Melanoma-associated antigens in esophageal adenocarcinoma: Identification of novel MAGE-A10 splice variants

Abstract

Abstract Introduction: The melanoma-associated antigen gene family (MAGE) encodes tumor-specific antigens recognized by cytotoxic T lymphocytes. In this study, expression of MAGE family A members was investigated during the development of esophageal adenocarcinoma as potential targets for immunotherapy. Methods: MAGE-A mRNA expression was evaluated in 46 samples including Barrett's metaplasia, dysplasia, and esophageal adenocarcinoma using oligonucleotide microarrays. Expression of MAGE-A proteins was confirmed by immunohistochemistry on tissue microarrays containing 59 esophageal adenocarcinoma, 11 dysplasia, and nine Barrett's metaplasia samples and by Western blot. To further evaluate MAGE-A10 expression, RT-PCR products were sequenced and protein expression was determined using a specific antibody. Results: Overexpression of MAGE-A1, 2b, 3, 4, 6, 9, 10, and 12 was found in esophageal adenocarcinomas relative to Barrett's metaplasia on oligonucleotide microarrays. MAGE-A3 overexpression was confirmed by Real-time RT-PCR in 21.4% (6/28) of esophageal tumors. Immunohistochemistry on tissue microarray revealed MAGE-A proteins in 20.3% (12/59) of esophageal adenocarcinomas and MAGE-A10 staining in 16.9% (10/59). MAGE-A expression was confirmed by Western blot in several esophageal tumors and in two esophageal adenocarcinoma cell lines, Flo-1 and Seg-1, while Flo-1 also expressed MAGE-A10. Tumors produced from these cell lines in nude mice retained MAGE-A expression. Interestingly, in primary tumors expressing MAGE-A10 protein, RT-PCR revealed the presence of additional PCR products that were identified as novel MAGE-A10 alternative splice variants using DNA sequencing. Conclusions: This is the first report of these MAGE-A10 alternative splice sequences, and characterization of MAGE-A expression may provide potential targets for immunotherapy in patients with esophageal adenocarcinoma.