Prospective Evaluation of WHO and European Clinical , Molecular and Pathological ( WHO-ECMP ) Criteria for Myeloproliferative Neoplasms ( MPN ) of Various Molecular Etiology : Characteristics of JAK 2 V 617 F , MPL 515 and CALR Mutated MPN

Volume 1 | Issue 1 Abstract The WHO defined JAK2V617F mutated myeloproliferative neoplasms (MPN) consist of normocellular essential thrombocythemia (ET), ET with features of early PV (prodromal PV), ET with hypercelular megakaryocytic granulocytic myeloproliferation (ET.MGM), and various stages of polycythemia vera (PV) when the WHO and European Clinical and Pathological (WHO-ECMP) criteria are applied. Bone marrow cellularities on histopathology are overlapping in normocellular ET, prodromal PV and PV carrying the JAK2V617F mutation. Increased erythrocyte counts at a cut off level of 5.8x1012/L separates JAK2 mutated ET and prodromal PV from overt PV. JAK2 wild type ET and MF carrying the MPL515 mutation is a distinct MPN entity featured by increase of clustered small and giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normal cellular bone marrow without features of PV. JAK2/MPL wild type ET and MF carrying one of the calreticulin (CALR) mutations is the third distinct entity associated with prefibrotic primary megakaryocytic and granulocytic myeloproliferation (PMGM) characterized by dense clustered, large, and immature dysmorphic megakaryocytes with bulky (bulbous) hyperchromatic nuclei, which are never seen in JAK2V617F mutated ET, prodromal PV, EMGM and PV, and also not in MPL515 mutated ET and MF.


Prospective Evaluation of WHO and European Clinical, Molecular and Pathological (WHO-ECMP) Criteria for Myeloproliferative Neoplasms (MPN) of Various Molecular Etiology: Characteristics of JAK2 V617F , MPL 515 and CALR Mutated MPN
As the PVSG classification provided no strict definitions for early stage essential thrombocythemia (ET) and polycythemia vera (PV), we defined in 1980 the Rotterdam Clinical and Pathological Criteria (RCP) for the diagnosis of ET and PV (Table 1) [1].Michiels & Thiele improved the RCP criteria for ET and PV and defined the European Clinical and Pathologic (ECP http://www.mpn-stichting.nl/doctors_brochure_2004.pdf) for ET, PV and hypercellular ET with features of prefibrotic myelofibrosis (MF) [2][3][4][5][6][7][8].Thiele joined the WHO to define the 2008 WHO classification of the myeloproliferative neoplasms (MPN) ET, PV and primary myelofibrosis (PMF) [9,10].In response to the significant shortcomings of the 2008 WHO classification, we could much better define the WHO and European Clinical Molecular and Pathological (WHO-ECMP) [11][12][13] criteria by the combined use of bone marrow histology, biological laboratory markers and JAK2 V617F mutation screening to distinguish the broad spectrum of JAK2 V617F mutated trilinear MPN entity (Tables 2 and 3) to clearly separate JAK2 mutated MPN from JAK2 wild type MPN carrying one of the MPL or calreticulin (CALR) mutations (Tables 4 and 5) [14,15].The 2008 WHO defined prefibrotic JAK2 V617F mutated myeloproliferative neoplasms (MPN) can be subclassified and staged as normocellular essential thrombocythemia (ET), ET with features of early PV (prodromal PV), ET with hypercelular megakaryocytic granulocytic myeloproliferation (ET.MGM), and various stages of polycythemia vera (PV) when the WHO-ECMP criteria are applied (Tables 2 and 3).In this report we could Journal of Hematology and Blood Disorders distinguish JAK2 V617F positive ET and prodromal PV from JAK2/wild type normocellular ET carrying the MPL 515 as a distinct MPN entity with no features of PV.JAK2/MPL wild type CALR mutated hypercellular ET is associated with prefibrotic primary megakaryocytic granulocytic myeloproliferation (PMGM) and appears to be the third MPN entity of ET and MF without features of PV.

A. The 1980 RCP major (A) and confirmative (B) criteria for prefibrotic ET [1]
Persistent platelet count in excess of 400x10 9 /L.A1 Increase and clustering of enlarged megakaryocytes in bone marrow biopsy.A2 No or slight increase of reticulin fibers (RF 0 or RF 1) A3 Presence of large platelets in a peripheral blood smear B1 Absence of any underlying disease for reactive thrombocytosis and normal ESR.B2 No or slight splenomegaly on palpation or scan (<15 cm) B3 Increase of LAP-score and no signs of fever or inflammation B4

Exclusion criterion
Ph+ chromosome and any other cytogenetic abnormality in blood or bone marrow cells

Normocellular ET ET
Predominant proliferation of enlargedmature megakaryocytes with hyperlobulated nuclei and mature cytoplasm, lacking conspicuous morphological abnormalities.No increase, proliferation or immaturity of granulopoiesis or erythropoiesis.Reticuline fibrosis (RF) 0 or 1 1.Platelet count of >350 x10 9 /l and the presence of large platelets in a blood smear 2. Presence of JAK2-V617 F mutation 3. Normal erythrocytes, haemoglobin (Hb) and hematocrit (ht)

Methods
Hemoglobin, hematocrit, erythrocytes, leukocytes, platelets, iron status and chemical parameters were routinely performed.Erythrocyte volume (red cell mass RCM) was measured using Cr51 (natriumchromate) labeled autologous erythrocyte and plasma volume was measures with J131-human serum albumin.The leukocyte-alkaline-phosphatase (LAP) stain was performed with Nanaphtyl and Fast Garnet G.B.C Salt.Iron stain of bone marrow smears were performed with Prussian blue reagens.Bone marrow biopsies from the iliac crest were stained with hematoxylin and eosin for histopathology evaluation.All bone marrow biopsies were evaluated by expert hematopathologists for morphology, grading of cellularity and scoring of reticulin fibers according to PVSG recommendations [16] (Table 1).Our pathologists used the collagen staining of Mason for objective detection of collagenisation of reticulin fibers to clearly distinguish between early stage reticulin fibrosis (RF) from advanced reticulin/collagen fibrosis for grading of myelofibrosis (MF, Table 1C).

Results
In the period of January 1975 to December 31, 1980, we prospectively studied 30 consecutive early prefibrotic stage MPD patients, who presented with erythromelalgic thrombotic thrombocythemia (ETT), 14 ET and 16 PV patients (Table 6).The mean age of 30 ETT patients (ET and PV) was 56.7 (range 33-96) years.Eleven of 14 ET patients had platelet counts below 1000x10 9 /L.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 /L) 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 clustered large pleomorphmegakaryocytes in bone marrow smears and biopsies was diagnostic for MPD in 14 ET and 16 PV patients (Table 6) (Figures 1 and 2).A normocellular bone marrow picture (cellularity <60%) with 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.A moderate increase of cellularity (1+ = 60-80%) in the bone marrow due to increased erythropoiesis leading to a ET/PV picture was seen in 3 ET and 4 PV patients (Figure 2).A typical PV bone marrow picture with pronounced increase of cellularity (2+ = 80-100%) due to predominantly increased erythropoiesis or trilinear hypercellular hematopoiesis due to erythro/megakaryo/ granulocytic myeloproliferation (EMGM) was seen in 2 of 14 ET patients and in 11 of 16 PV (Figure 2).These results indicate that bone marrow histopathology on its own is not reliable to differentiate between ET and PV but 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%.The morphology of large pleomorphic megakarocyteswere not different in ET and PV.Pleomorphism of megakaryocytes became more pronounced in the hypercellular (80-100%) bone marrows of advanced PV (Figure 2).RCP defined ET and PV patients show overlapping bone marrow pictures with increase of pleomorphic clustered megakaryocytes and varying degrees of increased erythropoisesis at time of diagnosis (Figures 1 and 2).The correlation curves between erythrocyte count, hemoglobin (Hb) or hematocrit (Ht) versus red cell mass (RCM) showed the best correlation between erythrocyte counts and RCM (Figure 3).Increased erythrocyte counts above 5.8x10 12 /L is diagnostic for PV (Table 1B) in the presence of large pleomorphic megakaryocytes in a bone marrow smears and biopsy.Increase of erythrocytes appears to be independent from the iron deficient status and persists in PV in a clinical remission obtained by repeated venesection (Figures 4 and 5).Two ET patients (A1 and A11, Table 6) had borderline increased RCM, which was associated with erythrocytes above 5.8x10 12 /L, increased LAP score, and a negative bone marrow iron stain.In retrospect both cases should be reclassified as PV patients.The ET case (A6) with an erythrocyte count of 6.0x10 12 /L and increased LAP 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.

Results period 1997→2007
Between 1997 and 2007 we prospectively studied 10 JAK2 V617F mutated patients, who presented with erythromelalgia or migrainelike microvascular cerebral ischemic attacks (MIA) and were referred for expert evaluation and treatment recommendation (Table 7).The clinical diagnoses were ET in 6 and PV in 4 without the use of bone marrow histopathology (Table 7A).The 6 ET were heterozygous for the JAK2 V617F mutation.Three PV patients were homozygous for the JAK2 V617F mutation (Case 7, 9 and 10, Table 10 in Table 7).All ET patients had an erythrocyte count below 5.8x10 12 /L and all PV had erythrocyte counts above 6x10 12 /L (Figures 4, 5 and 6) (Table 7A).7 with a typical trilinear PV bone marrow picture complicated by bleeding, poor response to pegylated interferon indicating the need to treat with hydroxyurea on top of low dose aspirin resulting in eliminating the thrombo-hemorrhagic diathesis and significant imporovement of quality of life during long-term follow-up (2006-2013)  The clinical diagnosis of the 10 JAK2 V617F positive patients without the use of bone marrow histology data was ET in 6 and PV in 4 cases (Figures 4, 5 and 6) (Table 7A).
The diagnosis of the 10 JAK2 V617F positive MPN patients based on bone marrow histology picture alone as blindly judged by pathologists and was consistent with ET in 3 and PV in 7 cases (Table 7B).The 3 ET patients diagnosed as PV bone marrow histology evaluation had very low serum EPO levels and EEC, but erythrocyte counts less than 5.8x10 12 /L consistent with the diagnosis of prodromal PV (Table 7B).
The diagnoses according to 2008 WHO criteria were ET in 5, MPN unclassifiable in 1 and PV in 4. The diagnoses according to the WHO-ECMP criteria for diagnosis and staging of ET and PV patients (Tables 2 and 3) were normocellular ET in 2 cases, prodromal PV (ET with low serum EPO, the presence of EEC and normal erythrocyte counts) in 3 cases, ET with a MGM bone marrow histopathology (ET.MGM Table 7B) with no leuko-erythroblastosis in 1 case, and acute onset PV in 4 patients (Cases 7, 8, 9 and 10, Table 7B).Examples of bone marrow histopathology of normocellular ET and prodromal PV are shown in figure 4 and of PV in figure 6 demonstrating that bone marrow histopathology alone cannot distinguish JAK2 V617F mutated ET, prodromal PV and PV.

Results period 2008→2012
JAK2 wild type ET carrying the MPL 515 mutation: In 2008 we studied bone marrow histopathology in 12 cases with JAK2 wild type ET carrying the MPL 515 mutation kindly provided by the courtesy of Dr. Vannucchi, Florence, Italy.Bone marrow histology from a recent patient with JAK2 wild type ET carrying the MPL 515 mutation consistently displayed clusters small and 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 as compared to JAK2 V617F ET with features of PV in the bone marrow (Figure 7).Bone marrow histopathology in MPL 515 mutated ET (Figures 8, 9 and 10) versus WHO-ECMP defined JAK2 V617F mutated ET were different on 4 points.
First, The presence of clustered small and giant megakaryocytes with deeply lobulated stag-horn like nuclei (Figures 7, 8 and  9) in ET carrying the MPL 515 mutation are not seen in JAK2 V617F positive ET, prodromal PV, ET.MGM and PV.The pleomorphic megakaryocytes in JAK2 V617F mutated ET in bone marrow smears and bone marrow biopsy were not larger but similar in size with small, medium and large megakaryocytes (pleomorphic) as in PV (Figure 4 and 6).
Second, there was local increase of erythropoiesis in areas of loose clustered pleiomorphicmegakaryoctyes in JAK2 V617F mutated normocelluar ET and in prodromal PV (Figure 7).Fourth, we observed significant increased reticulin fibrosis grade 2 to 3 in MPL 515 mutated ET in bone marrow areas of normocellularity but with dense clustered megakaryocytes (Figure 10), which is not seen in JAK2 V617F mutated normocellular ET, hypercellular prodromal and PV (Figure 10). .Such increase of RF 2 in a normocellular bone marrow is not seen in JAK2 V617F mutated ET and PV, and also not in JAK2 wild type PMGM(Figures 11 and 12) and CALR-MGM (Figures 13 and 14) Result 2014 → 2015 JAK2-MPL-wild type CALR mutated PMGM: From 1994 to 2006, we documented a case of JAK2 wild type ET with a PMGM bone marrow (Figure 11) in a 9-year-old boy (referred to us from Basel, Switserland) with a platelet count of 1596 to 1946x10 9 /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 a predominant prefibrotic primary megakaryocytic and granulocytic myeloproliferation (PMGM, Table 5), absence of reticulin fibers, loose to dense clustering of large dysmorphic megakaryocytes variable in size with cloud-like hypoploid nuclei.The dysmorphic megakaryocytes show definite abnormalities of maturation with bulky (bulbous) hyperchromatic nuclei and some disturbances of the nuclear cytoplasmic ratio (Figure 11, arrows), which are not seen in JAK2 wild type ET carrying the MPL 515 mutation and also not in prefibrotic JAK2 V617F mutated ET, prodromal PV and PV.Such bone marrow findings of PMGM are consistent with pronounced ET as the presenting feature of prefibrotic PMGM, and do not meet the criteria for fibrotic PMF according to the 2008 WHO classification (Table 5) [15,18].The 10 years follow-up from 1994 to 2004 showed normal blood cells counts, absence of the JAK2 V617F mutation, no evidence of myelofibrosis, and no splenomegaly on palpation (Figure 11).5) (Figure 12) in a 37-years old woman (asymptomatic except fatigue) with JAK2 wild type (anno 2006) hypercellular ET: platelets 1205 x10 9 /L, Hemoglobin 12.5 g/dl, leukocytes 18 x10 9 /L, borderline LDH, spleen size 13 cm on echogram (normal value <12cm) as the presenting features of primary megakaryocytic granulocytic myeloproiferation (PMGM, RF-1, Figure 12).This early fibrotic stage of JAK2 wild type hypercellular ET associated with PMGM developed anemia, significant splenomegaly and myelofibrosis during 10 years of follow-up and appeared to be CALR positive when tested in 2014.
In 2014/2015 we prospectively studied seven consecutive newly diagnosed CALR positive hypercellular ET or MF and found consistent bone marrow characteristics of hypercellular thrombocythemia as the presenting feature of prefibrotic and early fibrotic stages of JAK2 wild type PMGM.Four of these seven CALR mutated MPN patients presented with aspirin sensitive microvascular disturbances of erythromelalgic, cerebral and ocular ischemic manifestations (Sticky Platelet Syndrome) as the specific presenting manifestations of myeloproliferative thrombocythemia.Bone marrow histology in prefibrotic cases of CALR ET (Figure 13 left), and in early fibrotic stage CALR MF (Figure 13 right) 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 MPL 515 mutated ET and also not in JAK2 V617F mutated ET, prodromal PV and classical PV.Recently, we observed a case of aleukemic splenomegaly in a 67 year-old-man, who presented in January 2015 with anemia with normocytic anemia (hemoglobin 5.2 mol/l, hematocrit 0.25 and erythrocytes 3.1x10 12 /L, platelets 265x10 9 /L, leukocytes 6.5x10 9 /L) weight loss from 84 to 71 kg in the last 2 years, minor fatigue, no sweatings and asymptomatic splenomegaly on physical examination (Figure 14).The spleen length on echogram was 24 cm.Morphology of periperal blood smear showed anisoctytosis, trear drop erythrocytes and spherocytes, white blood cell differential counts was normal with the presence of banded (2-4%), blasts (1-2%), myelocytes (2-5%) and erythroblasts (3-4% of WBC).There was a drytap on bone marrow aspiration.Bone marrow biopsy revealed a strongly fibrosed hematopietic stroma with numerous dysmorphic dense clustered dysmorphic megakaryocytes, with remnants of previous erythropietic and myeloid precursor cells and reticulin fibrosis grade 3 to 4 (Figure 14), which could be diagnosed as JAK2 wild type calreticulin (CALR) mutated MF without a history of ET.This CALR mutated MF must have had a long history of subclinical CALR MPN as Michiels & Ten Kate already have demonstated in the 1980s (Table 8) [17].In 1992, we analysed the long-term follow-up of a 61-year-old man, who presented in 1971 with primary myeloid metaplasia of the spleen initially diagnosed as prefibrotic primary myeloproliferative disease (MPD) upon bone marrow histology (Table 8) [17].The spleen size increased from 18 cm to 23 cm length diameter on scan (normal value <12cm) in 1978 and 1988, but the peripheral blood counts remained normal for 14 years of follow-up (Table 8).From 1985 to 1989 he suffered from aspirin responsive erythromelalgia of fingers and toes at platelet counts of 421x10 9 /L.After 17 years of follow-up this case of PMD developed in 1988 anemia, thrombocythemia, and symptoms related to splenomegaly consistent with the diagnosis of agnogenic myeloid metaplasia and advanced myelofibrosis (MF 2/3).5) Right: Clinical case of CALR positive myelofibrosis (MF): hemoglobin 11.2 g/dL,hematocrit 0.33, leukocytes 9.2x10 9 /L, platelets 347x10 9 /L, LDH 1393 U/l, and the presence of tear drop erythrocytes, poikolocytosis and polychromasie in a peripheral blood smear, and hypercellular bone marrow with relative decrease of erythropoisis, dense cluster of immature megakaryocytes with hypolobulated nuclei consistent with PMGM, and reticulin fibrosis (RF) grade 2.The marrow histology features similar to WHO-defined primary myelofibrosis (PMF) and WHO-ECMP-defined PMGM or CALR-MGM, but distinct from JAK2 V617F mutated ET and PV, and distinct from MPL 515 mutated ET Figure 14: A 67 year old man presented in 2015 with normocytic anemia (hemoglobin 5.2 mol/l, hematocrit 0.25 and erythrocytes 3.1x10 12/L, platelets 265x10 9 /L, leukocytes 6.5x10 9 /L) weight loss from 84 to 71 kg in the last 2 years, minor fatigue, no sweatings, asymptomatic splenomegaly and dense clustered dysmorphic megakaryocytes, reticulin fibrosis grade 3 to 4, which was diagnosed as JAK2 wild type calcireticulin (CALR) mutated MF without a history of ET

Discussion
In 1940, Dameshek believed that the following minimal data should be present before a definite diagnosis of PV can be made: plethoric appearance, splenomegaly, definitely elevated erythrocyte count above 6x10 12 /L, elevated platelet count, and elevated hematocrit (Table 1) [18].The bone marrow is diagnostic showing increased trilinear hematopoiesis and large megakaryocytes.ET and PV patients present identical (typical) ET/PV or PV bone marrow histology pictures and the most objective criterion to differentiate ET from PV appeared to be the erythrocyte count at a cutoff level of 5.8x10/ 12 L obviating the need to measure red cell mass (RCM) (Tables 2 and 3) (Figures 3 and 5).We did not observe a significant difference in the morphology of pleomorphic megakaryocytes within the prefibrotic JAK2 V617F mutated MPNs normocellular ET, prodromal PV and PV.In JAK2 V617F positive EMGM (or masked PV), the pleomorphic megakaryocytes show dysmorphic features of nuclei (not cloud-like), which is clearly in between ET and post-ET myelofibrosis (Table 2).There is significant overlap of serum EPO levels in PV versus control and controls versus increased serum EPO in congenital erythrocytoses [11][12][13].A low serum EPO level has been described in about half of PVSG defined ET patients, who are predicted to carry the JAK2 V617F mutation and can be regarded as prodromal (forme frusta) PV [11,12].The diagnostic impact of low serum EPO (ELISA assay) below the normal range (<3.3IU/L) had a sensitivity, specificity and positive predictive value of 86%, 97% respectively for the diagnosis of PV [13].Piche, et al described the bone marrow histopathology findings in 59 JAK2 V617F positive ET and 44 JAK2 wild ET cases [16], which are completely in line with the present study that ET patients with JAK2 V617F mutation indeed have PV-like morphological bone marrow changes of pleomorphic large megakaryocytes.Our findings in WHO-ECMP defined ET, prodromal PV patients and PV patients with increased erythrocytic, megakaryocytic and granulocytic myeloroliferation (JAK2 V617F -EMGM) as well as increased serum LDH levels and spleen size were seen in more pronounced cases JAK2 V617F positive ET in particular at higher JAK2 V617F mutation load [16].Length diameter cm *N, normal ; ↓,decreased; ↑, increased; -, absent; +, present.One arrow slight, two arrows pronounced, and three exceptional.The peripheral blood and bone marrow findings are consistent with agnogenic myeloid metaplasia in case 1.aWhile on treatment with Hydroxyurea Source Michiels and Ten Kate, Amer J Hematol 1992; 39: 131-136 [17] Table 8: Hematological Findings in Cases of Erythromelalgia and Atypical Thrombocythemia* Our bone marrow findings of JAK2 wild type ET patients carrying the MPL 515 mutation displayed small and giant megakaryocytes with a greater number of large deeply lobulated stag-horn nuclei and more dense clustered megakaryocytes in a normocellular bone marrow with no increase of erythropoiesis.JAK2 wild type MPL 515 mutated ET has no clinical, laboratory and bone marrow features of prodromal PV at diagnosis and does not evolve into overt PV during follow-up [19][20][21].JAK2 wild types ET carrying the MPL 515 mutation have normal serum EPO and ferritin levels and no spontaneous endogenous erythroid colonies (EEC).The prevalence of ET or MF patients carrying the MPL 515 mutation may range from 5 to 10% of the JAK2 wild type MPN population.In a large collaborative European study, Jones, et al. [21] investigated a cohort of 176 MPN cases with the MPL 515 mutation: W515L in 110 and W515K in 58.The overall mutation levels were lower (25%) in W515L (N=106) than the level of 37% in cases with W515K (N = 32).Of the 138 cases (ET, N = 99; MF, N = 36), the median W515L mutation levels were significantly lower (21%) in ET than those (46%) in MF.The 29 homozygous MPL515 positive cases had a diagnosis of MF in 15 and ET in 12 patients.

Fine
MF  Thiele, et al.Grading reticulin fibrosis (RF)No reticulin fibers, occasional individual fibers or focal areas with tiny amount of reticulin fiber reticulin fiber network throughout much of section and no course reticulin fibers network with focal collections of thick course reticulin fibers and increase in reticulin with extensive intersections, and presence of collagen fibers and no or minor osteosclerosis+++ RCF = MF 2Marked increase BM dry tap RF 3 Diffuse and dense reticulin with coarse bundles of collagen associated with significant osteosclerosis

Figure 1 :
Figure 1: Presence of large platelets in peripheral (EDTA) blood smear (left top), increase of clustered enlarged megakaryocytes in a normocelluar ET bone marrow with stainable iron (left bottum).Local increase of erythropiesis (bold arrows) in areas of loose clustered pleomorphic megakaryocytes in patients with essential thrombocythemia: ET bone marrow picture [1]

Figure 2 :
Figure 2: ET (left upper), ET/PV (left right), and PV (left bottum) bone marrow features in ET and PV patients.Pleiomorphic megakaryocytes in ET (upper panels) have less hyperlobulated nuclei as compared to PV (left bottum).Dense clustered pleiomorphic megakaryocytes in PV/ RF (right bottum) in advanced PV show dysmorphic nuclei [1]

Figure 3 :
Figure 3: Correlation of erythrocyte counts and erythrocyte volume or red cell mass in 5 ET and 13 PV patients.Two ET patients with borderline increased RCM had erythrocyte counts above 5.8x10 12 /L associated with a typical PV bone marrow histology and should in fact be diagnosed as PV.Erythrocyte count at a cut off level of 5.8x10 12 /L differentiates between ET and PV even in iron deficient state of PV remission after phlebotomy

Figure 4 :Figure 5 :
Figure 4: Case 2 in Table 7 with a 10 to 5 years history of stable JAK2 V617F positive ET disease and ET bone marrow picture in 2006: normocellular ET stage 1 showing a typical ET bone marrow picture with cellularity of 60% due to slight increase of erythropiesis (upper panels).Case 6 in Table7with ET and a PV bone marrow in 1997, who developed PV seven years later in 2004 with a good response to interferon in 2005 (lower panels)

Figure 6 :
Figure 6: Rapid onset trilinear JAK2 V617F homozygous positive PV Case 9 in Table7with a typical trilinear PV bone marrow picture complicated by bleeding, poor response to pegylated interferon indicating the need to treat with hydroxyurea on top of low dose aspirin resulting in eliminating the thrombo-hemorrhagic diathesis and significant imporovement of quality of life during long-term follow-up(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)

Figure 9 :
Figure 9: JAK2 wild type, MPL 515 mutated ET with enlarged and giant mature megakaryocytes with loose clusters of hyperlobulated, "staghorn" hyperlobulated nuclei.Case 4, Dr. Vannucchi, upper panels.Case 5, Dr. Vannucchi, lower panels Third, JAK2 wild type ET carrying the MPL 515 mutation have no clinical, laboratory and bone marrow features of prodromal PV at diagnosis, do not evolve into overt PV during follow-up, and have normal serum EPO and ferritin levels.

Figure 10 :
Figure 10: Case of JAK2 wild type MPL 515 mutated ET with clustered large and giant megakaryocytes and increase of reticulin fibrosis grade 2 (RF 2).Such increase of RF 2 in a normocellular bone marrow is not seen in JAK2 V617F mutated ET and PV, and also not in JAK2 wild type PMGM(Figures 11 and 12) and CALR-MGM (Figures 13 and 14)

Figure 11 :
Figure11: Third MPN entity presenting with JAK2 wild type ET associated with prefibrotic primary megakaryocytic and granulocytic myeloproliferation (PMGM, colour picture), which is characterized by a hypercellular bone marrow due to dual myeloproliferation of granulopoiesis and dense clustered enlarged immature dysmorphic megakaryocytes with bulky (bulbous) hyperchromatic nuclei (arrows), which are never seen in JAK2 wild type MPL 515 mutated ET and also not in the prefibrotic JAK2 V617F mutated ET.During long-term follow-up, reduction of platelet count to normal or near normal by treatment with hydroxyurea in 1994 followed by anagrelide from 1995 to 1998 the bleeding manifestations did not recur.After discontinuation of anagrelide in 1998 the patient remained asymptomatic, the platelet counts were between 600 and 800x10 9 /L, which normalized after 8 years of follow-up.From 2001 to 2005 haemoglobin and hematocrit reached completely normal values Bone marrow histology in CALR mutated JAK2 wild type ET and MF: We prospectively observed in 2004 a case of WHO-ECMP defined PMGM (Table5) (Figure12) in a 37-years old woman (asymptomatic except fatigue) with JAK2 wild type (anno 2006) hypercellular ET: platelets 1205 x10 9 /L, Hemoglobin 12.5 g/dl, leukocytes 18 x10 9 /L, borderline LDH, spleen size 13 cm on echogram (normal value <12cm) as the presenting features of primary megakaryocytic granulocytic myeloproiferation (PMGM, RF-1, Figure12).This early fibrotic stage of JAK2 wild type hypercellular ET associated with PMGM developed anemia, significant splenomegaly and myelofibrosis during 10 years of follow-up and appeared to be CALR positive when tested in 2014.

Figure 13 :
Figure 13: Bone marrow features in newly diagnosed calreticulin (CALR) mutated essential thrombocythemia (ET, left) and early stage myelofibrosis (MF, right)Left: Clinical case of JAK2/MPL negative and calcireticulin (CALR) positive ET who presented with normal vlaues for hemogobin, hematocrit and erythrocytes, platelet count of 1832x10 9 /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 typical PMGM and no increase of reticulin fibrosis (Table5) Right: Clinical case of CALR positive myelofibrosis (MF): hemoglobin 11.2 g/dL,hematocrit 0.33, leukocytes 9.2x10 9 /L, platelets 347x10 9 /L, LDH 1393 U/l, and the presence of tear drop erythrocytes, poikolocytosis and polychromasie in a peripheral blood smear, and hypercellular bone marrow with relative decrease of erythropoisis, dense cluster of immature megakaryocytes with hypolobulated nuclei consistent with PMGM, and reticulin fibrosis (RF) grade 2.The marrow histology features similar to WHO-defined primary myelofibrosis (PMF) and WHO-ECMP-defined PMGM or CALR-MGM, but distinct from JAK2 V617F mutated ET and PV, and distinct from MPL 515 mutated ET /L is mandatory.2. Presence of JAK2-V617F mutation 3. Low serum EPO level and/or increased LAP score 4. Spontaneous EEC.

Table 4 :
2014 WHO Clinical Molecular and Pathological (2014 WHO-CMP) criteria for the diagnosis of normocelular ET carrying one of the MPL 515 mutations

Pathological ECP criteria of CALR MGM Clinical (CM) criteria JAK2 wild type PMGM P1 Primary
megakaryocytic granulocytic myeloproliferation (PMGM) and relative or absolute reduction of erythropoiesis and erythroid precursors.Abnormal dense clustering and increase in atypical medium sized, large to giant immature megakaryocytes containing bulbous (cloud-like) hypolobulated nuclei and definitive maturation defects

Table 7A :
Clinical features of 6 ET, and 4 patients carrying the JAK2 V617F mutation

Table 7B :
Bone marrow features of 10 JAK2 V617F mutated thrombocythemia patients with the clinical diagnosis of ET in 6 and PV in 4 cases