Analytical Validation of Ki67/CD8 Duplex IHC Assay Using Computational Tissue Analysis (cTA™)

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Using immunohistochemistry (IHC) for understanding the spatial relationships between immune cells and tumor cells in the context of cancer tissue is important for understanding and predicting therapeutic response to immune checkpoint inhibitors and other immuno-modulating therapies. While single marker predictors of clinical response can demonstrate the presence of the therapeutic target, the complexity of the tumor immune landscape often requires that several markers be evaluated for a single patient simultaneously in the same tissue section to avoid confounding analysis due to heterogeneity of the immune landscape. Multiplex assays that measure 2 or more analytes in a single tissue section allow direct investigation of the important spatial relationships. Fluorescent IHC multiplex assays provide this advantage, but requires specialized instruments, training, and interpretation of results; limiting their broad adoption by clinical diagnostic laboratories. Chromogenic assays, on the other hand, are standard methods that most of these laboratories have the equipment, expertise, and training to perform.

In this study, Flagship Biosciences analytically validated a chromogenic duplex IHC assay that quanti es Ki67 and CD8 in formalin- xed, para n-embedded non–small cell lung cancer (NSCLC) tissues. Similar to analytical validation studies for monoplex IHC assays, this study utilized an appropriate reference method and multiple days of staining. Distinct to this assay validation study, however, was inclusion of an additional step in the reference standard comparison due to the potential for false-positive and false-negative results linked to the dual-staining methodology. Speci cally, a reference method was used to qualify Ki67 and CD8 monoplex IHC assays that were then used as the reference standard to assess duplex assay performance. Five performance criteria were evaluated: reportable range, analytical sensitivity, analytical speci city, accuracy, and precision. These performance criteria were selected based on Clinical Laboratory Standards Institute guidelines.

To ensure accurate and consistent measurement of chromogenic IHC in the duplex setting, we quanti ed the percentage of cells positive for Ki67 nuclear staining and/or CD8 membrane staining, using our computational Tissue Analysis (cTA) approach, which can simplify high complexity staining and report precise, high content results about tissue biomarker context measurements (IHC-cTA). Performance of the Ki67/CD8 chromogenic duplex IHC-cTA based assay was considered acceptable for the performance criteria evaluated.

The multiplex setting requires additional assay performance assessments due to the complexity of the staining interpretation when multiple IHC stains are present on 1 tissue section. Flagship’s cTA approach allows for precise and consistent quanti cation of individual analytes on dual-stained tissue sections.

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