Dimitrios Mathios

Focal Radiation Therapy Combined with 4-1BB Activation and CTLA-4 Blockade Yields Long-Term Survival and a Protective Antigen-Specific Memory Response in a Murine Glioma Model

Background Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4) blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137) is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model. Methods GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors. Results Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4+ and CD8+ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4+ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8+ T cells had no effect on the treatment response. Conclusion Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy improves survival in an orthotopic mouse model of glioma by a CD4+ T cell dependent mechanism and generates antigen-specific memory.

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.

Therapeutic administration of IL-15 superagonist complex ALT-803 leads to long-term survival and durable antitumor immune response in a murine glioblastoma model.

Glioblastoma is the most aggressive primary central nervous system malignancy with a poor prognosis in patients. Despite the need for better treatments against glioblastoma, very little progress has been made in discovering new therapies that exhibit superior survival benefit than the standard of care. Immunotherapy has been shown to be a promising treatment modality that could help improve clinical outcomes of glioblastoma patients by assisting the immune system to overcome the immunosuppressive tumor environment. Interleukin‐15 (IL‐15), a cytokine shown to activate several effector components of the immune system, may serve as an excellent immunotherapeutic candidate for the treatment of glioblastoma. Thus, we evaluated the efficacy of an IL‐15 superagonist complex (IL‐15N72D:IL‐15RαSu‐Fc; also known as ALT‐803) in a murine GL261‐luc glioblastoma model. We show that ALT‐803, as a single treatment as well as in combination with anti‐PD‐1 antibody or stereotactic radiosurgery, exhibits a robust antitumor immune response resulting in a prolonged survival including complete remission in tumor bearing mice. In addition, ALT‐803 treatment results in long‐term immune memory against glioblastoma tumor rechallenge. Flow cytometric analysis of tumor infiltrating immune cells shows that ALT‐803 leads to increased percentage of CD8+‐cell infiltration, but not the NK cells, and IFN‐γ production into the tumor microenvironment. Cell depletion studies, in accordance with the flow cytometric results, show that the ALT‐803 therapeutic effect is dependent on CD4+ and CD8+ cells. These results provide a rationale for evaluating the therapeutic activity of ALT‐803 against glioblastoma in the clinical setting.

Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden

Significance Therapies that activate the host immune system have shown tremendous promise for a variety of solid tumors. However, in most cancer types, fewer than half of patients respond to these immunotherapies. We propose epigenetic therapy as a mechanism to sensitize tumors to immune checkpoint therapy. We have shown that inhibiting DNA methylation triggers a viral defense pathway in tumors. Here we show that epigenetic therapy in a mouse model of ovarian cancer increases the numbers of activated immune cells, and that this is dependent on the interferon antiviral response. The combination of epigenetic therapy and immune checkpoint blockade leads to the greatest reduction in tumor burden and increase in survival, and may hold the greatest promise for patients. Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

PD-1, PD-L1, PD-L2 expression in the chordoma microenvironment

Chordomas are rare malignant tumors that are postulated to arise from remnants of the notochord. Currently, the interaction between chordomas and the host immune system is poorly understood. The checkpoint protein, PD-1 is expressed by circulating lymphocytes and is a marker of activation and exhaustion. Its ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), are expressed on a variety of human cancers; however this pathway has not been previously reported in chordomas. We used flow cytometric and RT-PCR analysis in three established primary and recurrent chordoma cell lines (U-CH1, U-CH2, and JHC7) as well as immunohistochemical analysis of chordoma tissues from 10 patients to identify and localize expression of PD-1 pathway proteins. PD-1 ligands are not constitutively expressed by chordoma cells, but their expression is induced in the setting of pro-inflammatory cytokines in all cell lines examined. In paraffin embedded tissues, we found that tumor infiltrating lymphocytes expressed PD-1 in 3/6 cases. We also found that, although chordoma cells did not express significant levels of PD-L1, PD-L1 expression was observed on tumor-infiltrating macrophages and tumor infiltrating lymphocytes. Our study suggests that PD-1, PD-L1, and PD-L2 are present in the microenvironment of a subset of chordomas analyzed. Future studies are needed to evaluate the contribution of the PD-1 pathway to the immunosuppressive microenvironment of chordomas.

Accuracy of C2 pedicle screw placement using the anatomic freehand technique

OBJECTIVE The objective of this study is to evaluate the incidence and prognostic factors of breach rates following the placement of C2 pedicle screws using the anatomic, freehand technique. METHODS We retrospectively reviewed the medical records of all patients who underwent C2 transpedicular instrumentation over six years at a single institution. All intraoperative, image-guided techniques were excluded. Breaches were ascertained from immediate postoperative CT images. All images were analyzed by three independent reviewers. The screw length was correlated with (1) the breach rate and (2) the breach severity. Severity of the breached screws reflects the screw circumference (0-360°) perforating the pedicle wall (Grade 1-Grade 4). RESULTS Of the 341 C2 pedicle screws inserted in 181 patients, the average screw length was 22.93±3.7mm. The average distance from the foramen transversarium to the screw insertion point was 13.17±2.63mm. The distance from the medial rim of the pedicle to the dura of spinal cord was 3.53±1.57mm. Of the 341 screws, the overall breach rate was 17.3% (n=59). Of the 59 breaches, 89.83% of screws (n=53) breaching the spinal canal was statistically significantly higher than the 10.17% of screws (n=6) breaching the foramen transversarium (p<0.001). Moreover, 27 (45.8%) were Grade 1, 16 (27.1%) Grade 2, 6 (10.2%) Grade 3, and 10 (16.9%) Grade 4. None of the C2 breaches resulted in neurological sequela. No association was found between breach rate and gender, race or age. While the average screw length was 22.93±3.7mm [12-34mm], screw length did not predict a cortical violation (p=0.4) or severity of the breach (p=0.42) in a multiple regression model. CONCLUSIONS In this cohort study on the anatomic freehand placement of C2 pedicle screws, the breach rate was 17.3%. Lateral breaches were more common than medial breaches. Screw length was not statistically correlated with cortical violation or severity of breach. Therefore, screw length is not a prognostic factor for C2 pedicle screw misplacement.

Metastatic Melanoma to the Brain: Surgery and Radiation Is Still the Standard of Care

Opinion statementMalignant melanoma with brain metastases remains a difficult disease to treat. Patients presenting with disease affecting the central nervous system (CNS) have a poor prognosis. Treatment depends on a number of factors, including the size and number of lesions, performance status, comorbidities, and presenting symptoms. Physicians and patients must weigh risks and benefits of treatments, with the main goal of palliating symptoms and decreasing the risk of neurological death. Opinions throughout the country vary, but first-line treatment for brain metastases is local therapy involving either craniotomy or stereotactic radiosurgery (SRS) using CyberKnife or Gamma Knife, with or without whole brain radiation therapy (WBRT). Clinical trials remain another option for patients, with chemotherapy reserved for patients who have exhausted other options. There has been a recent surge in knowledge regarding the pathophysiology and treatment of metastatic melanoma leading to recent FDA approval in 2011 of new drugs: ipilimumab, a novel immune therapy, and vemurafenib, which blocks the MAP Kinase pathway. These drugs have the potential to treat patients with metastatic melanoma to the brain but are still undergoing clinical investigation. Despite these recent advances, the prognosis is poor, with few patients able to achieve durable and long-lasting response. Treatment for patients with brain metastases continues to lag behind treatment of other diseases, partly due to their exclusion from early clinical trials.

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.

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.