bluuebunny/crossref_metadata_2025 huggingface dataset:
EXTH-53. TUMOR TREATING FIELDS LEADS TO CHANGES IN MEMBRANE PERMEABILITY AND INCREASED PENETRATION BY ANTI-GLIOMA DRUGS
Authors: Chang Edwin , Patel Chirag , Beinat Corinne , Young Caroline , Flores Thomas , Zeng Yitian , Joubert Lydia , Arami Hamed , Natarajan Arutselvan , Sinclair Robert , Gambhir Sanjiv
Published: November 2019

BACKGROUND

Efforts are underway to uncover novel, unorthodox therapies against glioblastoma (GBM). Tumor treating fields (TTFields) disrupt mitotic spindle formation and stymie proliferation in tumor cells. Combining TTFields with Withaferin A, synergistically inhibited proliferation in GBM. We wished to uncover the relevant mechanism.

METHODS

Human and murine GBM cells (GBM2, GBM39, U87-MG, KR158B) were isolated from primary tumors. Cells were engineered to stably express firefly luciferase (fLuc). Proliferation was assessed by bioluminescence imaging (using D-Luciferin as a substrate for fLuc) or cell counting. Dextran-FITC binding and scanning electron microscopy (SEM) studies were used to probe effects on cellular membranes.

RESULTS

TTFields (200 kHz, 4 V/cm) significantly inhibited growth of cells (n=3/time point, p≤0.02; 2-way ANOVA, no TTFields vs. TTFields). Combination of Withaferin A with TTFields significantly inhibited growth of glioma cells synergistically beyond that of Withaferin A or TTFields alone (p< 0.01, at least n=3 experiments). Bioluminescence imaging suggested alterations in membrane configuration when cancer cells were exposed to TTFields. This was validated with observations of greater fluorescence of membrane-associating Dextran-FITC to U87-MG cells that were subjected to TTFields (p< 0.01, 2-way ANOVA, TTFields vs. no TTFields, n=3 experiments). SEM revealed significantly greater and larger number of holes on the membrane surface of TTFields-exposed U87-MG cancer cells (53.5±19.1 holes per field of view and mean size=240.6±91.7 nm2) compared to unexposed cells (23.9±11.0 holes per field of view and mean size=129.8±31.9 nm2, p< 0.005: TTFields exposed vs. non-exposed, n=3 samples, univariate Mann-Whitney test).

CONCLUSION

The findings suggest a novel combinatorial approach to treat GBM in a manner that is better than single treatment (drug or TTFields) alone and that is synergistic. Synergy is achieved through TTFields inducing increased permeability on membranes thus conferring greater accessibility to drug. Such a strategy is thus a promising candidate for future clinical translation in glioblastoma.
EXTH-56. MEDULLOBLASTOMAS RESPOND TO CDK4/6 INHIBITION WITH NANOPARTICLE-DELIVERED PALBOCICLIB WITH ALTERED S-PHASE PROGRESSION
Authors: Dismuke Taylor , Lim Chaemin , Gershon Timothy
Published: November 2019

CDK4/6 inhibition is a promising therapy for medulloblastoma, one of the most common malignant pediatric brain tumors. To improve pharmacokinetics, we developed a polyoxazoline nanoparticle-encapsulated formulation of the FDA-approved CDK4/6 inhibitor palbociclib (POx-palbo). We then administered POx-palbo to transgenic medulloblastoma-prone GFAP-Cre/SmoM2 mice, to determine the efficacy and mechanisms of action and resistance. We found that POx-palbo slowed tumor progression, but consistently failed to be curative. Further analysis showed that while CDK4/6 inhibition acutely blocked G1 cells from re-entering the cell cycle, this effect wore off within hours of drug administration. However, flow cytometric analysis of EdU uptake hours after palbociclib demonstrated aberrant S-phase with reduced rate of DNA synthesis. This POx-palbociclib-induced alteration of S-phase progression seems to remain true at later time points even when we observed that palbociclib G1/S inhibition began to decrease. Based on these data, we propose that the combinational therapy of POx-palbociclib and S-phase targeting agents will further improve treatment. Faulty tumor cell cycle progression in the presence of Pox-palbociclib may give increased window to target the S-phase for irreversible cell-cycle exit.
EXTH-63. LIPOSOMAL DELIVERY OF FERRITIN HEAVY CHAIN (FTH1) siRNA RESULTS IN INCREASED RADIATION SENSITIVITY IN PATIENT DERIVED GLIOMA INITIATING CELLS
Authors: Ravi Vagisha , Abraham Thomas , Madhankumar Achuthamangalam , Slagle-Webb Becky , Pandya Bhavyata , Connor James
Published: November 2019

Glioblastoma (GBM) is the most lethal and incurable brain cancer. Despite maximum treatment, high recurrence rates result in median survival of less than 2 years. A subset of treatment resistant cells within GBM known as glioma initiating cells (GICs) are implicated in recurrence and exhibit ferritin overexpression. High expression of ferritin is known to be associated with poor overall survival in GBM patients. Our previous finding showed that downregulation of the ferritin heavy chain subunit (FTH1) enhanced treatment sensitivity in astrocytoma cells. Recently, functional importance of FTH1 in GICs was demonstrated by shRNA silencing leading to ablation of tumorigenic ability. We therefore posited that disrupting iron metabolism through FTH1 knockdown would radiosensitize GICs. Thus we developed multivalent cationic liposomes that enabled delivery of FTH1 siRNA to patient derived GICs isolated from pro-neural (PN) and mesenchymal (Mes) GBM. In both subtypes, FTH1 loss led to increased apoptosis and reduced cell viability. When exposed to radiation, PN GICs were unable to repair and recover from DNA damage, leading to a further decrease in cell viability and a complete loss of reproductive clonogenic potential unlike their more radioresistant Mes counterparts. Moreover, expression of stem cell marker Nestin was markedly reduced in PN but not Mes GICs. Thus, FTH1 loss is detrimental to both GIC subtypes, it seems to be critical for maintaining stemness and radiation resistance of PN GICs. These findings further support the viability of FTH1 as a therapeutic target in GBM.
EXTH-70. ENHANCEMENT OF ANTITUMOR ACTIVITY BY USING 5-ALA–MEDIATED SONODYNAMIC THERAPY TO INDUCE APOPTOSIS AND NECROSIS IN A MOUSE GLIOMA MODEL
Authors: Suehiro Satoshi , Ohnishi Takanori , Inoue Akihiro , Yamashita Daisuke , Nishikawa Masahiro , Ohue Shiro , Kunieda Takeharu
Published: November 2019

OBJECTIVE

High invasiveness of malignant gliomas frequently causes local tumor recurrence. To control such recurrence, novel therapies targeted toward infiltrating glioma cells are required. Here, we examined cytotoxic effects of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas both in vitro and in vivo.

METHODS

In vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated.

RESULTS

The 5-ALA-SDT inhibited cell growth and changed cell morphology. Flow cytometric analysis indicated that 5-ALA-SDT induced apoptotic cell death. The 5-ALA-SDT generated higher ROS than in the control group, and inhibition of ROS generation completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact.

CONCLUSIONS

The 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.
EXTH-71. IND-ENABLING CHARACTERIZATION OF ONC206 AS THE NEXT BITOPIC DRD2 ANTAGONIST FOR NEURO-ONCOLOGY
Authors: Prabhu Varun , Rahman Kawakibi Abed , Madhukar Neel , Garnett Mathew , McDermott Ultan , Benes Cyril , Wechsler-Reya Robert , Elemento Olivier , Stogniew Martin , Oster Wolfgang , DeMorrow Sharon , Allen Joshua
Published: November 2019

ONC201 is the first clinical bitopic antagonist of DRD2, an oncogenic receptor in brain and neuroendocrine tumors. ONC206, a derivative of ONC201 that shares the imipridone core structure, is also a bitopic DRD2 antagonist that exhibits enhanced non-competitive effects, high specificity, nanomolar potency, and disruption of DRD2 homodimers. Broad nanomolar efficacy of ONC206 (GI50 < 78-889nM, CellTitre-Glo, 72h) was observed in >1,000 GDSC cancer cell lines. Maximal ONC206 sensitivity was observed in pheochromocytoma, high-grade gliomas, neuroblastoma, medulloblastoma, sarcoma and cholangiocarcinoma cell lines exhibiting a DRD2+/DRD5- RNA expression signature. An exposure time of 48h at nanomolar concentrations was sufficient for maximal inhibition of tumor cell viability. ONC206 reduced the viability of normal human fibroblasts at higher doses (GI50 > 5µM), suggesting a wide therapeutic window. Antitumor efficacy without body weight loss was observed with 50 mg/kg weekly oral ONC206 in a dopamine-secreting HuCCT1 cholangiocarcinoma subcutaneous xenograft model. Biodistribution studies in Sprague-Dawley rats revealed a ~12 µM plasma Cmax with a systemic terminal half-life of ~6 hours upon a single oral dose of 50 mg/kg. Additionally, 5–10 fold higher ONC206 concentrations were observed in adrenal gland, bile duct, brain and bone marrow relative to plasma. Nanomolar concentrations were also observed in the CSF above DRD2 antagonism thresholds, unlike ONC201. GLP toxicology studies with weekly oral ONC206 in Sprague-Dawley rats and beagle dogs revealed no dose-limiting toxicities. Mild and reversible decreased body weight and/or body weight gain with no effects on food consumption were observed at the highest evaluated dose in both species. The highest non-severely toxic dose (HNSTD) was ≥ 16.7 mg/kg in dogs and ≥ 50 mg/kg in rats that exceeds efficacious doses in preclinical models. Using standard allometric scaling, a 90 mg starting dose of ONC206 was selected for the first-in-human clinical trial in biomarker-defined adult recurrent CNS tumors.
GENE-03. SPECIFIC SIGNATURES OF microRNA IN CEREBROSPINAL FLUID OF PATIENTS WITH PRIMARY BRAIN TUMORS AND METASTASES
Authors: Jancalek Radim , Smrcka Martin , Kopkova Alena , Sana Jiri , Vecera Marek , Oppelt Jan , Trachtova Karolina , Fadrus Pavel , Slaby Ondrej
Published: November 2019

Cerebrospinal fluid (CSF) baths extracellular environment of the central nervous system, and thus, it is ideal source of tumor diagnostic biomarkers like microRNAs (miRNAs), short non-coding RNAs involved in the pathogenesis of many cancers. As dysregulated levels of brain tumor specific miRNAs have been already observed in CSF, analysis of CSF miRNAs in brain tumor patients might help to develop new diagnostic platform. Next-Generation sequencing (NGS) was performed for analysis of small RNAs in 89 CSF samples taken from 32 glioblastomas (GBM), 14 low-grade gliomas (LGG), 11 meningiomas, 13 brain metastases and 19 non-tumor donors. Subsequently, according to NGS results levels of 10 miRNAs were measured in independent set of CSF samples (41 GBM, 44 meningiomas, 12 brain metastases and 20 non-tumor donors) using TaqMan Advanced miRNA Assays. NGS analysis revealed 22, 12 and 35 CSF miRNAs with significantly different levels in GBM, meningiomas, and brain metastases (adj.p < 0.0005, adj.p < 0.01, and adj.p < 0.005) respectively, in comparison with non-tumor CSF samples. Subsequent validation of selected CSF miRNAs has confirmed different levels of 7 miRNAs in GBM, 2 in meningiomas, and 2 in brain metastases compared to non-tumors. Panel of miR-30e-5p and miR-140-5p was able to distinguish brain metastases with 65% sensitivity and 100% specificity compared to non-tumor samples (AUC = 0.8167); panel of miR-21-3p and miR-196-5p classified metastatic patients with 78% sensitivity and 92 % specificity in comparison to GBM (AUC = 0.90854) and with 75% sensitivity and 83% specificity compared to meningiomas (AUC = 0.84848). We have observed that CSFs from patients with various primary brain tumors and metastases are characterized by specific miRNA signatures. This work was supported by the Ministry of Health, Czech Republic grant nr. NV18-03-00398 and the Ministry of Education, Youth and Sports, Czech Republic under the project CEITEC 2020 (LQ1601).
GENE-10. A SIMPLE AND EFFICIENT METHOD FOR GENERATING MULTI-ALLELIC INSERTION-DELETION MUTATIONS AND PRECISE DELETIONS IN PRIMARY HUMAN CELL CULTURES
Authors: Hoellerbauer Pia , Kufeld Megan , Arora Sonali , Wu Hua-Jun , Feldman Heather , Paddison Patrick
Published: November 2019

CRISPR-Cas9-based technologies have revolutionized experimental manipulation of the human genome by enabling generation of site-specific genetic alterations, such as insertion-deletion (indel) mutations. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary human cells. Here, we present a simple and effective method for fast and penetrant induction of multi-allelic indels and near-precise deletions (ranging from ~50bp to >50kbp) in primary human neural stem/progenitor cell (NPC) and brain tumor-derived stem-like cell cultures using the CRISPR-Cas9 system. We report that mono-, bi-, or multi-allelic (depending on ploidy) indel efficiencies of >90% can be routinely achieved within 3 days, without the need for pre-engineering cells to express Cas9 or isolating clones. As a result, this method enables the quick generation of a series of knockouts at different genomic loci, which we illustrate by successively targeting TP53, CDKN2A, PTEN, and NF1 in human NPC populations, where ³95% loss of protein expression is observed. RNA-seq analysis of these NPC knockout pools confirmed gene expression changes including p53 transcriptional and Rb-axis targets, as well as NF1-dependent repression of major histocompatibility complex class II gene expression. Given its simplicity, this method is readily adaptable to other primary mammalian cell types.
GENE-32. SYNTHETIC LETHAL INTERACTIONS WITH IDH1R132H IN GLIOMA STEM-LIKE CELLS
Authors: Ventosa Maria , Kadiyala Padma , Carney Stephen , Castro Maria , Lowenstein Pedro
Published: November 2019

Gliomas are the most frequently diagnosed human primary brain tumors. Mutations in Isocitrate Dehydrogenase (IDH) 1 occur in the vast majority of low grade gliomas and secondary high grade glioblastomas. A single amino acid missense mutation in IDH1 at arginine 132 (R132H) is an early event in tumor development. IDH1R132H leads to the production of the oncometabolite 2-R-2-hydroxyglutarate. However the exact roles played by IDH1R132H in the development and malignant transformation of the tumors remain unclear. Further studies are required to determine optimal therapeutic strategies to target the IDH1 mutated subsets of gliomas. New generation high-throughput genetic perturbation technologies make it possible to systematically identify the genes and pathways required for the survival and proliferation of mammalian cells. Herein, we present preliminary results from a CRISPR-dCas9 derived activation to drive the transcriptional activation of more than 23,000 coding genes in both wild type and mutant IDH1 patient-derived pediatric glioma cells. Based on an average of three viability screens per cell type, we analyzed the sgRNA library representation in the IDH1 mutated and non-mutated glioma cultures after the genome wide activation. We identified 1553 candidate genes that upon gain of function trigger the death of glioma cells harboring the IDH1 mutation. The analysis of these results further pinpoints the activation of the Cyclin E1 (CCNE1), the BCL2 Antagonist/Killer 1 (BAK1) and the Homeobox B13 (HOXB13) as the most significant synthetic lethal targets in IDH1R132H glioma cells. Interestingly, results from RNAseq showed a decreased expression of these genes in IDH1 mutated compared to non-mutated glioma cells. Thus, this viability screening aims to elucidate genes that interact with IDH1R123H and play a role in tumor cell fitness. The functional analysis of these candidate genes will allow us to uncover their contribution to the progression of IDH1 mutated gliomas.
GENE-48. ANALYSIS OF HUMAN BRAIN TISSUE TRANSCRIPTOME TO IDENTIFY RISK GENES AND ALTERED MOLECULAR PATHWAYS IN GLIOMA-RELATED SEIZURES
Authors: Feyissa Anteneh , Carrano Anna , Wang Xue , Allen Mariet , Ertekin-Taner Nilufer , Rosenfeld Steven , Guerrero-Cazares Hugo , Quinones-Hinojosa Alfredo
Published: November 2019

OBJECTIVE

Our goal is to identify new pathways perturbed in and which may lead to GRS.

METHODS

We collected brain transcriptome levels from healthy controls (n=5), patients with GRS (n=9), glioma without seizures (non-GRS, n=8), and idiopathic temporal lobe epilepsy (iTLE, n=7) using Illumina-based poly-A capture high-throughput RNA sequencing approach. Differential gene expression analysis was conducted to ascertain expression differences between the four groups. We identified molecular pathways enriched for differentially expressed genes (DEG) using Ingenuity Pathway Analysis. DEGs were defined as those with log2 fold change ≥ 2 and q-value < 0.01 when compared to GRS.

RESULTS

Principal component analysis revealed that diagnostic grouping was the main determinant of the transcriptome variance. We identified 212 DEGs in the GRS vs. non-GRS analysis. Among these, 108 genes were up-regulated and 104 genes were down-regulated. Among the genes that were down‐regulated, there was significant enrichment for those involved in acute phase response signaling and immune cell trafficking (e.g. H19, BMP5, CXCL8). Compared to iTLE samples, there were 1015 DEGs in the GRS samples. Among these 877 genes were up-regulated and 138 genes were down-regulated. There was significant enrichment for genes involved in cell-to-cell signaling among those that were either uniquely up‐regulated (e.g. NRK, RGS10, SLC4A4, and SELPLG) or down‐regulated in the GRS samples (e.g. SLC9A3 –Na+/H+ exchanger; SLC5A5-sodium-glucose co-transporter family). Further, two of the most down-regulated genes in samples with GRS, SCLC17A6- vesicular glutamate transporter 2 and FOSB, have previously been shown to be involved in glutamatergic transmission.

CONCLUSIONS

Our findings suggest that inflammation-related processes and alterations in cell-to cell signaling may underlie the pathogenesis of GRS. Our results also provide support for perturbations in glutamatergic transmission in GRS. Our findings require replication in larger human cohorts and functional experiments to explore the function of the genes perturbed in GRS.
GENE-51. ROLE OF TUMOR ENDOTHELIAL MARKER 8 (TEM8) IN PRIMARY AND RECURRENT GLIOBLASTOMA.
Authors: Kundu Paramita , Jain Ruchi , Arimappamagan Arivazhagan , Santosh Vani , Kondaiah Paturu
Published: November 2019

Glioblastoma (GBM) is the most common primary malignant tumor of the adult central nervous system. It is highly invasive and almost inevitably recurs; leading to a median survival of 15 months. The recurrent tumor in most cases is radiotherapy and chemotherapy-resistant, making them quite challenging to treat. Several studies have elucidated key driver genes and molecular pathways involved in Gliomagenesis, but genes/pathways that might be involved in aiding relapse are not well elucidated. Interestingly, chemotherapy with Temozolomide and γ-irradiation have been documented to alter the DNA methylome of cells in vitro. A probable hypothesis could be that following surgical resection, therapy alters the epigenome of residual cancer cells giving rise to aggressively resistant recurrent tumors. To examine this possibility, 24 GBM biopsies (11 primary and 13 recurrent GBMs) were subjected to Methylation Bead Array to assess differential methylation of genes in recurrent GBM compared to primary tumors. This was correlated with RNA-seq data for recurrent tumors in TCGA database. A total of 1751 differentially methylated regions (DMRs) were identified, among which 205 differentially methylated genes (74 hypomethylated and 131 hypermethylated) were found to correlate with methylation and RNA-sequencing data of recurrent tumors in TCGA. Among these genes, ANTXR1/TEM8 (Tumor Endothelial Marker 8) was found to be overexpressed in recurrent tumors compared to primary tumors at both transcript and protein levels using RT-PCR and immunohistochemistry, respectively. When overexpressed in Glioma cell lines, TEM8 was found to upregulate phospho-GSK3β, phospho-AKT and lead to β-catenin translocation to the nucleus resulting in activation of Wnt signaling pathway. TEM8 overexpressing cells showed mild enhancement in proliferation and resistance to Temozolomide, Cisplatin and 5-fluoroUracil. Stem cells markers like Oct4 and Nanog are also upregulated. In conclusion, TEM8 may be involved in GBM recurrence and may have a role in conferring chemo- and radioresistance.
GENE-53. IDENTIFICATION OF EPITHELIAL MEMBRANE PROTEIN 2 (EMP2) AS A MOLECULAR MARKER AND THERAPEUTIC TARGET IN MENINGIOMA
Authors: Patel Kunal , Duong Courtney , Sun Matthew , Romiyo Prasanth , Kejriwal Sameer , Chan Ann , Gordon Lynn , Wadehra Madhuri , Yang Isaac
Published: November 2019

BACKGROUND

Intracranial meningiomas have been associated with a heterogenous set of genetic and molecular alterations. Despite these genetic associations, targeted therapies have been unable to increase survival in recurrent or residual meningiomas. There is a need for identification of therapeutic targets in meningioma.

MATERIALS & METHODS

Normal brain tissue from routine autopsy and tumor specimens from patients undergoing primary surgical resection of meningioma were analyzed. Pathologic diagnosis and grade were identified. Samples were stained with anti-EMP2 antisera and visualized with DAB (3,3’-Diaminobenzidine). Western blot analysis was carried out

RESULTS

In 17 patients with meningioma, there was statistically significant EMP2 staining in fresh brain tumor samples. EMP2 expression was confirmed and quantified with western blot which demonstrated significant positive expressivity. There were 12 grade I, 4 grade II, and 1 grade III meningiomas in our dataset and a significant trend towards increased EMP2 expression with higher grade. EMP2 expression was not identified in normal brain tissue samples from different locations including gray matter, white matter, and meninges in 3 control patients.

CONCLUSIONS

EMP2 is a protein associated with cell migration, invasion, and angiogenesis not identified in normal brain tissue but found in meningioma. EMP2 expression may be associated with increased tumor invasion and is a potential therapeutic target for residual or recurrent meningioma.
GENE-59. NOT ALL p53 MUTATIONS ARE CREATED EQUAL: A MURINE ASTROCYTE MODEL FOR HIGH-THROUGHPUT FUNCTIONAL ASSESSMENT OF p53 MISSENSE MUTATIONS
Authors: Rockwell Nathan , Staller Max , Cannella Maria , Cohen Barak , Rubin Joshua
Published: November 2019

The tumor suppressor TP53 (p53) is the most commonly mutated gene in cancer and among the most frequently mutated genes in glioblastoma (GBM). The majority of p53 mutations in GBM are missense mutations in the DNA binding domain that lead to the production of full length mutant p53 protein. In addition to the complete loss of tumor suppressor function, these mutations have gain-of-function (GOF) properties either through attenuation of wild-type function or neomorphic functions. The variability in GOF mutations results in heterogeneity in cancer phenotypes between mutants that remain poorly understood. Here, we developed a murine astrocyte model to functionally assess a library of p53 mutants in parallel. Primary astrocytes were isolated from postnatal day one pups possessing a single copy of wild-type p53 flanked by loxP sites (TRP53f/-). We then built a library of 17 individual alleles of recurring mutations in GBM with flanking loxP sites. When co-transfected into the mouse astrocytes with a plasmid expressing Cre recombinase, the endogenous WT p53 was excised and replaced with a single copy of the mutant allele. In this way, all astrocytes expressed a single copy of mutant p53 from the endogenous p53 locus. As the mutant p53 cells expanded, aliquots of cells were extracted for targeted genomic sequencing of the p53 allele. Comparing the allelic frequencies of each mutant overtime revealed a wide distribution of growth rates between mutants. To validate the screen results, wildtype astrocytes were transduced with mutant p53-IRES-eGFP retrovirus to overexpress one of three mutations with divergent growth phenotypes. As observed in the initial screen, equivalent overexpression of the different p53 mutants was sufficient to induce significant differences in growth phenotype, with astrocytes expressing the Y217C growing the fastest, R172H second, and Y202C growing the slowest. Ongoing studies are evaluating mutation-specific p53 binding partners and transcriptional outputs.
HOUT-15. IDENTIFICATION AND CHARACTERIZATION OF CARDIAC EVENT ABNORMALITIES IN PATIENTS WITH CRANIOPHARYNGIOMA
Authors: Seligman Christa , Harrison Rebecca , Hassan Saamir , Wu Jimin , DeGroot John , Raza Shaan
Published: November 2019

BACKGROUND

Craniopharyngiomas (CP) are frequently associated with a malignant clinical trajectory despite their benign histology. This population frequently has endocrinopathies and autonomic dysfunction due to tumour location, which may influence cardiac function and long term health outcome. In this study, we evaluated for clinical and paraclinical cardiac abnormalities in adult and pediatric patients with craniopharyngioma.

METHODS

We retrospectively identified patients in our neurosurgery database with a diagnosis of craniopharyngioma that received neurosurgery at MD Anderson Cancer Center. We queried the medical record for demographic and disease-related information, as well as clinical information regarding cardiac risk factors. Perioperative electrocardiograms (EKGs) were analyzed where available.

RESULTS

We identified 56 patients with craniopharyngioma, 27 (48.2%) of which were female. Average age was 39 years, and all patients had undergone subtotal (49.1%), gross total (32.7%), or near gross total (18.2%) resection. Over half (53.6%) had undergone radiation, none had received systemic therapy, and 1 (1.8%) intra-cystic chemotherapy. The majority had adamantanomatous-type histology (83%) and the remainder papillary (17%). At diagnosis, average BMI was elevated at 30.98. Hypertension (30.4%), diabetes mellitus (32.1%), and thyroid dysfunction (80%), were found in this cohort. Nearly a quarter (23.2%) were on statin therapy for dyslipidemia. Stimulant (14.3%) and neuropleptic (3.6%) use were also identified. Of patients with perioperative EKG performed (N= 39), 30 were available for review. Of these, patients were identified with abnormal rate (N=9, 30.0%), PR interval (N=2, 6.7%), QRS interval (N=4, 13.3%), and QTc interval (N=1, 3.3%). Some patients presented with a history of heart palpitations (1.8%) and syncopal episodes (3.6%), but not chest pain.

CONCLUSION

This analysis demonstrates frequent vascular and cardiac risk factors in patients with craniopharyngioma. We advocate for increased awareness of cardiac health in this young population. Further prospective evaluation is warranted to better understand cardiac health in this patient group.
IMMU-07. CELLULAR IMMUNOTHERAPY TO OVERCOME TEMOZOLOMIDE INDUCED T CELL EXHAUSTION IN GLIOBLASTOMA
Authors: Karachi Aida , Dastmalchi Farhad , O’Malley Ashley , Saia Megan , Mitchell Duane , Rahman Maryam
Published: November 2019

INTRODUCTION

We have previously demonstrated that standard dose (SD) temozolomide results in T cell exhaustion in glioblastoma. In this study, we hypothesized that cellular immunotherapies will prevent T cell exhaustion. We tested temozolomide treatment with adoptive T cell transfer alone, dendritic cell (DC) vaccines alone and T cell transfer in combination with DC vaccines.

METHOD

GL-261-gp100 tumor-bearing mice were treated with SD (50 mg/kg for 5 days) or metronomic dose (MD) (25 mg/kg for 10 days) temozolomide. CD3+ T cells were isolated from Pmel mice and infused intravenously. Antigen-specific DC vaccines (gp-100) were given intradermally at the concentration of 50×104. Peripheral blood T cell populations were evaluated by flow cytometry. IFN-gamma secretion was measured for evaluation of T cells function. Survival was compared between groups.

RESULTS

Survival analysis demonstrated that naïve T cell transfer alone and DC vaccine alone add no survival benefit to temozolomide treatment for tumor-bearing animals. There were no significant differences in the expression of exhaustion markers of Tim-3 and Lag-3 on T cells of animals that received a combination of activated T cells and DC vaccines in the context of temozolomide therapy compared to the animals that just received temozolomide. Combination of activated T cell transfer and DC vaccines did not increase IFN-gamma secretion from T cells compared to temozolomide treated animals. Combination of naïve T cell transfer and two DC vaccines increased IFN-gamma secretion from T cells of GL-261 gp100 tumor-bearing animals. However, naïve T cell transfer and two DC vaccines in the context of temozolomide was not potent enough to extend the survival of these tumor-bearing animals.

CONCLUSION

Combinatorial treatment with naïve antigen-specific T cells and three dendritic cell vaccine boosters in the context of temozolomide is a promising approach to preserve T cell function and avoid T cell exhaustion.
IMMU-11. SPATIOTEMPORAL IMMUNOGENOMIC ANALYSIS OF THE T-CELL REPERTOIRE IN IDH-MUTANT LOWER GRADE GLIOMAS
Authors: Zhang Michael , Hilz Stephanie , Martin Michael , Hong Chibo , Yu Yao , Bollam Saumya , Grimmer Matthew , Looney Timothy , Lowman Geoffrey , Ganpule Gauri , Phillips Joanna , Shai Anny , Chang Susan , Clarke Jennifer , Taylor Jennie , Butowski Nicholas , Ann Oberheim-Bush Nancy , Berger Mitchel , Aghi Manish , Hervey-Jumper Shawn , McDermott Mike , Theodosopoulos Philip , Okada Hideho , Costello Joseph
Published: November 2019

The design and evaluation of immunotherapies in IDH-mutant lower grade gliomas (LGG) is hindered by a poor understanding of the LGG T-cell repertoire. We present data on the temporal evolution, intratumoral spatial distribution, and prognostic value of the T-cell repertoire in IDH-mutant LGGs. We performed immunogenomic profiling using T-cell receptor beta-chain sequencing of 163 glioma and peripheral blood samples from 33 immunotherapy-naive glioma patients (22 astrocytomas, 11 oligodendrogliomas). T-cell repertoire evolution was analyzed in a subset of 26 patients (69 samples) with matched primary (WHO grade II) and recurrent (WHO grade II-IV) glioma samples. T-cell repertoire diversity was defined as the number of unique T-cell clonotypes by V-gene, J-gene, and CDR3 nucleotide sequences. Malignant transformed (Grade III or IV) recurrent gliomas demonstrated increased T-cell repertoire diversity compared to their patient-matched primary tumors (p=0.0023), but grade II recurrences did not show the same increased diversity (p=0.26). This increase in T-cell repertoire diversity was greater in patients who underwent transformation in the context of TMZ-associated hypermutation compared to spontaneously transformed counterparts (p=0.035). In grade II primary astrocytomas (n=17), T-cell repertoire diversity above the median (186 unique T-cell clonotypes per sample) was associated with worse transformation-free (HR=4.2, p=0.045) and overall survival (HR=6.4, p=0.025). Next, we evaluated intratumoral immune heterogeneity in 7 patients by sampling from up to 10 distinct and maximally-separated intratumoral sites per LGG (64 samples). Eighty-two to 96% of unique clonotypes within a given tumor were present only within a single sampled site. Despite this heterogeneity, six LGG patients harbored T-cell clonotypes present tumor-wide across all sampled sites within a given tumor. Ten of 24 (42%) tumor-wide T-cell clonotypes were enriched in the glioma compared to matched peripheral blood, suggesting glioma-specificity. Taken together, T-cell receptor profiling in LGGs may have utility both as a prognostic biomarker and to identify glioma-specific T-cells.
IMMU-12. IL13Ra2-CAR T CELLS STIMULATE ENDOGENOUS IMMUNE RESPONSES AGAINST MURINE GLIOBLASTOMAS
Authors: Alizadeh Darya , Wong Robyn , Pecoraro Joseph , Yang Xin , Forman Stephen , Brown Christine E
Published: November 2019

Malignant gliomas (MG) are one of the deadliest cancers with very limited therapeutic options. Chimeric antigen receptor (CAR)-T cell therapy has emerged as a powerful strategy for B-cell malignancies and may offer new opportunities to improve outcomes for patients with MGs. Our team is clinically evaluating IL13Rα2-targeted CAR-T cells for the treatment of recurrent IL13Rα2-positive MGs [NCT02208362]. While this trial is ongoing, we have previously reported that one patient with recurrent multifocal glioblastoma achieved a complete response post-IL13Rα2-CAR-T therapy despite the non-uniform expression of IL13Rα2 on the tumor. The therapeutic response against IL13Rα2-negative cells suggests CAR-T cells may stimulate endogenous immune responses. To study the interplay between CAR-T cells and host immune subsets, we have established a syngeneic immunocompetent glioma model, which recapitulates the tumor microenvironment (TME) of patients. Murine IL13Rα2-CAR-T cells mediate potent antitumor activity against IL13Rα2-engineered KR158, a highly invasive murine glioma model. Interestingly, mice “cured” from CAR-T therapy, after rechallenge, can successfully reject the tumors. Furthermore, we demonstrate comparable response rate in mice bearing gliomas with mixed antigen expression (50%IL13Rα2+/50%IL13Rα2-) vs 100% IL13Rα2+. Characterization of the TME post-CAR-T therapy indicates activation of endogenous cytotoxic CD8 T and myeloid cells, and decrease in the frequency of T regulatory cells. Further analyses reveal that tumor-associated macrophages (TAMs) may be reprogrammed during CAR-T therapy to exhibit tumoricidal activity and may promote the activation of endogenous T cells (CD4/CD8 T cells) resulting in enhanced antitumor activity. Current studies are focusing on the characterization of host immune cells to identify the mechanisms involved in induction of host immune responses mediated by CAR-T cell therapy. Our data thus strongly suggest that CAR-T therapy has the potential to reshape the glioma microenvironment creating a context permissible to elicit effective endogenous antitumor immunity.
IMMU-21. SEQUENTIAL TWO-RECEPTOR PRIMING CAR SYSTEM TO OVERCOME HETEROGENEOUS ANTIGEN EXPRESSION
Authors: Watchmaker Payal , Choe Joseph , Carrera Diego , Gilbert Ryan , Yu Wei , Downey Kira , Roybal Kole , Lim Wendell , Okada Hideho
Published: November 2019

Heterogeneous expression of target antigens allows tumor escape from chimeric antigen receptor-transduced T-cell (CART) therapy targeting a single antigen. While epidermal growth factor receptor (EGFR)vIII represents a glioblastoma (GBM)-specific antigen, its expression is heterogeneous within the tumor. On the other hand, most other antigens expressed more uniformly in GBMs are non-mutated, glioma-associated antigens (GAAs), such as EphA2. Although these GAAs are not expressed in the normal brain, they are expressed at low levels in other normal organs. As a way to safely target GAAs in the tumor without attacking normal cells expressing the same GAAs outside of the brain, we adapted a novel synthetic Notch (synNotch) receptor system, and established a “prime and kill” sequential two-receptor CAR circuit: the first is a transcriptional CAR against EGFRvIII, and the second is a CAR against a GAA (e.g. EphA2). When the first CAR binds to EGFRvIII, it induces the expression of the second CAR against GAA. While the first CAR does not trigger the cytotoxic function itself, the second CAR mediates the cytotoxicity upon recognition of the target GAA. We validated this system in vitro using the mixture of U87 GBM cells and those transduced with EGFRvIII. SynNotch CART effectively lysed both EGFRvIII+ and EGFRvIII-negative U87 cells when they are mixed, but never lysed EGFRvIII-negative cells in the absence of EGFRvIII+ cells. SynNotch CART cells exhibited precise local effect in vivo, sparing EGFRvIII-negative subcutaneous tumor while efficiently clearing intracranial EGFRvIII heterogeneous tumor. In the orthotopic GBM6 model, which is a patient-derived xenograft with heterogeneous expression of EGFRvIII, intravenous infusion of synNotch CART resulted in long-term (over 50 days) survival and eradication of the heterogeneous tumor in 6 of 6 mice, while the conventional anti-EGFRvIII CART failed to do so. These data provide a strong basis for developing synNotch CART therapy for GBM.
IMMU-35. COMPLEMENT SYSTEM AND GLIOBLASTOMA: AN INTRICATE RELATIONSHIP
Authors: Mittal Sandeep , Sengupta Shayak , Khan Sabbir , McGee Kain , Alfaro-Munoz Kristin , Balasubramaniyan Veerakumar , DeGroot John
Published: November 2019

The complement system is a vital part of the innate immune system which plays a critical role in immune surveillance and inflammatory processes. Malignant and host cells express various complement inhibitory proteins on their surface to protect against complement mediated cytotoxicity. Imbalanced complement activation triggers inflammation and alters the tumor microenvironment. Complement activation has been shown to induce proliferation and migration of breast, ovarian and lung cancer cells. At this time, the expression and functional role of the complement cascade in glioblastoma (GBM) remain elusive. Here, we investigated the role of complement proteins and their receptor expression in human primary glioma stem-like cells (GSCs) and human GBM tissues. Western blot data demonstrated a high level of expression of central complement components and their receptors in GSCs. RT-PCR data further confirmed the high expression of complement genes which was similar or higher to normal human astrocytes (HA), a cell with high baseline expression levels of complement genes. Flow cytometry analysis revealed that almost 95% of GSCs expressed the anaphylatoxin complement receptors C3aR and C5aR on their cell surfaces which was consistent with our immunohistochemistry analysis of freshly resected GBM tissues. Furthermore, anaphylatoxin C5a exposure increased the proliferation of a subgroup of GSCs while reduced in another subset. Interestingly, C5a exposure was found to increase the expression of various pro-inflammatory markers in GSCs with reduced proliferation. Fluorescence-activated cell sorting (FACS) analysis of freshly isolated human GBM tissue revealed predominant expression of C5aR on cancer cells (CD11b-/CD45- cells) rather than on immune cells. RT-PCR analysis also demonstrated high expression levels of complement genes with concomitant decrease in complement inhibitory genes in human GBM tissue. Evaluation of the differential role of the complement system in GSCs along with their role in in vivo glioma models is ongoing.
IMMU-38. CRISPR BASED GENOME EDITING OF HUMAN T CELLS TO TARGET H3.3K27M MUTATION IN GLIOMAS
Authors: Hegde Bindu , Roth Theodore , Nguyen David , Chheda Zinal , Apathy Ryan , Marson Alex , Okada Hideho
Published: November 2019

We recently identified an HLA-A*02:01-restricted CD8 T cell epitope encompassing the H3.3K27M mutation, which is common in diffuse midline glioma, and a corresponding high-affinity T cell receptor (TCR) that recognizes the epitope. While recombinant viral vectors have been widely used for genetic reprogramming of T cells, viral vectors are far from ideal as they typically integrate randomly into the genome and are not governed by the molecular regulatory mechanisms of the cell. We used a non-viral, CRISPR-Cas9-based approach to replace the endogenous TCR with H3.3K27M TCR at the TCR a constant region (TRAC) in human T cells. Co-electroporation of healthy donor-derived T cells with homology-directed repair (HDR) templates encoding the full-length sequence of H3.3K27M TCR along with CRISPR-Cas9 ribonucleoprotein (RNP) resulted in the integration of the new TCR into the TRAC locus by HDR. Antibody staining of TCR α/β and H3.3K27M dextramer showed replacement of endogenous TCR with H.3.3K27M TCR in ~5–10% of TCR+ CD8 T cells. Modifying the HDR template to include a binding site for Cas9, which contains the nuclear localization signal that acts as a “shuttle”, further enhanced the integration efficiency (~15% of TCR+ CD8 T cells). Furthermore, HLA-A2+ H3.3K27M TCR-engineered T cells selectively killed U87 glioma cells expressing the H3.3K27M epitope. In addition, the engineered T cells exhibited a stem memory-like phenotype when expanded in the presence of a cocktail of IL-2, IL-7 and IL-15. Taken together, these data provide evidence for non-viral genome editing as a strategy to engineer T cells with specific TCR for cancer immunotherapy.
IMMU-48. RATIONAL DEVELOPMENT AND CHARACTERIZATION OF MULTIVALENT TARGETING TANDEM AND PARALLEL CAR T CELLS FOR GLIOMA
Authors: Yin Yibo , Thokala Radhika , Zhang Logan , Kainth Devneet , Haddad Leila , Binder Zev , Lin Zhiguo , O’Rourke Donald
Published: November 2019

High grade glioma is the most common malignant primary brain tumor. Despite the best available therapy, the median survival is limited. Chimeric antigen receptor (CAR)-T cells effectively attack target positive tumor cells, demonstrating a promising possible treatment. Based on our previous clinical and pre-clinical studies, we utilized epidermal growth factor receptor variant III (EGFRvIII) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) targeting single-chain variable fragments (scFvs) to generate bi-specific tandem CAR T cells and bi-specific parallel CAR T cells to decrease the potential of antigen escape and tumor recurrence. 5, 10, 15 or 20 flexible amino acids were utilized to link scFvs in tandem CARs. Ribosomal “skipping” 2A peptides were utilized to generate parallel CAR T cells. Flow-based IL13Rα2 staining on glioma stem cells (GSCs) showed 7 out 11 cases positive (64%) heterogeneous intratumoral expression. Tandem CAR T cells and parallel CAR T cells upregulated CD69 expression (P< 0.0001), generated IFNγ, interleukin 2 and TNFα (P< 0.0001), as well as exhibiting cytotoxic activity (P< 0.0001) when compared against un-transduced T cells, in co-culture with either single- or double-target positive GSCs. There was no significant difference between different lengths of linker in tandem CAR T cells. Parallel CAR T cells generated a larger cytokine response (P< 0.0001) and more effectively killed target positive cells (P< 0.05) than tandem CAR T cells. In summary, glioma associated target expression is heterogeneous in GSCs. Multivalent tandem CAR T cells and parallel CAR T cells effectively respond to target expressing tumor cells. The function of tandem CAR T cells is not relative with the length of linker. Parallel CAR T cells are a more promising strategy in generating gene modified multivalent targeting T cells.
INNV-23. GLIOBLASTOMA AND FACEBOOK: AN ANALYSIS OF PERCEIVED ETIOLOGIES AND TREATMENTS.
Authors: Kumar Reddy Naveen , Blondin Nicholas
Published: November 2019

Facebook has become one of the most widely used platforms by patients and caregivers for information on GBM. As such, physicians treating GBM are challenged with reconciling their medical advice with online media sources. In many cases, the information from these online sources can run counter to the advice given by physicians.

OBJECTIVE

This study sought to understand the type of information being shared on a popular GBM Facebook community titled, “GLIOBLASTOMA SURVIVORS TO THRIVERS!” with regards to 1. The perceived causes of GBM and 2. The therapies that led to GBM remission.

METHODS

All the posts in a 30-day period (5/01/2019-6/01/2019) were screened for information on GBM etiologies and GBM therapies. Within each group, posts were sorted into distinct sub-categories with posts of similar content. The sub-categories were ranked to determine which etiologies and therapies were most commonly seen by group members.

RESULTS

A total of 83 posts were on the topic of “GBM Etiologies” and 80 on the topic of “GBM Therapies.” Within the “GBM Etiologies”, the reasons for developing GBM were due to 1. Unknown (31.3%) 2. Previous Radiation Exposure (24.1%) 3. Chemical Exposure (17%) 4. Genetic (12%) 5. Infectious Disease (6%) 6. Losartan/Valsartan (4.8 %). and 7. Head Trauma (2.4%) and Emotional Trauma (2.4%). Within the GBM Therapy category, the therapies that led to remission were 1. Standard of Care (36.3%) 2. CBD/THC (16.3%) 3. Ketogenic Diet (12.5%) 4. Avastin (7.5%) and Optune (7.5%) 5. IV Vitamin C (6.25%) and COC Protocol (6.25%) 6. Meditation/Yoga/Acupuncture (5.0%) and 7. Faith Healing (2.5%).

CONCLUSION

In the Facebook group titled, ““GLIOBLASTOMA SURVIVORS TO THRIVERS!,” the top three most commonly posted reasons for developing GBM were “Unknown” “Previous Radiation Exposure” and “Chemical Exposure.” The top three therapies that led to remission were “Standard of Care” “CBD/THC” and “Ketogenic Diet.”
INNV-27. THE IMPACT OF A DEDICATED MULTIDISCIPLINARY TUMOR BOARD ON CARE FOR PATIENTS WITH BRAIN METASTASES
Authors: Wang Nancy , Cohen Justine , Goss Nathaniel , Bertalan Mia , Keeley Maura , Parsons Michael , Nahed Brian , Cahill Daniel , Oh Kevin , Brastianos Priscilla
Published: November 2019

Brain metastases (BM) are the most common tumors to affect the central nervous system (CNS). Treatment options have recently evolved with the use of new targeted therapies, immune checkpoint inhibitors, and increased access to clinical trials. We describe our institutional experience with a weekly tumor board dedicated to BM.

METHODS

We conducted a single-institution cohort study at an academic hospital. Attendance at tumor board included representatives from neuro-oncology, medical oncology, radiation oncology, neurosurgery, neuropsychology, and neuroradiology. We prospectively gathered data on patient demographics, clinical history, and tumor board recommendations. Patients were followed to assess treatment course and survival. The Kaplan Meier method was used to calculate time to progression.

RESULTS

A total of 49 patients were presented over 2 months. The median age at presentation was 63 yrs with a median ECOG of 1. The primary malignancy was 35% melanoma, 29% lung, 23% breast, the remainder other. Most patients had advanced, heavily pretreated disease: 69% had Stage IV disease with a median of 2 prior lines of systemic therapy, 73% had multiple BM, 39% had prior surgical resection of BM, and 57% had prior CNS radiation. Change in management was recommended in 26/53 case presentations, with active BM-directed therapy (surgery, radiation, systemic therapy) recommended in 25/26 patients. The median time to start active therapy was 7 days. Only 3 patients have died at a median follow-up of 62 days. 9 patients have progressed, with a median time to progression of 57 days.

CONCLUSIONS

Multidisciplinary BM tumor board provides unique opportunities in the management of complex BM patients in an era of rapidly evolving therapeutic options. Additional follow-up is needed to assess long-term outcomes.