For immunohistochemistry, we used commercially available antibodies presented in Table 1 . Staining was performed in the BenchMark XT immunostainer (Ventana Medical Systems, Tuscon, AZ, USA). After antigen retrieval with Cell Conditioning 1 buffer (Ventana Medical Systems) at 95 °C for 30 min, the slides were incubated with primary antibodies against the antigens. The immunoreactivity was visualized using the iVIEW DAB Detection Kit (Ventana Medical Systems), according to the manufacturer’s instructions. Sections were counterstained with hematoxylin. Positive controls and negative controls omitting the primary antibodies were also included.
p16 immunohistochemistry was considered positive if strong and diffuse nuclear and cytoplasmic expression was found in at least 75% of the tumor [17 (link)].
PD-L1 immunohistochemistry was determined by using the combined positive score (CPS), defined as the number of PD-L1-staining cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen was considered to have PD-L1 expression if CPS ≥ 1 [18 (link)].
p16 immunohistochemistry was considered positive if strong and diffuse nuclear and cytoplasmic expression was found in at least 75% of the tumor [17 (link)].
PD-L1 immunohistochemistry was determined by using the combined positive score (CPS), defined as the number of PD-L1-staining cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen was considered to have PD-L1 expression if CPS ≥ 1 [18 (link)].
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