FUNCTIONAL DISSECTION OF THE SINGLE CELL TRANSCRIPTOMIC LANDSCAPE OF GENOTYPICALLY-IDENTIFIED POLYCLONAL PLASMACELLS IN MULTIPLE MYELOMA MICROENVIRONMENT

Introduction: Multiple Myeloma (MM) is a plasma cell (PC) dyscrasia usually preceded by asymptomatic stages defined as Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering MM (SMM). Progression is driven by cell-intrinsic factors in the clonal PCs population as well as by changes in the tumorigenic microenvironment (TME). Even if the role of immune and non-immune cells has been extensively studied, the disruption of polyclonal PCs and their role in contributing to TME dysregulation has never been deeply studied. Taking advantage of single-cell B-cell receptor (BCR) genotyping and scRNAseq in the same single cells, we aimed to dissect the transcriptional landscape and role of polyclonal PCs in asymptomatic PC dyscrasia progression. Methods: We performed scRNAseq and scBCR analysis to unequivocally identify polyclonal PCs within the CD138pos population in a cohort of n=7 MGUS, n=16 SMM and n=23 newly diagnosed (ND)MM patients. Cells derived from healthy donors (HDs) sourced both locally (n=1) and from publicly available repositories (n=17) were used as controls. InferCNV and MultinicheNET tools were applied to study the copy number alterations (CNA) and the PCs-TME interactions, respectively. Results: A total of n= 213,146 CD138pos PCs were analyzed, revealing the presence n= 170,428 clonal and n= 42,718 polyclonal cells in patients with PC dyscrasias. The polyclonal nature of the latter group was confirmed by lack of identity with the clonal BCR sequence at nucleotide level and by the lack of CNAs. As compared to PCs from HDs, polyclonal PCs from patients showed a peculiar transcriptomic profile across different stages. The supervised marker genes expression analysis revealed the upregulation of genes of TME and inflammation (i.e. ICAM1, TIMP1, ITGB7), autophagy (i.e. P62) and interferon pathway (i.e. IFI6, IFITM1) in polyclonal PCs derived from patients, as well as HLA-DPA1 and HLA-DOB, that are fundamental modulators of immune response. Gene Set Enrichment Analysis showed a climax of increasing inflammatory status from HD to NDMM. The HALLMARK TNF SIGNALING VIA NFkB was the only upregulated pathway in HD PCs, compared to the MGUS, SMM and NDMM ones. Inflammatory pathways as HALLMARK ALLOGRAFT REJECTION, HALLMARK INFLAMMATORY RESPONSE, IFN related pathways, resulted overexpressed in MGUS, SMM and NDMM as compared to HDs. Finally, we performed a network analysis between polyclonal PCs and TME in each patient, highlighting an increased suppressive interaction between polyclonal PCs and effector CD8 T cells, mainly in NDMM. Conclusions: Overall, our findings shed some light for the first time on the transcriptomic landscape of corrupted polyclonal PCs in patients with asymptomatic and symptomatic PCs dyscrasias, showing functional disruptions that may contribute to disease pathogenesis through promoting an inflammatory milieu, as well as explain the typical immunoparesis seen upon progression.