Abstract
BackgroundOne of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity. Although there is a great need for better clinical and biological markers to distinguish between tumours with different severity and to improve treatment, no clear-cut prognostic factors have been found. Also, no major NB tumour suppressor genes have been identified.MethodsIn this study we performed expression analysis by quantitative real-time PCR (QPCR) on primary NB tumours divided into two groups, of favourable and unfavourable outcome respectively. Candidate genes were selected on basis of lower expression in unfavourable tumour types compared to favourables in our microarray expression analysis. Selected genes were studied in two steps: (1) using TaqMan Low Density Arrays (TLDA) targeting 89 genes on a set of 12 NB tumour samples, and (2) 12 genes were selected from the TLDA analysis for verification using individual TaqMan assays in a new set of 13 NB tumour samples.ResultsBy TLDA analysis, 81 out of 87 genes were found to be significantly differentially expressed between groups, of which 14 have previously been reported as having an altered gene expression in NB. In the second verification round, seven out of 12 transcripts showed significantly lower expression in unfavourable NB tumours, ATBF1, CACNA2D3, CNTNAP2, FUSIP1, GNB1, SLC35E2, and TFAP2B. The gene that showed the highest fold change in the TLDA analysis, POU4F2, was investigated for epigenetic changes (CpG methylation) and mutations in order to explore the cause of the differential expression. Moreover, the fragile site gene CNTNAP2 that showed the largest fold change in verification group 2 was investigated for structural aberrations by copy number analysis. However, the analyses of POU4F2 and CNTNAP2 showed no genetic alterations that could explain a lower expression in unfavourable NB tumours.ConclusionThrough two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours. Four of the transcripts, CACNA2D3, GNB1, SLC35E2, and TFAP2B, have been observed in previous microarray studies, and are in this study independently verified. Our results suggest these transcripts to be markers of malignancy, which could have a potential usefulness in the clinic.
Highlights
One of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity
The transcript discriminating groups with the largest fold change in verification group 1 was POU4F2 (Table 2), showing an expression level of more than 1500 times lower (p = 0,011) in the unfavourable tumours compared to the favourable ones
Five tumours (4/5 unfavourable), did not express this gene at all (i.e. 25R9, 19R6, 9R9, 10R2, and 15R3). This absence of expression was verified in three rounds of individual quantitative PCR (QPCR) runs, and the Ct-values were set to 40 in these cases to enable calculations
Summary
One of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity. Neuroblastoma (NB) is the most common extracranial solid tumour in children and accounts for around 15% of all childhood cancer deaths It is a disease of the sympathetic nervous system and most often arises in the adrenal glands [1]. Among the most frequent changes, which are strongly associated with disease prognosis, are genomic amplification of MYCN (MNA), deletion of parts of chromosome arm 1p (del1p), partial deletion of 11q (del11q) and unbalanced gain of 17q. All these aberrations are correlated with poor outcome and clinical aggressiveness while whole chromosome gains or losses, and hyperdiploid/near-triploid cells define the more favourable tumour types [5]. ALK has been found to be altered in sporadic NB tumours through either mutations (approximately 10%) or amplifications (approximately 5%) [7,8,9,10,11]
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