【作者】 沈洁；

【导师】 刘宝瑞； 管文贤；

【作者基本信息】 南京大学 ， 内科学（肿瘤学）， 2013， 博士

【摘要】 第一部分 血浆游离TS mRNA与抗叶酸代谢类药物雷替曲塞敏感性关系的探索目的:雷替曲塞为新一代水溶性抗叶酸制剂;胸苷酸合成酶(Thymidylate Synthase,TS)是抗叶酸代谢途径的关键分子,是雷替曲塞等抗叶酸代谢类药物发挥抗肿瘤作用的最重要靶点之一。同时,近年来研究表明外周血浆游离mRNA来源于肿瘤组织,且包含着丰富的肿瘤信息。本研究从胃癌组织和血浆游离TS mRNA入手,初步探讨其与雷替曲塞药物敏感性之间的关系,为可能的实时个体化治疗提供客观依据。方法:系统收集125例经病理确诊的新鲜人胃癌标本和对应的术前血样。采用三维微组织块培养法(HDRA)行雷替曲塞的体外药物敏感试验;实时荧光定量PCR检测对应胃癌组织和术前血浆游离TS mRNA水平。分析肿瘤组织与血浆TS mRNA之间的相关性;TS mRNA水平与雷替曲塞药物敏感之间的相关性。结果:125例标本中,血浆TS mRNA在109人中顺利检出,检出率为87.2%。其中,Ⅰ、Ⅱ、Ⅲ和Ⅳ期患者分别为9例,26例,72例和2例。平均年龄为63岁,年龄范围29～83岁。其中男性77例,女性32例。病理类型全部为胃腺癌,其中粘液腺癌和印戒细胞癌各为13和10例。87例有淋巴结转移。新鲜胃癌组织对雷替曲塞的敏感性、胃癌组织和血浆游离TS mRNA水平均与临床特征无明显相关性。血浆游离TS mRNA水平与肿瘤组织TS mRNA水平呈正相关(rho= 0.653,P<0.001),且胃癌患者的TS mRNA 水平明显高于健康人群(肿瘤患者:1.20;正常人:0.31;P= 0.009)。雷替曲塞敏感组与耐药组的肿瘤组织TS mRNA相对表达量分别为 7.80(95%CI:6.14-10.20)和 12.59(95%CI:9.05-17.38,P=0.007),敏感组与耐药组的血浆游离TS mRNA相对表达量分别为0.91(95%CI:0.61-1.20)和1.62(95%CI:1.03-2.26,P = 0.013)。肿瘤组织TS mRNA预测雷替曲塞敏感性的灵敏度为70%,准确性为68%;血浆TS mRNA预测雷替曲塞敏感性的灵敏度为82%,准确性为60%。结论:本实验初步证实血浆游离TS mRNA水平与对应胃癌组织TS mRNA水平呈正相关,在一定程度上能反映肿瘤组织的生物学信息。胃癌组织和血浆游离TS mRNA水平与新鲜胃癌组织对雷替曲塞的敏感性呈负相关,是潜在的胃癌雷替曲塞疗效预测标志,为大样本临床研究的开展奠定基础。第二部分药物疗效相关分子在血浆中的表达与胃癌常用化疗药物敏感性关系目的:生物标志指导下的肿瘤个体化药物治疗是迄今提高治疗效果、减少无效治疗公认的最有价值的新的用药方式。然而,肿瘤治疗过程中可以出现新的突变及表观遗传学等的变化,引起肿瘤细胞生物学特性的变化,改变化疗敏感性。此外,在临床实践中,以肿瘤组织为检查对象存在组织难以获得、原发灶与转移灶存在部位和时间上差异等实际问题。因而迫切需要找到能实时有效地监测肿瘤药物敏感性状况的新的生物标志体系,从而达到在治疗过程中能适时调整治疗方案的目的。本部分研究拟以目前常用化疗药物(包括多西紫杉醇、顺铂、培美曲塞、吉西他滨、伊立替康)疗效相关性比较公认的生物标志为研究对象,探索与验证这些生物标志在血浆中表达水平与这些药物敏感性和疗效之间的关系。方法:系统收集150例经病理确诊的新鲜胃癌标本和对应的术前血样,采用三维微组织块培养法(HDRA)行多西紫杉醇、顺铂、培美曲塞、吉西他滨、伊立替康的体外药物敏感试验;实时荧光定量PCR检测胃癌石蜡组织和对应外周血中BRCA1,TS,ERCC1,hENT1,RRM1,Topo1,APTX mRNA水平。比较分析胃癌患者血浆和肿瘤组织中上述分子的表达水平之间相关性;并探讨血浆游离核酸表达水平与相应肿瘤组织药物敏感性的相关性。建立人原代胃癌荷瘤裸鼠模型并体内验证以上生物标志的疗效预测价值。结果:外周血浆游离BRCA1,ERCC1,hENT1,RRM1,TS,Topo1,APTX mRNA水平与对应肿瘤组织中相应基因水平呈正相关(P<0.001)。血浆/肿瘤组织BRCA1mRNA水平与新鲜胃癌组织的多西紫杉醇敏感性正相关(P<0.001);血浆/肿瘤组织TS mRNA水平与新鲜胃癌组织的培美曲塞敏感性负相关(P<0.001);血浆/肿瘤组织ERCC1 mRNA水平与新鲜胃癌组织的铂类敏感性负相关(血浆P=0.012;肿瘤P<0.001);血浆/肿瘤组织RRM1 mRNA水平与新鲜胃癌组织的吉西他滨敏感性负相关(血浆P=0.004;肿瘤P<0.001);血浆/肿瘤组织Topo1 mRNA水平与新鲜胃癌组织的伊立替康敏感性正相关(血浆P=0.015;肿瘤P =0.013)。血浆游离mRNA预测化疗敏感性具有较好的敏感性和特异性(P<0.001)。血浆ERCC1低表达的患者更易从含铂的化疗方案中获益(546天 vs.355天,HR:0.31,P = 0.003)。结论:本实验证实血浆游离mRNA水平与对应胃癌组织mRNA水平呈正相关,能反映肿关性,可通过简单无创的外瘤组织的生物学信息。药物疗效相关分子在血浆中的表达与胃癌常用化疗药物敏感性具有相周血获得与药物敏感性相关的分子表达信息。血浆游离生物标志是潜在的实时疗效预测标志,为胃癌实时个体化治疗提供理论基础。第三部分血浆游离生物标志组合对胃癌伊立替康敏感性的预测作用目的:伊立替康(Irinotecan,CPT-11)是大肠癌标准化疗最常用的药物之一,也是胃癌常用二线药物。CPT-11能与拓扑异构酶1(Topoisomerase1,Topo1)形成Topo1-药物-DNA复合物,抑制DNA重连并导致DNA双链断裂。Topo1诱导的DNA断裂的修复需要APTX,BRCA1和ERCC1等DNA修复通路上关键基因的参与,因此这些基因水平与CPT-11抗肿瘤效果可能具有潜在相关性。本研究从胃癌组织和外周血浆游离APTX,BRCA1,ERCC1,ISG15和Topo1 mRNA入手,初步探讨其与伊立替康药物敏感性之间的关系,为可能的胃癌伊立替康实时个体化治疗提供客观依据。方法:系统收集175例经病理确诊的新鲜人胃癌标本和对应的术前血样。采用三维微组织块培养法(HDRA)行伊立替康的体外药物敏感试验;实时荧光定量PCR检测胃癌组织和外周血浆游离APTX,BRCA1,ERCC1,ISG15和Topo1 mRNA水平。分析肿瘤组织和外周血浆游离APTX,BRCA1,ERCC1,ISG15和Topo1 mRNA水平与胃癌伊立替康药物敏感之间的相关性。通过多因素线性回归模型,建立胃癌伊立替康敏感性的疗效预测指数。构建胃癌荷瘤裸鼠模型,体内验证该疗效预测指数对伊立替康敏感性的预测价值。结果:175例患者中模型构建的测试组为135人,模型验证组为40人。患者平均年龄为63岁,年龄范围29～83岁,其中男性131例,女性44例。病理类型全部为胃腺癌,其中粘液腺癌和印戒细胞癌各为17和13例。绝大多数标本分化程度为中分化或者低分化;135例有淋巴结转移;Ⅰ、Ⅱ期患者为61例,Ⅲ、Ⅳ期患者为114例。伊立替康敏感组的患者其肿瘤组织APTX(P<0.001),BRCA1(P<0.001)和ERCC1(P<0.001)的水平明显低于耐药组,而ISG15(P=0.047)和Topo1(P= 0.002)的水平明显高于耐药组;伊立替康敏感组的患者其血浆APTX(P=0.006)和BRCA1(P = 0.019)的水平明显低于耐药组,ERCC1的水平有低于耐药组的趋势(P=0.14),而ISG15(P<0.001)和Topo1(P= 0.001)的水平明显高于耐药组。基于肿瘤组织各基因水平建立的伊立替康敏感性预测指数为Index =0.488-0.020× APTX mRNA表达水平+ 0.015× Topo1 mRNA表达水平-0.011 × BRCA1 mRNA表达水平。线性回归分析显示此Index与伊立替康抑制率呈显著相关(rho = 0.71,P<0.001)。ROC曲线分析,该Index预测伊立替康的敏感性的灵敏度为73%,特异性为86%,曲线下面积AUC达0.828(95%CI:0.755-0.901,P<0.001)。基于血浆各基因水平建立的伊立替康敏感性预测指数为Index =0.447-0.130× APTX mRNA 表达水平-0.051× BRCA1 mRNA 水平-0.166 ×ERCC1 mRNA 水平 + 0.026 × ISG15 mRNA 水平 + 0.110 × Topo1 mRNA 表达水平。血浆Index与伊立替康抑制率具有显著相关性(rho = 0.642,P<0.001)。ROC曲线分析,该Index预测伊立替康的敏感性的灵敏度为63%,特异性为87%,曲线下面积AUC达0.783(95%CI:0.702-0.865,P<0.001)。结论:本实验初步证实胃癌组织和血浆APTX,BRCA1和ERCC1 mRNA水平与伊立替康敏感性呈负相关,ISG15和Topo1 mRNA水平与伊立替康敏感性呈正相关。基于以上基因构建的伊立替康疗效预测指数能更精准地区分伊立替康敏感与不敏感的人群,是潜在的胃癌伊立替康疗效的预测标志,为大样本临床研究的开展奠定基础。更多还原

【Abstract】 Part Ⅰ Thymidylate synthase(TS)mRNA levels in plasma and tumor as potential predictive biomarkers for raltitrexed sensitivity in gastric cancerBackground:Different chemotherapeutic agents currently available are effective only in certain subsets of patients.Predictive biomarkers will be helpful in choosing those agents and can improve the clinical efficiency by a more personalized chemotherapeutic approach.Raltitrexed is a novel water-soluble quinazoline folate analogue and can improve the efficiency of gastric cancer treatment,but its predictive biomarker remains unclear.The aim of this study was to investigate the role of plasma and tumor TS mRNA levels as predictive biomarkers for raltitrexed in gastric cancer.Methods:125 freshly-removed gastric tumor specimens and corresponding blood samples before surgery were collected.Raltitrexed sensitivity was determined by histoculture drug response assay(HDRA)procedures.TS mRNA levels in tumor and plasma were determined by quantitative RT-PCR.Results:Plasma TS mRNA level in cancer patients was significantly higher than in healthy subjects(P = 0.009)and was significantly correlated with TS mRNA level in tumor tissues(r=0.665,P<0.001).Tumor and plasma TS mRNA expression levels were significantly lower in raltitrexed-sensitive group than in resistant group(P = 0.007,P = 0.013,respectively).The sensitivity and accuracy of raltitrexed sensitivity prediction based on plasma TS mRNA levels were 82%and 60%,respectively;whereas the prediction based on tumor TS mRNA reached 70%sensitivity and 68%accuracy.Conclusion:These results indicate that TS mRNA level in plasma can mirror tumor TS mRNA level,and both of them can be used to predict raltitrexed sensitivity in gastric cancer.Part Ⅱ Plasma mRNA expression levels as potential real-time predictive biomarkers for chemosensitivity in gastric cancerBackground:Personalized chemotherapy based on predictive biomarkers can maximize efficacy.However,tumor tissue obtained at the time of initial diagnosis will not reflect genetic alterations observed at the time of clinical progression.Biomarker from blood could be a useful and non-invasive tool to provide real-time information in the whole procedure of treatment.Methods:In 150 gastric cancer patients,mRNA levels of BRCA1,TS,ERCC1,hENTl,RRM1,Topol and APTX were assessed in plasma and paired formalin-fixed paraffin-embedded tumor tissue and correlated with chemosensitivity to docetaxel,pemetrexed,platinum,gemcitabine and irinotecan.Results:Significant correlations were observed between plasma and tumor mRNA levels of BRCA1,ERCC1,hENTl,RRM1,TS,Topol,and APTX(P<0.001 for all genes).Correlations were also observed between the mRNA expression of plasma/tumor BRCA1 levels and docetaxel sensitivity(P<0.001),plasma/tumor TS and pemetrexed sensitivity(P<0.001),plasma/tumor ERCC1 and platinum sensitivity(plasma,P = 0.012;tumor,P<0.001),plasma/tumor RRM1 and gemcitabine sensitivity(plasma,P = 0.004;tumor,P<0.001),and plasma/tumor Topol and irinotecan sensitivity(plasma,P = 0.015;tumor,P = 0.013).Plasma mRNA levels showed considerable sensitivity and specificity in predicting chemosensitivity(P<0.001).Gastric cancer patients with low levels of plasma ERCC1 mRNA could benefit from platinum-based chemotherapy(546 days vs.355 days,HR:0.31,P = 0.003).Conclusion:Plasma mRNA expression levels mirror those in the tumor and can be used as promising predictive biomarkers to predict chemosensitivity.Part Ⅲ Gene signature in plasma and tumor predicts irinotecan chemosensitivity in gastric cancerBackground:Personalized chemotherapy based on molecular biomarkers can maximum efficiency and reduce adverse effects.We investigated and validated predictive biomarkers for irinotecan and developed a three-gene signature that is closely associated with chemosensitivity to irinotecan in gastric cancer patients.Methods:We examined gene expression of APTX,BRCA1,ERCC1,ISG15 and Topol in plasma and paired formalin-fixed paraffin-embedded gastric cancer tissues from 175 patients and evaluated the association between gene expression levels and in vitro sensitivity to irinotecan.We used multiple linear regression analysis to develop a gene-expression model to predict irinotecan sensitivity in gastric cancer and validated this model in vitro and vivo.Results:The tumor gene expression levels of APTX(P<0.001),BRCA1(P<0.001)and ERCC1(P<0.001)were significantly lower in irinotecan-sensitive gastric cancer samples than those irinotecan-resistant samples,while ISG15(P = 0.047)and Topo1(P = 0.002)were significantly higher.The plasma gene expression levels of APTX(P = 0.006)and BRCA1(P = 0.019)were significantly lower in irinotecan-sensitive gastric cancer samples than those irinotecan-resistant samples,while ISG15(P<0.001)and Topo1(P = 0.001)were significantly higher.Based on those genes,plasma and tumor gene signature for irinotecan sensitivity prediction were established,respectively.The tumor gene signature was significantly associated with irinotecan sensitivity(rho = 0.71,P<0.001).The sensitivity and specificity for the prediction of irinotecan sensitivity based on the tumor gene signature reached 73%and 86%,respectively.We also validated the model in another independent set of gastric cancer samples and two cohorts of immunodeficient mice models with patient-derived gastric cancer xenografts.The plasma gene signature was significantly associated with irinotecan sensitivity(rho = 0.642,P<0.001).The sensitivity and specificity for the prediction of irinotecan sensitivity based on the plasma gene signature reached 63%and 87%,respectively.We also validated the model in another independent set of gastric cancer samples(65%vs.22%,P<0.001).Conclusions:The gene signatures established herein based plasma and tumor gene expression levels are closely associated with irinotecan sensitivity among patients with gastric cancer.更多还原