients suffering from bladder cancer with mutant FGFR3Ⅲb, and patients had bladder tumors with mutant P53had a relative risk of4.53(P=0.020;95%confidence interval [95%CI],1.273～16.110) for recurrence compared with patients had bladder tumors with wild P53.Conclusions:1. FGFR3gene mutation and H-ras gene mutation may play an important role in tumorigenesis of low grade and low stage bladder cancer, but P53 gene mutation and CD9mRN A levels may play an important role in tumorigenesis of high grade and high stage bladder cancer; The Logistic Regression analysis results reveal the mutation of FGFR3indicating a favorable prognosis while the mutation of P53indicating a poor prognosis.2. In low grade and stage tumors, FGFR3mut/TP53wt is the most prevalent genotype, but in high grade and stage tumors FGFR3wt/P53mt is the most prevalent genotype; It is becoming clear that the two urothelial tumour variants harbour distinctive genetic defects:Mutation of FGFR3indicates a favorable prognosis pathway while mutation of P53indicates a poor prognosis pathway.3. The molecular genetic characteristics are similar to high grade bladder cancer in the course of morphological changes from low grade to high grade.4. FGFR3transcript variant2, a soluble protein, plays an important role in the regulation of FGFRⅢb function.5. Mutations of the CD9gene may be the mechanism of decrease or loss of protein expression.6. COLD-PCR is a highly sensitive, inexpensive, and rapid clinical assay for mutation-detection.7. Silent mutations might play a role in in tumorigenesis of bladder cancer.