bluuebunny/crossref_metadata_2025 huggingface dataset:
ACTR-45. FATTY ACID SYNTHASE INHIBITOR TVB-2640 INCREASES PROGRESSION FREE SURVIVAL IN RECURRENT GBM
Authors: Konkel Brandon , Michalek Joel , Diaz Duque Enrique , Liu Qiangia , Caflisch Laura , Madhu Vinu , Brenner Andrew
Published: November 2019

BACKGROUND

Standard of care for glioblastoma (GBM) is surgical resection followed by temozolamide, with bevacizumab (bev) given at relapse. Responses to bev remain brief; resistance may involve overexpression of Fatty Acid Synthase (FASN).

METHODS

This is a prospective phase 2 study of bev with TVB-2640 in patients with rGBM at first relapse. Primary end point is progression free survival (PFS). Inclusion criteria are: age ≥ 18, ECOG 0 to 2, GBM progression following standard combined modality treatment. An exploratory phase randomization into 2 separate arms of single agent bev or in combination with TVB-2640 with MR-Spectroscopy (MRS) and serum sampling for exosome analysis obtained on all patients at day 1 and 28 of first cycle. Starting on cycle 2 day 1, all patients converged to a single arm and continue to receive bev in combination with TVB-2640. A total sample size of 24 patients will provide 90% power to detect a 4 month difference in PFS (3–4 months for Bev alone (historic controls) (i.e., a hazard ratio of 0.43) using a one-sided log-rank test with alpha=0.1.

RESULTS

Enrollment is complete with 28 patients to date. 3 failed screening. 21 came off study and 4 are active. No grade 4 or higher treatment-related AEs have occurred, with Grade 3 events included 3 cases of palmar-plantar erythrodysesthesia; 1 each of hypertension, stomatitis, optic neuritis, DVT, and wound infection. 95.2% of patients have achieved at least stable disease by RANO Criteria, with a 66.7% overall response rate (ORR). Median time to progression was 5.9, which is statistically superior to historical controls. Overall survival as well as biomarker analysis (exosome, MRS), is pending.

CONCLUSIONS

The combination of TVB2640 with bev appears well tolerated and with PFS6 significantly improved over historical controls. Final data analysis as well as exploratory biomarker analysis will be presented.
SURG-12. “NANOPASTE” THERAPY AS POTENTIAL TREATMENT OPTION FOR RECURRENT GLIOBLASTOMA
Authors: Schwake Michael , Grauer Oliver , Holling Markus , Brokinkel Benjamin , Müther Michael , Bruns Ann-Katrin , Wölfer Johannes , Stummer Walter
Published: November 2019

BACKGROUND

We recently showed that intracavitary thermotherapy with superparamagnetic iron-oxide nanoparticles can induce persistent inflammatory reactions which might lead to long-term stabilization in recurrent glioblastoma (GBM) patients.

METHODS

Here, we report further data from a series of ten recurrent GBM WHO IV patients (IDH WT, MGMT: methylated 30%, unmethylated 70%; median age: 59 years) who were treated with intracavitary thermotherapy after coating the resection cavity wall (“NanoPaste”) with NanoTherm® particles (MagForce AG, Berlin, Germany). All patients underwent six one-hour semi-weekly hyperthermia sessions in an alternating magnetic field (mean maximum temperature 52.3° C, SD +/- 6.0 °C). Six patients received concurrent radiotherapy at a dose of 39.6 Gy (5 x 1.8 Gy/week).

RESULTS

No major side effects were observed during active treatment. However, all patients developed cerebral edema and increasing clinical symptoms during treatment follow-up (median 92 days, range 73 to 144). Patients were treated with dexamethasone and, if necessary, underwent re-operation to remove nanoparticles (n=5). Histopathology revealed sustained necrosis and large amounts of nanoparticles without evidence for tumor activity as well as a prominent inflammatory reaction characterized by increased T-cell and myeloid cell infiltration. Median overall survival (mOS) for the study population was 10.1 months (CI 95% 8.0–12.2). A survival benefit was observed for patients treated at first recurrence (n=5) when compared to patients treated at second recurrence or later (mOS = 20.6 vs 9.4 months). Patients who received thermotherapy and concurrent radiotherapy (n=6) showed longer mOS than patients treated with thermotherapy alone (17.3 vs 8.6 months). Two patients had long-lasting treatment responses > 23 months with one patient still alive at 3.5 years after treatment without receiving any further therapy.

CONCLUSION

Our results warrant further investigations. A European clinical registry will be rolled out to further evaluate the potential of “NanoPaste” therapy for patients with recurrent glioblastoma.
SURG-22. CUSHING’S DISEASE: A DIFFICULT TO TREAT NEURO-ONCOLOGICAL CONDITION
Authors: Bora Santanu , Suri Ashish
Published: November 2019

BACKGROUND

Cushing disease (CD) comprises a spectrum of clinical manifestations secondary to hypercortisolism due to ACTH-secreting pituitary adenoma. Transsphenoidal adenomectomy remains the standard treatment. Because of the significant rate of recurrence or persistence of CD, it is of interest to determine factors that may correlate with long-term outcomes following surgical intervention.

OBJECTIVE

The objective of our study is to determine the remission rate after surgery with special emphasis on factors affecting remission.

METHODS

Data of all patients undergoing surgery for CD from 2009 to 2017 was analyzed retrospectively. Transphenoidal resection was the preferred treatment with a recent trend in favor of endonasal endoscopic skull base approach. Post-operative cortisol level of < 2 μg/dL was taken as remission and value between 2 and 5 μg/dL as possible remission.

RESULTS

104 patients operated primarily for CD were included for analysis. 47 patients underwent microscopic surgery, 55 endoscopic surgery and two were operated trans-cranially. Remission was achieved in 76.47% of patients. In univariate analysis, factors significantly associated with remission were (1) type of surgery (p=0.01); endoscopy (88.23% remission) better than microscopy (56.6% remission) (2) postoperative day-1 morning cortisol (p=0.004) and; (3) postoperative day-1 morning ACTH (p=0.015). In multivariate analysis, however only postoperative day-1 cortisol was found to be significant as predictor of remission (p=0.02).

CONCLUSION

Postoperative plasma cortisol level is a strong independent predictor of remission and value less than 10.7µgm/dl can be taken as cut off for predicting remission. Remission provided by endoscopy appears to be significantly better than microscopic approach.
TMIC-06. MYELOID POPULATIONS AND THE EFFECT OF NEOADJUVANT PD-1 INHIBITION IN THE GLIOBLASTOMA MICROENVIRONMENT: A SURFACEOMIC AND TRANSCRIPTOMIC DISSECTION AT THE SINGLE-CELL LEVEL
Authors: Mochizuki Aaron , Lee Alexander , Orpilla Joey , Kienzler Jenny , Galvez Mildred , Chow Frances , Davidson Tom , Wang Anthony , Everson Richard , Liau Linda , Cloughesy Timothy , Prins Robert
Published: November 2019

INTRODUCTION

Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a dismal prognosis. Neoadjuvant anti-PD-1 blockade has demonstrated efficacy in melanoma, non-small cell lung cancer and recurrent GBM; however, responses vary. While T cells have garnered considerable attention in the context of immunotherapy, the role of myeloid cells in the GBM microenvironment remains controversial.

METHODS

We isolated CD45+ immune populations from patients who underwent brain tumor resection at UCLA. We hypothesized that myeloid cells in glioblastoma contribute to T cell dysfunction; however, this immune suppression can be mitigated by neoadjuvant PD-1 inhibition. To test this, we utilized mass cytometry and single-cell RNA sequencing to characterize these immune populations.

RESULTS

Mass cytometry profiling of tumor infiltrating lymphocytes from patients with GBM demonstrated a preponderance of CD11b+ myeloid populations (75% versus 25% CD3+). At the transcriptomic level, myeloid cells in newly diagnosed GBMs exhibited decreased expression of CCL4 (loge fold change -1.18, Bonferroni-adjusted P = 1.62x10-254) and its ligands compared to anaplastic astrocytoma. In ranked gene set enrichment analysis, patients who received neoadjuvant pembrolizumab demonstrated enrichment in TNFα-, NFκB- and lipid metabolism-related gene sets by bootstrapped Kolmogorov-Smirnov test (Benjamini-Hochberg adjusted P = 4.74x10-3, 1.45x10-2 and 2.48x10-3, respectively) in tumor-associated myeloid populations. Additionally, single-cell trajectory analysis demonstrated increased CCL4 and decreased ISG15 with neoadjuvant checkpoint inhibition.

CONCLUSIONS

Here, we utilize mass cytometry and single-cell RNA sequencing to demonstrate the predominance and transcriptomic features of myeloid populations in GBM. Myeloid cells in patients who receive neoadjuvant PD-1 blockade re-express increased levels NFκB, TNFα and CCL4, a cytokine crucial for the recruitment of dendritic cells to the tumor for antigen-specific T cell activation. By delving into the GBM microenvironment at the single-cell level, we hope to better delineate the role of myeloid populations in this uniformly fatal tumor.
TMIC-08. IDENTIFICATION OF TUMOR SPECIFIC LYMPHOCYTES IN A MURINE GLIOMA MODEL
Authors: Shin David , Lamba Nikhil , Flores Catherine , Divita Bayli , Wildes Tyler , Grippin Adam , Fernandez Brandon , Dyson Kyle , Mitchell Duane
Published: November 2019

INTRODUCTION

One major challenge in the use of immunotherapy for glioblastoma is the identification of tumor specific lymphocytes within the tumor microenvironment. Previous studies in non-glioma cancer models have identified CD39 and PD1 as lymphocyte markers of tumor specificity. In this study, we demonstrate the existence of this specific T cell phenotype and show correlation with anti-tumor activity.

METHODS

We implanted KR158B murine glioma cells into right caudate nucleus into yellow fluorescent protein (YFP) interferon-gamma reporter mice for a total of 104 cells using stereotaxis. At 4 weeks, mice were sacrificed followed by organ harvest. Samples taken included tumor, cervical draining lymph nodes, spleen, and peripheral blood. These were processed into single cell suspensions and then stained for CD3, CD8, CD39, and PD1. Flow cytometry was performed on a BD FACSCanto II. Gating analysis was performed with FlowJo while data analysis was performed with GraphPad Prism.

RESULTS

Gating for CD3+ CD8+ on tumor lymphocytes reveals a significant population of CD39 and PD1 double positive (DP) cells (61.4%) not found in the peripheral organs (< 1.0%). Evaluation of these cells for YFP expression demonstrated significantly greater median fluorescence intensity (MFI) in the DP group as opposed to either of the single positive (SP) or double negative (DN) groups (DP = 225.4, SP-CD39 = 40.54, SP-PD1 = 82.7, DN = 29.5, p < 0.0001).

CONCLUSION

Here we demonstrate the existence of a unique phenotype of CD39+ and PD1+ expression on lymphocytes within the tumor microenvironment. These DP cells also had high levels of interferon gamma production compared SP and DN groups. This phenotype is not observed in peripheral circulating lymphocytes. Enrichment for this subpopulation will allow for delivery of purified lymphocyte-based immunotherapy in this murine glioma model.
TMIC-13. FEELING THE FORCE: DIFFERENCES IN THE MECHANOREGULATED MIGRATION OF HIGH GRADE GLIOMA BETWEEN INDIVIDUAL CELLS AND THOSE IN CONTACT WITH OTHER TUMOUR CELLS
Authors: Jegathees Thuvarahan , Prior Victoria , O’Neill Geraldine
Published: November 2019

A major limitation in the treatment of High Grade Gliomas (HGG) is their highly disseminating nature. While it is increasingly appreciated that the mechanical properties of the extracellular matrix, (measured as tissue elasticity, Young’s modulus, E), can independently cue cancer cell migration and invasion, to date there has been little consideration of this mechanism in HGG invasion. This is particularly important given that the brain parenchyma is a mechanically soft tissue (E values varying between 1 - 10 kPa). By measuring single cell migration we have previously demonstrated that molecular subclasses of HGGs exhibit different rigidity-sensitive and -insensitive migration. Following these findings, the present project aimed to determine whether these mechanosensitive phenotypes are maintained in the dissemination of multicellular tumour spheroids (MCTSs) that represent in vivo organisation of the primary tumour bulk. Therefore MCTSs composed of primary patient-derived HGG cells with pre-established single cell mechanosensitive phenotypes were cultured on mechanically tuneable polyacrylamide hydrogels, mimicking the range of physiological tissue rigidities. Bright-field time-lapse images were then captured over a period of 48 hours, 6 images per hour. In order to quantitate the migratory behaviours, we adapted a previously published automated image analysis program to segment the MCTS images into proliferative and migratory regions. Our analysis suggests that the cellular mechano-phenotype is affected by contact with neighbouring cells, as the migratory response to tissue stiffness is quantitatively different in the MCTSs. Our results highlight different migratory behaviour between HGG cells within the primary tumour mass versus individual cells that escape. Our results reveal the complex migratory behaviour of HGG cells and suggests that successful anti-invasive therapies will need different strategies depending on tumour cell location.
TMIC-14. PD-L1 EXPRESSION IS NEGATIVELY CORRELATED TO OUTCOMES IN PATIENTS WITH MGMT METHYLATED PROMOTERS IN GBM
Authors: Srivastava Siddhartha , Choi John , Lim Michael
Published: November 2019

BACKGROUND

Glioblastoma is a high-grade glioma and is considered the most aggressive primary brain tumor with a median overall survival of 11–15 months. Methylation of the promoter of the MGMT gene, which encodes a DNA repair protein, has been shown to be a good prognostic indicator for GBM survival. It is known that GBM has elevated expression of several immune checkpoint molecules that allow the tumor to evade anti-tumor immune response. Therefore, it is important to elucidate connections between prognostic factors like methylation status and immunologic markers in the tumor microenvironment (TME) that might lead to immunosuppression.

METHODS

Patients with resected GBM tumor samples were identified by pathology reports as having tumors with either methylated or unmethylated MGMT promoters. Corresponding patient tumor biopsy samples were embedded in paraffin, sectioned, and then stained using specific antibodies for known immune checkpoint molecules: PD-1, PD-L1, LAG-3, TIM-3, CSF1R, and IDO-1.

RESULTS

In our cohort of 34 patients, 17 had methylated MGMT promoter regions and 17 did not. We found no statistically significant difference between methylated and unmethylated MGMT promoter status for immune checkpoint expression. However, we did find a statistically significant negative correlation (P< 0.05) between the degree of PD-L1 expression in the TME and the progression free survival of patients with the MGMT promoter methylation phenotype.

CONCLUSION

Our study examined the relationship between immune checkpoint markers and methylation status of MGMT promoters in GBM. While there was no statistically significant difference between these two groups within the immunological framework of this study, there was a statistically significant negative correlation showing that increased PD-L1 expression in patients with methylated MGMT promoter tumors correlated to a worse progression free survival. Future studies are indicated to investigate the correlation between PD-L1 expression in relation to GBM treatment prognosis and its dependence on MGMT promoter methylation.
TMIC-20. ELIMINATION OF SENESCENT ASTROCYTES IN THE BRAIN TUMOR MICROENVIRONMENT ATTENUATES GLIOBLASTOMA RECURRENCE AFTER RADIOTHERAPY
Authors: Fletcher-Sananikone Eliot , Mukherjee Bipasha , Burma Sandeep
Published: November 2019

Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR) yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. During GBM therapy, the surrounding brain tissue is irradiated along with the tumor. IR induces senescence in multiple cell types, and senescent stromal cells are known to promote the growth of neighboring tumor cells by secreting cytokines which create a senescence-associated secretory phenotype (SASP). We hypothesize that IR-induced senescence of normal brain cells in the tumor microenvironment is a powerful driver of GBM recurrence. We intra-cranially irradiated C57BL/6J mice, and found evidence of widespread senescence, with the astrocytic population being highly susceptible. Genomic analyses of irradiated brains revealed an altered transcriptomic profile which included upregulation of CDKN1A (p21), a key enforcer of senescence, and increased expression of SASP proteins including HGF, the ligand for the RTK Met. We orthotopically implanted mock-irradiated or irradiated mice with a limiting number of syngeneic glioma cells. Pre-irradiation of mouse brains resulted in a striking increase in tumor growth and invasion driven by Met activation in the tumor cells. Importantly, irradiated p21-/- mouse brains did not exhibit SASP and failed to promote tumor growth. Irradiated primary astrocytes underwent senescence in vitro and promoted the migration of glioma cells, and this could be attenuated with HGF-neutralizing antibodies or by the Met inhibitor Crizotinib. These findings indicate that SASP factors (like HGF) in the irradiated brain microenvironment could drive GBM recurrence after radiotherapy via the activation of RTKs (like MET) in the tumor cells. Significantly, we found that senolytic drugs can selectively kill senescent astrocytes both in vitro and in vivo resulting in attenuated growth of glioma cells. These results are of great translational significance as they indicate that adjuvant therapy with senolytic drugs might attenuate GBM recurrence after radiotherapy.
TMIC-25. MODIFICATION OF EXTRACELLULAR MATRIX ENHANCES ONCOLYTIC ADENOVIRUS IMMUNOTHERAPY IN GLIOBLASTOMA
Authors: Kiyokawa Juri , Kawamura Yoichiro , Martuza Robert , Rabkin Samuel , Shah Khalid , Wakimoto Hiroaki
Published: November 2019

Complex interplay between heterogeneous cells and extracellular matrix (ECM) in the tumor microenvironment drives the pathogenesis of glioblastoma (GBM). Glycosaminoglycan hyaluronic acid (HA) is a major component of GBM ECM, and is associated with tumor growth, invasion, and resistance to treatments. Delta24-RGD oncolytic adenovirus is a powerful cancer therapeutic and is currently under clinical evaluation for GBM. We hypothesized that degradation of HA would enhance oncolytic adenovirus immunotherapy of GBM by overcoming the immunosuppressive functions of GBM ECM. Intratumoral injections of ICOVIR17, a delta24-RGD oncolytic adenovirus carrying hyaluronidase PH20 cDNA, increased animal survival, in comparison to its parental virus ICOVIR15, in an orthotopic GBM model generated with murine 005 stem-like GBM cells in immunocompetent C57/Bl6 mice. ICOVIR17 promoted the degradation of HA, and induced an increase in tumor infiltrating CD8+ T cells and macrophages, compared with ICOVIR15. This led to the upregulation of immune checkpoint PD-L1 on GBM cells and macrophages, while it downregulated immune checkpoints TIM3 and CTLA-4 on CD8+ T cells. The combination therapy of ICOVIR17 and systemic administration of anti-PD-1 antibody significantly prolonged the survival of mice bearing intracerebral GBM compared to no treatment control group and monotherapy groups, and achieved long-term remission in 21% of treated animals. Flow cytometry analysis showed that the combination treatment significantly increased the ratio of effector/regulatory (CD8/Treg) T cells in the brain, compared with all the other three groups. Furthermore, antibody and clodronate-mediated in vivo depletion of immune subsets revealed that CD4+, CD8+ T cells and macrophages were all necessary for combination therapy to be effective as any of these depletions abrogated efficacy. Our findings reveal that ICOVIR17-mediated degradation of HA within GBM dramatically alters the immune landscape of the tumor microenvironment, and offer a mechanistically rationale combination immunotherapy with PD-L1/PD-1 blockade that results in superior anti-GBM efficacy.
TMIC-26. PRECLINICAL MODEL SYSTEMS OF GLIOBLASTOMA REVEAL MICROENVIRONMENTAL PROGRAMS AND DEPENDENCIES IN PATIENT TUMORS
Authors: Bayley Nicholas , Tse Christopher , Baufeld Lynn , Gosa Laura , Yan Weihong , Zhu Henan , Balanis Nikolas , Cloughesy Timothy , Liau Linda , Graeber Thomas , Nathanson David
Published: November 2019

Patient-derived model systems serve as a platform for translational research representing the heterogeneity of human cancers, and their success in recapitulating disease-driving genomic alterations is well-documented. While recent studies have demonstrated genomic and functional divergence in patient-derived models with passaging, the need for accurate preclinical models remains. Glioblastoma (GBM) is the most common and aggressive primary brain tumor, and thus far preclinical models have failed to consistently replicate the responses found in patients. We therefore aimed to evaluate the multi-omic fidelity of low-passage GBM model systems across in vitro and in vivo environments and to elucidate the molecular features in which they differ. To this end we established a biobank of glioma direct-from-patient orthotopic xenograft (GliomaPDOX) models and primary gliomasphere cultures (GSCs) and performed whole-exome and RNA sequencing of over 40 purified patient tumors and their matched GliomaPDOXs and GSCs to facilitate paired comparisons across a gradient of full tumor microenvironment (TME) presence. We observed global genomic and transcriptomic fidelity in both systems, but specific programmatic gene expression differences associated with cell-cell interactions in the brain TME, glial cell identity, and in vitro GSC-forming ability. GSCs and GSC-forming ability are strongly associated with an astrocytic gene expression signature, while more stem-like and oligodendrocytic patient tumors including IDH- and H3F3A-mutant GBMs more successfully engraft in GliomaPDOXs. This result implicates the brain TME as a support system for these more stem/oligo-like tumors. Transcription factor network analysis identified regulators of the NOTCH and MYC pathways as strongly enriched in this subgroup of patient tumors and their derivative xenografts, and provides potential targets for therapeutic intervention in near future experiments. Collectively, these findings underline the critical role of the TME in defining GBM cell state, reveal the heterogeneity of TME dependence across patient tumors, and link this dependency to therapeutically actionable molecular features.
TMIC-27. GLUTAMATERGIC NEURON-GLIOMA SYNAPSES DRIVE BRAIN TUMOUR PROGRESSION
Authors: Venkataramani Varun , Tanev Dimitar , Strahle Christopher , Studier-Fischer Alexander , Fankhauser Laura , Kessler Tobias , Losada Perez Maria , Körber Christoph , Kardorff Markus , Ratliff Miriam , Xie Ruifan , Horstmann Heinz , Messer Mirko , Paik Sang , Knabbe Johannes , Sahm Felix , Kurz Felix , Acikgoez Azer , Herrrmannsdörfer Frank , Agarwal Amit , Bergles Dwight , Chalmers Anthony , Miletic Hrvoje , Turcan Sevin , Mawrin Christian , Hänggi Daniel , Liu Hai-Kun , Casas Tinto Sergio , Wick Wolfgang , Winkler Frank , Kuner Thomas
Published: November 2019

A network of communicating tumour cells established by tumour microtubes (TMs) is supposed to mediate relevant aspects of progression and resistance of incurable gliomas. Moreover, neuronal activity has been shown to foster malignant behavior of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here, we report an unexpected direct communication channel between neurons and glioma cells in multiple disease models as well as in astrocytomas and glioblastomas (GBs) of adult patients: functional bona fide chemical synapses formed between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses (NGS) show a typical synaptic ultrastructure, are located on TM networks, and produce depolarizing postsynaptic currents mediated by glutamate receptors of the AMPA subtype. AMPA-type glutamate receptors (AMPAR) are expressed by a molecularly and morphologically distinct subpopulation of network-integrated glioma cells. Increased neuronal activity under epileptic conditions ex vivo or neuronal optogenetic stimulation in vivo enhanced, while general anesthesia diminished synchronized calcium transients in TM-connected glioma networks. Accordingly, anesthesia reduced invasiveness of TM-positive tumour cells in mice. Genetic perturbation of AMPAR or chronic AMPAR inhibition by perampanel decreased glioma invasion and proliferation in mice and deletion of GluRII in Drosophila glioma increased survival. These findings reveal a hitherto unappreciated direct synaptic communication between neurons and glioma cells that appears relevant for brain tumour biology, implying new avenues for glioma treatment.
TMIC-28. LONG NON-CODING RNA EXPRESSION DIFFERS BETWEEN GLIOBLASTOMA PATIENT IMMUNE CELLS AND HEALTHY VOLUNTEERS
Authors: Nwankwo Anthony , Sanchez Victoria , Lynes John , Wang Xiang , Dominah Gifty , Obungu Arnold , Edwards Nancy , Sarpong Kwadwo , Ariyo Oluwatobi , Johnson Kory , K Nduom Edjah
Published: November 2019

To improve glioblastoma treatment, improved characterization of the tumor’s immune microenvironment is critical. Aberrant long non-coding RNA (LncRNA) expression has been implicated in the pathogenesis of many cancers, including glioblastoma. Other studies have discovered long non-coding RNAs which regulate immunity. However, lncRNA expression has not been systematically investigated glioblastoma patient immune cells. We aimed to investigate the differential expression of LncRNA and mRNs in the T lymphocytes and monocytes of glioblastoma patients when compared to healthy controls in an unbiased fashion. CD3 and CD14 cells were sorted from PBMC samples from 3 newly diagnosed glioblastoma patients and 3 normal donors, and RNA was extracted from them. These RNA samples were run on the Arraystar Human LncRNA microarray. We found 309 LncRNAs differentially expressed in glioblastoma patient T cells, and 316 in patient monocytes (fold change >2 or £0.5, P£0.05). A preliminary review of this data has revealed upregulation of lncRNAs that have previously been reported to play a role in tumorigenesis, such as MIF12-AS1, VIM-AS1, and WEE2-AS1. Additionally, lncRNAs such as CCDC26 and Hoxa10 that have been implicated in immune cell differentiation and hyperactivity also show differential expression between the two groups. We also found differential expression of 203 mRNA in T cells and 467 in monocytes (fold change ³2 or £0.5, P£0.05). An analysis based on the Kyoto Encyclopedia of Genes and Genomes identified 42 biological pathways that have enrichment of these differentially expressed mRNAs (P£0.05), including the RIG-I-like receptor pathway and toll-like receptor pathway, both implicated in innate immunity in T-cells. This array data will form the template for future single-cell RNA sequencing of tumor-infiltrating immune cells; peripheral immune cells from glioblastoma patients; and immune cells from non-tumorous brain (epilepsy tissue) and blood samples to investigate these differences in greater detail.
TMIC-30. MICROGLIA/BRAIN MACROPHAGES PROMOTE GLIOMA GROWTH BY EXPRESSING GLYCOPROTEIN NMB
Authors: Yalcin Fatih , Buonfiglioli Alice , Efecan Efe Ibrahim , Haneke Hannah , Kuhrt Leonard D , Synowitz Michael , Nickl Bernadette , Bader Michael , Kettenmann Helmut , Dzaye Omar
Published: November 2019

BACKGROUND

Glioma-associated microglia and blood-derived macrophages (GAMs) promote tumor growth in experimental mouse glioma models. Using microarray and RNA sequencing, we have previously shown that GAMs upregulate the expression of Glycoprotein NMB/Osteoactivin (GPNMB) when compared to naïve microglia. GPNMB is a type 1 transmembrane glycoprotein expressed intracellularly under healthy conditions. Malignancies such as glioma induce a translocation into the plasma membrane where the extracellular domain can be cleaved and released.

METHODS

We used qRT-PCR, immunocytochemistry, Western Blot and flow cytometry to determine the cellular localization of GPNMB expression in human and mouse glioblastoma. To test the impact of microenvironment-derived GPNMB on glioma growth, we inoculated GL261 and RCAS-PDGFb glioblastoma cells into organotypic brain slices obtained from wildtype and GPNMB-/- mice. In addition, we quantified glioma growth after injection of RCAS-PDGFb cells into wildtype and GPNMB-/- mice. The soluble extracellular domain of GPNMB was used to stimulate primary human glioblastoma and RCAS-PDGFb cells in vitro. SRB assays were performed to assess proliferation.

RESULTS

Our data indicate that GAMs are the predominant source of GPNMB in both human and mouse glioblastoma and that the levels of expression in GAMs in the tumor microenvironment is higher than in naïve microglia. In the organotypic brain slice model we found that tumors were significantly smaller in slices derived from GPNMB-/- mice as compared to wildtype. The tumor growth in vivo was nearly completely blocked in the absence of GPNMB. Stimulation of glioma cells with the extracellular domain of GPNMB did not increase proliferation.

CONCLUSION

Our results show that GPNMB is predominantly expressed in GAMs of human and murine samples. Loss of GPNMB impaired tumor growth ex vivo and glioblastoma progression in vivo. GPNMB seems to play a crucial role in the pro-tumorigenic activity of microglia and blood-derived macrophages in the tumor microenvironment.
ACTR-66. A PHASE 1, OPEN-LABEL, PERIOPERATIVE STUDY OF IVOSIDENIB (AG-120) AND VORASIDENIB (AG-881) IN RECURRENT IDH1 MUTANT, LOW-GRADE GLIOMA: UPDATED RESULTS
Authors: Mellinghoff Ingo , Cloughesy Timothy , Wen Patrick , Taylor Jennie , Maher Elizabeth , Arrillaga-Romany Isabel , Peters Katherine , Choi Changho , Ellingson Benjamin , Lin Alexander , Thakur Sunitha , Nicolay Brandon , Lu Min , Le Kha , Yin Feng , Tai Feng , Schoenfeld Steven , S Pandya Shuchi , Hassan Islam , Steelman Lori , Clarke Jennifer
Published: November 2019

BACKGROUND

Ivosidenib (AG-120, IVO) is a first-in-class oral inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1), and vorasidenib (AG-881, VOR) is an oral, potent, brain-penetrant inhibitor of mIDH1/2. Both have been evaluated in glioma patients in ongoing phase 1 studies. In orthotopic glioma models, IVO and VOR reduced 2-hydroxyglutarate (2-HG) levels by 85% and 98%, respectively, despite different brain-to-plasma ratios (< 0.04 vs 1.33).

METHODS

Patients with recurrent, nonenhancing, WHO-2016 grade 2/3, mIDH1-R132H oligodendroglioma or astrocytoma undergoing craniotomy were randomized 2:2:1 to IVO 500mg QD, VOR 50mg QD, or no treatment (cohort 1), or 1:1 to IVO 250mg BID or VOR 10mg QD (cohort 2), for 4 weeks preoperatively. Postoperatively, patients continued receiving IVO or VOR (control patients were randomized 1:1 to IVO or VOR). Tumors were assessed for mIDH1 status, cellularity, and 2-HG and drug concentrations. Treated subjects were compared with controls and mIDH1/wild-type banked reference samples. Primary endpoint: tumor 2-HG concentration following IVO or VOR.

RESULTS

As of March 1, 2019, 27 patients (18 men; 25/2 grade 2/3) were randomized preoperatively in cohort 1 (IVO 10, VOR 12, untreated 5): 27 received drug (IVO 13, VOR 14); 1 discontinued VOR postoperatively due to disease progression. Of 26 tumors analyzed, 22 were evaluable. Mean brain-to-plasma ratios: 0.13 IVO, 1.59 VOR. Relative to untreated samples, IVO and VOR reduced tumor 2-HG by 92.0% (95% CI 73.2, 97.4) and 92.5% (95% CI 78.1, 97.7), respectively. Common (≥ 4 patients) TEAEs (all cohort 1 patients, all grades): diarrhea (37.0%), constipation, hypocalcemia, and nausea (each 18.5%), anemia, hyperglycemia, pruritus, headache, and fatigue (each 14.8%). Cohort 2 has completed accrual, with analyses ongoing.

CONCLUSIONS

In cohort 1 of this phase 1 perioperative study, IVO and VOR demonstrated brain penetrance and lowered 2-HG compared with controls. Updated data from both cohorts will be presented.
TMIC-39. DEVELOPMENT OF CD11b TRACER FOR THE IMMUNE PET IMAGING IN GLIOBLASTOMA MODEL - COULD BE A GAME CHANGER FOR IMMUNOTHERAPY APPROACHES
Authors: Nigam Shubhanchi , McCarl Lauren , Kumar Rajeev , Anderson Carolyn , Edwards Barry , Kohanbash Gary
Published: November 2019

Glioblastoma is a lethal brain tumor, heavily infiltrated by tumor-associated myeloid cells (TAMCs). As up to 30% of a glioma cellular mass may be attributed to immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). TAMCs impede natural and immunotherapy-driven anti-tumor responses, they are a high-priority and promising therapeutic target currently being evaluated in clinical trials. Multiple preclinical and clinical trials have attempted to target these cells, however monitoring of biologic responses to therapy remains a challenge. Quantifying real time status of MDSCs and TAMs at the tumor site using non-invasive immunoPET could improve therapeutic response and allow for better patient stratification and monitoring of targeted treatment responses. TAMCs highly expressed the cell surface marker, integrin CD11b (Mac-1, αMβ2) and may be a highly effective imaging target for immunoPET strategies. The human/mouse cross-reactive anti-CD11b antibody (clone M1/70) was radiolabeled with 89Zr for PET imaging. PET/CT imaging, with or without a blocking dose of anti-CD11b Ab, was performed in mice bearing established orthotopic syngeneic GL261 gliomas. Flow cytometry and histology in tissues collected from post-imaging biodistribution validated targeting of CD11b+ MDSCs and TAMs. There was significant Zr-89-anti-CD11b Ab uptake in the tumor ipsilateral right brain (SUVmean = 2.6 ± 0.24) compared to contralateral left brain (SUVmean = 0.6 ± 0.11). Blocking with 10-fold lower specific activity 89Zr-anti-CD11b Ab reduced the SUV in right brain with (SUVmean = 0.11 ± 0.06). Immune rich organs spleen and lymph nodes showed high uptake. These results correlated with biodistribution analysis. CD11b expression in the right and left brain were validated using flow cytometry, H&E and IHC, showing high CD11b expression in the right brain. Imaging TAMs and MDSCs with 89Zr-labeled anti-CD11b Ab targeting was validated in a mouse model of malignant gliomas, demonstrating the feasibility of monitoring immune response during immunotherapy.
TMIC-50. 3D QUANTIFICATION OF DYNAMIC CYTOKINE GRADIENTS PRODUCED BY PRIMARY TUMOR MODELS
Authors: McGhee Eric , Uruena Juan , McGhee Alex , Mitchell Duane , Flores Catherine , Gregory Sawyer W
Published: November 2019

BACKGROUND & SIGNIFICANCE

The immune response is a coordinated effort directed by cytokine gradients and concentrations. High sensitivity and spatial resolution are necessary to resolve cytokine gradients in 3-dimensions. This system uses in situ confocal fluorescent microscopy with printed bead-based immunoassays. The combination of 3D printing of the beads and biofabrication of patient derived tumors allows for direct imaging, quantification, and movies of tumor cytokine secretion in response to challenges from immune cells, tumor associated fibroblasts, and chemotherapeutic agents. HYPOTHESIS: Cytokine dynamics can be measured in real-time by balancing concentrations of detection antibodies in solution with stationary immunoassay beads with measured capture and release rates. Favorable balancing of assay kinetics can facilitate measurements of concentrations of cytokines between 50 pg/mL to over 2,000 pg/mL.

METHODS

In situ confocal fluorescent microscopy identifies position, fluorescence, and type of bead relative to the in vitro tumor. Cytokines including IL2, IL6, IL8, IL11, and IFNg and growth factors such as VEGF and TGFb have been measured for a wide range of solid tumors (brain, sarcoma, and epithelial) under different stresses.

RESULTS

Dynamic gradients of IL8 were found across multiple tumor models showing local concentrations in excess of 2,000 pg/mL. Production rates were estimated to be over 1 protein per second for each cell, and inclusion of cancer associated support cells showed 10x increases in some cytokines. CD4+ cell production of IL2 was confirmed and quantified and showed strong sensitivity to tumor activity and antigen presentation. Multiplexed beads of CCL2, CCL5, CXCL8, CXCL9, and CXCL10 allow for simultaneous measurement of multiple chemokines. Uncertainties associated with fluorescence measurement and quantification of concentrations will be discussed.

CONCLUSIONS

This integrated system of 3D assay printing, biofabrication of tumors and immune cell constructs, and in situ confocal microscopy provides the first direct measurements of 3D cytokine gradients in response to tumor stress.
TMIC-59. INVESTIGATING PHYSIOLOGY OF THERAPY RESISTANT, TUMOR-INITIATING GBM CELLS IN HYPOXIC NICHES USING SPATIALLY-SENSITIVE HYPOXIA REPORTER SYSTEM AND SINGLE-CELL RNA SEQUENCING
Authors: Sattiraju Anirudh , Marallano Valerie , Friedel Roland , Zou Hongyan
Published: November 2019

Glioblastoma (GBM) is the most common and lethal brain cancer that invariably recurs after therapy due to presence of resistant GBM cells within hypoxic and peri-necrotic regions. Eradicating such GBM cells, which constitute a major source of tumor recurrence, is important to curb disease relapse. An endogenously expressed, spatially sensitive hypoxia reporter would therefore be a valuable tool to evaluate hypoxic zones in GBM in detail, and to measure the efficacy of hypoxia-activated drugs. For this purpose, we engineered a lentiviral vector that carries a hypoxia reporter, consisting of HIF response elements (HRE) that drive expression of UnaG fluorescent protein, which fluoresces independent of oxidative maturation. We validated the sensitivity of our reporter in vitro using U87MG, GBM2, and patient-derived GBM stem cell lines, and we performed intracranial transplantations of GBM cells in SCID mice to identify cells undergoing hypoxic stress in in vivo microenvironment. In addition, GL261 murine GBM cells with hypoxia reporter were intracranially implanted in C57BL/6 mice as syngeneic model for studies on immune responses. Brains from our transplant studies were dissociated and single-cell RNA sequencing (Drop-Seq) was performed to investigate heterogeneity in response to hypoxia within GBM cells and the cellular composition of microenvironment. We will also apply a hypoxia-activated prodrug, Evofosfamide (Evo), in our ongoing studies that can potentially eradicate hypoxic tumor cells and increase T cell infiltration and reverse immune suppression. As hypoxic niches are thought to confer resistance to radiation therapy (XRT), combining XRT with Evo could thus improve therapy efficacy. Our hypoxia gene reporter, combined with single-cell transcriptomics, could therefore serve as an effective tool to enable fundamental investigation of GBM microenvironment and could be used to evaluate therapies targeting tumor microenvironment to enhance GBM patient survival.
TMOD-13. MODELING THE GENETIC, TRANSCRIPTOMIC, AND CELLULAR HETEROGENEITY OF GLIOBLASTOMA USING TUMOR ORGANOIDS
Authors: Zhang Daniel , Jacob Fadi , Salinas Ryan , Nguyen Phuong , Ming Guo-li , Song Hongjun
Published: November 2019

Glioblastoma exhibits enormous genetic, transcriptional, and cellular heterogeneity at the macroscopic level across regions of the tumor as well as at the microscopic level between neighboring cells, all of which present significant challenges towards creating a definitive treatment for this devastating disease. We have developed a method of generating glioblastoma organoids (GBOs) from fresh tissue obtained directly from surgical resection and maintaining them in a defined medium without bFGF/EGF. Whole exome sequencing revealed that GBOs maintain the genomic landscape of their parent tumors. Somatic and copy number variants are present in the GBOs at similar allele frequencies or copy ratios as in the parent tumor, suggesting that the relative proportions of clonal populations are largely maintained in the organoids. Bulk transcriptomic analysis demonstrated strong gene expression correlations between the parent tumor and corresponding GBOs through 12 weeks of culture. Some tumors were sampled at multiple different anatomic regions, and the corresponding GBOs maintained region-specific gene expression signatures and genomic variants. EGFRvIII, a tumor-specific variant targeted in a number of emerging therapies, also remains present in the GBOs at similar transcript frequencies, reflecting the native heterogeneity of the parent tumor. Finally, we used single cell transcriptomics to examine cellular heterogeneity and find that GBOs contain many different cell types that exhibit similar gene expression profiles as the matching cell type in the corresponding parent tumor. Notably, these GBOs retain neoplastic as well as non-neoplastic cells, such as tumor associated macrophages / microglia, T-cells, endothelial cells, stromal cells, and oligodendrocytes. These GBOs preserve complex tumor heterogeneity an in vitro environment, creating opportunities for extended manipulation, characterization, and functional study for mechanistic investigation and therapeutic testing.
TMOD-22. LOSS OF PTEN AND TRP53 IN OLIGODENDROCYTE PROGENITORS LEADS TO GLIOMA FORMATION
Authors: Verma Ravinder , Lu Richard
Published: November 2019

Tumor suppressors PTEN and TP53 are the most commonly mutated anti-oncogenes in human gliomas after TERT mutation. 38% and 17% of total 4023 samples of gliomas in TCGA show TP53 and PTEN mutations respectively. TP53 signaling pathway plays an important role in the suppression of tumor progression PTEN negatively regulates PI3 triphosphate levels and suppresses AKT/PKB pathways. In-frame and missense mutations in PTEN constitutively activates the proliferative pathway and give a survival advantage. Mice with conditional mutation of PTEN and TP53 in neural stem cells directed by hGFAP-Cre develop high-grade gliomas. Our recent study indicates that primitive oligodendrocyte-progenitor intermediates (Pri-OPC) are abundant and hyperproliferative cells and can be progressively reprogrammed towards a stem-like state susceptible to malignant transformation. We hypothesize that mutations in PTEN and TP53 in pri-OPC lineage cells can lead to glioma formation. OPCs distribute across the whole adult brain and exhibit a much higher number than NSCs that are restricted to very small niche areas. Olig1 and Olig2 mark pri-OPC and oligodendrocyte lineage cells. In this study, we inactivated the PTEN/TP53 gene in mouse pri-OPCs using Olig1-Cre or Olig2tva-Cre. The incidence of glioma formation was 100%. Both the tumor formed in these models display multifocal, high-grade, diffuse and infiltrating gliomas. Though tumors are derived from similar cell sources, they vary in the expression and extent of oncogenic pathways. Our transcriptomics profiling at different stages of tumorigenesis reveals that progressive activation of oncogenic signaling pathways establishes a tumorigenic state to promote malignant transformation, suggesting a dynamic process of tumorigenesis caused by the cooperative action of the PTEN and TP53 loss in the pri-OPC lineage.
TMOD-34. A RADIOMIC SIGNATURE OF INFILTRATION IN PERITUMORAL EDEMA PREDICTS SUBSEQUENT RECURRENCE IN GLIOBLASTOMA
Authors: Rathore Saima , Shukla Gaurav , Akbari Hamed , Sako Chiharu , Bilello Michel , Mohan Suyash , Badve Chaitra , Barnholtz-Sloan Jill , Sloan Andrew , Lustig Robert , Davatzikos Christos
Published: November 2019

BACKGROUND

The malignant parenchyma in glioblastoma extends beyond the enhancing borders of tumor on postcontrast T1-weighted magnetic resonance imaging (MRI), which is the primary target of treatment. Such non-enhancing tumor invasion into the peri-tumoral edema (PED) is, however, not usually distinguishable on conventional MRI. The aim of this study was to evaluate pre-operative MRI in the PED to assess whether areas of tumor infiltration and early recurrence can be detected.

METHODS

A cohort of 90 de novo glioblastoma patients from a single institution (Penn) was selected. All included patients had preoperative multi-parametric MRI (mpMRI;T1,T1Gd,T2,T2-FLAIR,ADC), underwent initial gross-total-resection followed by standard chemoradiation, and had pathologically-confirmed recurrence. An extensive panel of handcrafted features, including shape, volume, intensity distributions, texture, was extracted from mpMRI scans. Predictive modeling for estimation of PED infiltration was performed using sequential feature selection approach designed with a support vector machine classifier and through a Leave-one-out cross-validation approach in the Penn cohort. Generalizability of the model was evaluated by applying it on a cohort of 20 patients from a second institution (Case), and predicted probability distributions in PED were compared in both the cohorts.

RESULTS

Spatial probability maps, representing the likelihood of tumor infiltration and eventual recurrence, were binarized at 50% cutoff, and compared with actual recurrence on post-recurrence scans. The cross-validated accuracy of our model within Penn cohort was 81.35% (odds-ratio=3.62, sensitivity/specificity=78.26/81.35). The model trained on the Penn cohort, when applied on the Case cohort, produced almost similar intensity distribution in the PED, suggesting that the method has potential for robust performance across institutions. The comparison of intensity distributions revealed higher ADC and T2-FLAIR in non-recurrent regions compared to recurrent ones.

CONCLUSION

Multi-parametric pattern analysis of mpMRI across multiple institutions generates similar, accurate estimates of spatial extent and patterns of recurrence in PED, which may guide strategies for treatment intensification in glioblastoma.
SCIDOT-17. UNDERSTANDING THE EFFECTS OF MOLECULAR SIZE ON VOLUME OF DISTRIBUTION IN CONVECTION-ENHANCED DELIVERY
Authors: Rechberger Julian S , Power Erica A , Zhang Liang , Olson Ian , Lu Victor M , Daniels David J
Published: November 2019

Diffuse midline gliomas harboring the H3K27M mutation, previously known as diffuse intrinsic pontine gliomas (DIPG), are rare and aggressive pediatric brain tumors without cure. One of the major challenge sin DIPG treatment is the effective delivery of therapeutic agents across the blood-brain barrier (BBB) to the tumor and surrounding infiltrating cells. Therefore, strategies that enhance drug delivery to the brain are of great interest. Convection-enhanced delivery (CED) is a technique that bypasses the BBB and increases drug distribution by applying hydraulic pressure to deliver compounds directly and evenly into a target region. However, knowledge in CED pharmacology and convective kinetics is still lacking. In an effort to characterize the feasibility, safety, and distribution in the brain based on molecular size of the delivered agent, we performed infusions of FITC-dextran (range 3,000 Da–150,000 Da) comparing CED and osmotic pump-based delivery into the brainstem of rodents. We calculated the area and volume of distribution (Vd) of the FITC-dextran throughout the brain. Our data showed that the Vd decreased exponentially with increased molecular weight of the FITC-dextran. Interestingly, the Vdcan maintain linearity at lower molecular weights. Maximal cross-sectional area and craniocaudal extension of fluorescence also decreased when lowering the infusate size. In addition, we developed a patient-derived DIPG orthotopic xenograft model and performed an image-guided CED cannula installation in the tumor bed. Using 3D bioluminescence imaging and computed tomography, we determined the tumor volume and the positioning of the cannula in the bulk tumor. The summation of these results supports CED as a promising technique for treating DIPG tumors. A better understanding of how drugs distribute by convection will allow us to optimize treatment regimens and, ultimately, offers hope to patients and families with this devastating disease.