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Background: Medulloblastoma is the most common malignant childhood brain tumour. Aberrant activation of the WNT/β-catenin pathway occurs in approximately 25% of medulloblastomas. However, its role in medulloblastoma pathogenesis is not understood.Methods: We have developed a model of WNT/β-catenin pathway-activated medulloblastoma. Pathway activation was induced in a Myc immortalised cerebellar progenitor cell line through stable expression of Wnt1. In vitro and in vivo analysis was undertaken to understand the effect of pathway activation and identify the potential cell of origin.Results: Tumours that histologically resembled classical medulloblastoma formed in vivo using cells overexpressing Wnt1, but not with the control cell line. Wnt1 overexpression inhibited neuronal differentiation in vitro, suggesting WNT/β-catenin pathway activation prevents cells terminally differentiating, maintaining them in a more 'stem-like' state. Analysis of cerebellar progenitor cell markers demonstrated the cell line resembled cells from the cerebellar ventricular zone.Conclusion: We have developed a cell line with the means of orthotopically modelling WNT/β-catenin pathway-activated medulloblastoma. We provide evidence of the role pathway activation is playing in tumour pathogenesis and suggest medulloblastomas can arise from cells other than granule cell progenitors. This cell line is a valuable resource to further understand the role of pathway activation in tumorigenesis and for investigation of targeted therapies. [ABSTRACT FROM AUTHOR]

Background: Over half of childhood intracranial ependymomas occur in children younger than 5 years. As an adjuvant treatment, radiotherapy can be effective, but has the potential to damage the child's developing nervous system at a crucial time-with a resultant reduction in IQ and cognitive impairment, endocrinopathy, and risk of second malignancy. We aimed to assess the role of a primary chemotherapy strategy in avoiding or delaying radiotherapy in children younger than 3 years with intracranial ependymoma.Methods: Between December, 1992, and April, 2003, we enrolled 89 children with ependymoma who were aged 3 years or younger at diagnosis, of whom nine had metastatic disease on pre-operative imaging. After maximal surgical resection, children received alternating blocks of myelosuppressive and non-myelosuppressive chemotherapy every 14 days for an intended duration of 1 year. Radiotherapy was withheld unless local imaging (ie, from the child's treatment centre) showed progressive disease.Findings: 50 of the 80 patients with non-metastatic disease progressed, 34 of whom were irradiated for progression. The 5-year cumulative incidence of freedom from radiotherapy for the 80 non-metastatic patients was 42% (95% CI 32-53). With a median follow-up of 6 years (range 1.5-11.3), overall survival for the non-metastatic patients at 3 years was 79.3% (95% CI 68.5-86.8) and at 5 years 63.4% (51.2-73.4). The corresponding values for event-free survival were 47.6% (36.2-58.1) and 41.8% (30.7-52.6). There was no significant difference in event-free or overall survival between complete and incomplete surgical resection, nor did survival differ according to histological grade, age at diagnosis, or site of disease. In 47 of 59 (80%) patients who progressed, relapse resulted from local control only. The median time to progression for the 59 patients who progressed was 1.6 years (range 0.1-10.2 years). The median age at irradiation of the whole group was 3.6 years (range 1.5-11.9). For the 80 non-metastatic patients, the 23 who achieved the highest relative dose intensity of chemotherapy had the highest post-chemotherapy 5-year overall survival of 76% (95% CI 46.6-91.2), compared with 52% (33.3-68.1) for the 32 patients who achieved the lowest relative dose intensity of chemotherapy.Interpretation: This protocol avoided or delayed radiotherapy in a substantial proportion of children younger than 3 years without compromising survival. These results suggest, therefore, that primary chemotherapy strategies have an important role in the treatment of very young children with intracranial ependymoma. [ABSTRACT FROM AUTHOR]

Survival and endocrine status in a cohort of boys with acute lymphoblastic leukaemia (ALL) who started treatment between 1972 and 1987 and subsequently developed a testicular relapse were analysed. During this period there was a significant improvement in the overall event free survival for boys, but no significant decrease in the testicular relapse rate. Thirty three boys had an apparently isolated testicular relapse, whereas 21 boys had a combined relapse. The event free survival for boys with an isolated testicular relapse was 59% at six years (95% confidence interval (CI) 42 to 74%). The event free survival for the 16 patients with a combined relapse who received a second course of treatment was 32% (95% CI 17 to 60%). Those patients receiving adequate second line treatment for an isolated testicular relapse whose first remission was longer than or equal to two years had an event free survival of 82% (95% CI 63 to 93%) at six years. No boy relapsing within two years from diagnosis has survived. Endocrine late effects are significant, with 82% of the boys requiring hormonal treatment at some stage for induction of puberty or continuing pubertal maturation, or both. It is concluded that, despite the increasing intensity of initial treatment for ALL, isolated testicular relapse is treatable by conventional means in most patients. Careful endocrine follow up of these patients is essential as most will require hormone replacement treatment. [ABSTRACT FROM AUTHOR]

Disturbances of growth and development in patients with thalassaemia receiving hypertransfusion programmes are well recognised and are most likely to be due to iron overload. The extent of endocrine dysfunction was investigated in a group of 18 patients thought to have been treated by acceptable modern standards, 11 of whom could be considered as well chelated. Assessment of growth and puberty showed a wide variation in height SD scores with five patients having significantly short stature. Most patients are progressing through puberty normally with the exception of two boys with marked pubertal delay. The most prominent finding was that growth hormone responses to glucagon stimulation were significantly impaired in all of the patients with iron overload. Basal endocrine assessment showed primary hypothyroidism in two patients aged 16.8 and 12.9 years with plasma thyroxine-concentrations of 86 and 59 nmol/l (normal range 65-165 nmol/l) and plasma thyroid stimulating hormone 10.2 and 30.3 mU/l (normal range 0.5-5 mU/l). One patient had diabetes mellitus. These results show that even when ideal management is sought a significant amount of endocrine damage occurs; surveillance of these patients is thus essential. [ABSTRACT FROM AUTHOR]

Altogether 156 children treated for Wilms' tumour (WT) between 1970 and 1998 were studied. Sixty-six children, selected only by their attendance at clinic, were carefully examined and the findings compared to those from a case note review of 90 children. Congenital abnormalities were present in 45% of the examined cohort, in 19% of the case notes review group and in 30% overall. Novel findings included the association of WT with Marshall Smith syndrome, developmental delay in 3 of 4 cases of WT (one bilateral) and 1 sibling from consanguineous Pakistani families and another sibling also had leukaemia. The possibility of rare DNA repair or cancer predisposing disorders among these 4 families requires further study. Careful examination and history taking of an unselected patient cohort revealed a higher than expected incidence of clinical abnormalities which may be overlooked if not specifically sought. [ABSTRACT FROM AUTHOR]

Proton magnetic resonance spectroscopy ( 1 H-MRS) is increasingly used for clinical brain tumour diagnosis, but suffers from limited spectral quality. This retrospective and comparative study aims at improving paediatric brain tumour classification by performing noise suppression on clinical 1 H-MRS. Eighty-three/forty-two children with either an ependymoma (ages 4.6 ± 5.3/9.3 ± 5.4), a medulloblastoma (ages 6.9 ± 3.5/6.5 ± 4.4), or a pilocytic astrocytoma (8.0 ± 3.6/6.3 ± 5.0), recruited from four centres across England, were scanned with 1.5T/3T short-echo-time point-resolved spectroscopy. The acquired raw 1 H-MRS was quantified by using Totally Automatic Robust Quantitation in NMR (TARQUIN), assessed by experienced spectroscopists, and processed with adaptive wavelet noise suppression (AWNS). Metabolite concentrations were extracted as features, selected based on multiclass receiver operating characteristics, and finally used for identifying brain tumour types with supervised machine learning. The minority class was oversampled through the synthetic minority oversampling technique for comparison purposes. Post-noise-suppression 1 H-MRS showed significantly elevated signal-to-noise ratios (P < .05, Wilcoxon signed-rank test), stable full width at half-maximum (P > .05, Wilcoxon signed-rank test), and significantly higher classification accuracy (P < .05, Wilcoxon signed-rank test). Specifically, the cross-validated overall and balanced classification accuracies can be improved from 81% to 88% overall and 76% to 86% balanced for the 1.5T cohort, whilst for the 3T cohort they can be improved from 62% to 76% overall and 46% to 56%, by applying Naïve Bayes on the oversampled 1 H-MRS. The study shows that fitting-based signal-to-noise ratios of clinical 1 H-MRS can be significantly improved by using AWNS with insignificantly altered line width, and the post-noise-suppression 1 H-MRS may have better diagnostic performance for paediatric brain tumours., (© 2024 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

1 H-magnetic resonance spectroscopy (MRS) has the potential to improve the noninvasive diagnostic accuracy for paediatric brain tumours. However, studies analysing large, comprehensive, multicentre datasets are lacking, hindering translation to widespread clinical practice. Single-voxel MRS (point-resolved single-voxel spectroscopy sequence, 1.5 T: echo time [TE] 23-37 ms/135-144 ms, repetition time [TR] 1500 ms; 3 T: TE 37-41 ms/135-144 ms, TR 2000 ms) was performed from 2003 to 2012 during routine magnetic resonance imaging for a suspected brain tumour on 340 children from five hospitals with 464 spectra being available for analysis and 281 meeting quality control. Mean spectra were generated for 13 tumour types. Mann-Whitney U-tests and Kruskal-Wallis tests were used to compare mean metabolite concentrations. Receiver operator characteristic curves were used to determine the potential for individual metabolites to discriminate between specific tumour types. Principal component analysis followed by linear discriminant analysis was used to construct a classifier to discriminate the three main central nervous system tumour types in paediatrics. Mean concentrations of metabolites were shown to differ significantly between tumour types. Large variability existed across each tumour type, but individual metabolites were able to aid discrimination between some tumour types of importance. Complete metabolite profiles were found to be strongly characteristic of tumour type and, when combined with the machine learning methods, demonstrated a diagnostic accuracy of 93% for distinguishing between the three main tumour groups (medulloblastoma, pilocytic astrocytoma and ependymoma). The accuracy of this approach was similar even when data of marginal quality were included, greatly reducing the proportion of MRS excluded for poor quality. Children's brain tumours are strongly characterised by MRS metabolite profiles readily acquired during routine clinical practice, and this information can be used to support noninvasive diagnosis. This study provides both key evidence and an important resource for the future use of MRS in the diagnosis of children's brain tumours., (© 2024 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Background: The malignant childhood brain tumour, medulloblastoma, is classified clinically into molecular groups which guide therapy. DNA-methylation profiling is the current classification 'gold-standard', typically delivered 3-4 weeks post-surgery. Pre-surgery non-invasive diagnostics thus offer significant potential to improve early diagnosis and clinical management. Here, we determine tumour metabolite profiles of the four medulloblastoma groups, assess their diagnostic utility using tumour tissue and potential for non-invasive diagnosis using in vivo magnetic resonance spectroscopy (MRS)., Methods: Metabolite profiles were acquired by high-resolution magic-angle spinning NMR spectroscopy (MAS) from 86 medulloblastomas (from 59 male and 27 female patients), previously classified by DNA-methylation array (WNT (n = 9), SHH (n = 22), Group3 (n = 21), Group4 (n = 34)); RNA-seq data was available for sixty. Unsupervised class-discovery was performed and a support vector machine (SVM) constructed to assess diagnostic performance. The SVM classifier was adapted to use only metabolites (n = 10) routinely quantified from in vivo MRS data, and re-tested. Glutamate was assessed as a predictor of overall survival., Findings: Group-specific metabolite profiles were identified; tumours clustered with good concordance to their reference molecular group (93%). GABA was only detected in WNT, taurine was low in SHH and lipids were high in Group3. The tissue-based metabolite SVM classifier had a cross-validated accuracy of 89% (100% for WNT) and, adapted to use metabolites routinely quantified in vivo, gave a combined classification accuracy of 90% for SHH, Group3 and Group4. Glutamate predicted survival after incorporating known risk-factors (HR = 3.39, 95% CI 1.4-8.1, p = 0.025)., Interpretation: Tissue metabolite profiles characterise medulloblastoma molecular groups. Their combination with machine learning can aid rapid diagnosis from tissue and potentially in vivo. Specific metabolites provide important information; GABA identifying WNT and glutamate conferring poor prognosis., Funding: Children with Cancer UK, Cancer Research UK, Children's Cancer North and a Newcastle University PhD studentship., Competing Interests: Declaration of interests HR holds stock options in Healx (AI drug discovery in rare diseases). PSM is unpaid co-chair of SIOP-Europe Brain Tumour Group Imaging Group. We declare no other competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Accurate identification of brain tumor molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European "Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)" study., Methods: Across 6 European BIOMECA laboratories, we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via fluorescent in situ hybridization (FISH) and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH., Results: DNA Methylation profiling classified 65.3% (n = 96/147) of cases as EPN-PFA and 15% (n = 22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99%-100% across 3 centers). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP-fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-center concordance and low sensitivity and specificity while MIP, MLPA, and DNA methylation-based approaches demonstrated greater accuracy., Conclusions: We confirm, in a prospective trial cohort, that H3K27me3 immunohistochemistry is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q, and CDKN2A loss while FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. Finally, we propose test sets to guide future diagnostic approaches., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown; however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this., Methods: In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence., Results: DNA methylome derived copy number variants (CNVs) revealed large-scale chromosome gains and losses at recurrence in PFA. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at first recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at first recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations., Conclusions: This study provides clinically and preclinically actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ependymoma (EPN) is a devastating childhood brain tumor. Single-cell analyses have illustrated the cellular heterogeneity of EPN tumors, identifying multiple neoplastic cell states including a mesenchymal-differentiated subpopulation which characterizes the PFA1 subtype. Here, we characterize the EPN immune environment, in the context of both tumor subtypes and tumor cell subpopulations using single-cell sequencing (scRNAseq, n = 27), deconvolution of bulk tumor gene expression (n = 299), spatial proteomics (n = 54), and single-cell cytokine release assays (n = 12). We identify eight distinct myeloid-derived subpopulations from which a group of cells, termed hypoxia myeloid cells, demonstrate features of myeloid-derived suppressor cells, including IL6/STAT3 pathway activation and wound healing ontologies. In PFA tumors, hypoxia myeloid cells colocalize with mesenchymal-differentiated cells in necrotic and perivascular niches and secrete IL-8, which we hypothesize amplifies the EPN immunosuppressive microenvironment. This myeloid cell-driven immunosuppression will need to be targeted for immunotherapy to be effective in this difficult-to-cure childhood brain tumor., Competing Interests: Competing financial interests: We, the authors and our immediate family members, have no financial interests to declare. Advisory/management and consulting positions: We, the authors and our immediate family members, have no positions to declare and are not members of the journal’s advisory board. Patents: We, the authors and our immediate family members, have no related patents to declare., (© 2023 The Author(s).)

Pediatric neoplasms in the central nervous system (CNS) show extensive clinical and molecular heterogeneity and are fundamentally different from those occurring in adults. Molecular genetic testing contributes to accurate diagnosis and enables an optimal clinical management of affected children. Here, we investigated a rare, molecularly distinct type of pediatric high-grade neuroepithelial tumor (n = 18), that was identified through unsupervised visualization of genome-wide DNA methylation array data, together with copy number profiling, targeted next-generation DNA sequencing, and RNA transcriptome sequencing. DNA and/or RNA sequencing revealed recurrent fusions involving the capicua transcriptional repressor (CIC) gene in 10/10 tumor samples analyzed, with the most common fusion being CIC::LEUTX (n = 9). In addition, a CIC::NUTM1 fusion was detected in one of the tumors. Apart from the detected fusion events, no additional oncogenic alteration was identified in these tumors. The histopathological review demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin, CD56 and CD99. All tumors were located in the supratentorial compartment, occurred during childhood (median age 8.5 years) and typically showed early relapses. In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusions involving the transcriptional repressor CIC. Downstream functional consequences of the fusion protein CIC::LEUTX and potential therapeutic implications need to be further investigated., (© 2023. The Author(s).)

Background: National advisory panels (NAPs) have been established for the care of children and young people (CYP) with cancer in the United Kingdom since 2011, with an increase in panel number in recent years. Their practice has not previously been reviewed; therefore, we sought to evaluate the role, practice and impact of six selected NAPs offering expertise in ependymoma, histiocytosis, leukaemia, neuroblastoma, renal tumours and sarcoma., Procedure: This service evaluation used mixed methodology, including review of NAP documentation, semi-structured interviews with the NAP chairs and an analysis of the cases referred for discussion., Results: Total 1110 referrals were analysed. Results demonstrated the significant scope and amount of work undertaken by the NAPs, largely testament to the commitment of the panel members. Specific roles fulfilled have been highlighted, and NAP recommendations have been shown to influence clinical decision-making and be implemented in the majority of cases. Despite widespread good practice, areas to address have been identified; these include clarity regarding NAP membership, consistency in recommendations, the consideration of holistic information to promote personalised management and the exploration of wider multidisciplinary team roles., Conclusions: In the context of increasing demand and the escalating number of NAPs, it is timely to consider how service improvement can be facilitated. Best practice guidelines have been formulated as a product of this study, to promote a sustainable and effective model for NAPs. Review and benchmarking national panel performance against these guidelines will drive high standards of care going forward and they should be embedded as standard practice., (© 2022 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

Objective: To evaluate a new protocol of risk stratification and early discharge for children with febrile neutropenia (FN)., Design: Prospective service evaluation from 17 April 2020 to 16 April 2021., Setting: 13 specialist centres in the UK., Patients: 405 children presenting with FN., Intervention: All children received intravenous antibiotics at presentation. Risk stratification was determined using the Australian-UK-Swiss (AUS) rule and eligibility for homecare assessed using criteria including disease, chemotherapy, presenting features and social factors. Those eligible for homecare could be discharged on oral antibiotics after a period of observation proportional to their risk group., Main Outcome Measures: Median duration of admission and of intravenous antibiotics, and percentage of patients with positive blood cultures, significant infection, readmission within 7 days of initial presentation, intensive care unit (ICU) admission, death from infection and death from other causes., Results: 13 centres contributed 729 initial presentations of 405 patients. AUS rule scores were positively correlated with positive blood cultures, significant infection, ICU admission and death. 20% of children were eligible for homecare with oral antibiotics, of which 55% were low risk (AUS 0-1). 46% low-risk homecare eligible patients were discharged by 24 hours vs 2% homecare ineligible. Homecare readmission rates were 14% overall and 16% for low-risk cases (similar to a meta-analysis of previous studies). No child eligible for homecare was admitted to ICU or died., Conclusions: Use of the AUS rule and homecare criteria allow for safe early outpatient management of children with FN., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Poor outcomes associated with diffuse high-grade gliomas occur in both adults and children, despite substantial progress made in the molecular characterisation of the disease. Targeting the metabolic requirements of cancer cells represents an alternative therapeutic strategy to overcome the redundancy associated with cell signalling. Cholesterol is an integral component of cell membranes and is required by cancer cells to maintain growth and may also drive transformation. Here, we show that removal of exogenous cholesterol in the form of lipoproteins from culture medium was detrimental to the growth of two paediatric diffuse glioma cell lines, KNS42 and SF188, in association with S-phase elongation and a transcriptomic program, indicating dysregulated cholesterol homeostasis. Interrogation of metabolic perturbations under lipoprotein-deficient conditions revealed a reduced abundance of taurine-related metabolites and cholesterol ester species. Pharmacological reduction in intracellular cholesterol via decreased uptake and increased export was simulated using the liver X receptor agonist LXR-623, which reduced cellular viability in both adult and paediatric models of diffuse glioma, although the mechanism appeared to be cholesterol-independent in the latter. These results provide proof-of-principle for further assessment of liver X receptor agonists in paediatric diffuse glioma to complement the currently approved therapeutic regimens and expand the options available to clinicians to treat this highly debilitating disease.

Background: Medulloblastoma, ependymoma, and pilocytic astrocytoma are common pediatric posterior fossa tumors. These tumors show overlapping characteristics on conventional MRI scans, making diagnosis difficult., Purpose: To investigate whether apparent diffusion coefficient (ADC) values differ between tumor types and to identify optimum cut-off values to accurately classify the tumors using different performance metrics., Study Type: Systematic review and meta-analysis., Subjects: Seven studies reporting ADC in pediatric posterior fossa tumors (115 medulloblastoma, 68 ependymoma, and 86 pilocytic astrocytoma) were included following PubMed and ScienceDirect searches., Sequence and Field Strength: Diffusion weighted imaging (DWI) was performed on 1.5 and 3 T across multiple institution and vendors., Assessment: The combined mean and standard deviation of ADC were calculated for each tumor type using a random-effects model, and the effect size was calculated using Hedge's g., Statistical Tests: Sensitivity/specificity, weighted classification accuracy, balanced classification accuracy. A P value < 0.05 was considered statistically significant, and a Hedge's g value of >1.2 was considered to represent a large difference., Results: The mean (± standard deviation) ADCs of medulloblastoma, ependymoma, and pilocytic astrocytoma were 0.76 ± 0.16, 1.10 ± 0.10, and 1.49 ± 0.16 mm 2 /sec × 10 -3 . To maximize sensitivity and specificity using the mean ADC, the cut-off was found to be 0.96 mm 2 /sec × 10 -3 for medulloblastoma and ependymoma and 1.26 mm 2 /sec × 10 -3 for ependymoma and pilocytic astrocytoma. The meta-analysis showed significantly different ADC distributions for the three posterior fossa tumors. The cut-off values changed markedly (up to 7%) based on the performance metric used and the prevalence of the tumor types., Data Conclusion: There were significant differences in ADC between tumor types. However, it should be noted that only summary statistics from each study were analyzed and there were differences in how regions of interest were defined between studies., Evidence Level: 1 TECHNICAL EFFICACY: Stage 3., (© 2021 International Society for Magnetic Resonance in Medicine.)

Despite significant improvements in treatment and survival in paediatric cancers, outcomes for children with brain tumours remain poor. Novel therapeutic approaches are needed to improve survival and quality of survival. Extracellular arginine dependency (auxotrophy) has been recognised in several tumours as a potential therapeutic target. This dependency is due to the inability of cancer cells to recycle or synthesise intracellular arginine through the urea cycle pathway compared to normal cells. Whilst adult glioblastoma exhibits this dependency, the expression of the arginine pathway enzymes has not been delineated in paediatric brain tumours. We used immunohistochemical (IHC) methods to stain for arginine pathway enzymes in paediatric high-grade glioma (pHGG), low-grade glioma (pLGG), ependymoma (EPN), and medulloblastoma (MB) tumour tissue microarrays (TMAs). The antibodies detected protein expression of the metaboliser arginase (Arg1 and Arg2); recycling enzymes ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS1), and argininosuccinate lyase (ASL); and the transporter SLC7A1. Deficiency of OTC, ASS1, and ASL was seen in 87.5%, 94%, and 79% of pHGG samples, respectively, consistent with an auxotrophic signature. Similar result was obtained in pLGG with 96%, 93%, and 91% of tumours being deficient in ASL, ASS1, and OTC, respectively. 79%, 88%, and 85% of MB cases were ASL, ASS1, and OTC deficient whilst ASL and OTC were deficient in 57% and 91% of EPN samples. All tumour types highly expressed SLC7A1 and Arginase, with Arg2 being the main isoform, demonstrating that they could transport and utilise arginine. Our results show that pHGG, pLGG, EPN, and MB demonstrate arginine auxotrophy based on protein expression and are likely to be susceptible to arginine depletion. Pegylated arginase (BCT-100) is currently in phase I/II trials in relapsed pHGG. Our results suggest that therapeutic arginine depletion may also be useful in other tumour types and IHC analysis of patient tumour samples could help identify patients likely to benefit from this treatment., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Eleanor Bishop et al.)

Background: The clinical management of ependymoma in childhood and adolescence is complex and the clinicobiopathological correlates of outcome remain poorly understood. This international SIOP Ependymoma II (SIOP EPII) trial aims to improve the outcome of patients with ependymoma., Methods: SIOP EPII includes any patient <22 years at diagnosis with ependymoma, stratified by age, tumor location, and outcome of the initial surgery. Centralized pathology and imaging is required for diagnosis confirmation. SIOP EPII included three randomized studies according to age, postoperative residue, and suitability to receive radiotherapy. Patients ineligible for interventional strata are followed-up in an observational study. The staging phase aims to determine if central neurosurgical and radiological postoperative MRI reviews increase the resection rate. Patients ≥ 12 months with (i) no residual disease are randomly assigned in a phase III trial to evaluate the efficacy of post-radiation 16-week chemotherapy (VEC + CDDP) on PFS (stratum I); (ii) centrally confirmed measurable inoperable residual disease are allocated to randomized frontline chemotherapy phase II study (VEC vs. VEC + high-dose methotrexate) and considered for a second-look surgery (stratum II). If second-look surgery is not feasible or tumor residuum remains, patients receive 8 Gy-boost radiotherapy after conformal radiotherapy (phase I). (iii) Patients < 12 months (18 months in the UK) or not eligible to receive radiotherapy are randomized in a phase II study to receive chemotherapy (alternated myelosuppressive and nonmyelosuppressive chemotherapy), with or without valproate (stratum III). To overcome the limitations encountered in the preliminary conclusions of the ACNS-0831 study, a SIOP EPII dedicated on-study amendment has been planned to definitively conclude the relevance of maintenance chemotherapy in stratum I. Secondary outcomes include overall survival, quality of life, neuropsychological and neuroendocrine outcomes, safety, and identification of key prognostic biomarkers (BIOMECA)., Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT02265770., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Leblond, Massimino, English, Ritzmann, Gandola, Calaminus, Thomas, Pérol, Gautier, Grundy and Frappaz.)

Background: SIOP Ependymoma I was a non-randomised trial assessing event free and overall survival (EFS/OS) of non-metastatic intracranial ependymoma in children aged 3-21 years treated with a staged management strategy. A further aim was to assess the response rate (RR) of subtotally resected (STR) ependymoma to vincristine, etoposide, and cyclophosphamide (VEC). We report final results with 12-year follow-up and post hoc analyses of recently described biomarkers., Methods: Seventy-four participants were eligible. Children with gross total resection (GTR) received radiotherapy, whilst those with STR received VEC before radiotherapy. DNA methylation, 1q, hTERT, ReLA, Tenascin-C, H3K27me3, and pAKT status were evaluated., Results: Five- and ten-year EFS was 49.5% and 46.7%, OS was 69.3% and 60.5%. GTR was achieved in 33/74 (44.6%) and associated with improved EFS (P = .003, HR = 2.6, 95% confidence interval (CI) 1.4-5.1). Grade 3 tumours were associated with worse OS (P = .005, HR = 2.8, 95%CI 1.3-5.8). 1q gain and hTERT expression were associated with poorer EFS (P = .003, HR = 2.70, 95%CI 1.49-6.10 and P = .014, HR = 5.8, 95%CI 1.2-28) and H3K27me3 loss with worse OS (P = .003, HR = 4.6, 95%CI 1.5-13.2). Methylation profiles showed expected patterns. 12 participants with STR did not receive chemotherapy; a protocol violation. However, best chemotherapy RR was 65.5% (19/29, 95%CI 45.7-82.1), exceeding the prespecified 45%., Conclusions: Participants with totally resected ependymoma had the best outcomes. RR of STR to VEC exceeded the pre-specified efficacy criterion. However, cases of inaccurate stratification highlighted the need for rapid central review. 1q gain, H3K27me3 loss, and hTERT expression were all associated with poorer survival outcomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: Relapsed ependymoma has a dismal prognosis, and the role of chemotherapy at relapse remains unclear. This study prospectively evaluated the efficacy of intensive intravenous (IV) etoposide in patients less than 21 years of age with relapsed intracranial ependymoma (NCT00278252)., Methods: This was a single-arm, open-label, phase II trial using Gehan's two-stage design. Patients received IV etoposide 100 mg/m 2 on days 1-3, 8-10, and 15-17 of each 28-day cycle, up to maximum of 6 cycles. Primary outcome was radiological response after 3 cycles. Pharmacokinetic analysis was performed in 10 patients., Results: Twenty-five patients were enrolled and included in the intention-to-treat (ITT) analysis. Three patients were excluded in per-protocol (PP) analysis. After 3 cycles of etoposide, 5 patients (ITT 20%/PP 23%) had a complete response (CR), partial response (PR), or objective response (OR). Nine patients (ITT 36%/PP 41%,) had a best overall response of CR, PR, or OR. 1-year PFS was 24% in ITT and 23% in PP populations. 1-year OS was 56% and 59%, 5-year OS was 20% and 18%, respectively, in ITT and PP populations. Toxicity was predominantly hematological, with 20/25 patients experiencing a grade 3 or higher hematological adverse event., Conclusions: This study confirms the activity of IV etoposide against relapsed ependymoma, however, this is modest, not sustained, and similar to that with oral etoposide, albeit with increased toxicity. These results confirm the dismal prognosis of this disease, provide a rationale to include etoposide within drug combinations, and highlight the need to develop novel treatments for recurrent ependymoma., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation., Significance: We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Children with cancer are not at increased risk of severe SARS-CoV-2 infection; however, adults with haematological malignancies have increased risk of severe infections compared with non-haematological malignancies., Methods: We compared patients with haematological and non-haematological malignancies enrolled in the UK Paediatric Coronavirus Cancer Monitoring Project between 12 March 2020 and 16 February 2021. Children who received stem cell transplantation were excluded., Results: Only 2/62 patients with haematological malignancy had severe/critical infections, with an OR of 0.5 for patients with haematological compared with non-haematological malignancies., Interpretation: Children with haematological malignancies are at no greater risk of severe SARS-CoV-2 infection than those with non-haematological malignancies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Medulloblastoma (MB) is a childhood malignant brain tumour but also occurs in teenagers and young adults (TYA). Considering that MB is heterogeneous, this study aimed to define the molecular landscape of MBs in TYAs. We collated more than 2000 MB samples that included 287 TYA patients (13-24 years). We performed computational analyses consisting of genome-wide methylation and transcriptomic profiles and developed a prognostics model for the TYAs with MB. We identified that TYAs predominantly comprised of Group 4 (40%) and Sonic Hedgehog (SHH)-activated (33%) tumours, with Wingless-type (WNT, 17%) and Group 3 (10%) being less common. TYAs with SHH tumours displayed significantly more gene expression alterations, whereas no gene was detected in the Group 4 tumours. Across MB subgroups, we identified unique and shared sets of TYA-specific differentially methylated probes and DNA-binding motifs. Finally, a 22-gene signature stratified TYA patients into high- and low-risk groups, and the prognostic significance of these risk groups persisted in multivariable regression models ( P = 0.001). This study is an important step toward delineating the molecular landscape of TYAs with MB. The emergence of novel genes and pathways may provide a basis for improved clinical management of TYA with MB.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients., (© 2021. The Author(s).)

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA-RELA or YAP1 -involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation-based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA -associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo , and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion-positive tumors. SIGNIFICANCE: ZFTA-RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion-positive tumors, such as GLI2. This article is highlighted in the In This Issue feature, p. 2113 ., (©2021 American Association for Cancer Research.)

Introduction: Survivors of childhood brain tumours have the poorest health-related quality of life of all cancer survivors due to the multiple physical and psychological sequelae of brain tumours and their treatment. Remotely delivered acceptance and commitment therapy (ACT) may be a suitable and accessible psychological intervention to support young people who have survived brain tumours. This study aims to assess the feasibility and acceptability of remotely delivered ACT to improve quality of life among these young survivors., Methods and Analysis: This study is a two-arm, parallel group, randomised controlled trial comparing ACT with waitlist control at 12-week follow-up as the primary endpoint. Seventy-two participants will be recruited, who are aged 11-24 and have completed brain tumour treatment. Participants will be randomised to receive 12 weeks of ACT either immediately or after a 12-week wait. The DNA-v model of ACT will be employed, which is a developmentally appropriate model for young people. Feasibility will be assessed using the proportion of those showing interest who consent to the trial and complete the intervention. Acceptability will be assessed using participant evaluations of the intervention, alongside qualitative interviews and treatment diaries analysed thematically. A range of clinical outcome measures will also assess physical and mental health, everyday functioning, quality of life and service usage at 12-week follow-up. The durability of treatment effects will be assessed by further follow-up assessments at 24 weeks, 36 weeks and 48 weeks., Ethics and Dissemination: Ethical approval was given by East Midlands, Nottingham 1 Research Ethics Committee (Reference: 20/EM/0237). Study results will be disseminated in peer-reviewed journals, through public events and relevant third sector organisations., Trial Registration: ISRCTN10903290; NCT04722237., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

We present here a novel surface mass spectrometry strategy to perform untargeted metabolite profiling of formalin-fixed paraffin-embedded pediatric ependymoma archives. Sequential Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) and liquid extraction surface analysis-tandem mass spectrometry (LESA-MS/MS) permitted the detection of 887 metabolites (163 chemical classes) from pediatric ependymoma tumor tissue microarrays (diameter: <1 mm; thickness: 4 μm). From these 163 classes, 60 classes were detected with both techniques, whilst LESA-MS/MS and 3D OrbiSIMS individually allowed the detection of another 83 and 20 unique metabolite classes, respectively. Through data fusion and multivariate analysis, we were able to identify key metabolites and corresponding pathways predictive of tumor relapse, which were retrospectively confirmed by gene expression analysis with publicly available data. Altogether, this sequential mass spectrometry strategy has shown to be a versatile tool to perform high-throughput metabolite profiling on sample-limited tissue archives.

Paediatric ependymomas are aggressive, treatment-resistant tumours with a tendency towards relapse, consistent with a sub-population of therapy-resistant cancer stem cells. These cells are believed to derive from brain lipid binding protein (BLBP)-expressing radial glia, hence we proposed that BLBP may be a marker for ependymoma therapy resistance. BLBP protein expression correlated with reduced overall survival (OS) in patients from two trials (CNS9204, a chemotherapy-led infant trial-5 y OS 45% vs. 80%, p = 0.011-and CNS9904, a radiotherapy-led trial-OS 38% vs. 85%, p = 0.002). All ependymoma cell lines examined by qRT-PCR expressed BLBP , with expression elevated in stem cell-enriched neurospheres. Modulation of BLBP function in 2D and 3D assays, using either peroxisome proliferator activated receptor (PPAR) antagonists or BLBP's fatty acid substrate docosahexaneoic acid (DHA), potentiated chemotherapy response and reduced cell migration and invasion in ependymoma cell lines. BLBP is therefore an independent predictor of poor survival in paediatric ependymoma, and treatment with PPAR antagonists or DHA may represent effective novel therapies, preventing chemotherapy resistance and invasion in paediatric ependymoma patients.

The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme-metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite (sequential solvent addition and shaking) single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue.

Background: Pediatric spinal ependymomas (SP-EPNs) are rare primary central nervous system tumors with heterogeneous clinical course. Considering that ependymomas in children are biologically distinct from their adult counterparts, this study aimed to define the molecular landscape of SP-EPNs in children., Methods: In this retrospective study, we have collected tumor samples from 27 SP-EPN patients younger than 18 years and carried out the histological review, DNA methylation, and gene expression profiling., Results: Unsupervised analyses with methylation profiles revealed 2 subgroups where all grade I tumors ( n = 11) were in Group 1, but the grade II/III tumors split into 2 groups ( n = 7 in Group 1 and n = 9 in Group 2). The Heidelberg classifier assigned Group 1 tumors as spinal myxopapillary ependymomas (SP-MPEs), 5 Group 2 tumors as SP-EPNs, and failed to classify 4 Group 2 tumors. Copy numbers derived from DNA methylation arrays revealed subgroup-specific genetic alterations and showed that SP-EPN tumors lack MYCN amplification. Gene expression profiling revealed distinct transcriptomic signatures, including overexpression of genes involved in oxidative phosphorylation in SP-MPEs that were validated by Western blot analysis. We discovered widespread decreases in DNA methylation at enhancer regions that are associated with the expression of oncogenic signaling pathways in SP-MPEs. Furthermore, transcription factor motifs for master regulators, including HNF1B , PAX3 , and ZIC3 , were significantly overrepresented in probes specific to distal regulatory regions in SP-MPEs., Conclusion: Our findings show substantial heterogeneity in pediatric SP-EPN and uncover novel enhancers and transcriptional pathways specific to the SP-MPE subgroup, providing a foundation for future therapeutic strategies., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)