The Genomic Profiling Of EGFR Rare Mutant NSCLC With Icotinib Resistance

BACKGROUND:At present,lung cancer is still the highest in all cancer morbidity and mortality worldwide,and non-small cell lung cancer(NSCLC)accounts for 80%-85%of all lung cancer.Some patients have lung cancer in the advanced stage when initially diagnosed.The traditional therapy for the advanced stage NSCLC is mainly systemic chemotherapy.As the tumor-driven effect of epidermal growth factor receptor(EGFR)gene mutation in lung cancer has been claimed,tyrosine kinase inhibitors(TKIs)have replaced traditional chemotherapy as standard first-line therapy for advanced stage NSCLC patients with EGFR-sensitive mutations.The research on the resistant mechanism of TKIs has also been hot spot.In addition to the common 19 exon deletion and the 21 exon L858R classical mutations,the efficacy and resistant mechanism of TKIs in EGFR rare mutations remain unclear.Icotinib belongs to the first-generation TKI which was developed in China and preserves the independent intellectual property and rights.The clinical efficacy was proved to be similar to other TKIs such as gefitinib and erlotinib in classical EGFR mutant NSCLC.Just like classical EGFR mutations,EGFR rare mutations NSCLC will still have primary or secondary resistant issues to TKIs.The mechanism of TKIs resistance in such rare EGFR mutant patients has also risen to be a difficult question in lung cancer research.In order to explore the resistance mechanism in EGFR rare mutant NSCLC patients treated by icotinib,it is necessary to first identify the resistant gene profiles and clinic-pathologic characteristics of those patients.Most clinical trials which exploring the efficacy of TKIs in EGFR mutant NSCLC patients did not enroll those with rare EGFR mutations.As far as we know that there is no large cohort of EGFR rare mutant NSCLC study in evaluating the efficacy and resistant genomic profiling of icotinib.OBJECTIVE:To explore the relationship between EGFR rare mutations,drug-resistant mutations and progression-free survival(PFS)by next-generation sequencing(NGS)method in EGFR rare mutant advanced NSCLC patients under icotinib therapy.To provide preliminary information for further research in overcoming drug resistance and developing new targeted therapies.METHODS:Primary in-and exclusion criteria:pathologically confirmed metastatic or advanced NSCLC patients with 18 y.o.or elder,complete clinic-pathologic and follow-up data;no previous TKIs targeting therapies including icotinib,and only EGFR exon 18,20,21 or other forms of rare mutation should be enrolled;Icotinib is prescribed as 125 mg orally,three times a day for 4 weeks or longer;those who had other malignant tumors or treated by anti-tumor therapies for other tumors or severe comorbidities which may affect their survival and tolerance in our study or pregnancy or liver and kidney dysfunction or treatment delayed more than 2 weeks or been interrupted up to 4 cycles or the quality of specimens did not meet with the standard should be excluded.1.Collecting the clinic-pathological and follow-up data and pathological specimens of EGFR rare-mutant NSCLC patients,using NGS to detect gene profiling of pre-existing EGFR rare mutations,and analyzing the general clinic-pathological features with PFS and EGFR rare mutations.2.Follow-up of these patients who developed drug resistance during therapy and dividing them into the primary resistance and secondary resistance groups.The drug-resistant lesions or circulating tumor DNA(ctDNA)were collected for corresponding primary and secondary resistance gene profiles detection by NGS method.The primary resistance was analyzed,and the relationships between secondary resistance gene profiling to icotinib and EGFR rare mutations and PFS were analyzed.3.The relationship between concomitant and primary/secondary-resistant mutations was analyzed and summarized in heat-maps.4.The categorical values were evaluated by the Pearson chi-square test,and Fisher's exact probability method was used if necessary.The continuous values were analyzed by T-test,and non-normal distribution data was analyzed by Rank-sum test.Kaplan-Meier plotting,log-rank test and COX regression for survival analysis.RESULTS:1.Among the 3117 patients with NSCLC from multi-center in China,196 patients with EGFR rare mutations(accounting for 10.88%of all EGFR mutations)were included.Compared with EGFR classical mutations,earlier age of onset and more adenocarcinoma types were found in EGFR rare mutation NSCLC patients(p<0.001).Ninety-eight patients of EGFR rare mutation who accepted icotinib therapy were enrolled for further analysis.EGFR rare mutations included mutations happened in EGFR exon 18,exon 20,exon 21 and other rare mutations.The single mutations with higher frequency included 20-Ins,L861Q,G719X,S768I,and T790M;the complex mutations with higher frequency included G719X+S7681 and T790M+ classical mutations.After a median follow-up of 6.2 months,69 patients(70.41%)had disease progression eventually,the objective rate(ORR)and disease control rate(DCR)was 13.27%and 29.59%respectively,and the median PFS was 5.5 months(95%CI:1.2-13 months).Subgroup analysis suggested that complex with classical mutations and single mutations have better PFS than complex mutations without classical mutations(P<0.05);patients harboring S768I mutation have worst PFS than other mutation subtypes(p<0.05);The highest incidence of side effects of icotinib was diarrhea(42.9%),but the incidence of all grade 3/4 side effects was relatively low(5.1%).2.Twenty-one patients(21.4%)developed primary resistance to icotinib therapy.In this group,patients harboring S768I,L861Q,G719X and 20-Ins accounted for 76.2%in all;the PFS of patients who have complex mutations without classical mutation inclined to be longer than single mutations but not statistically significant(2.3 vs 1.8 months,p=0.06).EGFR ECD mutations,BCL2L11 deletions,and MET amplification were the most common variations in the primary resistant gene profiling.Others included HER2 and MYC amplification,PTEN loss and PIK3CA mutations.There was no significant correlation between the primary resistance gene profiling and the EGFR rare mutation subtypes.3.Forty-eight patients(49%)developed secondary resistance to icotinb therapy.In this group,patients harboring G719X,S768I and L861Q accounted for 56.3%in all;the complex mutations accounted for 45.8%(22/48),including of 2 cases complexed with 19del and 10 cases complexed with L858R;the single mutations accounted for 54.2%(26/48).Median PFS in this group was 6.5 months,and comparing with the single mutation and complex with classical mutations,complex mutation without classical mutations patients had the shortest PFS(3.3 months,95%CI=2.53-4.08,p=0.00).Secondary resistance mutations can be divided into T790M acquired type(83.3%)and T790M wild type(16.7%),of which T790M+EGFR amplification accounted for 62.5%;T790M acquired patients tended to have longer median PFS than T790M wild patients but no significant differences between them(6.6 vs.5.3 months,p=0.06).4.The icotinib-related primary and secondary resistant mutations and accompanied mutant gene profiling were mapped and analyzed for corrections with each other.A total of 159 concomitant genomic mutations were detected in primary resistant patients,including TP53(61.9%),NF1(23.8%)and DNMT3A(19.04%)in most common.A total of 114 concomitant genomic mutations were detected in secondary resistant patients,including TP53(45.8%),SMARTA4(8.3%)and CDKN2A(8.3%)in most common.The mutation types of both groups were mainly deletion and nonsense mutations.Compared with secondary resistant patients,primary resistant patients had more accompanied mutant gene variations(p<0.001).CONCLUSION:In this study,almost 3/4 of advanced stage NSCLC patients with EGFR rare mutation developed resistance to icotinib during the follow-up period.The type of rare mutations in EGFR(especially whether complex or single mutations,complex mutations with classical mutations or not)may affect the PFS of those patients,meanwhile,the icotinib-resistance gene variations also had corrections with those pre-treated types.Primary and secondary icotinib-resistant gene spectral distributions were characteristically different.However,no significant correlation between the type of resistance genes and PFS of patients was found in our research.The primary resistance group carried more accompanied mutations than the secondary resistance group.In view of the strong heterogeneity of tumors in patients with EGFR rare mutations,it is necessary to further study the correlation among the EGFR rare mutations,TKIs-resistant gene expression and mechanism of causing resistance.Our purpose is to find the potential effective strategies or drugs to overcome drug resistance and prolong patients' overall survival.Searching for more effective therapeutic targets and optimal drugs or strategies in this particular advanced NSCLC patients is warranted.