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SUMMARYBackgroundInfiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo- and radio-therapy. A deeper understanding of the mechanisms invoked by GMB to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this.MethodsCRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterised EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived extracellular matrix (ECM) to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice.ResultsGBM cells expressing a p53 mutant (p53273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion ofPODXLopposes infiltration of GBMin vivo.ConclusionsThis work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue.KEY POINTSThe p53R273Honcogene encourages GBM cells to release EVs containing podocalyxin. Podocalyxin-containing EVs from GBM increase hyaluronic acid production by astrocytes. Hyaluronic acid production by astrocytes drives GBM migration.IMPORTANCE OF THE STUDYThe infiltrative behaviour of glioblastoma (GBM) leads to widespread dissemination of cancer cells throughout the brain. Thus, even following successful resection of the primary tumour these disseminated cells inevitably contribute to post-surgical relapse. In this study, we have discovered a new mechanism through which GBM can release small extracellular vesicles (EVs) to reprogramme extracellular matrix (ECM) production by astrocytes in a way that supports increased invasive behaviour of the GBM cells. Moreover, we have discovered several key components of the pathway which contribute to this EV-mediated GBM-glial cell communication. Principal amongst these, we show that a particular mutant of the p53 tumour suppressor, p53273Hdrives the release of EVs which foster the deposition of pro-invasive ECM by astrocytes. This study provides mechanistic insight into why brain tumours expressing p53273Hare associated with particularly poor patient survival and highlights the possibility of deploying agents which target astrocyte ECM deposition to reduce the morbidity of p53273H- expressing GBM.

Background Glioblastomas (GBM) have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist which counter GBM invasion. Here, we report that inhibition of Ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of GBM cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. Methods GBM motility and invasion was assessed both in vitro and in vivo in response to ATR inhibition (ATRi) and ATR over-expression using timelapse microscopy, two orthotopic GBM models and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high throughput, super resolution imaging and intravital imaging. Results High ATR expression was associated with significantly poorer survival in clinical datasets whilst GBM tumour specimens revealed higher ATR expression at infiltrative margins. ATRi opposed invasion of GBM cells due cytoskeletal dysregulation impacting integrin internalisation. Conclusion We demonstrate a novel role for ATR in determining invasion in GBM cells, and propose that pharmacological targeting of ATR could have far reaching clinical benefits.

BACKGROUND Currently approved Poly ADP-Ribose Polymerase (PARP) inhibitors (PARPi) selectively inhibit and trap both PARP1 and PARP2 (PARP1/2) at sites of single strand (ss) deoxyribonucleic acid (DNA) (ssDNA) damage, preventing ssDNA repair and leading to replication-dependent DNA double strand breaks. Recent data suggest that only inhibition of PARP1 is required for anti-proliferative effects, while PARP2 functions in the survival of haematopoietic stem and progenitor cells. These observations suggest that the inhibition and trapping of PARP 2 is not needed for anti-cancer effects, and may be a major driver of haematological toxicity observed. AZD9574 is a novel brain-penetrant PARPi that potently and selectively inhibits and traps PARP1, with the goal of delivering efficacious, less toxic, and more combinable PARPi. Furthermore, owing to its central nervous system penetration capability, AZD9574 may provide a new treatment option for patients with CNS malignancies or patients with brain metastases characterized by homologous recombination deficiency (HRD). METHODS This is a first-in-human modular study primarily designed to evaluate the safety and tolerability of AZD9574 as monotherapy and in combination with anti-cancer agents at increasing dose levels in patients with advanced solid malignancies, followed by expansion cohorts in specific indications. The study will also characterize the pharmacokinetics of AZD9574 and explore potential biological activity by assessing pharmacodynamic and exploratory biomarkers and anti-tumour activity. Module 1 will enrol patients with advanced breast, ovarian, pancreatic or prostate tumours harbouring homologous recombination deficiencies. Module 2 will enrol patients with isocitrate dehydrogenase (IDH)1/2 mutated glioma.

AIMS Gliomas have high levels of DNA replication stress and clinical trials of inhibitors of the key replication stress response protein ATR (ataxia telangiectasia and rad 3 related protein) as radiosensitisers are planned. We aimed to investigate the effect of ATR inhibition on the ability of glioma cells to infiltrate and invade the brain. METHOD Live cell imaging in a panel of primary glioma cultures following siRNA or pharmacological inhibition of ATR. Invasion following treatment of murine orthotopic gliomas was determined by immunohistochemistry. Intravital imaging of GFP expressing murine orthotopic xenografts via an intracranial window model of glioma was undertaken. RESULTS Invading margins of human glioma samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated reduced cell velocity following ATR inhibition (Berzosertib/BAY1895344) or siRNA. Cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures which engulf high molecular weight dextran, compatible with blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed sequestration of integrins within macropinosomes and reduced integrin cycling. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further in vivo study demonstrated a reduction in invading tumour cells. CONCLUSION We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin recycling in GBM cells which results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.

Period-colour and amplitude-colour (PCAC) relations can be used to probe both the hydrodynamics of outer envelope structure and evolutionary status of Cepheids and RR Lyraes. In this work, we incorporate the PCAC relations for RR Lyraes, BL Her, W Vir and classical Cepheids in a single unifying theory that involves the interaction of the hydrogen ionization front (HIF) and stellar photosphere and the theory of stellar evolution. PC relations for RR Lyraes and classical Cepheids using OGLE-IV data are found to be consistent with this theory: RR Lyraes have shallow/sloped relations at minimum/maximum light whilst long-period ($P>10$ days) Cepheids exhibit sloped/flat PC relations at minimum/maximum light. The differences in the PC relations for Cepheids and RR Lyraes can be explained based on the relative location of the HIF and stellar photosphere which changes depending on their position on the HR diagram. We also extend our analysis of PCAC relations for type II Cepheids in the Galactic bulge, LMC and SMC using OGLE-IV data. We find that BL Her stars have sloped PC relations at maximum and minimum light similar to short-period ($P, 20 pages, 14 figures. Accepted for publication in MNRAS

Talc slurry pleurodesis (TSP) prevents recurrence of symptomatic malignant pleural effusion (MPE) in 71% to 78% patients. Nonexpansile lung (NEL) frequently accounts for TSP failure but is often occult predrainage, impairing selection of patients. NEL is associated with high pleural elastance (PPatients with symptomatic MPE were randomized 1:1 between EDIT and standard care (TSP). EDIT involved PThirty-one patients were randomized (one allocation failure) over 12 months. PA phase 3 trial testing the effect of EDIT management on symptomatic MPE recurrence following TSP is feasible.ClinicalTrials.gov; No.: NCT03319186; URL: www.clinicaltrials.gov.

Introduction Non-expansile lung (NEL) is a common cause of talc pleurodesis (TP) failure in malignant pleural effusion (MPE), but is often occult prior to drainage. Reliable detection of NEL would allow patients to be allocated between intrapleural catheter (IPC) and TP. High pleural elastance (PEL) has been associated with NEL in observational studies. Pre-EDIT is a randomised feasibility trial of elastance-directed IPC or TP (EDIT) management using a novel, purpose-built digital pleural manometer (Rocket Medical, UK). Methods and analysis Consecutive patients with MPE without prior evidence of NEL or preference for IPC will be randomised 1:1 between EDIT management and standard care (an attempt at TP). The primary objective is to determine whether sufficient numbers of patients (defined as 30 within 12 months (or 15 over 6 months)) can be recruited and randomised to justify a subsequent phase III trial testing the efficacy of EDIT management. Secondary objectives include safety, technical feasibility and validation of study design elements, including the definition of PEL using 4D pleural MRI before and after fluid aspiration. EDIT involves PEL assessment during a large volume pleural fluid aspiration, followed by an attempt at TP or placement of an IPC within 24 hours. Patients will be allocated to IPC if the rolling average PEL sustained over at least 250 mL fluid aspirated (PEL250) is ≥ 14.5 cm H2O/L. Ethics and dissemination Pre-EDIT was approved by the West of Scotland Regional Ethics Committee on 8 March 2017 (Ref: 17/WS/0042). Results will be presented at scientific meetings and published in peer-reviewed journals. Trial registration number NCT03319186; Pre-results.

Glioblastoma (GBM) is one of the most common and aggressive forms of brain cancer. It is associated with poor survival rates, in part due to the infiltrative nature of GBM tumour cells. This allows GBM cells to disseminate through the brain via existing white matter tracts and perivascular spaces making complete surgical resection unachievable and contributing to high recurrence rates. For the majority of patients treatment of GBM includes radiotherapy. However, radiation resistance results in populations of surviving tumour cells with the potential for modified behaviour through radiation-induced alterations in signalling and gene expression profiles Myotonic dystrophy kinase-related CDC42- binding kinase (MRCK) is involved in regulating actin-myosin dynamics during cell migration through phosphorylation of its targets MLC2 and MYPT. Upon radiation of GBM cell lines in vivo we observed an increase in MRCK activity which was concomitant with a significant increase in GBM cell motility and invasion in in vitro and ex vivo assays. In support of these data we have observed increased MRCK activity at the invasive edges of both clinical GBM samples and an orthotopic GBM xenograft model that recapitulates key histological features of human GBM. Furthermore, therapeutic doses of irradiation were found to promote GBM invasion in vivo in this xenograft model, along with increased pMYPT. These observations suggest that MRCK activity may be required to promote invasion following irradiation and therefore may present a promising novel target for GBM treatment.In support of this, we found we were able to reverse the radiation dependent promotion of GBM cell motility via MRCK RNAi or by using a novel MRCK selective inhibitor, BDP-9066. These data suggest that MRCK activity is indeed required for GBM invasion and may offer a potential new target for GBM management.

INTRODUCTION: Based on imaging features, glioblastoma (GBM) can be classified as solitary, multifocal or multicentric. The incidence of multifocal/multicentric GBM has been reported to range from 0.5 to 35% and some studies have reported poorer survival in patients with multiple lesions. Two potential reasons have been proposed: (1) intrinsic differences in tumour biology; (2) failure to encompass the entire tumour within the radiotherapy planning tumour volume. To address this question we investigated clinical, imaging and genetic features in a cohort of GBM patients. METHODS: Imaging, clinical, treatment, MGMT methylation and outcome data were collected retrospectively from consecutive GBM patients treated in a single cancer centre between January 2011 and June 2012. Tumours were categorised as solitary, multifocal or multicentric by a consultant neuroradiologist. RESULTS: 122 patents with GBM were identified. Median age was 60 and male:female ratio was 2.1:1. MGMT promoter status was unmethylated in 48% of tumours, methylated in 37% and unknown in 15%. Preoperative imaging modality was CT in 70% and MRI in 30% of patients. Overall, the proportion of patients with solitary, multifocal and multicentric tumours was 78%, 15.5% and 6.5 % respectively, but in patients undergoing MR imaging these proportions were 60%, 26.5% and 13.5%. Gross total resection was performed in 65% of solitary compared with 42% of multifocal and 25% of multicentric cases, and radical chemoradiation was delivered to 50% of solitary, 58% of multifocal and 12% of multicentric cases. Overall, median survival was increased in solitary compared with multifocal/multicentric patients (9.9 vs. 6.7 months, p = 0.046). In patients receiving radical chemoradiation, however, there was no difference in overall survival between solitary and multifocal/multicentric patients (18.5 vs. 16.8 months, p=0.57). MGMT promoter methylation was associated with increased survival in patients with solitary tumours (14.6 vs. 8.5 months, p= 0.014) but not multifocal/multicentric tumours (3.8 vs. 6.9 months, p= 0.3). CONCLUSION: In this retrospective study the incidence of multifocal/multicentric GBM was in line with previous studies. Pre-operative CT imaging may underestimate the incidence of multifocal/multicentric disease. Our main finding was that multifocality or multicentricity did not affect survival in patients to whom radical chemo-radiotherapy could be delivered.

Background: A randomised study to investigate whether bilateral arm abduction is superior to unilateral abduction with respect to stability using shifts on treatment as the main outcome measure.Material and Methods: 50 consecutive female patients were randomised to ipsilateral or bilateral arm abduction. Central lung depth (CLD) and cranial caudal depth (CCD) on the simulator image was compared with that featured on three Electronic Portal Images (EPI) captured during treatment for each patient. Systematic and random errors were analysed with respect to the average translational displacement and standard deviation per patient and per population between the planning image and the EPI.Results: The CLD average translational displacement in the test group was −1.7 mm (95% CI = −5 to 1.6 mm) Σ pop = 2.3 mm, σ pop = 1.6 mm, and in the control group −1.9 mm (95% CI = −6 to 3 mm) Σ pop = 4 mm, σ pop = 2.1 mm. The average translational displacement of CCD in test group was 0 mm (95% CI = − 5.3 to 5.1 mm) Σ pop = 2.2 mm, σ pop = 2.6 mm. CCD translational displacement was greater in the unilateral arm abduction group at − 1.6 mm (95% CI = −6.7 to 3.4 mm) Σ pop = 3.6 mm, σ pop = 2.4mm.Conclusion: The reduction in systematic error and inter-patient variability observed in the test group is evidence that bilateral arm abduction is a more stable and reproducible position than unilateral arm abduction. The CCD translational data indicates that patients treated with unilateral arm abduction were moving inferiorly on the breast board. These results support the adoption of bilateral arm abduction as a standard technique.

HIV infection is likely to remain a significant medical and scientific problem well into the twenty-first century. During the first 15 years of the epidemic, much has been learned about the biology of HIV infection, but the majority of biomedical research has focused on the peripheral circulation. It is likely that the behavior of the virus within the unique immunologic environment of the intestinal mucosa differs from that which is observed in the periphery. Many clinical and epidemiologic features of HIV infection offer compelling reasons to encourage further examination of the mucosal immune system's role in AIDS pathogenesis. This article has touched on most of the significant observations concerning the mucosal immune system and HIV infection, and it is clear that much remains to be done. As mentioned earlier, the mucosal abnormalities observed in HIV infection are likely to have many causes. Careful evaluation of patients with early disease and fewer confounding variables may provide fresh insight into AIDS pathogenesis. Similarly, prospective evaluation of selected patient populations may be more informative in characterizing the progressive alterations in mucosal immune function than random cross-sectional studies of poorly defined groups. It is equally important for immunologic assessment to be correlated with nutritional and symptomatic evaluation. Finally, the success or failure of future antiretroviral therapies will be critically related to the impact of such agents on lymphoid reservoirs of HIV infection such as the gastrointestinal tract, which are at present refractory to treatment.