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

Abstract

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 falsedetection 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 MRDnegativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches. Leukemia (2007) 21, 956-964.

Chlorambucil plus rituximab with or without maintenance rituximab as first‐line treatment for elderly chronic lymphocytic leukemia patients

“…Biologic work-up, including minimal residual disease (MRD) in CR patients, is detailed in the Supporting Information [15][16][17][18][19][20][21][22].…”

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

“…Among proposed methods, calculating the ZAP70 T/B ratio is one of the earliest and most common approaches (12,(15)(16)(17)(18). Other methods based on ratios calculated on normal B-cell MFIs have drawbacks such as the very low percentage of residual B-cells or the difficulty of separating them from CLL cells by flow cytometry in the case of weak CD5 expression (11,13,32).…”

A single tube 10‐color flow cytometry assay optimizes detection of minimal residual disease in chronic lymphocytic leukemia

“…CD19 efluor 450 was purchased from eBioscience (Jomar Bioscience, Australia, New Zealand). The panels were constructed according to the published ISA approach (14), i.e., tube 1: CD45 FITC, CD14 PE, CD19 APC, CD3 PerCpCy 5.5; tube 2: CD81 FITC, CD22PE, CD19 APC, CD5 PerCpCy5.5; tube3: CD20 FITC, CD38 PE, CD19 APC, CD5 PerCpCy5.5; tube 4: CD43 FITC, CD79b PE, CD19 APC, CD5 PerCpCy5.5. The single tube 10-color panel consisted of the following antibodies CD81 FITC, CD22 PE, CD3 ECD, CD5 PerCpCy5.5, CD20 PECY7, CD79b APC, CD38 A700, CD43 APC Alexa 750, CD19 eFluor 450, and CD45 KO.…”

“…1A, plot 4, gate AD). These events (gate AD) were analyzed according to the ISA protocol (14) (Fig. 1B).…”

“…The authors reported a close correlation and 95% concordance with ASO-PCR for the detection of CLL above 0.01% (10 À4 ) using a method applicable to all sample types and therapeutic regimens (14). The CLL8 study of the German CLL Study Group identified three levels of MRD (<10 À4 , 10 À4 to 10 À2 and >10 À2 ) with prognostic significance following chemotherapy for previously untreated CLL (1).…”