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Journal of Clinical Oncology, Vol 23, No 34 (December 1), 2005: pp. 8919-8920 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.04.0170
Circulating MYCN DNA Predicts MYCN-Amplification in NeuroblastomaMolecular Oncology Unit, Centre Léon Bérard, Lyon, France
Department of Pediatry, Centre Léon Bérard, Lyon, France
Department of Pathology, Medical School, University of Valencia, Valencia, Spain
Molecular Oncology Unit, Centre Léon Bérard, Lyon, France To the Editor: We read with great interest the article by Gotoh et al published in the August 1, 2005, issue of the Journal of Clinical Oncology.1 The authors report on an analysis of serum DNA by real-time quantitative polymerase chain reaction (PCR) to predict MYCN amplification in neuroblastoma (NB). Their results are in fair agreement with our previous report of high levels of MYCN DNA in the peripheral blood of patients with MYCN amplified NB.2 Gotoh et al highlight their data by pointing out differences in the experimental procedures used in the two studies. In our initial work,2 we performed in parallel standard PCR amplification and real-time quantitative PCR using Taqman detection on the ABI Prism 7700 Sequence Detection system (Applied Biosystems, Foster City, CA). In contrast with the statement of Gotoh et al,1 control genes were used in both strategies (respectively GAPDH and ribonuclease P RNA component H1/RPPH1) to avoid any influence of the quality of the template DNA. Quantitative PCR showed that levels of MYCN DNA sequences in the peripheral blood of MYCN-amplified (MNA) neuroblastoma patients was 25 to 600 fold higher than in non-MYCN amplified patients or control individuals.2 Gotoh et al used DNA-based real-time quantitative PCR and a single copy reference gene (NAGK) located on 2p12. The location of NAGK, on chromosome 2 but at distance from the region usually affected by MYCN amplification, allows avoiding the influence of potential numerical changes of chromosome 2. We recently used a similar strategy to analyze, in a blind study, 104 new NB cases (serum DNA from 59 Spanish and 45 French NB patients). The study was conducted under research protocols approved by each institutional review board. The proIL1ß gene, located on 2q13-24, was used as a reference gene (sequences of primers were as follows: MYCN forward, 5'-CGGTCCCCCACCTCTCTT-3'; MYCN reverse, 5'-CGGTTTAGCCACCAACTTTCTC-3'; proIL1ß forward, 5'-AACAAGAGTGCTGGAGCGAT-3'; proIL1ß reverse 5'-GTCCTTCAAAGTCAGCAGCC-3'). MYCN DNA sequences were detected in 16 of 18 samples from patients with MNA tumors and in 2 of 86 samples from NB patients without MNA tumors (P < .0001), further confirming the potential of the clinical use of circulating MYCN DNA as a marker of MNA NB. The two cases described as false-positive could be explained by the heterogeneity of MYCN gene amplification in the tumors.3 The molecular biology of NBs has led to a biologic risk stratification in which MNA provides essential information but is not always easy to obtain. In the randomized study of 637 children with high-risk NBs reported by Matthay,4 26% of the patients had MNA NB while the MYCN status was unknown in 27% of the children. Today, the possibility of determining the MYCN status of NB patients using a simple blood sample should virtually eliminate cases with unknown MYCN status, allowing infants and children with previously undetermined MYCN status to benefit from more appropriate treatment. To illustrate this point, we report the case of a 13-day-old infant with typical 4S NB referred to our hospital with hepatomegaly, left adrenal tumor, and elevated urinary catecholamine metabolites. Thrombocytopenia (83 x 109/L) and hypofibrinogenemia (< 0.35 g/L) complicated the collection of tumor tissue by core biopsy or fine needle aspiration. Blood analysis revealed a high level of MYCN DNA sequences, thus providing evidence that this 4S NB could be a life-threatening disease. Although the patient scored 0 on the Philadelphia scale,5 chemotherapy (carboplatin-etoposide) was initiated and produced a significant response. Three weeks later, coagulation tests had reverted to normal and histological examination of a percutaneous core biopsy confirmed NB, with more than 50 MYCN copies, as evidenced by PCR analysis. The child achieved complete remission with chemotherapy alone and is healthy 1 year later, after intensive chemotherapy. This case demonstrates the usefulness of the approach, when no tumor sample is available. As suggested by Gotoh et al,1 it is now essential to standardize serum collection procedures and methodological strategies to ensure the accuracy and reproducibility of the assay. 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
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 3. Noguera R, Canete A, Pellin A, et al: MYCN gain and MYCN amplification in a stage 4S neuroblastoma. Cancer Genet Cytogenet 140:157-161, 2003[CrossRef][Medline]
4. Matthay KK, Villablanca JG, Seeger RC, et al: Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children's Cancer Group. N Engl J Med 341:1165-1173, 1999 5. Hsu LL, Evans AE, D'Angio GJ: Hepatomegaly in neuroblastoma stage 4s: Criteria for treatment of the vulnerable neonate. Med Pediatr Oncol 27:521-528, 1996[CrossRef][Medline]
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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