Circulating Endothelial Cells Values Research Articles

Distinctive phenogroup to differentiate diagnosis of cardiac myxoma vs cardiovascular disease examining blood-based circulating cell biomarkers

Cardiac myxoma (CM) is a potentially life-threatening disease because frequently asymptomatic or debuts with aspecific manifestations. Definitive diagnosis is established by histopathological assessment including tumor and endothelial cell markers. To derive a specific panel of circulating cells antigenically detectable, pre-surgery peripheral blood samples of CM patients were analyzed. Pre-surgery peripheral blood samples from patients with CM were simultaneously analyzed for Circulating tumor cells (CTCs) and circulating endothelial cells (CECs) that were matched with tumor tissue profiles and with patient-derived xenografts (PDXs) distinguishing tumor regions. Moreover, CECs values in CM patients were further matched with CEC’s levels in cardiovascular disease and control subjects. The blood-derived cytological specimens detected at least 1–3 CTCs/ml in 10 tested CM samples (p = 0.0001) showing specific CM features preserved in the central zones of the tumor. The central zone of the primary tumor, supported by a vessel density rate (55 ± 7%), with a proliferative profile of 32 ± 3% and a percentage of Calretininpos cells (p = 0.03), is the principal site of CTCs (r = 00) dissemination. The subsets of endothelial cells recognized in the blood were indifferent to their topological distribution within the tumor and corresponding PDXs. With further refinement and validation in large cohorts, multiparametric liquid biopsies can optimally integrate clinically informative datasets and maximize their utility in pre-surgery evaluation of CM patients. Blood-derived culture’s protocol provides a versatile method capable of viable analysis of CTCs of non-hematological rare tumors which conventional antibody-mediated analytical platform is unable to perform. Distinctive blood- based cell phenotype contributes to differentiate CM from other differentials assuring its prompt surgical resection by combining blood-based cell biomarkers integrated with clinically informative datasets.

Circulating Endothelial Cell Kinetic in Patients with Multiple Myeloma Who Receive Autologous Hematopoietic Stem Cell Transplantation

Introduction: Neoangiogenesis has a crucial role in multiple myeloma (MM), and circulating endothelial cells (CECs) contribute to neovascularization by inducing tumor progression and metastasis and by repairing damage to bone marrow vasculature after stem cell transplantation (HSC). We recently proved in a national multicenter study the possibility to reach a high-level standardization in CEC count and analysis based on a polychromatic flow cytometry Lyotube (BD). Our study aimed at assessing the kinetics of CECs in patients with MM undergoing autologous hematopoietic stem cell transplantation (Au-HSCT). Methods: Blood samples for analysis were collected at different time points before (T0, T1) and after (T2, T3, T4) Au-HSCT. For each sample, 20 × 106 leukocytes were processed as already described (Lanuti 2016 e 2018) through a multistep procedure. CECs were eventually identified as 7-ADDneg/Syto16pos/CD45neg/CD34pos/CD146pos. Results: Twenty-six MM patients were enrolled in the study. Overall, we observed a constant increase of CECs values from T0 to T3 (day of neutrophil engraftment) followed by decrease at T4 (100 days after transplantation). Using the median value of CECs at T3, we could define a cut-off concentration of 618/mL, with patients with more infective complications having CECs above that value (9/13 vs. 2/13; p = 0.005). Conclusion: CECs value may be a function of endothelial damage caused by conditioning regimen, as suggested by the increase of their level during the engraftment period. A more severe endothelial damage is reflected by the increase of infective complications in patients with higher CECs value at T3.

Circulating Endothelial Cells As a Marker of Vascular Injury in Pediatric Patients

<h3>Introduction</h3> Thrombotic microangiopathy (TMA), hepatic veno-occlusive disease (VOD) and GVHD are major causes of morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. Current strategies to identify these complications involve surveilling markers of immune activation and organ function. Unfortunately, these processes are frequently identified after organ damage has occurred, creating the need for a timelier marker of disease. Circulating endothelial cells (CECs) have been shown to directly reflect vascular injury and are a potential tool for identifying early evidence of endothelial damage. Our study quantifies and characterize CECs in pediatric patients undergoing HSCT and aims to identify the timing of peak vascular injury relative to transplant as well as the relationship between CECs and post-transplant vascular complications. <h3>Methods</h3> CEC analysis was performed prior to and after stem cell infusion. All patients had central venous catheters. CEC identification was done on whole blood using Invitrogen M-450 Tosylactivated Dynabeads coupled to a CD146 antibody. Confirmatory staining after immunomagnetic isolation was done using acridine orange and propidium iodide (AOPI). CEC identification criteria are shown in Figure 1. Two reviewers quantified CECs by morphology and bead number. Negative controls were done using healthy donor blood. <h3>Results</h3> CEC testing was done on 8 HSCT patients (Fanconi Anemia=1, AML=2, ALL=2, ICF-1 syndrome=1, Hemoglobin Hammersmith=1 and macrophage activation syndrome=1). 6 conditioning regimens were myeloablative and 2 were reduced intensity. CEC quantification for individual patients is shown in Figure 2. The vast majority of patients (n=7) had a peak CEC value prior to day 14, including 4 patients whose peak value occurred prior to stem cell infusion. Clinical complications are described in Figure 2. The two highest CEC values recorded were in patients with severe complications, including cardiac arrest (Patient 5, 124 CECs/mL) and death (Patient 7, 84 CECs/mL). The individual difference between lowest recorded CEC value and peak CEC value was also greatest in these patients (Patient 5, 120 CECs/mL; Patient 7, 80 CECs/mL). Mean CEC values for HSCT patients and controls are shown in Figure 3. <h3>Conclusion</h3> CECs reliably peaked within the first 2 weeks of HSCT suggesting marked vascular injury occurs in this timeframe. CEC count peaked prior to stem cell infusion in half of these patients, which implies a large component of vascular injury is driven by the conditioning process. As predicted, the highest CEC values recorded were in patients with grave complications, including one patient who died from multiorgan system failure. Continued data collection will be informative as to whether the nature of these peaks or characteristics of the CECs themselves will predict future vascular complications.

Counting circulating endothelial cells in allo-HSCT: an ad hoc designed polychromatic flowcytometry-based panel versus the CellSearch System

Physio-pathologic interrelationships between endothelial layer and graft-versus-host disease (GVHD) have been described leading to assess the entity “endothelial GVHD” as the early step for clinical manifestations of acute GVHD. The availability of the CellSearch system has allowed us to monitor Circulating Endothelial Cells (CEC) changes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) as useful tool to help clinicians in GVHD diagnostic definition. We have compared CEC counts generated by an ad hoc designed polychromatic-flowcytometry (PFC) Lyotube with those of the CellSearch system. CEC were counted in parallel at 5 timepoints in 50 patients with malignant hematologic disorders undergoing allo-HSCT (ClinicalTrials.gov, NCT02064972). Spearman rank correlation showed significant association between CEC values at all time points (p = 0.0001). The limits of agreement was demonstrated by Bland Altman plot analysis, showing bias not significant at T1, T3, T4, while at T2 and T5 resulted not estimable. Moreover, Passing Bablok regression analysis showed not significant differences between BD Lyotube and CellSearch system. We show that CEC counts, generated with either the CellSearch system or the PFC-based panel, have a superimposable kinetic in allo-HSCT patients and that both counting procedures hold the potential to enter clinical routine as a suitable tool to assist clinicians in GVHD diagnosis.

Circulating endothelial cells as biomarker for cardiovascular diseases

BackgroundEndothelial dysfunction is involved in several cardiovascular diseases. Elevated levels of circulating endothelial cells (CECs) and low levels of endothelial progenitor cells (EPCs) have been described in different cardiovascular conditions, suggesting their potential use as diagnostic biomarkers for endothelial dysfunction. Compared to typical peripheral blood leukocyte subsets, CECs and EPCs occur at very low frequency. The reliable identification and characterization of CECs and EPCs is a prerequisite for their clinical use, however, a validated method to this purpose is still missing but a key for rare cell events. ObjectivesTo establish a validated flow cytometric procedure in order to quantify CECs and EPCs in human whole blood. MethodsIn the establishment phase, the assay sensitivity, robustness, and the sample storage conditions were optimized as prerequisite for clinical use. In a second phase, CECs and EPCs were analyzed in heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction, in arterial hypertension (aHT), and in diabetic nephropathy (DN) in comparison to age‐matched healthy controls. ResultsThe quantification procedure for CECs and EPCs showed high sensitivity and reproducibility. CEC values resulted significantly increased in patients with DN and HFpEF in comparison to healthy controls. CEC quantification showed a diagnostic sensitivity of 90% and a sensitivity of 68.0%, 70.4%, and 66.7% for DN, HFpEF, and aHT, respectively. ConclusionA robust and precise assay to quantify CECs and EPCs in pre‐clinical and clinical studies has been established. CEC counts resulted to be a good diagnostic biomarker for DN and HFpEF.

Changes of Circulating Endothelial Cells and Microparticles after Conventional Chemotherapy in Patients with Extensive Small-Cell Lung Cancer: Relevance to Prognosis and Treatment Response

Objectives: Tumor angiogenesis is controlled by inhibitors and angiogenic factors. However, circulating endothelial cells (CECs) and microparticles (MPs) may be related to tumor angiogenesis in malignant tumors. We therefore investigated the significance of CECs and MPs variation for the prediction of treatment efficacy andprognosis in patients with extensive small-cell lung cancer (SCLC). Materials and methods: Isolation and quantification of CECs from the whole-blood was performed using immune magnetic separation (IMS) technique, while flow cytometry measurement (FCM) was used for enumerating MPs in plasma samples.Results: Twenty-six patients were included in this prospective study. The relative change in CEC and MP counts after treatment, correctly identified treatment response in 22/26 patients (84.6%) and 17/23 patients (73.9%) respectively. The difference in themedian duration of time to progression (TTP) between patient with high relative changes and those with low relative changes in CEC and MP values was not statistically significant (p=0.39 and p=0.2, respectively). By contrast, the increase in CEC and MP levels after chemotherapy tended to be inversely correlated with longer TTP duration (r²=0.111 and r²=0.171, respectively). However, onlym decreased MP values after chemotherapy have the trend to be correlated with longer TTP duration (r²=0.167).Conclusion: The relative change in CEC and MP values after treatment seems to be a useful biomarker for the evaluation of treatment response in patients with extensive SCLC. Moreover, this relative change in both biomarkers might be considered as a prognostic tool in SCLC.

Circulating endothelial cells and microparticles for prediction of tumor progression and outcomes in advanced non-small cell lung cancer.

Circulating endothelial cells (CECs) and microparticles (MPs) are proposed as useful biosensors for angiogenesis and membrane damage in cancer. We investigated their predictive value for progression disease (PD) and clinical outcomes in advanced non-small cell lung cancer (NSCLC) patients treated with cytotoxic chemotherapy. Peripheral blood samples were obtained from 60 patients. Immunomagnetic separation (IMS) and flow cytometry techniques were used to quantify CECs and MPs, respectively. Receiver operating characteristics (ROC) analysis was used to determine the optimal cutoff values for CECs and MPs counts according to their levels in patients with an objective response (OR) and non-responders after treatment. Baseline serum biomarkers levels and their kinetics after chemotherapy were correlated with tumor response and outcomes in advanced NSCLC patients. Forty-seven patients presented an OR after chemotherapy. Of these, 28 patients progressed within three months. Through an increase in their levels during or after chemotherapy, CECs and MPs correctly predicted PD in 57% and 61% of these patients, respectively. Regarding tumor stage, NSCLC patients with stage IV had significantly higher pretreatment CECs and MPs levels than stage III patients (p= 0.037 and 0.018, respectively). Moreover, progression-free survival (PFS) was significantly longer in patients with high baseline CECs levels than those with low pretreatment CECs values (p= 0.05). Moreover, patients with high percentage change in CECs count after chemotherapy had significantly longer time to progression (TTP) duration (p= 0.018). Our findings suggest the increase in CECs and MPs number during or after chemotherapy as predictive biomarkers of tumor progression in advanced NSCLC patients. An association of basal CECs and MPs values with tumor stage was also shown in advanced NSCLC patients. However, baseline CECs levels and their kinetics after chemotherapy seem to be prognostic factors in advanced NSCLC.

NCCTG N1174: Phase I/comparative randomized phase (Ph) II trial of TRC105 plus bevacizumab versus bevacizumab in recurrent glioblastoma (GBM) (Alliance).

2023 Background: TRC105 is a humanized antibody targeting CD105 (endoglin), a member of the TGFβ receptor superfamily. CD105 is expressed in glioma stem cells and circulating endothelial cells (CECs) in GBM patients (pts), increasing following VEGF inhibition. N1174 was conducted in recurrent bevacizumab (bev) naïve GBM pts to investigate the hypothesis that CD105 blockade+bev can delay development of bev resistance. Methods: After a ph I cohort (15 pts) established the ph II dose of TRC105 as 10 mg/kg IV over 4 hours every 7 days in combination with standard dose bev, the ph II trial used 1:1 randomization with 90% power and 0.10 type I error to detect a 3 month (mo) difference in progression-free survival (PFS) between the two arms. CECs including CD105 positive subsets were measured by flow cytometry at baseline and multiple time points. Results: Based on 101 evaluable ph II pts, there was no significant difference in PFS between TRC105+bev and bev only (2.9 vs 3.2 mo, respectively; HR=1.14, 95% CI 0.73-1.77, p=0.57), or overall survival (10.0 vs 7.4 mo; HR 1.02 95% CI 0.63-1.65, p=0.93). Response rate (complete or partial) was 12.2% (6/49) for TRC105+bev and 11.6% (5/43) for bev only. Overall incidence of grade (gr) 3+ toxicity was higher for TRC105+bev (67.3% vs 32.7%, p&lt;0.001) mainly due to 14 pts with gr 3 anemia in the TRC105+bev arm vs 0 in the bev only pts. Gr 3+ non-heme toxicities were higher in pts receiving TRC105+bev vs bev only (50% vs 30.6%, p=0.07), mainly due to headache (6 vs 1 pts) and hyperglycemia (3 vs 0 pts). CECs remained stable in combination treated pts, suggesting a possible impact of the anti-CD105 blockade, but increased at progression in the bev only arm. There was no association between PFS and initial changes in CEC values, independent of treatment received. Conclusions: TRC105 plus bev did not prolong median PFS vs single agent bev in recurrent GBM pts, although it was associated with a non-significant prolongation of OS. These data and associated correlative analysis of CECs from study pts point against endoglin being a clinically significant factor for the development of bev resistance. Support: U10CA180821, U10CA180882. Clinical trial information: NCT01648348.

Prospective assessment of circulating endothelial cells (CECs) as early markers of clear cell renal cell carcinoma (CCRCC) progression in first line setting: The Circles study (SOGUG 2011-01).

TPS4685 Background: Since the identification of Von Hippel Lindau (VHL) gene mutations as a critical step in the development of most Clear Cell Renal Cell Carcinomas (CCRCC), neoangionesis inhibition has become a cornerstone in their treatment. Despite the proven efficacy of antiangiogenic drugs, most patients will not achieve partial response by RECIST criteria. Thus, to accurately determine tumor progression is a challenging question incompletely answered by traditional radiological assessment. In recent years CECs counts have been proposed as surrogate biomarkers of angiogenesis that could be used for monitoring tumor evolution while on targeted therapies. We aim to figure out if CECs elevations could anticipate radiological progression in CCRCC. Methods: An observational prospective study is being performed in 10 institutions members of the Spanish Oncology Genitourinary group (SOGUG). Patients older than 18 years with histologically proven CCRCC on first line treatment with any targeted drug who have not progressed after 3 months of therapy are considered elegible. Recruitment begun on August the 1st 2011 and 15 of the 75 scheduled patients have been included so far. CECs are periodically collected in peripheral blood every 6 weeks for 15 months or radiological tumor progression, whatever occurs first. Median CEC values will be calculated and stated by descriptive statistics (Cellsearch, VERIDEX). To explore the evolution of CECs counts along treatment a linear model will be constructed. An association among CECs counts changes and time to progression will be analyzed with Cox model.

Abstract 372: Circulating endothelial progenitor cell levels predict survival benefit in metastatic renal cell cancer patients treated with antiangiogenic agents

Abstract Background: There is no validated biomarker to predict the efficacy of antiangiogenic therapy in patients with metastatic renal cell carcinoma (MRCC). We tested the hypothesis that circulating endothelial cells (CEC) and circulating endothelial progenitor cell (CEP) could predict clinical outcome in MRCC patients undergoing treatment with tyrosine kinase inhibitors (TKI). Methods: Fifty five patients (pts) with MRCC were monitored both at baseline and day 14 after anti-angiogenic treatment (46 pts received sunitinib and 9, sorafenib). CECs were measured in 1ml erythocyte lysed whole blood by four color flow cytometry (FCM) and identified by a CD45−CD31+CD1467-amino-actinomycin (7AAD)− phenotype. CEPs were measured in 10ml blood after progenitor cell enrichment and were identified by FCM as extremely rare events with a CD45dimCD34+VEGFR2+7AAD− phenotype. Relation between CEC and CEP levels and both response to TKI determined by RECIST criteria after 2 cycles with the Wilcoxon-Mann Whitney test, and progression free survival (PFS) and overall survival (OS) with the Cox model (Log-rank test). Results: CEC and CEP levels at both baseline and day 14 were not correlated to response to TKI. No correlation was found between baseline or day 14 CEC values and PFS or OS. However, pre-treatment CEP levels were correlated to PFS (p=0.01) and OS (p=0.01). Patients with elevated pre-treatment levels of CEP (&amp;gt; 2% of circulating CD34+ cells) had an increased risk of tumor progression and poor prognosis. Changes in CEP levels between day 1 and day 14 were also correlated with PFS (p=0.03). Conclusion: Our results indicate that pre-treatment CEP levels could accurately predict survival in MRCC patients treated with TKI. If these data are confirmed, measurement of CEP levels before TKI therapy in MRCC patients will allow to identify patients who will benefit most from treatment and those who will need to be rapidly switched to another treatment. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 372.

Levels of Circulating Endothelial Cells and Endothelial Progenitors Correlate with Disease Status and Treatment Response in Human Lymphoma Subtypes of CLL and MCL.

Abstract 3941Poster Board III-877 BACKGROUNDIncreases in the number of circulating endothelial cells (CECs) and endothelial progenitors (EPCs) have been reported in various pathological conditions including cancer. Both preclinical and clinical studies in a number of human malignancies have shown that the kinetics of CECs and EPCs correlate with clinical outcomes in patients who undergo anti-angiogenic treatment. Angiogenesis biomarkers in lymphoproliferative diseases have been undefined to date. We investigated the dynamics of circulating peripheral blood levels of endothelial cells and progenitors in lymphoma patients as potential biomarkers of disease status and treatment response. METHODSCirculating CD45-CD146+CD34+CD31+ mature endothelial cells (CECs) and CD45-/dimCD133+VEGFR2+ endothelial progenitors (EPCs) were measured by 4-color flow cytometry in 79 lymphoma patients with chronic lymphocytic lymphoma/leukemia (CLL, n=38) and mantle cell lymphoma (MCL, n=41). Normal volunteers served as controls (n=5). Non-parametric Kruskal-Wallis tests were performed to compare the difference in median marker values of different groups. Mann-Whitney tests were then used to compare marker values in pair-wise fashion between clinical groups. P-values were adjusted for multiple comparisons using the Bonferroni method. RESULTSIn CLL, the CECs were 525 ± 656/mL in previously untreated patients (n=27), and 50 ± 49/mL in patients with remission following fludarabine-based therapy (n=11), compared to 81 ± 51/mL in normal controls (n=5). Significant differences in CECs were observed in patients with untreated disease compared to patients in remission (p=0.0006) and normal controls (p=0.01). EPCs trended down during remission following treatment from 350 ± 932/mL to 45 ± 84/mL (p=0.052). In MCL, CECs were 397 ± 433/mL in previously untreated patients (n=11), 73 ± 85/mL in patients with remission following R-CHOP-based therapy (n=9), and 110 ± 215/mL in patients with relapse (n=21). A significant decline of CECs was detected in remission compared to pretreatment values (p=0.03). In addition, a significant difference in EPCs was noted comparing pre-therapy baseline values (383 ± 445) to normal controls (86 ± 53/mL) (p=0.03). EPCs trended down in remission from 383 ± 445/mL to 103 ± 138/mL (p=0.052). No significant differences were observed for baseline pretreatment CEC and EPC values between CLL and MCL. CONCLUSIONSDynamic levels of circulating endothelial cells and endothelial progenitors appear to correlate with disease status and treatment response in CLL and MCL. Large-scale studies are warranted to further evaluate the prognostic significance of these results, and to investigate whether circulating pro-angiogenic cell populations in selected lymphoma subtypes may be useful as biomarkers to select patients for and monitor response to anti-angiogenic therapy. Disclosures:No relevant conflicts of interest to declare.

Circulating Endothelial Cells in Non-small Cell Lung Cancer Patients Treated with Carboplatin and Paclitaxel

Circulating endothelial cells (CECs) increase in cancer patients and play an important role in tumor neovascularization. This study was designed to investigate the role of CEC as a marker for predicting the effectiveness of a carboplatin plus paclitaxel based first line chemotherapy in advanced non-small cell lung cancer (NSCLC). The CEC count in 4 ml of peripheral blood before starting chemotherapy (baseline value) was significantly higher in NSCLC patients, ranging from 32 to 4501/4 ml (n = 31, mean +/- SD = 595 +/- 832), than in healthy volunteers (n = 53, 46.2 +/- 86.3). We did not detect a significant correlation between the CEC count and estimated tumor volume. CECs were significantly decreased by chemotherapy as compared with pretreatment values (175.6 +/- 24 and 173.0 +/- 24, day +8, +22, respectively). We investigated the correlation between baseline CEC and the clinical effectiveness of chemotherapy. CEC values are significantly higher in patients with clinical benefit (partial response and stable disease, 516 +/- 458, 870.8 +/- 1215, respectively) than in progressive disease patients (211 +/- 150). Furthermore, a statistically significant decrease in CECs, on day 22, was observed only in patients with partial response. Patients who had a baseline CEC count greater than 400/4 ml showed a longer progression-free survival (>400, 271 days [range: 181-361] versus <400, 34 [range: 81-186], p = 0.019). CEC is suggested to be a promising predictive marker of the clinical efficacy of the CBDCA plus paclitaxel regimen in patients with NSCLC.

Circulating endothelial-cell kinetics and viability predict survival in breast cancer patients receiving metronomic chemotherapy

Antiangiogenic agents and therapeutic strategies have entered the clinical oncology arena. The classical tumor size measurements defined to monitor efficacy of chemotherapy, however, might not be appropriate for these newer therapeutics. We previously found that circulating endothelial cells (CECs) were increased in number and more viable in cancer patients compared with control subjects. We investigated the correlation between CEC kinetics and clinical outcome in patients with advanced breast cancer receiving metronomic chemotherapy, a therapeutic strategy associated with antiangiogenic activity and anticancer efficacy. CEC number and viability were measured by flow cytometry in patients and in preclinical models. CECs were decreased in patients for whom no overall clinical benefit (defined as a clinical response or a stable disease) was observed compared with those who had a clinical benefit (P = .015). This difference was due to an increased fraction of apoptotic CECs in patients with a clinical benefit. Univariate and multivariate analyses indicated that CEC values greater than 11/microL were associated with a longer progression-free survival (P = .001) and an improved overall survival (P = .005). Preclinical models indicated that the source of apoptotic CECs was most likely the tumor vasculature. CEC kinetics and viability are very promising as predictors of clinical response in patients undergoing metronomic chemotherapy.

Circulating endothelial cells as a novel marker of angiogenesis.

Measurement of tumor angiogenesis to predict and/or to assess the efficacy of antiangiogenic therapies is mainly based on the evaluation of microvessel density (MVD). We developed a novel flow cytometry procedure to measure circulating endothelial cells (CECs) and circulating endothelial cells progenitors (CECPs) in either preclinical and clinical studies. Preclinical studies were performed on an animal model of human lymphoma. A trend toward higher CECs values was observed on day 7 and 14 after transplant, and differences vs controls were highly significant on day 21 (p = 0.0061). A strong correlation was found between CECs and tumor volume (r = 0.942, p = 0.004) and between CECs and tumor-generated VEGF (r = 0.669, p = 0.02). In mice given cyclophosphamide, most of circulating apoptotic cells were hematopoietic and not endothelial. Conversely, in mice given endostatin, all of the increase in apoptotic cells was in the endothelial cell compartment. In a parallel study, we looked for CECs in the peripheral blood of 20 healthy controls and 76 newly diagnosed cancer patients by means of four-color flow cytometry. In breast cancer (n = 46) and lymphoma (n = 30) patients, both resting and activated CECs were increased by 5 fold (P < 0.0008 vs control). CECs significantly correlated with plasma levels of VCAM-1 and VEGF. Resting and activated CECs were similar to healthy controls in 7 lymphoma patients achieving complete remission after chemotherapy, and activated CECs were found to decrease in 13 breast cancer patients evaluated before and 24h after quadrantectomy. In conclusion, our findings indicate a close relation between CEC increase and tumor progression, and support CECs evaluation as a clinically relevant, non invasive angiogenesis marker. Furthermore, this assay offers insight into anti-angiogenic activity of different drugs.