Predictors Of EGFR-TKI Therapy In Chinese NSCLC And Mechanisms Of Downstream Signal Pathway In EGFR Family

Lung cancer is the leading cause of cancer deaths in China and worldwide. Non-small-cell lung cancer (NSCLC) accounts for about 85% in which 70-80% are advance cases. Despite treatment advances, chemotherapy is only marginally effective (approximately 30%). For those patients refractory to or intolerant of the current chemotherapy, treatment options are limited. Hence, more effective therapy with fewer side effects is needed.Epidermal growth factor receptor (EGFR) is one of the focus targets for molecular cancer therapy. The EGFR pathway plays a crucial role in tumor development and progression, including cell proliferation, regulation of apoptotic, angiogenesis, and metastatic spread. Ligand binding to EGFR induces the formation of receptor homo- and heterodimers and the activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for a range of proteins, the recruitment of which leads to the activation of intracellular signalling pathways including Src/STAT, PI3K/AKT and ras-raf-MEK-Erk/MAPK et al. Since EGFR is commonly overexpressed in NSCLC and the level of EGFR expression correlates with poor prognosis, EGFR inhibitors have been developed as novel therapy for NSCLC. Gefitinib, the first molecular targeted agent approved for the treatment of advanced NSCLC, is a highly effective EGFR tyrosine-kinase inhibitor (TKI) that selectively blocks the signal transduction pathways implicated in cancer growth, cell cycle arrest and apoptosis.In 2004, Lynch and Paez et al. reported almost simultaneously that mutations of EGFR might predict the sensitivity of NSCLC to gefitinib, which is regarded as a milestone for approaching individualized molecular targeted therapy for NSCLC. Subsequently, many publications reported data consistent with this finding. Phase II trials with EGFR-TKI in chemo-refractory NSCLC patients found response rates of 9%-19% and median survival ranging from 7.6 to 8.4 months. Furthermore, BR21, a phase III study of erlotinib showed a significant survival benefit compared with placebo. However, in a similarly designed study, ISEL, gefitinib did not show any survival gain over placebo. Subsets of patients, those who had never smoked and of Asian origin appeared to benefit from gefitinib. Whether gefitinib is less efficacious than erlotinib, or whether the study design, the patients enrolled, the tumor molecular characteristics, and the dose might have contributed to the different outcome remain unclear. One area of current research focuses on the identification of factors distinguishing those who are more likely to derive benefit from EGFR-TKIs therapy.China is the largest country in the world. However, data on EGFR mutations in Mainland China are remain scarce. Comprehensive review of existing information regarding EGFR mutations was essential for personalized therapy for advanced NSCLC. While data from clincal and epidemic resourse could only offer a trend and should be combined with the basic research theory, so there would be two part in our article:In the first part, we included all relevant trials from a comprehensive search to provide evidence on the relationship between EGFR mutations and gefitinib therapy in NSCLC cases from Mainland China, and baseline data from the natural history of large sample consecutive NSCLC patients would be evaluated.In the second part, RNAi tecnique was applied to explore the relatioship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 knocked down. PART 1: Clinical and Molecular Predictors of Response to EGFR-TKI Therapy in NSCLC PatientsCHAPTER 1: EGFR Mutations and Their Correlation with Gefitinib Therapy in Patients with NSCLC: Pooled-analysis Based on Updated Individual Patient Data in Mainland ChinaObjective:We aimed to investigate the relevance of demographic characteristics and EGFR mutations, correlations between the efficacy of gefitinib and EGFR mutations in NSCLC, and to identify individuals who would more likely benefit from gefitinib. Methods:A systematic search was used to identify all relevant trials about EGFR mutations in NSCLC in Mainland China from April 2004 to April 2007.Computerized bibliographic searches with PubMed, EMBASE, Cochrane Library, Chinese biomedical literature database, CNKI, ISI web of science and www.clinicaltrials.gov were supplemented with hand searches of conferences abstracts. Then quality of inclued studies was evaluated and a pooled-analysis based on updated individual patient data was performed. Outcome measures included the EGFR mutation status, demographic characteristics, response, and survival. The statistical analyses were performed using RevMan 4.2, SigmaPlot 10.0 with SigmaStat 3.5 integration, and STATA 8.0.Results:Totally, 834 NSCLC patients from 11 institutions in 30 articles matched the selection criteria. IPD were obtained from 7 institutions including 514 patients which 73 cases received gefitinib therapy and 57 cases have the full survival data. From South to North, cases in the 11 institutions orignated from 5 regions including Yunnan, Guangdong, East coast, Beijing and JiLin. The EGFR mutation rate was 30.6% in Chinese NSCLC. The exon 19 deletions and exon 21 L858R point muation totally accounts for 94.51% of all EGFR mutations cases. 21 types of exon 19 deletions, which account for 54% of total muations cases present difference of geographical distribution in China. Female patients with smoking history and adenocarcinoma had a higher mutation rate than male patients with no smoking history and non-adenocarcinoma. EGFR mutations rate in stageⅡis lower than stageⅣsignificantintly(16.1% vs. 40.4%, p=0.003). Multivariate analysis showed that"adenocarcinoma"and"non-smoker"were independent predictors of EGFR mutations. In a subgroup of gefitinib-treated NSCLC patients, response rate to gefitinib in the EGFR mutant group was 70.3%, significantly higher than that in wild-type EGFR group (16.7%) (p<0.0001)."EGFR mutation","adenocarcinoma"and"non-smoker"were independent predictors of response. Overall survival in the EGFR mutant group and the wild-type group did not differ significantly, (hazard ratio=0.60; 95% CI, 0.32-1.12; p=0.110). The prognosis of exon 19 deletions was better than the exon 21 L858R point muation (619天vs. 182 days; pooled HR 2.282; 95% CI 0.95- 5.49; p=0.057) after gefitinib therapy."Adenocarcinoma status"was an independent prognostic factor for survival (p<0.00001).Conclusions:In Mainland China, adenocarcinoma, non-smoker and female patients with NSCLC have a higher EGFR mutations rate. EGFR mutations rate is lower in an earlier TNM stage. Adenocarcinoma and smoking status are independent predictors for the EGFR mutations. Response to gefitinib therapy favors EGFR mutations group. The clinical selected populations for gefitinib are non-smokers with adenocarcinoma. The predominant type of EGFR mutations in China is exon 19 deletions; the prognosis of exon 19 delections after TKI therapy is better than missense mutation in exon 21 L858R. Therefore exon 19 delections is the most important target in Chinsese NSCLC population who receive gefitinib therapy. CHAPTER 2: Natural History of Peripheral Lung Adenocarcinomas in Female Non-smokersObjective:To evaluate the side effect and efficacy of peripheral lung adenocarcinomas in female non-smokers to chemotherapy, and the natural history after thoractomy; to offer a baseline data on the advantage population of TKI therapy and to display the internal relationship between EGFR muations and TKI therapy.Methods:A cohort of consecutive patients with NSCLC from June 1999 to June 2004 was included. All the cases were carefully staged, received standardized treatment strategy, closely followed up and recorded. Electronic medical records prospectively designed were finished by doctors in charge at the time of initial dignosis and followed up closely. Data were updated and input in the lung cancer ACCESS database from Sep 2004 to April 2007. IPD bilabelled with patients'name and admission number were exported from ACCESS to excel, filtered, sorted and imported to a single page of SPSS for analysis. Different subgroups of Gender, smoking status, histological type and tumor location were comprehensively assessed from toxicity and response to chemotherapy, complete resection rate, intrathoracic regional lymph node metastasis, postoperative complications, distant metastasis site, disease free survival and overall survival. Characteristics of the patients and treatments were compared by Fisher exact test or Pearson chi suquare test for categoric data, rank data by using non-parametric M-W U test, and t-test for continuous data. Survival estimates were derived by Kaplan-Meier analysis, and log-rank tests were used to assess differences in survival among the groups. Stratified log rank analyses and Cox proportional hazards modeling were used to investigate major prognostic factors. Analysis was performed using SPSS 13.0.Results:There were 616 patients inclued. Groups were similar for age, TNM stage and lesion locations between gender. Female had a higher incidence of non-smokers with adenocarcinoma while male had a higher incidence of smokers with squamous cell carcinoma. Adenocarcinoma and squamous cell carcinoma accounts for 42.6% and 33.88% respectively in the total cases. Peripheral and central lung cancer accounts for 50% and 44% respectively in the total cases. The non-hematologic toxicity induced by chemotherapy in male was milder than female (p=0.01); the postoperative complications rate in female is higher than male (p=0.011) and in smokers is higher than non-smokers (p=0.031). In survival analysis, DFS of adenocarcinomas is longer than non-adenocarcinomas (23.70 months vs. 11.37 months,p=0.028); the prognosis of lung adenocarcinomas in female non-smokers is better than non-adenocarcinomas in male smokers after thoracotomy (median survival being 43.9 months vs. 20.37 months, p=0.009; 52.17 months vs. 33.13 months,p=0.02;52.17 months vs. 33.13 months, p=0.006 repectively). Gender, smoking history and adenocarcinomas status were not independend prognositc factor in Cox regression analysis. Multivariate analysis for intrathoracic regional lymph node metastasis showed that retrotracheal (p=0.002), subaortic (p=0.002) and lobar lymph node (p=0.015) affecting DFS, while lobar lymph node(p=0.002) metastasis had influence on OS.Conclusions:Compared with their opposite features, peripheral lung adenocarcinomas in female non-smokers have not any advantage on chemotherapy toxicity, response, complete resection rate, complications in thoractomy, and no significant difference on mediastinal lymph node metastasis and distant metastases. While the DFS in adenocarcinomas is longer than non-adenocarcinomas, the prognosis of adenocarcinomas of female non-smokers is better than non-adenocarcinomas male somkers, these advantages present especially in the subgroup of adenocarcinomas in non-smokers. Combined with the conclusions draw from the previous chapter, we deduce adenocarcinomas in non-smokers are the advantage populations in TKI therapy, while EGFR gene mutation is the targeted populations in TKI therapy. PART 2: Effects of RNA Interference on EGFR/HER2 Expression in Non-Small-Cell Lung Cancer Cell LinesCHAPTER 1: RNA Interference on EGFR Gene Expression in A549 Cell LineObjectiveTo investigate whether RNA interference could induce gene silencing in human lung adenocarcinomas cell line A549, and to evaluate the degree of EGFR gene silencing and its effect on cell biological characteristics. Meterials and methods1. Cell culture: Recovery A549 lung adenocarcinoma cell line and maintained in RPMI 1640 medium with 10% fetal calf serum.2. Selection of sequence-specific siRNA-EGFR: Sequence-specific siRNA-EGFR were designed according to previous literatures, ensured by"Tuschl rules"and BLAST, and confirmed by the results of Western Blot after transfection.3. Construction and identification of Psilencer 2.0 U6-shRNA-EGFR: ShRNA-EGFR oligonucleotide was designed based on the sequence-specific siRNA-EGFR and the online sofeware offered by Ambion company. A series of procudures (including anneal the template oligonucleotides, ligate annealed shRNA template insert into Psilencer, transform E. coli with the ligation products, purify Psilencer plasmid for transfection and identify clones with the shRNA template insert through direct suquencing) were performed to construct and identify the Psilencer 2.0 U6-shRNA-EGFR.4. Transient transfection: Psilencer 2.0 U6-shRNA-EGFR and the control vector were transfected into A549 cell line with lipofectamine 2000.5. Effect of EGFR knocked down: Real time Quantitative RT-PCR was used to detect the silencing of the EGFR gene level. Western Blot was used to measure the reduction in the expression of the EGFR protein.6. Investigation of the alteration of biological features after EGFR RNA interference: MTT analysis, DAPI staining, Annexin V/PI double labeling and flow cytometry were used to evaluate cell proliferation, apoptosis and cell cycle distribution after RNA interference.Results1. Sequence-specific siRNA-EGFR: siRNA-EGFR-1'5 -CACAGUGGAGCGAAUUCCUtt-3'; siRNA-EGFR-2'5 -CUCUGGAGGAAAAGAAAGUtt-3'。2. Effect of EGFR knocked down: In A549 cell line, we displayed sequence specific silencing of the EGFR gene with 92.67%, 96.77% and 94.79% in shRNA-EGFR1, shRNA-EGFR2 and shRNA-EGFR3 based on the above sequence compared with the negative control through Real Time Quantitative RT-PCR. The EGFR protein expression was down regulated for 54% and 72% by shRNA-EGFR1and shRNA-EGFR2 respectively through Western Blot.3. Alteration of biological characteristics after EGFR RNA interference in A549 cells: MTT analysis detected growth inhibition of the cells dealt with shRNA-EGFR. Observation with fluorescent microscope revealed shrunk nuclei staining with DAPI, crimpled nuclear membrane as well as condensation and fragmentation of chromatin induced by shRNA-EGFR. Annexin V/PI double labeling found the sign of cell membrane stained with green fluorescent in the third phase was more significant in shRNA-EGFR group. Cell cycle analysis by flow cytometry showed that shRNA-EGFR induced accumulation of cells in phase G0-G1 with a decrease in the percentage of cells in phase S compared with the negative control.ConclusionThe sequence specific siRNA-EGFR and shRNA-EGFR showed a remarkable effect in downregulation of EGFR expression, inhibition of the cellular proliferation and motility, and arrestment of the cell cycle in A549 cells. The application of RNA interference to partly reverse the neoplastic phenotype of EGFR overexpressing cells might provide a new approach to gene therapy and research of NSCLC. CHAPTER 2: Relatioship between EGFR/HER2 Gene Knocked Down and Downstream Signal Pathway in EGFR FamilyObjectiveRNAi tecnique was applied to explore the relatioship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 RNA interference.Meterials and methods1. Cell culture: Recovery A549 lung adenocarcinoma, SPC-A-1 lung adenocarcinoma, L78 squamous cell carcinoma, H460 large cell lung cancer cell line and maintained in RPMI 1640 medium with 10% fetal calf serum.2. Cell Screening: The above 4-cell lines were screened by comparision with transfection efficiency, tolerance to transfection reagent and the level of EGFR/HER2 expression for further research on RNAi.3. Selection of sequence-specific siRNA for EGFR, HER2 and EGFR/HER2 joint interference: Sequence-specific siRNA for EGFR and HER2 were designed on the previous literature;"Tuschl rules" and BLAST on the full length of EGFR/HER2 cDNA were used to ensure the sequence-specific siRNA for EGFR/HER2 joint interference.4. Construction of recombinant vector: Based on the above sequence-specific siRNA, recombinant plasmids with GFP and neomycin resistance marker were constructed by company.5. Transient transfection: Six arms including mock, negative control, shRNA-EGFR, shRNA-HER2, shRNA-EGFR/HER2 and shRNA-EGFR+shRNA-HER2 were established.6. Effect of EGFR kncked down: Real time Quantitative RT-PCR was used to detect the silencing of the EGFR/HER2 gene level. Western Blot was used to measure the levels of EGFR/HER2 protein and protein phosphorylation expression. Transfected cells were stimulated with EGF 15mins before protein extraction. 7. Alteration of biological features: MTT assay and flow cytometry were used to evaluate the cell proliferation, apoptosis and cell cycle distribution after RNAi.8. Downstream signaling pathway protein detection: the protein expression level of downstream signaling pathway protein including Akt, p-Akt, p-Erk1/2, p-p38, were measured with Real time Quantitative RT-PCR PCR and Western Blot.9. Statistical Analysis: Randomized block analysis of variance and SNK methods were used to compare the differences between the groups. P<0.05 was considered significant. Analysis was performed using SPSS 13.0. Results6. SPC-A-1 was seleted for further research on RNAi based on its transfection efficiency, tolerance of transfection reagent and the level of EGFR/HER2 expression.7. Sequence-specific siRNA: siRNA-HER2'5 - GCATACGTGATGGCTGGTGTT-3'; siRNA- EGFR/HER2'5 - GTCTACATGATCATGGTCAATT-3'。8. Alteration of biological characteristics after EGFR RNA interference: MTT analysis found that cell proliferation was inhibited in the groups of shRNA-EGFR, shRNA-HER2, shRNA-EGFR+shRNA-HER2 and shRNA-EGFR/HER2. Cell cycle analysis by flow cytometry showed that apoptosis ratio in shRNA-HER2 (p=0.001), shRNA-EGFR/HER2 (p=0.005) and shRNA-EGFR+shRNA-HER2 (p=0.048) groups were significantly higher than negative control group, while there was no statistical difference between shRNA-EGFR and negative control (p=0.104), and that the distributions in phase G1 and phase S in shRNA-EGFR (p=0.007), shRNA-HER2 (p<0.001), shRNA-EGFR/HER2 (p< 0.001) and shRNA-EGFR+shRNA-HER2 (p=0.001) were significantly different compared with the negative control.9. Effect of EGFR/HER2 kncked down: The level of EGFR/HER2 protein and protein phosphorylation expression were down regulated.10. Downstream signaling pathway protein detection: The cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference have no significant relationship with downstearm signal pathway moleculars in EGFR family.ConclusionEGFR gene knockdown may not cause significant apoptosis in SPC-A-1. The variations of cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference were not found to have significant relationship with downstearm signal pathway molecules in EGFR family.