Aberrant Gene Promoter Methylation of Tumor Suppressor Genes in Plasma Cell Disorders.

There is increasing evidence that, in addition to genetic aberrations, epigenetic processes play a major role in carcinogenesis. Particularly, hypermethylation of CpG islands of the promoter regions of tumor suppressor genes (TSG) is now considered as an important epigenetic mechanism for gene inactivation. Multiple myeloma (MM) is characterized by neoplastic proliferation of monoclonal plasma cells. The natural course of disease may progress through monoclonal gammopathy of undetermined significance (MGUS) to MM. During this process, multiple genetic alterations are sequentially acquired and aberrant promoter hypermethylation might be one of the steps involved in this progression. In this study, we have evaluated methylation status of the following TSG: p15^INK4b, p16^INK4a, p14^ARF, SOCS-1, p27^KIP1, RASSF1A and p73 genes, in order to determine if they were involved in the evolution of MGUS to MM. Forty four MM (21 males; mean age 67.5 years; Durie-Salmon clinical stages: I: 20%, II:14% and III: 66%) and 21 MGUS patients (6 males; mean age 68 years) were study. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Peripheral blood samples from 10 normal individuals and CpGenome Universal Methylated DNA (Chemicon International) were used as negative and positive controls, respectively. DNA was extracted from bone marrow cells of patients and peripheral blood lymphocytes of controls using phenol/chloroform method. Methylation status was performed using Methylation Specific PCR (MSP) technique. For statistical analysis, Student t and Fisher exact tests were used. The methylation index (MI; ratio between the number of genes methylated and the number of genes analyzed) was also calculated. SOCS-1 gene methylation was significantly more frequent in MM (52%) than in MGUS patients (14%) (p=0,006). Frequencies of methylation of p14^ARF, p15^INK4b, p16^INK4a and RASSF1A were comparable in both entities: 29%, 32%, 7% and 2%, respectively, for MM; and 29%, 29%, 5% and 0%, respectively, for MGUS. TP73 gene showed a tendency of higher methylation in MM (45%) than in MGUS (33%). All patients lacked methylation at p27^KIP1 gene. Whereas the percentage of MM with at least one gene methylated (84%) did not showed differences to that of MGUS (66%), the mean MI of MGUS was lower (0.16; range 0.14-0.43) than that of MM (0.24; range 0.14-0.71) (p<0.05). None of the target genes were methylated in normal samples. No statistical significant correlation with clinical characteristics: gender, age, isotype, level of M-component, type of light chain, stage of the disease, haemoglobin, serum albumin level, calcium, β₂ microglobulin and LDH, were observed. To our knowledge, this is the first report of methylation in MM and MGUS from Argentina. The similar frequency of p14^ARF, p15^INK4b, p16^INK4a and RASSF1A gene methylation observed in MM and MGUS would suggest that they are probably not involved in the progression of MGUS. However, SOCS1 gene methylation was significantly more frequent in MM than in MGUS suggesting that methylation of this gene might be involved in clonal evolution of MGUS to MM. SOCS1 is a negative regulator of cytokine signaling, being important in normal lymphocyte development and differentiation. Silencing of SOCS1 may result in greater responsiveness to cytokines, which may favour the neoplastic development.