新辅助化疗对晚期卵巢癌组织PI3K及P-AKT表达的影响及其与耐药的关系
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摘要
本实验从蛋白水平分析了新辅助化疗患者化疗前后的肿瘤标本PI3K及P-AKT表达的变化,探求卵巢癌新辅助化疗是否会通过PI3K／AKT通道的激活产生耐药；并分析了新辅助化疗前后耐药相关基因MRP1及TopoⅡα表达的变化,通过这些指标确定新辅助化疗的可行性。
方法
一、标本的采集
标本来源于中国医科大学附属第一医院妇科2006年1月—2009年12月经手术病理证实的晚期上皮性卵巢癌标本共22例,年龄37—71岁,中位年龄54岁；FIGO分期：Ⅲ期16例,Ⅳ期6例；组织类型：浆液性囊腺癌15例,粘液性囊腺癌7例。患者术前均予新辅助化疗(1-3个疗程)后行肿瘤细胞减灭术,术后化疗6-8个疗程,化疗方案为CP或TP。术后随访：至截止点,22例患者中有5例死亡,11例生存,6例失访。所有病理组织均以福尔马林固定,石蜡包埋。
二、实验方法
采用免疫组化SP法对22例患者新辅助化疗前原发性腹水细胞沉渣石蜡标本,或超声引导下大网膜穿刺活检石蜡标本与化疗后手术切除卵巢癌组织石蜡标本进行自身对照研究。石蜡包埋组织以PI3K、P-AKT、TopoⅡα、MRP1标记检测,抗体的工作浓度为1：200,具体步骤严格按照S-P免疫组化试剂盒说明书的操作规范进行,并同时设有空白、阴性对照。
三、统计学分析
应用SPSS13.0统计软件分析。配对等级资料检验采用Wilcoxon符号秩和检验,生存率估计采用Kaplan-Meier法,等级资料相关分析采用Logistic回归,组间生存率比较采用Log-rank检验。假设检验显著水平P<0.05有统计学意义。
结果
1、新辅助化疗后卵巢癌组织中PI3K蛋白的表达水平明显低于化疗前,两者组之间差异有统计学意义(P<0.05)。
2、新辅助化疗后卵巢癌组织中P-AKT蛋白的表达水平明显低于化疗前,两者之间差异有显著统计学意义(P<0.01)。
3、新辅助化疗前后MRP1表达无明显变化,结果没有统计学意义(P>0.05)。
4、新辅助化疗后TopoⅡα表达较化疗前降低,结果有统计学意义(P<0.05)。但比较TopoⅡα表达降低者与表达无降低者的无进展生存率没有明显的变化(P>0.05)。
5、化疗前非耐药组的无进展生存期明显高于化疗前耐药组,两组之间差异有统计学意义(P<0.05)。
6、在晚期上皮性卵巢癌组织中PI3K及P-AKT表达均与MRP1有相关性(r分别为0.685,0.634,均P<0.05)。
结论
1、PI3K及P-AKT在化疗后的表达均低于化疗前。表明新辅助化疗后PI3K／AKT信号转导通路并没有出现异常的激活,可以推断短疗程的化疗可能未产生明显的耐药。
2、TopoⅡα化疗后表达较化疗前降低,但表达降低者的无进展生存期没有明显的缩短。表明在新辅助化疗后由于TopoⅡα表达改变有可能导致化疗耐药的出现。
3、化疗前非耐药组的无进展生存期明显高于化疗前耐药组。因此化疗前MRP1和TopoⅡα检测有一项提示耐药,则新辅助化疗需慎重对待。
4、P13K及P-AKT在卵巢癌组织中表达均与MRP1有相关性,提示PI3K／AKT通路的异常激活可能是卵巢癌耐药机制之一。
Objective
By testing pre and post neo-adjuvant chemotherapy, the expressions of PI3K, P-AKT in advanced epithelial ovarian cancer tissue, to investigate whether neo-adjuvant chemotherapy activates drug resistance through PI3K/AKT signal transduction pathway; and analyzed the expression of drug resistance associated genes MRP1 and TopoⅡαbefore and after neo-adjuvant chemotherapy. These indicators predict the feasibility of neo-adjuvant chemotherapy.
Methods
1. Tissue collection
Specimens from the First Affiliated Hospital of China Medical University gynecology surgery and pathology confirmed advanced epithelial ovarian cancer were 22 cases. Aged 37-71 years, median age 54 years; FIGO stage:StageⅢin 16 cases, StageⅣin 6 cases from January 2006 to December 2009; Tissue type:serous cystadenocarcinoma in 15 cases, mucinous cystadenocarcinoma in 7 cases; All patients underwent correspondingly preoperative neo-adjuvant chemotherapy (1-3 cycles), then 6-8 cycles of chemotherapy after cytoreductive surgery Chemotherapy for the CP or TP. Postoperative follow-up:to cut-off point,22 patients there were 5 deaths,11 patients survived,6 patients lost to follow. All organizations are formalin-fixed, paraffin-embedded.
2. Methods
Immunohistochemistry SP method in 22 patients with the paraffin-embedded specimens from primary ascites before neo-adjuvant chemotherapy, or ultrasound-guided omental biopsy specimens, and paraffin specimens from surgical resection in ovarian cancer tissue after chemotherapy were paraffin self-control study. Paraffin-embedded tissues for PI3K, P-AKT, TopoⅡα, MRP1 are labeled, antibody working concentration is 1:200, concrete steps can be operated in strict accordance with manual norms of SP immunohistochemistry kit, and at the same time with a blank, negative control.
3. Statistical Analysis
Results of application of SPSS13.0 statistical software analysis system. Grade data using paired Wilcoxon signed-rank test and test and grade data correlation analysis using logistic regression, the survival rate is estimated using Kaplan-Meier method was used to compare survival rates between the two groups log-rank test. Hypothesis testing the significance level P<0.05 statistically significant.
Results
1. After neo-adjuvant chemotherapy in ovarian cancer tissue, the expression level of PI3K protein were significantly lower than before chemotherapy, the difference between the two groups was significant (P<0.05).
2. After neo-adjuvant chemotherapy in ovarian cancer, P-AKT protein expression was significantly lower than that before chemotherapy, the difference between the two groups was significant (P<0.01).
3. Before and after neo-adjuvant chemotherapy, the expression of MRP1 was no significant changes, the result was not statistically significant (P> 0.05).
4. After neo-adjuvant chemotherapy compared with before chemotherapy, the expression of TopoⅡαdecreased, the result was statistically significant (P<0.05).but compared the decreased expression of TopoⅡαwith the no changed expression of TopoⅡα, the progression-free survival rate of those was not significant changes (P> 0.05).
5. The progression-free survival rate of non-resistant group before chemotherapy was significantly higher than resistance group, the differences between the two groups was statistically significant (P<0.05).
6. In advanced epithelial ovarian cancer P-AKT and PI3K expression with MRP1 expression exists correlation (r=0.685,0.634, both P<0.05).
Conclusions
1. PI3K and P-AKT expression after chemotherapy were lower than before chemotherapy. Shows that after neo-adjuvant chemotherapy PI3K/AKT signal transduction pathway did not appear to unusual activated, and short courses of chemotherapy can not be inferred to generate significant resistance.
2. The expression of TopoⅡαafter chemotherapy compared with chemotherapy decreased, but progression-free survival of decreased expression were not significantly reduced. Shows that after neo-adjuvant chemotherapy, because the changed expression of TopoⅡα, it may lead to the resistance to chemotherapy.
3. Before chemotherapy progression-free survival of non-resistant group was significantly higher than resistance group. Therefore, before chemotherapy, MRP1 or TopoⅡαis a prompt detection of drug resistance, then be careful of neo-adjuvant chemotherapy.
4. PI3K and P-AKT expression in ovarian cancer were correlated with the MRP1, suggesting that abnormal activation of PI3K/AKT pathway may be one of the mechanisms of drug resistance of ovarian cancer.
方法
一、标本的采集
标本来源于中国医科大学附属第一医院妇科2006年1月—2009年12月经手术病理证实的晚期上皮性卵巢癌标本共22例,年龄37—71岁,中位年龄54岁；FIGO分期：Ⅲ期16例,Ⅳ期6例；组织类型：浆液性囊腺癌15例,粘液性囊腺癌7例。患者术前均予新辅助化疗(1-3个疗程)后行肿瘤细胞减灭术,术后化疗6-8个疗程,化疗方案为CP或TP。术后随访：至截止点,22例患者中有5例死亡,11例生存,6例失访。所有病理组织均以福尔马林固定,石蜡包埋。
二、实验方法
采用免疫组化SP法对22例患者新辅助化疗前原发性腹水细胞沉渣石蜡标本,或超声引导下大网膜穿刺活检石蜡标本与化疗后手术切除卵巢癌组织石蜡标本进行自身对照研究。石蜡包埋组织以PI3K、P-AKT、TopoⅡα、MRP1标记检测,抗体的工作浓度为1：200,具体步骤严格按照S-P免疫组化试剂盒说明书的操作规范进行,并同时设有空白、阴性对照。
三、统计学分析
应用SPSS13.0统计软件分析。配对等级资料检验采用Wilcoxon符号秩和检验,生存率估计采用Kaplan-Meier法,等级资料相关分析采用Logistic回归,组间生存率比较采用Log-rank检验。假设检验显著水平P<0.05有统计学意义。
结果
1、新辅助化疗后卵巢癌组织中PI3K蛋白的表达水平明显低于化疗前,两者组之间差异有统计学意义(P<0.05)。
2、新辅助化疗后卵巢癌组织中P-AKT蛋白的表达水平明显低于化疗前,两者之间差异有显著统计学意义(P<0.01)。
3、新辅助化疗前后MRP1表达无明显变化,结果没有统计学意义(P>0.05)。
4、新辅助化疗后TopoⅡα表达较化疗前降低,结果有统计学意义(P<0.05)。但比较TopoⅡα表达降低者与表达无降低者的无进展生存率没有明显的变化(P>0.05)。
5、化疗前非耐药组的无进展生存期明显高于化疗前耐药组,两组之间差异有统计学意义(P<0.05)。
6、在晚期上皮性卵巢癌组织中PI3K及P-AKT表达均与MRP1有相关性(r分别为0.685,0.634,均P<0.05)。
结论
1、PI3K及P-AKT在化疗后的表达均低于化疗前。表明新辅助化疗后PI3K／AKT信号转导通路并没有出现异常的激活,可以推断短疗程的化疗可能未产生明显的耐药。
2、TopoⅡα化疗后表达较化疗前降低,但表达降低者的无进展生存期没有明显的缩短。表明在新辅助化疗后由于TopoⅡα表达改变有可能导致化疗耐药的出现。
3、化疗前非耐药组的无进展生存期明显高于化疗前耐药组。因此化疗前MRP1和TopoⅡα检测有一项提示耐药,则新辅助化疗需慎重对待。
4、P13K及P-AKT在卵巢癌组织中表达均与MRP1有相关性,提示PI3K／AKT通路的异常激活可能是卵巢癌耐药机制之一。
Objective
By testing pre and post neo-adjuvant chemotherapy, the expressions of PI3K, P-AKT in advanced epithelial ovarian cancer tissue, to investigate whether neo-adjuvant chemotherapy activates drug resistance through PI3K/AKT signal transduction pathway; and analyzed the expression of drug resistance associated genes MRP1 and TopoⅡαbefore and after neo-adjuvant chemotherapy. These indicators predict the feasibility of neo-adjuvant chemotherapy.
Methods
1. Tissue collection
Specimens from the First Affiliated Hospital of China Medical University gynecology surgery and pathology confirmed advanced epithelial ovarian cancer were 22 cases. Aged 37-71 years, median age 54 years; FIGO stage:StageⅢin 16 cases, StageⅣin 6 cases from January 2006 to December 2009; Tissue type:serous cystadenocarcinoma in 15 cases, mucinous cystadenocarcinoma in 7 cases; All patients underwent correspondingly preoperative neo-adjuvant chemotherapy (1-3 cycles), then 6-8 cycles of chemotherapy after cytoreductive surgery Chemotherapy for the CP or TP. Postoperative follow-up:to cut-off point,22 patients there were 5 deaths,11 patients survived,6 patients lost to follow. All organizations are formalin-fixed, paraffin-embedded.
2. Methods
Immunohistochemistry SP method in 22 patients with the paraffin-embedded specimens from primary ascites before neo-adjuvant chemotherapy, or ultrasound-guided omental biopsy specimens, and paraffin specimens from surgical resection in ovarian cancer tissue after chemotherapy were paraffin self-control study. Paraffin-embedded tissues for PI3K, P-AKT, TopoⅡα, MRP1 are labeled, antibody working concentration is 1:200, concrete steps can be operated in strict accordance with manual norms of SP immunohistochemistry kit, and at the same time with a blank, negative control.
3. Statistical Analysis
Results of application of SPSS13.0 statistical software analysis system. Grade data using paired Wilcoxon signed-rank test and test and grade data correlation analysis using logistic regression, the survival rate is estimated using Kaplan-Meier method was used to compare survival rates between the two groups log-rank test. Hypothesis testing the significance level P<0.05 statistically significant.
Results
1. After neo-adjuvant chemotherapy in ovarian cancer tissue, the expression level of PI3K protein were significantly lower than before chemotherapy, the difference between the two groups was significant (P<0.05).
2. After neo-adjuvant chemotherapy in ovarian cancer, P-AKT protein expression was significantly lower than that before chemotherapy, the difference between the two groups was significant (P<0.01).
3. Before and after neo-adjuvant chemotherapy, the expression of MRP1 was no significant changes, the result was not statistically significant (P> 0.05).
4. After neo-adjuvant chemotherapy compared with before chemotherapy, the expression of TopoⅡαdecreased, the result was statistically significant (P<0.05).but compared the decreased expression of TopoⅡαwith the no changed expression of TopoⅡα, the progression-free survival rate of those was not significant changes (P> 0.05).
5. The progression-free survival rate of non-resistant group before chemotherapy was significantly higher than resistance group, the differences between the two groups was statistically significant (P<0.05).
6. In advanced epithelial ovarian cancer P-AKT and PI3K expression with MRP1 expression exists correlation (r=0.685,0.634, both P<0.05).
Conclusions
1. PI3K and P-AKT expression after chemotherapy were lower than before chemotherapy. Shows that after neo-adjuvant chemotherapy PI3K/AKT signal transduction pathway did not appear to unusual activated, and short courses of chemotherapy can not be inferred to generate significant resistance.
2. The expression of TopoⅡαafter chemotherapy compared with chemotherapy decreased, but progression-free survival of decreased expression were not significantly reduced. Shows that after neo-adjuvant chemotherapy, because the changed expression of TopoⅡα, it may lead to the resistance to chemotherapy.
3. Before chemotherapy progression-free survival of non-resistant group was significantly higher than resistance group. Therefore, before chemotherapy, MRP1 or TopoⅡαis a prompt detection of drug resistance, then be careful of neo-adjuvant chemotherapy.
4. PI3K and P-AKT expression in ovarian cancer were correlated with the MRP1, suggesting that abnormal activation of PI3K/AKT pathway may be one of the mechanisms of drug resistance of ovarian cancer.
引文
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