Elias Sayour

Increased proportion of FoxP3+ regulatory T cells in tumor infiltrating lymphocytes is associated with tumor recurrence and reduced survival in patients with glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive malignancy associated with profound host immunosuppression mediated in part by FoxP3 expressing regulatory CD4+ T lymphocytes (Tregs) that down-regulate anti-tumor immunity. In order to assess whether FoxP3 was an independent driver differentially expressed in primary versus recurrent GBMs, we stained resected primary and recurrent GBM tumors for CD3, CD4, CD8 and FoxP3 expression using standard immunohistochemistry. Slides were scanned with a high-resolution scanner (ScanScope CS; Aperio), and image analysis software (Aperio ScanScope) was used to enumerate lymphocyte subpopulations allowing for high-throughput analysis and bypassing manual selection bias. As shown in previous studies, enumeration of individual lymphocyte populations did not correlate with clinical outcomes in patients with GBM. However, the CD4+ to regulatory FoxP3+ T cell ratio was diminished in recurrent disease, and increased CD3 and CD8+ to regulatory T cell ratios showed a positive correlation with survival outcomes in primary GBM. These results suggest that while absolute numbers of tumor infiltrating lymphocytes may not be informative for predicting clinical outcomes in patients with GBM, the effective balance of CD3, CD4 and CD8+ T cells to immunosuppressive FoxP3+ regulatory cells may influence clinical outcomes in this patient population.

Differential Immune Microenvironments and Response to Immune Checkpoint Blockade among Molecular Subtypes of Murine Medulloblastoma.

Purpose: Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma, the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and group 3 medulloblastoma for preclinical evaluation in immunocompetent C57BL/6 mice. Experimental Design and Results: Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid-derived suppressor cells, and tumor-associated macrophages in murine SHH model tumors compared with group 3 tumors. However, murine group 3 tumors had higher percentages of CD8+ PD-1+ T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial group 3 tumors compared with SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1+ peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3+ T cells within the tumor microenvironment. Conclusions: This is the first immunologic characterization of preclinical models of molecular subtypes of medulloblastoma and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment. Clin Cancer Res; 22(3); 582–95. ©2015 AACR.

BLyS levels correlate with vaccine-induced antibody titers in patients with glioblastoma lymphodepleted by therapeutic temozolomide

B lymphocyte stimulator (BLyS) is a cytokine involved in differentiation and survival of follicular B cells along with humoral response potentiation. Lymphopenia is known to precipitate dramatic elevation in serum BLyS; however, the use of this effect to enhance humoral responses following vaccination has not been evaluated. We evaluated BLyS serum levels and antigen-specific antibody titers in 8 patients undergoing therapeutic temozolomide (TMZ)-induced lymphopenia, with concomitant vaccine against a tumor-specific mutation in the epidermal growth factor receptor (EGFRvIII). Our studies demonstrate that TMZ-induced lymphopenia corresponded with spikes in serum BLyS that directly preceded the induction of anti-EGFRvIII antigen-specific antibody titers, in some cases as high as 1:2,000,000. Our data are the first clinical observation of BLyS serum elevation and greatly enhanced humoral immune responses as a consequence of chemotherapy-induced lymphopenia. These observations should be considered for the development of future vaccination strategies in the setting of malignancy.

Novel role of hematopoietic stem cells in immunologic rejection of malignant gliomas.

Adoptive cellular therapy (ACT) after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has been largely limited to melanoma due to a lack of reliable methods for expanding tumor-specific lymphocytes from the majority of other solid cancers. We have employed tumor RNA-pulsed dendritic cells (DCs) to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative ACT in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative (MA) conditioning, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed DC vaccines. Hematopoietic stem cells (HSCs), administered for salvage from MA conditioning, rapidly migrated to areas of intracranial tumor growth and facilitated the recruitment of tumor-specific lymphocytes through HSC-elaborated chemokines and enhanced immunologic rejection of intracranial tumors during ACT. Furthermore, HSC transplant under non-myeloablative (NMA) conditions also enhanced immunologic tumor rejection, indicating a novel role for the use of HSCs in the immunologic treatment of malignant gliomas and possibly other solid tumors.

The effects of new or worsened postoperative neurological deficits on survival of patients with glioblastoma

OBJECTIVE An increased extent of resection (EOR) has been shown to improve overall survival of patients with glioblastoma (GBM) but has the potential for causing a new postoperative neurological deficit. To investigate the impact of surgical neurological morbidity on survival, the authors performed a retrospective analysis of the clinical data from patients with GBM to quantify the impact of a new neurological deficit on the survival benefit achieved with an increased EOR. METHODS The data from all GBM patients who underwent resection at the University of Florida from 2010 to 2015 with postoperative imaging within 72 hours of surgery were included in the study. Retrospective analysis was performed on clinical outcomes and tumor volumes determined on postoperative and follow-up imaging examinations. RESULTS Overall, 115 patients met the inclusion criteria for the study. Tumor volume at the time of presentation was a median of 59 cm3 (enhanced on T1-weighted MRI scans). The mean EOR (± SD) was 94.2% ± 8.7% (range 59.9%-100%). Almost 30% of patients had a new postoperative neurological deficit, including motor weakness, sensory deficits, language difficulty, visual deficits, confusion, and ataxia. The neurological deficits had resolved in 41% of these patients on subsequent follow-up examinations. The median overall survival was 13.1 months (95% CI 10.9-15.2 months). Using a multipredictor Cox model, the authors observed that increased EOR was associated with improved survival except for patients with smaller tumor volumes (≤ 15 cm3). A residual volume of 2.5 cm3 or less predicted a favorable overall survival. Developing a postoperative neurological deficit significantly affected survival (9.2 months compared with 14.7 months, p = 0.02), even if the neurological deficit had resolved by the first follow-up. However, there was a trend of improved survival among patients with resolution of a neurological deficit by the first follow-up compared with patients with a permanent neurological deficit. Any survival benefit from achieving a 95% EOR was abrogated by the development of a new neurological deficit postoperatively. CONCLUSIONS Developing a new neurological deficit after resection of GBM is associated with a decrease in overall survival. A careful balance between EOR and neurological compromise needs to be taken into account to reduce the likelihood of neurological morbidity from surgery.

Severe Adverse Immunologic Reaction in a Patient with Glioblastoma Receiving Autologous Dendritic Cell Vaccines Combined with GM-CSF and Dose-Intensified Temozolomide

Mitchell and colleagues report the induction of a grade 3 immunologic reaction in a patient with glioblastoma receiving autologous RNA-pulsed dendritic-cell vaccines admixed with GM-CSF and coordinated cycles of temozolomide, highlighting the capacity for potent immunologic induction with this regimen. Therapeutic vaccination of patients with cancer-targeting tumor-associated antigens is a promising strategy for the specific eradication of invasive malignancies with minimal toxicity to normal tissues. However, as increasingly potent modalities for stimulating immunologic responses are developed for clinical evaluation, the risk of inflammatory and autoimmune toxicities also may be exacerbated. In this report, we describe the induction of a severe (grade 3) immunologic reaction in a patient with newly diagnosed glioblastoma (GBM) receiving autologous RNA-pulsed dendritic cell (DC) vaccines admixed with GM-CSF and administered coordinately with cycles of dose-intensified temozolomide. Shortly after the eighth administration of the admixed intradermal vaccine, the patient experienced dizziness, flushing, conjunctivitis, headache, and the outbreak of a disseminated macular/papular rash and bilateral indurated injection sites. Immunologic workup of patient reactivity revealed sensitization to the GM-CSF component of the vaccine and the production of high levels of anti–GM-CSF autoantibodies during vaccination. Removal of GM-CSF from the DC vaccine allowed continued vaccination without incident. Despite the known lymphodepletive and immunosuppressive effects of temozolomide, these observations demonstrate the capacity for the generation of severe immunologic reactivity in patients with GBM receiving DC-based therapy during adjuvant dose-intensified temozolomide. Cancer Immunol Res; 3(4); 320–5. ©2014 AACR.

FGFR1 N546K and H3F3A K27M mutations in a diffuse leptomeningeal tumour with glial and neuronal markers

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Bridging infectious disease vaccines with cancer immunotherapy: a role for targeted RNA based immunotherapeutics

Tumor-specific immunotherapy holds the promise of eradicating malignant tumors with exquisite precision without additional toxicity to standard treatments. Cancer immunotherapy has conventionally relied on cell-mediated immunity while successful infectious disease vaccines have been shown to induce humoral immunity. Efficacious cancer immunotherapeutics likely require both cellular and humoral responses, and RNA based cancer vaccines are especially suited to stimulate both arms of the immune system. RNA is inherently immunogenic, inducing innate immune responses to initiate cellular and humoral adaptive immunity, but has limited utility based on its poor in vivo stability. Early work utilized ‘naked’ RNA vaccines, whereas more recent efforts have attempted to encapsulate RNA thereby protecting it from degradation. However, feasibility has been limited by a lack of defined and safe targeting mechanisms for the in vivo delivery of stabilized RNA. As new cancer antigens come to the forefront with novel RNA encapsulation and targeting techniques, RNA vaccines may prove to be a vital, safe and robust method to initiate patient-specific anti-tumor efficacy.

Systemic activation of antigen-presenting cells via RNA-loaded nanoparticles

ABSTRACT While RNA-pulsed dendritic cell (DC) vaccines have shown promise, the advancement of cellular therapeutics is fraught with developmental challenges. To circumvent the challenges of cellular immunotherapeutics, we developed clinically translatable nanoliposomes that can be combined with tumor-derived RNA to generate personalized tumor RNA-nanoparticles (NPs) with considerable scale-up capacity. RNA-NPs bypass MHC restriction, are amenable to central distribution, and can provide near immediate immune induction. We screened commercially available nanoliposomal preparations and identified the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as an efficient mRNA courier to antigen-presenting cells (APCs). When administered intravenously, RNA-NPs mediate systemic activation of APCs in reticuloendothelial organs such as the spleen, liver, and bone marrow. RNA-NPs increase percent expression of MHC class I/II, B7 co-stimulatory molecules, and maturation markers on APCs (all vital for T-cell activation). RNA-NPs also increase activation markers on tumor APCs and elicit potent expansion of antigen-specific T-cells superior to peptide vaccines formulated in complete Freunds adjuvant. We demonstrate that both model antigen-encoding and physiologically-relevant tumor-derived RNA-NPs expand potent antitumor T-cell immunity. RNA-NPs were shown to induce antitumor efficacy in a vaccine model and functioned as a suitable alternative to DCs in a stringent cellular immunotherapy model for a radiation/temozolomide resistant invasive murine high-grade glioma. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a RNA-NP formulation that systemically activates host APCs precipitating activated T-cell frequencies necessary to engender antitumor efficacy. RNA-NPs can thus be harnessed as a more feasible and effective immunotherapy to re-program host-immunity.

Manipulation of Innate and Adaptive Immunity through Cancer Vaccines.

Although cancer immunotherapy has shown significant promise in mediating efficacious responses, it remains encumbered by tumor heterogeneity, loss of tumor-specific antigen targets, and the regulatory milieu both regionally and systemically. Cross talk between the innate and adaptive immune response may be requisite to polarize sustained antigen specific immunity. Cancer vaccines can serve as an essential fulcrum in initiating innate immunity while molding and sustaining adaptive immunity. Although peptide vaccines have shown tepid responses in a therapeutic setting with poor correlates for immune activity, RNA vaccines activate innate immune responses and have shown promising effects in preclinical and clinical studies based on enhanced DC migration. While the mechanistic insights behind the interplay between innate and adaptive immunity may be unique to the immunotherapeutic being investigated, understanding this dynamic is important to coordinate the different arms of the immune response in a focused response against cancer antigens.