Clinical Utility of LC3 and p62 Immunohistochemistry in Diagnosis of Drug-Induced Autophagic Vacuolar Myopathies: A Case-Control Study

University of Campinas, Brazil
PLoS ONE (Impact Factor: 3.23). 04/2012; 7(4):e36221. DOI: 10.1371/journal.pone.0036221
Source: PubMed


Some 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.
Microtubule-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.
In 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).
Immunohistochemistry 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.

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    • "We observed strong immunoreactivity of the autophagosome marker LC3B in the rimmed vacuoles and in the cytoplasm of very atrophic fibres (Fig. 4C). Accumulation of p62 and the autophagosome marker LC3B have been used to indicate abnormalities in the autophagic degradation pathways [34]–[36]. We observed p62 immunoreactivity in rimmed vacuoles and granular immunoreactive dots in many atrophic fibres (Fig. 4E). "
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    • "Immunoperoxidase staining for LC3 (mouse monoclonal antibody, clone 5F10, Nanotools; 1:100 dilution following antigen retrieval) and p62/SQSTM1 (guinea pig polyclonal antibody, Progen Biotechnik; 1:100 dilution following antigen retrieval) was performed on FFPE tissue samples using Ventana Benchmark XT automated slide preparation system at the UCSF Brain Tumor Research Center Tissue Core as described previously [18]. Immunoperoxidase staining for TDP-43 (rabbit polyclonal antibody, Proteintech Group, Chicago, IL) was performed on FFPE tissue samples either manually (1:3000 antibody dilution; no antigen retrieval) or using a Leica Bond automated slide preparation system (1:1000 antibody dilution; antigen retrieval for 20 min in a buffer with pH = 6.0 and at a temperature of up to 100°C); the two staining methods produced essentially identical results. "
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