Seizures are a frequent complication of adult-type diffuse gliomas, and are often difficult to control with medications. Gliomas with mutations in isocitrate dehydrogenase 1 or 2 (IDHmut) are more ...likely than IDH-wild type (IDHwt) gliomas to cause seizures as part of their initial clinical presentation. However, whether IDHmut is also associated with seizures during the remaining disease course, and whether IDHmut inhibitors can reduce seizure risk, are unclear. Clinical multivariable analyses showed that preoperative seizures, glioma location, extent of resection, and glioma molecular subtype (including IDHmut status) all contributed to postoperative seizure risk in adult-type diffuse glioma patients, and that postoperative seizures were often associated with tumor recurrence. Experimentally, the metabolic product of IDHmut, d-2-hydroxyglutarate, rapidly synchronized neuronal spike firing in a seizure-like manner, but only when non-neoplastic glial cells were present. In vitro and in vivo models recapitulated IDHmut glioma-associated seizures, and IDHmut inhibitors currently being evaluated in glioma clinical trials inhibited seizures in those models, independent of their effects on glioma growth. These data show that postoperative seizure risk in adult-type diffuse gliomas varies in large part by molecular subtype, and that IDHmut inhibitors could play a key role in mitigating such risk in IDHmut glioma patients.
Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified ...the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.
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•Myeloid cells in the hypoxic niche of GBM upregulate de novo creatine biosynthesis•TAMC-generated creatine is taken up by GBM cells under hypoxic stress•Creatine uptake by GBM tissue supports its growth, survival, and stem cell phenotypes•Inhibition of creatine uptake presents a potential therapeutic strategy for GBM
Glioblastoma (GBM), a deadly CNS malignancy, features profoundly hypoxic niches. Rashidi and Billingham et al. report that within these niches, myeloid cells upregulate creatine biosynthesis and “feed” it to tumor cells, which upregulate creatine import under metabolic stress. Therapeutic targeting of this axis provides a potential avenue for GBM treatment.
MGMT promoter methylation testing is required for prognosis and predicting temozolomide response in gliomas. Accurate results depend on sufficient tumor cellularity, but histologic estimates of ...cellularity are subjective. We sought to determine whether driver mutation variant allelic frequency (VAF) could serve as a more objective metric for cellularity and identify possible false-negative MGMT samples. Among 691 adult-type diffuse gliomas, MGMT promoter methylation was assessed by pyrosequencing (N = 445) or DNA methylation array (N = 246); VAFs of TERT and IDH driver mutations were assessed by next generation sequencing. MGMT results were analyzed in relation to VAF. By pyrosequencing, 56% of all gliomas with driver mutation VAF ≥ 0.325 had MGMT promoter methylation, versus only 37% with VAF < 0.325 (p < 0.0001). The mean MGMT promoter pyrosequencing score was 19.3% for samples with VAF VAF ≥ 0.325, versus 12.7% for samples with VAF < 0.325 (p < 0.0001). Optimal VAF cutoffs differed among glioma subtypes (IDH wildtype glioblastoma: 0.12-0.18, IDH mutant astrocytoma: ~0.33, IDH mutant and 1p/19q co-deleted oligodendroglioma: 0.3-0.4). Methylation array was more sensitive for MGMT promoter methylation at lower VAFs than pyrosequencing. Microscopic examination tended to overestimate tumor cellularity when VAF was low. Re-testing low-VAF cases with methylation array and droplet digital PCR (ddPCR) confirmed that a subset of them had originally been false-negative. We conclude that driver mutation VAF is a useful quality assurance metric when evaluating MGMT promoter methylation tests, as it can help identify possible false-negative cases.
OBJECTIVE Ki-67 immunohistochemistry is widely used as a prognostic marker in meningiomas, but visual estimations tend to be imprecise. Whether the average Ki-67 over an entire slide, a particular ...block, or areas of high staining (hotspots) is prognostic for recurrence-free survival (RFS) and overall survival (OS) is unknown. This study aimed to generate evidence-based recommendations for the optimal use of Ki-67 immunohistochemistry in the workup of meningiomas. METHODS All tissue blocks from a retrospective cohort of 221 patients with primary meningioma (141 WHO grade 1, 64 WHO grade 2, 16 WHO grade 3) were immunostained for Ki-67 and scanned using automated digital analysis software. QuPath was used to quantify the average Ki-67 proliferation index per slide as well as the Ki-67 hotspot in a 2.2-mm 2 area within each slide. The best block was defined subjectively by a neuropathologist as the most representative tissue block from each case. The pathology report Ki-67 was determined by visual estimation. Age, sex, WHO grade, extent of resection, tumor location, and quantitative Ki-67 labeling were tested to determine risk factors for RFS and OS. RESULTS Multivariable analyses demonstrated that WHO grade 2 (HR 2.42, p = 0.018), subtotal resection (HR 8.16, p < 0.0001), near-total resection (HR 2.24, p = 0.041), QuPath Ki-67 averaged across all blocks (HR per % increase = 1.72, p = 0.030), and pathology report Ki-67 (HR per % increase = 1.05, p = 0.0026) were associated with shorter RFS. The average Ki-67 in the best block, maximum across all slides, and maximum hotspot in the best block were not associated with RFS. Only male sex was independently associated with shorter OS (HR 8.52, p = 0.0003). The pathology report Ki-67 was, on average, 6.5 times higher than the QuPath average. CONCLUSIONS These data on Ki-67 in meningiomas indicate the following: 1) visual estimation substantially overestimates Ki-67, 2) digital quantification of average Ki-67 across all tissue blocks provides more prognostic information than small hotspot regions or an entire single block, and 3) Ki-67 is not informative for OS. The results suggest that best practices for incorporating Ki-67 into meningioma prognostication include digital quantification of average Ki-67 over multiple blocks.
Abstract
Introduction:
Immune surveillance, distribution, interactions, and antigen presentation within the tumor microenvironment (TME) may influence anti-tumor reactivity and survival.
Methods:
...Dichotomized newly diagnosed treatment-naïve IDH1 wild-type glioblastoma, negative MGMT promotor methylation (65%), short-term (STS; ≤ 6 months; mean age 66 years; n=10) or long-term (LTS; >2 years; mean age 50 years; n=10) survivors, were profiled using high dimensional fluorescence multiplex staining spatial analysis of the TME using a panel of 26 markers.
Results:
The amounts of specific immune cells (CD4 +, CD8 +, FOXP3 +, CD11c +, CD20 +, CD68 +, CD205 +, NKG2D +) were similar between cohorts, except STS were enriched with CD163 +expressing macrophages and LTS had more P2RY12 +microglia throughout the TME. There was minimal MHC-I expression and no difference in MHC-II on the myeloid populations (CD68 +, CD11c +, CD163 +, P2RY12 +, and/or in combination) in both cohorts. TIM-3 was the only immune regulator substantially expressed on P2RY12 +microglia and the other myeloid populations, irrespective of survival. Perivascular clustering of CD11c +CD163 +cells was enriched in STS cases. The TME in LTS cases demonstrated adaptive immune activation processes, including CD11c +P2RY12 +CD205 +microglia interacting with CD45RO −naïve, non-exhausted (PD-1 −, LAG3 −, Tim3 −) CD8 +T cells expressing the LCK +immune synaptic marker.
Conclusion:
Immune activation interactions between CD8+ T cells and microglia in the TME may help determine STS versus LTS in GBM patients.
Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, ...along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with N0TCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related N0TCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.
Abstract
MGMT promoter methylation predicts favorable temozolomide (TMZ) response in gliomas. Accurate test results depend on adequate tumor cellularity in analyzed samples, which is usually ...estimated (with sub-optimal accuracy) via light microscopy. We evaluated driver mutation variant allelic frequency (VAF) as a quantitative metric for tumor cellularity in MGMT promoter methylation testing. In a cohort of 691 adult-type diffuse gliomas, MGMT promoter methylation was tested by either pyrosequencing (N = 445) or DNA methylation array (N = 246). VAF of TERT and IDH driver mutations was quantified by next generation sequencing (NGS). We compared frequency of methylated (positive) and unmethylated (negative) results (Fisher’s exact test) and promoter methylation levels (unpaired t-test) according to VAF. We correlated MGMT promoter methylation, VAF, visually estimated tumor cellularity, and cellularity inferred from VAF via linear regression. We assessed survival (Log-Rank test) for 201 patients with IDH-wildtype GBM treated with TMZ. In samples with VAF <0.325 (identified by CutoffFinder), pyrosequencing yielded fewer positive results (37% versus 56%, p < 0.0001) and lower methylation levels (13% versus 19%, p < 0.0001) than samples with VAF≥0.325. Methylation array showed less frequent positive results at low VAF for IDH-mutant astrocytoma, but not for IDH-wildtype GBM. Microscopic examination tended to overestimate tumor cellularity, especially when VAF was low (R2 = 0.558). GBMs with unmethylated MGMT by pyrosequencing and TERT VAF <0.145 showed better responses to TMZ versus cases with VAF≥0.145 (median survival = 16.8 versus 13.3 months). Twelve pyrosequencing samples were re-tested by DNA methylation array and droplet digital PCR (ddPCR). RESULTS: changed for 2/6 samples with TERT VAF≤0.1, and 0/6 samples with VAF >0.1. Driver mutation VAF is useful for quality assurance in MGMT promoter methylation testing. Tumor samples with low VAF are at greater risk of false-negative results by pyrosequencing, which can be corrected by DNA methylation array and ddPCR.
STING agonists can reprogram the tumor microenvironment to induce immunological clearance within the central nervous system. Using multiplexed sequential immunofluorescence (SeqIF) and the Ivy ...Glioblastoma Atlas, STING expression was found in myeloid populations and in the perivascular space. The STING agonist 8803 increased median survival in multiple preclinical models of glioblastoma, including ĢPP8, an immune checkpoint blockade-resistant model, where 100% of mice were cured. Ex vivo flow cytometry profiling during the therapeutic window demonstrated increases in myeloid tumor trafficking and activation, alongside enhancement of CD8+ T cell and Nl< effector responses. Treatment with 8803 reprogrammed microglia to express costimulatory CD80/CD86 and ¡NOS, while decreasing immunosuppressive CD206 and arginase. In humanized mice, where tumor cell STING is epigenetically silenced, 8803 therapeutic activity was maintained, further attesting to myeloid dependency and reprogramming. Although the combination with a STAT3 inhibitor did not further enhance STING agonist activity, the addition of anti-PD-1 antibodies to 8803 treatment enhanced survival in an immune checkpoint blockade-responsive glioma model. In summary, 8803 as a monotherapy demonstrates marked in vivo therapeutic activity, meriting consideration for clinical translation.
Abstract
PURPOSE
To clarify the immunoreactivity and potential immunotherapeutic targets of pediatric low- and high-grade gliomas (HGG).
METHODS
Spatial sequential immunofluorescence (seqIFTM) of 24 ...protein markers, NanoString analysis of 770 immune genes, and scRNA sequencing were used to profile and characterize the tumor microenvironment (TME) of 13 newly diagnosed WHO grade 1 pilocytic astrocytomas (PA), 6 pediatric HGG, and 3 normal brain (NB) samples.
RESULTS
Relative to NB, HGGs were enriched with T cells, cancer-associated fibroblasts (CAF), CD11c+ antigen-presenting cells, and CD163+ macrophages. In contrast, PA showed more CD11c+P2RY12+ microglia and CD11c+CD205+ dendritic cells (DCs). TMEM119+ microglia were present in NB and brain adjacent to PA but were rare within the TME of all gliomas regardless of histology. PA uniquely contained naïve CD45RO-FoxP3-PD-1-TIM-3-LAG-3- T cells forming immunological synapses, as visualized by Lck+ expression at the membrane interfaces, during interaction with either microglia or DCs. This finding was not encountered in HGG. TIM-3 was the most frequently expressed immune modulatory target on all the myeloid populations, regardless of tumor histology. PD-L1 expression, especially on microglia, was more frequent in PA than in HGG. NanoString analysis revealed the pro-inflammatory and enabler of diapedesis IL-1 pathway genes (IL1RAP, TICAM2, and IRAK4) were upregulated in PA relative to the matched adjacent brain using unsupervised hierarchical clustering.
CONCLUSIONS
Based on multiple orthogonal analysis strategies, PAs display a more pro-inflammatory phenotype, in comparison to HGG, suggesting immunological surveillance that may be driven by IL-1, as a baseline feature at presentation.
