DNA methylation fingerprint of neuroblastoma reveals new biological and clinical insights.

Neuroblastoma (NB) is one of the most frequently occurring extracranial solid tumors of childhood (Maris et al., 2007 [1]; Brodeur, 2003 [2]). Probability of cure varies according to patient's age, extent of disease and tumor biology (Maris et al., 2007 [1]; Brodeur, 2003 [2]; Cohn et al., 2009 [3]). However, the etiology of this developmental tumor is unknown. Recent evidence has shown that pediatric solid tumors, including NB, harbor a paucity of recurrent genetic mutations, with a significant proportion of recurrent events converging on epigenetic mechanisms (Cheung et al., 2012 [4]; Molenaar et al., 2012 [5]; Pugh et al., 2013 [6]; Sausen et al., 2013 [7]. We have analyzed the DNA methylome of neuroblastoma using high-density microarrays (Infinium Human Methylation 450k BeadChip) to define the epigenetic landscape of this pediatric tumor and its potential clinicopathological impact. Here, we provide the detail of methods and quality control parameters of the microarray data used for the study. Methylation data has been deposited at NCBI Gene Expression Omnibus data repository, accession number GSE54719; superseries record GSE54721.

DNA methylation fingerprint of neuroblastoma reveals new biological and clinical insights.

AIM: To define the DNA methylation landscape of neuroblastoma and its clinicopathological impact. MATERIALS & METHODS: Microarray DNA methylation data were analyzed and associated with functional/regulatory genome annotation data, transcriptional profiles and clinicobiological parameters. RESULTS: DNA methylation changes in neuroblastoma affect not only promoters but also intragenic and intergenic regions at cytosine-phosphate-guanine (CpG) and non-CpG sites, and target functional chromatin domains of development and cancer-related genes such as CCND1. Tumors with diverse clinical risk showed differences affecting CpG and, remarkably, non-CpG sites. Non-CpG methylation observed essentially in clinically favorable cases was associated with the differentiation status of neuroblastoma and expression of key genes such as ALK. CONCLUSION: This epigenetic fingerprint of neuroblastoma provides new insights into the pathogenesis and clinical behavior of this pediatric tumor.

DNA hypomethylation affects cancer-related biological functions and genes relevant in neuroblastoma pathogenesis.

Neuroblastoma (NB) pathogenesis has been reported to be closely associated with numerous genetic alterations. However, underlying DNA methylation patterns have not been extensively studied in this developmental malignancy. Here, we generated microarray-based DNA methylation profiles of primary neuroblastic tumors. Stringent supervised differential methylation analyses allowed us to identify epigenetic changes characteristic for NB tumors as well as for clinical and biological subtypes of NB. We observed that gene-specific loss of DNA methylation is more prevalent than promoter hypermethylation. Remarkably, such hypomethylation affected cancer-related biological functions and genes relevant to NB pathogenesis such as CCND1, SPRR3, BTC, EGF and FGF6. In particular, differential methylation in CCND1 affected mostly an evolutionary conserved functionally relevant 3' untranslated region, suggesting that hypomethylation outside promoter regions may play a role in NB pathogenesis. Hypermethylation targeted genes involved in cell development and proliferation such as RASSF1A, POU2F2 or HOXD3, among others. The results derived from this study provide new candidate epigenetic biomarkers associated with NB as well as insights into the molecular pathogenesis of this tumor, which involves a marked gene-specific hypomethylation.

A three-gene expression signature model for risk stratification of patients with neuroblastoma.

PURPOSE: Neuroblastoma is an embryonal tumor with contrasting clinical courses. Despite elaborate stratification strategies, precise clinical risk assessment still remains a challenge. The purpose of this study was to develop a PCR-based predictor model to improve clinical risk assessment of patients with neuroblastoma. EXPERIMENTAL DESIGN: The model was developed using real-time PCR gene expression data from 96 samples and tested on separate expression data sets obtained from real-time PCR and microarray studies comprising 362 patients. RESULTS: On the basis of our prior study of differentially expressed genes in favorable and unfavorable neuroblastoma subgroups, we identified three genes, CHD5, PAFAH1B1, and NME1, strongly associated with patient outcome. The expression pattern of these genes was used to develop a PCR-based single-score predictor model. The model discriminated patients into two groups with significantly different clinical outcome [set 1: 5-year overall survival (OS): 0.93 ± 0.03 vs. 0.53 ± 0.06, 5-year event-free survival (EFS): 0.85 ± 0.04 vs. 0.042 ± 0.06, both P < 0.001; set 2 OS: 0.97 ± 0.02 vs. 0.61 ± 0.1, P = 0.005, EFS: 0.91 ± 0.8 vs. 0.56 ± 0.1, P = 0.005; and set 3 OS: 0.99 ± 0.01 vs. 0.56 ± 0.06, EFS: 0.96 ± 0.02 vs. 0.43 ± 0.05, both P < 0.001]. Multivariate analysis showed that the model was an independent marker for survival (P < 0.001, for all). In comparison with accepted risk stratification systems, the model robustly classified patients in the total cohort and in different clinically relevant risk subgroups. CONCLUSION: We propose for the first time in neuroblastoma, a technically simple PCR-based predictor model that could help refine current risk stratification systems.

Identification of tumoral glial precursor cells in neuroblastoma.

Neuroblastic tumors (NBT) are composed by neuroblasts and Schwannian-like stroma. The origin of these two cell subtypes remains unclear. In this study, we describe, a neuroblastic-like subpopulation in neuroblastoma (NB) coexpressing GD2 and S100A6, neuroblastic and glial lineage markers respectively. The GD2(+)/S100A6(+) neuroblastic subpopulation was found to be enriched in low risk NB, distributed around the perivascular niche. Some stromal bundles showed GD2(+)/S100A6 costaining. Metastatic bone marrow specimens also showed GD2(+)/S100A6(+) cells. During in vitro retinoic acid induced differentiation of NB cell lines, rare GD2(+)/S100A6 neuroblatic cells appeared. We conclude that GD2(+)/S100A6(+) neuroblasts may represent a tumoral glial precursor subpopulation in NBT.

Expression of the neuron-specific protein CHD5 is an independent marker of outcome in neuroblastoma.

BACKGROUND: The chromodomain, helicase DNA-binding protein 5 (CHD5) is a potential tumor suppressor gene located on chromosome 1p36, a region recurrently deleted in high risk neuroblastoma (NB). Previous data have shown that CHD5 mRNA is present in normal neural tissues and in low risk NB, nevertheless, the distribution of CHD5 protein has not been explored. The aim of this study was to investigate CHD5 protein expression as an immunohistochemical marker of outcome in NB. With this purpose, CHD5 protein expression was analyzed in normal neural tissues and neuroblastic tumors (NTs). CHD5 gene and protein expression was reexamined after induction chemotherapy in a subset of high risk tumors to identify potential changes reflecting tumor response. RESULTS: We provide evidence that CHD5 is a neuron-specific protein, absent in glial cells, with diverse expression amongst neuron types. Within NTs, CHD5 immunoreactivity was found restricted to differentiating neuroblasts and ganglion-like cells, and absent in undifferentiated neuroblasts and stromal Schwann cells. Correlation between protein and mRNA levels was found, suggesting transcriptional regulation of CHD5. An immunohistochemical analysis of 90 primary NTs highlighted a strong association of CHD5 expression with favorable prognostic variables (age at diagnosis <12 months, low clinical stage, and favorable histology; P < 0.001 for all), overall survival (OS) (P < 0.001) and event-free survival (EFS) (P < 0.001). Multivariate analysis showed that CHD5 prognostic value is independent of other clinical and biologically relevant parameters, and could therefore represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. The prognostic value of CHD5 was confirmed in an independent, blinded set of 32 NB tumors (P < 0.001).Reactivation of CHD5 expression after induction chemotherapy was observed mainly in those high risk tumors with induced tumor cell differentiation features. Remarkably, these NB tumors showed good clinical response and prolonged patient survival. CONCLUSIONS: The neuron-specific protein CHD5 may represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. Re-establishment of CHD5 expression induced by chemotherapy could be a surrogate marker of treatment response.