Noise suppression of proton magnetic resonance spectroscopy improves paediatric brain tumour classification.

Proton magnetic resonance spectroscopy (1H-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 1H-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 1H-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 1H-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 1H-MRS. The study shows that fitting-based signal-to-noise ratios of clinical 1H-MRS can be significantly improved by using AWNS with insignificantly altered line width, and the post-noise-suppression 1H-MRS may have better diagnostic performance for paediatric brain tumours.

Characterisation of paediatric brain tumours by their MRS metabolite profiles.

1H-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.

Metabolite profiles of medulloblastoma for rapid and non-invasive detection of molecular disease groups.

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.

Multi-omic approach identifies hypoxic tumor-associated myeloid cells that drive immunobiology of high-risk pediatric ependymoma.

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.

Significant increase of high-risk chromosome 1q gain and 6q loss at recurrence in posterior fossa group A ependymoma: A multicenter study.

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.

Evaluating the efficacy and safety of single-agent etoposide intra-CSF chemotherapy in children and young people with relapsed/refractory central nervous system tumours.

PURPOSE: The aim of the project was to evaluate intra-CSF etoposide administration in a palliative setting for children and young people with relapsed/refractory central nervous system (CNS) tumours, with the primary endpoints being overall survival and progression-free survival time. A safety endpoint was to assess the side effect profile and complications of intra-CSF etoposide. METHODS: Thirty-five patients under the age of 30 years (median age: 5.33 years) were enrolled onto the project. The cross-centre study was a service evaluation, with a data collection spreadsheet designed in Nottingham and completed by both Nottingham and Oxford centres. Data was analysed using SPSS, assessing the overall survival and progression-free survival times, as well as the 6-month and 1-year survival rates. RESULTS: The median overall survival and progression-free survival times were 10.97 and 5.91 months, respectively. The 6-month and 1-year overall survival rates were 67% and 48%, and the progression-free survival rates were 50% and 22%. Age at the start of intra-CSF therapy was significantly associated with overall survival (P = 0.046), with the 6 + age group having improved overall survival. Treatment type was significantly associated with overall survival (P = 0.012), with etoposide intra-CSF treatment being associated with improved overall survival. Treatment duration was significantly associated with both overall survival (P < 0.001) and progression-free survival (P < 0.001). CONCLUSION: Intra-CSF etoposide treatment has shown to increase both overall and progression-free survival significantly, whilst having few side effects and maintaining a good quality of life for patients, reflecting it as a beneficial therapy in the palliative setting.

Pediatric-type high-grade neuroepithelial tumors with CIC gene fusion share a common DNA methylation signature.

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.

Optimizing biomarkers for accurate ependymoma diagnosis, prognostication, and stratification within International Clinical Trials: A BIOMECA study.

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.

Transatlantic progress in measurement of cognitive outcomes in paediatric oncology trials.

The importance of measuring quality of survival within paediatric oncology trials is increasingly recognised. However, capturing neuropsychological outcomes and other aspects of quality of survival in the context of large or multinational trials can be challenging. We provide examples of protocols designed to address this challenge recently employed in clinical trials in the USA and Europe. We discuss their respective strengths and challenges, obstacles encountered and future opportunities for transatlantic collaboration.

Applying machine learning classifiers to automate quality assessment of paediatric dynamic susceptibility contrast (DSC-) MRI data.

OBJECTIVE: Investigate the performance of qualitative review (QR) for assessing dynamic susceptibility contrast (DSC-) MRI data quality in paediatric normal brain and develop an automated alternative to QR. METHODS: 1027 signal-time courses were assessed by Reviewer 1 using QR. 243 were additionally assessed by Reviewer 2 and % disagreements and Cohen's κ (κ) were calculated. The signal drop-to-noise ratio (SDNR), root mean square error (RMSE), full width half maximum (FWHM) and percentage signal recovery (PSR) were calculated for the 1027 signal-time courses. Data quality thresholds for each measure were determined using QR results. The measures and QR results trained machine learning classifiers. Sensitivity, specificity, precision, classification error and area under the curve from a receiver operating characteristic curve were calculated for each threshold and classifier. RESULTS: Comparing reviewers gave 7% disagreements and κ = 0.83. Data quality thresholds of: 7.6 for SDNR; 0.019 for RMSE; 3 s and 19 s for FWHM; and 42.9 and 130.4% for PSR were produced. SDNR gave the best sensitivity, specificity, precision, classification error and area under the curve values of 0.86, 0.86, 0.93, 14.2% and 0.83. Random forest was the best machine learning classifier, giving sensitivity, specificity, precision, classification error and area under the curve of 0.94, 0.83, 0.93, 9.3% and 0.89. CONCLUSION: The reviewers showed good agreement. Machine learning classifiers trained on signal-time course measures and QR can assess quality. Combining multiple measures reduces misclassification. ADVANCES IN KNOWLEDGE: A new automated quality control method was developed, which trained machine learning classifiers using QR results.

Can I go home now? The safety and efficacy of a new UK paediatric febrile neutropenia protocol for risk-stratified early discharge on oral antibiotics.

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.

National advisory panels for childhood cancer in the United Kingdom: An evaluation of current practice and a best practice statement for the future.

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.

Spatial transcriptomic analysis delineates epithelial and mesenchymal subpopulations and transition stages in childhood ependymoma.

BACKGROUND: The diverse cellular constituents of childhood brain tumor ependymoma, recently revealed by single cell RNA-sequencing, may underly therapeutic resistance. Here we use spatial transcriptomics to further advance our understanding of the tumor microenvironment, mapping cellular subpopulations to the tumor architecture of ependymoma posterior fossa subgroup A (PFA), the commonest and most deadly childhood ependymoma variant. METHODS: Spatial transcriptomics data from intact PFA sections was deconvoluted to resolve the histological arrangement of neoplastic and non-neoplastic cell types. Key findings were validated using immunohistochemistry, in vitro functional assays and outcome analysis in clinically-annotated PFA bulk transcriptomic data. RESULTS: PFA are comprised of epithelial and mesenchymal histological zones containing a diversity of cellular states, each zone including co-existing and spatially distinct undifferentiated progenitor-like cells; a quiescent mesenchymal zone population, and a second highly mitotic progenitor population that is restricted to hypercellular epithelial zones and that is more abundant in progressive tumors. We show that myeloid cell interaction is the leading cause of mesenchymal transition in PFA, occurring in zones spatially distinct from hypoxia-induced mesenchymal transition, and these distinct EMT-initiating processes were replicated using in vitro models of PFA. CONCLUSIONS: These insights demonstrate the utility of spatial transcriptomics to advance our understanding of ependymoma biology, revealing a clearer picture of the cellular constituents of PFA, their interactions and influence on tumor progression.

Lineage switching of the cellular distribution of BRAFV600E in multisystem Langerhans cell histiocytosis.

Most children with high-risk Langerhans cell histiocytosis (LCH) have BRAFV600E mutation. BRAFV600E alleles are detectable in myeloid mononuclear cells at diagnosis but it is not known if the cellular distribution of mutation evolves over time. Here, the profiles of 16 patients with high-risk disease were analyzed. Two received conventional salvage chemotherapy, 4 patients on inhibitors were tracked at intervals of 3 to 6 years, and 10 patients, also given inhibitors, were analyzed more than 2 years after diagnosis. In contrast to the patients responding to salvage chemotherapy who completely cleared BRAFV600E within 6 months, children who received inhibitors maintained high BRAFV600E alleles in their blood. At diagnosis, mutation was detected predominantly in monocytes and myeloid dendritic cells. With time, mutation switched to the T-cell compartment, which accounted for most of the mutational burden in peripheral blood mononuclear cells, more than 2 years from diagnosis (median, 85.4%; range, 44.5%-100%). The highest level of mutation occurred in naïve CD4+ T cells (median, 51.2%; range, 3.8%-93.5%). This study reveals an unexpected lineage switch of BRAFV600E mutation in high-risk LCH, which may influence monitoring strategies for the potential withdrawal of inhibitor treatment and has new implications for the pathogenesis of neurodegeneration, which occurred in 4 patients.

Impact of Tumour Epstein-Barr Virus Status on Clinical Outcome in Patients with Classical Hodgkin Lymphoma (cHL): A Review of the Literature and Analysis of a Clinical Trial Cohort of Children with cHL.

In this study, we have re-evaluated how EBV status influences clinical outcome. To accomplish this, we performed a literature review of all studies that have reported the effect of EBV status on patient outcome and also explored the effect of EBV positivity on outcome in a clinical trial of children with cHL from the UK. Our literature review revealed that almost all studies of older adults/elderly patients have reported an adverse effect of an EBV-positive status on outcome. In younger adults with cHL, EBV-positive status was either associated with a moderate beneficial effect or no effect, and the results in children and adolescents were conflicting. Our own analysis of a series of 166 children with cHL revealed no difference in overall survival between EBV-positive and EBV-negative groups (p = 0.942, log rank test). However, EBV-positive subjects had significantly longer event-free survival (p = 0.0026). Positive latent membrane protein 1 (LMP1) status was associated with a significantly lower risk of treatment failure in a Cox regression model (HR = 0.21, p = 0.005). In models that controlled for age, gender, and stage, EBV status had a similar effect size and statistical significance. This study highlights the age-related impact of EBV status on outcome in cHL patients and suggests different pathogenic effects of EBV at different stages of life.

Lipoprotein Deprivation Reveals a Cholesterol-Dependent Therapeutic Vulnerability in Diffuse Glioma Metabolism.

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.

Characterisation of Expression the Arginine Pathway Enzymes in Childhood Brain Tumours to Determine Susceptibility to Therapeutic Arginine Depletion.

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.

Response assessment in paediatric intracranial ependymoma: recommendations from the Response Assessment in Pediatric Neuro-Oncology (RAPNO) working group.

Response criteria for paediatric intracranial ependymoma vary historically and across different international cooperative groups. The Response Assessment in the Pediatric Neuro-Oncology (RAPNO) working group, consisting of an international panel of paediatric and adult neuro-oncologists, neuro-radiologists, radiation oncologists, and neurosurgeons, was established to address both the issues and the unique challenges in assessing the response in children with CNS tumours. We established a subcommittee to develop response assessment criteria for paediatric ependymoma. Current practice and literature were reviewed to identify major challenges in assessing the response of paediatric ependymoma to clinical trial therapy. For areas in which data were scarce or unavailable, consensus was reached through an iterative process. RAPNO response assessment recommendations include assessing disease response on the basis of changes in tumour volume, and using event-free survival as a study endpoint for patients entering clinical trials without bulky disease. Our recommendations for response assessment include the use of brain and spine MRI, cerebral spinal fluid cytology, neurological examination, and steroid use. Baseline postoperative imaging to assess for residual tumour should be obtained 24-48 h after surgery. Our consensus recommendations and response definitions should be prospectively validated in clinical trials.

Toward Improved Diagnosis Accuracy and Treatment of Children, Adolescents, and Young Adults With Ependymoma: The International SIOP Ependymoma II Protocol.

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.