ANALYSIS OF B-LYMPHOCYTE MATURATION KINETICS WITH A 10-COLOR BCP-ALL MINIMAL RESIDUAL DISEASE DETECTION TUBE BY FLOW CYTOMETRY: A COMPARISON OF REGENERATING BONE MARROW BEFORE AND AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

Abstract

Minimal residual disease (MRD) measurement in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been consolidated in the literature as the strongest adverse prognostic factor for overall and disease-free survival in both child and adult studies. However, only recently has multiparametric flow cytometry (MFC) been fully standardized with the addition of new markers and acquisition of a higher number of cellular events, which can improve the objectivity and sensitivity of the test. This prospective study aims to test a single 10-color flow cytometric assay compared to standardized 8-color two tubes in patients with BCP-ALL before bone marrow transplantation, follow by analysis of the kinetic of medullary B-cell recovery after transplant. Prospective analysis of 75 consecutive patients (pcts) transplanted between 2019/January and 2022/May, with MRD evaluation in the month before HSCT, day30, day100 and day360. Flow cytometry was performed on bone marrow samples after bulk-lysis with ammonium chloride when necessary, and subsequent staining with antibody panels. A 10-color tube included the backbone markers CD19, CD45, CD34, CD10, and CD20, in addition to CD38 and CD81 to improve separation between normal/reactive and malignant BCP, and four discriminatory markers placed in the same tube (CD66c and CD123 in PE, CD73 in BV605, and CD304 in APC-R700). This tube was compared to the standardized EuroFlow®method, which was composed of two 8-color tubes previously described. FACSCantoII™and FACSLyric™flow cytometry's were used. Data were analyzed with Infinicyt. The achieved sensitivity and percentages of lymphoblasts, normal mature and precursor B-cells, plasmatic cells and stromal cells were compared before and after transplantation. Statistical analyses were performed in SPSS Statistics v.20.0 software. 34 ALL (42.6%), being 26 BCP-ALL and 8 high-risk T-ALL, and 41 AML (57.4%), median age 31.6 (4.1-62.5 years) for ALL and 42.9 (0.6-74.2 years) for AML; 45(60%) in first complete remission (CR1), 21 CR2 (28%), one CR3 and 7 active diseases. Among 64 patients in morphologic remission, we found 38(59.4%) MRD negative and 26(40.6%) MRD positive before transplant. Pos-transplantation MRD and maturation analysis was possible in 26 pcts at d30, 24 at d60, 57 at d100 and 18 at d360. All but two samples had more than 9.000.000 cell events acquired, median lower limit of detection (LoD) was 0,0002% and lower limit of quantification (LoQ) was 0,0005% in both assays. All patients had the same percentages of lymphoblasts, normal mature and precursor B-cells, plasmatic cells, in the two different assays. The posttransplant B-cell recovery kinetic was similar between BCP-ALL, T-cell ALL and myeloid leukemia recovery after transplantation, and different between adults and children at day100 and day360, but not in day30. This study showed that this 10-color single tube strategy achieves the same sensitivity as the 8-color assay and allows correct identification of MRD, achieving maximum sensitivities and specificities in a single tube, and still maintaining the best discrimination between pathological B cells and normal B cell precursors. B-cell maturation kinetics were similar between leukemia types, and slightly different between adults and children. Additional studies and a larger sample size are needed to confirm these findings.