IHC assay (PDL-1, Clone 28-8)
tumour
TUMOUR
Staining of tumour tissue for PD-L1 was conducted in FFPE sections from tissue obtained at the three pre-determined time points (baseline/time point 1; Day 14/time point 2; surgery/time point 3) using automated immunostaining. A total of 46 samples from 21 patients were stained using clone 28-8 (cat# ab205921; Abcam).The immunohistochemistry protocol is briefly described here: tissue sections (4 μm) were stained in a Leica Bond Max automated stainer (Leica Biosystems, Vista, CA); the tissue sections were deparaffinised and rehydrated following the Leica Bond protocol. Antigen retrieval was performed for 20 min with Bond solution #2 (Leica Biosystems, equivalent EDTA, pH 9·0). The primary antibody (PDL-1, clone 28-8 [Abcam], dilution 1:100) was incubated for 15 min at room temperature and detected using the Bond polymer refine detection kit (Leica biosystems) with DAB as chromogen, the slides were counterstained with haematoxylin, dehydrated, and cover slipped.Analysis of the expression of PD-L1, was performed by a pathologist using a standard microscope approach. PD-L1 was evaluated in viable malignant cells and reported as percentage of malignant cells with any positive membrane expression, <1% was determined as PD-L1 negative; ≥1% was determined as PD-L1 positive. Samples with fewer than 100 malignant cells were considered inadequate for PD-L1 analysis. The optimal cut-off for identifying PD-L1 positivity in ccRCC has not been identified and is not used in clinical decision-making