Chronic Lymphocytic Leukemia

International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia

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The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01–0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

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Thanks to Florence Cymbalista, Remi Letestu, Sorina Tugulea, Chris Etzler, Fiona Bennett, Ruth de Tute, Brad Dickinson and Ludmila Bezdícková for much appreciated input into the project. This work was supported by Leukaemia Research Fund, UK; AIRC Milano, Italy; MIUR, Italy; National Cancer Institute Grant R21 CA101332 and U10 CA101140, US; Ben Eckenhoff fund, US; Instituto de Salud Carlos III, FIS 04/1051, Spain.

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Correspondence to A C Rawstron.

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On behalf of European Research Initiative on CLL ( and the CLL Research Consortium (

Supplementary Information accompanies the paper on the Leukemia website (

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  • CLL
  • MRD
  • flow cytometry

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