A Quantitative Exploration of Surface Antigen Expression in Common B-Cell Malignancies Using Flow Cytometry

Abstract

The use of flow cytometry to diagnose hematological malignancies has become routine due to its ability to often differentiate between morphologically similar diseases based on antigens expressed on the surface of malignant cells. In an attempt to expand on the utility of flow cytometry in the study of B-cell malignancies we have used the most reliable quantitative methodology, QIFI (quantitative indirect immunofluorescence assay), to study the expression of CD5, CD10, CD11c, CD19, CD20, CD22, CD23, and CD79b in 384 cases of several common B-lineage malignancies, including: B-ALL, CLL, SLL, hairy cell leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. The impetus behind this extensive, single institution study of surface antigens was two-fold: evaluating similarities and differences of antigen expression between B-cell neoplasms and finding additional clinical utility for the quantitative flow cytometric data generated. Our results show that each distinct malignant histology has its own quantitative pattern of surface antigen expression. In most cases, these quantitative patterns do not increase the ability of flow cytometry to distinguish between them. However, a high expression of specific antigens on a given B-cell malignancy may potentially identify optimal therapeutic targets for current and/or future monoclonal antibody-based therapies.

Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma

“…Previously published data suggest that the surface expression level of CD79b on follicular lymphoma and diffuse large B-cell lymphoma cells is near or slightly lower than on normal B cells but with a much broader range of expression levels. 11,12 Our data suggest that the surface copy number of CD79b on most NHLs will not be a limiting factor for the efficacy of these ADCs (Figure 1).…”

“…CD79 has the appropriate expression pattern, being expressed only on B cells and in most NHLs. [10][11][12] The molecule is a covalent heterodimer containing CD79a (Ig␣, mb-1) and CD79b (Ig-␤, B29); both subunits contain a single extracellular Ig domain, a transmembrane domain, and an intracellular signaling domain. The BCR is a complex between CD79 and surface Ig (sIg), and all of these components are required for surface expression of the BCR.…”

Variables affecting the quantitation of CD22 in neoplastic B cells

“…This can be accomplished using standard beads to construct calibrating curves for comparison to a cellular specimen (4). The quantitative indirect immunoflorescence (QIFI) assay is designed for indirect staining methods (4,5). The quantum simply cellular (QSC) and QuantiBRITE assays are designed for studying the binding of antibodies directly conjugated to fluorochromes.…”

Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells

“…Cell surface densities of CD33 and CD123 were measured by using a calibrated cytofluorimetric procedure as described. 60,68,85 For this purpose, a kit of fluorescent beads with known numbers of fluorescent chromophores per bead (QIFIKIT®; Agilent Technologies, cat. # K0078) was used for calibration purposes.…”

“…The procedure allows the investigator to express the measured fluorescence intensity of antibodies bound to a cell surface in terms of average number of antigen copies per cell. 60,68,85 PE-labeled mAbs specific for CD33 and CD123, calibrated for QIFI kit measurements of antigen densities on MOLM-13 AML cells, were used as a substitute for the less economic complete QIFI kits. MOLM-13 cells were used as standards because surface densities of CD33 and CD123 on these cells remained sufficiently constant over time to make these cells useful for calibration purposes.…”