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Supplementary Methods

Evidence supporting multiple lncRNA transcript isoforms mapping to chromosome 6p22.3.

List of imputation results at the 6p22.3 region.

rs6939340 genotype and expression of 6p22.3 gene products.

Supplementary Table 1. Functional prioritization of SNPs in linkage disequilibrium (r2=0.6) with significant rs4673067 at SCG2 locus. Supplementary Table 2. SNPs in LD (r2>0.60) with the SNP rs118727. Supplementary Table 3. SNPs in LD (r2>0.60) with the SNP rs196830. Supplementary Table 4. SNPs in LD (r2>0.60) with the SNP rs169061. Supplementary Table 5. SNPs in LD (r2>0.60) with the SNP rs11994014. Supplementary Table 6. SNPs in LD (r2>0.60) with the SNP rs17830286. Supplementary Table 7. Functional prioritization of SNPs in 3'UTR region of NEFL and in linkage disequilibrium (r2=0.6) with significant typed SNPs rs118727, rs196830, rs17830286, rs11994014. Supplementary Table 8. Prediction of mircoRNAs whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by mrSNP web tool. Supplementary Table 9. Prediction of transcriptional factors whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by HaploReg V2. Supplementary Table 10. List of UTR motifs analyzed for the SNPs rs3761, rs2979704, and rs1059111. Supplementary Table 11. Imputation results of SNPs at SCG2 and NEFL loci (+/- 1Kb) by using 1000 Genomes data. Supplementary Table 12. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in European American cohort. Supplementary Table 13. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in Italian cohort. Supplementary Figure 1. Linkage disequilibrium (LD) plot of NEFL gene (chr8: positions 24,845,004 to 24,887,674) by Haploview 4.2 for HapMap CEU subjects. Supplementary Figure 2. REST gene silencing in neuroblastoma cells. The efficiency of gene silencing mediated by lentiviral delivery of hairpin RNA directed against REST (shREST) in SH-SY5Y and SK-N-BE2c cell lines was assessed by western blotting (a-b). The bar graphs show integral optical density (OD) value for each band, normalized respect to β-Actin expression. The results are shown as mean of three experiments and are represented as fold respect to shCTR cells which are infected by lentivirus-mediated delivery of non-silencing hairpin RNA. Induction of NEFL and REST gene expression levels after REST silencing in SH-SY5Y (c) and SK-N-BE2c (d). *P

Depletion of the long isoform of CASC15 does not impact neuroblastoma cell viability.

Full Table for the CASC15-S Cox Statistical Model.

Additional expression data for CASC15 isoforms in neuroblastoma cell lines and patients.

Supplementary Figure Legends

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Bioinformatical support for CASC15-S as a long noncoding transcript.

Cell viability assays for neuroblastoma cell lines stably depleted of CASC15-S.

Results of Sanger sequencing from 5' and 3'-RACE products of CASC15-S.

TMEFF2, a transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is primarily restricted in expression to healthy brain and prostate and is enriched in prostate cancer. JNJ-70218902 (JNJ-902) is a bispecific antibody that engages TMEFF2-expressing tumor cells and CD3 on T cells causing exposure dependent pro-inflammatory responses and target tumor cell lysis. Here we report the preclinical characterization of JNJ-902 using in vitro and in vivo prostate cancer models. Expression of TMEFF2 transcript and protein in brain and prostate was confirmed in healthy human tissue, as was its elevated expression in advanced prostate cancer tumors, including in 41/45 (91%) metastatic castration-resistant prostate cancer (mCRPC) samples. JNJ-902 bound to TMEFF2-positive LNCaP-AR cells (androgen-sensitive human prostate cancer cell line) in a concentration-dependent manner (EC50 = 9.6 nM); no binding to TMEFF2-negative DU145 prostate cancer cells was observed. The binding affinity (KD) of JNJ-902 for CD3 on primary human T cells was determined to be ~151 nM. T cell-mediated killing, as measured by caspase-3 activity, was induced upon incubation of JNJ-902 with healthy human donor T cells (n = 5) and LNCaP-AR cells at a 3:1 effector to target ratio (EC50 = 1.4 nM). No cytotoxicity was detected using DU145 cells or with control antibodies. Corresponding T cell activation was confirmed by concentration-dependent increases in cell surface CD8+CD25+ expression and proinflammatory cytokine production (eg, GM-CSF, IFN-γ, IL-10, TNF-α). Antitumor activity of JNJ-902 at increasing concentrations up to 5 mg/kg was observed in T cell humanized NSG mice bearing LNCaP xenografts (tumor growth inhibition [∆%TGI] of 75-122%) and in LuCaP 86.2 patient-derived xenografts (∆%TGI of 72-88%) as compared with control-treated mice. JNJ-902-dependent intratumoral infiltration of CD8+ T cells was detected in both models with increases in frequency of CD8+granzymeB+ effector cells. The data summarized here are the first report on the pharmacological activity of a novel TMEFF2 × CD3 bispecific antibody, supporting further investigation of TMEFF2 in mCRPC given its expression in advanced prostate cancer. Targeting TMEFF2 with the TMEFF2 × CD3 bispecific antibody JNJ-902 elicited robust T cell-mediated responses in both prostate cancer cells and xenograft models. Based on these data, a phase 1 dose escalation trial of JNJ-902 in patients with mCRPC is currently ongoing (NCT04397276). Citation Format: Subhasree Basu, Mike Russell, Katrin Sproesser, Bethany Mattson, Cassandra L. Lowenstein, Sathya Venkataramani, Maura Diamond, Robin E. Ernst, Stephen Jarantown, Jackson Wong, Gerald Chu, Kathryn Packman, Shaozhou Ken Tian, Scott R. Brodeur, Margaret Yu, Brent A. Rupnow. Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB087.

MYCN amplification and 11q deletion are two inversely correlated prognostic factors of poor outcome in neuroblastoma. Here we identify common variants at 11q22.2 within MMP20 that associate with neuroblastoma cases harboring 11q deletion (rs10895322), using GWAS in 113 European-American cases and 5109 ancestry-matched controls. The association is replicated in 44 independent cases and 1902 controls. Our study yields novel insights into the genetic underpinnings of neuroblastoma, demonstrating that the inherited common variants reported contribute to the origin of intra-tumor genetic heterogeneity in neuroblastoma., Chromosomal abnormalities such as 11q deletion are associated with poor prognosis in neuroblastoma. Here, the authors perform a genome-wide association study and identify an association between a variant within a Matrix metalloproteinase (MMP) gene member, MMP20, and 11q-deletion subtype neuroblastoma.

Scientific Data 4:170033 doi: 10.1038/sdata.2017.33 (2017); Published 28 March 2017; Updated: 5 December 2017. The growth media for the cell lines CHP-212 and IMR-32 listed in Table 1 of this Data Descriptor were incorrectly stated. The cell line labelled as ‘NB-16’ in Fig. 2, Table 1, and Table 2 was incorrectly recorded.

Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma.

Neuroblastoma is a cancer of the developing sympathetic nervous system that most commonly presents in young children and accounts for approximately 12% of pediatric oncology deaths. Here, we report on a genome-wide association study (GWAS) in a discovery cohort or 2,101 cases and 4,202 controls of European ancestry. We identify two new association signals at 3q25 and 4p16 that replicated robustly in multiple independent cohorts comprising 1,163 cases and 4,396 controls (3q25: rs6441201 combined P = 1.2x10-11, Odds Ratio 1.23, 95% CI:1.16–1.31; 4p16: rs3796727 combined P = 1.26x10-12, Odds Ratio 1.30, 95% CI: 1.21–1.40). The 4p16 signal maps within the carboxypeptidase Z (CPZ) gene. The 3q25 signal resides within the arginine/serine-rich coiled-coil 1 (RSRC1) gene and upstream of the myeloid leukemia factor 1 (MLF1) gene. Increased expression of MLF1 was observed in neuroblastoma cells homozygous for the rs6441201 risk allele (P = 0.02), and significant growth inhibition was observed upon depletion of MLF1 (P < 0.0001) in neuroblastoma cells. Taken together, we show that common DNA variants within CPZ at 4p16 and upstream of MLF1 at 3q25 influence neuroblastoma susceptibility and MLF1 likely plays an important role in neuroblastoma tumorigenesis., Author summary Neuroblastoma is an embryonal tumor of the developing sympathetic nervous system that accounts for 12% of childhood cancer deaths. Approximately 1–2% of cases are inherited in an autosomal dominant fashion. These familial cases often harbor germline mutations in ALK or PHOX2B. However, the vast majority of neuroblastomas appear to arise sporadically. We are studying sporadic neuroblastoma through an ongoing genome-wide association study (GWAS). To date, this effort has identified single nucleotide polymorphisms (SNPs) within or upstream of CASC15 and CASC14, BARD1, LMO1, DUSP12, HSD17B12, DDX4/IL31RA, HACE1, LIN28B, and TP53, along with a common copy number variation (CNV) within NBPF23 at chromosome 1q21.1, each being highly associated with neuroblastoma. Here, we report on genome-wide association study (GWAS) comprising 3,264 neuroblastoma patients and 8,598 control subjects. We identify two new association signals at 3q25 and 4p16 (3q25: rs6441201 combined P = 1.2x10-11, Odds Ratio 1.23, 95% CI:1.16–1.31; 4p16: rs3796727 combined P = 1.26x10-12, Odds Ratio 1.30, 95% CI: 1.21–1.40). The 3q25 signal resides upstream of the MLF1 gene and the 4p16 signal maps to the CPZ gene. We further demonstrate that neuroblastoma cells homozygous for the risk allele at 3q25 express higher levels of MLF1 and that silencing of MLF1 in neuroblastoma cells results in significant growth inhibition.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers., National Human Genome Research Institute (U.S.) (Grant U54HG003067), National Cancer Institute (U.S.) (Contract HHSN261200800001E)

To investigate genetic predispositions for MYCN-amplified neuroblastoma, we performed a meta-analysis of three genome-wide association studies totaling 615 MYCN-amplified high-risk neuroblastoma cases and 1869 MYCN-nonamplified non-high-risk neuroblastoma cases as controls using a fixed-effects model with inverse variance weighting. All statistical tests were two-sided. We identified a novel locus at 3p21.31 indexed by the single nucleotide polymorphism (SNP) rs80059929 (odds ratio [OR] = 2.95, 95% confidence interval [CI] = 2.17 to 4.02, Pmeta = 6.47 × 10-12) associated with MYCN-amplified neuroblastoma, which was replicated in 127 MYCN-amplified cases and 254 non-high-risk controls (OR = 2.30, 95% CI = 1.12 to 4.69, Preplication = .02). To confirm this signal is exclusive to MYCN-amplified tumors, we performed a second meta-analysis comparing 728 MYCN-nonamplified high-risk patients to identical controls. rs80059929 was not statistically significant in MYCN-nonamplified high-risk patients (OR = 1.24, 95% CI = 0.90 to 1.71, Pmeta = .19). SNP rs80059929 is within intron 16 in the KIF15 gene. Additionally, the previously reported LMO1 neuroblastoma risk locus was statistically significant only in patients with MYCN-nonamplified high-risk tumors (OR = 0.63, 95% CI = 0.53 to 0.75, Pmeta = 1.51 × 10-8; Pmeta = .95). Our results indicate that common genetic variation predisposes to different neuroblastoma genotypes, including the likelihood of somatic MYCN-amplification.

Background: Neuroblastoma is an often fatal pediatric cancer more frequent in European-American than African-American children. African-American children, however, are at higher risk for the more severe form of neuroblastoma and have worse overall survival than European-American children. Genome-wide association studies (GWAS) have identified several single-nucleotide polymorphisms (SNP) associated to neuroblastoma in children of European descent. Knowledge of their association to neuroblastoma in African-American children is still lacking. Methods: We genotyped and imputed SNPs located in three gene regions reported to be associated to neuroblastoma in children of European descent, and tested them for association in 390 African-American patients with neuroblastoma compared with 2,500 healthy, ethnically matched controls. Results: SNPs in the BARD1 gene region show a similar pattern of association to neuroblastoma in African-American and European-American children. The more restricted extent of linkage disequilibrium in the African-American population suggests a smaller candidate region for the putative causal variants than previously reported. Limited association was observed at the other two gene regions tested, including LMO1 in 11p15 and FLJ22536 in 6p22. Conclusions: Common BARD1 SNPs affect risk of neuroblastoma in African-Americans. The role of other SNPs associated to neuroblastoma in children of European descent could not be confirmed, possibly due to different patterns of linkage disequilibrium or limited statistical power to detect association to variants with small effect on disease risk. Extension of GWAS to populations of African descent is important to confirm their results and validity beyond the European populations and can help to refine the location of the putative causal variants. Cancer Epidemiol Biomarkers Prev; 21(4); 658–63. ©2012 AACR.

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

We recently showed that relapsed neuroblastoma (NB) tumors harbor an increased somatic mutational burden, enriched for ALK or RAS-MAPK activating lesions, nominating targeted therapeutic strategies for a condition that is largely incurable. We also showed that these mutations can be present in subclonal tumor cell populations at diagnosis, suggesting that they may contribute to acquired chemoradiotherapy resistance. We hypothesize that subclonal mutations in NB oncogenes are common at diagnosis, are biomarkers for treatment failure, and can be targeted in conjunction with standard chemotherapy to improve patient survival. Here, we focus on the first aspect of this broad hypothesis by presenting the subclonal landscape of diagnostic, high-risk neuroblastomas. A meta-analysis of our previously published primary/relapse (N=23), whole exome (N=232) and whole genome (N=159) diagnostic data, and recent sequencing study of 78 diagnostic and 67 relapse cases were used to inform design of a custom amplicon panel for ultra-deep sequencing. The 28-gene panel covers 57 mutations in frequently mutated genes. Here, 250 high-risk primary tumors were sequenced to an average depth of 50,000X. More subclonal mutations were called (mean=36) compared to clonal alterations (mean=3), demonstrating the complex subclonal architecture in these tumors. We validated five clonal ALK mutations we originally discovered and identified 15 additional pathogenic ALK mutations with mutant allele frequencies (MAFs) ranging from 0.03-23%. Known ALK activating mutations were found at codons 1170, 1174, 1196, 1245, 1275 in 15 of 38 diagnostic primary tumors. This 39.5% frequency is much higher than the 14% frequency defined by us using Sanger or other sequencing methods. Improvement of subclonal mutation detection of actionable, relapse-specific driver mutations demonstrates the clinical utility of utilizing this sequencing methodology at diagnosis to enable timely improvement of outcomes for children with high-risk refractory NB. Citation Format: Jo Lynne Harenza, Maura A. Diamond, Derek A. Oldridge, Olivia Padovan-Merher, Pichai Raman, Yael P. Mosse, John M. Maris. Defining the subclonal landscape of high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2017-3000

Introduction: The 5 year mortality rate for stage 4 metastatic neuroblastoma (NBL) is ~50%; however, the stage 4S subset of NBL is typically classified as low-risk with tumors exhibiting spontaneous regression. To date, the underlying genetic mechanisms influencing this distinct phenotype remain unknown. Method: To identify genetic determinants specific to the 4S phenotype, we performed genome-wide association study (GWAS) analyses. Blood DNA samples from 6,195 NBL patients and 11,384 non-NBL controls were genotyped using Illumina SNP arrays. European ancestry subjects were selected for inclusion in a discovery [stage 4S (n=148), stage 4 (n=996), non-NBL controls (n=9,923)] or replication cohort [stage 4S (n=76), stage 4 (n=608), non-NBL controls (n=1,461)]. Following imputation using 1KG Phase 3, we conducted case:case comparisons including stage 4S vs. 4 and stage 4S vs. 1, followed by comparisons of NBL cases in each group to matched non-NBL controls. Variants uniquely associated with 4S NBL were defined as those meeting the following criteria: (1) stage 4S vs. 4 meta P < 5.0 x 10-5 and P < 0.005 in the discovery cohort (2) 4S vs. non-NBL control P < 0.05 and (3) either not significant (P > 0.05) or odds ratio in the opposite direction in 4 vs. non-NBL controls. Results: 4S-specific variants that met our criteria spanned multiple regions with several harboring genes important to neuronal function including 9q34 (SARDH/DBH; rs2502746), 1p32.2 (KCNH1; rs12122529), 1p34 (KCNQ4; chr 1:41317634), 7q34 (CNTNAP2; rs79403134) and 2q24.1 (KCNJ3; rs78829625). At the 9q34 locus, rs2502746 showed further evidence as an expression quantitative trait locus (eQTL) for Dopamine Beta-Hydroxylase Antisense RNA1( DBH-AS1 P = 1.9 x 10-5 sun-exposed skin) and Sarcosine Dehydrogenase SARDH expression (P = 2.5 x 10-6 whole blood). Other 4S-specific loci observed include 3q27.3 (rs113207181), 8p21.3 (rs12548388), and 15q12 (rs62189512). Notably, none of the previously identified NBL susceptibility loci were found to be associated with 4S NBL in the 4S vs. non-NBL control comparison, suggesting a unique genetic basis for this enigmatic subset. Discussion: Although a unifying mechanism underlying 4S NBL has yet to be identified, our results suggest a potential role for ion-channel and neuronal function genes. Ongoing efforts include investigating the role these genes and associated pathways play in NBL, including an integrative genomic, epigenomic and transcriptome analysis of stage 4 and 4S NBL. Citation Format: Zalman Vaksman, Lee D. McDaniel, Maura Diamond, John M. Maris, Sharon J. Diskin. Identifying the genetic basis of stage 4S neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 690. doi:10.1158/1538-7445.AM2017-690

TP53 is the most frequently mutated gene in human malignancies; however, de novo somatic mutations in childhood embryonal cancers such as neuroblastoma are rare. We report on the analysis of three independent case-control cohorts comprising 10290 individuals and demonstrate that rs78378222 and rs35850753, rare germline variants in linkage disequilibrium that map to the 3' untranslated region (UTR) of TP53 and 5' UTR of the Δ133 isoform of TP53, respectively, are robustly associated with neuroblastoma (rs35850753: odds ratio [OR] = 2.7, 95% confidence interval [CI] = 2.0 to 3.6, P combined = 3.43×10(-12); rs78378222: OR = 2.3, 95% CI = 1.8 to 2.9, P combined = 2.03×10(-11)). All statistical tests were two-sided. These findings add neuroblastoma to the complex repertoire of human cancers influenced by the rs78378222 hypomorphic allele, which impairs proper termination and polyadenylation of TP53 transcripts. Future studies using whole-genome sequencing data are likely to reveal additional rare variants with large effect sizes contributing to neuroblastoma tumorigenesis.

The genetic etiology of sporadic neuroblastoma is still largely obscure. In a genome-wide association study, we identified single-nucleotide polymorphisms (SNP) associated with neuroblastoma at the CASC15, BARD1, LMO1, DUSP12, HSD17B12, HACE1, and LIN28B gene loci, but these explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the multiple testing corrections. In this study, we evaluated eight additional genes selected as candidates for further study based on proven involvement in neuroblastoma differentiation. SNPs at these candidate genes were tested for association with disease susceptibility in 2,101 cases and 4,202 controls, with the associations found replicated in an independent cohort of 459 cases and 809 controls. Replicated associations were further studied for cis-effect using gene expression, transient overexpression, silencing, and cellular differentiation assays. The neurofilament gene NEFL harbored three SNPs associated with neuroblastoma (rs11994014: Pcombined = 0.0050; OR, 0.88; rs2979704: Pcombined = 0.0072; OR, 0.87; rs1059111: Pcombined = 0.0049; OR, 0.86). The protective allele of rs1059111 correlated with increased NEFL expression. Biologic investigations showed that ectopic overexpression of NEFL inhibited cell growth specifically in neuroblastoma cells carrying the protective allele. NEFL overexpression also enhanced differentiation and impaired the proliferation and anchorage-independent growth of cells with protective allele and basal NEFL expression, while impairing invasiveness and proliferation of cells homozygous for the risk genotype. Clinically, high levels of NEFL expression in primary neuroblastoma specimens were associated with better overall survival (P = 0.03; HR, 0.68). Our results show that common variants of NEFL influence neuroblastoma susceptibility and they establish that NEFL expression influences disease initiation and progression. Cancer Res; 74(23); 6913–24. ©2014 AACR.

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

Background. Neuroblastoma is a malignancy of the developing sympathetic nervous system which exacts significant morbidity and mortality in children. Large sequencing efforts have revealed a relative paucity of somatic point mutations in this and other childhood cancers. Structural variations (SVs) including translocations, inversions, deletions, duplications, and other complex events can occur in germline DNA or be acquired somatically in tumors. Recurrent somatic SVs affecting known genes are likely to be functional and may elucidate novel tumor suppressors or oncogenes. Methods. We are performing whole-genome sequencing (WGS) of 187 matched tumor-normal pairs from high-risk patients as part of the NCI-TARGET project (http://www.ocg.cancer.gov/programs/target) using Complete Genomics technology. High confidence somatic SVs were identified and a subset subsequently validated with Sanger sequencing; in vitro functional studies were performed using human derived neuroblastoma cell line models. Results. To date, we have analyzed WGS data from 106 matched stage 4 tumor-normal pairs. Among 2,770 high-confidence somatic SVs (average 26.1 per tumor, range 1-158), we observed recurrent focal deletions in ZFHX3 (n = 5) and EZH2 (n = 2), in addition to ATRX (n = 2), ARID1B (n = 2), and PTPRD (n = 5), each of the latter genes previously reported in neuroblastoma. A total of 210 inter-chromosomal translocations disrupting known genes were detected (average 2.0 per tumor, range 0-12). Notably, 14.4% of MYCN non-amplified stage 4 tumors (11/76) harbored SV breakpoints within SHANK2 at 11q13; all SHANK2 breakpoints were validated by Sanger sequencing. With the exception of a single deletion at the c-terminus, all breakpoints mapped within the long isoform of SHANK2 (transcript variant 1: NM_012309), and clustered within the ankyrin repeat domain which is involved in protein-protein interactions. Low SHANK2 expression in primary tumors obtained at diagnosis was associated with worse overall survival (p = 6.3 × 10−5), suggesting SHANK2, known to regulate neuronal differentiation, may function as a tumor suppressor. Consistent with this hypothesis, mRNA expression of SHANK2 was low in a panel of 27 neuroblastoma cell lines and further silencing with siRNA did not produce an observable phenotype. In contrast, forced expression of the long isoform of SHANK2 in three independent neuroblastoma cell lines with low endogenous SHANK2 levels resulted in profound reduction in cell growth (p < 0.0001) and viability (p < 0.0001) as measured by RTCES and CellTiter-glo assays respectively. Conclusion. A plethora of SVs exist in neuroblastoma, and unlike somatic point mutations, many are recurrent. Studies are ongoing to determine the mechanism by which SHANK2 suppresses neuroblastoma tumorigenesis and to understand the biological relevance of other recurrent coding and non-coding SVs in this childhood malignancy. Citation Format: Karina Conkrite, Nicole Ferraro, Lee McDaniel, Derek A. Oldridge, Edward Attiyeh, Shahab Asgharzadeh, Maura Diamond, Jaime Guidry Auvil, Tanja Davidsen, Malcom Smith, Wendy B. London, Robert Seeger, Javed Khan, Daniela S. Gerhard, John M. Maris, Sharon J. Diskin. Identification of SHANK2 as a tumor suppressor disrupted by recurrent somatic structural variation (SV) in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 475. doi:10.1158/1538-7445.AM2015-475

Background: Neuroblastoma, a clinically heterogeneous childhood tumor, presents as high-risk disease in 40% of diagnoses and is fatal in roughly half of these patients. In an effort to elucidate the genetic basis of high-risk disease, our lab previously undertook a genome-wide association study (2101 cases, 4202 controls), identifying a cluster of single-nucleotide polymorphisms on chromosome 6p associate with an increased risk of developing of high-risk disease (p < 1e-15). Here we utilized fine mapping to demonstrate that the highly most significant single nucleotide polymorphism (SNP) associations reside within a long intergenic non-coding RNA, CASC15, which we hypothesize plays a critical role in the development of high-risk neuroblastoma through the dysregulation of neuronal growth and differentiation pathways. Methods: CASC15 expression data was obtained from patient samples (n = 251, exon-level expression arrays), cell lines (qRT-PCR) and 108 primary neuroblastomas (RNA-Seq). Subcellular localization of CASC15-S was determined bioinformatically, as well as by RNA-FISH and 5′/3′ RACE. Depletion was carried out using both siRNA and shRNA constructs, and cell viability and growth kinetics were measured using the CellTiter-Glo and Xcelligence platforms. Gene expression analyses of neuroblastoma cell lines stably silenced for CASC15 was accomplished via Transcriptome 2.0 arrays followed by gene set enrichment and Ingenuity pathway analysis. Results: Regional imputation of the 6p22.3 locus refined our initial GWAS signal, and resulted in the validation CASC15-S as a functional gene isoform in neuroblastoma. Subsequent stratification of these imputed polymorphisms identified a functional SNP upstream of CASC15-S, rs9295534, which exhibits enhancer activity. Patients with high-risk disease express substantially less CASC15-S than low-risk patients (p < 0.0001), and lower CASC15-S levels correlate with poor overall survival (adj. p = 3.2e-06). CASC15-S depletion increases cellular proliferation, with ectopic overexpression of the CASC15-S transcript sufficient to revert this phenotype. Cells stably depleted of CASC15-S demonstrate overt morphological changes suggestive of a poorly differentiated phenotype, and gene expression pathway analyses corroborate these observations as cells significantly downregulated proneural gene pathways, while increasing cell motility and growth pathways. Lastly, consistent with lncRNA effects on proximal genes involved in development, CASC15-S expression is highly correlated with neighboring SOX4 mRNA levels (r = 0.76), and chromatin looping suggests putative interactions between these two genomic regions. Conclusions: These data suggest that common genetic variation at 6p22 influences CASC15-S expression, thus impacting neural growth and differentiation pathways as well as impacting neuroblastoma initiation and progression. Citation Format: Mike R. Russell, Annalise Penikis, Derek Oldridge, Juan R. Alvarez-Dominguez, Lee McDaniel, Maura Diamond, Olivia Padovan, Pichai Raman, Yimei Li, Jun Wei, Shile Zhang, Janahan Gnanachandran, Robert Seeger, Shahab Asgharzadeh, Javed Khan, Sharon Diskin, John Maris, Kristina Cole. CASC15 is a tumor suppressor lncRNA at the 6p22 neuroblastoma susceptibility locus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 144. doi:10.1158/1538-7445.AM2015-144

Background: Our ongoing genome-wide association study (GWAS) in neuroblastoma has identified multiple common and rare polymorphisms highly associated with disease susceptibility and phenotype. SNPs at the LMO1 locus showed one of the most robust associations and were associated with LMO1 overexpression and oncogenicity in preclinical models (Nature, 2011). Here we seek to define the DNA polymorphism(s) responsible for the GWAS association and determine the mechanism by which common variation determines tumor aggressiveness. Methods: Using 1000 Genomes data, we imputed all known variants within a 500-kilobase window around the LMO1 gene in a discovery set of 2,101 cases and 4,202 controls of European ancestry using the IMPUTE2 algorithm and performed association testing with SNPTEST. We further prioritized significant variants by evolutionary conservation based on comparative genome analysis of 46 mammalian species. Identification of non-coding variant regions with regulatory potential was performed through analysis of neuroblastoma-specific DNase I hypersensitivity mapping and chromatin immunoprecipitation (ChIP) sequencing data generated through the ENCODE consortium. Identification of canonical transcription factor binding motifs was performed using the MATCH-TM algorithm and JASPAR motif database. Independent replication of the top candidate causal SNP was performed by PCR-based genotyping in case series from Italy (420 cases, 751 controls) and the United Kingdom (369 cases, 1,108 controls). For statistical testing, homozygous and heterozygous odds ratios (OR) were computed from a comparison of (TT vs. GG) and (GT vs. GG) genotypes, respectively, and a single chi-squared p-value was computed from the 2x3 contingency table of phenotype vs. genotype. Results: The most significantly associated SNP at the LMO1 locus (rs2168101, homozygous OR=0.55, heterozygous OR=0.69, p-value=6.18x10-21) resides within an active enhancer region inferred by DNase I sequencing and histone modification ChIP-seq profiling in the SKNSH neuroblastoma cell line. Furthermore, rs2168101 lies within a GATA motif with perfect evolutionary conservation (5′-AGATAA-3′, phastCons score=100% across 46 mammalian species) that robustly binds to GATA2 and GATA3 transcription factors by ChIP-seq profiling. Consistent with an enhancer model, the risk “G” allele preserves the GATA motif and is associated with higher levels of LMO1 expression across a panel of 24 neuroblastoma cell lines (t-test p=0.047). Additionally, primary tumors with heterozygous G/T genotypes exhibit a greater degree of allele-specific expression imbalance relative to homozygous tumors by RNA-sequencing (t-test p=4.42x10-4), consistent with an effect in cis. In follow-up to imputation-based analysis, robust replication of the rs2168101 neuroblastoma association was observed by direct genotyping in an independent Italian cohort (homozygous OR=0.40, heterozygous OR=0.57, chi-squared p=9.94x10-6) and UK cohort (homozygous OR=0.31, heterozygous OR=0.51, chi-squared p=2.44x10-9). Furthermore, rs2168101 genotype was also associated with high-risk neuroblastoma and outcome (homozygous OR=0.67, heterozygous OR=0.74, p-value=2.16x10-4). Conclusions: Integrative genomic analysis provides mechanistic insight into how germline variation within non-coding DNA can impact neuroblastoma susceptibility and prognosis, supporting the hypothesis that the risk “G” allele at rs2168101 promotes oncogenic LMO1 overexpression through increased GATA transcription factor binding within an active enhancer region. Functional validation experiments are currently underway, including ChIP-PCR of the enhancer locus and targeted genome editing of the GATA binding site. Citation Format: Derek A. Oldridge, Andrew Wood, Cindy Winter, Maura Diamond, Ian Crimmins, Shile Zhang, Jun Wei, Javed Khan, Mario Capasso, Nazneen Rahman, Sharon J. Diskin, John M. Maris. A noncoding polymorphism in a GATA-containing enhancer element drives the association of LMO1 with neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A27.

Background: Neuroblastoma is a clinically heterogeneous childhood tumor that presents as high-risk disease in 40% of diagnoses, and is fatal in roughly half of these patients despite aggressive multi-modal therapy. In an effort to understand the etiology of high-risk neuroblastoma, our lab previously undertook a genome-wide association study (2101 cases, 4202 controls), which identified a cluster of single-nucleotide polymorphisms on chromosome 6p that correlated with a significantly increased chance of developing of high-risk disease (p < 1e-15). Here we present evidence that these polymorphisms fall within a long intergenic non-coding RNA (LINC00340) termed cancer-associated susceptibility 15 gene (CASC15), which we hypothesize plays a critical role in the development of high-risk neuroblastoma through the dysregulation of neuronal growth and differentiation pathways. Methods: CASC15 expression from patient samples (n=251) was obtained via Affymetrix Human Exon 1.0ST arrays, whereas expression in cell lines was assessed by qRT-PCR. CASC15 isoform sequences were validated through the use of 5′ and 3′ RACE, and subcellular localization was visualized using RNA-FISH. CASC15 depletion was carried out using both siRNA and shRNA constructs, and cell viability and growth kinetics were measured using the CellTiter-Glo and Xcelligence platforms. Gene expression analyses of neuroblastoma cell lines stably silenced for CASC15 was done through hybridization to Human Transcriptome 2.0 arrays followed by gene set enrichment and Ingenuity pathway analysis. Results: Our data demonstrates that patients with high-risk neuroblastoma express significantly less CASC15 than do those with low-risk disease (p < 0.0001), and that CASC15 expression inversely correlates with overall survival (bonf p = 3.1e-05). Expression of CASC15 is variable amongst neuroblastoma cell lines (n= 21), and is predominantly nuclear. Depletion of CASC15 increases the rate of substrate-dependent adherent cellular growth, but has no effect on overall cell number or viability. In contrast, ectopic overexpression of CASC15 in neuroblastoma cell lines is not tolerated. Subsequent observation of cells with stable CASC15 depletion shows overt morphological changes suggestive of a less neuronal phenotype. Gene set expression and pathway analyses confirm that these cells undergo a significant downregulation of neuronal markers, while upregulating cell adhesion molecules. Conclusions: These data suggest that CASC15 may be integral for proper neuronal development, and that loss of CASC15 may leads to a less differentiated cell type, thereby contributing to the tumorigenesis of high-risk neuroblastoma. Citation Format: Mike R. Russell, Annalise Penikis, Derek Oldridge, Maura Diamond, Sharon Diskin, John Maris, Kristina Cole. The long intergenic noncoding RNA LINC00340 is a neuroblastoma susceptibility gene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5237. doi:10.1158/1538-7445.AM2014-5237

Background: Neuroblastoma is a cancer of the sympathetic nervous system that most commonly affects young children and accounts for approximately 10% of all pediatric oncology deaths. We have reported common single nucleotide polymorphisms (SNPs) associated with neuroblastoma within LINC00340 (NEJM 2008), BARD1 (Nat Genet, 2009), LMO1 (Nature, 2010), DUSP12 and HSD17B12 (PLoS Genet, 2011), HACE1 and LIN28B (Nat Genet, 2012), and one common CNV at 1q21.1 within NBPF23 (Nature, 2009). Collectively, however, these variants still account for less than 10% of the risk for developing neuroblastoma. Based on this observation, along with the relative paucity of somatic mutations identified in this childhood cancer, we hypothesized that rare germline variants/mutations play a significant role in tumorigenesis. Methods and Results: We have taken a two-pronged approach to study both rare copy number variants (CNVs) and SNPs in neuroblastoma. First, we analyzed rare (< 1%) CNVs in a discovery cohort of 2,083 cases and 6,146 controls and identified several large (>500 Kb) CNVs associated with neuroblastoma, including a deletion at 16p11.2 (cases 0.43%, controls 0.04%; P = 4.0x10−4; OR: 8.9, 95% CI: 2.2-41.3). To date, we have replicated the 16p11.2 association in 1,167 neuroblastoma cases and 56,752 published controls (P=0.003; OR: 7.8, 95% CI: 2.3-23.6). In addition, we have validated by quantitative PCR several rare focal deletions of key developmentally regulated transcription factors, such as TBX2, detected in neuroblastoma patients but absent in 6,146 controls. In parallel, to identify relatively rare SNPs (0.5-5.0% minor allele frequency) influencing neuroblastoma susceptibility, we imputed genotypes using 1000 Genomes data in a discovery cohort of 2,101 neuroblastoma cases and 4,202 controls of European ancestry. We observed a strong association at rs78378222 (P = 6.4x10−9; OR: 2.1, 95% CI: 1.6-2.1) and rs35850753 (P = 2.2x10−9; OR: 1.9, 95% CI: 1.5-2.4), located in the 3' and 5' UTR of TP53 respectively. Both associations replicated robustly in our Italian and African-American case-series (combined rs78378222: P = 4.22 x 10−12, OR: 3.1, 95% CI: 2.1-10.1; rs35850753: P = 3.7 x 10−14; OR: 3.2, 95% CI: 2.0-9.7). These data add neuroblastoma to the subset of malignancies influenced by rs78378222, which disrupts the polyadenylation signal of TP53 having a hypomorphic effect and imparting cancer susceptibility (Nat Genet, 2011). Taken together, we show that rare CNVs and SNPs influence neuroblastoma susceptibility and have greater effect sizes than common variants reported thus far from GWAS. Efforts are ongoing to sequence the 16p11.2 region in deletion carriers and to understand the functional significance of the TP53 variants in neuroblastoma. Conclusions: Rare DNA variations at multiple loci have a significant influence on neuroblastoma tumorigenesis and begin to address the unexplained heritability of this cancer. Citation Format: Sharon J. Diskin, Mario Capasso, Maura Diamond, Kristopher Bosse, Hakon Hakonarson, Marcella Devoto, John M. Maris. Rare variants at 16p11.2 and within TP53 influence neuroblastoma susceptibility. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3811. doi:10.1158/1538-7445.AM2013-3811

Neuroblastoma is a cancer of the sympathetic nervous system that most commonly affects young children and accounts for approximately 10% of all pediatric oncology deaths. We have reported common SNPs associated with neuroblastoma within LINC00340 at 6p22 (NEJM 2008), BARD1 at 2q35 (Nature Genetics, 2009), LMO1 at 11p15 (Nature, 2010), DUSP12 at 1q23 (PLoS Genetics, 2011), and HSD17B12 at 11p11 (PLoS Genetics, 2011), and one common CNV at 1q21.1 within NBPF23 (Nature, 2009). We have further demonstrated that germline variation in LMO1 and BARD1 not only predisposes to neuroblastoma, but also plays an important role in tumor progression. To identify additional genetic risk factors for neuroblastoma, we expanded our previous genome-wide association study (GWAS) to include 2,841 patients and 7,482 control subjects from three independent case series including two cohorts of European ancestry and one African American cohort. Here, we report two new associations at 6q21, the first within the HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 gene (HACE1; rs4336470 combined P = 3.4 x 10−12; Odds Ratio: 1.27, 95% CI: 1.19-1.36), and the second within lin-28 homolog B (LIN28B; rs17065417 combined P = 8.4 x 10−9; Odds Ratio 1.38, 95% CI: 1.23-1.54). HACE1 is implicated in multiple human cancers as a critical tumor suppressor gene (Nature Genetics, 2009). LIN28B, on the other hand, acts as a repressor of the let-7 family of miRNA, is over-expressed in many human cancers, and promotes cellular transformation (Nature Genetics, 2009). More recently, LIN28B has also been identified as a key regulator of glucose homeostasis (Cell, 2011). Neuroblastoma cell lines homozygous for the LIN28B risk allele showed increased LIN28B mRNA and protein expression and down regulation of the let-7 family of micro RNAs, consistent with the risk allele conferring a growth advantage through increased LIN28B expression. Transcriptome analysis of 251 primary tumors showed that HACE1 is down regulated (p = 3.5 x 10−4) and LIN28B is up-regulated (P = 0.017) in high-risk neuroblastomas compared to more benign forms of the disease. Tumors over-expressing LIN28B showed increased activation of DNA damage response (P = 2.5 x 10−19) and metabolism (P = 3.5 x 10−25) pathways, independent of MYCN amplification. Taken together, we show that common DNA variants in HACE1 and LIN28B influence neuroblastoma susceptibility and our expression analyses suggest that both genes may play an important role in disease progression. Efforts are ongoing to understand the mechanism by which LIN28B and HACE1 influence neuroblastoma tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4871. doi:1538-7445.AM2012-4871

The NCI's Therapeutically Applicable Research to Generate Effective Targets (TARGET) initiative uses state-of-the-art genome-wide approaches to identify therapeutic targets for pediatric cancers. Applications of 2nd-generation sequencing technologies to the analysis of adult solid tumors and hematopoietic malignancies have led to novel, often clinically relevant insights into these diseases. The objective of this study is to utilize 2nd-generation sequencing approaches on a highly annotated set of ten high-risk neuroblastomas (NBLs). We performed whole genome shotgun sequencing of six stage 4 MYCN-non-amplified and four stage 4 MYCN-amplified NBL TARGET cases and matched peripheral blood, as well as whole transcriptome sequencing (RNA-Seq) of the corresponding tumor RNA. The tumor and normal genomes were sequenced to 30X haploid coverage, and an average 10.3 Gb of aligned sequence was generated for each tumor transcriptome. The level of sequencing redundancy allowed us to achieve at least 10X coverage across 90% of the genome enabling genome-wide detection of sequence and copy number changes in the tumor DNA. We used alignment and de novo assembly approaches to identify somatic and germline SNVs, indels, structural variants, regions of copy number gains and losses, and genome rearrangements. We used RNA-Seq data to determine whether the detected sequence changes were expressed, and to identify transcripts differentially or alternatively expressed between MYCN-amplified and non-amplified cases. Our analysis of tumor and normal genomes identified an average of 1664 candidate somatic mutations per case, the majority of which were SNVs and small indels falling within introns or intergenic sequence. We also detected two candidate germline mutations in the ALK oncogene, one of which was previously characterized in NBL. An average of 10% of candidate somatic mutations in coding sequence was expressed in the transcriptome representing candidate oncogenic events. In addition, we detected and validated using PCR 9 novel genomic rearrangements resulting in expressed products, 5 of which were somatic and 4 of which were germline. We report on two novel somatic gene fusions, between TRIM37 on chromosome 17 and RNF121 on chromosome 11, and between LSAMP and STAG1 on chromosome 3, previously uncharacterized in NBL. The fusions did not recur in our sample set. This work provides an initial genome-wide view of the landscape of somatic changes that occur in high-risk neuroblastoma and highlights novel candidate oncogenic events that may drive the malignancy. Ongoing efforts will catalogue discovered somatic mutation frequencies in a large set of cases and explore the role of germline DNA variations in NBL tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 926. doi:10.1158/1538-7445.AM2011-926

Neuroblastoma (NBL) is a cancer arising from the peripheral nervous system and is the most common solid tumor outside the brain in infants and children. Despite improved treatment over the past 20 years, only 45% of those with high-risk disease survive emphasizing the need for new molecular targets for therapy. To identify genetic mutations that encode such targets, we present an analysis of 162 whole exome sequences from 81 tumor/normal pairs from high-risk NBL patients (N=27 with MYCN amplification). From each sample, genomic DNA fragments encoding 185,961 exons from 18,380 genes were isolated using in-solution hybrid capture and sequenced using the Illumina sequencing platform to a median of 195X coverage. A wide diversity of somatic variation was observed with non-silent mutation counts ranging from 5 to 149 per tumor (median=24). Novel hyper-mutation phenotypes (> 125 candidate coding somatic mutations) were observed in four tumors including one case with both a deletion and a truncating mutation in MLH1. Several genes were mutated across the set at a statistically significant level including ALK (6 tumors, q=0.006) and PTPN11 (3 tumors, q=0.012). There are 27 other genes with candidate mutation frequencies in the 4-10% range, but no single gene identified to date with a mutation frequency >10%. Importantly, multiple paired blood samples (acquired at diagnosis) have sufficient numbers of circulating tumor DNA to potentially interfere with our SNV detection algorithm; refinement of the analytic pipeline using SNP-derived “contamination” metrics is ongoing. We anticipate validating candidate somatic candidates using an orthogonal genotyping method and further defining their true frequency in an expanded sample set that includes non-high-risk cases as well. Finally, we plan a concerted effort to catalogue potentially detrimental germline variations in order to further extend our understanding of the heritable component of NBL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4756. doi:10.1158/1538-7445.AM2011-4756

Neuroblastoma is a malignant neoplasm of the developing sympathetic nervous system that is notable for its phenotypic diversity. High-risk patients typically have widely disseminated disease at diagnosis and a poor survival probability, but low-risk patients frequently have localized tumors that are almost always cured with little or no chemotherapy. Our genome-wide association study (GWAS) has identified common variants within FLJ22536, BARD1, and LMO1 as significantly associated with neuroblastoma and more robustly associated with high-risk disease. Here we show that a GWAS focused on low-risk cases identified SNPs within DUSP12 at 1q23.3 (P = 2.07×10−6), DDX4 and IL31RA both at 5q11.2 (P = 2.94×10−6 and 6.54×10−7 respectively), and HSD17B12 at 11p11.2 (P = 4.20×10−7) as being associated with the less aggressive form of the disease. These data demonstrate the importance of robust phenotypic data in GWAS analyses and identify additional susceptibility variants for neuroblastoma., Author Summary Neuroblastoma is the most common solid tumor outside the central nervous system and is accountable for 10% of the mortality rate of all children's cancers. It has distinctive clinical behaviors and is categorized into different risk groups: high-risk, intermediate-risk, and low-risk. Genome-wide association studies have reported a number of genetic variations predisposing to high-risk neuroblastoma. This study focuses on the low-risk neuroblastoma group and identifies four novel genes (DUSP12, DDX4, IL31RA, and HSD17B12) at three distinct genomic positions that harbor disease-causing variants. This study also reports several gene sets that are enriched in overall neuroblastoma as well as in both high-risk and low-risk groups. Also of importance is that this study adopts a new computational method that identifies genes, instead of only one single nucleotide polymorphism, as disease-causing variants. Shown to have superior power of detection genome-wide association signals for neuroblastoma, the methodology presented in this study has great potential applications in case-control association studies in other diseases.

Genome-wide association studies have proven to be a powerful tool to identify susceptibility loci in cancer, however the molecular basis of a majority of these association signals remain undefined. We have previously reported nine common single nucleotide polymorphism (SNP) alleles at chromosome 2q35, within and 5′ upstream of the BRCA1-associated RING domain (BARD1) gene, to be significantly associated with high-risk neuroblastoma (Capasso, M et al., Nature Genetics 2009). Here, we focus on understanding the biological underpinnings of this genetic association by investigating how common variation at this locus correlates with changes in BARD1 expression in both the germline and in neuroblastoma samples, and further how these BARD1 alterations are involved in driving neuroblastoma tumorigenesis. We first showed that the neuroblastoma associated SNP variation at 2q35 is in linkage disequilibrium (LD) with multiple genomic areas dense with regulatory domains, including many putative binding sites for the neuronal splicing regulator, FOX2. We next demonstrated that in peripheral blood mononuclear cells, these SNPs correlate both with a trend towards increased overall BARD1 expression and with differential BARD1 exon expression. Specifically, a majority of the neuroblastoma associated SNPs were significantly associated with decreased expression of the RING domain forming BARD1 exons 2 and 3 (P = 0.0008-0.034). We have identified 16 unique BARD1 isoforms in the neuroblastoma tissue of origin, sympathetic fetal ganglia, with the β-BARD1 isoform specifically splicing out exons 2 and 3. β-BARD1 was found to be ubiquitously expressed in a panel of fetal sympathetic ganglia, neuroblastoma cell lines, and high-risk primary tumors, with its expression being significantly increased in cell lines harboring a homozygous BARD1 risk allele genotype at the most significantly associated 2q35 SNP, rs6435862 (N = 22; P = 0.04). Transient transfection of a specific siRNA targeting the alternatively spliced boundary of β-BARD1 in a panel of neuroblastoma cell lines (N = 12) showed that neuroblastoma cells harboring at least one risk allele at SNP rs6435862 and the highest β-BARD1 expression showed a significant inhibition of cell proliferation upon β-BARD1 knockdown (% growth inhibition = 45-81%, P = 0.0004-0.034), while six of seven cell lines with a homozygous major allele genotype at rs6435862 and lower β-BARD1 expression did not show significant growth inhibition. These data are consistent with β-BARD1 acting as an oncogene in neuroblastoma and we hypothesize that common genomic variation drives formation of this isoform, underlying neuroblastoma predisposition on 2q35. Ongoing work will focus on further defining the role of common variation in BARD1's RNA splicing patterns and identifying the molecular basis of β-BARD1's oncogenicity in neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3867.

Neuroblastoma is a pediatric malignancy of the developing sympathetic nervous system which is often lethal. Approximately 1% of cases are inherited in an autosomal dominant fashion, yet the remaining 99% appear to occur sporadically. We have previously demonstrated that common single nucleotide polymorphisms (SNPs) and common copy number variations (CNVs) at several loci are highly associated with neuroblastoma susceptibility (NEJM 2008, Nat Genet 2009, Nature 2009). By definition, common variants will each confer only a modest risk of developing a given disease. We hypothesized that rare variants/mutations play an important role in the etiology of sporadic neuroblastoma and have a strong influence on disease susceptibility. To test this hypothesis, we used data from our ongoing genome-wide association study (GWAS) to identify genes influenced by rare CNVs unique to neuroblastoma patients and test whether these genes are enriched in specific functional categories and/or pathways. We first identified a discovery set of 1,205 Caucasian neuroblastoma cases and 1,942 Caucasian healthy controls, each genotyped on the Illumina HumanHap550 or HumanHap610 SNP arrays. We observed large (> 1 Mb) CNVs in 4.1% of cases (median size = 1.3 Mb; range = 1.0 - 23.6 Mb), including two non-overlapping constitutional deletions greater than 5-Mb at 11q. Large CNVs (> 1 Mb) were significantly less common in controls (p = 3.5 × 10−4; 2.3% controls). We next mapped CNVs for each sample to known protein coding genes captured in the RefSeq database (NRefSeq ∼ 32,000). We identified 219 genes overlapping CNVs in multiple cases, but not detected in any controls; these CNVs were individually rare and occurred in < 1% of cases. To investigate the biological relevance of this gene set, we tested for enrichment in categories defined by Gene Ontology (GO), SwissProt keywords, Interpro protein domains, PIR (International Protein Sequence Database), and KEGG pathways. We observed significant enrichment in “pattern specification” (p = 4.3 × 10−10), “homeobox domain” (p = 4.7 × 10−7) and “developmental protein” (p = 1.3 × 10−6). Importantly, the set of genes with CNVs uniquely identified in controls (N = 529) was random and not enriched in any functional category. Finally, we replicated these findings in an independent set of 363 cases and 2,219 controls (p = 1.5 × 10−4 - 0.02). Several candidate neuroblastoma predisposition genes emerged from this study including homeobox genes HOXA10 and HOXA11 which have been implicated in other human cancers via hyper-methylation. We conclude that rare CNVs present in the constitutional DNA of neuroblastoma patients contribute to disease susceptibility. These CNVs can be very large and/or preferentially affect developmental genes, many of which have been implicated in cancer. Ongoing efforts are focused on validating the most common CNVs and determining whether they represent inherited genetic variation or de novo mutational events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5253.