Medulloblastoma is the most common malignant brain tumour of childhood. The identification of critical genes involved in its pathogenesis will be central to advances in our understanding of its molecular basis, and the development of improved therapeutic approaches.

Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.

Subunits of the SWI/SNF chromatin remodeling complex are mutated in a significant proportion of human cancers. Malignant rhabdoid tumors (MRTs) are lethal pediatric cancers characterized by a deficiency in the SWI/SNF subunit SMARCB1. Here, we employ an integrated molecular profiling and chemical biology approach to demonstrate that the receptor tyrosine kinases (RTKs) PDGFRα and FGFR1 are coactivated in MRT cells and that dual blockade of these receptors has synergistic efficacy. Inhibitor combinations targeting both receptors and the dual inhibitor ponatinib suppress the AKT and ERK1/2 pathways leading to apoptosis. MRT cells that have acquired resistance to the PDGFRα inhibitor pazopanib are susceptible to FGFR inhibitors. We show that PDGFRα levels are regulated by SMARCB1 expression, and assessment of clinical specimens documents the expression of both PDGFRα and FGFR1 in rhabdoid tumor patients. Our findings support a therapeutic approach in cancers with SWI/SNF deficiencies by exploiting RTK coactivation dependencies.

Medulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development.

The objective of the study is to conduct a systematic review to compare the effects of high-dose chemotherapy (HDCT) with autologous haematopoietic stem cell transplantation (HSCT) versus standard-dose chemotherapy (SDCT) in children with malignant central nervous system (CNS) tumours.

Although children with acute lymphoblastic leukemia (ALL) generally have a good outcome, some patients do relapse and survival following relapse is poor. Altered DNA methylation is highly prevalent in ALL and raises the possibility that DNA methylation-based biomarkers could predict patient outcome. In this study, genome-wide methylation analysis, using the Illumina Infinium HumanMethylation450 BeadChip platform, was carried out on 52 diagnostic patient samples from 4 genetic subtypes [ETV6-RUNX1, high hyperdiploidy (HeH), TCF3-PBX1 and dic(9;20)(p11-13;q11)] in a 1:1 case-control design with patients who went on to relapse (as cases) and patients achieving long-term remission (as controls). Pyrosequencing assays for selected loci were used to confirm the array-generated data. Non-negative matrix factorization consensus clustering readily clustered samples according to genetic subgroups and gene enrichment pathway analysis suggested that this is in part driven by epigenetic disruption of subtype specific signaling pathways. Multiple bioinformatics approaches (including bump hunting and individual locus analysis) were used to identify CpG sites or regions associated with outcome. However, no associations with relapse were identified. Our data revealed that ETV6-RUNX1 and dic(9;20) subtypes were mostly associated with hypermethylation; conversely, TCF3-PBX1 and HeH were associated with hypomethylation. We observed significant enrichment of the neuroactive ligand-receptor interaction pathway in TCF3-PBX1 as well as an enrichment of genes involved in immunity and infection pathways in ETV6-RUNX1 subtype. Taken together, our results suggest that altered DNA methylation may have differential impacts in distinct ALL genetic subtypes.

Somatic defects at five loci, WT1, CTNNB1, WTX, TP53 and the imprinted 11p15 region, are implicated in Wilms tumor, the commonest childhood kidney cancer. In this study we analysed all five loci in 120 Wilms tumors. We identified epigenetic 11p15 abnormalities in 69% of tumors, 37% were H19 epimutations and 32% were paternal uniparental disomy (pUPD). We identified mutations of WTX in 32%, CTNNB1 in 15%, WT1 in 12% and TP53 in 5% of tumors. We identified several significant associations: between 11p15 and WTX (P=0.007), between WT1 and CTNNB1 (P less than 0.001), between WT1 and pUPD 11p15 (P=0.01), and a strong negative association between WT1 and H19 epimutation (P less than 0.001). We next used these data to stratify Wilms tumor into three molecular Groups, based on the status at 11p15 and WT1. Group 1 tumors (63%) were defined as 11p15-mutant and WT1-normal; a third also had WTX mutations. Group 2 tumors (13%) were WT1-mutant. They either had 11p15 pUPD or were 11p15-normal. Almost all had CTNNB1 mutations but none had H19 epimutation. Group 3 tumors (25%) were defined as 11p15-normal and WT1-normal and were typically normal at all five loci (P less than 0.001). We also identified a novel clinical association between H19 epimutation and bilateral disease (P less than 0.001). These data provide new insights into the pattern, order, interactions and clinical associations of molecular events in Wilms tumor.

The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology.

Neuroblastoma (NB) originates from neural crest-derived precursors and represents the most common childhood extracranial solid tumour. MicroRNAs (miRNAs), a class of small non-coding RNAs that participate in a wide variety of biological processes by regulating gene expression, appear to play an essential role within the NB context. High-throughput next generation sequencing (NGS) was applied to study the miRNA transcriptome in a cohort of NB tumours with and without MYCN-amplification (MNA and MNnA, respectively) and in dorsal root ganglia (DRG), as a control. Out of the 128 miRNAs differentially expressed in the NB vs. DRG comparison, 47 were expressed at higher levels, while 81 were expressed at lower levels in the NB tumours. We also found that 23 miRNAs were differentially expressed in NB with or without MYCN-amplification, with 17 miRNAs being upregulated and 6 being downregulated in the MNA subtypes. Functional annotation analysis of the target genes of these differentially expressed miRNAs demonstrated that many mRNAs were involved in cancer-related pathways, such as DNA-repair and apoptosis as well as FGFR and EGFR signalling. In particular, we found that miR-628-3p negatively affects MYCN gene expression. Furthermore, we identified a novel miRNA candidate with variable expression in MNA vs. MNnA tumours, whose putative target genes are implicated in the mTOR pathway. The present study provides further insight into the molecular mechanisms that correlate miRNA dysregulation to NB development and progression.

The effectiveness of exercise and physical therapy for children with ataxia is poorly understood. The aim of this systematic review was to critically evaluate the range, scope and methodological quality of studies investigating the effectiveness of exercise and physical therapy interventions for children with ataxia. The following databases were searched: AMED, CENTRAL, CDSR, CINAHL, ClinicalTrials.gov, EMBASE, Ovid MEDLINE, PEDro and Web of Science. No limits were placed on language, type of study or year of publication. Two reviewers independently determined whether the studies met the inclusion criteria, extracted all relevant outcomes, and conducted methodological quality assessments. A total of 1988 studies were identified, and 124 full texts were screened. Twenty studies were included in the review. A total of 40 children (aged 5-18 years) with ataxia as a primary impairment participated in the included studies. Data were able to be extracted from eleven studies with a total of 21 children (aged 5-18 years), with a range of cerebellar pathology. The studies reported promising results but were of low methodological quality (no RCTs), used small sample sizes and were heterogeneous in terms of interventions, participants and outcomes. No firm conclusions can be made about the effectiveness of exercise and physical therapy for children with ataxia. There is a need for further high-quality child-centred research.

3T magnetic resonance scanners have boosted clinical application of 1 H-MR spectroscopy (MRS) by offering an improved signal-to-noise ratio and increased spectral resolution, thereby identifying more metabolites and extending the range of metabolic information. Spectroscopic data from clinical 1.5T MR scanners has been shown to discriminate between pediatric brain tumors by applying machine learning techniques to further aid diagnosis. The purpose of this multi-center study was to investigate the discriminative potential of metabolite profiles obtained from 3T scanners in classifying pediatric brain tumors.

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.

We reconstructed the natural history and temporal evolution of the most common childhood brain malignancy, medulloblastoma, by single-cell whole-genome sequencing (sc-WGS) of tumours representing its major molecular sub-classes and clinical risk groups. Favourable-risk disease sub-types assessed (MBWNT and infant desmoplastic/nodular MBSHH) typically comprised a single clone with no evidence of further evolution. In contrast, highest risk sub-classes (MYC-amplified MBGroup3 and TP53-mutated MBSHH) were most clonally diverse and displayed gradual evolutionary trajectories. Clinically adopted biomarkers (e.g. chromosome 6/17 aberrations; CTNNB1/TP53 mutations) were typically early-clonal/initiating events, exploitable as targets for early-disease detection; in analyses of spatially distinct tumour regions, a single biopsy was sufficient to assess their status. Importantly, sc-WGS revealed novel events which arise later and/or sub-clonally and more commonly display spatial diversity; their clinical significance and role in disease evolution post-diagnosis now require establishment. These findings reveal diverse modes of tumour initiation and evolution in the major medulloblastoma sub-classes, with pathogenic relevance and clinical potential.

Here, we present a protocol for deriving a continuum score for group 3 and 4 medulloblastoma tumor samples analyzed via RNA-sequencing or DNA methylation microarray. We describe steps for utilizing NMF-defined group 3/group 4 metagenes to calculate a continuum score between 0 and 1 that can be projected onto new sample data analyzed via RNA-sequencing. We then detail procedures for reverse engineering a continuum score for samples analyzed via DNA methylation microarray using a random forest classifier.

In 2012, an international consensus paper reported that medulloblastoma comprises four molecular subgroups (WNT, SHH, Group 3, and Group 4), each associated with distinct genomic features and clinical behavior. Independently, multiple recent reports have defined further intra-subgroup heterogeneity in the form of biologically and clinically relevant subtypes. However, owing to differences in patient cohorts and analytical methods, estimates of subtype number and definition have been inconsistent, especially within Group 3 and Group 4. Herein, we aimed to reconcile the definition of Group 3/Group 4 MB subtypes through the analysis of a series of 1501 medulloblastomas with DNA-methylation profiling data, including 852 with matched transcriptome data. Using multiple complementary bioinformatic approaches, we compared the concordance of subtype calls between published cohorts and analytical methods, including assessments of class-definition confidence and reproducibility. While the lowest complexity solutions continued to support the original consensus subgroups of Group 3 and Group 4, our analysis most strongly supported a definition comprising eight robust Group 3/Group 4 subtypes (types I-VIII). Subtype II was consistently identified across all component studies, while all others were supported by multiple class-definition methods. Regardless of analytical technique, increasing cohort size did not further increase the number of identified Group 3/Group 4 subtypes. Summarizing the molecular and clinico-pathological features of these eight subtypes indicated enrichment of specific driver gene alterations and cytogenetic events amongst subtypes, and identified highly disparate survival outcomes, further supporting their biological and clinical relevance. Collectively, this study provides continued support for consensus Groups 3 and 4 while enabling robust derivation of, and categorical accounting for, the extensive intertumoral heterogeneity within Groups 3 and 4, revealed by recent high-resolution subclassification approaches. Furthermore, these findings provide a basis for application of emerging methods (e.g., proteomics/single-cell approaches) which may additionally inform medulloblastoma subclassification. Outputs from this study will help shape definition of the next generation of medulloblastoma clinical protocols and facilitate the application of enhanced molecularly guided risk stratification to improve outcomes and quality of life for patients and their families.

Solid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. We aimed to elucidate the impact of hypoxia on the sensitivity of MB cells to chemo- and radiotherapy.

Low-grade gliomas (LGGs) account for about a third of all brain tumours in children. We conducted a detailed study of DNA methylation and gene expression to improve our understanding of the biology of pilocytic and diffuse astrocytomas. Pilocytic astrocytomas were found to have a distinctive signature at 315 CpG sites, of which 312 were hypomethylated and 3 were hypermethylated. Genomic analysis revealed that 182 of these sites are within annotated enhancers. The signature was not present in diffuse astrocytomas, or in published profiles of other brain tumours and normal brain tissue. The AP-1 transcription factor was predicted to bind within 200 bp of a subset of the 315 differentially methylated CpG sites; the AP-1 factors, FOS and FOSL1 were found to be up-regulated in pilocytic astrocytomas. We also analysed splice variants of the AP-1 target gene, CCND1, which encodes cell cycle regulator cyclin D1. CCND1a was found to be highly expressed in both pilocytic and diffuse astrocytomas, but diffuse astrocytomas have far higher expression of the oncogenic variant, CCND1b. These findings highlight novel genetic and epigenetic differences between pilocytic and diffuse astrocytoma, in addition to well-described alterations involving BRAF, MYB and FGFR1.

Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements in cure rates, prediction of disease outcome remains a major challenge and survivors suffer from serious therapy-related side-effects. Recent data showed that patients with WNT-activated tumors have a favorable prognosis, suggesting that these patients could be treated less intensively, thereby reducing the side-effects. This illustrates the potential benefits of a robust classification of medulloblastoma patients and a detailed knowledge of associated biological mechanisms.

The aim of this study is to use a systematic review framework to identify and synthesise the evidence on the use of proton beam therapy (PBT) for the treatment of children with CNS tumours and where possible compare this to the use of photon radiotherapy (RT).

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS.