This review aimed to evaluate the molecular mechanism underlying the development of myeloproliferative neoplasms (MPN) caused by mutant calreticulin (CALR). This mutation is found in a subset of patients with Philadelphia chromosome-negative MPNs, and it encodes a molecular chaperone. However, it is essentially impossible to elucidate the oncogenic property of mutant CALR from the wild-type CALR function. Studies have reported that mutant CALR forms a homomultimeric complex via intermolecular interaction between novel domains acquired due to a frameshift mutation, gains a high binding affinity for myeloproliferative leukemia protein (MPL), the thrombopoietin receptor, through a presumptive structural change, and acts as an agonist for MPL. In this review, I would like to describe the course of the discovery of this unique molecular mechanism and discuss future scope of research on mutant CALR.

Keywords: Calreticulin; Janus kinase 2; Myeloproliferative neoplasms; Thrombopoietin receptor.

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.

BACKGROUND

Primary myelofibrosis (PMF) is a chronic clonal myeloid disorder. Together with essential thrombocythemia (ET) and polycythemia vera (PV), it belongs to a group of Philadelphia chromosome-negative myeloproliferative neoplasms. Thrombotic events are serious complications negatively influencing the quality and length of these patients' lives. The confirmed risk factors for venous thromboembolism are age over 60 years, a positive history of thromboembolism, presence of common cardiovascular risks, JAK2 V617F mutation and, according to some authors, leukocytosis. Various opinions on the role of thrombocythemia have been published. The present study was undertaken to evaluate the benefit of thrombin generation test and its potential use in predicting the risk of thrombosis in MF patients.

METHODS

The analysis included plasma samples obtained from 36 patients diagnosed with MF in our center from 2004 to 2016 (JAK2 V617-positive 53%; CALR-positive 31%; MPL-positive 14%; triple negative 2%) and a control group comprising 20 healthy volunteer blood donors. Thrombin generation was measured in platelet-rich plasma using the TECHNOTHROMBIN® TGA kit (Technoclone, Austria) and the fully automated system Ceveron® Alpha (Technoclone). The results were correlated with clinical and laboratory parameters of the patients.

RESULTS

There were differences in thrombin generation as expressed by endogenous thrombin potential (ETP) between patients and healthy controls, with ETP being lower in the patient group (p = 0.0003). Analysis confirmed a significant correlation between thrombin generation and platelet counts, with higher thrombin generation in patients with thrombocythemia>> 400 x 109/L (p = 0.04). ETP values were consistently higher in earlier disease stages and lower in CALR-mutated myelofibrosis.

CONCLUSIONS

In MF patients, thrombin generation is mainly influenced by platelet counts and, to a lesser extent, by mutation status, activity, and progression of the disease. Thrombin generation test results have confirmed that thrombocythemia is a potential risk factor for thrombotic complications.

Essential thrombocythemia (ET) is a rare clonal myeloproliferative disorder with a prevalence rate of approximately 1-3 cases per 100 000 individuals per year. ET is characterized by a persistent increase in the platelet count with hyperplasia of bone marrow megakaryocytes. It is difficult to make a diagnosis of ET, because most thrombocythemia are reactive to certain disease conditions including iron deficiency anemia, infection, collagen diseases and malignant tumors. Mutation in the Janus kinase (JAK)2 gene is present in approximately 50-70% of ET patients, and somatic mutations in the calreticulin (CALR) gene were recently discovered in approximately 20-25% of sporadic patients with ET or primary myelofibrosis. Various cutaneous manifestations of ET often occur by microvascular thrombosis and precede severe arterial and venous thromboembolic events in other organs. Therefore, in order to prevent such severe events, it is important to make an early diagnosis of ET based on a number of cutaneous manifestations. Here, we report two cases of ET diagnosed based on livedo racemosa on feet with gene mutations in JAK2 and CALR, respectively, and show the pathological and immunohistological findings of the livedo resulting from platelet thrombosis rather than vasculitis. We also review the cutaneous manifestations in current published reports of Japanese ET patients. Our patients were successfully treated with low-dose aspirin, a vasodepressor and hydroxyurea, following regressed livedo and reduced platelet counts.

Myeloproliferative neoplasms (MPNs) are associated with the fewest number of mutations among known cancers. The mutations propelling these malignancies are phenotypic drivers providing an important implement for diagnosis, treatment response monitoring, and gaining insight into the disease biology. The phenotypic drivers of Philadelphia chromosome negative MPN include mutations in JAK2, CALR, and MPL. The most prevalent driver mutation JAK2V617F can cause disease entities such as essential thrombocythemia (ET) and polycythemia vera (PV). The divergent development is considered to be influenced by the acquisition order of the phenotypic driver mutation relative to other MPN-related mutations such as TET2 and DNMT3A. Advances in molecular biology revealed emergence of clonal hematopoiesis (CH) to be inevitable with aging and associated with risk factors beyond the development of blood cancers. In addition to its well-established role in thrombosis, the JAK2V617F mutation is particularly connected to the risk of developing cardiovascular disease (CVD), a pertinent issue, as deep molecular screening has revealed the prevalence of the mutation to be much higher in the background population than previously anticipated. Recent findings suggest a profound under-diagnosis of MPNs, and considering the impact of CVD on society, this calls for early detection of phenotypic driver mutations and clinical intervention.

Keywords: JAK2; cardiovascular disease; clonal hematopoiesis; mutation screening; myeloproliferative neoplasm; somatic mutations.

Essential thrombocythemia (ET) is a classical myeloproliferative neoplasm that is susceptible to hypercoagulable state due to impaired hemostatic system, so that thrombotic complications are the leading cause of mortality in ET patients. The content used in this article has been obtained by the PubMed database and Google Scholar search engine from English-language articles (2000-2019) using the following keywords: "Essential thrombocythemia," "Thrombosis," "Risk factors" and "Hemostasis. In this neoplasm, the count and activity of cells such as platelets, leukocytes, endothelial cells, as well as erythrocytes are increased, which can increase the risk of thrombosis through rising intercellular interactions, expression of surface markers, and stimulation of platelet aggregation. In addition to these factors, genetic polymorphisms in hematopoietic stem cells (HSCs), including mutations in JAK2, CALR, MPL, or genetic abnormalities in other genes associated with the hemostatic system may be associated with increased risk of thrombotic events. Moreover, disruption of coagulant factors can pave the way for thrombogeneration. Therefore, the identification of markers related to cell activation, genetic abnormalities, or alternation in the coagulant system can be used together as diagnostic and prognostic markers for the occurrence of thrombosis among ET patients. Thus, because thrombotic complications are the main factors of mortality in ET patients, a hemostatic viewpoint and risk assessment of cellular, genetic, and coagulation factors can have prognostic value and contribute to the choice of effective treatment and prevention of thrombosis.

Keywords: Essential thrombocythemia; Hemostasis; Risk factors; Thrombosis.

After the discovery of the JAK2V617F mutation in 2005, followed by the discovery of the MPL and JAK2 exon 12 mutations, and the CALR mutation in 2013, the majority of patients with Philadelphia negative myeloproliferative neoplasms (MPN) have been shown to display one of these somatic mutations. In regards to the pathogenesis of MPN, the CALR mutation has been shown to activate the JAK2 downstream cascade. However, the mechanism has yet to be elucidated. Nevertheless, these investigations have changed our understanding of MPN, especially in terms of diagnosis and treatment. Reports on the clinical effects of the different somatic mutations have revealed that MPN patients with CALR-mutated ET are less likely to have thrombosis, and that PMF patients with the CALR mutation have better overall survival. Hereafter, evaluations of the status of these gene mutations are necessary for the diagnosis of MPN, and it is important to consider how to put these genetic data to the best possible practical use.

Non-cirrhotic, non-malignant portal vein thrombosis (PVT) is commonly secondary to inherited or acquired prothrombotic states. However, even after extensive workup, 25% of patients with PVT have no apparent prothrombotic aetiology identified (idiopathic PVT). Inherited conditions include factor V Leiden, PT mutation and protein C/S/AT deficiency. Acquired conditions include APS, PNH and BCR-ABL 1-negative myeloproliferative neoplasms (MPN). BCR-ABL-1 negative MPNs are the most frequent underlying prothrombotic risk factor for PVT (15%-30%). However, peripheral blood counts often remain within normal ranges in these patients with MPN because of portal hypertension sequel. Despite suggestive features of MPN in bone marrow, these patients lack adequate diagnostic criteria and are classified as occult MPN. The discovery of recurrent molecular abnormalities such as CALR gene exon 9 mutation presented a crucial advance in the diagnosis of occult MPNs. In our patient, the diagnosis of MPN was made on this basis, despite lack of peripheral evidence of MPN.

BACKGROUND

The myeloproliferative neoplasms ET and PV are characterized by a high incidence of both arterial and venous thrombosis, and/or microcirculatory disturbances. Three somatic mutations, i.e. JAK2-V617F, Calreticulin (CalR) and MPL, commonly found in these diseases, correlate with different thrombotic risk levels.

OBJECTIVE

To analyze the influence of JAK2-V617F, CalR and MPL mutations on PLT adhesion, evaluated by a dynamic method under flow conditions in a group of patients with ET and PV.

METHODS

86 patients, i.e. 51 ET (19 M/32 F; age range 32-86 years) and 35PV (22 M/13 F; 41-83 yrs.), and 24 healthy controls (13 M/11 F; 28-61 yrs.) were enrolled upon informed consent. For the adhesion assay, peripheral venous whole blood was perfused over collagen for 4' at a 1,000 s-1 shear rate. PLTs were then stained with an anti-P-selectin-FITC antibody to evaluate PLT activation, and annexin V-AlexaFluor647 to detect procoagulant phosphatidylserine expression. Then, images of adherent PLTs in random fields were taken using phase contrast and fluorescence imaging by EVOS® fluorescence microscope. Results are mean±SEM of the % area covered by PLTs, or as the % of adherent PLTs positive for P-selectin or phosphatidylserine. Main hematological parameters and mutational status were recorded.

RESULTS

PLT adhesion was significantly (p<0.01) greater in ET (44.6±1.6%) and PV patients (49.0±1.9%) compared to controls (37.9±1.7%). In ET, PLT adhesion was highest in JAK2-V617F mutation carriers (n=23), followed by CalR-positive (n=16) and triple negative subjects (n=9), and lowest in the MPL-positive patients (n=3). In PV, no difference in PLT adhesion was observed between JAK2-V617F heterozygous and homozygous subjects. P-selectin expression by adherent PLTs was not statistically different between patients and controls. Differently, phosphatidylserine expression on adherent PLTs was significantly reduced (p<0.01) in both ET and PV compared to healthy subjects. In ET patients, a significant (p<0.05) correlation was found between PLT adhesion and PLT count in JAK2-V617F and CalR-positive mutation carriers. Multivariate regression analysis adjusted for age and sex, confirmed PLT count as a significant determinant of PLT adhesion in JAK2-V617F positive patients only.

CONCLUSIONS

ET and PV platelets show an increased adhesion to collagen in vitro, particularly in those carrying the JAK2-V617F mutation. A prospective study is ongoing to evaluate the predictive value of our PLT thrombus formation dynamic model for the thrombotic risk in ET and PV patients.

UNASSIGNED

Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.).

Bleeding and thrombosis represent common complications in myeloproliferative neoplasms (MPN) and significantly contribute to morbidity and mortality. Molecular markers, including CALR mutations, were considered not only as diagnostic markers, but also as risk factors for bleeding and thrombosis associated with MPN, especially for patients in remote primary hospitals. We sought to develop an easy-to-use assay for the rapid detection of CALR type 1 (CALR-1) and type 2 (CALR-2) mutations in Philadelphia chromosome-negative MPN patients. Peptide nucleic acid-locked nucleic acid (PNA-LNA) clamping loop-mediated isothermal amplification (LAMP) assays were established, which were integrated into a centrifugal compact disc (CD) microfluidic platform. A total of 158 clinical blood samples were tested simultaneously by this microfluidic platform and an in-house real time PCR assay. The detection performance of the LAMP arrays was validated and conflicting results were identified by Sanger sequencing. The results suggested that the LAMP methods we developed exhibited good sensitivity, specificity, and precision. By real time fluorescence assay the detection limit for CALR-1 and CALR-2 mutations could reach as low as 1% and 0.5% respectively, and 10% and 5% respectively by visual method. There were no nonspecific background amplifications among different detection systems. For the CALR-1 and CALR-2 LAMP detection systems, intra-batch CV values of 1% mutated plasmid were 10.56% and 10.51% respectively, and the inter-batch CV values were 19.55% and 18.39%, respectively. The products were all analyzed by melting curve analysis and electrophoresis followed by Sanger sequencing analysis, which were consistent with the database sequences. The microfluidic platform could complete rapid detection of CALR-1/2 mutations within 60 min. The results of clinical samples detected by our CD-like microfluidic chipLAMP assay and rtPCR assay suggested that 133 samples were CALR wild type, 15 were CALR-1 mutation type, and 9 were CALR-2 mutation type. The correlation coefficient value (Kendall's tau_b) of the two assays was 0.99. Interestingly, by the newly established detection platform, we were surprised to find that one patient of Chinese origin harbored both CALR-1 and CALR-2 mutations. This result was verified by Sanger sequencing analysis. The LAMP detection systems developed herein displayed good sensitivity, specificity, and stability. Additionally, the detection results could be directly judged by color changes of the reaction systems without any auxiliary equipment. Thus, the platform we developed has the potential of being widely used in remote and economically undeveloped areas in the future.

The clinical phenotypes and prognoses of CALR-mutant myeloproliferative neoplasms depend on the mutation type. The 2 most common mutations, type 1 (52-bp deletion) and type 2 (5-bp insertion), account for 85% of CALR-mutated neoplasms. The former confers a myelofibrotic phenotype, and the latter is associated with a low risk of thrombosis and an indolent clinical course. Individual case reports for patients with novel pathogenic CALR mutations are rare. Herein, we present the first case in the literature, to our knowledge, of a 63-year old ethnic Korean man with essential thrombocythemia who was diagnosed with a novel +1-bp frameshift mutation in CALR, which was predicted to exhibit a type 2-like phenotype.

Due to the central role of platelets in hemostasis, the clinical relevance of quantitative changes in platelet counts (< 150 G/l or>> 450 G/l) may be significant. Thrombopoesis (= production of platelets) occurs in the bone marrow, and the hormone thrombopoetin takes control on its regulation.In thrombocytosis, primary causes have to be distinguished from the far more common reactive (= secondary) reasons. The most important form of primary thrombocytosis occurs in myeloproliferative neoplasms especially in essential thrombocythemia (ET). Clinically, increased thrombophilia, microcirculatory disturbances as well as an increased hemorrhagic diathesis occur in patients with myeloproliferative neoplasms. According to the WHO diagnosis criteria 2016 standard diagnostic procedure in myeloproliferative neoplasms includes bone marrow biopsy and the detection of one of the acquired and typical MPN mutations in the JAK2, MPL or CALR gene.In contrast, patients with thrombocytopenia more often suffer from bleeding complications, however, in antiphospholipid syndrome or thrombotic microangiopathy (TMA) thrombotic events occur in spite of a low platelet count. Generally it makes sense to differentiate between pathological changes in thrombopoesis and the various causes of increased peripheral platelet turnover. Concerning differential diagnosis a careful anamnesis is very important in order to get hints like drugs associated with thrombocytopenia, signs of infection or autoimmune disorders. As an initial diagnostic approach we recommend examination of the blood smear in order to exclude pseudothrombocytopenia or disorderes like thrombotic microangiopathy, myelodysplasia or other hematological diseases.

Spliceosome genes (SF3B1, SRSF2, U2AF1 and ZRSR2) are commonly mutated in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS). JAK2, MPL and CALR mutations are associated with myeloproliferative neoplasms (MPN). Although SF3B1 and MPN-associated mutations frequently co-occur in the rare entity MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), myeloid neoplasms with concurrent spliceosome and MPN-associated mutations encompass many disease entities and are not well characterised.

Specimens from 2016 to 2019 with concurrent spliceosome and MPN-associated mutations were identified, and the clinicopathologic features were assessed.

RESULTS

The 36 cases were divided into mutational categories based on their spliceosome mutation. At diagnosis, cases with concurrent U2AF1 and MPN-associated mutations had lower leucocyte counts and platelet counts than did the other groups. Cases with mutant SRSF2 were more likely to have ASXL1 and IDH2 mutations, while U2AF1-mutated neoplasms were more likely to have an abnormal karyotype. The most common SF3B1 K700 and U2AF1 S34 mutational hotspots were underrepresented in our cohort of myeloid neoplasms with concurrent spliceosome and MPN-associated mutations, as SF3B1 and U2AF1 mutations tended to involve other codons. Numerous WHO-defined disease entities were represented in each spliceosome gene category; although MDS/MPN-RS-T were only identified in the group with SF3B1 mutations, they constituted only 1/4 of the neoplasms in the category.

Myeloid neoplasms with different mutant splicing factor and concurrent MPN-associated mutations demonstrate somewhat different clinical and pathologic features, but t he association between genotypes and phenotypes in these overlapping neoplasms is not straightforward.

Somatic CALR mutations have been identified in the majority of JAK2 mutation-negative essential thrombocythaemia (ET) and primary myelofibrosis. Almost all CALR mutations have been reported to typically generate a +1-base pair (bp) frameshift in the open reading frame. Here, we describe an ET patient with a +2-bp frameshift CALR mutation. A 41-year-old man was admitted because of headache, and diagnosed as JAK2-negative ET. After 4 years, his disease progressed to post-ET myelofibrosis, and CALR mutation analysis demonstrated a +2-bp frameshift CALR mutation caused by two different CALR mutations, c.1139_1151del and c.1211_1215delinsTTGA, on the same allele. The resultant mutant protein sequence shared 19 amino acids with those from type 1 and type 2 CALR mutations, but the downstream C-terminal sequences were different. To our knowledge, CALR double mutations causing +2-bp frameshift are extremely rare. Identification of this novel type of CALR mutation has potential implications for better understanding of CALR oncogenesis.

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.

Keywords: Essential thrombocythemia; Platelet; Plateletpheresis; Thrombocytapheresis; Thrombocytosis.

Essential thrombocytosis is also known as essential thrombocythemia (ET). It was first recognized in 1934; however, at that time, it was described as hemorrhagic thrombocythemia. Essential thrombocytosis is one of the myeloproliferative neoplasms. It was classified as a myeloproliferative neoplasm in 1951 by Damesheck. Myeloproliferative neoplasm includes polycythemia vera, primary myelofibrosis, and essential polycythemia. The three types of myeloproliferative neoplasm are similar as they share the same mutations. Approximately 55% of patients with essential thrombocytosis have the JAK2 mutation. Essential thrombocytosis is characterized by thrombocytosis with the presence of megakaryocytic hyperplasia in the bone marrow. Due to thrombocytosis, there are risks of vascular events such as thrombosis and hemorrhage and sometimes the conversion to a blast phase of myelofibrosis. According to the World Health Organization, essential thrombocytosis is a disease that occurs when the platelet count is more than 450000 with the presence of Janus kinase 2 (JAK2), Calreticulin (CALR) or myeloproliferative leukemia virus oncogene (MPL) mutation, lacking clonal or reactive causes. This review article will focus on reviewing the etiology, epidemiology, pathophysiology, evaluation, and treatment of this disease.

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

In 2003, a 60-year-old man presenting with thrombocytosis was referred to our hospital. Laboratory tests revealed normal white blood cell count and hemoglobin level. Bone marrow examination showed an increased number of megakaryocytes with dysplasia. G-banded karyotype analysis revealed del (5q). Initially, the patient was diagnosed with myelodysplastic/myeloproliferative neoplasm (MDS/MPN), and it was treated with aspirin and hydroxyurea. During the treatment course, fluorescence in situ hybridization for CSF1R and EGR1 was performed to detect del (5q), which showed negative results. In 2017, the patient had increased platelet count despite receiving treatment. A comprehensive genomic profiling revealed that the deleted region in this case was present in 5q14-5q23, which was different from the common deleted region of 5q- syndrome (5q32-5q33, where CSF1R was present) and that of high-risk MDS or acute myeloid leukemia (5q31, where EGR1 was present). Moreover, a CALR mutation was also detected. This case met the diagnostic criteria of essential thrombocythemia. The platelet count decreased with the administration of anagrelide. In conclusion, comprehensive genetic profiling is very important, and it leads to accurate diagnosis and therapy.

Recently, mutations have been identified in the calreticulin (CALR) gene in JAK2 or myeloproliferative leukemia negative patients with myeloproliferative neoplasm. A 49-year-old male patient with incidental thrombocytosis was investigated for CALR mutation by direct sequencing method. The patient carried two novel monoallelic somatic mutations, the L367fs*52 and the p.R368W in the CALR gen, which resulted in a novel C-terminal sequence. The absent endoplasmatic reticulum retention signal in the mutant CALR results in an altered subcellular localization of the mutant protein. The new positively charged C-terminal domain has an importance for oncogenicity, effecting different signaling pathways, activating the cytokine-independent growth of the cells and down-regulating the apoptotic signaling. But the new, alternative C-terminal domain offers an opportunity for immunologic therapy as it represents a cancer-specific epitope.

Essential thrombocythemia (ET) is an indolent myeloproliferative neoplasm characterized by megakaryocyte hyperplasia, thrombocytosis, thrombotic and hemorrhagic complications, and potential transformation into myelofibrosis and acute myeloid leukemia. The vast majority of cases are driven by a somatic mutation in JAK2, CALR, or MPL. CALR, a gene that codes for the calcium-binding chaperone calreticulin, is the predominant mutation in patients with non-mutated JAK2 essential thrombocythemia, accounting for 20-25% of the overall somatic mutation frequency in ET. In this brief review of ET, we introduce a rare CALR mutation through a case presentation of a 58-year-old man with diffuse pulmonary emboli in the setting of thrombocytosis. We subsequently characterize the main types of CALR mutations and their value in diagnosis and prognosis of disease course, and lastly discuss the current clinical approach to ET.

Testing for the CALR mutation is included in the updated WHO criteria for essential thrombocythaemia (ET) and primary myelofibrosis (PMF). We report on the application of the CAL2 monoclonal antibody, raised against the mutated CALR gene to myeloid cases. The immunostain was used on 116 acute myeloid leukaemias (AML) and 66 myeloproliferative neoplasms (MPN) or myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). None of AML cases was stained by the CAL2 antibody, while 20/66 MPNs and MDS/MPNs appeared positive. Fourteen of the latter cases were studied by molecular techniques, and all showed aberrations of the CALR gene. In addition, CAL2 positivity was found in some small-sized elements besides megakaryocytes. By double staining, these elements corresponded to small megakaryocytes as well as both erythroid and myeloid precursors. This finding suggests possible occurrence of CALR gene abnormalities in a stem cell.

Keywords: CAL2; CALR; essential thrombocythaemia; mutated calreticulin; myelofibrosis; myeloproliferative neoplasms.