Unsupervised Flow Cytometry Analysis Allows for an Accurate Identification of Minimal Residual Disease Assessment in Acute Myeloid Leukemia

Abstract

The assessment of minimal residual disease (MRD) is increasingly considered to monitor response to therapy in hematological malignancies. In acute myeloblastic leukemia (AML), molecular MRD (mMRD) is possible for about half the patients while multiparameter flow cytometry (MFC) is more broadly available. However, MFC analysis strategies are highly operator-dependent. Recently, new tools have been designed for unsupervised MFC analysis, segregating cell-clusters with the same immunophenotypic characteristics. Here, the Flow-Self-Organizing-Maps (FlowSOM) tool was applied to assess MFC-MRD in 96 bone marrow (BM) follow-up (FU) time-points from 40 AML patients with available mMRD. A reference FlowSOM display was built from 19 healthy/normal BM samples (NBM), then simultaneously compared to the patient’s diagnosis and FU samples at each time-point. MRD clusters were characterized individually in terms of cell numbers and immunophenotype. This strategy disclosed subclones with varying immunophenotype within single diagnosis and FU samples including populations absent from NBM. Detectable MRD was as low as 0.09% in MFC and 0.051% for mMRD. The concordance between mMRD and MFC-MRD was 80.2%. MFC yielded 85% specificity and 69% sensitivity compared to mMRD. Unsupervised MFC is shown here to allow for an easy and robust assessment of MRD, applicable also to AML patients without molecular markers.

Unsupervised flow cytometry analysis in hematological malignancies: A new paradigm

“…Of note, at this stage and as reported elsewhere, 45,55 an unsupervised analysis focused on the progenitor area ("bermudes") reduces the number of pathological nodes to be investigated, dubbed "nodes of interest," or NOI 43 (Figure 1B and C).…”

“…Statistical analyses on a large series of AML have determined a ratio of 1.9 (ie, 1.9 times more cells in the Dg node compared with the corresponding NBM node) as specific of an abnormal cluster. 55 This direct comparison combines the two congruent approaches of leukemia-associated immunophenotypes (LAIP) and different from normal (DfN). 56 In these cases, the immunophenotype will be similar between the FU and NBM samples, with potentially more cells in the FU node, but mostly these nodes will be lower than the threshold or even absent in the Dg file.…”

“…Further comparison with the FU files provides three types of important information: Disappearance or persistence of diagnostic subclones: The latter are qualified by their immunophenotypic features and size in terms of absolute number and percentage. Statistical analyses on a large series of AML have determined a ratio of 1.9 (ie, 1.9 times more cells in the Dg node compared with the corresponding NBM node) as specific of an abnormal cluster 55 . This direct comparison combines the two congruent approaches of leukemia‐associated immunophenotypes (LAIP) and different from normal (DfN) 56,57 …”

The Plasmacytoid Dendritic Cell CD123+ Compartment in Acute Leukemia with or without RUNX1 Mutation: High Inter-Patient Variability Disclosed by Immunophenotypic Unsupervised Analysis and Clustering

“…FlowSOM is one of the new types of software, developed in the Bioconductor R system [24]. This software has been applied to the definition of normal BM and hematological malignancies with myeloid, lymphoid, and erythroid antibody panels [25][26][27]. Here we explored how FlowSOM could dissect PDC-like populations in seven patients with leukemic disorders involving these peculiar cells.…”

Issue Highlights—September 2021

“…MRD remains a field where progress and harmonization are needed, especially because of the rapid answer that can be provided by flow cytometry. The large number of recent publications in various types of hematological malignancies highlight this need (Bayly et al, 2020; Bento et al, 2020; DiGiuseppe & Wood, 2019; Gudapati et al, 2020; Rossi et al, 2020; Soh et al, 2020; Vial et al, 2021; Zhou et al, 2019).…”