Effect In Tumorigenesis And Clincal Value Of FGFR3 And P53 Expression In Human Bladder Transitional Cell Carcinoma

Bladder cancer is one of the most commonly tumor in urinary system. More than260,000 cases are diagnosed worldwide each year, accounting for 3.2% of all newcancer cases. For 2002, 54,000 new cases and 12,000 deaths predicted for the UnitedStates make UCC the fifth most common cancer and the ninth leading cause of cancerdeath. In China, the BTCC was the eighth most common cancer in men and 25th inwomen. The worldwide prediction about bladder tumor recurrence and progressionhas performed.With the development of molecular biology and its application in study oftummorrigenesis, it has been demonstrated that the development of malignant tumorsis due to the activation of protooncogenes and inactivation of tumor suppressor genesleading to loss of control on cell proliferation and apoptosis. Cancer can be regardedas a gene disease, and will be treated in molecular level to correct the mistakes ofgenes in the tumor cell, through to restrain the expression of oncogene and to increasethe expression of antioncogene.Bladder tumors represent one of the rare examples in human cancer in which 2distinct oncogenic pathways have been clearly characterized. The first pathway,which was identified in 1993, results from alteration of the tumor suppressor geneTP53. P53 is located at 17q13.1 and comprises 11 exons and 10 introns spanning 20Kb. Over expression of p53 protein detected by immunohistochemistry and/orsomatic mutations of the gene have been detected in approximately 65% of carcinomain situ and muscle invasive tumors. The common biological effect of TP53 mutations is the inactivation of the transcriptional activity of the protein, which underlies therole of p53 in cell cycle regulation, apoptosis and DNA repair. The somatic mutationsin bladder tumors were localized in exon 5, 6, 7 and 8.The second pathway, which was identified more recently, involves somaticmutations of the FGFR3 gene, which encodes a transmembrane tyrosine kinasereceptor that is able to interact with FGF. These tyrosine kinase receptors regulateseveral cellular processes including cell growth, differentiation, migration, woundhealing, and angiogenesis, and this depends on the target cell type and thedevelopmental stage. FGFR3 is located at 4P16.3 and comprises 19 exons spanning16.5 Kb. The coding sequence of FGFR3 spanning exons 2-19 was investigatedpreviously, and somatic mutations in bladder tumors were localized in exons 7, 10,and 15. FGFR3 mutations have been found in most superficial papillary bladdertumors and recent studies suggested that in bladder oncogenesis inactivation of thep53 pathway and activation of the FGF pathways are mutually exclusive. There are 8mutation spots in bladder have been detected.In this study, protein overexpression of FGFR3 and p53 were detected byimmunohistochemistry for exploring tummorrigenesis and prognosis in protein level.We screened mutation for FGFR3 (exon 7, 10, 15) and P53 (exon 5-8) by DHPLCanalysis and sequencing in consecutive primary tumors. Thus, we could makefoundation n for detecting the value of FGFR3 and P53 mu tation in tummorrigenesisand prognosis and clinical prognosis. PartⅠStudy on the Expression of FGFR3, P53, Ki-67 in Tissue of BladderTransitional Cell Carcinoma and Their Clinical Biological BehaviorObjective: In this part we want to acquire the expression rate of FGFR3, p53 andKi-67 protein in bladder transitional cell carcinoma (BTCC) tissue and normalbladder epithelial tissue. At the same time to detect whether there were correlationamong the expression of FGFR3, p53 and Ki-67 protein expression and whether thesethree factors' expression have relationship with the biological behaviors of BTCC. Wecan understand the molecular mechanism of BTCC all the better from these resultsand at the same time evaluate the prognostic value of the three biotical markers of theBTCC.Methods: Immunohistochemistry was used to detect the protein expression ofFGFR3, p53 and Ki-67 in BTCC (n=108) and normal bladder mucosa (n=10);Immunohistochemistry result of Ki-67 was protocol by Ki-67 labeling index(LI).FGFR3 protein express was detected by western-blot in another 20 BTCC and 5normal bladder mucosa. Aim to evaluate the relationship between the proteinexpression of the three and the clinical pathological parameter.Results: Immunohistochemistry indicated that the expression rate of FGFR3 inBTCC (54.6%) was higher than normal bladder mucosa (0) (P＜0.01). The expressionrate of FGFR3 was strongly correlation with pathological grade (P＜0.01). The p53expression level between BTCC and normal bladder mucosa was 44.4% and 0,respectively (P＜0.01). With the higher of the pathological grade, the positiveexpression rate of p53 was higher meanwhile. (P＜0.01) The Ki-67LI expression inBTCC was 21.9±11.7 in average and normal bladder mucosa was 0 respectively (P＜0.01). The expression level of Ki-67 was strongly correlative with pathologicalgrade and clinical stage (P＜0.01). Nine tumors of 20 expressed FGFR3 bywestern-blot array, the normal bladder mucosa were negative expression.Conclusion:1. The expression of FGFR3, p53 and Ki-67 had significantly correlation with tumorstage and grade. All the three were valuable prognostic markers for BTCC.2. FGFR3 positive expression was frequently detected in low grade superficialBTCC, while p53 positive expression was detected in high grad invasive BTCC.The result suggested two distinct pathways of prognosis of bladder cancer.PartⅡStudy on the Mutation of FGFR3, P53 in Tissue of BladderTransitional Cell Carcinoma and Their Clinical Biological BehaviorObjective: In this part we detect the gene mutation rate of FGFR3 and p53 in bladdertransitional cell carcinoma (BTCC) tissue and normal bladder epithelial tissue. At thesame time to detect whether there were correlation among the mutation of FGFR3and P53 and the clinical biological behaviors of BTCC. We can understand themolecular mechanism of BTCC all the better from these results and at the same timeevaluate the prognostic value of the BTCC.Methods: DHPLC and PCR direct sequence were used to detect the mutation ofFGFR3 and P53 in BTCC (n=98) and normal bladder mucosa (n=10); Clinical resultsof the FGFR3 and P53 mutation were analyzed by Kaplan-Meier method and no recurrence survival rate was tested by log rank test. All the analysis were aim toexplore the clinical biological value of the mutation of FGFR3 and P53.Results: Genomic DNA of 98 BTCC was extracted. The exon 5-8 of P53 and theexon 7, 10, 15 were amplification by PCR. The production of PCR was screened byDHPLC to detect the mutation of the production. The production of the mutation wasdirected sequenced to detect the mutation spot. Mutation of FGFR3 in BTCC (44.9%)was higher than normal bladder mucosa (0) (P＜0.01). Among which there were 11transitions and 33 transversion. Mutation of P53 in BTCC (34.6%) was higher thannormal bladder mucosa (0) (P＜0.01). Among which there were 20 transitions and 14transversion. Kaplan-Meier method results revealed that mutation of FGFR3indicating a favorable prognosis pathway while mutation of P53 indicating a poorprognosis pathway. As to the analysis of genotype, the type of FGFR3mut/TP53wtrevealed a favorable pathway (P＜0.01).Conclusion:1. The mutation of FGFR3 and P53 had significantly correlation with tumor stageand grade.2. Mutation of FGFR3 indicated a favorable prognosis pathway of BTCC.3. Mutation of P53 indicated a poor prognosis pathway of BTCC.4. The genotype of FGFR3mut/TP53wt may be a favorite result of the prognosiswhich may be a potential recommendation for instrument of patients of BTCC inearly stage.PartⅢPrediction of Mutation p53 Protein 3D Structure in BTCCObjective: Our study was to detect the mutant p53 gene and the change of P53 protein 3D structure in bladder transitional cell carcinoma and the relationshipwith the the mechanism of carcinogenesis. Aim to make basis for the predictionand reconstruction of 3D structure of the protein of p53.Methods:DNA sequencing, computerized three-dimensional protein-modeling toolSWISS-MODEL, reconstitute P53 3D structure, related software analysis, were usedto analysis to mutation of the P53 in BTCC.Results:The difference in those cases with p53 gene mutation located in P53 protein 3Dstructure contribute to the different biological results of the BTCC.Conclusions:The change of the 3D structure of p53 protein have an important effect on thecarcinogenesis and progression of BTCC.