Understanding the patient’s tumor immune profile is key to providing the most effective immunotherapy treatment strategy. Multiplex immunohistochemistry (IHC) combines multiple biomarker assays on a single tissue section to examine multiple cell types or biological pathways that might be used in a single or combination therapeutic strategy. Notably, multiplex IHC assays allow for the assessment of end points that measure the spatial relationships between cell types expressing certain biomarkers, such as the colocalization of biomarkers or distance measurements between multiple biomarker-expressing cells of interest. Flagship’s Computational Tissue Analysis (cTA™) platform allows for the quantification of individual analytes on dual-stained tissue sections, enabling the investigation of complex biological questions of spatial relationships and biomarker colocalization that cannot be achieved with traditional tissue-based manual end points.
Analytical validation of multiplex IHC assays enables robust analysis of multiparametric end points in the clinical setting. In this study, a chromogenic duplex IHC assay that quantifies Ki67 and CD8 in formalin-fixed, paraffin-embedded non–small cell lung cancer (NSCLC) tissues using Flagship’s cTA platform (the “duplex IHC-cTA-based assay”) was analytically validated. Similar to analytical validation studies for monoplex IHC assays, this study utilized a reference method and multiple days of staining to assess 5 performance criteria for the assay: reportable range, analytical sensitivity, analytical specificity, accuracy, and precision. The percentage of cells positive for Ki67 nuclear staining or CD8 membrane staining was quantified using the cTA platform. Performance of the chromogenic IHC-cTA-based assay in quantifying Ki67 and CD8 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 and analysis of the spatial arrangement of immune cells relative to tumor cells.