Investigation Of The Efficacy And Predictive Marker Of Gefitinib In Patients With Non-Small Cell Lung Cancer And The Effect On Bleomycin-induced Lung Fibrosis In Mice

Objective:(1)The current study was undertaken to evaluate the efficacy and safety of Gefitinib monotherapy in patients with advanced non-small-cell lung cancer(NSCLC).(2)To analyze the correlation between the effectiveness of Gefitinib treatment and mutations of the kinase domain of EGFR and K-ras gene and aberrant promoter methylation of p16 gene.(3)To investigate the effect of Gefitinib on bleomycin-induced lung fibrosis in mice.Methods:(1)29 patients with advanced NSCLC verified by pathology or cytology were enrolled and received 250 mg of oral Gefitinib monotherapy daily. RECIST criteria were accepted to evaluate the efficacy of Gefitinib.(2)We obtained paraffin tissue blocks of primary tumors from 29 patients.EGFR gene in exon 18-21 and K-ras gene in exon 2 and the promoter methylation of pl 6 gene of these tumor samples were analyzed by genomic polymerase chain reaction and direct sequence.Then,the relationship between the mutants and methylation and the efficacy of Gefitinib was evaluated.(3)We examined the effect of Gefitinib,a selective EGFR tyrosine kinase inhibitor(TKI),on bleomycin-induced pulmonary fibrosis in mice.Animals were allocated to four groups,as follows:(1) intratracheal saline+vehicle,(2)intratracheal saline+200 mg/kg of oral gefitinib, (3)intratracheal bleomycin+oral vehicle,(4)intratracheal bleomycin +200mg/kg of oral gefitinib.C57BL/6 mice were administered a single intratracheal injection of bleomycin(5 units/kg)on day 1.Gefitinib(200 mg/kg)or vehicle alone were administered p.o.1 h before this injection and on days 1-5 each week for 3 weeks. Lung tissue was harvested on day 14 and 21.Lung histology and real-time PCR and western blot were performed to show the change between the four groups.Results:(1)29 patients were assessed for the efficacy and safety of Gefitinib. The response rate was 37.9%and the disease control rate was 72.4%.The median time to progression free survival(PFS)and the median overall survival(OS)were 10.0 and 16.8 months,respectively.The 1-year survival rate was 51.7%.Patients of female,adenocarcinomas and no smoking experienced statistically significant improvements in efficacy of Gefitinib compared with other group(P＜0.05).Grade 1 or 2 toxicities were observed in 79.3%patients.(2)EGFR mutation was detected in 11 of 29 tumors(37.9%),and K-ras mutation was in 3 tumors(13.8%). Gefitinib chemotherapy in patients with advanced NSCLC harboring EGFR mutations was highly effective.The overall survival of patients with K-ras mutation was shorter than that of patients without mutation(P＜0.05).The correlation between the mutations of EGFR and K-ras and the efficacy of gefitinib was statistically significant(P＜0.05).The results showed that 55.6%patients (15/29)with non-small cell lung cancer had the promoter hypermethylation of p16 gene.But there was no relationship verified between the promoter hypermethylation of p16 gene and the efficacy of Gefitinib.(3)Lung histology and the results of real-time PCR showed more severe fibrosis in the mice receiving bleomycin and the vehicle than in the mice receiving both bleomycin and gefitinib.Furthermore,in those mice that did not receive bleomycin treatment,gefitinib did not induce lung fibrosis.A western blotting analysis showed that phosphorylated EGFR and S100A4 were highly expressed by the regenerated epithelial cells in the mice treated with bleomycin and the vehicle.In contrast,the expression of these antigens was attenuated in the mice treated with bleomycin and gefitinib.Conclusions:(1)Gefitinib monotherapy in chemotherapy-naive advanced NSCLC patients was active,with acceptable toxicities.(2)The mutation of EGFR kinase domain was highly associated with response of gefitinib and prolonged overall survival.The mutation of K-ras has inverse correlation with the efficacy of gefitinib.The relationship between the promoter hypermethylation of p16 gene and the efficacy of gefitinib was not verified.(3)The point of view was not proved in our experiment,that gefitinib could augments bleomycin-induced lung fibrosis in mice.