CD34+CD19−, CD34+CD19+ and CD34−CD19+ Cells May All Have Leukemia-Initiating Potential in NOD/Scid Mice.

Current data on the leukemic stem cell (LSC) compartment in childhood acute lymphoblastic leukemia (ALL) are conflicting. The traditional hypothesis supposed that childhood ALL originates in a lymphoid progenitor cell and this is assumed to be consistent with the overall good treatment responses in pediatric patients. In accordance with this hypothesis, our previous studies failed to detect involvement of immature CD34+CD19− progenitor cells in ALL/t(12;21) (Hotfilder et al., Blood 2002) while high-risk ALL/t(9;22) and t(4;11) appears to originate in a more primitive CD34+CD19− cell (Hotfilder et al., Cancer Res 2005). In order to characterize the leukemia-initiating cell in vivo, we established a mouse xenograft model by serial intrafemoral transplantation of NOD/scid mice with flow sorted subpopulations from childhood ALL. Samples were taken from the bone marrow of children with ALL/t(12;21) (n=1), t(4;11) (n=3) and t(11;19) (n=1) and B-cell precursor ALL without a marker translocation (n=2). Primary transplantations were performed with freshly thawed unsorted cells, followed by secondary, tertiary and quaternary transplantations with flow sorted populations. Human leukemic engraftment was defined by a proportion of >5% human CD45+ cells in the murine bone marrow that simultaneously express CD34 and/or CD19. From the bone marrow of leukemic mice, we isolated different leukemic populations and successfully re-transplanted 2×10³ − 1×10⁵ CD34+CD19− cells, 2×10⁴ − 6×10⁶ CD34+CD19+ lymphoid progenitors and 3×10⁴ − 2×10⁶ more differentiated CD34−CD19+ blasts onto secondary, tertiary and quaternary mice (average purity after flow sorting: >96%). So far, we detected leukemic engraftment in 60 of 161 (37%) transplanted mice (with many mice - having only recently been transplanted - still alive). These include 7 of 36 (19%) mice engrafted with CD34+CD19− cells, 33 of 72 (46%) mice engrafted with CD34+CD19+ cells and 20 of 53 (38%) mice engrafted with CD34−CD19+ cells. With as few as 2 × 10³ CD34+CD19− cells being sufficient to re-initiate the leukemia, this intrafemoral ALL-NOD/scid mouse model represents a very sensitive functional assay for candidate LSC in childhood ALL. We have initiated limiting dilution experiments with the different subpopulations to quantify LSC frequency in the different compartments and to exclude that low levels of contaminating blasts with an immunophenotype different from the main transplanted cell population blurred the results. We are also currently investigating whether there is heterogeneity in the CD34+CD19− compartment in respect to standard and high-risk ALL. Altogether, our data indicate that all three subpopulations, CD34+CD19−, CD34+CD19+ and CD34−CD19+ cells, may have the capacity to transfer the leukemia onto NOD/scid mice and that lymphatic LSC may not loose their self-renewal potential with differentiation.