ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations.

// Simon Durand 1 , 2 , Cecile Pierre-Eugene 1 , 2 , Olivier Mirabeau 1 , 2 , Caroline Louis-Brennetot 1 , 2 , Valerie Combaret 3 , Leo Colmet-Daage 2 , 4 , Orphee Blanchard 1 , 2 , Angela Bellini 2 , 4 , Estelle Daudigeos-Dubus 5 , Virginie Raynal 1 , 2 , 6 , Gudrun Schleiermacher 2 , 4 , Sylvain Baulande 6 , Olivier Delattre 1 , 2 and Isabelle Janoueix-Lerosey 1 , 2 1 Institut Curie, PSL Research University, Inserm U830, Equipe Labellisee Ligue contre le Cancer, Paris F-75005, France 2 SIREDO: Care, Innovation, and Research for Children, Adolescents, and Young Adults with Cancer, Institut Curie, Paris F-75005, France 3 Centre Leon Berard, Laboratoire de Recherche Translationnelle, Lyon F-69008, France 4 Equipe SiRIC RTOP (Recherche Translationnelle en Oncologie Pediatrique), Institut Curie, Paris F-75005, France 5 Gustave Roussy, Vectorology and Anticancer Therapies, UMR 8203, CNRS, University Paris-Sud, Universite Paris-Saclay, Villejuif F-94805, France 6 Institut Curie Genomics of Excellence (ICGex) Platform, Institut Curie Research Center, Paris F-75005, France Correspondence to: Isabelle Janoueix-Lerosey, email: janoueix@curie.fr Keywords: neuroblastoma; ALK mutations; heterogeneity; clonal evolution; ALK inhibitors Received: March 26, 2019     Accepted: July 17, 2019     Published: August 13, 2019 ABSTRACT The ALK gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate in vitro and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. In vitro treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.