◥Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in highrisk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n ¼ 104) and patient-derived xenografts (n ¼ 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT þ , and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT þ , or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT-expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets.Significance: These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

SUMMARY Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)—many of which are refractory to current standard-of-care treatments—from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer.

Understanding the aberrant transcriptional landscape of neuroblastoma is necessary to provide insight to the underlying influences of the initiation, progression and persistence of this developmental cancer. Here, we present chromatin immunoprecipitation sequencing (ChIP-Seq) data for the oncogenic transcription factors, MYCN and MYC, as well as regulatory histone marks H3K4me1, H3K4me3, H3K27Ac, and H3K27me3 in ten commonly used human neuroblastoma-derived cell line models. In addition, for all of the profiled cell lines we provide ATAC-Seq as a measure of open chromatin. We validate specificity of global MYCN occupancy in MYCN amplified cell lines and functional redundancy of MYC occupancy in MYCN non-amplified cell lines. Finally, we show with H3K27Ac ChIP-Seq that these cell lines retain expression of key neuroblastoma super-enhancers (SE). We anticipate this dataset, coupled with available transcriptomic profiling on the same cell lines, will enable the discovery of novel gene regulatory mechanisms in neuroblastoma. Background & Summary An estimated 15,780 children in the United States will be diagnosed with cancer in 2019 1. While 80% of pediatric cancer patients overcome this disease, 20% of children do not survive, and survivors often have multiple side effects of therapy 1. Neuroblastoma accounts for more than 7% of malignancies in patients under 15 years of age and approximately 12% of all pediatric cancer-related deaths (for review see 2). Neuroblastoma shows wide phenotypic variability, with tumors arising in children diagnosed under the age of 18 months often spontaneously regressing with little or no treatment, but patients diagnosed at an older age or with unfavorable genomic features often showing a relentlessly progressive and widely metastatic disease pattern despite intensive, multimodal therapy (for review see 2-4). Ninety-eight percent of low-risk neuroblastoma disease are currently cured 5 , however, the survival rate for patients with high-risk neuroblastoma remains less than 50% 6. Relapsed high-risk neuroblastoma is typically incurable 7 , and thus these children require improved therapeutic options. A major prognostic factor predicting the severity, risk, and inferior outcome for neuroblastoma patients is amplification of the proto-oncogene MYCN. MYCN amplification occurs in nearly 20% of all neuroblastomas, and approximately 50% of patients with high-risk disease 8,9. It is a truncal genomic event, and typically stable across the spectrum of therapy and disease recurrence. MYCN, along with structural and binding homologues MYC and MYCL, are members of the MYC transcription factor family 10 and have been implicated in transcriptional regulation of proteins involved in cell growth 11 , proliferation 12 , and ribosome biogenesis 12. Mounting evidence has also indicated that MYCN and MYC are functionally redundant 13-15. However, the protein expression of MYC and MYCN appears to be mutually exclusive. For example, neuroblastoma tumors with MYCN amplification typically lack or have low MYC m...