Citations

Abstract Magnetic resonance imaging (MRI) is key to clinically managing brain tumor patients, however connecting the biology to imaging remains challenging. We previously developed a two-compartment model of MRI signal intensity to quantitatively estimate relative edema abundance from T2-weighted MRIs. Using this model, we previously identified the fatty acid metabolism (FAM) and oxidative phosphorylation (OxPhos) pathways as sex-distinct, with both pathways amplified for high edema in males and low edema in females. The purpose of this project was to further delineate sex-distinct biology associated with MRI-estimated brain tumor edema abundance. We analyzed 179 multiregional samples (Female: 75; Male: 104) from 55 high grade glioma patients (Female: 21; Male: 34) for bulk RNA-Seq. Patients’ pre-surgical multiparametric MRIs were preprocessed and segmented for abnormal regions and normal tissue. Utilizing the segmentations and preprocessed images we estimated the relative fractions of extracellular and intracellular space based on the edema mathematical model. Samples were characterized by their edema scores, which were analyzed using differential expression for high and low edema, gene set enrichment analysis (GSEA) using MSigDB hallmarks, and leading edge interpretation. Based on transcriptomic leading edge analyses of high and low edema samples from sex-separated cohorts, we identified common and sex-distinct enrichment of the FAM and OxPhos pathways underlying edema patterns. Of the OxPhos pathway leading edge genes, 45 were common, 36 were unique to females, and 66 were unique to males. Of the FAM pathway leading edge genes, 8 were common, 39 were unique to females, and 29 were unique to males. Notably, expression of both IDH1 and IDH2 were increased for males in regions of high edema in the OxPhos pathway. IDH3a was decreased for females in regions of low edema in the OxPhos pathway. These data suggest that there may be sex-distinct metabolism underlying MRI measurable edema formation.

Abstract BACKGROUND High-grade glioma (HGG) is the most common and aggressive form of primary brain cancer. Standard of care surgery employs Magnetic Resonance Imaging to identify the contrast enhancing (CE) core for resection, leaving a non-enhancing (NE) invasive rim of tumor which contributes to recurrence. The hypoxic tumor microenvironment is thought to drive invasion into healthy brain, and understanding cellular responses to hypoxia may provide insight into combatting recurrence. Here, we apply a graph network to integrate regionally heterogeneous data from a unique dataset of spatially-localized CE and NE biopsies from a large cohort of patients with HGG to identify gene networks regulating hypoxia response and invasion. METHODS RNA-sequencing of 1159 differentially expressed genes across 159 IDH-wt biopsies (74 patients) from NE/CE regions were represented as nodes in a Neo4j graph network. Candidate genes for network random walks occurred exclusively within NE communities detected by modularity optimization and were localized by cell-type using single-cell RNA-sequencing (scRNAseq). CSF2RA was selected as source (walk length = 5; 10,000 walks/source node). Frequently occurring genes (one-tailed test) in walks were ranked by correlation with CSF2RA expression in NE samples and analyzed by gene set enrichment analysis for biological processes (clusterProfiler). CSF2RA and hypoxia gene expression were visualized with DoHeatmap in composite NE/CE scRNAseq profiles. RESULTS CSF2RA, an immune receptor, was upregulated (logFC = 0.789) in NE samples, scRNAseq myeloid populations, and Glycolytic/Plurimetabolic glioma subtype. CSF2RA random walk genes (n=46) enriched metabolic/proliferative signatures. CSF2RA expression correlated negatively with hypoxia genes globally and regionally (NE/CE), while NE/CE scRNAseq revealed distinct cellular expression patterns of CSF2RA and hypoxia response genes. CONCLUSION CSF2RA expression may prime NE glioma cells for invasion by altering hypoxia response. The genetic network bridging CSF2RA and hypoxia response may be a targetable mechanism of tumor recurrence.

Abstract BACKGROUND High-grade glioma (HGG) is the most common and aggressive form of primary brain cancer. Standard of care surgery employs Magnetic Resonance Imaging to identify the contrast enhancing (CE) core for resection, leaving a non-enhancing (NE) invasive rim of tumor which contributes to recurrence. Recent multi-omics analyses identified predominant neuronal and immune signatures within NE tumor. Here, we apply a graph network to integrate regionally heterogeneous data from a unique dataset of spatially-localized CE and NE biopsies from a large cohort of patients with HGG. METHODS RNA-sequencing of 1159 differentially expressed genes across 159 IDH-wt biopsies (74 patients) from NE/CE regions were represented as nodes in a Neo4j network. Candidate genes for network random walks occurred exclusively within NE communities detected by modularity optimization and localized by cell-type using single-cell RNA-sequencing (scRNAseq). CSF2RA and RBFOX1 were selected as sources (walk length = 5; 10,000 walks/source node). Frequently occurring genes (one-tailed test) in walks were ranked by correlation with CSF2RA/RBFOX1 expression in NE samples and analyzed by gene set enrichment analysis for biological processes (clusterProfiler). Random walk genes were visualized (DoHeatmap) against composite NE/CE scRNAseq profiles. RESULTS CSF2RA and RBFOX1 were upregulated in NE samples (logFC = 0.789, 1.85). Upregulation of CSF2RA, an immune receptor, in the Glycolytic/Plurimetabolic and RBFOX1, a neurodevelopmental regulator, in the Neuronal glioma subtypes were observed in NE samples (ANOVA p < 0.05). CSF2RA walk genes (n=46) enriched metabolic/proliferative signatures, while RBFOX1 walk genes (n=201) showed neurodevelopmental upregulation. scRNAseq localized CSF2RA to monocytes/macrophages while RBFOX1 was expressed in distinct astrocyte/neuroepithelial populations. CONCLUSION CSF2RA and RBFOX1’s gene networks modulate immune and neurodevelopmental processes influencing glioma progression in NE tumor. Understanding the region-specific roles of these networks may offer strategies for targeting cells in the invasive glioma rim.

Abstract Heterozygous H3.3 K27M mutations are central to the pathogenesis of diffuse midline gliomas (DMG), causing a global reduction in H3K27 trimethylation (H3K27me3) and subsequent epigenetic dysregulation. H3.3 contains a unique Serine 31 (S31) phosphorylation site crucial for mitotic checkpoint regulation. In DMG cells, reduced mitotic S31 phosphorylation correlates with chromosomal instability and aneuploidy. Using a mouse model, we demonstrate that both H3.3K27M and H3.3S31A mutations promote high-grade glioma development, whereas wild-type H3.3 does not. Importantly, S31A, which maintains normal K27me, suggests that S31 phosphorylation loss alone is oncogenic. The H3.3K27M mutation disrupts epigenetic regulation by inhibiting the Polycomb Repressive Complex 2 (PRC2), leading to decreased H3K27me3 and unintended gene induction. Concurrently, K27M exerts a compensatory repressive effect on the transcriptome, mediated by reduced S31 phosphorylation, independent of PRC2 activity. Using CRISPR-engineered isogenic DMG cells and analyzing the transcriptome and chromatin accessibility with RNA-seq and ATAC-seq, we found that the K27M mutation produces a balanced gene expression profile, while PRC2 loss primarily leads to gene induction. Notably, cells with S31A mutations also maintain this balanced profile, linking the PRC2-independent effect of K27M to reduced S31 phosphorylation. Conversely, cells with wild-type K27 but S31A mutation show a repressive genomic state, evidenced by an up/down gene ratio of 0.75, owing to functional PRC2. We further show that the genes uniquely dysregulated by K27M or S31A, independent of PRC2, overlap significantly, with dysregulated genes displaying altered chromatin accessibility. This suggests that the effect of K27M and S31A mutations on gene regulation is primarily due to changes in chromatin state. This complex interplay points to targeting K27M-associated pathways, particularly S31 phosphorylation, as a promising therapeutic avenue in DMG, a malignancy with fewer effective treatments and dismal prognosis.

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by insufficient requisite circulating-tumor (ct) and cell-free (cf) DNA in blood due to blood-brain barrier (BBB). This limits the identification of blood-based tumor biomarkers along with biomarker-driven systemic therapies. Real-time image-guided low-intensity focused ultrasound (LIFU) plus IV microbubble oscillators, leads to non-invasive BBB disruption. METHODS LIBERATE is an ongoing, prospective, multi-center, self-controlled, pivotal trial investigating safety and technical efficacy of LIFU-based BBB disruption for increasing blood ctDNA and cfDNA levels in adult GBM patients (aged 18-80 years). Patients with suspected GBM planned for biopsy/resection at ten US centers are being enrolled. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV microbubbles for enhanced sonication effects, after which MR-guided BBB disruption using 220kHz LIFU device is performed with real-time acoustic-feedback-control for effective microbubble activation. Pre- and post-LIFU, phlebotomy and MRI brain are performed. Primary study endpoint is defined, per subject, as ratio between their blood cfDNA level 1-hour post-LIFU procedure compared to blood cfDNA pre-procedure. Primary study hypothesis is that BBB disruption leads to ≥2-fold rise in blood cfDNA. Secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in blood cfDNA sample obtained 1-hour post-LIFU and biomarker pattern in tumor tissue obtained later. Trial is powered to evaluate both primary and secondary hypotheses. Based on assumed true agreement rate of 91% and one-sided alpha=0.025, an exact test for binomial proportions provides N=50 with 84% power for secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of specific somatic mutations in ctDNA from blood collected pre- and post-LIFU, (2) ctDNA estimation in samples collected at 30-minutes, 1-hour, 2-hour, and 3-hour post-LIFU to determine time of greatest yield, (3) correlation of MRI parameters related to grading of BBB disruption and ctDNA-based biomarkers from post-LIFU blood samples. Patient enrollment commenced in 2022 and remains ongoing (NCT05383872).

Abstract BACKGROUND Pituitary adenomas are often effectively managed with medical therapy, surgery, and radiation therapy. However, up to 15% of pituitary neoplasms exhibit rapid growth, invasive behavior, or, rarely, metastasize despite optimal standard treatments. Temozolomide is associated with a survival benefit for patients with these aggressive pituitary tumors. METHODS We present a case series of 8 patients with an aggressive pituitary tumor (APT) or pituitary carcinoma (PC) treated with temozolomide. RESULTS Eight patients (3 female, median age at diagnosis 44) with APT (n=6) or PC (n=2) were treated. A variety of pituitary tumor types were represented (1 prolactinoma, 5 corticotroph adenomas, 1 gonadotroph adenoma, 1 thyrotropinoma). Prior to temozolomide initiation, patients underwent a median 5 treatments including medical management (n=4), surgery (n=7), radiation therapy (n=7). Temozolomide was started at median 3.5 recurrence. Median treatment duration was 6 cycles (n=6, 150-200 mg/m2 days 1-5, 28-day cycles; n=1, 75 mg/m2, days 1-21, 28-day cycles). Seven patients experienced progression after temozolomide treatment, median progression-free survival (PFS) was 4 months. One patient remains on treatment, progression-free at 5 months. MGMT methylation status was assessed for 3 tumors. Among 2 patients with MGMT promoter methylated tumors, PFS was 4 months for one patient, the other remains on treatment. For one patient with MGMT promoter unmethylated corticotroph adenoma, PFS was 9 months. No ≥ grade 4 toxicities were noted. Most common toxicities were nausea (n=5), thrombocytopenia (n=3), and fatigue (n=2). CONCLUSION In this group of heavily pretreated patients, temozolomide monotherapy was well-tolerated and associated with median PFS of 4 months. Interestingly, PC patients had longer PFS at 9 months and 24 months. Our findings support use of single-agent temozolomide chemotherapy for patients with APT/PC, but further prospective analysis is needed to identify patients more likely to benefit from treatment and optimal timing of chemotherapy initiation.

Abstract BACKGROUND The prognostic factors impacting the survival of grade 4 IDH-mutant astrocytoma treated with standard radiation/temozolomide are not well-described. METHODS A retrospective cohort was generated of all patients diagnosed at Mayo Clinic with grade 4 IDH-mutant astrocytoma (2021 WHO Classification) on initial diagnosis between 2002-2023, and who received standard radiation/temozolomide. The impact of patient, tumor and molecular variables on overall survival (OS) and progression-free survival (PFS) was evaluated using survival analyses and Cox regression models. RESULTS Fifty-eight patients were identified. The median age was 36 years, 64% were male and 93% were white. The median follow up was 3.3 years. The median OS and PFS were 6.9 years and 2.4 years, respectively. CDKN2A/B homozygous deletion significantly reduced both OS (2.5 years vs NR, HR=3.9, p=0.0168) and PFS (1.4 vs 4.5 years, HR=4.2, p=0.0005). Midline involvement (brainstem, basal ganglia, thalamus, cerebellum or corpus callosum) also significantly reduced both OS (2.4 vs 12.8 years, HR=7.0, p=0.0014) and PFS (1.4 vs 4.5 years, HR=4.8, p=0.0002). MGMT promoter hypermethylation showed a trend toward significance for PFS (p=0.082) but did not impact OS. Age, sex, tumor size, ATRX immunohistochemistry and tumor-treating field therapy did not impact survival outcomes. Multivariable analyses revealed that PFS remained significantly impacted by both CDKN2A/B homozygous deletion (HR=3.5, p=0.0051) and midline involvement (HR=3.1, p=0.0125). A significant association between CDKN2A/B homozygous deletion and midline involvement was unexpectedly observed (53% if midline, 24% if non-midline; χ2=4.3, p=0.037). Exploratory analyses revealed that 15/15 (100%) of patients with midline involvement demonstrated aberration in RB1 signaling (CDKN2A/B homozygous deletion, n=8; CDKN2A/B hemizygous deletion or loss of heterozygosity, n=6; CDK4 amplification, n=1). CONCLUSION CDKN2A/B homozygous deletion and midline involvement independently impact PFS in patients with grade 4 IDH-mutant astrocytoma treated with standard radiation/temozolomide. Furthermore, midline involvement may be associated with aberrant RB1 signaling.

Abstract Current standard of care for IDH-wildtype glioblastoma (GBM) includes maximal safe resection, adjuvant radiation, and chemotherapy with the alkylating agent temozolomide. A subset of gliomas treated with temozolomide develop somatic hypermutation with a predominance of C>T transitions due to the alkylation effects on DNA. However, the frequency of occurrence, clinical ramifications, and predictive biomarkers for developing temozolomide-induced hypermutation (TMZ-HM) in GBM are unknown. Here we performed comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent GBM from 106 patients. Seven patients (7%) developed hypermutation at time of first recurrence, while an additional five patients (5%) developed hypermutation at a subsequent recurrence. Patients who developed TMZ-HM had a significantly longer interval between initial surgery and first recurrence (27.8 vs. 9.9 months, p<0.001). In contrast to IDH-mutant gliomas where TMZ-HM is associated with poor prognosis, those patients with IDH-wildtype GBM who developed TMZ-HM had significantly longer overall survival from both initial surgery (67.9 vs. 20.3 months, p<0.001) and from time of recurrence after development of hypermutation (30.9 vs. 9.5 months, p=0.001). There was no difference in oncogenic alteration frequency in initial GBM that subsequently developed TMZ-HM. However, 4 specific CpG sites out of 12 total interrogated sites in the promoter region of MGMT (a DNA repair enzyme) and 5 specific CpG sites in the promoter region of KCNQ1DN (a negative regulator of c-Myc) were significantly more hypermethylated in those GBM that developed TMZ-HM. We then generated a weighted risk score across these CpG sites that can be used to prospectively identify GBM likely to develop TMZ-HM associated with favorable survival. In summary, patients with GBM that develop TMZ-HM follow a more favorable clinical course, and specific MGMT and KCNQ1DN promoter hypermethylation patterns could serve as a biomarker to identify these patients who may potentially benefit from extended alkylating chemotherapy treatment.

Abstract BACKGROUND Patients with meningiomas who have progressed after multimodal treatments face a significant shortage of viable therapeutic options. Recent advancements in the molecular understanding of meningiomas have identified the FAK and RAF/MEK signaling pathways as potential promising targets for progressive meningiomas. METHODS In vitro, we tested two human meningioma cell lines, IOMM-Lee (NF2 wild-type and CDKN2A loss) and CH157-MN (NF2 mutant). The cells were plated in Matrigel-coated wells, treated with defactinib (FAK inhibitor), VS-6766 (RAF/MEK inhibitor, avutometinib) and the combination over a 7-day period and assessed for cell viability using the CellTiter-Glo 3D assay. In vivo, we tested the efficacy of the FAK inhibitor VS-4718 (possessing favorable pharmacokinetic properties in mice) and RAF/MEK inhibitor VS-6766, both given daily via oral gavage, in athymic nude mice bearing IOMM-Lee or CH157-MN tumors. RESULTS In the matrigel assay, VS-6766 suppressed IOMM-Lee and CH157-MN cells in a dose dependent manner. The drug combination was mostly additive in IOMM-Lee cells. However, CH157-MN cells showed a strong synergistic response across all drug concentrations tested. In the IOMM-Lee flank tumor model, VS-4718 alone had no effect, while VS-6766 alone or in combination significantly reduced tumor growth and extended median survival from 25 days to 47 days (p < 0.05). In the CH157 flank tumor model, VS-4718 monotherapy did not affect tumor growth or survival, but VS-6766 monotherapy slowed tumor growth and increased median survival from 10 days to 31 days (p < 0.001). Combining both drugs further reduced tumor growth and extended median survival to 42 days (p < 0.001). CONCLUSION Treatment with FAK and RAF/MEK inhibitors showed synergistic or combination effects in the NF2 mutant CH157-MN model in vitro and in vivo. RAF/MEK inhibitor monotherapy was effective for the IOMM-Lee model (NF2 wild-type), without clear benefit of combining with the FAK inhibitor. Investigations in intracranial tumor models are underway.

Abstract Accurate response assessment in patients with high-grade gliomas (HGGs) is complicated by post-treatment radiographic changes that are equivocal for treatment effect versus progression. Plasma-based liquid biopsy assays for circulating tumor DNA (ctDNA) have had limited sensitivity to date, due in part to the presence of the blood-brain barrier that limits the diffusion of tumor-derived analytes out of the central nervous system. To address this challenge, we developed a ctDNA plasma-based assay to detect individualized tumor-derived amplified junctions in patients with HGGs. These junctions occur due to large-scale chromosomal rearrangements, such as those associated with epidermal growth factor receptor (EGFR) or cyclin-dependent kinase 4 (CDK4) amplification. To identify these amplified junctions, whole genome sequencing was performed on resected tissue. Primers were designed to flank junction breakpoints and were then utilized on each patient’s plasma samples to detect the amplified junction via qPCR. Tumor-specific amplified junctions were robustly detected in plasma from 14/14 (100%) patients with IDH-mutant or wild-type grade 4 astrocytomas whose tumor genomes demonstrated amplification-related junctions, located at chromosomes 7, 11, or 12. In comparison, ctDNA could not be detected in three control cases that did not have an amplification or did not have high copy number junctions. Importantly, serial plasma samples obtained prior to and at disease recurrence in 4 patients demonstrated an increasing abundance of amplified junctions that accurately correlated with disease progression in 4/4 patients (100%). In two of these patients, increasing abundance of amplified junctions preceded radiographic changes compatible with disease progression by approximately 2 or 4 months, respectively. In conclusion, tumor-specific amplified junctions could reliably be detected in plasma from patients with IDH-mutant or wild-type grade 4 astrocytomas and correlated with changes in disease burden. Further work is currently ongoing to evaluate these promising results in a larger cohort of patients with serial plasma samples.