Dataset Information

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

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.

SUBMITTER: Schilhabel A

PROVIDER: S-EPMC9562902 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Schilhabel Anke A Walter Peter Jonas PJ Cramer Paula P von Tresckow Julia J Kohlscheen Saskia S Szczepanowski Monika M Laqua Anna A Fischer Kirsten K Eichhorst Barbara B Böttcher Sebastian S Schneider Christof C Tausch Eugen E Brüggemann Monika M Kneba Michael M Hallek Michael M Ritgen Matthias M

Cancers 20221007 19

<h4>Background</h4>CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL).<h4>Methods</h4>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 measurement ...[more]

PMID: 36230840

Similar Datasets

Project description:Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Project description:IntroductionCommon Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients.MethodsIn this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD).ResultsThe Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21-CD24- (6.1 vs 0.74 cells/µl, p<0.0001) and CD21-CD24++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD+ (21 vs 32 cells/µl, p=0.03) and IgMD- (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%).ConclusionOur results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.

Dataset Information

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

Background

Methods

Results

Conclusions

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets