High VEGF Research Articles

Bidirectional crosstalk between periventricular endothelial cells and neural progenitor cells promotes the formation of a neurovascular unit

Interactions between neural progenitor cells (NPC) and endothelial cells (EC) from adult vascular beds have been well explored previously. However, the factors and signaling mechanisms that regulate neurogenesis and angiogenesis are most prevalent during embryonic development. This study aimed to determine whether embryonic brain endothelial cells from the periventricular region (PVEC) present an advantage over adult brain EC in supporting NPC growth and differentiation. PVEC were isolated from E15 mouse brains, processed, and sorted with immunomagnetic beads using antibodies against CD31/PECAM. On immunofluorescence (IF) staining, nearly all cells were positive for EC markers CD31 and CD144/VE-Cadherin. In proliferation studies, NPC proliferation was highest in transwell co-culture with PVEC, approximately 2.3 fold increase compared to baseline versus 1.4 fold increase when co-cultured with adult brain endothelial cells (ABEC). These results correlated with the PVEC mediated delay in NPC differentiation, evidenced by high expression of progenitor marker Nestin evaluated by IF staining. Upon further characterization of PVEC in an angiogenesis assay measuring cord length, PVEC exhibited a high capacity to form cords in basal conditions compared to ABEC. This was enhanced in the presence of NPC, with both cell types displaying a preferential structural alignment resembling neurovascular networks. PVEC also expressed high Vegfa levels at baseline in comparison to NPC and ABEC. Vegfa levels increased when co-cultured with NPC. We demonstrate that PVEC and NPC co-cultures act synergistically to promote the formation of a neurovascular unit through dynamic and reciprocal communication. Our results suggest that PVEC/NPC could provide promising neuro-regenerative therapies for patients suffering brain injuries.

The role of differential VE-cadherin dynamics in cell rearrangement during angiogenesis

Endothelial cells show surprising cell rearrangement behaviour during angiogenic sprouting; however, the underlying mechanisms and functional importance remain unclear. By combining computational modelling with experimentation, we identify that Notch/VEGFR-regulated differential dynamics of VE-cadherin junctions drive functional endothelial cell rearrangements during sprouting. We propose that continual flux in Notch signalling levels in individual cells results in differential VE-cadherin turnover and junctional-cortex protrusions, which powers differential cell movement. In cultured endothelial cells, Notch signalling quantitatively reduced junctional VE-cadherin mobility. In simulations, only differential adhesion dynamics generated long-range position changes, required for tip cell competition and stalk cell intercalation. Simulation and quantitative image analysis on VE-cadherin junctional patterning in vivo identified that differential VE-cadherin mobility is lost under pathological high VEGF conditions, in retinopathy and tumour vessels. Our results provide a mechanistic concept for how cells rearrange during normal sprouting and how rearrangement switches to generate abnormal vessels in pathologies.

Granulocyte/Macrophage Colony-Stimulating Factor Influences Angiogenesis by Regulating the Coordinated Expression of VEGF and the Ang/Tie System

Granulocyte/macrophage colony-stimulating factor (GM-CSF) can accelerate wound healing by promoting angiogenesis. The biological effects of GM-CSF in angiogenesis and the corresponding underlying molecular mechanisms, including in the early stages of primitive endothelial tubule formation and the later stages of new vessel maturation, have only been partially clarified. This study aimed to investigate the effects of GM-CSF on angiogenesis and its regulatory mechanisms. Employing a self-controlled model (Sprague-Dawley rats with deep partial-thickness burn wounds), we determined that GM-CSF can increase VEGF expression and decrease the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 in the early stages of the wound healing process, which promotes the degradation of the basement membrane and the proliferation of endothelial cells. At later stages of wound healing, GM-CSF can increase the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 and maintain a high VEGF expression level. Consequently, pericyte coverages were higher, and the basement membrane became more integrated in new blood vessels, which enhanced the barrier function of blood vessels. In summary, we report here that increased angiogenesis is associated with GM-CSF treatment, and we indicate that VEGF and the Ang/Tie system may act as angiogenic mediators of the healing effect of GM-CSF on burn wounds.

High Serum Levels of Vascular Endothelial Growth Factor-A and Transforming Growth Factor-β1 Before Neoadjuvant Chemoradiotherapy Predict Poor Outcomes in Patients with Esophageal Squamous Cell Carcinoma Receiving Combined Modality Therapy

This study was aimed at using proximity ligation assay (PLA) followed by enzyme-linked immunosorbent assay (ELISA) to identify serum biomarkers that predict treatment response and survival for patients with esophageal squamous cell carcinoma (ESCC) undergoing neoadjuvant concurrent chemoradiotherapy (CCRT) followed by esophagectomy. Seventy-nine patients with ESCC receiving CCRT of taxane-based/5-fluorouracil-based chemotherapy and 40 Gy followed by surgery were enrolled. Serum samples were collected before and <1 month after CCRT. Fifteen biomarkers were analyzed using PLA. Biomarkers significantly correlating with pathological response/survival were verified by ELISA. Associations of the serum level of biomarkers and clinical factors with pathological response, disease-free survival (DFS), and overall survival (OS) were evaluated by analysis of variance and log-rank tests. Thirty patients had complete response (38 %), 37 had microscopic residual disease (47 %), and 12 had macroscopic residual disease (15 %). With a median follow-up of 52.8 months, the median DFS was 43 months. Among the 15 biomarkers screened by PLA, vascular endothelial growth factor (VEGF)-A and transforming growth factor (TGF)-β1 were significantly associated with pathological response and/or DFS. These biomarkers were further analyzed by ELISA to confirm initial biomarker findings by PLA. After ELISA of these two markers, only VEGF-A levels were significantly correlated with pathological response. On multivariate analysis, patients with combined high pre-CCRT VEGF-A and TGF-β1 levels (greater than or equal to the median), independent of pathological response, had significantly worse DFS (11 months vs. median not reached; p = 0.007) and OS (16 vs. 46 months; p = 0.07). Pre-CCRT serum VEGF-A and TGF-β1 levels may be used to predict pathological response and survivals for ESCC patients receiving combined-modality therapy.

Assessment of IL-6, IL-8, bFGF, PDGF-BB, and VEGF-A as prognostic and predictive biomarkers for anti-VEGF in metastatic colorectal cancer (mCRC).

502 Background: There remains a need for predictive biomarkers in mCRC to better identify patients who clinically benefit from anti-VEGF therapy, in particular bevacizumab (BEV). The AGITG MAX trial compared capecitabine (C) with capecitabine (+/- mitomycin C (M)) and BEV and PFS was superior in the CB+/-M arm. Here we have taken a panel of pro-angiogenic proteins to assess whether there is a signal for a predictive factor for bevacizumab outcome in the MAX trial tissue population (TTP). Methods: Protein was isolated from FFPE tumour tissue and assayed using custom Bio-plex arrays to assess the expression levels of candidate tumour biomarkers IL-6, IL-8, bFGF, PDGF-BB and VEGF-A. As there are no accepted cutoff points for these analytes we chose the medians of distribution of each of these biomarkers as cutoff points for dichotomization into “high” v “low”. We then correlated levels of the biomarkers with objective response rate, disease-free and overall survival. Results: Total protein was isolated from FFPE tumour sections from 41.5% of patients (n=196) recruited into the MAX clinical trial and assayed for the proteins using a multiplex platform. Patients were generally balanced, tumour tissue population v intention to treat. Median follow up time is 30.2 mths. For patients receiving BEV there was no relationship between the level of IL-6, IL-8, bFGF, PDGF-BB and VEGF-A and PFS and OS. In patients treated with BEV/C vs. C alone, low tumor VEGF-A was predictive of better ORR as compared to those with a high level (ORR 53% vs 38%, p = 0.03). Multivariate analyses showed low VEGF-A expression level was a significant prognostic factor for longer progression free survival (median PFS 9.3 months low VEGF-A v 7.2 months high VEGF-A, multivariate adjusted HR 1.55 (95% CI 1.12-2.13), p = 0.008). Conclusions: We have shown that the chosen panel of pro-angiogenic proteins was not predictive for PFS or OS outcomes when B is added to C in mCRC. However low VEGF-A level did predict for higher ORR and was an independent prognostic marker for PFS in this cohort.

Abstract P6-05-17: A study of investigating biologic markers of anti-tumor effects of zoledronic acid and taxane-based chemotherapy for metastatic breast cancer in bone: A prospective, multi-center, non-randomized study (BEAT-ZO) (KCSG BR10-13)

Abstract Introduction Currently the predictive factors for taxane(T)-zoledronic acid(ZA) combination therapy in breast cancer patient with bone metastasis have not been established except tumor biology. The aim of this study is to investigate potential biologic markers of anti-tumor effects of and T-ZA for metastatic breast cancer(MBC) in bone. Methods Patients(pts) with MBC in bone being treated with docetaxel or paclitaxel based chemotherapy and ZA for the first time in metastatic setting were enrolled. Blood samples were collected serially at baseline, after 2 cycles to examine markers for angiogenesis(VEGF, VEGFR2, FGF-2, PDGF-AA), immune modulation (IL-2, IFN-γ, MCP-2, IL-10, TGF-β, IL-12, TNF-α, IL-17, IL-6) and apoptosis (TRAIL). Results Of enrolled total 58 pts, 31 pts (median age 49; ECOG 0-1 96.8%; menopause 58.1%; invasive ductal carcinoma 92.9%; ER-(+) 77.4%; HER2-(+) 35.5%; visceral metastasis 35.5%) were included in this preliminary analysis. Fifteen pts received docetaxel-based chemotherapy and the remainder were treated with paclitaxel-based chemotherapy. Median 6 (range: 1 – 23) cycles per pt were administered. In per-protocol analysis, overall RR was 55.6% [95% CI: 37.3 – 72.4]. After the median follow-up of 13.67 months(mo.), median PFS was 9.13 mths [95% CI: 3.25 – 15.02]. Osteonecrosis of the jaw was reported in only one patient (3.2%). In the baseline biomarker analysis, the pts with triple-negative breast cancer (TNBC) showed significantly higher VEGF level than hormone (+) or HER-2 (+) pts (518.7 vs 151.6 and 179.2 pg/ml, p = 0.041). Median baseline TRAIL was significantly higher in the postmenopausal women than the premenopausal women (52.0 vs 32.0 pg/ml, p = 0.038). For the group as a whole, there was a borderline significant reduction in median serum MCP-2 level (41.4 to 34.1 pg/ml, p = 0.066) and an increasing tendency in median serum TRAIL level (44.7 to 54.5 pg/ml, p = 0.080) after 2 cycles of treatment. Median percentage reduction in serum VEGF in the TNBC group was -50.0% compared with +37.7% in others (p = 0.099). Median changes in MCP-2 was -36.4% in hormone (+) group compared with +7.6% in others (p = 0.008). The pts who were progression free at 6 mths showed significant increase in median TNF-α after 2cycles of treatment, while the pts who experienced disease progression within 6 mths showed significant decrease in TNF-α level (p = 0.028) and there was a similar tendency in TRAIL level (p = 0.157). The pts with increase of serum TNF-α or TRAIL levels from baseline showed significant improvement of PFS comparing the pts with no change or decrease of TNF-α and TRAIL levels (13.3 vs 5.93 mths, p = 0.012). We are planning to perform additional analysis. The significance of serum TGF-β level on prognosis and the data of the remainder will be presented on the poster. Conclusion In this study, baseline levels and changes of biomarkers suggest potentially relevant interactions between menopausal status, tumor biology and treatment. Especially, TNF-α and TRAIL may be potential early marker for zoledronic acid and taxane-based chemotherapy for MBC in bone. Larger studies are needed to validate these complex interactions. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-17.

Clinical Implications of VEGF, TGF-beta1, and IL-1beta in Patients with Advanced Non-small Cell Lung Cancer

PurposeVascular endothelial growth factor (VEGF)-A, VEGF165b, interleukin (IL)-1β, and transforming growth factor (TGF)-β1 are known to influence tumor angiogenesis. Clinical implications of these cytokines need to be elucidated.Materials and MethodsUsing clinical data and baseline serum samples of 140 consecutive patients with advanced non-small cell lung cancer who received platinum-based combination chemotherapy, we investigated the association among serum cytokine levels, treatment outcomes, as well as leukocyte and platelet counts.ResultsThe median age of patients was 64 years (range, 26 to 86 years). The male to female ratio was 104:36. High TGF-β1 and IL-1β levels were associated with shorter progression-free survival, and high VEGF-A and IL-1β levels were associated with shorter overall survival in the univariate analysis. VEGF165b was not related to the treatment outcomes. Leukocytosis and thrombocytosis were associated with shorter overall survival. The multivariate analysis demonstrated that VEGF-A, IL-1β, and leukocytosis were significant prognostic factors (p=0.0497, p=0.047, and p<0.001, respectively). Leukocytosis was not associated with recent pneumonia (p=0.937) and correlated with VEGF-A (p<0.001) and TGF-β1 (p=0.020) levels.ConclusionSerum VEGF-A, TGF-1β, and IL-1β levels, in addition to leukocyte and platelet counts, are shown to be associated with clinical outcomes. Leukocyte and platelet counts are correlated with serum VEGF-A and TGF-β1 levels.

Spinal but not cortical microglia acquire an atypical phenotype with high VEGF, galectin-3 and osteopontin, and blunted inflammatory responses in ALS rats

Activation of microglia, CNS resident immune cells, is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting motor neurons. Despite evidence that microglia contribute to disease progression, the exact role of these cells in ALS pathology remains unknown. We immunomagnetically isolated microglia from different CNS regions of SOD1G93A rats at three different points in disease progression: presymptomatic, symptom onset and end-stage. We observed no differences in microglial number or phenotype in presymptomatic rats compared to wild-type controls. Although after disease onset there was no macrophage infiltration, there were significant increases in microglial numbers in the spinal cord, but not cortex. At disease end-stage, microglia were characterized by high expression of galectin-3, osteopontin and VEGF, and concomitant downregulated expression of TNFα, IL-6, BDNF and arginase-1. Flow cytometry revealed the presence of at least two phenotypically distinct microglial populations in the spinal cord. Immunohistochemistry showed that galectin-3/osteopontin positive microglia were restricted to the ventral horns of the spinal cord, regions with severe motor neuron degeneration. End-stage SOD1G93A microglia from the cortex, a less affected region, displayed similar gene expression profiles to microglia from wild-type rats, and displayed normal responses to systemic inflammation induced by LPS. On the other hand, end-stage SOD1G93A spinal microglia had blunted responses to systemic LPS suggesting that in addition to their phenotypic changes, they may also be functionally impaired. Thus, after disease onset, microglia acquired unique characteristics that do not conform to typical M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. This transformation was observed only in the most affected CNS regions, suggesting that overexpression of mutated hSOD1 is not sufficient to trigger these changes in microglia. These novel observations suggest that microglial regional and phenotypic heterogeneity may be an important consideration when designing new therapeutic strategies targeting microglia and neuroinflammation in ALS.

Prognostic Implication Of Circulating Vascular Endothelial Growth Factor In Patients With Diffuse Large B-Cell Lymphoma

BackgroundVascular endothelial growth factor (VEGF) plays an important role in angiogenesis and progression of cancer, and blood level of VEGF has been known to predict of outcome in several types of cancer. However, the impact of blood VEGF levels on prognosis of diffuse large B-cell lymphoma (DLBCL) has not been fully elucidated. Here we investigated the prognostic implication of circulating VEGF levels in patients with DLBCL. Patients and MethodsThe study involved 127 DLBCL patients treated by rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) at National Cancer Center, Korea. Both serum and plasma of the patients were obtained at diagnosis. VEGF were measured using ELISA kit (R&D systems) according to manufacturer's guidance. We investigated the correlation between clinical parameters and blood VEGF levels. Survival rates and hazard ratios (HRs) in terms of risk for overall survival were determined using Kaplan-Meier method and Cox proportional hazard regression analysis. ResultsThe median patient age was 56 years, and 75 (59%) patients were men. Clinical characteristics and international prognostic index (IPI), including age, performance, lactate dehydrogenase (LDH), Ann Arbor stage and extranodal involvement were evaluated. The mean (±SD) value of serum and plasma VEGFs were 713 (±599.8) and 107 (±164.4) pg/mL, and they were correlated as r=0.61 (p<0.001). Both serum and plasma VEGF showed the significant (p<0.01) correlation with serum LDH level, Ann Arbor stage and multiple extranodal involvement, but not with age, gender nor performance. IPI showed strong prediction for prognosis in our data set (HR 3.80, 95%CI 1.64-8.81, p=0.02), and the VEGF levels of high/high-intermediate IPI group were significantly higher than those of low/low-intermediate group [serum VEGF 1088(±838.0) and 557(±377.2), p<0.001 and plasma VEGF 79(±131.6) and 176(±212.2), p=0.002]. With the median follow-up of 44 months, high serum VEGF levels (higher than the median) were significantly associated with short survival (HR 2.74, 95%CI 1.13-6.60, p=0.025), though the plasma VEGF levels did not show the association similar to the serum samples (HR 1.40, 95%CI 0.63-3.12, p=0.414). The patients with higher serum VEGF than the median value showed significantly lower survival rate compared to the low group (3-year survival rates, 68.6% vs. 85.6%, p=0.019). ConclusionThese findings suggest that high serum VEGF levels can predict poor clinical outcome in patients with DLBCL. This study also represents the different type of specimen for VEGF measurement affected the results. We might have to select adequate specimen type for VEGF measurement, although further validation in large cohort and mechanism studies for this data are warranted. Disclosures:No relevant conflicts of interest to declare.

Efficacy and biomarker findings from AVaglio, a phase III trial of bevacizumab plus temozolomide and radiotherapy in newly diagnosed glioblastoma

WCN 2013 No: 1207 Topic: 36 — Other topic Efficacy and biomarker findings from AVaglio, a phase III trial of bevacizumab plus temozolomide and radiotherapy in newly diagnosed glioblastoma W. Wick, T. Cloughesy, W. Mason, R. Henriksson, F. Saran, R. Nishikawa, M. Hilton, J. Garcia, C. Pallaud, O. Chinot. University Medical Centre, Heidelberg, Germany; University of California, Los Angeles, CA, USA; Princess Margaret Hospital, University of Toronto, Toronto, ON, Canada; Regional Cancer Center Stockholm, Stockholm, Sweden; Umea University, Umea, Sweden; The Royal Marsden NHS Foundation Trust, Surrey, UK; International Medical Center, Saitama Medical University, Saitama, Japan; F. Hoffmann-La Roche, Basel, Switzerland; Aix-Marseille University, AP-HM, Service de Neuro-Oncologie, CHU Timone, Marseille, France Background: Glioblastoma has a high disease burden and poor prognosis. AVAgliowas the first double-blind, placebo-controlled phase III study evaluating bevacizumab in newly diagnosed glioblastoma. Objectives: To evaluate efficacy, safety and potential biomarkers (plasma VEGF-A/VEGFR-2 prioritised). Patients and methods: Patients received single-agent bevacizumab or placebo plus standard-of-care treatment (radiotherapy plus temozolomide) until progression/unacceptable toxicity. Co-primary endpoints were investigator-assessed PFS and OS. Secondary endpoints included health-related quality of life (HRQoL). Exploratory endpoints included correlative biomarker analysis, KPS, corticosteroid use. Results: Baseline characteristics were balanced for intent-to-treat (n = 921) and biomarker-evaluable (n = 571) populations. Bevacizumab significantly prolonged PFS (HR = 0.64, 95% CI 0.55– 0.74, p b 0.0001; median 10.6 vs 6.2 months) and delayed HRQoL time to definitive deterioration (p b 0.0001). Interim OS did not cross the threshold for significance (HR = 0.89, 95% CI 0.75–1.07, p = 0.2135). Functional independence (KPS ≥ 70%) was maintained during PFS in both arms (median bevacizumab vs placebo: 9 vs 6 months). Patients treated with bevacizumab had a diminished corticosteroid requirement. Patients with low and high baseline plasma VEGF-A concentrations derived similar PFS benefit with bevacizumab (p = 0.610): low VEGF-A (HR = 0.64, 95% CI 0.48– 0.84) versus high (HR = 0.59, 95% CI 0.45–0.78). Similarly, PFS benefit in patients with low and high baseline VEGFR-2 levels was comparable (p = 0.736): low VEGFR-2 (HR = 0.54, 95% CI 0.41– 0.71) versus high (HR = 0.66, 95% CI 0.50–0.87). Conclusion: Addition of bevacizumab to standard-of-care therapy provided a significant clinically meaningful PFS improvement with stable/improved HRQoL/KPS and reduced corticosteroid requirement. NoVEGF-A/VEGFR-2 predictive/prognostic effectwas observed. Interim OS analysis did not cross the threshold for significance. doi:10.1016/j.jns.2013.07.2268 Abstract — WCN 2013 No: 2103 Topic: 36 — Other topic Flow cytometric analysis of cerebrospinal fluid is low diagnostic yield without atypical morphology or prior history of hematologic malignancy WCN 2013 No: 2103 Topic: 36 — Other topic Flow cytometric analysis of cerebrospinal fluid is low diagnostic yield without atypical morphology or prior history of hematologic malignancy G.H.J. Stevens, A.M.B. Collie, B.T. Hill, K. Fenner, E. Gazdick, L.A. Rybicki, E.H. Hsi. Rose Ella Burkhardt Brain Tumor and Neuro-Oncology Center, Cleveland Clinic, Cleveland, OH, USA; Pathology, Cleveland Clinic, Cleveland, OH, USA; Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, USA; Quality Health Science, Cleveland Clinic, Cleveland, OH, USA Background: Flow cytometric analysis (FCA) of cerebrospinal fluid (CSF) has utility in detecting central nervous system (CNS) involvement by hematologic malignancies. Yet, the majority of samples are negative by FCA. Objective: Identify pre-test characteristics of CSF specimens that will allow the rational use of FCA in the diagnosis of hematologic malignancy in CSF. Design: Retrospective data was collected from the medical record and flow cytometric reports for all CSF samples submitted for FCA between 2007 and 2009. Patients: 423 patients for whom 501 CSF samples were submitted for FCA. Results: A positive diagnosis of a hematologic malignancy was made in 41 specimens (8.2%). The FCA-positive specimens showed atypical morphology, either blasts or atypical lymphocytes, in 98% of Abstracts / Journal of the Neurological Sciences e629 (2013) e629–e678 e654

Level of Vascular Endothelial Growth Factor Predicts Both Relapse and Nonrelapse Mortality after Allogeneic Hematopoietic Stem Cell Transplantation

Although the prognostic significance of vascular endothelial growth factor (VEGF) has been researched extensively in patients with hematologic malignancies undergoing chemotherapy, its role in allogeneic hematopoietic stem cell transplantation (HSCT) requires further investigation. The present study evaluated the associations between VEGF level and relapse rate and early complications after HSCT. VEGF levels were analyzed in 91 consecutive patients before the start of conditioning, on day 0, on the day of engraftment, and on the day of diagnosis of veno-occlusive disease (VOD). Compared with a normal level, an elevated high VEGF-A level before conditioning was associated with an increased 2-year relapse rate (55% versus 24%, P = .003; hazard ratio [HR], 3.25; 95% confidence interval [CI], 1.49 to 7.08) and decreased event-free survival (20% versus 44%; P = .022; HR, 2.03; 95% CI, 1.11 to 3.72). No association was found between VEGF level and the incidence of acute GVHD (P > .05). In patients with VOD, VEGF-A level was elevated on day 0 and on the day of VOD diagnosis (P < .05). A low VEGF-A level on day 0 was associated with reduced nonrelapse mortality (14% versus 35%; P = .048; HR, 0.32; 95% CI, 0.10 to 0.99). Our results indicate that a high VEGF-A level before HSCT increases the risk of relapse, and a high level after conditioning is associated with increased risks of early complications and nonrelapse mortality.

Prospective study assessing hypoxia-related proteins as markers for the outcome of treatment with sunitinib in advanced clear-cell renal cell carcinoma

BackgroundPrevious studies suggest that expression of hypoxia markers may be associated with response to antiangiogenic drugs. Thus, we aimed to identify predictors of sunitinib outcome in clear-cell renal cell carcinoma (ccRCC). Patients and methodsThe expression of eight key proteins related to hypoxia (CAIX, HIF1A, HIF2A, VEGFA, VEGFR1, VEGFR2, VEGFR3 and PDGFRB) and P-glycoprotein were assessed by immunohistochemistry in 67 primary ccRCC samples from prospectively recruited patients treated with first-line sunitinib. The proteins expression, VHL inactivation and EGLN3 mRNA content were compared with the patients' response to sunitinib. ResultsHigh expression of HIF2A and PDGFRB was associated with better sunitinib RECIST objective response (P = 0.024 and P = 0.026; respectively) and increased VEGFR3 expression was associated with longer progression-free survival (P = 0.012). VEGFR3 overexpression showed a negative correlation with VEGFR3 polymorphism rs307826 (P = 0.002), a sunitinib resistance predictor. With respect to overall survival (OS), high VEGFA was associated with short (P = 0.009) and HIF2A with long (P = 0.048) survival times. High EGLN3 mRNA content was associated with shorter OS (P = 0.023). ConclusionsWe found an association between several proteins involved in hypoxia and sunitinib efficacy. In addition, low VEGFR3 expression was associated with worse outcome and with VEGFR3 rs307826 variant allele, reinforcing VEGFR3 as a marker of sunitinib resistance.

Levels of Erythropoietin and Vascular Endothelial Growth Factor in Surgery-Required Advanced Neovascular Glaucoma Eyes Before and After Intravitreal Injection of Bevacizumab

To evaluate changes of levels of erythropoietin (EPO) and VEGF in aqueous humor before and after an intravitreal injection of bevacizumab (IVB) and determine the underlying correlation between them. This prospective study involved 21 eyes of 21 patients with surgery-required advanced neovascular glaucoma (NVG) and 20 control subjects from October 2011 through November 2012. The NVG eyes received the IVB treatment before antiglaucomatous surgery. Aqueous humor was collected at the time of the IVB injection (pre-IVB) and at the time of antiglaucomatous surgery (post-IVB). Aqueous humor and plasma VEGF and EPO levels were measured with ELISA and chemiluminescence methods, respectively. The mean aqueous humor EPO and VEGF concentrations in pre-ivb eyes were significantly higher than those of the control subjects (p 0.001), whereas plasma levels showed no significant difference. There was a statistically significant correlation between the aqueous humor epo and the VEGF concentration (R = 0.612; P = 0.003). The mean aqueous humor VEGF in post-ivb eyes dramatically decreased from 1704.83 757.82 TO 19.02 14.65 PG/ML (P 0.001). However, EPO remained almost the same: 326.60 ± 104.28 compared with 312.67 ± 103.23 mU/mL (P = 0.675). The NVG eyes showed high aqueous EPO and VEGF levels, and there was a positive correlation between them. However, levels of EPO did not change after post-IVB, whereas those of VEGF decreased.

Patterns of metastatic spread in early breast cancer

AimsThe aim of this study was to prospectively investigate metastatic pathways of spread to lymph node versus bone marrow and identify biological characteristics that determine these patterns in early invasive breast cancer. Patients and methodsIn all, 177 patients with early invasive breast cancer underwent surgical extirpation of the primary tumour with sentinel lymph node biopsy (SLNB). Bone marrow (BM) aspiration was performed to screen for cytokeratin-positive cells by immunocytochemistry. Lymphatic spread was assessed by histopathological examination of lymph nodes (LN). A representative subset of 87 tumours was analysed by tissue microarray (TMA) to evaluate expression of markers that potentially influence haematogenous vs. lymphatic spread. Patients were followed up for a median of 54.7 months. ResultsOf the 177 patients, 114 (64%) were BM−/LN−, 38 (22%) BM−/LN+, 19 (11%) BM+/LN− and 6 (3%) BM+/LN+. Multivariate analysis of histopathological characteristics revealed that increasing tumour size was significantly associated with both LN positivity (p = 0.003) and BM positivity (p = 0.01), the presence of lymphovascular invasion significantly correlated with LN+ (p = 0.01), whereas lower histological grade was significantly associated with BM+ (p = 0.03). LN+ and BM+ were non-significantly negatively related to each other. Univariate analysis of the TMA data showed differential expression patterns for several factors; significant differences between effects on the two metastatic pathways (lymphatic vs. haematogenous) were found for expression of CD54 (p = 0.03), osteopontin (p = 0.04), bone sialoprotein (p = 0.04) and CXCR4 (p = 0.009). High expression of CD54, osteopontin and bone sialoprotein (BSP) was positively associated with BM + but was either not associated, or negatively associated, with LN+. High CXCR4 expression was positively associated with LN+ and negatively with BM+. High VEGF–C expression was associated with both LN+ and BM+, although this did not attain statistical significance. Due to the small number of clinical events during clinical follow-up, no associations were identified between metastatic spread patterns, recurrence and/or death. ConclusionThese findings suggest that distinct lymphatic and haematogenous metastatic pathways exist in early breast cancer and that these pathways are governed by specific biological markers.

Visceral fat as a predictive marker of response to bevacizumab-based treatment in metastatic colorectal cancer.

e14665 Background: A large amount of visceral adipose tissue might be correlated with high VEGF levels and with resistance to bevacizumab-based regimens in metastasic colorectal cancer (mCRC). The aim is to evaluate that abdominal obesity can be a predictive marker of response to bevacizumab-based therapy in mCRC. Methods: From January 2005 to December 2010, we performed a retrospective analysis of 74 consecutive patients with mCRC received bevacizumab-based first line treatment. Pretreatment CT was used to measure visceral fat volume (VFV), subcutaneous fat volume (SFV) an waist circumference (WC) in 74 patients with mCRC who received bevacizumab-based first-line treatment (bevacizumab group, n=37) or chemotherapy alone (chemotherapy group, n=37). Associations linkingVFV, SFV and WC to tumor response, progression free survival (PFS) and overall survival (OS) were evaluated. For all analyses, VFV, SFV and WC were dichotomized using the median as the cut-off point. Results: In the bevacizumab group, median follow-up lasted for 25 months (7-47). VFV, SFV and WC values were not associated with response or OS. PFS was shorter in patients with high VFV (12.8 vs 7.7 months; p=0.04). By multivariate analysis, high VFA was independently associated with PFS (HR=4.32, p=0.045). In the chemotherapy group, median follow-up lasted for 26 months (2-68). VFV, SFV and WC were not associated with response, PFS or OS. Conclusions: Visceral fat volume plays a role of predictive marker of PFS to bevacizumab-based therapy for Japanese patients with mCRC.

Use of VEGF expression to predict for first-line treatment chemotherapy regimen in metastatic colorectal cancer.

e14695 Background: In metastatic colorectal cancer treatment more successful results are achieved by adding anti-angiogenic therapy to the chemotherapy. Over expression of VEGF in colorectal cancer is known to be a poor prognosis factor but its effects on designating the therapy is not clear. In this study, results of the patients receiving FOLFIRI + Bevacizumab or XELOX + Bevacizumab were compared for VEGF expression. Methods: VEGF was assessed immunohistochemically in primary tumors of the 53 metastatic colorectal cancers. Severity and conformity of VEGF expression was evaluated. Results were used to assess the association with the therapy response, progression free survival (PFS) and overall survival (OS). Results: The median age of the patients was 55 (range, 32-79). In combination with bevacizumab 38 patients received FOLFIRI and 15 patients received XELOX. In 30 patients (57%) there was a strong expression of VEGF and in 23 patients (23%) the expression was weak. In the high VEGF expression group, PFS was 11 months in FOLFIRI receiving patients and 8 months in XELOX receiving ones but in low expression group PFS was 8 months in patients receiving either one of the two treatment protocols. In high expression group, PFS in FOLFIRI-bevacizumab combination receiving patients was different and this difference was statistically significant (p=0.009). Overall survival in FOLFIRI receiving patients with high VEGF expression was 26 months and with low VEGF expression it was 16 months (p=0.04). Median OS was not reached in the XELOX receiving patient group. FOLFIRI regime clinical benefit was 58% in high VEGF expression group whilst it was 34% in low expression group. This difference was not statistically significant but it was close to the significance threshold (p=0.06). There was no association between the therapy responses and VEGF expression of the 15 patients who received XELOX. Conclusions: There is no difference in efficacy between the first line chemotherapy regimens of the metastatic colorectal cancer. In this study, in metastatic colorectal patients with over expressed VEGF first line FOLFIRI-bevacizumab combination led to better response and survival rates.

Comparison of the effects of bevacizumab (BV) in combination with chemotherapy (CT) and CT alone on the incidence of brain metastases (BM) in non-small cell lung cancer (NSCLC): A retrospective clinical analysis.

e19044 Background: The incidence of BM of advanced NSCLC is 20-40%, and there's a lack of effective treatment. This study aims to investigate the value of the angiogenesis marker VEGF in predicting the risk of BM in patients (pts) with NSCLC receiving CT+BV or CT alone and to explore the cumulative risk of BM of NSCLC with high VEGF expression that treated with BV. Methods: Pts with stage IIIB/IV NSCLC underwent CT and/or targeted therapy from 2008-1 to 2010-12 were included. Semi-quantitative detection of VEGF expression was carried out using conventional immunohistochemical staining of lung tissue specimens. Pts experienced BM prior to treatment were excluded. Pts underwent gemcitabine /carboplatin (GC) + BV treatment were included in observation group; pts underwent GC were included in control group. The cumulative risk of BM was calculated using death as a competing risk. Results: 177 pts were included (118 in BV+CT group; 59 in CT group). The median follow-up duration was 18.7 months. Up to 2012-12-31, BM occurred in 15 cases in BV+CT group and 17 in CT group (12.71% vs. 28.81%). 4 cases (33.89%) in BV+CT group survived, and 11 (18.64%) in CT group survived. In BV+CT group, the cumulative risks of BM at 6, 12, and 24 months were 0.86%, 7.52%, and 13.01%, respectively, and 6.74%, 18.88%, and 28.85% in CT group, respectively (P &lt; 0.001). HR of BM in BV+CT group was 0.048 (95% CI: 0.171-0.994) compared to CT group. In BV+CT group, the 6-, 12-, and 24-month cumulative risks of BM were 1.6%, 7.97%, and 12.63% in pts with VEGF(+) and 0, 7.24%, and 12.71% in pts with VEGF(-), respectively; in CT group, the 6-, 12-, and 24-month cumulative risks of BM were 9.35%, 27.04%, and 37.5% in pts with VEGF(+) and 3.52%, 10.71%, and 18.05% in pts with VEGF(-), respectively; there was no significant difference between them. Conclusions: BV+CT can effectively reduce the incidence of BM in pts with advanced NSCLC and improve their prognosis. This small data sample suggests that pts with VEGF(+) are more prone to BM than pts with VEGF(-). The incidence of BM in pts with VEGF(+) is reduced after BV treatment, though its specific mechanism still requires further study.

Expression of VEGF and Semaphorin Genes Define Subgroups of Triple Negative Breast Cancer

Triple negative breast cancers (TNBC) are difficult to treat due to a lack of targets and heterogeneity. Inhibition of angiogenesis is a promising therapeutic strategy, but has had limited effectiveness so far in breast cancer. To quantify heterogeneity in angiogenesis-related gene expression in breast cancer, we focused on two families – VEGFs and semaphorins – that compete for neuropilin co-receptors on endothelial cells. We compiled microarray data for over 2,600 patient tumor samples and analyzed the expression of VEGF- and semaphorin-related ligands and receptors. We used principal component analysis to identify patterns of gene expression, and clustering to group samples according to these patterns. We used available survival data to determine whether these clusters had prognostic as well as therapeutic relevance. TNBC was highly associated with dysregulation of VEGF- and semaphorin-related genes; in particular, it appeared that expression of both VEGF and semaphorin genes were altered in a pro-angiogenesis direction. A pattern of high VEGFA expression with low expression of secreted semaphorins was associated with 60% of triple-negative breast tumors. While all TNBC groups demonstrated poor prognosis, this signature also correlated with lower 5-year survival rates in non-TNBC samples. A second TNBC pattern, including high VEGFC expression, was also identified. These pro-angiogenesis signatures may identify cancers that are more susceptible to VEGF inhibition.

Sulodexide improves renal function through reduction of vascular endothelial growth factor in type 2 diabetic rats

AimsSulodexide is a promising therapeutic drug for the management of diabetic nephropathy. Although sulodexide has demonstrated a renoprotective effect through its ability to restore glomerular ionic permselectivity, the exact mechanism is still not clear. We investigated the effects of long-term sulodexide treatment on diabetic nephropathy in Otsuka-Long–Evans-Tokushima-Fatty (OLETF) rats. Main methodsDiabetic rats were treated with or without sulodexide at 10mg/kg/day in the drinking water for nine months. Renal morphology and changes in VEGF and p38 mitogen-activated protein kinase (p38 MAPK), urinary levels of albumin (UAE) and urinary VEGF excretion were determined. To define the direct effects of sulodexide, we performed an in vitro experiment using podocytes. Key findingsUAE was significantly higher in OLETF rats than in control LETO rats, and the sulodexide group showed significantly decreased UAE after six months of treatment. Interestingly, urinary VEGF levels were also significantly decreased in the sulodexide-treated group. In accordance with UAE and urinary VEGF changes, the renal expression of profibrotic molecules was significantly decreased after sulodexide treatment. In addition, the activation of p38 MAPK, assessed by measuring the level of phospho-specific p38 MAPK, increased in diabetic renal tissues and was markedly suppressed by sulodexide treatment. In cultured podocytes, sulodexide treatment significantly decreased high glucose-induced p38 MAPK activation and VEGF synthesis. SignificanceSulodexide directly suppresses VEGF synthesis through the p38 MAPK pathway in podocytes, and these results suggest that sulodexide may provide renoprotection via suppression of renal VEGF synthesis independently of glomerular basement membrane ionic permselectivity in type 2 diabetic rats.

Vascular endothelial growth factor in the CSF of elderly patients with ventriculomegaly: Variability, periodicity and levels in drainage responders and non-responders

ObjectivesThe aim of this study was to examine lumbar CSF-VEGF levels from elderly patients with ventriculomegaly to evaluate the possible circadian or periodic concentration profile and relevance to the prediction of drainage response. MethodsLumbar CSF samples were collected in 1-h interval over 35h from 22 patients with ventriculomegaly. CSF-VEGF levels were measured to elucidate the possible circadian or periodic concentration profiles. These VEGF levels were evaluated for correlations with clinical response to CSF drainage, ventricle size and other clinical information. ResultsThe 35-h CSF-VEGF levels demonstrated a periodic concentration pattern with significant episodic fluctuation with 3–5h intervals. CSF-VEGF levels in non-responder group in which patients did not show clinical improvement with CSF drainage were significantly higher than these in responder group. ConclusionVEGF variation in hydrocephalus patients suggests its possible pathophysiological role in hydrocephalus. The periodic concentration pattern of CSF-VEGF must be considered when choosing the most appropriate time for sample collection or clinical manipulation. Increased VEGF level in patients who showed no improvement with CSF drainage suggests that a possible greater ischemic or vascular injury may play a role in these patients. Pending further studies, these results suggest that high VEGF levels have a potential application in predicting non-responder patients with CSF drainage and so reducing the morbidity and cost of drainage and shunting in these patients.