The BM samples were obtained with informed consent. The study was approved by the Ethics Committee of the Peking University International Hospital and was in compliance with the guidelines of the Helsinki Declaration of 2008. A total of 220 longitudinal BM specimens from 98 adult AML patients (55 males and 43 females) were collected after chemotherapy from Peking University International Hospital between June 2017 and June 2019. Among the samples, 165 BM samples from 43 patients were collected at different time points after chemotherapy.
Fluorescent mouse anti-human monoclonal antibodies (mAbs) against the following markers were used: CD2, CD5, CD7, CD11b, CD13, CD14, CD15, CD19, CD25, CD33, CD34, CD36, CD38, CD41, CD45, CD56, CD61, CD64, CD71, CD117, CD123, CD235a, and HLA-DR (from BD Biosciences and Beckman Coulter). The MRD detection panels are listed in Additional file 1.
BM samples were collected in heparin anticoagulant tubes. A total of 5×105 white blood cells/tube were stained and detected within 12 hours after procurement. Data were acquired and analyzed using a BD FACSCalibur flow cytometer and Cell Quest software (from BD Biosciences).
MRD as determined by FC was defined as reported . CD25 expression was calculated as the MFIR as described previously . In MRD-negative BM samples, normal myeloblasts are positive for CD34, CD117, and CD45. Based on the expression profile of CD25, the MRD-positive BM samples were separated into two subgroups. If there was a CD25-positive subpopulation, the MFIR of CD25 applied only to this subpopulation instead of to the whole MRD cluster. In addition, the positive percentage of cells positive for CD25 in the MRD cluster was recorded. If the whole MRD cluster was positive for CD25, then the MFIR of CD25 applied to the whole cellular cluster.
All BM samples were analyzed by PCR. Five-milliliter BM samples were collected in in EDTA anticoagulant tubes. The PCR assay was performed based on previous reports [4,10,14] Fusion genes (PML-RARA, CBFB-MYH11, RUNX1-RUNX1T1, BCR-ABL1, MLLT3-KMT2A, and DEK-NUP214) and mutant genes (NPM1, CEBPA, RUNX1, FLT3, TP53, KIT, and ASXL1) were detected before treatment [8,21]. Samples from patients with mutant NPM1, PML-RARA, CBFB-MYH11, and RUNX1-RUNX1T1 mutations were subjected to PCR for MRD detection. MRD was defined based on previous reports [1,14]
The 2-tailed Student’s t-test was used to evaluate the difference in CD25 expression between the two different subgroups. A Receiver Operating Characteristic (ROC) Curve was used to set the CD25 MFIR cut-off value. All the statistical analyses were performed by using the GraphPad Prism 6 (GraphPad Software, San Diego, CA). The MFIR values are presented as the means ± standard errors of the mean (SEM).