Prognostic value of high-sensitivity measurable residual disease assessment after front-line chemoimmunotherapy in chronic lymphocytic leukemia.

Measurable residual disease (MRD) status is widely adopted in clinical trials in patients with chronic lymphocytic leukemia (CLL). Findings from FILO group trials (CLL2007FMP, CLL2007SA, CLL2010FMP) enabled investigation of the prognostic value of high-sensitivity (0.7 × 10-5) MRD assessment using flow cytometry, in blood (N = 401) and bone marrow (N = 339), after fludarabine, cyclophosphamide, and rituximab (FCR)-based chemoimmunotherapy in a homogeneous population with long follow-up (median 49.5 months). Addition of low-level positive MRD < 0.01% to MRD ≥ 0.01% increased the proportion of cases with positive MRD in blood by 39% and in bone marrow by 27%. Compared to low-level positive MRD < 0.01%, undetectable MRD was associated with significantly longer progression-free survival (PFS) when using blood (72.2 versus 42.7 months; hazard ratio 0.40, p = 0.0003), but not when using bone marrow. Upon further stratification, positive blood MRD at any level, compared to undetectable blood MRD, was associated with shorter PFS irrespective of clinical complete or partial remission, and a lower 5-year PFS rate irrespective of IGHV-mutated or -unmutated status (all p < 0.05). In conclusion, high-sensitivity (0.0007%) MRD assessment in blood yielded additional prognostic information beyond the current standard sensitivity (0.01%). Our approach provides a model for future determination of the optimal MRD investigative strategy for any regimen.

Evaluation of Fluorescently Labeled Aerolysin as a New Kind of Reagent for Flow Cytometry Tests: Optimization of Use of FLAER, Hints, and Limits.

OBJECTIVES: Diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH) are currently based on flow cytometry techniques. Typically, these tests use antibodies against glycosylphosphatidylinositol (GPI)-anchored proteins, but a new approach has been described recently, using a novel reagent named FLAER (fluorescently labeled aerolysin). In this work, we evaluate the performance and highlight the peculiarities of using this new reagent. RESULTS: We investigated the general conditions of staining and explored optimal labeling settings. We found that the kinetics of the FLAER labeling is slightly different from that of antibodies. Our results led us to select a 30-minute incubation period at room temperature using 50 nmol/L as a final concentration of FLAER. As the nonspecific binding was dependent on the balance between FLAER and its ligand, the number of target cells was also found critical. In addition, sample preparation affected FLAER staining, and the lyse-before-stain preparation was preferred. Interestingly, FLAER affinity seems restricted to certain types of GPI anchors, making it unsuitable for exploration of RBCs. Finally, we aimed to evaluate FLAER as a possible single diagnostic tool; we studied cellular background in non-PNH samples and found a limit of detection close to 0.01% in optimal conditions. CONCLUSIONS: The performance of the FLAER labeling on leukocytes proves that this reagent is a valuable tool for PNH diagnosis and particularly appropriate for high-sensitivity tests in laboratories aiming to detect minor PNH clones.