Efficacy And Mechanism Of Acquired Resistance Of The Second-Generation EGFR-TKIs In Advanced NSCLC With HER2 Mutation

Objective:In non-small cell lung cancer（NSCLC）,HER2 mutations in the tyrosine kinase domain（TKD）,especially exon 20 insertion mutation,have been widely reported before.With the increased accessibility of next-generation sequencing,more and more HER2 mutations in the non-tyrosine kinase domain（non-TKD）can be detected.However,the frequency and clinical significance of these non-TKD mutations remain unclear.The second-generation EGFR-TKIs was effective HER2-mutated NSCLC patients reported in early clinical trials and clinical reports,but the mechanism of drug resistance remains unknown.Therefore,this study aims to:firstly,summarize the mutational landscape of HER2 mutations in NSCLC;Secondly,explore the potential mechanism of drug resistance by the next-generation sequencing;Thirdly,validate the mechanism of drug-resistance caused by gene mutations through in vitro study.Methods:Part I:through the analysis of 1934 cases of NSCLC in the public database c Bio Portal for Cancer Genomics and 1895 cases of NSCLC in Guangdong Lung Cancer Institute,all of whom undergoing next-generation sequencing,this part systematically summarizes the HER2 mutational landscape and mutational characteristics.Part II:The second part analyzed the efficacy of second-generation EGFR-TKIs in advanced HER2-mutated NSCLC patients from the Guangdong Lung Cancer Institute.Moreover,the data of next-generation sequencing before EGFR-TKIs treatment and after disease progression were analyzed to explore potential drug-resistant mechanism in terms of gene mutations.Part III:cell experiments were designed to validate the mechanism of drug-resistance induced by gene mutations.Results:Part I:the frequency of HER2 mutation in the Chinese population was 4.0%,similar to that in the western population（cbioportal cohort,4.2%）.Eighty-one HER2-mutant subtypes were identified,among which exon 20 insertion mutation ranked top.HER2 mutations in the tyrosine kinase domain（TKD）and non-TKD accounted for 54.2%and 45.8%,respectively.Among the oncogenic mutations,up to 64.9%of the mutations occurred in non-TKD.Analysis of coexistence mutations showed that TP53 was the most common co-mutated gene,and the frequency of EGFR co-mutation in the non-TKD mutation group was significantly higher than that in the TKD group.Part II:The objective response rate of the second-generation EGFR-TKIs for HER2-mutated NSCLC was 21.4%,the disease control rate was 75%,and the median progression free survival（PFS）was 172 days.Five patients with non-TKD mutations were treated with the second-generation EGFR-TKIs,with an objective response rate of 20%and a median PFS of 113 days.The median PFS of HER2 non-TKD mutation was shorter than that of patients with TKD mutation（113 days vs.172 days）,but the difference was not statistically significant（P=0.058）.Acquired gene variations after drug resistance included HER2amplification,MTOR mutation,NRAS mutation,MYC amplification,CDK12 amplification,RICTOR amplification,CDKN2A deletion,and PTEN nonsense mutation,etc.Part III:we constructed NCI-H1781 cell line with HER2^{G776delins VC} and HER2^wild-type overexpression,respectively.Growth inhibition experiments exhibited that both of the NCI-H1781 cell lines with HER2^wild-type overexpression or HER2^{G776delins VC} overexpression were resistant to pyrotinib,compared with the negative control cell lines.However,these cell lines showed similar sensitivity to afatinib.Conclusion:（1）Mutation subtypes of HER2 are complex,which mainly include exon 20insertions.Non-TKD mutations also have a carcinogenic function.（2）The second-generation EGFR-TKIs are effective in advanced NSCLC patients with HER2mutations.The non-TKD mutations might be a therapeutic target for EGFR-TKIs.Acquired gene variation might be related to drug resistance of the second-generation EGFR-TKIs.（3）In vitro,cell experiments revealed that HER2 overexpression might be drug-resistant mechanism of pyrotinib but not afatinib in HER2-mutant NSCLC.