Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34(+) cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34(+) cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34(+) leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34(+) subfraction exhibiting the phenotype (CD34(+)/CD38(-)/CD123(+)/CD33(+)/CD90(-)) of leukemic stem cells. When transplanted into immunocompromised mice, CD34(+) cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34(+) fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34(-) cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Abstract Abstract 480 Acute myeloid leukemia with mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features [Falini B et al, Blood 2007;109:874-885] which include negativity for CD34 antigen expression at both immunohistochemistry and gene expression profiling. Flow cytometric analysis shows that, in most NPM1-mutated AML, percentages of CD34+ cells are in the low range (< 5-10%). Detection of NPM1 mutations by molecular techniques and/or immunohistochemistry and Western Blot analysis with specific antibodies provides an important tool for tracking the genetic lesion in leukemic cells at different hierarchical stage. We previously reported involvement by NPM1 gene mutation of the CD34+ cell fraction isolated from patients with NPM1-mutated AML, and, in one case, the involvement, in particular, of the early progenitor CD34+/CD38- [Martelli MP et al, Blood (ASH Annual Meeting Abstracts) 2008;112:307]. Here we expand and confirm our previous observation in 5 cases of CD34-negative NPM1-mutated AML. CD34+/CD38- cells were isolated by either FACS (3 cases, purity >98%) or MACS-sorting (2 cases, purity >92%) and analyzed by molecular analysis and Western Blot with a specific anti-NPM1 mutant antibody, respectively. The presence of either NPM1 gene mutation or mutant protein was demonstrated in all samples analyzed proving the CD34+/CD38- cells belong to the leukemic clone. This cell subpopulation displayed also immunophenotypic features classically associated to leukemic stem cells (LSCs) (CD123+/CD33+/CD90-) in all (16/16) samples analyzed, suggesting they might actually represent the LSCs in NPM1-mutated AML. Indeed, CD34+ cell fraction isolated from NPM1-mutated AML was able to generate leukemia in immunocompromised mice resembling the original patient's disease. However, there is experimental evidence that, at least in some CD34-negative AML, also the CD34- population may contain LSCs. Whether the CD34- cell compartment in NPM1-mutated AML is also able to engraft and outgrow into leukemia in mice remains to be clarified. For this purpose, we assessed the engraftment ability of CD34- cells from 5 NPM1-mutated AML patients. No engraftment was observed in one case. Interestingly, in three patients with myelomonocytic (M4, 2 cases) and myelocytic (M2, 1 case) AML, the CD34- fraction resulted into marrow engraftment by human CD45+/CD33+ myeloid cells that, at morphological and immunohistological grounds, consisted of a mixed population of macrophage cells expressing the CD68 (PG-M1) antigen and mature looking myeloperoxidase (MPO)-positive cells. This pattern possibly reflects short-term engraftment by leukemic cells devoid of self-renewal potential that differentiated into mature elements. However, the neoplastic nature of engrafted cells could be established with certainty only in one case by western blotting detection of NPM1 mutant protein. Immunohistochemistry could not help in these cases to establish the leukemic nature of human cells since terminally differentiated leukemic cells in NPM1-mutated AML show nucleus-restricted NPM1 positivity. In contrast, the pure CD34+ fraction (availabel for comparison in one of these three cases) engrafted as AML with clear blastic morphology and cytoplasmic dislocation of nucleophosmin. In a fourth patient, the highly purified CD34- fraction from relapsed NPM1-mutated AML engrafted in mice with a typical AML picture. These preliminary findings suggest that in general the CD34- fraction from NPM1-mutated AML may have more limited engraftment potential than the CD34+ fraction. Further studies are ongoing to address this issue. Disclosures: Falini: Xenomics: Patents & Royalties.