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As immunotherapy assumes a central role in the management of many cancers, ongoing work is directed at understanding whether immune-based treatments will be successful in patients with glioblastoma (GBM). Despite several large studies conducted in the last several years, there remain no FDA-approved immunotherapies in this patient population. Nevertheless, there are a range of exciting new approaches being applied to GBM, all of which may not only allow us to develop new treatments but also help us understand fundamental features of the immune response in the central nervous system. In this review, we summarize new developments in the application of immune checkpoint blockade, from biomarker-driven patient selection to the timing of treatment. Moreover, we summarize novel work in personalized immune-oncology by reviewing work in cancer immunogenomics–driven neoantigen vaccine studies. Finally, we discuss cell therapy efforts by reviewing the current state of chimeric antigen receptor T-cell therapy.

A recent meeting was held on March 22, 2019, among the FDA, clinical scientists, pharmaceutical and biotech companies, clinical trials cooperative groups, and patient advocacy groups to discuss challenges and potential solutions for increasing development of therapeutics for central nervous system metastases. A key issue identified at this meeting was the need for consistent tumor measurement for reliable tumor response assessment, including the first step of standardized image acquisition with an MRI protocol that could be implemented in multicenter studies aimed at testing new therapeutics. This document builds upon previous consensus recommendations for a standardized brain tumor imaging protocol (BTIP) in high-grade gliomas and defines a protocol for brain metastases (BTIP-BM) that addresses unique challenges associated with assessment of CNS metastases. The “minimum standard” recommended pulse sequences include: (i) parameter matched pre- and post-contrast inversion recovery (IR)–prepared, isotropic 3D T1-weighted gradient echo (IR-GRE); (ii) axial 2D T2-weighted turbo spin echo acquired after injection of gadolinium-based contrast agent and before post-contrast 3D T1-weighted images; (iii) axial 2D or 3D T2-weighted fluid attenuated inversion recovery; (iv) axial 2D, 3-directional diffusion-weighted images; and (v) post-contrast 2D T1-weighted spin echo images for increased lesion conspicuity. Recommended sequence parameters are provided for both 1.5T and 3T MR systems. An “ideal” protocol is also provided, which replaces IR-GRE with 3D TSE T1-weighted imaging pre- and post-gadolinium, and is best performed at 3T, for which dynamic susceptibility contrast perfusion is included. Recommended perfusion parameters are given.

Adoptive cellular therapy in the form of CAR T cells or TCR engineered T cells has emerged as a novel approach in the treatment of both solid and hematologic malignancies. Neoantigens generated by tumor somatic mutations represent potentially attractive therapeutic targets in this context owing to their tumor-specific expression and circumvention of immunological tolerance. However, existing cell therapy systems generally target self-proteins or virally overexpressed antigens that fail to recapitulate the features of endogenous tumor neoantigens. Thus, there exists a need for a model in which tumor-specific neoantigens can be targeted via adoptive cellular therapy. Prior work from our lab identified the Imp3D81N mutation (mImp3) within GL261 as a neoantigen recognized by CD8 T cells in both intracranial tumors and draining cervical lymph nodes. To generate a system for targeting this neoantigen, we isolated and cloned mImp3-specific TCRs through a single-cell sort followed by a nested multiplexed PCR reaction. The specificity and functionality of these isolated TCRs was determined through introduction into a T cell hybridoma, identifying a top candidate based upon a high degree of cytokine production and specificity for the mutant epitope. A TCR transgenic mouse was then generated in which more than 90% of all T cells were CD8 T cells bearing this mImp3-specific TCR. T cells isolated from this mouse display specificity for the mImp3 peptide and display in vitro reactivity to GL261 and other cell lines in a mImp3-dependent manner. Therefore, this model represents the first TCR transgenic targeting a brain tumor neoantigen, opening the door for further investigation into cell therapy against this class of antigens.

Background Although clinical trials testing immunotherapies in glioblastoma (GBM) have yielded mixed results, new strategies targeting tumor-specific somatic coding mutations, termed “neoantigens,” represent promising therapeutic approaches. We characterized the microenvironment and neoantigen landscape of the aggressive CT2A GBM model in order to develop a platform to test combination checkpoint blockade and neoantigen vaccination. Methods Flow cytometric analysis was performed on intracranial CT2A and GL261 tumor-infiltrating lymphocytes (TILs). Whole-exome DNA and RNA sequencing of the CT2A murine GBM was employed to identify expressed, somatic mutations. Predicted neoantigens were identified using the pVAC-seq software suite, and top-ranking candidates were screened for reactivity by interferon-gamma enzyme linked immunospot assays. Survival analysis was performed comparing neoantigen vaccination, anti-programmed cell death ligand 1 (αPD-L1), or combination therapy. Results Compared with the GL261 model, CT2A exhibited immunologic features consistent with human GBM including reduced αPD-L1 sensitivity and hypofunctional TILs. Of the 29 CT2A neoantigens screened, we identified neoantigen-specific CD8+ T-cell responses in the intracranial TIL and draining lymph nodes to two H2-Kb restricted (Epb4H471L and Pomgnt1R497L) and one H2-Db restricted neoantigen (Plin2G332R). Survival analysis showed that therapeutic neoantigen vaccination with Epb4H471L, Pomgnt1R497L, and Plin2G332R, in combination with αPD-L1 treatment was superior to αPD-L1 alone. Conclusions We identified endogenous neoantigen specific CD8+ T cells within an αPD-L1 resistant murine GBM and show that neoantigen vaccination significantly augments survival benefit in combination with αPD-L1 treatment. These observations provide important preclinical correlates for GBM immunotherapy trials and support further investigation into the effects of multimodal immunotherapeutic interventions on antiglioma immunity. Key Points 1. Neoantigen vaccines combined with checkpoint blockade may be promising treatments. 2. CT2A tumors exhibit features of human GBM microenvironments. 3. Differential scanning fluorimetry assays may complement in silico neoantigen prediction tools.

Background Diagnostic workflows for glioblastoma (GBM) patients increasingly include DNA sequencing-based analysis of a single tumor site following biopsy or resection. We hypothesized that sequencing of multiple sectors within a given tumor would provide a more comprehensive representation of the molecular landscape and potentially inform therapeutic strategies. Methods Ten newly diagnosed, isocitrate dehydrogenase 1 (IDH1) wildtype GBM tumor samples were obtained from 2 (n = 9) or 4 (n = 1) spatially distinct tumor regions. Tumor and matched blood DNA samples underwent whole-exome sequencing. Results Across all 10 tumors, 51% of mutations were clonal and 3% were subclonal and shared in different sectors, whereas 46% of mutations were subclonal and private. Two of the 10 tumors exhibited a regional hypermutator state despite being treatment naive, and remarkably, the high mutational load was predominantly limited to one sector in each tumor. Among the canonical cancer-associated genes, only telomerase reverse transcriptase (TERT) promoter mutations were observed in the founding clone in all tumors. Reconstruction of the clonal architecture in different sectors revealed regionally divergent evolution, and integration of data from 2 sectors increased the resolution of inferred clonal architecture in a given tumor. Predicted therapeutic mutations differed in presence and frequency between tumor regions. Similarly, different sectors exhibited significant divergence in the predicted neoantigen landscape. Conclusions The substantial spatial heterogeneity observed in different GBM tumor sectors, especially in spatially restricted hypermutator cases, raises important caveats to our current dependence on single-sector molecular information to guide either targeted or immune-based treatments.

BACKGROUND Lymphopenia is common after standard radiation therapy (RT) and temozolomide (TMZ) in high-grade gliomas (HGG) and correlates with shorter survival. Interleukin-7 (IL-7), a T-cell proliferation cytokine, is inappropriately low in HGG patients. Our previous preclinical studies demonstrated that NT-I7 (efineptakin alfa), a recombinant human IL-7, corrects lymphopenia and improves survival in murine glioma models. This study examines the safety and absolute lymphocyte counts (ALCs) following administration of NT-I7 patients with newly diagnosed HGG. METHODS Patients with HGG receiving RT/TMZ with ALC ≥600 were eligible. NT-I7 was administered intramuscularly within 1 week after completion of concurrent RT/TMZ and then every 12 weeks, for up to 4 total doses. Phase I examined 6 dose levels (60, 120, 240, 540, 720, and 960 mcg/kg) using the accelerated-titration design for the first 2 doses and 3 + 3 design thereafter to identify the maximum tolerated dose (MTD). Phase II is a double-blinded, placebo-controlled study with 10 HGG patients per arm comparing changes in ALC. Immune profiling will be performed with CyTOF and cytokine analysis. RESULTS Phase I is complete, with 19 patients (89% GBM, median age: 58 (range: 25-78), median baseline ALC: 1000 cells/mm3, median dexamethasone: 0 mg/day (range: 0-12)). The median number of NT-I7 doses administered was 2 (range: 2-4). The most common treatment-related adverse event was grade 1 or 2 injection site reactions (42%). Two patients had dose-limiting toxicities at 960 mcg/kg (grade 3 elevated alanine aminotransferase and grade 3 back pain), prompting selection of 720 mcg/kg for phase II. Dose-dependent increases in ALC were observed at 4 weeks ranging from 1.3x to 4.1x. Phase II enrollment is ongoing. CONCLUSIONS NT-I7 is well tolerated when administered after chemoradiotherapy for HGG patients with MTD of 720 mcg/kg and demonstrates dose-dependent increase of ALC. Phase II and immune profiling are ongoing. Clinical Trial ID: NCT03687957.

Pediatric brain tumors with replication repair deficiency (RRD) are hypermutant and may respond to immune checkpoint inhibition (ICI). We performed a consortium registry study of ICI in recurrent RRD cancers. Clinical and companion biomarkers were collected longitudinally on all patients. Biomarkers included tumor mutational burden (TMB), neoantigens and genetic signatures obtained from whole genome and exome sequencing. Immune inference was obtained by RNAseq and T cell rearrangement was collected in the tumor and in blood throughout treatment. Of the 46 tumors on the study, 32 were brain tumors with glioblastoma in 96%. Rapid, objective responses (>50%) were observed in 50% of glioblastomas. Three year overall survival for the whole cohort was 48+/-8% which compares favorably with historical controls. Brain tumors fared worse with OS of 39+/-10% and late recurrences observed even after 2 years of therapy (p=0.02). Tumor size and acute “flare” constitute poor outcome throughout all cancers. While all tumors are hypermutant, TMB and predicted neoantigens correlated with response to ICI (p=0.02). Specific signatures extracted from SNVs and total mutations predicted response to ICI and favorable outcome (p=0.005). RNA inference and TCR reveal that the FLARE phenotype is mostly acute nonspecific immune response and not true progression. Finally, glioblastomas (n=8) which failed single agent ICI had favorable responses to combinational immunotherapies with prolonged survival of 65%+/-8% at one year after failure vs 0 for other patients (p=0.01). RRD glioblastomas exhibit favorable outcome and responses to ICI. Combinational therapies based on tumor and immune signatures of these cancers are necessary.

While the degree of spatial genomic heterogeneity within primary glioblastoma (GBM) has been previously investigated, the extent of this heterogeneity in other malignant brain tumors and its connection to the local immune microenvironment remains unknown. To address this, we performed whole-exome, RNA, and TCR sequencing on multiple spatially distinct sectors from a cohort of malignant brain tumors comprised of both brain metastases and primary and recurrent GBMs. Our results suggest a striking difference in which a majority of mutations and predicted neoantigens are shared between spatially distinct regions of metastatic brain tumors in contrast to the spatial heterogeneity observed within both primary and recurrent GBMs. Additionally, despite substantial differences in immunotherapy responsiveness between brain metastases and GBM, we can detect significantly expanded T-cell clonotypes within both tumor types with some clonal frequencies exceeding 10% of intratumoral T-cells. Similar to the observed distribution of variants and neoantigens, the expanded clonotypes are more shared among spatially distinct sectors in metastatic brain tumors than in primary or recurrent GBMs. Interestingly, the frequencies of most of these enriched T-cell clones are significantly diminished within expanded tumor infiltrating lymphocyte (TIL) cultures, advising caution in the use of expanded TIL for the detection of tumor-specific responses. These results provide novel insight into the immunogenomic landscape of malignant brain tumors with implications for our understanding of tumor-immune interactions and the development of immunotherapies.

Cancer immunotherapy represents the biggest change in the cancer treatment landscape in the last several years. Indeed, the clinical successes in several cancer types have generated widespread enthusiasm that immune-based treatments may influence the management of patients with malignant brain tumors as well. A number of promising clinical trials in this area are currently ongoing in neuro-oncology, and a wave of additional efforts are sure to follow. However, the basic immunology underlying immunotherapy-and the nuances unique to the immunobiology in the central nervous system-is often not in the daily lexicon of the practicing neuro-oncologist and neurosurgeon. To this end, here we provide a timely and working overview of key principles of fundamental immunology as a pragmatic context for understanding where therapeutic efforts may act in the cellular dynamics of the immune response. Moreover, we review the issues of lymphatic drainage, antigen presentation, and the blood-brain barrier as considerations that are germane to thinking about immunity to tumors arising in the brain. Together, these topics will provide a foundation for the exciting efforts in immune-based treatments that will hopefully provide real benefit to brain tumor patients.

Cancer immunotherapy represents the biggest change in the cancer treatment landscape in the last several years. Indeed, the clinical successes in several cancer types have generated widespread enthusiasm that immune-based treatments may influence the management of patients with malignant brain tumors as well. A number of promising clinical trials in this area are currently ongoing in neuro-oncology, and a wave of additional efforts are sure to follow. However, the basic immunology underlying immunotherapy—and the nuances unique to the immunobiology in the central nervous system—is often not in the daily lexicon of the practicing neuro-oncologist and neurosurgeon. To this end, here we provide a timely and working overview of key principles of fundamental immunology as a pragmatic context for understanding where therapeutic efforts may act in the cellular dynamics of the immune response. Moreover, we review the issues of lymphatic drainage, antigen presentation, and the blood–brain barrier as considerations that are germane to thinking about immunity to tumors arising in the brain. Together, these topics will provide a foundation for the exciting efforts in immune-based treatments that will hopefully provide real benefit to brain tumor patients.

Diagnostic workflows for GBM patients increasingly include DNA sequencing-based analysis of a single tumor site following tumor biopsy or resection. Recent studies have highlighted the significant intratumoral heterogeneity of both treatment naïve and recurrent tumors in glioblastoma. We hypothesized that sequencing of multiple sectors within a given tumor would provide a more comprehensive representation of the molecular landscape than data from a single site and inform therapeutic strategies. We show that across 10 tumors subjected to whole-exome sequencing of multiple tumor sectors, 54% of mutations were shared between sectors (51% clonal and 3% subclonal), while 46% were subclonal and private. Among the canonical GBM-associated genes, only TERT promoter mutations were observed in the founding clone in all tumor sectors across all cases. Despite being treatment naïve, two of the 10 tumors exhibited a hypermutator state, which has been shown to predict response to immune checkpoint inhibitor therapy. Critically, the high mutational load was predominantly limited to one sector in both cases. Analysis of clonal architecture, inferred from variant allele frequencies of single nucleotide variants in copy-number neutral regions, demonstrated significant differences between sectors in 8 of 10 cases, suggesting regionally divergent evolution. Predicted therapeutically relevant mutations, based on exploratory analysis of the Drug Gene Interaction database (DGIdb), differed in presence and frequency between tumor sectors, with 74% of mutations across all tumors being private to one region. 80% of tumors had at least one private therapeutically relevant mutation. Similarly, different sectors exhibited significant divergence in the predicted neoantigen landscape, based on the pVAC-seq pipeline, with all 10 tumors harboring private neoantigens. Overall, 55% of predicted neoantigens were subclonal and private. These results suggest that multi-site analyses may be necessary to thoroughly characterize individual GBM tumors and precisely guide personalization of molecular and immune-based therapies.

High-grade Gliomas (HGG) are the most common primary brain tumors and continue to carry a poor overall survival. Despite extensive molecular characterization, the “mutation-to-drug” paradigm has not been successfully applied to patients with glioblastoma as it has in other cancers. Due to the (a) success of checkpoint blockade immunotherapy, such as inhibition of the PD-1/PD-L1 pathway, in other tumor types and (b) the known immunologic comprise these patients exhibit, there has been tremendous enthusiasm regarding immune-based treatments in this setting. Although clinical trials incorporating the inhibition of the PD-1/PD-L1 pathway are ongoing, the biology underlying PD-L1 expression in glioma remains unclear. Moreover, there is a limited understanding of the underlying functional consequences of the expression of the related PD-1 ligand, PD-L2. To focus exclusively on the cell autonomous regulation of these ligands, we interrogated the Cancer Cell Line Encyclopedia, a Broad Institute/Novartis compendium of passaged cell lines independent of effects of the tumor microenvironment. We found that a subset of glioma cell lines harbor high constitutive PD-L1 and PD-L2 mRNA levels. Consistent with the mRNA levels, we showed that both PD-L1 and PD-L2 cell surface protein is overexpressed by flow cytometry. Two subsequent studies are ongoing. First, to identify the transcriptional basis of ligand upregulation, we have identified a suite of transcription factors using in silico analysis that harbor consensus binding sites in suspected promoter/enhancer regions and have modeled this using endogenous promoter/enhancer luciferase assays. Secondly, to determine the functional consequences of PD-L1 and/or PD-L2 overexpression in the anti-glioma immune response, we ectopically expressed these ligands in the GL-261 murine glioma cell line and tested their influence on T cell recognition of a model antigen. Together, these studies will clarify the regulation of the PD-L1/PD-L2 ligands and identify additional methods with which to decrease their immunosuppressive functions.