Abstract
In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3Amut) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3Amut-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3Amut arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.
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Change history
16 April 2014
A Correction to this paper has been published: https://doi.org/10.1038/nature13190
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Acknowledgements
We thank all members of the Dick laboratory for critical assessment of this work, A. Khandani, P. Penttilä, N. Simard, T. Velauthapillai and the SickKids-UHN flow facility for technical support, J. Claudio for management of the HALT studies that enabled the genetic analysis described herein, and J. Cui and X.-Z. Yang for curating the human AML samples used in these studies. This work was supported by a Postdoctoral Fellowship Award from the McEwen Centre for Regenerative Medicine with funding made available through the Gentle Ben Charity (L.I.S.), a Canadian Institutes for Health Research (CIHR) fellowship in partnership with the Aplastic Anemia and Myelodysplasia Association of Canada and an award from Vetenskapsradet (S.Z.), and by grants from CIHR, Canadian Cancer Society, Terry Fox Foundation, Genome Canada through the Ontario Genomics Institute, Ontario Institute for Cancer Research with funds from the province of Ontario, a Canada Research Chair, and the Ontario Ministry of Health and Long Term Care (OMOHLTC). The views expressed do not necessarily reflect those of the OMOHLTC. This work was also supported by the Cancer Stem Cell Consortium with funding from the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-047), and through the Canadian Institutes of Health Research (CSC-105367). Contributors to the HALT Pan-Leukemia Gene Panel are listed in Supplementary Note 1.
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Contributions
L.I.S. and S.Z. designed and performed experiments, analysed data and wrote the manuscript; A.M., W.C.C screened AML engraftment in xenotransplantation assays; J.M.B., V.G., J.A.K., A.D.S., A.C.S., K.W.Y., M.D.M. collected AML samples and assembled clinical information; J.A.K. correlated xenotransplantation engraftment data with clinical information; J.L.M., M.D. performed xenotransplantation experiments; J.J.F.M., R.M. performed ddPCR; H.J.K., K.L. performed Sanger sequencing; J.D.M., T.J.H., supervised the targeted sequencing; A.M.K.B. and F.Y. performed and analysed targeted sequencing; Q.M.T., L.D.S. performed DNMT3A data mining. M.D.M. designed the study; J.C.Y.W. supervised AML xenotransplantation screening experiments, designed the study and wrote the manuscript; J.E.D. supervised the study and wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 FLT3-ITD is a late event in patients carrying DNMT3A mutation.
PCR analysis of FLT3-ITD50 in stem/progenitor, mature lymphoid and blast (CD45dim CD33+) cell populations from patient no. 13 (a) and no. 14 (b). FLT3-ITD was present in the blasts from both patients, and also in MLPs from patient no. 14. In contrast, DNMT3Amut without FLT3-ITD was detected in multiple non-blast cell populations (see Extended Data Fig. 2). HSC, haematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; MLP, multilymphoid progenitor; GMP, granulocyte monocyte progenitor; NK, natural killer cells.
Extended Data Figure 2 Frequent occurrence of DNMT3A mutation without NPM1 mutation in stem/progenitor and mature lymphoid cells in AML patients at diagnosis.
a, Summary of the allele frequency (%) of DNMT3A and NPM1 mutations in stem/progenitor, mature lymphoid, and blast (CD45dim CD33+) cell populations from 11 AML patient peripheral blood samples obtained at diagnosis, as determined by droplet digital PCR (ddPCR). Phenotypically normal cell populations were isolated by fluorescence activated cell sorting according to the strategy depicted in Fig. 2a. Mutant allele frequency ∼50% is consistent with a heterozygous cell population. Departures from 50% mutant allele frequency may be stochastic51, related to clonal heterogeneity, or due to the presence of copy number variations, for example loss of the wild type allele (loss of heterozygosity) or amplification of the mutant allele. NA, no cell population detected; HSC, haematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte erythroid progenitor; MLP, multilymphoid progenitor; GMP, granulocyte monocyte progenitor; NK, natural killer cells. Blank boxes indicate no DNMT3A or NPM1 mutation detected. b, Representative plots showing ddPCR analysis of DNMT3Amut and NPM1c allele frequency in sorted cell populations from patient no. 11. The mutant allele frequency (%) is indicated on each plot.
Extended Data Figure 3 Phenotypically normal stem/progenitor and mature cell populations are present in AML patient samples at diagnosis, remission and relapse.
Flow cytometric analysis showing the gating strategy used to isolate phenotypically normal stem/progenitor and mature lymphoid cell populations from AML patient samples. Diagnosis and relapse samples are from peripheral blood; remission samples are from bone marrow.
Extended Data Figure 4 Cells bearing mutations in DNMT3A but not NPM1 are present at diagnosis in AML patients and persist at remission and relapse.
Allele frequency of DNMT3A and NPM1 mutations of patients no. 28, 35, 55, and 57 in stem/progenitor, mature and blast (CD45dim CD33+) cell populations, as determined by droplet digital PCR (ddPCR). Cells were isolated from diagnosis (blue), early remission (white), relapse (red) or late remission (yellow) samples. At remission, CD33+ myeloid cells were also analysed. HSC, haematopoietic stem cell; MPP, multipotent progenitor; MLP, multilymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, megakaryocyte erythroid progenitor; NK, natural killer cells.
Extended Data Figure 5 PreL-HSCs in the peripheral blood of AML patients generate multilineage human grafts in immunodeficient mice.
Summary of results of limiting dilution experiments to assess frequency of pre-leukaemic HSCs generating multilineage grafts after xenotransplantation. Cohorts of NSG mice were transplanted intrafemorally with varying numbers of peripheral blood mononuclear cells from diagnostic samples of AML patient no. 11 (a) and no. 55 (b) and analysed after 8 or 16 weeks by flow cytometry. Engraftment was defined as >0.1% human CD45+ cells in the injected right femur. Shown is the number of mice with multilineage human grafts containing both CD33+ myeloid cells and CD33−CD19+ cells. The frequency of pre-leukaemic HSCs was calculated using the ELDA platform49.
Extended Data Figure 6 Frequent generation of non-leukaemic multilineage human grafts following xenotransplantation of peripheral blood cells from AML patients.
Summary of xenograft characteristics in 123 sublethally irradiated NSG mice transplanted intrafemorally with mononuclear peripheral blood cells from 20 AML patients at diagnosis and analysed after 8 weeks by flow cytometry. The proportion of myeloid (CD33+) and B-lymphoid (CD33−CD19+) cells in the human (CD45+) graft is shown. Leukaemic (AML) engraftment is characterized by a dominant myeloid (CD45dimCD33+) graft, whereas non-leukaemic multilineage grafts contain both lymphoid (predominantly CD33–CD19+ B cells) and myeloid (CD33+) cells. No leukaemic or multilineage graft could be detected in 65/123 mice (53%) in this cohort. Red box indicates AML grafts (27 mice, 22%); blue box indicates multilineage grafts (31 mice, 25%).
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Shlush, L., Zandi, S., Mitchell, A. et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature 506, 328–333 (2014). https://doi.org/10.1038/nature13038
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