Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells:A EuroFlow study

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating &amp; Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.</p

Benefits and risks of clofarabine in adult acute lymphoblastic leukemia investigated in depth by multi-state modeling

Background: We recently reported results of the prospective, open-label HOVON-100 trial in 334 adult patients with acute lymphoblastic leukemia (ALL) randomized to first-line treatment with or without clofarabine (CLO). No improvement of event-free survival (EFS) was observed, while a higher proportion of patients receiving CLO obtained minimal residual disease (MRD) negativity. Aim: In order to investigate the effects of CLO in more depth, two multi-state models were developed to identify why CLO did not show a long-term survival benefit despite more MRD-negativity. Methods: The first model evaluated the effect of CLO on going off-protocol (not due to refractory disease/relapse, completion or death) as a proxy of severe treatment-related toxicity, while the second model evaluated the effect of CLO on obtaining MRD negativity. The subsequent impact of these intermediate events on death or relapsed/refractory disease was assessed in both models. Results: Overall, patients receiving CLO went off-protocol more frequently than control patients (35/168 [21%] vs. 18/166 [11%], p = 0.019; HR 2.00 [1.13–3.52], p = 0.02), especially during maintenance (13/44 [30%] vs. 6/56 [11%]; HR 2.85 [95%CI 1.08–7.50], p = 0.035). Going off-protocol was, however, not associated with more relapse or death. Patients in the CLO arm showed a trend towards an increased rate of MRD-negativity compared with control patients (HR MRD-negativity: 1.35 [0.95–1.91], p = 0.10), which did not translate into a significant survival benefit. Conclusion: We conclude that the intermediate states, i.e., going off-protocol and MRD-negativity, were affected by adding CLO, but these transitions were not associated with subsequent survival estimates, suggesting relatively modest antileukemic activity in ALL.</p

A series of case studies illustrating the role of flow cytometry in the diagnostic work-up of myelodysplastic syndromes

Current guidelines recommend flow cytometric analysis as part of the diagnostic assessment of patients with cytopenia suspected for myelodysplastic syndrome. Herein we describe the complete work-up of six cases using multimodal integrated diagnostics. Flow cytometry assessments are illustrated by plots from conventional and more recent analysis tools. The cases demonstrate the added value of flow cytometry in case of hypocellular, poor quality, or ambiguous bone marrow cytomorphology. Moreover, they demonstrate how immunophenotyping results support clinical decision-making in inconclusive and clinically ‘difficult’ cases