Number Of Circulating Endothelial Cells Research Articles

Phase I Trial of Sorafenib Following Liver Transplantation in Patients with High-Risk Hepatocellular Carcinoma

Liver transplantation offers excellent long-term survival for hepatocellular carcinoma (HCC) patients who fall within established criteria. For those outside such criteria, or with high-risk pathologic features in the explant, HCC recurrence rates are higher. We conducted a multicenter phase I trial of sorafenib in liver transplantation patients with high-risk HCC. Subjects had HCC outside the Milan criteria (pre- or post-transplant), poorly differentiated tumors, or vascular invasion. We used a standard 3+3 phase I design with a planned duration of treatment of 24 weeks. Correlative studies included the number of circulating endothelial cells (CECs), plasma biomarkers, and tumor expression of p-Erk, p-Akt, and c-Met in tissue micro-arrays. We enrolled 14 patients with a median age of 63 years. Of these, 93% were men and 71% had underlying hepatitis C virus (HCV) and 21% had HBV. The maximum tolerated dose of sorafenib was 200 mg BID. Grade 3-4 toxicities seen in >10% of subjects included leukopenia (21%), elevated gamma-glutamyl transferase (21%), hypertension (14%), hand-foot syndrome (14%) and diarrhea (14%). Over a median follow-up of 953 days, one patient died and four recurred. The mean CEC number at baseline was 21 cells/4 ml for those who recurred, and 80 cells/4 ml for those who did not (p=0.10). Mean soluble vascular endothelial growth factor receptor-2 levels decreased after 1 month on sorafenib (p=0.09), but did not correlate with recurrence. There was a trend for tumor c-Met expression to correlate with increased risk of recurrence. Post-transplant sorafenib was found to be feasible and tolerable at 200 mg PO BID. The effect of post-transplant sorafenib on recurrence-free survival is potentially promising but needs further validation in a larger study.

CD34+/CD144+ Circulating Endothelial Cells as an Indicator of Carotid Atherosclerosis

The relationships between the number of circulating endothelial cells (CECs) or endothelial progenitor cells (EPCs) and indicators of carotid atherosclerosis, such as the intima-media thickness (IMT) and plaque score are not well characterized in patients with chronic ischemic stroke. The objective of this study was to investigate these relationships in patients with chronic ischemic stroke and in patients with risk factors for stroke. A total of 58 patients (69.6 ± 10.0 years, 21 females) with chronic ischemic stroke or with risk factors for stroke were included in this study. IMT was measured using an IntimaScope, and the numbers of CECs and EPCs were measured using flow cytometry. CECs and EPCs were defined as CD34+/CD144+ and CD34+/CD133+ cells, respectively. The number of CECs in patients with large artery atherosclerosis was higher than that in patients with cardioembolism or small vessel occlusion (P < .05). In contrast, there were no significant differences in the number of EPCs between groups. A positive correlation was also observed between the plaque score and the number of CECs (r(2) = .139, P < .05, n = 36). Moreover, the number of CECs in patients with moderate and severe atherosclerosis (.32 ± .11/μL, n = 22) was higher than that in patients with no plaque and mild atherosclerosis (.25 ± .07/μL, n = 34, P < .05). The number of CECs was high in patients with large artery atherosclerosis who experienced chronic ischemic stroke. And this number may reflect severity of carotid atherosclerosis.

Circulating Endothelial Cells Are Increased in Post-Thrombotic Syndrome Patients

BACKGROUNDPost-thrombotic syndrome (PTS) is a common complication presented by patients with lower limbs deep vein thrombosis (DVT) that is characterized by pain, edema, cramps, itching, paresthesia, hyperpigmentation, and/or wound. PTS results in significant disability, and patients with severe degrees have a quality of life comparable to patients with cancer. From 20-50% of proximal DVT patients develop PTS despite optimal anticoagulant therapy, and between 5 to 10% of them present severe PTS. The diagnosis is based on clinical signs and several clinical classifications have been applied to graduate its severity, whereas the ISTH recommends the use of Villalta scale. PTS physiopathology remains unclear, but the inflammatory response to acute thrombosis, as well as to the fibrosis of venous valves and walls, seem to play an important role. Circulating endothelial cells (CEC) are infrequently detected cells derived from vascular wall or recruited from bone marrow. They have been related to hemostasis, platelet and leukocytes interaction to the vessel wall, and endothelial injury. Increased numbers of CEC are observed in ischemia, vascular trauma and have been associated to angiogenic potential. The aim of this study was to analyze CEC numbers in patients with a history of previous DVT and PTS, comparing them with DVT patients without PTS and healthy controls. We also aimed to compare CEC levels among patients with different degrees of PTS severity. Plasma D-dimer and serum IL8 levels were also determined, respectively by turbidimetric and nephelometric methods. PATIENTS AND METHODSCEC percentage were determined in PTS patients (N=19), which were subdivided according to their score on Villalta scale; DVT patients with no post-thrombotic syndrome (N=9) and healthy controls (N=19). PTS patients and controls were matched by gender, ethnic origin and age ±5 years, and were also included. Blood (20mL) were collected in EDTA vacuum tubes, and 100μL of whole blood (with leukocyte counting between 5-10x103/μL) were used on flow cytometry analyzes. Different antibodies were included in order to better detect CEC, and two different panels were applied (panel 1: CD45-, CD144+, CD31+, CD133-; panel 2: CD45-, anti-VEGFR2+, CD31+, CD133-). RESULTSPatients with PTS presented higher percentage of CEC, comparing to the other groups. Mild PTS patients presented higher levels of CEC using both panels in comparison to patients without PTS (P= 0.015, P= 0.019 respectively) as well as to CTR (P= 0.077, P= 0.040 respectively). The moderate+severe PTS patients presented higher levels of CEC using panel 2 both in comparison to patients without PTS (P= 0.008) and CTR (P= 0.013). CD144+ cells were slightly increased in moderate+severe PTS patients comparing to patients without PTS (P= 0.054). Both IL8 (med= 21.85 vs med=13.84 pg/mL; P=0.011) and D-dimer (med= 0.475 vs med= 0.29mg/L; P=0.044) were increased in PTS patients in comparison to patients without PTS, respectively. As CEC could eventually be derived from the residual venous thrombosis (RVT), we also dichotomized patients between those with and without RVT. The levels of CEC were similar between patients with or without RVT. CONCLUSIONSOur results suggested that PTS may be associated with higher CEC levels. It is believed that CEC has angiogenic properties and that they may be increased on oxidative stress situations. In this context, our findings may suggest that CEC takes part on vascular-mediated PTS pathophysiology. Also the higher levels of D-dimer and IL8 may indicate that PTS patients were experiencing a pro-inflammatory condition. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of circulating endothelial cells in the rat after acute and fractionated whole-body gamma irradiation

Abstract Purpose: Damage to vascular endothelial cells is a well recognised complication of the irradiation. Our objective was to determine the gamma-irradiation effect on the rat circulating endothelial cells (CEC). Material and methods: Eight-week old rats were divided into four groups: group 1 - rats were exposed to acute whole- -body gamma irradiation with a wide range of single doses (0.5, 1, 2, 4 and 8 Gy), group 2 - rats were exposed to fractionated low doses of irradiation (0.1, 0.5 and 1 Gy) every three days for two months, group 3 as group 2, but followed by two months of rest, group 4 were control animals. CEC (CD146 positive cells) in group 1 were counted following CD146-based immuno-magnetic separation after one day and one week, as well as at the end of experiment in the other groups. Results: Quantified CEC showed that there was a dose-dependent reduction in CEC count in group 1 (one week after irradiation) and group 2. A partial re-population of CEC was observed at the end of experiment in both group 1 and group 2 compared to control group. Group 3 showed a significant increase in CEC levels as compared with group 2 without reaching the control level. Conclusion: The number of CEC (CD146 positive cells) in rats exposed to whole-body gamma irradiation was reduced in a dose-dependent manner and it partly recovered during the two-month interval after irradiation. We suggest that CEC count may be an indicator of the radiation-induced vascular damage.

Quantification of circulating endothelial cells as a predictor of response to chemotherapy with platinum and pemetrexed in patients with advanced non-squamous non-small cell lung carcinoma.

Circulating endothelial cells (CEC) play an important role in tumor neovascularization and may have prognostic value in cancer patients. This study was designed to investigate the role of CEC as a marker for predicting platinum plus pemetrexed first-line chemotherapy efficacy in advanced non-squamous non-small cell lung cancer (NSCLC). A prospective study was performed whose main objective was to study whether the numbers of CEC at baseline and prior to the second and third cycle of chemotherapy were response predictors. Sixty-nine patients received cisplatin plus pemetrexed, and peripheral blood samples were performed at baseline and after second and third cycle. Separation and CEC count were performed using inmunomagnetic separation (CellSearch). The CEC count in 4 mL of peripheral blood was obtained prior to the first, second, and third cycle of treatment. Baseline levels and evolution of CEC were correlated with response to treatment according to RECIST criteria after three cycles of treatment. Sixty-nine patients were included: 43 (64.2 %) received cisplatin/pemetrexed and 24 (35.8 %) carboplatin/pemetrexed. Range of baseline CEC: 8-965 (mean of 153 cel/4 mL). The results after 3 cycles were: 25 partial responses (36.2 %), 17 cases of stabilization of disease (24.6 %), 16 of progressive disease (23.2 %) and 11 non-evaluables (16 %). No significant relationship between the baseline CEC count and response was found (p value = 0.831). Increase >50 % between the first and second cycle was correlated significantly with progression disease (p = 0.008). Patients who had a baseline CEC count greater than the mean (>153 cells/4 mL) showed longer progression-free survival and global survival without statistical significance. In this homogeneous group of patients with NSCLC, there is no correlation between response to treatment and CEC baseline levels. The increase in CEC numbers after the first cycle could be a negative predictive factor.

Abstract 645: 20-HETE and Circulating Endothelial Cells, a Unique Vascular Profile Related to CVD in Morbidly Obese Appalachian Women

Background: A high BMI increases levels of metabolic syndrome biomarkers, including; Leptin in Appalachian women. Vascular dysfunction is persistent in the development of atherosclerosis and determined by the levels of inflammatory markers, circulating endothelial cells (CEC), 20-HETE (a powerful vasoconstrictor) and serum adiponectin levels. We have previously described the presence of a marked increase in CEC in both humans and mice with hypertension, obesity and diabetes. We hypothesize that a subject with high BMI would present with increased levels of CEC and 20-HETE concomitant with a marked reduction in serum adiponectin. Methods: Serum levels of adiponectin and 20-HETE were measured in female of Appalachian subjects. The effect of 20-HETE on 3T3 adipogenesis was also measured. Mature CEC were isolated from peripheral blood using anti-CD146-coated immunomagnetic Dynabeads™. Results: Subjects with a BMI between 45 to 72, had an elevated number of CECs (mean 79, range 49-116 cells/ml of peripheral blood) as compared to (mean 29, range 21-46 cells/ml peripheral blood , p&lt;0.05) subjects with BMI of 32-46 and in non-obese subjects with BMI of 17-32, (mean 3, range 2-9). Lipidomic analysis revealed females with high BMI (50-72) displayed a significant increase in the CYP-derived 20-HETE when compared to subject with lower BMI (32-45) (p = 0.005). Treatment of 3T3 cells with 20-HETE decreased serum adiponectin levels and increased TNF α and adipogenesis by 38% and 76%, respectively (p&lt;0.05). These results implicate both 20-HETE and oxidative stress as factors in the development and pathogenesis of BMI-related diseases in individuals with a high BMI. Conclusions: Increased BMI in Appalachian females is associated with an increase in CEC levels accompanied by an increase in 20-HETE expression and a decrease in adiponectin in peripheral blood. This represents a novel mechanism by which individuals with high BMI and controlled blood pressure remain sensitive to the development of atherogenesis and vascular dysfunction. It is the responsibility of the physician to recognize the medical problems that are endemic to the Appalachian population and to counsel the patient regarding these risks of CVD.

Effect of Scurrula atropurpurea on nitric oxide, endothelial damage, and endothelial progenitor cells of DOCA-salt hypertensive rats.

To know whether Scurrula atropurpurea is able to modulate total plasma nitrate/nitrite levels, decrease endothelial damage, and increase endothelial progenitor cells (EPCs) in hypertensive rats. The rats were divided in 5 groups: control (normotensive) group, Desoxy cortico sterone (DOCA)-salt hypertensive group, and three DOCA-salt hypertensive groups. All 5 groups received methanolic extract of S. atropurpurea (MESA) at a dosage of 50; 100; and 200 mg/KgBW. Serum nitric oxide (NO) was assayed by colorimetric. Circulating endothelial cells (CECs) and EPCs were assayed using flow cytometry. The administration of MESA100 and MESA200 elevated the total plasma nitrate/nitrite levels but cannot reach the level in control group. MESA100 and MESA200 also elevated the EPCs number compared with hypertensive group. The administration of MESA significantly (P< 0.05) decreased the CECs number compared to hypertensive groups. Methanolic extract of S. atropurpurea is able to modulate total plasma nitrate/nitrite levels and diminish endothelial damage via increasing EPCs.

Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay), to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs. Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined by positive staining for DAPI, CD146 and von Willebrand Factor and negative staining for CD45. In addition, CECs exhibited distinct morphological features that enable differentiation from surrounding white blood cells. CECs were found both as individual cells and as aggregates. CEC numbers were higher in MI patients compared with healthy controls. VS patients had lower CEC counts when compared with MI patients but were not different from healthy controls. Both HD-CEC and CellSearch® assays could discriminate MI patients from healthy controls with comparable accuracy but the HD-CEC assay exhibited higher specificity while maintaining high sensitivity. Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

Circulating endothelial cells and chronic kidney disease.

Endothelial dysfunction may play a crucial role in initiation of the pathogenesis of vascular disease and atherosclerosis. The identification and quantification of circulating endothelial cells (CEC) have been developed as a novel marker of endothelial function. We describe, in great detail, mechanisms of endothelial dysfunction and CEC detachment. We also review the relationship between numbers of CEC and disease severity and response to treatment. In addition, we describe the possible clinical use of CEC in chronic kidney disease (CKD) and kidney transplantation. In summary, CEC have been developed as a novel approach to assess the endothelial damage. Measurement of the CEC level would provide an important diagnostic and prognostic value on the endothelium status and the long-term outcome of vascular dysfunction.

Abstract P1-08-21: Detection and monitoring of circulating endothelial cells, circulating tumor cells and disseminated tumor cells during neoadjuvant breast cancer treatment including bevacizumab

Abstract Background: Results from studies with bevacizumab in addition to traditional neoadjuvant therapy (NAT) indicate a need for predictive biomarkers. As a sub-study of the NeoAva study (a neoadjuvant study), the aim was to investigate the potential association between the presence of circulating endothelial cells (CECs) in peripheral blood (PB), circulating tumor cells (CTCs) in PB and disseminated tumor cells (DTCs) in bone marrow (BM) at different time points during NAT +/- bevacizumab and at one year follow-up, and therapy response. Patients and methods: A total of 150 HER2-negative patients with cT2-4 (≥ 2.5cm) N0-3 M0 status were randomized to receive NAT with or without bevacizumab. In this sub-study, 90 patients have so far been analyzed. Of these, 82 received chemotherapy (FEC100→taxane) and 8 received endocrine therapy (letrozole) + / - bevacizumab. Therapy response was evaluated according to the RECIST criteria and achievement of pathological complete response (pCR). Number of CECs, CTCs and DTCs were assessed at baseline after 12 weeks, at surgery (after 24 weeks) and at one year follow-up. Blood samples were analyzed by CellSearch® to assess CEC and CTC counts. The detection of DTCs was performed by immunocytochemical analysis of 2 × 106 BM mononuclear cells. Results: The pathological complete response rate was 10 out of 90 (11.1%), eight of these patients received bevacizumab. For bevacizumab-treated patients with a change in CEC counts from baseline to time of surgery below median change (27 CECs), 35% (6/17) achieved pCR compared to 6% (1/18) in the group with a CEC count-increase above median change (p = 0.035). The corresponding pCR rates for patients not receiving bevacizumab (median CEC change 131 CECs) were 0% (0/15) and 13% (2/14), respectively. Stepwise testing of thresholds for CEC changes in the bevacizumab-arm revealed significant associations to pCR for change-values between 20 and 40. CTC- and DTC-status or -changes were not associated with tumor response or CEC changes. Conclusion: The presented results indicate that the level of change in the number of circulating endothelial cells during neoadjuvant therapy including bevacizumab is associated with the pathological complete response rate in breast cancer patients. This supports additional testing of CECs as a surrogate marker for response to this treatment. The analyses will be up-dated with results from the rest of the included patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-21.

Circulating Endothelial Cells As a Reliable Marker Of Endothelial Damage In Children Undergoing Hematopoietic Stem Cell Transplantation

The endothelial dysfunction associated with Hematopoietic Stem Cell Transplantation (HSCT) is responsible for pleomorphic complications that arise during the post-tranplant period: sinusoidal obstruction syndrome (SOS), pulmonary arterial hypertension (PAH), capillary leak syndrome (CLS) and transplantation-associated thrombotic microangiopathy (TA-TMA). Numerous factors may induce these endothelial damages. Circulating endothelial cells (CEC) emerge as a potent marker of endothelial lesion in a variety of disorders but their monitoring in HSCT has been little investigated so far. MethodsThirty-six pediatric patients treated in the department of Immuno-Hematology at the Necker-Enfants-Malades Hospital who received allogeneic HSCT after full intensity conditioning regimen for non-malignant disorders including inherited immunodeficiencies, metabolic diseases or hemoglobin disorders were enrolled in the study between April 2011 and December 2012. CEC were monitored at 5 time points: before conditioning (day -15), at time of transplant (day 0) and during the post-transplantation period (at day +15, day +30 and eventually day +60 if the patient was still hospitalized). Blood samples were not collected in period of sepsis to avoid any confounding factor7. ResultsAmong the thirty-six patient enrolled, four patients were excluded because of incomplete follow-up. Thirty-two patients completed the study, and their data were analyzed.Vascular complications occurred in 10 patients (31% patients) and are detailed in Figure 1C. Of note, all patients who developed SOS were under defibrotide prophylaxis. Two patients developed CLS in association with grade IV GVH. TA-TMA occurred in two patients and resolved in both after cyclosporine withdrawal.Patients with vascular events (VE) had significant higher CEC counts as compared to patients without endothelial complication at day +15 (p = 0.0122) and day +30 (p=0.0046). The increase of CEC counts was significantly higher in patients with VE during the first 15 days (p=0.0175) and during the first month post transplantation (p=0.0005) as compared to patient without VE. Strikingly, detection of high CEC counts in patients with major vascular events (ie. PAH, CLS and TA-TAM) was preceding the onset of clinical manifestations by a median of 18.5 days (range, 8-42).We didn't find any correlation on univariate analysis between CEC counts and the following variables: the underlying disease, the intensity of conditioning regimen, the existence of CMV reactivation, graft versus host disease or death. ConclusionWe observed that the occurrence of vascular complications is associated with an early rise (in the first 15 days) of CEC count. Interestingly, the increase in the number of CEC is preceding the onset of major endothelial complications. Thus, monitoring of CEC in routine could be a useful prognostic tool that could allow early therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Circulating endothelial cells in pediatric renal transplant recipients

An increase in the number of circulating endothelial cells (CEC) indicates endothelial damage and the risk of cardiovascular disease. The aim of our study was to investigate the association of CEC with various clinical parameters in pediatric renal transplant recipients. CEC, defined as CD45(-)CD146(+), were enumerated by flow cytometry from the peripheral blood of 50 pediatric renal transplant recipients and 20 healthy controls. Clinical parameters, including renal function tests, fasting blood glucose, serum cholesterol and triglyceride, cyclosporine A (CsA) (trough and 2nd-hour) and tacrolimus (tac) trough blood levels and their association with CEC numbers were analyzed. CEC numbers of patients were higher than those of controls (respectively, 128 ± 89 cells/ml (42-468 cells/ml), 82 ± 33 cells/ml (32-137 cells/ml), p = 0.024). There was a statistically significant negative correlation between CEC numbers and glomerular filtration rate (GFR) (r = -0.300, p = 0.012). There was also a statistically positive association between CEC numbers and transplant duration as well as cyclosporine trough level (respectively, r = 0.397, p = 0.004, r = 0.714, p = 0.004). CEC numbers in patients on tac and CsA were similar (p = 0.716). Our results demonstrate that renal transplant recipients with high CsA trough blood level, longer transplant duration, and lower GFR, are at greater risk of developing endothelial damage.

Abstract 386: Body Mass Index Exacerbates the Hypertension Mediated Increase in Endothelial Cell Sloughing and Suppression of Antioxidant Heme Oxygenase-1

Introduction: Endothelial Progenitor cells (EPCs) are bone marrow derived cells that migrate and differentiate into mature endothelial cells and play a significant role in the re-endothelialization and neovascularization of injured endothelium and restoration of vascular function. We examined whether obesity and hypertension exacerbates the levels of biomarkers including circulating endothelial cells (CEC), serum inflammatory cytokines, and the levels of heme oxygenase -1 (HO-1) in EPC. Methods: Peripheral blood from 10 normal, 15 obese, 12 hypertensive, 20 obese-hypertensive and 15 diabetic patients was analyzed for inflammatory cytokines, CEC number, adiponectin and HO-1 levels. Results: The levels of inflammatory cytokines increased with BMI and directly correlated with increasing obesity. Similarly, hypertensive patients have elevated CEC which are further increased in obese hypertensive patients (p&lt;.05). HO-1 was reduced (p&lt;.05) in both hypertensive and obese patients when compared to control. Similarly serum adiponectin levels were lower in hypertensive obese patients when compared to controls (p&lt;.01). Inflammatory cytokines IL-1, IL-6, MCP-1 and TNFα were elevated in obese hypertensive patients compared to non-obese hypertensive patients (p&lt;.05). Conclusion: We demonstrated in hypertension patients that obesity exacerbates vascular dysfunction and increases circulating endothelial cells and inflammatory cytokines. A reduction in the levels of HO-1 and adiponectin imply reduced antioxidant levels which are contributing factors towards vascular and adipocyte dysfunction in hypertension patients. Thus upregulation of HO-1 offers a potential therapeutic approach in the treatment of this population.

Pilot study of vascular health in survivors of osteosarcoma

Cardiovascular-related toxicities have been reported among survivors of osteosarcoma. Fasting blood samples from 24 osteosarcoma survivors were analyzed for high-sensitivity C-reactive protein (hsCRP), triglycerides, total cholesterol, high-density lipoprotein (HDL), apolipoprotein-ß, lipoprotein (a), fibrinogen, circulating endothelial cells (CECs), and surface expression of vascular cell adhesion molecule-1 (VCAM-1). Values were compared to subjects in the natural history Coronary Artery Risk Development in Young Adults (CARDIA) cohort study except for CECs and VCAM-1 expression, which were compared to controls studied at the University of Minnesota Lillehei clinical trials unit. Survivors (54.2% male), median age 18 years (9-32) at diagnosis, 36.5 years (20-56) at evaluation were treated with a variety of chemotherapeutic exposures, all but one were exposed to doxorubicin (median dose 450 mg/m(2) ; range: 90-645 mg/m(2)), 14 (58.3%) received cisplatin, and 3 (12.5%) were exposed to carboplatin. Two survivors (8.3%) received radiation therapy for disease relapse. Compared to CARDIA subjects, mean hsCRP (3.0 mg/L ± 2.0 vs. 1.6 ± 2.3), triglycerides (151 mg/dl ± 81.7 vs. 95.4 ± 101.3), lipoprotein (a) (34.9 mg/dl ± 17.7 vs. 13.8 ± 22.0), and fibrinogen (315.0 mg/dl ± 49.3 vs. 252.4 ± 61.7) were significantly elevated. The number of CECs (0.47 cells/ml ± 2.5 vs. 0.92 ± 2.5) did not differ while surface expression of VCAM-1 (86.4% ± 34.0 vs. 42.1 ± 33.8) was significantly elevated compared to controls. Among survivors of osteosarcoma, assessed a median of 14 years from diagnosis, there is evidence of vascular inflammation, dyslipidemia, and early atherogenesis.

A phase II clinical trial of neoadjuvant therapy with zoledronic acid for operable breast cancer.

e22027 Background: The neoadjuvant use of bisphosphonates has been reported to increase rates of pathological response in patients with operable breast cancer. We conducted a study to determine whether treatment with zoledronic acid (ZOL) would improve outcomes in such patients when it was added to standard neoadjuvant chemotherapy (NAC). Methods: From April 2010 to June 2012, 30 patients with HER2-negative operable breast cancer were eligible for the study. The patients received NAC (4 cycles of CEF: intravenous epirubicin 100mg/m2, cyclophosphamide 500mg/m2, and fluorouracil 500mg/m2 on day 1, then 12 cycles of weekly paclitaxel 80mg/m2) with ZOL. The ZOL was administered after each cycle of NAC in a 4-mg dose by intravenous infusion every 3 to 4 weeks. The primary end point of the study was pathological response (pPR) rate. The secondary end points were pathological complete response (pCR) rate, adverse effects (AEs) and the changes in the numbers of disseminated tumor cells (DTC), circulating tumor cells (CTC), and circulating endothelial cells (CEC). We evaluated the efficacy and safety of ZOL in the NAC in patients by use of Fisher’s exact test. Results: 29 patients completed surgery after NAC, and pathological analyses and the numbers of CTC and CEC were assessed. Two patients had progressive disease during NAC, with brain metastasis and bone metastasis, respectively. The pPR rate, pCR rate, was 31.0%, 10.4%, respectively. CTC were detected in 3 patients at baseline, and CTC had changed to positive in one patient in D15 of the first cycle, then after NAC, all four patients changed to negative. CTC were negative in two patients at baseline, but after NAC, changed to positive. There were no factors associated with the change rates of CTC. CEC after NAC increased from baseline significantly (p&lt;0.001). The increase rates of CEC were not significantly different among premenopausal and postmenopausal, PR and non-PR, and luminal and triple negative patients. There were no severe AEs associated with ZOL to discontinue treatment. Conclusions: These data suggested ZOL in combination with NAC might not have a significant direct anti-tumor effect. Change of CEC would be a biomarker for NAC, but further analyses would be necessary. Clinical trial information: 000003498.

Circulating endothelial cell kinetics and their potential predictive value during mobilization procedure

Circulating endothelial cells (CECs) in patients with hematological malignancies are assessed as a noninvasive marker of angiogenesis. The aim of this study was to evaluate the numbers of CECs and their subsets during mobilization of hematopoietic stem cells. Thirty-eight patients were enrolled to the study (19 females and 19 males) at median age of 56.5 years. The group consisted of patients with multiple myeloma (26), lymphoma (10), and acute myeloid leukemia (2). Blood samples were collected before chemotherapy (0), 1 day after chemotherapy (Cht+1), on the day G-CSF commenced (G0), after 1 day of G-CSF (G+1), and on the day of the first apheresis. CECs were evaluated by four-color flow cytometry. Circulating progenitor cells were defined as CD45-/CD34+/CD31+/CD133+. Apoptotic CECs (ApoCECs) were defined as CD146+/AnnexinV+. Median (Me) CECs number was 10.5/µl and it decreased after chemotherapy (Me = 8.3/µl, P < 0.001 when compared with baseline). Based on the number of aphereses needed to obtain 2 × 10(6)/kg CD34+ cells, patients were divided into "highly efficient" (one apheresis) and "poorly efficient" mobilizers (two or more aphereses). Median ApoCEC at Day G+1 was lower in highly efficient than in poorly efficient mobilizers (Me = 3.1/µl vs. Me = 5.1/µl, P = 0.02). ApoCEC at Day G+1 correlated with the number of aphereses (r = 0.48, P = 0.03). In multivariate analysis, ApoCEC at Day G+1 was an independent factor for successful mobilization during one apheresis. CECs and their subsets change significantly during mobilization of HSCs. ApoCECs measured at the time of G-CSF commencement can predict the efficacy of HSC collection.

The kinetics and apoptotic profile of circulating endothelial cells in autologous hematopoietic stem cell transplantation in patients with lymphoproliferative disorders

In neoplastic disorders, endothelial cells take part in tumor progression and also influence the recovery of hematopoiesis after high-dose chemotherapy. Measurements of circulating endothelial cells (CEC), their subsets and kinetics were taken in patients with lymphoid malignancies (37 multiple myeloma, ten lymphoma) during autologous hematopoietic stem cell transplantation (HSCT). CEC were evaluated by four-color flow cytometry at different time points. Additionally levels of angiopoietins 1 and 2 were evaluated by ELISA assay. The baseline number of CECs and their subsets in patients were higher than in the control group. The median CEC number dropped significantly after transplantation (from 9.5/μL to 6.2/μL, p < 0.001). Apoptosis of CECs 24 h after chemotherapy was enhanced in comparison to baseline values (median apoptotic CEC number 4.15/μL vs 3.1/μL; p < 0,001). The time for neutrophil engraftment was shorter for patients with a low apoptotic CEC count at baseline as compared to those with a high apoptotic CEC count (median time to engraftment 13 vs. 16 days respectively, p = 0.04). We observed an adverse correlation of progenitor CEC numbers measured 1 h after transplantation with the time to neutrophil engraftment (r = -0.49, p = 0.008). We also found a negative correlation between the number of CECs originating from microvessels measured 1 h after transplantation, and the time to neutrophil engraftment (r = -0.39, p = 0.04). Baseline angiopoietins 1 and 2 concentration did not influence the post-transplant regeneration time. CEC numbers significantly change during autologous HSCT. Our results suggest that progenitor CECs and CECs derived from microvessels both take part in successful engraftment.

Abstract 4424: Nonclinical safety evaluation of MEDI0639 (Anti-DLL4 Mab) to support first time In human: linking DLL4-notch signaling blockade to exaggerated pharmacology effects in cynomolgus monkeys.

Abstract Delta-like ligand 4 (DLL4)-Notch signaling regulates several key stages of angiogenesis. MEDI0639 is a human IgG1 antibody that blocks the binding of DLL4 to the Notch receptor and is being developed as a potential anticancer therapy for patients with solid tumors. Cynomolgus monkeys were selected as a pharmacologically relevant species for toxicologic evaluations of MEDI0639. The results of these studies showed dose-dependent MEDI0639-related serious adverse effects associated with possible gastrointestinal bleeding and heart failure. In general, these adverse effects were monitorable, reversible and consistent with the likely pharmacologic effects on vascular homeostasis. Pathologic changes were observed in liver, heart, lung, and thymus. Changes in clinical pathology parameters, such as decreased red blood cell (RBC) mass, were consistent with possible blood loss and responsive erythropoiesis. Elevations in levels of liver enzymes were indicative of hepatocellular injury and were consistent with histopathologic findings in liver. Due to the adverse cardiovascular (CV) findings from repeat-dose toxicity studies, the effects of MEDI0639 on the CV system were characterized in a dedicated CV safety pharmacology study in cynomolgus monkeys. Notable findings from this study were increased levels of C-reactive protein (CRP), blood pressure (BP), and heart rate (HR) which tended to return to baseline levels during an 8-week dose-free recovery period. In addition to the evaluation of these toxicity parameters, the potential pharmacodynamic (PD) effects of MEDI0639 on circulating endothelial cells (CECs) in whole blood and on RNA expression levels of selected genes in the DLL4 and angiogenesis pathways in samples of colon tissue were assessed. Results showed that mRNA expressions levels of DLL4 and Notch4 were up-regulated when normalized to housekeeping genes while expression levels of Jagged1, Hey1, and Hes1 were down regulated when normalized to the EC surface marker PECAM (CD31) in all MEDI0639-treated groups. The effects of MEDI0639 on EC were confirmed by the observed increase in expression of PECAM in colon tissue and the increased numbers of total, proliferating, and apoptotic CECs in whole blood of MEDI0639-treated animals. Overall, MEDI0639-related adverse effects were monitorable, reversible and consistent with exaggerated pharmacology effects - that is DLL4 blockade is expected to lead to the formation of immature, disorganized blood vessels with inadequate mural cell coverage. This data was used to determine an appropriate starting dose for a FTIH Phase 1 clinical study of MEDI0639 in cancer patients. Citation Format: Patricia C. Ryan, Jiaqi Huang, Haifeng Bao, Song Cho, Philip Brohawn, Patricia Burke, Kim Lehmann, Fernanda Pilataxi, Yihong Yao, Kathleen McKeever, Rakesh Dixit. Nonclinical safety evaluation of MEDI0639 (Anti-DLL4 Mab) to support first time In human: linking DLL4-notch signaling blockade to exaggerated pharmacology effects in cynomolgus monkeys. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4424. doi:10.1158/1538-7445.AM2013-4424

Circulating endothelial cell levels in psoriatic patients and their modification after an anti‐TNF‐alpha (Etanercept) treatment

Endothelial function in psoriatic patients has been mainly evaluated through a high-resolution ultrasound measurement of flow-mediated vasodilation in the brachial artery, which is an operator-dependent and technically demanding technique: this characteristic, together with different patient selection criteria, could account for the conflicting results emerging from different studies. Recently, Circulating Endothelial Cells (CECs) level has been suggested as a novel biomarker of vascular injury. The number of CECs was determined by a semi-automated immunomagnetic system (CellSearch system) in peripheral blood of psoriatic patients (n = 48) and healthy subjects (n = 50). In 15 patients, CEC level was also evaluated after 6 months of treatment with an anti-TNF-alpha agent, Etanercept. The plasma levels of high-sensitivity C-reactive Protein (CRP), E-selectin, VEGF and PAI-1 were measured by ELISA. The psoriasis severity was assessed by PASI score. A statistically significant difference (P = 0.001) was found between CEC level in psoriatic patients (10.6 ± 9.4 cells/mL) vs. the control group (3.9 ± 0.9 cells/mL). This count inversely correlated with sE-selectin levels (r(2) = 0.16; P = 0.03). After 6 months of therapy, patients experienced a significant (P < 0.05) decrease in CEC levels (3.4 ± 1.3 cells/mL) and in PASI score (from 11.7 ± 8.1 to 2.1 ± 4.0). The elevated CECs level that we found in a sample of high selected psoriatic patients could be expression of endothelial damage. Lowering of CECs count after treatment with Etanercept support the hypothesis that an effective systemic therapy of psoriasis may also improve the endothelial function.

Detection of vascular endothelial progenitor cells in patients with systemic lupus erythematosus

Objective To characterize the alteration in peripheral blood endothelial progenitor cells (EPCs) of patients with systemic lupus erythematosus (SLE).Methods Venous blood samples were obtained from 82 female patients with SLE aged (35 ± 10) years and 50 healthy female controls aged (35 ± 13) years.ACL 9000 automated coagulation analyzer was used to determine the level of Von Willebrand factor antigen (vWF Ag).Flow cytometry was performed to detect peripheral blood EPCs and circulating endothelial cells (CECs).Analysis of variance was performed to assess the differences in these parameters between patients with active and stable SLE and the controls,and Pearson correlation analysis was conducted to evaluate the relationship between these parameters.Results The number of CD34+ cells,CD133+ cells and CD34+CD133+cells per 200 000 peripheral blood mononuclear cells was 35.4 ± 16.7,86.5 ± 32.1 and 361.3 ± 176.4 in patients with active SLE,significantly higher than that in the patients with stable SLE (17.1 ± 10.9,28.7 ± 21.5,107.2 ±44.3,respectively,all P ＜ 0.01)) and the controls (13.8 ± 9.6,11.2 ± 5.5,92.3 ± 50.5,respectively,all P ＜0.01).The patients with active SLE exhibited an elevated level of vWF Ag (438.9％ ± 205.3％ vs.130.2％ ±51.5％,P ＜ 0.01),an increased number of EPCs (361.3 ± 176.4 vs.107.2 ± 44.3,P ＜ 0.01) but a similar number of CECs (127±51 vs.118 ± 39,P ＞ 0.05) per 200 000 peripheral blood mononuclear cells compared with the healthy controls.No significant differences were observed in these parameters between the patients with stable SLE and the controls (all P ＞ 0.05).The number of EPCs was positively correlated with the level of vWF Ag (r =0.67,P ＜ 0.01),but uncorrelated with the number of CECs (P ＞ 0.05) in patients with active SLE.Conclusions The quantity of EPCs in peripheral blood is closely correlated with the level of the vascular injury marker vWF Ag,hinting that the number of EPCs can serve as a useful marker of disease severity.