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1
Elala, Yoseph, Terra L. Lasho, Naseema Gangat, Christy Finke, A. Kamel Abou Hussein, Curtis A. Hanson, Rhett P. Ketterling, Animesh Pardanani, and Ayalew Tefferi. "Driver Mutations and Prognosis in 502 Patients with Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 1599. http://dx.doi.org/10.1182/blood.v126.23.1599.1599.
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Abstract Background : In essential thrombocythemia (ET) , ̴ 85% of patients harbor one of three "driver" mutations, with mutational frequencies of approximately 58%, 23% and 4%, for JAK2, CALR and MPL, respectively; ̴ 15% are wild type for all three mutations and are operationally referred to as "triple negative" (Blood. 2014;124:2507). In one of the original descriptions on CALR mutations, CALR -mutated patients with ET, compared to their JAK2-mutated counterparts, were reported to have better survival (NEJM. 2013;369:2379). However, this observation was not supported by subsequent studies while other reports suggested differential prognostic effect from distinct CALR variants in myelofibrosis (Blood. 2014;124:2465). In this study, we sought to clarify the impact of all three mutations, and CALR variants, on overall (OS), myelofibrosis-free (MFS) and leukemia-free (LFS) survival. Methods: Patientswere selected from our institutional database of myeloproliferative neoplasms, based on availability of mutational status inforomation. ET diagnosis was according to WHO criteria (Blood. 2009;114:937). Published methods were used for CALR, JAK2 and MPL mutation analyses and determination of CALR variants (Blood. 2014;124:2465). Kaplan-Meier survival analysis was considered from the date of diagnosis to date of death or last contact. MFS and LFS calculations considered fibrotic or leukemic transformation events as uncensored variables, respectively. Cox proportional hazard regression model was used for multivariable analysis. Results : A total of 502 patients (median age 59 year; 61% females) met study eligibility criteria. Median levels of hemoglobin, platelet count and leukocyte counts were 13.7 g/dL, 893 x 10 (9)/L and 8.8 x 10(9)/L, respectively. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 324 harbored JAK2, 111 CALR and 13 MPL mutations; 54 patients were triple-negative. The 111 CALR-mutated patients included type 1 (n=55), type 2 (n=41) or other (n=15) CALR variants. At a median follow-up time of 9.9 years, 172 (34.3%) deaths, 42 (8.4%) fibrotic progressions, 15 (3%) blast transformations and 12 (2.4%) polycythemic conversions were documented. In univariate analysis, survival data appeared significantly better in "triple negative" patients (median not reached) and inferior in MPL-mutated cases (median 8.5 years) whereas median survival times were similar for JAK2 (18.5 years) and CALR (22.1 years) mutated cases (Figure 1; p=0.0006). However, the difference in survival was no longer apparent (p=0.60) during multivariable analysis that included age and sex, which are known to differentially cluster with specific driver mutations; in the current study, median age/sex distributions for "triple-negative", CALR, JAK2 and MPL mutated cases were 44 years/72% females, 48 years/46% females, 60 years/65% females, 70 years/46% females, respectively (p=<0.0001/0.0007). Of note, both age and sex were independently predictive of shortened survival. OS data remained unchanged when CALR-mutated patients were further stratified into type 1 vs type 2 vs other CALR variants, with similar survival data between the three CALR mutation groups (p=0.98). In univariate analysis, MPL-mutated patients were significantly more prone to fibrotic progression (Figure 2; p=0.0083). The prognostic relevance of MPL mutations to MFS remained significant when age and sex were included in multivariable analysis (p=0.008). In the current cohort, univariate analysis identified lower hemoglobin and lower platelet count as the only other risk factors for fibrotic progression. Multivariable analysis confirmed the independent prognostic relevance of MPL mutations (p=0.003), lower hemoglobin level (p=0.0009) and lower platelet count (p=0.0094) for MFS. There was no significant difference in LFS among the four driver mutational categories (p=0.9): 9 events in JAK2, 6 in CALR, none in triple negative and none in MPL mutated cases. Among the 6 leukemic transformations in CALR-mutated cases, three were type 1, two type 2, and one other CALR variants. Conclusions : Age- and sex-adjusted survival is similar among ET patients with JAK2 vs CALR vs MPL vs "triple-negative" mutational status. Survival is also similar between patients with distinct CALR variants. MPL -mutated patients with ET might be at a higher risk of fibrotic progression. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.
2
Prejzner, Witold, Andrzej Mital, Maria Bieniaszewska, Aleksandra Leszczyńska, Agata Szymańska, Michał Czarnogórski, and Andrzej Hellmann. "Clinical characteristics of essential thrombocythemia patients depend on the mutation status." Acta Haematologica Polonica 51, no. 4 (December 1, 2020): 230–35. http://dx.doi.org/10.2478/ahp-2020-0040.
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AbstractThe impact of the mutation status on the clinical course and the outcome of essential thrombocythemia (ET) patients has not yet been completely established. A total of 171 patients with diagnosed ET were tested and subsequently grouped, according to their mutation status – Janus Kinase 2 (JAK2) – 112 patients, calreticulin (CALR) – 36 patients, and thrombopoietin receptor (MPL) – 5 patients. Moreover, 18 individuals were triple-negative (with non-mutated JAK2, CALR, and MPL). CALR-mutated patients preferentially were male, with higher platelets (PLT) counts (mean PLT = 1 002.3) and lower hemoglobin and hematocrit levels at the diagnosis, compared to the JAK2 (mean PLT = 933.6), MPL (mean PLT = 940.8) and triple-negative patients (mean PLT = 822.6) (p = 0.0035). The patients with CALR mutated, and the triple-negative ones had a lower risk of arterial and venous thrombosis (3% and 5.6% cases at the time of diagnosis, respectively) than the patients with JAK2 mutation (7.2%) (p = 0.9210). The overall survival rate did not differ statistically between the groups.
3
AL Assaf, Carla, Petros Papadopoulos, Laura Guttierez, Sanne Smits, Carlos Graux, Jan Emmerechts, Els Lierman, Timothy Devos, Lucienne Michaux, and Peter Vandenberghe. "MPL p.S204P Is a Recurrent Mutation in Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 2837. http://dx.doi.org/10.1182/blood.v126.23.2837.2837.
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Abstract Introduction: The JAK2 p.V617F, MPL p.W515K/L and CALR indels occur in a mutually exclusive pattern in 80-90% of cases with Essential Thrombocythemia (ET), but the driver mutations are unknown in the remaining 10-20%. In this study we aimed to identify driver mutations in the latter group of triple negative (TN) ET by exome sequencing of 10 such cases. Results: We found 27 somatic variants, including indels, in 6 out of 10 TN ET patients (range: 1-10 mutations/case; mean: 2,7 mutations/case), none of which were recurrent. In one case, we found a MPL c.610T>C (p.S204P) mutation, which is located in the extracellular domain of the MPLreceptor. By Sanger sequencing of MPL exon 4 in 20 additional TN ET cases, an additional patient with the MPL S204P mutation was identified. Moreover, this mutation was previously reported in one case with idiopathic myelofibrosis1. In order to study the effect of this mutation on the function of MPL, we produced stable Ba/F3 cell lines expressing MPL S204P, MPL W515K or MPL WT, and assessed the dependence of their growth on exogenous thrombopoietin (TPO). Only MPL W515K transduced Ba/F3 cells proliferated in the absence of TPO, but growth of MPL S204P Ba/F3 and of MPL WT Ba/F3 could be rescued by exogenous TPO, indicating the proper surface expression and the functionality of the transduced receptors. The levels of phospho-JAK2 and phospho-STAT5 were low in cytokine-deprived MPL S204P cells but increased upon TPO stimulation. In contrast, phospho-JAK2 and phospho-STAT5 were detectable in MPL W515K transduced Ba/F3 in the absence of cytokines as assessed by Western blotting. Culture of MPL S204P transduced Ba/F3 in the presence of TPO over a range of concentrations (0,01-10 ng/ml) yielded growth curves comparable with MPL WT transduced Ba/F3. Using flow cytometry, we also explored cell surface marker expression on peripheral blood platelets from the two MPL S204P ET patients. Data were compared with healthy donors or ET patients with JAK2 or CALR mutations. MPL S204P ET platelets displayed higher expression of CD61 than platelets from healthy donors or from JAK2 or CALR mutated ET (p<0,01). In addition, there was a trend for higher expression of KIT, CD36 and CD42b on platelets from the MPL S204P ET cases. Moreover, following platelet activation through the protease activated receptor 1, the degranulation response of platelets from MPL S204P ET was decreased in comparison with JAK2 or CALR mutated ET. Conclusion: The MPL S204P mutation is a recurrent mutation in TN ET, with a frequency of 7% (2/30) in this series, but this mutation does not induce TPO-independent growth nor increased TPO-sensitivity in Ba/F3 cells. However, preliminary phenotypic and functional evidence supports the notion that MPL S204P platelets display specific characteristics as compared with JAK2 or CALR mutated ET. The mechanisms by which the MPL S204P mutation influences megakaryopoiesis and platelet function remain to be elucidated. 1. Williams DM, et al. Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis. Exp Hematol 2007; 35: 1641. Disclosures Graux: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.
4
Rotunno, Giada, Carmela Mannarelli, Paola Guglielmelli, Annalisa Pacilli, Alessandro Pancrazzi, Lisa Pieri, Tiziana Fanelli, Alberto Bosi, and Alessandro M. Vannucchi. "Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia." Blood 123, no. 10 (March 6, 2014): 1552–55. http://dx.doi.org/10.1182/blood-2013-11-538983.
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Key Points CALR mutations occur in half of JAK2 and MPL wt patients with ET and associate with some distinctive phenotypic traits. Patients with ET harboring CALR mutations are at significantly lower risk of thrombosis compared with JAK2- and MPL-mutated patients.
5
Cazzola, Mario, and Robert Kralovics. "From Janus kinase 2 to calreticulin: the clinically relevant genomic landscape of myeloproliferative neoplasms." Blood 123, no. 24 (June 12, 2014): 3714–19. http://dx.doi.org/10.1182/blood-2014-03-530865.
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Abstract Our understanding of the genetic basis of myeloproliferative neoplasms began in 2005, when the JAK2 (V617F) mutation was identified in polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2 exon 12 and MPL exon 10 mutations were then detected in subsets of patients, and subclonal driver mutations in other genes were found to be associated with disease progression. Recently, somatic mutations in the gene CALR, encoding calreticulin, have been found in most patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. The JAK-STAT pathway appears to be activated in all myeloproliferative neoplasms, regardless of founding driver mutations. These latter, however, have different effects on clinical course and outcomes. Thus, evaluation of JAK2, MPL, and CALR mutation status is important not only for diagnosis but also for prognostication. These genetic data should now also be considered in designing clinical trials.
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Brown, Lilia, Ciaren Graham, and Ciro Rinaldi. "Calr Is up-Regulated in Patients with Essential Thrombocythemia Independently from Calr and JAK2 Mutations." Blood 124, no. 21 (December 6, 2014): 5581. http://dx.doi.org/10.1182/blood.v124.21.5581.5581.
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Abstract Somatic mutations in exon 9 of calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations, and absent in patient with polycythemia vera (PV). Among patients with ET or PMF with un-mutated JAK2 or MPL, CALR mutations were detected in 67% of those with ET and 88% of those with primary PMF. Several types of insertions or deletions were identified and all resulted in a frameshift in exon 9 generating a novel C-terminal peptide in the mutant CALR protein. Over expression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with myeloproliferative neoplasms carrying CALR mutations present with higher platelet counts and lower haemoglobin levels than patients with mutated JAK2. Studies suggest these patients have a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. We analysed by Real Time PCR, CALR expression in peripheral blood (PB) of 38 patients affected by ET, 17 JAK2 mutated (45%), 4 CALR (10.5%) mutated, 1 MPL mutated (3%) and 14 with no apparent molecular abnormalities. These were compared with a cohort of healthy volunteers. We found a significant over expression of CALR (median 5.15; range 1.13-270.08) comparing with controls (median 0.38, range 0.18-1). CALR mRNA expression is independent from the CALR mutational status. No significant difference was found comparing CALR expression in CALR mutated (median 4.9, range 1.51-37.14) and CALR/JAK2 un-mutated patients (4.68, range 1.51-28.71). CALR up-regulation is not mutually exclusive with JAK2 mutations; no difference was seen in CALR mRNA between JAK2 mutated (median 5.09, range 1.13-270) and wild type ET patients (median 5.08, range 1.51-37). There was no significant difference when we correlated CALR expression with PLT counts, spleen size or type of cytoreductive therapy. A larger cohort of patients is required to confirm these preliminary findings. Disclosures No relevant conflicts of interest to declare.
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Rashid, Munazza, Rifat Zubair Ahmed, Shariq Ahmed, Muhammad Nadeem, Nuzhat Ahmed, and Tahir Shamsi. "A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 5215. http://dx.doi.org/10.1182/blood.v126.23.5215.5215.
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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.
8
Tefferi, Ayalew, Terra L. Lasho, Paola Guglielmelli, Christy M. Finke, Giada Rotunno, Yoseph Elala, Annalisa Pacilli, et al. "Targeted deep sequencing in polycythemia vera and essential thrombocythemia." Blood Advances 1, no. 1 (November 22, 2016): 21–30. http://dx.doi.org/10.1182/bloodadvances.2016000216.
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Key Points More than half of patients with PV or ET harbor DNA mutations/variants other than JAK2/CALR/MPL. The presence of some of these mutations adversely affects overall, leukemia-free, or myelofibrosis-free survival.
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Yamanouchi, Jun, Takaaki Hato, Etsuko Matsubara, Taichi Azuma, Hiroshi Fujiwara, Yoshihiro Yakushijin, and Masaki Yasukawa. "Function of Integrin αIIbβ3 in Essential Thrombocythemia with Calreticulin Mutation." Blood 126, no. 23 (December 3, 2015): 4079. http://dx.doi.org/10.1182/blood.v126.23.4079.4079.
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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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Yamanouchi, Jun, Takaaki Hato, Etsuko Matsubara, Taichi Azuma, Hideyuki Nakanishi, Hiroshi Fujiwara, Yoshihiro Yakushijin, and Masaki Yasukawa. "Activation Status of Integrin αIIbβ3 in Essential Thrombocythemia with Calreticulin Mutation." Blood 124, no. 21 (December 6, 2014): 4571. http://dx.doi.org/10.1182/blood.v124.21.4571.4571.
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Abstract Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) lacking the JAK2V617F and MPLW515 mutations. 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 the effect of CALR mutations on integrin αIIbβ3 activation. 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 activation 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 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. 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 activation of integrin αIIbβ3 by CALR mutation, which may explain a clinical finding of a low risk for thrombosis in patients with CALR mutation. Disclosures No relevant conflicts of interest to declare.
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Benevolo, Giulia, Ludovica Riera, Barbara Nicolino, Andrea Evangelista, Eloise Beggiato, Chiara Aguzzi, Maura Nicolosi, Chiara Frairia, and Umberto Vitolo. "Prognostic Value of JAK2V617F, Calr and MPL Mutational Status on Outcome and Thrombotic Risk in a Retrospective Cohort of 138 Patients with Essential Thrombocythemia." Blood 124, no. 21 (December 6, 2014): 5553. http://dx.doi.org/10.1182/blood.v124.21.5553.5553.
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Abstract Background: Life expectancy in Essential Thrombocythemia (ET) patients is superimposed to normal population. The main causes of death are thrombotic events and evolution into myelofibrotic phase or secondary myelodisplasia/acute leukemia. Approximately 50% to 65% of patients with ET carry activating mutations in the Janus kinase 2 gene (JAK2), and an additional 5% in the thrombopoietin receptor gene (MPL) whereas 10 to 20% of patients have mutated calreticulin gene (CALR). Non-mutated JAK2, CALR and MPL ET (triple negative-TN) are about 10%. Patients and methods : In this study we analysed the prognostic value of JAK2V617F, CALR and MPL mutational status on outcome and thrombotic risk in a retrospective cohort of 138 ET patients defined according to WHO criteria, diagnosed from 1974 to 2013 in a single Italian centre (Turin). JAK2V617F mutation was assessed by Quantitative Real–Time PCR on DNA from peripheral blood or bone marrow samples. JAK2 negative cases were then analyzed for CALR mutations by Gene Scan Analysis in combination with direct sequencing or MPL W515L/K by Allelic Discrimination Real-Time PCR assay. Overall survival (OS), cumulative incidence of myelofibrotic transformations (CI-MT) and thrombosis (CI-TB) were calculated from the date of diagnosis. The between-group comparison for OS was performed with the log-rank test, whereas for CI-MT and CI-TB we using the Gray’s test considering death as competing event and adjusting for presence of cardiovascular risk factors . Results: Among 138 ET patients, 103 (74.6%) carried the JAK2V617F mutation, 3 (2.2%) carried activating mutations of MPL exon 10, 16 (11.6%) carried mutations of CALR exon 9, and only 16 patients (11.6%) had none of these markers (TN). An high incidence of elevated haemoglobin levels and/or haematocrit (males >16.5 g/dl or 49% and females >16.0 g/dl or 48%) was significantly associated with JAK2 positivity [13 pts JAK2+ (14.77%) vs 0 pts JAK2- (Fisher test p=0.019)]. Similarly, the CI-TB, analysed with a competing-risk approach, was higher in patients with a JAK2 mutation [(5-year CI-TB: 23.7% JAK2, 0% CALR, 0% MPL, 12.5% TN; P<0.001)]. With a median follow-up of 48 months (IQR: 27-78; range 1-269), we observed 4 (3%) deaths, 9 (7%) myelofibrotic transformations and none leukemic evolution. The overall survival at 5 years was 98.4% (95%CI: 89.1-99.8) and the cumulative incidence of myelofibrotic transformations was 2.1% (95%IC: 1.0-8.6). The CI-MT was significantly higher in MPL-positive patients [5-year CI-MT: 0% JAK2, 0% CALR, 33.3% MPL, 8.3% TN; (P=0.006)]. Not significant difference on OS was found [5-year OS: 100% JAK2, 100% CALR, 100% MPL, 85.7% TN; (P=0.66)]. We did not include leukemia transformation, because of lack of event. Conclusion: In our retrospective cohort of ET patients, we found a significant correlation between JAK2 positivity and high risk of thrombosis. We also found a significant incidence of elevated haemoglobin levels and/or haematocrit in JAK2-positive patients. These data support the correlation between JAK2 positivity and cumulative risk of transformation to Polycythemia Vera. The correlation between MPL mutational status and evolution to myelofibrosis found in our study needs to be confirmed in a larger series of patients. Disclosures No relevant conflicts of interest to declare.
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Poluben, L., L. Neumerzhytska, S. Klymenko, P. Fraenkel, C. Balk, and O. Shumeiko. "MOLECULAR GENETIC ABNORMALITIES IN THE GENOME OF PATIENTS WITH Ph-NEGATIVE MYELOPROLIFERATIVE NEOPLASIA AFFECTED BY IONIZING RADIATION AS A RESULT OF THE CHORNOBYL NUCLEAR ACCIDENT." Проблеми радіаційної медицини та радіобіології = Problems of Radiation Medicine and Radiobiology 25 (2020): 362–73. http://dx.doi.org/10.33145/2304-8336-2020-25-362-373.
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Objective. to determine the frequency of major somatic mutations in the JAK2, MPL and CALR genes in the genome of patients with Ph-negative myeloproliferative neoplasms that occur in individuals who have been exposed to ionizing radiation as a result of the Chornobyl accident. Materials and methods. Molecular genetic analysis of genomic DNA samples isolated from blood was performed in 90 patients with Ph-negative myeloproliferative neoplasia (MPN) with a history of radiation exposure and 191 patients with spontaneous MPN utilizing allele-specific polymerase chain reaction (PCR). Results. The presence of major mutations in the genes JAK2, CALR and MPL was revealed in patients with MPN with a history of radiation exposure with a frequency 58.9 % (53 of 90), 12.2 % (11 of 90), and 0 % respectively, and without exposure with frequency 75.4 % (144 of 191), 3.1 % (6 out of 191) and 1.6 % (3 out of 191) respectively. Mutations JAK2 V617F in patients with spontaneous MPN were observed in each clinical form: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations were detected exclusively in patients with PMF and ET, significantly more often in groups with a radiation exposure history (18.9 % and 33.3 %, vs. 4.2 % and 6.5 %) than without one. At the same time, the occurence of MPL mutations was determined only in patients with spontaneous MPN in 1.6 % of casees. Triple negative mutation status of genes JAK2, MPL and CALR prevailed in the group of patients with MPN with a history of radiation exposure and was 27.8 %, against 16.2 % in patients without radiation exposure (p = 0.05). Conclusions. Genomic research of patients with Ph-negative MPN revealed features of molecular genetic damage in those patients who were exposed to IR as a result of the Chornobyl accident and those with spontaneous MPN. The data obtained by determining of JAK2, MPL and CALR genes mutational status in the genome of patients with MPN is necessary to expand the understanding of the mechanism of leukogenesis, especially caused by radiation. Key words: myeloproliferative neoplasia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, JAK2 V617F, MPL and CALR, ionizing radiation.
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Tefferi, Ayalew, Terra L. Lasho, Christy Finke, Yoseph Elala, Daniela Barraco, Curtis A. Hanson, Rhett P. Ketterling, Animesh Pardanani, and Naseema Gangat. "Targeted Next-Generation Sequencing in Polycythemia Vera and Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 354. http://dx.doi.org/10.1182/blood.v126.23.354.354.
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Abstract Background : Polycythemia Vera (PV) is associated with JAK2 mutations. In essential thrombocythemia (ET), ̴ 85% of patients harbor one of 3 "driver" mutations, with frequencies of ̴ 58%, 23% and 4%, for JAK2, CALR and MPL, respectively; ̴ 15% are wild type for all three mutations and are referred to as "triple negative". We applied next-generation sequencing (NGS) with a 27-gene panel of myeloid malignancy-relevant genes, in order to describe the prevalence of "non-driver" mutations and their prognostic relevance in PV and ET. Methods: Targeted capture assays were carried out on bone marrow or whole blood DNA for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1.Paired-end indexed libraries were prepared using the NEBNext Ultra Library prep protocol (NEB, Ipswich, MA /Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter® software (PerkinElmer, Danvers, Massachusetts) was utilized to analyze targeted sequence data. Nucleotide variants were called using the Genome Analysis Toolkit (GATK -Broad Institute, Cambridge, MA). Specific variants were deemed as mutations if they are associated with a hematologic malignancy (COSMIC database), or if they have not been associated with a dbSNP. Results: 314 patients with PV (n =133; median age 64 years, 53% females) or ET (n =181; median age 58 years, 59% females) were evaluated. Median follow-up was 9.8 years for PV and 9.2 years for ET. During this time 59 (44%) deaths, 15 (11%) fibrotic and 5 (4%) leukemic transformations were documented for PV and the corresponding percentages for ET were approximately 33%, 8% and 2%. Driver mutation distribution was 98% JAK2 for PV and 52% JAK2, 24% CALR, 20% triple-negative and 4% MPL for ET. Polycythemia Vera Mutations other than JAK2, CALR or MPL were seen in 58 (44%) patients; 29% harbored one, 14% two and one 3 mutations. None of 3 JAK2 -unmutated cases expressed non-driver mutations. Mutational frequencies were 18% TET2, 11% ASXL1, 5% SH2B3, 3% SF3B1 and ≤2% SETBP1, IDH2, DNMT3A, CEBPA, CSF3R and SUZ12, SRSF2, ZRSR2, TP53, CBL, NRAS, RUNX1, KIT, PTPN11 and FLT3. "Number of mutations" significantly affected overall (OS; Figure 1) and myelofibrosis-free (MFS) survival; respective HR (95% CI) were 2.6 (1.3-5.2) and 1.7 (0.97-3.1) and 13.7 (2.9-63.4) and 5.1 (1.5-17.6) for ≥2 mutations and one mutation, respectively. OS was also adversely affected by SRSF2 (p=0.006) and RUNX1 (p=0.04) and borderline affected by TET2 (p=0.06), IDH2 (p=0.08), ASXL1 (p=0.19) and SF3B1 (p=0.19) mutations. In multivariable analysis, SRSF2 and RUNX1 retained significance whereas the others remained borderline significant. In both univariate and multivariable analyses, leukemia-free survival (LFS) was adversely affected by IDH2 and RUNX1 mutations. In univariate analysis, ASXL1, IDH2, RUNX1 and KIT mutations predicted fibrotic progression, whereas SETBP1 was of borderline significance (p=0.07); all, including SETBP1, were significant during multivariable analysis. Essential thrombocythemia Mutations other than JAK2, CALR or MPL were seen in 83 (46%) patients; 35% harbored one, 7% two and 4% three mutations; prevalence in JAK2, CALR, MPL mutated and triple-negative cases was 52%, 43%, 43% and 35%, respectively (p=0.52). Mutational frequencies were: 13% TET2, 11% ASXL1, 6% DNMT3A, 5% SF3B1, 4% CEBPA, 2% TP53, SH2B3, EZH2 and CSF3R and <2% for SETBP1, IDH2, SRSF2, ZRSR2, CBL, NRAS, RUNX1, U2AF1, KIT, PTPN11 and FLT3. "Number of mutations" significantly affected OS (Figure 2) but not MFS or LFS; HR (95% CI) for OS were 6.6 (2.5-17.8) for 3 mutations and 2.2 (1.3-3.9) for one or two mutations. In univariate analysis, survival was adversely affected by CBL, EZH2, SF3B1, SRSF2 and IDH2 mutations. In multivariable analysis, EZH2 and SF3B1 remained significant. Univariate analysis identified SETBP1 and SF3B1 mutations as risk factors for fibrotic progression and EZH2, TP53 and CSF3R mutations for leukemic transformation. Conclusions: "Non-driver" mutations occur in more than 40% of patients with PV or ET; the number of such mutations and presence of certain specific ones likely predict OS, MFS or LFS. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.
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Sekiya, Yuko, Yusuke Okuno, Hideki Muramatsu, Olfat Ismael, Nozomu Kawashima, Atsushi Narita, Xinan Wang, et al. "JAK2, MPL, and CALR mutations in children with essential thrombocythemia." International Journal of Hematology 104, no. 2 (May 21, 2016): 266–67. http://dx.doi.org/10.1007/s12185-016-2022-2.
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Yamamoto, Yukiya, Sachiko Iba, Akihiro Abe, and Nobuhiko Emi. "Elongation of MPL Transmembrane Domain Is a Novel Activating-Mutation in Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 1628. http://dx.doi.org/10.1182/blood.v126.23.1628.1628.
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Abstract Essential thrombocythemia (ET) is a clonal disease of hematopoietic stem cell. Somatic gene mutations including JAK2 or CALR are hallmark of diagnosis and molecular targets for developing novel therapies. However, non-mutated ET cases within JAK2 and CALR still exist. The third mutated gene of ET is MPL, which is known as encoding thrombopoietin receptor. MPL W515 and S505 are hot spot for missense mutations leading to constitutive active MPL signaling. These missense mutations are located within transmembrane or juxtamembrane domain of MPL. Here, we present a novel activating-mutation of MPL in an ET patient. The patient was a 54-year-old woman with occasional headache. A full blood count showed a hemoglobin level of 12.2 g/dL, a white blood cell count of 11.2 x 103/mL and a platelet count of 164 x 104/mL. A bone marrow sample was hypercellular, containing increased megakaryocytes. Chromosomal analysis showed normal karyotype. Further genetic analysis did not detect JAK2 V617F or CALR mutation. Finally, we directly sequenced MPL exon 10. The result showed MPL c.1496-1497AT>TGGGCCTCAGCTGGGCG (Figure 1). This mutation has been considered as an in-frame mutation, indicating MPL p.H499LGLSWA (reference: NP_005364). The amino acids insertion in transmembrane domain of MPL belongs to hydrophobic family, suggesting that MPL H499 mutation (H499ins) might construct stable structure. To investigate whether MPL H499ins is functionally active, we established stable BaF3/MPL H499ins cell lines. In contrast to BaF3/MPL wild-type, BaF3/MPL H499ins cells proliferate without WEHI3-conditioned medium as well as BaF3/MPL W515L or S505N. Western blot analysis showed BaF3/MPL H499ins cells constitutionally activate downstream signaling including JAK-STAT, MAPK and AKT. Furthermore, we established stable BaF3 cell lines with MPL H499 LGLSWALGLSWA (H499 insx2), MPL H499del and others. In contrast to BaF3/H499del, H499L, H499LG and H499insx3, BaF3/MPL H499insx2 cells proliferate without WEHI3-conditioned medium. This result suggests that elongation of MPL transmembrane domain is a novel oncogenic mechanism leading to constitutive active MPL signaling. Phosphorylation of MPL Y626 has significant role to transduce MPL signaling. To explore if constitutive activation of MPL H499ins depends on phosphorylation of MPL Y626, we established stable BaF3 cell lines with MPL H499insY626F. The BaF3 cells could not proliferate without WEHI3-conditioned medium. This result clearly shows phosphorylation of MPL Y626 has a pivotal role for constitutive activation of MPL H499ins. Finally, we examined potential effect to inhibit constitutive MPL signaling with JAK1/2 inhibitor, Ruxolitinib. In contrast to K562, growth of BaF3/MPL H499ins cells were inhibited with Ruxolitinib at half maximal effective concentration 113 nM as well as MPL W515L or S505N (Figure 2). In summary, elongation of MPL transmembrane domain is a novel oncogenic mechanism in ET patients. Ruxolitinib is also a potent inhibitor against MPL activating mutations as well as JAK2 V617F-associated myeloproliferative neoplasm. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
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Giona, Fiorina, Marica Laurino, Luciana Teofili, Sara Capodimonti, Maurizio Martini, Giovanna Palumbo, Maria Luisa Moleti, et al. "Primary Trombocythemia in Children and Adolescents Includes Different Subtypes Compared to Adult Essential Thrombocythemia." Blood 124, no. 21 (December 6, 2014): 1865. http://dx.doi.org/10.1182/blood.v124.21.1865.1865.
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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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Lim, Ken-Hong, Huan-Chau Lin, Yi-Hao Chiang, Wei-Ting Wang, Ching-Sung Lin, Yu-Cheng Chang, Nai-Wen Su, et al. "Calr Mutations in Essential Thrombocythemia: Frequency, Clinical Correlation and Screening By High-Resolution Melting Analysis." Blood 124, no. 21 (December 6, 2014): 1838. http://dx.doi.org/10.1182/blood.v124.21.1838.1838.
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Abstract Introduction: Essential thrombocythemia (ET) is a classic BCL-ABL1-negative chronic myeloproliferative neoplasm (MPN), and is associated with an increased risk of hemorrhagic and thrombotic complications and leukemic transformation. Recently, two research groups discovered a high frequency of somatic calreticulin (CALR) mutations in patients with JAK2 V617F/MPL-unmutated ET and primary myelofibrosis. The pattern of most CALR mutations in MPNs is indels in exon 9 causing one base pair reading frameshift. CALR mutations have been found to have important diagnostic and prognostic significances in patients with ET. High-resolution melting analysis (HRMA) is a well-established method for the detection of or screening for mutations. The aims of this study were to screen for CALR mutations and to determine its clinical correlation in a cohort of adult Taiwanese patients with ET, and to test for the feasibility of HRMA as a screening method for the detection of CALR exon 9 mutations. Methods: The screening for mutations in patients with hematologic neoplasms was approved by the Institutional Review Board of Mackay Memorial Hospital. 92 adult Taiwanese patients with ET were enrolled. Diagnosis of ET was established based on the 2008 WHO criteria. The clinical and laboratory characteristics of all patients at the time of diagnosis or referral were determined retrospectively by chart review. Genomic DNA derived from bone marrow, peripheral whole blood, and peripheral blood granulocytes or peripheral blood mononuclear cells were used for the screening. JAK2 V617F mutational status was screened by allele-specific polymerase chain reaction. CALR exon 9 mutations and MPL exon 10 mutations were screened by nucleotide sequencing on an ABI 3730 sequencer. The correlation between CALR mutational status and clinical characteristics was determined by using SPSS Statistics software (IBM, New York, USA). HRMA was performed with a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). Results: In this cohort of ET patients, 59 (64%) patients harbored the JAK2 V617F mutation and one (1%) patient harbored the MPL W515K mutation. By Sanger sequencing, 17 (18% overall and 50% in JAK2/MPL-unmutated cases) patients harbored 6 types of CALR exon 9 mutations: 5 type 1 (p.L367fs*46), 6 type 2 (p.K385fs*47), 2 type 3 (p.L367fs*48), 2 type 34 (p.K385fs*47), and 2 novel types (p.L367fs*43 and p.E369fs*50). One patient with JAK2 V617F mutation was found to harbor a single nucleotide polymorphism in CALR exon 9 (c.1142 A>C, rs143880510). One patient with JAK2 V617F mutation also harbored a CALR type 3 mutation (p.L367fs*48), and was excluded from the correlation study. When compared with JAK2 V617F mutated ET patients, the presence of CALR mutations correlated with higher platelet count (median 1379 x10^9/L vs. 880 x10^9/L; P<0.001), lower hemoglobin level (median 12.5 g/dL vs. 13.6 g/dL; P=0.032), and younger age at diagnosis (median 44.5-year-old vs. 55-year-old; P=0.025). There was no significant difference between CALR mutated and JAK2 V617F mutated ET patients with regard to the frequency of thrombotic and hemorrhagic complications. The HRM curves of these CALR mutated patients could be distinguished from wild-type curves (Figure 1). Interestingly, the HRM curves of another 5 patients were also suspected for the presence of CALR exon 9 type 2 mutations but their results of CALR exon 9 Sanger sequencing were all wild-type. These 5 patients were suspected to have low CALR mutant allele burden. The sensitivity of HRMA to detect CALR exon 9 type 2 mutations was evaluated by serial dilution of a sample with approximately 40% mutant allele burden. CALR type 2 mutant could be detected in up to 5% dilution (Figure 2). Conclusions: The frequency of CALR mutations and its phenotypic correlation in adult Taiwanese patients with ET is comparable with Western population. HRMA may be used as a rapid and relatively sensitive method for the screening of CALR mutations in patients with suspected MPN. Figure 1. Representative melting curves of six CALR exon 9 mutations, one single nucleotide polymorphism (SNP) and wild-type DNA. Figure 1. Representative melting curves of six CALR exon 9 mutations, one single nucleotide polymorphism (SNP) and wild-type DNA. Figure 2. HRM curves of serial dilution of a CALR exon 9 type 2 mutant in a background of wild-type DNA. Figure 2. HRM curves of serial dilution of a CALR exon 9 type 2 mutant in a background of wild-type DNA. Disclosures No relevant conflicts of interest to declare.
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AL Assaf, Carla, Els Lierman, Florence Van Obbergh, Timothy Devos, Johan Billiet, Carlos Graux, Lucienne Michaux, Petros Papadopoulos, and Peter Vandenberghe. "Analysis of Genotype, Phenotype and Outcome in a Belgian Cohort of Essential Thrombocythemia." Blood 124, no. 21 (December 6, 2014): 5584. http://dx.doi.org/10.1182/blood.v124.21.5584.5584.
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Abstract Bleeding and thrombotic events are major clinical complications of ET, together with progression to myelofibrosis and leukemic transformation (AML). The JAK2 V617F mutation, the thrombopoietin receptor mutation MPL W515K/L and the most recently discovered calreticulin (CALR) mutations are mutually exclusive in ET, account for up to 80-90 % of ET cases and support a novel molecular categorization of ET. In a retrospective study, we have examined the clinical phenotype and outcome of a Belgian cohort of 165 ET patients in relation to their mutational status. 38% of the patients were CALR mutated, 22% and 12% of whom carried Type 1 (p.L367fs*46) and Type 2 (p.K385fs*47) indels respectively. 35% were JAK2 V617F positive, 7% were MPL W515K/L positive, one patient was positive for both CALR and JAK2 V617F mutations, and 20% were triple negative (Fig. 1). We compared the hematological and clinical features between CALR mutant patients and JAK2 V617F positive patients. This revealed that CALR mutant ET is associated with younger age than JAK2 V617F positive ET (median age 56 y (range 23-84) versus 65 y (range 36-94), p<0.001), male gender (58% versus 39%, p=0.03), higher platelet count (988 ± 367*109/L versus 870 ± 291*109/L, p=0.04 (mean ± SD)), lower leukocyte count (9.1 ± 3.2*109/L versus 11.6 ± 5.8*109/L, p<0.001), lower erythrocyte count (4.36 ± 0.9*1012/L versus 4.98 ± 0.65*1012/L, p<0.001), hemoglobin (13.2 ± 1.8 g/dL versus 14.3 ±1.6 g/dL, p=0.001) and hematocrit (40 ± 6.2 % versus 44 ± 4.6 %, p<0.001). Analysis of the CALR mutant group according to the indel type showed that CALR Type 1 deletion is strongly associated with male gender (62%). Contrary to previously published findings, we did not find significant differences between CALR type I and II mutations with regard to age and platelet count.CALR mutant patients had a better overall survival than JAK2 V617F positive patients (mean survival 28 y versus 16 y, p=0.01). However, the better overall survival for CALR mutant ET was restricted to patients less than 60 years old (mean survival 29.2 y versus 18.5 y, p=0.02) while in the age group above 60 years, the overall survival was not significantly different (mean survival 18.3 y versus 13 y, p=0.32) (Fig. 2). In our cohort, no difference in myelofibrosis-free survival or leukemia-free survival was found between the molecular subtypes, although the risk of developing myelofibrosis was unexpectedly higher in the CALR mutant group (18% versus 4%, p=0.01). In contrast to other studies, we found no difference in the frequency of arteriovenous complications. In conclusion, this study on a Belgian cohort supports the notion that CALR mutant ET is phenotypically distinct from JAK2 V617F positive ET, with regard to the clinical and hematological presentation as well as the overall survival. In this cohort, the better overall survival was most marked in ET patients less than 60 y of age. Our study adds to the growing body of evidence that CALR mutant ET is a disease entity distinct from JAK2 V617F positive ET. Figure 1 Distribution of 165 ET patients according to their mutational status. Figure 1. Distribution of 165 ET patients according to their mutational status. Figure 2 Overall survival of CALR mutant and JAK2 V617F positive groups stratified according to age: older than 60 y versus younger than or equal to 60 y. Figure 2. Overall survival of CALR mutant and JAK2 V617F positive groups stratified according to age: older than 60 y versus younger than or equal to 60 y. Disclosures No relevant conflicts of interest to declare.
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Guglielmelli, Paola, Giada Rotunno, Giada Brogi, Annalisa Pacilli, Costanza Bogani, Carmela Mannarelli, Alessandro Pancrazzi, et al. "Calreticulin Mutation Is Associated with Milder Disease in Patients with Post Essential Thrombocythemia Myelofibrosis (PET-MF) Compared with JAK2V617F Mutation: A Study from the AGIMM Group." Blood 124, no. 21 (December 6, 2014): 3179. http://dx.doi.org/10.1182/blood.v124.21.3179.3179.
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Abstract Background: Mutations in the gene calreticulin (CALR) were recently discovered in 60-80% of patients (pts) with primary myelofibrosis (PMF) and essential thrombocythemia (ET) who were un-mutated for JAK2V617F and MPLW515. CALR mutated PMF pts had better overall survival (OS) compared with JAK2V617F or MPLW515 mutated while in ET CALR mutations were associated with lower incidence of thrombosis although the effect on survival was not significant. Conversely, there is no information concerning the impact of CALRmutation on disease phenotype and prognosis in post-essential thrombocythemia myelofibrosis (PET-MF). Aims: The aim of the study was to assess whether CALR mutational status and/or allele burden had clinical and/or prognostic relevance in PET-MF compared with JAK2, MPLmutated or triple-negative (TN) pts. Methods: ET and PET-MF were diagnosed by 2008 WHO and IWG-MRT criteria respectively; all pts provided an informed consent. Genotyping for CALR, JAK2V617F and MPLW515 was performed in granulocytes using allele specific RTQ-PCR (JAK2, MPL), capillary electrophoresis and direct sequencing (CALR, MPL). The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results: A series of 147 PET-MF pts from 4 Italian centres was collected. Pts median age was 63y. Median follow up from PET diagnosis was 3.2y (0.07-18.8y) and the median time from ET to PET diagnosis was 11.6y (0.9-30.6y). Death occurred in 38 pts (26%) and 14 pts (9.5%) developed acute leukemia (AML). The median OS in the entire series calculated from PET-MF diagnosis was 10.9y (7.1-14.7y). Frequency of mutations was: CALR 16%, JAK2V617F 77%; MPLW515 4.3%; TN 2.8%. The frequency of CALR mutations in PET-MF patients was superimposable to that observed in a control group of 576 ET patients from our Institution (15.5%) and slightly lower compared with other series (20-25%). Type of CALRmutations was: 59.6% type 1, 23.1%, type 2, 17.3% others, significantly different (P=0.023) from ET: 46% type 1, 38% type 2, 16% others. Median CALR allele burden in PET-MF was 56% (20%-100%) with no significant differences in the CALR mutation subtypes (57.5% in type 1, 47.5% in type 2 and 45.0% in others); however, the median mutant allele burden of CALR-mutated PET-MF patients was significantly higher than in ET patients (33%, range 2%-52%; n=100) (P<0.03) suggesting a role for mutated allele accumulation in evolution to PET-MF. Similarly, the median V617F allele burden in JAK2 mutated patients was 50.5% (range 5-100%) significantly greater than the value (24%; range, 1-87%) (P=0.02) in ET pts, confirming previous data that evolution to PET-MF is associated with accumulation of mutated JAK2allele. We then compared hematological and clinical characteristics of the patients who were categorized according to their JAK2V617F, MPLW515 and CALR mutation status. There was no statistically significant difference among the unique patient mutational groups regarding age, hemoglobin, leukocyte and platelet count, peripheral blasts, LDH, circulating CD34+ cells, abnormal karyotype, grade of bone marrow fibrosis and cellularity, and pruritus. However, JAK2+ pts showed an increased rate of large (>10 cm) splenomegaly (28.6% vs 14% in CALR+, 7.1 in MPL+ and 25% in TN pts; P=0.02) and constitutional symptoms (50% vs 18.8% in CALR+, 45% in MPL+ and 12.5% in TN pts; P=0.002). The interval from ET to PET-MF was significantly longer in CALR+ pts (14.5y) compared with JAK2+ (10.2y) and TN patients (11.0y; P=0.04 for both) and similar to MPL+ (14y). There was a reduced rate of death (13.5%) in CALR+ compared with JAK2+ (30.6%), MPL+ (21.4%) and TN (66.7%) pts (P=0.005), although Kaplan Meier estimates did not reach a statistically significant difference. Finally, there were less AML transformation in CALR+ pts (1.9%) compared with JAK2+ (13.9%), MPL+(7.1%) and TN (22.2%) (P=0.04). Conclusion: These results show that CALR mutation is associated with delayed transformation of ET to PET-MF, a milder disease in terms of splenomegaly and symptom burden and a reduced risk of death compared with JAK2V617F PET-MF pts and more in general with MPL mutated and TN pts.In addition, similar to findings in primary MF and unlike in ET, PET-MF is characterized by prevalence of type 1 CALR mutations. Disclosures No relevant conflicts of interest to declare.
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Tefferi, Ayalew, Paola Guglielmelli, Dirk R. Larson, Christy Finke, Emnet A. Wassie, Lisa Pieri, Naseema Gangat, et al. "Long-term survival and blast transformation in molecularly annotated essential thrombocythemia, polycythemia vera, and myelofibrosis." Blood 124, no. 16 (October 16, 2014): 2507–13. http://dx.doi.org/10.1182/blood-2014-05-579136.
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Key Points Survival in ET is superior to that of PV, regardless of mutational status, but remains inferior to the sex- and age-matched US population. JAK2/CALR/MPL mutational status is prognostically informative in PMF, regarding overall and leukemia-free survival.
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Zhang, Lei, Rongfeng Fu, Dongbing Liu, Shida Zhu, Hong Su, Xiuqing Zhang, Renchi Yang, and Tao Cheng. "Distinct Molecular Abnormalities Underlie Unique Clinical Features of Essential Thrombocythemia in Children." Blood 124, no. 21 (December 6, 2014): 4579. http://dx.doi.org/10.1182/blood.v124.21.4579.4579.
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Abstract Purpose Essential thrombocythemia (ET) is rare in children. The clinical course and pathogenesis of childhood ET is far less clear. We aimed to analyze the clinical and molecular characteristics of children diagnosed with ET. Patients and Methods Sixty-three children diagnosed with ET (age < 15 years) were enrolled in the study. JAK2 V617F mutation was detected using quantitative real-time polymerase chain reaction. MPL and CALR mutations were assessed using Sanger sequencing. Fifty-five genes associated with myeloid malignancies were analyzed using targeted next-generation sequencing in 25 children. Results The median follow-up time for 63 patients was 48 months (range, 12 to 302 months). Three children (4.8%) had thrombosis, and 2 (3.2%) progressed to myelofibrosis. One child developed both abdominal vein thrombosis and myelofibrotic transformation. JAK2 V617F mutation was found in 14 children (14/63, 22.2%). CALR mutations (1/49, 2.0%) and MPL mutations (0/49) were rarely detected. By targeted sequencing, somatic mutations were found in 13 genes and in 56.0% (14/25) of the patients (Figure 1A and 1B). After JAK2 V617F, the most frequently observed mutations were found in ASXL1 (4/25, 16.0%). Besides, mutations in U2AF1, FLT3, NRAS, MLL, GNAS, RUNX1, WT1, and ZRSR2 were first reported in childhood ET. These newly identified mutations, which are rarely seen in adult ET, are linked to more malignant diseases such as acute leukemia and myelodysplastic syndrome. The key pathway involved was the JAK2 signaling pathway, followed by epigenetic regulators. In 14 children with somatic mutations, 42.9% (6/14) had two or more mutations (Figure 1C). Co-occurrence of mutations in the JAK2 signaling pathway and epigenetic regulators was found in 4 children (4/14, 28.6%). Clonality assessment revealed that some children only had founding clones, but some had both founding clone and subclones (Figure 2). Analysis of allele frequencies showed that the JAK2 V617F mutation and CALR mutations were not the initial abnormalities in some children. Conclusion Childhood ET has a distinct molecular profile from that of adult patients. CALR and MPL are rarely mutated in childhood ET; instead, some previously undocumented mutations are detected. Genetic composition in childhood ET may be more complex than that in adults. The JAK2 V617F mutation and CALR mutations were not the initial abnormalities in some children. Whole exome sequencing would help to reveal the initial molecular events and to offer molecular targets for therapeutic intervention. Keywords Childhood; Essential thrombocythemia; Molecular markers Figure 1. Frequency and distribution of somatic mutations in children with essential thrombocythemia. (A) Number of patients and allele frequency of each mutated gene; (B) number of patients with JAK2 V617F mutation, with mutations except JAK2 V617F, and with no mutations; (C) number of patients with different number of somatic mutations. Figure 1. Frequency and distribution of somatic mutations in children with essential thrombocythemia. (A) Number of patients and allele frequency of each mutated gene; (B) number of patients with JAK2 V617F mutation, with mutations except JAK2 V617F, and with no mutations; (C) number of patients with different number of somatic mutations. Figure 2. Clonality assessment in two representative cases of essential thrombocythemia. (A) allele frequencies of mutations in Patient E1; (B) clusters of variants identify the founding clone in Patient E1; (C) allele frequencies of mutations in Patient E3; (D) clusters of variants identify the founding clone and subclones in Patient E3; in (B) and (D), the allele frequency is plotted versus the total number of sequencing reads covering the corresponding mutated nucleotide. Figure 2. Clonality assessment in two representative cases of essential thrombocythemia. (A) allele frequencies of mutations in Patient E1; (B) clusters of variants identify the founding clone in Patient E1; (C) allele frequencies of mutations in Patient E3; (D) clusters of variants identify the founding clone and subclones in Patient E3; in (B) and (D), the allele frequency is plotted versus the total number of sequencing reads covering the corresponding mutated nucleotide. Disclosures No relevant conflicts of interest to declare.
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Elhassan, Afra M., Arwa Alsaud, Mohamed A. Yassin, Mahmood Aldapt, Lubna Riaz, Firdous Ghori, Aiman Bin Ahmad, and Mohammad Abdulla. "Thrombocytapheresis in Patient with Essential Thrombocythemia: A Case Report." Case Reports in Oncology 13, no. 2 (June 16, 2020): 675–79. http://dx.doi.org/10.1159/000507651.
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Essential thrombocythemia (ET) is one of the myeloproliferative neoplasms, characterized by persistent thrombocytosis, platelets >450,000/μL, and evident clonal abnormalities like JAK2 V617F, MPL, CALR mutation and not fulfilling WHO criteria for MDS, CML, PV, and IDA. Here we report a 24-year-old female who presented with headache and was found to have thrombocytosis with a platelet count of 2,141 × 103/μL, diagnosed as ET as per WHO criteria 2008; she required ICU admission and thrombocytapheresis with a favorable outcome.
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Gisslinger, Heinz, Juergen Thiele, Bettina Gisslinger, Tiina Berg, Martin Schalling, Ashot S. Harutyunyan, Jelena D. Milosevic, et al. "Calreticulin Mutation Status Predicts Improved Disease Outcome in Prefibrotic Primary Myelofibrosis but Not in WHO-Defined Essential Thrombocythemia." Blood 124, no. 21 (December 6, 2014): 3167. http://dx.doi.org/10.1182/blood.v124.21.3167.3167.
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Abstract The genomic landscape of myeloproliferative neoplasms (MPNs) has altered dramatically due to the recent discovery of somatic mutations of calreticulin (CALR). This discovery enables for the first time molecular information, in addition to JAK2V617F, to be used in the majority of MPN patients as an affirmative variable to discriminate MPNs from reactive myeloid proliferations. The clinical course of essential thrombocythemia (ET) or primary myelofibrosis (PMF) in patients carrying the CALR mutation was reported to be more indolent than in JAK2 positive patients and was associated with increased survival. Our aim was to investigate whether the impact of CALR expression on prognosis and clinical outcome is different in prefibrotic/early PMF (prePMF) compared to WHO-ET. In a cohort of 348 adult patients with the clinical diagnosis of either ET or PMF mutational analysis for CALR was available. Eligibility criteria for the study included: availability of mutation analysis for JAK2, MPL and CALR; availability of representative, treatment-naive bone marrow biopsy (BM); availability of a histological and clinical consensus on the diagnosis; complete long-term documentation of clinical data and outcome. Consenting clinico-pathological findings in our cohort were consistent with 115 cases showing WHO-ET and 85 patients with prePMF. In comparison to WHO-ET, prePMF revealed minor/borderline age- and gender-matched anemia, slight increase in serum LDH level and leukocyte count, minor to slight splenomegaly, and an occasional left shift in granulo- and erythropoiesis with occurrence of a few myelo-and/or erythroblasts (table 1). An accurate differentiation between both MPN entities was shown to exert a significant difference in terms of overall and relative survival and hematologic transformation into overt PMF and AL. The present study revealed a different CALR mutation frequency in ET in contrast to most of the investigations published recently. We observed CALR mutations in 18% of WHO-ET; JAK2, MPL and CALR wildtype (wt) was observed in 13% of WHO-ET. The discrepancy in the frequencies of CALR positivity in our ET cohort to most of the recently published studies may be due to our strict adherence to the WHO criteria for diagnosis of ET. Regarding prePMF, we observed CALR mutations in 39% of the patients. 92% of the JAK2 and MPL wt subgroup carried the CALR mutation, with JAK2, MPL and CALR wt being observed in only 3% of prePMF. The most remarkable differences between WHO-ET and prePMF were seen in the comparison of the overall survival (figure 1). While the CALR mutation did not have any beneficial influence on survival in WHO-ET, it was associated with a superior overall survival in prePMF. Such a striking difference was not seen at the time of transformation into overt myelofibrosis, and there was only a slightly shorter time to progression to fibrosis in CALR wt prePMFs. There was a trend showing that CALR mutated prePMF patients have shorter thrombosis-free survival compared to CALR wt prePMF patients. There was no impact of the CALR mutations on thrombosis-free survival in WHO-ET. The present data confirm that WHO-ET and prePMF are biologically different sub-entities of MPNs. In prePMF, almost 100% of patients are now associated with a known disease-causing mutation. Our data support the classical clinical approach in the diagnosis of thrombocytosis, using BM histology to differentiate WHO-ET from prePMF and to estimate the outcome of the disease more accurately. Table 1:Total cohort (N=200)WHO-ET (N=115)prePMF (N=85)PAge at diagnosis, years0,04median58,8556,460,7range19-8819-8427-88Sex0,587male784335female1227250Hb, g/dL<0,001median14,114,313,5range8,1-17,68,8-17,68,1-16,6WBC, x109/l0,054median9,258,999,6range4,01-24,544,92-22,34,01-24,54Platelets, x109/l0,739median770770794range78-2530414-249078-2530Palpable splenomegaly<0,001No.51 (12 unk, 1 splenectomy)16 (7 unk, 1 splenectomy)35 (5 unk)%25,513,941,2Fibrotic transformations<0,001No.24 (10 unk)6 (1 unk)18 (9 unk)%125,221,2Thrombotic events0,439No.39 (1 unk)22 (1 unk)17%19,51920prev thrombosis0,042No.503416%2530,418,8JAK2 V617F pos0,08No.1207545%6065,252,9CALR pos0,001No.542133%2718,338,8MPL pos0,189No.844%43,54,7JAK2/MPL/CALR wt0,0029No.18153%9133,5 Figure 1 Figure 1. Disclosures Thiele: AOP Orphan Pharmaceuticals: Consultancy, Honoraria; Incyte Corporation: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria.
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Rizvi, Qurratulain, Uzma Zaidi, Saba Shahid, Shariq Ahmed, and Tahir Shamsi. "Homozygous CALR Mutation in Primary Myelofibrosis and Its Effect on Disease Phenotype: A Case Report and Review of the Literature." Case Reports in Hematology 2019 (January 20, 2019): 1–4. http://dx.doi.org/10.1155/2019/1430170.
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Somatic mutations in CALR gene have been reported in 60%–88% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) who are negative for JAK2 and MPL mutations. Most of the CALR mutations analyzed to date are heterozygous mutations in exon 9 of the gene. Homozygosity in CALR gene is rarely reported, and its association with clinical behavior of disease and impact on outcome of patients is not studied so far. We herein report a case of intermediate-2 risk PMF (according to IPSS) diagnosed with homozygous mutation (c.1139delA p.E380fs∗50) in CALR gene having severe disease manifestations at presentation.
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Wojtaszewska, Marzena, Małgorzata Iwoła, and Krzysztof Lewandowski. "Frequency and Molecular Characteristics of Calreticulin Gene (CALR) Mutations in Patients with JAK2-Negative Myeloproliferative Neoplasms." Acta Haematologica 133, no. 2 (October 16, 2014): 193–98. http://dx.doi.org/10.1159/000366263.
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In 2013, Nangalia et al. and Klampfl et al. found a recurrent and abundant mutation in the calreticulin gene (CALR), mutually exclusive with JAK2 and MPL alterations. At present, the data concerning the new mutation, i.e. its prevalence, allele burden and clinical significance, are scarce. We report the incidence and molecular characteristics of CALR mutations in a group of 184 Polish patients with myeloproliferative neoplasms (MPNs). Clinical data analysis revealed significant differences between JAK2 V617F-mutated and CALR-mutated groups. In essential thrombocythemia patients, hemoglobin levels and leukocyte counts were significantly higher in JAK2-positive than in CALR-positive patients (p = 0.023 and p = 0.017, respectively), but the CALR-positive patients had significantly higher platelet counts (p = 0.022). Patients harboring CALR mutations were also younger at the time of diagnosis (p = 0.039). In primary myelofibrosis patients, the degree of anemia was less severe in those who were CALR exon 9 mutation-positive than in those who were JAK2 V617F-positive (p = 0.048). © 2014 S. Karger AG, Basel
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Chang, Yu-Cheng, Ken-Hong Lim, Huan-Chau Lin, Yi-Hao Chiang, Ling Huang, Chen-Wei Kao, Chiao-Yi Chang, et al. "B Cell Immune Profiles in Essential Thrombocythemia with Calr Mutations: Clinical and Molecular Correlates." Blood 126, no. 23 (December 3, 2015): 1610. http://dx.doi.org/10.1182/blood.v126.23.1610.1610.
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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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Wang, Jing, Biao Zhang, Bing Chen, Rong-Fu Zhou, Qi-Guo Zhang, Juan Li, Yong-Gong Yang, et al. "JAK2, MPL, and CALR mutations in Chinese Han patients with essential thrombocythemia." Hematology 22, no. 3 (November 22, 2016): 145–48. http://dx.doi.org/10.1080/10245332.2016.1252003.
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Gasser, Klaus, Axel Muendlein, Elena Kinz, Andreas Leiherer, Michael Steurer, Peter Fraunberger, Heinz Drexel, and Alois Lang. "Determination of Driver Mutations in JAK2, MPL, and Calreticulin in Coronary Patients with Suspected Essential Thrombocythemia." Blood 124, no. 21 (December 6, 2014): 5552. http://dx.doi.org/10.1182/blood.v124.21.5552.5552.
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Abstract Patients with essential thrombocythemia (ET) are at increased risk towards atherothrombotic complications. The JAK2 V617F mutation is found in at least half of the patients with ET and screening for JAK2 V617F mutational status has been included in the World Health Organization diagnostic criteria for ET. Further somatic driver mutations including mutations in exon 12 of the JAK2 gene, MPL mutations W515L/W515K, as well as mutations in exon 9 of the calreticulin (CALR) gene have been suggested as diagnostic markers for ET. Due to the close relation of ET to thrombotic events, early diagnosis of ET is of high clinical relevance, especially in patients with increased cardiovascular risk, like coronary patients. However, elevated blood cell counts are often ascribed to a reactive genesis within an acute coronary event. For this reason mostly no further clarification concerning myeloproliferative neoplasms is done. In the present study, therefore, we determined the prevalence of JAK2 V617F, JAK2 exon 12, MPL W515L, MPL W515K, and CALR exon 9 mutations in a cohort of patients undergoing coronary angiography for the evaluation of suspected or established stable coronary artery disease and suspected diagnosis of ET. We aimed at identifying those with occult ET proposing need for additional treatment. From a total of 1,589 coronary patients, 12 patients had increased platelets (>450 x10 9/L) predisposing them to ET. Mutation analysis among these patients showed three individuals with the JAK2 V617F mutation and one with an ins/del mutation in exon 9 of the CALR gene confirming suspected diagnosis of ET. Consequently, frequency of ET was 0.25% among our coronary patients. For comparison, in the United States between 38 and 57 ET cases per 100,000 age-adjusted inhabitants have been reported. This corresponds to an up to 7-fold accumulation of ET cases in coronary patients compared to the general population. We conclude that the prevalence of ET is increased in coronary patients compared to the general population. For this reason comprehensive mutation analysis should be considered in all cardiovascular risk patients with persistent elevation of thrombocytes, not only to identify patient subgroups at high risk but also to individualize therapeutic strategies. Disclosures No relevant conflicts of interest to declare.
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Zulkeflee, Razan Hayati, Zefarina Zulkafli, Muhammad Farid Johan, Azlan Husin, Md Asiful Islam, and Rosline Hassan. "Clinical and Laboratory Features of JAK2 V617F, CALR, and MPL Mutations in Malaysian Patients with Classical Myeloproliferative Neoplasm (MPN)." International Journal of Environmental Research and Public Health 18, no. 14 (July 16, 2021): 7582. http://dx.doi.org/10.3390/ijerph18147582.
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Mutations of JAK2V617F, CALR, and MPL genes confirm the diagnosis of myeloproliferative neoplasm (MPN). This study aims to determine the genetic profile of JAK2V617F, CALR exon 9 Type 1 (52 bp deletion) and Type 2 (5 bp insertion), and MPL W515 L/K genes among Malaysian patients and correlate these mutations with clinical and hematologic parameters in MPN. Mutations of JAK2V617F, CALR, and MPL were analyzed in 159 Malaysian patients using allele-specific polymerase chain reaction, including 76 polycythemia vera (PV), 41 essential thrombocythemia (ET), and 42 primary myelofibrosis (PMF) mutations, and the demographics of the patients were retrieved. The result showed that 73.6% JAK2V617F, 5.66% CALR, and 27.7% were triple-negative mutations. No MPL W515L/K mutation was detected. In ET and PMF, the predominance type was the CALR Type 1 mutation. In JAK2V617F mutant patients, serum LDH was significantly higher in PMF compared to PV and ET. PV has a higher risk of evolving to post PV myelofibrosis compared to ET. A thrombotic event at initial diagnosis of 40.9% was high compared to global incidence. Only one PMF patient had a CALR mutation that transformed to acute myeloid leukemia. JAK2V617F and CALR mutations play an important role in diagnostics. Hence, every patient suspected of having a myeloproliferative neoplasm should be screened for these mutations.
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AL-Ansari, Rehab Y., Dena Al Otaibi, Nourah Al Hudaithi, and Leena Abdalla. "Isolated ten-eleven translocation 2 positive in triple negative essential thrombocythemia: Case report and literature review." SAGE Open Medical Case Reports 9 (January 2021): 2050313X2110320. http://dx.doi.org/10.1177/2050313x211032066.
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Essential thrombocythemia is one of the famous diseases under the category of myeloproliferative disorder. It is an end result of a genetic mutation of one or more of the most frequent oncogenes such as Janos kinase 2 (JAK2), MPL proto-oncogene, thrombopoietin receptor (MPL), and calreticulin (CALR). However, negative genetic markers, so-called (triple negative disease), can happen in the presence of other uncommon types of mutation. TET2 (ten-eleven translocation 2) positive as isolated genetic marker in triple negative essential thrombocythemia is uncommon genetic presentation. For that, we are reporting a 22-year-old lady who presented with a feature of dyspepsia and accidentally found to have persistently high platelet count, even after treating her mild iron deficiency anemia with no other secondary causes. Further investigations and bone marrow biopsy supported the diagnosis of isolated TET2 positive in triple negative essential thrombocythemia. We treated her conservatively with good hydration and low dose of aspirin. In conclusion, isolated TET2 positive in triple negative essential thrombocythemia at presentation is uncommon with no clear management or risk stratification guideline. However, it is hypothesized that TET2 mutation precedes JAK2; therefore, the detection of isolated TET2 in a triple negative essential thrombocythemia case should be closely followed for clonal evolution in long term. Further study and guidelines required in this area.
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Makarik, Tatiana V., Adhamjon O. Abdullaev, Sergei M. Kulikov, Elena E. Nikulina, Svetlana A. Treglazova, Irina N. Subortseva, Alla M. Kovrigina, Anait L. Melikyan, Andrey B. Sudarikov, and Valery G. Savchenko. "The Frequency of Calr and MPL Gene Mutations in Jak2 V617F - Positive Chronic Myeloproliferative Neoplasms in Russia." Blood 134, Supplement_1 (November 13, 2019): 5400. http://dx.doi.org/10.1182/blood-2019-124767.
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Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.
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Gadomska, Grażyna, Alicja Bartoszewska-Kubiak, Joanna Boinska, Karolina Matiakowska, Katarzyna Ziołkowska, Olga Haus, and Danuta Rość. "Selected Parameters of Angiogenesis and the JAK2, CALR, and MPL Mutations in Patients With Essential Thrombocythemia." Clinical and Applied Thrombosis/Hemostasis 24, no. 7 (February 1, 2018): 1056–60. http://dx.doi.org/10.1177/1076029617740222.
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The aim of the study was to evaluate selected angiogenic factors in patients with essential thrombocythemia (ET) depending on JAK2V617F, calreticulin gene (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations. Sixty ET patients and 20 healthy volunteers were enrolled in the study. The following tests were performed: vascular endothelial growth factor- A (VEGF-A), soluble vascular endothelial growth factor receptor-1 (sVEGFR-1),soluble vascular endothelial growth factor receptor-2 (sVEGFR-2), platelet-derived growth factor( PDGF-BB), and stromal-derived factor-1α (SDF-1α). We observed an increased PDGF-BB level in patients with ET compared to the controls. Patients with CALR mutation had significantly higher concentration of PDGF-BB and lower concentration of SDF-1α than patients with JAK2V617F mutation. High concentration of PDGF-BB and low concentration of SDF-1α in patients with CALR(+) ET may indicate a contribution of these chemokines in disturbed Ca2+ metabolism in platelets.
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Tokgoz, Huseyin, Umran Caliskan, Reyhan Kucukkaya, Ahmet Demir, and Veysel Sabri Hancer. "Two Pediatric Cases of Essential Thrombocytopenia Characterized By Extremely Rare Mutations (CALR and MPL W515K)." Blood 126, no. 23 (December 3, 2015): 4662. http://dx.doi.org/10.1182/blood.v126.23.4662.4662.
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Abstract Essential Thrombocythemia (ET) is an extremely rare disorder during childhood, characterized by clonal expansion of megakaryocytic and thrombocytic lineages in bone marrow, leading to a persistent increase in the number of circulating thrombocytes and thus increasing the risk for thrombotic and hemorrhagic events. Most of children with ET have JAK2 V617F mutation (1). MPL and CALR mutastions have been reported in a small proportion of adult ET patients. However, it is extremely rare in children. Herein we present two cases of pediatric ET with different rare mutations. Case 1: 15-year old female presented with Budd-Chiari syndrome and thrombocytosis (1500x109/L). Bone marrow smear and biopsy were consistent with ET. To elucidate the molecular mechanisms involved in this case of ET, we investigated candidate gene alterations. BCRABL, JAK2 V617F and calreticulin mutations were negative. Thus we analyzed codon 515 of MPL gene. The patient was found to have mutation for MPL W515K (figure 1). The patient who presented with BCS was diagnosed as ET with MPL W515K mutation. Hydroxyurea therapy at a dose of 20mg/kg/day was started. After 10 days, enoxaparin dose was reduced to single dose daily (1mg/kg/day) as a maintenance anticoagulation. The patient underwent liver transplantation at the end of the 3rd month of treatment. Warfarin treatment was started to patient after transplantation. Case 2: Nine year old female was applied to our policlinic because of thrombocytosis identified in routine control (1300x109/L). Secondary causes of thrombocytosis were excluded. Bone marrow smear and biopsy were consistent with ET. We investigated candidate gene alterations. BCRABL, JAK2 V617F and MPL mutations were negative. CALR type I mutation was identified (figure 2). Hydroxyurea treatment was successfully reduced the platelet count. Discussion: Thrombocytosis occurs in about 6-15% of children, but ET is very rare in children (2). Most of cases with pediatric ET have JAK2V617F mutation. Our first case has MPLW515K mutation. To our knowledge, this is the first pediatric patients of ET mutated W515K. The other patient has CALR mutation. This was also extremely rare mutation in children (3). We believe that our two cases will contribute to the literature. References 1. Fu R, Zhang L, Yang R. Paediatric essential thrombocythaemia: clinical and molecular features, diagnosis and treatment. Br J Haematol. 2013 Nov;163(3):295-302. 2. Hasle H. Incidence of essential thrombocythaemia in children. Br J Haematol. 2000 Sep;110(3):751. 3. Giona F, Teofili L, Capodimonti S, Laurino M, Martini M, Marzella D, et al. CALR mutations in patients with essential thrombocythemia diagnosed in childhood and adolescence. Blood. 2014 Jun 5;123(23):3677-9. Figure 1. W515K mutation first case with ET Figure 1. W515K mutation first case with ET Figure 2. CALR type I mutation in second case with ET. Figure 2. CALR type I mutation in second case with ET. Disclosures No relevant conflicts of interest to declare.
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Thorsten, Klampfl, Heinz Gisslinger, Ashot S. Harutyunyan, Harini Nivarthi, Elisa Rumi, Jelena D. Milosevic, Nicole C. C. Them, et al. "Frequent Mutations in the Calreticulin Gene CALR in Myeloproliferative Neoplasms." Blood 122, no. 21 (November 15, 2013): LBA—1—LBA—1. http://dx.doi.org/10.1182/blood.v122.21.lba-1.lba-1.
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Abstract The classical, BCR-ABL1 negative myeloproliferative neoplasms (MPN) are polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The most common genetic alteration in MPN is the JAK2-V617F mutation detected in 95% of PV patients and in 50-60% of patients with ET or PMF. Mutations in exon 12 of JAK2 and in the thrombopoietin receptor gene MPL are found in an additional 5-10% of the cases. In recent years a number of other genes were shown to be affected in MPN. However, these mutations are not mutually exclusive with JAK2 and MPL mutations and are also found in other myeloid malignancies. A specific molecular marker for the remaining 40% of ET or PMF patients with wild type JAK2 and MPL is still unknown. We used whole-exome sequencing to identify novel mutations in PMF patients with wild type JAK2 and MPL. The analysis revealed recurrent somatic insertions and deletions in CALR encoding for calreticulin. All detected mutations resulted in a frameshift and clustered in the last exon (exon 9) of the gene. Following up on this finding we developed a PCR based assay to screen 1107 MPN patients for insertion/deletion mutations in exon 9 of CALR. No mutations were detected in PV. In ET and PMF CALR mutations were mutually exclusive with mutant JAK2 and mutant MPL. Of the patients with wild type JAK2 and MPL, 67% ET and 88% PMF had mutant CALR. We also tested 19 patients with wild type CALR-exon 9 for mutations in the other exons of the gene, but all were negative. Furthermore we did not find CALR-exon 9 mutations in 254 patients with de novo acute myeloid leukemia, 45 with chronic myeloid leukemia, 73 with myelodysplastic syndrome or 64 with chronic myelomonocytic leukemia. Out of 24 patients with refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), 3 patients carried CALR mutations. These patients were wild type for JAK2 and MPL. In total we detected 36 different types of mutations in CALR. A 52 bp deletion and a 5 bp insertion were the most prominent types found in 53% and 32% of all cases with mutant CALR. All 36 types of mutations result in a frameshift to the same alternative reading frame, generating a novel C-terminus of the mutated protein. The wild type C-terminal region of CALR contains a high-capacity calcium-binding domain and is highly negatively charged. As a consequence of the frameshift mutations the negatively charged amino acids are replaced by both neutral and positively charged amino acids. In addition, an endoplasmic reticulum retention signal present in the wild type protein is lost in the mutant variants. Expression in HEK cells showed that wild type CALR localizes in the endoplasmic reticulum, whereas this localization is less prominent in cells expressing mutant CALR. This observation is in line with the loss of the endoplasmic reticulum retention signal in the mutant protein. Overexpression of the most common CALR mutation (a 52 bp deletion) in interleukin-3 (IL-3) dependent Ba/F3 cells led to IL-3-independent growth and hypersensitivity to IL-3. Cells overexpressing the mutant were sensitive to the JAK-family kinase inhibitor SAR302503 and showed elevated STAT5 phosphorylation in absence of IL-3. This indicates that JAK-STAT signaling is involved in the observed cytokine independent growth of mutant CALR expressing Ba/F3 cells. ET and PMF patients with mutant CALR present with lower white blood cell counts (P<0.001 for ET, P=0.027 for PMF) and elevated platelet levels (P<0.001 in both entities) compared to patients with mutant JAK2. In both disease entities patients with mutant CALR show significantly better overall survival than patients with mutant JAK2 (P=0.043 in ET, P<0.001 in PMF). ET patients with mutant CALR had a lower risk of thrombosis in comparison to those with mutant JAK2 (P=0.003). Mutant CALR is a novel, specific molecular marker detected in the majority of MPN patients negative for JAK2 and MPL mutations. Use of this marker in the clinic is expected to improve diagnostic and therapeutic decision-making in MPN. Disclosures: No relevant conflicts of interest to declare.
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Rumi, Elisa, and Mario Cazzola. "Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms." Blood 129, no. 6 (February 9, 2017): 680–92. http://dx.doi.org/10.1182/blood-2016-10-695957.
Full textAbstract:
Abstract Philadelphia-negative classical myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 revision of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues includes new criteria for the diagnosis of these disorders. Somatic mutations in the 3 driver genes, that is, JAK2, CALR, and MPL, represent major diagnostic criteria in combination with hematologic and morphological abnormalities. PV is characterized by erythrocytosis with suppressed endogenous erythropoietin production, bone marrow panmyelosis, and JAK2 mutation. Thrombocytosis, bone marrow megakaryocytic proliferation, and presence of JAK2, CALR, or MPL mutation are the main diagnostic criteria for ET. PMF is characterized by bone marrow megakaryocytic proliferation, reticulin and/or collagen fibrosis, and presence of JAK2, CALR, or MPL mutation. Prefibrotic myelofibrosis represents an early phase of myelofibrosis, and is characterized by granulocytic/megakaryocytic proliferation and lack of reticulin fibrosis in the bone marrow. The genomic landscape of MPNs is more complex than initially thought and involves several mutant genes beyond the 3 drivers. Comutated, myeloid tumor-suppressor genes contribute to phenotypic variability, phenotypic shifts, and progression to more aggressive disorders. Patients with myeloid neoplasms are at variable risk of vascular complications, including arterial or venous thrombosis and bleeding. Current prognostic models are mainly based on clinical and hematologic parameters, but innovative models that include genetic data are being developed for both clinical and trial settings. In perspective, molecular profiling of MPNs might also allow for accurate evaluation and monitoring of response to innovative drugs that target the mutant clone.
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Vainchenker, William, Stefan N. Constantinescu, and Isabelle Plo. "Recent advances in understanding myelofibrosis and essential thrombocythemia." F1000Research 5 (April 19, 2016): 700. http://dx.doi.org/10.12688/f1000research.8081.1.
Full textAbstract:
The classicBCR-ABL-negative myeloproliferative neoplasms (MPNs), a form of chronic malignant hemopathies, have been classified into polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). ET and PMF are two similar disorders in their pathogenesis, which is marked by a key role of the megakaryocyte (MK) lineage. Whereas ET is characterized by MK proliferation, PMF is also associated with aberrant MK differentiation (myelodysplasia), leading to the release of cytokines in the marrow environment, which causes the development of myelofibrosis. Thus, PMF is associated with both myeloproliferation and different levels of myelodysplastic features. MPNs are mostly driven by mutated genes called MPN drivers, which abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors. The recent discovery ofCALRmutations has closed a gap in our knowledge and has shown that this mutated endoplasmic reticulum chaperone activates the thrombopoietin receptor MPL and JAK2. These genetic studies have shown that there are two main types of MPNs: JAK2V617F-MPNs, including ET, PV, and PMF, and the MPL-/CALR-MPNs, which include only ET and PMF. These MPN driver mutations are associated with additional mutations in genes involved in epigenetics, splicing, and signaling, which can precede or follow the acquisition of MPN driver mutations. They are involved in clonal expansion or phenotypic changes or both, leading to myelofibrosis or leukemic transformation or both. Only a few patients with ET exhibit mutations in non-MPN drivers, whereas the great majority of patients with PMF harbor one or several mutations in these genes. However, the entire pathogenesis of ET and PMF may also depend on other factors, such as the patient’s constitutional genetics, the bone marrow microenvironment, the inflammatory response, and age. Recent advances allowed a better stratification of these diseases and new therapeutic approaches with the development of JAK2 inhibitors.
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Verger, Emmanuelle, Bruno Cassinat, Christine Dosquet, Marie-Helene Schlageter, Valérie Ugo, Jean-Christophe Ianotto, Mohamed A. Yassin, et al. "Impact of the Molecular Profile of Malignant Clones on the Response to Interferon Alpha (IFNa) Therapy in JAK2V617F-Negative Essential Thrombocythemia (ET)." Blood 124, no. 21 (December 6, 2014): 407. http://dx.doi.org/10.1182/blood.v124.21.407.407.
Full textAbstract:
Abstract Background: The majority of ET patients (pts) without JAK2 or MPL mutations present somatic mutations in the calreticulin gene (CALR). However, a series of mutations in other genes involved in the epigenome, the splicing machinery or leukemic transformation have been described in ET, but contrary to MF, their impact has not been clearly assessed. We have previously shown that IFNa may considerably reduce the JAK2-mutated clones, and we also observed that TET2 mutated clones are resistant to IFNa therapy in JAK2+TET2+ pts with polycythemia vera. As little is known about the IFNa response of clones harboring mutations other than JAK2V617F in ET, we took advantage of a cohort of pts without JAK2 mutation but CALRmutated and treated with IFNa (according to international and local guidelines) to assess the dynamics of the different MPN clones during therapy. Aims: 1) To determine, using a Next Generation Sequencing (NGS) approach, the molecular pattern of mutations in genes previously shown to have a prognostic impact in MPNs in a series of CALR-mutated ET pts; 2) To study the evolution of these mutational patterns during IFNa therapy. Methods: JAK2 and MPL-negative ET pts followed in our department fulfilling the following criteria were included in the study: presence of a CALR mutation; availability of at least 3 sequential blood samples including one taken before IFNa; IFNa therapy for at least 3 months; informed consent for molecular analysis. Total DNA was extracted from blood samples (Qiagen blood DNA mini kits) for molecular analyses. Mutations in TET2, ASXL1, EZH2, SRSF2, IDH1, IDH2, SH2B3 and CSF3R were searched through a NGS approach on a MiSeq instrument using a TruSeq custom amplicon approach (Illumina). CALRgene mutation detection was done by direct Sanger sequencing of exon 9, and mutant allele burden (%CALR) was estimated by fragment analysis (GeneMapper software , Life technologies) with a sensitivity of about 1%. Both sequencing and fragment analysis were performed on a 3500xL DX Genetic Analyser (Life technologies). Molecular response was defined as complete (CMR) when CALR mutation was no longer detectable, partial (PMR) when %CALR was decreased by >50%, minor (mMR) when %CALR was decreased by 25 to 49%, and non responders if %CALR was reduced by less than 25%. Results: Among 238 ET pts without JAK2 or MPL mutations, we identified 52 pts (22%) treated with peg-IFNa-2a, of whom 24 fulfilled the inclusion criteria. Median age was 52 years (range 31-68), 67% were women, median follow up was 12 years (range : 1.5 – 29) and median IFN treatment duration was 30 months (range: 12 – 102). 23/24 (96%) pts achieved complete or partial hematological response to IFNa. CALR mutations (present in all patients, quantifiable in all but 1 because of 1bp difference between mutated and WT) were of type 1 in 10 (42%), type 2 in 8 (33%) and of neither type in 6 (25%) pts, respectively. In addition to CALR, a second mutation was found in 8 pts (33%) by NGS, affecting ASXL1 (n=2), TET2 (n=2), IDH2 (n=2), CSF3R (n=1) and SH2B3 (n=1) genes. Comparison of sequential samples showed that the %CALR decreased from a median of 43% (range: 8-57) to 26% (range: 0-49) in the last sample (p= 0.018). In details, %CALR decreased in 13 (57%) pts, including 1 CMR, 7 PMR and 5 mMR. Interestingly, molecular response to IFNa of CALR+clones appeared poorer in pts with additional mutations (vs. pts with CALR mutation alone): 50% were non responders (vs. 40%), %CALR even increased during follow up in 25% (vs. 7%), and the median decrease in %CALR was 32% (vs. 45%). Dynamics of clones harboring additional mutated genes showed that, in contrast to CALR, mutant allele burden did not vary in 6/8 pts. However, an IDH2 mutation decreased from 9% to 1% in 1 pt, and a TET2 mutation increased from 1% to 21% in another (while %CALR remained unchanged in both pts, at 44% and 19% respectively). Of note, after 12 years of median follow-up, no emergence of new mutation was observed in any patient. Conclusion: In this cohort of JAK2V617F-negative ET, 96% of pts achieved hematological response with IFNa therapy. However, molecular profiling suggests that the mutational pattern found in malignant clones modulates the molecular response to IFNa: 1) Existence of more than 1 mutation induces poorer molecular response; 2) Clones with CALR mutation alone are sensitive to IFNa; 3) Clones harboring mutations in genes other than CALR seem less responsive to IFNa therapy. Disclosures Off Label Use: Interferon alpha was used off-label in selected ET patients according to local and international guidelines (Barbui et al., J Clin Oncol. 2011 Feb 20;29(6):761-70).
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Michiels, Jan Jacques, Zwi Berneman, Wilfried Schroyens, and Hendrik De Raeve. "Changing Concepts of Diagnostic Criteria of Myeloproliferative Disorders and the Molecular Etiology and Classification of Myeloproliferative Neoplasms: From Dameshek 1950 to Vainchenker 2005 and Beyond." Acta Haematologica 133, no. 1 (August 7, 2014): 36–51. http://dx.doi.org/10.1159/000358580.
Full textAbstract:
The Polycythemia Vera Study Group (PVSG) and WHO classifications distinguished the Philadelphia (Ph1) chromosome-positive chronic myeloid leukemia from the Ph1-negative myeloproliferative neoplasms (MPN) essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (MF) or primary megakaryocytic granulocytic myeloproliferation (PMGM). Half of PVSG/WHO-defined ET patients show low serum erythropoietin levels and carry the JAK2V617F mutation, indicating prodromal PV. The positive predictive value of a JAK2V617F PCR test is 95% for the diagnosis of PV, and about 50% for ET and MF. The WHO-defined JAK2V617F-positive ET comprises three ET phenotypes at clinical and bone marrow level when the integrated WHO and European Clinical, Molecular and Pathological (ECMP) criteria are applied: normocellular ET (WHO-ET), hypercellular ET due to increased erythropoiesis (prodromal PV) and hypercellular ET associated with megakaryocytic granulocytic myeloproliferation (EMGM). Four main molecular types of clonal MPN can be distinguished: JAK2V617F-positive ET and PV; JAK2 wild-type ET carrying the MPL515; mutations in the calreticulin (CALR) gene in JAK2/MPL wild-type ET and MF, and a small proportion of JAK2/MPL/CALR wild-type ET and MF patients. The JAK2V617F mutation load is low in heterozygous normocellular WHO-ET. The JAK2V617F mutation load in hetero-/homozygous PV and EMGM is clearly related to MPN disease burden in terms of splenomegaly, constitutional symptoms and fibrosis. The JAK2 wild-type ET carrying the MPL515 mutation is featured by clustered small and giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow (WHO-ET), and lacks features of PV. JAK2/MPL wild-type, CALR mutated hypercellular ET associated with PMGM is featured by dense clustered large immature dysmorphic megakaryocytes and bulky (cloud-like) hyperchromatic nuclei, which are never seen in WHO-ECMP-defined JAK2V617F mutated ET, EMGM and PV, and neither in JAK2 wild-type ET carrying the MPL515 mutation. Two thirds of JAK2/MPL wild-type ET and MF patients carry one of the CALR mutations as the cause of the third distinct MPN entity. WHO-ECMP criteria are recommended to diagnose, classify and stage the broad spectrum of MPN of various molecular etiologies.
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Usseglio, Fabrice, Nathalie Beaufils, Anne Calleja, Sophie Raynaud, and Jean Gabert. "Detection of CALR and MPL Mutations in Low Allelic Burden JAK2 V617F Essential Thrombocythemia." Journal of Molecular Diagnostics 19, no. 1 (January 2017): 92–98. http://dx.doi.org/10.1016/j.jmoldx.2016.08.006.
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Vainchenker, William, and Robert Kralovics. "Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms." Blood 129, no. 6 (February 9, 2017): 667–79. http://dx.doi.org/10.1182/blood-2016-10-695940.
Full textAbstract:
Abstract The genetic landscape of classical myeloproliferative neoplasm (MPN) is in large part elucidated. The MPN-restricted driver mutations, including those in JAK2, calreticulin (CALR), and myeloproliferative leukemia virus (MPL), abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors, more particularly the STATs. The most frequent mutation, JAK2V617F, activates the 3 main myeloid cytokine receptors (erythropoietin receptor, granulocyte colony-stimulating factor receptor, and MPL) whereas CALR or MPL mutants are restricted to MPL activation. This explains why JAK2V617F is associated with polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis (PMF) whereas CALR and MPL mutants are found in ET and PMF. Other mutations in genes involved in epigenetic regulation, splicing, and signaling cooperate with the 3 MPN drivers and play a key role in the PMF pathogenesis. Mutations in epigenetic regulators TET2 and DNMT3A are involved in disease initiation and may precede the acquisition of JAK2V617F. Other mutations in epigenetic regulators such as EZH2 and ASXL1 also play a role in disease initiation and disease progression. Mutations in the splicing machinery are predominantly found in PMF and are implicated in the development of anemia or pancytopenia. Both heterogeneity of classical MPNs and prognosis are determined by a specific genomic landscape, that is, type of MPN driver mutations, association with other mutations, and their order of acquisition. However, factors other than somatic mutations play an important role in disease initiation as well as disease progression such as germ line predisposition, inflammation, and aging. Delineation of these environmental factors will be important to better understand the precise pathogenesis of MPN.
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Wang, Yongbao, Albert K. Ho, Qiulu Pan, Frederick Karl Racke, and Dan Jones. "In-Frame Exon 9 CALR Deletions Co-Occur with Other Alterations in the JAK-STAT Pathway in Myeloproliferative Neoplasms." Blood 124, no. 21 (December 6, 2014): 4588. http://dx.doi.org/10.1182/blood.v124.21.4588.4588.
Full textAbstract:
Abstract Introduction: Mutations in the chaperone gene calreticulin (CALR) have been recently identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF), and are essentially mutually exclusive with JAK2 or MPL mutations. Normal and mutant CALR proteins may differentially affect the subcellular trafficking of JAK-STAT signaling components. CALR mutations previously reported in ET and PMF have been +1 frameshift (fs) mutations localized to exon (E) 9 that generate a novel C-terminal protein sequence with a shift from acidic to basic residues. CALR E9 in-frame (IF) deletions have been recently rarely reported as polymorphisms such as TMP_ESP_19_13054686_13054688 and TMP_ESP_19_13054650_13054658 (Ensembl database entries). We sought to determine the frequency and associated clinical features of CALR with E9 IF alterations in samples submitted for suspicion of a myeloproliferative neoplasm (sMPN). We also assessed whether CALR IF alterations are differentially associated with +1fs mutations or with JAK2 V617For other somatic mutations in MPN-associated genes. Materials and Methods: CALR mutation analysis of E9 was performed on genomic DNA extracted from blood, bone marrow (BM) aspirate or fixed BM biopsy sections using a Sanger sequencing assay with an analytic sensitivity of at least 15%. E9 IF cases were further assessed and mutations quantified by an Ion torrent sequencing panel assessing CALR, CSF3R, JAK2 and MPL, a second panel containing ASXL1, EZH2, IDH1, IDH2, KRAS, NRAS and TET2 and an Illumina MiSeq extended panel with 20 additional MPN-associated genes. These assays had a sensitivity of approximately 5%. JAK2 V617Fmutations were quantitated using a pyrosequencing assay with an analytic sensitivity of 1%. Results: We assessed CALR E9 mutation status in 733 sMPN samples that were negative for JAK2 V617F mutation. 148 (20.1%) had typical +1fs mutations (95 type 1 and variants, 53 type 2 and variants); 2 (0.3%) had point mutations (E381A and D7373M); 7 (1.0%) had IF deletions including E381_A382>A, D397_D400>D (n =4), D400_K401>D and E405_V409>V. All E9 IF deletions were present at ~50% of reads. Clinical diagnoses were cytopenia/BM fibrosis, ET, thrombocytosis/anemia, and sMPN unspecified. Mutation analysis for 27 additional MPN-associated genes revealed mutations in 5/7 (71.4%) IF deletion cases including in MPL (W515L,40%; D163Y,12%), CSF3R (A470T 46%), ASXL1 (D954fs*26, 45%) and ZRSR2 (S449_R450dup, 27%). No additional mutations were found in the 2 cases with non-synonymous CALR point mutations/SNPs. In a parallel set of 76 MPN samples that had JAK2 V617F at varying levels, we noted 1 E9 IF deletion (D397_D400>D) in a sMPN case with 21.6% JAK2 V617F, and a typical +1fs mutation (K385fs*47) in a case with low (4.2%) JAK2 V617F. All other JAK2 V617F cases had no E9 CALR alterations. Conclusions: CALR E9 in-frame deletions occur in up to 1% of sMPN samples and involve a variety of codons in the acidic domain. Therefore, sizing assays without DNA sequencing are not sufficient to unequivocally distinguish IF deletions from the characteristic +1 frameshift somatic mutations associated with ET and PMF. Given their level, these CALR IF deletions are likely germline sequence variants but are associated with a high frequency of somatic mutations in other MPN-associated genes but not with CALR +1fs mutations. Their co-occurrence with pathogenic somatic mutations in JAK2, MPL and CSF3R affecting the JAK-STAT pathway raises the possibility for a contributory role of altered CALR proteins produced by these E9 deletions in the pathogenesis of MPN. Disclosures Wang: Quest Diagnostics: Employment. Ho:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.
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Santoro, Marco, Vincenzo Accurso, Salvatrice Mancuso, Mariasanta Napolitano, Marta Mattana, Giorgia Vajana, Federica Russello, and Sergio Siragusa. "Triple-Negativity Identifies a Subgroup of Patients with Better Overall Survival in Essential Thrombocythemia." Hematology Reports 14, no. 3 (August 24, 2022): 265–69. http://dx.doi.org/10.3390/hematolrep14030037.
Full textAbstract:
Essential thrombocythemia, as defined by the WHO in 2016, is a Philadelphia-negative chronic myeloproliferative neoplasm showing a better prognosis than polycythemia vera and myelofibrosis. In a variable percentage, patients with essential thrombocythemia show none of the known driver-gene mutations that may occur on JAK2, CALR, and MPL genes. Such patients are classified as triple-negative and their clinical features and prognosis have not been described with precision yet. In this study, we evaluated some of the characteristics of this population by comparing them with those of patients with driver-gene mutated ET. Data from 266 consecutive essential thrombocythemia patients were analysed. Triple-negative patients had a significantly lower symptom load and a lower frequency of splenomegaly at diagnosis. The results show that the rate of thrombosis was equal in the two subgroups. Overall survival was slightly better in the triple-negative group of patients.
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Girodon, Francois, Julien Broseus, Ji-Hye Park-Alexandre, Sylvie Hermouet, and Serge Carillo. "Presence of Calreticulin Mutations in JAK2-Negative Polycythemia Vera." Blood 124, no. 21 (December 6, 2014): 1819. http://dx.doi.org/10.1182/blood.v124.21.1819.1819.
Full textAbstract:
Abstract Calreticulin (CALR) mutations have recently been reported in JAK2- and MPL-negative Myeloproliferative Neoplasms (MPN), particularly essential thrombocythemia (ET) and primary myelofibrosis (PMF).The clinical course of sporadic CALR-mutated patients seems to be more indolent than that of JAK2-mutated patients. In contrast, no CALR mutation has been found in the 647 published cases of Polycythemia Vera (PV) patients tested. Consequently, CALR mutations were considered exclusive to JAK2 and MPL mutations. Since 98% of PV patients harbor a JAK2 mutation (mostly the V617F mutation in exon 14 and more rarely, in exon 12), the absence of CALR mutations in PV seemed logical. Here, we describe two JAK2V617F-negative PV patients who presented with a CALR mutation at the time of diagnosis. Patient # 1 had hemoglobin at 168 g/L, hematocrit at 51.3%, and increased red cell mass (RCM) at 128% associated with a normal erythropoietin level. The bone marrow biopsy showed hypercellularity for age, panmyelosis associated with normal megakaryocytes and rare isolated abnormal enlarged forms. Using reticulin stain, no myelofibrosis was noted. Patient # 2 had hemoglobin at 194 g/L, hematocrit at 53% and low erythropoietin level without any dehydration. Both had moderately elevated platelet counts (658 and 575 x109/L respectively) with normal leukocyte counts. They were negative for BCR-ABL. No mutation was found in JAK2 exons 12, 13 and 14 by HRM and allele-specific real-time PCR or in MPL exon 10. Using HRM analysis, CALR mutations were suspected in both patients and confirmed using Sanger sequencing and product sizing analyses: CALR mutations were in both patients type 1 deletions (52-bp deletion; c.1092_1143del). To complete genomic tests made on peripheral blood granulocytes, we performed colony assays in methylcellulose and in collagen, picked single BFU-E colonies grown after 14 days in the presence of erythropoietin, and genotyped each colony individually for CALR. Of the 27 colonies genotyped, 6 had no PCR amplification and 21 harboured the same CALR mutation observed in peripheral blood granulocytes, i.e 52-bp deletion; c.1092_1143del. BFU-E were found heterozygous for CALR, with a mean allele burden of 49%. To our knowledge, these patients are the first cases of CALR-mutated PV to be reported. However, since a biclonalJAK2V617F and CALR MPN case recently reported, we cannot rule out the possibility of a biclonal disease involving a yet unknown mutation associated with a CALR mutation. On the other hand, the presence of a CALR mutation both in peripheral granulocytes and in BFU-E suggests that the CALR mutation plays a role in the polycythemia phenotype. Our observations highlight the fact that in the absence of JAK2 mutation, CALR mutations can also be associated with PV. In conclusion, our data indicate that testing JAK2-negative PV patients for CALR mutations may be useful. Disclosures No relevant conflicts of interest to declare.
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Guijarro-Hernández, Ana, and José Luis Vizmanos. "A Broad Overview of Signaling in Ph-Negative Classic Myeloproliferative Neoplasms." Cancers 13, no. 5 (February 26, 2021): 984. http://dx.doi.org/10.3390/cancers13050984.
Full textAbstract:
Ph-negative myeloproliferative neoplasms (polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF)) are infrequent blood cancers characterized by signaling aberrations. Shortly after the discovery of the somatic mutations in JAK2, MPL, and CALR that cause these diseases, researchers extensively studied the aberrant functions of their mutant products. In all three cases, the main pathogenic mechanism appears to be the constitutive activation of JAK2/STAT signaling and JAK2-related pathways (MAPK/ERK, PI3K/AKT). However, some other non-canonical aberrant mechanisms derived from mutant JAK2 and CALR have also been described. Moreover, additional somatic mutations have been identified in other genes that affect epigenetic regulation, tumor suppression, transcription regulation, splicing and other signaling pathways, leading to the modification of some disease features and adding a layer of complexity to their molecular pathogenesis. All of these factors have highlighted the wide variety of cellular processes and pathways involved in the pathogenesis of MPNs. This review presents an overview of the complex signaling behind these diseases which could explain, at least in part, their phenotypic heterogeneity.
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Ben Lassoued, Amin, Nathalie Beaufils, Caroline Bonnefoy, and Jean Gabert. "Calreticulin Gene Mutations in Suspicion of Myeloproliferative Neoplasms: A Single Institution Experience." Blood 124, no. 21 (December 6, 2014): 5569. http://dx.doi.org/10.1182/blood.v124.21.5569.5569.
Full textAbstract:
Abstract Recently, Klampfl et al. and Nangalia et al. reported recurrent somatic mutations in the calreticulin (CALR) gene in patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. All CALR mutations described to date are either deletions or insertions, and occur in exon 9 and result in a frameshift leading to a loss of KDEL sequence (endoplasmic reticulum retention motif) and multiple calcium biding sites with a new basic (instead of acidic) C terminal region. The aim of this work is to know how far the CALR mutations can fill the molecular diagnostic gap for myeloproliferative neoplasms (MPNs) left by JAK2 and MPL mutations. The study was approved by the local ethics committee. Our study population consists of 155 patients with platelet counts > 500 G/L sampled between 2012 and 2013 for suspicion of MPNs. Only JAK2 V617F negative patients were included. Analyses were performed using genomic DNA isolated from peripheral blood. Patients were screened for CALR mutations using a High Resolution Melting (HRM) and then the precise mutations were characterized by Sanger sequencing which is a new approach for this test. A total of 21 (13.5%) patients with CALR mutations were detected with 8 distinct variants. Among CALR mutations, type 1 (52 basepair deletion, c.1092_1143del) and type 2 (5 basepair insertion, c.1154_1155insTTGTC) were the most common (respectively 7 (33%) and 7 (33%) cases). Three patients had 2 concurrent mutations (insertion+substitution / Deletion+substitution / deletion +insertion). We found a deletion that has not been reported so far to our knowledge (c.1125_1147del). Sequencing is ongoing for 4 patients. Compared with negative patients, CALR-mutated patients demonstrated markedly higher platelet counts (median count: 820 vs 543 G/L). Disclosures No relevant conflicts of interest to declare.
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Rumi, Elisa, and Mario Cazzola. "How I treat essential thrombocythemia." Blood 128, no. 20 (November 17, 2016): 2403–14. http://dx.doi.org/10.1182/blood-2016-05-643346.
Full textAbstract:
Abstract Essential thrombocythemia (ET) is an indolent myeloproliferative neoplasm that may be complicated by vascular events, including both thrombosis and bleeding. This disorder may also transform into more aggressive myeloid neoplasms, in particular into myelofibrosis. The identification of somatic mutations of JAK2, CALR, or MPL, found in about 90% of patients, has considerably improved the diagnostic approach to this disorder. Genomic profiling also holds the potential to improve prognostication and, more generally, clinical decision-making because the different driver mutations are associated with distinct clinical features. Prevention of vascular events has been so far the main objective of therapy, and continues to be extremely important in the management of patients with ET. Low-dose aspirin and cytoreductive drugs can be administered to this purpose, with cytoreductive treatment being primarily given to patients at high risk of vascular complications. Currently used cytoreductive drugs include hydroxyurea, mainly used in older patients, and interferon α, primarily given to younger patients. There is a need for disease-modifying drugs that can eradicate clonal hematopoiesis and/or prevent progression to more aggressive myeloid neoplasms, especially in younger patients. In this article, we use a case-based discussion format to illustrate our approach to diagnosis and treatment of ET.
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Inano, Tadaaki, Marito Araki, Soji Morishita, Misa Imai, Yoshihiko Kihara, Maho Okuda, Masafumi Ito, et al. "Concomitant Occurrence of Polyclonal Hematopoiesis and Cell-Autonomous Megakaryopoiesis in Triple-Negative Essential Thrombocythemia." Blood 136, Supplement 1 (November 5, 2020): 27–28. http://dx.doi.org/10.1182/blood-2020-134494.
Full textAbstract:
Somatic mutations in JAK2, MPL, and CALR are found in approximately 80% of patients with essential thrombocythemia (ET), whereas the remaining patients are negative for disease-defining mutations and are defined as triple-negative (TN). Studies have shown that some patients with TN-ET harbor non-canonical mutations in JAK2 and MPL; however, the failure to identify recurrent mutations in most patients has made the pathogenesis of TN-ET ambiguous (Milosevic Feenstra et al. Blood 2016, Cabagnols et al. Blood 2016). In this study, we screened 483 patients suspected as having ET in a single center, performed mutation analysis for JAK2 V617F, CALR exon 9, and MPL exon 10, and centrally reviewed bone marrow specimens. We identified 23 patients with TN-ET based on the WHO 2016 criteria. Sequencing analysis of these patients revealed non-canonical mutations in JAK2 and MPL in 4 cases. Whole exome-sequencing analysis of genomic DNA from peripheral blood and CD3-positive cells from 9 patients revealed that 2 patients harbored somatic mutations in other genes; 7 patients showed no detectable somatic mutation. A STAT5 reporter assay revealed that unlike JAK2 V617F and MPL W515L, all non-canonical mutants of JAK2 or MPL activated STAT5 similar to wild-type proteins, suggesting that these mutations did not drive the disease. Statistical analysis of clinical records revealed that patients with TN-ET were mostly young (median age of 36.0 years), female (18/23, 78.3%), and had neither a history of thrombosis nor progression to secondary myelofibrosis and leukemia, demonstrating the unique characteristics of TN-ET. The presence of clonal hematopoiesis, analyzed using genomic DNA purified from granulocytes of peripheral blood from female patients in a human androgen receptor assay, revealed that only 1 out of 15 patients was clonal. Hypothesizing that TN-ET was reactive thrombocytosis, the concentrations of cytokines promoting platelet production such as thrombopoietin (TPO) and interleukin-6 (IL-6) in the serum were analyzed. However, no significant differences in concentrations were observed among ET with driver mutations, TN-ET, and healthy individuals. We next examined the capacity of hematopoietic stem cells from patients with TN-ET to form megakaryocytic colonies. CD34-positive cells purified from cryopreserved bone marrow cells were cultured in the absence or presence of TPO. CD34-positive cells derived from patients with TN-ET exhibited an equivalent capacity to form megakaryocytic colonies compared to those from patients with ET harboring a driver mutation, even in the absence of TPO (Figure 1). Thus, in TN-ET, megakaryopoiesis may have been induced in a cell-autonomous manner. In 10 patients with TN-ET with available blood count data, no sign of thrombocytosis was observed before ET development, indicating that thrombocytosis was not hereditary but rather occurred via an alternate mechanism, such as aberrations in epigenomic regulation that induced cellular transformation (Ohnishi et al, Cell 2015). Taken together, TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. Figure 1 Disclosures Komatsu: Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy.
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Abu-Tineh, Mohammad, Nancy Kassem, Mohammad Abdul-Jaber Abdulla, Omar Mohammad Ismail, Rola Ghasoub, Mahmood B. Aldapt, and Mohamed A. Yassin. "Outcome of Pregnancy in the Era of Pegylated Interferon Alpha 2a in Females with Essential Thrombocythemia: An Experience from Qatar." Case Reports in Oncology 13, no. 1 (March 26, 2020): 336–40. http://dx.doi.org/10.1159/000506447.
Full textAbstract:
Myeloproliferative neoplasms are a diversified group of diseases of the hematopoietic stem cell, such as essential thrombocythemia (ET) and polycythemia vera. They are mainly caused by mutations in the following genes: JAK2, CALR, and MPL. All carry an increased risk to transform into acute leukemia or chronic myelogenous leukemia along with thrombosis and hemorrhagic complications. Treatment of such disorders during pregnancy is a challenging footstep, given the high risk of complications for both the mother and the fetus. Here, we report about two pregnant females with ET that has been treated with pegylated interferon alpha with safe and effective outcome.
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Shide, Kotaro, Takuro Kameda, Masaaki Sekine, Ayako Kamiunten, Keiichi Akizuki, Yuki Tahira, Tomonori Hidaka, et al. "Physiological Expression of Calr Mutant Increases Cell Growth and Cytokine Independency in Human Cell Lines Expressing Mpl, and Develops Essential Thrombocythemia in Mice." Blood 128, no. 22 (December 2, 2016): 954. http://dx.doi.org/10.1182/blood.v128.22.954.954.
Full textAbstract:
Abstract Calreticulin (CALR) exon 9 mutations were reported in about two-thirds of JAK2 or MPL mutation negative ET and PMF patients. The mutations cause frameshifts that result in proteins with novel C-terminus.Retrovirus-mediated gene transfer into cell lines and mouse bone marrow (BM) cells is a common technique, but the expression level is very high compared to the physiological expression.We investigated the effects of physiological expression of mutant CALR using CRISPR/Cas9 gene editing techniques for cell lines, and as for the mouse model, we generated a transgenic mice (TG) expressing human CALR del52 mutant. We used two human cell lines expressing MPL: human acute megakaryoblastic leukemia cell line CMK11-5 which expressed endogenous MPL, and F-36P-MPL cell line which was generated by introducing MPL to GM-CSF-dependent erythroleukemia cell line F-36P. Plasmids coexpressing hCas9 and single-guide RNA were prepared by ligating oligonucleotides (5'-CACCGACAAGAAACGCAAAGAGGAGG-3', 5'-AAACCCTCCTCTTTGCGTTTCTTGTC-3') for the target sequence of human CALR exon 9 into pX330. The plasmids were introduced with a electroporator to each of the cell lines. After limiting dilution cloning, we identified cell lines which have indel mutation at the target site. We produced two types of CMK11-5 subline knocked in a CALR mutation, namely CALR del25 CMK cells and CALR del25/del17 CMK cells, respectively. The former lacks 25 bases in one CALR allele, causing a frameshift that results in a protein resembling human CALR mutant, while the latter lacks an additional 17 bases in another allele in CALR exon 9 and induces a frameshift that causes a deletion in CALR exon 9. Both kinds of CALR mutant CMK11-5 cells showed increased cell proliferation compared to WT cells. We also produced one type of F-36P-MPL subline, CALR del1/ins1 F-36P-MPL cells which had 1 base deletion in one CALR allele resembling human mutation and 1 base insertion in another allele. Though the growth of this subline in the presence of GM-CSF was comparable to WT cells, it showed GM-CSF independent autonomous cell growth. We generated TG mice expressing human CALR del52 mutant driven by the murine H2Kb promoter. We compared the expression level of human CALR mRNA in TG BM cells with the expression of endogenous WT CALR in human cell lines (CMK11-5, F-36P-MPL, CHRF288) using Rn18s as an endogenous control. The expression of human CALR in TG BM was approximately 0.6 times that of endogenous WT CALR in human cell lines, and the physiological expression level was obtained. They exhibited thrombocytosis, with platelet (PLT) counts as high as 2,000 x 109/L. Leukocyte number and the proportion of granulocytes and T and B lymphocytes, were comparable to WT mice. CALR mutation had no impact on Hb level or spleen weight. There was a striking difference in the number of megakaryocytes (Mgks), which was 2-fold higher in BM from TG mice than in WT mice. The TG Mgks were also more mature, with larger diameter, and contained higher number of alpha-granules compared to WT cells. In one year of observation, there is no fibrosis in BM. These observations showed that TG developed human ET-like disease. The survival of TG mice was comparable to that of WT mice. The disease phenotype was transplantable into WT recipient mice. To characterize in detail the impact of MPNs induced by the CALR del52 mutant, we evaluated the frequencies of HSCs and progenitors in BM. The frequency of both LT-HSC and ST-HSC in BM was higher inTG mice compared to WT mice. The frequencies of progenitors (CMP, MEP, and MKP) were also greater in BM from TG mice than from WT mice. However, BM cells did not have enhanced replating capacity. We next examined whether or not ruxolitinib (RUX) treatment ameliorated thrombocytosis in TG mice. Either 90 mg/kg bid of RUX or vehicle was administrated to TG mice for 4 weeks.TG mice treated with vehicle showed a mean 16% increase in PLT count during the treatment period, probably due to the disease progression. RUX treatment attenuated the increase in the number of PLTs in TG mice by a mean of 22%, but the overall count was still higher than that in WT mice. BM sections showed that RUX reduced the Mgks number in TG. In summary, physiological expression of CALR mutant increases cell growth and cytokine independency in human cell lines expressing MPL, and develops ET in mice. RUX therapy attenuated the increased numbers of peripheral blood PLTs and BM Mgks, and ameliorated CALR mutation-induced ET. Disclosures No relevant conflicts of interest to declare.
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Lim, Ken-Hong, Yu-Cheng Chang, Yi-Hao Chiang, Huan-Chau Lin, Ling Huang, Wei-Ting Wang, Ying-Wen Su, Ming-Chih Chang, Yi-Fang Chang, and Caleb Gon-Shen Chen. "Acquired Resistance to JAK Inhibitors in Calr-Mutated Myeloproliferative Neoplasms." Blood 134, Supplement_1 (November 13, 2019): 2970. http://dx.doi.org/10.1182/blood-2019-124420.
Full textAbstract:
Background: Calreticulin (CALR) mutations are one of the major driver mutations in BCL-ABL1-negative myeloproliferative neoplasm (MPN) and are frequently detected in JAK2/MPL-unmutated essential thrombocythemia and primary myelofibrosis. Mutant CALR activates JAK-STAT signaling through an MPL-dependent mechanism to mediate pathogenic thrombopoiesis in MPNs. Although JAK inhibitors such as ruxolitinib can provide important clinical benefits to MPN patients including those harboring CALR mutations, JAK inhibition does not preferentially target the MPN clone and acquired resistance develops over time. We aimed to characterize the mechanisms of acquired resistance to JAK inhibitors in CALR-mutated hematopoietic cells and to screen for novel therapeutic approaches specifically target CALR-mutant cells in this study. Methods: UT-7/TPO-derived cell lines expressing wild-type and type 1 and type 2 mutant CALR (CALRdel52 and CALRins5) were kindly provided by Drs. Komatsu and Araki. JAK2-inhibitor-resistant cells were generated by co-cultured with ruxolitinib and fedratinib (TG101348, a JAK2-selective inhibitor). JAK-STAT signaling was evaluated by Western blot on CALR-wild-type and mutated cells exposed to JAK2 inhibitor compared to untreated cells. For the detection of acquired secondary mutations in CALR-mutated cells exposed to JAK2 inhibitor, whole exome sequencing (WES) was performed using the BGISEQ-500 Sequencing platform (BGI, Shenzhen, China) with the 2 x 100 bp paired-end protocol. Genome Analysis Toolkit was used for variation detection. Reads were aligned to human reference genome hg19 using BWA version 0.7.15. Targeted resequencing was performed on leukocytes from patients with MPN who had been treated with ruxolitinib. Screening with chemical libraries/novel compounds will be conducted on UT7/TPO-CALR cell lines. Results: Compared to the parental cells, ruxolitinib-resistant UT7/TPO-CALR mutant cell lines have developed significant cross resistance to other JAK inhibitor as shown in the cell viability study. Signalling downstream of JAK2 in all 3 inhibitor-naïve UT-7/TPO/CALR parental cell lines was inhibited by acute treatment of ruxolitinib as shown on Western blot. Whereas, constitutive JAK2 activation was observed in all 3 inhibitor-resistant UT-7/TPO/CALR cell lines. No change in the expression of Epo and MPL receptors in these cell lines was found. Interestingly, constitutive JAK3 activation was also seen in inhibitor-resistant UT-7/TPO/CALR cells in comparison with parental cells. These findings indicated the presence of transphosphorylation by JAK3 in inhibitor-resistant UT-7/TPO/CALR cell lines. In addition, the results of WES identified several acquired secondary mutations in 3 inhibitor-resistant UT-7/TPO/CALR cell lines including SH2B1, ABCC1, HOXB3 and KRTAP4-5. No acquired secondary mutation was identified in CALR and other genes involved in JAK-STAT signaling. Acquired secondary mutation will be screened in primary MPN patients' samples treated with JAK inhibitor. Type II JAK inhibitor such as BBT-594 has been shown to inhibit JAK activation and signaling in JAK-persistent/resistant cells. Conclusions: Our results confirmed that the in vitro efficacy of JAK2 inhibition on CALR-mutant cells. Our data also suggested that JAK2 transphosphorylation and acquired secondary mutations could be underlying mechanisms for acquired resistance to JAK inhibitors in CALR-mutated cells. Novel therapeutics approaches should be developed to overcome acquired resistance in CALR-mutated cells. Disclosures No relevant conflicts of interest to declare.