Exploratory Study On EGFR Gene Mutation Status And EGFR-TKI Efficacy In Patients With LncRNAs With Advanced NSCLC

IntroductionAs we all know,lung cancer is the most malignant tumor with the highest morbidity and mortality in the world,which seriously threatens the health and life safety of all human beings.With the deterioration of the atmospheric environment,the abuse of food additives,and the rejuvenation of bad habits such as smoking and drinking,the incidence of lung cancer has gradually increased.GLOBOCAN data show that in 2001,there were 2.1 million new cases of lung cancer worldwide,accounting for 11.6% of all new cases(ranked first);1.8 million deaths,accounting for 18.4% of all tumor deaths(ranked first).In recent years,with the rapid development of immunotherapy and targeted therapy,the mortality rate of lung cancer has decreased,but compared with 20%of the 5-year survival rate of lung cancer in European countries,China can only reach about 18%.Fortunately,the mutation rate of the Asian population can be as high as 20%compared with the EGFR drug mutation of 5% in the Caucasian population,especially in women with no smoking history,even 50-60.% mutation rate.A number of studies have shown that this group of people can achieve 70%efficiency after receiving EGFR-TKI treatment,which greatly improves the patient's OS while improving the quality of life of lung cancer patients.The assessment of the EGFR gene mutation status in lung cancer tissues has important predictive value for the efficacy of EGFR-TKI.However,in patients with advanced NSCLC,the pathological diagnosis is often bronchoscopy or puncture,the sample size is usually small,not enough to detect EGFR mutation status,and even some patients can not obtain pathological tissue.As a non-invasive test,liquid biopsy represented by ct DNA can be used as a supplement to detect EGFR mutations.Its unique advantage is to overcome the heterogeneity of tumor to some extent.However,ct DNA detection still has some limitations,and it shows high accuracy for EGFR mutation status analysis,but the sensitivity range is low.The reason may be that the ability to detect gene mutations in plasma ct DNA is associated with tumor burden.In patients with small tumor burden,the false negative rate will increase significantly.Another reason may be that somatic mutations accumulated by senescent cells will be ct DNA.Practical Addition Limits [19-20] Benign gene mutations in adult stem cells are transmitted to the next generation as cells proliferate,and sometimes gene mutations in tumor-driven genes cause proliferation of stem cells carryingthese mutations.Faster,this may be mistaken for tumor proliferation..At the same time,a number of studies have shown that EGFR-TKI patients with EGFR-mutant NSCLC have an efficiency of only about 70%,which may be related to the activation of the bypass signaling pathway to produce primary resistance.However,there is currently no clinically relevant marker to predict the efficacy of EGFR-TKI.Therefore,it is necessary to find a group of tumor markers combined with ct DNA detection to predict EGFR mutation status,EGFR-TKI efficacy and to monitor the efficacy of EGFR-TKI therapy in patients with NSCLC.In the past studies,more attention was paid to the relationship between the coding protein gene mutation and the tumor.With the continuous understanding of lnc RNA,it is believed that the differential expression and mutation of lnc RNA play an important role in the malignant proliferation of tumor.Over the past decade,Lnc RNA has been found to be stably expressed in human blood,and circulating Lnc RNAs can serve as novel molecular biomarkers for diseases and cancers.Blood biomarkers have several significant advantages: they are easily obtained in a minimally invasive manner,can be measured repeatedly,and their expression levels can be compared longitudinally.Studies have shown that Lnc RNA is closely related to EGFR-TKI resistance,but most of the research is concentrated on the level of cell lines.The correlation between plasma Lnc RNA and EGFR mutation status and monitoring the efficacy of EGFR-TKI has not been carried out.Our aim was to identify a set of circulating Lnc RNAs to predict EGFR mutation status,and combined with plasma ct DNA detection to increase sensitivity,and we hope to explore the circulating Lnc RNA that can be predicted to predict EGFR-TKI efficacy in patients with positive EGFR mutations,and Explore the potential of this Lnc RNA group to monitor the therapeutic effects of EGFR-TKIs.Part ?: NP-positive mutations in advanced non-small cell lung cancer and differential expression of Lnc RNA in wild-type pleural effusion.Objective: To screen for differential expression of Lnc RNA in EGFR-positive mutations and wild-type pleural effusions in patients with NSCLC by Clariom D Human microarray analysis.Methods:The pathological tissues were diagnosed as lung adenocarcinoma,and the EGFR mutation status was detected by ARMS through lung cancer tissues and pleural effusion.Results:The results showed that EGFR positive mutations were consistent with the wild type and 3 cases were pleural effusions.Differential Lnc RNA andm RNAexpression profiles were established on samples using Clariom D Human chip technology.The molecular function of the differentially expressed Lnc RNA,the involved biological processes,and the disease pathway were then enriched by GO analysis.Further screening of EGFR-positive mutations and wild-type differential expression of Lnc RNA by GO analysis and FC values: In pleural effusion samples,EGFR-positive mutations were compared with EGFR wild-type,61 differentially expressed Lnc RNAs were screened,and 48 Lnc RNAs were significant.Up-regulated,13 Lnc RNAs were significantly down-regulated,including 25 new unreported lnc RNAs.GO analysis was performed on differentially expressed Lnc RNA.The results showed that Lnc RNA differentially expressed in EGFR mutation status can be enriched to 1142 molecular functions.Among them,SCARNA7,MALAT1,NONHSAT017369,NONHSAT051892 and FTH1P2 were significantly associated with EGFR mutation status.Conclusion: The EGFR mutation positive and EGFR wild-type differential LNc RNA expression profiles in pleural effusion were constructed.Twenty-five new unreported lnc RNAs were found in 61 differentially expressed lnc RNAs.Molecular functions,biological processes,and regulation of disease pathways involved in differential expression of LNC RNA were identified.Provide valuable data resources for oncology research and follow-up experiments.part ?: EGFR MU(+)and EGFR WT NSCLC patients with differential expression of Lnc RNA analysis and verificationObjective: q RT-PCR method to detect the expression of alternative Lnc RNA in pleural effusion in patients with advanced NSCLC,screening Specific Lnc RNAs that may be associated with EGFR-sensitive mutations.Methods: Combined with pleural effusion chip results,GO analysis and literature review,the selected target difference Lnc RNA was screened.In 77 patients with advanced NSCLS pleural effusion(35 patients with EGFR positive mutations,42 patients with EGFR wild type,all patients with EGFR mutation status confirmed by tissue testing,and tumor cells were found in the sample),using q RT-PCR Methods The alternative target differential Lnc RNA was validated and analyzed.Results: Combined with pleural effusion chip results,GO analysis,and literature review,SCARNA7,MALAT1,NONHSAT017369,NONHSAT051892,and FTH1P2 were significantly associated with EGFR mutation status.The q RT-PCR assay of 5 candidate Lnc RNAs showed no significant difference between NONHSAT051892 and FTH1P2 in EGFR-positive and EGFR wild-typepatients with pleural effusion samples.SCARNA7,MALAT1 and NONHSAT017369 were significantly up-regulated in the EGFR-positive mutant group compared to the EGFR wild-type.Part ? :Analysis and Validation of Differential Expression of Lnc RNA in Plasma Samples of EGFR MU(+)and EGFR WT NSCLC PatientsOBJECTIVE: To detect the expression level of Lnc RNA in plasma of patients with advanced NSCLC by q RT-PCR and to determine that Lnc RNA is associated with EGFR sensitive mutation.Methods: q RT-PCR was used to detect 72 cases of EGFR mutation positive in patients with advanced NSCLC and 7 cases of EGFR wild type in advanced NSCLC by ARMS.The detection target was 3 Lnc RNAs for pleural effusion verification..Correlation analysis of variance was performed on 3 differential Lnc RNAs between the EGFR mutation positive group and the EGFR wild type group using an unpaired T test.At the same time,the chi-square test was used to analyze the clinicopathological features of the patients enrolled.A single Lnc RNA predicted EGFR mutation status was predicted using a Receiving Operating Characteristics Curve(ROC)and Logistic Regression was used to assess the ability of multiple Lnc RNA combinations to predict EGFR mutation status.Kaplan-Meier survival analysis was used to analyze the initial expression level of plasma Lnc RNA and the PFS of patients with EGFR mutation-positive EGFR-TKI treatment.Multivariate analysis was performed between the clinicopathological features and the initial expression level of Lnc RNA and PFS in the enrolled patients.RESULTS: SCARNA7,MALAT1,and NONHSAT017369 showed consistent results with pleural effusions in plasma compared with EGFR wild-type,both up-regulated in the EGFR mutation-positive group,with ROC curves of 0.76,0.89,and 0.76,respectively.The specificity and specificity were 80.8%,79.2%,79.7% and 69.1%,85.7% and 63.6%,respectively.For the combined diagnosis model of 3 Lnc RNAs,the AUC=0.920,PPV and NPV were both over 82%.In addition,EGFR mutant subtype analysis showed that plasma MALAT1 was significantly associated with the EGFR 19 mutation of exon 19 deletion(19DEL).Plasma MALAT1 was significantly upregulated in patients with EGFR 19 DEL NSCLC.Multivariate analysis showed that smoking history was closely related to EGFR mutation status.The expression level of MALAT1 was significantly associated with PFS(m PFS: 11.6 vs 8.2 months,p = 0.020,HR: 0.431;95% CI,0.236-0.787).Multivariate analysis showed that MALAT1 and smoking historywere important predictors of efficacy in patients with NSCLC who were treated with EGFR-TKI.Conclusion: The circulating SCARNA7,MALAT1,and NONHSAT017369 are expected to be potential biomarkers for distinguishing EGFR mutation-positive and wild-type molecular typing;the expression level of MALAT1 is significantly correlated with PFS in patients receiving EGFR-TKI,and is expected to be accepted as an EGFR mutation-positive patient.Predictive markers of EGFR-TKI therapeutic efficacy;smoking history is not only associated with EGFR mutation status in patients,but also with PFS treated with EGFR-TKI.Part ?: Preliminary analysis of the dynamic changes of Lnc RNA before and after EGFR-TKI treatmentObjective: To explore the dynamic changes of circulating Lnc RNA in patients with EGFR mutation-positive NSCLC before,during and after EGFR-TKI treatment.METHODS: Thirty-six patients with EGFR 19DEL-sensitive mutations were enrolled and plasma samples from patients with EGFR-TKI before and after treatment were dynamically matched(16 patients received blood samples after disease progression).q RT-was used for the plasma samples.The expression of SCARNA7,MALAT1,and NONHSAT017369 was detected by PCR.Dynamic changes in ?Ct values before,during,and after EGFR-TKI treatment were analyzed.RESULTS: Of the 32 patients with EGFR-TKI benefit after EGFR-TKI treatment,27 had lower plasma MALAT1 levels(p = 0.020)and 23 had lower SCARNA7 levels(p=0.002),but no similar findings were observed in PD patients.Trends,at the same time,we also analyzed changes in plasma circulating Lnc RNA in 12 patients with EGFR 19 DEL who received blood samples at the time of disease progression,before,during,and after EGFR-TKI treatment,whether the short-term efficacy was remission,In patients who are stable or progressive,their plasma expression levels tend to return to pre-treatment levels after disease progression.Conclusions: Overall,studies of three Lnc RNAs have shown that changes in plasma SCARNA7 and MALAT1 levels before and after EGFR-TKI treatment have a good predictive value for EGFR-TKI therapy.