bluuebunny/crossref_metadata_2025 huggingface dataset:
GENE-30. PRELIMINARY STUDY ON WHOLE-EXOME SEQUENCING TECHNOLOGY IN THE GENETIC ANALYSIS OF PEDIATRIC PATIENTS WITH GLIOMAS
Authors: Lai Mingyao , Li Juan , Zhen Junjie , zhou jiangfen , Hu Qingjun , ye Minting , Li Shaoqun , Wang Lichao , Zhou Cheng , Zhou Zhaoming , Cai Linbo
Published: November 2019

OBJECTIVE

To analyze the genes related to the signaling pathways in pediatric gliomas and drug-related genes with whole-exome sequencing technology.

METHODS

The tumor tissues and matched blood samples of 17 enrolled patients were detected with whole-exome sequencing technology. There were 3 cases of diffuse midline gliomas, 2 cases of childhood glioblastomas, 3 cases of disffuse astrocytoma, 1 case of pleomorphic xanthoastrocytoma, 1 case of ganglioglioma, 6 cases of anaplastic ependymoma and 1 case of ependymoma in this study. All the enrolled patients who were no more than 14 years old received surgery in the Department of Neurosurgery, Guangdong Sanjiu Brain Hospital. The diagnosis was confirmed by pathological examination and the sample acquisition was approved by hospital ethics committee.

RESULTS

With the use of whole-exome sequencing technology, a total of 31 related genetic mutations were detected in 15 cases, while no genetic mutation was detected in the other 2 cases. The genes related to the signaling pathways in pediatric gliomas included ATRX, ASL1, BCOR, EP300, FGFR1, H3F3A, IGF1R, MED12, PIK3R1, PRKDC, RB1, SETD2, SMARCA4, SOX2, TGFBR2, and the drug-related genes included AKT1, BCL2, BRAF, BRCA2, CCND1, CCND2, CDK6, EGFR, FGF3, KRAS, MET, PDGFRA, PIK3CA, PTEN, TP53, TSC1. One patient only had genes related to the signaling pathways, and 14 patients had drug-related genes.

CONCLUSION

Applying whole-exome sequencing technology in the genetic analysis of pediatric patients with gliomas has remarkable guiding significance for revealing the mechanism of disease, searching for therapeutic targets and adopting individualized treatment, which can bring potential benefits to pediatric patients. However, more samples and further data analysis and verification are needed in future study.
GENE-36. INTEGRATING TRANSCRIPTOMICS AND KINOMICS IDENTIFIES SYNERGISTIC DRUG COMBINATIONS FOR GLIOBLASTOMA TREATMENT
Authors: Jermakowicz Anna , Stathias Vasileios , Suter Robert , Duncan James , Schürer Stephan , Ayad Nagi
Published: November 2019

Glioblastoma (GBM) is the most common and malignant adult brain tumor. Despite years of research, few advancements have been made in its management. One promising avenue of research has been treatment with BRD4 inhibitors, which decrease oncogene expression in GBM cells. However, resistance to these inhibitors is rapidly acquired. Kinome reprogramming is thought to underlie this resistance, suggesting a need for combination therapy with kinase inhibitors. The goal of this study is to determine whether transcriptomic and kinomic profiling of GBM tumors will identify synergistic drug pairs for GBM treatment. We profiled the active kinome on a set of three newly-diagnosed GBM patient-derived xenograft (PDX) tumors and three recurrent tumors using quantitative SILAC mass spectrometry. Additionally, kinome reprogramming following BET inhibition was profiled in vitro using the BET inhibitor JQ1. Kinome activity was uploaded into our novel computational platform, SynergySeq, to assess the synergistic potential of kinase inhibitors with JQ1. Additionally, single cell RNA-sequencing of a GBM tumor was used to determine the cell populations affected by each inhibitor. To quantify synergy in response to combination therapy, cells were treated with a combination matrix of JQ1 and a kinase inhibitor in variable concentrations and cell death was quantified via ATP levels. Our results showed that newly-diagnosed tumors were predicted to be sensitive to combined BET inhibition with Bcr/Abl, EGFR, and FGFR kinase inhibitors. Recurrent tumors were sensitive to combined Bcr/Abl and FGFR inhibitors but were not sensitive to EGFR inhibitors. We screened inhibitors in vitro and found a synergistic effect for the combination of JQ1 and TAS120, a pan-FGFR inhibitor. These data suggest that clinically a brain penetrant BET inhibitor should be effective in combination with a brain penetrant FGFR inhibitor. Importantly, our computational platform is a novel informatics-based approach for targeted therapy in a patient-specific and disease-specific manner.
GENE-40. CHARACTERIZING EPIGENETIC INTRATUMORAL HETEROGENEITY IN GLIOMA USING SINGLE-CELL BISULFITE SEQUENCING
Authors: Johnson Kevin C , Anderson Kevin , Courtois Elise , Barthel Floris , Samuels Michael , Robson Paul , Bulsara Ketan , Das Sunit , Verhaak Roel
Published: November 2019

Genetic and epigenetic alterations contribute to the observed intratumoral heterogeneity in adult glioma. Current glioma classification, based on genotype (e.g., IDH1 mutations) and DNA methylation profiles (e.g., glioma CpG Island Methylator Phenotype), can provide clinically relevant tumor subgroups. However, traditional bulk sampling fails to adequately capture the full complement of epigenomic heterogeneity, and may mask deadly features present in less abundant glioma cells. To more precisely characterize the glioma epigenome, we separately profiled single-cell DNA methylation (Reduced Representation Bisulfite Sequencing, RRBS), single-cell RNA expression (10X genomics), and bulk whole genome sequencing in nine gliomas. The genomic regions profiled by scRRBS were primarily gene promoters, but adequate coverage was also reached for glioma-specific enhancer elements and binding sites of chromatin remodelers. Unsupervised clustering of single-cell DNA methylation data revealed intratumoral variability in epigenetic classification and these cell types were distinguished by regulatory element DNA methylation. We further integrated single-cell epigenetic, single-cell transcriptomic, and genomic features to better understand gene regulation and reconstruct each tumor’s lineage history. Together, our study aims to generate a glioma cellular hierarchy shaped by the epigenetic programs that drive tumor growth.
GENE-41. UNDERSTANDING THE DIFFERENCES IN ADOLESCENT AND YOUNG ADULT (AYA) GLIOMAS AND OLDER ADULT (OA) GLIOMAS BASED ON FUNCTIONAL MOLECULAR SUBGROUPS
Authors: Bi Catherine , Subramaniam Ashwin , Xiu Joanne , Heimberger Amy , Michelhaugh Sharon , Mittal Sandeep , Sumrall Ashley , Subramaniam Deepa
Published: November 2019

BACKGROUND

Gliomas in the AYA population (15–39 years of age) have unique biological characteristics and need to be better characterized.

METHODS

Glioma tumors in AYA subjects and subjects >65 years of age (OA) were analyzed by next generation sequencing using a 592 gene panel. Pathogenic mutations were classified into five functional groups, viz. metabolic pathways genes (IDH1/2, FH), tumor suppressor genes (TP53, RB1, APC, NF1/2, PTEN, TSC1/2), genes involved in DNA repair (MMR genes, BRCA1/2, POLE, ARID1A, CHEK2, ATM, BLM, BRIP1, WRN, BARD1, POT1, MUTYH), oncogenes (BRAF, NRAS, HRAS, EGFR, PDGFRA, FGFR1, NOTCH1, MYCN), and genes involved in transcriptional regulation (SETD2, H3F3A, KMTD2A/2C/2D, KDM6A, PIK3CA). Mutation frequency in AYA tumors and OA tumors were compared using Chi-squared analysis (Pearson’s score χ2; likelihood ratio LR).

RESULTS

720 unique gliomas tumors were analyzed: 118 AYA, 602 OA; 420 males, 300 females. When both groups are considered together, glioblastoma was the most common histology (75%), followed by grade 3 astrocytoma (13%), glioma NOS (3.8%), oligodendrogliomas (3%), low grade gliomas (2.9%) and other (2.3%). AYA tumors harbored more metabolic pathway gene mutations (χ2 137.7, p< 0.0001) driven primarily by IDH1 mutations, while OA tumors had a higher mutation frequency in oncogenes (χ2 9.22, p=0.0024) driven by EGFR mutations (LR 27.567) and tumor suppressor genes (χ2 40.35, p< 0.0001) driven by NF1 (LR 18.147) and PTEN (LR 66.216). No significant differences were noted in mutation frequency in DNA repair or transcriptional regulation genes. However, AYA glioblastoma tumors had a significant increase in mutations in genes involved in chromatin remodeling, (χ2 11.43, p=0.0007) even after excluding H3F3A.

CONCLUSIONS

Functional genomic classification of AYA tumors may help develop better targeted therapies, especially focused on genes involved in metabolic pathways and transcriptional regulation.
GENE-54. INVESTIGATING THE ROLES OF NCOR1 AND NCOR2 IN METASTATIC INVASION INTO THE SPINE
Authors: Sultan Zain , Rocco Sabrina , Szerlip Nicholas , Calinescu Alexandra
Published: November 2019

Anywhere from 30–70% of osseous metastatic disease occurs in the spine across many different tumor types. These metastases lead to debilitating pain and neurologic dysfunction significantly reducing patients’ quality of life. Unfortunately, the cause of preferential metastatic growth to the spine is not understood. The purpose of this study was to identify the role of NCOR1 and NCOR2/SMRT, genes included in Cancer Gene Census (https://cancer.sanger.ac.uk/cosmic), in the promotion of metastatic spread to the spine. NCOR1, located on chromosome 17, and NCOR2, located on chromosome 12, encode for proteins that mediate ligand-independent transcription repression by forming a complex that activates HDAC3. Studies have shown that low expression of NCOR1 and NCOR2 is associated with decreased median survival in some cancers. Our lab collected tumors and blood/bone marrow samples from 9 patients with different primary tumors that metastasized to the spine, and sequenced their DNA. Based on preliminary bioinformatics analysis, we discovered a substitution of thymine to cytosine at chr17:16165160 in the NCOR1 sequence, which results in a nonsense mutation that ends transcription in the second exon. After Sanger sequencing validation, this mutation was found to be present in both the tumor and control samples of all patients, resulting in two possible interpretations: a germline mutation of NCOR1 sequence or the presence of tumors cells in the blood. In addition, by bioinformatics analysis, we identified a substitution of cytosine to thymine at chr12:124341916 in exon 34 of the NCOR2 sequence, resulting in a missense mutation that changes an alanine to threonine. Further experimentation must be done to determine the role of NCOR1 and NCOR2 in metastatic spread to the spine. The hope is to identify targetable molecules and pathways for the design of novel therapies aimed to improve the outcome of these patients.
HOUT-07. ASSOCIATION BETWEEN BASELINE BODY MASS INDEX (BMI) AND OUTCOMES FOR PATIENTS WITH GLIOBLASTOMA
Authors: Chew Jessica , Morin Olivier , Braunstein Steve
Published: November 2019

OBJECTIVES

This single institution retrospective study aims to evaluate the association between body mass index (BMI) and survival in glioblastoma (GBM).

METHODS

Patients were identified using a single institution cancer registry. Baseline height and weight were obtained at first clinical visit after diagnosis. Inclusion criteria were GBM diagnosis after 1999 with available height and weight data. Follow up was through July 2018.

RESULTS

779 patients were included who had available BMI data. Median age was 58 (IQR 48–66). 38% of patients were female and 62% were male. Median overall survival (OS) was 1.34 years (IQR 0.78–2.12) and median BMI was 26.01 (IQR 23.3–29.1). There was no strong linear correlation between OS and BMI using Pearson correlation (r=0.045). Patients with higher BMI had significantly improved OS when comparing the top 50% to bottom 50% (2.20 vs 1.89 years, p=0.03) and top 25% to bottom 25% (2.25 vs 1.84 years, p=0.03). When comparing patients classified as overweight or obese (BMI >/= 25) to those within normal range or below (BMI < 25), there was a trend towards increased OS (2.14 vs 1.90 years, p=0.075). There was no significant trend for BMI when comparing patients with higher OS with lower OS. There was also no significance for BMI on Cox proportional hazard multivariate analysis.

CONCLUSIONS

There was no significant linear correlation between BMI and survival for GBM patients, although there appears to be statistically significant improved survival benefit for patients with higher BMI compared to patients with lower BMI. Further investigation is warranted to explore this finding and if BMI could be used as a potential prognostic marker for GBM.
HOUT-08. PATIENT AND PHYSICIAN PERSPECTIVES ON LUMBAR PUNCTURE
Authors: Umemura Yoshie , Weill Benjamin , Khan Baber , Buthorn Justin , Ridder Andrew , Nevel Kathryn , Panageas Katherine , Boire Adrienne
Published: November 2019

OBJECTIVE

To investigate patients’ and physicians’ perceptions of lumbar puncture (LP) and tolerability.

METHODS

Adult patients were surveyed prior to and after the procedure regarding patient perceptions and actual experience of LP. Presence of symptoms pre- and post-LP were compared by McNemar test; symptom severity score was rated 0–10, compared by Wilcoxon test; and relative risks were compared by Fisher’s exact test. In addition to patient surveys, email requests to complete an eight-question survey regarding LP tolerability was sent to faculty physicians at two academic centers.

RESULTS

A total of 154 patients and 302 physicians completed the surveys during 1/2017-5/2019. Ninety-one patients (59%) reported anxiety prior to-LP compared to 52 (34%) who reported anxiety after the LP (p< 0.0001). Whereas 74% of patients predicted LP to be painful prior to LP, only 48% reported that LP was painful after the procedure (p< 0.0001). On an 11-point scale, patients anticipated significantly greater pain from LP than they reported after the procedure (mean 3.4 and 2.0 respectively, p< 0.0001). Patients who anticipated pain were more likely to experience pain (RR=1.65, 95% CI 1.07–2.73). There was no significant difference in symptoms such as headache, nausea, generalized pain, vision or gait disturbance before and after the procedure. After the LP, 117 patients (77%) answered they would repeat LP if recommended by their doctors. Out of 302 physicians (40% women), the majority (81%) felt that the LP induces anxiety or worry in patients, and 50% answered that the LP is painful for patients in general.

CONCLUSION

We observed that although anticipation of pain contributed to pre-procedure anxiety, LP was generally well-tolerated. The majority of patients experienced minimal pain. The results of this study suggest that measures to reduce pre-procedure anxiety may improve the tolerability of LP, an important diagnostic tool in neuro-oncology.
HOUT-14. PROGNOSTIC IMPACT OF FIRST PSEUDOPROGRESSION ON MRI IN GLIOBLASTOMA, AN 11 YEARS EXPERIENCE FROM A CANADIAN UNIVERSITY CENTER
Authors: Zeidan Elie , Assad Anis , Letourneau Guillon Laurent , Belanger Karl , Lemieux Blanchard Emilie , Lemieux Bernard , Bahary Jean-Paul , Roberge David , G Masucci Laura , Menard Cynthia , Lambert Carole , Moumdjian Robert , Berthelet France , Ben-Abdallah Meriem , De Guise Jacques , Florescu Marie
Published: November 2019

BACKGROUND

In glioblastomas (GBM) patients the first post-radiation MRI is usually difficult to interpret given the post-radiotherapy enhancement and possible pseudo-progression which is present in almost 50% of the patients.

METHODS

We retrospectively analyzed all patients with GBM treated between 2006 and 2017 at the CHUM (SARDO database). If the first brain MRI done within 3 months after the systemic treatment showed progression of contrast, these patients were considered pseudoprogressors (PsP) while the patients who had stable or response to treatment were the non-progressors (nP). If progression persisted in subsequent MRI with a change of treatment within 6 months, they were considered early progressors (eP). If subsequent MRI improved or was stable, they were classified as nP (or true pseudo-progression).

RESULTS

In our cohort of 470 patients with GBM, 57.7% were nP and 42.3% were PsP after the first post-treatment imagery. The median follow-up was 10 months. The nP had a longer mOS 15.3m vs 11.3m, p < 0.001, regardless of subsequent evolution. After the second assessment, 67.8% of PsP patients were then considered as eP and 36.4% of nP patients also progressed within 6 months. The nP either after the first or second evaluation had the same mOS (19.9m vs 18.3m), just like the eP (9.3m vs 8.6m), independently of the subsequent treatment. No demographic, molecular or clinical factor predicted PsP, except for tumor size (> 5cm, p=0.024). PsP incidence was similar between 2006–2011 (PsP 57.8%) and 2012–2017 (42.2%). The 1y OS with pseudo progression at the first MRI was 39.7% vs 54.8% with no progression (p=0.001) which has a meaningful impact for the patient.

CONCLUSION

Pseudo-progression is frequent (42%) in glioblastoma and predicts a poorer prognosis with 1y OS of 39,7%. In fact, PsP patients have more than two-thirds chance to progress precociously.
HOUT-18. TREATMENT STRATEGIES FOR GLIOBLASTOMA IN OLDER PATIENTS: AGE IS JUST A NUMBER
Authors: Youssef Michael , Ludmir Ethan , Mandel Jacob , Patel Akash , Jalali Ali , Treiber Jeffrey , Wu Jimin , McAleer Mary , DeGroot John
Published: November 2019

BACKGROUND

Optimal care for elderly patients with glioblastoma (GBM) remains in question due to their exclusion from and underrepresentation in clinical trials (including EORTC 22981) as well as their historically-poor overall survival.

METHODS

Retrospective chart review was conducted at a single high-volume cancer center for newly-diagnosed elderly (65 years old or older) GBM patients diagnosed from 2011 through 2017.

RESULTS

A total of 158 newly-diagnosed GBM patients aged 65 years and older were identified. 144 patients (91.1%) underwent radiation therapy. One-hundred thirty patient (90.3%) received concurrent temozolomide with radiotherapy. A minority of patients (23%) discontinued temozolomide during concurrent or adjuvant treatment due to side effects or complications of chemotherapy. Sixty-one patients (38.6%) completed concurrent chemoradiation and 6 cycles of adjuvant temodar. The median overall survival (OS) time for our cohort was 18.6 months, with estimated OS rates of 74.8%, 35.9%, and 9.5% at 1, 2, and 5 years, respectively. On multivariable analysis, higher KPS (p=0.002, HR 0.46; 95% CI: 0.63–0.82), completing planned course of radiation (p=0.01, HR 0.29; 95% CI: 0.11–0.75), and completing 6 cycles of adjuvant temozolomide (p=0.01, HR 2.62; 95% CI: 1.67–4.12) were associated with improved OS.

CONCLUSIONS

Our cohort of elderly GBM patients were predominately treated with a standard of care based on EORTC 22981. Despite their age, these patients tolerated treatment well and had a favorable overall survival compared to outcomes reported for patients treated on EORTC 22981. Using age alone as the reason to de-escalate treatment or as an exclusionary criteria in clinical trials should be discouraged.
HOUT-28. FIRST-LINE TEMOZOLOMIDE VS PCV FOR LOW GRADE GLIOMAS: A 11 YEARS REAL-WORLD DATA FROM CHUM, UNIVERSITY CENTER
Authors: Nait Ajjou Myriam , Assad Anis , Letourneau Guillon Laurent , Belanger Karl , Lemieux Blanchard Emilie , Lemieux Bernard , Bahary Jean-Paul , G Masucci Laura , Roberge David , Menard Cynthia , Lambert Carole , Moumdjian Robert , Berthelet France , Ben-Abdallah Meriem , De Guise Jacques , Florescu Marie
Published: November 2019

BACKGROUND

Before 2012, Temozolomide (TMZ) was used for low-grade gliomas (LGG) to avoid Lomustine toxicity. After 2012 RTOG data, Procarbazine, Lomustine and Vincristine (PCV) given sequentially with radiotherapy became standard treatment with the side effects burden associated.

METHODS

We retrospectively reviewed our SARDO clinical database of patients with low-grade glioma LGG grades 2 and 3 between 2006 and 2017 at CHUM University Health Center. Molecular profile for these tumors is reflex and standard in our institution since 2016.

RESULTS

A total of 123 patients were identified with grade 2 and grade 3 LGG; 37 (30%) treated with PCV and 86 (70%) received TMZ. Median follow up was 11mo for PCV 11mo vs 22mo TMZ. Both groups were balanced in terms of median age, sex, neurologic symptoms and surgery rate. 53% patients had tumor untested for IDH1-2 and codeletion1p19q because of diagnostic before 2016. Disparities were noted with a predominance of grade 3 in the TMZ group (74% vs 27%, p< 0.01). TMZ was the preferred regimen before 2012 (100% vs 43%) and PCV became the standard of care after 2012 (0% vs 57%). Radiation use as first line treatment was 90%. The 4y OS was not significantly longer for PCV 50% compared with TMZ 47% with mOS between both groups (PCV NR vs TMZ 39.9mo, p=0.158). When controlled for tumor grade, the 2y OS was 80% for PCV vs 64% for TMZ,p=0.542. The 4y PFS was trendly longer for PCV group 78% than TMZ group 45%, p=0.148.

CONCLUSION

As we are still waiting for the prospective ongoing prospective trials comparing these 2 regimens, PCV and radiation are still standard of care regimens for grade 2 and 3 LGG. This retrospective data is not reassuring for a replacement for PCV with TMZ.
IMMU-01. MYELOID-DERIVED SUPPRESSOR CELL HETEROGENEITY DRIVES GLIOBLASTOMA PROGRESSION IN A SEX-DEPENDENT MANNER
Authors: Bayik Defne , Silver Daniel , Park Chihyun , Watson Dionysios , Sorensen Mia , Roversi Gustavo , Lauko Adam , Alban Tyler , Lo Alice , Sims Peter , Barnholtz-Sloan Jill , Kristensen Bjarne , Hyun Hwang Tae , Khalil Ahmad , Iavarone Antonio , Cheng Feixiong , Lathia Justin
Published: November 2019

An immunosuppressive tumor microenvironment is a major factor facilitating glioblastoma (GBM) progression and therapeutic resistance. Immunotherapies have had variable success in improving the outcome of GBM patients, suggesting that there is a need to gain insight into the mechanisms of immunosuppression. Our group previously demonstrated that myeloid-derived suppressor cells (MDSCs) expand in GBM patients and infiltrate tumors, where they suppress the activity of cytotoxic cells. However, the mechanisms by which individual MDSC subsets promote tumorigenesis remain understudied. Using the syngeneic mouse glioma models GL261, CT-2A and SB28, we show that monocytic MDSCs (mMDSCs) are prevalent in tumors and that their frequency is significantly higher in males, who constitute 60% of GBM patients and have a worse prognosis than females. mMDSC abundance was further associated with poor survival, and male mice reached morbidity endpoint earlier. Consistent with preclinical observations, male GBM patient specimens had significantly more IBA+CD204+ immunosuppressive myeloid cells compared to female GBM tissue. In contrast, female tumor-bearing mice had a two-fold increase in circulating granulocytic MDSC (gMDSC) frequency, while this population remained unchanged in males. Female-to-male bone marrow chimeras demonstrated that intrinsic discrepancies in immune cell characteristics drive the sex differences in survival. Consistent with the differential MDSC accumulation pattern, targeting gMDSCs with anti-Ly6G neutralizing antibodies extended the lifespan of female mice without providing a survival advantage to males. However, mMDSCs were protected from the anti-Ly6C depletion strategy due to their systemic and local proliferation, as indicated by ex vivo Ki-67 staining and subsequently confirmed by gene expression analysis. Drug-prediction algorithms using the differential RNA sequencing profiles demonstrated that mMDSCs can be targeted by chemotherapeutics, while immunomodulatory drugs are effective against gMDSCs. Collectively, these findings indicate that MDSC subset variation might represent a therapeutic opportunity for improved therapeutic efficacy of immunotherapies while accounting for sex as a biological variable.
IMMU-03. SUPPRESSION OF ONCOMETABOLITE 2-HYDROXYGLUTRATE PRODUCED BY MUTANT IDH1 GLIOMA WITH AGI-5198 ENHANCES THE EFFICACY OF RADIOTHERAPY AND IMMUNE CHECKPOINT BLOCKADE ELICITING IMMUNOLOGICAL MEMORY
Authors: Kadiyala Padma , Nunez Felipe , Garcia-Fabiani Maria , Nunez Fernando , Li Dan , Schwendeman Anna , Lowenstein Pedro , Castro Maria
Published: November 2019

Patients with mutant IDH1 gliomas survive ~3.8 year longer than patients harboring wildtype IDH1 glioma, irrespective of histology or genetic lesions, making mutant IDH1 an essential prognostic indicator. Mutant IDH1 gliomas are highly infiltrative with a high incidence of relapse. We are studying a low grade glioma subtype, genetically characterized by inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) gene, TP53 and gain of function mutations in isocitrate dehyrdogenase 1 (mIDH1). Mutation in IDH1 converts α-ketoglutrate (αKG) to 2-hydroxyglutarate (2HG), an oncometabolite that inhibits histone and DNA demethylases, leading to a hypermethylated tumor phenotype [1]. This leads to epigenetic reprograming of the tumor transcriptome. The role of 2HG as an immune regulator in the mIDH1 glioma tumor microenvironment remains to be elucidated. Herein, we investigate whether 2HG produced by mIDH1 glioma in the tumor microenvironment mediating antitumor immunity. We used mIDH1 inhibitor AGI-5198 to abrogate the neomorphic enzymatic activity of IDH1R132H in a transplantable mIDH1 glioma mouse model. Animals were implanted with mIDH1 tumor neurospheres and they were systemically administered with AGI-5198. Our results indicate that pharmacological inhibition of IDH1R132H enzymatic function significantly prolongs the median survival (MS) of mIDH1 glioma bearing mice (~1.5-fold vs controls); eliciting strong antitumor activity, eradicating 40% of the established mIDH1 gliomas. Our results also demonstrate that antitumor immunity induced by 2HG inhibition is enhanced when combined with standard of care (temozolomide and radiation) and anti-PDL1 immune checkpoint blockade. In addition, when the long-term survivors (>90 days) were rechallenged with mIDH1 tumor cells in the contralateral hemisphere, without further treatment, all the mice remained tumor free, indicating the development of anti-glioma immunological memory. Collectively, these findings support the clinical testing of AGI-5198 as an adjuvant therapy and a novel immunotherapeutic strategy for treating patients with mIDH1 glioma.
IMMU-04. TEMPORAL AND SPATIAL MODULATION OF THE IMMUNE RESPONSE OF THE MURINE GL261 GLIOMA TUMOUR MICROENVIRONMENT
Authors: McKelvey Kelly , Hudson Amanda , Kumar Ramyashree , Long Georgina , Scolyer Richard , Wilmott James , Attrill Grace , Clarke Stephen , Wheeler Helen , Diakos Connie , Howell Viive
Published: November 2019

INTRODUCTION

Glioma is a debilitating and early fatal cancer arising in the glial cells of brain. Glioblastoma, the most aggressive form of gliomas, has a 5-year survival rate of 5% with treatment options limited to surgery, radiotherapy or chemotherapy with temozolomide.

OBJECTIVE

While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. In this study we quantified the temporal and spatial localisation of immune cell populations and mediators during glioma development.

METHODS

Mice were inoculated with 1x106 Gl261 cells at AP0.1mm, ML1.0mm, DV2.6mm Bregma. Tumour morphology, local and systemic immune cell populations were assessed at Day-0, 1, 3, 7, 14, and 21 post-inoculation by MRI, immunohistochemistry for Ki67+ proliferation and CD31+ vessel density; local immune infiltrate by multiplex immunofluorescence (VECTRA®;PerkinElmer); systemic immunity in spleen, bone marrow and peripheral blood by 16-parameter flow cytometry (LSRFortessa™;BD Biosciences); and multiplex immunoassay (Bio-Plex®;Bio-Rad) for plasma cytokines/chemokines.

RESULTS

From Day-3 tumours were distinguishable with high Ki67+ (> 30%) and increased tissue vascularisation (26.38±1.45 vessels/field; p< 0.05). Ki67 remained high until Day-21 with visible necrotic regions, increased vessel density and lumen area within tumour region. Increasing tumour proliferation/malignancy and vascularisation were associated with significant temporal changes in immune cell populations within the tumour (p< 0.05) and systemic compartments (p=0.02- p< 0.0001). Of note, at Day-14 NK, M1, PMN-MDSCs and M-MDSCs in the tumour infiltrate declined, coinciding with a decrease in 16/24 plasma cytokine/chemokines levels. Tumour infiltrating immune cell populations did not correlate with peripheral blood populations. However, a decrease in plasma cytokine/chemokine levels may indicate ‘immune exhaustion’.

CONCLUSIONS

The data derived provide baseline characteristics to study changes associated with multi-modal treatment strategies and to sequence therapies to maintain immune modulation. This information will contribute to the identification of novel combination therapies.
IMMU-06. TTFIELDS INDUCES IMMUNOGENIC CELL DEATH AND STING PATHWAY ACTIVATION THROUGH CYTOPLASMIC DOUBLE-STRANDED DNA IN GLIOBLASTOMA CELLS
Authors: Chen Dongjiang , Thomas Nagheme , Ren Jie , Le Son , Sebstian Mathew , Deng Changwang , Jin Dan , Tran David
Published: November 2019

OBJECTIVES

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults. Tumor Treating Fields (TTFields) was approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly. In many patients, a transient stage of increased peritumoral edema is often observed early during TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. We hypothesize that TTFields activate the immune system by triggering pyroptosis and type I Interferon (IFN) response.

METHODS

A panel of GBM cell lines were treated with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system. Cells were analyzed for the production of micronuclei and activation of both pyroptosis and STING pathways using immunostaining, quantitative PCR, ELISA and cytometry.

RESULTS

TTFields resulted in a significantly higher rate of micronuclei structures released into the cytoplasm, which were co-localized with two upstream dsDNA sensors AIM2 and cGAS. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway leading to the release of type I IFNs and pro-inflammatory cytokines downstream of the NFκB pathway. In a co-culture experiment of bone marrow cells with cells/supernatants obtained from GBM cells treated with TTFields, GBM cells depleted of AIM2 and STING failed to induce bone marrow cells.

CONCLUSIONS

These results provide compelling evidence that TTFields activates the innate immune system in GBM cells, and a strong rationale for combining TTFields with immune checkpoint inhibitors to create a therapeutic synergy.
IMMU-08. THE ANTIGENIC LANDSCAPE OF GLIOBLASTOMA - REFINING THE TARGETS FOR IMMUNOTHERAPY
Authors: Kapolou Konstantina , Katharina Freudenmann Lena , Friebel Ekaterina , Bichmann Leon , Becher Burkhard , Stevanović Stefan , Rammensee Hans-Georg , Weller Michael , Christoph Neidert Marian
Published: November 2019

We provide a comprehensive analysis of the antigenic landscape of glioblastoma using a multi-omics approach including ligandome mapping of the Human Leukocyte Antigen (HLA) ligandome, next generation sequencing (NGS) as well as an in-depth characterization of tumor-infiltrating lymphocytes (TIL) using mass cytometry and ultra-deep sequencing of the T-cell receptor (TCR). Tumor-exclusive HLA class I and class II ligands (immune precipitation and LC-MS/MS) of 24 isocitrate dehydrogenase 1 wild type glioblastoma samples and 10 autologous primary glioblastoma cell lines were defined in comparison to an HLA ligandome normal tissue reference database (n > 418). We found 11,496 glioblastoma exclusive HLA class I ligands (2,064 shared with cell lines; 3,754 on ≥ 2 glioblastoma samples). On the source protein level, 239 glioblastoma exclusive proteins were identified; among them 54 were also found in cell lines. For HLA class II ligands the analysis revealed 11,870 glioblastoma exclusive peptides (444 shared with cell lines; 3,420 on ≥ 2 glioblastoma samples) and 278 glioblastoma exclusive proteins; among which 18 were present also in cell lines. Moreover, whole-exome sequencing and whole RNA sequencing of 13 tumor samples was performed with the aim to predict neoantigens. On average 5,662 somatic missense effects were identified per patient (min: 4,258; max: 7,479). Candidate peptides are grouped into (i) in silico predicted neoepitopes, (ii) tumor-exclusivity on HLA, (iii) gene expression (e.g. cancer testis antigens). Top-ranking candidates from each group will be tested with regards to their immunogenicity in an autologous setting (TIL, peripheral blood mononuclear cells, patient derived tumor cells). Finally, the peptide and immunogenicity data is correlated with the immune phenotype of the TIL compartment as well as the TCR repertoire of the sample.
IMMU-15. A UNIQUE SELF-STIMULATIVE PROPERTY OF CD70 CAR ADVANCES T CELL FUNCTION AND ANTI-TUMOR RESPONSE IN GLIOBLASTOMA
Authors: Jin Linchun , Hou Alicia , Tao Haipeng , Karachi Aida , Na Meng , Lin Zhiguo , Deleyrolle Loic , Sayour Elias , Mitchell Duane , Huang Jianping
Published: November 2019

BACKGROUND

Glioblastoma (GBM) is a refractory brain tumor that desperately needs new therapeutic interventions. Our group identified CD70 as a novel target of CAR-T therapy for this malignancy. We demonstrate that CD70 is overexpressed by low-/high-grade gliomas and associated with poor survival for patients; CD70 promotes CD8 specific cell death and tumor-associated macrophage infiltration in gliomas. The CD70 CAR (using CD27, a natural costimulatory receptor of T cells as an antigen-binding region) T cells can efficiently eradicate CD70 positive tumors in syngeneic and xenograft mouse models.

OBJECTIVE

To evaluate the properties of CD70 CAR-transduced T cells in GBM treatment.

METHODS

CD70 CAR or IL13Rα2 CAR was linked with fluorescent reporter gene EGFP, and cloned into a retroviral vector (pMSGV8). In vitro T cell culture and flow cytometry were used to evaluate the self-enrichment property and susceptibility to TCR stimulation of the CAR T cells. KI67, Bcl-2, CD70 gene expression was tested by qPCR to measure the proliferation/apoptosis properties of the CAR T cells. Cytokine profile was analyzed by ELISA. The anti-tumor response was evaluated using Xenograft mouse models.

RESULTS

Compared with IL13Rα2 CAR T cells, the frequency of CD70 CAR T cells was significantly increased 3 weeks post transduction, and approximately 100 to 150-fold CD70 CAR T cell expansion without additional stimuli was achieved in vitro. The expanded CD70 CAR T cells were mostly (up to 85%) CD8+ T cells three weeks post CAR transduction. Enhanced proliferative capacity and production of IL-2, IFN-γ, and TNF-α of the CD70 CAR-transduced T cells upon anti-CD3/CD28 stimulation were also revealed. Results from animal models show that the CD70 CAR T cells present superior in vivo persistence and antitumor efficacy.

CONCLUSION

We show the auto-stimulative property, as well as superior T cell function and antitumor efficacy of CD70 CAR T cells in GBM models.
IMMU-17. TARGETING GLIOBLASTOMA WITH DNAM-1-BASED CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS
Authors: Weiss Tobias , Meister Hanna , Weller Michael , Sentman Charles , Roth Patrick
Published: November 2019

BACKGROUND

Genetically engineered T cells that express a chimeric antigen receptor (CAR) have shown powerful anti-tumor activity in extracranial malignancies. This concept is now also being explored against glioblastoma. However, many single target antigens used for CAR cell therapy are non-homogeneously expressed. We assessed the therapeutic potential of CAR T cells targeting 2 antigens which are homogeneously expressed by glioma cells which reduces the probability of tumor immune escape due to antigen loss.

METHODS

We analyzed the expression of CD112 and CD155, which are ligands to the activating immune cell receptor DNAX Accessory Molecule-1 (DNAM-1), in a panel of mouse and human glioma cell lines as well as in human glioblastoma samples and generated glioma cells with a CD112 or CD155 knock-out. To exploit the specific binding properties of DNAM-1, we generated first or second-generation CAR T cells that use DNAM-1 as an antigen-binding domain and investigated their anti-tumor activity in vitro and in vivo using syngeneic orthotopic mouse glioma models.

RESULTS

CD112 and CD155 are homogeneously expressed in mouse and human glioma cell lines as well as human glioblastoma tissue specimens. CRISPR/Cas9-mediated knock-out of CD112 or CD155 affected the migration of glioma cells, but had no impact on the proliferation or susceptibility to irradiation or temozolomide. DNAM-1-based CAR T cells exerted high cytolytic activity and secretion of various effector cytokines in vitro. Upon intravenous administration, DNAM-1-based CAR T cells did not exert significant toxicity, homed to the tumor site in the brain and prolonged the survival of orthotopic glioma-bearing mice with durable anti-tumor responses in a fraction of mice.

CONCLUSION

CD112 and CD155 represent attractive targets for glioma immunotherapy using genetically engineered immune cells. Based on the data obtained from our preclincal assessment of DNAM-1-based CAR T cells, this immunotherapeutic strategy might also be explored in human glioma patients.
IMMU-22. TARGETING NON-CANONICAL FUNCTION OF IDO1 IN GLIOBLASTOMA IMMUNOTHERAPY
Authors: Zhai Lijie , Bell April , Qian Jun , Ladomersky Erik , Lauing Kristen , Wainwright Derek
Published: November 2019

Recent failure of immune checkpoint blockade, anti-PD-1 therapy in patients suffering recurrent glioblastoma (GBM) indicates that development of novel immunosuppressive mediator-targeted therapeutic strategy is critical for more effective immunotherapies against GBM. Indoleamine 2, 3-dioxygenase 1 (IDO1) has been shown to mediate tumor-induced immunosuppression through its tryptophan catabolic activity. Surprisingly, pharmacological IDO1 enzyme inhibitors have not demonstrated any survival benefit in cancer patients either mono-therapeutically or in combination with other therapies. Previously we confirmed that tumor IDO1 mRNA expression is significantly upregulated in GBM patients compared to those from patients with lower grades gliomas. Furthermore, knockdown of IDO1 in mouse GBM cells significantly increased mouse survival and decreased tumor infiltrating Tregs, but did not affect tumor local tryptophan catabolism. Consistently, inhibition of IDO1 enzymatic activity on GBM-bearing mice did not yield any survival benefit. These findings indicate that IDO1 may suppress immune response through a non-enzymatic pathway. To test this hypothesis, IDO1-deficient mouse GBM cells were created and modified to stably express either wild-type (WT) or enzyme-null IDO1. Syngeneic mice intracranially engrafted with both WT and enzyme-null IDO1 overexpressing GBM cells showed significantly increase of tumor infiltrating Tregs and decreased survival in comparison to mice engrafted with IDO1-deficient GBM cells. In contrast, no difference was observed between WT-IDO1 GBM and enzyme-null IDO1 GBM groups. Additionally, when co-cultured with splenic monocytes, both WT and enzyme-null IDO1 overexpressing GBM cells induced more mature macrophages than IDO1-deficient GBM cells. Taken together, our data strongly imply an enzyme independent pathway associated with IDO1-driven immunosuppression in mouse GBM. Validation of this novel mechanism on human GBM cells is ongoing. A soluble molecule has been identified as a potential mediator for the non-enzymatic function of IDO1. Results from this study will provide innovative strategies for IDO1-based immunotherapy against GBM.
IMMU-28. TARGETING IMMUNOSUPPRESSIVE MYELOID DERIVED SUPPRESSOR CELLS VIA MIF/CD74 SIGNALING AXIS TO ATTENUATE GBM GROWTH
Authors: Alban Tyler , Bayik Defne , Otvos Balint , Grabowski Matthew , Ahluwalia Manmeet , Bucala Richard , Vogelbaum Michael , Lathia Justin
Published: November 2019

The immunosuppressive microenvironment in glioblastoma (GBM) enables persistent tumor growth and evasion from tumoricidal immune cell recognition. Despite a large accumulation of immune cells in the GBM microenvironment, tumor growth continues, and evidence for potent immunosuppression via myeloid derived suppressor cells (MDSCs) is now emerging. In agreement with these observations, we have recently established that increased MDSCs over time correlates with poor prognosis in GBM, making these cells of interest for therapeutic targeting. In seeking to reduce MDSCs in GBM, we previously identified the cytokine macrophage migration inhibitory factor (MIF) as a possible activator of MDSC function in GBM. Here, using a novel in vitro co-culture system to reproducibly and rapidly create GBM-educated MDSCs, we observed that MIF was essential in the generation of MDSCs and that MDSCs generated via this approach express a repertoire of MIF receptors. CD74 was the primary MIF receptor in monocytic MDSCs (M-MDSC), which penetrate the tumor microenvironment in preclinical models and patient samples. A screen of MIF/CD74 interaction inhibitors revealed that MN-166, a clinically relevant blood brain barrier penetrant drug, which is currently fast tracked for FDA approval, reduced MDSC generation and function in vitro. This effect was specific to M-MDSC subsets expressing CD74, and appeared as reduced downstream pERK signaling and MCP-1 secretion. In vivo, MN-166 was able reduce tumor-infiltrating MDSCs, while conferring a significant increase in survival in the syngeneic glioma model GL261. These data provide proof of concept that M-MDSCs can be targeted in the tumor microenvironment via MN-166 to reduce tumor growth and provide a rationale for future clinical assessment of MN-166 to reduce M-MDSCs in the tumor microenvironment. Ongoing studies are assessing the effects of MDSC inhibition in combination with immune activating approaches, in order to inhibit immune suppression while simultaneously activating the immune system.
IMMU-34. ATRX MUTATIONS PREDICT RESPONSE TO INNATE BASED THERAPY IN GLIOMA
Authors: Bowie Michelle , Hariharan Seethalakshmi , Hostettler Janell , Roso Kristen , He Yiping , Pirozzi Christopher , Roskoski Martin , Keir Stephen , Brown Michael , Zhang Gao , Gromeier Matthias , Yan Hai , Ashley David
Published: November 2019

BACKGROUND

Innate based immunotherapies are becoming increasingly important for treating brain tumor patients. Gliomas carry recurrent mutations in regulatory genes that control innate immune signaling responses. About 71% of adult WHO grade II and III gliomas and 57% of secondary glioblastomas also carry a loss-of-function mutation in the ATRX gene. ATRX is a SWI-SNF chromatin remodeling protein that has major roles in processes such as cell cycle regulation and maintenance of genomic stability. Recent studies have implicated ATRX in dysfunctional innate immune signaling in cancer cells. However, the role of ATRX in mediating innate immune responses has not been investigated in gliomas.

METHODS AND RESULTS

Human and mouse glioma cell lines from a variety of genetic contexts have been examined including models which carry IDH/ATRX mutations, IDH 1p-/19q- and ATRX -/- status. Additionally, using Crispr-Cas9 technology and cloning cell lines with ATRX deletions, we have derived a series of immune competent and nude mice models. Treating these cell lines with double-stranded RNA based innate stimuli led to an enhanced early induction in phospho-interferon regulatory factor 3 (IRF3) and late induction in phospho-STAT1 in the ATRX knockout (KO) cell lines. A differential increase in interferon-stimulated gene 15 (ISG15) release was also noted in the ATRX KO cell lines, further suggesting that ATRX deletion may enable a potent activation of type I interferon production. A combination of patient-derived glioma cell lines in xenograft models and syngeneic murine glioma models derived from ATRX KO cell lines and controls confirm a survival advantage in both immuno-competent mice and xenografts. Our models are under evaluation with PVSRIPO and other innate based RNA therapies.

CONCLUSION

Our data suggests that ATRX mutations may confer sensitivity to RNA-based innate immune signaling agonists in gliomas. This potential vulnerability can be targeted in future therapies.
IMMU-42. CD8+ T-CELLS MEDIATE IMMUNOEDITING, AND INFLUENCE GENOTYPE, TUMOR ONCOGENIC PATHWAYS AND MICROENVIRONMENT DURING PROGRESSION OF MURINE GLIOMAS
Authors: Robert Kane J , Zhao Junfei , Tsujiuchi Takashi , Laffleur Brice , Mahajan Aayushi , Rao Ganesh , Mela Angeliki , Dmello Crismita , Arrieta Victor A , Chen Li , Zhang Daniel Y , Lee-Chang Catalina , Xiao Ting , Rothschild Gerson , Basu Uttiya , Horbinski Craig , Lesniak Maciej S , Heimberger Amy , Rabadan Raul , Canoll Peter D , Sonabend Adam
Published: November 2019

Cancer immunoediting shapes tumor progression by the immunological selection of tumor cell variants that can evade immune recognition. Given the immune evasive cellular diversity of glioblastoma, we hypothesized that CD8+ T-cells mediate immunoediting in this tumor. We evaluated tumor progression in the absence of CD8+ T-cells by depleting this immune cell population in a transgenic murine glioma model. Tumors generated in the absence of CD8+ T-cells developed poorly in recipients with intact immunity, implying a more immunogenic profile. These tumors demonstrated increased chromosomal instability, gene fusions, MAPK signaling, and macrophage infiltration. These observations were stochastic, suggesting variability in the mode of tumor evolution in the absence of this immune effector. MAPK activation was correlated with macrophage recruitment in two transgenic murine models and the human disease. Our results indicate that CD8+ T-cells mediate a strong immunoediting selection in glioblastoma that protect against the hallmarks of cancer and drive immune evasion.