CD34⁺/CD38⁻ cells from AML with mutated NPM1 express mutated NPM1 gene and protein and display immunophenotypic features of LSCs. (A) CD34/CD38 flow cytometry staining patterns of peripheral blood MNCs (patients 2, 18, and 22) or whole bone marrow (patients 25 and 26) from the 5 NPM1-mutated AML patients studied for involvement of CD34⁺/CD38⁻ cells by NPM1 mutation. Flow cytometric analysis was performed on either FACSAria (BD Biosciences; patients 22, 25, and 26) or Cytomics FC500 cytometer (Beckman Coulter; patients 2 and 18), as indicated in “Flow cytometric immunophenotyping.” Concomitant expression of CD123 and CD33 on the CD34⁺/CD38⁻ cell population is shown in a representative example (patient 18, bottom right panels). Here, within the CD34⁺/CD38⁻ cell population, only a very small cell fraction (gate Q: ∼ 0.8%) was negative for both CD123 and CD33 that could represent normal residual HSCs. (B) WB showing expression of NPM1 mutant protein in MACS-sorted CD34⁺ cells from 2 selected cases from Figure 1 (patients 2 and 18) where CD34⁺/CD38⁻ cells represented almost the whole CD34⁺ cell population. Here, percentages of CD34⁺/CD38⁻ and CD34⁺/CD38⁺ cells are shown. (C-D) CD34⁺/CD38⁻ and CD34⁺/CD38⁺ cells were FACS-sorted from bone marrow of a patient (patient 25) with AML with cytoplasmic NPM1 with 0.3% CD34⁺ cells. Expression of CD123 and CD33 antigens in the 2 cell subpopulations is shown (C bottom panels). Molecular analysis for NPM1 and FLT3-ITD mutations was performed on genomic DNA by high-resolution fragment analysis (D). Results obtained on sorted CD34⁺/CD38⁻ (purity of 99%; i) and CD34⁺/CD38⁺ (purity of 99%; ii) cells were compared with the total bone marrow mononuclear cells (BM-MNC; iii). As shown in green, the typical double peak indicating the NPM1 mutation (green arrows) was detectable at similar levels in both populations as well as in the MNCs. Peaks indicating FLT3 gene status (wild-type [wt] and FLT3-ITD [ITD] mutation) are in blue. FLT3-ITD mutation/FLT3-wt ratio (mut/wt) is shown on the right. (E) Flow cytometric analysis of a representative NPM1-mutated AML patient sample (patient 30) showing an evident double-cell population with distinct immunophenotypic features within CD34⁺/CD38⁻ progenitor cells: (i) CD34⁺ cell percentage (2.93%) in the leukemic bulk from the patient's peripheral blood (MNCs); (ii) expression of CD38 on the purified CD34⁺ cells (CD34⁺ after sorting); and detection of 2 distinct cell subpopulations within CD34⁺/CD38⁻ cells: CD34^bright/CD38⁻/CD123⁻/CD33⁻ (iii) versus CD34^low/CD38⁻/CD123⁺/CD33⁺ (iv) cells.