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:

Minimal Residual Disease in ALL

Analysis of minimal residual disease in childhood acute lymphoblastic leukemia: comparison between RQ-PCR analysis of Ig/TcR gene rearrangements and multicolor flow cytometric immunophenotyping

Leukemia volume 18, pages 1630–1636 (2004)Cite this article

Abstract

Detection of minimal residual disease (MRD) in follow-up samples from patients with ALL is essential for evaluation of treatment response. We applied multicolor flow cytometry and real-time quantitative PCR (RQ-PCR) to compare MRD results in 71 follow-up samples from 22 children treated for ALL. When results obtained by flow cytometry and RQ-PCR were grouped into positive–negative categories, a significant level of agreement was found in 72% of samples (P<0.001). However, if a cutoff level of 0.01% was applied, the concordance was 89%. MRD could be quantified in 19 samples by both methods, showing a strong correlation (P<0.01). Nevertheless, MRD levels differed more than five-fold between both methods in 4/19 samples. In 20 (28%) samples, the two techniques showed discordant results. Most discordant results (17/20) were due to the limited sensitivity of flow cytometry analysis within the range 0.01–0.001%; remaining discordant results were due to the instable or subclonal IG/TCR gene rearrangements or a limited quantitative range of the applied RQ-PCR targets. Although concordant results could be obtained by flow cytometry and RQ-PCR analysis, MRD levels may differ. Therefore, MRD data obtained by these two techniques are not yet easily exchangeable.

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

Similar content being viewed by others

References

  1. Eckert C, Biondi A, Seeger K, Cazzaniga G, Hartmann R, Beyermann B et al. Prognostic value of minimal residual disease in relapsed childhood acute lymphoblastic leukaemia. Lancet 2001; 358: 1239–1241.

    Article  CAS  Google Scholar 

  2. Cave H, van der Werff ten Bosch J, Suciu S, Guidal C, Waterkeyn C, Otten J et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer – Childhood Leukemia Cooperative Group. N Engl J Med 1998; 339: 591–598.

    Article  CAS  Google Scholar 

  3. van Dongen JJ, Seriu T, Panzer-Grumayer ER, Biondi A, Pongers-Willemse MJ, Corral L et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998; 352: 1731–1738.

    Article  CAS  Google Scholar 

  4. Coustan-Smith E, Sancho J, Hancock ML, Boyett JM, Behm FG, Raimondi SC et al. Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood 2000; 96: 2691–2696.

    CAS  Google Scholar 

  5. Dworzak MN, Froschl G, Printz D, Mann G, Potschger U, Muhlegger N et al. Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood 2002; 99: 1952–1958.

    Article  CAS  Google Scholar 

  6. Bjorklund E, Mazur J, Soderhall S, Porwit-MacDonald A . Flow cytometric follow-up of minimal residual disease in bone marrow gives prognostic information in children with acute lymphoblastic leukemia. Leukemia 2003; 17: 138–148.

    Article  CAS  Google Scholar 

  7. Willemse MJ, Seriu T, Hettinger K, d'Aniello E, Hop WC, Panzer-Grumayer ER et al. Detection of minimal residual disease identifies differences in treatment response between T-ALL and precursor B-ALL. Blood 2002; 99: 4386–4393.

    Article  CAS  Google Scholar 

  8. Szczepanski T, Flohr T, van der Velden VH, Bartram CR, van Dongen JJ . Molecular monitoring of residual disease using antigen receptor genes in childhood acute lymphoblastic leukaemia. Best Pract Res Clin Haematol 2002; 15: 37–57.

    Article  CAS  Google Scholar 

  9. Szczepanski T, Orfao A, van der Velden VH, San Miguel JF, van Dongen JJ . Minimal residual disease in leukaemia patients. Lancet Oncol 2001; 2: 409–417.

    Article  CAS  Google Scholar 

  10. van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ . Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 2003; 17: 1013–1034.

    Article  CAS  Google Scholar 

  11. Vidriales MB, San Miguel JF, Orfao A, Coustan-Smith E, Campana D . Minimal residual disease monitoring by flow cytometry. Best Pract Res Clin Haematol 2003; 16: 599–612.

    Article  Google Scholar 

  12. Dworzak MN, Panzer-Grumayer ER . Flow cytometric detection of minimal residual disease in acute lymphoblastic leukemia. Leuk Lymphoma 2003; 44: 1445–1455.

    Article  Google Scholar 

  13. van der Velden VH, Wijkhuijs JM, Jacobs DC, van Wering ER, van Dongen JJ . T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis. Leukemia 2002; 16: 1372–1380.

    Article  CAS  Google Scholar 

  14. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999; 13: 1901–1928.

    Article  CAS  Google Scholar 

  15. Cazzaniga G, Rossi V, Biondi A . Monitoring minimal residual disease using chromosomal translocations in childhood ALL. Best Pract Res Clin Haematol 2002; 15: 21–35.

    Article  CAS  Google Scholar 

  16. Gabert J, Beillard E, van der Velolen 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  Google Scholar 

  17. Dworzak MN . Immunological detection of minimal residual disease in acute lymphoblastic leukemia. Onkologie 2001; 24: 442–448.

    CAS  PubMed  Google Scholar 

  18. Campana D, Coustan-Smith E . Advances in the immunological monitoring of childhood acute lymphoblastic leukaemia. Best Pract Res Clin Haematol 2002; 15: 1–19.

    Article  Google Scholar 

  19. Neale GA, Coustan-Smith E, Pan Q, Chen X, Gruhn B, Stow P et al. Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia 1999; 13: 1221–1226.

    Article  CAS  Google Scholar 

  20. Dworzak MN, Fritsch G, Panzer-Grumayer ER, Mann G, Gadner H . Detection of residual disease in pediatric B-cell precursor acute lymphoblastic leukemia by comparative phenotype mapping: method and significance. Leuk Lymphoma 2000; 38: 295–308.

    Article  CAS  Google Scholar 

  21. Malec M, Bjorklund E, Soderhall S, Mazur J, Sjogren AM, Pisa P et al. Flow cytometry and allele-specific oligonucleotide PCR are equally effective in detection of minimal residual disease in ALL. Leukemia 2001; 15: 716–727.

    Article  CAS  Google Scholar 

  22. Veltroni M, De Zen L, Sanzari MC, Maglia O, Dworzak MN, Ratei R et al. Expression of CD58 in normal, regenerating and leukemic bone marrow B cells: implications for the detection of minimal residual disease in acute lymphocytic leukemia. Haematologica 2003; 88: 1245–1252.

    PubMed  Google Scholar 

  23. Gustafsson G, Schmiegelow K, Forestier E, Clausen N, Glomstein A, Jonmundsson G et al. Improving outcome through two decades in childhood ALL in the Nordic countries: the impact of high-dose methotrexate in the reduction of CNS irradiation. Nordic Society of Pediatric Haematology and Oncology (NOPHO). Leukemia 2000; 14: 2267–2275.

    Article  CAS  Google Scholar 

  24. Jaffe E, Harris NL, Stein H, Vardiman J (eds). Tumours of Haematopoietic and Lymphatic Tissues. Lyon: IARC Press, 2001.

    Google Scholar 

  25. Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A et al. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia 1995; 9: 1783–1786.

    CAS  Google Scholar 

  26. Verhagen OJ, Willemse MJ, Breunis WB, Wijkhuijs AJ, Jacobs DC, Joosten SA et al. Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia. Leukemia 2000; 14: 1426–1435.

    Article  CAS  Google Scholar 

  27. van der Velden VH, Willemse MJ, van der Schoot CE, Hahlen K, van Wering ER, van Dongen JJ . Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR. Leukemia 2002; 16: 928–936.

    Article  CAS  Google Scholar 

  28. Pongers-Willemse MJ, Seriu T, Stolz F, d'Aniello E, Gameiro P, Pisa P et al. Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia. Leukemia 1999; 13: 110–118.

    Article  CAS  Google Scholar 

  29. Pongers-Willemse MJ, Verhagen OJ, Tibbe GJ, Wijkhuijs AJ, de Haas V, Roovers E et al. Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes. Leukemia 1998; 12: 2006–2014.

    Article  CAS  Google Scholar 

  30. Szczepanski T, van der Velden VHJ, van Dongen JJ . Real-time quantitative (RQ)-PCR for the detection of minimal residual disease in childhood acute lymphoblastic leukemia. Haematologica 2002; 87 (Suppl 1): 183–191.

    Google Scholar 

  31. Lucio P, Parreira A, van den Beemd MW, van Lochem EG, van Wering ER, Baars E et al. Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL. Leukemia 1999; 13: 419–427.

    Article  CAS  Google Scholar 

  32. Porwit-MacDonald A, Bjorklund E, Lucio P, van Lochem EG, Mazur J, Parreira A et al. BIOMED-1 concerted action report: flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL). Leukemia 2000; 14: 816–825.

    Article  CAS  Google Scholar 

  33. Nyvold C, Madsen HO, Ryder LP, Seyfarth J, Svejgaard A, Clausen N et al. Precise quantification of minimal residual disease at day 29 allows identification of children with acute lymphoblastic leukemia and an excellent outcome. Blood 2002; 99: 1253–1258.

    Article  CAS  Google Scholar 

  34. Borowitz MJ, Pullen DJ, Shuster JJ, Viswanatha D, Montgomery K, Willman CL et al. Minimal residual disease detection in childhood precursor-B-cell acute lymphoblastic leukemia: relation to other risk factors. A Children's Oncology Group study. Leukemia 2003; 17: 1566–1572.

    Article  CAS  Google Scholar 

  35. Coustan-Smith E, Sancho J, Hancock ML, Razzouk BI, Ribeiro RC, Rivera GK et al. Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood 2002; 100: 2399–2402.

    Article  CAS  Google Scholar 

  36. Coustan-Smith E, Behm FG, Sanchez J, Boyett JM, Hancock ML, Raimondi SC et al. Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 1998; 351: 550–554.

    Article  CAS  Google Scholar 

  37. Germano G, del Giudice L, Palatron S, Giarin E, Cazzaniga G, Biondi A et al. Clonality profile in relapsed precursor-B-ALL children by GeneScan and sequencing analyses. Consequences on minimal residual disease monitoring. Leukemia 2003; 17: 1573–1582.

    Article  CAS  Google Scholar 

  38. Li A, Zhou J, Zuckerman D, Rue M, Dalton V, Lyons C et al. Sequence analysis of clonal immunoglobulin and T-cell receptor gene rearrangements in children with acute lymphoblastic leukemia at diagnosis and at relapse: implications for pathogenesis and for the clinical utility of PCR-based methods of minimal residual disease detection. Blood 2003; 102: 4520–4526.

    Article  CAS  Google Scholar 

  39. Szczepanski T, Willemse MJ, Brinkhof B, van Wering ER, van der Burg M, van Dongen JJ . Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B-ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease. Blood 2002; 99: 2315–2323.

    Article  CAS  Google Scholar 

  40. Szczepanski T, van der Velden VH, Raff T, Jacobs DC, van Wering ER, Bruggemann M et al. Comparative analysis of T-cell receptor gene rearrangements at diagnosis and relapse of T-cell acute lymphoblastic leukemia (T-ALL) shows high stability of clonal markers for monitoring of minimal residual disease and reveals the occurrence of second T-ALL. Leukemia 2003; 17: 2149–2156.

    Article  CAS  Google Scholar 

  41. Bierings M, Szczepanski T, van Wering ER, Willemse MJ, Langerak AW, Revesz T et al. Two consecutive immunophenotypic switches in a child with immunogenotypically stable acute leukaemia. Br J Haematol 2001; 113: 757–762.

    Article  CAS  Google Scholar 

  42. Szczepanski T, der Velden VH, Hoogeveen PG, De Bie M, Jacobs DC, Van Wering ER et al. V{delta}2-J{alpha} gene rearrangements are frequent in precursor-B-acute lymphoblastic leukemia but rare in normal lymphoid cells. Blood 2003, (Epub ahead of print).

  43. Bruggemann M, van der Velden VH, Raff T, Droese J, Ritgen M, Pott C et al. Rearranged T-cell receptor beta genes represent powerful targets for quantification of minimal residual disease in childhood and adult T-cell acute lymphoblastic leukemia. Leukemia 2004; 18: 709–719.

    Article  CAS  Google Scholar 

  44. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 2257–2317.

    Article  CAS  Google Scholar 

  45. Coustan-Smith E, Sancho J, Behm FG, Hancock ML, Razzouk BI, Ribeiro RC et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood 2002; 100: 52–58.

    Article  CAS  Google Scholar 

  46. Campana D . Determination of minimal residual disease in leukaemia patients. Br J Haematol 2003; 121: 823–838.

    Article  Google Scholar 

  47. Neale GAM, Coustan-SMith E, Stow P, Pam Q, Chen X, Pui C-H et al. Comparative analysis of polymerase chain reaction and flow cytometry for the detection of miniomal residual disease in childhood acute lymphoblastic leukemia. Leukemia 2004; 18: 934–938.

    Article  CAS  Google Scholar 

  48. Basso G, Buldini B, De Zen L, Orfao A . New methodologic approaches in immunophenotyping acute leukemias. Hematologica 2001; 86: 675–692.

    CAS  Google Scholar 

  49. Szczepanski T, Pongers-Willemse MJ, Langerak AW, van Dongen JJ . Unusual immunoglobulin and T-cell receptor gene rearrangement patterns in acute lymphoblastic leukemias. Curr Top Microbiol Immunol 1999; 246: 205–213, discussion 214–215.

    CAS  PubMed  Google Scholar 

  50. Rosenquist R, Thunberg U, Li AH, Forestier E, Lonnerholm G, Lindh J et al. Clonal evolution as judged by immunoglobulin heavy chain gene rearrangements in relapsing precursor-B acute lymphoblastic leukemia. Eur J Haematol 1999; 63: 171–179.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by grants from The Swedish Children's Cancer Foundation and Stockholḿs County Council. The excellent technical assistance of Marianne Lestrin, Britt Lundh, Shalah Tarahumi, Margareta Söderqvist and Margareta Waern is gratefully acknowledged. We thank Professor Dr Jacques JM van Dongen for his continuous support and for critically reviewing the manuscript.

Author information

Authors and Affiliations

  1. Department of Pathology, Astrid Lindgren Child Hospital, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden

    M Malec, E Björklund & A Porwit-MacDonald

  2. Department of Hematology, Center for Molecular Medicine, Astrid Lindgren Child Hospital, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden

    M Malec & M Björkholm

  3. Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

    V H J van der Velden & J M Wijkhuijs

  4. Childhood Cancer Research Unit, Astrid Lindgren Child Hospital, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden

    S Söderhäll

  5. Department of Epidemiology, Institute of Mother and Child Health, Warsaw, Poland

    J Mazur

Authors
  1. M Malec
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V H J van der Velden
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E Björklund
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J M Wijkhuijs
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S Söderhäll
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J Mazur
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M Björkholm
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A Porwit-MacDonald
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M Malec.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Malec, M., van der Velden, V., Björklund, E. et al. Analysis of minimal residual disease in childhood acute lymphoblastic leukemia: comparison between RQ-PCR analysis of Ig/TcR gene rearrangements and multicolor flow cytometric immunophenotyping. Leukemia 18, 1630–1636 (2004). https://doi.org/10.1038/sj.leu.2403444

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2403444

Keywords

This article is cited by

Search

Advanced search

Quick links