Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma

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Authors	DECOCK Anneleen ONGENAERT Maté CANNOODT Robrecht VERNIERS Kimberly DE WILDE Bram LAUREYS Genevieve VAN ROY Nadine BERBEGALL Ana P. BIENERTOVÁ VAŠKŮ Julie BOWN Nick CLEMENT Nathalie COMBARET Valérie HABER Michelle HOYOUX Claire MURRAY Jayne NOGUERA Rosa PIERRON Gaelle SCHLEIERMACHER Gudrun SCHULTE Johannes H. STALLINGS Ray L. TWEDDLE Deborah A. DE PRETER Katleen SPELEMAN Frank VANDESOMPELE Jo
Year of publication	2016
Type	Article in Periodical
Magazine / Source	Oncotarget
MU Faculty or unit	Faculty of Medicine
Citation
Field	Oncology and hematology
Keywords	neuroblastoma; DNA methylation; prognosis; biomarker
Attached files	EL_1365447.pdf
Description	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.