A Study On The Expression And Distribution Of MAGE-A3, HLA-A1 And HLA-A2 In Esophageal Squamous Carcinoma

Tumor harm human's health and lives acutely, and malignant tumor posses fairly high lethality rate. The attack rate of esophageal carcinoma is 1%～2% of all malignant tumor and the 2nd high-incidence alimentary canal malignant tumor. Esophageal carcinoma is the sixth death cause in the word and high-incidence in China, about 9 millions residents face to threaten of esophageal carcinoma and 180 thousands residents die because of esophageal carcinoma. Three kinds of traditional therapeutic-surgery, radiotherapy and chems can't eradicate tumor completely and has its disadvantage respectively. With the development of molecular biology and gene engineering technique, bio-therapy gradually becomes a promising treatment. Immunotherapy as a part of comprehensive treatment to tumors has a tremendous development after the founding of tumor rejective antigens. Tumor genetic immunotherapy depend on the activation of cell immunity and humoral immunity, possess the feature of high degree of tumoral specificity ought to ensure the absence of damage on normal cells and tissues.The key point of genetic immunotherapy is tumor specific antigen. Melanoma associated antigens 3 (MAGE-A3) has been found to be highly expressed in a variety of primary tumors and cancer cell lines, for instance, melanoma, head and neck squamous carcinoma, esophageal carcinoma, hepatoma, lung cancer, gastric cancer and breast cancer, as a tumor specific antigen. MAGE-A3 gene is silent in normal tissues except for testis germ cells. Human leukocyte antigen (HLA) is one of the important ingredient of immune system, participate antigen transmission, determine the identify and killing that immunocell to tumor.MAGE-A3 antigens can be presented by HLA-I molecules, then stimulate the later's reproduction and activation, which represents attractive targets for tumor immunotherapy. There are a lot of antigenic determinant (epitope) in MAGE-A3 antigenic protein. They can be presented by different HLA-I molecules, and recognized by CTLS. Then the later performed it's function to kill the tumor cells expressing MAGE-A3 antigen effectively. So the success of specific immunotherapy mainly determined by HLA-I molecule. The T cell receptor (TCR) of effective T cells can bind with the complex of HLA-I molecule and MAGE-A3 related peptide. Only TCR-HLA-I molecule-MAGE-A3 epitope complex can activate CTLs effectively. HLA-A1 and HLA-A2 have the high distribution rate in China. So with the knowledge of MAGE-A3 antigenic protein expression and HLA-A1 and HLA-A2 distribution in esophageal carcinoma tissues, we can primarily estimate the ratio of esophageal carcinoma patients who can be treated with specific immunotherapy based on MAGE-A3 antigenic peptide.Methods and materials:1. Samples collection:60 samples of esophageal squamous carcinoma tissues,30 samples of esophageal atypical hyperplasia tissues and 30 samples of normal esophageal mucosa tissues. Samples of carcinoma tissues were flushed by zymo-water without RNA to clean up possible contaminant neoplastic cell. Samples of esophagesl atypical hyperplasia tissues and mormal esophagesl mucosa tissues were frozen in liquid nitrogen immediately after surgery and then stored at -80℃until the extraction of protein.2. MAGE-A3 mRNA expression in esphagedal carcinoma was assessed by RT-PCR. The full length of MAGE-A3 cDNA was cloned into pET30a(+) vector and transformed into BL21(DE3) E.coli. Positive clones were incluced with IPTG.Recombinant fusion proteins pET30a(+)-6His-MAGE-A3 was inditified by SDS-PAGE and westrn bolt. The proteins were purified by Ni-NTA superflow affinity chromatography and redolded by passing through Sephadex G100. The autologous antigen-presening cells pulsed with MAGE-A3 protein and these APCs were used to stimulate the autologous T lymphocytes.3. The expression of HLA-A1 and HLA-A2:Detecting and analysizing the expression of HLA-A1 and HLA-A2 in the paraffin sections of esophageal carcinoma by indirect peroxidase immunohistochemical method.4. Statistics method:All data was processed and analyzed by SPSS 13.0. Statistic methods such as t test, ANOVA, chi-square test and non-parametric test were employed to analyze the data. P value less than 0.05 was considered statistically significant.Results1. Expression of MAGE-A3 antigenic protein:of 120 samples, MAGE-A3 antigen are expressed in 21 esophageal carcinoma cases, and not expressed in 39 esophageal carcinoma cases. The ratio of MAGE-A3 antigen expression was 35% in esophageal carcinoma patients. MAGE-A3 antigen are expressed in 2 esophageal atypical hyperplasia tissues. The MAGE-A3 proteins was expressed effectively in E.coli with the expression vector pET30a(+), whose molecular weight was 48 kd. High purity of the recombinant soluble proteins had been obtained after denaturation, purification and restoration to original structure. MAGE-A3 antigen can be recognized by T lymphocytes of patients with esophageal carcinoma when presented by autologous APCs.2. The relationship between MAGE-A3 and esophageal carcinoma prognosis:The 18 months survival rate of esophageal carcinoma patients expressed MAGE-A3 (84.20%) was high than patients not expressed MAGE-A3 (57.60%).3. The distribution of HLA-A1 and HLA-A2 by DNA typing:The positive rates of HLA-A1 and HLA-A2 were 8.33%(5/60) and 53.33%(32/60) respectively in esophageal carcinoma patients; the positive rates of HLA-A1 and HLA-A2 were 6.66%(2/30) and 46.66%(14/30) respectively in esophageal atypical hyperplasia tissues; the positive rates of HLA-A1 and HLA-A2 were3.44% (1/29) and 43.33%(13/30) respectively in normal esophageal mucosa tissues. About 19.00%(MAGE-A3/HLA-A2,53.33%x35.00%) esophageal carcinoma patients suit specific immunotherapy of MAGE-A3/HLA-A2 antigen peptide.4. The relationship between the deletion of HLA-A1,-A2 and esophageal carcinoma:some HLA-A1,-A2 (one HLA-A1 and 10 HLA-A2) deleted in esophageal carcinoma tissues but existed in esophageal atypical hyperplasia and normal esophageal mucosa tissues. The deletion rates of HLA-A1 and HLA-A2 were 18.33%(11/60).Conclusion:1. The MAGE-A3 antigen is a promising target of esophageal carcinoma immunotherapy.2. The distribution of HLA-A1 and HLA-A2 molecule DNA typing shows that the result is similar with previously report. High positive rate of HLA-A2 exists in Chinese people, it can recognize and bind with MAGE-A3 antigenic peptide. All of these provide the feasibility of the designing of MAGE-A3 antigenic peptide related vaccine.3. We can estimate optimistically that there are about 19% esophageal carcinoma patients can be treated with tumor vaccine based on MAGE-A3 antigenic peptide.4. The molecular expression lose of HLA-A1 and HLA-A2 exists in a certain degree, which change is not conductive to T cell dependent immunotherapy.