Circulating Endothelial Cells Counts Research Articles

Evaluation of Circulating Endothelial Cells as Direct Marker of Endothelial Damage in Allo-Transplant Recipients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome

Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a rare but potentially fatal complication following allogenic hematopoietic cell transplantation (allo-HCT). Timely identification of SOS/VOD to allow for prompt treatment is critical, but identifying a VOD-predictive biomarker remains challenging. Given the pivotal role of endothelial dysfunction in SOS/VOD pathophysiology, the CECinVOD study prospectively evaluated levels of circulating endothelial cells (CECs) in patients undergoing allo-HCT with a myeloablative conditioning (MAC) regimen to investigate the potential of CEC level in predicting and diagnosing SOS/VOD. A total of 150 patients from 11 Italian bone marrow transplantation units were enrolled. All participants were age >18 years and received a MAC regimen, putting them at elevated risk of developing SOS/VOD. Overall, 6 cases of SOS/VOD (4%) were recorded. CECs were detected using the Food and Drug Administration-approved CellSearch system, an immunomagnetic selection-based platform incorporating ferrofluid nanoparticles and fluorescent-labeled antibodies, and were defined as CD146+, CD105+, DAPI+, or CD45-. Blood samples were collected at the following time points: before (T0) and at the end of conditioning treatment (T1), at neutrophil engraftment (T2), and at 7 to 10 days postengraftment (T3). For patients who developed VOD, additional samples were collected at any suspected or proven VOD onset (T4) and weekly during defibrotide treatment (T5 to T8). A baseline CEC count >17/mL was associated with an elevated risk of SOS/VOD (P = .04), along with bilirubin level >1.5 mg/mL and a haploidentical donor hematopoietic stem cell source. Postconditioning regimen (T1) CEC levels were elevated (P = .02), and levels were further increased at engraftment (P < .0001). Additionally, patients developing SOS/VOD after engraftment, especially those with late-onset SOS/VOD, showed a markedly higher relative increase (>150%) in CEC count. Multivariate analysis supported these findings, along with a high Endothelial Activation and Stress Index (EASIX) score at engraftment (T2). Finally, CEC kinetics corresponded with defibrotide treatment. After the start of therapy (T4), CEC levels showed an initial increase in the first week (T5), followed by a progressive decrease during VOD treatment (T6 and T7) and a return to pre-SOS/VOD onset levels at resolution of the complication. This prospective multicenter study reveals a low incidence of SOS/VOD in high-risk patients compared to historical data, in line with recent reports. The results from the CECinVOD study collectively confirm the endothelial injury in allo-HCT and its role in in the development of SOS/VOD, suggesting that CEC level can be a valuable biomarker for diagnosing SOS/VOD and identifying patients at greater risk of this complication, especially late-onset SOS/VOD. Furthermore, CEC kinetics may support treatment strategies by providing insight into the optimal timing for discontinuing defibrotide treatment.

Evaluation of Circulating Endothelial Cells (CECs) As Marker of Endothelial Damage in Allo-Transplanted Patients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome (VOD/SOS): The Cecinvod Study

INTRODUCTION Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a potentially fatal complication after allogeneic stem cell transplantation (alloSCT). Identifying a predictive biomarker for VOD has been challenging. Since endothelial injury is considered one of the main pathogenic factors for VOD onset, with the CECinVOD prospective study we aimed to evaluate Circulating Endothelial Cells (CEC) in allo-transplanted patients (pts) at higher risk to develop VOD. METHODS From October 2020 to November 2022, 150 pts have been enrolled in the CECinVOD study from 11 Italian Bone Marrow Transplantation Units. All pts must be older than 18 years and undergoing myeloablative alloSCT. CECs were detected using the CellSearch system, the FDA-approved immunomagnetic selection approach incorporating ferrofluid nanoparticles (anti CD146) and fluorophore-labelled antibodies (anti CD105, CD45 and DAPI). CEC were defined as CD146+, CD105+, DAPI+ and CD45-. CEC were collected at the following timepoints: before conditioning regimen (T0), at the end of conditioning regimen and before alloSCT (T1), at the time of neutrophils engraftment (T2), and 7-10 days after engraftment (T3). In pts who developed VOD, additional timepoints were collected as follows: at any time of suspected or proven VOD onset (T4), and then weekly during Defibrotide treatment (T5-T8). SOS/VOD was defined according to the 2016 European Group for Blood and Marrow Transplantation criteria. RESULTS Pts' main characteristics are summarized in Table 1. Six out of 150 pts (4%) developed VOD during the follow up (4 “severe”, and 2 “very severe”). All pts were treated with Defibrotide, obtaining a complete remission in 5 of them, while 1 pt died due to VOD complications. Pts receiving TBI-based regimen were more likely to develop VOD compared to those receiving Treosulfan (10 to 14 g/m2) or Busulfan ev (9.6 to 12.8 mg/kg) (p 0.08). Similarly, higher baseline levels of bilirubin were associated with a higher incidence of VOD (p 0.08). Considering the CECs analysis, 615 samples were evaluated. At the enrollment, CECs levels were not related with any of clinical characteristics analyzed, except for the number of previous treatments. Indeed, those pts with 2 or more previous lines of treatments had higher levels of CECs (OR=0.53; p&amp;lt;0.001). Considering the different timepoints, conditioning regimen and alloSCT result in higher levels of CECs. Thus, CEC were higher at T1 than at T0 (p 0.02), as well as they were even higher at T2 than at T1 (p&amp;lt;0.0001) (Fig.1). Conversely, no significant differences have been observed between T3 and T2. No other clinical characteristic was correlated with CEC counts at the different timepoints. Pts who developed VOD had higher median levels of CEC at all the timepoints analyzed, but this difference has not achieved statistical significance, probably due to the low number of pts in the VOD group. At VOD onset, the pts always had an increase in CEC levels compared with the previous timepoint. After defibrotide treatment, the CEC levels increased in the first week, while they progressively decreased during the VOD treatment (T6 and T7, -50,7% and -71,5%, respectively). Recently, another endothelial activation marker has been considered: the Easix score. We didn't find any relationship between the Easix score at transplant and the VOD onset, even if a trend may be observed. We also investigated whether the CEC levels may be related with Easix score, but we didn't find any relationship at each timepoint. Interestingly, in a subgroup of pts CECs have been observed as a cluster of multiple cells. This data is firstly described in allo-SCT. Its functional significate remains unclear, but we found a relationship between CECs cluster and the number of CECs (p &amp;lt; 0.001). CONCLUSIONS The incidence of VOD in our prospective study was low with respect to the one reported in the literature. This can be explained by the changes in alloSCT from the past (better selection of the pts, lower use of TBI, higher use of reduced intensity regimen) and the retrospective nature of most of the previous reports. We show that CECs can be considered reliable marker of endothelial damage in alloSCT pts, highlighting the impact of previous treatments, the conditioning regimen, and allo-SCT itself. Increased CEC level may be helpful to confirm VOD diagnosis, as well as their monitoring may be useful to evaluate the response to the treatment for VOD.

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.

The Vasculopathy of Juvenile Dermatomyositis: Endothelial Injury, Hypercoagulability, and Increased Arterial Stiffness.

Vasculopathy is considered central to the pathogenesis of juvenile dermatomyositis (DM) and is associated with severe extramuscular manifestations. We undertook this study to investigate the hypothesis that the vasculopathy of juvenile DM can be noninvasively tracked by examining biomarkers of endothelial injury, subclinical inflammation, hypercoagulability, and vascular arterial stiffness. The study population was a UK cohort of children with juvenile DM. Circulating endothelial cells (CECs) and microparticles (MPs) were identified using immunomagnetic bead extraction and flow cytometry, respectively. Plasma thrombin generation was determined using a fluorogenic assay. Cytokine and chemokine levels were measured by electrochemiluminescence. Arterial stiffness was assessed using pulse wave velocity (PWV). Results were expressed as the median and interquartile range (IQR), and statistical significance was assessed using nonparametric analyses. Ninety patients with juvenile DM and 79 healthy control subjects were included. The median age of the patients was 10.21 years (IQR 6.68-13.40), and the median disease duration was 1.63 years (IQR 0.28-4.66). CEC counts were higher in all patients with juvenile DM compared to controls (median 96 cells/ml [IQR (40-192] and 12 cells/ml [IQR 8-24], respectively; P < 0.0001). Circulating MP numbers were also significantly higher in patients with active juvenile DM compared to controls (median 204.7 × 103 /ml [IQR 87.9-412.6] and 44.3 × 103 /ml [IQR 15.0-249.1], respectively; P < 0.0001). MPs were predominantly of platelet and endothelial origin. Enhanced plasma thrombin generation was demonstrated in patients with active juvenile DM compared to those with inactive disease (P = 0.0003) and controls (P < 0.0001). Carotid-radial PWV adjusted for age was increased in patients with juvenile DM compared to controls (P = 0.003). We observed increased endothelial injury and increased levels of proinflammatory cytokines in patients with active juvenile DM. MP profiles reflected distinct disease activity status in juvenile DM and are markers of vascular pathology, platelet activation, and thrombotic propensity. Ongoing long-term vascular injury may result in increased arterial stiffness in patients with juvenile DM.

Role of circulating endothelial cells in assessing the severity of systemic sclerosis and predicting its clinical worsening

Endothelial damage and fibro-proliferative vasculopathy of small vessels are pathological hallmarks of systemic sclerosis (SSc). The consequence is the detachment of resident elements that become circulating endothelial cells (CECs). The aim of our study was to evaluate the potential of CECs as biomarker in SSc. We enrolled 50 patients with limited cutaneous (lcSSc) and diffuse cutaneous (dcSSc) subset of SSc, who underwent clinical evaluation to establish the organ involvement. CECs were measured by flow-cytometry utilizing a polychromatic panel. An evident difference was observed in CEC counts comparing controls to SSc patients (median 10.5 vs. 152 cells/ml, p < 0.0001) and for the first time, between the two subsets of disease (median lcSSc 132 vs. dcSSc 716 CEC/ml, p < 0.0001). A significant correlation was established between CECs and some SSc clinical parameters, such as digital ulcers, skin and pulmonary involvement, presence of Scl-70 antibodies, nailfold videocapillaroscopy patterns and EUSTAR activity index. After 12 months, CECs correlated with clinical worsening of patients, showing that a number higher than 414 CEC/ml is a strong negative prognostic factor (RR 5.70). Our results indicate that CECs are a direct indicator of systemic vascular damage. Therefore, they can be used as a reliable marker of disease severity.

The Role of Ganoderma Lucidum Polysaccharide Peptide in Endothelial Progenitor Cells and Circulating Endothelial Cells as anti Endothelial Dysfunction from Stable Angina Pectoris Patients

Objective: Endothelial dysfunction is the primary initial step for atherogenesis in cardiovascular disease. Stable angina pectoris is a stable form of cardiovascular disease that profoundly alters Endothelial Progenitor Cells (EPC) and Circulating Endothelial Cells (CEC). Both CEC and EPC have a significant role as native homeostasis biomarker of endothelial, which could initiate cytokine storm when homeostasis was altering. Ganoderma Lucidum is known for the antioxidative, anti-inflammatory, and anti-cancer properties and indirect anti endothelial dysfunction. The previous study has proven the Polysaccharide Peptide (PsP) of Ganoderma Lucidum as an effective antioxidant and anti-endothelial dysfunction in atherosclerosis rats and shows no toxicity in an animal model. This study goals to prove the effect of PsP in CEC and EPC in stable angina patients.&#x0D; Methods: This is a quasi-experimental trial of 35 Stable Angina patients, determined based on ESC Stable CAD Guidelines with pre and post-test design without a control group. The parameters are CEC and EPC counts. The patients were given PsP 750mg/day in 3 divided doses for 90 days. A paired t-test perform for normally distributed data, and the Wilcoxon test for not normally distributed data, and a significant level of p≤0,05.&#x0D; Results: CEC significantly reduced in stable angina patients, with p=0,001. EPC count significantly reduced in stable angina with p=0,001.&#x0D; Conclusion: Ganoderma Lucidum PsP is a potent anti-endothelial dysfunction against atherosclerosis's pathogenesis in stable angina.

Circulating Endothelial Cells as a Marker of Endothelial Injury in Severe COVID -19.

Beside the commonly described pulmonary expression of the coronavirus disease 2019 (COVID-19), major vascular events have been reported. The objective of this study was to investigate whether increased levels of circulating endothelial cells (CEC) might be associated with severe forms of COVID-19. Ninety-nine patients with COVID-19 were enrolled in this retrospective study. Patients in the intensive care units (ICU) had significantly higher CEC counts than non-ICU patients and the extent of endothelial injury was correlated with putative markers of disease severity and inflammatory cytokines. Altogether, these data provide in vivo evidence that endothelial injury is a key feature of COVID-19.

Abstract 6487: Circulating tumor cells, tumor- and endothelium- derived extracellular vesicles, but not circulating endothelial cells associate with poor prognosis in colorectal cancer

Abstract Elevated tumor-derived EV (tdEV) and Circulating Tumor Cell (CTC) load in patients with metastatic cancer is associated with poor clinical outcome. In this study, we investigated whether endothelium-derived extracellular vesicles (edEVs) can be detected in the blood of metastatic colorectal cancer (mCRC) patients and whether there is an association with prognosis. The open-source ACCEPT software was used to enumerate edEVs, tdEVs and other objects from digitally stored CellSearch® images obtained after Circulating Endothelial Cell (CEC) and CTC enrichment from blood of 395 mCRC patients, before the initiation of a new therapy. Patients had participated in the prospective phase III CAIRO2 study. ACCEPT classified objects, CTC and CEC counts and patient information were used in univariable and multivariable analysis to determine their relation with Progression-Free Survival (PFS) and Overall Survival (OS). edEVs are present in blood of mCRC patients at 5-10 fold higher frequencies than CECs. A significantly shorter PFS and OS was observed for patients with ≥ 3 CTCs/ 7.5 mL, ≥ 40 tdEVs/ 7.5 mL and ≥ 287 edEVs/ 4.0 mL. The Hazard Ratio HR (95% CI) of PFS for increased CTCs, tdEVs and edEVs was 1.4 (1.1-1.9), 2.0 (1.5-2.6) and 1.7 (1.2-2.5), respectively. Similarly, the HR of OS for increased CTCs, tdEVs and edEVs was 2.2 (1.7-3.0), 2.7 (2.0-3.5) and 2.1 (1.5-2.8), respectively. There was no cut-off value for CECs leading to a dichotomization of patients with a significant HR. Both tdEVs and edEVs remained significant predictors of OS in multivariable analysis. edEVs and tdEVs are independent prognostic factors for OS in mCRC patients. Funded by the NWO Applied and Engineering Sciences Cancer-ID project #14190. Citation Format: Afroditi Nanou, Linda Mol, Miriam Koopman, Cornelis J. Punt, Leon W. Terstappen. Circulating tumor cells, tumor- and endothelium- derived extracellular vesicles, but not circulating endothelial cells associate with poor prognosis in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6487.

Multiple functions of policosanol in elderly patients with dyslipidemia.

ObjectiveTo determine the multiple functions of policosanol in elderly dyslipidemia patients.Methodology: There were 294 elderly dyslipidemia patients enrolled into this clinical study. They were randomly divided into four groups, as follows: 20 mg policosanol (group A, n = 64); 10 mg policosanol (group B, n = 72); 20 mg atorvastatin (group C, n = 91); and 10 mg policosanol + 20 mg atorvastatin (group D, n = 62). Plasma platelet count, platelet aggregation rate, circulating endothelial cell (CEC) count, high sensitivity C-reactive protein (hs-CRP), and carotid intima–media thickness (IMT) were measured before the study (week 0) and at weeks 12, 24, and 52.ResultsIn group A, the platelet aggregation rate caused by adenosine diphosphate (ADP) after treatment was significantly decreased compared with before treatment (48.79% ± 20.29% vs. 40.37% ± 23.56%), but the arachidonic acid (AA)-induced platelet aggregation rates were similar. The platelet aggregation rates induced by AA and ADP in groups B, C, and D did not change significantly. CEC counts and hs-CRP and homocysteine levels in all groups after treatment were significantly lower compared with before treatment, but carotid IMTs were similar.ConclusionPolicosanol regulates blood lipid levels and improves endothelial cell function, and it could delay the progress of atherosclerosis.Trial registration number: ChiCTR-RRC-17013396 (retrospectively registered).

Prognostic plasma biomarkers of early complications and graft-versus-host disease in patients undergoing allogeneic hematopoietic stem cell transplantation.

Early complications post hematopoietic stem cell transplantation (HSCT) such as sinusoidal obstruction syndrome (SOS) and graft versus host disease (GVHD) can be life threatening. Although several biomarkers have been identified to correlate with these complications and their response to treatment, these are yet to be used in clinical practice. Here, we evaluated circulating endothelial cells (CECs) (n = 26) and plasma biomarkers (ST2, REG3α, VCAM1, ICAM1, TIM3) (N = 210) at early time points, to determine their association with early complications post‐HSCT. Elevated CEC counts at the end of conditioning was associated with GVHD, indicating endothelial damage during HSCT. Plasma levels of REG3α, VCAM1, ICAM1, and TIM3 on day 14 (D14) and D14 ICAM1 and D28 ST2 were significantly higher in patients with SOS and aGVHD, respectively. Upon sub‐group analysis, D28 ST2, D14/D28 REG3α, and D14 ICAM1 levels were significantly higher in patients with gastrointestinal GVHD, while D28 ST2 was higher in those with skin/liver GVHD. High ST2 levels on D28 was significantly associated with non‐relapse mortality (NRM) and overall survival. Our results suggest that elevated ST2 levels on D28 could predict the likelihood of developing aGVHD and could influence NRM and OS.

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.

CD276-Positive Circulating Endothelial Cells Do Not Predict Response to Systemic Therapy in Advanced Colorectal Cancer.

CD276 can discriminate between tumor derived and normal CECs (circulating endothelial cells). We evaluated whether CD276+CEC is a clinically relevant biomarker to predict response to palliative systemic therapy in patients with metastatic colorectal cancer (mCRC). Samples were prospectively collected from patients with mCRC enrolled in the ORCHESTRA trial (NCT01792934). At baseline and after three cycles of 5-fluorouracil/leucovorin and oxaliplatin ± bevacizumab, CECs were measured by flowcytometry (CD34+CD45negCD146+DNA+; and CD276+). A clinically relevant cut-off value of (CD276+)CECs was determined as 100% sensitivity (and 80% specificity in 95% confidence interval) identifying patients with progressive disease within 6 months. There were 182 baseline samples and 133 follow up samples available for analysis. CEC and CD276+CEC counts significantly increased during treatment from 48 to 90 CEC/4 mL (p = 0.00) and from 14 to 33 CD276+CEC/4 mL (p = 0.00) at baseline and at first evaluation, respectively. CEC and CD276+CEC counts were not predictive for poor response (area under the curve (AUC) 0.53 for CEC and AUC 0.52 for CD276+CEC). Despite numerical changes during therapy, CEC and CD276+CEC counts do not adequately predict poor response to first line palliative systemic therapy in patients with mCRC.

Prognostic value of CEC count in HER2-negative metastatic breast cancer patients treated with bevacizumab and chemotherapy: a prospective validation study (UCBG COMET).

Proof of concept studies has reported that circulating endothelial cell (CEC) count may be associated with the outcome of HER2-negative metastatic breast cancer (mBC) patients treated by chemotherapy and the anti-VEGF antibody bevacizumab. We report the results obtained in an independent prospective validation cohort (COMET study, NCT01745757). The main baseline criteria were HER2-negative mBC, performance status 0-2 and no prior chemotherapy for metastatic disease. CECs were detected by CellSearch® from 4ml of blood at baseline and after 4weeks of weekly paclitaxel and bevacizumab therapy. CEC counts (considered both as a continuous variable and using the previously described 20 CEC/4ml cutoff) were associated with clinical characteristics and progression-free survival (PFS). CEC count was obtained in 251 patients at baseline and in 207 patients at 4weeks. Median baseline CEC count was 22 CEC/4ml (range 0-2231). Baseline CEC counts were associated with performance status (p = 0.02). No statistically significant change in CEC counts was observed between baseline and 4weeks of therapy. High baseline CEC count was associated with shorter PFS in univariate and multivariate analyses (continuous: p < 0.001; dichotomized: HR 1.52, 95% CI [1.15-2.02], p = 0.004). CEC counts at 4weeks had no prognostic impact. This study confirms that CEC count may be associated with the outcome of mBC patients treated with chemotherapy and bevacizumab. However, discrepancies with previous reports in terms of both the timing of CEC count and the direction of the prognostic impact warrant further clinical investigation.

Single Cell Analysis of Circulating Endothelial Cells in Allogeneic Hematopoietic Stem Cell Transplant; To Whom Do They Belong: Host or Donor?

Background. We recently reported that Circulating Endothelial Cell (CEC) count changes represent a promising marker to monitor endothelial damage in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT), potentially becoming a valuable tool in the diagnostic definition of GVHD. Besides confirming an increase of CEC counts at GVHD onset, we repeatedly documented at time of engraftment statistically significant higher numbers of CEC in patients who will not manifest GVHD in comparison to patients in which GVHD will be diagnosed (Transplantation 2014,98:706-12; Bone Marrow Transplantation 2017,52:1637-42; Scientific Reports 2019,9:1-12). Recent knowledges in organ transplant pointed out that endothelial cells from the grafted organ, besides being a continuous source of alloantigens, can downregulate alloreactivity exerting tolerogenic responses. By inference to the allo-HSCT field, it could be envisaged that presence of donor CEC could induce protective effects on alloreactivity. Methods. We planned a study to test the hypothesis that at time of engraftment, CEC present in peripheral blood (PB), besides coming from cells shedding from patient vasculature, could partly belong to donor, originating from the cellular graft. Therefore, in an exploratory set, we performed FISH analysis on flowcytometry-sorted CEC (CD45neg/CD34bright/CD146pos, Lyotube #623920, BD Biosciences) (n=3) and on whole PB derived culture-expanded CEC (n=3) (EGM-2 BulletKit, Lonza), obtained at engraftment in sex-mismatched allo-HSCT. In the confirmatory set (n=15), single CEC were recovered from PB, at engraftment (T1) and at 90 days (T2) after allo-HSCT, through the DEPArrayTM technology (Menarini Silicon Biosystems), after preliminary bulk separation step carried out with the CellSearch® System. Single recovered CEC was whole genome amplified (Ampli1™ WGA Kit) and short tandem repeat (STR) profile determined (Ampli 1TM STR kit) on each single CEC. To confirm host/donor origin, single CEC STR profile was compared to that determined on patient and donor cells before allo-HSCT. Moreover, donor CEC presence was evaluated by CISH analysis on formaline fixed and paraffin-embedded biopsy sections obtained at least three months after sex mismatched allo-HSCT. Results. By positive findings of the exploratory set, we proved, at the single cell level in the confirmatory set, the presence of donor CEC at engraftment (T1) in 4 out of 15 patients (Table 1). Of them, 2 did not manifested GVHD, despite a GVHD risk score of 2, and the other 2 presented GVHD grade I. On the contrary, among the 10 patients in whom no donor CEC were detected, 6 experienced GVHD grade II-III, while 4 did not manifested GVHD, despite a 1-3 GVHD risk score. Conclusions. Our data represent the proof of principle that donor CEC may flow in host PB early on from hematopoietic recovery and seldom persist thereafter at steady-state conditions, being potentially embedded in host vascular wall. These puzzling findings suggest that neovascularization takes place in parallel with hematopoietic engraftment and could provide further clues on shedding light on tissue tolerance in the context of GVHD, opening up paradoxical scenarios on the protective role potentially played by donor CEC. Disclosures Fontana: Menarini Silicon Biosystem: Employment. Rotta:BD Biosciences Italia: Employment. Manaresi:Menarini SIlicon Biosystem: Employment, Membership on an entity's Board of Directors or advisory committees.

Circulating endothelial cells: Prognostic value in patients with glioblastoma.

e13517 Background: Glioblastoma (GB) is an aggressive tumor. Circulating Endothelial Cells (CECs) can be detected in peripheral blood and have been related to angiogenesis. CECs have an unclear prognostic value in patients with glioblastoma. The objective of this study was to quantify the presence and number of CECs in GB patients and determine its potential prognostic role. Methods: In this prospective, single center study, peripheral blood samples were obtained at the time of GB diagnosis. For the detection of CECs, 4 ml of blood was analyzed using the CellSearch system (Veridex). CEC blood samples were classified as CD146+, CD105+, CD45− and DAPI+. CEC detection was performed prior to surgery in a total of 26 patients and 22 patients with glioblastoma were included in the final analysis. We mesured progression free survival (PFS) and overall survival (OS). Results: Between 2014 and 2016, twenty two patients with histologically confirmed glioblastoma were studied. There were 14 males and 8 females with an average age of 63 years (range 42-81). A complete resection was achieved in 50% of cases, partial or subtotal resection in 31.8% and biopsy in 18%. The mean number of CECs was 59.3 cells/mL (range 0-954). Patients were classified into two groups depending on the number of CECs: group 1 had a CEC count below the mean and group 2 had a CEC count above the mean. A total of 17 patients (77.2%) were in group 1 and 5 patients (22.7%) were in group 2.The median OS was 14.4 months (range -0.36-33.36) for the entire study population. The median OS was 17.4 months (IC95% 11.47-23.37) for patients in group 1 and 8.12 months for patients in group 2 (IC 95% 4.38-11.85) p = 0.002. The median PFS was 8.94 m (IC95% 5.1-12.69) for group 1 and 3.95 for group 2 (IC 95% 2.91-4.98) p = 0.097. Conclusions: CECs can be detected in recently diagnosed GB patients. The present study demonstrates a possible prognostic value for CEC determination in GB cases at the time of diagnosis.

CD276-positive circulating endothelial cells in advanced colorectal cancer.

572 Background: The value of circulating endothelial cells (CECs) as predictive biomarker in metastatic colorectal cancer (mCRC) may be improved by using tumor-derived CECs. CD276 can discriminate between tumor derived and normal CECs. We evaluated whether CD276+CEC is a clinically relevant biomarker with 100% sensitivity to predict response to first line palliative combination systemic therapy with fluoropyrimidine and oxaliplatin in patients with mCRC. Methods: Samples were prospectively collected from patients with mCRC enrolled in the ORCHESTRA trial. At baseline and at first evaluation (after 4 cycles of 5-FU/LV or 3 cycles capecitabine with oxaliplatin ± bevacizumab), CECs were measured by flowcytometry with a previously validated panel (CD34+CD45-CD146+DNA+; and additional CD276+ for tumor derived CECs). A clinically relevant cut-off value of (CD276+) CECs was determined as 100% sensitivity (and 80% specificity in 95% CI) identifying patients with progressive disease within 6 months. Results: There were 182 baseline samples, from which 133 follow up samples were available for analysis. CEC and CD276+CEC counts significantly increased during treatment from 48 to 90 CEC/4 ml (p = 0.00) and from 14 to 33 CD276+CEC/4 ml (p = 0.00) at baseline and at first evaluation, respectively. In patients treated with bevacizumab, CEC counts were lower after three cycles compared to patients treated with chemotherapy alone 75.5 vs 131 CEC/4ml (p = 0.04), 28 vs 41 CD276+CEC/4 ml (p = 0.08). Baseline values nor changes in CEC and CD276+CEC counts were predictive for poor response (progressive disease within 6 months, AUC 0.53 for CEC and AUC 0.52 for CD276+CEC). Conclusions: Despite numerical changes during therapy, CEC and CD276+CEC counts do not adequately predict poor response to first line palliative systemic therapy in patients with mCRC. (NCT01792934.)

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.

Circulating endothelial progenitor cells and endothelial cells in moyamoya disease.

IntroductionThere is no well‐recognized biomarker for accurately predicting outcome in the presence of moyamoya disease (MMD), a progressive occlusive cerebrovascular disease of the internal carotid arteries or their branches. The aim of this study was to investigate the presence of endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) in MMD and correlate the findings with clinical features.MethodsPatients with MMD (n = 66) were compared with healthy controls (n = 81). Blood samples were obtained from an antecubital vein and analyzed using flow cytometry. EPCs were defined as CD31+ CD45dim CD34br CD133+ and CECs as CD31br CD45− CD34dim CD133−. Univariate and multivariate linear regression analyses were carried out.ResultsThe CEC counts were significantly higher in the patients than in the controls (p = 0.008). In multivariate analysis, EPC counts were independently associated with age of patients with MMD (p = 0.049) and CEC counts were independently negatively associated with concomitant disease such as hypertension, diabetes mellitus, and coronary heart disease (p = 0.034).ConclusionsThis is the first study to investigate the presence of CECs in the plasma of patients with MMD, and the amount of CECs was negatively correlated with concomitant disease in these patients.

Circulating endothelial cells and microparticles as diagnostic and prognostic biomarkers in small-cell lung cancer

ObjectivesIt has been proposed that circulating endothelial cells (CECs) and microparticles (MPs) may be useful for the assessment of patients with non-small-cell lung cancer (NSCLC). However, little is known about the potential clinical relevance of these biomarkers in small-cell lung cancer (SCLC). Therefore, we investigated the utility of baseline levels of CECs and MPs in SCLC patients. Materials and methodsAn immunomagnetic separation (IMS) technique was used to isolate and quantify CECs in the peripheral blood, while plasma samples were analyzed using flow cytometry for the measurement of circulating MPs. ResultsWe prospectively collected data from 56 patients and 41 healthy individuals. Forty-three patients presented at initial diagnosis and 13 patients presented at relapse. Baseline levels of CECs and MPs were significantly higher in SCLC patients either at initial diagnosis or at relapse than in healthy subjects (p < 0.0002 and p < 0.007, respectively). However, estimated tumor volume (ETV) was significantly correlated with basal MP values (p < 0.0001) but not with pretreatment CECs (p = 0.57). The amount of baseline CECs and MPs was significantly lower in patients with an objective response (OR, n = 23) than in those with progressive disease (PD, n = 15) after treatment (p = 0.016 and 0.05, respectively). With cut-off values of 110 cells/mL for CECs and 1257 events/μL for MPs according to receiver operating characteristics (ROC) analysis, baseline levels of these biomarkers were not significantly correlated with either progression-free survival (PFS) or overall survival (OS). However, patients with 6-month PFS displayed significantly decreased pretreatment CEC counts (p = 0.042), whereas basal MP values significantly increased in 1-year survivors compared with those in non-survivors (p = 0.05). ConclusionOur results suggest that baseline CECs and MPs may be predictive biomarkers of tumor response and long-term survival in SCLC patients.