Imperfect protection: NEPA at 35 years.

Background: Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands. Results: Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis. Conclusion: This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis. Published: 21 September 2006 Genome Biology 2006, 7:R84 (doi:10.1186/gb-2006-7-9-r84) Received: 6 July 2006 Revised: 17 August 2006 Accepted: 21 September 2006 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/9/R84 Genome Biology 2006, 7:R84 R84.2 Genome Biology 2006, Volume 7, Issue 9, Article R84 De Preter et al. http://genomebiology.com/2006/7/9/R84 Background Neuroblastoma is the most common and deadly extracranial solid childhood tumor, exhibiting remarkable variation in clinical presentation ranging from localized to highly metastatic disease. Despite multimodal therapies, survival rates for aggressive neuroblastomas are still disappointingly low. One possible approach to development of more efficient and less toxic therapies is to gain insight into the signaling pathways that are deregulated in neuroblastoma and to use this information in the design of molecular therapies. However, at present only two genes, namely MYCN and PHOX2B, have been directly linked to neuroblastoma development, although their exact role in oncogenesis is still unclear [1,2]. It is hoped that genome-wide gene expression studies will provide insights into the genes and molecular pathways that govern neuroblastoma pathogenesis. Thus far, no clear or consistent candidate genes or pathways have emerged from these analyses [3-5] (see Additional data file 3 for more references). Both for currently available expression data and forthcoming datasets, we anticipate that transcriptome information on the cells of origin of neuroblastoma (sympathetic nervous system progenitors) will be of crucial importance and could provide significant power on data mining strategies. The sympathetic nervous system is composed of sympathetic chain and truncus ganglia, paraganglia, and the adrenal gland. Ganglion cells (neuroblasts during development) are the major cell type of chain and truncus ganglia, and extraadrenal chromaffin cells form the paraganglia, whereas the adrenal gland is composed of adrenal chromaffin cells and, at least during development, sympathetic neuroblasts. The fate of the neuroblasts in the developing human adrenal gland is not clear; some or all may involute or mature as solitary intraadrenal neurons [6]. Evidence for the cellular origin of neuroblastoma is based on their occurrence in the adrenal gland or along the spinal cord in association with sympathetic ganglia, and on their neuroblastic phenotype that indicates that the tumor cells are derived from immature sympathetic nervous system cells of the ganglionic lineage [7]. Indeed, cells of adrenal neuroblastomas have neuroblastic morphology and do not express the adrenal chromaffin marker PNMT, but they share phenotypic characteristics with the immature sympathetic neuroblasts present as nests of cells in the developing adrenal gland. However, a small subset of neuroblastomas also contains cells with extra-adrenal chromaffin characteristics. In the present study we isolated and performed expression profiling of the human adrenal neuroblasts as they form monocellular structures during early fetal stages, which can be easily microdissected. In parallel, favorable and unfavorable neuroblastoma tumors were profiled on the same platform. Finally, our dataset was integrated in a meta-analytical data mining approach. Results Characterization, isolation, and gene expression profiling of fetal adrenal neuroblasts Prescreening of hematoxylin-eosin cryosections from 11 fetal adrenal glands demonstrated that large neuroblast clusters of more than 100 cells were predominantly found in adrenal glands at 19 and 20 weeks' gestational age (Figure 1a). To verify that these cell clusters indeed represent neuroblasts and to estimate the degree of intermingled chromaffin cells, cryosections were stained for the neuronal and chromaffin marker TH (tyrosine hydroxylase), the chromaffin marker CHGA (chromogranin A; which also has low expression in neuroblasts), and the neuronal markers BCL2 (B-cell CLL/lymphoma 2) and HNK1 (carbohydrate epitope) [8]. As shown in Figure 1, the clusters of neuroblastic cells stained positive for all markers and, in particular, these cells were positive for BCL2 and HNK1. The majority of chromaffin cells, identified by their strong CHGA and TH expression, were found to be scattered throughout the adrenal cortex (these cells coalesce and form large islands of chromaffin cells later during development), whereas a few cells were located in or adjacent to the neuroblast clusters. Neuroblast clusters and adjacent cortical cells (used as controls) were isolated using laser capture microdissection from stained cryosections from three different fetal adrenal glands (glands 1, 2 and 3, which were of gestational ages 20, 19 and 19 weeks, respectively) (Figure 2) and immediately lysed in RNA extraction buffer. In order to obtain a sufficient amount of good quality neuroblast RNA for oligonucleotide chip analyses, we applied a previously validated protocol for tissue sectioning, staining, and microdissection [9] (Additional data file 1(a)). By pooling different isolates of the same adrenal gland, between 2.5 and 15 ng total RNA could be obtained for each of the three neuroblast samples (Additional data file 1(b)). After two-round amplification and labeling of three neuroblast, three cortex, and 18 neuroblastoma RNA samples, hybridization was performed on HG-U133A Affymetrix oligonucleotide chips. Real-time polymerase chain reaction analysis of selected genes showed that there was no RNA amplification bias in the chip data (Additional data file 1(c)). Validation of the expression profile of fetal adrenal neuroblasts and cortex cells The expression profiles of the neuroblast and cortex samples were compared using the rank product nonparametric method, which is particularly suited for extracting significantly differentially expressed genes in a limited number of samples [10]. Two lists of 156 and 86 unique genes were established with significantly higher expression in neuroblast and adrenal cortex cells, respectively (multiple testing corrected P < 0.01; Additional data file 2). Gene Ontology (GO) analysis identified those classes of genes that are significantly over-represented in the cell specific gene lists (P < 0.01; Table 1). As expected, the neuroblast gene list is enriched for genes that are involved in catecholamine metabolism, neurogenesis Genome Biology 2006, 7:R84 http://genomebiology.com/2006/7/9/R84 Genome Biology 2006, Volume 7, Issue 9, Article R84 De Preter et al. R84.3