P.393 - 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.