Single-cell transcriptomic and clonotypic characterization of Waldenström macroglobulinemia reveals intra-patient monoclonal diversity with distinct differentiation states

Abstract Background: Waldenström macroglobulinemia (WM) is a rare, indolent B-cell lymphoma characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells and secretion of monoclonal IgM. A defining feature of WM is its hybrid phenotype, with tumor cells displaying varying degrees of differentiation from B cells to plasma cells (PCs). However, the extent of clonal complexity and cellular heterogeneity within individual patients remains poorly understood. Traditional bulk sequencing approaches lack the resolution to disentangle this intra-tumoral diversity. To address this limitation, we employed paired single-cell RNA sequencing (scRNA-seq) and B-cell receptor sequencing (scBCR-seq) to comprehensively dissect the transcriptional states, clonal architecture, and differentiation trajectories of WM and its precursor conditions. Methods: We analyzed 72 fresh BM aspirates and 1 peripheral blood sample from 70 patients spanning IgM monoclonal gammopathy of undetermined significance (MGUS, n=2), smoldering WM (SWM, n=12), untreated symptomatic WM (n=13), treated WM (n=43), and WM-transformed diffuse large B-cell lymphoma (DLBCL, n=3). Each sample was evenly divided for parallel CD19⁺ and CD138⁺ selection to isolate B cells and PCs. Both populations were used to generate cDNA libraries for 5' scRNA-seq and scBCR-seq. Data processing followed established pipelines (Dang et al., Cancer Cell, 2023). Mutation status for MYD88, CXCR4, TP53, DNMT3A, TET2, ASXL1, and IGLL5 was obtained from diagnostic testing, and treatment histories were documented for all previously treated patients. Results: We profiled 636,244 high-quality cells, including 321,646 B cells and 314,598 PCs. scBCR-seq data were recovered for 606,928 cells, of which 520,201 had paired transcriptomes. Clonotypes were categorized as monoclonal (≥5 cells), polyclonal (<5 cells), or undetected. Unsupervised clustering identified nine B-cell populations, including five precursor clusters (progenitor, proliferating progenitor, precursor, proliferating precursor, and immature), as well as naïve, activated, memory, and atypical B cells. Polyclonality predominated in early B-cell populations, while activated B cells were largely monoclonal. Among PCs, we identified seven clusters: two polyclonal IgG populations, one transitional B cell-like cluster, two proliferating clusters, and two IgM clusters. Most PC clusters, excluding the IgG ones, were monoclonal. All samples harbored both monoclonal B cells and PCs, often representing different stages of the same clone. scBCR-seq enabled identification of V(D)J usage and CDR3 sequences, revealing multiple monoclonal populations (with clone size > 50) in ~40% of samples. MGUS samples showed the highest clonal diversity (median = 4 clones), followed by SWM (median = 2.5) and WM/DLBCL (median = 1). Within individual patients, distinct clones exhibited divergent transcriptional profiles and differentiation states, ranging from B cell-like to PC-like. MGUS clones were largely PC-dominant (median = 92.1% PCs), while SWM clones had a more B cell-dominant phenotype (median = 39% PCs). In symptomatic WM, PCs fractions within clones increased (median = 63.3%) compared to SWM (p = 0.039), with a further increase following treatment (median = 94.6%, p = 1.2e-07 vs. SWM). Notably, TP53-mutant clones in untreated WM patients exhibited significantly higher PC fractions than TP53 wild-type clones (97% vs. 48%, p = 0.00054), suggesting a potential link between genetic lesions and plasma cell differentiation bias. Conclusions: This study presents the most comprehensive single-cell analysis to date of clonal and cellular heterogeneity in WM and its precursor stages. Our data reveal that multiple monoclonal populations frequently coexist within the same patient, each exhibiting distinct transcriptional profiles and differentiation states. These clones typically comprise a mixture of B cells and PCs, reflecting a complex and dynamic cellular landscape. Notably, we observed a stage-specific shift in clonal composition—from PC-dominant clones in MGUS, to B cell-dominant clones in SWM, and back toward PC-enriched clones in symptomatic WM and following treatment. These findings underscore the importance of dissecting clonal architecture and phenotypic plasticity in WM to better understand disease progression, therapeutic resistance, and potential vulnerabilities.