| Objective:To ananlysis the multiparameter flow cytometry(MFC)and WT1 expression in the first complete remission(CR1)defined by morphological in newly diagnosed atients with acute myeloid leukemia(AML),ananlysing the value of minimal residual disease(MRD)quantified by MFC and WT1 in relapse-free survival(RFS)and overall survival(OS),to guide the clinical follow-up treatment.Methods:To analyze the clinical data of patients with AML(except M3)who were newly diagnosed from October 2010 to October 2016 in the Department of Hematology,Second Hospital affiliated Shanxi Medical University.All patients were obtained CR / CRi / CRp,The leukemia associated immunophenotype(LAIP)by MFC and WT1 in predicting RFS and OS,guiding individualized treatment.1.Bone marrow(BM)come from patients with AML.Diagnostic and classification criteria based on the World Health Organization(2016).MFC-LAIP and WT1 expression were detected by BM of patients after diagnosis and CR1.This experiment was performed at the level of bone marrow cells.Bone marrow fluid come from the Department of Hematology,Second Hospital affiliated Shanxi Medical University due to the need for treatment(access to patients with informed consent).2.2-4 ml bone marrow from patients after diagnosis and complete remission,immunophenotype and blast cell count was detected by FC500(Beckman)flow cytometry.CXP software was used for data analysis.Every combination of compensation was setted with unlabeled isotype control.Monoclonal antibodies include CD45-PC7,CD38-FITC,CLL-1-PE,CD34-PC5,CD7-FITC,cMPO-PE,CD19-PC5,CD34-FITC,CD11b-PE,CD15-PC5,CD56-PE CD117-PC5.At the same time,real-time quantitative PCR(RT-qPCR)was used to quantify WT1.3.Forward scatter/side scatter(FSC/SSC)door was used to remove dead cells and debris in the MFC,at least 100,000 cells of every tube.Patients with bone marrow cell staining positive was antigen expression more than 20% of the CD45 / SSC cell population,the minimum effective cells was 40.The antibody expression intensity was determined by the mean fluorescence intensity(MFI)of the positive cell population,and the minimum effective cell count was 500.If there is still cells present in the region where the leukemia cells appear,and the phenotypic expression is abnormal,the residual leukemia cells are judged as the MRD.MRD quantitative ? 10-4 is a low level,? 10-4 is the high level.4.WT1 expression use the European leukemia network recognized ratio method,RT-qPCR determine the WT1 and ABL gene,ABL gene as the internal reference.WT1 / ABL × 10000> 60 is highly expressed,<60 is normal.Results:1.Among the 179 newly diagnosed AML patients,there were 88 males,the median age was 47 years(15-73 years).According to WHO(2016)subtype: AML with t(8;21)29 cases,with inv16 / t(16;16)9 cases,with inv(3)/ t(3;3)1 case,M0 5 cases,M1 12 cases,M2 15 cases,M4 53 cases,M4 EO 4 cases,M5 25 Cases,M6 4 cases,with myelodysplastic abnormalities associated with changes was 11 cases,treatment related to AML was 11 cases.low risk was 37 cases,intermediate risk was 117 cases,high risk was 25 cases.The median follow-up was 23 months(6-76months).A total of 100 patients were relapse,and relapse occurred within 2 years.62 cases were died,of which 55 died of relapse.Median RFS is 21 months(4-75months),median OS is 28 months(6-76months).2.Compared with 107 cases with MFC-LAIP negative,72 cases with positive has higher white blood cell count,a higher proportion of bone marrow,MFC and peripheral blast cells,and lower platelet count,mostly for the central nervous system leukemia(CNSL),secondary,high-risk patients,and the CR rate is lower,but there was no significant difference between the two groups(P>0.05).The RFS and OS of positive patients were significantly shorter(P<0.001).3.Based on the traditional immunological labeling,73 patients were analyzed for the role of C-type lectin-like receptor-1(CLL-1)in MRD.The expression of CLL-1 in leukemia cells,granulocytes and monocytes was 91.8% with AML,71.2% in CD34 + CD38-cells,CD34 + AML and CD34-AML patients had no difference(P=0.43),the expression was stable before and after CR,but not on CD34 + CD38-cells in contorls.4.Further analysis of the role of levels of MFC-LAIP in the prognosis of AML patients.The 5-year RFS rate was significantly lower in the MFC-LAIP?1.35% group compared with the <1.35% group(P<0.001),and the MFC-LAIP?1.70% group compared with the <1.70%,The OS was significantly lower(P<0.001).To analysis the different risk stratification,and the levels of MFC-LAIP were found to be independent of RFS in intermediate risk and high risk patients,and OS in intermediate risk patients,but not the independent factors of RFS and OS in low-risk patients.5.Among the 73 patients with AML,78.1% had higher expression of WT1,and 16 cases express normal.Among the latter,9 cases express higher after CR1,7 cases were early relapse,and mostly were high-risk,elderly patients.The level of WT1 expression was not associated with white blood cell count,hemoglobin concentration,platelet count,LDH,?2-MG,and risk stratification,but the early recurrence rate of WT1 overexpression was higher.WT1 expression level ?40 after CR predicts short-term RFS(P<0.001),the level of WT1 ?50 after CR showed shorter OS(P = 0.001).6.MFC combined the level of WT1 after CR can improve the sensitivity of AML patients to predict relapse,without affecting the specificity.Conclusions:1.MFC-LAIP after CR1 was independent factors of RFS and OS with AML,may play an important role in post-remission therapy or even the leading role,but in different risk stratification with different weights.CLL-1 can be used as a marker for AML diagnosis,MRD detection and targeted therapy.2.WT1 levels was ? 40 and 50 after CR1 indicates shorter RFS and OS;WT1 expression higher after CR1,mostly were high-risk,elderly patients,with higher relapse rate.3.The MFC and WT1 level can be used as a good method for detection of AML MRD,the combination can improve the sensitivity,does not affect the specificity. |
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