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Background: TOPACIO was a phase I/II study evaluating the PARP inhibitor (PARPi) niraparib in combination with the anti-PD-1 antibody pembrolizumab in patients with locally advanced and metastatic triple-negative breast cancer (TNBC, n=55) and ovarian cancer irrespective of BRCA mutation status. In the efficacy-evaluable population (n=47) the objective response rate (ORR) was 21% and disease control rate (DCR) 49%. Although activity was greater in patients with BRCA mutations (7/15, ORR=47% and 12/15, DCR=80%), durable clinical benefit was seen in patients with wild-type BRCA tumors (3/27, ORR=11% and 9/27, DCR=33%). In a limited cohort of 20 patients with durable clinical benefit, there were 8 BRCA wildtype patients, four of whom had mutations in genes associated with the homologous recombination repair and other DNA damage repair pathways. Pre-treatment tissues were collected and evaluated for tumor PD-L1 status. Patients with PD-L1 positive tumors (28/47, 60%) had a higher response rate (9/28, ORR=32%) than those with PD-L1 negative tumors (1/13, ORR=8%; 6 tumors had unknown PD-L1 status). It remains unstudied whether the tumor’s gene expression profile or immune status in baseline biospecimens is predictive of treatment response. In this study we conducted exploratory biomarker analyses to test the hypothesis that gene expression patterns and immune status are associated with treatment response. Methods: Transcriptional profiling of baseline samples was performed using the BC360 (n=41) and PanCancer IO360 (n=42) panels (Nanostring) and multigene signatures were used to measure tumor and immune activities as well as relative immune cell abundance. Transcriptional analysis was paired with high-dimensional, single-cell cyclic immunofluorescence (CyCIF) of samples that had adequate tissue for analysis (n=19) to characterize the composition and topology of the immune microenvironment at single-cell resolution. Results: Nanostring transcriptional analysis revealed that PAM50 genes stratified tumor samples into 4 subgroups with distinct histology as determined by CyCIF. Each subgroup was capable of responding to niraparib plus pembrolizumab. Multiple genes involved in WNT signaling (WNT5B, TANKS1, TANKS2, PARP4, and NET02) were associated with favorable clinical responses. Low neuropilin and tolloid-like protein 2 (NETO2) gene expression was strongly correlated with favorable progression free survival (PFS; R=-0.61, p=0.0008, Spearman’s correlation), suggesting it may be a predictive biomarker of therapeutic response. Nanostring gene expression signatures for tumor inflammation, apoptosis, and inflammatory chemokines also distinguished responders from non-responders (p Citation Format: Jennifer L Guerriero, Gregory J Baker, Jia-Ren Lin, Yu-An Chen, Ricardo Pastorello, Tuulia Vallius, Janae Davis, Clarence Yapp, Sarah E Church, Eric Miller, Anniina Färkkilä, Shaveta Vinayak, Melinda L Telli, Giulia Fulci, Alan D'Andrea, Geoffrey I Shapiro, Sara M Tolaney, Sandro Santagata, Peter K Sorger, Elizabeth A Mittendorf. High-dimensional, single-cell analysis and transcriptional profiling reveal novel correlatives of response to PARP inhibition plus PD-1 blockade in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-13. more...

Supplementary Figures S1-S3 from Distinguishing Inflammation from Tumor and Peritumoral Edema by Myeloperoxidase Magnetic Resonance Imaging

Purpose: Inflammation occurs routinely when managing gliomas and is not easily distinguishable from tumor regrowth by current MRI methods. The lack of noninvasive technologies that monitor inflammation prevents us to understand whether it is beneficial or detrimental for the patient, and current therapies do not take this host response in consideration. We aim to establish whether a gadolinium (Gd)-based agent targeting the inflammatory enzyme myeloperoxidase (MPO) can selectively detect intra- and peritumoral inflammation as well as glioma response to treatment by MRI.Methods: We carried out serial Gd-bis-5-HT-DTPA (MPO-Gd) MRI before and after treating rodent gliomas with different doses of oncolytic virus (OV) and analyzed animal survival. The imaging results were compared with histopathologic and molecular analyses of the tumors for macrophage/microglia infiltration, virus persistence, and MPO levels.Results: Elevated MPO activity was observed by MRI inside the tumor and in the peritumoral cerebrum at day 1 post–OV injection, which corresponded with activation/infiltration of myeloid cells inhibiting OV intratumoral persistence. MPO activity decreased, whereas tumor size increased, as the virus and the immune cells were cleared (days 1–7 post–OV injection). A 10-fold increase in viral dose temporally decreased tumor size, but augmented MPO activity, thus preventing extension of viral intratumoral persistence.Conclusions: MPO-Gd MRI can distinguish enhancement patterns that reflect treatment-induced spatiotemporal changes of intratumoral and intracerebral inflammation from those indicating tumor and peritumoral edema. This technology improves the posttreatment diagnosis of gliomas and will increase our understanding of the role of inflammation in cancer therapy. Clin Cancer Res; 17(13); 4484–93. ©2011 AACR. more...

Supplementary Figure 2 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 4 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 3 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma. [Cancer Res 2007;67(19):9398–406] more...

Supplementary Figure 1B from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 5 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Schwannomas are benign neoplasms that can cause gain‐ and loss‐of‐function neurological phenotypes, including severe, intractable pain. To investigate the molecular mechanisms underlying schwannoma‐associated pain we compared the RNA sequencing profile of painful and non‐painful schwannomas from NF2 patients. Distinct segregation of painful and non‐painful tumors by gene expression patterns was observed. Differential expression analysis showed the upregulation of fibroblast growth factor 7 (FGF7) in painful schwannomas. Behavioral support for this finding was observed using a xenograft human NF2‐schwannoma model in nude mice. In this model, over‐expression of FGF7 in intra‐sciatically implanted NF2 tumor cells generated pain behavior compared with controls. more...

Background There is a critical need to identify patient characteristics associated with long-term ovarian cancer survival. Methods Quality of life (QOL), measured by the Functional Assessment of Cancer Therapy-Ovarian-Trial Outcome Index (FACT-O-TOI), including physical, functional, and ovarian-specific subscales, was compared between long-term survivors (LTS) (8+ years) and short-term survivors (STS) ( Results QOL differed statistically significantly between STS (N = 1115) and LTS (N = 260) (P Conclusions Baseline and longitudinal QOL change scores distinguished LTS vs STS and are robust prognosticators for long-term survival. Results have trial design and supportive care implications, providing meaningful prognostic value in this understudied population. more...

BT8009 is a Bicycle® Toxin Conjugate (BTC™), a novel class of chemically synthesized molecules, comprising a bicyclic peptide targeting Nectin-4 tumor antigen, linked to cytotoxin (monomethyl auristatin E [MMAE]) via a valine-citrulline (val-cit) cleavable linker. Due to their small size, BTCs represent a unique therapeutic class, combining the pharmacology normally associated with a biologic with the pharmacokinetic properties of a small molecule. Here, we describe the preliminary monotherapy dose escalation results of the ongoing multicenter Phase I/II clinical trial (NCT04561362) that assesses the safety and tolerability of BT8009 administration in patients with advanced solid tumors associated with Nectin-4 expression, not exposed to enfortumab vedotin. Patients were treated until disease progression or intolerable toxicity, with tumor assessments for response per RECIST taken every two months. Nectin-4 expression levels were measured in tumor tissue. Thirty-four patients were initiated on weekly BT8009 (7 patients at 2.5 mg/m2, 20 patients at 5.0 mg/m2, and 4 patients at 7.5 mg/m2) or biweekly BT8009 (3 patient at 7.5 mg/m2). Baseline demographics were 62% patients were male, 44% and 56% were ECOG 0 and ECOG 1, respectively, median age was 65.5 years. BT8009 exhibits linear pharmacokinetics. The most common treatment-related adverse events were nausea (14 patients, 41%; ≥ G3 1 patient, 3%), fatigue (13 patients, 38%; ≥ G3 2 patients, 6%), anorexia (11 patients, 32%; ≥ G3 0 patients), constipation (10 patients, 29%; ≥ G3 1 patient, 3%) and anemia (10 patients, 29%; ≥ G3 3 patients, 9%). To date, BT8009 had low incidences of ocular disorders (3%), hyperglycemia (6%), neuropathy (6%), and rash (18%). There was 1 dose-limiting toxicity (G3 asthenia) reported at the 7.5 mg/m2 weekly dose level. Six patients (18%) experienced an SAE, with only 1 related SAE (vomiting). Treatment-emergent AEs resulting in treatment discontinuation, dose interruption, or reductions were observed in 2 patients (6%), 11 patients (32%), and 5 patients (15%), respectively. Preliminary confirmed responses observed in patients with urothelial carcinoma (UC) enrolled in the 5 mg/m2 cohort show 4/8 patients with a complete response (CR) or partial response (PR), including 1/8 patients with a CR and 3/8 patients with a PR, and 2/8 patients (25%) with stable disease (SD), reflecting a 50% overall response rate and a 75% disease control rate in UC patients for this cohort. BT8009 exhibits a promising preliminary tolerability profile and preliminary antitumor activity. The molecule will continue to be explored in the current dose-escalation/dose-expansion study of BT8009 monotherapy and in combination with nivolumab. Citation Format: Meredith McKean, Capucine Baldini, Loic Verlingue, Bernard Doger, Gerald Falchook, Antoine Italiano, Julia Lostes, Oscar Reig, Roshawn Watson, Sebastien Hazard, Dominic Smethurst, Giulia Fulci, Hongmei Xu, Phil Jeffery, Kevin Lee, Irene Braña, Sophie Cousin, Elisa Fontana, Hendrik-Tobias Arkenau. BT8009-100 phase I/II study of novel bi-cyclic peptide and MMAE conjugate BT8009 in patients with advanced malignancies associated with nectin-4 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT025. more...

Purpose Chemical exchange saturation transfer (CEST) MRI has shown promise in tissue characterization in diseases like stroke and tumor. However, in vivo CEST imaging such as amide proton transfer (APT) MRI is challenging because of concomitant factors such as direct water saturation, macromolecular magnetization transfer, and nuclear overhauser effect (NOE), which lead to a complex contrast in the commonly used asymmetry analysis (MTRasym). Here, we propose a direct saturation-corrected CEST (DISC-CEST) analysis for simplified decoupling and quantification of in vivo CEST effects. Methods CEST MRI and relaxation measurements were carried out on a classical 2-pool creatine-gel CEST phantom and normal rat brains (N = 6) and a rat model of glioma (N = 8) at 4.7T. The proposed DISC-CEST quantification was carried out and compared with conventional MTRasym and the original three-offset method. Results We demonstrated that the DISC-CEST contrast in the phantom had much stronger correlation with MTRasym than the three-offset method, which showed substantial underestimation. In normal rat brains, the DISC-CEST approach revealed significantly stronger APT effect in gray matter and higher NOE effect in white matter. Furthermore, the APT and NOE maps derived from DISC-CEST showed significantly higher APT effect in the tumors than contralateral normal tissue but no apparent difference in NOE. Conclusion The proposed DISC-CEST method, by correction of nonlinear direct water saturation effect, serves as a promising alternative to both the commonly used MTRasym and the simplistic three-offset analyses. It provides simple yet reliable in vivo CEST quantification such as APT and NOE mapping in brain tumor, which is promising for clinical translation. Magn Reson Med 78:2307-2314, 2017. © 2017 International Society for Magnetic Resonance in Medicine. more...

Background We evaluated apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) as a schwannoma tumor suppressor and explored its utilization in a schwannoma gene therapy strategy that may be translated to clinical use. Methods ASC protein expression and mRNA level were assessed in human schwannoma by immunohistochemistry and quantitative PCR, respectively. Methylation- specific PCR was used to assess ASC promoter methylation. The effect of ASC overexpression in schwannoma cells was evaluated through ATP-based viability, lactate dehydrogenase release, and apoptosis staining. Western blotting and colorimetric assay were used to test the effect of ASC overexpression on endogenous pro-apoptotic pathways. Bioluminescence imaging, behavioral testing, and immunohistochemistry in human xenograft and murine allograft schwannoma models were used to examine the efficacy and toxicity of intratumoral injection of adeno-associated virus (AAV) vector encoding ASC. Results ASC expression was suppressed via promoter methylation in over 80% of the human schwannomas tested. ASC overexpression in schwannoma cells results in cell death and is associated with activation of endogenous caspase-9, caspase-3, and upregulation of BH3 interacting-domain death agonist. In a human xenograft schwannoma model, AAV1-mediated ASC delivery reduced tumor growth and resolved tumor-associated pain without detectable toxicity, and tumor control was associated with reduced Ki67 mitotic index and increased tumor-cell apoptosis. Efficacy of this schwannoma gene therapy strategy was confirmed in a murine schwannoma model. Conclusion We have identified ASC as a putative schwannoma tumor suppressor with high potential clinical utility for schwannoma gene therapy and generated a vector that treats schwannomas via a novel mechanism that does not overlap with current treatments. more...

Currently, there is no stable and flexible method to label and track cytotoxic T-lymphocytes (CTLs) in vivo in CTL immunotherapy. We aimed to evaluate whether the sulfo-hydroxysuccinimide (NHS)-biotin-streptavidin (SA) platform could chemically modify the cell surface of CTLs for in vivo tracking. CD8+ T lymphocytes were labeled with sulfo-NHS-biotin under different conditions and then incubated with SA-Alexa647. Labeling efficiency was proportional to sulfo-NHS-biotin concentration. CD8+ T lymphocytes could be labeled with higher efficiency with sulfo-NHS-biotin in DPBS than in RPMI (P more...

In 2019 there will be 22,500 new cases of ovarian cancer, resulting in approximately 14,000 deaths. Ovarian cancer remains the highest case fatality rate of any gynecologic cancer. Despite significant improvement in the surgical and chemotherapeutic managements of ovarian cancer patients, the overall survival has not changed in 30 years. However, these new therapies have resulted in a significant improvement in median survival, resulting in increasing numbers of patients surviving greater than 10 years with disease. The clinical and molecular features of these unique patients remain uncharacterized. The Department of Defense Ovarian Cancer Research Program (DOD OCRP) funded the creation of our interdisciplinary Long-term Survivor Consortium (LTSC), composed of 6 research sites including MDACC, UAB, IU, UCI, Ludwig Cancer Research, and University Hospitals of Canton Vaud (CHUV) and INOVA, a bioinformatic center at DFCI, and administrative centers at UCI and UAB. The purpose of the consortium is to identify characteristics and predictors of long-term survival in advanced ovarian cancer patients. The consortium is overseen by both a scientific and advocacy advisory board to guide direction and efforts. 285 specimens of advanced-stage high-grade serous ovarian cancers with full clinical annotation including greater than 12 years of follow-up were analyzed for this project. In addition, 340 early-stage high-grade ovarian cancers were available for one of the AIMs of the project. These latter samples were obtained through the creation of an international consortium funded by the DOD OCRP. All specimens were first reviewed for tumor context by H&E staining to determine percentage of tumor. The scientific platforms include whole transcriptome, microRNAs, global proteomics, immune profiling, and methylation. All specimens were analyzed on each individual platform and the results will be integrated in a global process. In parallel with these efforts, a systematic analysis of quality of life of patients at the time of diagnosis and later during the course of their disease based upon patient-reported outcome surveys has been undertaken. These data will also be integrated with genomic results to produce signatures that can further characterize long-term survivors of ovarian cancer. Citation Format: Michael J. Birrer, Lari Wenzel, Austin Miller, Heather Lankes, Nilsa Ramirez, Giovanni Parmigiani, Samuel Mok, Kenneth Nephew, Thomas Conrads, George Coukos, George L. Maxwell, Giulia Fulci, Mary Scroggins. The Long-Term Ovarian Cancer Survivor Project: A Department of Defense initiative [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA06. more...

SUMMARY Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type., Graphical Abstract, In Brief Cancer cell-derived extracellular vesicles (EVs) can be used in diagnostics, but their enrichment remain s challenging. Zaborowski et al. identify membrane proteins enriched on the surface of cancer cells compared with normal tissues using TCGA, the Human Protein Atlas, and GTEx and present methods to measure immunocapture of cancer EVs in vitro and in animal models. more...

Intraperitoneal (IP) chemotherapy for ovarian cancer treatment prolongs overall survival by 16 months compared to intravenous chemotherapy but is not widely practiced due to catheter-related complications and complexity of administration. An implantable, nonresorbable IP microdevice was used to release chemotherapeutic agent at a constant rate of approximately 1.3 μg/h in vitro and 1.0 μg/h in vivo. Studies conducted in two orthotopic murine models bearing human xenografts (SKOV3 and UCI101) demonstrate that continuous dosing reduces tumor burden to the same extent as weekly IP bolus drug injections. Treatment-induced toxicity was quantified via body weight loss and complete blood count. The microdevice resulted in significantly less toxicity than IP bolus injections, despite administration of higher cumulative doses (total area under the concentration-time curve of 3049 ng day/mL with the microdevice vs. 2118 ng-day/mL with IP bolus injections). This preclinical study supports the concept that reduced toxicity with similar efficacy outcomes can be achieved by continuous dosing in ovarian cancer patients currently treated with IP therapy. more...

This study demonstrates the possibility of engineering an MR imaging reporter gene into oncolytic viruses that is detectable with chemical exchange saturation transfer MR imaging at acute stages of viral infection and does not interfere with viral replication or therapeutic effectiveness. more...

Bevacizumab (BEV) is an antiangiogenic drug approved for glioblastoma (GBM) treatment. However, it does not increase survival and is associated with glioma invasion. Angiostatin is an antiangiogenic polypeptide that also inhibits migration of cancer cells, but is difficult to deliver. Oncolytic viruses (OV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them and deliver anticancer agents to uninfected cells. We have tested a combination treatment of BEV plus an OV expressing angiostatin (G47δ-mAngio) in mice-bearing human GBM. Using a vascular intracranial human glioma model (U87) in athymic mice, we performed histopathological analysis of tumors treated with G47δ-mAngio or BEV alone or in combination, followed tumor response by magnetic resonance imaging (MRI), and assessed animal survival. Our results indicate that injection of G47δ-mAngio during BEV treatment allows increased virus spread, tumor lysis, and angiostatin-mediated inhibition of vascular endothelial growth factor (VEGF) expression and of BEV-induced invasion markers (matrix metalloproteinases-2 (MMP2), MMP9, and collagen). This leads to increased survival and antiangiogenesis and decreased invasive phenotypes. We show for the first time the possibility of improving the antiangiogenic effect of BEV while decreasing the tumor invasive-like phenotype induced by this drug, and demonstrate the therapeutic advantage of combining systemic and local antiangiogenic treatments with viral oncolytic therapy. more...

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma. [Cancer Res 2007;67(19):9398–406] more...

Schwannomas are tumors composed of Schwann-lineage cells that form along peripheral nerves. These tumors can cause pain, sensory/motor dysfunction, and death through compression of peripheral nerves, the spinal cord, and/or the brain stem. Management of these tumors-essentially restricted to operative resection and gamma-knife debulking- is limited in scope and efficacy and has significant associated morbidity. Chemotherapeutic treatment strategies have shown incomplete and transient tumor regression. Treatment of Schwannomas remains a major unmet clinical need.Schwannomas are appealing targets for gene therapy as they: 1) grow slowly, 2) have cellular and genetic homogeneity, and 3) can be readily localized using magnetic resonance and/or ultrasound imaging for direct intratumoral vector injection. Gene-therapy is potentially advantageous compared to resection as gene therapy is minimally invasive, kills tumor cells without damaging tumor-associated nerve, and may allow treatment of lesions not amenable to resection. We have previously published that schwannomas can be effectively treated with a gene therapy strategy that uses an adeno-associated virus (AAV) vector to deliver the apoptotic and inflammatory enzyme ICE (IL1-converting enzyme, aka caspase-1) under the Schwann cell specific promoter, myelin Protein 0 (P0). It efficiently debulkes schwannomas and resolves schwannoma associated pain, without any neuronal toxicity.However, the mechanisms by which AAV-P0-ICE selectively kills schwannomas without causing schwann-cell damage and associated neural demyelination are not clear. Understanding these mechanisms would provide the basis for broader utilization of schwannoma gene therapy and insight into how we might increase the efficacy of our current therapy.Through this project we have explored how the role of the P0 promoter and the inflammasome in regulating selective killing of schwannoma cells. Our results indicate that P0 is a highly stringent promoter as it is not expressed in neurons or even in schwannoma cells grown in vitro. P0 is active only in schwann and schannoma cells in vivo, and lack of P0 activity prevents any caspase-1 induced death in other cell types. Second, our data indicates that caspase-1 killing requires concomitant activation of the inflammasome. This pathway was active in 2 different schwannoma cell lines and may be the differentiating factor that prevents caspase-1 to cause axonal demyelination following AAV-P0-ICE injection into peripheral nerve. Finally, our data indicate that adjuvant treatment with factors that stimulate the inflammasome activity increases the efficacy of caspase-1 mediated schwannoma killing. Taken together, these data strongly support the safety of using the P0 promoter for the development of gene therapies for schwannomas and provide new, more efficient, venues for schwannoma capsase-1 mediated gene therapy. more...

Clinical trials are testing oncolytic viruses (OVs) as therapies for cancer. We have shown that animals that have brain tumors and are treated with a herpes simplex virus (HSV)-derived OV live significantly longer when cyclophosphamide (CPA) is preadministered. Here, we explore the mechanisms behind this finding. In a syngeneic rat glioma model, intratumoral HSV administration is associated with rapid increase of natural killer cells, microglia/macrophages (CD68+and CD163+), and IFN-γ. Pretreatment with CPA enhances HSV replication and oncolysis and reduces an HSV-mediated increase in CD68+and CD163+cells and intratumoral IFN-γ. Molecular imaging shows CPA pretreatment to inhibit HSV-induced infiltration of tumor-associated phagocytic cells. Our results reveal molecular and cellular mechanisms that inhibit intratumoral spread of HSV and suggest a therapeutic path for improving the efficacy of virotherapy as a treatment for cancer. more...

The oncolytic adenovirus Delta24-RGD represents a new promising therapeutic agent for patients with a malignant glioma and is currently under investigation in clinical phase I/II trials. Earlier preclinical studies showed that Delta24-RGD is able to effectively lyse tumor cells, yielding promising results in various immune-deficient glioma models. However, the role of the immune response in oncolytic adenovirus therapy for glioma has never been explored. To this end, we assessed Delta24-RGD treatment in an immune-competent orthotopic mouse model for glioma and evaluated immune responses against tumor and virus. Delta24-RGD treatment led to long-term survival in 50% of mice and this effect was completely lost upon administration of the immunosuppressive agent dexamethasone. Delta24-RGD enhanced intra-tumoral infiltration of F4/80+ macrophages, CD4+ and CD8+ T-cells, and increased the local production of pro-inflammatory cytokines and chemokines. In treated mice, T cell responses were directed to the virus as well as to the tumor cells, which was reflected in the presence of protective immunological memory in mice that underwent tumor rechallenge. Together, these data provide evidence that the immune system plays a vital role in the therapeutic efficacy of oncolytic adenovirus therapy of glioma, and may provide angles to future improvements on Delta24-RGD therapy. more...

TP53 is the most frequently mutated gene in cancer and about 40% of glioblastoma express mutated p53 protein. The effects of many anti-cancer agents are largely dependent on p53-mediated apoptosis at the G1/S cell cycle checkpoint subsequent to DNA damage. Therefore, TP53 status may help stratifying patients into subsets that may respond differently to treatments such as chemo- and radiotherapy.1,2 Since an inverse relationship between TP53 mutation and radiosensitivity has been observed in glioblastomas,3 it has been hypothesized that inactivation of TP53 may confer resistance to radio- and possibly chemotherapeutic agents.4,5 Clearly it is important to better understand the role of physiologic responses of p53 in these processes for developing optimal therapeutic strategies for glioma patients. For studying the role of p53 in these mechanisms, wild-type (WT) p53 function was restored in various glioblastoma cell lines by several gene transfer methods, including stable transfection of p53 constructs inducible by dexamethasone6 or tetracycline,7 an exogenous murine p53 Val 135 temperature-sensitive (ts) mutant,8 p53 expression from retroviruses9 and adenoviruses.10 Expression of p53 in glioblastoma cells from stably transfected clones or retrovirally transduced genes induced growth arrest or senescence, whereas expression from adenoviruses induced apoptosis. Reversibility of the growth arrest was not determined. The reasons for these uneven responses are unclear but may be linked to variable levels of p53 expression driven by the exogenous promoters used in all these constructs, perturbation of cellular status by the large difference of culture temperature (6°C) for the exogenous ts mutant, incomplete function of murine p53 in human cells, variation in p53 responses in different cell lines and synergy of p53 action with cellular responses to viral infection. To reassess glioblastoma cell responses to restoration of WT p53 activity close to physiologic levels, we have taken advantage of our discovery of a glioblastoma cell line (LN382) expressing a p53 mutant with ts properties in yeast.11 Here we demonstrate that this endogenous p53 mutant switches from mutant to WT activity over a narrow 3°C temperature range (37–34°C) in LN382 cells, using several assays for p53 activity. Using this cell line, we have subsequently analyzed changes in cell proliferation and apoptosis in response to p53 activation in the absence of genotoxic stress. The absolute advantage of using the intrinsic ts mutant over artificially transfected TP53 genes is that because the ts mutant is under the control of endogenous TP53 gene regulatory elements its expression level is expected to be in the physiologic range. Also, the results are not influenced by the selection of a particular clone resistant to cell-cycle arrest, senescence or apoptosis induced by p53, and does not undergo counterselection of mutant p53, which occurs rapidly after transfection of WT TP53 alleles.9,12 This cell line thus will be invaluable in permitting biochemical analyses of the molecular mechanisms underlying p53 action in a variety of situations. more...

Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, results from a disruption of the balance between stimulatory and inhibitory factors. Here, we show that anoxia reduces expression of thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, in glioblastoma cells. This suggests that reduced oxygen tension can promote angiogenesis not only by stimulating the production of inducers, such as vascular endothelial growth factor, but also by reducing the production of inhibitors. This downregulation may significantly contribute to glioblastoma development, since we show that an increase in TSP-1 expression is sufficient to strongly suppress glioblastoma cell tumorigenicity in vivo. more...

This article reviews the recent molecular and clinical studies that characterize the role of p53 in pathologies of the central nervous system, p53 has many important biological functions, notably, maintenance of DNA stability and regulation of apoptosis. These features are essential to avoid cellular transformation and ensure normal brain development. Lack of p53 function in the brain results in tumor formation in the astrocytic and lymphoid lineages and in severe neurodevelopmental diseases, such as exencephaly. more...

The p53 tumor suppressor gene (TP53) is the most frequently altered gene in human cancer and is also found mutated in several types of brain tumors. Loss of p53 function plays a central role in the development of cancer. The characterization of the biochemical pathways by which p53 alteration triggers tumorigenesis is the foundation for the design of novel therapeutic approaches. Investigations of the intracellular mechanisms at the origin of p53 tumor suppressive functions have shown that p53 is a transcription factor able to sense a variety of cellular insults and induce a dual response: cell growth arrest/senescence or apoptosis. Less well studied are p53′s influences on extracellular events such as tumor angiogenesis, immunology and invasion. Here, we review these findings and specifically discuss their implications for brain tumor genesis, molecular diagnosis and prognosis. Of clinical importance are the findings that brain tumors with wild type (wt) or mutant p53 status may respond differently to radiation therapy and that novel therapeutic strategies using TP53 gene transfer or specifically targeting tumor cells with mutated p53 are being evaluated in clinical trials. more...

Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene therapy for schwannomas, using a model in which immortalized human NF2 schwannoma cells expressing a fluorescent protein and luciferase are implanted in the sciatic nerve of nude mice. Direct injection of an adeno-associated virus (AAV) serotype 1 vector encoding caspase-1 (ICE) under the Schwann-cell specific promoter, P0, leads to regression of these tumors with essentially no vector-mediated neuropathology, and no changes in sensory or motor function. In a related NF2 xenograft model designed to cause measurable pain behavior, the same gene therapy leads to tumor regression and concordant resolution of tumor-associated pain. This AAV1-P0-ICE vector holds promise for clinical treatment of schwannomas by direct intratumoral injection to achieve reduction in tumor size and normalization of neuronal function. more...

Purpose: Inflammation occurs routinely when managing gliomas and is not easily distinguishable from tumor regrowth by current MRI methods. The lack of noninvasive technologies that monitor inflammation prevents us to understand whether it is beneficial or detrimental for the patient, and current therapies do not take this host response in consideration. We aim to establish whether a gadolinium (Gd)-based agent targeting the inflammatory enzyme myeloperoxidase (MPO) can selectively detect intra- and peritumoral inflammation as well as glioma response to treatment by MRI. Methods: We carried out serial Gd-bis-5-HT-DTPA (MPO-Gd) MRI before and after treating rodent gliomas with different doses of oncolytic virus (OV) and analyzed animal survival. The imaging results were compared with histopathologic and molecular analyses of the tumors for macrophage/microglia infiltration, virus persistence, and MPO levels. Results: Elevated MPO activity was observed by MRI inside the tumor and in the peritumoral cerebrum at day 1 post–OV injection, which corresponded with activation/infiltration of myeloid cells inhibiting OV intratumoral persistence. MPO activity decreased, whereas tumor size increased, as the virus and the immune cells were cleared (days 1–7 post–OV injection). A 10-fold increase in viral dose temporally decreased tumor size, but augmented MPO activity, thus preventing extension of viral intratumoral persistence. Conclusions: MPO-Gd MRI can distinguish enhancement patterns that reflect treatment-induced spatiotemporal changes of intratumoral and intracerebral inflammation from those indicating tumor and peritumoral edema. This technology improves the posttreatment diagnosis of gliomas and will increase our understanding of the role of inflammation in cancer therapy. Clin Cancer Res; 17(13); 4484–93. ©2011 AACR. more...

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.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.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).A single dose of angiostatic cRGD peptide treatment before oncolytic virus treatment enhanced the antitumor efficacy of oncolytic virus. more...

Approaches to improve the oncolytic potency of replication-competent adenoviruses include the insertion of therapeutic transgenes into the viral genome. Little is known about the levels and duration of in vivo transgene expression by cells infected with such "armed" viruses. Using a tumor-selective adenovirus encoding firefly luciferase (AdDelta24CMV-Luc) we investigated these questions in an intracranial mouse model for malignant glioma. Luciferase expression was detected by bioluminescence imaging, and the effect of the immunosuppressive agent cyclophosphamide (CPA) on transgene expression was assessed. Intratumoral AdDelta24CMV-Luc injection led to a localized dose-dependent expression of luciferase. Surprisingly, this expression decreased rapidly during the course of 14 days. In contrast, mice injected with nonreplicating Ad.CMV-Luc demonstrated stable transgene expression. Treatment of mice with CPA in combination with AdDelta24CMV-Luc retarded the loss of transgene expression. Staining of mouse brains for inflammatory cells demonstrated decreased tumor infiltration by immune cells in CPA-treated mice. Moreover, in immunodeficient NOD/SCID mice loss of transgene expression was less rapid and not prevented by CPA treatment. Together, our data demonstrate that transgene expression and viral replication decrease rapidly after intratumoral injection of oncolytic adenovirus in mouse brains and that treatment with the immunomodulator CPA prolongs viral-mediated gene expression. more...

Oncolytic viruses are genetically altered replication-competent viruses that infect, and reproduce in, cancer cells but do not harm normal cells. On lysis of the infected cells, the newly formed viruses burst out and infect other tumor cells. Experiments with injecting mutant herpes simplex virus 1 (hrR3) into glioma implanted in brains of rats show lack of efficacy in eradicating the cancer. This failure is attributed to interference by the immune system. Initial pretreatment with immunosuppressive agent cyclophosphamide reduces the percentage of immune cells. We introduce a mathematical model and use it to determine how different protocols of cyclophosphamide treatment and how increased burst size of the mutated virus will affect the growth of the cancer. One of our conclusions is that the diameter of the cancer will decrease from 4 mm to eventually 1 mm if the burst size of the virus is triple that which is currently available. The effect of repeated cyclophosphamide treatment is to maintain a low density of uninfected cells in the tumor, thus reducing the probability of migration of tumor cells to other locations in the brain. (Cancer Res 2006; 66(4): 2314-9) more...

Oncolytic viruses (OVs) are being used as anticancer agents in preclinical and clinical trials. Propagation of OVs inside infected tumors is critical to their efficacy and is mediated by the productive generation of progeny OVs within infected tumor cells. In turn, this progeny can spread the infection to other tumor cells in successive rounds of oncolysis. Previously, we had found that, in rats, cyclophosphamide (CPA) pretreatment increased infection of brain tumors by an intra-arterially administered herpes simplex virus type 1 OV, because it inhibited activation of complement responses, mediated by innate IgM. We also have previously shown that other pharmacologic inhibitors of complement, such as cobra venom factor (CVF), allowed for increased infection. However, in these studies, further inhibition of complement responses by CVF did not result in additional infection of brain tumor cells or in propagation of OV to surrounding tumor cells. In this study, we sought to determine if CPA did lead to increased infection/propagation from initially infected tumor cells. Unlike our results with CVF, we find that CPA administration does result in a time-dependent increase in infection of tumor cells, suggestive of increased propagation, in both syngeneic and athymic models of brain tumors. This increase was due to increased survival of OV within infected tumors and brain surrounding tumors. CPA's effect was not due to a direct enhancement of viral replication in tumor cells, rather was associated with its immunosuppressive effects. RT-PCR analysis revealed that CPA administration resulted in impaired mRNA production by peripheral blood mononuclear cells (PBMCs) of several cytokines (interferons alpha/beta, interferon gamma, TNFalpha, IL-15, and IL-18) with anti-HSV function. These findings suggest that the CPA-mediated facilitation of OV intraneoplastic propagation is associated with a general decrease of antiviral cytokines mRNAs in PBMCs. These findings not only suggest a potential benefit for the addition of transient immunosuppression in clinical applications of oncolytic HSV therapy, but also suggest that innate immunomodulatory pathways may be amenable to manipulation, in order to increase OV propagation and survival within infected tumors. more...

Little is known about the genetic and molecular events leading to the early stages of human astrocytoma formation. To examine this issue, we analyzed the significance of sequential accumulation of two somatic point mutations (R267W and E258D) in the TP53 gene during the initiation of astrocytoma in a patient born with a single germ-line p53 point mutation (R283H). We adapted a p53 transcriptional assay in yeast to establish the temporal occurrence and allelic distribution of the p53 mutations present in the patient and characterized these mutations through functional assays and structural modeling. Our results show that the first somatic mutation occurred at codon 267 on the p53 allele harboring the germ-line mutation R283H, whereas the second somatic mutation occurred in the remaining wild-type (wt) allele at codon 258. These two mutations induced the formation of tumor cells with the genotype p53(267W+283H/258D), which comprised 70% of the cells in the primary WHO grade II astrocytoma. Another 8% of cells within the tumor had the partially mutated genotype p53(267W+283H/WT) and represented the remnants of a clinically undetectable intermediate stage of astrocytic neoplastic transformation. The remaining 22% of cells had the constitutive p53(283H/WT) genotype and likely consisted of nontumor cells. Functional analysis of the p53 alleles present in the patient's tumor indicated that the germ-line p53(R283H) could transactivate the CDKN1A((p21, WAF1, cip1, SDI1)) but not the BAX gene and retained the ability to induce growth arrest of human glioblastoma cells. The p53(R267W+R283H) and p53(E258D) were incapable of transactivating either promoter or inducing growth arrest. Modeling of p53 interaction with DNA suggests that R283H mutation may weaken the sequence-specific interaction of p53 lysine 120 with the BAX gene but not the CDKN1A p53-responsive elements. Taken together, these results have characterized, for the first time, the genetic events defining a clinically undetectable precursor lesion leading to a grade II astrocytoma. They also suggest that astrocytoma initiation in this patient resulted from monoclonal evolution driven by a sequential loss of proapoptotic and growth arrest functions of p53. more...

Oncolytic viruses selectively lyse tumor cells, making these agents a promising treatment modality for glioma. Accumulating data suggest that the immune system plays an important role in the anti-glioma activity of oncolytic viruses. In an immune competent glioma model, the therapeutic effect of the oncolytic adenovirus Delta24-RGD was found to depend primarily on antitumor immune responses. more...

P16 and P14ARF are two tumor suppressors encoded by the locus ink4a-arf which is frequently deleted in human tumors. Recent experiments performed with mouse embryonic fibroblasts have shown that P14ARF is an upstream regulator of the P53 pathway. This raises the question as to whether in human tumors the loss of p14arf and mutation of p53 are mutually exclusive events which segregate with genetic alterations at other loci. To examine this question we performed a multigenic analysis on 29 gliomas. We analysed p53 and p14arf in relation with five other genetic loci encoding the most frequently mutated genes in human gliomas: cdkn2a, mdm2, egfr, pten and the chromosomal regions 10q23.3 and 10q25-26. Our study shows for the first time that p53 mutations and p14arf deletions appear mutually exclusive in human glioblastoma, suggesting that they may be functionally redundant in glioma tumorigenesis. The P53 pathway is, therefore, disrupted in 81.8% of malignant gliomas (WHO grades III and IV), either by mutation of the p53 gene (31.8%) or by p14arf deletion (54.5%). These tumors further showed MDM2 overexpression (9.1%), egfr oncogene amplification/egfr overexpression (50%), pten mutations (27.3%) and loss of heterozygosity (LOH) at the chromosomal regions 10q23.3 (86.4%) and 10q25-26 (100%). These alterations did not segregate with p53 mutations or p14arf deletions, while p14arf and cdkn2a were always deleted. more...

Background: Bevacizumab (Avastin), a monoclonal antibody that inactivates VEGF, was approved by US Food and Drug Administration as a single agent for treatment of recurrent glioblastomas (GBM). It prolongs progression-free survival and controls peritumoral edema. However, its effect on prognosis and overall survival remains to be determined. Angiostatin is a naturally occurring inhibitor of angiogenesis that was shown to inhibit growth of primary and metastatic tumors, but can not be delivered efficiently to GBM. Oncolytic viruses (OV) are promising agents for the treatment of GBM, whose therapeutic potential is not fully exploited due to infiltration of anti-viral macrophages. Treatment with anti-angiogenic factors was shown to increase replication and therapeutic capacity of OV. We hypothesized that combination of Avastin with an HSV OV (G47Δ) coding for angiostatin (G47Δ-angio) would enhance GBM treatment due to increased viral spread and delivery of angiostatin. Materials & Methods: G47Δ-angio was constructed using the “flip-flop” HSV-BAC system. Expression of angiostatin was confirmed by western blot; in vitro matrigel endothelial tube formation assay was performed to test its anti-angiogenic potential. We confirmed the capacity of this virus to infect cancer cells and inhibit tumor angiogenesis in vivo, and determined therapeutic efficacy using both a subcutaneous and an intracerebral glioma model in mice. A control virus not carrying the angiostatin gene (G47Δ-empty) served as the negative control for all experiments. Avastin (5mg/kg) was delivered by i.v injection in intracerebral glioma model, started at Day 5 and followed by twice a week for consecutive 4 weeks. Tumor angiogenesis was determined by CD31 and VEGF staining, macrophages infiltration was detected by F4/80 staining. The overall survival of intracerebral glioma mice was followed up in G47Δ-angio, Avastin and combination (G47Δ-angio + Avastin) treatments. Results: G47Δ-angio decreased endothelial tube formation in vitro and demonstrated potent antiangiogenic effects in vivo. In our subcutaneous tumor model, G47Δ inhibited tumor growth (n=7/group, P Conclusion: Our results demonstrate that G47Δ-angio could significantly enhance antitumor and antiangiogenic efficacy. Moreover, the combination treatment could further improve the antitumor and antiangiogenic efficacy in intracerebral glioma model in vivo. Hence, “Arming” HSV with an antiangiogenic gene that targets both the tumor cells and tumor vasculature is a promising therapy for use with Avastin treatment in clinical translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2568. doi:10.1158/1538-7445.AM2011-2568 more...

Top of pageAbstract Oncolytic viruses (OV) generate intratumoral progeny viruses that propagate the oncolytic effect throughout tumor cells in vitro. However, the generation of progeny viruses in vivo is suppressed by the host innate immune responses. We have demonstrated that, in rats harboring gliomas, pretreatment with cyclophosphamide (CPA), enhances OV replication. The use of CPA in viral therapy is limited by its broad immune-suppressive activity and toxicity. Our aim is to identify specific factors responsible for limiting OV survival in tumors in order to design pharmacological means that enhance OV replication in a selective fashion. We have used rats pretreated with either CPA or PBS to deliver OV intratumorally (CPA-/OV+ and CPA+/OV+) and analyze the immune pathways that are associated with viral proliferation. Our data show that OV has the same capacity to infect tumor cells in both CPA pre-treated and non pre-treated rats. After cell infection, OV increases the intratumoral levels of IFNg, a known inducer of macrophage/monocyte/microglia in the brain. This phenomenon is associated with a reduction of intratumoral viral titers within 76 hours from OV injection. In CPA pre-treated rats, IFNg levels remain low, macrophage intratumoral infiltration is suppressed and viral oncolysis is increased within 76 hours from OV injection. Pharmacologic depletion of macrophages enhances OV oncolysis and suppresses macrophage-induced immune responses in the tumor. Broad comparative analysis of gene expression in tumors of rats CPA-/OV+ and CPA+/OV+ using Affymetrix rat genome arrays has identified seven other molecular targets that are downstream effectors of IFNg-induced inflammation and are associated with viral proliferation. These data indicate that, selective temporary repression of inflammation leads to efficient viral replication and uncovers specific molecular targets that could be used to increase viral therapy. more...