Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia

Abstract

Background: Flow cytometric analysis of leukemia-associated immunophenotypes and polymerase chain reaction-based amplification of antigen-receptor genes rearrangements are reliable methods for monitoring minimal residual disease. The aim of this study was to compare the performances of these two methodologies in the detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Design and methods: Polymerase chain reaction and flow cytometry were simultaneously applied for prospective minimal residual disease measurements at days 15, 33 and 78 of induction therapy on 3565 samples from 1547 children with acute lymphoblastic leukemia enrolled into the AIEOP-BFM ALL 2000 trial.

Results: The overall concordance was 80%, but different results were observed according to the time point. Most discordances were found at day 33 (concordance rate 70%) in samples that had significantly lower minimal residual disease. However, the discordance was not due to different starting materials (total versus mononucleated cells), but rather to cell input number. At day 33, cases with minimal residual disease below or above the 0.01% cut-off by both methods showed a very good outcome (5-year event-free survival, 91.6%) or a poor one (5-year event-free survival, 50.9%), respectively, whereas discordant cases showed similar event-free survival rates (around 80%).

Conclusions: Within the current BFM-based protocols, flow cytometry and polymerase chain reaction cannot simply substitute each other at single time points, and the concordance rates between their results depend largely on the time at which they are used. Our findings suggest a potential complementary role of the two technologies in optimizing risk stratification in future clinical trials.

Figure 1.

Direct comparison of MRD estimates by PCR and FCM, at each time point (days 15, 33 and 78) in scatter plots, with the identity line, (A, B, C) and according to Bland- Altman (D, E, F) with the continuous line of zero difference and the dotted lines for the estimated mean difference ±2 standard deviations.

Figure 2.

Triple comparison of PCR-MRD (based on MNC) versus FCM-MRD (based on either MNC or NC).

Figure 3

Event-free survival in 1115 patients treated in the AIEOP BFM-ALL 2000 trial, stratified into four groups according to concordant or discordant MRD results obtained on day 33 bone marrow by the simultaneous application of both PCR and FCM.

Figure 4

Event-free survival in 328 patients treated in the AIEOPBFM ALL 2000 trial who had discordant MRD results by PCR and FCM in bone marrow at day 33. Four subgroups of patients with different levels of MRD by either PCR or FCM are represented. For clarity each subgroup can also be identified according to the level of MRD in the scatter plot (see also Figure 1, panel B).