Background
Deeper understanding of the interplay between tumor and immune cell infiltrates is critical for the development of next generation therapies. However, current immunohistochemistry protocols in clinical practice are limited to a small number of markers and rely on multiple rounds of antigen retrieval and sequential staining over two days. These steps are associated with a high risk for tissue and signal loss. We developed a clinical trial assay using 16 distinct antibodies (plus a nuclear counterstain) using the Orion platform (RareCyte) to enable deeper exploration of immune landscape of tumors in formalin-fixed paraffin-embedded (FFPE) tissues in a broad range of cancer subtypes, including breast, lung, prostate, melanoma, and colorectal cancers. The implementation of higher plex imaging will enable greater insight into immune surveillance, mechanisms of resistance, and patient stratification, while minimizing patient tissue required for analysis.
Methods
We designed a multiplexed fluorescence immunohistochemistry (mFIHC) assay with fluorescence conjugated antibodies to Granzyme B, Ki67, CD3e, CD20, CD4, CD163, CD8a, CD56, PDL1, PD-1, LAG3, FOXP3, CD11b, CD138, SOX10, Cytokeratin and nuclear stain (Hoechst) to profile the immune landscape in a broad range of tumor indications. We successfully validated a clinical grade mFIHC assay using automated staining (Biocare Medical, intelliPath FLX), imaging (RareCyte, Orion) and analysis (Indica Labs, HALO) workflows.
Results
Sensitivity and specificity were confirmed on known positive and negative controls. Reproducibility was observed across instruments, operators, and independent experiments for all markers. It is theoretically possible to report over 65,000 unique phenotypes from this assay.
Conclusions
The clinically validated 17-plex FIHC assay examines the tumor immune landscape across five cancer indications (breast, lung, prostate, melanoma, colorectal). Implementation of hi-plex FIHC assays coupled with robust image analysis may assist in risk stratification of patients or enable efficacy assessment of immunomodulating cancer therapies. This assay may be used to further understand the complex immune cell and tumor cell spatial biology in the context of clinical trials.