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Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management.Significance:Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.See related video: https://vimeo.com/438254885See related commentary by Szulzewsky and Cimino, p. 904.This article is highlighted in the In This Issue feature, p. 890

Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention. Significance: The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.

Figure S5Figure S5 and legend

Supplementary Figure S2 shows additional data obtained during aphidicolin studies detailed in Fig 1 and additional data detailing expression of genes associated with ongoing and stalled replication forks from figure 3.

Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention.Significance:The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.

Supplementary Table 1Table 1 and legend

Legends for supplementary figures and tables

Supplementary Table 2Table 2 and legend

Supplementary Table 3Table 3 and legend

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem–like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133− non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with “replication factories” and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro. These data identify RS as a cancer stem cell–specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM. Cancer Res; 78(17); 5060–71. ©2018 AACR.

Supplemental Table 1 Summary table of in vivo growth characteristics of E2, G7, R10, R15, R24, R9 and S2 GSC primary GBM cultures after intracranial injection in CD1 nude mice. Supplemental Table 2 List of primary antibodies utilised Supplemental Table 3 List of secondary antibodies utilised

Dual-emission fluorescence probes that provide high sensitivity are key for biomedical diagnostic applications. Non-toxic carbon dots (CDs) are a good alternative to traditional fluorescent probes, however robust and reproducible synthetic strategies are still needed to access materials with controlled emission profiles and improved fluorescence quantum yields (FQYs). Herein, we report a practical and general synthetic strategy to access dual-emission multicolour CDs with FQYs as high as 0.67 and green/blue, yellow/blue or red/blue excitation dependent emission profiles using common starting materials such as citric acid, cysteine and co-dopants to bias the synthetic pathway. Structural and physicochemical analysis is used to elucidate the material’s composition which is responsible for the unique observed photoluminescence properties. Moreover, the utility of the probes is demonstrated in the clinical setting by the synthesis of green/blue emitting antibody-CD conjugates which are used for the immunohistochemical staining of human brain tissues of glioblastoma patients, showing detection under two different emission channels.

Brain tumours are the biggest cancer killer in those under 40 and reduce life expectancy more than any other cancer. Blood-based liquid biopsies may aid early diagnosis, prediction and prognosis for brain tumours. It remains unclear whether known blood-based biomarkers, such as glial fibrillary acidic protein (GFAP), have the required sensitivity and selectivity. We have developed a novel in silico model which can be used to assess and compare blood-based liquid biopsies. We focused on GFAP, a putative biomarker for astrocytic tumours and glioblastoma multi-formes (GBMs). In silico modelling was paired with experimental measurement of cell GFAP concentrations and used to predict the tumour volumes and identify key parameters which limit detection. The average GBM volumes of 449 patients at Leeds Teaching Hospitals NHS Trust were also measured and used as a benchmark. Our model predicts that the currently proposed GFAP threshold of 0.12 ng ml −1 may not be suitable for early detection of GBMs, but that lower thresholds may be used. We found that the levels of GFAP in the blood are related to tumour characteristics, such as vasculature damage and rate of necrosis, which are biological markers of tumour aggressiveness. We also demonstrate how these models could be used to provide clinical insight.

Genetically predicted changes in molecular traits are associated with genetic liability to glioma risk. We sought to identify and provide evidence for the prioritisation of these traits using a combined two-sample Mendelian randomisation and colocalisation pipeline.We used genetic variants from genome-wide association studies (GWAS) to compare the effects of gene expression, protein abundance and splicing variation on genetic liability to glioma risk.We performed analysis of quantitative trait loci (QTL) (expression QTL [MetaBrain, n=4,119,012]; protein QTL [BrainQTL, n=786,632] and splicing QTL [GTEx, n=166,721]) derived from 15 different brain tissues and summary-level data from eight glioma GWAS (12,496 cases and 18,190 controls).We found robust evidence linking QTL for 25 genes in 15 loci with risk of glioma following relevant sensitivity analysis. We identified four novel genes putatively implicated in glioma risk: BTN3A2 (6p22.2), FAIM (3q22.3), GALNT6 (12q13.13) and GMPPB (3p21.31). Identification of these novel genes may improve understanding in pathogenic mechanisms underlying gliomagenesis and should be followed up in further studies, which may inform novel prevention studies following future clinical trials.

Background Ectopic intracranial schwannomas (those that do not arise from a named cranial nerve) are rare. They account for Case Description We report the case of a 14-year-old boy presenting with a left conductive hearing loss and temporal bone deformity. No facial or cranial nerve deficits were present. Cross-sectional imaging demonstrated a large expansile extra-axial temporal bone mass, extending into and distorting the middle cranial fossa. At surgical resection the tumor was functionally and anatomically distinct from the facial nerve or any other identifiable neural structure within the middle ear or temporal bone. Histology confirmed a World Health Organization grade 1 schwannoma. Conclusions This is the first reported case of a giant juvenile ectopic schwannoma within the temporal bone.

High-quality histopathology is essential for the success of clinical trials. Histopathologists have a detailed understanding of tumour biology and mechanisms of disease, as well as practical knowledge of optimal tissue handling and logistical service requirements for study delivery, such as biomarker evaluation, tissue acquisition and turnaround times. As such, histopathologist input is essential throughout every stage of research and clinical trials, from concept development and study design to trial delivery, analysis and dissemination of results. Patient recruitment to trials takes place among all healthcare settings, meaning that histopathologists make an invaluable contribution to clinical trials as part of their routine day-to-day work that often goes unrecognised. More complex evaluation of surgical specimens in the neoadjuvant setting and ever-expanding minimum data sets add to the workload of every histopathologist, not just academic pathologists in tertiary centres. This is occurring against a backdrop of increasing workload pressures and a worldwide shortage of histopathologists and biomedical scientists. Providing essential histopathology support for trials at grassroots level requires funding for adequate resources including histopathologist time, education and training, biomedical scientist and administrative support and greater recognition of the contribution made by histopathology. This paper will discuss the many ways in which histopathologists are involved in clinical trials and the challenges faced in meeting the additional demands posed by trial participation and potential ways to address this, with a special emphasis on the UK model and the Cellular-Molecular Pathology Initiative (CM-Path).

Background The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide (TMZ) chemotherapy in preclinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side effects of TMZ. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib and assess the safety and tolerability of its combination with TMZ. Methods Preclinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose TMZ in a 3 + 3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumor core and tumor margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. Results Olaparib was a substrate for multidrug resistance protein 1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients; median, 496 nM) and 21/21 tumor margin specimens (9 patients; median, 512.3 nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150 mg (3 days/week) with TMZ 75 mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression free at 6 months. Olaparib radiosensitized 6 glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. Conclusion Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better preclinical models.

Summary Background Marked variation exists in the use of genomic data in tumour diagnosis, and optimal integration with conventional diagnostic technology remains uncertain despite several studies reporting improved diagnostic accuracy, selection for targeted treatments, and stratification for trials. Our aim was to assess the added value of molecular profiling in routine clinical practice and the impact on conventional and experimental treatments. Methods This population-based study assessed the diagnostic and clinical use of DNA methylation-based profiling in childhood CNS tumours using two large national cohorts in the UK. In the diagnostic cohort—which included routinely diagnosed CNS tumours between Sept 1, 2016, and Sept 1, 2018—we assessed how the methylation profile altered or refined diagnosis in routine clinical practice and estimated how this would affect standard patient management. For the archival cohort of diagnostically difficult cases, we established how many cases could be solved using modern standard pathology, how many could only be solved using the methylation profile, and how many remained unsolvable. Findings Of 484 patients younger than 20 years with CNS tumours, 306 had DNA methylation arrays requested by the neuropathologist and were included in the diagnostic cohort. Molecular profiling added a unique contribution to clinical diagnosis in 107 (35%; 95% CI 30–40) of 306 cases in routine diagnostic practice—providing additional molecular subtyping data in 99 cases, amended the final diagnosis in five cases, and making potentially significant predictions in three cases. We estimated that it could change conventional management in 11 (4%; 95% CI 2–6) of 306 patients. Among 195 historically difficult-to-diagnose tumours in the archival cohort, 99 (51%) could be diagnosed using standard methods, with the addition of methylation profiling solving a further 34 (17%) cases. The remaining 62 (32%) cases were unresolved despite specialist pathology and methylation profiling. Interpretation Together, these data provide estimates of the impact that could be expected from routine implementation of genomic profiling into clinical practice, and indicate limitations where additional techniques will be required. We conclude that DNA methylation arrays are a useful diagnostic adjunct for childhood CNS tumours. Funding The Brain Tumour Charity, Children with Cancer UK, Great Ormond Street Hospital Children's Charity, Olivia Hodson Cancer Fund, Cancer Research UK, and the National Institute of Health Research.

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. Significance: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype. See related video: https://vimeo.com/438254885 See related commentary by Szulzewsky and Cimino, p. 904. This article is highlighted in the In This Issue feature, p. 890

The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters.Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems.

Codeletion of chromosomal arms 1p and 19q, in conjunction with a mutation in the isocitrate dehydrogenase 1 or 2 gene, is the molecular diagnostic criterion for oligodendroglioma, IDH mutant and 1p/19q codeleted. 1p/19q codeletion is a diagnostic marker and allows prognostication and prediction of the best drug response within IDH-mutant tumours. We performed a Cochrane review and simple economic analysis to establish the most sensitive, specific and cost-effective techniques for determining 1p/19q codeletion status. Fluorescent in situ hybridisation (FISH) and polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) test methods were considered as reference standard. Most techniques (FISH, chromogenic in situ hybridisation [CISH], PCR, real-time PCR, multiplex ligation-dependent probe amplification [MLPA], single nucleotide polymorphism [SNP] array, comparative genomic hybridisation [CGH], array CGH, next-generation sequencing [NGS], mass spectrometry and NanoString) showed good sensitivity (few false negatives) for detection of 1p/19q codeletions in glioma, irrespective of whether FISH or PCR-based LOH was used as the reference standard. Both NGS and SNP array had a high specificity (fewer false positives) for 1p/19q codeletion when considered against FISH as the reference standard. Our findings suggest that G banding is not a suitable test for 1p/19q analysis. Within these limits, considering cost per diagnosis and using FISH as a reference, MLPA was marginally more cost-effective than other tests, although these economic analyses were limited by the range of available parameters, time horizon and data from multiple healthcare organisations.

Background Detailed prevalence estimates of BRAFV600 mutations and BRAF inhibitor (BRAFi) treatment responses in V600-mutant glioma will inform trial development. Methods Our systematic review analyzed overall prevalence of BRAFV600 mutations in glioma and BRAFi treatment response. Results Based on 13 682 patients in 182 publications, the prevalence of BRAFV600 in epithelioid glioblastoma (eGBM) was 69% [95% CI: 45–89%]; pleomorphic xanthoastrocytoma (PXA): 56% [48–64%] anaplastic pleomorphic xanthoastrocytoma (aPXA): 38% [23–54%], ganglioglioma (GG): 40% [33–46%], and anaplastic ganglioglioma (aGG): 46% [18–76%]. Prevalence in astroblastoma was 24% [8–43%], desmoplastic infantile astrocytoma (DIA): 16% [0–57%], subependymal giant cell astrocytoma (SEGA): 8% [0–37%], dysembryoplastic neuroepithelial tumor (DNET): 3% [0–11%], diffuse astrocytoma (DA): 3% [0–9%], and pilocytic astrocytoma (PA): 3% [2–5%]. We reviewed 394 V600-mutant gliomas treated with BRAFi from 130 publications. One hundred and twenty-nine pediatric low-grade gliomas showed 4 (3.1%) complete response (CR); 53 (41.1%) partial response (PR); 64 (49.6%) stable disease (SD) and 8 (6.2%) progressive disease (PD). 25 pediatric high-grade gliomas showed CR; PR; SD; PD in 4 (16.0%); 10 (40.0%), 4 (16.0%); and 7 (28.0%) respectively. Thirty-nine adult low-grade gliomas showed CR; PR; SD; PD of 4 (10.3%); 17 (43.6%); 16 (41.0%) and 2 (5.1%) respectively. Ninety-seven adult high-grade gliomas showed CR; PR; SD; PD of 6 (6.2%); 31 (32.0%); 27 (27.8%); and 33 (34.0%) respectively. Conclusions BRAFV600 prevalence is highest in eGBM, PXA, aPXA, GG, aGG, and lower in astroblastoma, DIA, SEGA, DNET, DA, and PA. Our data provide the rationale for adjuvant clinical trials of BRAFi in V600-mutant glioma.

BackgroundThe DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of tumor cells to alkylating agents. It is a predictive biomarker in high-grade gliomas treated with temozolomide, however, there is no consensus on which test method, methylation sites, and cutoff values to use.MethodsWe performed a Cochrane Review to examine studies using different techniques to measure MGMT and predict survival in glioblastoma patients treated with temozolomide. Eligible longitudinal studies included (i) adults with glioblastoma treated with temozolomide with or without radiotherapy, or surgery; (ii) where MGMT status was determined in tumor tissue, and assessed by 1 or more technique; and (iii) where overall survival was an outcome parameter, with sufficient information to estimate hazard ratios (HRs). Two or more methods were compared in 32 independent cohorts with 3474 patients.ResultsMethylation-specific PCR (MSP) and pyrosequencing (PSQ) techniques were more prognostic than immunohistochemistry for MGMT protein, and PSQ is a slightly better predictor than MSP.ConclusionsWe cannot draw strong conclusions about use of frozen tissue vs formalin-fixed paraffin-embedded in MSP and PSQ. Also, our meta-analysis does not provide strong evidence about the best CpG sites or threshold. MSP has been studied mainly for CpG sites 76-80 and 84-87 and PSQ at CpG sites ranging from 72 to 95. A cutoff threshold of 9% for CpG sites 74-78 performed better than higher thresholds of 28% or 29% in 2 of the 3 good-quality studies. About 190 studies were identified presenting HRs from survival analysis in patients in which MGMT methylation was measured by 1 technique only.

Glioma stem-like cells (GSCs) were first described as a population which may in part be resistant to traditional chemotherapeutic therapies and responsible for tumour regrowth. Knowledge of the underlying metabolic complexity governing GSC growth and function may point to potential differences between GSCs and the tumour bulk which could be harnessed clinically. There is an increasing interest in the direct/indirect targeting or reprogramming of GSC metabolism as a potential novel therapeutic approach in the adjuvant or recurrent setting to help overcome resistance which may be mediated by GSCs. In this review we will discuss stem-like models, interaction between metabolism and GSCs, and potential current and future strategies for overcoming GSC resistance.

Aims Glioma is a fatal disease that causes significant years of life lost to an individual. Mutations in the driver gene BRAF, such as the V600 alteration, may contribute to gliomagenesis in adults and children through abnormal signaling causing uncontrolled cell proliferation. The use of BRAF-inhibitor drugs including Vemurafenib and Dabrafenib have shown a favorable response in 48% and 50% of melanoma patients with BRAF V600 mutations respectively. BRAF inhibitors and MEK inhibitors have shown efficacy in certain paediatric gliomas in the recurrent setting. Despite the potential benefit of BRAF inhibitors, the prevalence of BRAF V600 within primary gliomas is not fully discovered. Some studies identify the prevalence to be over 50%, while others find the prevalence to be around 1%. We performed a comprehensive systematic review to determine the prevalence of BRAF V600 within the adult and paediatric glioma population in different diagnostic groups. Method A systematic literature search was performed using Ovid MEDLINE and Embase from genesis to the 22nd October 2020. Studies were not restricted by language. Studies were eligible if patients were histologically diagnosed according to WHO guidelines as a primary glioma evaluating the prevalence of BRAF V600 and included ≥ 10 primary glioma patients. The review protocol was registered in PROSPERO (CRD42019127704). Search results were managed using Endnote. Two independent reviewers assessed the eligibility of the publications using Rayyan, conflicts were evaluated by a third reviewer. Included articles were extracted by one reviewer and confirmed by a second reviewer. Risk of bias assessments were conducted using Hoy et al’s risk of bias tool. Results were synthesized using “metaprop” in R. The meta-analysis was carried out in R which produced forest plots. Results Our cohort included 182 studies with a total of 13669 adult and paediatric glioma patients classified diagnostically according to WHO guidelines. Among 48 glioma entities, BRAF V600 was identified most commonly in epithelioid glioblastoma with a prevalence of 69% (95% confidence interval (CI): 45-89%), followed by pleomorphic xanthoastrocytoma with a prevalence of 56% (95% CI: 48-64%), anaplastic pleomorphic xanthoastrocytoma with a prevalence of 38% (95% CI: 23-54%), ganglioglioma with a prevalence of 40% (95% CI: 33-46%), and anaplastic ganglioglioma with a prevalence of 46% (95% CI: 18-76%). Other glioma entities were found to have a prevalence of BRAF V600, these include astroblastoma (24%), desmoplastic infantile astrocytoma (16%), subependymal giant cell astrocytoma (8%), dysembryoplastic neuroepithelial tumour (3%), diffuse astrocytoma (3%), and pilocytic astrocytoma (3%). Conclusion To our knowledge, this is the largest systematic review examining the prevalence of BRAF V600 in adult and paediatric glioma classified according to diagnostic WHO criteria. However, there were some limitations in this review. The sample sizes of some studies were very small, and the method of mutational analysis for BRAF V600 varied between papers. We found BRAF V600 in a significant prevalence of epithelioid glioblastoma, pleomorphic xanthoastrocytoma, anaplastic pleomorphic xanthoastrocytoma, ganglioglioma, and anaplastic ganglioglioma. Of interest, BRAF V600 mutation was found in a lower prevalence of astroblastoma, desmoplastic infantile astrocytoma, subependymal giant cell astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse astrocytoma, and pilocytic astrocytoma. Consideration of assessment of BRAF V600 mutation may enable further treatment options with BRAF and/or MEK inhibitors in these particular diagnostic entities.

Background: Glioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine–DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide.Objectives: To assess which way of measuring methylation of the MGMT promoter best predicts survival when people with glioblastoma are treated with temozolomide.Search methods: We searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies we additionally searched NHS Economic Evaluation Database (EED) up to December 2014.Selection criteria: Eligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiation therapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with su*icient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focus mainly on studies comparing two or more methods, and list brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies, and cost analysis.Data collection and analysis: All steps of the identification and data extraction process for multiple-method studies were undertaken by two reviewers independently. We assessed risk of bias and applicability using our own modified and extended version of the QUIPS tool. Comparisons of different techniques, exact promoter regions (CpG sites) and thresholds for interpretation were compared within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios, using an imputed value of the correlation between results based on the same individuals.Main results: We included 32 independent cohorts involving 3474 people in which two or more methods were compared. In meta-analyses of ratios of hazard ratios (RHR), we found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds: (RHR = 1.31; 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR = 1.36; 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR = 1.14; 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarized 190 articles describing a single method for measuring MGMT promoter methylation status.Authors' conclusions: Pyrosequencing and methylation-specific PCR appear to be more prognostic for overall survival than immunohistochemistry. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 was found to perform better than higher thresholds of 28% or 29% in two of three good quality studies making such comparisons.

There is an unmet need for the identification of biomarkers to aid in the diagnosis, clinical management, prognosis and follow-up of meningiomas. There is currently no consensus on the optimum management of WHO grade II meningiomas. In this study, we identified the calcium binding extracellular matrix glycoprotein, Fibulin-2, via mass-spectrometry-based proteomics, assessed its expression in grade I and II meningiomas and explored its potential as a grade II biomarker. A total of 87 grade I and 91 grade II different meningioma cells, tissue and plasma samples were used for the various experimental techniques employed to assess Fibulin-2 expression. The tumours were reviewed and classified according to the 2016 edition of the Classification of the Tumours of the central nervous system (CNS). Mass spectrometry proteomic analysis identified Fibulin-2 as a differentially expressed protein between grade I and II meningioma cell cultures. Fibulin-2 levels were further evaluated in meningioma cells using Western blotting and Real-time Quantitative Polymerase Chain Reaction (RT-qPCR), in meningioma tissues via immunohistochemistry and RT-qPCR, and in plasma via Enzyme-Linked Immunosorbent Assay (ELISA). Proteomic analyses (p &lt, 0.05), Western blotting (p &lt, 0.05) and RT-qPCR (p &lt, 0.01) confirmed significantly higher Fibulin-2 (FBLN2) expression levels in grade II meningiomas compared to grade I. Fibulin-2 blood plasma levels were also significantly higher in grade II meningioma patients compared to grade I patients. This study suggests that elevated Fibulin-2 might be a novel grade II meningioma biomarker, when differentiating them from the grade I tumours. The trend of Fibulin-2 expression observed in plasma may serve as a useful non-invasive biomarker.

Glioblastoma is the most common primary brain tumor in adults with poor overall outcome and 5-year survival of less than 5%. Treatment has not changed much in the last decade or so, with surgical resection and radio/chemotherapy being the main options. Glioblastoma is highly heterogeneous and frequently becomes treatment-resistant due to the ability of glioblastoma cells to adopt stem cell states facilitating tumor recurrence. Therefore, there is an urgent need for novel therapeutic strategies. The ubiquitin system, in particular E3 ubiquitin ligases and deubiquitinating enzymes, have emerged as a promising source of novel drug targets. In addition to conventional small molecule drug discovery approaches aimed at modulating enzyme activity, several new and exciting strategies are also being explored. Among these, PROteolysis TArgeting Chimeras (PROTACs) aim to harness the endogenous protein turnover machinery to direct therapeutically relevant targets, including previously considered “undruggable” ones, for proteasomal degradation. PROTAC and other strategies targeting the ubiquitin proteasome system offer new therapeutic avenues which will expand the drug development toolboxes for glioblastoma. This review will provide a comprehensive overview of E3 ubiquitin ligases and deubiquitinating enzymes in the context of glioblastoma and their involvement in core signaling pathways including EGFR, TGF-β, p53 and stemness-related pathways. Finally, we offer new insights into how these ubiquitin-dependent mechanisms could be exploited therapeutically for glioblastoma.

Genome-wide association studies (GWAS) have discovered 27 loci associated with glioma risk. Whether these loci are causally implicated in glioma risk, and how risk differs across tissues, has yet to be systematically explored. We integrated multi-tissue expression quantitative trait loci (eQTLs) and glioma GWAS data using a combined Mendelian randomisation (MR) and colocalisation approach. We investigated how genetically predicted gene expression affects risk across tissue type (brain, estimated effective n = 1194 and whole blood, n = 31,684) and glioma subtype (all glioma (7400 cases, 8257 controls) glioblastoma (GBM, 3112 cases) and non-GBM gliomas (2411 cases)). We also leveraged tissue-specific eQTLs collected from 13 brain tissues (n = 114 to 209). The MR and colocalisation results suggested that genetically predicted increased gene expression of 12 genes were associated with glioma, GBM and/or non-GBM risk, three of which are novel glioma susceptibility genes (RETREG2/FAM134A, FAM178B and MVB12B/FAM125B). The effect of gene expression appears to be relatively consistent across glioma subtype diagnoses. Examining how risk differed across 13 brain tissues highlighted five candidate tissues (cerebellum, cortex, and the putamen, nucleus accumbens and caudate basal ganglia) and four previously implicated genes (JAK1, STMN3, PICK1 and EGFR). These analyses identified robust causal evidence for 12 genes and glioma risk, three of which are novel. The correlation of MR estimates in brain and blood are consistently low which suggested that tissue specificity needs to be carefully considered for glioma. Our results have implicated genes yet to be associated with glioma susceptibility and provided insight into putatively causal pathways for glioma risk.

BackgroundIn vivo and observational studies suggest anti-hyperlipidaemic and -diabetic medications (fibrates and glitazones respectively) may have a role in primary prevention and progression of brain tumours by targeting PPAR-α and -γ, respectively.MethodsWe conducted a case-control and clinical cohort study within the UK Clinical Practice Research Datalink. We identified adults (aged 18 years+) with primary or secondary brain tumours diagnosed between 2000-2016 prescribed either fibrates or glitazones and identified four controls based on age, sex and drug exposure duration. Multivariable logistic regression analysis estimated an association between drug exposure and brain tumour status. Cox’s survival models were used to look at risk of mortality.Results1,916 cases were prescribed a fibrate and 445 cases a glitazone. Our analyses showed little evidence of an association between fibrates and either risk or mortality of brain tumours (adjusted odds ratio for ever exposed PPAR-α 0.98, 95% CI: 0.77, 1.23; adjusted hazard ratio for ever exposed 0.91; 95% CI: 0.76, 1.09). We observed a reduced risk with a per-year increase in exposure duration for glitazones (adjusted odds ratio 0.88, 95% CI: 0.81, 0.96, P=0.002) but no major mortality benefit (adjusted hazard ratio 0.99, 95% CI: 0.80, 1.23).ConclusionsOur results suggest longer duration exposure to glitazones is associated with a reduced risk of primary and secondary brain tumours but no large effect on mortality. We failed to find any strong evidence of a protective effect on risk or mortality for fibrate exposure. Further studies are required for replication and to provide more precise effect estimates.Key PointsRepurposing existing drugs that target PPAR-α and -γ receptors may have a role in prevention and progression of brain tumours.We observed a reduced risk between duration of glitazones and both primary and secondary brain tumours but no major reduction on mortalityWe observed no major reduction of risk or mortality with fibratesFuture studies need to be undertaken to ensure replication and obtain more precise estimatesImportance of StudyThe incidence of brain tumours appears to be increasing with a growing impact on years of life lost. Therapeutic options are limited, either for primary prevention or to prevent mortality of disease once developed. We investigated whether two commonly prescribed families of drugs (fibrates and glitazones) could offer potential drug-repurposing options in reducing brain tumour incidence or progression as informed by previous in vivo and observational studies. Our analyses suggest that glitazones are associated with a decreased brain tumour risk. These results can help guide future research into drug re-repurposing for brain tumour treatment.

Background: Meningiomas are the most common primary CNS tumors. According to the World Health Organization Classification (WHO), they are classified as benign (grade I), atypical (grade II), and anaplastic/malignant (grade III). Chemotherapy has proven ineffective in treating these tumors, which are primarily managed by surgery, radiotherapy, or a combination of them. Morbidity and mortality correlate with meningioma grade. Currently, risk assessment for treatment is based on the radiological assessment of tumor size, tumor growth rate, and/or clinical progression of symptoms. Methods: We performed a cancer miRNA array in an in vitro model of meningioma in order to identify circulating biomarkers in meningioma patients. We validated the miRNA biomarker candidate in cells and tissues and analyzed its regulation. We then investigated expression in tissues and blood. Results: We identified miR-9-1 as significantly overexpressed in atypical and anaplastic cells compared to benign. We further demonstrated that miR-9-1 overexpression is due to increased levels of FOS via upregulation of the EGFR receptor, and showed that miR-9-1 and FOS are upregulated in a cohort of higher-grade meningioma biopsies. Next, we isolated circulating exosomes from meningioma patients’ serum samples, and found higher levels of miR-9-1 in higher-grade compared to low-grade meningiomas patients. Conclusions: Overall, our study shows overexpression and the mechanism of miR-9-1 regulation and suggests miR-9-1 as a novel circulating biomarker candidate to identify tumor grade in meningioma.

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.

The majority of meningiomas are grade I, but some grade I tumours are clinically more aggressive. Recent advances in the genetic study of meningiomas has allowed investigation into the influence of genetics on the tumour microenvironment, which is important for tumorigenesis. We have established that the endpoint genotyping method Kompetitive Allele Specific PCR (KASP&trade, ) is a fast, reliable method for the screening of meningioma samples into different non-NF2 mutational groups using a standard real-time PCR instrument. This genotyping method and four-colour flow cytometry has enabled us to assess the variability in the largest immune cell infiltrate population, M2 macrophages (CD45+HLA-DR+CD14+CD163+) in 42 meningioma samples, and to suggest that underlying genetics is relevant. Further immunohistochemistry analysis comparing AKT1 E17K mutants to WHO grade I NF2-negative samples showed significantly lower levels of CD163-positive activated M2 macrophages in meningiomas with mutated AKT1 E17K, signifying a more immunosuppressive tumour microenvironment in NF2 meningiomas. Our data suggested that underlying tumour genetics play a part in the development of the immune composition of the tumour microenvironment. Stratifying meningiomas by mutational status and correlating this with their cellular composition will aid in the development of new immunotherapies for patients.

Background Meningiomas are the most frequent primary brain tumors of the central nervous system. The standard of treatment is surgery and radiotherapy, but effective pharmacological options are not available yet. The well-characterized genetic background stratifies these tumors in several subgroups, thus increasing diversification. We identified epidermal growth factor receptor–signal transducer and activator of transcription 1 (EGFR–STAT1) overexpression and activation as a common identifier of these tumors. Methods We analyzed STAT1 overexpression and phosphorylation in 131 meningiomas of different grades and locations by utilizing several techniques, including Western blots, qPCR, and immunocytochemistry. We also silenced and overexpressed wild-type and mutant forms of the gene to assess its biological function and its network. Results were further validated by drug testing. Results STAT1 was found widely overexpressed in meningioma but not in the corresponding healthy controls. The protein showed constitutive phosphorylation not dependent on the JAK–STAT pathway. STAT1 knockdown resulted in a significant reduction of cellular proliferation and deactivation of AKT and ERK1/2. STAT1 is known to be activated by EGFR, so we investigated the tyrosine kinase and found that EGFR was also constitutively phosphorylated in meningioma and was responsible for the aberrant phosphorylation of STAT1. The pharmaceutical inhibition of EGFR caused a significant reduction in cellular proliferation and of overall levels of cyclin D1, pAKT, and pERK1/2. Conclusions STAT1–EGFR-dependent constitutive phosphorylation is responsible for a positive feedback loop that causes its own overexpression and consequently an increased proliferation of the tumor cells. These findings provide the rationale for further studies aiming to identify effective therapeutic options in meningioma.