The Curative Effects Of EGFR-TKIs In Different EGFR Mutation Genotypes And Its Relationship With Expression Of AEG-1 In Advanced Lung Adenocarcinoma

Objective: Epidermal growth factor receptor(EGFR)mutation is one of the most important driver gene mutations in non-small cell lung cancer. Several clinical trials have indicated that EGFR mutation is distinctly relevant with the curative effects of EGFR tyrosine kinase inhibitors(EGFR-TKIs). Several researches have indicated that there are differences in different EGFR genotypes(EGFR exon 19 deletion, EGFR exon 21 point mutation and so on) on curative effects, nevertheless, the mechanism is still not clear. Astrocyte elevated gene-1(AEG-1), recognized as an oncogene, is an independent prognostic factor for many malignant tumors, and is related with PI3K/Akt pathway together with EGFR mutation. Questions still remain at home and abroad that if AEG-1 could be a prognostic indicator for EGFR targeted therapy in lung cancer. The aims of this study are to discuss the connection between EGFR-TKIs curative effects and different EGFR genotypes, clinicopathologic features, and prognosis of EGFR targeted therapy in advanced lung adenocarcinoma patients with EGFR mutations. Meanwhile, in this study we use immunohistochemistry to detect AEG-1 protein expression in different EGFR genotypes tissues, and discuss the relationships between AEG-1 protein expression and EGFR-TKIs curative effects, in order to provide referential indicators for predicting EGFR-TKIs efficacy and evaluating prognosis for advanced lung adenocarcinoma patients who have EGFR mutations.Methods: One hundred and fifteen patients were enrolled in this study. Data were pooled from the Fourth Hospital of Hebei Medical University from the May 2010 to October 2014. Patients all suffered from advanced lung adenocarcinoma with EGFR sensitive mutations and accepted EGFR-TKIs therapies. The clinical stages were ⅢB/Ⅳ. All of the pathological tissues had all been done the EGFR gene detection by quantitative fluorescence in situ hybridization and flow cytometry(flow-FISH), including exon 18, 19, 20, 21 sites, and proved to be mutated. All the patients were accepted EGFR-TKIs targeted therapy until disease progression or unacceptable toxic effects. TKIs were recommended as one of the following regimens: Gefitinib, 250mg/d; Erlotinib, 150mg/d; Icotinib, 250 mg, three times a day. The clinicalpathologic information of all these patients was recorded and all patients were followed up by telephone and out-patient review. Progression free survival(PFS) and overall survival(OS) were recorded and targeted lesions were evaluated according to Response Evaluation Criteria in Solid Tumors(RECIST) version 1.1. The follow-up period ended on September 30 th, 2015, or death, lost to date. It was considered complete data if patients appeared disease progression during the treatment of EGFR-TKIs or died of lung cancer related diseases; and it was considered censored value if patients did not appear disease progression, were still alive or died of other diseases. The main endpoint of this study is PFS, secondary endpoints includes OS, objective response rate(ORR) and disease control rate(DCR). The available tumor specimens of these patients were taken out from the pathology department. AEG-1 protein expression of these tumor specimens was detected by immunohistochemistry(IHC). Then the patients were divided into the low expression of AEG-1 group and high expression of AEG-1 group according to the 9 score of the median immunohistochemistry score.All the follow-up data would be proofread and input in SPSS21.0 software for statistical analysis. The clinicalpathologic features, such as gender, age, clinical stage, smoking history, EGFR mutation genotypes, choice of treatment beginning, EGFR-TKIs option and AEG-1 expression were chosen to analyze in these 112 patients. Chi-square test was used for enumeration data comparison. Kaplan-Meier was used for survival analysis in EGFR exon 19 deletion and EGFR exon 21 point mutation group, as well as in low expression and high expression of AEG-1 group. The univariate and multivariate analysis were performed by Cox proportional hazard model to find out independent prognostic determinants and the variables that P<0.10 in univariate analysis was brought into multivariate COX proportional hazard model analysis. P values less than 0.05 were considered statistically significant.Results: 115 patients were enrolled in this study. The median follow-up time was 21.2 months. During treatment or follow-up period, 65 patients were dead(56.5%) and 3 patients were lost(2.61%). The complete information of 112 patients was analyzed as follows.1、Clinicalpathologic features1.1 Patients with EGFR exon 19 deletion and EGFR exon 21 point mutation have no obvious difference in gender, age, clinical stage, smoking history, choice of treatment beginning and EGFR-TKIs option.1.2 The expression of AEG-1was analyzed by IHC in 89 advanced lung adenocarcinoma specimens: AEG-1 protein is all positive expressed. According to the median score 9 of the IHC staining scores, which was used as the cutoff point for the classification of the patients, 46 tumor specimens exhibited high expression of AEG-1 whereas 43 tumor specimens exhibited low expression of AEG-1. In AEG-1 high expression group, EGFR exon 21 point mutation is more commonly observed, however, in AEG-1 low expression group, EGFR exon 19 deletion is more commonly observed. The two groups have different EGFR mutation genotypes(P=0.003), but have no obvious difference in gender, age, clinical stage, smoking history, choice of treatment beginning and EGFR-TKIs option.2、EGFR-TKIs efficacy analysis and survival analysis2.1 Of all the 112 cases,there are one CR patient(1/112, 0.89%), 50 PR patients(50/112, 44.6%), 42 SD patients(42/112, 37.5%) and 19 PD patients(19/112, 17.0 %). ORR is 45.5%(51/112), DCR is 83.0%(93/112).Among the 56 patients with EGFR exon 19 deletion, there are no CR patient(0/56, 0%), 33 PR patients(33/56, 58.9%), 18 SD patients(18/56, 32.1%), 5 PD patients(5/56, 8.9%), ORR and DCR are 58.9%(33/56), 91.1%(51/56) respectively.Among the 48 patients with EGFR exon 21 point mutation, there are one CR patient(1/48, 2.08%), 17 PR patients(17/48, 35.4%), 19 SD patients(19/48, 39.6%), 11 PD patients(11/48, 22.9%), ORR is 37.5%(18/48), DCR is 77.1%(37/48). The TKIs efficacy for patients with EGFR exon 19 deletion is better than patients with EGFR exon 21 point mutation. There is statistically significance in ORR(P=0.029)and DCR(P=0.049)between two groups.Among the 8 rare EGFR mutation genotypes, there are 3 patients with EGFR exon 18 mutation, 2 of them are SD patients and one of them is a PD patient; There are 2 patients with EGFR exon 19 and 21 mutation, one patient is SD, the other is PD; There is a patient with EGFR exon 18 and 20 mutation, which is considered SD; There is a patient with EGFR exon 21 and 20 mutation, which is considered SD; There is a patient with EGFR exon 19 deletion and exon 20 T790 M resistant mutation, which is considered PD.2.2 PFS analysis: Of all the 112 patients under EGFR-TKIs treatment, median PFS is 11.8 months.2.2.1 PFS analysis for different EGFR mutation genotypes: Median PFS of patients with EGFR exon 19 deletion is 19 months, however median PFS of patients with EGFR exon 21 point mutation is 10 months. PFS is significantly different between the two groups(P<0.001).2.2.2 PFS analysis for different levels of AEG-1 expression: Of all the 89 tissue specimens analyzed by IHC, median PFS is 14 months. Median PFS of patients with high AEG-1 expression is 10 months, however, median PFS of patients with low AEG-1 expression is 19.9 months, which is significantly different between the two groups(P=0.01).2.2.3 COX analysis of PFS:COX univariate analysis indicates that PFS affecting factors include EGFR mutation genotypes(P < 0.001), AEG-1 expression level(P=0.012) and EGFR-TKIs option(P=0.005). COX multivariate analysis indicates that the independent PFS prognostic determinants include EGFR mutation genotypes and AEG-1 expression level.2.2.3.1 EGFR mutation genotypes: The disease progression risk of EGFR exon 21 point mutation group is 1.744 times of EGFR exon 19 deletion group in advanced lung adenocarcinoma. EGFR mutation genotype is an independent determinant for PFS(P=0.035).2.2.3.2 AEG-1 expression level: The disease progression risk of high AEG-1 expression group is 1.839 times of low AEG-1 expression group in advanced lung adenocarcinoma. AEG-1 expression level is another independent determinant for PFS(P=0.012).2.3 OS analysis: Of all the 112 patients under EGFR-TKIs treatment, median OS is 28 months.2.3.1 OS analysis for different EGFR mutation genotypes: Median OS of patients with EGFR exon 19 deletion is 32.8 months, while median OS of EGFR exon 21 point mutation is 21.6 months. OS is significantly different between the two groups(P=0.031).2.3.2 OS analysis for different levels of AEG-1 expression: Of all the 89 tissue specimens analyzed by IHC, median OS of patients with high AEG-1 expression is 22.9 months, but median OS of patients with low AEG-1 expression is 32.8 months. OS is also significantly different between the two groups(P=0.026).2.3.3 COX analysis of OS:COX univariate analysis indicates that the affecting factors of OS include EGFR mutation genotypes(P=0.020), AEG-1 expression level(P=0.029) and EGFR-TKIs options(P=0.027). COX multivariate analysis indicates that the independent prognostic determinant for OS is AEG-1 expression level. The mortality risk of high AEG-1 expression group is 1.975 times of low AEG-1 expression group in advanced lung adenocarcinoma. AEG-1 expression level is significantly correlated with OS(P=0.026),high AEG-1 expression indicates poor prognosis.Conclusions: 1. Patients with different EGFR mutation genotypes respond differently to EGFR-TKIs therapy:Patients with EGFR exon 19 deletion have higher ORR and DCR, longer PFS and OS with EGFR-TKI therapy compared to those who have EGFR exon 21 point mutation.2. There is a significant difference in different EGFR mutation genotypes on the matter of AEG-1 expression: AEG-1 expression level is higher in EGFR exon 21 point mutation tissue than that in EGFR exon 19 deletion tissue. Patients with high AEG-1 expression indicate poorer prognosis and have shorter PFS and OS than those with low AEG-1 expression.3. EGFR mutation genotypes as well as AEG-1 expression level can both evaluate prognosis(PFS and OS) for advanced lung adenocarcinoma patients with EGFR mutation.4.EGFR mutation genotypes and AEG-1 expression level can both recognised as the independent PFS prognostic determinants in individual EGFR-TKIs therapy for PFS while AEG-1 expression level has influence in OS which need to be verified in a large sample study.