Porter, Alyx B.

Abstract High-grade gliomas (HGGs) are aggressive tumors with poor outcomes and limited treatment options. Here, we combined genomic and transcriptomic tumor profiling with drug testing in a patient-derived 3-dimensional culture model to identify individualized treatments and predictive biomarkers. Activity of single agents targeting frequently dysregulated glioma pathways was relatively poor ex vivo and generally reflected historical patient data. However, compounds targeting PI3K, epigenetic, and survival/senescence signaling were effective in some cases. Drug sensitivity correlated with transcriptional rather than genomic features and suggested heterogeneity as a resistance mechanism. Bromodomain and extraterminal domain inhibition was particularly effective in tumors enriched in the mesenchymal transcriptional subtype, promoted proneural transition, and was overcome by upregulated PI3K signaling. Notably, combinations were largely effective, with 6 strategies exhibiting stronger efficacy than corresponding single agents in most cases (58-77%). This study identifies HGG vulnerabilities and associated biomarkers, resistance mechanisms, and effective combination strategies that warrant further clinical validation. Graphical Abstract

Abstract BACKGROUND The Response Assessment in Neuro-Oncology (RANO) was recently updated to RANO 2.0, which no longer requires follow-up imaging to confirm progression in recurrent glioma patients recieving immunotherapy, as previously recommended by iRANO. This study evaluates the utility of RANO 2.0 to determine whether radiographic progression on initial post-treatment MRI is sufficient to guide timely discontinuation of ineffective therapy. METHODS This retrospective study, conducted at Mayo Clinic, included patients ≥18 years receiving ≥2 cycles of off-label pembrolizumab for recurrent glioma between 1/1/2014 – 5/1/2025. Radiographic response was assessed using the first MRI performed ≥30 days after pembrolizumab initiation. Progression was defined as a ≥25% increase in the product of perpendicular diameters of the lesion, clear progression of non-measurable disease, new lesion appearance, clinical deterioration, or death. For suspected pseudo-progression, sequential MRIs were reviewed; if progression was confirmed, the original date was used. RESULTS Sixty-two patients were included (mean age: 46.1 years; 64.5% male), receiving pembrolizumab for a median duration of 2.8 (1.4 – 19.4) months. Median time to initial radiographic response assessment was 1.6 (1.1 – 3.5) months, and progression-free survival was 2.3 (0.7 – 15.2) months. Thirty-seven (59.7%) patients were identified with progression at initial evaluation. Among them, 11 were initially suspected of pseudo-progression, and continued pembrolizumab: 3 received additional radiation, 2 initiated bevacizumab, while 6 remained on their prior regimens [pembrolizumab only (3); pembrolizumab with bevacizumab (3)]. However, all were subsequently confirmed to have true progression, with a median delay of 1.4 (0.7 – 7.1) months until pembrolizumab discontinuation. No true pseudo-progression cases were observed. CONCLUSIONS Our findings support the clinical applicability of RANO 2.0 in recurrent glioma patients receiving pembrolizumab. Initial radiographic progression reliably predicted true progression, suggesting limited value in confirmatory follow-up imaging. Early recognition of treatment failure may reduce delays in initiating salvage therapies, along with associated toxicity and cost.

Abstract WSD0922-Fu is an oral, CNS-penetrant, small molecule EGFR inhibitor which potently inhibits EGFR aberrations specific to non-small cell lung cancer (NSCLC) and glioblastoma (GBM). MC1914 (NCT04197934) is a first-in-human phase 1 trial in patients with EGFR aberrant recurrent GBM and NSCLC. After determining the maximum tolerated dose, adults with recurrent GBM were treated in two expansion cohorts: (a) non-surgical cohort (6/12 patients thus far): EGFRvIII mutation, first recurrence; food-effect study included (b) surgical cohort (6/6 patients), any EGFR mutation, any recurrence; window-of-opportunity study (WOO) included. Among these 12 patients, median age = 60 years; 50% female; 58% ECOG 0). All patients received prior radiation and temozolomide. After the WOO or food-effect study, patients were dosed at 120 mg BID (5 days on, 2 days off), and escalated to 160 mg BID (5 days on, 2 days off) based on tolerability. Common adverse events (>40%) are comparable to other EGFR inhibitors (acneiform rash, diarrhea, fatigue, xerosis, pruritis). Treatment-related grade 3+ adverse events were limited to lymphopenia (n = 1). WSD0922-Fu exposure in the fed state exceeded the fasted state (median plasma Cmax and AUC0-24h are 1.6x and 1.3x, respectively) with a delayed Tmax (6.0 vs 1.8 hours). For the WOO, time between last dose administered to tumor collection ranged between 4 - 13.5 hours. Median tumor WSD0922-Fu concentration was 0.76 µg/g; estimated median unbound tumor concentration was 24.9 ng/g and within expectations for pharmacodynamic effect. Median brain/plasma ratio = 0.59; median unbound brain/unbound plasma = 0.88. Enrollment to the non-surgical cohort is ongoing, so formal efficacy data remain immature. However, examples of clinical efficacy will be shown along with data on recurrence patterns. Phase 2 clinical trial development is in progress.

Abstract BACKGROUND Pembrolizumab is FDA approved for solid tumors with high tumor mutation burden (TMB) and may thus have efficacy in hypermutant glioma. This study aimed to identify factors influencing efficacy of pembrolizumab in recurrent glioma. METHODS This retrospective study at Mayo Clinic included patients with adult-type diffuse gliomas who received pembrolizumab (≥2 cycles) for recurrent disease (01/01/2014–05/01/2025). The Mayo Clinic Solid Tumor Panel (MCSTP), which uses the Illumina TruSight Oncology 500 High-Throughput next-generation sequencing (NGS) assay, was also used to assess the molecular characteristics of the recurrent tumors. Responders to pembrolizumab were defined as patients who did not meet RANO 2.0 criteria for progressive disease on first radiographic response assessment. Progression-free survival (PFS) was analyzed using Kaplan-Meier and Cox-Regression analyses. RESULTS Sixty-two patients were identified (median age: 46 years; 65% male; 94% white). All patients received pembrolizumab and had prior alkylating chemotherapy. The median PFS from pembrolizumab initiation was 2.3 months. Twenty-five (40%) patients were responders. Patients with IDH-mutant, 1p/19q co-deleted gliomas (OLIGO, n=13) had a longer PFS (4.3 months) than IDH wildtype gliomas (IDH-WT, n=33, 2.2 months, p=0.012) and IDH-mutant, 1p/19q non-co-deleted (IDH-MUT, n=16, 1.7 months, p=0.0006). The proportion of responders was significantly greater in OLIGO patients (IDH-WT, 33%; IDH-MUT, 19%; OLIGO, 85%, p=0.0008). Overall PFS was not impacted by patient age, sex, or extent of initial resection. Twenty-nine percent of patient tumors tested thus far (6/21) had a high TMB (≥10 mut/Mb). Two-thirds (4/6) of high TMB patients were responders, while only one-third (5/15) of low TMB patients were responders. CONCLUSIONS Patients with recurrent OLIGO may have a longer PFS with pembrolizumab therapy than IDH-WT and IDH-MUT. Furthermore, high TMB may be associated with longer PFS with pembrolizumab therapy in glioma. Additional patients are being assessed using MCSTP (total expected n=43); complete data will be presented.

Abstract BACKGROUND Pembrolizumab elicits durable responses in many solid tumors, yet activity in gliomas has been limited. Lymphopenia has been reported in association with reduced survival and a shorter time to progression on checkpoint inhibition in other solid tumors. We evaluated whether low absolute lymphocyte count (ALC) is related to worse outcomes in pembrolizumab-treated patients with gliomas. METHODS In this single-center retrospective study, we identified adults with gliomas who received ≥1 dose of pembrolizumab and had available lymphocyte counts at our center between 05/01/2018 and 1/31/2025. Patients’ demographics, dexamethasone use, baseline ALC (before initiation of pembrolizumab), and clinical outcomes were collected. Overall Survival (OS) was estimated using Kaplan–Meier analysis from the date of starting pembrolizumab to the date of death. RESULTS Ninety patients met eligibility criteria (glioblastoma [GBM] n = 44, astrocytoma n = 20, oligodendroglioma n = 13, meningioma n = 8, other n = 5); 62% were male and 31% (n = 28) harbored IDH-mutant tumors. Across all histological subtypes, baseline ALC<750 cells/mm³ was associated with significantly shorter OS compared to baseline ALC≥750 cells/mm³ (3.7 vs 7.7 months, P = 0.0063) with similar dexamethasone exposure (63% vs 52%). In the recurrent GBM subgroup (n = 43; MGMT-methylated 26%), ALC≥750 cells/mm³ correlated with longer OS (8.5 vs 3.4 months, P = 0.0065) with a slightly lower proportion of patients on dexamethasone in high ALC group (52% vs 75%). Conversely, among patients with IDH-mutant gliomas (n = 28), no significant difference in OS was observed based on ALC levels (7.8 vs. 7.3 months, P = 0.58) with similar dexamethasone use (57% and 43% in high and low ALC groups, respectively). CONCLUSION Baseline lymphopenia preceding pembrolizumab treatment is associated with shorter OS in patients with GBM, but not in IDH-mutant gliomas. These associations require further validation in additional patient cohorts.

Therapeutic clinical trial enrollment does not match glioma incidence across demographics. Traditional statistical methods have identified independent predictors of trial enrollment; however, our understanding of the interactions between these factors remains limited. To test the interactive effects of demographic, socioeconomic, and oncologic variables on trial enrollment, we designed boosted neural networks (BNNs) for all glioma patients ( n = 1042), women ( n = 445, 42.7%), and minorities ( n = 151, 14.5%) and externally validated these models [whole cohort, n = 230; women, n = 89 (38.7%); minority, n = 66 (28.7%)]. For the whole-cohort BNN, the most influential variables on enrollment were oncologic variables, including KPS [total effect (TE), 0.327], chemotherapy (TE, 0.326), tumor location (TE, 0.322), and seizures (TE, 0.239). The women-only BNN exhibited a similar trend. Conversely, for the minority-only BNN, socioeconomic variables [insurance status (TE, 0.213), occupation classification (TE, 0.204), and employment status (TE, 0.150)] were most influential. These results may help prioritize patient-specific initiatives to increase accrual.

2053 Background: Recurrent gliomas are highly aggressive brain tumors, often resistant to conventional treatments. Immune checkpoint inhibitors (ICI) have emerged as promising therapeutic agents by targeting tumor cells through immune modulation. However, clinical trials have demonstrated limited efficacy in recurrent gliomas. This study aimed to identify potential factors influencing treatment efficacy of ICIs in recurrent gliomas. Methods: This retrospective study, conducted across the Mayo Clinic following IRB approval, included patients ≥ 18 years diagnosed with adult-type diffuse gliomas. Eligible patients received treatment with at least 2 cycles of ICI for recurrent glioma between 2014 – 2024. Patients treated with ICIs as initial therapy were excluded. Clinical, radiographic, histological, and molecular data were analyzed, with missing information excluded. Responders to ICI were defined as patients who did not meet iRANO criteria for progressive disease based on first radiographic response assessment (and confirmatory follow up imaging as needed for possible pseudo-progression). Survival outcomes [Progression-Free Survival (PFS) and Overall Survival (OS)] and potential predictive variables were analyzed using the Kaplan-Meier method and Cox-Regression Analyses. Results: 67 patients met eligibility criteria (mean age: 45.1 ± 15.0 years; 64.2% male; 94% white). 64 (95.5%) patients received Pembrolizumab, 2 (3%) Nivolumab, and 1(1.5%) combined Ipilimumab/Nivolumab, with a median treatment duration of 2.77 (1.39 – 19.4) months. All had prior alkylating chemotherapy. The OS (from diagnosis) for IDH wildtype (IDH-WT, n = 36), IDH mutant, 1p/19q non-co-deleted (IDH-MUT, n = 17) and IDH mutant, 1p/19q co-deleted (OLIGO, n = 14) gliomas were 3.1, 9.2, and 18.6 years, respectively. The median PFS from time of ICI was 2.23 (0.69 – 27.3) months. 24 (36.9%) patients were identified as Responders. PFS was not significantly different between patients with IDH-MUT and IDH-WT gliomas (2.30 vs 2.07 months, p = 0.593). However, patients with OLIGO gliomas had a significantly higher PFS compared to IDH-WT gliomas (5.16 vs 2.07 months, p = 0.021). The proportion of responders was greatest in OLIGO gliomas, however, did not reach statistical significance (IDH-WT, 31.4%; IDH-MUT, 29.4%; OLIGO, 61.5%, p = 0.120). Overall PFS was not impacted by patient age, sex, and extent of initial resection. When analyses were limited to Responders, the PFS for IDH-WT, IDH-MUT and OLIGO gliomas were 5.75, 7.01 and 10.8 months, respectively ( p = 0.434). Conclusions: Patients with recurrent OLIGO gliomas may have a longer PFS with ICI therapy compared with recurrent IDH-WT and IDH-MUT gliomas. However, there is significant variability in ICI treatment efficacy between patients. Further molecular profiling is in progress to evaluate additional predictive biomarkers of response.

Abstract Purpose: Monitoring disease progression in patients with high-grade gliomas (HGG) is challenging due to treatment-related changes in imaging and the requirement for neurosurgical intervention to obtain diagnostic tissue. DNA junctions in HGG often amplify oncogenes, making these DNA fragments potentially more abundant in blood than monoallelic mutations. In this study, we piloted a cell-free DNA approach for disease detection in the plasma of patients with HGG by leveraging patient-specific DNA junctions associated with oncogene amplifications. Experimental Design: Whole-genome sequencing of grade 3 or 4 isocitrate dehydrogenase–mutant or wild-type astrocytomas was utilized to identify amplified junctions. Individualized qPCR assays were developed using patient-specific primers designed for the amplified junction. ctDNA levels containing these junctions were measured in patient plasma samples. Results: Unique amplified junctions were evaluated by individualized semi-qPCR assays in presurgical plasma of 18 patients, 15 with tumor-associated focal amplifications and three without tumor-associated focal amplifications. high copy-number junctions were robustly detected in the plasma of 14 of 15 (93.3%) patients with amplified junctions and none of the controls. Changes in junction abundance correlated with disease trajectory in serial plasma samples from five patients, including increased abundance of amplified junctions preceding radiographic disease progression. Conclusions: In patients with grade 3 or 4 astrocytomas who had tumor-associated amplifications, patient-specific amplified junctions were successfully detected in assayed plasma from most patients. Longitudinal analysis of plasma samples correlated with disease trajectory, including cytoreduction and progression.

Abstract BACKGROUND Despite changes in national policy, women and minority patients remain under-accrued in glioma clinical trials. Supervised machine learning may serve as a novel approach to understanding which variables and their complex interplay most influence enrollment for underrepresented patients. We aimed to design and externally validate machine learning models to predict therapeutic clinical trial enrollment for a) all patients, b) women, and c) NIH-designated minority patients with low- and high-grade glioma. METHODS In a 20-year retrospective cohort of 1042 adult patients with low- and high-grade glioma, boosted neural network (BNN) models for the whole, women, and minority cohorts were used to analyze the combined effects of demographic, socioeconomic, and oncologic variables on clinical trial enrollment. These models were validated using an external cohort (n=103). RESULTS In the development cohort (445 [42.7%] women; 151 [14.5%] minorities; median age, 51.5 [interquartile range: 39.4-62.5] years), 350 patients (33.6%) enrolled in a therapeutic clinical trial. The whole cohort BNN (AUC: 0.827, misclassification rate (MR): 0.175), the women cohort BNN (AUC: 0.895, MR: 0.125), and the minority cohort BNN (AUC: 0.983, MR: 0.053) models accurately predicted trial enrollment on external validation. For the whole cohort and women models, several demographic (race, ethnicity, minority status, sex), socioeconomic (insurance, employment status), and oncologic variables (seizure, chemotherapy, KPS, WHO grade, tumor lobe) were equally influential (Main effect (ME): 1.000, Total effect (TE): 1.000) on enrollment. For the minority model, socioeconomic variables including insurance status (ME: 0.089, TE: 0.417) and occupation classification (ME: 0.087, TE: 0.381) drove enrollment. CONCLUSION We designed and externally validated the first machine learning model for predicting therapeutic clinical trial enrollment for patients with diffuse glioma. Model features driving trial enrollment varied for underrepresented patients. These results may help prioritize personalized patient-specific initiatives to address barriers to equitable patient accrual.

Abstract INTRODUCTION Lymphopenia attributed to postoperative chemoradiotherapy (CRT) can accelerate tumor recurrence and contribute to poor prognosis in GBM. We hypothesize that short course (1-2 weeks) CRT can mitigate lymphopenia compared to standard course and may provide immune benefits for future combination therapy. METHODS GBM patients were randomized on a multi-site phase II study comparing short course (35Gy/5Fx or 40Gy/10Fx depending on target volume) (Arm A) versus standard dose fractionation (60Gy/30Fx or 40Gy/15Fx, Arm B). All patients received concurrent and adjuvant temozolomide (TMZ). This unplanned interim analysis evaluates changes of absolute lymphocyte count (ALC), CD4 and CD8 prospectively at 3-timepoints: baseline (prior to CRT), end of CRT, and 1-month post-CRT. RESULTS To date, 72 patients enrolled (34 Arm A, 38 Arm B). Median age 66 (35-86), ECOG 0-2. Patients characteristics as followed in Arm A vs. Arm B, respectively: female 47% vs. 42%, MGMT promotor methylated 31% vs. 34%, steroid use 46% vs. 45%, ALC 1310 cells/mm3 (330 –9730) vs. 1480 (750 –7750), CD4 531 cells/mm3 (86 –1368) vs. 662 (318 –1671), and CD8 151 cells/mm3 (56 –940) vs. 272 (68 –875). Lymphocytes data were collected in 49 patients (21 Arm A, 28 Arm B). There were signficantly fewer reductions of ALC, CD4 and CD8 in Arm A. Median ALC changes were 95% and 99% of baseline (Arm A) vs 52% and 61% of baseline (Arm B) at the end of CRT and 1-month post-CRT, respectively (p<0.01). Median CD4 changes 93% and 96% of baseline (Arm A) vs 51% and 49% (Arm B), and median CD8 changes 97% and 98% of baseline (Arm A) vs 57% and 71% (Arm B) at the end of CRT and 1-month post-CRT, respectively (p<0.05). CONCLUSIONS This interim analysis suggests that short course RT could reduce treatment-related lymphopenia and immune suppression compared to standard RT, thus could be potentially beneficial to combine with immunotherapy.