Annals of Hematology

, Volume 66, Issue 5, pp 225–233 | Cite as

Proliferation patterns in acute myeloid leukemia: leukemic clonogenic growth and in vivo cell cycle kinetics

  • P. P. T. Brons
  • C. Haanen
  • J. B. M. Boezeman
  • P. Muus
  • R. S. G. Holdrinet
  • A. H. M. Pennings
  • H. M. C Wessels
  • T. de Witte
Original Article


In a prospective study of 33 newly diagnosed patients with acute myeloid leukemia (AML), we analyzed the relationship of proliferation parameters with clinical parameters, response to induction therapy, and survival. The median follow-up was 26 months. The proliferative capacity of the leukemic progenitor cells was studied using colony-forming assays (number of colonyforming units, growth pattern, and spontaneous clonogenic growth capacity). The cell kinetic parameters of the bone marrow blasts were determined by in vivo labeling with iododeoxyuridine and subsequent flow cytometry: labeling index (LI), DNA synthesis time (Ts), potential doubling time. No or only weak relationships were observed between the experimental and clinical parameters such as age, sex, % blasts, white blood cell count, FAB subtype, cytogenetics, and % CD 34+ cells. This suggests that clonogenic growth and cell cycle kinetics of bone marrow blasts are independent cell biologic properties of AML. No association between the proliferation parameters and induction response rate was noticed. Analysis of the overall survival and event-free survival revealed trends to longer survival rates in patients with a belowmedian LI (≤7.6%) and below-median Ts value (≤14.3 h). These trends were more pronounced in the group of de novo AML (n=23), where the prolonged event-free survival in patients with below-median Ts reached statistical significance (p=0.02). None of the other parameters appeared significantly correlated with survival, although there was a trend to longer survival rates in patients who had no spontaneous clonogenic growth capacity (p=0.13). In conclusion, proliferation parameters in leukemic cells provide additional information on the cell biologic characteristics of AML, and these parameters may have prognostic value for response and duration of survival in AML.

Key words

Flow cytometry Cell kinetics Iododeoxyuridine Clonogenic assay DNA synthesis time 


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  1. 1.
    Barlogie B, Maddox A, Johnston DA, Raber MN, Drewinko B, Keating MJ, Freireich EJ (1983) Quantitative cytology in leukemia research. Blood Cells 9: 35–55Google Scholar
  2. 2.
    Begg AC, McNally NJ, Shrieve DC, Kärcher H (1985) A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6: 620–626Google Scholar
  3. 3.
    Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1976) Proposals for the classification of the acute leukemias. Br J Haematol 33: 451–458Google Scholar
  4. 4.
    Beran M, Reizenstein P, Uden AM (1980) Response to treatment in acute nonlymphatic leukaemia: prognostic value of colony-forming and colony-stimulating capacities of bone marrow and blood cells compared to other parameters. Br J Haematol 44: 39–50Google Scholar
  5. 5.
    Bettelheim P, Valent P, Andreeff M, Tafuri A, Haimi J, Gorischek C, Muhm M, Sillaber C, Haas O, Vieder L, Maurer D, Schulz G, Speiser W, Geissler K, Kier P, Hinterberger W, Lechner K (1991) Recombinant human granulocyte-macrophage colony-stimulating factor in combination with standard induction chemotherapy in de novo acute myeloid leukemia. Blood 77: 700–711Google Scholar
  6. 6.
    Bokhari SAJ, Abbas A, Yousuf N, Mehdi A, Umerani A, Quadir K, Sheikh Y, Akhtar S, Chughtai S, Preisler H, Raza A (1992) Cell cycle parameters as biological predictors of prognosis in AML: a review and update of cell cycle kinetics and remission induction/duration in acute leukemia. Leukemia Lymphoma 6: 197–207Google Scholar
  7. 7.
    Brons PPT, Pennings AHM, Haanen C, Wessels HMC, Boezeman JBM (1990) Simultaneous measurement of DNA content and cell-surface immunofluorescence of human bone marrow cells using a single laser flow cytometer. Cytometry 11: 837–844Google Scholar
  8. 8.
    Brons PPT, Raemaekers JMM, Bogman MJJT, van Erp PEJ, Boezeman JBM, Pennings AHM, Wessels HMC, Haanen C (1992) Cell cycle kinetics in malignant lymphoma studied with in vivo iododeoxyuridine administration, nuclear Ki-67 staining, and flow cytometry. Blood 80: 2336–2343Google Scholar
  9. 9.
    Büchner T, Hiddemann W, Wörmann B, Rottmann R, Zühlsdorf M, Koeningsmann M, Buntkirchen K, Sander A, Frisch J, Schulz G (1991) Recombinant human GM-CSF priming and long-term administration together with multiple-course chemotherapy (CT) vs CT alone in newly diagnosed acute myeloid leukemia. Blood 78 [Suppl 1]: 163aGoogle Scholar
  10. 10.
    Biihring HJ, Asenbauer B, Katrilaka K, Hummel G, Busch FW (1989) Sequential expression of CD34 and CD33 antigens on myeloid colony-forming cells. Eur J Haematol 42: 143–149Google Scholar
  11. 11.
    Catovsky D, Matutes E, Buccheri V, Shetty V, Hanslip J, Yoshida N, Morilla R (1991) A classification of acute leukaemia for the 1990s. Ann Hematol 62: 16–21Google Scholar
  12. 12.
    Civin CI, Banquerigo ML, Strauss LC, Loken MR (1987) Antigenic analysis of hematopoiesis. VI. Flow cytometric characterization of My-10-positive progenitor cells in normal human bone marrow. Exp Hematol 15: 10–17Google Scholar
  13. 13.
    Clarkson B, Strife A, Fried J, Sakai Y, Ota K, Ohkita T, Masuda R (1970) Studies of cellular proliferation in human leukemia. IV. Behavior of normal hematopoietic cells in 3 adults with acute leukemia given continuous infusions of 3 H-thymidine for 8 or 10 days. Cancer 26: 1–19Google Scholar
  14. 14.
    Coiffier B, Bryon PA, Fière D, Felman P, Gentilhomme O, Vu Van H, Viala JJ, Germain D (1982) Agar culture of bone marrow cells in acute myeloid leukemia and dysmyelopoietic syndromes. Reevaluation of its prognostic value. Nouv Ref Fr Hematol 24: 13–18Google Scholar
  15. 15.
    Crowther D, Beard MEJ, Bateman CJT, Sewell RL (1975) Factors influencing prognosis in adults with acute myelogenous leukaemia. Br J Cancer 32: 456–464Google Scholar
  16. 16.
    Delmer A, Marie JP, Thevenin D, Cadiou M, Viguié F, Zittoun R (1989) Multivariate analysis of prognostic factors in acute myeloid leukemia: value of clonogenic leukemic cell properties. J Clin Oncol 7: 738–746Google Scholar
  17. 17.
    Dolbaere F, Gratzner H, Pallavicini MG, Gray JW (1983) Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci USA 80: 5573–5577Google Scholar
  18. 18.
    Dow LW, Mirro J, Dahl GV, Kalwinsky DK, Hancock ML, Nash MB, Roberson PK (1987) Comparison of two clonogenic assays for determining cell growth patterns in childhood acute myeloid leukemia: relationship to clinical findings and prognosis. Leukem Res 11: 565–573Google Scholar
  19. 19.
    Erp PEJ Van, Brons PPT, Boezeman JBM, De Jongh GJ, Bauer FW (1988) A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation. Cytometry 9: 627–630Google Scholar
  20. 20.
    Gratzner HG (1982) Monoclonal antibody to 5-bromoand 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218: 474–475Google Scholar
  21. 21.
    Greenberg ML, Chanana AD, Cronkite EP, Giacomelli G, Rai KR, Schiffer LM, Stryckmans PA, Vincent PC (1972) The generation time of human leukemic myeloblasts. Lab Invest 26: 245–252Google Scholar
  22. 22.
    Haanen C (1975) Pulse cytophotometry (leading article). Lancet 1: 435–436Google Scholar
  23. 23.
    Hart JS, George SL, Frei III E, Bodey GP, Nickerson RC, Freireich EJ (1977) Prognostic significance of pretreatment proliferative activity in adult leukemia. Cancer 39: 1603–1617Google Scholar
  24. 24.
    Herrmann F, Mertelsmann R (1989) Polypeptides controlling hematopoietic cell development and activation. I. In vitro results. Blut 58: 117–128Google Scholar
  25. 25.
    Hiddeman W, Büchner T, Andreeff M, Wörmann B, Melamed MR, Clarkson BD (1982) Cell kinetics in acute leukemia. A critical reevaluation based on new data. Cancer 50: 250–258Google Scholar
  26. 26.
    Hillen H, Wessels J, Haanen C (1975) Bone-marrow-proliferation patterns in acute myeloblastic leukaemia determined by pulse cytophotometry. Lancet 2: 609–611Google Scholar
  27. 27.
    Holdrinet RSG, v. Egmond J, Wessels JMC, Haanen C (1980) A method for quantification of peripheral blood admixture in bone marrow aspirates. Exp Hematol 8: 103–107Google Scholar
  28. 28.
    Holdrinet RSG, Pennings A, Drenthe-Schonk AM, v. Egmond J, Wessels JMC, Haanen C (1981) Flow cytometric determination of the S-phase compartment in bone marrow of adult patients with acute leukemia. Acta Haematol 70: 369–378Google Scholar
  29. 29.
    Kantarjian HM, Barlogie B, Keating MJ, Hall RR, Smith TL, McCredie KB, Freireich EJ (1985) Pretreatment cytokinetics in acute myelogenous leukemia. Age-related prognostic implications. J Clin Invest 76: 319–324Google Scholar
  30. 30.
    Killmann SA, Cronkite EP, Robertson JS, Fliedner TM, Bond VP (1963) Estimation of phases of the life cycle of leukemic cells from labeling in human beings in vivo with tritiated thymidine. Lab Invest 12: 671–684Google Scholar
  31. 31.
    Kinsella TJ, Dobson PP, Mitchell BS, Mitchell JB (1986) Interaction of iododeoxyuridine and its primary metabolite, iodouracil on radiation response. Int J Rad Oncol Biol Phys 12: 1519–1522Google Scholar
  32. 32.
    Koury MJ (1992) Programmed cell death (apoptosis) in hematopoiesis. Exp Hematol 20: 391–394Google Scholar
  33. 33.
    Lely N Van Der, Witte T De, Muus P, Raymakers R, Preijers F, Haanen C (1991) Prolonged exposure to cytosine arabinoside in the presence of hematopoietic growth factors preferentially kills leukemic versus normal clonogenic cells. Exp Hematol 19: 267–272Google Scholar
  34. 34.
    Lotem J, Cragoe EJ, Sachs L (1991) Rescue from programmed cell death in leukemic and normal myeloid cells. Blood 78: 953–960Google Scholar
  35. 35.
    Marie JP, Zittoun R, Thevenin D, Mathieu M, Viguie F (1983) In vitro culture of clonogenic leukaemic cells in acute myeloid leukaemia: growth pattern and drug sensitivity. Br J Haematol 55: 427–437Google Scholar
  36. 36.
    Matthews JH (1988) Cell cycle characteristics in acute nonlymphocytic leukemia. Blood 71: 532–534Google Scholar
  37. 37.
    Metcalf D (1991) Control of granulocytes and macrophages — molecular, cellular, and clinical aspects. Science 254: 529–533Google Scholar
  38. 38.
    Moore MAS, Spitzer G, Williams N, Metcalf D, Buckley J (1974) Agar culture studies in 127 cases of untreated acute leukemia: the prognostic value of reclassification of leukemia according to in vitro growth characteristics. Blood 44: 1–18Google Scholar
  39. 39.
    Morstyn G, Burgess AW (1988) Hematopoietic growth factors: review. Cancer Res 48: 5624–5637Google Scholar
  40. 40.
    Müller MR, Hofman V, Koller A, Erni J (1987) Prognostic significance of agar and liquid cultures in AML patients before treatment, early postinduction and in remission. Leukem Res 11: 17–23Google Scholar
  41. 41.
    Preisler HD, Azarnia N, Raza A, Grunwald H, Vogler R, Browman G, Goldberg J, Chervenick P, Miller K, Brennan J, Winton L, Joyce R, Gottlieb A (1984) Relationship between the percent of marrow cells in S-phase and the outcome of remission-induction therapy for acute nonlymphocytic leukaemia. Br J Haematol 56: 399–407Google Scholar
  42. 42.
    Raza A, Yasin Z, Grande C (1988) A comparison of the rate of DNA synthesis in myeloblasts from peripheral blood and bone marrows of patients with acute nonlymphocytic leukemia. Exp Cell Res 176: 13–19Google Scholar
  43. 43.
    Raza A, Preisler HD, Day R, Yasin Z, White M, Lykins J, Kukla C, Barcos M, Bennett J, Browman G, Goldberg J, Grun-wald H, Larson R, Vardiman J, Vogler R (1990) Direct relationship between remission duration in acute myeloid leukemia and cell cycle kinetics: a Leukemia Intergroup Study. Blood 76: 2191–2197Google Scholar
  44. 44.
    Riccardi A, Giordano M, Danova M, Girino M, Brugnatelli S, Ucci G, Mazzini G (1991) Cell kinetics with in vivo bromodeoxyuridine and flow cytometry: clinical significance in acute non-lymphoblastic leukaemia. Eur J Cancer 27: 882–887Google Scholar
  45. 45.
    Schattenberg A, De Witte T, Preijers F, Raemaekers J, Muus P, Van Der Lely N, Boezeman J, Wessels J, Van Dijk B, Hoogen-hout J, Haanen C (1990) Allogeneic bone marrow transplantation for leukemia with marrow grafts depleted of lymphocytes by counterflow centrifugation. Blood 75: 1356–1363Google Scholar
  46. 46.
    Smaaland R, Svardal AM, Lote K, Ueland PM, Laerum OD (1991) Glutathione content in human bone marrow and circadian stage relation to DNA synthesis. J Natl Cancer Inst 83: 1092–1098Google Scholar
  47. 47.
    Stryckmans P, Cronkite EP, Fache J, Fliedner TM, Ramos J (1966) Deoxyribonucleic acid synthesis time of erythropoietic and granulopoietic cells in human beings. Nature 211: 717–720Google Scholar
  48. 48.
    Terstappen LWMM, Safford M, Konemann S, Loken MR, Zurlutter K, Buchner T, Hiddemann W, Wormann B (1991) Flow cytometric characterization of acute myeloid leukemia. 2. Phenotypic heterogeneity at diagnosis. Leukemia 5: 757–767Google Scholar
  49. 49.
    Vanderlaan M, Watkins B, Thomas C, Dolbaere F, Stanker L (1986) Improved high-affinity monoclonal antibody to iododeoxyuridine. Cytometry 7: 499–507Google Scholar
  50. 50.
    Wickramasinghe SN, Chalmers DG, Cooper EH (1968) A study of ineffective erythropoiesis in sideroblastic anaemia and erythraemic myelosis. Cell Tissue Kinet 1: 43–50Google Scholar
  51. 51.
    Wiley J, Cebon J, Woodruff R, Jamieson G, Snook M, McKendrick J, Szer J, Dennington P, Sheridan W, Begley G, van der Weyden M, Fox R (1991) Granulocyte-macrophage colonystimulating factor followed by chemotherapy in the treatment of poor-prognosis acute myeloid leukemia (ANLL). Blood 78 [Suppl 1]: 42 aGoogle Scholar
  52. 52.
    Witte T De, Raymakers R, Plas A, Koekman E, Wessels H, Haanen C (1984) Bone marrow repopulation capacity after transplantation of lymphocyte-depleted allogeneic bone marrow using counterflow centrifugation. Transplantation 37: 151–155Google Scholar
  53. 53.
    Young DC, Griffin JD (1986) Autocrine secretion of GM-CSF in acute myeloblastic leukemia. Blood 68: 1178–1181Google Scholar
  54. 54.
    Yousuf N, Yanik GA, George BA, Masterson M, Mazewski CM, White LM, Miller MA, Lampkin BC, Raza A (1991) Comparison of two double-labeling techniques to measure cell cycle kinetics in myeloid leukemias. Anticancer Res 11: 1195–1199Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • P. P. T. Brons
    • 1
  • C. Haanen
    • 1
  • J. B. M. Boezeman
    • 1
  • P. Muus
    • 1
  • R. S. G. Holdrinet
    • 1
  • A. H. M. Pennings
    • 1
  • H. M. C Wessels
    • 1
  • T. de Witte
    • 1
  1. 1.Department of HematologyUniversity Hospsital NijmegenHB NijmegenThe Netherlands

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