PURPOSE: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome. PATIENTS AND METHODS: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed. RESULTS: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In FOXO1 fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. BCOR (15%), NF1 (15%), and TP53 (13%) mutations were found at a higher incidence than previously reported and TP53 mutations were associated with worse outcomes in both fusion-negative and FOXO1 fusion-positive cases. Interestingly, mutations in RAS isoforms predominated in infants < 1 year (64% of cases). Mutation of MYOD1 was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database ( ClinOmics ), containing all genomic variants, and clinical annotation including survival data. CONCLUSION: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.

Background and Objectives Opsoclonus-myoclonus-ataxia syndrome (OMAS) is a rare disorder of the nervous system that classically presents with a combination of characteristic eye movement disorder and myoclonus, in addition to ataxia, irritability, and sleep disturbance. There is good evidence that OMAS is an immune-mediated condition that may be paraneoplastic in the context of neuroblastoma. This syndrome may be associated with long-term cognitive impairment, yet it remains unclear how this is influenced by disease course and treatment. Treatment is largely predicated on immune suppression, but there is limited evidence to indicate an optimal regimen. Methods Following an international multiprofessional workshop in 2004, a body of clinicians and scientists comprising the International OMS Study group continued to meet biennially in a joint professionals and family workshop focusing on pediatric OMAS. Seventeen years after publication of the first report, a writing group was convened to provide a clinical update on the definitions and clinical presentation of OMAS, biomarkers and the role of investigations in a child presenting with OMAS, treatment and management strategies including identification and support of long-term sequelae. Results The clinical criteria for diagnosis were reviewed, with a proposed approach to laboratory and radiologic investigation of a child presenting with possible OMAS. The evidence for an upfront vs escalating treatment regimen was reviewed, and a treatment algorithm proposed to recognize both these approaches. Importantly, recommendations on monitoring of immunotherapy response and longer-term follow-up based on an expert consensus are provided. Discussion OMAS is a rare neurologic condition that can be associated with poor cognitive outcomes. This report proposes an approach to investigation and treatment of children presenting with OMAS, based on expert international opinion recognizing the limited data available.

Clinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind. To address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of low-frequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood. Analytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89-0.95]) and reproducible (>0.87 [95% CI: 0.87-0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples. This is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours.

Patient-derived preclinical models are now a core component of cancer research and have the ability to drastically improve the predictive power of preclinical therapeutic studies. However, their development and maintenance can be challenging, time consuming, and expensive. For neuroblastoma, a developmental malignancy of the neural crest, it is possible to establish patient-derived models as xenografts in mice and zebrafish, and as spheroids and organoids in vitro. These varied approaches have contributed to comprehensive packages of preclinical evidence in support of new therapeutics for neuroblastoma. We discuss here the ethical and technical considerations for the creation of patient-derived models of neuroblastoma and how their use can be optimized for the study of tumour evolution and preclinical therapies. We also discuss how neuroblastoma patient-derived models might become avatars for personalised medicine for children with this devastating disease.

We profiled a large heterogeneous cohort of matched diagnostic relapse tumor tissue and paired plasma-derived cell-free DNA (cfDNA) from patients with relapsed and progressive solid tumors of childhood. Tissue and cfDNA sequencing results were concordant, with a wider spectrum of mutant alleles and higher degree of intratumor heterogeneity captured by the latter, if sufficient ctDNA was present. Serial tumor sequencing identified putative drivers of relapse, with alterations in epigenetic drivers being a common feature. In keeping with epigenetic alterations being a common driver of many childhood cancers, fragmentomic analysis of cfDNA identified tumor-specific epigenetic states and transcription factor binding sites accessible in chromatin. This study leverages a large and well-annotated genomic dataset of aggressive childhood malignancies, identifies genomic and epigenetic drivers of childhood cancer relapse, and highlights the power and practicality of cfDNA analysis to capture both intratumoral heterogeneity and the epigenetic state of cancer cells. Significance: In tumors of childhood, we identify mutations in epigenetic genes as drivers of relapse, with matched cfDNA sequencing showing significant intratumor genetic heterogeneity and cell-state specific patterns of chromatin accessibility. This highlights the power of cfDNA analysis to identify both genetic and epigenetic drivers of aggressive disease in pediatric cancers.

Fragmentomics reveals disease specific differential accessibility at transcription factor binding sites.