WO2013002631A1

WO2013002631A1 - New flow cytometric method for detection of circulating endothelial cells

Info

Publication number: WO2013002631A1
Application number: PCT/NL2011/050468
Authority: WO
Inventors: Jaco KRAAN; Jan Willem GRATAMA; Stefan Sleijfer
Original assignee: Erasmus University Medical Center
Current assignee: Erasmus University Medical Center
Priority date: 2011-06-28
Filing date: 2011-06-28
Publication date: 2013-01-03
Anticipated expiration: 2013-12-28

Abstract

The invention comprises a method for detection of circulating endothelial cells (CECs) comprising: a. Lysis of erythrocyte cells in a blood sample; b. Subsequently adding a DNA dye and labelled antibodies directed at cell surface markers CD34, CD45 and CD146; c. Selecting cells positive for CD34; and d. Subsequently selecting cells containing DNA, positive for CD146 and negative for CD45. Such a method can preferably be used for diagnosis of a disease which involves vascular damage and/or for monitoring therapy effectiveness in such a disease.

Description

Title: New flow cytometric method for detection of circulating endothelial cells

FIELD OF THE INVENTION

The invention relates to the field of cardiology, oncology and diagnostics, more particularly diagnostics for conditions associated with vascular damage and as such especially suitable for monitoring drug efficiency of tumor drugs that target tumor angiogenesis.

BACKGROUND

.Circulating endothelial cells (CEC) are mature endothelial cells shed from injured vasculature and as such can serve as a biomarker for the presence of vascular disease (Blann, A.D. et al., 2005, Thromb. Haemost.

93:228-235; Delorme, B. et al., 2005, Thromb. Haemost. 94:1270-1279).

CECs have been implicated in cardiovascular (such as assessing cardiac risk), inflammatory and infectious diseases. Mature vascular endothelial cells exist in a variety of physiologic states ranging from quiescent to proliferative and activate to dysfunctional to terminal, whereupon they detach from the basement membrane and surrounding endothelial cells of the blood vessel and enter the circulation. As the cause, fate and role of CECs become better understood, the in vitro enumeration and characterization of CECs may offer a unique opportunity for diagnosis. Elevation of CECs has been observed in a large number of pathological conditions, such as cancer, cardiovascular, inflammatory, infectious and autoimmune diseases (Mutin, M. et al., 1999, Blood 93:2951-2958; WO 2004/045517). In particular, CEC levels may increase due to active tumor angiogenesis, vascular damage as result of tumor apoptosis or necrosis, or as a side effect of therapy on non-tumor vasculature. In recent years several methods have been applied to enumerate CECs. Unfortunately, there is no consensus between CEC phenotype and enumeration techniques and most methds have not been properly validated (Strijbos, M.H. et al., 2007, Ctometry B Clin. Cytom. 72:86-93). In combination with the relative rarity of CECs, this situation has resulted in a wide variation of reported CEC numbers, hindering further progress in the filed. The great variation in results is exemplified by the reported CEC numbers in healthy humans ranging from 4 to 1300 cells/mL (Furstenberger, G. et al., 2006, Br. J. Cancer 94:524-531; Goon, P.K. et al., 2006, Thromb. Haemost. 96:45-52;

Mancuso, P. et al., 2009, Clin. Cancer Res. 15:267-273; Widemann, A. et al.,2008, J. Thromb. Haemost. 6:869-876)).

Currently, most CEC enumeration tests rely on an enrichment step by immunomagnetic bead isolation (IB) using magnetic particles coupled to monoclonal antibody (mAb) targeting an endothelial antigen such as CD 146, followed by flow cytometry or visual counting with a fluorescent microscope to identify CEC on the basis of morphological or immunophenotypical criteria (Widemann, A. et al., 2008, supra; Dignat-George, F. et al., 2007, J. Clin. Oncol. 25:el-5). A major advantage of this latter approach is that it permits visual identification of CECs, which allows discrimination between CECs and endothelial micrparticles or platelets with strongly overlapping phenotypes. However, any enrichment step inevitable leads to cell loss and

underestimation of the actual CEC number, whilst manual bead-based isolation procedures are labor intensive and difficult to standardize.

A variant of manual IB enrichment techniques is the CellSearch® system (Veridex, Raritan, NJ). Initially designed to detect circulating tumor cells, the system provides a fully automated enrichment procedure that is followed by semi-automated imagecytometry. This assay has a high recovery and reproducibility. Importantly, CECs isolated in this way have been validated by global gene expression (Rowand, J.L. et al., 2007, Cytometry A 71:105-113; Smirnov, D.A. et al., 2006, Cancer Res. 66:2918-2922). Drawbacks of the CellSearch® system are the costly equipment and reagents, and the assay can not be customized. Also, the maximum number of eight samples that can be analyzed in a single run, combined with the relative long duration of a complete run (approximately 4 hours) does not allow high throughput analysis.

Therefore, there is still need for a quick, reliable, robust, validated and low cost CEC assay.

SUMMARY OF THE INVENTION

The inventors now have found an assay method for detection of circulating endothelial cells (CECs) comprising:

a. Lysis of erythrocyte cells in a blood sample;

b. Subsequently adding a DNA dye and labelled antibodies directed at cell surface markers CD34, CD45 and CD 146;

c. Selecting cells positive for CD34; and

d. Subsequently selecting cells containing DNA, positive for CD146 and negative for CD45.

Preferably in such a method the selection step is performed using a flow cytometrical method. In another preferred embodiment the DNA dye is 5-bis[2-(di-methylamino)ethyl] amino-4,8-dihydroxyanthracene-9,10-dione (DRAQ5). Also preferred is a method according to the invention wherein the antibodies directed to CD34, CD45 or CD 146 are monoclonal antibodies. More preferably, the antibodies directed to CD34, CD45 or CD 146 are labelled with three different fluorescent compounds.

Further preferred is a method wherein the lysis step is performed by adding ammoniumchloride to the sample. Also preferred is a method according to the invention wherein the blood sample contains peripheral blood and a method wherein between the lysis step and the staining step atypical Fc-receptor mediated antibody binding is prevented, preferably by adding antibodies against CD32. In a specific embodiment of the method of the invention the amount of CECs detected is measured as a percentage of total cells, and wherein the absolute amount of CECs is calculated by multiplication of the CEC percentage with the total CD34⁺ count obtained on the same blood sample using a flow cytometric assay.

Further part of the invention is a method for diagnosing a disease which involves vascular damage, by detection of CECs according to a method according to the invention. Preferably, the disease is selected from the group of cancer, cardiovascular, inflammatory, infectious and autoimmune disease.

In another embodiment, the invention comprises a method for monitoring therapy effectiveness in a patient by detection of CECs according to a method according to the invention 9 at least twice during therapy. Preferably said therapy is an angiogenesis inhibiting therapy against cancer.

LEGENDS TO THE FIGURES

Figure 1: Gating strategy for circulating endothelial cell (CEC, purple dots) analysis based on CD34 and CD 146 expression. Panel A-F: whole blood analysis for absolute counting of CD34 positive cells(red dots). Panel G-K: Flow cytometric analysis of 4 mL of blood using a CD34 threshold (panel G). Panel L shows the morphology and vWF expression of sorted CECs. Residual lymphocytes (CD45+, green dots) are serving as an internal (negative) control.

Figure 2: Flow cytometric analysis of endothelial cells isolated from a

dissected vene. Firstly, nucleated cells were selected based on DNA content using DRAQ5 expression (panel A). Secondly, endothelial cells were identified within the CD34 positive population (panel B, purple dots) expressing the pan- endothelial markers CD 146, CD144 and CD31 (panel C, D, H and I respectively), and subtype associated markers CD105, CD309 (VEGFR-2) and CD133 (panel E, F, G). Residual lymphocytes (CD45+, green dots) are serving as an internal (negative) control. Figure 3: Assay reproducibility. Correlation between duplicate samples assessed in two independently performed experiments. Regression analysis showed a best fit line with R2 of 0.96 and a slope of 1.01 (95% confidence interval (CI); 0.87 to 1.14) an Y-intercept of 1.51 (95% CI; -7.85 to 6.21) .

Figure 4: Correlation of CEC values from the two assays between the

number of events (red dots; R2 of 0.96 with a slope of 0.66 (95% CI; 0.61 to 0.71) and an intercept of 4.16 (95% CI; -1.95 to 10.26)) as well as the obtained absolute number (blue dots; R2 of 0.71 with a slope of

2.60 (95% CI; 1.92 to 3.27) and an intercept of 72.21 (95% CI = -7.88 to 152.3)).

Figure 5: Bland- Altman plots comparing the Cellsearch reference assay with the CD34- based FCM event counts and a absolute count assay.

The number of CEC using CD34-based event count analysis was significantly lower compared to the CellSearch reference assay (median; 20 versus 26, P=0.0065). suggesting a slightly lower recovery However, CEC numbers based on absolute numbers of CD34+ cells were significantly higher than the Cellsearch assay

(median; 129 versus 26, p<0.0001).

Figure 6: Prevalence of CEC using the CD34-based FCM assay in 4 mL of blood from 30 healthy individuals and significant increase in 55 metastatic carcinoma patients and 39 patients with a haematological malignancy in remission?. Solid lines indicate the median value for each group. DETAILED DESCRIPTION

In the following description and examples a number of terms are used. In order to provide a clear and consistent understanding of the

specification and claims, including the scope to be given to such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

A "DNA dye" is a compound that is able to stain cell organelles that contain DNA, especially DNA in the nucleus of the cells. Such dyes are commercially available. Examples are DAPI (4',6-diamidine-2-phenylindole), Hoechst 33258, Hoechst 33342, Syto 16 and DRAQ5.

"CD34" is a cell surface glycoprotein, which functions as an adhesion factor. It is expressed on hematopoietic cells, mesenchymal stem cells, endothelial progenitor cells, endothelial cells of blood vessels, mast cells and is some soft tissue tumors.

The "CD45" antigen is a protein tyrosine phosphatase involved in cell growth, differentiation and oncogenesis. It is present on differentiated hematopoietic cells, and several lymphomas and leukemia cells.

"CD 146" or the melanoma cell adhesion molecule is a marker for endothelial cell lineage. It is expressed on activated human T cells, endothelial progenitors such as angioblasts and mesenchymal stem cells, and strongly expressed on blood vessel endothelium and .

The invention now provides a method for detection of circulating endothelial cells (CECs) comprising:

a. lysis of erythrocyte cells in a blood sample;

c. selecting cells positive for CD34;

d. subsequently selecting cells containing DNA, positive for CD 146 and negative for CD45.

The choice of the marker specificity is derived from literature (Strijbos, M.H. et al., 2008, Br. J. Cancer 98:1731-1735), where it was discussed that clinical relevant CECs are characterized by the presence of a (DNA containg) nucleus and are characterized by being positive for the differentiation markers CD34 and CD 146, and negative for the differentiation marker CD45. Thus, in short, the cells can be characterized as DNA⁺, CD34⁺, CD45 , CD146⁺.

Mariucci, S. et al. (2008, Int. J. Lab. Hematol. 32:e 10-e 18) also used these differentiation markers in flow cytometry analysis for detection of CECs. However, it appears that the method as described by Mariucci et al., reveals only a very low amount of CECs, which corresponds to less than 1 per 100 μΐ blood.. The authors themselves indicate that a CEC analysis should only be considered informative when adequate numbers (approximately 1000 events) can be collected. Further, it is not clear whether all cell events counted by Mariucci et al. concern CECs. The present inventors have found previously (Strijbos, M.H. et al., 2007, Cytometry B Clin. Cytom. 72:86-93) that cells that seem to be CECs on basis of their surface marker distribution (CD31+, CD45-, CD 146+) and were further defined on basis of a scatter plot did not appear to be endothelial cells since they lacked a nucleus and/or DNA in the nucleus. Thus, apparently, platelets with similar differentiation markers will erroneously be counted as CECs unless these are discarded on basis of a selection for cells that contain nuclear DNA. It is submitted that in Mariucci et al., in which the cells have not been selected on the presence of nuclear DNA, many cells will be counted that would not classify as CECs.

A further difference with Mariucci et al. may be found in the fact that the blood sample in the present method is first subjected to mild lysis of the erythrocytes and only thereafter staining for the differentiation markers and the presence of DNA takes place, where in Mariucci et al. the sample is first stained and then lysed. In any case, due to the amount of (expensive) reagents needed, this necessitates a smaller sample size, which with the reported low numbers of CECs per sample increases the error margin in the analyses. In the present application, a relatively large (4 ml) sample is taken where after lysis this is condensed by centrifugation to an advantageous amount on which the staining and further flow cytometric analysis is performed. In the present invention, as compared with the method of Mariucci et al., thus an increase in the number of cells used for the flow cytometric analysis is achieved. The blood samples that are needed for the analysis may be taken in a regular manner (e.g. from the vena saphena in the arm) and should be prevented from clotting by adding EDTA, heparin, citric acid or any other clotting inhibitor. Then a mild lysis of the erythrocytes in the sample should be performed, which is advantageously done with an isoosmotic ammoniumchloride solution (concentration 0.15 M). However, also other methods, such as a hypotonic shock in distilled water, or lysis with diethylene glycol or other hypotonic agents may be used. After lysis the sample is enriched for the remaining cells by centrifugation and discarding the supernatant.

Before starting with the flow cytometric analysis, it is preferred to reduce aspecific binding of antibodies. Non specific binding can be a result of Fc receptor binding, binding to dead cells or improper use of reagents. In a lyse- stain-wash procedure, such as is used in the present invention, prevention of non specific binding can for instance be achieved by using only F(ab) or F(ab')2 antibody fragments for the identification of the cells that need to be analysed, but preferably this is achieved by Fc receptor blocking. A blocking reagent contains a high concentration of immunoglobulin that will bind to the Fc- receptors on cells like monocytes, thereby blocking the non-specific binding of the staining antibody reagents to these receptors. Specific antibodies directed against the Fc receptors of the cells in question are commercially available, such as the CD32 monoclonal antibody or other serum Fc receptor blocking agents, such as bovine serum albumin (BSA), normal serum diluted to 5% in PBS or commercially available Fc receptor blockers.

Further, false positives can be discarded on basis of DNA staining. Such a specific DNA stain is used to exclude platelets, aggregates and endothelial micro particles from the analysis. Dyes that also stain RNA, such as PI or LDS751 will fail to exclude platelets, since these have a relatively high expression of RNA (Strijbos, M.H. et al., 2007, Cytometry B Clin. Cytom. 72:86-93). For staining of the differentiation markers CD34, CD45 and CD146 preferably antibodies or antibody fragments, which are specific for these markers, are used. Such antibodies are commercially available. Further, it is necessary to label these antibodies or antibody fragments with a label, preferably a fluorescent label, and preferably each antibody with a different fluorescent label, to allow for a correct flow cytometric analysis. Also such labelled antibodies are commercially available as indicated in the experimental part herein.

Multiparameter flow cytometry analysis may be performed using standard apparatus and technology. The gating strategy that is preferably used is to select first for CD34+ cells in a fluorescence channel with minimal spectral overlap from other fluorescence channels used, to have optimal advantage of a threshold on CD34+ events to exclude majority of unwanted events. Finally CEC are identified as nucleated cells on basis of the DNA stain, CD 146+ and CD45 negative. The order in which the latter selection is performed is not essential.

Further, preferably the cell count obtained in the flow cytometry analysis is converted to an absolute cell count. Normally, the CEC count obtained in the above described analysis will be an underestimate, since a small degree of random cell loss will occur during the lysis, staining and washing procedure. A correction is preferably achieved by determining the absolute number of CD34⁺ cell counts obtained on the same blood sample using a single platform flow cytometry assay, such as described in literature (e.g. Sutherland, D.R. et al., 1996, J. Hematother. 5:213-226). After determining the absolute CD34⁺ cell count, the absolute numbers of CECs can be determined by multiplying the

CEC percentage obtained as a result from the flow cytometry analysis with the absolute CD34⁺ count.

The determination of the CECs in blood can be used for the diagnosis of diseases that are related to vascular damage. Since the variation in reported CEC counts of healthy humans is large, it is preferred to always include one or more healthy control samples for a reliable and statistically relevant analysis. The variation in healthy human subjects can be caused by the fact that obtaining the blood sample includes causing vascular damage, e.g. through the venipuncture by the needle that is used to collect the blood. It will be easily understandable that venipuncture is not standard and depends on many variables, such as the diameter of the needle, the poignancy of the needle, the elasticity of the vein, the position of the vein, the angle of insertion of the needle, etc., many of which are not under control of the experimenter.

Diseases that are accompanied by an elevated number of CECs are acute coronary syndrome, thromobocytopenic purpura, sickle cell disease, sepsis, lupus, nephritic syndromes, rejection of organ transplants, surgical trauma and cancer.

Except for the diagnosis of these diseases, the method of detection of CECs according to the invention can also be used to monitor progress of such a disease, or to monitor the effect of therapy of such a disease.

With respect to monitoring progress of the disease, the present method can also be used in assessing a favourable or unfavourable survival, and even preventing unnecessary therapy that could result in harmful side-effects whenm the prognosis is favourable. Thus, the present invention can be used for prognosis of any of a wide variety of disorders related to endothelial enumeration.

With respect to monitoring of therapy, the present invention is very well suited to monitor the effects of anti-angiogenic drugs that are especially used in cancer treatments. Antiangiogenic therapy inhibits the growth of new blood vessels. Because new blood vessel growth plays a critical role in many disease conditions, including disorders that cause blindness, arthritis, and cancer, angiogenesis inhibition is a "common denominator" approach to treating these diseases. Antiangiogenic drugs exert their beneficial effects in a number of ways: by disabling the agents that activate and promote cell growth, or by directly blocking the growing blood vessel cells. Angiogenesis inhibitory properties have been discovered in more than 300 substances, ranging from molecules produced naturally in animals and plants, such as green tea extract, to new chemicals synthesized in the laboratory.

The invention will be illustrated in the following Example(s), which is for illustrative purpose and not deemed to be limiting the invention as claimed.

EXAMPLE

Patients and blood collection

Peripheral blood (PB) was collected using EDTA- containing or CellSave™ tubes (Veridex) from healthy donors (n = 30), carcinoma patients (n = 51) and patients with hematological malignancies in remission (n = 66). All patients provided written informed consent and the study protocols were approved by the local research and ethics committee.

CEC isolation from dissected vein walls

Based on literature (Strijbos, M.H. et al., 2008, Br. J. Cancer 98:1731-1735), we defined CEC as CD34⁺, CD45^nes, CD146⁺ and DNA⁺ events. To confirm the CEC specificity of the combination of CD34 positivity, CD146 positivity and CD45 negativity, endothelial cells were isolated from dissections of vein walls using collagenase digestion. Vessel dissections (2-3 cm) were opened with sterile scissors, the opened vessel was placed in a petri dish with its endothelial side on a sterile cellulose tissue saturated with collagenase solution (Collagenase Type IV [Sigma C-5138]), 2 mg/mL in RPMI). After incubation for 60 min at 37°C, the vessel was placed in a petri dish containing RPMI after which cells from the endothelial side of the vessel were removed using a cell scraper. After the remainder of the vessel had been discarded, the content of the Petri dish was transported to a 50 mL conical tube and the cell suspension centrifuged at 1000 x g for 5 min. The resulting pellet was washed with 50 mL phosphate buffered saline (PBS) and concentrated at 1x106 cells/mL in PBS for flow cytometry.

CD34-based FCM assay

For CEC analysis, samples were prepared in a lyse-stain wash procedure with minimal sample handling. Four mL of PB were transferred into a 50 mL tube and 45 mL of ammoniumchloride 0.15M was added to induce red-cell lysis. After 20 min of lysis at room temperature (RT), the suspension was centrifuged for 5 min at 1000 x g and the supernatant was removed from the sample tube without disturbing the pellet. To reduce Fc receptor- mediated antibody binding, 50 pL of CD32 mAb (clone IV3, Stemcell

Technologies, Vancouver; 10 pg/mL in PBS containing 1% bovine serum albumin) was added and the pellet was carefully homogenized using a 100 pL pipette.

After 10 min of incubation, cells were stained using 50 pL of a mixture of the following monoclonal antibodies (mAb): fluorescein

isothiocyanate (FITC) conjugated CD34 (clone 8G12, BD Biosciences, San Jose, CA); R-phycoerythrin (PE) conjugated CD 105 (clone 1G2, IOTest, Marseille France); peridinin chlorophyll protein (PerCP) conjugated CD45 (clone 2D1, BD Biosciences); allophycocyanin (APC) conjugated CD146 (clone 541-10B2, Miltenyi Biotec), and 50 pL of the DNA dye 5-bis[2-(di-methylamino) ethyl] amino- 4, 8- dihydroxyanthracene-9,10-dione (DRAQ5, BioStatus Ltd, Leicestershire, UK). All reagents were diluted in PBS/BSA based on titration, i.e., absence of non-specific staining on negative populations and optimal discriminatory power between negative and positive populations. After 15 min of incubation in darkness at room temperature (RT), 45 mL of PBS were added and the suspension was centrifuged for 5 min at 1000 x g. After removal of supernatant the cells were resuspended in 500 pL of PBS and transferred to a standard 5 mL flow cytometry tube. The 50 mL tube was rinsed with 500 pL of PBS, and any remaining cells were also transferred to the cytometry tube to assure optimal recovery of cells. Samples were immediately acquired on a FACSCanto II flow cytometer with FACSDiva v6.1. software (BD Biosciences) or stored on ice for a maximum of 1 h. Data acquisition was started by collecting ungated data of 50,000 nucleated cells (DRAQ5+) at a low flow rate (10 pL/min) (Fig 1, panel A-F). Data acquisition was completed by acquiring the remainder of the sample at a high flow rata (120 pL/min) with a threshold (live-gate) on CD34+ events (Fig 1, panel G-I).

Absolute cell counting

Absolute counts were obtained by multiplying the CEC percentage within the CD34+ population in 4 mL of blood by the absolute CD34 counts obtained on the same blood sample using a single platform FCM assay according to ISHAGE guidelines 25-26. In brief, 200 pL of blood was incubated with CD34-FITC (clone 8G12), CD45-PErCP (clone 2D1) and DRAQ5. After 15 min incubation, 2 mL ammonium chloride lysing solution was added, followed by 100 pL Flow-Count beads. A minimum of 100 CD34+ events were acquired on a FASCCanto II flow cytometer (Fig 1, panel A-F).

Methods used for assay validation

Cell Sorting

Cells were sorted using a FACSAria Cell Sorting System (BD Biosciences) equipped with FACSDiva v6.1. software (BD Biosciences). For morphological and immunohistochemical analysis, cells were sorted on glass slides as spots of 50 cells. For quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). studies, cells were sorted in 2 mL Eppendorf tubes, lysed by adding 250 pL of Qiagen AllPrep DNA/RNA Micro Kit Lysis Buffer (RLT+ lysis buffer) (Qiagen BV, Venlo, The Netherlands) and stored immediately at -80°C until RNA isolation was performed with the AllPrep DNA/RNA Micro Kit (Qiagen) according to the manufacturer's guidelines.

Morphology

May-Grunwald-Giemsa (MGG) staining was performed using a standard eosin-methylene blue solution and Giemsa solution with an Autostainer XL (Leica Microsystems GmbH, Wetzlar, Germany). All images presented in this study were acquired on a DM2500 microscope equipped with a DC500 digital camera (both from Leica). The acquisition software (Leica IM1000 version 4.0) adjusted focus, brightness and contrast automatically.

Immunohistochemistry

Two-step horseradish peroxidase (HRP)-based

immunohistochemistry (IHC) was performed on sorted endothelial cells and lymphocytes using the ChemMate Envision Detection Kit (Dako, Glostrup, DK). Sorted populations were fixed in buffered formalin and stained using mouse-anti-human antibodies against CD31 and von Willebrand Factor (both from Dako). All Immunohistochemical stainings were evaluated by a pathologist.

qRT-PCR

The generation of pre- amplified cDNA from RNA and subsequent Taqman-based qRT-PCR analysis, as well as the validation procedures to ensure homogeneous amplification, were performed as described previously (Sieuwerts, A.M. et al., 2009, Breast cancer Res. Treat. 118:455-468). RT samples were diluted in nuclease-free ddF O and analyzed by real-time PCR in a 20 pL reaction volume in a Mx3000PTM Real-Time PCR System

(Stratagene, Amsterdam, The Netherlands). Negative controls included samples without reverse transcriptase and samples in which RNA and cDNA had been replaced with PCR-grade H2O or genomic DNA. Quantitative values were obtained from the threshold cycle (Ct) at which the increase in TaqMan probe fluorescent signal associated with an exponential increase of PCR products reached the fixed threshold value of 0.08; In all cases, this was at least ten times the standard deviation of the background signal.

Levels of HMBS, HPRT1 and GUSB were the reference genes used to control sample loading and RNA quality with Taqman assays-on-demand from Applied BioSystems (ABI, Nieuwerkerk a/d IJssel, The Netherlands) as described previously (Sieuwerts et al., 2009, supra). Protein tyrosine phosphatase receptor type C (PTPRC coding for CD45, ABI: Hs00236304_ml) was the control gene for leukocyte background and CDH5 (coding for CD 144, ABI: Hs00174344_ml), MCAM (coding for CD146, ABI: Hs00174838_ml), VEGFR2 (ABI: Hs00176676_ml) and VW (ABI: Hs00169795_ml) the controls for CEC detection. The delta Ct method was used to evaluate gene transcript levels relative to the reference genes.

CellSearch® Circulating Endothelial Cell reference assay CEC were enumerated using the CellSearch® Endothelial Cell Kit and CellTracks® AutoPrep® System (Veridex) according to the standard procedure (Rowland et al., 2007, supra). In brief, a blood sample of 4 mL was mixed with ferrofluid particles coated with anti-CD146 antibodies. After incubation, unlabeled cells and plasma were removed by magnetic separation. Subsequently, the isolated cells were stained with fluorescent monoclonal antibodies for endothelial cells (CD105-PE) and contaminating leucocytes (CD45-APC), and a nuclear staining dye (DAPI). During this sample

processing procedure, 4 mL of blood is reduced to ~ 700 pL containing enriched CEC, which is next transferred to a cartridge in a Magnest® device. Here, the strong magnetic field moves immunomagnetically labeled cells to the surface of the cartridge caused by the strong magnetic field in order to render them ready for analysis. All steps were performed fully automatically by the

CellTracks® AutoPrep® System.

The samples were read with the CellTracks Analyzer®, a semi- automated fluorescence microscope used to scan the entire surface of the cartridge at four different wavelengths in order to detect objects labeled with all fluorochromes used.

Captured images that contain objects fulfilling predetermined criteria were automatically presented in a gallery format from which events were classified by a trained operator. A CD146+ cell was classified as a CEC on the basis of its morphologic features and staining patterns (i.e., DAPI positive, CD 105 positive and CD45 negative). Statistical analysis

Statistical analysis was performed using Prism™ software (GraphPad Software, La Jolla, CA). To analyze the degree of agreement between the CD34-based FCM assay and the CellSearch reference assay, Bland-Altman plots were generated to relate the inter-assay difference with the mean CEC count of the two methods. The Mann-Whitney U-test was used to compare healthy control with patient samples. P < 0.05 was considered to be significant. RESULTS

Immunophenotypic characteristics of EC isolated from vein dissections.

We validated our phenotypic criteria to analyze CEC by FCM on isolated endothelial cells (EC) from vein wall dissections (n=3). EC were isolated and stained with the same mAb cocktail as used in the assay. As illustrated in Figure 2, panel A-C, isolated EC showed a high expression of CD34 and CD 146 and were negative for CD45 showing that isolated EC fulfill the phenotypic criteria for CEC and are selected by the Boolean gating strategy (DRAQ5+, CD34+, CD45-,CD146+) used in the FCM assay. Further we could demonstrate that isolated EC are positive for several other endothelial markers such as the pan- endothelial markers CD31 and CD144, and the subtype-associated markers CD105, CD309 (VEGFR-2) and CD133 (Figure 2, panel D-I).

Validation using morphology, IHC and gene expression

Firstly, CEC were sorted on glass slides and stained for MGG and by immunohistocytologyy using the endothelial specific markers CD31 and vWF. More than 95% of all sorted events showed specific EC morphology and were brightly positive for vWF (Figure 2, panel L) and CD31. In parallel lymphocytes and human stem cells (HSC) were sorted to serve as control cells. Lymphocytes were negative for vWF and partially weakly positive for CD31. HSC were negative for vWF and a subset was positive for CD31.

Secondly, sorted CEC were analyzed for mRNA expression by qRT- PCR of both endothelial specific genes and leukocyte specific genes in duplicate 5 assays. This RNA profiling by qRT-PCR showed the presence of endothelial specific markers (CDH5, MCAM, VWF) and absence of an hematopoietic marker (PTPRC coding for CD45) in our CEC sorted preparations (Table 1). This indicates an endothelial specific gene expression profile for the cells meeting the phenotypic criteria of the FCM assay.

Table 1: Gene transcripts measured in FCM sorted cells

undetectable

+/- detectable, less than 1-fold higher expressed relative to the reference gene

+ over 1-fold higher expressed relative to the reference gene

++ over 10-fold higher expressed relative to the reference gene

5 +++ over 100-fold higher expressed relative to the reference gene

Reproducibility of CEC measurements between duplicate samples

To measure the variability of CEC counts obtained from two 4 mL aliquots derived from a single blood draw, CEC were enumerated in duplicate using the blood in a single draw from 13 different samples. Regression analysis showed a best-fit line with a slope of 1.006 (95% confidence interval = 0.8702 to 1.141) an Y-intercept of 1.511 (95% confidence interval = - 7.854 to 6.206) and 5 an R2 of 0, 96 (Figure 3) .

Agreement with CellSearch assay We studied a total of 28 subjects (10 healthy controls and 18 patients with different metastatic carcinomas) for comparison of the two techniques. Figure 4 shows the correlation between the number of events (light grey dots) and the obtained absolute number (black dots) by the FCM method compared with the CellSearch reference assay. Regression analysis for the event count analysis (light grey dots) showed a best fit line with a slope of 0.66 (95% confidence interval = 0.61 to 0.71), an intercept of 4.16 (95% confidence interval = -1.95 to 10.26) and an R² of 0.96. Regression analysis for the absolute count analysis (black dots) showed a best fit line with a slope of 2.60 (95% confidence interval = 1.92 to 3.27), an intercept of 72.21 (95% confidence interval = -7.88 to 152.3 ) and an R² of 0.71. Figure 5 shows the corresponding Bland-Altman plots. The median number of CEC in the FCM-based event count analysis was significantly lower compared to the CellSearch reference assay (median 20 versus 26, p=0.0065), but significantly higher when expressing CEC as absolute numbers by multiplying the CEC percentage within the CD34+ population in 4 mL of blood by the absolute CD34 counts obtained on the same blood sample (median 129 versus 26, p<0.0001). This suggests a slightly lower recovery for counts but a significantly higher recovery when using the FCM absolute number method.

CEC in healthy individuals, patients with metastatic carcinoma and hematological malignancies in remission.

Figure 6 shows that CEC levels in healthy individuals ranged from 4 to 79 cells/4 mL (n = 30, median 18) and were significant higher in carcinoma patients (ranging from 5 to 354 (n = 51, median 35, p < 0.005)) and in patients with hematological malignancies (range: 4 to 653 (n = 66, median 37, p < 0.0005).

Claims

1. Method for detection of circulating endothelial cells (CECs) comprising:

a. Lysis of erythrocyte cells in a blood sample;

c. Selecting cells positive for CD34; and

2. Method according to claim 1, wherein the selection step is performed using a flow cytometrical method.

3. Method according to claim 1 or 2, wherein the DNA dye is 5-bis[2- (di-methylamino)ethyl]amino-4,8-dihydroxyanthracene-9,10-dione

(DRAQ5).

4. Method according to any of the preceding claims, wherein the antibodies directed to CD34, CD45 or CD 146 are monoclonal antibodies.

5. Method according to any of the preceding claims, wherein the antibodies directed to CD34, CD45 or CD 146 are labelled with three different fluorescent compounds.

6. Method according to any of the preceding claims, wherein the lysis step is performed by adding ammoniumchloride to the sample.

7. Method according to any of the preceding claims, wherein the blood sample contains peripheral blood.

8. Method according to any of the preceding claims, wherein the amount of CECs detected is measured as a percentage of total cells, and wherein the absolute amount of CECs is calculated by multiplication of the CEC percentage with the total CD34⁺ count obtained on the same blood sample using a flow cytometric assay.

9. Method according to any of the preceding claims, wherein between the lysis step and the staining step atypical Fc-receptor mediated antibody binding is prevented, preferably by adding antibodies against CD32.

10. Method for diagnosing a disease which involves vascular damage, by detection of CECs according to a method according to any of claims 1 - 9.

11. Method according to claim 10, wherein the disease is selected from the group of cancer, cardiovascular, inflammatory, infectious and autoimmune disease.

12. Method for monitoring therapy effectiveness in a patient by detection of CECs according to a method according to any of claims 1 - 9 at least twice during therapy.

13. Method according to claim 12, wherein said therapy is an

angiogenesis inhibiting therapy against cancer.