Abstract BACKGROUND Meningioma is the most common primary intracranial tumor. Although ~80% are benign some WHO grade I are clinically aggressive. Chemotherapies are ineffective and biomarkers for clinical management are lacking. Approximately 60% sporadic meningiomas harbor mutations in the NF2 gene andutations in TRAF7, KLF4, AKT1, SMO and PIK3CA have been identified in the majority NF2-positive tumors esp lower grade. However, the molecular mechanisms behind meningioma tumourigenesis is still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterizing the proteomic landscape. MATERIAL AND METHODS We analysed grade I, II and III frozen meningioma specimens and three different mutational groups: AKT1/TRAF7, KLF4/TRAF7 and NF2 -/- using LC-MS/MS to analyse global proteins, enriched phosphoproteins and phosphopeptides. Differential expression and functional annotation of proteins was completed using Perseus, IPA® and DAVID. For mutational subtypes quantitative phosphoproteomics was performed using TMT 10plex labeling approach followed by motif analysis using motif-X algorithm. We validated differential expression of proteins and phosphoproteins by Western blot and immunohistochemistry. RESULTS We quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades. Bioinformatics analysis revealed commonly upregulated (phospho)proteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation confirmed significant overexpression of proteins such as EGFR, CKAP4, the nuclear proto-oncogene SET, the splicing factor SF2/ASF as well as total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Hexokinase 2 was overexpressed in higher grades. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups. Analysis showed distinct proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. 14 and 7 proteins were commonly significantly upregulated in KLF4/TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 and Endoglin were verified via WB. Lastly, analyses of 6600 phosphosites predicted regulatory kinases CONCLUSION We show extensive proteomic and phospophoproteomics analysis of meningioma and suggest new therapeutic and biomarker candidates.

Introduction Meningioma is the most common primary brain tumour and is classified as benign (WHO I, 80%), atypical (WHO II, 15%–20%) and anaplastic (WHO III, 1%–3%). The 3 year recurrence rate in WHO I meningioma is ~50% and it is much greater in WHO II and III. Recent studies showed that cyclin D1 and E1 positively correlated with meningioma grade and higher recurrence rates, suggesting them as potential prognostic markers. Here, we show that cyclin D1-D2 and E1 overexpression in malignant meningioma is driven by downregulation of the miR-15 family members via GATA4, a transcription factor overexpressed in WHO III meningioma cells and tissues. Therefore, we propose GATA4 as a novel possible biomarker for monitoring meningioma progression. Material and methods Meningioma (MN) specimens were collected from consented patients according to the ethical approval for this study. All cell lines and primary MN cells were isolated from tumour specimens and cultured following recommended conditions. Real Time PCR was conducted using TaqMan reagents (Applied Biosystems), according to the manufacturer’s instructions following the 2-ΔΔCT method. In silico studies were conducted using TargetScanHuman7.1 to search for putative miRNA targets. P-values were calculated using the Student’s t-Test or the ANOVA one-way analysis of variance, led by the GraphPad Prism 5.01 and MS Excel 2016 software (p values<0.05±SEM). Results and discussions Proteomic analysis showed an increase of cyclin D1 in Ben Men-1, primary WHO I and KT21-MG1 cells, and an increase of cyclin D2 in KT21-MG1 cells only. In silico studies identified cyclin D1-D2-D3 and E1 as targets of the miR-15 family members. QPCR showed that miR-195 and −497 are downregulated in WHO II and III samples compared to WHO I (3.38 and 6.02 folds, respectively; p=0.02); these results were consistent in cell culture exosomes. Analysis of GATA4 revealed that the protein is highly overexpressed in KT21-MG1 cells but not in Ben Men-1 and primary meningioma cells (17940.19 and 4899.34 folds, respectively, p=0.03). These results were consistent in tissues (2640.53 folds). Conclusion Our data show that members of the miR-15 family are downregulated in WHO III meningioma cells and tissues, suggesting their contribution to control tumour progression. In addition, in WHO III cells and tissues the transcription factor GATA4, involved in miR-15 family regulation, is overexpressed. Ongoing studies will address GATA4 role in the biological progression of meningioma. (DB and COH contributed equally).

Meningioma is the most frequent primary intracranial tumour. Surgical resection remains the main therapeutic option as pharmacological intervention is still hampered by the poor knowledge of the molecular signature of these tumours In order to elucidate the proteomic profiling of meningiomas and identify proteins involved in their pathogenesis, we completed a comparative mass spectrometry analysis of meningioma tissue of all WHO grades, analysing global proteins, phosphoproteins and phosphopeptides. We performed differential expression analyses and functional annotation studies to identify commonly upregulated proteins and phosphoprotein in all grades of meningioma compared to meningeal tissue as well as grade-specific candidates relevant for tumour progression. Top candidates werevalidated by Western blotting and immunohistochemistry in an additional sample set We confirmed significant overexpression of proteins including EGFR, STAT2 and CKAP4 across all grades, as well as the aberrant activation of the downstream PI3K/AKT pathway, which seems differential between grades. Further, we validated upregulation in all grades of the total and activated phosphorylated form of the NIMArelated kinase, NEK9, involved in mitotic progression. Novel proteins identified in meningioma and validated as commonly overexpressed in all grades were the nuclear proto-oncogene SET and the splicing factor SF2/ASF, while another newly identified protein that was specific for higher grades was hexokinase-2, involved in cellular metabolism Overall, we generated a proteomic thesaurus of meningiomas in order to decipher aberrantly expressed proteins and activated pathways; this body of knowledge will eventually lead to the identification of relevant biomarkers and therapeutic targets.

Introduction Meningiomas are the most common primary intracranial brain tumour arising from meningeal tissue. Despite the majority of them displaying benign features, they can cause mild to severe morbidity. The current main therapeutic approach is complete tumour resection commonly with adjunct radiation therapy. However, tumour location can hamper complete resection and chemotherapies are ineffective. In this study we aim to elucidate the pathogenic signature of these tumours and identify novel molecular targets by deciphering the global proteome and phosphoprotein profile of different grades of meningiomas. Material and methods Tumour lysates were collected from grade I, II and III frozen meningioma specimens and three normal healthy human meninges. Phosphoprotein purification was performed using Qiagen® PhosphoProtein Purification Kit. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion. Extracted peptides were purified and analysed by electrospray ionisation LC-MS/MS. Raw mass spectrometry files were analysed using MaxQuantTM. Expression data were validated by Western blot and immunohistochemistry. In silico functional annotation of expression data was completed using Perseus 1.5.0.31 software suite, Ingenuity Pathway Analysis (IPA®) and DAVID 6.8. Results and discussions We have quantified 3888 proteins and 3074 phosphoproteins across all grades of meningioma and normal meninges. Comparative analysis identified 181 proteins and 338 phosphoproteins to be commonly significantly upregulated (log2 fold-change ≥1.5; p Conclusion In summary, we performed a comprehensive quantitative proteomic analysis from meningioma tissue of all WHO grades compared to healthy meninges and have identified several potential candidates that may hold therapeutic potential for targeted treatment of these tumours.

Aims: Sudden unexpected death in epilepsy (SUDEP) is one of the leading causes of death in people with epilepsy. For classification of definite SUDEP, a post mortem (PM), including anatomical and toxicological examination, is mandatory to exclude other causes of death. We audited PM practice as well as the value of brain examination in SUDEP. Methods: We reviewed 145 PM reports in SUDEP cases from four UK neuropathology centres. Data were extracted for clinical epilepsy details, circumstances of death and neuropathological findings. Results: Macroscopic brain abnormalities were identified in 52% of cases. Mild brain swelling was present in 28%, and microscopic pathologies relevant to cause or effect of seizures were seen in 89%. Examination based on whole fixed brains (76.6% of all PMs), and systematic regional sampling was associated with higher detection rates of underlying pathology (P < 0.01). Information was more frequently recorded regarding circumstances of death and body position/location than clinical epilepsy history and investigations. Conclusion: Our findings support the contribution of examination of the whole fixed brain in SUDEP, with high rates of detection of relevant pathology. Availability of full clinical epilepsy-related information at the time of PM could potentially further improve detection through targeted tissue sampling. Apart from confirmation of SUDEP, complete neuropathological examination contributes to evaluation of risk factors as well as helping to direct future research into underlying causes.

INTRODUCTION: Brain tumour tissue can be difficult for researchers to access. BRAIN UK is a collaborative virtual brain bank, facilitating access to under-utilised neuropathology archives for research. A review of National Health Service CNS biopsy archives revealed around 400,000 stored samples, accruing a further 18,500 annually. We aimed to determine how many of these samples were consented to use for research. Without obtaining this consent, it may not be possible to carry out testing on brain tissue samples using emerging diagnostic, prognostic or predictive tests which could improve disease management and access to new treatments. METHOD: 24 UK neuropathology centres were surveyed, by email, on their consent processes, recording and rates of consent. RESULTS: 23/24 centres participated in the audit, 16/23 (70%) obtained prospective informed consent but 6/23 (26%) did not. Of the 16 centres obtaining consent, 9/16 (56%) provided details of consent rates, 6 (37%) estimated their consent rates to be 95-100% of samples, 3 (19%) estimated rates around 10%. CONCLUSION: We estimated that only 30% of neurosurgical tissue samples had been consented to use for research. Robust systems for recording consent accurately were not consistent across the NHS and not all centres had ‘consent to researchu0027 procedures in place. Archived residual tissue could be a valuable research resource but could go unused due to lack of consent. This is despite research to suggest that patients are largely supportive of the use of their tissues in research.

Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell–matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1’s function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4DCAF1. Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.

Purpose: Current guidelines recommend adjuvant therapy with clopidogrel for 1 month following bare metal (BMS) and 6-12 months following drug eluting (DES) stent implantation. We investigated the effect of a delay in patients filling their first clopidogrel prescription after hospital discharge on clinical outcomes following coronary artery stenting. Methods: Hospital administrative, community pharmacy and cardiac revascularization data were linked to determine clopidogrel use and clinical outcomes for all patients receiving a coronary stent in British Columbia from 2004 - 2006 with follow-up to 5 years. Cox proportional hazard regression and propensity-matched analyses were performed to examine the effect of a delay of >3 days in filling a clopidogrel prescription following hospital discharge on clinical outcomes. Results: Of 15,629 patients, 3,599 patients received at least 1 DES whilst 12,030 received BMS alone. The median time to fill a clopidogrel prescription from hospital discharge was 1 day [IQR 1 to 3 days]. 1064 (30%) and 3758 (31%) patients in the DES and BMS groups respectively failed to fill a prescription within 3 days of discharge. Following regression analysis, a delay in filling a prescription for clopidogrel by >3 days was predictive of all-cause mortality (HR 1.9 95% CI [1.5-2.6] and HR 1.9; 95% CI [1.7-2.2]) and readmission for myocardial infarction (HR 1.7; 95% CI [1.3-2.2] and HR 1.5; 95% CI [1.3-1.8]) in the DES and BMS groups respectively. In propensity-matched analyses, patients who delayed filling their prescription had shorter survival (1722±386 days vs 1777±250 days, p=0.001 and 1658±472 days vs 1748±307 days, p<0.001) and were readmitted earlier with myocardial infarction (1705±423 vs 1748±331 days, p=0.029 and 1671±486 days vs 1736±360 days, p<0.001) in the DES and BMS groups respectively compared to those who filled their prescription within 3 days. Conclusions: A delay in patients filling their first prescription for clopidogrel following coronary stenting is common and is associated with serious adverse clinical outcomes, irrespective of stent type. Strategies to reduce delays in filling a prescription on discharge have the potential to improve clinical outcomes.

OBJECTIVES:To carry out a further survey of archived appendix samples to understand better the differences between existing estimates of the prevalence of subclinical infection with prions after the bovine spongiform encephalopathy epizootic and to see whether a broader birth cohort was affected, and to understand better the implications for the management of blood and blood products and for the handling of surgical instruments. DESIGN:Irreversibly unlinked and anonymised large scale survey of archived appendix samples. SETTING:Archived appendix samples from the pathology departments of 41 UK hospitals participating in the earlier survey, and additional hospitals in regions with lower levels of participation in that survey. SAMPLE:32,441 archived appendix samples fixed in formalin and embedded in paraffin and tested for the presence of abnormal prion protein (PrP). RESULTS:Of the 32,441 appendix samples 16 were positive for abnormal PrP, indicating an overall prevalence of 493 per million population (95% confidence interval 282 to 801 per million). The prevalence in those born in 1941-60 (733 per million, 269 to 1596 per million) did not differ significantly from those born between 1961 and 1985 (412 per million, 198 to 758 per million) and was similar in both sexes and across the three broad geographical areas sampled. Genetic testing of the positive specimens for the genotype at PRNP codon 129 revealed a high proportion that were valine homozygous compared with the frequency in the normal population, and in stark contrast with confirmed clinical cases of vCJD, all of which were methionine homozygous at PRNP codon 129. CONCLUSIONS:This study corroborates previous studies and suggests a high prevalence of infection with abnormal PrP, indicating vCJD carrier status in the population compared with the 177 vCJD cases to date. These findings have important implications for the management of blood and blood products and for the handling of surgical instruments.