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Chronic Lymphocytic Leukemia

Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL

Leukemia volume 27, pages 142–149 (2013)Cite this article

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Abstract

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10−4) level (n=59) and good linearity even at the 0.001–0.01% (10−5–10−4) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

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References

  1. Gribben JG, O’Brien S . Update on therapy of chronic lymphocytic leukemia. J Clin Oncol 2011; 29: 544–550.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Abrisqueta P, Pereira A, Rozman C, Aymerich M, Giné E, Moreno C et al. Improving survival in patients with chronic lymphocytic leukemia (1980–2008): the Hospital Clinic of Barcelona experience. Blood 2009; 114: 2044–2050.

    Article  CAS  PubMed  Google Scholar 

  3. Moreton P, Kennedy B, Lucas G, Leach M, Rassam SMB, Haynes A et al. Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival. J Clin Oncol 2005; 23: 2971–2979.

    Article  CAS  PubMed  Google Scholar 

  4. Hallek M, Fischer K, Fingerle-Rowson G, Fink AM, Busch R, Mayer J et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet 2010; 376: 1164–1174.

    Article  CAS  PubMed  Google Scholar 

  5. Böttcher S, Ritgen M, Fischer K, Stilgenbauer S, Busch RM, Fingerle-Rowson G et al. Minimal residual disease quantification is an independent predictor of progression-free and overall survival in chronic lymphocytic leukemia: a multivariate analysis from the randomized GCLLSG CLL8 trial. J Clin Oncol 2012; doi:10.1200/JCO.2011.36.9348.

    Article  PubMed  Google Scholar 

  6. Böttcher S, Ritgen M, Pott C, Brüggemann M, Raff T, Stilgenbauer S et al. Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation. Leukemia 2004; 18: 1637–1645.

    Article  PubMed  Google Scholar 

  7. Moreno C, Villamor N, Colomer D, Esteve J, Giné E, Muntañola A et al. Clinical significance of minimal residual disease, as assessed by different techniques, after stem cell transplantation for chronic lymphocytic leukemia. Blood 2006; 107: 4563–4569.

    Article  CAS  PubMed  Google Scholar 

  8. Rawstron AC, Villamor N, Ritgen M, Böttcher S, Ghia P, Zehnder JL et al. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia 2007; 21: 956–964.

    Article  CAS  PubMed  Google Scholar 

  9. Lenormand B, Bizet M, Fruchart C, Tilly H, Daliphard S, Thouret F et al. Residual disease in B-cell chronic lymphocytic leukemia patients and prognostic value. Leukemia 1994; 8: 1019–1026.

    CAS  PubMed  Google Scholar 

  10. Vuillier F, Claisse JF, Vandenvelde C, Travade P, Magnac C, Chevret S et al. Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements. Leuk Lymphoma 1992; 7: 195–204.

    Article  CAS  PubMed  Google Scholar 

  11. Robertson LE, Huh YO, Butler JJ, Pugh WC, Hirsch-Ginsberg C, Stass S et al. Response assessment in chronic lymphocytic leukemia after fludarabine plus prednisone: clinical, pathologic, immunophenotypic, and molecular analysis. Blood 1992; 80: 29–36.

    CAS  PubMed  Google Scholar 

  12. Tam CS, O’Brien S, Wierda W, Kantarjian H, Wen S, Do K-A et al. Long-term results of the fludarabine, cyclophosphamide, and rituximab regimen as initial therapy of chronic lymphocytic leukemia. Blood 2008; 112: 975–980.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hillmen P, Skotnicki AB, Robak T, Jaksic B, Dmoszynska A, Wu J et al. Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia. J Clin Oncol 2007; 25: 5616–5623.

    Article  CAS  PubMed  Google Scholar 

  14. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008; 111: 5446–5456.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Catovsky D, Richards S, Matutes E, Oscier D, Dyer MJS, Bezares RF et al. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial. Lancet 2007; 370: 230–239.

    Article  CAS  PubMed  Google Scholar 

  16. Kennedy B, Rawstron A, Carter C, Ryan M, Speed K, Lucas G et al. Campath-1H and fludarabine in combination are highly active in refractory chronic lymphocytic leukemia. Blood 2002; 99: 2245–2247.

    Article  CAS  PubMed  Google Scholar 

  17. Böttcher S, Stilgenbauer S, Busch R, Brüggemann M, Raff T, Pott C et al. Standardized MRD flow and ASO IGH RQ-PCR for MRD quantification in CLL patients after rituximab-containing immunochemotherapy: a comparative analysis. Leukemia 2009; 23: 2007–2017.

    Article  PubMed  Google Scholar 

  18. Bosch F, Abrisqueta P, Villamor N, Terol MJ, González-Barca E, Ferra C et al. Rituximab, fludarabine, cyclophosphamide, and mitoxantrone: a new, highly active chemoimmunotherapy regimen for chronic lymphocytic leukemia. J Clin Oncol 2009; 27: 4578–4584.

    Article  CAS  PubMed  Google Scholar 

  19. Bosch F, Ferrer A, Villamor N, González M, Briones J, González-Barca E et al. Fludarabine, cyclophosphamide, and mitoxantrone as initial therapy of chronic lymphocytic leukemia: high response rate and disease eradication. Clin Cancer Res 2008; 14: 155–161.

    Article  CAS  PubMed  Google Scholar 

  20. Marti GE, Rawstron AC, Ghia P, Hillmen P, Houlston RS, Kay N et al. Diagnostic criteria for monoclonal B-cell lymphocytosis. Br J Haematol 2005; 130: 325–332.

    Article  PubMed  Google Scholar 

  21. Böttcher S, Ritgen M, Buske S, Gesk S, Klapper W, Hoster E et al. Minimal residual disease detection in mantle cell lymphoma: methods and significance of four-color flow cytometry compared to consensus IGH-polymerase chain reaction at initial staging and for follow-up examinations. Haematologica 2008; 93: 551–559.

    Article  PubMed  Google Scholar 

  22. Perez-Andres M, Paiva B, Nieto WG, Caraux A, Schmitz A, Almeida J et al. Human peripheral blood B-cell compartments: a crossroad in B-cell traffic. Cytometry B Clin Cytom 2010; 78 (Suppl 1): S47–S60.

    Article  PubMed  Google Scholar 

  23. Bomberger C, Singh-Jairam M, Rodey G, Guerriero A, Yeager AM, Fleming WH et al. Lymphoid reconstitution after autologous PBSC transplantation with FACS-sorted CD34+ hematopoietic progenitors. Blood 1998; 91: 2588–2600.

    CAS  PubMed  Google Scholar 

  24. Rawstron AC, Kennedy B, Evans PA, Davies FE, Richards SJ, Haynes AP et al. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy. Blood 2001; 98: 29–35.

    Article  CAS  PubMed  Google Scholar 

  25. Le Roy C, Varin-Blank N, Ajchenbaum-Cymbalista F, Letestu R . Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism. Cytometry A 2009; 75: 882–890.

    Article  PubMed  Google Scholar 

  26. Hillmen P, Cohen DR, Cocks K, Pettitt A, Sayala HA, Rawstron AC et al. A randomized phase II trial of fludarabine, cyclophosphamide and mitoxantrone (FCM) with or without rituximab in previously treated chronic lymphocytic leukaemia. Br J Haematol 2011; 152: 570–578.

    Article  CAS  PubMed  Google Scholar 

  27. Logan AC, Gao H, Wang C, Sahaf B, Jones CD, Marshall EL et al. High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment. Proc Natl Acad Sci USA 2011; 108: 21194–21199.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pedreira CE, Costa ES, Almeida J, Fernandez C, Quijano S, Flores J et al. A probabilistic approach for the evaluation of minimal residual disease by multiparameter flow cytometry in leukemic B-cell chronic lymphoproliferative disorders. Cytometry A 2008; 73A: 1141–1150.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to BD Biosciences, particularly Frans Nauwelaers and Lucia Testolin, for provision of antibodies used in this study. PG supported by Cariplo Foundation (Milan, Italy), Program Molecular Clinical Oncology-5 per mille number 9965 and Investigator Grant, Associazione Italiana per la Ricerca sul Cancro (AIRC Milano, Italy), Progetti di Ricerca di Interesse Nazionale (PRIN - Ministry of education, University and Research - MIUR, Rome, Italy) and Ricerca Finalizzata 2010 (Ministry of Health, Rome, Italy); AR supported by LLR (www.leukaemialymphomaresearch.org.uk). We are grateful to Linda Falck, Elke Harbst, Jamileh Hanani, Lada Henseleit, Heike Hinrichs and Abdelmalek Dahmani for excellent technical support.

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Authors and Affiliations

  1. HMDS, St. James’s Institute of Oncology, Leeds Teaching Hospitals, Leeds, UK

    A C Rawstron, H Kartsios, R M de Tute & J Shingles

  2. Epidemiology and Cancer Statistics Group, University of York, York, UK

    A C Rawstron

  3. Second Department of Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

    S Böttcher, M Ritgen & M Kneba

  4. AP-HP, Hôpital Avicenne, Service d’Hématologie Biologique, Bobigny, France

    R Letestu & F Cymbalista

  5. Université Paris Nord, PRES Sorbonne Paris Cité, UMR Inserm U978 «Adaptateurs de Signalisation en Hématologie», UFR SMBH, Bobigny, France

    R Letestu & F Cymbalista

  6. Unitat d'Hematopatologia, Hospital Clínic and Institut d'investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

    N Villamor & E Montserrat

  7. Division of Molecular Oncology and Department of Onco-Hematology, Laboratory of B Cell Neoplasia and Unit of Lymphoid Malignancies, Università Vita-Salute San Raffaele and Ospedale San Raffaele, Milan, Italy

    C Fazi & P Ghia

  8. Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

    C Moreno

  9. Department of Hematology, Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, UK

    K Lin & A R Pettitt

  10. Klinik I für Innere Medizin, University of Cologne, Köln, Germany

    M Hallek

  11. Department of Clinical Haematology, St. James’s Institute of Oncology, Leeds Teaching Hospitals, Leeds, UK

    P Hillmen

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  1. A C Rawstron
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on behalf of the European Research Initiative in CLL (ERIC)

Corresponding author

Correspondence to A C Rawstron.

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Competing interests

The antibodies used for MRD analysis in this study were provided by BD Biosciences. AR has received royalties from BD for an unrelated product (IntraSure). Other companies were contacted at the time of publication of the international standardised approach8 but did not express an interest in providing antibodies for development of MRD detection. BD Biosciences had no scientific input into the project and there were no significant conflicts of interest that could be perceived to bias the work. The remaining authors declare no conflict of interest.

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Rawstron, A., Böttcher, S., Letestu, R. et al. Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL. Leukemia 27, 142–149 (2013). https://doi.org/10.1038/leu.2012.216

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