Abstract
BackgroundAccurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.ResultsTo identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma 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). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma 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.ConclusionsThis study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma 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 neuroblastoma.
Highlights
Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge
Integration with (NB) literature, using an in-house developed text-miningbased approach, and selection for genes located in genomic regions reported as recurrently affected by DNA copy number changes in NB, eventually led to the selection of 120 candidate biomarkers, comprising 30 novel candidate markers and 90 known methylation markers in other tumor types
In the NB cell lines, the DAC-treated samples show less methylation calls in comparison to untreated samples (130 methylation-specific PCR (MSP) assays (64%) are more frequently methylated in the untreated samples), and taking all MSP assays into account the average number of methylated samples per assay is 0.39 for the DACtreated cell lines versus 1.47 for the untreated cell lines (P = 0.0002), revealing dense methylation in genes upregulated upon DAC treatment and efficient demethylation by DAC
Summary
Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. Survival rates vary widely, depending on clinical features, such as age at diagnosis and tumor stage, as well as biological characteristics of the tumor. Amongst the latter, MYCN amplification has been used for many years as a genetic marker for therapy stratification [1]. In order to facilitate the comparison of risk-based clinical trials, a new consensus approach for pretreatment risk classification has been designed including genetic parameters [1,4] Despite this progress, additional markers for therapeutic stratification are warranted in order to avoid under- or overtreatment and to improve selection of ultra-high-risk patients for new experimental therapies. We propose that the use of DNA methylation markers is a new and promising method for prognostic classification
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