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1.  Targeted inhibition of VEGF Receptor-2: An update on Ramucirumab 
Expert opinion on biological therapy  2013;13(8):1187-1196.
Ramucirumab (IMC-1121B) is a fully humanized IgG1 monoclonal antibody targeting the extracellular domain of VEGF receptor 2 (VEGFR2). Numerous phase I-II trials with ramucirumab in various malignancies have shown promising clinical antitumor efficacy and tolerability. Most recently, the large phase III REGARD trial evaluated ramucirumab in patients with refractory metastatic gastric cancer. Patients receiving ramucirumab experienced a median overall survival of 5.2 months compared to 3.8 months on placebo.
Areas Covered
The purpose of this article is to review the preclinical motivation for VEGFR2 targeted therapies and survey recent data from clinical trials involving ramucirumab, as well as highlight ongoing studies.
Expert Opionion
Rational multi-target approaches to angiogenesis are needed to overcome resistance mechanisms. Predictive angiogenic biomarkers are also needed to optimize patient selection for novel anti-angiogenic agents.
PMCID: PMC4131847  PMID: 23803182
Ramucirumab; angiogenesis; cancer; VEGF; VEGFR2; monoclonal antibody; gastric cancer; adenocarcinoma; vascular endothelial growth factor
2.  Ramucirumab: preclinical research and clinical development 
OncoTargets and therapy  2014;7:1997-2006.
Ramucirumab (IMC-1121B, LY3009806), a fully humanized monoclonal antibody directed against the extracellular domain of vascular endothelial growth factor receptor 2 (VEGFR-2), is a new therapeutic option that selectively inhibits the human VEGFR-2 with a much greater affinity than its natural ligands. Based on the promising results of both preclinical and early clinical studies, ramucirumab has been tested in different tumor types either alone or in combination with chemotherapy. While it has recently been granted its first US Food and Drug Administration approval for use as a single agent in patients with advanced or metastatic gastric cancer or gastroesophageal junction carcinoma, its role for metastatic breast cancer or advanced non-small-cell lung cancer is still debated. The aims of this review are to recall and discuss the most significant preclinical and clinical studies that led to the development of ramucirumab and to present the results of the randomized clinical trials that have tested its efficacy in different malignancies, including gastric and lung cancer.
PMCID: PMC4218907  PMID: 25378934
ramucirumab; gastric cancer; lung cancer; breast cancer; antiangiogenic
3.  An Open-Label Phase II Study Evaluating the Safety and Efficacy of Ramucirumab Combined With mFOLFOX-6 as First-Line Therapy for Metastatic Colorectal Cancer 
The Oncologist  2014;19(4):350-351.
Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2) are believed to mediate angiogenesis in colorectal cancer (CRC). Ramucirumab (RAM; IMC-1121B) is a human IgG1 monoclonal antibody that inhibits VEGF ligand binding to VEGFR-2, inhibiting VEGFR-2 activation and signaling.
Patients with metastatic CRC, Eastern Cooperative Oncology Group performance status 0–1, and adequate organ function who had not received chemotherapy for metastatic disease received RAM and the modified FOLFOX-6 regimen every 2 weeks. Endpoints included progression-free survival (PFS), objective response rate, overall survival, and safety. The sample size was based on a potentially improved median PFS from 8 months to 11 months.
Forty-eight patients received therapy. Median PFS was 11.5 months (95% confidence interval [CI]: 8.6–13.1 months). The objective response rate was 58.3% (95% CI: 43.21–72.39). The disease control rate (complete or partial response plus stable disease) was 93.8% (95% CI: 82.8–98.7). Median overall survival was 20.4 months (95% CI: 18.5–25.1 months). The most frequent grade 3–4 adverse events included neutropenia (grade 3: 33.3%; grade 4: 8.3%), hypertension (grade 3: 16.7%), and neuropathy (grade 3: 12.5%). Two patients died during the study due to myocardial infarction and cardiopulmonary arrest.
RAM may enhance the efficacy of modified FOLFOX-6 chemotherapy with an acceptable safety profile in metastatic CRC.
PMCID: PMC3983832  PMID: 24674871
4.  Clinical advances in the development of novel VEGFR2 inhibitors 
Angiogenesis inhibitors have produced significant advances in the treatment of several tumors including colorectal, lung, ovarian and renal carcinomas. These agents, however, modestly impact on the overall cure rate, and their activity is often limited because of the early outbreak of redundant pathways or resistance mechanisms. Moreover, no clear predictive factor has been identified for treatment selection in the clinic. Preclinical evidence suggest that antibodies targeting the vascular endothelial growth factor (VEGF) axis may exert their activity throughout the inhibition of VEGF receptor 2 (VEGFR2) phosphorylation, a key factor in the cancer angiogenic process. Among other molecules, ramucirumab, an intravenously administered, fully humanized monoclonal antibody (mAb) targeting the extracellular domain of the receptor, and apatinib, a potent oral inhibitor of the intracellular domain, are emerging as original antiangiogenic opportunities. This up-to-date review focuses on the development of VEGFR2 inhibitors across multiple cancers and presents results of the most recent researches, ranging from early phase I studies to randomized phase III trials, in which those drugs have been tested as a single-agent or in combination with different chemotherapy regimens.
PMCID: PMC4260048  PMID: 25568876
Angiogenesis; vascular endothelial growth factor receptor 2 (VEGFR2); antibodies anti-VEGFR2; ramucirumab; apatinib; gastric cancer; lung cancer; breast cancer
5.  Anti-VEGF agents in metastatic colorectal cancer (mCRC): are they all alike? 
Bevacizumab is a monoclonal antibody that binds and neutralizes vascular endothelial growth factor (VEGF)-A, a key player in the angiogenesis pathway. Despite benefits of bevacizumab in cancer therapy, it is clear that the VEGF pathway is complex, involving multiple isoforms, receptors, and alternative ligands such as VEGF-B, and placental growth factor, which could enable escape from VEGF-A-targeted angiogenesis inhibition. Recently developed therapies have targeted other ligands in the VEGF pathway (eg, aflibercept, known as ziv-aflibercept in the United States), VEGF receptors (eg, ramucirumab), and their tyrosine kinase signaling (ie, tyrosine kinase inhibitors). The goal of the current review was to identify comparative preclinical data for the currently available VEGF-targeted therapies. Sources were compiled using PubMed searches (2007 to 2012), using search terms including, but not limited to: “bevacizumab,” “aflibercept,” “ramucirumab,” and “IMC-18F1.” Two preclinical studies were identified that compared bevacizumab and the newer agent, aflibercept. These studies identified some important differences in binding and pharmacodynamic activity, although the potential clinical relevance of these findings is not known. Newer antiangiogenesis therapies should help further expand treatment options for colorectal and other cancers. Comparative preclinical data on these agents is currently lacking.
PMCID: PMC3685399  PMID: 23807861
aflibercept; antiangiogenesis; metastatic colorectal cancer (mCRC); tyrosine kinase inhibitor (TKI); vascular endothelial growth factor (VEGF)
6.  A Phase Ib Study of Safety and Pharmacokinetics of Ramucirumab in Combination With Paclitaxel in Patients With Advanced Gastric Adenocarcinomas 
The Oncologist  2015;20(5):493-494.
Lessons Learned
The pharmacokinetic results of this phase Ib study of ramucirumab combined with paclitaxel as second-line therapy in Japanese patients with metastatic gastric or gastro-esophageal junction adenocarcinoma are in line with previous ramucirumab studies.
This combination at the doses and schedule given did not result in any dose-limiting toxicities and appeared to be safe and well tolerated.
This phase Ib study evaluated the tolerability and pharmacokinetics of ramucirumab, an anti-VEGFR-2 antibody, combined with paclitaxel as second-line therapy in Japanese patients with metastatic gastric or gastroesophageal junction adenocarcinoma after first-line therapy with fluoropyrimidines and/or platinum.
Patients received ramucirumab 8 mg/kg on days 1 and 15 and paclitaxel 80 mg/m2 on days 1, 8, and 15 of a 28-day cycle. Safety analyses included all patients (n = 6).
No dose-limiting toxicities occurred in the first cycle. All patients experienced ≥1 treatment-emergent adverse event (TEAE); 5 patients experienced grade ≥3 TEAEs. There were two deaths caused by disease progression. The best overall responses were stable disease (n = 5) and partial response (n = 1). Patients received ramucirumab and paclitaxel for a median of 12.5 weeks (range: 11.4–42.7 weeks) and 12.2 weeks (range: 11.0–41.0 weeks), respectively. Following a single dose of ramucirumab IV infusion 8 mg/kg, clearance was ∼0.017 L/hour, half-life (t1/2) was 138 to 225 hours, and steady-state volume of distribution (Vss) was ∼3 L.
The ramucirumab/paclitaxel combination appears to be well-tolerated in Japanese patients with advanced gastric adenocarcinomas. These results are in line with previous ramucirumab pharmacokinetic studies as anticipated.
PMCID: PMC4425390  PMID: 25888272
7.  Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1 
PLoS Medicine  2007;4(6):e186.
While vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial.
Methods and Findings
We investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope.
This study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.
Shalom Avraham and colleagues' study provides evidence of a survival system in breast cancer cells by which VEGF acts as an internal autocrine survival factor through its binding to VEGFR1.
Editors' Summary
One woman in eight will develop breast cancer during her lifetime. Most of these women live for many years after their diagnosis and many are cured of their cancer. However, sometimes the cancer grows inexorably and spreads (metastasizes) around the body despite the efforts of oncologists. Characteristics of the tumor known as prognostic factors can indicate whether this spreading is likely to happen. Large tumors that have metastasized have a poorer prognosis than small tumors that are confined to the breast. The expression of specific proteins within the tumor also provides prognostic information. One protein whose expression is associated with a poor prognosis is vascular endothelial growth factor (VEGF). VEGF stimulates angiogenesis—the growth of new blood vessels. Small tumors get the nutrients needed for their growth from existing blood vessels but large tumors need to organize their own blood supply. They do this, in part, by secreting VEGF. This compound binds to proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels), which then send a signal into the cell instructing it to make new blood vessels. Angiogenesis inhibitors, including molecules that block the activity of VEGF receptors, are being developed for the treatment of cancer.
Why Was This Study Done?
Some breast cancer cell lines (cells isolated from breast cancers and grown in the laboratory) make VEGF and VEGF receptors (VEGFR1, VEGFR2, and neuropilin 1 [NRP1]). But, although some studies have reported an association between VEGFR1 expression in breast tumors and a poor prognosis, other studies have found no expression of VEGFR1 in breast tumors. Consequently, the role of VEGF receptors in breast cancer is unclear. In this study, the researchers analyzed the expression and function of VEGF and its receptors in breast cancer cells to investigate whether and how VEGF helps these cells to survive.
What Did the Researchers Do and Find?
The researchers first examined the expression of VEGF receptors in several human breast cancer cell lines. All of them expressed VEGFR1, some expressed NRP1, but VEGFR2 expression was universally low. They then investigated the function of VEGF and its receptors in two human breast cancer cell lines (MDA-MB-231 and MCF-7). In both cell lines, blocking the expression of VEGF or of VEGFR1 (but not of the other two receptors) reduced cell survival by stimulating a specific process of cell death called apoptosis. Unexpectedly, adding VEGF to the cultures did not reverse the effect of blocking VEGF expression, a result that suggests that VEGF and VEGFR1 do not affect breast cancer cell survival by acting at the cell surface. Accordingly, when the researchers examined where VEGFR1 occurs in the cell, they found it on the membranes around the nucleus of the breast cancer cell lines and not on the cell surface; several primary breast tumors and normal breast tissue had the same localization pattern. Finally, the researchers showed that inhibitors of VEGF action that act at the cell surface did not affect the survival of the breast cancer cell lines.
What Do These Findings Mean?
These findings suggest that VEGF helps breast cancer cells to survive in a unique way: by binding to VEGFR1 inside the cell. In other words, whereas VEGF normally acts as a paracrine growth factor (it is released by one cell and affects another cell), in breast cancer cells it might act as an internal autocrine (intracrine) survival factor, a factor that affects the cells in which it is produced. These findings need confirming in more cell lines and in primary breast cancers but could have important implications for the treatment of breast cancer. Inhibitors of VEGF and VEGFR1 that act inside the cell (small molecule drugs) might block breast cancer growth more effectively than inhibitors that act at the cell surface (for example, proteins that bind to the receptor), because internally acting inhibitors might both kill the tumor directly and have antiangiogenic effects, whereas externally acting inhibitors could only have the second effect.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute information for patients and professionals on breast cancer (in English and Spanish) and on angiogenesis (in English and Spanish)
MedlinePlus Encyclopedia information for patients on breast cancer (in English and Spanish)
CancerQuest, information from Emory University on cancer biology and on angiogenesis and angiogenesis inhibitors (in several languages)
Wikipedia pages on VEGF (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC1885450  PMID: 17550303
8.  An updated review of gastric cancer in the next-generation sequencing era: Insights from bench to bedside and vice versa 
Gastric cancer (GC) is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. There is an increasing understanding of the roles that genetic and epigenetic alterations play in GCs. Recent studies using next-generation sequencing (NGS) have revealed a number of potential cancer-driving genes in GC. Whole-exome sequencing of GC has identified recurrent somatic mutations in the chromatin remodeling gene ARID1A and alterations in the cell adhesion gene FAT4, a member of the cadherin gene family. Mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) have been found in 47% of GCs. Whole-genome sequencing and whole-transcriptome sequencing analyses have also discovered novel alterations in GC. Recent studies of cancer epigenetics have revealed widespread alterations in genes involved in the epigenetic machinery, such as DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs and microRNAs. Recent advances in molecular research on GC have resulted in the introduction of new diagnostic and therapeutic strategies into clinical settings. The anti-human epidermal growth receptor 2 (HER2) antibody trastuzumab has led to an era of personalized therapy in GC. In addition, ramucirumab, a monoclonal antibody targeting vascular endothelial growth factor receptor (VEGFR)-2, is the first biological treatment that showed survival benefits as a single-agent therapy in patients with advanced GC who progressed after first-line chemotherapy. Using NGS to systematically identify gene alterations in GC is a promising approach with remarkable potential for investigating the pathogenesis of GC and identifying novel therapeutic targets, as well as useful biomarkers. In this review, we will summarize the recent advances in the understanding of the molecular pathogenesis of GC, focusing on the potential use of these genetic and epigenetic alterations as diagnostic biomarkers and novel therapeutic targets.
PMCID: PMC3983448  PMID: 24744582
Next-generation sequencing; Microsatellite instability; MicroRNA; Epigenetic field defect; Gastric washes; Insulin-like growth factor 1 receptor
9.  Second-line treatments for advanced gastric cancer: Interpreting outcomes by network meta-analysis 
AIM: To study the effectiveness of second-line treatments for advancer gastric cancer by application of Bayesian network meta-analysis.
METHODS: Our search covered the literature up to February 2015. The following 6 treatments were evaluated: (1) irinotecan (camptothecins); (2) paclitaxel (taxanes class); (3) docetaxel (taxanes); (4) everolimus (mammalian target of rapamycin inhibitors); (5) ramucirumab (vascular endothelial growth factor receptor 2 inhibitors); (6) ramucirumab + paclitaxel. Our methodology was based on standard models of Bayesian network meta-analysis. The reference treatment was best supportive care (BSC). The end-point was overall survival. Median survival was the outcome measure along with 95% credible intervals.
RESULTS: Our search identified a total of 7 randomized controlled trials. These trials included 2298 patients (in 15 treatment arms) in whom a total of 6 active treatments were evaluated as well as BSC. There were 21 head-to-head comparisons (6 direct, 15 indirect). The difference in survival between each of two active treatments (paclitaxel and ramucirumab + paclitaxel) vs BSC was statistically significant, while the other 4 showed no statistical difference. In the 6 head-to-head comparisons between active treatments, no significant survival difference was demonstrated.
CONCLUSION: Our results indicate that both paclitaxel monotherapy and ramucirumab + paclitaxel determine a significant prolongation in survival as compared with BSC.
PMCID: PMC4530381  PMID: 26266104
Meta-analysis; Bayesian methods; Advancer gastric cancer; Second line therapy; Paclitaxel; Irinotecan; Docetaxel; Ramucirumab
10.  Angiogenesis and lung cancer: ramucirumab prolongs survival in 2nd-line metastatic NSCLC 
In the REVEL trial, ramucirumab, a monoclonal antibody to VEGFR-2, improved overall survival in combination with docetaxel compared to docetaxel alone in the second-line setting of non-small cell lung cancer (NSCLC). Along with bevacizumab and nintedanib, ramucirumab is the third anti-angiogenic agent that has yielded positive overall survival results in a phase III trial of patients with advanced NSCLC. Given the lack of effective therapies in the relapsed setting and the disappointing results of many other VEGF-targeted agents in lung cancer, the results from REVEL are encouraging. One of the major remaining hurdles is the identification of reliable predictive biomarkers in order to predict which patients are most likely to benefit from anti-angiogenic therapies. Despite the positive results seen in REVEL, the exact role of ramucirumab in the treatment paradigm of lung cancer remains to be seen given the modest survival benefit of 1.4 months and the lack of predictive biomarkers at this time.
PMCID: PMC4367672  PMID: 25806332
Angiogenesis; ramucirumab; lung cancer; VEGF; docetaxel
11.  Functional Significance of VEGFR-2 on Ovarian Cancer Cells 
Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of the current study. By protein analysis, A2780-par and HeyA8 ovarian cancer cell lines expressed VEGFR-1 and HeyA8 and SKOV3ip1 expressed VEGFR-2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR-2 expression while only 15% showed moderate to high VEGFR-1 expression. By immunofluorescence, little or no VEGFR-2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR-2 specific antibody (1121B), murine VEGFR-2 specific antibody (DC101), or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR-1 antibody had no effect. Treatment with 1121B effectively blocked VEGF-induced phosphorylation of p130Cas. In vivo, treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p<0.001). We show functionally active VEGFR-2 is present on most ovarian cancer cells. The observed anti-tumor activity of VEGF-targeted therapies may be mediated by both anti-angiogenic and direct anti-tumor effects.
PMCID: PMC2668132  PMID: 19058181
VEGFR; angiogenesis; ovarian carcinoma
12.  VEGF-dependent tumor angiogenesis requires the inverse and reciprocal regulation of VEGFR1 and VEGFR2 
Cell death and differentiation  2009;17(3):499-512.
Vascular endothelial growth factor (VEGF) signaling is critical for tumor angiogenesis. However, therapies based on the inhibition of VEGF receptors have shown modest results in patients with cancer. Surprisingly little is known about mechanisms underlying the regulation of VEGFR1 and VEGFR2 expression, the main targets of these drugs. Here, analysis of tissue microarrays revealed an inversely reciprocal pattern of VEGF receptor regulation in the endothelium of human squamous cell carcinomas (high VEGFR1, low VEGFR2), as compared to the endothelium of control tissues (low VEGFR1, high VEGFR2). Mechanistic studies demonstrated that VEGF signals through the Akt/ERK pathway to inhibit constitutive ubiquitination and induce rapid VEGFR1 accumulation in endothelial cells. Surprisingly, VEGFR1 is primarily localized in the nucleus of endothelial cells. In contrast, VEGF signals through the JNK/c-Jun pathway to induce endocytosis, nuclear translocation, and downregulation of VEGFR2 via ubiquitination. VEGFR1 signaling is required for endothelial cell survival, while VEGFR2 regulates capillary tube formation. Notably, the antiangiogenic effect of Bevacizumab (anti-VEGF antibody) requires the normalization of VEGFR1 and VEGFR2 levels in human squamous cell carcinomas vascularized with human blood vessels in immunodeficient mice. Collectively, this work demonstrate that VEGF-induced angiogenesis requires the inverse regulation of VEGFR1 and VEGFR2 in tumor-associated endothelial cells.
PMCID: PMC2822115  PMID: 19834490
Angiogenesis; Apoptosis; Receptor Tyrosine Kinase; Differentiation; Tumor Microenvironment
13.  Anti-angiogenic therapies for advanced esophago-gastric cancer 
Neo-vascularization is a vital process for tumor growth and development which involves the interaction between tumor cells and stromal endothelial cells through several growth factors and membranous receptors which ultimately activate pro-angiogenic intracellular signaling pathways. Inhibition of angiogenesis has become a standard treatment option for several tumor types including colorectal cancer, glioblastoma and ovarian cancer. In gastric cancer, the therapeutic role of anti-angiogenic agents is more controversial. Bevacizumab and ramucirumab, two monoclonal antibodies, which target vascular endothelial growth factor-A and vascular endothelial growth factor receptor-2, respectively, have been demonstrated antitumor activity in patients with tumors of the stomach or esophagogastric junction. However, especially for bevacizumab, this antitumor activity has not consistently translated into a survival advantage over standard treatment in randomized trials. In this article, we provide an overview of the role of angiogenesis in gastric cancer and discuss the results of clinical trials that investigated safety and effectiveness of antiangiogenic therapies in this disease. A review of the literature has been done using PubMed, website and the ASCO Annual Meeting Library.
PMCID: PMC4264270  PMID: 25538401
Angiogenesis; bevacizumab; gastric cancer; esophageal cancer; ramucirumab
14.  Chemotherapy or Targeted Therapy as Second-Line Treatment of Advanced Gastric Cancer. A Systematic Review and Meta-Analysis of Published Studies 
PLoS ONE  2014;9(9):e108940.
Chemotherapy is a cornerstone in treatments of gastric cancer, but despite its benefit, less than 60% of patients receive salvage therapy in clinical practice. We performed a systematic review and meta-analysis based on trial data on the role of second-line treatment of advanced gastric cancer. MEDLINE/PubMed and Cochrane Library were searched for randomized phase III trials that compared active therapy to best supportive care in advanced gastric cancer. Data extraction was conducted according to the PRISMA statement. Summary HR for OS was calculated using a hierarchical Bayesian model and subgroup analysis was performed based on baseline Eastern Cooperative Oncology Group Performance Status (ECOG) performance status (0 vs. 1 or more). A total of 1,407 patients were evaluable for efficacy, 908 were treated in the experimental arms, with chemotherapy (231 pts) or with targeted therapies (677 pts). The risk of death was decreased by 18% (HR = 0.82; 95% CI, 0.79–0.85; posterior probability HR≥1: <0.00001) with active therapies. Chemotherapy and ramucirumab were able to decrease this risk by 27% and 22%, respectively. No differences were found between chemotherapy and ramucirumab. In patients with ECOG = 0 a greater benefit was found for chemotherapy with a reduction of the risk of death by 43% and no benefits were found for ramucirumab or everolimus. In patients with ECOG = 1 or more a significant reduction of the risk of death by 32% was reported in patients treated with ramucirumab, even if no significant difference was reported between chemotherapy and ramucirumab. This analysis reports that active and available therapies are able to prolong survival in patients with advanced gastric cancer with a different outcome based on initial patient’s performance status. New trials based on a better patient stratification are awaited.
PMCID: PMC4182573  PMID: 25268988
15.  Mechanism-related circulating proteins as biomarkers for clinical outcome in patients with unresectable hepatocellular carcinoma receiving sunitinib 
Several proteins that promote angiogenesis are overexpressed in hepatocellular carcinoma (HCC) and have been implicated in disease pathogenesis. Sunitinib has antiangiogenic activity and is an oral multitargeted inhibitor of vascular endothelial growth factor receptors (VEGFRs)-1, -2, and -3, platelet-derived growth factor receptors (PDGFRs)-α and -β, stem-cell factor receptor (KIT), and other tyrosine kinases. In a phase II study of sunitinib in advanced HCC, we evaluated the plasma pharmacodynamics of five proteins related to the mechanism of action of sunitinib and explored potential correlations with clinical outcome.
Patients with advanced HCC received a starting dose of sunitinib 50 mg/day administered orally for 4 weeks on treatment, followed by 2 weeks off treatment. Plasma samples from 37 patients were obtained at baseline and during treatment and were analyzed for vascular endothelial growth factor (VEGF)-A, VEGF-C, soluble VEGFR-2 (sVEGFR-2), soluble VEGFR-3 (sVEGFR-3), and soluble KIT (sKIT).
At the end of the first sunitinib treatment cycle, plasma VEGF-A levels were significantly increased relative to baseline, while levels of plasma VEGF-C, sVEGFR-2, sVEGFR-3, and sKIT were significantly decreased. Changes from baseline in VEGF-A, sVEGFR-2, and sVEGFR-3, but not VEGF-C or sKIT, were partially or completely reversed during the first 2-week off-treatment period. High levels of VEGF-C at baseline were significantly associated with Response Evaluation Criteria in Solid Tumors (RECIST)-defined disease control, prolonged time to tumor progression (TTP), and prolonged overall survival (OS). Baseline VEGF-C levels were an independent predictor of TTP by multivariate analysis. Changes from baseline in VEGF-A and sKIT at cycle 1 day 14 or cycle 2 day 28, and change in VEGF-C at the end of the first off-treatment period, were significantly associated with both TTP and OS, while change in sVEGFR-2 at cycle 1 day 28 was an independent predictor of OS.
Baseline plasma VEGF-C levels predicted disease control (based on RECIST) and were positively associated with both TTP and OS in this exploratory analysis, suggesting that this VEGF family member may have utility in predicting clinical outcome in patients with HCC who receive sunitinib.
Trial registration NCT00247676
PMCID: PMC3162912  PMID: 21787417
16.  Up-regulation of soluble vascular endothelial growth factor receptor-1 prevents angiogenesis in hypertrophied myocardium 
Cardiovascular Research  2010;89(2):410-418.
Inadequate capillary growth in pressure-overload hypertrophy impairs myocardial perfusion and substrate delivery, contributing to progression to failure. Capillary growth is tightly regulated by angiogenesis growth factors like vascular endothelial growth factor (VEGF) and endogenous inhibitors such as the splice variant of VEGF receptor-1, sVEGFR-1. We hypothesized that inadequate expression of VEGF and up-regulation of VEGFR-1 and its soluble splice variant, sVEGFR-1, restrict capillary growth in pressure-overload hypertrophy.
Methods and results
Neonatal New Zealand White rabbits underwent aortic banding. mRNA (qRT–PCR) and protein levels (immunoblotting) were determined in hypertrophied and control myocardium (7/group) for total VEGF, VEGFR-1, sVEGFR-1, VEGFR-2, and phospho-VEGFR-1 and -R-2. Free VEGF was determined by enzyme-linked immunoassay (ELISA) in hypertrophied myocardium, controls, and hypertrophied hearts following inhibition of sVEGFR-1 with placental growth factor (PlGF). VEGFR-1 and sVEGFR-1 mRNA (seven-fold up-regulation, P = 0.001) and protein levels were significantly up-regulated in hypertrophied hearts vs. controls (VEGFR-1: 44 ± 8 vs. 23 ± 1, P = 0.031; sVEGFR-1: 71 ± 13 vs. 31 ± 3, P = 0.016). There was no change in VEGF and VEGFR-2 mRNA or protein levels in hypertrophied compared with controls hearts. A significant decline in free, unbound VEGF was found in hypertrophied myocardium which was reversed following inhibition of sVEGFR-1 with PlGF, which was accompanied by phosphorylation of VEGFR-1 and VEGFR-2.
Up-regulation of the soluble VEGFR-1 in pressure-loaded myocardium prevents capillary growth by trapping VEGF. Inhibition of sVEGFR-1 released sufficient VEGF to induce angiogenesis and preserved contractile function. These data suggest sVEGFR-1 as possible therapeutic targets to prevent heart failure.
PMCID: PMC3020134  PMID: 20935166
Angiogenesis; Hypertrophy; Angiogenesis inhibitor; Placental growth factor
17.  Overexpression of vascular endothelial growth factor (VEGF) receptors on keratinocytes in psoriasis: regulated by calcium independent of VEGF 
Psoriasis is a common chronic inflammatory disease of the skin characterized by epidermal hyperplasia and angiogenesis. Recently, vascular endothelial growth factor receptors (VEGFRs, including VEGFR-1, VEGFR-2 and VEGFR-3) were found to be expressed in normal human epidermis and associated with proliferation and migration of keratinocytes. The purpose of this study is to investigate the expression of VEGFRs on psoriatic keratinocytes and the roles of calcium and VEGF in regulating VEGFR expression. Skin samples from 17 patients with chronic plaque psoriasis and 11 normal controls were included. The expression of VEGFRs in psoriatic keratinocytes at mRNA and protein levels was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Localization of the VEGFRs in skin lesions was determined by immuno-fluorescent method. Since keratinocyte proliferation and differentiation rely on calcium concentrations, and VEGF is overexpressed in psoriatic epidermis, we further investigated the roles of calcium and VEGF in regulating the expression of VEGFRs. Overexpression of VEGFR-1, VEGFR-2 and VEGFR-3 in psoriatic epidermis was demonstrated both at mRNA and protein levels in vitro. VEGFRs were strongly labeled in non-lesional, perilesional and lesional psoriatic keratinocytes in all viable epidermal stratums in vivo. Furthermore, both exogenous VEGF165 and calcium enhanced the expression of VEGFRs. Calcium also enhanced the expression of VEGF in non-lesional psoriatic keratinocytes, while targeted blockade of VEGF activity by bevacizum-ab could not inhibit calcium-induced up-regulation of protein levels of VEGFRs. We conclude from these results that VEGFRs are overexpressed in lesional psoriatic epidermal keratinocytes. Both calcium and VEGF regulate VEGFRs expression in psoriatic epidermis. More importantly, calcium is a potential regulator for VEGFR independent of VEGF.
PMCID: PMC3822550  PMID: 18419602
psoriasis; keratinocyte; VEGFR; VEGF; calcium
18.  Elevated VEGF Receptor-2 Abundance Contributes to Increased Angiogenesis in VEGF Receptor-1 Deficient Mice 
Circulation  2012;126(6):741-752.
Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. While germline Vegfr-1−/− embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain are apparently healthy.
Methods and Results
We carried out Cre-loxP mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice, and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell (EC) proliferation, and facilitated angiogenesis of blood vessels which matured and perfused properly. Vascular permeability was normal at the basal level, but elevated in response to high doses of exogenous VEGF-A. In the post-infarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/− heterozygosity into Vegfr-1 somatic knockout mice.
Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1 deficient mice.
PMCID: PMC3442373  PMID: 22753193
angiogenesis; vasculature; VEGFR-1; VEGFR-2; retina
19.  Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells 
BMC Cancer  2013;13:229.
Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells.
To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system.
Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines.
Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.
PMCID: PMC3658959  PMID: 23651517
20.  r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction 
PLoS ONE  2010;5(8):e12031.
Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF∶VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF∶VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors.
PMCID: PMC2917360  PMID: 20700512
21.  Targeted systemic therapies for hepatocellular carcinoma: Clinical perspectives, challenges and implications 
Hepatocellular carcinoma (HCC) is a lethal disease in most patients, due to its aggressive course and a lack of effective systemic therapies for advanced disease. Surgical resection and liver transplantation remain the only curative options for a small subset of patients. Few patients with HCC are diagnosed early enough to be eligible for curative treatment. Angiogenesis inhibition is a natural therapeutic target for all solid tumors, but particularly for the highly vascularized HCC tumors. With the approval of the targeted agent sorafenib, there are now additional options for patients with HCC. Although sorafenib does produce some improvement in survival in HCC patients, the responses are not durable. In addition, there are significant dermatologic, gastrointestinal, and metabolic toxicities, and, as importantly, there is still limited knowledge of its usefulness in special subpopulations with HCC. Other angiogenesis inhibitors are in development to treat HCC both in the first-line setting and for use following sorafenib failure; the furthest in development is brivanib, a dual fibroblast growth factor pathway and vascular endothelial growth factor receptor inhibitor. Additional agents with antiangiogenic properties also in phase II and III development for the treatment of patients with HCC include bevacizumab, ramucirumab, ABT-869, everolimus and ARQ 197.
PMCID: PMC3280394  PMID: 22363115
Hepatocellular carcinoma; Angiogenesis; Vascular endothelial growth factor; Fibroblast growth factor; Sorafenib; Tumor response; Brivanib
22.  Pazopanib exposure decreases as a result of an ifosfamide-dependent drug–drug interaction: results of a phase I study 
British Journal of Cancer  2013;110(4):888-893.
The vascular endothelial growth factor receptor (VEGFR) pathway plays a pivotal role in solid malignancies and is probably involved in chemotherapy resistance. Pazopanib, inhibitor of, among other receptors, VEGFR1–3, has activity as single agent and is attractive to enhance anti-tumour activity of chemotherapy. We conducted a dose-finding and pharmacokinetic (PK)/pharmacodynamics study of pazopanib combined with two different schedules of ifosfamide.
In a 3+3+3 design, patients with advanced solid tumours received escalating doses of oral pazopanib combined with ifosfamide either given 3 days continuously or given 3-h bolus infusion daily for 3 days (9 g m−2 per cycle, every 3 weeks). Pharmacokinetic data of ifosfamide and pazopanib were obtained. Plasma levels of placental-derived growth factor (PlGF), vascular endothelial growth factor-A (VEGF-A), soluble VEGFR2 (sVEGFR2) and circulating endothelial cells were monitored as biomarkers.
Sixty-one patients were included. Pazopanib with continuous ifosfamide infusion appeared to be safe up to 1000 mg per day, while combination with bolus infusion ifosfamide turned out to be too toxic based on a variety of adverse events. Ifosfamide-dependent decline in pazopanib exposure was observed. Increases in PlGF and VEGF-A with concurrent decline in sVEGFR2 levels, consistent with pazopanib-mediated VEGFR2 inhibition, were observed after addition of ifosfamide.
Continuous as opposed to bolus infusion ifosfamide can safely be combined with pazopanib. Ifosfamide co-administration results in lower exposure to pazopanib, not hindering biological effects of pazopanib. Recommended dose of pazopanib for further studies combined with 3 days continuous ifosfamide (9 g m−2 per cycle, every 3 weeks) is 800 mg daily.
PMCID: PMC3929878  PMID: 24366297
pazopanib; ifosfamide; drug–drug interaction; sarcoma; pharmacokinetics
23.  Cytotoxicity of VEGF121/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2 
BMC Cancer  2011;11:358.
The fusion protein VEGF121/rGel composed of the growth factor VEGF121 and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF121/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.
We investigated the binding, cytotoxicity and internalization profile of VEGF121/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis.
Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with 125I-VEGF121/rGel demonstrated binding specificity that was competed with unlabeled VEGF121/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF121/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC50 levels between PAE/VEGFR-2 (1 nM) and PAE/VEGFR-1 (100 nM) cells. VEGF121/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF121/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF121/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF121/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response.
Taken together, these data confirm the selectivity of VEGF121/rGel for VEGFR-2-overexpressing endothelial cells and represent the first analysis of genes governing intoxication of mammalian endothelial cells by a gelonin-based targeted therapeutic agent.
PMCID: PMC3176242  PMID: 21849059
24.  Changes in Vascular Permeability and Expression of Different Angiogenic Factors Following Anti-Angiogenic Treatment in Rat Glioma 
PLoS ONE  2010;5(1):e8727.
Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies.
Methodologies and Principal Findings
Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (Ktrans), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased Ktrans at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1α, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors.
These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.
PMCID: PMC2806917  PMID: 20090952
Vascular endothelial growth factor receptor 2 (VEGFR2) is a primary responder to vascular endothelial growth factor signal, and thereby regulates endothelial migration and proliferation. This receptor is expressed in endothelial cells and some vascular tumors, but many reports also detail its expression in carcinomas and lymphomas. VEGFR2 is a potential cell type marker, and data on VEGFR2 expression may also have therapeutic significance in view of recent availability of VEGFR2 inhibitors. In this study we immunohistochemically examined 262 vascular endothelial and 1640 non-vascular tumors and selected non-neoplastic tissues with a VEGFR2-specific rabbit monoclonal antibody 55B11. In early human embryo, VEFGR2 was expressed in endothelia of developing capillaries, thoracic duct, great vessels, hepatic sinusoids, epidermis, and mesothelia. In late first trimester fetus peripheral soft tissues, VEGFR2 was restricted to capillary endothelia, chrondrocytes, and superficial portion of the epidermis. In normal adult tissues, it was restricted to endothelia and mesothelia. VEGFR2 was consistently expressed in angiosarcomas, Kaposi sarcomas, and retiform hemangioendotheliomas. It was detected only in half of epithelioid hemangioendotheliomas (15/27), usually focally. VEGFR2 was strongly expressed in most capillary hemangiomas and weakly or focally in cavernous, venous, and spindle cell hemangiomas, and lymphangiomas. Malignant epithelial mesothelioma was found to be a unique epithelial neoplasm with a strong and nearly consistent VEGFR2 expression, including membrane staining (35/38). Approximately 10% of squamous cell carcinomas and 23% of pulmonary adenocarcinomas contained focal positivity. The only non-endothelial mesenchymal tumors found VEGFR2-positive were biphasic synovial sarcoma (focal epithelial expression), and chordoma. All melanomas and lymphomas were negative. VEGFR2 is a promising marker for malignant vascular tumors and malignant epithelioid mesothelioma. Expression in biphasic synovial sarcoma epithelium, chordoma, and some carcinomas has to be considered in differential diagnosis. Information on VEGFR2 tissue expression may be useful in development of targeted oncologic therapy via VEGFR2-specific tyrosine kinase inhibitors.
PMCID: PMC3310951  PMID: 22314185
angiosarcoma; Kaposi sarcoma; malignant mesothelioma; VEGFR2; endothelial cell; mesothelial cell; fetal tissue; adult tissue

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