Review Article
Change of the 2008/2016 WHO Criteria into European Clinical, Laboratory Molecular and Pathological (ECMP/CLMP) Classification of BRC/ABL Negative Myeloproliferative Neoplasms Caused by JAK2, MPL and CALR Driver Mutations
Jan Jacques Michiels1,9*, Timothy Devos2, Rik Schot3, Francisca Valster4, Vincent Potters5, Katrien Schelfout5, Jiri Schwarz6, Miroslav Penka7, Achille Pich8, Peter Te Boekhorst9, King Lam9 and Hendrik De Raeve10
1Jan Jacques Michiels, Internist Hematologist Goodheart Institute in Nature Medicine & Health, Erasmus Tower, Veenmos 13, 3069 AT Rotterdam, The Netherlands
2Timothy Devos, Internist Hematologist, Department of Hematology, University Hospital Leuven, Gasthuisberg Herenstraat 49, B-3000 Leuven, Belgium
3Rik Schot, Internist Hematologist, Department of Hematology, University Hospital Brussels, Laarbeeklaan 101, B-1090, Brussels, Belgium
4Fransje Valster, Internist Hematologist, Department of Internal Medicine, BRAVIS Hospital, Boerhaaveplein 1, 4624 VT, Bergen op Zoom, The Netherlands
5Vincent Potters, Pathologist, Department of Pathology, BRAVIS Hospital, Boerhaveplein 1, 4624 VT, Bergen op Zoom, The Netherlands
6Katrien Schelfout, Pathologist, Department of Pathology, BRAVIS Hospital, Boerhaveplein 1, 4624 VT, Bergen op Zoom, The Netherlands
7Jiri Schwarz and Miroslav Penka, Czech National Working Group on Myeloproliferative Disorders/ Myeloproliferative Neoplasms (MPD/MPN) of the Czech Hematology Society, The Czech Republic
8Achille Pich Department of Molecular Biotechnology and Health, Section Pathology, University of Turin, Italy
9Peter Te Boekhorst and King Lam, Department of Hematology, Erasmus Medical Center, Erasmus Univerisity Rotterdam , Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
10Hendrik De Raeve, Pathologist, Department of Pathology OLV Hospital Aalst OLV Ziekenhuis,Moorselbaan 164, B-9300 Aalst, Belgium and University Medical Center Brussels, Laarbeeklaan 101, B-1090, Brussels, Belgium
*Corresponding author: Jan Jacques Michiels, Internist Hematologist Goodheart Institute in Nature Medicine & Health, Erasmus Tower, Veenmos 13, 3069 AT Rotterdam, The Netherlands, Tel: +316 26970534; E-mail: goodheartcenter@outlook.com
Received: October 29, 2019; Accepted: December 12, 2019; Published: December 27, 2019
Abstract
Ninety five percent of Polycythemia Vera (PV) patients and half of patients with Essential Thrombocythemia (ET) patients have low serum Erythropoietin (EPO) levels, and carry the JAK2V617F mutation. The change of 2008/2016 WHO criteria for ET into the European Clinical, Molecular and Pathologic (ECMP) classification of the JAK2V617F positive ET patients refers to three phenotypes of ET. First, heterozygous JAK2V617F mutated normocellular ET life-long. Second, hypercellular ET due to increased erythropoiesis (prodromal PV) preceding classical PV. Third, hypercellular and ET with erythrocythic-megakaryocytic-granulocytic bone marrow proliferation not meeting the WHO criteria for PV (masked PV). JAK2V617F mutation load is low and stable around 30% in heterozygous normocellular ET but high and increasing from below 50% to 80-100% homozygous JAK2V617F mutated PV and masked PV. JAK2V617F mutation load is related to MPN disease burden in terms of leucocytosis, splenomegaly, constitutional symptoms and myelofibrosis. Five distinct JAK2V617F trilinear MPN stages can be distinguished: JAK2V617F heterozygous positive ET, JAK2V617F homozygous positive PV; benign JAK2exon 12 PV; MPL515 mutated ET without features of PV and CALR mutated ET without features of PV. CALR mutated thrombocythemia has been recognized and described in great detail by Georgii & Michiels between 1990 and 1997 as Primary Megakaryocytic Granulocytic Myeloproliferation (PMGM).
Bone marrow features in JAK2V617F mutated ET and PV are similar characterized by medium sized to large (pleomorphic) megakaryocytes with hyperlobulated nuclei. Bone marrow histology in MPL515 positive ET show clustered large to giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow. Bone marrow histology in CALR mutated ET patients is dominated by dense clusters of large immature dysmorphic megakaryocytes with bulky (cloud-like) hyperchromatic nuclei, which are never seen in JAK2V617F , JAK2exon12 and MPL515 mutated MPN. The 2008/2016 WHO defined terminology of ET and PMF can easily be replaced by ECMP defined CALR-, MPL515-and JAK2V617F-mutated thrombocythemia and secondary myelofibrosis (MF) with various degrees of splenomegaly, hypersplenism and myelofibrotic transformation of the bone marrow
Keywords
Bone marrow pathology; Essential thrombocythemia; JAK2 mutation; MPL mutation, calreticulin mutation; Myelofibrosis; Polycythemiavera; Primary megakaryocytic granulocytic myeloproliferation; Reticulin fibrosis
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 PVSG = WHO defined ET has been recognized by Thiele et al. in 1988 and 2005 as clearly distinct from PV [5,6]. The peripheral blood findings in JAK2 wild type ET carrying the MPL or CALR mutation are featured by high platelet counts, normal values for haemoglobin, haematocrit, erythrocyte, white blood cells, LAP score, serum EPO, LDH and no or minor splenomegaly despite platelet counts above 1000×109/L7. The megakaryocytes in JAK2 wild type ET carrying the MPL or CALR mutation are much larger to giant than the medium to large seized megakaryocytes in PV (Table 1) [5-7].
Bone marrow histology in JAK2V617F mutated PV is typically featured by medium sized to large pleomorphic megakaryocytes with hyperploid nuclei in a hypercellular bone marrow in a hypercellular bone marrow due to increased erythropoiesis or increased trilinearerythrocytic, megakaryocytic and granulocytic myeloproliferation [2-4]. Georgii et al 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) [8,9].
The Hannover Bone Marrow Classification of the MPDs distinguished three primary prefibrotic MPDs ET, PV and CMGM/PMGM from the advanced fibrotic stages of MPD8. 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 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 [8,9]. Michiels replaced in 1997 the term CMGM by essential or primary MGM (EMGM or PMGM) as the third JAK2 wild type MPN entity without features of PV or CML [10-12]. EMGM or PMGM (hypercellularJAK2 wild type ET) is the third MPN entity caused by the CALR driver mutation. ET associated with prefibrotic PMGM is dominated by an increase of clustered atypical dimorphic 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 JAK2V617F mutated ET and PV when the European Clinical Laboratory, Molecular and Pathological (ECMP/CLMP) criteria for BRC/ABL negative Myeloproliferative Disorders or Neoplasms (MPD/MPN, Tables 1 to 7) are applied [1-51].
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 PMGM 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 PMGM based on distinct bone marrow histology findings for each of the three MPDs ET, PV and PMGM [8-12]. Increased of clustered large megakaryocytes in bone marrow histology from biopsies is a pathognomonic clue to the diagnosis of in Ph-negative ET PV and PMGM. 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]. The Hannover Bone Marrow Classification distinguished the three primary prefibrotic MPDs ET, PV and PMGM from advanced fibrotic stages of MPD (Figure 1) [8,9,34,51]. Prefibrotic PMGM is the third distinct entity of primary MPD in the absence of reticulin or collagen fibrosis in bone marrow biopsy material [15]. Myelofibrosis (MF) is a secondary event in all variants of MPD. Consequently, the WHO defined Chronic Idiopathic Myelofibrosis (CIMF) or Primary Myelofibrosis (PMF) are a misconception. Georgii replaced the term CIMF and PMF by PMGM and used grading of Myelofibrosis (MF) (Table 1) for staging of the early, overt and advanced MPDs ET, PV and PMGM [8,9]. Prefibrotic 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 PMGM 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 PMGM 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 ECMP classification of myeloproliferative neoplasms (MPN) took over the CMGM concept and used the term primary megakaryocytic granulocytic myeloproliferation (PMGM, Table 1, Figure 1) [19,34,51].
The 1975 PVSG and the 2008/2016 classification of MPD and MPN 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 erythrocytosis (Table 1) [2,3]. The PV experts in the UK and France modified the PVSG criteria by including laboratory screening for serum EPO, spontaneous Erythroid Colony Formation (EEC) as clues for latent and classical PV but did not use bone marrow biopsy for the diagnostic differentiation between PV and primary or secondary erythrocytosis and therefore overlooked stage 1 erythrocythemic PV by definition [21,22]. In 1979 Pearson defined idiopathic erythrocythemia (IE) by increased RCM and not meeting the A and B criteria of the PVSG [21]. IE appeared to be an early stage of PV in about 10% to 15% at time of PV presentation [21-23]. A low serum EPO level has also been described about half of PVSG defined 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 large study of 241 patients, Mossuz et al. identified two thresholds, allowing a specific and direct diagnosis of 65.6% (65-99) of untreated PVSG-defined PV with very low serom EPO levels (EPO <1.4 U/L) and 19.7% (13 of 66) versus Primary Or Secondary Erythrocytosis (PSE) with increased serum EPO levels (EPO >13.7 U/L) [25]. About 50% of patients with absolute erythrocytosis could unequivocally diagnosed as PV or PSE by the combination of increased Red Cell Mass (RCM) and serum EPO levels.
JAK2V617F Mutated Trilinear MPNs in ET and PV: Dameshek-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 hypothesis of PV as a trilinear MPD has been proven to be correct by Vainchenker and Constantinescu by the discovery of the somatic JAK2V617F mutation as the driver cause of trilinear MPNs ET, PV and MF27. 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 pseudo-kinase part on the JH1 kinase part of JAK2, leading to enhanced activity of the normal JH1 kinase activity of JAK227. 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 1). Detection of JAK2V617F 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 MF17. The JAK2V617F mutation load is usually low in ET, less than 10 to 50% of the granulocytes are JAK2V617F positive (heterozygous) and either low or high in PV with less than 50% (heterozygous homozygous) or high between50 to 100% (homozygous) of the granulocytes positive for the JAK2V617F mutation. patients with hypercellular ET 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) and the percentage of JAK2V617F positive granulocytes in PV may range from rather low to 100% for JAK2V617F during the long-term follow-up [28-30,51]. At the bone marrow hematopoietic stem cell level, ET patients are heterozygous and PV patients hetero/homozygous or homozygous for the JAK2V617F mutation (Table 2, 3 and 4) [17,34,51].
The 2005 concept of Vainchenker & Michiels is that heterozygous JAK2V617F mutation is enough to constitutively activate TPO mediated megakaryopoiesis to induce ET with the production of constitutively activated (hypersensitive) platelets (Table 2) [17]. Homozygous JAK2V617F mutation is needed to more pronouncedly stimulate EPO mediated erythropoiesis compared to TPO mediated megakaryopoiesis. PV with allele load less than 50% indeed are hetero/homozygous at the EEC level in blood and bone marrow for the JAK2V617F mutation, whereas ET patients are heterozygous with a maximal JAK2V617F 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 JAK2V617F locus through mitotic amplification resulting in chromosome 9p loss of heterogeneity (9pLOH) indicating homozygous JAK2V616F mutation (Table 2). Godfrey et al studied the JAK2V617F mutation status of BFU-E grown in low erythropoietin conditions in 77 patients with PV or ET33. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, homozygous mutant colonies were absent or present in low percentages in JAK2V617F heterozygous ET, but prevalent and common in patients with homozygous JAK2V617F-positive 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 [33,34,51]. Combined heterozygous homozygous or homozygous JAK2V617F mutation is associated with pronounced constitutively activation of trilinear proliferation of megakaryopoiesis, erythropoiesis and granulopoiesis in the bone marrow as the cause of hyper cellular trilinear PV with a high risk of myelofibrotic progression.
According to WHO and ECMP criteria (Tables 3, and 4) [19,34,51], heterozygous JAK2V617F 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 2 and 3).
JAK2V617F positive ET with PV bone marrow features (prodromal PV) in 1997, who developed PV in 2004-2005 followed by a complete hematological response on pegylated interferon.
JAK2V617F mutated WHO defined PV uniformly reveal a hyper cellular bone marrow histology due to increase of trilinear hematopoiesis of megakaryopoiesis, erythropoiesis and granulopoiesis (panmyelosis [1,26;51]) and no or slight increase of reticuline fibers (Figures 3 and 4, Table 4) [4,19,34,51].
Middle panels. Typical PV picture in bone marrow biopsy specimen in a case of PV with rapid onset PV, increased red cell mass, and homozygous for the JAK2V617F mutation. Please note that loose clustered pleiomorphic enlarged megakaryocytes in this case are somewhat less enlarged and have hyperlobulated nuclei (middle panels) as in acase with classical PV with a hypercellular trilinear bone marrow proliferation (left bottum).
Lower panels. Comparison of bone marrow features in a case of classical PV with typical loose clusters of pleiomorphic small and large megakaryocytes (left bottum) as compared to small isolated normal sized megakaryocytes in bone marrow biopsy of a case with idiopathic erytrocytosis (right bottum).
The UK MPN Study Group assessed the clinical features in the cohort of 806 PVSG = 2008/2016 WHO defined ET patients [51] subdivided in 414 JAK2V617F positive and 362 JAK2 wild type ET and evaluated the bone marrow features in 393 ET patients [35,36]. 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 transformation into PV. PVSG = 2008/2016 WHO defined JAK2 wild type ET51 had significant higher platelet counts (962, range 668-1535×109/L) than JAK2V617F-positive ET (846, range 632-1222×109/L) [35]. In the UK Primary Thrombocythemia 1 (PT-1) study, bone marrow trephine of 209 JAK2V617F positive and 184 JAK2 wild type ET was independently assessed by 3 blinded pathologists who did not know the JAK2 mutation status36. The overall cellularity was significantly increased in JAK2V617F mutated ET as compared to JAK2 wild type ET, indicating that increased erythroid and granulocytic cellularity appears to be amain feature of prodromal PV, masked PV and classical PV [4,17,19,34,51].
JAK2exon 12 Mutated PV negative for the JAK2V617Fmutation
The 5% PV patients negative for JAK2V617F are frequently heterozygous for exon 12 JAK2 mutations and usually present with early stage PV with a favourable outcome and normal life expectancy [37-39]. JAK2exon12 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 JAK2exon12 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 JAK2exon12 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 5) [38,39]. Which differs from JAK2V617F mutated ET and PV (Figures 2, 3 and 4). At diagnosis, JAK2exon12 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 JAK2exon12 MPN cases lack the prominent clusters of large megakaryocytes with hyperlobulated nuclei that characterize JAK2V617F positive prodromal and classical PV. A spectrum of small to medium sized megakaryocyte is seen in JAK2exon12 PV bone marrows with a predominance of smaller forms of megakaryocytes with atypical nuclei with various degrees of monolobulated, normolobulated to hyperlobulated nuclei and abnormal chromatin distribution (Figure 5) [39].
Bone Marrow Histology in MPL515 Mutated Essential Thrombocythemia
Within the JAK2 wild type MPN, the prevalence of the MPL515 mutation as the cause of ET is 3% in the Vannucchi study [41], and 8.5% in the UK studies [42,43]. In the study of Vannucchi et al [41], 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 MPLW515L 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 6).
We recently described the essential differences in bone marrow histopathology features of differential diagnostic significance between patients with MPL515 mutated (N=12) versus JAK2V617F mutated MPN [34,51]. First, The presence of clustered small and giant megakaryocytes with deeply lobulated stag-horn like nuclei (Figure 6, Table 5).
In ET carrying the MPL515 mutation are not seen in JAK2V617F positive ET, prodromal PV, and classical PV. The pleomorphic medium to large megakaryocytes in JAK2V617F mutated ET and PV in bone marrow smears and bone marrow biopsy was comparable regarding size and degree of pleomorphy (Figures 2, 3 and 4). Second, there was local increase of erythropoiesis in areas of loose clustered pleiomorphic megakaryoctyes in normocelluar JAK2V617F mutated ET and prodromal PV, which is not seen in MPL515 mutated ET (Figure 6).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 JAK2V617F 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) [34,51].
Clinical, lzaboratory Features and Bone Marrow Histology in CALR Mutated ET and MF
Dr Kralovics and his team discovered Calreticulin (CALR) mutationin 78 of 311 (25%) ET patients, in 72 of 203 (35%) MF patients, in none of 382 PV patients [44]. 195 (67%) of 289 JAK2 wild type ET and 105 (80%) of 120 wild type MF carried one of the CALRmutations. Green and his team of MPN investigators in the UK found somatic CALR mutations in 110 of 158 JAK2 and MPL wild type MPN, including 80 of 112 (70%) ET patients, 18 of 32 (56%) MF patients [45]. CALR exon 9 mutations were found in 26 of 31 (84%) patients with JAK2/MPL wild type MF, were absent in all 120 patients who had JAK2 or MPL mutations, were present 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, JAK2V617F and MPL515 mutated MPN mutually exclude each other [44,45,51].
In eight newly diagnosed CALR positive ET cases in 2014 Michiels & De Raeve found consistent bone marrow characteristics of hypercellular ET as the presenting feature of prefibrotic and early fibrotic stages of PMGM. Bone marrow histology in typical prefibrotic CALR ET and in early fibrotic CALR myelofibrosis (MF) (Figure 7) show dysmorphic megakaryocytes with definite abnormalities of maturation with bulky (bulbous) hyperchromatic nuclei and some disturbances of the nuclear cytoplasmic ratio consistent with CALR mutated PMGM, which are not seen in MPL515 mutated ET (Figure 6) and also not in JAK2V617F mutated ET, prodromal PV and classical PV (Figures 2, 3 and 4). The JAK2/MPL wild type but CALR-mutated ET and MF patients appeared to become the third distinct MPN entity with typical characteristics of PMGM without features of PV when the ECMP criteria are applied (Figure 8). Absence of clinical, laboratory and bone marrow features of PV and no transformation into PV has been observed in CALR mutated patients [46-48,51].
The progression of ET disease burden complicated by splenomegaly and secondary myelofibrosis (MF) belong to the natural history of all molecular variants of the JAK2, MPL and CALR mutated MPNs. Life expectance was significantly longer in CALR mutated MF patients but the mean age of CALR mutated MPN was 10 years younger as compared to those with a JAK2V617F or MPL515 mutation. This Observation is a mystification or illusion in the mind of 2008/2016 WHO investigators simple because the CALR MF patients are 10 years younger at time of presentation whereas the overall survival of CALR mutated MF was 23 years as compared to 14.4 years of MF patients with the JAK2V617F or MPL515 mutation MF [46,47,51]. Patients with JAK2V617F mutated ET and PV had a similar high risk of aspirin responsive platelet mediated microvascular and major thrombosis as, which was twice that of thrombocythemia patients with CALR mutation [46,47,51]. The lower incidence of thrombotic complications in CALR mutated ET does indicate that CALR mutated are not constitutively activated but indolent similar as in Ph-positive ET. CALR mutated ET patients lack PV features with normal or low normal hemoglobin, normal LAP score, normal serum EPO levels and normal leukocyte counts ascompared to increased LAP score and decreased serum EPO levels in ECMP/CLMP defined JAK2V617F mutated ET and prodromal PVpatients (Table 3) [34,51].
Discussion
With the advent of the JAK2V617F mutation all latent, masked, early and overt stages of PV will be picked up more than 5 to 10 years earlier by the ECMP/CLMP criteria as compared to the PVSG criteria [34,51]. Heterozygous JAK2V617F mutated ET and hetero/homozygous JAK2V617F 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 8). In a prospective study of 59 JAK2V617F positive ET and 44 JAK2 wild ET cases, Pich et al. [40] described that JAK2V617F mutated ET patients have PV-like morphological bone marrow changes of medium sized to large pleomorphic megakaryocytes similar to our findings in newly diagnosed JAK2V617F mutated ET, prodromal PV patients and PV patients. JAK2V617F 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 JAK2V617F mutated ET (masked PV) at higher JAK2V617F mutation allele burden. Clustered large and giant megakaryocyte with hyperlobulated ‘staghorn’ 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, ferritin levels and LAP score [41,42,51]. 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 MF43. 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. CALR-mutated MF patients were about 10 years 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 but JAK2V617F mutated MF patients whether ASXL1-negative or positive [48].
The awareness of the molecular heterogeneity of the MPNs including JAKV617F, JAK2exonn12, MPL and CALR mutation on top of epigenetic factors reflect the funeral of the WHO defined term primary myelofibrosis (PMF) [49-51]. The 2008/2016 WHO defined Essential Thrombocythemia (ET) is not essential and primary myelofibrosis (PMF) is not primary anymore when the ECMP classification is applied [51]. The PVSG and 2008/2016 WHO defined term ET and PMF [16-18,49,51] can easily be replaced by CALR-, MPL515-and JAK2V617F-mutated thrombocythemia and secondary myelofibrosis (MF) with various degrees of splenomegaly, hypersplenism and myelofibrotic transformation of the bone marrow (Figure 8) [8,9,34,51].
Acknowledgement
We thank the molecular biology laboratory investigators Dr M. Bakkus, University Medical Center Brussels and Dr P.J.M. Valk, Erasmus University Medical Center (EUMC) Rotterdam for screening of MPN patients for the JAK2V617F, MPL and CALR mutations.
Figures
Figure 1: Hannover Bone Marrow Classification of MPD by Georgii199021 versus the 1975 PVSG, the 2008 WHO and the 2013 WHO CMP criteria for the three prefibrotic JAK2 positive ET, PV and ET.MGM (red), versus fibrotic primary myelofibrosis (PMF = PMGM = endstage MPD, black). The WHO-ECMP criteria distinguish two variants of prefibrotic hypercellular ET: JAK2V617F mutated ET.MGM (red) and JAK2/MPL wild type PMGM (blue).
Figure 2: JAK2V617F positive essential thrombocythemia (ET) withlong-term stable ET and ET bone marrow features 1996-2014.
Figure 3: Bone marrow features of JAK2V617F heterozygous positive essential thrombocythemia (ET, left) and JAK2V617F homozygous acute onset polycythemia vera (PV, right). Contribution of Drs Michiels and De Raeve, University Hospital Antwerp, Belgium.
Left: Presence of normal platelets in a peripheral blood smear (a) and normal size and morphology of megakaryocytes in a smear from an aspirated bone marrow (middle left) in a control person.Increase of platelets aggregates with the presence of large platelets in a peripheral blood smear (b) and the presence of large megakaryocytes with hyperlobulated nuclei (inset) in a smear of and aspirated bone marrow (middle right) of a case with ET featured by platelet count of 435×109/L and slight splenomegaly complicated by portal vein thrombosis. Bone marrow histology showing increased cellularity (70%, c left), due to increased erythropoiesis and pronounced increase of large pleomorphic megakaryocytes with hyperlobulated nuclei and no increase of bone marrow reticulin (c right) pathognomonic diagnostic for heterozygous mutated JAK2V617F ET (JAK2V617F mutation load 26%).
Right: Hypercellur bone marrow due to increased megakarypoiesis, erythropoiesis and granulopoiesis in a case with rapid onset trilinear JAK2V617F homozygous positive PV.
Figure 4: Morphology of bone marrow smears in control and polycythemia vera (PV) and bone marrow histology in PV versus idiopathic erythrocytosis. Upper panels. Bone marrow smears of PV with homozygous rapid onset PV show large megakaryocytes and hypercellularity (upper right) as compared to normal sized megakaryocytes and normal cellularity in a control Bmsmear (Upperleft).
Figure 5: Bone marrow histology in PV patients with JAK2exon12 mutations are featured by prominent erythroid hyperplasia meeting the criteria for Idiopathic Erythrocythemia (IE) or classical PV and megakaryocytic hyperplasia of atypical small to medium-sized large megakaryocytes with various degrees of monolobation to hyperlobation and abnormal chromatin distribution [39].
Figure 6: Bone marrow histology in essentialthrombocythemia (ET) consistent with normocellular ET carrying the MPLW515L mutation showing giant megakaryocytes with hyperlobulated stag-horn like nuclei charateristic for ET the MPLW515L/K mutation. Courtesy of Dr Schwarz, University Hospital Prague, Czech Republic.
Figure7: Bone marrow features in newly diagnosed calreticulin (CALR) mutated essential thrombocythemia (ET, left) and early stage myelofibrosis (MF, right).
Left: Clinical case of calcireticulin (CALR) positie ET who presented with normal values for hemogobin, hematocrit and erythrocytes, platelet count of 1832×109/L and slight splenomegaly (16 cm lenght diameter on echogram). Bone marrow histology is hypercellular with relative decrease of erythropoisis, dense cluster of immature megakaryocytes with hypolobulated nuclei consistent, and no increase of reticulin fibrosis consistent with a typical PMGM bone marrow. Contribution of Drs Valster, Potters and Schelfout, BRAVIS Hospital Bergen op Zoom, The Netherlands.
Right: Clinical case of CALR positive myelofibrosis (MF): hemoglobin 11.2 g/dL,hematocrit 0.33, leukocytes 9.2×109/L, platelets 347×109/L, LDH 1393 U/l, and the presence of tear drop erythrocytes, poikolocytosis and polychromasie in a peripheral bloodsmear, and hypercellular bone marrow with relative decrease of erythropoisis, dense cluster of immature megakaryocytes with hypolobulated nuclei consistent, and reticulin fibrosis (RF) grade 2 consistent with bone marrow histology features similar to WHO-defined primary myelofibrosis (PMF), but distinct from JAK2V617F mutated ET and PV, and distinct from MPL515 mutated ET. Contribution of Drs Schot and De Raeve, University Hospital, Brussels, Belgium.
Figure 8: Classification of five distinct ECMP34 and CLMP51 defined clonal myeloproliferative neoplams (MPN) and translational states of JAK2V617F mutated ET, prodromal PV, classical PV, Post ET and post-PV myelofibrosis (MF) and of MPL515 and CALR mutated Thrombocythemia and MF and triple negative MPN.
Tables
Clinical criteria JAK2V617F ET and PV Clinical criteria MPL515 ET Clinical criteria CALR PMGM |
Pathological criteria JAK2V617F PV A1 + B1 is idiopathic erythrocythemia: IE
Pathological criteria MPL515 ET
Pathological criteria CALR PMGM Staging of myelofibrosis: MF in ET, PV and PMGM |
Table 1: European Clinical, Molecular and Pathological (EMCP) criteria3 for diagnosis of myeloproliferative disorders (MPD) and myeloproliferative neoplasms (MPN) JAK2 polycythemia Vera (PV) [2-4], MPL ET [5-7] and CALR primary megakaryocytic granulocytic myeloproliferation (PMGM) [8-10]. Michiels, Georgii, Thiele, Schwarz & Penka [2-10].
Table 2: The Dameshek one cause hypothesis of trilinear PV and Vainchenker’s discovery of heterozygous and homozygous JAK2V617F somatic mutation as the driver cause of trilinear MPN with clinical and pathological manifestations of Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF).
Clinical and molecular (CLM) criteria |
Bone marrow pathology (P) criteria51 |
Prefibrotic ET |
Normocellular ET 51 |
|
Normocellular bone marrow (<60%), Megakaryocytic (M) proliferation of clustered of medium sized to large (pleomorphic) mature megakaryocytes in a normocellular bone marrow (<60%), no proliferation of erythropoiesis and granulopoiesis. Reticuline fibrosis (RF) 0 or 1 |
Prefibrotic prodromal PV |
ET with bone marrow features of PV51 |
Erythrocyte count below 5.8×1012/L males, below 5.6×1012/L females.
|
Increased cellularity (60-80%) due to increased erythrocytic, megakaryocytic (EM) proliferation or trilinearerythrocytic, megakaryocytic, granulocytic (EMG) proliferation. Increase of clustered medium sized to large (pleomorphic) mature megakaryocytes. Increased LAP score |
Prefibrotic hypercellular ET and masked PV |
Masked PV and advanced PV51 |
————————————————- |
Hypercellular ET due to increased Erythrocytic, Megakaryocytic and Granulocytic Myeloproliferation (EMG, masked PV, prefibrotic) or increased Megakaryocytic, Granulocytic (MG, fibrotic) proliferation with relative reduced erythroid precursors. Loose to dense clustering of more pleiomorphic megakaryocytes with hyperploid or clumpsy nuclei |
Table 3: European Clinical, Laboratory, Molecular and Pathobiological (ECMP/CLMP) criteria for diagnosis of JAK2V617F mutated ET, prodromal PV and masked PV [19,34,51]. ECMP criteria designed by Michiels 2005/2006.
ECMP/CLMP criteria |
Bone marrow pathology (P) criteria51 |
|
Major criteria for PV |
PV. Increased cellularity (60-100%) due to increased erythrocytic, megakaryocytic (EM) proliferation or trilinearerythrocytic, megakaryocytic and granulocytic (EMG) proliferation.
Grading of secondary reticulin fibrosis (RF), collagen fibrosis (RCF) and myelofibrosis (MF)51
Prefibrotic: RF-0/1 = MF-0 Early fibrotic: RF-2 = MF-1 Fibrotic: RCF 3 = MF-2 Post-PV MF: RCF 4 = MF-3 |
Table 4: European Clinical, Laboratory, Molecular and Pathological (ECMP/CLMP) criteria for the diagnosis of prodromal, masked and classical JAK2 mutated polycythemia Vera (PV) versus primary or secondary erythrocytoses [19,34,51]. ECMP criteria designed by Michiels 2005/2006.
ECMP/CLMP criteria MPL515 Thrombocythemia |
Bone marrow pathology (P) MPL515 MPN51 |
|
Clinical staging similar as in CALR thrombocythemia based on the degree of anemia, splenomegaly and myelofibrosis |
Megakaryocytic (M) proliferation in a normocellular (<60%) bone marrow featured by large to giant mature megakaryocyte with hyperlobulated, staghorn-like nuclei. No increase of erythropoiesis, and granulopoiesis51 |
Table 5a: European Clinical Laboratory, Molecular and Pathological (ECMP/CLMP) criteria for the diagnosis of normocellular ET carrying one of the MPL515 mutations [34,51]. ECMP criteria designed by Michiels between 2006 and 2014.
ECMP/CLMP criteria CALR thrombocythemia (ET) |
Pathological (P) criteria of CALR MPN51 MF Grading reticulin fibrosis (RF), myelofibrosis (MF) 51 |
Table 5b: European Clinical Laboratory, Molecular and Pathological (ECMP/CLMP) criteria for hypercellular ET associated with primary megakaryocytic, granulocytic myeloproliferation (PMGM) or JAK2 wild type Thrombocythemia caused by calreticulin (CALR) mutations[19,34,51]. ECMP criteria designed by Michiels between 2006 and 2014.
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Citation: Michiels JJ, Devos T, Schot R, Valster F, Potters V, et al. (2019) Change of the 2008/2016 WHO Criteria into European Clinical, Laboratory Molecular and Pathological (ECMP/CLMP) Classification of BRC/ABL Negative Myeloproliferative Neoplasms Caused by JAK2, MPL and CALR Driver Mutations. J Hematol Hemother 4: 006.
Copyright: © 2019 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.