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Immunotherapy has revolutionized cancer treatment in the past decade, but significant hurdles remain. Human studies with immune checkpoint inhibitors targeting programmed cell death protein have demonstrated suboptimal efficacy in the setting of low-grade gliomas (LGGs). Identification of mechanisms leading to inadequate anti-tumor immunity is paramount. The current study evaluates and validates barriers to immunotherapy using a novel machine learning algorithm.We utilized The Cancer Genome Atlas (TCGA) to generate expression levels of 28 immune genes related to known immunotherapeutic targets or lymphocyte cytolytic activity. We created training and testing groups and 3 machine learning models to determine the genes most highly correlated to cytolytic activity (CYT). The 3 models were run through multiple regression by exhaustive selection, LASSO, and random forest. We validated computational results by comparing expression of pertinent genes in patient-derived glioma samples.Our models demonstrated linearity, a low mean-squared error, and consistent results with respect to the most important variables. Expression of ICOS, IDO1, and CD40 were the most important variables in all models and demonstrated positive correlation with CYT. Other variables included TIGIT and CD137. Genetic analysis from 3 IDH-mutants (IDHm) and 3 IDH-wild type (IDHwt) patient-derived glioma samples validated TCGA data and demonstrated lower levels of CYT in IDHm gliomas compared with IDHwt.This novel methodology has elucidated 3 potential targets for immunotherapy development in LGGs. We also demonstrated a novel method of analyzing data using advanced statistical techniques that can be further used in developing treatments for other diseases as well.

Supplementary Table 2. Comparison of ELISPOT responses between the current study and our recent study in pediatric glioma patients

Supplementary Table 1. Associations between baseline IFN-γ ELISPOT values vs. PFS or subsequent IFN-γ ESLIPOT responses

Purpose: WHO grade 2 low-grade gliomas (LGG) with high risk factors for recurrence are mostly lethal despite current treatments. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in HLA-A2+ adults with high-risk LGGs in the following three cohorts: (i) patients without prior progression, chemotherapy, or radiotherapy (RT); (ii) patients without prior progression or chemotherapy but with prior RT; and (iii) recurrent patients.Experimental Design: GAAs were IL13Rα2, EphA2, WT1, and Survivin. Synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for eight courses with intramuscular injections of poly-ICLC, followed by q12 week booster vaccines.Results: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity was encountered except for one case with grade 3 fever, fatigue, and mood disturbance (cohort 1). ELISPOT assays demonstrated robust IFNγ responses against at least three of the four GAA epitopes in 10 and 4 cases of cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFNγ responses than cohort 3 patients. Median progression-free survival (PFS) periods since the first vaccine are 17 months in cohort 1 (range, 10–47+) and 12 months in cohort 3 (range, 3–41+). The only patient with large astrocytoma in cohort 2 has been progression-free for more than 67 months since diagnosis.Conclusion: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade 2 glioma patients. These results warrant further evaluations of this approach. Clin Cancer Res; 21(2); 286–94. ©2014 AACR.

Supplementary Figure 1. T2-Flair MRI images of 2 cases (Patient 9 in Cohort 1 and Patient 1 in Cohort 2)

Supplementary Figure 2. Immunohistochemistry evaluating expression of vaccine-targeted antigens in resected tumor tissues.

PDF file, 65K, mAb-mediated depletion of T-cells.

PDF file, 66K, Murine glioma-derived CD11b+ cells inhibit CD8+ T-cells in vitro.

PDF file, 129K, Critical role of IL-4Ralpha in T-cell-suppressing function of BM-derived myeloid cells.

PDF file, 65K, Confirmation of chimerism using enhanced green fluorescence protein (EGFP+) donor BM cells.

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Malignant gliomas are lethal cancers in the brain and heavily infiltrated by myeloid cells. Interleukin-4 receptor-α (IL-4Rα) mediates the immunosuppressive functions of myeloid cells, and polymorphisms in the IL-4Rα gene are associated with altered glioma risk and prognosis. In this study, we sought to evaluate a hypothesized causal role for IL-4Rα and myeloid suppressor cells in glioma development. In both mouse de novo gliomas and human glioblastoma cases, IL-4Rα was upregulated on glioma-infiltrating myeloid cells but not in the periphery or in normal brain. Mice genetically deficient for IL-4Rα exhibited a slower growth of glioma associated with reduced production in the glioma microenvironment of arginase, a marker of myeloid suppressor cells, which is critical for their T-cell inhibitory function. Supporting this result, investigations using bone marrow-derived myeloid cells showed that IL-4Rα mediates IL-13–induced production of arginase. Furthermore, glioma-derived myeloid cells suppressed T-cell proliferation in an IL-4Rα–dependent manner, consistent with their identification as myeloid-derived suppressor cells (MDSC). Granulocyte macrophage colony-stimulating factor (GM-CSF) plays a central role for the induction of IL-4Rα expression on myeloid cells, and we found that GM-CSF is upregulated in both human and mouse glioma microenvironments compared with normal brain or peripheral blood samples. Together, our findings establish a GM-CSF–induced mechanism of immunosuppression in the glioma microenvironment via upregulation of IL-4Rα on MDSCs. Cancer Res; 73(21); 6413–23. ©2013 AACR.

PDF file, 129K, IL-4Ralpha on CD11b+Gr1+ BILs and SPCs.

OBJECTIVES Although the impact of surgery in glioblastoma (GBM) is well-established, there is insufficient data to determine how surgical resection affects overall survival (OS). The impact of other GBM genomic alterations on EOR-induced survival remains poorly understood. Here, we leverage a large cohort of GBM patients with genomic data to establish the impact of genomic alterations on resection-dependent overall survival in GBM. METHODS 537 IDH WT GBMs from UPMC were analyzed using Glioseq next generation sequencing panel. Using molecular profiles derived from Glioseq data, semi-supervised Monte Carlo reference-based consensus clustering was used to define 5 distinct groups. Cox Proportional Hazards models were used to identify predictors of OS within molecular groups. RESULTS Clustering of 537 IDH wild-type GBM identified 5 distinct groups: 1: TERT mutated/MGMT methylated/EGFR mutated or gain; 2: TERT mutated /MGMT methylated/p53 mutated; 3: TERT WT; 4: TERT mutated/MGMT methylated/CDKN2A loss; 5: TERT mutated/MGMT unmethylated. Thresholds in postoperative residual contrast enhancing volume (rCEV) and residual non-contrast enhancing volume (rNCEV) were identified which marked significant differences in median overall survival (MOS) between molecular groups in rCEV -Group 1: 11.7cc, Group 2: 14.4cc, Group 3: 1.1cc, Group 4: 0.1cc, and Group 5 1.1cc. Sex, age, KPS >70, temozolomide, and radiation along with thresholded rCEV and rNCEV were used in multivariate Cox analyses. Groups demonstrated distinct predictors of OS: radiation only in Group 1 tumors, low rCEV in Group 2 tumors, younger age, temodar, low CEV, and KPS >70 in Group 3 tumors, female sex, younger age, radiation, KPS >70 low rCEV in Group 4 tumors, and KPS >70, temodar, and low rCEV in Group 5 tumors. CONCLUSIONS Clustering results suggest that there are clinically distinct molecular groups of IDH wild-type glioblastomas. Age, sex, KPS >70, temozolomide treatment, radiation therapy, and EOR have varying affects on these molecular groups.

BACKGROUND GBM genomic profiling relies on sequencing a limited tumor tissue sample, which is invasive and may underrepresent molecular heterogeneity. CSF, which can be obtained less invasively, is in intimate contact with tumor lesions and may better capture the full genomic profile of the tumor. However, datasets with contemporaneously collected CSF and tissue to support this claim have been lacking. To evaluate the performance of CSF NGS, we conducted a pilot study in patients with GBM undergoing a resection for suspected recurrence following first-line chemoradiotherapy. METHODS Paired CSF and tissue samples were sequenced using a hybrid capture-based NGS assay. Clinically meaningful variants were defined as those that are potentially targetable using off-label or clinical trial options, exhibit prognostic value, or may predict response or resistance to specific treatments. RESULTS Eighteen patients were enrolled, and 13 of 18 CSF samples (72.2%) were sequenced successfully. At least one variant was detected in all CSF samples analyzed. A median of 7 variants (range 1—67) was detected per sample across 54 genes. The median variant allele fraction was 0.6% (range 0.2—72.4%). Among 38 clinically meaningful genes, 102 variants were detected; 25 (24.5%) were detected in both tissue and CSF, while 60 (58.8%) were detectable solely in CSF. Hypermutation was detected by CSF in one patient. Of the 82 variants detected in this patient’s tumor, 15 (18.3%) were identified in both tissue and CSF, 15 (18.3%) were identified only in the tissue, and 52 (63.2%) were identified only in the CSF. CONCLUSIONS CSF NGS detects clinically meaningful variants at a substantial rate and frequently identifies mutations not detected by matched tissue NGS. These results suggest that CSF may be a suitable source material for tumor profiling, overcoming the limitations of tissue, and may also provide a more comprehensive tumor profile.

PURPOSE Differentiation of tumor progression (TP) from pseudoprogression (PsP) is a major unmet need in post-treatment glioblastoma (GBM). 18F-Fluciclovine is a synthetic amino acid PET radiotracer with higher uptake in tumor tissue vs. areas of treatment-related change. We investigated the value of 18F-Fluciclovine PET for differentiating PsP from TP independent from and in combination with multi-parametric MRI. METHODS We prospectively enrolled 30 patients with GBM with a new or enlarging contrast-enhancing lesion on MRI after chemoradiotherapy who were planned for surgical resection of the lesion. Patients underwent pre-operative 18F-Fluciclovine PET and multi-parametric MRI. Following surgery, the relative percentages of viable tumor and therapy-related changes observed in histopathology were quantified. Patients were categorized as TP if viable tumor represented ≥ 50% of the specimen, mixed TP if < 50% and > 10%, and PsP if ≤ 10%. RESULTS 18 patients had TP, 4 had mixed TP, and 8 PsP. Patients with TP/mixed TP had a significantly higher 40-50 minutes SUVmax (6.64 + 1.88 vs 4.11± 1.52, p=0.009) and an SUVmax cut-off of 4.66 provided 90% sensitivity and 83% specificity for differentiation of TP/mixed TP from PsP (AUC=0.856). A maximum cerebral blood volume (CBVmax) cut-off of 3.67 provided 90% sensitivity and 71% specificity for differentiation of TP/mixed TP from PsP (AUC=0.779). Combining a 40-50 minutes SUVmax cut-off of 4.662 and a relative CBVmax cut-off of 3.67 provided 100% sensitivity and 80% specificity for differentiating TP/mixed TP from PsP (AUC=0.95). The time activity curve patterns and time to peaks were not different between the groups. Normalization of PET parameters to normal brain parenchyma were not helpful to differentiate the groups due to variability in radiotracer uptake in normal brain between subjects. CONCLUSION 18F-Fluciclovine PET uptake can accurately differentiate PsP from TP in GBM patients, with even more accurate differentiation achieved when combined with MRI.

Diffuse gliomas are fatal, immunologically cold tumors characterized by mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) genes. IDH mutations have been shown to result in global genomic hypermethylation and drastically altered transcriptomic landscape. We and others have shown that IDH mutation is direct responsible for the immunological cold landscape observed in IDH mutated glioma. Using a combination of animal models and patient-derived glioma stem cells (GSCs), we identify retinoic acid signaling as a primary regulator of innate and adaptive immune activation in IDHm gliomas. Using the Sleeping Beauty (SB) orthotopic murine model of IDH glioma, which faithfully recapitulates the TME seen in patients, we show that intracellular accumulation of oncometabolite 2-HG leads to hypermethylation and transcriptional repression of Retinol Binding Protein-1 (RBP1), which encode CRBP1, a key chaperone protein required for intracellular all-trans-retinoic-acid biosynthesis in glial cells. Restoration retinoic acid signaling through the addition of all-trans retinoic acid (ATRA) resulted in spontaneous tumor regression and prolonged survival. ATRA administration is further correlated with increased CD8, NK, M1 macrophages and decreased Treg and MDSC populations within the TME. Single-cell sequencing of the TME shows increased type I interferon signaling, myeloid cell polarization, T cell activation, and TCR diversity. Using knockdown and overexpression techniques in patient-derived GSCs, we show that CRBP1 activity is critical for NK mediated immune surveillance and cytotoxicity through regulation of NKG2D ligand expression and immune cell migration through differential regulation of CXCL12 and CCL2. In summary, epigenetic silencing of RBP1 by 2-HG induced hypermethylation leads to a global immune dysregulation resulting in an immunologically cold landscape. Restoration of retinoic acid signaling is a promising immunotherapeutic approach for the treatment of IDHm gliomas.

BACKGROUND We evaluated the combination of retifanlimab, INCAGN01876, and FSRT in patients with recurrent GBM. METHODS Phase II, single-center, 2 cohort study (A: single-arm non-surgical cohort; B, two-arm neoadjuvant/surgical cohort). Cohort A patients received pre-FSRT one-time doses of retifanlimab (500mg) and INCAGN01876 (300mg), FSRT (8 Gy x 3 fractions), and post-FSRT 28-day treatment cycles (retifanlimab, day 1; INCAGN01876, days 1, 15). Cohort B patients received pre-surgery doses of retifanlimab + INCAGN01876; subsequently, patients either went directly to resection (Sub-Arm 1) or to FSRT followed by resection (Sub-Arm 2). All patients resumed immunotherapy post-operatively. Primary endpoint: ORR in Cohort A. Data cut-off for this analysis was June 30, 2022. RESULTS Thirty-two evaluable patients: Cohort A, n=16; Cohort B, n=16 (Sub-Arm 1, n=8, Sub-Arm 2, n=8). Median follow-up time: Cohort A, 13.6 months; Cohort B, 8.8 months. Forty-four percent women, median age 64 (IQR, 55–65), 56% MGMT unmethylated. Most common grade 3/4 treatment-related AEs included cerebral edema (25%), lymphopenia (16%), and cognitive disturbance (13%). Efficacy in Cohort A: no objective responses observed, best response of stable disease achieved in 9/16 patients (56%), median PFS 3.9 months (95% CI 2.1 – 6.2 months), median OS 9.8 months (95% CI 8.3 months – not reached [NR]). Efficacy in Cohort B: median PFS NR, median OS 15.1 months (95% CI 8.5 months – NR). Median PFS and OS are longer in Cohort B Sub-Arm 2 compared to Cohort B Sub-Arm 1 (PFS, NR vs. 2.2 months, p = 0.009; OS, NR vs. 8.5 months, p=0.026). Results of tissue and blood-based immune correlative analyses will be presented. CONCLUSIONS The combination of retifanlimab, INCAGN01876, and FSRT is generally well-tolerated in patients with recurrent GBM when administered with or without surgical resection. Survival outcomes in the neoadjuvant cohort are encouraging, largely driven by patients that received neoadjuvant FSRT.

INTRODUCTION Preoperative stereotactic radiosurgery (SRS) is an emerging therapeutic strategy for treatment of brain metastasis requiring surgical resection. Prior reports have been highly-controlled, excluding very large brain metastases. Here, we report a prospectively-collected preoperative series reflective of a real-world neurosurgical patient cohort, with a particular focus on resumption of immunotherapy. METHODS Our series included Nf27 prospective preoperative compared to concurrently collected Nf37 postoperative patients. Demographic, SRS volumes/dose, pre/postoperative immunotherapy, and clinical outcomes were collected. Subsequent univariate comparisons between groups were performed. RESULTS Pre/postoperative tumor volumes (19.1 [range: 2.4-65.1] vs. 12.5 [3.6-80.3]cc, p=0.20); local tumor control at 1 year (77.6% vs. 71.4%, p=0.616); overall survival (1.0 [0.8-2.2] vs. 1.3 [0.7-NR] years, p=0.8) were comparable between groups. Rates of LMD (3.7% vs. 8.0%, p=0.39) and subsequent WBRT (8.0% vs. 16.2%, p=0.46) were not statistically different. OR time (168 vs. 173 min, p=0.64), estimated blood loss (133 vs. 180 mL, p=0.31), and wound infections (1 vs. 1, p=1.00) were not significantly different.An ostensible advantage to preoperative SRS is that steroids may be weaned more quickly to allow resumption of immunotherapy. Median steroid taper length was significantly shorter (8 vs. 21.5 days, p=0.007), and median daily Dexamethasone dose at 1 week post-op was significantly lower in the preoperative group (1 vs. 7mg d, p=0.007). All preoperative patients eligible for resumption of immunotherapy (Nf5) resumed treatment within 6 weeks. Conversely, only 2/6 in postoperative patients resumed treatment within 6 weeks, (p=0.07), while 4 failed to resume immunotherapy altogether. In patients initiating immunotherapy after OR/SRS, time to initiation was non-significantly lower in the preoperative group (52[38-84] vs.163[108-208] days, p=0.06). CONCLUSIONS Preoperative SRS is safe and provides comparable local tumor control rate even in large tumors ( >20cc). In patients requiring resumption of immunotherapy, preoperative SRS may provide a faster pathway towards resumption of systemic therapy.

2015 Background: Glioblastoma (GBM) is associated with a high rate of venous thromboembolism (VTE), but there is little data to guide anticoagulation in GBM patients, in whom the risks of VTE must be balanced against the risk of intracranial hemorrhage (ICH). Methods: We performed a single-institution retrospective cohort study of patients with GBM diagnosed with VTE from 2014-2021 who were treated with low molecular weight heparin (LMWH) or a direct oral anticoagulant (DOAC). The cumulative incidence of ICH was compared between the LMWH and DOAC groups. The primary outcome was clinically relevant ICH within the first 30 days of anticoagulation, defined as any ICH that was fatal, symptomatic, required surgical intervention, and/or led to cessation of anticoagulation. Key secondary outcomes included clinically relevant ICH within 6 months, fatal ICH within 30 days and 6 months, any bleeding within 30 days and 6 months, and recurrent VTE within 6 months. Fisher’s exact test was used for comparison of primary and secondary endpoints between the two groups. Cumulative incidence curves were generated using the Kaplan-Meier method, and the cumulative incidence of clinically relevant ICH at both the 30-day timepoint and 6-month timepoint was compared between the DOAC and LMWH groups using the Gray test to account for death as a competing risk. Results: A total of 121 patients were identified in the primary cohort for 30-day outcome analyses (DOAC, n = 33; LMWH, n = 88). For 6-month outcome analyses, the cohort included only patients who were maintained on their initial anticoagulant (DOAC or LMWH) and did not switch anticoagulants during the 6 months following diagnosis of VTE (DOAC, n = 32; LMWH, n = 75). The cumulative incidence of clinically relevant ICH at 30 days was 0% (0/33) in the DOAC group and 9% (8/88) in the LMWH group (p = 0.11). The cumulative incidence of clinically relevant ICH at 6 months was 0% (0/32) in the DOAC group and 24% (18/75) in the LMWH group (p = 0.001), with 4 fatal ICHs in the LMWH group. Other outcomes are displayed in the Table. Conclusions: Our study suggests that DOACs are associated with a lower incidence of clinically relevant ICH in patients with GBM-associated VTE compared to LMWH. These data support the use of DOACs as a safe alternative to LMWH in patients with GBM.[Table: see text]

Purpose/Objective(s) Brain metastasis is a frequent cause of morbidity and mortality in stage IV cancer patients. Efforts to treat brain metastasis include surgical resection, whole brain radiation treatment (WBRT), and stereotactic radiosurgery (SRS). In contemporary times, SRS is deployed utilizing conformal, high dose radiation with rapid dose fall off to limit neurocognitive toxicity seen with WBRT. In the combined modality setting wherein surgical resection is required, post-operative SRS is utilized with excellent local control. However, several difficulties may arise from treatment in the post-operative setting. Changes to the tumor bed necessitating increased margin for uncertainty, increased treatment normal brain tissue, treatment in a hypoxic field may portend for less-than-ideal treatment. Therefore, pre-operative SRS is being explored due to potentially better-defined target, less target uncertainty, and reports of less radionecrosis and less leptomeningeal disease. Therefore, our group conducted a phase II clinical trial exploring the efficacy of the preoperative SRS approach in regard to local brain control, distant brain control, and overall survival. Materials/Methods A phase II institutional non-randomized clinical trial was initiated at UPMC Hillman Cancer Center enrolling patients with brain metastasis of any malignant histology. Lymphoma, leukemia, multiple myeloma, or germ cell tumors were excluded due to their radiosensitive nature. Patients were to receive SRS followed by surgical resection. Inclusion criteria included ≥18yo, KPS≥50 with a life expectancy of 12 weeks. Patients could have no more than four lesions measuring 1.5-4.0cm in size. Patients that had tumors Results A total of 24 patients were enrolled on study. One patient was deemed unresectable due to risk to the peri- rolandic location. Median number of brain metastasis treated was one (range 1-3). The most common treated histology was non-small cell lung carcinoma (NSCLC) (9). Median days from consult to SRS was 3.9 days. Median SRS dose and fraction was 16.5Gy [range: 15-24Gy] in a single fraction. The median number of surveillance MRIs performed was 6 [range 1-17]. Local brain control was 90%, 90%, and 74% at 6, 12, and 24 months. Distant brain control at 6, 12, and 24 months was 66%, 60%, and 54% respectively. Progression free survival (PFS) and overall survival (OS) at 6, 12, and 24 months were as followed [46%, 38%, 29%] and [66%, 58%, 50%] respectively. Conclusion Preoperative stereotactic radiosurgery appears to have excellent local control with a 6- and 12-month local control rates of 90%. Further study is warranted in the continued exploration of preoperative radiation therapy.

BACKGROUND CAN-2409 is a replication-deficient adenovirus that delivers HSV thymidine kinase to cancer cells, resulting in local conversion of orally administered valacyclovir into a toxic metabolite. Previously, a phase 1b/2 clinical trial of CAN-2409 combined with standard-of-care (SOC) demonstrated safety and improved survival in HGG patients. Addition of CAN-2409 to nivolumab has the potential to activate locally recruited lymphocytes and teach them to recognize tumor neoantigens, changing the ‘cold’ immunosuppressive tumor microenvironment, and synergizing with the activity mediated by immune checkpoint inhibitors. This notion is supported by preclinical experiments showing that the combination of CAN-2409 with anti-PD1 therapy was more effective than either treatment alone. METHODS This ongoing phase 1 clinical trial evaluates safety and initial efficacy of CAN-2409 combined with nivolumab and SOC in newly diagnosed HGG. CAN-2409 is injected during neurosurgery into the tumor bed, followed by 14-days of valacyclovir. Radiation starts within 8 (+/-4) days of surgery. Temozolomide is administered to MGMT-methylation positive patients only. Nivolumab is given every 2 weeks, up to 52-weeks. Deep immune profiling studies are ongoing and initial results will be available shortly. RESULTS From February 2019 to March 2021, 41 patients were enrolled and 35 were evaluable for safety from the combination of CAN-2409, nivolumab and SOC: 24 male and 11 female; 34 glioblastoma, 1 diffuse astrocytoma; 33 IDH-wildtype, 2 IDH-mutant; 15 MGMT-methylated, 20 unmethylated. Median age was 62-years (range 28-79), median KPS 90 (range 80-100). With 13 months median follow-up, no unexpected serious adverse events were observed, and 23 patients are still alive. The most frequent possibly related adverse events (>10%) were nausea, fatigue, fever, headache, and increased ALT. CONCLUSIONS The combination of CAN-2409 + nivolumab + SOC was well tolerated. Clinical follow-up and extensive biomarker analyses will provide a better understanding of the therapeutic potential of this approach.