The Correlation Between Crizotinib Efficacy And Molecular Heterogeneity By Next-generation Sequencing In Non-small Cell Lung Cancer

Objective:In recent years,with the concept of precision medicine,the treatment of non-small cell lung cancer has entered the age of molecular therapy.EML4-ALK fusion gene is a molecular subtype of non-small cell lung cancer,and it has carcinogenicity both in vivo and in vitro.Non-small cell lung cancer(NSCLC)patients harboring EML4-ALK fusion exhibited various duration of response to crizotinib.Molecular heterogeneity was also one of the factors that were resistant to crizotinib.This study investigated the relevance of molecular heterogeneity by next generation sequencing,with clinical efficacy of crizotinib.Methods:A total of 67 ALK-positive advanced NSCLC patients were screened,retrospectively.Screening criteria:Between May 2013 and June 2016,diagnosed as advanced ALK fusion positive NSCLC by three clinical institutions;CT showed measurable target lesions;age ? 18 years;ECOG score 0-1;normal liver,kidney,heart function;no other malignancy history;regular administration of crizotinib during treatment,250 mg,po bid,twenty-eight days were a treatment cycle,taking medication continuously until the disease progresses or meets termination criteria.We mainly counted the progression-free survival(PFS)of patients taking crizotinib.Paraffin sections were obtained from these 67 patients and second-generation sequencing technology was used to detect genetic heterogeneity.Among them,there are 15 samples of DNA fragments<170bp in length,suggesting DNA degradation of the sample.Another 4 samples failed to extract DNA,.Excluding these 19 patients,48 patients were eventually selected for in-depth sequencing.Statistical analysis was performed using SPSS version 21.0 software.The difference clinical characteristics between groups were compared using Independent Samples Test and Chi-square test(or Fisher's exact test).The survival probabilities were estimated using the Kaplan-Meier method,where differences in the variables were calculated using the log-rank test.All p values were two-sided,and p<0.05 was considered statistically significant.The genetic variation had been revealed by NGS.We identified different ALK fusion types,AF and additional coexisting mutations(ACMs),evaluated the correlation between above three factors and clinical response to crizotinib.The study design was approved by the ethics committee of Nanjing General Hospital,who waived the need for informed consent because of the non-invasive nature of the study and patient anonymity.Results:Among the group which was detected with ALK+ fusion by IHC,patients detected as ALK-fusion by NGS method associated with a shorter PFS,compared with ALK(+)patients by NGS.Moreover,for different ALK fusion types,the median PFS of variant 1/2/3,and other uncommon variants were 305 days,557 days,242 days and 370 days,respectively.Although no statistically significant difference,patients harboring ALK variant 2 seemed to display a longer PFS trend than other types of variants in this study.In addition,significant difference was not exist in the relationship of ALK fusion AF and PFS(p=0.639).Further exploration,we found that in the IHC+ group,IHC+/NGS-group and IHC+/NGS+ group,other coexisting mutations(ACMs)did not affect the efficacy of crizotinib(IHC+,IHCH+/NGS-,IHC+/NGS+,p= 0.738/0.801/0.550).Besides,the mutation frequency of TP53 gene and FAT3 gene in these patients was high,and unfortunately,they were not found to have a significant correlation with the efficacy of crizotinib.Finally,through second-generation sequencing,we observed that the six signaling pathways of ErbB,MAPK,Ras,FoxO,Rap1,and PI3K/Akt were closely related to the M staging of patients with advanced ALK-positive non-small cell lung cancer.Conclusions:Our study is the first report that investigated the relevance of ALK(+)by NGS and the clinical outcome in non-small cell lung cancer.Suggesting that a variety of detection methods can be used in combination to better understand ALK and the correlation between the different contents of heterogeneity of ALK and TKI effect is a promising research area.