Snk/Plk2 Methylation Is a Frequent Event in Patients with Multiple Myeloma

Background-Aim: The polo like kinases (Plk) are highly conserved in evolution and have critical functions in the regulation of proliferation and cell cycle checkpoints. We previously showed that polo like kinase 2 (Snk/Plk2) is subject to methylation-dependent transcriptional silencing in B lymphomas, with a very high frequency in Burkitt lymphomas, implying that Snk/Plk2 may have a tumour suppressor function in B lymphocytes. However, no study has examined epigenetic changes in plasma cell dyscrasias. Here, we have examined CpG methylation in Snk/Plk2 in a well-characterised series of multiple myeloma (MM) patients.

Patients and Methods: Bone marrow samples from individuals with MM were obtained at diagnosis and disease progression. Genomic DNA was isolated and bisulphite modification was performed using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). The methylation-specific polymerase chain reaction (MSP) with primers for methylated and unmethylated alleles of the Snk/Plk2 gene promoter was employed to study its methylation status. Control methylated (CpG Genome^™ Universal Methylated, Chemicon International) and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples from individuals with borderline thrombocytopenia, that proved to have no haematological malignancy, served as negative controls. Survival curves were generated using the method of Kaplan-Meier and compared with the log-rank test. Logistic regression analyses were also used to measure the association between gene methylation and the development of advanced disease (DS³II), extramedullary disease, bone disease, anemia (Hb≤10 mg/dl), serum albumin and beta 2 microglobulin levels.

Results: We analysed the methylation of Snk/Plk2 in 45 cases of multiple myeloma (MM) (24 male, 21 female, mean age 66.4 years ±12.4). Using the Durie and Salmon staging system MM patients’ disease stages were as follows: smoldering MM 7/45, IA 9/45, IIA 13/45 patients, IIIA 7/45 patients and IIIB 9/45 patients. Classical cytogenetic analysis was available in 30/45 patients and none of them was found to have chromosome 13 abnormalities. No sample from the control population was found methylated. The Snk/Plk2 promoter was found to be methylated in 27/45 (60%) MM patients. Median survival of patients in whom the Snk/Plk2 CpG island was methylated was 7.1 years compared to a median survival of 8.8 years for unmethylated. However Snk/Plk2 methylation was not a predictor of excess mortality (HR=0.6, p=0.5), bone lytic lesions (OR=0.56, p=0.3), anemia (OR=0.5, p=0.3) or advanced stage as defined above (OR=0.8, p=0.7). A trend was noted where patients with methylated Snk/Plk2 had a reduced risk of developing extramedullary disease (OR=0.3, p=0.1). No association was found between the methylation and serum albumin (p=0.3) or beta 2 microglobulin levels (p=0.8).

Conlusions: We examined for the first time the methylation status of Snk/Plk2 in MM patients and showed that it is a common event in these patients implying that loss of function in this gene is frequently involved in the pathogenesis of MM. However there was lack of association between the methylation status of the gene and relevant clinical parameters. Further evaluation in a larger sample of patients is needed in order to better define the prognostic and clinical value if any of Snk/Plk2 methylation in MM.