bluuebunny/crossref_metadata_2025 huggingface dataset:
BIOM-26. RAPID IDENTIFICATION OF IDH1-R132 VARIANTS IN GLIOMA WITH LOCKED NUCLEIC ACID LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LNA-LAMP)
Authors: Choate Kristian , Raack Edward , Line Veronica , Jennings Matthew , Belton Robert , Winn Robert , Mann Paul
Published: November 2023

Point mutations that impact the catalytic site of isocitrate dehydrogenase 1 (IDH1) are associated with an extended overall survival of approximately 3.8 years for glioma patients compared to 1 year for those with wild-type IDH1. The difference in survival benefit is associated with the maximal surgical resection of IDH1 mutant tumors, making intraoperative knowledge of the mutational status valuable. Unfortunately, current methods used to determine IDH1-R132 mutational status are incompatible with obtaining real-time results. We have developed a panel of assays that use LNA-LAMP to identify commonly encountered IDH1-R132 variants (R132H, R132C, R132G, R132S, R132L) within 40 minutes. These assays require little preanalytical processing, no complex equipment, and yield results which are easily interpreted by the naked eye. Tumor samples are initially subjected to a 5-minute alkaline digest, then added directly to LNA-LAMP reactions containing primers unique to each IDH1-R132 variant. Discrimination between the wild-type and mutant allele is achieved with the integration of locked nucleic acids within primers. A reaction with an IDH1-R132R specific primer serves as the sample positive control, as all tumor lysates are expected to contain wild-type IDH1 DNA. Lysates are incubated for 35 minutes in a hot water bath, followed by visual interpretation using a pH-based colorimetric indicator, where samples positive for the mutation appear a vibrant yellow while those lacking the mutation remain pink. This method has been rigorously tested with patient-derived tumor lysates, yielding reproducible and accurate results, and has an analytical sensitivity of about 20% mutant DNA in a background of wild-type DNA. This sensitivity is comparable to that of Sanger Sequencing while simultaneously offering a more cost-effective and efficient workflow. LNA-LAMP is amenable for use in the intraoperative window to allow patients with an IDH1-R132 mutation the benefit of a maximal surgical resection.
BIOM-33. TRNA FRAGMENTS IN PLASMA EXTRACELLULAR VESICLES FROM HIGH GRADE GLIOMA PATIENTS, POTENTIAL BIOMARKERS?
Authors: Weeks Adrienne , Phinney Thomson , Alwadei Ali , Wajnberg Gabriel , Han Jae , Chacko Simi , Atwood Kathleen , Roy Jeremy
Published: November 2023

Transfer RNA (tRNA) are non-coding RNAs responsible for bringing the amino acids to the ribosome during translation. Recent studies in cancer have shown that fragments (tRFs) derived from tRNA cleavage serve regulatory roles in epigenetics, signalling and translation. tRFs are also released from cells bound to proteins and can be found in extracellular vesicles to perform some aspects of paracrine signalling. Utilizing high throughput small RNA sequencing we have identified over 750 uniquely or multi-mapped (6.9% of total sRNA mapped reads) tRNA and tRFs in extracellular vesicles of high-grade glioma patient plasma (IDH-WT). Consistent with novel literature in other cancers there was a general trend to decreased expression of tRNA and tRFs (n = 76 with FDR < 0.05) in high grade glioma plasma EVs compared to controls. However, further analysis of tRFs reveals a much more complex story, as fragment distribution differs between cancer and controls. tRNAs and tRFs may serve as important biomarkers and regulators with therapeutic potential in the future.
BIOM-39. GENETIC VARIANTS ASSOCIATED WITH DISTANT RECURRENCE IN GLIOBLASTOMA PATIENTS TREATED WITH STANDARD THERAPY
Authors: Fougner Vincent , Chiranth Shivani , Yde Christina , Nørøxe Dorte , Christensen Ib , Hasselbalch Benedikte , Lassen Ulrik , Poulsen Hans , Urup Thomas
Published: November 2023

Infiltrative growth is a hallmark of glioblastoma and is a major factor in therapeutic failure. Genetic variants predictive of distant recurrence may serve as novel treatment targets. The aim is to identify genetic variants associated with distance recurrence in glioblastoma patients. METHODS: From a prospective cohort of consecutive, non-selected glioblastoma patients administered standard therapy between 2016-2021 at Rigshospitalet, all patients with a genomic tumor profile were included. Distant recurrence was defined as a new contrast-enhancing tumor > 2 cm from the initial lesion. Tumor tissue was analyzed by RNA- and DNA-sequencing (WES or WGS). Genetic variants were defined as pathogenic and likely-pathogenic variants based on genomic reports. Predefined candidate biomarkers included: i) the most common genetic variants in the cohort (mutational frequency > 2%), ii) the number of reported variants per sample, and iii) groups of genetic variants according to the common signaling pathways. Candidates were screened for association with time to distant recurrence using univariate Cox regression analysis. Biomarkers with p < 0.20 were considered for multivariate analysis adjusted for known prognostic factors with death as the competing risk. RESULTS: A total of 236 patients were included, and 208 patients (88%) were evaluable for recurrence pattern. Distant recurrence was observed in 40 patients (19%). Patients with distant and local recurrence showed no difference in progression-free survival (p = 0.58), while distant recurrence tended to have a poor overall survival (OS, p = 0.09). The OS from time of first recurrence was significantly poorer for patients with distant recurrence compared to local recurrence (p = 0.01) with a median OS of 7.2 months (95%CI: 5.9-8.5) and 10.9 months (95%CI: 9.8-12). The median frequency of called gene variants was 4 (range 0-11) per sample. CONCLUSION: Patients with distant recurrence have a poor prognosis. Final analyses will be presented.
BIOM-41. CITRULLINATED HISTONE H3 ELISA REVEALS NETOSIS AS A SOURCE OF PROGNOSTIC CIRCULATING CELL-FREE DNA IN GLIOBLASTOMA
Authors: Till Jacob , Ballinger Dominique , Wang Zhuoyang , Yazdani Zachariya , Macia Camilla , Yin Melinda , Abdalla Aseel , Nabavizadeh Ali , Desai Arati , Binder Zev , Gollomp Kandace , Zhou Wanding , Carpenter Erica , Bagley Stephen J
Published: November 2023

BACKGROUND

We have previously shown that higher concentrations of plasma cell free DNA (cfDNA), both at baseline and following chemoradiotherapy, are associated with worse survival in GBM. Because only scant amounts of GBM-derived cfDNA shed into the peripheral circulation, we sought to determine the predominant source of prognostic plasma cfDNA in these patients and hypothesized that this may help unravel the mechanism underlying the association of high cfDNA and survival.

METHODS

Plasma was obtained for 126 patients prior to resection of newly diagnosed GBM. A subset of 46 plasma samples had ccfDNA extracted, enzymatically converted, whole genome amplified, and analyzed on the Illumina Epic array for deconvolution of cfDNA methylation patterns. Based on these results demonstrating neutrophils as the predominant cfDNA source, we performed ELISA for citrullinated-histone 3 (citH3) in plasma from 113 of the patients as a specific marker of NETosis, a pro-tumor process of programmed neutrophil cell death during which DNA is released in web-like structures called neutrophil extracellular traps (NETs).

RESULTS

Deconvolution of cfDNA methylation patterns revealed that neutrophils were the dominant source of cfDNA (median percentage of cfDNA derived from neutrophils = 46% , range 17-67%) and correlated with baseline plasma cfDNA concentration (n = 46, r = 0.35, p = 0.017). Additionally, cfDNA concentration was significantly correlated with the concentration of citH3 (n = 113, r = 0.60, p < 0.0001), which was higher in GBM patients compared to 26 healthy controls (p < 0.0001).

CONCLUSION

Our findings suggest that NETosis may be the dominant source of plasma cfDNA, which we have previously shown to be highly prognostic in patients with GBM. Additional studies interrogating the tumor microenvironment for NETosis in GBM tumors are ongoing. These results lay the foundation for further exploration of NETosis as a therapeutic target and underscore the emerging importance of neutrophil biology in GBM.
BIOM-44. IMPROVED OVERALL SURVIVAL OF RECURRENT GLIOBLASTOMA (GBM) PATIENTS WITH EGFR AMPLIFICATION AND EGFR VIII MUTATIONS TREATED WITH OSIMERTINIB: A RETROSPECTIVE REVIEW
Authors: Nahm Joshua , Schumann Eva Highberg , Vu Mia , Sinha Mousumi , Ware Cornelius , Hsu Sigmund , Zhu Jay-Jiguang
Published: November 2023

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with an overall survival (OS) of 14.6-23 months in clinical trial patients and 11 months for the real world population. There is no uniform standard of care for recurrent GBM (rGBM) patients. EGFR amplification and EGFRvIII mutations are common in GBM and are potential targets with EGFR tyrosine kinase inhibitors (EGFR-TKI). Osimertinib, a third-generation EGFR-TKI, effectively penetrates the blood brain barrier and is FDA-approved for non-small cell lung cancer. We conducted a retrospective review of rGBM patients with EGFR amplification and EGFRvIII mutations who received osimertinib in addition to other therapies such as bevacizumab, irinotecan, tumor-treating fields, and salvage radiation at recurrence. The primary outcome was OS. Secondary outcomes included recurrence free survival (RFS), overall survival after initiation of osimertinib (rOS), and adverse events (AE). Statistical analysis utilized the Kaplan-Meier estimate. AE were defined by the Common Terminology Criteria for Adverse Events Version 5.0. Twenty-seven patients (17 males) with a median age of 59 years (range: 29-75) were identified. All except one patient were IDH1-wild type (92.9%). MGMT promoter statuses included ten methylated (35.71%) and 11 unmethylated patients (39.29%). The median time on osimertinib was 5.1 months. The median OS was 28 months (95% CI 24, NA). Following osimertinib initiation, the median RFS was 6.4 months (95% CI 5.1, NA), and the median rOS was 12 months (95% CI 8.4, NA). AEs included grade I/II rash (44.4%), grade I/II diarrhea (40.7%), grade III leukopenia (18.5%), and grade III thrombocytopenia (11.1%). The data suggests that osimertinib improves the OS of rGBM patients with manageable and reversible AEs. Despite the limitations of a retrospective study and small sample size, osimertinib is a promising targeted therapy for rGBM. Further research is warranted in a clinical trial to evaluate this treatment prospectively.
BIOM-48. A NOVEL LIQUID BIOPSY-BASED APPROACH TO ISOLATE AND CHARACTERIZE GLIOBLASTOMA-DERIVED SMALL EXTRACELLULAR VESICLES FROM BLOOD
Authors: He Yangen , Almousa Sameh , Kumar Ashish , Su Yixin , Sharma Mitu , Singh Sangeeta , Kim Susy , Lesser Glenn , Deep Gagan
Published: November 2023

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer characterized by a poor prognosis and limited treatment options. The absence of early diagnostic measures, prognostic biomarkers, and reliable methods for monitoring disease progression contribute to the high mortality rate among GBM patients. In recent years, liquid biopsy has emerged as a non-invasive tool for assessing cancer onset and therapeutic responses. Among the various liquid biopsy approaches, extracellular vesicles (EVs) have gained attention; as lipid bilayer membrane-delimited 30-150nm sized small extracellular vesicles (sEV) are released by all cell types. These vesicles carry molecular cargo reflecting the cell of origin and its pathophysiological state, making them a promising alternative to other liquid biopsy methods, such as circulating tumor cells and circulating nucleotides. However, the application of sEV-based liquid biopsy is hindered by the lack of methods to enrich GBM tumor-specific sEV in the blood. Our team has developed unique tools and expertise in isolating cell/tissue-specific sEV from biofluids. We have previously reported the isolation of sEV derived from specific brain cells in the blood, characterizing them for diagnosing neurodegeneration and predicting treatment response. In this study, we first isolated total sEV (TE) from the plasma of GBM patients (n = 11). Subsequently, we enriched GBM-specific sEV (sEVGBM) from TE using the astrocyte-specific surface marker glutamate aspartate transporter (GLAST). To achieve this, we utilized biotin-tagged GLAST antibody and streptavidin-coated magnetic beads for sEVGBM isolation. We then extensively characterized the size and concentration (number/ml) of sEVGBM by nanoparticle tracking analyses, while evaluating their purity through nanoflow cytometry for GLAST expression. Furthermore, we confirmed the expression of GBM-specific markers EGFRvIII, EPHB2 and IL-13Rα2 on the surface of sEVGBM (n = 3). Importantly, the expression of these markers was significantly enriched on sEVGBM compared to TE, underscoring the significance of our strategy as a potential liquid biopsy for GBM.
BIOM-51. IDENTIFICATION OF A LOW-FLIP ANGLE BASED CBV THRESHOLD FOR FRACTIONAL TUMOR BURDEN (FTB) MAPPING IN RECURRENT GLIOBLASTOMA
Authors: Anil Aliya , Stokes Ashley , Alhilali Lea , Karis John , Bell Laura , Eschbacher Jennifer , Hu Leland , Boxerman Jerrold , Schmainda Kathleen , Quarles C
Published: November 2023

INTRODUCTION

Differentiation of glioblastoma recurrence from post treatment radiation effects (PTRE) would improve patient management. Dynamic susceptibility contrast (DSC) MRI can be used to generate fractional tumor burden (FTB) maps, via application of regional cerebral blood volume (RCBV) thresholds, to spatially differentiate recurrence from PTRE. FTB maps derived from a DSC-MRI protocol that includes a contrast agent preload in addition to the bolus injection and a moderate-flip angle (MFA) been histologically validated. Recently, a single-dose, low-flip angle (LFA) protocol was shown to provide RCBV measurements consistent with the MFA protocol and has been recommended as a consensus protocol. This study aims to identify the RCBV thresholds for the single-dose, LFA protocol that generates FTB maps, concordant with those obtained from the reference double-dose, MFA protocol.

METHODS

Two sets of DSC-MRI data were collected sequentially from 52 patients using a single-dose, low-flip angle (30o) protocol and a double-dose, moderate flip angle (60o) protocol using TR = 1.5s and TE = 30ms. Standardized RCBV maps (sRCBV) were obtained and co-registered with the post-contrast T1-weighted images using the FDA-approved clinical software, IB Neuro™ and IB Delta Suite™, respectively. The ground truth MFA-based FTB maps were computed using multiple sRCBV thresholds (1.0 and 1.75). An ROC analysis was conducted to identify the optimal, voxel-wise LFA sRCBV thresholds to produce FTB maps that best match the reference maps. RESULTS/

CONCLUSIONS

The ROC analysis identified a lower LFA threshold of 1.0 to accurately distinguish PTRE from tumor recurrence and an upper threshold of 1.55 to differentiate aggressive tumor regions. The single-dose, LFA protocol eliminates variations in the contrast agent incubation time between the preload and bolus injection. This study provides motivation for the use of the single-dose LFA protocol in the management of patients with glioblastoma and is a reliable method for distinguishing tumor from PTRE.
BIOM-55. TARGETED GENE EXPRESSION PROFILING PREDICTS MENINGIOMA OUTCOMES AND RADIOTHERAPY RESPONSES
Authors: Chen William , Choudhury Abrar , Youngblood Mark W , Polly Mei-Yin , Lucas Calixto-Hope , Mirchia Kanish , Maas Sybren , Suwala Abigail , Won Minhee , Bayley James , Harmanci Akdes , Harmanci Arif , Klisch Tiemo , Nguyen Minh , Vasudevan Harish , McCortney Katy , Lam Tai-Chung , Pu Jenny Kan-Suen , Li Lai-Fung , Leung Gilberto Ka-Kit , Chan Jason , Perlow Haley , Palmer Joshua , Berghoff Anna , Preusser Matthias , Nicolaides Theodore , Mawrin Christian , Young Jacob , Boreta Lauren , Braunstein Steve , Schulte Jessica , Butowski Nicholas , Santagata Sandro , Bush Nancy Ann Oberheim , Villanueva-Meyer Javier , Chandler James P , Solomon David , Rogers Leland , Pugh Stephanie , Mehta Minesh , Sneed Penny , Berger Mitchel , Horbinski Craig , McDermott Michael , Perry Arie , Bi Wenya Linda , Patel Akash , Sahm Felix , Magill Stephen , Raleigh David
Published: November 2023

INTRODUCTION

Improvements in meningioma risk stratification are needed and current indications for postoperative radiotherapy are controversial. Here we report multicenter clinical and analytical validation of a gene expression biomarker using clinical and genomic data from 1856 meningiomas from 12 institutions across 3 continents.

METHODS

A previously-developed 34-gene expression biomarker was applied to 3 validation cohorts: (1) a multicenter retrospective clinical validation cohort (N = 6 centers, N = 866 meningiomas, median follow-up 5.2 years), (2) a prospective investigator-blinded clinical validation cohort from RTOG 0539 (N = 103 meningiomas, median follow-up 8.4 years), and (3) an analytical validation cohort for test/re-test and FFPE/frozen analyses, and comparison across platforms for gene expression quantification (N = 8 centers, N = 1219 meningiomas). Biomarker performance was compared to 9 other meningioma classification systems. Favorable versus unfavorable cases were defined as (1) gene expression low risk with any resection, or gene expression intermediate risk with GTR (favorable), versus (2) gene expression intermediate risk with STR, or gene expression high risk with any resection (unfavorable).

RESULTS

The biomarker improved on WHO 2021 grade (5-year local freedom from recurrence [LFFR] delta-AUC 0.11, 95% CI 0.07-0.17, p< 0.001) and all other classification systems for LFFR and OS on multivariate analysis, achieving a negative predictive value for recurrence at 5-years of 91.9%. The biomarker predicted LFFR after postoperative radiotherapy, with a hazard ratio of 0.33 for unfavorable primary WHO grade 2 meningiomas (95% CI 0.14-0.76, P = 0.009), and 0.54 for unfavorable propensity-matched meningiomas across all WHO grades (95% CI 0.37-0.78, p = 0.0001). The biomarker reclassified 39.8% of meningiomas from RTOG 0539, including downstaging 30.5% of patients who received postoperative radiotherapy, and was independently prognostic for overall survival on multivariate analysis.

CONCLUSIONS

A 34-gene expression biomarker improves prognostication of meningioma outcomes and predicts postoperative radiotherapy responses.
BIOM-59. FEASIBILITY AND UTILITY OF THE NOVEL CANCER COMPREHENSIVE GENOMIC PROFILING RNA-SEQ FOR THE IDENTIFICATION OF THERAPEUTIC TARGETS AND DIAGNOSTIC BIOMARKERS IN GLIOMA
Authors: Takahashi Masamichi , Kohsaka Shinji , Ichimura Koichi , Mano Hiroyuki , Narita Yoshitaka
Published: November 2023

BACKGROUND

The detection of novel therapeutic targets is increasingly important in oncology. The Todai OncoPanel (TOP) RNA panel is a custom target RNA-seq using the junction capture method to maximize the sensitivity in detecting of known 456 fusion gene transcripts, which is approved in Japan in 2022 and covered by insurance in later 2023. Above, TOP RNA panel interrogates aberrantly spliced transcripts and analyzes the expression profiles of 1,390 genes. Primary brain tumors require various molecular profiles to be classified based on WHO2021 classification and also necessitates new therapeutic targets. In this study, we aimed to classify gliomas and identify the molecular targets by TOP RNA panel.

METHODS

Among 131 frozen samples of malignant gliomas, 124 samples passed quality control and were subjected to TOP RNA panel. Fusion detection and RNA expression analysis were conducted with the original pipeline. Cluster analysis and Gene Set Enrichment Analysis (GSEA) were performed to identify gene sets specific for individual subtypes. Copy numbers of tyrosine kinase genes, MDM2 and CDK4 were evaluated by digital droplet PCR (ddPCR).

RESULTS

Among 55 cases of glioblastoma, gene fusions were detected in 11 cases (20.0%) including novel MET fusions, and overexpression of seven tyrosine kinase genes were observed in 15 cases (27.3%). Of the 34 overexpressed genes, gene amplification was observed in 17 genes (50%) by ddPCR. In contrast to IDH-wildtype glioblastoma, IDH-mutant tumors hardly harbored fusion genes or gene overexpression. GSEA identified that the gene sets related to 1p36 and 19q were relatively enriched in astrocytoma, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis.

CONCLUSION

TOP RNA panel detects more fusions than DNA panel and is utilized not only for the detection of molecular targets but also for the molecular classification of glioma, providing treatments optimized for the individual patients.
BIOM-62. DETECTION OF EXCLUSIVE MUTATIONS AND GENE-GENE FUSIONS IN GLIOBLASTOMA THROUGH ANALYSIS OF CIRCULATING CELL-FREE DNA (CFDNA) AS AN EMERGING DIAGNOSTIC TOOL
Authors: Frenkel-Morgenstern Milana , Giwa Olawumi , Baum Eliezer Gideon , Kaner Andrew , Laviv Yossef , Nof Sagi Har , Levy-Barda Adva , Siegal Tali
Published: November 2023

Tumor heterogeneity is a significant obstacle for developing tailored therapies for glioblastoma (GBM). We collected blood and tumors from 20 GBM patients at 4-time points and 20 blood samples from healthy controls. cfDNA was extracted from the plasma of GBM patients and healthy controls. Tumor DNA was extracted from fresh tumor samples. Extracted DNA was sequenced using a whole-genome sequencing procedure. We found that plasma cfDNA concentrations in GBM patients were significantly elevated (22.6 ± 5 ng·mL-1), as compared to healthy controls (1.4 ± 0.4 ng·mL-1). We identified unique mutations in cfDNA of GBM patients, including some of the most frequently mutated genes in GBM (TP53, 18.75%; EGFR, 37.5%; NF1, 12.5%; LRP1B, 25%; IRS4, 25%) and built a unique clinical panel for further patients monitoring. Using our gene fusion database, ChiTaRS 5.0, we identified gene fusions in both tumor DNA and cfDNA, such as KDR-PDGFRA and NCDN-PDGFRA, which correspond to previously reported alterations of PDGFRA in GBM (44% of all samples). Interestingly, the PDGFRA protein fusions can be targeted by tyrosine kinase inhibitors. We identified ROS1 fusions (CEP85L-ROS1 and GOPC-ROS1) in 8% of samples, and those might be targeted by crizotinib analogs. Moreover, we analyzed TCGA/ICGC data of 180 GBM samples and observed FGFR3-TACC3, EGFR-SEPT14 fusions in silico. Next, we measured cfDNA levels in vitro in response to Temozolomide treatment and discovered a positive correlation between unique 2000bp cfDNA fragments (R = 0.8961, P-value = 0.015632) and treatment responses in vitro. We extracted exosomes from the in-vitro experiments and validated the concentrations of both the encapsulated cfDNA as well as the free cfDNA. We found that half of cfDNA was exosome encapsulated and it varied according to the tumor stage. CONCLUSION: cfDNA may be used for the detection of exclusive patient mutations and gene fusions and is an emerging diagnostic tool in GBM.
BIOM-66. ESTABLISHMENT OF A PROGNOSTIC MODEL FOR GLIOBLASTOMA MULTIFORME MAGNETIC RESONANCE IMAGING BASED ON IMBALANCED CLASSES, CLASSIFICATION, AND KAPLAN-MEIER SURVIVAL CURVE LEARNING ALGORITHMS
Authors: Cheng Wen-Yu , Yang Chih-Chan , Kao JuHsuan , Shen Chiung-Chyi , Yang Yi-Chin , Yang Meng-Yin
Published: November 2023

Glioblastoma multiforme (GBM) is a rapidly invasive brain tumor with a high mortality rate. It is accompa-nied by peripheral edema. Physicians use magnetic resonance imaging (MRI) to diagnose each patient’s con-dition in clinical practice. The current use of machine learning to classify brain tumors has an accuracy of about 90% in most studies. Therefore, this study aimed to improve its prediction accuracy using machine learning models to predict GBM patient survival or non-survival. The study used MRI T1-related and T2-related sequences to obtain GBM tumor and edema area attributes. It then divided them into four da-tasets: maximum tumor area, maximum edema area, total tumor volume, and total edema volume. These four datasets were used as inputs for classification models based on support vector machines, decision tree algo-rithms, random forest algorithms, and multilayer perceptron (MLP) to predict patient survival. A confusion matrix was used to evaluate each model and determine the most suitable models for each dataset based on their accuracy. MLP was the most appropriate for predicting the overall edema volume dataset, with the highest accuracy in predicting GBM patient survival or non-survival at 96.97%. Hopefully, this study can assist physicians in assessing and diagnosing GBM patients’ survival status in the future. Keywords: Classification Models; Glioblastoma Multiforme; KaplanMeier Curve; Magnetic Resonance Imaging; Peritumoral Edema
BIOS-01. EFFICACY AMONG THERAPEUTICALLY EFFECTIVE ABSORBED DOSE PATIENTS IN RESPECT-GBM PHASE I OF RHENIUM-186 NANOLIPOSOME (186RNL) IN RECURRENT GLIOBLASTOMA (RGBM) COMPARED TO AN EXTERNAL CONTROL ARM (ECA)
Authors: Ensign Lisa , LaFrance Norman , Diao Lihong , Tummagunta Kala , Boisvert Denis , Michalek Joel , Brenner Andrew , Moore Melissa , Hedrick Marc , Davi Ruthanna
Published: November 2023

BACKGROUND

ReSPECT-GBM is a Phase 1/2 study of 186RNL administered by convection enhanced delivery (CED) in patients with rGBM. In Phase I, 15 of 24 patients have achieved a therapeutic absorbed dose defined as > 100 Gy. Based on prior work establishing the comparability of a bevacizumab (Bev) comparator to inform early-phase CED study results, OS of the 15 TAD patients were compared to a Bev ECA.

METHODS

Recent Bev-treated rGBM clinical trial patients meeting the same eligibility criteria as patients in the ReSPECT-GBM study were identified from the Medidata Enterprise Data Store of aggregated and deidentified patient-level historical clinical trial data. Propensity score (PS) methods were used to select the ECA and align the baseline characteristics of the Bev patients to the ReSPECT TAD cohort. OS estimates were generated using the weighted Kaplan-Meier method and compared with a weighted 2-sample 2-sided log-rank test at α = 0.05 based on a data cut-off of December 2022.

RESULTS

Baseline characteristics of the ReSPECT TAD and ECA patients were well-aligned after PS modeling. Although not achieving statistical significance due to small sample sizes (median OS 12.2 vs 10.1 months, hazard ratio 0.63, 95% CI: 0.26, 1.52), comparison of OS between the ReSPECT TAD and ECA patients demonstrate consistently longer OS at each 6-month interval: 6 months: 85.1% vs 73.3%, 12 mos: 51.1% vs 30.3%, 18 months: 30.6% vs 25.4%, 24 months: 30.6% vs 3.5%.

CONCLUSIONS

Single-arm studies can be challenging to interpret. By comparing the ReSPECT-GBM outcomes to an ECA of similar patients with balanced baseline characteristics, we can better appreciate the treatment effect of 186RNL administered via CED. The current results are encouraging, and updated results reflecting current follow-up will be presented at the meeting.
BIOS-03. COPY NUMBER VARIATION (CNV) AND CLINICAL OUTCOMES IN IDH-WILDTYPE VERSUS IDH-MUTANT GLIOMA
Authors: Chow Jocelyn , Bray David , Rupji Manali , Mannam Sai , Greenberg Ross , Dansby Kamaria , Neill Stewart , Switchenko Jeffrey , Bojanowski-Hoang Kimberly
Published: November 2023

Genomic classification of gliomas has the potential for improved prognostication and treatment guidance. However, much work remains to elucidate the full potential of genomics in glioma characterization. IDH mutant gliomas generally carry an improved survival compared to their wild-type counterparts, but their clinical course is significantly more variable and challenging to predict. The goal of this study is to characterize tumor genetic/genomic parameters associated with progression-free survival (PFS) and overall survival (OS) of both IDH-wildtype and IDH-mutant gliomas and compare the two subtypes for further stratification. We collected data for 203 patients diagnosed with IDH-wildtype gliomas (WHO Grade IV) and 88 patients with IDH-mutant gliomas (WHO Grade II-IV) from the Emory Healthcare system. Microarray single nucleotide polymorphism-copy number analysis was performed to detect total CNVs which are associated with tumor mutational burden. We analyzed total CNV based on the median, quartiles, and optimal cut points via the ideal cutpoint calculator, Cutoff Finder. We then estimated the correlation between % total CNV, PFS, and OS using the Pearson correlation coefficient. For IDH-wildtype gliomas, the mean CNV was 18.69 (standard deviation (SD): 9.62), and the median was 16.00 (IQR: 1-57). The optimal cutoff was defined as 39 for PFS analysis and 20.5 for OS analysis. For IDH-mutant gliomas, the mean CNV was 12.8 (SD: 11.36), and the median was 8.5 (IQR: 5 – 17.5). The optimal cutoff was defined as 10.5 for OS analysis and 7.5 for PFS analysis. Overall, higher CNV was associated with lower PFS and OS in both groups. However, the results were only statistically significant for OS at the optimal cutoff (20.5) for IDH-wildtype gliomas (p-value = 0.0379), and for PFS at the optimal cutoff (7.5) for IDH-mutant gliomas (p-value = 0.0420). These findings suggest that CNV may be useful as a potential prognosis factor for both tumor subtypes in different contexts.
CNSC-02. GLUTAMATERGIC SYNAPTIC INPUT DRIVES BRAIN METASTASIS OF BREAST CANCER AND MELANOMA
Authors: Karreman Matthia , Venkataramani Varun , Nguyen Linh C , Hebach Nils , Tehranian Cedric , Mayer Chanté , Meyer Lasse , Mughal Sadaf , Geckeler Lukas , Westphal Dana , Breckwoldt Michael , Wick Wolfgang , Kuner Thomas , Winkler Frank
Published: November 2023

The nervous system is implicated in pathology of both cranial and extracranial cancers. In glioblastoma, direct excitatory glutamatergic synapses between neurons and cancer cells were discovered. Importantly, these neuron-cancer contacts drive glioblastoma proliferation and invasion. However, such synapses have not yet been documented in other types of brain cancer. In the context of brain metastases, indirect, peri-synaptic contacts between breast cancer cells and neurons were observed, which promote brain metastatic growth through glutamatergic signaling via NMDA receptors on the neoplastic cells. Recently, two studies have demonstrated that neuronal activity supports the growth of both the primary tumor and brain metastases from neuroendocrine carcinoma small cell lung cancer. In the work presented here, we demonstrate direct neuron-cancer synapses in early-stage brain metastases originating from breast cancer and melanoma. These synapses exhibited the typical structural features observed in synaptic connections and induced postsynaptic currents in the cancer cells through the activation of AMPA subtype glutamate receptors. Importantly, genetic and pharmacological perturbation of these receptors reduces the number of brain metastases and the overall metastatic burden in preclinical models of breast cancer and melanoma. These results uncover a previously unknown form of direct communication between neurons and brain-metastatic breast cancer and melanoma tumor cells. This neuronal-tumor synaptic interaction demonstrates the significance of the neuronal microenvironment in the early stages of the brain metastatic cascade. Furthermore, it unveils a potential therapeutic vulnerability associated with the neuronal microenvironment, suggesting that targeting the synaptic communication between neurons and cancer cells could be a promising strategy to impede the progression of brain metastases.
CNSC-13. NEURODEVELOPMENTAL TRANSCRIPTION FACTORS PROMOTE PROLIFERATION, REGULATE CELL TYPE, AND PLAY OPPOSING ROLES IN CELLULAR MIGRATION IN GLIOBLASTOMA
Authors: Myers Bianca , Brayer Kathryn , Paez-Beltran Luis , Vue Tou Yia
Published: November 2023

Glioblastomas (GBM) make up ~50% of primary brain tumors and prognosis has not improved over the last 30 years. Despite high inter- and intratumoral heterogeneity, basic-helix-loop-helix (bHLH) transcription factors, ASCL1 and OLIG2, are present in the majority of tumors. Previously, we showed in patient-derived GBM xenograft (PDX-GBM) that ASCL1 binds to promoter and enhancer regions of cell cycle genes, as well as neurodevelopmental transcription factors including OLIG2. Similarly, we showed that OLIG2 binding overlaps with the majority (~90%) of ASCL1 binding sites, including at promoter and/or enhancer regions of ASCL1 and OLIG1/2 loci, illustrating their potential redundant and/or feed-forward function in GBM. It has been proposed that ASCL1 and OLIG2 contribute to the neural stem cell-like properties of tumor cells, which may promote tumor growth and progression but also the treatment resistivity and high recurrence rate of GBMs in patients. Using an immune competent glioma mouse model, we are able to efficiently induce tumors from glial progenitors surrounding the lateral ventricle while altering the levels of ASCL1 and OLIG2 to assess their combinatory roles in GBM progression. Remarkably, we found that loss of both Ascl1 and Olig2 prevents tumor formation in 80% of mice, whereas the loss of only Ascl1 resulted in reduced cellular migration from the tumor bulk while the loss of Olig2 promotes a highly migratory phenotype. Conversely, elevating the levels of Ascl1increased both tumor cell proliferation and migration similar to the loss of OLIG2. Using single cell RNA-sequencing, we found that tumor cells which express high levels of Ascl1 exhibit neural stem cell/astrocytic gene signatures, which supports ASCL1’s role as a marker of glioma-stem-cells. Collectively, these findings illustrate the role of ASCL1 and OLIG2 in regulating GBM initiation, proliferation, and migration where ASCL1 may directly be responsible for the highly invasive and proliferative phenotype of GBMs.
CNSC-17. WHOLE-BRAIN SPECTRAL CHANGE IN GLIOBLASTOMA PATIENTS: ASSOCIATIONS WITH CLINICAL COMORBIDITIES AND OVERALL SURVIVAL
Authors: Park Ki Yun , Snyder Abraham , Olufawo Michael , Trevino Gabriel , Luckett Patrick , Lamichhane Bidhan , Xie Tao , Lee John , Shimony Joshua , Leuthardt Eric
Published: November 2023

Glioblastoma, a highly aggressive form of brain tumor, is a brain-wide disease. We evaluated the impact of tumor burden on whole brain resting-state functional magnetic resonance imaging (rs-fMRI) activity. Specifically, we analyzed rs-fMRI signals in the temporal frequency domain in terms of the power-law exponent and fractional amplitude of low-frequency fluctuations (fALFF). We contrasted 189 patients with newly-diagnosed glioblastoma versus 189 age-matched reference participants. The patient and reference datasets were matched for age and head motion. The principal finding was markedly flatter spectra and reduced grey matter fALFF in the patients as compared to the reference dataset. We posit that the whole-brain spectral change is attributable to global dysregulation of excitatory and inhibitory balance and metabolic demand in the tumor-bearing brain. Additionally, we observed that clinical comorbidities, in particular, seizures, and MGMT promoter methylation, were associated with flatter spectra. Notably, the degree of change in spectra was predictive of overall survival. Our findings suggest that frequency domain analysis of rs-fMRI activity provides prognostic information in glioblastoma patients and offers a means of noninvasively studying the effects of glioblastoma on the whole brain.
CNSC-20. MGMT PROMOTER METHYLATED HIGH GRADE GLIOMA WITH DISTANT RECURRENCE AND STABLE ORIGINAL TUMOR SITE: CASE SERIES
Authors: Jani Ronak , Lee Jonathan , Jusue-Torres Ignacio , Germanwala Anand , Borys Ewa , Solanki Abhishek , Mallik Atul , Barton Kevin , Prabhu Vikram
Published: November 2023

Recurrence pattern in high grade glioma is classified as local and distant recurrence based on distance from original resection cavity and radiation field. We report the first series of three cases of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated high grade glioma with distant recurrence without local recurrence throughout the disease course. Patient 1: 65 year-old female with WHO grade 4 glioblastoma, isocitrate dehydrogenase enzyme (IDH) wild type (WT), MGMT methylated. She underwent gross total resection followed by Stupp protocol. Postoperative ischemic volume was < 1 ml. Distant recurrence occurred 4 months after completion of Stupp protocol. Patient 2: 55 year-old male with WHO grade 4 glioblastoma, IDH wild type, MGMT methylated. He underwent gross total resection followed by Stupp protocol. Postoperative ischemic volume was 18 ml. Distant recurrence occurred 5 months after completion of Stupp protocol. Patient 3: 31 year-old male with WHO grade 4 astrocytoma, IDH mutant, MGMT promoter methylated. He underwent gross total resection followed by Stupp protocol. Postoperative ischemic volume was 2 ml. Distant recurrence occurred 10 months after completion of Stupp protocol. Distant recurrence is more common in MGMT promoter methylated tumors. All 3 patients in our case series had poor outcomes after distant recurrence without local recurrence at the original tumor site. This observation indicates good local control in MGMT promoter methylated high grade glioma with current standard treatment. For one patient, next generation sequencing (NGS) was available for both original and recurrent tumor and did not reveal any major differences in the genetics of original tumor and distant recurrent tumor other than temozolomide induced high tumor mutational burden in the distant recurrent tumor. Understanding risk factors for distant recurrence in MGMT promoter methylated high grade gliomas and investigating correlations between recurrences will help plan therapeutic strategies to prevent distant recurrence and improve outcomes.
CNSC-26. GLIOBLASTOMA-NEURONAL CIRCUIT INTEGRATION IS MODULATED BY INTERLEUKIN-6
Authors: Daniel Andy , Krishna Saritha , Hervey-Jumper Shawn
Published: November 2023

The pleiotropic cytokine interleukin-6 (IL-6) is known to be involved in both pro- and anti-inflammatory signaling cascades. In glioblastoma studies using animal and human models, IL-6 promotes immunosuppression and tumor progression and is negatively associated with survival. Intriguingly, IL-6 also contributes to increased synaptogenesis during development and following focal brain injury. Glioblastoma remodeling of neuronal circuits influences patient survival; therefore, it is possible that IL-6 has mechanistic significance. Here, we investigated IL-6 as a driver of activity-dependent glioblastoma proliferation using patient-derived xenograft (PDX) mouse and human glioblastoma models. We assessed neuronal activity by microelectrode arrays and in vitro calcium imaging using weighted mean firing rate (WMFR), network burst frequency (NBF), and synchrony index. Next, we used bulk and single-cell RNA sequencing on 13,731 cells from ten tumors including pair-matched samples to identify differentially expressed genes in intratumoral regions with elevated functional connectivity and found IL6 to be highly upregulated. Mouse embryonic cortical neurons and cerebral organoids co-cultured with primary patient-derived glioblastoma cells demonstrated concentration-dependent neuronal hyperexcitability (increased WMFR and NBF) in IL-6-overexpressing conditions compared to the neuron-only condition. Pharmacological inhibition using the humanized IL-6 receptor antibody tocilizumab reduced network synchrony across models. These findings suggest that IL-6-induced glioma-neuronal hyperexcitability may be inhibited by the FDA-approved tocilizumab, thereby providing preclinical support for its use to treat activity-dependent mechanisms of glioblastoma proliferation. Future studies are needed to uncover mechanisms and clinical efficacy.
CNSC-28. CELLULAR AND FUNCTIONAL REMODELING OF HUMAN CEREBRAL ORGANOIDS BY GLIOBLASTOMA STEM-LIKE CELLS
Authors: Singh Sanjay , Milutinovic Bojana , Bronk Lawrence , Bronk Julianna , Shepherd Andrew , Grosshans David , Phillips Lynette , Lang Frederick
Published: November 2023

The burgeoning field of cancer neuroscience focuses on deciphering the role of normal neural cells in brain tumor growth. Until recently, representation of normal human neural cells in brain tumor model systems was lacking. By utilizing human induced-pluripotent stem cells derived cerebral organoids (COs) co-cultured with glioblastoma stem-like cells (RFP-luciferase tagged MDA-GSCs, n = 3 representing each glioblastoma molecular sub-type), we investigated the effects of GSCs on CO-derived neural cells and their activity. COs and CO+MDA-GSCs were subjected to single-cell RNA-sequencing (scRNA-seq). As per typical micro-anatomy of COs, neural stem/progenitor cells are interiorly distributed whereas differentiated neurons are positioned on the surfaces, enabling measurement of neuronal action potential on surfaces of COs by micro-electrode arrays (MEA). The scRNA-seq data from CO+MDA-GSC samples were subset into CO-derived (luciferase-ve) and GSCs-derived (luciferase+ve). Compared with COs, co-cultured samples had significantly higher percentage of non-telencephalon neural progenitor cells (ntNPCs) (CO_vs._CO+MDA-GSC(luciferase-ve): 15.6% vs. 47.2%-63.4%) while the percentages of cortical NPC (cNPCs) and intermediate progenitors (IPs) was lower (CO_vs._CO+MDA-GSC(luciferase-ve): 39.2% vs. 12.0%-20.5%). These alterations in NPC/IPs abundance were accompanied by decreased proportions of cortical excitatory neurons (CO_vs._CO+MDA-GSC(Luciferase-ve): 33.2% vs. 7.9%-23.4%). Among luciferase+ve MDA-GSCs-derived cells in co-culture samples, mesenchymal MDA-GSCs had higher abundance of ntNPCs versus cNPCs (30.4% versus 63.6%) whereas, among classical and proneural MDA-GSCs ntNPCs were 11.45-22.8 times higher. Interestingly, 14.9% of proneural MDA-GSCs derived cells displayed inhibitory neuron signatures. After 3 weeks, the mean firing rate measured by MEA was significantly lower in co-cultures compared to COs (CO_vs._CO+MDA-GSC: 0.052 Hz vs. 0.0081 Hz p-value 0.000219), suggesting functional remodeling. Our results show that cellular remodeling of normal cells resulted in progenitor-like cell-state whereas that of MDA-GSCs varied across molecular sub-type. These results suggest interactions between normal and tumor cells can potentially be leveraged to manage brain tumor growth and its impact on normal brain.
CNSC-29. NKCC1 SIGNALING IN GLIOBLASTOMA REGULATES NEURONAL HYPEREXCITABILITY THROUGH GABAERGIC TONE
Authors: Krishna Saritha , Daniel Andy , Ambati Vardhaan , Seibert Clara , Hervey-Jumper Shawn
Published: November 2023

Over 90% of patients with glioma experience tumor-associated epilepsy, however first-line treatment fails to control seizures in most patients. Thus, there is a critical need to identify novel drivers of glioblastoma-induced neuronal hyperexcitability. We therefore used high-density electrode arrays to record local field potentials in vivo from gliomas. We then developed cerebral organoid and mouse models using primary glioblastoma cells from patients and neurons derived from induced pluripotent stem cells. Single-cell RNA sequencing (13,730 cells analyzed) in this system identified genes elevated in glioma-intrinsic neurons, including the sodium-potassium-chloride co-transporter 1 (NKCC1), which mediates chloride (Cl-) influx into cells. This elevated NKCC1 expression and subsequent increase of intracellular Cl- sets the reversal potential of GABAAR-mediated currents promoting depolarizing excitatory GABAergic responses. Electrophysiological analysis of glioma-neuron co-cultures using multi-electrode array and live-cell calcium imaging demonstrated increased network burst synchrony in the presence of NKCC1-high expressing tumor cells. Single-nucleus sequencing (54,000 cells analyzed) of embryonic neurons in co-culture with GBM cells further confirmed the elevated expression of circuit assembly genes including neuronal NKCC1. Live-cell imaging of iPSC-induced neuron organoids co-cultured with NKCC1-high-and low-GBM cells revealed increased migration and integration of NKCC1-elevated tumor cells. Mechanistic and functional studies validating therapeutic vulnerabilities to NKCC1 silencing by shRNA knockdown and by the FDA-approved diuretic drug, bumetanide was performed in vitro. The reduction in neuronal synchrony induced by NKCC1 downregulation correlated with a decrease in extracellular GABA and glutamate, suggesting a strong association between excitatory GABAergic and glutamate signaling with neuronal hypersynchrony in glioblastoma. Patient-derived xenograft in vivo experiments evaluated the effect of NKCC1 on tumor growth and progression and NKCC1-high GBM cells exhibited greater tumor burden compared to control mice. Collectively, these findings reveal a novel mechanism driving neuronal hyperactivity in glioblastoma and identifies potential therapeutic vulnerabilities for the treatment of glioma-associated epilepsy.
CNSC-32. SIMULTANEOUS INVESTIGATION OF TUMOR CELL BEHAVIOR AND CONNECTIVITY WITH CORRELATIVE IN VIVO TWO-PHOTON AND EX VIVO EXPANSION MICROSCOPY
Authors: Reyhan Ekin , Wick Wolfgang , Venkataramani Varun
Published: November 2023

Glioblastomas are the most aggressive primary brain tumors, characterized by whole-brain colonization, high therapeutic resistance, and inevitable recurrence. It has previously been shown that glioblastoma cells can form functionally gap-junction connected tumor cell networks with other tumor cells and/or astrocytes, which play a crucial role in tumor progression and treatment response. Furthermore, there is increasing evidence that synaptic neuron-glioma networks promote tumor growth. However, methodologies to investigate the direct link between synaptic, astrocytic and tumor cell connectivity together with tumor cell behavior including invasion, proliferation and therapeutic resistance are yet lacking. We aimed to characterize neuron-tumor and tumor-tumor/astrocyte cell interactions on a single cell level by using correlative in vivo two-photon and ex vivo expansion microscopy with near synaptic-resolution. We studied glioblastoma cells with intravital two-photon microscopy using patient-derived glioblastoma mouse models and the fluorescent functional imaging dye Sulforhodamine 101 allowing to investigate intratumoral and astrocytic connectivity over an extended period up to weeks. Subsequently, these functional tumor cell subpopulations could be directly correlated ex vivo to identify neuron-glioma synaptic connectivity. We utilized machine learning based segmentation and a semi-automated analysis pipeline to explore the connection of different glioblastoma morphological and behavioral subtypes to direct and indirect neuronal input in multiple xenograft models. With this new correlative method, we were able to simultaneously evaluate tumor cell behavior, synaptic and tumor-tumor/astrocyte connections on a single cell level. In addition, we demonstrated that this pipeline can be used for patient samples, revealing its clinical-translational potential for cancer neuroscience. In summary, we have established a new correlative technology that interlinks two key aspects of brain cancer neuroscience: cancer cell behavior and brain tumor network connectivity. This methodology promises a more thorough understanding of glioblastoma biology, facilitating the discovery of cancer biology, including the role of brain tumor networks in therapeutic resistance.