Hans Wadenvik
email: hans.wadenvik@medic.gu.se

Bob Olsson

Moving towards a new era in the management of chronic immune thrombocytopenia

Keywords: Thrombopoietin mimetics, Romiplostim, Eltrombopag, Chronic immune thrombocytopenia H, Wadenvik (*) :

Immune thrombocytopenia (ITP) is an organspecific autoimmune disease in which a low concentration of plasma thrombopoietin (TPO) contributes to the thrombocytopenia. Functional thrombopoietin deficiency in response to thrombocytopenia is central to the pathophysiology of chronic ITP. Decreased platelet production in ITP patients has been described only in recent years, however. Following the development of TPO-mimetics, it has become clear that the augmentation of thrombopoiesis is a key therapeutic target. TPO mimetics are novel effective treatments providing durable platelet responses in ITP. Two agents have reached clinical practice, the 'peptibody' romiplostim (Nplate®) approved for treatment of thrombocytopenia in patients with chronic ITP in Europe, Canada, Australia and the USA and the non-peptide TPO mimetic, eltrombopag (Promacta®), approved in the USA. This review summarises the background to the development of these agents and presents an update on data from randomised phase III trials and open-label studies. These novel drugs provide a noteworthy treatment option for patients with chronic ITP, in whom thrombocytopenia and bleeding risk have not been controlled by standard treatments. The first candidates for treatment in clinical practice are undoubtedly refractory patients with lack of response to other therapies or at continued risk for bleeding despite treatment. Appropriate inclusion of TPO mimetics into the treatment paradigm will most likely have a positive impact on the long-term outcome of ITP and allow carefully monitored patients to remain well controlled, with good tolerability for prolonged periods.

Introduction

Chronic immune thrombocytopenia (ITP) is an acquired platelet-specific autoimmune disease characterised by low platelet counts, which can lead to life-threatening bleeding. In ITP, anti-platelet antibodies accelerate the destruction of platelets. In addition, bone marrow platelet production can be impaired [START_REF] Ballem | Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura. Evidence of both impaired platelet production and increased platelet clearance[END_REF][START_REF] Isaka | Platelet production, clearance and distribution in patients with idiopathic thrombocytopenic purpura[END_REF][START_REF] Stahl | Incomplete antigenic cross-reactivity between platelets and megakaryocytes: relevance to ITP[END_REF][START_REF] Gernsheimer | Mechanisms of response to treatment in autoimmune thrombocytopenic purpura[END_REF], and cytotoxic T lymphocytes have been shown to lyse platelets in vitro and in vivo [START_REF] Zhao | Increased cytotoxic T-lymphocyte-mediated cytotoxicity predominant in patients with idiopathic thrombocytopenic purpura without platelet autoantibodies[END_REF][START_REF] Olsson | T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura[END_REF][START_REF] Chow | Antibody-and cell-mediated immune thrombocytopenia are differentially sensitive to intravenous gammaglobulin therapy[END_REF]. Although the thrombocytopenia in ITP can be profound, in the majority of adult patients, signs of bleeding are typically mild to moderate. However, persistently low platelet counts (<20 × 10 9 /L) can contribute to a risk of serious bleeding, such as gastrointestinal and intracranial haemorrhages [START_REF] Butros | Intracranial hemorrhage in immune thrombocytopenic purpura: a retrospective analysis[END_REF][START_REF] Cines | Immune thrombocytopenic purpura[END_REF][START_REF] Lacey | Management of idiopathic thrombocytopenic purpura in the adult[END_REF]. When managing chronic ITP in adults, the ultimate goal is to maintain the platelet count at a haemostatically safe level to prevent bleeding with the least possible intervention and, therefore, low treatment-related toxicity [START_REF] Cines | How I treat idiopathic thrombocytopenic purpura (ITP)[END_REF].

Treatment remains challenging in many adult patients who relapse after therapy with corticosteroids and/or intravenous immunoglobulins and may ultimately require splenectomy [START_REF] Rodeghiero | Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group[END_REF][START_REF]British Committee for Standards in Haematology General Haematology Task Force (2003) Guidelines for the investigation and management of idiopathic thrombocytopenic purpura in adults, children and in pregnancy[END_REF][START_REF] Arnold | Current options for the treatment of idiopathic thrombocytopenic purpura[END_REF]. Approximately a third of adult patients relapse after splenectomy [START_REF] Kojouri | Splenectomy for adult patients with idiopathic thrombocytopenic purpura: a systematic review to assess long-term platelet count responses, prediction of response, and surgical complications[END_REF]. While current treatment options for refractory patients post-splenectomy now include immunomodulatory/immunosuppressive agents, e.g. intermittent immunoglobulins (IVIg), corticosteroids, azathioprin, cyclophosphamide, mofetil mycophenolate, vinca alkaloids and the B cell depleting agent rituximab, these generally generate low long-term response rates and significant toxicity [START_REF] Stasi | Rituximab chimeric anti-CD20 monoclonal antibody treatment for adults with chronic idiopathic thrombocytopenic purpura[END_REF][START_REF] Hou | Mycophenolate mofetil (MMF) for the treatment of steroidresistant idiopathic thrombocytopenic purpura[END_REF][START_REF] Cooper | The efficacy and safety of Bcell depletion with anti-CD20 monoclonal antibody in adults with chronic immune thrombocytopenic purpura[END_REF][START_REF] Zaja | A prospective randomised study comparing rituximab and dexamethasone vs dexamethasone alone in ITP: results of final analysis and long term follow up[END_REF].

Progress in uncovering an impaired bone marrow response to low platelet count in chronic ITP has made the augmentation of platelet production a key therapeutic target [START_REF] Gernsheimer | The pathophysiology of ITP revisited: ineffective thrombopoiesis and the emerging role of thrombopoie-tin receptor agonists in the management of chronic immune thrombocytopenic purpura[END_REF], with the result that novel effective treatments providing high durable response rates in patients with chronic ITP are now approved.

Thrombopoietin

The term thrombopoietin (TPO) was first used in 1958 to describe the humoral regulator of platelet production. Doubts surrounding its existence remained until the molecule was cloned [START_REF] De Sauvage | Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand[END_REF][START_REF] Lok | Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo[END_REF][START_REF] Kaushansky | Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin[END_REF]. TPO, the ligand for the cmpl proto-oncogene, is mainly expressed in liver and kidney [START_REF] De Sauvage | Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand[END_REF][START_REF] Lok | Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo[END_REF][START_REF] Stockelberg | Plasma thrombopoietin levels in liver cirrhosis and kidney failure[END_REF] and has been shown to influence all aspects of megakaryocyte development and platelet production [START_REF] Kelemen | Demonstration and some properties of human thrombopoietin in thrombocythaemic sera[END_REF]. Animal model data suggest a feedback mechanism whereby circulating platelet mass and megakaryocyte mass are inversely related to TPO levels [START_REF] Nagata | Serum thrombopoietin level is not regulated by transcription but by the total counts of both megakaryocytes and platelets during thrombocytopenia and thrombocytosis[END_REF][START_REF] Shivdasani | Regulation of the serum concentration of thrombopoietin in thrombocytopenic NF-E2 knockout mice[END_REF]. Several studies showed that circulating TPO is selectively cleared by platelets through receptor-mediated endocytosis and destruction [START_REF] Ulich | Megakaryocyte growth and development factor ameliorates carboplatin-induced thrombocytopenia in mice[END_REF][START_REF] Wendling | cMpl ligand is a humoral regulator of megakaryocytopoiesis[END_REF][START_REF] Kuter | Appearance of a megakaryocyte growth-promoting activity, megapoietin, during acute thrombocytopenia in the rabbit[END_REF][START_REF] Kuter | The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production[END_REF][START_REF] Stoffel | Thrombopoietin in thrombocytopenic mice: evidence against regulation at the mRNA level and for a direct regulatory role of platelets[END_REF]. Paradoxically, TPO levels appear normal or only slightly elevated in ITP. These patients uniformly display normal megakaryocyte numbers in bone marrow biopsies, further supporting plasma TPO level regulation by circulating platelets and bone marrow megakaryocytes [START_REF] Hou | Plasma thrombopoietin levels in thrombocytopenic states: implication for a regulatory role of bone marrow megakaryocytes[END_REF].

Early studies with pegylated and recombinant TPO in the late 1990s demonstrated platelet response in cancer patients with chemotherapy-induced thrombocytopenia [START_REF] Fanucchi | Effects of polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor on platelet counts after chemotherapy for lung cancer[END_REF][START_REF] Basser | Thrombopoietic effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in patients with advanced cancer[END_REF]. However, clinical evaluation of early TPO analogues, i.e. pegylated recombinant TPO, was halted because antibodies were formed that cross reacted with endogenous TPO, causing secondary and long-lasting thrombocytopenia and bleeding [START_REF] Li | Thrombocytopenia caused by the development of antibodies to thrombopoietin[END_REF]. This setback stimulated research for second-generation c-mpl agonists including peptide and non-peptide agonists [START_REF] Inagaki | Induction of megakaryocytopoiesis and thrombocytopoiesis by JTZ-132, a novel small molecule with thrombopoietin mimetic activities[END_REF][START_REF] Broudy | AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl[END_REF][START_REF] Erickson-Miller | Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist[END_REF][START_REF] Wang | Pharmacodynamics and pharmacokinetics of AMG 531, a novel thrombopoietin receptor ligand[END_REF][START_REF] Frederickson | A rationally designed agonist antibody fragment that functionally mimics thrombopoietin[END_REF].

TPO mimetics

The search for second-generation TPO analogues has focussed on agents capable of binding to the TPO receptor as strongly as TPO, without having cross-reactive immunogenicity against native TPO.

Two agents have reached clinical practice, the 'peptibody' romiplostim (Nplate®) approved for treatment of thrombocytopenia in patients with chronic ITP in Europe, Canada, Australia and the USA and the non-peptide TPO mimetic, eltrombopag (Promacta®), approved in the USA.

Romiplostim

The romiplostim 'peptibody', administered subcutaneously, consists of two covalently linked carrier-Fc domains, each attached to a polypeptide containing two c-mpl-activating sequences [START_REF] Broudy | AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl[END_REF][START_REF] Wang | Pharmacodynamics and pharmacokinetics of AMG 531, a novel thrombopoietin receptor ligand[END_REF]. Lack of sequence homology to endogenous TPO is clinically relevant as this reduces the risk of development of cross-reacting anti-TPO antibodies that can compromise treatment efficacy. Early studies in healthy adults showed that single injections of romiplostim induced a dose-dependent increase in platelet count, with no serious adverse events [START_REF] Wang | Pharmacodynamics and pharmacokinetics of AMG 531, a novel thrombopoietin receptor ligand[END_REF][START_REF] Kumagai | Pharmacodynamics and pharmacokinetics of AMG 531, a thrombopoiesis-stimulating peptibody, in healthy Japanese subjects: a randomised, placebo-controlled study[END_REF]. Platelet numbers peaked at 12-16 days, returning to baseline at 28 days [START_REF] Wang | Pharmacodynamics and pharmacokinetics of AMG 531, a novel thrombopoietin receptor ligand[END_REF]. Platelet response in adults with ITP and low platelet counts were first reported in a dose-finding assessment of romiplostim given as two injections separated by a 15-day interval [START_REF] Newland | An open-label, unit dosefinding study of AMG 531, a novel thrombopoiesis-stimulating peptibody, in patients with immune thrombocytopenic purpura[END_REF].

A randomised, double-blind, placebo-controlled phase I/ II US study investigating romiplostim in patients with ITP led to the establishment of individualised dosing based on platelet counts [START_REF] Bussel | AMG 531, a thrombopoiesis-stimulating protein, for chronic ITP[END_REF]. The phase II study population was 21 highly refractory adult ITP patients. Patients were randomised 4:1 to receive weekly doses of romiplostim (1, 3 or 6 μg/kg) or placebo, for 6 weeks, with follow-up (FU) up to day78. No dose adjustments were allowed, although doses were withheld when platelet count was >350 × 10 9 /L. The primary objectives were to evaluate safety and to identify a weekly dose that would give a platelet count within the target range of 50-450 × 10 9 /L and that was at least twice the baseline count. Platelet targets were reached or exceeded in 12 of 16 patients assigned to receive romiplostim (1 or 3 μg/kg). Rates of adverse events were similar across treatment arms, and no neutralising antibodies against romiplostim or endogenous TPO were detected. Transient post-treatment worsening of thrombocytopenia was observed in four patients.

Two double-blinded phase III trials, carried out at 35 sites in the USA and Europe, have investigated 6-month treatment with romiplostim [START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF]. The trials were of identical design, except for splenectomy status. Both studies enrolled adults with previously treated ITP with a mean platelet count of <30 × 10 9 /L. Patients were randomised to romiplostim or placebo (2:1). Weekly romiplostim was given for 24 weeks. Follow-up was 12 weeks. Patients were allowed to enter an extension study after 4 weeks of followup, or once platelet count fell below 50 × 10 9 /L. The starting dose was 1 µg/kg with subsequent dosing adjusted to achieve and maintain a target platelet count of 50-200 × 10 9 /L. The maximum allowed dose was 15 μg/kg.

Patients were allowed to receive concurrent and rescue ITP medication. The primary endpoint was a durable platelet response (weekly platelet response of 50 × 10 9 /L or more, for at least six out of the last 8 weeks of the study without use of rescue medication). Platelet responses that occurred within 8 weeks after rescue medication was used were not included. Secondary endpoints included transient and overall platelet response, number of weekly platelet responses and the proportion of patients requiring rescue medication.

The incidence of durable platelet response was significantly higher in patients treated with romiplostim compared with placebo, in both splenectomised and non-splenectomised patients [START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF]. Of splenectomised patients, 38% (16/42) receiving romiplostim showed a durable platelet response, compared with none of the 21 patients in the placebo group (p = 0.0013). Similarly, of non-splenectomised patients, 61% (25/41) receiving romiplostim showed a durable platelet response, compared with 5% (1/21) in the placebo group (p < 0.0001). A wide dose range was administered to responding patients, particularly in patients who had failed to respond to splenectomy, which highlights the heterogeneity seen in chronic ITP.

Benefits were also seen in romiplostim patients not achieving a durable platelet response, with 40% (17/42) of splenectomised patients and 27% (11/41) of nonsplenectomised patients achieving a transient platelet response (at least four weekly platelet responses from weeks2-25). The overall platelet response rate was 79% in the splenectomised group and 88% in the non-splenectomised group, compared with 0% (splenectomised) and 14% (non-splenectomised) of patients in the placebo group (durable plus transient platelet response). Median weekly platelet counts were higher in romiplostim-treated patients than placebo controls, with platelet increases first observed within 1-2 weeks and sustained over 24 weeks of treatment.

Significantly fewer patients receiving romiplostim required rescue medications than those receiving placebo (19/ 83 (22.7%) vs 25/42 (59.5%); romiplostim vs placebo, respectively, p < 0.0001) [START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF] or needed intervention with intravenous immunoglobulins [START_REF] Pullarkat | Reduction in immunoglobulin (IVIG or Anti D) use in patients with chronic immune thrombocytopenic purpura (ITP) receiving AMG 531: results from two phase 3 randomised placebo-controlled trials[END_REF]. Decreasing concurrent ITP treatments is a desirable aim, to reduce treatmentrelated side effects and improve quality of life. Also, romiplostim enabled many patients to discontinue all concurrent ITP medication (12/23, 52%) or reduce doses of one concurrent ITP drug by >25% (8/23, 35%). However, it should be borne in mind that the number of patients able to discontinue concurrent medication was limited by trial protocol, which did not allow discontinuation after week12 [START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF]. No patients tested positive for neutralising antibodies to romiplostim or TPO.

The incidence of adverse events was similar in the placebo and treatment arms, with 100% of patients receiving romiplostim reporting adverse events compared with 95% of patients receiving placebo. Most adverse events were mild to moderate and related to the underlying thrombocytopenia, being typical of events reported in the setting of chronic ITP (headache, fatigue, epistaxis, arthralgia and contusion bleedings). Two uncommon severe side effects were reported in the romiplostim-treated patient groups, with one non-responding patient developing bone marrow reticulin, which reversed on treatment discontinuation, and two cases of thromboembolism. Increased reticulin deposits have been infrequently reported in romiplostim clinical studies [START_REF] Kuter | Evaluation of bone marrow reticulin formation in chronic immune thrombocytopenia (ITP) patients treated with romiplostim[END_REF]. The reversible nature of reticulin formation in response to romiplostim has also been observed by retrospective analysis across romiplostim clinical trials [START_REF] Kuter | Evaluation of bone marrow reticulin formation in chronic immune thrombocytopenia (ITP) patients treated with romiplostim[END_REF].

Bleeding events (≥ grade 2) were less common in patients receiving romiplostim (14/84 vs 34/42, romiplostim vs placebo, respectively) [START_REF] Lyons | Evaluation of AMG 531 safety in splenectomised (S) and nonsplenectomised (NS) patients with chronic immune thrombocytopenic purpura (ITP) in two randomised placebo-controlled phase 3 studies[END_REF]. Significant bleeding events (defined as severe, life-threatening or fatal events) were less common in the treatment arm, with events reported in 7% (6/84) of the romiplostim group compared with 12% (5/41) of patients in the placebo group [START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF]. Two of the patients receiving placebo died during the study due to bleeding events (cerebral haemorrhage and pulmonary embolism following an episode of severe gastrointestinal haemorrhage). Another patient in the placebo group died from pneumonia complications presenting after an intracranial haemorrhage due to trauma. One patient who had received romiplostim died 1 day after study completion as a result of intracranial haemorrhage, which occurred after commencing aspirin treatment for thrombosis following discontinuation of romiplostim [START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF].

Patients who had been enrolled in the phase III studies [START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF], and other studies assessing romiplostim for treatment of ITP [START_REF] Newland | An open-label, unit dosefinding study of AMG 531, a novel thrombopoiesis-stimulating peptibody, in patients with immune thrombocytopenic purpura[END_REF][START_REF] Bussel | AMG 531, a thrombopoiesis-stimulating protein, for chronic ITP[END_REF], were eligible for a long-term extension open-label trial, irrespective of whether they had received romiplostim or placebo [START_REF] Bussel | Safety and efficacy of long-term treatment with romiplostim in thrombocytopenic patients with chronic ITP[END_REF]. The most recent reported data, (July 2008) is based on an Intent to Treat population of 223 patients; 99 of whom (44%) were splenectomised, and 34 patients (15%) were on concurrent ITP therapy at study entry [START_REF] Scott | Long term efficacy of romiplostim in patients with chronic immune thrombocytopenia (ITP): 4 year update from an open label extension study[END_REF][START_REF] Nelson | Long term safety of romiplostim in patients with chronic immune thrombocytopenia (ITP): 4 year update from an open label extension study[END_REF]. Previous treatment with romiplostim varied from no exposure to 24 weeks [START_REF] Newland | An open-label, unit dosefinding study of AMG 531, a novel thrombopoiesis-stimulating peptibody, in patients with immune thrombocytopenic purpura[END_REF][START_REF] Bussel | AMG 531, a thrombopoiesis-stimulating protein, for chronic ITP[END_REF][START_REF] Kuter | Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial[END_REF]. Dose adjustment was made according to platelet count, to a maximum of 10 μg/kg, weekly. In total, 215 patients received romiplostim, and 186 patients remain on study as of July 2008 [START_REF] Kuter | Long-term treatment with romiplostim in patients with chronic immune thrombocytopenic purpura (ITP): 3year update from an open-label extension study[END_REF]. The efficacy endpoint was platelet response (platelet count≥50 × 10 9 /L and double baseline) in the absence of rescue medication within the preceding 8 weeks [START_REF] Kuter | Long-term treatment with romiplostim in patients with chronic immune thrombocytopenic purpura (ITP): 3year update from an open-label extension study[END_REF].

Thirty percent of patients (61/207) responded after the first dose and 74% of patients (160/215) overall. Among patients receiving concurrent ITP medications at baseline, 74% (25/34) discontinued or reduced their dose by >25% [START_REF] Scott | Long term efficacy of romiplostim in patients with chronic immune thrombocytopenia (ITP): 4 year update from an open label extension study[END_REF]. Use of rescue medications decreased over time. Bleeding of moderate or greater severity declined from 14% of patients during the first 24 weeks to 10%, 7% and 5% of patients during subsequent 24-week periods [START_REF] Scott | Long term efficacy of romiplostim in patients with chronic immune thrombocytopenia (ITP): 4 year update from an open label extension study[END_REF].

This study also provided important safety data in patients treated for up to 204 weeks, with exposureadjusted incidence rates of adverse events showing no cumulative incidence and no cumulative toxicity [START_REF] Bussel | Safety and efficacy of long-term treatment with romiplostim in thrombocytopenic patients with chronic ITP[END_REF][START_REF] Nelson | Long term safety of romiplostim in patients with chronic immune thrombocytopenia (ITP): 4 year update from an open label extension study[END_REF][START_REF] Kuter | Long-term treatment with romiplostim in patients with chronic immune thrombocytopenic purpura (ITP): 3year update from an open-label extension study[END_REF]. Retrospective analysis across clinical trials (to February 2008) shows that bone marrow reticulin was confirmed in ten bone marrow samples [START_REF] Kuter | Evaluation of bone marrow reticulin formation in chronic immune thrombocytopenia (ITP) patients treated with romiplostim[END_REF]. Reticulin was also examined over time in selected patients who participated in a supplemental prospective analysis designed to evaluate the effect of romiplostim treatment on bone marrow morphology [START_REF] Bussel | Safety and efficacy of long-term treatment with romiplostim in thrombocytopenic patients with chronic ITP[END_REF]. Bone marrow biopsies were taken at baseline (before initiating romiplostim therapy) and after receiving romiplostim for 3 (n = 4) or 9 (n = 5) months. Of six patients with baseline and FU biopsies, reticulin staining following romiplostim treatment remained absent in five/six cases and in one patient treated with romiplostim for 3 months changed from none to mild.

Eltrombopag

The non-peptide, synthetic TPO-receptor agonist, eltrombopag (marketed as Promacta in the USA, but not licensed in the EU at the time of writing), is administered as a once daily oral tablet. Food interactions observed in pharmacokinetic studies indicate that eltrombopag should be administered to fasting patients or with low calcium foods [START_REF] Williams | Effects of food and antacids on the pharmacokinetics of eltrombopag in healthy adult subjects: two single-dose, open-label, randomised-sequence, crossover studies[END_REF]. It is a small non-peptide molecule and as such is unlikely to induce antibody formation. In a phase I clinical study, 73 healthy male subjects were randomised to receive eltrombopag (six dose groups of ∼9 patients each) or placebo (18 patients) for 10 days at doses of 5-75 mg. The mean change in platelet count for the 30-, 50-, and 75-mg dose levels was 24.1% (257.6 × 10 9 /L to 319.6 × 10 9 /L), 42.9% (254.3 × 10 9 /L to 363.4 × 10 9 /L) and 50.4% (235.9 × 10 9 /L to 354.9 × 10 9 /L), respectively. The highest individual platelet count recorded was 457 × 10 9 /L [START_REF] Jenkins | Phase 1 clinical study of eltrombopag, an oral, nonpeptide thrombopoietin receptor agonist[END_REF]. A consistent increase in platelet count began after 8 days of repeat dosing with eltrombopag, and the interval from first dose to peak platelet count was 16 days. By day22 (12 days after the last dose of eltrombopag), platelet count had returned to baseline values. These data are consistent with published platelet counts seen after the administration of recombinant TPO in humans [START_REF] Vadhan-Raj | Clinical experience with recombinant human thrombopoietin in chemotherapy-induced thrombocytopenia[END_REF]. In addition, there was no evidence of rebound thrombocytopenia. In a multicentre randomised trial, 118 adults with chronic ITP and platelet counts of less than 30 × 10 9 /L who had had relapses or whose platelet count was refractory to at least one standard treatment for ITP were randomly assigned to receive the oral thrombopoietin-receptor agonist eltrombopag (30, 50 or 75 mg daily) or placebo [START_REF] Bussel | Eltrombopag for the treatment of chronic idiopathic thrombocytopenic purpura[END_REF]. Of 109 patients receiving ≥1 dose, the primary endpoint (platelet count≥50 × 10 9 /L on day43) was achieved in 28%, 70% and 81% of patients for the respective 30, 50 and 75 mg doses compared with 11% for those receiving placebo. By day15, more than 80% of patients on the 50 and 75 mg doses had a raised platelet count with the median platelet count approaching the normal range. Eltrombopag was discontinued in 28 patients when platelet counts >200 × 10 9 /L were reported, with median platelet counts returning near to baseline within 2 weeks without rebound thrombocytopenia.

In a phase III, randomised, double-blind, placebocontrolled study, previously treated adult patients from 63 sites in 23 countries with chronic ITP and platelet counts less than 30 × 10 9 /L received standard care plus once daily eltrombopag 50 mg (n = 76) or placebo (n = 38) for up to 6 weeks [START_REF] Bussel | Effect of eltrombopag on platelet counts and bleeding during treatment of chronic idiopathic thrombocytopenic purpura: a randomised, double-blind, placebo-controlled trial[END_REF]. Forty-five patients (39%) had undergone splenectomy. Corticosteroid treatment was permitted, but other drugs for the disorder were stopped for at least 2 weeks before trial initiation. Patients were randomised (2:1, eltrombopag vs placebo). After 3 weeks, patients with platelet counts less than 50 × 10 9 /L could increase study drug to 75 mg, q.d. The primary endpoint was the proportion of patients achieving platelet counts 50 × 10 9 /L or more at day43 [START_REF] Bussel | Eltrombopag for the treatment of chronic idiopathic thrombocytopenic purpura[END_REF]. Of 110 patients receiving ≥1 dose, 43/73 (59%) eltrombopag patients and 6/37 (16%) placebo patients responded (odds ratio 9.61 (95% CI 3.31-27.86); p≤ 0.0001). Patient response was independent of sex, age, the use of concomitant ITP drugs, splenectomy status, baseline platelet count and number of previous ITP therapies. Responses

Chance of spontaneous recovery

Fig. 1 It is not controversial that corticosteroids and/or IVIg are the first-line therapy for patients with newly diagnosed ITP and in need for treatment, and it will presumably be that over the foreseeable future. Splenectomy is still the second-line treatment for patients with ITP at many centres. However, spontaneous recovery can be seen during the first year after diagnosis, and splenectomy are usually postponed for several months to avoid unneeded surgical procedures.

For patients who fail splenectomy, the therapy options are less uniform and include several treatment protocols with significant toxicity. The first candidates for treatment with TPO-mimetics in clinical practice are undoubtedly refractory ITP patients with lack of response to other therapies or at continued risk for bleeding despite treatment. It is likely that with increasing experience, these novel agents will be introduced earlier in the treatment algorithm, e.g. as steroid sparing agents pre-splenectomy baseline platelet counts 15 × 10 9 /L or less. Eight of the remaining 22 patients had clinically significant platelet increases. Significantly fewer patients in the eltrombopag group than in the placebo group had bleeding symptoms, as measured by the WHO bleeding scale, at day43 (20 (39%) vs 18 (60%); p = 0•029) as well as at any point in time during the course of treatment (46 (61%) vs 30 (79%); p = 0.021). Mean scores for health-related quality of life were not affected by treatment.

An extended phase III study has treated 109 patients with ITP given eltrombopag at 50 mg daily, adjusted, when necessary, to 25 or 75 mg according to platelet counts [START_REF] Bussel | Long-term safety and efficacy of oral eltrombopag for the treatment of idiopathic thrombocytopenic purpura (ITP) Haematologia[END_REF][START_REF] Bussel | Oral eltrombopag spares corticosteroids and reduces bleeding in patients with idiopathic thrombocytopenic purpura (ITP)[END_REF]. In this study, 78% of patients maintained a platelet count of >50 × 10 9 /L for more than half their time in the study (mean 194 days), and 35% of patients requiring ITP medication at baseline were able to stop concomitant medication. Thromboembolic events were noted in four patients, all of whom had risk factors for thrombosis. After 12 weeks, <5% of patients reported clinically significant bleeding vs >20% at baseline. Furthermore, eltrombopag has been investigated outside the context of primary chronic ITP in adults, with a report showing that eltrombopag administered to patients with hepatitis C-associated thrombocytopenia with cirrhosis produced a dose-dependent increase in platelet count [START_REF] Mchutchison | Eltrombopag for thrombocytopenia in patients with cirrhosis associated with hepatitis C[END_REF].

Current status and future trends

Insufficient production of thrombopoietin in response to thrombocytopenia is central to the pathophysiology of ITP. Despite increased knowledge of disease pathophysiology and advances in molecular therapy, the treatment algorithm for ITP remains largely based on modalities aimed at correcting autoantibody-induced platelet destruction. In refractory patients, the treatment has mainly been "on demand" due to the toxicity profile of the second and third line therapies. Now, with the availability of thrombopoietic agents, it may be the time to apply close scrutiny to current treatment efficacy and toxicities. If the promising safety data for the TPO-mimetics hold true in the long term, we might move into 'prophylactic treatment' of these patients, as has been implemented in the current management of other bleeding disorders, e.g. hemophilias. However, given the 5-day lag time for a platelet effect of these agents, they are not a substitute for platelet transfusions in the acute treatment of thrombocytopenia.

TPO-mimetics provide a noteworthy treatment option for patients with chronic ITP, in whom platelet count and bleeding have not been controlled by standard treatments, and the first candidates for treatment in clinical practice are undoubtedly refractory ITP patients with lack of response to other therapies or at continued risk for bleeding despite treatment. However, the development of TPO mimetics also has implications for the treatment algorithm in ITP (Fig. 1). In particular, TPO-mimetics could allow steroid-sparing treatment approaches to be implemented [START_REF] Michel | Reduced corticosteroid treatment in adults with ITP receiving romiplostim[END_REF]. Ultimately, if use of these novel drugs were to be extended to nonsplenectomised patients, this might allow surgical intervention to be postponed [START_REF] Michel | Immune thrombocytopenic purpura: epidemiology and implications for patients[END_REF].

Recent guidelines are already helping to provide consistency across ITP terminology for disease diagnosis, response and outcome [START_REF] Rodeghiero | Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group[END_REF]. Currently, platelet counts are a surrogate marker for the risk of bleeding events and are the basis on which treatment decisions are made. Sustained improvement of platelet counts by TPO-mimetics is therefore of clinical importance [START_REF] George | Improved quality of life for romiplostim-treated patients with chronic immune thrombocytopenic purpura: results from two randomised, placebo-controlled trials[END_REF] and can have additional quality-of-life implications for ITP patients [START_REF] Michel | Immune thrombocytopenic purpura: epidemiology and implications for patients[END_REF][START_REF] Mathias | Evaluating clinically meaningful change on the ITP-PAQ: preliminary estimates of minimal important differences[END_REF][START_REF] Mathias | A disease-specific measure of health-related quality of life in adults with chronic immune thrombocytopenic purpura: psychometric testing in an open-label clinical trial[END_REF]. For TPOmimetics to be implemented routinely, early in the treatment algorithm, supportive long-term safety data are needed.

In this promising era for ITP treatment, many questions remain to be answered regarding which patients should receive TPO-mimetics and exactly when this should occur. As these questions are answered, more patients will be considered for therapy with TPO-mimetics. Appropriate inclusion of TPO mimetics into the treatment paradigm will most likely have a positive impact on the long-term outcome of ITP and allow carefully monitored patients to remain well controlled, with good tolerability for prolonged periods. Furthermore, there are other thrombocytopenic conditions where the risks of short-term exposure with TPO-mimetics appear acceptable, e.g. hepatitis C-related thrombocytopenia, liver failure patients awaiting transplantation, thrombocytopenia in myelodysplastic syndromes, thrombocytopenia in presurgical patients and chemotherapy-induced thrombocytopenia. Even platelet apheresis donors might benefit from TPO mimetics. Post-marketing surveillance of the novel TPOmimetics and new studies in other thrombocytopenic conditions will increase our understanding of the correct use of these novel agents in haematology.

  to eltrombopag were seen in 16/38 patients with

		ITP treatment algorithm
		"Current and Future"
	1st line: Corticosteroids IVIg Anti-D	2nd line: Splenectomy Rituximab TPO-mimetics	3rd line: TPO-mimetics Rituximab Azathioprin Danazol
			Cyclophosphamide
			Mycophenolate
			in refractory ITP
	Newly Diagnosed ITP	Persistent ITP	Chronic ITP
	3 months	12 months
		Time after diagnosis

Ann Hematol (2010) 89 (Suppl 1):S87-S93

Acknowledgements This work was supported by grants from the Swedish Research Council (529-2004-6512; 521-2006-5103), the Swedish federal government under the LUA/ALF agreement, the Sahlgrenska University Hospital Foundation, the Foundations of the National Board of Health and Welfare, the Åke Wiberg Foundation, the Jeansson Foundations, the Tore Nilsson Foundation for Medical Research, the Magnus Bergvall Foundation and the Wilhelm and Martina Lundgren Science Foundation. Editorial support from Janet Stephenson of Bioscript Stirling Ltd., London, UK, was funded by Amgen.


Kenneth J Clemetson

Simon Panzer
email: simon.panzer@meduniwien.ac.at

The working group for "basic science" in ITP at the International Cooperative ITP Study Group (ICIS)

Keywords: Autoimmune thrombocytopenia, Basic science, Collaboration

published or not. The documents may come     The working group for "basic science" in ITP at the International Cooperative ITP Study Group (ICIS)

Introduction

The group discussed the origins of idiopathic thrombocytopenic purpura (ITP) and the feeling that it has a variety of etiologies, even if acute onset and chronic (in adulthood) are already considered separately. Further, this variety may persist after excluding "secondary" ITP in diseases like lupus erythematosus. The following suggestions for further directions of research may assist to characterize better the pathoetiology of the disease.

As acute ITP is thought to be associated with viral infections, characterization of cross-reacting viral and platelets epitopes was suggested [START_REF] Cines | Immune thrombocytopenic purpura[END_REF][START_REF] Provan | International consensus report on the investigation and management of primary immune thrombocytopenia[END_REF]. This could be accomplished by work on the 3-D structures of the epitopes on the one hand, and by the production of the relevant peptides and antibody binding studies on the other hand. However, none of the participants has the necessary expertise to carry on such investigations.

It is clear that the platelet count is only a surrogate marker for the risk of bleeding. However, some individuals hardly have bleedings at very low platelet counts of <20× 10 9 /l while others may bleed at a platelet count of <50× 10 9 /l. Therefore, a very good characterization of platelets, megakaryocytes, and plasma factors for coagulation active mechanisms was felt necessary.

(a) In particular, platelet and megakaryocyte apoptosis was suggested to be of importance [START_REF] Mason | Programmed anuclear cell death delimits platelet life span[END_REF][START_REF] Kile | The role of the intrinsic apoptosis pathway in platelet life and death[END_REF]. The group around Oliver Speer and Markus Schmugge (University Children's Hospital, Zürich, Switzerland) is interested in such studies and called for collaboration with other groups to provide carefully defined sera or plasma from patients as well as providing bone marrow material. Material obtained at various periods throughout the disease would be advantageous for such studies. A study proposal is shown at the end of this report. (b) A possible compensation of the coagulation system for the low platelet count that is met in some individuals, but not in others, may contribute to less bleeding. Therefore, microparticles and thrombin generation may serve to overcome the low platelet counts [START_REF] Smyth | for the 2009 platelet colloquium participants[END_REF]. Some groups have started to look into these mechanisms.

Another topic is cytokine regulation on serially obtained samples. John Semple (Toronto, Canada) would be interested in such studies. Such studies may advance our knowledge on T cell/B cell interaction during the course of the disease. Currently, the main body of data has been published on single observation snapshots [START_REF] Semple | Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity[END_REF][START_REF] Olsson | T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura[END_REF][START_REF] Zhou | BAFF and BAFF-R of peripheral blood and spleen mononuclear cells in idiopathic thrombocytopenic purpura[END_REF].

Familiar onset of ITP and genetic predisposition is a very interesting subject [START_REF] Gaiger | HLA class-I and -II antigens in chronic idiopathic autoimmune thrombocytopenia[END_REF][START_REF] Maia | Predisposition to idiopathic thrombocytopenic purpura maps close to the MICA gene[END_REF]. Johannes Rischewski, Basel, is collecting these samples and studies family genetics. Again, collaboration with others would improve the power of these investigations.

Conflict of interest

The authors have no conflict of interest to declare.


Elena Crisà

Ermanno Venturino

Roberto Passera

Marco Prina

Piercarla Schinco

Alessandra Borchiellini

Valentina Giai

Maria Ausilia

Ciocca Vasino

Mario Bazzan

Antonella Vaccarino

Mario Boccadoro

Dario Ferrero
email: dario.ferrero@unito.it

M A Ciocca Vasino

A retrospective study on 226 polycythemia vera patients: impact of median hematocrit value on clinical outcomes and survival improvement with anti-thrombotic prophylaxis and non-alkylating drugs

Keywords: Polycythemia vera, Thrombotic events, Chemotherapy, Hematocrit, Acute myeloid leukemia E, Crisà : P, Schinco : A, Borchiellini :

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A retrospective study on 226 polycythemia vera patients: impact of median hematocrit value on clinical outcomes and survival improvement with anti-thrombotic prophylaxis and non-alkylating drugs

Introduction

Polycythemia vera (PV) is the most common chronic myeloproliferative neoplasm, with an annual incidence of 2.3 per 100,000 [START_REF] Silverstein | Polycythemia vera, 1935-1969: an epidemiologic survey in Rochester, Minnesota[END_REF][START_REF] Ania | Trends in the incidence of polycythemia vera among Olmested Country, Minnesota residents, 1935-1989[END_REF], a reported median survival of 10-15 years [START_REF] Cervantes | Life expectancy and prognostic factors in the classic BCR/ABL-negative myeloproliferative disorders[END_REF][START_REF] Kiladjian | Long-term outcomes of polycythemia vera patients treated with pipobroman as initial therapy[END_REF][START_REF] Finazzi | The treatment of polycythaemia vera: an update in the JAK2 era[END_REF][START_REF]Polycythemia vera: the natural history of 1213 patients followed for 20 years[END_REF] and an overall mortality of 3.5 per 100 person/year [START_REF] Finazzi | The treatment of polycythaemia vera: an update in the JAK2 era[END_REF]. Thrombotic complications are the main cause of morbidity and mortality, occurring in more than one third of patients and causing 35-45% of deaths [START_REF] Finazzi | The treatment of polycythaemia vera: an update in the JAK2 era[END_REF][START_REF]Polycythemia vera: the natural history of 1213 patients followed for 20 years[END_REF]. Other possible adverse events are the evolution into secondary myelofibrosis (MF) or acute myeloid leukemia (AML) occurring in 10-15% [START_REF] Kiladjian | Long-term outcomes of polycythemia vera patients treated with pipobroman as initial therapy[END_REF][START_REF]Polycythemia vera: the natural history of 1213 patients followed for 20 years[END_REF][START_REF] Ellis | Studies of the bone marrow in polycythemia vera and the evolution of myelofibrosis and second hematologic malignancies[END_REF][START_REF] Silverstein | The evolution into and the treatment of late stage polycythemia vera[END_REF]] and 2-15% [START_REF] Kiladjian | Long-term outcomes of polycythemia vera patients treated with pipobroman as initial therapy[END_REF][START_REF] Silverstein | The evolution into and the treatment of late stage polycythemia vera[END_REF][START_REF] Finazzi | ECLAP Investigators (2005a) Acute leukemia in polycythemia vera: an analysis of 1638 patients enrolled in a prospective observational study[END_REF][START_REF] Berk | Increased incidence of acute leukemia in polycythemia vera associated with chlorambucil therapy[END_REF][START_REF] Of Cancer | Treatment of polycythaemia vera by radiophosphorus or busulphan: a randomized trial. "Leukemia and Hematosarcoma[END_REF][START_REF] Messinezy | Treatment of primary proliferative polycythaemia by venesection and low dose busulphan: retrospective study from one centre[END_REF][START_REF] Najean | Treatment of polycythemia vera: use of 32 P alone or in combination with maintenance therapy using hydroxyurea in 461 patients greater than 65 years of age[END_REF][START_REF] Najean | Treatment of polycythemia vera: the use of hydroxyurea and pipobroman in 292 patients under the age of 65 years[END_REF][START_REF] Najean | A very long-term evolution of polycythemia vera: an analysis of 318 patients initially treated by phlebotomy or 32 P between 1969 and 1981[END_REF][START_REF] Fruchtman | From efficacy to safety: a Polycythemia Vera Study Group report on hydroxyurea in patients with polycythemia vera[END_REF] of patients, respectively, depending on treatments and with increased incidence with long-standing disease.

Diagnostic criteria have been revised many times since their first publication by the Polycythemia Vera Study Group in 1975 [START_REF] Wasserman | Polycythemia Vera Study Group: a historical perspective[END_REF][START_REF] Mcmullin | General Haematology Task Force of the British Committee for Standards in Haematology (2005) Guidelines for the diagnosis, investigation and management of polycythaemia /erythrocytosis[END_REF]. Recently, the World Health Organization (WHO) proposed new criteria, in consideration of the discovery of JAK2 mutation role in PV [START_REF] Tefferi | Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel[END_REF][START_REF] Spivak | The revised World Health Organization diagnostic criteria for polycythemia vera, essential thrombocytosis, and primary myelofibrosis: an alternative proposal[END_REF]. However, before the discovery of JAK2 mutations [START_REF] Levine | Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis[END_REF][START_REF] Baxter | Cancer Genome Project (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders[END_REF][START_REF] Kralovics | A gain-of-function mutation of JAK2 in myeloproliferative disorders[END_REF][START_REF] Scott | Prevalence of JAK2 V617F and exon 12 mutations in polycythaemia vera[END_REF][START_REF] Skoda | The genetic basis of myeloproliferative disorders[END_REF], differential diagnosis between PV and secondary erythrocytosis was not always immediate and it is unclear how previous WHO diagnostic recommendations have been actually followed in general practice, outside of clinical trials.

Current therapy guidelines recommend phlebotomy as firstline treatment, in order to maintain the hematocrit (Hct) value below 0.45 and prevent cardiovascular accidents [START_REF] Mcmullin | General Haematology Task Force of the British Committee for Standards in Haematology (2005) Guidelines for the diagnosis, investigation and management of polycythaemia /erythrocytosis[END_REF]. Lowdose acetyl-salicylic acid (ASA) has been recommended too, unless contraindicated, in order to reduce thrombotic risk and mortality; conversely cytoreductive therapy is recommended only in the presence of poor tolerance to phlebotomy, symptomatic or progressive splenomegaly or evidence of high thrombotic risk or disease progression [START_REF] Mcmullin | General Haematology Task Force of the British Committee for Standards in Haematology (2005) Guidelines for the diagnosis, investigation and management of polycythaemia /erythrocytosis[END_REF][START_REF] Mcmullin | A review of the therapeutic agents used in the management of polycythaemia vera[END_REF][START_REF] Landolfi | European Collaboration on Low-Dose Aspirin in Polycythemia Vera Investigators (2004) Efficacy and safety of low-dose aspirin in polycythemia vera[END_REF][START_REF] Finazzi | Risk-adapted therapy in essential thrombocythemia and polycythemia vera[END_REF][START_REF] Finazzi | Evidence and expertise in the management of polycythemia vera and essential thrombocythemia[END_REF].

In spite of well-established therapeutic guidelines, some doubts still remain about the optimal management of PV patients. Indeed, a recent European survey on a very large casistics (ECLAP study) questioned the actual role of a low (<0.45) Hct value in reducing the thrombotic risk [START_REF] Finazzi | Evidence and expertise in the management of polycythemia vera and essential thrombocythemia[END_REF][START_REF] Nisio | European Collaboration on Lowdose Aspirin in Polycythemia Vera (ECLAP) Investigators (2007) The haematocrit and platelet target in polycythemia vera[END_REF]. Moreover, the same study evidenced that even in the setting of a clinical study, about 60% of patients did not reach or maintain the recommended Hct value [START_REF] Nisio | European Collaboration on Lowdose Aspirin in Polycythemia Vera (ECLAP) Investigators (2007) The haematocrit and platelet target in polycythemia vera[END_REF]. Therefore, it is unclear how the strict requirement of intensive Hct reduction has been actually satisfied outside of clinical trials. For chemotherapy-requiring patients, hydroxyurea (HU) is generally recommended as first-line agent because of its unproven leukemogenicity [START_REF] Finazzi | Risk-adapted therapy in essential thrombocythemia and polycythemia vera[END_REF]. However some alkylating agents like pipobroman and busulfan are still used, particularly in elderly patients [START_REF] Mcmullin | A review of the therapeutic agents used in the management of polycythaemia vera[END_REF].

In this study, we performed a retrospective analysis on 300 patients with a PV diagnosis, referred to a single blood bank for phlebotomy. The primary objectives of the study were: [START_REF] Silverstein | Polycythemia vera, 1935-1969: an epidemiologic survey in Rochester, Minnesota[END_REF] to evaluate in our casistics the controversial role of Hct value in survival and thrombosis prevention; (2) to verify whether the strategy for near all patients of anti-thrombotic prophylaxis and avoidance of alkylating chemotherapy might have improved survival and reduced AML and major thrombosis incidence, compared to literature data. As secondary objectives, we verified in our patients the actual adherence to published diagnostic and therapeutic guidelines in routine management of PV and the prognostic role of some known risk factors.

Methods

The present analysis is a retrospective study based on the database of polycythemic patients of S. Giovanni Battista Hospital transfusion center in Turin, Italy. Patients were followed in three different hematological institutions but all of them referred to this single transfusion center for phlebotomy. Patients' data were cumulatively evaluated anonymously, in agreement to local ethical rules. Only patients with a diagnosis of PV made from January 1, 1995 to March 31, 2008 were included. Each diagnosis was revised to include in the study only the cases that fully satisfied either 2001 or 2007 WHO diagnostic criteria for PV. Data files were reviewed before inclusion in the present analysis and the times of observation were censored on December 31, 2008. Primary end-points were overall survival (OS), defined as the time from diagnosis to death for any cause or last follow-up alive, time to occurrence of total thrombosis (TTT) and time to occurrence of major thrombosis (TMT). Major thrombotic events comprehended ischemic stroke, acute myocardial infarction, deep venous thrombosis and pulmonary embolism. Total thrombosis included all major events plus transient ischemic attacks, peripheral arterial thrombosis and superficial thrombophlebitis. Secondary end-points included the incidence and time to development of AML, solid tumors or secondary MF.

For each patient, the following parameters at diagnosis were evaluated: age; presence of cardiovascular risk factors such us hypertension, diabetes, cigarette smoking, dyslipidemia; history of thrombosis or bleeding. These parameters were used to define the thrombotic risk according to the recent definition by Finazzi and Barbui based on ECLAP data [START_REF] Finazzi | Risk-adapted therapy in essential thrombocythemia and polycythemia vera[END_REF][START_REF] Finazzi | Evidence and expertise in the management of polycythemia vera and essential thrombocythemia[END_REF] During the follow-up, data regarding thrombotic events, bleeding, cytoreductive treatments, anti-platelet or anticoagulant therapy and laboratory values were recorded. Median values of Hct, platelets and leucocytes during the observational time (with the exclusion of blood counts performed after occurrence of MF, AML, or other neoplasms) were used to estimate their impact on survival, TTT, TMT, and time to hematological transformation: these data were evaluated on 211 patients (93% of total) who had at least three complete blood counts performed per year (average 5/year). The prognostic role of Hct value was analyzed with two cutoffs: 0.45, corresponding to WHO recommendation, and 0.48, a value in the upper normal range. Higher cutoff values (0.49 or more) were not considered, due to the very low number of patients (<10%) maintained at so high Hct values. Cutoff values a Of tested cases b According to Finazzi and Barbui (32,33) c Percent of chemotherapy-treated patients; some patients received more than one treatment for platelet counts were 400×10 9 /l, corresponding to the normal upper range, and 600×10 9 /l, the thrombocytosis threshold previously used by WHO guidelines for the diagnosis of essential thrombocythemia [START_REF] Vardiman | WHO histological classification of chronic myeloproliferative diseases[END_REF]. A leukocyte cutoff value of 15×10 9 /l was chosen, since it has been previously reported to be prognostically relevant in terms of thrombosis-free survival [START_REF] Kiladjian | Long-Term outcome in Polycythemia Vera (PV): final analysis of a randomized trial comparing hydroxyurea (HU) to pipobroman (Pi)[END_REF].

Statistical methods

Patients' characteristics were compared using the Pearson Overall survival: all patients. b Overall survival for median hematocrit, under 0.45 vs. 0.45-0.48 vs. over 0.48 (p=0.001). c Overall survival for thrombotic risk according to Finazzi and Barbui [START_REF] Finazzi | Risk-adapted therapy in essential thrombocythemia and polycythemia vera[END_REF][START_REF] Finazzi | Evidence and expertise in the management of polycythemia vera and essential thrombocythemia[END_REF], high vs. low-intermediate (p=0.038) 0.76-13.78) and only three patients were lost to follow-up. Known risk factors at diagnosis comprehended history of previous thrombosis in 55 (24%; arterial thrombosis constituting 85% and venous thrombosis 15% of these thrombotic events) and concomitant cardiovascular risk factors in 137 (61%) patients. According to the thrombotic risk stratification, 39 patients were in the low-risk group, 29 in the intermediate one and 158 (70%) in the high-risk group.

χ
All patients received phlebotomy at least in the first month from diagnosis. Seventy-five patients proceeded with phlebotomy only, whereas 151 also received a cytoreductive treatment for at least 6 months (median 4 years). The main cytoreductive treatment was hydroxyurea, used as first-line therapy by 147 (97%) chemotherapy-treated patients. In particular, HU was the only cytoreductive agent for 126 patients (83%), whereas other patients received more than one cytoreductive treatment. Other drugs included pipobroman (19 patients), busulfan (five patients), and alpha interferon (two patients): they were used in the case of intolerance or suboptimal response to HU. Almost all patients (98.2%) received either anti-platelet (209) and/or anticoagulant (34) therapy.

Median Hct during follow-up was kept at the recommended value below 0.45 in 46/211 evaluable patients (22%). Sixty-two percent of patients maintained a median Hct value between 0.45 and 0.48, whereas in 16% the median Hct value was >0.48. Regarding WBC count, a median value above 15×10 9 /l was observed in 23 patients (11%). A median platelet value under 400×10 9 /l was maintained in 110 patients (52%), whereas values between 400×10 9 /l and 600×10 9 /l, or above 600×10 9 /l were observed in 74 patients (35%) and 27 patients (13%), respectively.

During follow-up, 25 patients (11%) died. Survival rate was 91.3% at the median follow-up of 5.8 years and is projected to 77% at the longest follow-up of 13.8 years (Fig. 1a). Cardiovascular and thrombotic mortality accounted for 32% of all deaths, while hematological transformation and solid tumors accounted for 24% of deaths each. Overall survival was negatively influenced by median Hct over 0.48 (p=0.001; Table 2 and Fig. 1b) and "high risk" score according to thrombotic risk stratification (p=0.038) (Table 2 and Fig. 1c), whereas sex had no prognostic value (Table 2). At multivariate OS analysis, the impact of median Hct over 0.48 and of high thrombotic risk on total mortality was confirmed, with a hazard ratio (HR) of 3.41 (p=0.004) and 1.56 (p=0.050), respectively (Table 3). However, due to the low number of dead patients no statistically significant difference was found in the causes of death according to median Ht value.

Thrombotic events occurred in 58 patients (25.7%) during follow-up, after a median time of 58 months (4.8 years). Nineteen percent of patients had a major thrombotic event. The main prognostic factors affecting time to thrombotic events were age over 65 (TTT p<0.001, TMT p=0.001), Hct over 0.48 (TTT p=0.020, TMT p= 0.002), and high thrombotic risk (TTT p=0.028; Table 4, Fig. 2). On the contrary, cytoreductive treatment resulted in a protective agent (TTT p<0.001, TMT p<0.001) (Table 4, Fig. 2). At multivariate analysis, major thrombosis were significantly associated with Hct over 0.48 (p=0.011) and inversely related to cytoreductive treatment (p<0.001; Table 3). Lower Hct values (≤0.45) did not significantly discriminate patients in terms of survival and time to thrombosis.

Follow-up WBC and PLT counts did not affect either overall or thrombosis-free survival.

Seventeen and five episodes of major and minor bleeding were observed, respectively. Sixteen events occurred during anti-platelet therapy but only four caused its Leukemic evolution occurred in six patients (2.7%), four of whom had received chemotherapy, after a median time of 7 years, with a cumulative incidence of 4.5% at 10 years. Secondary MF and neoplasia other than AML were observed in 11 (4.9%) and 15 (6.6%) patients, respectively. Evolution to MF occurred after a median time of 7 years and had a cumulative incidence of 7.95% at 10 years. Chemotherapy did not significantly affect either the hematological transformation or the occurrence of total malignancies (Table 5). Evolution in MF, at univariate analysis, was associated with high Hct levels (over 0.48; p=0.007; Table 5).

Discussion

The present study was aimed to evaluate the clinical management and outcome of PV patients in the past 14 years in our city, outside of clinical trials. In particular, we wished to ascertain, although retrospectively, a possible positive role on survival of the choice to give antithrombotic prophylaxis and to avoid alkylating drugs in 98% and 89% of patients, respectively. We also tried to ascertain the clinical impact of median Hct value during the follow-up. Considering the reported PV incidence [START_REF] Silverstein | Polycythemia vera, 1935-1969: an epidemiologic survey in Rochester, Minnesota[END_REF][START_REF] Ania | Trends in the incidence of polycythemia vera among Olmested Country, Minnesota residents, 1935-1989[END_REF], our casistics probably includes more than 50% of PV cases occurred during those years in the city of Turin. Follow-up was fairly accurate, as three patients only were lost and, moreover, all evaluated patients had at least three complete blood counts performed/year. About 25% of cases referred to our transfusion center for phlebotomy had a PV diagnosis that did not satisfy all WHO diagnostic criteria. Although in many of these cases the diagnosis might have been correct, according to their further clinical outcome, they have been excluded from further evaluations, wishing to restrict prognostic considerations to patients with a doubtless PV diagnosis. In all cases, the unconfirmed diagnosis was made before the analysis of JAK2 status entered the clinical practice. Conversely, 94% of patients with a PV diagnosis tested in the past 3 years for JAK2 status evidenced the V617F mutation, in agreement to the more recent literature data [START_REF] Skoda | The genetic basis of myeloproliferative disorders[END_REF]. These observations illustrate how PV diagnosis might be difficult before the advent of the molecular test and confirm the great impact of JAK2 analysis in improving the diagnostic accuracy.

The median age at diagnosis of our patients (66 years) was relatively advanced, slightly higher than the one previously reported for a large population of unselected Italian PV patients [START_REF]Polycythemia vera: the natural history of 1213 patients followed for 20 years[END_REF]. This certainly contributed to the high proportion (70%) of patients in the group of high thrombotic risk. In spite of these unfavorable prognostic features, the overall survival (91% at the median follow-up time of 5.84 years) compares favorably to that reported in many other studies [START_REF] Cervantes | Life expectancy and prognostic factors in the classic BCR/ABL-negative myeloproliferative disorders[END_REF][START_REF] Kiladjian | Long-term outcomes of polycythemia vera patients treated with pipobroman as initial therapy[END_REF][START_REF] Finazzi | The treatment of polycythaemia vera: an update in the JAK2 era[END_REF][START_REF]Polycythemia vera: the natural history of 1213 patients followed for 20 years[END_REF]. Although we did not perform a prospective, randomized study and the median follow-up is shorter than 10 years, the wide use in our patients of lowdose ASA and the strong limitation of alkylating cytotoxic therapy may have contributed to this more favorable outcome and to an incidence of leukemic transformation in the lowest range of values reported in literature [START_REF] Kiladjian | Long-term outcomes of polycythemia vera patients treated with pipobroman as initial therapy[END_REF][START_REF]Polycythemia vera: the natural history of 1213 patients followed for 20 years[END_REF][START_REF] Ellis | Studies of the bone marrow in polycythemia vera and the evolution of myelofibrosis and second hematologic malignancies[END_REF][START_REF] Silverstein | The evolution into and the treatment of late stage polycythemia vera[END_REF]. Indeed, a French randomized study with very long followup has recently demonstrated a lower incidence of leukemic evolution and improved survival with the use of hydroxyurea compared to pipobroman [START_REF] Kiladjian | Long-Term outcome in Polycythemia Vera (PV): final analysis of a randomized trial comparing hydroxyurea (HU) to pipobroman (Pi)[END_REF].

Low-dose ASA might have also contributed to the relatively low incidence of major thrombosis (18%), in the lower range of literature data [START_REF]Polycythemia vera: the natural history of 1213 patients followed for 20 years[END_REF][START_REF] Landolfi | European Collaboration on Low-Dose Aspirin in Polycythemia Vera Investigators (2004) Efficacy and safety of low-dose aspirin in polycythemia vera[END_REF], without increasing hemorrhagic complications compared to published observations [START_REF] Marchioli | Vascular and neoplastic risk in a large cohort of patients with polycythemia vera[END_REF]. Nevertheless, the high thrombotic risk according to Finazzi et al. [START_REF] Finazzi | Risk-adapted therapy in essential thrombocythemia and polycythemia vera[END_REF][START_REF] Finazzi | Evidence and expertise in the management of polycythemia vera and essential thrombocythemia[END_REF] confirmed its negative prognostic impact in terms of overall and thrombosis-free survival in both univariate and multivariate analysis.

Differently from what is reported in another study [START_REF] Landolfi | European Collaboration on Low-Dose Aspirin in Polycythemia Vera (ECLAP) (2007) Leukocytosis as a major thrombotic risk factor in patients with polycythemia vera[END_REF], WBC value during follow-up did not significantly affect survival and thrombotic complications. However, few patients (22:10.2%) maintained a value above the reported threshold of 15 ×10 9 /l: this may explain the lack of significant difference in thrombosis-free survival. Chemotherapy given before the occurrence of a thrombotic event significantly prolonged the freedom from major thrombosis but did not affect overall survival, in agreement to some previous reports [START_REF] Najean | Treatment of polycythemia vera: the use of hydroxyurea and pipobroman in 292 patients under the age of 65 years[END_REF][START_REF] Fruchtman | From efficacy to safety: a Polycythemia Vera Study Group report on hydroxyurea in patients with polycythemia vera[END_REF][START_REF] Finazzi | Risk-adapted therapy in essential thrombocythemia and polycythemia vera[END_REF].

Regarding Hct target, the need of keeping a value<0.45, as recommended by guidelines [START_REF] Mcmullin | General Haematology Task Force of the British Committee for Standards in Haematology (2005) Guidelines for the diagnosis, investigation and management of polycythaemia /erythrocytosis[END_REF][START_REF] Finazzi | Evidence and expertise in the management of polycythemia vera and essential thrombocythemia[END_REF][START_REF] Vannucchi | Advances in understanding and management of myeloproliferative neoplasms[END_REF] has been recently questioned by the ECLAP study [START_REF] Nisio | European Collaboration on Lowdose Aspirin in Polycythemia Vera (ECLAP) Investigators (2007) The haematocrit and platelet target in polycythemia vera[END_REF]. Unfortu- nately, few patients (22% only) in our survey were actually maintained at an Hct value<0.45, this suggesting the difficulties for patients and/or physicians to comply with published guidelines, in agreement to other reports [START_REF] Nisio | European Collaboration on Lowdose Aspirin in Polycythemia Vera (ECLAP) Investigators (2007) The haematocrit and platelet target in polycythemia vera[END_REF][START_REF] Streiff | The diagnosis and management of polycythemia vera in the era since the Polycythemia Vera Study Group: a survey of American Society of Hematology members' practice patterns[END_REF]. Although this group of patients had a favorable outcome, no significant differences could be detected in terms of overall or thrombosis-free survival compared to the larger group of patients who maintained median Hct values in the range of 0.45 -0.48. Conversely, the few patients (16%) with a poor control of erythrocytosis (median Hct value> 0.48) had a significantly worst outcome in terms of both overall survival and time to major thrombosis, both by univariate and multivariate analysis.

In conclusion, although our study is retrospective and the casistic is not very large, the results obtained on unselected PV patients receiving routine care are in agreement to the already reported positive roles of antiplatelet therapy and of hydroxyurea as a cytoreductive drug. The role of JAK2 status analysis in improving the diagnostic accuracy has been also confirmed, as well as the negative impact of belonging to the high thrombotic risk category. Other known risk factors, such as leukocytosis and prior thrombosis could not be confirmed in our casistic, probably because of the relatively limited number of patients. Regarding one of the most controversial point in the last years, the Hct value to be pursued, we believe on the basis of our results that a good control of erythrocytosis, with an Hct value below 0.48 may be advisable, although definitive conclusions on the optimal value have not been reached. A randomized study is ongoing in Italy comparing different Hct targets and hopefully should provide more sound information about the optimal value to be achieved.

Fig. 1

 1 Fig. 1 Overall survival of 226 patients with polycythemia vera. a

Fig. 2

 2 Fig. 2 Major thrombosis-free survival of 226 patients with polycythemia vera. a Major thrombosis-free survival for median hematocrit under 0.45, between 0.45 and 0.48 and over 0.48 (p=0.002 ). b Major thrombosis-free survival for cytoreductive treatment, yes vs. no (HR 0.31, p<0.001)

Table 1

 1 Patients' general features are illustrated. The 3 groups of patients divided according to median Hct values include 211 patients only, who had at least 3 blood count/year of follow-up

	Main clinical features of 226 patients with PV
			All the patients	Median HCT<0.45	Median HCT 0.45-0.48	Median HCT >0.48
			No	%	No	%	No	%	No	%
	No		226		46	22	131	62	34	16
	Males		124	54.9	10	21.7	79	60.3	26	76.4
	Females		102	45.1	36	78.3	52	39.7	8	23.6
	Age at diagnosis-years (range)
	Median	66 (18-92)	67 (25-87)		64 (18-83)		69 (31-87)
	over 65		117	51.8	28	60.9	61	46.6	20	58.8
	under 65		109	48.2	18	39.1	70	53.4	14	41.2
	JAK2 V216F mutated
	Positive		102	93.6 a	24	96.0 a	63	92.6 a	10	90.9 a
	Negative		7	6.4 a	1	4.0 a	5	7.4 a	1	9.1 a
	not determined		117	51.8	21	45.7	63	48.1	23	67.6
	Thrombotic risk b
	Low		39	17.3	13	28.3	18	13.7	7	20.6
	Intermediate		29	12.8	1	2.2	24	18.3	2	5.9
	High		158	69.9	32	69.6	89	67.9	25	73.5
	Cardivascular risk factors		137	60.6	25	54.3	84	64.1	18	52.9
	Prior thrombosis		55	24.3	10	21.7	33	25.2	11	32.4
	Prior major thrombosis		34	15.0	7	15.2	21	16.0	9	26.5
	Median follow-up-years (range)	5.83 (0.76-13.78)	5.24		6.60		5.58
	Cytoreductive treatment		151	66.8	31	67.4	85	64.9	20	58.8
	Hydroxyurea		147	97.4 c	29	93.6 c	85	100 c	18	90.0 c
	Alkylants		24	15.9 c	5	16.1 c	11	12.9 c	4	20.0 c
	Alpha interferon		2	1.3 c	0	0.0 c	2	2.36 c	0	0.0 c
	Anti-platelet drugs		209	87.2	43	93.5	123	93.9	30	88.2
	Anticoagulants drugs		34	13.3	6	13.0	20	15.3	5	14.7
	Death causes
	AML		6	24.0	2	100	3	23.1	1	11.1
	Thrombotic and CV event		8	32.0	0	0.0	3	23.1	4	44.4
	Tumors (except AML)		6	24.0	0	0.0	4	30.8	2	22.2
	Others		4	16.0	0	0.0	3	23.1	1	11.1
	Unknown		1	4.0	0	0.0	0	0.0	1	11.1
	Events rate (Patients/events)
	Rate of death	25/226 (11.1%)	2/46 (4.3%)		13/131 (9.9%)		9/34 (26.5%)
	Rate total thrombosis	58/226 (25.7%)	12/46 (26.0%)		30/131 (22.9%)		12/34 (35.3%)
	Rate of major thrombosis	43/226 (19.0%)	8/46 (17.4%)		21/131 (16.0%)		11/34 (32.4%)
	Rate of tumors (AML excluded) 15/226 (6.6%)	1/46 (2.2%)		10/131 (7.6%)		3/34 (8.8%)
	Rate of AML		6/226 (2.7%)	1/46 (2.2%)		4/131 (3.0%)		1/34 (2.9%)
	Rate of MF		11/226 (4.9%)	2/46 (4.3%)		5/131 (3.8%)		4/34 (11.8%)

Table 4

 4 Prognostic factors for time to occurrence of thrombotic events by univariate Cox model

	Time to total thrombosis		Time to major thrombosis
	HR	95%CI	p value	HR	95%CI	p value

a Time-dependent covariate

Table 3

 3 Prognostic factors for overall survival and time to occurrence of thrombotic events by multivariate Cox model Multivariate analysis tested the following variables that have been demonstrated significative in univariate analysis: Hct value and thrombotic risk (high vs. medium/low) for overall survival; Hct value, thrombotic risk and cytoreductive treatment for time to total thrombosis; Hct value and cytoreductive treatment for time to major thrombosis. Values in italics indicate significant variables

	HR	95%CI	p value

a Time-dependent covariate

Table 5

 5 Prognostic factors for occurrence of acute myeloid leukemia. Solid tumors or myelofibrosis by univariate Cox model

	Time to AML or solid tumors		Time to myelofibrosis
	HR	95%CI	p value	HR	95%CI	p value

a Time-dependent covariate

Acknowledgment This study was in part supported by the Università degli Studi di Torino (ex 60% grants)