The European clinical, molecular and pathological (EurocmpCMP) and WHO-CMP criteria of the myeloproliferative disorders 1940-2008 and myeloproliferative neoplasms 2008-2015

The 1990 Hannover Bone Marrow Classification separated Ph1+ CML from the Ph1myeloproliferative neoplasms (MPN) essential thrombocythemia (ET), polycythemiavera (PV) and chronic or primary megakaryocytic granulocytic myeloproliferation (CMGM/PMGM). The 2000-2008 European Clinical Molecular and Pathological (ECMP) criteria discovered 3 variants of thrombocythemia: ET with features of PV (prodromal PV) versus “true” normocellular ET and hypercellular ET associated with CMGM/PMGM without features of PV, MDS or CML. The 2008 World Health Organization (WHO)-ECMP and 2007-2015 WHOECMP MPN classifications defined three phenotypes of JAK2V617F mutated ET: normocellular ET (WHO-ET), hypercelluar ET due to increased erythropoiesis (prodromal PV) and ET with hypercellular megakaryocytic-granulocytic myeloproliferation (ET.MGM) or masked PV. JAK2 wild type MPL515 mutated ET is the second distinct thrombocythemia featured by clustered giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow consistent with the diagnosis of “true” ET. JAK2/MPL wild type hypercellular ET is the third distinct thrombocythemia characterized by clustered larged immature dysmorphic megakaryocytes and bulky (bulbous) hyperchromatic nuclei consistent with CMGM/PMGM. Correspondence to: Jan Jacques Michiels, MD, PhD, Investigator, Senior Internist, International Hematology and Bloodcoagulation Research Center, Goodheart Institute and Foundation in Nature Medicine, European Working Group on Myeloproliferative Neoplasms, Erasmus Tower, Veenmos 13, 3069 AT Rotterdam, The Netherlands, Tel: +31-62-6970534; E-mail: goodheartcenter@upcmail.nl


Introduction
In this historical appraisal of the myeloproliferative disorders

Polycythemia vera, a trilinear MPD
A definite diagnosis of PV can be made: plethoric appearance, splenomegaly, definitely elevated erythrocyte count above 6 × 10 12 /L, elevated platelet count, and elevated hematocrit [1]. The bone marrow is pathognomonic diagnostic showing a panmyelosis (increased trilinear hematopoiesis) and large megakaryocytes (Table 1) [1,2]. The majority of PV patients have a long life span and every attempt should be made to keep the treatment of PV as physiologic as possible by venesection aiming at haematocrit of 0.40 as a satisfactory method resulting in a state of iron deficiency [1][2][3]. During complete remission of PV by phlebotomy alone red cell count remains elevated above 6 × 10 12 /L due to microcytosis of red cells, haemoglobin and hematocrit levels remain low for periods of months to years and the PV patient is reasonable asymptomatic indicating that the best index of phlebotomy therapy is the hematocrit value and haemoglobin concentration [2,3]. It is possible to control PV patients by phlebotomy alone for several up to ten to fifteen years and such PV are in as good health as comparable persons of the same age group [3][4][5].
PV as a total marrow disorder in which peripheral blood erythrocytosis, leukocytosis and thrombocytosis are all simultaneously present and the bone marrow is featured by a trilinear myeloproliferaive disease (MPD) of erythrocythemic, thrombocythemic and granulocythemic myeloproliferation (Figures 1-4) [2,3]. PV is complicated by primary myeloid metaplasia of the spleen with increasing degree of splenomegaly, myelofibrosis and the development of anemia in about one third of the cases after long-term follow-up of about 15 to 30 years. The one cause hypothesis cause of Dameshek for PV as a trilinear myeloproliferative disease (MPD) either the presence of excessive bone marrow stimulation by an unknown factor or the lack or diminution of an inhibitory factor has been confirmed by Vainchenker's discovery of the acquired somatic JAK2 V617F mutation as the cause of three phenotypes of MPD essential thrombocythemia (ET), PV, myeloid neoplasia of the spleen (MNS) and secondary myelofibrosis (MF) [2,3]. In 1951 Dameshek speculated on an unifying theory that erythroleukemia, chronic granulocytic leukemia (CML), PV, idiopathic or agnogenic myeloid metaplasia (AMM) of the spleen, and The diagnostic criteria for polycythemiavera proposed by the polycythemiavera study group (PVSG)

A3
Splenomegaly on palpation B3 Raised neutrophil alkaline phosphate score > 100 or raised B 12 ( > 900 ng/L) or raised unsaturated Note: RCM does not distinguish between PV and primary or secondary erythrocytosis. RCM does not distinguish between PV and inapparent PV with splenomegaly in splanchnic vein thrombosis (IPV, table 3). In IPV RCM is increased due to splenomegaly with absence of hypervolumemic symptoms. Bone marrow histology distinguishes PV from all variants of erythrocytosis with a sensitivity and specificity of 100%. Table 1. PVSG criteria for PV [10] and diagnostic differentiation of PV from all variants of primary and secondary erythrocytoses by bone marrow histology [17].
No reticulin fibers, occasional individual fibers or focal areas with tiny amount of reticulin fiber network.   megakaryocytic leukemia (ML) [6,7]. It is possible that these various 'myeloproliferative disorders" are all somewhat variable manifestations of proliferative activity of the bone marrow cells, or may represent one myeloproliferative activity of bone marrow cells due to a hitherto undiscovered hypothetical stimulus [6]. This concept of MPD is the basis of which the Polycythemia Vera Study Group (PVSG) defined in The 1980 RCP major (A) and confirmative (B) criteria for prefibrotic ET.

A2
Increase and clustering of enlarged megakaryocytes in bone marrow biopsy.

A3
No or slight increase of reticulin fibers (RF 0 or RF 1).

B1
Presence of large platelets in a peripheral blood smear.

B3
Increase of LAP-score and no signs of fever or inflammation.

B4
Absence of Ph + chromosome or any other cytogenetic abnormality in nucleated blood or bone marrow cells.
The 1980 RCP major (A) and minor (B) criteria for prefibrotic PV.

A2
Slight, moderate or marked increase in bone marrow biopsy of clustered, enlarged pleomorphic megakaryocytes with hyperlobulated nuclei and moderate to marked increase cellularity of megakaryopoiesis/erythropoiesis or typically trilinearerythrocytic, megakaryocytic and granulopocytic myeloproliferation (EMGM). A typical PV bone marrow excludes erythrocytosis with a sensitivity and specificity of 100%. No or presence of reticuline fibers and no collagen fibers (no dry tap).

B4
Splenomegaly on palpation or on isotope/ultrasound scanning.

A3
Normal LAP score, normal ESR, and increased MPV.

B2
No proliferation or immaturity of granulopoiesis or erythropoiesis.

A5
No signs or cause of RT. B3 No or only borderline increase inreticulin.

A6
No preceding or allied other subtype of MPD, CML, or MDS.

A7
Absence of the Philadelphia chromosome.

A1
No preceding or allied other subtype of MPD, CML, OR MDS B1 Megakaryocytic and granulocytic myeloproliferation and relative reduction of erythroid precursors. Abnormal clustering and increase in atypical giant to medium-sized megakaryocytes contacting bulbulous (cloud-like) hypolobulated nuclei and definitive maturation defects.

A4
Advanced clinical stage Anemia grade III: hemoglobin < 10 g/L. One or more adverse signst.

A1
Persistent increase of platelet count: in excessof 400 × 10 9 /L. B1 Predominant proliferation of dispersed or loosely clustered enlarged to giant A2 Normal spleen or only minor splenomegaly on echogram.

A3
Normal or increased LAP-score and normal ESR.

B2
No or only borderline increase in reticulin.

A5
No signs or cause of reactive thrombocytosis (RTh).

A6
No preceding or allied other subtype of MPD, CML OR MDS.

A7
Absence of the Ph-Chromosme.

A4
Granulocytes > 10 × 10 9 /l and/or raised LAP-score in the absence of fever or infection.

A5
Absence of any cause of secondary erythrocytosis.

MF 3
Advanced myelofibrosis in PV.

B2
Granulocytes > 10 ×10 9 /L or raised LAP-score or increased PRV-1 expression in the absence of fever or infection.

P3
Grading of reticulin fibrosis (RF) post PV-MF-2 and MF-3. Platelet count of > 350 × 10 9 /l and the presence of large platelets in a blood smear (also stage 2 and 3).
Absence bone features consistent with congenital polycythemia and secondary erythrocytosis. No borderline increase in reticulin. RF-01 2 Presence of JAK2-V617F mutation. 3 Low serum EPO level and/or increased LAP score. 4 Spontaneous EEC.

ET stage 3 ET MGM 1
Platelet count of > 350 × 10 9 /l and no signs of leukoerythroblastosis.
Increased cellularity due to megakaryocytic granulocytic myeloproliferation (MGM). Normal or reduced erythropoiesis. Loose to dense clustering of enlaredpleiomorphic megakaryocytes with hyperploid nuclei and the presence of pleiomorphic megakaryocytes with clumsy dysmorphic lobulated nuclei. No or borederline increase in reticulin RF0-1 2 No or slight splenomegay on ultrasound. 3 No anemia with Hb and Ht in the normal or lower range of normal: > 12g/dl. 4 Presence of JAK2-V617F mutation. 5 No proceeding or allied of CML, PV, RARS-T OR MDS.

Clinical and molecular criteria WHO bone marrow criteria ET ET
1. Platelet count > 350-400 × 10 9 /l. Presence of large platelets in blood smear.
Predominant proliferation of small, large and giant megakaryocyte with hyperlobulated, staghorn-like nuclei lacking morphological abnormalities. No increase, proliferation or immaturity of granulopoiesis or erythropoiesis.

No or slight splenomegaly on ultrasound.
Increased cellularity due to chronic megakaryocytic and granulocytic myeloproliferation and reduced erythroid precursors.
Loose to dense clustering of small, large and giant megakaryocytes and the presence of some dysmatureclumpsy lobulated nuclei. Increase in reticulin: RF 2,3 or 4.     and AML [13,14] This concept has been used by the WHO fort he classification of MDS.  1975 the authorative criteria for the diagnosis of PV (Table 1), AMM and primary hemorrhagic thrombocythemia (PHT) and primary myelofibrosis (PMF) [8][9][10]. PMF or AMM is a clinicopathological entity not preceded by any other PVSG defined MPD PHT or ET, PV, CML or preleukemia with myelodysplastic features. CML is leukemia or neoplasia that destroys normal hematopoiesis whereas ET, PV and AMM form a benign proliferation of trilinear hematopoietic proliferation in the bone marrow (myeloproliferation) and extramedullary hematopoiesis in the spleen [8].

PVSG clinical criteria for PV diagnosis: RCM vs. bone marrow histology
The PVSG defined laboratory findings of 325 PV patients in the PVSG 01 study all PV patients had increased red cell mass by definition and showed an increase in hematocrit > 0.52 in 92%, white cells > 12 × 10 9 /L in 43%, platelets > 400 × 10 9 /L in 63%, leukocyte alkaline phosphatase (LAP) score > 100 in 70% and increased spleen size on palpation (splenomegaly due to myeloid metaplasia) in 70% [10]. Absence of splenomegaly was noted in about 30% of cases and leucocytosis and platelet counts can remain normal in early stage PV with typical PV bone marrow histology . Such masked cases of PV with normal platelets, leukocytes and spleen were labeled in 1979 as idiopathic erythrocythemia (IE) by Pearson and Wetherley-Mein [42]. The 1975 PVSG criteria for PV in Table 2 exclude per definition IE (stage 1 PV) . IE is featured by increased red cell mass, normal spleen size, normal leukocyte and platelet counts and no clinical or laboratory evidence of primary or secondary erythrocytosis [42]. Leucocyte alkaline phosphatase (LAP) scoring [8,17], in vitro cultures of erythroid progenitors (EEC) [43] and radioimmunoassay of erythropoetin (EPO) do contribute to differentiate PV from all variants erythrocytosis. These assays are useful when there is only isolated elevation of the red cell mass and all the usual causes of secondary polycythemia have been excluded. The characteristic histology findings in bone marrow biopsies of 155 evaluable PV patients with a documented increased RCM in the PVSG 01 study revealed a broad spectrum of bone marrow cellularity from 50% to 60% in 10 cases, from 60% to 80% in 45 cases, and from 80% to 100% in 100 cases ( Figure  5) [41]. Silver stained reticulin fiber content was normal (RF-0 and 1 = prefibrotic) in 94 cases, slightly increased (RF-2 = early fibrotic) in 40 cases, and moderately to marked increased (RF-3) in 21 cases. The bone marrow histology diagnoses in the PVSG-01 study [11] could roughly be interpreted as typical for normocellular ET in 10, for PV (hypercellular 60%-80%) in 45, for trilinear PV in 70 and for PV/RF-3 or 4 in 13 PV patients (Table 2, Figure 5).The bone marrow histology data of megakaryopoiesis in PVSG PV and PHT studies were identical in appearance, and the condition PV versus ET could not be distinguished on megakaryocyte histology [39-41]. Increased bone marrow cellularity due to increased erythropoiesis and/or myelopoesis in PVSG defined PV and PHT or ET was identical. The PVSG  concluded that the condition PV versus ET could not be distinguished on the basis of bone marrow histopathology. Consequently, the PVSG only used increase red cell mass (RCM) and did not include bone marrow histology as the determinative major inclusion criterion for the diagnosis of PV, and to separate ET from PV [10,[39][40][41]. RCM is insensitive and not specific for the diagnostic differentiation of PV, IE, SE and inapparent PV (Table 1) [17]. In contrast, EEC and bone marrow histology are specific clues for the diagnosis of PV since the early 1970s [1][2][3][41][42][43].
From 1988 to 1994, Lamy et al., [44] measured RCM in 103 consecutive PV patients seen in a single center diagnosed as inapparent PV (IPV) in 18 patients (17%) and as PV with increased hemoglobin (Hb) and hematocrit (Ht) defined, respectively, by Hb > 18 g/dL, Ht > 0.52 in males and Hb > 16 g/dL, Ht > 0.47 in females (Table 3) [44]. IPV or masked PV was defined by a normal Hb and Ht value at diagnosis. In the IPV group, the reasons to perform RCM were as follows: splenomegaly associated with increased platelets and/or leucocytes counts (n = 8), portal vein thrombosis (n = 5), increased platelets or leucocytes counts without splenomegaly (n = 3), and isolated splenomegaly (n = 2) [22]. The two groups were balanced in terms of age, sex, leucocyte, serum iron, and platelet level. Hemoglobin, Ht levels, red cell counts, and plasma volumes were significantly different between the two groups ( Table 3). Red cell mass was increased in the two groups due to hypervolumemia in PV, but caused by splenomegaly in cases with IPV, whereas the erythrocyte counts were increase in the majority of 85 PV patients, but completely normal in 18 IPV patients except one (Table 3). Consequently IPV with normal or decreased hemoglobin, hematocrit and erythrocytes in Table 3 cannot become candidates for phlebotomy because of absence of hypervolumemic symptoms. Treatment will hydroxyurea carry the great danger of inducing relative anemia and acceleration of myelofibrosis. In the context of splanchnic vein thrombosis (portal or splenic vein thrombosis), and hepatic vein thrombosis (Budd Chiari syndrome), RCM and plasma volume are increased due to splenomegaly as the cause of IPV or masked PV [45].
The 1975 PVSG defined clinical criteria for PV are relatively simple to implement but rather crude thereby overlooking prodromal and masked PV because bone marrow histology as a pathognomonic clue to PV and ET was not considered ( Table 1). The PVSG 01 study randomized 431 PV patients for phlebotomy in 134, chlorambucil in 141 and P 32 in 156, there was a significant loss of survival of PV patients due to major thrombotic complications during the first 3 years in the phlebotomy arm due to uncontrolled thrombocythemia and aiming at a too high haematocrit just below 0.50 [9,10,49,50]. In retrospect this would not be the case with the recommendation of phlebotomy aiming at a haematocrit of 0.40 according to Dameshek and Pearson et al., [42] on top of aspirin in the United Kingdom and The Netherlands since 1985 [51]. There was a striking increased incidence of overall malignant complications in PV patients with P 32 and chorambucil as compared to the phlebotomy-treated PV patients during long-term treatment [49,50]. The overall incidence of leukemia/lymphoma and cancer after 10 to 11 years follow-up was 25% in the phlebotomy arm, 40% in the P 32 arm and 67% in the clorambucil arm. The increased incidence of malignancies of bone marrow, lymphoid tissue, skin, and gastrointestinal tract highlights mutagenic effects of chronic myelosuppressive agents in particular when treatment is already started in newly diagnosed early and overt stages of PV. The PVSG 01 trial confirmed the hypothsis of Dameshek [3,50,51] that P 32 is leukemogenic when used as the first line myelosuppressive treatment in early and overt stage PV indicating the need to postpone myelopsuppresive therapy in PV as long as possible [52][53][54][55]. A large group of low risk PV patients included in the PVSG 01 study were exposed to the leukemogenic agents P 32  . According to the intention to treat (ITT), the median survival was 17 years for the whole cohort, 20.3 years for the HU arm, and 15.4 for the pipobroman arm (P = 0.008). At 10, 15 and 20 years, the cumulative incidence (probability) of AML/MDS was 6.6%, 16.5% and 24% in the HU arm versus 13%, 34% and 52% in the pipobroman arm. The cumulative incidence (probability) of myelofibrosis (MF) at 10, 15 and 20 years was 15%, 24% and 32% in the HU arm versus 5%, 10% and 21% in the pipobroman arm (P = 0.02) [56]. Results from PV patients who received only one treatment during the entire period (HU n = 94, Pipobroman n = 130) the cumulative incidence of AML/MDS at 10, 15 and 20 years was 7.3%, 10 [17,65]. Current risk stratification in PV and ET should not anymore be based on age above 60 years and history of thrombosis [55], but on real life MPN disease burden using objective

The 1975 PVSG criteria for ET
Primary hemorrhagic thrombocythemia (PHT) has already been defined in 1960 by Gunz as clinical syndrome of recurrent spontaneous hemorrhages often preceded by thromboses, extremely high platelet count in excess of 1000 × 10 9 /L, frequently splenomegaly, and hypochromic anemia with a tendency towards polycythemia between hemorrhages [37]. Mucocutaneous bleeds from nose gums and gastrointestinal tract were most frequent followed by bruises and bleedings after trauma or surgery [38]. Accordingly, the PVSG used from 1975 to 1986 a minimum platelet count of 1000 × 10 9 /L for the diagnosis for PHT without features of PV or AMM [8,[38][39][40]. The PVSG inclusion and exclusion criteria in 1975 for the diagnosis of PHT or essential thrombocythemia (ET) were very crude [8]: (1) A platelet count in excess of 1000 × 10 9 /L and a bone marrow smear which shows marked megakaryocytic hyperplasia and abundant platelet clumps; (2) Absence of polycythemia vera as defined by the PVSG (normal red cell mass (RCM) [10]; (3) Absence of the Philadelphia chromosome to exclude CML [16]; and (4) Absence of significant reticulin fibrosis (myelofibrosis) with dry tap on bone marrow aspiration, and no signs of preleukemia erythroleukemia or trilinear myelodysplastic syndrome [8,39,40].
PVSG defined PHT 8 labeled as ET [39,40] is featured by platelet counts between 1000 and 3000 × 10 9 /L, splenomegaly in about 80%, autoinfarction of the spleen in 20%, and iron deficient microcytic anemia in 60% [39,40]. In 40% of PHT patients gastrointestinal roentgenograms suggest duodenal ulcer caused by small infarcts in the duodenal mucosa resulting from the high platelet count [40]. In the first prospective evaluation of PVSG defined PHT, 37 evaluable ET patients with platelet counts between 1000 to 2650 × 10 9 /L suffered from thrombohemorrhagic events at presentation including mild bleedings in 5 epistaxis in 5, ecchymoses in 2, pelvic, buccal, fundal or urinary tract hemorrhage in 6, melena with a fall in hemoglobin of 7 gm/dL in 1 and massive postoperative bleeding in 1 case [39,40]. Eleven ET patients experienced acroparesthesias (numbness), including burning sensations, usually in hand or feet (suggestive for erythromelalgia), 9 had dizziness, light-headedness or syncope, 7 had visual disturbances such as scotomas and transient dimming or blurred vision. Catastrophic complications (severe hemorrhages, myocardial infarction, stroke) in 6 (16%) were observed. In this study of 37 untreated PHT or ET patients, bone marrow cellularity was normal in 11%, increased between 50% to 90% in 78% and greater than 90% in 11% [39,40]. Two-thirds of diagnostic bone marrow biopsies showed marked megakaryocyte hyperplasia with atypical large megakaryocytes. Reticulin content was essentially normal in 90% indicating prefibrotic MPD. MPD features in ET and PV were quiete similar: leukocytosis was common in PHT (ET) and PV, LAP scores over 100 were seen in 42% of PHT, and in 70% of PV patients; pruritis was observed in 14% in PHT and 43% in PV patients; the spleen was palpable in 38% of PHT and 70% of PV patients, and when enlarged in PHT the spleen was palpable 2 to 4 cm below the costal margin [8]. Since 1975 we discovered a causal relation between erythromelalgic microvasclur disturbances and thrombocythemia in early early stage MPD disease at platelet counts above 400 × 10 9 /L in symptomatic ET and PV patients with persistent increased platelet count in excess of 400 × 10 9 /L and recognized that the increase of clustered large pleomorphic megakaryocytes in bone marrow biopsies was a pathognomonic clue to myeloproliferative thrombocythemia in ET and PV (Table 4).

The 1980 Rotterdam clinical and pathological (RCP) criteria for ET and PV
As the 1975 PVSG criteria for ET (PTH) are crude, we could recognize since 1975 the existence of erythromelalgic thrombotic thrombocythemia (ETT) in early stage MPD with a persistent increase of platelet count in excess of 400 × 10 9 /L [17,81,82]. At that time we followed since 1975 the definition of PV according to Dameshek as a trilinear MPD [2,3] of which the bone marrow features were described as erythrocytic megakaryocytic granulocytic myeloproliferation (EMGM) by Kurnick et al., [83] in 1972 and Ellis et al., [41] in 1975 ( Figure 6). The trephine biopsy in our ET cases showed a proliferation of large mature megakaryocytes in a normal cellular bone marrow with normal erythropoiesis and granulopoiesis (Table 4, Figure 7). In PV increased erythropoiesis was most prominent [76] together with variable degrees of increased platelets (> 400 × 10 9 /L) [10], erythrocytes (> 6 × 10 12 /L) [2,3] and granulocytes in the peripheral blood in the absence of the Ph-chromosome (Table 4) and could document distinct bone marrow features as the pathognomonicclue to very early stage of ET ( Figure  7). The 1980 RCP criteria of ET and PV were determined by careful prospective documentation of peripheral blood and bone marrow smears and bone marrow biopsy material (Table 4). Platelets in excess of 400 × 10 9 /L, and an increase of clustered enlarged megakaryocytes in a bone marrow biopsy material was found to be pathognomonic diagnostic for ET and excluded reactive thrombocytosis. The combination of bone marrow histology and erythrocyte count above 6 × 10 12 /L according to Dameshek [1][2][3] appeared to be specific clues to the diagnosis of PV (Table 3) (Table 4). First, the major criterion O 2saturation of > 92% is replaced by absence of primary or secondary erythrocytosis by clinical and laboratory tests. Second; splenomegaly is replaced by bone marrow histology as a major criterion (A3). Third, the 1980 RCP diagnostic set used splenomegaly as a minor criterion (Table  4). Fourth, we skipped raised B12 (> 900 ng/L) or raised B12 binding capacity (> 2200 ng/L) as completely irrelevant for the diagnosis of early and overt stage PV (Table 4).
Between 1975 and 1980, we prospectively evaluated the RCP criteria in 30 consecutive early prefibrotic stage patients, who presented with erythromelalgic thrombotic thrombocythemia (ETT), 14 ET and 16 PV patients [84]. The mean age of 30 ETT patients (ET and PV) was 56.7 (range 33-96) years. Eleven of 14 ET patients had platelet counts below 1000 × 10 9 /L, in whom the diagnosis of ET would have been overlooked by the crude PVSG criteria at that time between 1975-1985. Spleen size on scan was slightly increased in 5 of 14 ET and in 13 of 16 PV patients. Leukocyte count counts was increased (> 10 × 10 9 /L) in 5 out of 14 patients with ET and in 14 of 16 PV patients. LAP score was increased (> 100) in 12 out of 14 ET and in all PV patients (Figure 8). All PV patients with erythrocytes above 6 × 10 12 /L had increased red cell mass (manuscript in preparation). Increased erythrocyte counts above 6 × 10 12 /L and increase of large pleomorphic megakaryocytes in bone marrow smear (Dameshek) [2,3] and biopsy is diagnostic for PV [17]. Erythrocyte count at a cutoff level of 6 × 10/ 12 (Table 4). Increased erythrocytes above 6 × 10 12 /L persists in PV in a comple hematological remission by repeated venesection [2,3,77,78].
The presence of clustered large pleomorph megakaryocytes in bone marrow smears and biopsies is diagnostic clue for ET and PV (Table 4, Figures 6 and 7). An ET bone marrow picture with increase of clustered pleomorphic megakaryocytes and no increase of cellularity (Figure 7) was seen in 7 of 14 ET and only in 1 of 16 PV patients [83,84]. A moderate increase of cellularity (60%-80%) in the bone marrow due to increased erythropoiesis (= decreased M/E ratio) consistent with early stage PV was seen in 3 ET and 4 PV patients. A typical PV hypercellular (80%-100%) bone marrow due to megakaryo/ erythro/granulocytopoiesis ( Figure 6) was seen in one ET patients and in the majority of PV [83,84]. These results indicate that bone marrow histopathology on its own is characteristic for MPN but not fully reliable to differentiate between ET and PV [77,78]. The peripheral blood findings in 30 ET (primary thrombocythemia; PT) and 20 PV) seen between 1975 and 1985 are shown in Figure 7 [84]. Hemoglobin levels are normal in ET and elevated in PV, leukocytes were normal or elevated in ET and PV [84]. Out of 30 ET patients 24 had an increased and 6 had a normal LAP scores (Figure 8). Platelet counts were were between 400 × 10 9 /L and 1000 × 10 9 /L in the majority of the 30 ET patients. Serum uric acid levels were normal in ET and frequently elevated in PV. Increased LAP score in the absence of infection and normal erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) is indicative for MPN PV or ET. LAP score was much more elevated in all 20 PV than in ET [84]. As leucocyte alkaline phosphatase (LAP) scoring is becoming increasingly rare in common practice, it can easily be replaced by CD11b neutrophil expression   and unclassifiable MPD [35][36][37]. Reticulin and collagen fibrosis is a reactive feature consecutive to myeloproliferation [37]. The reliable distinction within the three Ph-negative MPDs and its variations appears to be problematic caused by an overlapping cytomorphology of megakaryocyte and within the three MPDs thrombocythemia, PV and CMGM. According to Georgii & Michiels an MPD classification system should be focused cytomorphology of megakaryocytes from bone marrow smears and histopathology from bone marrow biopsies [30][31][32]35,36]. Megakaryocyte morphology and bone marrow histology had become a hallmark of distinction for the diagnostic differentiation of thrombocythemia (ET), PV versus CMGM in the Hannover Institute of Pathology since 1980 ( Figure 5) [67,68]. For the understanding of MPD classification bone marrow histology should distinguish between primary prefibrotic and advanced diseases [35,36]. Primary prefibrotic MPDs according to the Hannover Bone Marrow Classification in 1990 include PV, normocellular thrombocythemia and hypercellular thrombocythemia associated with CMGM without any feature of PVSG defined PV [35,36].  (Tables 8-11).

The 2000-2005 ECP of thrombocthemia in various MPDs
In 2002 Michiels and Thiele defined "true" ET and differentiated "true" ET from hypercellular ET associated with prefibrotic CIMF/ PMGM (Table 5AB, Figure 9) [82]. In "true" ET megakaryocytes display large to giant megakaryocytes showing hyperlobulated staghorn-like nuclei in a normocellular bone marrow, (Table 6, Figure 9) [78,80]. PV was typically featured by large pleomorphic megekaryocytes with hyperploid nuclei in a hypercellular bone marrow due to increased erythropoiesis or increased erythrocytic-megakaryocytic-granulocytic myeloproliferation. Interestingly the megakaryocytes in "true" ET were larger than in PV (Table 5A, Figure 9) [1]. Hypercellular ET associated with prefibrotic CIMF ("false" ET = CMGM = PMGM, (Table 5B, Figures 5 and 9) is dominated by an increase of clustered atypical dysmorphic megakaryocytes due to increases of cellular and nuclear size and bulky nuclei with clumsy lobuli and irregular roundish shaped form (so-called cloud-like nuclei, Figures 5 and 9), which are never described in ET and PV 2 . Normocellular "true" ET according to the 2002 ECP criteria is featured by normal LAP scores (normal CD11b neutrophil expression), higher platelet counts and large to giant megakaryocytes with multilobulated stag-horn like nuclei in a completely normocellular bone marrow (Table 5A) [90,91].
In "true" ET the values for hemoglobin, erythrocytes, and LAP scores (CD11b neutrophil expression) were completely normsal (Table 5A). In contrast, normocellular ET and prodromal PV with increased LAP score (Figure 8) show the presence of pleomorphic megakaryocytes ( Figure 6) low serum EPO levels, slight splenomegaly, and spontaneous EEC [17,84]. This point to the existance of at least two phenotypes of normocelluar ET: ET with features of early PV (prodromal PV) versus "true" ET without features of PV. These differences in RCP defined ET (Table 1) criteria and ECP-defined "true" ET and false ET (Table 5A) are related to a selection bias of patients. In 1988, Thiele as a pathologist selected 25 cases with 1975 PVSG defined normocellular ET (minimum platelet count of 1000 × 10 9 /L) who had pronounced thrombocythemia and normocellular bone marrow without PV features and this was associated with normal LAP score (CD11b neutrophil expression) [87]. Focussing on aspirin-sensitive erythromelalgic inflammatory and ischemic complications as pathognomonic presenting symptoms of early ET or PV patients, the Rotterdam MPD Working group studied a selected and biased group of early stage myeloproliferative ET and PV at platelet counts above 400 × 10 9 /L [84]. Only symptomatic ET and PV patients were included in our 1975-1985 studies because of erythromelalgic ischemic digital circulation disturbances [44].

The 2006-2015 WHO-ECMP Criteria for MPD/MPN
Myelofibrosis is not specific for a disease and can be observed in patient with hairy cell leukemia, Ph-positive CML and in the Phnegative MPDs , CMGM, PV, ET and .many other conditions as well [35][36][37]92,93]. An increase of reticulin in the silver impregnation stain of bone marrow biopsies can be graded as fine or coarse fibers and is a secondary event relevant for prognosis assessment (  (Tables 8-11).With the advent of the JAK2 mutation for the trilinear MPNs we separated the JAK2 V617F positive ET, PV (or EMGM) from the JAK2-negative normocellular ET and hypercellular ET associated with PMGM (Tables 8-11) [77,78,94-96]. Myelofibrosis (MF) itself is the reflection of MPN disease progression but not a disease entity on its own because reticulin and collagen fibrosis are produced by polyclonal fibroblasts in response to cytokines released from the clonal granulocytic and megakaryocytic proliferative cells in both PV and ET of various molecular etiology [69][70][71][72][73][74]. The presence of reticuline fibrosis is well documented in all variants of ET, PV, PMGM, CML and in many other conditions. An increase of reticulin fibrosis is rare in WHO normocellular ET will occur in about one third of PV and will occur in the majority of patients with PMGM during long-term follow-up [35,36,89].
The WHO bone marrow features and the variable phenotypes of thrombocythemias according to the 2000-2005 ECP and the 2006/2007 WHO-ECMP criteria for ET do in fact distinguish within the JAK2 V617F mutated MPN normocellular (WHO-ET, < 60%) and hypercellular ET (prodromal PV, 60%-90%) due to increased erythropoiesis (prodromal PV), from JAK2 wild type normocellular 'true' ET (Tables 5A) and from MPL 515 -mutated normocellular ET (Table 10). We separated JAK2 mutated MPN from JAK2 wild type hypercellular ET associated with a granulocytic myeloproliferation (PMGM, Table 11, Figure  10) [94-96] JAK2 V617F mutated normocellular ET and prodromal PV patients had increased LAP score similar as in PV (Figure 8). Bone marrow histopathology on its own was not reliable to differentiate between JAK2 V617F positive ET, prodromal PV and classical PV. The 200-2005 ECP criteria had defined "true" ET with normal LAP score and giant megakaryocytes with staghorn-like nuclei in a normocellular bone marrow (Table 5) proved to be consistent with MPL515 mutated normocellular thrombocythemia (Table 10). Prefirotic CIMF = PMF = PMGM (Table 11) belonged since 2005 to the JAK2/MPL wild type MPD/MPN (Tables 5B, Table 11, Figure 10) [64], and proved to become thecalreticulin (CALR) mutated Thrombocythemia and MF in the 2015 WHO-CMP claasification (  [77,78]. Bone marrow histology separates idiopathic erythrocytosis with increased RCM from early erythrocythemic stage 1 PV, and do detect "masked" ET, "masked" PV overlooked by the PVSG and WHO criteria with a sensitivity and specificity of 100% if the trephine biopsy is of good quality [77,78]. A bone marrow biopsy is mandatory for grading cellularity in prefibrotic stages and for grading reticulin and collagen fibrosis (Tables 1 and 7)  The 2008 WHO investigators were persistently confronted with unsolved problems and pitfalls regarding in ET of various MPNs and did not recognize the distinct differences in bone marrow histology between JAK2 mutated ET and PV and JAK2 wild type ET without features of PV. The 2008 WHO recognized the differences in megakaryocyte morphology in CML, MDS as compared to PV, but did not pick up the potential importance of significant differences of megakaryocyte morphology related to JAK2 and MPL mutated versus JAK2/MPL wild type ET and MF [77,78]. The 2007-2015 WHO and European clinical, molecular and pathological (EuroCMP) classification of the prefibrotic MPNs distuinguishes JAK2 V617F mutated trilinear ET, prodromal PV, masked PV and classical PV ( Figure 11). Based on distinct megakaryocyte morphology features JAK2 wild type "true" ET (Table 10) carrying the MPL mutation clearly differs from JAK2/MPL wild type hypercelluar ET associated with CMGM/PMGM (Table 11, Figure 10). JAK2 wild type MPL 515 mutated ET is the second distinct thrombocythemia featured by clustered giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow consistent with the diagnosis of "true" ET. JAK2/MPL wild type hypercellular ET is the third distinct thrombocythemia characterized by clustered larged immature dysmorphic megakaryocytes and bulky (bulbous) hyperchromatic nuclei consistent with CMGM/PMGM.   The 2015 WHO-CMP criteria define three phenotypes of JAK2 V617F mutated Myeloproliferative Neoplasms (MPNs) essential thrombocythemia (ET), prodromal polycythemiavera (PV), prodromal PV, classical PV or erythrocytic, megakaryocytic granulocytic myeloproliferation (EMG) and hypercellular ET associated with EMG (masked PV) versus the JAK2 exon 12 mutated idiopathic eryhrocythemia (IE) and PV ( Figure 12) [74,75]. MPL 515 mutated JAK2 wild type ET and MF is a distinct thrombocythemia without features of PV in blood and bone marrow ( Figure 12). Calreticulin mutated Thrombocythemia and MF is the third thrombocythemia entity with characteristic features of primary megakaryocytic granulocytic myeloproliferation (PMGM) in the bone marrow, which are not seen in JAK2 and MPL mutated MPNs ( Figure 12) [103,104]. MPN disease burden in JAK2, MPL and CALR mutated MPN is best reflected by the degree of anemia, splenomegaly, mutation allele burden, bone marrow cellularity and myelofibrosis ( Figure 12) [103,104].