Epigenetic Regulation of Cancer-Testis Antigen Expression in Multiple Myeloma and Correlation with High-Risk Cytogenetics.

Abstract 2923

Cancer-testis (C-T) antigens are often expressed in advanced multiple myeloma and therefore represent attractive potential targets for immunotherapy with vaccines or adoptive T-cell transfer. To estimate the fraction of myeloma patients that might be eligible for C-T antigen-specific immunotherapy, we prospectively collected bone marrow samples from 21 multiple myeloma patients at different stages of disease, and assessed expression of 20 C-T genes in CD138-enriched mononuclear cells from these samples by real-time PCR. The 20 C-T genes selected for analysis have all been shown to encode T-cell epitopes that elicit CD8⁺ and/or CD4⁺ T-cell responses in cancer patients. Unsupervised cluster analysis of the C-T gene expression profiles revealed two distinct groups. One group, comprising 71% of the samples, showed expression of most or all of the C-T genes tested at levels comparable to that observed in testis. The remaining 29% of samples evaluated showed expression of a minority of the 20 C-T genes, typically at lower levels than those observed in the first group. Five of the 21 myeloma patients studied had high-risk cytogenetic abnormalities at the time the marrow samples were obtained, and all 5 patients were assigned to the cluster characterized by high-level C-T gene expression.

The expression of C-T genes is controlled, at least in part, by methylation of CpG islands in their promoter regions. To determine if promoter methylation might also regulate expression of C-T genes in primary myeloma cells, we used methylation-specific PCR on bisulfite-treated genomic DNA from the CD138-enriched samples to assess the degree of methylation of the NY-ESO-1 and MAGE-A3 promoters. The expression of these genes was correlated with the methylation status of their promoters.

Epigenetic modulation of C-T gene expression in myeloma cells could potentially broaden the applicability of C-T antigen-specific immunotherapy to a larger proportion of myeloma patients. To test the feasibility of this concept, we evaluated the effect of decitabine exposure on C-T gene expression on a panel of cell lines including four myeloma cell lines. High-level expression was seen at baseline in the U266, UM-9, and RPMI-8226 myeloma lines, and was marginally enhanced by 48-hour decitabine exposure. The L363 myeloma line, the HL-60 promyelocytic leukemia line, and the SW480 colon carcinoma line, in contrast, showed low-level C-T expression at baseline that was strongly enhanced by decitabine, and associated with significant demethylation of the NY-ESO-1 and MAGE-A3 promoters.

In summary, C-T genes that are known to encode CD8⁺ or CD4⁺ T-cell epitopes are expressed in CD138-enriched bone marrow mononuclear cells from a majority of patients with multiple myeloma. Coordinated expression of multiple C-T genes is commonly observed and correlates with the presence of high-risk cytogenetic abnormalities and with incomplete promoter methylation. Epigenetic modulation with agents such as decitabine may increase the proportion of myeloma patients who are eligible for C-T antigen-specific immunotherapy.