Approach to patients with essential thrombocythaemia and very high platelet counts: what is the evidence for treatment?

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1 review Approach to patients with essential thrombocythaemia and very high platelet counts: what is the evidence for treatment? Lorenzo Falchi, 1 Prithviraj Bose, 2 Kate J. Newberry 2 and Srdan Verstovsek 2 1 Division of Hematology/Oncology, Columbia University Medical Center, New York, NY, and 2 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston,TX, USA Summary Treatment of essential thrombocythaemia (ET) is directed at decreasing the risk of complications of the disease, including arterial and venous thrombosis and bleeding episodes. Established risk factors for vascular events in patients with ET include advanced age (>60 years) and prior history of thrombosis or haemorrhage. The role, if any, of other potential risk factors, including cardiovascular risk factors, leucocytosis, high haematocrit, and JAK2 V617F has been analysed in multiple studies. The impact of thrombocytosis on the risk of vascular events has also been investigated. Many clinicians consider an elevated platelet count to be a risk factor for thrombosis or, when extreme, bleeding and utilize this as a criterion to start cytoreductive therapy. However, the relationship between thrombocytosis and vascular events is controversial and solid evidence to support the use of cytoreductive therapy in ET patients who have no other risk factors is lacking. In this review, we discuss current treatment recommendations for patients with ET, the biology underlying vascular events and risk factors thereof. We then review the evidence on the management of patients with ET and extreme thrombocytosis. Keywords: essential thrombocythaemia, platelet, thrombosis, bleeding, hydroxycarbamide. Essential thrombocythaemia (ET) is a myeloproliferative neoplasm (MPN) characterized by clonal proliferation of the megakaryocytic lineage within the bone marrow and, phenotypically, by an elevated platelet count in peripheral blood. According to the 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukaemia (Arber et al, 2016), diagnostic criteria for ET include (i) an elevated platelet count (i.e., /l); (ii) bone marrow biopsy showing proliferation, mainly of the Correspondence: Srdan Verstovsek, Leukemia Department, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 428, Houston, TX 77030, USA. sverstov@mdanderson.org megakaryocyte lineage, without any significant increase or left shift in neutrophil granulopoiesis or erythropoiesis and very rarely minor (grade 1) increase in reticulin fibres; (iii) failure to meet diagnostic criteria for other MPNs, myelodysplastic syndromes (MDS) or other myeloid neoplasms; (iv) demonstration of clonal markers, such as the Janus kinase (JAK)2 V617F (hereafter referred to as JAK2), calreticulin (CALR) or myeloproliferative leukaemia virus oncogene (MPL) mutations. The diagnosis of ET requires meeting all four of the major criteria above or the first three major criteria and a minor criterion: presence of a clonal marker or absence of evidence for reactive thrombocytosis. The incidence rate for ET is 02 25/ people/year based on registry data from the United States and European Union (Moulard et al, 2014; Titmarsh et al, 2014). The prevalence is higher, at 38 57/ people (Stein et al, 2015), because of the long life expectancy (in excess of 20 years) of patients with ET (Wolanskyj et al, 2006). In the absence of disease-eradicating therapies, the indication for, and goals of therapy are to reduce the risk of life-threatening complications, such as venous or arterial thrombosis or severe bleeding, rather than induce remission or disappearance of the neoplastic clone. Treatment may also help control vasomotor symptoms, such as headaches, acroparesthesias, erythromelalgia, peripheral ischaemia, transient ischaemic attacks, amaurosis fugax or scotomas (Michiels et al, 2006a). In patients with ET, the overall risk of thrombosis is 1 3% per patient-year [77% in high-risk, JAK2-mutated patients (Vannucchi et al, 2007)]. The rates of non-fatal arterial events and venous thrombosis were 12% patients-years and 06% patients-years, respectively (Passamonti et al, 2004; Casini et al, 2013). On the other hand, the reported incidence of bleeding episodes is less consistent across studies. In an observational study, the rate of major bleeding events was 079% per year (Finazzi et al, 2012). In other reports, the overall risk was estimated to be in the 5 30% range (Casini et al, 2013). Much work has been devoted to identifying ET patients at higher risk of developing thrombotic events. As a result, the current risk stratification of ET patients is based on two factors: age equal to or greater than 60 years and prior history of thrombosis. While low-dose aspirin (ASA) is indicated in First published online 16 December 2016 doi: /bjh ª 2016 John Wiley & Sons Ltd

2 all patients with ET, the presence of either one constitutes an indication for the addition of cytoreductive therapy, regardless of the presence of other potential risk factors. Hydroxycarbamide (HC, also termed hydroxyurea, HU) is considered the first-line cytoreductive therapy and it is usually titrated to maintain platelet counts below /l (Barbui et al, 2011). A major advantage of this risk classification is that it is very easy to implement in clinical practice. Risk factors for bleeding have also been identified but these have not been solid enough to warrant systematic pre-treatment risk stratification and clear treatment recommendations. Over the years, a number of additional risk factors for both thrombosis and haemorrhage in patients with ET have been proposed and tested, but their ultimate prognostic significance has remained elusive (Carobbio et al, 2011). In particular, extreme thrombocytosis has been invoked as a risk factor for both thrombosis and bleeding (Alvarez-Larran et al, 2010; Palandri et al, 2012). Although frequently used, the term extreme thrombocytosis may be misleading, and its definition has varied in the literature. Many authors have used a platelet count of over /l as the threshold to ascertain the risk of vascular events associated with very high platelet counts. Intuitively, a higher number of platelets may result in increased risk of thrombosis. On the other hand, platelet counts greater than /l have been associated with acquired von Willebrand syndrome (AVWS), which can increase the risk of haemorrhagic complications (Elliott & Tefferi, 2005). For this reason, ET patients with extreme thrombocytosis often represent a clinical dilemma. Despite the scarcity of available evidence, there is a tendency to utilize thrombocytosis as a criterion to initiate patients on cytoreductive therapy. However, several questions remain unanswered. For example, should cytoreduction with HC be offered to otherwise low-risk patients with very high platelet counts as a thrombotic/haemorrhagic risk-reducing therapy? Should ASA be withheld in the presence of extreme thrombocytosis due to the risk of haemorrhage? In this review, we provide an overview of the current treatment recommendations for patients with ET, as well as the biological basis for thrombotic and haemorrhagic complications and associated risk factors. We then review available evidence on the management of patients with ET and extreme thrombocytosis. Current treatment recommendations Table I summarizes pertinent recommendations from various scientific societies regarding risk assessment and indications for antiplatelet and cytoreductive treatment of patients with ET. Of note, specific recommendations regarding cytoreductive therapy for reducing risk of bleeding are either lacking or based on limited, poor-quality evidence. The European LeukaemiaNet (ELN) largely based their recommendations on the report by Cortelazzo et al (1995), which showed a decreased risk of thrombotic events in patients treated with HC compared with those who received only antiplatelet therapy (ASA or ticlopidine). Their recommendations also took into account the observation that the risk of thrombotic events in patients with low-risk disease parallels that of the general population (Cortelazzo et al, 1990; Ruggeri et al, 1998). Therefore, the panel recommended that ASA be given to all ET patients (but withdrawn in case of major bleeding) and HC to high-risk patients of any age. In addition, the panel recommended considering treatment with platelet-lowering agents for platelet counts /l (Barbui et al, 2011). The latter recommendation and the specific choice of a platelet count threshold of /l were largely based on pathophysiologic considerations (van Genderen et al, 1997a). While the recognized goal of therapy is to decrease the risk of vascular events, the endpoint of treatment in clinical studies is the achievement of a so-called clinicohaematological response. However, the correlation between the former and the latter has remained unproven (Barosi et al, 2012). Similarly, no clinical evidence was provided for defining the criteria for treatment failure proposed by the panel (Barbui et al, 2011). To address some of these gaps, the ELN Consortium and the International Working Group for Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) jointly revised the criteria for complete response to include absence of any haemorrhagic or thrombotic events, among others (Barosi et al, 2013). Recommendations from the British Committee for Standards in Haematology (Harrison et al, 2010) are similar to those made by the ELN in terms of risk stratification and treatment indications. In particular, a platelet count > /l was considered an indication for cytoreductive therapy to reduce the haemorrhagic risk based on level III, grade B clinical evidence (i.e., derived from non-controlled studies or case series). The panel also stated that it may be reasonable to increase this threshold in younger (< 40 years), asymptomatic individuals. Caution (but not withdrawal) is recommended in using ASA in patients with history of bleeding or platelet count in excess of /l (Harrison et al, 2010). In a joint statement, the German and Austrian Haematology and Oncology Societies made specific recommendations for the prophylaxis and treatment of venous thromboembolism in patients with MPNs. One notable and unique recommendation is to perform a functional characterization of von Willebrand factor (VWF), including Ristocetin Cofactor activity (VWF:RCo), VWF activity and VWF collagen binding activity (VWF:CB), etc., in patients with more than /l platelets in order to assess the extent of secondary VWF deficiency. ASA should be avoided if platelet count is > /l or if the VWF:RCo is 30% (Kreher et al, 2014). Of note, the ability of VWF testing to reliably predict the risk of bleeding is limited. Interestingly, AVWS was found to occur at near-normal platelet counts in a patient with polycythaemia vera (Tefferi et al, 2010). ª 2016 John Wiley & Sons Ltd 353

3 Table I. Key recommendations from selected scientific Societies on the management of essential thrombocythaemia. Scientific society (References) Indication to start treatment Recommended treatment Goals of therapy (Response criteria) European Leukaemia Network (Barbui et al, 2011; Barosi et al, 2013) History of thrombosis or haemorrhage Age >60 years Platelet count > /l Low-dose aspirin unless contraindicated HC Anagrelide or IFNa in patients intolerant of, or resistant to HC Platelet-lowering agents in patients with platelet count > /l Platelet count /l No disease-related symptoms Normal spleen size on imaging WBC count /l Absence of haemorrhagic or thrombotic events Disappearance of megakaryocyte hyperplasia and reticulin fibrosis grade 1 British Committee for Standards in Haematology (Harrison et al, 2010) History of thrombosis or haemorrhage Age >60 years Platelet count > /l Low-dose aspirin unless contraindicated HC IFN-a in patients < 40 years Anagrelide as second-line therapy Platelet count < /l Absence of haemorrhagic or thrombotic events German and Austrian Haematology and Oncology Society (Kreher et al, 2014) History of thrombosis or haemorrhage Age >60 years Platelet count > /l Low-dose aspirin unless platelet counts > /l or VWF:RCo 30% HC, anagrelide or IFN-a Platelet count < /l Absence of haemorrhagic or thrombotic events HC, hydroxycarbamide; IFN-a, interferon-alpha; VWF:RCo, ristocetin co-factor activity; WBC, white blood cell. 354 ª 2016 John Wiley & Sons Ltd

4 The European Society of Medical Oncology (ESMO) also expressed concern regarding bleeding in ET patients with extreme thrombocytosis (> /l). The panel recommends ASA for all low-risk ET patients (although the benefit may be restricted to those with cardiovascular risk factors or JAK2) and weighing the benefits of anti-platelet agents against the risk of haemorrhage in the presence of platelet counts > /l, due to the occurrence of AVWS (Vannucchi et al, 2015). Biology of thrombosis and bleeding in ET The genesis of thrombosis in MPN is multifactorial and encompasses patient-related characteristics, such as age, history of thrombosis and common cardiovascular risk factors, as well as disease-related parameters, such as leucocytosis, thrombocytosis and splenomegaly. Moreover, prothrombotic changes are demonstrated in both blood and endothelial cells of patients with MPN. Platelets are found in an activated status in patients with MPN (Falanga et al, 2005a, 2007; Arellano-Rodrigo et al, 2006). Moreover, increased levels of ß-thromboglobulin and platelet factor 4 were found in the plasma and of the thromboxane A2 metabolites, 11-dehydro- TxB2 and 2,3-dinor-TxB2, in the urine of MPN patients (Jensen et al, 2000). In a vicious cycle, activated platelets enhance the generation of thrombin (also increased in MPN patients), which in turn amplifies their own activation. This phenomenon is more pronounced in patients who have the JAK2 mutation (Panova-Noeva et al, 2011a). Moreover, MPN patients (especially those with the JAK2 mutation) exhibit an increased fraction of immature, reticulated platelets, which are more strongly haemostatic (Harrison et al, 1997; Panova-Noeva et al, 2011b). Red cells also have a role in clot formation, both by inducing hyperviscosity and by displacing platelets toward the vessel wall, thus facilitating their activation and aggregation. Changes in the cell membrane and intracellular contents induce the formation of red cell aggregates, further contributing to thrombus formation (Turitto & Weiss, 1980). Finally, activated neutrophils are known to generate a prothrombotic state in patients with MPN by releasing proteolytic enzymes and reactive oxygen species (Falanga et al, 2000). Moreover, they overexpress CD11b, which can further activate platelets and endothelial cells. The latter then detach from the basal membrane and alter the regulation of thrombus formation (Falanga et al, 2005b). In MPN patients, the adhesion of platelets to leucocytes forms thrombogenic platelet leucocyte aggregates (Marchetti & Falanga, 2008). Thrombocytosis with platelet counts > /l has been associated with AVWS type (Federici et al, 2000), resulting in increased bleeding tendency. The international registry on AVWS reports this condition to be associated with MPN in 11 15% of cases (Federici et al, 2000). Of note, haemorrhagic complications occur much less commonly in individuals younger than 60 years of age and even less frequently for patients younger than 40 years. Increased platelet number is accompanied by increased proteolysis of VWF by ADAMTS13. The concomitant decrease in large VWF multimers is directly correlated with a decrease in VWF:RCo activity and inversely correlated with increased platelet counts. The VWF antigen (VWF:Ag) levels remain normal, resulting in a decreased VWF:RCo/VWF:Ag ratio. This VWF abnormality has been thought to be due to adsorption of the highest molecular weight multimers of VWF by protein surface membrane receptors. On chromatographic analysis, the abnormality of VWF structure resembles that of congenital von Willebrand disease. Usually, the integrity of the multimers is restored if the platelet counts are reduced to less than /l [reviewed by (Michiels et al, 2006b)]. As mentioned above, neutrophils release pro-inflammatory mediators of tissue destruction, which can damage the endothelium and have been reported to impair the bloodbrain barrier, leading to intracerebral haemorrhage, or to mediate intestinal ulceration and bleeding in animal models (Fang et al, 2010; Moxon-Emre & Schlichter, 2011). Risk factors for thrombosis or haemorrhage in patients with ET Small early studies attempted to characterize vascular events in patients with ET. The vast majority of patients had one or more vasomotor symptoms during follow-up. The incidence of thrombotic episodes ranged from 22% to 63% and the incidence of bleeding episodes from 11% to 21%. However, the proportions of patients treated, treatment types and outcomes were heterogeneous (Lahuerta-Palacios et al, 1988; Millard et al, 1990; McIntyre et al, 1991; Regev et al, 1997). Notably, in one study, 22 of 35 transient ischaemic attacks occurred at platelet counts above /l and the remaining 13 between 650 and /l (Lahuerta-Palacios et al, 1988). In a subsequent report, 35 young patients with ET (median age 38 years) were treated with anagrelide and followed for a median of 108 years. Twenty per cent of them had a history of thrombosis and 26% a history of haemorrhage. During follow-up, 20% had a thrombotic event and 20% a haemorrhagic one. All complications occurred at platelet counts > /l (Storen & Tefferi, 2001). More recent analyses were carried out through collaborative national and/or international efforts and have included larger numbers of patients. These studies have been more specifically focused on identifying risk factors for vascular events (see below). Patient characteristics A number of comorbid conditions can increase the risk of vascular events in patients with ET. These include hypercholesterolemia, hypertension, smoking and diabetes mellitus (Besses et al, 1999; Jantunen et al, 2001; Alvarez-Larran et al, ª 2016 John Wiley & Sons Ltd 355

5 2007, 2010; Carobbio et al, 2011). For example, tobacco smoking was associated with a nearly 2-fold increased risk of thrombosis [Hazard ratio (HR) = 191; P = 0007] (Carobbio et al, 2011) and a shorter 10-year thrombosis-free survival in younger patients (72% vs. 90% in non-smokers, P = 002) (Alvarez-Larran et al, 2007). Dyslipidaemia has also been found to increase the risk of arterial events in patients with ET [HR 47; 95% confidence interval (CI) ; P < 0001] (Besses et al, 1999). Although the independent contribution of cardiovascular risk factors in patients with ET has been difficult to isolate, these have recently been included as key variables in prognostic scores (see below). As a general rule, every ET patient should be counseled regarding smoking and cardiovascular risk factors (Barbui et al, 2011). Finally, although rare, conditions of inherited thrombophilia may further increase the thrombotic risk in patients with ET (De Stefano et al, 2009). Leucocytosis The degree of leucocytosis has been shown to correlate with the risk of vascular events in patients with MPN, including ET, in a number of studies. Three large retrospective studies found a direct correlation between the degree of leucocytosis and the incidence of vascular events in patients with ET. In the first (N = 1063) the risk of vascular events was increased (relative risk = 243) for patients with platelet count < /l and white blood cell (WBC) count > /l (Carobbio et al, 2008). In the second (N = 532) a WBC count > /l correlated with a higher risk of thrombosis in high-risk patients and worse survival in a cohort of patients with a median follow-up of 76 years (Palandri et al, 2011). In the third study, a WBC count > /l was linearly correlated with the incidence of thrombosis (P = 005) and major haemorrhage (P = 001) (Campbell et al, 2012). More recently, an analysis by Lim et al (2015) in 102 patients (69 with ET) using time-weighted averages of blood cell counts showed that WBC > /l was associated with a 3-fold higher risk of vascular events. Furthermore, WBC and platelet counts were significantly higher during the 3 months preceding a vascular event. The impact of high WBC count was greater on the risk of haemorrhage than on that of thrombosis (P = 0003 vs. P = 0076) (Lim et al, 2015). Mutations of JAK2, CALR, and c-mpl In recent years, recurrent molecular lesions have been identified in the vast majority of patients with MPN. While the pathogenetic role of these mutations, particularly CALR, has not been completely elucidated, somewhat distinct clinical phenotypes have been described in patients bearing each of these lesions. For example, in ET patients, both the lack of JAK2, CALR and MPL mutations (i.e., triple-negative status) and the presence of isolated CALR mutations have been associated with younger age, higher platelet count, lower WBC count, male gender, lower haemoglobin level, and lower risk of thrombosis compared with JAK2-mutated cases (Rotunno et al, 2014; Rumi et al, 2014). In a study that included 300 ET patients from the Mayo Clinic and 102 from Italy, triplenegative status was associated with longer thrombosis-free survival when age and thrombosis history were controlled for in multivariate analyses. The presence of CALR mutation was associated with longer thrombosis-free survival in patients younger than 60 years when thrombosis history was included in the model (Gangat et al, 2015). Among 217 young patients with WHO-defined ET or early primary myelofibrosis (PMF) followed for a median of 102 years, the 15-year cumulative incidence of thrombosis and haemorrhage was 166% and 86%, respectively. Although patients with a CALR mutation had higher platelet counts than those with the JAK2 mutation, the latter had a significantly higher 15- year cumulative incidence of thrombosis (217% vs. 91%, P = 004), and JAK2 allele burden >50% was independently associated with a higher risk of thrombosis. No thrombotic or haemorrhagic event was registered in the 28 (13%) patients who were triple-negative (Palandri et al, 2015). A recent study suggested that increased leucocyte activation in JAK2-positive ET might lead to an increased risk of thrombosis or haemorrhage compared with JAK2-negative ET. No such difference was found between CALR-positive and CALR-negative ET (Torregrosa et al, 2016). Is thrombocytosis per se a risk factor for vascular complications in patients with ET? Evidence for Table II summarizes the clinical evidence supporting thrombocytosis as a risk factor for vascular complications in patients with ET. In a seminal randomized study, 114 patients with ET (67% women, median age 68 years) were randomly assigned to either observation or HC treatment with the goal of maintaining the platelet count below /l. Eighty-five per cent of the patients were >60 years old and 46% had had a prior episode of thrombosis. Notably, all patients had platelet counts < /l. Antiplatelet prophylaxis with ASA or ticlopidine was allowed. At a median follow-up of 27 months, patients treated with HC had platelet counts maintained at < /l compared with /l in the control group. Two patients in HC group had a thrombotic event (36%, 1 stroke, 1 myocardial infarction) versus 14 patients (24%) in the control group (P = 0003). Thrombosis-free survival was also longer in the intervention arm (P = 0005). Both patients in the HC group and 8 of 14 in the observation group were on antiplatelet agents at the time of thrombosis. Platelet counts were /l and (a median of) /l, respectively. Antiplatelet drugs had no significant effect on the outcome on multivariate analysis (Cortelazzo et al, 1995). 356 ª 2016 John Wiley & Sons Ltd

6 Table II. Summary of the evidence supporting a relationship between the degree of thrombocytosis and the risk of vascular events. References Study type Patients (n) Median age, years (range) Median platelet count, /l (range) Median leucocyte count, /l (range) Cytoreductive therapy, n (%) Median follow-up time (range) Antiplatelet therapy, n (%) Incidence of thrombosis, n (%) Incidence of major bleeding, n (%) Cortelazzo et al (1995) van Genderen et al (1997b) Storen and Tefferi (2001) *De Stefano et al (2008) Alvarez-Larran et al (2010) Harrison et al (2005) Palandri et al (2012) Prospective, Randomized 56 (HC) 58 (control) 68 (40 85) 788 ( ) HC: 88 (5 217) Control: 90 (53 20) Retrospective Bu., 32 47%) HC, 20 (29%) IFN-a, 1 (%) P, 1 (%) Retrospective (17 48) 1075 ( ) Retrospective 259 (ET) 235 (PV) Retrospective 300 Obs: 35 (5 59) APA: 41 (13 59) Prospective, Randomized 809 HC: 62 (21 88) Anagrelide: 61 (23 88) NR Anagrelide 35 (100) 61 (19 88) NR NR Cyto: 195 (75%) Obs: 817 ( ) APA: 767 ( ) HC: 947 Anagrelide: 930 Obs: 86 (41 188) APA: 91 (39 165) HC: 104 Anagrelide: 100 HC, 56 (49%) 27 months (range HC: 25 42; range control: 38 40) All: 419 Obs: 127 ASA: 139 Cyto 113 ASA + Cyto 40: ASA, 50 (44) Ticlopidine, 29 (25) HC: 39 (70) Control: 40 (69) All, 57 (84) Obs, 4 (6) ASA: 20 (84) Cyto, 7 (10) ASA + Cyto: 37 (54) All: 16 (14) APA: 10 (88) No APA: 6 (53) HC: 2 (36) Control: 14 (24) All: 19 (28) Obs: 323/100 pr-years ASA: 36/100 pr-years Cyto: 89/100 pr-years ASA + Cyto: years 0 7 pts (20) 7 pts (20) 53 years (01 262) None Obs: 848 APA: 802 HC: 404 (50) Anagrelide: 405 (50) Retrospective (16 95) Cyto (86%) 78 years (05 325) 5(55) All occurred on APA All: 14 (21) Obs: 16/100 pr-years ASA: 72/100 pr-years Cyto: 18/100 pr-years ASA + Cyto: 10/100 pr-years 194 (75) 56/100 09/100 ASA: 185 Dipyridamole: 10 Triflusal: 2 Ticlopidine: 1 Obs: 15 (177) APA: 17 (212) 39 months ASA, 809 (100) HC: arterial = 17, (4) venous = 14 (3) Anagrelide: arterial = 37 (9); venous = 3(<1) ASA (70) Ticlopididine (14) Indobuphene (11) Obs: 6/1000 pr-years APA: 10 (126) HC: 8 (2) Anagrelide: 22 (5) NR 139/1000 pr-years pt(s), patient(s); pr(s), person(s); Cyto, cytoreductive therapy; HC, hydroxycarbamide; IFN-a, interferon-alpha; Bu, busulfan; 32 P, Phosphorus-32; ASA, low-dose aspirin; APA, antiplatelet agents; Obs, observation, NR, not reported. *Data are reported patients with polycythaemia vera (PV) and essential thrombocythaemia (ET), all of whom had experienced a previous thrombotic or haemorrhagic event. ª 2016 John Wiley & Sons Ltd 357

7 In a retrospective analysis of 68 patients with ET, 60% presented with (mostly arterial) thrombosis and 7% with bleeding. The median platelet count was /l. Platelet and WBC counts were higher in patients who presented with bleeding than in those with thrombosis. History of thrombosis was the only significant risk factor for thrombosis at presentation. During follow-up, the incidence of thrombosis was higher in patients on no cytoreduction or incomplete control of platelet counts. Additionally, the rate of thrombosis was significantly lower in patients receiving ASA, either alone or in conjunction with cytoreduction. Twenty-one per cent of patients had a bleeding episode. In this group, the mean platelet count was /l in patients on ASA alone. Most of these patients were receiving ASA at doses of 500 mg daily or higher (van Genderen et al, 1997b). In another small series, 35 ET patients aged years were treated with anagrelide. Of 33 responding patients, 27 remained on therapy for a median of 108 years. Seven (20%) patients had 10 thrombotic episodes and 7 (20%) had a major haemorrhagic event, one of which was fatal. All events occurred at platelet counts > /l and 8 of the 10 thrombotic events at > /l. All haemorrhages occurred with platelet counts between 400 and /l (Storen & Tefferi, 2001). A potential benefit of controlling the platelet count was also suggested in a retrospective review of 494 patients, 259 of whom had ET. Cytoreductive therapy halved the risk of recurrent thrombosis. The addition of oral anticoagulants and/or antiplatelet agents further increased the protective effect (De Stefano et al, 2008). The haemorrhagic risk associated with marked thrombocytosis was also explored in a retrospective analysis of 300 patients with low-risk ET, of whom 198 were treated with antiplatelet therapy and 102 observed. The follow-up times were 802 and 848 person-years, respectively. Rates of thrombotic events were similar in the two groups (212 and 177 per 1000 person-years, respectively, P = 06). A platelet count > /l was associated with increased bleeding, but not thrombosis, on multivariate analysis. Of note, among patients who were observed, JAK2 mutation and cardiovascular risk factors significantly increased the risk of venous and arterial thrombosis, respectively (Alvarez-Larran et al, 2010). In the Primary Thrombocythaemia (PT)-1 prospective randomized study, patients with ET received risk-adapted therapy. High-risk patients were treated with ASA and randomized to HC or anagrelide. Intermediate-risk patients (40 60 years of age without vascular risk factors) received ASA with or without HC, and low risk patients (<40 years of age without vascular risk factors) were treated with ASA alone. The median follow up was 36 months (Harrison et al, 2005). A post-hoc analysis of 776 patients in the high-risk group failed to reveal an association between blood counts at the time of diagnosis and future vascular events. However, platelet counts outside of the normal range during follow-up were associated with an immediate risk of major haemorrhage (HR 37, P = 00005), but not thrombosis (P = 07). Of note, platelet counts up to around /l were not associated with an increased risk of thrombosis (Campbell et al, 2012). Adding to these data, 565 patients with ET were followed at one institution for a median of 78 years (Palandri et al, 2012). Platelet counts were > /l in 20% of the patients and WBC counts were > /l in 25%. Eighty-six per cent of patients received cytoreductive therapy and 95% antiplatelet drugs. After 4433 person-years, 62 (11%) had suffered one or more haemorrhages. Median platelet count at the time of bleeding was /l ( /l). On multivariate analysis, splenomegaly, platelets > /l, and leucocytes > /l were associated with a higher risk of haemorrhage. However, the effect of antiplatelet therapy on the risk of bleeding in this study was unclear, as only 45% had never received an antiplatelet agent (Palandri et al, 2012). One potential variable adding to the bleeding risk in some of the above analyses is anagrelide. In the PT-1 study the incidence of major bleeding was higher in patients receiving anagrelide [OR 261 (95% CI ) P = 0008] (Harrison et al, 2005). In the large phase 4 observational EXELS study (Birgegard et al, 2015), patients treated with single-agent anagrelide as cytoreductive therapy (N = 804) had a higher rate of major bleeding events (089/100 patient-years) compared with those (N = 2666) receiving non-anagrelide-containing therapies (043/100 patient-years). However, only history of vascular events and age 65 years were identified as being independent predictors of thrombohaemorrhagic events at multivariate analysis (HR 191) (Birgegard et al, 2015). Whether or not anagrelide per se represents a risk factor for bleeding in ET patients remains to be ascertained. Evidence against Table III summarizes the clinical evidence suggesting that thrombocytosis may not be a risk factor for vascular complications in patients with ET. A prospective observational cohort study of 65 patients with low-risk ET, without history of thrombosis or haemorrhage and platelet counts < /l, showed that the incidence of thrombosis was similar to that of an age- and sex-matched control population (adjusted risk rate ratio = 143, 95% CI ) (Ruggeri et al, 1998). In general, various retrospective studies have failed to show a direct correlation between a higher platelet count and the incidence of vascular events. Some of them have been discussed above and will be put in perspective in this section. In an early report, 44 young ET patients (median age 30 years) were followed for 6 months to 10 years. Mean platelet count at presentation was /l and 36% had platelets > /l. There was no significant leucocytosis or erythrocytosis. Platelet count was not predictive of vascular complications (Mitus et al, 1990). In another study, Tefferi and colleagues followed 99 consecutive patients with low-risk ET, but with platelet counts /l. 358 ª 2016 John Wiley & Sons Ltd

8 Table III. Evidence suggesting lack of relationship between thrombocytosis and vascular events. References Study type Patients, n (%) Median age, years (range) Median plt count, /l (range) Median WBC count, /l (range) Median follow-up time (range) Cytoreductive therapy, n (%) Antiplatelet therapy, n (%) Incidence of thrombosis, n (%) and/or per ptor pr-years Incidence of major bleeding, n (%) and/or per pt- or pr-years Ruggeri et al (1998) Mitus et al (1990) Tefferi et al (2006) Carobbio et al (2008) Carobbio et al (2011) Finazzi et al (2012) Prospective, observational ET: 65 Controls: 65 ET: 41 (18 58) Controls: 39 (19 58) ET: 823 ( ) Controls: 268 ( ) ET: 89 (4 31) Controls: ND Retrospective (9-44) 1005 (mean) NR 6 months 10 years Retrospective All: (76) Cyto after event: 24 (24) All: 43 (16 59) 45 (16 59) Cyto after event: 31 (16 55) All: 1323 ( ) 1338 ( ) Cyto after event: 1280 ( ) All: 96 (4 192) 9(4 182) Cyto after event: 109 (4 192) 41 years (1 12) None ASA, 25 (38%) ET: 191/ 100 Controls: 150/100 All: 147 months (3 397) 174 months Cyto after event: 88 months None None 14 (32) 6 (14) HC or anagrelide upfront: 75 (76) Hu or anagrelide after event: 24 (24) Retrospective (8 95) 806 ( ) 88 (33 35) 48 years HC or Bu: 546 (51) All: ASA, 46 (46) 33 (44) Cyto after event: 13 (54) All: 21 (21) 015/100 pt years Cyto after event: 0 13/100 ASA, 703 (66) 118 pts (23/100 ) Plt > /l + WBC < /l = 159%/pt-year (26% JAK2- positive) Plt < /l + WBC > /l = 259%/pt-year (77% JAK2- positive) Retrospective (13 91) >1000 in 21% of pts >11 in 21% of pts 62 years (0 27) 507 (57) ASA, 602 (68) 109 (12) 17/100 ET: 112/100 (on ASA) Controls: 0 All: 7 (7) 005/ 100 Cyto after event: 004/ pts (076/100 ) 55 (6) 079/100 ª 2016 John Wiley & Sons Ltd 359

9 Table III. (Continued) Incidence of major bleeding, n (%) and/or per pt- or pr-years Incidence of thrombosis, n (%) and/or per ptor pr-years Antiplatelet therapy, n (%) Cytoreductive therapy, n (%) Median follow-up time (range) Median WBC count, /l (range) Median plt count, /l (range) Median age, years (range) References Study type Patients, n (%) All: 12 (5) Cyto + ASA: 144/100 Cyto only: 014/100 ASA, 168 (68) All: 28 (11) Cyto + ASA: 144/100 Cyto only: 248/100 pt-year Cyto, 247 (100) All: 75 years (01 26) Cyto + ASA: 763 pr-years Cyto only: 685 pr-years Cyto + ASA: 88 (39-24) Cyto only: 9(44 24) Cyto + ASA: 734 ( ) Cyto only: 775 ( ) Cyto + ASA: 66 (28 90) Cyto only: 65 (18 93) Retrospective All: 247 Cyto + ASA: 168 Cyto only: 79 Alvarez-Larran et al (2013) ET, essential thrombocythaemia; plt, platelets; WBC, white blood cell; Cyto, cytoreductive therapy; pt(s), patient(s); pr(s), person(s); HC, hydroxycarbamide; Bu, busulfan; ASA, low-dose aspirin; NR, not reported. Seventy-five of them (median age 45 years) received cytoreductive therapy upfront, whereas 24 (median age 31 years, P < 0001) received therapy after the first vascular event. The median follow-up times were 174 and 88 months (P = 0005), respectively, and all other characteristics were well balanced. When discrepancies in follow-up time or age were controlled for, there was no significant difference in rate of thrombosis or haemorrhage between treated and untreated patients (Tefferi et al, 2006). Interestingly, in a large retrospective analysis of patients with ET (N = 1063), platelet counts > /l were independently associated with a significantly lower rate of thrombosis. Instead, patients with leucocytosis, lower platelet count and JAK2 mutation at diagnosis had a higher risk of thrombosis. Specifically, the thrombotic rate was 159% per patient-years for patients with platelets > /l and WBC < /l and 295% of patient-years for those with platelets < /l and WBC > /l. A JAK2 mutation was found in 26% of patients in the former group compared with 77% in the latter group (Carobbio et al, 2008). Although one could speculate that AVWS or other acquired platelet defect may have a direct role in decreasing the thrombotic risk in patients with extreme thrombocytosis, this idea remains to be further corroborated. Similarly, in another retrospective study in 891 patients with WHO-defined ET followed for a median of 62 years, independent predictors of arterial, but not venous thrombosis included age >60 years, history of thrombosis, cardiovascular risk factors, leucocytosis and presence of the JAK2 mutation (only male gender predicted venous thrombosis). Platelet count > /l was associated with a lower risk of arterial thrombosis (HR = 04, P = 0007) and had no impact on the risk of venous thrombosis in either JAK2-positive or -negative cases (Carobbio et al, 2011). In addition, a multivariate analysis of bleeding risk using the same cohort of WHO-defined ET patients (N = 891), plus 180 patients with pre-fibrotic primary PMF, showed that predictors of haemorrhage included a diagnosis of prefibrotic PMF, leucocytosis, prior bleeding, and ASA use, but not platelet count. When the analysis was restricted to patients with WHO-defined ET, only previous haemorrhage and ASA therapy emerged as independent risk factors for bleeding (Finazzi et al, 2012). Similarly, in a retrospective study of high-risk ET patients performed by the Spanish group, extreme thrombocytosis (platelet count > / l) did not increase the risk of vascular events, whereas antiplatelet therapy was an independent risk factor for bleeding (Alvarez-Larran et al, 2013). In other studies that showed an association between higher platelet counts and bleeding tendency, the possible interaction between marked or extreme thrombocytosis and antiplatelet therapy could not be appropriately addressed because only a handful of patients did not receive ASA (Alvarez-Larran et al, 2010; Campbell et al, 2012; Palandri et al, 2012). Despite the above-mentioned seemingly negative studies, one should exercise caution in 360 ª 2016 John Wiley & Sons Ltd

10 dismissing a relationship between high, or very high, platelet count and vascular events in ET patients, given the lack of a consistent platelet count threshold across studies, the fact that platelet counts were considered as continuous variable in some controlled analyses, and, perhaps more importantly, the generally small number of patients not treated with ASA. Relationship between achievement of haematologic response and vascular events Despite the original finding that control of thrombocytosis resulted in a decreased risk of vascular events, (Cortelazzo et al, 1995) the majority of (retrospective) studies aimed at establishing a relationship between haematological response and clinical outcomes in patients with ET failed to show such benefit. For example, in a study by Palandri et al (2009), 338 of 386 patients with ET (88%) received cytoreductive therapy and 86% of them obtained a response. However, controlling thrombocytosis did not appear to reduce the incidence of thrombosis. Only a prior history of vascular event was associated with a higher incidence of thrombosis (Palandri et al, 2009). Similarly, among 416 ET patients treated with HC (overall and complete response rate 83% and 25%, respectively), and followed for a median of 39 years, age over 60 years, previous thrombosis and leucocytosis (> /l), but not achievement of an ELN-defined response or a higher platelet count, increased the risk of thrombosis in a controlled analysis (Carobbio et al, 2010). Finally, among 154 ET patients treated with anagrelide for a median of 29 years, the ELN-defined overall and complete response rates were 865% and 56%, respectively (only 25% achieved a sustained complete response). The incidence of thrombosis and haemorrhage was independent of complete response status, which was also not predictive of survival (Hernandez-Boluda et al, 2013). Modern risk stratification of ET patients: the IPSET-thrombosis score In an effort to improve the current dichotomous risk stratification of ET patients (low- and high-risk, depending on age and/or history of thrombosis), risk factors for vascular events were searched for in 891 patients with WHO-defined ET (Barbui et al, 2012). The International Prognostic Score of Thrombosis for ET (IPSET-thrombosis) included cardiovascular risk factors (1 point) and JAK2 mutation (2 points) in addition to age >60 years (1 point) and history of thrombosis (2 points). In this 3-tiered model, the thrombosis risk was 103%, 235%, and 356% patients/year for patients in the low, intermediate and high-risk group, respectively. This score predicted the risk of vascular events more accurately than the classic 2-tiered model (Barbui et al, 2012). The IPSET-thrombosis was further validated in a population of 970 Chinese patients with the additional observation that only patients in the intermediateand high-risk group derived significant clinical benefit from cytoreductive agents (Fu et al, 2014). The IPSET-thrombosis score was recently revised. In patients of any age with a history of thrombosis, the cooccurrence of JAK2 mutations and/or cardiovascular risk factors did not significantly increase the risk of vascular events, whereas in patients older than 60 years, but with no thrombosis history, the presence of JAK2 mutations did increase the risk. Moreover, given the very low thrombotic risk in low-risk patients without JAK2 mutations (105% and 044% patient-years in the presence and absence of cardiovascular risk factors, respectively) patients previously adjudicated lowrisk were divided into low-risk and very low-risk, based on the presence or absence, respectively, of JAK2 mutations. The revised IPSET-thrombosis, therefore, identified four risk categories: high risk (thrombosis history or age >60 years with JAK2 mutation); intermediate risk (no thrombosis history, age >60 years and JAK2-unmutated); low risk (no thrombosis history, age 60 years and JAK2-mutated); and very low risk (no thrombosis history, age 60 years and JAK2-unmutated). For the last group, ASA might be unnecessary (unless indicated for cardiovascular risk factors) (Barbui et al, 2015). The revised IPSET-thrombosis was also externally validated in a cohort of 585 ET patients. In that study, only JAK2 mutations and a history of thrombosis were independently predictive of thrombotic events (Haider et al, 2016). Conclusion While the role of antiplatelet and cytoreductive therapy in patients with high-risk ET defined by age >60 and/or history of thrombosis is firmly established, the evidence regarding the use of cytoreductive therapy with the mere goal of controlling the platelet count remains controversial, regardless of risk category. In addition, the risk/benefit ratio of ASA in low- or very low-risk patients is unclear, especially in the presence of extreme thrombocytosis, in which case the risk of bleeding might outweigh that of thrombosis. The only two prospective randomized studies (Cortelazzo et al, 1995; Harrison et al, 2005; Campbell et al, 2012) that have shown a benefit from reducing platelet counts had insufficient numbers of low-risk patients, making it difficult to draw firm conclusions regarding this group. On the other hand, larger analyses suggesting no correlation between platelet counts and the risk of vascular events are retrospective and therefore have inherent methodological weaknesses, such as the potential lack of adequate power to detect significant associations between variables (including blood counts) and clinical outcomes and the overlap between the populations described in some of the studies. Although a level of /lhasbeenutilizedasacategorical variable in several of the studies mentioned, the platelet count threshold to be considered (or not considered) a risk factor is, itself, a moving target. Patients with baseline platelet count > /l (theoretically at higher risk to have bleeding from AVWS) are rarely studied in isolation and many of them have received antiplatelet agents. Therefore, an operational, ª 2016 John Wiley & Sons Ltd 361

11 clinically helpful definition of extreme thrombocytosis did not emerge from the literature and the indiscriminate use of this term appears unwarranted at this time. In our practice,we do not use thrombocytosis per se as a criterion to initiate cytoreductive therapy in patients with no history of bleeding. In otherwise low-risk ET patients, we do not test for AVWS and do not hold ASA unless the platelet count is /l or greater. In the recently identified very low risk patients, it might be reasonable to lower the platelet threshold for holding ASA and testing for AVWS to /l. However, given that clear-cut, prospective data to support or dismiss thrombocytosis as risk factor for vascular events in ET patients are lacking, prospective studies of risk-adapted cytoreductive and/or antiplatelet therapy based on modern thrombotic risk scores, such as the revised IPSET-thrombosis score (Barbui et al, 2015) will certainly inform clinical practice and are eagerly awaited. Acknowledgements LF and SV designed and performed the research; LF and KJN performed the literature search and selection; LF, KJN and PB critically analysed the data; all authors wrote the paper and approved the final and submitted version. SV received research support for conduct of clinical studies by Incyte Corporation, Astrazeneca, Lilly Oncology, Roche, Geron, NS Pharma, Bristol Myers Squibb, Celgene, Infinity Pharmaceuticals, Gilead, Seattle Genetics, Promedior, Cell Therapeutics, Inc., Galena BioPharma, Pfizer. LF, PB and KJN have no competing interests. References Alvarez-Larran, A., Cervantes, F., Bellosillo, B., Giralt, M., Julia, A., Hernandez-Boluda, J.C., Bosch, A., Hernandez-Nieto, L., Clapes, V., Burgaleta, C., Salvador, C., Arellano-Rodrigo, E., Colomer, D. & Besses, C. (2007) Essential thrombocythemia in young individuals: frequency and risk factors for vascular events and evolution to myelofibrosis in 126 patients. 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