IN PEDIATRIC LYMPHOBLASTIC LEUKEMIA OF B CELL ORIGIN A SMALL POPULATION OF PRIMITIVE BLAST CELLS IS NON-CYCLING, CONSISTENT WITH LEUKEMIA STEM CELLS
A Hirt, AM Schmid, R Ammann, K Leibundgut
Background and Aims. In pediatric precursor-B cell acute lymphoblastic leukemia (PBC ALL), the majority of blast cells express the B cell antigen CD19. However, a minor fraction of blast cells lack CD19 expression corresponding to a more primitive cell population. Investigations on cell proliferation in pediatric ALL are usually based on the entire leukemic cell population reflecting the proliferative behavior of the predominant CD19+ blast cells. In light of the ongoing debate on the immunophenotype of leukemia stem cells in pediatric PBC ALL, we analyzed the proliferative characteristics of ALL cells at different stages of immunophenotypic maturation. Methods. Isolated mononuclear bone marrow cells of 16 children with untreated PBC ALL were stained with monoclonal antibodies against CD19 and CD3 and then separated by fluorescence-activated cell sorting (FACScan Becton-Dickinson). The sorted cells were incubated in vitro with bromo-deoxy-uridine (BrdU) and, after preparation of cytocentrifuge smears, stained with an anti-BrdU antibody to enable detection of cells in the S phase of the cell cycle (BrdU-LI). In addition, individual smears of sorted CD19-CD3- cells were stained with monoclonal antibodies against myeloperoxidase (MPO), glycophorin A (GLA), CD34 or CD10. Because the data were non-normally distributed median and interquartile ranges (IQR) were calculated. Results. Bone marrow cells were sorted into CD19–CD3+, CD19+CD3– and CD19–CD3– populations. Whereas the CD19+CD3– cell compartment represented a pure blast cell population, the CD19–CD3– compartment was composed of both leukemic blast cells and residual normal hematopoietic cells. Therefore, smears were stained for MPO and GLA to exclude normal hematopoietic cells. By staining of sorted CD19–CD3– cells for CD34 or CD10 the compartment of these blast cells was further subdivided. The relative frequencies and the BrdU-LI of these subpopulations are shown in the Table. Conclusions. In untreated pediatric PBC ALL the continuous expansion of the leukemic cell population is exclusively based on the proliferative activity of CD19+ blast cells. The very low proliferative activity of primitive CD19– blast cells is consistent with self-renewal but not with expansion. Due to their immature immunophenotype and their low proliferative activity, CD19– blast cells may represent leukemia-initiating cells. Whereas actively proliferating CD19+ cells may be killed by current treatment approaches, the primitive non-cycling population may survive and subsequently contribute to disease relapse.