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Journal of Hematology & Thromboembolic Diseases

Michiels et al., J Hematol Thromb Dis 2014, 2:6 http://dx.doi.org/10.4172/2329-8790.1000172

Research Article

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2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms Jan Jacques Michiels1*, Karel Forstier2, Fransje Valster3, Vincent Potters3, Katrien Schelfout3 and Hendrik De Raeve4,5 International Collaboration and Research on Myeloproliferative Neoplasms: ICAR. MPN, Goodheart Insititute in Nature Medicine and Health, The Netherlands Department of Hematology, Rotterdam, The Netherlands 3 Department of Internal Medicine, University Hospital Brussels, Belgium 4 Departments of Pathology, Lievensberg Hospital Bergen op Zoom, OLV Hospital Aalst, The Netherlands 5 Departments of Pathology, University Hospital Brussels, Belgium 1 2

Abstract Somatic mutations in the JAK2, MPL and calreticulin (CALR) genes are the driver causes of clonal myeloproliferative neoplasms (MPN). Applying the WHO Clinical, Molecular and Pathologic (WHO-CMP) classification of MPN, the JAK2V617F positive ET patients comprise three phenotypes of ET: normocellular ET, hypercellular ET due to increased erythropoiesis (prodromal PV) and ET with hypercellular megakaryocytic-granulocytic myeloproliferation (ET.MGM or masked PV). The percentage of JAK2V617F mutation load is low and stable in heterozygous normocellular ET and increasingly high in hetero/homozygous PV and masked PV. The JAK2V617F allele burden is related to MPN disease burden in terms of splenomegaly, constitutional symptoms and myelofibrosis. Five distinct clonal MPNs can be distinguished: JAK2V617F mutated ET and PV; JAK2 exon 12 PV and the JAK2 wild type ET and MF caused by the somatic mutations MPL515 or CALR. JAK2 mutated trilinear MPN reflects a broad spectrum of ET, prodromal or masked PV and classical PV, but the JAK2 wild type MPL or CALR positive ET and MF lack features of PV at diagnosis and during follow-up. Bone marrow features in JAK2V617F mutated ET and PV are similar and featured by medium sized to large (pleomorphic) megakaryocytes with only a few giant forms. Bone marrow histology in MPL515 mutated ET and MF is featured by clustered small and giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow with no features of PV. Bone marrow histology in CALR mutated ET and MF is featured by dense clustered large immature dysmorphic megakaryocytes and bulky (cloude-like) hyperchromatic nuclei similar as described in primary megakaryocytic granylocytic myeloproliferation (PMGM), which are never seen in JAK2V617F, JAK2 exon 12 and MPL515 mutated MPN.

Keywords: Myeloproliferative neoplasms; Essential thrombocythemia; Polycythemia vera; Primary megakaryocytic granulocytic myeloproliferation; Myelofibrosis; JAK2V617F mutation; MPL515 mutation; Calreticulin; CALR mutation; Bone marrow pathology Introduction Polycythemia vera (PV) are described by Vaquez [1], Osler [2,3] and essential thrombocythemia (ET) and PV has been delineated as distinct clinical disease entities when the 1980 Rotterdam Clinical and Pathological (RCP) criteria are applied (Table 1) [4,5]. Osler’s description in 1903 of PV is charaterized by chronic cyanosis, erythrocythemia, and moderate enlargment of the spleen [2]. Osler interpreted in 1908 red painfull ‘neuralgias’in the extremities as erythromelalgia [3]. Michiels discovered between 1975 and 1980 that aspirin responsive erythromelalgia is caused by platelet-mediated arteriolar inflammation and thrombosis in thrombocythemia of patients with ET and PV [4,5]. Park-Weber and Watson [6] recognized that chronic polycythemia with enlarged spleen was a disease of the bone marrow. The oldest treatment of PV is phlebotomy mentioned by Osler in 1903, and 1908 [4,5]. The relationship to PV was described In 1923 Minot and Buckman described the evolution of myelofibrosis and myeloid metaplasia in 3 PV patients, who developed anemia and splenomegaly after 5-20 years follow-up [7]. The original diagnostic criteria and treatment of PV of Dameshek in 1940 and 1950 has been completely overlooked by all leading PVSG and WHO MPN investigators in the USA [8-13]. Dameshek and Henthell described between 1928 and 1937 twenty PV cases and proposed a definite set of diagnosistic criteria for PV by the presence of plethoric appearance, splenomegaly, definitely elevated erythrocyte count (>6 x 1012/L), elevated platelet count, elevated hematocrit and a hypercellular bone J Hematol Thromb Dis ISSN: 2329-8790 JHTD, an open access journal

marrow morphology with increased erythro-megakaryo-grabulocytosis in smears of aspirated bone marrow from the iliac crest or sternum [9].

1975 PVSG versus 1980 RCP criteria for ET and PV The 1975 PVSG clinical criteria used a minimal platelet count of 1000 x 109/L for the diagnosis of ET [10]. The 1975 PVSG diagnostic criteria of PV did not use bone marrow histology and excluded by definition the erythrocythemic stage 1 PV (idiopathic erythrocythemia: IE) with normal platelets, leukocytes and spleen size [11-13]. The 1980 RCP criteria of ET and PV were determined between 1975 and 1980 by careful prospective documentation of peripheral blood and bone marrow smears and bone marrow histology in the late 1970s (Table 1) [5,14]. The PVSG reduced in 1986 the minimum platelet count for the diagnosis of ET from 1000 x 109/L to 600 x 109/L [15] as the consequence of two evidence-based studies by Michiels et al. [5] and Van De Pette et al. [16]. Lengfelder et al. [17] demonstrated in 1998

*Corresponding author: Jan Jacques Michiels, Goodheart Institute, Bloodcoagulation and Vascular Medicine Center, Erasmus Tower, Veenmos 13, 3069 AT Rotterdam, The Netherlands, Tel: +81-22-717-7312; Fax: +81-22-717-7316; E-mail: [email protected] Received October 08, 2014; Accepted November 10, 2014; Published November 17, 2014 Citation: Michiels JJ, Forstier K, Valster F, Potters V, Schelfout K, et al. (2014) 2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms. J Hematol Thromb Dis 2: 172. doi: 10.4172/23298790.1000172 Copyright: © 2014 Michiels JJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Citation: Michiels JJ, Forstier K, Valster F, Potters V, Schelfout K, et al. (2014) 2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms. J Hematol Thromb Dis 2: 172. doi: 10.4172/2329-8790.1000172 Page 2 of 13 A. The 1980 RCP major (A) and confirmative (B) criteria for prefibrotic ET A1 Persistent platelet count in excess of 400 x 109/L [4,5]. 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 B2 Absence of any underlying disease for reactive thrombocytosis and normal ESR. B3 No splenomegaly (32 ml/kg: PVSG 1971-1975 [12,13] A3 Increase in bone marrow biopsy of clustered, large pleomorphic megakaryocytes with hyperlobulated nuclei and increased cellularity due to increased megakaryopoiesis erythropoiesis or typically trilinear mega-erythro-granulopoiesis. typical PV bone marrow excludes erythrocytosis. B1 Thrombocythemia, persistant increase of platelet >400 x 109/L [4,5] B2 Leukocytosis, leucocyte count >109/L and low erythrocyte sedimentation rate (ESR) B3 Raised leukocyte alkaline phosphatase (LAP) score >100, absence of fever or infection B4 Splenomegaly on palpation or on isotope/ultrasound scanning A1 or A2 plus A3 and none of B establishes erythrocythemic PV A1 or A2 plus A3 plus one of B establishes PV and excludes erythrocytosis Table 1: The Rotterdam Clinical and Pathological (RCP) criteria for Essential Thrombocythemia (ET) and Polycythemia Vera (PV) 1975-1980.

Table 2: The Dameshek one cause hypothesis of trilinear PV [33] in 1950 and Vainchenker’s discovery of heterozygous and homozygous JAK2V617F somatic mutations [36] in 2005 as the driver cause of trilinear MPN with clinical and pathological manifestations of Essential Thrombocythemia (ET), Polycythemia Vera (PV) and ‘Primary’ Megakaryocytc Granulocytic Myeloproliferation (PMGM) and secondary Myelofibrosis (MF) [27,31] Designed by Michiels in 2005 [31].

that PVSG defined ET at platelet count above 600 x 10 /L overlooks 30% of early stage ET as compared to the cut-off level of 400 x 109/L (Table 1). Platelets in excess of 400 x 109/L, and increase of clustered larged megakaryocytes in a bone marrow biopsy material were found to be diagnostic for ET and excluded reactive thrombocytosis [5,17]. Between 1975 and 1980, we routinely used bone marrow histopathology and erythrocyte count above 6 x 1012/L according to Dameshek in 1940 [9] as specific clues to the diagnosis of PV to clearly differentiate PV from all variant of primary and secondary erythrocytosis (Table 1). The RCP modifications of PVSG criteria for PV include 4 changes (Table 1). The major criterion O2-saturation of >92% is deleted and replaced by bone biopsy as a major criterion (A3) to differentiate between PV and secondary erythrocytosis. Splenomegaly is used as a minor criterion (Table 1). Raised B12 (>900 ng/L) or raised B12 binding capacity (>2200 ng/L) is skipped as completely irrelevant for the diagnosis of PV J Hematol Thromb Dis ISSN: 2329-8790 JHTD, an open access journal

(Table 1). Bone marrow histology has a specificity and sensitivity near to 100% to differentiate between the MPDs ET and PV from reactive thrombocytosis and primary or secondary erythrocytoses [14,17]. Idiopathic erythrocythemia (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 and a typical PV bone marrow histology (Table 1). The PV experts in the UK and France did not use bone marrow biopsy for the diagnostic differentiation between PV and primary or secondary erythrocytosis and therefore overlooked stage 1 erythremic PV by definition [18,19]. IE represent a significant number of early stage erythremic PV of about 10% to 15% at time of PV presentation [18,19]. The diagnostic difficulties regarding the original PVSG criteria without the use of bone marrow pathology were solved by Tom Pearson by applying on top of PVSG criteria low serum erythropoietine (EPO) levels and

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Citation: Michiels JJ, Forstier K, Valster F, Potters V, Schelfout K, et al. (2014) 2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms. J Hematol Thromb Dis 2: 172. doi: 10.4172/2329-8790.1000172 Page 3 of 13

spontaneous endogenous erythrocyte colony formation (EEC) as specific clues to PV [20]. About half of ET patients are ECC positive and have decreased or low serum EPO levels and are in fact prodromal phases (forme frusta) of PV. Standardized and easy-to-perform commercial serum EPO assays are used for the differential diagnosis of either erythrocytosis or PV [21,22]. In a multicenter study on 241 patients, Mossuz et al. identified two thresholds of serum EPO levels, allowing a specific and correct diagnosis in 65.6% (65 out of 99 PV patients) of PVSG-defined PV patients with serum EPO levels below 1.4 U/L and in 19.7% (13 of 66 SE patients) of secondary erytrocytosis (SE) with serum EPO levels above 13.7 U/L) [21]. Consequently, about 50% of patients with increased RCM could not be diagnosed as PV or erythrocytosis indicating the needto peform a bone marrow biopsy to distinguish PV from SE (Table 1) [5,17].

Bone Classification and WHO criteria for ET, PV and PMGM Georgii et al. discovered in 1980 prefibrotic chronic megakaryocytic gramulocytic myeloproiferation (CMGM) as the third distinct entity of primary MPD in the absence of reticulin or collagen fibrosis in bone marrow biopsy material [23]. In 1987 Michiels et al. defined strict morphological, biochemical, and cytogenetic criteria for BCR/ ABL-positive ET and chronic myeloid leukemia (CML) as a separate malignant and individual entity, whereas ET, PV and CMGM form a chronic proliferation of three hematopoietic cell lines [24]. Michiels et al. [24] and Georgii et al. (Hannover Bone Marrow Classification of CML and MPD, Table 1) [25,26] separated the Ph-positive or BCR/AB -positive CML and ET from the Ph- or BCR/ABL-negative MPDs ET, PV and CMGM based on distinct bone marrow histology findings for each of the three MPDs ET, PV and CMGM [23-28]. The difference in

Hannover Bone Marrow classification ET

size and morphology of small monolobulated megakaryocytes in Phpositive CML and ET from the large pleomorphic megakaryocytes in the Ph-negative MPDs ET and PV is so obvious that cytologists and pathologists can easily distinguish [24,25]. The 1990 Hannover Bone Marrow Classification distinguished three primary prefibrotic MPDs ET, PV and chronic megakaryocytic granulocytic myeloproliferation (CMGM) from advanced fibrotic stages of MPD (Table 2) [25,26]. As myelofibrosis (MF) is a secondary event in all variants of MPD and the terms chronic idiopathic myelofibrosis (CIMF) and primary myelofibrosis (PMF) are a misconception, Georgii consequently replaced the terms CIMF and PMF by CMGM and used grading of reticulin fibrosis (RF) and increase of reticulin and collagen myelofibrosis (MF) for staging of prefibrotic, early fibrotic and overt and advanced fibrotic MPDs ET, PV and CMGM [8,9]. Prefibrotic CMGM is the third MPD entity without features of ET, PV or CML and its diagnosis is based on the presence of loose to dense clustering of large megakaryocytes with immature cytoplasm and cloud-like nuclei not seen in ET, PV and CML [25-28]. The term CMGM of the Hannover Bone Marrow Classification has illogically replaced again by Thiele and Vardiman by chronic idiopathic myelofibrosis (CIMF) in the 2001 WHO classification [29], and as PMF by Tefferi and Thiele in the 2008 WHO classification (Figure 1) [30]. The diagnosis of prefibrotic CMGM is based on the association of hypercellular ET with the presence of large immature megakaryocytes with immature cytoplasm and cloud-like nuclei not seen in ET and PV (Table 2) [31]. The WHO Clinical, Molecular and Pathological (2014-CMP) classification of Michiels et al. of the myeloproliferative neoplasms extended the CMGM concept of Georgii et al. and replaced the term CMGM by primary megakaryocytic granulocytic myeloproliferation (PMGM, Figure 1) [32,33].

PV

ET prefibrotic PMGM

Georgii et al1990 ET RF 0 PV Prefibrotic PMF MF-0,1 Thiele 1988-2008 PVSG 2008 WHO 2008 WHO ET PV Prefibrotic PMF -----------------------------------------------------------------------------------------------------------------------------------------------------PVSG Classification ET PV Prefibrotic MF ↓ translation ↓ 2008 WHO-ECMP

Fibrotic AMM Fibrotic AMMPMF Fibrotic PMF Fibrotic AMM/PMF

Hypercelluar ET

3 stages of ET

PMGM

2008 WHO-ECMP

JAK2V617F

Bone marrow Michiels et al 2006/2014 Cellularity % Bone marrow CALR 2014 Megakaryocytes

ET stage1

ET stage2

ET picture ET/PV picture PV picture 1000 -/2006

ET.MGM

mature

Pleomorph

Pleomorph

Enlarged/clusters Erythropoiese Granulocytosis

+/↑ N/N N/N

+/↑ ↑ N/↑

+/↑↑ ↑↑ ↑↑

Dysmorph nucleci +/↑↑ N/↓ ↑↑

Hemoglobin N Platelets >400 x109/L JAK2V617F 2005

N/N +/+ +/-

0.51 + +/++

N + -/+/++

Conclusion: The 2014 WHO-CMP MPN classification and staging separates JAK2V617F positive classical trilinear PV and ET stage 1, 2 and 3 (in red) from JAK2 wild type PMGM = CALR mutated ET and MF (blue) Figure 1: Hannover Bone Marrow Classification (1990) versus 2008 WHO and 2014 WHO-CMP criteria for the three prefibrotic MPNs ET, PV and PMGM versus primary advanced AMM or endstage MPD of various MPNs diagnosed between 1975 and 2008 in large cohorts of PVSG defined ET, PV, and AMM and its trans;ation into JAK2V617F mutated trilinear MPN (red) and JAK2 wild type CALR mutated ET and MF (Blue). MPL515 mutated ET is not included.

J Hematol Thromb Dis ISSN: 2329-8790 JHTD, an open access journal

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Figure 2: Heterozygous JAK2V617F positive Essential thrombocythemia (ET) stage 1 showing a typical ET bone marrow picture with slightly increased cellularity (60%) due to increased erythropiesis in a case with a 20 years history of stable ET disease.

JAK2V617F mutated trilinear MPNs in ET and PV: DameshekVainchenker’s Disease In 1950, Dameshek (1900-1969) proposed two highly speculative possibilities as the cause of trilinear PV (erythrocythemia, thrombocythemia, granulocythemia: either excessive bone marrow stimulation by an unknown factor, or the lack or diminution of an inhibitory factor [34,35]. This original observation of PV as a trilinear MPD has been proven to be correct by Vainchenker’s discovery in 2005 of the somatic JAK2V617F mutation as the driver cause of he trilinear MPNs ET, PV and MF [36]. This has been rapidly confirmed by Green UK, Kralovics Europe and Levine USA [37-39]. On position 617 of the JAK2 JH2 domain Valine (V) is replaced by Fenylalanine (F) in the JAK2V617F mutation and induces a loss of inhibitory activity of the JH2 pseudokinase part on the JH1 kinase part of JAK2, leading to enhanced activity of the normal JH1 kinase activity of JAK2[36]. The JAK2V617F makes the mutated hematopoietic stem cells hypersensitive to hematopoietic growth factors TPO EPO, IGF1, SCF and GCSF, resulting in PV as a trilinear MPN (Table 2) [31]. Detection of JAK2V617F has become the first intention diagnostic test for ET and PV (Tables 3 and 4) [31]. The prevalence of the JAK2 V617F mutation in PVSG defined PV is 95% and about 50% in ET and MF [31]. The JAK2V617F mutation load in gramulocytes is usually low in heterozygous ET, less that 10 to maximal 50% and either low with less than 50% (heterozygous homozygous) or high between 50 to 100% (homozygous) in PV [40,41]. Patients with hypercellular ET, masked PV and PV homozygous for the JAK2V617F mutation patients are at high risk for myeloid metaplasia of the spleen with splenomegaly and bone marrow transformation into myelofibrosis (MF) [42]. The 2005 concept according to Vainchenker and Michiels is that heterozygous JAK2V617F mutation leading to constitutively activated megakaryocytes with increased sensitivity to TPO and EPO is enough to induce ET with J Hematol Thromb Dis ISSN: 2329-8790 JHTD, an open access journal

the production of constitutively activated (hypersensitive) platelets (Table 2) [31]. So-called heterozygous PV with allele load less than 50% appeared to be hetero/homozygous for the JAK2V617F mutation at the EEC level in blood and bone marrow for the JAK2V617F mutation, whereas ET patients are heterozygous for the JAK2V617F mutation at the EEC level with a maximal JAK2V617F mutation load ranging from low to maximal 50% [43,44]. The change from heterozygous ET into homozygous masked or overt PV is due to the loss of 9p heterogeneity (9P LOH) of the JAK2V617F locus through mitotic amplification resulting in homozygosity of JAK2V617F somatic mutation on chromosome 9p (Table 2) [25]. Godfrey et al. studied the JAK2 mutation status of BFU-E grown in low erythropoietin conditions in 77 patients with PV or ET [45]. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, homozygous JAK2V617F mutant colonies were absent or present in low percentages in heterozygous ET, but prevalent and common in patients with JAK2V617F-positive PV45. PV was distinguished from ET by expansion of a dominant homozygous JAK2V617F subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Hetero/homozygous or homozygous JAK2V617F mutation is associated with pronounced constitutively activation and genetic instability of megakaryopoiesis, erythropiesis and granulopoiesis in the bone marrow as the cause of hypercellular trilinear PV with a high risk of myelofibrosis (Table 2). According to WHO [30] and WHO-CMP criteria (Tables 3 and 4) [32,33], heterozygous JAK2V617F positive ET is defined by a normocellular bone marrow histology with slight increase of erythropoiesis (Figure 2) or with a hypercellular bone marrow histology due to increased erythropoiesis (prodromal PV or masked PV). JAK2V617F positive hypercellular ET associated with prefibrotic megakaryocytic granuloctic myeloproliferation and (relative) reduction of erythropoiesis is consistent with hypercellular ET associated with

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Citation: Michiels JJ, Forstier K, Valster F, Potters V, Schelfout K, et al. (2014) 2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms. J Hematol Thromb Dis 2: 172. doi: 10.4172/2329-8790.1000172 Page 5 of 13 Clinical and molecular criteria

Bone marrow pathology criteria (WHO)

ET

Normocellular ET

1. Platelet count of >350 x109/l and the presence of large platelets in a blood smear 2. Heterozygous JAK2V617F mutation, low mutation load 3. Normal erythrocytes 5.8 x 1012/L males >5.6 x 1012/L females A 2. Presence of heterozygous and/or homozygous JAK2V617F or JAK2 exon 12 mutation A 3. Low serum Epo level Minor B 1. Persistent increase of platelet count x109/L: grade I: 400-1500, grade II: >1500. B 2. Granulocytes >10 x109/l or Leukocytes >12 x109/l and raised LAP-score or increased CD11b expression in the absence of fever or infection B 3. Splenomegaly on ultrasound echogram (>12 cm length in diameter) or on palpation. B 4. Spontaneous endogenous erythroid colony (EEC) formation (optional)

P1. Bone marrow pathology: increased cellularity (60-100%) due to trilinear increase of erythropoiesis, megakaryopoiesis and granulopoiesis and clustering of small to giant (pleomorph) megakaryocytes with hyperlobulated nuclei. Absence of stainable iron. No pronounced inflammatory reaction P2. Erythrocytosis. Normal erythropoiesis, normal granulopoiesis and megakaryocytes of normal size, morphology and no clustering Grading of reticulin fibrosis (RF) and reticulin collagen myelofibrosis (MF) Prefibrotic: RF-0/1=MF-0 Early fibrotic: RF-2=MF-1 Fibrotic: RCF 3=MF-2 Post-PV MF: RF 4=MF-3

A2 + B1 + P1 establish early PV (mimicking ET) prodromal PV CMP stage 0 A1 + A2 + A3 + P1and none of B establish idiopathic erythrocythemia (IE) or stage 1 PV A1 + A2 + A3 + P1 and one or more of B establish classic stages of PV stage 2 and 3 A2 + B3 + P1 detect masked cases of PV with splenomegaly and hypersplenism to be labelled as Inapparent PV (IPV) frequently seen Budd-Chiari syndrome or splanchnic vein thrombosis Table 4: 2014 WHO Clinical Molecular andPathological (WHO-CMP) criteria for the diagnosis of prodromal, masked and classical JAK2 mutated polycythemia vera (PV) versus primary or secondary erythrocytoses [32,33].

a MGM bone marrow (EMGM or masked PV, Table 3) [32,33]. JAK2V617F mutated WHO defined PV typically shows a hypercellular bone marrow histology due to increased trilinear hematopoiesis of megakaryopoiesis, erythropoiesis and granulopoiesis (panmyelosis of Dameshek [34]) and no or slight increase of reticuline fibers (Figure 3 and Table 4). The UK MPN Study Group assessed the clinical features in the cohort of 806 PVSG defined ET patients subdivided in 414 JAK2V617F positive and 362 JAK2 wild type ET and evaluated the bone marrow features in 393 ET patients [46,47]. JAK2V617F positive ET patients had multiple features of PV such as a significantly higher hemoglobin, lower serum EPO and ferritin, higher neutrophils, bone marrow erythrocytosis and granulocytosis, more venous thrombosis and a higher rate of polycythemic transformation. PVSG defined JAK2 wild type ET had significant higher platelet counts (962, range 668-1535x109/L) than JAK2V617F-positive ET (846, range 632-1222x109/L) [46]. In the UK MPN (Primary Thrombocythemia 1 (PT-1) study, bone marrow trephine of 209 JAK2V617F positive and 184 JAK2 wild type ET was independently assessed by 3 hematopathologists who did not know the JAK2 mutation status [47]. The overall cellularity was significantly


increased in JAK2V617F mutated ET as compared to JAK2 wild type ET, indicating that increased erythroid and/or granulocytic cellularity are features of prodromal PV or masked PV [47]. Pich et al. prospectively analyzed histological changes in diagnostic bone marrow biopsy from 2006-2010 of 103 newly diagnosed WHO defined ET patients [48]. Bone marrow features in 44 JAK2 wild ET cases revealed prominent clusters of large megakaryocytes with staghorn nuclei, less micromegakaryocytes and no or minor erythroid hyperplasia as compared to JAK2V617F positive ET [48]. In contrast, 59 JAK2V617F positive ET patients revealed a typical PV bone marrow histology with higher hemoglobin, hematocrit, erythrocytes and increased bone marrow due to hyperplasia of erythroid and myeloid lineages and the presence of pleomorphic megakaryocytes very similar as in WHO-CMP defined ET and PV (Tables 3 and 4). The mean and median JAK2V617F mutation burden in 2008 WHO defined ET was 14.4% and 8.7% respectively [48]. Interestingly LDH (604+132) and spleen size (15.4+4.9 cm on echogram) in 16 ET cases with a JAK2V617F mutation load above 12.5% were significantly increased as compared to normal LDH (386+94) and normal spleen size (11.2 ± 2.1 cm on echogram) in 37 ET cases with a JAK2V617F mutation load below 12.5% [48].

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Figure 3: Homozygous JAK2V617F positive polycythemia vera (PV) with hypercellular bone marrow histology due to increased erythropoisesis and granulopoiesis (A,B) with pleomorphic megakaryocytes (C ) PV bone marrow histology with a hypercellular bone marrow picture due to increased trilinear hematopoiesis of megakaryopoiesis, erythropoiesis and granulopoiesis and slight increase of reticuline fibers grade 1 (D).

Figure 4: Bone marrow histology in seven PV patients with JAK2 exon 12 mutations showed prominent erythroid hyperplasia meeting the criteria for idiopathic erythrocythemia (IE) in 4 and classical PV in 2, and hyperplasia of atypical small to medium-sized large megakaryocytes with various degrees of monolobation to hyperlobation and abnormal chromatin distribution [51].

JAK2 exon 12 mutations as cause of IE and PV The finding of the JAK2 exon 12 mutations in PV patients negative for the JAK2V617F mutation further confirms the strong association between the JAK2 mutations and MPN [49,50]. The 5% PV patients negative for JAK2V617F are frequently heterozygous for exon 12 JAK2 mutations and usually present with early stage PV or idiopathic erythrocythemia J Hematol Thromb Dis ISSN: 2329-8790 JHTD, an open access journal

(IE) with favourable outcome and normal life expectancy. The UK MPN Study Group identified JAK2 exon 12 mutations in 10 JAK2V617F – negative PV patients with increased red cell mass, which according to PVSG criteria could diagnosed as PV in 6 and IE in 4 cases [49]. Pre-treatment bone marrow biopsies in 5 patients carrying one of the JAK2 exon 12 mutations showed characteristic erythroid hyperplasia

Volume 2 • Issue 6 • 1000172

Citation: Michiels JJ, Forstier K, Valster F, Potters V, Schelfout K, et al. (2014) 2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms. J Hematol Thromb Dis 2: 172. doi: 10.4172/2329-8790.1000172 Page 7 of 13 Clinical and molecular JAK2 wild type ET

Bone marrow pathology criteria (WHO)

1. Platelet count >350 x 109/L and presence of large platelets in blood smear 2. Hemoglobin, haematocrit and erythrocyte count in the normal range 3. Presence of MPL515 mutation and JAK2 wild type 4. Normal serum EPO 5. Normal LAP score and CD11b expression 6. No or slight splenomegaly 7. No leukoerythroblastosis 8. No preceding or allied CML, PV, RAS-T or MDS

P1 . Proliferation of large to giant mature megakaryocyte with hyperlobulated, staghorn-like nuclei in a normocellular bone marrow (12 g/dL, slight to moderate splenomegaly, thrombocytosis around or above 1000 x 109/L, normal LAP score

MF Grading reticulin fibrosis (RF), and reticuln colloagen myelofibrosis (MF)

A2 Presence of CALR mutation and JAK2 wild type

C2. Intermediate clinical stage: slight anemia Hb 10 g/dL, decreasing platelet count, splenomegaly, increased LDH and definitive tear drop erythrocytes C3. Advanced stage: anemia Hb 350 x109/l or clinical suspicion on masked MPN (splanchnic vein thrombosis)

Bone marrow biopsy (BMB) and JAK2V617F mutation screening

BMB: ET, early PV, MGM and RF 1-4 or MF 0-3

JAK2V617F(+) ET, prodromal PV, EMGM grading RF Thrombocythemia: Platelet count:

Masked Normal -

JAK2V617F(-) MPL515 or CALR ET vs MGM MF grading RF

Early 400

Both normal: no MPN

RARS-T: JAK2V617F +/Atypical MPN/MDS Overt

600

>1500 x109/L PVSG 1975 (*70%/>99%) WHO 2008 (*80%/>99%)

Bone marrow biopsy (*>99%/>99%) including grading of reticuln fibrosis: RF and MF JAK2V617F (*60%/100%) MPL515 (0.70

IE (15%)

PVSG (*65%/95%) WHO (*80%/>99%)

Bone marrow biopsy (*>99%/>99%) including grading of reticulin fibrosis: RF and MF JAK2V617F (*95%/100%) JAK2 exon12 (3%) 2014 WHO-CMP criteria for polycythemia vera versus erythrocytosis 99%/100% *: sensitivity/specificity

Figure 9: Diagnostic algorithm for the diagnosis and staging of JAK2 mutated Polycythemia vera (PV) and diagnostic sensitivity and specificity of the 1975 PVSG, the 2008 WHO, bone marrow biopsy and the 2014 WHO-CMP criteria for the diagnosis of JAK2 mutated PV.

Normocellular ET Heterozygous JAK2 V617F+

Prodromal PV JAK2 V617F+

Prefibrotic EMGM ,Masked PV JAK2 V617F++

Hereditary ET: HET Germline Gain of function TPO, JAK2 or MPL gene Acute onset PV JAK2 V617F++

PV JAK2 V617F+/++ JAK exon12+ IE/PV

Increasing splenomegaly RF-1, 2, 3, 4

Post-PV & Post-ET MF-1 MF-2, MF-3 JAK2 V617F ++

JAK2 wild type Normocellular ET MPL515 mutated

MF RF 1, 2, 3 RCF JAK2 neg

Hypercellular ET CALR mutated MGM

Figure 10: The Spectrum of MyeloproliferativeNeoplasms ET, PV andMF according to 2014 WHO-CMP Classification, Staging and Translational states of congenital heterozygous JAK2 mutated essential thrombocythemia (ET) without features of polycythemia vera (PV) [64-66], acquired JAK2 mutated ET, PV and myelofibrosis (MF) [31,33,36] versus MPL mutated ET and MF [32,33] and CALR mutated ET and MF [58-62].

to 100% (Figures 8-10) [67,68]. Within the JAK2 mutated ET and PV at time of diagnosis the megakaryocytes are similar large and pleiomorphic with normocellular bone marrow in heterozygopus JAK2 mutated ET and increased cellularity of 60-80% due to increased erythropoiesis=prodromal PV and strongly increased 90-100% cellularity in more advanced EMGM=masked PV and in classical


prefibrotic PV due to increased trilinear hematopoiesis of increased erythropoiesis, megakaryooiesis and granulopoisesis (Tables 3 and 4) [14,27-35,67,68]. Staging of MPN disease burden in the trilinear MPN phenotypes of JAK2 mutated ET, masked PV and overt PV is based on grading of splenomegaly, LDH, leukocyte count and grading of reticulkin fibrosis on top of JAK2 allele burden (Figures 8-10).

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Exon 12 JAK2 mutated PV reflects a more favorable presentation of mainly erythrocythemia and and early stage PV with no or slight increase of platelets and leukocytes during long-term follow-up as compared to JAK2V671F mutated masked and classical PV. JAK2 wild type ET carrying the MPL or CALR mutation do not show PV features in blood and bone marrow during the prefibrotic and early stages of reticulin fibrosis (myelofibrosis=MF) at diagnosis and during longterm follow-up. CALR mutated ET present with high platelet counts around 1000x109/L which after longterm follow-up tend to decline, which is related to progressive splenomegaly and increased MF (Figures 8-10). CALR mutated ET and MF is featured by hyeprcellular bone marrow due to dual primary megakaryocytiv granulocytic myeloproliferation (PMGM) with relative or absolute reduction of erythropoiesis and the presence of loose to dense clustered more or less immature large megakaryocytes with immature cloud-like nuclei, which are not seen in JAK2 mutated ET and PV and also not in MPL mutated ET (Figures 8-10). Bone Marrow Pathology (BMP) has a specificity and sensitivity near to 100% to differentiate between all molecular variants of the the MPNs ET, PV and early stage MF from reactive thrombocytosis and primary or secondary erythrocytoses [67-70]. Bone marrow histology alone has a near to 100% accuracy to distinct MPN from CML and MDS (RARS-T and 5q-minus syndrome). The sensitivity and specificity to distinguish JAK2 on one hand vs MPL vs CALR mutated ET on the other hand just based on bone marrow histology alone surely will not always be possible [69,70]. The distinction of JAK2, versus MPL versus CALR mutated ET based on megakaryocyte morphology and backgound increase of cellularity is predicted to have an estimated accuracy of 70 to 80% in pretreatment diagnostic bone marrow biopsies at time of diagnosis [67-70]. The diagnostic differentiation and staging of the early and fibrotic stages of JAK2, MPL and CALR mutated MPNs should be based on bone marrow morphology on top of quantitaive measurement of JAK2, MPL and CALR mutation load related to the degree of anemia and splenomegaly to validate the natural history of each of the MPN disease burden prospectively during evolution of all molecular variants of ET into MF [25-28,67-70]. References 1. Vaquez MH (1892) Sur uneforme speciale de cyanose sa™accompagnant da ™hyperglobulie excessive et persistante. Comptes rendues des seances de la siciete de Biologie 44: 384-388. 2. Osler W (2008) Chronic cyanosis, with polycythaemia and enlarged spleen: a new clinical entity. 1903. Am J Med Sci 335: 411-417. 3. Osler W (1908) A clinical lecture on erythhaemia. Polycythemia vera with cyanosis. Maladie du Vaquez. Lancet 1: 143-145. 4. Michiels JJ (1997) Erythromelalgia and thrombocythemia: a disease of platelet prostaglandin metabolism--thesis, Rotterdam, 1981. Semin Thromb Hemost 23: 335-338. 5. Michiels JJ, Abels J, Steketee J, van Vliet HH, Vuzevski VD (1985) Erythromelalgia caused by platelet-mediated arteriolar inflammation and thrombosis in thrombocythemia. Ann Intern Med 102: 466-471. 6. Weber FP, Watson JH (1904) Chronic polycythemia with enlarged spleen, probably a disease of the bone marrow. Br Med J 1: 729. 7. Minot GR, Buckman THE (1923) Erythremia (polycythemia rubra vera). Am J Med Sci 166: 469-489. 8. Tefferi A (2008) The history of myeloproliferative disorders: before and after Dameshek. Leukemia 22: 3-13. 9. Dameshek W, Henstell HH (1940) The diagnosis of polycythemia. Ann Intern Med 13: 1360-1387. 10. Laszlo J (1975) Myeloproliferative disorders (MPD): myelofibrosis, myelosclerosis, extramedullary hematopoiesis, undifferentiated MPD and hemorrhagic thrombocythemia. Semin Hematol 12: 409-432. 11. Wasserman LR Berk PD, Berlin NI (1995) Polycythemia vera and the myeloproliferative disorders. WB Saunders Philadelphia.


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Citation: Michiels JJ, Forstier K, Valster F, Potters V, Schelfout K, et al. (2014) 2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms. J Hematol Thromb Dis 2: 172. doi: 10.4172/2329-8790.1000172 Page 13 of 13 32. Michiels JJ, De Raeve H, Hebeda K, Lam KH, Berneman Z, et al. (2007) WHO bone marrow features and European clinical, molecular, and pathological (ECMP) criteria for the diagnosis of myeloproliferative disorders. Leuk Res 31: 1031-1038. 33. Michiels JJ, Berneman Z, Schroyens W, De Raeve H (2015) Changing concepts on the diagnostic criteria of myeloproliferative disorders and the molecular etiology and classification of myeloproliferative neoplasms. From Dameshek 1950 to Vainchenker 2005 and beyond. Acta Haematol (2014 online) 133: 3651. 34. DAMESHEK W (1950) Physiopathology and course of polycythemia vera as related to therapy. J Am Med Assoc 142: 790-797. 35. Michiels JJ, Institute And Foundation G, Education Thrombocythemia Vera Study Group FO, Ewg Mpn TA (2013) Physiopathology, etiologic factors, diagnosis, and course of polycythemia vera as related to therapy according to william dameshek, 1940-1950. Turk J Haematol 30: 102-110. 36. James C, Ugo V, Le Coudic JP, Staerk J, Delhommeau F, (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature 434: 1144-1148. 37. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, et al. (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365: 1054-1061. 38. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, et al. (2005) A gain-offunction mutation of JAK2 in myeloproliferative disorders. N Engl J Med 352: 1779-1790. 39. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, et al. (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia and myeloid metaplasia with myelofibrosis. Cancer cells 7: 387-397. 40. Antonioli E, Guglielmelli P, Pancrazzi A, Bogani C, Verrucci M, et al. (2005) Clinical implications of the JAK2 V617F mutation in essential thrombocythemia. Leukemia 19: 1847-1849. 41. Vannucchi AM, Antonioli E, Guglielmelli P, Longo G, Pancrazzi A, et al. (2007) Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden. Leukemia 21: 1952-1959. 42. Passamonti F, Rumi E, Pietra D, Matteo G, Porta D, et al. (2006) Relation between JAK2 V617F mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders. Blood 107: 3676-3682.

51. Lakey MA, Pardanani A, Hoyer JD, Nguyen PL, Lasho TL, et al. (2010) Bone marrow morphologic features in polycythemia vera with JAK2 exon 12 mutations. Am J Clin Pathol 133: 942-948. 52. Ding J, Komatsu H, Wakita A, Kato-Uranishi M, Ito M, Satoh A, Tsuboi K, Nitta M, Miyazaki H, Iida S, Ueda R (2004) Familial essential thrombocythemia associated with a dominant-positive activating mutation of the c-MPL gene, which encodes for the receptor for thrombopoietin. Blood 103: 4198-4200. 53. Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M, et al. (2006) MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood 108: 3472-3476. 54. Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, et al. (2006) MPLW515L is a novel somatic activation mutation in myelofibrosis with myeloid metaplasia. PLOS Med 3: e270. 55. Vannucchi AM, Antonioli E, Guglielmelli P, Pancrazzi A, Guerini V, et al. (2008) Characteristics and clinical correlates of MPL 515W>L/K mutation in essential thrombocythemia. Blood 112: 844-847. 56. Beer PA, Campbell PJ, Scott LM, Bench AJ, Erber WN, et al. (2008) MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort. Blood 112: 141-149. 57. Jones AV, Campbell PJ,Beer PA, Schnittger, Vannucchi AM, Zoi K, Percy MJ, McMullin MF, Scott LM, Tapper W, Silver RT, Oscier D, Harrison CN, Grallert H, Kisialiou A,Strike P, Chase AJ, Green AR, Cross NCP (2010) The JAK2 46/1 haplotype predisposes to MPL-mutated myeloproliferative neoplasms. Blood 115: 4517-4523. 58. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, et al. (2013) Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 369: 2379-2390. 59. Nangalia J, Massie CE, Baxter J, Nice FL, Gundem G, et al. (2013) Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2. N Engl J Med 369: 2391-2405. 60. Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, et al. (2014) Jak2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcome. Blood 123(10): 1544-1551. 61. Rumi E, Pietra D, Pascutto C, Guglielmelli P, Martinez-Trillos A, et al. (2014) Clinical effects of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis. Blood 124(7): 1062-1069. 62. Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, et al. (2014) CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 28: 1472-1477.

43. Scott LM, Scott MA, Campbell PJ, Green AR (2006) Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera, but not essential thrombocythemia. Blood 108: 2435-2437.

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