Systems pathology by multiplexed immunohistochemistry and whole-slide digital image analysis.

The paradigm of molecular histopathology is shifting from a single-marker immunohistochemistry towards multiplexed detection of markers to better understand the complex pathological processes. However, there are no systems allowing multiplexed IHC (mIHC) with high-resolution whole-slide tissue imaging and analysis, yet providing feasible throughput for routine use. We present an mIHC platform combining fluorescent and chromogenic staining with automated whole-slide imaging and integrated whole-slide image analysis, enabling simultaneous detection of six protein markers and nuclei, and automatic quantification and classification of hundreds of thousands of cells in situ in formalin-fixed paraffin-embedded tissues. In the first proof-of-concept, we detected immune cells at cell-level resolution (n = 128,894 cells) in human prostate cancer, and analysed T cell subpopulations in different tumour compartments (epithelium vs. stroma). In the second proof-of-concept, we demonstrated an automatic classification of epithelial cell populations (n = 83,558) and glands (benign vs. cancer) in prostate cancer with simultaneous analysis of androgen receptor (AR) and alpha-methylacyl-CoA (AMACR) expression at cell-level resolution. We conclude that the open-source combination of 8-plex mIHC detection, whole-slide image acquisition and analysis provides a robust tool allowing quantitative, spatially resolved whole-slide tissue cytometry directly in formalin-fixed human tumour tissues for improved characterization of histology and the tumour microenvironment.