Molecular markers are important in guiding treatment and predicting outcome in the genomic era. Meta-analysis of molecular markers in myelofibrosis through a search of PubMed and Medline through October 31, 2017 was performed. Markers with more than 3 studies that compared overall survival (OS) and leukemia-free survival (LFS) were analyzed. A total of 16 studies were included. Hazard ratios (HRs) for OS were as follows: IDH 2.65 (95% confidence interval [CI], 1.66-4.21), SRSF2 2.12 (95% CI, 1.18-3.79), high-risk myeloma 2.11 (95% CI, 1.70-2.61), ASXL1 1.92 (95% CI, 1.60-2.32), EZH2 1.88 (95% CI, 1.32-2.67), JAK2 1.41 (95% CI, 1.04-1.93) in the univariate analysis and 1.49 (95% CI, 0.42-5.30) in the multivariate analysis. LFS of JAK2 and SRSF2 had HRs of 1.81 (95% CI, 0.42-5.30) and 0.36 (95% CI, 0.02-6.48), respectively. In conclusion, mutations in IDH, SRSF2, and ASXL1 had worse prognosis in OS with HRs around 2. JAK2 and SRSF2 mutation were not associated with increased leukemia transformation. The adverse effect of triple-negative, which was often compared with CALR mutation, needs to be explored.

OBJECTIVE

We investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis (PMF).

METHODS

Pyrosequencing was used to detect JAK2V617F mutational status and measure allele burden, while MPL (exon 10) mutations were analysed by PCR assay and then by direct sequencing. CALR exon 9 mutations were first screened for length changes by GeneScan followed by sequencing. The allele burden of the mutated CALR gene was measured by pyrosequencing. The EEC assay was conducted using a serum-free culture system.

RESULTS

The frequencies of the three driver mutations and triple-negative status were similarly distributed between pre-PMF and overt PMF patients, except that pre-PMF patients had a higher incidence of CALR type 2/type-2 like mutations and a lower JAK2V617F allele burden. EEC growth and CALR mutations conferred favourable overall survival (OS). A lower JAK2V617F allele burden and grade 3 bone marrow fibrosis were associated with shorter OS and decreased leukaemia-free survival (LFS). Type 2/type 2-like CAL mutations were associated with better LFS compared with type1/type 1-like mutations. Patients with triple-negative mutation status had significantly worse OS and LFS. The allele burden of CALR mutations remained unchanged, while some JAK2V617F mutations showed clonal expansion in patients during secondary acute myeloid leukaemia transformation.

CONCLUSIONS

Our study showed that EEC growth, a higher JAK2V617F allele burden and CALR mutations, especially type 2, were independent predictors for better outcomes in PMF. The allele burden of CALR mutations remained stable, but the allele burden of JAK2V617Fmutations was variable during leukaemia transformation.

Myeloproliferative neoplasms (MPNs) are a group of hematologic disorders characterized by clonal proliferation of myeloid lineage cells. The diagnostic criteria are based on morphological features of bone marrow and peripheral blood cells but also include specific genomic mutations. In some patients, co-occurrence of hematologic and rheumatic diseases could be observed. To date, most of the reported cases concerned patients with myelodysplastic syndrome (MDS) or essential thrombocythemia (ET). In this paper, we present a case of a patient with a complicated diagnostic process leading to the diagnosis of unclassified MPN and giant cell arteritis (GCA). Routine tests did not reveal any mutations typical for MPNs such as JAK-2, CALR, MPL or BCR-ABL. Targeted next-generation sequencing (NGS) helped to confirm the diagnosis by demonstrating the presence of heterozygous ASXL1, TET2, SRSF2, and CBL mutations. The second important issue was the overlapping of symptoms of MPN and seronegative rheumatic disease, which finally was diagnosed as GCA. Leukocytosis and musculoskeletal pain, which were present at the time of diagnosis, resolved after allogeneic hematopoietic stem cell transplantation but recurred after a few months along with decreasing donor cell chimerism. Differentiation of the causes of recurrence of the symptoms was an important issue. This case shows the diagnostic challenge posed by co-incidence of MPN and rheumatic disease, especially its atypical variants.

Keywords: allogeneic stem cell transplantation; giant cell arteritis; myeloproliferative neoplasm; next-generation sequencing.

The identification of driver mutations in Janus kinase (JAK) 2, calreticulin (CALR), and myeloproliferative leukemia (MPL) has contributed to a better understanding of disease pathogenesis by highlighting the importance of JAK signal transducer and activator of transcription (STAT) signaling in classical myeloproliferative neoplasms (MPNs). This has led to the therapeutic use of novel targeted treatments, such as JAK2 inhibitors. More recently, with the development of next-generation sequencing, additional somatic mutations, which are not restricted to MPNs, have been elucidated. Treatment decisions for MPN patients are influenced by the MPN subtype, symptom burden, and risk classification. Although prevention of vascular events is the main objective of therapy for essential thrombocythemia (ET) and polycythemia vera (PV) patients, disease-modifying drugs are needed to eradicate clonal hematopoiesis and prevent progression to more aggressive myeloid neoplasms. JAK inhibitors are a valuable therapeutic strategy for patients with myelofibrosis (MF) who have splenomegaly and/or disease-related symptoms, but intolerance, refractory, resistance, and disease progression still present challenges. Currently, allogeneic stem cell transplantation remains the only curative treatment for MF, but it is typically limited by age-related comorbidities and high treatment-related mortality. Therefore, a better understanding of the molecular pathogenesis and potential new therapies with the aim of modifying the natural history of the disease is important. In this article, I review the current understanding of the molecular basis of MPNs and clinical studies on potential disease-modifying agents.

Keywords: Essential thrombocythemia; Myelofibrosis; Myeloproliferative neoplasms; Polycythemia vera.

Major disease complications for patients with essential thrombocythemia (ET) include thrombosis and fibrotic or leukemic transformation. Calreticulin (CALR) mutation type 1 frequencies in ET are estimated between 7% and 11% and ET patients carrying CALR type 1 mutation are associated with lower risk of thrombosis but higher risk of myelofibrosis transformation compared to ET patients with JAK2 mutation. Leukemic transformation rates at 20 years are estimated at less than 5% for ET and risk factors for leukemic transformation are advanced age, thrombosis history, leukocytosis, and anemia. Amongst the subtypes of blast phase myeloproliferative neoplasms, acute promyelocytic leukemia is extremely rare. Herein, we present a case of a promyelocytic blast crisis of post-ET myelofibrosis with associated life-threatening splanchnic vein thrombosis. This case suggests that inflammation plays a key role in thrombotic events and fibrotic/leukemic transformation in ET patients, regardless the molecular landscape.

Keywords: Acute promyelocyte leukemia; Blast-phase myeloproliferative neoplasm; Essential thrombocytemia; Myelofibrosis; Splanchnic vein thrombosis.

Somatic mutations associated with oncological diseases, including Ph-myeloproliferative neoplasms (Ph-MPN), are very diverse, occur with different frequencies and different allelic burden levels. Therefore, at the initial stage of performing molecular-genetic diagnostic procedures, it is desirable to be able to conduct screening tests in the laboratory. This is especially important when analyzing rare and diverse mutations. Analysis of high resolution melting curves (HRM analysis), which has high sensitivity and is suitable for screening all types of mutations, in a number of studies is proposed for the analysis of Ph-MPN associated mutations in the JAK2 and CALR genes. For analysis of somatic mutations in the majority of literature sources that we reviewed, the authors use the LightCycler (Roche) thermocycler and much rarely the CFX96 (Bio-Rad), which is often presented in Russian scientific and practical and medical organizations. The aim of the study was to screen the somatic JAK2 and CALR mutations by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN. In the present research, HRM analysis was conducted on the DNA samples from patients with mutations in the JAK2 or in the CALR gene. The Precision Melt Analysis software identified all variants of the analyzed mutations, both a single nucleotide substitution in the JAK2 gene (with allelic burden level in the range of 5-40%), and various indel mutations in the CALR gene (with allelic burden level in the range of 40-50%) Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 14 of the JAK2 gene and in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.

Keywords: CALR; JAK2; Ph-myeloproliferative neoplasms; high-resolution melt curve analysis; pyrosequencing.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are clonal disorders of a hematopoietic stem cell, characterized by an abnormal proliferation of largely mature cells driven by mutations in JAK2, CALR, and MPL. All these mutations lead to a constitutive activation of the JAK-STAT signaling, which represents a target for therapy. Beyond driver ones, most patients, especially with myelofibrosis, harbor mutations in an array of "myeloid neoplasm-associated" genes that encode for proteins involved in chromatin modification and DNA methylation, RNA splicing, transcription regulation, and oncogenes. These additional mutations often arise in the context of clonal hematopoiesis of indeterminate potential (CHIP). The extensive characterization of the pathologic genome associated with MPN highlighted selected driver and non-driver mutations for their clinical informativeness. First, driver mutations are enlisted in the WHO classification as major diagnostic criteria and may be used for monitoring of residual disease after transplantation and response to treatment. Second, mutation profile can be used, eventually in combination with cytogenetic, histopathologic, hematologic, and clinical variables, to risk stratify patients regarding thrombosis, overall survival, and rate of transformation to secondary leukemia. This review outlines the molecular landscape of MPN and critically interprets current information for their potential impact on patient management.

Keywords: CALR; JAK-STAT pathway; JAK2; MPL; additional mutations; essential thrombocythemia; myelofibrosis; myeloproliferative neoplasms; polycythemia vera; prognosis.

Morbidity and mortality of Philadelphia-chromosome negative myeloproliferative neoplasms (MPN) are mainly determined by thromboembolic complications. Thrombus formation is facilitated by a neutrophil-specific form of cell death linked to Neutrophil Extracellular Trap (NET) formation (NETosis). Pre-clinical and clinical data suggested a potential link between NETosis and thrombosis in MPN. In this study we aimed at defining the impact of NETosis on clinical endpoints in a large MPN cohort. NETosis was induced in vitro by Ionomycin and quantified by ELISA-based nucleosome release assays as well as fluorescent staining of free DNA in 103 samples from MPN patients and 28 healthy donors. NETosis rate was correlated to a broad set of clinical data, such as MPN subtype, mutational status, laboratory variables, history of thrombotic events and treatment types. Triggered NETosis levels were clearly higher in MPN patients when compared to healthy donors. Both, positivity for JAK2 V617F or exon 12 as well as CalR mutations correlate with increased NET formation. However, neither JAK2 allelic burden nor history of thromboembolic complication nor the presence of other risk factors for thrombosis (e.g. leukocytosis) were associated with the rate of NETosis. In addition, none of the analyzed laboratory parameters nor the type of treatment significantly impacted the rate of NETosis formation. MPN biology impacts NET formation as genetic driver mutations favor NETosis induction. However, this seems not to translate into important clinical endpoints such as thromboembolic complications. Therefore, NETosis may play a role in facilitating thrombosis but is not a sole causative determinant in MPN-associated thrombophilia.

OBJECTIVE

In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function.

UNASSIGNED

We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER.

CONCLUSIONS

Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (EC) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 weeks of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex-vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.

Myeloproliferative neoplasms (MPN) have been characterized by their clinical and histological patterns and have also been accordingly subclassified. Several specific genomic abnormalities are identifiable, raising the possibility of introducing a new era of genotype classification for an updated MPN classification. MPN classified by clinical and hematological data have specific histopathological characteristics of JAK2, MPL, and CALR abnormalities. Herein, the author will endeavor to devise an algorithm for MPN diagnosis based on bone marrow histology. Cellularity, erythroid islet formation, size, nuclear morphology, and the distribution pattern of megakaryocytes are the specific findings indicative of differences in genotype abnormalities.

Mutations in JAK2, MPL and CALR are regarded as driver mutations, and are mutually exclusively detected in more than 90% of myeloproliferative neoplasms (MPNs). In addition, mutations in epigenetic regulator genes such as TET2 or DNMT3A are detected in MPNs. Although the roles of mutations in epigenetic regulator genes were clarified in normal hematopoiesis, their roles have remained unclear in malignant hematopoiesis of MPNs. We analyzed three lines of mutant mice: mice with JAK2V617F, a representative of driver gene mutations; mice with loss of TET2, a representative of epigenetic abnormalities; and mice with both. We thereby clarified two roles of loss of TET2 in malignant hematopoiesis of JAK2-mutated MPNs: one is "disease initiator and sustainer" via reinforcing the function of JAK2-mutated hematopoietic stem cells, and the other is "disease accelerator". New strategies in risk assessment or treatment are required, considering not only single but also multiple mutations.

JAK2, MPL and CALR gene mutations play an important role in the onset of myeloproliferative disease(MPD). The latest researches show that the difference of ATP binding ability between the wild type JAK2 protein and mutated JAK2 protein can help us understand the pathogenesis of the MPD further, and the clinical manifestation is related to the mutation burden of the JAK2. In some ET and PMF patients, research find the expression of MPL mutation, which can affects the progress of the disease by collaborating with the JAK2 mutation. CALR mutation is a gene related with the MPD that has been found recently. The pathogenesis of the CALR is similar to that of the JAK2, while there are some features in clinical manifestation comparing with the other mutations.

The Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) share similar molecular characteristics in that they frequently harbor hotspot mutations in JAK2, CALR or MPL, leading to activated JAK/STAT signaling. However, these MPN have distinct symptoms, morphology, and natural histories, including different tendencies to progress to fibrosis. Although the role of inflammation in tissue fibrosis is well recognized, inflammatory gene expression in bone marrows involved by MPN has been understudied. We analyzed the expression of inflammatory genes by directly measuring RNA transcript abundance in bone marrow biopsies of 108 MPN patients. Unsupervised analyses identified gene expression patterns that distinguish prefibrotic (grade 1-2) MPN from overtly fibrotic (grade 2-3) MPN with high sensitivity and specificity in two independent cohorts. Furthermore, prefibrotic and overtly fibrotic MPN are separable into subsets with different activities in biological pathways linked to inflammation, including cytokines, chemokines, interferon response, and toll-like receptor signaling. In conclusion, this study demonstrates that MPN with overt fibrosis is associated with significant inflammatory gene upregulation in the bone marrow, revealing potential roles for multiple pro-inflammatory signaling networks in the development of myelofibrosis and suggesting potential therapeutic mechanisms to alleviate this process in the bone marrow microenvironment.

Autoimmune myelofibrosis is a rare, distinct clinicopathological entity that can occur in isolation (primary) or in association with systemic autoimmune disorders (secondary), such as systemic lupus erythematosus and Sjogren's syndrome. This disease is characterized by isolated or combined chronic cytopenias associated with autoimmune phenomena and bone-marrow fibrosis. Due to the rarity of this disease, patients are frequently misdiagnosed as having primary myelofibrosis, the most common form of bone-marrow fibrosis. Distinguishing between both disease entities is essential given the drastic therapeutic and prognostic differences between both disorders. We report a case of primary autoimmune myelofibrosis presenting with severe isolated anemia refractory to multiple lines of therapy. This patient was initially misdiagnosed as primary myelofibrosis. The absence of the characteristic features of primary myelofibrosis and the lack of a clonal abnormality on cytogenetic and molecular studies, particularly JAK2, CALR, and MPL mutation analyses, confirmed the absence of an aberrant neoplastic process. Furthermore, the presence of monoclonal T-cell receptor gamma gene rearrangements delineated the presence of an autoimmune disorder supporting our diagnosis of primary autoimmune myelofibrosis.

BACKGROUND

- Detection of variants in hematologic malignancies is increasingly important because of a growing number of variants impacting diagnosis, prognosis, and treatment response, and as potential therapeutic targets. The use of next-generation sequencing technologies to detect variants in hematologic malignancies in a clinical diagnostic laboratory setting allows for efficient identification of routinely tested markers in multiple genes simultaneously, as well as the identification of novel and rare variants in other clinically relevant genes.

OBJECTIVE

- To apply a systematic approach to evaluate and validate a commercially available next-generation sequencing panel (TruSight Myeloid Sequencing Panel, Illumina, San Diego, California) targeting 54 genes. In this manuscript, we focused on the parameters that were used to evaluate assay performance characteristics.

METHODS

- Analytical validation was performed using samples containing known variants that had been identified previously. Cases were selected from different disease types, with variants in a range of genes. Panel performance characteristics were assessed and genomic regions requiring additional analysis or wet-bench approaches identified.

CONCLUSIONS

- We validated the performance characteristics of a myeloid next-generation sequencing panel for detection of variants. The TruSight Myeloid Sequencing Panel covers more than 95% of target regions with depth greater than 500×. However, because of unique variant types such as large insertions or deletions or genomic regions of high GC content, variants in CEBPA, FLT3, and CALR required supplementation with non-next-generation sequencing assays or with informatics approaches to address deficiencies in performance. The use of multiple bioinformatics approaches (2 variant callers and informatics scripts) allows for maximizing calling of true positives, while identifying limitations in using either method alone.

In 2013, two seminal studies identified gain-of-function mutations in the Calreticulin (CALR) gene in a subset of JAK2/MPL-negative myeloproliferative neoplasm (MPN) patients. CALR is an endoplasmic reticulum (ER) chaperone protein that normally binds misfolded proteins in the ER and prevents their export to the Golgi and had never previously been reported mutated in cancer or to be associated with hematologic disorders. Further investigation determined that mutated CALR is able to achieve oncogenic transformation primarily through constitutive activation of the MPL-JAK-STAT signaling axis. Here we review our current understanding of the role of CALR mutations in MPN pathogenesis and how these insights can lead to innovative therapeutics approaches.

Mesenchymal stromal cells (MSCs) represent an essential component of the bone marrow (BM) niche and display disease-specific alterations in several myeloid malignancies. The aim of this work was to study possible MSC abnormalities in Philadelphia-negative myeloproliferative neoplasms (MPNs) in relationship to the degree of BM fibrosis. MSCs were isolated from BM of 6 healthy donors (HD) and of 23 MPN patients, classified in 3 groups according to the diagnosis and the grade of BM fibrosis: polycythemia vera and essential thrombocythemia (PV/ET), low fibrosis myelofibrosis (LF-MF), and high fibrosis MF (HF-MF). MSC cultures were established from 21 of 23 MPN patients. MPN-derived MSCs did not exhibit any functional impairment in their adipogenic/osteogenic/chondrogenic differentiation potential and displayed a phenotype similar to HD-derived MSCs but with a decreased expression of CD146. All MPN-MSC lines were negative for the patient-specific hematopoietic clone mutations (JAK2, MPL, CALR). MSCs derived from HF-MF patients displayed a reduced clonogenic potential and a lower growth kinetic compared to MSCs from HD, LF-MF, and PV/ET patients. mRNA levels of hematopoiesis regulatory molecules were unaffected in MSCs from HF-MF compared to HD. Finally, in vitro ActivinA secretion by MSCs was increased in HF-MF compared to LF-MF patients, in association with a lower hemoglobin value. Increased ActivinA immunolabeling on stromal cells and erythroid precursors was also observed in HF-MF BM biopsies. In conclusion, higher grade of BM fibrosis is associated with functional impairment of MSCs and the increased secretion of ActivinA may represent a suitable target for anemia treatment in MF patients.

Keywords: ActivinA; Mesenchymal stromal cells; Myelofibrosis; Myeloproliferative neoplasms.

Objective: To investigate the pathological characteristics of megakaryocytes in myeloproliferative neoplasms(MPN)and their correlations with driver gene mutations. Methods: Trephine specimens administered for 160 patients with MPN from February 2012 to October 2017 were reevaluated according to the World Health Organization(WHO)'s(2016)diagnostic criteria. Results: This cohort of patients included 72(45.0%)men, with the median age of 59(range, 13-87)years, comprising 39 with polycythemia vera(PV), 33 with essential thrombocythemia(ET), 37 with prefibrotic/early-primary myelofibrosis(pre-PMF), 37 with overt PMF, 1 with post-ET MF, 2 with post-PV MF, and 11 with MPN-unclassifiable(MPN-U)after the re-diagnosis. With PV, ET, pre-PMF, and overt PMF changes, proportions of dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes gradually increased, whereas erythropoiesis gradually decreased. Proportions of reticulin, collagen, and osteosclerosis grades of ≥1 also increased. Dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes were negatively correlated with erythropoiesis and positively correlated with granulopoiesis and fibrosis. In patients with pre- and overt PMF, dense clusters and naked nuclei of megakaryocytes were positively correlated with fibrosis. Patients with JAK2V617F MPN had significantly increased erythropoiesis(P=0.022). Patients with CALR-mutated MPN were characterized by increased loose and dense clusters; paratrabecular distribution and naked nuclei of megakaryocytes(P=0.055, P=0.002, P=0.018, P=0.008); and increased reticulin, collagen, and osteosclerosis(P=0.003, P<0.001, P=0.001). In patients with pre- and overt PMF, patients with JAK2V617F had increased cellularity(P=0.037). CALR-mutated patients had increased dense clusters and giant sizes of megakaryocytes, collagen, and osteosclerosis(P=0.055, P=0.059, P=0.011, P=0.046). Conclusion: Megakaryocytes showed abnormal MPN morphology and distribution, which were related to fibrosis. CALR mutation was probably associated with abnormal morphology and distribution of megakaryocytes and fibrosis.

目的： 探讨骨髓增殖性肿瘤(MPN)巨核细胞病理特征及其与起始基因突变的相关性。 方法： 收集2012年2月至2017年10月于中国医学科学院血液病医院就诊的160例初诊MPN患者，根据世界卫生组织(WHO)2016年MPN诊断标准对患者骨髓活检组织切片进行重新评估。 结果： 160例患者中男72例(45.0%)，女88例(55.0%)，中位年龄59(13~87)岁。重新评估后真性红细胞增多症(PV)39例，原发性血小板增多症(ET)33例，纤维化前/早期原发性骨髓纤维化(pre-PMF)37例，明显期原发性骨髓纤维化(overt PMF)37例，真性红细胞增多症后骨髓纤维化(post-PV MF)2例，原发性血小板增多症后骨髓纤维化(post-ET MF)1例，MPN-未分类(MPN-U)11例。按PV、ET、pre-PMF及overt PMF疾病亚型顺序，密集成簇分布、少分叶核及裸核巨核细胞逐渐增加，红系增生程度正常及增高的比例逐渐降低，1级及以上网状纤维、胶原及骨硬化的比例逐渐升高。相关性分析示密集成簇分布、少分叶核及裸核巨核细胞占比与红系增生程度呈负相关，与粒系增生程度及纤维化程度呈正相关。对pre-PMF及overt PMF患者病理特征的相关性分析示密集成簇分布及裸核巨核细胞占比与纤维化程度呈正相关。JAK2V617F突变MPN患者红系增生程度明显增高(P=0.022)，CALR突变MPN患者疏松成簇、密集成簇、骨小梁旁分布及裸核巨核细胞明显增多(P=0.055，P=0.002，P=0.018，P=0.008)，1级及以上网状纤维、胶原及骨硬化比例增高(P=0.003，P<0.001，P=0.001)。伴JAK2V617F突变pre-PMF及overt PMF患者骨髓增生程度较高(P=0.037)，伴CALR突变患者巨大体积及密集成簇分布巨核细胞明显增多(P=0.059，P=0.055)，1级及以上胶原及骨硬化的比例明显增高(P=0.011，P=0.046)。 结论： 不同亚型MPN患者骨髓巨核细胞病理改变特征各异，其病理异常特征与纤维化水平相关。CALR突变可能与巨核细胞病理异常特征及骨髓纤维化水平相关。.

Keywords: Driver gene mutations; Histopathology; Megakaryocyte; Myeloproliferative neoplasms.