Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15-CD11b- was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.

UNASSIGNED: Philadelphia-chromosome negative myeloproliferative neoplasms (MPN) exhibit phenotypic similarities with JAK/STAT-unmutated idiopathic erythrocytosis and thrombocytosis (IE/IT). We aimed to develop a clinical diagnostic model to discern MPN and IE/IT.
UNASSIGNED: A retrospective study was performed on 77 MPN patients and 32 IE/IT patients in our center from January 2018 to December 2023. We investigated the role of hemogram, cytokine and spleen size in differentiating MPN and IE/IT among newly onset erythrocytosis and thrombocytosis patients. Independent influencing factors were integrated into a nomogram for individualized risk prediction. The calibration and discrimination ability of the model were evaluated by concordance index (C-index), calibration curve.
UNASSIGNED: MPN had significantly higher TNF-α level than IE/IT, and the TNF-α level is correlated with MF-grade. Multivariable analyses revealed that TNF-α, PLT count, age, size of spleen were independent diagnostic factors in differentiating MPN and IE/IT. Nomograms integrated the above 4 factors for differentiating MPN and IE/IT was internally validated and had good performance, the C-index of the model is 0.979.
UNASSIGNED: The elevation of serum TNF-α in MPN patients is of diagnostic significance and is correlated with the severity of myelofibrosis. The nomogram incorporating TNF-α with age, PLT count and spleen size presents a noteworthy tool in the preliminary discrimination of MPN patients and those with idiopathic erythrocytosis or thrombocytosis. This highlights the potential of cytokines as biomarkers in hematologic disorders.

Myeloproliferative neoplasm (MPN) usually has an adverse prognosis, progressing to acute leukemia or splanchnic vein thromboses (SVTs). Therefore, early diagnosis and intervention are significantly important. Clinically, the burden of JAK2V617F is a vital diagnostic basis, which can be detected during the early stage of MPN. Thus, an accurate and rapid detective technique is urgently required. In recent years, real-time quantitative PCR (qPCR) has primarily been applied to detect the copies of JAK2V617F, whereas droplet digital PCR (ddPCR), a novel and promising detective tool, can conduct precise and repeatable quantification of nucleic acid copies without relying on the standard curve. In our study, both qPCR and ddPCR are used to evaluate the mutation burden of JAK2V617F in a series of gradient diluted standards and clinical JAK2V617F-positive MPN patients\' bone marrow samples collected, while using next-generation sequencing technology (NGS) as a contrast. With the help of statistical methods, our study concluded that ddPCR had a better performance in accuracy, sensitivity, and stability, especially in a low burden. Regarding the accuracy, ddPCR showed a better linearity (Pearson R2 = 0.9926; P < 0.0001) than qPCR (Pearson R2 = 0.9772; P < 0.0001). What is more, ddPCR showed lower intra-assay and inter-assay CVs and the limit of detection (LOD) for the series of diluted standards than qPCR, demonstrating better stability and lower LOD. In a nutshell, ddPCR is a more promising technique for the detection and quantification of JAK2V617F.

UNASSIGNED: Myeloproliferative neoplasm (MPN) is a heterogeneous group of hematopoietic stem cell disorders characterized by clonal proliferation of one of more of the hematopoietic stem cell lineages. Clinical manifestations result from uncontrolled myeloproliferation, extramedullary hematopoiesis with splenomegaly and excessive inflammatory cytokine production. Currently available therapy improves hematologic parameters and symptoms but does not adequately address the underlying neoplastic biology. Bomedemstat has thus far demonstrated clinical efficacy and tolerability in the treatment of MPNs with recent evidence of impacting the malignant stem cell population.
UNASSIGNED: This review summarizes the mechanisms of action, pharmacokinetics and pharmacodynamics, safety and efficacy of bomedemstat in MPN with specific emphasis on essential thrombocythemia (ET) and myelofibrosis (MF).
UNASSIGNED: In patients with MPNs, bomedemstat appears effective and well tolerated. The signs and symptoms of these diseases are managed as a reduction in the frequency of mutant cells was demonstrated in patients with ET and MF. Ongoing and planned studies of bomedemstat in MPN will establish the position of bomedemstat in MPNs and may help to redefine treatment endpoints of MPNs in the future.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized clinically by the proliferation of one or more hematopoietic lineage(s). The classical Philadelphia-chromosome (Ph)-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The Asian Myeloid Working Group (AMWG) comprises representatives from fifteen Asian centers experienced in the management of MPN. This consensus from the AMWG aims to review the current evidence in the risk stratification and treatment of Ph-negative MPN, to identify management gaps for future improvement, and to offer pragmatic approaches for treatment commensurate with different levels of resources, drug availabilities and reimbursement policies in its constituent regions. The management of MPN should be patient-specific and based on accurate diagnostic and prognostic tools. In patients with PV, ET and early/prefibrotic PMF, symptoms and risk stratification will guide the need for early cytoreduction. In younger patients requiring cytoreduction and in those experiencing resistance or intolerance to hydroxyurea, recombinant interferon-α preparations (pegylated interferon-α 2A or ropeginterferon-α 2b) should be considered. In myelofibrosis, continuous risk assessment and symptom burden assessment are essential in guiding treatment selection. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in MF should always be based on accurate risk stratification for disease-risk and post-HSCT outcome. Management of classical Ph-negative MPN entails accurate diagnosis, cytogenetic and molecular evaluation, risk stratification, and treatment strategies that are outcome-oriented (curative, disease modification, improvement of quality-of-life).

Sperm-associated antigen 6 (SPAG6) has been identified as an oncogene or tumor suppressor in various types of human cancer. However, the role of SPAG6 in BCR::ABL1 negative myeloproliferative neoplasms (MPNs) remains unclear. Herein, we found that SPAG6 was upregulated at the mRNA level in primary MPN cells and MPN-derived leukemia cell lines. The SPAG6 protein was primarily located in the cytoplasm around the nucleus and positively correlated with β-tubulin expression. In vitro, forced expression of SPAG6 increased cell clone formation and promoted G1 to S cell cycle progression. Downregulation of SPAG6 promoted apoptosis, reduced G1 to S phase transition, and impaired cell proliferation and cytokine release accompanied by downregulated signal transducer and activator of transcription 1 (STAT1) expression. Furthermore, the inhibitory effect of interferon-α (INF-α) on the primary MPN cells with high SPAG6 expression was decreased. Downregulation of SPAG6 enhanced STAT1 induction, thus enhancing the proapoptotic and cell cycle arrest effects of INF-α both in vitro and in vivo. Finally, a decrease in SPAG6 protein expression was noted when the STAT1 signaling was blocked. Chromatin immunoprecipitation assays indicated that STAT1 protein could bind to the SPAG6 promoter, while the dual-luciferase reporter assay indicated that STAT1 could promote the expression of SPAG6. Our results substantiate the relationship between upregulated SPAG6, increased STAT1, and reduced sensitivity to INF-α response in MPN.

The translocation t(8;9) produces the fusion gene PCM1-JAK2, resulting in the continuous activation of the JAK2 tyrosine kinase. Myelodysplastic/myeloproliferative neoplasms are the most common disease with t(8;9)/PCM1-JAK2. Individuals with this abnormality have similar features, and JAK2 kinase inhibitor (ruxolitinib) is an effective treatment of the condition. The long-term remission results of ruxolitinib are varied. It is important to determine the response to ruxolitinib. Here, we describe a patient who has been diagnosed with eosinophilia-associated myeloproliferative neoplasm with t(8;9)(p21;p24). This patient has achieved sustained response for >1 year since the administration of ruxolitinib.

OBJECTIVE: To investigate the expression properties, structural features and function of CALR E381A in myeloproliferative neoplasms (MPN) patients.
METHODS: In this retrospective study, 435 MPN patients admitted to the Department of Hematology, Ningbo First Hospital from July 2015 to July 2021 were selected as the study subjects. Mutations in CALR exon 9 from genomic DNA samples were identified by PCR, followed by Sanger sequencing. The physicochemical properties of the wild-type calreticulin and the p.E381A variant, and the structural information of the p.E381A variant were analyzed by using the bioinformatics databases. Growth assay of UT-7/mpl cells with CALR E381A was used for the functional analysis of CALR E381A.
RESULTS: The predominant types of CALR variants were identified as follows: p.L367fs*46 (38.1%), p.K385fs*47 (25.8%) and p.E381A (19.6%). Notably, the frequency of the p.E381A variant (c.1142A >C) in polycythemia vera or essential thrombocythemia was significantly higher than the frequency of that as a single nucleotide polymorphism (SNP) in the East Asian population. Furthermore, CALR E381A coexisted with other genetic variants, of which JAK2 V617F was more common. Bioinformatics analysis confirmed that CALR E381A did not change the physicochemical properties of the calreticulin protein, but did change the electrical charge, energy state and steric hindrance of amino acid residues at site 381. UT-7/mpl cells harboring CALR E381A overexpression did not exhibit altered cell growth, which is distinctly different from the stereotypical frameshift mutation.
CONCLUSIONS: CALR E381A is not a driver mutation for the development of MPN but may be a risk SNP implying an inherited predisposition for MPN disease in East Asian populations.

Objective: To evaluate the clinical characteristics and prognostic factors of patients with Philadelphia-negative myeloproliferative neoplasm-accelerated phase/blast phase (MPN-AP/BP) . Methods: A total of 67 patients with MPN-AP/BP were enrolled from February 2014 to December 2021 at the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences. Their clinical features and prognostic factors were analyzed retrospectively. Results: ① Sixty-seven patients with MPN-AP/BP with a median age of 60 (range, 33-75) years, including 31 males (46.3% ) and 36 females (53.7% ) , were analyzed. Forty-eight patients progressed from primary myelofibrosis (PMF) , and 19 progressed from other myeloproliferative neoplasms (MPNs) , which included polycythemia vera, essential thrombocythemia, and MPN unclassifiable. Patients who progressed from PMF had higher lactate dehydrogenase (LDH) levels than those who progressed from other MPNs (925.95 vs. 576.2 U/L, P=0.011) , and there were higher proportions of patients who progressed from PMF with splenomegaly (81.4% vs. 57.9% , P=0.05) , a myelofibrosis grade of ≥2 (93.6% vs. 63.2% , P=0.004) , and a shorter duration from diagnosis to the transformation to AP/BP (28.7 vs. 81 months, P=0.001) . ② JAK2V617F, CALR, and MPLW515 were detected in 41 (61.2% ) , 13 (19.4% ) , and 3 (4.5% ) patients, respectively, whereas 10 (14.9% ) patients did not have any driver mutations (triple-negative) . Other than driver mutations, the most frequently mutated genes were ASXL1 (42.2% , n=27) , SRSF2 (25% , n=16) , SETBP1 (22.6% , n=15) , TET2 (20.3% , n=13) , RUNX1 (20.3% , n=13) , and TP53 (17.2% , n=11) . The ASXL1 mutation was more enriched (51.1% vs. 21.1% , P=0.03) , and the median variant allele fraction (VAF) of the SRSF2 mutation (median VAF, 48.8% vs. 39.6% ; P=0.008) was higher in patients who progressed from PMF than those who progressed from other MPNs. ③ In the multivariate analysis, the complex karyotype (hazard ratio, 2.53; 95% confidence interval, 1.06-6.05; P=0.036) was independently associated with worse overall survival (OS) . Patients who received allogeneic stem cell transplantation (allo-HSCT) (median OS, 21.3 vs. 3 months; P=0.05) or acute myeloid leukemia-like (AML-like) therapy (median OS, 13 vs. 3 months; P=0.011) had significantly better OS than those who received supportive therapy. Conclusion: The proportions of patients with PMF-AP/BP with splenomegaly, myelofibrosis grade ≥2, a higher LDH level, and a shorter duration from diagnosis to the transformation to AP/BP were higher than those of patients with other Philadelphia-negative MPN-AP/BP. The complex karyotype was an independent prognostic factor for OS. Compared with supportive therapy, AML-like therapy and allo-HSCT could prolong the OS of patients with MPN-AP/BP.
目的： 分析Ph染色体阴性骨髓增殖性肿瘤加速期/急变期（MPN-AP/BP）患者的临床特征及预后因素。 方法： 收集2014年2月至2021年12月期间就诊于中国医学科学院血液病医院的67例Ph染色体阴性MPN-AP/BP患者的病例资料，回顾性分析其临床特征及预后因素。 结果： ①全部67例MPN-AP/BP患者中男31例（46.3%），女36例（53.7%），中位年龄为60（33~75）岁；原发性骨髓纤维化（PMF）进展的患者48例（PMF-AP/BP组），由真性红细胞增多症（PV）、原发性血小板增多症（ET）、骨髓增殖性肿瘤不能分类（MPN-U）进展的患者19例（其他MPN-AP/BP组）。PMF-AP/BP组与其他MPN-AP/BP组比较，乳酸脱氢酶（LDH）水平较高（925.9 U/L对576.2 U/L，P＝0.011)，脾肿大患者占比较高（81.4%对57.9%，P＝0.05），骨髓网状纤维≥2级者占比较高（93.6%对63.2%，P＝0.004），进展为AP/BP时间较短（28.7个月对81.0个月，P＝0.001）。②67例Ph染色体阴性MPN-AP/BP患者中，41例（61.2%）检出JAK2V617F突变，13例（19.4%）检出CALR外显子9突变（1型CALR突变11例，2型CALR突变2例），3例（4.5%）检出MPLW515突变，JAK2、MPL和CALR基因突变均未检出10例（14.9%）；非驱动基因突变检出率较高的依次为ASXL1（42.2%，27例）、SRSF2（25.0%，16例）、SETBP1（22.6%，15例）、TET2（20.3%，13例）、RUNX1（20.3%，13例）和TP53基因（17.2%，11例）。PMF-AP/BP组ASXL1基因突变检出率（51.1%对21.1%，P＝0.03）及SRSF2基因突变频率（VAF）高于其他MPN-AP/BP组（48.8%对39.6%，P＝0.008）。③多因素分析示复杂染色体核型是影响MPN-AP/BP患者总生存（OS）期的独立不良预后因素（HR＝2.53，95%CI 1.06~6.05，P＝0.036）。接受异基因造血干细胞移植或白血病样化疗的患者与接受支持治疗的患者比较，OS期较长[（21.3（95%CI 10.2~32.3）个月对3.0（95%CI 2.3~3.7）个月，P＝0.05；13.0（95%CI 8.3~17.7）个月对3.0（95%CI 2.3~3.7）个月，P＝0.011]。 结论： 与其他Ph染色体阴性MPN-AP/BP患者比较，PMF-AP/BP患者具有较高的脾肿大和骨髓网状纤维≥2级发生率、较高的LDH水平，进展为AP/BP时间也较短。复杂染色体核型是影响MPN-AP/BP患者OS期的独立不良预后因素。异基因造血干细胞移植和白血病样化疗可延长MPN-AP/BP患者OS期。.

The classic BCR-ABL1-negative myeloproliferative neoplasm (MPN) is a highly heterogeneous hematologic tumor that includes three subtypes, namely polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). Despite having the same JAK2V617F mutation, the clinical manifestations of these three subtypes of MPN differ significantly, which suggests that the bone marrow (BM) immune microenvironment may also play an important role. In recent years, several studies have shown that peripheral blood monocytes play an important role in promoting MPN. However, to date, the role of BM monocytes/macrophages in MPN and their transcriptomic alterations remain incompletely understood. The purpose of this study was to clarify the role of BM monocytes/macrophages in MPN patients with the JAK2V617F mutation. MPN patients with the JAK2V617F mutation were enrolled in this study. We investigated the roles of monocytes/macrophages in the BM of MPN patients, using flow cytometry, monocyte/macrophage enrichment sorting, cytospins and Giemsa-Wright staining, and RNA-seq. Pearson correlation coefficient analysis was also used to detect the correlation between BM monocytes/macrophages and the MPN phenotype. In the present study, the proportion of CD163+ monocytes/macrophages increased significantly in all three subtypes of MPN. Interestingly, the percentages of CD163+ monocytes/macrophages are positively correlated with HGB in PV patients and PLT in ET patients. In contrast, the percentages of CD163+ monocytes/macrophages are negatively correlated with HGB and PLT in PMF patients. It was also found that CD14+CD16+ monocytes/macrophages increased and correlated with MPN clinical phenotypes. RNA-seq analyses demonstrated that the transcriptional expressions of monocytes/macrophages in MPN patients are relatively distinct. Gene expression profiles of BM monocytes/macrophages suggest a specialized function in support of megakaryopoiesis in ET patients. In contrast, BM monocytes/macrophages yielded a heterogeneous status in the support or inhibition of erythropoiesis. Significantly, BM monocytes/macrophages shaped an inflammatory microenvironment, which, in turn, promotes myelofibrosis. Thus, we characterized the roles of increased monocytes/macrophages in the occurrence and progression of MPNs. Our findings of the comprehensive transcriptomic characterization of BM monocytes/macrophages provide important resources to serve as a basis for future studies and future targets for the treatment of MPN patients.