Jacek Treliński

Rozrolimupab, a mixture of 25 recombinant human monoclonal RhD antibodies, in the treatment of primary immune thrombocytopenia

Rozrolimupab, a recombinant mixture of 25 fully human RhD-specific monoclonal antibodies, represents a new class of recombinant human antibody mixtures. In a phase 1 or 2 dose escalation study, RhD(+) patients (61 subjects) with primary immune thrombocytopenia received a single intravenous dose of rozrolimupab ranging from 75 to 300 μg/kg. The primary outcome was the occurrence of adverse events. The principal secondary outcome was the effect on platelet levels 7 days after the treatment. The most common adverse events were headache and pyrexia, mostly mild, and reported in 20% and 13% of the patients, respectively, without dose relationship. Rozrolimupab caused an expected transient reduction of hemoglobin concentration in the majority of the patients. At the dose of 300 μg/kg platelet responses, defined as platelet count ≥ 30 × 10(9)/L and an increase in platelet count by > 20 × 10(9)/L from baseline were observed after 72 hours and persisted for at least 7 days in 8 of 13 patients (62%). Platelet responses were observed within 24 hours in 23% of patients and lasted for a median of 14 days. Rozrolimupab was well tolerated and elicited rapid platelet responses in patients with immune thrombocytopenia and may be a useful alternative to plasma-derived products. This trial is registered at www.clinicaltrials.gov as #NCT00718692.

Circulating endothelial cells in essential thrombocythemia and polycythemia vera: correlation with JAK2-V617F mutational status, angiogenic factors and coagulation activation markers

Angiogenesis plays an important role in the biology of hematological malignancies, including essential thrombocythemia (ET) and polycythemia vera (PV). Some data suggests that it has a role in the pathogenesis of thrombosis, the major clinical problem in ET and PV. The number of different subpopulations of circulating endothelial cells (CECs), plasma levels of vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor 1 and 2 (sVEGFR-1,2) and placenta growth factor (PlGF) were determined in 30 patients with ET and 16 patients with PV. Correlations between angiogenesis and JAK2-V617F mutational status, risk factors for thrombosis and coagulation activation markers were also assessed. The number of CEC subpopulations, were markedly higher in ET and PV patients, irrespective of JAK2-V617F status, when compared to the control group. The median values of activated CECs were markedly higher in PV patients with WBC >8.7 (×109/l). Significantly higher VEGF plasma levels were found in ET patients and a similar trend was seen in PV patients in relation to healthy volunteers. The plasma levels of sVEGFR-1 were significantly higher, and PlGF levels markedly lower, in the ET and PV group than in controls. Our study also demonstrated markedly increased levels of D-dimer and TAT complexes in the patient groups. In conclusion, we found that angiogenesis, as measured by CEC numbers, is increased in ET and PV patients regardless of JAK2-V617F mutational status. Our results demonstrated that angiogenic cytokines interact with known thrombotic risk factors. We confirmed the coagulation activation in ET and PV patients but found no differences in levels of coagulation activation markers in relation to JAK2-V617F mutational status.

Successful treatment of refractory autoimmune thrombocytopenia with rituximab and cyclosporin A in a patient with chronic granulomatous disease.

Dear Editor, Although immunodeficiency and autoimmunity seem to be on the opposite sides of the immune response in many cases, they are associated to each other [1]. We present the first case of female with coexistence of chronic granulomatous disease (CGD) and refractory autoimmune thrombocytopenia (ITP), successfully treated with rituximab and cyclosporin A (CyA). The patient was diagnosed with CGD in 1999 at the age of 14. Thrombocytopenia with platelets (PLT) 9×10/l was first observed in June 2002, and the patient was given prednisone as the initial treatment. The response was good but thrombocytopenia (PLT<20×10/l) relapsed when the dosage of prednisone was tapered below 20 mg/day. In March 2003 the patient received high doses IVIg but during that treatment, aggravation of the haemorrhagic diathesis with epistaxis, menorrhea and secondary anaemia (Hb 8.2 g/dl) was observed. Due to severe diathesis, methylprednisolone 1,000 mg i.v. for 7 days was introduced. The platelet count increased to 340×10/l within few days, and all symptoms of bleeding retreated completely. The patient was discharged from hospital on 60 mg oral prednisone with the recommendation of reducing the dose. She was also given oral contraception. In January 2004 the patient was admitted to our Department because of epistaxis, presence of petechiae on both lips, upper palate and lower extremities. The patient’s complete blood count revealed Hb 11.2 g/dl, red blood cells 4.03×10/l, white blood cells 9.07×10/l and PLT count 3×10/l. The patient received puls of dexamethasone 40 mg i.v. for 4 days [2]. The maximum elevation of platelet count to 77×10/l was achieved, but no consent for splenectomy obtained. After 3 weeks, thrombocytopenia PLT 3×10/l relapsed, but the next dexamethasone course was not effective. The patient was then qualified for rituximab treatment whichwas administered inMay 2004 four times on aweekly basis at 375 mg/m [3, 4]. During that period, platelet and blood transfusions were required. Platelet transfusions were not effective in increasing platelet level, but there was a significant clinical response in terms of diminishing bleeding symptoms. There was also a short episode of fever with good reaction to antibiotic therapy, but neither clinical, radiological nor laboratory signs of infectionwere observed. Twoweeks after the last dose of rituximab, shewas admitted to the hospital suffering from partial blindness due to both eyes retinal haemorrhage, haematuria and profound anaemia (Hb 6.5 g/dl) caused by extensive epistaxis and menorrhea with platelet count of 2×10/l. After RBC and PLT transfusions, therapy with cyclosporinA (2.5mg/kg of bodyweight per day) was introduced [5]. The patient was discharged from the hospital after 10 days completely recovered from blindness with the platelet count of 7×10/l, but 3 days later she was presented with strong headache, nausea and general weakness. CT scan of head showed no signs of cerebral bleeding. The cyclosporin level was within therapeutic ranges, and no renal or hepatic damage was observed. Two months after the last dose of rituximab and 1.5 months after cyclosporin A was introduced, the patient required no blood transfusions and was in stabile medical state without haemorrhagic diathesis symptoms. For the next 2 months, her PLT count varied between 11 and 29×10/l with no signs of bleeding and significant clinical improvement. The dosage of CyAwas reduced to 1 mg/kg per day and continued at that level with only slight changes since it was enough to maintain a therapeutic serum level between 200 and 400 ng/ml. Renal and hepatic functions as well as blood cells counts were checked every 4 weeks. In January 2005, 7 months after CyAwas started, the platelet count was 84×10/l, while in March 2005 it reached J. Treliński . K. Chojnowski . M. Kasznicki . T. Robak (*) Department of Haematology, Medical University of Lodz and Copernicus Memorial Hospital, Lodz, Poland e-mail: robaktad@csk.umed.lodz.pl Tel.: +48-42-6895191 Fax: +48-42-6895192

JAK Inhibitors: Pharmacology and Clinical Activity in Chronic Myeloprolipherative Neoplasms

The Janus family kinases (JAKs), JAK1, JAK2, JAK3, and TYK2, are involved in cell growth, survival, development, and differentiation of a variety of cells, particularly immune cells and hematopoietic cells. They form a subgroup of the non-receptor protein tyrosine kinases. Activating mutations within each of the JAKs is associated with malignant transformations; the most common are mutations of JAK2 in polycythemia vera (PV) and other myeloproliferative neoplasms (MPN). Identification of the V617F mutation of the JAK2 gene (JAK2 V617F) led to an important breakthrough in the understanding of MPN disease pathogenesis. The JAK2 V617F mutation is present in the majority of PV patients, and about 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) are affected. This mutation leads to hyperactivation of JAK2, cytokine-independent signaling, and subsequent activation of downstream signaling networks. JAK2 ATP-competitive inhibitors that indirectly inhibit the JAK-STAT pathway are new candidates for the treatment of MPN. JAK2 inhibitors in development for the treatment of MPN have demonstrated clinical activity with minimal toxicity. These agents consistently alleviate constitutional symptoms and reduce spleen size in PMF and other MPN. However, some of these inhibitors have additional unique effects. Ruxolitinib causes a significant reduction in the level of pro-inflammatory cytokines. Another inhibitor, CYT387, improves anemia. Many other JAK2 inhibitors such as TG101348 or SAR302503, SB1518, CEP701 and LY2784544 are now under investigation for MPN development. In contrast tasocitinib, a predominantly JAK3 inhibitor, is being evaluated in a number of inflammatory and immunological diseases, including rheumatoid arthritis, psoriasis, ulcerative colitis, dry eye disease and in kidney transplant patients. In conclusion the use of JAK inhibitors in MPN and some of the immune-mediated disorders is a promising new strategy for therapy. However, definitive data from ongoing and future preclinical and clinical trials will aid in better defining the status of these drugs in the treatment of these diseases.

Effects of Rivaroxaban Therapy on ROTEM Coagulation Parameters in Patients with Venous Thromboembolism

BACKGROUND Rivaroxaban (Xarelto) does not require routine coagulation monitoring; however, in certain clinical situations (overdose, drug accumulation, urgent surgery) measurement of its plasma concentration is highly recommended. Currently, there is no single hemostasis test that shows a direct correlation between rivaroxaban plasma levels and anticoagulant efficacy. OBJECTIVES This study was intended to assess the value of ROTEM in determining rivaroxaban administration. MATERIAL AND METHODS Thirteen patients with venous thromboembolism and 13 healthy volunteers were compared with regard to certain ROTEM parameters and anti-FXa activity. The tests were done before the administration of 20 mg rivaroxaban (i.e. 24 h after previous administration) and 2.5 h afterwards. RESULTS The study group demonstrated residual activity of rivaroxaban in plasma (20 ± 11.3 ng/mL) 24 h following the previous administration, which did not cause marked changes in clotting assays compared to controls. In the group, 2.5 h after rivaroxaban administration, prolongation of PT (PTratio 1.51 ± 0.22), APTT (APPTratio: 1.30 ± 0.14) and ROTEM CT (CTratio - EXTEM: 2.45 ± 1.06, CTratio - INTEM 1.32 ± 0.21) were observed. The cut-off values for particular tests were created to determine if the patient had achieved desirable anticoagulant effect after rivaroxaban administration. The mean anti-FXa values were significantly lower in patients before rivaroxaban dosing than after. CONCLUSIONS PT demonstrated better diagnostic value than APTT in rivaroxaban administration. The ROTEM clotting time (CT) according to EXTEM may be used to determine the anticoagulation effect of rivaroxaban, but is not sensitive enough to measure the residual activity of this drug.

The influence of low molecular weight heparin on the intravascular activation of the coagulation system in patients with acute leukemia during induction chemotherapy: Report of a prospective randomized study

Intravascular activation of the coagulation system can already be observed in the majority of patients with acute leukemia (AL) at the time of diagnosis. This activation can be further enhanced by chemotherapy. The study comprised of 46 patients with AL, randomly divided into two groups. Twenty-three patients received prophylactic doses of nadroparin (Fraxiparine). Thrombin-antithrombin complexes (TAT), prothrombin fragment (F1+2), D-dimer (DD), plasmin-antiplasmin complexes (PAP) and antithrombin III (AT III) activity were determined in all patients. The tests were performed before treatment, and on the 3rd and 8th days of chemotherapy. The TAT, F1+2, DD and PAP concentrations were found to be elevated in 83% of patients already at the time of diagnosis. No significant difference between either groups test results was noted when either tested before treatment or on the third day of therapy. DIC (disseminated intravascular coagulation) syndrome appeared in two patients receiving heparin prophylaxis and in three patients to whom this treatment was not administered. The concentrations of activation markers on the eighth day of chemotherapy were lower than at the beginning of treatment in most of the patients receiving nadroparin. At this time there were no laboratory signs of DIC in any of the patients receiving heparin prophylaxis. In conclusion: although the application of prophylactic doses of nadroparin does not prevent DIC syndrome during first days of chemotherapy, it may limit the intravascular activation of the coagulation system during later chemotherapy.

The influence of low-dose aspirin and hydroxyurea on platelet-leukocyte interactions in patients with essential thrombocythemia.

Essential thrombocythemia is associated with an increased risk of thromboembolic complications. Recently, there has been a growing evidence that platelet–leukocyte interactions may contribute to pathogenesis of thrombosis in essential thrombocythemia. Low-dose aspirin (ASA) is generally recommended in the therapy of low-risk patients for thrombosis, whereas hydroxyurea in high-risk patients. The aim of the present study was to determine the effect of ASA and hydroxyurea on platelet, leukocyte functions and on formation of platelet/leukocyte conjugates in vivo in patients with essential thrombocythemia. Markers of platelet and leukocyte activation were assessed in 40 patients with essential thrombocythemia at diagnosis and in 20 controls using flow cytometry assays. In second part of the study, the tests were repeated after either ASA treatment (in 25 low-risk patients) or hydroxyurea therapy (in 15 high-risk patients). On diagnosis, significantly elevated expression of P-selectin on platelets (4.98 ± 3.31 vs. 0.99 ± 0.69 P < 0.001) and increased percentage of platelet–polymorphonuclear leukocyte CD11b/CD42b conjugates [10.12 (4.21–31.22) vs. 3.17 (1.43–5.99) P < 0.001] and platelet–monocyte CD11b/CD14/CD61 conjugates [36.62 (12.23–51.62) vs. 13.86 (7.14–23.51) P < 0.001] were found in essential thrombocythemia group as compared with the healthy control group. Therapy with ASA significantly reduced platelet–polymorphonuclear leukocyte [10.72 (4.21–26.97) vs. 8.12 (1.13–26.94) P < 0.05] and platelet–monocyte conjugates [38.6 (13.45–51.62) vs. 25.76 (13.52–45.02) P < 0.05]. Surprisingly, therapy with hydroxyurea was poorly effective in reduction of platelet/leukocyte conjugates. These data document an increased platelet and leukocyte activation at the time of diagnosis. This is the first report showing enhanced platelet–leukocyte aggregate formation in low-risk essential thrombocythemia patients and the efficacy of ASA in its reduction.

Fostamatinib for the treatment of adult persistent and chronic immune thrombocytopenia: Results of two phase 3, randomized, placebo‐controlled trials

Spleen tyrosine kinase (Syk) signaling is central to phagocytosis‐based, antibody‐mediated platelet destruction in adults with immune thrombocytopenia (ITP). Fostamatinib, an oral Syk inhibitor, produced sustained on‐treatment responses in a phase 2 ITP study. In two parallel, phase 3, multicenter, randomized, double‐blind, placebo‐controlled trials (FIT1 and FIT2), patients with persistent/chronic ITP were randomized 2:1 to fostamatinib (n = 101) or placebo (n = 49) at 100 mg BID for 24 weeks with a dose increase in nonresponders to 150 mg BID after 4 weeks. The primary endpoint was stable response (platelets ≥50 000/μL at ≥4 of 6 biweekly visits, weeks 14‐24, without rescue therapy). Baseline median platelet count was 16 000/μL; median duration of ITP was 8.5 years. Stable responses occurred in 18% of patients on fostamatinib vs. 2% on placebo (P = .0003). Overall responses (defined retrospectively as ≥1 platelet count ≥50 000/μL within the first 12 weeks on treatment) occurred in 43% of patients on fostamatinib vs. 14% on placebo (P = .0006). Median time to response was 15 days (on 100 mg bid), and 83% responded within 8 weeks. The most common adverse events were diarrhea (31% on fostamatinib vs. 15% on placebo), hypertension (28% vs. 13%), nausea (19% vs. 8%), dizziness (11% vs. 8%), and ALT increase (11% vs. 0%). Most events were mild or moderate and resolved spontaneously or with medical management (antihypertensive, anti‐motility agents). Fostamatinib produced clinically‐meaningful responses in ITP patients including those who failed splenectomy, thrombopoietic agents, and/or rituximab. Fostamatinib is a novel ITP treatment option that targets an important mechanism of ITP pathogenesis.

Long-lasting extreme anemia during the therapy of acute lymphoblastic leukemia in a Jehovah's Witness patient.

The treatment of patients with acute leukemia, who due to their religious beliefs refuse to accept blood transfusion, is a great challenge for hematologists.

Assessment of rotation thromboelastometry parameters in patients with essential thrombocythemia at diagnosis and after hydroxyurea therapy.

Patients with essential thrombocythemia suffer from thrombotic complications that are the main source of mortality. Due to its complex pathogenesis, no existing single laboratory method is able to identify the patients at highest risk for developing thrombosis. Twenty patients with essential thrombocythemia at diagnosis, 15 healthy volunteers and 20 patients treated with hydroxyurea were compared with regard to certain rotation thromboelastometry parameters. Clotting time (CT), clot formation time (CFT), &agr;-angle, and maximum clot firmness (MCF) were assessed by using the INTEM, EXTEM, FIBTEM, and NATEM tests. Patients with essential thrombocythemia at diagnosis demonstrated significantly higher mean platelet count and markedly lower mean red blood count than controls. CT and CFT readings were found to be markedly lower in essential thrombocythemia patients at diagnosis than in the control group according to the EXTEM test. Patients at diagnosis had markedly lower CT values (EXTEM, FIBTEM) than patients on hydroxyurea therapy. Alpha angle values were markedly higher in essential thrombocythemia patients at diagnosis than in controls, according to the EXTEM, FIBTEM and NATEM tests. MCF readings were significantly higher in essential thrombocythemia patients at diagnosis than in controls according to EXTEM, INTEM, FIBTEM, and NATEM tests. Patients on hydroxyurea therapy had markedly lower MCF values according to EXTEM test than patients at diagnosis. Patients with essential thrombocythemia demonstrate a prothrombotic state at the time of diagnosis, which is reflected in changes by certain rotation thromboelastometry parameters. The hydroxyurea therapy induces downregulation of the prothrombotic features seen in essential thrombocythemia patients at diagnosis.