A Novel EGFR 19、21 Exon Mutation Level Detection Based On Fluorescence Polarization In Lung Cancer Tissue Samples

【Background】 Over the years, lung cancer has been the leading cause of cancer death among males in the world. And according to the latest statistic survey, lung cancer has already excessed breast cancer’s mortality in developed countries to become the first major cancer death in women. Human epidermal growth factor receptor tyrosine inhibitor(EGFR-TKIs) is a small molecule inhibitor for lung cancer which could express abnormal EGFR to activate the signaling pathways of the downstream. It is a chemical synthesis of targeted therapies, cancer in non-small-cell lung cancer, especially in patients with sensitive EGFR mutations contain will have a more significant effect. It has been approved as monotherapy in advanced metastatic non-small-cell lung cancer in the second or third-line therapy, or the first-line therapy in some patients. However, with the recent incidence of drug resistance and efficacy differences having been reported, many sensitive and resistant predictors of these drugs have gradually been discovered and applied in clinical patient screening. For example, in spite of containing the same EGFR mutation, different individuals also mayshowed differences in the efficacy of EGFR-TKIs in clinical treatment. Based on this phenomenon, we propose hypothesis: whether mutations have different levels in tumor tissues, and this heterogeneity may cause differences in the efficacy of targeted therapies? Fluorescence polarization technique is a fluorescent marker detection technology that can detect different wavelengths of the fluorescence intensity values, particularly suitable for mutation and mutation abundance detection aspects. At present, as most EGFR mutation detection techniques fail to detect mutation in the tumor abundance, so as can’t provide information for the mutation content levels. For this purpose, we have designed a new method basing on the advantages of fluorescence polarization detection technology in terms of gene mutation to detect mutations levels in the lung pathology specimens for the aim to apply in the clinical treatment work.【Purposes】 Establish a rapid, sensitive, and specific detection technology system based on fluorescence polarization. And apply the method to detect the EGFR 19、21 mutation containing in the lung cancer tissues, for a more comprehensive and accurate targeting predictor of drug efficacy and clinical rational use of targeted drugs. Acceptance of gefitinib treated patients were followed up, statistical response rate, and survival further prove the predictor function of the EGFR mutation abundance detection.【Methods】 The first step is to establish a standard fluorescence polarization detection system. Extract wild-type genome DNA and mutation ones of EGFR 19、21 exons from the patients. Design primers and specific fluorescently labeled probes. Then, construct four kinds of wild-type and mutant recombinant plasmid standard substances. Finally, after fluorescence polarization PCR reaction, obtained the amplified products and detected FP values using fluorescence polarization. Make a liner equation according to the FP value and corresponding standard quality copy number. After further stability, specificity, and the sensitivity verification, we make it as a quantitative criteria system. Pathological specimens from lung cancer are detected in the second part of the study for the mutationabundance of EGFR 19, 21 mutations. After ordinary PCR reaction, send the products for a sequencing, therefore, we can check the two method exactly. In addition, follow the 58 cases of patients who receive a targeted therapy, and use statistical methods to compare the relationship between the two groups.【Results】 Standard system established in this study has been fully validated with a good stability, high sensitivity, exact specificity. And the minimum detectable concentration is 40 copies/ul, the minimum detectable content is to 10%. The test results for samples is basically the same with the direct sequencing. The abundance of mutations in patient using Gefitinib is proportionable to its efficacy and survival. And the differences between the two groups are significant(p<0.05).【Conclusion】 Fluorescence polarization(FP) detection technique is a fluorescent labeling detection technology that can detect the fluorescence intensity at different wavelengths, especially for the detection of gene mutation and mutation abundance aspects. Because it is easy to operate, and can provides a comprehensive analysis of intratumoral heterogeneity. Therefore, this study will provide a new way to detect the mutation levels in the cancer tissue, and it will also guide individual treatment of lung cancer with a good technology platform.