PD-1T TILs as precision biomarker in NSCLC 45 2 partitioned the patient population into four groups: (1) PD-L1 low (<50% or <1%)+PD1T low, (2) PD-L1 low+PD-1T high, (3) PD-L1 high (≥50% or ≥1%)+PD-1T low and (4) PD-L1 high+PD-1T high (Fig. S5B). We observed an enrichment of PD patients in the <50% PD-L1+PD-1T low group (35/38 when assessing DC 6m as clinical outcome, 38/38 for DC 12m). Patients with DC 6m or 12m were distributed over all 4 groups and 3 out of 4 groups, respectively (Fig. S5C,D). A similar patient distribution was found for the combination with 1% PD-L1 TPS (Fig. S5E,F). The predictive value of PD-1T+PD-L1 at 50% cut-off was comparable to PD-1T alone with a few percent increase in sensitivity at the cost of a slightly lower specificity (Fig. S5G, Table S9, for details on prediction model see Methods section). The sensitivity of PD-1T+PD-L1 at 1% cut-off was similar to PD-1T+PD-L1 50%, but the specificity of this combination was below 50% (Fig. S5G, Table S9). Thus, the predictive accuracy of PD-1T alone is not increased by parallel quantification of PD-L1 levels. PD-1T TILs and tertiary lymphoid structures Tertiary lymphoid structures (TLS) are immune cell aggregates that form in the context of chronic inflammation and have been described in many cancer types, including NSCLC34–36. A number of recent studies have shown that TLS and B cells as one of their main cellular components are associated with response to ICB in melanoma, renal cell carcinoma and sarcoma14–16. Moreover, we previously showed in a small number of NSCLC samples that PD-1T TILs appear to predominantly localize in TLS and constitutively secrete CXCL13, a chemoattractant that is crucial for the formation of TLS10. For 91 of our pretreatment samples for which additional FFPE material was available, we assessed whether TLS and B cells were present. To this end, CD20/CD3 double IHC staining were performed to identify TLS by the presence of B cell clusters and T cell zones, as described previously10,16,22,23,35. A CD3+CD20+ area was defined as TLS when its size was more than 60,000 µm2 in the annotated tumor area, and as lymphoid aggregate (LA) when between 10,000 and 60,000 µm2. To estimate the presence of B cells, we quantified the CD20-positive area per mm2 (Fig. S6A). This analysis revealed that TLS and TLS and/or LA (referred as TLS+LA) were present in 30/91 (33%) and 46/91 (51%) of tumors, respectively. B cells were found in 86/91 (95%) of tumors, suggesting that the presence of these cells does not always relate to TLS and LA (Fig. 5A). However, in most of the 40 samples without TLS+LA, CD20-positive area per mm2 was low (Fig. S6B). Next, we wanted to assess the localization of PD-1T TILs in relation to TLS. To improve the accuracy of this analysis we focused on tumor resections (n=32), for which the annotated TLS areas based on the CD20/CD3 double staining were copied to a consecutive slide stained for PD-1 to calculate the frequency of PD-1T TILs inside TLS (Fig. 5B-C). The frequency of PD-1T TILs per mm2 was significantly higher inside
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