Defining the Cell Surface Landscape in B-Cell Malignancies: Comprehensive Profiling of Surface Antigens for Biomarker Discovery and Patient Stratification

Flow cytometric analysis of surface marker expression is a fundamental component of diagnosis, classification, and monitoring for many hematologic malignancies. However, most clinical implementations of surface immunophenotyping focus on only a handful of markers. A comprehensive survey of CD markers and cell-surface receptors could provide a higher resolution and more accurate characterization of cancer lineages, as well as enabling new insights into the cellular changes resulting from disease progression or therapeutic intervention. To this end, we collated and evaluated antibodies to over 400 known markers and receptors. To establish the baseline and variability of each marker in major B cell subsets within normal human blood, a protocol was established to co-stain each sample for CD19, CD20, CD27, IgD, CD38, and CD24 simultaneously with each of the 400 markers. Antigen abundance and expression frequency were evaluated to establish a surface marker profile for each B-cell subset and to establish the variation inherent in each marker across the normal human population. To validate the application of this panel for cancer immunophenotyping, 20 malignant B-cell lines were selected for comprehensive surface characterization. Prior to staining, each cell line was barcoded with a combination of fluorescent dyes to generate a unique fluorescent signature. This approach allowed multiple cell lines to be combined into a single well for staining and analysis, enabling the rapid evaluation of hundreds of surface antigens using only 1-2 million cells from each line. Hierarchical clustering of data from cancer lines and normal B cell subsets allowed the identification of markers useful for discriminating between B-cell cancer subtypes, as well as markers that differed between normal and malignant B cells. These studies have resulted in the optimization of a protocol and antibody panel for use in profiling surface protein expression on human blood cells and cell lines. Application of this procedure to clinical samples may provide information useful for biomarker discovery, improved stratification of hematologic malignancies, and the identification of new drug targets.