Clinical Application Of Radiotherapy Combined With EGFR-TKI In NSCLC And Radiobiology Study Of Multi-Beam Irradiated Egfr Mutant Cell Lines

Background and objective Non-small cell lung cancer(NSCLC)is a common malignant tumor that seriously threatens human life and health.Its morbidity and mortality locate at the first in the world and China,causing serious medical and health burden.Radiotherapy and targeted therapy are the common main treatment methods for inoperable NSCLC.Radiotherapy plays an important role in improving local control rate,prolonging the overall survival(OS)and progression-free survival(PFS).However,the best combination strategy of locally advanced or metastatic NSCLC based on different epidermal growth factor receptor(EGFR)mutation status is unclear,the current situation of thoracic radiotherapy(TRT)combined with EGFR tyrosine kinase inhibitors(TKI)is not yet definite,the clinical benefit of radiotherapy combined with EGFR-TKI versus EGFR-TKI alone remains controversial.In consideration of above problems,we conducted this systematic research aimed to supply more evidence.Firstly,we used evidence-based medicine(EBM)method to comprehensive search relative clinical studies worldwide of TRT combined with EGFR-TKI in NSCLC and performed a Meta-analysis,in order to evaluate effectiveness and safety of this combined treatment modality.Secondly,a retrospective clinical cohort study was conducted to compare the clinical efficacy of Icotinib alone versus Icotinib combined with TRT for stage III/IV or post-operative recurrence and metastatic EGFR-positive NSCLC.Lastly,we conducted a vitro cell experiment to explore the radiobiological differences of different dose carbon ion beam versus X-ray on two kinds of EGFR mutation cell line PC-9(19 DEL)and H1975(21 L858 R and 20 T790 M mutation)cell line.It is expected to provide theory basis on application of radiotherapy in NSCLC with different EGFR mutation type.Methods(1)Evidence-based medicine research: Computer retrieval method was used to search foreign language database including MEDLINE,EMBASE and Cochrane Library databases,Chinese database including Chinese journal full-text database(CNKI)and Chinese biomedical literature database(CBM),respectively.We comprehensively screened relative literatures through reading title,abstract and fulltext and then to perform Meta-analysis.The main measure outcomes included response rate(RR),overall survival(OS),progression-free survival(PFS),and safety outcomes.(2)Retrospective cohort study: According to inclusion/exclusion criteria,we retrospectively analyzed patients with EGFR mutation positive NSCLC who first line using Icotinib combined with TRT or Icotinib alone in Gansu provincial cancer hospital during Jan 2012 to Dec 2017.Using propensity score matching(PSM)to match baseline data between two groups.The main endpoints included lung primary lesion RR,PFS and OS,the secondary endpoints were Icotinib resistance duration and total application time,AEs incidence,and clinical factors of influence survival.SPSS 17.0 software was used for Statistical analysis.Independent samples t test and chi-square test was used to compare general characteristics of two groups.The differences of RR,incidence of AEs,and progress patterns were calculated using chi-square test.The log-rank test of kaplan-meier method estimated differences of PFS and OS.COX multi-factor regression analysis and Pearson correlation analysis were used to analyze clinical factors affecting survival.(3)Vitro cell experiment: Two kinds of EGFR mutation cell line PC-9 and H1975 were cultured in vitro.Cells in logarithmic growth stage were selected for experiments.After irradiation with carbon ion beam and X-ray of same hierarchical dose,the cells survival fraction(SF)was calculated in the cloning formation experiment and fitted survival curve to observe radiosensitivity.Cell cycle was detected by flow cytometry.Hoechst staining was used to detect apoptosis.Hypoxic induction factor-1a(HIF-1α)and vascular endothelial growth factor(VEGF)mRNA were extracted by RT-PCR,and the expression of VEGF and HIF-1α protein was detected by Western blot(WB).SPSS 17.0 and GraphPad Prism 5 software were used for statistical analysis and diagramming.Results(1)A total of 16 prospective single-arm studies and 14 clinical controlled studies were included in Meta-analysis.12 single-arm studies(including 446 patients)reported RR and survival outcomes of TRT combined with EGFR-TKI for NSCLC with unknown EGFR status,the pooled CR,PR,SD and PD respectively were 0.06(95%CI 0.03-0.09),0.44(95%CI 0.38-0.49),0.29(95%CI 0.24-0.34)and 0.15(95%CI 0.11-0.19).1-and 2-year pooled OS were 0.52(95%CI 0.44-0.60)and 0.26(95%CI 0.18-0.33)respectively.4 single-arm studies(including 182 patients)reported RR and survival outcomes of CRT combined with EGFR-TKI for NSCLC with unknown EGFR status,the pooled CR,PR,SD,and PD respectively were 0.12(95% CI 0.02-0.22),0.41(95% CI 0.27-0.55),0.31(95% CI 0.16-0.46),and 0.14(95% CI 0.01-0.30).1-and 2-year pooled OS were 0.83(95%CI 0.71-0.94)and 0.67(95%CI 0.54-0.81),respectively.There was no severe AEs reported in these studies using TRT or CRT combined with EGFR-TKI.7 clinical controlled studies(including 695 patients)reported the results of TRT+TKI vs.TKI alone in the treatment of EGFR mutant NSCLC.The pooled results showed that Relative risk(RR)(95%CI)and p values of Objective response rate(ORR)and Disease control rate(DCR)respectively were [1.69(1.44-1.98),p<0.00001] and [1.35(1.25-1.46,p<0.00001].RR(95%CI)and p values of 1-and 2-year pooled OS were [1.22(1.06-1.41),p=0.007] and [1.56(1.09-2.23),p=0.02],respectively.There were statistical differences between two groups to above results,TRT+TKI group superior to TKI alone group.In terms of safety,there were statistical differences in interstitial pneumonia,radiation esophagitis,nausea and vomiting,leukopenia and liver function abnormal between two groups,TRT+TKI group was higher than TKI alone group in pooled AEs incidence.7 clinical controlled studies(including 493 patients)reported the results of TRT+TKI vs.TRT in the treatment of EGFR mutant NSCLC.The pooled results showed that RR(95%CI)and p values of ORR and DCR were [1.40(1.22-1.61),p<0.00001] and [1.13(1.05-1.21,p=0.0007],respectively.In terms of survival rate,RR(95%CI)and p values of 1-year and 2-year OS were [1.42(1.16-1.74),p=0.0006] and [1.71(1.15-2.54),p=0.008],respectively.There was a statistical difference between two groups in above results,and the TRT+TKI group was superior to the TRT group.In terms of safety,there were statistical differences in rash and diarrhea between two groups,TRT+TKI group was higher than TRT group in pooled AEs incidence.(2)In retrospective cohort study,according to the inclusion/exclusion criteria and propensity score matching,a total of 56 patients with EGFR mutation positive NSCLC were included.28 in Icotinib combined TRT group and 28 in Icotinib alone group.There was a statistically difference in lung primary lesion RR between two groups,especially in ORR(p=0.019),the Icotinib +TRT group was superior than Icotinib alone group(p=0.002).There were no statistical differences in terms of 1-and 2-year PFS and 1-year OS(p >0.05),while there was statistical difference in 2-year OS(p=0.032).The median OS and median PFS were 40.8 month(m)vs.25.7m and 20.3m vs.13.3m.Subgroup analysis showed that Icotinib+TRT group have more obvious benefits in ECOG 2(HR 0.43,95%CI 0.03-0.79),adenocarcinoma(HR 0.44,95%CI 0.02-0.81),EGFR 19 DEL mutation(HR 0.43,95%CI 0.08-0.93)and pre-inclusion chemotherapy(HR 0.65,95%CI 0.15-0.97).Multi-factor analysis showed that Icotinib resistance duration and total application time were positively correlated with PFS and OS(p=0.000),and ORR was also closely correlated with PFS and OS(p=0.000).In terms of toxicity,there were statistically differences between the two groups in acute radiation pneumonia,radiation esophagitis,leukopenia,nausea and vomiting(p<0.05),Icotinib +TRT group was higher than Icotinib alone group in AEs incidence.(3)In vitro experiment,cell SF and proliferation suppression effect of carbon ions were significantly greater than that of X-ray,this effect was significantly correlated with the irradiation dose(p< 0.05).At the same dose,carbon ion had a greater suppression on the SF of PC-9 than H1975,but in X-ray group,the SF of two types of cell were similar.The RBEs of carbon ion fitted by cell survival curve on PC-9 and H1975 were 2.86 and 1.85,respectively.The apoptosis induced by carbon ion was more significant than that of X-ray,and the difference was more significant with the increase of irradiation dose.Comparing two cells,PC-9 cell had higher apoptosis rate than H1975,especially in carbon ion group,along with the dose increase,the more significant difference.Irradiation mainly caused G2/M phase arrest,with the increase of irradiation dose,G2/M phase cells proportion increased significantly.Comparing 0Gy group,there were statistical differences in 1Gy,2Gy,and 4Gy of carbon ion irradiation and 4Gy of X-ray irradiation(p<0.05).And there was no significant difference between the two cells in each dose group.RT-PCR results showed that mRNA level of HIF-1α in two cells did not decrease significantly in carbon ion and X-ray group at any dose group,and there was not statistical difference compared with 0Gy group(p>0.05),there was also no statistical difference between carbon ion and X-ray in each dose group(p>0.05).Carbon ion had a certain down-regulation effect on VEGF mRNA level in PC-9 and H1975 cells,and the down-regulation result was most significant in the 4Gy dose group(p<0.05),especially on H1975 cell.Comparing two cells,the downregulation effect of carbon ion on VEGF mRNA level in H1975 was more significant than that in PC-9 cell under the 4Gy dose group(p<0.05).Compared with the 0Gy,the HIF-1α protein expression level of two cell lines in each dose group of each radiation was not significant(p>0.05).The down-regulated effect of VEGF protein by carbon ion was the most significant in 2Gy and 4Gy dose groups compare to 0Gy(p<0.05),and by X-ray,only in 4Gy dose group had a statistically significant compare to 0Gy(p<0.05).The decrease of VEGF protein in H1975 cell line was more significant than that in PC-9 cell line in carbon ion irradiation,especially in 2 and 4 Gy groups(p<0.05).Conclusion(1)Based on the current limited evidences,Meta-analysis indicated that comparing other treatments,such as TKI alone,TRT,CRT or chemotherapy based on best regimen,TRT combined with EGFR-TKI can significantly improve response rate and survival rate in patients with EGFR mutant locally advanced or metastatic NSCLC,even in patients with EGFR mutations status unknown.This combined treatment modality does not increase the TKI-related AEs.(2)Comparing Icotinib alone,Icotinib combined with TRT increased response rate of primary lung lesion,improved PFS and OS,but accordingly added the risks of radiotherapy related acute radiation pneumonia and esophagitis.In clinical practice,we should lay emphasis on individual patient selection based on the specific clinical characteristics and pay close attention to treatment-related adverse reactions.(3)Carbon ion have higher radiobiological effects compared to X-ray for EGFR mutation cell lines PC-9 and H1975,which could significantly reduce cell clone survival rate,inhibit cell proliferation,induce apoptosis,promote the G2/M cell cycle arrest.Furthermore,radiosensitivity of PC-9 was higher than that of H1975 on carbon ion and hypofraction X-ray irradiation.The carbon ion significantly reduced VEGF level in H1975 cell line,suggesting that carbon ion may reverse EGFR-TKI resistance and improve tumor chemotherapy sensitivity by down-regulating VEGF level in tumor tissues.