Characterization of BCMA Expression in Circulating Rare Single Cells of Patients with Plasma Cell Neoplasms

Abstract

B-cell maturation antigen (BCMA), a key regulator of B-cell proliferation and survival, is highly expressed in almost all cases of plasma cell neoplasms and B-lymphoproliferative malignancies. BCMA is a robust biomarker of plasma cells and a therapeutic target with substantial clinical significance. However, the expression of BCMA in circulating tumor cells of patients with hematological malignancies has not been validated for the detection of circulating plasma and B cells. The application of BCMA as a biomarker in single-cell detection and profiling of circulating tumor cells in patients' blood could enable early disease profiling and therapy response monitoring. Here, we report the development and validation of a slide-based immunofluorescence assay (i.e., CD138, BCMA, CD45, DAPI) for enrichment-free detection, quantification, and morphogenomic characterization of BCMA-expressing cells in patients (N = 9) with plasma cell neoplasms. Varying morphological subtypes of circulating BCMA-expressing cells were detected across the CD138(+/-) and CD45(+/-) compartments, representing candidate clonotypic post-germinal center B cells, plasmablasts, and both normal and malignant plasma cells. Genomic analysis by single-cell sequencing and correlation to clinical FISH cytogenetics provides validation, with data showing that patients across the different neoplastic states carry both normal and altered BCMA-expressing cells. Furthermore, altered cells harbor cytogenetic events detected by clinical FISH. The reported enrichment-free liquid biopsy approach has potential applications as a single-cell methodology for the early detection of BCMA+ B-lymphoid malignancies and in monitoring therapy response for patients undergoing anti-BCMA treatments.

Keywords: BCMA; HDSCA; bone marrow aspirate; circulating plasma cells; liquid biopsy; morphogenomics; multimodal data; multiple myeloma; peripheral blood; rare single cell.

Figure 1

Assay rationale and Immunofluorescence targeting: (A) B-cell differentiation and BCMA expression during the B-cell maturation spectrum from GC B to mature plasma cells [7,8]. (B) Clinical studies involving BCMA as a therapeutic target of interest. Data from ClinicalTrials.gov (accessed on 1 May 2022) with “BCMA” as the search key term, as of April 2022. “Not Applicable” = study phase explicitly marked as “not applicable”. “Unknown” = no data entered for study phase. The low count in the year 2020 reflects the effect of the COVID-19 pandemic on clinical enrollment. (C) Immunofluorescence targeting with different antibodies for the detection of BCMA+ and CD138+ cells in the enrichment-free HDSCA workflow with histological features of plasma cells and B cells. Large CD138+ cells with eccentric nuclei represent the plasma cell compartment, while small CD45+ and BCMA+CD138- cells represent the B-cell fraction. Gt = goat, Ms = mouse, Rb = rabbit, Ig = immunoglobulin, A555 = Alexa Fluor 555 dye, A488 = Alexa Fluor PLUS 488 dye, A647 = Alexa Fluor 647 dye.

Figure 2

Morphological characterization of BCMA+ cells in PB samples: (A) Representative candidate rare cells with comparative BCMA and CD138 expression across different cell sizes. (B) UMAP projection of all candidate cells using all 761 single-cell morphology and marker intensity features, colored by CD138 expression. (C) UMAP projection, colored by BCMA expression. (D) UMAP projection, colored by CD45 expression. (E) Density plots showing channel intensities for each disease state.

Figure 3

Quantitative enumeration of circulating rare and BCMA+ cells: (A) UMAP projection, colored by manual cell classification of rare cell groups. (B) The total cell count per immunofluorescence intensity classification. (C) Proportions of cells across patients. (D) Enumeration by channel type. The color codes in panels (C,D) are the same channel-type color codes in the legend shown in panel (B). (E) Total counts of BCMA+ cells across disease states. The boxplot colors are as shown in the diagnosis color scheme in panel D.

Figure 4

scCNV analysis for genomic validation of candidate aberrant cells: (A) Representative CNV profiles along with cellular morphology for each single cell. Dashed rectangles mark diagnostic cytogenetic events also detected in scCNV; red = gain, blue = loss. (B) Distribution of sequenced cell counts grouped by normal and altered scCNV profiles across patients. (C) Distribution of total counts of sequenced cells across diseases states (and NDs), grouped by normal and altered scCNV. (D) Distribution of total counts of sequenced cells across morphological groups, grouped by normal and altered scCNV. The color code is by channel type (as shown in panel (F)). (E) Clinical cytogenetic results from FISH and karyotyping for 12 common genomic events used for the diagnosis of myeloma. (F) Intersection plot showing total counts of altered single cells harboring alterations detected by clinical cytogenetics and corresponding morphological classification.