Multiplex Imaging Analysis in Pathology: a Comprehensive Review on Analytical Approaches and Digital Toolkits

Conventional histopathology has long been essential for disease diagnosis, relying on visual inspection of tissue sections. Immunohistochemistry aids in detecting specific biomarkers but is limited by its single-marker approach, restricting its ability to capture the full tissue environment. The advent of multiplexed imaging technologies, like multiplexed immunofluorescence and spatial transcriptomics, allows for simultaneous visualization of multiple biomarkers in a single section, enhancing morphological data with molecular and spatial information. This provides a more comprehensive view of the tissue microenvironment, cellular interactions, and disease mechanisms - crucial for understanding disease progression, prognosis, and treatment response. However, the extensive data from multiplexed imaging necessitates sophisticated computational methods for preprocessing, segmentation, feature extraction, and spatial analysis. These tools are vital for managing large, multidimensional datasets, converting raw imaging data into actionable insights. By automating labor-intensive tasks and enhancing reproducibility and accuracy, computational tools are pivotal in diagnostics and research. This review explores the current landscape of multiplexed imaging in pathology, detailing workflows and key technologies like PathML, an AI-powered platform that streamlines image analysis, making complex dataset interpretation accessible for clinical and research settings.

PD-L1 Classification of Weakly-Labeled Whole Slide Images of Breast Cancer

Specific and effective breast cancer therapy relies on the accurate quantification of PD-L1 positivity in tumors, which appears in the form of brown stainings in high resolution whole slide images (WSIs). However, the retrieval and extensive labeling of PD-L1 stained WSIs is a time-consuming and challenging task for pathologists, resulting in low reproducibility, especially for borderline images. This study aims to develop and compare models able to classify PD-L1 positivity of breast cancer samples based on WSI analysis, relying only on WSI-level labels. The task consists of two phases: identifying regions of interest (ROI) and classifying tumors as PD-L1 positive or negative. For the latter, two model categories were developed, with different feature extraction methodologies. The first encodes images based on the colour distance from a base color. The second uses a convolutional autoencoder to obtain embeddings of WSI tiles, and aggregates them into a WSI-level embedding. For both model types, features are fed into downstream ML classifiers. Two datasets from different clinical centers were used in two different training configurations: (1) training on one dataset and testing on the other; (2) combining the datasets. We also tested the performance with or without human preprocessing to remove brown artefacts Colour distance based models achieve the best performances on testing configuration (1) with artefact removal, while autoencoder-based models are superior in the remaining cases, which are prone to greater data variability.