Using Target Capture Next Generation Sequencing To Detect Driver Gene Mutations As Predictive Factor For Targeted Therapies In Non-small Lung Cancer

The incidence of cancer in China is increasing year by year,and lung cancer is the most common incident cancer and the leading cause of cancer death.More and more lung cancer related mutations have been discovered,and pharmaceutical companies have developed a series of targeted drugs target on these mutations.The drug efficacy is affected by the gene mutations on downstream of the pathways or gene mutations on other related signal pathways.The signal pathway of each gene mutation and downstream or other drug pathway gene mutation effect has a relationship,therefore,to choose appropriate targeted drugs,it is necessary to do a comprehensive evaluation on all the related targeted genes based on tumor cells.At present,the two main testing technology are fluorescent quantitative PCR and Sanger sequencing,which all can only be used for single gene detection of the mutation of known genes.The aim of this study is to develop a panel by target capture next generation sequencing(sequencing by synthesis)for detecting all genes in a single test,which are common driver genes of lung cancer and related to the efficacy of targeted drugs in lung cancer.According to the NCCN guideline,FDA official website,COSMIC database,etc.,a total of 8 genes,EGFR,ALK,ROS1,MET,RET,HER2,BRAF,KRAS were selected.The IDT captured probes were synthesized which involved all the exons and part of intron regions of 8 genes,including ends of the extension of the 10 bp region.In this study,the mutant standard HD733 and wild type standard HD752 of Horizon company and the clinical samples of known results were selected as the experimental objects.These two standards contain a number of lung cancer related genes,known mutation sites and frequencies.Mutations of HD733 is different with HD752.To evaluate the performance of the panel affected by different DNA initial amount,HD733 standard test were designed for detection gradient initial amount,100 ng and 30 ng respectively.After DNA extraction,DNA library and high-throughput sequencing,sequencing depth chose more than 300.The experimental data showed that the ratio of on target was more than 53% and the ratio of Uniformity%(>30%mean)in more than 99.9%.The data indicated that the sequencing uniform quality of designed probe was fine.The mutant contained in the standard were detected,and no false positive sites were detected,the sensitivity and specificity meet the demand,the performance of panel was well qualified.The genetic variations of HD752 and HD733 only have point mutation and deletions,therefore in subsequent experiments,another 3 samples with known results were used as control standard which contained clinical positive mutations of ALK fusion gene and EGFR mutation,Also,mutations in the corresponding panel were detected.In this study,there is no copy number variation of the sample,but also need to be improved in subsequent studies and find samples to verify.In the standard tests,using the panel can detect low mutation frequency at about 2%.The new panel,which has been improved,may also be used to detect ctDNA of cancer patients.The liquid technology enables a comprehensive assessment and real-time detection of tumor development.