Part 1 Features And Clinical Significance Of Gene Mutations In Patients With Myelodysplastic Syndromes With Ring Sideroblasts Part 2 Origination Of Myofibroblasts And Fibrocytes In Persons With Primary Myelofibrosis And Persons With Myelodysplastic Syndro

BackgroundGenetic mutations were prevalent in myelodysplastic syndromes(MDS),and might influence the prognosis of MDS subjects.MDS subjects harboring SF3B1 mutation presented with higher marrow ring sideroblasts(RS)percentage and a better overall survival.According to WHO(2016)classification,a diagnosis of MDS with ring sideroblasts(MDS-RS)might be made if RS comprise as few as 5%of nucleated erythroid cells in MDS subjects with SF3B1 mutation and without excess blasts(<5%).ObjectiveTo explore the features and clinical significance of gene mutations in patients with myelodysplastic syndromes with MDS-RS.MethodsA total of 255 newly diagnosed primary MDS-RS patients were retrospectively reviewed from our center from January 2001 to June 2019.SF3B1 gene mutations were detected by Sanger sequencing in 129 patients,and next generation sequencing(NGS)was performed in the other 126 patients using a set of selected 112-genes.Results193(75.7%)patients presented with SF3B1 mutation,predominantly mutant at amino acid position 700(K700E)(n=147,76.2%).The frequencies of various type of SF3B1 mutation had no significant difference between 5%?RS<15%and RS>15%groups(all P value>0.05).Non-SF3B1 gene mutations were TET2(16.7%),ASXL1(14.3%),U2AF1(11.1%),TP53(7.9%),SETBP1(6.3%)and RUNX1(6.3%).5%?ring sideroblasts(RS)<15%patients had a higher SETBP1 mutation frequency than RS?15%patients(21.4%vs 4.5%,P=0.044),mutation frequencies of other genes were similar in both groups(all P vale>0.05).SF3B1 variant allele frequencies(VAF)had positive correlation with marrow RS percentage but without statistical significance in 5%?RS<15 group(P=0.078,r=0.486).SF3B1 mutant patients presented with higher marrow RS percentage compared to wild-type patients[40.0%(15.0?80.0%)vs 25.5%(15.0?82.0%),P<0.001],and SF3B1 VAF positively correlated with RS percentage(P=0.009,r=0.261)in RS?15%group.Age,ANC,PLT,mean RBC corpuscular volume,RS percentage,IPSS-R cytogenetics and IPSS-R risk score were significantly different between patients with SF3B1 mutations and wild-type SF3B1 in all patients or RS?15%patients(all P value<0.05).Clinical and laboratory characteristics of 5%<RS<15%patients were almost similar to those with RS?15%.There was no significant difference for overall survival(OS)between patients with 5%?RS<15%and RS?15%(P=0.314).Univariable and multivariable survival analyses adjusted by age and IPSS-R cytogenetics displayed that SF3B1 mutation was an independent favorable prognostic factor(HR=0.265,95%CI 0.077-0.917,P=0.036),and TP53 mutation was an adverse variable independent of SF3B1 mutation(HR=6.272,95%CI 1.725?22.809,P=0.005).According to the mutant status of SF3B1 and TP53,MDS-RS patients were categorized into four groups,namely group with SF3B1 and TP53 mutation,with wild type SF3B1 and TP53,with wild type SF3B1 but TP53 mutation and with SF3B1 mutation but wild type TP53.There was significant difference for OS among these four groups(P<0.001).The former three groups showed no significant difference in OS in multiple comparison.However,the SF3B1 mutation but wild type TP53 group had a better OS than wild type SF3B1 but TP53 mutation group and wild type SF3B1 and TP53 group,whereas a similar OS compared to SF3B1 and TP53 mutation group.ConclusionSF3B1 mutations were prevalent in MDS-RS patients with the most common mutation at amino acid position 700(K700E).5%?RS<15%and RS?15%patients had similar mutation spectrum,clinical features and prognosis.SF3B1 mutation was an independent favorable prognostic variable,whereas TP53 mutation was an independent adverse variable.SF3B1 mutation could coordinate with TP53 mutation for more sophisticated prognosis stratification in MDS-RS patients.BackgroundThe pathogenesis of myelofibrosis(MF)in persons with primary myelofibrosis(PMF)and persons with myelodysplastic syndrome with myelofibrosis(MDS-F)still remains unclear.Inflammation,megakaryocytes,remodeling of stromal cells and genetic mutation might involve in this process.Fiber-producing fibrocytes expanded with higher expression of pro-inflammatory cytokines in the bone marrow of PMF subjects.Researchers have documented the transformation of Gli1+ and LeptinR+mesenchymal stem cells(MSC)towards myofibroblasts in PMF mouse models.But how these fibrotic populations affect MF in PMF and MDS subjects and whether the affections are analogous have not been studied.ObjectiveTo compare origination of myofibroblasts and fibrocytes in persons with PMF and persons with MDS with MF.Methods?Bone marrow(BM)biopsy sections of PMF and MDS patients were stained with specific immunofluorescence antibodies to lable Glil,LeptinR,?-SMA,CD45 and ProcollagenI.Images captured by confocal microscopy were ananlyzed by Fiji-ImageJ to calculate cell counts of Gli1+,LeptinR+,?-SMA,?-SMA/Glil+,?-SMA/LeptinR+,?-SMA/Glil+or LeptinR+and ProcollagenI+/CD45+cells.?Genomic DNA was extracted from bone marrow cells of subjects for targeted next-generation sequencing.?Correlations between fibrosis-relevant cell counts(including ?-SMA,?-SMA/Gli1+,?-SMA/LeptinR+,?-SMA/Gli1+ or LeptinR+and ProcollagenI+/CD45+cells)and MF grade and other clinical and laboratory characteristics were analyzed.?Our 30 PMF and 30 MDS subjects were used as discovery cohorts,and two independent datasets of subjects with PMF(n=210)and MDS(n=884)from our center as validation cohorts.We explored howed genetic mutations correlated with fibrotic cell counts(including ?-SMA,?-SMA/Gli 1+,?-SMA/LeptinR+,?-SMA/Gli1+or LeptinR+and ProcollagenI+/CD45+cells)and MF grade in discovery cohorts,which was then validated in validation cohorts.The differences in fibrotic mutaitons between PMF and MDS subjects were also investigated.Results?Subjects with PMF were more likely to have splenomegaly,higher haemoglobin,WBC,granulocyte and platelet concentrations but a lower percentage of bone marrow blasts and a lower mean corpuscular volume compared with subjects with MDS.Abnormal cytogenetics was twice as common in MDS subjects compared with PMF subjects.Median survival from the time of diagnosis for PMF subjects(not reached)was longer compared with MDS subjects,20 months(range,1-26 months;P=0.016).?We sequenced 114 myeloid neoplasm-relevant genes in the 60 subjects with targeted next-generation sequencing and detected 111 variants.JAK2 and CALR mutations were more frequent in PMF subjects,and TP53 mutations more frequent in MDS subjects.?Subjects with PMF and MF-2/3 had higher Gli1+,LeptinR+and?-SMA counts than subjects with MF-0/1.Similar results were seen in MDS subjects except for Glil+MSCs.Sub-group analyses showed more?-SMA/Gli1+ ?-SMA/LeptinR+and?-SMA/Gli1+ or LeptinR+cells in MF-2/3 subjects with PMF compared to those with MF-0/1.Comparable data were seen in MDS subjects except for ?-SMA/LeptinR+cells which were similar between subjects with MF-0/1 and MF-2/3.?We found a significant increase of ProcollagenI+/CD45+fibrocytes in PMF subjects with MF-2/3 compared to subjects with MF-0/1.Data from MDS subjects were similar to those with PMF.?We compared fibrotic cells including ?-SMA,?-SMA/Gli1+,?-SMA/LeptinR+,?-SMA/Gli1+ or LeptinR+and ProcollagenI+/CD45+cells in PMF subjects to those with MDS.PMF subjects presented with similar fibrotic cell counts with MDS subjects except for the higher fibrocyte counts.In subjects with MF-0/1,fibrotic cells did not differ between PMF and MDS.But fibrocyte counts were higher in PMF subjects with MF-2/3 compared to those with MDS and MF-2/3.?Fibrotic cell counts correlated with peripheral reticulocyte percentage in PMF.In MDS,BM blasts percentage,IPSS cytogenetics group and peripheral reticulocyte percentage were associated with fibrotic cell counts.Logistic multivariate analyses displayed that DAPI/?-SMA cells were independent factor correlated with MF-grade in PMF and MDS.?In discovery cohorts,PMF subjects with?1 mutant genes in this PMF gene-set,but not subjects with MDS,had higher myo-fibroblasts counts.MDS subjects with mutations in? 1 genes of MDS gene-set,but not subjects with PMF,were more likely to have higher myo-fibroblasts counts.?We interrogated correlations between gene-set mutations and MF-grade in validation cohorts.In PMF subjects but not in MDS subjects,? 1 mutant genes in the PMF gene-set was associated with worsening myelofibrosis.Similarly,in MDS subjects but not in PMF subjects,? 1 mutant genes in the MDS gene-set was associated with worsening myelofibrosis.ConclusionDifferent mechanisms contributed to the myelofibrosis in subjects with PMF and MDS.Upon cellular level,myo-fibroblasts contributed to the development of myelofibrosis in PMF and MDS persons with expansion of different types of fibrosis-driving cells.At molecular level,correlations between mutation signature and MF-grade in PMF persons differed from those in MDS persons with myelofibrosis.