Chapter 2 46 TLS compared to tumor areas outside of TLS (P<0.0001) (Fig. 5D). We found similar results when performing the same analysis including TLS+LA (Fig. S6C). Of note, samples without TLS or LA showed only very low frequencies of PD-1T TILs (Fig. 5D, Fig. S6C). Next, we investigated whether the number of TLS was associated with clinical benefit to PD-1 blockade in NSCLC as has been shown in other tumor types14–16. The ranges of TLS and TLS+LA per mm2 are shown in Fig. 5E and Fig. S6D (DC 12m) and Fig. S6E,F (DC 6m). The AUC of TLS per mm2 was 0.62 (95% CI: 0.47-0.76) for DC 12m (Fig. 5F) and 0.62 (95% CI: 0.49-0.76) for DC 6m (Fig. S6G), respectively, indicating a lower predictive performance than PD-1T TILs in the same sample set. Similar results were observed for TLS+LA per mm2 and CD20-positive area per mm2 (Fig. 5F, Fig. S6G). As PD-1T TILs predominantly localize in TLS, this observation would possibly be consistent with subtypes of TLS that differ in the number of PD-1T TILs. To investigate this, we quantified the frequency of TLS and of PD-1T TILs inside and outside TLS in PD-1T high (n=13) and PD-1T low resected samples (n=10). Resected samples with no TLS were excluded from this analysis (n=9). We found that TLS numbers did not significantly differ between both groups (Fig. 5G). However, tumors in the PD-1T high group had significantly higher numbers of PD-1T TILs inside TLS (Fig. 5H). In addition, in PD-1T high tumors also significantly more PD-1T TILs were present outside of TLS (Fig. 5I) compared to the PD-1T low group. Notably, PD-1T lymphocytes were only sparsely present in the tumor parenchyma of PD-1T low tumors (Fig. 5I). Altogether, these data suggest that in tumors responding to PD-1 blockade PD-1T TILs not only infiltrate TLS in higher numbers, but also expand in the tumor parenchyma. Figure 4. Association of PD-L1 with long-term benefit and survival compared to PD-1T TILs. (A) Immunohistochemical (IHC) analysis of PD-L1. Example of NSCLC tumors with ≥50%, ≥1% and 0% PD-L1 expression (PD-L1 TPS), respectively. (B) Percentage of patients with ≥50% (n=8), 1-50% (n=19) and 0% PD-L1 TPS (n=50) in pretreatment samples showing DC 12m or PD in the validation set (n=77). (C) ROC curve for predictive value of PD-L1 TPS for DC 12m (AUC 0.68; 95% CI: 0.51-0.86) in the validation set (n=77). (D) Sensitivity and specificity of PD-L1 TPS 50% and 1% for DC 6m and 12m in comparison to PD-1T 90 per mm2 in the validation set (n=77). (E) PFS (HR 0.36; 95% CI: 0.18-0.70, * P=0.03) and (F) OS (HR 0.40; 95% CI: 0.20-0.80, P=0.06) of patients with ≥50% versus <50% PD-L1 TPS in pretreatment samples in the validation set (n=77). (G) PFS (HR 0.63; 95% CI: 0.38-1.03, P=0.08) and (H) OS (HR 0.67; 95% CI: 0.40-1.11, P=0.14) of patients with ≥1% versus <1% PD-L1 TPS in pretreatment samples in the validation set (n=77). Tick marks represent data censored at the last time the patient was known to be alive and without disease progression or death. P-value was determined by log-rank test. ▶
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