Detection of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia by 4color Cytometry in International Multicenter Trial Minimini.

FC is still not employed in MRD based treatment protocols. One problem is lack of standardization suitable for prospective trials involving multiple clinical centers and FC laboratories. Therefore, we established a MiniMini Project, an international collateral study within the ALL IC BFM 2002 treatment protocol for childhood acute lymphoblastic leukemia (ALL). The MiniMini Project provides a mainframe of minimal panel of monoclonal antibody combinations to evaluate MRD by FC. Patients (pts) are stratified according non-MRD criteria (prednisone response at day 8 in peripheral blood (PB), percentage of blasts at day 15 and day 33 in bone marrow (BM), leukocytosis, and age at diagnosis and presence of BCR/ABL or MLL/AF4 fusions). Identical immunophenotypic populations are reported in all pts regardless presenting phenotype. Each laboratory investigates at least 2 pts with B lineage ALL by the T ALL combinations and vice versa. These “cross-lineage controls” together with data on subpopulations that are negative at diagnosis were used to set the specificity cutoff values at each time point (diagnosis, day 8 BM and PB, day 15, day 33 day 52 BM). MRD levels obtained by Ig/TCR rearrangements RQ-PCR in 32 pts (24 pts BCP ALL, 8 pts T ALL) were used to define specificity thresholds. 185 pts were investigated in the participating laboratories. We used data from first Czech cohort of pts (92 pts in total, 16 pts T lineage, 74 pts B lineage, in standard risk group (SRG), n=36, IRG, n=40 and HRG, n=16) in whom clinical data as well as standard FC analysis results were available. We compared morphological percentage of blasts (used for stratification) to a level of residual disease by FC. There was high concordance in SRG of both methods, except 1 patient redirected into IRG group (M3 BM vs. only 14% of blasts by FC). In IRG, concordance was in 92.5% of pts, 3 pts should be placed in HRG group according FC. 98.9% of pts morphologically in complete remission at day 33 were confirmed by FC. Although FC data confirm a significant difference between PGR and PPR in PB specimens at day 8 (p=0.0014), there is an overlap in percentage of leukemic cells between these categories. In total, MRD level above 0.1% was observed in BM of 100, 99, 84, 32 and 3.5 % pts in days 0, 8, 15, 33 and 52, respectively and in PB of 95% pts at day 8. Our first results show feasibility of FC standardization. The choice of subpopulations and the cutoff points will be validated in an independent cohort within the same Project.