ALBERT

Description: Background: Venous Thromboembolism (VTE) is a common complication of cancer and is strongly associated with early all-cause mortality during the course of cancer chemotherapy (Kuderer et al. ASCO 2008). A clinical model for predicting the risk of VTE in cancer patients initiating chemotherapy has been recently developed and validated (Khorana et al. Blood 2008). Risk of VTE in low (group I), intermediate (group II) and high risk patients (group III) was 0.8%, 1.8% and 7.1%, respectively. The aim of current study is to evaluate the ability of the VTE risk model to predict disease progression and early all-cause mortality. Methods: A prospective study of 4,458 adult cancer patients with solid tumors or malignant lymphoma initiating a new chemotherapy regimen was conducted between 2002 and 2006 at 115 randomly selected practice sites throughout the USA. Demographic, clinical and treatment-related information was captured prospectively at baseline and during the first four cycles of chemotherapy, including rates of documented VTE, disease recurrence and deaths from all causes. Progression-free survival (PFS) and overall survival (OS) within 4 months of starting chemotherapy were estimated by the method of Kaplan-Meier and adjusted hazard ratios (HR ± 95% CI) were estimated by a Cox regression model, incorporating VTE as a time-dependent covariate. Results: Patient age ranged from 18–97 with a mean of 60 years. VTE occurred in 3% of patients by 4 months with a median of 38 days following initiation of chemotherapy. The HR for VTE occurrence among risk score groups II and III, compared to group I, were 3.07 [1.39–6.77] and 11.73 [5.22–16.37], (P

Description: Introduction: The fibrinolytic activity has been shown to be reduced in many vascular diseases, including hepatic veno-occlusive disease (VOD) after stem cells transplantation (SCT). Defibrotide (DF) is a polydisperse oligonucleotide with antithrombotic, profibrinolytic, anti-ischemic, and anti-adhesive properties. Numerous clinical studies have shown promising results with DF in the treatment of VOD, with minimal toxicity. In laboratory studies, DF has been shown to decrease plasminogen activator inhibitor-1 (PAI-1), increase tissue plasminogen activator (t-PA) levels and increase the overall plasma fibrinolytic activity in patients with VOD. Similar results have been observed in pre-clinical studies with endothelial cells stimulated by lipopolysaccharide. Plasmin is a potent and nonspecific serine protease, generated from the proenzyme plasminogen by plasminogen activators. Plasmin plays a pivotal role in fibrinolysis by virtue of its ability to effectively degrade fibrin clots. The purpose of this study was to investigate whether DF is capable of modulating the activity of plasmin in different in vitro studies. Method: Plasmin activity was measured in the presence and absence of DF, at different concentrations, by following the cleavage of the chromogenic substrate S-2251. S-2251 contains the preferential cleavage site for plasmin and mimics the fibrin polymer substrate present in fibrin clots. In addition, we evaluated the activity of plasmin generated by t-PA and urokinase-plasminogen activation as well as the plasmin activity in plasma in the presence and absence of DF. Further, plasmin activity generated in fibrin clot plate was measured in the presence and absence of DF. Finally, the inhibition of plasmin by aminocaproic acid was tested in these systems. Results: DF increased the activity of plasmin, to hydrolyze its substrate, in a dose dependent manner. Similar concentration-dependent effects of DF were observed when plasmin was generated by t-PA or urokinase activation of plasminogen and on plasmin in plasma samples (p

Description: We used the thrombin generation assay to evaluate the hypercoagulable state according to JAK2V617F mutational status in essential thrombocythemia (ET) and polycythemia vera (PV) patients. Thrombin generation was determined in the presence and absence of activated protein C (APC), and APC resistance was expressed as normalized APC sensitivity ratio (nAPCsr). Tissue factor pathway inhibitor (TFPI), total and free protein S (PS), prothrombin (FII), factor V (FV), and neutrophil elastase were measured in plasma; CD11b was measured on neutrophils. Compared with normal controls, patients had a lower endogenous thrombin potential in the absence of APC but had a higher endogenous thrombin potential in the presence of APC, showing the occurrence of APC resistance. The nAPCsr increased in JAK2V617F carriers compared with noncarriers and was highest in JAK2V617F homozygous patients. FII, FV, free PS, and TFPI levels were reduced in patients, mainly in JAK2V617F carriers. Multiple regression analysis indicated the low free PS level as major determinant of the increased nAPCsr. Elastase was increased in patients and inversely correlated with free PS. In conclusion, these data indicate the occurrence of acquired APC resistance in ET and PV patients, probably because of a reduction in free PS levels. The APC-resistant phenotype is influenced by the JAK2V617F mutational load.

Description: Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET). Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay. Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p

Description: Abstract 3047 Poster Board II-1023 Introduction A beneficial effect of low molecular weight heparins (LMWH) on the survival of cancer patients is suggested by clinical data and has been attributed to a broad range of activities, including the capacity to affect the hemostatic and angiogenic properties of endothelial cells. We have previously observed that unfractionated heparin and LMWHs with a mean molecular weight (MW) of 4.5 to 5.7 KDa can effectively antagonise tumor cell-induced angiogenesis in a microvascular endothelium-based Matrigel assay (Marchetti et al, Thromb Res 2008). Aim Since this anti-angiogenic activity of heparins may depend on the mean MW and the composition of their polysaccharidic chains, this study was designed to evaluate the capacity of ‘second generation’ LMWHs, with a lower MW, to counteract the pro-angiogenic stimulus of tumor cells: i.e. the LMWH Bemiparin, that possesses the lowest MW among commercially available LMWH (i.e. 3.6 KDa), and of its ultra low MW derivative RO-14 (MW=2.5 KDa, Laboratorios Farmaceuticos Rovi S.A., Madrid, Spain). Methods Tumor cell conditioned media (TCM) were obtained from three human cell lines of different tumor types, i.e. H69 (small cell lung cancer), MDA.MB.231 (breast cancer), and NB4 (acute promyelocytic leukemia). Human microvascular endothelial cells (HMEC-1) were incubated for 24h in Matrigel with TCM, or purified proangiogenic factors (VEGF, FGF-2), in the absence or presence of increasing concentrations (0.01-0.1-1 IU/ml) of Bemiparin or RO-14. Tubule formation was photographed under inverted microscopy and quantified by an image analysis software. The same heparins were tested for their capacity to counteract endothelial migration elicited by TCM in the wound healing assay. The levels of the pro-angiogenic factors VEGF, FGF-2 and IL-1beta in TCM were quantified by ELISA. Results All three TCM induced a significant (p

Description: Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

Description: The pathogenesis of the thrombotic diathesis of patients with ET is not completely clarified. Activation of polymorphonuclear leukocyte (PMN) occurs in these patients and is associated with a hypercoagulable state and increased number of circulating platelet/PMN aggregates. Further, increased procoagulant Tissue Factor (TF) expression in ET PMN is also reported. Recently, a gain-of-function JAK2 mutation (V617F) has been described in a high proportion of bcr/abl-negative myeloproliferative disorders. Specifically, in subjects with ET the mutation is present in about 50% of cases and retrospective data suggest an association with a higher rate of complications, including thrombosis. Aim of this study was to evaluate whether ET patients carrying the JAK2 mutation possess PMN with different hemostatic characteristics compared to ET subjects without JAK2 mutation and healthy controls. Twenty ET patients, 10 with and 10 without JAK2 mutation (median age: 50 years, range 32–61; platelet count: median 782 x 109/L, range 474–1,565 x 109/L), not receiving cytoreductive therapy (classified as low risk group), were included in the study; 16 age matched healthy subjects acted as controls. Expression of CD11b, TF and fibrinogen on PMN surface as well as PMN-platelet mixed aggregates (defined as the percentage of CD11b-positive PMN co-expressing a platelet-specific marker, i.e. CD42b or CD62P) were evaluated by whole blood flow-cytometry in both basal condition and after in vitro PMN stimulation by f-MLP. In washed isolated PMN samples the level of TF-mRNA was determined by RT-PCR. In basal conditions, significantly (p

Description: Background: the Registro Italiano Trombocitemia, that is a GIMEMA project, has been activated to registry Italian Essential Thrombocythemia (ET) patients, to improve the diagnosis appropriateness (WHO criteria), to verify the prognostic value of the clinical and biological parameters, to evaluate the compliance to the therapeutical Italian guidelines (1), and to create a network for activation of new studies. Objective: this analysis is mainly devoted to describe the ET patients registered in the RIT and to evaluate the therapeutic approach adopted in the 102 participating hematological centers. Material and methods: two thousand and fifteen ET patients have been registered after the written informed consent was obtained, and data validation by various expert panels is in progress. This preliminary report considers 1785 patients, diagnosed mainly (1078, 60.4%) since the publication in the year 2004 of the ET therapy Italian guidelines (1). Results: the patients, 678 (38%) males and 1107 (62%) females, showed at diagnosis: age 60.3 ± 16.8 years with higher values in males than in females (61.7 ± 15.3 vs. 59.4 ± 17.7, p

Description: Acute promyelocytic leukemia (APL) is associated with severe hemorrhagic coagulopathy, which is the main cause of early death. The introduction of ATRA ameliorated the complete remission rate and improved the overall survival compared to treatment with chemotherapy alone. However, the pioneristic large randomized trials comparing ATRA alone versus chemotherapy alone in previously untreated patients failed to demonstrate a significant reduction of early hemorrhagic deaths, notwithstanding ATRA capability to quench the ongoing coagulopathy. Nevertheless, the incidence of such very early deaths remains unsettled, leading to a bias in the evaluation of real impact of ATRA in clinical outcome of APL. We have retrospectively collected and examined a total of 31 very early deaths from APL from the database of 7 Italian Centres. Very early death was defined as death occurring within 72 hours from the first clinical observation. The incidence was 9.7% (31 of 318 APL enrolled in the same period in GIMEMA APL trials). Median age was 53 years (21–88), M/F ratio 0.9, 13 were variant forms of APL, median peripheral blood cell count was: WBC 41.2 ×109/L (0.68–181), with 94% of blast cells, platelets 24 ×109/L (6–96); BCR type 1, 2, 3 were observed in 10, 2, and 7 cases, respectively; clinical features of disseminated intravascular coagulopathy (DIC) were present in all but two patients, while median WHO grade of hemorrhage was 3. Median time from symptoms onset to diagnosis and from diagnosis to death was respectively 72 and 44 hours. One third of the patient did not receive any therapy, the others received some doses of ATRA with or without chemotherapy. The majority of these patients was not enrolled in any clinical trial. These preliminary data suggest that a relevant percentage of APL patients died before starting any effective treatment. Every clinical trial should include this slice of patients to give realistic clinical outcome of APL therapies. Seventy-two percent of patients showed WBC≥10×109/L and variant form was more frequent (42%) than expected (usually 20–25% of APL). Based on the possible confused morphological features with monocytic leukaemia, diagnosis required rapidly confirmative molecular methods and heavy supportive and transfusional therapies. Moreover, underlying APL should be considered as a differential diagnosis when apparently spontaneous cerebral hemorrhage occurs; in some instance, a prompt supportive and differentiating therapy could be lifesaving.

Description: Abstract 3986 Poster Board III-922 Introduction A deregulated expression of procoagulant and anticoagulant activities characterises the phenotype of APL cells and contributes to the coagulopathy of this disease. The APL molecular remission induced by differentiation therapy with ATRA or ATO significantly affects specific cellular hemostatic properties of APL blasts. It is not known whether these variations translate into a comparable reduction of the overall procoagulant activity of APL cells. In this study we characterize the overall APL cell hemostatic potential by the calibrated automated method (CAT), a standardized global assay which reflects the net results of pro- and anti-coagulant forces. The endogenous thrombin potential (ETP), measured as the area under the thrombin generation (TG) curve, is a good indicator of overall plasma prothrombotic and hemorrhagic tendency. We evaluated 1) the sensitivity of NB4 cell TG potential to treatment with ATRA or ATO; 2) the correlation of CAT parameters to the levels of two known procoagulants, i.e. tissue factor (TF) and cancer procoagulant (CP), and anticoagulants (i.e. thrombomodulin, TM) and 3) the association of global TG to cell differentiation, proliferation, and apoptosis/necrosis. Methods NB4 cells were incubated 24h with either 0.1 μM ATO, or 1 μM ATRA, or the combination 0.1 μM ATO/1 μM ATRA, or the vehicle (control). The TG potential of NB4 cells was evaluated in normal pool plasma (NPP) by CAT assay; TF by chromogenic, ELISA, and cytofluorimetric assays; TFmRNA by RT-PCR; CP activity by chromogenic assay; TM by ELISA, cell differentiation by cytofluorimetric analysis of surface CD11b expression; and cell apoptosis/necrosis by annexin V/propidium iodide staining. Results TG potential of control NB4 cells (1350±70 nM*min) was significantly higher compared to normal granulocytes (p