Chapter 3 86 previous work25. In 27 patients, none of the biomarkers could be assessed because samples did not contain tumor tissue. In one sample no tumor tissue was left for CD8 and PD-1T TIL analysis, and five samples had insufficient tumor tissue for CD3 TIL, TLS and CD20+ B cell analysis (Fig. S1). An additional number of 32 patients were excluded for PD-1T TIL analysis based on the following criteria: samples contained less than 10,000 cells (n=12), were obtained from endobronchial lesions (n=16), contained abundant normal lymphoid tissue (n=1) and showed fixation and/or staining artefacts (n=2) (Fig. S1). As described before, we excluded bronchial biopsies because they frequently showed unspecific antibody staining due to mechanical damage, and lymph node resections due to presence of PD-1+ T cells in normal abundant lymphoid tissue, which could potentially lead to false positive results25. One sample was excluded for CD8 TIL, CD3 TIL, TLS, CD20+ B cell and PD-L1 analysis because of fixation/staining artefacts. One sample contained less than 2,000 cells and was excluded for CD8 TIL, CD3 TIL, TLS and CD20+ B cell analysis. A total of 67 patients (41%) were excluded for mRNA expression analysis due to low RNA yield and/or low RNA quality (Fig. S1). Immunohistochemistry The CD8 immunostaining of samples was executed using the BenchMark Ultra autostainer Instrument (Ventana Medical Systems) on 3 µm paraffin sections from FFPE blocks. Initially, sections were baked at 75°C for 28 minutes and deparaffinized in the instrument with EZ prep solution (Ventana Medical Systems). Heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 32 minutes. CD8 was detected using clone C8/144B (1/200 dilution, 32 minutes at 37°C, Agilent/DAKO). Bound antibody was detected using the OptiView DAB Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems). Immunostaining for PD-1 was carried out using clone NAT105 (Roche Diagnostics), for PD-L1 using clone 22C3 (Agilent/DAKO), and for CD68 using clone KP1 (Agilent/ DAKO). For the double staining of CD20 (yellow) followed by CD3 (purple), clone L26 (Agilent/DAKO) (CD20) and clone SP7 (Thermo Fisher) (CD3) were used. All immunostainings were performed as described previously25. The immunostained slides for CD8, PD-1, PD-L1 and CD68 were subjected to scanning at a magnification of x20 with a resolution of 0.50 per µm2 using an Aperio slide AT2 scanner (Leica Biosystems). Immunostained slides for CD20CD3 were scanned at x20 magnification with a resolution of 0.24 per µm2 using a
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