Sensitivity but not specificity of minimal residual disease by 4-color flow cytometry depends on the time point during the treatment of childhood B lineage ALL. (2006)

Several studies showed a good correlation between minimal residual disease (MRD) obtained by flow cytometry (FC) and by PCR (Ig/TCR rearrangements). However, before FC is widely applied for therapeutic decisions we need exact, objective and standardized criteria. Therefore, we pre-defined B-lineage cell subsets using 4- and 3-color FC and we established background levels for each subset in pre-defined time-points on therapy. Investigators’ subjective bias was minimized. Values above the background are compared to PCR- based MRD. Patients and methods. Children with ALL were treated by ALL IC BFM2002 protocol. This international flow cytometric trial MiniMini contained 4 laboratories (Croatia 36 patients, Israel 48, Hong Kong 33 and Czech Republic 136). 442 samples were measured simultaneously by PCR and 4color FC. Pre-defined subsets (28 values) were measured in all patients at 5 time points (d0, d8, d15, d33 and week 12). Cut off for positivity and negativity of individual subpopulations were assessed as follows: 1) negative samples by PCR for respective time point were randomly divided into training and testing cohort 2) 98.5 percentile of individual subpopulation in training cohort was selected as a cut off for positivity and negativity. 3) cut off values were tested on testing cohort and PCR positive samples (total 337 samples), sample was considered positive when having at least one value above cut off (when more values were above cut off, highest value was considered as MRD) Results. When all time points were analyzed together, sensitivity of FC MRD was 82% and specificity 88% There are no negative patients at day 8 in bone marrow by FC or by PCR. 5 samples at day 15 were positive by PCR (<=10^-4) and negative by FC, no sample was false positive by FC, sensitivity was 98%, no sample was negative by both methods. 29 samples at day 33 were positive by PCR and negative by FC, overall sensitivity was 34% and specificity 81%. At day 33 and at week 12 sensitivity dramatically dropped to 34 and 13%, specificity wasat both time points above 80%. We asked whether FC MRD can predict positivity by PCR at day 33 and at week 12. Univariate analysis showed strong correlation between MRD by FC at day 8 and/or day 15 and day 33 and/or week 12 by PCR. At day 8 in bone marrow patients with more than 50% of blasts by FC are significantly more often positive by PCR at day 33 and at week 12 (Fischer test p=0, 0013 and p=0, 04). Similar relation was observed between day 15 and day 33, week 12 using cut off 5% by FC (p=0, 0015 and p=0, 0036). Conclusion. Dynamics of MRD by FC at early time points day 8 and 15 correlates with MRD at day 33 and at week 12 by PCR. The prognostic significance of MRD at these time points must be tested on cohort with longer follow up. At day 33 and at week 12 FC is less sensitive method. Non leukemic background influences sensititivity of FC MRD at day 33 and at week 12, complex approach using 6 and more color combinations is needed for precise separation of leukemic and non leukemic B cells.