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Background: Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer related deaths. Targeting neuroblastoma with immunotherapies has proven challenging due to a paucity of immune cells in the tumor microenvironment and the release of immunosuppressive cytokines by neuroblastoma tumor cells. We hypothesized that combining an oncolytic Herpes Simplex Virus (oHSV) with natural killer (NK) cells might overcome these barriers and incite tumor cell death., Methods: We utilized MYCN amplified and non-amplified neuroblastoma cell lines, the IL-12 expressing oHSV, M002, and the human NK cell line, NK-92 MI. We assessed the cytotoxicity of NK cells against neuroblastoma with and without M002 infection, the effects of M002 on NK cell priming, and the impact of M002 and priming on the migratory capacity and CD107a expression of NK cells. To test clinical applicability, we then investigated the effects of M002 and NK cells on neuroblastoma in vivo., Results: NK cells were more attracted to neuroblastoma cells that were infected with M002. There was an increase in neuroblastoma cell death with the combination treatment of M002 and NK cells both in vitro and in vivo. Priming the NK cells enhanced their cytotoxicity, migratory capacity and CD107a expression., Conclusions: To the best of our knowledge, these investigations are the first to demonstrate the effects of an oncolytic virus combined with self-maintaining NK cells in neuroblastoma and the priming effect of neuroblastoma on NK cells. The current studies provide a deeper understanding of the relation between NK cells and neuroblastoma and these data suggest that oHSV increases NK cell cytotoxicity towards neuroblastoma., (© 2024. The Author(s).)

Since the discovery that cGAS/STING recognizes endogenous DNA released from dying cancer cells and induces type I interferon and antitumor T cell responses, efforts to understand and therapeutically target the STING pathway in cancer have ensued. Relative to other cancer types, the glioma immune microenvironment harbors few infiltrating T cells, but abundant tumor-associated myeloid cells, possibly explaining disappointing responses to immune checkpoint blockade therapies in cohorts of patients with glioblastoma. Notably, unlike most extracranial tumors, STING expression is absent in the malignant compartment of gliomas, likely due to methylation of the STING promoter. Nonetheless, several preclinical studies suggest that inducing cGAS/STING signaling in the glioma immune microenvironment could be therapeutically beneficial, and cGAS/STING signaling has been shown to mediate inflammatory and antitumor effects of other modalities either in use or being developed for glioblastoma therapy, including radiation, tumor-treating fields, and oncolytic virotherapy. In this Review, we discuss cGAS/STING signaling in gliomas, its implications for glioma immunobiology, compartment-specific roles for STING signaling in influencing immune surveillance, and efforts to target STING signaling - either directly or indirectly - for antiglioma therapy.

Rationale: Novel immune-activating therapeutics for the treatment of glioblastoma multiforme (GBM) have shown potential for tumor regression and increased survival over standard therapies. However, immunotherapy efficacy remains inconsistent with response assessment being complicated by early treatment-induced apparent radiological tumor progression and slow downstream effects. This inability to determine early immunotherapeutic benefit results in a drastically decreased window for alternative, and potentially more effective, treatment options. The objective of this study is to evaluate the effects of combination immunotherapy on early CD8 + cell infiltration and its association with long term response in orthotopic syngeneic glioblastoma models. Methods: Luciferase positive GBM orthotopic mouse models (GSC005-luc) were imaged via [ 89 Zr]-CD8 positron emission tomography (PET) one week following treatment with saline, anti-PD1, M002 oncolytic herpes simplex virus (oHSV) or combination immunotherapy. Subsequently, brains were excised, imaged via [ 89 Zr]-CD8 ImmunoPET and evaluated though autoradiography and histology for H&E and CD8 immunohistochemistry. Longitudinal immunotherapeutic effects were evaluated through [ 89 Zr]-CD8 PET imaging one- and three-weeks following treatment, with changes in tumor volume monitored on a three-day basis via bioluminescence imaging (BLI). Response classification was then performed based on long-term BLI signal changes. Statistical analysis was performed between groups using one-way ANOVA and two-sided unpaired T-test, with p < 0.05 considered significant. Correlations between imaging and biological validation were assessed via Pearson's correlation test. Results: [ 89 Zr]-CD8 PET standardized uptake value (SUV) quantification was correlated with ex vivo SUV quantification (r = 0.61, p < 0.01), autoradiography (r = 0.46, p < 0.01), and IHC tumor CD8 + cell density (r = 0.55, p < 0.01). Classification of therapeutic responders, via bioluminescence signal, revealed a more homogeneous CD8 + immune cell distribution in responders (p < 0.05) one-week following immunotherapy. Conclusions: Assessment of early CD8 + cell infiltration and distribution in the tumor microenvironment provides potential imaging metrics for the characterization of oHSV and checkpoint blockade immunotherapy response in GBM. The combination therapies showed enhanced efficacy compared to single agent immunotherapies. Further development of immune-focused imaging methods can provide clinically relevant metrics associated with immune cell localization that can inform immunotherapeutic efficacy and subsequent treatment response in GBM patients., Competing Interests: Competing Interests: James M. Markert has the following relationships which may pose or be perceived as posing a financial conflict of interest. He is a board and equity holding member, in Aettis, Inc. and may receive royalties. The company holds frozen oncolytic viral stocks. Mustang Bio Tech is licensing the IP of C134 an oncolytic viral therapy. Markert is blinded to the conditions for the C134 clinical trials. He is a shareholder for this privately held Small Business Innovation Research LLC, Treovir, Inc., concerning G207 oncolytic viral therapy now in clinical trial. Merck, Inc. provides industry grant support by providing Keytruda (pembrolizumab) for a clinical trial of M032 oncolytic virotherapy now in clinical trial. Markert is a listee on Intellectual Property, related to a cancer immunotherapy system filed by in8Bio, formerly Incysus, Ltd. This IP has royalty earning potential., (© The author(s).)

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Despite standard therapies, including resection and chemoradiation, recurrence is virtually inevitable. Current treatment for recurrent glioblastoma (rGBM) is rapidly evolving, and emerging therapies aimed at targeting primary GBM are often first tested in rGBM to demonstrate safety and feasibility, which, in recent years, has primarily been in the form of immunotherapy. The purpose of this review is to highlight progress in clinical trials of immunotherapy for rGBM, including immune checkpoint blockade, oncolytic virotherapy, chimeric antigen receptor (CAR) T-cell therapy, cancer vaccine and immunotoxins. Three independent reviewers covered literature, published between the years 2000 and 2022, in various online databases. In general, the efficacy of immunotherapy in rGBM remains uncertain, and is limited to subsets/small cohorts of patients, despite demonstrating feasibility in early-stage clinical trials. However, considerable progress has been made in understanding the mechanisms that may preclude rGBM patients from responding to immunotherapy, as well as in developing new approaches/combination strategies that may inspire optimism for the utility of immunotherapy in this devastating disease. Continued trials are necessary to further assess the best therapeutic avenues and ascertain which treatments might benefit each patient individually.

Purpose: Oncolytic virotherapy with herpes simplex virus-1 (HSV) has shown promise for the treatment of pediatric and adult brain tumors; however, completed and ongoing clinical trials have utilized intratumoral/peritumoral oncolytic HSV (oHSV) inoculation due to intraventricular/intrathecal toxicity concerns. Intratumoral delivery requires an invasive neurosurgical procedure, limits repeat injections, and precludes direct targeting of metastatic and leptomeningeal disease. To address these limitations, we determined causes of toxicity from intraventricular oHSV and established methods for mitigating toxicity to treat disseminated brain tumors in mice., Experimental Design: HSV-sensitive CBA/J mice received intraventricular vehicle, inactivated oHSV, or treatment doses (1×107 plaque-forming units) of oHSV, and toxicity was assessed by weight loss and IHC. Protective strategies to reduce oHSV toxicity, including intraventricular low-dose oHSV or interferon inducer polyinosinic-polycytidylic acid (poly I:C) prior to oHSV treatment dose, were evaluated and then utilized to assess intraventricular oHSV treatment of multiple models of disseminated CNS disease., Results: A standard treatment dose of intraventricular oHSV damaged ependymal cells via virus replication and induction of CD8+ T cells, whereas vehicle or inactivated virus resulted in no toxicity. Subsequent doses of intraventricular oHSV caused little additional toxicity. Interferon induction with phosphorylation of eukaryotic initiation factor-2α (eIF2α) via intraventricular pretreatment with low-dose oHSV or poly I:C mitigated ependyma toxicity. This approach enabled the safe delivery of multiple treatment doses of clinically relevant oHSV G207 and prolonged survival in disseminated brain tumor models., Conclusions: Toxicity from intraventricular oHSV can be mitigated, resulting in therapeutic benefit. These data support the clinical translation of intraventricular G207., (©2022 American Association for Cancer Research.)

Glioblastoma (GBM) is the most common malignant adult brain and has a poor prognosis. Routine post-treatment MRI evaluations are required to assess treatment response and disease progression. We present a case of an 83-year-old female who underwent MRI assessment of post-treatment GBM after intravenous iron replacement therapy, ferumoxytol. The brain MRI revealed unintended alteration of MRI signal characteristics from the iron containing agent which confounded diagnostic interpretation and subsequently, the treatment planning. Ferumoxytol injection prior to contrast enhanced MRI must be screened in post-treatment GBM patients to accurately evaluate tumor activity.

Purpose: Previously, clinical trials of experimental virotherapy for recurrent glioblastoma multiforme (GBM) demonstrated that inoculation with a conditionally replication-competent Δγ 1 34.5 oncolytic herpes simplex virus (oHSV), G207, was safe. Following the initial safety study, a phase Ib trial enrolled 6 adult patients diagnosed with GBM recurrence from which tumor tissue was banked for future studies., Patients and Methods: Here, we analyzed tumor RNA sequencing (RNA-seq) data obtained from pre- and posttreatment (collected 2 or 5 days after G207 injection) biopsies from the phase Ib study patients., Results: Using a Spearman rank-order correlation analysis, we identified approximately 500 genes whose expression pattern correlated with survival duration. Many of these genes were enriched for the intrinsic IFN-mediated antiviral and adaptive immune functional responses, including immune cell chemotaxis and antigen presentation to T-cells. Furthermore, we show that the expression of several T-cell-related genes was highest in the patient with the longest survival after G207 inoculation., Conclusions: Our data support that the oHSV-induced type I IFN production and the subsequent recruitment of an adaptive immune response differed between enrolled patients and showed association with survival duration in patients with recurrent malignant glioma after treatment with an early generation oHSV., (©2022 The Authors; Published by the American Association for Cancer Research.)

Cancer is the leading cause of death by disease in children, and over 15% of pediatric cancer-related mortalities are due to neuroblastoma. Current treatment options for neuroblastoma remain suboptimal as they often have significant toxicities, are associated with long-term side effects, and result in disease relapse in over half of children with high-risk disease. There is a dire need for new therapies, and oncolytic viruses may represent an effective solution. Oncolytic viruses attack tumor cells in two ways: direct infection of tumor cells leading to cytolysis, and production of a debris field that stimulates an anti-tumor immune response. Our group has previously shown that M002, an oncolytic herpes simplex virus (oHSV), genetically engineered to express murine interleukin-12 (mIL-12), was effective at targeting and killing long term passage tumor cell lines. In the current study, we investigated M002 in three neuroblastoma patient-derived xenografts (PDXs). PDXs better recapitulate the human condition, and these studies were designed to gather robust data for translation to a clinical trial. We found that all three PDXs expressed viral entry receptors, and that the virus actively replicated in the cells. M002 caused significant tumor cell death in 2D culture and 3D bioprinted tumor models. Finally, the PDXs displayed variable susceptibility to M002, with a more profound effect on high-risk neuroblastoma PDXs compared to low-risk PDX. These findings validate the importance of incorporating PDXs for preclinical testing of oncolytic viral therapeutics and showcase a novel technique, 3D bioprinting, to test therapies in PDXs. Collectively, our data indicate that oHSVs effectively target high-risk neuroblastoma, and support the advancement of this therapy to the clinical setting.

Chemotherapeutic drugs such as the alkylating agent Temozolomide (TMZ), in addition to reducing tumor mass, can also sensitize tumors to immune recognition by transient upregulation of multiple stress induced NKG2D ligands (NKG2DL). However, the potential for an effective response by innate lymphocyte effectors such as NK and γδ T cells that recognize NKG2DL is limited by the drug's concomitant lymphodepleting effects. We have previously shown that modification of γδ T cells with a methylguanine DNA methyltransferase (MGMT) transgene confers TMZ resistance via production of O 6 -alkylguanine DNA alkyltransferase (AGT) thereby enabling γδ T cell function in therapeutic concentrations of TMZ. In this study, we tested this strategy which we have termed Drug Resistant Immunotherapy (DRI) to examine whether combination therapy of TMZ and MGMT-modified γδ T cells could improve survival outcomes in four human/mouse xenograft models of primary and refractory GBM. Our results confirm that DRI leverages the innate response of γδ T cells to chemotherapy-induced stress associated antigen expression and achieves synergies that are significantly greater than either individual approach., (© 2021. The Author(s).)

Determination of treatment response to immunotherapy in glioblastoma multiforme (GBM) is a process which can take months. Detection of CD8 + T cell recruitment to the tumor with a noninvasive imaging modality such as positron emission tomography (PET) may allow for tumor characterization and early evaluation of therapeutic response to immunotherapy. In this study, we utilized 89 Zr-labeled anti-CD8 cys-diabody-PET to provide proof-of-concept to detect CD8 + T cell immune response to oncolytic herpes simplex virus (oHSV) M002 immunotherapy in a syngeneic GBM model. Immunocompetent mice (n = 16) were implanted intracranially with GSC005 GBM tumors, and treated with intratumoral injection of oHSV M002 or saline control. An additional non-tumor bearing cohort (n = 4) receiving oHSV M002 treatment was also evaluated. Mice were injected with 89 Zr-labeled anti-CD8 cys-diabody seven days post oHSV administration and imaged with a preclinical PET scanner. Standardized uptake value (SUV) was quantified. Ex vivo tissue analyses included autoradiography and immunohistochemistry. PET imaging showed significantly higher SUV in tumors which had been treated with M002 compared to those without M002 treatment (p = 0.0207) and the non-tumor bearing M002 treated group (p = 0.0021). Accumulation in target areas, especially the spleen, was significantly reduced by blocking with the non-labeled diabody (p < 0.001). Radioactive probe accumulation in brains was consistent with CD8 + cell trafficking patterns after oHSV treatment. This PET imaging strategy could aid in distinguishing responders from non-responders during immunotherapy of GBM., (© 2021. The Author(s).)

Objective: The diagnosis of glioma remains disheartening in the clinical realm. While a multitude of studies and trials have shown promise, improvements in overall survival have been disappointing. Modeling these tumors in the laboratory setting has become increasingly challenging, given their complex in situ behavior and interactions for therapeutic evasion. Dogs, particularly brachycephalic breeds, are known to spontaneously develop gliomas that resemble human gliomas both clinically and pathophysiologically, making canines with sporadic tumors promising candidates for study. Typically, survival among these dogs is approximately 2 months with palliation alone., Methods: The authors have completed the first stage of a unique phase I dose-escalating canine clinical trial in which the safety and tolerability of M032, a nonneurovirulent oncolytic herpes simplex virus-1 vector genetically engineered to express interleukin-12, are being studied in pet dogs with gliomas undergoing maximum safe tumor resection and inoculation of the cavity with the viral infusate., Results: Twenty-five canine patients were enrolled between January 2018 and August 2020. One patient was electively withdrawn from the trial by its owner, and 3 did not receive the virus. For the 21 dogs that remained, 13 had high-grade gliomas, 5 had low-grade gliomas, and 3 were undetermined. According to histopathological analysis, 62% of the tumors were oligodendrogliomas. At the time of this report, the median overall survival from the date of treatment was 151 days (± 78 days). No significant adverse events attributable to M032 or dose-limiting toxicities have been observed to date., Conclusions: In this largest study of oncolytic viral therapy for canine brain tumors to date, treatment with M032 did not cause harm and the combination of surgery and oncolytic viral therapy may have contributed to prolonged survival in pet dogs with spontaneous gliomas. Forthcoming in-depth radiographic, immunohistochemical, and genetic analyses will afford a more advanced understanding of how this treatment impacts these tumors and the immune system. Our goal is to utilize these findings bitranslationally to inform human studies and refine therapies that will improve outcomes in both humans and pet dogs with gliomas.

Brain tumors represent the most common pediatric solid neoplasms and leading cause of childhood cancer-related morbidity and mortality. Although most adult brain tumors are supratentorial and arise in the cerebrum, the majority of pediatric brain tumors are infratentorial and arise in the posterior fossa, specifically the cerebellum. Outcomes from malignant cerebellar tumors are unacceptable despite aggressive treatments (surgery, radiation, and/or chemotherapy) that are harmful to the developing brain. Novel treatments/approaches such as oncolytic virotherapy are urgently needed. Preclinical and prior clinical studies suggest that genetically engineered oncolytic herpes simplex virus (HSV-1) G207 can safely target cerebellar malignancies and has potential to induce an antitumor immune response at local and distant sites of disease, including spinal metastases and leptomeningeal disease. Herein, we outline the rationale, design, and significance of a first-in-human immunotherapy Phase 1 clinical trial targeting recurrent cerebellar malignancies with HSV G207 combined with a single low-dose of radiation (5 Gy), designed to enhance virus replication and innate and adaptive immune responses. We discuss the unique challenges of inoculating virus through intratumoral catheters into cerebellar tumors. The trial utilizes a single arm open-label traditional 3 + 3 design with four dose cohorts. The primary objective is to assess safety and tolerability of G207 with radiation in recurrent/progressive malignant pediatric cerebellar tumors. After biopsy to prove recurrence/progression, one to four intratumoral catheters will be placed followed by a controlled-rate infusion of G207 for 6 h followed by the removal of catheters at the bedside. Radiation will be given within 24 h of virus inoculation. Patients will be monitored closely for toxicity and virus shedding. Efficacy will be assessed by measuring radiographic response, performance score, progression-free and overall survival, and quality of life. The data obtained will be invaluable in our efforts to produce more effective and less toxic therapies for children with high-grade brain tumors.

Purpose: Prior studies have mixed conclusions about the efficacy and central nervous system (CNS) toxicity profile of combining radiosurgery with anti-programed cell death 1 (PD-1) immune checkpoint inhibition (ICI) for brain metastases. This study evaluates the safety and efficacy of combined radiosurgery and anti-PD-1 ICI for melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC) brain metastases (BM)., Methods and Materials: Forty-one patients with 153 radiation naïve melanoma BM and 33 patients with 118 BM of NSCLC and RCC origin from 2014 through 2019 received radiosurgery and either anti PD-1 receptor inhibition or anti PD-L1 inhibition targeting the PD-1 ligand with less than 4 months separating either therapy. Similar to Radiation Therapy Oncology Group 9005, high-grade CNS toxicity was defined as irreversible grade 3 or any grade 4/5 neurologic event. Salvage resection revealing necrosis and viable tumor was considered grade 4 toxicity and local failure. An increase in greatest cross-sectional diameter of 25% on contrasted magnetic resonance imaging was designated as a local failure., Results: Median follow-up was 10 months (range, 1-41 months). Local control was estimated to be 90.3% at 1 year. Distant control was 38.8% at 1 year, and neither local nor distant control were significantly influenced by limiting steroids to the day of treatment ( P = .55, .52 respectively). One-year freedom from high-grade toxicity was 90.4% for patients and 94.6% for tumors. Though melanoma accounted for 41 (55%) patients and 153 (56%) tumors, it accounted for all high-grade toxicities ( P = .03). These patients had some combination of high tumor burden, aggressive steroid taper, and treatment with ipilimumab., Conclusions: Stereotactic radiosurgery combined with anti-PD-1 ICI appears to result in a high rate of local tumor control and a low rate of high-grade CNS toxicity, comparable to historical series with radiosurgery alone. High-grade toxicity is more likely in melanoma than RCC and NSCLC. Coming prospective studies will shed light on further questions about treatment timing, steroids, and response., (© 2020 Published by Elsevier Inc. on behalf of American Society for Radiation Oncology.)

As the most common and deadly of primary brain tumors, malignant gliomas have earned their place within one of the most multifaceted and heavily-funded realms of medical research. Numerous avenues of pre-clinical investigation continue to provide valuable insight, but modeling the complex evolution and behavior of these tumors within a host under simulated circumstances may pose challenges to extrapolation of data. Remarkably, certain breeds of pet dogs spontaneously and sporadically develop high grade gliomas that follow similar incidence, treatment, and outcome patterns as their human glioma counterparts. The most malignant of these tumors have been refractory to limited treatment options despite aggressive treatment; outcomes are dismal with median survivals of just over 1 year in humans and 2 months in dogs. Novel treatments are greatly needed and combination therapies appear to hold promise. This clinical protocol, a dose-escalating phase I study in dogs with sporadic malignant glioma, represents a first in comparative oncology and combination immunotherapy. The trial will evaluate M032, an Interleukin-12 expressing Herpes Simplex virus, alone and combined with a checkpoint inhibitor, Indoximod. Extensive pre-clinical work has demonstrated safety of intracranial M032 administration in mice and non-human primates. M032 is currently being tested in humans with high-grade malignant gliomas. Thus, in a novel fashion, both canine and human trials will proceed concurrently allowing a direct "head-to-head" comparison of safety and efficacy. We expect this viral oncolytic therapy to be as safe as it is in human patients and M032 to (a) infect and kill glioma cells, producing a virus and tumor cell antigen-rich debris field; (b) provide an adjuvant effect due to liberation of viral DNA, which is rich in unmethylated CpG sequences that "toggle" TLR-9 receptors; and (c) express IL-12 locally, stimulating induction of TH1 lymphocytes. The resultant immune-mediated anti-viral responses should, through cross-epitope spreading, translate into a strong response to tumor antigens. The ability to compare human and dog responses in real time affords the most stringent test of suitability of the dog as an informative model of human brain tumors. Subsequent studies will allow canine trials to properly inform the design of human trials., (Copyright © 2020 Chambers, Bentley, Crossman, Foote, Koehler, Markert, Omar, Platt, Self, Shores, Sorjonen, Waters, Yanke and Gillespie.)

Epidermal Growth Factor Receptor variant III (EGFRvIII) is an active mutant form of EGFR that drives tumor growth in a subset of glioblastoma (GBM). It occurs in over 20% of GBMs, making it a promising receptor for small molecule targeted therapy. We hypothesize that poor penetration of the blood-brain barrier by previously tested EGFR-tyrosine kinase inhibitors (EGFR-TKIs) such as afateninb, erlotinib, gefitinib, and lapatinib played a role in their limited efficacy. The present study examined the effects of osimertinib (previously known as AZD9291) on EGFRvIII+ GBM models, both in vitro and in vivo . Therefore, a panel of six GBM stem cells (GSCs) expressing EGFRvIII+ was evaluated. The EGFRvIII+ GSC differed in the expression of EGFRvIII and other key genes. The GSC line D317, which expresses high levels of EGFRvIII and has robust tyrosine kinase activity, was selected for assessing osimertinib's efficacy. Herein, we report that osimertinib inhibits the constitutive activity of EGFRvIII tyrosine kinase with high potency (<100 nM) while also inhibiting its downstream signaling. Further, osimertinib inhibited D317's growth in vitro and in both heterotopic and orthotopic xenograft models. Additional preclinical studies are warranted to identify EGFRvIII+ GBM's molecular signature most responsive to osimertinib., Competing Interests: CONFLICTS OF INTEREST The authors report no conflicts of interest related to the data presented. A part of the study was presented at the 2017 meeting of the Society of Neuro-oncology in San Francisco, CA., (Copyright: © 2020 Chagoya et al.)

Purpose: Interest and application of stereotactic radiosurgery for multiple brain metastases continue to increase. Various planning systems are available for linear accelerator (linac)-based single-isocenter multiple metastasis radiosurgery. Two of the most advanced systems are BrainLAB Multiple Metastases Elements (MME), a dynamic conformal arc (DCA) approach, and Varian RapidArc (RA), a volumetric modulated arc therapy (VMAT) approach. In this work, we systematically compared plan quality between the 2 techniques., Methods and Materials: Thirty patients with 4 to 10 metastases (217 total; median 7.5; V min = 0.014 cm 3 ; V max = 17.73 cm 3 ) were planned with both Varian RA and MME at 2 different institutions with extensive experience in each respective technique. All plans had a single isocenter and used Varian linac equipped with high-definition multileaf collimator. RA plans used 2 to 4 noncoplanar VMAT arcs with 10 MV flattening filter-free beam. MME plans used 4 to 9 noncoplanar DCAs and 6 MV flattening filter-free beam, (minimum planning target volume [PTV min] = 0.49 cm 3 ; PTV max = 27.32 cm 3 ; PTV median = 7.05 cm 3 ). Prescriptions were 14 to 24 Gy in a single fraction. Target coverage goal was 99% of volume receiving prescription dose (D99% ≥ 100%). Plans were evaluated by Radiation Therapy Oncology Group/Paddick conformity index (CI) score, 12 Gy volume (V 12Gy ), V 8Gy , V 5Gy , mean brain dose (D mean ), and beam-on time., Results: Conformity was favorable among RA plans (median: MME CI RTOG = 1.38; RA CI RTOG = 1.21; P < .0001). V 12Gy and V 8Gy were lower for RA plans (median: MME V12 = 23.7 cm 3 ; RA V12 = 19.2 cm 3 ; P = .0001; median: MME V 8Gy = 53.6 cm 3 ; RA V 8Gy = 44.1 cm 3 ; P  = .024). V 5Gy was lower for MME plans (median: MME V 5Gy = 141.4 cm 3 ; RA V 5Gy = 142.8 cm 3 ; P = .009). Mean brain was lower for MME plans (median: MME D mean = 2.57 Gy; RA D mean = 2.76 Gy; P < .0001)., Conclusions: For linac-based multiple metastasis stereotactic radiosurgery, RapidArc VMAT facilitates favorable conformity and V 12Gy /V 8Gy volume compared with the MME DCA plan. MME planning facilitates reduced dose spill at levels ≤V 5Gy ., (© 2019 The Authors.)

Immunotherapy with oncolytic herpes simplex virus-1 therapy offers an innovative, targeted, less-toxic approach for treating brain tumors. However, a major obstacle in maximizing oncolytic virotherapy is a lack of comprehensive understanding of the underlying mechanisms that unfold in CNS tumors/associated microenvironments after infusion of virus. We demonstrate that our multiplex biomarker screening platform comprehensively informs changes in both topographical location and functional states of resident/infiltrating immune cells that play a role in neuropathology after treatment with HSV G207 in a pediatric Phase 1 patient. Using this approach, we identified robust infiltration of CD8 + T cells suggesting activation of the immune response following virotherapy; however there was a corresponding upregulation of checkpoint proteins PD-1, PD-L1, CTLA-4, and IDO revealing a potential role for checkpoint inhibitors. Such work may ultimately lead to an understanding of the governing pathobiology of tumors, thereby fostering development of novel therapeutics tailored to produce optimal responses., (© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Purpose: To evaluate the results of the randomized, double-blind, placebo-controlled phase II clinical trial of ICT-107 in patients with newly diagnosed glioblastoma., Patients and Methods: We conducted a double-blinded randomized phase II trial of ICT-107 in newly diagnosed patients with glioblastoma (GBM) and tested efficacy, safety, quality of life (QoL), and immune response. HLA-A1 + and/or -A2 + -resected patients with residual tumor ≤1 cm 3 received radiotherapy and concurrent temozolomide. Following completion of radiotherapy, 124 patients, randomized 2:1, received ICT-107 [autologous dendritic cells (DC) pulsed with six synthetic peptide epitopes targeting GBM tumor/stem cell-associated antigens MAGE-1, HER-2, AIM-2, TRP-2, gp100, and IL13Rα2] or matching control (unpulsed DC). Patients received induction ICT-107 or control weekly × 4 followed by 12 months of adjuvant temozolomide. Maintenance vaccinations occurred at 1, 3, and 6 months and every 6 months thereafter., Results: ICT-107 was well tolerated, with no difference in adverse events between the treatment and control groups. The primary endpoint, median overall survival (OS), favored ICT-107 by 2.0 months in the intent-to-treat (ITT) population but was not statistically significant. Progression-free survival (PFS) in the ITT population was significantly increased in the ICT-107 cohort by 2.2 months ( P = 0.011). The frequency of HLA-A2 primary tumor antigen expression was higher than that for HLA-A1 patients, and HLA-A2 patients had higher immune response (via Elispot). HLA-A2 patients achieved a meaningful therapeutic benefit with ICT-107, in both the MGMT methylated and unmethylated prespecified subgroups, whereas only HLA-A1 methylated patients had an OS benefit., Conclusions: PFS was significantly improved in ICT-107-treated patients with maintenance of QoL. Patients in the HLA-A2 subgroup showed increased ICT-107 activity clinically and immunologically., (©2019 American Association for Cancer Research.)

Purpose: Surgery is often used for large or symptomatic brain metastases but is associated with risk of developing leptomeningeal dissemination. Emerging data suggest that fractionated stereotactic radiation therapy (FSRT) is an effective management strategy in large brain metastases. We sought to retrospectively compare leptomeningeal disease (LMD) and local control (LC) rates for patients treated with surgical resection followed by radiosurgery (S + SRS) versus FSRT alone., Methods and Materials: We identified all patients with a brain metastasis ≥3 cm in diameter treated from 2004 to 2017 with S + SRS or FSRT alone (25 or 30 Gy in 5 fractions) who had follow-up imaging. LMD was defined as focal or diffuse leptomeningeal enhancement that was >5 mm from the index metastasis. Categorical baseline characteristics were compared with the χ 2 test. LMD and LC rates were evaluated by the Kaplan-Meier (KM) method, with the log-rank test used to compare subgroups., Results: A total of 125 patients were identified, including 82 and 43 in the S + SRS and FSRT alone groups, respectively. Median pretreatment Graded Prognostic Assessment in the S + SRS and FSRT groups was 2.5 and 1.5, respectively ( P  < .001). Median follow-up was 7 months. The KM estimate of 12-month LMD rate in the S + SRS and FSRT groups was 45% and 19%, respectively ( P  = .048). The KM estimate of 12-month local control in the S + SRS and FSRT groups was 70% and 69%, respectively ( P  = .753). The 12-month KM estimate of grade ≥3 toxicity was 1.4% in S + SRS group versus 6.3% in the FSRT alone group ( P  = .248). After adjusting for graded prognostic assessment (GPA), no overall survival difference was observed between groups ( P  = .257)., Conclusions: Surgery is appropriate for certain brain metastases, but S + SRS may increase LMD risk compared with FSRT alone. Because S + SRS and FSRT seem to have similar LC, FSRT may be a viable alternative to S + SRS in select patients with large brain metastases., (© 2019 The Author(s).)

Malignant tumors of the central nervous system (CNS) continue to be a leading cause of cancer-related mortality in both children and adults. Traditional therapies for malignant brain tumors consist of surgical resection and adjuvant chemoradiation; such approaches are often associated with extreme morbidity. Accordingly, novel, targeted therapeutics for neoplasms of the CNS, such as immunotherapy with oncolytic engineered herpes simplex virus (HSV) therapy, are urgently warranted. Herein, we discuss treatment challenges related to HSV virotherapy delivery, entry, replication, and spread, and in so doing focus on host anti-viral immune responses and the immune microenvironment. Strategies to overcome such challenges including viral re-engineering, modulation of the immunoregulatory microenvironment and combinatorial therapies with virotherapy, such as checkpoint inhibitors, radiation, and vaccination, are also examined in detail.

Imaging plays a central role in evaluating responses to therapy in neuro-oncology patients. The advancing clinical use of immunotherapies has demonstrated that treatment-related inflammatory responses mimic tumor growth via conventional imaging, thus spurring the development of new imaging approaches to adequately distinguish between pseudoprogression and progressive disease. To this end, an increasing number of advanced imaging techniques are being evaluated in preclinical and clinical studies. These novel molecular imaging approaches will serve to complement conventional response assessments during immunotherapy. The goal of these techniques is to provide definitive metrics of tumor response at earlier time points to inform treatment decisions, which has the potential to improve patient outcomes. This review summarizes the available immunotherapy regimens, clinical response criteria, current state-of-the-art imaging approaches, and groundbreaking strategies for future implementation to evaluate the anti-tumor and immune responses to immunotherapy in neuro-oncology applications., Competing Interests: Competing Interests: Anna Wu is a founder, board member, and consultant to ImaginAb, Inc., and a consultant to Avidity Biosciences. James Markert has received funding from a structured buyout of Catherex, Inc., and holds <8% equity interest in Aettis, Inc. Both companies had/have interests in oncolytic virotherapy.

Oncolytic viral therapy has gained significant traction as cancer therapy over the past 2 decades. Oncolytic viruses are uniquely designed both to lyse tumor cells through their replication and to recruit immune responses against virally infected cells. Increasingly, investigators are leveraging this immune response to target the immunosuppressive tumor microenvironment and improve immune effector response against bystander tumor cells. In this article, we review the spectrum of preclinical, early-stage clinical, and potential future efforts with cytokine-secreting oncolytic viruses, with a focus on the treatment of brain tumors and solid tumors.

Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133 + PN signature, transition to a CD109 + MES subtype in a C/EBP-β-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133 - /CD109 + cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Pediatric high-grade brain tumors and adult glioblastoma are associated with significant morbidity and mortality. Oncolytic herpes simplex virus-1 (oHSV) is a promising approach to target brain tumors; oHSV G207 and M032 (encodes human interleukin-12) are currently in phase I clinical trials in children with malignant supratentorial brain tumors and adults with glioblastoma, respectively. We sought to compare the sensitivity of patient-derived pediatric malignant brain tumor and adult glioblastoma xenografts to these clinically-relevant oHSV. In so doing we found that pediatric brain tumors were more sensitive to the viruses and expressed significantly more nectin-1 (CD111) than adult glioblastoma. Pediatric embryonal and glial tumors were 74-fold and 14-fold more sensitive to M002 and 16-fold and 6-fold more sensitive to G207 than adult glioblastoma, respectively. Of note, pediatric embryonal tumors were more sensitive than glial tumors. Differences in sensitivity may be due in part to nectin-1 expression, which predicted responses to the viruses. Treatment with oHSV resulted in prolonged survival in both pediatric and adult intracranial patient-dervied tumor xenograft models. Our results suggest that pediatric brain tumors are ideal targets for oHSV and that brain tumor expression of nectin-1 may be a useful biomarker to predict patient response to oHSV.

Purpose: The study aimed to develop and demonstrate a standardized linear accelerator multileaf collimator-based method of delivering small, spherical dose distributions suitable for radiosurgical treatment of small targets such as the trigeminal nerve., Methods and Materials: The virtual cone is composed of a multileaf collimator-defined field with the central 2 leaves set to a small gap. For 5 table positions, clockwise and counter-clockwise arcs were used with collimator angles of 45 and 135 degrees, respectively. The dose per degree was proportional to the sine of the gantry angle. The dose distribution was calculated by the treatment planning system and measured using radiochromic film in a skull phantom for leaf gaps of 1.6, 2.1, and 2.6 mm. Cones with a diameter of 4 mm and 5 mm were measured for comparison. Output factor constancy was investigated using a parallel-plate chamber., Results: The mean ratio of the measured-to-calculated dose was 0.99, 1.03, and 1.05 for 1.6, 2.1, and 2.6 mm leaf gaps, respectively. The diameter of the measured (calculated) 50% isodose line was 4.9 (4.6) mm, 5.2 (5.1) mm, and 5.5 (5.5) mm for the 1.6, 2.1, and 2.6 mm leaf gap, respectively. The measured diameter of the 50% isodose line was 4.5 and 5.7 mm for the 4 mm and 5 mm cones, respectively. The standard deviation of the parallel-plate chamber signal relative to a 10 cm × 10 cm field was less than 0.4%. The relative signal changed 32% per millimeter change in leaf gap, indicating that the parallel-plate chamber is sensitive to changes in gap width., Conclusions: The virtual cone is an efficient technique for treatment of small spherical targets. Patient-specific quality assurance measurements will not be necessary in routine clinical use. Integration directly into the treatment planning system will make planning using this technique extremely efficient.

Malignant gliomas are the most common primary brain tumor and are characterized by rapid and highly invasive growth. Because of their poor prognosis, new therapeutic strategies are needed. Oncolytic virotherapy (OV) is a promising strategy for treating cancer that incorporates both direct viral replication mediated and immune mediated mechanisms to kill tumor cells. C134 is a next generation Δγ 1 34.5 oHSV-1 with improved intratumoral viral replication. It remains safe in the CNS environment by inducing early IFN signaling which restricts its replication in non-malignant cells. We sought to identify how C134 performed in an immunocompetent tumor model that restricts its replication advantage over first generation viruses. To achieve this we identified tumors that have intact IFN signaling responses that restrict C134 and first generation virus replication similarly. Our results show that both viruses elicit a T cell mediated anti-tumor effect and improved animal survival but that subtle difference exist between the viruses effect on median survival despite equivalent in vivo viral replication. To further investigate this we examined the anti-tumor activity in immunodeficient mice and in syngeneic models with re-challenge. These studies show that the T cell response is integral to C134 replication independent anti-tumor response and that OV therapy elicits a durable and circulating anti-tumor memory. The studies also show that repeated intratumoral administration can extend both OV anti-tumor effects and induce durable anti-tumor memory that is superior to tumor antigen exposure alone., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigated the effects of multiple planning factors on normal brain dose for single-isocenter VMAT stereotactic radiosurgery (SRS). Ten patients were retrospectively planned using a standardized objective function and all 16 combinations of 2 versus 4 arcs, collimator angle 45° versus selected per beam to minimize area of normal brain exposed in the beams-eye-view, fixed jaw versus following the trailing MLC leaf, and a 2 Gy mean dose objective for healthy brain versus no low dose objective. Limiting the normal brain mean dose in the optimization objective function significantly reduced the low dose spill into the normal brain without changing target coverage. Jaw tracking and appropriate selection of collimator also reduced the low dose volume, but to a lesser extent. To reduce low dose spill into normal brain for single isocenter VMAT radiosurgery of multiple targets, it is important to incorporate a limit on low dose spill into the objective function. This study has implications beyond single-isocenter VMAT radiosurgery. When comparing different inverse-planned treatment techniques, metrics that are important for evaluation of plan quality must be included the objective function., Competing Interests: Authors’ disclosure of potential conflicts of interest Dr. Fiveash reports grants and other support from Varian Medical Systems, outside the submitted work. Dr. Popple reports grants and speaking fees from Varian Medical Systems, outside the submitted work. Dr. Thomas reports speaking honoraria from Varian Medical Systems, outside the submitted work. Drs Clark, Markert, and Yuan have nothing to disclose.

Despite advances in conventional chemotherapy, surgical techniques, and radiation, outcomes for patients with relapsed, refractory, or metastatic soft tissue sarcomas are dismal. Survivors often suffer from lasting morbidity from current treatments. New targeted therapies with less toxicity, such as those that harness the immune system, and immunocompetent murine sarcoma models to test these therapies are greatly needed. We characterized two new serendipitous murine models of undifferentiated sarcoma (SARC-28 and SARC-45) and tested their sensitivity to virotherapy with oncolytic herpes simplex virus 1 (HSV-1). Both models expressed high levels of the primary HSV entry molecule nectin-1 (CD111) and were susceptible to killing by interleukin-12 (IL-12) producing HSV-1 M002 in vitro and in vivo. M002 resulted in a significant intratumoral increase in effector CD4 + and CD8 + T cells and activated monocytes, and a decrease in myeloid-derived suppressor cells (MDSCs) in immunocompetent mice. Compared to parent virus R3659 (no IL-12 production), M002 resulted in higher CD8:MDSC and CD8:T regulatory cell (Treg) ratios, suggesting that M002 creates a more favorable immune tumor microenvironment. These data provide support for clinical trials targeting sarcomas with oncolytic HSV-1. These models provide an exciting opportunity to explore combination therapies for soft tissue sarcomas that rely on an intact immune system to reach full therapeutic potential.

Purpose: Limited data exist on fractionated stereotactic radiation therapy (FSRT) for brain metastases. We sought to evaluate the safety and efficacy of FSRT and further define its role in brain metastasis management., Methods and Materials: A total of 72 patients were treated with linear accelerator-based FSRT to 182 previously untreated, intact brain metastases. Targets received 25 or 30 Gy in 5 fractions. All targets within the same course received the same prescription regardless of size. Toxicity was recorded per Radiation Therapy Oncology Group central nervous system toxicity criteria., Results: The median follow-up was 5 months (range, 1-71 months). The Kaplan-Meier estimate of 12-month local control was 86%. Tumors <3 cm in diameter demonstrated improved 12-month local control of 95% compared with 61% in tumors ≥3 cm ( P < .001). The Kaplan-Meier estimate of 12-month local control was 91% in tumors treated with 30 Gy and only 75% in tumors treated with 25 Gy ( P = .015). Tumor diameter ≥3 cm resulted in increased local failure, and a 30 Gy prescription resulted in decreased local failure on multivariate analysis (hazard ratio [HR], 8.11 [range, 2.09-31.50; P = .003] and HR, 0.26 [range, 0.07-0.93; P = .038]). Grade 4 central nervous system toxicity occurred in 4 patients (6%) requiring surgery, and no patient experienced irreversible grade 3 or 5 toxicity. Increasing tumor diameter was associated with increased toxicity risk (HR, 2.45 [range, 1.04-5.742; P = .04])., Conclusions: FSRT for brain metastases appears to demonstrate a high rate of local control with minimal risk of severe toxicity. Local control appears to be associated with smaller tumor sizeand a higher prescription dose. FSRT is a viable option for those who are poor single-fraction candidates.

Vaccines and immunotherapeutic approaches to cancers with the advent of immune checkpoint inhibitors and chimeric antigen receptor-modified T cells have recently demonstrated preclinical success and entered clinical trials. Despite advances in these approaches and combinatorial therapeutic regimens, depending on the nature of the cancer and the immune and metabolic landscape within the tumor microenvironment, current immunotherapeutic modalities remain inadequate. Recent clinical trials have demonstrated clear evidence of significant, and sometimes dramatic, antitumor activity, and long-term survival effects of a variety of oncolytic viruses (OVs), particularly oncolytic herpes simplex virus (oHSV). Acting as a multifaceted gene therapy vector and potential adjuvant-like regimens, oHSV can carry genes encoding immunostimulatory molecules in its genome. The oncolytic effect of oHSV and the inflammatory response that the virus stimulates provide a one-two punch at attacking tumors. However, mechanisms underlying oHSV-induced restoration of intratumoral immunosuppression demand extensive research in order to further improve its therapeutic efficacy. In this review, we discuss the current OV-based therapy, with a focus on the unique aspects of oHSV-initiated antiviral and antitumor immune responses, arising from virus-mediated immunological cell death to intratumoral innate and adaptive immunity.

Malignant glioma is the most common primary brain tumor and carries a grim prognosis, with a median survival of just over 14 months. Given the poor outcomes with standard-of-care treatments, novel treatment strategies are needed. The concept of virotherapy for the treatment of malignant tumors dates back more than a century and can be divided into replication-competent oncolytic viruses and replication-deficient viral vectors. Oncolytic viruses are designed to selectively target, infect, and replicate in tumor cells, while sparing surrounding normal brain. A host of oncolytic viruses has been evaluated in early phase human trials with promising safety results, but none has progressed to phase III trials. Despite the 25 years that has passed since the initial publication of genetically engineered oncolytic viruses for the treatment of glioma, much remains to be learned about the use of this therapy, including its mechanism of action, optimal treatment paradigm, appropriate targets, and integration with adjuvant agents. Oncolytic viral therapy for glioma remains promising and will undoubtedly impact the future of patient care.

Oncolytic herpes simplex virus (oHSV) type I constructs are investigational anti-neoplastic agents for a variety of malignancies, including malignant glioma. Clinical trials to date have supported the safety of these agents even when directly administered in the CNS. Traditional pre-clinical US Food and Drug Administration (FDA) toxicity studies for these agents have included the use of two species, generally including murine and primate studies. Recently, the FDA has decreased its requirement of non-human primates as an animal model for ethical reasons, especially for established viral systems where there are good alternative model systems. Here we present data demonstrating the safety of C134, a chimeric oHSV construct, in CBA mice as well as in a limited number of the HSV-sensitive non-human primate Aotus nancymaae as a proposed agent for clinical trials. These data, along with the previously conducted clinical trials of oHSV constructs, support the use of the CBA mouse model as sufficient for the pre-clinical toxicity studies of this agent. We summarize our experience with different HSV recombinants and differences between them using multiple assays to assess neurovirulence, as well as our experience with C134 in a limited number of A. nancymaae .

Pediatric brain and extracranial solid tumors are a diverse group of malignancies that represent almost half of all pediatric cancers. Standard therapy includes various combinations of surgery, cytotoxic chemotherapy, and radiation, which can be very harmful to a developing child, and survivors carry a substantial burden of long-term morbidities. Although these therapies have improved survival rates for children with solid tumors, outcomes still remain extremely poor for subsets of patients. Recently, immunosuppressive checkpoint molecules that negatively regulate immune cell function have been described. When found on malignant cells or in the tumor microenvironment, they contribute to immune evasion and tumor escape. Agents designed to inhibit these proteins have demonstrated significant efficacy in human adult solid tumor studies. However, there is limited research focusing on immune checkpoint molecules and inhibitors in pediatric solid tumors. In this review, we examine the current knowledge on immune checkpoint proteins with an emphasis on cytotoxic T lymphocyte antigen-4 (CTLA-4); programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1); OX-2 membrane glycoprotein (CD200); and indoleamine 2,3-dioxygenase (IDO). We review T-cell signaling, the mechanisms of action of these checkpoint molecules, pediatric preclinical studies on checkpoint proteins and checkpoint blockade, pediatric checkpoint inhibitor clinical trials conducted to date, and future immunotherapy opportunities for childhood cancers. Clin Cancer Res; 23(2); 342-50. ©2016 AACR., Competing Interests: authors have no conflicts of interest, (©2016 American Association for Cancer Research.)

Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, is the most common sarcoma of childhood. Despite multidrug chemotherapy regimens, surgical intervention, and radiation treatment, outcomes remain poor, especially in advanced disease, and novel therapies are needed for the treatment of these aggressive malignancies. Genetically engineered oncolytic viruses, such as herpes simplex virus-1 (HSV), are currently being explored as treatments for pediatric tumors. M002, an oncolytic HSV, has both copies of the γ 1 34.5 gene deleted, enabling replication in tumor cells but thwarting infection of normal, postmitotic cells. We hypothesized that M002 would infect human RMS tumor cells and lead to decreased tumor cell survival in vitro and impede tumor growth in vivo. In the current study, we demonstrated that M002 could infect, replicate in, and decrease cell survival in both embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) cells. Additionally, M002 reduced xenograft tumor growth and increased animal survival in both ARMS and ERMS. Most importantly, we showed for the first time that repeated dosing of oncolytic virus coupled with low-dose radiation provided improved tumor response in RMS. These findings provide support for the clinical investigation of oncolytic HSV in pediatric RMS., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Interferon-stimulated genes (ISG) encode diverse proteins that mediate intrinsic antiviral resistance in infected cells. Here it was hypothesized that malignant peripheral nerve sheath tumor (MPNST) cells resist the productive infection of oncolytic herpes simplex virus (oHSV) through activation of the JAK/STAT1 pathway and resultant upregulation of ISGs. Multiple human and mouse MPNST cells were used to explore the relationship between STAT1 activation and the productive infection of Δγ134.5 oHSVs. STAT1 activation in response to oHSV infection was found to associate with diminished Δγ134.5 oHSVs replication and spread. Multiday pretreatment, but not cotreatment, with a JAK inhibitor significantly improved viral titer and spread. ISG expression was found to be elevated prior to infection and downregulated when treated with the inhibitor, suggesting that the JAK/STAT1 pathway is active prior to infection. Conversely, upregulation of ISG expression in normally permissive cells significantly decreased oHSV productivity. Finally, a possible link between NF-κB pathway activation and ISG expression was established through the expression of inhibitor of kB (IκB) which decreased basal STAT1 transcription and ISG expression. These results demonstrate that basal ISG expression prior to infection contributes to the resistance of Δγ134.5 oHSVs in MPNST cells., Implications: Although cancer-associated ISG expression has been previously reported to impart resistance to chemotherapy and radiotherapy, these data show that basal ISG expression also contributes to oncolytic HSV resistance. Mol Cancer Res; 14(5); 482-92. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: To determine if temozolomide reduces the risk of distant brain failure (DBF, metachronous brain metastases) in patients with 1 to 4 brain metastases treated with radiosurgery without whole-brain radiation therapy (WBRT)., Methods and Materials: Twenty-five patients with newly diagnosed brain metastases were enrolled in a single institution phase 2 trial of radiosurgery (15-24 Gy) and adjuvant temozolomide. Temozolomide was continued for a total of 12 cycles unless the patient developed DBF, unacceptable toxicity, or systemic progression requiring other therapy., Results: Twenty-five patients were enrolled between 2002 and 2005; 3 were not evaluable for determining DBF. Of the remaining 22 patients, tumor types included non-small cell lung cancer (n = 8), melanoma (n = 7), and other (n = 7). Extracranial disease was present in 10 (45%) patients. The median number of tumors at the time of radiosurgery was 3 (range, 1-6). The median overall survival was 31 weeks. The median radiographic follow-up for patients who did not develop DBF was 33 weeks. Six patients developed DBF. The 1-year actuarial risk of DBF was 37%., Conclusions: In this study, there was a relatively low risk of distant brain failure observed in the nonmelanoma subgroup receiving temozolamide. However, patient selection factors rather than chemotherapy treatment efficacy are more likely the reason for the relatively low risk of distant brain failure observed in this study. Future trial design should account for these risk factors.

Over the past 1-2 decades we have witnessed a resurgence of efforts to therapeutically exploit the attributes of lytic viruses to infect and kill tumor cells while sparing normal cells. We now appreciate that the utility of viruses for treating cancer extends far beyond lytic cell death. Viruses are also capable of eliciting humoral and cellular innate and adaptive immune responses that may be directed not only at virus-infected cells but also at uninfected cancer cells. Here we review our current understanding of this bystander effect, and divide the mechanisms into lytic, cytokine, innate cellular, and adaptive phases. Knowing the key pathways and molecular players during virus infection in the context of the cancer microenvironment will be critical to devise strategies to maximize the therapeutic effects of oncolytic viroimmunotherapy.

Background: Childhood medulloblastoma is associated with significant morbidity and mortality that is compounded by neurotoxicity for the developing brain caused by current therapies, including surgery, craniospinal radiation, and chemotherapy. Innate therapeutic resistance of some aggressive pediatric medulloblastoma has been attributed to a subpopulation of cells, termed cancer-initiating cells or cancer stemlike cells (CSCs), marked by the surface protein CD133 or CD15. Brain tumors characteristically contain areas of pathophysiologic hypoxia, which has been shown to drive the CSC phenotype leading to heightened invasiveness, angiogenesis, and metastasis. Novel therapies that target medulloblastoma CSCs are needed to improve outcomes and decrease toxicity. We hypothesized that oncolytic engineered herpes simplex virus (oHSV) therapy could effectively infect and kill pediatric medulloblastoma cells, including CSCs marked by CD133 or CD15., Methods: Using 4 human pediatric medulloblastoma xenografts, including 3 molecular subgroup 3 tumors, which portend worse patient outcomes, we determined the expression of CD133, CD15, and the primary HSV-1 entry molecule nectin-1 (CD111) by fluorescence activated cell sorting (FACS) analysis. Infectability and cytotoxicity of clinically relevant oHSVs (G207 and M002) were determined in vitro and in vivo by FACS, immunofluorescent staining, cytotoxicity assays, and murine survival studies., Results: We demonstrate that hypoxia increased the CD133+ cell fraction, while having the opposite effect on CD15 expression. We established that all 4 xenografts, including the CSCs, expressed CD111 and were highly sensitive to killing by G207 or M002., Conclusions: Pediatric medulloblastoma, including Group 3 tumors, may be an excellent target for oHSV virotherapy, and a clinical trial in medulloblastoma is warranted., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We designed multimodal tumor vaccine that consists of irradiated tumor cells infected with the oncolytic IL-12-expressing HSV-1 virus, M002. This vaccine was tested against the syngeneic neuroblastoma mouse model Neuro 2a injected into the right caudate nucleus of the immunocompetent A/J mice. Mice were vaccinated via intramuscular injection of multimodal vaccine or uninfected irradiated tumor cells at seven and 14 days after tumor establishment. While there was no survival difference between groups vaccinated with cell-based vaccine applied following tumor injection, a premunition prime/boost vaccination strategy produced a significant survival advantage in both groups and sustained immune response to an intracranial rechallenge of the same tumor. The syngeneic but unrelated H6 hepatocellular tumor cell line grew unrestricted in vaccinated mice, indicative of vaccine-mediated specific immunity to Neuro 2a tumors. Longitudinal analyses of tumor-infiltrating lymphocytes revealed a primary adaptive T cell response involving both CD4+ and CD8+ T cell subsets. Spleen cell mononuclear preparations from vaccinated mice were significantly more cytotoxic to Neuro 2a tumor cells than spleen cells from control mice as demonstrated in a four-hour in vitro cytotoxicity assay. These results strongly suggest that an irradiated whole cell tumor vaccine incorporating IL-12-expressing M002 HSV can produce a durable, specific immunization in a murine model of intracranial tumor.

Progress for improving outcomes in pediatric patients with solid tumors remains slow. In addition, currently available therapies are fraught with numerous side effects, often causing significant life-long morbidity for long-term survivors. The use of viruses to kill tumor cells based on their increased vulnerability to infection is gaining traction, with several viruses moving through early and advanced phase clinical testing. The prospect of increased efficacy and decreased toxicity with these agents is thus attractive for pediatric cancer. In part I of this two-part review, we focus on strategies for utilizing oncolytic engineered herpes simplex virus (HSV) to target pediatric malignancies. We discuss mechanisms of action, routes of delivery, and the role of preexisting immunity on antitumor efficacy. Challenges to maximizing oncolytic HSV in children are examined, and we highlight how these may be overcome through various arming strategies. We review the preclinical and clinical evidence demonstrating safety of a variety of oncolytic HSVs. In Part II, we focus on the antitumor efficacy of oncolytic HSV in pediatric tumor types, pediatric clinical advances made to date, and future prospects for utilizing HSV in pediatric patients with solid tumors.

Oncolytic engineered herpes simplex viruses (HSVs) possess many biologic and functional attributes that support their use in clinical trials in children with solid tumors. Tumor cells, in an effort to escape regulatory mechanisms that would impair their growth and progression, have removed many mechanisms that would have protected them from virus infection and eventual virus-mediated destruction. Viruses engineered to exploit this weakness, like mutant HSV, can be safely employed as tumor cell killers, since normal cells retain these antiviral strategies. Many preclinical studies and early phase trials in adults demonstrated that oncolytic HSV can be safely used and are highly effective in killing tumor cells that comprise pediatric malignancies, without generating the toxic side effects of nondiscriminatory chemotherapy or radiation therapy. A variety of engineered viruses have been developed and tested in numerous preclinical models of pediatric cancers and initial trials in patients are underway. In Part II of this review series, we examine the preclinical evidence to support the further advancement of oncolytic HSV in the pediatric population. We discuss clinical advances made to date in this emerging era of oncolytic virotherapy.

Background: Nearly all patients with malignant glioma will have disease recurrence. Our purpose was to define the treatment toxicity and efficacy of concurrent bevazicumab (BVZ) with hypofractionated stereotactic radiosurgery (SRS) of relatively larger targets for patients with recurrent MG., Methods: A retrospective review of 21 patients with recurrent malignant glioma (18 glioblastoma, 3 WHO grade III glioma), treated at initial diagnosis with surgery and standard chemoradiation, was performed. All patients had concurrent BVZ with hypofractionatedSRS, 30 Gy in 5 fractions, with or without concurrent chemotherapy (temozolomide or CCNU)., Results: Median patient age was 54 years, median Karnofsky Performance Status was 80, and median target size was 4.3 cm (range, 3.4-7.5 cm). Eleven patients (52%) had previously failed BVZ. One patient had grade 3 toxicities (seizures, dysphasia), which resolved with inpatient admission and intravenous steroids/antiepileptics. Treatment-related toxicities were grade 3 ( n = 1), grade 2 ( n = 9), and grade 0-1 ( n = 11). Kaplan-Meier median progression-free survival and overall survival estimates (calculated from start of SRS) for GBM patients ( n = 18) were 11.0 and 12.5 months, respectively. Concurrent chemotherapy did not appear to show any statistically significant efficacy benefit or have any propensity for toxicity., Conclusion: BVZ concurrent with hypofractionated SRS was well tolerated by this cohort of patients with relatively larger targets. Ongoing randomized trials with more moderate radiotherapy dosing may help establish the efficacy of this regimen, though intricacies of this approach, including patient selection, radiation target volume delineation/size, and optimal radiation dose, will need further evaluation.