Detection Of Circulating Tumor DNA Of Lung Cancer Through Next Generation Sequencing

Background:Lung cancer is one of the most morbidity disease in the world,and it is one of the most serious malignancies that seriously threaten people's health.And mortality rate of lung cancer is highest in males.At present,radiotherapy,chemotherapy and molecular targeted therapy are the main ways for lung cancer treatment.Since 2000,targeted therapies have developed rapidly.Targeted therapies broadly employed has resulted in a significant increase for patients' overall survival.The application of targeted therapy is depended on the achievement of the molecular biological information of tumors.At present,the main samples for gene detection are mainly from tissue biopsy.However,tissue biopsy sometimes may not be able to meet the requirements of complicated gene mutation detection.Otherwise,lung cancer patients often have multiple gene mutations.Circulating tumor DNA(ctDNA),derived from tumor cells,has the same gene altered information as tumor tissues.Therefore it is considered to be the best alternative object for mutation gene detection instead of tumor tissue in patients with carcinomia.However,the content of ctDNA in the blood is very low,only accounting for about 1%of the cell free DNA(cfDNA).Therefore,high-precision methods must be found for the detection of ctDNA.Next generation sequencing(NGS)has the characteristics of high throughput,high precision,and high depth,which is very suitable for the detection of ctDNA.The purpose of this assay is to construct a high-throughput sequencing-based way for the detection of ctDNA in lung cancer.And I hope that this way would able to detect lung cancer ctDNA for obtaining the molecular biological information of tumors,and to provide help for targeted therapy.Method:1,Through literature review,a multiplex PCR primer covering multiple gene loci was designed.2.Using the different types of cfDNA reference standard set as objects,next generation sequencing technology was used to detect the reference standard sets to obtain information about standard quality control for high-throughput sequencing.3.The 10 cases of ctDNA of lung cancer with a known gene mutations information was used as the objects,and High-throughput sequencing technology was used to sequence the ctDNA samples.The sequencing quality must meet the quality control standards established above.And digital PCR technology was used to verified the sequencing results.4,Using next generation sequencing technology to detect 74 cases of lung cancer ctDNA,and obtaining statistics of the sequencing results,the analysis of whether the detection rate of mutant genes is related to tumor types and stage and metastasis and operative treatment was performed.Results:1,The results of next generation sequencing technology to detect cfDNA reference standard sets showed that when the sequencing depth is more than 12,000X,all mutation frequency of 0.1%can be detected.According to the results,the standard quality control of high-through technology for ctDNA detection was established.2.The High-throughput sequencing results of 10 cases of lung cancer ctDNA showed that 5 cases were positive and other 5 cases were negative.The consistency with the tissue test results was 70%(7/10),and the specificity was 100.%(2/2)with a sensitivity of 62.5%(5/8).3.The sequencing results of 74 cases of lung cancer ctDNA showed that 36 mutations of 8 genes were detected,of which the positive rate of TP53 mutation was the highest,reaching 52.7%.Statistical analysis of the results revealed that the positive rate of each mutation gene was not related to the stage of the tumor and the tumor metastasis.And the detection rate of EGFR mutant gene was higher in adenocarcinoma than squamous.Conclusion:In summary,we successfully established a high-throughput sequencing technology to detect lung cancer ctDNA samples.And this method was successfully applied to the clinical detection of lung cancer ctDNA,and the message of gene mutation in 74 cases were obtained and analyzed.