Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system

Background: Neuroblastoma is the most common extracranial malignant solid t umor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers wi th 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 an real-time quantitative PCR with the TaqMan(TM) reaction. The degree of MYCN amplification was derived from the ratio of t he MYCN oncogene and the single-copy reference gene, beta-actin. The absolu te abundance of these two genes in tumor sample DNA was obtained by extrapo lation 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). MYC N amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplifi cation is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than pat ients with low (<9; P = 0.02) and high (greater than or equal to 9; P = 0.0 3) oncogene amplification. Conclusion: The assay is rapid and reproducible and does not require any po st-PCR analytical procedure. (C) 1999 American Association for Clinical Che mistry.