Investigation On The Mechanism Of PIK3CA Mutation To EGFR-TKIs Resistance In Non-small Cell Lung Cancer

Objective:(1)To screen the optimal targeted therapy for patients with advanced non-small cell lung cancer by targeted high-throughput sequencing technology,explore whether the tumor mutational burden(TMB)obtained by targeted high-throughput sequencing technology can be used as a predictive marker for immunotherapy.Analyze the feasibility of targeted high-throughput sequencing technology as a clinical dynamic monitoring and guidance of targeted therapy and immunotherapy for NSCLC patients,so as to provide a basis for rapid,accurate and individualized treatment of NSCLC patients.(2)To analyze the correlation between PIK3 CA mutation types and clinicopathological characteristics in patients with non-small cell lung cancer,and Analyze the prognosis of patients with PIK3 CA and EGFR mutations coexisting NSCLC receiving epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs),revealing the predictive value of PIK3 CA mutation in the prognosis of NSCLC patients with EGFR mutation And the correlation with EGFR mutation resistance,Provide new targets for effective treatment of patients with advanced NSCLC,and provide new ideas for the reversal of drug resistance in patients with advanced NSCLC who are resistant to EGFR-TKIs;(3)To explore the effect of PI3K/Akt pathway inhibitor LY294002 on the sensitivity of EGFR-TKIs-resistant non-small cell lung cancer cells to erlotinib and analyze its intrinsic molecular mechanism.providing a theoretical basis for the clinical treatment of EGFR-TKIs combined with PI3 K inhibitors to reverse the resistance of NSCLC.Methods:(1)To retrospectively analyze the clinicopathological data of 533 newly diagnosed and recurrent non-small cell lung cancer patients,Extract DNA from patients’ blood or tumor tissues and perform targeted high-throughput sequencing.Targeted sequencing genes include 34 common tumor drug-related genes including EGFR,TP53,ALK,ROS1,BRAF,KRAS,ERBB2,and PIK3 CA.analyze the correlation between EGFR,BRAF,ALK,ROS1 mutations and clinicopathological characteristics by chi-square test.Calculate TMB value using bioinformatics method.Detection of PD-L1 expression level on the surface of tumor tissues in patients with non-small cell lung cancer by immunohistochemistry;Analyze the correlation between TMB and PD-L1 expression level by R software version 3.5.3,Using the average value of TMB as the cut-off value,patients with non-small cell lung cancer receiving immunotherapy were divided into two groups with high TMB(TMB-H)and low TMB(TMB-L),and Kaplan-Meier survival analysis was used to analyze the progression-free survival,PFS and draw the survival curve.(2)The clinicopathological characteristics of 29 patients with PIK3 CA mutation non-small cell lung cancer were retrospectively analyzed.Chi-square test was used to analyze the correlation between different types of PIK3 CA mutations and clinicopathological characteristics.analyze other driver gene mutation types that coexist with PIK3 CA mutations by targeted highthroughput sequencing technology;According to whether PIK3 CA and EGFR co-mutate,patients with advanced non-small cell lung cancer who received EGFR-TKIs treatment were divided into PIK3 CA and EGFR co-mutation group and EGFR mutation group only,and Kaplan-Meier survival analysis was used to analyze the progression-free survival and draw survival curve,analyze whether PIK3 CA mutation affects the effect of EGFR-TKIs treatment in patients with EGFR-mutated non-small cell lung cancer.(3)The effect of different concentrations of LY294002 and erlotinib on the cell proliferation of two non-small cell lung cancer cell lines NCI-H460 and NCI-H661 was analyzed by MTT experiment,and the effect of combining EGFR-TKIs erlotinib and PI3 K inhibitor LY294002 on Inhibition of growth of two NSCLC cells;Using flow cytometry to analyze the effects of erlotinib or LY294002 and the combination of erlotinib and LY294002 on the apoptosis of two NSCLC cells NCI-H460 and NCI-H661;The total protein of NCI-H460 and NCI-H661 cells treated with erlotinib or LY294002 and the combination of erlotinib and LY294002 was extracted,and the PI3K/AKT signaling pathway related proteins p70S6 K and p-p70S6 K and were detected by Western Blot.Results:(1)(1)Among 533 patients with non-small cell lung cancer,347 patients had at least one EGFR,KRAS,ALK,PIK3 CA,ROS1,BRAF,ERBB2,MET,and TP53 gene mutations,and the driver gene mutation rate was 65.10%,186 cases No mutations in related driver genes were detected in patient samples.The high mutation frequency are EGFR,TP53,KRAS,PIK3 CA,ALK,BRAF,MET,ERBB2,ROS1 in descending order.(2)EGFR is the most common mutation in NSCLC patients,accounting for 73.20%of all mutation types,EGFR mutations are mainly concentrated in exons 18-21,among which exon 19 deletion mutation and exon 21 L858 R mutation are the main types of EGFR mutations.EGFR 21 exon mutations have a higher incidence in women and adenocarcinoma(P < 0.001);EGFR19 exon mutations have a higher incidence in women under 60 years of age,women,with family history,adenocarcinoma,and newly diagnosed patients(P<0.05);Exon 20 mutations include insertion mutations and T790 M point mutations.Among them,T790 M coexists with other EGFR mutation types,and compared with EGFR exon 21 mutations,the co-mutation rate of T790 M and EGFR exon 19 is higher.Significant(P<0.05),In addition to the common EGFR mutations,the sequencing results also detected rare EGFR mutation types and their distribution in newly diagnosed and recurrent non-small cell lung cancer patients was different.20 mutations occur more frequently in women,adenocarcinoma,and relapsed patients(P<0.05);The E746 site mutation in exon 19,the L747 site mutation in exon 19,and the L861 Q mutation in exon 21 appeared in newly diagnosed and relapsed patients.However,the P753 A mutation in exon 19,the R836 H mutation in exon 21,the G719 A mutation in exon 18,the G719 C mutation in exon 18,and the E709 K mutation in exon 18 only appeared in newly diagnosed patients.Exon 19I740 delins IPVAIKL insertion mutation and The L718 M mutation in exon 18 is only found in relapsed patients;(3)The incidence of EML4-ALK fusion mutation was 2.25%.Analysis of clinicopathological characteristics of NSCLC patients found that ALK rearrangement occurred more frequently in newly diagnosed patients younger than 60 years old,which was statistically significant(P<0.05);A p.L1152 R point mutation in exon 20 of ALK was detected in a second-line crizotinib and third-line blind ceritinib for relapsed patients;besides that,the ALK mutation did not coexist with other gene mutations.(4)The BRAF gene mutation rate was 1.69%,and no correlation with clinicopathological characteristics was detected;the main BRAF gene mutations all occurred in exon 15,and the V600 E mutation accounted for 66.67%of the total BRAF mutations.In addition,there are also D594 G and A1801 G mutation types on 15 and one case of G469 A point mutation on BRAF exon 11.(5)The incidence of ROS1 fusion mutation is 0.94%.The fusion genes of ROS1 are CD47,TPM3 and EZR.(6)The average TMB of the patients in this study was 7.13 Mut/Mb,the highest value was 44 Mut/Mb,and the lowest value was 0.7 Mut/Mb;there was no significant difference between the TMB of newly diagnosed patients and relapsed patients(P=0.473);There was no significant difference in TMB between patients with EGFR activating mutations,mutations other than EGFR,and patients without gene mutations;there was no statistically significant difference between the TMB values of the PD-L1 positive group and the PD-L1 negative group(P=0.345),indicating that the level of TMB has no correlation with the expression level of PD-L1;The survival statistics of NSCLC patients receiving immunotherapy found that the median value of PFS in the TMB-H group was 15 months,and the median value of PFS in the TMB-L group was 13 months,but there was no significant difference in survival between the two groups(P=0.449).(2)(1)Among 533 patients with non-small cell lung cancer,29 patients with PIK3 CA mutations were detected,with an incidence rate of 5.44%;PIK3CA mutations include PIK3 CA amplification and PIK3 CA mutations,the incidence of PIK3 CA amplification was 0.56%,and the incidence of PIK3 CA mutation was 4.88%.(2)PIK3CA mutation has a higher incidence in patients with early stage squamous cell carcinoma and non-small cell lung cancer(P<0.001).The 3 patients with PIK3 CA amplification in this study were all lung squamous cell carcinoma.(3)PIK3CA point mutations mainly occur in exons 4,7,9 and 20 of PIK3CA.There are two types of mutations,E545 K and E542 K in exon 9.The E545 K mutation has the highest mutation frequency in PIK3 CA mutation type,which is 34.48%,while the E542 K mutation frequency is 10.34%;The main types of mutations in exon 20 are H1047R,H1047 L,H1047Y,H1047 Q and M1043 I,among which the most frequent mutation is H1047 R mutation,which accounts for 20.69%of the total mutation frequency;This study also detected two rare mutations of PIK3 CA into exon 4(p.N345K)and exon 7(p.E453K).(4)PIK3CA mutations also coexist with EGFR mutations,ROS1 mutations,and MET mutations.Patients with PIK3 CA and EGFR double mutations account for the highest PIK3 CA mutation rate of 41.38%;In addition to double-gene mutations,6 patients also had the coexistence of three-gene mutations.Among them,PIK3 CA mutation + TP53 mutation + EGFR mutation accounted for 17.24%of the total PIK3 CA mutations,and PIK3 CA + BRAF + KRAS mutations accounted for 3.45%of the total mutations;(5)The results of survival analysis showed that the median PFS of the EGFR gene mutation group was 12 months,while the median PFS of the PIK3 CA and EGFR co-mutation group was 9months.The survival time of PIK3 CA and EGFR co-mutation patients was shorter than that of EGFR mutation patients(P=0.0083).(3)(1)MTT results showed that the IC50 value of erlotinib on NCI-H460 cells was 12.43μM and the IC50 value of NCI-H661 cells was 13.97μM.Both erlotinib and LY294002 had strong inhibitory ability on NCI-661 cells.(2)Compared with erlotinib single agent,erlotinib combined with LY294002 significantly improved the inhibitory ability of erlotinib on NCI-H460(P<0.05)and NCI-H661(P<0.05).(3)The results of flow cytometry showed that compared with erlotinib and LY294002 single agent acting on NCI-H661 cells,the combined drug significantly improved the ability of erlotinib to induce apoptosis of NCI-H661 cells(P < 0.05).(4)Western Blot results showed that compared with the effects of single-agent erlotinib and LY294002,the combined medication significantly reduced the expression level of p-p70S6 K in NCI-H661 cells,but the expression level of p-p70S6 K in NCI-H460 cells did not change much.Conclusion:(1)(1)Targeted high-throughput sequencing can be used as a tool to dynamically guide targeted therapy for patients with non-small cell lung cancer.(2)There is no correlation between the expression levels of TMB and PD-L1.TMB has the potential as a predictive marker for immunotherapy.However,the targeted sequencing platform panel in this study is small and requires a larger targeted panel to verify.(3)Targeted high-throughput sequencing technology can replace traditional molecular diagnostic methods as a detection tool that simultaneously guides targeted therapy and immunotherapy.(2)The PIK3 CA co-mutation may suggest that patients with EGFR-mutant non-small cell lung cancer receiving EGFR-TKIs targeted therapy have a poor prognosis.(3)(1)Combined with LY294002 can improve the sensitivity of erlotinib to non-small cell lung cancer,but it inhibits the growth of PIK3 CA wild-type NSCLC cells more significantly.(2)The combination of LY294002 and erlotinib can induce apoptosis in PIK3 CA wild-type NSCLC cells.(3)Combining PI3K-AKT signaling pathway inhibitors may provide new treatment strategies for patients with EGFR-TKI-resistant non-small cell lung cancer.