Michiel H. Strijbos

Circulating endothelial cells in oncology: pitfalls and promises

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.

Cells meeting our immunophenotypic criteria of endothelial cells are large platelets

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.

A new approach for rapid and reliable enumeration of circulating endothelial cells in patients

Background: Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4–1300 per mL).

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

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: a potential parameter of organ damage in sickle cell anemia?

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.

On the origin of (CD105+) circulating endothelial cells

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.

Levels of circulating endothelial cells in normotensive and severe preeclamptic pregnancies

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.

Quantification of circulating endothelial cells by flow cytometry

To the Editors: We read with great interest the article by Mancuso et al. ([1][1]) in the January edition of Clinical Cancer Research . In this article, the authors describe the validation of a novel multicolor flow cytometrical assay that allows the quantification of both circulating endothelial

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

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.

Potential biomarkers in metastatic gastrointestinal stromal tumors.

To the Editor: We read with great interest the article by Norden-Zfoni and colleagues in the May issue of Clinical Cancer Research . In this article, the investigators aimed to identify potential biomarkers in the blood of patients with metastatic gastrointestinal stromal tumors treated with