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Abstract BACKGROUND Understanding long-term sequelae of cancer care is critical. Cognitive impairment is a common and disabling survivorship issue. Because sex influences tumor biology, therapy exposure, and dementia risk, we examined whether cancer care for a prior malignancy alters late-life cognitive outcomes differently in males and females. METHODS We assessed adults ≥50 years, cognitively normal at study entry, from a longitudinal, population-based cohort (Mayo Clinic Study of Aging). Participants were linked to our institutional cancer registry (excluding non-melanoma skin cancers). Mild cognitive impairment (MCI) and dementia were adjudicated by consensus. We fitted multivariable Cox proportional-hazards models to estimate how prevalent cancer diagnosis influenced the risk of developing MCI/dementia. Sex-specific effects were determined by incorporating a cancer×sex interaction term and by generating separate hazard ratios. Analyses were controlled for age, APOE4 status, education, and presence of vascular/metabolic conditions. RESULTS 5793 MCSA participants (female: 49.4%, APOE4 positive: 27.5%) were included in the study, with a mean age of 73 years at study entry. Incident MCI was recorded in 866 participants, and 737 were diagnosed with incident dementia. About 20% (n=1176) of the cohort had a cancer diagnosis, most commonly prostate (n=423) and breast (n=277). There was no independent relationship between history of cancer and risk for MCI or dementia. We found that males with cancer had an increased risk for MCI (HR 1.28, 95% CI 1.04-1.57, p=0.003) and dementia (HR 1.29, 95% CI 1.04-1.6, p=0.053). Conversely, females with cancer had a lower risk for MCI (HR 0.79, 95% CI 0.61-1.01, p=0.063), although this was not statistically significant. CONCLUSIONS Our data suggests male cancer survivors may be more susceptible to cognitive impairment, independent of age, APOE4 status, education, and vascular/metabolic burden. Elucidating the hormonal, treatment-related, and systemic pathways driving this should be a priority for future mechanistic studies.

Abstract Zanubrutinib is a second-generation Bruton Tyrosine Kinase (BTK) inhibitor, FDA-approved for Mantle Cell Lymphoma (MCL), designed to be more selective and potentially better tolerated than first-generation agents like Ibrutinib. Progressive multifocal leukoencephalopathy (PML), a rare but severe complication, has not previously been reported with Zanubrutinib. We report a 74-year-old woman with a germline CHEK2 mutation and asymptomatic MCL (CD5+, cyclin D1+, Ki67 10–20%) diagnosed in 2016, managed with surveillance. In 2023, she was also diagnosed with localized breast cancer treated with lumpectomy and exemestane. Later that year, she developed new headaches. Brain MRI revealed diffuse bilateral pachymeningeal enhancement, suspicious for lymphomatous spread, without parenchymal involvement. By early 2024, follow-up imaging showed stable pachymeningeal enhancement with new scattered punctate right frontal subcortical lesions, coinciding with systemic MCL progression. Spinal imaging revealed epidural and bone marrow involvement; CSF was negative for malignancy. Zanubrutinib was initiated in March 2024. By August 2024, new bilateral frontal and parietal fuzzy hyperintense and faintly enhancing white matter lesions emerged, while prior punctate lesions regressed. Biopsy confirmed PML with JC virus DNA by in situ hybridization and absence of B-cell clonality. Zanubrutinib was discontinued. Follow-up imaging showed regression of lesions without new enhancement, consistent with immune reconstitution. The initial subcortical punctate lesions remained stable. In 2025, she was diagnosed with localized pancreatic adenocarcinoma, precluding cytotoxic therapy initially due to concern for PML reactivation. However, as of May 2025, neuroimaging remains stable, and patient was started on palliative gemcitabine/paclitaxel. This is the first reported case of PML associated with Zanubrutinib. It underscores the importance of vigilance for PML even in patients without overt immunosuppression, especially those with overlapping malignancies and treatment exposures in the correct clinical context.

Abstract Recent studies demonstrate that platelet factor 4 (PF4), an abundant platelet-derived chemokine in young plasma, can reduce neuroinflammation, enhance cognition, and rejuvenate peripheral immunity in aged mice. Intriguingly, neuroimmune markers modulated by PF4—such as Iba1, CD68, and C1q—also contribute to glioma progression. Age-associated immune shifts, including increased myeloid-to-lymphocyte ratios, T cell depletion, and systemic inflammation, are similarly implicated in cancer development. Here, we show that systemic administration of PF4 significantly extends survival in aged (18–23 month-old) glioma-bearing mice and trends toward improved outcomes in young (2–3 month-old) counterparts. Longitudinal IVIS imaging revealed reduced tumor-associated luminescence in PF4-treated animals, particularly in aged males and females. Histological analysis demonstrated downregulation of microglial and macrophage activation markers within the tumor microenvironment. Ongoing studies utilizing flow cytometry, cytokine profiling, and novel PF4-delivery platforms aim to further elucidate its effects on both central and peripheral immunity. These findings suggest that age-related immune modulation may be leveraged to delay glioma progression, positioning PF4 as a promising therapeutic at the intersection of aging, immunology, and neuro-oncology.

Abstract BACKGROUND Glioblastoma (GBM) is a universally lethal brain cancer with limited durable responses to immunotherapy, largely due to its immunosuppressive tumor microenvironment (TME). Ephrin receptor A3 (EphA3), a receptor tyrosine kinase minimally expressed in normal tissue, is enriched on GBM cells, glioma stem cells (GSCs), and M2-like tumor-associated macrophages (TAMs). We hypothesize that dual targeting of TAMs and tumor cells by EphA3-CAR-T is a viable treatment strategy. METHODS We developed EphA3-directed second-generation CAR-T cells (EphA3-CAR-T) incorporating the single-chain variable fragment of ifabotuzumab, an anti-EphA3 monoclonal antibody, achieving >90% transduction efficiency. We evaluated antitumor efficacy in vitro; the functional activities of EphA3-CAR-T on GSCs were assessed via selective cytotoxicity, T cell proliferation, degranulation assays, and depletion of M2-like macrophages. In vivo, 1 × 10⁶ EphA3⁺ U251 MG cells and/or 1 × 10⁶ ex vivo–polarized M2-like macrophages were implanted in the flanks of NOD-SCID-γ⁻/⁻ mice. Comparative studies with EGFRvIII-directed CAR-T cells were also performed. RESULTS Immunohistochemistry confirmed EphA3 expression across both tumor and immune compartments in patient-derived specimens. EphA3-CAR-T cells demonstrated potent, selective cytotoxicity (p<0.001), T cell proliferation (p<0.001), and CD107α degranulation (p<0.001) against EphA3⁺ GBM and GSCs, while also reversing TAM-mediated immunosuppression via depletion of ex vivo–polarized M2-like macrophages (p<0.05), compared to other validated CAR-Ts. In vivo, EphA3-CAR-T significantly reduced tumor volume (p<0.001) and improved survival (p<0.01), in the presence of TAMs. In contrast, EGFRvIII CAR-T cells were inactivated under the same TME conditions. EphA3-CAR-T cells remained active in models co-engrafted with macrophages, suggesting preserved function within the immunosuppressive TME. CONCLUSIONS EphA3-CAR-T therapy addresses a major barrier to CAR-T success in GBM: TME-mediated immunosuppression. This tumor- and TME-targeting approach demonstrated robust preclinical activity. These findings support the clinical translation of EphA3-CAR-T cells as a next-generation immunotherapy for GBM.

Abstract Glioblastoma (GBM) is the most common malignant tumor of the CNS, accounting for 80% of brain tumors. Recent advances in gene editing and immunology have led to the development of chimeric antigen receptors (CARs), including CARs incorporating chlorotoxin (CLTX) receptor domains for GBM targeting. While CAR-engineered neutrophils have shown promise, their systemic administration can induce severe off-target effects. Extracellular vesicles (EVs), secreted by these cells, retain properties of their parent cells—including CAR expression and tumor-lytic activity—yet their potential has not been explored in CAR Neutrophils. To address this, CAR-CLTX DNA was inserted into the AAV1 safe harbor locus of induced pluripotent stem cells, which were subsequently differentiated into neutrophils. Conditioned media were collected during differentiation and processed using extraPEG-based EV isolation. EVs were quantified via nanoparticle tracking analysis, electron microscopy, and western blotting. Proteomic and microRNA (miRNA) profiling of EV cargo was conducted at days 12 and 21 of differentiation. Cytotoxic effects of EVs were assessed in U87MG and LN229 glioblastoma cells in 2D culture, 3D organoids, and in vivo models. EVs were secreted in high quantities within the 100–200 nm size range, expressed canonical exosomal markers, and exhibited characteristic morphology. Proteomic analysis identified neutrophil-associated proteins involved in migration, chemotaxis, and degranulation. miRNA sequencing revealed high levels of tumor-suppressive miRNAs (e.g., miR-182) implicated in apoptotic signaling, T cell receptor pathways, cytokine-cytokine receptor interactions, Jak-STAT, Hedgehog, and NK cell-mediated cytotoxicity. EV uptake by glioma cells was demonstrated in vitro. Cytotoxic effects of CAR Neutrophil EVs were observed across glioblastoma cell lines, organoids, and in vivo. These findings demonstrate that CAR Neutrophil-derived EVs carry anti-tumor protein and miRNA cargo, are efficiently taken up by GBM cells, and mediate glioma cell cytotoxicity. This supports the feasibility of correlating EV cargo composition with function in GBM treatment.

Abstract Secondary central nervous system lymphoma (SCNSL) is defined as the diagnosis of CNS lymphoma diagnosed concurrently with systemic lymphoma or after prior treatment. Due to the poor prognosis associated with SCNSL, the lack of randomized trial data, and the need for international consensus guidelines, there is a high unmet need for effective therapy options for SCNSL. Here we present safety and efficacy data on 2 SCNSL patients treated with the combination of emavusertib and ibrutinib. Both patients had directly progressed on their prior BTKi treatment. One patient achieved an unconfirmed complete response (CRu) and remained on treatment for over 4 mos, while the other patient maintained stable disease (SD) with a 38% reduction in disease burden from baseline and was on treatment for more than 8 mos. The patient with a durable SD, who received 5 prior lines of therapy along with an ASCT, presented with 3 related AEs Grade ≥ 3 that were reversible and manageable. These included elevated ALT, AST, and bilirubin levels that resolved. The patient received emavusertib 300 mg BID and ibrutinib 560 mg QD. The CRu patient received 6 prior lines of therapy and experienced CK elevation, fatigue, hypoglycemia, sepsis, urinary retention, neutropenia, and constipation that resolved with either a dose reduction and/or supportive care. The initial dose for this patient was 150 mg emavusertib BID and ibrutinib 560 mg QD. Subsequently, the dose of emavusertib was decreased to 150 mg BID due to Grade 4 neutropenia, then increased to 200 mg AM and 150 mg PM once resolved. This patient achieved CRu within 60 days on treatment. The 2 patients presented here are supportive data for using the combination of emavusertib and ibrutinib in SCNSL.

Abstract There are no approved treatments for relapsed/refractory (R/R) PCNSL. We show promising efficacy in the salvage setting using emavusertib in combination with ibrutinib, which targets the BCR and TLR pathways. These 2 major pathways are associated with activation of NF-κB. Updated safety and efficacy data of this combination in R/R PCNSL from the ongoing Phase 1/2 TakeAim Lymphoma trial (NCT03328078) will be presented. As of 01 May 2025, 33 patients with R/R PCNSL were treated with emavusertib in combination with ibrutinib. Eligible patients receive emavusertib in combination with ibrutinib until tumor progression or unacceptable toxicity. Median age was 63 years (range 43-81); 16 patients were male. Median prior anti-cancer therapy was 2 (range 1-5); 26 (78.8%) patients had prior BTKi exposure. Median duration of emavusertib/ibrutinib treatment was 51 days (range 5-798). Grade ≥ 3 treatment-related adverse events (TRAEs) were reported in 11 (33.3%) patients; neutropenia in 5 (15.2%) patients, amylase increased, lipase increased, and leukopenia in 2 (6.1%) patients each. The majority of Grade ≥ 3 TRAEs were reversible and manageable. Among the 19 BTKi-experienced patients treated for ≥ 28 days, there were 5 CRs and 2 PRs, with an ORR of 36.8%. Three patients with CR were on treatment for 10+ mos, with 1 CR patient ongoing with DOT of 26.2+ mos. Among the 5 BTKi-naïve patients treated for ≥ 28 days, 1 achieved CR and 3 had PR, with ORR of 80%; the CR patient is ongoing with DOT of 18.1+ mos. The ORR in the combined population was 45.8% (6 CRs; 5 PRs). The combination of emavusertib and ibrutinib demonstrates promising clinical activity and an acceptable safety profile in patients with R/R PCNSL. This regimen appears capable of overcoming resistance to prior BTKi therapy. Patient enrollment in this trial continues.

Abstract PCNSL is a rare and aggressive NHL with no approved treatments for R/R patients, representing a critical unmet need. MyD88 mutations in ~70% of PCNSL patients drive IRAK4 activation, promoting NF-κB signaling, inflammation, and tumor progression. Emavusertib, a potent oral IRAK4 inhibitor, crosses the blood-brain barrier and shows preclinical synergy with BTKis, and has the ability to re-sensitize BTKi-resistant cell lines. This study evaluates the molecular and PK data associated with responses to emavusertib + ibrutinib combination therapy in R/R PCNSL patients. The safety, clinical activity, and potential biomarkers of emavusertib in R/R PCNSL are being investigated in the ongoing TakeAim Lymphoma trial (NCT03328078). Pre-dose and 1.5-hour post-dose plasma samples were collected on Day 1 of Cycles 3, 5, and 7. CSF samples were obtained via a lumbar puncture within 1.5 hrs of collection of the post-dose plasma PK sample on Cycle 3 Day 1. As of 06 December 2024, CSF concentration was available for 7 patients. The mean emavusertib concentration in CSF was 81.3 ng/ml (54.7-104.0) in patients receiving 100 mg emavusertib BID (n=4). In patients receiving 200 mg emavusertib BID (n=3), the mean emavusertib concentration in CSF was higher at 175.7 ng/ml (114.8-209.4), which is 2.2 × the mean value in patients who received 100 mg emavusertib BID (p-value = 0.02). MyD88 mutation status was available for 7 patients of which all had prior BTKi exposure. Among these patients, 6 had MyD88 mutations of which 4 had responded (3 CRs and 1 PR) to the combination with DOR up to 18.9 mos with data collection ongoing. Preliminary CNS PK data demonstrate that emavusertib concentration in CSF increases with increasing emavusertib dose. Patients with MyD88 mutations showed expected promising preliminary efficacy to emavusertib + ibrutinib combination and may overcome BTKi resistance. Enrollment is ongoing.

Abstract BACKGROUND The phase III ECOG-ACRIN E3F05 trial tested whether combined therapy with radiation and concomitant followed by adjuvant temozolomide is more effective than radiation alone in patients with grade II gliomas. The study incorporated 1p/19q analysis but predated assessment of IDH mutational status. We previously reported that the addition of temozolomide to radiotherapy significantly improved overall survival in grade II gliomas, with similar hazard ratios in co-deleted (0.56) and non-codeleted (0.53) tumors. Herein we incorporate the results of DNA methylation profiling to examine outcomes in IDHmt, non-codeleted subjects as well as co-deleted ones. METHODS All 172 subjects underwent 1p/19q co-deletion analysis as a stratification factor, 1p and 19q deletion analysis was performed on formalin-fixed paraffin-embedded (FFPE) gliomas by Fluorescent In Situ Hybridization (FISH) following standard methods, 97/172 (56.3%) had tissue with 60% or more tumor content available for DNA methylation profiling using Heidelberg classifier v12 and Bethesda Classifier 2.0 to ascertain classification as IDH mutant glioma and alternative evaluation of 1p/19q co-deletion status. RESULTS Of the 172 subjects, 74 (43.0%) had 1p/19q codeletion by FISH, 38 (22.1%) were IDHmt without codeletion, 14 (8.1%) were IDHwt, and 46 (26.7%) were non-codeleted with unknown IDH status (these include n=4 pts originally classified as 1p/19q undetermined). With a median follow-up of 130 months, the addition of TMZ to RT improved OS in patients with IDHmt, non-codeleted tumors (HR 0.15 (0.03-0.74)) and 10-year OS from 39% to 80%. In codeleted tumors, the addition of TMZ improved OS (HR 0.51 (0.19-1.42)) and 10-year OS from 68% to 90%. CONCLUSION The addition of temozolomide to radiation improves overall survival in both co-deleted and IDHmt, non-co-deleted grade II gliomas.

Abstract BACKGROUND STELLAR was a phase III randomized, open-label trial of eflornithine (ornithine decarboxylase inhibitor) with lomustine versus lomustine alone in patients with recurrent anaplastic astrocytoma (AA). We previously reported an overall survival (OS) and progression free survival (PFS) of the combination in the patients with grade 3 IDH-mutant astrocytoma. Here we report updated OS and PFS outcomes based on additional molecular classification and blinded independent central review (BICR). METHOD Eligibility included: age ≥ 18 years, AA (2016 WHO criteria) with first recurrence ≥ 6 months after radiation, KPS ≥ 70, and no imaging findings consistent with grade 4 glioblastoma. Patients received eflornithine (2.8 g/m2 TID Q 2/3 weeks) with lomustine (90 mg/m2 Q 6 weeks) (Arm A) or lomustine alone (110 mg/m2 Q 6 weeks) (Arm B). Primary endpoint was OS. RESULTS Updated molecular data resulted in re-classification of the 343 AA patients enrolled into 3 WHO 2021 defined tumor subtypes, 196 tumors were IDHmut, CDKN2A/B intact, consistent with diagnosis of astrocytoma, IDHmut, WHO grade 3, 33 tumors had CDKN2A/B loss consistent with astrocytoma, IDHmut, WHO grade 4, 106 tumors were IDHwt consistent with diagnosis of glioblastoma, IDHwt, WHO grade 4. Updated results strengthen initial conclusion that eflornithine plus lomustine resulted in significant survival benefit in the astrocytoma, IDHmut, WHO grade 3 group (mOS 34.9 vs, 23.5 months, HR = 0.64, stratified log rank p =0.0136). PFS with eflornithine was also improved (15.8 vs, 7.2 months, HR = 0.57, stratified log rank p =0.0113). BICR demonstrated a high correlation between investigator and centrally determined PFS (p=0.0305), and high correlation between OS and PFS (p=.0001). CONCLUSION The clinically meaningful OS and PFS benefits observed with eflornithine in the molecularly defined 2021 WHO CNS grade 3 astrocytomas is further confirmed and expanded based on additional molecular classification and BICR of PFS.