bluuebunny/crossref_metadata_2025_split huggingface dataset:
IMMU-01. SINGLE CELL SEQUENCING OF MELANOMA BRAIN METASTASES UNVEILS HETEROGENEITY OF THE TUMOR MICROENVIRONMENT IN RESPONSE TO IMMUNE CHECKPOINT BLOCKADE
Authors: Alvarez-Breckenridge Christopher , Markson Samuel , Stocking Jackson , Lastrapes Matt , Nayyar Naema , Bertalan Mia , Kaplan Alexander , Alvarez Maria Martinez-Lage , Izar Benjamin , Suva Mario , Sullivan Ryan , Cahill Daniel , Carter Scott , Brastianos Priscilla 
Published: December 2020

Abstract
Immune checkpoint inhibitors (ICI) have revolutionized oncologic treatment for metastatic melanoma. With improved systemic control, there has been increasing prevalence of patients with brain metastases. Recent evidence has demonstrated intracranial responses in a subset of these patients treated with ICI. We hypothesize that the response to ICI in melanoma brain metastases (MBM) is reflective of unique features within the tumor microenvironment of the brain. A cohort of 27 patients, encompassing 8 pre- and 19 post-immunotherapy MBM underwent single cell RNA sequencing (Smart-Seq2). The cohort includes patients with longitudinal cranial resections and simultaneously resected, spatially distinct tumors. Each tumor underwent unsupervised transcriptomic analysis, differential gene expression, inferred copy number variation, and T-cell receptor (TCR) clonotyping. Published extracranial melanoma single cell datasets were used to compare the tumor microenvironment of the brain and periphery in response to ICI. A total of 14,027 cells (6,189 malignant, 7,838 non-malignant) were sequenced. Brain metastases demonstrated a heterogeneous distribution of macrophage states. Intracranial macrophages were found to be more tumor-supportive than their extracranial counterparts. MBM also included a distribution of reactive neutrophils and astrocytes. Analysis across pre- and post-treatment MBM demonstrated an increase in clonally expanded T cells in patients responding to ICI. Across longitudinal brain metastases collected from the same patients, there was evidence of identical T cell clones across timepoints and locations. Single cell sequencing of MBM provides insights into the cellular composition of the tumor and microenvironment. Our data suggest the cellular heterogeneity within MBM is unique when compared to extracranial disease. Additionally, T cell clonal expansion is found following ICI and T cells of the same clonotype infiltrate spatially and temporally separated brain metastases. These findings raise potential therapeutic implications as we learn to target the differential features of the innate and adaptive immune system within brain metastases and their extracranial counterparts.
IMMU-15. HEPARIN INHIBITS THE EXTRACELLULAR VESICLE-MEDIATED INDUCTION OF IMMUNOSUPPRESSIVE MONOCYTES IN GLIOBLASTOMA
Authors: Himes Benjamin , Fain Cori , Tritz Zachariah , Li Helen , Geiger Philipp , Peterson Timothy , Parney Ian 
Published: December 2020

Abstract
Glioblastoma (GBM) is the most common and fatal primary brain tumor in adults. The development of novel therapies is critical, as little has changed regarding the standard of care in nearly two decades. Immunotherapy holds much promise, as treatments including chimeric antigen receptor (CAR) T cells and immune checkpoint blockade inhibitors have transformed the treatment of a number of cancers in recent years. However, GBM patients exhibit profound immunosuppression, limiting the efficacy of these therapies. Understanding the mechanisms of GBM-mediated immunosuppression is critical to overcoming this barrier. GBM-derived extracellular vesicles (EVs) have been shown to mediate the induction of immunosuppressive monocytes, which may point to a mechanism of immunosuppression. EVs make initial contact with target cells through interactions between heparan sulfate proteoglycans, and soluble heparin has been shown to inhibit these interactions in some models. We demonstrate that soluble heparin inhibits the binding of GBM-derived EVs to monocytes in a dose-dependent manner, and that heparin treatment reduces the induction of immunosuppressive monocytes upon in vitro conditioning of monocytes with GBM-derived EVs (p&lt; 0.01). Further, we demonstrate that heparin treated EV-conditioned monocytes are functionally less immunosuppressive than untreated EV-conditioned monocytes as measured by T cell proliferation in co-culture studies (p&lt; 0.05). Taken together, these findings underscore the import of tumor-derived EVs in immunosuppression in GBM, and demonstrate the feasibility of targeting EV-monocyte interactions in treating GBM-mediated immunosuppression.
IMMU-22. EARLY RELEASE OF TYPE I INTERFERON DRIVES BRAIN CANCER IMMUNOGENICITY AND RESPONSE TO IMMUNOTHERAPY
Authors: Wummer Brandon , Qdaisat Sadeem , Grippin Adam , Karachi Aida , Weidert Frances , Huang Jianping , Rahman Maryam , Mitchell Duane , Mendez-Gomez Hector , Sayour Elias 
Published: December 2020

Abstract

BACKGROUND
Molecular drivers of cancer immunogenicity in brain tumors are still being unraveled. While BATF3 expression, STING, and interferon response factors (IRFs) are necessary for cancer immunogenicity, the presence of type I interferon (IFN-I) is contextual having been reported to elicit both anti-tumoral and pro-tumoral effects. A better understanding of IFN-I signaling mechanisms is necessary to elucidate drivers of brain cancer immunogenicity and resistance.

OBJECTIVE
We sought to assess the role of IFN-I signaling in brain tumor immunogenicity and response to immune checkpoint inhibitors (ICIs) in ICI sensitive brain tumor models (i.e. GL261). We then sought to develop strategies to reset IFN-I signaling in ICI resistant brain tumor models (i.e. KR158b).

METHODS
To reset IFN-I signaling in immunologically ‘cold’ tumors unresponsive to ICIs, we developed lipid-nanoparticles (NPs) to deliver mRNA payloads to the brain tumor microenvironment (TME).

RESULTS
In immune-sensitive GL261 tumors, we showed that early release of IFN-I unlocks cancer immunogenicity and ICI response. Blockade of IFN-I during tumorigenesis (within 24h, but not days later) increases tumorigenicity and abrogates ICI activity in sensitive tumors. In ICI resistant KR158b tumors, we show that systemic administration of tumor-derived RNA-NPs localize to myeloid cells within the TME for simultaneous activation of multiple innate pathways including BATF3 (necessary for effector DCs), IRF5 (necessary for M2 to M1 macrophage reprograming), and IRF7 (necessary for IFN-I production). These RNA-NPs induce near-immediate release of IFN-I (within hours), reprogram the brain TME in an IFNAR1 (IFN-I receptor) dependent manner, and elicit significant anti-KR158b efficacy as a monotherapy. Following IFNAR1 blockade, RNA-NP mediated anti-tumor efficacy was abrogated. We demonstrated safety of tumor-specific RNA-NPs (derived from KR158b) in acute/chronic GLP toxicity studies without normal-brain cross-reactivity, and confirmed feasibility/safety and immunologic activity in large-animal studies.

FUTURE DIRECTIONS
We have since received FDA-IND approval for first-in-human trials (IND#BB-19304) in glioblastoma patients.
IMMU-24. TEMOZOLOMIDE INDUCED IMMUNE DYSFUNCTION REQUIRES THE PRESENCE OF INTRACRANIAL TUMORS
Authors: Karachi Aida , Dastmalchi Farhad , O’Malley Ashley , Yang Changlin , Mitchell Duane , Rahman Maryam 
Published: December 2020

Abstract
Temozolomide was recently shown to cause peripheral and intra-tumoral T cell dysfunction in a dosing schedule dependent fashion. Standard dose (SD) temozolomide (TMZ) resulted in T cell dysfunction precluding response to immune checkpoint inhibition that was avoided with a metronomic dosing (MD) schedule. Building on these studies, we investigated the TMZ-induced immune changes in tumor and non-tumor bearing models to understand the interaction of an intracranial tumor on host immunity. C57BL/6 mice underwent intracranial implantation of GL-261 tumor cells. Tumor bearing animals and naïve animals with no tumor were treated with standard dose (50 mg/kg x 5 days) or metronomic dose (25mg/kg x 10 days) of TMZ. Peripheral blood and spleens were collected for flow cytometry, ELISA and luciferase killing assay. Tumor bearing animals treated with SD TMZ demonstrated an increase in circulating myeloid derived suppressor cells (MDSCs), an upregulation of exhaustion markers on endogenous host CD8 T cells (TIM3, LAG3) and a decrease in IFN-gamma secretion from adoptively transferred T cells (tested via ELISA). The cell killing capability of adoptively transferred T cells was not reduced after exposure to a TMZ treated host. Non-tumor bearing animals treated with SD TMZ did not demonstrate an increase in circulating MDSCs or exhaustion markers on endogenous T cells. IFN-gamma secretion from adoptively transferred T cells was still reduced in these animals. The host immune dysfunction induced by TMZ is dependent on the presence of an intracranial malignancy. The mechanisms causing these changes are under active investigation.
IMMU-25. SEVERE AND MULTIFACETED SYSTEMIC IMMUNOSUPPRESSION CAUSED BY EXPERIMENTAL CANCERS OF THE CENTRAL NERVOUS SYSTEM REQUIRES RELEASE OF NON-STEROID SOLUBLE MEDIATORS
Authors: Ayasoufi Katayoun , Pfaller Christian , Evgin Laura , Khadka Roman , Tritz Zachariah , Goddery Emma , Fain Cori , Yokanovich Lila , Himes Benjamin , Jin Fang , Zheng Jiaying , Schuelke Matthew , Hansen Michael , Tung Wesley , Parney Ian , Pease Latty , Vile Richard , Johnson Aaron 
Published: December 2020

Abstract
Immunosuppression of unknown etiology is a hallmark feature of glioblastoma (GBM) and is characterized by decreased CD4 T cell counts and down regulation of MHC class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for GBM. We recapitulated the immunosuppression observed in GBM patients in the C57BL/6 mouse and investigated the etiology of low CD4 T cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of CNS cancer, including mice harboring GL261 glioma, B16 melanoma, and in a spontaneous model of Diffuse Intrinsic Pontine Glioma (DIPG). In addition to thymic involution, we determined that tumor growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC class II expression on hematopoietic cells, and a modest increase in bone marrow resident CD4 T cells with a naïve phenotype. Using parabiosis we report that thymic involution, declines in peripheral T cell counts, and reduced MHC class II expression levels were mediated through circulating blood-derived factors. Conversely, T cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is nonsteroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the aforementioned immunosuppression was not unique to cancer itself, but rather occurs in response to CNS injury. Noncancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that CNS cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.
IMMU-27. SINGLE CELL RNA-SEQUENCING IDENTIFIES NOVEL BONE MARROW DERIVED MYELOID CELLS IN GLIOBLASTOMA ASSOCIATED WITH TUMOR AGGRESSION
Authors: Jackson Christina , Cherry Christopher , Bom Sadhana , Zhang Hao , Choi John , Yegnasubramanian Vasan , Lim Michael , Elisseeff Jennifer , Pardoll Drew 
Published: December 2020

Abstract

BACKGROUND
Glioma associated myeloid cells (GAMs) can be induced to adopt an immunosuppressive phenotype that can lead to inhibition of anti-tumor responses in glioblastoma (GBM). Understanding the composition and phenotypes of GAMs is essential to modulating the myeloid compartment as a therapeutic adjunct to improve anti-tumor immune response.

METHODS
We performed single-cell RNA-sequencing (sc-RNAseq) of 435,400 myeloid and tumor cells to identify transcriptomic and phenotypic differences in GAMs across glioma grades. We further correlated the heterogeneity of the GAM landscape with tumor cell transcriptomics to investigate interactions between GAMs and tumor cells.

RESULTS
sc-RNAseq revealed a diverse landscape of myeloid-lineage cells in gliomas with an increase in preponderance of bone marrow derived myeloid cells (BMDMs) with increasing tumor grade. We identified two populations of BMDMs unique to GBMs; Mac-1and Mac-2. Mac-1 demonstrates upregulation of immature myeloid gene signature and altered metabolic pathways. Mac-2 is characterized by expression of scavenger receptor MARCO. Pseudotime and RNA velocity analysis revealed the ability of Mac-1 to transition and differentiate to Mac-2 and other GAM subtypes. We further found that the presence of these two populations of BMDMs are associated with the presence of tumor cells with stem cell and mesenchymal features. Bulk RNA-sequencing data demonstrates that gene signatures of these populations are associated with worse survival in GBM.

CONCLUSION
We used sc-RNAseq to identify a novel population of immature BMDMs that is associated with higher glioma grades. This population exhibited altered metabolic pathways and stem-like potentials to differentiate into other GAM populations including GAMs with upregulation of immunosuppressive pathways. Our results elucidate unique interactions between BMDMs and GBM tumor cells that potentially drives GBM progression and the more aggressive mesenchymal subtype. Our discovery of these novel BMDMs have implications in new therapeutic targets in improving the efficacy of immune-based therapies in GBM.
IMMU-34. LANDSCAPE OF GLIOBLASTOMA-ASSOCIATED IMMUNITY BY INTEGRATION OF scRNA-SEQ AND SPATIAL TRANSCRIPTOMICS
Authors: Ravi Vidhya , Neidert Nicolas , Joseph Kevin , Beck Juergen , Schnell Oliver , Heiland Dieter 
Published: December 2020

Abstract
The diversity of molecular states and cellular plasticity of immune cells within the glioblastoma (GBM) environment remain poorly investigated. Here, we conduct deep transcriptional profiling of lymphoid and myeloid cell populations by scRNA-sequencing, map potential cellular interactions and cytokine responses that lead to the dysfunctional and exhausted phenotype of T cells. We identified Interleukin 10 (IL-10) response during T cell activation, which lead to a dysfunctional state of T cells. By the use of a novel method: The nearest functionally connected neighbor (NFCN), an in-silico model to explore cell-cell interaction, the dysfunctional/exhausted phenotype was found to be driven by subset of myeloid cells defined by high expression of HMOX1. By using spatial transcriptomic RNA-sequencing, we identified a correlation between T cell exhaustion and colocalized mesenchymal gene expression. We found that HMOX1 expressing myeloid cells occupying regions marked by T cell exhaustion. Using a human neocortical slice model with myeloid cell depletion we confirmed the functional interaction of myeloid and lymphoid cell leading to the dysfunctional state of T cells. A comprehensive understanding of cellular states and plasticity of lymphoid cells in GBM aids in providing successful immunotherapeutic approaches.
IMMU-36. HIGH AFFINITY CHIMERIC ANTIGEN RECEPTOR WITH CROSS-REACTIVITY TO CLINICALLY-RELEVANT EGFR MUTATED PROTEINS
Authors: Thokala Radhika , Binder Zev , Yin Yibo , Milone Michael , O’Rourke Donald M 
Published: December 2020

Abstract
Tumor heterogeneity is one of the key reasons for therapeutic failure in Glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell clinical trial (NCT02209376) against Epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood brain barrier into GBM active tumor sites. However, CART cell infiltration was associated with a selective loss of EGFRvIII+ tumor cells and upregulation of immunosuppressive molecules. Post-CAR T treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations. To study this further We generated two structurally different CARs by fusing the scFv of mAb806 to 4-1BB and Killer immunoglobulin like receptor (KIR) co-stimulatory domains. Both 4-1BB and KIR based EGFR806 CAR T cells specifically lysed tumor cells and secreted cytokines when co-cultured with U87-MG (expressing low levels of EGFR) and U87-MG EGFR (U87-MG transduced with EGFR wild type for amplified background) cell lines and engineered to ectopically express targeted EGFR-ECD missense mutations and EGFRvIII. Unlike EGFR-specific cetuximab based CAR, EGFR-806CART cells did not kill EGFR wild type expressing fetal brain primary astrocytes and keratinocytes in vitro. We further evaluated the in vivo efficacy of 806-CARs and EGFRvIII CART currently in clinic in NSG mice. While EGFRvIII CART cells cleared EGFRvIII+ tumors alone 806 CART cells controlled EGFRvIII, amplified EGFR and EGFRR108K-mutant bearing tumors. KIR-CAR treated tumors are GVHD free and enhanced survival compared to 4-1BB based CARs. The broad specificity of EGFR806 CART cells to amplified EGFR and its mutant variants gives us the potential to clear various forms of EGFR. The enhanced anti-tumor efficacy by KIR based CAR in vivo setting provides us with additional therapeutic options.
IMMU-53. CHARACTERIZATION OF THE GENOMIC AND IMMUNOLOGICAL DIVERSITY OF MALIGNANT BRAIN TUMORS THROUGH MULTI-SECTOR ANALYSIS
Authors: Schaettler Max , Richters Megan , Griffith Malachi , Dunn Gavin 
Published: December 2020

Abstract
While the degree of spatial genomic heterogeneity within primary glioblastoma (GBM) has been previously investigated, the extent of this heterogeneity in other malignant brain tumors and its connection to the local immune microenvironment remains unknown. To address this, we performed whole-exome, RNA, and TCR sequencing on multiple spatially distinct sectors from a cohort of malignant brain tumors comprised of both brain metastases and primary and recurrent GBMs. Our results suggest a striking difference in which a majority of mutations and predicted neoantigens are shared between spatially distinct regions of metastatic brain tumors in contrast to the spatial heterogeneity observed within both primary and recurrent GBMs. Additionally, despite substantial differences in immunotherapy responsiveness between brain metastases and GBM, we can detect significantly expanded T-cell clonotypes within both tumor types with some clonal frequencies exceeding 10% of intratumoral T-cells. Similar to the observed distribution of variants and neoantigens, the expanded clonotypes are more shared among spatially distinct sectors in metastatic brain tumors than in primary or recurrent GBMs. Interestingly, the frequencies of most of these enriched T-cell clones are significantly diminished within expanded tumor infiltrating lymphocyte (TIL) cultures, advising caution in the use of expanded TIL for the detection of tumor-specific responses. These results provide novel insight into the immunogenomic landscape of malignant brain tumors with implications for our understanding of tumor-immune interactions and the development of immunotherapies.
INNV-01. NEUROLOGIC IMPROVEMENT WITH CORTICOSTEROIDS PRIOR TO SURGERY IS PREDICTIVE OF DURABLE POST-OPERATIVE IMPROVEMENT IN MOTOR DEFICITS DUE TO BRAIN METASTASES
Authors: Bowden Stephen , Cheaney Barry , Yaghi Nasser , Munger Daniel , Ramos Christian Lopez , Richie Emma , Wood Matthew , Raslan Ahmed , Cetas Justin , Dogan Aclan , Han Seunggu 
Published: December 2020

Abstract
Corticosteroids reduce vasogenic cerebral edema and are thought to improve related neurologic deficits or symptoms of increased intracranial pressure. Brain metastases are typically associated with a large amount of edema and, consequently, come with a disproportionate degree of mass effect that may cause such deficits. There remains no standard approach to pre-operative corticosteroid therapy, nor is it understood what clinical characteristics are associated with a neurologic response to pre-operative steroids. We examined characteristics of steroid responders versus non-responders and, further, evaluated whether a response to preoperative steroids is predictive of durable improvement in neurologic function. Patients with pathology-proven brain metastases who underwent open surgical resection between 2009 and 2019 were identified from departmental records. Charts were reviewed to identify patients with motor dysfunction who received corticosteroids prior to surgery. Multiple patient and clinical characteristics were extracted and compared using student t-, chi-square, and Fisher’s exact tests. 90 patients exhibited pre-operative motor deficits, 69 of whom received corticosteroids prior to surgery (dose 2 – 112 mg; median 25 mg). 34 patients neurologic function improved prior to surgery, whereas 35 patients had no demonstrable improvement. All 34 patients (100%) whose motor function improved pre-operatively with steroids had sustained improvement at follow-up, whereas 27 of 35 (77%) patients who did not improve pre-operatively were better at follow-up (p = 0.005). All other clinical characteristics were similar between responders and non-responders. All motor deficits related to brain metastases that responded to steroids prior to surgery demonstrated durable improvement at follow-up, suggesting such an improvement portends a favorable long-term functional outcome. Conversely, a failure to improve with steroid therapy confers a more guarded prognosis.
INNV-04. CONGENITAL SPINAL GLIOBLASTOMA WITH SUSTAINED RESPONSE TO TROPOMYOSIN RECEPTOR KINASE (TRK) INHIBITION: A CASE REPORT
Authors: Coleman Abadi Christina , Sun Peter , Hastings Caroline 
Published: December 2020

Abstract
Large scale genomic efforts have shown that a significant percentage of infant high-grade gliomas (HGG) carry targetable gene fusions. Given the rarity of congenital spinal HGGs, little is known about the molecular features of these tumors and their response to treatment. We describe a case of a patient with congenital spinal glioblastoma. A full-term female infant with symmetric IUGR was noted to have weakness with little movement of her upper extremities. MRI of the spine showed an infiltrative and hemorrhagic intraspinal cervicothoracic neoplasm with a large hemorrhagic intramedullary component. A biopsy was done and revealed a high-grade glial neoplasm with pan-TRK expression. Next generation sequencing (NGS) confirmed a MEF2D-NTRK1 gene fusion. Prior to the result of her molecular testing, the patient clinically deteriorated with decreased movement and increased apneic episodes despite steroid treatment. She was then started on treatment with carboplatin and etoposide per Macy et al 1. After one cycle of chemotherapy and given the results of NGS, targeted therapy with larotrectinib was added to her treatment. The patient showed a response (clinical and imaging) following cycle 1 with chemotherapy, and a marked response following cycle 2 with both chemotherapy and targeted TRK inhibition. The patient completed six cycles of chemotherapy and has continued on daily oral larotrectinib. She continues to tolerate the targeted therapy well and currently is 8 months old, growing and developing well. She has regained much of her movement and is eating and growing with apparent normal development. This case provides an example of a patient with congenital glioblastoma with TRK gene fusion that showed an impressive response to targeted therapy combined with more conventional chemotherapy. 1Macy M, et al. Clinical and molecular characteristics of congenital glioblastoma. Neuro Oncol. Jul;14 (7) 931–941, 2012).