INTRODUCTION

While the genetic drivers of Waldenström macroglobulinemia (WM) are well characterized, the contribution of the bone marrow (BM) microenvironment to disease initiation and progression is less well understood. Prior studies have relied on BM aspirates, which are prone to dilution and fail to capture spatial context. In this study, we used spatial proteomics to profile the BM microenvironment across patients with IgM monoclonal gammopathy of undetermined significance (MGUS), asymptomatic WM (AWM), and symptomatic WM, aiming to identify spatial features linked to disease progression.

METHODS We analyzed 80 bone marrow biopsies from 70 patients with IgM gammopathies enrolled in the PCROWD study at Dana-Farber Cancer Institute. Spatial proteomic profiling was performed using imaging mass cytometry (IMC; Hyperion XTi, Standard BioTools). Cell segmentation was performed using cellpose-sam. Phenotype markers were used to annotate 31 cell types in 733,010 single cells. Cellular neighborhoods, defined as the 10-μm radius around each cell, were calculated on a spatial distance graph and grouped using K-means clustering. Clinical data were integrated, including MYD88 mutational status (assessed from peripheral blood via institutional panel). P values were adjusted using FDR correction.

RESULTS The study included 44 patients with IgM MGUS, 34 with AWM, and 2 with symptomatic WM at initial diagnosis. The median age was 65 years, and 49% were female. Among IgM MGUS patients, 43% were MYD88-mutated, 46% wild-type, and 11% untested. In AWM, 68% were mutated, 12% wild-type, and 21% unknown. Over a median follow-up of 8.2 years, 30% of patients progressed to symptomatic disease (IgM MGUS: 10%; AWM: 41.2%). The median time from diagnosis to BM sampling was 6.1 months (IQR 1.0–28.9). At the time of collection, 5 IgM MGUS patients had progressed to AWM, and 2 IgM MGUS and 4 AWM patients had developed symptoms. Pathologist-reported BM infiltration was similar in AWM and WM (median 40%) and strongly correlated with IMC-based quantification of CD45⁺CD19⁺ WM cells (R=0.78, p<0.001).

Analysis of the overall BM composition revealed a significantly higher fraction of WM cells in AWM compared to IgM MGUS (q<0.001), accompanied by lower proportions of myeloid-lineage cells (q<0.001), erythroid-lineage cells, and hematopoietic stem cells (both q=0.002). These findings aligned with higher hemoglobin levels in IgM MGUS vs AWM (p<0.01) at sample collection, reflecting the preserved hematopoiesis in precursor states. Surprisingly, despite greater lymphomatous involvement, the AWM samples exhibited a higher abundance of T cells (q=0.02), predominantly driven by the CD8⁺ fraction (q=0.004). Phenotypic analysis in AWM showed increased Tregs (q=0.002); activated CD4⁺ T cells (q=0.02); early activated (q=0.007) and exhausted CD8⁺ T cells (Tex) (q=0.03); and reduced GZB⁺Ki67⁺ CD8⁺ effectors (q=0.003), suggesting a progressively immunosuppressed T cell microenvironment in later disease stages.

Cell neighborhood analysis revealed two spatially adjacent but distinct WM neighborhoods within the same samples. The two WM neighborhoods exhibited similar compositions across most cell types but differed in their proportions of WM and T cells: one was characterized by a higher WM cell content and lower T cell infiltration (62% WM, 17% T cells), while the other showed reduced WM density alongside increased T cell presence (52% WM, 23% T cells). WM cells in the T cell–enriched neighborhood displayed elevated expression of Ki-67 (q=0.004), HLA-DR (q=0.0005), and PD-L1 (q=0.001). Within the T cell compartment, this neighborhood was enriched for activated CD8⁺ and CD4⁺ T cells (q<0.0001 and q=0.0009), CD8⁺ Tex (q=0.0002), and Treg (q=0.002). Notably, the fraction of HLA-DR–expressing WM cells correlated with the abundance of CD4⁺ Tregs (R=0.46, p=0.004) and CD8⁺ Tex cells (R=0.57, p<0.0001), supporting the notion that the T cell–enriched WM neighborhood represents a “hot” immune microenvironment. Patients with above-median enrichment of this neighborhood showed a trend toward higher progression to symptomatic WM (p=0.06), whereas the T cell–low WM neighborhood was not associated with progression risk.

CONCLUSIONS By mapping spatial microenvironmental changes across IgM gammopathies, this study reveals a T cell–enriched WM niche associated with disease progression, supporting a model in which localized tumor–immune interactions shape the course of WM pathogenesis.

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2025
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