Study On The MAGE-A3 Polymorphism/Mutation And MRNA Expression Levels In NSCLC Patients

Lung cancer is among the most common malignancies, and remains, at the same time, among the most difficult to treat. Despite recent developments of early diagnosis and treatment, the statistics are sobering:lung cancer is by far the most common cancer worldwide in men and in women. Less than 15% of those diagnosed with lung cancer are alive 5 years following their diagnosis. Inability to predict which patients will benefit from which treatment results in significant morbidity and mortality. Increasing understanding of tumour biology, and in particular of the molecular pathways contributing to tumour growth, has led to the development of innovative medications for the treatment of NSCLC.MAGE-A3 is a member of the cancer/testis gene family. This human gene is silent in all normal tissues except the testis but with no antigen presentation because of the lack of classⅠpresenting molecules in the testis cells expressing the gene. TheMAGE-A3 protein is thus considered as a truly tumor-specific antigen with expression in a variety of tumors such as melanoma, NSCLC, bladder and head and neck cancer, but also squamous esophageal cancer and hepatocarcinoma. Recently, clinical investigation of cancer immunotherapy has been very active and several approaches have been evaluated in PhaseⅢtrials. In particular, the characterisation at the molecular level of tumour- specific antigens MAGE-A3 has led to develop Antigen-Specific Cancer Immunotherapeutic(ASCI) against NSCLC.MAGRIT is a PhaseⅢtrial has thus been designed to evaluate the efficacy of the MAGE-A3 immunotherapeutic in patients with MAGE-A3-positive, completely resected stageⅠB,ⅡorⅢA NSCLC, and who received or did not receive adjuvant chemotherapy. From interim premature data of MAGRIT trial, MAGEA3 expression levels were mainly different between histology (24% in AC vs 45% in SCC) as well as regional population (East Asian vs Europe and America). MAGEA3 expression is positive about 35.7% in Europe vs 26% in international groups, which further showed that MAGEA3 is positive in Taiwan (13%), Singapore (11%) and Hongkong (20%) that is lower than that in Korea and Japan groups (both～30%).Which reason underlying the differential MAGE A3 expression between regions is unknown. From point of gene expression regulation, potential differential polymorphisms in MAGE A3 gene between racial populations may contribute to differential gene expression regulation and the relevant mRNA expression level. For clinical patients selection, the relationship between MAGEA3 expression and EGFR mutation status and expression is unknown. As EGFR mutation has been greatly implicated in patient selection for specific EGFR inhibitors like Gefitinib or Erlotinib in clinical setting, one question to be asked is if MAGEA3 targeted patients are mutually exclusive or inclusive to EGFR expression/muation? Answer to this question will help to determine the extent of MAGEA3 used in combination with EGFR inhibitors.Chapter 1 Prognosis of MAGE-A3 mRNA expression levels and the realtionship with the clinical parameters of lung cancer patientsMethodsReverse transcriptase and real-time polymerase chain reaction (RT-PCR) were used to evaluate the MAGE-A3 mRNA expression levels in 212 frozen tissue samples (112 adenocarcinoma,100 squamous carcinoma) of stageⅠB,ⅡorⅢA NSCLC patients. A tumor sample was considered MAGE-3 positive when its MAGE-3 expression level after beta-actin normalization was equal or greater than the cut-off value determined by 1% Gerl (GSK company). The MAGE-A3 mRNA expression positive rate and clinical parameters were analyzed by Chi Square test. The Kaplan-Meier method and the log-rank test were used for survival analysis. ResultsOf these 212 patients,100 were squamous cell carcinoma,110 were adenocarcinoma; 102 wereⅠB stage,63 wereⅡB stage, and 47 wereⅢA stage; 151 were male,61 were female; The mean age of these patients was 60.91 years, range from 26-82 years. The MAGE-A3 positive rate is 27.4%(58/212) in all of these patients. Of them, the positive rate is higher in squamous cell carcinoma than in adenocarcinoma (37.0% vs 18.8%, X2=8.854, P=0.003), higher in male than in female (32.5% vs 14.8%, X2=6.846, P=0.009), and higher in smokers than in non-smokers (36.3% vs 19.1%, X2=7.863, P=0.005). Our results provided no evidence for a relationship between MAGEA3 expression and tumor stage, or age. However, with regard to certain subgroups, such as smokers, male, squamous cell carcinoma samples, MAGE-A3 gene expression positive rate was significantly higher than others (45.2% vs 20.0%, X2=13.974, P<0.005).The MAGE-A3 expression showed significant association with good survival(P=0.047). A multivariate analysis enabled us to conclude that the TNM staging is the only independent predictive factor.Conclusion The present study showed that the MAGE-A3 is highly positive expressed in male, smokers and squamous cell histology of patients with non-small cell lung cancer. We conclude that the MAGE-A3 expression is associated with good prognosis in NSCLC.Chapter 2 Study on the polymorphisms and mutations of MAGE-A3 gene in NSCLC patientsMethodsPotential MAGE-A3 gene polymorphisms analysis by PCR-base sequencing. Six pairs of primers were designed for amplifying fragments of MAGEA3 gene. PCR products will be purified and labeled with ABI BigDye 3.1 agents, and then for sequencing on the ABI 3130 genetic analyzer platform. Sequence results were collected and compared with a MGEA3 reference sequence in NCBI GenBank. The SNPs'genotype and clinical parameters were analyzed by Chi Square test. The Kaplan-Meier method and the log-rank test were used for survival analysis.ResultsThe sequences of SNPs' sites (rs5970360,rs5925210,rs5970361,rs5925211,rs35123853) of MAGE-A3 gene in 201cases NSCLC patients were assayed by PCR and DNA sequencing methods. There are no relationship of sites' genotype of rs5970360,rs5925210,rs5970361 with MAGE-A3 mRNA expression (P=0.035,0.134,0.153). MAGE-A3 mRNA expression level were associated with the 7-bases "cattccc" inserting at site420, the frequency of PCR products as MAGE-A3^ MAGE-A3 and -A6 and MAGE-A6 were 27.6%,5.9%,37.6% respectively(P=0.035). There were no relationship between these SNPs'genotype (rs5970360[CC/CT],rs5925210[CG/CC],rs5970361[CC/CA],rs5925211[AT/AA]) and survival, the 7-bases inserting at site 420 as the same(P=0.434,0.370,0.895,0.212,0.940 respectively).ConclusionThere were no relationships between the polymorphisms and MAGE-A3 mRNA expression, and no relationship with survival.Chapter 3 Relationship between EGFR expression level and it's mutation and MAGE-A3 mRNA expression in NSCLC patientsMethodsEGFR mutation status containing Exons 18,19,20 and 21 were amplified using 4 pairs of primers respectively, and then run PCR reactions, PCR products were subject to sequencing as mentioned above for MAGE A3 polymorphism analysis. EGFR expression level:The same cDNA samples used for MAGE A3 expression tests will be tested for EGFR expression as well. Specific primers and TaqMan probe for EGFR will be used for quantifying EGFR expression at mRNA level by real-time qRT-PCR reactions. Delta Ct will be acquired by subtraction of Ct values of EGFR and endogenous gene beta-actin.ResultsTotal 206 samples finished the detecting of EGFR mutation and EGFR RT-PCR. Of them, the positive rate is higher in adenocarcinoma than in squamous cell carcinoma (46.2% vs 6.0%, P<0.001), higher in female than in male (43.3% vs 19.9%, P=0.001), and higher in non-smokers than in smokers (38.5% vs 13.4%, P<0.001). Our results provided no evidence for a relationship between MAGEA3 expression and EGFR mutation along with EGFR mRNA expression, also no relationship between EGFR mutation/expression and survival. With regard to certain subgroups, such as 55 cases EGFR mutation patients(P=0.096) and 106 cases lower EGFR expression level patients(P=0.093), survival was prolonged in MAGE-A3 positive expression group. A multivariate analysis enabled us to conclude that the smoking status and histologic type is the independent predictive factor of EGFR mutation.ConclusionThe present study showed that the EGFR is highly positive expressed in female, non-smokers and adenocacinoma histology of patients with non-small cell lung cancer. A multivariate analysis was to conclude that the smoking status and histologic type is the independent predictive factor of EGFR mutation. MAGE-A3 expression got no relationship with EGFR mutation and mRNA expression.