Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Accurate outcome prediction in neuroblastoma (NB), which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve NB patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers. To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight NB cell lines. Specifically, we used re-expression profiling upon 5-aza-2’-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Candidate methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR (MSP) on 20 primary NB tumors, as well as through MBD-seq in combination with publicly available NB tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput MSP on an independent cohort of 89 primary NB tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival. In conclusion, this study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in NB performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in NB.