The findings of this study have demonstrated that there are significant ultrastructural morphological differences between MYCN-amplified and non-MYCN-amplified neuroblastomas, including a statistically significant reduction in diameter of NT, and significantly more NSG and NT in the cell body of MYCN- amplified tumours. These differences may be explained by the finding that MYCN amplified cells appear less differentiated, and therefore the NSG and NT in the cell bodies have not yet extended into cell processes. Additionally, since MYCN-amplified tumours appear less differentiated, the smaller size of their NT may represent tubules in these tumours being less well-developed.
MYCN is involved in the expression of many target genes, which regulate a range of cellular processes, including cell growth, proliferation, differentiation, and apoptosis . MYCN is primarily an embryonic developmental gene, whose normal expression is specifically restricted (in humans and mice) to certain tissues in the developing embryo. MYCN is specifically found in the neuroepithelium, and also in the developing lung, heart kidney and intestine [7, 20]. Primary functions of MYCN are proposed to be related to control of cell proliferation and enabling cells to remain in relatively undifferentiated states . Over expression of MYCN in the neural crest results in an increased generation of neurons but down regulation of MYCN is required for these neurons to become terminally differentiated [21, 22]. In combination, these findings suggest that MYCN signalling is important in maintaining cells in an undifferentiated state and that down regulation of MYCN can lead to neuronal differentiation . The present ultrastructural findings support these suggested roles since MYCN amplified cells had a morphologically less differentiated appearance, with associated morphometric changes.
Increased MYCN expression keeps cells in an undifferentiated and proliferative state . Therefore, the hypothesis has been raised that blocking MYCN expression or its action could lead to less aggressive tumours, and lead to new therapies for high-risk patients . Differentiation as a target for therapy has been investigated and several groups are attempting to down regulate MYCN expression as a treatment in high-risk MYCN amplified neuroblastoma. Several agents have been investigated, of which 13-cis-retinoic acid shows the most promise, showing neurite outgrowth and differentiation of human neuroblastoma cells in vitro and in vivo. These results are in accordance with the current ultrastructural findings, that lower-risk tumours show substantial neurite outgrowth and were morphologically more differentiated, despite being within the poorly differentiated light microscopy subgroup of neuroblastomas.
The present study demonstrated a significant difference in both the size and number of neurotubules within MYCN amplified tumours compared to the non-MYCN amplified tumours, which has not been reported previously. Microtubules and intermediate filaments, of which neurotubules are an example, are involved in intracellular transport and it can be hypothesised that if a cell has a more patent network of tubules and filaments then drug transport may occur more efficiently. This remains entirely speculative but may offer a possible explanation regarding the association of MYCN amplified tumours with adverse outcome.
Whilst markers of differentiation, such as dense core neurosecretory granules and neuritic processes, may be identified, and the neuropil seen on light microscopy is identified as neuritic processes with NSG, poorly differentiated neuroblastomas have previously been reported to generally show no major ultrastructural variations related to outcome not identified by light microscopy, although there may be an association between low numbers of NSG and adverse clinical course [10–13].