Malignant Tumors Research Articles

PATH-66. MOLECULAR FEATURES RELATED TO LONG-TERM SURVIVAL IN GLIOBLASTOMA

Abstract Glioblastoma Multiforme (GBM) remains the most common malignant primary brain tumor in adults, with a dismal prognosis that rarely exceeds beyond 2 years of survival despite multimodal treatment. While small fractions of GBM patients seem to display extended survival, the underlying mechanisms for this peculiarity remain largely unknown. Previously published investigations of long-term survivors (LTS) usually incorporate small numbers of patients, non-uniform definition of LTS and rarely provide a comprehensive molecular analysis. With advances in multi-omics technologies and their integration with disease diagnostics, numerous prognostic molecular markers have been proposed. So far, only O6-methylguanine-DNA-methyltransferase (MGMT) gene promotor hypermethylation was linked to outcome and therapy response. We aim to investigate omics characteristics associated with LTS. Molecularly confirmed Isocitrate Dehydrogenase (IDH) wildtype GBM patients, >18 years old, operated and treated at our institution between 2013 and 2020 and living ≥5 years post-diagnosis (LTS = 62) or 1-2 years (STS = 62) were identified. Clinical and demographic characteristics were matched. Tumor tissues was analyzed with targeted next generation sequencing (NGS) and compared between LTS and STS. In addition, methylation analysis was done for all LTS. Results will be presented. We hope to shed a broader light on the molecular drivers behind the LTS in GBM patients, that will provide prognostic markers and potential targets for novel treatments.

QLTI-20. BRIDGING THE GAP: UNDERSTANDING THE CURRENT TREATMENT RESPONSES TO ENHANCE THE STANDARD OF CARE AND OUTCOMES FOR MALIGNANT PERIPHERAL NERVE SHEATH TUMORS

Abstract Malignant peripheral nerve sheath tumor (MPNST) are a rare and aggressive cancer that make up 5-10% of all sarcomas. There is currently no effective targeted therapy to treat MPNST and surgical resection remains the mainstay of treatment, though often not feasible due to location, size of the tumor, or presence of metastases. Evaluation of MPNST treatment response to palliative chemotherapy regimens could serve as a reference point for target response rates in clinical trials going forward. We performed a retrospective electronic health record analysis of patients with biopsy-proven diagnoses of MPNST at Washington University and Johns Hopkins University. We evaluated tumor characteristics along with treatment response, overall survival (OS), and progression free survival (PFS) rates for different chemotherapeutic regimens. The cohort comprises 140 patients, with ages at diagnosis ranging from 21 to 40 years. Most patients had tumors located in the extremities, with sizes ranging from 5-10 cm at the time of diagnosis. We identified 66 patients who received adjuvant chemotherapy, 44 received neoadjuvant chemotherapy and 28 did not receive any chemotherapy. We will present objective response rate (ORR) and clinical benefit rate (CBR) for neoadjuvant chemotherapy. Among patients with metastatic disease (n=53), 12 chemotherapy regimens were assessed. All regimens other than gemcitabine-docetaxel chemotherapy regimen performed better than the median PFS of 1.77 months which has been observed in clinical trials of targeted agents to date with the most compelling response to vincristine-irinotecan-temozolomide regimen. Despite advancements in our understanding of MPNST pathogenesis, these tumors carry a poor prognosis. Our study has identified several systemic therapies which should be further evaluated prospectively to improve treatment options and outcomes for patients with this aggressive sarcoma. These results can serve as a benchmark for future clinical trials.

EXTH-50. THYROID HORMONE-INDUCED DIFFERENTIATION: A PROMISING THERAPEUTIC STRATEGY FOR MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children, consisting of four major subgroups (WNT, SHH, Group 3, and Group 4) that differ in genetic/epigenetic profiles and prognoses. Despite current therapy, a significant proportion of patients still succumb to MB. Moreover, survivors often suffer severe side effects from aggressive treatments. Therefore, improved strategies to treat MB are urgently needed. MB tumor cells retain the capacity for differentiation and can undergo terminal differentiation, after which they lose their proliferative and tumorigenic capacity. This suggests that MB can potentially be treated by inducing tumor cell differentiation. However, the mechanisms underlying MB tumor cell differentiation remain unclear. Our recent studies reveal that thyroid hormone (TH) plays a critical role in promoting tumor cell differentiation in MB. Reduced TH levels free the TH receptor, TRα1, to bind to EZH2 and repress the expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH levels reverse the EZH2-mediated repression of NeuroD1 by disrupting the binding between EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, triiodothyronine (T3, active form of TH) inhibits the growth of both SHH- and Group 3 tumors, suggesting that the tumor-inhibitory effect of T3 is not restricted by MB subgroups. Additionally, this effect is significantly enhanced by concurrent chemotherapy. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising therapeutic modality for MB treatment.

TMOD-14. ESTABLISHING A CLINICALLY RELEVANT MOUSE MODEL TO STUDY NECROSIS AS A PRIMARY VARIABLE IN GLIOBLASTOMA

Abstract All glioblastoma (GBM) molecular subsets share the common trait of accelerated progression following necrosis, which cannot be adequately explained by cellular proliferation arising from accumulated genetic alterations. We suggest that necrosis is much more than a passive phenomenon related to rapid growth but rather is a driving force causing tumor microenvironment (TME) restructuring and biologic progression. yet mechanisms remain poorly understood due to a lack of animal models to study necrosis as a primary variable. In GBM, the most malignant primary brain tumor, vascular pathology and central necrosis precede rapid, radial expansion. To reveal spatio-temporal changes directly following vascular damage in the brain, we developed methodology to induce clinically relevant thrombosis vaso-occlusion within GBMs in immunocompetent RCAS/tv-a mouse model to study TME restructuring by intravital microscopy and demonstrate its impact on glioma progression in real-time. We found that following Rose Bengal photothrombosis, GBMs rapidly expanded radially, with glioma migration away from central necrosis and tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) increased dramatically in the peri-necrotic zones. Through the single cell tracking analysis, we found that TAMs displaying increased displacement toward necrosis and greater overall migration distances. Collectively, this model introduces necrosis as the primary variable and captures glioma growth dynamics and TME restructuring in a manner that will facilitate the development of therapies that antagonize these mechanisms to improve outcomes.

DDDR-25. IBUDILAST AND ANTI-PD-L1 COMBINATION TREATMENT SIGNIFICANTLY IMPROVES SURVIVAL IN A SEX-DEPENDENT MANNER IN A MURINE MODEL OF GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) is the most common primary malignant brain tumor, and there remains a significant need for new treatments. Previous trials studying immune checkpoint inhibitors (ICI) as single agents have been negative. Ibudilast is a brain penetrant inhibitor of the macrophage migration inhibitory factor (MIF)-CD74 interaction that sensitizes GBM to temozolomide in vitro and alters myeloid-derived suppressor cells in vitro and in vivo. The purpose of this study was to investigate whether ibudilast increases ICI efficacy in a murine model of GBM. METHODS 6-week-old male and female C57BL/6 mice were implanted with SB28 cells intracranially. One week after implantation, each group was randomized to receive intraperitoneal treatment with either: ibudilast (50 mg/kg), anti-PD-L1 (1mg/kg), combination, or vehicle control. Mice were euthanized at neurological endpoint. Survival was modeled with Cox proportional hazards regression. RESULTS Among females, there was a significant difference in survival among treatment groups (p=0.0002). Specifically, combination treatment led to significantly longer survival compared to ibudilast (p=0.004) and control (p=0.03), but not anti-PD-L1 (p=0.12). Among males, there was no significant difference in survival among groups (p=0.40). Males had significantly shorter median survival compared to females (overall: 18 versus 22 days, combination treatment: 20 days versus 32 days, anti-PD-L1: 14 versus 22 days, ibudilast: 17 versus 17 days, control: 18 versus 22 days, p=0.02). CONCLUSIONS Ibudilast with anti-PD-L1 is a promising combination treatment against GBM. Future research is needed to identify the mechanisms underlying this treatment regimen and its sex-dependent effect. These results may inform the development of a combination new clinical trial, as ibudilast is currently being evaluated in the newly-diagnosed and recurrent GBM setting with temozolomide.

EPCO-56. INHERITED PREDISPOSITION TO ADULT GLIOMAS: OVERVIEW OF THE CURRENT CLINICAL PRACTICE AND FUTURE RECOMMENDATIONS

Abstract Gliomas are the most common adult primary malignant brain tumor and occur in ages 45-65 years old. While most gliomas are sporadic in nature, a small portion is related to cancer predisposition syndromes (CPS), such as Li Fraumeni or Lynch syndromes. Given that 5–10% of patients will have a family history of glioma, there is currently an effort in identifying germline variants associated with gliomas. Additionally, with the advent of somatic tumor testing, it is possible to pick up suspected germline mutations in genes that cause CPS. Our experience includes a 32-year-old patient diagnosed with astrocytoma IDH mutant, Grade 2 who was found to have a BRAC1 mutation on somatic brain tumor testing. 10 years later, the patient was found with metastatic ovarian cancer and a germline BRAC1 mutation. Early identification of CPS can lead to early detection or prevention of malignancies in affected patients and relatives. However, gliomas are rarely the primary or presenting tumor of CPS, and guidance of CPS screening for these individuals is still lacking. Due to the increasing interest and importance of CPS testing in patients with glioma we have developed a collaborative protocol between our neuro-oncologists and genetic counselors aso that patients have access to germline testing. We propose that all patients with glioma at age of 50 or under, patients with strong family history of cancer including gliomas, and patients with suspected germline mutations based on tumoral somatic mutational profile should be referred to the genetic counselors. Our preliminary findings include 8 patients who completed germline genetic testing. Of these, one patient tested positive for a BRCA1 pathogenic variant. We are planning to continue to collect this data to better inform clinical practice.

TMIC-65. USING BIOINFORMATICS TO INVESTIGATE THE TUMOR MICROENVIRONMENT OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas, H3K27-altered (DMG) are highly malignant brain tumors with a 2-year survival rate of less than 10 percent. These tumors are primarily localized to the pons and thalamus regions of the brain. The cellular compartment of the tumor microenvironment (TME) which surrounds a tumor is composed of tumor and non-tumor cells, such as immune cells and oligodendrocytes. Communication between tumor cells and non-tumor cells in the TME can promote tumor growth and targeting communication networks could improve patient outcomes. Using a bioinformatics approach and publicly available pediatric DMG single-cell RNA-sequence (scRNA-seq) data, several methods of communication (secreted signaling, cell-cell contact, and extracellular matrix) within the DMG TME were identified. Based on the high communication probability, outgoing and incoming networks from non-tumor cells and tumor cells were inferred, such as the Complement, Galectin, and CD96 pathways. Specific ligand-receptor interactions between non-tumor and tumor cells were also found, including LGALS9-HAVCR2, PDGFB/PDGFRA, and COL4A5/ITGAV. A subsequent literature review was performed to validate these identified pathways in other tumors, including gliomas, and inform future in vitro experiments with primary DMG cell lines.

RBIO-08. INVESTIGATING THE EFFICACY AND MECHANISMS OF TUMOR TREATING FIELDS IN RADIATION-RESISTANT GLIOBLASTOMA MODELS

Abstract Glioblastoma (GBM) is an aggressive malignant brain tumor with poor prognosis despite current treatment regimens involving surgical resection, radiation, temozolomide, and the newly approved use of Tumor Treating Fields (TTFields; a noninvasive, transdermal, administration of alternating low-intensity, intermediate-frequency electrical fields). Acquired radiation resistance, thought to be partially driven by brain tumor-initiating cells (BTICs), underscores therapeutic challenges in GBM. While clinical evidence supports the therapeutic benefit of TTFields, its precise mechanisms of action are poorly understood, and TTFields impact on radiation-resistant GBM is unknown. To better understand the effect and mechanism of TTFields in radiation-resistant GBM, we utilized patient-derived xenolines (PDX) and their acquired radio-resistant pairs to assess changes in viability and molecular signaling. PDX were previously developed by subcutaneous implantation of patient GBM cells into immunodeficient (nu/nu) mice. Acquired radiation-resistant PDX were generated through serial in vivo radiation. TTFields were administered with the Novocure inovitroTM device in a cooling incubator to maintain 37oC sample temperature in the ceramic well plates of the device. PDX were cultured on geltrex-coated coverslips under serum-free conditions. Cytotoxicity was quantified 72h post TTFields with crystal violet. Molecular signaling was captured with PamChip peptide-based kinase activity arrays and RayBiotech proteomic arrays. We found that TTFields inhibit cell growth in both parental and acquired radiation-resistant PDX. Differences in molecular signatures between the PDX lines suggest molecular mechanisms of TTFields sensitivity signatures and interactions with acquired radio-resistant PDX. We are currently trying to generate TTFields-resistant PDX lines through serial treatment with plans to examine BTIC populations relative to TTFields efficacy. We also plan to explore targetable TTFields-induced alterations in the future.

CNSC-54. CENTRAL AND BOUNDARY-DRIVEN GROWTH PATTERNS DOMINATE RESPECTIVELY IDH WILD-TYPE AND MUTANT GLIOMAS

Abstract Diffuse gliomas are the most prevalent primary malignant brain tumors in adults and are characterized by invasive growth and poor prognosis. Tumor cells infiltrate the brain parenchyma and spread to distant sites. The aim of this study is to characterize the spatial heterogeneity, mode, and direction of tumor development to identify key areas for localized treatment. Neuronavigation was employed to collect n=134 image-guided samples from n=16 adult patients with diffuse gliomas, including n=7 IDH wild-type (IDHwt) and n=9 IDH mutant (IDHmut) tumors from regions with and without MRI abnormalities. Collected samples were subjected to targeted and shallow whole-genome sequencing. Somatic variants were used to determine the evolutionary processes governing heterogeneity. Examination of the dN/dS ratio, Tajima’s D score, and MOBSTER model suggested that all tumors are consistent with neutral evolution, as previously reported. Considering that genetic heterogeneity was a result of stochastic mutations, we used phylogeographic relationships between samples to dissect the spatiotemporal development of these tumors. Evolutionary (patristic) and cartesian (Euclidean) distances between sample pairs from the same patient were correlated in n=2/9 IDHmut and n=2/7 IDHwt patients, suggesting that migratory or punctuated evolutionary processes play a major role in tumor diffusion. An examination of spatial diffusion revealed that the latest descendant appeared peripherally in n=5/9 IDHmut patients, suggesting boundary-driven growth. In the remaining cases, three patients exhibited centrally-driven growth, whereas a fourth patient displayed a mixed pattern. In contrast, in n=4/7 IDHwt patients, descendants appeared closer to the tumor center, suggesting centrally-driven growth. In the remaining three IDHwt patients, one patient showed a diffusion that was consistent with boundary-driven growth, while the other two patients showed a mixed pattern. Taken together our data suggest that IDHwt tumors tend to develop centrally, whereas IDHmut tumors preferably develop outward. Our data could aid in tailoring localized treatment based on projected growth patterns.

CNSC-67. ANATOMICAL ORGANIZATION AND ELECTRICAL PROPERTIES OF GLIOMA-INNERVATING NEURONS

Abstract Gliomas are the most common malignant primary brain tumors and are often associated with severe neurological deficits and mortality. Unlike many cancers, gliomas rarely metastasize outside the brain, indicating a possible dependency on the brain’s unique microenvironment. Recent discoveries have highlighted the existence of neuron-glioma synapses, suggesting that glioma cells depend on neuronal inputs and AMPA-receptor-mediated glutamate signaling for their proliferation. Yet, the specific locations and properties of the neurons that innervate gliomas remain elusive. In this study, we utilized transsynaptic tracing with a pseudotyped, glycoprotein-deleted rabies virus to specifically infect TVA and glycoprotein-expressing human glioblastoma cells in an orthotopic xenograft model. This approach allowed us to identify the presynaptic neuronal inputs to the glioma. Comprehensive whole-brain mapping and quantification revealed that these glioma-innervating neurons, predominantly glutamatergic, exhibit a consistent input pattern influenced by the tumor’s locations and engage selective neuromodulatory pathways. Notably, the electrophysiological properties of these neurons mirror those innervating the cortex in vivo. Our study introduces a novel method for investigating glioma-innervating neurons and reveals that these neurons are consistently integrated into the glioma network, displaying distinctive location-dependent neuromodulatory patterns. Our findings offer a promising scaffold for future interventions aimed at disrupting these critical neuronal inputs, potentially redefining therapeutic strategies against brain tumors.

DDDR-05. SPATIALLY RESOLVED DRUG SENSITIVITY PROFILING IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is the most common malignant primary brain tumor in adults, characterized by poor overall survival and an urgent need for more effective treatment strategies. Macroscopic heterogeneity, as observed through MR imaging, divides the tumor into distinct regions: a tumor-cell dense contrast-enhancing tumor core and a non-contrast-enhancing infiltration zone that appears hyperintense on T2/FLAIR imaging. The infiltration zone contains both infiltrating tumor cells and non-malignant cells from the local brain microenvironment. Although genomic and transcriptomic differences between the tumor core and the infiltration zone have been reported, functional differences, such as differential drug sensitivities, remain largely unknown. METHODS To investigate this, we collected region-specific glioblastoma patient tissue samples from both the contrast-enhancing tumor core and the non-contrast-enhancing but 5-aminolevulinic acid (5-ALA) fluorescent infiltration zone during surgery. Using pharmacoscopy, a microscopy-based single-cell drug screening platform, we assessed drug responses across regions and patients. RESULTS To determine whether drug responses within a single tumor region were more similar compared to responses across different regions we collected multiple tissue samples from each tumor region in seven patients. We identified region-specific drug responses for temozolomide and lomustine, among other drugs, in some of the patients. However, drug sensitivities were not consistent within the same region across different patients. Thus, across patients, the region-specific drug responses were obscured by patient heterogeneity. To address intra- and inter-tumor drug response heterogeneity, we developed a complementarity score based on region-specific drug response data of 60 drugs across 23 paired samples. The complementarity score identified promising drug combinations that demonstrated strong anti-tumor activity across tumor regions and patients. CONCLUSIONS Monotherapy approaches have largely failed in glioblastoma, despite extensive molecular profiling efforts aimed at identifying tumor-specific vulnerabilities. Identifying drug combinations that synergistically target the locoregional heterogeneity of glioblastoma may open up new avenues of personalized therapy.

TMIC-87. ICAM-1 REGULATE EXTRACELLULAR VESICLE-MEDIATED IMMUNOSUPPRESSIVE MONOCYTE IN HUMAN GLIOBLASTOMA CELL

Abstract Glioblastoma is the most common malignant primary brain tumor and is universally fatal. Treatment involves maximal safe resection with adjuvant chemoradiation. Poor prognosis in glioblastoma is multifactorial, and local and systemic immunosuppression is an important factor. Extracellular vesicles (EVs) are small, membrane-bound particles released by cells into the external environment. They play a key role in intercellular communication. In glioblastomas, EVs regulate immunosuppression through induction of myeloid-derived suppressor cells (MDSCs) in a process dependent on the PD-1/PD-L1 and IL-6 axes. Primary cilia are microtubule-based organelles that play crucial roles in various cellular processes and signal transduction cascades. We recently showed that primary cilia modulate IL-6 expression and consequently, MDSC induction. Comparative proteomic analysis of EVs derived from human glioblastoma cells devoid of cilia showed loss of ICAM-1 expression compared to control glioblastoma cells. ICAM-1 (Intercellular Adhesion Molecule-1) is a cell surface glycoprotein that mediates cell-cell adhesion and immune responses. In this study, we showed that depletion of primary cilia in human glioblastoma cells result in increased PD-L1 but loss of IL-6 and ICAM-1. RNAi-meidated loss of ICAM-1 was synergistic with primary cilia depletion in preventing MDSC inductionn normal human monocytes exposed to glioblastoma-derived EVs. Exogenous IFN-g was sufficient to restore IL-6 levels and MDSC induction despite loss of primary cilia and ICAM-1 suggesting that primary cilia act upstream of IFN-g, IL-6 and ICAM-1 in glioblastoma-mediated MDSC induction. This study is the first to connect primary cilia signal transduction to IL-6 and ICAM-1 expression and to MDSC induction. The evidence suggests a potential parallel primary cilia regulatory cascade independent of the PD-L1 axis that controls tumor-mediated immunosuppression. These results may inform novel therapeutic strategies aimed at increasing efficacy of immunotherapy in the glioblastoma and other neoplastic processes.

DDDR-38. TRANSLATIONAL STEPS OF THE ANTITUMOR PEPTIDE TAT-CX43266-283 AS A TREATMENT FOR GLIOBLASTOMA: EGFR AS A TREATMENT RESPONSE BIOMARKER AND RESULTS OF COMBINATION THERAPY IN A PRECLINICAL RESECTION MODEL

Abstract Glioblastomas (GBM) are the most malignant primary brain tumors, and they remain incurable. Despite the current standard of care (surgical resection of the tumor and administration of chemotherapy and radiotherapy) some GBM stem cells (GSCs) remain in the brain parenchyma causing tumor recurrence. The Src-inhibitory peptide TAT-Cx43266-283 has shown promising antitumor results in GBM preclinical models, reducing GSC viability and increasing survival in GBM-bearing mice. Here we have investigated some of the main gaps to overcome when translating preclinical results to the clinic, to promote the progress of TAT-Cx43266-283 as a clinical therapy to fight GBM. First, we investigated biomarkers of treatment response to design a more targeted therapy. We assessed cell viability after treatment with TAT-Cx43266-283 in 13 patient-derived GSC lines and we found a stronger treatment response in those GSCs that had alterations in the Epidermal Growth Factor Receptor (EGFR). This was further confirmed in several murine GBM lines. Importantly, we found that treatment with TAT-Cx43266-283 was more effective in vitro than temozolomide or the classical EGFR inhibitor, erlotinib. Moreover, we showed that EGFR participates in TAT-Cx43266-283 response together with Src, by decreasing EGFR and EGFRvIII activity in some GSCs and increasing survival in mice bearing tumors with EGFR alterations. Next, we studied the effect of TAT-Cx43266-283 in a clinical context by administering it in a murine model of tumor resection. We analyzed the histopathology of this GBM model, uncovering histological features typical of human GBM. We found that resection alone improved, although not significantly, GBM-bearing mice survival. However, tumors that regrew after resection exhibited more aggressive features. Importantly, the combination of tumor resection and TAT-Cx43266-283 achieved better survival outcomes and showed reduced invasive features. Altogether, these results serve as a base for future clinical investigations and support the therapeutic potential of TAT-Cx43266-283 as a treatment for GBM.

TMIC-34. INFILTRATIVE BEHAVIOR OF PATIENT-DERIVED GLIOBLASTOMA CELL LINES IN REGION-SPECIFIC HUMAN BRAIN ORGANOIDS

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults. GBMs are known for their highly invasive and proliferative properties and are associated with a dismal prognosis (~12-18 months post-diagnosis). Neuroanatomical disparities in the occurrence of GBMs suggest that in addition to microenvironmental impacts, tumor cells are impacted by the cellular and molecular diversity across varying regions of this nervous system. This idea has been difficult to study due to the lack of human-specific models that effectively recapitulate the biological behaviors of GBMs in region-specific microenvironments. To address these challenges, we engrafted GFP-tagged patient-derived glioma cell lines into human induced pluripotent stem cell (hiPSC)-derived organoids patterned to mimic the most forebrain (dorsal cortex) and hindbrain (spinal cord) regions of the nervous system. Immunostaining assays at 2, 7, 14, and 21 days post-engraftment compared the extent of infiltration, as measured by the distance traveled by glioma cells, in both regions. We found that glioma cells infiltrated a significantly greater distance in the dorsal cortical organoids compared to the spinal cord organoids. This suggests a more conducive environment for glioma infiltration in the forebrain vs. hindbrain at baseline. We are currently determining if these regional biases in infiltration capacity are modified with either optogenetic stimulation of the host brain organoids or in the presence of interregional interactions in an assembloid system. Thus, using this fully human-specific platform, we aim to gain insights into the infiltrative behaviors of GBM cells, which will advance our understanding of this devastating disease.

QOL-31. RESULTS OF A TELEHEALTH-BASED PSYCHOLOGICAL INTERVENTION FOR CAREGIVERS OF PATIENTS WITH PRIMARY MALIGNANT BRAIN TUMORS: A MODERATION ANALYSIS OF A RANDOMIZED CONTROLLED TRIAL

Abstract BACKGROUND In a recent randomized controlled trial, caregivers of patients with primary malignant brain tumors (PMBT) who received NeuroCARE—a six-session, telehealth-based psychological intervention—demonstrated improved anxiety and depression symptoms, coping, and self-efficacy at 11-weeks compared to usual care (UC) participants. We now explore how sociodemographic variables, disease-specific factors, and baseline mood symptoms moderate intervention effects on anxiety and depression symptoms. METHODS We examined data from 120 enrolled caregivers (Mage=53 years; 83% female, 92% White, 70% spouses/partners) of PMBT patients (84% glioblastoma). Eligible caregivers had elevated anxiety (Generalized Anxiety Disorder-7 ≥ 5). Participants were randomized 1:1 to NeuroCARE or UC and completed baseline sociodemographic questionnaires and the Hospital Anxiety and Depression Scale at baseline and 11-weeks. We used linear regression to evaluate whether selected sociodemographic characteristics (gender, age, relationship to patient), tumor characteristics (tumor location/laterality, IDH mutation status), and baseline mood symptoms moderated intervention effects on anxiety and depression symptoms. We report the group-by-moderator (b) effects, using a 0.20 significance level. RESULTS The beneficial effects of NeuroCARE on reducing 11-week anxiety symptoms were greater in women compared to men (b=-2.63, CI: -6.17, 0.91, p=0.14) and greater in caregivers who reported higher baseline anxiety symptoms (b=-0.43, CI: -0.78, -0.08, p=0.02) and depression symptoms (b=-0.56, CI: -0.96, -0.17, p=0.01). The beneficial effects of NeuroCARE on reducing 11-week depression symptoms were greater in caregivers of patients with IDH-wildtype tumors compared to caregivers of patients with IDH-mutant tumors (b=2.49, CI: -0.26, 5.24, p=0.08). Caregiver age, relationship to patient, and tumor location/laterality did not significantly moderate NeuroCARE effects on mood. CONCLUSIONS The positive effects of NeuroCARE were particularly salient for caregivers with elevated anxiety and/or depression symptoms and those caring for patients with IDH-wildtype tumors. Content may be further tailored to account for the unique needs of men and caregivers of patients with IDH-mutant tumors.

INNV-06. ADULT GLIOBLASTOMA WITH MISMATCH REPAIR DEFICIENCY FORMS A DISTINCT HIGH-RISK MOLECULAR SUBGROUP FOR WHICH TREATMENT WITH IPILIMUMAB-NIVOLUMAB SHOWS SURVIVAL ADVANTAGE

Abstract Glioblastoma is the most frequent and malignant primary brain tumor. Although the survival is generally dismal for glioblastoma patients, risk stratification and the identification of high-risk subgroups is important for prompt and aggressive management. The G1-G7 molecular subgroup classification based on the MAPK pathway activation has offered for the first time a non-redundant, all-inclusive classification of adult glioblastoma. Five patients from the large, 220-patient prospective cohort showed germline mutations in mismatch repair (MMR) genes and significantly worse median survival (3.25 months post-surgery) than those from other subgroups or from the rest of the cohort adjusted for age. These tumors were assigned to a new subgroup, G3/MMR, a spin-off from the major G3/NF1 subgroup, as they generally show genomic alterations leading to RAS activation. An integrated clinical, histological, immunohistochemical and molecular analysis of the G3/MMR tumors showed distinct characteristics as compared to other glioblastomas, including those with iatrogenic high tumor mutation burden (TMB), warranting a separate subgroup. Among these, prior history of cancer, midline location or multifocality, presence of multinucleated giant cells, positive p53 and MMR immunohistochemistry, and specific molecular characteristics, including high TMB, alert to this high-risk G3/MMR subgroup. High TMB constitutes the criterium for treatment with immune checkpoint inhibitors in solid cancers. The FDA-approved first-line therapy for advanced non-small cell lung cancer and high-TMB colorectal cancer is with the dual ipilimumab-nivolumab regimen. One of the five G3/MMR subgroup patients received the dual ipilimumab-nivolumab regimen and survived much longer than the rest of the G3/MMR patients, setting a proof-of-principle example for the treatment of these very aggressive G3/MMR glioblastomas.

STEM-18. A HIGH THROUGHPUT NEUROSPHERE BASED COLONY FORMATION ASSAY TO VALIDATE DRUG AND RADIATION EFFECTIVENESS IN PATIENT DERIVED XENOLINES

Abstract Glioblastoma (GBM) is a primary malignant brain tumor developed from normal astrocytes. GBM patients’ median survival is less than 2 years despite safe surgical resection, fractionated conventional radiation, and temozolomide (TMZ) therapy. Few currently used drugs are successful in GBM although there are many FDA approved blood-brain barrier (BBB) penetrant drugs, for other uses that have not been screened in GBM. We utilized a panel of 734 of these BBB penetrant drugs in different GBM patient derived xenolines (PDX) along with acquired radio-resistant derivatives (-RT) including X1441, JX14P, JX14P-RT, JX39P, JX39P-RT and normal human astrocytes (NHA) as control and screened for viability with Cell Titer-Glo. We identified multiple candidate drugs that preferentially killed GBM PDX cells at 10 µM relative to NHA control. As we used stem cell media (serum free) in our drug screening study, it indicates that the potent drugs are also effective in killing cancer stem cells or brain tumor initiating cells. These drugs had targets ranging from PDGFR, DNA/RNA synthesis, as well as other kinase inhibitors. We developed a high throughput (HT), neurosphere based colony formation assay (CFA) in 96-well plate to screen multiple drugs and doses in combination with radiation. For visualizing the colonies, we use a Cytation 5 imager at 4X resolution and count the colonies based on parameters including object size, width, intensity and others. We plan on validating our drug screen and HT-CFA assay with high throughput PamChip kinase activity assays and with in vivo orthotopic tumor PDX models.

EPCO-45. COMPLETE CHROMOSOME-SCALE DIPLOID-PHASED ASSEMBLY OF A NORMAL HUMAN ASTROCYTE CELL LINE

Abstract Genomic studies of the malignant brain tumor glioma invariably rely on aligning sequencing reads to a reference genome such as HG38, even though no single reference can capture the diversity of an individual patient or cell line. To begin addressing these biases in genomics research, we sought to construct a chromosome-scale diploid-phased assembly of a normal human astrocyte (NHA) cell line that is frequently used as a basis for developing isogenic glioma cell models. To achieve this, we generated 45x Pacific Biosciences HiFi data (N50: 16kb), 26x Oxford Nanopore Technologies ultra-long data (N50: 80kb), and 2 billion paired-end reads of Hi-C data. The assembler verkko2 was used to integrate these data and build a phased diploid assembly. We achieved an assembly size of 6.01 Gbp, with 17 gapless chromosomes. The two haplotypes contain 214 and 182 contigs, respectively. Our assembly includes 76 fully assembled telomeres and 20 fully assembled centromeres. The contiguity of our assembly is 130 Mbp, which vastly exceeds that of HG38 (N50: 68 Mbp) and approaches that of the recent telomere-to-telomere CHM13 assembly (N50: 155 Mbp). We ran BUSCO on each haplotype of the assembly using the metazoa database to quantify the single-copy genes that are expected to be present in every animal genome. The haplotypes of our assembly contain the complete sequence of 98.6% and 99.9% of these genes respectively. Finally, we used Merqury to perform a k-mer based evaluation of the two haplotypes and found quality values of 59.09 and 59.75 respectively. This diploid genome assembly of NHA will serve as a resource for researchers that wish to develop NHA-derived glioma models. It can be used to call indels or structural variants in a haplotype-specific manner which will further elucidate genotypes underlying glioma phenotypes. Most importantly, it sets the stage for personalized genome assembly and tackling the lack of diversity in genomic studies of glioma to date.

INNV-01. METASTATIC GLIOBLASTOMA TO THE LUNG TREATED WITH LENVATINIB

Abstract Glioblastoma is the most common malignant brain tumor. Despite its invasive nature, extra-cranial glioblastoma metastases are rare. We present a case of a 63 year-old woman with metastatic glioblastoma to the lung. Sarcomatous histology, a reported risk factor for disseminated disease, was found. Genomic alterations of TP53 mutation, TERT mutation, PTEN mutation, and +7/-10 were also uncovered. Early evidence suggests these molecular aberrations are common in metastatic glioblastoma. Treatment with third-line lenvatinib resulted in a mixed response. This case contributes to the growing body of evidence for the role of genomic alterations for predictive risk in metastatic glioblastoma. There remains an unmet need for treatment of metastatic glioblastoma.

DNAR-15. GROUP 3 MEDULLOBLASTOMAS EXPLOIT A TRANSIENT MECHANISM OF TELOMERE REPAIR MEDIATED BY ZSCAN4 WHICH IS TARGETABLE FOR THERAPEUTIC BENEFIT WITH BRAIN-PENETRANT DRUGS

Abstract All cancers need some mechanism of telomere repair. However, it is unknown how Group 3 (G3) medulloblastomas (MB) repair their telomeres, even though MB is the most common malignant brain tumor in kids and G3 is its most aggressive subtype. With rare exceptions, G3 MBs lack telomerase gain-of-function alterations or evidence of constitutive alternative-lengthening-of-telomeres pathway activation. We profiled tumors from N=38 MB patients from UCSF via single-nucleus RNA sequencing; 13 cases were also subjected to single-cell assay for transposase-accessible chromatin by sequencing, and 4 were subjected to single-cell cleavage under targets and tagmentation for H3K27ac. These data identified a network of gene enhancers that were specifically active in rhombic-lip progenitor-like MB cells, the putative G3 MB cell of origin. This program was driven by zinc finger and SCAN domain containing 4 (ZSCAN4), a gene reported to drive transient telomere repair during zygotic genome activation in early embryos. CRISPR inhibition of ZSCAN4 led to significant telomere shortening, telomeric DNA damage, telomere fusions, decreased proliferation, and increased survival of orthotopic xenografts. Conversely, ZSCAN4 over-expression induced significant telomere elongation, increased proliferation, and decreased survival in vivo. Analysis of endogenous-reporter cells indicated that ZSCAN4 is transiently expressed in response to telomere shortening and increases global histone acetylation by inducing chromatin-remodeling factors. Rebastinib targeted ZSCAN4, histone acetylation, and telomere repair, was brain penetrant, and significantly enhanced orthotopic-xenograft survival with no toxicity. Acetyltransferase inhibitor CPI-1612 was brain penetrant and extended xenograft survival with no toxicity. Our data support the hypothesis that G3 MBs exploit a transient mechanism of telomere repair mediated by ZSCAN4. Targeting ZSCAN4 or downstream histone acetylation is therapeutically viable. Pan-cancer whole-genome sequencing data showed that MBs have lower telomeric content than most other cancers. Thus, MBs may be particularly vulnerable to therapeutic strategies that inhibit telomere repair.