Relationship between myeloma cell genomic alternations and tumor immune microenvironment in bone-related extramedullary disease of newly diagnosed multiple myeloma

Abstract Bone-related extramedullary disease (EMD-B) are a common extramedullary manifestation of multiple myeloma (MM), typically reflecting the characteristics of the immune microenvironment in MM. TP53 abnormalities represent one of the most critical high-risk features in MM and are clearly associated with drug resistance and disease relapse. However, the relationship between genetic alterations in MM bone-related extramedullary disease and the regulation of the immune microenvironment remains unexplored.In this study, fresh tissue samples from 28 MM patients with bone-related lesions were collected. A portion of the samples was used for DNA extraction and targeted DNA sequencing, while another portion was processed into single-cell suspensions for immune microenvironment analysis via CyTOF to determine cellular composition. These findings were further integrated with fluorescence in situ hybridization (FISH) results (1q21 amplification, IGH rearrangements, and 17p deletion) from CD138-sorted bone marrow specimens. Single-cell RNA sequencing was performed on seven patient samples, which were stratified based on the presence or absence of TP53 abnormalities (including TP53 mutations and 17p deletion). The primary objective was to investigate the correlation between genetic alterations in myeloma cells and the immune microenvironment in newly diagnosed MM bone lesions, aiming to identify potential novel targeted therapeutic strategies. Additionally, whether there is consistency between the immune microenvironment characteristics identified by CyTOF and those identified by single-cell sequencing.Results:CyTOF analysis results：The immune microenvironment of NDMM EMD-B lesions was predominantly composed of CD8+ T cells and NK cells, with other immune cell populations including CD4+ T cells, γ/δ T cells, monocytes/macrophages, neutrophils, and B cells. The immune microenvironment profiles of the KRAS/NRAS mutation-only group showed no significant differences compared to the mutation-negative group, suggesting that KRAS and NRAS mutations do not influence the immune microenvironment. In contrast, the TP53 mutation group exhibited a significantly reduced proportion of CD8+ effector T (Teff) cells (CD45RA+ and CD62L- or CCR7-) and an increased proportion of exhausted CD8+ cells (PD1+ and TIM3+ and LAG3+). Compared to the FISH-negative group, both the 1q21 amplification and 17p deletion groups showed reduced NK cell proportions (clusters C9, C10, and C11), with a more pronounced reduction in the 17p deletion group. Similarly, CD8+ Teff cell proportions were decreased in both the 1q21 amplification and 17p deletion groups, with a more significant reduction in the 17p deletion group. Conversely, CD8+ Tna cell proportions were elevated in these groups, with the 17p deletion group showing a more marked increase. Single cell sequencing results: Comparative analysis revealed that the TP53-abnormal group exhibited a significantly higher proportion of regulatory T cells (Tregs) and exhausted CD8+ T cells (Exhausted CD8T) compared to the TP53-normal group. Conversely, the proportion of effector CD8+ T cells (effector CD8T) was lower in the TP53-abnormal group, though this difference did not reach statistical significance. Furthermore, T cells in the TP53-abnormal microenvironment displayed elevated expression of inhibitory molecules such as PDCD1, CTLA4, CD274 (PD-L1), TIGIT, and LAG3 compared to those in the TP53-normal group. These findings suggest that TP53 abnormalities in NDMM EMD-B lesions foster an immunosuppressive microenvironment characterized by increased inhibitory T cells and reduced effector T cells, thereby impairing immune-mediated tumor cell clearance. Additionally, comparative analysis of therapeutic target molecules on plasma cells revealed that the TP53 mutation group exhibited significantly reduced expression of GPRC5D and SLAMF7 but elevated CD38 expression, with no significant change in BCMA expression. This suggests that CD38 and BCMA may be more suitable therapeutic targets than GPRC5D and SLAMF7 in TP53-aberrant NDMM EMD-B lesions.CyTOF can serve as a convenient and efficient alternative method for detecting the immune microenvironment in multiple myeloma.Conclusion: In newly diagnosed MM patients with bone-related extramedullary disease, TP53 abnormalities contribute to the formation of an immunosuppressive microenvironment. CD38 may serve as a promising immunotherapeutic target for NDMM with bone-related lesions.