Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.

The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its ∼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.

Divergent transcriptomic signatures from putative mesenchymal stimuli in glioblastoma cells.

In glioblastoma, a mesenchymal phenotype is associated with especially poor patient outcomes. Various glioblastoma microenvironmental factors and therapeutic interventions are purported drivers of the mesenchymal transition, but the degree to which these cues promote the same mesenchymal transitions and the uniformity of those transitions, as defined by molecular subtyping systems, is unknown. Here, we investigate this question by analyzing publicly available patient data, surveying commonly measured transcripts for mesenchymal transitions in glioma-initiating cells (GIC), and performing next-generation RNA sequencing of GICs. Analysis of patient tumor data reveals that TGFß, TNFα, and hypoxia signaling correlate with the mesenchymal subtype more than the proneural subtype. In cultured GICs, the microenvironment-relevant growth factors TGFß and TNFα and the chemotherapeutic temozolomide promote expression of commonly measured mesenchymal transcripts. However, next-generation RNA sequencing reveals that growth factors and temozolomide broadly promote expression of both mesenchymal and proneural transcripts, in some cases with equal frequency. These results suggest that glioblastoma mesenchymal transitions do not occur as distinctly as in epithelial-derived cancers, at least as determined using common subtyping ontologies and measuring response to growth factors or chemotherapeutics. Further understanding of these issues may identify improved methods for pharmacologically targeting the mesenchymal phenotype in glioblastoma.

Phase I study targeting newly diagnosed grade 4 astrocytoma with bispecific antibody armed T cells (EGFR BATs) in combination with radiation and temozolomide.

PURPOSE: The purpose of this study was to determine the safety, feasibility, and immunologic responses of treating grade 4 astrocytomas with multiple infusions of anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) armed T cells (EGFR BATs) in combination with radiation and chemotherapy. METHODS: This phase I study used a 3 + 3 dose escalation design to test the safety and feasibility of intravenously infused EGFR BATs in combination with radiation and temozolomide (TMZ) in patients with newly diagnosed grade 4 astrocytomas (AG4). After finding the feasible dose, an expansion cohort with unmethylated O6-methylguanine-DNA methyltransferase (MGMT) tumors received weekly EGFR BATs without TMZ. RESULTS: The highest feasible dose was 80 × 109 EGFR BATs without dose-limiting toxicities (DLTs) in seven patients. We could not escalate the dose because of the limited T-cell expansion. There were no DLTs in the additional cohort of three patients with unmethylated MGMT tumors who received eight weekly infusions of EGFR BATs without TMZ. EGFR BATs infusions induced increases in glioma specific anti-tumor cytotoxicity by peripheral blood mononuclear cells (p < 0.03) and NK cell activity (p < 0.002) ex vivo, and increased serum concentrations of IFN-γ (p < 0.03), IL-2 (p < 0.007), and GM-CSF (p < 0.009). CONCLUSION: Targeting AG4 with EGFR BATs at the maximum feasible dose of 80 × 109, with or without TMZ was safe and induced significant anti-tumor-specific immune responses. These results support further clinical trials to examine the efficacy of this adoptive cell therapy in patients with MGMT-unmethylated GBM. CLINICALTRIALS: gov Identifier: NCT03344250.

Understanding current experimental models of glioblastoma-brain microenvironment interactions.

Glioblastoma (GBM) is a common and devastating primary brain tumor, with median survival of 16-18 months after diagnosis in the setting of substantial resistance to standard-of-care and inevitable tumor recurrence. Recent work has implicated the brain microenvironment as being critical for GBM proliferation, invasion, and resistance to treatment. GBM does not operate in isolation, with neurons, astrocytes, and multiple immune populations being implicated in GBM tumor progression and invasiveness. The goal of this review article is to provide an overview of the available in vitro, ex vivo, and in vivo experimental models for assessing GBM-brain interactions, as well as discuss each model's relative strengths and limitations. Current in vitro models discussed will include 2D and 3D co-culture platforms with various cells of the brain microenvironment, as well as spheroids, whole organoids, and models of fluid dynamics, such as interstitial flow. An overview of in vitro and ex vivo organotypic GBM brain slices is also provided. Finally, we conclude with a discussion of the various in vivo rodent models of GBM, including xenografts, syngeneic grafts, and genetically-engineered models of GBM.

The semaphorin 3A/neuropilin-1 pathway promotes clonogenic growth of glioblastoma via activation of TGF-ß signaling.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence; thus, understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the CNS, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF-ß receptor 1 (TGF-ßR1), which in turn leads to activation of canonical TGF-ß signaling in both GSCs and NPCs. TGF-ß signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A/NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF-ß pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A/NRP1/TGF-ßR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

Tissue factor is a critical regulator of radiation therapy-induced glioblastoma remodeling.

Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated ß-galactosidase (SA-ßGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-ßGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse in vivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.

Dopamine receptor D2 regulates glioblastoma survival and death through MET and death receptor 4/5.

Recent studies indicate that signaling molecules traditionally associated with central nervous system function play critical roles in cancer. Dopamine receptor signaling is implicated in various cancers including glioblastoma (GBM) and it is a recognized therapeutic target, as evidenced by recent clinical trials with a selective dopamine receptor D2 (DRD2) inhibitor ONC201. Understanding the molecular mechanism(s) of the dopamine receptor signaling will be critical for development of potent therapeutic options. Using the human GBM patient-derived tumors treated with dopamine receptor agonists and antagonists, we identified the proteins that interact with DRD2. DRD2 signaling promotes glioblastoma (GBM) stem-like cells and GBM growth by activating MET. In contrast, pharmacological inhibition of DRD2 induces DRD2-TRAIL receptor interaction and subsequent cell death. Thus, our findings demonstrate a molecular circuitry of oncogenic DRD2 signaling in which MET and TRAIL receptors, critical factors for tumor cell survival and cell death, respectively, govern GBM survival and death. Finally, tumor-derived dopamine and expression of dopamine biosynthesis enzymes in a subset of GBM may guide patient stratification for DRD2 targeting therapy.

A multi-institutional phase II trial of bevacizumab for recurrent and refractory meningioma.

Background: Systemic therapies for refractory meningiomas are limited with no FDA-approved therapeutics. Vascular endothelial growth factor (VEGF) is a signaling protein associated with neovascularization, peritumoral edema, and meningioma tumorigenesis. Methods: This phase II study investigates the efficacy of bevacizumab (BEV), a VEGF binding monoclonal antibody, in patients with progressive Grade I (G1M), Grade II (G2M), Grade III (G3M) meningioma, and other non-parenchymal tumors including vestibular schwannoma (n = 4) and hemangiopericytoma (n = 4) with the primary endpoint of progression-free survival rate at 6-months (PFS-6). Non-meningiomas were included with the respective meningioma grade in the analysis. Secondary endpoints include median overall survival (mOS) and response rate. Results: Fifty Patients (26 women; median age 54 years; range 23-81), 42 with progressive meningioma were treated: 10 G1M, 20 G2M, and 12 G3M. Prior treatments include surgical resection (41 patients), radiosurgery (24 patients), external beam radiotherapy (28 patients), and chemotherapy (14 patients). Median infusions administered were 16 (range, 2-68). Response was graded using the Macdonald's criteria. PFS-6, median PFS, and mOS were 87%, 22 months, 35 months for G1M; 77%, 23 months, 41 months for G2M; and 46%, 8 months, 12 months for G3M. Best radiographic responses include stable disease (G1M: 100%; G2M: 85%; G3M: 82%); partial response (G1M: 0%; G2M: 5%; G3M: 0%) and progressive disease (G1M: 0%; G2M: 10%; G3M:18%). The most common toxicities were hypertension (n = 19, 42.2%), proteinuria (n = 16, 35.6%), and fatigue (n = 14, 31.1%). Conclusion: This study showed BEV is well tolerated and appears to be a promising systemic treatment option for patients with recurrent and refractory meningiomas.

The landscape of brain tumor mimics in neuro-oncology practice.

BACKGROUND AND OBJECTIVE: Differentiating neoplastic and non-neoplastic brain lesions is essential to make management recommendations and convey prognosis, but the distinction between brain tumors and their mimics in practice may prove challenging. The aim of this study is to provide the incidence of brain tumor mimics in the neuro-oncology setting and describe this patient subset. METHODS: Retrospective study of adult patients referred to the Division of Neuro-oncology for a presumed diagnosis of brain tumor from January 1, 2005 through December 31, 2017, who later satisfied the diagnosis of a non-neoplastic entity based on neuroimaging, clinical course, and/or histopathology evaluation. We classified tumor mimic entities according to clinical, radiologic, and laboratory characteristics that correlated with the diagnosis. RESULTS: The incidence of brain tumor mimics was 3.4% (132/3897). The etiologies of the non-neoplastic entities were vascular (35%), inflammatory non-demyelinating (26%), demyelinating (15%), cysts (10%), infectious (9%), and miscellaneous (5%). In our study, 38% of patients underwent biopsy to determine diagnosis, but in 26%, the biopsy was inconclusive. DISCUSSION: Brain tumor mimics represent a small but important subset of the neuro-oncology referrals. Vascular, inflammatory, and demyelinating etiologies represent two-thirds of cases. Recognizing the clinical, radiologic and laboratory characteristics of such entities may improve resource utilization and prevent unnecessary as well as potentially harmful diagnostic and therapeutic interventions.

Delivering Glioblastoma a Kick-DGKα Inhibition as a Promising Therapeutic Strategy for GBM.

Diacylglycerol kinase α (DGKα) inhibition may be particularly relevant for the treatment of glioblastoma (GBM), a relatively common brain malignancy incurable with current therapies. Prior reports have shown that DGKα inhibition has multiple direct activities against GBM cells, including suppressing the oncogenic pathways mTOR and HIF-1α. It also inhibits pathways associated with the normally treatment-resistant mesenchymal phenotype, yielding preferential activity against mesenchymal GBM; this suggests possible utility in combining DGKα inhibition with radiation and other therapies for which the mesenchymal phenotype promotes resistance. The potential for DGKα inhibition to block or reverse T cell anergy also suggests the potential of DGKα inhibition to boost immunotherapy against GBM, which is generally considered an immunologically "cold" tumor. A recent report indicates that DGKα deficiency increases responsiveness of macrophages, indicating that DGKα inhibition could also have the potential to boost macrophage and microglia activity against GBM-which could be a particularly promising approach given the heavy infiltration of these cells in GBM. DGKα inhibition may therefore offer a promising multi-pronged attack on GBM, with multiple direct anti-GBM activities and also the ability to boost both adaptive and innate immune responses against GBM. However, both the direct and indirect benefits of DGKα inhibition for GBM will likely require combinations with other therapies to achieve meaningful efficacy. Furthermore, GBM offers other challenges for the application of DGKα inhibitors, including decreased accessibility from the blood-brain barrier (BBB). The ideal DGKα inhibitor for GBM will combine potency, specificity, and BBB penetrability. No existing inhibitor is known to meet all these criteria, but the strong potential of DGKα inhibition against this lethal brain cancer should help drive development and testing of agents to bring this promising strategy to the clinic for patients with GBM.

Identification of ritanserin analogs that display DGK isoform specificity.

The diacylglycerol kinase (DGK) family of lipid enzymes catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Both DAG and PA are lipid signaling molecules that are of notable importance in regulating cell processes such as proliferation, apoptosis, and migration. There are ten mammalian DGK enzymes that appear to have distinct biological functions. DGKα has emerged as a promising therapeutic target in numerous cancers including glioblastoma (GBM) and melanoma as treatment with small molecule DGKα inhibitors results in reduced tumor sizes and prolonged survival. Importantly, DGKα has also been identified as an immune checkpoint due to its promotion of T cell anergy, and its inhibition has been shown to improve T cell activation. There are few small molecule DGKα inhibitors currently available, and the application of existing compounds to clinical settings is hindered by species-dependent variability in potency, as well as concerns regarding isotype specificity particularly amongst other type I DGKs. In order to resolve these issues, we have screened a library of compounds structurally analogous to the DGKα inhibitor, ritanserin, in an effort to identify more potent and specific alternatives. We identified two compounds that more potently and selectively inhibit DGKα, one of which (JNJ-3790339) demonstrates similar cytotoxicity in GBM and melanoma cells as ritanserin. Consistent with its inhibitor profile towards DGKα, JNJ-3790339 also demonstrated improved activation of T cells compared with ritanserin. Together our data support efforts to identify DGK isoform-selective inhibitors as a mechanism to produce pharmacologically relevant cancer therapies.

Loss of Diacylglycerol Kinase α Enhances Macrophage Responsiveness.

The diacylglycerol kinases (DGKs) are a family of enzymes responsible for the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). In addition to their primary function in lipid metabolism, DGKs have recently been identified as potential therapeutic targets in multiple cancers, including glioblastoma (GBM) and melanoma. Aside from its tumorigenic properties, DGKα is also a known promoter of T-cell anergy, supporting a role as a recently-recognized T cell checkpoint. In fact, the only significant phenotype previously observed in Dgka knockout (KO) mice is the enhancement of T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to diverse stimuli that yield different phenotypes, including LPS, IL-4, and the chemoattractant MCP-1. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also demonstrated increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains. Taken together, these findings identify a novel immune-regulatory checkpoint function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings.

Technical choices significantly alter the adaptive immune response against immunocompetent murine gliomas in a model-dependent manner.

PURPOSE: Due to the recent rise in immunotherapy research to treat glioblastoma (GBM), immunocompetent mouse models have become increasingly crucial. However, the character and kinetics of the immune response against the most prevalent immunocompetent GBM models, GL261 and CT2A, have not been well studied, nor has the impact of commonly-used marker proteins and foreign antigens. METHODS: In this study, we compared the immune response in these models using flow cytometry and immunohistochemistry as well as investigated several factors that influence the immune response, including kinetics, tumor size, and expression of commonly-used marker proteins and foreign antigens. We hypothesize that these factors influence the immune response enough to warrant consideration when studying new immunotherapeutic approaches for GBM. RESULTS: CT2A-Luc, but not GL261-Luc2, drastically increased the number of T cells in the brain compared with wild-type controls, and significantly altered CT2A's responsiveness to anti-PD-1 antibody therapy. Additionally, a larger cell inoculum size in the GL261 model increased the T cell response's magnitude at day 28 post-injection. CT2A and GL261 models both stimulate a peak T cell immune response at day 21 post-injection. CONCLUSIONS: Our results suggest that the impact of foreign proteins like luciferase on the intracranial immune response is dependent upon the model, with CT2A being more sensitive to added markers. In particular, luciferase expression in CT2A could lead to meaningful misinterpretations of results from immune checkpoint inhibitor (ICI) studies.

Data-Driven Computational Modeling Identifies Determinants of Glioblastoma Response to SHP2 Inhibition.

Oncogenic protein tyrosine phosphatases have long been viewed as drug targets of interest, and recently developed allosteric inhibitors of SH2 domain-containing phosphatase-2 (SHP2) have entered clinical trials. However, the ability of phosphatases to regulate many targets directly or indirectly and to both promote and antagonize oncogenic signaling may make the efficacy of phosphatase inhibition challenging to predict. Here we explore the consequences of antagonizing SHP2 in glioblastoma, a recalcitrant cancer where SHP2 has been proposed as a useful drug target. Measuring protein phosphorylation and expression in glioblastoma cells across 40 signaling pathway nodes in response to different drugs and for different oxygen tensions revealed that SHP2 antagonism has network-level, context-dependent signaling consequences that affect cell phenotypes (e.g., cell death) in unanticipated ways. To map specific signaling consequences of SHP2 antagonism to phenotypes of interest, a data-driven computational model was constructed based on the paired signaling and phenotype data. Model predictions aided in identifying three signaling processes with implications for treating glioblastoma with SHP2 inhibitors. These included PTEN-dependent DNA damage repair in response to SHP2 inhibition, AKT-mediated bypass resistance in response to chronic SHP2 inhibition, and SHP2 control of hypoxia-inducible factor expression through multiple MAPKs. Model-generated hypotheses were validated in multiple glioblastoma cell lines, in mouse tumor xenografts, and through analysis of The Cancer Genome Atlas data. Collectively, these results suggest that in glioblastoma, SHP2 inhibitors antagonize some signaling processes more effectively than existing kinase inhibitors but can also limit the efficacy of other drugs when used in combination. SIGNIFICANCE: These findings demonstrate that allosteric SHP2 inhibitors have multivariate and context-dependent effects in glioblastoma that may make them useful components of some combination therapies, but not others.

ERK-dependent suicide gene therapy for selective targeting of RTK/RAS-driven cancers.

Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a chimeric protein and prodrug, as highlighted by a series of in vitro and in vivo examples explored here.

Genesis of Quality Measurements to Improve the Care Delivered to Patients With Brain Metastases.

PURPOSE: High-value and high-quality health care requires outcome measurements to inform treatment decisions, but, to our knowledge, no standardized measurements exist to evaluate brain metastases (BMs) care. We propose a set of measurements and report on their implementation in the care of patients with BMs. METHODS: On the basis of a stakeholders' needs assessment and review of the literature, we identified outcome and process measurements to assess the care of patients with BMs according to treatment modality. Retrospectively, we applied these indicators of care to all patients diagnosed and treated at our institution over 2 years. RESULTS: We ascertained 5 outcome and 6 process measurements of relevance in the care of BMs. When applied to 209 patients (89.7%) who received cancer treatment, 77% were alive > 90 days after diagnosis. The proportion alive at 90 days after surgery, whole-brain radiation therapy (WBRT), and stereotactic radiosurgery (SRS) was 82%, 59%, and 81%, respectively. Other performance measurements included 30-day postoperative readmission rate (6%), SRS within 30 days of surgery (79%), use of memantine with WBRT (41%), advance directives within 6 months of diagnosis (53%), and palliative care consultation for patients with poor prognosis or receiving WBRT (45%). Measurements for the 24 patients (10.3%) receiving best supportive care were advance directives documentation (67%) and referral to palliative or hospice care (83%). CONCLUSION: We propose a set of measurements to apprise quality improvement efforts, inform treatment decision-making, and to use in evaluation of the performance of interdisciplinary BMs programs. Their refinement can potentially enhance the quality and value of care delivered to patients with BMs.

Glioblastoma Cell Resistance to EGFR and MET Inhibition Can Be Overcome via Blockade of FGFR-SPRY2 Bypass Signaling.

SPRY2 is a purported tumor suppressor in certain cancers that promotes tumor growth and resistance to receptor tyrosine kinase inhibitors in glioblastoma. Here, we identify a SPRY2-dependent bypass signaling mechanism in glioblastoma that drives resistance to EGFR and MET inhibition. In glioblastoma cells treated with EGFR and MET inhibitors, SPRY2 expression is initially suppressed but eventually rebounds due to NF-κB pathway activation, resultant autocrine FGFR activation, and reactivation of ERK, which controls SPRY2 transcription. In cells where FGFR autocrine signaling does not occur and ERK does not reactivate, or in which ERK reactivates but SPRY2 cannot be expressed, EGFR and MET inhibitors are more effective at promoting death. The same mechanism also drives acquired resistance to EGFR and MET inhibition. Furthermore, tumor xenografts expressing an ERK-dependent bioluminescent reporter engineered for these studies reveal that this bypass resistance mechanism plays out in vivo but can be overcome through simultaneous FGFR inhibition.

Checkpoint inhibitor failure in hypermutated and mismatch repair-mutated recurrent high-grade gliomas.

BACKGROUND: Recurrent high-grade gliomas in adults remain a deadly cancer with median survival of less than 1 year. In the absence of effective agents, immunotherapy with checkpoint inhibitors has been adopted as a potentially beneficial next step for recurrences with hypermutated or mismatch repair-mutated phenotypes. The rationale for their use, however, is based on case reports and studies with other types of cancer. METHODS: We reviewed 4 cases of hypermutated or mismatch repair-mutated recurrent high-grade gliomas treated with checkpoint inhibitors. RESULTS: All cases had recurrent high-grade glioma that harbored either a hypermutated phenotype and/or a mismatch repair mutation. Treatment with checkpoint inhibitor therapy resulted in no significant response. CONCLUSIONS: In our experience, hypermutated or mismatch repair-mutated high-grade gliomas in adults do not respond to checkpoint inhibitors alone. This lack of efficacy is in agreement with underwhelming results of clinical trials examining checkpoint inhibitors in high-grade gliomas. The case reports of responders have been in pediatric patients with glioma and are likely a different subtype altogether.