New markers for minimal residual disease detection in acute lymphoblastic leukemia

Abstract

To identify new markers for minimal residual disease (MRD) detection in acute lymphoblastic leukemia (ALL), we compared genome-wide gene expression of lymphoblasts from 270 patients with newly diagnosed childhood ALL to that of normal CD19⁺CD10⁺ B-cell progenitors (n = 4). Expression of 30 genes differentially expressed by ≥ 3-fold in at least 25% of cases of ALL (or 40% of ALL subtypes) was tested by flow cytometry in 200 B-lineage ALL and 61 nonleukemic BM samples, including samples containing hematogones. Of the 30 markers, 22 (CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, CD49f) were differentially expressed in up to 81.4% of ALL cases; expression of some markers was associated with the presence of genetic abnormalities. Results of MRD detection by flow cytometry with these markers correlated well with those of molecular testing (52 follow-up samples from 18 patients); sequential studies during treatment and diagnosis-relapse comparisons documented their stability. When incorporated in 6-marker combinations, the new markers afforded the detection of 1 leukemic cell among 10(5) BM cells. These new markers should allow MRD studies in all B-lineage ALL patients, and substantially improve their sensitivity.

Figure 1

Markers expressed at significantly different levels in ALL cells and in CD19⁺CD10⁺ B-cell progenitors as determined by flow cytometry. Shown are mean fluorescence intensity (MFI) values obtained in CD19⁺ leukemic lymphoblasts from BM samples of patients with newly diagnosed ALL (“L”) and BM CD19⁺CD10⁺ cells from healthy donors or from patients with leukemia in remission and no evidence of MRD (“N”). Each symbol indicates results of one sample; horizontal bars correspond to median values within each group.

Figure 2

Markers expressed at levels not significantly different (P > .05) in ALL cells and CD19⁺CD10⁺ B-cell progenitors as determined by flow cytometry. Shown are mean fluorescence intensity (MFI) values obtained in CD19⁺ leukemic lymphoblasts from BM samples of patients with newly diagnosed ALL (“L”) and BM CD19⁺CD10⁺ cells from healthy donors or from patients with leukemia in remission and no evidence of MRD (“N”). Each symbol indicates results of one sample; horizontal bars correspond to median values within each group.

Figure 3

Relation between results of MRD monitoring with the new markers and those of standard MRD assays. (A) Comparison with flow cytometry using standard markers. The top 16 differentially expressed markers listed in Table 2 were included for a total of 258 tests (number of tests for each marker: CD44, 15; BCL2, 18; HSPB1, 32; CD73, 15; CD24, 7; CD123, 34; CD72, 10; CD86, 21; CD200, 29; CD79b, 5; CD164, 7; CD304, 12; CD97, 20; CD102, 10; CD99, 8; CD300A, 15). Spearman regression analysis of positive MRD results by both methods: r = 0.9816, P < .0001). (B) Comparison with PCR amplification of Ag-receptor genes performed in a subset of the samples (r = 0.8178, P < .0001). Each symbol represents results obtained by 4-color flow cytometry including Abs against CD19, CD10, CD34 and one of the new markers. The dashed line is the line of identity.

Figure 4

Expression of the new markers before, during and at the end of remission induction therapy. Symbols indicate mean fluorescence intensity (MFI) of each marker as measured on ALL cells at diagnosis and on residual leukemic lymphoblasts detected on days 19 and 42 of remission induction therapy. At all time points, all markers were expressed at levels that exceeded those measured in normal CD19⁺CD10⁺ cells.

Figure 5

Differential expression of the new markers according to the genetic subtype of ALL. Heatmap shows the percentage of cases among the main genetic subtypes of childhood ALL in which the markers studied were differentially expressed by flow cytometry. Percentages refer to cases in which each marker was overexpressed in ALL cases compared with CD19⁺CD10⁺ cells from nonleukemic BM samples; markers underexpressed in ALL cells are indicated by “u.” All markers differentially expressed in at least 20% of cases were included.

Figure 6

Additional leukemia-associated markers improve resolution of MRD. Files containing flow cytometric data from 5 nonleukemic BM samples cells (gray dots) and one diagnostic ALL sample (black dots) were merged and analyzed as radial plots using the Kaluza software after selecting all CD19⁺CD10⁺CD34⁺ cells. Mixtures containing 0.01% ALL cells (top row) and 0.001% ALL cells (bottom row) are shown. Underexpression of CD38 alone, a standard MRD marker, could not discriminate well between ALL cells and normal CD19⁺ CD10⁺ CD34⁺ cells (left panels) in this case; the discrimination was improved by analyzing expression of CD97 (middle panels), and further improved by the inclusion of CD86 and the use of a 3-dimensional space (right panels).