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Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies-as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

Copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) are two important potential sources of phenotypic variation in humans. Until now, only SNPs have been associated with cancer, but the increasing recognition that germline DNA dosage is a critical component of human diversity raises the possibility that CNVs might also influence susceptibility to this cancer. Diskin et al. now report that a common CNV at chromosome 1q21.1 is associated with the childhood cancer neuroblastoma, and that a transcript within this CNV, the previously unknown neuroblastoma breakpoint family gene NBPF23, is involved in the early stages of tumorigenesis. Copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) are two important potential sources of phenotypic variation in humans; however, only SNPs have been associated with cancer. Here, a CNV at 1q21.1 is shown to be associated with neuroblastoma, and a transcript within this CNV, NBPF23, is implicated in early tumorigenesis of the disease. 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 human cancer, we performed a genome-wide association study of CNVs in the childhood cancer neuroblastoma, a disease in which single nucleotide polymorphism variations are known to influence susceptibility1,2. We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy 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. Here we describe the identification of a common CNV at chromosome 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets. This CNV was validated by quantitative polymerase chain reaction, fluorescent in situ hybridization and analysis of matched tumour specimens, and was shown to be heritable in an independent set of 713 cancer-free parent–offspring trios. We identified a previously unknown transcript within the CNV that showed high sequence similarity to several neuroblastoma breakpoint family (NBPF) genes3,4 and represents a new member of this gene family (NBPF23). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and the 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 previously unknown neuroblastoma breakpoint family gene in early tumorigenesis of this childhood cancer.

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

Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells. Here we investigate the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation and transcription factor binding sites. We show that SNP rs2168101 G>T is the most highly associated variant (combined P = 7.47 × 10(-29), odds ratio 0.65, 95% confidence interval 0.60-0.70), and resides in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G allele that is associated with tumour formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P = 0.028) in neuroblastoma primary tumours, and ablates GATA3 binding (P

Neuroblastoma is a malignant condition of the developing sympathetic nervous system that most commonly affects young children and is often lethal. Its cause is not known.We performed a genomewide association study by first genotyping blood DNA samples from 1032 patients with neuroblastoma and 2043 control subjects of European descent using the Illumina HumanHap550 BeadChip. Samples from three independent groups of patients with neuroblastoma (a total of 720 patients) and 2128 control subjects were then genotyped to replicate significant associations.We observed a significant association between neuroblastoma and the common minor alleles of three consecutive single-nucleotide polymorphisms (SNPs) at chromosome band 6p22 and containing the predicted genes FLJ22536 and FLJ44180 (P=1.71x10(-9) to 7.01x10(-10); allelic odds ratio, 1.39 to 1.40). Homozygosity for the at-risk G allele of the most significantly associated SNP, rs6939340, resulted in an increased likelihood of the development of neuroblastoma (odds ratio, 1.97; 95% confidence interval, 1.58 to 2.45). Subsequent genotyping of the three 6p22 SNPs in three independent case series confirmed our observation of an association (P=9.33x10(-15) at rs6939340 for joint analysis). Patients with neuroblastoma who were homozygous for the risk alleles at 6p22 were more likely to have metastatic (stage 4) disease (P=0.02), amplification of the MYCN oncogene in the tumor cells (P=0.006), and disease relapse (P=0.01).A common genetic variation at chromosome band 6p22 is associated with susceptibility to neuroblastoma.

The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study, we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma. Cancer Res; 72(8); 2068–78. ©2012 AACR.

Neuroblastoma is a cancer of the sympathetic nervous system that accounts for approximately 10% of all pediatric oncology deaths. Here, we report a genome-wide association study of 2,817 neuroblastoma cases and 7,473 controls. We identified two new associations at 6q16, the first within HACE1 (rs4336470; combined P=2.7×10(-11); odds ratio 1.26, 95% confidence interval (CI) 1.18-1.35) and the second within LIN28B (rs17065417; combined P=1.2×10(-8); odds ratio 1.38, 95% CI 1.23-1.54). Expression of LIN28B and let-7 miRNA correlated with rs17065417 genotype in neuroblastoma cell lines, and we observed significant growth inhibition upon depletion of LIN28B, specifically in neuroblastoma cells that were homozygous for the risk allele. Low HACE1 and high LIN28B expression in diagnostic primary neuroblastomas were associated with worse overall survival (P=0.008 and 0.014, respectively). Taken together, these data show that common variants in HACE1 and LIN28B influence neuroblastoma susceptibility and indicate that both genes likely have a role in disease progression.

A genome-wide association study (GWAS) has shown that single nucleotide variants within the LMO1 locus are associated with inherited susceptibility to neuroblastoma, a childhood cancer of the sympathetic nervous system. LMO1 encodes a transcriptional regulator previously linked to cancers. Acquired structural variation in the same locus is common in patients with neuroblastoma, suggesting that loci identified through GWAS approaches might also be prone to somatic alterations that influence tumour progression. Such studies could help to identify potential therapy targets and/or biomarkers of cancer aggressiveness. Here, single nucleotide variants within the LMO1 locus are shown to be associated with inherited susceptibility to neuroblastoma, a childhood cancer of the sympathetic nervous system. Acquired structural variation in the same locus was also frequently found in neuroblastoma patients, leading to the suggestion that loci identified through genome-wide association studies might be also prone to somatic alterations and therefore identify potential therapy targets and/or biomarkers of tumour aggressiveness. Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P