膀胱癌和乳腺癌相关分子标志物的研究
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摘要
这一关于恶性肿瘤分子标志物的研究课题由相对独立的两部分工作构成。
第一部分：以DNA拷贝数异常改变作为膀胱癌诊断相关分子标志物的研究
膀胱癌在发生发展过程中常见基因组DNA结构的异常改变,这些分子遗传学改变既可以为膀胱癌发生发展的分子机制研究指引方向,也能够为膀胱癌相关分子标志物的发现与鉴定提供信息。本实验室的前期工作采用array CGH (array comparative genome hybridization)技术对膀胱尿路上皮癌组织进行分析,初步获得了膀胱癌特征性DNA拷贝数变化谱型。为了将这组通过高通量技术获得的膀胱癌相关分子遗传学改变的研究结果进行可能的临床应用转化,本课题将上述膀胱癌特征性DNA拷贝数异常改变的检测手段从array CGH技术平台过渡到实时定量PCR (real time PCR)技术平台。经过对array CGH实验数据再挖掘、候选DNA片段/基因筛选与验证,旨在获得一组具有临床应用前景的膀胱癌诊断相关DNA分子标志。
采用生物信息学方法,通过对前期array CGH实验的数据再挖掘,将芯片上基因/区段拷贝数改变按发生频率进行排序,首先挑选出DNA拷贝数改变频率最高的、将用于本项研究Real-time PCR分析验证的11个候选基因(A_14_P139169、CEP63、CLK1、FOSL2、GHR、LILRA3、PAQR6、PARVA、 SFRS8、ZFAND3、ZNF76),以及无拷贝数改变的2个基因(TBP和TMEM39A)作为Real-time PCR分析内参。
通过对array CGH实验分析过的56例膀胱癌“起始’样品进行Real-time PCR检测,以“跨技术平台核查”的方式,从上述11个候选基因中筛选出5个(CEP63、 FOSL2、GHR、PAQR6和ZFAND3)候选目的基因用于后续验证。5个候选目的基因的验证在3组独立样本中进行：分别为58例开放性手术切除的膀胱癌冻存组织、62例甲醛固定石蜡包埋(FFPE)膀胱癌组织和43例经尿道切除(TURBT)膀胱癌组织。
结果显示,与对照组30例正常人外周血白细胞基因组DNA比较,CEP63和PAQR6两个基因在全部3组膀胱癌组织样本中均出现拷贝数增加的趋势,其差别具有显著性；而FOSL2和GHR基因拷贝数增加只出现在"FFPE"样本中；ZFAND3基因的拷贝数显著减少可见于"FFPE"样本和"TURBT"样本。值得注意的是,与正常对照组相比,"FFPE'’样本中5个候选目的基因均显示了与芯片结果趋势一致的显著性拷贝数改变。并且,这种改变从正常对照到非浸润性再到浸润性膀胱癌组织中有渐进性的趋势,尤其以CEP63、PAQR6和ZFAND3表现明显。以膀胱癌组织验证结果为基础,进一步分析了这5个基因在膀胱癌患者尿脱落细胞中的拷贝数改变。与对照组患者尿脱落细胞DNA样品相比,PAQR6和FOSL2基因在膀胱癌病例组中拷贝数显著增加,结果与array CGH芯片分析结果一致；但是ROC曲线分析其诊断效能尚不足。
上述研究资料表明,膀胱癌中PAQR6和CEP63等基因的拷贝数改变是在膀胱尿路上皮癌病变组织基因组DNA中的大概率分子事件；这些具有统计学意义的DNA分子改变可在膀胱癌患者尿脱落细胞中检出。若将这些异常改变转化为膀胱癌诊断相关分子标志物,还有待于进一步的缜密分析和大样本多中心验证。
第二部分：乳腺癌新辅助化疗组织病理学治疗反应相关分子标志物的研究
新辅助化疗已成为针对局部进展期的乳腺癌综合治疗中的重要环节；治疗后达到组织病理学完全缓解的患者可长期生存。肿瘤对化疗的反应性是选择治疗方案最重要的依据。传统的病理类型、病理分级等形态学指标尚不足以全面有效地评估新辅助化疗后的组织病理学治疗反应；相关分子标志表达谱对于准确预测新辅助化疗后的组织病理学治疗反应具有重要的辅助意义。
本项研究选择近年报道的与乳腺癌新辅助化疗组织病理学治疗反应有关的蛋白分子CK5/6、EGFR、cyclin B1、14-3-3σ.14-3-3ζ,结合传统病理学分子标志物ER、PR、HER2,联合检测这8个蛋白在新辅助化疗前乳腺癌核芯针穿刺活检组织中的表达情况。对215例浸润性乳腺癌新辅助化疗前的活检穿刺获得的甲醛固定石蜡包埋组织切片进行这8个蛋白的免疫组化染色。收集入组患者手术切除肿瘤组织,进行组织病理学治疗反应的MP分级(Miller&Payne病理分级)。统计分析患者年龄、月经状态、临床分期、病理分级、病理类型,以及上述8个蛋白表达与MP分级的关系。将结果进行Logistic回归分析,建立分子联合检测模型,并绘制ROC曲线；分析候选蛋白分子标志物评估/预测乳腺癌新辅助化疗组织病理学反应的效能。
研究结果显示,8个候选蛋白在215例乳腺癌患者新辅助化疗前核芯针穿刺活检的肿瘤组织中的阳性表达率分别为：ER42.8%. PR42.8%, HER234.0%、 CK5/627.9%、EGFR55.8%、cyclin B147.9%、14-3-3σ28.8%、14-3-3ζ79.5%。 HER2主要表达于绝经后组中,与绝经前组比较,其表达差别具有显著性。ER、PR同步表达,并主要表达于分化较好的肿瘤组织中。而CK5/6主要表达于分化较差的肿瘤组织中。与MP分级(有效/无效)显著相关的病理特征只有组织学分级。蛋白分子标志物之中,ER、PR、CK5/6、EGFR、14-3-3σ在MP有效组和无效组之间的表达具有显著性差异,其P值分别为ER(P<0.001). PR (P=0.001)、CK5/6(P<0.001)、EGFR(P=0.024).14-3-3σ (P=0.017)。经Logistic回归分析建立预测模型,其中ER和14-3-36进入模型。经ROC曲线分析结果显示ER和14-3-3σ联合分析的预测效能显著高于单个分子(ER、PR、CK5/6、EGFR以及14-3-3σ)对新辅助化疗组织病理学治疗反应(MP分级)的预测效能。
本项研究获得的结果为寻找临床适用的乳腺癌新辅助化疗组织病理学治疗反应预测分子标志谱提供了重要线索,为后续大样本多中心研究奠定了工作基础。
This PhD degree project is composed of two parts of study, on molecular markers for bladder cancer and breast cancer.
Part I:Study on DNA copy number aberrations in the genome as molecular biomarkers of bladder urothelial carcinoma
Abnormal change of the DNA structure is common in the carcinogenesis and malignant progression of bladder. These molecular genetic aberrations provide an insight for the molecular mechanism on development of the bladder cancer; and also benefit for discovery and identification of biomarkers for the disease. The purpose of this study was to identify the characteristic DNA copy number aberrations (CNA) in the genome that could serve as molecular biomarkers of bladder urothelial carcinoma.
We previously generated a genome-wide DNA copy number aberration profile in bladder cancer by using the array comparative genome hybridization (array CGH) approach. In this study, we selected11DNA fragments/genes (A_14_P139169, CEP63, CLK1, FOSL2, GHR, LILRA3, PAQR6, PARVA, SFRS8, ZFAND3and ZNF76) with high frequencies of CNA in the genome of bladder cancer, by re-mining the array-CGH data. The copy number aberration of the11genes was firstly examined by real time polymerase chain reaction in56tumor samples that were used for the array CGH analysis. Among the11genes,5(CEP63, FOSL2, GHR, PAQR6and ZFAND3) were verified, for further validation. Three independent sample sets of bladder cancer were applied for the validation; they were58freshly frozen tumor tissues derived from patients of bladder cancer with treated partial cystectomy,62formalin-fixed paraffin-embedded (FFPE) tumor tissues, and43freshly frozen tumor tissues derived from patients treated with transurethral resection of bladder tumor (TURBT). Comparing with30peripheral leukocyte genomic DNA samples of healthy individuals, gain of copy number of both CEP63and PAQR6was detected in all three sample sets of bladder cancer tissues; whereas for FOSL2and GHR, gain of copy number was only observed in the 'FFPE' samples. While loss of copy number for ZFAND3was found in the 'FFPE' samples and 'TURBT' samples. It is noteworthy that, the copy number aberrations of all5genes (CEP63, FOSL2, GHR, PAQR6and ZFAND3) were identified by real-time PCR in the 'FFPE' samples consisting with that of array CGH analysis. Based on data derived from the tumor tissues, we examined5genes in urine sediment samples of123patients with bladder cancer, and found gain of copy number of PAQR6and FOSL2in the bladder cancer group, comparing with patients with tumor of head and neck.
In conclusion, copy number aberration of the5candidate genes could be common molecular events, and can be detected in urine sediments of bladder cancer patients. However, further multicentre study is needed for developing them as diagnostic biomarkers for bladder cancer.
Part Ⅱ:Study on molecular biomarkers of histopathological response to neoadjuvant chemotherapy for breast cancer
Neoadjuvant chemotherapy (NAC) has become an integral part in the comprehensive treatment for breast cancer. Those patients achieve complete pathological response or significant pathological response have a longer disease-free survival and overall survival. To properly estimate pathological response to neoadjuvant chemotherapy is important for choosing therapeutic schedule. The purpose of this study was to identify appropriate molecular biomarkers of histopathological response to neoadjuvant chemotherapy for breast cancer.
A panel of protein markers composed of ER, PR, HER2, CK5/6, EGFR, cyclin B1,14-3-3σ and14-3-3ζ was examined by immunohistochemical (IHC) staining, on core needle biopsy tissue samples collected from215patients with invasive breast cancer, prior to neoadjuvant chemotherapy. With Miller&Payne (MP) criterion, the histopathologic response to NAC was classified upon the tissue section of surgical resected tumor after neoadjuvant chemotherapy. The results of the IHC analysis showed that positive staining rates were, ER42.8%, PR42.8%, HER234.0%, CK5/627.9%, EGFR55.8%, cyclin B147.9%,14-3σ28.8%and14-3-3ζ79.5%. Among them, HER2mainly expressed in the postmenopausal group; ER and PR mainly expressed in the tumors with low grade, whereas CK5/6mainly expressed in the tumors with high grade. Between the groups of MP good response and poor response, there were significantly differential expression of ER (P<0.001), PR (P=0.001), CK5/6(P<0.001), EGFR (P=0.024), and14-3-3σ (P=0.017). The logistic regression analysis showed that only ER and14-3-3σ were included in the prediction model for histopathological response to NAC. Joint detection of ER and14-3-3σ have more power to estimate the pathological response than that any single protein marker did.
第一部分：以DNA拷贝数异常改变作为膀胱癌诊断相关分子标志物的研究
膀胱癌在发生发展过程中常见基因组DNA结构的异常改变,这些分子遗传学改变既可以为膀胱癌发生发展的分子机制研究指引方向,也能够为膀胱癌相关分子标志物的发现与鉴定提供信息。本实验室的前期工作采用array CGH (array comparative genome hybridization)技术对膀胱尿路上皮癌组织进行分析,初步获得了膀胱癌特征性DNA拷贝数变化谱型。为了将这组通过高通量技术获得的膀胱癌相关分子遗传学改变的研究结果进行可能的临床应用转化,本课题将上述膀胱癌特征性DNA拷贝数异常改变的检测手段从array CGH技术平台过渡到实时定量PCR (real time PCR)技术平台。经过对array CGH实验数据再挖掘、候选DNA片段/基因筛选与验证,旨在获得一组具有临床应用前景的膀胱癌诊断相关DNA分子标志。
采用生物信息学方法,通过对前期array CGH实验的数据再挖掘,将芯片上基因/区段拷贝数改变按发生频率进行排序,首先挑选出DNA拷贝数改变频率最高的、将用于本项研究Real-time PCR分析验证的11个候选基因(A_14_P139169、CEP63、CLK1、FOSL2、GHR、LILRA3、PAQR6、PARVA、 SFRS8、ZFAND3、ZNF76),以及无拷贝数改变的2个基因(TBP和TMEM39A)作为Real-time PCR分析内参。
通过对array CGH实验分析过的56例膀胱癌“起始’样品进行Real-time PCR检测,以“跨技术平台核查”的方式,从上述11个候选基因中筛选出5个(CEP63、 FOSL2、GHR、PAQR6和ZFAND3)候选目的基因用于后续验证。5个候选目的基因的验证在3组独立样本中进行：分别为58例开放性手术切除的膀胱癌冻存组织、62例甲醛固定石蜡包埋(FFPE)膀胱癌组织和43例经尿道切除(TURBT)膀胱癌组织。
结果显示,与对照组30例正常人外周血白细胞基因组DNA比较,CEP63和PAQR6两个基因在全部3组膀胱癌组织样本中均出现拷贝数增加的趋势,其差别具有显著性；而FOSL2和GHR基因拷贝数增加只出现在"FFPE"样本中；ZFAND3基因的拷贝数显著减少可见于"FFPE"样本和"TURBT"样本。值得注意的是,与正常对照组相比,"FFPE'’样本中5个候选目的基因均显示了与芯片结果趋势一致的显著性拷贝数改变。并且,这种改变从正常对照到非浸润性再到浸润性膀胱癌组织中有渐进性的趋势,尤其以CEP63、PAQR6和ZFAND3表现明显。以膀胱癌组织验证结果为基础,进一步分析了这5个基因在膀胱癌患者尿脱落细胞中的拷贝数改变。与对照组患者尿脱落细胞DNA样品相比,PAQR6和FOSL2基因在膀胱癌病例组中拷贝数显著增加,结果与array CGH芯片分析结果一致；但是ROC曲线分析其诊断效能尚不足。
上述研究资料表明,膀胱癌中PAQR6和CEP63等基因的拷贝数改变是在膀胱尿路上皮癌病变组织基因组DNA中的大概率分子事件；这些具有统计学意义的DNA分子改变可在膀胱癌患者尿脱落细胞中检出。若将这些异常改变转化为膀胱癌诊断相关分子标志物,还有待于进一步的缜密分析和大样本多中心验证。
第二部分：乳腺癌新辅助化疗组织病理学治疗反应相关分子标志物的研究
新辅助化疗已成为针对局部进展期的乳腺癌综合治疗中的重要环节；治疗后达到组织病理学完全缓解的患者可长期生存。肿瘤对化疗的反应性是选择治疗方案最重要的依据。传统的病理类型、病理分级等形态学指标尚不足以全面有效地评估新辅助化疗后的组织病理学治疗反应；相关分子标志表达谱对于准确预测新辅助化疗后的组织病理学治疗反应具有重要的辅助意义。
本项研究选择近年报道的与乳腺癌新辅助化疗组织病理学治疗反应有关的蛋白分子CK5/6、EGFR、cyclin B1、14-3-3σ.14-3-3ζ,结合传统病理学分子标志物ER、PR、HER2,联合检测这8个蛋白在新辅助化疗前乳腺癌核芯针穿刺活检组织中的表达情况。对215例浸润性乳腺癌新辅助化疗前的活检穿刺获得的甲醛固定石蜡包埋组织切片进行这8个蛋白的免疫组化染色。收集入组患者手术切除肿瘤组织,进行组织病理学治疗反应的MP分级(Miller&Payne病理分级)。统计分析患者年龄、月经状态、临床分期、病理分级、病理类型,以及上述8个蛋白表达与MP分级的关系。将结果进行Logistic回归分析,建立分子联合检测模型,并绘制ROC曲线；分析候选蛋白分子标志物评估/预测乳腺癌新辅助化疗组织病理学反应的效能。
研究结果显示,8个候选蛋白在215例乳腺癌患者新辅助化疗前核芯针穿刺活检的肿瘤组织中的阳性表达率分别为：ER42.8%. PR42.8%, HER234.0%、 CK5/627.9%、EGFR55.8%、cyclin B147.9%、14-3-3σ28.8%、14-3-3ζ79.5%。 HER2主要表达于绝经后组中,与绝经前组比较,其表达差别具有显著性。ER、PR同步表达,并主要表达于分化较好的肿瘤组织中。而CK5/6主要表达于分化较差的肿瘤组织中。与MP分级(有效/无效)显著相关的病理特征只有组织学分级。蛋白分子标志物之中,ER、PR、CK5/6、EGFR、14-3-3σ在MP有效组和无效组之间的表达具有显著性差异,其P值分别为ER(P<0.001). PR (P=0.001)、CK5/6(P<0.001)、EGFR(P=0.024).14-3-3σ (P=0.017)。经Logistic回归分析建立预测模型,其中ER和14-3-36进入模型。经ROC曲线分析结果显示ER和14-3-3σ联合分析的预测效能显著高于单个分子(ER、PR、CK5/6、EGFR以及14-3-3σ)对新辅助化疗组织病理学治疗反应(MP分级)的预测效能。
本项研究获得的结果为寻找临床适用的乳腺癌新辅助化疗组织病理学治疗反应预测分子标志谱提供了重要线索,为后续大样本多中心研究奠定了工作基础。
This PhD degree project is composed of two parts of study, on molecular markers for bladder cancer and breast cancer.
Part I:Study on DNA copy number aberrations in the genome as molecular biomarkers of bladder urothelial carcinoma
Abnormal change of the DNA structure is common in the carcinogenesis and malignant progression of bladder. These molecular genetic aberrations provide an insight for the molecular mechanism on development of the bladder cancer; and also benefit for discovery and identification of biomarkers for the disease. The purpose of this study was to identify the characteristic DNA copy number aberrations (CNA) in the genome that could serve as molecular biomarkers of bladder urothelial carcinoma.
We previously generated a genome-wide DNA copy number aberration profile in bladder cancer by using the array comparative genome hybridization (array CGH) approach. In this study, we selected11DNA fragments/genes (A_14_P139169, CEP63, CLK1, FOSL2, GHR, LILRA3, PAQR6, PARVA, SFRS8, ZFAND3and ZNF76) with high frequencies of CNA in the genome of bladder cancer, by re-mining the array-CGH data. The copy number aberration of the11genes was firstly examined by real time polymerase chain reaction in56tumor samples that were used for the array CGH analysis. Among the11genes,5(CEP63, FOSL2, GHR, PAQR6and ZFAND3) were verified, for further validation. Three independent sample sets of bladder cancer were applied for the validation; they were58freshly frozen tumor tissues derived from patients of bladder cancer with treated partial cystectomy,62formalin-fixed paraffin-embedded (FFPE) tumor tissues, and43freshly frozen tumor tissues derived from patients treated with transurethral resection of bladder tumor (TURBT). Comparing with30peripheral leukocyte genomic DNA samples of healthy individuals, gain of copy number of both CEP63and PAQR6was detected in all three sample sets of bladder cancer tissues; whereas for FOSL2and GHR, gain of copy number was only observed in the 'FFPE' samples. While loss of copy number for ZFAND3was found in the 'FFPE' samples and 'TURBT' samples. It is noteworthy that, the copy number aberrations of all5genes (CEP63, FOSL2, GHR, PAQR6and ZFAND3) were identified by real-time PCR in the 'FFPE' samples consisting with that of array CGH analysis. Based on data derived from the tumor tissues, we examined5genes in urine sediment samples of123patients with bladder cancer, and found gain of copy number of PAQR6and FOSL2in the bladder cancer group, comparing with patients with tumor of head and neck.
In conclusion, copy number aberration of the5candidate genes could be common molecular events, and can be detected in urine sediments of bladder cancer patients. However, further multicentre study is needed for developing them as diagnostic biomarkers for bladder cancer.
Part Ⅱ:Study on molecular biomarkers of histopathological response to neoadjuvant chemotherapy for breast cancer
Neoadjuvant chemotherapy (NAC) has become an integral part in the comprehensive treatment for breast cancer. Those patients achieve complete pathological response or significant pathological response have a longer disease-free survival and overall survival. To properly estimate pathological response to neoadjuvant chemotherapy is important for choosing therapeutic schedule. The purpose of this study was to identify appropriate molecular biomarkers of histopathological response to neoadjuvant chemotherapy for breast cancer.
A panel of protein markers composed of ER, PR, HER2, CK5/6, EGFR, cyclin B1,14-3-3σ and14-3-3ζ was examined by immunohistochemical (IHC) staining, on core needle biopsy tissue samples collected from215patients with invasive breast cancer, prior to neoadjuvant chemotherapy. With Miller&Payne (MP) criterion, the histopathologic response to NAC was classified upon the tissue section of surgical resected tumor after neoadjuvant chemotherapy. The results of the IHC analysis showed that positive staining rates were, ER42.8%, PR42.8%, HER234.0%, CK5/627.9%, EGFR55.8%, cyclin B147.9%,14-3σ28.8%and14-3-3ζ79.5%. Among them, HER2mainly expressed in the postmenopausal group; ER and PR mainly expressed in the tumors with low grade, whereas CK5/6mainly expressed in the tumors with high grade. Between the groups of MP good response and poor response, there were significantly differential expression of ER (P<0.001), PR (P=0.001), CK5/6(P<0.001), EGFR (P=0.024), and14-3-3σ (P=0.017). The logistic regression analysis showed that only ER and14-3-3σ were included in the prediction model for histopathological response to NAC. Joint detection of ER and14-3-3σ have more power to estimate the pathological response than that any single protein marker did.
引文
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