International Journal of Bone Marrow Research

Research Article

European Clinical Laboratory, Molecular and Pathological (ECMP) criteria for prefibrotic JAK2^V617F-Thrombocythemia and Polycythemia Vera versus MPL⁵¹⁵- and CALR-Thrombocythemia and Myelofibrosis: From Dameshek to Michiels 1950-2018

Jan Jacques Michiels^1,2,5*, Zwi Berneman², Wilfried Schroyens², Fibo W J ten Kate³, King Lam³ and Hendrik De Raeve^4,5

¹Multidisciplinary Internist, Scientific Investigator, Department of Hematology & Blood Coagulation, Erasmus Free University Network, Europe, Rotterdam, Netherlands
²Hematologist, Internist, Department of Hematology, University Hospital, Antwerp, Belgium
³Pathologist, Department of Pathology, Erasmus University Medical Center Rotterdam, Netherlands
⁴Pathologist, Department of Pathology, OLV Hospital Aalst and Free University Hospital, Brussels, Belgium
⁵European Working Groups on Myeloproliferative Neoplasms (EWG.MPN 1998-2018), Good heart Institute & Foundation, Freedom of Science & Education Rotterdam, Netherlands

*Address for Correspondence: Jan Jacques Michiels, Multidisciplinary Internist & Scientific Investigator, European Working Group on Myeloproliferative Neoplasm EWG.MPN 1998-2018, Goodheart Institute in Nature Medicine, Rotterdam, Europe, Erasmus Tower, Veenmos 13, 3069 AT Rotterdam, Netherlands; Tel: +316-26970534; Email: goodheartcenter@outlook.com

Dates: Submitted: 18 February 2019; Approved: 28 February 2019; Published: 01 March 2019

How to cite this article: Michiels JJ, Berneman Z, Schroyens W, J ten Kate FW, Lam K, et al. European Clinical Laboratory, Molecular and Pathological (ECMP) criteria for prefibrotic JAK2^V617F-Thrombocythemia and Polycythemia Vera versus MPL⁵¹⁵- and CALR-Thrombocythemia and Myelofibrosis: From Dameshek to Michiels 1950-2018. Int J Bone Marrow Res. 2019; 2: 001-017. DOI: 10.29328/journal.ijbmr.1001002

Copyright License: © 2019 Michiels JJ, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Focusing since 1975 on the discovery and elucidation of erythromelalgia caused by platelet-mediated inflammatory and thrombosis in thrombocythemia in ET and PV patients [1-3], and on the association of migraine-like microvascular cerebral transient ischemic attacks (MIA) as specific presenting symptoms of ET [4], we were able to document the early pre-fibrotic stages of ET and PV by the combined use of clinical, laboratory and bone marrow histopathology features (Figures 1-5, Tables 1,2) [4,5]. As no definition was available for early stage ET at that time, we prospectively performed clinical and basic research studies on the causal relationship between aspirin-sensitive erythromelalgia and atypical MIA in thrombocythemia of ET and PV patients in the period of 1975 and 1998. Between 2000 and 2008 we performed prospective studies on the clinical and pathobiological features of pre-fibrotic JAK2^V617F mutated ET and PV and JAK2 wild type ET patients.

During the years, ET and PV patients were diagnosed according to the Rotterdam Clinical and Pathologic (1980 RCP) [1-5], the European Clinical and Pathologic (2002-2005 ECP [6] http://www.mpn-stichting.nl/doctors_brochure_2004.pdf) and the European Clinical Molecular and Pathological (2007 ECMP) [7], criteria for the classification of pre-fibrotic ET and PV and ET with a hypercellular megakaryocytic granulocytic (MG) bone marrow histology. In this contribution, we present the updated results of our prospective studies in view of a critical approval of the literature. This report provide good evidence that the characteristic features of pre-fibrotic myeloproliferative neoplasms (MPN) can be subclassified as JAK2^V617F mutated ET, ET with early features of PV (prodromal PV), classical PV, and ET with hypercellular megakaryocytic granulocytic proliferation as the three main JAK2^V617F mutated variants of pre-fibrotic MPN (Figures 1 to 10) [1-48]. According to Dameshek (1950) [23], PV is a trilinear myeloproliferative disease (MPD) caused by the JAK2^V617F mutation (Vainchenker and his team 2005, Figure 6) [32,33,36]. According to Dameshek (1951), PV is clearly distinct from megakaryocytic leukemia (ML) [1,9] or primary thrombocythemia hemorrhagica (PTH) without features of PV (Laszlo 1975) [8]. Here we demonstrate that ML or PTH belong to the JAK2 wild type variants of pre-fibrotic ET carrying one of the MPL [42,43] or CALR [44] mutations. JAK2 wild type hypercellular ET associated with primary megakaryocytic granulocytic myeloproliferation (PMGM) [26,45,46}, proved to be CALR mutated [44,47,48], as the third distinct MPN when the Clinical Laboratory Molecular and Pathologic (CLMP) criteria are applied (Tables 3-5) [49,50].

In1975 the minimum criterion for the diagnosis of ET proposed by the Polycythemia Vera Study Group (PVSG) was 1000x10^9/. ET at platelet count above 1000x10^9/ usually presented with the clinical picture of hemorrhagic thrombocythemia (HT) [8,9]. In view of our discovery of platelet-mediated erythromelalgia as a specific and early presenting symptoms of thrombocythemia in ET we were able to document the very early stage of ET at platelet counts between 400 and 1000x10^9/ by the use of the Rotterdam Clinical and Pathological (1980 RCP) criteria for ET (Table 1) [2,3]. The 1980 RCP criteria of ET were determined by careful prospective documentation of peripheral blood and bone marrow smears and bone marrow biopsy material [2+4]. Platelets in excess of 400x10^9/, and an increase of clustered enlarged megakaryocytes in a bone marrow biopsy material was found to be diagnostic for ET and excluded reactive thrombocytosis. On top of the clinical PVSG criteria for PV [8-10] we used since 1980 bone marrow histopathology and erythrocyte count above 6x10^12/L to confirm PV and exclude all variants of primary and secondary erythrocytosis [11,12]. The 1980 RCP criteria for PV replaced O2-saturation of >92% by bone marrow biopsy, changed splenomegaly by bone marrow biopsy as a major criterion, and used splenomegaly as a minor criterion (Table 1) [1-3]. Raised B12 (>900 ng/L) or raised B12 binding capacity (>2200 ng/L) were skipped as completely irrelevant for the diagnosis of early stage PV. Raised score for leukocyte alkaline phosphatase (LAP) stain and normal erythrocyte sedimentation rate (ESR) are specific features of ET and PV [13,14]. Applying the 1980 RCP criteria we could detect masked and prodromal cases of ET and PV. In 2002 Michiels & Thiele proposed the European Clinical and Pathological (ECP, http://www.mpn-stichting.nl/doctors_brochure_2004.pdf) criteria for ET, PV and defined ET with a hypercellular bone marrow defined as PMGM [15-17]. In this report we extended the RCP and ECP (Table 1) into the 2006-2018 European Clinical, Molecular and Pathological (ECMP) for the diagnosis and classification of JAK2^V617F mutated ET, prodromal PV, classical PV and masked PV as clearly distinct from MPL⁵¹⁵ mutated ET and of JAK2/MPL wild type ET associated with CALR mutated PMGM (Tables 1,2) [13-16,34,46-50].

Bone marrow biopsies from the iliac crest were stained with hematoxylin and eosin for histopathology evaluation. All bone marrow biopsies were evaluated by expert pathologists for morphology, grading of cellularity and scoring of reticulin fibers using the silver impregnation stain according to PVSG and ECP recommendations (Ellis et al., Table 2) [11]. Additional screening was performed for endogenous erythroid colony (EEC), formation of nucleated peripheral blood or aspirated bone marrow cells, serum erythropoietin (EPO) levels [19-21] and JAK2^V617F mutation using the PCR test according to Baxter et al. [18]. With the advent of JAK2^V617F as the cause of trilinear MPN [32,33], MPL⁵¹⁵ mutations [42,43] as another cause of ET and MF and JAK2/MPL wild type CALR mutated PMGM [26,34,44-46] we extended the ECMP criteria [47,48], into the Clinical Laboratory Molecular and Pathobiological (CLMP) features [49,50], for classification and staging three distinct JAK2^V617F ET, PV and MF, JAK2 exon 12 PV versus MPL⁵¹⁵ and CALR mutated thrombocythemia and myelofibrosis without features of PV (Tables 3-5) [46-50].

ET and PV result analysis using the RCP, ECMP and CLMP criteria

Between 1975 to 1985, we prospectively studied 30 consecutive thrombocythemia patients diagnosed as ET in 14 and PV in 16. The clinical features of erythromelalgia in ET and PV cases have been reported in 1985 [3]. Eleven of 14 ET patients had platelet counts below 1000x10^9/, in whom the diagnosis of ET would have been overlooked by the PVSG criteria at that time (1975-1985). Spleen size on scan was slightly increased in 5 of 14 ET and in 13 of 16 PV patients. Leukocyte count counts was increased (>10x10^9/) in 5 out of 14 patients with ET and in 14 of 16 PV patients. LAP score was increased (>100) in 12 out of 14 ET and in all PV patients. Increase of clustering of enlarged megakaryocytes and a normal erythropoiesis was present in bone marrow smears and biopsies of 14 ET and 16 PV patients. Increase of clustered pleomorphic megakaryocytes and no increase of cellularity (Figure 1) was seen in 7 of 14 ET and in one of 16 PV patients. Increase of bone marrow cellularity (1+ = 60-80%) due to increased erythropoiesis leading to an ET/PV picture was seen in 3 ET and 4 PV patients (Figure 1). Pronounced increase of cellularity (2+ =80-100%) due to trilinear erythrocytic, megakaryocytic, granulocytic (EMG) bone marrow proliferation described as panmyelosis by Dameshek [1,24]) was seen in 2 of 14 ET patients and in 11 of 16 PV and (Figure 1). Bone marrow histopathology appeared to be a powerful tool to differentiate ET and PV from all variants of primary or secondary erythrocytosis and reactive thrombocytosis with a sensitivity and specificity near to 100% [6,11,15,16,34]. The morphology of megakaryocytes were not different in ET and PV in the Rotterdam study (Figure 1, Tables 1-3). ET and PV patients in the Rotterdam ET/PV and the PVSG-01 PV studies show overlapping bone marrow pictures with increase of pleiomorphic clustered megakaryocytes (Figures 2,3).

Increased erythrocyte counts above 6x10^12/L and increase of large pleiomorphic megakaryocytes in hypercellular bone marrow smears according to Dameshek (1950) [1,23] and bone marrow biopsy according to Michiels 1980 [2-3,34], appeared to a pathognomonic clue diagnostic for classical PV (Table 1). Increase of erythrocytes above 6x10^12/L persisted during the iron deficient status in PV in remission by repeated venesection [23]. Two of our ET patients had borderline increased RCM, which was associated with erythrocytes above 6.0x10^12/L, increased LAF score, and a negative bone marrow iron stain and should be diagnosed as PV. One ET case had erythrocyte count of 5.8x10^12/L and increased LAF score developed PV within 2 years. Bone marrow iron stain was negative in all PV patients (N=16) and in 4 out of 12 evaluable ET patients and therefore cannot be used as diagnostic for PV. The most objective criterion to differentiate ET from PV (both may present typical ET/PV or PV. In recent studies we produced good evidence that the combination of bone marrow and erythrocyte count above 5.8X10/12L is diagnostic for PV (Table 3) [1,22].

European CLMP versus PVSG and WHO classification of the MPNs

Analysis of BM biopsies of 155 evaluated PV patients with documented increased RCM in the PVSG 01 study by Michiels using RCP and ECP criteria (Table 1) showed a spectrum of no, slight, moderate to marked (>80%) increase of bone marrow cellularity (Figures 2,3, Ellis 1986) [11], in ranges from <50-60% in 10, from 60-80% in 45 and from 80-100% in 100 cases (Figure 2). The silver stained reticulin fiber content was normal (RF 0 and 1) in 94, slightly increased (RF 2) in 40, and moderately to marked increased (RF 3 and 4) in 21 (Figure 2). Diagnosis according to bone marrow cellularity and morphology were typical for ET in 10, for ET/PV in 45, for PV in 70 and for PV/RF 3 or 4 in 13 PV patients when the RCP criteria were applied [11].

Between 2000 and 2015 we studied 10 JAK2^V617F mutated patients with early stage ET or newly diagnosed PV who presented with migraine-like microvascular cerebral ischemic attacks (MIA) who were referred from various European countries to the Antwerp University Hospital for expert evaluation and treatment recommendation (Table 6). The PVSG diagnosis of 10 JAK2^V617F mutated patients in the second international prospective clinical research study without use of bone marrow histopathology was ET in 6 and PV in 4 (Table 6). The 6 JAK2^V617F mutated ET patients had erythrocyte counts below 6x10^12/L. Three ET and 4 PV fulfilled the histological bone marrow features of typical PV according to ECMP bone marrow criteria [22,23]. Three ET patients with PV bone marrow features developed overt PV after long-term follow-up of 8, 9 and more than 10 years (slow onset PV, Figure 5, Table 6). Bone marrow cellularity was normal in 2 of 10 and increased (range 60-90%) due to increased erythropoiesis in 4 of 10 and increased erythropoiesis granulopoiesis in another 4 of 10 patients. Myelofibrosis (MF) MF-0 (RF 0/1) in 8 (5 ET, 3 PV), and MF-1 (RF 2) in MF-2 (1 ET, 1 PV) MPN patients. The diagnoses according to 2008 WHO criteria [24] were ET in 5, MNP unclassifiable in 1 and PV in 4. The diagnoses according to 2006-2015 ECMP criteria [34,46,47] (Table 6), were normocellular ET in 2, ET with features of early PV: low serum EPO, the presence of EEC and normal values for hemoglobin, hematocrit and erythrocyte counts (prodromal PV) in 3, hypercellular trilinear ET showing no leuko-erythroblastosis in 1, and acute onset PV not preceded by ET in 4 patients (Figure 5, Table 6).

There are significant differences in blood and bone marrow features between ECMP defined ET compared to PVSG defined primary thrombocythemia hemorrhagica (PTH) at platelet counts above 1000x10/L (PTH) [8] described by Thiele (1988) in a cohort of 25 patients (Table in Figure 7) [25]. In PTH the values for hemoglobin, erythrocytes, leukocytes and LAP scores are normal [25], whereas LAP score were increased in RCP (Table 1) and ECMP defined ET [34,46,47] but in the normal range in PVSG defined PTH (Table in Figure 7). The megakaryocytes in PTH are large to giant with hyper-lobulated nuclei (Figure 7), whereas in RCP and ECMP defined JAK2^V617F mutated ET patients the size of pleiomorphic megakaryocytes are medium similar to early and classical PV (Figure 4). In 2008 we studied bone marrow histopathology in 12 cases with JAK2 wild type ET carrying the MPL⁵¹⁵ mutation (Table 4) kindly provided by the courtesy of Dr Vannucchi, Florence, Italy, Europe. Bone marrow pathology (P, Table 4) from patients with JAK2 wild type MPL⁵¹⁵ mutated ET consistently displayed clusters small and large megakaryocytes with a greater number of giant megakaryocytes with hyper-lobulated stag-horn nuclei in a normal cellular bone marrow and no increase of erythropoiesis (Figure 8) [47].

From 1994 to 2006, we observed a case of JAK2 wild type hemorrhagic thrombocythemia (HT) in a 9-year-old boy with a platelet count of 1596 to 1946x109/l, no splenomegaly on palpation, white blood differential count (metamyelocytes 0.5%, banded forms 1%, segmented granulocytes 52%, basophiles 2.5%, lymphocytes 35% monocytes 6%), low LAP score, and a hypercellular (80-100%) bone marrow with pre-fibrotic primary megakaryocytic and granulocytic myeloproliferative (PMGM) disease, absence of reticulin fibers, loose to dense clustering of enlarged dysmorphic megakaryocytes variable in size from large to giant with hyperploid and some naked nuclei (Figure 9). The dysmorphic megakaryocytes in this (Figure 9) and another case of PMGM (Figure 10) show definite abnormalities of maturation with bulky (bulbous) hyperchromatic nuclei and some disturbances of the nuclear cytoplasmic ratio, which are not seen in MPL⁵¹⁵ mutated normocellular ET (Figure 8) and also not in pre-fibrotic JAK2^V617F mutated ET, ET/PV, PV and EMGM (masked PV) (Figure 5). Characteristic bone marrow findings of JAK2/MPL wild type, CALR mutated PMGM are consistent with pronounced thrombocythemia at platelet counts around 1000x10^9/ mimicking PTH as the presenting feature of pre-fibrotic PMGM [26,45,46], and do not meet the 2008 WHO criteria for AMM, or PMF [24]. The long-term follow-up of a typical case of PMGM showed normal blood cells counts, absence of the JAK2^V617F mutation, without features of PV and myelofibrosis, and no splenomegaly on palpation (Figure 9). We observed similar cases of ECMP defined JAK2/MPL wild type CALR mutated PMGM (Figure 10), which in retrospect has been misinterpreted by Thiele and Vardiman as pre-fibrotic PMF [6,13,16] in the 2001 WHO classification. Tefferi and Thiele persisted to use the term pre-fibrotic and fibrotic stages of PMF without features of PVSG defined PV in the 2008 and 2016 WHO classifications [24,34].

Dameshek and Henthell (1940) [22] defined the diagnostic criteria for true polycythemia based on the description of 20 PV cases seen between 1928 and 1937, and proposed the historic set of symptoms, signs and laboratory tests for the diagnosis of PV¹:

Dameshek (1940, 1950, 1951) demonstrated that the combination of plethoric appearance, splenomegaly, definitely elevated erythrocyte count in excess of 6x10^12/L, elevated platelet count, elevated hematocrit is consistent with a definite diagnosis of PV¹. The bone marrow is pathognomonic diagnositic for PV showing the combination of large megakaryocytes and panmyelosis (Dameshek) of increased trilinear erythrocytic, megakaryocytic and granulocytic (EMG) bone marrow proliferation without the need to measure blood volume [1,23]. According to Damehek in 1950 PV is a trilinear myeloproliferative disease (MPD) of erythrocythemia, thrombocythemia and granulocythemia in early pre-fibrotic stage of MPD disease, and complicated by spent phase PV and myeloid metaplasia of the spleen (increasing splenomegaly), and myelofibrosis in about one third of the cases after long-term follow-up of about 15 to 30 years [1,23].

The finding of increased LAF score and platelet count between 400 and 1000x10^9/ in the majority of our ET patients is a consistent finding in our ET and PV patients between 1975 to 2015 belong to the same JAK2^V617F mutated MPN, whereas leukocyte count in ET usually normal or slightly increased. The combined use of bone marrow histology, blood cell (including erythrocyte) counts, EEC, serum EPO levels appeared to be highly sensitive and specific enough to distinguish the 3 stages of JAK2^V617F mutated ET: normocellular ET, ET with features of PV (prodromal PV), and advanced ET with trilinear EMG bone marrow proliferation (Table 2). Interestingly, three cases of prodromal PV (ET with features of PV) indeed developed overt PV after long-term (around 10 years) follow-up (slow onset PV preceded by erythromelalgia or migraine-like atypical TIAs for more than 5 to10 years (Table 6). Four of 10 JAK2^V617F mutated cases in our pilot study presented with acute onset PV not preceded by ET (Table 6).

The JAK2^V617F mutated Thrombocythemia in trilinear MPN (Table 3), MPL⁵¹⁵ mutated Thrombocythemia [34,46,47] and CALR Thrombocythemia associated with PMGM without features of PV clearly differ when the ECMP and 2018 CLMP criteria [51,52], are applied (Tables 4,5). The morphology of pleiomorphic megakaryocytes are not different between the JAK2^V617F mutated MPNs in the three stages of normocellular ET, prodromal PV and masked PV (Figures 1,2,4,5, Table 3). The more the cellularity is increased, the more dysmorphic the megakaryocytes were in JAK2^V617F mutated ET and PV. In JAK2^V617F mutated hypercellular ET (EMGM or masked PV) pleiomorphic megakaryocytes show dysmorphic features of nuclei (not cloud-like) with a back ground of masked PV bone marrow histology [34,46,47]. Michiels et al., recognized that the MPL⁵¹⁵ mutation Thrombocythemia is characterized by clusters of medium, large and giant megakaryocytes with a greater number of large deeply lobulated staghorn like nuclei, which are not seen in JAK2^V617F mutated ET and PV patients [47]. Pich et al., described in 2012 characteristic bone marrow histopathology findings in 59 JAK2^V617F positive ET and 44 JAK2 wild PT cases [27]. The bone marrow histology findings in the studies of Pich et al. [27], and Michiels et al. [34,46,47], in JAK2^V617F mutated ET indeed have PV-like hypercellular morphological bone marrow changes showing pleiomorphic medium sized large mature megakaryocytes similar to our first 1980 cohort ET and PV (Figure 1) and also in PV patients of the PVSG 01 study (Figure 2) [11]. Clusters of large to giant megakaryocyte with staghorn nuclei and platelet count increase are more pronounced in MPL⁵¹⁵ mutated normocellular Thrombocythemia with the complete absence of PV features [47]. ET with increased cellularity due to increased erythropoiesis is consistent with JAK2^V617F mutated prodromal PV [34,46,47]. The prognosis of JAK2^V617F mutated normocellular ET and prodromal PV is favorable and to be treated with low aspirin. Additional phlebotomy on top of low dose aspirin in early PV to maintain the hematocrit (Ht) around 0.40 in man and women is warranted for the cure and prevention of microvascular and major thrombosis [14,16]. The real field experiences from our prospective clinical observations in the 1980 and 1990s are completely in line with subsequent studies on patients with JAK2^V617F mutated ET, prodromal PV and PV observed between 2005 and 2018 [27,34-46-50].

The 2008 and 2016 WHO-classifications of myeloproliferative neoplasms (MPN) are the direct extension of the 1975 PVSG classification and only based on clinical and laboratory features without the use of erythrocyte counts and bone marrow histology. The PVSG and WHO classification criteria are too crude to detect the early stages and of ET and PV and do not differentiate between normo-cellular ET, ET/PV (forme fruste PV) [39], classical PV and trilinear EMG (masked PV) bone marrow proliferation complicated by myelofibrosis [47-50]. The combined use of erythrocytes counts above 5.8x10^12/L and bone marrow pathologic in the ECMP and CLMP classification is a pathognomonic clue to JAK2^V617F mutated PV without the need of red cell mass (RCM) measurement. With the advent of JAK2^V617F, MPL⁵¹⁵ and CALR mutations as driver causes of three distinct MPN disease entities, ET is not essential and primary myelofibrosis (PMF) is not a primary disease anymore. MF is a secondary event in all molecular variants of MPD, MPN, CML and in hairy cell leukemia as well. Advanced MPN is featured by the combination of anemia, splenomegaly increased CD34 circulating cells, and myelofibrosis grade MF-2. MF-3 and MF-4 [6,16,17,24,29]. As 2006/2016 ECMP defined MF is a secondary event in advanced stages of all variant of MPN [26,34,50], this novel insight refers to the compelling need to better defined the natural history of JAK2^V617F vs MPL⁵¹⁵ vs CALR mutated MPN in large prospective clinical and basic research studies using the updated CLMP criteria including next generation sequencing and bone marrow pathology evaluation by expert pathologists. The JAK2^V617F mutated ET, PV and MF appear to be a broad spectrum of sequential distinct trilinear MPN entities at the blood and bone marrow level during life-long follow-up [27,31-36,47-50]. By investigating the bone marrow features these JAK2^V617F mutated cases in the initial pre-fibrotic stages some of them had a unique mono-linear megakaryocytic proliferation in a normocellular bone marrow lifelong in all three molecular variants of MPN. JAK2^V617F mutated MPN had in addition a discrete expansion of the erythropoiesis in prodromal PV, and evolve into hypercellular trilinear megakaryocytic erythrocytic and granulocytic in classical, masked and advanced PV as can be documented by bone marrow proliferation and associated clinical features [32-37,47-50]. Increased erythrocytic bone marrow cellularity (60%-80%) in ET/PV patient was very well known to European pathologists [30,31]. Thiele et al., described the so-called initial (latent) stage of PV as ET mimicking PV [37]. The laboratory data of 23 cases diagnosed as initial or latent PV not meeting the 1975 PVSG and 2008 WHO criteria for PV did have increased erythrocyte count above 6x10^12/L in men and above 5.5x10^12/L in female, which indeed is consistent with the diagnosis of overt PV according to 1980 RCP and 2012-2018 ECMP criteria [37]. JAK2^V617F mutated ET and prodromal PV are featured by increased LAF score, EEC+, low serum EPO and a normal erythrocyte count (<6x10^12/L) [46,47].

The 2005 concept of Vainchenker & Constantinescu [33] is that heterozygous JAK2^V617F mutation leading to constitutively activated megakaryocytes due to increased sensitivity to TPO is enough to induce ET phenotype [32,33,36]. The production of hyperreactive (sticky) platelet in thrombocythemia of ET and PV patients is the cause of platelet mediated arteriolar inflammation and thrombosis in ET and PV patients [34,35], (platelet thrombophilia Dames (1940) [1] or Sticky Platelet Syndrome, Michiels 1985) [2-4]. Thrombocythemia is associated with no or slight increased erythropoiesis in ET (ET stage 1, cases 1 and 2 of table 6), or definite increase of erythropoiesis in ET with features of PV (prodromal PV, cases 3 and 4 of table 6). Early stage erythromelalgic thrombotic JAK2^V617F mutated ET with low JAK2^V617F mutation load does exist [38] can now be diagnosed at as normocellular ET without progression into pro-dromal PV or classical PV during long-term follow-up of more than 20 years (Figure 5A). Homozygous JAK2^V617F mutated hematopoietic progenitor cells are hypersensitive to EPO and TPO and do induce trilinear erythropoiesis, megakaryopoiesis and granulopoiesis (trilinear EMG) bone marrow proliferation. Such hypercellular trilinear EMG bone marrow pictures are the pathognomonic diagnostic clue for JAK2^V617F mutated PV, masked PV or post-PV myelofibrosis without the need to measure red cell mass (RCM) [34-36,47-50]. The sequential occurrence of heterozygous/WT into homozygous JAK2^V617F mutation due to mitotic recombination of chromosome 9p can readily explain the evolution of ET and prodromal PV into slow onset PV or masked PV with subsequent evolution into post-PV and post-ET myelofibrosis and splenomegaly due to myeloid metaplasia of the spleen (Table 6, Figure 6) [1,23,34,36,46-52]. Lifelong follow-up studies of slow onset PV and acute onset PV are lacking. There is a broad spectrum of transitional states between JAK2^V617F mutated prodromal, masked and overt PV, its early reticulin fibrotic (RF) and reticulin collagen fibrotic (RCF) stages on one hand and post-ET and post-PV-myelofibrosis during long-term follow-up of 10 to even more than 25 years of follow-up were already defined by Dameshek (1950) [1,23]. The sequential use of RCP, ECP and 2006 ECMP and 2018 CLMP criteria did pick the early latent stages of all clonal variants and overt stages of MPN of various molecular etiologies [1,22,23,34,39-41,47,48].

We produced good evidence that ML (megakaryocytic leukemia) defined by Dameshek (1951) consist of MPL⁵¹⁵ and CALR mutated Thrombocythemia without features of PV at diagnosis and during follow-up [42-44,48]. JAK2/MPL wild type cases of pre-fibrotic PMGM first defined by Georgii in 1990 [26] has recently been discovered by Michiels et al., as CALR mutated Thrombocythemia associated with PMGM [44,47,48]. Both MPL⁵¹⁵ and CALR mutated thrombocythemia have no features of PV including low LAP score, whereas the incidence of secondary myelofibrosis is similar as in trilinear PV. In contrast myelofibrosis and palpable splenomegaly is rare in heterozygous JAK2^V617F mutated ET [26,34,45-50] and much more prevalent in heterozygous/homozygous or homozygous JAK2^V617F mutated hypercellular PV. The three main distinct MPNs at the level of bone marrow characteristics combined with MPN-specific clinical and laboratory features EEC, EPO levels in JAK2^V617F mutated ET, prodromal PV and overt PV versus MPL⁵¹⁵ and CALR mutated Thrombocythemia and Myelofibrosis without features of PV mutually exclude each other when the 2006/2018 ECMP [47-49] and 2018 CLMP [51,52], criteria are applied [47-52].

Contribution of authors

JJM, KL and FWJTK designed the study and JJM wrote the manuscript. JJM, ZB and WS collected the clinical data between 1975 and 2015. FWJTK evaluated the bone marrow biopsy specimens from the study in 1975-1985 and KL and HDR performed the bone marrow pathology studies between 2000 and 2018.

Dr. Juergen Thiele, Cologne, Germany as a most respected active member of the EWG.MPD (1998-2018) significantly contributed to the formulation of the ECP and ECMP criteria for the MPDs ET, PV, and PMGM or PMF and its differentiation from Ph+ ET, Ph+ CML, and the Ph-negative CMML, MDS and CNL just by bone marrow histopathology alone.