Strijbos, M.H.

External quality assurance of circulating tumor cell enumeration using the CellSearch® system: A feasibility study (Article)

2011-03-01T00:00:00Z

Background: Circulating tumor cells (CTCs) are cells that have detached from solid tumors and entered the blood. CTCs can be detected, among others, by semi-automated immunomagnetic enrichment and image cytometry using CellSearch® (Veridex, Raritan, NJ). We studied the feasibility of external quality assurance (EQA) of the entire CellSearch procedure from blood draw to interpretation of results in multiple laboratories. Methods: Blood samples from six cancer patients and controls were distributed to 14 independent laboratories to test between-laboratory, between-assay, and between-instrument variation. Additionally, between-operator variability was assessed through the interpretation of blinded images of all blood samples on a website. Results: Shipment and storage of samples had no influence on CTC values. Between-instrument (coefficient of variation (CV) < 12%) and between-assay variation was low (CV ≤ 20%), indicating high reproducibility. However, between-laboratory CV ranged from 45 to 64%. Although inter-operator agreement on image interpretation (Fleiss' I° statistics) ranged from " to "almost perfect," image interpretation, particularly of samples containing high numbers of apoptotic cells, was the main contributor to between-laboratory variation. Conclusions: This multicenter study shows the feasibility of an EQA program for CTC detection in patient samples, and the importance of continuation of such a program for the harmonization of CTC enumeration.

Levels of circulating endothelial cells in normotensive and severe preeclamptic pregnancies (Article)

2010-11-01T00:00:00Z

Background: Preeclampsia is a disease hypothesized to originate from widespread endothelial dysfunction or damage. This study investigated whether circulating endothelial cells (CEC) can serve as a surrogate marker for disease severity in patients with preeclampsia, and if their number correlates to serum endothelial biomarkers for activation, dysfunction, or damage of those cells. Methods: Blood was drawn consecutively from 30 patients admitted with a diagnosis of severe preeclampsia. Thirty healthy, normotensive, patients matched for age, body mass index, and gestational age served as a control group. We determined the number of CEC and serum concentrations of biomarkers indicative of endothelial damage (thrombomodulin) and activation (E-selectin), and the antiangiogenic protein (endoglin), which reflects endothelial dysfunction. Results: Median CEC counts did not differ significantly between preeclamptic patients and the control group (median 5.3 vs. 3.5 CEC/mL, respectively) and were mostly within the normal range (i.e., <20 CEC/mL). However, serum concentrations of thrombomodulin (median 3.6 vs. 5.2 ng/mL; P = 0.006), E-selectin (median 32.0 vs. 42.9 ng/mL; P = 0.02), and especially endoglin (median 5.0 vs. 76.2 ng/mL; P < 0.0001) were significantly increased in severe preeclamptic patients. CEC counts did not correlate with any of the clinical parameters or routinely determined laboratory indices. Conclusion: Preeclampsia is characterized by endothelial dysfunction and activation rather than actual endothelial damage as characterized by increased CEC counts.

Levels of circulating endothelial cells in normotensive and severe preeclamptic pregnancies (Article)

2010-11-01T00:00:00Z

mRNA levels of CD31, CD144, CD146 and von Willebrand factor do not serve as surrogate markers for circulating endothelial cells (Article)

2010-08-01T00:00:00Z

Circulating endothelial cells (CEC) are considered a promising marker to determine the extent of vascular damage. However, currently available and validated CEC enumeration assays are laborious, time consuming and costly, which limits their clinical utility. Here, we evaluated the feasibility of quantifying mRNA levels of the endothelium-associated markers CD31, CD144, CD146 and von Willebrand factor (vWf) in peripheral blood (PB) of healthy donors, patients, and human umbilical veins by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and their use as surrogate markers for CEC. Whole blood samples and CD146+ cell-enriched fractions were assessed for mRNA and protein expression of CD31, CD144, CD146 and vWf by RT-PCR and flow cytometry, respectively. We showed the feasibility to detect endothelial mRNA isolated from HUVEC numbers as low as 10. However, no endothelial mRNA could be measure in whole blood samples, and only low levels of CD31 and CD146 mRNA were detected in suspensions of isolated CEC with numbers up to 4,450 CEC per sample. We conclude that mRNA levels of CD31, CD144, CD146 and vWf in whole blood as detected by real time RT-PCR cannot be used as biomarkers for end-stage endothelial cells such as CEC.

Circulating endothelial cells, circulating tumour cells, tissue factor, endothelin-1 and overall survival in prostate cancer patients treated with docetaxel (Article)

2010-07-01T00:00:00Z

Purpose: We investigated whether serum markers of angiogenesis endothelin-1 (ET-1) and tissue factor (TF), and/or markers of vascular damage such as circulating endothelial cells (CECs), or their relative changes during treatment, were prognostic for overall survival (OS) in castration resistant prostate cancer (CRPC) patients. Additionally, we combined these markers with circulating tumour cells (CTCs) to construct a predictive nomogram for treatment outcome. Patients and methods: One hundred and sixty two CRPC patients treated with a docetaxel containing regimen had blood drawn before and at 2-5 weeks and 6-8 weeks after treatment start. Prospectively determined CTC and CEC levels, and retrospectively measured serum concentrations of ET-1 (pg/mL) and TF (pg/mL) were evaluated to determine their prognostic value for OS. Results: Baseline CEC, TF and ET-1 were not prognostic for OS. A ≥3.8-fold increase in CEC 2-5 weeks after treatment initiation was associated with decreased OS (median 10.9 versus 16.8 months; P = 0.015), as was any decrease in TF levels compared to baseline levels (median 11.9 versus 21.5 months; P = 0.0005). As previously published, baseline and CTC counts ≥5 at 2-5 weeks were also predictive of decreased OS. Combining CTC with changes in TF and CEC 2-5 weeks after treatment initiation yielded four groups differing in OS (median OS 24.2 versus 16.0 versus 11.4 versus 6.1 months; P < 0.0001). Conclusion: CEC, CTC and TF levels alone and combined can predict early on OS in CRPC patients treated with docetaxel-based therapy. A prospective study to confirm the use of these markers for patient management is needed.

Quantification of Circulating Ednothelial Cells as Surrogate Marker for Vascular Damage (Doctoral Thesis)

2009-11-27T00:00:00Z

The inner lining of our vascular system consists of approximately 1.2 trillion endothelial cells. Rather than being a mere wall between the blood and organs, the endothelium is a highly dynamic structure involved in many aspects of vascular biology. Endothelial cells play an important role in regulation of vasomotor tonus, hemostasis and coagulation, fluid and electrolyte transport, inflammation, and angiogenesis. Understanding the biology of the endothelium in specific diseases that can be associated with vascular damage could be of great interest for physicians. Quantification of specific endothelial features, such as the degree of proliferation (angiogenesis) or the extent of endothelial injury, could be of predictive or prognostic value, or could enable physicians to monitor the response to treatment. A novel endothelial biomarker currently under investigation, is the number of circulating endothelial cells (CEC). CEC are endothelial cells detached from the vessel wall, and as such are considered a promising marker for endothelial damage Increased numbers of CEC have been observed in diseases with documented vascular involvement such as vasculitis, sickle cell anemia, and cancer. Also, their number correlates to putative plasma markers of endothelial injury.

On the origin of (CD105+) circulating endothelial cells (Article)

2009-08-01T00:00:00Z

Cells designated by the CellSearch™ assay as circulating endothelial cells (CEC) (CD146+/CD105+/ CD45- nuclear cells) are thought to derive from damaged vasculature. As CD105 has been suggested to be expressed by endothelial cells from malignant vasculature particularly, it is currently unknown whether this assay is suitable to determine CECs in non-malignant diseases. Also, more insight is needed whether CECs as detected by this assay predominantly measures CECs or also endothelial progenitor cells (EPCs), which originate from the bone marrow and reflect angiogenesis rather than vascular damage. CEC counts were determined in nine patients treated with isolated limb perfusion with tumour necrosis factor (TNF) a and melphalan, and in 10 healthy donors. Given the severe vascular damage caused by venesection and cannulation of the main vessels, we expected a significant increase in CEC counts in case CEC were of vascular rather than of bone marrow origin. Additionally, this finding, as well as the presence of CD105+CEC in the blood of healthy controls, would confirm that healthy endothelial cells express CD105. Numbers of CD146+/CD105+/CD45-nuclear CEC increased significantly after venesection and cannulation. After administration of TNF, a large fraction of non-intact, possibly apoptotic CEC appeared. This study shows that the Cell-Search™ assay detects CECs originating from damaged vasculature. Furthermore, CD105 expression is found on CEC from damaged normal vasculature rendering further exploration of the value of CEC determined by this assay worthwhile not only in malignant diseases but also in non malignant disorders characterised by vascular damage.

Circulating endothelial cells: A potential parameter of organ damage in sickle cell anemia? (Article)

2009-07-01T00:00:00Z

Objective laboratory tools are needed to monitor developing organ damage in sickle cell disease (SCD). Circulating endothelial cells (CECs) are indicative of vascular injury. We determined whether elevated CEC can be detected in asymptomatic SCD with the CellSearch system and whether the CEC count is related to clinical and blood-based biomarkers of disease severity. Fifteen consecutive clinically asymptomatic HbSS patients and 15 matched HbAA controls were analyzed for CEC counts, laboratory parameters of disease severity (Hb, leukocyte counts, HbF%), plasma levels of markers for endothelial activation (sVCAM-1, VWF:Ag) and of endogenous inhibitors of nitric oxide synthase (asymmetrical dimethylarginine [ADMA]). CEC counts were significantly higher in patients (12 cells/mL, IQR 8-29) as compared to controls (4 cells/mL, 3-10) (P = 0.007). CEC counts were significantly higher in patients with pulmonary hypertension (PHT) (P = 0.015), and increased with increasing number of affected organs (0-4 involved organs, P = 0.002). No significant correlations between CEC and any other laboratory parameter were detected. In conclusion, CECs could prove to be an important new tool for assessing developing vasculopathy and organ damage in SCD.

Quantification of circulating endothelial cells by flow cytometry (Article)

2009-05-15T00:00:00Z

Correlation between circulating endothelial cell counts and plasma thrombomodulin levels as markers for endothelial damage (Article)

2008-10-01T00:00:00Z

Increased numbers of circulating endothelial cells (CEC) in peripheral blood have been observed in diseases with vascular involvement, and are considered a promising surrogate marker for vascular damage. It was the objective of this study to evaluate the correlation between putative soluble markers of endothelial injury, activation, and endothelial proliferation, and absolute numbers of CEC. CEC were evaluated in 125 healthy donors and 40 patients with metastatic carcinoma by automated CD146 driven immunomagnetic isolation. Plasma concentrations of E-selectin, endoglin, and thrombomodulin were assessed by ELISA in plasma obtained from 40 healthy donors and 40 patients. CEC numbers in blood were positively correlated with plasma thrombomodulin levels, but not with levels of E-selectin and endoglin. Multivariate analysis demonstrated a significant increase in CEC numbers with age. The levels of plasma biomarkers were not influenced by age. Higher levels of thrombomodulin and E-selectin were observed in males when compared to females. In conclusion, CEC numbers correlate positively with plasma levels of thrombomodulin.

Circulating endothelial cells in oncology: Pitfalls and promises (Article)

2008-06-03T00:00:00Z

Adequate blood supply is a prerequisite in the pathogenesis of solid malignancies. As a result, depriving a tumour from its oxygen and nutrients, either by preventing the formation of new vessels, or by disrupting vessels already present in the tumour, appears to be an effective treatment modality in oncology. Given the mechanism by which these agents exert their anti-tumour activity together with the crucial role of tumour vasculature in the pathogenesis of tumours, there is a great need for markers properly reflecting its impact. Circulating endothelial cells (CEC), which are thought to derive from damaged vasculature, may be such a marker. Appropriate enumeration of these cells appears to be a technical challenge. Nevertheless, first studies using validated CEC assays have shown that CEC numbers in patients with advanced malignancies are elevated compared to healthy controls making CEC a potential tool for among other establishing prognosis and therapy-induced effects. In this review, we will address the possible clinical applications of CEC detection in oncology, as well as the pitfalls encountered in this process.

Potential biomarkers in metastatic gastrointestinal stromal tumors (Article)

2007-10-15T00:00:00Z

Response to Goon et al. (Article)

2007-09-01T00:00:00Z

Cells meeting our immunophenotypic criteria of endothelial cells are large platelets (Article)

2007-03-15T00:00:00Z

Background: Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)low-to-intermedate, sideward light scatter (SSC)low, CD45-, CD31++and CD146+is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658-3661). Here, we set out to confirm the endothelial nature of these cells. Methods: We isolated cells with a FSClow-to-intermediate, SSClow, CD31++, CD45dimimmunophenotype (termed "cells meeting our immunophenotypic criteria for endothelial cells" [CMOIC]) from healthy donors to study the expression of endothelium-associated markers using several techniques. Special attention was paid to reagents identifying the endothelial cell-specific marker CD146. We compared antigen expression patterns of CMOIC with those of the HUVEC endothelial cell line and lymphocytes. Electron microscopy was used to detect the presence of endothelial cell-specific Weibel-Palade bodies in the sorted cells. Results: CD146 expression was negative on CMOIC for all tested CD146 mAbs, but positive on HUVEC cells and a minor subset of T lymphocytes. Using flow cytometry, we found no expression of any endothelium-associated marker except for CD31 and CD34. HUVEC cells were positive for all endothelial markers except for CD34. Evaluation of CMOIC morphology showed a homogenous population of cells with a highly irregular nucleus-like structure and positive endothelial immunohistochemistry. CMOIC contained neither nuclei nor DNA. Electron microscopy revealed the absence of a nucleus, the absence of endothelial specific Weibel-Palade bodies, and revealed CMOIC to be large platelets. Conclusion: The vast majority of cells with the immunophenotype FSClow-to-intermediate, SSClow, CD45-, CD31++do not express CD146 and are large platelets rather than endothelial cells.