1. Detection of minimal residual disease in CLL using multiparametric flow cytometry
JONCKHEERE, Stijn 1; STEUR, Elise 1; BILLIET, Johan 1
1 Dept. Laboratory Medicine, AZ Sint-Jan Brugge, Ruddershove 10, 8000 BRUGGE
Introduction
Achievement of minimal residual disease (MRD) eradication at the end of induction therapy in patients with CLL is a desirable therapeutic goal as this is clearly associated with a longer progression free survival, as compared to patients with MRD positivity. MRD analysis can be done by laborious PCR techniques but the emerge of multiparametric flow analysis has created a more convenient solution. However, there is no consensus of which combination of markers is superior. Here we evaluate two flow cytometric panels to detect MRD.
Materials and Methods
In preliminary experiments, expression of different lymphocytic markers (CD5, CD10, CD20, CD23, CD43, CD79b, CD81, CD200 and FMC7) was compared between normal B-lymphocytes (22 patients) and CLL-cells (33 patients). These markers were chosen based on the EuroFlow protocol. Then, aberrant marker expression was used to compose two combinations of markers to detect MRD on an 8-color flow cytometer (FACS Canto II, BD). Gating strategy was based on intersected gates. A series of dilutions (undiluted, 10-2, 10-3, 10-4 and 10-5) of CLL-cells in WBC of healthy donors were analyzed to determine the sensitivity. A total of 10 samples (5 for each panel) were analyzed and a median of WBC were measured in the highest dilution. All samples were blinded for the observer.
Results
1. Preliminary results
The preliminary experiments showed significant differences (p<0.001) in marker expression (expressed as median fluorescence intensity) between normal B-lymphocytes and CLL-cells for CD5 (449 vs 2769), CD10 (128 vs 57), CD20 (20708 vs 2021), CD23 (724 vs 1582), CD43 (358 vs 1535), CD79b (3013 vs 181), CD81 (1008 vs 205), CD200 (2696 vs 8100) and FMC7 (4387 vs 393) (see figure 1).
Based on this data, two panels of markers were composed:
panel 1: CD20(V450) / CD45(V500) / mlambda(FITC) / mkappa(PE) / CD5(PerCP-Cy5.5) / CD19(PE-Cy7) / CD200(APC) / CD43(APC-H7)
panel 2: CD20(V450) / CD45(V500) / CD81(FITC) / CD79b(PE) / CD5(PerCP-Cy5.5) / CD19(PE-Cy7) / CD200(APC) / CD43(APC-H7)
Fig. 1: Box and whisker plots of the marker expression (expressed as median fluorescence intensity). Statistically significant differences were found for all the shown markers between normal B-cells and CLL-cells.
2. MRD experiments
Both panels were able to detect residual CLL-cells until a level of 10-4 in all samples. However, panel 1 failed to detect MRD at a level of in 4 of the 5 samples, whereas panel 2 detected 10-5 in all 5 samples (p<0.05 according to the Fisher’s exact test). All blanks were negative with both protocols.
3. Recovery of CLL-cells
Both panels showed a good correlation between the expected and measured events over the whole range of dilutions. This implies that the quantitative results of MRD can be reported.
Fig. 2: Correlation between the expected and measured events over the whole range of dilutions.
Conclusions
In conclusion, multi-parametric 8-color flow cytometry is a simple and quick method to detect MRD in CLL patients. The proposed panel (panel 2 in which mkappa and mlambda is replaced by CD79b and CD81) is able to detect such a low level as 10-5.