Abstract
Immune checkpoint inhibitors, including those concerning programmed cell death 1 (PD-1) and its ligand (PD-L1), have revolutionised the cancer therapy approach in the past decade. However, not all patients benefit from immunotherapy equally. The prediction of patient response to this type of therapy is mainly based on conventional immunohistochemistry, which is limited by intraobserver variability, semiquantitative assessment, or single-marker-per-slide evaluation. Multiplex imaging techniques and digital image analysis are powerful tools that could overcome some issues concerning tumour-microenvironment studies. This novel approach to biomarker assessment offers a better understanding of the complicated interactions between tumour cells and their environment. Multiplex labelling enables the detection of multiple markers simultaneously and the exploration of their spatial organisation. Evaluating a variety of immune cell phenotypes and differentiating their subpopulations is possible while preserving tissue histology in most cases. Multiplexing supported by digital pathology could allow pathologists to visualise and understand every cell in a single tissue slide and provide meaning in a complex tumour-microenvironment contexture. This review aims to provide an overview of the different multiplex imaging methods and their application in PD-L1 biomarker assessment. Moreover, we discuss digital imaging techniques, with a focus on slide scanners and software.