bluuebunny/crossref_metadata_2025 huggingface dataset:
EPCO-10. INTRATUMORAL HETEROGENEITY OF ENVIRONMENT-INDUCED EXPRESSION PROGRAMS IN GLIOMA PATIENTS AND DERIVED MODEL SYSTEMS
Authors: Bayley Nicholas , Tse Christopher , Zhu Henan , Ta Lisa , Baufeld Lynn , Gosa Laura , Yan Weihong , Prins Robert , Yong William , Cloughesy Timothy , Liau Linda , Graeber Thomas , Nathanson David
Published: November 2021

Bulk tumor and single-cell RNA sequencing have revealed the remarkable molecular heterogeneity and plasticity of gliomas. This has led to the creation of tumor subtypes and cellular states describing inter- and intra-tumoral heterogeneity, respectively. While there has been great interest in creating and revising new classifications, the biological reasons for this degree of heterogeneity and the selective pressures driving it remain to be fully described. The brain tumor microenvironment (TME) is a complex, regionally heterogeneous ecosystem of communicating normal and malignant cell types and scavenge-able nutrients and metabolites. We hypothesized that distinct cellular interactions and metabolic flux in the TME may drive the inter- and intra-tumoral heterogeneity of gliomas. To identify tumorigenic programs impacted by environmental context we performed bulk RNA sequencing of over 35 triplets of patient glioma samples and their matched derivative models established in direct orthotopic mouse xenografts (DPDOX) and conventional gliomasphere cultures (GS). This analysis revealed environment-specific programs including in vivo immune and neuroglial signaling, in vitro lipid metabolism, and cell migration altered in model systems. These environmental programs are enriched in specific tumor subtypes and neuroglial signaling programs lost in vitro are dynamically upregulated upon re-transplantation in vivo. To further investigate associations between tumor cellular state and environment-driven programs we performed single-cell RNA sequencing of 3 patient and model system triplets. By annotating with brain cell atlases from previous single-cell characterizations, 6 major clusters and over 20 sub-clusters of tumor cell states and hybrid states were identified. Overlaying results from bulk sequencing revealed cell state-specific expression of environment-induced programs. Further, these cellular state “niches” diverge in model environments suggesting that environmental factors modulate not only the composition of cellular states but also their biological roles within gliomas.
EPCO-13. MULTIOMIC SINGLE NUCLEUS RNA- AND ATACseq PROFILING REVEALS REGULATORS OF GLIOMA CELL STATE DIVERSITY
Authors: Anderson Kevin , Johnson Kevin , Varn Frederick , Bessonett Shannon , Gujar Amit , Flynn Bill , Courtois Elise , Hau Ann-Christin , Golebiewska Anna , Paek Sun Ha , Niclou Simone , Samuels Michael , Robson Paul , Verhaak Roel
Published: November 2021

Multiomic single nucleus RNA- and ATACseq profiling reveals regulators of glioma cell state diversity. The extensive intra- and intertumoral heterogeneity observed in glioma reflects the resistance to therapy and poor prognosis observed clinically. Single-cell sequencing studies have highlighted that glioma heterogeneity reflects the co-existence of cell subpopulations with distinct cell states. Prior studies have also shown that EGFR-amplifying extrachromosomal DNA (ecDNA) elements in IDH-wild-type gliomas can contribute to heterogeneity by driving oncogene amplification through long range chromatin contacts. However, single cell studies have largely focused on analyses of transcriptional profiles, and the epigenetic mechanisms underlying the contribution of ecDNA elements to tumor cell state diversity remain poorly understood. To further our understanding of the regulatory programs that contribute to transcriptional diversity and mediate the distribution of tumor cell states, we profiled primary-recurrent tumor pairs from 18 patient samples with multiomic single-nucleus RNA- and ATACseq, resulting in 86,135 cells identified with linked chromatin accessibility and gene expression profiles. Integrative clustering of the tumor cells identified tumor cell states ranging from a stem-like to differentiated- phenotype that were also associated with differences in chromatin accessibility and inferred transcription factor binding activity. Analyses of chromatin accessibility resulted in the identification of ecDNA, and integrative clustering of ecDNA+ cells highlighted distinct cell states with increased copy number burden, oncogene amplification, and differential chromatin accessibility. These results suggest that a better understanding of extrachromosomal contributions to tumor diversity would aid in development of more efficient therapies.
EPCO-16. ONCOHISTONE INTERACTOME PROFILING UNCOVERS MECHANISMS OF CHROMATIN DISRUPTION AND IDENTIFIES POTENTIAL THERAPEUTIC TARGETS IN PEDIATRIC HIGH-GRADE GLIOMA
Authors: Siddaway Robert , Canty Laura , Pajovic Sanja , Coyaud Etienne , Milos Scott , Sbergio Stefanie-Grace , Lubanszky Evan , Yun Haley , Carette Sheyenne , Portante Alessia , Raught Brian , Campos Eric , Hawkins Cynthia
Published: November 2021

Mutations in histone H3 at amino acids 27 (H3K27M) and 34 (H3G34R) occur with high-frequency in pediatric high-grade glioma. H3K27M mutations have been shown to lead to global disruption of H3K27me3 through dominant negative PRC2 inhibition with accompanying gains in H3K36me3, while H3G34R mutations lead to local losses of H3K36me3 through inhibition of SETD2. However, the mechanism of action of these mutants on the broader landscape of chromatin-associated proteins remains unknown. Importantly, proteins with differential associations with oncohistones could be targeted therapeutically. Here we profiled the interactomes of the H3.1K27M, H3.3K27M and H3.3G34R oncohistones using BioID to gain an unbiased measure of their interaction landscapes. Among the differential interactors all 3 mutants lost interaction with H3K9 methyltransferases, while H3G34R also had reduced interaction with DNA methyltransferases accompanied by genome-wide DNA hypomethylation. In contrast, H3K27M mutants had increased association with transcription factors, consistent with the activation of transcription induced by the global loss of H3K27me3. H3K9me3 was reduced in H3K27M-containing nucleosomes, and cis-H3K9 methylation was required for H3K27M to exert its effect on global H3K27me3. Depletion of H3K9 methyltransferases with shRNA or treatment with H3K9 methyltransferase inhibitors was lethal to H3.1K27M, H3.3K27M and H3.3G34R mutant pHGG cell lines, underscoring the importance of H3K9 methylation for oncohistone-mutant gliomas and suggesting it could make an attractive therapeutic target.
EPCO-20. PEDIATRIC HIGH-GRADE GLIOMA EXHIBITS DISTINCT 3D GENOME STRUCTURE THAT IMPACTS TRANSCRIPTION REGULATION
Authors: Huang Tina , Wang Juan , Hu Ye , Piunti Andrea , Bartom Elizabeth , Shilatifard Ali , Yue Feng , Saratsis Amanda
Published: November 2021

INTRODUCTION

Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are highly morbid brain tumors. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding Histone H3. To investigate whether the H3K27M mutant protein is associated with distinct chromatin structure affecting transcription regulation, we generated the first high-resolution Hi-C and ATAC-Seq maps of pHGG cell lines, and integrated these with tissue and cell genomic data.

METHODS

We generated sequencing data from patient-derived cell lines (DIPG n=6, GBM n=3, normal n=2) and frozen tissue specimens (DIPG n=1, normal brainstem n=1). Analyses included cell line RNA-Seq, ChIP-Seq (H3K27ac, H3K27me3, H3K27M) and genome-wide chromatin conformation capture (Hi-C), as well as tissue ATAC-Seq. Publicly available pediatric glioma tissue ChIP-Seq data was integrated with cell data. CRISPR knock-down of target enhancer regions was performed.

RESULTS

We identified tumor-specific enhancers and regulatory networks for known oncogenes in DIPG and GBM. In DIPG, FOX, SOX, STAT and SMAD families were among top H3K27Ac enriched motifs. Significant differences in Topologically Associating Domains (TADs) and DNA looping were observed at OLIG2 and MYCN in H3K27M mutant DIPG, relative to wild-type GBM and normal cells. Pharmacologic treatment targeting H3K27Ac (BET and Bromodomain inhibition) altered these 3D structures. Functional analysis of differentially enriched enhancers in DIPG implicated SOX2, SUZ12, and TRIM24 as top activated upstream regulators. Distinct genomic structural variations leading to enhancer hijacking and gene co-amplification were identified at A2M, JAG2, and FLRT1.

CONCLUSION

We show genome structural variations enhancer-promoter interactions that impact gene expression in pHGG in the presence and absence of the H3K27M mutation. Our results imply that tridimensional genome alterations may play a critical role in the pHGG epigenetic landscape and thereby contribute to pediatric gliomagenesis. Further studies examining the impact of the alterations is therefore underway.
EPCO-22. INHERITED POLYMORPHISM IN CHROMOSOME 8Q24 COOPERATES WITH MUTANT IDH1, Trp53 AND ATRX LOSS TO INDUCE LOW-GRADE GLIOMA
Authors: Yanchus Connor , Drucker Kristen , Kollmeyer Thomas , Abyzov Alexej , Lachance Daniel , Eckel-Passow Jeanette , Mak Tak , Taylor Michael , Zadeh Gelareh , Dirks Peter , Jenkins Robert , Schramek Daniel
Published: November 2021

Establishing causal links between genetic polymorphisms and increased heritable risk of developing brain cancer is a major challenge. The non-coding single nucleotide polymorphism rs55705857 (A >G) is associated with a ~6-fold increased risk to develop IDH-mutant low-grade glioma (LGG). The rs55705857 G allele has a minor allele frequency of only ~5% in the general population but is found in ~40% of patients with IDH-mutant LGG and patients carrying risk-alleles are diagnosed on average 7-12 years earlier than those carrying non-risk A alleles. This makes rs55705857 one of the highest reported genetic associations with cancer, comparable with inherited BRCA1 gene mutations and the risk of developing breast cancer or other familial glioma genes such as NF1/2, CDKN2A or p53. To generate a LGG mouse model, we combined clonal activation of IDH1R132H with mutations of Trp53 and Atrx, which resulted in the development of LGG-like brain tumors in 25% of mice. Mutating the highly conserved, orthologous mouse rs55705857 locus to mimic the human risk allele dramatically accelerated tumor development from 463 to 172 days and increased penetrance to 75%. The resulting tumors exhibit elevated R-2-hydroxyglutarate levels, well-differentiated fibrillary neoplastic histology and metabolic rewiring, recapitulating histopathological and molecular hallmarks of human LGG. Mechanistically, we show that the rs55705857 locus resides within a brain-specific enhancer, which shows enhanced activity in IDH-mutant tumors. In addition, we found that the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. The hyperactive chromatin status combined with the tissue specificity of this enhancer explains the cooperativity between mutant IDH and rs55705857 and why rs55705857 is associated specifically with IDH-mutant glioma, but not other cancers. Overall, we generated new LGG mouse models, which provide insights into the pathophysiology of this deadly disease and shed light into the heritable predisposition to LGG development.
EPCO-23. A SINGLE-CELL BASED PRECISION MEDICINE APPROACH USING GLIOBLASTOMA PATIENT-SPECIFIC MODELS
Authors: Feroze Abdullah , Park James , Emerson Samuel , Mihalas Anca , Keene Dirk , Cimino Patrick , de Lomana Adrian Lopez Garcia , Kannan Kavya , Wu Wei-Ju , Turkarslan Serdar , Baliga Nitin , Patel Anoop
Published: November 2021

Glioblastoma is a heterogeneous tumor made up of cell states that evolve over time. We modeled tumor evolutionary trajectories during standard-of-care treatment using multimodal single-cell analysis of a primary tumor sample, corresponding mouse xenografts subjected to standard of care therapy, and recurrent tumor at autopsy. We mined the multimodal data with single cell SYstems Genetics Network AnaLysis (scSYGNAL) to identify a network of 52 regulators that mediate treatment-induced shifts in xenograft tumor-cell states that were also reflected in recurrence. By integrating scSYGNAL-derived regulatory network information with transcription factor accessibility deviations derived from single-cell ATAC-seq data, we developed consensus networks that regulate subpopulations of primary and recurrent tumor cells. Finally, by matching targeted therapies to active regulatory networks underlying tumor evolutionary trajectories, we provide a framework for applying single-cell-based precision medicine approaches in a concurrent, neo-adjuvant, or recurrent setting. Our proof-of-concept work herein provides the basis for the development of a modeling and analytical system that enables single-cell characterization of an individual patient’s tumor and inferred therapeutic vulnerabilities. Although further validation is required, in the form of in vivo studies of these putative druggable targets, our preliminary analysis and results suggest that systems biology techniques can be used to infer and predict therapeutic vulnerabilities that are either selected or induced during standard-of-care treatment. Ultimately, the information gathered from such systematic modeling and analysis of individual tumors may inform clinical treatment in a more targeted manner and enable a rational, tailored precision medicine that accounts for intratumoral cell heterogeneity.
EPCO-30. MACHINE-LEARNING PREDICTIVE MODELS BASED ON DNA METHYLATION SIGNATURES DETECTED IN LIQUID BIOPSY SPECIMENS ACCURATELY PREDICT THE DIAGNOSIS AND PROGNOSIS OF MENINGIOMAS
Authors: Herrgott Grayson , Sabedot Thais , She Ruicong , Wells Michael , Asmaro Karam , Malta Tathiane , Mosella Maritza , Nelson Kevin , deCarvalho Ana , Poisson Laila , Robin Adam , Lee Ian , Snyder James , Walbert Tobias , Kalkanis Steven , Rock Jack , Noushmehr Houtan , Castro Anavaleria
Published: November 2021

BACKGROUND

Detection of distinct epigenetic features in circulating cell-free DNA (cfDNA) of liquid biopsy specimens (e.g. blood) provides an opportunity to diagnose and prognosticate central nervous system (CNS) tumors. Utilization of distinctive cell-type genome-wide DNA methylation patterns allows for development of machine-learning (ML) models with the ability to predict tumor diagnosis and prognosis, and remains widely unexplored in meningiomas using LB.

METHODS

We profiled the cfDNA methylome (EPIC array) in serum specimens from patients with meningiomas, other CNS tumors and nontumor conditions (n= 63, 190 and 18, respectively) and harnessed internal and external meningioma tissue methylome data. For diagnostic model development, we identified “meningioma specific” signatures through comparison of meningioma and non-meningioma serum specimens (Wilcoxon rank-sum test) which exhibited tissue methylation similarities named Meningioma- epigenetic Liquid Biopsy (MeLB), and were then employed to develop and cross-validate a Random-Forest derived “MeLB” score to discriminate meningiomas from the other conditions. To predict recurrence risk (RR), we classified a meningioma tissue cohort as ‘favorable’ or ‘unfavorable’ (low and high RR, respectively), using a validated ML outcome model and identified outcome-specific methylation markers with serum subgroup specificities used as input to train a Random-Forest to predict RR in serum-based specimens.

RESULTS

Prediction models based on meningioma-specific methylation markers detected in the serum presented a high accuracy in classification of samples as meningioma or not (Accuracy: 89.6%, Sensitivity: 80%, Specificity: 93.8%). The prognostic model using tissue-serum matching methylation markers was validated across an independent tissue-based cohort (Accuracy: 88%, Sensitivity: 86%, Specificity: 88%) and allowed for classification of serum samples according to RR.

CONCLUSIONS

Machine-learning models using DNA methylation markers identified in the serum can accurately diagnose and predict prognosis in patients with meningioma. After validation in an external cohort, these approaches may improve presurgical diagnosis and therapeutic management of patients with this type of tumor.
EPCO-31. GERMLINE AND SOMATIC MUTATIONS IN PEDIATRIC GERM CELL TUMORS
Authors: Song Dong , Zhao Yang , Mu Quanhua , Liang Zhuangzhuang , Wang Jiajia , Cao Liangliang , Chen Luonan , ma jie , Wang Jiguang
Published: November 2021

Pediatric germ cell tumors (pGCTs), a group of germ-cell-originated neoplasms, currently lack sufficient study. Although somatic activation of KIT/AKT and RAS/MAPK pathways have been revealed, no driver mutations have been detected in most of GCT cases. To extensively explore germline and somatic mutations, we analyzed genomic and/or transcriptomic sequencing data of 236 pGCTs, including data from not only public datasets but also newly collected whole-genome-sequencing data of tumor samples together with case-unaffected-parents trios collected in Shanghai Xinhua Hospital. A new computational pipeline was developed and carried out to identify functional germline and somatic variants. Overall, > 30,000 germline and > 100,000 somatic mutations were detected and prioritized. Particularly, we found germline trisomy 21 in three cases, germline XXY in five cases, and one germline XO, demonstrating the enrichment of germline chromosomal abnormality in pGCTs (p-value < 0.0001). We identified 196 loss-of-function-like inherited germline mutations involving CBL (4/212), PTEN (3/212), TEX11 (2/212), and ATM (1/212). In addition, we detected 2,160 de novo germline mutations (DNMs), and showed an average of 69.7 DNMs per proband, which is compatible to the previously reported incidence. Moreover, we discovered recurrent somatic mutations in known driver genes such as KIT (27/110), KRAS (10/110), NRAS (4/110), MTOR (5/110), PTEN (3/110), and CBL (2/110). Interestingly, somatic hotspot RRAS2 mutations were detected in seven of 110 cases. Subsequent clinical association analysis showed that patients who harbored RRAS2 mutations were younger than those harboring KRAS mutations (p-value < 0.05). Somatic copy number changes were frequently observed, including chr12p+ (47/110), chr21q+ (47/110), chrX+ (34/110), chr13q- (27/110), and chr20q+ (26/110). Furthermore, we identified chromoplexy in nine out of 62 cases, and this alteration is significantly enriched in yolk sac tumors with the occurrence 7/11. Collectively, portraying the mutational landscape of pGCTs, we revealed its disease etiology and potential new drug targets.
BIOM-16. NTRK (NEUROTROPHIC TYROSINE RECEPTOR KINASE) FUSIONS IN GLIOMA PATIENTS: A PROMISING MOLECULAR TARGET?
Authors: Vernadou Anastasia , Argyrakos Theodoros , Papadopoulou Eirini , Rigakos Georgios , Tsoukalas Konstantinos , Labropoulos Stefanos , Avgoustidou Maria , Razis Evangelia
Published: November 2021

BACKGROUND

NTRK gene fusions are rare in gliomas (less than 2%). The use of TRK inhibitors in NTRK-fused solid tumors has generated significant clinical interest. It is likely that the unmet need for effective therapies in glioma will lead to routine testing for NTRK fusions.

METHODS

We performed a retrospective review of 22 patients with malignant gliomas accrued between 2007 and 2021. We collected tumor samples from those patients and NTRK fusions were tested using either immunohistochemistry or FISH or NGS. We then evaluated their association with clinical characteristics, histology and other markers (IDH1/2 mutation, MGMT methylation, BRAF mutation, EGFR expression, ATRX expression, TERT mutation).

RESULTS

Median age at diagnosis was 50 years. NTRK1 translocation was detected in 3 out of 22 patient tumors, while NTRK1 duplication was present in 1 patient (probably related to a translocation, but not proven). All NTRK1 translocations were not found in glioblastomas and their median survival was 15.8 months. On the other hand, median survival of gliomas without NTRK translocations was 11 months. In one NTRK1-translocated patient a TRK inhibitor (entrectinib) was used (for a few days), but we cannot evaluate its efficacy as the patient deteriorated and died. As far as the other biomarkers are concerned, 2 out of 3 NTRK1-translocated gliomas were MGMT methylated.

CONCLUSION

NTRK fusions are rare in gliomas, as confirmed in our small sample analysis. Although adult NTRK-fused gliomas are considered to predominantly involve high-grade histology, in our study all patients had initially lower grade gliomas. NTRK alterations can be detected by different laboratory assays, but each of these approaches has specific advantages and limitations and more data are needed in order to identify the best method. For glioblastomas, NTRK fusions and TRK inhibitors are potentially a new targeted therapeutic strategy but more data are needed.
BIOM-17. DIFFERENCES IN THE IMMUNE MICROENVIRONMENT OF GLIOMAS HARBORING IDH2 VERSUS IDH1 MUTATIONS
Authors: Ostrom Quinn , Xiu Joanne , lopez Giselle , Sumrall Ashley , Aulakh Sonikpreet , Glantz Michael , Pan Edward , Khasraw Mustafa , Ashley David M , Walsh Kyle
Published: November 2021

INTRODUCTION

IDH mutations are a defining feature of lower-grade glioma and secondary glioblastoma. Approximately 95% of glioma-associated IDH mutations are in codon 132 of IDH1, but a small proportion are in IDH2. IDH mutations produce the oncometabolite 2-hydroxyglutarate, which induces global DNA hypermethylation and is associated with an immunosuppressive tumor microenvironment. IDH1 is localized in the cytosol while IDH2 is found in the mitochondrial matrix, and mutations in these genes may have differing effects on the tumor microenvironment.

METHODS

Formalin-fixed, paraffin-embedded tissue from 633 IDH-mutant gliomas (615 IDH1-mutant, 18 IDH2-mutant) underwent whole-exome and whole-transcriptome sequencing at Caris Life Sciences (236 grade 2/3 astrocytoma, 158 grade 2/3 oligodendroglioma, 202 IDH-mutant glioblastoma, 37 glioma, NOS). QuantiSEQ was used to infer tumor-infiltrating immune cell populations from RNAseq data, and gene-set enrichment analyses (GSEA) were performed using Wikipathway.

RESULTS

IDH1-mutant gliomas had higher levels of pro-inflammatory M1 macrophages (P=0.04), modestly higher levels of monocytes (P=0.08), and lower levels of neutrophils (P=0.04) – typically considered immunosuppressive – compared with IDH2-mutant gliomas. No differences were observed in levels of B cells, dendritic cells, NK cells, or T cell subsets (Treg, CD4+, CD8+). IDH2-mutant gliomas were enriched for hallmark oligodendroglioma mutations (TERT promoter, CIC, FUBP1), while IDH1-mutant gliomas were enriched for hallmark astrocytoma mutations (ATRX, TP53). However, associations with tumor-infiltrating immune cells persisted after excluding 1p/19q co-deleted oligodendroglioma from analyses. GSEA revealed upregulation of the microglial TYROBP signaling pathway, the microglial phagocytic pathway, and of Type II Interferon signaling in IDH1-mutant gliomas versus IDH2-mutant gliomas.

CONCLUSIONS

Although IDH2 mutations are generally thought to function similarly to IDH1 mutations, we observe differences in tumor-infiltrating immune cells across groups. IDH2-mutant gliomas appeared to have a more immunosuppressive tumor microenvironment than their IDH1-mutant counterparts. Early-phase immunotherapy trials should consider covariate-adaptive randomization approaches to equally allocate IDH2-mutant gliomas across treatment arms.
BIOM-18. METHYLATION ANALYSIS TO REVEAL THE CELLULAR ORIGIN OF PROGNOSTIC CIRCULATING CELL-FREE DNA IN GLIOBLASTOMA
Authors: Till Jacob , Abdalla Aseel , Wang Zhuoyang , Zhou Wanding , Nabavizadeh S Ali , Binder Zev , Hussain Jasmin , Black Taylor , Yee Stephanie , Desai Arati , Bagley Stephen J , Carpenter Erica
Published: November 2021

We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.
BIOM-20. TUMOR-INTRINSIC AND PERIPHERAL FEATURES ASSOCIATE WITH SURVIVAL AFTER POLIO VIROTHERAPY IN RECURRENT GBM
Authors: Brown Michael , Zhang Gao , Stevenson Kevin , Lin Xiang , Chen Yeqing , Wei Zhi , Beaubier Nike , Yan Hai , He Yiping , Desjardins Annick , Herndon James , Varn Frederick , Verhaak Roel , Zhao Junfei , Friedman Allan , Friedman Henry , McSherry Frances , Muscat Andrea , Lipp Eric , Khasraw Mustafa , Peters Katherine , Randazzo Dina , Sampson John , McLendon Roger , Bigner Darell , Gromeier Matthias , Nair Smita , Ashley David M
Published: November 2021

BACKGROUND

PVSRIPO is a live-attenuated recombinant rhino:poliovirus that mediates antitumor efficacy by engaging antitumor immunity. A subset (~20%) of patients with recurrent GBM (rGBM) survive >24 months after therapy. We previously reported that low tumor mutation burden (TMB) is associated with longer survival after PVSRIPO and immune checkpoint blockade therapy in rGBM, and that low TMB associates with higher inflammatory gene expression signatures in rGBM tumors.

METHODS

Clinical features were tested for association with survival after PVSRIPO therapy. Whole exome sequencing and RNA-sequencing of tumors were used to correlate mutational landscape, tumor mutation burden (TMB), and gene expression signatures of patient tumors with survival. An in vitro assay that measures inflammatory responses of patient PBMCs to PVSRIPO was performed. An independent cohort of paired primary and recurrent GBM tumors was used to assess longitudinal changes in TMB and gene expression signatures after standard of care treatment.

RESULTS

A short time to recurrence and low TMB associated with longer survival after PVSRIPO therapy; these features were not prognostic for longer survival in immunotherapy naïve rGBM cohorts. Unexpectedly, higher pre-treatment polio neutralizing antibody titers were also associated with longer survival after PVSRIPO therapy in two independent clinical cohorts. PBMCs from patients surviving longer after PVSRIPO therapy mounted higher TNF, but lower IFN-a responses after in vitro challenge with PVSRIPO. In analysis of paired primary vs recurrent GBM tumors, we discovered that patients with low TMB upon recurrence were more likely to experience increased tumor inflammation and suppression of overall TMB. Low TMB in rGBM tumors was also associated with neoantigen depletion. Collectively, these observations imply that patients with low TMB and/or shorter duration of standard of care therapy may have intact immune surveillance, and that pre-treatment immunological status may dictate survival response to polio virotherapy.
BIOM-29. ASSOCIATION OF PLASMA microRNA-21 EXPRESSION WITH KARNOFSKY PERFORMANCE SCALE SCORES IN GLIOMA PATIENTS
Authors: Malueka Rusdy , Dwianingsih Ery , Alethea Maria , Wicaksono Adiguno , Dananjoyo Kusumo , Asmedi Ahmad , Hartanto Rachmat
Published: November 2021

Gliomas are one of the most common primary brain tumors. MicroRNA-21 (miRNA-21) has been shown in previous studies to be associated with prognosis in glioma patients. However, similar studies in the Asian population, particularly in Indonesia, are very limited. This study aimed to find the association of plasma miRNA-21 expression with functional status measured by Karnofsky Performance Scale (KPS) in Indonesian glioma patients. The patients were enrolled from a neuro-oncology referral center in Yogyakarta, Indonesia. MiRNA-21 expression from plasma was measured by real-time quantitative PCR. Clinical data were obtained from medical records. KPS scores were classified as low (< 70) and high (> 70). In total, 50 patients were included in this study. Most patients were diagnosed with WHO grade IV gliomas (30.4%), followed by grade II (30.4%), grade III (21.4%), and grade I (5.4%). Most patients (64%) have low KPS scores (< 70). Patients in the low KPS scores group have significantly higher miRNA-21 expression compared to patients in the high KPS scores group (2-∆CT 4.26 vs. 0.68, p=0.002). In conclusion, higher expression of plasma miRNA-21 is associated with worse functional status in glioma patients as measured by KPS.
BIOM-30. BLOOD COUNTS THROUGH TREATMENT COURSE IN NEWLY DIAGNOSED GLIOBLASTOMA PATIENTS RECEIVING CHEMORADIATION
Authors: Deng Davy , Hammoudeh Lubna , Cagney Daniel , McFaline-Figueroa J Ricardo , Chukwueke Ugonma , Reardon David , Lee Eudocia Quant , Nayak Lakshmi , Lim-Fat Mary Jane , Ligon Keith , Bi Wenya Linda , Arnaout Omar , Alexander Brian , Wen Patrick , Rahman Rifaquat
Published: November 2021

BACKGROUND

Glioblastoma (GBM) patients are treated with radiation therapy (RT), temozolomide, and corticosteroids which can affect hematologic and immunologic parameters. We examined lymphocytes, neutrophil-to-lymphocyte ratio and platelet measurements and their association with progression-free survival (PFS) overall survival (OS).

METHODS

We identified 759 newly diagnosed adult GBM patients treated at our institution in the temozolomide (TMZ) era with blood counts that could be automatically extracted from the electronic medical record during chemoradiation (CRT, defined as within 42 days of RT) and at first recurrence. Linear regression and Cox modeling were used to evaluate outcomes.

RESULTS

Median age was 60.3 years; 87% had KPS ≥ 70, 37.5% had gross total resection, and 90% received TMZ. Prior to RT, 56.4% (375/665) patients had a lymphocyte measurement < 1.0 × 1000 cells [K]/μL. Within 42 days of CRT, 81.7% (536/656) had a lymphocyte measurement < 1.0 K/μL, 37.8% (248/656) < 0.5 K/μL. 10.7% (58/544) patients developed grade 2 or higher neutropenia, 9.1% (50/547) patients developed grade 2 or higher thrombocytopenia. On multivariable analysis (MVA), older age (AHR1.03, p< 0.001), unmethylated MGMT status (AHR2.56,p< 0.001), lower RT dose (<54Gy, AHR 3.45, p< 0.001), male sex (AHR1.45, p=0.02), non-gross total resection (AHR1.63, p< 0.001), lymphopenia during CRT (AHR0.63, p=0.008) and higher NLR during CRT (AHR1.02, p=0.001) were significantly associated with worse OS.

Older age (AHR1.01, p=0.02), unmethylated MGMT status (AHR2.44, p< 0.001), lower RT dose (AHR1.82, p=0.02), higher NLR during CRT (AHR1.03, p < 0.001) were significantly associated with worse PFS on MVA. At first recurrence, median lymphocyte count was 0.7 K/μL with 74% (348/468) patients < 1.0 K/μL and 27% < 0.5 K/μL.

CONCLUSION

Lymphopenia and higher neutrophil-to-lymphocyte ratio are associated with inferior outcomes. Persistent lymphopenia at time of first recurrence may have implications for clinical trial eligibility and immunotherapy approaches in recurrent GBM.
BIOM-37. ELUCIDATION OF THE TRANSCRIPTOMIC CONSEQUENCES OF KLF4 AND TRAF7 MUTATIONS IN SECRETORY MENINGIOMAS
Authors: Michaelsen Signe , Cloos Paul , Broholm Helle , Johansen Jens , Ahmedov Ulvi , Mathiesen Tiit , Kristensen Lasse , Kristensen Bjarne , Hansen Klaus
Published: November 2021

Secretory meningioma (SM) is a benign histological meningioma subtype frequently located in the central skull-base, complicating surgical removal, highlighting the need of gentle targeting treatment alternatives. SM tumors arise from the arachnoid layer in the meninges and are associated with a hotspot p.K409Q mutation in KLF4 combined with different mutations in TRAF7. The objective of this study was to identify how KLF4 and TRAF7 mutations alter signaling in SM tumors. The study includes 16 FFPE samples from SM tumors and three arachnoid cysts as normal-tissue reference. All tissues were histologically confirmed, followed by genomic sequencing of KLF4, TRAF7, NF2 and TERT-promoter (C220T/C228T) using a customized NGS panel. Gene expression profiles were analyzed using NanoString Pan-cancer Progression and Pan-cancer Pathways panels, which resulted in measurement of 1,202 unique transcripts over background level. All 16 SM tumors presented the KLF4p.K409Q mutation, of which 12 had mutation in TRAF7 (four being p.N520S), while no cysts had mutations in these genes. Neither tumors nor cysts had NF2 or TERT-promoter mutations. PCA analysis showed different RNA expression profiles for SM tumors and cysts, while TRAF7 mutation was without influence on the overall gene expression patterns in the tumors. Sub-analysis found increased KLF4 expression in TRAF7-mutated SM tumors versus non-mutated tumors and cysts at the RNA level, while TRAF7 expression was stable in all samples. In total 98 genes were found differentially expressed (54 up- and 44 downregulated) with a log2-fold change over 1 and an adjusted p-value under 0.05 in tumors versus cysts. Further analyses are planned to explore and validate these results. In conclusion, we find that the KLF4p.K409Q mutation correlates more strongly with SM histology, than the TRAF7 mutation, and that SM tumors have gene expressions profiles that are distinct from normal arachnoid tissue for a number of disease relevant genes.
BIOM-43. CROSS-PLATFORM ROBUSTNESS IN THE GLUCOCORTICOID RESPONSE PHARMACODYNAMIC BIOMARKER
Authors: Tang Emily , Wiencke John , Warrier Gayathri , Hansen Helen , McCoy Lucie , Rice Terri , Bracci Paige , Wrensch Margaret , Taylor Jennie , Clarke Jennifer , Koestler Devin , Salas Lucas , Christensen Brock , Kelsey Karl , Molinaro Annette
Published: November 2021

The neutrophil dexamethasone methylation index (NDMI) is an algorithm-based biomarker to assess individuals’ exposures to dexamethasone, a synthetic glucocorticoid commonly administered for inflammation. Cortisol is the main endogenous glucocorticoid that controls vital processes including the immune response and lipid and carbohydrate metabolism. Variations in the NDMI score reflect individuals’ sensitivities of exposures to both exogenous and endogenous glucocorticoids, and this biomarker was trained using elastic net regression on Illumina’s most recent DNA methylation beadarray, the EPIC array, which contains 850,000 cytosine-guanine (CpG) sites. While technology for microarray research continues to advance over time, researchers are capable of conducting more comprehensive epigenome-wide association studies (EWAS). However, many studies are still run and archived using Illumina’s historical 450K platform with approximately 450,000 CpGs, and there are fewer published databases using the 850K EPIC array. To evaluate the cross-platform bioinformatic comparability, we performed elastic net regression modeling using predictors available in the 450K to train the NDMI. Among the 135 pre-surgery glioma cases from the UCSF Immune Profiles Study (IPS), NDMI scores between the 450K and 850K model were strongly correlated (r = 0.99, p < 0.0001). In the 311 controls from the UCSF Adult Glioma Study (AGS), similar correlations were observed (r = 0.96, p < 0.0001). We observe that NDMI remains a robust tool using historical 450K data and conclude that this algorithmic tool is capable of detecting the variations in individuals’ responses to dexamethasone.
STEM-15. SerpinB3 DRIVES CANCER STEM CELL SURVIVAL IN GLIOBLASTOMA
Authors: Lauko Adam , Turaga Soumya M , Volovetz Josephine , Bayik Defne , Bao Shideng , Yu Jennifer , Lathia Justin
Published: November 2021

Despite therapeutic interventions for glioblastoma (GBM), self-renewing, therapy-resistant populations of cells referred to as cancer stem cells (CSCs) drive recurrence. Previously, we identified the unique expression of junctional adhesion molecule-A (JAM-A) on CSCs and demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. Moreover, we determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the entire signaling network has yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from patient-derived GBM CSCs and performed mass spectrometry to determine JAM-A binding proteins. This led to the identification of the cysteine protease inhibitor SerpinB3 as a putative JAM-A binding partner. Using in vitro CSC functional assays, we show that SerpinB3 is necessary for CSC maintenance and survival. In an in vivo orthotopic xenograft model, knockdown of SerpinB3 extended survival. Mechanistically, knockdown of SerpinB3 led to decreased expression of TGF-β, Myc, WNT, and Notch signaling, known regulators of the CSC state. Additionally, knockdown of SerpinB3 increases susceptibility to radiation therapy. SerpinB3 is essential for buffering cells against cathepsin-mediated cell death, and we found that elevated lysosomal membrane permeability after radiation leads to cathepsin release into the cytoplasm. As a result, SerpinB3 knockdown cells have a diminished capacity to inhibit cathepsin-driven cell death after radiation. The addition of the cathepsin inhibitor E64D partially rescues the SerpinB3 knockdown, however, SerpinB3 mutants that are unable to inhibit cathepsins fail to do the same. Taken together, our findings, identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to increase the efficacy of standard of care GBM therapies against therapy-resistant CSCs.
STEM-20. THE ROLE OF HUMAN BETA DEFENSINS IN THE CLONOGENICITY OF GLIOBLASTOMA MULTIFORME
Authors: Harris Peggy , Kerstetter-Fogle Amber , Sloan Anthony , Raghavan Alankrita , Hoffman Harry , Barnholtz-Sloan Jill , Couce Marta , Jin Ge , Weinberg Aaron , Sloan Andrew
Published: November 2021

INTRODUCTION

Glioblastoma (GBM) is the most common primary malignant brain tumor with a median overall survival of 12-15months. GBM aggressiveness and poor response to treatment are often attributed to a small population of stem-like cells referred to as glioma stem cells (GSCs). Human Beta-Defensins (HBDs), a family of small molecules initially thought to function as anti-microbials, have been implicated in various cancers with functions that are cancer type specific, including proinflammatory and immunosuppressive roles. GOAL: This study aimed to elucidate HBDs expression in GSCs and differentiated gioma cells (DGSs).

METHODS

We identified HBD2 and HBD3 in glioma and GSCs and DGSs by immunofluorescence (IF) and immunohistochemistry (IHC). We also assessed the role HBD2/3 play in GBM oncogenesis by investigating their effects on the self-renewal capacity of GSCs.

RESULTS

HBD2 and HBD3 are found in both bulk tumor and GSCs by IHC and IF. Expression of HBD2 and HBD3 mRNA levels are elevated in GBM patient tissue in comparison to lower grade gliomas by 16.2 & 1.9 fold (respectively; p < 0.0001). Additionally, transcriptional expression of HBD2 and HBD3 are increased by 0.68 and 1.15 fold respectively in GSCs versus autologous DGCs (p < 0.05; p< 0.005 respectively), suggesting a role for HBD 2/3 in oncogenesis. Interestingly however, HBD2 and HBD3 pretreatment of GSC cell lines showed decreased self-renewal capacity by 34.2 and 66.4% (p < 0.001), determined by the reduced number of large neurospheres ( >250mm).

CONCLUSIONS

Our results demonstrate the presence of HBD2/3 in GBM samples by IHC and IF with increased HBD2/3 mRNA expression in GBM samples. Interestingly DGCs contain less HBD2/3 than their GCS counterparts, and clonogenicity assays demonstrate a decrease in oncogenesis, suggesting that HBD’s role in proliferation and clonogenicity of DGCs and GSCs may be context dependent, leading to additional questions and future studies.
STEM-22. TARGETING REPLICATION STRESS RESPONSE FOR GLIOMA STEM CELL SPECIFIC CYTOTOXICITY
Authors: Clough Emily , Ballinger Tracy , Semple Colin , Strathdee Karen , carruthers ross
Published: November 2021

BACKGROUND

Evidence suggests treatment resistant glioma stem cells (GSCs) drive glioblastoma (GBM) recurrence. Current treatments fail to eradicate GSC and novel GSC targeting therapies are a priority. GSC exhibit elevated DNA replication stress (RS) versus non GSC tumour cells driving constitutive DNA damage response (DDR) activation and efficient DNA repair. We previously demonstrated that targeting RS response with combined ATR and PARP inhibition (CAiPi) (VE821 and Olaparib) provides potent GSC specific cytotoxicity. In this study we investigated the underlying DDR phenotype which determines this vulnerability.

RESULTS

Paired GSC enriched (‘GSC’) and GSC depleted, differentiated (‘bulk’) populations were cultured from GBM specimens in neurobasal media with growth factors or serum containing media respectively. GSC exhibited reduced survival following exposure to CAiPi versus bulk. CAiPi significantly increased 53BP1 G1 phase nuclear bodies (53BP1NBs) in GSC, which are known to shield under-replicated DNA in actively transcribed genes. Mapping the genomic distribution of endogenously occurring replication dependent DNA double strand breaks via Breaks Ligation In Situ Sequencing (BLISS), revealed reduced intragenic DSB in long actively transcribed genes in GSC versus bulk at baseline, suggesting a reliance upon transcription coupled DSB repair in GSC. RNA seq demonstrated CAiPi-induced transcriptomic alterations in GSC including replication regulation and initiation. DNA fibre assay showed that CAiPi increased GSC new origin firing which correlated with PARP trapping. GSCs were rescued from CAiPi by roscovitine induced inhibition of excess origin firing. CAiPi is potently radiosensitizing by clonogenic assay and we demonstrated murine blood brain barrier penetration of CAiPi utilising VE822 and pamiparib in vivo.

CONCLUSION

Dysregulation of origin firing by CAiPi exposes a GSC specific vulnerability which results in DNA under-replication and abrogation of proficient DNA repair seen at long actively transcribed genes and has potential to be clinically translated as a GSC specific cytotoxic therapy.
CBIO-17. A NOVEL ROLE FOR ATR KINASE DETERMINES GLIOBLASTOMA TUMOUR CELL INVASION
Authors: carruthers ross , Derby Sarah , Strathdee Karen , Chalmers Anthony , Norman Jim , Birch Joanna
Published: November 2021

BACKGROUND: Widespread contamination of the brain with malignant cells is a predominant feature of glioblastoma (GBM) and fatal brainstem infiltration is frequently observed at autopsy. Whilst radiotherapy improves survival, irradiation increases GBM cell invasion, resulting in sublethal dose to cells migrating outside the irradiated volume. Tumour cell invasion should be a therapeutic priority if survival is to be improved. The responsible molecular mechanisms are key to improving outcomes but remain enigmatic. Ataxia telangiectasia and rad3-related (ATR) is a DNA damage response (DDR) kinase involved in DNA replication stress (RS) response and is an established therapeutic target for GBM. In this study we demonstrate a novel role for ATR kinase in facilitating malignant cell invasion. METHODS AND RESULTS: Invading margins of human GBM samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated a reduction in cell velocity following ATR inhibition (ATRi; VE822) or ATR siRNA, and a retraction defect was evident in vitro. Extensive cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures on electron microscopy which could engulf high molecular weight dextran, suggesting blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed integrin sequestration within macropinosomes and reduced integrin internalisation respectively. Interrogation of a published ‘ATR interactome’ revealed ATR targets with functions in endocytic vesicle trafficking. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further study demonstrated reduced invading tumour cells following ATRi in intracranial xenografts. CONCLUSION: We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin internalisation. ATRi results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.
CBIO-27. INTEGRATED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES THERAPEUTIC VULNERABILITIES IN GLIOBLASTOMA
Authors: Fernandez Elizabeth , Mia Wilson , Bayley Nicholas , Zhu Henan , Tse Christopher , Liau Linda , Cloughesy Timothy , Nathanson David
Published: November 2021

Molecular profiling frequently fails to predict therapeutic outcomes in cancer. This is in part due to the myriad of genetic alterations comprising a tumor which enable rapid rewiring of cancer signaling pathways. Functional profiling ascertains signaling dynamics and has emerged as an alternative method to predict drug responses. It is unknown whether incorporating molecular with functional profiling offers superior insight into specific tumor dependencies and consequently therapeutic susceptibilities. Here we performed integrated molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in 50 GBM patient specimens. We found that treatment with standard of care (e.g., temozolomide or radiation) rewired the apoptotic machinery in a p53 dependent manner, eliciting an exclusive survival dependency on BCL-xL in a genetically defined subset of GBM tumors. However, functional BH3 profiling of ‘apoptotic priming’ was required to predict response to combined IR/TMZ and BCL-xL inhibition in p53 WT GBM tumors. Accordingly, a composite of genetic and functional profiling predicted the response of patient derived GBM models to IR/TMZ with a novel GBM specific BCLxL antagonist. Collectively, these studies identify the mechanisms of intrinsic apoptosis resistance in GBM and demonstrate how functional and molecular data can be complementary to robustly predict therapy-induced cell death.