Clone sp263

The H&E-stained specimens in each case were microscopically reviewed and each case was pathologically diagnosed according to the 2015 WHO classification [5 ]. An appropriate FFPE block containing the tumor in each case was selected by reviewing H&E-stained specimens and each FFPE block was sliced into 3-μm-thick tissue sections. The tissue sections were deparaffinized and subjected to immunostaining with the following four anti-PD-L1 antibodies: clone SP142 (Ventana Medical Systems, Tucson, AZ, USA), clone SP263 (Ventana Medical Systems), clone 22C3 (Dako, Carpinteria, CA, USA), and clone 28-8 (Dako). SP142 and SP263 immunostaining was carried out with a Bond-Max autoimmunostainer (Leica Microsystems, Wetzlar, Germany) and a Bond polymer-refine detection kit (Leica Microsystems). 22C3 and 28-8 immunostaining was carried out with a Dako autostainer Link48 system (Dako) and a PD-L1 PharmDx kit (Dako).

An appropriate formalin-fixed and paraffin-embedded (FFPE) block containing the tumor in each case was selected by reviewing H&E-stained specimens, and each FFPE block was sliced into 3-μm-thick tissue sections. The tissue sections were deparaffinized and subjected to immunostaining with the following for anti-PD-L1 antibodies: clone SP142 (Ventana Medical Systems, Tucson, AZ, USA), clone SP263 (Ventana Medical Systems), clone 28-8 (Dako, Carpentaria, CA, USA), and clone 22C3 (Dako). SP142 and SP263 immunostaining was carried out with a Bond-Max autoimmunostainer (Leica Microsystems, Wetzlar, Germany) and a Bond polymer-refine detection kit (Leica Microsystems). 28-8 and 22C3 immunostaining was carried out with a Dako autostainer Link48 system (Dako) and a PD-L1 PharmDx kit (Dako).

Formalin-fixed, paraffin-embedded (FFPE) tumor samples were collected at time of surgical resection before start of systemic treatment in melanoma and UC patients. 4 µm-thick FFPE tissue sections were subjected to heat-induced antigen retrieval and incubated with primary monoclonal antibodies against PD-L1: clone SP263 (Ventana Medical Systems Inc., Tucson, AZ, USA) for melanoma samples and clone 22C3 (Agilent Technologies, Santa Clara, CA, USA) for UC samples. Samples were visualized with 3,3′-diaminobenzidine (DAB) chromogen and hematoxylin counterstain and cover-slipped for review. Scoring of PD-L1 was conducted by 2 pathologists blinded to patient characteristics. In melanoma sections, the percentage of tumor cells with membranous PD-L1 staining was scored (0–100%). In UC sections, the percentage of tumor cells and any tumor infiltrating mononuclear inflammatory cells with membranous PD-L1 staining was scored (0–100%).
The abundance of intraepithelial TILs was determined on H&E stained sections. This morphological assessment of TILs within tumor nests was evaluated semi quantitatively: 1+, sporadic TILs; 2+, moderate number of TILs; 3+, abundant occurrence of TILs. For dichotomization, the TILs score was categorized into ‘low’ (1+ or 2+) and ‘high’ (3+). TILs were assessed on 19 melanoma patients as the only available specimens for the 20th patient was a cytological sample.

For immunohistochemical analyses, the TMA blocks were cut at 4 µm thickness. All PD-L1 immunohistochemical staining was carried out on a Benchmark Ultra System (Ventana Medical Systems, Tucson, AZ). Clone SP142 (Ventana Medical Systems; retrieval: CC1 48′; incubation: 16′; RTU dilution) and clone SP263 [Ventana Medical Systems; retrieval: CC1 40′; incubation: 32′; ready to use (RTU) dilution] were used in accordance with the manufacturer’s guidelines. The SP142 assay used tonsil tissue, and the SP263 assay used placenta tissue as control tissue. Two control tissues were used for each staining run, one as a positive control using an antibody reagent and one as a negative control using a negative reagent.
Additional immunohistochemical staining for CK5/6, CK14, GATA3, FOXA1, and CK20 was performed using a Ventana Benchmark XT automated staining system (Ventana Medical Systems). Mouse monoclonal antibodies against CK5/6 (1:100; D5/16 B4; Dako, Glostrup, Denmark), CK14 (1:300; LL002; Cell Marque, Rocklin, CA), GATA3 (1:500; L50-823; Cell Marque), FOXA1 (1:500; PA5-27157; Thermo Fisher, Waltham, MA), and CK20 (1:50; Ks 20.8; Dako) were used.

The PD-L1immunohistochemistry of 77 cases of non-small cell carcinomas of the lung diagnosed over a period of two years were reviewed and analyzed (2018-2020). All biopsies/ specimens were fixed in 10% neutral buffered formalin and processed by standard methods and stained with the Hematoxylin and Eosin stain. PD-L1 staining by immunohistochemistry was carried out on 4 μm sections, using the Ventana SP263 clone (10 (link)), OptiView Amplification Kit, and OptiView DAB IHC Detection Kit on Ventana Benchmark XT equipment. Hematoxylin was used as a counterstain. The tumor proportion score (TPS) of PD-L1 was calculated as the percentage of viable tumor cells with membrane positivity. Tumors with ≥50% TPS were considered to have high expression of PD-L1. Tumors with ≥ 1 to <50% TPS were considered to have low expression of PD-L1. Tumours with no or <1% TPS were considered negative.
All cases of non-small cell carcinomas of the lung primary/metastatic, received in the department of pathology of Apollo hospitals during the period 2018-2020 with PD-L1 immunohistochemistry were reviewed. Exclusion criteria included non-small cell carcinomas of the lung without PD-L1 immunohistochemistry or PD-L1immunohistochemistry in carcinomas of other sites.

Three-micrometer-thick sequential histologic tumor sections were obtained from representative formalin-fixed paraffin-embedded tumor blocks (whole-face or TMA) and used for IHC analysis. IHC was performed using an automated staining system (Ventana BenchMark) with antibodies against PD-L1 (SP263 clone, Ventana, CC1 pre-treatment for 64 minutes, Ventana Optiview detection protocol) or using a Dako automated platform with antibody to the 22C3 clone of PD-L1. Both systems used a diaminobenzidine reaction to detect antibody labeling and hematoxylin counterstaining.