David Coope

Microglial activation in normal-appearing brain regions of patients with cerebral glioma: a cross-sectional study

Abstract Background Translocator protein (TSPO) is overexpressed mainly in activated microglia under disease conditions. [ 11 C]-(R)PK11195 is a radioligand for TSPO widely applied in PET studies. Previously we have found upregulated TSPO in neoplastic cells and its correlation with malignant transformation in human gliomas using [ 11 C]-(R)PK11195 PET and neuropathological assessment. We aimed to investigate TSPO expression and microglial activation in normal-appearing brain regions of patients with glioma. Methods 14 patients with low-grade glioma (before tumour biopsy or debulking) and ten healthy controls underwent MRI and PET scans. Epilepsy history was reviewed. Binding potential (BP ND ) of [ 11 C]-(R)PK11195 was calculated by the simplified reference tissue model. BP ND of normal-appearing brain regions was compared with that in controls. Post-mortem brains with treatment-naive low-grade glioma were assessed for TSPO and microglia by immunohistochemistry. Findings BP ND of [ 11 C]-(R)PK11195 in low-grade gliomas was lower than in normal-appearing cerebral regions in the contralateral hemisphere (mean −0·108 [SD 0·148] vs 0·047 [0·101], p=0·0006). Compared with controls, BP ND in patients normal-appearing brain regions was increased (0·065 [0·062] vs 0·002 [0·024], p=0·001), being more prominent in the tumour-bearing hemisphere than in the tumour-free hemisphere (0·081 [0·064] vs 0·057 [0·063], p=0·002). The extratumoral BP ND correlated with the length of epilepsy history (Spearman rank correlation coefficient r =0·5, p=0·016). An ipsilateral pattern of increased extratumoral BP ND was seen in patients with partial seizures, whereas a bilateral pattern of increase was seen in those with generalised seizures. Post-mortem brain tissue showed a 10-fold increase in microglia and elevation in TSPO-expressing microglia in the gyri adjacent to the tumour and in the contralateral tissue compared with normal brains. Interpretation [ 11 C](R)PK11195 PET and neuropathological assessment revealed widespread microglial activation in normal-appearing brain regions of patients with glioma. That the magnitude of activation was associated with length of epilepsy history and seizure type suggests that modulation of microglial activation could be a novel target for seizure control in these patients. TSPO PET provides an in-vivo demonstration of this inflammatory response, which is undetectable by structural MRI. Funding European Unions Seventh Framework Programme (FP7/2007-2013) under grant agreement INMiND (HEALTH-F2-2011-278850), Engineering and Physical Science Research Council grant MIMIT (EP/G041733/1), Astro Brain Tumour Fund (1133561).

In vivo imaging of translocator protein expression in gliomas by Positron Emission Tomography (PET).

INTRODUCTION: In the past 25 years only modest advancements in glioma treatment have been made, with patient prognosis and median survival time following diagnosis only increasing from 3 to 7 months. A substantial body of clinical and preclinical evidence has suggested a role for aspirin in the treatment of cancer with multiple mechanisms of action proposed including COX 2 inhibition, down regulation of EGFR expression, and NF-κB signaling affecting Bcl-2 expression. However, with serious side effects such as stroke and gastrointestinal bleeding, aspirin analogues with improved potency and side effect profiles are being developed. METHOD: Effects on cell viability following 24 hr incubation of four aspirin derivatives (PN508, 517, 526 and 529) were compared to cisplatin, aspirin and di-aspirin in four glioma cell lines (U87 MG, SVG P12, GOS – 3, and 1321N1), using the PrestoBlue assay, establishing IC50 and examining the time course of drug effects. RESULTS: All compounds were found to decrease cell viability in a concentration and time dependant manner. Significantly, the analogue PN517 (IC50 2mM) showed approximately a twofold increase in potency when compared to aspirin (3.7mM) and cisplatin (4.3mM) in U87 cells, with similar increased potency in SVG P12 cells. Other analogues demonstrated similar potency to aspirin and cisplatin. CONCLUSION: These results support the further development and characterization of novel NSAID derivatives for the treatment of glioma.INTRODUCTION: Cortical mapping can maximize tumour resection whilst preserving eloquent cortex in glioma surgery. Direct cortical stimulation (DCS) during awake craniotomy remains the current ?gold standard?. Functional MRI (fMRI) presents an alternative but raises theoretical concerns relating to neurovascular de-coupling close to the tumour. Comparative studies are limited by difficulties in accurately recording the locations at which DCS is performed. We present the initial evaluation of a system to facilitate the use of commercial neuro-navigation technology to record DCS outcomes.nnMETHOD: Three patients underwent pre-operative neuropsychological assessment and motor / language fMRI on a Phillips 3T Achieva scanner. fMRI data was available for surgical planning but the surgeon was blinded during DCS to minimise bias. During awake craniotomy, a bipolar cortical stimulator was tracked using a BrainLab VectorVision system networked with a laptop using the VVLink interface. DCS was performed whilst motor and language function was assessed by a neuropsychologist and outcomes recorded by the surgeon using a panel of footswitches to control a custom logging module implemented within the Slicer software package.nnRESULTS: A mean of 103 unique data-points were recorded (range 73-136) with minimal additional operative time. Mean number of areas tested for language function at the highest required stimulation level was 51 (range 26-74). Mean distance to the nearest fMRI activation did not differ between positive and negative DCS (6.8 vs 6.1mm).nnCONCLUSION: Use of fMRI/neuro-navigation to record DCS outcomes is feasible and enables detailed study of the correlation between pre- and intra-operative techniques for cortical mapping.

Preliminary evaluation of [11C]-(R)PK11195 kinetics in low-grade gliomas on the high resolution research tomograph

Times Cited: 0 8th International Symposium on Functional Neuroreceptor Mapping of the Living Brain Jul 22-24, 2010 Glasgow, SCOTLAND

Quantitative analysis of brain tumour interactions with white matter tracts using Diffusion Tensor Imaging. In: Abstracts from the BNOS meeting, 23-25 June 2010, Glasgow.

INTRODUCTION: We previously reported expression of CD44(Hyaluronic acid/lymphocyte homing receptor) and CD155 (Poliovirus receptor) on established cell lines and early passage cultures of biopsyderived glioma using immunocytochemistry, TIRF microscopy and flow cytometry. Both receptors have been reported to facilitate glioma invasion. METHODS: Antibody blocking and siRNA silencing of CD44 and CD155 were assessed using Transwell assay and live cell imaging for motility and invasion. A ECM cell adhesion array was used to determine adhesive potential of wild type cells versus silenced cells. BrdU cell proliferation assay was carried out to assess proliferative rate of cells following siRNA knockdown of both CD44 and CD155. Interaction and localisation of CD44 and CD155 with F-actin and integrins (b, avb, and avb3) were shown by confocal and TIRF microscopy. RESULTS: CD44 antibody blocking and gene silencing resulted in a higher level of inhibition of invasion than for CD155. Interference with combined CD44/CD155 resulted in 100% inhibition of invasion. Live cell imaging showed reduced speed of motility of knockdown cells over their controls. Wild type cells adhered most efficiently to laminin whereas siRNA-treated cells showed decreased adhesive potential on most of the ECMs used. A higher proliferative rate of siRNA CD44 and siRNA CD155 treated cells was inversely correlated with the reduced invasion of these cells. Confocal microscopy showed distinct overlapping of CD155 and the integrins on filopodia while TIRF microscopy allowed high signal/low noise imaging of double labelled cells. CONCLUSIONS: Joint CD44/CD155 approaches may merit further study in targeting infiltrating glioma cells in therapeutic protocols.Times Cited: 0 Meeting of the British-Neuro-Oncology-Society JUN 23-25, 2010 Glasgow, SCOTLANDINTRODUCTION: The Axl receptor tyrosine kinase (RTK) and its ligand Gas6, have previously been shown to regulate neoplastic cell motility, migration, invasion and proliferation. The aim of this study was to determine the role of Gas6/Axl on migration of human glioma cells. METHODS: Three human glioblastoma derived cell lines of differing origin and degree of heterogeneity,were screened for Axl protein expression by western blot. For investigation of cell migration, serum-starved cells were stimulated with recombinant human Gas6, and scratch wound assays coupled with live cell imaging were performed to monitor the migration rates of these cells. The cells were also analysed for intracellular signalling effects on Axl and phospho-Axl by western blot and protein tyrosine kinase assays. RESULTS: We detected expression of Axl protein in SNB-19 (homogeneous) and UPAB (heterogeneous) cell lines, although not in IN699 (paediatric). It was furthermore demonstrated that Gas6 stimulated the migration of SNB-19 cells over 36 h by a two-fold increase as compared to control-treated cells, resulting in a migration speed of 11.650+0.564 mm/h compared to 6.861+1.264 mm/h. Moreover, a Gas6 concentration-dependent response was observed, revealing a specific promotion of migration (p , 0.05). CONCLUSIONS: Stimulation of the Axl RTK with its ligand Gas6 stimulates migration of human glioma cells, a mechanism that remains to be fully understood. These data can help to understand the mechanisms of local invasion in brain tumours. We wish, therefore, to further investigate Axl activation as well as related signalling pathway activities in glioma cells to help understand the molecular basis of glioma migration and invasion.

Evaluation of multiple tumour parameters to improve the non-invasive classification of primary brain tumours with 11C-methionine PET

Background: Gliomas demonstrate significant heterogeneity both in terms of outcome and imaging characteristics within histological grades and particularly between histopathological and genetic sub-types. Methionine uptake as measured with positron emission tomography (PET) has been shown to correlate with histological grade but interpretation of the results is difficult unless the histological sub-type is known. We hypothesized that imaging biomarkers of other features known to reflect malignancy including tumour volume, shape and heterogeneity could be measured from methionine PET data to support this interpretation. Method: Methionine PET scans performed in 39 individuals from 2003-4 were identified from the database at the Max-Planck-Institute for Neurological Research in Cologne. Scans were analysed using the ratio to a normal uptake map as previously described. A software application was developed in R to enable standardised, highly reproducible image analysis. Tumour x91masksx92 were defined using a 3D region-growing technique constrained by voxel connectivity. Several statistical measures were recorded for this tumour mask including the volume, characteristics of the border and variability of the methionine uptake including texture measures. Receiver operating characteristic (ROC) analysis was performed for each of these parameters to evaluate accuracy in the discrimination of (1) grade II vs. III/IV and (2) grade III vs. IV tumours. Results: Ten factors were selected including peak methionine uptake, the volume of the tumour mask, measures relating to irregularity of the tumour border / shape, heterogeneity within the tumour volume and the distribution of uptake values within adjacent brain. Each of these features produced significant results on ROC analysis (p=0.000 to 0.010) with the volume of the region of increased methionine uptake being most effective in separating low and high-grade gliomas. Threshold values were defined from the ROC curves to identify results with high specificity for low-grade tumours. 10 of the 13 (77%) grade II tumours analysed exhibited 3 or more of these characteristics as compared to 2 of the 26 (8%) grade III/IV tumours. A score derived from these features demonstrated a greater area under the ROC curve (AUC=0.88) than any of the individual measures in isolation. Conclusion: PET with amino-acid tracers enables multiple tumour measures to be derived from a single diagnostic investigation. Combined evaluation of multiple parameters in an individual tumour may be used to improve accuracy in the non-invasive characterisation of primary brain tumours.