Expression of LAG-3, TIM-3, and VISTA on Tumor-Infiltrating Lymphocytes in Advanced Laryngeal Squamous Cell Carcinoma and Their Association with CD8+ TIL Density

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Abstract
Background: Alternative immune checkpoints such as lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin-domain-containing-3 (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA) have emerged as potential modulators of tumor immune escape. However, their expression patterns and prognostic significance in advanced laryngeal squamous cell carcinoma (LSCC) remain insufficiently characterized. This study aimed to evaluate LAG-3, TIM-3, and VISTA expression on tumor-infiltrating lymphocytes (TILs), examine their association with CD8+ TIL density and clinicopathological features, and determine their impact on survival outcomes. Methods: In this retrospective observational cohort study, 132 patients who underwent total or partial laryngectomy for stage III–IV LSCC were included. Tissue microarrays were constructed using three 2 mm cores per case. Immunohistochemical expression of LAG-3, TIM-3, VISTA, and CD8 was assessed exclusively on TILs. Survival outcomes were analyzed using Kaplan–Meier and Cox proportional hazards models. Results: LAG-3, TIM-3, and VISTA positivity rates were 26.5%, 51.5%, and 53.8%, respectively. High CD8+ TIL density was observed in 69.7% of cases. Significant positive correlations were identified among checkpoint markers (all p < 0.05); VISTA positivity did not significantly correlate with CD8 infiltration (r = 0.149, p = 0.088). LAG-3 positivity was associated with lower thyroid cartilage invasion (p = 0.006). Kaplan–Meier analysis demonstrated no significant differences in overall survival (OS) or disease-free survival (DFS) according to checkpoint expression or CD8 status. In multivariable analysis, extranodal extension (HR = 2.719, p = 0.005) and thyroid cartilage invasion (HR = 1.970, p = 0.043) were independent predictors of worse OS. CD8 negativity showed a trend toward adverse OS (HR = 1.825, p = 0.084). None of the immune markers independently predicted DFS. Conclusions: In advanced surgically treated LSCC, LAG-3, TIM-3, and VISTA expression correlate with CD8+ TIL density but do not independently predict survival outcomes. These findings suggest that alternative immune checkpoint expression reflects immune engagement rather than intrinsic tumor aggressiveness and may hold greater predictive than prognostic relevance in LSCC.
Funding Information
  • Adnan Menderes Üniversitesi (TPF-23013)

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