J Korean Med Sci.  2016 Sep;31(9):1392-1396. 10.3346/jkms.2016.31.9.1392.

Detection of MYCN Amplification in Serum DNA Using Conventional Polymerase Chain Reaction

• Ma Y
• Lee JW
• Park SJ
• Yi ES
• Choi YB
• Yoo KH
• Sung KW
• Koo HH

• KMID: 2468270
• DOI: http://doi.org/10.3346/jkms.2016.31.9.1392

Abstract

Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and is characterized by a wide range of clinical behaviors. Amplification of MYCN is a well-known poor prognostic factor in NB patients. As the MYCN amplification status is usually tested using tumor specimens, lengthy and invasive procedures are unavoidable. To evaluate the possibility of detecting MYCN amplification without invasive procedure, we performed conventional polymerase chain reaction (PCR) analysis to identify MYCN amplification using the preserved serum DNA. PCR of serum DNA was done in 105 NB patients whose MYCN status had been confirmed by fluorescence in situ hybridization. MYCN amplification was evaluated as the ratio of signal intensities between MYCN and NAGK (M/N ratio). When regarding the tissue FISH results as a reference, 10 patients had MYCN-amplified (MNA) NB, and 95 had non-MNA NB. The M/N ratio of the MNA group (median 2.56, range 1.01-3.58) was significantly higher than that of the non-MNA group (median 0.97, range 0.67-5.18) (P < 0.001). In the receiver operating characteristic curve analysis, the area under the curve was 0.957 (95% confidence interval 0.898-1.000; P < 0.001), and it showed 90.9% sensitivity and 97.9% specificity with the selected cut-off value set as 1.6. The detection of MYCN amplification using conventional PCR analysis of serum samples seems to be a simple and promising method to evaluate the MYCN status of NB patients. Further study with a larger set of patients is needed to confirm the accuracy of this result.

Keyword

Neuroblastoma; MYCN Amplification; Polymerase Chain Reaction; Serum DNA

MeSH Terms

Adolescent
Area Under Curve
Child
Child, Preschool
DNA/*blood/isolation & purification/metabolism
Female
Humans
In Situ Hybridization, Fluorescence
Infant
Male
N-Myc Proto-Oncogene Protein/*genetics/metabolism
Neoplasm Staging
Neuroblastoma/*diagnosis/genetics/pathology
Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism
Polymerase Chain Reaction
ROC Curve
Sensitivity and Specificity
Young Adult
N-Myc Proto-Oncogene Protein
DNA
Phosphotransferases (Alcohol Group Acceptor)

Figure

• 

  Fig. 1 Distributions of MYCN/NAGK (M/N) ratios in MYCN-amplified (MNA) and non-MNA tumors. The M/N ratio of serum DNA according to the MYCN tissue status is shown. The line in the middle indicates cut off level (1.6).

• 

  Fig. 2 Detection of MYCN amplification in serum using PCR analysis. Patients A and B had MYCN-amplified (MNA) tumors, while patients C and D had non-MNA tumors. The MYCN/NAGK (M/N) ratios of patient A, B, C and D were 3.758, 3.527, 0.977, and 0.907, respectively. The very left lane is ladder marker DNA.

• 

  Fig. 3 Receiver operating characteristic (ROC) curve constructed using the MYCN/NAGK (M/N) ratio. In the ROC curve analysis, the area under the curve was 0.957 (95% confidence interval, 0.898–1.000; P < 0.001), and it showed 90.9% sensitivity and 97.9% specificity with the selected M/N ratio cut-off value set of 1.6.

Reference

1. Cohn SL, Pearson AD, London WB, Monclair T, Ambros PF, Brodeur GM, Faldum A, Hero B, Iehara T, Machin D, et al. The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report. J Clin Oncol. 2009; 27:289–297.

2. Owens C, Irwin M. Neuroblastoma: the impact of biology and cooperation leading to personalized treatments. Crit Rev Clin Lab Sci. 2012; 49:85–115.

3. Park JR, Bagatell R, London WB, Maris JM, Cohn SL, Mattay KK, Hogarty M; COG Neuroblastoma Committee. Children’s Oncology Group’s 2013 blueprint for research: neuroblastoma. Pediatr Blood Cancer. 2013; 60:985–993.

4. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science. 1984; 224:1121–1124.

5. Janoueix-Lerosey I, Schleiermacher G, Michels E, Mosseri V, Ribeiro A, Lequin D, Vermeulen J, Couturier J, Peuchmaur M, Valent A, et al. Overall genomic pattern is a predictor of outcome in neuroblastoma. J Clin Oncol. 2009; 27:1026–1033.

6. Combaret V, Audoynaud C, Iacono I, Favrot MC, Schell M, Bergeron C, Puisieux A. Circulating MYCN DNA as a tumor-specific marker in neuroblastoma patients. Cancer Res. 2002; 62:3646–3648.

7. Gotoh T, Hosoi H, Iehara T, Kuwahara Y, Osone S, Tsuchiya K, Ohira M, Nakagawara A, Kuroda H, Sugimoto T. Prediction of MYCN amplification in neuroblastoma using serum DNA and real-time quantitative polymerase chain reaction. J Clin Oncol. 2005; 23:5205–5210.

8. Combaret V, Hogarty MD, London WB, McGrady P, Iacono I, Brejon S, Swerts K, Noguera R, Gross N, Rousseau R, et al. Influence of neuroblastoma stage on serum-based detection of MYCN amplification. Pediatr Blood Cancer. 2009; 53:329–331.

9. Kojima M, Hiyama E, Fukuba I, Yamaoka E, Ueda Y, Onitake Y, Kurihara S, Sueda T. Detection of MYCN amplification using blood plasma: noninvasive therapy evaluation and prediction of prognosis in neuroblastoma. Pediatr Surg Int. 2013; 29:1139–1145.

10. Shimada H, Ambros IM, Dehner LP, Hata J, Joshi VV, Roald B, Stram DO, Gerbing RB, Lukens JN, Matthay KK, et al. The International Neuroblastoma Pathology Classification (the Shimada system). Cancer. 1999; 86:364–372.

11. Ambros PF, Ambros IM, Brodeur GM, Haber M, Khan J, Nakagawara A, Schleiermacher G, Speleman F, Spitz R, London WB, et al. International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee. Br J Cancer. 2009; 100:1471–1482.

12. Kimoto T, Inoue M, Tokimasa S, Yagyu S, Iehara T, Hosoi H, Kawa K. Detection of MYCN DNA in the cerebrospinal fluid for diagnosing isolated central nervous system relapse in neuroblastoma. Pediatr Blood Cancer. 2011; 56:865–867.

13. Sauvat F, Sarnacki S, Brisse H, Medioni J, Rubie H, Aigrain Y, Gauthier F, Audry G, Helardot P, Landais P, et al. Outcome of suprarenal localized masses diagnosed during the perinatal period: a retrospective multicenter study. Cancer. 2002; 94:2474–2480.

14. Maki E, Oh K, Rogers S, Sohaey R. Imaging and differential diagnosis of suprarenal masses in the fetus. J Ultrasound Med. 2014; 33:895–904.

15. Hero B, Simon T, Spitz R, Ernestus K, Gnekow AK, Scheel-Walter HG, Schwabe D, Schilling FH, Benz-Bohm G, Berthold F. Localized infant neuroblastomas often show spontaneous regression: results of the prospective trials NB95-S and NB97. J Clin Oncol. 2008; 26:1504–1510.

16. Nuchtern JG, London WB, Barnewolt CE, Naranjo A, McGrady PW, Geiger JD, Diller L, Schmidt ML, Maris JM, Cohn SL, et al. A prospective study of expectant observation as primary therapy for neuroblastoma in young infants: a Children’s Oncology Group study. Ann Surg. 2012; 256:573–580.

17. Minard V, Hartmann O, Peyroulet MC, Michon J, Coze C, Defachelle AS, Lejars O, Perel Y, Bergeron C, Boutard P, et al. Adverse outcome of infants with metastatic neuroblastoma, MYCN amplification and/or bone lesions: results of the French Society of Pediatric Oncology. Br J Cancer. 2000; 83:973–979.

18. Canete A, Gerrard M, Rubie H, Castel V, Di Cataldo A, Munzer C, Ladenstein R, Brichard B, Bermúdez JD, Couturier J, et al. Poor survival for infants with MYCN-amplified metastatic neuroblastoma despite intensified treatment: The International Society of Paediatric Oncology European Neuroblastoma Experience. J Clin Oncol. 2009; 27:1014–1019.

19. Di Cataldo A, Dau D, Conte M, Parodi S, De Bernardi B, Giuliano M, Pession A, Viscardi E, Luksch R, Castellano A, et al. Diagnostic and prognostic markers in infants with disseminated neuroblastoma: a retrospective analysis from the Italian Cooperative Group for Neuroblastoma. Med Sci Monit. 2009; 15:MT11–8.

20. Tonini GP, Boni L, Pession A, Rogers D, Iolascon A, Basso G, Cordero di Montezemolo L, Casale F, Pession A, Perri P, et al. MYCN oncogene amplification in neuroblastoma is associated with worse prognosis, except in stage 4s: the Italian experience with 295 children. J Clin Oncol. 1997; 15:85–93.

21. Yoo SY, Kim JS, Sung KW, Jeon TY, Choi JY, Moon SH, Son MH, Lee SH, Yoo KH, Koo HH. The degree of tumor volume reduction during the early phase of induction chemotherapy is an independent prognostic factor in patients with high-risk neuroblastoma. Cancer. 2013; 119:656–664.

22. Swinkels DW, Wiegerinck E, Steegers EA, de Kok JB. Effects of blood-processing protocols on cell-free DNA quantification in plasma. Clin Chem. 2003; 49:525–526.