Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. Experimental Design: The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. Results: ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. Conclusions: ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants. Clin Cancer Res; 16(17); 4353–62. ©2010 AACR.

ALK (anaplastic lymphoma kinase) is oncogenic in several tumours and has recently been identified as a predisposition gene for familial NB (neuroblastoma) harbouring mutations in the TKD (tyrosine kinase domain). We have analysed a large set of sporadic human NB primary tumours of all clinical stages for chromosomal re-arrangements using a CGH (comparative genomic hybridization) array (n=108) and mutations of the ALK gene (n=90), and expression of ALK and related genes (n=19). ALK amplification or in-gene re-arrangements were found in 5% of NB tumours and mutations were found in 11%, including two novel not previously published mutations in the TKD, c.3733T>A and c.3735C>A. DNA mutations in the TKD and gene amplifications were only found in advanced large primary tumours or metastatic tumours, and correlated with the expression levels of ALK and downstream genes as well as other unfavourable features, and poor outcome. The results of the present study support that the ALK protein contributes to NB oncogenesis providing a highly interesting putative therapeutic target in a subset of unfavourable NB tumours.

Analysis of chromosomal aberrations is used to determine the prognosis of neuroblastomas (NBs) and to aid treatment decisions. MYCN amplification (MNA) alone is an incomplete poor prognostic factor, and chromosome 11q status has recently been included in risk classification. We analyzed 165 NB tumors using high-density SNP microarrays and specifically compared the high-risk groups defined by MNA ( n = 37) and 11q-deletion ( n = 21). Median patient age at diagnosis was 21 months for MNA tumors and 42 months for 11q-deletion tumors, and median survival time after diagnosis was 16 months for MNA and 40 months for 11q deletion. Overall survival (at 8 years) was ∼35% in both groups. MNA and 11q deletion were almost mutually exclusive; only one case harbored both aberrations. The numbers of segmental aberrations differed significantly; the MNA group had a median of four aberrations, whereas the 11q-deletion group had 12. The high frequency of chromosomal breaks in the 11q-deletion group is suggestive of a chromosomal instability phenotype gene located in 11q; one such gene, H2AFX , is located in 11q23.3 (within the 11q-deletion region). Furthermore, in the groups with segmental aberrations without MNA or 11q deletion, the tumors with 17q gain have worse prognosis than those with segmental aberrations without 17q gain, which have a favorable outcome. This study has implications for therapy in different risk groups and stresses that genome-wide microarray analyses should be included in clinical management to fully evaluate risk, aid diagnosis, and guide treatment.

Purpose: In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution. Methods: We analyzed ALK mutations in 54 paired diagnosis–relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000× deep sequencing of the relevant hotspot, with a sensitivity of 0.17%. Results: All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%). Conclusion: In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.