THOR METHYLATION PROVIDES INSIGHT INTO THE TELOMERE MAINTENANCE LANDSCAPE OF MALIGNANT GLIOMASTabori, Uri; Castelo-Branco, Pedro; Lee, Donghyun; Gallo, Marco; Limpan, Tatiana; Mangerel, Joshua; Price, Aryeh; Remke, Marc; Zhang, Cindy; Heidari, Abolfazl; Wani, Khalida; Vanner, Robert; Zadeh, Gelareh; Karamchandani, Jason; Das, Sunit; Taylor, michael; Hawkins, Cynthia; Yan, Hai; Aldape, Kenneth; Dirks, Peter B.
doi: 10.1093/neuonc/nou208.29pmid: N/A
BACKGROUND: Gliomas are a deadly group of childhood and adult cancers associated with high relapse rate following current therapies. Limitless self-renewal is a hallmark of cancer recurrence and is controlled by telomerase activation and telomere maintenance. We have recently uncovered THOR (TERT Hypermethylated Oncological Region) which is paradoxically hypermethylated in gliomas with telomerase activation. In order to further explore the biological impact of THOR hypermethylation on self renewal and telomere maintenance of gliomas we undertook a stepwise approach. METHODS: RESULTS: Mapping of the human TERT promoter reveals that THOR spans 432 BP and comprises 52 CG sites. In contrast, the area where mutations in TERT promoter were uncovered is permanently hypomethylated. Promoter driven expression was analysed through luciferase assays and unveiled a repressive effect of THOR on the TERT promoter. Moreover, TERT mutations promoted hyperactivation of the reporter gene providing explanations for the methylation pattern observed in malignant gliomas. THOR methylation increases in gliomas as they transform from low to high grade and from primary tumor to established cell lines (p < 0.001). Analysis of allelic Tert expression reveals that THOR is initally methylated in the mutant allele and throughout tumor progression, the other allele eventually becomes methylated. This correlates with higher TERT expression. In contrast, tumors utilizing alternative lengthening of telomeres (ALT) lack THOR methylation and TERT mutations. Glioma stem cells (n = 32) are addicted to telomerase and have higher THOR methylation than the bulk tumor. Strikingly, glioblastomas which activate ALT lack this phenotype in their stem cells compartment. Whole exome sequencing reveals multiple ALT related mutations (TP53 and ATRX) which are present in the mature tumor cells subpopulation and absent in the TERT expressing stem cell subpopulation. THOR demethylation with Decitabine results in loss of telomerase activation only in tumor cells and not in normal and embryonic stem cells which lack THOR methylation. Combining telomerase inhibition with decitabine result in permanent loss of self renewal capacity of patient derived cell lines in vitro and lack of tumor formation in vivo. CONCLUSIONS: We offer a model and a novel classification of gliomas based on THOR hypermethylation and alterations in the telomere maintenance pathway. We also suggest that intratumoral heterogeneity in telomere maintenance might be the result of secondary hits in the non-stem cell compartment Finally, we propose that THOR demethylation can be a safe and viable option for exhausting the self renewal capacity of gliomas. SECONDARY CATEGORY: Pediatrics.
THE IMPACT OF OXYGEN TENSION ON CD8+ T CELL FUNCTION AND INTERACTION WITH GLIOMA CELLSWalker, Paul R.; de Silly, Romain Vuillefroy; Derouazi, Madiha
doi: 10.1093/neuonc/nou208.64pmid: N/A
BACKGROUND: (blind field) METHODS: We studied a clonal population of gp100 antigen-specific CD8+ T cells from pmel-1 mice and the assays were performed in vitro in a hypoxia work station. 5% O2 corresponds to the expected oxygen fraction in lymph nodes where most T cells are physiologically primed. Interactions with glioma were modelled using gp100-expressing GL261 glioma cells. RESULTS: In vitro activation of CD8+ T cells at physiologic O2 fraction generated cytotoxic T cells (CTLs) with higher effector functions (cytotoxicity and IFN-γ secretion) than at the 21% used in conventional culture. After activating CD8+ T cells at different O2 fractions, we then studied their function under hypoxia (1% O2), mimicking conditions after migration and infiltration of the glioma. Co-culture of CTLs and GL261 glioma cells at 1% O2 resulted in efficient tumor cell lysis, indicating that in the short term, hypoxia does not inhibit the killing capacities of CTLs, and that glioma cells are susceptible to immune-mediated elimination in this microenvironment. However, we also measured negative effects of hypoxia on anti-tumour immunity: CTL secretion of the immunosuppressive cytokine IL-10 and reduced T cell proliferation. CONCLUSIONS: Overall, these results validate the potential of T cell-based glioma therapy, even for glioma cells in hypoxic regions of the tumor. However, if sustained T cell responses are required for clinical efficacy, local amplification of anti-glioma effector cells may be limited by oxygen availability in the microenvironment, with the risk of accumulation of the immunosuppressive cytokine IL-10. SECONDARY CATEGORY: n/a.
INCIDENCE AND OUTCOMES IN GLIOMAS FROM BRAIN TUMOR REGISTRY OF JAPAN 2001-2004Narita, Yoshitaka; Shibui, Soichiro
doi: 10.1093/neuonc/nou206.9pmid: N/A
BACKGROUND: (blind field). METHODS: Data on primary or metastatic brain tumor cases that were newly diagnosed from 2001 to 2004 were collected from 2009 until the end of 2012. Approximately 330 neurosurgeons, medical doctors, and staff from 109 institutions collected 16,338 data from clinical records. These institutions included most universities, colleges, and hospitals with Japan Neurosurgical Society-authorized boards. These data were collected via the Internet Data and Information Center for Medical Research. Data, including the birthplace, age, tumor sites, diagnostic method, therapy (surgery, radiation, or chemotherapy), and the outcome of these treatments, according to the World Health Organization (WHO) 2007 brain tumor classification were collected. Survival data were analyzed using the Kaplan-Meier method. RESULTS: Overall survival (OS, months), progression free survival (PFS, months), 5-year OS, 5-year PFS of each grade of gliomas are as follows; pilocytic astrocytoma; NR (not reached), NR, 92.1%, 73.8%, diffuse astrocytoma: NR, 84.1, 75%, 57%, grade II oligodendroglial tumor: NR, NR, 90%, 74.6%, anaplastic astrocytoma: 38, 19, 41.1%, 28.7%, grade III anaplastic oligodendroglial tumor: NR, 71, 68.2%, 54%, glioblastoma: 15, 8.1, 10.1%, 9.2%. CONCLUSIONS: The 5-year OS of grade II and III gliomas improved compared to that in 1997-2000 but that of glioblastoma did not. Based on the data of BTRJ 2001-2004, trends and problems in treatment and outcomes of gliomas will be presented and discussed. SECONDARY CATEGORY: n/a.
OPTIMIZATION OF DEMETHYLATING THERAPY FOR IDH1 MUTANT GLIOMASRiggins, Gregory J.; Borodovsky, Alexandra
doi: 10.1093/neuonc/nou208.32pmid: N/A
BACKGROUND: Mutations in Isocitrate Dehydrogenase 1 (IDH1) are found in the majority of grade II and III gliomas and the subsequent progressive GBMs. The mutant IDH1 enzyme produces 2-hydroxyglutarate (2-HG), an “oncometabolite” that inhibits α-ketogluterate dependent histone and DNA demethylases resulting in characteristic hypermethylation of genomic DNA and suppression of cellular differentiation as a key mechanism of tumor progression and maintenance. METHODS: To accurately test therapeutics for IDH1 mutant gliomas, we developed a patient derived IDH1 mutant anaplastic astrocytoma model which produces 2-HG and exhibits characteristic DNA hypermethylation. This IDH1 mutant glioma grows only as a xenograft and not in cell culture. We measured tumor growth using a flank model in 5-azacytidine treated vs. untreated animals, passaging both sets of tumors from animal to animal in order to increase the exposure time to the drug. Tumor size was measured for treated vs. untreated mice and tumors were analyzed for gene expression, differentiation markers and promoter DNA methylation. Methylation status was by pyrosequencing analysis of bisulfate-converted genomic DNA at five genetic loci, with subsequent studies assessing global gene promoter methylation status. RESULTS: Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and significantly reduced tumor growth. Nearly complete tumor regression was observed by 14 weeks, resulting in a sustainable response and significant survival benefit. When therapy was discontinued, tumors did not show any signs of re-growth for at least an additional 8 weeks. Statistical analyses were performed using a student t-test for comparisons between the treatment groups. Following one cycle of 5-azacytidine treatment, GFAP expression was strongly increased and protein levels were maintained throughout subsequent cycles. 5-azacytidine treatment significantly reduced the fraction of Ki-67 positive cells in a time dependent manner and loss of DNMT1 expression in vivo following one treatment cycle. CONCLUSIONS: Using this genetically and molecularly accurate model we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine in vivo. The goal of this work is to optimize treatment efficacy and to precisely define the molecular alterations responsible for successful therapy. Long term administration of 5-azacytidine resulted in tumor regression acting through the expected mechanism of reversal of DNA methylation, and promoting a more normal cellular differentiation process. Optimization of demethylating therapy shows promise for IDH1 mutant gliomas. SECONDARY CATEGORY: Tumor Biology.
PREOPERATIVE DYNAMIC CONTRAST-ENHANCED MRI CORRELATES WITH MOLECULAR MARKERS OF HYPOXIA AND VASCULARITY IN SPECIFIC AREAS OF INTRATUMORAL MICROENVIRONMENT AND IS PREDICTIVE OF PATIENT OUTCOMEJensen, Randy; Salzman, Karen; Schabel, Mattias
doi: 10.1093/neuonc/nou206.47pmid: N/A
BACKGROUND: Measures of tumor vascularity and hypoxia have been correlated with glioma grade and outcome. Dynamic contrast-enhanced MRI (DCE-MRI) can noninvasively map multiple parameters of tumor hypoxia, blood flow, and vascularity. In this prospective observational cohort pilot study, our goal was to use preoperative imaging to correlate specific glioma samples with molecular markers of hypoxia, tumor vascularity, and tumor proliferation along with progression-free and overall patient survival. METHODS: Pharmacokinetic modeling methods were used to generate maps of tumor blood flow (F), extraction fraction (E), permeability-surface area product (PS), transfer constant (Ktrans), washout rate (kep), interstitial volume (v_e), blood volume (vb), capillary transit time (tc), and capillary heterogeneity (α(-1)) from preoperative DCE-MRI data in human glioma patients. Tissue samples were obtained using an MRI-guided intraoperative navigation system from areas of peritumoral edema (PE), active tumor (AT), hypoxic penumbra (HP), and necrotic core (NC). Each sample was evaluated for tumor vascularity, proliferation and expression of hypoxia-regulated molecules including hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF). Mean DCE-MRI parameter values corresponding to tissue sample sites and marker expression were compared with imaging results. RESULTS: Patient survival correlated with DCE parameters in two cases: α(-1) in AT and ve in areas of PE. Statistically significant correlations between DCE parameters and tissue markers were also observed. Specifically, 1) Vb correlated with VEGF (r = 0.604, p = 0.032) and HIF-1 (r = 0.747, p = 0.043) expression in AT; 2) tc correlated with HIF-1 in AT (r = 0.659, p = 0.017), HP (r = 0.761, p = 0.023), and PE (r = 0.697, p = 0.031) and with VEGF in AT (r = 0.588, p = 0.034) and HP (r = 1.00, p = 0.003); and 3) α(-1) correlated with VEGF in AT (r = 0.599, p = 0.034) and PE (r = 0.973, p < 0.0001). kep correlated with VEGF in PE (r = 0.761, p = 0.0065) and NC (r = 0.931, p = 0.007) and E correlated with HIF-1 expression in NC (r = 0.943, p = 0.017). In addition, MIB-1 index was found to be predictive of OS (p = 0.044) and is correlated with VEGF expression in HP (r = 0.7933, p = 0.0071) and PE (r = 0.4546). Increased microvascular density (MVD) found to correlate with worse patient outcome (p = 0.026). With these results we describe a new clinical trial that we will implement to use these biomarkers in guiding therapeutic interventions for patients with malignant gliomas. CONCLUSIONS: Our findings suggest that it may be possible to use DCE-MRI to make noninvasive preoperative predictions of areas of tumor with increased hypoxia and proliferation. This has the potential to both make unprecedented prognostic decisions and to guide therapies to specific tumor areas SECONDARY CATEGORY: Imaging.
STEM- AND PROGENITOR-LIKE CELL CONTRIBUTION TO MALIGNANT ASTROCYTOMA HETEROGENEITYPetritsch, Claudia; Lerner, Robin; Lewis, Kate; Andor, Noemi; Ozawa, Tomoko; Yoshida, Yasuyuki; McMahon, Martin; Nicolaides, Theo; David James, C.
doi: 10.1093/neuonc/nou206.76pmid: N/A
BACKGROUND: Stem-like tumor propagating cells (TPCs) isolated from malignant astrocytoma (MA) are reportedly therapy-resistant. In contrast, progenitor-like TPCs with loss of asymmetric cell division (Cancer Cell. 20:328-40; 2011) are sensitive to temozolomide (Cancer Cell. 18:669-82; 2010). In a pediatric MA subset, an activating BRAFT1799A mutation leads to expression of hyperactivated BRafV600E kinase, and occurs with deletion of the cell cycle inhibitor CDKN2A (Cancer Res. 70:512-9; 2010). BRAFV600E-targeted therapies, including PLX4720, retard the growth of intracranial BRAFV600E MA xenografts (Clin. Cancer Res. 17:7595-604; 2011). Studies suggest that TPCs defined by current methods are not a homogeneous population and may contain subpopulations that respond differently to treatment. The divergent effects of targeted therapies on heterogeneous TPCs have yet to be explored. METHODS: Stem-like (CD133+) and progenitor-like (NG2+) cells were isolated from human MA cell lines and murine neurosphere cells with BRafV600E expression and Ink4a/Arf (CDKN2A) deletion. CD133+ and NG2+ cells labeled with proliferation markers were investigated for heterogeneity in cell cycle dynamics and division modes. Viability assays were used to determine PLX4720 IC50s, and RT-PCR was used to analyze cell cycle and polarity regulator gene expression. Pharmacological modulators of genes altered in NG2+ and CD133+ cells were tested alongside PLX4720 for anti-tumor efficacy in intracranial BRAFV600E MA xenografts. RESULTS: In murine CD133+ cells with BRafV600E expression and CDKN2A deletion, a low proliferation frequency and high rate of asymmetric division associate with reduced sensitivity to PLX4720-induced G1 arrest. Human CD133+ but not isogenic NG2+ cells have differential mRNA expression of G2-specific cell cycle regulators. Concurrently, CD133+ cells exhibit enhanced sensitivity to blockade of polo-like-kinase1 (PLK1), a cell cycle and polarity regulator. Results from ongoing analyses of the distinct proliferative capacities, cell cycle dynamics, cell division mode and responses to targeted BRafV600E and PLK1 inhibition of CD133+ and NG2+ MA cells will be presented. CONCLUSIONS: Our studies revealed that murine malignant astrocytoma TPCs are phenotypically and functionally heterogeneous. We further discovered a TPC-subpopulation specific therapy response, where CD133+ cells from malignant astrocytoma cell lines are more resistant to BRafV600E targeted inhibition but more sensitive to G2/M checkpoint kinase inhibition than NG2+ cells. This provides a potential mechanistic basis for the differential responses of stem and progenitor-like TPCs to treatment. Understanding why current treatment strategies preferentially target only certain cell-types will allow intelligent design of chemotherapeutic strategies with greater anti-tumor effects. SECONDARY CATEGORY: n/a.
HIGHLY SELECTIVE INTRA-ARTERIAL CHEMOTHERAPY FOR THE TREATMENT OF PROGRESSIVE DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPG)Cohen, Kenneth J.; Jones, Amber; Raabe, Eric; Pearl, Monica
doi: 10.1093/neuonc/nou208.24pmid: N/A
BACKGROUND: DIPG remains a uniformly fatal diagnosis with most children dying of disease between six months and two years from initial diagnosis. Radiation therapy remains the only intervention that alters the natural history of the disease, but is not curative. Given the frequent imaging feature of a non-contrast enhancing mass at diagnosis, the extent of systemic delivery of chemotherapeutics to the pons is uncertain. Attempts have been made to circumvent this issue by various methods including convention-enhanced delivery utilizing catheters inserted into the pons, blood-brain barrier disruption, and intra-arterial (IA) administration of chemotherapy into the vertebral circulation. This pilot study explores the safety, tolerability, and initial efficacy of highly selective intra-arterial administration of melphalan via the basilar artery at the time of clinical progression following initial radiotherapy for DIPG. METHODS: Subjects with a radiologic or biopsy proven diagnosis of DIPG are eligible at the time of symptom and/or radiographic progression in the months following initial irradiation. Consensus of the multidisciplinary Pediatric Neuro-Oncology conference faculty is required. Subjects with a documented hypercoagulable disorder or vasculopathy are not eligible, nor are subjects who have been re-irradiated. Two cycles of IA chemotherapy are planned 4 weeks apart. Under general anesthesia, the vertebral artery (right or left) is catheterized with a 4-French guide catheter. Following a baseline angiogram, a 1.5-French microcatheter is advanced into the mid basilar artery. After angiographic confirmation of positioning, IA delivery of 4 or 6 mg of melphalan is delivered over 30 minutes. Following completion of the procedure, subjects are observed overnight in the Pediatric Intensive Care prior to discharge the following day. Subjects are monitored for toxicity between, and following, IA administrations. RESULTS: To date, 2 children, of a planned cohort of 5 children, have been treated with IA melphalan. Both children tolerated the actual IA administrations without any procedural associated toxicity including the absence of stroke, hemorrhage, or other technical complications. The first child received one of two planned IA administrations but clinically deteriorated shortly before the second IA infusion was planned. The second child received both planned IA administrations and is in early follow-up. CONCLUSIONS: Highly selective IA administration of melphalan to the basilar artery is feasible and acutely tolerated. Updated experience and follow-up with enrolled patients will be presented. SECONDARY CATEGORY: Clinical Neuro-Oncology.
PROFICIENCY PERFORMANCE BENCHMARKS FOR REMOVAL OF SIMULATED BRAIN TUMORS USING NEUROTOUCHDel Maestro, Rolando; Al Zhrani, Gmaan; Azarnoush, Hamed; Winkler-Schwartz, Alexander; Alotaibi, Fahad; Lajoie, Susanne P.
doi: 10.1093/neuonc/nou209.25pmid: N/A
BACKGROUND: Objective assessment of neurosurgical technical skills involved in the resection of cerebral tumors in operative environments is complex. Educators emphasize the need to develop and use objective and meaningful assessment tools that are reliable and valid for assessing trainees' progress in acquiring surgical skills. Novel technologies, such as virtual-reality simulation, have the potential to play important roles in the training of neurosurgeons. The purpose of this study was to develop benchmarks for a newly proposed set of objective measures (metrics) of neurosurgical technical skills performance during simulated brain tumor resection using a new virtual reality simulator (NeuroTouch). METHODS: A total of 31 participants were recruited including 16 ‘experts’ (neurosurgery staff) and 15 neurosurgery residents (‘novices’, 7 junior and 8 senior). Each participant performed 18 simulated brain tumor resections utilizing the NeuroTouch platform. The metrics for assessing surgical performance were computed using the NeuroTouch simulator and consisted of 1) Safety metrics including, volume of surrounding normal tissue removed, maximum force applied and sum of forces utilized during tumor resection 2) Quality of Operation metric which involved the percentage of tumor removed and 3) Efficiency metrics including duration for task completion, instrument path lengths and pedal activation frequency. RESULTS: The results demonstrated that ‘expert’ neurosurgeons (neurosurgery staff) resected less surrounding simulated normal brain tissue and less tumor tissue then residents. This data is consistent with the concept that ‘experts’ focused more on safety of the surgical procedure compared to novices. By analyzing experts' neurosurgical technical skills performance on these different metrics we were able to establish benchmarks for goal proficiency-based training of neurosurgery residents. CONCLUSIONS: Examining ‘expert’ neurosurgical performance in simulated settings such as NeuroTouch provides researchers with novel metrics for assessment of technical skills and development of proficiency based benchmarks. Identification of expert proficiency can led to improvements in resident training and assessment SECONDARY CATEGORY: n/a.