Cell-free DNA (cfDNA) are short acellular fragments of DNA found in the circulation. It has emerged as a promising diagnostic tool for the early detection and follow-up of tumor-derived biomarkers in many cancer types, but primary brain tumors remain challenging to monitor using this approach. Allegedly limited by the blood-brain-barrier, detection rates of tumor-derived cfDNA range from 25-50% in plasma and 60-80% in CSF samples using conventional read alignment and variant calling approaches, sowing doubt in the utility of cfDNA-based monitoring of primary brain tumors. In this study, we sought to investigate the effectiveness of a novel alignment-free read classifier to detect tumor-derived cfDNA (ctDNA) sequencing reads and quantify tumor burden. Our dataset comprises plasma (n = 101) and CSF (n = 15) samples collected from n = 24 patients with primary brain tumors, including both adult (n = 8) and pediatric (n = 16) patients. In our cohort, n = 6 patients (25%) were diagnosed with a diffuse midline glioma, n = 13 (54%) high-grade glioma, n = 4 (17%) medulloblastoma, and n = 1 (4%) ependymoma. Applying read classification, we detected ctDNA reads at varying abundances across all cfDNA samples examined (n = 116/116, 100%), highlighting the potential to track sequence-based tumor signals from liquid biopsies using our approach. At several plasma timepoints across patients, an increase in ctDNA signal correlated with clinical progression and a decrease in signal aligned with the absence of residual tumor. In several patient datasets, ctDNA signal was higher in CSF samples compared to plasma, consistent with findings reported in the literature. Our method has the potential to accurately and specifically quantify tumor signals in cfDNA, offering a sensitive and mutation-agnostic approach for tracking tumor dynamics across various brain tumor subtypes. Further investigations with larger patient cohorts and more comprehensive data analysis to validate these findings are actively underway.

Abstract Glioblastoma (GBM) is the most prevalent type of malignant tumor within the central nervous system. There is a growing demand to develop and adapt new therapeutic options in order to combat the five-year survival rate of 6.8% and improve patient quality of life. Arsenic trioxide (ATO) is the standard of care for myelodysplasia and relapsed or refractory acute promyelocytic leukemia. While currently being classified to have an idiopathic mechanism of action, two specific cytotoxic consequences of ATO have emerged: MNK1 activity and oxidative stress. Across six GBM PDX models a 20-fold difference in sensitivity manifests, indicative of underlying innate sensitivity or resistance to ATO. To correlate this to the proposed mechanisms of action, we observed ATO’s effect on MNK1 activity through eIF4E phosphorylation and glutathione (GSH) levels following treatment. A subset of GBM models exhibited a marked upregulation of eIF4E phosphorylation following treatment with 2 uM ATO. Of the six models, three were chosen to undergo further testing to observe whether this upregulation showed a consistent dose-dependent relationship across the models and correlated with vulnerability to ATO. Combination therapy with the MNK1 inhibitor, ETC-206, displayed mild to moderate synergy on overall cell viability and strong synergy when observing its effects on the glioma stem cell population. This preferential effect on the GSC populations suggests that MNK1i based combination strategies may show greater effectiveness in vivo than indicated by our previous in vitro studies. Nutraceuticals targeting GSH synthesis, Chrysin and Silibinin, displayed a 0.95 to 1.4-fold decrease in ATO IC50 values across all 6 models. To observe whether each nutraceutical impacted GSH levels consistently across all of the tested models, we measured GSH levels following treatment for each individual compound and their combination treatments. These findings further implicate MNK1 activity and cellular response to oxidative stress as markers of ATO sensitivity, however they are not the sole determinates of response. Understanding of the mechanism of action for idiopathic compounds may allow for the discovery of molecular signatures of sensitivity, improving patient selection for clinical trials and the development of new combination approaches to combat resistance in other tumor types. Citation Format: Charles Shaffer, Nanyun Tang, Yue Hao, Karen Fink, George Snipes, Bruce Mickey, Michael Berens. Reclaiming a dirty drug: What is the context of vulnerability to arsenic trioxide in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4718.

Objective Dizziness is one of the most prevalent and debilitating symptoms associated with vestibular schwannoma (VS), and there are little data on contributing or alleviating factors in the perioperative setting. In this study, we aimed to evaluate whether vestibular nerve sectioning or labyrinthectomy concomitant with surgical resection would improve dizziness in the postoperative period. Methods This is a retrospective study of a consecutive series of VS patients who underwent resection at a large tertiary care center between 2009 and 2023. Dizziness at 6 months was the primary endpoint, whereas facial nerve and hearing outcomes were secondary endpoints. Fisher's exact test was used to identify significant differences between categorical variables, and multivariate logistic regression analysis was performed to identify predictors of dizziness as well as facial nerve and hearing outcomes. Results A total of 333 patients underwent resection of VS at our institution. There was no significant difference in binary reported dizziness based on surgical approach (p = 0.14). However, patients reported significantly less dizziness at discharge (p < 0.01) as well as 6-month (p = 0.02) and 1-year (p < 0.01) follow-up in the trans-labyrinthine group. On the other hand, patients who underwent labyrinth-sparing approaches reported a significant increase in dizziness that remained up to 1 year from the time of surgery. Conclusions Our data suggest that labyrinthectomy may improve dizziness symptoms in patients with VS. Selective VN sectioning does not appear to affect dizziness or hearing outcomes. Finally, surgical approach does not affect facial nerve outcomes.

Abstract Glioblastoma (GBM) multiforme is the most aggressive type of brain cancer. These lethal brain tumors are characterized by the inter- and intra-tumor molecular heterogeneity and demonstrated cell plasticity. A male:female distribution of 1.6:1; survival outcome from treatment (debulking, radiation, and temozolomide) benefits females nearly twice that for males. Through spatial transcriptomics, we interrogated the immune landscape of GBM, seeking to tease out whether distinct biological and therapeutic niches in GBM were evident, and to understand how the tumor microenvironments contributed to the heterogeneity of GBM. We designed a set of RNA hybridization probes specifically for portraying the cell identities and cell functional states within GBM tumor. The probe set was used to profile six human GBM samples from male and female cohorts on the Vizgen MERSCOPE platform. An integrated analysis of 505,614 cells from the six samples yielded a rich and varied spatial cell atlas of glioblastoma. Our preliminary findings depict distinct spatial niches of different tumor cell subpopulations that are associated with local environmental factors such as hypoxia. We identified a small cluster of cells that are in neuronal-like state, which could potentially drive invasion. We found spatial proximity of CD4+ cells and cells that express HIF1A, suggesting the dynamic interactions between tumor cells, regulatory T cells and tumor microenvironment. We also observed significant difference in the degree of CD8+ T-cell infiltration in female samples versus male samples. The average CD4+/CD8+ T-cell ratio in males is twice as high as that of females, suggesting intrinsic sex-based differences in immune response to tumor. Expansion of these studies in additional specimens will further illuminate microenvironmental influences on glioma progression and may instruct specific interventions leading to more effective and predictable immune-oncology of this disease. Citation Format: Yue Hao, George Reid, Angela Baker, Karen L. Fink, George J. Snipes, Bruce E. Mickey, Andrew E. Sloan, Jill S. Barnholtz-Sloan, Michael Berens. Spatially resolved immune landscape revealed sex-biased T-cell infiltration in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3764.

Abstract Glioblastoma (GBM) is characterized by inter- and intra-tumor molecular heterogeneity and demonstrated cell plasticity. Sex differences have been observed in various aspects: a male:female GBM incidence ratio is 1.6:1; standard of care treatments (debulking, radiation, and temozolomide) benefit females more than males. 14 human GBM samples from two study sites were subject to spatial profiling on the Vizgen Merscope platform to examine whether distinct biological and therapeutic niches in GBM were evident, to understand how the tumor microenvironments (TME) contributed to the heterogeneity of GBM, and to interrogate sex difference in the immune landscape of GBM. Multiple sets of spatial probes were designed to capture the cell identities (cancer stem cells, astrocytes, oligodendrocytes, microglia, macrophages, etc.) and the spatially-resolved expression patterns of genes with different functions. Two of the 14 GBM samples were also profiled on another FISH-based spatial platform (Veranome). The data from the two platforms exhibited a linear correlation. We explored the gene-gene co-expression network, and calculated the interaction probabilities between genes which were then compared for sex difference. We found that female samples showed significantly lower (two-fold) CD4+/CD8A+ T-cell count ratios than male samples. We also observed sample-specific spatial niches of different GBM cell subtypes and immune cell populations. Our large-scale spatial transcriptomics study revealed intrinsic sex-based differences in immune response to tumor and illuminated microenvironmental influences on GBM.

Glioblastoma (GBM) multiforme is the most aggressive type of brain cancer. These lethal brain tumors are characterized by the inter- and intra-tumor molecular heterogeneity and diverse cell plasticity. We sought to interrogate the immune landscape of GBM, tease out the distinct biological and therapeutic niches in GBM and to understand how the tumor microenvironments mediated the heterogeneity of GBM. We have collected six human GBM samples for spatial profiling and single-nucleus RNA sequencing. Multiple sets of spatial gene panels were designed to capture the cell identities (cancer stem cells, astrocytes, oligodendrocytes, microglia, macrophages, endothelial cells, etc.) and the spatially-resolved expression patterns of genes indicative of different functional states. We also aim to find altered neurovascular units by examining the spatial correlation between cells that highly express angiogenesis markers such as VEGFA with tumor-associated macrophages, which could be recruited to the angiogenic niche by GBM tumor cells. From the spatial RNA dataset of the first two samples, we observed close proximity of tumor-associated macrophages and endothelial cells that form the microvasculature. Our preliminary results from the single-nucleus RNA sequencing and spatial transcriptomics of two samples shed light on the complex cell landscape and the dynamic interaction between tumor cells and infiltrating or resident immune cells in the GBM tumor tissue. Citation Format: Yue Hao, Angela Baker, Brian Hilbush, Marcos Otero, Chris Streck, Karen L. Fink, George J. Snipes, Bruce E. Mickey, Michael Berens. Spatial and single-nucleus transcriptomics of human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6779.

Supplementary Figures 1-5 from RIP1 Activates PI3K-Akt via a Dual Mechanism Involving NF-κB–Mediated Inhibition of the mTOR-S6K-IRS1 Negative Feedback Loop and Down-regulation of PTEN

. The erratum corrects a Python code error in the originally published article; corrected accuracies from the TCI/TCGA data set are reported.

Supplementary Figures 1-3, Tables 1-4 from The Receptor Interacting Protein 1 Inhibits p53 Induction through NF-κB Activation and Confers a Worse Prognosis in Glioblastoma

[This corrects the article DOI: 10.1117/1.JMI.9.1.016001.].

Supplementary Data from The Telomerase Antagonist, Imetelstat, Efficiently Targets Glioblastoma Tumor-Initiating Cells Leading to Decreased Proliferation and Tumor Growth

Abstract Glioblastoma (GBM) is the most prevalent type of malignant tumor within the central nervous system. Possessing a five-year survival rate of 6.8%, new therapeutic options are a necessity in order to increase patient quality of life and survival outcomes. Arsenic trioxide (ATO) is an FDA approved drug for the treatment of relapsed or refractory acute promyelocytic leukemia. Clinical trials of ATO in combination with radiation and temozolomide show therapeutic effects in a modest subset of glioblastoma patients. ATO resistance in GBM cell lines is associated with MNK1-eIF4E upregulation. Additionally, diminished response to ATO is linked to resistance to oxidative stress via increased glutathione levels and NrF2 pathway expression. We sought to target each of these proposed mechanisms of resistance in order to develop a suite of compounds for use in combination therapies, with the goal of sensitizing GBM models to ATO. Six GBM PDX models were dosed with ATO at varying concentrations and monitored for the IC50 value of treatment. The cell lines exhibited a 20-fold difference in sensitivity, indicative of an underlying innate resistance to ATO. We performed drug dose response studies of patient-derived glioma cells treated with ATO in combination with the nutraceutical compounds Chrysin and Silibinin. Combination treatments using Chrysin and Silibinin showed potentiation of the cytotoxic effects of ATO. Confirmation of the mechanism for induced sensitivity to ATO will be explored by measuring shifts in glutathione content and susceptibility to oxidative stress in response to treatment using nutraceutical compounds. In addition, drug dosing with combination therapies utilizing the MAPK interacting serine/threonine kinase 1 (MNK1) inhibitors eFT-508 and EFT-206 also show synergistic effects in GBM cell lines. The eIF4E phosphorylation response to treatment of both ATO alone ATO MNK1i combination therapies will be monitored and analyzed for potential translational effects. Each of these compounds were selected based on safe use determined in clinical trials (MNK1 inhibitors) or over-the-counter availability (nutraceuticals). All four companion approaches show low pharmacological liabilities in crossing the blood brain barrier. Future studies involving these compounds will consist of testing these suggested combination therapies in vivo by observing their effectiveness in orthotopic, GBM tumor bearing mice. Identification and development of sensitization targets for ATO will allow for greater effectiveness of treatment and a greater potency of the drug in combating resistance which arises in response to treatment. Discovery of a predictive molecular signature of synergy from ATO and other targeted agents may pave the way for a successful clinical trial of these combinations in an identifiable subpopulation of GBM patients. Citation Format: Charles Shaffer, Nanyun Tang, Yue Hao, Karen Fink, George Snipes, Bruce Mickey, Michael Berens. Potentiation of arsenic trioxide in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1601.

Selective vascular access to the brain is desirable in metabolic tracer, pharmacological and other studies aimed to characterize neural properties in isolation from somatic influences from chest, abdomen or limbs. However, current methods for artificial control of cerebral circulation can abolish pulsatility-dependent vascular signaling or neural network phenomena such as the electrocorticogram even while preserving individual neuronal activity. Thus, we set out to mechanically render cerebral hemodynamics fully regulable to replicate or modify native pig brain perfusion. To this end, blood flow to the head was surgically separated from the systemic circulation and full extracorporeal pulsatile circulatory control (EPCC) was delivered via a modified aorta or brachiocephalic artery. This control relied on a computerized algorithm that maintained, for several hours, blood pressure, flow and pulsatility at near-native values individually measured before EPCC. Continuous electrocorticography and brain depth electrode recordings were used to evaluate brain activity relative to the standard offered by awake human electrocorticography. Under EPCC, this activity remained unaltered or minimally perturbed compared to the native circulation state, as did cerebral oxygenation, pressure, temperature and microscopic structure. Thus, our approach enables the study of neural activity and its circulatory manipulation in independence of most of the rest of the organism.

OBJECTIVE:Meningioma prognostication and treatment continues to evolve with an increasing understanding of tumor biology. In this study, the authors aimed to test conventional predictors of meningioma recurrence, histopathology variables for which there exists some controversy (brain invasion), as well as a novel molecular-based location paradigm.METHODS:This is a retrospective study of a consecutive series of patients with WHO grade I-III meningioma resected at The University of Texas Southwestern Medical Center between 1994 and 2015. Time to meningioma recurrence (i.e., recurrence-free survival [RFS]) was the primary endpoint measured. Kaplan-Meier curves were constructed and compared using log-rank tests. Cox univariate and multivariate analyses were performed to identify predictors of RFS.RESULTS:A total of 703 consecutive patients with meningioma underwent resection at The University of Texas Southwestern Medical Center between the years 1994 and 2015. A total of 158 patients were excluded for insufficient follow-up (< 3 months). The median age of the cohort was 55 years (range 16-88 years) and 69.5% (n = 379) were female. The median follow-up was 48 months (range 3-289 months). There was not a significantly increased risk of recurrence in patients with evidence of brain invasion, in patients with otherwise WHO grade I meningioma (Cox univariate HR 0.92, 95% CI 0.44-1.91, p = 0.82, power 4.4%). Adjuvant radiosurgery to subtotally resected WHO grade I meningiomas did not prolong the time to recurrence (n = 52, Cox univariate HR 0.21, 95% CI 0.03-1.61, p = 0.13, power 71.6%). Location (midline skull base, lateral skull base, and paravenous) was significantly associated with RFS (p < 0.01, log-rank test). In patients with high-grade meningiomas (WHO grade II or III), location was predictive of RFS (p = 0.03, log-rank test), with paravenous meningiomas exhibiting the highest rates of recurrence. Location was not significant on multivariate analysis.CONCLUSIONS:The data suggest that brain invasion does not increase the risk of recurrence in otherwise WHO grade I meningioma. Adjuvant radiosurgery to subtotally resected WHO grade I meningiomas did not prolong the time to recurrence. Location categorized by distinct molecular signatures did not predict RFS in a multivariate model. Larger studies are needed to confirm these findings.

Gliomas demonstrate diverse imaging features, variable response to therapy, and differences in prognosis. This is largely a function of genetic heterogeneity. Several key mutations serve as therapeutic and prognostic markers such as isocitrate dehydrogenase (IDH) mutation status, O6-methyl guanine-DNA methyltransferase (MGMT) promoter status, and 1p/19q co-deletion status. Currently, the gold standard for molecular marker determination requires tissue from either an invasive brain biopsy or surgical resection. Here we describe our work in developing highly accurate simultaneous deep learning segmentation and classification approaches for noninvasive profiling of molecular markers using T2-weighted magnetic resonance images only.

The epidermal growth factor receptor (EGFR) is a prime oncogene that is frequently amplified in glioblastomas. Here we demonstrate a new tumour-suppressive function of EGFR in EGFR-amplified glioblastomas regulated by EGFR ligands. Constitutive EGFR signalling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumours and decreased survival in orthotopic models. Ligand-activated EGFR promotes proliferation and surprisingly suppresses invasion by upregulating BIN3, which inhibits a DOCK7-regulated Rho GTPase pathway, resulting in small hyperproliferating non-invasive tumours and improved survival. Data from The Cancer Genome Atlas reveal that in EGFR-amplified glioblastomas, a low level of EGFR ligands confers a worse prognosis, whereas a high level of EGFR ligands confers an improved prognosis. Thus, increased EGFR ligand levels shift the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastomas by suppressing invasion. The tumour-suppressive function of EGFR can be activated therapeutically using tofacitinib, which suppresses invasion by increasing EGFR ligand levels and upregulating BIN3.

Objective: The midline location and relatively large size of olfactory groove meningiomas at diagnosis present a unique surgical challenge, with transcranial subfrontal, both unilateral and bilateral, and endoscopic endonasal approaches employed to access these tumors. While endoscopic approaches have been well-explored in the recent literature, few studies directly explore the difference between bilateral and unilateral approaches in accessing and treating olfactory groove meningiomas. In this study, we aim to compare extent of resection and clinical outcomes in patients treated for OGMs with bilateral and unilateral subfrontal approaches.

Objective This study aimed to assess the durability of audiological outcomes after radiation and surgery in the management of vestibular schwannoma. Study Design Retrospective review. Setting Tertiary academic center. Patients Adults with sporadic vestibular schwannoma and serviceable hearing at the time of intervention. Interventions Gamma Knife, middle cranial fossa, or retrosigmoid approaches. Main Outcome Measures Pure-tone audiometry and speech discrimination scores. Results Postintervention serviceable hearing (class A/B) was preserved in 70.4% (n = 130; mean follow-up, 3.31 yr; range, 0–15.25 yr). Of the 49 patients treated with radiation, 19 (39.6%) had serviceable hearing at last follow-up, compared with 38 (46.9% of 81) who underwent retrosigmoid (n = 36 [44.4%]) and middle cranial fossa (n = 45 [55.6%]) approaches (odds ratio [OR], 1.40; 95% confidence interval [CI], 0.67–2.82; p = 0.47). A matched analysis by age, tumor volume, and preintervention hearing (n = 38) also found no difference in hearing preservation (HP) likelihood between surgery and radiation (OR, 2.33; 95% CI, 0.24–35.91; p = 0.59). After initial HP, 4 (9.5%) surgical versus 10 (37.0%) radiated patients subsequently lost residual serviceable (A/B) hearing (OR, 0.18; 95% CI, 0.06–0.69; p = 0.01) at a mean 3.74 ± 3.58 and 4.73 ± 3.83 years after surgery and radiation, respectively. Overall, 5- and 10-year HP rates (A/B) after initially successful HP surgery were 84.4 and 63.0%, respectively. However, survival estimates declined to 48.9% at 5 years and 32.7% at 10 years when patients with immediate postoperative serviceable hearing loss were also included, which were comparable to radiation-HP rates at 5 and 10 years of 28.0 and 14.2%, respectively (p = 0.75). Conclusions After vestibular schwannoma intervention, overall HP was similar between radiated and surgical cohorts. However, when successful, surgical approaches offered more durable hearing outcomes at long-term follow-up.