13 | Spatial contexture of tertiary lymphoid structures as a predictor of recurrence in early-stage melanoma

Background: Tertiary Lymphoid Structures (TLS) are organized aggregates of immune cells that form in non-lymphoid tissues in response to chronic inflammation, including cancer. In melanoma, TLS presence within the tumor microenvironment is associated with a pre-existing anti-tumor immunity and linked to improved immunotherapy responses. However, their role in predicting recurrence, particularly in early-stage melanoma, remains unclear.Methods: We conducted a retrospective analysis of archival primary tumor samples from 65 patients with stage I-II melanoma and negative sentinel lymph node (Table 1). Tumor immune infiltrates and TLS were initially assessed using four-marker immunohistochemistry (IHC) panels targeting CD20, CD4, CD8, and Podoplanin (PDPN), a marker of stromal cells. TLS were objectively identified using the HDBSCAN AI-based clustering algorithm. Tumor samples were further analyzed for high-dimensional phenotyping and spatial analysis using a 17 markers panel with hyperplexed immunofluorescence imaging (HIFI). Associations between TLS features and clinical outcomes were evaluated.Results: IHC revealed that 88% of patients had peritumoral T cell infiltration, and TLS were detected in 54% of cases. TLS-positive patients had improved disease-free survival (DFS, p=0.2) and overall survival (OS, p=0.2) (Figure 1A, B). To investigate further, HIFI was performed on 8 patients (4 recurrent, 4 non-recurrent), revealing 13 distinct cell types: stromal (n=6), tumor (n=2), and immune (n=5) (Figure 1C). Spatial analysis identified 9 recurrent spatial modules (SM), each representing a distinct pattern of interaction between cell types. One SM, enriched for PD-L1⁺ stromal cells, was significantly associated with recurrence (p=0.019) (Figure 1D).Conclusions: TLS presence associates with improved DFS and OS in early-stage melanoma. However, beyond their mere presence, TLS spatial organization and local stromal-immune context could influence clinical outcomes. Our data demonstrated that a specific spatial module—defined by the proximity of PD-L1⁺ stromal cells—is associated with disease recurrence. Table 1. All p-values are calculated in relation to recurrence status. Significant associations were identified for Sex (Recurrence less frequent among female patients) and for Ulceration (more common in recurrent cases). Figure 1A-D.