Doctor's Brochure Myeloproliferative Disorders: MPDs. Essential Thrombocythemia: ET. Polycythemia Vera: PV

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1 Doctor's Brochure 2004 Myeloproliferative Disorders: MPDs Essential Thrombocythemia: ET Polycythemia Vera: PV Chronic Idiopathic Myelofibrosis: IMF Jan Jacques Michiels Goodheart Institute Rotterdam, MPD Center Europe Department of Hematology, Antwerp University Hospital Hans Michael Kvasnicka and Juergen Thiele Institute of Pathology, University of Cologne This Doctor's MPD Brochure provides basic information for MPD patients and their doctors to better find their way in order to improve proper diagnosis and management of the Ph 1- MPDs: ET, PV and IMF MPD Stichting NL MPD Foundation NL Grondelsloot BT Zoetermeer Phone Website:

2 Chronic Ph 1- negative Myeloproliferative Disorders (MPDs): A concise update Jan Jacques Michiels Goodheart Institute Rotterdam, MPD Center Europe Department of Hematology, Antwerp University Hospital Hans Michael Kvasnicka Institute of Pathology, University of Cologne Juergen Thiele Institute of Pathology, University of Cologne page Introduction Definition of chronic myeloproliferative disorders (MPDs) Tools to diagnose, characterize and stage MPDs Thrombocytosis in various MPDs Diagnostic criteria of essential thrombocythemia (ET) Morphological features of ET Presenting features of ET The paradox of thrombotic and bleeding complications in ET Risk stratification and treatment of ET patients Platelet lowering agents in ET Diagnostic criteria of polycythemia rubra (PV) Morphological features PV Signs and symptoms of PV The microvascular syndrome of thrombocytosis Hypervolemic symptoms Vascular complications Treatment of PV patients Phlebotomy and low dose aspirin Hydroxyurea Interferon-alpha First and second treatment options in PV Diagnostic criteria of prefibrotic and (classical) chronic idiopathic myelofibrosis (IMF) or myelofibrosis with myeloid metaplasia (MMM)/agnogenic myeloid metaplasia (AMM) Morphological features - problems to recognize prefibrotic/early IMF Clinical impact of prefibrotic early IMF Cytogenetic findings in IMF Signs and symptoms of classical IMF Prognosis in IMF Management of IMF patients Therapy of thrombocytosis Management of symptomatic anemia Treatment of splenomegaly References

3 Introduction About thirty years ago the philosopher Ivan Illich accused the medical establishment of being a major threat to health [91]. He argued that it was responsible for a growing epidemic of iatrogenic morbidity, and referred to this as a form of medical nemesis. Today he would doubtless express his opinion that little has changed, and, as far as the Philadelphia chromosome negative (Ph 1- ) chronic myeloproliferative disorders (MPDs) are concerned, he would be right. It is evident that clinicians and hematopathologists may often make matters confounding and worse in the way they manage this condition by lack of available knowledge and ill-defined criteria for the diagnosis of patients presenting with essential thrombocythemia (ET), polycythemia vera (PV) and chronic idiopathic myelofibrosis (IMF). In this context doctors may also offer self-defensive and over-cautious advice on risk management, creating a desperate state in some MPD patients by applying under - and over - treatment regimens and even improper medical therapeutic strategies. Patients are still too often taken through despair, hope, confusion, and back to an overall desperate mood because they do not understand or do not get the proper information or what an MPD really implies or what is going to happen to their lives. The MPD support groups founded by concerned patients are doing much to improve this overall adverse situation. Through its website, seminars, and newsletters, MPD patients now feel less lonely, know more about their disease and are taking a greater part in self management and in deciding how to handle their manifold emerging problems more or less adequately. This, in turn, is the beginning of a change in the medical practice by informing and educating the MPD patients and their attending physicians. This approach, aiming at a better understanding and advance of knowledge, works for hemophilia, it works for leukemia and there are reasons enough to believe that it would also work for MPDs. Definition of chronic myeloproliferative disorders (MPDs) Blood consists of red blood cells (erythrocytes), white blood cells (myelocytes - leukocytes, granulocytes, monocytes and lymphocytes), platelets (thrombocytes) and plasma. The bone marrow is the source for generating all blood cells and platelet formation (hematopoiesis). Hematopoiesis includes erythro-, myelo-, megakaryo- and finally lymphocytopoiesis. The erythropoiesis consists of nucleated red blood cell precursors which loose their nuclei and mature into red blood cells. The myelopoiesis or precursors of the granulopoiesis differentiate into a variety of nucleated white blood cells (leukocytes) while megakaryopoiesis consists of large cells with lobulated nuclei that produce platelets. Red blood cells transport oxygen from the lungs to all tissues of the body. The optimal hematocrit to transport oxygen from the lungs to tissues is Leukocytes play a major role in inflammation and the defence against micro-organism like bacteria. Platelets are involved in blood coagulation and lymphocytes mediate various immunological process. A MPD is featured by the proliferation of one, two or three hematopoietic cell lineages: megakaryopoiesis in ET, erythropoiesis and megakaryopoiesis in initial and early PV plus additional granulopoiesis in progressive (polycythemic) PV or megakaryopoiesis and granu- Fig. 1 Comparison of diagnostic classification systems for MPDs and accordingly calculated disease specifc loss of life expectancy [236]. Clinical presumptive ET 75.2% (n=631) IMF classical 14.9% (n=125) PV 9.9% (n=83) Differentiation of Ph 1- MPDs with thrombocythemia (n=839) PVSG criteria ET 76.6% (n=483)? UC 23.4% (n=148) Histopathology (WHO criteria) ET 33.5% (n=162) IMF % (n=184) IMF % (n=137) IMF-2 (n=84) IMF-3 (n=41) PV 37.5% 40 Loss of life expectancy in Ph 1- MPDs with thrombocythemia (n=839) 16.5% 20.3% PVSG criteria ET IMF classical 10 Loss of life expectancy PV Histopathology (WHO criteria) ET IMF-0 IMF-1 8.9% 21.6% 32.3% IMF-2 & IMF-3 PV 20.3% Loss of life expectancy 37.5% 2

4 lopoiesis with suppression of erythropoiesis in IMF [51,52, 70,227]. ET is a dominated by a proliferation of mature megakaryocytes and clinically characterized by a normal life expectancy when properly treated to prevent thrombotic and bleeding symptoms. PV consists of a trilineage proliferation including megakaryocytes, erythro- and granulopoiesis (panmyelosis) with a normal survival rate in the majority of the cases when properly treated to prevent thrombotic complications. Thrombocytosis is usually associated with early stage PV and progresses during the advanced stages. About 30% of PV patients develop a significant proliferation of granulopoiesis in the bone marrow and myelofibrosis leading to so-called postpolycythemic myeloid metaplasia within 10 to 15 years [61]. The latter feature indicates endstage disease and therefore worsening of prognosis. Pronounced thrombocytosis is usually present in the prefibrotic and early fibrotic stages of IMF and frequently mimicks ET, but platelet counts decrease when early stage IMF progresses to classical myelofibrosis with myeloid metaplasia [223, 226,229,234,236]. Classical IMF is a relative slowly progressive MPD featured by overt reticulin and collagen fibrosis of the bone marrow, splenomegaly (large spleen), anemia, thrombocytopenia and a shortened life expectancy [11,13,34,35]. Regarding the disease-specific loss of life expectancy for the Ph 1- negative MPDs which is adjusted to age and gender [83,84,103,104] a value ranging from 8% to 38% is observed. Impact of disease is significantly higher in elderly patients (Figure 1), especially in IMF and PV. In contrast, patients with true ET reveal no relevant reduction of life expectancy [236]. Thrombocytosis in various MPDs Thrombocytosis may occur in association with all variants of MPDs [75,172,252]. The majority of PV patients do present with an elevated platelet count [23,190]. Pronounced thrombocytosis is frequently observed in prefibrotic and early fibrotic IMF and often regarded as ET [226,235,236]. Thrombo-cytosis may preceed or accompany Ph-positive chronic myeloid leukemia (CML) or myelodysplastic syndrome (MDS) including the 5q-minus syndromes [27,49,121,251] and thus again may be confused with (true ET) as so-called ET with ringed sideroblasts [186]. In contrast with the typically enlarged and giant megakaryocytes in ET and PV [149,218], the megakaryocytes in so-called Ph 1+ (bcr/abl-positive) thrombocythemia [25,120,145,194] are significantly smaller than normal with rounded nuclei [132,143] consistent with features observed in CML [29,68,213,217]. Therefore the term Ph 1+ ET is a misnomer and should be discarded because it significantly adds to the confusion associated with Tools to diagnose, characterize and stage the MPDs Careful registration of complaints, signs and symptoms characteristic for ET, PV and/or IMF. Physical examination to document all findings related to ET, PV and/or IMF. Measurement of spleen size by palpation and more preferably in length diameter on ultrasound echogram. Routine measurement of peripheral blood parameters including hemoglobin, hematocrit, mean red cell volume, erythrocytes, leukocytes, white blood cell differential count, leukocyte alkaline phosphates (LAP) score, platelet count, serum lactate dehydrogenase activity (LDH), creatinine, uric acid, bilirubine and liver enzymes Peripheral blood and bone marrow smears morphology and differential counts. In any case where an MPD is suspected, representative pretreatment bone marrow biopsy (minimal length 15 cm) for histopathology and immunohistochemistry in search for characteristic morphological features of ET, PV or IMF using a proper evaluation form for a clear-cut documentation of findings (Figure 2). Biological markers like erythropoietin (EPO) level and optional parmeters like spontaneous endogenous erythroid colony formation (EEC), PRV-1 gene expression (PRV-1 positivity), clonality studies (women only), cmpl (thrombopoietin receptor expression) expression of peripheral blood platelets and bone marrow megakaryocytes. Cytogenetic analysis of peripheral blood and bone marrow nucleated cells. 3

5 Fig. 2 Cologne bone marrow evaluation sheet for standardized diagnosis of MPDs a clear-cut diagnosis of this condition [44,62]. The megakaryocytes in MDS show overt dysplastic features and are different from ET and CML [220]. On the other hand, essential thrombocythemia (ET) is due to a proliferation of only one of the three major hematopoietic cell lineages in the bone marrow, the megakaryocytes leading to a persistent increase in the number of platelets (thrombocytes) in the peripheral blood (thrombocythemia ). The megakaryocytes in ET are significantly enlarged and mature with hyperlobulated (staghorn-like) nuclei 4

6 [69,70,223,226,235]. ET should be clearly distinguished from reactive thrombocytosis (RT). RT is usually transient [75] and associated with diseases like chronic infections and inflammatory systemic diseases, malignancies, iron deficiency and may occur after splenectomy (surgical removal of the spleen). The megakaryocytes in RT are increased, but usually exhibit a normal size [140,226]. RT is not associated with an increased risk for thrombosis or bleeding. Diagnostic criteria of essential thrombocythemia (ET) Strict inclusion and exclusion criteria for the diagnosis of ET have been established by the Polycythemia Vera Study Group (PVSG) [ , ,205]. In addition to platelet count greater than 600 x10 9 /l megakaryocytic hyperplasia was an absolute requirement. For protocols, involving myelosuppressive therapy a platelet count greater than 1,000 x10 9 /l was generally required. A normal red cell mass or a hemoglobin (Hb) of less than 13 g/dl was required to exclude overt PV. No stainable marrow iron, and no more than 1g/dl increase in Hb after iron therapy. Collagen fibrosis more than one-third the cross-sectional area of marrow biopsy, and only mild fibrosis in patients that presented with splenectomy and a leukoerythroblastic reaction. Absence of the Philadelphia chromosome. It has to be emphasized that the PVSG criteria do not distinguish between true ET, prefibrotic or early IMF without a leukoerythroblastic peripheral blood picture [140,142,216,223, 226,228,235,236,240] and also fail to regard initial (latent) PV [127,150,162] with thrombocy- European clinical and pathological (ECP) criteria for the diagnosis of ET [140] Clinical criteria Pathological criteria A1 A2 Persistent increase of platelet count: in excess of 400 x 10 9 /l Normal spleen or only minor splenomegaly on echogram B1 Predominant proliferation of enlarged to giant megakaryocytes with hyperlobulated nuclei and mature cytoplasm, lacking conspicuous cytological abnormalities. No proliferation or immaturity of granulopoiesis or erythropoiesis A3 Normal or increased LAPscore, and normal ESR B2 No or only borderline increase in reticulin A4 Spontaneous megakaryocyte colony formation (CFU-Meg) and or endogenous erythroid colony formation (EEC) A5 No signs or cause of reactive thrombocytosis (RT) A6 No preceding or allied other subtype of MPD, CML or MDS A7 Absence of the Phchromosome A1 and B1 + B2 establish (true) ET. Any other additional A criterion confirms ET. ET: Grade I: platelet counts between 400 and 1,000 x10 9 /l; Grade II: platelet counts 1,000-1,500 x10 9 /l; and Grade III: platelet counts above 1,500 x10 9 /l. 5

7 Table 2. Clinical, biological and some pathological features of conditions that may mimic (true) ET, in particular when following the PVSG criteria [129,147,148,163,165] Features Hereditary ET True ET (WHO-criteria) Initial PV (false ET) False ET (IMF) Estimated incidence (%) < < Blood: Thrombocytes / / / / Erythrocytes N N N/ Hematocrit N N N/ Bone marrow: Erythropoiesis N N N/ Megakaryocytes Features Normal large/giant mature abnormal Myelofibrosis /+ Splenomegaly - - -/+ +/++ EEC - -/ PVR-1 0 -/ /++ = increased, slight, moderate, pronounced. = decreased, slight, moderate, pronounced N= normal, - = absent, + = present, + slight, ++ moderate, +++ pronounced Fig. 3 Morphological features of ET 6

8 tosis that may mimick ET, however, later show full-blown PV [147,233]. As compared with the WHO classification [92,236] and the clinicopathological MPD criteria [136,137,140,226,227], the PVSG criteria include true ET (Table 1) as well as thrombocythemias associated with initial-early stage IMF and even PV (Table 2). Moreover, symptomatic ET patients with microvascular circulation disturbances and increased platelet counts above normal and between 400 and 600 x10 9 /l [114,140,184] are disregarded and overlooked by the PVSG criteria [ ,165]. These obvious shortcomings prompted us to incorporate the WHO bone marrow criteria [92] by the combined use of the European pathological criteria and clinical (ECP) criteria as specific clues for the differential diagnosis of true ET, and thrombocythemias associated with PV and IMF [140,226,227,236]. Finally, the term hyperplasia should never be used in conjunction with a MPD, because in general pathology this implies a reactive increase in number. Morphological features of ET (Figure 3) Megakaryocytes in true ET are characterized by an enlarged to giant size and exhibit deeply lobulated nuclei with mature cytoplasm randomly dispersed or loosely clustered in a normocellular bone marrow (Table 1). There is no hypercellular bone marrow according to an agematched population and contrasting PV [231] no increase or left-shifting of the nucleated erythroid precursors or granulocytes is detectable [17,29,69,70,140,223,226,235]. Reticulin myelofibrosis is extremely rare in true ET at presentation and very few ET patients develop MF during long-term follow-up [30,70,234] indicating that true ET is the most benign variant of MPD with a normal life expectancy [236]. Moreover, there are very rare cases of congenital (familiar, hereditary) ET [170] and a minority of young ET patients that have polyclonal hematopoiesis [71,243,258,259]. By definition polyclonal ET is not a MPD since its cause lies outside the hematopoietic stem cell and is based on a mutation of the thrombopoietin gene[71,243,258]. The majority of ET and all PV patients have clonal hematopoiesis consistent with a manifest MPD. About half of the ET and nearly all PV patients exhibit spontaneous EEC [54,63,96,113,253]. In this regard, the development of a sensitive and specific assay for the EPO is more important. While a significantly elevated EPO level suggests tissue hypoxia as the (reactive) cause for erythrocytosis, a normal or slightly raised value does not exclude this pathomechanism [24,32,76,128,255]. Altogether ET according to the PVSG criteria [ ,165] may present as true ET, however, in the majority of cases of ET as prefibrotic and early IMF [236], and finally as ET with features of latent or initial PV that is difficult to discriminate [90,94,147,181] when not applying modern means of bone marrow processing and staining techniques [233]. This salient point has been repeatedly demonstrated by a scrutinized evaluation of bone marrow trephine biopsy specimens showing a heterogenous picture that was significantly associated with prognostic impact [7, ]. Over-expression of the mrna of a novel gene, designated as PRV-1 was identified in mature peripheral blood neutrophil leukocytes and hailed with great enthusiasm as a molecular marker of PV [156,157,212]. Recently expectations were deflated by the demonstration that PVR-1 is constitutively expressed in bone marrow cells and therefore, does not distinguish PV from reactive and other MPDs [26]. This statement was supported by the finding that some patients with EEC formation and clinical as well as laboratory characteristics of PV failed to exhibit raised mrna levels of PVR-1 [117]. Finally, a prospective study where real-time PCR-based assay showed a PVR-1 expression across the MPDs and also in secondary (reactive) polycythemia and therefore it was concluded that quantifying PRV-1 mrna is not self-sufficient for the diagnosis of PV [211].The suggestion that PVR- 1 positive cases with ET comprise a distinctive subgroup [78] should therefore be discussed with caution, because it is tempting to speculate that these patients may actually present with an initial [233] or latent PV [181] that is accompanied by an elevated platelet count, but does not fulfill the relevant criteria yet [128,150,162]. This heterogeneity of so-called ET as diagnosed by the stringent postulates of the PVSG is shown in Table 2. Although in PV onset is difficult to recognize, it is reasonable to assume that a number of patients formerly described as idiopathic erythrocytosis [127,150,162] may fall into this group of initial PV that occasionally present with an excess in platelets and may mimick ET [140,233]. 7

9 Presenting features of ET ET may be diagnosed in asymptomatic individuals and picked up by routine blood testing. The majority of ET individuals present with typical platelet-mediated microvascular circulation disturbances [38,40,75,89,93,172,173]. As prospective studies are lacking, the relative incidence and the spectrum of microvascular disturbances, major thrombosis and bleeding complications in true ET and variants preceding or associated with initial (latent) PV and IMF are unknown. Mean age of ET patients at time of diagnosis is about 50 to 60 years. About 20 to 25% of ET patients are younger than 40 years of age. ET occurs 1½ to 2 times more frequent in women than in men. This female preponderance is more pronounced at young ages. The signs and symptoms of platelet-mediated microvascular disturbances are located in the end- arterial circulation of the peripheral, cerebral, coronary, skin and abdominal microcirculation [38,40-42,74,98,130,131,133,134,139,144,172,185,244,246]. The typical ET-related microvasular disturbances in the peripheral circulation include a broad spectrum of acropresthesias, erythromelalgia and its ischemic complications of acrocyanosis or even gangrene of one or more toes or fingertips [130,131,144]. Erythromelalgia is characterized by warm, red, congested extremities and painful burning sensations [130,131]. Acroparesthesias like tingling, pins and needles sensations and numbness in the toes or fingers usually precede the disabling burning distress. Warmth intensifies the discomfort and cold provides relief. If left untreated erythromelalgia may lead to painful acrocyanosis and gangrene of one or more toes or tips of the fingers. The burning distress is always associated with local swelling with mottled redness and blue spots. At more advance stage intense burning, throbbing and aching with peeling of the skin of the affected toes or fingers that become cold and ischemic. When erythromelalgia progress to ischemic acrocyanosis or gangrene of a toe or fingertip, the peripheral arterial pulsations are usually normal [130,131,144]. The typical ET-related microvascular disturbances of the cerebral and ocular circulation include a broad spectrum of migraine like headache, atypical transient ischemic attacks (TIAs), typical TIAs, occasionally minor stroke, and rarely major stroke [40,98,133,134]. ETpatients may present focal symptoms: transient monocular blindness, transient mono- or hemiparesis, both of these, migraine accompaniments, and partial stroke. Non-focal symptoms presenting ET-patients include transient attacks of postural instability, dysarthria, and scintillating scotomas. The transient focal and non-focal neurological and visual symptoms all display a sudden onset, usually occur with a march rather than all at one time, lasted a few seconds to several minutes and were usually associated with or followed by a dull or pulsatile migraine-like headache. This clinical presentation is very atypical for transient ischemic attacks caused by atherosclerosis, but the striking similarity with migraine accompaniments supports the crucial of platelets in the pathogenesis of ischemic neurological disturbances in essential thrombocythemia. Acute coronary syndromes, including myocardial infarction and unstable angina pectoris, have been described as the presenting symptom of ET like a thunder clap at the blue sky [185]. The paradox of thrombotic and bleeding complications in ET In 809 ET patients from 11 retrospective studies, 36% were asymptomatic, 58% experienced thrombotic and 17% bleeding symptoms while not on aspirin. The arterial thrombotic manifestations were described as microvascular disturbances in 41% involving the extremities (24%) or cerebral circulation (17%), and as major thrombosis in 20% [40-42,74,173]. The paradoxical occurrence of microvascular disturbances and mucocutaneous bleedings is usually seen at platelet counts between 1,000 and 2,000 x10 9 /l. At increasing platelet counts from below 1,000 to in excess of 2,000 x10 9 /l, the arterial thrombophilia of thrombocythemia vera changes into a spontaneous bleeding tendency as the consequence of a platelet-mediated increased proteolysis of the large von Willebrand factor (VWF) multimers leading to a type II acquired von Willebrand syndrome [139]. At platelet counts between 1,000 and 2,000 x10 9 /l, thrombosis (erythromelalgic thrombotic thrombocythemia - ETT) and bleeding (Figure 4) frequently occur in sequence or paradoxically and low dose aspirin does prevent thrombot- 8

10 ic complications but aggravates or may elicit bleeding symptoms. In this situation aspirin should not be discontinued, but there is an urgent need to reduce the platelet count to below 1,000 x10 9 /l by anagrelide, interferon or hydroxyurea (Figure 4). Hemorrhagic thrombocythemia (HT) is a spontaneous bleeding tendency that occurs at extremely high platelet counts far in excess of 1,000x 10 9 /l. HT is a clinical syndrome of recurrent spontaneous mucocutaneous bleedings (including bruises and hematomas of the skin, epistaxis, gum bleeding, silent gastrointestinal bleeding leading to iron deficiency) and secondary hemorrhages after surgery and trauma. Frequency and severity of hemorrhages in patients with HT is directly related to the pronounced increased number of circulating platelets. HT is caused by an acquired VWF deficiency type II at increasing platelet counts in excess of 1,000 to 2,000 x10 9 /l. Fig. 4 The paradox phenomenon of bleeding and thrombosis in ET related to platelet count and ensuing therapeutic strategies (Rotterdam concept) The laboratory features of acquired von Willebrand syndrome in reported cases of HT are characterized by: a very high platelet count (range 1,285 to 5,860 x10 9 /l), a prolonged Ivy or Simplate bleeding time, a normal factor VIII coagulant activity and VWF antigen (VWF:Ag) concentration, a very low VWF-ristocetine cofactor activity (VWF:RCo) and vwf-collagen binding activity (VWF:CB) and absence of large VWF multimers simulating a type II von Willebrand disease. Reduction of the platelet count to below 1,000 x10 9 /l by platelet lowering agents usually results in the disappearance of the bleeding tendency and improvement of the von Willebrand syndrome, but the thrombotic tendency persists as long as platelet counts are between 400 and 1,000 x10 9 /l. The microvascular disturbances already occur at platelet counts between 400 and 1,000 x10 9 /l [114,184]. Low-dose aspirin (50 to 100 mg/day) is highly effective and safe in the cure and prevention of thrombotic and ischemic events and does not elicit bleedings at platelet counts below 1,000 x10 9 /l (Figure 4). Relief of microvascular circulation disturbances in ET is not obtained with dipyridamol, ticlopedine, coumadin or heparin. Risk stratification and treatment of ET patients The main challenge for treating ET patients is to select cohorts who will benefit form a cytoreductive or antiplatelet therapy, because it remains doubtful whether the positive effects of treatment outweigh the potential hazards in all cases [77] The Bergamo criteria for thrombotic risk stratification are derived from a cohort of 100 ET patients (mean platelet count 1,135, age below 40 in one third, between 40 and 60 in one third and above 60 years in nearly one third) treated in the 1980s with busulfan (short-remitters and long-remitters) and who did not receive aspirin [41]. Platelet counts in the ET patients at time of thrombotic event or recurrence were increase platelet counts between normal and more than 3,000 indicating that 9

11 Table 3. Risk stratification of ET patients: therapeutic implications [40-42, ] Low thrombotic and bleeding risk Age up to 80 years, No vascular risk factor or previous thrombosis No associated disease and normal life expectancy No history or signs of bleeding Platelet count between 400 and <1,500 x10 9 /l and completely asymptomatic in whom the use and indication of aspirin is uncertain. Platelet count between 400 and <1,000 x10 9 /l and symptoms for microvascular disturbances with a clear indication for low dose aspirin mg/day Intermediate thrombotic and bleeding risk Age up to 80 years. *Symptomatic for microvascular disturbances and platelet count between 1,000 and 1,500 x10 9 /l with a clear indication for low dose aspirin 100 mg/day. No history of bleeding or major arterial or venous thrombosis. Absence of any vascular risk factor. At platelet counts in excess of 1,000 x 10 9 /l aspirin may elicit bleeding symptoms, which will disappear after reduction of platelet counts to below 1,000 x10 9 /l. High thrombotic and/or bleeding risk Clear indication for platelet reduction plus low dose aspirin except when contraindicated Platelets >1,500 x 10 9 /l History of major thrombosis ( myocardial infarction, stroke, peripheral vascular diseas e) Presence of vascular risk factor (hypertension, diabetes, hypercholesterolemia etc) History or presence of spontaneous or major bleedings. Bleedings elicited by low dose aspirin at platelets <1,500x10 9 /l. Progression from low risk to high risk ET-patients. Platelet lowering agents: Anagrelide or interferon below the age of 65 years Hydroxyurea above the age of 65 years excess in thrombocytes is the determinative factor of thrombotic events with a statistical higher incidence in ET patients above 60 years as compared to less than 40 years in ET patients not in remission and not on aspirin. Low-risk ET patients are below 60 years of age, have no history or manifestations of major thrombosis or bleeding symptoms, and exhibit platelet counts <1,500 x10 9 /l. All four features must be present to establish a low risk group. High risk ET is defined by age above 60, a history of Fig. 5 Prognostic factors in ET major thrombosis, spontaneous bleeding mani- 100 festations and/or platelet counts in excess of 1,500 x /l [41,42]. Only one criterion need be present to define high risk. 60 However, the majority of ET patients above the age of 60 years do have 40 platelet counts below 1,500 x10 9 /l, and do not have a history of major 20 thrombosis/bleeding with ET no history of thrombosis or hemorrhage the absence of a vascular risk factor, which ET history of thrombosis or hemorrhage ET total 0 according to Rotterdam criteria should be consid- % Survival 10 Years after diagnosis

12 ered as low risk (platelet <1,000) or intermediate risk (platelets 1,000-1,500) [244,246]. This constellation is regarded in Table 3. However, regarding the age- and sex-adjusted relative survival rate [83,84,104], age at diagnosis has no significant impact on the disease progress, but only a history of thrombosis or hemorrhage in the prediagnostic period. These patients reveals a significant higher rate of clinical complications which is responsible for the overall worsening of prognosis (Figure 5). Besses et al retrospectively assessed the clinical and hematological factors predictive for the occurrence and recurrence of microvascular disturbances and macrovascular thrombosis in 148 consecutive ET patients [22]. Aspirin was given in only 61 for secondary prevention of thrombotic complications. Among 63 (43%) symptomatic patients at presentation, 43 (29%) experienced microvascular disturbances at a mean platelet count of 1,050 ± 556 x10 9 /l, and 9 (6%) with hemorrhagic complications were registered at a mean platelet count of 1,325 ± 146 x10 9 /l. During follow-up while not on aspirin in the majority of them, 73 (49%) ET patients were symptomatic for microvascular disturbances in 41 (28%), macrovascular thrombosis in 33 (22%) and bleeding complications in 17 (11%). A total of 37 (25%) ET patients had major vascular complications with a mean platelet count of 956 ± 321 x10 9 /l, and 2 (1.3%) patients died of major thrombosis. At multivariate analysis, age >60 years, history of major thrombosis and hypercholesterolemia were the variables associated with increased risk of major thrombosis in symptomatic ET patients while not on aspirin in the majority of them [22]. ET patients with symptoms or a history of microvascular disturbances in the absence of vascular disease and risk factors are candidates for low dose aspirin at platelet counts between 400 and 1,500 x10 9 /l [244,246]. The main question to be answered is whether low dose aspirin at platelet count between 400 and 1,500 x10/l versus correction of platelet counts to normal by platelet lowering agents are equally effective in preventing minor and major thrombotic events in ET patients with symptoms or a history of TIAs, minor stroke, or myocardial infarction in the absence of vascular disease and risk factors [22,41,42,244,246]. In the Italian prospective trial, 144 high risk ET patients with a mean platelet count of 788 x10 9 /l, range 53-1,240 x10 9 /l were randomized for hydroxyurea and placebo [42]. In this study 70% in the hydroxyurea arm and 69% in the placebo arm received antiplatelet drugs, aspirin or ticlopedine. Two of 56 (3.6%) patients in the hydroxyurea arm developed major thrombosis, and 14 of 58 patients (24%) in the placebo arm developed ischemic events: major thrombosis 2 (3.4%: 1 stroke, 1 deep vein thrombosis), and microvascular thrombosis in 12 (20,6%: 5 TIAs, 5 digital artery occlusion and 2 superficial thrombophlebitis). At least 10 of these 14 symptomatic ET patients in the placebo arm were not on aspirin at time of ischemic event. Therefore, a direct comparison of hydroxyurea versus low dose aspirin in high risk ET at platelet count between 400 and 1,500 x10 9 /l would predict to be equally effective [244,246]. Table 4. Proposed risk stratification of ET patients: therapeutic implications Platelets: x10 9 /l x10 9 /l x10 9 /l > 1500 x10 9 /l Symptoms: Completely Microvascular Major Irrespective of Asymptomatic disturbances only* Thrombosis age and symptoms No vascular risk No vascular risk Bleeding Symptoms Age < 60 All ages All ages All ages Aspirin uncertain Low dose aspirin Continue aspirin Platelet reduction to Wait and see If aspirin side effects Platelet reduction* <1000 plus aspirin *At platelet counts in excess of 1000 x10 9 /l aspirin will usually elicit bleeding symptoms, which disappear after reduction of platelet counts to below 1000 x10 9 /l with continuation of aspirin. Age > 60, no vascular risk Platelets < 1000: aspirin only Platelets > 1000: aspirin plus platelet reduction from above to below 1000 Age >60 and presence of vascular risk factor: aspirin plus platelet reduction to normal Vascular risk include: atherosclerosis, hypertension, hypercholester olemia, diabetes. 11

13 The Bergamo and Rotterdam criteria for thrombotic risk assessment are very similar [41,42,244,246], but differ with regard to age and clear indication for aspirin in low and clear indications for platelet lowering agents in intermediate and high risk ET. In our experience true ET patients above the age of 60 years, platelet counts below 1,000, asymptomatic or microvascular symptoms, but with no history of major thrombosis or bleeding and absence of vascular risk factors are not at high risk for a first major thrombotic event while on low dose aspirin (Table 4). The risk assessment in the UK PT-1 study is problematic [164]. In the UK PT-1 study, low risk ET is defined by age below 40 years and completely asymptomatic and randomized for placebo versus low dose aspirin. The intermediate risk group is defined age 40 to 60, completely asymptomatic and randomized for aspirin versus aspirin plus hydroxyurea, which according to the Bergamo and Rotterdam criteria [41,42,244,246] means over-treatment. According to the Bergamo and Rotterdam criteria the intermediate risk group in the UK PT-1 study is in fact low risk and should have been included in the low risk for the randomization placebo versus low dose aspirin. In the UK PT-1 study, symptomatic ET patients including erythromelalgia, TIAs, major thrombosis, and asymptomatic ET patients with the presence of vascular risk factors or age above 60 are labelled as high risk (Table 4). The high risk of the UK PT-1 study consists of low, intermediate and high risk ET according to the Rotterdam criteria [244,246]. The Rotterdam criteria recognize an intermediate group with increased risk on bleeding and thrombosis who may be candidate for platelet lowering therapy and this group of patients is labelled as low risk by the Bergamo criteria [41,42] and as high risk by the UK PT-1 criteria. In this context it should not be overlooked that inconsistencies in diagnostic criteria significantly incfluence risk calculations in ET [236]. Improvement of the diagnostic reliability for ET seems to be largely based on the recognition of BM histopathology [235] that has been explicitly accomplished by the WHO classification [92,230] and thus represents a major advance in comparison to the PVSG criteria [148]. By separating IMF from (true) ET it has been realized that outcome is also significantly different in both series of patients (Figure 1). This impression of a heterogeneity of ET patients diagnosed according to PVSG with respect to their survival pattern has been recently confirmed [7,235,240]. Evaluation of certain BM features revealed that in 40 of the 93 patients from the Italian ET Study Group [38] an increased cellularity was present including myeloid precursors, a higher content of fibers and trapped dysplastic megakaryocytes [7]. These results fit well with other descriptions of corresponding BM findings in initial and early IMF [70][30,31]. These studies provide persuasive evidence that a considerable number of patients assumed to present with ET according to the PVSG criteria [102,147,148] are more likely to have IMF and consequently are characterized by an unfavorable prognosis. Platelet lowering agents in ET Anagrelide is a selective inhibitor of megakaryocyte differentiation (endoreduplicative activity) [237], but does not reduce the number of megakaryocytes [242]. By this pathomechanism this agent lowers platelet production without a significant effect on bone marrow erythro- and myelopoiesis, and without an increased risk concerning development of myelofibrosis and leukemia [2,28,73,167,189,199,235,260]. The average dose to control thrombocytosis is 2.0 to 2.4 mg/day [205]. The overall response for both complete and partial response in a large group of thrombocythemic patients was 76 to 79% [2,73,166]. The most common adverse event of anagrelide was headache in 37%. It was controlled by acetaminophen and lasted <2 weeks. Palpitations were reported in 26%, diarrhoea accounted for 25%, and fluid retention was seen in 22% [166,195,199,202]. Other less frequent adverse effects caused withdrawal side effects included nausea, abdominal pain, asthenia, flatulentia and bleeding [5,107,168,195]. Adverse side effects caused 13% of these patients to withdraw anagrelide [195,198,199]. Interferon is effective in the treatment of ET to reduce the excessive platelet count without an increased risk on cancer and leukemia [56,198]. A relative low dose of 3 to 5 million units subcutaneously 3 times a week will induce a complete and partial response in 70 to 80%. Adverse 12

14 side effects will cause withdrawal of interferon in 20 to 25%. The new PEG-interferon to be given subcutaneously once a week has less side effects and is predicted to be as effective as interferon [77]. Hydroxyurea is currently the palliative treatment of choice for the MPDs including ET. Hydroxyrea is an effective cytostatic agent to reduce not only platelet but also erythrocyte and leukocyte counts in MPD patients without side effect during the first 5 years of follow-up, but the degree of increased risk on leukemia and cancer after long-term follow-up of more than 10 to 15 years in ET patients is unknown [77]. There is a legitimate concern regarding the leukemogenic potential of hydroxyurea [67,88,116,118,171]. In a large study, 251 of 357 ET patients were treated with hydroxyurea either alone or in combination with other agents and followed for 8 years [193]. The comparative rates of evolution into acute leukemia or myelodysplastic syndromes were 3.5% for hydroxyurea alone, 14% for hydroxyurea in combination with other agents busulfan or 32 P, 7% for 32 P, and 3% for busulfan [193]. This study showed a high frequency of 17p deletions in the patients with acute transformations treated with hydroxyurea. There has been no randomized study with an appropriate control arm which implicates hydroxyurea as being leukemogenic in previously untreated ET patients with adequate baseline clinical, pathological, cytogenetic and molecular examinations of blood and bone marrow. The current availability of non-leukemogenic platelet-lowering agents (anagrelide and PEG-interferon) provides the opportunity to investigate the very long-term safety (more than 10 to 15 years) of hydroxyurea in a controlled fashion, but is very difficult to realize. Alternatively, large scale observational studies of ET patients treated with hydroxyurea alone for more than 10 to 15 years are lacking and should be initiated. Hydroxyurea and busulfan are the two candidates for a first choice treatment option in high risk ET patients older than 65 to 70 years [202,205]. If anagrelide and interferon fail in high risk ET patients younger than 65 years, hydroxyurea but not busulfan should be the choice. Radioactive phosphor, 32 P, should not be used for the treatment of ET and PV because of its high risk of leukemia and cancer after long-term follow-up. In a recent retrospective study, 259 MPD patients (183 PV and 76 ET with a mean age 72 years, range years) were treated with 32 P in a regional nuclear medicine department [9]. After a median follow-up of 5 years (range 2 to 11 years) 18 (7.6%) developed leukemia and cancer arose in 19 (8%) patients [9]. Diagnostic criteria of polycythemia rubra (PV) Strict inclusion and exclusion criteria for the diagnosis of PV were established by the PVSG in 1975 [20,21,23,129,148,202]. Three major and a few minor criteria were postulated (Table 5) to ensure that patients who entered into the PVSG prospective trials were indeed suffering from that disease and not from secondary erythrocytosis-polycythemia (SP) to be treated with potential leukemogenic drugs. However, the original set of PVSG criteria failed to add bone marrow histopathology proposed by the German pathologists as a specific pathognomonic criterion for PV and the differentiation between PV and SP [17,29,68-70,214,227,231,233]. The original PVSG criteria in Table 5 overlook by definition the latent and the early stage of PV [233], the so-called idiopathic erythrocytosis characterized by a mild increase in hemoglobin (Figure 6) or red cell mass, normal leukocyte count, no splenomegaly, and absence of any cause of SP [127,138,150,162]. This comprises about 15 to 30% of early stage 1 and 2 PV patients featured by EEC and frequent PVR-1 positivity, and a typical PV bone marrow picture [140,233]. These Table 5. The PVSG diagnostic criteria for PV [21,23,148] A. Major criteria A1.Raised red cell mass. Male > 36 ml/kg, female >32 ml/kg A2. Normal arterial oxygen saturatin >92%. A3. Splenomegaly on palpation. B. Minor criteria B1. Thrombocythemia. Platelet count >400 x10 9 /l B2. Leukocytosis >12 x10 9 /l (no fever or infection) B3. Raised leukocyte alkaline phosphatase (LAF) score >100 or raised B 12 (>900 ng/l), or raised unsaturated B 12 binding capacity (>2,200 ng/l) 13

15 Fig. 6 Comparative laboratory parameters in patients with ET and PV early stages of PV are not treated, but at very high risk for potential fatal macrovascular complications. Based on new laboratory investigations including spontaneous EEC [256], serum EPO levels [24,43], clonality markers and spleen size on echogram, Pearson proposed his modification of the PVSG criteria, however, again without reference to bone marrow morphology (Table 6) [129,163,165]. Spontaneous EEC and especially EPO levels [24,32,43,53, 128, 129] have outstanding diagnostic specificity and sensitivity to differentiate between PV and and SP comparable to characteristic bone marrow features [231,233], while the discriminating impact regarding the distinctive power of the PVR-1 positive expression remains debatable [39,65, 78,97,99,117, 156, 211, 212]. Altogether their positive predictive and diagnostic value is far less as compared to the WHO classification [169] and the new European clinical and pathological criteria (ECP) for PV (Table 7). Therefore, we recently introduced the ECP criteria that are essential based on the WHO guidelines as the current gold standard for the diagnosis of PV and its differentiation from SP [138,140]. The quantitative measurement of the PRV-1 gene RNA expression reflects the polyclonal marker expression of activated granulocytes [26] very similar to the leukocyte alkaline phosphatase (LAP) score (Figure 6), whereas the autonomous EEC indicates the spontaneous growth of monoclonal erythroid precursor cells which are hypersensitive to EPO [24,43,163]. The EEC-positive and PRV-1-positive MPDs consist of a broad spectrum of disorders including ET, early and advanced stage PV and IMF [158]. About half of the ET patients according to the WHO and ECP criteria are predicted to be EEC-negative and PRV-1 negative [78,99], but we do not know whether this EEC/PRVnegative ET is polyclonal or monoclonal and whether these are true ET or variants of ET as the presenting symptom of prefibrotic or early fibrotic IMF, because a considerable fraction of patients with (classical) IMF do exhibit PRV-1 positivity [158]. Therefore this finding is not unexpected and again demonstrates the pitfalls when relying alone on this ill- Table 6. Modified PVSG criteria for the diagnosis of PV [129,163,165] A. Major criteria A1. Raised red cell mass (>25% above mean normal value) A2. Absence of secondary polycythemia A3. Palpable splenomegaly. A4. Clonality marker e.g. abnormal marrow kar yotype B. Minor criteria B1. Thrombocythemia. Platelet count >400 9 /l B2. Neutrophil leukocytosis (neutrophil count >10 x10 9 /l) B3. Splenomegaly on isotope/ultrasound scanning B4. Characteristic spontaneous EEC or reduced serum EPO level A1 + A2 + A3 or A4 establishes PV, and A1 + A2 + two of B establishes PV 14

16 Table 7. European clinical and pathological (ECP) criteria for the diagnosis of Polycythemia Vera (PV) [140] Clinical criteria Pathological criteria A1 Erythrocytes >6 x10 12 /l, hemoglobin: male >18.5 g/dl, female >16.5 g/dl and hematocrit: male >0.51 %, female >0.48 %). Raised red cell mass (optional) RCM: male >36 ml/kg, female >32 ml/kg. B1 Increased cellularity with trilineage myeloproliferation (i.e. panmyelosis). Proliferation and clustering of small to giant (pleiomorphic) megakaryocytes. Absence of stainable iron. No pronounced inflammatory reaction (plasmacytosis, cellular debris). A2 Persistent increase of platelet count: grade I: 400-1,500, grade II: >1,500. B2 Spontaneous erythroid colony formation. A3 Splenomegaly on palpation or on ultrasound or CT (>12 cm length diameter). Grading of myelofibrosis in (MF) PV A4 Granulocytes >10 x10 9 /l MF 0 prefibrotic stage PV and/or raised LAP-score in the absence of fever or infection. MF 1 early fibrotic stage PV A5 Absence of any cause of secondary erythrocytosis. MF 2 MF 3 manifest myelofibrosis in PV advanced myelofibrosis in PV A6 Low plasma EPO level MF >3 osteosclerosis, decreased cellularity A1 + B1 + B2 establishes Polycythemia Vera (PV) - any other criterion confirms PV defined polyclonal molecular marker that is also expressed in reactive inflammatory conditions and resembles very strikingly the activity of the LAP. Regarding prognosis an overall loss of life expectancy of about 20% is observed (Figure 1), however disease impact is significantly higher in older patients [105]. Beside the age at diagnosis, signs of generalisation of disease like liver size and increase in leukocytes are generally associated with a worsening of prognosis. Based on these findings a simplified prognostic score can be calculated [105], which helps to discriminate into low- and high risk patients (Figure 7). Fig. 7 Prognostic factors in PV 100 Prognostic staging for PV: Cologne score % Survival PV total Age < 60 yrs. Age 60 yrs. Liver size 2 cm WBC 20 x 10 9 /L Years after diagnosis Parameter Age (years) Liver size (cm) Leukocytes ( 10 9 /l) Prognostic impact > 60 2 > 2 1 >

17 Morphological features of PV (Figure 8) In contrast to the predominant megakaryocytic proliferation in a normocellular bone marrow in ET, PV usually shows a slight to moderate hypercellularity and a marked proliferation of all cell lineages termed panmyelosis [17,69,70,231], but especially of erythroid precursors [221,227], even in the latent (subclinical) or initial or early stages [233] as shown in Tables 7 and 8. In PV, megakaryocytes are either dispersed or loosely grouped, especially in the patients presenting with elevated platelet counts [59-61,68-70]. Unlike those in SP, megakaryocytes in PV show a pleiomorphous aspect featured by a loose clustering of giant to small megakaryocytes displaying regularly lobulated nuclei, and lack of maturation defects of nuclei and cytoplasm [70,231]. There is always a proliferation and left-shifting of the neutrophil granulopoiesis and especially erythroid precursor cells [221,227] with formation of extended sheets. Contrasting SP, a lack of iron-laden macrophages or inflammatory reactions of the myeloid stroma (perivascular plasmacytosis), eosinophils, accumulation of cell debris is remarkable [227,231,233]. Endstages of PV [61] reveal predominant granulocytic proliferation associated with grossly abnormal megakaryocytes and reticulin-collagen myelofibrosis consistent with the so-called post-polycythemic myelofibrosis/metaplasia and splenomegaly (Tables 7 and 8). Whether EEC/PRV-1-positive and EEC/PRV-1-negative IMF patients are linked to this kind of myelofibrosis and agnogenic myeloid metaplasia respectively remains to be elucidated [211]. Using the ECP criteria including bone marrow morphology and repeatedly performed trephine biopsies [30,70] and the biological markers EEC, PRV-1 and serum EPO levels, we are able to distinguish at least 6 stages of PV with typical bone marrow features (Table 8). These are grossly reflecting the dynamics of this condition starting with an initial (latent) phase and finally terminating into a fibro-osteosclerotic stage consistent with postpolycythemic myeloid metaplasia.. However, chemo- or interferon therapy was not able to inhibit the progress of myelofibrosis [100] comparable to IMF [238,239,241]. Fig. 8 Morphological features of PV 16

18 Table 8. The six stages of polycythemia vera according to Wasserman and Michiels Stage ET PV PV PV PostPVMF Spent Hemoglobin mmol/l N/ N/ Erythrocytes N/ N/ Hematocrit N/ 0.50 >0.60 >0.60 Thrombocytes x10 9 /l </>400 <400 >400 >1000 variabel N/ Leukocytes x10 9 /l N N N >15 variabel >20 Spleen on echogram <15 <12 <15 >15 > 20 >20 cm Bone marrow: Cellularity, erythropiesis Megakaryocytes Myelofibrosis (MF) grade 0 0 0/1 1/2 2/3 3 Spontaneous EEC PVR-1 gene expression Serum Epo N/ N/? N= normal, - = absent, + = present, = increased, = pronounced increased. = decreased. < = less than. > = more than. Signs and symptoms of PV The microvascular syndrome of thrombocytosis As shown in Figure 7 at time of diagnosis, PV is associated with thrombocytosis in about 65 to 75% of the cases [23,56,129,160,165,202]. Nearly all PV patients will develop an elevated platelet count during follow-up. Complete remission of polycythemia by bloodletting alone aiming at a hematocrit near to 0.40 is featured by persistence or appearance of thrombocytosis [131,144]. Signs and symptoms of the latter feature in PV patients in complete remission by phlebotomy as only treatment exhibit exactly the same broad spectrum of platelet-mediated microvascular disturbances as described above for ET [131,135,144]. Hypervolemic symptoms On top of the microvascular syndrome of thrombocytosis PV patients have typical hypervolemic symptoms due to the increased red cell mass, hematocrit and associated increase whole blood viscosity. Complaints of hypervolemia include fatigue, dizziness, apathy, dullness, headache, dyspnea, lack of attention, no drive to work or joy in life [129,190,202]. Signs of hypervolemia are a plethoric face, red handpalm, fingers, toes and footsole. Acrocyanosis of the tip of nose and of toes and fingers may be present. Vascular complications Between 30 to 50% of PV patients present with minor and major vascular complications. These occur both in arteries and in veins [53,165]. If PV patients remain untreated, the median survival is only 18 months with the majority dying of vascular events. The distribution of arterial complications in PV is different from age-matched population with atherosclerosis without this disorder. However, in the atherosclerosis group males predominate but there is equal gender distribution in PV. The intrinsic blood changes of increased red cells and platelets are responsible for this altered distribution of vascular complications in PV [144]. Cerebral artery complications are far more common than coronary artery conditions in PV compared with non-polycythemic atherosclerosis patients [10,79,126,135,161]. The thrombot- 17

19 ic events in PV patients include transient ischemic attacks (TIAs), facial weakness or aphasia, TIAs followed by stroke, attacks of transient blindness (amaurosis fugax), acute coronary artery disease, sudden ischemia of a toe or finger leading to amputation of one or more digits, femoral artery occlusion, superficial thrombophlebitis, deep vein thrombosis, splanchnic vein thrombosis, pulmonary embolism and priapism. Arterial complications occurred in about one third and venous thromboembolism in about one fourth of the PV patients at time of presentation [21,23,190]. Itching Itching in particular after a warm bath is very typical of PV, is difficult to treat, cryptoheptadine may help, and interferon may have a favorable response. Gout Gout is related due to increased uric acid levels in PV patients (Figure 6). Treatment of PV patients Phlebotomy and low dose aspirin Phlebotomy is the cornerstone of therapy for PV. Before the use of phlebotomy, survival of symptomatic PV patients was curtailed to less than two years as a result of premature death from major thrombotic complications including stroke, myocardial infarction [45,179,192]. With the use of phlebotomy alone as initial therapy, the estimated median survival is between 12.5 to 13.5 years. The PVSG 01 study showed that overall survival was significantly compromised due to excess of leukemia and cancer when the alkylating agents chlorambucil or 32 P was added to phlebotomy as initial therapy with a median survival of 9.1 and 10.9 years compared with 12.6 years for phlebotomy alone [19]. The risk of leukemia was 12% for chorambucil, 9.6% for 32 P and less than 1.5% for phlebotomy alone during long-term follow-up of more than 10 years [19,66]. In the PVSG 01 study approximately 35% of PV patients in the phlebotomyonly arm aiming at a hematocrit of less than 0.50 and not on aspirin experienced major thrombotic events in the first three years [66]. This high incidence of thrombotic events in PVSG 01 study can readily be explained by the poor control of the hematocrit and the persistence of thrombocytosis after bloodletting alone without the use of aspirin. A positive correlation has been found between the hematocrit level and the incidence of major arterial and venous thrombotic complications [141,161]. The risk of major vascular episodes was highest at hematocrits above 0.50, moderately increased at hematocrits between 0.45 and 050 and low at hematocrits between 0.40 and 0.45 [141,161]. The optimal hematocrit in the treatment of PV for the prevention of major thrombotic events appeared to be much lower (below 0.45 and near to 0.40) than was assumed ( ) [10,19,66,79,126,141,161]. Recent clinical studies on primary and secondary secondary intervention and prevention studies on vascular events in thrombocytosis associated with PV in remission by bloodletting alone have clearly shown that low dose aspirin 50 to 100 mg/day is highly effective in terms of platelet COX 1 inhibition and prevention of microvascular circulation disturbances [74,80,98,108,109,131, 133,134,144,178,185, ]. Low dose aspirin 50 to 100 mg is associated with a very low gastrointestinal bleeding and toxicity rate [80,109]. Therefore, the microvascular ischemic syndrome of thrombocytoisis in polycythemia vera in remission after bloodletting is best controlled by low dose aspirin (40 to 100 mg/day) or reduction of platelet count to completely normal levels (<400 x10 9 /l) by anagrelide, interferon or hydroxyurea (Figure 6) [144]. The European collaboration on low dose aspirin in PV (ECLAP) recruited 1,638 PV patients diagnosed and followed up in a qualified network of 94 European In the observational study of 1,120 PV patients, 66% had a clear indication for low dose aspirin, 23% displayed a contraindication for aspirin, and 18% belonged to the group unwilling or unable to follow the protocol recommendations. In the randomized trial of 580 patients (32%), who exhibited no clear indication and no contra-indication to aspirin, these patients were randomly allocated to 18

20 aspirin 100 mg/day or placebo [109]. Treatment of PV was according to the generally accepted recommendation and included hydroxyurea in 44%, pipobroman in 5.4% interferon in 4.2% and phlebotomy alone or as an adjuvant in 72% of the randomized PV patients. Median follow-up was three years. Mean values were 0.45 for hematocrit and 330 x10 9 /l for platelet count. On top of this, treatment with low dose aspirin as compared to placebo significantly reduced the overall risk of a combined end-point of microvascular and major vascular complications including cardiac death, no fatal myocardial infarction, no fatal stroke, pulmonary embolism and major venous thrombosis from 15.1 % to 6.7% [109]. Major, total and gastrointestinal hemorrhages were slightly increased in the aspirin group without reaching a statistical significance. These data confirm the Rotterdam concept, that low dose aspirin should be included on top of treatment in early stage PV patients by bloodletting alone, but also on top of treatment in advanced stage PV patients with hydroxyurea [74,80,98,108,109,131,133,134, 144,178,185, ]. Hydroxyurea Because of its potential leukemogenic risk, cytoreductive agents like hydroxyurea and busulfan [64,67,88,122,152,153,171,193,206,210,254] should be used with caution and withheld as long as the combination of bloodletting and low-dose aspirin is effective in preventing thrombotic events in the early stages of PV without significant myelofibroic splenomegaly [138,151,190,191,196]. The shortcomings of phlebotomy alone include the lack of effect in controlling red cell mass, splenomegaly, itching, and increased proliferative activity such as leukocytosis, thrombocytosis, postpolycythemic myelofibrosis and spent phase PV. Retrospective analysis of newly diagnosed 114 PV patients (mean age 63 years) initially randomized to phlebotomy alone showed that 50% by the 5 th year and 90% by the 10 th year had received myelosuppression either 32 P or hydroxyurea because of progressive myeloproliferative disease [138]. The median survival of these 114 PV patients was 18 years as compared to 20 years of similar age indicating a 10% loss of life expectancy. In a recent update, patients with PV that entered into the PVSG 08 and who received hydroxyurea and no prior therapy were compared retrospectively to PV patients included in the phlebotomy arm of the PVSG 01. At a median follow up of 8.6 years and a maximum of 15 years, 5.9% of the hydroxyurea treated and 1.5% of the phlebotomised patients developed acute leukemia; On the other hand, 7.8% of the hydroxyurea treated and 11.2% of phlebotomised treated patient developed spent phase by 15 years. By 15 years 31% of hydroxyurea treated and 40% of phlebotomy treated patients had died. These data are evidence-based and not statistically different, but a paradoxical trend for increased leukemia and improved survival in PV patients treated with hydroxyurea is evident. This paradox may be explained by the observation that hydroxyurea therapy of stage 3 symptomatic PV patients delays the development of postpolycythemic myelofibrosis and spent phase PV [109]. The French prospective PV study evaluated hydroxyurea with regard to clinical safety, hematological efficacy, frequency of progression to myeloid metaplasia and myelofibrosis or spent phase and risk of carcinoma or leukemia in a long-term follow-up study of 133 previously untreated PV-patients below the age of 65 [151]. Complete remission was defined by a hematocrit lower than 0.50 and platelet count lower than 400 x10 9 /l. The long-term use of aspirin was let free at the decision of the physician. Toxicity of hydroxyurea was observed in 29% of 133 patients, which was limited to dry skin and acne in 7%, gastric pain/diarrhea in 9%, aphthous ulcers in the mouth in 10%, and leg ulcers in 9%. Leg ulcers only healed after discontinuation of hydroxyurea; these complications appear generally late (5 years or more after initial treatment). Dry skin in 1, aphthous stomatitis in 4 and leg ulcers in 10 cases were reasons to replace hydroxyurea by pipobroman in 9%. Leg ulcers generally resolved when hydroxyurea was replaced by pipobroman. Efficient control of hematocrit <0.50 and platelet count <400 x10 9 /l was obtained by hydroxyurea in 82% and 55% respectively. The frequency of progression to (postpolycythemic) myeloid metaplasia and overt myelofibrosis (MMM) or spent phase in the hydroxyurea treated PV patients was 17% at 10 years and 40% at 16 years. The high incidence of MMM or spent phase in the hydroxyurea treated arm was only slightly different from those patients treated by phlebotomy alone in a previous study. Seventy-six per- 19

21 Table 9. Proposed clinical trial in previously untreated PV patients Stage 1 and 2 Age yr Stage 3 Age < 50 yr Age yr Age > 65 yr Bloodletting plus low dose aspirin Anagrelide optional to control platelet number Interferon first choice Randomize for interferon versus hydroxyurea Hydroxyurea first choice If side effects interferon change to hydroxyurea If side effect s hydroxyurea change to interferon cent of cases who developed MMM had a permanent high platelet count in excess of 400 x10 9 /l despite maintenance of myelosuppressive treatment. The incidence of leukemia was about 10% at 13 years in hydroxyurea treated PV patients. Life expectancy was 70% at 14 years as compared to 83.7% in age-matched controls. In another study, 71 newly diagnosed PV patients were phlebotomized to a hematocrit of <0.45 and then therapy was started with hydroxyurea daily [197]. Median duration of the disease was 10.9 years and the median duration of treatment with hydroxyurea was 7.3 years. Maintaining the hematocrit under 0.45 and platelet count below 400 x10 9 /l, the incidence of acute leukemia was 4 in 71 patients (5.6%). As the life expectancy of newly diagnosed and properly treated PV patients is completely normal for the first decade but compromised after more than 10 years follow-up due to progression to postpolycythemic mylofibrosis, spent phase, or leukemia [180], there is a need to compare hydroxyurea with the non-leukemogenic agent interferon-alpha in terms of side effect, toxicity and very long-term survival. In a proposed trial, clear indications for the start of interferon or hydroxyurea in stage 3 PV include: Uncontrolled platelet count of >1,000 x10 9 /l during aspirin therapy. Side effects of aspirin or anagrelide, symptomatic and platelet count >600 x10 9 /l. Increase spleen size more than 2 cm per year, or exceeding 15 cm length diameter. Symptomatic large spleen (splenomegaly). PV-related constitutional symptoms including pruritis etc. Leukocyte count in excess of 25 x10 9 /l. Leukoerythroblastic blood picture and signs of myeloid metaplasia. Major arterial or venous thromboembolic complications. Symptomatic iron deficiency. High frequency of phlebotomy of >8 per year to maintain the hematocrit normal (<0.45). Interferon-alpha Therapeutic benefits reported include induction of hematological remission, significant improvement in the platelet counts, iron status, and leukocytosis, resolution of disease-associated symptoms in particular, relief from thrombohemorrhagic events and refractory pruritis, and resolution of splenomegaly [187]. Hematological remission has been reported differently by authors, but in general, a complete response (CR) implies maintenance of a hematocrit less than 0.45 without the need of phlebotomy and partial response (PR) refers to maintenance of a hematocrit between 0.45 and 0.50 with a 50% reduction in phlebotomy requirements to reduce the hematocrit to below 0.45 [115]. Overall, hematological responses were achieved in 76% of cases within 6 months (range 3 to 12 months). Approximately 15% of patients failed to gain any benefit from IFN-alpha therapy. In those studies that clearly defined a CR as described above, this response was achieved in approximately 60% of cases. Doses of IFN-alpha used to obtain hematological responses varied between 4.5 and 25 million units weekly, with a majority of patients receiving 9 million units weekly, generally given three times a week. IFN-alpha seems to be most effective when started in the early plethoric stage of PV. 20

22 The required IFN-alpha dose in early stage PV is lower and less toxic and does not interfere with the working and recreation ability. Side-effects remain a significant problem, occurring in over 30% of patients, and may be related to the high mean age of the patients. Approximately 20% of patients discontinued IFN-alpha therapy due to intolerance. Rational for using IFN-alpha for the treatment of PV include: 1.) Abatement of constitutional symptoms; 2.) Maintenance of hematocrit and platelet suppression in the normal or near normal range; 3.) Avoidance of phlebotomy, iron deficiency, and macrocytosis due to hydroxyurea; 3.) Lack of mutagenicity; 4.) May prevent or delay postpolycythemic myelofibrosis if used early in the course of the disease; 4.) Effectiveness/toxicity (risk/reward) ratio is positive. The rational for using PEG-Intron is its much favorable efficacy, safety and toxicity profile. Therefore, an open label randomized treatment program is warranted to establish the efficacy and safety of PEG-Intron concerning the induction of complete haematological remission as compared to standard phlebotomy/low dose aspirin in previously untreated patients with early stage PV. First and second treatment options in PV A primary rigid venesection regimen to maintain the hematocrit below 0.45 near to 0.40 is currently accepted as a nonleukemogenic approach and first choice treatment in newly diagnosed PV patients [41,74,98,134,139,185,202]. The microvascular syndrome of thrombocythemia in PV in remission by bloodletting is easily and best controlled by low dose aspirin (50 to 100 mg/day) [42,244]. Anagrelide can be considered to control extreme thrombocytosis (>1,000 x10 9 /l) particularly when associated with microvascular or bleeding complications [198]. The combination of bloodletting plus low dose aspirin (ASA) has recently become the recommended first line treatment option for PV stage 1 and 2 for as long as possible until progression to stage 3 PV occurs. Many stage 1 and stage 2 PV patients require no other treatment for many years (up to more than 10 to 15 years); stage 3 PV calls for the introduction of cytoreductive therapy. Hydroxyurea is the less expensive and most frequently used cytoreductive agent, althought serious doubts persist about its long-term toxicity and leukemogenicity [64,67,88,122,152, 153,171,193,254]. Interferon-alpha is a promising alternative in case of no or minor side effect, but it is expensive and its side-effects can be unbearable. A reasonable approach could be to reserve interferon for younger patients below the age of 50 to 55 as long as it works without significant side effects. Hydroxyurea is a good choice for older patients over 65 and 70 years of age. Whether interferon or hydroxyurea at ages between 50 and 65 in terms of quality of life and life expectancy is the best option remains uncertain and should be addressed in a randomized clinical trial. We propose to perform a large scale prospective randomized trial using a flexible study design to compare the efficacy, toxicity, leukemogenicity of hydroxyurea versus interferon-alpha in newly diagnosed and previously untreated PV patients. Diagnostic criteria of prefibrotic and (classical) chronic idiopathic myelofibrosis (IMF) or myelofibrosis with myeloid metaplasia (MMM) / agnogenic myeloid metaplasia (AMM) The third category of MPD is usually termed agnogenic myeloid metaplasia (AMM) or idiopathic myelofibrosis (IMF), but various other designations have been used, such as primary myelofibrosis, myelosclerosis, osteomyelofibrosis, and myelofibrosis with myeloid metaplasia (MMM) [13,200,209]. This disorder is generally defined as a clinicopathological entity not preceeded by any other or allied haematological disorder. Diagnostic features of classical IMF usually include anemia, splenomegaly, leukoerythroblastic blood picture, tear drop erythrocytosis, and varying degrees of bone marrow reticulin and collagen fibrosis [11,13,14]. Different sets of prognostic and clinical parameters of IMF have been proposed [11,13,34,35,103,104, 140,182,226,227,250], but all agree on the clinical scoring system in Table 10 [130,140], that 21

23 Table 9. European clinical and pathological (ECP) criteria for the diagnosis of agnogenic myeloid metaplasia (AMM) or idiopathic myelofibrosis (IMF) [140] A1 C1 C2 C3 Clinical criteria No preceding or allied other subtype of myeloproliferative disorders CML or MDS. C: Clinical stages Early clinical stages Normal hemoglobin or slight anemia, grade I: hemoglobin >12 g/dl Slight or moderate splenomegaly on ultrasound scan or CT Thrombocytosis, platelets in excess of 400, 600 or even 1,000 x109/l Intermediate clinical stage Anemia grade II: hemoglobin >10g/dl Definitive leuko-erythroblastic blood picture and/or tear drop erythrocytes Splenomegaly Advanced clinical stage Anemia grade III: hemoglobin <10 g/l Splenomegaly, thrombocytopenia, leukocytosis, leukopenia B1 Pathological criteria Megakaryocytic and granulocytic myeloproliferation and relative reduction of erythroid precursors. Abnormal clustering and increase in atypical giant to medium sized megakaryocytes containing clumsy (cloud-like) hypolobulated nuclei and definitive maturation defects. Staging of myelofibrosis (MF) MF 0 MF 1 MF 2 MF 3 MF >3 prefibrotic stage IMF: no reticulin fibrosis early IMF: slight reticulin fibrosis manifest IMF: marked increase in reticulin and slight to moderate collagen fibrosis overt IMF: advanced collagen fibrosis osteosclerosis (endophytic bone formation) and decreased cellularity The combinations of A1 + B1 establish IMF - any other criterion confirms IMF/AMM is essentially based on scrutinized follow-up studies including sequential bone marrow biopsies [31,70,239]. Anemia at a cut off for hemoglobin of 10 g/dl, has been consistently associated with a shorter survival and yields the basis of several useful prognostic systems. The Lille scoring system [55], based on two adverse prognostic factors, namely hemoglobin <10g/dl and leukocytes <4 or >30 x10/l, is able to separate patients with classical IMF into 3 groups with low (0 factor), intermediate (1 factor) and high risk (2 factors) associated with a median survival of 7.75, 2.2 and 1.1 years respectively. The Sheffield score, by comparing hemoglobin, age, and karyotype, identifies patient groups with median survival times that vary from 180 months (15 years = good risk) to 16 months (1 year and 4 months = poor risk) [174]. IMF patients with intermediate and high risk of compromised survival may be considered as candidates for bone marrow transplantation. Morphological features - problems to recognize prefibrotic/early IMF A prefibrotic form of IMF (Figure 9) and its differentiation from true ET has been recognized by Thiele et al. [225,226,228,232,235,236,240] and recently validated by the WHO classification [230]. It has been estimated that about 25 % of patients with IMF initially present with a hypercellular stage characterized by a prominent granulocytic and megakaryocytic myeloproliferation and no or borderline increase in reticulin (IMF-0). The prevalence of the (left shifted) neutrophil granulocytic and megakaryocytic lineage with reduction and maturation arrest of erythroid precursors [222] was previously termed chronic megakaryocytic -granulocytic myelosis - CMGM [30,68-70]. Most conspicuous, however, was the megakaryopoiesis characterized not only by a disturbance of BM histotopography (loose to dense clustering and translocation to the endosteal borders), but also by striking abnormalities of maturation. The 22

24 Fig. 9 Morphological features of prefibrotic IMF (IMF-0) latter consisted of variations in size including giant forms and deviations of the nuclear cytoplasmic ratio accompanied by bulbous and hyperchromatic cloud-like nuclei. In addition to the disorganized nuclear lobulation there were many naked (bare) nuclei detectable [30,68,70, 215,225,230,248]. Although progression of IMF is unpredictable the search for factors indicating a rapid transition from the prefibrotic into manifest fibrosclerotic stages revealed that increasing megakaryocyte maturation defects (dysplasia) exert a predicative value. It should be explicitly emphasized that according to morphology, the megakaryocytes in prefibrotic, early fibrotic and fibrotic IMF are more atypical than those in the other subtypes of MPDs and consequently present the major discriminating diagnostic hallmark (Table 9). As in CML there is always a close correlation between megakaryocytes and fibers recognizable [224] that accounts for the generation of myelofibrosis [110,112]. With increase in reticulin (IMF-1) and collagen fibers (IMF-2) (Figure 10) BM cellularity becomes variable and occasionally displays a patchy pattern. The overt fibro-osteosclerotic stages of disease are characterized by coarse bundles of collagen fibers with optional osteosclerosis (endophytic bone formation) and areas of hematopoiesis that may be separated by regions of fat cells, i.e. progressive hypoplasia [31,51,52,68-70,215,238,239]. Most strikingly expressed are dilated marrow sinuses [106,219] with a prominent intraluminal hematopoiesis. However, comparable with the initial prefibrotic stages (IMF-0) atypical megakaryocytes were again a diagnostic hallmark and arranged in sizable clusters surrounded by a residual left-shifted granulopoiesis and small groups of (macrocytic) erythroid precur- 23

25 Fig. 10 Morphological features of manifest IMF (IMF-2) sors that are reduced in this condition [222]. It is noteworthy that even without previously applied cytoreductive therapy mild to moderately expressed myelodysplastic changes may occur in the natural course of the disease process [238,241], occasionally initiating an insiduous transition into an acceleration and blastic crisis (terminal stage). In these advanced stages of IMF that are consistent with MMM a marked collagen fibrosis of the bone marrow is a constant finding (Figure 11). Consequently, the peripheral blood shows a striking leukoerythroblastosis and poikilocytosis with many tear drop-shaped erythrocytes accompanied by anemia and pronounced splenomegaly, i.e. the classical, generally accepted features of fullblown IMF [11,13,14,34,35,52,85,249]. The recognition of prefibrotic (IMF-0)and early stage IMF (IMF-1) and its differentiation from true ET and PV as indicated by the WHO classification [230]completely alters both the diagnostic and prognostic criteria of this disease and therefore continues to be a matter of discussion and evaluation. Recognition of prefibrotic and early stage IMF largely depends on careful evaluation of bone marrow histopathology and associated clinical data [68-70,223,227,229,232,240]. This has been accomplished by an improved set of and the clinical and pathological criteria for the diagnosis and staging of IMF (Table 9) [142,223,226,229,235].Altogether, classical IMF can now be redefined as intermediate and advance stage IMF [150,226,227]. Clinical impact of prefibrotic early IMF Prefibrotic and early IMF is usually associated with pronounced thrombocythemia with platelet counts above 1,000 x10 9 /l, borderline therapy-refractory anemia, slight splenomegaly, normal or slightly elevated LDH, but absence of a leukoerythroblastic blood picture [223,227,229, 232,240]. Comparing the PVSG criteria and the WHO guidelines, ET was diagnosed in 483 patients according to the PVSG, but applying the WHO criteria only 162 could be diagnosed as true ET, and 321 were diagnosed as prefibrotic or early fibrotic IMF [36]. Loss of life expectancy was minor (8.9%) in true ET, but ranged up to about 32% in thrombocythemia associated with prefibrotic or early fibrotic IMF [236]. In another study of 142 adult ET patients according to the PVSG were reclassified by the WHO criteria as ET in 26%, prefibrotic IMF in 30%, early fibrotic IMF in 34% and classical IMF in 10% (personal communication V. Franco). In a retrospective study of 195 ET patients according to the PVSG 13 cases developed clas- 24

26 Fig. 11 Morphological features of advanced osteosclerotic stage of IMF (IMF-3) sical IMF (anemia in 13, splenomegaly in 7, leukoerythroblastosis in 9 and increased LDH in 13) after a median follow-up of 8 years (range 3.6 to 20 years) [36]. In a large cohort of 575 true ET patients, myelofibrosis is minor at time of diagnosis and during long-term follow-up [30,69,70]. Moreover, of the 93 patients derived from the Italian ET Study, elaborate bone marrow re-evaluation revealed that 40 patients showed features in keeping with early stage IMF and an unfavorable survival [7]. As prospective studies are lacking, the natural history of prefibrotic and early fibrotic is not yet known, and discussion and controversy exists regarding the stepwise evolution of the disease process in IMF. However, prelimnary data indicate that progress to overt and advanced IMF may be very slow to more or less rapid. Comparison of so-called early hypercellular stage without reticulin fibrosis, MF score 0 (prefibrotic IMF), versus stages with minimal to gross reticulin or/and collagen fibrosis, MF score 1 to 3, in the study of Thiele et al [223] reveals significant differences with median survival of 9.2 versus 4.3 years. Risk of progress of prefibrotic into advanced IMF in 500 cases with prefibotic IMF (MF grade 0) was only about 30% with stable disease in 70% after 6 to 7 years follow-up [70]. Cytogenetic findings in IMF Approximately one third of classical IMF patients have cytogenetic abnormalities of nucleated peripheral blood cells, a figure that increases if follow-up analysis is performed [18,50,203]. 25

27 At least several cytogenetic anomalies are pathogenetically important, namely, del (13 q ), del (20 q ), +8 or partial trisomy 1 q and finally abnormalities of chromosomes 7 and 9 [177,188,203,204]. The presence of a cytogenetic abnormality makes the prognosis of IMF unfavorable [177,261]. The Sheffield scoring system, by combining haemoglobin, age and karyotype, identifies patient groups with median survivals times that vary from 180 months (good risk), about 75 months (intermediate risk), to 16 months (poor risk) [175,176]. Dupriez et al reported that cytogenetics retained prognostic value in their low-risk group : patients with normal karyotype had a mean survival of 9.3 years, whereas those with an abnormal karyotype had a median survival of 4.2 years [55]. On the other hand, only 15% of patients with PV carry detectable cytogenetic abnormalities at diagnosis, which consist primarily of complete or partial +9 and +8 [37,257]. In addition, 13q- and 20q- anomalies in PV are infrequently detected at diagnosis but their incidence increases with longer duration of disease, and the presence of 13q- and 20q- has been loosely linked to immanent myelofibrosis [18,50]. These observations suggest a potential link between some of the cytogenetic markers and the disease phenotype of idiopathic and postpolythemic myelofibrosis. Signs and symptoms of classical (advanced) IMF (Figure 11) Contrasting prefibrotic and early fibrotic IMF where patients are usually asymptomatic at presentation except for some fatigue, due to mild anemia, ocasionally microvascular disturbances or bleeding that may eventually occur with associated thrombocytosis, classical IMF typically present with on or more of the following symptoms [13,55,174,200,223]: severe fatigues, abdominal distress due to splenomegaly, unexplained weight loss, history of thrombosis, fever (night sweats), easy bruising, gastric ulcers, frequent infections, bone tenderness. Typically laboratory features of classical IMF are anemia <12 g/dl, splenomegaly with spleen size of >2cm below the costal margin, hepatomegaly with liver size >1 cm below the costal margin, elevated LDH, leukoerythroblastosis, thrombocytosis, normal platelet count or thrombocytopenia [11,13,14,55,174,200,223]. The severity and number of complaints increases in the advanced stages of IMF. Prognosis in IMF A wealth of data has been accumulated in recent years about the extremely heterogeneity of survival patterns in IMF applying univariate [6,86,119] and multivariate [11,55,174,182,249, 250] evaluation methods to identify independent prognostic features (Table 10). However, this large body of predictive data has been partly obscured by relative small study populations and ill-defined series with altering diagnostic criteria. In particular, patients with other causes of myelofibrosis, i.e. postpolycythemic myeloid metaplasia and acute (malignant) myelofibrosis [11,86,154], were not always strictly excluded. Moreover, only a few series comprised the full spectrum of initial early (prefibrotic) and hypercellular to advanced full-blown osteosclerotic disease stages into prognostic classification [101,103,104]. Despite of these inconsistencies in most cohorts degree of anemia [11,33,55,86,101,103,104,154,155,182,250], age at diagnosis [11,33,103,174,249], peripheral blood precursors [11,33,250], and platelet as well as leukocyte counts [55,103,174,250] were found to exert a prognostic impact in IMF. Furthermore, in recently published series cytogenetic abnormalities were additional indicators for a worsening of prognosis [47,174]. Hoewever, in most studies, evolution of bone marrow fibrosis revealed no significant influence on survival [6,103,249]. Since in the prevailing senescent population of IMF patients mortality resulting from causes other than the underlying disorder has to be regarded, most of these prognostic calculations are seriously impaired, resulting in an overestimation of disease-associated mortality [ ]. In IMF bone marrow morphology accompanied by clinical and hematological data on admission is consistent with a static view of an ongoing and stage like disease process. Additional characteristics concerning the dynamics of disease should substantially improve prognostic 26

28 Table10: Prognostic models for IMF Study group No. of patients early stages of disease included Secondary myelofibrosis included (i.e. PPMM) Parameters indicating worsening of prognosis Barosi et al., Age Hemoglobin Myeloblasten Erythroid iron turnover Visani et al Hemoglobin Leukozyten Myeloblasts prediagnostic period Rupoli et al., Hemoglobin Dupriez et al., Hemoglobin Leukocytes Reilly et al., Age Hemoglobin Thrombocytes Leukocytes Cytogenetic abnormalities Cervantes et al., Hemoglobin constitutional sysmptoms Myeloblasts Okamura et al., Age Hemoglobin Thrombocytes Myeloblasten Tefferi et al., Age Hemoglobin Kvasnicka et al., Age Hemoglobin Leukocytes Thrombocytes Kvasnicka et al., Age Hemoglobin Leukocytes Thrombocytes peripheral blasts Apotosis and proliferation Kreft et al., Hemoglobin Age Grade of myelofibrosis efficiency and stratification of patients subgroups [104,106]. Reduction of proliferative activity together with a lower rate of apoptosis was reported to be associated with a worsening of prognosis in the latter series. It may be speculated that a reduced proliferation rate in combination with a decrease in apoptosis reflects the failure of regenerative capacity of hematopoiesis, especially in early hypercellular stages [105]. Considering the well-known heterogeneity of survival patterns which is mainly influenced by early signs of myeloid metapla- 27

29 Table 11. Proposal for a simple synthesis prognostic staging system in IMF: The Cologne prognostic score Parameter Prognostic Impact Age (years) > 70 2 Hemoglobin (g/dl) < 10 2 Thrombocytes (x 10 9 /l) < Leucocytes (x 10 9 /l) or: Myeloblasts (%) Erythroblasts (%) > 20 > 2 > 2 1 Risk group Score low risk < 2 intermediate risk 2 and < 4 high risk 4 Fig. 12 Prognostic factors in early and advanced stages of IMF % Survival Risk group low (n=346) intermediate n=277) high (n=103) Years after diagnosis sia [103], a simplified synthesis score (Table 11, Figure 12) enabled a clear-cut separation of patient prognosis [ ]. Management of IMF patients No medical therapy has proven to have an effect on overall patient survival, justifying the common attitude toward conservative management of IMF patients [13,55,125,174,176,200,208]. Asymptomatic good risk IMF patients with hemoglobin above 10 g/dl and no adverse signs and symptoms may not require any therapy but should be observed at periodic intervals. The majority of IMF patients remain incurable, and current management is directed towards the alleviation of constitutional symptoms, anemia and symptomatic splenomegaly. High dose chemotherapy and allogeneic peripheral blood or bone marrow stem cell transplantation (SCT) offers the only chance to achieve a cure in patients with classical IMF, but is associated with an overall 30% death rate within 1 year, and a median survival of about 5 years [3,4,46,48,81,82,87,95,203]. Patients with prefibrotic and early fibrotic IMF with no or slight anemia, and no or slight splenomegaly and a survival probability of about 10 years or more 28

30 are not to be considered for allogeneic SCT. Low, intermediate and high risk patients with classical IMF, in particular young age, may be candidates for allogeneic SCT before treatment for complaints and complications has to be started. For classical IMF patients with a median survival of about 5 years or less, allogeneic SCT seems to be the treatment of choice if a suitable donor is available. In a recent study of 55 patients who received an allogeneic transplant for classical IMF, the 5 year probability of survival was 55% if there was grade 1 or 2 bone marrow fibrosis, and 38% if there was grade 3 marrow fibrosis (osteosclerosis). According to the Lille score, the 5-year probability of survival was 83% for the patients in the low risk group; 43% for the intermediate risk group; and 31% in the high risk group [82]. The 5-year probability of survival was 76% for patients with haemoglobin >10 g/dl or with haemoglobin <10 g/dl and no red blood cell transfusion before transplant; and 23% for patients with haemoglobin <10 g/dl receiving pre-transplant red blood cell transfusion [82]. Another study of 56 patients with classical IMF undergoing allogeneic SCT using an improved conditioning regimen showed similar results and demonstrated that patients with lower Lille score, less severe marrow fibrosis, platelet count above 100 x10 9 /l and normal karyoptype fared better than patients with more advanced and treated disease [46]. These two studies show that allogeneic SCT offers curative therapy for patients with classical IMF, that the results with HLA identical unrelated donors are comparable with those with related donors, and that in patients, who are interested in pursuing transplantation, allogenic SCT should be carried out before severe grade III marrow fibrosis, clonal cytogenetic abnormalities, and severe abnormalities of hematologic parameters develop [46,82]. Patients with classical IMF and candidate for allogeneic SCT have to be fully informed of the high risk of immediate death along with a possibility of cure for more than 5 years [3,4,46,48,81,82,87]. New therapeutic strategies include imatinib mesylate [207] and thalidomide [15,16,208]. Therapy of thrombocytosis Treatment of thrombocytosis related microvascular disturbances and clear indications for platelet lowering agents like bleeding symptoms and are similar as has been described for true ET [12,199,200,208]. Management of symptomatic anemia Hydroxyurea remains the drug of choice for those symptomatic IMF patients to control leukocytosis, thrombocytosis and progressive splenomegaly that may be associated with control or some improvement of anemia. Interferon may have similar cytoreductive effect similar to hydroxyurea, but it may not be as well tolerated [13,55,174,176,200,208]. Because interferon preferentially inhibits the proliferation of the megakaryocytic cell lineage and consequently interferes with the cytokine-mediated generation of myelofibrosis [111] this agent seemed to be particularly suitable for treatment [72]. However, compared to hydroxyurea as the drug of choice, interferon according to clinical data was not shown to exert a clear-cut beneficial effect on the regression or inhibition of myelofibrosis [8,12,159,183]. As has been demonstrated in longitudinal follow-up studies including sequential trephine biopsies, in most patients a progressively developing bone marrow fibrosis could be observed in IMF that was not influenced by any treatment modalities, especially not by interferon [239]. This result is in keeping with data from another study on IMF including 109 patients of whom 48 received either busulfan or hydroxyruea or combination therapies [31]. On the other hand, according to repeatedly performed bone marrow biopsies during the course of IMF borderline to mild maturation defects developed in about 23 % of patients that received only symptomatic treatment, contrasting the significantly expressed myelodysplastic changes in 3.3 % patients of the hydroxyurea arm that were not present after interferon treatment [238,241]. It is reasonable to assume that minor to moderate maturation defects may develop during the normal course of disease even without interference by cytoreductive treatment. Nutricial deficiencies of folate or iron are easily diagnosed by serum levels and therapeutic responses, and relate to gastrointestinal or other blood loss and increased folate requirement. 29

31 Patients with normal red cell mass and marked increase in plasma vlomue have a dilutional form of anemia due to spelenomegaly and require no treatment. Androgen therapy improves impaired marrow function in approximately 40% of patients, with optimal responses being observed in patients lacking massive splenomegaly, or who have had a splenectomy, and in those with normal karyotype.. The drug of choice is oxymethalon, with intial doses of 50 mg one to three times a day, which should be prescribed for at least 3 to 6 months. Side effects include fluid retention, increased libido, hirsutism and abnormal liver function. The combination of thalidomide and prednisone is a promising alternative to improve anemia. the use of human recombinant erythropoietin (repo) in myelofibrosis usually failed to observe an effect, but prolonged transfusion-free periods or improved haemoglobin levels have been observed. Its use should be restricted in only a few patients that have low serum EPO levels. Treatment of splenomegaly Hydroxyurea may have a favorable response in symptomatic splenomegaly associate with leukocytosis in decreasing spleen size considerably together with improvement of anemia for as long as several months to years without significant side effects. Interferon is a valuable alternative to hydroxyurea. Hematologic responses have been seen in approximately 50% of assessable patients, usually with a so-called hyperproliferative type (leukocytosis, thrombocytosis and splenomegaly). Responses of hydroxyurea or interferon in cases with pancytopenia and severe anemia are not to be expected. Irradication of a large spleen is usually not an option because of only a slight and transient response, and may interfere with an impaired outcome of splenectomy in the future [57,58]. Advanced IMF is featured by transfusion dependent anemia. Main causes of death include thrombocytopenic bleeding, infection, leukemic transformation, and cachectic endstage disease. Splenectomy should be timely prepared in patients with advanced IMF. Clear indications for splenectomy are the following [1,13,123,124,201]: Hydroxyurea and interferon resistant symptoms and complaints of greatly enlarged spleen. Mechanic complaint of a very large spleen with portal hypertension due to increased blood flow in the absence of a Budd-Chiari syndrome, and portal or renal vein thrombosis. Transfusion dependent anemia and severe thrombocytopenia associated with bleeding symptoms. Mechanical complaints of a very large spleen plus complaints of fatigue, night sweat and weight loss. The chance to survive and the risk of death during the peri-operative period of splenectomy is 91% and 9% respectively. The risk of bleeding, infection or thrombosis during and after splenectomy is about 31%. After splenectomy there is a risk of progressive thrombocythemia in 22%, progressive hepatomegaly in 16%, and transformation to acute leukaemia in 16%. IMF patients after splenectomy may survive for a several months to a few to several years [13,57,58,123,201]. In conclusion, a synoptical approach towards a clear-cut diagnosis of MPDs is recommended, but especially the inclusion of morphological criteria as authorized in the WHO and ECP guidelines contrasting the outdated PVSG postulates. Therefore, a representative pretreatment bone marrow biopsy has to be always performed as a basic, although by many patients not readily appreciated procedure. In this context, clinical features and bone marrow morphology remain the two supporting pillars of diagnosis, since until now biological markers have an only limited value regarding their diagnostic impact or predicting power concerning prognosis. 30

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