Table 2.
Recommendations for optimal conditions for PD-L1 immunohistochemistry.
| Specimen Selection | Recommendations for Optimum Conditions * |
|---|---|
| Site of biopsy | Use biopsies from a sample relevant to the disease stage, e.g., TURBS for MIBC, primary/metastatic for mUC. |
| Specimen age | Use formalin-fixed, paraffin-embedded sample blocks which have not been subjected to warm or fluctuating temperatures. Use the most recent sample proximal to starting therapy (maximum of 1 year old) [55]. When using approved assays follow the manufacturer’s instructions as cut slide stability can range from 1 to 6 months (PD-L1 IHC pharmDx 22C3 PI [51] and VENTANA PD-L1 (SP263) PI [58]). |
| Specimen type | Score the whole slide. Select a tissue specimen with invasive disease. ** TURB samples may be considered, however, only if they contain the invasive disease. Avoid highly necrotic samples where possible. Use positive controls for PD-L1; lymphatic tonsil tissue is recommended as optimal, with positive staining for macrophages, dendritic cells, and lymphocytes. Do not use cytology/smears for scoring ICs, as tissue architecture is necessary to understand if ICs are tumor infiltrating. It is currently not generally advised to use samples of bone metastases. There is however some evidence that bone decalcification using EDTA solution may yield good results and could possibly be used for PD-L1 IHC. Validation is required. |
| Sample preparation/fixation | Use 10% neutral buffered formalin in a quantity > 10 times the volume of the specimen. Sample should be placed in formalin as soon as possible (<30 min) and for a period of 12–24 h. Longer fixation may cause diffuse staining patterns. For immunohistochemistry, fixation should be performed for a minimum of 6 h and no more than 72 h. Large samples should be excised to allow for sufficient penetration of the fixative. Fixative penetrates about 1 mm/h with slight variation across different types of tissues. i.e., for cystectomy samples, the tumor should be excised, or the bladder opened to allow fixative to penetrate. Avoid decalcified tissue or tissue processed with other fixatives. Use on-slide positive and negative controls from the same institution or manufacturer, especially if an automated stainer is not used. Section specimens into a thickness of 3 or 4 µmm (as specified in manufacturers instruction). |
* Author recommendations only; please refer to the manufacturers’ package insert instructions for the licensed use (VENTANA PD-L1 (SP263) Assay PI [58] and VENTANA PD-L1 (SP142) Assay PI [57]). ** As most IO agents are currently only approved for locally advanced or metastatic UC (except for pembrolizumab, which is also approved for non–muscle-invasive bladder cancer in the USA), we recommend selecting a tissue specimen with invasive disease, rather than TURB.