Both Methylation and Copy Number Variation Participated in the Varied Expression of PRAME in Multiple Myeloma.

ABSTRACT

PURPOSE: The cancer-testis antigen, which is a preferentially expressed antigen of melanoma (PRAME), is an ideal target for immunotherapy and cancer vaccines. Since the expression of this antigen is relevant to therapy responses, the heterogeneity in its expression and the underlying mechanism need to be investigated. PATIENTS AND METHODS: Plasma cell sorting was performed in 48 newly diagnosed multiple myeloma (MM) patients. Real-time quantitative PCR was performed to examine the PRAME transcript levels and gene copy numbers. Bisulfate clone sequencing of the PRAME promoter and exon 1b regions was performed in 4 patients. Quantitative methylation-specific PCR of the +287 CpG site was performed for all patients. The human MM cell lines RPMI8226, LP-1 and MOLP-2 were treated with 5-azacytidine. RESULTS: The median PRAME transcript level was 3.1% (range 0-298.3%) in the plasma cells sorted from the 48 MM patients. Eleven (22.9%) and 37 (77.1%) patients were individually categorized into the PRAME low- and high-expression groups according to the cut-off value of 0.05%. The methylation ratios of the promoter and the 3' region of exon 1b region were both negatively related to the transcript levels. The degrees of methylation at the +287 CpG site were significantly negatively related to the transcript levels in all 48 patients (r=-0.44, P=0.0018), and those in the high-expression group (r=-0.69, P<0.0001) but not those in the low-expression group (r=-0.27, P=0.43). All 5 patients with homozygous deletions were categorized into the low-expression group. There were no significant differences in the PRAME transcript levels between the hemizygous deletion (n=8) and no deletion (n=35) groups (P=0.40). Furthermore, the PRAME transcript levels significantly increased in the MM cell lines after treatment with 5-azacytidine. CONCLUSION: Both methylation and copy number variation may participate in the regulation of PRAME expression in MM; in patients with no homozygous deletion, PRAME expression is mainly controlled by methylation, and a proportion of fairly low expression is caused by homozygous deletion.