Neuroblastoma genetics and phenotype: A tale of heterogeneity

Seminars in Cancer Biology 21 (2011) 238–244

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Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer

Review

Neuroblastoma genetics and phenotype: A tale of heterogeneity Frank Speleman ∗ , Katleen De Preter, Jo Vandesompele Center for Medical Genetics, Ghent University Hospital (MRB), De Pintelaan 185, B-9000 Ghent, Belgium

a r t i c l e Keywords: Neuroblastoma Genetics Genomics

i n f o

a b s t r a c t Cancer is a complex disease driven by multiple genetic and epigenetic alterations. Understanding the (epi-)genetic changes and consequent deregulation of regulatory networks controlling the various normal critical cellular phenotypes that are perturbed in cancer cells can provide clues to new therapeutic opportunities. Moreover, such insights into the molecular pathology of a given cancer type can offer clinical relevant genetic markers or molecular signatures for assessment of prognosis and response to therapy, and prediction of risk for relapse. Therefore, as for many other tumour entities, neuroblastoma (NB) has been the subject of intensive ongoing genomic research. Here we will summarize the current state-of-the-art of these studies with focus on genome wide DNA copy number and gene expression analyses in relation to the relevance for present and future clinical management of NB patients. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Cancer is a complex disease driven by multiple genetic and epigenetic alterations. Understanding the (epi-)genetic changes and consequent deregulation of regulatory networks controlling the various normal critical cellular phenotypes that are perturbed in cancer cells can provide clues to new therapeutic opportunities. Moreover, such insights into the molecular pathology of a given cancer type can offer clinical relevant genetic markers or molecular signatures for assessment of prognosis and response to therapy, and prediction of risk for relapse. Therefore, as for many other tumour entities, neuroblastoma (NB) has been the subject of intensive ongoing genomic research. Here we will summarize the current state-of-the-art of these studies with focus on genome wide DNA copy number and gene expression analyses in relation to the relevance for present and future clinical management of NB patients.

2. Genome wide analyses of DNA copy number alterations: deciphering tumour heterogeneity MYCN amplification was discovered in the early 1980s and ever since has remained a mainstay in neuroblastoma (NB) research and therapeutic stratification [1,2, Wilhelm et al., in this issue]. NB cell lines with evidence of MYCN amplification also showed the presence of deletions and structural alterations leading to loss of the distal part of the short arm of chromosome 1 [3]. Despite the difficulties in karyotyping, some early studies reported near-triploidy

∗ Corresponding author. Tel.: +32 93322451. E-mail address: [email protected] (F. Speleman). 1044-579X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2011.07.003

in tumours with patterns of numerical changes only. This pattern was found only in low stage tumours with favorable outcome suggesting the existence of distinct genetic subsets of near-triploid and near- or pseudo-diploid or hypo-tetraploid tumours reflecting the observed clinical heterogeneity for this tumour [4]. This observation was later supported by DNA ploidy studies [5]. The introduction of molecular cytogenetics in the 1990s uncovered a third highly recurrent structural alteration within the subgroup of high stage aggressive NBs leading to gain of a large distal segment of the long arm of chromosome 17 [6,7]. Molecular cytogenetics paved the way for the development of chromosomal comparative genomic hybridization (cCGH) which had the advantage that living dividing cells were not required, thus opening the way for broader analysis of all subtypes of NBs. Using this approach, the highly recurrent nature of the chromosome 17q gain was confirmed [8]. Remarkably, whole chromosome 17 gain was almost invariably present in near triploid low stage tumours with favorable prognosis [9]. In addition, other recurrent patterns of chromosomal gains and losses emerged, with amongst others frequent loss of chromosomes 3 and 11 and gain of chromosome 7. Chromosomal CGH analyses also allowed the delineation of the second group of aggressive high stage NBs without MYCN amplification. As it turned out, the majority of these tumours exhibited deletions of the long arm of chromosome 11, often in association with 3p deletion and, like MYCN amplified tumours, almost always showing distal 17q gain [10]. Taken together, genome wide analyses in NB identified three major genomic subtypes [11]. As cCGH required analysis of normal metaphase chromosomes as template for read out of the comparative hybridization, the procedure was tedious and time consuming. This situation dramatically changed with the introduction of DNA arrays using bacterial artificial chromosomes (BACs) as reporters and subsequently, improved

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commercial high-resolution oligonucleotide array-CGH (aCGH) platforms. This offered unprecedented speed and resolution and provided a detailed view of the NB genomic landscape [12,13]. Three major findings have emerged from these aCGH studies. First, the previously established patterns of DNA copy number gains and losses were reinforced based on larger series of hundreds of tumours samples and further underlining the genomic heterogeneity of this disease. Secondly, extensive prognostic analysis of large tumour series with well-annotated clinical data have underscored that the presence of whole-chromosome copy number variations only, were associated with excellent survival. Importantly, the presence of segmental alterations with or without MYCN amplification was the strongest predictor of relapse in a multivariate analysis and patients with both numerical and segmental abnormalities clearly shared the higher risk of relapse of segmental-only patients [11,14]. Thirdly, several studies revealed the occurrence of high-level ALK amplification, albeit in rare cases [15]. Subsequently, activating mutations in the tyrosine kinase domain of this transmembrane receptor were described while an independent linkage study of familial cases marked ALK as the major NB predisposing gene [15–18, Mossé et al., in this issue]. In depth analyses of high-resolution aCGH analyses have further contributed to the molecular portrait of NB. Using an oligonucleotide platform with a median probe spacing of 6 kb, Stallings et al. identified likely somatically acquired alteration including genes involved in tumourigenesis, apoptosis, or neural cell differentiation. The most frequently observed microdeletion involved the protein phosphatase PTPRD with possible tumour suppressor function [19]. A recent study from our group has confirmed the occurrence of similar rare but recurrent gains and losses [20]. Moreover, it became evident that several of such targeted DNA copy number alterations affect genes implicated in MYCN signaling, with the genomic defect reinforcing the direction of MYCN regulation. These observations are also in line with the rare amplification of direct MYCN target genes such as TERT, MDM2 and CDK4. This pattern of both common and rare recurrent genomic alterations is reminiscent to those described for mutations detected by exome wide sequencing analysis [21]. This illustrates the dynamic adaptation of tumour cells exploiting a variety of genetic alterations converging to central deregulated signaling pathways and thus may have implications towards future patient tailored molecular therapies [22]. In a recent study by Carén et al. [23], more in depth study of subtype 2A tumours with 11q deletion revealed an increased number of segmental aberrations as compared to MYCN amplified tumours, suggesting increased genomic instability in 11q deleted tumours. An alternative explanation is that in the absence of MYCN amplification, additional genetic events are required to attain the full oncogenic potential. Interestingly, within this subset of 11q deleted tumours, we found a new recurrent deletion targeting the distal end of the long arm of chromosome 1 which encompasses two bona fide tumour suppressor genes implicated in familial cancers, PHD2 and FH [24]. Massive parallel sequencing technology also holds promise to determine DNA copy number alterations as well as to characterize up to the base pair level copy number neutral genomic alterations such as balanced inter- and intrachromosomal rearrangements. This will soon further define the complex genomic landscape of NB [25–27].

3. Oncogenes: beyond MYCN As pointed out above, MYCN amplification is one of the first discovered genetic defects in NB and the gene has been intensively studied ever since. MYCN is member of the MYC family of basic helix–loop–helix transcription factors that control a broad reg-

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ulatory network implicated in cell cycle, DNA damage response, differentiation and apoptosis. Unraveling the pathways controlled by MYCN is essential for understanding how this transcription factor contributes to NB pathogenesis and might offer new therapeutic targets [28]. Currently only a limited number of bona fide upregulated target genes were identified, including ODC1, MDM2, MCM7 and TERT [29–31]. MYCN can also function as a transcriptional repressor of genes such as NTRK1 (TRKA) and NGFR (p75NTR). Here, MYCN associates with transcription factors MIZ1/SP1 followed by a recruitment of HDAC1, thereby inducing a repressed chromatin state [32]. Typically, high levels of MYCN expression are associated with aggressive tumour behaviour and poor survival. Interestingly, recent studies have indicated that high MYCN/c-MYC target gene expression is a hallmark of malignant NB progression, with high cMYC levels being detected in stage 4-non-amplified tumours [33]. This finding was further supported through the identification of a MYCN/c-MYC signature of MYC regulated miRNAs in aggressive NB tumours [34]. The emerging insight that (oncogenic) transcription factors form an intricate network with both protein coding and non-coding regulatory miRNAs has been pioneered through the study of MYCN/c-MYC and more particular also through analysis of MYCN regulated miRNAs in the context of NB [35,36]. Further details on MYCN regulation and differentiation are reviewed in this issue by Wilhelm et al. ALK is another important oncogene that only recently came under the attention in NB. In retrospect, a number of independent observations were in line with a putative role of ALK in NB such as its developmental expression pattern, differential expression of NB tumours compared to normal fetal neuroblasts and rare amplifications in primary tumours. In particular the latter observation together with an independent search for predisposing loci in familial NB lead to the discovery of both germline and somatic activating ALK mutations [15,16]. Two hotspot mutations were detected in sporadic NB and further analysis of a large tumour cohort revealed a particular association between the ALK F1174 mutation hotspot and MYCN amplification. Moreover, we showed that this particular mutation has a stronger oncogenic effect than the other hotspot mutation at position R1275 [37]. Further unraveling of the ALK and MYCN controlled regulatory networks and in vivo modeling will shed light on the possible cooperative effect between both oncogenes in NB. For further review of current insights into ALK biology in NB we refer to the review of Mossé et al. (in this issue). The high incidence of 17q gain in NB is strongly suggestive for a role of one or more genes on the duplicated segment as a result of increased gene dosage or perhaps mutation followed by copy number increase as described for certain other oncogenes. The region of recurrent 17q-gain has been narrowed down to 17q21-qter [38], in which several important candidate genes such as BIRC5 (Survivin) [39], PPM1D [40] and NME1 and NME2 [41] are located. Also the WSB1 gene, located at 17q11 was put forward as interesting candidate gene on 17q [42,43]. However, so far for none of these genes, the ultimate proof for involvement in neuroblastoma aggressiveness through 17q gain is present,

4. Tumour suppressor genes: hide and seek? The search for tumour suppressor genes in NB has been long, intense and little rewarding. So far no examples have been found of typical two hit mechanisms as a frequent recurrent mechanism of tumour suppressor gene inactivation. It remains to be seen if certain candidates will emerge from ongoing exome/whole genome sequencing efforts. Although TP53 mutations may occur in relapsed NB, mutations in primary tumours are extremely rare suggesting other genetic mechanism are perturbing normal TP53

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function [44]. As was previously described, MYCN overexpression can trigger apoptosis which may—at least in part—result from direct up regulation of TP53 by MYCN [45]. Detailed insight into how NB cells evade apoptosis are lacking but successful treatment with the MDM2 inhibitor Nutlin-3a suggests that MDM2 mediated control of TP53 function is essential in this respect [46–49]. Moreover, MDM2 has been described as a direct transcriptional target of MYCN and is amplified in some cases [44,50]. The TP53 pathway may also be disrupted through loss of CHD5 expression as a result from 1p36 deletions and methylation [51]. CHD5 belongs to a family of chromatin remodeling proteins and [52] controls proliferation, senescence and apoptosis through the p19Arf-p53 pathway [13]. Other genes on the short arm of chromosome 1 such as KIF1B [53,54], CAMTA1 [55,56] and ELAVL4 [20] may exert tumour suppressor function and contribute to the NB phenotype upon hemizygous or homozygous loss but in vivo modeling will be required to understand their role in more detail. Ochiai et al. [54] reported that candidate tumour suppressors KIF1B on 1p and CADM1 on 11q (see below) are repressed by BMI1 which is a direct target of MYCN. Similarly, a recent study showed CAMTA1 down regulation through the MYCN up regulated miR-17-92 cluster in glioblastoma, a mechanism which may also play a role in further inactivation of CAMTA1 in chromosome 1p-deleted MYCN amplified cells [34,57]. In keeping with this, miR-17-92 has been shown to be a master effector of MYCN controlled down regulation of several tumour suppressor genes [15,35,58]. In collaboration with Versteeg et al., our team performed an epic search for a presumed tumour suppressor gene through positional cloning of a de novo translocation 1;17 in a NB patient [6]. The cloning was hampered by the complex and repetitive nature of the 1p36 breakpoint region, which forced us to clone the gene through mapping of the 17q21 breakpoint. Ultimately, a new gene coding for a presumed tumour suppressor gene was identified, coined NBPF1 for NB breakpoint family member 1, which forms a family of recently duplicated genes located in segmental duplications on chromosome 1 [59]. Evidence was obtained that NBPF1 has a growth-suppressing role in colorectal carcinoma cells and more recently renewed attention for this gene family was triggered by the finding of a common CNV at chromosome 1q21.1 associated with NB with a previously unknown transcript showing high sequence similarity to several NB breakpoint family (NBPF) genes and representing a new member of this gene family (NBPF23) [60]. This particular transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and the expression level was strictly correlated with CNV state in NB cells. For neither NBPF1 nor NBPF23, the pathogenetic role in NB is currently clear. In a recent study, Vandepoele et al. [59] described interaction between Chibby (CBY1), NBPF1 and clusterin (CLU), the latter also being proposed as a NB tumour and metastasis suppressor gene [61]. Of further interest, clusterin is one of the miR-17-92 regulated tumour suppressor genes thus again linking MYCN with down regulation of tumour suppressors in NB and further underlining the possible role of the enigmatic NBPF1 gene. Another major target of ongoing searches for a tumour suppressor is the long arm of chromosome 11. We and others have provided evidence for CADM1 (IGSF4/TSCL1) as haploinsufficient tumour suppressor gene [62,63]. CADM1 is a transmembrane glycoprotein that forms dimers both within a cell and between adjacent cells to promote cell–cell adhesion [64] and interacts with the actin cytoskeleton affecting cell motility [65]. CADM1 has been reported as tumour suppressor gene in other tumour entities. Thus far, no recurrent inactivating mutations have been described but high MYCN/MYC expression in aggressive NBs may cause BMI1 mediated down regulation of CADM1 (see also above). Interestingly, mutations in CADM2 were detected in prostate cancer as revealed in a recent genome sequencing effort [66] further underlining the

potential role of these proteins in cancer development. Other candidate tumour suppressors residing in the gene rich 11q23–24 distal segment are MLL and CHK1. MLL is a histone-lysine Nmethyltransferase gene and a frequent fusion partner in leukemia. Family members MLL2 and MLL3 are mutated in 16% of medulloblastoma patients [67]. CHK1 is a known haploinsufficient tumour suppressor gene [68] and is a cell cycle checkpoint protein that is recently identified through an RNAi screen as a therapeutic target for NB [69]. In addition to the above-mentioned genetic defects implicated in NB cells, epigenetic modifications can also be expected to contribute to the tumour phenotype. Several abnormally methylated genes have been described in NB encoding proteins that are involved in various cellular pathways, including apoptosis (several genes, involved in the death-receptor pathway with central molecule the protease CASP8 [70–76]), the cell cycle (the cyclindependent kinase inhibitor CDKN2A [73], the cyclin CCND2 [70,77] and stratifin (SFN) [78,79]), differentiation (the retinoic acid receptor RARB2 [77]), and invasion and metastasis (the cadherin CDH1 [75]). Some significant associations of epigenetic signatures with clinical and biological parameters have been found. Besides single gene associations, there are several ‘DNA-methylation signatures’ described, where a combination of a limited number of DNA-methylation markers is associated with stage, MYCN amplification, age at diagnosis, risk and event-free or overall survival [70,71,74,77–81]. Recently, the presence of methylation markers in serum of NB patients has been described as an indicator of prognosis and therapy efficacy [76,82]. These findings have demonstrated the clinical relevance of promoter hypermethylation in the pathogenesis of NB. Global profiling of gene promoter hypermethylation to identify genome-wide aberrantly methylated genes is therefore warranted in order to further understand NB pathogenesis and to identify new prognostic methylation markers for the optimization of therapeutic strategies.

5. Germline predisposition to neuroblastoma Germline mutations in familial forms of NB have been identified in genes PHOX2B and ALK [15,16,83,84]. Germline missense or frameshift mutations in the homeodomain of PHOX2B account for a minority of familial NB tumours but given the central role of this transcription factor in differentiation of autonomic neurons further studies of the role of this gene in NB are warranted in order to understand the exact contribution of mutant PHOX2B to tumour development. For the moment, a classical tumour suppressor role is unclear as PHOX2B mutated (missense and frameshift) tumours do not show loss of the second allele and heterozygous deletion of the locus does not predispose to tumour development. Also, a recent study showed high expression levels of PHOX2B in sympathetic neuronal progenitors in TH-MYCN mice and suggested a role as lineage survival oncogene [85]. The majority of familial NB cases can be explained by the presence of activating ALK mutations [16] and will be discussed in further detail in this issue (Mossé et al., in this issue). In addition to germline mutations, genome wide association studies identified genetic polymorphisms that are associated with a predisposition to sporadic NB [86–88]. In total, 7 predisposition loci were identified through SNP genotyping of a large cohort of patients and controls. A first locus encompassed two overlapping genes, FLJ22536 and FLJ44180. Homozygosity for these risk alleles was significantly associated with high-risk features including MYCN amplification, metastases and poor survival. In a subset of patients with high risk disease, several SNPs located within the BARD1 gene were identified. This gene is assumed to play an impor-

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tant role in the normal function of the tumour suppressor gene BRCA1 and risk alleles are associated with over-expression of an oncogenic isoform of the BARD gene. In an expanded study, yet another predisposition locus at 11p15, within the LIM domain only 1 (LMO1) gene, was detected and tumour promoting capacities in NB could be shown to this gene [87]. Finally, upon enrichment of the GWAS study for low-risk NB cases four more loci were identified. In addition to SNPs, also CNVs were investigated as a normal source of variation linked to tumour predisposition. As already pointed out above, increased risk for NB was linked to a CNV at 1q21 encompassing the NBPF23 gene. An excellent and more in depth review of genetic predisposition in NB is available from [89].

6. Prognostic mRNA signatures Similar as in many other tumour entities, access to platforms for genome wide transcriptome analysis was exploited in order to establish prognostic mRNA signatures. Several gene-expressionbased classifiers for improved outcome prediction were established [90–98]. Overall, many of these classifiers showed a similar performance but remarkably the number of overlapping genes between each of the classifiers was rather limited. A similar observation has been made for other prognostic classifiers and in particular for breast cancer this has been thoroughly investigated in order to exclude influence of external parameters such as type of platform, RNA isolation protocol, etc. [99]. One possible drawback of transcriptome analysis on limited number of samples is data-overfitting as the number of measured features is much larger than the number of cases studied. To circumvent this possible pitfall, we have used an innovative data-mining approach to establish a 42-gene expression signature using expression profiles of four published microarray studies [100]. This classifier was validated on three independent and unpublished datasets. The gene set was extended by an extensive literature screening for single candidate prognostic genes, yielding a list of 59 potential prognostic genes in total. By measuring gene expression using reverse transcription quantitative PCR, this set of 59 genes was profiled in 579 NBs of all risk and age groups, the largest series so far [101]. This showed that the signature was a strong independent risk predictor and could identify patients with increased risk in the current risk groups. This survival classifier will definitely help to identify patients with increased risk in the current treatment groups and to make a better choice of risk-related therapy. For example, low-risk patients with high molecular risk might benefit from more aggressive treatment protocols, whereas more intensive follow-up and new experimental therapies might be considered for high-risk patients with high molecular risk. In addition to a mRNA prognostic signature, we were able to establish a miRNA prognostic classifier in the largest NB miRNA expression study so far, including more than 500 NB patients [92,102,103]. We established and validated a robust miRNA classifier, able to identify NB patients with increased risk for adverse outcome. Some of the possible benefits of miRNA based signatures are their lower sensitivity to RNA degradation opening the possibility for implementation on paraffin embedded tumour material as well as serum detection for non invasive molecular risk assessment. Having a molecular risk classifier established, the major challenge now is to implement this in the clinic. The recently introduced International Neuroblastoma Risk Group (INRG) staging system for clinical decision making includes DNA index, MYCN oncogene amplification status and the presence of copy number aberrations at chromosome arm 1p, 11q and 17q together with patient age, tumour stage, histology and grade of differentiation [104]. The advantage of the selected biological markers is that they can be established by well-validated assays on stable DNA. Introducing molecular risk signatures based on mRNA analysis poses new

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challenges in tumour sampling and processing. Ideally, a primary tumour biopsy should be available, which may not be necessarily the case for high stage metastatic tumours, unless enriched tumour cells isolated from bone marrow biopsies can be used [105]. Further processing of samples should be done according to standard operating procedures with proven efficacy. Although this may seem challenging for the moment, tumour sampling and in particular mRNA profiling may also become critically important for future more in depth analysis of perturbed signaling as a prelude to patient tailored molecular therapies (see below). 7. The neuroblastoma cell-of-origin transcriptome Most of the gene expression studies in NB have not fully exploited the available transcriptome data in order to unravel the underlying molecular pathogenesis. In order to facilitate this aim, we assumed that access to the transcriptome profile of the presumed NB progenitor cells would provide extra power for data mining efforts. To achieve this aim we isolated clusters of human neuroblasts from adrenal medullas of human fetuses. To this purpose, a protocol was optimized in order to procure intact mRNA followed by linear RNA amplification and gene expression profiling [106]. In order to verify whether the proper cells were microdissected, expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts was evaluated. The similarities in expression profile between normal neuroblasts and malignant NBs provided strong evidence for the neuroblast origin hypothesis of NB. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus NB tumours. This approach efficiently sifted out genes previously reported in NB expression profiling studies. Most importantly, it also highlighted a series of genes and pathways previously not mentioned in NB biology but that are now assumed to be involved in NB pathogenesis, including ALK, PTPRD [107], CADM1, CDK4, CAMTA1, amongst others [43]. One of the tools that was used to pinpoint interesting candidate genes is our in-house developed neuroblastoma gene server [108]. This database and analysis tool contains manually curated lists of differentially expressed genes retrieved from all major published high-throughput NB gene expression profiling studies. The users data can be mapped against the database to explore and calculate the overlap with the existing gene lists. 8. Serial transcriptome analysis and cross-species integration Although the isolation of the fetal neuroblasts from a given gestation time of human embryos has further contributed to our insights in NB development, an important limitation of this approach is the absence of dynamic data in relation to the differentiation of these cells. Further mouse modeling using tissue and developmental time specific promoters followed by sorting of the cells of interest could partly resolve this issue as indicated by similar successful studies in pediatric brain tumours [109]. Also, in vitro culture or induction of mouse or human neural crest cells [110,111] and subsequent differentiation along the sympathetic neuronal lineage might generate richer and more dynamic data sets for further data mining and identification of critical components in development and NB oncogenesis. Another alternative to study tumour formation over time was exploited by Maris and co-workers using the TH-MYCN transgenic model in order to identify oncogenic drivers for therapeutic targeting [112]. To this purpose, the transcriptome of nine hyperplastic ganglia harvested at three time points and four tumour cohorts of progressively larger size in mice homozygous for the TH-MYCN

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transgene was determined. A total of 93 genes were identified with linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumours. Subsequent crossspecies integration identified 24 genes that were highly expressed in human MYCN-amplified NBs. Through further prioritization centromere-associated protein E (Cenpe) was selected as a molecular target and tested for the small molecular inhibitor GSK923295. This inhibitor caused inhibition of in vitro proliferation in NB cells and delayed tumour xenograft growth.

9. From mRNA to miRNA and beyond With more and more mRNA targets being detected for miRNAs, integrated regulatory mRNA/miRNA networks are being established at an increasing pace in normal developing and cancer cells [113,114]. Presently, aCGH, mRNA and miRNA profiles are available for more large cohorts of NBs. In addition, a growing set of mRNA and miRNA profiles are present or being generated for particular in vitro cellular model systems with perturbations of specific genes of interest. This unique resource is currently being explored in relation to ongoing research project of the different partners but will also be exploited in a broader context using the appropriate bio-informatic tools. One such tool is the miRNA body map (http://www.mirnabodymap.org) which allows, amongst others, to predict perturbed cellular functions based upon matching mRNA and miRNA expression. This might be used as a tool for generating hypotheses for further functional testing [102]. The recent emerging insights into NB oncogenesis and the identification of genes implicated in the disease are offering immediate possibilities for design of new therapies as illustrated by the ongoing evaluation of ALK inhibitors and also opens the way to further in vitro and in vivo modeling and generation of complex datasets including proteome profiles. Such multidimensional and cross species integrated information will be exploited using high end bio-informatic tools and systems biology approaches in order to unravel the various implicated perturbed signaling pathways and complex interactions and cross talk between critical nodes within these networks. Understanding such regulatory networks at play and in particular possible compensatory interactions may also be crucial for developing the appropriate therapeutic strategies and to anticipate to and design strategies against new forms of therapy resistance.

Acknowledgments We acknowledge the excellent editorial assistance of Anneleen Decock. K.D.P. is a postdoctoral fellow of the Research Foundation – Flanders (FWO). This work is further supported by the Belgian program of Interuniversity Poles of Attraction, initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming; Fund for Scientific Research (FWO grant number G.O198.08) and GOA (grant number 01G01910). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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