Aneuploidy is ubiquitous in cancer and has been linked to cell proliferation, cell differentiation and prognosis. The karyotypes of most tumours are aneuploid, meaning that chromosomes, which carry thousands of genes, are structurally rearranged, duplicated, broken or entirely missing.
Gain of chromosome 17 is one of the most frequent genetic abnormalities observed in NBTs, and may involve either the entire chromosome or partial gain of the distal segment 17q21-qter [17
]. Unbalanced translocations, characteristic of near diploid NBTs or tumours with structural rather than numerical chromosome aberrations, are thought to arise from DNA double strand breaks repaired erroneously, suggesting an impaired DNA maintenance or repair pathway [18
]. On the other hand, abnormalities in the mitotic segregation of chromosomes are thought to underlie the numerical aberrations characteristic of near-triploid, good prognostic, NBTs. Both mechanisms define the type of aneuploidy behind each of the subgroups of NBTs, determining the kind of genetic aberrations as well as the biological behaviour of each NBT subtype.
Gene expression profiling of NBTs with different ploidy status, near-triploid or near-diploid/tetraploid, enabled us to identify distinct expression profiles associated with each subgroup. Interestingly, a statistically significant proportion of genes shown to be differentially expressed mapped to chromosomes described to be recurrently altered in NBTs, chromosomes 1 and 17 [17
]. Chromosomal region specificity was also observed for these differentially expressed genes since the majority spread predominantly over the chromosomal regions 1p36-p22.1 and 17p13-17q21. Besides, over 90% of these genes displayed higher expression levels in near-triploid tumours. Only two genes mapping to chromosome 17, MAC30 and NME1, exhibited a higher expression level in near-diploid/tetraploid NBTs. MAC30 gene encodes for a meningioma-associated protein, highly expressed in several types of tumours, but, with unknown clinicopathological and biological significance. The product of the NME1 gene, the nm23A protein, is a nucleoside diphosphate kinase, whose expression has been related to cell proliferative activity [19
]. Whereas reduced expression of NME1 is associated with a high potential for metastasis in some tumour types, like breast cancer and melanoma, its expression is increased in aggressive NBTs [20
Genome array CGH, together with FISH and Q-PCR results, confirmed the association of specific chromosomal abnormalities with each of the NBTs subgroups. Therefore, it is not unreasonable to assume that these specific chromosomal alterations are associated with the observed gene expression profiles. The highly significant and strikingly persistent chromosomal localization of the differentially expressed genes made us hypothesize about which transcriptional regulation mechanisms can underlie these gene expression patterns. As a result of aneuploidy, cells possibly produce imbalanced expression of large sets of genes that are amplified or lost. Such gross imbalances would inevitably disrupt critical cellular circuits and destabilize regulatory pathways and cellular structures. It has been assumed that gene dosage effects may play a role in the pathogenesis of malignant diseases. Variations of the transcriptome due to alterations of the gene dosage have been described in vitro
], in vivo
] and in human pathologies such as trisomies 13 and 21 [23
]. In our hands, when comparing gene expression levels with gene copy number of a set of differentially expressed genes located at chromosomes 1p36 and 17q13-q21, we observed a concordance between copy number and mean expression values in all those analyzed genes that displayed in the microarray analysis higher expression levels in near-triploid NBTs. In contrast, NME1 gene, as from microarray results, showed low expression values, close to the disomic reference sample expression, in near-triploid NBTs, and high fold increase in mRNA levels in near-diploid/tetraploid cases. NME1 gene has been identified as one of the MYCN targets. Correlation between MYCN overexpression and upregulation of NME1 expression has been reported both in NBTs and neuroblastoma cell lines [24
]. In our experience, all MYCN amplified NBTs, displaying MYCN overexpression, as well as near-diploid cases with increased copy number of chromosome 17q, showed high NME1 expression levels. However, NME1 overexpression was also observed in 2 near-diploid MYCN single copy cases, with low MYCN expression and no 17q gain. This suggests that in NBTs NME1 gene expression is only partly dependent on gene copy number and MYCN expression, and therefore implies the existence of other mechanisms of NME1 transcriptional regulation.
Recently, we reported that clonal ploidy heterogeneity is present in virtually every single loco-regional, near-triploid NBT, and detected the existence of clonal DNA content heterogeneity and evolution [25
]. In this report our results underscore the clonal heterogeneity of all NBTs, with a marked complexity in the near-diploid/tetraploid tumours. Furthermore, clonal variations reflected in the array CGH plots as copy-number alterations with varying log2
values, could unveil the presence of subpopulations emerged during tumour development. These cellular subpopulations are likely to be the cause of the high cell heterogeneity also observed in the FISH analyses. These findings are important in emphasizing the cellular heterogeneity and karyotypic complexity (aneuploidy) generally associated with malignant tumours, but need a more detailed understanding of their significance.