The Expression And Clinical Significance Of Human MAGE-A3Gene In Lung Cancer Patients

Lung cancer is one of the most common cancers and a major cause of mortality globally. In American, the mortality of lung cancer is the first in malignant tumor.There were219440new cases of lung cancer and159390death cases in2009. In most developing countries, especially in China, the incidence rate of lung cancer has been rising constantly. If it is indifferent to control smoking and air pollution, the number of lung cancer patients of our country will surpass one million by2025. China will be the first large lung cancer country in the worldwide by the time. Non small cell lung cancer (NSCLC) accounts for approximately80%of all cases of lung cancer. Although the diagnosis and treatment of the NSCLC have been constantly updated,5-year survival rate is50%for stage I patients,30%for stage II patients, and still less than15%for advanced stage patients. The leading cause of deaths for NSCLC patients is recurrence and metastasis.With the development of molecular biology, the biochemical markers play vital role in the auxiliary diagnosis, prognosis and monitoring anti-neoplastic therapy of NSCLC. Therefor, more and more studies tried to find the maker with high sensitivity and specificity. Melanoma-associated antigen-A3(MAGE-A3) is one of the cancer/testis antigens, which was first found in Melanoma cells. As a human tumor-specific antigen, the MAGE-A3gene is silent in normal tissues except for the testis and placenta, but they are widely expressed in many types of human malignancies including melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung cancer, gastric cancer and breast cancer. It was reported that approximately30-50%of lung cancers express MAGE-A3, which shows the MAGE-A3may play a vital role in the pathology of human tumorigeness, cancer cell proliferation, development, metastasis and progression.Objective:1. To study the expression of MAGE-A3in peripheral blood of patients with lung cancer, to analyze the relationship between the expression of MAGE-A3and clinical pathological feature in lung cancer, to investigate the diagnostic value of MAGE-A3in lung cancer.2. To assess the predictive and prognostic roles of peripheral blood of MAGE-A3during chemotherapy in patients with lung cancer.3. To construct the recombinant lentiviral vector containing human MAGE-A3gene and measure the expression level of MAGE-A3in lung cancer cell line of A549.Materials and Methods:1. Subjects (divide into two parts):Part Ⅰ: Patient with lung cancer, separate into three groups.Part Ⅱ: NSCLC cell A549.Peripheral blood samples from three different groups of patients were collected. The first group was a cohort of120lung cancer patients with different histology. Patients with previously untreated Ⅰ-Ⅳ stage lung cancer who received standard first-line combination chemotherapy were enrolled prospectively into this study at a single institution (Jinling Hospital, Nanjing, China) between June2009and September2011. To be eligible for first-line combination chemotherapy, patients were required to have cytologically or histologically proven lung cancer; an Eastern Cooperative Oncology Group (ECOG) performance status (PS) from0to2; normal hepatic-, renal-and hematologic function and no concomitant serious co-morbidities. The second group had39patients. All cases were diagnosised as benign pulmonary lesions according to clinical symptom, chest CT and histological examination.involving pneumonia (n=25), tuberculosis (n=l), bacteremia (n=l), atelectasis (n=2), interstitial pneumonia (n=l), chronic obstructive pulmonary diseases (n=6), and bronchiectasis with infection (n=2). The third group comprised healthy adults as control, who were volunteers (n=32).2. Methods(l)Peripheral blood samples were obtained immediately before the first cycle of therapy and before the third chemotherapy course at the time of clinical response evaluation. The haemocyte were prepared immediately after collection and frozen at-80°C until analysis. Peripheral blood MAGE-A3was measured with the SYBR Green real time PCR assay systems bought from Takara (Dalian, China). The total RNA were extracted by TRIzol law from peripheral blood samples, and tested the total RNA concentration and quality. Then the total RNA were reverse transcriptased to cDNA by reverse transcription polymerase chain reaction (reverse transcription PCR, RT-PCR) method, and the kit were bought from Formentas Life Sciences companies(USA).(2) We successfully constructed the lentiviral recombinant eukaryotic expression plasmid pLV-MAGE-A3-GFP by recombinant DNA technology. The resultant lentivirus were confirmed by restriction enzyme digestion and DNA sequencing. Recombinant lentivirus PLV-MAGE-A3-GFP were produced from293TAB by transient calcium-phosphate co-transfection with pCMV-dR8.91and pCMV-VSV-G. A549cells were infected by PLV-MAGE-A3-GFP lentivirus and the expression of MAGE-A3was confirmed by Western blotting.3. The assessing criteria of tumor responseEligible patients were required to have received at least2cycles of platimum-based combination chemotherapy. Tumor response was assessed according to the RECIST criteria1.1. In this study, patients who achieved a complete response (CR) or partial response (PR) were classified with OR, and all remaining patients (stable disease [SD] and progressive disease [PD]) were considered as non-responders. CR: Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10mm. PR: At least a30%decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. PD: At least a20%increase in the sum of diameters of target lesions, taking the smallest sum on study as reference (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of20%, the sum must also demonstrate an absolute increase of at least5mm (Note: the appearance of one or more new lesions is also considered progression). SD: Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking the smallest sum diameters while on study as reference.4. Follow-upWe made overall survival (OS) as the endpoint of follow-up. OS is defined as referring to the start from randomization to death due to any cause. And7patients were lost to follow.5. Statistical analysisThe results were statistically evaluated using SPSS17.0statistics software. Quantitative data was described using the number of cases, the median, interquartile. The two groups were compared using Kruskal-Wallis H test. If the homogeneity of variance, the two groups before and after treatment were compared using t test, or else using Wilcoxon test. Qualitative data was described using the number of cases and percentage. Comparing between the two groups used Chi-Square test. Ranked data was described using Kruskal-Wallis H test. Survival of lung cancer patients with different baseline levels of MAGE-A3was described using Kaplan-Meier analysis, survival of the two groups using the log-rank test. The influential factors of the survival of lung cancer patients was analyzed using Cox regression model.P≤0.05was considered statistically significant.Results:1. The concentration of MAGE-A3in patients with lung cancer, benign lung diseases and healthy controls were0.156,0.010,0.001, respectively. The peripheral blood MAGE-A3concentration in lung cancer group was significantly higher than that of benign lung disease group (P<0.001) and healthy controls (P<0.001). The peripheral blood level of MAGE-A3in benign lung disease group was significantly higher than that of healthy control group (P<0.001).2. A ROC curves analysis was performed to find out the cut-off value of distinguishing malignant from benign and normal tissues. For MAGE-A3, the AUC was0.860(95%CI:0.809-0.902). When the cut-off value of MAGE-A3was0.115, the efficacy of sensitivity (SEN) and specificity (SPE) were both the best, with a SEN of53.3%and a SPE of100%.3. We divided the patients with lung cancer into two groups according to the age (<60versus>60), sex, the degree of cell differentiation, histopathology (adenocarcinoma and squamous cell carcinoma versus small cell lung cancer), stage, lymph nodes metastasis, the position of primary lesion (left lung versus right lung), distant metastasis and history of smoking, then conformed the comparion between the two groups.(1) The peripheral blood level of MAGE-A3had no relation to age, histopathology, lymph nodes metastasis, the degree of cell differentiation and the position of primary lesion.(2) The peripheral blood level of MAGE-A3in lung cancer with distant metastasis positive was higher than those of negative (0.407,0.024)(PO.001).(3) The peripheral blood level of MAGE-A3in NSCLC at the stage of Ⅲ-Ⅳ was higher than those of at the stage of Ⅰ-Ⅱ (0.238,0.003)(P=0.005). The peripheral blood level of MAGE-A3in patients with smoking was lower than that in patients without smoking (0.062,0.528; P<0.001). The peripheral blood level of MAGE-A3in male was lower than that in female (0.062,0.559; P<0.001).These results indicated that the high expression of MAGE-A3in the peripheral blood of lung cancer patients has relation to the cell proliferation and metastasis.4.61of120patients were evaluable for peripheral blood MAGE-A3before the first cycle chemotherapy and before the third cycle chemotherapy, we observed the dynamic change of peripheral blood MAGE-A3during the process of treatment. The level of MAGE-A3decreased obviously after treatment. Based on the ROC, a post-treatment92.9%reduction in peripheral blood MAGE-A3was calculated as cut-off level for defining a response. The patients who had MAGE-A3-response and no MAGE-A3-response had significantly difference in objective response (OR) to treatment (P<0.001).5. For the whole group, the median OS was10.630months. Based on the results of survival analysis, for OS, the patients with the low level expression of peripheral blood MAGE-A3(MAGE-A3<0.115) was longer than that with high expression (MAGE-A3≥0.115)(P<0.001)o6. Recombinant eukaryotic lentivirus PLV-MAGE-A3-GFP were successfully constructed. Green fluorescent state and Western blot analysis revealed that the MAGE-A3gene can be correct transcripted and translated in A549cell which infected by LV-MAGE-A3-GFP. MAGE-A3could be stablely expressed and promote the proliferation of A549cell.Conclusion:1. The overexpression of MAGE-A3in lung cancer patients indicates that MAGE-A3is related with the occurrence of lung cancer.2. The overexpression of MAGE-A3in NSCLC was closely related to the stage of tumor, distant metastasis, which indicated that MAGE-A3plays an important role in cell proliferation, invasion and matastssis in NSCLC.3. Detecting MAGE-A3was helpful to the diagnosis of lung cancer.4. MAGE-A3can probably serve as a peripheral blood marker of monitoring therapeutic efficacy of treatment and prognostic in patients with lung cancer.5. We successfully constructed the lentiviral recombinant eukaryotic expression plasmid pLV-MAGE-A3-GFP by recombinant DNA technology. And MAGE-A3gene can be correct transcripted and translated in A549cell which infected by pLV-MAGE-A3-GFP.We detected the expression of MAGE-A3in peripheral blood of patients with lung cancer, benign lung diseases and healthy controls by the method of Real time PCR, to analyze the relationship between the expression of MAGE-A3and clinical pathological feature in lung cancer. And we assessed the predictive and prognostic roles of peripheral blood of MAGE-A3during chemotherapy in patients with lung cancer.In addition,we constructed the recombinant lentiviral vector containing human MAGE-A3gene and measure the expression level of MAGE-A3in lung cancer cell line of A549. Western blot analysis revealed that the MAGE-A3gene can be correct transcripted and translated in A549cell which infected by LV-MAGE-A3-GFP. The recombinant lentiviral vector pLV-MAGE-A3-GFP was constructed successfully it could be used to transfect lung carcinoma cells. MAGE-A3could be stablely expressed and promote the proliferation of A549cell. And it provided an experimental basis for further immunotherapy of non-small cell lung cancer.MAGE-A3is a tumor specific associated antigen, widely expressed in many tissues, with a variety of biological functions.The high expression in the Peripheral blood of patients with advanced NSCLC may be associated with tumor proliferation, invasion and metastasis. MAGE-A3appeared to be prognostic factors in patients with advanced NSCLC, and probably serve as a Peripheral blood marker of monitoring therapeutic efficacy of treatment. The in-depth research about the expression, function and regulation of MAGE-A3in NSCLC is helpful for further understanding the molecular features of development and metastasis of NSCLC. MAGE-A3may be a new therapeutic target for human NSCLC.