Melanoma-associated antigens in esophageal adenocarcinoma: identification of novel MAGE-A10 splice variants.
OBJECTIVE
The melanoma-associated antigens (MAGEs) are tumor-specific antigens recognized by cytotoxic T lymphocytes. In this study, expression of MAGE family A members was evaluated during the development of esophageal adenocarcinoma (EA) as potential targets for immunotherapy.


METHODS
MAGE-A mRNA expression was evaluated in 46 samples including Barrett's metaplasia (BM), dysplasia, and EA using oligonucleotide microarrays. Expression of MAGE-A proteins was confirmed by immunohistochemistry on tissue microarrays containing 59 EA, 11 dysplasia, and 9 BM samples and by Western blot. To further evaluate MAGE-A10 expression, reverse transcription-polymerase chain reaction (RT-PCR) products were sequenced, and protein expression was determined using a specific antibody.


RESULTS
Overexpression of MAGE-A1, MAGE-A2b, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, and MAGE-A12 was found in EAs relative to BM on oligonucleotide microarrays. MAGE-A3 overexpression was confirmed by real-time RT-PCR in 21.4% (6 of 28) of esophageal tumors. Immunohistochemistry on tissue microarray revealed MAGE-A proteins in 20.3% (12 of 59) of EAs and MAGE-A10 staining in 16.9% (10 of 59) of EAs. MAGE-A expression was confirmed by Western blot in several esophageal tumors and in two EA cell lines, Flo-1 and Seg-1, whereas Flo-1 also expressed MAGE-A10. Tumors produced from these cell lines in nude mice retained MAGE-A expression. Interestingly, RT-PCR in primary tumors expressing MAGE-A10 protein revealed 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 EA.
Lin
Jules
J
Department of Surgery, Section of General Thoracic Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Lin
Lin
L
Thomas
Dafydd G
DG
Greenson
Joel K
JK
Giordano
Thomas J
TJ
Robinson
Gregory S
GS
Barve
Ruteja A
RA
Weishaar
Frank A
FA
Taylor
Jeremy M G
JM
Orringer
Mark B
MB
Beer
David G
DG
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017398:Alternative Splicing; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D001471:Barrett Esophagus / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques; D058950:Melanoma-Specific Antigens; D008679:Metaplasia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C119317:MAGE-A10 antigen; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D012333:RNA, Messenger