Prognostic Significance of Promoter DNA Methylation in Patients with Childhood Neuroblastoma

Purpose: To characterize the clinical significance of promoter methylation in a cohort of primary neuroblastoma tumors and investigate the association between DNA methylation and clinical outcome. Experimental Design: A customized Illumina GoldenGate methylation assay was used to assess methylation status of 96 CpG sites within 48 candidate genes in primary neuroblastoma tumors obtained from 131 children diagnosed in Australia. Genes were selected on the basis of previous reports of altered DNA methylation in embryonal cancers. Levels of DNA methylation were validated in a subset of 48 patient samples using combined bisulfite restriction analysis (CoBRA) and bisulfite sequencing. A Cox proportional hazards model was used to investigate the association between promoter hypermethylation and the risk of relapse/death within 5 years of diagnosis, while adjusting for known prognostic factors including MYCN amplification, age, and stage at diagnosis. Results: Levels of promoter methylation of DNAJC15, neurotrophic tyrosine kinase receptor 1 or TrkA (NTRK1), and tumor necrosis factor receptor superfamily, member 10D (TNFRSF10D), were higher in older patients at diagnosis (P < 0.01), whereas higher levels of methylation of DNAJC15, NTRK1, and PYCARD were observed in patients with MYCN amplification (P < 0.001). In multivariate analysis, hypermethylation of folate hydrolase (FOLH1), myogenic differentiation-1 (MYOD1), and thrombospondin-1 (THBS1) remained significant independent predictors of poorer clinical outcome after adjusting for known prognostic factors (P <= 0.017). Moreover, more than 30% of patients displayed hypermethylation in 2 genes or more and were at least 2 times more likely to relapse or die (HR - 2.72, 95% confidence interval 1.55-4.78, P = 0.001), independent of MYCN status, age, and stage at diagnosis. Conclusions: Our findings highlight the potential use of methylation profiling to identify additional prognostic markers and detect new therapeutic targets for selected patient subsets. Clin Cancer Res; 18(20); 5690-700. (C) 2012 AACR.