Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10−2 versus 5.2 × 10−3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10−4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.


PCR sensitivity
All values are given as median (IQR) Supplementary

Supplementary Table S4 -Review of discrepantly stratified cases at end of induction by FCM and PCR
Causes for discrepant FCM and PCR results in 44 patients at end of induction. Discrepancy of FCM-and PCR-MRD at day 29 was defined as either a discordant detectable/undetectable or discrepant stratification by the day 29 cut-off level of 10 -3 . The former was defined as an undetectable MRD by one method but detectable ≥10 -4 by the other with a >2.5-fold higher value than the detection limit of the method with an undetectable MRD to account for individual variation in the detection limit of the two. Marker modulation with loss of LAIP included KMT2A-r with loss of CD19 lineage marker (illustrated in Figure 3A). FCM technical limitations included hemodilution, many regenerating granulocytes, suboptimal panel (incl. 4 colours), and insufficient red cell lysis in membrane tubes. PCR technical limitations included a sensitivity >10 -3 and the presence of a CD34pos subpopulation at diagnosis, not detected by PCR, expanding at MRD timepoints leading to MRD underestimation (illustrated in Figure 3B).

Supplementary figure S1 -Patients with discrepant detectable/undetectable MRD by FCM and PCR
The were acquired at diagnosis although, if material was available, 100,000 events per marker-combination was recommended to ensure optimal identification of subpopulations. Normal cells present in BM samples served as positive and negative controls of antibody-reagent performance. The antigen expression of each marker was recorded as positive when >20% marker positive blasts were found and additionally recorded as dim, normal/medium, or bright, when fluorescence intensity (FI) was weaker, equal to or stronger compared to normal BM cells. In addition, immunophenotypic subpopulations were documented by recording bimodal or heterogenous marker expression. A leukemia-associated immunophenotype (LAIP) was categorized according to the following guidelines: If at least one marker was aberrantly expressed by > 90% of the leukemic BCP-lymphoblasts, the LAIP was considered as fully informative; if one or more markers were aberrantly expressed on part of the leukemic BCP-lymphoblasts or only on some but not all subpopulations of the leukemic BCP-lymphoblasts the LAIP were considered as partially informative. Cases in which no aberrant marker expression was detected at diagnosis were considered as non-informative.
BM samples taken at the MRD timepoints were analyzed using the standardized MRD antibody combinations. If cross-lineage antigen expression (e.g. myeloid or T-lineage marker expression) was detected at diagnosis, MRD monitoring was supplemented with a patient-tailored antibody combination. At MRD time points, at least 300.000 events, but preferably 1 million events, per antibody combination were analyzed when sufficient material was available.
The gating strategy according to NOPHO guidelines was as follows: firstly dead cells/debris were excluded were considered more quantifiable if 30-40 LAIP events were detected, and more credible when comparable results were identified in more than one antibody combination. If the data were equivocal, the reason for doubts should be reported. The final MRD value was considered as the highest of the values obtained from the results of the most informative antibody combinations, clearly resolving abnormal blasts from normal BM cells. All data were reported into the NOPHO ALL2008 MRD database.