Phenotypic evolution of circulating plasma cells from early precursor stages to multiple myeloma

Circulating tumour plasma-cells (CTPCs) have emerged as valuable diagnostic and prognostic marker in multiple myeloma (MM), with their presence linked to progression from precursor stages and poorer outcomes in newly diagnosed MM (NDMM). While quantitative CTPC enumeration is increasingly validated, comprehensive phenotypic profiling across disease stages remains lacking. We applied 36-parameter spectral flow cytometry to 113 PBMC samples of MGUS (n=42), SMM (n=22), NDMM (n=15), treated MM (n=24), and healthy controls (n=10), alongside paired bone marrow (BM) samples from six NDMM patients. CTPCs were defined phenotypically as CD45-CD38highCD56+ events without assessment of clonality. Phenotypic profiling of the CD56+ CTPC-like subset across disease stages revealed alterations in canonical and lineage-atypical surface markers. Progression to NDMM was characterised by upregulation of CD38 and CD56 with concomitant loss of B- (CD19), myeloid- (CD14, CD16), and T-cell-associated (CD45RA) markers. In addition, HLA-ABC, interleukin receptor subunits (CD25, CD123), and chemokine receptors (CCR6, CCR7) were upregulated in NDMM. In treated MM, a reversed pattern was observed with lower CD25 and CD123 expression and increased levels of exhaustion markers (TIGIT, PD-1). Paired BM samples showed a different tissue-residency marker expression, characterised by higher CD69 and CCR6 and lower CD138, along with changes in cell-survival related markers, including increased CD25 and CD123. Both BCMA and CD307e were more highly expressed on BM plasma-cells than CTPCs. This study is among the first to provide a comprehensive phenotypic characterisation of CD56+ CTPCs across the MM spectrum, including checkpoint and chemokine receptors, treated disease cases, and paired BM samples for NDMM.