Myeloproliferative neoplasms (MPNs) are characterized by activation of the JAK-STAT pathway due to driver mutations including JAK2V617F and MPLW515K/L, as well as to mutations in CALR. Driver mutations phosphorylate multiple STAT proteins that lead to proliferations, differentiations and cytokine secretions of various hematopoietic cells. However, hematopoietic cells carrying JAK2V617F, which causes excessive cellular proliferation and differentiation, do not necessarily have a clonal growth advantage in terms of hematopoietic repopulation. Alterations of epigenetic modifiers involving histone modifications and DNA methylations, which often co-exist with driver mutations and eventually upregulate several oncogenes, may play crucial roles in long-term clinical courses characterized by progression to myelofibrosis or acute leukemia in MPNs. In addition to JAK2 inhibition, molecules altered by abnormal epigenetic modifications may be worth exploring as potential new therapeutic targets in MPNs.

Calreticulin (CALR) and JAK2-V617F gene mutations, which are major genetic mutations in patients with primary myelofibrosis (PMF) and essential thrombocythemia (ET), exert different effects on the clinical features and outcomes of these diseases. We analyzed 88 and 9 patients with ET and PMF, respectively, and determined the differences in the clinical characteristics of ET patients with JAK2-V617F compared with CALR mutations. The frequency of the JAK2-V617F and CALR mutations were 64 and 22 %, respectively. Patients with CALR mutations were younger, had a lower white blood cell count, and had a lower rate of thrombotic events than patients with the JAK2 mutation. The neutrophil alkaline phosphatase (NAP) score of 16 patients with CALR mutations was significantly lower than the normal controls, which was mainly due to the high proportion of NAP-negative neutrophils. This is the first report to show an association between CALR mutations in patients with myeloproliferative neoplasms (MPN) and the NAP score. Although the mechanism is unclear, the NAP score could be a useful and reliable biochemical marker to discriminate the mutational status of MPN patients. Further investigation is warranted to determine whether these characteristics contribute to the pathogenesis of MPN and the NAP score.

Motility is important for virulence, biofilm formation, and the environmental adaptation of many bacteria. Vibrio parahaemolyticus (V. parahaemolyticus) contains two flagellar systems that are responsible for motility, and are tightly regulated by transcription regulators and sigma factors. In this study, we identified a novel transcription factor, VPA1701, which regulates the swarming motility of V. parahaemolyticus. The VPA1701 deletion mutant (ΔVPA1701) eliminated the swarming motility on the surface of BHI agar plates and reduced colonization in infant rabbits. RNA-seq assays, confirmed by qRT-PCR, indicated that VPA1701 regulated the expression of lateral flagellar cluster genes. Further analyses revealed that VPA1701 directly binds to the promoter region of the flgBCDEFGHIJKL cluster to regulate the expression of lateral flagellar genes. CalR was originally identified as a repressor for the swarming motility of V. parahaemolyticus, and it was inhibited by calcium. In this study, we found that VPA1701 could inhibit the expression of the calR gene to increase the swarming motility of V. parahaemolyticus. Calcium downregulated the expression of calR, indicating that calcium could increase swarming motility of ΔVPA1701 by inhibiting calR. Thus, this study illustrates how the transcription factor VPA1701 regulates the expression of lateral flagellar genes and calR to control the swarming motility of V. parahaemolyticus.

Objective: To analyze the genetic mutations and clinical features of the subtypes of classical BCR-ABL-negative myeloproliferative neoplasm (MPN) . Methods: Mutations of 108 newly diagnosed BCR-ABL-negative MPN patients [including 55 patients with essential thrombocytopenia (ET) , 24 with polycythemia vera (PV) , and 29 with primary myelofibrosis (PMF) ] were identified using next-generation sequencing with 127-gene panel, and the relationship between gene mutations and clinical features were analyzed. Results: Total 211 mutations in 32 genes were detected in 100 MPN patients (92.59% ) , per capita carried (1.96±1.32) mutations. 85.19% (92/108) patients carried the driver gene (JAK2, CALR, MPL) mutations, 69.56% (64/92) of these patients carried at least 1 additional gene mutation. In descending order of mutation frequency, the highest frequency was for activation signaling pathway genes (42.2% , 89/211) , methylation genes (17.6% , 36/211) , and chromatin-modified genes (16.1% , 34/211) . There was a significant difference in the number of mutations in the activation signaling pathway genes, epigenetic regulatory genes, spliceosomes, and RNA metabolism genes among the three MPN subgroups. The average number of additional mutations in PMF patients was higher than that in ET and PV patients (1.69±1.39, 0.67±0.70, 0.87±1.22, χ(2)=13.445, P=0.001) . MPN-SAF-TSS (MPN 10 score) (P=0.006) and myelofibrosis level (P=0.015) in patients with ≥ 3 mutant genes were higher and the HGB level (P=0.002) was lower than in those with<3 mutations. Twenty-six patients (24.1% ) carried high-risk mutation (HMR) , and patients with HMR had lower PLT (P=0.017) , HGB levels (P<0.001) , and higher myelofibrosis level (P=0.010) and MPN10 score (P<0.001) . The frequency of ASXL1 mutations was higher in PMF than in PV patients (34.5% vs. 4.2% , P=0.005) . PMF patients with ASXL1 had lower levels of PLT and HGB (P=0.029 and 0.019) . Conclusion: 69.56% of MPN patients carry at least one additional mutation, and 24.1% patients had HMR. Each subgroup had different mutation patterns. PMF patients had a higher average number of additional gene mutations, especially a higher frequency of ASXL1 mutation; PLT and HGB levels were lower in ASXL1 mutation PMF patients.

目的： 分析经典型BCR-ABL阴性骨髓增殖性肿瘤（MPN）的基因突变及临床特征。 方法： 应用二代测序方法检测108例初诊BCR-ABL阴性MPN患者[原发性血小板增多症（ET）55例，真性红细胞增多症（PV）24例，原发性骨髓纤维化（PMF）29例]与疾病相关的127个基因突变情况，并分析基因突变与临床特征间的关系。 结果： 108例MPN患者中，100例（92.59％）检出32种基因共211个突变，人均检出（1.96±1.32）个突变。85.19％（92/108）的患者检出驱动基因（JAK2、CALR、MPL）突变，其中69.56％（64/92）的患者至少检出1种附加基因突变。按突变频率由高到低依次为激活信号通路基因（42.2％，89/211）、DNA甲基化基因（17.6％，36/211）、染色质修饰基因（16.1％，34/211）。PV、ET、PMF三组间激活信号通路基因、表观遗传调节基因、剪接体和RNA代谢基因突变个数差异均有统计学意义，PMF附加基因平均突变数目高于ET及PV患者（1.69±1.39、0.67±0.70、0.87±1.22，χ(2)＝13.445，P＝0.001）。检出≥3个突变基因患者MPN总症状评估量表（MPN 10评分）（P＝0.006）、骨髓纤维化程度（P＝0.015）均较检出<3个突变基因患者高，且HGB水平更低（P＝0.002）；26例（24.1％）患者检出高危基因突变（HMR），HMR突变的患者具有更低的PLT（P＝0.017）和HGB水平（P<0.001），更高的骨髓纤维化程度（P＝0.010）、MPN10评分（P<0.001）。PMF患者ASXL1突变频率高于PV患者（37.9％对4.2％，P＝0.005），ASXL1突变的PMF患者具有更低的PLT和HGB水平（P＝0.029、0.019）。 结论： 近七成MPN患者检出至少1种附加基因突变，24.1％检出HMR突变，各亚组有不同的基因突变模式；PMF患者附加基因平均突变数目更多，ASXL1突变频率更高。ASXL1突变的PMF患者PLT和HGB水平更低。.

Keywords: ASXL1; Driver gene; JAK2; Myeloproliferative neoplasm; Next-generation sequencing.

Philadelphia chromosome-negative myeloproliferative neoplasms are hematopoietic stem cell disorders characterized by dysregulated proliferation of mature myeloid blood cells. They can present as polycythemia vera, essential thrombocythemia, or myelofibrosis and are characterized by constitutive activation of JAK2 signaling. They share a propensity for thrombo-hemorrhagic complications and the risk of progression to acute myeloid leukemia. Attention has also been drawn to JAK2 mutant clonal hematopoiesis of indeterminate potential as a possible precursor state of MPN. Insight into the pathogenesis as well as options for the treatment of MPN has increased in the last years thanks to modern sequencing technologies and functional studies. Mutational analysis provides information on the oncogenic driver mutations in JAK2, CALR, or MPL in the majority of MPN patients. In addition, molecular markers enable more detailed prognostication and provide guidance for therapeutic decisions. While JAK2 inhibitors represent a standard of care for MF and resistant/refractory PV, allogeneic hematopoietic stem cell transplantation remains the only therapy with a curative potential in MPN so far but is reserved to a subset of patients. Thus, novel concepts for therapy are an important need, particularly in MF. Novel JAK2 inhibitors, combination therapy approaches with ruxolitinib, as well as therapeutic approaches addressing new molecular targets are in development. Current standards and recent advantages are discussed in this review.

Myeloproliferative neoplasms (MPN), are clonal hematopoietic stem cell (HSC) disorders driven by gain-of-function mutations in JAK2 (JAK2-V617F), CALR or MPL genes. MPN treatment options currently mainly consist of cytoreductive therapy with hydroxyurea and JAK2 inhibitors such as ruxolitinib and fedratinib. Pegylated interferon-alpha can induce complete molecular remission (CMR) in some MPN patients when applied at early stages of disease. The ultimate goal of modern MPN treatment is to develop novel therapies that specifically target mutant HSCs in MPN and consistently induce CMR. Basic research has identified a growing number of candidate drugs with promising effects in vitro. A first step on the way to developing these compounds into drugs approved for treatment of MPN patients often consists of examining the effects in vivo using pre-clinical mouse models of MPN. Here we review the current state of MPN mouse models and the experimental setup for their optimal use in drug testing. In addition to novel compounds, combinatorial therapeutic approaches are often considered for the treatment of MPN. Optimized and validated mouse models can provide an efficient way to rapidly assess and select the most promising combinations and thereby contribute to accelerating the development of novel therapies of MPN.

Background: Essential thrombocythemia (ET) is one of the "classic" Philadelphia chromosome negative (Ph-) myeloproliferative neoplasms characterized by sustained thrombocytosis, increased megakaryopoiesis and high risk of vascular complications. ET is very rare in childhood. The annual incidence is approximately 1 per 10,000,000 in children less than 14 years, and about 60 times lower than adults. The genetic landscape and clonal features in childhood ET has not been well defined. There is no evidence-based guidance on the diagnosis of childhood ET.

Methods: Medical records of 28 pediatric patients (age ≤ 14 years at diagnosis) with ET were reviewed and evaluated to characterize the different mutation profiles and to evaluate the treatment modalities used and the potential long-term outcome.

Results: More than half of the patients were found to have positive history of parental consanguinity (57.1%) whereas positive family history was documented for more than a quarter of our patients (28.6%). Janus kinase 2 gene (JAK2) V617F mutation was positive in two of 26 patients (7.7%). Myeloproliferative leukemia virus oncogene (MPL) exon 10 and calreticulin (CALR) mutations were tested in eight patients, which were negative for all of them. Treatment included low-dose aspirin (LDA) in seven patients (50%), combination of LDA with hydroxyurea in three patients (21.4%), hydroxyurea in two patients (14.3%), combination of platelets apheresis with LDA and anagrelide in one patient each (7.1%). During the treatment, two patients experienced stroke (7.1%), one patient developed Budd-Chiari syndrome (3.6%) and one patient developed azoospermia (3.6%).

Conclusions: The incidence of ET in children is extremely low in Saudi Arabia. Most of the children with ET were asymptomatic, and thrombocytosis was often discovered incidentally. JAK2 V617F mutation has no known impact on the prognosis or on the outcome of the disease in the pediatric age group that is in contrast to the adult ET. Children less than 1 year are at high risk for complications particularly during acute precipitating infectious episode. The potential complications and clinical course of pediatric ET are unpredictable.

Keywords: Essential thrombocythemia; JAK2 mutation; Pediatric ET.

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.

Keywords: JAK2V617F; MPL; calreticulin; chronic myeloproliferative neoplasm; essential thrombocythemia; hematologic malignancies; molecular biology; polycythemia vera; primary myelofibrosis.

Primary aldosteronism (PA) is characterized by inappropriate aldosterone production. Chronic aldosterone excess has detrimental effects on cardiovascular system, including endothelial dysfunction and vascular inflammation. Circulating extracellular vesicles (EVs) are central players in the crosstalk between endothelium, vascular structures, and inflammatory cells. The aim of the study was to assess the potential role of EVs in aldosterone-related vascular damage by evaluating a comprehensive panel of 37 EV surface antigens. Serum EVs were isolated by immunocapture from 32 patients with PA, 29 patients with essential hypertension and from 22 normotensive controls. EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. Particle concentration was higher and diameter lower in patients with PA compared with controls and the number of particles decreased after unilateral adrenalectomy. Nineteen EV surface antigens were differentially expressed in patients with PA compared with patients with essential hypertension or normotensive controls, including markers of activated platelets, endothelial and immune/inflammatory cells. The specific EV surface signature discriminated patients with PA from controls, whereas after specific PA treatment the profile became similar to essential hypertension. Stimulation of human endothelial cells with PA-derived EVs resulted in the overexpression of 5 selected genes (AKT1-CALR-CSNK2A1-FN1-PIK3R1), previously identified by bioinformatic analysis as targets of differentially expressed EV antigens. Overexpression of CALR and FN1 was confirmed also at protein level. Our data suggest that EVs represent biomarkers of vascular inflammation and endothelial dysfunction in patients with PA and also potential biovectors contributing to accelerated organ damage by multiple signaling processes.

Keywords: aldosterone; extracellular vesicles; hyperaldosteronism; inflammation; nanoparticles.

Introduction: In 1991, we reported 18 persons with a clinical-pathologic entity and termed atypical myeloproliferative disorder because they did not meet the contemporary diagnostic criteria for a myeloproliferative neoplasm. We sought to gain further knowledge on this disease entity.

Methods: This retrospective cohort study included consecutive subjects registered in the database of the Center for the Study of Myelofibrosis in Pavia, Italy, from 1998 to 2020 (June), and diagnosed with atypical myeloproliferative disorder according to our adjudicated criteria. We studied clinical, histological, cytogenetic, and molecular covariates and risks of thrombosis, disease progression, and death. Data were compared with those of concurrent subjects with prefibrotic myelofibrosis.

Results: Fifteen new subjects with atypical myeloproliferative disorder were identified. Seven were male. Median age was 50 years (IQR, 41-54 years). Thirteen were diagnosed with a synchronous symptomatic or incidentally detected thrombotic event. The bone marrow showed megakaryocyte hyperplasia with dysplasia. JAK2V617F was present in 10 subjects and CALR mutation in one. No other somatic mutations were identified in next generation sequencing. After a median follow-up of 101 months (IQR, 40-160 months), no subject had disease progression or blast transformation. Incidence of post-diagnosis or recurrent thrombosis was 3.9 events (95% confidence interval, 3.5-4.0) and 5.0 events (4.6-5.6) per 100 person-years. Features of subjects with atypical myeloproliferative disorder differed markedly from those of 546 subjects with prefibrotic myelofibrosis.

Conclusion: Our data indicate that these 15 persons have a distinct myeloproliferative neoplasm. We propose naming this new disorder clonal megakaryocyte dysplasia with normal blood values.

Keywords: Atypical myeloproliferative disorder; Megakaryocyte dysplasia; Myelofibrosis; Myeloproliferative neoplasm; Prefibrotic myelofibrosis.

miR-1307 is highly expressed in liver cancer and inhibits methyltransferase protein8. Thereby, miR-1307 inhibits the expression of KDM3A and KDM3B and increases the methylation modification of histone H3 lysine 9, which enhances the expression of endoplasmic-reticulum-related gene CALR. Of note, miR-1307 weakens the binding ability of OSTC to CDK2, CDK4, CyclinD1, and cyclinE and enhances the binding ability of CALR to CDK2, CDK4, CyclinD1, and cyclinE, decreasing of p21WAF1/CIP1, GADD45, pRB, and p18, and decreasing of ppRB. Furthermore, miR-1307 increases the activity of H-Ras, PKM2, and PLK1. Strikingly, miR-1307 reduces the binding ability of OSTC to ATG4 and enhances the binding ability of CALR to ATG4. Therefore, miR-1307 reduces the occurrence of autophagy based on ATG4-LC3-ATG3-ATG7-ATG5-ATG16L1-ATG12-ATG9- Beclin1. In particular, miR-1307 enhances the expression of PAK2, PLK1, PRKAR2A, MYBL1, and Trim44 and inhibits the expression of Sash1 and Smad5 via autophagy. Our observations suggest that miR-1307 promotes hepatocarcinogenesis by CALR-OSTC-endoplasmic reticulum protein folding pathway.

Keywords: Cancer; Cell biology; Molecular biology.

Calreticulin (CALR) is a chaperone present in the endoplasmic reticulum, which is involved in the quality control of N-glycosylated proteins and storage of calcium ions. In 2013, the C-terminal mutation in CALR was identified in half of the patients with essential thrombocythemia and primary myelofibrosis who did not have a JAK2 or MPL mutation. The results of 8 years of intensive research are changing the clinical practice associated with treating myeloproliferative neoplasms (MPNs). The presence or absence of CALR mutations and their mutation types already provide important information for diagnosis and treatment decision making. In addition, the interaction with the thrombopoietin receptor MPL, which is the main mechanism of transformation by CALR mutation, and the expression of the mutant protein on the cell surface have a great potential as targets for molecular-targeted drugs and immunotherapy. This chapter presents recent findings on the clinical significance of the CALR mutation and the molecular basis by which this mutation drives MPNs.

Keywords: CALR mutations; Calreticulin; Essential thrombocythemia; Immunotherapy; MPL; Mouse models; Primary myelofibrosis.

Myeloproliferative neoplasms (MPN) patients share driver mutations in JAK2, MPL or CALR genes leading to the activation of the thrombopoietin receptor (TPOR) and downstream signaling pathways. JAK2 mutation drives all the three major entities of MPN (Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis) through the constitutive activation of TPOR, erythropoietin (EPOR) and colony stimulating factor 3 receptor (CSF3R) signaling. MPL is a proto-oncogene encoding for TPOR, the hematopoietic growth factor receptor of myeloid stem cells. MPL mutants induce the stable dimerization of TPOR that in turn activate JAK2 and the thrombopoietin pathway. The thrombopoietin pathway plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of hematopoietic stem cells. Little wonder therefore that mutations of MPL result in thrombocytosis, leading to an abnormal MPL trafficking or receptor activation. Finally, some extremely rare germline genetic variants in MPL can induce MPN-like hereditary disease. Against this molecular background, TPOR is a key actor in the MPN development and MPL mutations are of major relevance to fully elucidate the molecular mechanisms underlying the clinical manifestations of MPN and to arrange novel therapeutic strategies aiming to disrupt the dysegulated signaling cascade. This chapter will focus on the role MPL in the pathogenesis of MPN and in familial thrombocytosis and will review these different subtypes of somatic and germline genetic variants by dissecting how they impact clinical phenotype.

Keywords: Hereditary thrombocytosis; MPL; Mutations; Myeloproliferative Neoplasms; Thrombocytosis; Thrombopoietin receptor.

Progression in Ph-chromosome-negative myeloproliferative neoplasms (MPN) develops with variable incidence and time sequence in essential thrombocythemia, polycythemia vera, and primary myelofibrosis. These diseases show different clinic-pathologic features and outcomes despite sharing deregulated JAK/STAT signaling due to mutations in either the Janus kinase 2 or myeloproliferative leukemia or CALReticulin genes, which are the primary drivers of the diseases, as well as defined diagnostic criteria and biomarkers in most cases. Progression is defined by the development or worsening of marrow fibrosis or the progressive increase in the marrow blast percentage. Progression is often related to additional genetic aberrations, although some can already be detected during the chronic phase. Detailed scoring systems for clinical usage that are mostly applied in patients with primary myelofibrosis have been defined, and the most recent ones include cytogenetic and molecular parameters with prognostic significance. Additional different clinic-pathologic changes have been reported that may occur during the course of the disease and that are, at present, classified as WHO-defined types of progression, although they likely represent such an event. The present review is meant to provide an updated overview on progression in Ph-chromosome-negative MPN, with a major focus on the pathologic side.

Keywords: WHO classification; fibrosis; leukemia; myeloproliferative neoplasms; progression.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm due to the clonal proliferation of a hematopoietic stem cell. The vast majority of patients harbor a somatic gain of function mutation either of JAK2 or MPL or CALR genes in their hematopoietic cells, resulting in the activation of the JAK/STAT pathway. Patients display variable clinical and laboratoristic features, including anemia, thrombocytopenia, splenomegaly, thrombotic complications, systemic symptoms, and curtailed survival due to infections, thrombo-hemorrhagic events, or progression to leukemic transformation. New drugs have been developed in the last decade for the treatment of PMF-associated symptoms; however, the only curative option is currently represented by allogeneic hematopoietic cell transplantation, which can only be offered to a small percentage of patients. Disease prognosis is based at diagnosis on the classical International Prognostic Scoring System (IPSS) and Dynamic-IPSS (during disease course), which comprehend clinical parameters; recently, new prognostic scoring systems, including genetic and molecular parameters, have been proposed as meaningful tools for a better patient stratification. Moreover, new biological markers predicting clinical evolution and patient survival have been associated with the disease. This review summarizes basic concepts of PMF pathogenesis, clinics, and therapy, focusing on classical prognostic scoring systems and new biological markers of the disease.

Keywords: chronic myeloproliferative neoplasms; inflammation; primary myelofibrosis; prognosis.

Classical Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell-derived disorders characterized by uncontrolled proliferation of differentiated myeloid cells and close pathobiologic and clinical features. According to the 2016 World Health Organization (WHO) classification, MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 revision aimed in particular at strengthening the distinction between masked PV and JAK2-mutated ET, and between prefibrotic/early (pre-PMF) and overt PMF. Clinical manifestations in MPNs include constitutional symptoms, microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, cytopenia-related symptoms, and progression to overt MF and acute leukemia. A dysregulation of the JAK/STAT pathway is the unifying mechanistic hallmark of MPNs, and is guided by somatic mutations in driver genes including JAK2, CALR and MPL. Additional mutations in myeloid neoplasm-associated genes have been also identified, with established prognostic relevance, particularly in PMF. Prognostication of MPN patients relies on disease-specific clinical models. The increasing knowledge of MPN biology led to the development of integrated clinical and molecular prognostic scores that allow a more refined stratification. Recently, the therapeutic landscape of MPNs has been revolutionized by the introduction of potent, selective JAK inhibitors (ruxolitinib, fedratinib), that proved effective in controlling disease-related symptoms and splenomegaly, yet leaving unmet critical needs, owing the lack of disease-modifying activity. In this review, we will deal with molecular, clinical, and therapeutic aspects of the three classical MPNs aiming at highlighting either shared characteristics, that overall define a continuum within a single disease family, and uniqueness, at the same time.

Keywords: Essential thrombocythemia; Gene mutations; JAK-STAT pathway; Myelofibrosis; Myeloproliferative neoplasm; Polycythemia vera.

Purpose of review: Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic stem cell neoplasms comprising of polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) that share driver mutations (JAK2/CALR/MPL) resulting in constitutive activation of JAK/STAT and other signaling pathways. Patients with MPN have shortened survival and an inherent risk for leukemic evolution. Prognostically relevant clinical and genetic parameters have been incorporated into mutation-enhanced scoring systems (MIPSS70-plus version 2.0, MIPSS-ET/PV). In the current review, we describe clinical and pathological features along with prognostic significance of MPN with monocytosis.

Recent findings: Monocytosis, defined by an absolute monocyte count (AMC) ≥ 1 × 10 ⁹/L, is a typical manifestation of chronic myelomonocytic leukemia (CMML) but is also associated with 21% and 17% of PV and PMF patients, respectively. Recent studies on the subject have reported that MPN patients with monocytosis are older and present with concomitant leukocytosis. In regard to PV, patients with monocytosis harbor unfavorable cytogenetic abnormalities including +8, 7/7q, i(17q), 5/5q-,12p-, inv(3), or 11q23 rearrangement and SRSF2 mutations, whereas PMF patients with monocytosis had significant thrombocytopenia, higher circulating blasts, higher symptom burden, and ASXL1 mutations. Moreover, presence of monocytosis predicted inferior survival in both PV and PMF. Monocytosis in MPN is associated with a distinct clinical and genetic profile and may serve as a marker of aggressive disease biology.

Keywords: Chronic myelomonocytic leukemia; Monocytosis; Myeloproliferative neoplasm; Prognosis.

Objective: To evaluate the prognostic value of MIPSS70-plus in Chinese patients with primary myelofibrosis (PMF) . Methods: A total of 113 Chinese patients with PMF were retrospectively analyzed. The Kaplan-Meier method, Log-rank test, and Cox proportional hazard regression model were performed to evaluate the prognostic factors. The likelihood ratio test was used to evaluate the predictive power between MIPSS70-plus and DIPSS systems. Results: The median age of the Chinese patients was 55 (range: 20-70) years, including 71 males and 42 females. According to the standard of MIPSS70-plus system, 99 patients (79.6% ) had a favorable karyotype and 23 patients (20.4% ) had an unfavorable karyotype. JAK2V617F in 55.8% (n=63) , CALR exon9 in 17.7% (including 15 CALR type 1 and 5 CALR type 2, n=20) , MPLW515 in 4.4% (n=5) , and triple negative (no detectable JAK2, MPL, and CALR mutations) in 22.1% of patients in our cohort were found by target-specific next-generation sequencing approach. At least one high-molecular risk mutations were presented in 45.1% (n=51) of patients, with ASXL1 in 38.9% (n=44) , SRSF2 in 7.1% (n=8) , IDH1/2 in 4.4% (n=5) , and EZH2 in 3.5% (n=4) of patients. A total of 28 patients (26.7% ) were in low risk, 20 (19.0% ) in intermediate risk, 41 (39.0% ) in high risk, and 16 (15.3% ) in very-high risk categories, which were delineated for the MIPSS70-plus model. A 2-year OS was 100% in low risk, 89.7% (95% CI 76.2% -100.0% ) in intermediate risk, 64.8% (95% CI 47.0% -82.6% ) in high risk, and 35.0% (95% CI 10.3% -59.7% ) in very-high risk categories, which had a significant difference (P<0.001) . A significantly higher predictive power for survival of the MIPSS70-plus group was observed compared with the DIPSS group (P=0.001, -2 log-likelihood ratios of 86.355 vs 95.990 for the MIPSS70-plus and DIPSS systems, respectively) . Conclusion: The MIPSS70-plus had significantly higher predictive power than the DIPSS.

目的： 评价MIPSS70-plus预后积分系统对中国原发性骨髓纤维化（PMF）患者的预后评估价值。 方法： 回顾性分析113例PMF患者的临床资料，应用Log-rank和COX回归模型进行预后相关因素分析；应用似然比检验比较MIPSS70-plus和动态国际预后积分系统（DIPSS）的预后评估效能。 结果： 113例PMF患者中男71例，女42例，中位年龄55（20～70）岁。依据MIPSS70-plus染色体核型分组标准，染色体核型预后良好组90例（79.6%），预后不良组23例（20.4%）。二代测序基因突变检测结果示，JAK2V617F突变63例（55.8%），CALR外显子9突变20例（17.7%）（其中1型CALR突变15例，2型CALR突变5例），MPLW515突变5例（4.4%），25例（22.1%）未检测到JAK2、MPL和CALR基因突变（三阴性）。高分子风险（HMR）突变检出率依次为ASXL1突变44例（38.9%）、SRSF2突变8例（7.1%）、IDH1/2突变5例（4.4%）、EZH2突变4例（3.5%）；51例患者（45.1%）有1种以上高危基因突变。MIPSS70-plus预后积分低危组、中危组、高危组、极高危组分别为28例（26.7%）、20例（19.0%）、41例（39.0%）、16例（15.3%），2年预期总生存率分别为100%、89.7%（95%CI 76.2%～100.0%）、64.8%（95%CI 47.0%～82.6%）、35.0%（95%CI 10.3%～59.7%）（P<0.001）。MIPSS70-plus的-2log似然比显著低于DIPSS（86.355对95.990，P＝0.001），表明MIPSS70-plus较DIPSS有更准确的预后分组预测效能。 结论： MIPSS-70plus较DIPSS预后积分系统对中国PMF患者有更好的预后评估效能。.

Keywords: Gene mutation; Karyotype; Primary myelofibrosis; Prognosis; Prognostic scoring system.

Background and purpose: When choosing a cytoreduction method for patients suffering from essential thrombocythemia (ET), it is important to know the safety profile of the medicine used. Few articles have been published about the effects of hydroxycarbamide (hydroxyurea, HU) and anagrelide (ANA) on renal function in ET patients. This study is the largest analysis of nephrotoxicity of cytoreductive drugs used in ET therapy so far, which additionally includes risk factors for the progression of kidney disease and coexisting genetic mutation.

Experimental approach: The retrospective study included 310 patients diagnosed with ET. Demographic data, comorbidities, Cr, and estimated glomerular filtration rate (eGFR) were all taken into account prior to diagnosis and after 6 months of HU and ANA treatment.

Key results: A statistically significant difference was found between Cr and eGFR levels at baseline and after 6 months of treatment (p < 0.001). The applied treatment (HU and ANA) had the greatest impact on kidney function. ANA significantly increased the risk of worsening renal function in contrary to hydroxycarbamide after 6 months of treatment (eGFR change: median +1 mL/min/1.73 m2 [interquartile range (IQR) (-4)-(+7)] in the HU group vss. median -13 mL/min/1.73 m2 [IQR (-18)-(-6)] in the ANA group, odds ratio [OR] 7.92 95% confidence interval [95% CI] [4.17-15.08], p < 0.001). Lowering of eGFR <60 mL/min/1.73 m2 occurred in 31 patients (31.0%) from the ANA group and 10 people (4.8%) treated with HU (p = 0.000). In 1 patient from the ANA group, >50% decrease in eGFR was observed. The chance for an increase in Cr levels was higher in people with pre-existing arterial hypertension (OR 1.92 CI = 95% [1.21-3.05], p = 0.006). Sex, type of mutation found (JAK2 V617F or CALR), and previous renal impairment did not affect renal function after 6 months of treatment. In addition, there was no difference in the efficacy of ET treatment between HU and ANA (p = 0.998).

Conclusions and implications: The observations indicate that ANA should be used in patients with ET with great caution and taking into account the risk of worsened kidney function.

Keywords: Anagrelide; Essential thrombocythemia; Hydroxycarbamide; Patient safety; Renal function.

Increased exposure of calreticulin (CALR) on malignant cells is associated with therapy-relevant adaptive immune responses and superior therapeutic outcome in solid tumors and haemato-oncological diseases, because surface-exposed CALR acts as an 'eat-me' signal facilitating the phagocytosis of stressed and dying cancer cells by immature dendritic cells, thus favoring antitumor immune responses. On the contrary, mutations of the CALR gene that cause the omission of the C-terminal KDEL endoplasmic reticulum retention motif from CALR protein, resulting in its secretion from cells, act as oncogenic drivers in myeloproliferative neoplasms via the autocrine activation of the thrombopoietin receptor. We recently showed that soluble CALR inhibited the phagocytosis of cancer cells by dendritic cells, thus dampening anticancer immune responses. Furthermore, systemic elevations of soluble CALR that is secreted from tumors or that is artificially supplied by injection of the recombinant protein decreased the efficacy of immunotherapy. Thus, depending on its location, CALR can have immunostimulatory or immunosuppressive functions.

Keywords: Immunosuppression; calreticulin secretion; immune checkpoint blockade.

Gastric cancer is a leading cause of cancer death worldwide. Endoplasmic reticulum (ER) stress-induced apoptosis has been confirmed to be important in the treatment of gastric cancer. MiR-637 has recently been found to exert inhibitory effects on gastric cancer, and this study aimed to investigate whether miR-637 could regulate apoptosis through ER stress. The results showed that tunicamycin (TM) induced downregulation of miR-637 in gastric cancer cells (AGS) and increase of apoptosis and ER stress. Overexpression of miR-637 promoted TM-induced apoptosis and expression of ER stress associated proteins (GRP78 and CHOP), but inhibited expression of Calreticulin. MiR-637 could bind with the 3'-UTR of CALR, and negatively regulated the expression of CALR. The co-transfection of miR-637 and CALR in AGS cells show that, CALR overexpression could reverse the pro-apoptosis effects of miR-637 in TM-treated cells. In conclusion, the present study suggests that miR-637 participates in ER stress-induced apoptosis in gastric cancer cells by suppressing CALR expression. miR-637 or CALR may be a future potential target for gastric cancer treatment.

Keywords: Calreticulin; MiR-637; apoptosis; endoplasmic reticulum stress; gastric cancer.

Background: Severe hyperlipemia (SHLE) has an impact on the results of many kinds of laboratory tests. Complete blood count (CBC) examination by automated blood cell counter (ABCC) is a quick and convenient measurement for screening abnormalities of blood cells that are triggered by various pathogenic insults in disease diagnosis and for monitoring changes in the treatment of existing hematological conditions. However, CBC results are frequently affected by many intrinsic and extrinsic factors from blood samples, such as in the setting of hypergammaglobulinemia and certain anticoagulants. SHLE could also affect CBC results.

Case summary: A 33-year-old Chinese male presented with painful foot numbness and abdominal pain. He was initially misdiagnosed as having a myeloproliferative neoplasm (MPN) because of the marked abnormalities in CBC examination by the ABCC. Morphological evaluation of the bone marrow smears and biopsy showed no evidence of MPN. Gene mutations in Breakpoint cluster regions-Abelson murine leukemia viral oncogene homologue 1 (BCR-ABL1), Janus kinase 2 (JAK2), calreticulin (CALR), myeloproliferative leukemia virus (MPL), and colony-stimulating factor 3 receptor (CSF3R) were negative. Having noticed the thick chylomicron layer on blood samples and the dramatically fluctuating CBC results, we speculated that the fat droplets formed by shaking the blood samples in the setting of SHLE were mistakenly identified as blood cells due to the limited parameters of ABCC. Therefore, we removed a large part of the chylomicron layer and then reexamined the CBC, and the CBC results, as we expected, differed significantly from that of the sample before the chylomicron layer was removed. These significant differences had been validated by the subsequently repeated laboratory tests by measuring dual blood samples that the chylomicron layer was removed in one sample and was not in another, and comparing the CBC results. Computerized tomography reexamination of the upper abdomen revealed an exudative lesion surrounding his pancreas. After intensive consultation, definitive diagnosis was made as recurrent pancreatitis, hyperlipemia and pseudoerythrocytosis.

Conclusion: SHLE may become a potential cause of misdiagnosis of hyperlipemia-related diseases as MPNs and the resultant mistreatment. It may also lead to the misinterpretation of transfusion indications in patients with hematological disorders who critically need blood transfusion for supportive treatment.

Keywords: Blood transfusion indication; Case report; Fat droplet; Hyperlipemia; Pancreatitis; Pseudoerythrocytosis.