Leukemia Initiating Cells: New Markers for Minimal Residual Disease Monitoring in B-Precursor Acute Lymphoblastic Leukemia

Minimal residual disease(MRD) is currently the most powerful prognostic indicator in childhood ALL as well as in adult ALL. Using neonatal NOD/SCID/IL2rγ^null xenotransplantation model, we previously demonstrated that CD34⁺CD38⁺CD19⁺ cells as well as CD34⁺CD38⁻CD19⁺ cells have the capacities to initiate B-ALL in vivo and to self-renew, that is, CD34⁺CD19⁺ cells are leukemia initiating cells(LICs) in human B-precursor ALL (B-ALL)(Kong Y et al. Leukemia 2008; 22: 1207–1213). Nevertheless, immunophenotypic differences between B-ALL initiating cells and normal progenitor B cells have not been clearly clarified. Especially, whether the LICs identified in xenotransplantation assay are clinically useful for routine MRD monitoring in B-ALL patients is largely unknown.

To compare phenotypic characteristics of LICs in B-ALL patients to that of normal progenitor B cells in healthy donors. To evaluate clinical significances of LICs as novel MRD markers in predicting relapses in human B-precursor ALL.

To identify new markers for MRD detection in B-ALL, we compared phenotypic characterization of LICs from 40 patients with newly diagnosed B-ALL and 40 patients with relapsed B-ALL to that of normal progenitor B cells from 40 healthy donors by seven-color flow cytometry (FCM). Comparative analysis was performed at mean fluorescence intensity (MFI) of CD38, CD45, CD58 and CD123 on CD34⁺CD19⁺ cells among the above three groups. Subsequently, the most promising markers were examined in detail for their usefulness as MRD markers by seven-color FCM at different time points in 823 patients (including pediatric and adult patients) with B-ALL from January 2010 to June 2011 at Peking University Institute of Hematology. A total of 1,050,000 events were routinely collected. More than 0.001% of LICs with aberrant highly expression of CD123 and/or CD58 in bone marrow samples detected by seven-color FCM were defined as FCM positive (FCM+). FCM positivity at any time was defined as MRD positive (FCM MRD+), all others cases were defined as MRD negative (FCM MRD−). Real-time quantitative polymerase chain reaction (RQ-PCR) was applied concurrently to evaluate MRD in Philadelphia chromosome positive ALL (Ph⁺ ALL) patients. The value of BCR/ABL equal to 0 was defined as RQ-PCR negative (RQ-PCR-). The results of MRD studies by the two methods were recorded independently.

MFI of CD58(3.46±1.85 vs.3.82±1.83 vs.1.35±0.77, p<0.001) and CD123(6.37±3.47 vs.7.56±3.90 vs. 3.66±1.94, p<0.001) was significantly higher on LICs from newly diagnosed B-ALL and relapsed B-ALL than that of their normal counterparts. Meanwhile, MFI of CD38(8.09±6.36 vs.11.23±8.59 vs. 37.89±7.91, p<0.05) and CD45(7.42±5.14 vs.9.91±7.72 vs.13.50±8.20, p<0.05) was significantly lower on LICs than that of their normal counterparts. Further ANOVA analysis by LSD method demonstrated MFI of CD38, CD45, CD58 and CD123 on LICs differed significantly from that of normal progenitor B cells, whereas the phenotypic differences on LICs from newly diagnosed B-ALL or relapsed B-ALL showed no statistically significance.

In the sequential MRD follow up, 185 samples from110 cases of B-ALL were detected as FCM MRD+. Among them, LICs from 56 cases(51%) with aberrant highly CD58 expression, 106 cases(96%) with aberrant highly CD123 expression and 40 cases(36%) with simultaneously aberrant highly expressions of CD58 and CD123. Twenty-two cases relapsed during MRD monitoring, of them 19 cases were detected as FCM MRD+ at a median time of 57 days (13–90days) before their recurrence. The left 3 relapsed cases did not monitor MRD status for about 3 months as we expected. MRD analysis was performed concurrently by both RQ-PCR and FCM in 85 follow-up bone marrow samples from 23 Ph⁺ ALL patients. A good correlation was found between RQ-PCR and FCM (Spearman r=0.883, p< 0.001).

Mean fluorescence intensity of CD38, CD45, CD58 and CD123 antigens distinguished B-ALL initiating cells from their normal counterparts, CD34⁺CD19⁺ normal progenitor B cells. The B-ALL initiating cells with aberrant highly expression of CD123 and/or CD58 detected by seven-color FCM may serve as promising markers for MRD monitoring in B-ALL.

No relevant conflicts of interest to declare.