Spatial transcriptomics reveals profound subclonal heterogeneity and T-cell dysfunction in extramedullary myeloma

• EMD mimics the architectural complexity of solid tumors, marked by diverse microenvironments, multiclonality, and TNFRSF17 and GPRC5D levels.

• EMD shows infiltration of active T cells spatially confined to niches segregated from MM cells, potentially affecting the therapeutic response.

Extramedullary disease (EMD) is a high-risk feature of multiple myeloma (MM) and remains a poor prognostic factor, even in the era of novel immunotherapies. Here, we applied spatial transcriptomics (RNA tomography for spatially resolved transcriptomics [tomo-seq] [n = 2] and 10x Visium [n = 12]) and single-cell RNA sequencing (n = 3) to a set of 14 EMD biopsies to dissect the 3-dimensional architecture of tumor cells and their microenvironment. Overall, infiltrating immune and stromal cells showed both intrapatient and interpatient variations, with no uniform distribution over the lesion. We observed substantial heterogeneity at the copy number level within plasma cells, including the emergence of new subclones in circumscribed areas of the tumor, which is consistent with genomic instability. We further identified the spatial expression differences between GPRC5D and TNFRSF17, 2 important antigens for bispecific antibody therapy. EMD masses were infiltrated by various immune cells, including T cells. Notably, exhausted TIM3⁺/PD-1⁺ T cells diffusely colocalized with MM cells, whereas functional and activated CD8⁺ T cells showed a focal infiltration pattern along with M1 macrophages in tumor-free regions. This segregation of fit and exhausted T cells was resolved in the case of response to T-cell–engaging bispecific antibodies. MM and microenvironment cells were embedded in a complex network that influenced immune activation and angiogenesis, and oxidative phosphorylation represented the major metabolic program within EMD lesions. In summary, spatial transcriptomics has revealed a multicellular ecosystem in EMD with checkpoint inhibition and dual targeting as potential new therapeutic avenues.