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. 2012 Sep;14(Suppl 2):ii1–ii12. doi: 10.1093/neuonc/nos198

Abstracts from the 2012 BNOS Conference

PMCID: PMC3431946
Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

01. COMBINED BOSWELLIC ACID AND TEMOZOLOMIDE TREATMENT SELECTIVELY INDUCES APOPTOSIS AND REDUCES PROLIFERATIVE BEHAVIOUR IN GLIOBLASTOMA IN VITRO

Suzanne Birks 1, Melis Altinkaya 1, Ali Altinkaya 1, Geoff Pilkington 1

Abstract

INTRODUCTION: Boswellia derivatives possess pro-apoptotic potential towards glioblastoma in vitro and inhibit cell growth. We investigated whether combining boswellic acid with either clomipramine or temozolomide has synergistic therapeutic potential and whether this would reduce viability of the cancer stem cell (CSC) population. METHOD: Standard and CSC enriched human glioblastoma cell cultures were grown in normoxic and hypoxic conditions. 50 µg/ml of two boswellic acid isomers was administered for 24, 48 and 72 hours before treating with either clomipramine (50 µg/ml for 2 hours) or temozolomide (50 µg/ml for 4 hours). Viability, apoptosis and cell cycle analysis were carried out along with Western blots for cMYC and p21 and ELISAs for Stat3 activation and Bcl-2 levels. RESULTS: In CSC enriched cultures, viability was reduced to around ∼56% after addition of boswellic acid alone and ∼31% in combination with temozolomide but no reduction was observed after treatment with clomipramine and boswellic acid. Cell cycle arrest and apoptosis was induced in up to 95% of glioblastoma cells in vitro while levels of pStat3 were reduced from 83% to 26% in CSC rich cultures. Western blots and ELISAs showed a reduction in cMYC, p21and Bcl-2 levels. CONCLUSION: While addition of clomipramine surprisingly reduced activity of boswellic acid, boswellic acid in combination with temozolomide had an additive effect and reduced cell viability through induction of apoptosis and cell cycle arrest. We reduced viability of CSCs and this may be due to activation of the sustained DNA damage response. These novel findings demonstrate an important therapeutic strategy for exploitation.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

04. A COMBINED APPROACH WITH R132H MUTATION SPECIFIC IDH1 ANTIBODY FOLLOWED BY IDH1/IDH2 SEQUENCING IS IDEAL FOR THE DETECTION OF IDH1/2 MUTATIONS

Kathreena M Kurian 1, Charlene Crosby 1, Kirsten Hopkins 1, Maggie Williams 1

Abstract

INTRODUCTION: Recent research suggests there is a high rate of IDH1/2(isocitrate dehydrogenase) mutations in low grade gliomas and in the high grade gliomas deriving from them. We compared antibody and sequencing techniques for the detection of these mutations METHOD: In total 74 cases were examined (n= 13 WHO Grade 2 gliomas (diffuse astrocytomas, oligodendroglioma,, or oligoastrocytoma), n = 33 WHO Grade 3 gliomas (anaplastic astrocytomas, anaplastic oligoastrocytomas or anaplastic oligodendrogliomas) and n= 28 Grade 4 glioblastomas). 4 cases were excluded from antibody testing due to insufficient tumour, 19 cases had insufficient DNA for sequencing . For immunohistochemistry: sections were stained with anti-IDH1R132H antibody mIDH1R132H (clone H14). For sequencing: DNA was extracted from fresh, frozen tissue. Bi-directional Sanger sequencing of the R132 region of IDH and the R172 region of IDH2 was carried out on 3730 DNA analyser. RESULTS: In total 20/70 (29%) were positive for the R132H IDH1 mutation by antibody. Four of these cases 4/20 (20%) were not detected by sequencing as insufficient DNA was available for testing. In total 21/55 (38%) of the samples tested positive by IDH1 sequencing, and 0/55 (0%) tested positive for IDH2 sequencing. 5/21 (24%) showed rarer forms of IDH1 mutation (Arg/Cys, Arg/Gly) which are not detectable by the R132H IDH1 antibody.16/16 (100%) cases tested both by R132H antibody and sequencing showed concurrent results. CONCLUSION: The antibody for the common R132H IDH1 mutation detected more IDH1 mutations than sequencing. However sequencing demonstrates rare IDH1 mutations (Arg/Cys, Arg/Gly) which are not detectable by monoclonal antibody.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

05. INDUCTION OF THE ASTROCYTIC LINEAGE PATHWAY SELECTIVELY ENHANCES THE CHEMOTHERAPEUTIC POTENTIAL OF TEMOZOLOMIDE AND CLOMIPRAMINE IN STEM-CELL ENRICHED GLIOBLASTOMA

Laura Donovan 1, Suzanne Birks 1, Amy Eason 1, Viktoria Bosak 1, Geoffrey Pilkington 1

Abstract

INTRODUCTION: The treatment of glioblastomas remains a considerable therapeutic challenge due to their heterogenous nature and ability to evade current treatment regimens. The cancer stem cell (CSC) population is emerging as a critical target for successful chemotherapeutic treatments as this small subset of cells show an increased resistance to radio- and chemo- therapeutic agents. The induction of astrocytic differentiation plus sequential mitochondrial-mediated or DNA-alkylating therapy is an attractive method which may render such CSCs more susceptible to treatment. METHOD: From three preliminary differentiation approaches, dbcAMP and theophylline delivered the greatest effect on cell differentiation towards an astrocytic phenotype. Using an early passage paediatric glioblastoma, a highly established adult glioblastoma, and a non-neoplastic astrocytic cell culture under normoxic and hypoxic conditions we induced differentiation over 72 hours with dbcAMP (1mM) and theophylline (1mM) before analysing the expression of stem and differentiation markers by flow cytometry. Viability, apoptosis and proliferation assays and live cell imaging for cell migration were conducted downstream of Temozolomide and Clomipramine treatments. RESULTS: Differentiation along the astrocytic lineage not only resulted in arbitration and complexity of morphology but also decreased the percentage gated expression and fluorescence intensity of all stem cell antigens analysed. Significantly, this treated population of cells showed enhanced response to Clomipramine and Temozolomide in terms of apoptotic cell death. CONCLUSION: Reducing the stem-like phenotype within glioblastoma enhances the therapeutic potential of mitochondrially-mediated and DNA-alkylating agents.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

06. TUMOUR-ASSOCIATED MACROPHAGES PROMOTE INVASIVE POTENTIAL IN GLIOBLASTOMA

Suzanne Birks 1, Jonathon Holliday 1, Ivason Corbett 1, Geoffrey Pilkington 1

Abstract

INTRODUCTION: It has recently been suggested that tumour-associated macrophages (TAMs) may stimulate tumour growth and metastasis through secretion of matrix metalloproteinases, cathepsins and other factors. We firstly investigated whether glioblastoma cell cultures with high numbers of TAMs show enhanced invasive potential and secondly whether macrophages isolated from peripheral blood monocytes (PBMCs) are capable of stimulating invasion when co-cultured with glioblastoma. METHOD: CD68 expression was first determined in human glioblastoma cell cultures before carrying out Transwell™ invasion and BrdU cell proliferation assays. CD14+ PBMCs were isolated from bloods corresponding to each cell culture and differentiated into macrophages using CSF-1. Conditioned media was used to culture glioblastoma cells before determining effect on invasion and proliferation as described above. Conditioned media from glioblastoma cell cultures was also used to culture the PBMC macrophages and phenotypic changes assessed using flow cytometry for IL-10, IL-12, IL-23 and IFN-γ. RESULTS: Between 30 and 70% CD68 expression was detected in glioblastoma cell cultures. Cultures with high CD68 expression demonstrated enhanced invasive potential while glioblastoma cells cultured with conditioned media from macrophages isolated from PBMCs showed reduced proliferation and increased invasive potential. CONCLUSION: Emerging evidence suggests invasive glioblastoma cells display macrophage/microglia characteristics. Identifying the mechanisms behind macrophage phenotypic changes and effects on tumour behaviour may lead the way to possible therapeutic intervention.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

07. PSYCHOLOGICAL OUTCOMES OF PEOPLE DIAGNOSED WITH LOW GRADE TUMOURS: THE ROLE OF ILLNESS PERCEPTIONS

Melanie Keeling 1, Jacki Bambrough 1, Jane Simpson 1

Abstract

INTRODUCTION: Individuals diagnosed with a low-grade brain tumour experience a range of emotional, behavioural and psychosocial consequences. Biomedical factors associated with psychological distress are generally not amenable to psychological intervention. This study draws on the self regulation model to explore biopsychosocial factors associated with distress and examines the relationships between illness perceptions, coping and psychological outcomes. METHOD: Seventy-four people diagnosed with a low-grade brain tumour completed a tumour adapted version of the Illness Perceptions Questionnaire-Revised. Participants also completed self report measures of coping and psychological outcomes. Data were also collected on clinical and demographic variables previously associated with psychological distress in neuro-oncology populations. RESULTS: Hierarchical multiple regression analyses demonstrated that a biopsychosocial causal attribution was a significant independent predictor of anxiety and depression. Furthermore, illness coherence and illness identity trended towards significant predictors of anxiety and depression, respectively. Coping through venting and acceptance also contributed to the prediction of anxiety scores. No illness perceptions variables significantly predicted positive affect. CONCLUSION: Individuals' illness perceptions play a significant role in psychological distress experienced by people with low-grade brain tumours. Results suggest psychological interventions targeted at modifying illness perceptions may reduce psychological distress.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

10. CHARACTERISATION OF A HUMAN GLIOBLASTOMA AND THE INFLUENCE OF SERUM SUPPLEMENTATION

Samantha Higgins 1, Harjot Dogra 1, Geoffrey Pilkington 1

Abstract

INTRODUCTION: The most frequent approach to cell culture is to use a standard medium which is usually supplemented with foetal bovine serum (FBS) to provide essential growth factors required for sufficient growth in vitro. Since FBS is by definition not of human origin and may contain a different range of bio-active growth factors at different concentrations from human serum (HS), we studied the influence of FBS and HS supplemented DMEM and serum free media (SFM) on the growth kinetics, adhesion properties and expression of glioma-associated antigens. METHOD: The growth kinetics of the human glioblastoma cell line UPAB were evaluated, population doubling times and proliferation indices determined using growth curves and a BrdU assay; morphology of cells was monitored with phase contrast microscopy and immunoreactivity was evaluated by both immunocytochemistry and flow cytometry. RESULTS: The addition of HS resulted in changes in cellular morphology, adhesion, longer population doubling times and lower proliferation indices compared with FBS and SFM. Expression rates for PDGFαR, GD3, Musashi 1 and Olig 2 were highest in FBS conditions, Vimentin and Nestin, in HS. Interestingly, GFAP expression was lowest in HS and highest in SFM. CONCLUSION: When utilising human derived cell lines, supplementation with human serum may better reflect antigen expression and biological activity in situ, and the possibility may arise that culturing cells in FBS supplemented media may give rise to artificial results.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

11. IMAGING AND BIOPSY CHARACTERISTICS OF PRIMARY THALAMIC GIOMAS WITH CLINICAL OUTCOME

Kathreena M Kurian 1, Yuening Zhang 1, Marcus Bradley 1

Abstract

INTRODUCTION: Primary thalamic gliomas (excluding childhood pilocytic astrocytoma) are rare, with an incidence quoted as between 1% and 5% of all primary brain tumours. METHOD: We present the imaging and biopsy characteristics of 25 primary thalamic gliomas and compare with clinical outcome. RESULTS: 3/25 (12%) had non-diagnostic biopsies.10/22 (45%) were WHO Grade 4 Glioblastomas, 9/22 (41%) were Grade 3 (Anaplastic Astrocytomas n = 7, Anaplastic Oligoastrocytoma n = 2), 3/22 (14%) were Grade 2 (Diffuse Astrocytoma n = 2 and Central Neurocytoma n = 1) on neuropathology. 16/19 (84%) suspected high grade (Grade 3 or 4) on neuroradiology were WHO Grade 3 or 4 on neuropathology. 3/19 (16%) were suspected low grade (Grade 2) on neuroradiology, but were Grade 3 or 4 on neuropathology because they were contained within the thalamus with minimal enhancement, lack of necrosis or oedema. 3/3 (100%) suspected intermediate grade (Grade 3) on neuroradiology, but were Grade 2 gliomas on neuropathology (1 of these was a central neurocytoma and 1 showed piloid features). Neuropathology was a better predictor of clinical outcome than neuroradiology, with 9/10 (90%) WHO 4 gliomas and 8/9 (88%) Grade 3 gliomas on neuropathology, dead between 3-7 years after diagnosis. 3/3 (100%) Grade 2 gliomas on neuropathology were alive 3 years or more after diagnosis. 2/3 (67%) of the non-diagnostic cases were alive 3 years or more after biopsy. In 1/3 (33%) of the non-diagnostic cases the outcome was unknown. CONCLUSION: Diagnosis of thalamic glioma is challenging, requiring a multidisciplinary approach. Neuropathology is the best predictor of final clinical outcome.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

12. TAM RECEPTOR FAMILY EXPRESSION AND ACTIVATION OF THE AKT SIGNALLING PATHWAY IN GLIOMA CELLS

Claudia Schmidberger 1, Sassan Hafizi 1

Abstract

INTRODUCTION: The receptor tyrosine kinase Axl, a member of the TAM receptor family (Tyro3, Axl and Mer), is strongly expressed in some human brain tumours. Furthermore, we have reported that Gas6, the ligand for TAM receptors, stimulates brain tumour cell migration. The aim of the present study was to determine TAM receptor expression profile in human glioma cells, and the signalling pathways they mediate in response to Gas6. METHOD: The cultured adult human glioma cell line SNB-19 was used in this study. Expression of proteins and phosphoproteins was determined by western blot and immunofluorescence confocal microscopy. Tyro3 phosphorylation and Axl-Tyro3 interaction were analysed by coimmunoprecipitation (coIP) and western blot. RESULTS: Both Axl and Tyro3 were strongly expressed in SNB-19 cells. Addition of exogenous Gas6 to cells caused a rapid increase in tyrosine phosphorylation of both receptors, peaking at 30 min. Furthermore, Gas6 stimulated a two-fold increase in levels of phosphorylated Akt and GSK3, both elements of the Akt signalling pathway. Moreover, Axl and Tyro3 were both present in coIP pulldowns from cell lysates. CONCLUSION: Having previously observed Gas6 as a promoter of human glioma cell migration, we have now probed the signalling proteins involved. We have shown that SNB-19 cells express more than one TAM receptor, both of which are activated by Gas6. Furthermore, Gas6 stimulates the Akt intracellular signalling pathway, and potentially TAM receptor heterodimerisation, in these cells. This data furthers our knowledge of the TAM receptor family and involvement of its signalling pathways in glioma cell function.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

13. THE METHODOLOGY OF STUDIES OF INVASION MECHANISMS FOR HIGH-GRADE GLIOMAS

Imran Noorani 1, Stephen Price 1

Abstract

INTRODUCTION: Over the last few decades, there has been great interest in invasion mechanisms of glioblastoma multiforme (GBM), yet we are only beginning to understand this invasion process. We performed a descriptive study of the methodology in other studies on GBM invasion mechanisms to clarify our current level of understanding human GBM invasion. METHOD: A Medline search was performed using 26 key terms related to GBM invasion, including ‘glioblastoma’, ‘invasion’, ‘migration’, ‘angiogenesis’, ‘extracellular matrix’, ‘cytokine’, ‘chemokine’, and MeSH terms. Papers were excluded based on these criteria: articles discussing signalling pathways only; imaging studies; studies relating to genetic susceptibility to GBM; general cancer articles; articles on treatment of GBM that provide no insight into GBM invasion. RESULTS: There were 848 articles published between 1991 – 2011. 162 original articles were included (see exclusion criteria). 80 studies (49.4%) were solely in vitro, 11 (6.8%) used in vivo animal models only, 36 (22.2%) used human specimens alone, and the remainder of studies used a combination of at least two of these techniques. Overall, 58 (36.4%) of studies involved some work directly on human GBM tissue (from biopsy or autopsy). CONCLUSION: Our study highlights that the most of our knowledge of glioblastoma invasion is still from in vitro or animal in vivo studies. Though these studies have provided great insight over the last two decades, much more work is needed to confirm and extend our understanding of invasion of human glioblastomas in vivo, such as by use of biopsy or autopsy specimens or biopsy microdialysates.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

14. MITOCHONDRIAL GENETIC ABNORMALITIES ARE RELATED TO THE DEVELOPMENT AND PROGRESSION OF HUMAN GLIOMA

Aurore Dubocq 1, Tomasz Jaunky 1, Clémence Chatelain 1, Luke Evans 1, Tanja Gaißmaier 1, Geoffrey J Pilkington 1, Qian An 1

Abstract

INTRODUCTION: Mitochondria play critical roles in tumourigenesis and recently these organelles have been evaluated as novel therapeutic targets. Compared with other solid cancers, the biological function of mitochondria in human glioma is under-investigated. The present study aims to investigate the possible role of mitochondria in glioma development and progression. METHODS: Low-passage grade I-IV glioma cells and the normal human astrocytes, CC2565, were cultured in DMEM / 10% FCS. The whole mitochondrial genome was amplified by PCR using 9 pairs of overlapping primers. Mitochondrial DNA (mtDNA) mutations were analysed by sequencing the purified PCR products. Mitochondrial copy number change in tumour cells was investigated by SYBR Green qRT-PCR, in comparison to CC2565. RESULTS: mtDNA mutations were detected in all glioma cells with increased mutation rates in higher grade tumours. Mutations occurred in D-loop (the known ‘hot-spot’ region) and mitochondrial genes which encode complex I, III, IV proteins as well as ATP synthase 6 (ATP6). Furthermore, mitochondrial copy number decreased significantly in glioma cells on average by 83%, compared with CC2565 (p < 0.001). However there was no clear correlation between mtDNA copy number change and the grading of gliomas. CONCLUSION: The present study indicates that mtDNA mutations play an important role in the development and progression of human glioma. The clinical significance of mtDNA copy number change in glioma cells requires further investigation with expanded samples. Our future work will also focus on the possible roles of ROS in glioma progression, particularly with regard to glioma invasion.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

15. DRAMATHERAPY – A UNIQUE SURVIVORSHIP INTERVENTION

V Hurwitz 1, J Logan 1, R Bhangoo 1, K Ashkan 1, A Gullan 1, R Beaney 1, L Brazil 1, S Kokkinos 1, R Blake 1, A Singleton 1

Abstract

INTRODUCTION: Neuro-oncology patient outcomes have improved creating a shift towards a survivorship agenda. Dramatherapy has been recognised as a profession since the 1970's and produced positive outcomes in both palliative care and mental health. Due to commonalities between these patient groups and Neuro-oncology patients it was an area worth further exploration. Macmillan Cancer Support provided funding for this pilot. METHOD: The intervention consisted of six weekly sessions. There were four intakes comprised of up to five people per group. Each group followed a structured programme to explore their personal attributes, aims and goals. The group offered a safe space to share feelings surrounding diagnosis and treatment. Qualitative data collection tools were used including questionnaires, holistic Needs Assessment, anxiety squares and informal interviews. RESULTS: 50 % of patients experienced a decrease in their distress levels following the intervention. • “The group helped me a great deal to put my life back on track and move forward” Group 1 • “I am so grateful for being put forward for this intervention it has really helped me come to terms with my diagnosis and I've met some good friends in the group who I will keep in touch with”. Members of each group chose to stay in contact and provide peer support allying feelings of isolation. CONCLUSION: • Brain tumour patients are now surviving longer and Neuro-oncology services need to adapt what they offer patients to reflect and acknowledge this. Dramatherapy is a positive intervention for brain tumour patients.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

18. REBIOPSY RATE IN CNS LYMPHOMA

Abigail Shaw 1, Venkat Iyer 1, Kathreena M Kurian 1

Abstract

INTRODUCTION: CNS lymphoma can be difficult to diagnose and rebiopsy in these cases presents a significant cost to the NHS METHOD: We present the rebiopsy rate, steroid use and neuropathological diagnosis of CNS lymphoma over a 10 year period RESULTS: We reviewed 102 cases of suspected primary CNS lymphoma over a 10 year period, of which 96/102 (94%) were histologically diagnosed and 6/102 (6%) did not achieve a histological diagnosis. 81/96 (84%) were brain lymphoma and 15/96 (16%) were spinal lymphoma. 67/81 (82%) were primary brain lymphoma and 14/81 (17%) were secondary lymphomas to the brain. In total 78/96 (81%) of the diagnosed CNS lymphoma cases had received steroids prior to biopsy and 18/96 (19%) had not received steroids prior to biopsy. 9/102 (9%) required rebiopsy and 1/102 (1%) was not rebiopsied despite a non-diagnostic result. 5/9 (56%) rebiopsied cases did not achieve histological diagnosis despite rebiopsy (4 cases had 2 non-diagnostic biopsies, 1 had 3 non-diagnostic biopsies). Of these 2/5 (40%) had received steroids prior to biopsy, 3/5 (60%) had not received steroids. 4/9 (44%) cases were diagnostic on rebiopsy and of these 2/4 (50%) had steroids and 2/4 (50%) had no steroids prior to rebiopsy. None of the rebiopsied cases had been sent for PCR analysis of clonality. CONCLUSION: The rebiopsy rate in CNS lymphoma is high and represents a significant cost to the NHS. In our cohort prebiopsy steroid use does not affect diagnostic yield.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

21. DNA METHYLATION PROFILING OF LOW GRADE ASTROCYTOMAS USING THE ILLUMINA 450K BEADCHIP REVEALS CHANGES IN GENES INVOLVED IN BRAIN DEVELOPMENT AND DRUG RESISTANCE

Jennie N Jeyapalan 1, Isabel CF Morley 1, Alfred A Hill 1, Muhammad A Mumin 1, Ruth G Tatevossian 1, Ibrahim Qaddoumi 1, David W Ellison 1, Denise Sheer 1

Abstract

INTRODUCTION: Low grade astrocytomas are the most common central nervous system tumours in childhood. RAF gene fusions predominate in pilocytic astrocytomas (grade I), but are not found in diffuse astrocytomas (grade II) and little is currently known about genetic changes implicated in the development of these tumours. The aim of this study was to understand the role of epigenetics in the development of low grade astrocytomas. METHOD: DNA methylation was analysed using the new Illumina Human methylation 450K array, which covers 450,000 CpG sites across the genome. The tumour cohort consisted of 20 pilocytic astrocytomas, 10 diffuse astrocytomas and normal brain controls. The findings were validated in a larger tumour cohort consisting of grade I and grade II astrocytomas and compared with gene expression data. RESULTS: Unsupervised hierarchical clustering grouped the samples into tumour-types and controls. Pilocytic astrocytomas located in the cerebral cortex did not group with those in the cerebellum, but grouped instead with the diffuse astrocytomas, all of which were located in the cerebral cortex. This may reflect site-specific regulatory mechanisms in the putative cells of origin of these tumours. Changes associated with the different tumour types were identified in genes involved in brain development, cell cycle regulation and drug resistance. CONCLUSION: This study is the first to report genome-wide DNA methylation analysis on paediatric low grade astrocytomas. Our findings indicate that methylation changes in specific genomic regions, rather than the wide spread methylation seen in some other solid tumours, are involved in the aetiology of these tumours.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

22. A PROSPECTIVE EVALUATION OF BARRIERS TO PATIENT RECRUITMENT IN NEURO-ONCOLOGY TRIALS

A Frary 1, S Price 1, S Jefferies 1, F Harris 1, N Burnet 1, R Jena 1, C Watts 1

Abstract

INTRODUCTION: The latest survival trends for patients with CNS malignancies have remained largely static reflecting the lack of therapeutic options for patients with these cancers. There is a recognised need to recruit more patients into clinical trials. However, the barriers to patient participation remain poorly understood. We have established a rigorous process of MDT referral for all patients with CNS malignancies within the Anglian Cancer Network allowing enhanced opportunities for trial recruitment. We prospectively evaluated trial recruitment. METHOD: We prospectively evaluated trial recruitment over a 20 month period from June 2010 to February 2012. Patient recruitment into 8 NCRI portfolio studies was evaluated using screening logs. RESULTS: 17 trials were identified that were available for adult patients with CNS malignancy. 8 have been opened at Addenbrooke's. Reasons for trials not being opened at the centre included: • Additional Imaging Costs not being funded • Bureaucratic issues & need for research nurse support Of the 8 trials open to recruitment – 80 patients have been recruited in this time period. Recurrent themes preventing patient uptake into clinical trials include: • Proximity to a trial accredited centre • Evaluation of Eligibility Criteria prior to recruitment • Capped travel expense funding CONCLUSION: To improve patient recruitment: • Decentralisation from major centres: all cancer network neuro-oncology units need to be resourced to conduct research • Trial paperwork needs to be streamlined • Imaging costs not covered by the trial must be financed CLRN support for research nurse involvement in each neuro-oncology centre within the network is essential.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

24. LONG TERM SURVIVAL IN GLIOBLASTOMAS TREATED WITH CHEMORADIOTHERAPY: A SINGLE INSTITUTION STUDY

Brian Haylock 1, Sophie Leow-Dyke 1, Nitika Rathi 1, Helen Wong 1, Julie Dunn 1, Atik Baborie 1, Daniel Crooks 1, David Husband 1, Adi Shenoy 1, Andrew Brodbelt 1, Carol Walker 1

Abstract

INTRODUCTION: Following the success of the EORTC/NCIC Phase III trial, concomitant and adjuvant temozolomide and radiotherapy was adopted in the U.K. NICE guidelines for good performance glioblastoma patients, but its full impact in the routine clinic is still being assessed. In this study, we present 5-year survival data for patients treated in a single U.K. centre. METHOD: Clinical data for all glioblastoma patients treated using the Stupp protocol between June 2004 and September 2007 was collected (n = 110). MGMT methylation was determined by pyrosequencing. IDH1 mutation was assessed using immunohistochemistry. RESULTS: At study, 7 patients were alive (4 progression-free) with median follow-up 5 years (range 4.2-5.7). The median progression-free survival and overall survival (OS) for 108 newly-diagnosed glioblastomas was 9.7 and 12.4 months. Two, 3, 4 and five year survival was 19, 9, 6 and 3% respectively. No unifying clinical characteristics identified patients with survival >2 years, but 17/17 had methylated MGMT, whereas 2/16 were IDH1 immunopositive. Median OS for MGMTmethylated/IDH1+ve, MGMTmethylated/IDH1−ve and MGMTunmethylated/IDH1−ve cases was 19.7, 15.5 and 11.1 months respectively. In multivariate Cox Regression analysis, independent prognostic factors included surgery (P = 0.0002), gender (P = 0.017), steroid dose at start of radiotherapy (P = 0.005), MGMT methylation (P = 0.0001) and IDH1 immunopositivity (P = 0.015). Two secondary glioblastomas, both MGMTmethylated, one IDH1+ve, survived >4 years. CONCLUSION: The study demonstrates the efficacy of chemoradiotherapy in the routine clinic with 9% of patients surviving >3 years and shows the benefits of MGMT methylation and IDH1 mutation as prognostic biomarkers.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

26. TRANSFORMATION OF LOW-GRADE GLIOMA ASSESSED BY VOLUMETRIC 3T MRI

A Bahl 1, JJ Larsen 1, I Craven 1, P Metherall 1, FM McKevitt 1, CAJ Romanowski 1, N Hoggard 1, D A Jellinek 1

Abstract

INTRODUCTION: The optimal management of presumed low-grade glioma (LGG) remains controversial. Standard imaging may not detect biological change in tumour behaviour. We present 69 patients with an initial diagnosis of LGG who have undergone 234 3Tesla(3T) MRI scans over 36-months. METHOD: Semi-automated measurement of tumour volume was performed on each MRI. All patients who had undergone at least two sequential 3T MRI and had a diagnosis of LGG on biopsy and/or presumed radiologically were included. Case notes and imaging were reviewed retrospectively. Any patient with debulking surgery or radiotherapy prior to 3T MRI was excluded. Clinical data with associated volume change on serial MRI were analysed. RESULTS: 69 patients (41M,28F) with an average age of 41.7 years (16-72) were divided into two subgroups: 19 patients (Group A) underwent histological examination after a reported increase in annualised growth rate(84%), increase in enhancement(11%) or patient choice(5%). Although 90% of these patients presented with seizures, only two had exhibited an increase in seizure frequency. These patients proceeded to definitive treatment after the confirmed histological transformation. Tumour volume at presentation was 50.7mls. Mean duration from symptom onset to suspicion of transformation was 32.2 months. Mean duration of symptoms preceding diagnosis was 5.9 months. 50 patients (Group B) of whom 63% presented with seizure remain under surveillance. Volume at presentation was 24.4mls. Mean duration of symptoms preceding diagnosis was 42.9 months. CONCLUSION: Change in volume on 3TMRI prompted intervention prior to change in symptoms. Tumours with larger presentation volume may have higher risk of transformation.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

30. EVIDENCE FOR IMPROVED OUTCOME IN GERMINAL CENTRE VERSUS NON-GERMINAL CENTRE PHENOTYPE IN PRIMARY CNS LYMPHOMA

S Bell 1, E Murray 2, R Muirhead 2, A James 2, Z Hanzely 1, R Jackson 3, W Stewart 1

Abstract

INTRODUCTION: The majority of Primary CNS Lymphomas (PCNSL) are diffuse large B cell lymphomas (DLBCL). There is limited evidence suggesting a correlation between pathological subtypes and outcome in PCNSL. We identified all patients diagnosed with PCNSL over a 10 year period and reviewed clinicopathological features with respect to outcomes. METHOD: A consecutive cohort of patients diagnosed with PCNSL between 1st January 2001 and 31st December 2010 were identified from a search of the regional neuropathology and neuro-oncology databases. Full clinical information was retrieved and and the pathology reviewed with reference to pathological subtype. RESULTS: Fifty diagnoses of PCNSL were made with median age 59.5 years (range 42-81) and median overall survival 8 months. 44 had full pathology and clinical outcome data available. There was a wide variation in anatomical site of tumour presentation with just over half of cases presenting as either multifocal (n = 10), frontal (n = 9) or periventricular disease (n = 9). On reviewing the pathology 34 (73%) were found to be of non-germinal centre (NGC) phenotype and 10 germinal centre (GC) phenotype. Median OS was 16 months in the GC group versus 7 months in the NGC group. Though not statistically significant, the data suggests a trend towards more favourable outcome with GC immunophenotype. CONCLUSION: This review confirms a poor prognosis in patients diagnosed with PCNSL, though there may be an association between OS and pathology subtype. Specifically, similar to experience in systemic DLBCL, patients with a GC phenotype may have a more favourable outcome than those with a NGC phenotype.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

31. EXPERIENCE OF MOLECULAR NEUROPATHOLOGY REFERRALS IN THE WEST OF SCOTLAND REGIONAL SERVICE

A O'Brien 1, A Young 1, S Bell 1, Z Hanzely 1, W Stewart 1

Abstract

INTRODUCTION: With increasing demand for diagnostic molecular neuropathology, there is awareness that referral to laboratories with experience and access to a range of investigations may be appropriate. The West of Scotland neuropathology service offers these investigations and we have reviewed the experience in our practice. METHOD: All external cases received for molecular investigations between January 2007 and December 2011 were identified from a search of the neuropathology reporting archive. The reports were interrogated and information collated on the reasons for referral, referral diagnosis, type of molecular tests performed, the results and where appropriate a change in diagnosis. RESULTS: Over the period examined the service received 173 referrals from 15 centres: 10 from the UK, 4 from Europe and 1 outside Europe. In total these cases generated 301 investigations (133 1p-19q studies, 132 MGMT and 36 IDH-1) with the number of investigations increasing each year. There were a variety of tumours referred for assessment, the commonest being oligodendrogliomas (37%), followed by astrocytomas (26%) and glioblastomas (24%). This compares to the local laboratory practice where the overwhelming majority of cases are glioblastomas. Though all local oligodendroglial tumours are assessed they form a comparatively smaller proportion of the molecular workload. CONCLUSION: Referral for specialist molecular neuropathology investigations is increasingly common. However, from our experience there appears to be a difference between cases generating molecular investigations and local laboratory practice. Whilst the final diagnosis is rarely altered following molecular review the results undoubtedly influence patient management. Wider utilisation of molecular investigations may be merited.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

32. ‘NAVIGATION’ TO SUPPORT DECISION MAKING FOR PATIENTS WITH A HIGH GRADE BRAIN TUMOUR. A QUALITATIVE EVALUATION

SC Shepherd 1,2, D Cavers 3, LM Wallace 2, B Hacking 1,2, SE Scott 1,2, DJ Bowyer 1,2

Abstract

INTRODUCTION: Provision of tailored information and support for shared decision-making is essential in adjustment for brain tumour patients. This study compares patients receiving Navigation to a prior cohort of usual care patients with a high grade brain tumour. Navigation: Involved planning, summarising and recording. Prior to oncology consultations, Navigators helped patients create a key information and question list to use in the consultation. Subsequently, patients were given a recording of the consultation and sent a summary documenting key points discussed. Navigation was repeated for 3 oncology consultations. METHOD: Patients (n = 26) attending the Edinburgh Centre for Neuro-oncology being diagnosed with a glioma took part in serial interviews to explore illness experiences and support care needs. Subsequently, patients (n = 15) being diagnosed with a glioma were invited to participate in Navigation and serial evaluation interviews. Interviews were analysed using grounded theory. RESULTS: Comparing navigated to non-navigated patients: • Patients attributed knowledge and understanding of diagnosis and treatment to feeling better able to cope. Those less informed described higher levels of distress. • Navigated patients felt by preparing and using question lists in consultations a discussion of personalised key issues, broader than the prime focus of the consultation, resulted. • Recordings and summaries aided navigated patient's understanding and memory of consultation information. Summaries were shared with family and friends. • Navigated patients and clinicians welcomed the service. CONCLUSION: Navigated patients felt more informed and utilised navigation materials to aid memory, information gathering and understanding.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

33. GLIOBLASTOMA WITH OLIGODENDROGLIAL COMPONENT HAS THE SAME CLINICAL PHENOTYPE AS GLIOBLASTOMA

Ashraf Elmahdi 1, Amy J Frary 1, Dominic G O'Donovan 1, Stephen J Price 1

Abstract

INTRODUCTION: Glioblastomas are the commonest primary brain tumour and one of the most heterogeneous tumour types. The introduction of a glioblastoma with oligodendroglial component (GBM + O) in the latest WHO Classification of Tumours of the Central Nervous System was to help with this. There has been conflicting evidence as to whether this tumour conferred a better prognosis than classical glioblastoma (GBM). The aim of this study was to study the clinical phenotype of GBM + O tumours and compare it to classical GBM. METHOD: All patients with histological evidence of a glioblastoma between 1st January 2007 and 31st January 2011 were identified from the Neuropathology Database. Clinical and radiological details were obtained for all patients. The overall survival of patients treated with chemoradiotherapy were obtained and the GBM + O cohort compared to the classical GBM cohort. RESULTS: 396 patients with newly diagnosed glioblastomas were identified, 294 (74.2%) were classical GBM and 102 (25.6%) GBM + O. The two cohorts presented at a similar age (61.1 years GBM + O vs. 63.2 years GBM; P = 0.09) and were matched for sex and side of the tumour. GBM + O were more likely to be located in the frontal lobes (38.2% for GBM + O vs. 27.2% for GBM: P = 0.04). In the group that were treated with chemoradiotherapy the overall survival was similar (median survival GBM + O 361 days vs. 379 days GBM; Log Rank 0.61, P = 0.43). CONCLUSION: The presence of an oligodendroglial component does not confer any improvement in survival and has a similar clinical phenotype to classical GBMs.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

34. DUAL SYSTEMIC TUMOUR TARGETING WITH LIGAND-DIRECTED PHAGE AND GRP78 PROMOTER INDUCES REGRESSION OF GLIOBLASTOMA

Azadeh Kia 1, Justyna M Przystal 1, Nastasia Nianiaris 1, Nicholas D Mazarakis 1, Paul J Mintz 1, Amin Hajitou 1

Abstract

INTRODUCTION: The effects of current therapies against glioblastoma are limited and novel therapeutic approaches are required. Malignant brain tumours were one of the earliest targets for gene therapy and the failure of most clinical gene therapy seems to be mainly due to low tumour transduction. Integration of both ligand-directed tropism and transcriptional targeting into a single platform might facilitate clinical cancer gene therapy. The promoter of the glucose-regulated protein 78 (Grp78) and the ligand RGD that targets αvintegrins specifically expressed in tumours, offer excellent candidates. METHOD: The stress-inducible Grp78 shows specific over-activation in aggressive tumours and encodes for an anti-apoptotic protein involved in tumour survival and drug resistance of glioblastoma. However, the clinical benefit of Grp78 promoter in gene therapy has been hindered by lack of efficacy via the systemic non-invasive route. We have generated a dual tumour targeted phage chimera containing the RGD ligand and Grp78 promoter. RESULTS: The double-targeted phage provides persistent gene expression in glioblastoma cells invitro and in subcutaneous glioblastoma invivo after intravenous administration. Next, we found a systemic antitumor effect of Grp78-driven HSVtk therapeutic gene against recurrent glioblastoma invivo and uncovered a novel mechanism of Grp78 promoter activation by HSVtk and ganciclovir therapy. Finally, treatment of glioblastoma cells with temozolomide and cisplatin enhances HSVtk expression from Grp78 promoter. CONCLUSION: Our data prove the potential of Grp78 promoter against glioblastoma and indicate that combination of chemotherapeutic drugs and the double-targeted phage should be considered in cancer patients.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

36. EXPRESSION OF BEX1 IS DOWN-REGULATED BY HYPERMETHYLATION IN PAEDIATRIC EPENDYMOMA

K Karakoula 1, KP Phipps 1, W Harkness 1, R Hayward 1, D Thompson 1, TS Jacques 1, B Harding 1, J Darling 1, T Warr 1

Abstract

INTRODUCTION: Promoter hypermethylation is a common epigenetic mechanism implicated in the repression of gene transcription in cancer, including malignant brain tumours. We examined whether gene silencing due to aberrant methylation is the predominant mechanism driving downregulation of gene expression in paediatric ependymoma by determining global gene expression changes in ependymoma cells following treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC). METHOD: Using Affymetrix U133 plus2.0 arrays, we identified epigenetically silenced genes in 3 short-term ependymoma cell cultures. Genes showing a ≥3-fold increase in expression levels in 5-Aza-dC treated cells compared to untreated control cells were selected for further analysis. The methylation status of candidate genes was assessed by Combined Bisulfite Restriction Analysis (COBRA) and direct bisulphite sequencing in 45 ependymoma samples. Decreased expression levels of candidate genes were also confirmed by real-time quantitative PCR. RESULTS: Microarray analysis revealed 40 silenced genes reactivated by 5-Aza-dC treatment in ependymoma cultures including genes involved in Wnt signalling, apoptosis, cell differentiation and p53 pathway. CoBRA analysis and sequencing determined that the most frequently hypermethylated genes were BEX1 (28/45 cases, 62%), BAI2 (22/45, 49%), CCND2 (20/45, 44%) and CDKN2A (15/45, 33%). Bex1 (brain expressed X-linked 1) has tumour suppressor activity in adult GBM and is also involved in response to chemotherapy-induced apoptosis. CONCLUSION: DNA hypermethylation makes a significant contribution to the downregulation of gene expression in paediatric ependymoma. Inactivation of Bex1 has not previously been reported in ependymoma and further investigations of its role in chemoresistance may have important implications for their clinical management.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

37. INTRAOPERATIVE IMAGE-GUIDED SAMPLING TO IDENTIFY HYPOXIC REGIONS WITHIN GLIOBLASTOMAS

Sophie Leow-Dyke 1, Nitika Rathi 1, Brain Haylock 1, Daniel Crooks 1, Michael Jenkinson 1, Carol Walker 1, Andrew Brodbelt 1

Abstract

INTRODUCTION: The hypoxic microenvironment of glioblastomas (GBM) may contribute to tumour invasiveness and resistance to therapy. Previous studies have correlated cellular heterogeneity with hypoxia using image-guided samples. The aim of this pilot study was to determine the feasibility of image-guided surgery to sample regions of hypoxia within GBMs in our institute. METHOD: Tumour samples from four GBMs were obtained from hypointense (core), hyperintense periphery (edge) and intermediate regions in contrast-enhanced T1-MR using burr hole biopsy or ultrasound-guided excision during surgical resection. Analysis of histological features in H&E sections and immunohistochemical expression of hypoxia-inducible factor-1α (HIF-1α), carbonic anhydrase-9 (CAIX) and Ki67 was performed. Samples were cultivated in vitro. RESULTS: The extent of necrosis within core samples ranged from 10 to 100%. HIF-1α was expressed in tumour cells from the core but not in those from the periphery. Immunoreactivty of CAIX correlated to that of HIF-1α. Neurones and astrocytes and proliferating Ki67+ve cells were more common in peripheral samples. Samples from core and periphery were in general homogenous in their histological features and expression of hypoxia markers. 80% and 72% of core and edge image-guided samples showed histological features consistent with their respective location on imaging. Neurospheres were cultured from core and peripheral samples. CONCLUSION: Using intraoperative image-guided sampling we are able to target hypoxic and non-hypoxic regions and establish neurosphere cultures. This will facilitate molecular and cell biological studies to further investigate the role of hypoxia in therapy resistance.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

39. MERLIN DEFICIENT HUMAN TUMOURS SHOW LOSS OF CONTACT INHIBITION, ACTIVATION OF WNT/β-CATENIN SIGNALING LINKED TO THE PDGFR/SRC AND RAC/PAK PATHWAYS

Lu Zhou 1, Emanuela Ercolano 1, Sylwia Ammoun 1, M Caroline Schmid 1, Magdalena Barczyk 1, C Oliver Hanemann 1

Abstract

INTRODUCTION: Merlin loss causes benign tumours in the nervous system including schwannomas, meningiomas and ependymomas. We previously demonstrated that adherens junctions (AJs) are impaired in schwannoma cells due to ubiquitous, upregulated Rac activity. However, the mechanism by which loss of contact inhibition leads to proliferation remains obscure in merlin-deficient tumours METHOD: Following methods were used: Primary human cell cultures, lentiviral infections, co-immunopecipitation, Western Blotting, immunocytochemistry and proliferation assays RESULTS: We show that proliferative Wnt/β-catenin signaling is elevated as active β-catenin (dephosphorylated at serine 37 and threoine 41) localizes to the nucleus and the Wnt targets genes c-myc and cyclin D1 are upregulated in confluent human schwannoma cells. We demonstrate that Rac effector p21 activated kinase 2 (PAK2) is essential for the activation of Wnt/β-catenin signaling as depletion of PAK2 suppressed active β-catenin, c-myc, and cyclin D1. Most importantly, loss of the AJ complex and the increased proliferation in human schwannoma cells are connected by Src and PDGFR induced tyrosine 654 phosphorylation on β-catenin and associated with degradation of N-cadherin. We also demonstrate that active merlin maintains β-catenin and N-cadherin complex at the plasma membrane through direct regulation. Finally, we demonstrate that phosphorylation of tyrosine 654 is critical for the increased proliferation in human schwannoma cells, as overexpression of a Y654F mutant β-catenin reduces hyperproliferation of schwannoma cells. CONCLUSION: We suggest a model that these pathways are coordinated and relevant for proliferation in merlin deficient tumors.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

40. SILENCING OF CA2 + /CAM-STIMULATED PHOSPHODIESTERASE 1C (PDE1C) EXPRESSION REDUCES PROLIFERATION AND INVASION IN ADULT HIGH GRADE ASTROCYTOMA CELLS

FB Rowther 1, T Dawson 1, K Ashton 1, J Darling 1, T Warr 1

Abstract

INTRODUCTION: Cyclic nucleotides (cAMP & cGMP) are critical intracellular second messengers involved in the transduction of a diverse array of stimuli. Their catabolism is mediated by phosphodiesterase (PDEs) which also have a putative role in control of cell cycle and invasion. Previously we have demonstrated genomic amplification of PDE1C in >90% high grade astrocytoma (HGA) cultures using CGH analysis. This suggested that PDE1C could be involved in the development of HGA and have potential as a novel therapeutic target. METHOD: Real-time Q-PCR analysis was performed to determine PDE1C expression levels in 15 short-term HGA cultures with known PDE1C copy number status. PDE1C was knocked down in 6 cultures using siRNA alongside a non-targeting siRNA control. Functional studies were employed to compare the capacity of PDE1C non-expressing (PDE1C-) & expressing (PDE1C+) cells to proliferate, invade and migrate. Intracellular levels of cAMP & cGMP were determined in PDE1C- and PDE1C+ populations. The anti-proliferative activity of a chemical inhibitor to PDE1, Vinpocetine, was assessed in the HGA cultures. RESULTS: >90% depletion of PDE1C mRNA levels was achieved by siRNA. We detected up to 54% reduction in proliferation of PDE1C- cells. In addition, PDE1C- cells showed less migratory and invasive capacity (>45% reduction) compared to the PDE1C+ cells. These effects were also associated with accumulation of intracellular cAMP and cGMP levels. Treatment with Vinpocetine resulted in reduced proliferation of cells (EC50: 40-100 µM). CONCLUSION: The potential of PDE1C as a novel therapeutic target in HGA was confirmed. Whole genome expression analysis post-PDE1C depletion is underway.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

41. DYSREGULATED EXPRESSION OF GENES IMPLICATED IN CANCER METASTASIS TO THE BRAIN

Zaynah Maherally 1, Kathryn E Hatherell 1, Karolin Kroese 1, Sassan Hafizi 1, Geoffrey J Pilkington 1

Abstract

INTRODUCTION: 20-40% of cancers are reported to metastasise into the brain at some stage, thereby adversely affecting the prognosis. These brain secondaries are, due to their location within the brain and multiple nature, difficult to treat and bring with them a reduced quality of life. Here we report the expression level and possible role of a panel of genes which may be implicated in cancer cell metastasis to the brain. METHOD: Immunocytochemical (qualitative), Western blotting (semi-quantitative) and qRT-CR (quantitative) expression of COX2, HB-EGF, ST6GALNAC5, AEG-1, Tensin 3, Axl and CD15 were investigated in cell cultures derived from primary melanoma and lung cancer (HT-144 and NCI-H1299) respectively as well as melanoma and lung cancer brain metastases (UPMH and UPCM) respectively. RESULTS: Brain metastatic cell lines generally showed an upregulation of the genes compared with their primaries with the exception of ST6GALNAC5 which showed a decrease in its mRNA level from 1.4 fold change (HT-144) to 0.75 fold change (UPMH) detected by qRT-PCR. ICC results revealed even distribution of most gene products across the cell surface while some accumulated at the leading edge of the brain metastatic cells. CONCLUSION: Based upon their respective reported roles in cancer metastasis, this pilot study suggests that upregulation of these genes may play critical roles in brain metastasis. We are now looking for a link between level of expression and ability of cancer cells to cross a 3D in vitro ‘all human’ model of the blood-brain barrier using shRNA gene silencing.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

42. PREDICTORS OF STEM CELL-MEDIATED RADIATION RESISTANCE IN HUMAN GLIOBLASTOMAS – AN IMMUNOHISTOCHEMICAL STUDY

P Singh 1, S McQuaid 1, S Al-Rashid 1, K Prise 1, B Herron 1, E Healy 1, A Shoakazemi 1, M Donnelly 1, R McConnell 1, J Harney 1, D Conkey 1, E McGrath 1, LD Lunsford 1, D Kondziolka 1, A Niranjan 1, H Kano 1, R Hamilton 1, T Flannery 1

Abstract

INTRODUCTION: Glioblastomas (GBMs) are the most common and aggressive primary malignant brain tumour (median survival of 12-14 months). Although GBMs respond to radiotherapy, subsequent recurrence is inevitable, suggesting suboptimal killing of tumour cells – likely to be mediated by glioma stem-like cells. While a range of glioma stem cell markers have been reported their relative importance in radiation resistance remains to be determined. This study aims to quantify immunohistochemical stem cell expression in GBM specimens pre- and post-radiation treatment to determine which markers are most predictive of radiation-resistance. METHOD: In collaboration with the University of Pittsburgh, formalin-fixed paraffin-embedded (FFPE) GBM specimens obtained from cases subjected to prior multiple radiation treatments (both fractionated radiotherapy and stereotactic radiosurgery) were used for analysis. FFPE-sections were immunostained for a panel of stem cell markers (CD133, A2B5, Nestin, Notch). Fields of view were image-captured and quantified using a “percentage-positive field” and intensity scoring system by two independent reviewers. RESULTS: Nestin expression was highest in the majority of cases analysed with Notch expression less pronounced and CD133 and A2B5 expression minimal and absent respectively. Irradiated specimens indicated hallmarks of radiation effect including radiation necrosis and hyalinization of blood vessels (particularly in the re-irradiated Pittsburgh series). CONCLUSION: This study suggests that selection of Nestin/Notch-positive tumour cells from prospective tumour biopsies may be most relevant with which to study mechanisms of radiation resistance in GBMs.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

45. CHEMOKINE EXPRESSION IN PAEDIATRIC HIGH GRADE GLIOMA IS INFLUENCED BY THE MICROENVIRONMENT AND DIFFERS FROM ADULT HIGH GRADE GLIOMA

Yvonne Majani 1, Stuart Smith 1, Richard Grundy 1, Ruman Rahman 1

Abstract

INTRODUCTION: Chemokines are chemotactic cytokines involved in high grade glioma (HGG) angiogenesis, proliferation and invasion. We investigated whether chemokine biology differs between paediatric/adult HGG in 2D/3D culture and effects of hypoxia. METHOD: Primary tumours, KNS42 (paediatric HGG) and U87 (adult HGG) cells cultured as 2D monolayers in normoxic or hypoxic (1% oxygen) conditions or as tumour aggregates in a dynamic 3D-culture system. RNA extraction was performed, followed by real-time array-PCR to analyze 84 chemokine-related genes. RESULTS: CXCL1, CXCL2, CXCL3. CXCL8, CCL1, CCL2, CCL3, CCL4, CCL8, CCR6 were upregulated in KNS42 3D cultures compared to 2D cells, whilst CXCL11 was downregulated (p < 0.02). There was no difference in chemokine expression of KNS42 2D normoxia and hypoxia. CXCL8 was upregulated in U87 2D hypoxia compared to normoxia, whilst CCL2 was downregulated (p < 0.05). In normoxia, CXCL6, CXCL11, CCL2 were upregulated in KNS42 2D compared to U87 2D, whilst CX3CL1, CXCL3, CXCL8 were downregulated. (p < 0.05). In hypoxia, CXCL6, CXCL10, CXCL11, CCL2, CCL7, CXCR4, CCR10 were upregulated in KNS42 2D compared to U87 2D, whilst CXCL1, CXCL2, CXCL8, CXCL12, CX3CL1 were downregulated (p < 0.01). Many chemokines were downregulated when compared to a cohort of primary paediatric HGG. CONCLUSION: Chemokine expression in HGG is likely influenced by intrinsic cell-cell interactions and hypoxic gradients within the tumour. 3D brain tumour cultures may reflect more physiologically relevant chemokine expression patterns. Adult and paediatric HGG exhibit differential chemokine and receptor expression. Correlation between chemokine expression in primary tumours and 2D/3D cultures +/- hypoxia will be discussed.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

46. A LARGE LONG TERM RADIOLOGICAL STUDY OF MENINGIOMAS, INCLUDING THE FIRST LONG TERM REPORT OF ANTI-PROGESTERONE THERAPY WITH GESTRINONE

Sarvinder Saini 1, Gregory Hall 1, Charles Davis 1

Abstract

INTRODUCTION: We present a long term study of patients attending a meningioma clinic including a group treated with gestrinone. METHOD: Data regarding diagnosis, management and radiological or clinical progression was collected and analysed. 93 meningiomas were identified in 89 patients, median age of 64 (35-107); follow up of 45 months (3-226). Meningiomas were grouped according to management: total excision (TE) (n = 31), subtotal excision (SE) (n = 14), radiosurgery (n = 6) and conservative management (CM) (n = 42). 19 patients with 20 meningiomas, prescribed gestrinone, had a median age of 82 (56 -97), and follow up of 86 months (13-191). These were separated into groups: TE and gestrinone (n = 2), SE and Gestrinone (n = 7), radiosurgery and gestrinone (n = 1) and gestrinone only (n = 10). RESULTS: 16.1% of the TE group recurred after a median interval of 27 (3-56) months. 50% of the SE group progressed after 38 (7-137) months. 40% of the CM group progressed after a period of 31 (8-99) months. 16.7% in the radiosurgery group progressed after 57 months. No recurrence was seen in the SE and gestrinone group. Of the SE and gestrinone group, 42.9% progressed after 74 (8-107) months. 30% of the CM and gestrinone group progressed after 38 months (31-57). There was no progression in the radiosurgery and gestrinone group. CONCLUSION: Few patients with progression reported clinical symptoms, suggesting that conservative management may be an alternative for some patients. Due to the small sample size in this study, it is not possible to accurately evaluate the effectiveness of gestrinone in the management of meningioma.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

47. NEW MULTI-THERAPEUTIC TARGETS FOR THE TREATMENT OF HIGH GRADE ASTROCYTOMA

FB Rowther 1, T Lawson 1, K Ashton 1, N Potter 1, E Goessl 1, J Darling 1, T Warr 1

Abstract

INTRODUCTION: Targeting multiple pathways within tumour cells is often necessary to evade development of resistance and effect tumour cell death when more than one mechanisms are compromised. Due to its selective targeting of tumour cells, TNF-related apoptosis ligand (TRAIL) has generated extensive interest in cancer therapy. TRAIL acts primarily through the extrinsic apoptosis pathway while chemo- and radiotherapy uses the intrinsic mitochondrial pathway. On the other hand, DNMT (DNA methyltransferase) and histone deacetylases inhibitors (HDI) help restore deregulated gene expression in cancer. HDIs such as Panobinostat (PAN) and Valproic acid are being evaluated in clinical trials for glioma. Therefore, combining PAN with TRAIL could be an effective treatment strategy for high grade astrocytoma (HGA). METHOD: Real-time Q-PCR analysis was performed to determine expression of TRAIL death (TNFRSF10A,10B) and decoy receptors (10C,10D,11B) in 15 HGA short-term cultures. Drug synergy assays were then carried out in these cultures to assess the anti-proliferative effects of TRAIL and PAN alone and in combination, using a SRB cytotoxic assay. RESULTS: Significant levels of expression of TNFRSF10B, D and TNFRSF11B (10-1000 fold higher) compared to TNFRSF10A and C were detected. Drug synergy analysis revealed that all 15 HGA cultures were sensitive to TRAIL (EC50 3-50ng/mL) and PANO (EC50 6-120nM). However in combination with TRAIL (1-3ng/mL), the PAN EC50 was significantly reduced to 3-53nM. CONCLUSION: These results demonstrate that by combining a non-effective dose of TRAIL with PAN regimen significantly reduces the dose required to effect cell killing and may help circumvent the clinical side-effects of PAN.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

48. MAGNETIC RESONANCE IMAGING CHARACTERISTICS OF GLIOBLASTOMAS TREATED WITH TEMOZOLOMIDE AND RADIOTHERAPY

Andrew Brodbelt 1, Michael Jenkinson 1, Carol Walker 1, Sophie Leow-Dyke 1, Brian Haylock 1, Julie Dunn 1, Simone Wilkins 1, Trevor Smith 1

Abstract

INTRODUCTION: Previous studies of glioblastomas (GBM) have suggested that proximity to the lateral ventricles and subventricular zone influences tumour phenotype and patient survival and that molecular genetics may be associated with some MR characteristics and tumour location. METHOD: Neuroradiological features (21 parameters) in presurgery T1 and T2 MR scans for 63 GBM patients treated in a single centre with concurrent and adjuvant temozolomide and radiotherapy (June 2004-September 2007) were assessed by a panel of experienced neurosurgeons and a neuroradiologist. O6methylguanine-DNA-methyltransferase (MGMT) methylation was determined by pyrosequencing. IDH1 mutation was assessed using immunohistochemistry. Clinical outcome data was collected retrospectively. RESULTS: No relationships between any of the neuroradiological features and MGMT methylation or IDH1 immunopositivity were observed, including tumour location, necrosis and contrast-enhancement. Proximity of contrast-enhancing lesions to the lateral ventricle or involvement of the subventricular zone did not influence survival. Multifocal tumours seen in 5 cases were associated with short survival (P = 0.000). Extension of the contrast enhancing lesion into the cortex (P = 0.004), surgical resection (P = 0.02) and MGMT methylation (P = 0.000) indicated good prognosis. In Cox Regression multivariate analysis multifocal tumours, biopsy versus resection and MGMT methylation were significant independent prognostic factors. CONCLUSION: In this cohort treated with a uniform therapeutic protocol, neuroimaging features could not be utilized for non-invasive prediction of MGMT status and proximity to the lateral ventricles and subventricular zones were unrelated to outcome.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

49. ANTI-CANCER EFFECTS AND MECHANISM OF ACTIONS OF ASPIRIN ANALOGUES IN THE TREATMENT OF GLIOMA CANCER

V Petinou 1, ID Nicholl 1, J Singh 1, RW Lea 1, PJ Welsby 1

Abstract

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.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

50. TUMOUR-INITIATING CELLS IN HUMAN GLIOBLASTOMA ARE HETEROGENEOUS POPULATIONS WHICH RESIDE IN DISTINCT ENVIRONMENTAL NICHES

Inma Spiteri 1, Andrea Sottoriva 1, Nicholas Marko 1, Simon Tavare 1, Peter Collins 1, Stephen J Price 1, Colin Watts 1

Abstract

INTRODUCTION: We previously demonstrated that it is possible to objectively interrogate human glioblastoma (GBM) by using 5-aminolevulinic acid (5-ALA), an endogenous intermediate of the porphyrin biosynthesis pathway. Tumour tissue resection is performed on a fluorescence-guided basis allowing direct visualization of tumour tissue. This approach gave us the possibility to identify and characterize tumour compartments in GBM. METHOD: The tissue was collected in GBM debulking operations in accordance with local ethical guidelines. Patients were administered with 5-ALA 3 hours before surgery as oral dose of 20 mg/kg. Primary culture, culture propagation and immunofluorescences were performed according to the Cambridge protocol. Intracerebral transplantation of 3X105 cells into the right striatum of Nod/Scid mice was carried out according to the Home Office guidelines. Gene expression profiling was determined using standard array Illumina platform (HumanWG6_V3). Quality of RNA was evaluated by running tests. SNP analysis was performed using the AB7900 system. RESULTS: Our results demonstrate that: 1) cells residing in the tumour margin do not resemble the “cancer stem cell” phenotype but are tumorigenic in vivo, 2) fluorescence material is present in the sub-ventricular zone (SVZ) of 65% of our GBM (n = 65 patients), 3) it is possible to isolate tumour-initiating cells from SVZs. These cells are phenotypically similar to those isolated from the mass and possess the “cancer stem cell” molecular signature, 4) SNP analysis of SVZ tissues reveals distinct genetic aberrations. CONCLUSION: Fluorescence-guided tumour sampling identifies heterogeneous tumour-initiating cell populations residing in distinct environmental niches in GBM.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

51. IN VIVO IMAGING OF TRANSLOCATOR PROTEIN EXPRESSION IN GLIOMAS BY POSITRON EMISSION TOMOGRAPHY (PET)

ZJ Su 1, A Gerhard 1, R Hinz 1, F Roncaroli 1, D Coope 1, G Thompson 1, K Karabatsou 1, A Sofat 1, J Leggate 1, D du Plessis 1, F Turkheimer 1, A Jackson 1, A Brodbelt 1, MD Jenkinson 1, K Das 1, D Crooks 1, K Herholz 1

Abstract

INTRODUCTION: The translocator protein (TSPO) is a 18 kDa mitochondrial molecule associated with neuroinflammation and over-expressed in several brain diseases. 11C-(R)PK11195 is a specific ligand of TSPO for PET studies. We investigated the in vivo TSPO expression in high- and low-grade gliomas using 11C-(R)PK11195 PET, and applied it in guiding tumour biopsies. METHOD: 24 patients underwent volumetric MRI and dynamic 11C-(R)PK11195 PET scans. Reference tissue input function was obtained from the grey matter of cerebellum to determine the 11C-(R)PK11195 uptake in the tumours and the mirrored regions of interest (ROIs) in the contralateral hemispheres. Co-registered MR/PET images were used to guide tumour biopsies prior to surgical debulking, with high and/or low 11C-(R)PK11195 uptake foci defined as biopsy targets. Biopsy specimens were assessed for TSPO expression by immunohistochemical staining. RESULTS: In low-grade gliomas (14 cases), 11C-(R)PK11195 uptake within the tumours was lower than the contralateral ROIs (p = 0.001); sporadic high uptake foci were found in 8 tumours. In high-grade gliomas, 7 out of the 10 tumours showed higher uptake than the contralateral ROIs (p = 0.016), 4 of which displayed intensive 11C-(R)PK11195 signal despite only minor contrast enhancement on the MRI. There was low 11C-(R)PK11195 uptake and no contrast enhancement in 3 anaplastic asctrocytomas. Immunostains on tissue sections confirmed variable TSPO expression in neoplastic cells and activated microglia/macrophages. CONCLUSION: TSPO expression is decreased in low-grade gliomas and increased in most high-grade gliomas. TSPO expression within gliomas is inhomogeneous as detected by 11C-(R)PK11195 PET and confirmed by immunohistochemistry.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

52. NICE GUIDANCE ON THE USE OF CARMUSTINE WAFERS IN HIGH GRADE GLIOMAS: A NATIONAL STUDY ON VARIATION IN PRACTICE

Stephen J Price 1, Ian R Whittle 1, Keyoumars Ashkan 1, Paul Grundy 1, Garth Cruickshank 1

Abstract

INTRODUCTION: Multidisciplinary team (MDT) working in oncology aims to improve outcomes for patients with cancer. One role is to ensure the implementation of best practice and National Institute for Health and Clinical Excellence (NICE) guidance. In this study we have assessed the role of MDT in implementing the TA121 appraisal of the use of Carmustine wafers in high grade gliomas. METHOD: 296 patients with high-grade glioma suitable for maximal resection were recruited from 17 Neurosurgical Centres. The number of patients treated with Carmustine wafers and reasons for not using this were recorded. Complications at 48 hours post-operatively and at 6 weeks post-radiotherapy were recorded. RESULTS: 94/296 (32%) of suitable patients received Carmustine wafers. In 55% of cases Carmustine was not used due to either surgeon preference or a lack of an MDT decision. There was no increased complication rate with Carmustine use at either 48 hours post-surgery or at 6 weeks post radiotherapy. Use of Carmustine wafers did not decrease access to and use of chemoradiotherapy. CONCLUSION: One third of patients suitable for Carmustine wafers received them. Their use was neither associated with more frequent complications, nor decreased use of chemoradiotherapy. Implementation of NICE TA121 Guidance is extremely variable in different MDTs across the UK.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

53. EXPRESSION OF CRMP2 WITHIN GLIAL TUMOURS

VL Berry 1, DJE Elder 2, V Iyer 3, K Hopkins 4, NR Cohen 1, JM Tavaré 2

Abstract

INTRODUCTION: Collapsin-response mediator protein-2 (CRMP2) has roles in neuronal cell migration, development and mitotic spindle formation. It is phosphorylated on T514 by GSK-3β, associated with its deactivation. METHOD: Ten anonymised glial tumours from patients operated and treated within the Bristol NeuroOncology Group were subjected to western blot and immunohistochemical analysis with antibodies to total and T514 phosphorylated CRMP2, with appropriate ethical approval and consent. Samples from the densest part of the tumour and its periphery were examined for protein expression, whereas the diagnostic sample was examined immunohistochemically, usually involving dense tumour and its surround. RESULTS: CRMP2 was found in tumours as 64kDa and 58kDa forms, the former being processed to the 58kDa form by removal of the C-terminus. The 58kDa form was highly phosphorylated and was the predominant form found in lower grade glial tumours. By contrast the 64kDa form was most highly expressed in higher grade tumours. CRMP2 was expressed in neuronal cell bodies and in tumour cells that infiltrated CNS tissue, and within webs of glial tumour cytoplasm. Occasional mitotic figures in high and low grade glial tumours also show CRMP2 immunopositivity, implying a multifaceted function of this protein. CONCLUSION: Our data reveal changes in the processing of CRMP2 during gliomagenesis, and demonstrate the principally cytoplasmic location of CRMP2 in tumour cells in both the body of the tumour and in infiltrating tumour cells. The data suggest CRMP2 is activated during gliomagenesis but further work is required to understand the role of this pathway in the disease.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

55. AKT INDEPENDENT GLIOMA SUPPRESSION BY PTEN

Georgios Zilidis 1, Priyanka Tibarewal 1, Laura Spinelli 1, Nick R Leslie 1

Abstract

INTRODUCTION: Almost all glioblastomas display loss of at least one copy of the PTEN tumour suppressor gene and activation of the PI 3-kinase signalling pathway of which PTEN is part. Evidence for the importance of AKT and mTOR as downstream mediators of PI3K/PTEN driven tumorigenesis has motivated the development of drugs targeting AKT and mTOR that are now in clinical trials. METHOD: PTEN and PTEN mutants were re-expressed in PTEN null glioma cells using polyclonal unselected physiological level lentiviral methods. Functional cell based assays then used biochemical analyses including expression microarrays, and studies of cell proliferation, 3D morphology, 3D invasion and in vivo intracranial xenografts. RESULTS: We identify eleven PTEN mutations originating in human tumours, including seven from gliomas, that retain the ability to suppress cellular AKT phosphorylation, yet fail to suppress glioma cell invasion. We also show that a mutant selectively lacking protein phosphatase activity, PTEN Y138L, which is able to suppress AKT phosphorylation, fails to suppress xenograft tumour formation in vivo, in contrast to wild-type PTEN. Finally, we identify gene expression signatures that are regulated by PTEN either correlating with, or independently of, AKT and are highly significantly represented in human glioblastoma samples. CONCLUSION: In many gliomas, the regulation of invasion and identifiable gene expression changes, but not AKT, correlate with PTEN-mediated tumour suppression. This rationale explains why not all PTEN mutant gliomas are suitable for treatment with drugs targeting AKT, PDK1 or mTOR and the identified gene expression signatures should provide predictive biomarkers for these treatments.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

57. INTRA-OPERATIVE RECORDING OF DIRECT CORTICAL STIMULATION IN LOW-GRADE GLIOMAS USING NEURO-NAVIGATION FOR CROSS-VALIDATION OF PRE-OPERATIVE MOTOR AND LANGUAGE FUNCTIONAL MRI: FEASIBILITY AND INITIAL PILOT DATA

David John Coope 1, Konstantina Karabatsou 1, Sophie Green 1, Gemma Wall 1, Jacki Bambrough 1

Abstract

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. METHOD: 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. RESULTS: 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). CONCLUSION: 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.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

58. A CENTRAL ROLE FOR PTEN AND ERBB2 IN A NOVEL GENETICALLY ENGINEERED MOUSE MODEL OF MALIGNANT PERIPHERAL NERVE SHEATH TUMOUR

PM Brennan 1, J Baily 1, M Diaz 1, J Ironside 1, O Sansom 1, V Brunton 1, M Frame 1

Abstract

INTRODUCTION: Neurofibromatosis type 1 (NF1) is the most common nervous system genetic disease (1 in 3500). Malignant peripheral nerve sheath tumours (MPNST) are the most common malignancy in these patients. Even with optimal treatment 5 year survival is ∼50%. Existing pre-clinical models focus on the role of NF1 mutations and have failed to adequately elucidate disease pathogenesis and select effective chemotherapeutic agents that translate into the clinic. Analysis of human tissue and existing transgenic models has however implicated a number of other mutations in MPNST disease pathogenesis which might be therefore utilised for in vivo modelling. METHOD: We have developed a novel Cre-Lox model of MPNST where PTEN and ErbB2 transgenes are expressed specifically in Schwann cells under a 3-cyclic nucleotide 3-phosphodiesterase (CNP) promoter cre. At least nine mice in each genotype group were observed for clinical evidence of tumour development and the tumours subjected to histological and molecular analysis. RESULTS: Mice heterozygous for PTEN deletion and ErbB2 overexpression (ErbB2+/-PTEN+/-; CNP-Cre) develop single malignant tumours at an average of 198 days. In contrast, mice with both wild type alleles preserved in either gene develop occasional dermal lesions, but not malignant tumour, and survive an average 326 days (p = 0.0001). Histological and molecular analysis of the malignant tumours characterises them as MPNST. CONCLUSION: Our model provides significant progress in the understanding of the cell of origin and pathogenesis of MPNST. It follows that existing chemotherapies developed for tumours characterised by similar PTEN or ErbB2 mutations might bring benefit to patients with MPNST.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

59. CHARACTERISATION OF THE METABOLIC CHANGES AT THE DIFFUSION TENSOR DEFINED INVASIVE MARGINS OF GLIOBLASTOMA USING MAGNETIC RESONANCE SPECTROSCOPY

AMH Young 1, OM Thomas 1, LA Mohsen 1, AJ Frary 1, VC Lupson 1, MA McLean 1, SJ Price 1

Abstract

INTRODUCTION: Diffusion tensor imaging (DTI) can demonstrate accurate discrimination of the invasive margin of gliomas. Nevertheless, little is known about the metabolic activity of these regions. In this study, we have used magnetic resonance spectroscopy (MRS) to characterise the metabolic profiles associated with DTI-defined tumour invasion. METHOD: The study group comprised 40 patients with newly diagnosed, subsequently histologically confirmed glioblastomas (28 males, 12 females; mean age 55, range 28–75 years). Preoperatively, all patients underwent MR imaging at 3T (Siemens TrioTim) which included DTI and multivoxel, short echo time (TE = 35ms) MRS sequences and routine clinical imaging. The isotropic (p) and anisotropic (q) components of the DTI were calculated as previously described. Comparing these parameter maps, MRS voxels of peritumoral invasion were compared to normal-appearing adjacent and contralateral white matter. RESULTS: Seven metabolites demonstrated significant concentration differences in the invasive region compared to normal-appearing white matter. The invasive margin demonstrated a reduction in N-Acetylaspartate (NAA; p = 0.03), Myo-inositol (Ins; p = 0.007), Creatine (Cr; p = 0.008), Glutamate (Glu; p = 0.008) and the glutamate/glutamine cycle (Glx; p = 0.008). Comparisons between normal-appearing adjacent and contralateral white matter demonstrated no difference. CONCLUSION: We have observed a number of metabolic changes within the invasive tumour margin as defined by DTI imaging. These suggest underlying alterations in neuron and immune cell density as well as shifts in signalling metabolites fundamental to neuronal function. These changes were only seen in invasive regions and not in non-invasive regions.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

60. IDENTIFICATION OF GLIOMA SPECIFIC TARGETS FOR USE AS BIOMARKERS IN CLINICAL DIAGNOSIS

Mohit Arora 1, Lisa Shaw 1, Clare Lawrence 1, Jane Alder 1, Tim Dawson 1, Greg Hall 1

Abstract

INTRODUCTION: Gliomas are primary brain tumours with poor patient survival. Not many markers are known for brain tumours. Therefore, identification of new and reliable biomarkers can be of both diagnostic and clinical relevance. Aptamers are oligomer nucleic acids which can act as “antibodies” and help in detecting cellular target molecules. METHOD: Aptamers were identified which were specific for glioma cell lines by selecting them from publications and generating them de novo in the lab. U87, 1321N1,T98 and SVGP12 cell lines were used including bladder and breast tumour cell lines as control and then tested on primary tissue cultures for specificity. RESULTS: Aptamer binding to glial cell lines and primary tissue was identified using confocal microscopy and found to have significantly greater binding to gliomas compared to foetal astrocytes and control cell lines. These aptamer sequences with biotin attached, was isolated on streptavidin gel to identify the cell protein responsible for binding to glial cell. A few bands were formed on the gel electrophoresis, all of which are not know yet. These are being investigated further with mass spectrometry which might enable us to identify new, novel glioma cell markers. CONCLUSION: Gliomas remain an aggressive and incurable tumour and identification of new markers will enable better prognostication and diagnosis, opening up possible new channels of treatment. It is unknown how many different molecular structures are still to be found and aptamer technology is promising to forward our fight towards brain tumours.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

61. A NOVEL SEGMENTATION ALGORITHM FOR GLIOMAS MEASUREMENTS

Lavdie Rada 1, Ke Chen 1

Abstract

INTRODUCTION: Malignant brain tumours result in the highest loss of potential years of patients' life. The difficulties in segmentation of brain tumours include complexity of tumours surfaces, different tumour size, shape and location, contrast variability between tumours, presence of neighbouring regions with approximately the same density/values and possible presence of many small metastases. Accurate and reliable segmentation techniques are essential for tumour volume measurement and staging analysis. Many existing techniques struggle for low contrast segmentation due to difficulty in differentiating one feature from similar ones nearby. METHOD: In this talk we first present a new mathematical model (based on a dual level-set selective segmentation, DLSS, framework) where we combine the edge detection and geometric distance functions to differentiate two objects having similar or almost identical intensities. Apart from defining some geometrical markers near the legion region of interest, our method is fully automatic and minimal user involvement is required. RESULTS: The two level-sets of our DLSS model are a global level-set, which segments all boundaries, and a local level-set which finds the boundary of the object closest to the geometric constraints (markers). Test results will show that DLSS leads to better results in term of accuracy and robustness than previous work. CONCLUSION: The new model delivers similar results for easier problems to old models and equally reliable results for harder problems where old models fail.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

62. EXPRESSION OF C-JUN & SOX-2 IN HUMAN SCHWANNOMAS AND TRAUMATIC NEUROMAS

A Shivane 1, S Ammoun 1, DB Parkinson 1, CO Hanemann 1

Abstract

INTRODUCTION: Schwann cells myelinate axons of the peripheral nervous system. This process of myelination is regulated by various transcription factors. c-Jun and Sox-2 are negative regulators of myelination and control Schwann cell demyelination/ de-differentiation. Schwannoma cells are in a de-differentiated state with an ability to proliferate and avoid cell death. In vitro experiments in our lab have shown that c-Jun and Sox-2 are extensively co-regulated and that some of the inhibitory effects of c-Jun on myelination may be channelled through Sox-2. In this study, we look at the expression of these two negative regulators of myelination (c-Jun, Sox-2) in human schwannoma and traumatic neuroma. METHOD: 6 cases of schwannoma, 2 cases of traumatic neuroma and 1 control peripheral nerve were selected from the archives of the Neuropathology Department. The tissue was consented for use in research and had Ethics Committee approval. Immunohistochemistry was performed on 4 µm sections using antibodies to c-Jun and Sox-2. Western blot analysis was also carried out using anti-c-Jun antibodies. RESULTS: The schwannoma cells showed strong nuclear labelling with c-Jun and Sox-2. The c-Jun staining on traumatic neuroma reveals nuclear labelling. The Sox-2 staining was patchy and cytoplasmic in a neuroma. The control nerve did not stain with c-Jun and Sox-2 antibodies. Western blot of 3 schwannoma cases displayed increased c-Jun expression compared to normal tissues. CONCLUSION: Our data shows evidence of increased expression of c-Jun and Sox-2 in schwannomas compared to traumatic neuroma and normal peripheral nerve.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

65. IDENTIFYING EPIGENETIC DETERMINANTS OF METASTATIC BRAIN TUMOURS

Rajendra P Pangeni 1, Tracy J Warr 1, Mark R Morris 1

Abstract

INTRODUCTION: Brain metastases account for ∼ 40% of all secondary tumours. Primary tumours differ in their acquisition of metastatic traits and in many cases disseminated tumour cells must acquire further adaptive changes to enable intracranial growth. The underlying genomic alterations that provide tumours with the capacity to metastasis to the brain are not well defined. We have analysed the methylation status of candidate genes in metastatic brain tumours derived from primary tumour that commonly metastasise to the brain. Here we report our preliminary analysis. METHOD: The Cancer Genome Atlas (TCGA; http://cancergenome.nih.gov/) methylation array data has been interrogated to identify genes with differential promoter methylation in primary tumour types that most frequently metastasise to the brain. Promoter methylation was analysed in metastatic brain tumours (derived from these tumour types) by Combined Bisulphite and Restriction Analysis (CoBRA). RESULTS: We have screened 37 metastatic brain tumours, derived from lung, breast, melanoma or renal tumours, for 15 candidate genes. We have identified genes frequently methylated in brain metastases that are infrequently methylated in the originating primary tumours (CCDC8, HOXB13, COL14A). We have also found the absence of gene promoter methylation in brain metastases where in primary tumours these genes are frequently methylated (KLHL35, ANK3). CONCLUSION: These findings indicate that genes differentially regulated by promoter methylation may contribute to brain metastasis development. This is a preliminary analysis, we intend to extend our study to include further genes in a larger cohort of metastatic tumours.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

66. PRE-TREATMENT ASSESSMENT CLINIC FOR GLIOMA PATIENTS: AN UPDATE

Mairi Mackinnon 1, Aoife Williamson 1, Allan James 1, Anthony Chalmers 1

Abstract

INTRODUCTION: In patients with high grade glioma (HGG), the period between neurosurgery and starting oncological treatment is a particularly vulnerable time. With this in mind, a Pre-Treatment Assessment Clinic (PTAC) was implemented at the Beatson WoS Cancer Centre (September 2010) with the aim of conducting more detailed clinical and psychosocial assessments of patients in whom radical treatment (radiotherapy and/or chemotherapy) had been recommended at their first oncology appointment. The PTAC was considered a vital component in optimising recruitment to phase 1 studies in newly diagnosed patients requiring multiple assessments in a short period of time. METHOD: The PTAC was staffed by an experienced Specialist Nurse and Radiographer with a Consultant available if required. Performance status, steroid requirement, current medication, physical and psychosocial symptoms were reviewed and recorded as baseline measurements. The continued suitability of the intended treatment plan was assessed, potential toxicities discussed and informed consent obtained. Numbers of patients screened and entering studies was audited. RESULTS: Over 12 months, 52 patients with HGG considered suitable for radical treatment at first consultation attended the PTAC within 2 weeks. 30% of these were amended to a palliative schedule, usually because of deteriorating performance status. 30 patients were screened (10 for >1 study) and 15 recruited to phase 1 studies. CONCLUSION: The value of this service is apparent in ensuring that: • accurate assessments of patient needs and performance status are carried out • treatments slots are utilised appropriately • quality of life considered • clinical trials discussed • informed consent obtained.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

76. IS PERFORMANCE STATUS AN ACCURATE INDICATOR OF PATIENT-REPORTED QUALITY OF LIFE?

VL Beckett 1, AJ Joannides 1, R Brock 1, K McCarthy 1, SJ Price 1

Abstract

INTRODUCTION: Performance status (PS) is an important determinant for guiding surgical management of neuro-oncology patients. However its relationship to subjective, patient-reported quality of life (QoL) is largely unclear. We compared clinician-reported PS with patient-reported QoL in pre-operative neuro-oncology patients. METHOD: Pre-operative patients with primary brain tumours were included. Patients completed EORTC QLQ-C30 assessment and BN20 disease-specific module in clinic. The reviewing clinician determined WHO PS. For analysis, patients were stratified into two groups; PS 0-1 and PS 2-4 and statistically compared with each QLQ-30 domain using Mann-Whitney-U analysis. Average QLQ-30 scores were compared to EORTC reference ranges. RESULTS: 76 patients (37 female, 39 male) were recruited. Mean age at presentation was 59 years (95% CI: 57.7-61.2). Patients with PS 0-1 had statistically higher global QoL scores on QLQ-30 compared to the PS 2-4 group (mean scores 61.1 and 39.9 respectively, Z = 3.05, p = 0.002). Similarly, the PS 0-1 group displayed statistically higher levels of physical, role, cognitive and social functioning on QLQ-30 compared to PS 2-4 patients (p < 0.05). Emotional functioning was not correlated with PS. Mean QLQ-30 scores for cognitive and social functioning were significantly lower than reference ranges (p < 0.01), whilst global, role and emotional QoL scores were not significantly different. Main presenting symptoms were fatigue (88%), insomnia (62%) and pain (55%). CONCLUSION: There is a significant correlation between patient and clinician assessment of functionality. Patients with no/few symptoms reported a statistically higher global QoL compared to those with moderate/severely disabling symptoms. PS is therefore a valuable method of assessing pre-operative QoL.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

78. HIGH-DENSITY ARRAY COMPARATIVE GENOMIC HYBRIDISATION OF LOW-GRADE GLIOMAS IN ADULTS

A Singh 1, K Karakoula 1, T Dawson 1, K Ashton 1, J Darling 1, T Warr 1

Abstract

INTRODUCTION: Low-grade gliomas (LGG) (WHO Grade II) are a heterogeneous group of slow-growing, infiltrative tumours that have a tendency to transform into anaplastic astrocytoma (WHO Grade III), anaplastic oligodendroglioma (WHO Grade III) and glioblastoma multiforme (WHO Grade IV). We have used high-density array comparative genomic hybridisation (aCGH) to identify non-random copy number aberrations (CNAs) in these tumours. METHOD: Whole genome analysis using the Agilent 244K high-density oligonucleotide array was performed to determine copy number aberrations in 11 LGG comprising of 8 astrocytoma, 2 oligodendroglioma and 1 oligoastrocytoma. There were 3 fresh frozen biopsies and 8 formalin fixed, paraffin embedded samples. Data analysis was performed using BioDiscovery softwares, ImaGene 8.0 and Nexus 5.1. RESULTS: The frequency of CNAs per tumour ranged from 12-41 and the majority of these were small interstitial gains and losses. Aneuploidies were present in only 2 tumours (+7 and -15, respectively). 1p/19q deletion was observed in only 1 case, an oligodendroglioma. Losses were significantly more prevalent than gains. The most common deletions encompassed PRIM2 at 6p11 (10/11 cases), CDKN1C, CD81, DUSP8 and RASSF7 at 11p15 (7/11 cases) and PDCD6 at 5p15 (5/11 cases). These genes all negatively regulate cell proliferation through DNA replication, signal transduction, transcription and apoptosis. CONCLUSION: The genomic heterogeneity in this small cohort of LGG was striking. However, we have identified a number of genes with known or putative tumour suppressor activity which are deleted in >50% of samples. Importantly, the most frequently deleted gene, PRIM2, has not previously been associated with gliomagenesis.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

80. EPIGENETIC SILENCING OF LHX9 AND DIO1 EXPRESSION IN PAEDIATRIC MALIGNANT ASTROCYTOMA

H Kardooni 1, M Morris 1, F Rowther 1, J Darling 1, T Warr 1

Abstract

INTRODUCTION: Brain tumours are the most frequent type of solid tumours among children. Studies have shown that tumours can initiate not only by genomic alterations but also through epigenetic changes in tumour suppressor genes. Previously, we have used Affymetrix U133A arrays to determine global gene expression patterns in paediatric malignant astrocytoma (WHO Grades III and IV). Ten genes which were downregulated ≥3-fold in the malignant astrocytoma compared to normal brain (including TAZ, MTSS1, BHLHE41, KLHL35, DIO1, PTGDS, EPHX1, MCJ, LHX9 and PGRMC1) were selected to determine whether promoter hypermethylation is the predominant mechanism for their decreased expression. METHOD: The methylation status of the candidate genes was assessed using Combined Bisulfite Restriction Analysis (CoBRA) in 11 short-term cell cultures derived from paediatric malignant astrocytoma (9 glioblastoma multiforme (GBM) and 2 anaplastic astrocytoma). Gene expression was assessed by semi-quantitative reverse transcriptase (RT)-PCR. RESULTS: CoBRA results revealed LHX9 was methylated in all samples (11/11 cases), DIO1 was methylated in 45% (5/11 cases) and MCJ in 27% (3/11 cases). Methylation of KLHL35 and PTGDS was detected in 1 and 2 samples respectively. All 5 genes were methylated in the paediatric GBM cell culture, IN699. Re-expression was confirmed for all these genes following treatment,of IN699 with the demethylating agent 5-aza-2′deoxycytidine. The remaining 5 genes were found to be un-methylated (0/11 cases). CONCLUSION: LHX9 has been reported to be frequently methylated in paediatric astrocytoma. However, although DIO1 is commonly underexpressed in renal and thyroid tumours, it has not previously been associated with the development of astrocytoma.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

81. A BIDIRECTIONAL NON-HIERARCHICAL RELATIONSHIP EXISTS IN NG2-EXPRESSING CELLS IN GLIOBLASTOMA

Colin Watts 1

Abstract

INTRODUCTION: We have recently demonstrated that the GBM cells expressing the proteoglycan NG2 (GBM NG2+ cells) are more proliferative and tumourigenic compared to the GBM NG2- cells. However, the hierarchical relationship between the GBM NG2+ cells and GBM NG2- cells from the same tumour remains to be elucidated. METHOD: We used FACS sorting to separate the GBM NG2+ and GBM NG2- cells. Individual cells were followed for changes in NG2 expression, clonogenicity, proliferative activity and in vivo tumourigenicity. RESULTS: Single cell analysis showed that the sorted GBM NG2+ and GBM NG2- populations recapitulated the unsorted parent cell population in terms of NG2 expression. The GBM NG2- cells began to express NG2 de novo, which was associated with acquisition of the proliferative dominance. Intracerebral implants of the GBM NG2+ cells generated tumours containing GBM NG2+ and GBM NG2- cells. Intracerebral implants of the GBM NG2- cells were less tumourigenic and generated mixed tumours in a similar manner. Survival analysis indicated the proliferative dominance of the GBM NG2+ GBM cells was maintained in vivo and resulted in more aggressive tumour growth. CONCLUSION: The expression of NG2 does not appear to mark a unidirectional hierarchy. Expression of NG2 is associated with a more aggressively proliferative state in GBM in vivo. Our data create a platform to explore new model of the GBM growth and maintenance that is distinct from the rigid limitations of the CSC hypothesis. The data also has potential significance clinically in terms of prospective therapeutic modalities and recurrence.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

82. TRANSCRIPTIONAL SILENCING DISRUPTS TWO LEVELS OF ARGININE BIOSYNTHESIS IN GLIOBLASTOMA MULTIFORME (GBM): A NOVEL, TARGETED THERAPEUTIC STRATEGY FOR GBM

N Syed 1, F Roncaroli 1, K Janczar 1, P Singh 1, K O'Neil 1, C Lo Nigro 1, L Lattanzio 1, HM Coley 1, E Hatzimichael 1, J Bomalaski 1, P Szlosarek 1, T Crook 1

Abstract

INTRODUCTION: Changes in structure and expression of enzymes involved in key metabolic pathways (such as isocitrate dehydrogenase) have been identified in glial brain tumours. To seek novel candidate brain tumour suppressors, we have performed pharmacological methylation reversal. METHOD: Methylation reversal was performed in GBM cell lines using 5′ azacytidine (5′AZA), coupled with micro-array analysis. Gene expression was analysed using qPCR and Western blotting. Methylation was analysed using methylation specific PCR and quantitative pyrosequencing. RESULTS: Arginine deprivation, using ADI-PEG20 induces adaptive transcriptional up-regulation of ASS1 and ASL in glioblastoma multiforme which is blocked by neoplasia-specific CpG island methylation, causing arginine auxotrophy and cell death. ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to the cytotoxic effects of ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show that epigenetic changes in the two key genes of arginine biosynthesis occur in human cancer and confer sensitivity to therapeutic autophagy due to arginine deprivation. Moreover, we demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to autophagy. CONCLUSION: Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood:brain barrier.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

85. IDENTIFICATION OF POTENTIAL SIGNALLING PATHWAYS ASSOCIATED WITH MATRIX METALLOPROTEINASE-1 MEDIATED GLIOMA ANGIOGENESIS

Nicholas A Pullen 1, Monika Anand 1

Abstract

INTRODUCTION: Recent in vivo functional analysis of MMP-1 in a glioma model established that MMP-1 over expression enhanced formation of glioma tumours and resulted in a dramatic increase in CD31 expression suggesting a role in glioma angiogenesis. Silencing of MMP-1 resulted in a significant decrease in tumour formation. While MMP-1 is well known to be involved in tumour invasion, MMP-1 may also play a role in angiogenesis in gliomas. Proteomic analysis of selected angiogenesis markers revealed that TIMP-4, known for its ability to inhibit microvessel growth was upregulated when MMP-1 was inhibited. Observations were confirmed in vitro using a GBM cell induced 3-D angiogenesis model. The purpose of this study was to identify potential downstream signalling pathways associated with MMP-1 expression and glioma angiogenesis. METHOD: Human phospho-kinase arrays were utilized. Multiple RIPA lysates of glioma cell lines with or without modulation of MMP-1 were pooled and analyzed. RESULTS: Several phospho-signalling proteins were identified in preliminary analysis. In U251 MMP-1 over expressing cell lines Fyn, Pyk2, STAT1, TOR, and MEK1/2 were among several detected. Lck, B-catenin, CREB, FAK, STAT6 and Lyn were found to be inversely correlated with MMP-1 over-expression. Interestingly converse changes in B-catenin and CREB were detected (an increase in MMP-1 knockdown in T98G cells and an associated decrease in MMP-1 over-expression). CONCLUSION: The role of MMP-1 in human glioma pathology may not be limited to tumour cell invasion. These preliminary data represent an initial analysis of potential signalling pathways involved in MMP-1 mediation of angiogenesis in gliomas.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

86. IS THE STIMULATION OF ANGIOGENESIS ASSOCIATED WITH MMP-1 EXPRESSION MEDIATED VIA THE MMP-1/PAR1AXIS AND DOWNSTREAM CYTOKINES?

Suzanne Birks 1, Timothy Van Meter 1, Nicholas A Pullen 1, Monika Anand 1

Abstract

INTRODUCTION: There is compelling evidence that MMP-1 can mediate angiogenesis in certain cancers via its unique interactions with the protease activated receptor1. PAR1, a G-protein coupled receptor that carries its own ligand remains cryptic until unmasked by proteases, contributes to the induction of pro-angiogenic genes and in cytokine expression. For example SDF1 and CXCR4, which in turn promote endothelial progenitor cell proliferation. We have shown that MMP-1 over expression in vivo leads to a dramatic increase in recruitment of endothelial cells. METHOD: We utilized a 3-D glioma-angiogenesis model where MMP-1 expression was manipulated. PAR1 and MMP-1 expression and localisation in human glioma biopsies was evaluated. Genomic profiling studies were conducted on human tissue isolated from tumour core (viable hypoxic regions with angiogenic gene expression) and non-hypoxic periphery tissue from same patients. RESULTS: Endothelial cell projection number was increased in the presence of glioma cells forced to overexpress MMP-1 while this number decreased with silencing of MMP-1 in glioma lines known to express high amounts of MMP-1. Immunohistochemical stains for MMP-1 decorated both tumour cells and vascular endothelial cells of human GBM, whereas PAR1 staining was encountered only in the GBM vascular endothelial cells. PAR1 transcript expression was not elevated, but its downstream mediators were elevated in tumour core, consistent with the hypothesis that SDF1 and CXCR4 downstream of cleavage mediated PAR1 activation could lead to angiogenesis. CONCLUSION: These preliminary data support a role for the MMP-1/PAR1 axis and their downstream targets in the induction of angiogenesis in human glioma.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

87. GSK-3 AS A MULTIFUNCTIONAL TARGET FOR GLIOBLASTOMA TREATMENT; HITTING INVASION AND ANGIOGENESIS WITH A SINGLE DRUG

Shante Williams 1, Marjorie Boissinot 1, Lynette Steele 1, Shante Williams 1, E Antonio Chiocca 1, Sean Lawler 1

Abstract

INTRODUCTION: Previous studies have shown that inhibition of GSK-3 (glycogen synthase kinase-3) can specifically block glioblastoma cell migration in vitro. Here we have tested this approach using in vivo orthotopic models of glioblastoma, and show a significant increase in survival in animals treated with GSK-3 inhibitors of the indirubin family. The data suggests that this is due to a combination of anti-angiogenic and anti-invasive effects. METHODS: A panel of indirubin derivatives was examined in glioblastoma migration assays. A lead compound (bis-indirubin-acetoxime – BIA), was delivered by intraperitoneal injection in three separate orthotopic xenograft models (GBM9 glioblastoma stem cells, U87ΔEGFR, and X12 cells) and survival was monitored. Tumour sections were examined for the extent of invasion and blood vessel morphology. RESULTS: Three indirubin derivatives specifically and potently blocked glioblastoma cell migration in vitro in spheroid assays. These drugs blocked GSK-3 activity in treated cells as confirmed by Western blotting for β-catenin, and by a luciferase reporter assay. Treated animals survived twice as long as controls. Analysis of tumours revealed reduced invasion across the corpus callosum, and marked reduction in haemorrhage of treated tumours. Further examination showed a marked alteration in blood vessel morphology and density. Finally, we showed that indirubins block endothelial cell migration in vitro. CONCLUSION: Indirubins have anti-invasive effects on glioblastoma cells in vitro and in vivo. Treatment also has pronounced effects on tumour-associated endothelium. Thus we have identified an approach that combines anti-angiogenic and anti-invasive elements – a highly desirable combination of effects, and the subject of our ongoing investigations.

Neuro Oncol. 2012 Sep;14(Suppl 2):ii1–ii12.

88. MODELS OF BYSTANDER SIGNALLING BETWEEN PRIMARY ASTROCYTES AND GLIOMA CELLS: EFFECTS ON SURVIVAL AND MIGRATION/INVASION

Shahnaz T Al Rashid 1, Stephanie Mashal 1, Laura Taggart 1, Ellen Clarke 1, Thomas Flannery 1, Kevin M Prise 1

Abstract

INTRODUCTION: The goal of radiotherapy is to cause cell death in the targeted tumour field and minimize toxicity to the surrounding normal tissue. However, malignant gliomas are highly resistant to ionizing radiation (IR). Recent data suggest migration/invasion is up-regulated as a counter-productive effect of IR. In addition to direct IR effects, non-targeted radiation-induced bystander effects (RIBE), observed beyond the targeted field, can cause significant cell death of the tumour and surrounding normal tissues. Little is known of how RIBE modulates glioma migration/invasion. METHODS: RIBE of 10 astrocytes and glioma cells grown as single and co-cultures in 2D (monolayer) and 3D matrices was investigated, using media transferred from irradiated cultures and in half-exposed/shielded cultures. Nuclear 53BP1 foci were used as a surrogate DNA damage marker and transwell assays to quantitate migration/invasion. Two 3D models were developed to study gliospheres in: (i) collagen matrices with/without astrocytes and (ii) mouse brain slice cultures. RESULTS: RIBE signals from IR and non-IR glioma cultures both decreased astrocyte clonogenic survival. We observed increased DNA damage in astrocyte and glioma bystander cells in 2D and 3D cultures. Bystander cells had increased p38-MAPK, Connexin 43, and COX-2 signalling. Direct IR induced an increase and RIBE a decrease in transwell migration. Experiments of gliosphere migration/invasion in 3D models are underway. CONCLUSIONS: These results may have implications for tumour-normal tissue margin interactions. By comparing differential molecular factors involved in the RIBE, we aim to find targets to enhance glioma cell death and inhibit migration/invasion, while preserving normal tissue.

Articles from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Oxford University Press

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