title: Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers.
abstract: BACKGROUND
  Accurate 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.
  
  
  RESULTS
  To 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.
  
  
  CONCLUSIONS
  This 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.
authors.forename: Anneleen
authors.initials: A
authors.lastname: Decock
authors.forename: Maté
authors.initials: M
authors.lastname: Ongenaert
authors.forename: Jasmien
authors.initials: J
authors.lastname: Hoebeeck
authors.forename: Katleen
authors.initials: K
authors.lastname: De Preter
authors.forename: Gert
authors.initials: G
authors.lastname: Van Peer
authors.forename: Wim
authors.initials: W
authors.lastname: Van Criekinge
authors.forename: Ruth
authors.initials: R
authors.lastname: Ladenstein
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Schulte
authors.forename: Rosa
authors.initials: R
authors.lastname: Noguera
authors.forename: Raymond L
authors.initials: RL
authors.lastname: Stallings
authors.forename: An
authors.initials: A
authors.lastname: Van Damme
authors.forename: Geneviève
authors.initials: G
authors.lastname: Laureys
authors.forename: Joëlle
authors.initials: J
authors.lastname: Vermeulen
authors.forename: Tom
authors.initials: T
authors.lastname: Van Maerken
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D002864:Chromogranins; D019175:DNA Methylation; D030541:Databases, Genetic; D000077209:Decitabine; D057890:Epigenomics / Q000379:methods*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D015894:Genome, Human; D045325:HCT116 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009447:Neuroblastoma / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic; D012307:Risk Factors; D017422:Sequence Analysis, DNA; D016019:Survival Analysis
chemical_list: D014408:Biomarkers, Tumor; D002864:Chromogranins; D000077209:Decitabine; C406732:GNAS protein, human; D019205:GTP-Binding Protein alpha Subunits, Gs; D001374:Azacitidine