Bone marrow spatial architecture predicts response duration in smoldering multiple myeloma patients receiving checkpoint inhibitor therapy

Abstract Introduction While spatial analysis has identified immune features predicting immunotherapy response in solid tumors, spatial information remains unexplored in hematologic malignancies. Previous studies of the tumor microenvironment in blood cancer have relied on bone marrow (BM) aspirates, which lack spatial architecture. Our recent work developing spatial methods for BM biopsies demonstrated that spatial organization associated with chimeric antigen receptor (CAR) T cell therapy outcomes in patients with relapsed/refractory multiple myeloma (RRMM). However, the role of BM spatial immune features in patients with smoldering multiple myeloma (SMM) treated with checkpoint inhibitors remains unknown. Methods We analyzed 15 BM trephine biopsies from high-risk SMM patients enrolled in a phase II trial of nivolumab combined with lenalidomide and dexamethasone (NCT02903381). The primary outcome was progression-free survival (PFS) at 2 years. Patients received nivolumab, lenalidomide, and dexamethasone for 12 months total. Biopsies were collected pre- and post-induction therapy (7 pre-treatment; 8 post-treatment). We assessed spatially orchestrated cellular niches using imaging mass cytometry (IMC) with a 32-antibody immune panel (Hyperion, Standard BioTools). Cells were segmented and annotated based on marker expression. Cellular neighborhoods (CNs), defined as the microenvironment within a 10-μm radius, were identified using clustering algorithms. Results Clinical Outcomes: Eight high-risk SMM patients (median age 60 years, 62% male) were enrolled with median follow-up of 38.7 months. The combination was well-tolerated with no grade 4-5 toxicities; the most common adverse events were fatigue and neutropenia (75% each). Seven patients achieved minimal response or better, with 5 achieving very good partial response or better. Four patients eventually progressed, with a median duration of response of 908 days. At 2 years, 4 of 8 patients (50%) remained progression-free, with median PFS of 33.8 months and overall survival (OS) not reached. Cellular Composition Analysis: We detected 70,390 single cells across all samples representing 27 cell subtypes, including plasma cells, regulatory T cells (Treg), exhausted CD8 T cells, and M1/M2-like macrophages. IMC-measured plasma cell infiltration strongly correlated with pathologists' reports (r=0.92, p0.01). Pre- and post-treatment comparison revealed decreased plasma cell proportion (p=0.016), increased monocyte proportion (p=0.016), and elevated M2-like macrophages among total macrophages (p=0.016). Baseline exhausted CD8 T cell proportion positively correlated with treatment duration (r=0.78, p=0.039), while longer responders showed higher post-treatment M2-like macrophage proportions (p=0.03). These findings suggest that optimal treatment response requires pre-existing inflammatory conditions characterized by elevated exhausted CD8 T cells at baseline, followed by adequate tissue repair responses corresponding to treatment-induced immune activation. Spatial Analysis: Spatial clustering identified 13 distinct CNs with varying compositions. Only the CN3 proportion showed significant positive correlation with treatment duration (r=0.792, p=0.034). CN3 was enriched with M1-like macrophages, plasma cells, and Tregs, and depleted of progenitor cells, dendritic cells, and intermediate monocytes (adjusted p0.001). Within CN3, M1-like macrophages were significantly more proximate to exhausted CD8 T cells within 10μm compared to other CNs, while classical monocytes and M1-like macrophages showed reduced proximity (adjusted p0.001). CN3 demonstrates strategic positioning of immune cells with controlled spacing between inflammatory populations, providing spatial evidence of a “regulated inflammatory state.” Our findings indicate that spatial orchestration of immune cells serves as a critical biomarker of therapeutic response in checkpoint inhibitor therapy. Conclusions Our findings suggest that baseline spatial organization, particularly the CN3 neighborhood characterized by strategic positioning of M1-like macrophages and exhausted CD8 T cells, may be associated with treatment duration. While limited by sample size, these preliminary results suggest that spatial analysis of BM biopsies could provide novel insights into immune dynamics in multiple myeloma and may inform future therapeutic strategies in this patient population.