Reconstructing clonal evolution from dysplasia to AML. (A) Study design. BM aspirates collected from 358 patients with newly diagnosed AML were analyzed using MFC. In 21 patients, leukemic cells were isolated using FACS according to patient-specific aberrant phenotypes and so were cells of the neutrophil (Neutro), monocytic (Mono), and erythroid (Erythro) lineages whenever dysplastic phenotypes were observed in 1 or more lineages. T cells were systematically isolated for germline DNA. NGS of genes frequently mutated in myeloid neoplasms was performed in all cell types available in each patient. (B) Mutational status of 33 genes in T cells (T), neutrophils (N), monocytes (M), erythroblasts (E), and blasts (B) isolated from 21 patients at the onset of AML; cell types not available for NGS are represented with gray lines. Mutated genes were colored in a gradient of red according to the variant allele frequency (VAF). Three models were identified: (1) stable transition according to identical mutational landscapes in blasts and dysplastic cells; (2) branching evolution with blasts originating from leukemic stem cells other than the ones driving dysplasia, due to mutations absent in blasts and present in dysplastic cells; and (3) clonal evolution with new mutations in blasts onto mutations shared between these and dysplastic cells. (C) Percentage of mutations grouped according to functional categories that were simultaneously mutated in mature dysplastic cells and blasts (gray), or that were private in the former (blue) and the latter (purple). Functional categories with significantly different distributions were highlighted in bold.