Parney, Ian F.

Abstract Background While serial sampling of glioma tissue is rarely performed prior to recurrence, cerebrospinal fluid (CSF) is an underutilized longitudinal source of candidate glioma biomarkers for understanding therapeutic impacts. However, the impact of key variables to consider in longitudinal CSF samples for monitoring biomarker discovery, including anatomical location and post-surgical changes, remains unknown. Methods Aptamer-based proteomics was performed on 147 CSF samples from 74 patients; 71 of whom had grade 2–4 astrocytomas or grade 2–3 oligodendrogliomas. This included pre- versus post-resection intracranial CSF samples obtained at early (1–16 days; n = 20 patients) or delayed (86–153 days; n = 11 patients) time points for patients with glioma. Paired lumbar versus intracranial glioma CSF samples were also obtained (n = 14 patients). Results Significant differences were identified in the CSF proteome between lumbar, subarachnoid, and ventricular CSF in patients with gliomas. Importantly, we found that resection had a significant, evolving longitudinal impact on the CSF proteome, with distinct sets of proteins present at different time points since resection. Our analysis of serial intracranial CSF samples suggests the early potential for disease monitoring and evaluation of pharmacodynamic impact of targeted therapies, such as bevacizumab and immunotherapies. Conclusions The intracranial glioma CSF proteome serves as a rich and dynamic reservoir of potential biomarkers that can be used to evaluate the effects of resection and other therapies over time. All data within this study, including detailed individual clinical annotations, are shared as a resource for the neuro-oncology community to collectively address these unanswered questions and further understand glioma biology through CSF proteomics.

Abstract Cerebrospinal fluid (CSF) has emerged as a valuable liquid biopsy source for glioma biomarker discovery and validation. CSF produced within the ventricles circulates through the subarachnoid space, where the composition of glioma-derived analytes is influenced by the proximity and anatomical location of sampling relative to tumor, in addition to underlying tumor biology. The substantial gradients observed between lumbar and intracranial CSF compartments for tumor-derived analytes underscore the importance of sampling site selection. Moreover, radiographic features, such as tumor-CSF contact and blood-brain barrier disruption, are critical covariates that may affect biomarker detection and the abundance of plasma-derived analytes in CSF, respectively. Longitudinal intracranial CSF sampling, enabled by access devices like Ommaya reservoirs, may offer a window into treatment response and disease progression, though variability in analyte yield, sample volumes, and the dynamic effects of surgical resection pose challenges. This review critically evaluates the anatomic, radiographic, and longitudinal factors, or “time-space continuum,” that impact glioma CSF biomarker abundance. Practical considerations for longitudinal CSF biobanking, including access device placement and collection, are also reviewed. Key takeaways and recommendations for CSF glioma biomarker discovery and validation are provided as a “hitchhiker’s guide” based on our collective experience, along with resources for investigators aiming to develop CSF biobanking at their institutions.

Abstract Glioblastoma (GBM), the most common and deadly primary brain tumor, exerts profound local and systemic immunosuppressive effects through a variety of mechanisms. Tumor-derived extracellular vesicles (EVs) are increasingly appreciated as critical mediators of these effects. In surveying the tumor-immune microenvironment in samples of ultrasonic aspirate collected during surgical resection of GBM (n=13), we identified several key immunomodulatory markers expressed across a range of myeloid cell populations. Particularly, we found TIGIT, classically understood as a T cell inhibitory protein, expressed across a range of myeloid cell populations in patient samples, including in myeloid-derived suppressor cells (MDSCs, 46.84±16.78% of total monocytic MDSCs). Using EVs collected from patient-derived cell lines, we were able to recapitulate these effects upon co-culture with monocytes derived from the peripheral blood of healthy donors (n=7). Furthermore, Induced MDSCs significantly inhibit T cells proliferation (70.97±8.454%, compared to control 19.4±5.71; p<0.03), concomitant with a surge in Th2 immune responses via TIGIT/CD155 axis. This effect was reversed after TIGIT silencing, indicating a TIGIT-dependent immune suppression in the GBM microenvironment. Our study indicates EV-induced TIGIT upregulation in myeloid cells as a crucial immunosuppressive mechanism in glioblastoma, with potential therapeutic applications.

Abstract BACKGROUND Lynch syndrome is caused by pathogenic germline variants in the DNA mismatch repair (MMR) genes, predisposing individuals to malignancies, including glioblastoma (GBM). MMR-deficient tumors are resistant to monofunctional alkylators such as temozolomide but not to bifunctional alkylators such as the nitrosourea lomustine. Anti-programmed cell death protein 1 (PD1) immune checkpoint inhibitor (ICI) pembrolizumab represents another treatment option, which is approved by the United States Food and Drug Administration (FDA) for the treatment of mismatch repair deficient cancers. METHODS We present a patient with multifocal GBM and Lynch syndrome who experienced rapid progression following standard-of-care chemoradiation, followed by durable responses to lomustine and then to pembrolizumab. RESULTS A 58-year-old man with Lynch syndrome (MSH2 mutation) and prior history of colon cancer presented with seizures. Magnetic resonance imaging (MRI) brain identified right frontal and left parietal masses. Right frontal tumor was resected; pathology was consistent with GBM, IDH wildtype, MGMT-unmethylated. Both lesions were treated with radiation therapy (60 Gy, 30 fractions) with concurrent and adjuvant temozolomide. After two cycles of adjuvant temozolomide, his left parietal tumor progressed and was subtotally resected, followed by four cycles of lomustine (stopped due to myelotoxicity). He remained progression-free for 15 months. Surveillance MRI identified new enhancement involving the left frontal lobe, left periventricular white matter, and right internal auditory canal, concerning for leptomeningeal dissemination. MRI spine identified no additional lesions. Cerebrospinal fluid cytology was negative for tumor cells on two occasions. Patient was treated with pembrolizumab. He remains on treatment with stable disease four months after ICI initiation and 31 months after GBM diagnosis. CONCLUSIONS Patients with MMR-deficient GBM resist standard chemoradiation. Alternatives such as nitrosourea or ICI therapy may achieve durable responses. While data is insufficient to administer these agents as upfront therapy in this specific setting, both should be considered as options for recurrent disease.

Abstract Glioblastoma is the most common malignant primary brain tumor and is universally fatal. Treatment involves maximal safe resection with adjuvant chemoradiation. Poor prognosis in glioblastoma is multifactorial, and local and systemic immunosuppression is an important factor. Extracellular vesicles (EVs) are small, membrane-bound particles released by cells into the external environment. They play a key role in intercellular communication. In glioblastomas, EVs regulate immunosuppression through induction of myeloid-derived suppressor cells (MDSCs) in a process dependent on the PD-1/PD-L1 and IL-6 axes. Primary cilia are microtubule-based organelles that play crucial roles in various cellular processes and signal transduction cascades. We recently showed that primary cilia modulate IL-6 expression and consequently, MDSC induction. Comparative proteomic analysis of EVs derived from human glioblastoma cells devoid of cilia showed loss of ICAM-1 expression compared to control glioblastoma cells. ICAM-1 (Intercellular Adhesion Molecule-1) is a cell surface glycoprotein that mediates cell-cell adhesion and immune responses. In this study, we showed that depletion of primary cilia in human glioblastoma cells result in increased PD-L1 but loss of IL-6 and ICAM-1. RNAi-meidated loss of ICAM-1 was synergistic with primary cilia depletion in preventing MDSC inductionn normal human monocytes exposed to glioblastoma-derived EVs. Exogenous IFN-g was sufficient to restore IL-6 levels and MDSC induction despite loss of primary cilia and ICAM-1 suggesting that primary cilia act upstream of IFN-g, IL-6 and ICAM-1 in glioblastoma-mediated MDSC induction. This study is the first to connect primary cilia signal transduction to IL-6 and ICAM-1 expression and to MDSC induction. The evidence suggests a potential parallel primary cilia regulatory cascade independent of the PD-L1 axis that controls tumor-mediated immunosuppression. These results may inform novel therapeutic strategies aimed at increasing efficacy of immunotherapy in the glioblastoma and other neoplastic processes.

Abstract BACKGROUND Glioma radiotherapy planning requires accurate tumor delineation. While T1- contrast-enhanced-MRI (T1-CE-MRI) is the standard, due to blood-brain-barrier breakdown dependencies, T1-CE-MRI fails to reflect the extent of glioma. Studies using amino acid PET tracers report glioma uptake up to 3cm-4cm beyond T1-CE. In this study, amino acid tracer 18F-fluoro-dihydroxyphenylalanine (18F-DOPA) uptake was correlated with histopathological features to determine tumor thresholds. METHODS Using registered 18F-DOPA-PET and T1-CE-MRI images in the Stealth Neuronavigation system targeting concordant and discordant regions, 181 stereotactic biopsy samples (newly-diagnosed=74; recurrent=107) were obtained from 59 enrolled astrocytoma patients across two prospective phase II clinical trials between 2010-2019. An experienced neuropathologist reviewed each sample with hematoxylin and eosin stain and assigned an alphanumeric score: mitotic activity (0=no tumor; 1=no mitosis; 2=few/scattered mitosis; 3=frequent mitosis) and cellular density (A=<25%; B=25%-50%; C=50%-75%; D=>75%). Samples assigned 1C-1D, 2B-2D, or 3A-3D were categorized as histopathological high-score (aggressive disease), remaining samples as low-score. Relative mean (rFDOPA_mean) tumor-to-background uptake ratios were computed for 5mm uniformly expanded volumes from each Stealth biopsy coordinate using the crescent and wedge normalization methods. Relative uptake was compared overall and separately within newly-diagnosed/recurrent disease subgroups. An optimal rFDOPA_mean threshold to delineate high- from low-score was determined using the maximal Youden index of ROC curves. RESULTS Overall, high-score samples (n=116) had higher median (IQR) rFDOPA_mean uptake than low-score samples (n=65): high-score rFDOPA:mean=1.70(1.16-2.26), low-score rFDOPA:mean=1.16(0.83-1.49), Kruskal-Wallis p<0.001. The uptake difference between high- vs. low-score samples was observed within newly-diagnosed and recurrent subgroups, but was only significant for newly-diagnosed (p<0.001). For newly-diagnosed, an rFDOPA:mean=1.49 cutoff between high- and low-score groups shows 69% accuracy, 63% sensitivity, 80% specificity (AUC=0.782). Using the wedge normalization method, rFDOPA_mean values were 0.2 higher than crescent method values. CONCLUSIONS This work presents histologically verified 18F-DOPA-PET thresholds for aggressive disease in newly-diagnosed astrocytoma patients. Separate thresholds will need to be determined for recurrent glioma patients.

Abstract BACKGROUND Mouse double minute 2 (MDM2) inhibits the tumor suppressor protein 53 (p53). Brigimadlin, a potent oral MDM2-p53 antagonist, restores wild-type (wt) p53 function and has shown early efficacy in patients with solid tumors. Glioblastoma (GBM) is an area of unmet need with 5-year survival at <10%. In p53 wt GBM patient-derived xenograft models, brigimadlin promotes tumor cell apoptosis and extends survival in combination with radiotherapy (RT). METHODS NCT05376800 is a Phase 0/Ia non-randomized, open-label, single-arm trial that aims to measure brigimadlin concentration in brain tumor tissue in patients with histologically or radiologically newly diagnosed GBM eligible for resection (Phase 0), and determine the maximum tolerated dose of brigimadlin plus RT in patients with TP53 wt, IDH wt, MGMT promoter unmethylated GBM (Phase Ia). In Phase 0, patients received one brigimadlin dose (30 mg or 45 mg) ~12-24 hours before resection. Phase 0 primary endpoints are the measured total concentration and the calculated unbound concentration of brigimadlin in brain tissue homogenate from non-contrast enhancing (NCE) and contrast enhancing (CE) regions. The predefined threshold for trial continuation is 0.5 nmol/L (corresponding to IC50 in GBM cell lines) unbound brigimadlin in CE samples in ≥50% of patients. Brigimadlin concentration was measured using liquid chromatography-mass spectrometry and corrected for amount of brigimadlin in residual blood. Unbound concentration was calculated using an in vitro estimate of unbound fraction (fu): 0.654% (rat brain slice). fu in human plasma was 0.22%. Ratio of unbound concentration in brain versus plasma (Kp,uu) was calculated. Biomarker testing was performed. RESULTS Data are available for 11 patients (brigimadlin 30 mg: n=6; 45 mg: n=5). In the 30 mg group, median total brigimadlin concentration in NCE samples was 267 nmol/L (4 samples, range 86-316 nmol/L) and 332 nmol/L (6 samples, range 272-952 nmol/L) in CE samples. In the 45 mg group, median total brigimadlin concentration in NCE samples was 197 nmol/L (5 samples, range 140-347 nmol/L) and 603 nmol/L (5 samples, range 441-905 nmol/L) in CE samples. Unbound concentration exceeded the 0.5 nmol/L threshold in most cases. Post-brigimadlin, an increase in selected p53 target gene expression was observed in CE versus NCE tissue. CONCLUSION Unbound brigimadlin concentrations in CE regions in all patients receiving the low dose of 30 mg brigimadlin exceeded the 0.5 nmol/L threshold. Kp,uu in most patients was close to 1 in CE regions. Biomarker data support target engagement in brain tissue. Our findings support continued investigation of brigimadlin in GBM. Recruitment is ongoing. Updated data will be presented.The trial is funded by Boehringer Ingelheim.

OBJECTIVE Following resection of posterior superior frontal gyrus (PSFG) tumors, patients can experience supplementary motor area (SMA) syndrome consisting of contralateral hemiapraxia and/or speech apraxia. Given the heterogeneity of PSFG tumors, the authors sought to determine the risk of postoperative deficits and assess predictors of outcomes for all intraparenchymal PSFG tumors undergoing surgery (biopsy or resection), regardless of histology. METHODS This was a retrospective single-center cohort study of adult PSFG-region tumors undergoing biopsy or resection by a single surgeon. RESULTS A total of 106 consecutive patients undergoing 123 procedures (21 biopsies, 102 resections) fulfilled inclusion and exclusion criteria. Anaplastic astrocytomas were the most frequent among resected tumors (39% vs 29%), while glioblastomas were most common among biopsies (38% vs 27%) (p < 0.0001). The biopsy cohort was more likely to have tumor involvement outside the PSFG (90% vs 62%) (p = 0.011), most commonly in the motor cortex (67% vs 31%) (p = 0.005). Seizures were the most common presenting symptom in the resection cohort (p = 0.017), while motor deficits were more common in the biopsy cohort (58% vs 29%) (p < 0.001). Immediate postoperative neurological deficits occurred in 71 cases (58%), but only 3 of the deficits were permanent at 6 months of follow-up (2%). Postoperative SMA syndrome occurred in 48 cases (47%) and was significantly associated with involvement of the motor cortex (p = 0.018) or cingulate gyrus (p = 0.023), which were also significant in multivariate analysis as risk factors for SMA syndrome. However, postoperative SMA syndrome was not significantly associated with overall survival (p = 0.51). There were no perioperative deaths, but corpus callosum involvement (p < 0.001), contrast enhancement (p = 0.003), and glioblastoma pathology (p = 0.038) predicted worse overall survival in patients undergoing resection. CONCLUSIONS Nearly half of all patients undergoing resection of PSFG-region tumors experience a postoperative SMA syndrome. Individuals with corpus callosum and/or motor cortex involvement may be at an increased risk of experiencing SMA syndrome. However, these deficits are usually transient, and the risk of permanent new deficits is very low (3%). Preoperative characteristics including corpus callosum involvement and tumor enhancement—in addition to pathology—might serve as predictors of overall survival within this patient population.

2017 Background: MDM2 inhibits tumor suppressor p53. Brigimadlin, a potent MDM2–p53 antagonist, restores wild-type (wt) p53 function and has shown early efficacy in pts with solid tumors (LoRusso et al Cancer Disc 2023). GBM is an area of unmet need with 5-year survival <10%. In p53 wt GBM pt-derived xenograft models, brigimadlin promotes tumor cell apoptosis and extends survival in combination with RT. Methods: NCT05376800 is a Ph 0/Ia open-label, single-arm trial that aims to measure brigimadlin concentration in brain tumor tissue in pts with histologically or radiologically newly diagnosed GBM eligible for resection (Ph 0) and determine the maximum tolerated dose of brigimadlin plus RT in pts with TP53wt, IDH wt, MGMT promoter unmethylated GBM (Ph Ia). In Ph 0, pts received one brigimadlin dose (30 mg or 45 mg) ~12–24 h before resection. Ph 0 primary endpoints are the measured total concentration and the calculated unbound concentration of brigimadlin in brain tissue homogenate from non-contrast enhancing (NCE) and contrast enhancing (CE) regions. The predefined threshold for trial continuation is 0.5 nmol/L (corresponding to IC50 in GBM cell lines) unbound brigimadlin in CE samples in ≥50% of pts. Brigimadlin concentration was measured using LC/MS and corrected for amount of brigimadlin in residual blood. Unbound concentration was calculated using an in vitro estimate of unbound fraction (fu): 0.654% (rat brain slice). fu in human plasma was 0.22%. Kp,uu (ratio of unbound concentration in brain vs plasma) was calculated. Biomarker testing was performed. Results: Data are available for 11 pts (brigimadlin 30 mg: n=6; 45 mg: n=5). In the 30 mg group, median total brigimadlin concentration in NCE samples was 267 nmol/L (4 samples, range 86–316 nmol/L) and 332 nmol/L (6 samples, range 272–952 nmol/L) in CE samples. In the 45 mg group, median total brigimadlin concentration in NCE samples was 197 nmol/L (5 samples, range 140–347 nmol/L) and 603 nmol/L (5 samples, range 441–905 nmol/L) in CE samples. Unbound concentration exceeded the 0.5 nmol/L threshold in most cases (Table). Post-brigimadlin, an increase in selected p53 target gene expression was observed in CE vs NCE tissue. Conclusions: Unbound brigimadlin concentrations in CE regions in all pts receiving the low dose of 30 mg brigimadlin exceeded the 0.5 nmol/L threshold. Kp,uu in most patients was close to 1 in CE regions. Biomarker data support target engagement in brain tissue. Our findings support continued investigation of brigimadlin in GBM. Recruitment is ongoing. Updated data will be presented. Clinical trial information: NCT05376800 . [Table: see text]

Dilated perivascular spaces (PVSs) are common and easily recognized on imaging. However, rarer giant tumefactive PVSs (GTPVSs) can have unusual multilocular cystic configurations, and are often confused for other pathologic entities, including neoplasms, cystic infarctions, and neuroepithelial cysts. Because GTPVSs are scarcely encountered and even more infrequently operated upon, many radiologists are unaware of the imaging and pathologic features of these lesions. Here, a case of a resected GTPVS is presented, highlighting both its radiologic and histologic characteristics, and discussing how such lesions can be differentiated from their closest mimickers on imaging.