Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia - PubMed (original) (raw)

Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia

Max Jan et al. Sci Transl Med. 2012.

Abstract

Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions.

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Conflict of interest statement

COMPETING INTERESTS.

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Prospective separation of residual HSCs from leukemia cells

(A) Flow cytometry analysis of samples from AML cases SU008 and SU048 indicating lineage negative cells (left panels) and Lin−CD34+CD38− subsets (right panels) further analyzed for expression of CD99 and/or TIM3. Leukemia cells (red gate) and putative residual HSCs (blue gate) were each purified via two rounds of FACS (Supplemental Figure 1). (B) Lin−CD34+CD38−CD99− cells (6000 cells) isolated from case SU008 or Lin−CD34+CD38−TIM3−CD99− cells (450 cells) isolated from case SU048 were transplanted into newborn NSG mice. 12 weeks later, bone marrow engraftment was analyzed by flow cytometry for the presence of human hematopoietic cells (hCD45+) (left panel), further subdivided into lymphoid (CD19+) and myeloid (CD33+) subsets (right panel). (C) The indicated cells were further analyzed for the presence of the _FLT3_-ITD mutation by PCR. Leukemia cells and HSCs from each case corresponded to the red and blue gates in panel A, respectively, and the analysis also included bone marrow from mice engrafted with the residual HSCs. (D) Experimental scheme for identification of pre-leukemic mutations and clonal evolution in de novo AML.

Figure 2

Figure 2. Targeted sequencing identifies pre-leukemic HSCs

FACS-purified leukemia cells and residual HSCs from AML cases SU008, SU014, SU030, SU048, and SU070 were analyzed by targeted deep sequencing for the presence of patient-matched somatic mutations in genes with detectable mRNA expression identified in leukemia cells by exome and transcriptome sequencing. The percentage of mutant allele reads is indicated in each case. The dashed red line indicates the threshold of 1% for variant allele detection as determined by sequencing of defined mixtures of normal and leukemic DNA (fig. S6). Details of sequencing reads are presented in table S2.

Figure 3

Figure 3. Functional HSCs contain pre-leukemic mutations

FACS-purified hCD45+, hCD45+CD19+, and/or hCD45+CD33+ cells isolated from the bone marrow of mice engrafted with residual HSCs from AML cases SU008 (n=4), SU048 (n=5), and SU070 (n=3) were analyzed by targeted deep sequencing for patient-matched somatic mutations. The percentage of mutant allele reads is indicated in each case. The dashed red line indicates the threshold of 1% for variant allele detection as determined by sequencing of defined mixtures of normal and leukemic DNA (fig. S6).

Figure 4

Figure 4. Single cell analysis identifies sequential mutation acquisition in pre-leukemic HSCs

(A) The genotype of 546 myeloid colonies derived from clone sorted single residual HSCs from case SU008 were determined by multiplexed custom Taqman SNP assays for mutations identified by exome analysis to be present in each population. Selected assays are shown with the full data presented in fig. S8. Each colony is represented by a single dot in graphs for each mutation tested; colonies are colored according to genotype (see sure key). (B) 3-D plots illustrate the genotype of each colony. In both panels, the yellow dot indicates the genotype of patient T cells, and the red dot indicates the genotype of patient leukemia cells. (C) Shown is a model for the proposed clonal evolution of AML in patient SU008.

Figure 5

Figure 5. Single cell analysis identifies sequential driver mutation acquisition in pre-leukemic HSCs

(A, D) The genotype of 81 myeloid colonies derived from clone sorted single residual HSCs from AML case SU048 (A) or 189 colonies derived from single HSCs from AML case SU070 (D) were determined by multiplexed custom Taqman SNP assays for mutations identified by exome analysis to be present in each population. Selected assays are shown with the full data presented in fig. S10 (SU048) and fig. S11 (SU070). Each colony is represented by a single dot in graphs for each mutation tested; colonies are colored according to genotype (see figure key). (B, E) 3-D plots illustrating the genotype of each colony from SU048 (B) and SU070 (E). In both panels, the yellow dot indicates the genotype of patient T cells, and the red dot indicates the genotype of patient leukemia cells. (C, F) Models for the proposed clonal evolution of SU048 (C) and SU070 (F) are presented.

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