Quantification of MYCN, DDX1, and NAG Gene Copy Number in Neuroblastoma Using a Real-Time Quantitative PCR Assay

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 timeconsuming 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.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

“…Moreover, during the evaluation process, we managed to visually discern the concentration of MYCN, facilitating its semi-quantification without the need for densitometry. Consistent with the results of a previous study (8), our results from the absolute and relative real-time quantification of MYCN from tumor samples were identical. Comparison of real-time quantitative PCR with SQ-PCR demonstrated that both assays perform equally well in determining MYCN molecule number and that the SQ-PCR method defined MYCN molecule number with an accuracy approaching that of real-time quantitative PCR.…”

“…We also evaluated the relative quantification power of the test by calculating the MYCN molecules based on the observed C T values. This was done by transforming the difference in C T values between the tumor sample and the calibrator to a copy number ratio (8).…”

International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee

“…Frequently, adjacent or more distantly located genes, such as DDX1, NAG, and ALK, are coamplified with MYCN, but amplification events in the absence of MYCN amplification are rare. Whether these coamplifications have a prognostic impact awaits clarification (Manohar et al, 1995;Squire et al, 1995;George et al, 1997;De Preter et al, 2002;Weber et al, 2004).…”

Expression of aspartyl beta-hydroxylase and its clinicopathological significance in hepatocellular carcinoma

“…The real-time RT-PCR cycle started with the initial denaturation at 951C for 10 min, followed by 45 cycles of denaturation at 951C for 10 s, annealing at 611C for 10 s, and elongation at 721C for 10 s. As an internal quantitative control of the gene expression, GAPDH gene expression was determined as reported previously. [19][20][21] The aspartyl beta-hydroxylase and GAPDH gene expressions of all cDNA samples were determined by fluorescence from SYBR green using the Light Cycler software Version 3.5 (Roche Diagnostics), and the ratios of aspartyl beta-hydroxylase and GAPDH gene expressions represented the normalized relative levels of aspartyl betahydroxylase expressions. 19,22 A no-template negative control was also included in each experiment.…”