Abstract
BackgroundNeuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. However, by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. In the current study we characterized these different subgroups in more detail, with a specific focus on the fourth divergent tumour subgroup (r4).MethodsExpression microarray data from four international studies corresponding to 148 neuroblastic tumour cases were subject to division into four expression subgroups using a previously described 6-gene signature. Differentially expressed genes between groups were identified using Significance Analysis of Microarray (SAM). Next, gene expression network modelling was performed to map signalling pathways and cellular processes representing each subgroup. Findings were validated at the protein level by immunohistochemistry and immunoblot analyses.ResultsWe identified several significantly up-regulated genes in the r4 subgroup of which the tyrosine kinase receptor ERBB3 was most prominent (fold change: 132–240). By gene set enrichment analysis (GSEA) the constructed gene network of ERBB3 (n = 38 network partners) was significantly enriched in the r4 subgroup in all four independent data sets. ERBB3 was also positively correlated to the ErbB family members EGFR and ERBB2 in all data sets, and a concurrent overexpression was seen in the r4 subgroup. Further studies of histopathology categories using a fifth data set of 110 neuroblastic tumours, showed a striking similarity between the expression profile of r4 to ganglioneuroblastoma (GNB) and ganglioneuroma (GN) tumours. In contrast, the NB histopathological subtype was dominated by mitotic regulating genes, characterizing unfavourable NB subgroups in particular. The high ErbB3 expression in GN tumour types was verified at the protein level, and showed mainly expression in the mature ganglion cells.ConclusionsConclusively, this study demonstrates the importance of performing unsupervised clustering and subtype discovery of data sets prior to analyses to avoid a mixture of tumour subtypes, which may otherwise give distorted results and lead to incorrect conclusions. The current study identifies ERBB3 as a clear-cut marker of a GNB/GN-like expression profile, and we suggest a 7-gene expression signature (including ERBB3) as a complement to histopathology analysis of neuroblastic tumours. Further studies of ErbB3 and other ErbB family members and their role in neuroblastic differentiation and pathogenesis are warranted.
Highlights
Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups
By expression analysis of histopathology categories (i.e. NBs, GNBs, and GNs) we found the r4 subgroup to show an identical expression profile to GNB/GN types, and overexpression of ErbB3 was confirmed at the protein level in GN tumours
Thirty-seven tumour cases from three studies were pre-processed in two separate data sets, and both data sets were divided into four r-subgroups based on rules according to the previously described 6-gene signature (6-GeneSig, Additional file 1) [12]
Summary
Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. Neuroblastoma exhibit an extreme clinical and biological heterogeneity, and patients are assigned to risk groups based on several criteria including stage [2,3], age [4], histological category and grade of tumour differentiation (histopathology) [5], the status of the MYCN oncogene [6], chromosome 11q status [7], and DNA ploidy [8] as the most highly statistically significant and clinically relevant factors [9]. 7-9% of sporadic NB cases show inherent ALK mutations [22,23], and ALK overexpression, both in its mutated and wild type form, is demonstrated to define a poor prognosis in NB patients [24] In relation to this our previous findings suggests the Type 2A (r2) and Type 2B (r3) subgroups, which both display high ALK expression, to be driven by the ALK pathway. The r4 subgroup displaying low expression of all six genes of the signature, is suggested to be driven by an alternative oncogenesis pathway
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