Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia

Abstract

Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

FCM analysis of MRD samples using the protocol-adjusted CD38-tube. All dot plots are pre-gated for Syto41- and CD19-positivities. (A–C) BCP-ALL samples with different levels of B-cell regeneration (green dots) and MRD (red dots). (D) T-ALL sample with a high level of B-cell regeneration (green dots) as cross-lineage negative control staining.

Figure 2.

Box plot presentation of marker expression in hematogones (white) and BCP-ALL cells (grey). The markers are ordered in descending order of mean expression values in hematogones.

Figure 3.

Histograms displaying the distribution of different MRD levels within testing series. The height of the bar (y-axis) corresponds to the relative frequency of the samples falling within the indicated MRD interval (x-axis). The series using experimental CD58-tubes comprised 159 samples, the series using the CD38-tube comprised 104 samples.

Figure 4.

Quantitative comparison of MRD estimates by FCM and PCR using experimental CD58-tubes (A) and the protocol-adjusted CD38-tube (B). The diagonal is the 1:1 identity line.

Figure 5.

Bland-Altman plot of the difference of PCR-MRD and FCM-MRD (LDIFF) against the mean of the PCR-MRD and FCM-MRD (LMEAN) after logarithmic transformation. The lines indicate the estimated mean LDIFF (continuous line) and the upper and lower limits of agreement, mean LDIFF ± 1.96 SD (dotted lines).