157 | Assessing Synovial Tissue Heterogeneity in Rheumatoid Arthritis

Aims: Three distinct histopathological synovial tissue (ST) pathotypes have been established in new-onset rheumatoid arthritis (RA) cohorts with implications for treatment response. Heterogeneity in synovial inflammation across joints and technical limitations of sampling techniques limits accuracy of pathotyping individual fragments. We aimed to assess variability within individual new-onset RA patients, using conventional approaches to determine the minimum number of fragments required to accurately assign a pathotype. Additionally, using spatial transcriptomics we aimed to determine variability between fragments. Methods: We performed blinded assessment of ST biopsy H&E samples from knee arthroscopies, assigning pathotypes to each fragment. We investigated heterogeneity between assigned and final pathotype (using H&E, IHC) to determine within-patient fragment concordance and minimum number of fragments required for accurate pathotype. We analysed 18 fragments from 7 RA patients with Xenium spatial transcriptomics, quantifying cellular composition, gene expression variability and cellular distance shifts to determine variability between fragments. Results: 86 fragments were analysed from 12 arthroscopies (9 RA, 3 healthy controls); median fragment size was 5.96 mm2 (IQR 3.38–8.44 mm2). Lympho-myeloid (LM) pathotype had high within-patient agreement (median pairwise agreement 0.83; modal proportion 0.92; entropy 0.41), compared to diffuse myeloid (DM) and pauci-immune (PI) pathotypes (DM: 0.32, 0.50, 1.41; PI: 0.52, 0.71, 0.86, respectively). A minimum of 10 fragments was required to achieve 90% accuracy. Across fragments that were spatially imaged using Xenium, there was striking heterogeneity for cell-type proportions and gene expression. Significant regional variation was observed between fragments: median significant cell-type pair (e.g.: T cell--> fibroblast, myeloid-->mural) and distance shifts ranged from 9.29%–50.35% and 2.82–8.12 μm, respectively. Conclusion: Within an individual RA patient, there is significant ST heterogeneity when assessed by histology, cellular composition, gene expression or cell-cell distance. This emphasises the importance of selecting an appropriate number of representative ST samples for analysis.