Neuroblastoma with the TaqMan Detection System

Claudia Casini Raggi¹, Maria Letizia Bagnoni¹, Gian Paolo Tonini³, Mario Maggi², Giovanna Vona¹, Pamela Pinzani¹, Katia Mazzocco³, Bruno De Bernardi⁴, Mario Pazzagli¹ and Claudio Orlando^1,a

1.        Clinical Biochemistry

2.        Andrology Units, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy.

3.        Unit of Solid Tumor Biology, Advanced Biotechnology Centre

4.        Giannina Gaslini Children’s Hospital, 16132 Genoa, Italy.

Background: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients.

Methods: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqMan^TM reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, ß-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA.

Results: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0.987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (<9; P = 0.02) and high (9; P = 0.03) oncogene amplification.

Conclusion: The assay is rapid and reproducible and does not require any post-PCR analytical procedure.

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