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

Anke Schilhabel; Peter Jonas Walter; Paula Cramer; Julia von Tresckow; Saskia Kohlscheen; Monika Szczepanowski; Anna Laqua; Kirsten Fischer; Barbara Eichhorst; Sebastian Böttcher; Christof Schneider; Eugen Tausch; Monika Brüggemann; Michael Kneba; Michael Hallek; Matthias Ritgen

doi:10.3390/cancers14194917

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

Cancers (Basel). 2022 Oct 7;14(19):4917. doi: 10.3390/cancers14194917.

Authors

Anke Schilhabel¹, Peter Jonas Walter¹, Paula Cramer², Julia von Tresckow³, Saskia Kohlscheen¹, Monika Szczepanowski¹, Anna Laqua¹, Kirsten Fischer², Barbara Eichhorst², Sebastian Böttcher⁴, Christof Schneider⁵, Eugen Tausch⁵, Monika Brüggemann¹, Michael Kneba¹, Michael Hallek², Matthias Ritgen¹

Affiliations

¹ Laboratory for Specialized Hematological Diagnostics, Medical Department II, University Hospital Schleswig-Holstein, 24105 Kiel, Germany.
² Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, German CLL Study Group, University Hospital Cologne, 50924 Cologne, Germany.
³ Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany.
⁴ Hematology, Oncology, Palliative Care, Clinic for Internal Medicine III, Rostock University Medical School, 18057 Rostock, Germany.
⁵ Department III of Internal Medicine, Ulm University, 89081 Ulm, Germany.

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.

Keywords: CD20 expression; MRD; flow cytometry normalization.

Grants and funding

E.T. received funding for molecular genetics analyses from Else Kröner-Fresenius-Stiftung (2010_Kolleg24) and German Research Foundation DFG (SFB1074 Project B2). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.