Emergence of New ALK Mutations at Relapse of Neuroblastoma Gudrun Schleiermacher, Niloufar Javanmardi, Virginie Bernard, Quentin Leroy, Julie Cappo, Thomas Rio Frio, Gaelle Pierron, Eve Lapouble, Vale ´rie Combaret, Frank Speleman, Bram de Wilde, Anna Djos, Ingrid Øra, Fredrik Hedborg, Catarina Tra ¨ger, Britt-Marie Holmqvist, Jonas Abrahamsson, Michel Peuchmaur, Jean Michon, Isabelle Janoueix-Lerosey, Per Kogner, Olivier Delattre, and Tommy Martinsson Author affiliations appear at the end of this article. Published online ahead of print at www.jco.org on July 28, 2014. In France, this study was supported by the Annenberg Foundation. Funding was also obtained from Site de Recherche Integre ´ en Cance ´ rologie/Institut National du Cancer (Grant No. INCa-DGOS-4654) and from the Comite ´ d’e ´ valuation et suivi des projets de Recherche de Transfert of Institut Curie. This study was also funded by the Associa- tions Enfants et Sante ´ , Association Hubert Gouin Enfance et Cancer, Les Bagouz a ` Manon, and Les Amis de Claire. Next- generation sequencing (NGS) experiments were conducted on the Institut Curie’s ICGex NGS platform funded by the EQUIPEX Investissements d’Avenir program (Grant No. ANR-10-EQPX-03) and Grant No. ANR10-INBS-09-08 from the Agence Nationale de le Recherche and by the Canceropo ˆ le Ile-de-France. In Sweden, this work was supported by grants from the Swedish Cancer Society (Grant No. 12-817 TM), the Swedish Children’s Cancer Foundation (Grant No. 10-129 TM), and the Swedish state through the LUA/ALF agreement. The Swedish part of the project was also supported by BioCARE, a National Strategic Research Program at the University of Gothenburg. In Belgium, this work was supported by the Fund for Scientific Research (Grants No. G.0198.08 and 31511809), the Euro- pean Network for Cancer Research in Chil- dren and Adolescents: Seventh European Union Framework Programme (NoE No. 261474; Analyzing and Striking the Sensitiv- ities of Embryonal Tumors: Seventh Euro- pean Union Framework Programme, CP No. 259348), and the National Cancer Plan KPC_29_010 (Integrated biology-driven diagnostic and therapeutic management of neuroblastoma). This funding provided the necessary resources for the experimental phase of the study and for data analysis. Both O.D. and T.M. contributed equally as senior authors of this study. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article. Corresponding author: Gudrun Schleierm- acher, MD, PhD, Department of Pediatric Oncology, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France; e-mail: gudrun.schleiermacher@curie.net. © 2014 by American Society of Clinical Oncology 0732-183X/14/3225w-2727w/$20.00 DOI: 10.1200/JCO.2013.54.0674 A B S T R A C T Purpose In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution. Methods We analyzed ALK mutations in 54 paired diagnosis–relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000 deep sequencing of the relevant hotspot, with a sensitivity of 0.17%. Results All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%). Conclusion In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions. J Clin Oncol 32:2727-2734. © 2014 by American Society of Clinical Oncology INTRODUCTION Current treatment approaches in cancer often lead to initial response followed by secondary progres- sion that presents a therapeutic challenge because of resistance to conventional chemotherapy treat- ment. 1 Thus, genetic characterization of cancer cells provides invaluable information for the identifica- tion of molecular therapeutic targets. Importantly, particular genetic alterations may be selected for or emerge during treatment. 2,3 Subclonal driver muta- tions might play a role in tumor progression, and the presence of driver mutation– harboring subclones at diagnosis, which might expand at relapse, has been linked to adverse outcomes. 2-5 In neuroblastoma, the most frequent extracra- nial solid cancer of early childhood, tumor progres- sion is often associated with limited therapeutic possibilities, underlining the need of molecular analyses. 6 Genetic alterations in neuroblastoma at diagnosis mainly concern copy number alterations, with MYCN amplification in 20% to 25% of cases, and other copy number changes over extensive chromosome regions. 6-11 Only a few recurrently al- tered genes, such as chromatin-remodeling or neu- ritogenesis genes, have been reported, targeted by either small interstitial structural alterations or mutations. 8-10 Activating point mutations in the ty- rosine kinase domain of ALK, the most frequent mu- tations in neuroblastoma, are detected at diagnosis in approximately 8% to 10% of patients and play an im- portant role in neuroblastoma oncogenesis. 12-17 These alterations can be targeted using ALK inhibitors, and in vitro and in vivo models have JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T VOLUME 32  NUMBER 25  SEPTEMBER 1 2014 © 2014 by American Society of Clinical Oncology 2727 Downloaded from ascopubs.org by 3.80.241.116 on June 15, 2022 from 003.080.241.116 Copyright © 2022 American Society of Clinical Oncology. All rights reserved.