The Effect Of EGFR Mutation On First-line Chemotherapy For Lung Adenocarcinoma And Related Molecular Mechanism

BackgroundThe most important progresses in the field of advanced lung adenocarcinoma are related to the study of tumor-driving genes and targeted therapy. The first and best known example is the discovery of epidermal growth factor receptor (EGFR) mutations, which has provided an important basis in determining therapeutic schedule for the patients with advanced and inoperable lung adenocarcinoma.Studies found that EGFR mutation status would probably affect the response to chemotherapy for the patients with advanced lung adenocarcinoma. Many researchers suggested that EGFR-mutated patients would be much easier to benefit from chemotherapy than EGFR-wild patients. There were also researchers claimed the advantage of EGFR-mutant patients in term of survival. Nevertheless, a few of studies reported that the response rate to chemotherapy and survival time were not significantly different between EGFR-mutant and wild patients. Another investigator indicated that the survival time of EGFR-mutant patients was probably much longer. Therefore, it’s still not clear whether the response rate and survival for EGFR-mutant patients is superior to EGFR-wild patients, and more evidence-based researches are required to assess the response rate and survival benefits.Some researchers pointed out that there is an association between EGFR mutation status and DNA damage response. DNA damage response is related to chemotherapy resistance, so we can infer that EGFR mutation status is associated to the efficacy of chemotherapy and it may be relevant with DNA damage response and chemotherapy resistance. Platinum-based chemotherapy is considered as the standard first-line treatment, and paclitaxel-platinum is the best known chemotherapy regimen. The main target of taxanes is beta-tubulin and studies found a negative correlation between class III beta-tubulin (TUBB3) protein and response to paclitaxel chemotherapy in patients with non-small lung cancer. Platinum acts as a cytotoxic agent by inducing DNA damage. Structure-specific endonuclease excision repair cross complementing 1 (ERCC1) and breast cancer susceptibility gene 1 (BRCA1) both act as pivotal roles in repairing DNA damage induced by platinum. Some clinical studies certified that the patients with ERCC1 or BRCA1 gene low-expressed would tend to benefit from platinum-based chemotherapy.This research selects EGFR-wild lung adenocarcinoma cell line A549 and EGFR-mutant lung adenocarcinoma cell line HCC827 as the test subjects. The purpose of the research is to compare the killing activity of paclitaxel and cisplatin against A549 cells and HCC827 cells, detecte the related molecular mechanism, and state the relation between EGFR mutation status and to chemotherapy response of lung adenocarcinoma. In the retrospective analysis, the clinical data of 56 patients with advanced lung adenocarcinoma, who accepted the first-line taxane plus platinum chemotherapy were collected. The effect of EGFR mutation status to outcome of first-line chemotherapy is detected, which will provide more predictive evidences for the response of advanced lung adenocarcinoma.Chapter one Study on molecular mechanism of paclitaxel and cisplatin killing activity against EGFR-wild and mutant lung adenocarcinoma cellsObjectiveTo compare the killing activity of paclitaxel and cisplatin against EGFR-wild lung adenocarcinoma cell line A549 and EGFR-mutant lung adenocarcinoma cell line HCC827 and detect its molecular mechanism, to compare the expression of chemotherapy-related genes between A549 and HCC827 cells and explore its relevance to EGFR mutation status and its impacts on chemotherapy response.Methods1. The median inhibitory concentration (IC50) of paclitaxel and cisplatin against A549 and HCC827 cells in 24 hours was measured by MTT assay.2. Cytotoxicity of paclitaxel, cisplatin and their combination against A549 and HCC827 cells was measured and compared by MTT assay. The combination index (Q) of paclitaxel plus cisplatin was analysed by Jin’s formula.3. The expression of chemotherapy-related genes (BRCA1, ERCC1, TUBB3) of A549 or HCC827cells were detected by real-time fluorescent quantitative PCR.4. Statistical analyses:The SPSS 17.0 software was used for data analyses. All statistical data were presented as mean ± standard deviation (x±s). The differences of killing rate between paclitaxel and cisplatin against A549 and HCC827 cells were tested by independent t test. The differences of chemotherapy-related genes expression before and after chemotherapy treatment were tested by paired t test. All statistical analyses were two-sided and P＜0.05 was considered significant difference.Resultsl.IC50 of paclitaxel and cisplatin in 24 hoursThe IC50 of paclitaxel against A549 and HCC827 cells were 100μg/ml and 70μ.g/ml respectively, while the IC50 of cisplatin were 40μg/ml and 50μg/ml respectively.2. Comparison of killing activity between paclitaxel and cisplatin against A549 or HCC827 cellsThe killing rate (%) of paclitaxel at the some concentration (100μg/ml) against A549 and HCC827 cells was 47.33±3.79,69.33±2.08 respectively. The cytotoxicity of paclitaxel was much stonger against HCC827 cells (P=0.001). The killing rate (%) of cisplatin at the some concentration (40μg/ml) against A549 and HCC827 cells was 51.67±1.53,42.00±4.58 respectively. The cytotoxicity of cisplatin was significantly higher against A549 (P=0.026).3. Comparison of paclitaxel plus cisplatin killing activity against A549 and HCC827 cellsThe killing rate (%) of paclitaxel plus cisplatin against A549 and HCC827 cells was 82.00±5.33,88.96±1.87 respectively. The cytotoxicity of paclitaxel plus cisplatin was not significantly different anginst A549 and HCC827 cells (P=0.100). The combination index (Q) of paclitaxel plus cisplatin was 1.03 and 1.06 respectively, which showed additive effect.4. Comparison of chemotherapy-related gene expressionThe expression of BRCA1 in HCC827cells was much higher than A549cells (P=0.021), but the expression of ERCC1 and TUBB3 was not significantly different (P=0.317; P=0.115). The expression of ERCC1 was up-expressed after treated with cisplatin in A549 cells CP=0.010), but the expression of BRCA1 and TUBB3 was not significantly different (P=0.904; P=0.417). The expression of ERCC1, BRCA1 and TUBB3 after treated with paclitaxel was not significantly different in A549 cells (P=0.516; P=0.434; P=0.417). The expression of ERCC1, BRCA1 and TUBB3 after treated with cisplatin was not significantly different in HCC827 cells (P=0.962; P=0.873; P=0.615). Similarly, the expression of ERCC1, BRCA1 and TUBB3 after treated with paclitaxel was not significantly different in HCC827 cells (P=0.556; P=0.673;P=0.582).Conclusion1. The cytotoxicity of paclitaxel was much stonger against EGFR-mutant lung adenocarcinoma cells, while the cytotoxicity of cisplatin was significantly higher against EGFR-wild cells. The expression of BRCA1 in EGFR-mutant cells was much higher than EGFR-wild cells. We can infer that the lung adenocarcinoma cells with BRCA1 low-expressed is resistant to cisplatin but sensitive to paclitaxel.2. The expression of ERCC1 was up-expressed after treated with cisplatin in EGFR-wild lung adenocarcinoma cells, which was not significantly different in EGFR-mutant cells. ERCC1 gene is a resistant gene of cisplatin. We can infer that the EGFR-wild lung adenocarcinoma cells are much easier to appear cisplatin-resistance than mutant phenotype.3. The killing activity of paclitaxel plus cisplatin was not significantly different against EGFR-wild and mutant lung adenocarcinoma cells.Chapter two Observation on response to first-line chemotherapy in EGFR-mutant and wild patients with advanced lung adenocarcinomaObjectiveTo compare the response to first-line chemotherapy between EGFR-mutant and wild patients with advanced lung adenocarcinoma, to explore the prognostic implication of EGFR gene mutation status.Methods1. The clinical data of 56 patients with stage IIIB/IV lung adenocarcinoma, who accepted the first-line taxane plus platinum chemotherapy from January 1,2010 to May 31,2015 in the department of oncology of Zhengzhou People’s Hospital Affiliated to Southern Medical University were collected. The histopathology and EGFR gene mutation were confirmed. The relationships between EGFR mutation status and clinical characteristics of patients, and the differences of objective response rate (ORR), disease control rate (DCR), and progression free survival (PFS) between EGFR-mutant and wild patients were analyzed.2. Statistical analyses:The SPSS 17.0 software was used for data analyses. The relationships among EGFR mutation status, clinical characteristics of patients, and response to chemotherapy were analyzed by Pearson x2 test or Fisher’s exact test. The PFS was analyzed by Kaplan-Meier survival curve, together with Log-Rank test. The COX regression model was established for multivariate analysis. All statistical analyses were two-sided and P＜0.05 was considered significant difference.Results1. Data of clinical casesThere were 56 cases were selected among the patients with advanced lung adenocarcinoma, who accepted chemotherapy from January 1,2010 to May 31,2015. Among the 56 patients,32 cases were male (57.1%) while 24 cases were female (42.9%). The patients’age ranged from 36 to 75 years (median 53).46 cases were less than 65 years (82.1%), while 10 cases were 65 years or older (17.9%).5 cases’ clinical stage were IIIB (8.9%), while 51 cases’stage were IV (91.1%).63 cases’PS score were less than 2 (96.4%), while 2 cases’PS score was 2 (3.6%).24 cases were smokers (42.9%), while 32 cases were non-smokers (57.1%).19 cases were EGFR-mutant type (33.9%), while 37 cases were in EGFR-wild status (66.1%). The age, clinical stage and PS score of patients were all not significantly different between the EGFR-mutant and wild group (P>0.999; P=0.846; P=0.544). The EGFR-mutant rate in female cases was much higher than male cases (P=0.028). The EGFR-mutant rate in smokers was significantly higher than non-smokers (P=0.018).2. Comparison of ORR and DCR between EGFR-mutant and wild groupAmong the 56 cases,0 case was evaluated as CR,21 cases were evaluated as PR (37.5%),20 cases evaluated as SD (35.7%) and 15 cases were evaluated as PD (26.8%) according to the Response Evaluation Riteria in Solid Tumors (RICIST), so we can infer that ORR was 37.5%and DCR was 73.2%. In the EGFR-mutant group, 10 cases were evaluated as PR (52.6%),5 cases were evaluated as SD (26.3%) and 4 cases were evaluated as PD (21.1%), so we can infer that ORR was 52.6%and DCR was 78.9%. In the EGFR-wild group,11 cases were evaluated as PR (29.7%),15 cases were evaluated as SD (40.5%) and 11 cases were evaluated as PD (29.7%), so we can infer that ORR was 29.7%and DCR was 70.3%. The ORR in the EGFR-mutant and wild group was not significantly different (P=0.094), and there were no significant relationships between gender, age, clinical stage, PS score, smoking status and ORR, respectively (P=0.577; P= 0.368; P>0.999; P=0.523; P=0.577). The DCR in the EGFR-mutant and wild group was not significantly different (P=0.384), and there were no significant relationships between gender, age, clinical stage, PS score, smoking status and DCR, respectively (P=0.384; P>0.999; P>0.999; P=0.468; P=0.384).3. Comparison of PFS between EGFR-mutant and wild groupThe clinical cases were followed up until December 31,2015. The median PFS was 4.9 months in the 65 cases. The median PFS in EGFR-mutant group was much longer than EGFR-wild group, which was 6.0 months versus 3.5 months (P=0.001). The median PFS of the cases less than 65 years was much longer than the cases equal to or older than 65 years, which was 5.4 months versus 3.9 months (P=0.009). The median PFS of the cases with PS score less than 2 was significantly longer than the cases with PS score equal to 2, which was 5.1 months versus 0.7 months (P＜0.001). The effects of gender, clinical stage or smoking status to PFS were not statistically significant (P=0.442;P=0.449; P=0.275). The multivariate analysis indicated that EGFR mutation was a dependent influencing factor for PFS of the advanced lung adenocarcinoma patients (HR=0.309,95%CI:0.150-0.637, P=0.001).ConclusionThe results indicate that ORR and DCR of the advanced lung adenocarcinoma patients receiving first-line taxane-platinum chemotherapy were not significantly affected, but PFS was significantly influenced. The PFS of EGFR-mutant lung adenocarcinoma patients was probably much longer than EGFR-wild patients. EGFR mutation is a dependent influencing factor for PFS of the advanced lung adenocarcinoma patients.