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1
Clinical Biochemistry and
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.
a Address correspondence to this author at: Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. Fax 39-55-4377290; e-mail c.orlando{at}dfc.unifi.it
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 TaqManTM 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.
The following articles in journals at HighWire Press have cited this article:
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  T. Gotoh, H. Hosoi, T. Iehara, Y. Kuwahara, S. Osone, K. Tsuchiya, M. Ohira, A. Nakagawara, H. Kuroda, and T. Sugimoto Prediction of MYCN Amplification in Neuroblastoma Using Serum DNA and Real-Time Quantitative Polymerase Chain Reaction J. Clin. Oncol., August 1, 2005; 23(22): 5205 - 5210. [Abstract] [Full Text] [PDF]
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 L. H. J. Lambooy, C. E. M. Gidding, L. P. van den Heuvel, C. A. Hulsbergen-van de Kaa, M. Ligtenberg, J. P. M. Bokkerink, and R. A. De Abreu Real-Time Analysis of Tyrosine Hydroxylase Gene Expression: A Sensitive and Semiquantitative Marker for Minimal Residual Disease Detection of Neuroblastoma Clin. Cancer Res., February 1, 2003; 9(2): 812 - 819. [Abstract] [Full Text] [PDF]
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 J. M. Nigro, M. A. Takahashi, D. G. Ginzinger, M. Law, S. Passe, R. B. Jenkins, and K. Aldape Detection of 1p and 19q Loss in Oligodendroglioma by Quantitative Microsatellite Analysis, a Real-Time Quantitative Polymerase Chain Reaction Assay Am. J. Pathol., April 1, 2001; 158(4): 1253 - 1262. [Abstract] [Full Text] [PDF]
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 J. Wilhelm, M. Hahn, and A. Pingoud Influence of DNA Target Melting Behavior on Real-Time PCR Quantification Clin. Chem., November 1, 2000; 46(11): 1738 - 1743. [Abstract] [Full Text] [PDF]
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