Vaubel, Rachael A.

Abstract Pleomorphic xanthoastrocytomas (PXAs) harbor CDKN2A homozygous deletion in >90% of cases, resulting in loss of p16 expression by immunohistochemistry. Considering the proximity of MTAP to CDKN2A and their frequent concurrent deletions, loss of MTAP expression may be a surrogate for CDKN2A homozygous deletion. We evaluated p16 and MTAP expression in 38 patient PXAs (CNS WHO grade 2: n = 23, 60.5%; grade 3: n = 15, 39.5%) with available chromosomal microarray data to determine whether MTAP can be utilized independently or in combination with p16 to predict CDKN2A status. CDKN2A, CDKN2B, and MTAP homozygous deletion were present in 37 (97.4%), 36 (94.7%), and 25 (65.8%) cases, respectively. Expression of p16 was lost in 35 (92.1%) cases, equivocal in one (2.6%), and failed in 2 (5.3%), while MTAP expression was lost in 27 (71.1%) cases, retained in 10 (26.3%), and equivocal in one (2.6%). This yielded a sensitivity of 94.6% for p16 and 73.0% for MTAP in detecting CDKN2A homozygous deletion through immunohistochemistry. MTAP expression was lost in the 2 cases with failed p16 staining (combined sensitivity of 100%). Our findings demonstrate that combined p16 and MTAP immunostains correctly detect CDKN2A homozygous deletion in PXA, while MTAP expression alone shows reduced sensitivity.

Abstract Background The optimal diagnosis and management of patients with brain tumors currently uses the 2021 WHO integrated diagnosis of histomorphologic and molecular features. However, neuro-oncology practice in resource-limited settings usually relies solely on histomorphology. This study aimed to classify glioma cases diagnosed in the Department of Anatomic and Molecular Pathology, Lagos University Teaching Hospital, using the 2021 WHO CNS tumor classification. Methods Fifty-six brain tumors from 55 patients diagnosed with glioma between 2013 and 2021 were reevaluated for morphologic diagnosis. Molecular features were determined from formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) for IDH1-R132H, ATRX, BRAF-V600E, p53, Ki67, and H3-K27M, OncoScan chromosomal microarray for copy number, targeted next generation sequencing for mutation and fusion and methylation array profiling. Results Of 55 central nervous system tumors, 3 were excluded from histomorphologic reevaluation for not being of glial or neuroepithelial origin. Of the remaining 52 patients, the median age was 20.5 years (range: 1 to 60 years), 38(73%) were males and 14(27%) were females. Seventy-one percent of the gliomas evaluated provided adequate DNA from archival FFPE tissue blocks. After applying the 2021 WHO diagnostic criteria the initial morphologic diagnosis changed for 35% (18/52) of cases. Diagnoses of 5 (9.6%) gliomas were upgraded, and 7 (14%) were downgraded. Conclusions This study shows that the incorporation of molecular testing can considerably improve brain tumor diagnoses in Nigeria. Furthermore, this study highlights the diagnostic challenges in resource-limited settings and what is at stake in the global disparities of brain tumor diagnosis.

2071 Background: Brigimadlin (BI 907828) is a potent small molecule inhibitor of the p53-MDM2 pathway currently in a phase 0/1 clinical trial in combination with radiotherapy in newly diagnosed MGMTunmethylated glioblastoma (GBM). To inform development of brigimadlin for GBM, we developed a pharmacokinetic (PK)/ pharmacodynamic (PD)/ efficacy model using GBM patient derived xenografts (PDXs). Methods: Brigimadlin PK parameters were evaluated in FVB wild-type and knockout (Mdr1a/b-/-Bcrp1-/-) mice and in athymic nude mice. Drug binding was measured by rapid equilibrium dialysis, and drug levels quantified by LC/MS-MS. Dose-response relationships were evaluated in subcutaneous and orthotopic PDXs across a range of doses. Response to brigimadlin (2 mg/kg weekly) +/- RT (2 Gy x 10 fractions) was evaluated in orthotopic PDXs. Results: Brigimadlin significantly delayed median time to regrowth of GBM108 ( MDM2amplified) subcutaneous tumors (vs. placebo; p<0.05) at dose levels of 0.25 mg/kg (1.3-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (2.6-fold), and 2 mg/kg (5.3-fold). A single dose of brigimadlin resulted in corresponding intra-tumoral drug levels 24 hours post-dose: 84 nM (0.25 mg/kg), 175 nM (0.5 mg/kg), 398 nM (1 mg/kg) and 924 nM (2 mg/kg) and dose-dependent upregulation of p53 target genes (p21 and PUMA). In contrast, GBM14 ( MDM2non-amplified) showed regrowth delay (1.6-fold) only at 2 mg/kg (p=0.008). Brigimadlin was highly bound (fraction unbound = 0.0006 in brain; 0.0017 in GBM tumor tissue), and CNS distribution was limited by efflux; in wild-type mice Kp,uu_brain = 0.002 ± 0.003; in Mdr1a/b-/-Bcrp1-/- mice Kp,uu = 0.043 ± 0.025. Efficacy of brigimadlin was compared in orthotopic GBM108 PDXs grown in athymic nude mice vs. immuno/efflux-deficient (Rag2-/-Mdr1a-/-Bcrp1-/-) mice. In nude mice, median survival was extended at 2 mg/kg brigimadlin (1.4-fold, p=0.009), with corresponding intra-tumoral drug levels of 29 nM. In efflux deficient mice, median survival was extended (p<0.05) at 0.25 mg/kg (1.5-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (1.8-fold), and 2 mg/kg (>4-fold). Measurement of intra-tumoral drug levels is in progress. In combination with RT in GBM108, brigimadlin (2 mg/kg x 2 weeks) extended median survival vs. vehicle (66 vs. 50 days; p=0.012) and enhanced median survival following RT (128.5 vs. 82 days; p=0.009). In GBM14, 2 mg/kg brigimadlin (2 mg/kg x 12 weeks) extended median survival (39.5 vs. 32.5 days; p=0.0004) and enhanced survival following RT (97 vs. 81 days; p<0.0001). Conclusions: Efficacy of brigimadlin is dependent on adequate CNS delivery. Studies in subcutaneous PDX demonstrated intra-tumoral total drug levels in the mid-nanomolar range provide meaningful tumor effects in a highly sensitive, MDM2 amplified PDX. In orthotopic PDXs with and without MDM2 amplification, brigimadlin provided further benefit in combination with RT. These data support ongoing clinical evaluation of brigimadlin in GBM.

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]

2012 Background: Navtemadlin (nvtm) is a potent, selective, orally available small molecule inhibitor of murine double minute 2 homologue (MDM2), which has completed a Ph 0 surgical window of opportunity study in glioblastoma, IDH-wildtype (GBM; Lee SNO 2022). To optimally interpret the clinical data, an extensive evaluation of nvtm PK, PD, and efficacy was performed in GBM patient derived xenografts (PDXs). Methods: Response to nvtm +/- radiotherapy (RT) was characterized in vitro and in vivo across a panel of GBM PDXs with various TP53 and MDM2 alterations. Efficacy, PK, and PD were evaluated in flank or orthotopic PDX models. p53 pathway activation was evaluated by qPCR, Western blot, and IHC. Nvtm brain penetration was evaluated in immunodeficient abcb1a/b-/- (P-glycoprotein; P-gp), abcg2-/- (BCRP), or double-knockout mice. Binding was assessed by rapid equilibrium dialysis, and nvtm concentrations by LC/MS-MS and MALDI-MSI. Results: Nvtm decreased in vitro viability of TP53WT GBM PDX explant cultures with IC50s <100 nM, but was ineffective in TP53-mutant GBM. In flank PDX models, nvtm (100 mg/kg daily for 4 weeks) delayed tumor regrowth by 1.9 to 4.8-fold in TP53 WT PDXs without MDM2amplification (n=9 models), and more markedly by 20.1 to 29.4-fold in MDM2amplified models (n=4; 2 PDXs did not recur). In a flank PDX dose-response study, nvtm delayed the growth of MDM2amplified GBM108 at 25 mg/kg vs. placebo (61 vs. 11 days; p=0.014; mean intra-tumoral nvtm = 590 nM). In contrast, growth of MDM2 non-amplified GBM14 was delayed only at 100 mg/kg (37 vs. 15 days; p=0.082; mean intra-tumoral nvtm = 2990 nM). In both flank models, p53 target gene upregulation was similar between 25 and 100 mg/kg and did not correlate with regrowth. Nvtm was highly bound (fraction unbound = 0.090 in culture media and 0.014 in GBM tissue homogenate). In mice after oral dosing, distribution to CNS was limited by P-gp, with a brain to plasma ratio of 0.009 and unbound ratio of 0.005, whereas BCRP deficiency did not affect CNS distribution. With orthotopic GBM108, nvtm efficacy was evaluated in nude vs. efflux-deficient mice. In nude mice, nvtm was ineffective at doses up to 100 mg/kg; however, in efflux deficient mice, the mean intra-tumoral nvtm level at 25 mg/kg was similar to flank tumors (690 nM) and survival was significantly extended (134 vs. 62 days, p=0.0002) with additional benefit at 100 mg/kg (>300 vs. 62 days, p=0.002). Conclusions: Nvtm showed significant efficacy in TP53 WT GBM flank PDX models but efficacy in orthotopic PDX was highly dependent on CNS penetration. Targeting nvtm levels based on the in vitroIC50 may significantly underestimate the in vivo effective concentration . In vivo studies showed lower intra-tumoral nvtm levels are required to significantly inhibit growth of MDM2amplified compared with non-amplified tumors.

Tumor evolution is driven by genetic variation; however, it is the tumor microenvironment (TME) that provides the selective pressure contributing to evolution in cancer. Despite high histopathological heterogeneity within glioblastoma (GBM), the most aggressive brain tumor, the interactions between the genetically distinct GBM cells and the surrounding TME are not fully understood. To address this, we analyzed matched primary and recurrent GBM archival tumor tissues with imaging-based techniques aimed to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix (ECM) organization, TERT promoter mutational status, and several oncogenic amplifications on the same slide and location. We found that the relationships between genetic and TME diversity are different in primary and matched recurrent tumors. Interestingly, the texture of the ECM, identified by label-free reflectance imaging, was predictive of single-cell genetic traits present in the tissue. Moreover, reflectance of ECM revealed structured organization of the perivascular niche in recurrent GBM, enriched in immunosuppressive macrophages. Single-cell spatial transcriptomics further confirmed the presence of the niche-specific macrophage populations and identified interactions between endothelial cells, perivascular fibroblasts, and immunosuppressive macrophages. Our results underscore the importance of GBM tissue organization in tumor evolution and highlight genetic and spatial dependencies.

Abstract Purpose: Antibody-drug conjugates (ADC) are targeted therapies with robust efficacy in solid cancers, and there is intense interest in using EGFR-specific ADCs to target EGFR-amplified glioblastoma (GBM). Given GBM’s molecular heterogeneity, the bystander activity of ADCs may be important for determining treatment efficacy. In this study, the activity and toxicity of two EGFR-targeted ADCs with similar auristatin toxins, Losatuxizumab vedotin (ABBV-221) and Depatuxizumab mafodotin (Depatux-M), were compared in GBM patient-derived xenografts (PDX) and normal murine brain following direct infusion by convection-enhanced delivery (CED). Experimental Design: EGFRviii-amplified and non-amplified GBM PDXs were used to determine in vitro cytotoxicity, in vivo efficacy, and bystander activities of ABBV-221 and Depatux-M. Nontumor-bearing mice were used to evaluate the pharmacokinetics (PK) and toxicity of ADCs using LC-MS/MS and immunohistochemistry. Results: CED improved intracranial efficacy of Depatux-M and ABBV-221 in three EGFRviii-amplified GBM PDX models (Median survival: 125 to &gt;300 days vs. 20–49 days with isotype control AB095). Both ADCs had comparable in vitro and in vivo efficacy. However, neuronal toxicity and CD68+ microglia/macrophage infiltration were significantly higher in brains infused with ABBV-221 with the cell-permeable monomethyl auristatin E (MMAE), compared with Depatux-M with the cell-impermeant monomethyl auristatin F. CED infusion of ABBV-221 into the brain or incubation of ABBV-221 with normal brain homogenate resulted in a significant release of MMAE, consistent with linker instability in the brain microenvironment. Conclusions: EGFR-targeting ADCs are promising therapeutic options for GBM when delivered intratumorally by CED. However, the linker and payload for the ADC must be carefully considered to maximize the therapeutic window.

Abstract Ganglioglioma (GG) with anaplasia (anaplastic ganglioglioma) is a rare and controversial diagnosis. When present, anaplasia involves the glial component of the tumor, either at presentation or at recurrence. To date, most published cases lack molecular characterization. We describe the histologic and molecular features of 3 patients presenting with BRAF p. V600E-mutant GG (CNS WHO grade 1) with high-grade glial transformation at recurrence. The tumors occurred in pediatric patients (age 9–16 years) with time to recurrence from 20 months to 7 years. At presentation, each tumor was low-grade, with a BRAFV600E-positive ganglion cell component and a glial component resembling pleomorphic xanthoastrocytoma (PXA) or fibrillary astrocytoma. At recurrence, tumors resembled anaplastic PXA or high-grade astrocytomas without neuronal differentiation. CDKN2A homozygous deletion (HD) was absent in all primary tumors. At recurrence, 2 cases acquired CDKN2A HD; the third case showed loss of p16 and MTAP immunoexpression, but no CDKN2A/B HD or mutation was identified. By DNA methylation profiling, all primary and recurrent tumors either grouped or definitely matched to different methylation classes. Our findings indicate that malignant progression of the glial component can occur in GG and suggest that CDKN2A/B inactivation plays a significant role in this process.

Abstract Glioblastoma (GBM) is a highly aggressive and infiltrative primary malignant brain tumor. Current standard-of-care for newly diagnosed GBM patients includes maximal safe surgical resection, followed by concurrent radiation with temozolomide (TMZ), and adjuvant TMZ chemotherapy. However, despite aggressive treatments, GBM patients have a poor prognosis, with a median survival of 14.6 months and a 5-year survival rate of 6.8%. The p53 protein is a critical tumor suppressor in the cell cycle. The expression level of p53 is tightly controlled by its negative regulator, murine double minute 2 (MDM2). Amplification of the MDM2 gene, found in 14 % of GBMs, can inactivate p53, leading to loss of tumor suppressor functions. Therefore, we are investigating a potent MDM2 antagonist – BI-907828 in GBM to restore the p53 tumor suppressor function by inhibiting the p53/MDM2 interactions. We aim to evaluate the PK/PD of BI-907828 and demonstrate a new translational paradigm to rethink the “Delivery – Potency – Efficacy” relationship in drug discovery and development for central nervous system (CNS) tumors. BI-907828 was evaluated for cytotoxicity and the inhibition effect to MDM2-p53 pathway. Systemic PK and transport mechanisms controlling CNS distribution were evaluated in wild-type and transgenic mice that lack of efflux transporters P-gp and BCRP. BI-907828 unbound fractions in cell media, mouse and human specimens were determined by using rapid equilibrium dialysis to evaluate “active” concentrations. Efficacy measures, including survival and free drug distribution in tumor were assessed in mouse orthotopic GBM xenografts. BI-907828 exhibited potent inhibition of MDM2-p53 pathway and promoted cell death in GBM TP53 wild-type cells. MDM2-amplified cells are highly sensitive to BI-907828, with an effective unbound concentration of 0.1 nM. The CNS distribution of BI-907828 is limited by BBB efflux mediated by P-gp, resulting in a brain free partition coefficient Kp,uu of 0.002. Despite this seemingly “poor” BBB penetration, weekly administration of 10 mg/kg BI-907828 significantly extended median survival of orthotopic GBM108 xenografts to 218 days compared to 28 days with vehicle treatment (P &lt; 0.0001). These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is severely limited by BBB efflux. In another words, in this case, this little drug distribution to brain is “enough” for extending survival of orthotopic GBM108 xenografts. Therefore, a comprehensive understanding of drug “Delivery – Potency – Efficacy” relationship is warranted in drug discovery development, especially for treatment of CNS tumors. Citation Format: Wenjuan Zhang, Rachael Vaubel, Juhee Oh, Ann Mlade, Surabhi Talele, Wenqiu Zhang, Katie Waller, Danielle Burgenske, Jann Sarkaria, William Elmquist. How much is enough? BI-907828: a MDM2-p53 antagonist with limited BBB penetration but potent efficacy in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B025.

ATM is a key mediator of radiation response, and pharmacological inhibition of ATM is a rational strategy to radiosensitize tumors. AZD1390 is a brain-penetrant ATM inhibitor and a potent radiosensitizer. This study evaluated the spectrum of radiosensitizing effects and the impact of TP53 mutation status in a panel of IDH1 wild-type (WT) glioblastoma (GBM) patient-derived xenografts (PDXs). AZD1390 suppressed radiation-induced ATM signaling, abrogated G 0 -G 1 arrest, and promoted a proapoptotic response specifically in p53-mutant GBM in vitro. In a preclinical trial using 10 orthotopic GBM models, AZD1390/RT afforded benefit in a cohort of TP53 -mutant tumors but not in TP53 -WT PDXs. In mechanistic studies, increased endogenous DNA damage and constitutive ATM signaling were observed in TP53 -mutant, but not in TP53 -WT, PDXs. In plasmid-based reporter assays, GBM43 ( TP53 -mutant) showed elevated DNA repair capacity compared with that in GBM14 (p53-WT), whereas treatment with AZD1390 specifically suppressed homologous recombination (HR) efficiency, in part, by stalling RAD51 unloading. Furthermore, overexpression of a dominant-negative TP53 (p53DD) construct resulted in enhanced basal ATM signaling, HR activity, and AZD1390-mediated radiosensitization in GBM14. Analyzing RNA-seq data from TCGA showed up-regulation of HR pathway genes in TP53 -mutant human GBM. Together, our results imply that increased basal ATM signaling and enhanced dependence on HR represent a unique susceptibility of TP53 -mutant cells to ATM inhibitor–mediated radiosensitization.