bluuebunny/crossref_metadata_2025 huggingface dataset:
EPCO-25. MULTI-OMICS DISEASE STRATIFICATION IN PATIENTS WITH IDH-WILDTYPE GLIOBLASTOMA: SYNERGISTIC VALUE OF CLINICAL MEASURES, CONVENTIONAL AND DEEP RADIOMICS, AND GENOMICS FOR PREDICTION OF OVERALL SURVIVAL
Authors: Kazerooni Anahita Fathi , Saxena Sanjay , Tu Danni , Toorens Erik , Bashyam Vishnu , Akbari Hamed , Mamourian Elizabeth , Sako Chiharu , Koumenis Costas , Shinohara Russell T , Bagley Stephen J , Desai Arati , Lustig Robert A , O’Rourke Donald M , Ganguly Tapan , Bakas Spyridon , Nasrallah MacLean , Davatzikos Christos
Published: November 2021

PURPOSE

Multi-omics data integration captures tumor characteristics at multiple scales [i.e., microscopic (genomics and epigenetics), macroscopic (radiomics), clinical manifestation], provides a more comprehensive assessment of patient’s risk, and facilitates personalized therapies. In this work, we investigated the synergistic value of such multiple data sources for risk stratification and prediction of overall survival in IDH-wildtype glioblastoma tumors.

METHODS

Quantitative conventional and deep radiomics were extracted from pre-operative multi-parametric structural MRI (T1, T1Gd, T2, T2-FLAIR) of 501 patients with newly diagnosed glioblastoma. 389/501 and 112/501 patients formed our discovery and replication cohorts, respectively. Conventional radiomics were extracted from CaPTk, and deep radiomics from a pre-trained VGG-19 model. Multivariate SVM classification was performed on the discovery cohort to stratify patients in high, medium, and low-risk groups, using recursive feature elimination and 5-fold cross-validation. This model was independently tested on the replication cohort, and a radiomic-based survival prediction index (SPIradiomics) was calculated for each patient. Multi-stage integration of omics data, i.e., clinical (age, gender, extent of resection (EOR)), SPIradiomics, epigenetics (MGMT promoter methylation), and genomics (27 clinically relevant gene mutations via next-generation sequencing (NGS)), was performed using multivariate Cox proportional hazards (Cox-PH) model for stratification of the risk in the replication cohort.

RESULTS

Cox-PH modeling resulted in a concordance index (c-index) of 0.65 (95% CI:0.6–0.7) for clinical data, 0.67 (95% CI:0.62–0.72) for clinical and epigenetics, 0.70 (95% CI:0.65–0.75) for clinical and radiomics, 0.72 (95% CI:0.68–0.77) for clinical, epigenetics, and radiomics, and 0.75 (95% CI:0.71 – 0.78) for the multi-omics combination of all data; highlighting the added value of each layer of information in prediction of the patient’s risk.

CONCLUSION

Our results reinforce the synergistic value of integrated diagnostic methods for improving risk assessment of patients with glioblastoma that may pave the path towards a more personalized treatment planning.
BIOM-04. SENSITIVE DETECTION OF LEPTOMENINGEAL DISEASE USING CELL-FREE DNA FROM CEREBROSPINAL FLUID
Authors: White Michael , Klein Robert , Shaw Brian , Kim Albert , Subramanian Megha , Mora Joana , Giobbie-Hurder Anita , Nagabhushan Deepika , Jain Aarushi , Singh Mohini , Kuter Benjamin , Nayyar Naema , Stocking Jackson , Markson Samuel , Lastrapes Matthew , Alvarez-Breckenridge Christopher , Cahill Daniel , Mahar Maura , Rhoades Justin , Rotam Denisse , Bertalan Mia , Gydush Greg , Adalsteinsson Viktor , Kaplan Alexander , Oh Kevin , Sullivan Ryan , Gerstner Elizabeth , Carter Scott , Brastianos Priscilla
Published: November 2021

Leptomeningeal disease is a devastating complication of cancer that is frequently underdiagnosed due to the low sensitivity of cerebrospinal fluid cytology, the current gold-standard diagnostic method. We performed genomic sequencing on cerebrospinal fluid specimens obtained from patients with suspected or confirmed leptomeningeal disease to identify tumor-derived cell-free DNA. From the same fluid draw, cerebrospinal fluid cytology was assayed for comparison. 30 patients underwent cytology and cell-free DNA analysis. This study consisted of two patient populations: 22 patients with cytology-confirmed leptomeningeal disease without parenchymal tumors abutting their cerebrospinal fluid and 8 patients with parenchymal brain metastases with no evidence of leptomeningeal disease. The primary outcome was the diagnostic accuracy of cell-free DNA, defined as the number of correct diagnoses out of the total number of tests assayed. A total of 30 patients, 23 female and 7 male, with a median age of 51 participated in this study. Participants mostly presented with metastatic solid malignancies. In patients previously diagnosed with leptomeningeal disease via cytology with no parenchymal tumor abutting cerebrospinal fluid, cell-free DNA was accurate in diagnosis of leptomeningeal disease in 45 of 48 follow-up samples (94%; 95% CI, 83%-99%). Cytology was accurate in 36 of 48 follow-up samples (75%; 95% CI, 60%-86%). Cell-free DNA was significantly more accurate (P=.02) and sensitive (P=.02) than cytology in patients without parenchymal tumors abutting the cerebrospinal fluid. In three patients with parenchymal brain metastases abutting the cerebrospinal fluid and no suspicion for leptomeningeal disease, cytology was negative in all three patients; whereas, cell-free DNA was positive in all three. This study demonstrates the improved sensitivity and accuracy of cell-free DNA in diagnosing leptomeningeal disease with the exception of parenchymal tumors abutting cerebrospinal fluid. Overall, these results will lead to improved diagnosis of leptomeningeal disease and potentially earlier intervention and clinical trial enrollment.
BIOM-05. CASE SERIES OF MULTI-INSTITUTIONAL UTILITY OF CNSide TO MANAGE LEPTOMENINGEAL DISEASE IN PATIENTS WITH METASTATIC BREAST CANCER
Authors: Kumthekar Priya , Blouw Barbara , Mayer Julie , Fisher Deanna , Dugan Michael , Azadi Amir , Blondin Nicholas
Published: November 2021

INTRODUCTION

Leptomeningeal Disease (LMD) occurs in 5% of breast cancer patients. Diagnosing LMD remains challenging. Current standard of care has limited sensitivity and is inadequate for monitoring treatment response. Biocept’s CNSide™ is a proprietary assay utilizing a 10-antibody capture cocktail with microfluidic chamber that quantitatively detects tumor cells in the cerebrospinal fluid (CSF). We present a case series using CNSide to manage LMD of 4 unique breast cancer patients treated at three different institutions and demonstrate its impact on clinical management.

METHODS

Patients were treated at Smilow Cancer Hospital at Yale-New Haven (1 patient), Northwestern Medicine Lou and Jean Malnati Brain Tumor Institute (1 patient) and Barrow Neurological Institute (2 patients). All patients received intrathecal treatment (IT) via an Ommaya Reservoir. CSF tumor cells were detected via cytology and CNSide at diagnosis (3 patients) and throughout treatment (4 patients).

RESULTS

At diagnosis, CNSide detected tumor cells in 3/3 patients, vs 2/3 patients for cytology. The fourth patient was diagnosed with LMD before CNSide was available. CNSide detected CSF tumor cells in 9/9 (100%) of measurements, vs 4/9 (44%) for cytology for samples analyzed in parallel. Throughout treatment, CNSide was able to track the quantitative LMD response and showed a decrease in CSF tumor cells in all four patients, ranging from 99.7% (from 773 to 2 cells, 1 patient) to 100% (from 4447 to 0 cells; and from 33 to 0 cells, 2 patients).

CONCLUSION

Intrathecal treatment of LMD via Ommaya reservoir is not widely adopted across the US. Our experience suggests that using CNSide for quantitative CSF tumor cell detection may aid in diagnosing LMD, as well as in quantifying response to treatment particularly in the setting of intrathecal therapy. However, larger prospective clinical trials are needed to establish the role of CNSide in the diagnosis and management of LMD.
BIOM-06. MOLECULAR SIGNATURES UNDERLIE THE DISTINCT FAILURE PATTERNS OF MALIGNANT GLIOMAS
Authors: Choi Seung Won
Published: November 2021

BACKGROUND

Malignant gliomas exhibit varied failure patterns upon recurrence; however, genomic backgrounds of these distinct phenotypes have not been evaluated. We aimed to explore the genomic traits associated with distinct failure patterns in malignant glioma patients.

METHODS

This study involved 272 malignant glioma patients. Failure pattern was defined for the spatial relationship between recurrent tumor and the original tumor as follows: local recurrence (LR), remote recurrence, leptomeningeal seeding (LMC), and combined failure pattern. Molecular characteristics underlying different failure patterns were investigated for the mutation profile, copy number variation (CNV), and transcriptomic signatures.

RESULTS

Local recurrence was the most prominent failure pattern (62.9%), followed by combined recurrence (22.8%). Multivariate Cox regression analysis confirmed failure pattern as one of the independent prognostic factors. Patients with combined failure patterns exhibited the worst prognoses, whereas patients with remote recurrence exhibited the most favorable outcomes (median overall survival = 11.4 and 25.2 months, respectively). In IDH1-wild type glioblastoma (GBM) patients, TERT and PIK3CA mutation were significantly associated with the development of combined failure pattern and leptomeningeal seeding, respectively (p-value=0.015 & p-value=0.004, respectively). Transcriptomic analysis exhibited that inter-neuronal synaptic transmission was enriched in GBMs with combined failure pattern and this finding was further validated in proteomic analysis; neuronal myelination and synaptic transmission-related pathways were upregulated in GBMs which exhibited combined failure pattern.

CONCLUSIONS

Collectively, we demonstrated that the inherent molecular characteristics of the tumors might contribute to the eventual relapse patterns; tracking their evolutionary pathways may unravel novel therapeutic vulnerabilities of these tumors.
BIOM-08. DNA METHYLATION-BASED SUBGROUPING PREDICTS SURVIVAL BENEFIT FROM LOMUSTINE/TEMOZOLOMID COMBINATION THERAPY IN MGMT PROMOTOR-METHYLATED GLIOBLASTOMA
Authors: Weller Johannes , Waha Andreas , Schneider Matthias , Seidel Clemens , Steinbach Joachim , Hau Peter , Schlegel Uwe , Tonn Jörg-Christian , Grauer Oliver , Sabel Michael , Krex Dietmar , Schnell Oliver , Ringel Florian , Tabatabai Ghazaleh , Goldbrunner Roland , Radbruch Alexander , Schuss Patrick , Güresir Erdem , Vatter Hartmut , Glas Martin , Schmid Matthias , Schäfer Niklas , Tzaridis Theophilos , Giordano Frank , Zeyen Thomas , Schaub Christina , Németh Robert , Pietsch Torsten , Herrlinger Ulrich
Published: November 2021

BACKGROUND

The CeTeG/NOA-09 trial showed that lomustine/temozolomide chemotherapy prolongs survival for newly diagnosed MGMT-methylated glioblastoma patients. Previous reports on temozolomide monotherapy suggested, that the survival benefit of temozolomide in MGMT-methylated tumors may be restricted to the RTK II methylation subgroup and absent in RTK I and MES subgroups. To identify patients with a particularly strong benefit from CCNU/TMZ, we explored the association of methylation subgroups with outcome after lomustine/temozolomide therapy.

METHODS

All patients from the CeTeG/NOA-09 trial with sufficiently available tumor tissue (n = 98) underwent 850K methylation array analysis of their tumor and methylation subgroup annotation (Heidelberg brain tumor methylation classifier v11b4; calibrated score > 0.5 required). Overall survival (OS) was compared between a pooled cohort of tumors of the RTK I/MES subgroups and RTK II tumors.

RESULTS

In the CCNU/TMZ arm of CeTeG/NOA-09, OS was prolonged in RTK I/MES (n = 16; median not reached, 4-year OS 69%) as compared to RTK II patients (n = 14; median 20.6 months, 4-year OS 23%; p = 0.004 logrank test). In the standard temozolomide arm of CeTeG/NOA-09, OS tended to be shorter in RTK I/MES (n = 7; median 23.7 months, 4-year OS 17%) as compared to RTK II patients (n = 17; median 35.2 months; 4-year OS 38%, p = 0.15).

CONCLUSION

The CCNU/TMZ-dependent survival prolongation in patients with RTK I/MES tumors as opposed to RTK II seen in CeTeG/NOA-09 suggests that methylation-based subgrouping could be predictive for CCNU/TMZ efficacy in newly diagnosed MGMT-methylated glioblastoma.
BIOM-19. DECIPHERING THE METHYLATION SIGNATURE OF CIRCULATING EXTRACELLULAR VESICLE DNA FOR CNS TUMOR CLASSIFICATION
Authors: Ricklefs Franz , Ricklefs Tammo , Maire Cecile , Salviano da Silva Amanda , Wollman Kathrin , Sauvigny Thomas , Dührsen Lasse , Schüller Ulrich , Westphal Manfred , Lamszus Katrin
Published: November 2021

Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. We previously showed that extracellular vesicle (EV) DNA faithfully reflects the tumor methylation class, including information on the IDH mutation and MGMT promoter methylation status. Furthermore we showed that circulating plasma EVs are elevated in CNS tumor patients in comparison to non-tumor donors (HD) controls with tumor related protein profiles. We now investigated, whether the methylation signatures of circulating DNA (both EV and cfNDA) can be used in liquid biopsy approaches for CNS tumor detection and classification. We isolated DNA from circulating EVs (n=27), cfDNA (n=27) and tumor tissue DNA (n=90) of patients with glioblastoma (GBM), meningioma (MGN) and cerebral metastases (CM). Patients undergoing epilepsy surgery as well as aneurysm clipping were used as non-tumor controls (HD, n= 7). EVs were classified by nanoparticle analysis, immunoblotting, imaging flow cytometry and electron microscopy. Isolated EV-DNA comprised many sorts of molecular weight (up tp >10Kb) in comparison to cfDNA (130-140bp). Healthy donors and tumor patients showed not differences in their DNA size profiles. We performed genome-wide methylation profiling by 850k Illumina EPIC arrays for all DNA analytes and tumor entities. Linear models and empirical Bayes methods identified significant differentially methylated CpGs (GBM vs. HD, MGN, vs HD, CM vs. HD), that revealed tumor specific signatures to detect and discriminate different CNS tumor entities. Visualization of differentially methylated CPGs by dimension reduction (PCA, t-SNE, Umap) verified tumor specific clusters. cfDNA and EV-DNA exhibited distinctive individual CpG profiles. Our study shows that the methylation signature of circulating EV DNA and cfDNA can be used to separate healthy individuals from tumor patients and could potentially complement standard-of-care imaging to improve tumor detection, classification and surveillance.
BIOM-24. PROTEIN SURFACE SIGNATURE ON SERUM EXTRACELLULAR VESICLES FOR NON-INVASIVE DETECTION OF TUMOR PROGRESSION IN GLIOBLASTOMA PATIENTS
Authors: Tzaridis Theophilos , Weller Johannes , Bachurski Daniel , Shakheri Farhad , Schaub Christina , Hau Peter , Buness Andreas , Schlegel Uwe , Steinbach Joachim , Seidel Clemens , Goldbrunner Roland , Schäfer Niklas , Wechsler-Reya Robert , Scheffler Björn , Glas Martin , Haeberle Lothar , Herrlinger Ulrich , Coch Christoph , Reiners Katrin , Hartmann Gunther
Published: November 2021

INTRODUCTION

Detection of tumor progression in glioblastoma patients remains a major challenge for clinicians due to equivocal MRI results. Extracellular vesicles (EVs) are potential biomarkers and can be detected in the blood of tumor patients. In this study, we evaluated the potential of serum-derived EVs from glioblastoma patients to serve as a marker for tumor progression in adjunction with MRI assessment.

METHODS

Glioblastoma patients from two independent cohorts, one from the multicenter Phase III CeTeG/NOA-09 trial (n=36) and the other from patients treated at the University of Bonn (n=31), were included in this study. EVs from serum of glioblastoma patients and healthy volunteers were separated by size exclusion chromatography and ultracentrifugation. EV markers were defined by using a proximity-extension assay and bead-based flow cytometry. Tumor progression was defined according to modified RANO criteria.

RESULTS

EVs from the serum of glioblastoma patients (n=67) showed an upregulation of CD29 (p=0.08), CD44 (p< 0.0001), CD81 (p< 0.0001), CD146 (p< 0.0001), C1QA (p=0.003), and histone H3 (p< 0.0001) as compared to serum EVs from healthy volunteers. For both independent cohorts of glioblastoma patients, we noted upregulation of C1QA, CD44, and histone H3 upon tumor progression, but not in patients with stable disease. Notably, six patients with worse survival compared to the median survival of the cohort did not fulfill RANO criteria at the time of suspected progression, yet showed an elevation of at least one out of these three markers. In a multivariable logistic regression analysis, a combination of CD29, CD44, CD81, C1QA, and histone H3 correlated with RANO-defined tumor progression with an AUC of 0.76.

CONCLUSION

Measurement of CD29, CD44, CD81, C1QA, and histone H3 in serum-derived EVs of glioblastoma patients, along with standard MRI assessment, could improve detection of true tumor progression and thus be a useful tool for clinical decision making.
BIOM-33. EV-ASSOCIATED IL13Ra2 AS A LIQUID BIOPSY BIOMARKER FOR GBM
Authors: Khristov Vladimir , Nesterova Darya , Zacharia Brad , Connor James , Madhankumar Achuthamangalam
Published: November 2021

Glioblastoma (GBM) is both the most common and deadly malignant primary brain tumor with a 1-year survival of 37.2% and a 5-year survival of just 5.1%. Despite advances in imaging technology, distinguishing between pseudoprogression and pseudoresponse remains problematic and there is a dire need for additional diagnostic methods. Tumor-derived extracellular vesicles are found in a variety of biofluids and are good candidates for a liquid biopsy. A unique characteristic of GBM is the overexpression of the α2 variant of IL-13 receptor (IL13Rα2) in 78-96% of patients while virtually undetectable in normal brain tissue. We hypothesize that biofluids of patients with GBM contains sufficient IL13Rα2-positive extracellular vesicles (EV) to inform the clinical picture regarding disease status such as tumor recurrence and response to therapy. We have measured the levels of IL13Rα2 in the EV fraction of plasma from GBM patients and correlated these levels to patient survival, and patient clinical characteristics (n=35). We have demonstrated that the EV fraction of some patients with GBM is enriched for the IL13Rα2 receptor. The plasma level of IL13Rα2 could also predict the level of IL13Rα2 in the GBM tumor (n=9). Further, IL13Rα2 positive EVs from patient-derived GBM cell lines could be immunoprecipitated for further interrogation of genomic and proteomic molecular markers. These exciting findings lay the groundwork for a GBM liquid biopsy based on tumor-specific EVs in patient biofluids.
BIOM-34. GENOMIC PROFILING IDENTIFIED COPY NUMBER VARIATIONS OF CDK4/6 AS A NOVEL PROGNOSTIC BIOMARKER FOR THALAMIC GLIOMA
Authors: Lai Mingyao , Zhou zhaoming , Li Hainan , Zhou Jiangfen , Li Juan , Zhen junjie , Zhou Cheng , Cai Linbo
Published: November 2021

BACKGROUND

Thalamic glioma is a rare tumor, which is poorly understood in adults. The genetic variation of this tumor is still unknown. In this study, we investigated the mutation landscape of thalamic glioma and compared the clinical outcomes between different mutation situations in thalamic glioma.

METHODS

Next-generation sequencing targeting 425 cancer-relevant genes was performed with 34 thalamic glioma tissue samples. Gene mutations and copy number variations were investigated for prognostic effect with overall survival data.

RESULTS

Several diagnostic and prognostic biomarkers appeared in our thalamic glioma cohort, including TP53 (56%), EGFR (41%), TERT (35%), M CL1 (26%), PDGFRA (26%), PTEN (26%), CDK4/6 (24%), POLE (24%), PIK3CA (24%), NF1 (21%), ATR (21%), ATRX (18%), BRAF (15%), and ROS1 (12%). Among all genetic aberrations with a more than 10% occurrence rate, two mutations (TERT and PTEN) were associated with poor overall survival and one copy number variation (CDK4/6) was associated with favorable overall survival (univariate P < 0.1). Among these genes, CDK4/6 copy number variations (hazard ratio [HR], 0.16; 95% confidence interval [CI], 0.035–0.704; P = 0.016) remained significant survival associated in multivariate analyses. Copy number variations of CDK4/6 was seldom reported as a prognostic biomarker for glioma, especially for thalamic glioma in public databases. Besides, several gene mutations (BRIP1, MRE11A, MAP2K1, ROS1, MUTYH, JARID2, CTCF, and EGFR) were found positively associated with CDK4/6 copy number variations. Gene enrichment analysis demonstrated that those genes were related to astrocyte differentiation.

CONCLUSIONS

In our study, CDK4/6 copy number variation was determined as a favorable overall survival biomarker for thalamic glioma, and CDK4/6 copy number variation associated mutant genes were related to astrocyte differentiation, which could be the potential therapeutic targets for thalamic glioma.
BIOM-44. PRE-SURGICAL ADVANCED MRI IS USEFUL FOR FORECASTING DRUG DISTRIBUTION IN BRAIN TUMORS
Authors: Jackson Pamela , Kim Minjee , Hawkins-Daarud Andrea , Singleton Kyle , Mohammad Afroz , Burns Terence , Parney Ian , Hu Leland , Kaufmann Timothy , Elmquist William , Sarkaria Jann , Swanson Kristin
Published: November 2021

Choosing effective chemotherapies for intravenous delivery to brain tumors is challenging, especially given the protective nature of the blood brain barrier (BBB). Connecting drug distribution to non-invasive, pre-surgical magnetic resonance imaging (MRI) could allow for predictive insight into drug distribution. In a previous study, we found that T2Gd images were predictive of a low BBB penetrant drug (Cefazolin), and FLAIR images were predictive of a high BBB penetrant drug (Levetiracetam). While these results are promising, we further seek to explore how advanced MRI sequences might inform image-based models of drug distribution. Prior to surgery, we acquired advanced dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) MRI sequences for eight brain tumor patients (7 gliomas and 1 metastatic adenocarcinoma) in addition to the anatomic MRIs. All resulting quantitative maps and acquired images were co-registered. Prior to incision, patients received injections of cefazolin and levetiracetam. Next, multiple blood samples and biopsies were collected during surgery. Biopsies and plasma samples were analyzed for drug concentration using liquid chromatography mass spectrometry (LCMS), and biopsy drug levels were reported as Brain-Plasma Ratio (BPR). Mean image intensity was extracted from a 15x15 voxel window surrounding the biopsy location. We performed linear regression analyses to determine which combination of images were predictive of BPR. We found that considering quantitative imaging improved our initial ability to predict BPR for both drugs. For cefazolin, the third diffusion tensor eigenvalue (L3) map was significantly correlated with BPR (p< 0.001, R2= 0.36). For levetiracetam, the best model consisted of a combination of images and maps with the L3 map and the isotropic diffusion map (P) being the most influential (p= 0.001, R2= 0.63). Advanced MRI-based modeling is a promising tool for forecasting drug distribution in brain tumors and could be of great importance for understanding efficacy and selecting therapeutic strategies.
STEM-01. PHAGE DISPLAY BIOPANNING IDENTIFIES AMOT REGULATING CELL MOTILITY OF BRAIN METASTATIC STEM-LIKE CELLS
Authors: She Chunhua , Kim JongMyung , Potez Marine , Liu James
Published: November 2021

OBJECTIVE

Metastatic brain tumors (MBTs) are the most common type of malignant brain tumors. Due to the deviation of MBTs from the parental tumors, the effective therapies for primary tumors often are not useful in brain metastases. Even more new intracranial lesions were developed though the primary lesion was under control. The model of brain metastatic stem-like cells (BMSCs) could partially explain the progression of intracranial lesions. Here we aimed to explore the biological behavior in cell motility of BMSCs and understand the potential mechanisms.

METHODS

In vitro and in vivo phage display biopanning strategies were used to isolate dodecapeptides that specifically target BMICs by selecting against primary lung cancer cells and normal brain cells. In silico analysis was used to derive specific protein targets in BMICs. Potential targets were narrowed down through analysis in patient databases and verified for their presence in BMIC through RT-PCR. Cell migration and adhesion in BMICs were analyzed using Transwell, scratch, and adhesion assays. Protein expression and cell morphology were detected by immunofluorescence. Immune blot was performed to detect the epithelial-mesenchymal related molecules and explore protein-protein interactions.

RESULTS

In silico analysis of BMSCs specific peptides revealed Angiomotin (Amot) as a potential target in BMSCs. Amot was found to be overexpressed in BMSCs compared to primary lung cancer cells. Kaplan-Meier analysis demonstrated Amot was negatively correlated with overall survival among lung adenocarcinoma patients. Functionally, knockdown of AMOT in BMSCs decreased the capability of cell migration and adhesion by reduced active Cdc42/Rac1 signals caused by downregulation of E-Cadherin. Amot was found to maintain the E-Cadherin in BMSCs through reducing ubiquitination of E-Cadherin.

CONCLUSIONS

Amot plays a role in promoting migration and adhesion in BMSCs through preservation of E-cadherin.
STEM-07. PRC2 MAINTAINS A CANCER STEM CELL PHENOTYPE IN GLIOBLASTOMA MULTIFORME VIA CHROMATIN MODIFICATION OF H3K27me3
Authors: Korleski Jack , Shudir Sweta , Caputo Christopher , Lal Bachchu , Rui Yuan , Green Jordan , Lopez-Bertoni Hernando , Laterra John
Published: November 2021

Multi-potent stem-like cells (i.e. cancer stem cells, CSCs) are critical determinants of tumor propagation, therapeutic resistance, and recurrence in glioblastoma (GBM). Modifications in chromatin architecture play a fundamental role in the tumor cell phenotype of GBM. The polycomb repressor complex 2 (PRC2) is a key histone modifier that supports multi-potency and oncogenesis via H3K27 trimethylation (H3K27me3). Understanding how these epigenetic modifications cooperatively drive cancer cell stemness should unveil new targets for therapeutic development in GBM. Using a combination of next-generation sequencing, bioinformatics, and molecular approaches we identified EZH2, the catalytic domain of the PRC2 complex, as a critical mediator of reprograming events in GBM cells. We found that EZH2 is highly induced in response to transgenic Oct4/Sox2 with global increases in H3K27me3. Pharmacological inhibition of EZH2 diminishes self-renewal capacity of GBM neurospheres concurrent with a reduction in gene expression levels of markers and drivers of stemness. Furthermore, we identified and validated a set of 6 putative tumor suppressor genes repressed by Oct4 and Sox2 in a PRC2-dependent manner. We identified miR-217 as an EZH2 regulator in GBM cells and miR-217 reconstitution using advanced nanoparticle formulations re-activates the PRC2-repressed tumor suppressors, inhibited tumor growth and enhanced the effects of ionizing radiation in an orthotopic model of GBM. Taken together, these data show that PRC2-mediated chromatin changes in H3K27me3 help regulate the stem-cell phenotype induced by Oct4 and Sox2 in GBM cells and predict that targeting EZH2 could have therapeutic benefit in GBM.
STEM-11. HYDROGEN SULFIDE FUNCTIONS AS A TUMOR SUPPRESSION IN GLIOBLASTOMA
Authors: Silver Daniel J , Roversi Gustavo , Bithi Nazmin , Wang Sabrina , Troike Katie M , Neumann Chase K , Ahuja Grace , Reizes Ofer , Brown J Mark , Hine Christopher , Lathia Justin
Published: November 2021

Glioblastoma (GBM) cancer stem cells (CSCs) respond to a variety of stimuli within their immediate surroundings. However, little is known about the lifestyle factors that alter CSC enrichment and function within the tumor microenvironment (TME). To examine the consequences of diet-induced obesity on the progression of GBM, we interrogated tumor growth using patient-derived and syngeneic GBM models implanted into the brains of mice fed either an obesogenic high-fat diet (HFD) or a low-fat, control diet. HFD consumption resulted in an accelerated disease trajectory, presenting significantly shortened overall survival. HFD reshaped the TME altering the lipid landscape, enhancing the CSC phenotype, stimulating tumor cell proliferation, and protecting from necrotic cell death. Similar results were not observed in metabolically obese, leptin-deficient (ob/ob) mice. We simultaneously identified a potent suppression of the gasotransmitter, hydrogen sulfide (H2S). H2S functions principally through protein S-sulfhydration and regulates multiple programs including bioenergetics, metabolism, and immune response. Inhibition of H2S increased tumor cell proliferation and chemotherapy resistance, whereas treatment with H2S donors reduced tumor cell fitness in vitro and attenuated GBM growth in vivo. Exogenous treatment with H2S donors also rescued the lipid-mediated increase in tumor cell proliferation. As H2S exerts its action though protein S-sulfhydration, we confirmed that HFD-fed mice, which experienced decreased H2S synthesis, presented a severely depleted S-sulfhydrated protein landscape. Loss of this post-translational modification was confirmed in GBM patient tissues compared to age- and sex-matched controls. Taken together, our findings provide evidence that H2S functions as a tumor suppressor in GBM. Our observations highlight a new mechanism for tumor growth dynamics that can be leveraged for new therapeutic strategies focused on boosting H2S. Finally, our findings indicate that lifestyle factors can have pleiotropic effects on GBM progression through concomitant mechanisms involving tumor metabolism, modifications to the TME, and regulation over the CSC phenotype.
STEM-19. NON-REDUNDANT, ISOFORM-SPECIFIC ROLES OF HDAC1 IN THE REGULATION OF THE GLIOMA STEM CELL PHENOTYPE
Authors: Cascio Costanza Lo , McNamara James , Melendez Ernesto Luna , Lewis Erika , Dufault Matthew , Sanai Nader , Plaisier Christopher , Mehta Shwetal
Published: November 2021

Glioblastoma (GBM) is characterized by an aberrant yet druggable epigenetic landscape. One major family of epigenetic regulators, the Histone Deacetylases (HDACs), are considered promising therapeutic targets for GBM due to their repressive influences on transcription. Although HDACs share redundant functions and common substrates, the unique isoform-specific roles of different HDACs in GBM remain unclear. There is a temporal and cell-type specific requirement of HDAC1 and 2 during normal brain development, with HDAC2 being indispensable in neural stem cells. Here, we specifically investigated the functional importance of HDAC1 in glioma stem cells, an HDAC isoform whose expression increases with brain tumor grade and is correlated with decreased survival. Using cell-based and biochemical assays, transcriptomic analyses and patient-derived xenograft models, we report that knockdown of HDAC1 alone has profound effects on the glioma stem cell (GSC) phenotype and survival in a p53-dependent manner. HDAC1 is the essential class I deacetylase in glioma stem cells, and its loss is not compensated for by its paralogue HDAC2 or other HDACs. Loss of HDAC1 expression significantly suppresses viability of GSCs harboring functional p53, and that HDAC2 expression is completely dispensable in GSCs. In addition, HDAC1 silencing but not HDAC2, stabilizes and acetylates p53 in GSCs, resulting in upregulation of key p53 target genes and induction of programmed cell death. Furthermore, ablation of HDAC1 function alone results in histone hyperacetylation and a collapse of the stemness state in GSCs. We demonstrate significant suppression in tumor growth upon targeting of HDAC1 and identify compensatory pathways that provide insights into combination therapies for GBM. Our study highlights the importance of HDAC1 in GBM and the need to develop isoform-specific HDAC inhibitor drugs.
STEM-24. INTERFERON SIGNALING REGULATES THE PROLIFERATION AND MESENCHYMAL PHENOTYPE OF GLIOBLASTOMA STEM CELLS
Authors: Khan Sabbir , Mahalingam Rajasekaran , Sen Shayak , Gandy Kaitlin , Alfaro-Munoz Kristin , Majd Nazanin , Balasubramaniyan Veerakumar , DeGroot John
Published: November 2021

Interferon (IFN) signaling contributes to stemness, cell proliferation, cell death, and cytokine signaling in cancer and immune cells; however, the role of IFN signaling in glioblastoma (GBM) and GBM stem-like cells (GSCs) is unclear. This study aimed to investigate the cancer cell-intrinsic IFN signaling in tumorigenesis and malignant phenotype of GBM. We characterized cell-intrinsic IFN signaling in The Cancer Genome Atlas, patient-derived cohorts of GSCs, and published single-cell RNA sequencing datasets by in-silico analyses. The in-silico findings were further validated by evaluating the cytokine secretion and using pharmacological activators and blockers of IFN/transducer and activator of transcription 1 (STAT1) signaling. We found that GSCs and GBM tumors exhibited differential cell-intrinsic IFN signaling, and high IFN/STAT1 signaling is associated with mesenchymal phenotype and poor survival outcomes. Ruxolitinib, a pharmacological inhibitor of IFN/STAT1, abolished the IFN/STAT1 signaling in GSCs with intrinsically high IFN signaling. IFN-γ treatment for 1 week promotes the mesenchymal phenotype in GSCs with low IFN signature. In addition, chronic inhibition of IFN/STAT1 signaling with ruxolitinib decreased cell proliferation and mesenchymal signatures (CD44, YKL40, and TIMP1) in GSCs with intrinsically active IFN/STAT1 signaling. Publicly available human glioma single-cell RNA-seq (scRNA-seq) datasets analyses showed that both tumor and nontumor cells expressed IFN signaling genes, and the mesenchymal signature was highly expressed in the same cluster where IFN signaling genes were upregulated. We demonstrated that cell-intrinsic IFN signaling in GSCs and GBM tumors is associated with mesenchymal signatures and cell proliferation. Our study provides evidence for the possibility of targeting IFN signaling in a specific group of GBM patients.
CBIO-02. IN VITRO TUMOR TREATING FIELDS (TTFields) REDUCE PROLIFERATION AND ALTER MLH1 EXPRESSION IN TEMOZOLOMIDE RESISTANT (TMZR) PATIENT-DERIVED GLIOBLASTOMA (GBM) CELLS
Authors: Michelhaugh Sharon , Degen Katherine , Walker Blake , Mittal Sandeep
Published: November 2021

BACKGROUND

TTFields therapy is approved as a monotherapy for the treatment of recurrent GBM, tumors that often have acquired resistance to TMZ. To examine TTFields efficacy in the context of TMZR in vitro, TTFields were applied to TMZR GBM cells to assess proliferation, clonogenicity, and changes in expression of O6-methylguanine-DNA methyltransferase (MGMT) that confers TMZ resistance and DNA Mismatch Repair Protein Mlh1 (MLH1) that may confer TMZ sensitivity.

METHODS

15-037 GBM cells were isolated from a newly-diagnosed patient tumor (IDH-WT, unmethylated MGMT promoter. Cells were grown in DMEM/F12 media with 10% FBS and gentamicin. U-251 MG cells and 15-037 cells were incubated with 100 µM TMZ for three 5-day cycles to establish TMZR. TMZR cells were plated on coverslips (1×104cells/coverslip) and incubated overnight prior to TTFields application. TTFields were applied at 200 kHz (field intensity of ~1.6 V/cm) for 14 days. Then coverslips (n=3/group) were harvested, counted, and replated for clonogenic assay at 1000 cells/35mm2 well. Clonogenic assay plates were stained with crystal violet, imaged, and colonies counted. Additional coverslips (n=3/group) were harvested and MGMT and MLH1 expression were assessed by qRT-PCR with the delta-delta Ct method using GAPDH as housekeeping control. Cell counts and colony counts were compared with two-tailed t-test with significance at p< 0.05.

RESULTS

In U-251 MG and 15-037 TMZR cells, TTFields application significantly reduced both cell counts and clonogenic assay colony counts compared to untreated controls (U-251 MG: 45.4% and 26.5%; 15-037: 96.2% and 60.9%, respectively, p< 0.05). In both TMZR cell lines, MGMT expression was unchanged by TTFields, but MLH1 expression increased 2.2 fold in U-251 MG cells and 7.1 fold in 15-037 cells after TTFields application.

CONCLUSIONS

Not only are TMZR GBM cells sensitive to TTFields in vitro, but TTFields increased expression of MLH1 which may be able to reduce resistance to TMZ.
CBIO-07. ST-401: A BRAIN-PENETRANT MICROTUBULE-TARGETING AGENT THAT KILLS CANCER CELLS IN INTERPHASE
Authors: Ruiz Juan Jesus Vicente , Wordeman Linda G , Stella Nephi
Published: November 2021

Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of cancers, and evidence suggests that glioblastoma is sensitive to disruptions in microtubule (MT) functions; but most MTAs do not cross the blood brain barrier. To address this limitation, we developed a brain-penetrant MTA, ST-401, that inhibited the growth of human glioblastoma in culture at nanomolar concentrations (Horne et al. (2020) Neuro-Onc Adv. 3(1): vdaa165). Of note, ST-401 binds to the colchicine site, inhibits tubulin assembly and reversibly reduces MT dynamics in cells with a milder activity compared to the reference MTA, nocodazole. Pharmacokinetic analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 demonstrated significant antitumor and radiosensitizing activities. Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity 1) did not correlate with any of the compounds tested thus far and 2) only weakly correlated with MTAs that act through alternate mechanisms [Taxol and Vinblastine]. Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function that is different from MTAs tested thus far on the NCI 60 cancer cell panel. As initial step to discover this novel mechanism of action, we studied the anti-tumor activity of ST-401 and nocodazole in HCT116 cells in culture. As expected, low concentrations (30 and 100 nM) of nocodazole killed HCT116 cells in mitosis and increased p-P53; by contrast these low concentrations of ST-401 killed HCT116 cells in interphase and did not increase p-P53. Our study shows that the brain-penetrant MTA, ST-401, kills cancers in interphase, a response that contrasts with the activity of the reference MTA nocodazole. ST-401 represents a promising chemical scaffold to develop brain-penetrant therapeutics for the treatment of patients diagnosed with glioblastoma.
CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS
Authors: Kitange Gaspar , Vaubel Rachael , Sarkaria Jann
Published: November 2021

Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.
CBIO-14. SINGLE CELL RNA SEQUENCING ANALYSIS OF HUMAN GLIOBLASTOMA STEM-LIKE CELL CULTURES AND XENOGRAFT TUMORS
Authors: Arora Sonali , Mihalas Anca , Bassett John , Patel Anoop , Paddison Patrick
Published: November 2021

Single cell RNA-seq (scRNA-seq) studies for glioma have yielded critical insight into intratumoral heterogeneity and developmental gene expression patterns for primary gliomas. One key conclusion from these studies is that each tumor represents a complex, yet maligned, neuro-developmental ecosystem, harboring diverse cell types, which presumably contribute to tumor growth and homeostasis in specific ways (e.g., vascular mimicry, immune evasion, recreating NSC niches, neural injury responses, etc.). Here, to better understand experimental models of human glioblastoma (GB), we performed single cell RNA-seq analysis of human GB stem-like cells (GSCs) of distinct tumor subtypes (mesenchymal and proneural) during their in vitro culture in serum-free conditions and also during tumor formation in immunocompromised mice. This analysis revealed surprising differences between in vitro and in vivo grown GSCs. Among our results, we find that in vivo mesenchymal GSCs are capable of transitioning to proneural-like states, while proneural GSCs are capable of transitioning to mesenchymal states. We characterize cycling cells based on expression of and G2/M and S phase makers, estimate RNA velocity, and examine different developmental trajectories arising in vitro and in vivo. We also compare and discuss different analysis pipelines for scRNA-seq data.
CBIO-15. LOSS OF WILLIAMS SYNDROME TRANSCRIPTION FACTOR (WSTF) LEADS TO IMPROVED TEMOZOLOMIDE SENSITIVITY IN HUMAN GLIOBLASTOMA CELLS IRRESPECTIVE OF MGMT EXPRESSION
Authors: Sarnow Katharina , Schwab Stephanie , Rio Oline , Mukherjee Joydeep , Bjerkvig Rolf , Johannessen Tor-Christian
Published: November 2021

BACKGROUND

The prognosis for glioblastoma multiforme (GBM) patients is poor with a median survival of approximately 15 months. The DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) counteracts the effects of temozolomide (TMZ) chemotherapy and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its functions in the DDR in GBMs, is so far unknown. In this study, we investigated whether a loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment.

METHODS

We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA.

RESULTS

WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p< 0.0001, U-251: Clone 7/35.1/70.2 vs control: p< 0.0001/p= 0.0107/p= 0.0119).

CONCLUSION

WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.
CBIO-16. DOSE INTENSIFIED TEMOZOLOMIDE IMPROVES INFILTRATION OF CD70 CAR T CELLS INTO THE GBM TUMOR MICROENVIRONMENT
Authors: Karachi Aida , Huang Jianping , Tao Haipeng , Dastmalchi Farhad , Jin Linchun , Mitchell Duane , Rahman Maryam
Published: November 2021

CD70 CAR T cells developed by our group have anti-tumor efficacy in syngeneic murine GBM model and are currently being developed for first-in-human testing. The objective of this study was to evaluate the impact of temozolomide (TMZ) in various dosing strategies on the expansion, persistence and function of the CD70 CAR T cells after infusion. C57BL/6 mice underwent intracranial implantation of KR-158 overexpressing CD70 tumor cells. Tumor bearing animals were treated with standard dose (SD) (50 mg/kg x 5 days), metronomic dose (MD) (25mg/kg x 10 days), or dose intensified (DI) (75mg/kg x 5 days) of TMZ, followed by 10×106 CAR T cells infusion. Peripheral blood was collected to monitor the persistence of CAR T cells in the systemic blood circulation. Six weeks post treatments, spleens and tumors were collected and CAR T cells abundance and function were measured. TMZ preconditioning resulted in the expected lymphopenia in animals by generation of dose dependent lymphopenia. Circulating CD70 CAR T cells peaked in the systemic blood 2 weeks after infusion (3-fold, p< 0.0001). Markers of T cell exhaustion including PD-1 and TOX expression on CAR T cells were not different between the three TMZ treatment groups compared to control. Six weeks post treatment, CD70 CAR T cells were found to be highly infiltrated within the tumor microenvironment in the DI TMZ group compared to the other groups (4-fold,p < 0.0001). Tumor infiltrating CD70 CAR T cells in the DI TMZ group did not have an increase in PD-1 and TOX expression which was seen in the MD and SD TMZ groups. DI TMZ preconditioning results in greater CD70 CAR T cell trafficking to the tumor without T cell exhaustion compared to lower doses of TMZ. The affects of TMZ on the immune microenvironment to enhance CAR T cell regimens warrants further study.