A Novel Generative Artificial Intelligence Method for Interference Study on Multiplex Brightfield Immunohistochemistry Images

Multiplex brightfield imaging offers the advantage of simultaneously analyzing multiple biomarkers on a single slide, as opposed to single biomarker labeling on multiple consecutive slides. To accurately analyze multiple biomarkers localized at the same cellular compartment, two representative biomarker sets were selected as assay models - cMET-PDL1-EGFR and CD8-LAG3-PDL1, where all three biomarkers can co-localize on the cell membrane. One of the most crucial preliminary stages for analyzing such assay is identifying each unique chromogen on individual cells. This is a challenging problem due to the co-localization of membrane stains from all the three biomarkers. It requires advanced color unmixing for creating the equivalent singleplex images from each triplex image for each biomarker. In this project, we developed a cycle-Generative Adversarial Network (cycle-GAN) method for unmixing the triplex images generated from the above-mentioned assays. Three different models were designed to generate the singleplex image for each of the three stains Tamra (purple), QM-Dabsyl (yellow) and Green. A notable novelty of our approach was that the input to the network were images in the optical density domain instead of conventionally used RGB images. The use of the optical density domain helped in reducing the blurriness of the synthetic singleplex images, which was often observed when the network was trained on RGB images. The cycle-GAN models were validated on 10,800 lung, gastric and colon images for the cMET-PDL1-EGFR assay and 3600 colon images for the CD8-LAG3-PDL1 assay. Visual as well as quantified assessments demonstrated that the proposed method is effective and efficient when compared with the manual reviewing results and is readily applicable to various multiplex assays.