| Objectives:Non-small cell lung cancer(NSCLC)is a malignant tumor with extremely high morbidity and mortality.Targeted therapy based on molecular type of tumor tissue is a common treatment for NSCLC.However,patients’ tumor tissues are difficult to obtain in most cases,and the following genetic tests are not eligible to perform.In this study,plasma circulating tumor DNA(ctDNA)was used instead of tumor tissue DNA for gene mutation detection.And a simple,rapid and non-invasive method was established,which could apply to personalized targeted treatment for more patients with non-small cell lung cancer.Methods:(1)External standard detection method was established to evaluate the extraction efficiency of ctDNA extraction kit,the effect of centrifugation conditions in ctDNA extraction,the ctDNA stability of freeze-thaw,delayed extraction and long-term storage.And a novel standard ctDNA sample protocol was set as data suggest.(2)A novel ctDNA detection technology was established by combining one-tube nested PCR with dual PNA clamp reaction(LNA-dPNA PCR clamp),and then we evaluated the performance of sensitivity,specificity,accuracy.(3)Four EGFR hotspot mutation sites in 132 non-small cell lung cancer plasma and matched tissue samples were detected using the established novel ctDNA detection technique to evaluate their consistency.Results:(1)Simgen Circulating Nucleic Acid Kit was selected for ctDNA extraction with large volume plasma.Data suggested whole blood samples for ctDNA detection could not be frozen-thawn,placed at 4℃ for less than 24 h,and placed at room temperature for less than 12 h.The ctDNA in plasma was stable within 3 freeze-thaw cycles.It could be stored for less than 24 h at-20℃ or 4℃,and less than 4 h at room temperature.Nevertheless,it will gradually degrade at-80℃ for a long time.The ctDNA in buffer AE was stable within 3 freeze-thaw cycles.It could be stored for less than 48 h at-20℃ or 4℃,and less than 12 h at room temperature.However,it will gradually degrade at-80℃for a long time.(2)The amplification sensitivity of LNA-dPNA PCR clamp was 100 copies/reaction,which was 10 times higher than ddPCR;the linear range was 100～106-107,which was 2-3 orders of magnitude wider than ddPCR;the LOD of mutation detection was 0.01%-0.1%,similar to ddPCR.LNA-dPNA PCR clamp was universal that could achieve high sensitivity detection for EGFR L858R,EGFR Exon 19 deletion,EGFR T790M,EGFR C797S.In addition,LNA-dPNA PCR clamp also achieved absolute quantification of ctDNA,the quantitative results were similar to ddPCR(r=0.9782,p<0.0001).Furthermore,it was found that the cost of LNA-dPNA PCR clamp was 29.0%lower than ddPCR($39.68 vs.$56.04)and consuming-time reduced by 31.3%(2.75 h vs.4.0 h).(3)EGFR L858R mutation was detected using LNA-dPNA PCR clamp and ddPCR in both plasma ctDNA and DNA of tumor tissues matched in 132 NSCLC patients,and the consistency of the results was compared.It showed that the sensitivity of the two methods in evaluating plasma ctDNA replacing DNA of tumor tissue was 19.6%and 21.7%,and the specificity was 94.2%and 91.9%.The overall consistency of two methods was 68.2%and 67.4%.Further analysis found that the consistency between plasma ctDNA and tumor tissue was lower in early NSCLC(stage Ⅰ)and higher in advanced stage(stage Ⅱ-Ⅳ).Therefore,we recommend that plasma ctDNA can be used instead of tumor tissue for gene mutation detection in NSCLC patients with advanced stage.On this basis,we conducted a preliminary evaluation of the correlation between plasma ctDNA test results and TKI efficacy by case analysis,and initially demonstrated that personalized treatment of targeted drugs based on plasma ctDNA results allows NSCLC patients to benefit from TKI therapy.Conclusions:This study established a new method to detect scarce ctDNA in plasma,which is highly sensitive,highly specific,simple and rapid,eco-friendly and low-cost.This investigation provided NSCLC patients with a new tool to individualize their treatments. |
PDF Full Text Request