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    Myeloma Genomics and Microenvironment and immune profiling

    (OA-27) Composition and Functional State of T and NK Cells in Extramedullary Myeloma Tumor Microenvironment

    Wednesday, September 17, 2025
    16:30 - 16:40 East Coast USA Time
    Location: Room 718
    Introduction: Extramedullary disease (EMD) is an aggressive manifestation of multiple myeloma (MM), when clonal plasma cells (PCs) become independent of the bone marrow (BM) microenvironment (TME) and invade distant tissues and organs. The incidence of EMD is increasing and is associated with drug resistance and poor prognosis. There is limited knowledge about the composition of the EMD TME and the fitness of its effector immune cells, potentially responsible for treatment efficacy.

    Methods: Biopsy of 23 EMD soft tissue tumor was performed in relapsed/refractory MM (RRMM) patients and samples were processed immediately after surgery. Single-cell RNAseq (scRNAseq) was performed using Chromium GEM Single Cell 3' reagent kit v3.1 (10x Genomics) and cell suspensions from the samples. The computational pipeline combined scanpy python framework and R packages enabling comparison of paired and unpaired EMD vs BM samples. For flow cytometry (FC) assessment, samples were stained using 8-color panels to distinguish key subsets.

    Results:

    ScRNAseq and FC yielded comparable results, confirming that EMD tumor is primarily composed of PCs and the effector:tumor (E:T) ratio is dramatically lower compared to BM.  

                      Semi-automated cell type annotation resulted in the identification of 9 distinct T cell and 2 NK cell clusters. CD8+ T cell clusters exhibited markedly lower cytotoxicity and higher exhaustion scores in EMD compared to BM. This trend was attributed to different composition of T cells between the groups. EMD had the highest proportion of cells in the most exhausted tumor-reactive CD8_exhausted-like_TOX cluster, while clusters with high cytotoxicity score, were less prevalent in EMD. Accordingly, using FC we found an increased percentage of T cells positive for exhaustion marker PD-1 in EMD compared to EMD_BM and RRMM_BM. In addition, we observed a substantially lower number of CD4+ T cells in EMD compared to BM resulting in a significantly lower CD4+/CD8+ ratio.
                      Analysis of NK cell compartment revealed significantly higher proportion of less cytotoxic CD16- NK cells in EMD (73.4% vs 0.7%; p=0.01, and 7.9%; p=0.01 in EMD_BM and RRMM_BM, respectively), which are typically enriched in solid tumors (Rebuffet et al. 2024, Nature Immunology), compared to BM samples. This finding was further confirmed by FC as well as in an independent validation cohort of 8 EMD samples using spatial transcriptomics. Importantly, CD16- NK cells from EMD exhibited significantly higher expression of inhibitory receptor NKG2A compared to EMD_BM.

    Conclusions: In this study, using scRNAseq and FC data, we revealed that EMD TME is characterized by a high proportion of exhausted tumor-reactive CD8+ T cells and CD16- NK cells. Additionally, we demonstrated that EMD CD8+ T and NK cells overexpress immune checkpoints like PD-1 and NKG2A, suggesting therapeutic opportunity with checkpoint inhibitors.