Activating ALK Tyrosine Kinase Domain Mutations in Neuroblastoma
LJ Tafe, I Yilmaz, A Luina-Contreras, T Mitchell, SC Jhanwar, M Ladanyi, K Nafa. Memorial Sloan-Kettering Cancer Center, New York, NY
Background: Several groups have recently reported germline and somatic mutations in the anaplastic lymphoma kinase (ALK) gene in familial and sporadic cases of neuroblastoma (NB), respectively, with the mutation prevalence ranging from 6 to 11% in sporadic NB. Activating ALK mutations were located in the tyrosine kinase domain (TKD) encoded by exons 20 through 25. Codons F1174 in exons 23 and R1275 in exon 25 were targeted by 78% of the mutations in the initial reports. The ALK locus has also been previously reported to be amplified in NB with or without MYCN amplification. Both MYCN and ALK are located on the short arm of chromosome 2, at 2p24 and 2p23.2, respectively.
Design: We searched for mutations in exons 20 through 25 of ALK in a panel of 191 NB, none of which were known to be familial. PCR amplification of the 6 exons encoding the ALK TKD was performed in 2 multiplex PCRs. PCR products were purified and sequenced with forward primers. All positive cases were re-amplified and sequenced in both forward and reverse directions. Normal tissue from 17 ALK mutation positive patients was also tested to investigate the somatic vs. germline origin of the mutation.
Results: We identified ALK mutations in 24 cases (12.6%) (Table 1). The P1191H mutation has not been previously reported and was found to be a germline heterozygous mutation in this patient. The other 16 normal tissues tested were negative for mutations, supporting ALK mutations as somatically acquired in these patients. No mutations were found in exons 20, 21 or 22. MYCN copy number data were available in all cases, of which 41/191 (21.5%) were amplified. Seven of the 24 ALK mutant cases had MYCN amplification (p=n.s.).
Conclusions: Our results confirm the presence of ALK mutations in a significant minority of NB, including a novel germline P1191H mutation. ALK mutations show no relationship to MYCN amplification. Testing for ALK mutations will help identify patients with NB who may be candidates for trials of ALK-directed kinase inhibitors.
|ALK mutation||Number of Cases|
|Exon 23 F1174L (TTC>CTC)||2 (8.3%)|
|Exon 23 F1174C (TTC>TGC)||1 (4.1%)|
|Exon 23 F1174L (TTC>TTA)||8 (33.3%)|
|Exon 23 F1174L (TTC>TTG)||2 (8.3%)|
|Exon 23 P1191H (CCC>CAC)||1 (4.1%)|
|Exon 24 F1245V (TTC>GTC)||1 (4.1%)|
|Exon 24 F1245C (TTC>TGC)||1 (4.1%)|
|Exon 25 R1275Q (CGA>CAA)||7 (29.2%)|
|Exon 25 R1275L (CGA>CTA)||1 (4.1%)|