Background: Neuroblastoma is a genetically heterogenic tumor diagnosed in childhood which exhibits broad clinical diversity ranging from rapid disease progression to spontaneous regression. Although amplification of the MYCN oncogene is one of the most prominent prognostic markers of this disease it is only present in around 25% of patients. Hence, additional molecular markers are needed for patients lacking MYCN amplification to enable better stratification of patient risk groups.
To date, genome wide association studies have identified few gene candidates in neuroblastoma patients that can be further developed for prognostic use in the clinic. DNA methylation is a major epigenetic mechanism for gene silencing in a wide range of human cancers and methylation sites may reveal new prognostic targets in neuroblastoma. Recent studies have suggested that the methylation status of specific gene promoters may be useful for clinically discriminating subgroups of children diagnosed with neuroblastoma. To date, these investigations have assessed DNA methylation using variable techniques; focused on small numbers of candidate genes and; examined few primary tumor samples. The aims of our study were to characterize the clinical significance of promoter methylation in a large cohort of primary neuroblastoma tumors and investigate the association between DNA methylation and clinical outcome.
Methods: 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 between 1985 and 2000. Candidate genes were selected on the basis of previous reports of altered DNA methylation in embryonal cancers. Levels of DNA methylation were quantified and confirmed in a subset of 48 patient samples using combined bisulphite restriction analysis (CoBRA). A Cox proportional hazards model was used to investigate the association between promoter hypermethylation and the risk of relapse or death within 5 years of diagnosis, while adjusting for known prognostic cofactors such as MYCN amplification, age and stage at diagnosis.
Results: The level of promoter methylation of DNAJC15, NTRK1 and TNFRSF10D were significantly higher in older patients at diagnosis (p<0.01), while higher levels of promoter methylation of DNAJC15, NTRK1 and PYCARD were observed in patients with MYCN amplification (p<0.001). In multivariate analysis, promoter hypermethylation of FOLH1, MYOD1 and THBS1 remained significant independent predictors of poorer clinical outcome, after adjusting for MYCN amplification, age at diagnosis and tumor stage (p≤0.017). Around a third of patients displayed promoter hypermethylation in 2 or more of these genes and were almost 3 times more likely to relapse or die than those who did not display promoter hypermethylation (HR=2.72; 95%C1=1.55-4.78; p=0.001).
Conclusion: DNA methylation of FOLH1, MYOD1 and THBS1 appears to have independent prognostic value in children diagnosed with neuroblastoma. While the functional consequences of promoter hypermethylation in individual neuroblastoma patients remains to be fully determined, these investigations show how epigenetic profiling of tumor tissue can provide additional genetic markers for predicting treatment response in the clinical setting and has the potential to reveal important new therapeutic targets for selected patient subsets.
Citation Format: Diana T. Lau, Luke B. Hesson, Murray D. Norris, Glenn Marshall, Michelle Haber, Lesley J. Ashton. Beyond GWAS: The prognostic significance of promoter DNA methylation in patients with neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 68.