Spatial Architecture of Tumor-Infiltrating Lymphocytes (TILs) Is an Independent Prognostic Marker in Vulvar Squamous Cell Carcinoma (VSCC): A Multi-Institutional Assessment from the Female Lower Genital Tract Cancer Consortium (FLGTCC)

Summary: Background: High TIL density is suggested to predict survival outcomes in various solid tumors. Little is known about the clinical implications of spatial interplay between TILs and tumor cells in VSCC. We aimed to examine and validate the prognostic significance of TIL spatial architecture in VSCC using an H&E-based image analysis algorithm. Design: VSCC cases (total n=242) from 3 academic institutions were centrally reviewed and used as independent training (St; n=123) and validation (Sv; n=119) cohorts for analyses. TIL features were computationally assessed using H&E whole slide images including their (i) overall density and (ii) spatial organization based on 350 characteristics encompassing TIL clustering properties, interactions with tumor cells, neighborhood relationships among TILs and non-TILs, as well as density and network-based measures. Predictive features were selected using the Boruta algorithm and Cox regression models to generate binary spatial TIL (spaTIL) risk scores, with high score (spaTILH) reflecting increased spatial TIL disorganization. Association of TIL features with overall survival (OS) and recurrence-free survival (RFS) was evaluated with adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs). Results: St and Sv cohorts shared similar baseline features. SpaTILH was more often seen in VSCC with tumor buds, larger tumor size, more keratinization, as well as VSCC without p16 expression or peritumoral tertiary lymphoid structures. Worse OS was associated with spaTIL H in both St (aHR: 3.7; CI: 1.8-7.8) and Sv (aHR: 3.1; CI: 1.9-5.1) cohorts after controlling for p16 status, FIGO stage, and other tumor features. Decreased RFS also correlated with spaTILH in St (aHR: 1.9; CI: 1.1-3.4) and Sv (aHR: 1.9; CI: 1.1-3.6) cohorts with confounding adjustment. When analyses were restricted to those receiving adjuvant radiation therapy, the associations remained robust with spaTILH portending increased hazards for both local (HR: 2.2; CI: 1.1-4.5) and regional (HR: 4.2; CI: 1.4-12.7) recurrences. Higher overall TIL density showed borderline associations with OS (HR: 0.5; CI: 0.2-1.0) and RFS (HR:1.9; CI: 1.0-3.5) in univariable models but lost statistical significance after controlling for confounding factors in cohorts. Conclusion: Our data support the utility of quantifying TIL spatial disorganization as an independent predictor of worse VSCC prognostic outcomes, warranting further evaluation of its applicability in clinical risk stratification.