Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Despite the power of next-generation DNA sequencing to describe AML genomes and to identify recurrent mutations, our fundamental understanding of the genomics of leukemogenesis is incomplete. Founder mutations in the majority of AML cases are unknown because pre-leukemic cells are clinically silent and are outcompeted by their malignant descendants. Our limited knowledge of founder mutations comes from infrequent cases of AML arising secondary to antecedent clonal bone marrow disorders or rare instances of inherited syndromes. Importantly, recent advances in AML stem cell biology now enable the prospective separation of residual non-leukemic hematopoietic stem cells (HSC) from AML cells. We previously showed that non-leukemic HSC contain clonal antecedents of AML in patients in long-term remission post-therapy, and have proposed a model in which serial acquisition of mutations occurs in self-renewing HSC. Here, we investigated this model and the nature of founder mutations through the genomic analysis of de novo AML and patient-matched residual non-leukemic HSC. Using exome sequencing, we identified mutations present in eight individual AML genomes (mean 10 mutations per patient) and screened for them in the residual HSC. We identified several mutations present in residual HSC that retained normal multilineage differentiation in vivo. These putative “early” mutations detected in residual HSC include NPM1c, mutations in TET2, and novel AML mutations in genes involving the cell cycle and mRNA biogenesis. Putative “late” mutations absent from residual HSC and only found in leukemic cells include FLT3 ITD and IDH1 R132H. Finally, through single cell analysis, we determined that as we hypothesized, a clonal progression of multiple mutations occurs in non-leukemic HSC. Quantitative genetic analyses of the HSC compartment enabled us to reconstruct the subclonal architecture of normal and pre-leukemic stem cells. Consistent with mouse models, pre-leukemic HSC with TET2 mutations outcompeted their normal counterparts. However, in most cases, normal HSC were 6-50 times more numerous than pre-leukemic HSC. In two cases where sequential populations of pre-leukemic HSC were identified, the less mutated pre-leukemic HSC population was 10-25 times more numerous than its more mutated descendent. This result contrasts with the classical model of a linear succession of dominant pre-leukemic subclones. These pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations, suggest a potential mechanism contributing to relapse, and constitute a cellular reservoir that may need to be targeted for more durable remissions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3303. doi:1538-7445.AM2012-3303