Karlijn Hummelink

A PD-1T signature as clinical applicable biomarker in NSCLC 139 4 Regularized regression analysis (LASSO) yielded a 12-gene PD-1T signature (STAT1, OAS1, TAP1, HEY1, CXCL13, IFIT2, IL6, TDO2, CD6, CTLA4, CD274, LAG3) as being most predictive (Table 1). All genes in the signature were positively associated with DC 12m, except for HEY1, which showed a negative association (Fig. 2C,D). In line with the selection strategy of the samples, we observed a high correlation between the number of PD-1T TILs assessed by IHC and the PD-1T signature score within samples (R2=0.603; P<0.0001; Fig. 2E). The PD-1T signature was able to separate the preselected PD‑1T IHC high DC 12m group from the preselected PD-1T IHC low PD group with high significance (P<0.0001) (Fig. 2D,F). The area under the ROC curve (AUC) was 0.97 (95% CI: 0.93-1.00) (Fig. 2G). We aimed for a sensitivity of ≥90% to minimize the risk of undertreatment and a specificity of ≥50%, a strategy that was previously used for the PD-1T TIL IHC biomarker7. A probability score of 0.35 was selected as optimal cutoff (Fig. 2F). This cut-off resulted in a sensitivity of 92%, specificity of 93%, positive predictive value (PPV) of 85% and NPV of 96% (Table 2). PD-1T signature validation Next, the predictive performance of this PD-1T signature was validated in an independent cohort of 42 patients with advanced stage NSCLC treated with nivolumab. 6/42 (14%) patients showed DC 12m and 36/42 (86%) showed PD (Fig. 1). In contrast to the training cohort, tumor samples were not preselected. None of the clinicopathological characteristics differed significantly between training and validation set, except for the performance score as more patients in the validation cohort showed a higher performance score (P<0.01) (Table S3). In the validation cohort, in line with previously observed patterns of PD-1T TILs quantified by IHC, PD-1T signature scores were significantly higher in the DC 12m group than in the PD group (P<0.01) (Fig. 3A). A high PD-1T signature score (≥0.35) correctly identified 6/6 patients with DC 12m (sensitivity for treatment benefit 100%), and a low score (<0.35) identified 23/36 patients with PD (specificity for no treatment benefit 64%), yielding a PPV of 32%, and an NPV of 100%, with an AUC of 0.87 (95% CI: 0.74-0.99) (Fig. 3A,B and Table 2). Similar to the training cohort, most signature genes were overexpressed in the PD-1T signature high DC 12m group (n=6), compared to the PD-1T signature low PD group (n=23), and the HEY1 gene was expressed at lower levels. The PD-1T signature high patients with PD (n=13) showed a similar gene expression profile as the PD-1T signature high DC 12m group (Fig. 3C).

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