CD34⁺ cells from AML with mutated NPM1 express mutated NPM1 gene and protein. (A) WB analysis of CD34⁺ cells isolated from 13 NPM1-mutated AML patient samples. Patient sample codes are indicated on the left (Table 1). Lysates from 1 to 2 × 10⁶ cells of either leukemic bulk (Bulk) or CD34⁺ MACS sorted (Sorted CD34⁺) cell population were loaded and run on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, transferred to polyvinylidene difluoride membrane, and probed with an anti-NPM mutant (anti-NPMm) rabbit polyclonal antibody. A specific band corresponding to NPM mutant protein is detected at 37-kDa molecular weight (MW) in both bulk and CD34⁺ cell populations from patients. Columns on the right show percentage of CD34⁺ cells in the original patient sample and in the CD34-sorted cell population. Lysates from human myeloid leukemic cell lines OCI/AML3 and U937 were used, respectively, as positive and negative control for NPM1 mutant protein expression. (B) AML patient sample dilution test for WB with anti-NPM mutant specific antibody. Unpurified cell fraction from 3 representative NPM1-mutated AML patient samples (patients 30, 32, and 33) was progressively diluted (100%, 50%, 25%, 20%, 15%, 10%, 5%, and 0%) with cells of AML with unmutated NPM1. Lysates from a total of 2 × 10⁶ cells were loaded in each lane and checked for NPM1 mutant protein detection by WB (anti-NPMm, top panels). Progressive dilution of signal indicates that saturation for NPM1 mutant protein detection is not reached in our experimental conditions. Signal is not anymore detectable when NPM1-mutated AML sample is less than 15% to 10% of the original sample. Equal protein lysate loading was demonstrated by blotting the same membranes with an anti–β-tubulin monoclonal antibody (bottom panels). (C) Chromatograms of direct sequencing of the NPM1 gene exon 12 (forward sequence reading) showing both wild-type sequence (coming from the wild-type allele) and TCTG insertion (type A mutation; coming from the mutated allele) in CD34⁺ cells isolated from bone marrow of 1 patient with NPM1-mutated AML (patient 19; bottom panel) and in the relative controls (leukemic bulk and CD34⁻ cell populations; top and middle panels, respectively). Percentage of CD34⁺ cells in each sample is shown on the right. Pt. code indicates patient code; and mut A = NPM1 gene mutation A.