The Research Of Real-time PCR In Companion Diagnostics Of Human Cancer Therapies

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.