Flex polymer system

Formalin‐fixed, paraffin‐embedded (FFPE) tissue blocks were deparaffinized and rehydrated with serial passage through changes of xylene and graded ethanols for PD‐L1 and PD‐1 IHC. All slides were subjected to heat‐induced epitope retrieval in Envision FLEX Target Retrieval Solution, High pH (Dako Corporation, Carpinteria, Calif). Endogenous peroxidase in tissues was blocked by incubation of slides in 3% hydrogen peroxide solution before incubation with the primary antibody (anti‐PD‐L1 clone 22C3 [Merck Research Laboratories, Palo Alto, Calif] or anti‐PD‐1 clone NAT105 [Cell Marque, Rocklin, Calif]) for 60 minutes. Antigen‐antibody binding was visualized with the FLEX + polymer system (Dako) and application of 3,3' diaminobenzidine chromogen (Dako). Stained slides were counterstained with hematoxylin and coverslipped for review and scoring. Scoring was conducted by a pathologist (J.H.Y.) who was blinded to all patient clinical information using a semiquantitative scale of 0 to 5, in which positive cell frequency within the tumor tissue was grouped into the following categories: 0 indicates negative, 1 indicates rare, 2 indicates low, 3 indicates moderate, 4 indicates high, and 5 indicates very high. These patterns are illustrated for both PD‐1 and PD‐L1 in Supporting Information Figure 1.

IHC for ICAM-1 was conducted on 5-μm sections of FFPE tissue. In brief, tissue sections were deparaffinized and rehydrated with serial passage through changes of xylene and graded ethanols. All slides were subjected to heat-induced epitope retrieval in Envision FLEX Target Retrieval Solution, High pH (Dako Corporation, Carpinteria, CA) in a PT Link unit (Dako). Endogenous peroxidase in tissues was blocked by incubation of slides in 3% hydrogen peroxide solution before incubation with primary antibody (anti-ICAM-1 clone E3Q9N, Cell Signaling Technology, Danvers, MA) for 60 min. Antigen-antibody binding was visualized with the FLEX + polymer system (Dako) and application of 3,3′ diaminobenzidine chromogen (Dako). Stained slides were counterstained with hematoxylin and coverslipped for review and scoring. Scoring was conducted by a pathologist using a semiquantitative scale of 0–5, in which positive tumor and immune cell frequency within the tumor region was grouped into the following categories: 0 indicates negative, 1 indicates rare, 2 indicates low, 3 indicates moderate, 4 indicates high, and 5 indicates very high.