Chromogenic staining of formalin-fixed paraffin embedded (FFPE) tissue biopsy samples aims to explore spatial location of defined cells and proteins in the tumor microenvironment and to phenotype specific cell types based on the expression of different markers. This assay can also be used to identify cellular events in the tumor tissue and its microenvironment, such as apoptosis, proliferation, etc.
Staining of tumor tissue for PD-L1 and other immune markers (T cell, B cell, myeloid cell, immune checkpoints, etc.) may be conducted at the different time points of the study (baseline, during treatment, and progression/recurrence) and will be performed using automated immunostaining. Briefly, 4µm-thick histology sections will be stained using a Leica Bond-Max automated IHC stainer (Leica Biosystems, Vista, CA). All antibodies have been optimized for IHC by examination of positive and negative controls, and antibody specificity has been confirmed by Western blotting.
Antibody labeling will be detected with a diaminobenzidine reaction and hematoxylin counterstaining using a Novocastra™ Bond Polymer Refine Detection kit (Leica Microsystems, Buffalo Grove, IL). To quantify marker expression, all IHC slides will be scanned into a digital IF image scanner (Aperio™ AT Turbo, Leica Biosystems, Buffalo Grove, IL), and analyzed using the Aperio Image Genie Toolbox™ software (Leica Biosystems, Buffalo Grove, IL). For immune cell markers (such as CD8, CD3, KI68, CD68, etc.) a threshold for positivity will be determined based on algorithm application. Three to five representative one-square-millimeter areas within the tumor region will be selected for analysis. The expression of markers in malignant cells, such as PD-L1, will be evaluated using a microscope by a trained pathologist and/or the Aperio™ digital imaging system to quantify the percentage of positive cells (0 to 100%) and, if required, intensity (0 to 3+). Quantification of markers expressed in immune cells will be expressed as cell density (number of cells per mm square).
IHC assays are available at all CIMAC sites.