1.ASXL1 Down-regulation May Contribute To The Abnormal Migration Of SET2 And UKE1 Cells 2.Validation Of The WHO 2016 Proposals For Myelodysplastic Syndromes Patients With The Presence Of Ring Sideroblasts But Without Excess Blasts 3.Prognostic Evaluation

Background and ObjectiveASXL1 plays a vital role in regulation of the histone methylation,previous studies indicate that ASXL1 acts as a tumor suppressor.Majority of ASXL1 mutations are frame-shift and nonsense mutations.The most frequent mutation is C.1934dupG(P.Gly646TrpfsX12)with a loss of PHD domain and truncated protein formed.Reduced normal ASXL1 protein expression may promote myeloid transformation.ASXL1 mutations change the transcription of certain oncogenes and result in dysfunction of tumor stem cells.Previous studies indicate that ASXL1 mutated patients were more likely to present splenomegaly which suggests potential links during the disease course.Our previous data showed that ASXL1 mutations were identified in 28.2%(31/110)PMF patients,JAK2V617F mutated patients with ASXL1 mutations were more likely to present splenomegaly,while in JAK2V617F negative patients,the connections were gone.There is evidence that SDF-la/CXCR4 axis plays an important role in the adhesion of leukemic cells and bone marrow stromal cells.Abnormal CXCR4 expression may contributed to constitutive migration of CD34+ cells in PMF.Accordingly,we supposed that on the basis of JAK2V617F mutation,mutated ASXL1 acts on the SDF-la/CXCR4 axis leading to the stem cell mobilization and certain symptoms.The present study is aimed to investigate ASXL1 mutation in PMF patients and its clinical significance;aimed to explore the biological roles and molecular mechanism of ASXL1 mutation in the JAK2V617F mutated UKE1 and SET2 cell lines and JAK2V617F negative HL-60 cell line.Methods1.A retrospective study of 182 PMF cases was performed.The clinical and laboratory features were analyzed between ASXL1 mutated patients and wild type patients.2.ShRNA expressing plasmids specifically targeting ASXL1 were constructed.These plasmids were transfected into UKE1,SET2 and HL-60 cells and stable clones were selected by puromycin.The efficiency of ASXL1 knockdown and CXCR4 level were detected by Real time quantitative PCR and Western Blot.3.CCK8 assay was used to detect the cell proliferation ability.Cell colony formation was performed to detect the monoclonal formation ability.4.Transwell assay was performed to detect the migration ability.Flow cytometry was used to detect the membrane CXCR4 expression.5.Using mRNA expression Microarray in SET2 cells to compare the differences of certain gene expression between ASXL1 knockdown cells and ASXL1 wild type cells.Results1.ASXL1 mutation status was related to patients’ spleen size(P=0.005),JAK2V617F mutated patients with ASXL1 mutations were more likely to present splenomegaly,while in JAK2V617F negative patients,the connections were gone.2.Interference plasmids were structured.The expression of ASXL1 in UKE1,SET2 and HL-60 cells were remarkably reduced.In UKE1 and SET2 cells,ASXL1 knockdown accompanied with the elevation of CXCR4 mRNA and protein levels;while in HL-60 cell,ASXL1 knockdown accompanied with the decreased expression of CXCR4.3.CCK8 Assay indicated that there were no difference in cell proliferation whether ASXL1 protein down regulated or not in all 3 cell lines.While in colony formation assay,suppressed colony formation efficiencies were shown in ASXL1 knockdown cells in all 3 cell lines.4.In UKE1 and SET2 cells,ASXL1 knockdown cells showed obvious promotion in migration,while CXCR4 antagonist AMD3100 could disrupt this effect;In HL-60 cells,down regulated ASXL1 expression accompanied with declined migration ability.Flow cytometry showed increased membrane CXCR4 expression in UKE1 and SET2 cells.5.mRNA expression Microarrary results showed in ASXL1 knockdown SET2 cells,the function of cell adhesion ability,cell-matrix adhesion and leukocyte migration ability were up regulated.ConclusionIn JAK2V617F mutated ASXL1 knockdown UKE1 and SET2 cells,CXCR4 expressions were up regulated and cell migration abilities were promoted,which may indicated that ASXL1 mutation may act on SDF-1/CXCR4 axis.We speculated that with the JAK2V617F mutated background,ASXL1 knockdown may promote the migration ability of tumor stem cells and formation of tumor microenvironment through acting on SDF-1/CXCR4 axis and result in certain symptoms such as splenomegaly and extramedullary hematopoiesis in MPN patients.This study elucidated potential molecular mechanisms involved in MPN disease course.Background and ObjectiveRecently,a revision of the 4th edition of the WHO 2008 Classification of myelodysplastic syndromes(MDS)was proposed on the 57th American Society of Hematology Meeting.One of its major changes is the division of MDS with ring sideroblasts(RS):patients with at least 5%RS but not meeting the 15%threshold used to define refractory anemia with ring sideroblasts(RARS)plus an SF3B1 mutation will be named as ’MDS with single lineage dysplasia and ring sideroblasts(MDS-RSSLD)’ or ’MDS with multilineage dysplasia and ring sideroblasts(MDS-RSMLD)’ depending on the isolated erythroid or multi-lineage dysplasia.The aim of this research was to assess the validity of the WHO 2016 classification for MDS patients with the presence of RS and without excess blasts(<5%).MethodA total of 230 consecutive MDS patients with the presence of at least 1%RS and without excess blasts(<5%)were collected between January 2003 and December 2014.We amplified the genomic DNA corresponding to the mutation hotspots of SF3B1(exonl2-16)by PCR using the primers.Identification of the mutation status was performed by direct sequencing.A retrospective study of the patients was reviewed according to the WHO 2016 classification.Statistic analysis of clinical and laboratory characteristic and prognosis were performed accordingly.ResultMedian age was 51(16-84)years old of the 230 patients,with 154 males(67.0%).SF3B1 mutations were identified in 44.8%patients(103/230),all of which were heterozygous missense mutations with K700E the most frequent one(69.9%).The SF3B1 mutation frequencies were 2 of 12(16.7%)for patients with<5%RS,16 of 74(21.6%)for patients with 5-15%RS and 85 of 144(59.0%)for patients with ≥15%RS with a positive correlation between SF3B1 mutation frequencies and RS ratios.Follow-up data was available for 216 subjects,with a median follow-up of 35 months(range 2-126 months).In both uni-variate and multi-variate analyses,SF3B1 mutation was independent protective factor for MDS patients.In patients with 5～15%RS,a better overall survival was seen in subjects with SF3B1 mutations compared to those without(median survival not reached vs 46months,P=0.033),although no significant difference in clinical and hematological features was found.More importantly,patients with 5～15%RS with SF3B1 mutations seem to share no significant difference in survival with subjects>15%RS irrespective of the SF3B1 mutation status(median survival not reached vs 66 months,P=0.230).Furthermore,subjects with or without SF3B1 mutations in patients with>15%RS appear to have no significant difference in survival(median survival 71months vs 53months,P=0.067).Patients were then re-classified according to the WHO 2016 proposals,survival analyses showed that subjects with MDS-RSSLD or MDS-RSMLD had significantly better survival than those with MDS-MLD(median survival 68months vs 48months,P=0.028).Conclusion Integrating the SF3B1 mutation status with the proportion of RS,as proposed for the WHO 2016 classification,indeed allows more accurate diagnosis of MDS.Background and ObjectiveMyelodysplastic syndromes(MDS)comprise a heterogeneous group of acquired clonal malignancies.More than 50%of de novo MDS patients have more than one kind of comorbidities.In recent years,we have seen the increased emphasis on prognostic value of comorbidities.The aim of this study was to discuss the impacts of comorbidities onthe outcome of MDS patients.MethodThe clinical information of 676 MDS patients with detailed comorbidities evaluations was analyzed retrospectively.ResultThere were 395/676 cases(58.4%)with comorbidities,281/676 cases(41.6%)without.Significant differences were seen in the distribution of age(≥ 60y),bone marrow blasts,abnormal karyotype,WHO 2008 subtypes and IPSS-R risk cohorts(P<0.05)between the two groups.While gender,hemoglobin concentrations,white blood cell levels,platelet levels and serum ferritin were not significantly different(P>0.05).Independent prognostic significance of comorbidities was seen in both uni-variate and multi-variate analyses(P<0.001).According to MDS-specific comorbidity index(MDS-CI),the median survival time were 32 months,19 months and 13 months in the low-risk,intermediate-risk and high-risk cohorts respectively,while 96 months in cohorts without any comorbisities,of which significant differences were seen(P<0.001).The MDS-CI allows further stratification in the IPSS-R low-risk,intermediate-risk and high-risk cohorts(P<0.001).ConclusionComorbidities provide prognostic stratification independently of IPS S-R for MDS patients...