Multiplex Immunohistochemistry for Image Analysis of Tertiary Lymphoid Structures in Cancer

Multiplex immunohistochemistry allows the demonstration of multiple protein antigens in individual histological sections of formalin-fixed paraffin-embedded tumors or other types of tissue. Carefully designed and optimized immunohistochemistry (IHC) assays not only maximize the information available from limited tissues, but also enable a higher level interpretation of that information by demonstrating the histo-anatomical relationships among key cell types which express the included biomarkers. Programmable automated IHC instruments support the development and application of complicated multiplex IHC protocols, help save time and effort, and enhance immunostaining quality and reproducibility. Simple data can be extracted from immunostained tissues to include qualitative (descriptive) findings and semiquantitative analysis. The value of multiplex IHC can be increased further by the utilization of image analysis software either to better visualize multiple markers or by applying suitable digital scoring solutions to capture data (automated pathology).Here, we describe a five-marker multiplex based on application of two individual assays to serial sections of non-small cell lung carcinoma (NSCLC). We use this assay to label PD1, PD-L1, CD3, CD68, and cytokeratins in relation to tertiary lymphoid structures (TLS) and other regions of the tumor microenvironment. We illustrate how visualization of the immunostaining results can be used to understand TLS organization and other aspects of the tumor microenvironment, and briefly consider means to further yield additional information.