Reduced expression of CD20 antigen as a characteristic marker for chronic lymphocytic leukemia

Almasri, Nidal M.; Duque, Ricardo E.; Iturraspe, Jose; Everett, Eric T.; Braylan, Raul C.

doi:10.1002/ajh.2830400404

Cited by 164 publications

(105 citation statements)

References 15 publications

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“…At present, flow cytometry has been used to establish an immunophenotypic profile of B lymphocytes, that is characteristic of CLL, involving coexpression of CD5, CD19, CD20, and CD23 with low to absent expression of CD79b and FMC7. Low surface Ig expression and downregulation of CD20 are consistent finding in B-CLL (1)(2)(3)(4). However, the reported number of CD20 receptors per cell varies by more than 1 order of magnitude.…”

supporting

confidence: 58%

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Wang

Abbasi

Gaigalas

et al. 2006

Cytometry Part B Clinical

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Methods: We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE TM PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression.Results: On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV 6 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV 6 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 6 10.4)%, and it did not differ significantly in CLL samples.Conclusion: The expression of CD20 on normal and B-CLL lymphocytes can be quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies. q

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supporting

confidence: 58%

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Wang

Abbasi

Gaigalas

et al. 2006

Cytometry Part B Clinical

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“…The characteristic low intensity expression of CD20 in CLL is well established (26)(27)(28)(29). We also confirmed that the level of CD20 molecule numbers on CLL cells was low, being only 10% that of normal control B cells, as estimated by all standards tested.…”

Section: Discussionmentioning

confidence: 99%

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia

Rossmann

Lenkei

Lundin

et al. 2007

Cytometry Part B Clinical

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Background: The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors.Methods: The performance of three different types of microbeads was compared, i.e. Quantum molecules of equivalent soluble fluorochrome (Q-MESF), Quantum simply cellular (QSC), and QuantiBRITE TM (QB). As all PE-conjugates had a F/P ratio of 1:1, the MESF units represented also the antibody binding capacity (ABC).Results: The ABCs of CD4 and CD20 antigens estimated with QSC (ABC QSC ) were higher than those assigned with QB (ABC QB ) with an average difference 49%. Higher numbers of antigenic sites were obtained with Q-MESF than with QSC for CD20 antigen. On the contrary, CD4 antigenic sites numbers estimated with QSC were higher than those estimated with Q-MESF. ABC values estimated with Quantum MESF PE (ABC Q-MESF ) were *15% higher than ABC QSC , whereas ABC Q-MESF was *49% higher than ABC QB INTRODUCTIONQuantitative measurements of fluorescence intensity by flow cytometry (QFCM) assess the numbers of monoclonal antibody (mAb) molecules bound to a cell. The fluorescence intensity (FI) of staining correlates to the number of fluorochrome conjugated mAb molecules bound to the cell, and consequently, the amount of receptors on the cell. Thus, the number of antigen receptors can be determined by QFCM, through estimation of the exact numbers of bound fluorescent antibody molecules, provided that appropriate standards and fluorescence controls are tested in parallel and reagents in saturating amounts are used. In addition, the process of quantitation facilitates standardization and quality control (1).

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“…However, extensive in vitro and preclinical studies suggest that Ab-dependent cell-mediated cytotoxicity (10,11), complement-dependent cytotoxicity (12)(13)(14)(15), and direct effects of transmembrane signaling leading to apoptosis and cell growth arrest (16 -18) may be responsible for the observed clinical effects of rituximab. Both in vitro and preclinical data suggest that decreased CD20 expression may be responsible for the lack of response observed in certain patients (12,19,20).…”

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confidence: 99%

Enhanced Expression of CD20 in Human Tumor B Cells Is Controlled through ERK-Dependent Mechanisms

Wojciechowski

Marshall

et al. 2005

The Journal of Immunology

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Rituximab, a chimeric Ab directed against CD20, induces apoptosis in targeted cells. Although the majority of B cell malignancies express the CD20 Ag, only ∼50% of patients will respond to single-agent rituximab. The available data suggest that a decreased CD20 expression could account for the lack of response observed in some patients treated with rituximab. Despite the potential critical role of CD20 in the biology of B cell malignancies, the mechanisms controlling its expression are poorly understood. We evaluated the effect of the immune modulator agent bryostatin-1 on the expression of CD20 in non-Hodgkin’s lymphoma cells. Using the B cell lines, DB and RAMOS, as well as tumor cells derived from a chronic lymphocytic leukemia patient, we demonstrated that bryostatin-1 enhanced the expression of both CD20 mRNA and protein. The enhanced expression of CD20 was associated with increased transcriptional activity of the CD20 gene, whereas the stability of CD20 mRNA was not affected. The effect of bryostatin-1 on CD20 expression in non-Hodgkin’s lymphoma cells was mediated through the MAPK kinase/ERK signal transduction pathway and involved protein kinase C, but was independent of p38 MAPK and was insensitive to dexamethasone. Cells pretreated with bryostatin-1 were more susceptible to the proapoptotic effect of anti-CD20 Ab. Overall, these data demonstrate for the first time that ERK phosphorylation is required for the up-regulated expression of CD20 on B cell malignancies. The findings also suggest that bryostatin-1 and rituximab could be a valuable combined therapy for B cell malignancies.

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Reduced expression of CD20 antigen as a characteristic marker for chronic lymphocytic leukemia

Cited by 164 publications

References 15 publications

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia

Enhanced Expression of CD20 in Human Tumor B Cells Is Controlled through ERK-Dependent Mechanisms

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