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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.)

Objective: The engineered herpes simplex virus-1 G207, is a promising therapeutic option for central nervous system tumors. The first-ever pediatric phase 1 trial of continuous-infusion delivery of G207 via intratumoral catheters for recurrent or progressive malignant brain tumors is ongoing. In this article, we describe surgical techniques for the accurate placement of catheters in multiple supratentorial locations and perioperative complications associated with such procedures., Methods: A prospective study of G207 in children with recurrent malignant supratentorial tumors is ongoing. Preoperative stereotactic protocol magnetic resonance imaging was performed, and catheter trajectories planned using StealthStation planning software. Children underwent placement of 3-4 silastic catheters using a small incision burr hole and the Vertek system. Patients had a preinfusion computed tomography scan to confirm correct placement of catheters., Results: Six children underwent implantation of 3-4 catheters. Locations of catheter placement included frontal, temporal, parietal, and occipital lobes, and the insula and thalamus. There were no clinically significant perioperative complications. Postoperative computed tomography scans coupled with preoperative MRI scans demonstrated accurate placement of 21 of 22 catheters, with 1 misplaced catheter pulled back to an optimal location at the bedside. One patient had hemorrhage along the catheter tract that was clinically asymptomatic. Another patient had cerebrospinal fluid leak from a biopsy incision 9 days after surgery that was oversewn without complication., Conclusions: The placement of multiple intratumoral catheters in pediatric patients with supratentorial tumors via frameless stereotactic techniques is feasible and safe. Intratumoral catheters provide a potentially effective route for the delivery of G207 and may be employed in other trials utilizing oncolytic virotherapy for brain tumors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: IDH1 mutation has been identified as an early genetic event driving low grade gliomas (LGGs) and it has been proven to exerts a powerful epigenetic effect. Cells containing IDH1 mutation are refractory to epigenetical reprogramming to iPSC induced by expression of Yamanaka transcription factors, a feature that we employed to study early genetic amplifications or deletions in gliomagenesis., Methods: We made iPSC clones from freshly surgically resected IDH1 mutant LGGs by forced expression of Yamanaka transcription factors. We sequenced the IDH locus and analyzed the genetic composition of multiple iPSC clones by array-based comparative genomic hybridization (aCGH)., Results: We hypothesize that the primary cell pool isolated from LGG tumor contains a heterogeneous population consisting tumor cells at various stages of tumor progression including cells with early genetic lesions if any prior to acquisition of IDH1 mutation. Because cells containing IDH1 mutation are refractory to reprogramming, we predict that iPSC clones should originate only from LGG cells without IDH1 mutation, i.e. cells prior to acquisition of IDH1 mutation. As expected, we found that none of the iPSC clones contains IDH1 mutation. Further analysis by aCGH of the iPSC clones reveals that they contain regional chromosomal amplifications which are also present in the primary LGG cells., Conclusions: These results indicate that there exists a subpopulation of cells harboring gene amplification but without IDH1 mutation in the LGG primary cell pool. Further analysis of TCGA LGG database demonstrates that these regional chromosomal amplifications are also present in some cases of low grade gliomas indicating they are reoccurring lesions in glioma albeit at a low frequency. Taken together, these data suggest that regional chromosomal alterations may exist prior to the acquisition of IDH mutations in at least some cases of LGGs.

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.

Background: Procalcitonin, a bloodstream inflammatory biomarker, has proven useful in the diagnosis of sepsis in critically ill patients treated in medical and surgical intensive care units. This study aims to further our understanding of the significance of procalcitonin levels in neuroscience intensive care unit (NICU) patients., Methods: Neurosurgical patients who underwent a procalcitonin assay in an NICU over a 2-year period were included in our analysis. A procalcitonin level ≥0.2 ng/mL was considered a positive result. Infection was defined by clinical and/or microbiological diagnosis. Sensitivity, specificity, positive predictive value, and negative predictive value in the diagnosis of clinically and/or microbiologically identified infection were calculated for procalcitonin level ≥0.2 ng/mL., Results: The study cohort comprised 203 patients, including 63 with a positive procalcitonin assay (31%). Meeting the criteria for SIRS was the most common reason for a procalcitonin draw (35.5%). A procalcitonin level >0.2 ng/mL was not significantly associated with infection (P = 0.25). With a 37.4% false-negative rate and a 10.8% false-positive rate, the sensitivity of a procalcitonin level >0.2 ng/mL was 35.0%, specificity was 74.4%, the positive predictive value was 65.1%, and the negative predictive value was 45.7%. A receiver operating characteristic analysis revealed an area under the curve of 0.61., Conclusions: Although the utility of procalcitonin in sepsis and bacterial pneumonia has been amply demonstrated, this biomarker shows limited utility in diagnosing infection in our cohort, emphasizing the importance of cautious and selective use of procalcitonin assays in NICU patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

In patients undergoing surgical resection of a metastatic brain tumor, whole brain radiation therapy reduces the risk of recurrence and neurologic death. Focal radiation has the potential to mitigate neurocognitive side effects. We present an institutional experience of postoperative radiosurgery for the treatment of brain metastases. A retrospective review of a prospectively maintained institutional radiosurgery database was performed for the years 2005-2015 identifying all adult patients treated with postoperative radiosurgery to the tumor bed. Primary endpoints include local recurrence and postoperative LMD. Kaplan-Meier curves and Cox regression were used to evaluate time to local recurrence and postoperative LMD. Ninety-one patients received adjuvant focal radiation for a brain metastasis. Median radiographic follow-up among patients who had not developed a local failure was 9 months. Of the 91 patients, 20 (22%) developed local recurrence and 32 (35%) experienced postoperative LMD. Freedom from local recurrence and LMD at 1 year was 84% and 69%, respectively. In multivariable models, predictors of local failure included the presence of more than one brain metastasis (HR = 2.65, p = .04) with a preoperative tumor diameter of >3 cm (HR = 4.16, p = .06) trending toward significance. There was a trend to a higher risk of LMD with >1 tumor (HR 2.07, p = .06) and breast cancer (HR 2.37, p = .07). More than one metastasis is an independent predictor of local and leptomeningeal failure following postoperative radiosurgery. The high rate of LMD was likely related to the liberal definition of LMD to include focal dural recurrences., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

The true incidence of light chain deposition disease (LCDD) restricted to the central nervous system (CNS) is unknown. To our knowledge only 7 cases of LCDD restricted to the brain have been previously reported. We herein describe an unusual example. A 44-year-old man presented with a history of ischemic retinopathy in 2004 and left lower extremity hypoesthesia in 2007 that progressed gradually to left-sided weakness and numbness in the 2 years prior to his hospitalization in 2015. A stereotactic brain biopsy was performed, displaying nonspecific hyaline deposits of amorphous "amyloid-like" material involving deep brain white matter and vessels. These were Congo red negative and were accompanied by a sparse lymphoplasmacytic infiltrate. Plasma cells demonstrated kappa light chain class restriction by chromogenic in situ hybridization (CISH). There was patchy reactivity with kappa immunohistochemistry in the amorphous deposits. A diagnosis of light chain deposition disease was made. Subsequent systemic myeloma and lymphoma workups were negative. Previously reported cases have included men and women, spanning the ages of 19 and 72 years, often presenting with hemiparesis, hypoesthesia, or seizures. Deposits have been reported in the cerebrum and cerebellum. T2/FLAIR (fluid attenuation inversion recovery) changes are usual, but lesions may or may not produce contrast enhancement. The light chain deposition may be of kappa or lambda class. Most lesions have been accompanied by local lymphoid and/or plasma cell infiltrates exhibiting light chain restriction of the same class as the deposits. In summary, LCDD limited to the CNS is a rare lesion consisting of deposition of amyloid-like, but Congo red-negative monotypic light chain usually produced by local lymphoplasmacytic infiltrates.

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.

OBJECTIVE Many neurosurgical training programs have moved from a 24-hour resident call system to a night float system, but the impact on outcomes is unclear. Here, the authors compare length of stay (LOS) for neurosurgical patients admitted before and after initiation of a night float system at a tertiary care training hospital. METHODS The neurosurgical residency at the University of Alabama at Birmingham transitioned from 24-hour call to a night float resident coverage system in July 2013. In this cohort study, all patients admitted to the neurosurgical service for 1 year before and 1 year after this transition were compared with respect to hospital and ICU LOSs, adjusted for potential confounders. RESULTS A total of 4619 patients were included. In the initial bivariate analysis, night float was associated with increased ICU LOS (p = 0.032) and no change in overall LOS (p = 0.65). However, coincident with the transition to a night float system was an increased frequency of resident service transitions, which were highly associated with hospital LOS (p < 0.01) and ICU LOS (p < 0.01). After adjusting for resident service transitions, initiation of the night float system was associated with decreased hospital LOS (p = 0.047) and no change in ICU LOS (p = 0.35). CONCLUSIONS This study suggests that a dedicated night float resident may improve night-to-night continuity of care and decrease hospital LOS, but caution must be exercised when initiation of night float results in increased resident service transitions.

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.

Primary central nervous system tumors are the most common solid neoplasm of childhood and the leading cause of cancer-related death in pediatric patients. Survival rates for children with malignant supratentorial brain tumors are poor despite aggressive treatment with combinations of surgery, radiation, and chemotherapy, and survivors often suffer from damaging lifelong sequelae from current therapies. Novel innovative treatments are greatly needed. One promising new approach is the use of a genetically engineered, conditionally replicating herpes simplex virus (HSV) that has shown tumor-specific tropism and potential efficacy in the treatment of malignant brain tumors. G207 is a genetically engineered HSV-1 lacking genes essential for replication in normal brain cells. Safety has been established in preclinical investigations involving intracranial inoculation in the highly HSV-sensitive owl monkey (Aotus nancymai), and in three adult phase 1 trials in recurrent/progressive high-grade gliomas. No dose-limiting toxicities were seen in the adult studies and a maximum tolerated dose was not reached. Approximately half of the 35 treated adults had radiographic or neuropathologic evidence of response at a minimum of one time point. Preclinical studies in pediatric brain tumor models indicate that a variety of pediatric tumor types are highly sensitive to killing by G207. This clinical protocol outlines a first in human children study of intratumoral inoculation of an oncolytic virus via catheters placed directly into recurrent or progressive supratentorial malignant tumors.

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.)

OBJECT Handoffs and services changes are potentially modifiable sources of medical error and delays in transition of care. This cohort study assessed the relationship between resident service handoffs and length of stay for neurosurgical patients. METHODS All patients admitted to the University of Alabama at Birmingham neurosurgical service between July 1, 2012, and July 1, 2014, were retrospectively identified. A service handoff was defined as any point when a resident handed off coverage of a service for longer than 1 weekend. A conditional probability distribution was constructed to adjust length of stay for the increasing probability of a random handoff. The Student t-test and ANCOVA were used to assess relationships between resident service handoffs and length of hospital stay, adjusted for potential confounders. RESULTS A total of 3038 patients met eligibility criteria and were included in the statistical analyses. Adjusted length of hospital stay (5.32 vs 3.53 adjusted days) and length of ICU stay (4.38 vs 2.96 adjusted days) were both longer for patients who experienced a service handoff, with no difference in mortality. In the ANCOVA model, resident service handoff remained predictive of both length of hospital stay (p < 0.001) and length of ICU stay (p < 0.001). CONCLUSIONS Occurrence of a resident service handoff is an independent predictor of length of hospital and ICU stay in neurosurgical patients. This finding is novel in the neurosurgical literature. Future research might identify mechanisms for improving continuity of care and mitigating the effect of resident handoffs on patient outcomes.

M032 is a second-generation oncolytic herpes simplex virus (oHSV) that selectively replicates in tumor cells. M032 kills tumor cells directly through oncolytic replication and then proceeds to infect tumor cells in proximity, continuing the process of tumor destruction. In addition to this direct oncolytic activity, the virus carries a therapeutic payload-thus acting as a gene therapy vector-and causes the tumor cell to synthesize and secrete the immunity-stimulating protein interleukin-12 (IL-12) before cell death. (1) Human IL-12 is expressed and promotes an immune response against surviving tumor cells, increasing the antitumor effect of the therapy. IL-12 also produces an antiangiogenic effect, by interfering with the production of new tumor blood vessels necessary for tumor growth. Thus, M032 oHSV exerts antitumor effects through three distinct potential mechanisms. The virus has also been genetically engineered to minimize toxic effects for the patient. Preclinical animal models support the safety of intracranial inoculation with M032 in two relevant species (mouse and nonhuman primate). This clinical protocol outlines the dose-escalating phase I study for evaluation of M032 in patients with recurrent or progressive malignant glioma.

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.

Background: Cerebral blastomycosis is a rarely reported disease, and in the absence of associated, underlying systemic infection, poses a great diagnostic difficulty. Magnetic resonance imaging can sometimes provide equivocal information when trying to pinpoint a diagnosis. Classically, cerebral blastomycosis has been treated with amphotericin B. Voriconazole is a newer triazole antifungal with potential as a follow-up treatment of blastomycosis of the central nervous system after initial therapy with amphotericin B., Case Description: We describe one such case of a cerebral blastomycotic abscess, presenting in the absence of any systemic disease, which was initially thought to be a neoplasm. It was successfully treated by surgical resection followed by sequential amphotericin B and voriconazole. The patient did well with voriconazole therapy and was followed for voriconazole tolerance with liver function tests, which continued to be stable at 8 months past the initiation of therapy. At 12 months postoperatively, the patient was doing well and showed gradual improvement in a visual field cut, with no sign of recurrent infection., Conclusions: Isolated cerebral blastomycosis can present a diagnostic challenge. In the absence of systemic infection, surgical resection followed by antifungal therapy is a logical treatment plan.

Bevacizumab is widely used for treatment of high-grade gliomas and other malignancies. Because bevacizumab has been shown to be associated with neurocognitive decline, this study is designed to investigate whether prolonged treatment with bevacizumab is also associated with brain atrophy. We identified 12 high-grade glioma patients who received bevacizumab for 12 months at the first recurrence and 13 matched controls and blindly compared the volumes of the contralateral hemispheres and contralateral ventricle in these two groups at baseline and after 12 ± 2 months of the baseline scan by two independent analyses. The volumes of the contralateral hemispheres and ventricles did not differ significantly between the two groups at baseline. Whereas, in the control group the volumes of the contralateral hemisphere changed subtly from baseline to follow-up (p = 0.23), in the bevacizumab-treated group the volumes significantly decreased from baseline to follow-up (p = 0.03). There was significant increase in the contralateral ventricle volume from base line to follow-up scans in both the control group (p = 0.01) and in the bevacizumab group (p = 0.005). Both the absolute and the percentage changes of contralateral hemisphere volumes and contralateral ventricular volumes between the two patient groups were statistically significant (p < 0.05). Results of this study demonstrate prolonged treatment with bevacizumab is associated with atrophy of the contralateral brain hemisphere.

The standard treatment for glioblastoma multiforme (GBM) remains maximal safe surgical resection. Here, we evaluated the ability of a systemically administered antibody-dye probe conjugate (cetuximab-IRDye 800CW) to provide sufficient fluorescent contrast for surgical resection of disease in both subcutaneous and orthotopic animal models of GBM. Multiple luciferase-positive GBM cell lines (D-54MG, U-87MG, and U-251MG; n = 5) were implanted in mouse flank and tumors were fluorescently imaged daily using a closed-field near-infrared (NIR) system after cetuximab-IRDye 800CW systemic administration. Orthotopic models were also generated (n = 5), and tumor resection was performed under white light and fluorescence guidance using an FDA-approved wide-field NIR imaging system. Residual tumor was monitored using luciferase imaging. Immunohistochemistry was performed to characterize tumor fluorescence, epidermal growth factor receptor (EGFR) expression, and vessel density. Daily imaging of tumors revealed an average tumor-to-background (TBR) of 4.5 for U-87MG, 4.1 for D-54MG, and 3.7 for U-251MG. Fluorescence intensity within the tumors peaked on day-1 after cetuximab-IRDye 800CW administration, however the TBR increased over time in two of the three cell lines. For the orthotopic model, TBR on surgery day ranged from 19 to 23 during wide-field, intraoperative imaging. Surgical resection under white light on day 3 after cetuximab-IRDye 800CW resulted in an average 41% reduction in luciferase signal while fluorescence-guided resection using wide-field NIR imaging resulted in a significantly (P = 0.001) greater reduction in luciferase signal (87%). Reduction of luciferase signal was found to correlate (R (2) = 0.99) with reduction in fluorescence intensity. Fluorescence intensity was found to correlate (P < 0.05) with EGFR expression in D-54MG and U-251MG tumor types but not U-87MG. However, tumor fluorescence was found to correlate with vessel density for the U-87MG tumors. Here we show systemic administration of cetuximab-IRDye 800CW in combination with wide-field NIR imaging provided robust and specific fluorescence contrast for successful localization of disease in subcutaneous and orthotopic animal models of GBM.

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.

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.

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.

Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using γ134.5-attenuated (Δγ134.5) oHSVs and a γ134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for Δγ134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve Δγ134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved Δγ134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for Δγ134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.

: The Division of Neurosurgery at the University of Alabama at Birmingham was formally founded in 1954 under the leadership of James Garber Galbraith. The following 60 years would see neurosurgery at the forefront of the development of a nationally recognized medical center in the heart of Birmingham, Alabama. The Department of Neurosurgery now employs 14 faculty members, performs more than 4500 neurosurgical procedures annually, is active in clinical and laboratory research, and boasts a contemporary, comprehensive residency training program.

Background: Volumetric modulated arc therapy (VMAT) has been shown to be feasible for radiosurgical treatment of multiple cranial lesions with a single isocenter., Objective: To investigate whether equivalent radiosurgical plan quality and reduced delivery time could be achieved in VMAT for patients with multiple intracranial targets previously treated with Gamma Knife (GK) radiosurgery., Methods: We identified 28 GK treatments of multiple metastases. These were replanned for multiarc and single-arc, single-isocenter VMAT (RapidArc) in Eclipse. The prescription for all targets was standardized to 18 Gy. Each plan was normalized for 100% prescription dose to 99% to 100% of target volume. Plan quality was analyzed by target conformity (Radiation Therapy Oncology Group and Paddick conformity indices [CIs]), dose falloff (area under the dose-volume histogram curve), as well as the V4.5, V9, V12, and V18 isodose volumes. Other end points included beam-on and treatment time., Results: Compared with GK, multiarc VMAT improved median plan conformity (CIVMAT = 1.14, CIGK = 1.65; P < .001) with no significant difference in median dose falloff (P = .269), 12 Gy isodose volume (P = .500), or low isodose spill (P = .49). Multiarc VMAT plans were associated with markedly reduced treatment time. A predictive model of the 12 Gy isodose volume as a function of tumor number and volume was also developed., Conclusion: For multiple target stereotactic radiosurgery, 4-arc VMAT produced clinically equivalent conformity, dose falloff, 12 Gy isodose volume, and low isodose spill, and reduced treatment time compared with GK. Because of its similar plan quality and increased delivery efficiency, single-isocenter VMAT radiosurgery may constitute an attractive alternative to multi-isocenter radiosurgery for some patients.

Background: To analyze patterns of failure in patients with glioblastoma multiforme (GBM) treated with limited-margin radiation therapy and concurrent temozolomide. We hypothesize that patients treated with margins in accordance with Adult Brain Tumor Consortium guidelines (ABTC) will demonstrate patterns of failure consistent with previous series of patients treated with 2-3 cm margins., Methods: A retrospective review was performed of patients treated at the University of Alabama at Birmingham for GBM between 2000 and 2011. Ninety-five patients with biopsy-proven disease and documented disease progression after treatment were analyzed. The initial planning target volume includes the T1-enhancing tumor and surrounding edema plus a 1 cm margin. The boost planning target volume includes the T1-enhancing tumor plus a 1 cm margin. The tumors were classified as in-field, marginal, or distant if greater than 80%, 20-80%, or less than 20% of the recurrent volume fell within the 95% isodose line, respectively., Results: The median progression-free survival from the time of diagnosis to documented failure was 8 months (range 3-46). Of the 95 documented recurrences, 77 patients (81%) had an in-field component of treatment failure, 6 (6%) had a marginal component, and 27 (28%) had a distant component. Sixty-three patients (66%) demonstrated in-field only recurrence., Conclusions: The low rate of marginal recurrence suggests that wider margins would have little impact on the pattern of failure, validating the use of limited margins in accordance ABTC guidelines.

Reovirus, an oncolytic RNA virus exhibiting antiglioma activity, was shown in a previous single institution phase 1 study found that the inoculation of the virus to be well tolerated in patients with recurrent malignant glioma (MG). The goals of multicenter study reported herein were to determine the dose-limiting toxicity, maximum tolerated dose, and target lesion response rate when reovirus was administered in a novel fashion via intratumoral infusion for 72 hours in patients with recurrent malignant glioma. Fifteen adult patients were treated in a dose escalation study ranging from 1 × 10(8) to 1 × 10(10) tissue culture infectious dose 50, tentimes the dose achieved in the previous trial. Neurological, functional examinations, and imaging studies were completed pre- and postinfusion. There was one grade 3 adverse event (convulsions) felt to be possibly related to treatment, but no grade 4 adverse events considered probably or definitely related to treatment. Dose-limiting toxicity were not identified and a maximum tolerated dose was not reached. Evidence of antiglioma activity was seen in some patients. This first report of intratumoral infusion of reovirus in patients with recurrent malignant glioma demonstrated the approach to be safe and well tolerated, warranting further studies.

G207, a mutant herpes simplex virus (HSV) type 1, is safe when inoculated into recurrent malignant glioma. We conducted a phase 1 trial of G207 to demonstrate the safety of stereotactic intratumoral administration when given 24 hours prior to a single 5 Gy radiation dose in patients with recurrent malignant glioma. Nine patients with progressive, recurrent malignant glioma despite standard therapy were included. Patients received one dose of G207 stereotactically inoculated into the multiple sites of the enhancing tumor margin and were then treated focally with 5 Gy radiation. Treatment was well tolerated, and no patient developed HSV encephalitis. The median interval between initial diagnosis and G207 inoculation was 18 months (mean: 23 months; range: 11-51 months). Six of the nine patients had stable disease or partial response for at least one time point. Three instances of marked radiographic response to treatment occurred. The median survival time from G207 inoculation until death was 7.5 months (95% confidence interval: 3.0-12.7). In conclusion, this study showed the safety and the potential for clinical response of single-dose oncolytic HSV therapy augmented with radiation in the treatment of malignant glioma patients. Additional studies with oncolytic HSV such as G207 in the treatment of human glioma are recommended.

New therapies are needed for metastatic breast cancer patients. Oncolytic herpes simplex virus (oHSV) is an exciting therapy being developed for use against aggressive tumors and established metastases. Although oHSV have been demonstrated safe in clinical trials, a lack of sufficient potency has slowed the clinical application of this approach. We utilized histone deacetylase (HDAC) inhibitors, which have been noted to impair the innate antiviral response and improve gene transcription from viral vectors, to enhance the replication of oHSV in breast cancer cells. A panel of chemically diverse HDAC inhibitors were tested at three different doses (<,  = , and >LD50) for their ability to modulate the replication of oHSV in breast cancer cells. Several of the tested HDAC inhibitors enhanced oHSV replication at low multiplicity of infection (MOI) following pre-treatment of the metastatic breast cancer cell line MDA-MB-231 and the oHSV-resistant cell line 4T1, but not in the normal breast epithelial cell line MCF10A. Inhibitors of class I HDACs, including pan-selective compounds, were more effective for increasing oHSV replication compared to inhibitors that selectively target class II HDACs. These studies demonstrate that select HDAC inhibitors increase oHSV replication in breast cancer cells and provides support for pre-clinical evaluation of this combination strategy.

Herpes simplex virus type 1 (HSV-1) mutants lacking the γ(1)34.5 neurovirulence loci are promising agents for treating malignant glioma. Arming oncolytic HSV-1 to express immunostimulatory genes may potentiate therapeutic efficacy. We have previously demonstrated improved preclinical efficacy, biodistribution, and safety of M002, a γ(1)34.5-deleted HSV-1 engineered to express murine IL-12. Herein, we describe the safety and biodistribution of M032, a γ(1)34.5-deleted HSV-1 virus that expresses human IL-12 after intracerebral administration to nonhuman primates, Aotus nancymae. Cohorts were administered vehicle, 10(6), or 10(8) pfu of M032 on day 1 and subjected to detailed clinical observations performed serially over a 92-day trial. Animals were sacrificed on days 3, 31, and 91 for detailed histopathologic assessments of all organs and to isolate and quantify virus in all organs. With the possible exception of one animal euthanized on day 16, neither adverse clinical signs nor sex- or dose-related differences were attributed to M032. Elevated white blood cell and neutrophil counts were observed in virus-injected groups on day 3, but no other significant changes were noted in clinical chemistry or coagulation parameters. Minimal to mild inflammation and fibrosis detected, primarily in meningeal tissues, in M032-injected animals on days 3 and 31 had mostly resolved by day 91. The highest viral DNA levels were detected at the injection site and motor cortex on day 3 but decreased in central nervous system tissues over time. These data demonstrate the requisite safety of intracerebral M032 administration for consideration as a therapeutic for treating malignant brain tumors.

Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ134.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.

Despite intensive research efforts and therapeutic advances over the last few decades, the pediatric neural crest tumor, neuroblastoma, continues to be responsible for over 15% of pediatric cancer deaths. Novel therapeutic options are needed for this tumor. Recently, investigators have shown that mice with syngeneic murine gliomas treated with an engineered, neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ 1 34.5 gene, enabling replication in tumor cells but precluding infection of normal neural cells. We hypothesized that M002 would also be effective in the neural crest tumor, neuroblastoma. We showed that M002 infected, replicated, and decreased survival in neuroblastoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly decreased tumor growth, and that this effect was augmented with the addition of ionizing radiation. Importantly, survival could be increased by subsequent doses of radiation without re-dosing of the virus. Finally, these studies showed that the primary entry protein for oHSV, CD111 was expressed by numerous neuroblastoma cell lines and was also present in human neuroblastoma specimens. We concluded that M002 effectively targeted neuroblastoma and that this oHSV may have potential for use in children with unresponsive or relapsed neuroblastoma.

Patients with primary glioblastoma multiforme (GBM) have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO) promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%), and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.