Clonal Profile Analysis of Leukemic Progenitors and Diagnosis Blast Cells in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Recent studies suggest that the majority of malignant cells found in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) arise from a rare population of leukemic progenitors. Little information is available on the presence of clonal rearrangements in cells at the stage of early precursor. To address this issue we analyzed clonality profile of early leukemic precursors sorted by flow-cytometry. Leukemic cells were obtained from bone marrow samples collected at diagnosis from 6 patients with childhood BCP-ALL. Furthermore, bone marrow cells were collected from 3 healthy children who were harvested for bone marrow donation. Three subpopulations of leukemic cells were investigated: total unsorted blasts, the sorted CD34+/CD38−/CD19+, and the sorted CD34+/CD38−/CD19− cells. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were screened by polymerase chain reaction (PCR) in the sorted populations and in the bulk leukemic cells in order to identify molecular markers of clonal evolution. Sequence analysis was then performed on the N-region. Overall, a total of 38 different Ig/TCR gene rearrangements were identified in the 3 cell populations under study (total blasts, CD34+/CD38−/CD19+, and CD34+/CD38−/CD19−). Of them, 13 (34%) were found in the three populations; 12 (31%) were found in two of the three populations: 7 in total blasts and CD19+ subset, 3 in total blasts and CD19−, 2 in CD19− and CD19+; finally, 13 were found only in one subpopulation: 4 in total blast cell, 5 in CD19+, 4 in CD19−. In all the six patients studied, BCP-ALL progenitors CD34+/CD38−/CD19− and CD19+ and the bulk tumor blasts shared at least one Ig/TCR gene clonal rearrangement with the same N-region. In 5 out of 6 patients at least one rearrangement detected in the BCP-ALL progenitors was undetectable in total blasts. Conversely, in 3 patients the clonal rearrangement observed in the bulk leukemic cells was not identified in any of the two sorted ALL precursor populations. Clonal rearrangement was never detected in the samples from healthy bone marrow donors. Our findings confirm that clonal rearrangement may be detected at the stage of early B-lineage precursor CD34+/CD38−/CD19−, suggesting that leukemic transformation may occur at this stage or even before in BCP-ALL. We plan to extend this observation by repopulating studies in NOD/SCID mice.