PD-L1 Immunohistochemistry Analysis in Cancers

The immunohistochemistry reaction was performed using BenchMark Ventana Ultra™ (Ventana, Tucson, AZ, USA) platform, through multimer linked to horse radish peroxidase, to detect PD-L1 protein, as previously reported [25 (link)]. The anti-PD-L1 (E1L3N®) XP® Rabbit mAb, Cell Signaling Technology, was used as primary antibody and we used the OptiView DAB IHC Detection Kit, following manufacturer’s guidelines. Placental syncytiotrophoblast was used as positive control tissue. The combined positive score (CPS) was used to measure the expression of PD-L1. CPS corresponds to the ratio between the total of PD-L1 positive cells (tumor cell, lymphocytes and macrophages) and the total of viable tumor cells, multiplied by 100 [26 (link)]. Considering the experience of appropriateness of CPS cutoff in other types of tumors and no agreement of CPS cutoff for CUPs, we used CPS ≥ 1 in our study [27 (link)].