RESEARCH ARTICLE Serum-Based Quantification of MYCN Gene Amplification in Young Patients with Neuroblastoma: Potential Utility as a Surrogate Biomarker for Neuroblastoma Shigeki Yagyu 1 , Tomoko Iehara 1 , Shiro Tanaka 2 , Takahiro Gotoh 1 , Akiko Misawa- Furihata 1 , Tohru Sugimoto 1 , Wendy B. London 3 , Michael D. Hogarty 4 , Satoshi Teramukai 5 , Akira Nakagawara 6,7 , Eiso Hiyama 8 , John M. Maris 4 , Hajime Hosoi 1 * 1 Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2 Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Kyoto, Japan, 3 Division of Hematology/Oncology, Children's Hospital Boston/Dana- Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 5 Department of Biostatistics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan, 6 Division of Biochemistry and Innovative Cancer Therapeutics, Chiba Cancer Center, Chiba, Japan, 7 Saga Medical Cancer KOSEIKAN, Saga, Japan, 8 Department of Pediatric Surgery, Hiroshima University Hospital, Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan * hhosoi@koto.kpu-m.ac.jp Abstract We previously developed a method for determining MYCN gene amplification status using cell-free DNA fragments released from cancer cells into the blood of patients with neuroblas- toma (NB). Here, we analyzed the relationship between MYCN amplification (MNA) status and neuroblastoma prognosis. We screened serum samples from 151 patients with NB for MNA, using real-time quantitative PCR, and compared the results with MYCN status deter- mined using paired tumor samples. We additionally investigated whether MNA status corre- lates with patient survival. When a cut-off value of 5 was used, serum-based MNA analysis was found to show good sensitivity (86%) and very high specificity (95%). The sensitivities for stage 1 and 2 might be acceptable, even though it is not as good as for stage 3 and 4 (67% for stage 1 and 2, 92% for stage 3, and 87% for stage 4). MNA status correlated with overall survival in our cohort of 82 patients, with survival data available (p < 0.01). The hazard ratio of MNA status was 4.98 in patients diagnosed at less than 18 months of age (95% confidence interval, 1.00–24.78), and 1.41 (95% confidence interval, 0.63–3.14) for those diagnosed at 18 months of age or older. Serum-based MNA analysis is rapid and non-invasive compared with tumor-based MNA analysis, and has potential to predict tumor MNA status. There is still a room to improve the sensitivity of the test for tumors of stages 1 and 2, nonetheless this assay might help to determine therapeutic strategies prior to tumor biopsy, especially for patients with a life-threatening condition, as well as for patients of less than 18 months of age whose risk-grouping and treatment allocation depends on their MNA status. PLOS ONE | DOI:10.1371/journal.pone.0161039 August 11, 2016 1 / 11 a11111 OPEN ACCESS Citation: Yagyu S, Iehara T, Tanaka S, Gotoh T, Misawa-Furihata A, Sugimoto T, et al. (2016) Serum- Based Quantification of MYCN Gene Amplification in Young Patients with Neuroblastoma: Potential Utility as a Surrogate Biomarker for Neuroblastoma. PLoS ONE 11(8): e0161039. doi:10.1371/journal. pone.0161039 Editor: Pierre Busson, Gustave Roussy, FRANCE Received: December 15, 2015 Accepted: July 28, 2016 Published: August 11, 2016 Copyright: © 2016 Yagyu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI, Grant Number 25253095, (https://www. jsps.go.jp/english/e-grants/); the Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and development (AMED), (http:// www.amed.go.jp/en/); Kyoto-Funding for Innovation in Health-related R&D Fields Grant Number 88, (http:// www.astem.or.jp/english/); Adaptable and Seamless Technology Transfer Program through Target-driven