Heterogeneity of clonal expansion and maturation-linked mutation acquisition in hematopoietic progenitors in human acute myeloid leukemia

Abstract

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML), but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34(+)/CD33(-) cells revealed polyclonal growth in highly curable AMLs, suggesting that mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34(+)/CD33(-) cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications.

Figure 1. Experimental Strategy

(A) Diagram depicting the experimental strategy pursued with primary human AML specimens to assess bulk AML cells for the disease-associated X chromosome inactivation pattern and presence of somatic mutations, to isolate phenotypically immature cell populations based on expression of CD34 and CD33 with subsequent direct clonal/molecular analysis, and to subject these isolated cell populations to short- and long-term in vitro E4ORF1⁺ EC co-culture with SR1 in the presence of hypoxia, with subsequent CFC assays and clonal/molecular analyses of individual CFU-GMs. (B) Scatter and histogram plots from 3 representative primary AML specimens illustrating the sorting strategy pursued to isolate CD34⁺/CD33⁻ and CD34⁺/CD33⁺ cells. Fluorescence intensity of CD34 and CD33 is shown in black/bold, whereas staining with isotype control antibodies is shown in grey/dashed. FSC, forward scatter; SSC, side scatter.

Figure 2. Clinical Significance of AML Progenitor Cell Heterogeneity

(A) Overall survival and (B) relapse-free survival of adult patients with newly diagnosed non-APL AML undergoing standard induction chemotherapy, stratified based on polyclonal vs. clonal growth of CFU-GMs derived from FACS-isolated CD34⁺/CD33⁻ cells after long-term (≥4 weeks) in vitro co-culture.