Study On The Efficacy And Safety Of Crizotinib In Patients With Advanced NSCLC And Preliminary Research On Its Drug-resistance Mechanism

Objective:Study on the clinical efficacy and safety of crizotinib in Chinese patients with advanced NSCLC,the relationship between Css and AEs,also screening lncRNAs related to crizotinib resistance.Methods:1.Inclusion criteria were made for screening suited patients into our study.Efficacy of crizotinib in Chinese patients with advanced NSCLC was evaluated according to RECICT 1.1 and all adverse events were graded by attending doctors according to CTCAE v.4.0.Difference of clinical efficacy was compared with Fisher’s exact probability,Kaplan-Meier curve and Log-rank test were used for survival analysis.2.HPLC-MS/MS method was established for determining the concentration of crizotinib in human plasma and the trough concentration of patients were measured.The incidence of AEs were assessed at one month interval of sampling,and all AEs were graded according to CTCAE v.4.0.To assess the correlation on the number of AEs with crizotinib trough concentration,the trough concentrations were divided into a higher trough concentration group and a lower trough concentration group delimited by the median concentration.3.The human lung adenocarcinoma cell line H3122 was treated with ENU and also exposed to crizotinib of a high and then increasing concentration to establish crizotinib resistance cell line H3122/CR.Differentially expressed lncRNAs between H3122 and H3122/CR were identified using lncRNA gene chip.Target lncRNAs were selected according to the fold change and database query,and then validated by RT-PCR.Result:1.A total of 32 NSCLC patients were enrolled in this study.26 patients were ALK-positive,4 patients were c-MET gene amplification（one of them was also EGFR-positive）,2 patients were ROS1-positive and another one did not do any gene detection.21 patients were male,11 patients were female,the median age was49,the objective response rate was 71.0%,disease control rate was 93.6%,median PFS was 7 months.Survival analysis showed that there was a difference in PFS between patients with or without pleural metastasis at baseline.（9 VS 3 months,P< 0.05）.The adverse events included nausea,vomiting,elevated ALT/AST,diarrhea,visual disorder,anorexia,constipation,fatigue,and neutropenia,and most of them were mild adverse events of grade 1²,the safety was favorable.2.The HPLC-MS/MS method for determining the concentration of crizotinib in human plasma was successfully established.The standard curve of crizotinib was y=3.91×10-3x+1×10-3（r=0.9994）,and in 5-1000 ng·m L-1it showed good linear relationship.The lowest concentration was 5 ng·m L-1.Intra-day and inter-day RSD of blood samples were less than 15%,the average recovery was > 95%,and stability are good.The method is simple,rapid,sensitive and accurate,specific,suitable for the determination of crizotinib concentration in human plasma.The trough concentration of eight ALK-positive NSCLC patients were measured and median Css was 352.65 ng m L-1.The trough concentrations were divided into a Css higher group（n = 4）and a Css lower group（n = 4）delimited by the median concentration（352.65 ng m L-1）.In this study,adverse events of grade 3 were only observed in Css higher group,adverse events in Css lower group were all grade 1²,and the number of AEs in Css higher group was a little more than Css lower group.3.H3122/CR was able to grow normally in 1200 nmol·L-1crizotinib after induction for 3 months.The IC50 of H3122/CR was 1672 nmol·L-1and H3122 was just 309nmol·L-1.The resistance index could reach 5.41.Eight lncRNAs related crizotinib resistance were selected according to lncRNA gene chip and RT-PCR,four of them were up-regulated（ENST00000414416.2,ENST00000512067.1,TCONS₀0005147,TCONS₀0015171）,and four of them were up-regulated（ENST00000426615.2,ENST00000562613.1,ENST00000566951.1,ENST00000568189.1）.Conclusion:The clinical efficacy and safety of crizotinib were confirmed in this study.It was very effective for the treatment of ALK-positive/c-MET gene amplification/ROS1-positive advanced NSCLC,ORR and DCR were increased,PFS was prolonged,and adverse reactions were mild.Patients were well tolerated.The study on the relationship between trough concentration and adverse events indicated that the high level of trough concentration may be an important risk factor for the occurrence of severe adverse event.For inevitable crizotinib-resistance,eight lncRNAs related crizotinib resistance were selected for further functional and mechanism studies.