Abstract / Summary
Abstract Introduction: Spatial organization and functionality of tumor-infiltrating lymphocytes may provide prognostic information beyond immune-cell abundance alone in advanced non-small cell lung cancer (NSCLC). We investigated whether multiplex immunophenotyping of tumor-proximal CD8 + and CD4 + immune engagement, according to naive (CD45RA+) or memory-like (CD45RO+) cell states, identify clinically relevant immune architectures in patients treated with first-line immune checkpoint inhibitor (ICI)-based therapy. Methods This retrospective cohort included 39 patients with stage IIIB–IV NSCLC treated between 2019 and 2022 with first-line pembrolizumab monotherapy or chemo-immunotherapy. Formalin-fixed paraffin-embedded tumor samples were analyzed by multiplex immunophenotyping (CellScape™) for CD4, CD8, CD45RO, CD45RA, granzyme B and PanCK-based tumor compartments. Patient-level immune-cell densities and nearest PanCK-distance metrics were quantified after core aggregation. A tumor engagement index (TEI) was defined as phenotype density divided by the upper-quartile nearest PanCK distance, integrating immune abundance with tumor proximity. Patients were stratified by predefined progression-free survival (PFS; 6-month cutoff) and overall survival (OS; 12-month cutoff). Results CD8 + CD45RO+ cells showed the strongest tumor-proximal immune phenotype and the most consistent association with favorable outcome. Higher final CD8 + CD45RO + TEI was associated with prolonged PFS and OS in Kaplan–Meier analyses (median PFS, 17 vs 6 months; median OS, 48.0 vs 15.4 months). Neither CD4+, nor CD45RA+ phenotypic dominance showed significant influence on clinical outcomes. In Cox models adjusted for sex, treatment group, metastatic burden and PD-L1 status, spatial TEI remained independently associated with both PFS (HR 0.09, 95% CI 0.02–0.44, p = 0.003) and OS (HR 0.10, 95% CI 0.02–0.42, p = 0.002). Elastic-net Cox analysis supported CD8 + CD45RO + TEI as the dominant stable spatial feature, with high bootstrap selection frequency for both PFS and OS. Conclusion A tumor-proximal CD8 + CD45RO+ immune architecture identifies a favorable spatial immune phenotype far exceeding the predictive power of mere CD8 + density and PD-L1 expression in advanced NSCLC treated with first-line immunotherapy-based regimens.