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. 2022 Oct 7;14(19):4917.
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

Anke Schilhabel  1 , Peter Jonas Walter  1 , Paula Cramer  2 , Julia von Tresckow  3 , Saskia Kohlscheen  1 , Monika Szczepanowski  1 , Anna Laqua  1 , Kirsten Fischer  2 , Barbara Eichhorst  2 , Sebastian Böttcher  4 , Christof Schneider  5 , Eugen Tausch  5 , Monika Brüggemann  1 , Michael Kneba  1 , Michael Hallek  2 , Matthias Ritgen  1
Affiliations

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 et al. Cancers (Basel). .

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.

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Conflict of interest statement

A.S., P.J.W., S.K., M.S., A.L., and M.K. declare no conflict of interest. B.E.: Consulting or Advisory Boards for Janssen, Novartis, AbbVie, BMS, ArQule, AstraZeneca, Oxford Biomedica (UK), MSD, Miltenyi, Lilly, Speaker / Speaker’s Bureau: Janssen, Roche, AbbVie, Novartis, Celgene, AstraZeneca, Adaptive Biotechnologies, Hexal; Research funding: Janssen, Gilead, Roche, AbbVie, BeiGene, Astra Zeneca; Travel support: Janssen, Roche, AbbVie. P.C.: research funding by Acerta, AstraZeneca, Beigene, F. Hoffmann-LaRoche, Gilead, Janssen-Cilag, and Novartis, honoraria for scientific talks by AbbVie, AstraZeneca, F. Hoffmann-LaRoche and Janssen-Cilag, advisory boards by AbbVie, AstraZeneca and BeiGene, travel grants by AbbVie, AstraZeneca, F. Hoffmann LaRoche, and Janssen-Cilag. M.H.: Honoraria (speaker’s bureau and/or advisory board) by Roche, Gilead, Janssen, Bristol Myers Squibb, AbbVie, AstraZeneca, Research support by Roche, Gilead, Janssen, Bristol Myers Squibb, AbbVie, AstraZeneca. J.v.T.: advisor or consultant for AbbVie, AstraZeneca, Janssen, and Roche; honoraria from AbbVie, AstraZeneca, Janssen, and Roche, research funding from Janssen and Roche and travel support from AbbVie, AstraZeneca, Janssen, and Roche. K.F.: Honoraria by AbbVie, Roche, Consulting or Advisory Role by AbbVie, AstraZeneca, Other (Travel) by Roche. S.B.: Research funding from AbbVie, Celgene, Janssen, and Roche; consultancy or advisory role for AbbVie, AstraZeneca, Celgene, Roche, Amgen, and Janssen. C.S: Honoraria (Speaker’s bureau) by AstraZeneca and AbbVie. E.T.: Research support by Roche, Abbvie and Gilead and honoraria, advisory board, speakersbureau with Roche, Abbvie, Beigene and AstraZeneca. M.B.: Consultancy or Advisory Boards: Incyte, Amgen; Speaker’s Bureau: Janssen, Amgen; Research funding: Amgen, Roche, Pharma AG; Reference diagnostics: Affimed, Regeneron, Amgen; Travel support: Amgen. M.R.: Consultancy: MSD, Chugai, Abbvie, Roche; Travel support: MSD, Abbvie, Roche, Celgene; Research funding: Abbvie, Roche.

Figures

Figure 1
Figure 1
Distribution of patients of the CLL2-BXX trials among the defined MRD response groups (see Table 1 for scoring to MRD response groups); BCG, BAG, BIG, BIO: patients with deep (uMRD) MRD response under the respective trial schemes (B: Bendamustine; I: Ibrutinib; A: Venetoclax; C: Idelalisib; G: Obinutuzumab; O: Ofatumumab), IR: intermediate MRD response; LR: limited MRD response; NR: Non-Responders; IR/LR/NR include patients from the different trial schemes.
Figure 2
Figure 2
Variability of daily quality control measurements of peak-7 Rainbow beads’ MFI differences to the target values on different flow cytometry instruments (CIIA, CIIB: CantoII; LA, LB: Lyric) under equal environmental conditions during a time period of 8 months.
Figure 3
Figure 3
Measured (colored) and normalized (grey) MFIs of peak 2 of the Rainbow beads for instrument CantoIIA during an 8-month time period (July 2018 to April 2019).
Figure 4
Figure 4
Correlation plot of measured and normalized MFIs for CD19-PerCP Cy5.5 and CD20-FITC in 196 samples prior to therapy (black) and 166 on-therapy (grey) samples from 196 patients enrolled in the CLL2-BXX trials. Linear regression (red) is close to 1 (R2 CD19: 0.997; R2 CD20: 0.999), as NF used for normalization was between 0.9 and 1.13.
Figure 5
Figure 5
CD20 expression on CLL cells of patients in the MRD response groups prior to therapy start. Number of patients in each group is given above. Reported p-values for group differences were obtained from Dunn’s test. BCG, BAG, BIG, BIO: patients with deep (uMRD) MRD response under the respective trial schemes (B: Bendamustine; I: Ibrutinib; A: Venetoclax; C: Idelalisib; G: Obinutuzumab; O: Ofatumumab), IR: intermediate MRD response; LR: limited MRD response; NR: Non-Responders; IR/LR/NR include patients from the different trial schemes. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Figure 6
Figure 6
Observation map of mixed principal component analysis of pre-treatment samples using age and CD20 expression levels as continuous variables and Binet stage, IGHV mutational status, trisomy 12, and therapy strata as categorical variables.
Figure 7
Figure 7
Expression of CD20 (MFI) on CLL cells in diagnostic (DX) and on-therapy samples (final restaging: RE; maintenance cycle 3 or 5: M3, M5) for patients in the CLL2-BIO and CLL2-BIG trials, scored according to their MRD response. MRD response categories include patients with medium and weak responses according to the therapy scheme (BIG_IR/LR, BIO_IR/LR), the non-responders in the BIO trial (BIO_NR), and patients with uMRD response (BIG, BIO).
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