Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia (original) (raw)

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References

  1. Fialkow, P. J. et al. Clonal development, stem-cell differentiation, and clinical remissions in acute nonlymphocytic leukemia. N. Engl. J. Med. 317, 468–473 (1987)
    Article CAS Google Scholar
  2. McCulloch, E. A., Howatson, A. F., Buick, R. N., Minden, M. D. & Izaguirre, C. A. Acute myeloblastic leukemia considered as a clonal hemopathy. Blood Cells 5, 261–282 (1979)
    CAS PubMed Google Scholar
  3. Vogelstein, B., Fearon, E. R., Hamilton, S. R. & Feinberg, A. P. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science 227, 642–645 (1985)
    Article ADS CAS Google Scholar
  4. Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013)
    Article ADS CAS Google Scholar
  5. Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306–313 (2012)
    Article ADS CAS Google Scholar
  6. Yates, L. R. & Campbell, P. J. Evolution of the cancer genome. Nature Rev. Genet. 13, 795–806 (2012)
    Article CAS Google Scholar
  7. Anderson, K. et al. Genetic variegation of clonal architecture and propagating cells in leukaemia. Nature 469, 356–361 (2011)
    Article ADS CAS Google Scholar
  8. Campbell, P. J. et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature 467, 1109–1113 (2010)
    Article ADS CAS Google Scholar
  9. Ding, L. et al. Genome remodelling in a basal-like breast cancer metastasis and xenograft. Nature 464, 999–1005 (2010)
    Article ADS CAS Google Scholar
  10. Ding, L. et al. Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature 481, 506–510 (2012)
    Article ADS CAS Google Scholar
  11. Notta, F. et al. Evolution of human BCR_–_ABL1 lymphoblastic leukaemia-initiating cells. Nature 469, 362–367 (2011)
    Article ADS CAS Google Scholar
  12. Shah, S. P. et al. Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature 461, 809–813 (2009)
    Article ADS CAS Google Scholar
  13. Yachida, S. et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 467, 1114–1117 (2010)
    Article ADS CAS Google Scholar
  14. Mullighan, C. G. et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 322, 1377–1380 (2008)
    Article ADS CAS Google Scholar
  15. Shlush, L. I. et al. Cell lineage analysis of acute leukemia relapse uncovers the role of replication-rate heterogeneity and microsatellite instability. Blood 120, 603–612 (2012)
    Article CAS Google Scholar
  16. Sgroi, D. C. Preinvasive breast cancer. Annu. Rev. Pathol. 5, 193–221 (2010)
    Article CAS Google Scholar
  17. Wistuba, I. I., Mao, L. & Gazdar Smoking molecular damage in bronchial epithelium. Oncogene 21, 7298–7306 (2002)
    Article CAS Google Scholar
  18. Balaban, G. B., Herlyn, M., Clark, W. H., Jr & Nowell, P. C. Karyotypic evolution in human malignant melanoma. Cancer Genet. Cytogenet. 19, 113–122 (1986)
    Article CAS Google Scholar
  19. Vogelstein, B. et al. Genetic alterations during colorectal-tumor development. N. Engl. J. Med. 319, 525–532 (1988)
    Article CAS Google Scholar
  20. Walter, M. J. et al. Clonal architecture of secondary acute myeloid leukemia. N. Engl. J. Med. 366, 1090–1098 (2012)
    Article CAS Google Scholar
  21. Doulatov, S., Notta, F., Laurenti, E. & Dick, J. E. Hematopoiesis: a human perspective. Cell Stem Cell 10, 120–136 (2012)
    Article CAS Google Scholar
  22. Raza, A. & Galili, N. The genetic basis of phenotypic heterogeneity in myelodysplastic syndromes. Nature Rev. Cancer 12, 849–859 (2012)
    Article CAS Google Scholar
  23. Shih, A. H., Abdel-Wahab, O., Patel, J. P. & Levine, R. L. The role of mutations in epigenetic regulators in myeloid malignancies. Nature Rev. Cancer 12, 599–612 (2012)
    Article CAS Google Scholar
  24. Busque, L. et al. Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis. Nature Genet. 44, 1179–1181 (2012)
    Article CAS Google Scholar
  25. Fialkow, P. J., Gartler, S. M. & Yoshida, A. Clonal origin of chronic myelocytic leukemia in man. Proc. Natl Acad. Sci. USA 58, 1468–1471 (1967)
    Article ADS CAS Google Scholar
  26. Jan, M. et al. Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Sci. Transl. Med. 4, 149ra118 (2012)
    Article Google Scholar
  27. Miyamoto, T., Weissman, I. L. & Akashi, K. AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc. Natl Acad. Sci. USA 97, 7521–7526 (2000)
    Article ADS CAS Google Scholar
  28. The Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N. Engl. J. Med. 368, 2059–2074 (2013)
  29. Yan, X. J. et al. Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nature Genet. 43, 309–315 (2011)
    Article CAS Google Scholar
  30. Ley, T. J. et al. DNMT3A mutations in acute myeloid leukemia. N. Engl. J. Med. 363, 2424–2433 (2010)
    Article CAS Google Scholar
  31. Patel, J. P. et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N. Engl. J. Med. 366, 1079–1089 (2012)
    Article CAS Google Scholar
  32. Krönke, J. et al. Clonal evolution in relapsed _NPM1_-mutated acute myeloid leukemia. Blood 122, 100–108 (2013)
    Article Google Scholar
  33. Doulatov, S. et al. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nature Immunol. 11, 585–593 (2010)
    Article CAS Google Scholar
  34. Notta, F. et al. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science 333, 218–221 (2011)
    Article ADS CAS Google Scholar
  35. Laurenti, E. et al. The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment. Nature Immunol. 14, 756–763 (2013)
    Article CAS Google Scholar
  36. Kim, H. J. et al. Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates. Blood 96, 1–8 (2000)
    CAS PubMed Google Scholar
  37. Fialkow, P. J., Janssen, J. W. & Bartram, C. R. Clonal remissions in acute nonlymphocytic leukemia: evidence for a multistep pathogenesis of the malignancy. Blood 77, 1415–1417 (1991)
    CAS PubMed Google Scholar
  38. Eppert, K. et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nature Med. 17, 1086–1093 (2011)
    Article CAS Google Scholar
  39. Jankowska, A. M. et al. Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood 118, 3932–3941 (2011)
    Article CAS Google Scholar
  40. Kikushige, Y. et al. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell 20, 246–259 (2011)
    Article CAS Google Scholar
  41. Walter, M. J. et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia 25, 1153–1158 (2011)
    Article CAS Google Scholar
  42. Chan, S. M. & Majeti, R. Role of DNMT3A, TET2, and IDH1/2 mutations in pre-leukemic stem cells in acute myeloid leukemia. Int. J. Hematol. 98, 648–657 (2013)
    Article CAS Google Scholar
  43. Challen, G. A. et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nature Genet. 44, 23–31 (2012)
    Article CAS Google Scholar
  44. Tadokoro, Y., Ema, H., Okano, M., Li, E. & Nakauchi, H. De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells. J. Exp. Med. 204, 715–722 (2007)
    Article CAS Google Scholar
  45. Kim, S. J. et al. A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells. Blood 122, 4086–4089 (2013)
    Article CAS Google Scholar
  46. Clappier, E. et al. Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse. J. Exp. Med. 208, 653–661 (2011)
    Article CAS Google Scholar
  47. Inaba, H., Greaves, M. & Mullighan, C. G. Acute lymphoblastic leukaemia. Lancet 381, 1943–1955 (2013)
    Article Google Scholar
  48. Yasuda, T. et al. Leukemic evolution of donor-derived cells harboring IDH2 and DNMT3A mutations after allogeneic stem cell transplantation. Leukemia http://dx.doi.org/10.1038/leu.2013.278 (15 October 2013)
  49. Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009)
    Article CAS Google Scholar
  50. Kottaridis, P. D. et al. Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors. Blood 100, 2393–2398 (2002)
    Article CAS Google Scholar
  51. Heinrich, V. et al. The allele distribution in next-generation sequencing data sets is accurately described as the result of a stochastic branching process. Nucleic Acids Res. 40, 2426–2431 (2012)
    Article CAS Google Scholar

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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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Author notes

  1. Liran I. Shlush, Sasan Zandi and The HALT Pan-Leukemia Gene Panel Consortium: These authors contributed equally to this work.

Authors and Affiliations

  1. Princess Margaret Cancer Centre, University Health Network (UHN), Toronto, Ontario M5G 2M9, Canada,
    Liran I. Shlush, Sasan Zandi, Amanda Mitchell, Weihsu Claire Chen, Joseph M. Brandwein, Vikas Gupta, James A. Kennedy, Aaron D. Schimmer, Andre C. Schuh, Karen W. Yee, Jessica L. McLeod, Monica Doedens, Jessie J. F. Medeiros, Rene Marke, Mark D. Minden, Jean C. Y. Wang & John E. Dick
  2. Department of Medicine, University of Toronto, Toronto, Ontario M5S 2J7, Canada,
    Joseph M. Brandwein, Vikas Gupta, Aaron D. Schimmer, Andre C. Schuh, Karen W. Yee, Mark D. Minden & Jean C. Y. Wang
  3. Division of Medical Oncology and Hematology, UHN, Toronto, Ontario M5G 2M9, Canada,
    Joseph M. Brandwein, Vikas Gupta, Aaron D. Schimmer, Andre C. Schuh, Karen W. Yee, Mark D. Minden & Jean C. Y. Wang
  4. Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada,
    Aaron D. Schimmer, John D. McPherson, Thomas J. Hudson & Mark D. Minden
  5. Radboud University, Nijmegen Medical Centre, Nijmegen 6500 HB, The Netherlands,
    Rene Marke
  6. Chonnam National University Hwasun Hospital, Genome Research Center for Hematopoietic Diseases, Gwangju 519-809, South Korea,
    Hyeoung Joon Kim & Kwon Lee
  7. Ontario Institute for Cancer Research, Toronto, Ontario M5G 0A3, Canada,
    John D. McPherson, Thomas J. Hudson, Andrew M. K. Brown, Fouad Yousif, Quang M. Trinh & Lincoln D. Stein
  8. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada,
    Thomas J. Hudson, Lincoln D. Stein & John E. Dick
  9. †Lists of participants and their affiliations appear in Supplementary Information.,
    The HALT Pan-Leukemia Gene Panel Consortium

Authors

  1. Liran I. Shlush
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  2. Sasan Zandi
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  3. Amanda Mitchell
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  4. Weihsu Claire Chen
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  5. Joseph M. Brandwein
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  6. Vikas Gupta
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  7. James A. Kennedy
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  8. Aaron D. Schimmer
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  9. Andre C. Schuh
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  10. Karen W. Yee
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  11. Jessica L. McLeod
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  12. Monica Doedens
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  13. Jessie J. F. Medeiros
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  14. Rene Marke
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  15. Hyeoung Joon Kim
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  16. Kwon Lee
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  17. John D. McPherson
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  18. Thomas J. Hudson
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  19. The HALT Pan-Leukemia Gene Panel Consortium
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  20. Andrew M. K. Brown
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  21. Fouad Yousif
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  22. Quang M. Trinh
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  23. Lincoln D. Stein
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  24. Mark D. Minden
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  25. Jean C. Y. Wang
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  26. John E. Dick
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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.

Corresponding author

Correspondence toJohn E. Dick.

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Competing interests

The authors declare no competing financial interests.

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, DNMT3A mut 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 DNMT3A mut 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%).

Supplementary information

Supplementary Information

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Supplementary Table 1

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Supplementary Table 2

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Supplementary Table 3

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Supplementary Table 4

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Supplementary Table 5

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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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