Spatial determinants of TILs in immunotherapy sensitivity of patients with non-small cell lung cancer (NSCLC)

Immune checkpoint blockers (ICB) targeting T-cell co-inhibitory receptors such as PD-1 and CTLA-4 provide clinical benefit to a subset of patients with non-small cell lung cancer (NSCLC). The abundance and functional state of tumor-infiltrating lymphocytes (TILs) have been proposed as immunotherapy biomarkers. However, the role of the spatial arrangement and distribution of TILs in immune evasion and immunotherapy sensitivity remains less explored. We used multiplexed quantitative immunofluorescence to measure and spatially map major TIL subpopulations (CK, CD4, CD8 and CD20), candidate tumor-antigen specific T-cells (CK, CD8, CD39 and PD-1) and T-cell functional differentiation states (CD4, CD8, TOX, TCF7) in consecutive tissue microarray sections from baseline NSCLCs in three retrospective cohorts treated with frontline chemotherapy (CTX, n=156), first line ICB (1L-ICB, n=56) or ICB as second or subsequent treatment line (2L+-ICB, n=68). We studied the spatial characteristics of TILs using computational metrics of cell heterogeneity and clustering, and their association with clinicopathologic variables and treatment-specific outcomes. We identified five spatial metrics significantly associated with overall survival (OS) only in the 1L-ICB patient population. A high Morisita-Horn Index (spatial diversity) of CD8+ or CD20+ TILs and CK+ cancer cells in the stromal tissue compartment, and a high Ripley's k (Rk) function (spatial clustering) of CD8+ T-cells in the total tissue area were associated with shorter OS (HR=1.57, CI:1.09-2.28; HR=1.42, CI:1-2.01; HR=1.38, CI: 1.03-1.83; respectively). In contrast, a high Rk function of CD8+ T-cells and the Moran's Index (MI, spatial autocorrelation) of CD20+ TILs measured within the cancer cell nests were significantly associated with longer OS (HR=0.58, CI:0.35-0.95; HR=0.44, CI:0.23-0.84, respectively). Tumors with high Rk values for CD8+ TILs in the cancer cell compartment had a higher density of inactive effector T-cells (CD8+ PD1- CD39-) and bystander-like effector T-cells (CD8+ PD1+ CD39-) than tumors with low CD8+ Rk. Tumors with high MI of CD20+ TILs in the cancer cell area showed higher densities of inactive effector T-cells, progenitor-like effector T-cells (CD8+ TOX+ TCF7+) and lower levels of candidate tumor antigen-specific effector T-cells (CD8+ PD1+ CD39+) than tumors with low MI of CD20+ TILs. There was a significant association between higher MI of CD20+ TILs and advanced age (≥65). The spatial arrangement of TILs in baseline tumor samples is associated with distinct local T-cell responses and frontline immunotherapy outcomes in patients with NSCLC. Increased clustering of CD8+ and CD20+ TILs within the cancer cell nests is independently associated with favorable survival in this population. This supports a strong biomarker potential for the integrated analysis of TILs incorporating spatial features.