Circulating Endothelial Cells and Procoagulant Microparticles in Patients with Glioblastoma: Prognostic Value
et al. (2013) Circulating Endothelial Cells and Procoagulant Microparticles in Patients with
Glioblastoma: Prognostic Value. PLoS ONE 8(7): e69034. doi:10.1371/journal.pone.0069034
Circulating Endothelial Cells and Procoagulant Microparticles in Patients with Glioblastoma: Prognostic Value
Gaspar Reyne s 0
Virtudes Vila 0
Tania Fleitas 0
Edelmiro Reganon 0
Jaime Font de Mora 0
Mara Jorda 0
Vicenta Martnez-Sales 0
Benjamin Edward Rich, Dana-Farber Cancer Institute, United States of America
0 1 Servicio de Oncolog a Me dica, Hospital Universitari i Polite`cnic La Fe , Valencia , Spain , 2 Centro de Investigaci o n, Hospital Universitari i Polite`cnic La Fe , Valencia , Spain , 3 Servicio de Hematolog a y Oncolog a Me dica, Hospital Cl nico Universitario , Valencia , Spain , 4 Instituto de Investigaci o n Sanitaria Hospital La Fe , Valencia , Spain , 5 Servicio de Anatom a Patolo gica, Hospital Universitari i Polite`cnic La Fe , Valencia , Spain
Aim: Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter. Methods: Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification. Results: Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p,0.001). After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p,0.004). Conclusion: Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.
Funding: This work was supported in part by SAF2012-37330 grant from the Spanish Ministry of Economy and Competitiveness. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The current standard of care for newly diagnosed glioblastoma
is surgery, radiotherapy, and concomitant daily temozolomide,
followed by cycles of this drug given for five days every four weeks.
Despite treatment, most patients die within two years of surgery
. The proportion of patients who benefit from this therapy is
determined only partially by the methylation status of the
O6alkylguanine-DNA-methyltransferase (MGMT) gene promoter,
which is considered a prognostic factor rather than a predictor
of response . Assessment of response in glioblastoma patients is
difficult because radiochemotherapy modifies vascular
permeability in the tumor area. This alteration can lead to
pseudoprogression, an increase in contrast tumor enhancement that mimics the
true progression . Therefore, it is crucial to identify biomarkers
that may help establish the prognosis of patients with glioblastoma
and predict their response to treatment.
Glioblastoma is a highly vascularized tumor that displays active
angiogenesis ; thus, drugs with antiangiogenic properties, such
as bevacizumab and cilengitide, are being tested for use with
radiotherapy and temozolomide [5,6]. Nevertheless, continuous
temozolomide, as described for other metronomic chemotherapy
regimens, might have antiangiogenic activity by itself, mediated in
part by a direct effect on tumor vessel endothelium .
Circulating endothelial cells (CECs) consist of at least
endothelial progenitor cells (EPCs) that originate in the bone marrow,
mature endothelial cells shed from vessel walls, apoptic endothelial
cells and some cells with endothelial function from cancerous cells
. Recently, circulating endothelial cells (CECs) have been
established as markers of endothelial damage or dysfunction .
CEC levels increase in many kinds of disorders such as
cardiovascular [10,11], autoimmune , and infectious diseases
, as well as in cancer . In cancer patients, CEC number
correlates with tumor progression  and constitutes a promising
tool for monitoring disease activity, with potential for the
assessment of prognosis and response to treatment. In patients
with non-small cell lung cancer (NSCLC), we observed an
association between elevated CEC numbers and decreased overall
survival (OS) , although in a study by Kawaishi et al. ,
high CEC numbers were associated with longer progression-free
survival (PFS). In patients with breast cancer treated with
metronomic chemotherapy, CEC levels after two months
treatment were associated with prolonged PFS ; in another trial
with metronomic chemotherapy and bevacizumab, baseline CEC
levels were also associated with PFS . It has been suggested
that quantification of CECs is useful to identify patients who might
benefit from antiangiogenic treatments . Batchelor et al., in a
series of patients with glioblastoma treated with AZD2171, a
panVEGF receptor tyrosine kinase inhibitor, found that viable CEC
number increased when tumors escaped treatment .
Microparticles (MPs) are small vesicles (100 nm1 mm) which
directly bud from the plasma membrane of different cells,
including blood, endothelial and tumor cells [22,23]. During MP
formation, phosphatidylserine (PS) is transferred from the inner to
the outer leaflet of the membrane; this externalization of PS
facilitates the assembly of components of the clotting cascade, thus
increasing the procoagulant activity of MPs . The
procoagulant MP levels increase in cancer patients . In patients with
castration-resistant prostate cancer, high platelet-derived MP
number is associated with shorter survival . However, in
patients with NSCLC we observed an association between
elevated total MP count and increased OS . Nevertheless,
the potential prognostic value of MPs in glioblastoma patients
The aim of this study was to evaluate the potential prognostic
value of CECs, MPs and MP-mediated procoagulant activity in
patients with newly diagnosed glioblastoma. In addition, we have
analyzed the methylation status of MGMT promoter in tumor
Materials and Methods
Study Design and Patients
This prospective study included consecutive patients with newly
diagnosed, histologically proven glioblastoma who received
standard treatment  at La Fe University Hospital. The control
group comprised healthy subjects matched for sex and age with
the patients. The study was conducted in accordance with the
principles outlined in the Declaration of Helsinki. All participants
gave written informed consent. The study was approved by the
institutional Biomedical Research Ethics Committee.
After surgery, patients received radiotherapy to a total dose of
60 Gy in 30 fractions given five days per week, plus concomitant
temozolomide at a daily dose of 75 mg/m2. After a four-week rest,
adjuvant temozolomide was administered at a dose of 150 to
200 mg/m2 for five days every four weeks until progression,
unaccepable toxicity or other reasons that hinder treatment.
Patients were assessed by magnetic resonance imaging (MRI) at
baseline. Subsequent imaging assessments were performed within
72 hours after surgery, to check the extent of tumor resection and
to rule out postsurgical complications, and every three cycles of
temozolomide thereafter. Perfusion, diffusion, and spectroscopy
MRI procedures were performed when indicated. At progression,
patients amenable for second-line treatment received bevacizumab
plus irinotecan, fotemustine or rechallenge with temozolomide.
Blood sampling. Venous blood samples were obtained from
patients within two weeks before the start of radiochemotherapy
and during the last week of this treatment. The initial 3 mL of
blood was discarded to avoid contamination with endothelial cells
from the puncture wound of the vein. Blood for quantification of
CECs was collected in a tube containing
ethylenediaminetetraacetic acid (1.8 mg/mL). For the determination of MP levels and
MP-mediated procoagulant activity, blood was collected in a tube
containing sodium citrate (129 mM) at a ratio of 1:9 (v/v, sodium
citrate/blood). We have previously studied pre-analytical
conditions to analyze MP count and pro-coagulant activity, and
centrifugation at 15006g, for 30 min, at 4uC and analysis on
frozen plasma samples have been applied. Plasma was stored at
80uC to allow later batch analysis.
Quantification of circulating endothelial cells. The
isolation and quantification of CECs was performed by
immunomagnetic technique following a consensus protocol . In brief,
cells were isolated from whole blood at 4uC by means of an
endothelial cell specific monoclonal antibody sEndo1 (BioCytex,
Marseille, France) raised against the endothelial antigen CD146,
coupled to micromagnetic beadsPan-Mouse M450
Dynabeads.Dynal, Oslo, Norway. To avoid nonspecific binding of leukocytes
to CD146-coated beads, cells were incubated after
immunomagnetic isolation of CECs with fluorescein isotiocyanate-conjugated
(FITC)-Ulex europaeus lectin-1 (UEA1). UEA-1 lectin
(SigmaAldrich, Inc., Saint Louis, MO, USA) is a good histologic marker
for endothelium in human, and constitutes a specific and sensitive
additional tool in demonstrating endothelial cells and endothelial
derivation of human tumors. After incubation, samples were
washed, suspended in buffer, and counted with fluorescence
microscopy using a Nageotte chamber. The size of the CEC
population often exceeds 10 mm, which is not compatible with the
typical size of endothelial progenitor cells. In addition, the
morphology of our cells indicates considerable damage or even
necrosis. Nucleated cells .10 mm in length with more than eight
immunomagnetic beads attached and positive UEA1 staining were
regarded as CECs. Conglomerates were counted as one cell. The
number of CECs was expressed as cells/mL of blood.
Reproducibility was tested by performing six replicates of 10 different
samples; the coefficient of variation was 12%.
Quantification of total microparticles. Plasma MPs were
quantified by flow cytometry in an EPICS XL-cytometer
(Beckman Coulter, Brea, CA, USA) at high flow rate. Plasma was
incubated with FITCAnnexin V conjugate (TACS Annexin V;
Trevigen Inc. Gaithersburg, MD, USA) to detect accessible
phosphatidylserine on MP membranes. Standard fluorescent
beads of different diameters were used for size calibration (0.5
3.0 mm, Megamix; BioCytex, Marseille, France) and to set the gate
for MP detection at a diameter of 0.51 mm following a consensus
guideline on MP measurement . The number of FITC
Annexin V-positive MPs was calculated and expressed as events/
mL of plasma.
Assessment of MP-mediated Procoagulant Activity
The MP-mediated procoagulant activity of plasma was analyzed
by thrombin generation (TG) assay without added exogenous
tissue factor or phospholipids (Calibrated automated
thrombogram, CAT; Thrombinoscope BV, Paris, FranceG). Under these
conditions, the assay was critically dependent on MPs present in
plasma. Curves were calculated using the Thrombinoscope
software and the results were expressed as the thrombin peak
(nM). MP activity depends on the exposure of anionic
phospholipids that provide a surface for the assembly of the tenase and
prothrombinase complexes. To measure this activity the
procoagulant phospholipid-dependent clotting time (PPLCT) assay was
also analyzed (STA-Procoag-PPL; Diagnostica Stago, Paris,
MGMT methylation analysis. Formalin-fixed,
paraffinembedded tumor samples were subjected to careful histological
assessment in order to select tumor areas. Three non-tumor areas
of brain tissue were further isolated and independently mounted in
paraffin blocks. DNA from 10 mm sections of each tumor and
from the three controls was extracted with the deparaffination
solution (Qiagen, Venlo, The Netherlands), followed by its
purification with the DNA Investigator kit (Qiagen). Methylation
status of MGMT promoter was determined by multiplex
ligationdependent probe amplification (MLPA) following the
manufacturers protocol (ME011-B1 MLPA, MRC-Holland). A
methylation-sensitive restriction enzyme, HhaI (New England BioLabs),
which cuts unmethylated GCGC sites, was applied to each set of
samples. Reaction products were resolved on ABI3700 automated
DNA sequencer and quantification of the methylation status of
MGMT promoter was performed by Coffalyser software
The Kolmogorov-Smirnov test was used to evaluate whether
each parameter came from a normal distribution. Statistical
analyses were performed using a non-parametric test among
sample types (control, pre-treatment and post-treatment)
(KrusalWallis test) and for the independent relationship of the control
samples with respect to patient groups (Chi-Square test with Yates
correction). Bivariate correlation was performed using Spearmans
correlation test. OS and PFS were analyzed by Kaplan-Meyer
method and survival curves of subgroups were compared using the
log-rank test. CEC values were dichotomized as greater than 99%
confidence interval in healthy controls (CEC = 20 cells/ml). All
statistical calculations were performed using SPSS software (v.
15.0; SPSS Inc., Chicago, IL, USA).
Twenty-two patients and forty healthy subjects were included in
the study during a period of 18 months. Patients characteristics
are shown in Table 1. All patients had histologically confirmed
glioblastoma. Median PFS was 30 weeks (9135) and median OS
was 33 weeks (10146).
Circulating Marker and Correlation Analysis
Levels of CECs, MPs, TG and PPLCT in patients and controls
are shown in Figure 1. Compared with the healthy control group,
mean pre-treatment levels of CECs and MPs were significantly
higher (p,0.001). Post-treatment levels of CECs remained
significantly higher in patients than in controls, while levels of
MPs and TG decreased, and PPLCT increased significantly after
treatment. Significant correlations were found between both
preand post-treatment levels of TG and MPs (p,0.01), while PPLCT
inversely correlated with pre-treatment levels of MPs (p,0.01) and
TG (p,0.05) (Table 2). Platelet and leukocyte count significantly
decreased after treatment (platelet: 283 vs 1666103, p,0.0001;
leukocyte: 8.6 vs 6.26103, p = 0.021). Platelet count significantly
correlated with TG (r = 0.52, p,0.001), MPs (r = 0.39, p = 0.013)
and PPLCT (r = 20.49, p,0.01).
Patients (n = 22)
KPS: Karnofsky performance scale; S. Biopsy: stereotactic biopsy.
Circulating Markers, MGMT Status and Clinical Outcome
The analysis of the associations between circulating markers and
clinical outcome showed that pre-treatment CEC levels .20 cells/
mL (corresponding to the 99th percentile in controls) were
associated with poor OS (19 vs. 72 weeks; Log rank 4.566;
p = 0.033) (Figure 2). No such association was found for pre- or
post-treatment levels of MPs, TG and PPLCT.
MGMT promoter was methylated in 27% of tumor samples.
MGMT status significantly influenced median OS, which was 66
weeks (95% CI, 44.6 to 87.4 weeks) in MGMT promoter
methylated patients and 30 weeks (95% CI, 14.4 to 45.5 weeks)
in MGMT promoter unmethylated patients (P,0.004), although
its influence on PFS was not statistically significant. The difference
in OS observed according CEC pre-treatment levels and TG
posttreatment levels did not reach statistical significance when
analyzed separately in methylated and unmethylated patients.
Our results show that pre-treatment CEC levels were
significantly elevated in patients with glioblastoma compared with
controls, a finding that is consistent with those of previous studies
in different types of cancer . We have found an association
between higher basal CEC count (.99th percentile of the CEC
count in controls) and poor survival. In other tumors, the
association between baseline CEC count and clinical outcome is
conflicting [1517,29]. Specific tumor characteristics and the
variety of methods being used to identify CECs may explain these
discrepancies. The standard treatment received by all patients in
the present study includes low-dose, daily temozolomide along
with radiation therapy. A study using a murine model reported
that low-dose; continuous (metronomic) chemotherapy leads to
apoptosis of endothelial cells within the tumor bed, resulting in
increased apoptosis of tumor cells . In patients with breast
cancer receiving metronomic chemotherapy with methotrexate
and cyclophosphamide with or without thalidomide, an increase in
CEC count after two months was associated with a better PFS
. Similar results (i.e., an association between good outcome
and an increase in CEC count after several treatment cycles) have
been found in patients with cancer receiving antiangiogenic drugs
[31,32]. Nevertheless, a recent study in patients with colorectal
Figure 1. Pre- and posttreatment levels of biomarkers in patients and in controls. A: Circulating endothelial cells (CECs), B: Microparticles
(MPs), C: Endogen thrombin generation (TG); D: Procoagulant phospholipid-dependent clotting time (PPLCT). Marker levels and their standard
deviations are shown for pre-t: pretreatment; post-t: posttreatment; c: controls. Logarithmic transformation of data was made to normalize the
distributions. NS: no significant.
cancer concluded that high viable CEC count both at baseline and
after the first cycle of chemotherapy plus bevacizumab, was
associated with a worse outcome . In the present study, the
CEC levels were higher than in controls; however, they did not
increase significantly after radiochemotherapy. The impact of
radiation therapy on CEC number has not been studied fully, and
any effect could have influenced the post-treatment CEC count in
We found that the pretreatment MP count was significantly
elevated in glioblastoma patients compared with controls. In
agreement with our findings, microvesicles together with exosomes
have also been reported to be elevated in glioblastoma patients
and decrease upon temozolomide treatment .
Another finding of the present study is the decrease of MP and
TG levels, and the increased PPLCT observed after treatment, an
effect that could be explained by a decrease in the levels of their
parent cells or by an inhibition of MP release induced by
radiochemotherapy; although leukocytes and platelets decreased
after treatment, only platelet count significantly correlated with
TG, MPs and PPLCT. In a recent work by Sartori et al. , the
procoagulant activity of annexin V-positive MPs was analyzed in
61 patients with glioblastoma at different times in their evolution;
in accordance with the present study, they found that MP activity
became significantly lower 1 and 4 months after surgery, though
only in patients achieving complete surgical resection. As
expected, in the present study OS was higher in patients with
methylated MGMT promoter.
In summary, this exploratory study suggests an association
between postsurgical higher CEC count and shorter survival in
patients with glioblastoma. We believe that these findings warrant
further investigation with a larger number of patients.
The authors thank Josefa Llorens, Ursula Salinas, and David Mesado for
their technical assistance. We also thank Adela Ma nez for her assistance in
blood sampling and David Hervas at Unidad de Bioestadstica La Fe for
reviewing the statistical analysis.
Conceived and designed the experiments: GR VV VM-S. Performed the
experiments: VV JFdM MJ VM-S. Analyzed the data: VV TF ER JFdM
VM-S. Wrote the paper: GR VV TF JFdM VM-S.
1. Stupp R , Mason WP , van den Bent MJ , Weller M , Fisher B , et al. ( 2005 ) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma . N Engl J Med 352 : 987 - 996 .
2. Hegi ME , Diserens AC , Gorlia T , Hamou MF , de Tribolet N , et al. ( 2005 ) MGMT gene silencing and benefit from temozolomide in glioblastoma . N Engl J Med 352 : 997 - 1003 .
3. Brandsma D , Stalpers L , Taal W , Sminia P , van den Bent MJ ( 2008 ) Clinical features, mechanisms, and management of pseudoprogression in malignant gliomas . Lancet Oncol 9 : 453 - 461 .
4. Kargiotis O , Rao JS , Kyritsis AP ( 2006 ) Mechanisms of angiogenesis in gliomas . J Neurooncol 78 : 281 - 293 .
5. Stupp R , Hegi ME , Neyns B , Goldbrunner R , Schlegel U , et al. ( 2010 ) Phase I / IIa study of cilengitide and temozolomide with concomitant radiotherapy followed by cilengitide and temozolomide maintenance therapy in patients with newly diagnosed glioblastoma . J Clin Oncol 28 : 2712 - 2718 .
6. Lai A , Tran A , Nghiemphu PL , Pope WB , Solis OE , et al. ( 2011 ) Phase II study of bevacizumab plus temozolomide during and after radiation therapy for patients with newly diagnosed glioblastoma multiforme . J Clin Oncol 29 : 142 - 148 .
7. Kim JT , Kim JS , Ko KW , Kong DS , Kang CM , et al. ( 2006 ) Metronomic treatment of temozolomide inhibits tumor cell growth through reduction of angiogenesis and augmentation of apoptosis in orthotopic models of gliomas . Oncol Rep 16 : 33 - 39 .
8. Ricci-Vitiani L , Pallini R , Biffoni M , Todaro M , Invernici G , et al. ( 2010 ) Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells . Nature 468 : 824 - 828 .
9. Erdbruegger U , Dhaygude A , Haubitz M , Woywodt A ( 2010 ) Circulating endothelial cells: markers and mediators of vascular damage . Curr Stem Cell Res Ther 5 : 294 - 302 .
10. Boos CJ , Lip GY , Blann AD ( 2006 ) Circulating endothelial cells in cardiovascular disease . J Am Coll Cardiol 48 : 1538 - 1547 .
11. Martnez-Sales V , Sanchez-Lazaro I, Vila V , Almenar L , Contreras MT , et al. ( 2011 ) Circulating endothelial cells in patients with heart failure and left ventricular dysfunction . Disease Markers 31 : 75 - 82 .
12. Attia FM , Maaty A , Kalil FA ( 2011 ) Circulating endothelial cells as a marker of vascular dysfunction in patients with systemic lupus erythematosus by real-time polymerase chain reaction . Arch Pathol Lab Med 135 : 1482 - 1485 .
13. Muntunga M , Fulton B , Bullock R , Batchelor A , Gascoigne A , et al. ( 2001 ) Circulating endothelial cells in patients with septic shock . Am J Respir Crit Care Med 163 : 195 - 200 .
14. Mancuso P , Burlini A , Pruneri G , Goldhirsch A , Martinelli G , et al. ( 2001 ) Resting and activated endothelial cells are increased in the peripheral blood of cancer patients . Blood 97 : 3658 - 3661 .
15. Beerepoot LV , Mehra N , Vermaat JS , Zonnenberg BA , Gebbink MF , et al. ( 2004 ) Increased levels of viable circulating endothelial cells are an indicator of progressive disease in cancer patients . Ann Oncol 15 : 139 - 145 .
16. Fleitas T , Martnez-Sales V , Vila V , Reganon E , Mesado D , et al. ( 2012 ) Circulating endothelial cells and microparticles as prognostic markers in advanced non-small cell lung cancer . PLoS One 7 : e47365 . Available: http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC3471832/.
17. Kawaishi M , Fujiwara Y , Fukui T , Kato T , Yamada K , et al. ( 2009 ) Circulating endothelial cells in non-small cell lung cancer patients treated with carboplatin and paclitaxel . J Thorac Oncol 4 : 208 - 213 .
18. Mancuso P , Colleoni M , Calleri A , Orlando L , Maisonneuve P , et al. ( 2006 ) Circulating endothelial-cell kinetics and viability predict survival in breast cancer patients receiving metronomic chemotherapy . Blood 108 : 452 - 459 .
19. Calleri A , Bono A ( 2009 ) Predictive Potential of Angiogenic Growth Factors and Circulating Endothelial Cells in Breast Cancer Patients Receiving Metronomic Chemotherapy Plus Bevacizumab . Clin Cancer Res 15 : 7652 - 7657 .
20. Mancuso P , Calleri A , Bertolini F ( 2012 ) Circulating endothelial cells and circulating endothelial progenitors . Recent Results Cancer Res 195 : 163 - 170 .
21. Batchelor TT , Sorensen AG , di Tomaso E , Zhang WT , Duda DG , et al. ( 2007 ) AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients . Cancer Cell 11 : 83 - 95 .
22. Morel O , Toti F , Hugel B , Freyssinet JM ( 2004 ) Cellular microparticles: a disseminated storage pool of bioactive vascular effectors . Curr Opin Hematol 11 : 156 - 164 .
23. Cocucci E , Racchetti G , Meldolesi J ( 2009 ) Shedding microvesicles: artefacts no more . Trends Cell Biol 19 : 43 - 51 .
24. Owens AP 3rd, Mackman N ( 2011 ) Microparticles in hemostasis and thrombosis . Circ Res 108 : 1284 - 1297 .
25. Thaler J , Ay C , Weinstabl H , Dunkler D , Simanek R , et al. ( 2011 ) Circulating procoagulant microparticles in cancer patients . Ann Hematol 90 : 447 - 453 .
26. Helley D , Banu E , Bouziane A , Banu A , Scotte F , et al. ( 2009 ) Platelet microparticles: a potential predictive factor of survival in hormone-refractory prostate cancer patients treated with docetaxel-based chemotherapy . Eur Urol 56 : 479 - 484 .
27. Woywodt A , Blann AD , Kirsch T , Erdbruegger U , Banzet N , et al. ( 2006 ) Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol . J Thromb Haemost 4 : 671 - 677 .
28. Lacroix R , Robert S , Poncelet P , Kasthuri RS , Key NS , et al ( 2010 ) Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop . J Thromb Haemost 8 : 2571 - 2574 .
29. Manzoni M , Mariucci S , Delfanti S , Rovati B , Ronzoni M , et al. ( 2012 ) Circulating endothelial cells and their apoptotic fraction are mutually independent predictive biomarkers in Bevacizumab-based treatment for advanced colorectal cancer . J Cancer Res Clin Oncol 138 : 1187 - 1196 .
30. Browder T , Butterfield CE , Kraaling BM , Marshall B , O'Reilly MS , et al. ( 2000 ) Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer . Cancer Res 60 : 1878 - 1886 .
31. Vroling L , van der Veldt AA , de Haas RR , Haanen JB , Schuurhuis GJ , et al. ( 2009 ) Increased numbers of small circulating endothelial cells in renal cell cancer patients treated with sunitinib . Angiogenesis 12 : 69 - 79 .
32. Gruenwald V , Beutel G , Schuch-Jantsch S , Reuter C , Ivanyi P , et al. ( 2010 ) Circulating endothelial cells are an early predictor in renal cell carcinoma for tumor response to sunitinib . BMC Cancer 10 : 695 .
33. Malka D , Boige V , Jacques N , Vimond N , Adenis A , et al. ( 2012 ) Clinical value of circulating endothelial cell levels in metastatic colorectal cancer patients treated with first-line chemotherapy and bevacizumab . Ann Oncol 23 : 919 - 927 .
34. Shao H , Chung J , Balaj L , Charest A , Bigner DD , et al. ( 2012 ) Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy . Nat Med 18 : 1835 - 1840 .
35. Sartori MT , Della Puppa A , Ballin A , Saggiorato G , Bernardi D , et al. ( 2011 ) Prothrombotic state in glioblastoma multiforme: an evaluation of the procoagulant activity of circulating microparticles . J Neurooncol 104 : 225 - 231 .