Ruff, Michael W.

Abstract Purpose: Current methods for glioma response assessment are limited. This study aimed to assess the technical and clinical feasibility of molecular profiling using longitudinal intracranial cerebrospinal fluid (CSF) from patients with gliomas. Experimental Design: Adults with gliomas underwent longitudinal intracranial CSF collection via Ommaya reservoirs or ventriculoperitoneal shunts. Cell-free DNA (cfDNA) was extracted and analyzed using PredicineCARE for cancer variant profiling and/or PredicineSCORE for low-pass whole-genome sequencing. Results: Five patients (two females and three males; median age, 40 years; range, 32–64 years) underwent longitudinal intracranial CSF collection via Ommaya reservoirs (n = 4) or ventriculoperitoneal shunts (n = 1). In total, 47 CSF samples were obtained (median volume, 4.00 mL; 0.5–5 mL). Forty-one samples (87.2%) yielded sufficient cfDNA for testing. Patient-specific tumor-associated variant allelic frequencies (VAF), and thus tumor fraction, decreased in pre- versus postchemoradiation samples, including through pseudoprogression. These also increased with radiographic progression in three patients, although identifying the time of definitive disease progression from MRIs was a significant limitation. In two patients with isocitrate dehydrogenase (IDH)–mutant gliomas, decreasing IDH1 VAF after resection and chemoradiation correlated with decreased CSF D-2-hydroxyglutarate levels (0.64× and 0.62×, respectively, for the first patient and 0.01× and 0.07× for the other patient), although D-2-hydroxyglutarate and IDH1 VAF were not concordant in one patient thereafter. Moreover, the copy-number burden decreased below the limit of quantification during treatment and increased above the limit at progression. Conclusions: Longitudinal intracranial CSF cfDNA can be obtained in patients with gliomas during their disease course. However, before deploying this technique, numerous questions and challenges should be answered.

Despite the high initial responses to chimeric antigen receptor T (CART) cell therapy, most patients relapse within the first 1-2 years. T cell quality influences the success of CART cell therapies greatly, and T cells from cancer patients are prone to transition into dysfunctional states, such as exhaustion or senescence. The role of exhaustion in CART cell failures has been studied extensively; however, the role of T cell senescence has remained mostly unexplored. In this study, we aimed to determine the development of CART cell senescence and its impact on CART therapeutic efficacy. First, we studied the development of CART cell senescence using an in vitro model to induce CART cell dysfunction. Here, we cyclically activated CD19-directed chimeric antigen receptor T (CART19) cells (either costimulated via 4-1BB (BBζ) or CD28 (28ζ)). Activation cycles are designed to mimic recurrent activation of CART cells to study the impact of T cell activation on CART cell fates throughout treatment. We added a rest time in between activation cycles to focus on senesced CART cell populations since, unlike exhaustion, senescence is irreversible even after rest. We examined CART immunophenotype, cell cycle regulators, transcriptomic profile, senescence marker gene expressions and T cell effector functions on days 0 (D0-T cell), 8 (D8-baseline CART), and 15 (D15-CART cell after one activation/rest cycles). Following an activation/rest cycle, T cell exhaustion markers CTLA4, LAG3, and PD1 returned to basal levels after an initial increase due to activation, while only TIM-3 levels remained elevated (BBζ: p=0.0090, 28ζ: p=0.0394). Increased TIM-3 expression is associated with T cell senescence, which prompted us to further investigate the development of a senescence phenotype. We found a prominent T cell senescence phenotype in BBζ compared to 28ζ, as evident by increased expression of p53 (BBζ: p=0.0248), p21 (BBζ: p=0.0483), CD27 (BBζ: p=0.0080), CD28 (BBζ: p=0.0327), uPAR (BBζ vs. 28ζ: p<0.0001), and KLRG1 (BBζ vs. 28ζ: p=0.0029) in recurrently activated-rested CART cells. Next, we evaluated the transcriptomic signature of recurrently activated-rested CART cells. Gene set enrichment analysis (GSEA) indicated enrichment of gene sets related to senescent T cells as well as senMayo, a novel gene set which is reported to be selectively enriched in senescing cells or tissues, in BBζ, but not in 28ζ, compared to T cell D0 or in BBζ D15 compared to 28ζ D15 (p=0.00014 and p=0.089, respectively). MYC-related gene sets were found to be significantly enriched in recurrently activated/rested BBζ and depleted in 28ζ (p<0.1). MYC expression is increased in BBζ (p= 0.0010) while decreased in 28ζ (p=0.0036) upon recurrent activation and is higher in BBζ (D8: p=0.0018, D15: p= 0.0022) compared to 28ζ at indicated time points by qPCR. Given these results, we induced senescence in CART cells using two alternative strategies: MYC-T58A activation (oncogene-induced senescence) and irradiation. Senescence induced by MYC-T58A overexpression ameliorated only BBζ killing (p=0.0057), whereas senescence induced by irradiation improved 28ζ killing (p= 0.0086), indicating versatile impact of senescence on treatment outcome diverse CART cell therapies. Finally, analysis with two independent patient data sets correlated the emergence of senescence with contradicting clinical outcomes to BBζ and 28ζ treatments. The presence of senescent 28ζ cells in the peripheral blood of 28ζ-treated patients was reported to be associated with complete response in the clinic in an independent analysis. In another cohort, 28ζ-treated patients with higher senescence score were correlated with longer disease-free survival (p=0.0029), while senMayo gene set enrichment in BBζ-treated patients was associated with non-response in the clinic (p=0.014). In the same cohort, MYC-related gene sets-the same genes sets enriched in our recurrently activated/rested BBζ-are also enriched in nonresponsive BBζ-treated patients. In summary, our study indicates that (i) CART cells are susceptible to a state of senescence, as demonstrated using three independent approaches (repeated activation/rest, irradiation, and MYC modulation), (ii) CART senescence is more prominent in BBζ than 28ζ, and (iii) senescence is associated with worse outcomes following BBζ but not 28ζ treatment.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies. However, CAR T-cell therapy still has low durable response rates and common associated toxicities such as cytokine release syndrome (CRS). We recently reported that sequential therapy with the CD19 monoclonal antibody tafasitamab and CD19-directed CAR T (CART19, 41BBζ costimulated) cells significantly ameliorated CAR T-cell apoptosis and tumor cell pyroptosis, improving CAR T safety and efficacy (PMID: 37879074). Building on these findings, we hypothesized that controlling massive initial CAR T-cell stimulation by diminishing extensive tumor cell pyroptosis is critical to improve CAR T safety and durability. To test this hypothesis, we evaluated several caspase (Casp) inhibitors, including Casp1, Casp3, Casp7, and Casp11, in combination with CAR T cells. We found that the Casp1 inhibitor VX765 did not inhibit CAR T antigen-specific cytotoxicity and thus focused on VX765 in subsequent assays. We first assessed CAR T proliferation upon stimulation with the CD19+ JeKo-1 mantle cell lymphoma cell line in the presence of VX765. CAR T with vehicle control showed greater proliferation at day 3, but CAR T with VX765 showed higher proliferation at day 5 (p=0.03, absolute CD3+ counts by flow cytometry). We then co-cultured CAR T and JeKo-1 for 24 hours and observed a significant reduction in apoptotic CAR T (Annexin V+ and 7-AAD- via flow cytometry) in VX765-treated conditions (p=0.05). Additionally, there was significantly reduced pyroptosis (Annexin V+ 7-AAD+) (p=0.02) but higher apoptosis (Annexin V+ 7-AAD-) in JeKo-1 in VX765-treated conditions (p=0.04). VX765-treated CAR T showed higher Bcl2 expression via flow cytometry (p=0.03). We also studied tumor cell pyroptosis by measuring the level of HMGB-1. CART19, JeKo-1, and VX765 or vehicle control were co-cultured for 24 hours, and the level of HMGB-1 was found to be significantly reduced in VX765-treated conditions, indicating reduced tumor pyroptosis (p=0.006). We then assessed the impact of Casp1 inhibition in our in vitro model of CAR T exhaustion. We stimulated CAR T with CD19+ JeKo-1 every other day for 2 weeks and then assessed CCR7 and CD45RA expression on CAR T. VX765-treated CAR T showed higher central memory (p=0.08) and naïve (p=0.02) and lower terminally differentiated effector memory phenotypes (p=0.03). We next studied the effect of Casp1 inhibition with VX765 in vivo. To recapitulate CAR T-cell toxicities, including CRS, we utilized an immunocompetent mouse model. First, we generated murine CART19 (mCART19, CD28ζ costimulated) cells. Recipient mice were then engrafted with luciferase+ murine CD19+ TBL12 cells, a murine lymphoma cell line. Mice were treated 12 days later with cyclophosphamide (300 mg/kg, ip) and 24 hours after mice were imaged and randomized to receive 1 x 107, 1) untransduced T cell control (UTD), 2) mCART19 + vehicle control, or 3) mCART19 + VX765 (25 mg/kg). VX765 was given immediately before the CART19 injection and on day 2 post-CAR T injection. Mice were serially monitored and weighed to assess CAR T-cell toxicity. Tumor burden was assessed via weekly bioluminescent imaging. Mice treated with vehicle control showed a significant reduction in weight compared to the UTD control (p=0.02), while mice treated with VX765 maintained their weight and remained active without signs of CRS, including motor weakness and hunched body. UTD mice reached humane endpoints within 3 weeks after T-cell injection. Three satellite mice from each group were euthanized and spleens were harvested. Flow cytometric analysis showed a significant reduction in apoptotic CAR T (p=) and pyroptotic tumor cells in mice treated with VX765 (p=0.03). Western blot showed lower cleaved GSDME and GSDMD in mice treated with VX765. VX765-treated mice demonstrated prolonged CAR T persistence with durable anti-tumor activity. In summary, Casp1 inhibition with VX765 significantly reduced CAR T apoptosis, improved memory phenotype, and increased delayed CAR T proliferation. VX765 also reduced tumor cell pyroptosis and CRS incidence without impacting CAR T anti-tumor activity in vivo. Given the favorable preclinical outcomes and existing clinical data on VX765, this approach holds substantial translational potential for enhancing the safety and efficacy of CAR T-cell therapy in patients with large B-cell lymphoma.

Brexucabtagene autoleucel (brexu-cel), a CD28-costimulated CD19-targeted chimeric antigen receptor T cell (CART19) product, was FDA-approved in 2020 after demonstrating remarkable efficacy in the pivotal ZUMA-2 trial of patients with mantle cell lymphoma (MCL). However, despite the impressive initial activity of CART19 cell therapy in MCL, most patients relapse within the first 3 years following treatment. Myeloid cells, such as monocytes and macrophages, have been demonstrated to be major inhibitors of other immune cells, including T cells. Growing evidence shows that monocytes and macrophages contribute to CART cell failure, although the exact mechanisms are not fully understood. To better understand the mechanisms contributing to CART treatment resistance in MCL, we first utilized brexu-cel products from patients with MCL on the ZUMA-2 trial to uncover their transcriptomic features by single cell RNA sequencing (scRNAseq) analysis. To understand if myeloid cells had negative impacts on clinical outcomes, we then analyzed autologous baseline peripheral blood mononuclear cell (PBMC) samples by scRNAseq. Our analyses of the brexu-cel samples revealed reduced response to IL-1β and its downstream signaling pathway as well as impaired T cell functions in non-responders. Transcriptomic analyses of the autologous baseline monocyte populations showed downregulation of IL-1β production and its associated pathways as well as upregulation of M2 phenotype-associated genes and IL-1 receptor antagonist (IL-1ra) in non-responder monocytes, implying a potential role of myeloid cells in CART resistance through impairment of IL-1 signaling in brexu-cel products. To validate our findings from the ZUMA-2 samples, we further studied the interactions between macrophages, MCL cells, and CART19 cells in preclinical models. First, CD28-costimulated CART19 cells generated from healthy donors were cocultured with JeKo-1, a CD19+ MCL cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression by M2-like macrophages was contact-independent. Cytokine profile analysis of coculture supernatants showed significant elevation of IL-1ra in M2-like macrophage cocultures (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292). This led us to hypothesize that M2-like macrophages inhibit CART19 by secreting IL-1ra to block IL-1 signaling in CART19. To test this hypothesis, we interrogated the role of IL-1ra in CART19. CART19 + JeKo-1 cocultures were supplemented with IL-1β + IL-1ra or IL-1β + IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 proliferation was improved by IL-1β (vehicle control vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra, p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Next, we assessed the impact of IL-1ra on CART functions in mouse MCL xenografts in the presence of M2-like macrophages. NOD-SCID-γ-/- (NSG) mice were subcutaneously injected with 0.5 million of human macrophages and 1 million of luciferase+ JeKo-1. Mice were then randomized once tumor engraftment was confirmed by bioluminescence imaging (BLI), followed by treatment with 2 million CART19 with 10 mg/kg IL-1ra neuAb or control IgG for 3 weeks. Combination of CART19 cells and IL-1ra neuAb led to improved antitumor activity based on BLI measurements compared to control IgG (IL-1ra neuAb vs control IgG, p=0.0058). Additionally, expression of IL-1RI, receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which might potentially desensitize CART response to IL-1β, resulting in further suppression in CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Overall, our study elucidates a potential mechanism of IL-1 pathway modulation mediated by myeloid cells in contributing to CART19 failure in MCL.

CD19-targeted chimeric antigen receptor T (CART19) cell therapy has shown outstanding clinical success. However, patients can develop CART-induced toxicities, including cytokine release syndrome (CRS) and neurotoxicity. Studies have found that activation of myeloid cells by granulocyte monocyte-colony stimulating factor (GM-CSF) can contribute to toxicity development. GM-CSF depletion leads to reduced myeloid activation, prevention of CART-induced toxicities, and improved CART efficacy. CART cells upregulate cytokine receptors upon activation, and our results indicate that CART19 cells significantly upregulate GMCSFR upon antigen-specific stimulation (<10% vs >30%, p<0.01). This led us to hypothesize that GMCSFR signaling contributes to CART cell activity. To understand the role of GM-CSFR upregulation on CART cell functions, we compared the impacts of neutralizing GM-CSF or blocking GM-CSF receptor alpha (GM-CSFRα) on CART19. First, 4-1BB-costimulated CART19 cells generated from healthy donors were stimulated with CD3/CD28 beads at a 1:3 ratio for 6 days. CART cells were maintained at 1 x 106/mL throughout the culture and supplemented with IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade at 10 μg/mL, 20 μg/mL, or 100 μg/mL. On day 6, cells were debeaded and rested for 2 days. Then, CART cells were cocultured with NALM6, a CD19+ acute lymphoblastic leukemia cell line, at a ratio of 1:1 for 3 days. The cocultures were supplemented with the same doses of antibodies listed above. Antigen-specific proliferation of CART cells was measured by flow cytometry. Our data suggested that CART19 proliferated is not impacted by GM-CSF neutralization but is inhibited by GM-CSFRα blockade (IgG isotype vs 10 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 20 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 100 μg/mL GM-CSFRα blockade, p=0.009). CART cells were assessed in a cytotoxicity assay with NALM6 luciferase+ cells as well, which did not show significant impacts from GM-CSF neutralization or GM-CSFRα blockade. To evaluate the impacts of GM-CSF neutralization and GM-CSFRα blockade in a mouse model, we engrafted NSG mice with 1 million NALM6 luciferase+ cells intravenously. Mice were then treated with 1.4 million CART19 plus 10 mg/kg IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade intraperitoneally. GM-CSFRα blockade drastically suppressed antitumor activities of CART (CART19+GM-CSFRα blockade vs CART19+IgG isotype control, p<0.0001; CART19+GM-CSFRα blockade vs CART19+GM-CSF neutralizing antibody, p<0.0001; CART19+ GM-CSFRα blockade vs untransduced T cell, n.s.). Serum cytokines were analyzed 2 days after CART treatment, showing that mice with GM-CSFRα blockade had significantly lower levels of human IFNγ (p=0.0189) and IL-2 (p=0.0121) compared to IgG isotype control group, while GM-CSF neutralization did not show significant impacts. Mice treated with CART plus IgG isotype control or GM-CSF neutralizing antibody showed prolonged survival compared to mice treated with GM-CSFRα blockade or untransduced T cells (p=0.0285). To unravel the mechanisms behind functional differences between GM-CSFRα blockade and GM-CSF neutralization, CART cells (n = 3 biological replicates) were cocultured with NALM6 at a 1:1 ratio supplemented with 10 μg/mL GM-CSF neutralizing antibody, GM-CSFRα blockade, or IgG isotype control overnight, followed by T cell isolation, bulk RNA isolation, and RNA sequencing (RNAseq) analysis. Our RNAseq data indicated significant upregulation (Log2FC>0.5, padj. <0.05) of T cell exhaustion- and dysfunction-associated genes (RGS16, PDCD1, CTLA4, NR4A3, CD101, LAG3) as well as negative regulators of T cell functions (G0S2, PIK3IP1, LGALS3) in the GM-CSFRα blockade group compared to GM-CSF neutralizing antibody or IgG isotype control groups. T cell activation-associated pathways were enriched in the GM-CSFRα blockade group. Overall, our data show that GM-CSFRα blockade impacts CART functions differently from GM-CSF neutralization. Specifically, GM-CSFRα blockade inhibits CART proliferation in vitro and antitumor activity in vivo. Interestingly, GM-CSFRα blockade leads to enrichment of T cell exhaustion- and dysfunction-associated genes in CART19, which is worth further investigation. These findings may be applicable to other cytokine receptors upregulated on activated CART cells.

Abstract BACKGROUND Pineocytoma is a rare pineal parenchymal WHO grade 1 tumor, typically well-managed with surgical resection alone. With gross total resection (GTR), 91% 1-year and 84% 5-year survival rates have been reported. Recurrent pineocytoma is rare and optimal management in that setting is unknown, in part owing to the rarity of the condition. METHODS We report an atypical course of pineocytoma in a 58-year-old male involving metastatic disease. RESULTS A pineal mass was identified upon MRI of the brain obtained for vision changes. Patient underwent GTR, pathology was consistent with pineocytoma, WHO grade 1. Ki-67 labeling index was low at 1-2%. Despite low-grade pathology, two years later, surveillance imaging demonstrated recurrence in the pineal region and a second lesion in the right thalamus. The patient underwent gamma knife surgery (GKRS) to both lesions. Three years later, he developed three new lesions in the right cerebellum and again underwent GKRS. Subsequent surveillance imaging identified new lesions in the right cerebellum and along ventricular walls, treated with further GKRS. Nine years after initial diagnosis, focal nodular leptomeningeal metastases were discovered, along with progression at the primary tumor site. Patient was treated with GKRS to the nodular deposits followed by pembrolizumab. Further disease progression as noted despite immunotherapy. Patient was treated with 6 cycles of temozolomide chemotherapy and at further progression 3 cycles of lomustine. Upon additional progression, further radiation therapy was administered with bevacizumab to reduce risk of radiation necrosis. Patient remains on surveillance 13 years after initial diagnosis with visual changes and mild headache representing only clinical issues. CONCLUSIONS Although exceedingly rare, pineocytoma is capable of metastasizing to brain parenchyma and the leptomeninges, as demonstrated in our patient. However, our case demonstrates prolonged survival is possible even in the setting of relentlessly progressive metastatic disease with utilization of multimodality treatment.

Abstract Improved response assessment methods are needed for patients with gliomas. Intracranial cerebrospinal fluid (CSF) is longitudinally accessible and provides an abundance source of candidate biomarkers, such as cell-free DNA (cfDNA), for disease monitoring. To evaluate the clinical feasibility of molecular profiling from longitudinal intracranial CSF, CSF was acquired from patients with CSF access devices, including Ommaya reservoirs and ventriculoperitoneal shunts. cfDNA was extracted and analyzed via PredicineCARE (Next Generation sequencing) or PredicineSCORE (low pass whole genome sequencing). Five patients (2 females, 3 males; median age: 40 years, range 32-64 years) underwent longitudinal intracranial CSF collection via Ommaya reservoirs (n=4) or VP shunt (n=1). In total, forty-eight CSF samples were obtained (median volume: 4.00 mL; 0.5-5 mL), with 39 samples (81.3%) yielding sufficient cfDNA for variant profiling. Our initial findings suggest that tumor fraction increased by 6.28x, 2.87x, and 8.31x with radiographic progression in three patients. Tumor fraction decreased by 0.08x and 0.04x in two patients with paired immediate pre-versus-post chemoradiation samples. Despite ongoing pseudoprogression in one patient, tumor fraction decreased to unmeasurable levels from a post-resection baseline of 0.26. Patient-specific tumor-associated variant allelic frequencies (VAFs), including TP53, PTEN, TERT, and CDKN2A/B, decreased within individual patients after resection and chemoradiation. In two patients with isocitrate dehydrogenase (IDH) mutant gliomas, decreasing IDH1 VAF after resection and chemoradiation correlated with decreased CSF D-2-hydroxyglutarate (D-2-HG) levels (0.64x and 0.62x, respectively, for the first patient, and 0.01x and 0.07x for the other patient). Both CSF IDH1 VAF and CSF D-2-HG increased a patient who had radiographic progression (2.56x and 9.21x, respectively). Moreover, CNB decreased below the limit of quantification during treatment and increased above the limit at progression. In conclusion, longitudinal CSF cfDNA can feasibly be obtained via CSF access devices in patients with gliomas during their disease course toward evaluating candidate monitoring biomarkers.

Abstract BACKGROUND BRAF pathway alterations are frequent in glial/neuroepithelial tumors. BRAF-targeted therapy with BRAF inhibitors (BRAFi) and/or MEK inhibitors (MEKi) has proven efficacy in clinical trials, though mainly enrolling pediatric patients. We sought to investigate real-world data for adult patients with BRAF-pathway mutant glial/neuroepithelial neoplasms treated with BRAFi/MEKi. METHODS Adults with BRAF-pathway mutated glial/neuroepithelial neoplasms treated with BRAFi/MEKi were retrospectively reviewed. RESULTS Five patients (n=4 female, median age at diagnosis 36) were identified (tumor types: glioblastoma n=1, high-grade astrocytoma with epithelioid features n=1, low-grade neuroepithelial tumor n=1, pilocytic astrocytoma n=1, pleomorphic xanthoastrocytoma n=1). Four had BRAF V600E and one had NF1 p.S2687Cfs*5 mutant tumors. Prior to targeted therapy initiation, patients underwent a median 2.4 treatments including surgery (n=4), radiation therapy (n=4), chemotherapy (temozolomide n=4; carboplatin/vincristine n=1). BRAF-targeted therapies included dabrafenib/trametinib (n=4), binimetinib/encorafenib (n=2), binimetinib (n=2). One NF1-mutant glioblastoma received binimetinib followed by binimetinib/bevacizumab. Median BRAFi/MEKi treatment was 15.2 months (range: 6-36 months). Toxicities included leukopenia (n=3), peripheral edema (n=1), reduced ejection fraction (n=1), skin rash (n=5), transaminitis (n=1) with all patients requiring dose reductions and 3 cases requiring discontinuation. Among patients where BRAFi/MEKi was discontinued due to toxicity, 2 received other treatments at progression, then switched to a different BRAFi/MEKi combination, which they tolerated (duration 6-10months). Best radiographic response was partial response, noted in 3 patients treated with dabrafenib/trametinib. Four patients remain alive and continue BRAFi/MEKi. CONCLUSIONS In this cohort of heavily-pretreated patients with glial/neuroepithelial neoplasms, BRAF-targeted therapy was associated with durable responses. Interestingly, two patients who were unable to tolerate one combination of BRAFi/MEKi were able to tolerate a different combination with sustained response. Our findings support ongoing use of BRAF targeted therapy for CNS tumors. Optimal timing and duration of treatment requires further prospective evaluation in adult patients.

Abstract Von Hippel-Lindau (VHL) disease is caused by pathogenic variants in the VHL gene. Abnormal function of VHL protein leads to accumulation of HIF-α and the engagement of hypoxia-sensitive genes with oncogenic effects. VHL is associated with tumors in multiple organs. Belzutifan is an oral selective inhibitor of hypoxia-inducible factor 2 alpha. Belzutifan was approved in 2021 as a targeted therapy for VHL-associated CNS hemangioblastomas, renal cell carcinomas, and pancreatic neuroendocrine tumors. Here, we report on our institutional clinical experience with belzutifan for VHL-associated CNS hemangioblastomas. From October 2021 to May 2024, thirteen adults (9 female, 4 male, median age 40) were treated. Median duration of therapy was 24 months. Seven had a partial radiographic response after median of 3 months; one patient had a partial response; 1 patient had progressive disease on belzutifan. Four patients have been taking belzutifan for less than 3 months, and do not have follow-up imaging to assess response. The most common adverse effect was fatigue (7 patients). Four patients experienced anemia and 2 experienced hypoxia. Three patients discontinued belzutifan due to adverse effects. One patient had to take multiple medication pauses, due to seizure and kidney injury, but resumed treatment with dose reduction. Nine patients remain on therapy. Based on this data, belzutifan is often well-tolerated and associated with durable radiographic responses. Toxicity profile among our patients was similar to that encountered in the clinical trial leading to approval of the agent. These findings provide real-world data supporting continued use of belzutifan in this patient population, with an awareness of potential adverse reactions. Future studies should assess continued radiographic response, sex differences, and effects after long-term use.

Abstract BACKGROUND Pituitary adenomas are often effectively managed with medical therapy, surgery, and radiation therapy. However, up to 15% of pituitary neoplasms exhibit rapid growth, invasive behavior, or, rarely, metastasize despite optimal standard treatments. Temozolomide is associated with a survival benefit for patients with these aggressive pituitary tumors. METHODS We present a case series of 8 patients with an aggressive pituitary tumor (APT) or pituitary carcinoma (PC) treated with temozolomide. RESULTS Eight patients (3 female, median age at diagnosis 44) with APT (n=6) or PC (n=2) were treated. A variety of pituitary tumor types were represented (1 prolactinoma, 5 corticotroph adenomas, 1 gonadotroph adenoma, 1 thyrotropinoma). Prior to temozolomide initiation, patients underwent a median 5 treatments including medical management (n=4), surgery (n=7), radiation therapy (n=7). Temozolomide was started at median 3.5 recurrence. Median treatment duration was 6 cycles (n=6, 150-200 mg/m2 days 1-5, 28-day cycles; n=1, 75 mg/m2, days 1-21, 28-day cycles). Seven patients experienced progression after temozolomide treatment, median progression-free survival (PFS) was 4 months. One patient remains on treatment, progression-free at 5 months. MGMT methylation status was assessed for 3 tumors. Among 2 patients with MGMT promoter methylated tumors, PFS was 4 months for one patient, the other remains on treatment. For one patient with MGMT promoter unmethylated corticotroph adenoma, PFS was 9 months. No ≥ grade 4 toxicities were noted. Most common toxicities were nausea (n=5), thrombocytopenia (n=3), and fatigue (n=2). CONCLUSION In this group of heavily pretreated patients, temozolomide monotherapy was well-tolerated and associated with median PFS of 4 months. Interestingly, PC patients had longer PFS at 9 months and 24 months. Our findings support use of single-agent temozolomide chemotherapy for patients with APT/PC, but further prospective analysis is needed to identify patients more likely to benefit from treatment and optimal timing of chemotherapy initiation.