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.

  • Original Manuscript
  • Published:

MRD

Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method

Leukemia volume 18, pages 1981–1988 (2004)Cite this article

Abstract

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100–1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01–0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Burnett AK, Goldstone AH, Stevens RM, Hann IM, Rees JK, Gray RG et al. Randomised comparison of addition of autologous bone-marrow transplantation to intensive chemotherapy for acute myeloid leukaemia in first remission: results of MRC AML 10 trial. UK Medical Research Council Adult and Children's Leukaemia Working Parties. Lancet 1998; 351: 700–708.

    Article  CAS  PubMed  Google Scholar 

  2. Campana D, Coustan-Smith E . Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 1999; 38: 139–152.

    Article  CAS  PubMed  Google Scholar 

  3. Orfao A, Schmitz G, Brando B, Ruiz-Arguelles A, Basso G, Braylan R et al. Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: current status and future directions. Clin Chem 1999; 45: 1708–1717.

    CAS  PubMed  Google Scholar 

  4. San Miguel JF, Ciudad J, Vidriales MB, Orfao A, Lucio P, Porwit-MacDonald A et al. Immunophenotypical detection of minimal residual disease in acute leukemia. Crit Rev Oncol Hematol 1999; 32: 175–185.

    Article  CAS  PubMed  Google Scholar 

  5. van der Pol MA, Pater JM, Feller N, Westra AH, van Stijn A, Ossenkoppele GJ et al. Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia. Leukemia 2001; 15: 1554–1563.

    Article  CAS  PubMed  Google Scholar 

  6. Venditti A, Tamburini A, Buccisano F, Del Poeta G, Maurillo L, Panetta P et al. Clinical relevance of minimal residual disease detection in adult acute myeloid leukemia. J Hematother Stem Cell Res 2002; 11: 349–357.

    Article  PubMed  Google Scholar 

  7. Venditti A, Maurillo L, Buccisano F, Del Poeta G, Mazzone C, Tamburini A et al. Pretransplant minimal residual disease level predicts clinical outcome in patients with acute myeloid leukemia receiving high-dose chemotherapy and autologous stem cell transplantation. Leukemia 2003; 17: 2178–2182.

    Article  CAS  PubMed  Google Scholar 

  8. Feller N, van der Pol MA, van Stijn A, Weijers G, Westra AH, Evertse BW et al. MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukemia. Leukemia 2004; 18: 1380–1390.

    Article  CAS  PubMed  Google Scholar 

  9. van Stijn A, Kok A, van der Pol MA, Feller N, Roemen GM, Westra AH et al. A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia. Leukemia 2003; 17: 780–786.

    Article  CAS  PubMed  Google Scholar 

  10. van Stijn A, Kok A, van Stalborch MA, 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.

    Article  CAS  PubMed  Google Scholar 

  11. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G . Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002; 30: e57.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Eldering E, Spek CA, Aberson HL, Grummels A, Derks IA, de Vos AF et al. Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 2003; 31: e153.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Feller N, Schuurhuis GJ, van der Pol MA, Westra G, Weijers GW, van Stijn A et al. High percentage of CD34-positive cells in autologous AML peripheral blood stem cell products reflects inadequate in vivo purging and low chemotherapeutic toxicity in a subgroup of patients with poor clinical outcome. Leukemia 2003; 17: 68–75.

    Article  CAS  PubMed  Google Scholar 

  14. Spek CA, Verbon A, Aberson H, Pribble JP, McElgunn CJ, Turner T et al. Treatment with an anti-CD14 monoclonal antibody delays and inhibits lipopolysaccharide-induced gene expression in humans in vivo. J Clin Immunol 2003; 23: 132–140.

    Article  CAS  PubMed  Google Scholar 

  15. Bustin SA . Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25: 169–193.

    Article  CAS  PubMed  Google Scholar 

  16. Pallisgaard N, Clausen N, Schroder H, Hokland P . Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. Genes Chromosomes Cancer 1999; 26: 355–365.

    Article  CAS  PubMed  Google Scholar 

  17. Degan M, Mazzocco FT, Di Francia R, Rossi FM, Pinto A, Gattei V . Normalizing complementary DNA by quantitative reverse transcriptase-polymerase chain reaction of beta2-microglobulin: molecular monitoring of minimal residual disease in acute promyelocytic leukemia. Diagn Mol Pathol 2000; 9: 98–109.

    Article  CAS  PubMed  Google Scholar 

  18. Radich JP, Gooley T, Bryant E, Chauncey T, Clift R, Beppu L et al. The significance of bcr-abl molecular detection in chronic myeloid leukemia patients ‘late,’ 18 months or more after transplantation. Blood 2001; 98: 1701–1707.

    Article  CAS  PubMed  Google Scholar 

  19. Gabert J, Beillard E, Van Der Velden VH, Bi W, Grimwade D, Pallisgaard N et al. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – a Europe Against Cancer program. Leukemia 2003; 17: 2318–2357.

    Article  CAS  PubMed  Google Scholar 

  20. Tamaki H, Mishima M, Kawakami M, Tsuboi A, Kim EH, Hosen N et al. Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1). Int J Hematol 2003; 78: 349–356.

    Article  CAS  PubMed  Google Scholar 

  21. Beillard E, Pallisgaard N, Van Der Velden VH, Bi W, Dee R, Van Der Schoot E et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) – a Europe against cancer program. Leukemia 2003; 17: 2474–2486.

    Article  CAS  PubMed  Google Scholar 

  22. Miller RA, Galecki A, Shmookler-Reis RJ . Interpretation, design, and analysis of gene array expression experiments. J Gerontol A Biol Sci Med Sci 2001; 56: B52–B57.

    Article  CAS  PubMed  Google Scholar 

  23. Lacombe F, Durrieu F, Briais A, Dumain P, Belloc F, Bascans E et al. Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia 1997; 11: 1878–1886.

    Article  CAS  PubMed  Google Scholar 

  24. Vermes I, Haanen C, Reutelingsperger C . Flow cytometry of apoptotic cell death. J Immunol Methods 2000; 243: 167–190.

    Article  CAS  PubMed  Google Scholar 

  25. Breit S, Nees M, Schaefer U, Pfoersich M, Hagemeier C, Muckenthaler M et al. Impact of pre-analytical handling on bone marrow mRNA gene expression. Br J Haematol 2004; 126: 231–243.

    Article  CAS  PubMed  Google Scholar 

  26. Tanner MA, Berk LS, Felten DL, Blidy AD, Bit SL, Ruff DW . Substantial changes in gene expression level due to the storage temperature and storage duration of human whole blood. Clin Lab Haematol 2002; 24: 337–341.

    Article  CAS  PubMed  Google Scholar 

  27. Almeida A, Paul TJ, Magdelenat H, Radvanyi F . Gene expression analysis by real-time reverse transcription polymerase chain reaction: influence of tissue handling. Anal Biochem 2004; 328: 101–108.

    Article  CAS  PubMed  Google Scholar 

  28. Kern W, Danhauser-Riedl S, Ratei R, Schnittger S, Schoch C, Kolb HJ et al. Detection of minimal residual disease in unselected patients with acute myeloid leukemia using multiparameter flow cytometry for definition of leukemia-associated immunophenotypes and determination of their frequencies in normal bone marrow. Haematologica 2003; 88: 646–653.

    PubMed  Google Scholar 

  29. 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.

    CAS  PubMed  Google Scholar 

  30. San Miguel JF, Martinez A, Macedo A, Vidriales MB, Lopez-Berges C, Gonzalez M et al. Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acute myeloid leukemia patients. Blood 1997; 90: 2465–2470.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are indebted to Dr J Berkhof for statistical assistance.

Author information

Authors and Affiliations

  1. Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands

    C J Hess, F Denkers, G J Ossenkoppele, Q Waisfisz & G J Schuurhuis

  2. MRC-Holland, Hudsonstraat, Amsterdam, The Netherlands

    C J McElgunn & J P Schouten

  3. Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands

    E Eldering

Authors
  1. C J Hess
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F Denkers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G J Ossenkoppele
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Q Waisfisz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C J McElgunn
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. E Eldering
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J P Schouten
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. G J Schuurhuis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G J Schuurhuis.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hess, C., Denkers, F., Ossenkoppele, G. et al. Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method. Leukemia 18, 1981–1988 (2004). https://doi.org/10.1038/sj.leu.2403520

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2403520

Keywords

This article is cited by

Search

Advanced search

Quick links