VEGF Therapy Research Articles

Inhibition of autophagy potentiated the anti-tumor effects of VEGF and CD47 bispecific therapy in glioblastoma.

Glioblastoma, characterized by extensive microvascular proliferation and invasive tumor growth, is one of the most common and lethal malignancies in adults. Benefits of the conventional anti-angiogenic therapy were only observed in a subset of patients and limited by diverse relapse mechanism. Fortunately, recent advances in cancer immunotherapy have offered new hope for patients with glioblastoma. Herein, we reported a novel dual-targeting therapy for glioblastoma through simultaneous blockade of VEGF and CD47 signaling. Our results showed that VEGFR1D2-SIRPαD1, a VEGF and CD47 bispecific fusion protein, exerted potent anti-tumor effects via suppressing VEGF-induced angiogenesis and activating macrophage-mediated phagocytosis. Meanwhile, autophagy was activated by VEGFR1D2-SIRPαD1 through inactivating Akt/mTOR and Erk pathways in glioblastoma cells. Importantly, autophagy inhibitor or knockdown of autophagy-related protein 5 potentiated VEGFR1D2-SIRPαD1-induced macrophage phagocytosis and cytotoxicity against glioblastoma cells. Moreover, suppression of autophagy led to increased macrophage infiltration, angiogenesis inhibition, and tumor cell apoptosis triggered by VEGF and CD47 dual-targeting therapy, thus eliciting enhanced anti-tumor effects in glioblastoma. Our data revealed that VEGFR1D2-SIRPαD1 alone or in combination with autophagy inhibitor could effectively elicit potent anti-tumor effects, highlighting potential therapeutic strategies for glioblastoma through disrupting angiogenetic axis and CD47-SIRPα anti-phagocytic axis alone or in combination with autophagy inhibition.

Vitreous changes after intravitreal bevacizumab monotherapy for retinopathy of prematurity: a case series

PurposeReporting a special clinical finding after intravitreal bevacizumab monotherapy for retinopathy of prematurity.MethodsIn a retrospective case series, the clinical courses of five premature infants with similar vitreous changes after a single dose of intravitreal bevacizumab (IVB) injection without additional laser therapy were reported.ResultsThe mean post-conceptional age at IVB injection was 39.8 ± 2.2 (range 37–43) weeks. Localized vitreous syneresis and linear fibrotic vitreous condensation occurred 8.2 ± 2.3 weeks after IVB monotherapy in our patients (15.5% of injections). The mean last post injection visit was 61.6 ± 5.3 weeks (post-conceptional age). Further regression and complete retinal vascularization occurred in all patients.ConclusionsThread-like vitreous condensation with localized vitreous liquefaction may be related to involutional ROP disease itself, combined to anti VEGF therapy and may be a predictor factor for further regression and retinal vascularization. The case series describes a successful response to anti-VEGF monotherapy with no further complications.

Rekombinan Vascular Endothelial Growth Factor-121 Menekan Ekspresi Heat Shock Protein-70 pada Plasenta Mencit Model Preeklampsia (VASCULAR ENDOTHELIAL GROWTH FACTOR-121 RECOMBINANT DEPRESSIS HEAT SHOCK PROTEIN-70 EXPRESSION IN PREECLAMPSIA MICE PLACENTA

Preeclampsia has remained a major cause of morbidity and maternal-perinatal mortality. Oxidative stress that occurs in preeclampsia increases HSP-70 expression. Therefore, VEGF therapy is expected to recover this oxidative stress. This research aimed to determine the role of VEGF-121 recombinant on HSP-70 expression in mice model of preeclampsia. This research was an explanatory study conducted at Animal Cage Experiment, Faculty of Veterinary Medicine, Airlangga University. The samples were 30 mice that were divided into 3 groups, namely a group of 10 normal pregnant mice, a group of 10 mice model of preeclampsia, and a group of 10 mice model of preeclampsia with VEGF-121 recombinant therapy. On the 16th day of gestation, HSP-70 expressions in the placenta of all mice were examined using immunohistochemical methods. The data were analyzed using Kruskall-Wallis test of SPSS program. The mean of HSP-70 expression in normal pregnancy group was 1.69 ± 0.68, in preeclampsia model group was 3.50 ± 0.95 with p = 0.00, and in preeclampsia model group with VEGF-121 recombinant therapy was 2.24 ± 0.84 with p = 0.00. In short, VEGF-121 recombinant has a role in lowering HSP-70 expression in mice placenta model of preeclampsia

Progression and Incidence of Geographic Atrophy in Patients with Wet Age-Related Macular Degeneration undergoing Anti- VEGF Therapy

Importance: Age Related Macular Degeneration (AMD) is one of the leading causes of blindness in the developed world. There are two phenotypes of AMD as defined by the United Kingdom Department of Health: neovascular AMD (nAMD) and geographic atrophy (GA). GA can develop after nAMD as well. There is recent evidence of the development of GA after anti-vascular endothelial growth factor (anti-VEGF) treatment. Our study aims to assess the percentage, risk factors and characteristics of patients developing GA after the different anti-VEGF medications for nAMD. Observations: We retrospectively reviewed case records of 1415 eyes diagnosed with nAMD treated with anti-VEGF for a period of 60 months. Pre and post anti-VEGF treatment characteristics were recorded. Main outcome measures were the number of patients that developed GA after treated for nAMD with aflibercept, ranibizumab and bevaciumab. Secondary outcome measures included the following: number of injections per type, mean change in the best corrected visual acuity (BCVA), area of GA, central macular thickness (CMT), and the proportion of patients showing change in intra- retinal fluid (IRF), sub-retinal fluid (SRF) and pigment epithelial detachment (PED). 109 eyes (7.7%) developed GA after the last assessment. The number of injections was significantly related to the development of GA, R2=0.000246358, P<0.01. Other studied variables could not significantly predict development or progression of GA. Conclusion and relevance: In the era of antiVEGF for nAMD treatment, we need to take into account the risk of excessive treatment. Considering GA treatment is still under research the perfect balance between treatment and withholding still needs to be found. In addition other associations or risk factors for GA development after anti-VEGF need to be researched.

The advance and application of radiotherapy in exudative age-related macular degeneration

Exudative or wet age-related macular degeneration (AMD) is characterized by the progressive growth of choroidal neovascularization (CNV). Anti-vascular endothelial growth factor (VEGF) drugs have been used in the control of the development of CNV and vision improvement, but there are still defects like frequent injections, drug resistance and so on. Radiotherapy can deactivate local inflammatory cell populations, and make CNV unstable in the absence of pericytes and VEGF stimulation, which induce apoptosis of the vascular endothelial cells. Therefore, radiotherapy is considered as a potential adjuvant treatment of anti- VEGF therapy. The current clinical approaches include epimacular brachytherapy (EMBT) and long-range stereotactic radiotherapy (SRT). SRT may be a preferred adjuvant treatment for patients receiving anti-VEGF therapy. Knowing the progress of radiotherapy for the treatment of exudative AMD may help us to fully understand the pathogenesis of wAMD in China Key words: Wet macular degeneration/therapy; Radiotherapy; Angiogenesis inhibitors; Review

Abstract 5215: Time dependent blood-brain barrier modulation by vascular endothelial growth factor to improve CNS drug delivery

Abstract Introduction: Efforts to transiently increase blood brain barrier permeability would facilitate improved CNS delivery of chemotherapy agents. Previous studies demonstrated that vascular endothelial factor (VEGF) increases permeability in traumatic brain injury and ischemic brain disease. Exogenously administered VEGF increases barrier permeability in the non-diseased CNS. In our study, we investigated the role for exogenous VEGF therapy to modulate blood-brain barrier permeability Methods: For in vitro studies, confluent mouse brain endothelial cells (BEND.3) were treated with VEGF165. Trans-endothelial electrical resistance measured cell impedance changes and immunofluorescence was used to assess qualitative expression of tight and adherens junction proteins at key time points. Western blotting and qPCR were done to evaluate the effect of VEGF on expression of protein and RNA associated with tight and adherens junctions. Results: Confluent endothelial cells treated with rVEGF165 (200ng/mL) evaluated by TEER show a significant drop in cell impedance approximately 6 hours post treatment, compared to control (more than 10-fold) reflecting increased permeability of the monolayer. Treatment with progressively higher doses of VEGF (200, 400, and 600ng/mL) showed a progressively greater drop in cell impedance. Immunofluorescence of endothelial cells treated with rVEGF165 (200ng/mL) shows disassembly of both tight junctions and adherens junctions between 4 and 8 hours post-treatment compared to controls. Western blots and qPCR demonstrate that rVEGF165 treatment induces down-regulation of genes associated with tight junctions, such as Claudin5, beginning as early as 4 hours post treatment and continuing up to 24 hours post treatment. Conclusion: Our results suggest that VEGF is capable of inducing transient breakdown of the blood-brain barrier, which may facilitate improved CNS delivery of chemotherapeutic agents. Our in vitro data warrants further investigations of the applicability of VEGF therapy to induce endothelial cell permeability in animal tumor models, potentially improving delivery of chemotherapeutic drugs. Citation Format: Matthew McCord. Time dependent blood-brain barrier modulation by vascular endothelial growth factor to improve CNS drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2017-5215

Neutrophil lymphocyte ratio (NLR) as a clinical biomarker predictive of outcomes with immune checkpoint inhibitor therapy in genitourinary cancers.

453 Background: Immune checkpoint inhibitors (ICI) have been approved in genitourinary cancers (GU) such as renal cell carcinoma (RCC) and urothelial carcinoma (UC). There is an unmet need to determine factors predictive of response, to guide therapeutic selection in these cancers. We evaluated NLR as a predictor of response, progression free survival (PFS), and overall survival (OS) in patients treated with ICI. Other known prognostic clinical factors assessed were age, race, and smoking status and for RCC the prognostic score per MSKCC (Memorial Sloan Kettering) and Heng criteria. Methods: Regulatory approval was obtained. A retrospective chart review of RCC and URC patients at Karmanos Cancer Institute, treated with ICI based therapy was conducted. Data was collected on demographics, smoking status, prognostic scoring, NLR pretherapy, and post 4 doses of ICI. Correlation with clinical PFS and OS was conducted by univariable and multivariable analyses. Log-rank test was used to compare PFS and OS. Results: 57 pts were evaluated with median age 62 yrs (range, 24-85). 11 (19%) were African American (AA) and 31 (54%) were smokers. Pretherapy NLR&lt;4 and ≥4 was seen in 38 (67%) and 19 (33%) pts respectively. 13 (31%) RCC pts were treated with &gt; 1 VEGF therapy and 24(57%) pt were treated for &gt; 6 mths and 14/15 UC pts were pretreated. RCC pts treated with &gt; 6 mths and &gt; 12 mths of VEGF therapy had a shorter PFS (HR =2.31, p= 0.028; HR = 2.075, p= 0.051 respectively). AA had shorter PFS and OS with ICI in RCC but not in UC (HR=3.72, p=0.001; HR= 40.8; p=0.001; HR=0.49, p=0.5; HR=0.85, p=0.88, respectively). Conclusions: Pretherapy NLR ≥4 was a statistically significant predictor of shorter PFS and OS with ICI therapy in RCC. NLR is an easily applicable clinical predictive factor that can help guide therapy, after validation of these findings in a larger population dataset. [Table: see text]

Clinical analysis of the interaction and synergistic effect of anti VEGF therapy with retinal photocoagulation

Clinical analysis of the interaction and synergistic effect of anti VEGF therapy with retinal photocoagulation

Anti-Vegf in Management of Macular Edema in Retinal Disease

Abstract Aim. To present new opportunities, clinical implications and benefits of the available VEGF therapy as a treatment of macular edema, which is a result of venous vascular occlusions, diabetic macular edema in diabetic retinopathy and age-related macular degeneration. Background. The pathophysiology of macular edema is complex and various processes are involved in its development. It is actually an abnormal retinal capillary permeability and a disorder in the blood retinal barrier, which only increases the vascular permeability. This causes an expansion of the extracellular spaces, which leads to fluid accumulation, which additionally leads to macular thickening and eventual vision loss. Methods. The studies included 40 patients, of whom17 was diagnosed with macular edema in diabetic retinopathy and were treated with anti-VEGF therapy. Also, there were 11 patients diagnosed with wet form of AMD, and 12 cases diagnosed with macular edema secondary to vein occlusion. This retrospective study of 18 months monitored the effects of visual acuity on Snellen chart and the effects of macula anatomy using Optical Coherent tomography /OCT/. All patients received intravitreal injection of Bevacizumab /Avastin/ of 1.25mg /0.04ml/ and were evaluated monthly or every 4 to 8weeks. We monitored the potential ocular and systematic side effects in all our cases. Results. In the first group which included patients with edema due to venous vascular occlusion improvement of visual acuity in 58.33% patients, 25.0% showed no change in visual acuity and 16.66% showed slight worsening of 0.029 and regression of CMT entirely to 393.22 after 4.6 intravitreal injections on average. In the second group there was no improvement of VA 0.172 and reducing central macular thickness for 218.34μm by 5.6 intravitreal applications. The third group, 17 patients with macular edema due to diabetic retinopathy had stabilization of visual acuity, i.e. slight improvement in 8 of them by 0.14; and, in 9 and improvement of 0.21 and regression CMT, an average of 174.3 μm. Although it has been shown that benefit of intravitreal use of Bevacizumab and improvement of visual acuity has not been always change hand in hand with the reduction of macular edema, the need for this kind of treatment in certain cases are needed to maintain stable CMT and VA in such patients. Conclusion. Over the last few years monoclonal antibodies have become a standard therapy in treatment of wet form of AMD. Switch on anti-VEGF drugs has shown significant results in clinical and visual out-comes in patients with changes of the macula as a result of other disease. In fact, they caused a revolution in the treatment of refractory macular edema.

MOLECULAR MARKERS OF ANGIOGENESIS AS A BASIS FOR THE USE OF VEGF BLOCKADE IN PATIENTS WITH LOCALIZED EWING’S SARCOMA

Background . Chemoresistance to modern basic regimens of therapy is observed in nearly 30–35% of patients with localized (non-metastatic) Ewing’s sarcoma. Earlier we have established the possibility to predict poor outcomes basing on mRNA expression levels of both TFPI2 (tissue factor pathway inhibitor) and VEGFA165/VEGFA189 (vascular endothelial growth factor isoforms) ratio in pretreatment tumor tissue. Objektive of the study was to administer anti VEGF therapy for treatment enhancement in patients with poor prognosis based on angiogenesis markers. Мethods . The study enrolled 123 patients with localized ES (29 were included in the main group, 94 — in control group). Angiogenesis markers were determined in the patients of the main group at the stage of initial diagnosis; when the patients with poor prognosis were revealed the standard therapeutic plan was strengthened through bevacizumab (VEGF inhibitor). Results . 5-year event-free survival rate (82.8% vs 60.1%; p<0.05), overall survival rate (84.4% vs 64.8%; p<0.05), and cumulative incidence of recurrence (13.8% vs 37.1%; p<0.05) were better for the main group compared with the patients to whom this technology has not been applied (control group). Conclusion . The individual use of VEGF blockade in cases with predicted chemoresistance (based on molecular angiogenesis markers) can improve 5-year survival rate in pediatric patients with non-metastatic Ewing’s sarcoma. Anti- angiogenic therapy may be effective in patients with poor prognosis (based on angiogenesis markers in pretreatment tumor tissue).

Abstract LB-347: Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Abstract OBJECTIVE: We aimed to enhance the efficacy of anti-VEGF therapy in glioblastoma (GBM) through additional inhibition of Angiopoietin-2 (Ang-2), a potential mediator of resistance to antiangiogenic therapy using VEGF inhibition. INTRODUCTION: Glioblastoma (GBM) is a uniformly lethal primary brain tumor affecting more than 12.000 patients every year in the US alone. The standard therapy regimen for this highly angiogenic tumor entity comprises maximal safe resection and chemoradiation with temozolomide. The addition of antiangiogenic (anti-VEGF) therapy to the standard of care regimen improved progression-free survival, but failed to improve overall survival of GBM patients. Preclinical and clinical data suggest that resistance to anti-VEGF therapy in GBM is mediated by Ang-2, making this pathway a potential target. EXPERIMENTAL DESIGN: We tested the effect of dual Ang-2/VEGF blockade with A2V on mouse survival using a syngeneic (Gl261) model and a human xenograft (MGG8) model, compared to anti-VEGF antibody therapy (B20). In addition, we used blood-based Gaussian Luciferase (GLUC) assays, immunohistochemistry and flow cytometry to measure changes in tumor growth, microvessel density (MVD), and immune microenvironment, respectively. RESULTS: Gl261 tumors have a highly abnormal tumor vasculature. In this model, treatment with A2V reduced MVD compared to B20. The decrease in MVD was due to a reduction in pericyte-low tumor vessels, while pericyte-high vessels were unaffected. These vascular changes were accompanied by reduced tumor burden and enhanced survival. Interestingly, in the MGG8 tumors, which have a vasculature similar to the normal brain, we detected no change in MVD after A2V treatment. Nevertheless, we found a reduced tumor burden and prolonged animal survival in the MGG8 model. Since vascular normalization may impact immune cell infiltration and function in tumors, we next evaluated these cell populations. We found that A2V therapy reduced pro-tumor M2 polarization of macrophages and microglia and reprogrammed these cells toward the M1 phenotype in both the Gl261 and MGG8 models. Collectively, our data indicate that therapy-induced anti-tumor immunity is mediated by M1-type macrophages but not by T-cell infiltration or function. CONCLUSION: Dual Ang-2/VEGF therapy with A2V reprogrammed macrophages and microglia from pro-tumor M2 toward the anti-tumor M1 phenotype in two GBM models, in addition to normalizing vasculature in tumors with abnormal vessels. These data indicate that dual anti-angiogenic therapy has the potential to overcome resistance to anti-VEGF therapy and confer clinical benefits in GBM patients through vascular and immuno-modulatory effects. Citation Format: Jonas Kloepper, Lars Riedemann, Zohreh Amoozgar, Giorgio Seano, Katharina H. Susek, Veronica Yu, Nisha Dalvie, Robin L. Amelung, Meenal Datta, Jonathan W. Song, Vasileios Askoxylakis, Jennie W. Taylor, Christine Lu-Emerson, Ana Batista, Nathaniel D. Kirkpatrick, Keehoon Jung, Matija Snuderl, Alona Muzikansky, Kay G. Stubenrauch, Oliver Krieter, Hiroaki Wakimoto, Lei Xu, Lance L. Munn, Dan G. Duda, Dai Fukumura, Tracy T. Batchelor, Rakesh K. Jain. Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-347.

Clinicopathologic characteristics and survival of patients with gynecologic malignancies metastatic to the brain

ObjectiveNo standardized treatment strategies exist for patients with gynecologic malignancies complicated by brain metastases. Identification of poor outcome characteristics, long-term survival indicators, and molecular markers could help individualize and optimize treatment. MethodsThis retrospective cohort study included 100 gynecologic cancer patients with brain metastases treated at our institution between January 1990 and June 2009. Primary outcome was overall survival (OS) from time of diagnosis of brain metastases. We used univariate and multivariate analyses to evaluate associations between OS and clinical factors. We used immunohistochemistry to examine expression of five molecular markers in primary tumors and brain metastases in a subset of patients and matched controls. Statistical tests included the Student's paired t-test (for marker expression) and Kaplan-Meier test (for correlations). ResultsOn univariate analysis, primary ovarian disease, CA-125<81units/mL at brain metastases diagnosis, and isolated versus multi-focal metastases were all associated with longer survival. Isolated brain metastasis remained the only significant predictor on multivariate analysis (HR 2.66; CI 1.19–5.93; p=0.017). Expression of vascular endothelial growth factor A (VEGF-A) was higher in metastatic brain samples than in primary tumors of controls (p<0.0001). None of the molecular markers were significantly associated with survival. ConclusionsMulti-modality therapy may lead to improved clinical outcomes, and VEGF therapy should be investigated in treatment of brain metastases.

Abstract B32: Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab

Abstract Despite significant success, the response of Her2+ patients to trastuzumab (TzMAb, Herceptin ®) is varied, with many still experiencing tumor progression. We have used 3D tissue, tumor xenograft and metastatic models to examine the microregional distribution of TzMAb when administered alone or in combination with bevacizumab (BvMAb). Methods: Her2 positive (Her2+) SKOV-3 ovarian and MDA361, JIMT-1, BT474 mammary cancer cells were grown as 3D tissue discs, spheroids and as xenografts. Her2 expression was ranked as SKOV3 &amp;gt; BT474 &amp;gt; JIMT-1 &amp;gt; MDA361. Variable concentrations (25-100 µg/mL for in vitro; 2.5-10 mg/kg ip q3d for in vivo) of TzMAb, BvMAb or isotype IgG control antibodies were administered and tissues collected. Multiplexed immunohistochemistry generated maps of whole tissue sections for quantitative and qualitative analysis. In addition to direct visualization of fluorescent-tagged antibody therapeutics, features including Her2 expression (Her2), hypoxia (pimonidazole), blood vessels (CD31), vascular perfusion (carbocyanine fluorescent dye), pericytes (SMA, desmin), basal lamina (CIV) and tight junctions (ZO-1) were mapped relative to each other. Findings: In vitro: The rate of TzMAb distribution through 3D Her2+ tissue models was not significantly different to that of BvMAb or an isotype control (IgG). Inter-model variability was not correlated with the degree of Her2 expression. In vivo: All the xenograft models exhibited highly heterogeneous distribution of TzMAb, with variation at inter-vessel, inter-tumor and intra-tumor levels. There was no discernible pattern in the deposition of TzMAb; it was not limited to tumor margins or the central core, and was often bound up to 200 µm away from the nearest blood vessels. Other areas containing perfused vessels had little to no binding. Areas of limited TzMAb binding persisted after repeat dosing. SKOV-3 and BT474 metastatic lesions collected from liver and lung tissues had on average greater TzMAb staining intensity than was seen in subcutaneous tumors. However, there was a significant range in the ability of TzMAb to access and bind Her2+ve cells. Lesions ranged from less than 150 µm to several mm in diameter, with some metastases showing bound TzMAb on every cell and others showing no observable bound drug despite containing perfused vessels or being surrounded by highly perfused normal lung or liver tissue. For both subcutaneous tumors and metastatic lesions, no consistent, quantifiable difference or correlation was found between the amount of bound TzMAb and the presence of tight junctions, the density of nearest CD31 vessels, the location of hypoxic cells or the fraction of perfused, mature or immature vessels. Pretreatment with BvMAb for 24-72h resulted in significant reductions (up to 90% reduced) in tumor accumulation of TzMAb in all tumor models examined, including lung and liver metastases, despite the continued presence of perfused vessels. Summary: The extravascular distribution of TzMAb in Her2-overexpressing xenografts and metastases is heterogeneous and is not explained simply by the presence or absence of functioning vasculature. The relatively good distribution of TzMAb through 3D tissue models suggest that tight or irreversible binding to Her2 may not adequately explain the limited distribution of TzMAb seen in vivo. Pre-treatment with BvMAb dramatically decreases the access of TzMAb to the tumor microenvironment. Persistence of metastases and tissues with poor access to TzMAb suggests the possibility of inadequate drug exposure as a mechanism for resistance to TzMAb activity that could apply to additional targeted monoclonal antibody therapeutics. This resistance may be exacerbated when TzMAb or other antibodies are administered in combination with anti VEGF therapy. Citation Format: Jennifer H.E. Baker, Alastair H. Kyle, Jordan Cran, Haley Patrick, Maria-Jose Gandolfo, Andrew I. Minchinton. Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B32.

Enhanced Anticancer Activity of PF-04691502, a Dual PI3K/mTOR Inhibitor, in Combination With VEGF siRNA Against Non-small-cell Lung Cancer.

Lung cancer is the leading cause of cancer deaths in both men and women in the United States accounting for about 27% of all cancer deceases. In our effort to develop newer therapy for lung cancer, we evaluated the combinatory antitumor effect of siRNA targeting VEGF and the PI3K/mTOR dual inhibitor PF-04691502. We analyzed the anticancer effect of siRNA VEGF and PF-04691502 combination on proliferation, colony formation and migration of A549 and H460 lung cancer cells. Additionally, we assessed the combination treatment antiangiogenic effect on human umbilical vein endothelial cells. Here, we show for the first time that the antiangiogenic siRNA VEGF potentiates the PF-04691502 anticancer activity against non–small-cell lung cancer. We observed a significant (P < 0.05) decrease in cell viability, colony formation, and migration for the combination comparing with the single drug treatment. We also showed a significant (P < 0.05) enhanced effect of the combination treatment inhibiting angiogenesis progression and tube formation organization compared to the single drug treatment groups. Our findings demonstrated an enhanced synergistic anticancer effect of siRNA VEGF and PF-04691502 combination therapy by targeting two main pathways involved in lung cancer cell survival and angiogenesis which will be useful for future preclinical studies and potentially for lung cancer patient management.

Abstract POSTER-THER-1406: Tissue factor-factor VIIa complex triggers vascular endothelial growth factor-a secretion in epithelial ovarian cancer cell lines via a protease activated receptor-2 dependent mechanism

Abstract Purpose of study: Epithelial ovarian cancer (EOC) is highly sensitive to initial platinum-based chemotherapy, but ultimately re-growth of the tumor occurs in the majority of patients with development of platinum resistance. Identification of: 1) the major mechanisms leading to regrowth, and 2) potential drug targets for maintenance therapy to prevent regrowth/relapse of EOC would potentially have a dramatic impact on patient outcomes. Progression of EOC is often associated with a procoagulant phenotype, characterized by increased tissue factor (TF) expression and enhanced metastasis. TF-factor VIIa (FVIIa) complex on the cell surface activates protease activated receptor-2 (PAR-2) directly, and PAR-1 activation indirectly via triggering local thrombin generation. PAR-2 activation is associated with an induction in growth factor release in renal cell carcinoma. We hypothesize that TF-FVIIa dependent activation of PAR-2 on EOC cells up-regulates expression of angiogenic mediators (VEGF-A) that modify the peritoneal microenvironment, enhancing vascular leakage and ascites formation. Incorporation of anti–VEGF therapy in standard treatment of EOC has been shown to prolong progression-free survival, but the underlying mechanisms are incompletely understood. The objective of this study is to determine the role TF-FVIIa-PAR-2 axis plays in EOC progression. Methods: TF antigen expression in EOC cell lines (CaOV-3, SKOV-3 &amp; OvCAR-3) was determined by western blot (WB) analysis. TF-bearing microparticles were isolated from conditioned media by differential centrifugation (20,800 g) and TF coagulant activity was detected by factor X activation. VEGF-A levels in conditioned media were determined by ELISA. Proliferation of the SKOV-3 and OVCAR-3 cell lines in response to thrombin (PAR-1 agonist) was determined via direct cell count. Results: WB analysis of EOC cell line lysates demonstrated PAR-2 expression (CaOV-3&amp;gt;OvCAR-3&amp;gt;&amp;gt;SKOV-3), and exposure of SKOV-3 and OVCAR-3 cells to 50 nM FVIIa resulted in 3-fold induction of VEGF-A release over 48 hr relative to untreated cells (p&amp;lt;0.05). TF-FVIIa complex was a more potent inducer of VEGF-A when compared to the PAR-2 peptide agonist (SKIGKL-NH2). Pre-incubation with the PAR-2 antagonist ENMD-1068 reduced VEGF-A to basal levels in the presence of TF-FVIIa; further supporting PAR-2 dependent induction of VEGF-A. Pan-expression of PAR-1 was observed in EOC cell lines by WB. Thrombin (0.1-1U/mL), an established PAR-1 agonist, induced dose-dependent proliferation of SKOV-3 and OVCAR-3 cell lines under serum free conditions (p&amp;lt;0.001). Pre-incubation with the PAR-1 antagonist vorapaxar reduced proliferation to basal levels in the presence of thrombin. Conclusion: TF expression and coagulant activity is up regulated in EOC. FVIIa triggers a PAR-2 dependent induction of VEGF-A release from EOC cell lines. Similarly, the PAR-1 agonist thrombin triggered a significant increase in EOC cell proliferation. These results suggest that interaction of EOC with the coagulation system in the tumor microenvironment may enhance both PAR-1 dependent cellular proliferation and PAR-2 dependent release of VEGF, a potent regulator of angiogenesis. Thus, PAR-1 and PAR-2 may represent pharmacologically relevant drug targets to block ovarian cancer progression via inhibition of growth and angiogenesis. Citation Format: Alice Chanakira PhD, John P. Sheehan MD. Tissue factor-factor VIIa complex triggers vascular endothelial growth factor-a secretion in epithelial ovarian cancer cell lines via a protease activated receptor-2 dependent mechanism [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1406.

Vascular Endothelial Growth Factor Enhanced the Angiogenesis Response of Human Umbilical Cord-Derived Mesenchymal Stromal Cells in a Rat Model of Radiation Myelopathy.

The present study was designed to examine a synergistic role for VEGF and human umbilical cord-derived MSCs (UC-MSCs) therapy in a rat model of radiation myelopathy. UC-MSCs and VEGF were injected through the tail vein at 90, 97, 104 and 111days post-irradiation. Behavioral tests were performed, and histological damage was examined. The microcirculation in the spinal cord was assessed using von Willebrand factor immunohistochemical analysis and laser-Doppler flowmetry. The microenvironment in the spinal cord was determined by measuring the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in the serum and the anti-inflammatory cytokines brain-derived neurotrophic factor and glial cell-derived neurotrophic factor in the spinal cord. The UC-MSCs processed with VEGF, including VEGF165-induced UC-MSC (iUC), VEGF and UC-MSCs (VEGF-UC), increased the endothelial cell density and the microvessel density in the white matter and gray matter of the spinal cord, raised the relative magnitude of spinal cord blood flow compared to UC-MSCs treatment alone. Our data provided the first evidence that vascular endothelial growth factor enhanced the angiogenesis response of human umbilical cord-derived mesenchymal stromal cells in a rat model of radiation myelopathy.

Angiotensin system inhibitors and survival outcomes in patients with metastatic renal cell carcinoma.

The renin-angiotensin system may play a role in carcinogenesis. The purpose of this study was to evaluate the impact of angiotensin system inhibitors (ASI) on outcomes in metastatic renal cell carcinoma (mRCC) patients treated in the targeted therapy era. We conducted a pooled analysis of mRCC patients treated on phase II and III clinical trials. Statistical analyses were performed using Cox regression adjusted for several risk factors and the Kaplan-Meier method. A total of 4,736 patients were included, of whom 1,487 received ASIs and 783 received other antihypertensive agents. Overall, ASI users demonstrated improved overall survival (OS) compared with users of other antihypertensive agents (adjusted HR, 0.838, P = 0.0105, 26.68 vs. 18.07 months) and individuals receiving no antihypertensive therapy (adjusted HR, 0.810, P = 0.0026, 26.68 vs. 16.72 months). When stratified by therapy type, a benefit in OS was demonstrated in ASI users compared with nonusers in individuals receiving VEGF therapy (adjusted HR, 0.737, P < 0.0001, 31.12 vs. 21.94 months) but not temsirolimus or IFNα. An in vitro cell viability assay demonstrated that sunitinib in combination with an ASI significantly decreased RCC cell viability compared with control at physiologically relevant doses. This effect was not observed with either agent alone or with other non-ASI antihypertensives or temsirolimus. In the largest analysis to date, we demonstrate that ASI use improved survival in mRCC patients treated in the targeted therapy era. Further studies are warranted to investigate the mechanism underlying this interaction and verify our observations to inform clinical practice.

Phase II trial of dovitinib in recurrent glioblastoma.

2050 Background: Glioblastoma (GBM) is a vascular tumor and bevacizumab (a monoclonal antibody against VEGF-A) is FDA-approved in recurrence. Mechanisms of resistance to anti VEGF therapy include u...

Incidence of endophthalmitis after intravitreal application of anti VEGF therapy at the University Clinical Center in Tuzla, Bosnia and Herzegovina.

To report the incidence of endophthalmitis following the use of intravitreal injection of anti- vascular endothelial growth factor (anti VEGF) therapy. In this retrospective study a total of 986 intravitreal bevacizumab injections were applied between January 2008 and April 2015 at the University Clinical Center Tuzla, Bosnia and Herzegovina (BiH). Since January 2012, a total of 55 intravitreal ranibizumab injections were applied and since October 2014, 60 intravitreal aflibercept injections were applied to patients. Two cases of endophthalmitis following intravitreal injection of bevaciuzumab occurred and none after ranibizumab or aflibercept. The overall incidence of clinical endophtahlmitis was 0.2%. The results suggest that a low rate of endophthalmitis can be achieved by means of a protocol. This is a very important study as it is the first of this kind in BiH that documents the incidence of endophthalmitis after intravitreal application. Currently, bevacizumab in BiH is most frequently used intravitreal anti-vascular endothelial growth factor due to very low price.

AT-04 * A RANDOMIZED PHASE II TRIAL OF VANDETANIB IN COMBINATION WITH CARBOPLATIN VERSUS CARBOPLATIN ALONE FOLLOWED BY VANDETANIB ALONE IN ADULTS WITH RECURRENT ANAPLASTIC ASTROCYTOMA

BACKGROUND: Vandetanib is a selective VEGFR and EGFR inhibitor that targets these key pathways implicated in gliomagenesis. While single-agent vandetanib has not shown significant clinical activity in high grade glioma but preclinical experience supports potential synergistic efficacy in combination with carboplatin. METHODS: This randomized non-comparative phase-II trial evaluates the efficacy and safety of vandetanib (300 mg/d NEIAED, 400 mg/d EIAED) in combination with carboplatin (AUC 6 q 4 weeks) versus carboplatin alone (AUC 6 q 4 weeks) in adults with recurrent anaplastic glioma. Upon progression with single-agent carboplatin, single-agent vandetanib was offered. Contrast-enhanced-MRI before subsequent cycles were compared to baseline and response assessed using RANO criteria. Primary end-point was progression free survival at 6 months (PFS-6). RESULTS: 23 patients were accrued to each group (n = 46). Gender, median age, performance status, number of recurrences, 1p/19q co-deletion status, and baseline steroid doses were comparable. 60% of patients in each group had failed prior VEGF-inhibitor therapy. Most frequent treatment related grade ≥3 toxicities were lymphopenia (n = 7), thrombocytopenia (n = 4), neutropenia (n = 3), hypertension (n = 3) and transaminitis (n = 3). All grade 4 toxicities were observed in the combination-group. There were 3 partial responses in the combination group, and 1PR in the sequential-group. PFS-6 was 8.7% (95% CI: 2.3%, 32.7%) and 17.4 % (95% CI: 7.1%, 42.4%), median-PFS (months) was 1.8 (95% CI: 1.15, 2.66) and 0.95 (95% CI:0.92,2.04)while median-OS (months) was 6.5 (95% CI: 5.42, 11.7) and 9.27 (95% CI: 5.52, 43.86) in combination-group and sequential-group respectively. CONCLUSION: Although this study was not powered to detect differences between groups and did not complete accrual as planned, no meaningful differences in PFS-6, PFS or OS were observed compared to historical controls. Majority of patients were VEGF therapy resistant, which may have limited efficacy. Combination therapy was more toxic.