209P - Mapping the landscape of immune cells for optimal index calculation using AI-powered image analysis of multiplexed immunohistochemistry in breast cancer patients

Background

Immune-cell indices of immunohistochemistry (IHC) are calculated in countless research studies; often to identify prognostic or predictive biomarkers in cancer patients. The study aimed to investigate how the location of immune cells and the amount of tumor epithelia may affect their calculation in breast cancer (BC) patients whilst exploring multiple sample techniques for IHC immune-cell quantification.

Methods

Formalin-fixed, paraffine-embedded pretreatment tumor tissue (N=227; full-cut slides) from BC patients was stained with two multiplexed chromogenic IHC using CK7/19, CD8, CD4, FOXP3, CD20, CD66b, and CD68. AI-powered image analysis (U-net) determined their area-based indices in the reference spaces: total tumor area, tumor stroma, tumor epithelia, a stromal tumor brim, and a fixed area for each lesion using multiple subsamples (mean no., 100) within tumor stroma (in total, 1 mm²) and epithelia (in total, 1 mm²).

Results

Immune cells were predominantly situated in tumor stroma and rarely epithelia (P<0.01). The most infiltrative cell type was CD68 with a mean index of 3% for epithelia, yet 32% for stroma. In remainder cells, indices were <0.7% for epithelia. The amount of epithelia ranged from 1-60%, which strongly affected index calculations that included the total area of epithelia (P<0.01). In addition, solidity of tumor islands was highly associated with the number of infiltrative CD8, CD66b, and CD68 cells (P<0.01).

Conclusions

The location (stroma vs epithelia) of each immune cell of interest should be considered in connection to its index calculation because the amount of tumor epithelia may skew the result. Alternatively, the novel, unbiased subsampling developed in this study could be used. High solidity meant fewer infiltrating cells, which, conceivably, may affect both prognostic and predictive utility of immune cells.

Legal entity responsible for the study

The authors.

Funding

Danish Cancer Society.

Disclosure

All authors have declared no conflicts of interest.