A Novel Method For Detection Of EGFR And KRAS Mutations In Lung Cancer Using Droplet Digital PCR Of CfDNA

Lung cancer is the first leading cause of cancer mortality in China.The study found that the mutation rate of EGFR was 40%,KRAS was 10%.However,it is highly challenging to develop tissue sections to continuously monitor the progress of the disease.Therefore,a non-invasive detection method is needed.Liquid biopsy is a good diagnostic method,which can be used to monitor the progression of the disease.Plasma free DNA(cfDNA)is mainly in the blood,synovial fluid and other body fluids,cfDNA analysis is a basic tool for clinicians to select targeted therapy,and it is becoming a powerful method to predict prognosis.Many highly sensitive and specific techniques have been developed to assess changes in cancer disease by capturing and analyzing plasma free DNA.Droplet digital PCR(DDPCR)is a sensitive method for quantitative detection,which can be detected with only a very small number of samples.This method does not require reference to standard or endogenous control,high sensitivity and required sample size Less,greatly satisfying the clinical need for rare samples such as puncture samples,pleural effusion,and peripheral blood target sequences.Objective: The aim of this study was to develop a plasma free DNA mutant gene detection technique based on the digital PCR for minimally invasive detection of lung cancer.Detection of KRAS and EGFR mutations by collecting plasma samples from patients.Methods: Design special primers and probes to the five mutations of EGFR and KRAS,and then use the positive cell line DNA and standard for testing.Established a method for detection of EGFR18/19/20/21 and KRAS mutations.Finally in 351 cases of clinical samples for validation,and compare its consistency with the second generation sequencing.Results: First,The reaction system of EGFR and KRAS mutations in plasma DNA was established used the cell line DNA and positive standard by DDPCR method.Second,according to the established methods for the standard detection,the result show that the lowest can detect 0.04%,the accuracy is 100%.Third,The second generation of sequencing as a gold standard,used the methods which has been established by digital PCR to detect the plasma free DNA samples in 351 patients with lung cancer.The results showed that the sensitivity of E18 \ E19 \ E20 \ E21 and KRAS respectively could reach 100%,96%,97%,100% and 93%.The consistency of the second generation sequencing can reach more than 96%.Conclusion: A method for the detection of EGFR and KRAS mutations in free DNA was established,and a rapid detection technique of plasma free DNA mutant gene based on digital PCR was developed in this study.It was proved that Droplet digital PCR was used to detect plasma free DNA with High specificity and sensitivity.This method provides a noninvasive,rapid and absolute quantitative method for dynamic monitoring of EGFR and KRAS gene mutations in lung cancer patients.This technique can not only monitor the mutation rate of EGFR and KRAS sensitive mutations,but also detect the emergence of drug resistance mutations in time.