Optimizing the use of Ki-67 proliferative index as a prognostic biomarker in meningiomas using digital analysis.

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-mm2 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.

Prohibitin Expression in Antigen-Presenting Cells: Implications for Inciting Trigger in CNS IgG4-Related Disease.

Immunoglobulin G4-related disease (IgG4-RD) is a rare autoimmune disorder with an unknown etiology. Using orthogonal immune profiling and automated sequential multiplexing, we found an enhanced frequency of activated circulating B cells, antigen-presenting myeloid cells in peripheral blood, and a distinct distribution of immune cells within the CNS lesions. Prohibitin-expressing CD138+ plasma B cells and CD11c+ dendritic cells have been found interacting with T cells resulting in irmnune cell activation within the lesion. The data implicate prohibitin as a potential triggering antigen in the pathogenesis of IgG4-RD and shed light on the cellular dynamics and interactions driving IgG4-RD in the central nervous system, emphasizing the need for further studies corroborating these findings.

Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice.

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 NOTCH3 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 NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

STING agonist 8803 reprograms the immune microenvironment and increases survival in preclinical models of glioblastoma.

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 QPP8, 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 NK effector responses. Treatment with 8803 reprogrammed microglia to express costimulatory CD80/CD86 and iNOS, 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.

Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth.

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.

Variant allelic frequencies of driver mutations can identify gliomas with potentially false-negative MGMT promoter methylation results.

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.

Docetaxel targets aggressive methylation profiles and serves as a radiosensitizer in high-risk meningiomas.

BACKGROUND: Meningioma is the most common primary intracranial tumor in adults. A subset of these tumors recur and invade the brain, even after surgery and radiation, resulting in significant disability. There is currently no standard-of-care chemotherapy for meningiomas. As genomic DNA methylation profiling can prognostically stratify these lesions, we sought to determine whether any existing chemotherapies might be effective against meningiomas with high-risk methylation profiles. METHODS: A previously published dataset of meningioma methylation profiles was used to screen for clinically significant CpG methylation events and associated cellular pathways. Based on these results, patient-derived meningioma cell lines were used to test candidate drugs in vitro and in vivo, including efficacy in conjunction with radiotherapy. RESULTS: We identified 981 genes for which methylation of mapped CpG sites was related to progression-free survival in meningiomas. Associated molecular pathways were cross-referenced with FDA-approved cancer drugs, which nominated Docetaxel as a promising candidate for further preclinical analyses. Docetaxel arrested growth in 17 meningioma cell sources, representing all tumor grades, with a clinically favorable IC50 values ranging from 0.3 nM to 10.7 mM. The inhibitory effects of this medication scaled with tumor doubling time, with maximal benefit in fast-growing lesions. The combination of Docetaxel and radiation therapy increased markers of apoptosis and double-stranded DNA breaks, and extended the survival of mice engrafted with meningioma cells relative to either modality alone. CONCLUSIONS: Global patterns of DNA methylation may be informative for the selection of chemotherapies against meningiomas, and existing drugs may enhance radiation sensitivity in high-risk cases.

Postoperative risk of IDH-mutant glioma-associated seizures and their potential management with IDH-mutant inhibitors.

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.