Characterizing the autoreactome in thymoma-associated myasthenia gravis

Thymic epithelial tumors (TETs), including thymomas (TMMs), are rare anterior mediastinal malignancies arising from the thymus, an organ essential for early-life T-cell maturation. Paraneoplastic autoimmune disorders (ADs) frequently accompany TMMs, likely due to the tumor's retained, but dysregulated capacity for T-cell processing, leading to defective negative selection and emergence of autoreactive T cells. Myasthenia gravis (MG) occurs in approximately 30-50% of patients with TMM, and nearly all individuals with TMM-associated MG (TAMG) harbor acetylcholine receptor (AChR) antibodies, often accompanied by reactivity against additional skeletal muscle proteins such as titin and ryanodine receptors. Although TAMG is characterized by a complex network of autoreactive immune elements, the breadth and functional relevance of associated autoantibodies remain poorly understood. We hypothesized that TAMG patients exhibit greater diversity of circulating autoantibodies compared with non-autoimmune controls. Using biospecimens from the Indiana University Simon Comprehensive Cancer Center and the George Washington University Myasthenia Gravis Research Laboratory, we performed autoreactome profiling of sera from TAMG patients and age-matched healthy controls. Samples were interrogated using Molecular Indexing of Proteins by Self-Assembly (MIPSA; Infinity Bio), a high-throughput DNA-barcoded antigen display platform. The screening encompassed >16,000 full-length proteins and ~400,000 peptide sequences. Autoreactome analysis was completed for 40 TAMG patients and 14 controls. The TAMG cohort had a mean age of 58.7 years; 22 (55%) were female. Twenty-six (65%) patients were AChR-antibody positive, none had MuSK antibodies, one (2.5%) was seronegative, and 13 (32.5%) had unknown serologic status. WHO TET subtypes included A (2.5%), AB (15%), B1 (12.5%), B2 (40%), B3 (20%), metaplastic (2.5%), and unknown (7.5%). Overall, 4,724 proteins and 6,553 peptides were identified as autoantibody targets, with 2,688 proteins and 4,890 peptides uniquely enriched in TAMG. Although TAMG patients demonstrated a higher number of unique autoantibodies than controls, this difference did not reach statistical significance. Notably, interferon-alpha (IFN-alpha) autoreactivity was observed in 23 (57.5%) TAMG subjects, including 14 with corresponding IFN-alpha peptide reactivity. Among 20 patients with IFN-alpha antibodies, we identified novel reactivity against P2X4 receptor and TRIM46, proteins implicated in immune regulation and neuromuscular signaling. These findings suggest a broader and previously unrecognized autoantibody landscape in TAMG. Ongoing studies will evaluate the specificity of these signatures, incorporate TMM patients without MG, and correlate autoreactive patterns with MG-related clinical features.