bluuebunny/crossref_metadata_2025_split huggingface dataset:
IMMU-09. HUMAN MICROBIOTA INFLUENCE THE EFFICACY OF IMMUNOTHERAPY IN A MOUSE MODEL OF GLIOBLASTOMA
Authors: Dees Kory , Koo Hyunmin , Humphreys James , Hakim Joseph , Crossman David , Crowley Michael , Nabors L Burt , Benveniste Etty , Morrow Casey , McFarland Braden 
Published: November 2021

Abstract
Although immunotherapy works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has unfortunately not demonstrated efficacy in humans. In melanoma and other cancers, the composition of the gut microbiome has been shown to determine responsiveness or resistance to immune checkpoint inhibitors (anti-PD-1). Most pre-clinical cancer studies have been done in mouse models using mouse gut microbiomes, but there are significant differences between mouse and human microbial gut compositions. To address this inconsistency, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a pre-clinical mouse model of GBM. We used five healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate five independent humanized mouse lines (HuM1-HuM5). Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. Interestingly, we found that the HuM lines responded differently to anti-PD-1. Specifically, we demonstrate that HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls, while HuM1, HuM4, and HuM5 mice are resistant to anti-PD-1. These mice are genetically identical, and only differ in the composition of the gut microbiome. In a correlative experiment, we found that disrupting the responder HuM2 microbiome with antibiotics abrogated the positive response to anti-PD-1, indicating that HuM2 microbiota must be present in the mice to elicit the positive response to anti-PD-1 in the GBM model. The question remains of whether the “responsive” microbial communities in HuM2 and HuM3 can be therapeutically exploited and applicable in other tumor models, or if the “resistant” microbial communities in HuM1, HuM4, and HuM5 can be depleted and/or replaced. Future studies will assess responder microbial transplants as a method of enhancing immunotherapy.
IMMU-11. SELECTIVE ENRICHMENT OF SUPPRESSED IMMUNE CELLS IN THE TUMOR MICROENVIRONMENT CORRELATES WITH ESTABLISHMENT OF DISTINCT IMMUNOLOGIC NICHES IN HUMAN GLIOBLASTOMAS CONTACTING THE LATERAL VENTRICLE
Authors: Bartkowiak Todd , Brockman Asa , Mobley Bret , Mistry Akshitkumar , Barone Sierra , Hayes Madeline , Roe Caroline , Sinnaeve Justine , Leelatian Nalin , Greenplate Allison , Chambless Lola , Thompson Reid , Weaver Kyle , Irish Jonathan , Ihrie Rebecca 
Published: November 2021

Abstract
Glioblastomas (GBM) account for 60% of adult primary brain tumors. With few advances in therapeutics, median overall survival remains 15-months post-diagnosis. Immunotherapies may provide therapeutic benefit in GBM patients; however, no predictive immune features currently inform therapeutic stratification in GBM. We have shown that, independently of known prognosticators, radiographic tumor contact with the lateral ventricle (C-GBM) correlates with 7-months worse prognosis compared to patients with ventricle non-contacting GBM (NC-GBM). This study sought to characterize the GBM immune microenvironment and identify targetable mechanisms of immunosuppression correlating with worse outcomes in C-GBM. Primary glioblastoma specimens were resected in accordance with the Declaration of Helsinki (IRB #131870). Twelve patients presented with C-GBM and thirteen with NC-GBM. Machine learning tools applied to mass cytometry data characterized tumor-infiltrating immune populations and identified biomarkers correlating with C-GBM and patient survival. C-GBM tumors were enriched in blood-derived macrophages compared to NC-GBM (19 ± 8% vs. 6 ± 2%; p&lt; 0.001) and depleted in lymphocytes (2.9 ± 1% vs. 7.6 ± 2%; p&lt; 0.001) and tissue-resident microglia (1.8 ± 0.3% vs. 7 ± 3%; p&lt; 0.001). Further, T cells in C-GBM co-expressed the checkpoint receptors PD-1 and TIGIT, suggesting acute T cell exhaustion. Multiplex immunohistochemistry (mxIHC) on matched FFPE tissue provided spatial context to risk-stratifying immune populations, and defined structured immunological niches within the TME. Macrophage-tumor niches were most common (36%), followed by T cell-microglia-tumor niches (26%). Within niches, T cell-T cell interactions were more prevalent (log odds ratio = 0.90) whereas T cell-macrophage interactions were less prevalent (log odds ratio = -1.61). These findings suggest that factors within the periventricular space may influence antitumor immunity within tumors, and identify clinically targetable immune biomarkers in glioblastoma. Notably, radiologic assessment of lateral ventricle contact by standard-of-care MRI may guide clinical trial design for immunotherapies in neuro-oncology.
IMMU-15. INVESTIGATING CD8 T CELL EXHAUSTION STATES GLIOBLASTOMA
Authors: Polania Jessica Waibl , Tomaszweski William , Hoyt-Miggelbrink Alexandra , Woroniecka Karolina , Fecci Peter 
Published: November 2021

Abstract

BACKGROUND
Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Median survival remains a bleak 15-17 months from time of diagnosis, and current immunotherapeutic efficacy continues to be hindered by the robust immunosuppression present in the GBM microenvironment. T cells, critical for tumor clearance, are particularly affected, and many take on a functionally exhausted phenotype within the tumor. Importantly, two exhaustion states, progenitor and terminal, have been identified in models of chronic infection and cancer. This distinction is particularly relevant, as progenitor exhausted T cells can respond favorably to immune checkpoint blockade, while terminally exhausted T cells are resistant. To date, the dynamics and characteristics of these exhausted populations in GBM remain unclear.

RESULTS
In an orthotopic murine model of GBM, progenitor and terminal exhausted CD8 T cells were identified by flow cytometry as PD1+SLAMF6+ and PD1+TIM3+, respectively. Using a time-course approach, we detected progenitor exhaustion by day 8 in the tumor, but not in draining lymph nodes. Additionally, we show that the frequency of progenitor exhaustion is highest during early tumor progression, while terminal exhaustion is the most abundant in more advanced tumors ( &gt;14 days). Functional differences between subsets were evaluated via intracellular staining of IFNγ, TNFα, granzyme B, and Ki67. Terminally exhausted T cells displayed higher cytotoxic molecule expression than progenitor exhausted T cells, similar to what has been documented in melanoma models.

CONCLUSIONS
Our findings identify T cell exhaustion subsets within GBM that require further investigation and may be relevant to overcome current barriers to immunotherapeutic efficacy.
IMMU-27. LONG TERM STABILIZATION OF RECURRENT HIGH-GRADE GLIOMA WITH PD-1 INHIBITOR PEMBROLIZUMAB IN TWO CASES
Authors: Moldenhauer Matthew , Du Steven , Fu Dan , Myung Joon , Bota Daniela , Kong Xiao-Tang 
Published: November 2021

Abstract

INTRODUCTION
Despite PD-1 inhibition having success in many cancers, it has uncertain effects in brain tumors. We report two cases of recurrent high-grade gliomas that have remained stable for over one year since starting pembrolizumab.

CASE REPORTS
Case 1: A 59-year-old male was diagnosed with glioblastoma (GBM) without MGMT methylation or IDH mutation in late 2018 after surgery. He received radiation and temozolomide (TMZ) followed by adjuvant TMZ before tumor progression. He underwent second tumor debulking with recurrent GBM on pathology with negative PD-L1 expression. He started carboplatin. Progression was noticed after 7 to 8 cycles. Pembrolizumab was added. Tumor was stabilized. Carboplatin was completed after total 12 cycles and the patient has continued single agent of pembrolizumab for more than one year with stable brain MRIs. The patient has survived for 24 months since recurrence and 30 months since diagnosis. Case 2: A 53-year-old male had a brain tumor discovered on MRI in 2012 and received no treatment until resection in 2014. In 2016, he underwent second tumor debulking and was diagnosed with anaplastic oligodendroglioma with negative PD-L1 expression. He received radiation followed by PCV regimen. 17 months since diagnosis, he had first tumor progression on PCV. TMZ was started. 22 months since diagnosis, bevacizumab was initiated due to further growth. 33 months since diagnosis, pembrolizumab was added due to new lesions after 12-months of bevacizumab therapy. His tumor was stabilized. Bevacizumab was eventually discontinued. He has continued single agent pembrolizumab for 6 months so far. His tumor has been stable for 22 months since starting pembrolizumab. Survival has been 38 months from first recurrence and 7 years since tissue diagnosis.

DISCUSSION
These cases demonstrate the potential effects of anti-PD-1 immunotherapy in stabilizing recurrent high-grade glioma with combination of other treatment agents followed by single agent as maintenance therapy.
IMMU-39. EVALUATION OF CD317-TARGETING CAR T CELLS AS A NOVEL IMMUNOTHERAPEUTIC STRATEGY AGAINST GLIOBLASTOMA
Authors: Hänsch Lena , Peipp Matthias , Myburgh Renier , Silginer Manuela , Weiss Tobias , Gramatzki Dorothee , Vasella Flavio , Manz Markus , Weller Michael , Roth Patrick 
Published: November 2021

Abstract
Glioblastoma remains one of the deadliest cancers despite aggressive treatment, which is why novel therapeutic approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy has demonstrated significant success in the field of hematological malignancies. However, treating glioblastoma with this type of therapy is more difficult for several reasons such as the lack of suitable target antigens. Here, we generated a second-generation CAR construct targeting the transmembrane protein CD317 (BST-2, HM1.24), which is expressed by human glioma cell lines in vitro as well as in vivo. We demonstrate strong anti-glioma activity of CD317-CAR T cells against different glioma target cells with varying CD317 expression levels. Glioma cells harboring a CRISPR/Cas9-mediated CD317 knockout were not susceptible for these CAR T cells, demonstrating their target antigen-specificity. CD317 is also expressed on T cells and transduction with a CD317-directed CAR impaired expansion of T cells due to residual CD317 expression and subsequent fratricide. Therefore, we silenced CD317 in the transduced T cells by co-expressing the CAR construct with a specific shRNA, which significantly increased the viability, proliferation and cytotoxicity of the CAR T cells. Finally, we observed strong anti-glioma activity of CD317-CAR T cells in clinically relevant orthotopic xenograft glioma mouse models resulting in prolonged survival of CAR T cell-treated animals. Taken together, these data reveal a promising role of CD317 as a novel target for CAR T cell therapy in glioblastoma and warrant further evaluation of this strategy in clinical neuro-oncology.
IMMU-40. IMMUNE PROFILING OF MATCHED PRIMARY AND RECURRENT GLIOBLASTOMA TUMORS IDENTIFIED CD64 AS A NEGATIVE PREDICTOR FOR SURVIVAL
Authors: Shekarian Tala , Ritz Marie-Françoise , Martins Tomàs , Schmassmann Philip , Hutter Gregor 
Published: November 2021

Abstract

BACKGROUND
To target tumor-induced immune evasion mechanisms of glioblastoma for clinical research, we aimed at characterizing the immunological changes in patient-matched treatment-naïve and recurrent GBM samples.

MATERIAL AND METHODS
Proteins and total RNA were extracted from 15 patient-matched treatment-naïve (primary) and recurrent GBM fresh frozen tumor samples. The expression of genes involved in brain immune responses were compared using Nanostring panels encompassing Cancer and Neuroinflammation genes. Correlation analysis using log2-transformed patient-matched recurrent/primary expression ratio (log2(P/R)) and clinical outcome data (time to relapse and overall survival (OS)) was conducted. Proteomic was conducted to compare total protein expressions. Moreover, immunostaining was performed on a tissue microarray of matched tumor samples to visualize CD64+ cells, CD47+ tumor cells and microglia in order to assess quantitative transcriptome of these specific cell types using GeoMX technology. Functional assays are designed for deciphering phagocytosis induction by factors produced by primary and recurrent tumors.

RESULTS
Genes significantly differentially expressed between primary and recurrent tumors revealed “Antigen presentation” and “Microglia Pathogen Phagocytosis” as the most significant pathways enriched in recurrent tumors. Considering individual genes, a strong negative correlation (R2&gt;0.4) between log2(R/P) and time to relapse and OS was found for 2 genes, FCGR1A (CD64) and CD47. We observed that only CD64 expression levels in recurrent samples also negatively correlated with OS. CD64 and CD47 proteins were quantified by proteomic analysis and log2 (R/P) of their levels also negatively correlated with time to relapse. Immunostaining revealed numerous CD64-positive cells in tumor biopsies resembling bushy or amoeboid microglia.

CONCLUSION
Upregulation of CD64 and CD47 expressions in recurrent GBM was identified as a negative predictor for time to relapse and OS. This suggests an important role of CD64 and CD47 and related inflammation dysregulation in tumor resistance and regrowth. Interfering with CD64-induced functions may represent a novel therapeutic option for GBM.
IMMU-41. NEOADJUVANT PD-1 BLOCKADE INDUCES T CELL AND CDC1 ACTIVATION BUT FAILS TO OVERCOME THE IMMUNOSUPPRESSIVE TUMOR ASSOCIATED MACROPHAGES IN RECURRENT GLIOBLASTOMA
Authors: Lee Alexander , Sun Lu , Mochizuki Aaron , Reynoso Jeremy , Orpilla Joey , Chow Frances , Kienzler Jenny , Everson Richard , Nathanson David , Liau Linda , Cloughesy Timothy , Hugo Willy , Prins Robert 
Published: November 2021

Abstract
In a small phase I clinical trial, neoadjuvant anti-PD-1 (neo-aPD1) improved survival in glioblastoma patients (GBM) and was associated with increased interferon-γ-related gene expression and an associated decrease in cell cycle-related gene expression in the tumor. Despite this, neo-aPD1 was not curative. To better understand how neo-aPD1 alters the GBM tumor microenvironment and to discover new therapeutic axes, we used CyTOF and single-cell RNAsequencing to analyze the tumor-infiltrating immune populations of 70 GBM patients, 20 of whom had received neo-aPD1. In patients treated with neo-aPD1 the proportion and number of tumor-infiltrating T cells increased and these T cells had increased expression of the cytokines CCL5 and XCL1, which promote dendritic cell migration. Among the infiltrating T cells was a population of progenitor exhausted CD8+ T cells expressing the genes TCF, SLAM6, CCR7, IL7R, and GZMB. Single-cell TCR analysis revealed that these CD8+ T cells arose from a population of early activated, cytotoxic T cells that had expanded in the peripheral blood and trafficked into the tumor. Within the tumor-infiltrating myeloid cells, we found that neo-aPD1 upregulated genes in the interferon-γ response pathways; namely genes related to T cell trafficking and immune suppression, such as CD274 (PD-L1), TREM2, GPNMB, and IL10, indicating a maladaptive response by some myeloid cells to the immune response triggered by neo-aPD1. Finally, we identified a new migratory and interferon-γ activated tumor-infiltrating conventional dendritic cell population that was increased with neo-aPD1 and that notably expressed XCR1, the receptor for XCL1. Additionally, our results provide future basis to study if modalities that enhance the T cell:DC axis or that inhibit the inhibitory myeloid populations may improve survival in patients with GBM when used in conjunction with neo-aPD1. In sum, we characterized the immune landscape in GBM and how it changes with neo-aPD1 at single cell resolution.
IMMU-42. OPTIMIZING IMMUNOTHERAPY FOR GLIOMA USING CYTOKINE-ACTIVATED NATURAL KILLER CELLS
Authors: Kerstetter-Fogle Amber , Otegbeye Folashade , Soler David , Harris Peggy , Raghavan Alankrita , Sloan Andrew 
Published: November 2021

Abstract

INTRODUCTION
Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy associated with a 12-15 month survival after surgery and radio-chemotherapy. Utilizing adoptive cellular immunotherapy using natural killer (NK) cells has developed over the past two decades for a variety of hematologic malignancies. This approach in solid malignancies is limited by questions of cell dose versus tumor burden, insufficient tumor infiltration, and a tumor microenvironment that suppresses NK cell function.

METHODS
We isolated NK cells from healthy volunteers and activated them using IL-2, -15, -12, -18, then perform cytotoxic assays in the presence of glioma stem cells. We also tested the efficacy of the NK cells with intracranial delivery in a pre-clinical murine model of glioma. We tested various concentrations of IL-2 and IL-15 on the cytokine culture platform.

RESULTS
In this study, we demonstrate human NK cells, activated using a cytokine cocktail of interleukins-2, -15, -12, and -18, exert strong cytotoxic events against glioma cell lines. To further examine the efficacy of activated NK cells in vitro, we utilized intracranially xenografted glioma lines and demonstrated a survival benefit with tumor bed injections of these cytokine-activated NK cells (p=0.0089). We were able to confirm that NK cells cultured with low doses (200u IL2; 50ng/ml IL15) of both cytokines are just as effective as higher doses. This is important, as in vivoexhaustion of NK cells stimulated with high doses of either cytokine has been well validated. We also found that low-dose irradiation (4Gy) of glioma cells prior to co-culture with cytokine-activated NK cells promoted increased targeted glioma cell killing within 4 hours(32% cell killing).

CONCLUSIONS
These findings suggest that in a clinical study, injection of cytokine-activated NK cells into the glioblastoma tumor bed could be used as adjuvant treatment following either stereotactic radiation or surgical resection.
IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM
Authors: Martins Tomás A , Ritz Marie-Françoise , Shekarian Tala , Schmassmann Philip , Kaymak Deniz , Bicvic Valentina , Hutter Gregor 
Published: November 2021

Abstract
The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.
IMMU-49. DYNAMIC CHANGES AND PROGNOSTIC VALUE OF PERIPHERAL BLOOD LYMPHOCYTE SUBSETS IN INTRACRANIAL GERM CELL TUMORS
Authors: Wang Hairong , Guo Cheng-Cheng , Chen Hongyu , Qun-ying Yang , Chen Zhong-ping 
Published: November 2021

Abstract

OBJECTIVE
This study was designed to retrospectively analyze the dynamic changes of peripheral blood lymphocyte subsets and prognosis among patients with intracranial germ cell tumors.

METHODS
A total of 150 intracranial germ cell tumors patients diagnosed between June 2011 till November 2019 were retrospectively investigated. Peripheral blood total T lymphocytes (CD3+) percentage, T helper/inducible lymphocytes(CD3+CD4+)percentage, T inhibitory/toxic lymphocytes (CD3+CD8+) percentage, B lymphocyte (CD19+) percentage, NK lymphocyte (CD3/CD16+CD56+) percentage, regulatory T cells (CD4+CD25+,CD8+CD25+), and T helper/toxic lymphocyte ratio (CD4+/CD8+ ratio) were quantified by flow cytometry analysis. Clinical information was extracted from the database in Sun Yat-sen University Cancer Center and survival data was confirmed through outpatient visits and telephone follow-up.

RESULTS
T lymphocytes population was increased after anti-tumor treatment, with significant difference of total T lymphocytes (CD3+), inhibitory/toxic T cells (CD3+CD8+) and regulatory T cells (CD4+CD25+ and CD8+CD25+), (p=0.008, p=0.000, p=0.008 and p=0.001 respectively), while B lymphocytes(CD19+) decreased after chemotherapy(p=0.003). The dynamic levels of T lymphocyte and B lymphocyte subpopulation after chemotherapy did not present significant differences between gender, age, and locations of tumors (p &gt;0.05), except CD4+CD25+ T cells in younger children (age&lt; 16 years older) increased significantly than the elder (age &gt;16), p=0.04. Patients with increased CD19+ B cells presented significant suboptimal outcomes compared with the no increased (p=0.024). Similarly, increased CD3+ T cells, CD3+CD8+ T cells, CD4+CD25+ T cells reduced the risk of death (p=0.006, p=0.019, p=0.042 respectively). Multivariate Cox Regression analysis showed: increased CD19+ B cells, p=0.04, HR=1.688, 95%CI=1.025-2.779.

CONCLUSION
After anti-tumor treatment, cell-mediated immunity activated, enhanced, and dominated in anti-tumor response. An increased level of CD19+ subsets was an independent predictor for inferior overall survival. Systemic circulating T cells immunity played an important role and mediating antitumor responses may pave the road for new immunity strategies.