Identification of Cytogenetically Normal Human CD34⁺CD38⁺ Hematopoietic Stem/Progenitor Cells from Inv(16)⁺ Leukemic Bone Marrow

For many subtypes of AML including cases with the inv(16), mutations that give rise to the leukemic phenotype occur, at least in part, in the hematopoietic stem/progenitor (HSPC) cell subset, as suggested by studies showing that primitive CD34⁺ CD38^- bone marrow cells can function as leukemia-initiating cells (LIC) when transferred into immunodeficient mice. A significant challenge has been that LIC share many of the same cell-surface markers as their normal HSPC counterparts, thus making it difficult to purify and functionally characterize either subset from the bulk bone marrow of leukemia patients. Here we report the FACS analysis of several previously reported human LIC markers on bone marrow samples from inv(16) AML patients and show that a combination of TIM3, CLL1, and CD33 can significantly enrich for a rare population of CD34⁺ CD38^- cells that lack the inv(16) fusion mRNA when tested by nested RT-PCR. Heterogeneous expression of these markers among different patient samples often causes incomplete elimination of the fusion mRNA when FACS-sorting the CD34⁺ CD38^- population as single TIM3^-, CLL1^-, or CD33^- subsets. The combination of TIM3 with CLL1 and/or CD33 leads to a more consistent elimination of the fusion mRNA from the FACS-sorted CD34⁺ CD38^- subsets. Results from methylcellulose assays showed that the TIM3^- CLL1^- CD33^- subset of CD34⁺CD38^- cells could form multiple colony types, including CFU-GEMM, that were all negative for the fusion mRNA by RT-PCR. In contrast, colonies derived from bulk bone marrow were all positive for the fusion mRNA. The TIM3^- CLL1^- CD33^- subset of CD34⁺CD38^- cells displayed greater than 600-fold enrichment for progenitor activity compared to bulk bone marrow but did not form additional colonies upon serial re-plating. These results have important implications for the therapeutic targeting of inv(16)⁺ hematopoietic stem/progenitor cells in patients with relapsed and refractory disease and for purification of normal HSPC from leukemic bone marrow samples.