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 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, beta-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.


CONCLUSIONS
The assay is rapid and reproducible and does not require any post-PCR analytical procedure.
Raggi
C C
CC
Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy.
Bagnoni
M L
ML
Tonini
G P
GP
Maggi
M
M
Vona
G
G
Pinzani
P
P
Mazzocco
K
K
De Bernardi
B
B
Pazzagli
M
M
Orlando
C
C
D002138:Calibration; D002648:Child; D002675:Child, Preschool; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D009447:Neuroblastoma / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase; D013997:Time Factors
D004273:DNA, Neoplasm; D016271:Proto-Oncogene Proteins c-myc; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase