LETTERS Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma Isabelle Janoueix-Lerosey 1,2 , Delphine Lequin 1,2 , Laurence Brugie `res 3 , Agne `s Ribeiro 4 , Loı ¨c de Pontual 5 , Vale ´rie Combaret 6 , Virginie Raynal 1,2 , Alain Puisieux 6,7,8 , Gudrun Schleiermacher 1,2,9 , Gae ¨lle Pierron 4 , Dominique Valteau-Couanet 3 , Thierry Frebourg 10 , Jean Michon 9 , Stanislas Lyonnet 5 , Jeanne Amiel 5 & Olivier Delattre 1,2,4 Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in child- hood 1,2 . It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with con- genital malformations of the neural crest being linked to germline mutations of the PHOX2B gene 1–4 . Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) 5 tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indi- cating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments 6,7 . In a survey of the amplicons of neuroblastoma 8 , we recently observed that high-level amplification (.10 copies) of ALK, a tyrosine kinase receptor (RTK) gene preferentially expressed in the central and peripheral nervous systems 5 , was associated with MYCN amplifica- tion, the most frequent amplicon in neuroblastoma 1,2,9 . The expres- sion level of ALK was strongly correlated with copy number 8 , hence confirming and extending previous findings of ALK amplifications and overexpression in neuroblastoma 10–12 . To document more precisely ALK genomic status, we evaluated copy number variation at this locus in a series of 592 neuroblastomas that were investigated by bacterial artificial chromosome (BAC)-array com- parative genomic hybridization (CGH). A total of 26 cases (4.4%) demonstrated higher than twofold copy number increases and an addi- tional 135 cases (22.8%) presented lower level gains, therefore indicating that more that 25% of neuroblastomas harbour a significant copy num- ber increase at the ALK locus (Fig. 1a). We also analysed the expression pattern of ALK across a series of paediatric tumours and normal tissues. This clearly showed that, whereas other tumour types occasionally expressed ALK, neuroblastoma consistently showed much higher levels of expression (Fig. 1b). In the course of this genomic analysis, a subset of cases without MYCN amplification was further studied by 100K single- nucleotide polymorphism (SNP) arrays. One particularly informative case was identified (NB-99) harbouring a unique and very high-level amplicon that was strictly limited to the ALK gene (Fig. 1c). The obser- vation of such a focal and high-level amplicon suggested that ALK may have a role on its own in neuroblastoma. Because RTK may be activated in human malignancies by genomic amplification, chromosome translocation or point mutations 13 , we wondered whether ALK gene alterations, different from gene amplifica- tion, may be observed in neuroblastoma. We therefore searched for mutations in a first series of 28 cell lines. Single-nucleotide substitu- tions leading to changes of highly conserved amino acid residues were found and prompted us to extend the analysis to frozen tumour sam- ples (115 cases). Altogether, the 16 identified mutations could be grouped into two main hotspots (Table 1). Notably, these changes were not observed in matched constitutional DNAs that were available for nine cases, hence demonstrating their somatic occurrence (Table 1 and Supplementary Fig. 1). In case CLB-GE that combined gene amplifica- tion and somatic mutation of the ALK gene, sequence analysis showed that the amplified and consequently overexpressed allele was the one with the F1174V change (Table 1), therefore suggesting that amplifica- tion and mutation may exert synergistic roles. In other cases, polymer- ase chain reaction with reverse transcription (RT–PCR) analysis indicated that both wild type and mutant alleles were expressed (Table 1). Although a F1174 mutation was observed in one primary tumour (NB-512), changes involving this amino acid were more fre- quent in cell lines, suggesting that they may provide a selective in vitro growth advantage (Table 1). We also investigated the sequence of the kinase domain in six families presenting at least two siblings with neuroblastoma in the absence of malformation phenotype and in three cases associating neuroblastoma and Hirschprung’s disease. A R1275Q heterozygous mutation was identified in one family. It was observed in the two affected children and inherited from the mother who was asympto- matic at 39 years of age (Fig. 1d). A R1192P heterozygous mutation was detected in another family. In this family, three out of six muta- tion carriers developed neuroblastic tumours, being ganglioneuroma in one case and stage 4s neuroblastomas in two other cases (Fig. 1d). No ALK mutation was detected in the other neuroblastoma families nor in Hirschprung’s disease–neuroblastoma associations. 1 Institut Curie, Centre de Recherche, 2 Inserm, U830, 26 rue d’Ulm, Paris F-75248, France. 3 Institut Gustave Roussy, De ´partement de pe ´diatrie, 39 rue Camille Desmoulins, 94805 Villejuif, France. 4 Institut Curie, Unite ´ de Ge ´ne ´tique Somatique, Paris F-75248, France. 5 De ´partement de Ge ´ne ´tique, Universite´ ParisDescartes, Faculte ´ de Me ´decine et INSERM-U781, Ho ˆpital Necker-Enfants Malades, 149, rue de Se `vres, 75743 Paris Cedex 15, France. 6 Centre Le ´on Be ´rard, FNCLCC, Laboratoire de Recherche Translationnelle. 7 Inserm, U590, 8 Universite ´ de Lyon, Lyon1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon F-69008, France. 9 Institut Curie, De ´partement de Pe ´diatrie, Paris F-75248, France. 10 Service de Ge ´ne ´tique, CHU de Rouen et Inserm U614, Faculte ´ de Me ´decine et de Pharmacie, 76183 Rouen Cedex, France. Vol 455 | 16 October 2008 | doi:10.1038/nature07398 967 ©2008 Macmillan Publishers Limited. All rights reserved