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.

Bioinformatics Institute Ghent From Nucleotides to Networks De Pintelaan Ghent Belgium

Cancer Research Institute Ghent De Pintelaan Ghent Belgium

Center for Medical Genetics Ghent University De Pintelaan Ghent Belgium

Centre Léon Bérard Laboratoire de Recherche Translationnelle rue Laennec Lyon France

Children's Cancer Institute Lowy Cancer Research Centre UNSW Randwick NSW Australia

DAMBI VIB Inflammation Research Center Technologiepark Ghent Belgium

Department of Molecular and Cellular Therapeutics Royal College of Surgeons in Ireland York House Dublin Ireland

Department of Pathological Physiology Department of Pediatric Oncology Masaryk University Černopolní Brno Czech Republic

Department of Pathology Medical School University of Valencia and Health Research Institute INCLIVA Blasco Ibañez Valencia Spain

Department of Pediatric Hematology and Oncology Ghent University Hospital De Pintelaan Ghent Belgium

Department of Pediatric Oncology and Hematology University Children's Hospital Essen Hufelandstraße Essen Germany

Department of Pediatric Oncology Institut Curie rue d'Ulm Paris France

Department of Respiratory Medicine Ghent University De Pintelaan Ghent Belgium

National Children's Research Centre Our Lady's Children's Hospital Crumlin Dublin Ireland

Newcastle Cancer Centre Northern Institute for Cancer Research Newcastle University Framlington Place Newcastle upon Tyne United Kingdom

Northern Genetics Service Institute of Genetic Medicine Central Parkway Newcastle upon Tyne United Kingdom

Pediatric Hemato oncology CHR Citadelle Liège Belgium

U830 INSERM Recherche Translationelle en Oncologie Pédiatrique and Department of Pediatric Oncology Institut Curie rue d'Ulm Paris France

Unité de Génétique Somatique Institut Curie rue d'Ulm Paris France

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