SUMMARY Survival rates for the childhood cancer neuroblastoma have not substantively improved despite dramatic escalation in chemotherapy intensity. Like most human cancers, this embryonal malignancy can be inherited, but the genetic etiology of familial and sporadically occurring neuroblastoma was largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase gene (ALK) explain the majority of hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at the short arm of chromosome 2 (maximum nonparametric LOD=4.23 at rs1344063) using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate missense mutations in the tyrosine kinase domain of ALK (G1128A, R1192P and R1275Q) that segregated with the disease in eight separate families. Examination of 491 sporadically occurring human neuroblastoma samples showed that the ALK locus was gained in 22.8%, and highly amplified in an additional 3.3%, and that these aberrations were highly associated with death from disease (P=0.0003). Resequencing of 194 high-risk neuroblastoma samples showed somatically acquired mutations within the tyrosine kinase domain in 12.4%. Nine of the ten mutations map to critical regions of the kinase domain and were predicted to be oncogenic drivers with high probability. Mutations resulted in constitutive phosphorylation consistent with activation, and targeted knockdown of ALK mRNA resulted in profound growth inhibition of 4 of 4 cell lines harboring mutant or amplified ALK, as well as 2 of 6 wild type for ALK. Our results demonstrate that heritable mutations of ALK are the major cause of familial neuroblastoma, and that germline or acquired activation of this cell surface kinase is a tractable therapeutic target for this lethal pediatric malignancy.

Summary Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples; at three hotspots plus 13 minor sites – and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from non-oncogenic mutations and allowed robust computational prediction of their effects. We also established differential in vitro crizotinib sensitivity of mutated variants. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma, and will allow tailoring of ALK-targeted therapy to specific mutations.

Common copy number variations (CNVs) represent a significant source of genetic diversity, yet their influence on phenotypic variability, including disease susceptibility, remains poorly understood. To address this problem in cancer, we performed a genome-wide association study (GWAS) of CNVs in the childhood cancer neuroblastoma, a disease where SNP variations are known to influence susceptibility 1,2 . We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy Supplementary Information is linked to the online version of the paper at www.nature.com/nature Author Contributions S.J.D and J.M.M. designed the study and drafted the manuscript. C.H., C.K., and H.H. performed the genotyping. S.J.D. analyzed SNP data and performed CNV association study. J.B., S.F.A.G., H.H., and H.L. performed the corrections for population stratification. S.J.D., E.F.A. and Y.P.M. analyzed and interpreted SNP data for tumor specimens. K.W. and S.J.D. analyzed SNP data for second replication set. J.G. analyzed SNP data from trios for inheritance estimates. C.H., S.J.D., A.W. and E.R.R. performed and/or analyzed qPCR experiments. E.A.G., K.C., and T.H.S. performed FISH experiments. S.J.D., M.L., K.B., K.P., M.D, and E.R.R. designed and/or performed experiments to identify and sequence transcript within 1q21.1 CNV. J.E.L., C.W., S.J.D. and E.R.R. performed and/or analyzed expression experiments. P.M. and W.L. analyzed clinical covariates. A.I.B. provided detailed endpoints for 1q21.1 CNV in an independent analysis of healthy controls using a custom high-resolution Agilent array. M.D., H.L., and HH contributed to overall GWAS study design. All authors commented on or contributed to the current manuscript. Author InformationThe authors declare no competing interests. Correspondence and requests for materials should be addressed to J.M.M. (maris@chop.edu). Caucasian controls at 550,000 single nucleotide polymorphisms, and performed a CNV-based test for association. We then replicated significant observations in two independent sample sets comprised of a total of 595 cases and 3,357 controls. We identified a common CNV at 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets (P combined = 2.97 × 10 −17 ; OR = 2.49, 95% CI: 2.02 to 3.05). This CNV was validated by quantitative PCR, fluorescent in situ hybridization, and analysis of matched tumor specimens, and was shown to be heritable in an independent set of 713 cancer-free trios. We identified a novel transcript within the CNV which showed high sequence similarity to several "Neuroblastoma breakpoint family" (NBPF) genes 3,4 and represents a new member of this gene family (NBPFX). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and expression level was strictly correlated with CNV state in neuroblastoma cells. These data demonstrate that inherited copy number variation at 1q21.1 is associated with neuroblastoma and implicate a novel NBPF gene in early tumorigenesis of th...

Background-Neuroblastoma is a malignancy of the developing sympathetic nervous system that most commonly affects young children and is often lethal. The etiology of this embryonal cancer is not known.

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-offunction screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC 50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC 50 values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease. N euroblastoma is an embryonal tumor of early childhood thought to arise from fetal sympathetic neuroblasts (1). Children with localized neuroblastoma can be cured with surgery and/or chemotherapy. About half of children with neuroblastoma have high-risk disease, however, characterized by widespread disease dissemination at diagnosis. For these children, current treatment consists of chemotherapy, surgery, external beam radiation therapy, myeloablative chemotherapy with stem cell rescue, and a maintenance therapy regimen combining retinoids and anti-GD2-based immunotherapy (2). Despite the intense multimodality therapy, at least half of high-risk patients will experience relapse that is almost always fatal and survivors show significant morbidity (1).To address the unmet need of identifying bona fide molecular targets for drug development in neuroblastoma, we and others have undertaken comprehensive characterization of the neuroblastoma genome, leading to the identification of mutations in the anaplastic lymphoma kinase gene (ALK) in 10% of newly diagnosed cases (3, 4). Ongoing comprehensive resequencing efforts will likely discover other potential drug targets in this disease. Although these data may identify the critical genes and pathways for drug development efforts, it is also possible that ot...

We conducted a SNP-based genome-wide association study (GWAS) focused on the high-risk subset of neuroblastoma1. As our previous unbiased GWAS showed strong association of common 6p22 SNP alleles with aggressive neuroblastoma2, we now restricted our analysis to 397 high-risk cases compared to 2,043 controls. We detected new significant association of six SNPs at 2q35 within the BARD1 gene locus (Pallelic = 2.35×10−9 − 2.25×10−8). Each SNP association was confirmed in a second series of 189 high-risk cases and 1,178 controls (Pallelic = 7.90×10−7 − 2.77×10−4). The two most significant SNPs (rs6435862, rs3768716) were also tested in two additional independent high-risk neuroblastoma case series, yielding combined allelic odds-ratios of 1.68 each (P = 8.65×10−18 and 2.74×10−16, respectively). Significant association was also found with known BARD1 nsSNPs. These data show that common variation in BARD1 contributes to the etiology of the aggressive and most clinically relevant subset of human neuroblastoma.

Merlin-rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 30:97-101 Cordell HJ (2004) Bias toward the null hypothesis in modelfree linkage analysis is highly dependent on the test statistic used.

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