The Relationship Between Neutrophils And Immunosuppressive Therapy For Aplastic Anemia

Objective:To testify whether absolute neutrophil count(ANC)response to pre-immunosuppressive-therapy(pre-IST)granulocyte-stimulating factor(G-CSF)treatment can predict early response to IST in severe aplastic anemia(SAA).Methods:Clinical data and hematologic response of 125 SAA patients treated with front-line antithymocyte globulin(r-ATG)combined with cyclosporine were retrospectively analyzed.ANC response to pre-IST G-CSF treatment was defined as max?ANC?0.5×109/L while receiving G-CSF treatment.Patients were categorized into ANC responders and ANC non-responders according to the above standard.Correlation of ANC response to pre-IST G-CSF treatment and early response to IST were statistically analyzed.To optimize,ANC response at day 5,day7,day 10,day 13,day 14,day 16 and day21 were analyzed,repectively,seeking to find correlation of ANC response at corresponding time point with early response to IST.Receiver operating characteristic(ROC)curve was used to estimate the value of increased ANC(AANC)in predicting early IST response.Results:The hematologic response(HR)rate to IST in ANC reponded patients was significantly higher than non-respond group(3 month HR 49%vs 28.9%,P=0.023;6 month HR 61.2%vs 40.8%,P=0.026).With ?ANC?0.5×109/L as cutoff level,the best time to predict early IST response was 10 days after G-CSF(d 10).Response of ANC to pre-IST G-CSF treatment at d 10 is among the independent factors of predicting response to IST after 3-month(P=0.004),but not for 6-month.The overall 5-year survival rate was 92.8%and 69.5%in ANC responded and non-response groups,respectively(P=0.025).Conclusions:Responding to pre-IST G-CSF treatment reflects the residual bone marrow hematopoiesis,and can act as a convenient and practical predictor to early IST response as well as long-term survival in severe aplastic anemia.Objective:To analyze early hematopoietic response and long-term survival of very severe aplastic anemia(VSAA)patients with different absolute neutrophil counts(ANC)after frontline immnunosuppressive therapy(IST),so as to provide evidence for optimizing the frontline regimen for patients with VSAA.Methods:Clinical data and outcome of 145 VSAA patients treated with rabbit antithymocyte globulin combined with cyclosporine were retrospectively analyzed.Hematopoietic responses to IST and long-term survival were statistically analyzed for VSAA patients in different ANC subgroups.Results:Both early hematopoietic response rate and long-term survival rate of VSAA patients were significantly lower than that of SAA patients.Pre-IST ANC>0.5×109/L acted as the best cutoff level to predict IST response at 6 months as well as good response at 12 months.For VSAA patients with extremely low ANC(?0.05×109/L),early death rate was 12/130(9.2%),accounting for 91.7%(11/12)of all the early death of VSAA patients;hematopoietic response rates to IST was only 18/82(22.0%)at three months and 28/82(34.1%)at six months,respectively;the overall five-year survival rate was only(62.5±5.4)%and five-year event-free survival rate was(42.3±5.5)%,respectively.Conclusions:VSAA patients with extremely low ANC(<0.05×109/L)had high early death rate and with very low response rate to frontline IST and poor survival.Objective:ASXL1 mutations are frequent in the entire myeloid malignances and among the most prevalent in patients with aplastic anemia,grouped with "unfavorable mutations" that confer leukemic transformation and adverse survival.Previous studies have reported that ASXL1 acts as a regulator of cell survival and myeloid differentiation by epigenetic modification.However,molecular mechanisms of malignant transformation caused by mutated ASXL1 are not well-defined.For this purpose,our study is to explore molecular mechanisms of ASXL1-mutations in human leukemic cells,as well as role of ASXL1 and related mechanisms in malignant transformation.Methods:The CRISPR/Cas9 gene editing system was employed to obtain ASXL1-knockout clones from the U937 human leukemic cell line.After transfection,single cells were sorted by flow cytometry,single cell clones were generated.ASXL1 mutations were assessed by Sanger sequencing.Several clones with c.594insA,p.Ser199Glufsx55(three heterozygous and three homozygous)mutations,as well as ASXL1-wild type parental bulk U937 cells,and two transfected wild type single cell clones were used for further experiments.Characteristic features of both mutated and wild type clones were examined by:cell morphology by Wright-Giemsa staining;karyotype analysis by G-banding;and cell cycle,apoptosis,and cell differentiation by flow cytometry.5-fluoruracil induced cell apoptosis and inhibition of cell growth were observed.PMA was used to induce monocytic/phagocyte differentiation of U937 cells.RNA sequencing(RNA-Seq)was performed to screen differentially expressed genes between wild-type and ASXL1-mutated clones,followed by validation of gene expression using reverse transcription quantitative PCR(RT-qPCR).Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were performed to analyze pathways or gene sets disturbed by ASXL1 mutation.Results:We obtained six ASXL1-MT+/-and eleven ASXL1-MT-/-U937 single cell clones with the same c.594insA(Ser199Glufsx55)mutation which translated a short truncated protein followed by 55 additional amino acids,due to a premature termination codon(Figure la),and 56 WT clones(transfected but not ASXL1-mutated).Three ASXL1-MT+/-(MT1,MT2 and MT3)clones,three ASXL1-MY-/-(MT11,MT12 and MT13)clones,two wild-type clones(WT1 and WT2 derived from transfected ones)andWTblk cells were used for further experiments.We assessed cell proliferation and cell cycle,ASLX1-mutated U937 cell lines showed comparable growth curves and cell cycle with wild type.There were fairly variable in 5-FU-induced growth inhibition and apoptosis rates among individual ASLX1-MT cells or compared to ASLX1-WT cells,even though their average rates among the three groups were not significantly different.After treated with PMA at different concentrations for 96 hours,U937 cells adhered to bottom of culture chambers,differentiated to macrophages,with larger sizes and plenty of cytoplasm.Cells were harvested fand subjected to flow cytometry analysis,both CDllb-positive cell percentages and mean fluorescence intensity(MFI)of CD11b were lower in MT1 and MT12 cells,relative to both WTblk and WT1 cells,at most concentrations used,which means that ASLX1-MT U937 cells were less responsive to PMA-induced differentiation.RNA sequencing revealed 15 downregulated and 2 upregulated genes were found in the MT+/-group while 5 downregulated and 22 upregulated genes in the MT-/-group,in which again only CYBB was differentially expressed(downregulated)in both the MT+/-and MT-/-groups relative to the WT group.GSEA showed that gene sets related to cell development and survival as well as cell-to-cell signaling and interaction were massively dysregulated in both MT+/-and MT-/-groups.GSEA showed a tendency of downregulation of myeloid cell differentiation gene sets,among genes involved in myeloid differentiation,two genes(CYBB and CLEC5A indicated in Figure 5b)reported to be highly related to U937 cell differentiation were downregulated.qRT-PCR confirmed significantly reduced expression levels of genes essential to myeloid differentiation,such as CYBB and CLEC5A,and genes involved in cell death and survival,including NAIP,CACNA2D3,ACTL8,CTSG,OXR1 and CSPG4,in MT cells compared with WT cells.When compared with WTblk,chromosome alterations were observed in MT cells,such as chromosome 11q deletion in three out of four ASXL1-MT cell lines,additional chromosome 15p in MT1 and MT12,and additional chromosome 17 in MT11,indicating that ASXL1 mutations appeared to lead to chromosomal instability,especially of chromosome 11.Conclusions:ASXL1 mutations perturbed monocytic/phagocyte differentiation,which is a hallmark of myeloid malignancies,by downregulating genes essential to myeloid differentiation,including CYBB and CLEC5A,also massively affected multiple gene sets involving in cell survival.Additionally,ASXL1 mutations induce genomic instability.