Minimal residual disease monitoring in chronic lymphocytic leukaemia patients. A comparative analysis of flow cytometry and ASO IgH RQ‐PCR

Abstract

SummaryMinimal residual disease (MRD) is becoming increasingly important in chronic lymphocytic leukaemia (CLL) as treatment strategies are progressively improving. The primary objective of this study was to compare the applicability of three different flow cytometric approaches: basic 4-colour analysis, European Research Initiative in CLL (ERIC) consensus method and 8-colour analysis. Secondly, we investigated the sensitivity and specificity of flow cytometry (FC) compared to molecular analyses for MRD detection. A total of 462 CLL samples were evaluated by basic FC; in 143, ERIC consensus method was also performed and all three FC methodologies were applied in a subgroup of 10 cases. No discordance in defining MRD-positive/negative samples was observed between the FC methods; within positive samples, the ERIC consensus method and 8-colour analysis showed the most accurate results. MRD was analysed by FC and polymerase chain reaction (PCR) in 243 cases: concordant results were obtained in 199/243 samples (81Á9%); 42/243 were FCÀ/PCR+. Overall, the sensitivity and specificity of FC compared to PCR was 96Á5% and 77Á2%, respectively. Both FC and PCR proved suitable for the detection of MRD and prediction of progression-free survival, which was significantly reduced in MRD-positive patients, regardless of the methodology. These results offer the rationale for a strategy to monitor MRD in CLL patients.

Minimal Residual Disease Surveillance in Chronic Lymphocytic Leukemia by Fluorescence-Activated Cell Sorting

“…The study by Raponi et al22 conducted a comparative analysis of FLC and real-time ASO-PCR for MRD monitoring in CLL. This was done by monitoring blood and bone marrow samples from 98 patients, establishing a consensus of 82% between the two methods.…”

“…Raponi et al22 also addressed this question and found an approximately 92% concordance between results in bone marrow and PB; however, among the discordant 8% almost all were positive in bone marrow and negative in PB, implying that true MRD negativity cannot be unequivocally determined using PB only.…”

“…Raponi et al22 examined an even more complex eight-color panel, including CD81/CD38/CD20/CD43/CD5/CD45/CD19/CD3, in a very limited number of patients and compared it to the ERIC consensus panel with a good overlap of MRD results.…”

Achieving Minimal Residual Disease Negative Status in CLL - a Good Point to Start Thinking about Life without Treatment: How to Check MRD Status?

“…Stehlíková et al and Raponi et al suggested an 8-color labeling antibodies methodology application for the FC in order to enhance detection of MRD as well as to reduce the difficulties associated with the preparation of samples for laboratory analysis [17,35]. Stehlíková has used the regime with two tubes which seems to be easier and less time-consuming then 4-color labeling method utilizing 4 tubes.…”

“…Another difficulty is the necessity of designing PCR primers for each patient. Raponi et al have taken into consideration the direct sequencing of DNA before designing primers for ASO IGH RQ-PCR reaction [35]. This approach is expensive, and the validation of the method for the implementation to the daily clinical practice is extremely difficult.…”