Novel European Asiatic Clinical, Laboratory, Molecular and Pathobiological (2015-2020 CLMP) criteria for JAK2 V617F trilinear polycythemia vera (PV), JAK2 exon12 PV and JAK2 V617F , CALR and MPL 515 thrombocythemias: From Dameshek to Constantinescu-Vainchenker, Kralovics and Michiels

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The Hannover Bone Marrow criteria proposed by Georgii, et al. [11], translated the PVSG criteria in the Hannover BM criteria for ET, PV and PMGM and stages of each by grading of myelo ibrosis (MF) as a secondary event in advanced stages of MPDs complicated by anemia, splenomegaly and ibrosis in the bone marrow [11][12][13][14]16].Michiels drew attention to the importance of bone marrow histology as a pathognomonic clue to each of the MPDs ET, PV and PMGM [14][15][16][17].The number and size of mature megakaryocytes in bone marrow biopsies are typically increased in ET and PV.Large megakaryocytes with mature cytoplasm and multilobulated nuclei and the tendency to cluster in small groups close to the sinuses represent the hallmark feature of ET (Figure 2).The histologic background of hematopoiesis in ET at platelet counts above 400x10 9 /L is one of normal cellularity in the early stage [14,17] (Table 3).A slight to moderate increased cellularity due to increased erythropoiesis may be seen in ET with increasing platelet counts between 400 to above 1000x10 9 /L against a background of normally maturing granulopoiesis and eryrhropoesis comparable with the early stage of PV [18][19][20].Increase in number and size of clustered large megakaryocytes comparable to ET and a moderate to marked increased cellularity due to increased erythropoiesis/megakaryopoiesis (EM) and erythro-megakaryo-granulopoiesis (EMG) are the diagnostic features of untreated PV [14,17] (Figure 3, Table 4).Increase of large megakaryocytes with mature cytoplasms and multilobulated nuclei in a hypercellular bone marrow is even more conspicuously altered in PV than in ET or early prodromal stage PV.The megakaryocytes in PV usually have a pleomorphic appearance with a wide range of megakaryocyte sizes including small, medium sized and large forms (Tables 3,4) as can be demonstrated in immune stained bone marrow biopsies using monoclonal antibodies against platelet glycoprotein.The characteristic increase and clustering of large megakaryocytes and proliferation of erythropoiesis with hyperplasia of dilatated sinuses are the diagnostic hallmark of untreated PV to distinguish it from secondary erythrocytosis [12,15,17,18], from Ph+ chronic granulocytic leukemia and Ph+ ET [13,15] and most importantly from PMGM [12,15,16].Bone marrow histology in PMGM is dominated by atypical immature megakaryocytes, which are conspciously large due to increase of nuclear as well as cellular size.The nuclei of megakaryocytes in PMGM are bulky with lobuli becoming clumsy.The lightly stained chromatin and irregular roundish nuclear forms give rise to the co-called cloud-like nuclei, which are almost never seen in ET and PV [11,12,14,16,21].
Within the European Working Group on myeloproliferative Disorders (EWG.MPD founded by Dr. Michiels in 1994).Michiels, et al. [14], translated the Hannover Bone Marrow Classi ication of Georgii, et al. [11] in a new set of the Rotterdam Clinical and Pathological (RCP) criteria for the diagnosis of ET and PV and chronic, essential or primary

Abstract
The Myeloproliferative Neoplasms (MPN) of trilinear polycythemia vera (PV) and megakaryocytic leukemia (ML = primary megakaryocytic granulocytic myeloproliferation: PMGM) and Essential Thrombocythemia (ET) in the studies of Dameshek and Michiels are caused by the MPN driver mutations JAK2 V617F , JAK2 exon12 , CALR and MPL 515 discovered by Constantinescu-Vainchenker, Green and Kralovics.The JAK2 V617F mutated trilinear myeloproliferative neoplasms (MPN) include a broad spectrum of clinical laboratory and bone marrow features in essential thrombocythemia (ET), prodromal PV and erythrocythemic PV, classical PV and advanced stages of masked PV and PV complicated by splenomegaly and secondary myelofi brosis (MF).Heterozygous JAK2 V617F mutated ET is associated with low JAK2 allele and MPN disease burden and normal life expectance.In combined heterozygous and homozygous or homozygous JAK2 V617F mutated trilinear PV, the JAK2 mutation load increases from less than 50% in prodromal PV and classical PV to above 50% up to 100% in hypercellular PV, advanced PV and PV with MF.Bone marrow histology show diagnostic features of eryhrocytic, megakaryocytic and granulocytic (EMG) myeloproliferation in JAK2 V617F mutated trilinear MPN, which clearly diff ers from monolinear megakaryocytic (M) myelproliferation in MPL and CALR thrombocythemia and dual megakaryocytic granulocytic (MG) myeloproliferation in CALR mutated thrombocythemia.The morphology of clustered large pleomorphic megakaryocytes with hyperlobulated nuclei are similar in JAK2 V617F thrombocythemia, prodromal PV and classical PV patients.Monolinear megakaryocytic (M) myeloproliferation of large to giant megakaryocytes with hyperlobulated staghorn-like nuclei is the hallmark of MPL 515 mutated normocellular thrombocythemia.CALR mutated thrombocythemia usually presents with high platelet count around 1000x10 9 /l and normocellular megakaryocytic (M) proliferation of immature megakaryocytes with cloud-like hyperchromatic nuclei followed by dual megakaryocytic granulocytic (MG) myeloproliferation followed by various degrees of bone marrow fi brosis.Natural history and life expectancy of MPN patients are related to the response to treatment and the degree of anemia, splenomegaly, myelofi brosis and constitutional symptoms.The acquisition of epigenetic mutations at increasing age on top of MPN disease burden independently predict unfavorable outcome in JAK2      (Michiels, et al. 2006ab [8,9], Bellucci & Michiels, 2006 [21] on the molecular etiology of JAK2 V617F mutated hypersensitive platelets and platelet-mediated arteriolar erythromelalgic arterial (Michiels, et al. 1984(Michiels, et al. [86], 1985(Michiels, et al. , 1993) ) in heterozygous essential thrombocythemia (ET) and major microvascular thrombosis in mixed heterozygous and homozygous or homozygous thrombocythemia and polycythemia vera (TPV) complicated by splenomegaly due to myeloid metaplasia of the spleen and secondary bone marrow fi brosis according to the dosage hypothesis of Constantinescu & Vainchenker (James, et al. 2005  megakaryocytic granulocytic myeloproliferation PMGM as the third distinct MPD [11,12,[14][15][16][17]22] (Table 2).The present appraisal of the myeloproliferative neoplasms (MPN) from Dameshek to Michiels review the clinical laboratory molecular Pre bro c MGM Figure 3: According to Constantinescu-Vainchenker low V617F constitutional kinase activity in heterozygous mutated JAK2 V617F mutated patients is enough to produce the ET phenotype via the MPL signalling pathway and that higher V617F constitutional kinase activity in JAK2 V617F mutated, heterozygous/homozygous or homozygous mutated patients is needed to produce the sequential stages of prodromal, classical and advanced (masked) PV phenotypes via activation of both the EPO and pathways of hematopietic progenitors cells (Table 1).The JAK2 V617F dosage hypothesis has been confi rmed at the bone marrow haematopoietic stem cell level by the demonstration that endogenous erythroid colonies (EEC) from ET patients are mainly heterozygous for the JAK2 V617F mutation, whereas all PV patients are either hetero/homozygous or mainly homozygous for the JAK2 V617F mutation (Figure 8).and pathological (CLMP) characteristic of the MPNs caused by the driver mutations JAK2 V617F , MPL 515 , JAK2 exon12 , CALR and MPL 515 discovered by Constantinescu-Vainchenker, Pardani, Green and Kralovics respectively (Tables 1,2, Figure 1).
Moderate myeloid neoplasia of the spleen splenomegaly
Erythrocytosis.Normal erythropoiesis, normal granulopoiesis and megakaryocytes of normal size, morphology and no clustering.
Platelet count > 350x10 9 /L and presence of large platelets in blood smear.
Clinical staging similar as in CALR thrombocythemia based on the degree of anemia, splenomegaly and myelofi brosis.
No increase of erythropoiesis, and granulopoiesis.
No or slight increase in reticulin RF 0/1.
In the late 1970s, the London PV study Group of Pearson, Messinezy, Thomas and Weitherley-Mein demonstrated that on top of the microvascular disease of thrombocythemia, the incidence of major arterial and venous episodes in PV correlated positively with increased haematocrit level [46][47][48][49].A mean haematocrit of 0.60 and a mean platelet count of 512x10 9 /L at time of diagnosis of PV were associated microvascular ischemic events and major thrombosis in 49%.The risk of major vascular episodes were the lowest at hematocrits below 0.44, higher at hematocrits above 0.45 and the highest at hemotocrits above 0.50 as was the case in the PVSG 01 study when not on low dose aspirin for the prevention of platelet-mediated microvascular ischemic distubances, TIAs and acute coronary syndromes [36,50,51].Low dose aspirin at hematocrits of around 0.40 in JAK2 V617F mutated ET and PV signi icantly reduces the incidences of both microvascular as well as major vascular events as compared to not using aspirin in randomized clinical trials [30,36,52].Phlebotomy on top of low dose aspirin (40 to 80 mg OD) is the cornerstone of treatment of newly diagnosed PV patients with low, intermediate and high MPN disease burden [34][35][36]53].

JAK2 V617F mutated trilinear MPN
EPO-independent progenitor colony-forming uniterythroid (CFU-E) and burst forming unit-erythroid (BFU-E) labelled as spontaneous endogenous erythroid colony formation (EEC) became the hallmark of PV [54].Analysis of about 500 PV patients from 26 studies indicated that EEC in expert hematological laboratories has a near 100% diagnostic speci icity for overt and masked PV and ET mimicking PV or latent PV [54,55].The 9p loss of heterogeneity (9pLOH) due to mitotic recombination of chromosome 9p is the most frequent chromosomal lesion described in PV (∼33%) not detectable by cytogenetic analysis [56].Kralovics, et al. sequenced 19 candidate genes mutations within the 9pLOH region and no mutations were found in the Janus kinase (JAK) gene, but Kralovics did not screen the JH2 pseudokinase gene thereby overlooking the JAK2 V617F in the JH2 pseudokinase gene (Figure 1).Constantinescu & Vainchenker searched for a mutation in the 9pLOH region of the complete JAK2 gene and did found the JAK2 V617F in the JAK2 pseudogene of 3 PV and 2 controls by detection of a G-to-T mutation at nucleotide 1849 in exon 12 leading a substitution of valine to phenylalanine at position 617 (V617F) in the JAK2 pseudo-gene.This V617F substitution of the JAK2 V617F mutation was present in 40 of 45 PV patients, in 9 of 21 ET patients and in 3 of 7 MF patients [57], (Figure 1).The JAK2 V617F substitution was absent in patients with secondary erythrocytosis (N = 35) and 15 controls [57].Thirty percent of PV patients are homozygous for JAK2 V617F mutation without socalled 9pLOH due to mitotic recombination, whereas heterozygous JAK2 V617F mutated ET and PV patients showed the presence of 9pLOH [57,58] (Table 1, Figure 1).[48] and Messinazy, et al. [49] of the London PV Study Group [46,47].Repeated venesections for more than 1 year (from August 2012 to September 2013) was needed to induce a complete hematological remission (CHR) reaching the desired plateau of Ht 0.45, Hb 9.0 mmol/L, and MCV of 66 fL.While on low dose aspirin neither microvascular nor major thrombosis did occur.Once the iron defi ciency state is reached the erythrocytes remain microcytic and reach values of 7.0 to 7.2x10 12 /L without further need of phlebotomy due to the persistence of the iron defi cienct state [2,30].Correction of the Ht from 0.63 to below 0.45 is associated with reduction of major venous and arterial thrombotic events [8,9,48,51], but the microvascular thrombotic syndrome of associated thrombocythemia persisted.Low dose aspirin in ET in the Dutch Collaborative Low dose Aspirin in Thrombocythemia (Dutch CLAT) Van Genderen studies [89][90][91]99] and low dose aspirin on top of phlebotomy or hydroxurea in the European Collaborative Low dose Aspirin in PV (ECLAP, [50]) reduced the incidences of microvascular disturbances and major thrombosis from above 50% per 100 pt/yr to less than 3% per 100 pt/yr in both ET and PV [91,94], but does not prevent the progression of JAK2 mutated MPN disease in terms of JAK2 mutation load, MPN disease burden like progressive leukocythemia, thrombocythemia, splenomegaly and constitutional symptoms [6,7,34].Constantinescu & Vainchenker, [59] demonstrated that the acquisitions of heterozygous, hetero-homozygous and homozygous due to mitotic recombination JAK2 V617F mutation on chromosome 9p are the driver causes of sequential megakaryocytic (M), erythrocytic megakaryocytic (EM) and erythro-megakaryo-granulocytic (EMG) myeloproliferations seen in normocellular ET, prodromal PV and classical and advanced PV in trilinear MPN (Figures 5,6), [21,57,60], (Table 1, Figure 1).The JAK2 V617F mutation as the driver cause of trilinear MPN was immediately con irmed in three large groups of ET, PV and MF patients due to inside information during the peer review process in 2004 [58,59,61].JAK2 V617F mutation induces a loss of inhibitory activity of the JAK2 pseudokinase part on the JAK2 JH kinase part leading to enhanced activated of the normal JAK2 JH1 kinase activity, which makes the TPO, EPO and granulocyte growth factor receptors on the hematopoietic progenitor cells hypersensitive to their growth factors TPO, EPO and granulocyte growth factor (Figures 5,6).The JAK2 V617 mutated hematopietic cells produce Contitutively activated platelets and leukocytes (increased leukocyte alkaline phosphatase: LAP) and quantitative increase of platelets erythrocytes and granulocytes (Table 1).The sequential occurrence of low heterozygous, combined heterozygous and homozygous and high homozygous JAK2 V617F allele load can readily explain the sequential occurrence of ET, prodromal PV, classical PV and advanced PV followed by secondary MF in trilinear MPN during lifelong follow-up.JAK V617F trilinear MPN is clearly differentfrom JAK2 wild type hypercellular ET associated with PMGM [11,12,14,16] as the third distinct entity of MPD without features of PV (Figure 2, Tables 12-15 in Michiels, et al. [8]).According to 2006 ECMP criteria the sequential transitional states of JAK2 V617F disease entity ranged from heterozygous normocellular ET and latent PV mimicking ET labelled as prodromal PV or forme fruste PV followed by heterozygous/homozygous mutated erythrocythemic and early PV, and homozygous nutated advanced PV and post-PV myelo ibrosis (Figures 1,2, Table 1), [8,21,62].The JAK2 V617F mutated trilinear MPN phenotypic expression includes normocellular ET, prodromal PV, erythrocythemic PV with normal platelet and leukocyte count, classical PV, masked PV, and various degrees of splenomegaly and myelo ibrosis (MF) [57,59] (Figure 1, Table 1).The quantitative JAK2 V617F allele burden in neutrophils and in CD34+ cells from the same blood sample in 96 JAK2-positive MPN patients (17 ET, 64 PV and 15 MF mean follow-up 7 years) were below 50% in the majority of ET and about half of the PV patients [63].The JAK2 V617F -CD34 + allele burden were above 50% in about half of the PV patients and the majority of MF indicating advanced trilinear MPN disease burden.The neutrophil JAK2 V617F allele burden is usually below 50% in ET and above 50% in PV and MF.The CD34 + allele burden is much lower than the neutrophil JAK2 V617F allele burden in ET and early stage PV with no splenomegaly [64].This is completely in line with the concept that some maturation of JAK2 V617F mutated hematopoietic stem cells is needed to neoproliferate because of hypersensitivity megakaryopoesis to TPO and erythropiesis to EPO [64,65] (Figures 5,6, Table 1).The neutrophil JAK2 V617F allele burden alone can overestimate the MPN disease burden at the bone marrow progenitor cell level in early stage ET and PV neoproliferative disease [63,64].
The UK MPN Study Group [71,78] elegantly con irmed the Vainchenker's 'dosage' concept at the biological EEC level by studying the genotype of individual BFU-E in a crossectional cohort of 29 JAK2 V617F mutated ET and 30 JAK2 V617F mutated PV patients (Figure 8).The JAK2 mutation load was expressed as a percentage (%) of EEC colonies genotyped as homozygous (red), heterozygous (purple) or wild type [78] (Figure 8).All 29 JAK2 V617F positive ET patients have heterozygous JAK2 mutated EEC colonies: 9 of them have a low percentage (< 10%) of homozygous JAK2 mutated colonies.Out of 30 JAK2 V617F positive PV patients, 8 have heterozygous JAK2 mutated EEC, 13 have homozygous EEC colonies of more than 50% and 7 of less than 50%.Homozygous EEC colonies were absent or rare in heterozygous ET, but prevalent in JAK2 V617Fpositive PV [78] (Figure 6).These observations are completely in line with Vainchenker's "dosage" concept (Figures 5,6, Table 1) [8,9,21,60,72].Additional cytogenetic [79], genetic or epigenetic alterations in PV and MF patients are of huge prognostic signi icance [80][81][82][83][84].The presence of epigenetic factors like TET2 or ASXL1 etc on top of the JAK2, MPL and CALR driver mutations of MPN is associated with impaired prognosis in MPN, MDS and other myeloid malignacies as well.The targeted search for epigenetic factors will become hugely important to the understanding of differences in biology, prognosis and outcome of MPN patients [81][82][83][84].Using next generation sequencing (NGS) on top of the JAK2 or CALR mutation, the Swiss MPN investigators in Basel found one, two or more epigenetic somatic mutations in 65 (33%) of 197 WHO de ined MPN patients (94 PV, 69 ET, 34 MF) [82].Seventeen of 69 (25%) ET patients, 11 of 34 (32%) MF and none (0%) of 94 PV patients carried mutations in CALR.In addition to JAK2 V617F and CALR, the most frequently observed epigenetic somatic mutations affecting the biology and natural history of MPN disease included TET2, ASXL1, DNMT3A, EZH2, and IDH1 [82][83][84].Rare epigenetic mutations were NF1, NFE2, and RUNX1.The presence of one, two or more somatic mutations appeared to impair prognosis in JAK2 and CALR mutated MPN [82].Tefferi, et al. [83,84], con irmed the Lundberg observations in large scale retrospective studies in WHO de ined ET, PV and MF patients demonstrating that epigenetic somatic mutation detection on top of the JAK2, CALR and MPL mutational load and subtype MPN characteristization is far superior to classify the distinct MPN diseases as compared to the crude WHO classi ication, that cannot clearly distinct between ET, prodromal overt and masked PV and PV with MF.Dr. Green, addressed the key question whether the sequence of acquisition of somatic mutations can be inferred from the genotypes of detectable subclones [85].For instance, if some tumor cells have JAK2V 617F , and others from the same patient bear JAK2 V617F with an additional somatic mutation, then this indicates that JAK2 V617F came irst.Genotyping individual hematopoietic colonies has shown that the order of acquisition of JAK2 V617F , relative to mutations in TET2 or DNMT3A, in luences subclonal composition within HSPCs and mature cell compartments, disease presentation, and clinical outcome.In JAK2-irst patients, the HSC compartment is dominated by double-mutant cells, and such patients present at a younger age, often with PV.Conversely, in TET2-irst patients, the HSC compartment is dominated by single mutant cells, and such patients present at an older age, usually with ET.JAK2-irst patients had a greater likelihood of presenting with PV than with ET, had an increased risk of thrombosis, and an increased sensitivity of JAK2 mutant progenitors to ruxolitinib in vitro.

Erythromelalgic microvascular circulation distubances or platelet thrombophilia in PV and ET: From Dameshek to Michiels & Van Vliet
Dameshek & Henthel, [1] described the presenting clinical manifestations in 20 newly diagnosed PV patients including quite severe headaches in 17, attacks of migraine in 14, visual disturbances, particularly spots before the eyes and coloured scotomas in 6, paresthesias numbing and tingling Figure 8: Proportions of JAK2 genotypes in BFU-Es from 59 patients with JAK2 V617F -mutated essential thrombocythemia (ET) and polycythemia vera (PV) [78].Each vertical bar represents 1 patient, divided according to the proportion of wild-type, heterozygous, and homozygous-mutant colonies obtained, with the absolute colony numbers shown above: (wild type white), heterozygous (purple) homozygous (red).Results of EEC colony genotypes are presented for 29 JAK2 V617F -positive ET (B) patients (total 2277 colonies; mean 79 per patient) and for30 JAK2 V617F -positive PV (A) patients (total 2287 colonies; mean 76 colonies per patient).All 29 JAK2 V617F positive ET patients have heterozygous JAK2 mutated EEC colonies and less than 10% homozyous colonies in 9 and 20% in 1 of them.Out of 30 JAK2 V617F positive PV patients 8 have heterozygous JAK2 mutated EEC, 13 have homozygous EEC colonies of more than 50% and 7 of less than 50%.
In total 30 ET patients: all are predomominant heterozygous (low allele burden) for the JAK2 V617F mutation but half of them do have a minor clone of homozygous mutated BFU-Es.B. In total 18 JAK2 exon 12 mutated PV: all are predominany heterozygous (low allele burden) for the JAK2 exon 12 mutation, but 7 of them had a minor clone of homozygous mutated BFU-Es.C and D. EEC colony genotypes for 18 patients with JAK2 exon 12 mutated PV (total 1931 colonies; mean 107 per patient (C).D show example sequence traces for patients with patients with homozygous JAK2 exon12 mutations in colonies.In total, 16 patients (5 "heterozygous-only" JAK2 V617F -positive PV patients, 4 JAK2 V617F positive PV patients with homozygous and heterozygous clones, 3 JAK2 V617F positive ET patients with small homozygous clones, and 4 JAK2 exon 12 mutated PV patients with homozygous clones) were assessed in this way (mean time between experiments, 13 months; range, 2-32 months) and showed reproducibility of proportions of heterozygous and homozygous-mutant colonies.

JAK2 exon12 mutations as cause of Isolated Erythrocythemia and PV
The inding of the JAK2 exon12 mutations in the 5% PV patients negative for JAK2 V617F usually present with early stage PV or isolated erythrocythemia (IE, Figure 8) with increased red cell mass but normal leukocytes and platelets and no palpable spleen [95][96][97][98].T he frequency of JAK2 exon12 mutations among all PV patients is estimated around 3% [95,98].JAK2 N542-E543del is the most frequent among the different reported exon 12 mutations.JAK2 exon12 mutated MPN patients with increased erythrocytes above 6.0x10 12 /L and a typical PV bone marrow histology are diagnosed as benign IE or PV with a favourable outcome and normal life expectancy [95,96,98,99].Pre-treatment bone marrow histology in JAK2 exon 12 mutated PV or IE showed characteristic erythroid hyperplasia with minor and distinct histology changes of the megakaryocytic lineage, which are not seen in primary or secondary erythrocytoses (PE and SE) [95].Cases of JAK2 exon12 mutated IE or PV have erythrocytes above 6x10 12 /L [100], normal platelet and leukocyte counts, no or palpable spleen and a typical hypercellular bone histopathology predominantly due to erythroid hyperplasia and clusters of large megakaryocytes with hyperploid nuclei [95,98] (Figure 8).Bone marrow histology in 7 cases (4 IE, 3 PV) of JAK2 exon12 mutated MPN in the pathology study of Lakey, et al. [97], showed prominent hyperplasia of erythropoiesis and atypical small to medium-sized large megakaryocytes (Figure 8).A low percentage of homozygosity was found for the JAK2 K539L-type and E543del-type exon 12 mutations (Figure 8) [78].Godfrey, et al. [78], assesed the colony genotypes for 18 patients with JAK2 exon 12 -mutated PV in a total of 1931 colonies; mean 107 per patient (Figure 8C).
Example sequence traces for patients with patients with homozygous JAK2 exon12 mutations in colonies are shown in igure 8D.In total, 16 patients (5 "heterozygous-only" JAK2 V617Fpositive PV patients, 4 JAK2 V617F -positive PV patients with homozygous and heterozygous clones, 3 JAK2 V617F -positive ET patients with small homozygous clones, and 4 JAK2 exon12 mutated PV patients with homozygous clones showed reproducibility of proportions of heterozygous and homozygous-mutant colonies (Figure 8D).

Megakaryocyte Leukemia (ML) and CALR mutated Thrombocythemia: From Dameshek 1951 to Kralovics 2013 and Michiels 2015
According to Dameshek, [10] mega karyocyte leukemia (ML) is de ined by platelet counts around and above 1000x10 9 /L without features of PV in blood and bone marrow smear and biopsy.The traditional classi ication of the myeloproliferative disorders (MPD) by the PVSG and used in textbooks was revised in the Hannover Bone Marrow classi ication to include PV, primary thrombocythemia (PTH), and hyper cellular thrombocythemia related to primary megakaryocytic https://www.heighpubs.org/hbmr012 https://doi.org/10.29328/journal.ijbmr.1001011myeloprliferation (PMGM, Table 7) without features of PV [11,12,14,16,104].The discovery of the calreticulin (CALR) as the main cause of JAK2/MPL 515 wild type thrombocythemia and PMF by Kralovics and his team [105] was identi ied by Michiels & De Raeve [31,32] as the driver cause of pre ibrotic and ibrotic stages of PMGM without features of PV.This led to the second ground breaking event in the molecular landscape of the MPNs that induced a complete revision of all MPN classi ications of the PVSG, WHO into the current Clinical Laboratory, Genetic and Pathobiological (2018 CLMP) criteria for JAK2 V617F trilinear MPN (Tables 3 and 4), and JAK2 exon 12 PV as compared to two distinct MPL 515 (Table 6) and CALR thrombocythemias and myelo ibrosis (Table 7) without features of PV.
Kralovics performed targeted whole-exome seqencing in 6 cases of WHO de ined JAK2/MPL wild type PMF patients and found somatic calreticulin (CALR) mutations of 52-bp deletion in 1, of 1bp deletion in 1 and recurrent 5-bp insertion in 4 https://doi.org/10.29328/journal.ijbmr.1001011MF patients.The CALR somatic mutation was subsequently discovered as the driver cause of thrombocythemia in 78 of 311 (25%) ET patients and in 72 of 203 (35%) MF patients [105].The CALR mutation was detected in none of 382 PV, 45 CML, 73 MDS, and 64 chronic myelomonocytic leukemia (CMML) patients.Three (12%) of 24 RARS-T cases were positive for both the SF3B1 and CALR mutation.A subsequent Italian-Austrian study of 1235 WHO-de ined ET and MF patients detected the JAK2 V617F , MPL 515 and CALR mutation in 63.3%, 23.5% and 4.4% respectively with 8.8% being negative for all three mutations [76] (Figure 6).Evolution into MF during follow up was as high in CALR mutated ET as in JAK2 V617F mutated PV (about 20% after 20 years).CALR mutated MPN patients lacked features of PV (normal erthrocytes and hematocrit), had higher platelet counts and a lower incidence of major thrombosis compared to JAK2 V617F positive ET [76,105].The large UK study con irmed the presence of the somatic CALR driver mutations in 80 of 112 (70%) JAK2/MPL wild type ET patients, and in 18 of 32 (56%) JAK2/MPL wild type MF patients and in none of 120 JAK2 V617F or MPL 515 mutated MPN patients [106].CALR mutations were detected in 10 of 120 (8%) MDS patients (RA in 5 of 53, RARS in 3 of 27 and RAEB-T in 2 of 27), and in one patient each with CMML and atypical CML.CALR mutations were not found in control samples, lymphoid cancers, solid tumors, or cell lines [106].A third large Italian study found CALR mutations in 15.5% of 576 WHO-de ined ET and in 48.9% of JAK2/MPL wild type ET patients [107].The distribution of the JAK2 V617F , CALR and MPL 515 mutations or triple negative cases in 254 WHO-de ined MF patients was 58%, 25%, 8.3% and 8.7% with median overall survival of 8.2, 4.1, 4.3 and 2. 5 years respectively re lecting advanced or end stage MPN disease [39].
The biological and clinical features of WHO-de ined ET carrying the JAK2 V617F and CALR mutation ET clearly differ [76].The mutant allele burden was lower in JAK2 V617F mutated than in CALR mutated ET (Figure 7).JAK2 V617F ET patients were older, had higher hemoglobin and white blood cell counts but lower platelet counts.Serum erythropoietin levels are lower and frequently decreased in JAK2 V617F ET but normal in CALR thrombocythemia.The cumulative risk of WHOde ined ET carrying the JAK2 V617F mutation to transform into WHO-de ined PV was 29% after 15 years but transformation into PV was never observed in CALR thrombocythemia.With the advent of the CALR mutation as the main driver cause of JAK2/MPL wild type ET, hypercellular ET associated with PMGM [8,11,12,14,15] and CALR thrombocythemia and myelo ibrosis appeared to be the same distinct MPN entity without features of PV (Table 7), [31][32][33].JAK2 V617F mutated ET and PV patients had a similar two times higher risk of major thrombosis than that of CALR mutated thrombocythemia patients.CALR-mutated ET patients were more frequently male, had higher platelet counts, lower hemoglobin and leukocyte count and showed a lower risk of major thrombosis than JAK2 mutated ET patients in two large studies [107,108].

Incorporation of 2008 WHO into 2020 clinical, laboratory, molecular and bone marrow pathology (CLMP) classifi cation of myeloproliferative neoplasms
Bone marrow histology indings in 59 WHO-de ined JAK2 V617F positive ET and 44 JAK2 wild ET cases in the study of Pich, et al. [66], (Figure 11) revealed PV-like hypercellular morphological bone marrow changes of pleiomorphic enlarged megakaryocytes in JAK2 V617F mutated ET similar as described previously (Figure 9), [8,15].Various stages erythropoiesis and or myelopoiesis with megakaryocyte proliferation as well as LDH and spleen size are more pronounced in PV-like phenotype in JAK2 V617F mutated ET in particular at higher JAK2 mutation load (Figure 9), [66].WHO de ined JAK2 V617F positive ET showing increased cellularity due to increased erythropoiesis is consistent with prodromal PV [62].The prognosis of JAK2 V617F mutated ET and prodromal PV is favorable and to be treated with low aspirin and additional phlebotomy in early PV to maintain ht below 0.45 in man and below 0.42 in women.This concept based on prospective clinical observations are completely in line with the present study of patients with JAK2 V617F mutated ET, prodromal PV and PV.
Bone marrow histology analysis of bone marrow biopsies by Michiels & De Raeve from the Vannucchi's study on WHO de ined MPL 515 mutated ET revealed that clustered large to giant maure megakaryocyte with staghorn nuclei and platelet count increase in a normocellular bone marrow are characteristic for JAK2 wild type ET carrying the MPL 515 mutation [31][32][33].JAK2/CALR wild type ET carrying the MPL 515 mutation indeed displayed clustered large and giant mature megakaryocytes with a greater number of large deeply lobulated 'staghorn' nuclei in a normocellular bone marrow as the hallmark of MPL 515 thrombocythemia (Figure 10), [6,7,31,32].

Figure 1 :
Figure 1: Upper part: The discovery by Constantinescu & Vainchenker of the JAK2 V617F somatic mutation as the cause of trilinear myeloproloferative neoplasms and the sequential occurrence of heterozygous JAK2 V617F mutation in essential thrombocythemia (ET) and homozygous JAK2 V617F mutation in trilinear polycythemia vera (PV) which does explain the occurrence of three sequential phenotypes of ET, PV and myelofi brosis (MF) during life long follow-up in the studies of Dameshek and Michiels.Lower part: Concept of Michiels & De Raeve on the dynamics of the JAK2 V617F disease processes in trilinear MPN ranged from normocellular ET and prodromal PV mimicking ET with normal erythrocyte count below 5.8x10 12 /L to defi ntive increase in peripheral blood erythrocytes above 5.8x10 12 /L) in PV followed by masked PV, advanced PV complicated by fi brosis and splenomegaly, spentphase PV and blast transformation of post -PV myelofi brosis.Designedby Michiels 2020.

Figure 2 :
Figure 2: The heterozygous JAK2 V617F mutated acquired thrombocythemia and germline TPO and JAK2 V671I or JAK2 Q534R mutated hereditary thrombocythemia as well as acquired MPL 515 and congenital hereditary MPL 505 mutated thrombocythemia (ET) are driven by indirect cytokine activation heterozygous JAK2 V617F STAT5, germline JAK2 or TPOTpoR = MPL.Direct binding of TPO to the D3D4 domain of TpoRor direct cytokine activation (MPL 515 and MPL 505 ) cytokine receptor activation induce an ET phenotype of MPN without features of PV (EEC negative) Michiels, et al. 2014 [27].CALR mutants Type 1 and 2, but not wild type CALR, did induce STAT5 activation via TpoR (MPL) and GCSFR, but not via EpoR.The STAT5 activation via GSCFR was much weak erthan via TpoR (MPL).The extracelllar domain of TpoR (MPL), but not of EpoR, was indispensible for CALR mutant induced activity and the D1D2 distal part of the extracelluar TpoR domain and its associated N-glycosylation sites but not the TPO binding site in the D3D4 domain Araki, et al.Vainchenker and Kralovics 2017) [65].

Figure 4 :
Figure4: Megakaryocytic (M) proliferation in a normocellular bone marrow (60%) with increase of large mature megakaryocytes, multilobulated nuclei and the tendency to cluster in small groups close to the sinuses is the hallmark of JAK2 V617F mutated ET.Pathology Laboratory Lam Rotterdam and De Raeve, Brussels.

Figure 6 :
Figure 6: State of the art treatment according to Dameshek [2,4] of a newly diagnosed PV patient (Ht 0.63, Hb 12.9 mmol/L, erythrocytes 7.1 x1012/L and MCV 89 fL) with repeated vene sections as confi rmed by Pearson & Wetherley-Mein[48] and Messinazy, et al.[49] of the London PV Study Group[46,47].Repeated venesections for more than 1 year (from August 2012 to September 2013) was needed to induce a complete hematological remission (CHR) reaching the desired plateau of Ht 0.45, Hb 9.0 mmol/L, and MCV of 66 fL.While on low dose aspirin neither microvascular nor major thrombosis did occur.Once the iron defi ciency state is reached the erythrocytes remain microcytic and reach values of 7.0 to 7.2x1012 /L without further need of phlebotomy due to the persistence of the iron defi cienct state[2,30].Correction of the Ht from 0.63 to below 0.45 is associated with reduction of major venous and arterial thrombotic events[8,9,48,51], but the microvascular thrombotic syndrome of associated thrombocythemia persisted.Low dose aspirin in ET in the Dutch Collaborative Low dose Aspirin in Thrombocythemia (Dutch CLAT) Van Genderen studies[89][90][91]99] and low dose aspirin on top of phlebotomy or hydroxurea in the European Collaborative Low dose Aspirin in PV (ECLAP,[50]) reduced the incidences of microvascular disturbances and major thrombosis from above 50% per 100 pt/yr to less than 3% per 100 pt/yr in both ET and PV[91,94], but does not prevent the progression of JAK2 mutated MPN disease in terms of JAK2 mutation load, MPN disease burden like progressive leukocythemia, thrombocythemia, splenomegaly and constitutional symptoms[6,7,34].

Figure 9 :
Figure 9: Upper left: Prodromal PV with hypercellular bone marrow (70%) due to increased erythropoiesis proliferation of mature medium to large pleomorphic megakaryocytes with variable degrees of hyperlobulated nuclei.Courtesy of Pathology Laboratory Pich.Upper right: Pre-treatment bone marrow histology in JAK2 exon 12 mutated PV or E showed characteristic erythroid hyperplasia with distinct histology changes of the megakaryocyte lineage with no or minor lobulation of nuclei[97].Lower left and right: Pleiomorphic small to large megakaryocytes with hyperlobulated nuclei in JAK2 V617F positive ET (200x and right 400x Pich, et al.[66]).

Figure 10 :
Figure 10: Dense clustering of mature, large to gaint megakaryocytes with hyperlobulated staghorn-like nuclei in a normocellular bone marrow of a symptomatic (aspirin responsive microvascular disturbances) case of MPL 515 mutated thrombocythemia (platelet 1239x10 9 /L) without features of PV in blood and bone marrow.(Pathology Laboratory Brussels, Dr De Raeve).

Figure 11 :
Figure 11: Loose dense clustering of large immature megakaryocytic (M) proliferation with immature 'cloud'-like nuclei in a normocellular bonemarrow of an asymptomatic case of prefi brotic CALR thrombocythemia at platelet count of 879x10 9 /L, minor splenomegaly on echogram and no features of PV in blood and bone marrow.(Pathology Laboratory Brussels, Dr De Raeve).
Kim, et al. was divided into minimal ibrosis MF 0/1 and overt ibrosis MF 2/3[7,11,12,21,109].The frequency of overt ibrosis in JAK2 V617F -and CALR-mutated and triple-negative MPN patients was 22.2%, 27.1% and 29.3%, respectively.JAK2-GM and CALR-GM showed a high rate of overt ibrosis (46.0 and 42.1%), followed by JAK2-M (17.5%),CALR-M (17.2%) and JAK2-EMG) (10.4%; p < 0.001).None of the JAK2-EM ('forme fruste', early and overt PV and exon 12 PV) patients presented overt ibrosis.The overall bone marrow histology indings of erythroid, granulocytic and/or megakaryocytic hyperplasia in JAK2 V617F mutated MPN, and of granulocytic and/or megakaryocytic hyperplasia in CALR mutated MPN patients in the Seoul study are completely in line with the 2015-2020 CLMP classi ication of six distinct MPN disease entities and transitional MPN states.Comparing the survival curves of 2008/2016 WHO de ined PV, ET and PMF versus the 2015-2020 CLMP de ined JAK2 V617F , JAK2 exon12 , CALR and MPL 515 de ined MPN without ibrosis versus with ibrosis strongly suggest that bone marrow ibrosis (BMF) grade MF 0/1 versus grade 2/3 appeared to be main adverse prognostic factor when associated with JAK2 V617F and triple negative MPN disease (Figures 13,14), [100].Conclusion The present insight review is a strenuous joint effort by a multicentre MPN European Asiatic collaborative study group to demonstrate that scrutinized and integral clinical, laboratory, genetic and pathological (2015-2020 CLMP) approaches and intense communications amongst clinicians, scientist, molecular biologists, and pathologists are warranted to more precisely diagnose and treat each MPN patient before avoidable major complications had occurred.The change of 2008/2016 WHO into the 2015-2020 CLMP criteria in table 8 incorporate the established 1975 PVSG and 2001/2008/2016 WHO classi ications.The novel 2015-2020 CLMP criteria for at least ive distinct clonal MPNs are in urgent need of validation in well designed large clinical prospective unmet need (PUN) studies within the context of the International Collaborations and Academic Research on MPN (ICAR.MPN 2015 founded and chaired by Dr. Michiels Europe and Dr.

Table 1 :
The 2006 concept of Michiels

Table 6 :
2015-2020 Clinical Laboratory, Molecular and Pathobiology (CLMP) criteria for the diagnosis of normocelular ET carrying one of the MPL 515 mutations.

Table 8 )
. The bone marrow (BM) lineage proliferation class in MPN including 101 PV, 95 ET and 78 PMF WHO de ined patients MPN consisted of M (WHO-ET) in 80; EM and EMG in 116 consistent with prodromal and classical PV; and GM myelo ibrosis in 72.The mean JAK2 V617F mutation load was high 69 to 80% in EM, EMG and 69% in MG bone marrow class, but low (37%) in M class ET patients (Table