Evaluation of immunologic parameters in canine glioma patients treated with an oncolytic herpes virus.

Aim: To molecularly characterize the tumor microenvironment and evaluate immunologic parameters in canine glioma patients before and after treatment with oncolytic human IL-12-expressing herpes simplex virus (M032) and in treatment naïve canine gliomas. Methods: We assessed pet dogs with sporadically occurring gliomas enrolled in Stage 1 of a veterinary clinical trial that was designed to establish the safety of intratumoral oncoviral therapy with M032, a genetically modified oncolytic herpes simplex virus. Specimens from dogs in the trial and dogs not enrolled in the trial were evaluated with immunohistochemistry, NanoString, Luminex cytokine profiling, and multi-parameter flow cytometry. Results: Treatment-naive canine glioma microenvironment had enrichment of Iba1 positive macrophages and minimal numbers of T and B cells, consistent with previous studies identifying these tumors as immunologically "cold". NanoString mRNA profiling revealed enrichment for tumor intrinsic pathways consistent with suppression of tumor-specific immunity and support of tumor progression. Oncolytic viral treatment induced an intratumoral mRNA transcription signature of tumor-specific immune responses in 83% (5/6) of canine glioma patients. Changes included mRNA signatures corresponding with interferon signaling, lymphoid and myeloid cell activation, recruitment, and T and B cell immunity. Multiplexed protein analysis identified a subset of oligodendroglioma subjects with increased concentrations of IL-2, IL-7, IL-6, IL-10, IL-15, TNFα, GM-CSF between 14 and 28 days after treatment, with evidence of CD4+ T cell activation and modulation of IL-4 and IFNγ production in CD4+ and CD8+ T cells isolated from peripheral blood. Conclusion: These findings indicate that M032 modulates the tumor-immune microenvironment in the canine glioma model.

Repeat gamma knife stereotactic radiosurgery in the treatment of trigeminal neuralgia: A single-center experience and focused review of the literature.

OBJECTIVES: Repeat Gamma Knife stereotactic radiosurgery (GKSR) for refractory trigeminal neuralgia (TGN) is an increasingly common practice. Prior studies have reported varying success rates and incidence of trigeminal nerve dysfunction following repeated GKSR. We report treatment outcomes and toxicity in patients following repeat GKSR for TGN at the University of Alabama at Birmingham (UAB) with a focused review of the literature. METHODS: We retrospectively reviewed medical records of 55 TGN patients re-treated with radiosurgery using the Leksell Gamma Knife® at the University of Alabama at Birmingham between 1996 and 2012. Outcomes were defined using the Modified Marseille Scale. Demographics, prior treatments and symptom duration were correlated with outcomes. RESULTS: Eighteen patients (33%) achieved Marseille Class I or II, 14 (25%) Class III or IV, and 23 (42%) Class V at a mean follow-up of 14.4 months. Twenty-five patients (45%) developed new trigeminal nerve dysfunction after re-treatment. Of these, four (16%) did not develop dysfunction until subsequent microvascular decompression (MVD) for inadequate symptom relief. CONCLUSIONS: Although more than half of the patients undergoing repeat GKSR for refractory TGN maintained excellent or good outcomes (Marseille classes I-IV) at an average follow-up of 14.4 months, neither age, gender, nor pre-treatment duration of symptoms or interval between treatments had a statistically significant effect on outcomes. Following repeat GKSR, patients have increased risk for new-onset trigeminal nerve dysfunction and those undergoing MVD after repeat GKSR may have an increased risk for new-onset trigeminal nerve dysfunction.

γ134.5-deleted HSV-1-expressing human cytomegalovirus IRS1 gene kills human glioblastoma cells as efficiently as wild-type HSV-1 in normoxia or hypoxia.

Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

Assessment of oncolytic HSV efficacy following increased entry-receptor expression in malignant peripheral nerve sheath tumor cell lines.

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.

Increased oncolytic efficacy for high-grade gliomas by optimal integration of ionizing radiation into the replicative cycle of HSV-1.

Oncolytic viruses have been combined with standard cancer therapies to increase therapeutic efficacy. Given the sequential activation of herpes viral genes (herpes simplex virus-1, HSV-1) and the temporal cellular changes induced by ionizing radiation, we hypothesized an optimal temporal sequence existed in combining oncolytic HSV-1 with ionizing radiation. Murine U-87 glioma xenografts were injected with luciferase encoding HSV-1, and ionizing radiation (IR) was given at times before or after viral injection. HSV-1 replication and tumor-volume response were followed. Radiation given 6-9 h after HSV-1 injection resulted in maximal viral luciferase expression and infectious viral production in tumor xenografts. The greatest xenograft regression was also seen with radiation given 6 h after viral injection. We then tested if HSV-1 replication had a dose response to ionizing radiation. HSV-1 luciferase expression exhibited a dose response as xenografts were irradiated from 0 to 5 Gy. There was no difference in viral luciferase expression as IR dose increased from 5 Gy up to 20 Gy. These results suggest that the interaction of IR with the HSV-1 lytic cycle can be manipulated for therapeutic gain by delivering IR at a specific time within viral replicative cycle.

CRAdRGDflt-IL24 virotherapy in combination with chemotherapy of experimental glioma.

Malignant forms of glioma, the most common primary brain tumors, remain poorly responsive to multimodality therapeutic interventions, including chemotherapy. Suppressed apoptosis and extraordinary invasiveness are important distinctive features that contribute to the malignant phenotype of glioma. We have developed the vascular endothelial growth factor receptor 1 (VEGFR-1/flt-1) conditional replicating adenoviral vector (CRAdRGDflt-IL24) encoding the interleukin-24 (IL-24) gene. We investigated whether a combination of CRAdRGDflt-IL24-mediated oncolytic virotherapy and chemotherapy using temozolomide (TMZ) produces increased cytotoxicity against human glioma cells in comparison with these agents alone. Combination of CRAdRGDflt-IL24 and TMZ significantly enhanced cytotoxicity in vitro, inhibited D54MG tumor growth and prolonged survival of mice harboring intracranial human glioma xenografts in comparison with CRAdRGDflt-IL24 or TMZ alone. These data indicate that combined treatment with CRAdRGDflt-IL24-mediated oncolytic virotherapy and TMZ chemotherapy provides a promising approach for glioma therapy.

Enhanced antiglioma activity of chimeric HCMV/HSV-1 oncolytic viruses.

Oncolytic herpes simplex virus (HSV)-1 gamma(1)34.5-deletion mutants (Deltagamma(1)34.5 HSV) are promising agents for tumor therapy. The attenuating mutation renders the virus aneurovirulent but also limits late viral protein synthesis and efficient replication in many tumors. We tested whether one function of gamma(1)34.5 gene, which mediates late viral protein synthesis through host protein kinase R (PKR) antiviral response evasion, could be restored, without restoring the neurovirulence. We have previously reported the construction of two chimeric Deltagamma(1)34.5 HSV vectors (chimeric HSV), C130 and C134, which express the human cytomegalovirus (HCMV) PKR-evasion genes TRS1 and IRS1, respectively. We now demonstrate the following. The HCMV/HSV-1 chimeric viruses (i) maintain late viral protein synthesis in the human malignant glioma cells tested (D54-MG, U87-MG and U251-MG); (ii) replicate to higher titers than Deltagamma(1)34.5 HSV in malignant glioma cells in vitro and in vivo; (iii) are aneurovirulent; and (iv) are superior to other Deltagamma(1)34.5 HSV with both improved reduction of tumor volumes in vivo, and improved survival in two experimental murine brain tumor models. These findings demonstrate that transfer of HCMV IRS1 or TRS1 gene into Deltagamma(1)34.5 HSV significantly improves replication in malignant gliomas without restoring wild-type neurovirulence, resulting in enhanced tumor reduction and prolonged survival.

Necrotizing neurosarcoid: three cases with varying presentations.

BACKGROUND: Neurosarcoid affects approximately 5% of patients with sarcoidosis. A significantly more rare entity, necrotizing sarcoidosis affecting the central nervous system, has been confirmed previously in only three case reports. This paper documents three additional cases of necrotizing neurosarcoid, involving a wide spectrum of central nervous system (CNS) locations. RESULTS: One patient presented to the emergency department after being found unresponsive. The second patient was referred due to hearing loss and the third patient sought care due to weakness and numbness of his left lower extremity. Locations of involvement were diverse and included diffuse leptomeningeal involvement, a cerebellopontine angle mass and a thoracic spinal cord lesion. All patients eventually underwent surgical biopsy, and histologic review of tissue samples revealed necrotizing granulomatous inflammation. Serum ACE levels were available for two of the patients and were within normal limits. Once the diagnosis of necrotizing neurosarcoid was confirmed, all patients were treated with systemic corticosteroid therapy; one patient was also treated with an immunosuppressive agent. CONCLUSIONS: Necrotizing neurosarcoid may occur more commonly than previously described and should be considered in the differential diagnosis of patients without systemic manifestations of sarcoidosis.

Engineered herpes simplex virus expressing bacterial cytosine deaminase for experimental therapy of brain tumors.

Lack of effective therapy of primary brain tumors has promoted the development of novel experimental approaches utilizing oncolytic viruses combined with gene therapy. Towards this end, we have assessed a conditionally replication-competent, gamma(1)34.5-deleted herpes simplex virus type 1 (HSV-1) expressing cytosine deaminase (CD) for treatment of malignant brain tumors. Our results are summarized as follows: (i) a recombinant HSV (M012) was constructed in which both copies of the gamma(1)34.5 gene were replaced with the bacterial CD gene, under the control of the cellular promoter Egr-1; (ii) M012-infected cells in vitro efficiently convert 5-fluorocytosine (5-FC) to 5-fluorouracil, thereby enhancing cytotoxicity of neighboring, uninfected cells; (iii) both direct and bystander cytotoxicity of murine neuroblastoma and human glioma cell lines after infection with M012 were demonstrated; (iv) direct intracerebral inoculation of A/J mice demonstrated lack of neurotoxicity at doses similar to G207, a gamma(1)34.5-deleted HSV with demonstrated safety in human patient trials and (v) intratumoral injection of M012 into Neuro-2a flank tumors in combination with 5-FC administration significantly reduced tumor growth versus tumors treated with R3659 combined with 5-FC, or treated with M012 alone. Thus, M012 is a promising new oncolytic HSV vector with an enhanced prodrug-mediated, antineoplastic effect that is safe for intracranial administration.

The use of a genetically engineered herpes simplex virus (R7020) with ionizing radiation for experimental hepatoma.

The herpes simplex virus (HSV) recombinant virus R7020 is an attenuated virus designed as a candidate for immunization against both HSV-1 and HSV-2 infections. It was extensively tested in an experimental animal system and in a healthy human adult population without significant untoward effects. We report on the use of R7020 with ionizing radiation as an oncolytic agent for hepatomas. Two hepatoma cell lines were studied, Hep3B and Huh7. R7020 replicated to higher titers in Hep3B cells than in Huh7 cells. Tissue culture studies correlated with hepatoma xenograft responses to R7020. R7020 was more effective in mediating Hep3B tumor xenograft regression compared with Huh7. Ionizing radiation combined with R7020 also showed differential results in antitumor efficacy between the two cell lines in tumor xenografts. Ionizing radiation enhanced the replication of R7020 in Hep3B xenografts. Moreover, the combination of ionizing radiation and virus caused a greater regression of xenograft volume than either R7020 or radiation alone. Ionizing radiation had no effect on the replication of R7020 virus in Huh7 xenografts. These results indicate that a regimen involving infection with an appropriate herpesvirus such as R7020 in combination with ionizing radiation can be highly effective in eradicating certain tumor xenografts.

pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers.

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).

Differential gene expression profiling in human brain tumors.

Gene expression profiling of three human temporal lobe brain tissue samples (normal) and four primary glioblastoma multiforme (GBM) tumors using oligonucleotide microarrays was done. Moreover, confirmation of altered expression was performed by whole cell patch clamp, immunohistochemical staining, and RT-PCR. Our results identified several ion and solute transport-related genes, such as N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 receptors, GABA(A) receptor subunits alpha3, beta1, beta2, and beta3, the glutamate transporter, the glutamate/aspartate transporter II, the potassium channel K(V)2.1, hK(V)beta3, and the sodium/proton exchanger 1 (NHE-1), that are all downregulated in the tumors compared with the normal tissues. In contrast, aquaporin-1, possibly aquaporins-3 and -5, and GLUT-3 message appeared upregulated in the tumors. Our results also confirmed previous work showing that osteopontin, nicotinamide N-methyltransferase, murine double minute 2 (MDM2), and epithelin (granulin) are upregulated in GBMs. We also demonstrate for the first time that the cytokine and p53 binding protein, macrophage migration inhibitory factor (MIF), appears upregulated in GBMs. These results indicate that the modulation of ion and solute transport genes and heretofore unsuspected cytokines (i.e., MIF) may have profound implications for brain tumor cell biology and thus may identify potential useful therapeutic targets in GBMs.

Genetically engineered human herpes simplex virus in the treatment of brain tumours.

Central nervous system malignancies--particularly glioblastoma multiforme--pose significant problems for the development of novel therapeutics. In the absence of advances with standard surgical and chemotherapeutic approaches, the utilization of genetically engineered viruses--both as direct oncolytic agents (virus therapy) and for the delivery of foreign proteins (gene therapy)--represents a significant advance in the experimental approach to the management of patients with incurable tumours. Among other viruses, herpes simplex virus (HSV) offers an opportunity to influence the replication of tumour cells directly within the central nervous system. The propensity for HSV to replicate in tumour cells, and its large coding capacity, provide an experimental model for the development of novel therapeutics. The status of these experimental approaches and Phase I studies are summarized.

Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.

G207 is a conditionally replicating derivative of herpes simplex virus (HSV) type-1 strain F engineered with deletions of both gamma(1)34.5 loci and a lacZ insertion disabling the UL39 gene. We have demonstrated the efficacy of G207 in treating malignant glial tumors in athymic mice, as well as the safety of intracerebral G207 inoculation in mice and in Aotus nancymai. We sought to determine the safety of G207 inoculation into cerebral malignant glial tumors in humans. Criteria for inclusion into this dose-escalation study were the diagnosis of histologically proven malignant glioma, Karnofsky score > or = 70, recurrence despite surgery and radiation therapy, and an enhancing lesion greater than 1 cm in diameter. Serial magnetic resonance images were obtained for volumetric analysis. The trial commenced at a dose of 10(6) plaque forming units (p.f.u.) inoculated at a single enhancing site and was completed when the 21st patient was inoculated with 3x10(9) p.f.u. at five sites. While adverse events were noted in some patients, no toxicity or serious adverse events could unequivocally be ascribed to G207. No patient developed HSV encephalitis. We found radiographic and neuropathologic evidence suggestive of anti-tumor activity and long-term presence of viral DNA in some cases.

Genetically engineered HSV in the treatment of glioma: a review.

Central nervous system malignancies, particularly glioblastoma multiforme, pose significant problems for the development of novel therapeutics. In the absence of advances with standard surgical and chemotherapeutic approaches, the utilisation of genetically engineered viruses, both as direct oncolytic agents as well as for the delivery of foreign proteins, represents a significant advance in the experimental approach to management of patients with these incurable tumours. Among other viruses, HSV offers an opportunity to directly influence the replication of tumour cells within the central nervous system. Because of its propensity to replicate in neuronal tissue as well as its large coding capacity, it provides an experimental model for the development of novel therapeutics. The status of these experimental approaches will be summarised in this review.

Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors.

Genetically engineered, neuroattenuated herpes simplex viruses (HSVs) expressing various cytokines can improve survival when used in the treatment of experimental brain tumors. These attenuated viruses have both copies of gamma(1)34.5 deleted. Recently, we demonstrated increased survival of C57BL/6 mice bearing syngeneic GL-261 gliomas when treated with an engineered HSV expressing IL-4, as compared with treatment with the parent construct (gamma(1)34. 5(-)) alone or with a virus expressing IL-10. Herein, we report construction of a conditionally replication-competent mutant expressing both subunits of mIL-12 (M002) and its evaluation in a syngeneic neuroblastoma murine model. IL-12 induces a helper T cell subset type 1 response, which may induce more durable antitumor effects. In vitro studies showed that, when infected with M002, both Vero cells and murine Neuro-2a neuroblastoma cells produced physiologically relevant levels of IL-12 heterodimers, as determined by ELISA. M002 was cytotoxic for Neuro-2a cells and human glioma cell lines U251MG and D54MG. Neurotoxicity studies, as defined by plaque-forming units/LD(50), performed in HSV-1-sensitive A/J strain mice found that M002 was not toxic even at high doses. When evaluated in an intracranial syngeneic neuroblastoma murine model, median survival of M002-treated animals was significantly longer than the median survival of animals treated with R3659, the parent gamma(1)34.5(-) mutant lacking any cytokine gene insert. Immunohistochemical analysis of M002-treated tumors identified a pronounced influx of CD4(+) T cells and macrophages as well as CD8(+) cells when compared with an analysis of R3659-treated tumors. We conclude that M002 produced a survival benefit via oncolytic effects combined with immunologic effects meditated by helper T cells of subset type 1.

Ionizing radiation improves survival in mice bearing intracranial high-grade gliomas injected with genetically modified herpes simplex virus.

Malignant gliomas remain incurable with current interventions. Encouraging investigational approaches include the use of genetically modified herpes simplex-1 (HSV-1) viruses as direct cytotoxic agents. Combining attenuated HSV-1 with standard therapy, human U-87 malignant glioma xenografts grown in the hind limb or intracranially in athymic nude mice were exposed to ionizing radiation, inoculated with genetically modified HSV R3616, or received both virus and radiation. The combination of virus with fractionated ionizing radiation suggests a synergistic action and results in reduced tumor volumes and longer survivals when compared with treatment with either modality alone.

Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors.

A genetically engineered, nonneurotropic herpes simplex virus (R7020) with a proven safety profile in both animals and humans was found effective in the treatment of large xenotransplanted tumors arising from a radiation- and chemotherapy-resistant human epidermoid carcinoma and a hormone-refractory prostate adenocarcinoma. R7020 replicated to high titer and caused rapid regression of the human tumor xenografts. Tumor destruction was accelerated in animals given both R7020 and fractionated ionizing radiation. Tumors arising from cells surviving one treatment with R7020 were fully susceptible to a second dose of virus. We conclude R7020 is an effective antitumor agent for non-central nervous system tumor xenografts with an excellent safety profile.

Treatment of intracranial gliomas in immunocompetent mice using herpes simplex viruses that express murine interleukins.

This report describes a test of the hypothesis that the oncolytic effect of genetically engineered, replication competent herpes simplex viruses (HSV) depends both on cell destruction by the virus and an immune response to the tumor cells induced in an immunocompetent animal system. The oncolytic vector was a HSV recombinant virus in which both copies of the gamma 1 34.5 gene were replaced with the murine genes encoding the cytokine interleukin-4 (IL-4) or interleukin-10 (IL-10). The hypothesis predicted that if an immune response plays a role in survival following intratumoral treatment of tumor-bearing animals with HSV, expression of IL-4 should prolong survival whereas expression of IL-10 should reduce it. The results are that (1) these cytokines can be expressed by HSV in productively infected cells both in vitro and in vivo; (2) HSV-expressing IL-4 or IL-10 genes were able to infect and destroy glioma cells in vitro; (3) intracerebral inoculation of HSV expressing either IL-4 or IL-10 into syngeneic murine glioma GL-261 cells implanted in the brains of immunocompetent C57BL/6 mice produced dramatically opposite physiologic responses. The IL-4 HSV significantly prolonged survival of tumor bearers, whereas tumor-bearing mice that received the IL-10 HSV had a median survival that was identical to that of saline treated controls; (4) immunohistochemical analyses of mouse brains at 3 and 7 days after virus inoculation showed marked accumulation of inflammatory cells composed primarily of macrophages/microglia, with various proportions of CD8+ and CD4+ T cells, but few B lymphocytes. We conclude that the cytokines expressed from genes encoded in the viral genome influence HSV therapy of tumors and this is probably due to the host immune response. Thus, cytokine expression may be an important adjunct to tumor therapy utilizing genetically engineered HSV.