Evaluation of Minimal Residual Disease in Blood and Bone Marrow for CLL as Assessed by 6 Color Flow Cytometry.

MRD evaluation is becoming critical in CLL patients since the new therapeutic approaches lead to higher and higher biological response. Multicolor flow cytometry is demonstrated to be one of the best technique for assessing MRD. In a recent work, Moreno et al (Blood, 2006) suggested that peripheral blood could be sufficient for MRD evaluation. We here report our experience which compares peripheral blood and bone marrow aspirate for MRD assessment using a 6 color flow cytometry (FC) method. Two months after the end of the treatment, blood and bone marrow were harvested at the same time for 64 CLL patients in complete remission according to the NCI criteria. Patients were treated by chemotherapy followed or not by autologous stem cell transplantation (ASCT). None of them received immunotherapy. Until now 30 out of the 64 couples of blood and marrow samples have been studied. Immunophenotyping was performed using the FacsCanto Device (Becton Dickinson) and the following monoclonal antibody (moAb) combinaisons: k-FITC / l-PE / CD19-PerCP-Cy5.5 / CD5-PE-Cy7 / CD79b-APC / CD20-APC-Cy7; FMC7-FITC / CD38-PE / CD19PerCP-Cy5.5 / CD5-PE-Cy7 / CD20-APC-Cy7 for peripheral blood assessment and k-FITC / l-PE / CD45-PerCP-Cy5.5 / CD5-PE-Cy7 / CD19-APC / CD20-APC-Cy7; FMC7-FITC / CD19-PE / CD45PerCP-Cy5.5 / CD5-PE-Cy7 / CD79b-APC / CD20APC-Cy7 ; CD38-FITC / CD23-PE / CD45-PerCP-Cy5.5 / CD5-PE-Cy7 / CD19-APC. In the bone marrow, we have discriminated hematogons from CLL cells by using moAb combinations containing CD45 and CD38. Between 4500 and 5500 lymphocytes were analyzed in each test. By using a method of serial dilutions, we demonstrated a sensitivity of 10⁻⁴ for our 6-color FC technique. CLL cells were defined as CD19+CD5+CD79b ^low CD20^low lymphocytes with low expression of monotypic Ig light chain. Results were expressed as a percentage of leukemic cells among total B lymphocytes. We showed a complete correlation between blood and bone marrow for MRD evaluation using this technique. A complete phenotypic remission defined by the absence of leukemic cells in the blood and in the bone marrow, was observed in 17 out of 30 cases. In the other 13 patients, persistent leukemic cells were detected in the marrow as well as in the peripheral blood with a median rate of 1.56 % [0.15 – 41] and 0.56% [range 0.06 – 36] respectively. Our results showed that, by using 6 color FC, blood assessment for detecting MRD in CLL after chemotherapy or ASCT could provide a simple surrogate to bone marrow assessment. Analysis of the remaining couples of samples for the last 34 patients is ongoing.