Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones

Abstract

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

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

“…The biomarker-based classified groups represented candidate PCs, committed clonotypic B cells, and plasmablasts ( Figure 3 A). Quantification of the different circulating rare cell groups across patients showed that CD138+ cells were more abundant in neoplastic myeloma and not in NDs, with a higher proportion being found in NDMM than in precursor states (SMM and MGUS) ( Figure 3 B–D), consistent with our prior observations [ 19 , 20 ]. BCMA+ cells were detected in all samples, including NDs, confirming that the expression of BCMA in early B cells, although suggested to be low, is detectable in our assay.…”

“…Technical validation for CD138, CD45, and DAPI cells in HDSCAs has been previously reported [ 19 , 20 ]. In titration experiments with U266, MM.1S, and control Jurkat cells, BCMA expression was consistently higher in U266 and MM.1S across all concentration conditions, while no BCMA expression was detected in control Jurkat cells, as expected ( Figure S1A ).…”

“…The rationale for developing the BCMA assay is grounded in BCMA’s progressive expression across the B-cell differentiation spectrum ( Figure 1 A) and the increasing interest in this marker as a therapeutic target, as demonstrated by the rise in clinical studies over the past 9 years ( Figure 1 B). The selection criteria for CD138, CD45, and DAPI have been previously described in two validated myeloma assays for morphogenomic differentiation of malignant and normal PCs and identification of rare cell clones [ 19 , 20 ]. Here, we substituted CD56 with BCMA for a new immunofluorescence staining panel (CD138, BCMA, CD45, DAPI), with the primary goal of targeting key cells of interest ( Figure 1 C) and characterizing circulating B lymphocytes expressing BCMA in the PB of patients with PC neoplasms.…”

“…In this study, we report the technical development and initial genomic and clinical validation of a new slide-based, enrichment-free immunofluorescence assay (i.e., BCMA, CD138, CD45, DAPI) for the detection of circulating rare cells and morphogenomic profiling of BCMA+ cells in PC malignancies, hereafter referred to as the “BCMA assay”. The technical methodology is built on the established “no-cell-left-behind” approach of the high-definition single-cell assay (HDSCA) workflow—previously validated clinically for various pathologies, including breast cancer [ 14 ], myocardial infarction [ 15 ], melanoma [ 15 ], prostate cancer [ 16 ], bladder cancer [ 17 ], colorectal cancer [ 18 ], and multiple myeloma [ 19 , 20 ]—and has been optimized for the detection of BCMA expression for the characterization of MM precursor cells in PC neoplasia.…”

Circulating Tumour Cells, Cell Free DNA and Tumour-Educated Platelets as Reliable Prognostic and Management Biomarkers for the Liquid Biopsy in Multiple Myeloma

“…In a different study, researchers used an enrichment-free four-plex immunofluorescence technique and single-cell DNA sequencing for identification and genomic classification of plasma cells to distinguish normal and malignant plasma cells in blood and BM aspirates from patients with newly diagnosed myeloma and monoclonal gammopathy of undetermined significance (MGUS) [ 33 ]. The results confirmed that peripheral blood plasma cells presented the same genomic modifications that were present in BM plasma cells.…”