Both the 113 primary CRC tumours with 105 liver and 59 lung metastases were included in analogous immunohistochemical analyses. Formalin-fixed paraffin-embedded whole tissue sections of 3 μm thickness were used. Mismatch repair status was determined by immunohistochemical analysis for the expression of MLH1, PMS2, MSH2, and MSH6 as described previously [47 (link)]. Staining for PD-1 (The Human Genome Organization (HUGO) name PDCD1) and PD-L1 (HUGO name CD274) was conducted with anti-PDCD1 (SP269, 1:50; Spring Bioscience, Pleasanton, CA, USA) and anti-CD274 (E1L3N, 1:100; Cell Signaling Technology, Danvers, MA, USA) antibodies, using a BOND-III stainer (Leica Biosystems, Buffalo Grove, IL, USA). Staining for CD3 and CD8 was conducted with anti-CD3 (LN 10, 1:200; Leica Biosystems, Newcastle, UK) and anti-CD8 (SP16, 1:400; Thermo Scientific, Fremont, CA, USA) antibodies, using a Lab Vision Autostainer 480 (ImmunoVision Technologies Inc., Brisbane, CA, USA). Signal visualization was done by diaminobenzidine and sections were counterstained with haematoxylin. Slides were scanned with a NanoZoomer-XR (Hamamatsu Photonics, Hertfordshire, UK) at ×20 magnification.
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