PF236 IDENTIFICATION OF ACUTE MYELOID LEUKAEMIA STEM CELLS AT DIAGNOSIS REVEALS PHENOTYPIC HETEROGENEITY AND IS FEASIBLE IN A CLINICAL LABORATORY
Authors: Lee S.Y. , Li J.B. , Chng W.J. , Liu T.C. 
Published: June 2019

Background:Acute myeloid leukemia (AML) stem cells are postulated to be leukemic counterparts of Lin‐CD34+CD38‐ hematopoietic stem cells (HSC). Several primitive AML compartments with leukemia‐initiating potential have been identified, increasing the complexity of their identification. Identification of AML stem cells have applications in therapeutic targeting and minimal residual disease monitoring.Aims:The aim of our study was to characterise the phenotypic landscape of AML stem cells and to assess the feasibility in a clinical laboratory.Methods:Whole marrow samples from normal controls and AML cases at diagnosis were analysed by flow cytometry using surface stain lyse/wash method on BD FACSCantoII. Fluorescence conjugated antibodies were used in combinations of CD34/CD38/CD45 with CD45RA/CLL1/CD135, CD25/CD44/CD90, CD47/CD96/CD32 or TIM3/CD123. Events with CD34+CD38‐ phenotype were selected for analysis. CD38‐ was defined by mean fluorescence intensity (MFI) below expression level of 99% of CD38‐ erythroblasts. MFI and qualitative expression of each parameter was collected. Erythroblasts were used as reference controls for most markers except CD34, CD45, CD47 (lymphocytes) and CD44 (unstained controls as erythroblasts and lymphocytes had variable CD44 expression). Positive expression was taken as >20% positivity above negative control. The normal control mean/SD or median/absolute deviation for each parameter was used to calculate Z‐scores (or modified Z‐scores) for each parameter on each population (z = [x–μ]/σ).Results:Eleven normal controls and 33 AML cases were analysed. A mean of 5.2x106 events were collected across all tubes. In normal controls, mean (SD) frequency of HSCs was 0.020% (0.017%) and mean frequency out of CD34+ events was 5.37% (4.43%). Normal HSCs were found to be CD45RA‐CLL1‐CD135+loCD90+loCD44+CD47+CD45+25–32–96–123‐TIM3‐. All AML cases had at least one abnormal population. Two and three abnormal populations were observed in 4 and 1 cases, giving a total 39 abnormal populations. In 22/33 AML cases, a population with normal HSC phenotype was also observed. Mean (SD) frequency of phenotypically normal HSCs was 0.013% (0.009%), while that of abnormal stem cells was 0.91% (2.51%). Of the markers tested, CD45RA was positive in 30/39 (77%) abnormal populations, CLL1+ in 82%, TIM3+ in 33%, CD123+ in 59%, CD25+ in 41%, CD32+ in 41% and CD96+ in 59%. The CD45RA negative abnormal populations had aberrancies in other markers. One, two, three, four and >four aberrancies occurred in 4, 5, 7, 12 and 11 cases (Figure 1A). Analysis of the populations by hierarchical clustering did not reveal any obvious clusters, likely because the composite phenotype was highly variable. Z‐score >2.5 was significantly more frequent in abnormal populations for CD45RA, CLL1, CD135, CD44, CD32, CD123 (p < 0.05 for all), translating to significantly higher Z‐scores compared with normal populations/controls (Figure 1B). CD47 MFI on normal stem cells was consistently lower than lymphocytes, whereas 20/38 abnormal populations had higher MFI (p = 0.0003). However, high baseline CD44 and CD47 in cases and controls, and similarity with lymphocytes reduced their discriminating power. In contrast, CD34 was frequently lower in abnormal poulations (p = 0.002). The scatter characteristics of phenotypically normal stem cells in AML cases were similar or lower compared with normal controls (Figure 1B).Summary/Conclusion:Despite phenotypic heterogeneity of AML stem cells, CD45RA and CLL1 emerge as the more informative markers at diagnosis. In many AML cases, phenotypically normal stem cells are retained.image