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

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.

Increased autophagy accelerates colchicine-induced muscle toxicity

“…Thus, the emerging hypothesis is that colchicine-induced muscle toxicity is a result of autophagic vacuole build-up due to enhanced autophagosome formation and impaired autophagic degradation; this possibility is supported by the fact that autophagic markers LC3 and SQSTM1 robustly accumulate in the muscle of patients with colchicine-induced autophagic vacuolar myopathy. 23 Colchicine and chloroquine have both been associated with toxic vacuolar myopathies. 1 In the case of chloroquine, lysosomal de-acidification is the presumed mechanism by which autophagic vacuoles accumulate and fail to be degraded.…”

“…Immunoperoxidase staining for LC3 (mouse monoclonal antibody, clone 5F10, Nanotools, 0231-100) and SQSTM1 (guinea pig polyclonal antibody, Progen Biotechnik, GP62) was performed on formalin-fixed, paraffin-embedded tissue samples using the Ventana Benchmark XT automated slide preparation system as described previously. 23 Images were taken with a DP72 digital camera on a BX41 bright-field light microscope using cellSens Entry 1.6 software (all by Olympus) and were edited with Adobe Photoshop CS5 Version 12.1.32.…”

“…A detailed quantification of the percentage of LC3-and SQSTM1-immunopositive myofibers in these human specimens has been previously reported. 23 We therefore reasoned that statin medications, like rapamycin, may accelerate and/or potentiate colchicine toxicity.…”

Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity

“…58 In addition, the airway epithelium had elevations in SQSTM1, a multifunction scaffolding protein that functions as an autophagy cargo receptor and accumulates in tissues during in vivo autophagy. 59 Morphometric evaluation provided quantitative evidence that ubiquitin frequently colocalized with SQSTM1, LAMP1, and LAMP2 to confirm autophagy. We were, furthermore, able to use confocal microscopy to provide three-dimensional spatial confirmation of these autophagyassociated interactions.…”

Sequential changes in autophagy in diabetic cardiac fibrosis

“…The upregulation of autophagy was further indicated by the downregulation in the expression of P62 (Fig. 3C), which is known as an important measurement in evaluating autophagy pathway efficacy (15)(16)(17). In addition, the mRNA expression levels of Beclin1 were examined using RT-qPCR analysis.…”