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Overwhelming evidence has shown the significant role of the tumor microenvironment (TME) in governing the triple-negative breast cancer (TNBC) progression. Digital pathology can provide key information about the spatial heterogeneity within the TME using image analysis and spatial statistics. These analyses have been applied to CD8+ T cells, but quantitative analyses of other important markers and their correlations are limited. In this study, a digital pathology computational workflow is formulated for characterizing the spatial distributions of five immune markers (CD3, CD4, CD8, CD20, and FoxP3) and then the functionality is tested on whole slide images from patients with TNBC. The workflow is initiated by digital image processing to extract and colocalize immune marker-labeled cells and then convert this information to point patterns. Afterward invasive front (IF), central tumor (CT), and normal tissue (N) are characterized. For each region, we examine the intra-tumoral heterogeneity. The workflow is then repeated for all specimens to capture inter-tumoral heterogeneity. In this study, both intra- and inter-tumoral heterogeneities are observed for all five markers across all specimens. Among all regions, IF tends to have higher densities of immune cells and overall larger variations in spatial model fitting parameters and higher density in cell clusters and hotspots compared to CT and N. Results suggest a distinct role of IF in the tumor immuno-architecture. Though the sample size is limited in the study, the computational workflow could be readily reproduced and scaled due to its automatic nature. Importantly, the value of the workflow also lies in its potential to be linked to treatment outcomes and identification of predictive biomarkers for responders/non-responders, and its application to parameterization and validation of computational immuno-oncology models.

Digital Pathology Analysis Quantifies Spatial Heterogeneity of CD3, CD4, CD8, CD20, and FoxP3 Immune Markers in Triple-Negative Breast Cancer