Mutations in the nucleophosmin (NPM) gene are found in about 30% of cases of acute myeloid leukemia (AML) and lead to a dislocation of the nucleophosmin protein from the nucleus to the cytoplasm (NPMc+ AML). NPMc+ AML shows distinctive biological and clinical features, including a unique gene expression profile, a distinct microRNA signature, low percentage of CD34+ cells, increased incidence of Flt3-ITD (about 40% of cases), good response to induction chemotherapy and (in the absence of Flt3-ITD) a favourable prognosis. Despite significant progress in the characterization of the NPMc+ AML subgroup, questions remain about the leukemia-initiating cell. Distinct features of NPMc+ AML, including multilineage involvement and overexpression of HOX-genes, may point to an early progenitor as the leukemia-initiating cell, but the characteristic low percentage of CD34+ cells may point to a more differentiated leukemic stem cell in NPMc+ AML. To gain more insight in the leukemia-initiating cell in AML with mutated NPM, NPMc+ AML cells were sorted based on the expression of CD34 (n=8, the percentage of CD34+ in the total AML fraction varied between 0.06 and 37%). Western blotting, using an antibody that specifically recognizes the nucleophosmin mutant protein revealed that the NPM mutant protein is expressed in both CD34+ and CD34− cells, proving that the CD34+ NPMc+ AML cells belong to the leukemic clone. This was verified by sequencing the NPM gene in CD34+ and CD34− AML cells. Importantly, culture of sorted CD34+ and CD34− NPMc+ AML cells on a stromal layer revealed that the CD34+ but not the CD34− cells of NPMc+ AML were capable of expanding and initiating long-term growth. In the first 5 weeks of culture an at least 16 fold (range 16–208) expansion of CD34+ AML cells was seen in 5 out of 6 NPMc+ AML cases. This expansion was associated with the formation of cobblestone areas (CAs) under the stromal layer within 3 weeks after plating. The NPMc+ AML cells which expanded in culture were able to expand further after replating in 4 out of 5 investigated cases (fold expansion range 1.6–2.5), indicative of the self renewal capacity of these CD34+ NPMc+ AML cells. Gene expression analysis of CD34+ and CD34− NPMc+ AML cells of 4 cases analyzed thus far revealed the presence of the characteristic HOX-overexpression profile in both CD34+ and CD34− NPMc+ AML cells. In summary, this study shows that the NPM mutation is not only present in CD34−, but also in CD34+ cells of NPMc+ AML and that the properties of long-term expansion and self renewal belong exclusively to the CD34+ subfraction of NPMc+ AML.

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