N-MYC amplification in neuroblastomas is of utmost interest with regards to the prognosis of disease. In fact, in clinical practice, N-MYC gene expression correlates with both advanced disease stage and rapid tumor progres-sion.
Several methods have been used for the detection of N-MYC detection mainly based on Southern or dot blot, quantitative PCR and fluorescent in situ hybridization techniques. However, the use of most PCR methods is restricted by the fact that end-point measurements are used for quantitation. Therefore, Raggi et al. introduced a TaqMan real-time base method for the determination of N-MYC amplification in neuroblastomas. The authors demonstrate a precise assay with an interassay coefficient of variation of 13% and an intraassay coefficient of variation of 11%. The threshold cycle for the detection of N-MYC correlates in an inverse linear way with the logarithm of the input of genomic DNA molecules. There is a good linear relationship between the N-MYC amplification measured by TaqMan real-time PCR and competitive PCR. Using Kaplan-Meier survival curves, the authors showed that the amplification of N-MYC as assessed by TaqMan real-time PCR is closely linked to cumulative survival as this has already been demonstrated with several other techniques for the quantitation of N-MYC amplification.
Similar results were obtained by others using the LightCycler approach for the quantitation of N-MYC amplification.1-15-1
Lately, the application of real-time PCR has focused on the detection of MRD of neuroblastoma. A number of groups used tyrosine hydroxylase—the key enzyme for the synthesis of catecholamines as a marker for the detection of neuroblastoma cells in the blood and bone marrow.
One group depicts that as few as 70 transcripts per milliliters can be measured. There were no false-negative samples in control samples. Similar results with regards to the use of tyrosine hydroxylase for the detection of MRD were reported by another group, which described the sensitivity of the assay as 1:106 white blood cells. Again, real-time PCR was found to be more sensitive than qualitative PCR in the detection of MRD. However, one pitfall in the use of tyrosine hydroxylase might be its association with a higher degree of differentiation of the neuroblastoma. Therefore, less differentiated tumors might be detected at higher threshold levels, or could even be missed.
A different surrogate parameter for the quantitation of MRD in neuroblastoma patients is GD2 synthase—the key enzyme for the synthesis of p1,4-N-acetylgalactosa-minyltransferase. The measurements proved to have clinical relevance in evaluating adjuvant therapy with anti-GD2 antibodies for bone marrow purging in neuro-blastoma by detecting a subclinical level of tumor cells in bone marrow aspirates and was predictive of long-term outcome.
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