The Study Of Molecular Genetics,immune Characteristics,and The Mechanisms Of Ruxolitinib Improving Myelofibrosis In Patients With Myeloproliferative Neoplasms

Background and Objective:Among the pathogenesis of myeloproliferative neoplasms(MPNs),driver mutations,cytokines,and fibroblasts are involved in the evolution of MPNs.Myelofibrosis(MF)is a common disease feature of MPNs subtypes and is related to bone marrow mesenchymal cell(BMMSC),tumor fibroblasts(CAF)and cytokines.However,its exact mechanism has not been elucidated.In this study,we investigate the heterogeneity of MPNs by detecting mutations,T lymphocyte subsets and cytokines.And then we explore the possible relationship among CAF,LOXL-2 and MF evolution by analyzing the changes of MPNs cells after the treatment with ruxolitinib(RUX)to provide a strong theoretical basis for the treatment of MPNs patients.Methods:1.Applying next-geineration sequencing(NGS)based 127-gene panel to detect the 125 MPNs patients and analyze the mutation status.2.A total of 33 cytokines and T lymphocyte subsets were detected in 123 MPNs patients by high-thr-oughput protein chip.ELISA test to review MF related cytokines.3.Detection of normal and MPNs before and after RUX treatment,including 1)mononuclear cell CAF markers(a-SMA,FAP)and serum LOXL-2 mRNA and protein in bone marrow sample;2)LOXL-2 in bone marrow or peripheral blood;3)a-SMA,FAP in BMMSC.4.BMMSCs from normal humans,MPNs,and RUX+MPNs were cocultured to analyze cell morphology,immunophenotype,osteogenesis,and adipogenic capacity,and to detect a-SMA,FAP mRNA and protein levelResult:1.JAK2,CALR,TET2,ASXL1 and DNMT3A were the five most common mutations.According to the classification of gene function,the mutations of signaling pathways and epigenetic regulators were the most frequent mutations.Additional mutations have distinct heterogeneity.2.The CD3+ and CD4+ T cells of MPNs decreased in ET,PV and PMF(P<0.05),and PMF decreased significantly compared with ET and PV(P<0.05).CD3+ and CD4+ T cells in JAK2 mutation are lower than those in control group(P<0.05).And the CD8+CD28-cells of JAK2 and CALR mutations are lower than the control group(P<0.05),too.CD3+,CD4+ and CD8+CD28-cells with and without additional mutation MPNs decreased compared with the control group,but the latter decreased more significantly(P<0.05).3.Compared with the control group,12 cytokines MPNs are increased(including 2 MF--related cytokines IL-8 and VEGF)(P<0.01),while IL-15 decreased(P<0.01).IL-8 and VEGF are higher in overt myelofibrosis group than ino overt group(P<0.05).The ELISA method was used to review IL-8 and VEGF,and the results were consistent with the results of high-throughput protein array detection.4.Compared with the control group,it is a high expression of a-SMA,FAP and LOXL-2 mRNA and protein levels in bone marrow of MPNs and LOXL-2 in bone marrow and peripheral blood,(P<0.05)while decreased expression after RUX treatment(P<0.05).5.It’s not significantly different from those in normal groups that the immunophenotype and a-SMA and FAP mRNA levels of BMMSCs in MPNs before and after RUX treatment(P>0.05);the protein levels of a-SMA and FAP before RUX treatment were significantly higher than those of normal(P<0.05).It is a decline after ruxolitinib treatment(P<0.05).Conclusion:1.It is limited that the number of driver mutation types of MPNs.The heterogeneity of additional mutations according to functional classification displayed a diverse mutation pattern,which is of great value in judging prognosis and disease evolution.Epigenetic regulators mutation was the most common additional mutation types in MPNs.2.This study detected rare mutations in MPNs patients.SH2B3 and GNAS may be involved in the onset of MPNs;CUX1 and IKZF1 may be related to the progression of MPNs to leukemia;CEBPA,STAG2,NF1 and PTEN may play a role in the development of MPNs;the role of KMT2C,PTPN11,ZRSR2 and IL7R in the evolution of MPNs disease is unknown.;ZRSR2 and IL7R are the first discovered in patients with MPNs,providing clues for further research.3.It is a clinical value that dynamic monitoring of lymphocyte subsets,IL-8 and VEGF in MPNs in understanding disease progression and myelofibrosis extent and judging prognosis.4.RUX can decrease the LOXL-2 of MPNs and the ability of BMMSC to activate CAF.It is speculated that under hypoxic niche,RUX may delay the MF process of MPNs by lowering LOXL-2 level and reducing the stimulation of MSC convert to CAF.