实时荧光PCR在肿瘤护理诊断中的应用研究
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摘要
肿瘤的发生机制和治疗一直是医疗卫生领域的难题之一。随着分子生物学技术的发展,肿瘤的发生机制逐渐被揭示,为肿瘤的预防,控制和治疗提供了新的依据和途径。肿瘤是一种高度异质性的疾病,从分子生物学角度来讲,涉及非常多的基因改变,包括基因突变,表达,表观遗传学等多个领域。不同的肿瘤类型,不同的肿瘤患者,不同的发病部位,不同的组织学类型都涉及了不同基因的不同改变形式,而这些改变形式与肿瘤患者的预后及对治疗的反应性相关。因此,针对肿瘤异质性,以患者分子生物学状态为依据的个体化治疗应运而生。从肿瘤的预防,早期诊断到肿瘤的控制和治疗等一系列过程中都需要对患者的分子生物学状态进行深入的分析后才能给出具体的方案。而这一过程的实施主要依赖于肿瘤的护理诊断。通过对肿瘤特异性的生物标记物进行检测和分析,得到肿瘤的相关分子生物学信息后,依据生物学信息制定个体化的治疗方案。因此,快速准确地检测这些标记物在临床上十分重要。本论文以实体瘤个体化治疗中涉及的生物标记物为研究对象,以实时PCR技术为平台,建立相应分子诊断技术用于临床中基因突变和基因表达的检测和定量,服务于肿瘤的诊断,预后评估和靶向治疗。
第一章绪论
本章回顾了肿瘤的个体化治疗以及护理诊断的概念,总结了目前在肿瘤护理诊断中研究相对透彻的生物标记物及其诊断方法,着重讲述了实时PCR技术的原理和应用,最后提出了本论文的研究目的,内容及意义。
第二章探针溶解曲线技术检测非小细胞肺癌EGFR基因突变
非小细胞肺癌EGFR基因突变状态与靶向药物吉非替尼和厄罗替尼的药物疗效相关。中国人群中EGFR基因突变率约为30%,因此对EGFR基因突变状态的检测可以有效筛选药物受益人群。本研究针对EGFR基因突变数量多(29种突变),突变类型复杂(包括单碱基替换,插入,缺失三种类型突变)的情况,利用探针熔解曲线技术,并结合突变富集技术,实现了6管反应体系对29种突变类型的同时检测,经优化后体系对突变的检测能力高达0.1%,即可以检测到1000个细胞中混有的1个突变细胞。此外,该检测体系通过对141份肺癌组织标本进行检测发现阳性标本38份,阳性率为27.0%,并经过测序验证,与已有文献报道一致。实时PCR技术的应用,缩短了检测时间,只需经过1.5个小时的PCR扩增和30分钟的熔解曲线分析即可得到检测结果,多重体系的使用简化了操作,节约了试剂和标本用量,也大大降低了检测成本。总之,该检测体系具有快速,简便,准确,成本低等优点,具有较大的临床应用价值。
第三章多重定量PAP技术用于EGFR基因和KRAS基因突变检测
肿瘤体细胞突变检测面临的最大挑战是需要从大量的野生背景DNA中检测到微量的突变DNA,因此要求检测技术具有较强的抗干扰能力和高选择性,以实现稀有突变检测。本研究立足于稀有突变的检测要求,在传统PAP技术的基础上,在PAP引物5'端引入一段标签序列,作为DNA探针结合序列,并加入DNA探针检测实现实时PCR技术与PAP技术的结合,建立具有高选择能力的标签双PAP技术用于检测低突变含量的标本。该技术首先应用于肺癌EGFR基因突变检测,然后进一步用于结直肠癌KRAS基因突变检测,检测结果证明该技术解决了传统PAP技术需要PCR产物后处理的问题,闭管操作防止污染,同时简化了操作步骤；此外,该检测体系保持了传统PAP技术对突变的富集能力,选择性高达0.01%,非常适合稀有突变的检测；最后,标签序列及其检测探针的引入实现了多重扩增体系的建立,通过在体系中引入内控,使该检测体系具备对突变含量定量的能力,可以为临床提供更多信息帮助医生制定有效的治疗方案。
第四章多重qRT-PCR技术用于乳腺癌复发风险相关21基因定量
基因表达作为生物标记物用于指导用药和预后已经越来越广泛的被应用到临床中。本研究基于21基因表达谱对早期乳腺癌患者复发风险的评估作用,以实时PCR技术为平台,建立多重qRT-PCR技术实现6管反应体系对21个基因表达水平的同时定量。该定量体系通过设计小片段扩增产物实现对发生部分降解的FFPE标本RNA的定量；扩增引物5'端同源加尾设计使21基因可以同时被一条引物扩增,实现不同基因间扩增效率的一致性(同一反应体系中各基因扩增效率差不超过5%)；通过对不同扩增产物检测探针标记不同荧光基团实现了一个反应体系中多个基因的同时扩增和检测。该体系经过稳定性评估其扩增Cq值的批间差不超过5%,稳定性好。此外,多重qRT-PCR检测体系与免疫组化结果有非常好的一致性,证明了该体系的定量准确性。最后,我们用70份乳腺癌FFPE标本对多重qRT-PCR检测体系在乳腺癌复发风险预测上的作用进行了评估,结果证明该体系通过对21基因定量结果进行分析后计算复发分数将患者分为高复发风险和低复发风险两组,两组的实际复发率差异显著(P<0.05)。多重qRT-PCR检测体系减少了反应数,简化了操作,节约标本用量,降低了成本,通过与免疫组化技术的比对证明其定量的准确性,非常适合临床使用。
Tumorigenesis and treatment is one of the difficult issue for medical treatment and public health system. As the development of molecular biology, the mechanism of tumorigenesis is discovered gradually and provide new approach for the prevention, control and therapy of tumor. Tumor is a kind of disease with high heterogeneity, in the field of molecular biology, it refers to lots of gene variations including gene mutaion, expression, epigenetics and so on. Different tumor types, patients, tumor locations and histologic types contain different kinds of gene variations, and all these gene variations are related to patients prognosis and response to therapy. Therefore, the personalized medicine based on molecular biology comes with the tumor heterogeneity. The therapy decision should be made based on the individualized situation in the course of tumor prevention, early diagnosis, control and therapy. This process can be achieved only by companion diagnosis. Companion diagnosis could give doctor more information by detecting tumor biomakers. Therefore, it is important to detect these biomakers rapidly and accurately. Our study established molecular diagnostic technology based on real-time PCR to detect gene mutation and expression.
In chapter one, we firstly recalled the concept of personalized medicine and companion diagnosis in tumor, review the currently used biomakers which have been studied clearly and corresponding detecting technologies. We then illustrated the prineciple and application of real-time PCR. Finally, the objective and content of this dissertation were proposed.
In chapter two, a multiplex real-time PCR system based on probe melting curve technology was developed for screening of29mutations in EGFR gene. Approximately30%of patients with lung cancer contained EGFR gene mutaions and these mutations were related to the efficacy of gefitinib and erlotinib. Our detection system used6reactions to detect29mutations simultaneously and had a high selectivity up to0.1%which means1mutaion cell exist in1000cells can be detected by our detection system. In the141lung cancer samples we detected,38samples were found to be mutaion positive and validated by DNA sequencing which was consistent with previous reports. The application of multiplex PCR in the system makes the detection can be done within2hours and save reaction reagent and sample, reduce the cost. It is a rapid, simple, accurate and cost-effective way to apply to the clinical.
In chapter three, we established a new technology called real-time Tag-Bi-PAP based on traditional PAP technology to detect the rare mutaion from wild type backgroud. A tag sequence were added to the5'terminus of PAP primers to hybridize with DNA probe and make the PAP can be done in a real-time way. This new method was fisrtly used to detect EGFR mutations and then apply to KRAS gene mutations. The results showed that real-time Tag-Bi-PAP could give a selectivity of0.01%. There is no need to make PCR products postprocessing compared with the traditional PAP, so it is simple and rapid. Furthermore, the adding of internal control gene in the reaction system endowed the technology with the ability of quantifing the mutation content and provided more information to help doctor make a decision.
In chapter four, we developed a multiplex qRT-PCR system to quantify mRNA levels of21genes.21-gene was a expression profile used for evaluating the recurrence risk of patients with breast cancer. We used6reactions to detect the21gene expression simultaneously based on real-time PCR (multiplex qRT-PCR). This multiplex qRT-PCR system can be used to detect FFPE sample which was fragmented due to degradation by designing short amplification products. Different genes had a similar amplification efficiencies by using only one primer to amplify all the21genes(the difference of amplification efficiencies between genes were within5%). The interassay variances (%CV) of Cq values for each gene were all within5%, indicating stability of the multiplex qRT-PCR system. Furthermore, the quantitative results from multiplex qRT-PCR system were consistent with the results from immunohistochemistry which demonstrated the accuracy of this system. Finally,70FFPE samples were used to validate whether the21-gene proflie quantified by multiplex qRT-PCR system had a predictive function on recurrence risk of breast cancer in china. The results showed a significant difference in recurrence rate between high risk recurrence group and low risk recurrence group (P<0.05). The multiplex qRT-PCR system reduced the reation numbers, simplfied the manipulation, saved the samples and cost.
第一章绪论
本章回顾了肿瘤的个体化治疗以及护理诊断的概念,总结了目前在肿瘤护理诊断中研究相对透彻的生物标记物及其诊断方法,着重讲述了实时PCR技术的原理和应用,最后提出了本论文的研究目的,内容及意义。
第二章探针溶解曲线技术检测非小细胞肺癌EGFR基因突变
非小细胞肺癌EGFR基因突变状态与靶向药物吉非替尼和厄罗替尼的药物疗效相关。中国人群中EGFR基因突变率约为30%,因此对EGFR基因突变状态的检测可以有效筛选药物受益人群。本研究针对EGFR基因突变数量多(29种突变),突变类型复杂(包括单碱基替换,插入,缺失三种类型突变)的情况,利用探针熔解曲线技术,并结合突变富集技术,实现了6管反应体系对29种突变类型的同时检测,经优化后体系对突变的检测能力高达0.1%,即可以检测到1000个细胞中混有的1个突变细胞。此外,该检测体系通过对141份肺癌组织标本进行检测发现阳性标本38份,阳性率为27.0%,并经过测序验证,与已有文献报道一致。实时PCR技术的应用,缩短了检测时间,只需经过1.5个小时的PCR扩增和30分钟的熔解曲线分析即可得到检测结果,多重体系的使用简化了操作,节约了试剂和标本用量,也大大降低了检测成本。总之,该检测体系具有快速,简便,准确,成本低等优点,具有较大的临床应用价值。
第三章多重定量PAP技术用于EGFR基因和KRAS基因突变检测
肿瘤体细胞突变检测面临的最大挑战是需要从大量的野生背景DNA中检测到微量的突变DNA,因此要求检测技术具有较强的抗干扰能力和高选择性,以实现稀有突变检测。本研究立足于稀有突变的检测要求,在传统PAP技术的基础上,在PAP引物5'端引入一段标签序列,作为DNA探针结合序列,并加入DNA探针检测实现实时PCR技术与PAP技术的结合,建立具有高选择能力的标签双PAP技术用于检测低突变含量的标本。该技术首先应用于肺癌EGFR基因突变检测,然后进一步用于结直肠癌KRAS基因突变检测,检测结果证明该技术解决了传统PAP技术需要PCR产物后处理的问题,闭管操作防止污染,同时简化了操作步骤；此外,该检测体系保持了传统PAP技术对突变的富集能力,选择性高达0.01%,非常适合稀有突变的检测；最后,标签序列及其检测探针的引入实现了多重扩增体系的建立,通过在体系中引入内控,使该检测体系具备对突变含量定量的能力,可以为临床提供更多信息帮助医生制定有效的治疗方案。
第四章多重qRT-PCR技术用于乳腺癌复发风险相关21基因定量
基因表达作为生物标记物用于指导用药和预后已经越来越广泛的被应用到临床中。本研究基于21基因表达谱对早期乳腺癌患者复发风险的评估作用,以实时PCR技术为平台,建立多重qRT-PCR技术实现6管反应体系对21个基因表达水平的同时定量。该定量体系通过设计小片段扩增产物实现对发生部分降解的FFPE标本RNA的定量；扩增引物5'端同源加尾设计使21基因可以同时被一条引物扩增,实现不同基因间扩增效率的一致性(同一反应体系中各基因扩增效率差不超过5%)；通过对不同扩增产物检测探针标记不同荧光基团实现了一个反应体系中多个基因的同时扩增和检测。该体系经过稳定性评估其扩增Cq值的批间差不超过5%,稳定性好。此外,多重qRT-PCR检测体系与免疫组化结果有非常好的一致性,证明了该体系的定量准确性。最后,我们用70份乳腺癌FFPE标本对多重qRT-PCR检测体系在乳腺癌复发风险预测上的作用进行了评估,结果证明该体系通过对21基因定量结果进行分析后计算复发分数将患者分为高复发风险和低复发风险两组,两组的实际复发率差异显著(P<0.05)。多重qRT-PCR检测体系减少了反应数,简化了操作,节约标本用量,降低了成本,通过与免疫组化技术的比对证明其定量的准确性,非常适合临床使用。
Tumorigenesis and treatment is one of the difficult issue for medical treatment and public health system. As the development of molecular biology, the mechanism of tumorigenesis is discovered gradually and provide new approach for the prevention, control and therapy of tumor. Tumor is a kind of disease with high heterogeneity, in the field of molecular biology, it refers to lots of gene variations including gene mutaion, expression, epigenetics and so on. Different tumor types, patients, tumor locations and histologic types contain different kinds of gene variations, and all these gene variations are related to patients prognosis and response to therapy. Therefore, the personalized medicine based on molecular biology comes with the tumor heterogeneity. The therapy decision should be made based on the individualized situation in the course of tumor prevention, early diagnosis, control and therapy. This process can be achieved only by companion diagnosis. Companion diagnosis could give doctor more information by detecting tumor biomakers. Therefore, it is important to detect these biomakers rapidly and accurately. Our study established molecular diagnostic technology based on real-time PCR to detect gene mutation and expression.
In chapter one, we firstly recalled the concept of personalized medicine and companion diagnosis in tumor, review the currently used biomakers which have been studied clearly and corresponding detecting technologies. We then illustrated the prineciple and application of real-time PCR. Finally, the objective and content of this dissertation were proposed.
In chapter two, a multiplex real-time PCR system based on probe melting curve technology was developed for screening of29mutations in EGFR gene. Approximately30%of patients with lung cancer contained EGFR gene mutaions and these mutations were related to the efficacy of gefitinib and erlotinib. Our detection system used6reactions to detect29mutations simultaneously and had a high selectivity up to0.1%which means1mutaion cell exist in1000cells can be detected by our detection system. In the141lung cancer samples we detected,38samples were found to be mutaion positive and validated by DNA sequencing which was consistent with previous reports. The application of multiplex PCR in the system makes the detection can be done within2hours and save reaction reagent and sample, reduce the cost. It is a rapid, simple, accurate and cost-effective way to apply to the clinical.
In chapter three, we established a new technology called real-time Tag-Bi-PAP based on traditional PAP technology to detect the rare mutaion from wild type backgroud. A tag sequence were added to the5'terminus of PAP primers to hybridize with DNA probe and make the PAP can be done in a real-time way. This new method was fisrtly used to detect EGFR mutations and then apply to KRAS gene mutations. The results showed that real-time Tag-Bi-PAP could give a selectivity of0.01%. There is no need to make PCR products postprocessing compared with the traditional PAP, so it is simple and rapid. Furthermore, the adding of internal control gene in the reaction system endowed the technology with the ability of quantifing the mutation content and provided more information to help doctor make a decision.
In chapter four, we developed a multiplex qRT-PCR system to quantify mRNA levels of21genes.21-gene was a expression profile used for evaluating the recurrence risk of patients with breast cancer. We used6reactions to detect the21gene expression simultaneously based on real-time PCR (multiplex qRT-PCR). This multiplex qRT-PCR system can be used to detect FFPE sample which was fragmented due to degradation by designing short amplification products. Different genes had a similar amplification efficiencies by using only one primer to amplify all the21genes(the difference of amplification efficiencies between genes were within5%). The interassay variances (%CV) of Cq values for each gene were all within5%, indicating stability of the multiplex qRT-PCR system. Furthermore, the quantitative results from multiplex qRT-PCR system were consistent with the results from immunohistochemistry which demonstrated the accuracy of this system. Finally,70FFPE samples were used to validate whether the21-gene proflie quantified by multiplex qRT-PCR system had a predictive function on recurrence risk of breast cancer in china. The results showed a significant difference in recurrence rate between high risk recurrence group and low risk recurrence group (P<0.05). The multiplex qRT-PCR system reduced the reation numbers, simplfied the manipulation, saved the samples and cost.
引文
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