Abstract Introduction. Circulating tumor plasma cells (CTPCs) have emerged as a powerful diagnostic and prognostic tool in multiple myeloma (MM), with their presence linked to progression in monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) as well as poorer outcomes in newly diagnosed MM (NDMM). While quantitative CTPC enumeration is increasingly validated, comprehensive phenotypic profiling of CTPCs across disease stages is lacking. In this study, we used 36-parameter spectral flow cytometry in a cohort of MGUS, SMM, NDMM, and treated MM patients to delineate CTPC phenotypic evolution across disease progression and to advance our understanding of myeloma biology. Methods. Peripheral blood (PB) mononuclear cells (PBMCs) of patients with MGUS (n=42), SMM (n=22), NDMM (n=15), treated MM (n=30) and healthy controls (n=10) were examined using a 36-parameter spectral flow cytometry panel for normal (=NPCs, defined as CD45-CD38+)and aberrant CPCs (=CTPCs, CD45-CD38+CD56+). Moreover, paired bone marrow (BM) samples of six NDMM patients were studied for differences in marker expression on aberrant plasma cells (PC) in comparison to PB. Results. CTPC counts increased markedly with disease progression, whereas NPC frequencies remained unchanged. The percentage of CTPCs (of all CPCs) rose from MGUS (median 5.62%) to SMM (13.01%) and NDMM (32.41%). Phenotypic profiling of CTPCs across disease stages revealed distinct shifts in marker expression, which encompassed both canonical and lineage-atypical surface markers. Disease progression was marked by a gradual increase in CD38 and CD56, paired with a loss of B-lineage (CD19) as well as myeloid-lineage markers (CD14, CD16), thereby mirroring potential progression of plasma-cell maturation and malignant transformation. Markers of early activation (CD69) and exhaustion (TIGIT), typically T cell associated markers, decreased in NDMM compared to MGUS and SMM, which possibly reflects a shift toward more immune-resistant clones. Furthermore, CTPCs in NDMM displayed a higher expression of HLA-DR and HLA-ABC, which could enable a higher antigen-presentation potential with subsequent T-cell tolerance. Several interleukin receptor subunits that are involved in the STAT5 survival pathway (CD25, CD123, CD127) showed a heightened expression in NDMM compared to precursor states, which could promote interleukin responsiveness and cancer cell proliferation. Moreover, the chemokine receptors CCR6 and CCR7, that are not typically associated with a CPC phenotype, were upregulated on CTPCs in NDMM, which possibly reflects a different homing pattern. In the treated myeloma cohort, we observed a partial reversion of the CTPC phenotypic profiles. Specifically, CD25, CD123 and CD127 were downregulated, whereas TIGIT and PD-1 were upregulated, which mimics several characteristics of exhausted T cells. Expression of CD19 increased again on CTPCs as a possible sign of enrichment for less differentiated PC subclones or plasmablastic phenotypes due to therapy-induced pressure, with similar trends seen for CD14. CD45RA, typically lost on PCs, showed a strong increase in the treated cohort, a behavior reminiscent of TEMRA. Lastly, although BCMA expression remained largely constant during disease progression, it markedly increased after treatment. In contrast, FcRL5/CD307e peaked in SMM and declined to its lowest level post-treatment. Comparison of paired PB and BM samples of patients with NDMM revealed distinct marker patterns. BM malignant plasma cells exhibited markedly higher CD69 levels than their CTPC counterparts, probably in analogy to its role in tissue residency for T cells. Furthermore, CTPCs showed a significantly higher expression of CD127 than aberrant BM PC, which indicates the use of different survival pathways. Conclusion Our study provides novel insights into the phenotypic evolution of CTPCs from precursor lesions to NDMM, as well as partial reversals following treatment. Notably, the striking expression of non-canonical CTPC markers further deepens our understanding of disease biology and could enable new strategies for disease monitoring.
Beer et al. (Mon,) studied this question.