Chronic myeloid leukemia (CML) typically causes leukocytosis rather than thrombocytosis. We encountered two women in their thirties with remarkable thrombocytosis, whose platelet counts were over 3,000×103/µl, and without significant leukocytosis. Although their clinical findings resembled that of essential thrombocythemia (ET), they were diagnosed with CML because of the presence of Philadelphia chromosome. JAK2, CALR, and MPL were unmutated. On fluorescence in situ hybridization analysis, only 19.8% of granulocytes in case 2 were found to be BCR/ABL positive in peripheral blood (PB). We reviewed 11 CML cases whose platelet counts were over 2,000×103/µl, but their WBC counts were not significantly elevated (<12,000/µl). Most of them were young females with a normal or a high neutrophil alkaline phosphatase score and without immature myeloid cells in PB. These findings suggested that there is a subgroup of CML patients with marked thrombocytosis and without significant leukocytosis, which may be misdiagnosed as ET.

Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P-, and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion, and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed, and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehlis' gland, prostate gland, and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed.

Primary Myelofibrosis (PMF) is a myeloproliferative disorder associated with JAK2V617F, Calreticulin (CALR) indels, and MPLW515L/K mutations activating the tyrosine kinase JAK2 and its downstream signaling pathway. The nature of signaling abnormalities in primary cells from PMF patients is poorly understood, since most of the work has been performed in cell lines or animal models. By flow cytometry we measured constitutive and cytokine induced phosphorylation of STAT5, STAT3, and ERK1/2 in circulating CD34+ cells from 57 patients with PMF (20 with prefibrotic-PMF) and 13 healthy controls (CTRLs). Levels of constitutive and TPO induced p-STAT5, and IL6 induced p-STAT3 were higher in patients than in CTRLs. Constitutive p-STAT5 values were lower in CALR than homozygous JAK2V617F mutated CD34+ cells from PMF patients. Moreover, constitutive p-STAT5 and IL6 induced p-STAT3 values correlated directly with circulating CD34+ cell number/L, and inversely with the frequency of circulating CD34+ cells expressing CXCR4. Constitutive p-STAT5 values of CD34+ cells were also inversely correlated with hemoglobin levels. When the patients were divided according with presence/absence of JAK2V617F mutation, all the correlations described characterized the JAK2V617F+ patients with prefibrotic-PMF (P-PMF). In conclusion, increased constitutive p-STAT5 and IL6 induced p-STAT3 values in circulating CD34+ cells characterize patients with PMF. Constitutive p-STAT5 and IL6 induced p-STAT3 values correlate with circulating CD34+ cell number/L, the frequency of circulating CD34+ cells expressing CXCR4 and hemoglobin levels within the prefibrotic JAK2V617F+ patient population. Our data point toward a complex activation of STAT5-dependent pathways in the stem/progenitor cell compartment, that characterize the phenotypic diversity of PMF.

The classical myeloproliferative neoplasms (MPNs) consist of chronic myelogenous leukemia (CML) and the non-CML MPNs, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Molecular testing plays a crucial role in each of these disease entities. In this review, we discuss the role and caveats of BCR-ABL1 fusion transcript evaluation in CML diagnosis and monitoring, as well as ABL1 kinase mutation testing in the setting of tyrosine kinase inhibitor resistance. We also focus on JAK2, MPL, and CALR mutations in PV, ET, and PMF.

Objective: To explore the utility of circulating tumor DNA detection in early breast cancer by using next-generation sequencing. Methods: This exploratory study of circulating tumor DNA detection is for early invasive breast cancer patients treated in Breast Disease Center, Peking University First Hospital from December 2015 to July 2016. Plasma samples were collected and were used to isolate plasma cell-free DNA.Exons or hotspots of 247 cancer related genes were sequenced by next-generation sequencing. Mutations and their correlation with clinic-pathological factors were analyzed. The correlation between mutations and clinic-pathological factors was evaluated by χ(2) test or Fisher's exact test. Results: Seventy-five patients were enrolled in this study. All patients were female and aged from 31 to 88 years with median age of 58 years. All patients' clinic-pathological records were complete. Sixty-four mutations in 18 genes (ALK, BCR, ERBB2, ROS1, PDGFRA, EGFR, FGFR2, CYP1B1, CALR, CASP7, BRAF, FGFR1, FGFR3, MET, NRAS, PTEN, KIT, SOD2) were detected in 47 (62.7%) among all 75 patients.Exons were captured in 10 genes, and mutations in 2 of 3 genes analyzed were clustered. Gene mutations were not correlated with menopausal status, histological type, primary tumor (T), regional lymph nodes (N), TNM stage, histological grade, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 status, Ki-67 and molecular subtype (all P>0.05). Conclusion: Circulating tumor DNA sequencing by next-generation sequencing was useful for detecting breast cancer-related mutations.

In the 2016 revision of the World Health Organization (WHO) classification, the categories of myeloid neoplasms have not been revised significantly from the 2008 fourth edition. However, recent discovery of molecular abnormalities provides a new perspective regarding the diagnostic and prognostic markers. In myeloproliferative neoplasms, the identification of CALR gene mutation, in addition to the JAK2 and MPL mutations, has impacted the diagnostic criteria. In myelodysplastic syndromes and acute myeloid leukemia, in addition to alterations in the transcription factors and signal transduction pathways, discovery of gene mutations in the epigenetic regulators that are involved in DNA methylation, histone modification, cohesin complex, and RNA splicing, by comprehensive genetic analyses, has improved our understanding of the pathobiology of these diseases. Moreover, recent large-scale sequencing studies have revealed the acquisition of clonal somatic mutations, in the myeloid neoplasm-associated genes of the hematopoietic cells. Such mutations were detected in people with normal blood cell counts, without any apparent disease. Presence of these mutations confers an increased risk for subsequent hematological neoplasms, indicating the concept of clonal hematopoiesis of indeterminate potential. This updated WHO classification incorporates the criteria of new clinical, prognostic, morphologic, immunophenotypic, and genetic findings in myeloid neoplasms.

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are often characterized by specific somatic mutations in any of the three genes: JAK2, CALR, or MPL. A single nucleotide polymorphism (SNP), rs2736100, in the reverse transcriptase gene (TERT) and a germline JAK2 46/1 haplotype have been associated with MPNs in North American and European patients. We examined 201 Japanese MPN patients, including 52 with PV, 131 with ET, and 18 with PMF, as well as 366 control individuals for TERT rs2736100 and JAK2 rs10974944, a tagging SNP of the 46/1 haplotype. Furthermore, correlations between the JAK2 V617F allele burden at diagnosis and TERT rs2736100 or JAK2 rs10974944 were evaluated using a digital PCR assay for accurate quantitation. The JAK2 46/1 haplotype, but not the TERT rs2736100 SNP, was correlated to the JAK2 V617F mutant allele burden in JAK2 V617F-positive MPN patients. In conclusion, we demonstrated that both TERT rs2736100_C and JAK2 46/1 haplotype are predisposing factors for MPNs in Japanese patients. While TERT rs2736100_C tended to have a more general, non-specific effect on all MPNs, the JAK2 46/1 haplotype was essentially predisposed to the JAK2 V617F-positive MPNs.

Calreticulin (CALR) mutations are present in 50% to 85% of JAK2/MPL wild-type (wt) myeloproliferative neoplasms (MPNs). The histopathologic features of CALR-mutated MPNs are unknown.

We identified 71 patients with essential thrombocythemia (ET), primary myelofibrosis (PMF), and post-essential thrombocythemia myelofibrosis (post-ET MF) with available CALR status. CALR was assessed using capillary electrophoresis followed by Sanger sequencing confirmation. CALR status was correlated with histopathologic features.

The megakaryocytes of CALR-mutated PMF more often were hyperchromatic (20/21) compared with CALR-wt cases (10/14) (P = .05). CALR-mutated ET showed more megakaryocytic clustering (7/7) compared with CALR-wt cases (5/9) (P = 03). Megakaryocytes of CALR-mutated post-ET MF (8/8) had a predominance of convoluted nuclei compared with CALR-wt cases (2/4) (P = .03). CALR mutations were more frequent in post-ET MF compared with ET (P = .04).

CALR-mutated MPNs have a higher frequency of megakaryocytic aberrancies compared with CALR-wt cases. Patients with CALR-mutated ET appear to be more likely to develop myelofibrosis compared with patients with wt CALRUpon completion of this activity you will be able to: describe morphologic features that are associated with CALR-mutated myeloproliferative neoplasms.examine cases of essential thrombocythemia and primary myelofibrosis and predict which cases are more likely to be CALR-mutated based on histopathologic features.initiate CALR mutation testing for cases likely to have mutations. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Exam is located at www.ascp.org/ajcpcme.

Introduction: Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

Methods: Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing.

Results: CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2.

Conclusion: CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

Keywords: AS-PCR; calreticulin; conformation sensitive gel electrophoresis; myeloproliferative neoplasms.

Background: Diabetes mellitus (DM) is characterized by hyperglycemia (high blood glucose levels) which is due to the destruction of insulin producing β-cells in the islets of Langerhans in the pancreas. It is associated with oxidative and endoplasmic reticulum stress. The plant alkaloid Palmatine has been previously reported to possess antidiabetic and antioxidant properties as well as other protective properties against kidney and liver tissue damage.

Objective: Here, we investigated the ability of Palmatine to reduce the up-regulation of chaperone proteins glucose regulatory protein 78 (GRP78), and calreticulin (CALR) protein in a streptozotocin (STZ)-induced diabetic rat model.

Method: Streptozotocin (STZ) induced diabetes sprague dawley rats treated with 2mg/kg of Palmatine for 12 weeks after elevation of plasma glucose levels above 11mmol/L post-STZ administration. Proteins were extracted from pancreas after treatment and Two-dimensional gel electrophoresis (2-DE), PD Quest software genomic solutions and mass spectrometer were used to analyze differentially expression protein. Mass spectrometry (MS/MS), multidimensional protein identification technology (MudPIT) was used for protein identification.

Results: There was an up-regulation of the expression of chaperone proteins CALR and GRP78 and down-regulation of the expression of antioxidant and protection proteins peroxidoxin 4 (Prdx4), protein disulfide isomerase (PDIA2/3), Glutathione-s-transferase (GST), and Serum Albumin (ALB) in non-diabetic rats. Palmatine treatment down regulated the expression of chaperone proteins CALR and GRP78 and up-regulated the expression of Prdx4, PDIA2/3, GST, and ALB.

Conclusion: Palmatine may have activated antioxidant proteins, which protected the cells against reactive oxygen species and endoplasmic stress. The result is in consonance with our previous report on Palmatine.

Keywords: 2-DE; CALR; GRP78; Streptozotocin; diabetes mellitus; palmatine.

The construction of protein abundance profiles helps to interpret the clinical applications of stem cells. Dental pulp stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) can be isolated from teeth and used as a highly convenient clinical potential material. Here, we aimed to explore commonalities and differences of DPSCs and GMSCs at the protein level. TMT-based quantitative proteomics and two-dimensional gel electrophoresis technology were used in combination to describe the protein profile of DPSCs and GMSCs extracted from the same donor. A total of 2821 proteins were identified by LC-MS/MS, of which 248 differentially abundant proteins (DAPs) were highly expressed in GMSCs while 782 proteins were highly expressed in DPSCs. The biological functions and molecular pathways of DAPs were annotated with GO enrichment and KEGG analysis. The relationship between molecular abundance and cell characteristics including source, proliferation, angiogenesis and inflammation were connected by WGCNA. Special markers, including Calreticulin (CALR), Annexin A5 (ANXA5) and Rho GDP dissociation inhibitor alpha (GDIR1), were proposed to distinguish DPSCs from GMSCs. Our results provide a molecular basis for in-depth understanding of the protein composition and special functions of dental stem cells, and promote the potential clinical application.

Keywords: Calreticulin; Dental stem cells; Special markers; WGCNA.

Myeloproliferative neoplasms (MPNs) are a class of hematologic diseases characterized by aberrant proliferation of one or more myeloid lineages and progressive bone marrow fibrosis. In 2005, seminal work by multiple groups identified the JAK2V617F mutation in a significant fraction of MPN patients. Since that time, murine models of JAK2V617F have greatly enhanced the understanding of the role of aberrant JAK-STAT signaling in MPN pathogenesis and have provided an in vivo pre-clinical platform that can be used to develop novel therapies. From early retroviral transduction models to transgenics, and ultimately conditional knock-ins, murine models have established that JAK2V617F alone can induce an MPN-like syndrome in vivo. However, additional mutations co-occur with JAK2V617F in MPNs, often in proteins involved in epigenetic regulation that can dramatically influence disease outcomes. In vivo modeling of these mutations in the context of JAK2V617F has provided additional insights into the role of epigenetic dysregulation in augmenting MPN hematopoiesis. In this overview, early murine model development of JAK2V617F is described, with an analysis of its effects on the hematopoietic stem/progenitor cell niche and interactions with downstream signaling elements. This is followed by a description of more recent in vivo models developed for evaluating the effect of concomitant mutations in epigenetic modifiers on MPN maintenance and progression. Mouse models of other driver mutations in MPNs, including primarily calreticulin (CALR) and Tpo-receptor (MPL), which occur in a significant percentage of MPN patients with wild-type JAK2, are also briefly reviewed. © 2017 by John Wiley & Sons, Inc.

Polycythemia vera and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms associated with thrombotic or hemorrhagic complications, and increased risk of transformation to myelofibrosis and acute myeloid leukemia. The main goal of therapy is aimed at preventing vascular events that are the leading cause of morbidity and mortality in these patients. Accordingly, risk stratification is the basis for deciding when to treat a patient with cytoreductive therapy. The European LeukemiaNet has developed a series of management recommendations for front-line and second-line therapy to provide the optimal treatment for the individual patient. There is still controversy about the efficacy and safety of several modalities of cytoreductive treatment in the long-term for both diseases and in the use of antiplatelet therapy in ET. The presence of JAK2V617F and CALR mutations in patients with ET has been related to different thrombotic risks, and this will probably lead to different therapeutic approaches in the near future. On the other hand, the near normal life expectancy of these patients makes a careful analysis of the benefits and risks associated with treatment essential. This review provides our current management strategy of patients with polycythemia vera and ET.

Mutations in the gene for calreticulin (CALR) were identified in the myeloproliferative neoplasms (MPNs) essential thrombocythaemia (ET) and primary myelofibrosis (MF) in 2013; in combination with previously described mutations in JAK2 and MPL, driver mutations have now been described for the majority of MPN patients. In subsequent years, researchers have begun to unravel the mechanisms by which mutant CALR drives transformation and to understand their clinical implications. Mutant CALR activates the thrombopoietin receptor (MPL), causing constitutive activation of Janus kinase 2 (JAK2) signaling and cytokine independent growth in vitro. Mouse models show increased numbers of hematopoietic stem cells (HSCs) and overproduction of megakaryocytic lineage cells with associated thrombocytosis. In the clinic, detection of CALR mutations has been embedded in World Health Organization and other international diagnostic guidelines. Distinct clinical and laboratory associations of CALR mutations have been identified together with their prognostic significance, with CALR mutant patients showing increased overall survival. The discovery and subsequent study of CALR mutations have illuminated novel aspects of megakaryopoiesis and raised the possibility of new therapeutic approaches.

Among classical BCR-ABL-negative myeloproliferative neoplasms (MPN), primary myelofibrosis (PMF) is the most aggressive subtype from a clinical standpoint, posing a great challenge to clinicians. Whilst the biological consequences of the three MPN driver gene mutations (JAK2, CALR, and MPL) have been well described, recent data has shed light on the complex and dynamic structure of PMF, that involves competing disease subclones, sequentially acquired genomic events, mostly in genes that are recurrently mutated in several myeloid neoplasms and in clonal hematopoiesis, and biological interactions between clonal hematopoietic stem cells and abnormal bone marrow niches. These observations may contribute to explain the wide heterogeneity in patients' clinical presentation and prognosis, and support the recent effort to include molecular information in prognostic scoring systems used for therapeutic decision-making, leading to promising clinical translation. In this review, we aim to address the topic of PMF molecular genetics, focusing on four questions: (1) what is the role of mutations on disease pathogenesis? (2) what is their impact on patients' clinical phenotype? (3) how do we integrate gene mutations in the risk stratification process? (4) how do we take advantage of molecular genetics when it comes to treatment decisions?

Keywords: mutation; myelofibrosis; myeloproliferative.

Non-small cell lung cancer (NSCLC) is highly prevalent and needs novel therapies. Melanoma-associated antigen 3 (MAGE-A3) is a lung cancer antigen and calreticulin (CALR) can modulate immune responses. Our previous study has shown that up-regulated MAGE-A3 and CALR expression inhibits the proliferation and invasion of glioma cells. In this study, we examined the effect of adenovirus (Ad)-mediated MAGE-A3 and/or CALR expression on the proliferation, invasion, and apoptosis of human NSCLC cells and on the vascular tube formation of human endothelial cells as well as on dendritic cell (DC) activation and induced CD8(+) cytotoxic T lymphocyte (CTL) activity in vitro. We found that low levels of CALR and MAGE-A3 were expressed by A549 cells, but only very low CALR was expressed by DC. Up-regulated CALR and MAGE-A3 expression by infection with Ad-CALR/MAGE-A3 significantly inhibited the proliferation and invasion, but promoted the apoptosis of A549 cells. Up-regulated CALR and MAGE-A3 expression significantly inhibited cyclin D1 expression and the AKT, ERK1/2 and NF-κB expression and phosphorylation in A549 cells. Up-regulated CALR expression inhibited the tube formation in human endothelial cells. Up-regulated CALR and MAGE-A3 expression synergistically enhanced classical DC activation by enhancing IL-12, but reducing IL-10 secretion. Furthermore, CTLs induced by up-regulated CALR and MAGE-A3 expressing DCs synergistically triggered A549 cell apoptosis, which was abrogated by treatment with anti-HLA I, but not anti-HLA II antibodies. Moreover, CTLs induced by CALR and MAGE-A3-expressing DCs had a higher frequency of A549-specific IFN-γ-secreting T cells. Our data indicated that up-regulated CALR and MAGE-A3 expression inhibited the carcinogenesis of NSCLC by modulating the AKT, ERK MAPK and NF-κB signaling and enhanced classical DC activation and MAGE-A3-specific CTL cytotoxicity. Therefore, our findings may provide new insights in understanding the role of CALR in modulating antigen-specific T cell immunity and may aid in the design of new therapies for NSCLC.

Primary myelofibrosis (PMF) is a chronic myeloproliferative disorder in human bone marrow. Over 50% of patients with myelofibrosis have mutations in JAK2, MPL, or CALR. However, these mutations are rarely detected in children, suggesting a difference in the pathogenesis of childhood PMF. In this study, we investigated the response to drug treatment of a monozygotic twin pair with typical childhood PMF. The twin exhibited different clinical outcomes despite following the same treatment regimen. The transcriptomic profiles of patient samples after drug treatment (E2 and Y2) were significantly different between the twin pair, which is consistent with the observation that the drug treatment was effective only in the younger brother, despite the twin being genetically identical. Bioinformatics analysis of the drug-responsive genes showed that the JAK-STAT pathway was activated in the cured younger brother, which is opposite to the pathway inhibition observed in adult PMF cases following treatment. Moreover, apoptosis and cell cycle processes were both significantly influenced by drug treatment in the sample of younger brother (Y2), implying their potential association with the pathogenesis of childhood PMF. Gene mutations in JAK2, MPL, or CALR were not observed; however, mutations in genes including SRSF2 and SF3B1 occurred in this twin pair with childhood PMF. Gene fusion events were extensively screened in the twin pair samples and the occurrence of IGLV2-14-IGLL5 gene fusion was confirmed. The current study reported at transcriptomic level the different responses of monozygotic twin brothers with childhood PMF to the same androgen/prednisone treatment regimen providing new insights into the potential pathogenesis of childhood PMF for further research and clinical applications.

Drug resistance substantially limits the curative capability of chemotherapy in head and neck cancers such as oral squamous cell carcinoma. Immunosuppression is considered a potential cause of drug resistance. A key discovery in the past decade is that chemotherapeutics can alter tumor cell immunogenicity via inducing release of damage-associated molecular patterns (DAMPs), including ecto-calreticulin (ecto-CALR), high mobility group box 1 (HMGB1) and ATP, causing tumor cells to die in a manner known as bona fide immunogenic apoptosis or immunogenic cell death (ICD). Intriguingly, JQ1 was found in this study to exhibit therapeutic potential in tongue squamous cell carcinoma (TSCC) by inducing ICD. JQ1 induced significant release of calreticulin (CALR), HMGB1 and ATP from Cal27 and SCC7 cells in vitro. Immature dendritic cells (Im-DCs) cocultured with JQ1-pretreated Cal27 cells exhibited significant upregulation of mature markers on their surface and an increase in the secretion of cytokines. In vivo experiments demonstrated that JQ1-pretreated dying SCC7 cells protected immunocompetent mice from rechallenge of SCC7 cells. Intravenous injection of JQ1 efficiently reduced tumor growth and increased tumor-infiltration of CD3⁺/CD8⁺ T cells in C3H mice.

In this study, we investigate in detail the morphological, clinical and molecular features of 71 consecutive patients with a diagnosis of myeloproliferative neoplasms, unclassifiable. We performed a meticulous morphological analysis and found that most of the cases displayed a hypercellular bone marrow (70%) with normal erythropoiesis without left-shifting (59%), increased granulopoiesis with left-shifting (73%) and increased megakaryocytes with loose clustering (96%). Megakaryocytes displayed frequent giant forms with hyperlobulated or bulbous nuclei and/or other maturation defects. Interestingly, more than half of the cases displayed severe bone marrow fibrosis (59%). Median values of hemoglobin level and white blood cells count were all within the normal range; in contrast, median platelets count and lactate dehydrogenase were increased. Little less than half of the patients (44%) showed splenomegaly. JAK2V617F mutation was detected in 72% of all patients. Among the JAK2-negative cases, MPLW515L mutation was found in 17% and CALR mutations in 67% of the investigated cases, respectively. Finally, by multiple correspondence analysis of the morphological profiles, we found that all but four of the cases could be grouped in three morphological clusters with some features similar to those of the classic BCR-ABL1-negative myeloproliferative neoplasms. Analysis of the clinical parameters in these three clusters revealed discrepancies with the morphological profile in about 55% of the patients. In conclusion, we found that the category of myeloproliferative neoplasm, unclassifiable is heterogeneous but identification of different subgroups is possible and should be recommended for a better management of these patients.

The myeloproliferative neoplasms (MPNs) are acquired hematological stem cell neoplasms characterized by driver mutations in JAK2, CALR, or MPL. Additive mutations may appear in predominantly epigenetic regulator, RNA splicing and signaling pathway genes. These molecular mutations are a hallmark of diagnostic, prognostic, and therapeutic assessment in patients with MPNs. Over the past decade, next generation sequencing (NGS) has identified multiple somatic mutations in MPNs and has contributed substantially to our understanding of the disease pathogenesis highlighting the role of clonal evolution in disease progression. In addition, disease prognostication has expanded from encompassing only clinical decision making to include genomics in prognostic scoring systems. Taking into account the decreasing costs and increasing speed and availability of high throughput technologies, the integration of NGS into a diagnostic, prognostic and therapeutic pipeline is within reach. In this review, these aspects will be discussed highlighting their role regarding disease outcome and treatment modalities in patients with MPNs.

Keywords: early treatment; essential thrombocythemia; gene mutations; myelofibrosis; myeloproliferative neoplasms; next generation sequencing; outcome; polycythemia vera; prognosis; treatment modalities.

Essential thrombocythaemia (ET) is driven by somatic mutations involving the JAK2, CALR and MPL genes. Approximately 10% of patients lack driver mutations and are referred as 'triple-negative' ET (TN-ET). The diagnosis of TN-ET, however, relies on bone marrow examination that is not always performed in routine practice, and thus in the real-world setting, there are a group of cases with suspected TN-myeloproliferativeneoplasm.In this real-world cohort, patients with suspected TN-ET were initially rescreened for JAK2, CALR and MPL and then targeted next-generation sequencing (NGS) was applied.The 35 patients with suspected TN-ET had a median age at diagnosis of 43 years (range 16-79) and a follow-up of 10 years (range 2-28). The median platelet count was 758×10⁹/L (range 479-2903). Thrombosis prior to and following diagnosis was noted in 20% and 17% of patients. Six patients were JAKCALR positive on repeat screening. NGS results showed that 24 of 27 patients harboured no mutations. Four mutations were noted in three patients.There was no evidence of clonality for the majority of patients with suspected TN-ET with targeted NGS analysis. Detection of driver mutations in those who were previously screened suggests that regular rescreening is required. This study also questions the diagnosis of TN-ET without the existence of a clonal marker.

Keywords: diagnosis; molecular biology; myeloproliferative disorders.

BACKGROUND

The prevalence of calreticulin (CALR) mutations in splanchnic vein thrombosis (SVT) varies among studies. The role of routine screening for CALR mutations in SVT patients remains a debate.

OBJECTIVE

To synthesize the prevalence of CALR mutations according to the different types (i.e., Budd-Chiari syndrome [BCS] and portal vein thrombosis [PVT]) and characteristics (i.e., with and without myeloproliferative neoplasms [MPNs] and JAK2V617F mutation) of SVT patients.

METHODS

Eligible studies were searched by the PubMed and Embase databases. The study quality was assessed according to the STROBE checklist. The proportion of CALR mutations was pooled by using a random-effects model. The heterogeneity and publication bias were calculated.

RESULTS

Eleven papers were included. The study quality was moderate to high. The pooled proportion of CALR mutations was 1.21%, 1.41%, and 1.59% in SVT, BCS, and PVT patients, respectively; 1.52%, 1.03%, and 1.82% in these patients without JAK2V617F mutation, respectively; 3.71%, 2.79%, and 7.87% in these patients with MPN, respectively; and 15.16%, 17.22%, and 31.44% in these patients with MPN but without JAK2V617F mutation, respectively. Only the meta-analysis examining the prevalence of CLAR mutations in BCS patients with MPN but without the JAK2V617F mutation showed statistically significant heterogeneity. Statistically significant publication bias was seen only in the meta-analysis examining the prevalence of CALR mutations in SVT patients without the JAK2V617F mutation.

CONCLUSIONS

Screening for CALR mutations may have a role in SVT patients with a high probability of MPN in whom the JAK2V617F mutation has been excluded.