FGFR3和PIK3CA基因在膀胱肿瘤中的作用和临床生物学价值的研究
发布时间：2018-09-10 18:25
【摘要】：第一章膀胱癌中FGFR3和PIK3CA基因突变与预后关系的研究目的:检测膀胱癌组织、配对癌旁组织及良性肿瘤组织中FGFR3基因和PIK3CA基因突变,同时分析FGFR3基因和PIK3CA基因突变与临床病理参数之间的关系及相关性,为膀胱肿瘤的病因学研究提供一定的理论依据,并探寻FGFR3基因和PIK3CA基因突变是否可作为膀胱肿瘤的有效治疗靶点及预测肿瘤复发、肿瘤预后的标志物。方法:分别从34例膀胱癌组织、7例配对癌旁组织、6例良性肿瘤组织中提取DNA,以FGFR3基因外显子7、PIK3CA基因外显子9、20设计引物,经PCR扩增,扩增产物经电泳确认目的产物。应用Sanger基因检测方法检测PCR扩增产物中FGFR3和PIK3CA基因突变,探讨膀胱癌组织中FGFR3和PIK3CA基因突变与临床病理参数之间的关系,FGFR3和PIK3CA基因突变与膀胱癌的预后关系采用Kaplan-Meier生存曲线分析,单因素和多因素COX模型分析影响膀胱癌预后的各个因素。结果:FGFR3基因在膀胱癌中的突变是17.65%(6/34),癌旁组织是28.57%(2/7),良性肿瘤未见突变,三组FGFR3基因突变两两比较,差异无统计学意义(p0.05)。PIK3CA基因在膀胱癌中的突变是5.88%(2/34),癌旁组织中未见突变,良性肿瘤未见突变,膀胱癌组织分别与癌旁组织和良性肿瘤比较,差异无统计学意义(p0.05)。FGFR3基因突变在膀胱癌中,6例都是7号外显子S249C位点突变,错义突变;癌旁组织中,2例都是7号外显子S249C位点突变,错义突变,其中1例癌组织和配对癌旁组织都发生了7号外显子S249C位点突变。PIK3CA基因突变在膀胱癌中,2例均为9号外显子E545K位点,错义突变,未见外显子20突变。FGFR3和PIK3CA基因突变与临床病理分级分期都没有相关性。34例膀胱癌中,术后复发5例,其中6例FGFR3基因突变的膀胱癌,术后复发3例,皮尔生相关系数(r)是0.46(p=0.01),FGFR3基因突变与肿瘤复发呈正相关。在FGFR3和PIK3CA基因突变对膀胱癌生存影响的研究中,通过Kaplan-Meier生存曲线进行分析,FGFR3基因突变型和野生型生存期比较,差异无统计学意义(p0.05),而PIK3CA基因突变型和野生型生存期比较,差异有显著的统计学意义(p0.05)。影响膀胱癌预后因素的单因素COX分析中,年龄是影响膀胱癌预后的重要因素(p0.05),而影响膀胱癌预后的因素进行多因素COX分析,年龄也是影响膀胱癌预后的独立因素(p0.05),与性别、肿瘤大小、数目、分期、分级以及FGFR3和PIK3CA蛋白表达都没有相关性。结论:1.在膀胱肿瘤中PIK3CA/E545K是较常见基因突变类型,且提示是一个不利的预后因素。2.在膀胱肿瘤和癌旁组织中均存在FGFR3/S249C基因突变类型,故其突变不能作为诊断膀胱癌的指标,但可以预测肿瘤复发。第二章膀胱癌中FGFR3、PIK3CA蛋白表达的临床生物学意义目的:探讨膀胱癌、配对癌旁组织和良性肿瘤中FGFR3和PIK3CA蛋白表达情况,同时分析FGFR3和PIK3CA的表达与临床病理参数之间的关系及其相互之间的关联性,揭示FGFR3和PIK3CA蛋白在膀胱癌中表达的临床意义,为进一步探讨膀胱肿瘤发生发展的分子机制奠定基础。方法:应用免疫组织化学的检测42例膀胱癌组织、7例配对癌旁组织、5例良性膀胱肿瘤组织中FGFR3和PIK3CA蛋白表达情况,并计算蛋白表达强度。探讨膀胱肿瘤组织中FGFR3和PIK3CA蛋白表达之间的关系及两者与各临床病理学参数之间的关系,FGFR3和PIK3CA蛋白表达与膀胱癌的预后关系采用Kaplan-Meier生存分析,单因素和多因素COX模型分析影响膀胱癌预后的因素。结果:FGFR3蛋白在膀胱癌中的表达是50.00%(21/42),癌旁组织是14.29%(1/7),良性肿瘤是20.00%(1/5),癌组织分别与癌旁组织和良性肿瘤组织比较FGFR3蛋白表达,差异无统计学意义(p0.05)。PIK3CA蛋白在膀胱癌中的表达是64.28%(27/42),癌旁组织是14.29%(1/7),而良性肿瘤是20%(1/5),癌组织与癌旁组织比较差异有统计学意义(p0.05),而癌组织与良性肿瘤组织比较,差异无统计学意义(p0.05)。FGFR3蛋白表达在膀胱癌不同分期中,Ta-T1期是55.56%(10/18),T2-T4是45.83%(11/24),虽Ta-T1期蛋白表达要高于T2-T4期,但差异没有统计学意义(P0.05)。FGFR3蛋白表达在膀胱癌不同病理分级中,低级别是53.85%(7/13),高级别是48.28%(14/29),低级别膀胱癌FGFR3蛋白表达高于高级别,但差异没有统计学意义(P0.05)。PIK3CA蛋白表达在膀胱癌不同分期中,Ta-T1期是83.33%(15/18),T2-T4期是50.00%(12/24),Ta-T1期蛋白表达明显高于T2-T4期,并且差异有显著的统计学意义(p0.05)。PIK3CA蛋白表达在膀胱癌不同病理分级中,低级别是76.92%(10/13),高级别为58.62%(17/29),低级别膀胱癌PIK3CA蛋白表达高于高级别,差异没有统计学意义(p0.05)。膀胱癌中FGFR3蛋白表达强度以2分为主,占57.14%(12/21);PIK3CA蛋白表达强度以3分为主,占50.00%(14/28)。FGFR3蛋白表达与PIK3CA蛋白表达呈正的中等线性相关(r=0.845,p=0.001),随着FGFR3蛋白表达和强度增加,PIK3CA表达和强度也随之增加。FGFR3和PIK3CA蛋白表达与肿瘤复发无相关性。Kaplan-Meier生存曲线分析FGFR3和PIK3CA蛋白表达对生存期影响,FGFR3蛋白表达差异无统计学意义(p0.05),而PIK3CA蛋白表达差异有统计学意义(p0.05)。多因素COX分析FGFR3和PIK3CA基因突变和蛋白表达对膀胱癌预后影响,PIK3CA基因突变和蛋白表达对膀胱癌的预后影响,差异有统计学意义(p0.05);而FGFR3基因突变和表达对膀胱癌预后的影响,差异无统计学意义(p0.05)。影响膀胱癌预后因素的单因素COX分析中,年龄和PIK3CA蛋白表达强度是影响膀胱癌预后的因素(p0.05),PIK3CA蛋白表达可以作为影响膀胱癌预后的因素(p=0.05)。影响膀胱癌预后的多因素COX分析,PIK3CA蛋白表达是影响膀胱癌预后的因素(p0.05)。结论:1.FGFR3和PIK3CA蛋白表达不能作为膀胱癌诊断、治疗、复发的指标。2.FGFR3蛋白表达与PIK3CA蛋白表达呈线性相关。3.PIK3CA蛋白表达与肿瘤的分期相关,PIK3CA蛋白表达是膀胱癌预后的一个不利因素。
[Abstract]:Chapter I Mutations of FGFR3 and PIK3CA genes in bladder cancer and their relationship with prognosis Objective: To detect mutations of FGFR3 and PIK3CA genes in bladder cancer tissues, matched adjacent tissues and benign tumor tissues, and to analyze the relationship between mutations of FGFR3 and PIK3CA genes and clinicopathological parameters. Methods: DNA was extracted from 34 cases of bladder cancer, 7 cases of matched adjacent tissues, 6 cases of benign tumors, and exon 7 of FGFR3 and PIK3CA genes were used as the effective therapeutic targets and prognostic markers. The primers of exon 9 and 20 were designed and amplified by PCR. The target products were identified by electrophoresis. The mutations of FGFR3 and PIK3CA genes in the PCR products were detected by Sanger gene detection method. The relationship between the mutations of FGFR3 and PIK3CA genes and clinicopathological parameters in bladder cancer tissues was discussed. The relationship between the mutations of FGFR3 and PIK3CA genes and the prognosis of bladder cancer was also discussed. Results: The mutations of FGFR3 gene in bladder cancer were 17.65% (6/34), 28.57% (2/7) in adjacent tissues and 28.57% (2/7) in benign tumors. There was no significant difference in the mutations of FGFR3 gene between the three groups (p0.05). There was no mutation in adjacent tissues and benign tumors. There was no significant difference between adjacent tissues and benign tumors (p0.05). The mutation of FGFR3 gene was found in 6 cases of bladder cancer. The mutation of S249C in exon 7 was missense in 2 cases of adjacent tissues. It is a mutation at exon 7, S249C, with missense mutation. In one case, the mutation at exon 7, S249C, occurred in both cancer tissues and matched adjacent tissues. In bladder cancer, the mutation of PIK3CA gene was found at exon 9, E545K in both cases. There was no mutation at exon 20. There were no mutations in FGFR3 and PIK3CA genes and no clinical pathological grading and staging. Correlation. Of 34 cases of bladder cancer, 5 had recurrence, 6 had recurrence, 3 had recurrence, the Pearson correlation coefficient (r) was 0.46 (p = 0.01), and the mutation of FGFR3 gene was positively correlated with recurrence. There was no significant difference in survival time between R3 mutant and wild type (p0.05), but there was significant difference in survival time between PIK3CA mutant and wild type (p0.05). Age was an important prognostic factor in univariate COX analysis of prognostic factors of bladder cancer (p0.05). Multivariate COX analysis showed that age was also an independent prognostic factor for bladder cancer (p0.05). There was no correlation between age, sex, tumor size, number, stage, grade, and the expression of FGFR3 and PIK3CA protein. The mutation of FGFR3/S249C gene can not be used as a marker for the diagnosis of bladder cancer, but can predict the recurrence of bladder cancer. Chapter 2 The clinical biological significance of the expression of FGFR3 and PIK3CA proteins in bladder cancer. The expression of fibroblast growth factor receptor 3 and PIK3CA in bladder cancer was analyzed. The correlation between the expression of fibroblast growth factor receptor 3 and PIK3CA and clinicopathological parameters was analyzed. The clinical significance of the expression of fibroblast growth factor receptor 3 and PIK3CA protein in bladder cancer was revealed. The expression of FGFR3 and PIK3CA protein in tissues, 7 matched paracancerous tissues and 5 benign bladder tumors was measured. The relationship between the expression of FGFR3 and PIK3CA protein in bladder tumors and the clinicopathological parameters was investigated. The relationship between the expression of FGFR3 and PIK3CA protein and the prognosis of bladder cancer was studied. Results: The expression of FGFR3 protein in bladder cancer was 50.00% (21/42), 14.29% (1/7) in adjacent tissues, and 20.00% (1/5) in benign tumors. There was no statistical difference in the expression of FGFR3 protein between adjacent tissues and benign tumors. The expression of PIK3CA protein in bladder cancer was 64.28% (27/42), paracancerous tissue was 14.29% (1/7), and benign tumor was 20% (1/5). There was significant difference between cancer tissue and paracancerous tissue (p0.05). There was no significant difference between cancer tissue and benign tumor tissue (p0.05). The expression of FGFR3 protein in different stages of bladder cancer. The expression of Ta-T1 was 55.56% (10/18) and T2-T4 was 45.83% (11/24). Although the expression of Ta-T1 was higher than that of T2-T4, the difference was not statistically significant (P 0.05). The expression of FGFR3 protein was 53.85% (7/13) in low grade and 48.28% (14/29) in high grade of bladder cancer. The expression of FGFR3 protein in low grade bladder cancer was higher than that in high grade. The expression of PIK3CA protein in different stages of bladder cancer, Ta-T1 stage was 83.33% (15/18), T2-T4 stage was 50.00% (12/24), the expression of Ta-T1 stage was significantly higher than that in T2-T4 stage, and the difference was statistically significant (p0.05). The expression of PIK3CA protein in different pathological grades of bladder cancer was 76.92% (10/13) in low grade and 58.92% (10/13) in high grade. The expression of PIK3CA protein in low grade bladder cancer was higher than that in high grade bladder cancer (p 0.05). The expression of FGFR3 protein in bladder cancer was mainly 2 points (57.14% (12/21); the expression of PIK3CA protein was mainly 3 points (50.00% (14/28). The expression of FGFR3 protein was positively correlated with the expression of PIK3CA protein (r = 0.845, p = 0.0). There was no correlation between the expression of FGFR3 and PIK3CA protein and tumor recurrence. Kaplan-Meier survival curve showed that the expression of FGFR3 and PIK3CA protein had no significant effect on survival, but the expression of PIK3CA protein had no statistical difference (p0.05). Multivariate COX analysis showed that mutation and protein expression of FGFR3 and PIK3CA gene had significant effect on the prognosis of bladder cancer, and mutation and protein expression of PIK3CA gene had significant effect on the prognosis of bladder cancer (p0.05). However, mutation and expression of FGFR3 gene had no significant effect on the prognosis of bladder cancer (p0.05). Age and PIK3CA protein expression were the prognostic factors of bladder cancer (p0.05). PIK3CA protein expression could be a prognostic factor of bladder cancer (p = 0.05). Multivariate COX analysis showed that PIK3CA protein expression was a prognostic factor of bladder cancer (p0.05). Conclusion: 1. The expression of 3CA protein can not be used as an indicator of diagnosis, treatment and recurrence of bladder cancer. 2. The expression of FGFR3 protein is linearly correlated with the expression of PIK3CA protein. 3. The expression of PIK3CA protein is correlated with the stage of bladder cancer. The expression of PIK3CA protein is a disadvantageous factor for the prognosis of bladder cancer.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14
[Abstract]:Chapter I Mutations of FGFR3 and PIK3CA genes in bladder cancer and their relationship with prognosis Objective: To detect mutations of FGFR3 and PIK3CA genes in bladder cancer tissues, matched adjacent tissues and benign tumor tissues, and to analyze the relationship between mutations of FGFR3 and PIK3CA genes and clinicopathological parameters. Methods: DNA was extracted from 34 cases of bladder cancer, 7 cases of matched adjacent tissues, 6 cases of benign tumors, and exon 7 of FGFR3 and PIK3CA genes were used as the effective therapeutic targets and prognostic markers. The primers of exon 9 and 20 were designed and amplified by PCR. The target products were identified by electrophoresis. The mutations of FGFR3 and PIK3CA genes in the PCR products were detected by Sanger gene detection method. The relationship between the mutations of FGFR3 and PIK3CA genes and clinicopathological parameters in bladder cancer tissues was discussed. The relationship between the mutations of FGFR3 and PIK3CA genes and the prognosis of bladder cancer was also discussed. Results: The mutations of FGFR3 gene in bladder cancer were 17.65% (6/34), 28.57% (2/7) in adjacent tissues and 28.57% (2/7) in benign tumors. There was no significant difference in the mutations of FGFR3 gene between the three groups (p0.05). There was no mutation in adjacent tissues and benign tumors. There was no significant difference between adjacent tissues and benign tumors (p0.05). The mutation of FGFR3 gene was found in 6 cases of bladder cancer. The mutation of S249C in exon 7 was missense in 2 cases of adjacent tissues. It is a mutation at exon 7, S249C, with missense mutation. In one case, the mutation at exon 7, S249C, occurred in both cancer tissues and matched adjacent tissues. In bladder cancer, the mutation of PIK3CA gene was found at exon 9, E545K in both cases. There was no mutation at exon 20. There were no mutations in FGFR3 and PIK3CA genes and no clinical pathological grading and staging. Correlation. Of 34 cases of bladder cancer, 5 had recurrence, 6 had recurrence, 3 had recurrence, the Pearson correlation coefficient (r) was 0.46 (p = 0.01), and the mutation of FGFR3 gene was positively correlated with recurrence. There was no significant difference in survival time between R3 mutant and wild type (p0.05), but there was significant difference in survival time between PIK3CA mutant and wild type (p0.05). Age was an important prognostic factor in univariate COX analysis of prognostic factors of bladder cancer (p0.05). Multivariate COX analysis showed that age was also an independent prognostic factor for bladder cancer (p0.05). There was no correlation between age, sex, tumor size, number, stage, grade, and the expression of FGFR3 and PIK3CA protein. The mutation of FGFR3/S249C gene can not be used as a marker for the diagnosis of bladder cancer, but can predict the recurrence of bladder cancer. Chapter 2 The clinical biological significance of the expression of FGFR3 and PIK3CA proteins in bladder cancer. The expression of fibroblast growth factor receptor 3 and PIK3CA in bladder cancer was analyzed. The correlation between the expression of fibroblast growth factor receptor 3 and PIK3CA and clinicopathological parameters was analyzed. The clinical significance of the expression of fibroblast growth factor receptor 3 and PIK3CA protein in bladder cancer was revealed. The expression of FGFR3 and PIK3CA protein in tissues, 7 matched paracancerous tissues and 5 benign bladder tumors was measured. The relationship between the expression of FGFR3 and PIK3CA protein in bladder tumors and the clinicopathological parameters was investigated. The relationship between the expression of FGFR3 and PIK3CA protein and the prognosis of bladder cancer was studied. Results: The expression of FGFR3 protein in bladder cancer was 50.00% (21/42), 14.29% (1/7) in adjacent tissues, and 20.00% (1/5) in benign tumors. There was no statistical difference in the expression of FGFR3 protein between adjacent tissues and benign tumors. The expression of PIK3CA protein in bladder cancer was 64.28% (27/42), paracancerous tissue was 14.29% (1/7), and benign tumor was 20% (1/5). There was significant difference between cancer tissue and paracancerous tissue (p0.05). There was no significant difference between cancer tissue and benign tumor tissue (p0.05). The expression of FGFR3 protein in different stages of bladder cancer. The expression of Ta-T1 was 55.56% (10/18) and T2-T4 was 45.83% (11/24). Although the expression of Ta-T1 was higher than that of T2-T4, the difference was not statistically significant (P 0.05). The expression of FGFR3 protein was 53.85% (7/13) in low grade and 48.28% (14/29) in high grade of bladder cancer. The expression of FGFR3 protein in low grade bladder cancer was higher than that in high grade. The expression of PIK3CA protein in different stages of bladder cancer, Ta-T1 stage was 83.33% (15/18), T2-T4 stage was 50.00% (12/24), the expression of Ta-T1 stage was significantly higher than that in T2-T4 stage, and the difference was statistically significant (p0.05). The expression of PIK3CA protein in different pathological grades of bladder cancer was 76.92% (10/13) in low grade and 58.92% (10/13) in high grade. The expression of PIK3CA protein in low grade bladder cancer was higher than that in high grade bladder cancer (p 0.05). The expression of FGFR3 protein in bladder cancer was mainly 2 points (57.14% (12/21); the expression of PIK3CA protein was mainly 3 points (50.00% (14/28). The expression of FGFR3 protein was positively correlated with the expression of PIK3CA protein (r = 0.845, p = 0.0). There was no correlation between the expression of FGFR3 and PIK3CA protein and tumor recurrence. Kaplan-Meier survival curve showed that the expression of FGFR3 and PIK3CA protein had no significant effect on survival, but the expression of PIK3CA protein had no statistical difference (p0.05). Multivariate COX analysis showed that mutation and protein expression of FGFR3 and PIK3CA gene had significant effect on the prognosis of bladder cancer, and mutation and protein expression of PIK3CA gene had significant effect on the prognosis of bladder cancer (p0.05). However, mutation and expression of FGFR3 gene had no significant effect on the prognosis of bladder cancer (p0.05). Age and PIK3CA protein expression were the prognostic factors of bladder cancer (p0.05). PIK3CA protein expression could be a prognostic factor of bladder cancer (p = 0.05). Multivariate COX analysis showed that PIK3CA protein expression was a prognostic factor of bladder cancer (p0.05). Conclusion: 1. The expression of 3CA protein can not be used as an indicator of diagnosis, treatment and recurrence of bladder cancer. 2. The expression of FGFR3 protein is linearly correlated with the expression of PIK3CA protein. 3. The expression of PIK3CA protein is correlated with the stage of bladder cancer. The expression of PIK3CA protein is a disadvantageous factor for the prognosis of bladder cancer.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14
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