The aim of this study was to
The aim of this study was to evaluate the role of aberrant methylation of promoter regions of tumor suppressor genes in the clonal evolution from MGUS to MM. Thus, we analyzed in MGUS, SMM, and symptomatic MM patients, the methylation status of 4 genes—p15, p16, p53, and DAPK—whose promoter hypermethylation was previously found to be associated with transcriptional silencing.4, 15 We also aimed to explore possible correlations between the clinical and laboratorial features of MGUS, SMM, and MM and the methylation pattern of the 4 evaluated genes at diagnosis. A third objective was to evaluate the impact of the methylation profile of p15, p16, p53, and DAPK, individually or combined, on progression-free and overall survivals.
Patients and Methods
Discussion Epigenetic modifications have been recognized as playing a relevant role in the critical points of cancer initiation and progression. One of the most common and well-defined epigenetic modifications involves methylation of CpG dinucleotides in the promoter regions of tumor suppressor genes associated with gene silencing. Although it is known that MM is a highly heterogeneous disease resulting from the accumulation of several genetic and epigenetic events, published data regarding epigenetic events in monoclonal gammopathies, namely, the methylation profile of tumor suppressor genes, are limited and controversial. In this study, we examined the role of aberrant methylation of promoter regions of tumor suppressor genes (p15, p16, p53, and DAPK) and their methylation status in the clonal evolution from MGUS to MM. In all cases, promoter hypermethylation of these genes was previously found to be associated with transcriptional silencing.4, 15, 31, 32 We used methylation-specific PCR analysis because it is a sensitive and quick method, better suited to the analysis of large numbers of samples.27, 33, 34 MSP has been used to detect methylation in both unsorted bone marrow mononuclear BMS 753 and sorted CD138-positive cells (which includes the tumor clone).15, 18, 35 Several authors have studied the methylation of a number of genes in unsorted mononuclear cells18, 33, 36 and sorted CD138-positive cells in MM and other B-cell malignancies, with comparable methylation frequencies of p15 and p16, which indicates that the technique can be performed in mononuclear cells. Overall, we identified at least 1 hypermethylated gene in 52% of patients with monoclonal gammopathy, with patients with MM presenting a significantly higher frequency of hypermethylation (63%) compared with those with MGUS (39%) and no aberrant methylation status in controls. These results confirm that aberrant methylation of tumor suppressor genes is a frequently observed event in patients with monoclonal gammopathies and involves a number of genes that control different pathways in the development and progression of MGUS to a more aggressive symptomatic phase, in agreement with previous studies.14, 15, 16, 17, 26 Our study also showed that aberrant methylation of p15, p16, p53, and DAPK is already present in patients with MGUS (15%, 15%, 2%, and 19%, respectively) and increases in symptomatic MM (21%, 32%, 5%, and 39%, respectively). However, only DAPK methylation showed a statistically significant increase in patients with MM compared with patients with MGUS (P < .05). These observations support the hypothesis that aberrant methylation of p15, p16 p53, and DAPK constitutes an early event in the pathogenesis and development of plasma cell disorders, consistent with the results previously published by Stanganelli et al and Ng et al. A wide range of frequencies (5.9%-77%) of DAPK methylation was reported in MM at diagnosis15, 16, 18, 26 and in other neoplasms (16%-81%),38, 39, 40 with some studies reporting that the loss of DAPK may confer a selective advantage during the multistep process of metastasis. Thus, the loss of DAPK expression provides a unique mechanism that links suppression of apoptosis to metastasis and may be involved in the clonal progression from MGUS to MM.