Mass Cytometry Analysis Shows That a Novel Memory Phenotype B Cell Is Expanded in Multiple Myeloma

Abstract

It would be very beneficial if the status of cancers could be determined from a blood specimen. However, peripheral blood leukocytes are very heterogeneous between individuals and thus high resolution technologies are likely required. We used cytometry by time-of-flight (CyTOF) and next generation sequencing to ask whether a plasma cell cancer (multiple myeloma) and related pre-cancerous states had any consistent effect on the peripheral blood mononuclear cell phenotypes of patients. Analysis of peripheral blood samples from 13 cancer patients, 9 pre-cancer patients, and 9 healthy individuals revealed significant differences in the frequencies of the T, B, and natural killer cell compartments. Most strikingly, we identified a novel B-cell population that normally accounts for 4.0±0.7% (mean±SD) of total B cells and is up to 13-fold expanded in multiple myeloma patients with active disease. This population expressed markers previously associated with both memory (CD27+) and naïve (CD24loCD38+) phenotypes. Single-cell immunoglobulin gene sequencing showed polyclonality, indicating that these cells are not precursors to the myeloma, and somatic mutations, a characteristic of memory cells. SYK, ERK, and p38 phosphorylation responses, and the fact that most of these cells expressed isotypes other than IgM or IgD, confirmed the memory character of this population, defining it as a novel type of memory B cells.

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis

“…+ follicular Th cells (33), B cells (27,(34)(35)(36), and vaccines (18,37). The benefits of larger Ab panels include the discovery of new subsets (31,35), the identification of coexpressed markers (27), and of signatures for differentiation, activation, and homing status of cells.…”

“…The benefits of larger Ab panels include the discovery of new subsets (31,35), the identification of coexpressed markers (27), and of signatures for differentiation, activation, and homing status of cells. Novel findings that are generated by high-dimensional data are also expected to facilitate the discovery of biomarkers or cell subsets with predictive capacity in terms of vaccine and immunotherapies efficacy, and unravel phenotypic progressions (27,31,38) and new mechanisms of induced immune responses that could guide future pharmaceutical developments (39)(40)(41)(42)(43)(44).…”

“…The CyTOF panel was designed to include B cell-associated markers with the capacity to characterize vaccine-induced populations using high-dimensional data analysis. Whereas previous multidimensional studies have focused on comparing different tissue compartments (27,33), healthy versus diseased samples (35), or Ag-specific responses (18,31), we developed a data mining and analysis pipeline to identify and deeply characterize the phenotype of vaccine-induced B cells that changed in frequency over time and those that correlated with the magnitude of the antivaccine Ab response.…”

Identifying Cell Populations in Flow Cytometry Data Using Phenotypic Signatures

“…This is because existence of the unknown subtypes whose identification can be important in generating new knowledge in the field. One reason is that some diseases may change the cellular composition and introduce additional cell population(s) which must be taken into account [3]. Also, the prior knowledge are not comprehensive and discovering new knowledge is possible using such analyses [2].…”

Systems immune monitoring in cancer therapy

“…Several recent studies have demonstrated that overcoming the practical challenges of mass cytometry data acquisition and analysis in longitudinal immune monitoring can unlock biologic and clinical insights in tumor immunology (34, 52, 53). A comprehensive and dynamic picture of anti-tumor immunity can be achieved by using cutting edge, single cell tools on samples collected longitudinally, over the course of therapy, or after specific perturbation.…”