WHO defined polycythemia vera (PV) and half of patients with essential thrombocythemia (ET) patients have low serum erythropoietin (EPO) levels, and carry the JAK2^V617F mutation. Clinical, laboratory, molecular and pathologic (CLMP) criteria for the JAK2^V617F positive ET patients comprise three phenotypes of ET: normocellular ET (WHO-ET), hypercellular ET due to increased erythropoiesis (prodromal PV) and ET with hypercellular megakaryocytic-granulocytic myeloproliferation (EMGM or masked PV). JAK2^V617F mutation load is low and stable in heterozygous normocellular ET and high in heterozygous homozygous JAK2 mutated PV and masked PV. JAK2^V617F mutation load is related to MPN disease burden in terms of splenomegaly, constitutional symptoms and myelofibrosis. Five distinct clonal MPNs can be distinguished: JAK2^V617F-positive ET and PV; JAK2 exon 12 PV; JAK2 wild type ET and MF carrying the MPL⁵¹⁵; JAK2 wild type ET caused by somatic mutations in the calreticulin (CALR) gene. Bone marrow features in JAK2^V617F mutated ET and PV are similarly featured by medium sized to large (pleomorphic) megakaryocytes with only a few giant forms. Bone marrow histology in MPL⁵¹⁵ positive ET and MF show 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 the novel third MPN entity of CALR mutated ET and MF patients is dominated by dense clusters of large immature dysmorphic megakaryocytes with bulky (cloud-like) hyperchromatic nuclei, which are described in primary megakaryocytic granylocytic myeloproliferation (PMGM), and  never seen in JAK2^V617F , JAK2 exon 12 and MPL⁵¹⁵ mutated MPN.

INTRODUCTION

The clinical and pathological features for prodromal, erythrocythemic and polycythemic stages of PV are variable and featured by increased erythrocytes above 6 × 10¹²/L, increased leukocyte alkaline phosphatase (LAP) score (increased CD11bexpression), normal or increased platelets, leukocytes and spleen size, and by characteristic bone marrow features with increased pleomorphic large megakaryocytes and erythropoiesis (Table 1) [1-4]. The clinical and bone marrow histology features of “true” ET has been recognized by Thiele et al. [5,6] in 1988 and 2005 as clearly distinct from PV. The peripheral blood findings in “true” ET are featured by high platelet counts, normal values for haemoglobin, haematocrit, erythrocyte, white blood cells, LAP score, LDH and no or minor splenomegaly despite platelet counts above 1000 × 10⁹/L [7]. The megakaryocytes in “true” ET are larger than in PV (Table 1) [5-7]. Michiels and Thiele [7] have defined in 2002 normocellular “true” ET as a distinct MPN entity different from PV and hypercellular ET associated with prefibrotic primary myelofibrosis (pPMF) or primary megakaryocytic granulocytic myeloproliferation (PMGM, Table 1). Bone marrow histology in PV is typically featured by large pleomorphic megakaryocytes with hyperploid nuclei in a hypercellular bone marrow due to increased erythropoiesis or increased trilinear erythrocytic, megakaryocytic and granulocytic myeloproliferation [2-4]. Georgii et al. [8,9] discovered a third entity of primary myeloproliferative disease (MPD) characterized by chronic or primary megakaryocytic granulocytic myelosis (CMGM/PMGM) in the absence of reticulin or collagen fibrosis in bone marrow biopsy material (Figure 1). The Hannover Bone Marrow Classification of the MPDs distinguished three primary prefibrotic MPDs ET, PV and CMGM from the advanced fibrotic stages of MPD [8]. Myelofibrosis (MF) is a secondary event in all variants of MPD, the labeling of chronic idiopathic myelofibrosis or primary myelofibrosis (CIMF or PMF) is a contradiction of terms [8,9]. Georgii et al. [8,9] replaced the term CIMF and PMF by CMGM and used grading of myelofibrosis (MF) for staging of early, overt and advanced stage of MF in patients with ET, PV and CMGM. Michiels replaced in 1997 the term CMGM by essential or primary MGM (PMGM) as the third JAK2 wild type MPN entity without features of PV or CML [10-12]. EMGM or PMGM (hypercellular JAK2 wild type ET) is the third MPN entity 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), which are never described in JAK2 mutated ET and PV [8-12].

HANNOVER BONE CLASSIFICATION AND WHO CRITERIA FOR THE MPDS ET, PV AND PMGM

According to strict morphological, biochemical and cytogenetic criteria for BCR/ABL-positive ET and CML is a separate malignant and individual entity, whereas ET, PV and CMGM form a chronic proliferation of three hematopoietic cell lines [13]. The Hannover Bone Marrow Classification of MPD (Table 1) [8,9,13] 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 [8-12]. The difference in size and morphology of small monolobulated megakaryocytes in Ph-positive 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 [13,14]. Prefibrotic CMGM/PMGM is the third distinct entity of primary MPD in the absence of reticulin or collagen fibrosis in bone marrow biopsy material [15]. The Hannover Bone Marrow Classification distinguished the three primary prefibrotic MPDs ET, PV and CMGM/PMGM from advanced fibrotic stages of MPD (Figure 1) [8,9]. Myelofibrosis (MF) is a secondary event in all variants of MPD. Consequently, the terms chronic idiopathic myelofibrosis (CIMF) or primary myelofibrosis (PMF) are a misconception. Georgii replaced the term CIMF and PMF by CMGM and used grading of myelofibrosis (MF, Table 1) for staging of the early, overt and advanced MPDs ET, PV and CMGM [8,9]. Prefibrotic CMGM/PMGM 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 [8-15]. The term CMGM of the Hannover Bone Marrow Classification of the MPDs is illogically replaced in the 2001 WHO classification into chronic idiopathic myelofibrosis (CIMF) [16,17] and has been labeled as PMF in the 2008 WHO classification (Figure 1) [18]. 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 1). The 2015 ECMP classification of myeloproliferative neoplasms (MPN) took over the CMGM concept and used the term primary megakaryocytic granulocytic myeloproliferation (PMGM, Table 1 and Figure 1) [19].

The 1975 PVSG criteria exclude stage 1 idiopathic erythrocythemia (IE) and do not recognize prodromal PV by definition [20]. According to ECP criteria 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 PV bone marrow histology. ECP defined bone marrow histology has a specificity and sensitivity near to 100% to differentiate between the MPDs ET and PV from reactive thrombocytosis and all variants of primary or secondary erythrocytoses (Table 1) [2,3]. 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 [21,22]. In 1979 idiopathic erythrocythemia (IE) has been defined by increased RCM and not meeting the A and B criteria of the PVSG [21]. IE represent a significant number of early stage erythremic PV of about 10% to 15% at time of PV presentation [21-23]. A low serum EPO level has also been described about half of ET patients, which are to be regarded as prodromal phases (forme frusta) of PV [17,23]. Standardized and easy-to-perform commercial serum EPO assays provide a reliable and accurate criterion in support of the diagnosis of either erythrocytosis or PV and ET [24,25]. In a multicenter study on 241 patients, Mossuz et al. [25] identified two thresholds, allowing a specific and direct diagnosis of 65.6% (65-99) of untreated PVSG-defined PV (EPO<1.4 U/L) and 19.7% (13 of 66) of SP (EPO>13.7 U/L) [25]. About 50% of patients with absolute erythrocytosis could unequivocally diagnose as PV or erythrocytosis by the combination of increased red cell mass (RCM) and serum EPO levels.

JAK2^V617F MUTATED TRILINEAR MPNS IN ET AND PV: VAINCHENKER’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 [26]. This one cause hypothesis of PV as a trilinear MPD has been proven to be correct by Vainchenker in France of the somatic JAK2^{Vainchenker617Franc} mutation as the driver cause of trilinear MPNs ET, PV and MF [27]. On position 617 of the JAK2 JH2 domain Valine (V) is replaced by Fenylalanine (F) in the JAK2^V617F 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 JAK2²⁷. The JAK2^V617F 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 1). Detection of JAK2^V617F has become the first intention diagnostic test for erythrocytosis [17]. The prevalence of the JAK2 ^V617F mutation in PVSG defined PV is 95% and about 50% in ET and MF [17]. The JAK2^V617F mutation load is usually low in ET, less than 10 to 50% of the granulocytes are JAK2^V617F positive (heterozygous) and either low or high in PV with less than 50% (heterozygous homozygous) or high between 50 to 100% (homozygous) of the granulocytes positive for the JAK2^V617F mutation [28-30]. Patients with hypercellular ET and PV homozygous for the JAK2^V617F mutation patients are at high risk for myeloid metaplasia of the spleen with splenomegaly and bone marrow transformation into myelofibrosis (MF) and the percentage of JAK2^V617F positive granulocytes in PV may range from rather low to 100% for JAK2^V617F during the long-term follow-up [28-30]. At the bone marrow hematopoietic stem cell level, ET patients are heterozygous and PV patients hetero/homozygous or homozygous for the JAK2^V617F mutation (Figure 2) [17]. The 2005 concept of Vainchenker and Michiels [17] is that heterozygous JAK2^V617F mutation is enough to constitutively activate TPO mediated megakaryopoiesis to induce ET with the production of constitutively activated (hypersensitive) platelets (Figure 2) [17]. Homozygous JAK2^V617F mutation is needed to more pronouncedly stimulate EPO mediated erythropoiesis compared to TPO mediated megakaryocpoiesis. PV with allele load less than 50% indeed are hetero/homozygous at the EEC level in blood and bone marrow for the JAK2^V617F mutation, whereas ET patients are heterozygous with a maximal JAK2^V617F mutation load of 50% [31,32]. The second molecular hit of the loss of 9p heterogeneity (9P LOH) is due to the amplification of the JAK2^V617F locus through mitotic amplification resulting in chromosome 9p loss of heterogeneity (9pLOH) indicating homozygous JAK2^V616F mutation (Figure 2). Godfrey et al. studied the JAK2 mutation status of BFU-E grown in low erythropoietin conditions in 77 patients with PV or ET [33]. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, homozygous mutant colonies were absent or present in low percentages in heterozygous ET, but prevalent and common in patients with JAK2^V617F-positive PV and JAK2 exon 12-mutated PV. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions [33]. Combined heterozygous homozygous or homozygous JAK2^V617F 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 myelofibrotic progression.

According to WHO [18] and ECMP criteria (Figures 3 and 4) [19,34], heterozygous JAK2^V617F positive ET is defined by a normocellular with slight increase of erythropoiesis in the bone marrow or with a hypercellular bone marrow due to increased erythropoiesis (Figures 5 and 6). JAK2^V617F mutated WHO defined PV typically shows a hypercellular bone marrow histology due to increase of trilinear hematopoiesis of megakaryopoiesis, erythropoiesis and granulopoiesis (panmyelosis of Dameshek^1,26) and no or slight increase of reticuline fibers (Figures 3 and 5 and 7) [4,19,34]. The UK MPN Study Group assessed the clinical features in the cohort of 806 PVSG defined ET patients subdivided in 414 JAK2^V617F positive and 362 JAK2 wild type ET and evaluated the bone marrow features in 393 ET patients [35,36]. JAK2^V617F positive ET patients had multiple features of PV such as 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 668-1535 × 10⁹/L) than JAK2^V617F-positive ET (846, range 632-1222 × 10⁹/L) [35]. In the UK Primary Thrombocythemia 1 (PT-1) study, bone marrow trephine of 209 JAK2^V617F positive and 184 JAK2 wild type ET was independently assessed by 3 blinded hematopathologists who did not know the JAK2 mutation status [36]. The overall cellularity was significantly increased in JAK2^V617F mutated ET as compared to JAK2 wild type ET, indicating that increased erythroid and granulocytic cellularity appears to be a main feature of prodromal PV, masked PV (ET.MGM) and classical PV [4,17,19,34].

JAK2^exon12 mutated PV negative for the JAK2^V617F mutation

The finding of the JAK2^exon12 mutations in patients with JAK2^V617F negative PV or idiopathic erythrocytosis further confirms the strong association between the JAK2 mutations and MPN [37-39]. The 5% PV patients negative for JAK2^V617F are frequently heterozygous for exon 12 JAK2 mutations and usually present with early stage PV with a favourable outcome and normal life expectancy. JAK2^exon12 mutations in 10 idiopathic erythrocytosis (IE) patients showed increased red cell mass and could be diagnosed in 6 patients as PV in 6 and IE in 4 cases [37]. Bone marrow biopsies in 5 JAK2^exon12 positive patients showed characteristic erythroid hyperplasia with some morphological abnormalities of the megakaryocyte and normal granulopoiesis in bone marrow biopsy specimens clearly different from primary or secondary erythrocytosis. The bone marrow histology in 7 cases of JAK2^exon12 mutated MPN (IE in 4, PV in 2, MF in 1) revealed hyperplasia of atypical small to medium-sized large megakaryocytes was present in all (Figure 8) [38,39], which differs from JAK2^V617F mutated ET and PV (Figures 4-6). At diagnosis, JAK2^exon12 mutated IE or PV patients presented aquagenic pruritis and/or erythromelalgia in 3 and microvascular events including headache, dizziness, blurred vision and distal extremity numbness (aspirin responsive platelet thrombophilia or sticky platelet syndrome) in 4 at platelet counts between 152 and 790 × 10⁹/L (of whom 5 below and 2 above 300 × 10⁹/L) [38,39]. The JAK2^exon12 MPN cases lack the prominent clusters of large megakaryocytes with hyperlobulated nuclei that characterize JAK2^V617F-positive prodromal and classical PV. A spectrum of small to medium sized megakaryocyte is seen in JAK2^exon12 PV bone marrows with a predominance of smaller forms with atypical nuclei with various degrees of monolobation to hyperlobation and abnormal chromatin distribution (Figure 8) [39].

BONE MARROW HISTOLOGY IN MPL⁵¹⁵ MUTATED THROMBOCYTHEMIA

Within the JAK2 wild type MPN, the prevalence of the MPL⁵¹⁵ mutation as the cause of ET is 3% in the Vannucchi study [30] and 8.5% in the UK studies. In the study of Vannucchi et al. [30], patients with JAK2 wild type ET carrying the MPL⁵¹⁵ mutation present with typical microvascular erythromelalgic acrocyanosis and migraine-like ocular or cerebral ischemic events (Sticky Platelet Syndrome) but have no clinical, laboratory and bone marrow features of prodromal PV at diagnosis, do not evolve into overt PV during follow-up, have normal serum EPO, normal ferritin levels, absence of spontaneous endogenous erythroid colonies (EEC). Bone marrow histology from a patient with JAK2 wild type ET carrying the MPL^W515L mutation displayed clusters large megakaryocytes with a greater number of giant megakaryocytes with hyperlobulated stag-horn nuclei in a normal cellular bone marrow and no increase of erythropoiesis (Figure 9). We recently described the essential differences in bone marrow histopathology features of differential diagnostic significance between patients with MPL⁵¹⁵ mutated (N=12) versus JAK2^V617F mutated MPN [34]. First, the presence of clustered small and giant megakaryocytes with deeply lobulated stag-horn like nuclei (Figures 9-11) in ET carrying the MPL⁵¹⁵ mutation are not seen in JAK2^V617F positive ET, prodromal PV and classical PV. The pleomorphic medium to large megakaryocytes in JAK2^V617F mutated ET and PV in bone marrow smears and bone marrow biopsies were comparable regarding size and degree of pleomorphy (Figures 4-6). 

Second, there was local increase of erythropoiesis in areas of loose clustered pleiomorphic megakaryoctyes in normocelluar JAK2^V617F mutated ET and prodromal PV, which is not seen in MPL⁵¹⁵ mutated ET (Figure 9). Third, JAK2 wild type MPL⁵¹⁵ mutated ET have no clinical, laboratory and bone marrow features of prodromal PV at diagnosis (Table 1), do not evolve into PV during follow-up and have normal LAP score, serum EPO and ferritin levels. Laboratory and bone marrow  histology evaluations have the diagnostic potential to separate the JAK2^V617F mutated ET and prodromal PV with increased LAP score, low serum EPO and pleomorphic megakaryocyte morphology from MPL⁵¹⁵ mutated ET with normal LAP score and serum EPO and giant megakaryocytes with staghorn-like nuclei similar to “true” ET (Table 1) [34].

DISCUSSION AND CONCLUSION

With the advent of the JAK2^{Vainchenker617France} mutation all latent, masked, early and overt stages of PV will be picked up more than 5 to 10 years earlier by the CLMP criteria as compared to the PVSG criteria. Heterozygous JAK2^V617F mutated ET and hetero/homozygous JAK2^V617F mutated PV and post-ET MF or post-PV MF represent different phenotypes of a single distinct MPN. JAK2 wild type ET and MF carrying one of the MPL⁵¹⁵ mutations is the second distinct MPN without features of PV at diagnosis and during follow-up (Figure 13). In a prospective study of 59 JAK2^V617F positive ET and 44 JAK2 wild ET cases, Piche et al. [40] described that JAK2^V617F mutated ET patients have PV-like morphological bone marrow changes of medium sized to large pleomorphic megakaryocytes similar to our findings in newly diagnosed JAK2^V617F mutated ET, prodromal PV patients and PV patients. JAK2^V617F positive ET and prodromal PV patients usually have low serum EPO, increased LAP score and slight to moderate increased bone marrow cellularity due to increased erythropoiesis. Increase of bone marrow erythropoiesis, granulopoiesis and serum LDH levels and spleen size are more pronounced in advanced JAK2^V617F mutated ET (EMGM or masked PV) at higher JAK2^V617F mutation allele burden. Clustered large and giant megakaryocyte with hyper lobulated ‘stag horn’ nuclei are rare in JAK2 mutated MPN, but typically  present in MPL⁵¹⁵ mutated ET patients with no features of PV in the bone marrow consistent with the diagnosis of ‘true ’ET and normal blood values for serum EPO, ferritine levels and LAP score [41,42]. The prevalence of MPL⁵¹⁵ mutated ET or MF patients ranges from 5 to 10% of the JAK2 wild type MPN population [41,42]. In the large collaborative European study of 176 MPN cases with the MPL⁵¹⁵ mutations W515L and W515K were detected in 110 and in 58, respectively [43]. The overall MPL mutation allele levels in granulocytes were lower (25%) in W515L (N=106) than in W515K (37%, N=32). Of the 138 cases (ET, N=99; MF, N=36), the median W515L mutation allele levels were significantly lower (21%) in ET than those (46%) in MF [43]. In 254 WHO-defined PMF patients the JAK2-, MPL- and CALR-mutations were detected in 58%, 8.3 and 25% respectively and 8.7% were triple negative [48]. The median overall survival (OS) among 253 WHO-defined PMF patients in 83 CALR-, 21 MPL- and 147 JAK2-mutated cases and in 22 triple negative cases was 8,2, 4.1, 4.3 and 2.5 years. As compared to CALR wild type MF, CALR-mutated MF patients were younger, had higher platelet count, lower leukocyte count, higher hemoglobin (less anemic) and lower DIPSS-plus score. CALR-mutated MF patients had a favorable impact on median survival as compared to CALR-negative MF patients whether ASXL1-negative or positive [48]. The etiology of triple JAK/MPL/CALR negative MF remains elusive whether they represent MPN or MDS. The awareness of the molecular heterogeneity of the MPNs including JAK, MPL and CALR mutation on top of epigenitic factors reflect the funeral of the term primary myelofibrosis (PMF) [49,50]. The PVSG and WHO defined term PMF [16-18,49] can easily be replaced by CALR-, MPL⁵¹⁵-and JAK2^V617F-ET and secondary myelofibrosis (MF) with various degrees of splenomegaly, hypersplenism and myelofibrotic transformation of the bone marrow (Figure 13) [8,9,34].

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