CD20 Expression as a Possible Novel Prognostic Marker in CLL: Application of EuroFlow Standardization Technique and Normalization Procedures in Flow Cytometric Expression Analysis

SIMPLE SUMMARY: Up to 50% of patients with chronic lymphocytic leukemia relapse within four years after anti-CD20-directed treatment with a need for a subsequent treatment, which can potentially include anti-CD20 agents again. To retrospectively study the influence of CD20 expression in CLL patients receiving anti-CD20 directed therapy on therapy response, we designed and evaluated a bead-based normalization approach for the analysis of CD20 expression levels to reduce variation of flow cytometric measurements due to technical issues. Normalization significantly reduced the variability of median fluorescence intensities of fluorochrome-conjugated beads without artificially rendering the instruments’ performances, and longitudinal biological variations of marker expression based on MFI values could be robustly assessed. In a cross-trial comparison, strong MRD response correlated with the CD20 expression level before therapy started in patients receiving Ofatumumab, but not in patients receiving Obinutuzumab. ABSTRACT: Background: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL). Methods: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents. Results: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy. Conclusions: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab.