title: Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma.
abstract: Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature.
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.
authors.forename: Anneleen
authors.initials: A
authors.lastname: Decock
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.
authors.forename: Maté
authors.initials: M
authors.lastname: Ongenaert
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.|Bioinformatics Institute Ghent From Nucleotides to Networks (BIG N2N), De Pintelaan, Ghent, Belgium.|DAMBI, VIB Inflammation Research Center, Technologiepark, Ghent, Belgium.|Department of Respiratory Medicine, Ghent University, De Pintelaan, Ghent, Belgium.
authors.forename: Robrecht
authors.initials: R
authors.lastname: Cannoodt
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Verniers
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.|Department of Pediatric Hematology and Oncology, Ghent University Hospital, De Pintelaan, Ghent, Belgium.
authors.forename: Bram
authors.initials: B
authors.lastname: De Wilde
authors.affiliation: Department of Pediatric Hematology and Oncology, Ghent University Hospital, De Pintelaan, Ghent, Belgium.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Laureys
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.
authors.forename: Nadine
authors.initials: N
authors.lastname: Van Roy
authors.affiliation: Department of Pathology, Medical School, University of Valencia, and Health Research Institute INCLIVA, Blasco Ibañez, Valencia, Spain.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Berbegall
authors.affiliation: Department of Pathological Physiology, Department of Pediatric Oncology, Masaryk University, Černopolní, Brno, Czech Republic.
authors.forename: Julie
authors.initials: J
authors.lastname: Bienertova-Vasku
authors.affiliation: Northern Genetics Service, Institute of Genetic Medicine, Central Parkway, Newcastle upon Tyne, United Kingdom.
authors.forename: Nick
authors.initials: N
authors.lastname: Bown
authors.affiliation: Department of Pediatric Oncology, Institut Curie, rue d'Ulm, Paris, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Clément
authors.affiliation: Centre Léon Bérard, Laboratoire de Recherche Translationnelle, rue Laennec, Lyon, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Combaret
authors.affiliation: Children's Cancer Institute, Lowy Cancer Research Centre, UNSW, Randwick NSW, Australia.
authors.forename: Michelle
authors.initials: M
authors.lastname: Haber
authors.affiliation: Pediatric Hemato-oncology, CHR Citadelle, Liège, Belgium.
authors.forename: Claire
authors.initials: C
authors.lastname: Hoyoux
authors.affiliation: Children's Cancer Institute, Lowy Cancer Research Centre, UNSW, Randwick NSW, Australia.
authors.forename: Jayne
authors.initials: J
authors.lastname: Murray
authors.affiliation: Department of Pathology, Medical School, University of Valencia, and Health Research Institute INCLIVA, Blasco Ibañez, Valencia, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Noguera
authors.affiliation: Unité de Génétique Somatique, Institut Curie, rue d'Ulm, Paris, France.
authors.forename: Gaelle
authors.initials: G
authors.lastname: Pierron
authors.affiliation: U830 INSERM, Recherche Translationelle en Oncologie Pédiatrique (RTOP) and Department of Pediatric Oncology, Institut Curie, rue d'Ulm, Paris, France.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Schleiermacher
authors.affiliation: Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Hufelandstraße, Essen, Germany.
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Schulte
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.|Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, York House, Dublin, Ireland.
authors.forename: Ray L
authors.initials: RL
authors.lastname: Stallings
authors.affiliation: Newcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Tweddle
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.|Bioinformatics Institute Ghent From Nucleotides to Networks (BIG N2N), De Pintelaan, Ghent, Belgium.
authors.forename: Katleen
authors.initials: K
authors.lastname: De Preter
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.affiliation: Center for Medical Genetics, Ghent University, De Pintelaan, Ghent, Belgium.|Cancer Research Institute Ghent (CRIG), De Pintelaan, Ghent, Belgium.|Bioinformatics Institute Ghent From Nucleotides to Networks (BIG N2N), De Pintelaan, Ghent, Belgium.
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
mesh_terms: D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis*; D015331:Cohort Studies; D019295:Computational Biology; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D009367:Neoplasm Staging; D009447:Neuroblastoma / Q000175:diagnosis* / Q000235:genetics*; D011379:Prognosis; D060888:Real-Time Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D015415:Biomarkers; D004273:DNA, Neoplasm
keywords: DNA methylation; biomarker; neuroblastoma; prognosis