A PD-1T signature as clinical applicable biomarker in NSCLC 141 4 ◀ Figure 2. PD-1T signature development for prediction of non-response to PD-1 blockade. (A) PD-1T TILs per mm2, as measured by digital IHC algorithm-based quantification, in pretreatment samples from patients with disease control at 12 months (DC 12m) (n=12) and progressive disease (PD) (n=46) in the training cohort (n=58). Only patients with available PD1T IHC and gene expression data are included. Dashed line indicates a cut-off of 90 PD-1T TILs per mm2. Medians, interquartile ranges and minimum/maximum shown in boxplots, **P<0.01 by Mann Whitney U-test. The red colored dots indicate PD-1T IHC high (≥90 per mm2) patients with DC 12m (n=12) and the blue colored dots indicate PD-1T IHC low (<90 per mm2) patients with PD (n=29). (B) Gene set enrichment analysis displaying gene sets that are significantly enriched in PD-1T IHC high tumors from patients with DC 12m (n=12). Pathways are ordered by P value (log transformed), P values were calculated by Fisher exact test. (C) Volcano plot showing the differentially expressed genes between PD-1T IHC high tumors from patients with DC 12m and PD-1T IHC low tumors from patients with PD (n=29) in the training cohort (n=41). 56 genes reached statistical significance. The red line indicates a P-value <0.05. P-values were computed by moderated t-statistics. D) Heatmap showing the expression of the 12-gene PD-1T signature in pretreatment samples from PD-1T IHC high patients with DC 12m (n=12) and PD1T IHC low patients with PD (n=29) in the training cohort (n=41) ordered by probability score. Each column represents one patient (blue: PD-1T signature high (score of ≥0.35) and light grey: PD-1T signature low (score of <0.35), green: PD-1T IHC high patients with DC 12m, dark grey: PD-1T IHC low patients with PD) and rows display genes. Positive values (red) indicate higher gene expression and negative values (blue) indicate lower gene expression. (E) Correlation of PD-1T TILs and the PD-1T signature in the training cohort (n=41), R2=0.603, ****P<0.0001. R2 and P-values were calculated using linear regression analysis. (F) PD-1T signature scores in pretreatment samples from PD-1T IHC high patients with DC 12m (n=12) and PD-1T IHC low patients with PD (n=29) in the training cohort (n=41). The dashed line indicates a cut-off score of 0.35. Medians, interquartile ranges and minimum/maximum shown in boxplots, ****P<0.0001 by Mann Whitney U-test. (G) Receiver operating characteristic (ROC) curve for predictive value of the PD-1T signature for DC 12m (AUC 0.97; 95% CI: 0.93-1.00) in the training cohort (n=41). Table 1. Overview of the up- and downregulated PD-1T signature genes. PD-1T signature genes STAT1 UP OAS1 UP TAP1 UP HEY1 DOWN CXCL13 UP IFIT2 UP IL6 UP TDO2 UP CD6 UP CTLA4 UP CD274 UP LAG3 UP
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