Immune Marker Spatial Distribution and Clinical Outcome after PD-1 Blockade in Mismatch Repair–deficient, Advanced Colorectal Carcinomas

Purpose: Targeting the programmed cell death protein 1 (PD-1)/ programmed cell death ligand 1 (PD-L1) interaction has led to durable responses in fewer than half of patients with mismatch repair–deficient (MMR-d) advanced colorectal cancers. Immune contexture, including spatial distribution of immune cells in the tumor microenvironment (TME), may predict immunotherapy outcome. Experimental Design: Immune contexture and spatial distribution, including cell-to-cell distance measurements, were analyzed by multiplex immunofluorescence (mIF) in primary colorectal cancers with d-MMR (N = 33) from patients treated with anti–PD-1 antibodies. By digital image analysis, density, ratio, intensity, and spatial distribution of PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFβR2, MHC-I, CD14, B2M, and pan-cytokeratin were computed. Feature selection was performed by regularized Cox regression with LASSO, and a proportional hazards model was fitted to predict progression-free survival (PFS). Results: For predicting survival among patients with MMR-d advanced colorectal cancer receiving PD-1 blockade, cell-to-cell distance measurements, but not cell densities or ratios, achieved statistical significance univariately. By multivariable feature selection, only mean number of PD-1⁺ cells within 10 μm of a PD-L1⁺ cell was significantly predictive of PFS. Dichotomization of this variable revealed that those with high versus low values had significantly prolonged PFS [median not reached (>83 months) vs. 8.5 months (95% confidence interval (95% CI), 4.7–NR)] with a median PFS of 28.4 months for all patients [adjusted HR (HR_adj) = 0.14; 95% CI, 0.04–0.56; P = 0.005]. Expression of PD-1 was observed on CD8⁺ T cells; PD-L1 on CD3⁺ and CD8⁺ T lymphocytes, macrophages (CD68⁺), and tumor cells. Conclusions: In d-MMR colorectal cancers, PD-1⁺ to PD-L1⁺ receptor to ligand proximity is a potential predictive biomarker for the effectiveness of PD-1 blockade.