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In 2016 there were 5250 brain cancer deaths in the UK. Standard treatment is surgical resection followed by chemo-radiotherapy. In most cases of diffuse glioma, complete tumour resection is not feasible. Many chemotherapy drugs have untested penetration through the blood brain barrier, potentially leading to sub-therapeutic concentrations in the tumour. There is need to refine current treatment strategies in relation to the understanding of tumour biology, and rapidly introduce and evaluate novel therapeutic approaches and agents through delivering rigorous clinical trials. The TESSA JOWELL BRAIN MATRIX Study will evaluate the feasibility of delivering precision medicine for brain cancer patients within the NHS. A multicentre, platform feasibility study of 1200 patients with diffuse glioma will build on the 100,000 genome project to develop and evaluate an infrastructure to collect and integrate: 1) real time comprehensive integrated molecular analysis, including whole genome sequencing and epigenetic classification; 2) serial sampling and annotation of tumours; 3) collection of matched clinical data; 4) assessment of patient quality of life; 5) centralised radiological review and response assessment as per RANO criteria. Once developed this will allow rapid introduction of therapeutic trials to specific patient groups. Secondary objectives include: understanding the association between extent of resection and molecular stratification to refine the role of surgery; optimisation and harmonisation of protocols to best collect, manage and store tissue, clinical data, and radiological images in order to provide a resource for researchers, both within and outside of the study. Improve patient recruitment by identifying and removing recruitment barriers and improve the information and consent processes for patients. Promote the development of a national network with expertise in brain cancer. Enrolment of the first patient is expected in late 2019. For further information, please contact the Brain Matrix Trial Office BrainMatrix@trials.bham.ac.uk.

Recent findings from our group, and the wider community, show that standard treatment does not impose an apparent bottleneck on the clonal evolution of adult glioblastoma (GBM), implying a lack of direct therapeutic opportunity. This does not negate the possibility that multiple treatment-resistance mechanisms co-exist in tumours, repeated across patients, making a combination of targeted therapies a potentially effective approach. We investigated whether treatment resistance may be driven by selection of cellular properties conferred above the level of the genome. Differential expression analysis was performed on 23 pairs of primary and recurrent tumours from patients who received standard treatment and had a local recurrence treated by surgery and second line chemotherapy. This revealed a treatment-induced shift in cell states linked to normal neurodevelopment. The latter is orchestrated by cascades of transcription factors. We, therefore, applied a bespoke gene set enrichment analysis to our paired expression data to investigate whether any factors were implicated in co-regulation of the genes that were altered through therapy. This identified a specific chromatin remodelling machinery, instrumental in normal neurogenesis. We validated our results in an independent cohort of 22 paired GBM samples. Our results suggest that the chromatin remodelling machinery is responsible for determining transcriptional hierarchies in GBM, shown elsewhere to have different treatment sensitivities such that their relative abundances are altered through treatment.

BackgroundMicroRNAs play an important role in the regulation of mRNA translation, and have therapeutic potential in cancer and other diseases.MethodsTo profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen using a synthetic oligonucleotide library representing all known human microRNAs in adult and pediatric GBM cells. Bio-informatics analysis were used to refine this list and the top seven microRNAs were validated in a larger panel of cells by flow-cytometry, and RTqPCR. The downstream mechanism of the strongest and most consistent candidate was investigated by siRNAs, 3’UTR luciferase assays and Western Blotting.ResultsOur screen identified ∼100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 expressing cells and characterized the mechanism of action as cytokinesis failure followed by apoptosis, which was observed in an extended GBM cell panel including two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In glioblastoma cells, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target of miR-1300. ECT2 siRNA phenocopied the effects of miR-1300, and its overexpression led to a significant rescue of miR-1300 induced binucleation.ConclusionMiR-1300 was identified as a novel regulator of endomitosis with translatable potential for therapeutic application. The datatasets will be a resource for the neuro-oncology community.Key points (2 or 3 key points 85 characters plus spaces each)70% of cytotoxic microRNAs were shared between adult and pediatric glioblastoma cellsMiR-1300 expression is restricted to endomitosis within megakaryocyte differentiationMiR-1300’s ectopic expression is a potent and promising therapeutic tool in cancerImportance of StudyPrevious functional studies of microRNAs involved in the regulation of glioblastoma cell proliferation and/or survival have focused on adult glioblastoma alone and are restricted to only a few microRNAs at a time. Our study provides the first encompassing landscape of potent cytotoxic microRNAs in pediatric and adult glioblastoma.Not only, does our data provide an invaluable resource for the research community but it also revealed that 70% of microRNAs with significant cytotoxicity were shared by adult and pediatric cells. Finally, we identified and characterized the previously undescribed role of microRNA-1300 in the tight regulation of megakaryocyte differentiation into platelets and how, when expressed outside of this context, miR-1300 consistently causes cytokinesis failure followed by apoptosis, and thus represents a powerful cytotoxic tool with potential for translation towards therapeutic applications.

BACKGROUND Glioblastoma (GBM) tumours recur following standard treatment in almost all cases. We use ‘omics technologies to simultaneously profile pairs of primary and matched recurrent GBM to specifically identify and characterise the cells that resisted treatment, with the aim of determining how to more effectively kill them. MATERIAL AND METHODS We have analysed high coverage RNAseq data from pairs of GBM tumours: primary de novo tumour and matched local recurrence from patients that underwent standard therapy. Our original cohort constituted 23 pairs and our validation cohort was an additional 22 pairs. We also cultured two plates of spheroids directly from a patient’s GBM, treating one with radiation and temozolomide. We monitored growth and captured and sequenced RNA from single cells at two time-points: one week post-treatment when the deviation between untreated and treated spheroid growth curves was most pronounced; and three weeks post-treatment when the growth rate of treated spheroids had recovered. We investigated differential gene expression between primary and recurrent pairs, and single cells pre- and post-treatment, and performed a bespoke per patient gene set enrichment analysis. RESULTS Differential gene expression analysis in 23 tumour pairs indicated a treatment-induced shift in cell states linked to normal neurogenesis and prompted us to develop a novel gene set enrichment analysis approach to identify gene regulatory factors that may orchestrate such a shift. This revealed the significant and universal dysregulation of genes, through therapy, that are targeted by a specific chromatin remodeling machinery. This finding was validated in an independent cohort of 22 further GBM pairs. To understand the therapeutic potential of this finding we must determine whether genes are dysregulated through therapy owing to a) their fixed expression in inherently treatment resistance cells in the primary tumour which get selected during therapy to increase the signal of that profile, or b) changes in expression during the process of cells acquiring treatment resistance. To inspect this, we analysed single cell gene expression data from GBM spheroids pre- and post-treatment. We found that there was significant dysregulation of the genes associated with the chromatin remodeling complex but only at the three-week post-treatment time-point. CONCLUSION Our results indicate that GBM cells are being transcriptionally reprogrammed in response to treatment; the mechanism of which may represent a therapeutic opportunity.

BackgroundGlioblastoma (GBM) is a fatal and incurable brain cancer with a dismal prognosis. In order to impact on this disease, we need to understand how infiltrating, non resectable tumour cells resist chemoradiation and facilitate disease recurrence. To this end, we generated or acquired bulk tumour RNA sequencing data from 45 paired primary and locally recurrent GBM tumours (split into original and validation cohorts) from patients that received standard treatment. We also generated DNA methylation profiles for 9 pairs and sequenced RNA from single cells isolated from a patient derived GBM spheroid model at different timepoints following in vitro chemoradiation.ResultsWe have identified a set of genes with Jumonji and AT-Rich Interacting Domain 2 (JARID2) binding sites in their promoters that are universally dysregulated in post-standard treatment recurrent GBMs compared to the primary tumour. The direction of dysregulation is patient-dependent and not associated with differential promoter DNA methylation. Our in vitro experiments suggest that this dysregulation occurs dynamically following treatment as opposed to resulting from selection of cells with specific expression profiles.ConclusionJARID2 is an accessory protein to a chromatin remodeling complex, responsible for histone modifications observed during cell state transitions in both normal brain and GBM. We propose that JARID2 facilitates GBM recurrence following treatment by indirect transcriptional reprogramming of surviving cells in whichever manner is needed to reproduce the phenotypic heterogeneity required for tumour regrowth in vivo. The mechanism of this reprogramming may present a therapeutic vulnerability for more effective treatment of GBM.

The heterogeneous and invasive nature of pediatric gliomas poses significant treatment challenges, highlighting the importance of identifying novel chemotherapeutic targets. Recently, recurrent Fibroblast growth factor receptor 1 (FGFR1) mutations in pediatric gliomas have been reported. Here, we explored the clinical relevance of FGFR1 expression, cell migration in low and high grade pediatric gliomas and the role of FGFR1 in cell migration/invasion as a potential chemotherapeutic target. A high density tissue microarray (TMA) was used to investigate associations between FGFR1 and activated phosphorylated FGFR1 (pFGFR1) expression and various clinicopathologic parameters. Expression of FGFR1 and pFGFR1 were measured by immunofluorescence and by immunohistochemistry (IHC) in 3D spheroids in five rare patient-derived pediatric low-grade glioma (pLGG) and two established high-grade glioma (pHGG) cell lines. Two-dimensional (2D) and three-dimensional (3D) migration assays were performed for migration and inhibitor studies with three FGFR1 inhibitors. High FGFR1 expression was associated with age, malignancy, tumor location and tumor grade among astrocytomas. Membranous pFGFR1 was associated with malignancy and tumor grade. All glioma cell lines exhibited varying levels of FGFR1 and pFGFR1 expression and migratory phenotypes. There were significant anti-migratory effects on the pHGG cell lines with inhibitor treatment and anti-migratory or pro-migratory responses to FGFR1 inhibition in the pLGGs. Our findings support further research to target FGFR1 signaling in pediatric gliomas.

Patients with glioblastoma (GBM) have a poor prognosis, and inefficient delivery of drugs to tumors represents a major therapeutic hurdle. Hematopoietic stem cell (HSC)-derived myeloid cells efficiently home to GBM and constitute up to 50% of intratumoral cells, making them highly appropriate therapeutic delivery vehicles. Because myeloid cells are ubiquitously present in the body, we recently established a lentiviral vector containing matrix metalloproteinase 14 (MMP14) promoter, which is active specifically in tumor-infiltrating myeloid cells as opposed to myeloid cells in other tissues, and resulted in a specific delivery of transgenes to brain metastases in HSC gene therapy. Here, we used this novel approach to target transforming growth factor beta (TGFβ) as a key tumor-promoting factor in GBM. Transplantation of HSCs transduced with lentiviral vector expressing green fluorescent protein (GFP) into lethally irradiated recipient mice was followed by intracranial implantation of GBM cells. Tumor-infiltrating HSC progeny was characterized by flow cytometry. In therapy studies, mice were transplanted with HSCs transduced with lentiviral vector expressing soluble TGFβ receptor II–Fc fusion protein under MMP14 promoter. This TGFβ-blocking therapy was compared with the targeted tumor irradiation, the combination of the two therapies, and control. Tumor growth and survival were quantified (statistical significance determined by t-test and log-rank test). T cell memory response was probed through a repeated tumor challenge. Myeloid cells were the most abundant HSC-derived population infiltrating GBM. TGFβ-blocking HSC gene therapy in combination with irradiation significantly reduced tumor burden as compared with monotherapies and the control, and significantly prolonged survival as compared with the control and TGFβ-blocking monotherapy. Long-term protection from GBM was achieved only with the combination treatment (25% of the mice) and was accompanied by a significant increase in CD8+ T cells at the tumor implantation site following tumor rechallenge. We demonstrated a preclinical proof-of-principle for tumor myeloid cell-specific HSC gene therapy in GBM. In the clinic, HSC gene therapy is being successfully used in non-cancerous brain disorders and the feasibility of HSC gene therapy in patients with glioma has been demonstrated in the context of bone marrow protection. This indicates an opportunity for clinical translation of our therapeutic approach.

The treatment of primary brain tumours remains a significant clinical challenge and developing novel treatment approaches is a major unmet need. The results from recent studies investigating novel molecular targeting agents have been disappointing and this is thought to reflect significant intra-tumoral heterogeneity, tumour cell plasticity and redundancy in targetable pathways. Improved understanding of the underlying biology that drives hallmarks of this disease including angiogenesis, invasion and stem-like behaviour is necessary to identify new approaches to intervention. The identification of prognostic and predictive biomarkers that reflect this biology is also an unmet need in progress towards individualised treatment.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. The current standard of care includes surgery followed by radiotherapy (RT) and chemotherapy with temozolomide (TMZ). Treatment often fails due to the radiation resistance and intrinsic or acquired TMZ resistance of a small percentage of cells with stem cell-like behavior (CSC). The NOTCH signaling pathway is expressed and active in human glioblastoma and NOTCH inhibitors attenuate tumor growth in vivo in xenograft models. Here we show using an image guided micro-CT and precision radiotherapy platform that a combination of the clinically approved NOTCH/γ-secretase inhibitor (GSI) RO4929097 with standard of care (TMZ + RT) reduces tumor growth and prolongs survival compared to dual combinations. We show that GSI in combination with RT and TMZ attenuates proliferation, decreases 3D spheroid growth and results into a marked reduction in clonogenic survival in primary and established glioma cell lines. We found that the glioma stem cell marker CD133, SOX2 and Nestin were reduced following combination treatments and NOTCH inhibitors albeit in a different manner. These findings indicate that NOTCH inhibition combined with standard of care treatment has an anti-glioma stem cell effect which provides an improved survival benefit for GBM and encourages further translational and clinical studies.

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of Glioblastoma Multiforme (GBM), the most aggressive type of brain cancer in adults. We show that small molecule inhibition of RHO-associated serine/threonine kinase (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo-induced cellular network (iNet) ‘webs’, which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range calcium signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.

SummaryTumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of Glioblastoma Multiforme (GBM), the most aggressive type of brain cancer in adults. We show that small molecule inhibition of RHO-associated serine/threonine kinase (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formedde novo-induced cellular network (iNet) ‘webs’, which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range calcium signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet websex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.

INTRODUCTION: Gliomas are intrinsic brain tumours characterised by their highly invasive, malignant and aggressive nature. Persistently poor prognoses highlight the urgent clinical need to identify novel approaches and therapeutic targets to improve glioma management. Recently we reported a correlation of fibroblast growth factor receptor 1 (FGFR1) expression with malignancy, tumour grade and location in paediatric gliomas. There is evidence that the L1 cell adhesion molecule (L1CAM) potentiates FGFR1 signalling. L1CAM is a transmembrane glycoprotein associated with poor clinical outcomes in various cancers promoting cell motility. Here, following our initial studies on FGFR1, we investigated FGFR1 in its activated phosphorylated form (pFGFR1) as well as L1CAM expression in our cohort and association with various clinicopathological parameters. METHODS. A commercially available tissue microarray (CNS2081, US Biomax) was stained for pFGFR1 and L1CAM expression and data was scored using manual and digital assessment (QuPath). Scores were separately dichotomised into low and high L1CAM and pFGFR1 expression using the median value in SPSS. A two-sided Pearson’s chi squared statistical test was performed using GraphPad Prism where p

BACKGROUND: Treating elderly patients with glioblastoma remains challenging and alternatives to conventional cytotoxics, particularly in patients with unmethylated tumours are required. Pre-clinical data suggest that autophagy inhibition sensitizes gliomas to radiotherapy and chemotherapy MATERIAL AND METHODS: We conducted an open-label multicentre randomized phase-II study of patients aged >=70, performance status 0–1 with newly diagnosed high-grade glioma considered suitable for short course radiotherapy. They were randomized (1:2) to a calibration arm given radiotherapy (RT) alone (30Gy;5Gy/6fractions over 2 weeks), or an experimental arm given RT plus hydroxychloroquine (HCQ, 200mg orally twice daily) from 7 days prior to RT until progression/toxicity. The primary endpoint was one-year overall survival (OS; target 34%). Secondary endpoints included progression free survival (PFS), quality of life and toxicity. RESULTS: Fifty-three patients with median age 75 were included May 2013 to Oct 2016 (n=35 RT+HCQ, n=18 RT alone). One-year survival was 20.3% (95%CI 8–36) in the experimental arm and median OS was 7.9 months. In the calibration arm, one-year survival was 47.6% (95%CI 2–71) and median OS was 11.8 months. Six-month PFS was 25.2% vs 36.5%. Grade 3–5 toxicities occurred in 60% (RT+HCQ) and 38.8% (RT alone), which was not significantly different, with no obvious imbalance in specific events. Common toxicities included lethargy and pulmonary infections. QoL did not differ between arms. The Data Monitoring Committee stopped recruitment early because the experimental arm could not reach its target 1-year OS CONCLUSION: Despite growing interest in autophagy inhibition as a potential mechanism for treating cancer, giving HCQ with short course radiotherapy did not improve outcomes compared to radiotherapy alone. Although median OS was similar to data from other studies for this patient group, it was worse than the calibration arm. Future opportunities could include using higher doses of HCQ, or definition of selective biomarkers for response to autophagy inhibition.

Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.

Introduction UK Neuro-Oncologists and multidisciplinary colleagues are developing one of the first randomised clinical trials of proton beam radiotherapy(PBT) to compare quality of life(QOL), cognitive function and other late effects in adults with good prognosis glioma following either PBT or photon radiotherapy. The feasibility of running randomised studies with PBT is an important consideration, particularly in respect of participants’ views of a randomised design requiring treatment at national centres. We sought patient and carer engagement on our proposals to ensure we incorporate their views. Methods To explore these issues, we invited patients who had previously completed radiotherapy for oligodendroglioma and their carers to attend a focus group in Manchester in November 2018. Fifteen participants attended. We sought views on our trial proposal through small group discussions centred around 5 questions, led and facilitated by neuro-oncologists, a research radiographer, neuro-psychologist and statistician. Results Participants strongly endorsed the trial proposal and positively highlighted the opportunity to access PBT within a clinical trial and the group recognised and supported the need for randomisation and stated this should be 1:1. Patients disliked some traditional terminology such as ‘trial’ and ‘neurocognitive tests’ and preferred ‘research study’ and ‘neurocognitive assessments’. Patient and carers expressed the need for careful consideration of issues around travel and accommodation during PBT away from home. Interestingly, participants considered that standard QOL questionnaires fail to address some important areas reflecting daily wellbeing. Conclusion We acknowledge and will now incorporate these important patient and carer observations to strengthen our study and add validity to the key study endpoints.

Background Systemic delivery of a complementary cDNA library expressed from the vesicular stomatitis virus (VSV) treats tumors by vaccinating against a wide range of tumor associated antigens (TAAs). For subcutaneous B16 melanomas, therapy was achieved using a specific combination of self-TAAs (neuroblastoma-Ras, cytochrome c, and tyrosinase-related protein 1) expressed from VSV. However, for intracranial B16 tumors, a different combination was therapeutic (consisting of VSV-expressed hypoxia-inducible factor [HIF]–2α, Sox-10, c-Myc, and tyrosinase-related protein 1). Therefore, we tested the hypothesis that tumors of different histological types growing in the brain share a common immunogenic signature which can be exploited for immunotherapy. Methods Syngeneic tumors, including GL261 gliomas, in the brains of immune competent mice were analyzed for their antigenic profiles or were treated with systemic viroimmunotherapy. Results Several different histological types of tumors growing intracranially, as well as freshly resected human brain tumor explants, expressed a HIF-2αHi phenotype imposed by brain-derived CD11b+ cells. This location-specific antigen expression was exploited therapeutically against intracranial GL261 gliomas using systemically delivered VSV expressing HIF-2α, Sox-10, and c-Myc. Viroimmunotherapy was enhanced by immune checkpoint inhibitors, associated with the de-repression of antitumor T-helper cell type 1 (Th1) interferon-γ and Th17 T cell responses. Conclusions Since different tumor types growing in the same location in the brain share a location-specific phenotype, we suggest that antigen-specific immunotherapies should be based upon expression of both histological type–specific tumor antigens and location-specific antigens. Our findings support clinical application of VSV-TAA therapy with checkpoint inhibition for aggressive brain tumors and highlight the importance of the intracranial microenvironment in sculpting a location-specific profile of tumor antigen expression.

We present a retrospective single-centre study to determine whether delays in diagnosis of high-grade glioma (HGG) impact on overall survival (OS).Consecutive patients diagnosed with HGG at a single neuroscience centre in 2011 were reviewed. Route of referral and time from initial presentation to diagnosis were analysed and correlated with OS.118 patients were studied - 92 patients with glioblastoma (GBM). Diagnosis of GBM in patients presenting to emergency services was quicker than that through outpatients (8 days vs. 26 days, p0.0001), but these patients had significantly worse OS (181 days vs. 386 days p = 0.0075). This trend was observed for the whole cohort (Grade III and GBM), with OS 278 days in patients presenting to emergency services compared with 423 days for patients presenting via outpatients (p = 0.0034). Patients presenting to outpatients were younger (median age: 54 years) compared with patients presenting to emergency services (median age: 62.5 years) (p = 0.0106). There were no other differences between the two groups with respect to the nature of presenting symptoms.Earlier diagnosis is paradoxically associated with a worse OS in GBM. An 'aggressive' phenotype with rapid symptomatic deterioration and hence emergency presentation is a poor prognostic factor not influenced by earlier diagnosis.

Immune checkpoint inhibitors, including those targeting programmed cell death protein 1 (PD-1), are reshaping cancer therapeutic strategies. Evidence suggests, however, that tumor response and patient survival are determined by tumor programmed death-ligand 1 (PD-L1) expression. We hypothesized that preconditioning of the tumor immune microenvironment using targeted, virus-mediated interferon (IFN) stimulation, would upregulate tumor PD-L1 protein expression and increase cytotoxic T cell infiltration, improving the efficacy of subsequent checkpoint blockade. Oncolytic viruses (OVs) represent a promising form of cancer immunotherapy. For brain tumors, almost all studies to date have used direct intralesional injection of OV, because of the largely untested belief that intravenous (i.v.) administration will not deliver virus to this site. Here we show, in a window-of-opportunity clinical study, that i.v. infusion of oncolytic human Orthoreovirus (referred to herein as reovirus), leads to infection of tumor cells subsequently resected as part of standard clinical care, both in high-grade glioma (HGG) and in brain metastases, and increases cytotoxic T cell tumor infiltration relative to patients not treated with virus. We further show that reovirus upregulates IFN-regulated gene expression, as well as the PD-1/PD-L1 axis in tumors, via an IFN-mediated mechanism. Finally, we show that addition of PD-1 blockade to reovirus enhances systemic therapy in a preclinical glioma model. These results support the development of combined systemic immunovirotherapy strategies for the treatment of both primary and secondary tumors in the brain.

Background: The EGF-receptor variant III (EGFRvIII) is expressed in approximately 30% of all primary glioblastoma (GBM). Although several anti-EGFRvIII strategies have already been tested, the importance of EGFRvIII expression on treatment response is still under debate. Our aim was to clarify the relevance of EGFRvIII for GBM treatment using uni- and multivariate analysis of clinical data and preclinical studies. Methods: We performed retrospective analysis of 338 IDH1 wild type GBM patients treated either at the University of Pennsylvania or North Bristol NHS Trust and included data from the TCGA database. Survival was stratified according to MGMT promoter methylation status, EGFRvIII expression and EGFR gene amplification. The effect of EGFRvIII expression was also analyzed at the cellular and tumor level using isogenic pairs of EGFRvIII-negative and -positive GBM cells and xenograft tumors. Findings: EGFRvIII expression was associated with a significantly prolonged median overall survival but only for GBM patients with MGMT promoter methylated tumors. Importantly, this effect was independent of EGFR gene amplification. We also observed increased temozolomide (TMZ) sensitivity of EGFRvIII positive cells, which translated into improved survival in xenograft experiments. This increase in TMZ sensitivity was associated with an elevated DNA damage induction accompanied by an increased expression of DNA mismatch repair (MMR) proteins. In line with this finding, knock down of MMR proteins or EGFRvIII restored TMZ resistance. Interpretation: EGFRvIII expression is of high relevance for the prediction of GBM therapy outcome. Due to EGFRvIII-dependent upregulation of MMR standard of care treatment is most efficient for GBM displaying both MGMT promoter methylation and EGFRvIII expression. Funding Statement: This work was supported by the Brigitte and Dr. Konstanze Wegener-Stiftung (M.K.), Wilhelm-Sander-Stiftung (M.K.), Forschungsforderungsfonds des Fachbereichs Medizin des Universitatsklinikums Hamburg-Eppendorf (N.S.) and the Federal Ministry of Education and Research (BMBF grant 02NUK032; M.K. K.R.), Cancer Research UK (CRUK/21600; S.C.S, T.B.), The Brain Tumour Charity (TBTC13/192; S.C.S., T.B.), Medical Research Council (MR/K015214/1; S.C.S., T.B.), University of Leeds Academic Fellowship (L.F.S), The Templeton Family Research Fund (Z.A.B) and the National Institutes of Health (NIH 2R01-NS042645-11A1; D.O.R.). U.S. is supported by the Fordergemeinschaft Kinderkrebs-Zentrum Hamburg Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Animal Use: All work was performed in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986), under the authority of project licence 70/7340.

Background: Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed. Methods: Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays. Results: All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging. Conclusions: Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

Despite many clinical efforts, the prognosis of GBM patients has not improved in the last 10 years. Epigenetic silencing of the O6-methylguanine-DNA-methyltransferase (MGMT) gene is the only predictive biomarker that is associated with temozolomide (TMZ) response. The establishment of new biomarkers, for personalized treatment is of significant interest. The epidermal growth factor receptor (EGFR) variant III (EGFRvIII) is expressed in approximately 20–30% of GBM. The aim of this study was to investigate if EGFRvIII is a predictive biomarker for TMZ response by analyzing the TMZ sensitivity of isogenic cell cultures and tumour xenografts with/without endogenous EGFRvIII expression. Using two, unique isogenic pairs of MGMT deficient GBM cells we observed that endogenous EGFRvIII expression was associated with a significant increase in TMZ sensitivity in vitro and in vivo. Knockdown of EGFRvIII restored TMZ resistance, confirming EGFRvIII dependence. We observed prolonged accumulation in S/G2-phase in EGFRvIII+ cells after TMZ treatment, indicating increased replication stress induced by TMZ and a significantly higher number of DNA double strand breaks. Because TMZ sensitivity in MGMT negative cells is predominantly determined by functional mismatch repair (MMR), we next analyzed MMR protein expression. We observed that EGFRvIII expression was associated with upregulated MMR proteins and that conversely, even a moderate decrease in MSH6 and MLH1 expression yielded a pronounced increase in TMZ resistance. In accordance with our findings, analysis of clinical data revealed that EGFRvIII positive GBM patients displaying MGMT promotor methylation have significantly better outcomes than EGFRvIII negative patients. In summary, we have shown that MGMT negative EGFRvIII expressing GBM cells and tumours are significantly more sensitive to TMZ compared to isogenic EGFRvIII negative counterparts due to upregulated MMR. Our data highlight a new mechanism of TMZ sensitivity and we conclude that EGFRvIII is a predictive biomarker for response to TMZ in MGMT methylated GBM patients.

Pharmacological inhibition of uncontrolled cell growth with small molecule inhibitors is a potential strategy against glioblastoma multiforme (GBM), the most malignant primary brain cancer. Phenotypic profiling of the neurogenic small molecule KHS101 revealed the chemical induction of lethal cellular degradation in molecularly-diverse GBM cells, independent of their tumor subtype, whereas non-cancerous brain cells remained viable. Mechanism-of-action (MOA) studies showed that KHS101 specifically bound and inhibited the mitochondrial chaperone HSPD1. In GBM but not non-cancerous brain cells, KHS101 elicited the aggregation of an enzymatic network that regulates energy metabolism. Compromised glycolysis and oxidative phosphorylation (OXPHOS) resulted in the metabolic energy depletion in KHS101-treated GBM cells. Consistently, KHS101 induced key mitochondrial unfolded protein response factor DDIT3 in vitro and in vivo, and significantly reduced intracranial GBM xenograft tumor growth upon systemic administration, without discernible side effects. These findings suggest targeting of HSPD1-dependent oncometabolic pathways as an anti-GBM therapy.

Purpose: The DNA mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations; however, it is becoming increasingly clear that a therapy targeting MMR deficiency in general would be clinically very beneficial. Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivo. Results: The selective tumor cell cytotoxicity of triamterene occurs through its antifolate activity and depends on the activity of the folate synthesis enzyme thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double-strand breaks. Conclusions: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR deficiency in a range of different tumor types and could significantly improve patient survival. Clin Cancer Res; 23(11); 2880–90. ©2016 AACR.

Background:Immobilisation for patients undergoing brain or head and neck radiotherapy is achieved using perspex or thermoplastic devices that require direct moulding to patient anatomy. The mould room visit can be distressing for patients and the shells do not always fit perfectly. In addition the mould room process can be time consuming. With recent developments in three-dimensional (3D) printing technologies comes the potential to generate a treatment shell directly from a computer model of a patient. Typically, a patient requiring radiotherapy treatment will have had a computed tomography (CT) scan and if a computer model of a shell could be obtained directly from the CT data it would reduce patient distress, reduce visits, obtain a close fitting shell and possibly enable the patient to start their radiotherapy treatment more quickly.Purpose:This paper focuses on the first stage of generating the front part of the shell and investigates the dosimetric properties of the materials to show the feasibility of 3D printer materials for the production of a radiotherapy treatment shell.Materials and methods:Computer algorithms are used to segment the surface of the patient’s head from CT and MRI datasets. After segmentation approaches are used to construct a 3D model suitable for printing on a 3D printer. To ensure that 3D printing is feasible the properties of a set of 3D printing materials are tested.Conclusions:The majority of the possible candidate 3D printing materials tested result in very similar attenuation of a therapeutic radiotherapy beam as the Orfit soft-drape masks currently in use in many UK radiotherapy centres. The costs involved in 3D printing are reducing and the applications to medicine are becoming more widely adopted. In this paper we show that 3D printing of bespoke radiotherapy masks is feasible and warrants further investigation.

BACKGROUND: Dysregulated, stem cell-like self-renewal has been implicated in glioma treatment resistance and tumour recurrence. Drugs that eliminate tumour cells possessing this malignant characteristic are urgently needed. It remains, however, an experimental challenge to link heterogeneous glioma genotypes to drug response at scale. To this end, we successfully derived patient-specific induced pluripotent stem cell (iPSC) models from both low- (LGG) and high-grade gliomas (HGG) and developed an initial drug discovery application. METHODS: Brain tumour tissue, acquired at surgery, was reprogrammed. Derived iPSC models were characterised using pluripotency markers, tri-germinal layer differentiation, gene expression, karyology and deep whole genome sequencing (WGS, iPSC versus parental tumour). Glioma iPSC differentiation in 2-dimensional (adherent, optically clear 96-well imaging plates) and 3-dimensional (organoid) culture was carried out. Gene expression of neural induction and neuronal differentiation was analysed using mRNA-seq. Neural cancer stem cells from each glioma iPSC line were orthotopically implanted in vivo. RESULTS: Reprogrammed cells were confirmed as fully-reprogramed/stable iPSCs, with mutational variants (SNPs, CNVs) preserved as compared to the parental tumours. Glioma iPSC maturation and quantification of TUJ-1 expression indicated a ‘differentiation block’ in the HGG iPSC models. This phenotype was concordant in HGG iPSC-derived tumour organoids which displayed SOX2/MKI67-positive neural rosettes. Consistently, mice developed xenograft tumours with GBM histopathological characteristics. Expression profiling during neuronal differentiation (from iPSC to neural stem cells to neurons) has revealed candidate genes that may be responsible for the phenotypic differences between HGG and control/LGG iPSC models. CONCLUSIONS: Our adherent, organoid and in vivo iPSC models may uncover genetic mutations and regulatory networks underlying glioma stem cell self-renewal capability, and provide a basis for industrial-scale drug discovery. Here we have successfully implemented the first stages towards this development (in a 96-well assay format). Ultimately, our patient-derived iPSC-based approach may enable personalised precision medicine strategies against glioma.

One of the biggest challenges when treating brain tumours is achieving efficient delivery of therapeutic agents to the brain and more specifically to the cancer cells. MicroRNA-1300 was identified in our group as a very promising therapeutic microRNA given its cytotoxic effect when introduced in both established as well as cancer-stem-like patient-derived glioblastoma cultures, while not affecting differentiated glioblastoma cells. We are now collaborating to assess the potential efficiency of the natural biopolymer chitosan to form nanocomplexes containing the mature form of microRNA-1300 for delivery. Chitosan has been established as a highly attractive biocompatible polymer to deliver both in vitro and in vivo therapeutic nucleotides intracellularly. In previous studies, we have shown chitosan’s efficacy to form spherical nanocomplexes with microRNA and apply them to the downregulation of JAMA-A mRNA in MCF-7 breast cancer cells. Chitosan can also be chemically conjugated to introduce affinity towards a wide range of cellular targets (e.g. with aptamers). Methods We have optimised of the composition and characterised the biophysical properties of chitosan-microRNA nanocomplexes of varying (+/-) charge ratios using both a control nontargeting microRNA coupled to a fluorochrome (CS-miRdy547, efficiency of cell entry) and mature microRNA-1300 (CS-mi1300, efficient release and biological effect). We have tested the nanocomplexes in 2D monolayers and 3D spheroid cultures on established U251 as well as two patient-derived cultures. Reverse transfection was used as positive control. Results The control nanoparticles of CS-miRdy547 are taken up by the patient-derived cultures in 2D and 3D. Analysis is ongoing using the CS-miR-1300 nanoparticles.

Background A neuro-oncology research radiographer was included in the multi-disciplinary study team (MDT) from the set-up stage of a new advanced imaging study in glioblastoma, as it was envisaged that this would enhance progress and provide better patient-centred care. Aims This pilot study aimed to include 12 participants. This specialist radiographer role was to support recruitment, co-ordinate appointments and monitor patients’ progress. We aimed to ensure that optimal treatment and patient-centred care remained at the forefront of the study pathway. Method Patients were identified and approached by the radiographer prior to consent by a trial investigator. Additional scans were planned prior to radiotherapy (RT), 2 weeks into RT and 6 weeks post RT. The radiographer ensured streamlined appointments and was present during each study visit to liaise with health professionals, provide a specific point of contact with the patient and to ensure continuity. Results 6 patients have been recruited since November 2018. The specialist radiographer has attended 28 study scans to date. Over 40 face-to-face interactions have been undertaken, 30 email communications and 18 telephone consultations. Patient feedback has been extremely positive, all patients convey that they feel supported and value continuity throughout the pathway. Enhanced communication across the entire study team has also guaranteed efficient recruitment. Conclusion The research radiographer has a key role in the multi-disciplinary team delivering this study. Effective liaison between the radiotherapy and imaging departments has been crucial and patients value a consistent point of contact through the study and treatment pathways.

BACKGROUND Glioblastoma multiforme (GBM) carries a poor prognosis, partly due to biological and anatomical heterogeneity. Although radiotherapy (RT) is effective, high doses damage surrounding healthy tissues. Multimodal imaging with Magnetic Resonance (MRI) and Positron Emission Tomography (PET) may represent a useful approach for identifying GBM heterogeneity and visualising metabolic tumour properties. PET radiotracer [18F]-fluciclovine is preferentially accumulated in gliomas compared to healthy brain tissue via the cellular transport systems, LAT1 and ASCT2. In this study the effect of fractionated RT using multimodal imaging including [18F]-fluciclovine uptake and immunohistochemistry (IHC) in a GBM preclinical model will be validated. MATERIAL AND METHODS Two C57BL/6J mice cohorts were injected intracranially (i.c.) with murine CT2A-luc cells and subsequently submitted to multiparametric MRI and [18F]-fluciclovine PET imaging during hemi-brain RT (3Gy on 2 days/each week) for maximum 25 days after i.c. injection. Brains were collected for IHC characterization including LAT1 and ASCT2 staining. RESULTS Preliminary data showed that both MRI and PET were effective modalities to track tumour growth in this model. PET data revealed up to greater than 3-fold increase in SUVmax from regions of interest around the tumour site compared to healthy brain tissue. Time activity curves showed a steady increase in tumour uptake over 90 minutes. MRI showed a 25% increase in T2 values in tumours relative to unaffected contralateral regions. Confirmation of treatment response through matched imaging and IHC are ongoing, from which changes in glioma cell biology as well as amino acid transporter protein levels will be analysed. CONCLUSION These preliminary results show that multimodal imaging presents novel data in the assessment of treatment response in this model and will permit parallel IHC analyses to better define GBM tumour heterogeneity aligned with imaging changes. These data will also inform an on-going clinical study using the same imaging modalities. Work at authors’ labs are supported by an Investigator initiated project from Blue Earth Diagnostics (AS, SCS) and a University of Leeds Biswas studentship (SCS, DS). Daniela Salvatore is also supported by a Scholarship provided by Molecular and Translational Medicine Doctorate School of University of Milan (Italy).

BACKGROUND Gliomas are associated with great societal burden through both direct (health and social care) and indirect (e.g., productivity loss) costs, but socioeconomic reports are scarce. We describe costs in a sample of glioma patients at high risk for depression and their family caregivers, and explore relationships between costs and treatable symptoms (depression; fatigue; cognitive complaints). MATERIAL AND METHODS Data from a nationwide randomised trial comparing internet-based therapy for depressive symptoms with waitlist controls were used. Healthcare utilization, medication use, and productivity loss based on the Trimbos/iMTA questionnaire for Costs associated with Psychiatric Illness (TIC-P) were described and costs in the past 4 weeks were calculated. Caregivers reported on their own costs and wellbeing We used generalized linear regression models to predict costs using multiple observations per participant and robust standard errors. We included depressive symptoms (CES-D), fatigue (CIS), cognitive complaints (MOS), tumour grade (low-/high-grade), and disease status (stable/progression/active treatment). RESULTS Data from 91 glioma patients and 46 caregivers were used with multiple assessments from baseline through 12 months follow-up. Baseline data showed that over a 4 week period, 64.8% of patients (M=€394.99, sd=856.83) and 41.3% of caregivers (M=€131.11, sd=392.89) had used healthcare services. Medication was used by 90.1% of patients (M=€100.83, sd=191.07) and 43.5% of caregivers (M=€13.19, sd=21.03). Productivity loss resulted in M=€1231.19, sd=2185.35 for patients and M=€310.92, sd=841.36 for caregivers. In total, mean direct and indirect costs were €1632.22 (sd=2314.25) for patients and €447.85 (sd=1002.94) for caregivers. In patients, more depressive symptoms and cognitive complaints were associated with increased healthcare use costs; higher tumour grade and active disease were linked with higher medication costs (all p CONCLUSION Direct and indirect costs are substantial in glioma patients and caregivers. This indicates that the true cost of brain tumours is a burden shared between patients, caregivers, the healthcare system, and society more widely. Tentative links between costs and symptoms of depression, fatigue, and cognitive complaints indicate that adequate support may lead to cost reductions. Multivariable regression models are currently being prepared.

BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

Background External beam radiotherapy (EBRT) is the gold standard adjuvant treatment after breast conserving surgery for localised breast cancer. A recent phase 3 trial has shown the non-inferiority of intraoperative radiotherapy (IORT) compared with EBRT in terms of short-term safety and efficacy (TARGIT A Trial; Lancet 2010). IORT also has advantages in cost saving and patient convenience. Radiation exposure of the heart and cardiac vessels is associated with an increase in morbidity and mortality following EBRT for breast cancer and has been shown to increase the rate of major coronary events by 7.4% per gray of exposure to the heart (Darby; NEJM 2013). IORT uses low energy x-rays (50 kV) and is likely to reduce the radiation exposure of the cardiovascular system compared with EBRT. We have used γ-H2AX foci formation in peripheral blood lymphocytes as a surrogate marker of radiation dose to the heart and great vessels. The phosphorylated histone H2AX protein (γ-H2AX) is expressed after induction of DNA double strand breaks caused by ionising radiation, created as the lymphocytes pass through and adjacent to the irradiated field. Methods 34 patients were recruited, having either EBRT or IORT as part of a randomised controlled trial. The main inclusion criteria were adult females with early breast cancer suitable for breast conserving surgery and the main exclusion criteria were previous malignancy, recent exposure to radiation (excluding CT planning scan) and prior chemotherapy. Blood samples were taken immediately prior to and 30 minutes after either first fraction of EBRT or after IORT treatment, and then rapidly processed to allow quantification of the γ-H2AX biomarker in lymphocytes (Rothkamm; Radiology 2007). This study had approval from the Local Research and Ethics Committee. Results Data were available for 31 patients. Means and standard deviations for the change in γ-H2AX foci number per cell for each group are summarised in table 1. Following IORT there was an increase of 0.203 foci per cell (range -1.436 to 1.275) compared with an increase of 0.935 foci per cell (range -0.679 to 2.216) in the EBRT group; this difference was highly significant (p = 0.009). Table 1: γ-H2AX foci per cell IORTEBRTMean0.2030.935SD0.6330.764n1318 Conclusions These data show a significantly greater change in γ-H2AX foci number per cell following one fraction of EBRT compared to IORT. IORT is a single treatment (20Gy at the applicator surface) whereas EBRT is repeated 15 times (2.67Gy x 15 fractions) multiplying the effect on the patients’ cardiovascular system. It may be hypothesised that this reduced radiotherapy dose from IORT will reduce the risk of cardiovascular morbidity and mortality compared to EBRT. This is the first study to demonstrate the real time effect of radiotherapy to the heart and great vessels using a biomarker and demonstrates a proof of concept methodology for similar applications. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-13.

We used sequentially transformed mesenchymal stem cells to investigate how the events that lead to tumorigenicity influence the cellular response to radiation. Bone marrow derived SH2(+), SH4(+), Stro-1(+) mesenchymal stem cells (MSC) were transformed stepwise by retroviral transfection of hTERT, HPV-16 E6 and E7, SV40 small T antigen and oncogenic H-ras. Cells at three different stages of transformation were irradiated and compared using assays for cytotoxicity, apoptosis, DNA double-strand break (DSB) repair and checkpoint signaling. The effects of inhibition of cell cycle checkpoint signaling on radiosensitivity were investigated using RNA interference. During stepwise transformation, specifically after HPV-16 E6 and E7 transduction, MSCs became more sensitive to radiation. This was associated with increased residual DNA DSB at 24 h and increased apoptosis. Enhanced checkpoint signaling occurred during transformation and there was a differential effect of checkpoint targeting in cells at different stages; Chk1 knockdown enhanced radiosensitivity in all cells while Chk2 knockdown only affected non-transformed cells. These data show that transformation of MSC is associated with a reduction in DNA DSB repair capacity and increased radiosensitivity. Up-regulation of checkpoint signaling does not overcome this and the effect of checkpoint inhibition may change with transformation status.

Brain tumours are one of the most aggressive forms of cancer with limited treatment options. Current treatment options include surgical resection of the main tumour mass, radiation and chemotherapy treatments. Surgical resection will inadvertently leave ~ 5 % of cancer cells within difficult anatomical locations in the brain, while radiation and chemotherapy treatments will often lead to unwanted systemic toxicities. In an attempt to minimise systemic toxicities, we aim to develop a targeted approach for the delivery of therapeutic agents using haematopoietic stem cells (HSCs) as cellular delivery vehicles. We first evaluated the suitability of HSCs as drug delivery vehicles for brain tumours in a syngeneic mouse model. Murine Sca1+ HSCs isolated from mouse bone marrow (BM) were transduced with the lentiviral vector pFUGW expressing green fluorescent protein (GFP) and transplanted into lethally irradiated C57BL/6J mice. This resulted in successful BM engraftment as confirmed by flow cytometry. Subsequently, brain tumours in these mice were generated by intracranial implantation of primary glioma cell lines (GL261 and CT-2A) to address primary brain tumours or the murine breast carcinoma cell line, EO771, to address breast cancer brain metastases. Our studies show that the progeny of GFP-tagged HSCs strongly infiltrated primary brain tumours and brain metastases, with the main immune cell infiltrating population being CD11b+/F4/80+ macrophages. To address the issue of targeted therapy delivery specifically to brain lesions, we sought to identify gene promoters whose activity is specifically upregulated in the HSC progeny that infiltrates brain tumours. Gene expression analysis identified 3 genes whose expression is specifically upregulated in brain tumour infiltrating macrophages as opposed to macrophages infiltrating other tissues, namely bone marrow and spleen. To validate promoter specificity in our mouse models, we generated promoter:reporter constructs to drive GFP expression downstream of these 3 identified promoters. To this end, C57BL/6J mice were lethally irradiated and reconstituted with genetically modified murine Sca1+ HSCs expressing GFP downstream of the 3 identified promoters. Following successful bone marrow reconstitution, we tested promoter specificity in mouse models of breast cancer brain metastases. Our results show that 2 of the promoters display specific activity in the brain as opposed to other tissues (blood, bone marrow, spleen, lungs, liver, heart), suggesting that HSC progenitors can drive delivery of therapeutics specifically to the brain downstream of these 2 promoters. We now plan to test the delivery of therapeutic agents downstream of these tissue-specific promoters. This study demonstrates a proof-of-concept for using HSCs as cellular delivery vehicles for the targeted delivery of therapeutics to the brain to overcome the blood-brain barrier and has potential for combination therapy approaches in the future.

INTRODUCTION: High-grade gliomas feature prominently among the most devastating cancers due to their prevalence in the younger population, highly invasive nature and low 5-year survival. Despite a combined treatment regime of surgical resection, radiation therapy and chemotherapy most patients die within 1 year of initial diagnosis. Alternative, novel ways to treat these tumours are urgently required. Histone deacetylase (HDAC) are important regulators of chromatin remodelling and gene expression and have been shown to be involved in cancer. There is an increasing interest in the study and development of HDAC inhibitors towards clinical application. Moreover, they have been recently reported to inhibit cell migration due to their modulation of tubulin acetylation. Thus HDAC inhibitors represent attractive candidates for chemotherapeutics especially when considering the infiltrative nature of high-grade gliomas. Here, the specific HDAC2/3 inhibitor MI192 developed at Leeds University was investigated for its ability to inhibit 2D and 3D migration of two established adult glioma cell lines. METHODS: U87 and U251 cell lines were used for migration and cytoskeletal studies in 2D and 3D assays. The HDAC inhibitor MI192 was assessed for its ability to inhibit migration alongside LiCl and Bio-Indirubin, two anti-migratory drugs commonly used in glioma in vitro studies. RESULTS: In random migration (2D) the MI192 inhibitor adversely affected the migratory ability of U87 and U251 as evidenced by reduced cell velocity and also cell displacement. This effect was more pronounced in U251 than U87. Immunofluorescence studies of the MI192-treated cells revealed an increase in the acetylation of tubulin in U87, whilst proliferation and apoptosis status remained unchanged. U251 did not appear to show increased levels of tubulin acetylation. In a 3D spheroid invasion assay U87 migration was inhibited by MI192 at 1 uM concentration, but not at 0.1 or 0.01 uM, whereas we did not see a significant change in U251. Immunohistochemistry of the treated spheroids revealed that proliferation was adversely affected in both the core and migratory cells in U87 spheroids with MI192 at a concentration of 1 uM but not in U251 spheroids. Tubulin acetylation appeared more pronounced in U251 after MI192 treatment. In addition, SOX-2 (as a marker of stemness) was reduced in both U251 cores and migratory cells with MI192. CONCLUSION: In addition to its activity as a chromatin remodelling inhibitor, MI192 appears to inhibit glioma cell migration via hyperacetylation of tubulin and possibly microtubule stabilisation in a cell line and topography dependent manner. Thus, the HDAC2/3 inhibitor MI192 may prove useful in targeting migration in certain subtypes of glioma cell.

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are invasive tumors with poor survival. Oncolytic virotherapy, initially devised as a direct cytotoxic treatment, is now also known to act via immune-mediated mechanisms. Here we investigate a previously unreported mechanism of action: the inhibition of migration and invasion in pediatric brain tumors. We evaluated the effect of oncolytic herpes simplex virus 1716 (HSV1716) on the migration and invasion of pHGG and DIPG both in vitro using 2D (scratch assay, live cell imaging) and 3D (spheroid invasion in collagen) assays and in vivo using an orthotopic xenograft model of DIPG invasion. HSV1716 inhibited migration and invasion in pHGG and DIPG cell lines. pHGG cells demonstrated reduced velocity and changed morphology in the presence of virus. HSV1716 altered pHGG cytoskeletal dynamics by stabilizing microtubules, inhibiting glycogen synthase kinase-3, and preventing localized clustering of adenomatous polyposis coli (APC) to the leading edge of cells. HSV1716 treatment also reduced tumor infiltration in a mouse orthotopic xenograft DIPG model. Our results demonstrate that HSV1716 targets the migration and invasion of pHGG and DIPG and indicates the potential of an oncolytic virus (OV) to be used as a novel anti-invasive treatment strategy for pediatric brain tumors.

A clinical SPECT insert for a commercial MRI scanner has been developed within the INSERT project, allowing simultaneous SPECT/MRI studies of the human brain. Here we present preliminary experimental results. The reconstructed resolution was 6-11 mm and the sensitivity 280-440 s-1/MBq. The INSERT is the first clinical SPECT prototype for simultaneous SPECT/MRI.

The higher specificity of amino-acid positron emission tomography (AA-PET) in the diagnosis of gliomas, as well as in the differentiation between recurrence and treatment-related alterations, in comparison to contrast enhancement in T1-weighted MRI was demonstrated in many studies and is the rationale for their implementation into radiation oncology treatment planning. Several clinical trials have demonstrated the significant differences between AA-PET and standard MRI concerning the definition of the gross tumor volume (GTV). A small single-center non-randomized prospective study in patients with recurrent high grade gliomas treated with stereotactic fractionated radiotherapy (SFRT) showed a significant improvement in survival when AA-PET was integrated in target volume delineation, in comparison to patients treated based on CT/MRI alone. This protocol describes a prospective, open label, randomized, multi-center phase II trial designed to test if radiotherapy target volume delineation based on FET-PET leads to improvement in progression free survival (PFS) in patients with recurrent glioblastoma (GBM) treated with re-irradiation, compared to target volume delineation based on T1Gd-MRI. The target sample size is 200 randomized patients with a 1:1 allocation ratio to both arms. The primary endpoint (PFS) is determined by serial MRI scans, supplemented by AA-PET-scans and/or biopsy/surgery if suspicious of progression. Secondary endpoints include overall survival (OS), locally controlled survival (time to local progression or death), volumetric assessment of GTV delineated by either method, topography of progression in relation to MRI- or PET-derived target volumes, rate of long term survivors (>1 year), localization of necrosis after re-irradiation, quality of life (QoL) assessed by the EORTC QLQ-C15 PAL questionnaire, evaluation of safety of FET-application in AA-PET imaging and toxicity of re-irradiation. This is a protocol of a randomized phase II trial designed to test a new strategy of radiotherapy target volume delineation for improving the outcome of patients with recurrent GBM. Moreover, the trial will help to develop a standardized methodology for the integration of AA-PET and other imaging biomarkers in radiation treatment planning. The GLIAA trial is registered with ClinicalTrials.gov ( NCT01252459 , registration date 02.12.2010), German Clinical Trials Registry ( DRKS00000634 , registration date 10.10.2014), and European Clinical Trials Database (EudraCT-No. 2012-001121-27, registration date 27.02.2012).

Purpose We investigated whether microRNA expression data from glioblastoma could be used to produce a profile that defines a bevacizumab responsive group of patients. Patients and methods TCGA microRNA expression data from tumors resected at first diagnosis of glioblastoma in patients treated with bevacizumab at any time during the course of their disease were randomly separated into training (n = 50) and test (n = 37) groups for model generation. MicroRNA-seq data for 51 patients whose treatment included bevacizumab in the BELOB trial were used as an independent validation cohort. Results Using penalized regression we identified 8 microRNAs as potential predictors of overall survival in the training set. We dichotomized the response score based on the most prognostic minimum of a density plot of the response scores (log-rank HR = 0.16, p = 1.2e−5) and validated the profile in the test cohort (one-sided log-rank HR = 0.34, p = 0.026). Analysis of the profile using all samples in the TCGA glioblastoma dataset, regardless of treatment received, (n = 473) showed that the prediction of patient benefit was not significant (HR = 0.84, p = 0.083) suggesting the profile is specific to bevacizumab. Further independent validation of our microRNA profile in RNA-seq data from patients treated with bevacizumab (alone or in combination with CCNU) at glioblastoma recurrence in the BELOB trial confirmed that our microRNA profile predicted patient benefit from bevacizumab (HR = 0.59, p = 0.043). Conclusion We have identified and validated an 8-microRNA profile that predicts overall survival in patients with glioblastoma treated with bevacizumab. This may be useful for identifying patients who are likely to benefit from this agent.

MicroRNAs are proving to be potent tumour suppressors in cancer. Many microRNAs are in pre-clinical and clinical validation.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with an estimated 5-year survival of less 10%. The current standard of care involves maximal tumor resection followed by radiation and chemotherapy with the alkylating agent temozolomide (TMZ). Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) gene predicts response to TMZ therapy, but does not explain all of the heterogeneity in responses observed in the clinic. The establishment of additional molecular biomarkers, is therefore of significant interest.

High grade gliomas (HGGs) are characterized by resistance to radiotherapy and chemotherapy. Targeting Rad51-dependent homologous recombination repair may be an effective target for chemo- and radiosensitization. In this study we assessed the role of Rad51-dependent repair on sensitivity to radiation and temozolomide (TMZ) as single agents or in combination. Repair protein levels in established glioma cell lines, early passage glioblastoma multiforme (GBM) cell lines, and normal human astrocytes (NHAs) were measured using western blot. Viability and clonogenic survival assays were used to measure the effects of Rad51 knockdown with radiation (XR) and TMZ. Immunocytochemistry was used to evaluate kinetics of Rad51 and γ-H2AX repair foci. Immunohistochemistry was used to assess Rad51 protein levels in glioma specimens. Repair proteins including Rad51 are upregulated in HGG cells compared with NHA. Established glioma cell lines show a dose-dependent increase in Rad51 foci formation after XR and TMZ. Rad51 levels are inversely correlated with radiosensitivity, and downregulation markedly increases the cytotoxicity of TMZ. Rad51 knockdown also promotes more residual γ-H2AX foci 24 h after combined treatment. Newly established GBM cell lines also have high Rad51 levels and are extremely sensitive to Rad51 knockdown. Clinical samples from recently resected gliomas of varying grades demonstrate that Rad51 levels do not correlate with tumor grade. Rad51-dependent repair makes a significant contribution to DNA repair in glioma cells and contributes to resistance to both XR and TMZ. Agents targeting Rad51-dependent repair would be effective adjuvants in standard combination regimens.

BACKGROUND: Three-dimensional tumour spheroid models are increasingly used in cancer research. However, imaging for quantification analysis of spheroids in drug testing has remained primarily based on 2D methodologies. Imaging 3D structures in 2D can cause discrepancies and introduce interpretive bias. We previously described a novel method to create high-resolution 3D spheroid images using histopathological sectioning. We compare for the first time 3D histopathological images and data analysis of glioma spheroid models in drug studies to commonly used imaging techniques, highlighting the advantages of 3D imaging over 2D methodologies. METHODS: U251 glioma spheroids embedded in collagen were untreated or treated with inhibitors 6-Bromoindirubin-3’-oxime (BIO) or MI-192. Spheroids were imaged at 24-hour time points over 72 hours using light-microscopy and confocal laser-microscopy and then subsequently embedded in paraffin and sectioned at 5µm. Slides were H&E stained and serially scanned at 20x magnification. Custom software was used to digitally reconstruct the spheroid in 3D. For details at the single cellular level, 70µm spheroids were also imaged at 72 hours at high-magnification using an Instant Structured illumination microscope (iSIM). RESULTS: Superior detail of core cellular components and migratory single cells was achieved by 3D histopathological reconstruction compared to all other methods. Migration indices were similar to 3D confocal-generated reconstructions demonstrating promoted migration with MI-192 and reduced migration with BIO. Detailed morphological analysis of migratory cells was achieved at 40x optical magnification using our 3D histology method. However, greater morphological detail was obtained using high-magnification iSIM. Failure to capture migratory cells in the z-plane using 2D techniques lead to considerable differences between 3D and 2D imaging data. CONCLUSION: We have shown that 3D histopathological image reconstruction is a preferable method for cell migration and morphological analyses of 3D cancer spheroid models. This method can be used for high-resolution analysis of both core and migratory cells.