Frequencies, clinical characteristics, and outcome of somatic CALR mutations in JAK2-unmutated essential thrombocythemia

Abstract Introduction: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm (MPN), and is characterized by increased number of mature megakaryocytes (MKs) in the bone marrow and sustained thrombocytosis in the peripheral blood. We have reported that activated B cells are increased in patients with essential thrombocythemia, and can facilitate platelet production mediated by cytokines, such as interleukin-1beta (IL-1β) and interleukin-6 (IL-6) regardless JAK2 V617F mutational status (Thromb Haemost. 2014, 112: 537). Recently, Calreticulin (CALR) mutations were discovered in JAK2/MPL-unmutated essential thrombocythemia (ET) and primary myelofibrosis. Although CALR mutations may be associated with activated JAK-STAT signaling pathway, its exact molecular pathogenesis remains elusive in MPN. Interestingly, in vitro study has shown that CALR is capable of driving B cells activation through the toll-like receptor 4 (TLR4) pathway (J Immunol.2010; 185: 4561). Here we sought to evaluate the association between CALR mutations and B cell immune profiles in ET patients. Methods: Fifty-four patients diagnosed with ET based on the 2008 WHO classification were enrolled into this study. CALR mutations were screened by high-resolution melting analysis and nucleotide sequencing. JAK2 V617F and MPL mutations were screened by allele-specific PCR and nucleotide sequencing, respectively. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) and CALR levels, B cells TLR4 expression and intracellular levels of IL-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF and plasma CALR concentrations were measured by ELISA. Forty-eight healthy adults and 17 patients with reactive thrombocytosis were used for comparison. The association between clinical, laboratory and molecular characteristics were studied. Statistical significance was defined as a two-sided p value <0.05 and SPSS version 22.0 (IBM, New York, USA) was used for all analyses. Results: In this series, 19 (35.2%) patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2 V617F mutations. Compared to JAK2 V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Among all ET patients, CALR mutations correlated with significantly lower serum BAFF level (median 1.6 ng/mL, p =0.049) and higher fraction of B cells with TLR4 expression (median 11.3%, p=0.021). Compared to healthy adults, patients with ET had statistically significant higher serum BAFF concentrations and higher mBAFF levels on both granulocytes and monocytes, and higher fraction of B cells with TLR4 expression and higher fractions of B cells with intracellular IL-1β and IL-6 expression irrespective of their genotypes. ET patients with both JAK2 and CALR mutations had statistically higher number of CD69-positive and CD86-positive activated B cells when compared with healthy adults. Among the three mutational groups of ET patients, there were no significant differences in granulocytes/monocytes mBAFF, in the fraction of B cells with intracellular IL-1β or IL-6 expression, and the numbers of CD80-positive and CD86-positive activated B cells. Granulocyte membrane-bound CALR levels were highest in patients with reactive thrombocytosis. Plasma CALR concentrations were highest in patients with reactive thrombocytosis (mean +/- SE: 9.04 +/- 0.59) and lowest in CALR -mutated ET patients (5.35 +/- 0.90, p <0.001). Conclusions: Activation of B cells is universally present in ET. Both granulocyte membrane-bound CALR levels and plasma CALR concentrations were lower in CALR-mutated ET patients suggesting that CALR may not play a major role in the activation of B cells in these patients. Disclosures No relevant conflicts of interest to declare.

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No increase of JAK2 46/1 haplotype frequency in essential thrombocythemia with CALR mutations: Functional effect of the haplotype limited to allele with JAK2V617F mutation but not CALR mutation

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2015.03.009 ◽

2015 ◽

Vol 55 (1) ◽

pp. 36-39 ◽

Cited By ~ 2

Author(s):

Jyh-Pyng Gau ◽

Chih-Cheng Chen ◽

Yi-Sheng Chou ◽

Chia-Jen Liu ◽

Yuan-Bin Yu ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Haplotype Frequency ◽

Jak2v617f Mutation ◽

Functional Effect ◽

Calr Mutations ◽

Calr Mutation

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Primary Trombocythemia in Children and Adolescents Includes Different Subtypes Compared to Adult Essential Thrombocythemia

Blood ◽

10.1182/blood.v124.21.1865.1865 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1865-1865

Author(s):

Fiorina Giona ◽

Marica Laurino ◽

Luciana Teofili ◽

Sara Capodimonti ◽

Maurizio Martini ◽

...

Keyword(s):

Children And Adolescents ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Correct Diagnosis ◽

Young Patients ◽

Antiplatelet Agents ◽

Wild Type ◽

Primary Thrombocythemia ◽

Calr Mutations

Abstract The recent discovery of various mutations of the CALR gene that are mutually exclusive with JAK2 and MPL mutations has allowed a correct diagnosis in about 90% of adult cases of essential thrombocythemia (ET). Moreover, the mutation status of JAK2 and CARL defines subtypes of ET in adults with a substantially different clinical course and outcome. Based on our experience, we suggested that primary thrombocythemia (PT) in children is characterized by subtypes that differ from those found in adult ET. The present study was carried out in children and adolescents with PT in order to (a) characterize the various subtypes of the disease and (b) analyze their clinical and biologic features, treatment approach and outcome. PT patients aged <20 years (yrs) at diagnosis (dx) were evaluated for mutations of JAK2, thrombopoietin (TPO) and its receptor (MPL) and CALR genes, and for clonal hematopoiesis (females). The presence of MPLS505A (confirmed on DNA from buccal swabs) defined a hereditary thrombocytosis (HT). ET was diagnosed according to WHO 2008 criteria. For wild type patients, an additional inclusion criteria was a follow-up >24 months. Among 58 PT patients (males: 23; females: 35; median age at dx: 14.4 yrs), 21 (36%) had HT due to MPLS505A, 14 were JAK2V617F-mutated (24%), 9 (16%) harbored CALR mutations and 14 (24%) were wild type for JAK2, CALR and MPL (Fig 1). JAK2- and CALR-mutated were older than those with wild type ET or with HT (median age, 17.6 and 16.1 vs 10.4 and 13.7 yrs, p .028). As to the hematologic findings, HT patients showed both hematocrit values (median, 36.3%) and leukocytes counts (median, 9.53 x109/L) significantly lower than ET patients, whatever the subtypes (median, 41.2% and 11.2 x109/L, p .006 and p .029, respectively). No differences were found with regard to platelets both between HT and ET and among the different ET subtypes. JAK2-mutated patients exhibited more frequently symptoms (69%) compared to CALR-mutated (22%), wild-type ET (14%) and HT (14%) patients (p. 0057). Splenomegaly at diagnosis was recorded more frequently in JAK2-mutated than in CALR-mutated or wild type-ET or HT (50%, 33% 21% and 14% , respectively, p .122). Antiplatelet agents, mostly acetylsalicylic acid (ASA), were started less frequently in HT than in ET patients, irrespective of the subtypes (57% vs 81%, p .05). The use of ASA progressively decreased over the time; at the last follow-up, 2 patients with HT, 2 CALR-mutated and 1 JAK2-mutated patients were still receiving ASA, while no wild type ET patient was on treatment. Cytoreductive agents, hydroxyurea and/or interferon and/or anagrelide, were used in a minority of HT patients (19%) in comparison with ET patients (65%), p .001, mainly with those wild-type (78%, p <.001). At the last observation, one HT patient was still receiving cytoreductive agents compared to 30% of ET patients whatever the subtypes (p .024). After a median follow-up of 196 months (similar in the different subtypes), all patients are alive. On the whole, 5 thrombotic events were recorded in 3 patients with HT and in 2 ET patients (1 JAK2-mutated and 1 JAK2 and CALR wild-type), without any significant thrombophilic abnormalities during treatment with ASA and/or cytoreductive agents. A progressive splenomegaly was recorded in 9 (15%) patients (2 HT, 4 JAK2-mutated, 3 CALR-mutated) and it was combined with grade ≥2 medullar fibrosis in 2/4 JAK2-mutated and in 2/3 CALR-mutated patients. None of the JAK2 and CALR wild-type patients had spleen enlargement or reticulin fibrosis (p .022). Two untreated patients (1 HT and JAK2 and CALR wild-type) developed malignancies. On the whole, these data emphasize that in young patients with PT, hereditary forms can be frequently observed. Thrombotic events, recorded mainly in HT patients despite treatment with ASA, were probably due to a MRP4 protein overexpression that was found in our MPLS505A HT. Moreover, our observations highlight that, in contrast to adult ET, more than one third of young ET patients have no JAK2 or CALR mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Essential Thrombocythemia Patients with CALR Mutations Have Increased Platelet Activation Markers Compared to JAK2V617F Mutated Patients

Blood ◽

10.1182/blood.v124.21.3223.3223 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3223-3223

Author(s):

Maya Koren-Michowitz ◽

Hagit Hauschner ◽

Yulia Shuly ◽

Meital Nagar ◽

Elena Ribakovsky ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Absolute Number ◽

Median Number ◽

Healthy Controls ◽

Activation Markers ◽

Increased Risk ◽

Platelet Aggregates ◽

Calr Mutations

Abstract Essential thrombocythemia (ET) is associated with an increased risk for thrombo-hemorrhagic complications. The presence of the JAK2V617F mutation, found in approximately 50% of ET patients, has been associated with increased indices of platelet (PLT) activation suggesting its casual role in thrombus formation. Mutations in CALR were recently described in the majority of JAK2V617F negative ET patients, and are associated with a decreased rate of thrombotic events. This has led us to hypothesize that CALR mutations have a different influence on PLT activation compared to JAK2V617F. To evaluate the PLT activation state, surface expression of two PLT activation markers - p-selectin (CD62P) and PAC1 was studied using specific antibodies. MFI was analyzed by flow cytometry at baseline, as well as following ADP addition to PLT rich plasma. Monocyte-platelet aggregates were studied in whole blood samples by gating CD45+/CD14+ cells and calculating the percentage of CD41+ cells in the monocytes population. The immature PLT fraction (IPF) was analyzed with the XE-5000 hematology analyzer (Sysmex UK Ltd., Milton Keynes, UK), and the absolute number of immature PLT (nIP) was calculated from the total PLT count. Low risk ET patients (N-13, M/F-5/8) and healthy controls (N-10, M/F-4/6) are included in this analysis. JAK2V617F and CALR mutations were present in 8 and 5 patients, respectively; low dose aspirin (range 75-100mg) was taken by 85% of patients and 90% of controls. Median PLT count in CALR mutated, JAK2V617F mutated and healthy subjects was 913, 579 and 247 K/uL, respectively (p=0.0002), and it was higher in CALR compared to JAK2V617F positive patients (p=0.09). Both patient subgroups had a lower baseline MFI of p-selectin and PAC1 compared to healthy controls (p-selectin: 2.8, 3 and 4.5 for JAK2V617F [p=0.01], CALR [p=0.05] and controls; PAC1: 3, 3.3 and 5.2 for JAK2V617F [p=0.01], CALR [p=0.02] and controls, respectively) with no difference between CALR and JAK2V617F mutated patients. CALR compared to JAK2V617F mutated patients had higher median number of immature PLT (30 and 10.6 K/uL, p=0.04), and a higher fraction of monocyte- platelet aggregates (90 and 58%, p=0.05). nIP and monocyte- platelet aggregates were also significantly higher in CALR mutated but not in JAK2V617F mutated patients compared to healthy controls. Interestingly, there was no difference in post ADP PLT activation (post/baseline ratio) between ET patients and healthy controls. Finally, there were correlations between the PLT counts and nIP (R=0.8, p<0.0001), monocyte- platelet aggregates (R=0.5, p=0.02), baseline p-selectin MFI (R=-0.5, p=0.02) and PAC1 MFI (R=-0.5, p=0.01). Our preliminary results suggest a correlation between PLT activation markers and the PLT numbers, which can explain why CALR mutated patients in our cohort had higher nIP and monocyte- platelet aggregates fractions. The absence of an increased ADP induced PLT activation between patients and controls in this cohort compared with previous reports could be explained by the use of aspirin in the majority of patients and the high ADP concentration used for PLT activation. These results will be further studied in a lager cohort of patients. Disclosures No relevant conflicts of interest to declare.

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A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽

10.1182/blood.v126.23.5215.5215 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5215-5215

Author(s):

Munazza Rashid ◽

Rifat Zubair Ahmed ◽

Shariq Ahmed ◽

Muhammad Nadeem ◽

Nuzhat Ahmed ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Diagnostic Algorithm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Common Mutation ◽

Hypochondriac Region ◽

Exon 9 ◽

Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

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Function of Integrin αIIbβ3 in Essential Thrombocythemia with Calreticulin Mutation

Blood ◽

10.1182/blood.v126.23.4079.4079 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4079-4079

Author(s):

Jun Yamanouchi ◽

Takaaki Hato ◽

Etsuko Matsubara ◽

Taichi Azuma ◽

Hiroshi Fujiwara ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Lower Risk ◽

Healthy Subjects ◽

Thrombotic Event ◽

Jak2 V617f ◽

Β1 Integrin ◽

Wild Type ◽

Integrin Αiibβ3 ◽

Calr Mutations ◽

Calr Mutation

Abstract Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia and it has been turned out that CALR mutated patients have a lower risk of thrombosis than JAK2 V617F patients. However, the molecular mechanism for this differential risk remains obscure. It has been reported that CALR is a potential regulatory protein of integrin activation based on the interaction between CALR and a conserved sequence of GFFKR in the integrin α cytoplasmic tails. Recent studies suggest that calreticulin activates β1 integrin and modulates integrin-associated signaling. In this study, we examined if the mutant CALR proteins observed in patients with ET affect integrin αIIbβ3 activation which plays a crucial role on thrombus formation. We first identified mutations of JAK2, MPL, and CALR genes in 37 patients with WHO defined ET and explored clinical characteristics of patients with CALR mutation. The patients with JAK2 V617F were 22 (59%), MPL W515L was 1 (3%), and CALR mutations were 10 (27%). The two types of CALR mutations were found; deletion (52-bp deletion; c.1092_1143del) and insertion (5-bp insertion; c.1154_1155insTTGTC) mutations. The patients with CALR mutations had lower hemoglobin and leukocyte count compared with JAK2 V617F patients, but platelet count did not have a difference between the CALR and JAK2 mutation groups. Nine (41%) of 22 patients with JAK2V617F had a thrombotic event while 1 (10%) of 10 patients with CALR mutation did (p<0.05), suggesting that patients with CALR mutation had a lower risk of thrombosis than JAK2 V617F patients. Two patients with CALR mutations developed myelofibrosis while no patient with JAK2V617F did. One patient with CALR mutation developed acute myeloid leukemia, with persistence of the CALR mutation in his leukemic cells. To see if the CALR mutation affects functional status of αIIbβ3, we examined the binding of PAC1, a monoclonal antibody recognizing the active conformation of αIIbβ3, to platelets from 5 patients with CALR mutation and 12 patients with JAK2V617F in the presence or absence of ADP. Platelets from all the 5 patients with CALR mutation showed the same level of PAC1 binding as platelets from healthy subjects. Overexpression of recombinant CALR proteins in Chinese Hamster Ovary (CHO) cells expressing αIIbβ3 by transfection of a protein-expression vector containing wild-type, deletion, or insertion mutant CALR had no effect on PAC1 binding. We further examined adhesive function of CHO cells stably expressing αIIbβ3 and mutant or wild-type CALR to various concentrations of immobilized fibrinogen. Expression of wild-type or mutant CALR had no effect on αIIbβ3-mediated cell adhesion to fibrinogen. Moreover, each cell adherent to fibrinogen showed apparently the similar extent of spreading. On the other hand, platelets from 4 of 12 patients with JAK2V617F had an increase in PAC1 binding in the presence and absence of ADP compared with platelets from healthy subjects. All 4 (100%) of 4 patients with increased PAC1 binding had a thrombotic event while 4 (30%) of 13 patients with normal PAC1 binding did. Our study suggests no functional activation of integrin αIIbβ3 by CALR mutation, which is contrary to a recent finding that CALR activates β1 integrin. Nonetheless, our finding is rather in line with a clinical finding of a low risk for thrombosis in patients with CALR mutation and may provide the molecular basis for the differential thrombotic risk between the patient with CALR and JAK2 mutations. Disclosures Fujiwara: Celgene: Honoraria, Other: Travel, Acomodations, Expenses.

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