Karlijn Hummelink

Chapter 4 148 This is in line with the notion that the predictive capacity is specifically driven by the PD-1T TIL subset, which reflects a tumor-reactive population that is likely crucial for response to PD-1 blockade, and not simply by the presence of PD-1 positive T cells8. The development of predictive mRNA signatures that characterize a broader transcriptomic immune profile of the TME has gained interest. For example, the commercially available Tumor Inflammation Signature (TIS) has shown to predict clinical benefit in different cancer types, including NSCLC11–13. We correlated the PD1T signature scores to the TIS scores in samples from the validation cohort (n=42). We observed a good correlation between the signatures (R2=0.705, P<0.0001), in line with a partial overlap in three genes (STAT1, CD274, LAG3) (Fig. S3). While the small sample size limits conclusions that can be made from this analysis, it may further support the notion that these types of mRNA signatures can robustly detect tumor immune environments that are responsive to immune checkpoint inhibitors (ICI). In the future, it will be interesting to compare the performance of the signatures in a larger patient cohort to understand whether they report on similar features of the TME or whether there may be possible additive value for a subgroup of patients. While the analysis of samples from two distinct expert centers on NSCLC ICI therapy strengthens the results of the current study, a number of limitations should be noted: First, the sample size was low in both the training and the validation cohorts. Thus, additional studies with higher patient numbers are needed. Second, this is a retrospective study which makes further validation in prospective studies necessary. Third, different PD-L1 IHC antibodies were used in the validation cohort which could potentially have introduced analytical differences19,20. Finally, an important emerging question in the clinical treatment of NSCLC is how to preselect patients for therapy with either single agent PD-1 blockade or in combination with chemotherapy. As we developed the PD-1T biomarker aimed at high sensitivity and NPV, the signature is currently not designed to make such predictions. Nevertheless, retraining of the biomarker for high specificity and PPV in a suitable patient cohort could be considered for this purpose. Such an approach, however, may be potentially limited by the number of false positives driven by a relatively large fraction of mixed responses in the biomarker high, PD group as shown previously7. Taken together, in the present study we developed a PD-1T gene expression signature with high sensitivity and NPV that can be used as a predictive biomarker for nonresponse to PD-1 blockade in NSCLC. Our data demonstrate, that our digital imagebased IHC assay can reliably be replaced by a matching gene expression signature

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