tbuckley/cabot-search huggingface dataset:
TMIC-78. CHEMOTHERAPY INDUCED IMMUNOGENIC RESPONSE IN GLIOMA AND IMPLICATIONS FOR THERAPEUTIC STRATEGIES
Authors: Damian Teasley, Nicholas Dadario, Liang Lei, Colin Sperring, Nadine Khoury, Nathaniel Rolfe, Melanie Kristt, Angeliki Mela, Nkechime Ifedoria, Corina Kotidis, Wenting Zhao, Julia Furnari, Aayushi Mahajan, Brianna Pereira, Michael Argenziano, Nathan Winans, Nina Yoh, Nelson Humala, Athanassios Dovas, Charles Karan, Brian Gill, Peter Sims, Jeffrey Bruce, Peter Canoll
Journal: Neuro-Oncology (2024)

Abstract INTRODUCTION Many glioblastoma (GBM) treatments fail because they treat tumor cells in isolation and ignore the surrounding microenvironment. In our phase 1b clinical trial of delivery of Topotecan (TPT) via Convection Enhanced Delivery (CED) in glioma, we demonstrated effective tumor cell elimination accompanied by a robust inflammatory response. We hypothesized that TPT induces an immunogenic response resulting in an unfavorable inflammatory landscape. METHODS To characterize chemotherapy’s effect on myeloid cells, we analyzed pre- and post-treatment biopsies from our trial using bulk RNA sequencing to measure canonical myeloid markers. We validated survival benefit of CED in vivo in our tumor model and characterized post-treatment tissue using single cell RNA sequencing (scRNA seq) after 7 days of treatment. We treated slice cultures generated from surgical samples of GBM with TPT and performed scRNA seq and histologic analysis to assess microenvironment changes after 24 hours of treatment. We used several in vitro assays to characterize the direct and indirect mechanisms of myeloid activation. RESULTS Bulk RNA sequencing of post treatment biopsies demonstrates upregulation of markers associated with activated and exhausted immune cell populations such as IBA1, CD68, MSR1, MARCO and loss of markers associated with homeostatic microglia such as P2RY12 and TMEM119. Similar results are found within 7 days of treatment in vivo, however, are not found in human derived samples treated acutely. Treatment of in vitro mono and co-culture systems demonstrate a tumor cell death dependent activation of myeloid cell populations via immunogenic molecules. CONCLUSIONS Chemotherapies eliminate proliferating glioma cells but also induce inflammation. While many investigators have tried to leverage this activation, our results suggest that chronic inflammation contributes to poor prognosis and recurrence. The identification of molecules responsible for this activation point to new therapeutic targets aimed at controlling treatment-induced inflammation, as part of a combinatorial approach to treat GBM.
EPCO-50. MOLECULAR DETERMINANTS OF CENTRAL NERVOUS SYSTEM TROPISM AND PROGRESSION IN COLORECTAL CANCER
Authors: Chengcheng Gui, Henry Walch, Kirin Mueller, Anna Skakodub, Emily Miao, Ishaani Khatri, Rahul Kumar, Michel Padilla Mazzeo, Junchao Shen, Claire Cooper, Audree Hsu, Roshal Patel, A Turan Ilica, Jordan Eichholz, Kenny Yu, Jessica Wilcox, Paolo Manca, Yao Yu, Yoshiya Yamada, Brandon Imber, Steven Maron, Michael Foote, Walid Chatila, Rona Yaeger, Nikolaus Schultz, Luke Pike
Journal: Neuro-Oncology (2024)

Abstract BACKGROUND Brain metastases (BM) from colorectal cancer (CRC) are associated with dismal prognosis, but little is understood regarding their molecular characteristics. We identified genomic features that predispose to brain tropism, differentiate BM from extracranial disease, and predict early intracranial progression (IP) after BM-directed therapy. METHODS We analyzed next-generation sequencing (NGS) of tumor biopsies from three subgroups of CRC patients: patients with BM (BM+, n=379), patients with extracranial metastases (EM) but without BM (BM-/EM+, n=3968), and patients without metastatic disease (BM-/EM-, n=1061). NGS was performed with MSK-IMPACT, a targeted panel of &amp;gt;300 genes. First, we compared genomic alterations in primary tumors (PT) among the BM+, BM-/EM+, and BM-/EM- subgroups. Second, we focused on patients in the BM+ subgroup who received stereotactic radiosurgery (SRS) for limited BM, comparing tumor genomic alterations from different anatomic sites: BM (n=32), EM (n=62), and PT (n=68). Third, we focused on the 32 resected BM, identifying genomic alterations that predicted early IP, defined as new BM, leptomeningeal disease, or enlargement of existing BM, within 6 months of postoperative SRS. RESULTS PT of BM+ patients demonstrated lower rates of microsatellite instability, increased frequency of BRAF alterations, and fewer APC alterations compared with PT of BM-/EM+ and BM-/EM- patients. Among BM+ patients, SMAD4, MYC, and CDK8 alterations were enriched among resected BM specimens, compared with EM and PT specimens. Resected BM of patients with early IP demonstrated increased frequency of SMAD4 alterations, particularly biallelic SMAD4 loss, and decreased frequency of MYC alterations, compared with resected BM of patients without early IP. CONCLUSION Alterations in key driver genes are associated with initial development and progression of CRC BM. BRAF alterations are enriched in PT of patients who develop BM. SMAD4 alterations are enriched in BM compared to extracranial disease and furthermore predict early IP after BM-targeted therapy.
TMIC-73. SEX DIFFERENCES IN INTERFERON-GAMMA RESPONSES IN GLIOMA MICROENVIRONMENT
Authors: Jia-Shiun Leu, Han Nhat Tran, Taha S Ismail, Kyuson Yun
Journal: Neuro-Oncology (2024)

Abstract IFNγ is a cytokine with both pro- and anti-tumorigenic effects and its role in glioma immunotherapy resistance is not well understood. In the tumor microenvironment, T and NK cells produce IFNγ, which affects cellular phenotypes in most of the cancer and stromal cells since IFNγ receptor is ubiquitously expressed. IFNγ induces expression of molecules that confer resistance to immunotherapies, such as PDL1, PDL2, IDO1, and iNOS. The goal of this study is to elucidate cell type-specific responses to IFNγ in immune competent glioma models we developed that are resistant to immunotherapy. We intracranially injected male and female murine glioma cells into B6 and Ifng-/- honst mice to measure overall survival and perform immune phenotyping and single cell RNA-sequencing analyses. Ifnγ-/- hosts exhibited a shorter survival, and the proportion of GBM cells is higher in Ifng-/- hosts compared to wild type, indicating that the net effect of IFNγ signaling in glioma is anti-tumorigenic. In a recent study, we published that IFNγ-response pathway is enriched in the mesenchymal-like (MES-like) subtype of human GBM cells, compared to neutral-progenitor-like (NPC-like) and oligodendrocyte-progenitor-like (OPC-like) cells (Abdelfattah et al., 2022). To test our hypothesis that IFNγ signaling induces MES transition in glioma cells in vivo, we compared fractions of glioma cell subtypes in wildtype and Ifng-/- male and female hosts. In female Ifng-/- mice, MES-like GBM cell fraction was reduced while OPC-like fraction was increased, supporting our hypothesis. However, there was no difference in male hosts. Furthermore, gliomas grown in female B6 hosts showed higher infiltration of immune cells (CD45+), lymphocytes (CD3+) and fewer resident microglia cells (CD11b+CD45int), compared with Ifng-/- female hosts. In contrast, no significant difference in immune cell infiltration was observed between B6 vs Ifng-/- males. These results suggest an unanticipated and complex context-dependent role of IFNγ signaling in GBM, which include sex differences in its role in immune cell recruitment and MES transition in glioma cells.
EPCO-53. CHARACTERIZING THE FUNCTIONAL IMPACT OF GERMLINE-INHERITED RISK ALLELES IN GLIOMA EVOLUTION
Authors: Bhargavi Brahmendra Barathi, Prit Malgulwar, Yang Liu, Kasthuri Kannan, Jason Huse
Journal: Neuro-Oncology (2024)

Abstract Efforts to understand etiology of glioma led researchers to investigate associated genetic factors. Genome Wide Association Studies (GWAS) conducted to that end, uncovered an association of various single nucleotide polymorphisms (SNP) among hallmark oncogenes and tumor suppressors as risk variants in glioma incidence. Here, we perform in vitro modeling of four risk alleles/SNPs((rs723527 (EGFR), rs55705857 (MYC), rs7572263 (IDH1), rs78378222 (TP53)) mapped to non-coding regulatory regions of the genome, to assess their causal contribution to glioma evolution. To model the aforementioned risk alleles in an in-vitro system, we used a CRISPR-knock in strategy in human induced pluripotent stem cells (hiPSCs) and verified by Sanger sequencing. To study the effects of the risk alleles on brain cell types, we generated a cerebral organoid model from the edited and control lines and subjected them to downstream morphological and transcriptomic analyses. While we did not see morphological changes in the histological sections relative to controls at the early 14-day timepoint, we observed increased differentiation in both control and risk cohorts, along with an absence of neural rosettes, at the late 90-day timepoint. Bulk RNA-seq of the organoids at 14-days resulted in positive enrichment of oncogenic signaling pathways and negative enrichment of DNA repair pathways in the risk allele cohorts. Furthermore, single cell RNA seq of 14 and 90-day samples revealed a sharp decrease in neural stem cell clusters in both IDH and TP53 cohorts, while control organoids demonstrated robust production of immature neurons at 90 days. Overall, we observed a significant increase in differentiation into mature cell clusters in the control, relative to the risk allele organoids. Ongoing efforts are aimed at understanding this apparent differentiation block in the IDH and TP53 compartments, relative to control. Further analysis will lead to addressing key understudied areas of glioma etiology and associated mechanisms of risk incidence.
EPCO-38. IDENTIFYING THE MOLECULAR SIGNATURE OF INFILTRATING EDGE IN GLIOBLASTOMA AS DRIVERS OF TUMOUR INVASION AND RECURRENCE
Authors: Alyona Ivanova, Shamini Ayyadhury, Megan Wu, David G Munoz, Trevor Pugh, Sunit Das
Journal: Neuro-Oncology (2024)

Abstract Complete tumour resection in glioblastoma patients is not possible. Residual therapy-resistant edge-derived cells drive tumour recurrence and infiltrative expansion in glioblastoma. Features that are specific to malignant edge-derived cells may serve as predictive biomarkers to allow individual tailoring of the treatment plan to slow down tumour invasion and prevent tumour recurrence in GBM patients following standard therapy. Intratumoural spatial heterogeneity facilitates therapeutic resistance and recurrence in glioblastoma. Our knowledge on glioblastoma heterogeneity is mostly restricted to the surgically resectable tumour core, while the functional characterization of tumour cells at the infiltrating edge remains largely elusive due to the presence of normal functional brain tissue in the peritumoural lesion. Edge-derived cells exhibit larger capacity for infiltrative expansion and are the main drivers of treatment failure and tumour recurrence, making them action targets for novel treatment approaches in glioblastoma. To resolve the transcriptional heterogeneity of GBM within the spatial context, we profiled gene expression of different tumour regions (“edge” and “core”) obtained at initial surgical resection from primary and recurrent IDH-WT GBM patients with Visium 10x and GeoMx. We show that infiltrative edge-derived cells are spatially segregated and are characterized by regionally shared distinct genomic and transcriptomic signatures that promote invasiveness and underly disease recurrence. Tumour edge is enriched for neuronal signatures, while tumour core displays larger transcriptional subpopulation diversity and abundance of proliferative cells with a high capacity for self-renewal, consistent with the proliferative and cancer stem cell-enriched phenotype in early GBM progressors. Upregulated DEGs of tumour edge cells are significantly associated with ion regulation of transport, chemical synaptic transmission, and nervous system development. These modules may represent tumour cell hijacking of neuronal programs specifically at the tumour periphery as described in the context of glioma-neuron synaptic communication and formation of neurite-like microtubes. Upregulation of ion regulation transport at the tumour periphery indicates enhanced neuronal activity and excitability driving infiltrating growth.
EPCO-15. INTRA-TUMORAL HETEROGENEITY OF DNA METHYLATION PROFILING AND CELLULAR COMPOSITION IN GLIOBLASTOMA
Authors: Yuji Matsumoto, Omkar Singh, Jose Garcia, Sunwoo Kwak, Zied Abdullaev, Nelson Freeburg, Dana Silverbush, Kenneth Aldape, Christos Davatzikos, MacLean Nasrallah
Journal: Neuro-Oncology (2024)

Abstract BACKGROUND DNA methylation profiling has become an important diagnostic and exploratory tool in neuro-oncology. Intra-tumoral molecular heterogeneity is a crucial factor in the treatment resistance of IDH-wildtype glioblastoma (GBM). However, the intra-tumoral heterogeneity of methylation profiles in GBM remains poorly elucidated. This study aimed to investigate the intra-tumoral heterogeneity of DNA methylation subclasses and deconvolve methylation profiles to infer the cellular composition of the tumor. METHODS We conducted genome-wide DNA methylation analysis and applied the DKFZ/Heidelberg CNS tumor classifier to FFPE tissue samples from a multi-sampling cohort with multiple samples per patient and a single-sampling cohort with one sample per patient. Only samples classified as GBM were included. We conducted two different methylation-based deconvolution analyses. Deconvolution 1 inferred the fractions of tumor subtypes (RTK_I, RTK_II, MES_TYP, and MES_ATYP) and cell types in the microenvironment. Deconvolution 2 estimated the abundance of malignant cell states (stem-like and differentiated cell components) and cell types in the microenvironment. RESULTS The multi-sampling cohort included 32 patients with 113 samples, and the single-sampling cohort included 87 patients with 87 samples. In the multi-sampling cohort, 8 patients (25%) exhibited intra-patient heterogeneity in methylation subclasses across their samples. Deconvolution 1 indicated that each tumor subclass component was heterogeneously distributed within samples, and the abundance of the myeloid cell component correlated with the MES_TYP component across samples. Deconvolution 2 similarly revealed heterogeneity in the immune component abundance across samples within patients. Integrating the results of Deconvolution 1 and Deconvolution 2 using the entire cohort revealed significant correlations between RTK_I and stem-like, RTK_II and differentiated, and MES_TYP and immune components. These findings were also replicated in TCGA dataset. CONCLUSIONS Our findings underscore the importance of considering intra-tumoral heterogeneity in methylation profiles and the biological characteristics of each methylation subclass in developing novel GBM therapies.
EPCO-37. LONGITUDINAL TUMOR-WIDE SAMPLING OF GLIOBLASTOMA REVEALS EARLY EVOLUTIONARY DIVERGENCE OF RECURRENCE AND CLUES TOWARDS IDENTIFYING THE BIOLOGICAL TUMOR CENTER
Authors: Benjamin Lerman, Chibo Hong, Patrick Schupp, Lee Chen, Daniel Zamler, Ivan Smirnov, Joanna Phillips, Anny Shai, Janine Lupo, Michael Oldham, Joseph Costello
Journal: Neuro-Oncology (2024)

Abstract Therapy resistance in glioblastoma is in part attributed to intratumoral heterogeneity and treatment-resistant cell populations. Understanding heterogeneity requires more complete tumor sampling at diagnosis and at recurrence. We used a whole-tumor sampling approach to obtain 43 spatially mapped biopsies from 3 glioblastomas at diagnosis and recurrence, Pyclone and ClonEvol imputed clonal evolution from exome data, and weighted gene co-expression network analysis inferred gene expression programs from RNA sequencing. Across the cohort, tumor-wide clonal alterations representing evolutionarily early expansions included canonical changes (i.e. Chr7 gain, EGFR amplification) and a diverse set of copy number variations (Chr19/20 gain), driver mutations (i.e. PTEN, KDR), and fusions (LIMCH1::UCHL1, KANK::DOCK8). In 2/3 patients, the founding clone was the only subclone common to primary and recurrence samples and 37% of cancer drivers across the cohort appeared after evolutionary divergence of the primary and recurrent tumors. We identified thirty-two transcriptional programs, six of which were differentially expressed between timepoints. Programs enriched for mitochondrial activity, endothelium/pericytes and classical glioblastoma subtype signature were more highly expressed in primary samples. Programs enriched for mesenchymal-like state, oligodendrocytes and inhibitory interneurons were more highly expressed in recurrence samples. At diagnosis, 11/16 samples showed higher expression of the classical subtype program compared to mesenchymal-like, but at recurrence the mesenchymal program dominated in 18/18 samples. Expression of the programs correlated more strongly with position relative to the contrast-enhancing rather than T2 tumor centroid (p&amp;lt;0.001). These findings reveal novel diversity and complexity in the genetic roots of individual glioblastoma. Recurrences arose from subclones diverging early in evolution of the primary tumor and contained clonal drivers not detected in the primary. Expression data revealed transition from an intratumorally heterogeneous mixture of classical and mesenchymal signatures to tumor-wide mesenchymal at recurrence and suggests that the contrast enhancing centroid may serve as the biological center of the tumor.
EPCO-24. WHAT SCRNA SEQUENCING TAUGHT US ABOUT MGMT EXPRESSION IN GLIOBLASTOMA
Authors: Iyad Alnahhas, Wenyin Shi
Journal: Neuro-Oncology (2024)

Abstract INTRODUCTION The promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMTp) is an established predictive and prognostic marker in GBM. Previous studies showed that the expression of MGMT based on immunohistochemistry was variable and lacked association with survival. This in part is because non-tumor cells including endothelial cells and macrophages express MGMT. Advanced technologies such as single-cell RNA (scRNA) sequencing have helped to elucidate the cellular composition of cancer and its microenvironment. scRNA sequencing allows to assess gene expression level in tumor cells specifically. METHODS We used publicly available data from two recent IDHwt GBM scRNA studies that included MGMTp methylation status data to explore and uncover details about MGMT expression at the single-cell level: CPTAC (13 primary samples) and Neftel (20 primary samples). RESULTS In the CPTAC study, MGMT gene expression ranged from 0.19%-1.43% in the MGMTp methylated group (median 0.82%), and from 2.17%-28.36% in the MGMTp unmethylated group (median 5.7%). It therefore appears that 2% is a reasonable expression cutoff to predict the MGMTp methylation status based on scRNA data. In the Neftel study, MGMT expression ranged from 0-1.26% in the MGMTp methylated group (median 0.59%), and from 0.3-27.67% in the MGMTp unmethylated group (median 12.44%). Three unmethylated samples (out of 16) did not follow the 2% rule. It is unclear if this is due to simply inaccurate annotation of these samples. Moreover, the Neftel paper did not specify the method used to detect MGMTp methylation. Alternatively, could it be that truly MGMTp unmethylated samples can have low MGMT expression? Could this explain why some unmethylated MGMTp GBM patients surpass the expected survival? Interestingly, gene set enrichment analysis shows that MGMT expressing cells are enriched with mesenchymal genes, whereas MGMT negative cells are enriched with proneural genes. CONCLUSION Fewer than 2% of GBM cells express MGMT if MGMTp is methylated.
TMIC-83. ABERRANT EGFR ACTIVATION SUPPORTS ENHANCED TUMOR-MYELOID CROSSTALK AND IMPAIRS EFFECTOR T CELL INFILTRATION AND FUNCTION IN GLIOBLASTOMA
Authors: Marissa Pioso, Marissa Li, Julio Sanchez, Andrea Tamayo, Katie Grausam, Joshua Breunig, David Nathanson, Robert Prins
Journal: Neuro-Oncology (2024)

Abstract Glioblastoma (GBM) is known to display a highly suppressive tumor immune microenvironment (TIME) dominated by myeloid cells with low T cell infiltration. Aberrant epidermal growth factor receptor (EGFR) activation is observed in ~50% of GBM patients via amplification and the constitutively active EGFRvIII variant, however the impact of EGFR activation on the brain TIME and T cell-mediated anti-tumor immunity remains poorly understood. Single cell transcriptomic and receptor-ligand interaction analyses of paired newly diagnosed GBM tumor and intratumoral CD45+ immune cells revealed increased crosstalk between EGFR activated tumors and EGFR ligand-expressing tumor-associated myeloid cells. Therefore, a novel orthotopic murine EGFRvIII-expressing glioma model wherein the mEGFRvIII gene is under a tetracycline-off system (MADR-mEGFRvIII) was leveraged to identify how EGFR signaling impacts overall TIME composition and the phenotype of intratumoral and systemic T cells in vivo. Temporal flow cytometric immunophenotyping following tumor or mock (saline) implantation identified no significant shift in T cell infiltration within the brain or cervical lymph nodes (cLNs). In contrast, one week of doxycycline-mediated genetic EGFR ablation conferred a significant survival benefit and was associated with enhanced intratumoral T cell infiltration as well as diminished tumor-associated F4/80+ myeloid cells compared to vehicle treated animals. Further characterization revealed genetic EGFR ablation enhanced recruitment of CXCR3+, TNFα+, and TIGIT+ CD4 T cells to the tumor, while the prevalence of these populations contracted in CD4 and CD8 T cells within the draining cLNs. In vitro, pharmacologic EGFR inhibition enhanced antigen-specific Pmel-1 T cell killing of gp100-expressing MADR-mEGFRvIII tumor cells and was associated with increased IFNγ secretion by ELISA. Collectively, our work demonstrates that aberrant EGFR signaling promotes an immunosuppressive brain TIME with reduced T cell infiltration potentially via tumor-myeloid signaling. Targeting this oncogenic axis may therefore reprogram the TIME and directly enable enhanced trafficking of effector T cells to the tumor.