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AIMS Identifying significant miRNAs that are either under or overexpressed in hypoxia compared to normoxia in glioma cells. Further exploring the effect and role of hypoxia on miR-92a-3p and miR-149-5p in gliomas in apoptotic and cellular senescence pathways. METHOD A range of glioma cells were used for screening, including primary cell lines: GIN28 and GIN31; low-grade cell lines: LGG19 and LGG24; a paediatric cell line: SF188 and a commercially available glioblastoma cell line U87. These cells were cultured at both 1% (hypoxia) and 20% (normoxia) oxygen levels. Screening of miRNAs was achieved by quantitative polymerase chain reaction (qPCR) using miRNA specific primers. Knockdowns/in were achieved by transfecting with miR-92a-3p and miR-149-5p mimics and inhibitors. Caspase-glo assay was used to assess the effect of hypoxia on apoptosis. RESULTS A range of miRNAs were differentially expressed in hypoxia in a range of glioma cell lines. miRNA 92a-3p and miR-149-5p were found to be downregulated and upregulated respectively in primary gliomablastoma cells in hypoxia compared to normoxia. These particular miRNAs are also found to have multiple targets in apoptosis and cellular senescence. CONCLUSION The screening of 90 miRNAs among the different categories of gliomas highlighted multiple miRNAs that were significant in hypoxia compared to normoxia. Using primary cell lines have identified hypoxia-affected miRNAs in glioblastomas. This discovery will increase our knowledge and understanding of the hypoxia effect on miRNAs which may aid and direct targeted therapy to conquer hypoxia in glioblastomas.

Background: Therapeutic intervention in metastatic medulloblastoma is dependent upon elucidating the underlying metastatic mechanism. We investigated whether an epithelialmesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. Methods: A 3D Basement Membrane Extract (3D-BME)-model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and morphology assessed by time-lapse imaging. Gene expression was analysed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil and TWIST1 expression was reduced by either Harmine or shRNA. Results: Metastatic cells migrated to form large metabolically active aggregates, whereas nontumorigenic/ non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (p=0.041 and p=0.04 respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knock-down in cell lines was associated with reduced cell migration (p

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.

Background Glioblastoma (GBM) is a highly aggressive brain tumor with rapid subclonal diversification, harboring molecular abnormalities that vary temporospatially, a contributor to therapy resistance. Fluorescence-guided neurosurgical resection utilizes the administration of 5-aminolevulinic acid (5-ALA) generating individually fluorescent tumor cells within a background population of non-neoplastic cells in the invasive tumor region. The aim of the study was to specifically isolate and interrogate the invasive GBM cell population using a novel 5-ALA-based method. Methods We have isolated the critical invasive GBM cell population by developing 5-ALA-based metabolic fluorescence-activated cell sorting. This allows purification and study of invasive cells from GBM without an overwhelming background “normal brain” signal to confound data. The population was studied using RNAseq, real-time PCR, and immunohistochemistry, with gene targets functionally interrogated on proliferation and migration assays using siRNA knockdown and known drug inhibitors. Results RNAseq analysis identifies specific genes such as SERPINE1 which is highly expressed in invasive GBM cells but at low levels in the surrounding normal brain parenchyma. siRNA knockdown and pharmacological inhibition with specific inhibitors of SERPINE1 reduced the capacity of GBM cells to invade in an in vitro assay. Rodent xenografts of 5-ALA-positive cells were established and serially transplanted, confirming tumorigenicity of the fluorescent patient-derived cells but not the 5-ALA-negative cells. Conclusions Identification of unique molecular features in the invasive GBM population offers hope for developing more efficacious targeted therapies compared to targeting the tumor core and for isolating tumor subpopulations based upon intrinsic metabolic properties.

Paediatric spinal ependymomas are important, albeit uncommon, malignant central nervous system tumours. Unlike adults, children with these tumours are likely to experience a more aggressive disease course, with a higher rate of local failure and a higher rate of metastases. The clinical and molecular factors underlying these differences remain poorly characterized. We analyzed spinal ependymoma (SEPN) tumour samples from 27 paediatric patients (female: 11, male: 15; age range: 4–18 years) using genome-wide DNA methylation profiling, copy-number analysis, as well as transcriptome profiling. Using DNA methylation profiles, two distinct unsupervised consensus-clustering approaches, hierarchical clustering and non-negative matrix factorization reliably identified two subgroups. These subgroups were designated as Myxopapillary ependymomas (SP-MPE) and spinal ependymomas (SP-EPN) based on the online Classifier tool (MNP2.0). The genome-wide copy-number analysis showed differences in numbers and pattern of copy-number alterations between these groups. The gain of chromosome 20 (39%) followed by loss of chromosomes 6 (28%), 10 (28%), and 13 (28%) were detected in the SP-MPE group, whereas loss of chromosome 22 was frequent (60%) in the SP-EPN group. Transcriptomics analysis showed that genes associated with oxidative phosphorylation, TCA cycle components, electron transport, and Interferon-gamma production characterize the SP-MPE group whereas potassium ion import and regulation of astrocyte differentiation characterize the SP-EPN group. Western blot analysis validated the increased protein expression of oxidative phosphorylation complexes in SP-MPE. With this study, we provide a foundation for further molecular characterization of pediatric SEPN subgroups. Our results suggest that mitochondrial oxidative phosphorylation may drive the regulation of energy metabolism of SP-MPE tumours.

Ependymoma is the third commonest childhood brain tumour. Relapse is frequent, often fatal and current therapeutic strategies are inadequate. Previous ependymoma research describes an immunosuppressive environment with T-cell exhaustion, indicating a lack of response to T-cell directed immunotherapy. Understanding the immune microenvironment is therefore critical. We present a computational analysis of ependymoma, gene expression derived, immune profiles. Using 465 ependymoma samples from gene expression datasets (GSE64415, GSE50385, GSE100240) and two RNA-seq databases from UK ependymomas, we applied bulk tumour deconvolution methods (CIBERSORT and xCell) to infer immune cell populations. Additionally, we measured checkpoint blockade related mRNAs and used immunohistochemistry to investigate cell populations in ependymoma sections. CIBERSORT indicated high proportions of M2-like macrophages and smaller proportions of activated natural killer (NK) cells, T follicular helper cells, CD4+ memory T-cells and B-cells. xCell overlapped with the M2-like macrophage and CD4+ memory T-cell signatures seen in CIBERSORT. On immunohistochemistry, T and B cells were scarce, with small numbers of CD8+, CD4+ and CD20+ cells in the parenchyma but greater numbers in surrounding regions. CD68 was more highly expressed in the parenchyma. Analysis of nine checkpoint ligands and receptors demonstrated only the TIM3/GAL9 combination was reliably detectable. GAL9 is implicated in tumour interactions with T-cells and macrophages elsewhere, possibly contributing to poorer outcomes. Our study supports the presence of myeloid cells being leading contributors to the ependymoma immune microenvironment. Further work will delineate the extent of myeloid contribution to immunosuppression across molecular subtypes. Modulation of tumour immunity may contribute to better clinical outcomes.

There have been no significant improvements in the treatments for childhood High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), which continue to have a very poor prognosis. These cancers harbor mutations affecting histone 3 (H3) proteins; 80% of DIPGs with histone H3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. We have generated and validated a histone 3.3 G34R mutant-specific antibody and investigated the downstream effects of H3.3 G34R mutations in pHGG. In order to identify the genes that may be deregulated by G34R mutant histone expression, we have performed chromatin immunoprecipitation (ChIP) assays with our H3.3 G34R and wild type H3 antibodies, using pHGG H3 G34R mutant and wild-type cell lines. Initial analyses of ChIP data have implicated deregulation of cell-signaling pathways including Notch1, Hedgehog, PPAR-1, PLC-beta and Androgen, in H3 G34R mutated pHGG. We are currently determining the effects of altered expression of Notch1 pathway components on tumorigenesis of H3 G34R mutated pHGG, through gene and protein expression and inhibition assays. Specifically we find that the Notch1 pathway component HES1 shows increased expression in G34R mutant cells compared to controls, directing our evaluation of the utility of gamma-secretase inhibitors as potential therapeutics. These analyses may underpin development of novel treatment strategies for H3 mutated pHGG.

Background and aim: Extrahepatic cholangiocarcinoma (CCA) typically presents as biliary strictures. Endoscopic ultrasound (EUS)-fine needle aspiration (FNA) may contribute to the diagnosis of CCA as the etiology of extrahepatic biliary strictures. Our aim was to study the uselfulness of EUS-FNA in diagnosing CCA as the etiology of biliary strictures. Patients and methods: In this meta-analysis, PUBMED and EMBASE databases were examined to find studies published to April 2014 where diagnostic correlation of CCA was available. Studies reporting only “positive for malignancy” were included in our analysis. The main outcome measurements were sensitivity, specificity and likelihood ratio. Results: Six studies were included, covering 196 patients. The overall pooled sensitivity and negative likelihood ratio (LR-) of EUS-FNA for diagnosis of CCA were 66% [95% confidence interval (CI) 57–74%] and 0.34 (95% CI 0.26–0.43), respectively. In five studies (146 patients), where a mass lesion was detected during EUS, the pooled sensitivity and LR- of EUS-FNA for diagnosis of CCA were 80% [95% CI 72–87%] and 0.20 (95% CI 0.13–0.28), respectively. In the 49 patients with a negative brush cytology, the pooled sensitivity and LR- of EUS-FNA for diagnosis of CCA were 59% [95% CI 44–73%] and 0.41 (95% CI 0.27–0.56), respectively. Conclusions: Our study suggests that EUS-FNA is useful in the evaluation of CCA as the etiology of biliary strictures. EUS-FNA may improve the diagnosis of CCA in patients with negative cytology and no mass on cross-sectional imaging.

In contrast, compared to adults, spinal ependymomas (SEPN) are less common in childhood and adolescents. Children with these tumours are likely to experience a more aggressive disease course, with a higher rate of local failure, and a higher rate of metastasis. Presently the molecular basis of SEPN is poorly characterized. Therefore, we have analyzed 29 SEPN tumour samples from pediatric patients (female: 11, male: 15; age range: 4 – 21 years) and performed DNA methylation (n=28) and transcriptome profiling (n=29). Unsupervised analysis of methylation data reliably separated these tumours into two distinct groups: one group covering all myxopapillary ependymomas (MPE) and a second group dominated by grade II SPENs (SP-EPN). We identified 242 differentially methylated regions between these two groups, of which 56% showed high methylation levels in MPE, including 22 regions localized on chromosome 6. Genome-wide copy number analysis using methylation data showed differences in numbers and pattern of DNA copy number alterations between these groups. Gain of chromosome 20 (39%) followed by loss of chromosomes 6 (28%), 10 (28%), and 13 (28%) were detected in the MPE group, whereas loss of chromosome 22 was frequent (60%) in the SP-EPN group. Transcriptomic analysis showed that genes associated with oxidative phosphorylation, TCA cycle components, electron transport, and Interferon-gamma production characterize the MPE group whereas potassium ion import and regulation of astrocyte differentiation characterize the SP-EPN group. Taken together, this data suggest that mitochondrial oxidative phosphorylation may drive the regulation of energy metabolism of MPE tumours.

INTRODUCTION: We previously described separating invasive tumor cells from within normal brain using 5ALA based fluorescent activated cell sorting (FACS). Using RNAseq on these cells we identify new potential therapeutic targets whose expression is elevated in tumor cells invading brain parenchyma. METHOD: 5-aminolevulinic acid (5-ALA) is a clinically used drug for fluorescence guided resection of GBM. Tumors from 11 patients were dissociated and FACS used to separate invasive fluorescent cancer cells from within normal brain. FACS sorted and unsorted mixed samples from tumor core, rim and invasive margin were compared. Gene expression was analyzed by RNA-seq and validated by qPCR, IHC and in vivo xenografts. RESULTS: Differential expression analysis identified 2567 genes with differences between core and invasive margin, and 78 genes with differences between 5-ALA FACS positive and FACS negative. Interestingly SERPINE1 expression is reduced in the unsorted invasive margin but expression remains high within sorted invasive tumor cells. Pathway analysis identified a predominance of immune system pathway changes between core and invasive margin. The differential expression of SERPINE1, VEGF, CHI3L1 and RTN1 in qPCR and IHC validated the same changes observed from the RNA-seq data. The invasive and tumorigenic capacity of 5-ALA positive sorted tumor cells was confirmed by enhanced engraftment in a mouse flank model compared to unsorted cells, whereas 5-ALA negative sorted cells failed to engraft. SERPINE1 knockdown had no effect on GBM cell proliferation but significantly reduced the ability of GBM cells to invade in an in vitro assay. CONCLUSION: This study has demonstrated that 5-ALA fluorescent sorting of the invasive region can identify new targets such as SERPINE1, whose high expression in invasive tumor cells would otherwise be overlooked. Our approach gives hope that we can interrogate true residual disease, identifying more relevant therapeutic targets.

Ependymoma is the second most common posterior fossa tumour in children and causes significant neurological morbidity and mortality. Although these tumours are morphologically similar, recent advances in genomics have revealed that posterior fossa ependymoma (PF-EPN) is actually comprised of three distinct biological entities. Despite these differences, PF-EPN commonly resists chemotherapy and majority of them will recur. Recurrent ependymoma is fatal in children, and yet little is known about its biology. We hypothesized that post-transcriptional changes such as alternative splicing could reveal the underlying mechanisms and uncover genes that would inform actionable targets for therapy. To this end, we mapped the alternative splicing landscape in 17 matched paired samples of primary-recurrent PF-EPNs in children using RNA-seq. We identified 582 differential alternative splicing (AS) events between primary and recurrent PF-EPN, covering five basic types of AS patterns with cassette exon skipping being the majority of the AS events affected (~ 63%). These AS events were enriched for cancer hallmarks important for sustained proliferation and invasion, and affecting key regulators of invasive phenotype such as CD44 and TNC. In addition, immune system genes were enriched among AS events that show shift in inclusion levels in recurrent PF-EPN. Strikingly, genes with AS events did not show significant expression changes between primary and recurrent PF-EPN at the whole-transcript level, suggesting, AS events are an independent and informative measure for defining recurrent ependymoma. Collectively, our data provides evidence for widespread splicing dysregulation in PF-EPN, and suggests the role of aberrant AS in the pathogenesis of ependymoma.

Molecular subgroup is now influencing risk stratification and disease management in medulloblastoma. Tissue metabolite profiles have shown promise in identifying the four consensus subgroups. A smaller number of metabolites can be measured non-invasively in patients using magnetic resonance spectroscopy (MRS). We investigated if a classifier constructed from tissue metabolites could be applied to in-vivo metabolite profiles to accurately predict subgroup non-invasively. Machine learning was used to construct a classifier with tissue concentrations from 10 metabolites reliably detected in-vivo. Retrospectively acquired diagnostic in-vivo MRS was available for 37 cases from four treatment centres. Although we identified WNT tumours by the presence of GABA with 100% accuracy in tissue, GABA was not reliability quantified in this in-vivo dataset. Therefore the classifier was developed using tissue profiles of known molecular subgroup (determined using DNA methylation array) from group 3(n=20), group 4(n=34) and SHH(n=23). The cross-validated accuracy of the tissue classifier was 86%. When applied to in-vivo metabolite profiles, subgroup was predicted non-invasively with an overall accuracy of 76%. Group 3 had the highest proportion of incorrectly classified cases (4/11), followed by SHH (2/10), and group 4 (3/16), largely due to differences in measuring lipids, glutamate, glutamine and hypotaurine in-vivo. We have established the feasibility of non-invasive metabolite profiling to identify medulloblastoma subgroups. With the ongoing optimization of MRS to target specific metabolites including GABA, we can further improve accuracy. Rapid, non-invasive preoperative diagnosis of subgroup will offer opportunities to stratify early therapeutic intervention especially surgery, avoiding long-term sequalae and improving quality of survival.