Essential thrombocythaemia: challenges and evidence-based management

Transcription

1 review Essential thrombocythaemia: challenges and evidence-based management Claire N. Harrison Department of Haematology, St Thomas Hospital, Lambeth Palace Road, London, UK Summary Essential thrombocythaemia was first described over 70 years ago. This condition is dominated by thrombotic and haemorrhagic complications and, in the long-term, by risk of transformation to myelofibrosis and/or acute leukaemia. However, it is heterogeneous both clinically and biologically. Here, a review of current concepts in disease aetiology and management is offered with reference to recent focused reviews where appropriate. In addition, five specific areas are discussed in detail: the role of the trephine biopsy, the disease entity prefibrotic myelofibrosis; the recently described Janus kinase 2 (JAK2) mutations; the leukaemogenicity of hydroxyurea (hydroxycarbamide); and lastly, the implications of the results of the Medical Research Council Primary Thrombocythaemia 1 study are explored. Keywords: essential thrombocythaemia, hydroxyurea, trephine biopsy, MRC-PT1. Essential thrombocythaemia (ET) is probably the commonest myeloproliferative disorder; despite this, our current understanding of its biology and therapy is limited. Clinical heterogeneity in this condition is familiar to many clinicians and has been well documented in historical literature. Research has identified biological heterogeneity but this has not correlated with clinical features (reviewed in Harrison, 2002). This is also a controversial field, as has been emphasized by the recent results of the Medical Research Council Primary Thrombocythaemia 1 (MRC-PT1) study in high-risk patients (Harrison et al, 2005). This article reviews ET from clinical and biological perspectives and controversial areas are discussed. These particular areas include: the evidence for the existence of a separate clinical entity prefibrotic myelofibrosis and what the significance of this might be; the recent identification of Janus kinase 2 (JAK2) mutations in myeloproliferative disorders (MPDs) (Baxter et al, 2005; James et al, 2005); a review of Correspondence: Claire N. Harrison, Department of Haematology, St Thomas Hospital, Lambeth Palace Road, London SE1 7EH, UK. claire.harrison@gstt.nhs.uk the evidence for the leukaemogenicity of hydroxyurea (or hydroxybarbamide) and the implications of the results of the MRC PT1 study. Molecular biology and pathogenesis Almost 95% of ET patients will have normal cytogenetics but, when present, any karyotypic abnormalities are highly variable. Identification of the Philadelphia chromosome or detection of BCR-ABL rearrangement by fluorescent in situ hybridaization (FISH) or reverse transcription polymerase chain reaction (RT-PCR) effectively excludes a diagnosis of classical ET. There has been some confusion about terminology for those patients who do not display any other features of chronic myeloid leukaemia (CML). Also confusion arises in the context of those patients who have only a low level of BCR-ABL transcripts detected by RT-PCR (Heller et al, 2001). However, both a recent review of over 20 cases demonstrating a high risk of transition to blastic phase or myelofibrosis and the advent of imatinib therapy for CML argues strongly that these patients should effectively be managed as CML (Michiels et al, 2004). Of interest are patients in whom the disease may have arisen separately or for whom the thrombocytosis is not adequately controlled by imatinib (Curtin et al, 2005) suggesting, perhaps, clonal evolution. Particular karyotypic abnormalities have been reported in association with progression to myelofibrosis or leukaemia (Sessarego et al, 1989) but this remains to be confirmed. Abnormalities of 17p have been found in patients treated with hydroxyurea who developed acute leukaemia (Sterkers et al, 1998; Bernasconi et al, 2002) and, whilst it is pertinent that this region of chromosome 17 is the location of p53, studies of this and other oncogenes and tumour suppressor genes have failed to identify candidate target genes (Neri et al, 1996; Gaidano et al, 1997; Mavrogianni et al, 2002). Essential thrombocythaemia was assumed to be a clonal disorder but it is now well recognized that a significant proportion, up to 50% of patients, do not appear to have clonal haemopoiesis when assessed using X-chromosome inactivation patterns (el-kassar et al, 1997; Harrison et al, 1999a; Chiusolo et al, 2001; Shih et al, 2002). However, this ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, doi: /j x

2 may reflect the limited ability of current techniques to detect a low disease burden against a polyclonal background rather than being a true feature of the biology of this disease. Agerelated skewing or finding a clonal pattern in apparently haematologically normal females further limits the wider application of these assays. However, an intriguing correlation between monoclonal ET patients and increased risk thrombotic complications (Harrison et al, 1999a; Shih et al, 2002) implies that their role in ET possibly merits further investigation in large prospective studies. The identification of thrombopoietin (TPO) stimulated investigation of this cytokine and its receptor c-mpl in ET. In contrast to what has been described for polycythaemia vera (PV) and CML, where erythropoietin and granulocyte colonystimulating factor (G-CSF), respectively, are reduced; TPO levels are paradoxically elevated not only in ET and reactive thrombocytosis, but also in the other MPDs (Cerutti et al, 1997; Harrison et al, 1999b). An explanation for inappropriately elevated TPO levels in ET has yet to be defined. Mutations causing elevated TPO levels have been identified in some kindreds with familial ET (Jorgensen et al, 1997; Kondo et al, 1998; Wiestner et al, 1998; Ghilardi & Skoda, 1999) but they have not been found in classical ET (Harrison et al, 1998; Allen et al, 2001). As downregulation following binding to its receptor c-mpl is a mechanism for controlling TPO levels reduced megakaryocyte and platelet expression of c-mpl as shown in some patients with ET may be pertinent (Horikawa et al, 1997; Moliterno et al, 1998; Harrison et al, 1999b). Indeed C-MPL itself is a candidate oncogene but although it has been linked to familial thrombocytosis in one kindred (Ding et al, 2004) no mutations have as yet been identified in classical ET. Interestingly, a significant subgroup of ET patients (up to 30%) may have reduced erythropoietin levels and an increased tendency to macrovascular complications (Messinezy et al, 2002). Clonality and erythropoietin are reported to be independent of each other (Andreasson et al, 2003). Further molecular markers for PV, including PRV1, a member of the upar receptor superfamily, and the growth of spontaneous erythroid colonies have been investigated in ET. Increased levels of PRV1 mrna have consistently been described in granulocytes derived from patients with PV (Temerinac et al, 2000) and in up to 50% of patients with ET. Preliminary reports suggest that those ET patients with normal PRV1 expression and no detectable spontaneous erythroid colonies are more likely to have polyclonal haemopoiesis (Kralovics et al, 2003; Liu et al, 2003). However, the situation is less clear for ET patients with monoclonal haemopoiesis. As yet we have no clear understanding of the molecular pathogenesis of ET but the components of postreceptor signalling pathways are likely candidates, a summary of potential pathogenic mechanisms is shown in Fig 1. A major breakthrough in our understanding of the myeloproliferative disorders has come with the very recent identification of an acquired mutation of JAK2 in a substantial proportion of MPD patients (Baxter et al, 2005; James et al, 2005). An identical 1 hit Stem cell 2 hits e.g. drugs MPD stem cell Acute Leukaemia Clonal or polyclonal?? Red cell EEC++, EPO reduced Neutrophil PRV1 mrna ++ MK/ platelet cmpl-- MYELOPROLIFERATION MK +/or monocyte stromal Enhanced stimulatory interaction signals e.g. TPO, PTP Meg, JAK/STAT Reduced inhibition eg Myelofibrosis resistance to TGF β,? SMAD defect Fig 1. Candidate pathogenic mechanisms in ET. mutation has been found in the vast majority of patients with PV as well as approximately half those with ET or idiopathic myelofibrosis (IMF). The mutation (V617F) is located in the negative regulatory JH2 domain and replaces a highly conserved valine with a bulky phenylalanine. As predicted by previous structural and biochemical studies, the consequence of this mutation is increased tyrosine kinase activity of JAK2. JAK2 plays a central role in signal transduction from multiple growth factors, and so its activation is consistent with the growth factor independent phenotype. Sequence analysis of peripheral blood granulocytes detected the mutation in 12% of patients with ET, but over 50% of patients were positive by allele-specific PCR. These results demonstrate that in some patients, the mutation is present in only a minority or peripheral blood granulocytes. The mutation was present in both erythroid and granulocytemacrophage colonies, demonstrating that it arose in a multipotent progenitor. Moreover, whereas both mutant and normal colonies were able to grow in the presence of erythropoietin, only mutant colonies were present in its absence. This observation reveals a clear link between the JAK2 mutation and growth factor independence. Furthermore, the data from James et al (2005) supports the association of the mutation with erythrocytosis in the mouse model. These exciting results demonstrate that ET can be divided into JAK2-positive and -negative subgroups, and it seems likely that the former subgroup will prove to include patients positive for other markers associated with PV, such as PRV1 and erythropoietin-independent colonies. The molecular basis for JAK2-negative ET remains obscure. It is also unclear how the same mutation can give rise to PV, ET and IMF. Potential explanations include inter-individual differences in genetic background, in additional acquired mutations or in the target cell for transformation. This data clearly requires confirmation, however, JAK2 data has emerged simultaneously from four groups internationally (Baxter et al, 2005; James et al, 2005; unpublished data) it is the authors understanding that at least one of these reports explores more comprehensively the functional consequences of the mutation but that none have yet have discovered a significant clinical correlation. 154 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130,

3 Diagnostic criteria Both a raised platelet count and megakaryocyte hyperplasia are found in reactive thrombocytosis; thrombocytosis because of other myeloproliferative disorders; and myelodysplasia with thrombocytosis. Hence a major difficulty in achieving a diagnosis of ET is the lack of a definitive positive criterion analogous to the presence of BCR-ABL transcripts in CML. The diagnostic criteria in widespread use for ET remain those proposed by the PV study group (PVSG) (Murphy, 1997, Table I). The more recently published World Health Organization (WHO) criteria (Vardiman et al, 2002, Table II) have yet to gain widespread acceptance. A major obstacle to general use of the WHO criteria is the significant emphasis placed upon histological evaluation of the trephine biopsy, as Table I. PVSG criteria for the diagnosis of ET (Murphy, 1997). (1) Platelet count > /l (2) Haematocrit <40 or normal red cell mass (males <36 ml/kg, females<32 ml/kg) (3) Stainable iron in the marrow or normal RBC mean corpuscular volume* (4) No Philadelphia chromosome or BCR-ABL gene rearrangement (5) Collagen fibrosis of the bone marrow A: Absent or B: Less than one third of the biopsy area without both marked splenomegaly and a leucoerythroblastic blood film (6) No cytogenetic or morphological evidence for a myelodysplastic syndrome (7) No cause for a reactive thrombocytosis *If these measurements suggest iron deficiency, PV cannot be excluded unless a trial of iron therapy fails to increase the red cell mass into the polycythaemic range. Table II. WHO diagnostic criteria for ET (Vardiman et al, 2002). Positive criteria (1) Sustained platelet count > /l (2) Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryocytes Negative criteria (1) No evidence of polycythaemia vera Normal red cell mass or Hb <18Æ5 g/dl in men, 16Æ5 g/dl in women Stainable iron in marrow, normal serum ferritin or normal MCV If the former condition is not met, failure of iron trial to increase red cell mass or Hb levels to the PV range (2) No evidence of CML No Philadelphia chromosome and no BCR-ABL fusion gene (3) No evidence of chronic idiopathic myelofibrosis Collagen fibrosis absent and reticulin fibrosis minimal or absent (4) No evidence of myelodysplastic syndrome No del(5q), t(3;3)(q21;q26), inv(3)(q21q26) No significant granulocytic dysplasia, few if any micromegakaryocytes (5) No evidence that thrombocytosis is reactive due to: Underlying inflammation, infection, neoplasm or prior splenectomy discussed later. During the past decade, several additional diagnostic tests have been proposed including culture of megakaryocyte colonies, clonal analysis using X-chromosome inactivation patterns and megakaryocyte morphology in trephine biopsies but, for the most part, as discussed, these are of limited or unproven diagnostic potential. The finding of an acquired mutation in JAK2 is likely, once substantiated, to be a useful diagnostic marker (Baxter et al, 2005; James et al, 2005), further data is anticipated. The diagnostic utility of clonogenic assays for spontaneous megakaryocyte colonies has been proposed by many workers and is analogous to the use of endogenous erythroid colonies in the diagnosis of PV. Unfortunately, significant difficulties arise in the standardisation of these assays; furthermore, spontaneous megakaryocytic colonies have also been reported in reactive thrombocytosis (reviewed in Westwood & Pearson, 1996). The increased sensitivity of colonies to TPO has also been evaluated and, although most ET patients displayed hypersensitivity, so did patients with reactive thrombocytosis (Axelrad et al, 2000). Heterogeneity of the data in relation to clonogenic assays in ET may reflect more complex underlying biological heterogeneity. Clonal analysis using X-chromosome inactivation patterns, as discussed above, are unlikely to be useful diagnostically for most patients. Until the advent of the WHO criteria (Vardiman et al, 2002) the trephine biopsy was principally used to exclude myelofibrosis. Trephine histology has now been proposed as a tool to positively to establish the presence of ET. However, both megakaryocyte morphology and reticulin grading are notoriously difficult to assess in a reproducible manner and detailed studies of inter-observer variation are lacking. It is, therefore, not yet clear whether the WHO criteria are robust enough to be widely applied outside specialized centres. However, the proposal that interpretation of the trephine could be used to facilitate earlier diagnosis in those patients with a platelet count of /l (Lengfelder et al, 1998; Sacchi et al, 2000) is interesting and, if validated, would be potentially useful in symptomatic patients with borderline platelet counts. Two recent reports have also suggested that trephine features can identify subgroups of patients with a worse prognosis. In the study of Annaloro et al (1999) reticulin abnormalities, increased and dysplastic granulopoiesis, hypoplastic erythropoiesis, trapped but not large megakaryocytes, and aggregates of immature precursors were associated with significantly worse event-free and overall survival, with most of the difference in event-free survival attributable to thrombotic events. The seminal work in this field has been produced by Thiele et al (2000), who provided elegant descriptions of histological subtypes of ET and myelofibrosis (true ET, prefibrotic myelofibrosis and early myelofibrosis). These descriptions, however, vary from those produced by other workers, as described above. This group has also reported these histologically distinct subgroups (true ET, prefibrotic myelofibrosis and early myelofibrosis) with different prognoses (loss of life ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130,

4 expectancy of 10Æ9%, 29Æ6% and 51Æ3%, respectively). Further clinical data are awaited to confirm precisely the events that underpin these prognostic differences and larger scale studies are required to demonstrate critical aspects, such as reproducibility, longitudinal data and validation from other centres. Given the high probability that several conditions are currently classed as ET it is important to utilize differences that can be readily identified and may provide meaningful classification. Hence, for example, if prefibrotic myelofibrosis indeed exists as a separate diagnostic entity amongst those patients with an apparent diagnosis of ET, it will be important to identify its biological basis. Furthermore, complex management issues will inevitably arise as to whether these different entities require specifically tailored treatments to prevent their separate biological destinies. Clinical features A significant number of ET patients are asymptomatic and may continue in this fashion. For the remaining symptomatic patients the predominant clinical feature is thrombosis; haemorrhage also occurs but less frequently. In a proportion of patients, the disease will transform to myelofibrosis or acute myeloid leukaemia (AML) and, in a lesser number, a condition akin to PV occurs. The literature is variable with respect to frequency of thrombotic and haemorrhagic complications, reflecting case selection and variable definitions of clinical events. The pathogenesis and elements of the management of thrombosis and haemorrhage in ET have recently been reviewed (Elliott & Tefferi, 2005). Sadly, although our understanding of some of the basic biology and the pathogenesis of inherited and acquired thrombophilia has advanced in recent times, this has not been reflected in the myeloproliferative field. In parallel with future advances in our knowledge of the molecular pathogenesis we need to understand the mechanisms of thrombosis and better target therapy thereafter. It seems likely that elevated platelet counts are implicated in thrombotic events in ET but the degree of elevation does not appear to be important. In fact, it is well documented that thrombosis may occur at relatively low platelet counts (Lengfelder et al, 1998). Dominant risk factors for thrombosis are age over 60 years and/or a prior thrombotic or haemorrhagic event, as identified in the hallmark study from Cortelazzo et al (1990) and subsequently verified by others (Lengfelder et al, 1998; Besses et al, 1999). A number of welldefined conventional risk factors for cardiovascular disease are also variably implicated including smoking, hyperlipidaemia, hypertension and diabetes (Cortelazzo et al, 1990; Besses et al, 1999). Preliminary studies have also correlated platelet glycoprotein polymorphisms (Afshar-Kharghan et al, 2004), platelet activation and platelet leucocyte aggregates, as well as increased expression of leucocyte adhesion molecules, with thrombosis (Falanga et al, 2000; Jensen et al, 2001). The presence of antiphospholipid antibodies principally of IgM subclass (Harrison et al, 2002), monoclonal haemopoiesis (Harrison et al, 1999a), reduced expression of c-mpl in megakaryocytes (Yoon et al, 2000), atypical trephine biopsy features (Annaloro et al, 1999), overexpression of PRV1 (Johansson et al, 2003), reduced erythropoietin levels (Messinezy et al, 2002) and heterozygosity for factor V-Leiden (Ruggeri et al, 2002) have all also been correlated with thrombosis. The major risk factor for haemorrhage in ET appears to be a platelet count exceeding /l that may be accompanied by a reduction in higher molecular weight multimers of von Willebrand factor and hence acquired von Willebrand s disease. To date, analyses of a broad array of specific structural, biochemical, and metabolic platelet defects have failed to correlate them with bleeding (reviewed in Elliott & Tefferi, 2005). Although an early PVSG study demonstrated excessive haemorrhage occurring in patients treated with aspirin, this probably resulted from the high doses used (900 mg/d) (Tartaglia et al, 1986). Subsequent studies utilizing lower doses (100 mg) and, indeed, the recently published European Collaboration on Low-dose Aspirin in Polycythemia vera (ECLAP) study (Landolfi et al, 2004), support the safety of low dose aspirin and have not reported associated excess haemorrhage. Patients with ET have an inherent risk of developing myelodysplastic syndrome (MDS) or AML since there have been several reports of treatment-naïve patients suffering from this complication (Andersson et al, 2000) Undoubtedly, this risk is significantly affected by several treatments, especially sequential therapy with more than one potentially leukaemogenic agent. This may reflect additive leukaemogenicity or that these patients requiring multiple therapy have more aggressive or unstable disease. This is discussed in greater detail below. In a recent study of 195 ET patients with median follow-up of 7 years the incidence of myelofibrosis was 8% at 10 years (Cervantes et al, 2002). It is important that the diagnosis of myelofibrosis is not based solely upon histological evaluation of the trephine biopsy. Criteria developed in evaluating the MRC PT1 trial (Harrison et al, 2005) were based upon a modification of the Italian criteria for de novo myelofibrosis (Barosi, 1999). The increase in collagen fibrosis and clinical features of myelofibrosis may relate to release of humoral factors from cells including megakaryocytes and monocytes. Candidate factors include the alpha granule contents, plateletderived growth factor (PDGF), platelet factor 4 (PF-4), transforming growth factor b (TGFb), basic fibroblast growth factor (b-fgf) and calmodulin (Le Bousse-Kerdiles & Martyre, 2001). It has also been reported that abnormal subcellular location of P-Selectin within megakaryocytes correlates with emperipoiesis, thence disrupting megakaryocyte organelles and causing leakage of alpha granule contents (Schmitt et al, 2002). Monocytes activated following contact with the extracellular matrix upregulate CD25 expression, and increase production of TGFb and interleukin-1, which have pro-fibrotic potential. It has been suggested that myelofibrotic transformation may be predicted on the basis of trephine biopsy appearances and 156 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130,

5 that those patients with true ET are very unlikely to suffer this complication (Thiele et al, 2000). This demands validation. Other than histological parameters, no other consistent risk factors for myelofibrotic transformation have been identified. Different therapeutic modalities have anecdotally and variably been reported to have variable effects upon features of myelofibrosis; the first conclusive data come from the MRC PT-1 study (see below). Prognosis Four large cohort studies report conflicting data in relation to prognosis in ET. Rozman et al (1991) compared survival to Spanish life tables with no significant loss of life expectancy over a period of 10 years, however, a study from Olmsted county suggested the opposite (Mesa et al, 1999) Given that evolution to myelofibrosis or acute leukaemia might not be anticipated until at least 10 years and risk of major thrombotic events accumulates with time, studies with longer follow up, that are limited to younger patients might be more informative. Such studies have shown either equivalent survival (Tefferi et al, 2001) or a fourfold increase in the relative risk of death (Bazzan et al, 1999). A retrospective analysis of 148 patients identified that only the number of thrombotic or haemorrhagic events occurring during follow-up continued to have a significant impact upon survival on multivariate analysis (Lengfelder et al, 1998). This study provides further support for the use of therapies to reduce thrombotic risk. The challenge for the future is to gather high quality clinical data that can be used to inform ET patients in relation to their future life expectancy and to make more balanced decisions about the benefits or risks of therapy. This data could also be used to confirm or refute fascinating data in relation to histological features and loss of life expectancy. Therapeutic options Although a number of treatment options for ET are available, they have a limited evidence base. As a minimum, patients should have an assessment and modulation of identified risk factors for vascular disease including, for example, hyperlipidaemia, and smoking amongst others. Treatment options include aspirin (or an equivalent therapy) and the possible addition of cytoreductive therapy to control the platelet count. The main cytoreductive drugs to consider are hydroxyurea (hydroxycarbamide), anagrelide, or interferon, which are considered in detail below. Aspirin Although there is no randomized data demonstrating that aspirin reduces major thrombotic events in ET, several lines of argument justify the use of low dose aspirin in this condition. It has been known for some time that, microvascular events, principally erythromelalgia and transient neurological manifestations, are alleviated by aspirin (Michiels et al, 1993; Griesshammer et al, 1997). Most recently, a randomised study in PV (ECLAP) provided clear evidence that, for this group of patients, low dose aspirin reduced a combined primary endpoint of non-fatal myocardial infarction, non-fatal stroke, major venous thrombosis or death from cardiovascular cause (Landolfi et al, 2004). In ET, low dose aspirin ( mg/d) has been demonstrated to be safe in two studies (if platelets are less than /l and no prior haemorrhage) that also suggested aspirin reduced thrombotic complications (van Genderen et al, 1995; van Genderen et al, 1997). Preliminary evidence supports an emerging role for the thienopyridines (antiplatelet agents), such as clopidogrel or ticlopidine (Nurden et al, 1996), particularly for patients with ongoing thrombotic events suggesting in vivo aspirin resistance. But Ruggeri et al (1993) compared ticlopidine with aspirin for the management of myeloproliferative disorders with thrombocytosis and found no benefit, indeed microcirculatory events appeared to be refractory to ticlopidine. Further data is required to inform management in this area. For those patients developing gastrointestinal symptoms while treated with aspirin, a recent study in an unrelated patient cohort suggested that aspirin and a proton pump inhibitor was superior to clopidogrel (Chan et al, 2005). Hydroxyurea (hydroxybarbamide) Hydroxyurea, now known as hydroxycarbamide, has emerged as the treatment of choice in ET patients who are at high risk of complications because of its proven efficacy (Cortelazzo et al, 1995) and rare acute toxicity. Major short-term toxic effects are dose limiting haemopoietic impairment and, less frequently, oral or leg ulcers and other skin lesions. The most controversial aspect of hydroxyurea therapy is whether or not it is leukaemogenic. A number of long-term cohort studies have documented that, whilst leukaemia does occur in patients treated with hydroxyurea, it also occurs in untreated ET patients. Leukaemia has rarely been reported in patients with sickle cell disease who are treated with hydroxyurea (Halsey & Roberts, 2003); overall there have only been two cases of AML in this patient population. ET is a condition with an innate tendency to leukaemia and here the question is whether the innate leukaemic risk is increased. Sadly, the literature is variable in this respect and not all studies that have been reported are sufficiently powered to assess whether hydroxyurea is truly leukaemogenic. For example, the mean time from presentation to leukaemia is approximately 6Æ5 years (Barbui et al, 2004), hence, only long-term studies will be sufficiently robust. In addition, patients requiring cytotoxic therapy should be assessed with those not requiring any therapy, as disease biology may be an important determinant of leukaemic risk. However, the reported risk of developing leukaemia after hydroxyurea therapy alone is variable and, in several recent studies, not statistically different from the inherent risk of ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130,

6 leukaemia in untreated patients. Finazzi et al (2003) described a study of 25 ET patients aged less than 50 years followed for a mean of 8 years (range 5 14 years), none of whom developed acute leukaemia or other malignancy. In a retrospective study of 2316 patients with ET the rate of transformation to MDS/ AML was 1Æ3%, a similar rate was found for the 741 patients treated with hydroxyurea alone (0Æ7%) or 159 with interferon only (1Æ2%) in comparison with 4% of patients treated with alkylating therapy (Gugliotta et al, 1997). In 1638 PV patients from the ECLAP study with shorter follow-up (median 2Æ8 years), older age but not hydroxyurea was reported to be associated with acute leukaemia (Finazzi et al, 2005); however, the value of this data is probably limited by the relatively short median follow up. Furthermore, some biological studies have failed to show an increase in DNA mutations in patients with myeloproliferative disorders treated with hydroxyurea (Hanft et al, 2000). A randomized study is required to definitively address this issue. The early closure of the high-risk arm of MRC PT1 will prevent an answer from this source; however, the intermediate risk arm of PT1 where patients are randomized to either aspirin alone or aspirin with hydroxyurea ( may produce this data. Treatment of patients with multiple cytotoxic agents that have leukaemogenic potential has been repeatedly shown to be associated with high leukaemic risk. For example, in the longterm follow-up of a randomized study comprising 112 patients, rates of leukaemia were 0 of 20 in untreated patients, three of 77 (3Æ7% P ¼ n.s.) in those treated with hydroxyurea alone, but five of 15 (33%, P <0Æ0001) for those given hydroxyurea with busulphan (Finazzi et al, 2000). In 357 patients followed for a median of 8 years, Sterkers et al (1998) reported that 14% of those receiving multiple therapies including hydroxyurea developed leukaemia. This group and others also identified a chromosome 17p deletion in a high proportion of patients with leukaemia and a prior history of hydroxyurea (Sterkers et al, 1998; Bernasconi et al, 2002). Those patients requiring treatment with multiple agents may have biologically more aggressive disease and are perhaps inherently more likely to transform. An alternative theory proposed by Murphy (1997) is that one drug may potentiate the leukaemogenic effect of a subsequent drug. Whatever the cause, the importance of this data should not be underestimated for patients who become refractory or intolerant to hydroxyurea as the implications for choice of a second agent are very significant. Anagrelide Anagrelide, an imidazo quinazinoline derivative, reduces the platelet count in a species-specific manner, and at high doses anagrelide also inhibits platelet aggregation. In the largest study to date, 934 ET patients were evaluated for efficacy and 2251 for safety (Fruchtman et al, 2002). With a maximum follow-up of 7 years there was no evidence of increased risk of leukaemia; it is desirable to have longer follow-up to definitively exclude associated leukaemogenicity. Major side effects of anagrelide are consistent with its action as a phosphodiesterase inhibitor: palpitations (or forceful heartbeats), headaches, non-cardiac oedema and congestive cardiac failure (Anagrelide Study Group, 1992). Cardiac side effects have been a concern; indeed, a series of patients with anagrelide-induced cardiomyopathy have recently been reported (Jurgens et al, 2004). Although anagrelide appears to be effective at controlling the platelet count and is relatively well tolerated, two studies suggest it does not represent a panacea for treatment of ET. First, a study including 37 consecutive young ET patients, with median follow-up of 10Æ7 years, reported that 20% had thrombotic, and a similar proportion major haemorrhagic, complications (Storen & Tefferi, 2001). These events occurred when the platelet count was above /l, perhaps suggesting control of the platelet count to below /l might mitigate against this risk. However, the results of the MRC PT1 trial suggest that anagrelide is less effective at reducing thrombotic (particularly arterial thrombotic) and haemorrhagic events than hydroxyurea (Harrison et al, 2005) despite equivalent long-term platelet control. Furthermore, the excess rate of myelofibrotic transformation in the anagrelide arm of MRC PT1 is also of concern. Further data may be available from a number of on-going studies, including the non-randomized Evaluation of Xagrid Ò Efficacy and Longterm Safety (EXELS) postapproval non-randomized study including anagrelide- and hydroxyurea-treated patients. Interferon Interferon-a therapy may achieve a satisfactory response in more than 80% of ET patients (reviewed in Elliott & Tefferi, 1997) but it has many side effects and at least 20% of patients may be intolerant. Interferon-a has been used safely in pregnant women (Delage et al, 1996) and many regard it as the cytoreductive treatment of choice for high-risk women contemplating pregnancy (reviewed in Harrison, 2005). This drug has not been trialled in randomized studies either with aspirin or in comparison with hydroxyurea and may, in fact, be inferior to other conventional therapies. Interesting data from phase II studies of pegylated interferon-a were presented at the recent American Society of Hematology meeting by three groups, reporting a total of 112 patients: the discontinuation rate was 25 52% and one patient had a cerebrovascular accident (Langer et al, 2004; Samuelsson et al, 2004; Verstovsek et al, 2004). An Italian phase II study also reported that after 2 years the bone marrow biopsy from patients treated with pegylated interferon revealed reduced cellularity, increased dysplasia and fibrosis although the clinical significance of this is unclear (Gugliotta et al, 2004). Other histological studies have also demonstrated an increase in reticulin in marrows of patients with CML treated with conventional interferon-a (Thiele & Kvasnicka, 2001) but again, evidence of clinical significance is lacking. Collection of 158 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130,

7 further data will be important; the results of the Dutch Hovon study comparing anagrelide with pegylated interferon-a in ET are awaited. Alternative cytoreductive therapy Piprobroman is a generally well-tolerated piperazine derivative with a structural resemblance to alkylating agents; it appears to competitively inhibit pyrimidines. In 118 ET patients at high risk for thrombosis treated with piprobroman, the reported 10-year cumulative risks were 14% thrombosis and 3% acute leukaemia (Passamonti et al, 2002). Studies in PV also document its efficacy, for example, in a French study, 292 patients with PV were randomized to receive treatment with either hydroxyurea or piprobroman. Survival was similar in the two arms and shorter than that of the reference population. The risk of leukaemia was approximately 10% at the 13th year, with no significant difference between the two arms (Najean & Rain, 1997). Busulphan and radioactive phosphorous are alternative cytoreductive treatments for ET. Both can result in prolonged control of the platelet count but are also leukaemogenic. The leukaemogenic risk associated with low dose busulphan is probably small, and this drug can be useful in patients that are intolerant of hydroxyurea (Van de Pette et al, 1986). Radioactive phosphorus is definitely leukaemogenic but may have a role in very elderly patients for whom clinic attendance or regular blood counts are difficult. Platelet apheresis is usually effective in reducing the platelet count but only in the shortterm. Its use should be restricted to rare life-threatening situations where waiting for onset of cytoreductive therapy would be unacceptable, or in the event of a complication occurring in pregnancy with either the need for rapid count control or intolerance of interferon-a. The management would then be for daily platelet apheresis in combination with cytoreductive therapy. Splenectomy would only be indicated in patients with massive symptomatic splenomegaly, often accompanying myelofibrotic transformation. In this situation careful management is required to avoid postsplenectomy thrombosis. Allogeneic bone marrow transplantation would generally only be indicated for those patients who have transformed to high risk myelofibrosis and should, even in this context, be regarded as experimental therapy despite encouraging results (Cervantes, 2005; Kroger et al, 2005). Recently, a clinical trial of combined therapy with imatinib and anagrelide was attempted but failed to demonstrate adequate control of the platelet count in ET (Tsimberidou et al, 2003). Clinical trials There has been a dearth of prospective randomized studies in ET and only two have been published. A decade ago, the results of the first study in which 114 patients with high-risk ET (age >60 years or prior thrombosis) were randomized to either hydroxyurea or no cytoreductive agent was reported (Cortelazzo et al, 1995). The patients treated with hydroxyurea to a target platelet count of less than /l had significantly fewer thrombotic events (P ¼ 0Æ003). This landmark study provided the first clear demonstration that cytoreductive therapy reduces thrombotic events in ET. The second randomized study, MRC PT1, has three arms based upon thrombotic risk (Harrison et al, 2005). The low (aspirin only, observation) and intermediate risk arms (randomization aspirin versus aspirin and hydroxyurea) of this study continue to recruit patients (information about this on-going study is available at The high-risk arm, comprising a randomization between low dose aspirin and anagrelide or low dose aspirin and hydroxyurea, closed in 2003 because of an excess of adverse events in the anagrelide arm. The results demonstrated that more aspirin- and anagrelide-treated patients reached a composite primary end point of major thrombosis (arterial or venous), major haemorrhage or death from a vascular cause (P ¼ 0Æ03). When individual endpoints were assessed, arterial thrombosis (in particular transient ischaemic attacks), major haemorrhage and myelofibrosis were significantly more frequent for patients treated with anagrelide (P ¼ 0Æ004, 0Æ008 and 0Æ01, respectively). Paradoxically, venous thrombosis was much less frequent in patients treated with anagrelide (P ¼ 0Æ006). As venous events are less common than arterial thrombosis, this reduced, but did not reverse the trend of the composite endpoint. Long-term control of the platelet count was similar for both arms of the study. Of interest, at 2 years the prevalence of thrombotic events in the hydroxyurea-treated patients was the same for both these clinical trials, at 4% (Cortelazzo et al, 1995). Importantly, anagrelide and aspirin seems to offer at least partial protection from thrombosis as the prevalence of thrombotic events for anagrelide-treated patients (8%) was significantly less than the control arm of the previous study (24%). The results of MRC PT1 are important for many reasons, not least because they confirmed that control of the platelet count is not always a reliable indicator of reduced thrombotic risk. The success of hydroxyurea is hence likely to reflect the importance of additional factors, such as the reduction of the haematocrit, and/or leucocyte or neutrophil count or possibly subtle effects upon the endothelium. The increased risk of major haemorrhage in PT1 was principally gastrointestinal and was not anticipated. Interestingly, a previous long-term followup (median 10Æ7 years) study of 35 young patients (<50 years old) treated with anagrelide also documented a 20% incidence of major haemorrhage (Storen & Tefferi, 2001). It seems likely that these haemorrhagic events may result from some subtle effect of anagrelide upon platelet function that is possibly accentuated by aspirin or, alternatively, some local effect upon the gastrointestinal tract may have potentiated these events. Myelofibrotic transformation occurred in 16 patients on the anagrelide arm of the PT1 study in comparison with five on the hydroxyurea arm. The aetiology of myelofibrotic transformation in ET remains unclear but, as discussed earlier, pro- ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130,

8 fibrotic cytokines released from megakaryocytes or monocytes have been implicated. It is tempting to speculate that anagrelide may be less effective at suppressing this release or interaction than hydroxyurea, an agent that reduces the numbers of most haemopoietic cells. Increased myelofibrotic transformation has never previously been reported in the large cohorts of patients treated with anagrelide, however, the median follow-up was not long, patient numbers variable and there was no comparator arm in previous studies (Silverstein & Tefferi, 1999; Storen & Tefferi, 2001; Thiele et al, 2003; Petrides, 2004). The diagnostic criteria used in PT1 were akin to those of the PVSG; it is likely that some of the 809 patients in the high-risk arm would now be assigned a diagnosis of early or prefibrotic myelofibrosis. However, as the distribution of histological sub types is likely to be equal in both arms of PT1 this is unlikely to be the cause of this result and further data is likely to emerge. Myelofibrotic transformation is a rare complication of ET that may be subtle in its presentation, hence the data emerging from PT1 merit further investigation. However, the incidence of myelofibrosis in the anagrelide arm (3Æ95% at median follow-up of 39 months) is approximately in accordance with that previously noted, i.e. 0Æ9% per annum (Cervantes et al, 2002), supporting the view that hydroxyurea (incidence 1Æ2%) may be more effectively suppressing myelofibrosis. Management The concept of risk-stratified management is generally accepted in ET. Unfortunately the criteria used to allocate risk remain varied and under debate. For comparison, the MRC PT1 risk stratification is given in Table III with those proposed in the recent Italian Society of Haematology practice guidelines (Barbui et al, 2004) and recent publications (Elliott & Tefferi, 2005). Whilst consensus is apparent for high-risk patients there is a lack of homogeneity in the low and intermediate risk groups. It is the opinion of the author that sufficiently robust data to facilitate clear evidence-based stratification is lacking. There has also been considerable debate about the risks of disease-related events for low risk patients (Ruggeri et al, 1998; Table III. Risk stratification for treatment of patients with ET. MRC PT1 criteria Low risk Patients aged less than 40 years with all of the following: NO prior thrombosis NO hypertension or diabetes Platelet count < /l Italian Society of Haematology practice guidelines (Barbui et al, 2004) Patients <40 years AND platelet count < /l AND no prothrombotic comorbidity or years AND platelet count /l NO vascular risk factors/familial thrombophilia or years AND platelet count < /l NO vascular risk factors/familial thrombophilia Elliott and Tefferi (2005) None of factors below Recommendation: aspirin alone Recommendation: no cytoreductive therapy Recommendation: nil Intermediate risk Patients aged years with all of the following: NO prior thrombosis NO hypertension or diabetes years AND platelet count < /l AND vascular risk factors/familial thrombophilia or <60 years, NO thrombosis + either platelet count > /l or cardiovascular risk factor (e.g. smoking, diabetes) Platelet count < /l Recommendation: randomise aspirin versus HU + aspirin Recommendation: no consensus on treatment Recommendation aspirin + no consensus High risk Patients either age >60 years, or with one of the following: Prior thrombosis or haemorrhage Hypertension or diabetes Platelet count > /l (1500 <60 years) Recommendation: HU + aspirin for most patients ANY of age >60 years, prior thrombosis/ haemorrhage, platelet count > /l OR <40 years AND prothrombotic comorbidity AND platelet count < /l OR years, platelet count /l AND vascular risk factor/familial thrombophilia Recommendation HU (if >60 years OR years AND major thrombosis with aspirin) Anagrelide/Interferon (if age <40 years OR years NO major thrombosis) Age >60 years OR thrombosis Recommendation >40 years HU <40 years HU or interferon 160 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130,

9 Pearson et al, 1999), with concern that the control population reported by Ruggeri et al (1998) had an unduly high rate of thrombotic events. It is likely that crucial observations about disease biology will come from the low and intermediate risk arms of PT1. Until such time as these data become available, recruitment of patients into this and other studies is to be encouraged. Contrasting opinion in respect of aspirin therapy is also apparent from Table III. The Italian Guidelines (Barbui et al, 2004) recommend aspirin for patients with microcirculatory disturbance, recent major arterial vascular event or for whom there is laboratory evidence of coronary artery disease provided no clear contraindication to aspirin therapy is present. No consensus was reached concerning the use of anti-platelet therapy in patients over the age of 40 years who had one or more cardiovascular risk factors (cigarette smoking, arterial hypertension, diabetes mellitus, hypercholesterolaemia) the literature is variable in respect to the contribution of these factors to thrombotic risk. The results of PT1 suggest that aspirin and anagrelide should only be used together with caution, weighing the relative risks of haemorrhage against arterial thrombosis on an individual basis; if aspirin is used with anagrelide the platelet count should ideally be maintained within the normal range although it is not clear that this will reduce haemorrhagic risk. The MRC PT1 trial provides some clarity in relation to the choice of cytoreductive therapy for most high-risk patients as hydroxyurea has proven efficacy in comparison with anagrelide. No analysis of interferon relative to either hydroxyurea or anagrelide has been made. Ultimately, for the majority of patients, hydroxyurea will be the treatment of choice and the decision to use either anagrelide or interferon will depend upon a number of factors, including age of the patient and the perceived risk of leukaemogeniciy of hydroxyurea. For those patients intolerant or refractory to primary therapy it would seem prudent to avoid the addition of a potentially leukaemogenic drug where the use of combined hydroxyurea and anagrelide or interferon alone may be beneficial. Logically, patients should be monitored for myelofibrotic transformation, however, it must be acknowledged that there is no conclusive evidence that treatment other than transplantation can reverse or cure myelofibrosis. Transplantation is not indicated in ET other than for patients who have symptomatic transformation to myelofibrosis. The reader is directed to recent reviews considering the management of children with ET (Randi & Putti, 2004); pregnancy (Harrison, 2005) and surgery (Elliott & Tefferi, 2005). It is likely that we are on the brink of major molecular advances in our knowledge of ET and other myeloproliferative diseases. Major therapeutic advances are likely to depend upon further definition of the molecular basis of this condition. Recognition of the importance of MPDs and international collaboration in clinical trials, linked with well-designed scientific studies is required. References Afshar-Kharghan, V., Lopez, J.A., Gray, L.A., Padilla, A., Borthakur, G., Roberts, S.C., Pruthi, R.K. & Tefferi, A. (2004) Hemostatic gene polymorphisms and the prevalence of thrombotic complications in polycythemia vera and essential thrombocythemia. Blood Coagulation and Fibrinolysis, 15, Allen, A.J., Gale, R.E., Harrison, C.N., Machin, S.J. & Linch, D.C. (2001) Lack of pathogenic mutations in the 5 -untranslated region of the thrombopoietin gene in patients with non-familial essential thrombocythaemia. European Journal of Haematology, 67, Anagrelide Study Group (1992) Anagrelide, a therapy for thrombocythemic states: experience in 577 patients. American Journal of Medicine, 92, Andersson, P.O., Ridell, B., Wadenvik, H. & Kutti, J. (2000) Leukemic transformation of essential thrombocythemia without previous cytoreductive treatment. Annals of Hematology, 79, Andreasson, B., Harrison, C., Lindstedt, G., Linch, D. & Kutti, J. (2003) Monoclonal myelopoiesis and subnormal erythropoietin concentration are independent risk factors for thromboembolic complications in essential thrombocythemia. Blood, 101, 783. Annaloro, C., Lambertenghi, D.G., Oriani, A., Pozzoli, E., Lambertenghi, D.D., Radaelli, F. & Faccini, P. (1999) Prognostic significance of bone marrow biopsy in essential thrombocythemia. Haematologica, 84, Axelrad, A.A., Eskinazi, D., Correa, P.N. & Amato, D. (2000) Hypersensitivity of circulating progenitor cells to megakaryocyte growth and development factor (PEG-rHu MGDF) in essential thrombocythemia. Blood, 96, Barbui, T., Barosi, G., Grossi, A., Gugliotta, L., Liberato, L.N., Marchetti, M., Mazzucconi, M.G., Rodeghiero, F. & Tura, S. (2004) Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation. Haematologica, 89, Barosi, G. (1999) Myelofibrosis with myeloid metaplasia: diagnostic definition and prognostic classification for clinical studies and treatment guidelines. Journal of Clinical Oncology, 17, Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N., Swanton, S., Vassiliou, G.S., Bench, A.J., Boyd, E.M., Curtin, N., Scott, M.A., Erber, W.N. & Green, A.R. (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 365, Bazzan, M., Tamponi, G., Schinco, P., Vaccarino, A., Foli, C., Gallone, G. & Pileri, A. (1999) Thrombosis-free survival and life expectancy in 187 consecutive patients with essential thrombocythemia. Annals of Hematology, 78, Bernasconi, P., Boni, M., Cavigliano, P.M., Calatroni, S., Brusamolino, E., Passamonti, F., Volpe, G., Pistorio, A., Giardini, I., Rocca, B., Caresana, M., Lazzarino, M. & Bernasconi, C. (2002) Acute myeloid leukemia (AML) having evolved from essential thrombocythemia (ET): distinctive chromosome abnormalities in patients treated with pipobroman or hydroxyurea. Leukemia, 16, Besses, C., Cervantes, F., Pereira, A., Florensa, L., Sole, F., Hernandez- Boluda, J.C., Woessner, S., Sans-Sabrafen, J., Rozman, C. & Montserrat, E. (1999) Major vascular complications in essential thrombocythemia: a study of the predictive factors in a series of 148 patients. 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