POSTER: Computational Alignment of Duplex Immunohistochemically-Stained Muscle Sections in Support of Therapies for Duchenne Muscular Dystrophy

Abstract: Continuous expression of utrophin protein by a utrophin modulator could be a disease modifying treatment for Duchenne muscular dystrophy (DMD) patients regardless of their dystrophin mutation. At least 3 biomarkers (a fibre identification biomarker, a regeneration biomarker, and utrophin biomarker) are needed to separate utrophin expression resulting from therapeutic intervention with a utrophin modulator from the pathophysiologic utrophin expression associated with muscle fibre regeneration.

Technical challenges of multiplex assay development and analytical validation for 3 or more biomarkers limit the utility of such assays in clinical trials. Having successfully completed the analytical validation of utrophin-laminin α2 (merosin) and developmental myosin heavy chain (MHCd)-laminin α2 duplex immunohistochemical-Computational Tissue Analysis (IHC-cTA™) based assays, we have developed a fibre-based image analysis software to co-register consecutive and non-consecutive serial biopsy sections. The co-registration assay was developed utilising DMD, Becker muscular dystrophy (BMD), and control muscle biopsy sections obtained from the Wellstone Muscular Dystrophy Cooperative Research Center stained with each duplex assay highlighted above. Co-registration quality was numerically evaluated, and visually assessed by a veterinary pathologist. We demonstrate that the cTA™
approach reproducibly detects biomarker signal features (e.g., mean signal intensities), tissue morphometrics (e.g., fibre minimum diameter), and fibre classification (e.g., utrophin+/MHCd-) in whole-slide images of muscle cryosections at an individual muscle fibre resolution. This facilitates understanding of architectural and functional relationships between multiple biomarkers throughout biopsy specimens, and may provide valuable assessment of therapeutic efficacy in on-going and future clinical trials.


  • Coregistration analysis facilitates biomarker quantification in consecutive and nonconsecutive tissue sections, enabling evaluation of biomarkers in the same myofibres when multiplex assays are infeasible or impossible.
  • This cTA™ method can be used to follow changes in biomarker expression along the length of individual myofibres, thereby addressing questions of heterogeneity, localized events (eg. damage, regeneration), and drug effects (eg. extent and uniformity of an effect throughout the tissue).
  • Simultaneous evaluations of utrophin and MHCd in CTRL, DMD, and BMD samples show that (1) utrophin and MHCd expression overlap but not completely, and that (2) utrophin+/MHCd– and utrophin–/MHCd+ subpopulations are differentially represented in diseased muscle. These observations suggest that the regulation of utrophin and MHCd may differ temporally during regeneration, and that fibres may be retaining utrophin expression in DMD/BMD post-maturation, as there is no binding competition with dystrophin.
  • Coregistration analysis is supporting the evaluation of ezutromid in ongoing and planned DMD clinical trials. This analysis may be complex due to potential therapeutic effects on a number of fibres needing to regenerate and/or on the extent of maintenance of utrophin expression in more mature fibres: it will be facilitated by the ability to recognize populations expressing one marker, both markers, or neither marker.
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