A. Rousseau

Impact of breast cancer stage, time from diagnosis and chemotherapy on plasma and cellular biomarkers of hypercoagulability

BackgroundIn breast cancer patients routine thromboprophylaxis is not recommended but individualized risk assessment is encouraged. The incorporation of hypercoagulability biomarkers could increase the sensitivity of risk assessment models (RAM) to identify patients at VTE risk. To this aim we investigated the impact of cancer-related characteristics on hypercoagulability biomarkers.MethodsThrombin generation (TG) assessed with the Thrombogramme-Thrombinoscope®, levels of platelet derived microparticles (Pd-MP) assessed with flow cytometry, procoagulant phospholid dependent clotting time (PPL-ct) measured with a clotting assay and D-Dimers (were assessed in a cohort of 62 women with breast cancer and in 30 age matched healthy women.ResultsPatients showed significantly higher TG, Pd-MP, D-Dimers levels and shortened PPL-ct compared to the controls. The PPL-ct was inversely correlated with the levels of Pd-MP, which were increased in 97% of patients. TG and D-Dimers were increased in 76% and 59% of patients respectively. In any stage of the disease TG was significantly increased as compared to the controls. There was no significant difference of TG in patients with local, regional of metastatic stage. There was no significant difference in Pd-MP or Pd-MP/PS+ between the subgroups of patients with local or regional stage of cancer. Patients with metastatic disease had significantly higher levels of Pd-MP and Pd-MP/PS+ compared to those with regional stage. The D-Dimers increased in patients with metastatic stage. In patients on chemotherapy with less than 6 months since diagnosis TG was significantly higher compared to those on chemotherapy who diagnosed in interval > 6 months. Patients with metastatic disease had significantly higher levels of Pd-MP and D-Dimers compared to those with non-metastatic disease.ConclusionIn breast cancer patients the stage, the time elapsed since the diagnosis and the administration of chemotherapy are determinants of cellular and plasma hypercoagulability. The levels and the procoagulant activity of Pd-MP are interconnected with the biological activity and the overall burden of cancer. TG reflects the procoagulant properties of both breast cancer and chemotherapy in the initial period of cancer diagnosis. Thus the weighted incorporation of the biomarkers of cellular and plasma hypercoagulabilty in RAM for VTE might improve their predictive value.

Cancer cells BXPC3 and MCF7 differentially reverse the inhibition of thrombin generation by apixaban, fondaparinux and enoxaparin.

INTRODUCTIONnCancer cells may alter the efficiency of the antithrombotic agents. To explore this possibility, the present study compared the capacity of the LMWH enoxaparin and the specific inhibitors of Xa (apixaban and fondaparinux) to inhibit thrombin generation triggered by pancreas adenocarcinoma cells (BXPC3) and human breast carcinoma cells (MCF7).nnnMATERIALS AND METHODSnSamples of platelet poor (PPP) or platelet rich plasma (PRP) spiked with apixaban, fondaparinux or enoxaparin were added in micro wells carrying cancer cells and assessed for thrombin generation. In the control experiment thrombin generation was triggered with tissue factor reagent.nnnRESULTSnThe three antithrombotics inhibited thrombin generation in a concentration dependent manner. The BXPC3 and MCF7 cells reversed in a different intensity the effect of the studied agents. According to the histological type of the cancer the antithrombotic efficiency of apixaban was preserved or partially reversed. Fondaparinux, was more vulnerable to the presence of cancer cells as compared to apixaban. The effect of BXCP3 or MCF7 cells on the antithrombotic potency of enoxaparin was of similar magnitude as that on apixaban.nnnCONCLUSIONSnThe type of cancer cells is determinant for the antithrombotic efficiency of the specific factor Xa inhibitors. In contrast it does not significantly influence the potency of enoxaparin. The present study shows that the impact of the type of cancer cells on the antithrombotic activity of the specific Xa inhibitors should not be neglected. This has to be taken into consideration for the design of dose-finding studies of the direct orally active FXa inhibitors in patients with different histological types of cancer.

Differential contribution of tissue factor and Factor XII to thrombin generation triggered by breast and pancreatic cancer cells

Most cancer cells trigger thrombin generation (TG) to various extent. In the present study, we dissected the mechanisms responsible for the procoagulant activity of pancreatic adenocarcinoma cells (BXPC3), a highly thrombogenic cancer type, and breast cancer cells (MCF7), a less thombogenic tumor type. TG of normal plasma was assessed by the Thrombinoscope (CAT®) in the presence or absence of cancer cells. TG was also assessed in plasma depleted of clotting factors, in plasma spiked with tissue factor (TF) and/or procoagulant phospholipids, in plasma spiked with an anti-TF monoclonal antibody or with corn trypsin inhibitor (CTI). The presence of alternatively spliced TF (asTF), TF activity (TFa) and cancer procoagulant (CP) levels were determined. TFa and asTF were highly expressed by BXPC3 cells, compared to MCF7 cells, while CP levels were higher in MCF7 cells. BXPC3 cells had a stronger effect on TG than MCF7 cells. Accordingly, anti-TF had more inhibitory activity on TG triggered by BXPC3 cells while CTI had more pronounced inhibitory effect on TG triggered by MCF7 cells. TG enhancement by both BXPC3 and MCF7 cells was mediated by FVII and intrinsic tenase while FXII and FXI were also important for MCF7 cells. The induction of TG by BXPC3 cells was mainly driven by the TF pathway while TG generation triggered by MCF7 cells was also driven by FXII activation. Therefore, hypercoagulability results from a combination of the inherent procoagulant properties of cancer cell-associated TF as well as of procoagulant phospholipids in the plasma microenvironment.

Procoagulant microparticles derived from cancer cells have determinant role in the hypercoagulable state associated with cancer

Hypercoagulablity is a common alteration of blood coagulation in cancer patients. However, the procoagulant activity of cancer cells is not sufficient to induce hypercoagulability. The present study was aimed to identify the mechanism with which hypercoagulabilty is produced in the presence of cancer cells. We focused on the analysis of the procoagulant elements carried by cancer cell-derived microparticles (CaCe-dMP) and we evaluated the impact of microparticles associated with the cancer cells from which they stem on thrombin generation. CaCe-dMP from the cancer cells were isolated from the conditioned medium and analyzed for tissue factor (TF) and procoagulant phospholipid expression. Thrombin generation of normal plasma was assessed by the Thrombinoscope (CAT®) in the presence or absence of pancreas adeno-carcinoma cells (BXPC3) or breast cancer MCF7 cells supplemented with the respective CaCe-dMP. Both BXPC3 and MCF7 cells express abundant amounts of active TF. Phosphatidylserine was identified on the surface of CaCe-dMP, unlike the cancer cells themselves. The expression of TFa by the microparticles was significantly higher to that observed on the cancer cells. Culture of the cancer cells with their microparticles resulted in thrombin generation significantly higher as compared to the upper normal limit. In conclusion, cancer cells enrich the microenvironment with procoagulant elements, especially procoagulant micro-particles which express TF and procoagulant phospholipids. The association of cancer cells with procoagulant microparticles is necessary for a state of hypercoagulability, at the level of the tumoral microenvironment. The intensity of the hypercoagulability depends on the histological type of the cancer cells.

OC-03 - Modelisation of the procoagulant properties of cancer cells and their capacity to alter the antithrombotic efficiency of LMWHs and specific inhibitors of factor Xa.

INTRODUCTIONnThe risk of venous thromboembolism varies according to the histological type of cancer. The failure of antithrombotic treatment is more frequent in cancer patients as compared to non-cancer ones.nnnAIMnWe aimed to elucidate the mechanism of activation of blood coagulation induced by cancer, the impact of chemo-resistance phenotype on the capacity of cancer cells to trigger thrombin generation and the alterations of the efficiency of LMWHs and the specific inhibitors of factor Xa (fondaparinux and apixaban) in the presence of cancer cells.nnnMATERIALS AND METHODSnThrombin generation of human plasma was assessed in the presence of various cancer cell lines. The model of cancer-induced hypercoagulability was coupled to the research for the expression of procoagulant molecules by cancer cells.nnnRESULTSnThe pancreatic adenocarcinoma cells BXPC3 and the breast adenocarcinoma cells MCF7 were initially tested. The BXPC3 cells induce significantly higher thrombin generation as compared to the MCF7 cells. In the same line Marchetti et al. showed that malignant hematologic cells (NB4, HEL, and K562) and H69 small cell lung cells express different procoagulant potential on triggering thrombin generation of human plasma. The comparison of the procoagulant activity has been extended to cancer cell lines from various cancers (i.e. colon, ovarian and prostatic cancer) as well as to different cell lines of the same type of cancer. The differences of the cancer cell lines to trigger thrombin generation are mainly due to the expression of TF. The acquisition of chemoresistant phenotype by cancer cells is correlated with increased TF expression and enhancement of theit procoagulant activity. The ability of cancer cells to activate FXII is an alternative pathway of significant importance for some cancer cell lines (i.e. MCF7). Clinically relevant concentrations of LMWH and specific direct and indirect inhibitors of FXa (apixaban and fondaparinux) inhibit thrombin generation induced by cancer cells. The synergy between the anti-Xa and anti-IIa activities of LMWHs rather than the AT-dependent selective inhibition of FXa results in profound inhibition of thrombin generation induced by BXPC3 cells. This experimental model allowed the functional distinction between the two specific FXa inhibitors (apixaban and fondaparinux).nnnCONCLUSIONSnThe cancer cell-based model of hypercoagulability is suitable for the identification of the prothrombotic fingerprint of various cancer types. This experimental model allows to perform pharmacological studies for the evaluation of the efficiency of the antithrombotic drugs in cancer-induced hypercoagulability. It is suitable for the study of the impact of anticancer drugs on the procoagulant properties of the cancer cells.

O.04b Effect of the dose of oral treatment with 17beta-estradiol associated with dydrogesterone on thrombin generation in healthy postmenopausal women: a randomized double-blind placebo-controlled study

s / Thrombosis Research 127 (2011) S123–S150 S125 O.04b Effect of the dose of oral treatment with 17beta-estradiol associated with dydrogesterone on thrombin generation in healthy postmenopausal women: a randomized double-blind placebo-controlled study G. Gerotziafas *, V. Galea, A. Rousseau, D. Torchin, K.F. Zannad, K. Lacut, J. Demolis, T. Simon, I. Elalamy. ER2UPMC, Faculte de Medecine Pierre et Marie Curie, Universite Paris 6, Hopital Tenon, APHP, Paris, Clinical research Unit, Department of Clinical Pharmacology, APHP, Saint-Antoine hospital, Paris 6 University, Paris, Department of Gynaecology, APHP, Saint-Antoine hospital, Paris, Clinical Investigation Center, CHU, Nancy, Clinical Investigation Center, CHU, Brest, Clinical Investigation Center, Department of Clinical Pharmacology, AP-HP Saint-Antoine hospital, Paris 6 University, Paris, France Introduction: Increased risk of venous thrombosis during oral hormone therapy in postmenopausal women is related to estrogen-induced hypercoagulable state. Lower estrogen dose is recommended, but the effect of hormone dose optimisation on haemostasis has not been studied. Aim of the study: To evaluate the influence of two different doses of daily oral 17beta-estradiol therapy compared with placebo, on thrombin generation (TG) in healthy postmenopausal women. Materials-Methods: Seventy two menopausal women were randomized to receive daily 1mg (n =24) or 2mg (n =26) of 17beta-estradiol associated with 10mg dydrogesterone or placebo (n =22). TG was monitored before and 2 months after treatment using the Calibrated Automated Thrombogram® (Stago, France). Results: At baseline parameters of TG were not statistically different in the three study groups. Two months after randomisation, lag time and time to reach the peak of thrombin were decreased while the mean rate index and the peak of thrombin were significantly increased in the two groups of hormone-treated women as compared to placebo group. Endogenous thrombin potential remained unchanged. No difference was observed in TG parameters between women receiving lower and higher dose of 17betaestradiol. Conclusions: Oral hormone therapy with 2mg of 17beta-estradiol for two months induced acceleration and increase of TG. Lowering the dose by 1mg did not attenuate this effect. On the contrary endogenous thrombin potential was not influenced in any dose. The clinical significance of the profile of TG of postmenopausal women being on hormone therapy remains to be explored. Table 1: Parameters of thrombin generation 2 months after randomisation (Treatment with two different doses of 17beta-estradiol associated with dydrogesterone or placebo) placebo 17beta-estradiol

P.323 - Cancer du pancréas et maladie veineuse thromboembolique : implication du facteur tissulaire ?

Introduction Le cancer du pancreas est l’un des cancers le plus fortement associe a la maladie veineuse thrombo-embolique (MVTE) [1]. Les cellules tumorales, en particulier celles du pancreas, sont capables d’exprimer le facteur tissulaire (FT), l’initiateur de la coagulation. Dans cette etude, nous avons compare les taux circulants de FT chez les patients atteints de cancer du pancreas a ceux mesures chez les patients avec un cancer d’un autre site. Patients et Methodes Entre le 1er janvier 2005 et le 31 aout 2007, 50 patients consecutifs [27 hommes (54 %), âge moyen 71,6xa0±xa011,7 ans] ayant participe a l’etude EDITH pour une MVTE symptomatique secondaire a un cancer actif ont ete inclus. Le FT a ete mesure dans les 7 jours suivant le diagnostic de MVTE. Resultats 7 patients (14 %) avaient une MVTE secondaire a un cancer du pancreas, 14 (28 %) a un cancer genito-urinaire, 9 (18 %) a une tumeur bronchopulmonaire, 5 (10 %) a un lymphome, 15 (30 %) a une tumeur d’autre localisation. Il n’y avait pas de difference d’âge (68,3xa0±xa011,8 ans vs 72,2xa0±xa011,7 ans, pxa0=xa00,68) et de sexe (57 % d’hommes vs 53 %, pxa0=xa01) entre les patients avec un cancer du pancreas et les patients ayant un autre cancer. Le cancer du pancreas etait associe a des taux moyens de FT significativement superieurs a ceux des cancers d’une autre localisation (27,10xa0±xa030,60 pmol/l vs 6,04xa0±xa08,60 pmol/l, pxa0=xa00,002). Discussion Une surexpression de FT lors du cancer du pancreas pourrait expliquer l’incidence plus elevee de MVTE associee a ce cancer. Les nouveaux anticoagulants ciblant le FT pourraient etre plus efficaces que les anticoagulants « classiques » pour la prevention primaire et secondaire de la MVTE chez les patients atteints de cancer du pancreas. Conclusion De futures etudes devront confirmer que l’elevation des taux de FT au cours du cancer du pancreas est bien associee a une augmentation de l’incidence de la MVTE.