PD-1T TILs as precision biomarker in NSCLC 51 2 allowed to correctly classify a sample as PD-1T high or low, suggesting that PD-1T TIL infiltration can be reliably captured in a relatively small area of the tumor. An additional level of heterogeneity may result from, for instance, sample type, sampling site and/or the time between sampling and initiation of treatment. Thus, we explored the impact of these potential confounding factors on the predictive value of PD-1T TILs as a biomarker. We observed a trend towards increased predictive accuracy of PD-1T TILs when measured in tumor resections as compared to biopsies. In contrast, no difference in predictive value of the biomarker was found between primary tumors and metastases. Hong and colleagues previously observed differences in PD-L1 expression levels in distinct anatomical sites and showed that high PD-L1 was associated with better clinical outcome in lung and distant metastases, but not in lymph node biopsies40. The low number of lymph node biopsies in our sample set precluded the separate analysis of lymph node and organ metastases, thus a possible difference in predictive value between these sample sites should be further explored in future work. Finally, we observed that samples taken immediately before start of PD-1 blockade were more accurate for prediction of clinical benefit, as shown by the higher AUC, reaching significance for prediction of DC 12m. Collectively, these data suggest that heterogeneity in PD-1T TIL infiltration across and within lesions exist, and that PD-1T TILs may therefore be reflective of the capacity of locally residing T cells to control tumor growth upon anti-PD-1. Hence, it will be important in future studies to address the mechanistic basis of this heterogeneity, such as differences in local antigen availability, HLA expression or else. A further aim of the study was to explore the association of PD-1T TILs to other immune-related biomarkers, such as PD-L1 and TLS. PD-1T TILs performed superior to PD-L1, since both 50% and 1% PD-L1 TPS showed substantially lower sensitivity and NPV. Predictive performance could be improved when PD-L1 TPS and PD-L1 IC were combined, but remained below that observed with PD-1T TILs. Notably, whereas previous studies showed an additive value of PD-L1 to TMB30,33 and PD-L1 to CD831,32, the combination of either 50% or 1% PD-L1 TPS with PD-1T TILs did not improve predictive accuracy. However, since the ≥50% PD-L1 group only comprised 10% of the samples in the validation set, which is different from previous reported percentages1,2, additional studies with subgroups that are more balanced or including PD-L1 IC should validate these findings. As another immune-related marker, TLS have recently been associated with response and survival benefit to ICB in multiple cancer types13–16, and in previous work we observed that PD-1T TILs predominantly localize in TLS10. In the present study we found a lower predictive accuracy of TLS and LA compared to PD-1T TILs. The observation that tumors with high and low PD-1T TIL count do not show substantially different frequencies of TLS, but vary in the number
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