Debebe Theodros

Combination therapy with anti-PD-1, anti-TIM-3, and focal radiation results in regression of murine gliomas

Purpose: Checkpoint molecules like programmed death-1 (PD-1) and T-cell immunoglobulin mucin-3 (TIM-3) are negative immune regulators that may be upregulated in the setting of glioblastoma multiforme. Combined PD-1 blockade and stereotactic radiosurgery (SRS) have been shown to improve antitumor immunity and produce long-term survivors in a murine glioma model. However, tumor-infiltrating lymphocytes (TIL) can express multiple checkpoints, and expression of ≥2 checkpoints corresponds to a more exhausted T-cell phenotype. We investigate TIM-3 expression in a glioma model and the antitumor efficacy of TIM-3 blockade alone and in combination with anti-PD-1 and SRS. Experimental Design: C57BL/6 mice were implanted with murine glioma cell line GL261-luc2 and randomized into 8 treatment arms: (i) control, (ii) SRS, (iii) anti-PD-1 antibody, (iv) anti-TIM-3 antibody, (v) anti-PD-1 + SRS, (vi) anti-TIM-3 + SRS, (vii) anti-PD-1 + anti-TIM-3, and (viii) anti-PD-1 + anti-TIM-3 + SRS. Survival and immune activation were assessed. Results: Dual therapy with anti-TIM-3 antibody + SRS or anti-TIM-3 + anti-PD-1 improved survival compared with anti-TIM-3 antibody alone. Triple therapy resulted in 100% overall survival (P < 0.05), a significant improvement compared with other arms. Long-term survivors demonstrated increased immune cell infiltration and activity and immune memory. Finally, positive staining for TIM-3 was detected in 7 of 8 human GBM samples. Conclusions: This is the first preclinical investigation on the effects of dual PD-1 and TIM-3 blockade with radiation. We also demonstrate the presence of TIM-3 in human glioblastoma multiforme and provide preclinical evidence for a novel treatment combination that can potentially result in long-term glioma survival and constitutes a novel immunotherapeutic strategy for the treatment of glioblastoma multiforme. Clin Cancer Res; 23(1); 124–36. ©2016 AACR.

Anti–PD-1 antitumor immunity is enhanced by local and abrogated by systemic chemotherapy in GBM

Local chemotherapy synergizes with immunotherapy against glioblastoma, whereas systemic chemotherapy abrogates its antitumor effect. Combining drugs as the doctor ordered Cancer immunotherapy is rapidly increasing in prominence and being applied for a growing number of cancer types. Chemotherapy is still the mainstay of cancer treatment, however, and it can be difficult to find good ways to combine the two approaches. Mathios et al. addressed this problem by systematically evaluating the effectiveness of local or systemic chemotherapy given before or after immune checkpoint inhibition in mouse models of glioblastoma. The authors demonstrated that local chemotherapy was particularly effective in combination with checkpoint inhibition, whereas systemic chemotherapy was too damaging to the immune system to make for useful combinations. The immunosuppressive effects of chemotherapy present a challenge for designing effective cancer immunotherapy strategies. We hypothesized that although systemic chemotherapy (SC) exhibits negative immunologic effects, local chemotherapy (LC) can potentiate an antitumor immune response. We show that LC combined with anti–programmed cell death protein 1 (PD-1) facilitates an antitumor immune response and improves survival (P < 0.001) in glioblastoma. LC-treated mice had increased infiltration of tumor-associated dendritic cells and clonal expansion of antigen-specific T effector cells. In comparison, SC resulted in systemic and intratumoral lymphodepletion, with decreased immune memory in long-term survivors. Furthermore, adoptive transfer of CD8+ cells from LC-treated mice partially rescued SC-treated mice after tumor rechallenge. Last, the timing of chemo- and immunotherapy had differential effects on anti–PD-1 efficacy. This study suggests that both mode of delivery and timing have distinct effects on the efficacy of anti–PD-1. The results of this work could help guide the selection and scheduling of combination treatment for patients with glioblastoma and other tumor types.

Pituitary adenomas: historical perspective, surgical management and future directions

Pituitary adenomas are among the most common central nervous system tumors. They represent a diverse group of neoplasms that may or may not secrete hormones based on their cell of origin. Epidemiologic studies have documented the incidence of pituitary adenomas within the general population to be as high as 16.7%. A growing body of work has helped to elucidate the pathogenesis of these tumors. Each subtype has been shown to demonstrate unique cellular changes potentially leading to tumorigenesis. Surgical advancements over several decades have included microsurgery and the employment of the endoscope for surgical resection. These advancements increase the likelihood of gross-total resection and have resulted in decreased patient morbidity.

Reduced CSF leak in complete calvarial reconstructions of microvascular decompression craniectomies using calcium phosphate cement.

OBJECT Calcium phosphate cement provides a biomaterial that can be used for calvarial reconstruction in a retrosigmoid craniectomy for microvascular decompression (MVD). This study evaluates the outcomes of postoperative CSF leak and wound infection for patients undergoing a complete cranioplasty using calcium phosphate cement versus incomplete cranioplasty using polyethylene titanium mesh following a retrosigmoid craniectomy for MVD. METHODS The authors evaluated 211 cases involving patients who underwent first-time retrosigmoid craniectomies performed by a single attending surgeon fortrigeminal neuralgia from October 2008 to June 2014. From this patient population, 111 patients underwent calvarial reconstruction after retrosigmoid craniectomy using polyethylene titanium mesh, and 100 patients had reconstructions using calcium phosphate cement. A Pearsons chi-square test was used to compare postoperative complications of CSF leak and wound infection in these 2 types of cranioplasties. RESULTS The polyethylene titanium mesh group included 5 patients (4.5%) with postoperative CSF leak or pseudomeningocele and 3 patients (2.7%) with wound infections. In the calcium phosphate cement group, no patients had a CSF leak, and 2 patients (2%) had wound infections. This represented a statistically significant reduction of postoperative CSF leak in patients who underwent calcium phosphate reconstructions of their calvarial defect compared with those who underwent polyethylene titanium mesh reconstructions (p = 0.03). No significant difference was seen between the 2 groups in the number of patients with postoperative wound infections. CONCLUSIONS Calcium phosphate cement provides a viable alternative biomaterial for calvarial reconstruction of retrosigmoid craniectomy defects in patients who have an MVD. The application of this material provides a biocompatible barrier that reduces the incidence of postoperative CSF leaks.

Successful anterior fusion following posterior cervical fusion for revision of anterior cervical discectomy and fusion pseudarthrosis

Pseudarthrosis occurs after approximately 2-20% of anterior cervical discectomy and fusion (ACDF) procedures; it is unclear if posterior or anterior revision should be pursued. In this study, we retrospectively evaluate the outcomes in 22 patients with pseudarthrosis following ACDF and revision via posterior cervical fusion (PCF). Baseline demographics, preoperative symptoms, operative data, time to fusion failure, symptoms of pseudarthrosis, and revision method were assessed. Fusion outcome and clinical outcome were determined at last follow-up (LFU). Thirteen females (59%) and 9 (41%) males experienced pseudarthrosis at a median of 11 (range: 3-151)months after ACDF. Median age at index surgery was 51 (range: 33-67)years. All patients with pseudarthrosis presented with progressive neck pain, with median visual analog scale (VAS) score of 8 (range: 0-10), and/or myeloradiculopathy. Patients with pseudarthrosis <12 months compared to >12 months after index surgery were older (p=0.013), had more frequent preoperative neurological deficits (p=0.064), and lower baseline VAS scores (p=0.006). Fusion was successful after PCF in all patients, with median time to fusion of 10 (range: 2-14)months. Eighteen patients fused both anteriorly and posteriorly, two patients fused anteriorly only, and two patients fused posteriorly only. Median VAS neck score at LFU significantly improved from the time of pseudarthrosis (p=0.012). While uncommon, pseudarthrosis may occur after ACDF. All patients achieved successful fusion after subsequent posterior cervical fusion, with 91% fusing a previous anterior pseudarthrosis after posterior stabilization. Neck pain significantly improved by LFU in the majority of patients in this study.

Efficacy of primary microvascular decompression versus subsequent microvascular decompression for trigeminal neuralgia.

OBJECTIVE Trigeminal neuralgia (TN) is characterized by intermittent, paroxysmal, and lancinating pain along the distribution of the trigeminal nerve. Microvascular decompression (MVD) directly addresses compression of the trigeminal nerve. The purpose of this study was to determine whether patients undergoing MVD as their first surgical intervention experience greater pain control than patients who undergo subsequent MVD. METHODS A retrospective review of patient records from 1998 to 2015 identified a total of 942 patients with TN and 500 patients who underwent MVD. After excluding several cases, 306 patients underwent MVD as their first surgical intervention and 175 patients underwent subsequent MVD. Demographics and clinicopathological data and outcomes were obtained for analysis. RESULTS In patients who underwent subsequent MVD, surgical intervention was performed at an older age (55.22 vs 49.98 years old, p < 0.0001) and the duration of symptoms was greater (7.22 vs 4.45 years, p < 0.0001) than for patients in whom MVD was their first surgical intervention. Patients who underwent initial MVD had improved pain relief and no improvement in pain rates compared with those who had subsequent MVD (95.8% and 4.2% vs 90.3% and 9.7%, respectively, p = 0.0041). Patients who underwent initial MVD had significantly lower rates of facial numbness in the pre- and postoperative periods compared with patients who underwent subsequent MVD (p < 0.0001). The number of complications in both groups was similar (p = 0.4572). CONCLUSIONS The results demonstrate that patients who underwent other procedures prior to MVD had less pain relief and a higher incidence of facial numbness despite rates of complications similar to patients who underwent MVD as their first surgical intervention.

Dendritic cell activation enhances anti-PD-1 mediated immunotherapy against glioblastoma

Introduction The glioblastoma (GBM) immune microenvironment is highly suppressive as it targets and hinders multiple components of the immune system. Checkpoint blockade (CB) is being evaluated for GBM patients. However, biomarker analyses suggest that CB monotherapy may be effective only in a small fraction of GBM patients. We hypothesized that activation of antigen presentation would increase the therapeutic response to PD-1 blockade. Results We show that activating DCs through TLR3 agonists enhances the anti-tumor immune response to CB and increases survival in GBM. Mice treated with TLR3 agonist poly(I:C) and anti-PD-1 demonstrated increased DC activation and increased T cell proliferation in tumor draining lymph nodes. We show that DCs are necessary for the improved anti-tumor immune response. Conclusions This study suggests that augmenting antigen presentation is an effective multimodal immunotherapy strategy that intensifies anti-tumor responses in GBM. Specifically, these data represent an expanded role for TLR3 agonists as adjuvants to CB. Methods Using a preclinical model of GBM, we tested the efficacy of combinatorial immunotherapy with anti-PD-1 and TLR3 agonist, poly(I:C). Characterization of the immune response in tumor infiltrating immune cells and in secondary lymphoid organs was performed. Additionally, dendritic cell (DC) depletion experiments were performed.

Stereotactic radiotherapy increases functionally suppressive regulatory T cells in the tumor microenvironment

Stereotactic irradiation of implanted murine tumors increased proliferation of regulatory T cells (Tregs) within tumors. These cells were functionally more suppressive than Tregs derived from unirradiated tumors, highlighting a potential counterbalance to the immunogenic effects of radiotherapy. Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992–1004. ©2017 AACR.

TNFα and Radioresistant Stromal Cells Are Essential for Therapeutic Efficacy of Cyclic Dinucleotide STING Agonists in Nonimmunogenic Tumors

Stromal and immune cells are required for effective responses to intratumoral cyclic dinucleotide therapy. Responses leading to productive innate and adaptive antitumor responses are demonstrated and highlight the cooperation between the tumor stroma and immune compartments during immunotherapy. The cGAS–STING cytosolic DNA sensing pathway may play an integral role in the initiation of antitumor immune responses. Studies evaluating the immunogenicity of various cyclic dinucleotide (CDN) STING agonists administered by intratumoral (i.t.) injection showed potent induction of inflammation, tumor necrosis, and, in some cases, durable tumor-specific adaptive immunity. However, the specific immune mechanisms underlying these responses remain incompletely defined. The majority of these studies have focused on the effect of CDNs on immune cells but have not conclusively interrogated the role of stromal cells in the acute rejection of the CDN-injected tumor. Here, we revealed a mechanism of STING agonist-mediated tumor response that relied on both stromal and immune cells to achieve tumor regression and clearance. Using knockout and bone marrow chimeric mice, we showed that although bone marrow–derived TNFα was necessary for CDN-induced necrosis, STING signaling in radioresistant stromal cells was also essential for CDN-mediated tumor rejection. These results provide evidence for crosstalk between stromal and hematopoietic cells during CDN-mediated tumor collapse after i.t. administration. These mechanistic insights may prove critical in the clinical development of STING agonists. Cancer Immunol Res; 6(4); 422–33. ©2018 AACR.

TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM

ABSTRACT The use of inhibitory checkpoint blockade in the management of glioblastoma has been studied in both preclinical and clinical settings. TIGIT is a novel checkpoint inhibitor recently discovered to play a role in cancer immunity. In this study, we sought to determine the effect of anti-PD-1 and anti-TIGIT combination therapy on survival in a murine glioblastoma (GBM) model, and to elucidate the underlying immune mechanisms. Using mice with intracranial GL261-luc+ tumors, we found that TIGIT expression was upregulated on CD8+ and regulatory T cells (Tregs) in the brain compared to draining cervical lymph nodes (CLN) and spleen. We then demonstrated that treatment using anti-PD-1 and anti-TIGIT dual therapy significantly improved survival compared to control and monotherapy groups. The therapeutic effect was correlated with both increased effector T cell function and downregulation of suppressive Tregs and tumor-infiltrating dendritic cells (TIDCs). Clinically, TIGIT expression on tumor-infiltrating lymphocytes was shown to be elevated in patient GBM samples, suggesting that the TIGIT pathway may be a valuable therapeutic target. Expression of the TIGIT ligand, PVR, further portended a poor survival outcome in patients with low-grade glioma. We conclude that anti-TIGIT is an effective treatment strategy against murine GBM when used in combination with anti-PD-1, improving overall survival via modifications of both the T cell and myeloid compartments. Given evidence of PVR expression on human GBM cells, TIGIT presents as a promising immune therapeutic target in the management of these patients.