Oral Presentation Advances in Neuroblastoma Research Congress 2016

Frequency of high and low level clonal ALK mutations in high risk neuroblastoma patients. A SIOPEN study. (#84)

Angela Bellini 1 , Virginie Bernard 2 , Eve Lapouble 3 , Nathalie Clement 4 , Sylvain Baulande 5 , Gaelle Pierron 3 , Mathieu Chicard 1 , Inge Ambros 6 , Katleen De Preter 7 , Nadine Van Roy 7 , Ales Vicha 8 , Valérie Combaret 9 , David Betts 10 , Marta Jeison 11 , Smadar Avigad 11 , Martina Morini 12 , Luigi Varesio 12 , Raffaella Defferrari 12 , Katia Mazzocco 6 , Barbara Marques 13 , Annick Muhlethaler 14 , Rosa Noguera 15 , Ana Berbegall 15 , Jaime Font de Mora 16 , Angharad Humphreys , Peter Ambros 6 , Ruth Ladenstein 6 , Ulrike Poetschger 6 , Dominique Valteau-Couanet 17 , Jean Michon 18 , Olivier Delattre 19 , Nick Bown 20 , Deborah Tweddle 21 , Gudrun Schleiermacher 22
  1. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique" and INSERM U830, Institut Curie, Paris, France
  2. Plateforme ICGEX and U900 INSERM, Institut Curie, Paris, France
  3. Unité de Génétique Somatique, Institut Curie, Paris, France
  4. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique", Institut Curie, Paris, France
  5. Plateforme de Séquençage ICGEX, Institut Curie, Paris, France
  6. Children's Cancer Research Institute, Vienna, Austria
  7. Ghent University, Ghent, Belgium
  8. Charles University, Prague, , Czech Republic
  9. Centre Léon Bérard, Lyon, France
  10. Our Lady's Children's Hospital,, Dublin, Ireland
  11. Schneider Children's Medical Center of Israel, Petach Tikvah, Israel
  12. IRCCS Istituto Giannina Gaslini, Genoa, Italy
  13. Instituto Nacional de Saúde Dr. Ricardo Jorge, , Lisboa, Portugal
  14. University Hospital CHUV, Lausanne, Switzerland
  15. University of Valencia, Valencia, Spain
  16. La Fe Hospital Research Institute, Valencia, Spain
  17. Gustave Roussy, Villejuif, France
  18. Institut Curie, Paris, France
  19. U830 INSERM, Institut Curie, Paris, France
  20. Northern Genetics Service, Newcastle, United Kingdom
  21. Northern Institute for Cancer Research Medical School , Newcastle, United Kingdom
  22. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique", INSERM U830 and Department of Pediatric Oncology, Institut Curie, Paris, France

Background: In neuroblastoma (NB), activating ALK receptor tyrosine kinase point mutations are detected in 8-10% of patients at diagnosis using conventional sequencing. To determine the occurrence and the prognostic impact of both high and low level clonal ALK mutations in high-risk NB patients we studied ALK variation frequencies using targeted deep sequencing in samples of patients enrolled in the HR-NBL1/SIOPEN trial.

 

Methods: Diagnostic NB samples from 524 high-risk NB patients were analyzed, focusing on the exons 23, 24 and 25 containing the F1174, F1245 and R1275 hotspots respectively. DNA was amplified via a two-step PCR approach, the second step consisting of addition of sample-specific barcodes for targeted resequencing in a single experiment. Amplicon sequencing (Illumina HiSeq2500) achieved an extremely high depth of coverage (80,000X). The background base variability (error rate) in 32 control samples was 0.017%+/-0.010; thus a base frequency >0.06% was significantly different from background noise (Fisher’s exact test). Mutated allele fractions (MAF) <20% were defined as low level clonal.

 

Results: At the F1174 hotspot, mutations were observed in 28/524 samples, 15 being high (MAF was >20%) and 13 low level clonal (MAF: 0.123%-11.688%). At the R1275 hotspot, mutations were observed in 31/524 samples, 18 high and 13 low level clonal. Finally, at the F1245 hotspot, mutations were observed in 3/524 samples, 2 high and 1 low level clonal. All clonal mutations were validated by Sanger sequencing. Validation of all other events and analysis of additional samples from collaborating groups is ongoing.

 

A correlation between ALK mutations and MYCN amplification was detected (chi-square, p=0.0124). No statistically significant difference in survival of patients with ALK wild-type versus ALK-mutated NB was observed.

 

Conclusion: Our study documents a high frequency of both high level (6.6%) and low level clonal ALK mutations (4.9%) in high-risk NB patients. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution, and the possibility of ALK-targeted therapy.