Bone marrow histology in CALR mutated thrombocythemia and myelofi brosis: results from two cross sectional studies in 70 newly diagnosed JAK2/MPL wild type thrombocythemia patients

The clinical phenotypes in 268 JAK2V617F mutated MPN patients in the Seoul study were PV in 101, ET in 95 and MF in 78 and 56 CALR mutated MPN consisted of PV in none, ET in 40 and MF in 16 cases. CALR mutated MPN patients were younger than JAK2V617F mutated MPN patients (mean ages 57.5 and 66 years), had lower values for values for leukocytes (8.6 vs 11.9x109/L) and higher values for platelets (898 vs 643x109/L respectively). Bone marrow histopathology in 268 JAK2V617F mutated MPN patients in the Seoul study was featured by an increased erythropoiesis and megakaryopoiesis (EM) in 13.5%, an increased erythropoiesis, megakaryopoiesis and granulopoiesis (EMG) in 31.3%, a normocellular megakaryocytic (M) proliferation in 29,1%, a megakaryocytic and granulocytic (MG) proliferation with a relative reduction of erythropoiesis in post-ET and Post-PV myelofi brosis in 26.2%. The bone marrow histology in 56 cases of CALR mutated MPN show a predominantly increased megakaryopoiesis (M) in two thirds and an increased megakaryopoiesis and granulopoiesis (MG) with a decreased erythropoiesis in one third.


Introduction
Polycythemia vera (PV) is a trilinear myeloproliferative disorder (MPD) of erythroid (E), megakaryocytic (M) and granulocytic (G) bone marrow proliferation irst described by Dameshek (1950) and subsequently con irmed by the 1975 PVSG, 2001 WHO classi ications of the myeloproliferative disorders (MPD) and the 2008 WHO classi ication of the myeloproliferative neoplasms (MPN) [1][2][3][4][5][6][7]. The minute Phchromosome in chronic myeloid leukemia (CML) originates from a translocation of chromosome 9 and 22 (t9: 22), which results in a BRC/ABL fusion gene on chromosome 22 with high tyrosine kinase activity and CML-transforming capacity [8,9]. Michiels (1987) separated the Ph-chromosome BCR/ABL positive ET and CML from the Phchromosome BRC/ABL negative MPDs PV, ET or megakaryocytic leukemia (ML) without features of PV. The Ph + / BRC/ABL positive ET and CML belong to one malignant disease with a rapidly progressive splenomegaly and myelo ibrosis. The megakaryocytes are smaller than normal with mono-or binucleated nuclei in Ph + / BCR/ABL positive ET and thrombocythemia associated with CML.
The discovery in 2005 of the heterozygous and homozygous JAK2 V617F mutation ( Figure 1) as the driver cause of the trilinear MPNs in ET and PV patients by Constantinescu & Vainchenker [27,28] can easily explain the sequential occurrence of normocellular ET, prodromal PV, classical, masked and advanced PV complicated by secondary MF during lifelong follow-up. The JAK2 V617F mutation load is low in heterozygous mutated ET, intermediate in heterozygous/homozygous prodromal PV and high in homozygous JAK2 V617F mutated hypercellular and advanced PV [16][17][18][19][20][21][22][23][24][25][26]. Increase in large megakaryocytes with mature cytoplasm and multilobulated nuclei in a hypercellular bone marrow are more conspicuously altered in JAK2 V617F mutated PV than in ET and prodromal stage PVm, MPL 515 mutated ET is the second distinct MPN entity. The discovery of the calreticulin (CALR) somatic mutation in JAK2 V617F /MPL 515 wild ET and MF patients by Kralovics in 2013 [29], as the driver cause of ET associated with PMGM without features of PV ( Figure 1, Table 1). CALR mutated hypercellular ET associated with PMGM has been recognized by Michiels & De Raeve in 2014 as the third distinct MPD entity without features of PV [20][21][22][23][24][25][26]. In this cross-sectional Belgian Dutch Korean (Seoul) collaborative study of 70 CALR mutated MPN cases we describe the clinical presentation, laboratory features and bone marrow characteristics in JAK2V617F mutated trilinear MPN and in CALR mutated thrombocythemia and myelo ibrosis without features of PV.

Bone marrow histology methods
Bone marrow biopsies were performed from the iliac crest with an orthograde directed trephine which is a main prerequisite for bone marrow diagnosis of myeloproliferative neoplasms (MPNs) [14,15]. Fixation of the specimens is usually carried out in 4 % formalin. For achievement of optimal quality for enzyme-and or immunochemistry, any acid medium for decalci ication has to be avoided. The next step in the present study consisted of paraf in embedding and employment of Giemsa, hematoxylin and eosin (H&E) staining, and silver impregnation method (Gomori's techniques) [14,15]. De ining the early and advanced stages in each of the MPNs subtypes in routine practice include bone marrow cellularity related to various degrees of increased/decreased erythropoiesis and/or granulopoiesis, and more importantly the diagnostic differences of megakaryocyte number, size, morphology and clustering in the various clonally mutated MPNs ET, PV and PMGM [20][21][22][23][24][25][26]. The reticulin iber content and pattern of iber density in each patient at time of irst diagnosis and in subsequent trephine biopsies is of utmost prognostic importance [9,10,14,15]. Bone marrow ibrosis (myelo ibrosis = MF) is a secondary to the myeloproliferative transformation of bone marrow hematopoiesis in clonal JAK2 V617F mutated ET and PV, MPL 515 mutated ET and CALR ET / PMGM characteristics without features of PV ( Figure  2) [9,30,31]. Myelo ibrosis (MF) is according to Table 2 [14,15]. The blood and bone marrow features have been explicitly described in great detail by the ECP and ECMP classi ications between 2005 and 2017 for the each of the JAK2 V617F mutated ET and PV and MPL 515 and for CALR mutated pre ibrotic thrombocythemia and myelo ibrosis ( Table 2) [16][17][18][19][20][21][22][23][24][25][26]. Each of the MPDs JAK2 V617F , MPL 515 and CALR mutated ET, PV and PMGM as well as Ph + ET and Ph + CML are associated with various degrees of myeloid metaplasia of the spleen and secondary myelo ibrosis [9,30,31].

Laboratory and bone marrow fi ndings in JAK2 versus CALR MPN in the Seoul study
In the Seoul study of 407 MPN a driver mutation JAK2, CALR or MPL was detected in 82.7% with a mutation distribution of JAK2 V617F in 275 (67.5%) and CALR in 55 (13.7%) and MPL in 6 (1.5% [30]). The clinical presentation and bone marrow features of the 6 MPL 515 cases (ET in 3 and MF in 3) has been recently described by Michiels et al. 2018) [31]. The distribution of clinical phenotypes in 268 evaluable JAK2 V617F mutated MPN in the Seoul study were PV in 101, ET in 95 and MF in 78 ( Table 3). The distribution of clinical phenotypes in 56 CALR mutated MPN were PV in none, ET in 40 and MF in 16 cases (Table 3). The mean age of 56 CALR mutated MPN patients (57.5 years) was 8.5 years younger than in JAK2 V617F mutated MPN patients (66 years). JAK2 V617F mutated MPN had signi icantly higher values for leukocytes (11.9 x10 9 /L) compared to CALR MPN (8.6x10 9 /L) and lower values for platelets (643x10 9 /L) compared to CALR MPN (898x10 9 /L) ( Table 3). Major thrombosis was recorded in 23 (7%) JAK2 V617F mutated cases and in none of CALR mutated MPN (    ( Table 3). CALR mutated MPN patients presented with decreased to normal values for hemoglobin, hematocrit and erythrocyte counts did not exceed the upper limit of normal.
The bone marrow lineage proliferation pro ile in 268 evaluable cases of JAK2 V617F mutated MPN featured dual increased proliferation of erythropoiesis and megakaryopoiesis (EM) in 13.5%, trilinear increased proliferation of erythropoiesis, megakaryopoiesis and granulopoiesis (EMG) in 31.3%, monolinear megakaryocytic proliferation (M) consistent with WHO de ine ET in 29,1% and dual megakaryocytic and granulocytic (MG) advanced hypercellular MPN with relative reduction of erythropoiesis mimicking PMGM in 26.2% (Table 3) [30]. The erythropoiesis was normal, relatively decreased or signi icantly decreased in pre ibrotic and ibrotic stages of CALR mutated MPN ( Table 3). The histology in 56 cases of CALR mutated MPN revealed a predominant increase in megakaryopoiesis (M) in a normal cellular bone marrow consistent with ET in two thirds and a dual increase in megakaryopoiesis and granulopoiesis (MG) consistent with PMGM in one third ( Table 3).
The Clinical, Laboratory, Molecular and Pathologic (CLMP) characteristics of 268 evaluable JAK2 V617F mutated patients and 56 CALR mutated patients in the cross sectional Seoul MPN study of Kim et al. 2016 [30], revealed that the frequency of myelo ibrosis grade MF2/3 in 285 JAK2 V617F was associated with an EMGM bone marrow in 22.2% and with a GM bone in 27.1% in 56 CALR-mutated PMGM patients. (Table 3). Myelo ibrosis MF2/3 was present in 46% of advanced JAK2 V617F + GM and in 42% of advanced CALR-GM patients. Myelo ibrosis MF2/3 was much lower in early stage JAK2 V617F -M (17.5%) in JAK2 V617F -EM (10.4%) patients and in early stage CALR-M (17.2%) (P<0.001)). None of the cases with initial stages of JAK2 V617F -M normocellular ET or early stage prodromal PV patients with M or EM histology presented with MF2/3 grade ibrosis in the bone marrow (Table 3). The correlations of bone marrow histology, grading of myelo ibrosis, WHO clinical diagnosis and allele burden in 208 evaluable JAK2 V617F mutated MPN patients of the Seoul study, table 4 shows that the majority of 57 JAK2 M patients have no or minimal ibrosis and allele burden, all 21 JAK2 EM PV patients have minimal ibrosis and high allele burden, 67 JAK2 EMG patients had minimal ibrosis and a high allele burden and about half of 63 JAK2 post ET/PV MF patients had an overt myelo ibrosis at increased allele burden. These data in table 4 indicate that the assessment of the allele burden is an independent and important factor for MPN disease burden, whereas overt myelo ibrosis at the bone marrow level occurred in about 30% of 208 evaluable JAK2 MPN patients, which is consistent with advanced post ET/PV myelo ibrosis in the Seoul study.

Results bone marrow histology of CALR thrombocythemia in the Seoul study
Bone marrow histology of WHO de ined normocellular ET patients with typical pre ibrotic CALR normocellular ET (CALR thrombocythemia) are featured by loose to dense clusters of large megakaryocytes with hypolobulated or hyperlobulated cloudlike nuclei, normal erythropoiesis and no increase in reticulin ibers RF grade 0. Bone marrow histology of four cases of CALR mutated normocellular thrombocythemia (ET) are shown in igures 4 and 5. A ifth case of CALR thrombocythemia diagnosed as ET showed a typical PMGM hypercellular bone marrow due to dual megakaryocytic granulocytic myeloproliferation with the presence of isolated large megakaryocytes, reduced erythropoiesis and reticulin ibrosis RF grade 1 (Figure 6 upper panels). A case of CALR mutated myelo ibrosis (MF) showed a typical PMGM bone marrow featured by dense clusters of dysmorphic large megakaryocytes and cloud-like nuclei, reduced erythropoiesis and increase in reticulin ibers RF grade 2/3 ( Figure 6 lower    Table 4 and large immature megakaryocytes with irregular roundish nuclear forms lobuli becoming clumsy give rise to the co-called cloud-like nuclei in CALR mutated Thrombocythemia in two asymptomatic cases of ET case 11 and 12 (lower panels), Table 4 with persistent increased platelet counts above as the only clinical presentation.

Results of CALR thrombocythemia in the Belgian-Dutch study
Clinical and laboratory indings. Our series of 14 Belgian-Dutch consecutive JAK2 wild type MPN cases were recruited from the Antwerp region Flandria Belgium and South West Netherlands and diagnosed as CALR thrombocythemia in 13 and MPL thrombocythemia in one ( Table 5). The CALR mutated thrombocythemia patients presented with pre ibrotic ET associated with a typical PMGM bone marrow histology (ET/PMGM table 5) in 11 cases (85%). Two MF/PMGM cases (15%) (Case 5 and case 9, Table 4) presented with fatigue only. All 11 ET/PMGM and the 2 MF/PMGM were relative asymptomatic and not suffering from constitutional symptoms (Table 5). Two ET/PMGM cases presented with hemorrhagic manifestation at platelet count above 1000x10 9 /L ( Table 5). Case 2 presented with easy bruising at the age of 24 associated        with documented acquired von Willebrand syndrome. Case 13 presented with subdural hematoma at platelet count of 1148x10 9 /L, which was successfully treated by operative drainage and reduction of platelet count by hydroxyurea. The hemorrhagic   thrombocythemia in these two CALR thrombocythemia patients was not preceded or followed by erythromelalgic microvascular circulation disturbances. Platelet counts in ET/PMGM ranged from 536 to 1306x10 9 /L at time of irst presentation and the two MF/PMGM cases had platelet counts of 265 and 347x10 9 /L respectively. The values for hemoglobin, erythrocytes and white blood cells were in the normal range before and after follow-up in all cases of ET/PMGM (Table 1). Case 5 presented with advanced MF complicated by anemia and splenomegaly.
Bone Marrow Histology. Three cases of CALR mutated thrombocythemia (ET/ PMGM case 3, 6, and 14) showed loose clusters of large megakaryocytes with bulky, cloud-like nuclei with a relative reduction of erythropoiesis and no increase in reticulin ibers grade 0 (RF 0) in a normocellular bone marrow with an abnormally increased megakaryopoiesis (M) indicative for MPN (Figures 8, 9). Two cases of CALR mutated thrombocythemia (ET/PMGM case 11 and 12, Figure 3) showed loose clustered dysmorphic megakaryocytes with the presence of ine reticulin ibers grade 1 (RF 1) in a normocellular M bone marrow in one (case 11) and in a hypercellular MG bone marrow in the other (case 12). Case 8 of CALR mutated ET/PMGM showed moderately dense clusters of medium-sized to large megakaryocytes with partly cloud-like nuclei with an area without reticulin ibers (RF 0) in a normocellular M bone marrow and an area with an increase in reticulin ibers (RF 2) in the hypercellular MG bone marrow area ( Figure 11). Two cases of CALR mutated thrombocythemia (ET/PMGM case 3 after 12 years follow-up and case 9) showed an increase in reticulin ibers grade 2 (RF 2, Figures 12-14) and clusters of large megakaryocytes with dysmature, bulky, cloud-like nuclei in a slightly increased cellular bone marrow in case 9 and in the follow-up hypercellular MG bone marrow in case 3 ( Figure 15). One case of CALR MF (MF/PMGM case 13) showed dense clustered large dysmorphic megakaryocytes with hyperlobulated nuclei in a hypercellular MG bone marrow with increased reticulin ibers (RF 2, Figure 14). The second case of CALR mutated MF (MF/PMGM case 5) presented with asymptomatic splenomegaly, no constitutional symptoms and symptomatic anemia associated with advanced myelo ibrosis (RF 4, MF 3, Figure  16) in a hypocellular bone marrow showing dysmorphic megakaryocytes in ibrotic parts of the bone marrow. The transfusion dependent anemia responded to EPO, which improved the anemia from a Hb 4.2 Hb 5,7 mmol/L without the need of blood transfusion during follow-up.

Discussion
Newly diagnosed JAK2 V617F positive ET and prodromal PV patients usually have low serum EPO, increased LAP score, and slight to moderate increased bone marrow cellularity due to an increased megakaryopoiesis (M) in a normocellular bone marrow or present with increased bone marrow cellularity due to increased erythropoiesis and megakaryopoiesis (EM). Bone marrow hypercellularity due to trilinear increase in erythropoiesis, megakaryopoiesis and granulopoiesis (EMG) is the hallmark of classical PV and in advanced JAK2 V617F mutated ET (masked PV). This is combined with increased serum LDH levels, high scores for leukocyte alkaline phosphatase stain (LAP score), splenomegaly and high JAK2 V617F mutation allele burden between 50% and 100%. Clustered large and giant mature megakaryocyte with hyperlobulated 'staghorn' nuclei -which are rare in JAK2 V617F mutated MPN -typically present in MPL 515 mutated ET patients with no features of PV [30]. The prevalence of MPL 515 mutated ET or MF patients ranges from 5 to 10% of the JAK2 wild type MPN population [30].
The Hannover Bone Marrow classi ication distinguished three MPD disease entities of ET, PV and hypercellular thrombocythemia related to PMGM without features of PV (Table 1) [9,10]. The discovery by Kralovics et al. in 2013 [29], of CALR as the driver cause of JAK2/MPL 515 wild type thrombocythemia and PMF led to the second ground breaking event in the molecular landscape of the MPNs that induced in the minds of and extended experiences by Michiels & De Raeve a complete revision and integration of the PVSG/WHO classi ications into the current Clinical Laboratory, Molecular and Pathobiological (2018 CLMP) [22][23][24][25][26], criteria for JAK2 V617F trilinear MPN and JAK2 exon12 PV on the one hand and two distinctive MPL 515 and CALR thrombocythemias and myelo ibrosis without features of PV on the other hand ( Figure 3) [30][31][32]. CALR mutated thrombocythemia is the third distinct MPN entity featured by bone marrow indings of dysmorphic large megakaryocytes associated with pre ibrotic and ibrotic stages of PMGM during lifelong follow-up. This CALR MPN entity has -in contrast to the JAK2-mutated MPNs -no features of PV at time of diagnosis and during follow-up. The clinical and bone marrow histology features of CALR mutated thrombocythemia patients are phenotypically identical to hypercellular ET associated with PMGM de ined by Georgii in the Hannover Bone Marrow (BM) Classi ication (1990) [9] and do belong to the original description of megakaryocytic leukemia (ML) in 1951 by Dameshek [2].