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Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19(–) subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)(–) deep remission, whereas 67 patients had a recurrence after achieving a MRD(–) deep remission: 28 patients with CD19(+) leukemia and 39 patients with CD19(–) leukemia. Return of CD19(+) leukemia was associated with loss of CAR T-cell function, whereas CD19(–) leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19(–) events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD(–) remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.

Background Neuroblastoma is a biologically and clinically heterogeneous disease. Based on recent studies demonstrating an association between the primary tumor site, prognosis, and commonly measured tumor biological features, we hypothesized that neuroblastomas arising in different sites would show distinct genomic features reflective of the developmental biology of the sympathicoadrenal nervous system. Methods We first compared genomic and epigenomic data of primary diagnostic neuroblastomas originating in the adrenal gland (n = 646) compared to thoracic sympathetic ganglia (n = 118). We also evaluated association of common germline variation with these primary sites in 1027 European-American neuroblastoma patients. Results We observed higher rates of MYCN amplification, chromosome 1q gain, and chromosome 11q deletion among adrenal tumors, which were highly predictive of functional RNA signatures. Surprisingly, thoracic neuroblastomas were more likely to harbor ALK driver mutations than adrenal cases among all cases (odds ratio = 1.89, 95% confidence interval = 1.04 to 3.43), and among cases without MYCN amplification (odds ratio = 2.86, 95% confidence interval = 1.48 to 5.49). Common germline single nucleotide polymorphisms (SNPs) in BARD1 (previously associated with high-risk neuroblastoma) were found to be strongly associated with predisposition for origin at adrenal, rather than thoracic, sites. Conclusions Neuroblastomas arising in the adrenal gland are more likely to harbor structural DNA aberrations including MYCN amplification, whereas thoracic tumors show defects in mitotic checkpoints resulting in hyperdiploidy. Despite the general association of ALK mutations with high-risk disease, thoracic tumors are more likely to harbor gain-of-function ALK aberrations. Site of origin is likely reflective of stage of sympathetic nervous system development when malignant transformation occurs and is a surrogate for underlying tumor biology.

Molecular profiling of glioblastoma has revealed complex cytogenetic, epigenetic, and molecular abnormalities that are necessary for diagnosis, prognosis, and treatment. Our neuro-oncology group has developed a data-driven, institutional consensus guideline for efficient and optimal workup of glioblastomas based on our routine performance of molecular testing. We describe our institution’s testing algorithm, assay development, and genetic findings in glioblastoma, to illustrate current practices and challenges in neuropathology related to molecular and genetic testing. We have found that coordination of test requisition, tissue handling, and incorporation of results into the final pathologic diagnosis by the neuropathologist improve patient care. Here, we present analysis of O 6 -methylguanine-DNA-methyltransferase promoter methylation and next-generation sequencing results of 189 patients, obtained utilizing our internal processes led by the neuropathology team. Our institutional pathway for neuropathologist-driven molecular testing has streamlined the management of glioblastoma samples for efficient return of results for incorporation of genomic data into the pathological diagnosis and optimal patient care.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to other antigenically distinct common seasonal human coronaviruses (hCoVs) before the COVID-19 pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that ∼20% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection., Analysis of human serum samples before and after the onset of the COVID-19 pandemic show that antibodies against common seasonal human coronaviruses are cross-reactive against SARS-CoV-2 but do not confer cross-protection against infection or hospitalization.

The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor–armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. Significance: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms. Cancer Discov; 5(12); 1282–95. ©2015 AACR. See related commentary by Jackson and Brentjens, p. 1238. This article is highlighted in the In This Issue feature, p. 1225

SummaryA more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.

The majority of neuroblastoma patients have tumors that initially respond to chemotherapy, but a large proportion of patients will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas showed clonal evolution from the diagnostic tumor with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK signaling pathway. Seven events were detected only in the relapse tumor while the others showed clonal enrichment. In neuroblastoma cell lines we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18, 61%) and these lesions predicted for sensitivity to MEK inhibition in vitro and in vivo. Our findings provide the rationale for genetic characterization of relapse neuroblastoma and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

Background Prior studies suggest that neuroblastomas that do not accumulate metaiodobenzylguanidine (MIBG) on diagnostic imaging (MIBG non-avid) may have more favorable features compared with MIBG avid tumors. We compared clinical features, biologic features, and clinical outcomes between patients with MIBG nonavid and MIBG avid neuroblastoma. Procedure Patients had metastatic high- or intermediate-risk neuroblastoma and were treated on Children's Oncology Group protocols A3973 or A3961. Comparisons of clinical and biologic features according to MIBG avidity were made with chi-squared or Fisher exact tests. Event-free (EFS) and overall (OS) survival compared using log–rank tests and modeled using Cox models. Results Thirty of 343 patients (8.7%) had MIBG nonavid disease. Patients with nonavid tumors were less likely to have adrenal primary tumors (34.5 vs. 57.2%; P = 0.019), bone metastases (36.7 vs. 61.7%; P = 0.008), or positive urine catecholamines (66.7 vs. 91.0%; P < 0.001) compared with patients with MIBG avid tumors. Nonavid tumors were more likely to be MYCN amplified (53.8 vs. 32.6%; P = 0.030) and had lower norepinephrine transporter expression. Patients with MIBG nonavid disease had a 5-year EFS of 50.0% compared with 38.7% for patients with MIBG avid disease (P = 0.028). On multivariate testing in high-risk patients, MIBG avidity was the sole adverse prognostic factor for EFS identified (hazard ratio 1.77; 95% confidence interval 1.04–2.99; P = 0.034). Conclusions Patients with MIBG nonavid neuroblastoma have lower rates of adrenal primary tumors, bone metastasis, and catecholamine secretion. Despite being more likely to have MYCN-amplified tumors, these patients have superior outcomes compared with patients with MIBG avid disease.

Contains fulltext : 177057.pdf (Publisher’s version ) (Closed access) A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dbetah promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

[This corrects the article DOI: 10.1371/journal.pone.0147682.].

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.

In cancer, clonal evolution is characterized based on single nucleotide variants and copy number alterations. Nonetheless, previous methods failed to combine information from both sources to accurately reconstruct clonal populations in a given tumor sample or in a set of tumor samples coming from the same patient. Moreover, previous methods accepted as input all variants predicted by variant-callers, regardless of differences in dispersion of variant allele frequencies (VAFs) due to uneven depth of coverage and possible presence of strand bias, prohibiting accurate inference of clonal architecture. We present a general framework for assignment of functional mutations to specific cancer clones, which is based on distinction between passenger variants with expected low dispersion of VAF versus putative functional variants, which may not be used for the reconstruction of cancer clonal architecture but can be assigned to inferred clones at the final stage. The key element of our framework is QuantumClone, a method to cluster variants into clones, which we have thoroughly tested on simulated data. QuantumClone takes into account VAFs and genotypes of corresponding regions together with information about normal cell contamination. We applied our framework to whole genome sequencing data for 19 neuroblastoma trios each including constitutional, diagnosis and relapse samples. We discovered specific pathways recurrently altered by deleterious mutations in different clonal populations. Some such pathways were previously reported (e.g., MAPK and neuritogenesis) while some were novel (e.g., epithelial-mesenchymal transition, cell survival and DNA repair). Most pathways and their modules had more mutations at relapse compared to diagnosis.

The detection of copy number variants (CNV) by array-based platforms provides valuable insight into understanding human diversity. However, suboptimal study design and data processing negatively affect CNV assessment. We quantitatively evaluate their impact when short-sequence oligonucleotide arrays are applied (Affymetrix Genome-Wide Human SNP Array 6.0) by evaluating 42 HapMap samples for CNV detection. Several processing and segmentation strategies are implemented, and results are compared to CNV assessment obtained using an oligonucleotide array CGH platform designed to query CNVs at high resolution (Agilent). We quantitatively demonstrate that different reference models (e.g. single versus pooled sample reference) used to detect CNVs are a major source of inter-platform discrepancy (up to 30%) and that CNVs residing within segmental duplication regions (higher reference copy number) are significantly harder to detect (P < 0.0001). After adjusting Affymetrix data to mimic the Agilent experimental design (reference sample effect), we applied several common segmentation approaches and evaluated differential sensitivity and specificity for CNV detection, ranging 39–77% and 86–100% for non-segmental duplication regions, respectively, and 18–55% and 39–77% for segmental duplications. Our results are relevant to any array-based CNV study and provide guidelines to optimize performance based on study-specific objectives.

Purpose: Dihydrotestosterone (DHT) is an important factor in prostate cancer (PCA) genesis and disease progression. Given PCA's strong genetic component, we evaluated the possibility that variation in genes involved in DHT metabolism influence PCA risk. Experimental Design: We investigated copy number variants (CNV) and single nucleotide polymorphisms (SNP). We explored associations between CNV of uridine diphospho-glucuronosyltransferase (UGT) genes from the 2B subclass, given their prostate specificity and/or involvement in steroid metabolism and PCA risk. We also investigated associations between SNPs in genes (HSD3B1, SRD5A1/2, and AKR1C2) involved in the conversion of testosterone to DHT, and in DHT metabolism and PCA risk. The population consisted of 426 men (205 controls and 221 cases) who underwent prostate-specific antigen screening as part of a PCA early detection program in Tyrol, Austria. Results: No association between CNV in UGT2B17 and UGT2B28 and PCA risk was identified. Men carrying the AA genotype at SNP rs6428830 (HSD3B1) had an odds ratio (OR) of 2.0 [95% confidence intervals (95% CI), 1.1-4.1] compared with men with GG, and men with AG or GG versus AA in rs1691053 (SRD5A1) had an OR of 1.8 (95% CI, 1.04-3.13). Individuals carrying both risk alleles had an OR of 3.1 (95% CI, 1.4-6.7) when compared with men carrying neither (P = 0.005). Controls with the AA genotype on rs7594951 (SRD5A2) tended toward higher serum DHT levels (P = 0.03). Conclusions: This is the first study to implicate the 5α-reductase isoform 1 (SRD5A1) and PCA risk, supporting the rationale of blocking enzymatic activity of both isoforms of 5α-reductase for PCA chemoprevention. Cancer Epidemiol Biomarkers Prev; 19(1); 229–39

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

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries and is useful for general investigations of the bacterial infection process. However, the study of the molecular pathogenesis of EPEC has been hampered by the lack of genetically tractable, convenient animal models. We have therefore developed the use of the nematode Caenorhabditis elegans as a small animal model of infection for this diarrheal pathogen. We found that nematodes died faster on nematode growth medium in the presence of EPEC pathogens than in the presence of the laboratory control strain MG1655. Increased numbers of pathogens in the gut, determined by standard plate count assays and fluorescence microscopy using green fluorescent protein-expressing bacteria, correlated with killing. Deletion of the gene encoding the global regulator Ler severely reduced the ability of EPEC to colonize the nematode gut and could be complemented by providing the ler gene on a multicopy plasmid in trans . Neither the type III secretion system nor the type IV bundle-forming pilus was required for colonization. Combined, the similarities and distinct differences between EPEC infection of nematodes and that of humans offer a unique opportunity to study several stages of the infection process, namely, attachment, colonization, and persistence, in a genetically tractable, inexpensive, and convenient in vivo system.

High-throughput sequencing of DNA coding regions has become a common way of assaying genomic variation in the study of human diseases. Copy number variation (CNV) is an important type of genomic variation, but detecting and characterizing CNV from exome sequencing is challenging due to the high level of biases and artifacts. We propose CODEX, a normalization and CNV calling procedure for whole exome sequencing data. The Poisson latent factor model in CODEX includes terms that specifically remove biases due to GC content, exon capture and amplification efficiency, and latent systemic artifacts. CODEX also includes a Poisson likelihood-based recursive segmentation procedure that explicitly models the count-based exome sequencing data. CODEX is compared to existing methods on a population analysis of HapMap samples from the 1000 Genomes Project, and shown to be more accurate on three microarray-based validation data sets. We further evaluate performance on 222 neuroblastoma samples with matched normals and focus on a well-studied rare somatic CNV within the ATRX gene. We show that the cross-sample normalization procedure of CODEX removes more noise than normalizing the tumor against the matched normal and that the segmentation procedure performs well in detecting CNVs with nested structures.

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

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.

Arising from the sympathetic nervous system, neuroblastoma (NBL) is the most common extracranial solid tumor in children. Although the 5-year survival rate for children in the low and intermediate risk groups ranges between 90 to over 95%, children with high-risk disease have only a 40-50% likelihood of survival and relapse of high-risk disease is currently largely incurable. The best-characterized genetic determinant of high-risk disease is amplification of MYCN. Members of the MYC protein family are highly homologous and share redundant biological functions, each able to form heterodimers with MAX at consensus E-box motifs (CANNTG) to regulate transcription. While MYCN is known to transcriptionally activate genes involved in malignancy and repress expression of genes that antagonize metastases, amplification of MYCN alone is likely not sufficient for transformation. To identify MYCN and MYC DNA binding partners and ultimately, therapeutic targets in the high-risk NBL subset, we performed chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq) for MYCN, MYC, and MAX across three MYCN amplified and three non-MYCN amplified cell lines (n = 2 ChIPs per protein). Peak calling was performed using MACS2 and annotated using bedtools and/or ChIPseeker. MYCN displays genome-wide binding in all three amplified cell lines, suggesting global transcriptional regulation in NBL. ChIPenrich and TOPPgene were used to determine enrichment of peaks based on gene ontology. As expected, we show MYCN ChIP peaks were significantly enriched for MYC and MAX interactions, as well as DNA-binding, in the MYCN amplified subset of cell lines. Genes containing peaks within 1 kb of gene promoters were intersected with genes differentially expressed based on MYCN amplification status in high-risk NBL cases based on RNA-Seq analysis. This set of over 4000 genes was significantly enriched (p < 1E-10) for multiple categories of RNA binding and ribosomal proteins, as well as interactions with known oncogenes and/or cooperating partners of MYCN or MYC: CUL7, VCAM1, SUMO2, and CDK2 (p < 1E-20). Finally, we performed de novo motif discovery on MYCN ChIP peaks using Homer2 and found significant enrichment (p < 1E-50) of 15-20 motifs, including the canonical MYCN basic helix loop helix E-box motif (CCACGTGGCN, ∼23% enriched), an ELK1 ETS motif (∼44%), an AHR motif (∼29%), and a SMAD4 (MAD) motif (∼21%). To our knowledge, this combination of RNA- and ChIP-Seq represents the first global epigenomic analysis of MYCN, MYC, and MAX binding across multiple NBL cell lines, revealing MYCN-targeted transcriptional upregulation and/or putative repression. This and further epigenomic characterization of known and novel MYC and MYCN transcriptional targets will enable prioritization of potential therapeutic targets for treatment of the high-risk NBL subset. Citation Format: Jo Lynne Harenza, Robyn Sussman, Derek Oldridge, John Maris. Genome-wide mapping of MYCN, MYC, and MAX binding across neuroblastoma cell lines identifies novel transcriptional targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2425.

Copy number variants (CNVs) are a recently recognized class of human germ line polymorphisms and are associated with a variety of human diseases, including cancer. Because of the strong genetic influence on prostate cancer, we sought to identify functionally active CNVs associated with susceptibility of this cancer type. We queried low-frequency biallelic CNVs from 1,903 men of Caucasian origin enrolled in the Tyrol Prostate Specific Antigen Screening Cohort and discovered two CNVs strongly associated with prostate cancer risk. The first risk locus ( P = 7.7 × 10 −4 , odds ratio = 2.78) maps to 15q21.3 and overlaps a noncoding enhancer element that contains multiple activator protein 1 (AP-1) transcription factor binding sites. Chromosome conformation capture (Hi-C) data suggested direct cis -interactions with distant genes. The second risk locus ( P = 2.6 × 10 −3 , odds ratio = 4.8) maps to the α-1,3-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase C ( MGAT4C ) gene on 12q21.31. In vitro cell-line assays found this gene to significantly modulate cell proliferation and migration in both benign and cancer prostate cells. Furthermore, MGAT4C was significantly overexpressed in metastatic versus localized prostate cancer. These two risk associations were replicated in an independent PSA-screened cohort of 800 men (15q21.3, combined P = 0.006; 12q21.31, combined P = 0.026). These findings establish noncoding and coding germ line CNVs as significant risk factors for prostate cancer susceptibility and implicate their role in disease development and progression.

Background: High throughput sequencing of DNA coding regions has become a common way of assaying genomic variation in the study of cancer. Copy number aberration (CNA) is an important type of genomic change, but detecting and characterizing CNA from whole-exome sequencing (WES) is challenging due to the high level of biases and artifacts. Methods: We propose CODEX, a normalization and CNA calling procedure for WES data. The Poisson latent factor model in CODEX includes terms that specifically remove biases due to GC content, exon capture and amplification efficiency, and latent systemic artifacts. CODEX also includes a Poisson likelihood-based recursive segmentation procedure that explicitly models the count-based WES data. CODEX can be used to detect both germline and somatic CNAs in cancer samples with or without matched normal. Results: Compared to existing approaches, CODEX is shown to be more effective in removing the biases in WES, and attains better sensitivity and specificity in detecting copy number aberrations by in silico spike-in studies. We further evaluate performance on 222 neuroblastoma samples with matched normal from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Project. We carry out systematic genome-wide analysis and detailed characterization of both germline and somatic copy number events. With a focus on a well-studied rare somatic CNV within the ATRX gene, we show that the cross-sample normalization procedure of CODEX is more effective in removing noise than the standard pipeline of normalizing the tumor against the matched normal, and that the segmentation procedure performs well in detecting CNVs with recurrent complex nested structures. For detecting germline mutations, CODEX is compared to existing methods on a population analysis of HapMap samples from the 1000 Genomes Project, and shown to be perform well on three microarray-based validation data sets. Conclusions: The cross-sample normalization procedure of CODEX, when applied to the matrix of tumor and normal samples, is more effective in reducing noise than normalizing each tumor to its matched normal. The somatic deletions in the ATRX region have a nested structure, which CODEX was able to recover. Through multiple types of validation, CODEX is shown to be applicable to a wide range of study designs for copy number estimation using WES data. Citation Format: Yuchao Jiang, Derek A. Oldridge, Sharon J. Diskin, Nancy R. Zhang. CODEX: a normalization and copy number variation detection method for whole-exome sequencing. [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 4868. doi:10.1158/1538-7445.AM2015-4868

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 childhood cancer of the sympathetic nervous system, accounts for approximately 10-15% of pediatric oncology deaths. The genetic basis of neuroblastoma has grown clearer, with genome-wide association studies performed by our laboratory uncovering CASC15, BARD1, NBPF23, LMO1, HACE1, TP53 and LIN28B as susceptibility genes, with many (if not all) playing a major role in tumorigenesis. Here we focus on LIN28B, which binds mRNAs directly and is a master regulator of the let-7 family of tumor suppressor microRNAs, as we previously showed that high LIN28B expression is associated with advanced stage disease and worse patient outcome. Methods: To discover LIN28B-associated pathways in neuroblastoma, we performed gene set enrichment analysis (GSEA) on mRNA expression datasets and analyzed SNP-array based DNA copy number datasets. We used siRNAs, shRNAs, and microRNA mimetics to perturb transcripts of interest in neuroblastoma cells and measured effects on downstream signaling, protein-protein interactions, and proliferation. Results: We applied GSEA to mRNA expression profiles from 250 neuroblastoma tumors and found LIN28B expression to be robustly correlated with several biologically relevant gene sets, including “RAN signaling.” We focused on RAN signaling as RAN is a member of the Ras family of GTPases implicated in the pathogenesis of several malignancies and we demonstrated a strong positive correlation between LIN28B and RAN expression, most strikingly in the MYCN-amplified context (p = 2.2×10−10). We next analyzed 374 high-risk neuroblastoma tumors and found that 28% of them displayed recurrent somatic copy number gain of chromosome 12q24, the genomic location of RAN, which was associated with increased RAN expression (p = 0.0004) and was inversely related to MYCN amplification (p = 0.0021). Increased RAN expression was associated with stage 4 disease (p = 0.0047) and decreased overall survival (p = 0.0002). To further dissect the LIN28B-RAN relationship, we depleted LIN28B using shRNAs, showing that it reduced RAN RNA and protein levels. LIN28B directly bound RAN mRNA, likely enhancing its translation. As RAN promotes the phosphorylation and activation of Aurora kinase A (AURKA), we then demonstrated that LIN28B leads to AURKA activation via RAN. Moreover, we demonstrated that AURKA is a direct let-7 target, defining a separate mechanism by which LIN28B/let-7 influences AURKA expression. Finally, we showed that RAN depletion resulted in decreased neuroblastoma proliferation, phenocopying LIN28B depletion. Conclusions: These results demonstrate that enhanced LIN28B expression and chromosome 12q24 gain each independently promote RAN expression and that LIN28B and RAN signaling further converge on AURKA. Collectively, our studies support LIN28B as a master regulator of multiple oncogenes implicated in neuroblastoma pathogenesis, nominating it as a candidate for therapeutic targeting. Citation Format: Robert W. Schnepp, Priya Khurana, Edward F. Attiyeh, Sara Chodosh, Pichai Raman, Derek A. Oldridge, Maria E. Gagliardi, Karina Conkrite, Shahab Asgharzadeh, Robert C. Seeger, Blair Madison, Anil Rustgi, John M. Maris, Sharon J. Diskin. A LIN28B/RAN/AURKA signaling network promotes neuroblastoma tumorigenesis. [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 4734. doi:10.1158/1538-7445.AM2015-4734

Background The majority of high-risk neuroblastomas initially respond to chemotherapy, but over half of patients experience therapy-resistant relapses. The molecular defects driving relapse and drug resistance are unknown. Methods We performed Illumina or Complete Genomics whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas, and corresponding normal lymphocyte DNA, to define genetic alterations associated with relapse. A panel of 18 neuroblastoma cell lines was analyzed for the presence of RAS-MAPK mutations and sensitivity to small molecule inhibitors of this pathway. Results Neuroblastomas that relapsed after chemotherapy showed dramatic clonal evolution, with only 33% of primary tumor mutations also detected at relapse. In 21 out of 23 patients, more somatic coding mutations were observed at relapse (median: 29 unique to relapse, range: 4-129). Unbiased pathway analysis of the somatic mutations detected in the relapse tissues identified a strong enrichment in genes associated with RAS-MAPK signaling (p = 6.1×10−7). 18 of the 23 cases (78%) showed somatic mutations (N = 15) or structural alterations (N = 3) predicted to activate the MAPK pathway, and these were mutually exclusive: ALK (N = 10), NRAS (N = 1), KRAS (N = 1), HRAS (N = 1), BRAF (N = 1), PTPN11 (N = 1), FGRF1 (N = 1) and NF1 (N = 2). These RAS-MAPK mutations were clonally enriched at relapse and exist within clonal or major subclonal tumor populations. Seven of these RAS-MAPK mutations were detected only in the relapse tumor by whole genome sequencing (∼50X coverage), and only 2 of these 7 mutations were detectable in the primary tumor with targeted detection methods (104-105X coverage). Similar MAPK pathway mutations were detected in 11 of 18 human neuroblastoma-derived cell lines, and these lesions are predicted to be sensitive to small molecule inhibition of MEK in vitro (p Conclusions In this study of 23 neuroblastoma cases selected based solely on having diagnostic-relapse specimens available for analysis, MAPK pathway mutations were highly enriched in the relapsed genomes, providing a potential biomarker for new therapeutic approaches to chemotherapy refractory disease. The fact that several ALK-RAS-MAPK mutations were found in the relapse but not in the corresponding primary tumors favors a model in which rare subclones with secondary driver mutations expand over time. However, it remains to be determined whether these mutations occurred de novo after treatment, were present in rare subclones below detection limits, or were undetectable due to spatial heterogeneity of the primary tumor, which will impact the clinical utility of targeted sequencing at diagnosis. Our study provides strong rationale for performing biopsies on relapse neuroblastoma tumors in order to comprehensively characterize the molecular lesions that underlie treatment-refractory disease, determine their prognostic relevance, and guide treatment decisions for patients. Citation Format: Derek A. Oldridge, Thomas F. Eleveld, Virginie Bernard, Jan Koster, Leo C. Daage, Sharon J. Diskin, Linda Schild, Nadia B. Bentahar, Angela Bellini, Mathieu Chicard, Eve Lapouble, Valérie Combaret, Patricia Legoix-Né, Jean Michon, Trevor J. Pugh, Lori S. Hart, JulieAnn Rader, Edward F. Attiyeh, Jun S. Wei, Shile Zhang, Arlene Naranjo, Julie M. Gastier-Foster, Michael D. Hogarty, Malcolm A. Smith, Jaime G. Auvil, Thomas B. K. Watkins, Danny A. Zwijnenburg, Marli E. Ebus, Peter van Sluis, Anne Hakkert, Esther van Wezel, C. Ellen van der Schoot, Ellen M. Westerhout, Johannes H. Schulte, Godelieve A. Tytgat, M. Emmy M. Dolman, Isabelle Janoueix-Lerosey, Daniela S. Gerhard, Huib N. Caron, Olivier Delattre, Javed Khan, Rogier Versteeg, Gudrun Schleiermacher, John M. Maris, Jan J. Molenaar. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations. [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 2980. doi:10.1158/1538-7445.AM2015-2980

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

CD19 is expressed on most B-cell neoplasms, including acute lymphoblastic leukemias (B-ALL). Chimeric antigen receptor (CAR)-armed T cells targeting CD19 (CART-19) yield complete remission rate of 70-90% in B-ALL patients; the bispecific anti-CD19/CD3 antibody blinatumomab also shows clinical efficacy. Yet relapses due to the apparent CD19 loss have been observed following both therapies. Here we investigated the molecular mechanisms of epitope loss using five B-ALL samples obtained after CD19-directed therapy. These samples were matched, when available, with CD19-positive pre-treatment leukemias. Although the CD19 epitope recognized by CART-19 was undetectable in relapsed specimens, in all cases the CD19 genomic sequence was retained and the mRNA levels were only slightly reduced, suggesting regulation at the level of transcript processing. Indeed, using RT-PCR and RNA-Seq we detected several alternatively spliced CD19 mRNA species lacking exons 5-6 and/or exon 2 (Δex2), which encodes the cognate CART-19 epitope. In the sets of matched samples, the relative abundance of the Δex2/5-6 mRNA isoforms was increased post-treatment, and accordingly truncated CD19 protein variants were detectable using antibodies recognizing alternative epitopes. In one relapsed leukemia with a nonsense mutation in exon 2 of CD19, Δex2 was the only expressed isoform. To determine the contribution of alternative splicing to immune escape, we introduced CD19-expressing retroviruses into murine neoplastic B-cells, which had lost endogenous CD19 expression following silencing of its main transcriptional regulator Pax5. While skipping of exon 2 made the resultant protein invisible to CART-19, it partly rescued the defects in cell proliferation associated with CD19 loss. Collectively, these data suggest the existence of a novel mechanism of resistance to immunotherapy, based not on mutations in the coding sequence but rather on selection for alternatively spliced target protein isoforms (e.g., CD19 Δex2). Our findings could lead to the development of additional CD19-targeting CARTs for treating relapsed patients. Citation Format: Elena Sotillo, David Barrett, Aseb Bagashev, Kathryn Black, Caludia Lanauze, Derek Oldridge, Robyn Sussman, Colleen Harrington, Elaine Y. Chung, Ted J. Hofmann, Shannon L. Maude, Nicole M. Martinez, Pichai Raman, Marco Ruella, David Allman, Elad Jacoby, Terry Fry, Yoseph Barash, Kristen W. Lynch, Crystal Mackall, John Maris, Stephen A. Grupp, Andrei Thomas-Tikhonenko. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope and contributes to treatment failure. [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 3143. doi:10.1158/1538-7445.AM2015-3143

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

Structural variants which cause changes in copy numbers constitute an important component of genomic variability. They account for 0.7% of genomic differences in two individual genomes, of which copy number variants (CNVs) are the largest component. A recent population-based CNV study revealed the need of better characterization of CNVs, especially the small ones (