Molecular analysis of single colonies reveals a diverse origin of initial clonal proliferation in B-precursor acute lymphoblastic leukemia that can precede the t(12;21) translocation

The pathogenesis of pediatric B-precursor acute lymphoblastic leukemia is l argely unknown, and even with nonrandom chromosomal translocations present, the precise order of clonal molecular events is undefined. We developed an in vitro system using cytokines interleukin (IL)-3, IL-7, IL-10, and FMS-l ike tyrosine kinase 3 ligand with CD40 ligand-expressing fibroblasts to obt ain single blast colonies from which clonal immunoglobulin heavy chain (IgH ), T-cell receptor delta gene rearrangements, and, in t(12;21)-positive cas es, TEL-AML1 fusion transcripts could be simultaneously PCR amplified. The proliferation of early tumor progenitors increased subclone detection enabl ing us, in seven diagnostic samples, to determine the stage of differentiat ion at which each leukemia occurred. Four were derived from the stage befor e initiation of IgH rearrangement, one during recombination of variable, jo ining, and diversity segments of the heavy chain gene VDJ(H), and two after completion of IgH rearrangement. Furthermore, analysis of a t(12;21)-posit ive leukemia with unusually late onset, identified both TEL-AML1 -positive and -negative colonies carrying a clonal T-cell receptor delta rearrangemen t, inferring the presence of clonal expansion before the occurrence of the t(12;21). In contrast, in a typical, early onset t(12;21) -positive leukemi a, the t(12;21) appeared to be the first clonal event. In both leukemias, t he t(12;21) occurred before recombination of variable, joining and diversit y segments of the heavy chain gene VDJ.