Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay.
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.
De Preter
Katleen
K
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
Speleman
Frank
F
Combaret
Valérie
V
Lunec
John
J
Laureys
Geneviève
G
Eussen
Bert H J
BH
Francotte
Nadine
N
Board
Julian
J
Pearson
Andy D J
AD
De Paepe
Anne
A
Van Roy
Nadine
N
Vandesompele
Jo
J
D053487:DEAD-box RNA Helicases; D005784:Gene Amplification; D018628:Gene Dosage; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009363:Neoplasm Proteins / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics / Q000473:pathology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D020365:RNA Helicases / Q000235:genetics*; D015203:Reproducibility of Results; D016019:Survival Analysis
C000715470:MAS1 protein, human; C117339:NBAS protein, human; D009363:Neoplasm Proteins; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases