The First Part Is The Study Of The Specific Changes And Mechanism Of The Bone Marrow Hematopoietic Microenvironment In Patients With Essential Thrombocythemia. The Second Part Is The Evaluation Of The Symptom Burden Of 173 Chinese Patients With Essential

Objectives:To understand the changes and underlined mechanism of the bone marrow niche in JAK2V617F-positive essential thrombocythemia(ET).Method:In total,87 never treated or currently untreated patients with JAK2V617F-positive ET and 48 healthy donors(HDs)were included in this study.We isolated and amplified mesenchymalmal stromal cells(MSCs)from healthy donors and patients with JAK2V617F-positive ET,and identified them according to the minimal criteria for defining multipotent mesenchymal stromal cells stated by the International Society for Cellular Therapy position.[1]We performed RNA sequencing in combination with GO(gene ontology),KEGG(Kyoto Gene and Genomic Encyclopedia)analysis and GSEA(gene set enrichment analysis)on MSCs from HDs and patients with JAK2V617F-positive ET.Based on the differences in transcriptome,we performed a comparative study of MSCs function,including detection of proliferation of MSCs using Cell Counting Kit-8(CCK-8),detection of apoptosis of MSCs using Annexin V and propidium iodide staining,detection of senescence of MSCs using β-galactosidase staining,detection of differentiation of MSCs using oil red O,alizarin red and Alcian blue staining,detection of MSCs cell cycle using propidium iodide staining,detection of normal hematopoietic support capacity of MSCs by long-term culture system,detection of hematopoietic-associated cytokine levels in bone marrow extracts by enzyme-linked immunosorbent assay(ELISA),detection of CD4-positive T cell proliferation in bone marrow using a cell proliferation assay kit,detection of CD4-positive T cell activation and the number of T helper cells 1/2/17(Thl/2/17)and regulatory T cell(Treg)subsets in bone marrow,detection of inflammation-related cytokine levels in bone marrow extracts by ELISA,detection of the immunomodulatory capacity of MSCs using co-culture systems and detection of the sympathetic nerve fibers and Schwann cells in bone marrow using immunohistochemical staining.Based on transcriptome and functional studies,we used gene overexpression and knockdown techniques in combination with signaling pathway inhibitors to further exploit the molecular mechanisms underlying these differences.Results:Here,we observed multilevel defects in the hematopoietic niche in JAK2V617F-positive essential thrombocythemia,including deficient function of MSCs,immune imbalance,and sympathetic-nerve damage.MSCs from patients with JAK2V617F-positive ET showed transformed transcriptome,enhanced proliferation,decreased apoptosis and senescence,attenuated ability to differentiate into adipocytes and osteoblasts,reduced inhibition of CD4-positive T cell proliferation and activation and secretion of the inflammatory factor soluble CD40-ligand,decreased induction of mostly immunosuppressive T helper 2(Th2)cell formation and secretion of the anti-inflammatory factor interleukin-4,and insufficient support for normal hematopoiesis.Furthermore,we identified WDR4 as a potent protein with low expression and correlated with increased proliferation,reduced senescence and differentiation,and insufficient support of normal hematopoiesis in MSCs from patients with JAK2V617F-positive ET.We also showed that loss of WDR4 triggers decreased production of interleukin-6 and found a novel link between WDR4 and ERK-GSK3β-CREB signaling in regulating interleukin-6(IL-6)expression and secretion in MSCs in vitro.Conclusions:Multilevel changes occur in the bone marrow niche and low expression of WDR4 in MSCs may be critical for inducing the failure of normal hematopoiesis via IL-6 modulation in JAK2V617F-positive ET.Objectives:To systematically analyze the symptom burden and its clinical value in Chinese ET patients.Method:In total,173 Chinese ET patients were selected and grouped based on disease characteristics(mutation status,treatment regimen,and sex).Results:All subgroups showed low-to-high symptom burden,with the highest mean in the Hu-used subgroup(total symptom score[TSS],14.7;range,7.6-14.7).The JAK2V617F-positive,Hu-used,and female subgroups had higher TSS and independent symptom scores than the control group.The CALR-positive and IFN-α-used subgroups had lower overall and individual scores than subgroups lacking the corresponding characteristics.As the number of features(JAK2V617F-positive,Hu-used,and female)increases,the severity of symptoms gradually increased.Conclusions:We revealed that different characteristics have various effects on symptom burden in ET patients.The accumulation of certain characteristics will lead to more severe symptom burden,significantly guiding the choice of clinical treatment.