ABSTRACT
Background: Amplification of the MYCN (myc myelocytomatosis viral related oncogene, neuroblastoma derived) gene in neuroblastoma is associated with a poor prognosis. Methods for estimating MYCN gene copy number that are based on pooled cells do not address copy number heterogeneity at the cell level and can underestimate or even miss amplification. MYCN copy number can be directly assessed by fluorescence in situ hybridization, but evaluation of tissue histology is difficult if not impossible. Objective: This paper reviews chromogenic in situ hybridization (CJSH) as it applies to the MYCN gene in neuroblastoma. We compare this technique to other methods for determining gene copy number and highlight the advantages of CISH. Methods: We have developed a chromogenic method for in situ hybridization (CISH) that enables us to determine MYCN copy number on an individual cell basis. This technique uses light microscopy on routine paraffin sections, and therefore allows simultaneous assessment of tumour histology. Results: In a previous study, CISH identified 100 % of the cases that were known to be amplified by other techniques and proved to be more sensitive than Southern blotting or the quantitative DNA polymerase chain reaction. The MYCN copy number is generally believed not to vary within a tumour, nor between tumour samples, including primary vs. metastases, and pre-and post-treatment specimens. However, we found heterogeneity from cell to cell, with ~30 % of amplified tumours showing >50 % variation in MYCN copy between cells. Conclusion: For detection of gene amplification, CISH has all the advantages of FISH but in addition, needs no special microscopy or image capturing systems, and preparations are permanent. In the case of neuroblastoma, CISH has disclosed considerable heterogeneity in MYCN copy number between cells in a tumour. Heterogeneity reflects different tumour clones and its role has been under-recognized in neuroblastoma biology. Additional studies are needed to investigate the significance of tumour heterogeneity in neuroblastoma, and whether the aggressive (i.e., MYCN-amplified) clones are more likely to metastasize, survive treatment modalities, and ultimately kill the patient.