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(ERIC) Update on the International Harmonised Approach for Flow Cytometric Residual Disease Monitoring In Leukemia (2013) 27, 142–149 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL AC Rawstron1,2,SBo¨ ttcher3, R Letestu4,5, N Villamor6, C Fazi7, H Kartsios1, RM de Tute1, J Shingles1, M Ritgen3, C Moreno8, K Lin9, AR Pettitt9, M Kneba3, E Montserrat6, F Cymbalista4,5, M Hallek10, P Hillmen11 and P Ghia7 on behalf of the European Research Initiative in CLL (ERIC) 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/k/l 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/k/l thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/k/l 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 À 4) level (n ¼ 59) and good linearity even at the 0.001–0.01% (10 À 5–10 À 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. Leukemia (2013) 27, 142–149; doi:10.1038/leu.2012.216 Keywords: chronic lymphocytic leukaemia; minimal residual disease; flow cytometry INTRODUCTION This method is not directly quantitative and has variable sensitivity The outcome of chronic lymphocytic leukaemia (CLL) has changed and specificity. However, in some situations the use of highly remarkably during the last decades1,2 with intensive and/or sensitive MRD flow cytometry may not be necessary, for example, combination therapies now capable of inducing long-lasting when there are still proportionally high levels of CLL cells in the clinical remissions in the majority of patients. As many patients presence of a normal lymphocyte count. In such situations CD19/ achieve complete remissions, the quantification of minimal CD5/k/l alone may be already informative. We therefore aimed at residual disease (MRD) gained importance. MRD levels during determining whether and when CD19/CD5/k/l analysis is and after therapy were shown to be independent predictors of sufficient to definitively confirm or exclude the presence of progression-free and overall survival in CLL.3–5 residual disease in situations where a quantitative result is not Quantification of MRD can be achieved using RQ-ASO IgH-PCR required. Moreover, with MRD data using CD19/CD5/k/l analysis or multi-parameter flow cytometry.6,7 An international available from recent clinical trials and series, it is important to standardised approach for flow cytometric four-CLR analysis has firmly establish the relationship between those analyses and the been developed that is applicable to all sample types and quantitative MRD methods. treatment regimes.8 However, the procedure is time consuming, Conversely, in those cases requiring a comprehensive MRD may be unnecessary for many patients with obvious CLL present evaluation, the increasing availability of cytometers that can and the interpretation of results may be difficult for non- analyse six or more fluorochromes in parallel makes it possible to specialised laboratories. reduce the cost and complexity by developing a protocol using six A simpler flow cytometric approach for MRD detection would or more CLRs. To this purpose we aimed at developing and include an initial screening for CD19/CD5 coexpression analysis standardising a six-CLR antibody panel to reduce sample coupled with clonality assessment using surface k/l expression.9–13 requirements and time taken for acquisition and analysis. 1HMDS, St. James’s Institute of Oncology, Leeds Teaching Hospitals, Leeds, UK; 2Epidemiology and Cancer Statistics Group, University of York, York, UK; 3Second Department of Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; 4AP-HP, Hoˆpital Avicenne, Service d’He´matologie Biologique, Bobigny, France; 5Universite´ Paris Nord, PRES Sorbonne Paris Cite´, UMR Inserm U978 )Adaptateurs de Signalisation en He´matologie*, UFR SMBH, Bobigny, France; 6Unitat d’Hematopatologia, Hospital Clı´nic and Institut d’investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 7Laboratory of B Cell Neoplasia and Unit of Lymphoid Malignancies, Division of Molecular Oncology and Department of Onco-Hematology, Universita` Vita-Salute San Raffaele and Ospedale San Raffaele, Milan, Italy; 8Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 9Department of Hematology, Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, UK; 10Klinik I fu¨r Innere Medizin, University of Cologne, Ko¨ ln, Germany and 11Department of Clinical Haematology, St. James’s Institute of Oncology, Leeds Teaching Hospitals, Leeds, UK. Correspondence: Dr AC Rawstron, HMDS, St. James’s Institute of Oncology, Leeds Teaching Hospitals, Bexley Wing, Beckett Street, Leeds LS1 3EX, UK. E-mail: [email protected] Received 3 May 2012; revised 18 June 2012; accepted 25 June 2012; accepted article preview online 31 July 2012; advance online publication, 5 October 2012 ERIC update on CLL MRD AC Rawstron et al 143 PATIENTS AND METHODS leukocytes, (ii) the percentage of CD19 þ cells expressing CD5, (iii) the Patients percentage of CD19 þ and CD19 þ CD5 þ cells lacking surface immunoglo- bulins and (iv) the k/l ratio on CD19 þ and CD19 þ CD5 þ cells. For A total of 1155 samples from CLL patients were assessed in this study: 784 specificity/sensitivity analysis, the four-CLR flow cytometry MRD assay samples from patients during or after treatment for comparison of CD19/ quantitative result was used as the gold standard, with a CLL cell CD5/k/l analysis with four-CLR MRD flow cytometry; 304 samples from À 4 patients at presentation or relapse sent for routine diagnostic immuno- percentage of leukocytes at or above 0.01% (10 ) classified as positive. phenotyping (270 for hierarchical cluster analysis of surface antigen The PPV was therefore defined as the number of cases above the CD19/ expression, 6 samples for dilution studies to investigate the impact of CD3 CD5/k/l threshold for predicting presence of residual disease that had X0.01% (10 À 4) CLL detected by the four-CLR MRD flow panel (that is, true in different fluorochrome combinations, 22 samples for optimising positive) as a proportion of the total number of cases above the CD19/ fluorochromes used and 6 samples for dilution studies to compare four- k l CLR and six-CLR analysis); and 67 samples from patients with CLL after CD5/ / threshold for predicting presence of residual disease, indepen- treatment for comparison of four-CLR and six-CLR MRD flow cytometry. All dent of the four-CLR MRD flow panel result (that is, true positive þ false patients were diagnosed according to the iwCLL/NCI-WG criteria.14 positive). Patients were included before, during and after treatment with For evaluation of the requirement to use CD3 to exclude contamination with different antibody combinations (CD19/CD5/CD3 combined with chlorambucil, fludarabine-based combinations þ / À rituximab, 3,7,15–19 CD20/CD79b/CD38, or CD81/CD22/CD43, or CD81/CD79b/CD43), compar- alemtuzumab and autologous/allogeneic transplantation. All samples were taken with full-informed patient consent and analysis was ison of the numbers of events classified as CLL from FCS files without CD3 performed according to the requirements of the local ethics review data (electronically removed) against those with CD3 data was performed committee. using a paired t-test analysis. For assessment of the limit of detection in dilution studies, the error was defined as the difference between observed number of CLL cell events and Flow cytometry actual number of CLL cells events as a percentage of actual CLL cell events Samples were prepared using participating laboratories standard proce- where the latter was calculated from the known CLL dilution. dures as reported previously, either using ammonium chloride prestaining or FACSlyse post-staining.3,5,7,8,17–19 In six CLL cases, dilution studies have been carried on to assess the limit of detection by diluting CLL cells into RESULTS bone marrow or peripheral blood containing only normal B cells. An initial CD19/CD5 and clonality assessment for the quick identification of 1:10 dilution of CLL cells into normal blood/marrow and then six further samples with residual disease: a poor negative predictive value serial 1:3 dilutions were made to obtain a series of 42 samples with known concentration of CLL cells ranging from 0.001 to 1% of leukocytes. Peripheral blood and bone marrow samples from 784 patients For evaluation of the requirement to use CD3 to exclude contamination with CLL after treatment were assessed by a basic analysis with different antibody combinations, simulated minimal disease samples comprising antibodies to CD19, CD5, k and l immunoglobulin were prepared by diluting six CLL cases into bone marrow or peripheral light chains and compared with results derived from the complete blood containing only normal B cells. The cells were incubated with CD19/ harmonised four-CLR CLL MRD panel reported previously.8 The CD5/CD3 coupled with either CD20/CD79b/CD38, or CD81/CD22/CD43, or assessment of k and l ratios were standardised as outlined in CD81/CD79b/CD43.
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