title: Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2'-deoxycytidine.
abstract: Cancer/testis antigens (CTA) are suitable targets for immunotherapy of human malignancies, and clinical trials are mainly focusing on MAGE-A3. However, the heterogeneous intratumor expression of CTA may hamper the effectiveness of CTA-directed vaccination through the emergence of CTA-negative neoplastic clones. We investigated the intratumor heterogeneity of CTA in human melanoma and the underlying molecular mechanism(s) at clonal level using 14 single cell clones generated from the melanoma lesion Mel 313. Reverse transcription-PCR revealed a highly heterogeneous expression of MAGE-A1, -A2, -A3, -A4, -A6, GAGE 1-6, SSX 1-5, and PRAME among melanoma clones. Only nine clones expressed MAGE-A3 and competitive reverse transcription-PCR identified relative differences in the number of mRNA molecules of up to 130-fold between clones 5 and 14. This clonal heterogeneity of MAGE-A3 expression correlated with the methylation status of specific CpG dinucleotides in MAGE-A3 promoter: i.e., hypomethylated CpG dinucleotides at positions -321, -151, -19, -16, -5, -2, +21, and +42 were found in clones expressing high but not low levels of MAGE-A3. Supporting the role of DNA methylation in generating the intratumor heterogeneity of CTA, the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA-dCyd) invariably induced their expression in all CTA-negative clones. Furthermore, 5-AZA-dCyd-treatment reduced to 6 folds the differential expression of MAGE-A3 between clones 5 and 14, which became recognized to a similar extent by T cells specific for a MAGE-A-encoded peptide. These findings identify promoter methylation as directly responsible for the intratumoral heterogeneity of therapeutic CTA in melanoma and foresee the use of 5-AZA-dCyd to overcome the limitations set by their intratumor heterogeneous expression to CTA-based vaccine therapy.
authors.affiliation: Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Sigalotti
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Fratta
authors.forename: Sandra
authors.initials: S
authors.lastname: Coral
authors.forename: Silvia
authors.initials: S
authors.lastname: Tanzarella
authors.forename: Riccardo
authors.initials: R
authors.lastname: Danielli
authors.forename: Francesca
authors.initials: F
authors.lastname: Colizzi
authors.forename: Ester
authors.initials: E
authors.lastname: Fonsatti
authors.forename: Catia
authors.initials: C
authors.lastname: Traversari
authors.forename: Maresa
authors.initials: M
authors.lastname: Altomonte
authors.forename: Michele
authors.initials: M
authors.lastname: Maio
mesh_terms: D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D015235:HLA-B Antigens / Q000235:genetics / Q000276:immunology; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000276:immunology*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000235:genetics / Q000276:immunology*
chemical_list: D000951:Antigens, Neoplasm; D015235:HLA-B Antigens; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins; D000077209:Decitabine; D001374:Azacitidine