MPL overexpression induces a high level of mutant-CALR/MPL complex: a novel mechanism of ruxolitinib resistance in myeloproliferative neoplasms with CALR mutations

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

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CALR mutations in a cohort of JAK2 V617F negative patients with suspected myeloproliferative neoplasms

Scientific Reports ◽

10.1038/s41598-019-56236-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Tanja Belcic Mikic ◽

Tadej Pajic ◽

Matjaz Sever

Keyword(s):

Sanger Sequencing ◽

Blood Count ◽

Mutation Detection ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Frameshift Mutations ◽

Secondary Conditions ◽

Non Invasive ◽

Calr Mutations

AbstractSuspicion of myeloproliferative neoplasms (MPNs) and especially essential thrombocythemia (ET) in primary care is often based solely on blood counts, with patients referred to a haematologist without a thorough evaluation. We retrospectively assessed the role of calreticulin gene (CALR) mutations in the diagnosis of MPN in this population. We studied CALR mutations in 524 JAK2 V617F-negative patients with suspected MPN. Uncommon CALR mutations were confirmed by Sanger sequencing and searched for in the COSMIC or HGMD database. Mutations were defined as frameshift or non-frameshift mutations. CALR mutations were detected in 23 patients (23/524 = 4.4%). Four mutations detected in our study were newly identified mutations. Non-frameshift mutations were detected in two patients. Most patients (380/524 = 72.5%) were diagnosed with secondary conditions leading to blood count abnormalities such as iron deficiency, inflammatory and infectious diseases, malignancy and hyposplenism. Nine patients (9/23 = 39%) were retrospectively diagnosed with ET based on CALR mutation confirmation. Two patients with non-frameshift CALR mutations were diagnosed with reactive thrombocytosis and MPN unclassifiable, respectively. Our study showed that CALR mutations are important, non-invasive diagnostic indicators of ET and can aid in its diagnosis. Moreover, the type of CALR mutation must be accurately defined, as non-frameshift mutations may not be associated with ET. Finally, CALR mutation detection should be reserved for patients with high suspicion of clonal haematological disease.

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Bioinformatic analyses of CALR mutations in myeloproliferative neoplasms support a role in signaling

Leukemia ◽

10.1038/leu.2014.190 ◽

2014 ◽

Vol 28 (10) ◽

pp. 2106-2109 ◽

Cited By ~ 10

Author(s):

L Eder-Azanza ◽

D Navarro ◽

P Aranaz ◽

F J Novo ◽

N C P Cross ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Calr Mutations

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Mutation Type As a Major Determinant of Clinical Phenotype in Myeloproliferative Neoplasms Associated with Mutant Calreticulin

Blood ◽

10.1182/blood.v124.21.3215.3215 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3215-3215 ◽

Cited By ~ 1

Author(s):

Daniela Pietra ◽

Elisa Rumi ◽

Chiara Milanesi ◽

Christian A Di Buduo ◽

Marta Bellini ◽

...

Keyword(s):

Amino Acids ◽

Clinical Phenotype ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Charged Amino Acids ◽

Calr Mutations ◽

Mutation Type ◽

Calr Mutation

Abstract About 25% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) carry a somatic mutation of CALR, the calreticulin gene [N Engl J Med. 2013;369:2379-90]. So far, more than 50 different indels in CALR exon 9 have been found, but a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2) are the most common lesions. All indels generate a novel C-terminus of the mutant protein, in which the endoplasmic reticulum retention signal KDEL is lost and the negatively charged amino acids are replaced by neutral and positively charged amino acids, disrupting the Ca-binding site. This suggests that both cellular dislocation and impaired Ca-binding activity may be involved in the abnormal proliferation of cells expressing a mutant calreticulin. It is still unclear, however, why the same mutant gene is associated with 2 different disease phenotypes (ET and PMF). In particular, little in known about the effect of the mutant protein on megakaryocyte biology and bone marrow collagen deposition. We studied the relationships between CALR mutation type, megakaryocyte biology, and clinical phenotype in patients with myeloproliferative neoplasms. According to the 2008 WHO criteria, 716 out of 892 patients had ET and 176 had PMF. Overall, 578 (65%) patients carried JAK2 (V617F), 230 (26%) had a CALR indel, and 84 (9%) had nonmutated JAK2 and CALR. Patients with MPL mutations were excluded. Twenty-six different types of CALR lesions were identified: 120 (52%) patients had type 1 mutation, 75 (33%) had type 2, and 35 (15%) carried other indels. The frequency of type 1 mutation was significantly higher in PMF than in ET (71% vs 46%, P=.004). All these variants involved 3 different stretches of negatively charged amino acids, with an increase in the isoelectric points (pI) of the mutant protein. As type 1 and type 2 mutations affected stretch I and III, respectively, the 26 indels were categorized into 3 groups on the basis of the stretch they affected: i) type 1-like (61%), affecting stretch I; ii) type 2-like (36%), stretch III; iii) and other types (3%), stretch II. The pI values were significantly different in the 3 groups (P<.001). The frequency of type-1 like mutations was significantly higher in PMF than in ET (82% vs 55%, P=.001). In vitro differentiated megakaryocytes from CALR-mutant patients displayed a significant increase in the extent of both intracellular Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry inside the cytoplasm, as compared with healthy controls. Megakaryocytes carrying type 1-like CALR mutations exhibited the highest amplitude of Ca2+ flows regardless of the type of disease. In ET, impaired Ca2+ homeostasis was accompanied by atypical proplatelet architecture (ie, more branches and bifurcations). With respect to clinical phenotype at diagnosis, ET patients with type 2-like CALR mutation showed a trend towards higher PLT count (P=.063) and lower age (P=.053), and significantly lower LDH values (P=.021) than those with type 1-like mutation. In a hierarchical cluster analysis including demographic, clinical and molecular data, CALR mutation type (1 vs 2) identified the 2 clusters with the highest dissimilarity. Considering all patients, those with type 2-like CALR lesions had a better survival than those with JAK2 (V617F) (96.1% vs 84.4% at 10 years, P=.039), while no difference was found between the 2 CALR mutation types. ET patients with type 2-like CALR mutations showed a lower risk of thrombosis than those with JAK2 (V617F) (P=.010). By contrast, ET patients with type 1-like CALR mutations had a higher risk of myelofibrotic transformation that those with type 2-like CALR mutations (P=.029) and especially those with JAK2 (V617F) (P=.011). Finally, PMF patients with type 1-like CALR variants had a better survival than those with JAK2 (V617F) (80.1% vs 48% at 10 years, P=.008). In summary, abnormalities in megakaryocyte calcium metabolism and proplatelet architecture are found in patients with CALR-mutant myeloproliferative neoplasms, and their extent is related to mutation type. Type 2-like CALR mutations are more likely to be associated with isolated thrombocytosis without bone marrow fibrosis, ie, with an ET phenotype. By contrast, type 1-like CALR mutations are generally associated with bone marrow fibrosis, ie, with a PMF phenotype. Thus, in CALR-mutant myeloproliferative neoplasms, the mutation type is a major determinant of the clinical phenotype. Disclosures No relevant conflicts of interest to declare.

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Calreticulin Mutations in JAK2-Negative Myeloproliferative Neoplasms. Experience in Our Center

Blood ◽

10.1182/blood.v124.21.5586.5586 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5586-5586

Author(s):

Maria Jose Penalva Moreno ◽

Carolina Martinez-Laperche ◽

Santiago Osorio Prendes ◽

Elena Buces Gonzalez ◽

Jose Luis Diez-Martin ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Statistical Correlation ◽

Type I ◽

Type Ii ◽

Multifunctional Protein ◽

High Clinical Suspicion ◽

Fluorescent Pcr ◽

Calr Mutations

Abstract Introduction: Calreticulin (CALR) is a multifunctional protein regulated by calcium that is located in the endoplasmic reticulum. Recently, mutations in the calreticulin gene have been described in patients with the diagnosis of essential thrombocytemia (ET) and primary myelofibrosis (PMF), mainly in JAK2-negative cases. CALR mutations are localized to exon 9 and generate deletions or insertions that lead to a frameshift change resulting in a mutant protein. The detection of these mutations helps in the actual diagnosis of JAK2 mieloproliferative syndromes (MPN). Our aim is to assess the utility of the determination of these mutations in the management of patients with diagnosis of MPN in our center. Patients and methods: This study includes 94 patients with diagnosis of JAK2-negative MPN retrospectively selected following clinical and analytical criteria between 2008 and 2014 in our center (Table 1, 2). CALR mutations were performed with the use of fluorescent PCR following the methods described by Klampf et al. (NEJM, 2013). Results: 94 patients were analyzed, 77 of them had the diagnosis of TE, 8 of PMF and 9 of others disorders of myelodisplastic/mieloproliferative. 22% of the cases of ET had mutations in CALR (Table 1). In these mutations, a total of 53% were type I mutations (52-bp deletion) and 47% were type II mutations (5-bp insertion). Only one mutation was infrequent, a 46-pb deletion. We have found statistical correlation in the number of platelets depending on the presence of the mutation and in the largest number of platelets in type II mutations. 33% of the cases of PMF had mutations in CALR, all of them type I. Among other diseases not included in MPN, one of them had a type I mutation (data not shown). Conclusions: Our results are close to recently published results regarding the frequency of mutation and as the largest number of platelets in type II mutations with respect to mutation type I. This study confirms the importance of CALR mutations determination in the diagnosis of JAK2-negative ET and PMF with high clinical suspicion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v126.23.5215.5215 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5215-5215

Author(s):

Munazza Rashid ◽

Rifat Zubair Ahmed ◽

Shariq Ahmed ◽

Muhammad Nadeem ◽

Nuzhat Ahmed ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Diagnostic Algorithm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Common Mutation ◽

Hypochondriac Region ◽

Exon 9 ◽

Calr Mutations

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

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Physical Interaction Between Mutant Calreticulin and the Thrombopoietin Receptor Is Required for Hematopoietic Transformation

Blood ◽

10.1182/blood.v126.23.lba-4.lba-4 ◽

2015 ◽

Vol 126 (23) ◽

pp. LBA-4-LBA-4 ◽

Cited By ~ 1

Author(s):

Shannon Elf ◽

Nouran Abdelfattah ◽

Edwin Chen ◽

Javier Perales-Patón ◽

Emily Rosen ◽

...

Keyword(s):

Tertiary Structure ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Gene Set Enrichment Analysis ◽

Common Mutation ◽

Physical Interaction ◽

C Terminus ◽

Thrombopoietin Receptor ◽

Arginine Residues ◽

Calr Mutations

Abstract Somatic mutations in calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, are found in up to 40% of patients with myeloproliferative neoplasms (MPN). All pathologic CALR mutations are out-of-frame insertion and/or deletions (indels) in exon 9, generating a 1 base-pair (bp) frame shift and a common mutant-specific C-terminus, with the most common mutation being a 52 bp deletion (del52). The observation that CALR mutations are mutually exclusive with other MPN-initiating mutations such as JAK2V617F suggests a key pathogenic role for mutant CALR. To determine if mutant CALR alone is sufficient to induce MPN we began by over-expressing CALR-del52 in a retroviral bone marrow transplant (BMT) mouse model. We found that CALR-del52-expressing mice develop thrombocytosis and megakaryocytic hyperplasia, recapitulating the megakaryocyte-specific phenotype of CALR-mutant MPN patients. These findings suggest that the thrombopoietin receptor, MPL plays a key role in the pathogenesis of mutant CALR-driven MPN. To evaluate the role of MPL in mutant CALR driven oncogenesis, we over-expressed CALR-del52 in interleukin-3 (IL-3)-dependent Ba/F3 hematopoietic cells. We found that CALR-del52 over-expression results in transformation to IL3-independent growth only in Ba/F3 cells co-expressing MPL, but not in parental Ba/F3 cells or Ba/F3 cells co-expressing the EPO receptor (EPOR) or the G-CSF receptor (GCSFR). We found similar results in human cytokine-dependent UT-7 cells. We also introduced +1 frameshift mutations into the endogenous Calr locus in Ba/F3-MPL cells using CRISPR/Cas9 gene editing and successfully engendered IL-3 independent growth, indicating that endogenous levels of mutant Calr expression are sufficient for transformation. Together, these data indicate that MPL is specifically required for the transforming capacity of mutant CALR. Using RNA-sequencing followed by gene set enrichment analysis (GSEA), we confirmed that mutant CALR transformed Ba/F3-MPL cells display strong enrichment of Stat5 and Stat3 gene expression signatures. Concordantly, we also saw differential phosphorylation of Stat5 and Stat3 in these cells. Furthermore, we found that the IL-3 independent proliferation of mutant CALR expressing Ba/F3-MPL cells is decreased upon shRNA-mediated knockdown of Jak2, and that differential activation of Stat5 and Stat3 is abrogated by the JAK2 inhibitor, ruxolitinib. Together, these data demonstrate that mutant CALR signals through the JAK/STAT axis downstream of MPL. We next sought to define the specific domains within mutant CALR required for oncogenic transformation. We found that neither expression of the mutant C-terminus alone nor expression of CALR lacking the C-terminus leads to cytokine-independent growth, suggesting that the novel C-terminus is necessary (but not sufficient) for transformation. We therefore generated an extensive series of truncation, domain deletion and point mutations within the C-terminus and assessed their respective transforming capabilities. Surprisingly, we found that the oncogenic activity of mutant CALR is not encoded within a specific sequence or domain of the mutant C-terminus. Rather, we found that the positive electrostatic charge of the mutant C-terminus is critical for its transforming capacity. Mutagenizing all 18 lysine/arginine residues (positively charged) within the C-terminus to a neutral glycine residue abrogates CALR-del52 transformation activity. In contrast, mutagenizing the 18 non-lysine/arginine residues within the C-terminus to glycine does not affect transforming activity, a remarkable finding considering that, in this mutant, 50% of the amino acids have been modified. Finally, using co-immunoprecipitation assays we found that mutant CALR, but not wild-type CALR, physically interacts with MPL, and that neither the mutant C-terminus alone nor mutant CALR lacking the C-terminus can bind to MPL. This suggests that the tertiary structure of mutant CALR is required for binding to MPL. Moreover, we found that the ability of our engineered CALR mutants to bind MPL perfectly correlates with their ability to mediate transformation, suggesting that the interaction with MPL is critical for mutant CALR-mediated transformation. Together, our findings elucidate a novel mechanism of pathogenesis in MPN and provide insights into how CALR mutations drive the development of MPN. Disclosures: No relevant conflicts of interest to declare.

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Germline Calr Mutation and Thrombocytosis Presenting with Concomitant BCR-ABL1+ CML

Blood ◽

10.1182/blood.v128.22.5494.5494 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5494-5494 ◽

Cited By ~ 1

Author(s):

Linda B. Baughn ◽

Scott Gilles ◽

Elizabeth L. Courville ◽

Andrew C. Nelson ◽

Zohar Sachs

Keyword(s):

Platelet Count ◽

Allele Frequency ◽

Peripheral Blood ◽

Myeloproliferative Neoplasms ◽

Germline Mutations ◽

Myelogenous Leukemia ◽

Base Pair Deletion ◽

Exon 9 ◽

Calr Mutations ◽

Calr Mutation

Abstract CALR mutations are present in 70-84% of JAK2 wild-type myeloproliferative neoplasms (MPN) and 67% and 88% of essential thrombocytopenia (ET) and primary myelofibrosis (PMF) respectively. Most cases of MPN are apparently sporadic, but 7-11% have evidence of familial predisposition. While germline mutations in ET-associated genes, MPL and JAK2, have been described in hereditary thrombocytosis, germline mutations in CALR have not been described in any setting. Two types of CALR mutations are common in MPN: a 52-base pair deletion (bp) and a 5 bp insertion, both in exon 9. With rare exceptions, CALR mutations are generally mutually exclusive with JAK2 or MPL mutations and have very rarely been reported in conjunction with the BCR-ABL1translocation. Here, we report a patient with a germline CALR mutation, thrombocytosis, and subsequent development of BCR-ABL+ CML. A 67-year-old female with no significant medical history presented with severe abdominal pain and nausea. Peripheral blood analysis revealed a marked leukocytosis composed of 66% neutrophils, 16% myelocytes, 6.5% monocytes, 3.5% basophils, 2.5% promyelocytes, 2.5% metamyelocytes, 1.5% lymphocytes, 1.5% blasts, and no eosinophils. The patient was non-anemic and had a normal platelet count (340,000/mm3). Bone marrow biopsy revealed a hypercellular marrow with myeloid predominant trilineage hematopoiesis and 1-2% blasts with morphology consistent with chronic myelogenous leukemia (CML). Fluorescence in-situ hybridization analysis of peripheral blood identified a BCR-ABL1fusion in 98.5% of interphase cells. After 3 months of standard imatinib therapy, quantitative RT-PCR showed a reduction of BCR-ABL1/ABL1 in the peripheral blood, however platelet count was elevated at 539,000/mm3. Thrombocytosis persisted over 2 years with a maximal platelet count of 584,000/mm3. Given the patient's thrombocytosis, her peripheral blood was subjected to a next generation sequencing of JAK2, MPL, and CALR genes. A 52-bp out-of-frame deletion in exon 9 of the CALR gene was detected (52% allele frequency) in peripheral blood. In addition, the same 52-bp CALR deletion (63% allele frequency) was present at the time of diagnosis and within a buccal specimen (47% allele frequency) when the BCR-ABL1 transcript was 1% in the peripheral blood. Immunostain of the buccal sample was strongly positive for cytokeratin (CK) AE1/AE3 but CD45 was not detected indicating no leukocyte contamination. This case reports the first instance of a germline CALR mutation associated with thrombocytosis and is the fourth report of the co-occurrence of BCR-ABL1 and CALR mutation in a single patient. Evolution to BCR-ABL1+ CML suppressed the CALR-mutant thrombocytosis phenotype, emphasizing the effect of these genes on lineage determination in abnormal myeloid proliferation. Disclosures No relevant conflicts of interest to declare.

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Calreticulin mutations Are Present in Polyclonal As Well As Clonal ET

Blood ◽

10.1182/blood.v124.21.4590.4590 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4590-4590

Author(s):

Xylina Gregg ◽

Sabina Swierczek ◽

Soo Jin Kim ◽

Josef T. Prchal

Keyword(s):

T Cells ◽

X Chromosome ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Calr Mutations ◽

Calr Mutation

Abstract First and second authors contributed equally During female embryogenesis, most of the genes in either the maternal or paternal X-chromosome are randomly inactivated in each cell, a process that remains remarkably constant in their progeny. X-chromosome inactivation has been used to define clonality in myeloproliferative neoplasms (MPNs) such polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET). One such method to determine clonality uses a quantitative, transcriptional clonality assay based on conservative exonic polymorphisms in five X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD). Females who are heterozygous for any of these polymorphisms are considered “informative” and can be studied for clonality by interrogating their platelets’ and granulocytes’ RNA allelic usage ratio. JAK2 mutations occur in >95% of PV and 50-60% of ET and PMF; cMPL mutations are found in another 5-10% of ET and MF. Somatic calreticulin (CALR) mutations have been identified in a majority of patients with ET and MF who lack JAK2 and cMPL mutations. CALR mutations are reported to be associated with a more favorable prognosis and are believed to be acquired early in the disease course. More than 30 CALR mutations have been described, but type 1 (52-bp deletion; c.1092_1143del) and type 2 (5-bp insertion; c.1154_1155insTTGTC) mutations are the most frequent. We analyzed 61 females informative for a transcriptional clonality assay and 44 males with unexplained thrombocytosis or marrow fibrosis and no detectable JAK2 or cMPL mutations for CALR mutations in their granulocytes. With the exception of an absence of a clonal marker, these patients met WHO criteria for ET or PMF. A CALR mutation (20 type 1 and 17 type 2) was present in 37 of these 105 patients (22 females and 15 males). One of the CALR mutated females had a paternal grandmother with JAK2V617F –positive PV, confirming a previous report that, in familial clustering of MPNs, affected individuals may carry different disease-defining somatic mutations. In those CALR positive patients who had available T cells, no detectable CALR mutations were found in their T cells. In one of these subjects, CD34+ cells were available and had a similar mutation level as in the granulocytes. Of the 22 females with a CALR mutation, 19 had clonal hematopoiesis, but 3 had polyclonal hematopoiesis; all 3 had previously unexplained thrombocytosis. None of these patients had any prior treatment for thrombocytosis. Clonal hematopoiesis was present in 26 of the 39 females without a CALR mutation. All female patients with myelofibrosis had clonal hematopoiesis, regardless of CALR mutation status. In contrast to the polyclonal hematopoiesis seen in some CALR positive ET patients, 166 informative PV and JAK2V617F-positive ET or PMF females all had clonal hematopoiesis. We report that CALR mutations are associated with polyclonal hematopoiesis in some ET patients. This finding differs from JAK2V617F-positive ET and PMF and PV females, where clonal hematopoiesis was always seen. This indicates that CALR mutated clones have a weaker suppressive effect on residual normal hematopoietic stem cells than JAK2 mutated clones and may contribute to the possibly more benign course of CALR mutated ET. The CALR mutation was not detected in T cells, which also differs from JAK2V617F mutated MPNs, where a small level of the JAK2 mutation is often detected in T cells. Similar to other reports, we found a lower prevalence of the CALR mutation in JAK2 or cMPL non-mutated ET and PMF than initially described. Disclosures No relevant conflicts of interest to declare.

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