Chromogenic multiplex immunohistochemistry (IHC) assays enable investigation of the spatial relationships between tumor cells and immune cells, which are thought to be important for understanding and predicting the therapeutic response. The development and analytical validation of multiplex IHC assays enables the use of such assays to simultaneously investigate multiple biomarkers as predictors of clinical response. In this study, we analytically validated a chromogenic duplex IHC assay that quantifies Ki67 and CD8 in formalin-fixed, paraffin-embedded non–small cell lung cancer tissue samples. Five performance criteria were selected and evaluated based on the Clinical and Laboratory Standards Institute guidelines: reportable range, analytical sensitivity, analytical specificity, accuracy, and precision. Similar to analytical validation studies for monoplex IHC assays, this study utilized a reference method and required multiple days of staining. The percentage of cells positive for Ki67 nuclear staining and CD8 membrane staining were quantified using our Computational Tissue Analysis (cTA) technology. Performance of the Ki67/CD8 chromogenic duplex IHC assay was considered acceptable for the 5 criteria evaluated.
Once the performance of the assay was established, additional exploratory cTA-based end points were examined, including the quantification of each biomarker in the tumor compartment and tumor microenvironment (TME) and an analysis of the spatial arrangement of immune cells relative to tumor cells. In conclusion, Flagship’s cTA technology allows for more consistent quantification of individual analytes on dual-stained tissue sections, enabling the investigation of complex biological questions that cannot be answered with traditional tissue-based manual end points.