E1l3n xp rabbit mab

Formalin-fixed, paraffin-embedded (FFPE) samples from the 48 enrolled patients were retrospectively included in the study. The FFPE tissue slides from the 48 samples were immunostained by a two-step method. Anti-PD-L1 antibodies ((E1L3N) XP Rabbit mAb, 1:200; Cell Signaling Technology, Danvers, MA, USA), anti-PD-1 antibodies ((D4W2J) XP Rabbit mAb, 1:200; Cell Signaling Technology, Danvers, MA, USA), anti-L1CAM antibodies ((OTI10C12) XP Mouse mAb, 1:50; OriGene Technologies, Rockville, MD, USA), anti-Ki-67 antibodies ((MIB-1) XP Mouse mAb, 1:200; OriGene Wuxi Biotechnology Co, Wuxi, Jiangsu, CHN), and a DAB Detection Kit (Polymer) (PV-6000-D, ZSGB-BIO, Beijing, China) were used. Five fields were selected randomly in each case by ×400 magnification, and then the percentage of positive cells (PPC) and staining intensity (SI) were assessed using Image-pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The immunoreactive score (IRS) was determined based on the PPC and SI as follows: IRS = PPC × SI. The PPC scores were as follows: 0%, 0; 0–25%, 1; 25–50%, 2; 50–75%, 3; and 75–100%, 4. The SI scores were as follows: absent, 0; weak, 1; moderate, 2; and strong, 3. The expression levels of PD-L1, PD-1 and L1CAM were calculated as the mean IRSs of 5 fields, and Ki-67 was defined by the mean percentage of stained nuclei in 5 random fields.

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)].

PD-L1 expression was assessed by IHC staining, using SP263 (Ventana Benchmark Ultra, Tuscon, AZ, USA), the 22C3 pharmDx kit (Agilent Technologies, Santa Clara, CA, USA), or E1L3N XP Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA). If more than 1% of viable tumor cells had PD-L1, they were considered PD-L1-positive [18 (link)]. The tumor proportion score (TPS) was defined as the percentage of viable tumor cells showing partial or complete membrane-staining at any intensity [19 (link)]. The combined positive score (CPS) was defined as the number of PD-L1-positive cells (tumor cells, lymphocytes, and macrophages), divided by the total number of viable tumor cells, and multiplied by 100 [20 (link)].
The microsatellite stability was assessed using antibodies specific for mismatch repair proteins, including mutL homolog 1 (1:10; clone G168-15; BD Pharmingen, San Jose, CA, USA), mutS homolog (MSH)-2 (1:100; clone FE11; Calbiochem, San Diego, CA, USA), MSH6 (1:100; clone EP49; Novus Biologicals, Centennial, CO, USA), and PMS1 homolog 2 (1:50; clone A16-4; BD Pharmingen).