Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Acute Leukemias

Analysis of factors that affect in vitro chemosensitivity of leukaemic stem and progenitor cells to gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukaemia

Abstract

Relapse in acute myeloid leukaemia (AML) is considered to result from the persistence of drug-resistant leukaemic stem and progenitor cells (LSPC) within a bone marrow ‘niche’ microenvironment. Identifying novel agents that have the potential to target these LSPC in their niche microenvironment will aid in the characterization of candidate agents for post-remission chemotherapy. Using an in vitro model, we found that 48-h culture with gemtuzumab ozogamicin (Mylotarg) resulted in a 34% reduction in CD34+CD38CD123+ LSPC number, whereas normal CD34+CD38 haemapoietic stem cells were insensitive to this agent. As there was considerable heterogeneity in LSPC response to Mylotarg treatment, various factors potentially underpinning the differential response were assessed. LSPC that overexpressed CD33 (P=0.01), which were P-glycoprotein-negative (P=0.008) and with internal tandem duplication (ITD) of the FLT3 gene (FLT3/ITD) status (P=0.006) responded better to Mylotarg treatment. LSPC from patient samples that have these combined characteristics as well as low LSPC burden showed significantly more chemosensitivity to Mylotarg compared with all other cases (P=0.002). In multivariate analysis, LSPC burden and FLT3 status were found to be predictors of LSPC chemosensitivity to Mylotarg treatment (P<0.0001). In conclusion, we have shown heterogeneity in the LSPC compartment of AML patients underpinning differential in vitro sensitivity to Mylotarg.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

References

  1. 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.

    CAS  Article  Google Scholar 

  2. Florian S, Sonneck K, Hauswirth AW, Krauth MT, Schernthaner GH, Sperr WR et al. Detection of molecular targets on the surface of CD34+/CD38− stem cells in various myeloid malignancies. Leuk Lymphoma 2006; 47: 207–222.

    CAS  Article  Google Scholar 

  3. Hauswirth AW, Florian S, Printz D, Sotlar K, Krauth MT, Fritsch G et al. Expression of the target receptor CD33 in CD34+/CD38−/CD123+ AML stem cells. Eur J Clin Invest 2007; 37: 73–82.

    CAS  Article  Google Scholar 

  4. 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; 2: 1700–1707.

    Article  Google Scholar 

  5. Mony U, Jawad M, Seedhouse C, Russell N, Pallis M . Resistance to FLT3 inhibition in an in vitro model of primary AML cells with a stem cell phenotype in a defined microenvironment. Leukemia 2008; 22: 1395–1401.

    CAS  Article  Google Scholar 

  6. Stasi R, Evangelista ML, Buccisano F, Venditti A, Amadori S . Gemtuzumab ozogamicin in the treatment of acute myeloid leukemia. Cancer Treat Rev 2008; 34: 49–60.

    CAS  Article  Google Scholar 

  7. Dinndorf PA, Andrews RG, Benjamin D, Ridgway D, Wolff L, Bernstein ID . Expression of normal myeloid-associated antigens by acute leukemia cells. Blood 1986; 67: 1048–1053.

    CAS  Google Scholar 

  8. Griffin JD, Linch D, Sabbath K, Larcom P, Schlossman SF . A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells. Leuk Res 1984; 8: 521–534.

    CAS  Article  Google Scholar 

  9. 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.

    CAS  Article  Google Scholar 

  10. Linenberger ML, Hong T, Flowers D, Sievers EL, Gooley TA, Bennett JM et al. Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood 2001; 98: 988–994.

    CAS  Article  Google Scholar 

  11. Zein N, Sinha AM, McGahren WJ, Ellestad GA . Calicheamicin gamma 1I: an antitumor antibiotic that cleaves double-stranded DNA site specifically. Science 1988; 240: 1198–1201.

    CAS  Article  Google Scholar 

  12. Burnett AK, Kell WJ, Goldstone AH, Milligan D, Hunter A, Prentice AG et al. The addition of gemtuzumab ozogamicin to induction chemotherapy for AML improves disease free survival without extra toxicity: preliminary analysis of 1115 patients in the MRC AML15 trial. Blood 2006; 108 (Abstracts, 13).

  13. Walter RB, Gooley TA, van der Velden VH, Loken MR, van Dongen JJ, Flowers DA et al. CD33 expression and P-glycoprotein-mediated drug efflux inversely correlate and predict clinical outcome in patients with acute myeloid leukemia treated with gemtuzumab ozogamicin monotherapy. Blood 2007; 109: 4168–4170.

    CAS  Article  Google Scholar 

  14. Pallis M, Syan J, Russell NH . Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34. Cytometry 1999; 37: 308–313.

    CAS  Article  Google Scholar 

  15. Pallis M, Das-Gupta E . Flow cytometric measurement of functional and phenotypic P-glycoprotein. Meth Mol Med 2005; 111: 167–181.

    CAS  Google Scholar 

  16. Seedhouse CH, Hunter HM, Lloyd-Lewis B, Massip AM, Pallis M, Carter GI et al. DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412. Leukemia 2006; 20: 2130–2136.

    CAS  Article  Google Scholar 

  17. Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001; 98: 1752–1759.

    CAS  Article  Google Scholar 

  18. Gilliland DG, Griffin JD . The roles of FLT3 in hematopoiesis and leukemia. Blood 2002; 100: 1532–1542.

    CAS  Article  Google Scholar 

  19. Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukaemia. Leukemia 1996; 10: 1911–1918.

    CAS  Google Scholar 

  20. Levis M, Murphy KM, Pham R, Kim KT, Stine A, Li L et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood 2005; 106: 673–680.

    CAS  Article  Google Scholar 

  21. 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.

    CAS  Google Scholar 

  22. Guzman ML, Rossi RM, Neelakantan S, Li X, Corbett CA, Hassane DC et al. An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells. Blood 2007; 110: 4427–4435.

    CAS  Article  Google Scholar 

  23. Walter RB, Raden BW, Hong TC, Flowers DA, Bernstein ID, Linenberger ML . Multidrug resistance protein attenuates gemtuzumab ozogamicin-induced cytotoxicity in acute myeloid leukemia cells. Blood 2003; 102: 1466–1473.

    CAS  Article  Google Scholar 

  24. Morris KL, Adams JA, Liu JA . Effect of Gemtuzumab Ozogamicin on acute myeloid leukemia blast cells in vitro, as a single agent and combined with other cytotoxic cells. Br J Haematol 2006; 135: 509–512.

    CAS  Article  Google Scholar 

  25. Pallis M, Turzanski J, Higashi Y, Russell N . P-glycoprotein in acute myeloid leukaemia: therapeutic implications of its association with both a multidrug-resistant and an apoptosis-resistant phenotype. Leuk Lymphoma 2002; 43: 1221–1228.

    CAS  Article  Google Scholar 

  26. 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.

    CAS  Article  Google Scholar 

  27. Walter RB, Raden BW, Kamikura DM, Cooper JA, Bernstein ID . Influence of CD33 expression levels and ITIM-dependent internalization on gemtuzumab ozogamicin-induced cytotoxicity. Blood 2005; 105: 1295–1302.

    CAS  Article  Google Scholar 

  28. Jedema I, Barge RM, van der Velden VH, Nijmeijer BA, van Dongen JJ, Willemze R et al. Internalization and cell cycle-dependent killing of leukemic cells by gemtuzumab ozogamicin: rationale for efficacy in CD33-negative malignancies with endocytic capacity. Leukemia 2004; 18: 316–325.

    CAS  Article  Google Scholar 

  29. Sievers EL, Larson RA, Stadtmauer EA, Estey E, Löwenberg B, Dombret H et al. Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. J Clin Oncol 2001; 19: 3244–3254.

    CAS  Article  Google Scholar 

  30. van Der Velden VH, te Marvelde JG, Hoogeveen PG, Bernstein ID, Houtsmuller AB, Berger MS et al. Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells. Blood 2001; 97: 3197–3204.

    CAS  Article  Google Scholar 

  31. van Stijn A, Kok A, van Stalborch MA, van Der Pol MA, Feller N, Westra AH et al. Minimal residual disease cells in AML patients have an apoptosis-sensitive protein profile. Leukemia 2004; 18: 875–877.

    CAS  Article  Google Scholar 

  32. Leone G, Rutella S, Voso MT, Fianchi L, Scardocci A, Pagano L . In vivo priming with granulocyte colony-stimulating factor possibly enhances the effect of gemtuzumab-ozogamicin in acute myeloid leukemia: results of a pilot study. Haematologica 2004; 89: 634–636.

    CAS  Google Scholar 

  33. Rutella S, Bonanno G, Procoli A, Mariotti A, Lucia MB, Contemi AM et al. Granulocyte colony-stimulating factor enhances the in vitro cytotoxicity of gemtuzumab ozogamicin against acute myeloid leukemia cell lines and primary blast cells. Exp Hematol 2006; 34: 54–65.

    CAS  Article  Google Scholar 

  34. Balaian L, Ball ED . Cytotoxic activity of gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia correlates with the expression of protein kinase Syk. Leukemia 2006; 20: 2093–2101.

    CAS  Article  Google Scholar 

  35. Jawad M, Russell N, Pallis M . Azacytidine and gemtuzumab ozogamicin co-operate to inhibit the Wnt pathway and increase cytotoxicity in AML. Blood 2008 (ASH Abstract, 2991).

Download references

Acknowledgements

This study was funded by a project grant from the Leukaemia Research Fund. We thank the NCRN AML Working Group for permission to use trial samples.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to M Jawad.

Additional information

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jawad, M., Seedhouse, C., Mony, U. et al. Analysis of factors that affect in vitro chemosensitivity of leukaemic stem and progenitor cells to gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukaemia. Leukemia 24, 74–80 (2010). https://doi.org/10.1038/leu.2009.199

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2009.199

Keywords

  • AML
  • LSPC
  • Mylotarg
  • FLT3
  • chemosensitivity
  • CD33

This article is cited by

Search

Quick links