Abstract T025

Characterization of cancer-associated fibroblasts and their spatial architecture reveals heterogeneity and survival associations in classical Hodgkin lymphoma

Background: Cancer-associated fibroblasts (CAFs) are a heterogeneous population of stromal cells, which modulate the immune system and can have both pro- and anti-tumorigenic effects. The impact of CAFs in shaping the tumor microenvironment (TME) has been recognized in solid tumors, but in classical Hodgkin lymphoma (cHL), their role has remained largely undefined. We aimed to characterize distinct CAF subsets and their interactions with other TME cells and associate the findings with clinical characteristics and outcomes of patients with primary cHL. Methods: CAFs, macrophages, other leukocytes and Hodgkin Reed-Sternberg (HRS) cells were characterized using multiplexed immunofluorescence imaging in two independent cHL patient cohorts (n=131 and n=166). Image processing and quality control were performed by Ilastik and CellProfiler softwares, and a pretrained deep learning segmentation model was applied to segment the nuclei. Single cell features were extracted using histoCAT software. Phenograph clustering algorithm was utilized for cell phenotyping, and permutation tests by histoCAT and Scimap for interaction and neighborhood analysis. Results: We identified a total of 952,099 and 2,2x10 6 single cells in the discovery and validation cohorts, respectively. These were split into distinct phenotype metaclusters spanning CAFs, macrophages, leukocytes, and HRS cells. In both cohorts, the median proportion of all CAFs was approximately 20%, being higher in nodular sclerosis (NS) compared to other subtypes. Higher proportions of all CAFs, and more specifically fibroblast activation protein (FAP)-positive CAFs, were associated with favorable outcomes independent of the histological subtype, age, and stage. In contrast, a subset of CD45+ immune cells with strong FAP-positivity, classified as macrophages, was less abundant in the NS subtype and associated with worse outcomes. Neighborhood analysis allowed for the identification of colocalization or regional exclusion of phenotypically defined cell types and recurrent cellular neighborhoods. Despite the positive impact of CAFs on survival, patients with enrichment of platelet-derived growth factor-beta (PDGFRb)-positive CAFs in the vicinity of HRS cells had worse survival in both cohorts, independent of the clinical determinants (Figure 1). Conclusion: Our findings distinguish various subsets of CAFs and macrophages impacting survival in cHL and underscore the importance of the spatial arrangements in the TME.