title: Deep Sequencing Reveals Occurrence of Subclonal ALK Mutations in Neuroblastoma at Diagnosis.
abstract: OBJECTIVE
  In neuroblastoma, activating ALK receptor tyrosine kinase point mutations play a major role in oncogenesis. We explored the potential occurrence of ALK mutations at a subclonal level using targeted deep sequencing.
  
  
  METHODS
  In a clinically representative series of 276 diagnostic neuroblastoma samples, exons 23 and 25 of the ALK gene, containing the F1174 and R1275 mutation hotspots, respectively, were resequenced with an extremely high depth of coverage.
  
  
  RESULTS
  At the F1174 hotspot (exon 23), mutations were observed in 15 of 277 samples (range of fraction of mutated allele per sample: 0.562%-40.409%). At the R1275 hotspot (exon 25), ALK mutations were detected in 12 of 276 samples (range of fraction of mutated allele: 0.811%-73.001%). Altogether, subclonal events with a mutated allele fraction below 20% were observed in 15/27 ALK-mutated samples. The presence of an ALK mutation was associated with poorer 5-year overall survival (OS: 75% vs. 57%, P = 0.0212 log-rank test), with a strong correlation between F1174 ALK mutations and MYCN amplification being observed.
  
  
  CONCLUSIONS
  In this series, deep sequencing allows the detection of F1174 and R1275 ALK mutational events at diagnosis in 10% of cases, with subclonal events in more than half of these, which would have gone undetected by Sanger sequencing. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution and relapse. These findings also demonstrate the importance of deep sequencing techniques for the identification of patients especially when considering targeted therapy.
authors.affiliation: Equipe SiRIC RTOP Recherche Translationelle en Oncologie Pédiatrique, Institut Curie, Paris, France. INSERM U830, Laboratoire de Génétique et Biologie des Cancers, Institut Curie, Paris, France.
authors.forename: Angela
authors.initials: A
authors.lastname: Bellini
authors.affiliation: Plateforme de Séquençage ICGex, Institut Curie, Paris, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Bernard
authors.affiliation: Plateforme de Séquençage ICGex, Institut Curie, Paris, France.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Leroy
authors.affiliation: Plateforme de Séquençage ICGex, Institut Curie, Paris, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rio Frio
authors.affiliation: Unité de Génétique Somatique, Institut Curie, Paris, France.
authors.forename: Gaelle
authors.initials: G
authors.lastname: Pierron
authors.affiliation: Laboratoire de Recherche Translationnelle, Centre Léon-Bérard, Lyon, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Combaret
authors.affiliation: Unité de Génétique Somatique, Institut Curie, Paris, France.
authors.forename: Eve
authors.initials: E
authors.lastname: Lapouble
authors.affiliation: Equipe SiRIC RTOP Recherche Translationelle en Oncologie Pédiatrique, Institut Curie, Paris, France. Département de Pédiatrie, Institut Curie, Paris, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Clement
authors.affiliation: Unite d'Hemato-Oncologie, Hôpital des Enfants, Toulouse, France.
authors.forename: Herve
authors.initials: H
authors.lastname: Rubie
authors.affiliation: Service d'Hémato-Oncologie Pédiatrique, CHU Nantes, Nantes, France.
authors.forename: Estelle
authors.initials: E
authors.lastname: Thebaud
authors.affiliation: Service d'Immuno-Hémato-Oncologie, Hôpital d'Enfants, Nancy, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Chastagner
authors.affiliation: Service d'Oncologie Pédiatrique, Centre Oscar Lambret, Lille, France.
authors.forename: Anne Sophie
authors.initials: AS
authors.lastname: Defachelles
authors.affiliation: Institut d'Hematologie et d'Oncologie Pédiatrique, Centre Léon Bérard, Lyon, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Bergeron
authors.affiliation: Service d'Immuno-Hémato-Oncologie Pédiatrique, Hôpital Charles Nicolle, Rouen, France.
authors.forename: Nimrod
authors.initials: N
authors.lastname: Buchbinder
authors.affiliation: Service d'Hématologie et de Cancérologie, CHU Hôpital Sud, Rennes, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Taque
authors.affiliation: Service d'Hématologie et d'Oncologie Pédiatrique, Hôpital d'Enfants Armand-Trousseau, Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Auvrignon
authors.affiliation: Departement de Pédiatrie, Institut Gustave Roussy, Villejuif, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Valteau-Couanet
authors.affiliation: Département de Pédiatrie, Institut Curie, Paris, France.
authors.forename: Jean
authors.initials: J
authors.lastname: Michon
authors.affiliation: INSERM U830, Laboratoire de Génétique et Biologie des Cancers, Institut Curie, Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Janoueix-Lerosey
authors.affiliation: INSERM U830, Laboratoire de Génétique et Biologie des Cancers, Institut Curie, Paris, France. Unité de Génétique Somatique, Institut Curie, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Delattre
authors.affiliation: Equipe SiRIC RTOP Recherche Translationelle en Oncologie Pédiatrique, Institut Curie, Paris, France. INSERM U830, Laboratoire de Génétique et Biologie des Cancers, Institut Curie, Paris, France. Département de Pédiatrie, Institut Curie, Paris, France. gudrun.schleiermacher@curie.fr.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Schleiermacher
mesh_terms: D000293:Adolescent; D000328:Adult; D000483:Alleles; D000077548:Anaplastic Lymphoma Kinase; D002648:Child; D002675:Child, Preschool; D060965:Clonal Evolution / Q000235:genetics*; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D005784:Gene Amplification; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009364:Neoplasm Recurrence, Local; D009367:Neoplasm Staging; D009447:Neuroblastoma / Q000175:diagnosis / Q000235:genetics* / Q000401:mortality; D011379:Prognosis; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D055815:Young Adult
chemical_list: C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases