Adult brain tumour research in 2024: Status, challenges and recommendations.

In 2015, a groundswell of brain tumour patient, carer and charity activism compelled the UK Minister for Life Sciences to form a brain tumour research task and finish group. This resulted, in 2018, with the UK government pledging £20m of funding, to be paralleled with £25m from Cancer Research UK, specifically for neuro-oncology research over the subsequent 5 years. Herein, we review if and how the adult brain tumour research landscape in the United Kingdom has changed over that time and what challenges and bottlenecks remain. We have identified seven universal brain tumour research priorities and three cross-cutting themes, which span the research spectrum from bench to bedside and back again. We discuss the status, challenges and recommendations for each one, specific to the United Kingdom.

Calcifying pseudoneoplasms of the neuraxis (CAPNON) in foramen magnum (cervicomedullary junction) region: a case report and review of the literature.

BACKGROUND AND IMPORTANCE: Calcifying pseudoneoplasms of the neuraxis (CAPNON) is an extremely rare tumor, with nearly 150 cases have been reported in the literature. We present a case of CAPNON at foramen magnum (cervicomedullary junction). We also discuss the histological and radiological features of this rare pathology. CLINICAL PRESENTATION: A 35-year-old male patient presented to our center complaining of neck pain for the last 6 months and for the last 3 months has had headaches associated with nausea, blurred vision and papilledema. The patient's magnetic resonance imaging showed the presence of right craniocervical junction intra-dural extramedullary tumor. Neuro examination showed no neurological deficits. He underwent midline suboccipital craniotomy with C1 laminectomy. The mass was completely resected, and histopathology confirmed it to be a CAPNON. CONCLUSION: We presented a new case of CAPNON at cervicomedullary junction. The tumor was radically resected, without any complications. Resection of this kind of tumor is curative, and the need for post-operative chemo or radiotherapy is unnecessary, which depends on the outcome of future research and the long-term follow ups.

Diagnostic accuracy of 1p/19q codeletion tests in oligodendroglioma: A comprehensive meta-analysis based on a Cochrane systematic review.

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.

Diagnostic test accuracy and cost-effectiveness of tests for codeletion of chromosomal arms 1p and 19q in people with glioma.

BACKGROUND: Complete deletion of both the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q), known as 1p/19q codeletion, is a mutation that can occur in gliomas. It occurs in a type of glioma known as oligodendroglioma and its higher grade counterpart known as anaplastic oligodendroglioma. Detection of 1p/19q codeletion in gliomas is important because, together with another mutation in an enzyme known as isocitrate dehydrogenase, it is needed to make the diagnosis of an oligodendroglioma. Presence of 1p/19q codeletion also informs patient prognosis and prediction of the best drug treatment. The main two tests in use are fluorescent in situ hybridisation (FISH) and polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) assays (also known as PCR-based short tandem repeat or microsatellite analysis). Many other tests are available. None of the tests is perfect, although PCR-based LOH is expected to have very high sensitivity. OBJECTIVES: To estimate the sensitivity and specificity and cost-effectiveness of different deoxyribonucleic acid (DNA)-based techniques for determining 1p/19q codeletion status in glioma. SEARCH METHODS: We searched MEDLINE, Embase and BIOSIS up to July 2019. There were no restrictions based on language or date of publication. We sought economic evaluation studies from the results of this search and using the National Health Service Economic Evaluation Database. SELECTION CRITERIA: We included cross-sectional studies in adults with glioma or any subtype of glioma, presenting raw data or cross-tabulations of two or more DNA-based tests for 1p/19q codeletion. We also sought economic evaluations of these tests. DATA COLLECTION AND ANALYSIS: We followed procedures outlined in the Cochrane Handbook for Diagnostic Test Accuracy Reviews. Two review authors independently screened titles/abstracts/full texts, performed data extraction, and undertook applicability and risk of bias assessments using QUADAS-2. Meta-analyses used the hierarchical summary ROC model to estimate and compare test accuracy. We used FISH and PCR-based LOH as alternate reference standards to examine how tests compared with those in common use, and conducted a latent class analysis comparing FISH and PCR-based LOH. We constructed an economic model to evaluate cost-effectiveness. MAIN RESULTS: We included 53 studies examining: PCR-based LOH, FISH, single nucleotide polymorphism (SNP) array, next-generation sequencing (NGS), comparative genomic hybridisation (CGH), array comparative genomic hybridisation (aCGH), multiplex-ligation-dependent probe amplification (MLPA), real-time PCR, chromogenic in situ hybridisation (CISH), mass spectrometry (MS), restriction fragment length polymorphism (RFLP) analysis, G-banding, methylation array and NanoString. Risk of bias was low for only one study; most gave us concerns about how patients were selected or about missing data. We had applicability concerns about many of the studies because only patients with specific subtypes of glioma were included. 1520 participants contributed to analyses using FISH as the reference, 1304 participants to analyses involving PCR-based LOH as the reference and 262 participants to analyses of comparisons between methods from studies not including FISH or PCR-based LOH. Most evidence was available for comparison of FISH with PCR-based LOH (15 studies, 915 participants): PCR-based LOH detected 94% of FISH-determined codeletions (95% credible interval (CrI) 83% to 98%) and FISH detected 91% of codeletions determined by PCR-based LOH (CrI 78% to 97%). Of tumours determined not to have a deletion by FISH, 94% (CrI 87% to 98%) had a deletion detected by PCR-based LOH, and of those determined not to have a deletion by PCR-based LOH, 96% (CrI 90% to 99%) had a deletion detected by FISH. The latent class analysis suggested that PCR-based LOH may be slightly more accurate than FISH. Most other techniques appeared to have high sensitivity (i.e. produced few false-negative results) for detection of 1p/19q codeletion when either FISH or PCR-based LOH was considered as the reference standard, although there was limited evidence. There was some indication of differences in specificity (false-positive rate) with some techniques. Both NGS and SNP array had high specificity when considered against FISH as the reference standard (NGS: 6 studies, 243 participants; SNP: 6 studies, 111 participants), although we rated certainty in the evidence as low or very low. NGS and SNP array also had high specificity when PCR-based LOH was considered the reference standard, although with much more uncertainty as these results were based on fewer studies (just one study with 49 participants for NGS and two studies with 33 participants for SNP array). G-banding had low sensitivity and specificity when PCR-based LOH was the reference standard. Although MS had very high sensitivity and specificity when both FISH and PCR-based LOH were considered the reference standard, these results were based on only one study with a small number of participants. Real-time PCR also showed high specificity with FISH as a reference standard, although there were only two studies including 40 participants. We found no relevant economic evaluations. Our economic model using FISH as the reference standard suggested that the resource-optimising test depends on which measure of diagnostic accuracy is most important. With FISH as the reference standard, MLPA is likely to be cost-effective if society was willing to pay GBP 1000 or less for a true positive detected. However, as the value placed on a true positive increased, CISH was most cost-effective. Findings differed when the outcome measure changed to either true negative detected or correct diagnosis. When PCR-based LOH was used as the reference standard, MLPA was likely to be cost-effective for all measures of diagnostic accuracy at lower threshold values for willingness to pay. However, as the threshold values increased, none of the tests were clearly more likely to be considered cost-effective. AUTHORS' CONCLUSIONS: In our review, most techniques (except G-banding) appeared to have good sensitivity (few false negatives) for detection of 1p/19q codeletions in glioma against both FISH and PCR-based LOH as a reference standard. However, we judged the certainty of the evidence low or very low for all the tests. There are possible differences in specificity, with both NGS and SNP array having high specificity (fewer false positives) for 1p/19q codeletion when considered against FISH as the reference standard. The economic analysis should be interpreted with caution due to the small number of studies.

Prognostic value of test(s) for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation for predicting overall survival in people with glioblastoma treated with temozolomide.

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 determine which method for assessing MGMT methylation status best predicts overall survival in people diagnosed with glioblastoma who 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 radiotherapy/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 sufficient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focused mainly on studies comparing two or more methods, and listed 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: Two review authors independently undertook all steps of the identification and data extraction process for multiple-method studies. We assessed risk of bias and applicability using our own modified and extended version of the QUality In Prognosis Studies (QUIPS) tool. We compared different techniques, exact promoter regions (5'-cytosine-phosphate-guanine-3' (CpG) sites) and thresholds for interpretation 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 (RHR), using an imputed value of the correlation between results based on the same individuals. MAIN RESULTS: We included 32 independent cohorts involving 3474 people that compared two or more methods. 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 summarised 190 articles describing a single method for measuring MGMT promoter methylation status. AUTHORS' CONCLUSIONS: PSQ and MSP appear more prognostic for overall survival than IHC. 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 performed better than higher thresholds of 28% or 29% in two of three good-quality studies making such comparisons.

Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner.

Epigenetic changes are frequently observed in cancer. However, their role in establishing or sustaining the malignant state has been difficult to determine due to the lack of experimental tools that enable resetting of epigenetic abnormalities. To address this, we applied induced pluripotent stem cell (iPSC) reprogramming techniques to invoke widespread epigenetic resetting of glioblastoma (GBM)-derived neural stem (GNS) cells. GBM iPSCs (GiPSCs) were subsequently redifferentiated to the neural lineage to assess the impact of cancer-specific epigenetic abnormalities on tumorigenicity. GiPSCs and their differentiating derivatives display widespread resetting of common GBM-associated changes, such as DNA hypermethylation of promoter regions of the cell motility regulator TES (testis-derived transcript), the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), and many polycomb-repressive complex 2 (PRC2) target genes (e.g., SFRP2). Surprisingly, despite such global epigenetic reconfiguration, GiPSC-derived neural progenitors remained highly malignant upon xenotransplantation. Only when GiPSCs were directed to nonneural cell types did we observe sustained expression of reactivated tumor suppressors and reduced infiltrative behavior. These data suggest that imposing an epigenome associated with an alternative developmental lineage can suppress malignant behavior. However, in the context of the neural lineage, widespread resetting of GBM-associated epigenetic abnormalities is not sufficient to override the cancer genome.

Fibulin-2: A Novel Biomarker for Differentiating Grade II from Grade I Meningiomas.

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 < 0.05), Western blotting (p < 0.05) and RT-qPCR (p < 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.

The important role of the histopathologist in clinical trials: challenges and approaches to tackle them.

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).

shRNA-mediated PPARα knockdown in human glioma stem cells reduces in vitro proliferation and inhibits orthotopic xenograft tumour growth.

The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD-SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα-expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c-Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα-target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self-renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A Rapid Robust Method for Subgrouping Non-NF2 Meningiomas According to Genotype and Detection of Lower Levels of M2 Macrophages in AKT1 E17K Mutated Tumours.

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™) 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.

Symptoms in primary care with time to diagnosis of brain tumours.

Background: Brain tumours often present with varied, non-specific features with other diagnoses usually being more likely. Objective: To examine how different symptoms and patient demographics predict variations in time to brain tumour diagnosis. Methods: We conducted a secondary analysis of brain tumour cases from National Audit of Cancer Diagnosis in Primary Care. We grouped neurological symptoms into six domains (headache, behavioural/cognitive change, focal neurology, 'fits, faints or falls', non-specific neurological, and other/non-specific) and calculated times for patient presentation, GP referral, specialist consultation and total pathway interval. We calculated odds ratios (ORs) for symptom domains comparing the slowest to other quartiles. Results: Data were available for 226 cases. Median (interquartile range) time for the total pathway interval was 24 days (7-65 days). The most common presentation was focal neurology (33.2%) followed by 'fits, faints or falls' and headache (both 20.8%). Headache only (OR = 4.11, 95% CI = 1.10, 15.5) and memory complaints (OR = 4.82, 95% CI = 1.15, 20.1) were associated with slower total pathway compared to 'fits, faints or falls'. GPs were more likely to consider that there had been avoidable delays in referring patients with headache only (OR = 4.17, 95% CI = 1.14, 15.3). Conclusion: Patients presenting to primary care with headache only or with memory complaints remain problematic with potentially avoidable delays in referral leading to a longer patient pathway. This may or may not impact on the efficacy and morbidity of therapies. Additional aids are required to help doctors differentiate when to refer headaches and memory complaints urgently for a specialist opinion.

DNA methylation-based classification and grading system for meningioma: a multicentre, retrospective analysis.

BACKGROUND: The WHO classification of brain tumours describes 15 subtypes of meningioma. Nine of these subtypes are allotted to WHO grade I, and three each to grade II and grade III. Grading is based solely on histology, with an absence of molecular markers. Although the existing classification and grading approach is of prognostic value, it harbours shortcomings such as ill-defined parameters for subtypes and grading criteria prone to arbitrary judgment. In this study, we aimed for a comprehensive characterisation of the entire molecular genetic landscape of meningioma to identify biologically and clinically relevant subgroups. METHODS: In this multicentre, retrospective analysis, we investigated genome-wide DNA methylation patterns of meningiomas from ten European academic neuro-oncology centres to identify distinct methylation classes of meningiomas. The methylation classes were further characterised by DNA copy number analysis, mutational profiling, and RNA sequencing. Methylation classes were analysed for progression-free survival outcomes by the Kaplan-Meier method. The DNA methylation-based and WHO classification schema were compared using the Brier prediction score, analysed in an independent cohort with WHO grading, progression-free survival, and disease-specific survival data available, collected at the Medical University Vienna (Vienna, Austria), assessing methylation patterns with an alternative methylation chip. FINDINGS: We retrospectively collected 497 meningiomas along with 309 samples of other extra-axial skull tumours that might histologically mimic meningioma variants. Unsupervised clustering of DNA methylation data clearly segregated all meningiomas from other skull tumours. We generated genome-wide DNA methylation profiles from all 497 meningioma samples. DNA methylation profiling distinguished six distinct clinically relevant methylation classes associated with typical mutational, cytogenetic, and gene expression patterns. Compared with WHO grading, classification by individual and combined methylation classes more accurately identifies patients at high risk of disease progression in tumours with WHO grade I histology, and patients at lower risk of recurrence among WHO grade II tumours (p=0·0096) from the Brier prediction test). We validated this finding in our independent cohort of 140 patients with meningioma. INTERPRETATION: DNA methylation-based meningioma classification captures clinically more homogenous groups and has a higher power for predicting tumour recurrence and prognosis than the WHO classification. The approach presented here is potentially very useful for stratifying meningioma patients to observation-only or adjuvant treatment groups. We consider methylation-based tumour classification highly relevant for the future diagnosis and treatment of meningioma. FUNDING: German Cancer Aid, Else Kröner-Fresenius Foundation, and DKFZ/Heidelberg Institute of Personalized Oncology/Precision Oncology Program.

The transcription factor PPARα is overexpressed and is associated with a favourable prognosis in IDH-wildtype primary glioblastoma.

AIMS: PPARα agonists are in current clinical use as hypolipidaemic agents and show significant antineoplastic effects in human glioblastoma models. To date however, the expression of PPARα in large-scale glioblastoma datasets has not been examined. We aimed to investigate the expression of the transcription factor PPARα in primary glioblastoma, the relationship between PPARα expression and patients' clinicopathological features and other molecular markers associated with gliomagenesis. METHODS AND RESULTS: With protein immunoblotting techniques and reverse transcription quantitative real-time PCR, PPARα was found to be significantly overexpressed in glioblastoma compared with control brain tissue (P = 0.032 and P = 0.005). PPARA gene expression was found to be enriched in the classical glioblastoma subtype within The Cancer Genome Atlas (TCGA) dataset. Although not associated with overall survival when assessed by immunohistochemistry, cross-validation with the TCGA dataset and multivariate analyses identified PPARA gene expression as an independent prognostic marker for overall survival (P = 0.042). Finally, hierarchical clustering revealed novel, significant associations between high PPARA expression and a putative set of glioblastoma molecular mediators including EMX2, AQP4, and NTRK2. CONCLUSIONS: PPARα is overexpressed in primary glioblastoma and high PPARA expression functions as an independent prognostic marker in the glioblastoma TCGA dataset. Further studies are required to explore genetic associations with high PPARA expression and to analyse the predictive role of PPARα expression in glioblastoma models in response to PPARα agonists.

Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology.

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n = 22), diffuse oligodendroglial tumors (d-OTs; n = 20), diffuse astrocytomas (DAs; n = 17), angiocentric gliomas (n = 15), and gangliogliomas (n = 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87 %), and MYB fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78 % of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.

EGFR and EGFRvIII analysis in glioblastoma as therapeutic biomarkers.

INTRODUCTION: EGFR and EGFRvIII analysis is of current interest because of new EGFRvIII vaccine trials opened in the UK. EGFR activation promotes cellular proliferation via activation of MAPK and PI3K-Akt pathways. EGFRvIII is the most common variant resulting from an in-frame deletion of 801bp, leading to constitutively active EGFR. METHOD: 51 glioblastoma samples from a cohort of 50 patients were tested for EGFR amplification by FISH and immunohistochemistry and EGFRvIII expression by reverse-transcriptase PCR (RT-PCR), and immunohistochemistry. EGFR and EGFRvIII expression was compared with Overall Survival in the cohort. RESULTS: Overall 22/51 samples (43%) were positive for EGFR, 16/51 (31%) were positive for EGFRvIII and 13/51 (25%) were positive for both. 9/51 cases (18%) were positive for EGFR alone, and 3/51 (6%) were positive for EGFRvIII alone. Of the EGFR positive cases, 22/51 (43%) were positive by FISH, 24/51 (47%) were positive by IHC and 2/51 (4%) were discrepant between methods (positive by IHC but non-amplified by FISH). Of the EGFRvIII positive cases, 16/51 (31%) were positive by RT-PCR, 17/51 (33%) were positive by IHC and 1/51 (2%) sample was discrepant (positive by IHC but not by RT-PCR). Neither EGFRvIII or EGFR are predictive of overall survival in this cohort. CONCLUSION: In our cohort, 25/51 (49%) of GBM showed EGFR alterations, including 16/51 (31%) with EGFRvIII. There was high concordance between IHC and FISH (96%) and IHC and RT-PCR (98%) as diagnostic methods. Neither EGFR or EGFRvIII is predictive of overall survival in this cohort. These results are key for selecting patients for novel individualised anti-EGFR therapies.

Diagnosis of primary cerebral lymphomas: possible value of PCR testing in equivocal cases requiring rebiopsy.

INTRODUCTION. Rebiopsy rates as high as 12% have been reported in previous studies of Primary Central Nervous System Lymphoma (PCNSL). This can lead to secondary operations, increasing risks of morbidity to the patient and costs for the NHS. Polymerase Chain Reaction (PCR) testing for clonality in haematological malignancies has been applied to cases of lymphoma outwith the central nervous system (CNS), but is less commonly used in the diagnosis of CNS lymphomas. Clonality in B- and T-cell populations may indicate the presence of malignancy. We aimed to identify factors to reduce the rebiopsy rate in PCNSL. METHODS. We examined a cohort of 102 suspected cerebral lymphoma cases biopsied at Frenchay Hospital, Bristol over a 10-year period (2000-2010). Clinical data, including age, sex, location, pre-biopsy steroid use, the need for rebiopsy and histological diagnosis, were collected. We retrospectively reviewed rebiopsied cases and they subsequently underwent PCR testing for clonality. RESULTS. Overall, 96/102 (94%) cases achieved a histological diagnosis after one or more biopsies. 81/96 (84%) of these were lymphomas involving the brain and 15/96 (16%) were spinal lymphomas. The majority of these were B-cell lymphomas (95/96 (99%)), with one case of peripheral T-cell lymphoma (1/96 (1%)). Due to insufficient histological evidence of PCNSL after the first biopsy, 9/102 (9%) of cases had required rebiopsy. In 7/9 (78%) of these cases, we undertook PCR testing for clonality on tissue from the first biopsy. We found 3/7 (43%) cases were monoclonal for B or T populations, raising the possibility of PCNSL. CONCLUSIONS. We recommend that all CNS lymphoproliferative lesions be assessed by haematopathologists, with the inclusion of PCR testing particularly in equivocal cases. This would reduce the number of patients going for rebiopsy and reduce the patient morbidity and costs for the NHS.

Evaluating circulating tumour cell enrichment techniques to establish an appropriate method for clinical application in glioblastomas.

Brain tumours reduce life expectancy for an average of 20 years per patient, the highest of any cancer. A third of brain tumour patients visit their GP at least five times before diagnosis and many of those are diagnosed late through emergency departments. A possible solution to this challenge is to utilise a "liquid biopsy" blood test designed for circulating tumour cells (CTCs). Such a test could be applied at a primary healthcare centre, contributing to informed decision making for diagnostic imaging referrals. Furthermore, it could also be applied at secondary health care centres for the ongoing monitoring of disease recurrence. There is increased interest in CTC enrichment methods as a potential approach for faster diagnosis and monitoring of disease progression. The aim of this review to compare four CTC enrichment methods - OncoQuick®, Screen Cell®, pluriBead® and Cell Search® - with the objective of identifying a suitable method for application in the clinical setting for the isolation of CTCs from glioblastomas.

Pre-diagnostic blood biomarkers for adult glioma.

Glioma is one of the most common malignant primary brain tumours in adults, of which, glioblastoma is the most prevalent and malignant entity. Glioma is often diagnosed at a later stage of disease progression, which means it is associated with significant mortality and morbidity. Therefore, there is a need for earlier diagnosis of these tumours, which would require sensitive and specific biomarkers. These biomarkers could better predict glioma onset to improve diagnosis and therapeutic options for patients. While liquid biopsies could provide a cheap and non-invasive test to improve the earlier detection of glioma, there is little known on pre-diagnostic biomarkers which predate disease detection. In this review, we examine the evidence in the literature for pre-diagnostic biomarkers in glioma, including metabolomics and proteomics. We also consider the limitations of these approaches and future research directions of pre-diagnostic biomarkers for glioma.

Insulin-like growth factor binding protein-2 and glucose-regulated protein 78 kDa: Potential biomarkers affect prognosis in IDH-wildtype glioblastoma patients.

BACKGROUND: The overall survival of IDH-wildtype glioblastoma patients is poor despite best available treatments. There is an urgent need for new biomarkers to inform more precise disease stratification. Previous studies have identified insulin-like growth factor binding protein-2 (IGFBP-2) as a potential biomarker for glioblastoma diagnosis and therapeutic targeting. Other studies have indicated links between the insulin-like growth factor (IGF) axis and tumorigenic functions of a molecular chaperone glucose related protein of 78 kDa (GRP78). We aimed to interrogate the oncogenic effects of IGFBP-2 and GRP78 in our glioma stem cell (GSC) lines and clinical cohort. METHODS: Immunoblotting, reverse transcription quantitative real-time PCR were used to quantify protein and mRNA levels derived from GSCs and non-malignant neural stem cells (NSCs). Microarray analysis was used to compare the differences in IGFBP-2 (IGFBP-2) and GRP78 (HSPA5) transcript expression between NSCs, GSCs and adult human cortex samples. Immunohistochemistry was used to quantify IGFBP-2 and GRP78 expression in IDH-wildtype glioblastoma tissue sections (n = 92) and clinical implications assessed using survival analysis. Finally, the relationship between IGFBP-2 and GRP78 was further explored molecularly using coimmunoprecipitation. RESULTS: Here, we demonstrate that IGFBP-2 and HSPA5 mRNA is overexpressed in GSCs and NSCs in comparison to non-malignant brain tissue. We also determined a relationship in which G144 and G26 GSCs expressed higher IGFBP-2 protein and mRNA than GRP78, and this was reversed in mRNA isolated from adult human cortex samples. Clinical cohort analysis revealed that Glioblastomas with high IGFBP-2 protein expression paired with low GRP78 protein expression were significantly associated with a much shorter survival time (Median = 4 months, p = 0.019) compared with 12-14 months for any other combination of high/low protein expression. CONCLUSIONS: Inverse levels of IGFBP-2 and GRP78 may be adverse clinical prognostic markers in IDH-wildtype glioblastoma. Further interrogation of the mechanistic link between IGFBP-2 and GRP78 may be important for rationalisation of their potential as biomarkers and therapeutic targets.