Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia

Abstract 4

Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Despite the power of next-generation genome sequencing to describe AML genomes and to identify recurrent mutations, our fundamental understanding of the genomics of leukemogenesis is incomplete. Founding mutations in the majority of AML cases are largely unknown because pre-leukemic cells are clinically silent and are outcompeted by their malignant descendants. Our limited knowledge of founding mutations comes from infrequent cases of AML arising secondary to antecedent clonal bone marrow disorders or rare instances of inherited syndromes, but this does not include the large majority of de novo AML cases. Previously, we showed that non-leukemic hematopoietic stem cells (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. More recently, we demonstrated the prospective separation of residual HSC from AML cells, based on differential expression of surface markers such as CD47 and TIM3.^1,2  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, speculating that these residual non-leukemic HSC might in fact constitute a reservoir of pre-leukemic HSC harboring founder mutations, but lacking the complete complement of abnormalities required to generate AML. Using exome sequencing, we identified mutations present in multiple individual AML genomes (mean 10 mutations per patient) and screened for them in the residual HSC. In most cases, we identified several mutations present in residual HSC that retained normal multilineage differentiation in vivo. These “early” mutations include NPM1c and novel AML mutations in genes involved in 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. Next, using custom-designed SNP Taqman genotyping assays, we analyzed single residual HSC for the presence of the identified “early” mutations. As hypothesized, we determined that a clonal progression of mutations occurs in non-leukemic HSC, based on the identification of individual cells containing subsets of these “early” mutations. Quantitative genetic analyses of the HSC compartment enabled us to reconstruct the subclonal architecture of normal and pre-leukemic stem cells. In all cases, normal HSC were 6–50 times more numerous than pre-leukemic HSC, and in one case where we identified two sequential populations of pre-leukemic HSC, the less mutated population was 25 times more numerous than its more mutated descendent. This result contrasts with the classical model of a linear succession of increasingly dominant pre-leukemic subclones, suggesting that the relationship between subclone size and clonal progression may be complex. In summary, our results show that pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations. Ultimately, these clonal antecedents of leukemia may prove to be clinically important. Indeed, some cases of relapsed pediatric ALL have been shown to arise from a clone ancestral to the presenting leukemia. The same may be true in AML, in which relapsed disease develops from a pre-leukemic HSC clone that acquires additional novel mutations resulting in a genetically divergent leukemic relapse. This possibility suggests that pre-leukemic HSC constitute a cellular reservoir that may need to be targeted for more durable remissions.