Single-cell DNA profiling unveils clonal evolution and identifies mechanisms of therapeutic resistance and progression in monoclonal gammopathies

Abstract INTRODUCTION Monoclonal gammopathies –including MGUS, smoldering multiple myeloma (SMM), and multiple myeloma (MM)– are driven by clonal expansion of malignant plasma cells. Understanding their clonal architecture and the genomic landscape at single-cell resolution is essential to unravel disease progression and guide treatment strategies, particularly in the context of novel immunotherapies. In this study, single-cell DNA sequencing, was combined with surface protein profiling, to comprehensively dissect somatic mutations, copy number variations (CNVs), clonotypes, and immunophenotypes across disease stages and treatment responses. This novel single cell multi-omics approach provides a powerful perspective on the genomic and functional heterogeneity of plasma cell disorders, offering key insights for precision medicine and therapeutic stratification. METHODS CD138+ were isolated from bone marrow aspirates of 11 patients (21 samples) with MGUS, SMM, or MM using AutoMACS sorting. Patient samples included diagnostic, progression, relapse, and treatment-resistant stages. Single-cell DNA sequencing was performed using a panel of 839 amplicons, combined with surface protein profiling via a 20-antibody cocktail. Clonal architecture was inferred by integrating somatic mutations, clonotyping and CNVs. Surface protein expression was used to correlate genotypic features with targetable phenotypes at single cell resolution. RESULTS Clonal complexity and genomic architecture showed substantial heterogeneity across disease stages and therapeutic contexts. In early disease, such as MGUS and SMM, some cases already exhibited complex CNV profiles or early driver mutations, indicating subclinical clonal evolution. Several patients displayed stable CNV landscapes over time, while mutational profiles fluctuated, suggesting a selection or suppression of cell-specific subclones rather than wholesale genomic shifts. In relapsed/refractory multiple myeloma, branched clonal evolution was frequently observed, with sequential acquisition of mutations affecting genes such as TP53, FRG1, and TRAF2 in specific cells. In some cases, dual-clone coexistence with distinct genomic and immunoglobulin rearrangement profiles was evident, reflecting convergent tumor evolution. Furthermore, certain patients exhibited a distinct pattern of surface antibody expression, indicating the presence of plasma cells co-expressing both major therapeutic targets in MM, GPRC5D and BCMA, while other plasma cell populations expressed only one or neither of these targets. A key finding emerged when integrating genomic data with clinical outcomes: patients harboring monoallelic deletions and/or mutations in GPRC5D or BCMA (TNFRSF17) consistently showed limited or no clinical response to corresponding targeted therapies. As an example, one patient with biallelic inactivation of GPRC5D in near 90% of the cells prior to TALQUETAMAB treatment, failed to respond, correlating with absent membrane expression of the target. Conversely, a separate patient who progressed on anti-GPRC5D therapy demonstrated a favorable clinical response to BCMA-directed CAR-T cells, consistent with preserved BCMA expression and copy number in all the cells. These findings indicate that the genomic integrity and surface expression of immunotherapeutic targets strongly influence treatment efficacy. Importantly, discrepancies between gene copy number and surface protein levels — such as cases with BCMA amplification but heterogeneous expression — highlight post-transcriptional regulation as an additional layer of resistance. CONCLUSIONS Single-cell DNA and protein co-profiling in monoclonal gammopathies provides critical insights into clonal evolution, therapeutic resistance, and the dynamic expression of immunotherapeutic targets. Specifically, GPRC5D genomic alterations and reduced protein expression may compromise response to targeted therapies, highlighting the need for functional biomarker screening. Our data support the clinical relevance of integrating single-cell genomics in the management of plasma cell dyscrasias to tailor therapy and anticipate resistance. Our results suggest that performingsingle-cell genomic and proteomic profiling prior to treatment initiation may improve therapeutic selection and predict response, particularly in the era of targeted immunotherapies.