Single–cell analysis of BCR and transcriptome uncovers plasma cell clonal dynamics and immune exhaustion linked to daratumumab response in primary light–chain amyloidosis Free

Primary light–chain amyloidosis (pAL) is a systemic disorder driven by slowly proliferative clonal plasma cells that secrete misfolded free light chains, which deposit in organs and cause progressive damage. While the anti–CD38 monoclonal antibody Daratumumab (Dara) has shown promising efficacy as a first–line anti–plasma cell therapy for pAL, approximately 20–30% of patients fail to achieve a hematologic complete response (CR) or very good partial response (VGPR). The cellular mechanisms underlying Dara resistance within the bone marrow and immune microenvironment remain poorly understood.

To investigate the link between cell biology and clinical response, we performed 10X Genomics 3‘ single–cell RNA sequencing on whole bone marrow aspirates and 10X Genomics 5‘ V(D)J and RNA sequencing on CD138⁺ magnetic bead–enriched plasma cells from matched samples. We analyzed a cohort of 30 patients with newly diagnosed pAL treated with first–line daratumumab–bortezomib–dexamethasone (Dara–Vd) regimen as reported (Shen et al., Haematologica, 2024). All patients had cardiac involvement; 23.3% had κ light chains, 46.7% harbored the t(11;14) translocation, and 20.0% had 1q21 gain. With a median follow–up of 34 months, 62.9% of patients achieved a best hematologic response of ≥VGPR. Among these 30 patients, 17 patients contributed baseline–only samples, 11 had paired samples collected pre– and post–treatment, and 2 who achieved partial response (PR) provided post–treatment samples.

Using our PRISM (Plasma cell disease Repertoire Identification and Single–cell Mapping) bioinformatics framework, we generated a bone marrow atlas of 630,111 single cells from 78 samples, including 182,000 BCR–defined clonal plasma cells. Non–negative matrix factorization identified pAL plasma cell gene programs (Wang et al., SOHO 2025, Houston, TX) associated with treatment response: a mitosis and somatic hypermutation gene program was highly expressed in patients with hematologic responses <VGPR (p=0.053), while cell adhesion (p=0.002) and endopeptidase/protein hydrolysis (p=0.031) gene programs were enriched in those achieving ≥VGPR. Mitotic plasma cell subclones sharing BCR clonality with the dominant pAL clone, expressing low levels of CD38, persisted as residual disease. Optimal transport–based clonal dynamics revealed that in patients achieving PR, these mitotic subclones gave rise to novel, Dara–resistant plasma cells. Conversely, patients who achieved CR or VGPR exhibited complete or almost complete eradication of clonal plasma cells with stable transcriptomic profiles.

Dara mediates anti–plasma cell effects by binding CD38 on plasma cells and immune cells in the bone marrow microenvironment, facilitating T cell–mediated cytotoxicity and antibody–dependent cellular cytotoxicity (ADCC). We observed T cell exhaustion that emerged following treatment exclusively in patients failing to achieve ≥VGPR: upregulation of LAG3 and HAVCR2 in CD8⁺ effector memory cells in patients achieving PR or no response (NR), and TIGIT and CTLA4 in CD4⁺ effector memory cells in patients showing NR. Natural killer (NK) cells, as key mediators of ADCC, exhibited variable expression levels of TIGIT, LAG3, HAVCR2, and CD96 in pre– or post–treatment samples. However, this NK cell exhaustion was not associated with hematologic response and was attenuated following Dara treatment.

In summary, hematologic responses <VGPR are associated with the mitosis gene program and the persistence of mitotic plasma cell subclones that escape Dara and gives rise to Dara–resistant plasma cells. T cell exhaustion arises following Dara treatment specifically in patients with hematologic responses <VGPR and may contribute to the resistance to Dara. Although NK cell exhaustion is present, it is not associated with treatment outcomes. By integrating single-cell and clinical data through the PRISM framework, we aim to continue translating insights into plasma cell biology and the immune microenvironment into clinically relevant knowledge for researchers in the field of pAL.