PURPOSE:There is a need for biomarkers that may help in selecting the most effective anticancer treatments for each patient. We have investigated the prognostic value of a set of angiogenesis, inflammation and coagulation markers in patients treated for advanced non-small cell lung cancer.PATIENTS AND METHODS:Peripheral blood samples were obtained from 60 patients before first line platinum-based chemotherapy ± bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Angiogenesis, inflammation and coagulation markers vascular endothelial growth factor (VEGF), their soluble receptors 1 (VEGFR1) and 2 (VEGFR2), thrombospondin-1 (TSP-1), interleukin-6 (IL6), sialic acid (SA) and tissue factor (TF) were quantified by ELISA.RESULTS:Except for TSP-1, pre- and post-treatment levels of all markers were higher in patients than in controls (p < 0.05). There was a positive and significant correlation between VEGF and VEGFR2 before treatment. VEGF also correlated with inflammatory markers IL-6 and SA. Moreover, there was a positive and significant correlation between levels of VEGFR1 and TF. Decreased levels of TSP-1 and increased levels of VEGF were associated with shorter survival. Bevacizumab significantly modified angiogenesis parameters and caused a decrease of VEGF and an increase of TSP-1.CONCLUSION:Angiogenesis, inflammation and coagulation markers were increased in NSCLC patients. Increased levels of VEGF and low levels of TSP-1 correlated with a poor prognosis.

Measuring von Willebrand factor (VWF) activity is essential for the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra‐ and inter‐assay variabilities. A new automated chemiluminescent immunoassay VWF activity has recently become commercially available (HemosIL AcuStar von Willebrand Factor Ristocetin Cofactor Activity). The main objective of this study was to evaluate this new method and to compare it with the VWF:RCo assay as the reference method. We studied 91 samples, 18 healthy volunteers samples and 73 samples from patients (VWF:RCo level <50 IU dL−1): 29 type 1 VWD, 13 type 2A, 5 type 2B, 5 type 2M, 3 type 2N, 5 type 3, 4 type 3 under treatment, 5 type 3 carriers and 4 samples with other pathologies. The HemosIL AcuStar VWF:RCo assay was 96% sensitive and 100% specific for detecting VWF abnormalities. The good analytical performance, and the sensitivity and specificity of HemosIL AcuStar VWF:RCo to detect VWF deficiency renders it a suitable method for VWD screening.

The severity of haemophilia A has traditionally been classified by the dosage of factor VIII (FVIII) by one-step coagulation tests. However, an homogeneous group of patients with similar FVIII levels show clinical heterogeneity and 10-15% of the patients classified as severe haemophilia do not have a severe bleeding phenotype. Traditional tests used for measuring FVIII are not capable of detecting other prohaemorrhagic or prothrombotic factors. Global tests as the thrombin generation assay (TGA) may detect these haemostatic factors. So TGA may be an additional tool for classifying the actual severity of haemophilia. Our group is carrying out correlation tests between FVIII and TGA in platelet-poor and -rich plasmas (PPP and PRP, respectively). PRP has the inconvenience that must be done freshly soon after blood extraction. Our aim is to study the differences between TGA performed with fresh and frozen PRP and PPP and its implementation in multicenter studies. We included 70 patients with severe haemophilia A in prophylactic treatment. Venous blood drawing was obtained prior to administration of FVIII, at the trough levels. FVIII measurement and TGA were performed in fresh and frozen PRP and PPP. The platelet absence caused a significant decrease in TGA although PPP and PRP correlated well. Frozen samples gave different results in PPP, but there were no significant differences between fresh and frozen PRP. This fact enables using frozen PRP in multicenter studies with a TGA-specialized laboratory for reclassifying haemophilia severity and for pharmacokinetic studies with TGA.

7583 Background: Circulating endothelial cells (CECs), endothelial progenitor cells (EPCs), and microparticles (MPs) have prognostic value in cancer, and might be predictors of response to chemotherapy and antiangiogenic treatments. We investigated the pre- and post-treatment levels of CECs, EPCs MPs and inflammation and coagulation markers in patients with stage IIIB and IV non-small cell lung cancer (NSCLC). Methods: We studied 60 patients, before first line taxane and platinum chemotherapy +/- bevacizumab and sixty controls. Quantification of CECs was carried out by an immunomagnetic technique. The number of EPCs (CD34/CD133/CD309) and MPs-phosphatidylserine-positive (0.5-1μm) in blood was evaluated by flow cytometry. Markers of inflammation: interleukin-6 (IL-6), coagulation: tissue factor (TF) and angiogenesis vascular endothelial growth factor (VEGF) and their receptors VEGFR1 and VEGFR2 were quantified by ELISA. Results: Mean number of CECs was significantly increased in patients before and after treatment as compared to controls p<0.0001 and p=0.01 respectively). Levels of EPCs before treatment were similar to those of controls (p=0.05) but they were significantly higher after treatment (p=0.01). Levels of MPs were significantly higher in patients before and after treatment than in controls (p< 0.0001). Levels of IL-6, TF were significantly higher in patients before and after treatment than in controls (p<0.0001 and p<0.001 respectively). Levels of VEGF and their receptors were significantly higher in patients before treatment and after treatment than in controls (p<0.001). The Pearson correlation between VEGF and MPs was positive and statistically significant (r=0.40, p<0.001). There was an association between increased levels of MPs before treatment and survival >15 months (p<0.05), as well as an increased levels of IL6 and TF in stage IIIB patients (p<0.01). Conclusions: CECs, EPCs and MPs, as well as a set of markers of inflammation and coagulation, are strongly elevated in patients with advanced NSCLC and still elevated after treatment. The influence of treatment on CEC, EPC and MP levels is unclear and needs further study.

2086 Background: Circulating endothelial cells (CECs) have prognostic and predictive value in breast cancer patients treated with metronomic chemotherapy. High levels of microparticles (MPs) are associated to shorter survival in prostate cancer. We investigated the pre- and posttreatment levels of CECs, MPs and several inflammation and coagulation markers in patients with newly diagnosed glioblastoma (GB) who received protracted temozolomide (PTMZ) and radiotherapy (RT). Methods: Peripheral blood was obtained from 14 patients with GB before and 4-5 weeks after beginning of treatment. Healthy subjects (n = 22) were used as controls. Isolation and quantification of CECs were determined by an immunomagnetic technique using CD146 and lectin-1 of Ulex Europaeus combined with fluorescein. Flow cytometry of plasma was used for detection of total phosphatidylserine-positive MPs, labeled with FITC-Annexin V. Endogen thrombin generation (ETG) was measured by the calibrated automated thrombogram method (CAT). Prothrombin fragment 1+2 (F1+2) and interleukin-6 (IL-6) were measured by ELISA. Results: Mean number of CECs was significantly increased in patients before (112 ± 65 cells/mL) and after treatment (122 ± 80 cell/mL) as compared to controls (10 ± 5 cell/mL; p < 0.0001). Mean number of MPs was significantly increased in patients before (794 ± 573 MPs/μ L) and after treatment (408 ± 207 MPs/μ L) as compared to controls (234 ± 174 MPs/μ L; p < 0.05). Levels of IL-6 were significantly higher in patients before and after treatment (IL6, 8.3 ± 14.3 pg/mL; 3.2 ± 3.1 pg/mL) than in controls (0.9 ± 0.8 pg/mL). Levels of coagulation markers were significantly higher in patients before treatment (F1+2, 0.33 ± 0.18 pmol/mL; ETG, 234 ± 106 nM). However, levels of both markers decreased near normal values (F1+2, 0.22 ± 0.07 vs. 0.17 ± 0.04 pmol/mL; ETG, 169 ± 146 vs. 162 ± 72 nM) after treatment. Conclusions: CECs and MPs, as well as a set of markers of inflammation and coagulation, are strongly elevated in patients with GB. Coagulation markers decrease after treatment with PTMZ+RT. The influence of treatment on CEC and MP levels is unclear and needs further study. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Schering-Plough Schering-Plough

It is not established whether there is an association between erythrocyte aggregation and AB0 blood type, as glycophorins carry sialic acid which is responsible for the negative erythrocyte surface charge and the antigenicity for AB0 blood groups. We have determined erythrocyte aggregation by means of the Myrenne aggregometer in 114 healthy volunteers, along with plasma lipids, fibrinogen and AB0 blood groups. No differences in erythrocyte aggregation (EA0 and EA1) were observed when subjects with 0 (n = 45) and non-0 (n=69) blood group were compared (P = 0.624 and P = 0.838, respectively). Fibrinogen was statistically lower in 0 group compared with non-0 group (P = 0.012). Erythrocyte aggregation (EA0 and EA1) correlated significantly with both lipids and fibrinogen (P < 0.01). When erythrocyte aggregation was dichotomized as EA1 > or = 8, no association was found with 0 and non-0 blood groups (P > 0.05) but it was associated with high lipid levels: T-Chol > 220 mg/dl, TG > 175 mg/dl and high fibrinogen levels > 300 mg/dl (P = 0.035; P = 0.030; P = 0.010, respectively). Erythrocyte aggregation does not seem to be associated with blood groups, but rather with plasma lipids and fibrinogen.

Abstract: Introduction:  Acute cellular rejection is a major cause of graft loss in heart transplantation (HT). Endomyocardial biopsy remains the gold standard for its diagnosis, but it is an invasive procedure not without risk. A proinflammatory state exists in rejection that could be assessed by determining plasma levels of inflammatory biomarkers.

There are conflicting results regarding the erythrocyte membrane cholesterol and phospholipid content in patients with primary hypercholesterolemia (PHC), due to methodological problems in obtaining haemoglobin-free ghosts. At the same time, the different units used and the fact that the cholesterol and phospholipids are not expressed in relation with integral protein membrane content, produces contradictory results. We have analysed in 33 patients with PHC (12 male, 31 female) aged 43+/-12 years and in 33 healthy normolipaemic volunteers (9 male, 24 female) aged 43+/-13 years plasma lipids, along with, erythrocyte membrane cholesterol, phospholipids and integral proteins. PHC patients showed increased erythrocyte membrane cholesterol: 0.36+/-0.15 mg/mg when compared with controls: 0.29+/-0.75 mg/mg; p=0.018. Phospholipid membrane content, although higher in the cases, did not reach statistical significance (PHC patients: 0.38+/-0.15 mg/mg vs. 0.33+/-0.72 mg/mg; p=0.098). The cholesterol/phospholipids ratio (Chol/Ph) was 0.99+/-0.22 in PHC patients versus 0.92+/-0.28 in controls; p=0.127. Our results suggest that there is a slight increase in erythrocyte membrane cholesterol in patients with PHC. Given the increasing importance of erythrocyte membrane cholesterol in the stability of the atheroma plaque due its possible contribution to the clinical signs of ischaemic heart disease, it seems relevant to determine this parameter in risk populations. Therefore, a simple and reproducible method needs to be standardised which would enable comparisons between laboratories and facilitate further studies aimed to it as a marker of acute coronary syndromes.

Erythrocyte deformability (ED) has been scarcely evaluated in obese patients without other concomitant cardiovascular risk factors and contradictory results have been published regarding the influence of plasma lipids on the erythrocyte membrane lipid composition and insulin resistance on this rheological parameter. In 67 severe or morbid obese patients without other cardiovascular risk factors (51 women and 11 men, aged 34+/-11 years) and in 67 controls (45 women and 22 men, aged 32+/-10 years), ED has been determined by ektacytometric techniques in a Rheodyn SSD, the elongation index (EI) being measured at 12, 30 and 60 Pa, along with plasma lipids, red blood cell membrane lipids (cholesterol and phospholipids) and insulin resistance indexes in basal conditions and after a three month diet period. No significant differences were obtained in the EI between obese patients and the control group at any of the shear stresses tested (P>0.05). The cholesterol and phospholipid content of the red blood cell membrane did not significantly differ between cases and controls (P>0.05). Obese patients with metabolic syndrome showed lower EI at 30 and 60 Pa than those without metabolic syndrome (P=0.014 and P=0.031 respectively). Weight loss was not accompanied by any changes in these rheological parameters. Obesity itself does not seem to modify ED. However, metabolic syndrome seems to decrease ED, possibly through insulin resistance.

BACKGROUND Endometriosis is a highly prevalent, benign disease in which the angiogenic, fibrinolytic and metalloproteinase (MMP) systems may be implicated. The objective of this study is to analyse mRNA expression and protein levels of several angiogenic factors and to correlate them with several components of the fibrinolytic and MMP systems in samples from 71 women with endometriosis and 50 controls. METHODS AND RESULTS Eutopic endometrium showed higher mRNA expression of vascular endothelial growth factor (VEGF) in patients than in controls. However, ovarian endometrioma had lower VEGF mRNA levels than did the eutopic endometrium of patients. Similar results were obtained for VEGF protein levels. On the other hand, a significant increase in thrombospondin-1 (TSP-1) levels was observed in ovarian endometrioma than in eutopic endometrium. The peritoneal fluid from women with endometriosis showed a significant increase in VEGF, urokinase-type plasminogen activator (uPA) and MMP-3 levels than that of controls. A significant correlation was observed between the levels of VEGF and uPA in endometrium and in peritoneal fluid. CONCLUSIONS Endometrium and peritoneal fluid from women with endometriosis have increased levels of VEGF, uPA and MMP-3 levels. Therefore, the development of endometriotic implants at ectopic sites may be facilitated, promoting the progress of the endometriosis.