BACKGROUND: In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. METHODS: The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. RESULTS: Patients <18 months (18 m) showed a better outcome in all stages as compared to older patients (5-year OS in localised stages: <18 m: 0.95 ± 0.04, >18 m: 0.67 ± 0.14, p = 0.011; metastatic: <18 m: 0.76 ± 0.15, >18 m: 0.28 ± 0.09, p = 0.084). The genomic 'background', but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. CONCLUSIONS: This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.

Cancer Cytogenetic and Molecular Cytogenetic Laboratory Schneider Children's Medical Center of Israel 49202 Petach Tikva Israel

Centre Léon Bérard Laboratoire de Recherche Translationnelle 28 rue Laennec Lyon 69008 France

Ciberonc Madrid Spain

Department of Paediatric Haematology Oncology Agia Sofia Children's Hospital Athens 11528 Athens Greece

Department of Paediatric Haematology Oncology Aix Marseille University and APHM Hôpital d' Enfants de La Timone 13385 Marseille France

Department of Paediatric Haematology Oncology Schneider Children's Medical Center of Israel 49202 Petach Tikva Israel

Department of Paediatric Medicine Rikshospitalet Oslo University Hospital 0372 Oslo Norway

Department of Paediatrics Medical University Vienna Vienna Austria

Department of Pathology Gaslini Institute Largo G Gaslini 5 16147 Genoa Italy

Department of Pathology Medical School University of Valencia INCLIVA Biomedical Research Institute 46010 Valencia Spain

Department of Pediatric Hematology and Oncology Charles University Prague 2nd Faculty of Medicine and University Hospital Motol 15006 Prague Czech Republic

Department of Tumour Biology CCRI Children's Cancer Research Institute St Anna Kinderkrebsforschung 1090 Vienna Austria

Institute of Clinical Medicine Faculty of Medicine University of Oslo and Department of Pathology Norwegian Radium Hospital Oslo University Hospital 0372 Oslo Norway

Institute of Clinical Pathology Medical University Vienna Vienna Austria

Laboratory of Molecular Biology Gaslini Institute Largo G Gaslini 5 16147 Genoa Italy

Northern Genetics Service The Newcastle upon Tyne Hospitals NHS Foundation Trust Institute of Genetic Medicine Central Parkway Newcastle upon Tyne NE1 3BZ UK

Our Lady's Children's Hospital Crumlin Dublin D12 N512 Ireland

Pediatric Oncology Unit Hospital Universitari i Politècnic La Fe 46026 Valencia Spain

S2IRP Studies and Statistics for Integrated Research and Projects CCRI Children's Cancer Research Institute St Anna Kinderkrebsforschung 1090 Vienna Austria

St Anna Children's Hospital and Department of Paediatrics of the Medical University 1090 Vienna Austria

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Schwab M, et al. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature. 1983;305:245–248. doi: 10.1038/305245a0. PubMed DOI

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. doi: 10.1126/science.6719137. PubMed DOI

Cohn SL, et al. The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report. J. Clin. Oncol. 2009;27:289–297. doi: 10.1200/JCO.2008.16.6785. PubMed DOI PMC

Maris JM. Recent advances in neuroblastoma. N. Engl. J. Med. 2010;362:2202–2211. doi: 10.1056/NEJMra0804577. PubMed DOI PMC

Matthay KK, et al. Neuroblastoma. Nat. Rev. Dis. Prim. 2016;2:16078. doi: 10.1038/nrdp.2016.78. PubMed DOI

Ambros PF, 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. doi: 10.1038/sj.bjc.6605014. PubMed DOI PMC

Cheung NK, et al. Association of age at diagnosis and genetic mutations in patients with neuroblastoma. JAMA. 2012;307:1062–1071. doi: 10.1001/jama.2012.228. PubMed DOI PMC

Schleiermacher G, et al. Segmental chromosomal alterations have prognostic impact in neuroblastoma: a report from the INRG project. Br. J. Cancer. 2012;107:1418–1422. doi: 10.1038/bjc.2012.375. PubMed DOI PMC

Villamon E, et al. Genetic instability and intratumoral heterogeneity in neuroblastoma with MYCN amplification plus 11q deletion. PLoS ONE. 2013;8:e53740. doi: 10.1371/journal.pone.0053740. PubMed DOI PMC

Cheung NK, et al. Murine anti-GD2 monoclonal antibody 3F8 combined with granulocyte-macrophage colony-stimulating factor and 13-cis-retinoic acid in high-risk patients with stage 4 neuroblastoma in first remission. J. Clin. Oncol. 2012;30:3264–3270. doi: 10.1200/JCO.2011.41.3807. PubMed DOI PMC

Pinto NR, et al. Advances in risk classification and treatment strategies for neuroblastoma. J. Clin. Oncol. 2015;33:3008–3017. doi: 10.1200/JCO.2014.59.4648. PubMed DOI PMC

Ladenstein R, et al. Busulfan and melphalan versus carboplatin, etoposide, and melphalan as high-dose chemotherapy for high-risk neuroblastoma (HR-NBL1/SIOPEN): an international, randomised, multi-arm, open-label, phase 3 trial. Lancet Oncol. 2017;18:500–514. doi: 10.1016/S1470-2045(17)30070-0. PubMed DOI

Squire JA, et al. Identification of MYCN copy number heterogeneity by direct FISH analysis of neuroblastoma preparations. Mol. Diagn. 1996;1:281–289. doi: 10.1016/S1084-8592(96)70010-3. PubMed DOI

Lorenzana AN, et al. Heterogeneity of MYCN amplification in a child with stroma-rich neuroblastoma (ganglioneuroblastoma) Pediatr. Pathol. Lab. Med. 1997;17:875–883. doi: 10.1080/15513819709168751. PubMed DOI

Ambros PF, et al. Intratumoural heterogeneity of 1p deletions and MYCN amplification in neuroblastomas. Med. Pediatr. Oncol. 2001;36:1–4. doi: 10.1002/1096-911X(20010101)36:1<1::AID-MPO1002>3.0.CO;2-L. PubMed DOI

Kerbl R, et al. Neuroblastoma with focal MYCN amplification and bone marrow infiltration: a staging and treatment dilemma. Med. Pediatr. Oncol. 2002;38:109–111. doi: 10.1002/mpo.1281. PubMed DOI

Noguera R, et al. MYCN gain and MYCN amplification in a stage 4S neuroblastoma. Cancer Genet. Cytogenet. 2003;140:157–161. doi: 10.1016/S0165-4608(02)00677-5. PubMed DOI

Ambros IM, et al. Quality assessment of genetic markers used for therapy stratification. J. Clin. Oncol. 2003;21:2077–2084. doi: 10.1200/JCO.2003.03.025. PubMed DOI

Valent A, et al. Alternative pathways of MYCN gene copy number increase in primary neuroblastoma tumors. Cancer Genet. Cytogenet. 2004;153:10–15. doi: 10.1016/j.cancergencyto.2003.12.007. PubMed DOI

Spitz R, Hero B, Skowron M, Ernestus K, Berthold F. MYCN-status in neuroblastoma: characteristics of tumours showing amplification, gain, and non-amplification. Eur. J. Cancer. 2004;40:2753–2759. doi: 10.1016/j.ejca.2004.05.002. PubMed DOI

Thorner PS, Ho M, Chilton-MacNeill S, Zielenska M. Use of chromogenic in situ hybridization to identify MYCN gene copy number in neuroblastoma using routine tissue sections. Am. J. Surg. Pathol. 2006;30:635–642. doi: 10.1097/01.pas.0000202163.82525.5c. PubMed DOI

Sano H, et al. A case of composite neuroblastoma composed of histologically and biologically distinct clones. Pediatr. Dev. Pathol. 2007;10:229–232. doi: 10.2350/06-06-0117.1. PubMed DOI

Cañete A, 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. doi: 10.1200/JCO.2007.14.5839. PubMed DOI

Theissen J, et al. Heterogeneity of the MYCN oncogene in neuroblastoma. Clin. Cancer Res. 2009;15:2085–2090. doi: 10.1158/1078-0432.CCR-08-1648. PubMed DOI

Bishop MW, et al. Management of stage 4S composite neuroblastoma with a MYCN-amplified nodule. J. Pediatr. Hematol. Oncol. 2014;36:e31–e35. doi: 10.1097/MPH.0b013e3182847376. PubMed DOI

Berbegall AP, et al. Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma versus aggressive genetic profile neuroblastic tumors. Oncogene. 2016;35:1423–1432. doi: 10.1038/onc.2015.200. PubMed DOI

Berbegall AP, Navarro S, Noguera R. Diagnostic implications of intrapatient genetic tumor heterogeneity. Mol. Cell. Oncol. 2016;3:e1079671. doi: 10.1080/23723556.2015.1079671. PubMed DOI PMC

Bogen D, et al. The genetic tumor background is an important determinant for heterogeneous MYCN-amplified neuroblastoma. Int. J. Cancer. 2016;139:153–163. doi: 10.1002/ijc.30050. PubMed DOI PMC

Marrano P, Irwin MS, Thorner PS. Heterogeneity of MYCN amplification in neuroblastoma at diagnosis, treatment, relapse, and metastasis. Genes Chromosomes Cancer. 2017;56:28–41. doi: 10.1002/gcc.22398. PubMed DOI

Defferrari R, et al. Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification. Br. J. Cancer. 2015;112:290–295. doi: 10.1038/bjc.2014.557. PubMed DOI PMC

Mehes G, et al. Automatic detection and genetic profiling of disseminated neuroblastoma cells. Med. Pediatr. Oncol. 2001;36:205–209. doi: 10.1002/1096-911X(20010101)36:1<205::AID-MPO1050>3.0.CO;2-G. PubMed DOI

Scaruffi P, et al. Identification and characterization of DNA imbalances in neuroblastoma by high-resolution oligonucleotide array comparative genomic hybridization. Cancer Genet. Cytogenet. 2007;177:20–29. doi: 10.1016/j.cancergencyto.2007.05.002. PubMed DOI

Combaret V, et al. Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results. Cancer Genet. 2012;205:657–664. doi: 10.1016/j.cancergen.2012.11.002. PubMed DOI

Ambros IM, Brunner C, Abbasi R, Frech C, Ambros PF. Ultra-high density SNParray in neuroblastoma molecular diagnostics. Front. Oncol. 2014;4:202. doi: 10.3389/fonc.2014.00202. PubMed DOI PMC

Beiske K, et al. Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force. Br. J. Cancer. 2009;100:1627–1637. doi: 10.1038/sj.bjc.6605029. PubMed DOI PMC

Burchill SA, et al. Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma; on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group. Cancer. 2016;123:1095–1105. doi: 10.1002/cncr.30380. PubMed DOI

Marubini E, Valsecchi MG. Analysing Survival Data from Clinical Trials and Observational Studies. 1st edn. United Kingdom: John Wiley & Sons; 2004.

London WB, et al. Evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children’s Oncology Group. J. Clin. Oncol. 2005;23:6459–6465. doi: 10.1200/JCO.2005.05.571. PubMed DOI

Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a competing risk. J. Am. Stat. Assoc. 1999;94:14. doi: 10.1080/01621459.1999.10474144. DOI

Bedard PL, Hansen AR, Ratain MJ, Siu LL. Tumour heterogeneity in the clinic. Nature. 2013;501:355–364. doi: 10.1038/nature12627. PubMed DOI PMC

Gay L, Baker AM, Graham TA. Tumour cell heterogeneity. F1000Res. 2016;5:F1000 Faculty Rev-238. doi: 10.12688/f1000research.7210.1. PubMed DOI

McGranahan N, Swanton C. Clonal heterogeneity and tumor evolution: past, present, and the future. Cell. 2017;168:613–628. doi: 10.1016/j.cell.2017.01.018. PubMed DOI

Mengelbier LH, et al. Intratumoral genome diversity parallels progression and predicts outcome in pediatric cancer. Nat. Commun. 2015;6:6125. doi: 10.1038/ncomms7125. PubMed DOI

Cresswell GD, et al. Intra-tumor genetic heterogeneity in Wilms tumor: clonal evolution and clinical implications. EBioMedicine. 2016;9:120–129. doi: 10.1016/j.ebiom.2016.05.029. PubMed DOI PMC

Bellini A, et al. Deep sequencing reveals occurrence of subclonal ALK mutations in neuroblastoma at diagnosis. Clin. Cancer Res. 2015;21:4913–4921. doi: 10.1158/1078-0432.CCR-15-0423. PubMed DOI

Abbasi MR, et al. Impact of disseminated neuroblastoma cells on the identification of the relapse-seeding clone. Clin. Cancer Res. 2017;23:4224–4232. doi: 10.1158/1078-0432.CCR-16-2082. PubMed DOI PMC

Williamson D, et al. Relationship between MYCN copy number and expression in rhabdomyosarcomas and correlation with adverse prognosis in the alveolar subtype. J. Clin. Oncol. 2005;23:880–888. doi: 10.1200/JCO.2005.11.078. PubMed DOI

Pfister S, et al. Outcome prediction in pediatric medulloblastoma based on DNA copy-number aberrations of chromosomes 6q and 17q and the MYC and MYCN loci. J. Clin. Oncol. 2009;27:1627–1636. doi: 10.1200/JCO.2008.17.9432. PubMed DOI

Williams RD, et al. Molecular profiling reveals frequent gain of MYCN and anaplasia-specific loss of 4q and 14q in Wilms tumor. Genes Chromosomes Cancer. 2011;50:982–995. doi: 10.1002/gcc.20907. PubMed DOI

Theriault BL, Dimaras H, Gallie BL, Corson TW. The genomic landscape of retinoblastoma: a review. Clin. Exp. Ophthalmol. 2014;42:33–52. doi: 10.1111/ceo.12132. PubMed DOI PMC

Caren H, et al. High-risk neuroblastoma tumors with 11q-deletion display a poor prognostic, chromosome instability phenotype with later onset. Proc. Natl. Acad. Sci. USA. 2010;107:4323–4328. doi: 10.1073/pnas.0910684107. PubMed DOI PMC

De Bernardi B, et al. Treatment of localised resectable neuroblastoma. Results of the LNESG1 study by the SIOP Europe Neuroblastoma Group. Br. J. Cancer. 2008;99:1027–1033. doi: 10.1038/sj.bjc.6604640. PubMed DOI PMC

Quetglas IM, Moeini A, Pinyol R, Llovet JM. Integration of genomic information in the clinical management of HCC. Best Pract. Res. Clin. Gastroenterol. 2014;28:831–842. doi: 10.1016/j.bpg.2014.08.004. PubMed DOI

Vance GH, et al. Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines. Arch. Pathol. Lab. Med. 2009;133:611–612. PubMed

Seol H, et al. Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance. Mod. Pathol. 2012;25:938–948. doi: 10.1038/modpathol.2012.36. PubMed DOI

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1)