MRD-Detection by Multiparametric Flow Cytometry in Childhood Precursor B-Cell All:Predictive Impact of Early Blast Reduction Kinetics in Peripheral Blood (PB) on MRD-Level in Bone Marrow (BM) at Day 33 of the All-BFM2000 Potocol.

Detection of minimal residual disease in childhood acute lymphoblastic leukemia (ALL) has been proven to be of crucial importance for relapse prediction. However, MRD assessment, usually performed at postinduction time points, does not allow early stratification of induction therapy. Here, we address the predictive impact of early blast reduction in pB on BM MRD-levels at the end of induction therapy phase I (d33), quantified by multiparametric flow cytometry (MFC-MRD). Absolute blast cell counts (BCC, blasts/μl) in pB at diagnosis and day 8 as well as MRD-levels in BM and pB at day 15 and 33 were determined in a series of 77 consecutive patients (pts.) enrolled in the AIEOP-BFM-FCM-MRD pilot study (BM d33:MRD+ n=29, MRD- n=48). Blast cells were detected using a two platform method and a 4-color antibody panel with three follow-up tubes:1) CD20-FITC/CD10-PE/CD34-PC5/CD19-PC7, 2) CD58-FITC/CD10-PE/ CD34-PC5/CD19-PC7, 3) CD10-FITC/CD11a-PE/CD45-PC5/CD19-PC7. Additionally, staining with the cell permeant live-cell nucleic acid fluorochrome SYTO 16 combined with CD19 or CD3 and CD45 were used to exclude residual non-nucleated erythroid cells, platelets or debris from absolute blast calculations. We found that BCC in BM and pB at d15 but not at diagnosis and after prednisone prephase at day 8 correlated with MRD-levels in BM at d33 (Spearman correlation coefficient rs=0.41, p<0.001 and rs=0,30, p<0,01). In order to further investigate the impact of early blast reduction between day 0 and day 8 we calculated the relative blast reduction rate on day 8 (BRRd8 = 1-BCCd8/BCCd0). In the group of MRD-negative patients the mean values of BRRd8 were found to be significantly higher than in the group of MRD-positive pts. (98,9% vs. 88,0%, p< 0,05). Moreover, the BRRd8 correlated with MRD-levels in BM at day 33 (Spearman correlation coefficient rs=−0.25, p<0.05). In the current series of pts. maximally selected log-rank statistics were performed for the BRRd8 and the MRD-level in BM at d33 and provided a cut-off value of 97% separating MRD-positive and MRD-negative pts. In conclusion, the assessment of the dynamics of early blast reduction with the parameter of BRRd8 is highly predictive for the outcome of induction phase I of the ALL-BFM2000 protocol.