Technology And Application Of EGFR Mutation Detection In Non-small Cell Lung Cancer Based On Droplet Digital PCR

Background:Targeted therapy based on epidermal growth factor receptor(EGFR)mutations has gradually become part of comprehensive treatment for non-small cell lung cancer(NSCLC).In order to provide personalized treatment and clinical decision guidance,there is an urgent need for EGFR mutation detection technology to identify patients who are sensitive to targeted therapy,and to monitor the emergence of genetic mutations after drug resistance.Compared with tissue biopsy,cell-free DNA(cfDNA)is highly consistent with the genetic mutation information contained in the tumor,which can comprehensively reflect all the genetic information of the tumor in real time.Therefore,blood has also become a routine test material for EGFR mutation detection.At the same time,saliva provides high-quality genomic DNA and has the same genetic information as blood.Because of the unique advantages of convenient,non-invasive sampling and avoidance of blood-borne infections,it is expected to be ideal for EGFR mutation detection.However,due to the low content of cfDNA in blood and saliva and the low frequency of gene mutations,more sensitive methods are needed for EGFR mutation detection.The detection of EGFR mutation requires more sensitive methods.Absolute quantitative droplet digital PCR(ddRCR)technology has higher accuracy,higher resolution and higher sensitivity,which can realize low mutation frequency detection of cfDNA and meets the current clinical needs for the detection of EGFR mutationMethods:Based on ddPCR,the relevant probes and primers were designed for EGFR mutation detection technologies(Exonl9Dels,T790M and L858R).The ddPCR reaction system was established and optimized,the optimal annealing temperature was established,and the relevant sensitivity was analyzed.On this basis,we mainly performed preliminary performance verification of the EGFR-T790M mutation detection technology and confirmed the clinical utility.It mainly included linear range,blank detection limit,specificity analysis,sensitivity analysis,repeatability,inter-assay precision and accuracy.Real-time fluorescent PCR was used to detect the content of saliva cfDNA in NSCLC patients,and the ddPCR technology established above was used to detect the EGFR mutations in the paired plasma and saliva samples,and the consistency between them was analyzedResults:The ddPCR reaction systems for EGFR mutations(Exonl9Dels,T790M and L858R)were established and optimized,the optimal annealing temperatures were 60.1℃,58.2℃ and 58.2℃,respectively.For sensitivity analysis,the limits of mutation detection can determined to be at least 0.5%,0.05%and 0.1%.On this basis,using ddPCR,the linear evaluation showed that R2>0.99 between the actual detection result of the technology and the expected mutation ratio.The blank detection limit was determined to be the numbers of mutant droplets>2.The specificity of this technology was 100%.In the repeatability and inter-assay precision tests,the results of CV<20%were considered to meet the requirements.The relative deviation of the results was within the range of±10%for accuracy analysis.Using the established T790M mutation detection technology,72 cases of NSCLC patients were tested for genetic mutations in cfDNA samples,and the overall agreement with Bole ddPCR for mutation detection was 91.67%(k=0.749;P<0.001).There was no significant difference in scfDNA levels between NSCLC patients and healthy volunteers or benign patients;saliva cfDNA concentrations were significantly lower than plasma cfDNA concentrations,and there was also no significant difference in correlation(Spearman rank correlation r=-0.123,P=0.269).In 37 paired plasma and saliva specimens,the overall consistency of the three EGFR mutations was 83.78%(31/37;k=0.602;P<0.001)Conclusion:Based on ddPCR,detection technologies for EGFR mutations(Exonl9Dels,T790M and L858R)were successfully established.The above three mutation tests have confirmed that they have good sensitivity,which fully meets the needs of clinical samples.On this basis,the established T790M mutation detection technology was applied to perform a series of performance verifications.It shows that the technology has good specificity,sensitivity,repeatability,inter-assay precision and accuracy,and confirms the clinical utility of the technology,indicating that it has broad clinical application and market development prospects.In addition,although the level of saliva cfDNA was not applicable for the diagnosis of NSCLC,the above ddPCR technologies can be used for salivary cfDNA-EGFR mutation detection,which found that the high coincidence rate of EGFR mutation detection between saliva and plasma cfDNA.