Flow minimal residual disease monitoring of candidate leukemic stem cells defined by the immunophenotype, CD34+CD38lowCD19+ in B-lineage childhood acute lymphoblastic leukemia

Abstract

Flow cytometric minimal residual disease (MRD) monitoring could become more powerful if directed towards the disease-maintaining leukemic stem cell (LSC) compartment. Using a cohort of 48 children with B-lineage acute lymphoblastic leukemia (ALL), we sought the newly proposed candidate-LSC population, CD34(+)CD38(low)CD19(+), at presentation and in end of induction bone marrow samples. We identified the candidate LSC population in 60% of diagnostic samples and its presence correlated with expression of CD38, relative to that of normal B-cell progenitors. In addition, the candidate LSC was not detectable in all MRD positive samples. The absence of the population in 40% of diagnostic and 40% of MRD positive samples does not support the use of this phenotype as a generic biomarker to track LSCs and suggests that this phenotype may be an artifact of CD38 underexpression rather than a biologically distinct LSC population. ClinicalTrials.gov Identifier: NCT00222612.

Figures

Figure 1.

(A) Diagnostic ALL samples were investigated for the presence of the CD34+CD38LowCD19+ population. Contour plots shown are already gated on a lymphoid light scatter profile and display CD34 and CD38 expression. The CD34+CD38Low population has then been gated as shown to display CD19 expression in histogram form. Plot 1: normal bone marrow showing the normal pattern of maturation and the absence of the aberrant CD34+CD38LowCD19+ population. Marker lines show the boundary of CD19 positive and negative events. The same gates were then used to assess diagnostic leukemic samples. Plot 2: ALL (CD38 normal expression) with no detectable CD34+CD38LowCD19+ population (<0.1%). Plot 3: ALL (CD38 overlap expression) with low but detectable numbers of CD34+CD38LowCD19+ cells (0.3%). Plots 4 and 5: ALL samples (CD38 underexpression) that clearly show the CD34+CD38LowCD19+ population at higher levels (12.3% and 53.4%, respectively). (B) Bar chart showing the incidence of the candidate LSC across the 3 major cytogenetic subgroups.

Figure 2.

(A) CD38 expression in ALL. Diagnostic samples were assessed for CD38 expression relative to that of normal B-cell progenitors using multiparameter flow cytometry. The dot plots show CD38 versus CD10 expression of events resulting from the sequential gating strategy; lymphoid scatter gate, then a CD19+, low side scatter gate and finally a CD19+CD34+ gate. Plot 1 shows the CD38 expression levels of normal hematopoietic CD19+CD34+ B-cell progenitors outlined with a rectangle region which served as a reference template. Plot 2: a diagnostic ALL with CD38 expression similar to that of normal progenitors (Normal). Plot 3: an ALL in which a proportion of the population has underexpression. Plot 4: an ALL in which the bulk of the population shows CD38 underexpression (Under). (B) CD38 expression and cytogenetic subgroup. Also shown is the presence (dark shade) or absence (light shade) of the candidate LSC. (C) The incidence of the candidate-LSC population in relation to CD38 expression. The median percentage of the candidate-LSC within the blast population is also shown.

Figure 3

The CD34+CD38lowCD19+ population in diagnostic and MRD positive samples. (A) Row 1 plots show flow cytometric data of diagnostic samples. Plot 1: CD34/CD19 expression of diagnostic blasts already gated on a lymphoid scatter gate. Plot 2: CD38 versus CD10 expression of cells gated by the region shown in the CD34/CD19 plot with a rectangle showing the area in which normal progenitor cells are found. Plot 3: a contour plot of cells already gated on a lymphoid light scatter profile and CD34 and CD38 expression is shown. The CD34+CD38low population is gated as shown to display CD19 expression in histogram form (Plot 4). Row 2 plots show data from the same patient analyzed at day 28 in which the MRD blasts (marked with arrow) are clearly identifiable but because they are CD34 negative, there is no evidence of the candidate LSC population. Similarly, Rows 3 and 4 show data from a second patient, gated in the same way which shows an increase in the candidate LSC population in the day 28 samples due to a decrease in CD38 expression in the MRD compared to the diagnostic blasts.