Abstract
BackgroundSome patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity.MethodologyMicrotubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria.Principal FindingsIn all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p≤0.001).SignificanceImmunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these markers will improve the speed and accuracy of diagnosis, resulting in significantly improved clinical care.
Highlights
Macroautophagy is an evolutionarily conserved mechanism for degradation of cytoplasmic components, which at baseline contributes to cellular homeostasis by enabling routine protein and organelle turnover
Histologic and ultrastructural findings On light microscopy, we identified several histologic patterns suggestive of autophagic vacuolar myopathy
We demonstrate that immunohistochemistry for light chain 3 (LC3) and/ or p62 can be used to detect autophagosome accumulation by light microscopy, providing a valuable diagnostic tool for this group of disorders
Summary
Macroautophagy (hereafter called autophagy) is an evolutionarily conserved mechanism for degradation of cytoplasmic components, which at baseline contributes to cellular homeostasis by enabling routine protein and organelle turnover (reviewed in [1,2,3]). Steps include the formation of an isolation membrane (the phagophore), which engulfs proteins and organelles destined for degradation. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian orthologue of yeast ATG8, is commonly used as a marker of autophagosome formation [6,7]. Cytosolic in its precursor form (LC3-I), LC3 undergoes proteolytic cleavage of the C-terminal end upon autophagy induction, resulting in exposure of a glycine residue and subsequent lipidation with a phosphatidylethanolamine group. This modified form, termed LC3-II, is associated with autophagic membranes and is preferentially detected by LC3 immunohistochemistry. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity
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