| Objectives1.Explore the clinical features,efficacy of immunosuppressive therapy and prognosis in patients with acquired pure red cell aplasia(aPRCA).2.Explore the efficacy,safety and prognosis of sirolimus for refractory/relapsed/cyclosporin A-intolerant aPRCA patients.3.Explore the regulatory mechanism of sirolimus in erythroid hematopoiesis and erythroid differentiation.MethodsWe retrospectively analyzed the clinical characteristics,treatment,prognosis and outcome of newly diagnosed aPRCA patients collected in Peking Union Medical College Hospital from August 2015 to August 2020,to evaluate the effectiveness and safety of different initial induction therapy by immunosuppressive treatments for aPRCA,and analyze the factors that may affect the efficacy.Meanwhile,we collected aPRCA patients,who were refractory,relapsed or intolerant to cyclosporine A(CsA)/other immunosuppressants,diagnosed in Peking Union Medical College Hospital from April 2017 to August 2020 according to the inclusion/exclusion criteria.We treated them prospectively with sirolimus at a dose of 1-3mg/d,and adjusted the dose according to the plasma concentration by laboratory test every 3 months,to evaluate the efficacy,safety and prognosis of sirolimus.The human leukemia cell line K562 was used as the research object.After induction by different concentration of sirolimus,we detected the hemoglobin level,globin mRNA level and cell proliferation in K562 by benzidine staining,qRT-PCR and CCK-8,respectively.We used transcriptome sequencing to detect different-expression genes in K562 cells induced by sirolimus,and carried out and verified the possible mechanism gene by GO-analyses and qRT-PCR,respectively.After knocking down the mechanism molecule by siRNA in K562 cells,we detected changes of the benzidine staining positive cells,globin mRNA level and mechanism molecule protein level again in K562 cells induced by sirolimus.Results1.Among 121 cases newly diagnosed aPRCA retrospectively collected in our center,males accounted for 52.9%(64),and the median age was 59y(18-89y).67.8%(82)cases were primary aPRCA,and 32.2%(39)cases had secondary factors.Among secondary aPRCA,large granular lymphocytic leukemia(LGLL,58.5%,15 cases)and thymoma(20.5%,8 cases)were the main secondary factors.At the baseline,the hemoglobin level was 65.9 ± 15.3 g/L,the absolute count of reticulocytes was 1.12±1.12%,and the positive rate of STAT3 mutation was 14.8%(9/61).As for initial therapy,84(69.4%)were single CsA,12(9.9%)were CsA combined with corticosteroid(CS),and the other(25 cases,20.7%)were sirolimus due to the renal failure or intolerance for CsA(durable time<1 month).During the median follow-up period of 14(6-68)months,the overall response(OR)rate of 121 patients was 81.0%and the complete response(CR)rate was 68.6%,with the median onset time 3(1-12)months.At the 3rd,6th,12th month and the end of follow-up,the OR rates were 55.4%,76.9%,78.6%and 46.3%,and the CR rates were 46.3%,65.3%,71.4%and 38.0%,respectively.During the median follow-up of 24(6-68)months,42.9%(42/98)of patients with OR relapsed.51 patients had adverse reactions mainly related to creatinine increasing(58.8%)and infection(15.7%).One patient died due to severe pneumonia during treatment,and other patients did not die or undergo clonal evolution.Multivariate analysis showed that the initial agents were independent risk factor to OR rate of the initial treatment(p=0.024),but not the CR rate.Other factors such as age,gender,secondary factors,initial hemoglobin level and STAT3 mutation were not correlated with the initial treatment efficacy.During the median follow-up period of 16(6-68)and 11.5(6-68)months,the median onset time of initial treatment was 3(1-12)and 3(1-7)months in CsA group and CsA+CS group respectively(p=0.332).At the time of 6th and 12th month,the OR rate of CsA group was significantly higher than that of CsA+CS group(81.0%vs.33.3%,p=0.001;85.7%vs.22.2%,p<0.001),and the CR rate was also significantly higher than that in CsA+CS group(71.4%vs.33.3%,p=0.022;79.4%vs.22.2%,p<0.001).The recurrence rate at 12th and 24th month was significantly lower in CsA group than those in CsA+CS group(12m:15.3%vs.83.3%,p<0.001;24m:46.0%vs.100.0%,p=0.037).The median relapse-free survival(RFS)of CsA group was better than that of CsA+CS group(32 vs.7.5m,p=0.003).Except for age mixing,the median onset time of CsA group and sirolimus group was 3(1-12)and 3(1-7)months(p=0.332)during the median 16(6-68)and 9(6-33)months of follow-up period,respectively.There was no significant difference in OR rate and CR rate at the 3rd,6th and 12th months,and recurrence rate at the 12th and 24th month between such two groups,respectively.The incidence of adverse reactions in CsA group was 50.0%(42/84).including elevated creatinine levels(27 cases)and infection(4 cases).The incidences of adverse reactions in sirolimus group and CsA+CS were 24.0%(6/25)and 25.0%(3/12),respectively.CsA group had no significant difference in the incidences of adverse reactions and renal dysfunction compared with CsA+CS group(50.0%vs.25.0%,p=0.130;32.1%vs.16.7%,p=0.337),but had significant difference than those in sirolimus group(50.0%vs.24.0%.p=0.022;32.1%vs.4.0%,p=0.036).2.64 aPRCA patients who were refractory,relapsed or intolerant to first-line therapy were included in sirolimus clinical trial.These patients included 56.3%(36)males with 64(18-85)years at baseline.62.5%(40 cases)were primary pathogenic,and LGLL(6 cases),thymoma(5 cases)and autoimmune disease(5 cases)were the main secondary factors.The median dose of sirolimus is 1(0.5-3)mg/d,and the median response time is 3(1-6)months.During the median follow-up time 12(6-47)months,the OR rates at the 3rd,6th,12th months and end point of the follow-up period were 60.9%,84.4%,73.5%,and 71.9%,respectively;and the CR rates were 50.0%,65.6%,66.0%,and 67.2%,respectively.At the end point,the patient’s hemoglobin level was higher than baseline(p<0.001),creatinine level was lower than pre-treatment(p=0.041),and ferritin level was lower than baseline(p=0.013).Gender,age,secondary factors,initial induction therapy efficacy,hemoglobin level before treatment,and STAT3 mutations were all not related to the efficacy of sirolimus.29.7%of patients had adverse reactions,and the most common adverse reaction was infection(5/19).During a follow-up of 16(6-47)months,18.6%(8/43)of the patients relapsed,and no patient died or had clonal evolution.3.After K562 cells were treated with different concentrations of sirolimus for 24 h,48 h and 72 h,the proportion of benzidine staining positive cells and the expression of CD71 and α/β/γ globin mRNA in K562 cells were up-regulated,but the cell proliferation was inhibited.The above effects were not related to the concentration of sirolimus.Transcriptome sequencing analysis showed that under the induction of sirolimus,K562 cells differentially expressed erythroid regulatory genes,which were mostly related to biological processes or functional components such as hemoglobin complex,oxygen carrier activity,iron molecule binding and oxygen molecule transport.Sirolimus could induce the expression of ALAS2 mRNA in K562 cells.Knock-down ALAS2 expression in K562 cells could significantly inhibit the proportion of benzidine positive cells and the expression of α,β,γ globin mRNA in K562 cells induced by sirolimus.Conclusions1.The initial induction therapy of aPRCA has a high remission rate,but the combination of corticosteroid can reduce the efficacy of CsA and increase the risk of long-term recurrence.Renal dysfunction and infection are the main adverse reactions during treatment.2.Sirolimus has high effective rate and mild adverse reactions in refractory/relapsed aPRCA or those intolerant to first-line therapy,accompanied with low recurrence rate in long-term follow up.3.Sirolimus can promote globin expression and erythroid differentiation in K562 cells,and this regulatory function in erythroid hematopoiesis may be related to the function of ALAS2 gene. |
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