Differential Expression of Type I MAGE in New and Relapsed Multiple Myeloma: Evidence for Association with Proliferation and Progression of Disease.

The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis family of tumor-associated antigens and are found in a broad range of solid and hematologic malignancies. We previously showed that the type I MAGE proteins CT7 (MAGE-C1) and MAGE-A3 were commonly detected in primary myeloma by both RT-PCR and immunohistochemistry (IHC). Higher levels of MAGE protein expression had a positive correlation with abnormally elevated proliferation as measured by the Plasma Cell Proliferation Index (PCPI, percentage of Ki-67+ cells in the CD138+ myeloma cell compartment). These findings suggest that MAGE may play a role in abnormal cell cycle regulation in myeloma. We explored this hypothesis by examining type I MAGE gene expression and proliferation by IHC in 46 newly-diagnosed, untreated and 35 relapsed myeloma patients, based on the clinical observation that relapsed patients exhibit lower response rates to therapy and shorter time to progression, indicative of more aggressive disease. PCPI was significantly higher in relapsed patients (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p<0.0002). Expression of CT7 and CT10 (MAGE-C2), a type I MAGE not previously associated with myeloma, was stable between newly-diagnosed and relapsed patients (76.0% of new samples vs. 77.1% of relapsed for CT7, 48.5% vs. 50.0% for CT10). In contrast, MAGE-A3 was detected in a significantly greater percentage of relapsed patients (77.1%) compared to newly diagnosed (35.6%, p=0.0003). The link between MAGE expression and unrestricted proliferation was further supported by in vitro studies with human myeloma cell lines. Proliferating myeloma cells were metabolically labeled with the nucleotide analog bromodeoxyuridine (BrdU) followed by intracellular staining and flow cytometry. This assay demonstrated that proliferating myeloma cells that incorporated BrdU into their genomic DNA expressed higher levels of type I MAGE protein compared to non-proliferating cells. Arresting cells at the G1-S interface by double thymidine blockade lead to the accumulation of cells expressing high levels of MAGE, which rapidly entered S phase and incorporated BrdU upon release from block. These results strongly suggest that expression of CT7 and CT10 are relatively early and stable events in the pathogenesis of myeloma, whereas activation of MAGE-A3 expression is associated with disease progression. Furthermore, MAGE expression is correlated with abnormal proliferation in vitro and in vivo, suggesting a potential functional role in the dysregulation of the cell cycle that is a hallmark of this disease. These results support the further exploration of the role of type I MAGE expression in myeloma and the development of therapeutic agents targeting them, especially tumor vaccines.