A PD-1T signature as clinical applicable biomarker in NSCLC 135 4 PD-L1 immunostaining of samples from the CERTIM cohort was performed using a different anti-PD-L1 antibody (E1L3N, Cell Signaling, catalog no. AB_2687655) on a Bond automat (Leica Biosystems) as described previously by Adam et al14. PD-1 IHC slides were scanned at x20 magnification with a resolution of 0.50 per µm2 using an Aperio slide AT2 scanner (Leica Biosystems). Digital quantification of PD-1T TILs In 11/94 (12%) pretreatment samples automated quantification of PD-1T TILs was performed using an image analysis algorithm with a cut-off of 0.25 optical density (OD) of PD-1 staining as described previously7. In 83/94 (88%) samples PD-1T TIL numbers were used from a previously published cohort and quantified using the same approach7. PD-1T TIL numbers of 58 samples were used to develop the signature and are provided in Supplementary Table S1. PD-L1 scoring Tumor PD-L1 expression in pretreatment FFPE samples was assessed in the NKIAVL training (n=58) and validation cohort (n=21) using the clinical grade LDT IHC assay with the 22C3 DAKO clone (Agilent) as described previously7. For 35/37 (95%) samples in the CERTIM cohort, PD-L1 tumor proportion score (TPS) data has been reported before13. In 2/37 (5%) the PD-L1 status was unknown. The expression levels were scored using a different anti-PD-L1 antibody (E1L3N, Cell Signaling, catalog no. AB_2687655) as previously described and validated by the PATTERN French thoracic pathologists’ group14. PD-L1 TPS data is provided in Supplementary Table S1. RNA extraction and hybridization to nCounter tagset RNA of pretreatment FFPE samples from the NKI-AVL cohorts were isolated with the AllPrep DNA/RNA FFPE isolation kit (#80234, Qiagen) according to the recommendations of the manufacturer and quantified by Tapestation (Agilent). RNA from the CERTIM cohort was extracted with High Pure FFPE RNA Isolation Kit (Roche Diagnostics) according to the recommendations of the manufacturer and quantified using fluorimetry with Qubit RNA XR Assay Kit (Invitrogen, Thermo Fisher). 200 to 300 ng RNA from the NKI-AVL cohorts and 30-100 ng RNA from the CERTIM cohort were hybridized to Nanostring PanCancer IO 360 Panel code set (Nanostring), according to the recommendations of the manufacturer. After hybridization non-bound probes were washed off and the RNA-probe complex was bound to the cartridge on the Nanostring Flex Prep Station (Nanostring) according to manufacturing protocol. The cartridge was sealed and transferred to the Digital Analyzer for imaging.
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