BLEACH&STAIN 15-marker Multiplexed Imaging in 3,098 Human Carcinomas Reveals Six Major PD-L1–driven Immune Phenotypes with Distinct Spatial Orchestration

Abstract Multiplex fluorescence IHC (mfIHC) approaches were yet either limited to six markers or limited to a small tissue size that hampers translational studies on large tissue microarray cohorts. Here we have developed a BLEACH&STAIN mfIHC method that enabled the simultaneous analysis of 15 biomarkers (PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, and CD31) in 3,098 tumor samples from 44 different carcinoma entities within one week. To facilitate automated immune checkpoint quantification on tumor and immune cells and study its spatial interplay an artificial intelligence–based framework incorporating 17 different deep-learning systems was established. Unsupervised clustering showed that the three PD-L1 phenotypes (PD-L1+ tumor and immune cells, PD-L1+ immune cells, PD-L1−) were either inflamed or noninflamed. In inflamed PD-L1+patients, spatial analysis revealed that an elevated level of intratumoral M2 macrophages as well as CD11c+ dendritic cell (DC) infiltration (P < 0.001 each) was associated with a high CD3+ CD4± CD8± FOXP3± T-cell exclusion and a high PD-1 expression on T cells (P < 0.001 each). In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival (OS; AUC, 0.72, P < 0.001) compared with the commonly used percentage of PD-L1+ tumor cells (AUC, 0.54). In conclusion, our deep-learning–based BLEACH&STAIN framework facilitates rapid and comprehensive assessment of more than 60 spatially orchestrated immune cell subpopulations and its prognostic relevance. Implications: The development of an easy-to-use high-throughput 15+1 multiplex fluorescence approach facilitates the in-depth understanding of the immune tumor microenvironment (TME) and enables to study the prognostic relevance of more than 130 immune cell subpopulations.