Pharmdx assay

TMAs were sectioned at a thickness of 4-μm and stained using the Dako pharmDx assay. Briefly, the slides were stained with anti-PD-L1 22C3 mouse monoclonal primary antibodies with the EnVision FLEX visualization system on a Dako Autostainer Link 48 instrument (Carpinteria, CA, USA), along with negative control reagents and cell line run controls, as per the manufacturer’s instructions. The IHC slides were scored independently by two pathologists (H.J. Kwon and H. Kim). PD-L1 was considered positive in tumor cells only in cases of at least 100 viable tumor cells, if membranous staining alone or membranous and cytoplasmic staining together was present. Membranous staining in tumor cells directly adjacent to immune cells was not considered positive if the surface touching immune cells was the only stained part. The percentage of stained cells in the overall area of the tumor (Tumor Proportion Score) was scored regardless of intensity [6 (link)]. Cases were then classified by two different cut-off values, 1% and 50%, based on the published association of this cut-off with anti-PD-1 therapeutic response [6 (link)].

Formalin-fixed, paraffin-embedded tissue sections of biopsy or surgical specimens were used for IHC. The PharmDx assay (Dako, Carpinteria, CA, USA) was utilized, using an anti-PD-L1 22C3 mouse monoclonal primary antibody and EnVision FLEX visualization system (Agilent, Santa Clara, CA, USA) on an Autostainer Link 48 system (Dako, Carpinteria, CA, USA) along with negative control reagents and cell line run controls, as per the manufacturers’ instructions^{23 (link),24 (link)}. PD-L1 staining was defined as complete or partial circumferential linear cellular membrane staining at any intensity that could be differentiated from the background as well as diffuse cytoplasmic staining^{23 (link),25 (link)}. Tonsillar tissue was used as a positive internal control^{23 (link)}. Positivity of PD-L1 status was determined based on a 1% threshold in tumor cells. A TPS was recorded as the overall percentage of PD-L1 stained tumor cells relative to the entire tumor area^{26 (link)}. A CPS was recorded based on the number of PD-L1 positive tumor and immune cells in relation to total tumor cells^{26 (link)}.

PD-L1 expression was measured by immunohistochemistry pharmDx assay (Agilent Technologies, Santa Clara, CA, USA) on archive HCC tissues for 18 patients. The anti-PD-L1 28-8 antibody was used for nivolumab-treated HCC, and anti-PD-L1 22C3 antibody was applied for pembrolizumab-treated HCC [7 (link),8 (link)]. Expression levels were reported by tumor proportion score (TPS) and/or combined positive score (CPS), respectively [7 (link),8 (link)].