In Infant ALL with t(4;11)/MLL-AF4 Multiple and Phenotypically Distinct CD19+ BM Subsets Initiate Leukemia in NOD/SCID Mice..

Abstract 1432

Poster Board I-455

Whether a stochastic or hierarchical model best reflects the identity of leukemia initiating cell is still an open debate. In the case of acute lymphoblastic leukemia (ALL) little is known about the identity and the origin of leukemic stem cells (LSCs). In this study we focused on infant ALL carrying the t(4;11)/MLL-AF4, the most frequent and aggressive form of leukemia occurring within the first year of life. Using fluorescence-activated cell sorting (FACS) combined with fluorescence in situ hybridization (FISH) analysis, in vivo repopulating assay and clonal analysis, we investigated whether different BM subsets, sorted from infant ALL diagnostic samples on the bases of their CD34/CD19/NG2 phenotype, have similar or distinct LSC properties. Our studies demonstrate that in t(4;11)/MLL-AF4 infant ALL the 34+19- hematopoietic stem/progenitor cell pool is intact (MLL-AF4 negative by FISH), and that the LSC is not CD19-, as CD19- cells lacked repopulating ability (CD34-) or gave rise exclusively to normal multilineage reconstitution (CD34+). In contrast, CD19+ LSCs rapidly engraft and establish leukemia in NOD/SCID mice, with features recapitulating the human disease, and possess self-renewal potential in serial transplantation, regardless of CD34 and NG2 expression. However, although multiple leukemic subsets possess some “stemness”, we did observe differences in the kinetics and immunophenotypes of engraftment between phenotypically distinct LSC subpopulations, suggesting that in t(4;11)/MLL-AF4 infant ALL the LSCs are diverse but distinct. These findings might reconcile conflicting results with regard to previously proposed stochastic versus hierarchical models for LSCs. By designing patient-specific primers and probes on the junctional regions, we analyzed by RQ-PCR the clonal contribution to the leukemic grafts and found that, as in infant patients, ALL grafts in mice were oligoclonal, as several independent clones were able to sustain engraftment in recipient mice. However, among the engrafting clones, we observed different behaviours: either major or minor clones in the patient dominate in the graft; conversely some other clones which were predominant in the diagnostic sample failed to sustain leukemia in vivo. Specifically we observed that dominant clones in primary recipients reconstituted with leukemia can either persist over time or extinguish (or become dormant) in serial passages; while other clones, which were quiescent in primary recipents, never-the-less persisted, reactivated and became dominating in secondary recipients. In conclusion we provided evidences that LSC in infant ALL with t(4;11) is restricted to the CD19+ fraction and, among this, multiple phenotypically distinct BM subsets can initiate leukemia in NOD/SCID mice, with different kinetics and immunophenotypes of engraftment. Moreover, our clonal analysis results point out a further heterogeneity within purified LSC subsets and implicate the co-existence and competition between multiple clones.