Abstract
Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34+CD38− leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Pabst T, Vellenga E, van Putten W, Schouten HC, Graux C, Vekemans M-C et al. Favorable effect of priming with granulocyte colony-stimulating factor in remission induction of acute myeloid leukemia restricted to dose escalation of cytarabine. Blood 2012; 119: 5367–5373.
Terwijn M, van Putten WLJ, Kelder A, van der Velden VHJ, Brooimans RA, Pabst T et al. High prognostic impact of flow cytometric minimal residual disease detection in acute myeloid leukemia: data from the HOVON/SAKK AML 42 A study. J Clin Oncol 2013; 31: 3889–3897.
Feller N, van der Pol MA, van Stijn A, Weijers GWD, Westra AH, Evertse BW et al. MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia. Leukemia 2004; 18: 1380–1390.
Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S, Haferlach T . Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004; 104: 3078–3085.
Venditti A, Buccisano F, Del Poeta G, Maurillo L, Tamburini A, Cox C et al. Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood 2000; 96: 3948–3952, Presented in part at the 41st Annual Meeting of the American Society of Hematology, 3–7 December 1999, New Orleans, LA, USA.
San Miguel JF, Vidriales MB, López-Berges C, Díaz-Mediavilla J, Gutiérrez N, Cañizo C et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood 2001; 98: 1746–1751.
Freeman SD, Virgo P, Couzens S, Grimwade D, Russell N, Hills RK et al. Prognostic relevance of treatment response measured by flow cytometric residual disease detection in older patients with acute myeloid leukemia. J Clin Oncol 2013; 31: 4123–4131.
Taussig DC, Vargaftig J, Miraki-Moud F, Griessinger E, Sharrock K, Luke T et al. Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(−) fraction. Blood 2010; 115: 1976–1984.
Terwijn M, Zeijlemaker W, Kelder A, Rutten AP, Snel AN, Scholten WJ et al. Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia. PLoS One 2014; 9: e107587.
Goardon N, Marchi E, Atzberger A, Quek L, Schuh A, Soneji S et al. Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia. Cancer Cell 2011; 19: 138–152.
Martelli MP, Pettirossi V, Thiede C, Bonifacio E, Mezzasoma F, Cecchini D et al. CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice. Blood 2010; 116: 3907–3922.
Sarry J-E, Murphy K, Perry R, Sanchez PV, Secreto A, Keefer C et al. Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice. J Clin Invest 2011; 121: 384–395.
Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 2007; 25: 1315–1321.
Bonnet D, Dick JE . Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–737.
Guzman ML . Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells. Blood 2001; 98: 2301–2307.
Costello RT, Mallet F, Gaugler B, Sainty D, Arnoulet C, Gastaut JA et al. Human acute myeloid leukemia CD34+/CD38− progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities. Cancer Res 2000; 60: 4403–4411.
Bradbury C, Houlton AE, Akiki S, Gregg R, Rindl M, Khan J et al. Prognostic value of monitoring a candidate immunophenotypic leukaemic stem/progenitor cell population in patients allografted for acute myeloid leukaemia. Leukemia 2014; 9: 1–4.
Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 2011; 17: 1086–1093.
Horton SJ, Huntly BJP . Recent advances in acute myeloid leukemia stem cell biology. Haematologica 2012; 97: 966–974.
Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly G et al. Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia. Blood 2005; 106: 4086–4092.
Jordan CT, Upchurch D, Szilvassy SJ, Guzman ML, Howard DS, Pettigrew AL et al. The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia 2000; 14: 1777–1784.
Van Rhenen A, van Dongen GAMS, Rombouts EJ, Feller N, Moshaver B, Walsum MS et al. The novel AML stem cell—associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells. Blood 2007; 110: 2659–2666.
Van Rhenen A, Moshaver B, Kelder A, Feller N, Nieuwint AW, Zweegman S et al. Aberrant marker expression patterns on the CD34+CD38− stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission. Leukemia 2007; 21: 1700–1707.
Hosen N, Park CY, Tatsumi N, Oji Y, Sugiyama H, Gramatzki M et al. CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia. Proc Natl Acad Sci USA 2007; 104: 11008–11013.
Jan M, Chao MP, Cha AC, Alizadeh AA, Gentles AJ, Weissman IL et al. Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker. Proc Natl Acad Sci USA 2011; 108: 5009–5014.
Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE . Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med 2006; 12: 1167–1174.
Schuurhuis GJ, Meel MH, Wouters F, Min LA, Terwijn M, de Jonge NA et al. Normal hematopoietic stem cells within the AML bone marrow have a distinct and higher ALDH activity level than co-existing leukemic stem cells. PLoS One 2013; 8: e78897.
Gerber JM, Smith BD, Ngwang B, Zhang H, Vala MS, Morsberger L et al. A clinically relevant population of leukemic CD34+CD38- cells in acute myeloid leukemia. Blood 2012; 119: 3571–3578.
De Leeuw DC, Denkers F, Olthof MC, Rutten AP, Pouwels W, Schuurhuis GJ et al. Attenuation of microRNA-126 expression that drives CD34+38− stem/progenitor cells in acute myeloid leukemia leads to tumor eradication. Cancer Res 2014; 74: 2094–2105.
Moshaver B, van Rhenen A, Kelder A, van der Pol M, Terwijn M, Bachas C et al. Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia. Stem Cells 2008; 26: 3059–3067.
Zeijlemaker W, Kelder A, Wouters R, Valk PJM, Witte BI, Cloos J et al. Absence of leukaemic CD34(+) cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered. Br J Haematol 2015; e-pub ahead of print 24 June 2015 doi:10.1111/bjh.13572.
Majeti R, Chao MP, Alizadeh AA, Pang WW, Gibbs KD Jr, Van Rooijen N et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 2009; 138: 286–299.
Bachas C, Schuurhuis GJ, Assaraf YG, Kwidama ZJ, Kelder A, Wouters F et al. The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse. Leukemia 2012; 26: 1313–1320.
Ding L, Ley TJ, Larson DE, Miller CA, Koboldt DC, Welch JS et al. Clonal evolution in relapsed acute myeloid leukemia revealed by whole genome sequencing. Nature 2012; 481: 506–510.
Welch JS, Ley TJ, Link DC, Miller CA, Larson DE, Koboldt DC et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 2012; 150: 264–278.
Zeijlemaker W, Gratama JW, Schuurhuis GJ . Tumor heterogeneity makes AML a “moving target” for detection of residual disease. Cytometry B Clin Cytom 2014; 86: 3–14.
Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature 2014; 506: 328–333.
Corces-Zimmerman MR, Majeti R . Pre-leukemic evolution of hematopoietic stem cells: the importance of early mutations in leukemogenesis. Leukemia 2014; 28: 2276–2282.
Valent P, Bonnet D, De Maria R, Lapidot T, Copland M, Melo JV et al. Cancer stem cell definitions and terminology: the devil is in the details. Nat Rev Cancer 2012; 12: 767–775.
Pandolfi A, Barreyro L, Steidl U . Concise review: preleukemic stem cells: molecular biology and clinical implications of the precursors to leukemia stem cells. Stem Cells Transl Med 2013; 2: 143–150.
Van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 2005; 11: 6520–6527.
Corces-Zimmerman MR, Hong W-J, Weissman IL, Medeiros BC, Majeti R . Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proc Natl Acad Sci USA 2014; 111: 2548–2553.
Jan M, Snyder TM, Corces-Zimmerman MR, Vyas P, Weissman IL, Quake SR et al. Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia. Sci Transl Med 2012; 4: 149ra118.
Van der Pol MA, Feller N, Roseboom M, Moshaver B, Westra G, Broxterman HJ et al. Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells. Haematologica 2003; 88: 983–993.
Wulf GG, Wang R, Kuehnle I, Weidner D, Marini F, Brenner MK et al. A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood 2001; 98: 1166–1174.
Acknowledgements
We thank all participating study centers for including their patients in the HOVON trials.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
Financial support for part of this research was received by BD Biosciences.
Additional information
Supplementary Information accompanies this paper on the Leukemia website
Supplementary information
Rights and permissions
About this article
Cite this article
Zeijlemaker, W., Kelder, A., Oussoren-Brockhoff, Y. et al. A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia. Leukemia 30, 439–446 (2016). https://doi.org/10.1038/leu.2015.252
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/leu.2015.252
This article is cited by
-
Immunophenotypic aberrant hematopoietic stem cells in myelodysplastic syndromes: a biomarker for leukemic progression
Leukemia (2023)
-
Genomic analysis of cellular hierarchy in acute myeloid leukemia using ultrasensitive LC-FACSeq
Leukemia (2021)
-
Flow Cytometric Minimal Residual Disease Analysis in Acute Leukemia: Current Status
Indian Journal of Hematology and Blood Transfusion (2020)
-
Liquid biopsy for minimal residual disease detection in leukemia using a portable blast cell biochip
npj Precision Oncology (2019)