Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single nucleotide polymorphism (SNP) associations reside within CASC15, a long non-coding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.

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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

The presentation of abdominal arteriovenous fistulas is classically described as a triad of a pulsatile abdominal mass with a bruit, high-output heart failure, and regional venous hypertension with primarily open operative therapy. In the following case, we present the treatment of a patient who arrived with acute right heart failure and renal failure due to an arteriovenous fistula and who was successfully treated with endovascular repair.

<p>Supplementary Methods</p>