Mapping the neuroblastoma epigenome: perspectives for improved prognostic biomarkers

Background: Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. Current risk assessment schemes unfortunately result in a significant proportion of patient misclassifications, leading to under - or overtreatment. Clearly, a more objective and accurate classifier is needed for improved outcome prediction. Next to genomic changes, epigenetic alterations have been described as well. Most of these methylation markers are found using ‘candidate gene’ approaches and the methylation frequencies are usually very low. Methods: In order to find novel methylation markers that can be used for improved prognosis, we applied a whole-genome methylation screen. This technique relies on capturing with the MBD2 protein, containing a methyl-binding domain, with a very high affinity towards methylated genomic regions. In an initial phase, MBD2-seq was performed on 8 NB cell lines (for which mRNA profiles before and after treatment with the demethylating agent DAC were also available). Results: An integrated analysis (MBD2-sequencing, re-expression analysis, analysis of public expression data) led to the selection of 43 candidate biomarkers, which were validated using real-time methylation-specific PCR in 89 primary NB patients in three prognostic groups. More than ten novel biomarkers in NB were found to be related with classical NB risk factors and survival. MBD-sequencing data on 45 primary NB tumors of the same three prognostic groups are underway, allowing us to make a genome-wide map of the epigenome of NB. An integrated data-analysis across the genomic (exon-arrays and array CGH) and epigenomic information, will lead to the identification of highly sensitive and specific biomarkers for improved risk classification and treatment. Conclusions;: Mapping the complete neuroblastoma methylome gives insight in NB biology and opens perspectives for improved prognosis