Hypermethylation of DAP-K Is an Adverse Prognostic Factor in Patients with Multiple Myeloma (MM).
Authors: Braggio Esteban , Gouveia Maria E. , Magalhaes Roberto , Souto Joao T. , Garnica Marcia , Nucci Marcio , Maiolino Angelo , Zalcberg Ilana R. 
Published: October 2019

Abstract
Aberrant methylation in promoter-associated CpG islands has been recognized as a major mechanism for silencing tumor suppressor genes in many malignant diseases. In multiple myeloma (MM), a limited number of studies of gene methylation have been reported, with conflicting results. We determined the methylation status of 9 suppressor tumor genes in 68 newly diagnosed MM patients by methylation specific PCR (MSP). Ten DNA of normal healthy donors were used as controls. The target genes chosen are involved in many cellular pathways as DNA repair (MGMT), cell cycle regulation (p15INK4b and p16INK4a), cell-cell adherence (E-cadherin), apoptosis (DAP-k and BNIP3), hormonal response (RARb and ER) and Jak/STAT3 signaling pathway (SHP1). An association between hypermethylation and loss of expression of these genes has been demonstrated. The correlation between methylation status and risk factors was assessed by Fisher exact test or Chi-square test (categorical variables) or Student’s t-test (continuous variables). Overall survival (OS) was estimated using the Kaplan-Meier method. Differences between survival curves were estimated by the log-rank test. Multivariate analysis of factors associated with OS was performed by Cox regression. With a median follow-up of 15.5 months, 16 patients died (23%). Healthy donor samples were negative for methylation in all 9 genes tested. Hypermethylation was detected in 50% of patients for E-cadherin, 43% for p16, 16% for p15, 15% for SHP1, 13% for ER and BNIP3, 12% for RARb, 6% for DAP-k, and 0% for MGMT. Overall, 54 patients (79%) presented at least one hypermethylated gene (1 in 19 patients, 2 in 17 patients, 3 in 12 patients, 4 in 5 patients, and 5 in 1 patient). By univariate analysis, hypermethylation of DAP-k (p<0.001) and RARb (p=0.01) genes, platelet counts <100,000/mm3 (p<0.001) and serum calcium >9.5 mg/dL (p=0.03) were identified as adverse prognostic features. The median OS of patients with hypermethylation in DAP-k (4 months) and RARb (34 months) were considerably lower compared to patients without aberrant methylation (median survival not reached, p<0.001 and p=0.01, respectively). Patients with hypermethylation of DAP-k were more likely to have a serum creatinine >2.0 mg/dL (p=0.006), serum calcium >9.5 mg/dL (p=0.05), and Durie-Salmon stage III (p=0.04). No correlation was observed between methylation status of any gene and the presence of chromosome 13 abnormalities, t(4;14)(p16;q32), or t(11;14)(q23;q32). By multivariate analysis, hypermethylation of DAP-k (odds ratio [OR] 5.56, 95% confidence interval [95% CI] 1.4 – 22, p=0.01) and platelet counts <100,000/mm3 (OR 4.13, 95% CI 1.32 – 12.8, p=0.01) were associated with poor prognosis. Our data suggest that hypermethylation of DAP-k is an independent prognostic factor in MM. The impact of these features in identifying a group of poor prognosis beyond the classical prognostic factors warrants a higher sample size and longer follow-up.