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Analytical Validation (Based on CLIA & CLSI Standards) of Utrophin–Laminin α2 (Merosin) and MHCd-Laminin α2 Duplex Immunohistochemical Assays Using Computational Tissue Analysis (cTA™) for Evaluation of Duchenne Muscular Dystrophy Therapeutics

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Abstract: Tissue–based assays provide insight for understanding therapeutic response for Duchenne muscular dystrophy (DMD) therapies within the context of intact muscle. This necessitates that at least two biomarkers are evaluated simultaneously in the same tissue section, one for fibre identification and one for biomarker quantification. While fluorescent multiplex assays are feasible, they present many technical challenges for analytical validation. Chromogenic assays are not subject to the same technical limitations and are commonly used in the clinical space to provide robust data, enabling confident interpretation of therapeutic effect.

In this study, two chromogenic duplex IHC assays were analytically validated. These assays identify fibres via laminin α2 (merosin) expression and quantify either utrophin or developmental myosin heavy chain (MHCd) expression in acetone–fixed, frozen skeletal muscle sections (DMD, Becker muscular dystrophy, and control) obtained from the Wellstone Muscular Dystrophy Cooperative Research Center. Five performance criteria were evaluated: reportable range, analytical sensitivity and specificity, accuracy, and precision, based on Clinical Laboratory Improvements Amendments standards. The duplex assays were compared to analytically validated monoplex IHC assays for each biomarker.

To ensure accurate and repeatable measurement of IHC in the duplex setting, we quantified the percentage of fibres positive for utrophin membrane staining and MHCd cytoplasmic staining, using Flagship’s Computational Tissue Analysis (cTA™) approach, which can simplify high complexity staining and precisely report novel endpoints while maintaining context of the surrounding tissue (IHC–cTA™). Performance of the utrophin–laminin α2 and MHCd–laminin α2 duplex IHC–cTA™ based assays were considered acceptable for all performance criteria evaluated. Both assays are supporting Summit Therapeutics’ ongoing clinical trial for evaluation of the utrophin modulator ezutromid in DMD.

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