1. ¹Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
2. ²Department of Pediatric Hemato-Oncology, Ghent University Hospital, Ghent, Belgium
3. ³Molecular Oncology Unit, Centre Léon Bérard, Lyon, France
4. ⁴Cancer Research Unit, University of Newcastle, Newcastle upon Tyne, United Kingdom
5. ⁵Department of Child Health, University of Newcastle, Newcastle upon Tyne, United Kingdom
6. ⁶Department of Clinical Genetics, Erasmus University, Rotterdam, The Netherlands
7. ⁷Department of Pediatrics (Hemato-Oncology section), Cliniques Saint-Joseph Espérance, Montegnée, Belgium

Correspondence: Frank Speleman, Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, 9000 Ghent, Belgium. e-mail: franki.speleman@rug.ac.be; fax: 32-(0)9-2404970.

Accepted 11 December 2001.

Abstract

Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Consequently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantitative PCR (Q-PCR) assay as an alternative for time-consuming Southern blot analysis (SB), and as a second independent technique in parallel with fluorescence in situ hybridization (FISH) analysis. Advantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two different detection chemistries i.e., a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e., the kinetic method and the comparative C_T method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined using a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis.