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In Reply:

Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kamigyo-ku, Kyoto, Japan

Akira Nakagawara

Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan

We appreciate the encouraging comments about our article¹ by Dr Combaret et al. We realized that our statement that Combaret et al² did not use a reference gene is incorrect. In fact, their assay was based in part on duplex real-time polymerase chain reaction with a reference gene Ribonuclease P RNA component H1, which is on chromosome 14. Thus, their method was similar to ours, except that we used a reference gene on chromosome 2 (NAGK, 2p12). Using a reference gene on the same chromosome as MYCN (2p24) is better because then, the ratio of MYCN to the reference gene gives the MYCN copy number, even if there is a change in the number of copies of chromosome 2. Changes in chromosome numbers are common in neuroblastomas. Dr Combaret et al say that they recently determined MYCN status in 104 neuroblastoma cases by using a reference gene on chromosome 2 (proIL1ß, 2q13-24) and had two false negatives and two false positives. However, the duplex real-time polymerase chain reaction method is so precise that it can detect a gain of only a few extra copies of a gene as well as amplification,^1,3 and so their false negatives and false positives are puzzling. It would help to know the distribution of MYCN/proIL1ß ratios in their MNA and nonMNA groups, and the cutoff value that they used. The false-negative cases might be caused by contamination of serum DNA with cellular DNA. Recently, we found that contaminating WBCs could be removed either by an additional high-speed centrifugation (15,000 x g for 10 minutes) or by filtration with a 0.45 µm filter unit (MILLEX-HA; Millipore, Cork, Ireland). The false-positive cases may be heterogeneous cases, as they speculated. A fluorescence in situ hybridization examination of tumor sections of the false-positive cases would determine whether these cases had focal lesions with MYCN amplification. A better understanding of the causes of false-negative and -positive cases is needed to open the way to using a serum MYCN assay as a diagnostic test.

Authors' Disclosures of Potential Conflicts of Interest

The authors indicated no potential conflicts of interest.

REFERENCES

1. Gotoh T, Hosoi H, Iehara T, et al: Prediction of MYCN amplification in neuroblastoma using serum DNA and real-time quantitative polymerase chain reaction. J Clin Oncol 23:5205-5210, 2005[Abstract/Free Full Text]

2. Combaret V, Audoynaud C, Iacono I, et al: Circulating MYCN DNA as a tumor-specific marker in neuroblastoma patients. Cancer Res 62:3646-3648, 2002[Abstract/Free Full Text]

3. Morowitz M, Shusterman S, Mosse Y, et al: Detection of single-copy chromosome 17q gain in human neuroblastoma using real-time quantitative polymerase chain reaction. Mod Pathol 16:1248-1256, 2003[CrossRef][Medline]

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Related Correspondence

• Circulating MYCN DNA Predicts MYCN-Amplification in Neuroblastoma
  Valérie Combaret, Christophe Bergeron, Rosa Noguera, Isabelle Iacono, and Alain Puisieux
  JCO 2005 23: 8919-8920 [Full Text]

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