Lupus Double-Negative B Cells Harbor Expanded Somatic Mutations in Lymphoma-Relevant Pathways

Abstract

Objectives Systemic lupus erythematosus (SLE) patients have a 2–7-fold increased risk of hematologic malignancies, particularly B-cell non-Hodgkin lymphoma (B-NHL).[1] Although germline susceptibility shared between SLE and B-NHL is minimal,[2] somatic evolution of autoreactive B cells has been implicated as a precursor to malignant transformation in autoimmune syndromes.[3] We therefore aimed to characterize somatic alterations across SLE B-cell subsets, with an emphasis on double-negative (DN) B cells, a central autoreactive population. We hypothesized that DN cells would accrue increased somatic mutation burden, show evidence of clonal expansion, and harbor lymphoma-relevant alterations. Methods In a cohort of 35 SLE patients, we performed whole exome sequencing on FACS-isolated naïve (CD19+, IgD+), memory (MBC; CD19+, IgD−, CD27+), and DN B-cells (CD19+, IgD−, CD27−) from peripheral blood. Patient-matched buccal DNA was used to filter out germline variants, and somatic mutations detected by ≥2 callers (Mutect2, Strelka2, and CaVEMan) were retained. Variant allele frequencies (VAFs) were compared across subsets to assess clonal dynamics. Associations with disease activity (SLEDAI-2 KG), time since diagnosis, and age were evaluated. Pathway enrichment and lymphoma-associated mutations were evaluated by interrogating mutations detected in MBC and DN cells across all patients. Results DN and MBC proportions correlated with disease activity (ρ=0.443, p=0.008; ρ=0.354, p=0.037). DN and MBC had significantly higher somatic mutation burdens than naïve cells (p=0.001 and p=0.017), consistent with expansion of antigen-experienced and/or autoreactive compartments under chronic inflammation (Figure 1A-B). Mutation burden in DN correlated with time since diagnosis, but not age, suggesting disease-associated acquisition and/or selective growth of mutated clones (Figure 1C). Among mutations shared between MBC and DN, a disproportionate fraction expanded in DN (Figure 1D), consistent with a competitive advantage of mutated DN subclones. Shared mutations expanded in DN were enriched for B-cell activation and proliferation pathways, including NF-κB and B-cell receptor signaling (FDR<0.05; Figure 1E). In patients with high mutation burden, both MBC and DN harbored nonsynonymous variants in lymphoma-associated genes, including IGLL5 and CARD11 (Figure 1F-H). Conclusion Our results identify DN B-cells as a clonally evolving population that acquires a high burden of somatic mutations under chronic inflammatory conditions, with some variants affecting genes and pathways relevant to malignant transformation. These results highlight DN cells as a plausible cell of origin for transformation in SLE and underscore the value of DN-focused molecular profiling to identify patients with high-risk clonal features and improve monitoring for progression and lymphoma transformation potential. References [1.] Bernatsky S. J Autoimmun 2013;42:130-5. [2.] Din L. Genet Epidemiol 2019;43:844-63. [3.] Singh M. Cell 2020;180:878-94.