bluuebunny/crossref_metadata_2025_split huggingface dataset:
IMMU-02. NEOANTIGENS ARISING FROM ALTERNATIVE SPLICING EVENTS MAY BE TARGETED BY TUMOR INFILTRATING LYMPHOCYTES IN GLIOBLASTOMAS
Authors: Lee Alexander , Pan Yang , Mochizuki Aaron , Galvez Mildred , Chow Frances , Nathanson David , Wang Anthony , Everson Richard , Liau Linda , Xing Yi , Prins Robert 
Published: November 2019

Abstract

INTRODUCTION
Alternative splicing, the cellular process that converts premature mRNA to mature mRNA and allows for single genes to produce multiple protein products, is frequently dysregulated in many cancers, including glioblastoma. However, along with non-synonymous mutations in the DNA, altered splicing mechanisms in cancers may produce novel antigens (so-called neoantigens) that distinguish cancer cells from healthy cells and can thus be targeted by the immune system.

METHODS
We developed a new computation pipeline (IRIS – Isoform peptides from RNA splicing for Immunotherapy targets Screening) that took bulk RNA-sequencing data from 23 glioblastoma patient tumor samples and predicted neoantigens that may arise from alternative splicing events. We prioritized predicted neoantigens that arose in HLA*A02:01 and HLA*A03:01 patients and selected 8 potential neoantigens to generate peptide:MHC Class 1 dextramers. We tested PBMCs and/or ex vivo expanded tumor infiltrating lymphocytes (TIL) from 6 of our glioblastoma patients against these dextramers, sorted for any neoantigen-reactive T cells, and performed single-cell RNAsequencing on the sorted population to determine the TCR sequence.

RESULTS
Among the 8 predicted neoantigens tested, 7 of the neoantigens were recognized by at least 1 patient’s T cells. 1 HLA*A03:01 epitope was recognized in 3 of the 4 HLA*A03:01 patients tested and this epitope was highly positive in an expanded TIL population, representing 1.7% of all CD3+ CD8+ cells. When we sorted for those neoantigen reactive T cells from the expanded TIL population and performed single-cell RNAsequencing, we found 325 unique T cell clonotypes, but the top 10 clonotypes represented 83.6% of all TCR clonotypes. The most frequent TCR clonotype represented 39.1% of the repertoire and suggests that clonal expansion of a select few TCR clones occurred within the tumor.

CONCLUSIONS
In total, our data indicates that neoantigens arising from alternative splicing events may represent a potential target for immunotherapy in glioblastoma.
IMMU-10. ESTABLISHING EFFECTIVE MODELS FOR IMMUNOTHERAPY IN GBM
Authors: Zamler Daniel , Yen Er-Yen , Shingu Takashi , Ren Jiangong , Kassab Cynthia , Liu Jintan , Heimberger Amy , Hu Jian , Draetta Giulio , Curran Michael 
Published: November 2019

Abstract
The introduction of immunotherapies has been paradigm shifting for cancers that were previously a death sentence. However, preclinical/clinical studies on glioblastoma (GBM) have generated mixed outcomes in patients, likely due to its great heterogeneity of immune microenvironment, particularly the myeloid cell populations. Primary patient studies have been limited by a difficulty in performing longitudinal studies, uncontrolled environmental conditions, and genetic variability. There is also, unfortunately, a paucity of mouse models that effectively re-capitulate the immune microenvironment of the human disease. To address these difficulties, we have established the Qk/p53/Pten (QPP) triple knockout mouse model established in our lab. The QPP model uses a cre-lox system to induce Qk deletion on a Pten−/−; p53−/− background which helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2;QkiL/L PtenL/L p53L/L mice, which develops glioblastoma with survival of ~105 days. We have preliminarily assessed the QPP tumors as a faithful model to study the immune response to GBM and found them to recapitulate human GBM with respect to differential response to checkpoint blockade therapy and myeloid and T-cells histopathologically, particularly regarding upregulation of Arginase-1 (Arg1). Arg1 is the canonical marker for tumor-associated macrophages (TAMs), which is a major population of myeloid cells that greatly infiltrate in human GBM, sometimes making up more than ~30% of all GBM cells. Given TAMs’ prevalence in the tumor microenvironment and their upregulation of Arg1 in both human GBM and our QPP model, we are testing whether manipulation of Arg1 will impact TAM function and influence GBM growth. We are also evaluating arginine metabolism in TAMs effect on T cell function in GBM. Lastly, we have developed a genetically engineered mouse model to study the role of Arg1 knockout in a GBM context in-vivo. Our studies suggest that Arg1 plays an important role in GBM immune interaction.
IMMU-18. IMMUNOGENOMIC RESPONDER PHENOTYPE FROM A PHASE I TRIAL OF ANTI-LAG3 OR ANTI-CD137 ALONE AND IN COMBINATION WITH ANTI-PD-1 IN PATIENTS WITH RECURRENT GBM
Authors: Jackson Christina , Choi John , Zhang JiaJia , Piotrowski Anna , Walbert Tobias , Desai Arati , Ahluwalia Manmeet , Nabors Burt , Ye Xiaobu , Desideri Serena , Fisher Joy , Wen Patrick , Grossman Stuart , Smith Kellie , Pardoll Drew , Lim Michael 
Published: November 2019

Abstract

BACKGROUND
Immune checkpoint inhibitors (ICIs) are not uniformly effective in glioblastoma treatment. Immunogenomic determinants may identify patients who are most likely to benefit from these therapies. Therefore, we compared the immunogenomic phenotype of a responder to combination anti-LAG-3 and anti-PD-1 therapy to non-responders.

METHODS
We performed T cell receptor (TCR) sequencing and gene expression analysis on pre-treatment, post-chemoradiation, and post-immunotherapy tumor specimens of glioblastoma patients treated with anti-LAG3 in combination with anti-PD-1 after first recurrence (NCT02658981, ongoing). We evaluated T cell clonotypes and immunophenotype of serially collected peripheral blood mononuclear cells (PBMCs) during treatment using multi-parametric flow cytometry.

RESULTS
To date, six patients have been enrolled in the initial anti-LAG-3 and anti-PD-1 cohort. One patient demonstrated complete response, one had stable disease, and four had progressive disease by radiographic evaluation. The responder demonstrated substantially higher TCR clonality in the resected tumor at initial diagnosis compared to non-responders (mean 0.028 vs. 0.005). Shared tumor infiltrating clonotypes with pre-immunotherapy PBMCs exhibited an increase in frequency from initial resection (6.8%) to resection at recurrence (20%). The responder’s tumor at initial resection exhibited increased gene signatures of PD1low CD8+ T cells, chemokine signaling, and interferon gamma pathways. On PBMC phenotypic analysis, the responder demonstrated significantly higher percentages of CD137+ CD8+T cells (median 8.38% vs 3.24%, p=0.02) and lower percentages of Foxp3+CD137+ CD4+T cells compared to non-responders (median 18.5% vs. 38.5%, p=0.006). Interestingly, dynamic analysis of PBMCs showed that the responder demonstrated a lower percentage of PD1+ CD8+ T cells pre-immunotherapy (median 2.5% vs.12.4%, p=0.002), with persistent decrease over the course of treatment while non-responders showed no consistent pattern.

CONCLUSION
Our preliminary results demonstrate significant differences in tumor and peripheral blood immunogenomic characteristics between responder and non-responders to anti-LAG3 and anti-PD-1 therapy. These immunogenomic characteristics may help stratify patients’ response to combination ICIs.
IMMU-20. ADVANCED AGING INCREASES IMMUNOSUPPRESSIVE IDO1 LEVELS THAT ARE UNINHIBITED BY IDO1 ENZYME INHIBITOR TREATMENT IN MODELS OF GLIOBLASTOMA
Authors: Ladomersky Erik , Zhai Lijie , Lauing Kristen , Qian Jun , Otto-Meyer Sebastian , Savoor Rohan , Wainwright Derek 
Published: November 2019

Abstract
Immunotherapy has failed to improve the overall survival (OS) of adults with GBM in all phase III clinical trials to-date. Somewhat less appreciated is that, de novo GBM is a disease associated with a median age of diagnosis at 64 years old. Because of the strong association between advanced aging and human subjects presenting with GBM, we further investigated our previously described observation of a higher mortality rate in elderly C57BL/6 wild-type (WT) mice with intracranial GL261 as compared to young counterparts after treatment with radiotherapy (RT) and anti-PD-1 (PD1)/IDO1 enzyme inhibitor (IDO1i) (Ladomersky et al., 2018; Clinical Cancer Research). Since immunosuppressive IDO1 expression significantly increases in both the mouse and human brain during advanced aging (Ladomersky et al., 2019; Frontiers in Pharm.), we decided to study how IDO1 and aging interact during immunotherapy of GBM. We found that the pretreatment effects of an IDO1 enzyme inhibitor improve OS in young but not elderly WT mice with intracranial GL261 (n=12–15/group; p< 0.05). Strikingly, in the setting of advanced aging, IDO1-/- mice with GBM show improved OS as compared with WT mice (p< 0.01) independent of treatment with an IDO1 enzyme inhibitor. Similarly stunning, 74–80 week old IDO1-/- mice with GBM treated at 14 days post-ic. with RT and PD1/IDO1i survive significantly longer as compared to elderly WT mice administered the same treatment (n=20–22/group; p< 0.05). These results collectively suggest that, the therapeutic effects of IDO1 enzyme inhibition is less effective in the setting of old age, and provides rationale for the development of new approaches that inhibit IDO1-mediated immunosuppression independent of its association with enzyme activity for eventually improving immunotherapeutic efficacy in human subjects with malignant glioma.
IMMU-24. A COMPREHENSIVE IN SILICO APPROACH TO DISCOVERING TUMOR REJECTION ANTIGENS IN MALIGNANT BRAIN TUMORS
Authors: Yang Changlin , Dyson Kyle , Yegorov Oleg , Mitchell Duane 
Published: November 2019

Abstract

BACKGROUND
Proteins that can serve as effective tumor rejection antigens within brain tumors are poorly characterized. Current prediction algorithms relying on identification of mutated epitopes as neoantigens are limited in low mutational burden tumors. We have developed a multi-faceted computer algorighm for identifying tumor rejection antigens called the Open Reading Frame Antigen Network (ORAN). This pipeline provides a comprehensive solution for predicting brain tumor immunogenic targetable epitopes/transcripts.

METHODS
Human and murine RNASeq and WES data were QCed. Patients individual HLA-I and HLA-II haplotypes were predicted by highly customized Optitype and Phlat Algorithms. SNPs and Indels were called from tumors WES and read through matched RNASeq data. 19,131 transcripts expression were counted per TOIL algorithm. Tumor associate antigens(TAA) of individual patient or murine tumors were selected by setting a cutoff of Transcripts Per Million (TPM) > 1 on individual patient’s tumor, while RNA Seq data from 7000 normal tissues was used to identify tumor unique transcripts. Actual sequence of HLA and SNPed Consenses TAA(CTAA) were called. Only expressed mutations and personalized TAAs were used for antigenic epitope predictions. All neoepitopes were screened against a human reference proteomic library to guarantee that epitopes were not shared by other expressed isoforms or genes. In silico validation were preformed to cross validate predictions made by ORAN.

RESULTS
In medullobastoma (N=121 samples), ORAN gives an average of 15.6 MHC class I restricted neoepitopes,11.9 epitopes encoded by Indels and with 33.2 MHC class II restricted neoepitopes and 16.2 Indel antigens per patient. The TAAs of each patient reaches average 256 antigenic epitopes per patient. ORAN also predicts the exact HLA and neo-antigens from a validated neoantigens vaccine dataset (Gros A Nat Med 2016).

CONCLUSION
ORAN accurately identifies validated neoantigens and provides a comprehensive list of potential tumor rejection antigens within human and murine brain tumors.
IMMU-31. DRIVER GENE MUTATIONS DICTATE THE COMPOSITION OF THE IMMUNE LANDSCAPE OF GLIOBLASTOMA AND CONFER SELECTIVE RESPONSE TO IMMUNOTHERAPY
Authors: Yeo Alan , Delcuze Bethany , Strauss Laura , Boussiotis Vassiliki , Charest Al 
Published: November 2019

Abstract
The characterization of the immune landscape of glioblastoma multiforme (GBM) is rapidly emerging. Current immunotherapeutic efforts for GBM would benefit from a comprehensive and mechanistic understanding of the relationship between distinct driver gene mutations and the composition and function of the immune tumor microenvironment. The majority of GBM tumors overexpress EGFR and EGFRvIII and ~40% are mutated for PTEN. Using genetically engineered and accurate preclinical mouse models of GBM built on combinations of these driver genes, we demonstrate that the compositions of the immune landscape and the response to checkpoint blockade immunotherapy vary according to the genotype of the GBM. Mechanistically, we show that signaling networks downstream of EGFR and PTEN establish chemokine and cytokine profiles that parallel the levels of intra-tumoral polymorphonuclear myeloid-derived suppressor cells and regulatory T cells. Additionally, tumor-associated microglia/macrophages number, function and polarization are heavily influenced by specific driver-gene mutation combinations. Furthermore, we demonstrate that efficacy of checkpoint blockade therapy using anti PD-1 as a single agent or in combination with anti CTLA-4 is dependent on EGFR and PTEN status. Taken together, our findings demonstrate that important components of the tumor immune microenvironment are influenced by specific driver gene mutations. Our results suggest that stratifying patients based on tumor genotype or signaling events may be informative for selection of appropriate candidates for checkpoint blockade therapy and present an opportunity to pharmacologically modulate GBM signaling with targeted therapeutics to sensitize patients to immunotherapy.
IMMU-33. IMMUNE PROFILING REVEALS INHIBITORY MACROPHAGES AND A DISTINCT SPATIAL DISTRIBUTION OF IMMUNE CELLS IN DIFFERENT TYPES OF BRAIN METASTASES
Authors: Kienzler Jenny , Reynoso Jeremy , Owens Geoffrey , Orpilla Joey , Lee Alexander , Mochizuki Aaron , Yong William H , Liau Linda , Prins Robert 
Published: November 2019

Abstract

BACKGROUND
Current treatment modalities, including surgical resection, radiosurgery and immune checkpoint inhibitors, have improved local control rates of brain metastases, but overall prognosis is still poor. AIM: To investigate the immune microenvironment in brain metastases for better understanding of treatment failures.

METHODS
We identified 42 patients with melanoma (M), breast (B) and lung (L) brain metastases and performed multiplex immunofluorescent staining for immune markers (CD4, CD8, CD45, PD1, PD-L1) and quantitative analysis with HALO software. NanoString nCounter mRNA gene expression assay was used for identification of immune pathways. CyTOF and single cell RNA sequencing were done on 12 different brain metastases and their matched peripheral blood samples.

RESULTS
The median CD8, CD4 and PD1 density (as a total number of cells) was 0.24%, 0.13%, and 0.07% for M, 0.24%, 0.51% and 0.08% for B, and 0.87%, 1.36%, 0.67% for L. CD4 and CD8+PD1+ expression was significantly different between all groups (p=0.01), CD4+PD1+ in M vs. L (p=0.03) and PD1 in B vs. L (p=0.03). No significant difference was found in terms of CD45 positive cells (M 2.13%, B, 1.99%, L 4.95%). PD-L1 expression was significantly different between histologies; the highest expression was present in breast metastases (M: 1.51%, B: 49.98%, L: 31.90%, p=0.0002). Spatial distribution revealed a characteristic pattern with a peritumoral border infiltrate in M, and predominant intratumoral distribution in B and L (M 0.2%, B 2.6%, L 3.6%, p=0.02). CyTOF clustering demonstrated that macrophages are the most prevalent immune population overall (83.08%), with an inhibitory phenotype and gene expression pattern present in different histologies (S100A9, CCL20, IFI16, MARCO). T-cells (predominantly CD4+, 3.6%), and B-cells (1.5%) comprise the remaining immune cell populations.

CONCLUSION
Neoadjuvant targeted treatment approaches may be needed to alter predominance of inhibitory macrophages and recruit activated T lymphocytes in brain metastases.
IMMU-39. RNA LOADED LIPID-NANOPARTICLES FUNCTION AS CUSTOMIZABLE IMMUNOTHERAPEUTIC VEHICLES AGAINST MALIGNANT GLIOMAS
Authors: Grippin Adam , Wummer Brandon , Mendez-Gomez Hector , Stover Brian , Huang Jianping , Rinaldi Carlos , Mitchell Duane , Dobson Jon , Sayour Elias 
Published: November 2019

Abstract

BACKGROUND
While dendritic cell (DC) vaccine therapy has shown considerable promise for glioblastoma (GBM) patients (Mitchell et al. Nature, 2015), their advancement into human clinical trials has been fraught with challenges in the development, manufacturing, and marketing of successful cancer immunotherapies. To circumvent the challenges associated with cell therapy, we have developed a new platform technology consisting of tumor derived mRNA complexed into lipid-nanoparticles (RNA-NPs) for systemic delivery to DCs in vivo and induction of antigen specific T cell immunity against GBM. OBJECTIVES/

METHODS
We sought to assess if surface and charge modifications to our custom lipid-NP could facilitate its localization to lymphoid organs and the brain tumor microenvironment.

RESULTS
We demonstrate that intravenous administration of our unmodified custom RNA-NPs mediate systemic activation of DCs; these include activation of CD11c+ cells in the brains of animals with intact blood brain-barriers (BBBs). RNA-NPs mediate antigen specific T cell immunity and anti-tumor efficacy with increased tumor infiltrating lymphocytes against a NF-1/p53 mutant glioma that recapitulates features of human GBM in immunocompetent mice. Modification of surface charge could direct these RNA-NPs to lymphoid organs (e.g. spleen, lymph nodes) while modification of the lipid backbone (with cholesterol) enhances localization to innate immune cells in NF-1/p53 mutant and GL261 gliomas. We therefore assessed if this customizable lipid-NP could be leveraged for delivery of immune checkpoint inhibitors (ICIs) (i.e. PD-L1 siRNA) to the brain tumor microenvironment. Compared with scrambled siRNA-NPs in combination with ICIs, surface modified siRNA-NPs (antagonizing PD-L1) in combination with ICIs mediated significant antitumor efficacy with 37% long term survivors in an otherwise fatal brain tumor model.

CONCLUSION
We designed multifunctional RNA-NPs with a simple, scalable synthesis method that enables delivery of nucleic acids to innate immune cells in lymphoid organs and brain tumors.
IMMU-41. RNA-MODIFIED T CELLS AS PROFESSIONAL ANTIGEN PRESENTING CELLS
Authors: Pohl-Guimarães Fernanda , Mitchell Duane 
Published: November 2019

Abstract

INTRODUCTION
With the lack of antigen presenting cells (APCs) in the central nervous system (CNS), anti-tumor immune responses from intracranial tumors are believed to be initiated in the peripheral lymph nodes. Recently, studies have shown that intratumoral dendritic cells (DCs), a well-known professional APC, play a crucial role at inducing anti-tumor immunity. Although DCs have been tested in cancer immunotherapy clinical trials for two decades, DC delivery strategies have limited access to the CNS, mainly due to the blood brain barrier (BBB). To bypass the limitations of the lack of intracranial APCs, we investigate the HYPOTHESIS that RNA-modified T cells can express and cross-present single tumor antigens locally within the CNS tumor microenvironment. Since activated T cells have an inherent ability to cross the BBB and can be genetically modified, this strategy makes them an attractive professional antigen presenting cells for cancer cell therapy.

METHODS
Using electroporation to deliver messenger RNA (mRNA) encoding a model tumor antigen to T cells, we evaluated the expression and cross-presentation capability of RNA-modified T cells.

RESULTS
We demonstrated that activated T cells can be genetically modified to express tumor antigens in vitro while retaining their inherent effector functions. Compared to unmodified T cells, T cells modified with tumor antigen encoding RNA showed high expression of inflammatory cytokines, including IFN gamma and TNF alpha and enhanced cellular proliferation and IL-2 secretion. Tumor antigen RNA-modified T cells demonstrated potent antigen presentation in vitro and importantly were significantly superior to RNA-loaded DCs in antigen presenting capacity.

CONCLUSIONS
This study strongly supports the implementation of a non-invasive and efficacious strategy to deliver “APC-like” T cell locally to brain tumors, thereby bypassing the lack of APCs within CNS malignancies.
INNV-02. ARE WE AGGRESSIVE WITH OUR GLIOBLASTOMA PATIENTS: A PROPOSED TOOL TO EVALUATE CENTERS ATTITUDES IN TREATMENT OF GLIOBLASTOMA
Authors: Al Abdulsalam Hissah , Alhothali Wajda , Alkhalidi Hisham , Orz Yasser , Alobaid Abdullah , Bafaqeeh Mohammed , Shaffi Ahamed Shaik , Hussain Sajjad , Albader Fahad , AlSaeed Eyad , Ibrahim Ahmed , Abdel Warith Ahmed , Alomair Ameen , Altewaijri Ikhlas , Elwatidy Sherif , Al-Habib Amro , Ajlan Abdulrazag 
Published: November 2019

Abstract

INTRODUCTION
Glioblastoma is the most common malignant primary brain tumor in adults. Optimal treatment for glioblastoma requires a multidisciplinary approach and knowledge of complications from both the disease and its treatment. Despite modern advances in the standards of care, the outcome in patients with Glioblastoma remains poor. Thus, it is crucial to optimize treatment strategies to improve over-all survival in patients with Glioblastoma, with the therapeutic challenge of “how aggressive a physician should be?” OBJECTIVES: We herein propose a novel method to compare aggressiveness in the standard of care between multiple centers, using a novel tool, and provide a more precise cut-off definition of aggressiveness.

METHOD
Multiple centers were included. A novel case-based questionnaire was generated which included only certain parameters, and then sent and answered jointly by tumor board members from each center. We then stratified centers into more aggressive vs. less aggressive based on our proposed Aggressiveness (AS) score.

RESULTS
In our novel cases, center A scored an AS score of 44 (Mean ± SD; 3.67 ± 1.07), while center B scored 49 (Mean ± SD; 4.08 ± 0.90) In real cases, center A scored 40 (Mean ± SD; 3.25 ± 1.42), while center B scored 44 (Mean ± SD; 3.75 ± 1.29). Our results showed consistency between centers evaluated using our tool. Age and presence of comorbidities were the most influential parameters.

CONCLUSION
In the current study, we present a novel and a reliable tool that can be easily used and replicated to compare aggressiveness in the treatment of Glioblastoma between multiple centers. The methodology we used has never been previously proposed and can serve as a novel concept to compare aggressiveness in treatment of all incurable diseases that require a multidisciplinary approach.