A supervised learning method for classifying methylation disorders.

BACKGROUND: DNA methylation is one of the most stable and well-characterized epigenetic alterations in humans. Accordingly, it has already found clinical utility as a molecular biomarker in a variety of disease contexts. Existing methods for clinical diagnosis of methylation-related disorders focus on outlier detection in a small number of CpG sites using standardized cutoffs which differentiate healthy from abnormal methylation levels. The standardized cutoff values used in these methods do not take into account methylation patterns which are known to differ between the sexes and with age. RESULTS: Here we profile genome-wide DNA methylation from blood samples drawn from within a cohort composed of healthy controls of different age and sex alongside patients with Prader-Willi syndrome (PWS), Beckwith-Wiedemann syndrome, Fragile-X syndrome, Angelman syndrome, and Silver-Russell syndrome. We propose a Generalized Additive Model to perform age and sex adjusted outlier analysis of around 700,000 CpG sites throughout the human genome. Utilizing z-scores among the cohort for each site, we deployed an ensemble based machine learning pipeline and achieved a combined prediction accuracy of 0.96 (Binomial 95% Confidence Interval 0.868[Formula: see text]0.995). CONCLUSION: We demonstrate a method for age and sex adjusted outlier detection of differentially methylated loci based on a large cohort of healthy individuals. We present a custom machine learning pipeline utilizing this outlier analysis to classify samples for potential methylation associated congenital disorders. These methods are able to achieve high accuracy when used with machine learning methods to classify abnormal methylation patterns.

Immunovirotherapy with measles virus strains in combination with anti-PD-1 antibody blockade enhances antitumor activity in glioblastoma treatment.

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome. Methods: In vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy. Results: In vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells. Conclusions: This report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment.

BAI1 Orchestrates Macrophage Inflammatory Response to HSV Infection-Implications for Oncolytic Viral Therapy.

Purpose: Brain angiogenesis inhibitor (BAI1) facilitates phagocytosis and bacterial pathogen clearance by macrophages; however, its role in viral infections is unknown. Here, we examined the role of BAI1, and its N-terminal cleavage fragment (Vstat120) in antiviral macrophage responses to oncolytic herpes simplex virus (oHSV).Experimental Design: Changes in infiltration and activation of monocytic and microglial cells after treatment of glioma-bearing mice brains with a control (rHSVQ1) or Vstat120-expressing (RAMBO) oHSV was analyzed using flow cytometry. Co-culture of infected glioma cells with macrophages or microglia was used to examine antiviral signaling. Cytokine array gene expression and Ingenuity Pathway Analysis (IPA) helped evaluate changes in macrophage signaling in response to viral infection. TNFα-blocking antibodies and macrophages derived from Bai1-/- mice were used.Results: RAMBO treatment of mice reduced recruitment and activation of macrophages/microglia in mice with brain tumors, and showed increased virus replication compared with rHSVQ1. Cytokine gene expression array revealed that RAMBO significantly altered the macrophage inflammatory response to infected glioma cells via altered secretion of TNFα. Furthermore, we showed that BAI1 mediated macrophage TNFα induction in response to oHSV therapy. Intracranial inoculation of wild-type/RAMBO virus in Bai1-/- or wild-type non-tumor-bearing mice revealed the safety of this approach.Conclusions: We have uncovered a new role for BAI1 in facilitating macrophage anti-viral responses. We show that arming oHSV with antiangiogenic Vstat120 also shields them from inflammatory macrophage antiviral response, without reducing safety. Clin Cancer Res; 23(7); 1809-19. ©2016 AACR.

The Impact of Macrophage- and Microglia-Secreted TNFα on Oncolytic HSV-1 Therapy in the Glioblastoma Tumor Microenvironment.

PURPOSE: Oncolytic herpes simplex viruses (oHSV) represent a promising therapy for glioblastoma (GBM), but their clinical success has been limited. Early innate immune responses to viral infection reduce oHSV replication, tumor destruction, and efficacy. Here, we characterized the antiviral effects of macrophages and microglia on viral therapy for GBM. EXPERIMENTAL DESIGN: Quantitative flow cytometry of mice with intracranial gliomas (±oHSV) was used to examine macrophage/microglia infiltration and activation. In vitro coculture assays of infected glioma cells with microglia/macrophages were used to test their impact on oHSV replication. Macrophages from TNFα-knockout mice and blocking antibodies were used to evaluate the biologic effects of TNFα on virus replication. TNFα blocking antibodies were used to evaluate the impact of TNFα on oHSV therapy in vivo. RESULTS: Flow-cytometry analysis revealed a 7.9-fold increase in macrophage infiltration after virus treatment. Tumor-infiltrating macrophages/microglia were polarized toward a M1, proinflammatory phenotype, and they expressed high levels of CD86, MHCII, and Ly6C. Macrophages/microglia produced significant amounts of TNFα in response to infected glioma cells in vitro and in vivo. Using TNFα-blocking antibodies and macrophages derived from TNFα-knockout mice, we discovered TNFα-induced apoptosis in infected tumor cells and inhibited virus replication. Finally, we demonstrated the transient blockade of TNFα from the tumor microenvironment with TNFα-blocking antibodies significantly enhanced virus replication and survival in GBM intracranial tumors. CONCLUSIONS: The results of these studies suggest that FDA approved TNFα inhibitors may significantly improve the efficacy of oncolytic virus therapy.

SapC-DOPS-induced lysosomal cell death synergizes with TMZ in glioblastoma.

SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.

NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors.

The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1(-/-) mice and a Ncr1(-/-) NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy.

Phase IB study of gene-mediated cytotoxic immunotherapy adjuvant to up-front surgery and intensive timing radiation for malignant glioma.

PURPOSE: Despite aggressive therapies, median survival for malignant gliomas is less than 15 months. Patients with unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) fare worse, presumably because of temozolomide resistance. AdV-tk, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, plus prodrug synergizes with surgery and chemoradiotherapy, kills tumor cells, has not shown MGMT dependency, and elicits an antitumor vaccine effect. PATIENTS AND METHODS: Patients with newly diagnosed malignant glioma received AdV-tk at 3 × 10(10), 1 × 10(11), or 3 × 10(11) vector particles (vp) via tumor bed injection at time of surgery followed by 14 days of valacyclovir. Radiation was initiated within 9 days after AdV-tk injection to overlap with AdV-tk activity. Temozolomide was administered after completing valacyclovir treatment. RESULTS: Accrual began December 2005 and was completed in 13 months. Thirteen patients were enrolled and 12 completed therapy, three at dose levels 1 and 2 and six at dose level 3. There were no dose-limiting or significant added toxicities. One patient withdrew before completing prodrug because of an unrelated surgical complication. Survival at 2 years was 33% and at 3 years was 25%. Patient-reported quality of life assessed with the Functional Assessment of Cancer Therapy-Brain (FACT-Br) was stable or improved after treatment. A significant CD3(+) T-cell infiltrate was found in four of four tumors analyzed after treatment. Three patients with MGMT unmethylated glioblastoma multiforme survived 6.5, 8.7, and 46.4 months. CONCLUSION: AdV-tk plus valacyclovir can be safely delivered with surgery and accelerated radiation in newly diagnosed malignant gliomas. Temozolomide did not prevent immune responses. Although not powered for efficacy, the survival and MGMT independence trends are encouraging. A phase II trial is ongoing.

Enhanced antitumor efficacy of vasculostatin (Vstat120) expressing oncolytic HSV-1.

Oncolytic viral (OV) therapy is a promising therapeutic modality for brain tumors. Vasculostatin (Vstat120) is the cleaved and secreted extracellular fragment of brain-specific angiogenesis inhibitor 1 (BAI1), a brain-specific receptor. To date, the therapeutic efficacy of Vstat120 delivery into established tumors has not been investigated. Here we tested the therapeutic efficacy of combining Vstat120 gene delivery in conjunction with OV therapy. We constructed RAMBO (Rapid Antiangiogenesis Mediated By Oncolytic virus), which expresses Vstat120 under the control of the herpes simplex virus (HSV) IE4/5 promoter. Secreted Vstat120 was detected as soon as 4 hours postinfection in vitro and was retained for up to 13 days after OV therapy in subcutaneous tumors. RAMBO-produced Vstat120 efficiently inhibited endothelial cell migration and tube formation in vitro (P = 0.0005 and P = 0.0184, respectively) and inhibited angiogenesis (P = 0.007) in vivo. There was a significant suppression of intracranial and subcutaneous glioma growth in mice treated with RAMBO compared to the control virus, HSVQ (P = 0.0021 and P < 0.05, respectively). Statistically significant reduction in tumor vascular volume fraction (VVF) and microvessel density (MVD) was observed in tumors treated with RAMBO. This is the first study to report the antitumor effects of Vstat120 delivery into established tumors and supports the further development of RAMBO as a possible cancer therapy.

Oncolytic HSV-1 infection of tumors induces angiogenesis and upregulates CYR61.

Oncolytic viral therapy is under evaluation for toxicity and efficacy in clinical trials relating to several different tumors. We report a significant increase in the angiogenic index of oncolytic virus (OV)-treated glioma-matrigel implants (2.83-fold, P < 0.02). In a rat intracranial glioma model, large tumors from OV-treated animals were significantly more angiogenic than the phosphate-buffered saline (PBS)-treated control tumors (OV: 101 +/- 21.6; PBS: 19.8 +/- 10; P = 0.0037). Transcript profiling of OV-treated tumors revealed dysregulation of several transcripts involved in glioma angiogenesis. OV-mediated induction of CYR61 gene expression (8.94-fold, P = 0.001) correlated significantly with the presence of OV in tumor tissue in vivo (R = 0.7, P < 0.001). Further, induction of CYR61 mRNA and protein were confirmed in multiple human cancer cell lines and primary human tumor-derived cells in vitro, and in tumor lysate and cerebrospinal fluid (CSF) in vivo. Finally, we show that treatment of glioma cells with Cilengitide, known to counter CYR61-induced integrin activation, significantly suppressed the proangiogenic effect of OV treatment of gliomas (P < 0.05).

Effect of tumor microenvironment modulation on the efficacy of oncolytic virus therapy.

BACKGROUND: The tumor microenvironment is being increasingly recognized as an important determinant of tumor progression as well as of therapeutic response. We investigated oncolytic virus (OV) therapy-induced changes in tumor blood vessels and the impact of modulating tumor vasculature on the efficacy of oncolytic virus therapy. METHODS: Rat glioma cells (D74/HveC) were implanted intracranially in immune-competent rats. Seven days later, the rats (groups of 3-7 rats) were treated with oncolytic virus (hrR3), and, 3 days later, brains were harvested for evaluation. Some rats were treated with angiostatic cRGD peptide 4 days before oncolytic virus treatment. Some rats were treated with cyclophosphamide (CPA), an immunosuppressant, 2 days before oncolytic virus treatment. Changes in tumor vascular perfusion were evaluated by magnetic resonance imaging of live rats and by fluorescence microscopy of tumor sections from rats perfused with Texas red-conjugated lectin immediately before euthanasia. Leukocyte infiltration in tumors was evaluated by anti-CD45 immunohistochemistry, and the presence of oncolytic virus in tumors was evaluated by viral titration. Changes in cytokine gene expression in tumors were measured by quantitative real-time polymerase chain reaction-based microarrays. Survival was analyzed by the Kaplan-Meier method. All statistical tests were two-sided. RESULTS: Oncolytic virus treatment of experimental rat gliomas increased tumor vascular permeability, host leukocyte infiltration into tumors, and intratumoral expression of inflammatory cytokine genes, including interferon gamma (IFN-gamma). The increase in vascular permeability was suppressed in rats pretreated with cyclophosphamide. Compared with rats treated with hrR3 alone, rats pretreated with a single dose of cRGD peptide before hrR3 treatment had reduced tumor vascular permeability, leukocyte infiltration, and IFN-gamma protein levels (mean IFN-gamma level for hrR3 versus hrR3 + cRGD = 203 versus 65.6 microg/mg, difference = 137 microg/mg, 95% confidence interval = 72.7 to 202.9 microg/mg, P = .006); increased viral titers in tumor tissue; and longer median survival (21 days versus 17 days, P<.001). CONCLUSIONS: A single dose of angiostatic cRGD peptide treatment before oncolytic virus treatment enhanced the antitumor efficacy of oncolytic virus.

Oncolytic viruses driven by tumor-specific promoters.

Oncolytic viruses can selectively replicate in and lead to tumor cell lysis with minimal infection/replication potential in adjoining non-neoplastic tissue. Because of paramount safety concerns, first-generation oncolytic viruses were designed to be significantly attenuated in their lytic potential. Results from recent clinical trials have revealed the safety of this approach, but have underscored the urgency for design and testing of more tumor-selective and -potent viruses to realize the full therapeutic potential of this revolutionary treatment modality. With the discovery of various molecular/genetic changes associated with neoplasia, tumor-specific transcriptional targeting of viral virulence is being tapped to generate tumor- and tissue-specific variants. This review will focus on the various strategies exploited to generate viruses whose virulence is governed by tumor-specific transcriptional events.