Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer.

BACKGROUND: Tumor infiltrating lymphocytes (TILs) reflect adaptive antitumor immune responses in cancer and are generally associated with favorable prognosis. However, the relationships between TILs subsets and their spatial arrangement with clinical benefit from immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC) remains less explored. METHODS: We used multiplexed quantitative immunofluorescence panels to determine the association of major TILs subpopulations, CD8+ cytotoxic T cells, CD4+ helper T cells and CD20+ B cells, and T cell exhaustion markers, programmed cell death protein-1 (PD-1),lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin mucin-3 (TIM-3) with outcomes in a multi-institutional cohort of baseline tumor samples from 179 patients with NSCLC treated with ICI. The analysis of full-face tumor biopsies including numerous fields of view allowed a detailed spatial analysis and assessment of tumor immune heterogeneity using a multiparametric quadratic entropy metric (Rao's Q Index (RQI)). RESULTS: TILs were preferentially located in the stromal tissue areas surrounding tumor-cell nests and CD8+ T cells were the most abundant subset. Higher density of stromal CD8+ cytotoxic T cells was significantly associated with longer survival, and this effect was more prominent in programmed death ligand-1 (PD-L1) positive cases. The role of baseline T cell infiltration to stratify PD-L1 expressing cases was confirmed measuring the T cell receptor-burden in an independent NSCLC cohort studied with whole-exome DNA sequencing. High levels of LAG-3 on T cells or elevated RQI heterogeneity index were associated with worse survival in the cohort. CONCLUSION: Baseline T cell density and T cell exhaustion marker expression can stratify outcomes in PD-L1 positive patients with NSCLC treated with ICI. Spatial immune heterogeneity can be measured using the RQI and is associated with survival in NSCLC.

Spatial Analysis and Clinical Significance of HLA Class-I and Class-II Subunit Expression in Non-Small Cell Lung Cancer.

PURPOSE: To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell-specific defects, and clinicopathologic/survival associations of ß2 microglobulin (ß2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II ß chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts. RESULTS: Cancer cell-specific downregulation of HLA markers was identified in 30.4% of cases. ß2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of ß2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in <5% of lung malignancies. Tumors with cancer cell-specific ß2M downregulation displayed reduced T cells and increased natural killer (NK)-cell infiltration. Samples with cancer cell HLA-A downregulation displayed modest increase in CD8+ T cells and NK-cell infiltration. Samples with cancer cell-selective HLA-B,-C or HLA class-II downregulation displayed reduced T cells and NK-cell infiltration. There was limited association of the markers with clinicopathologic variables and KRAS/EGFR mutations. Cancer cell-selective downregulation of the HLA subunits was associated with shorter overall survival. CONCLUSIONS: Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival.