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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Available from: Andrew W Bollen, Oct 13, 2015
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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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    ABSTRACT: Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCP's accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres.
    PLoS ONE 03/2014; 9(3):e90819. DOI:10.1371/journal.pone.0090819 · 3.23 Impact Factor
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    • "Induction of macro-autophagy is another phenotypic response to PI3K pathway inhibitors [26], [27]. To assess whether differences in the ability to undergo functional autophagy explained the observed differential sensitivity of SCCHN cell lines to SAR245408 and MK2206, we first examined LC3B, a commonly used marker of autophagosome formation and autophagic flux [28]. Interestingly, we observed marked differences in baseline autophagic activity between different SCCHN cell lines (Figure 1B). "
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    ABSTRACT: The phosphoinositol-3 kinase (PI3K) pathway is highly dysregulated in squamous cell carcinoma of the head and neck (SCCHN). While inhibitors of the PI3K/AKT pathway are being developed in cancer, their efficacy does not appear to be related to the presence of mutations or amplification in pathway genes. The PI3K pathway is a major regulator of macro-autophagy, an evolutionarily conserved catabolic process that degrades cellular materials to promote cellular homeostasis and survival under stress. Employing a panel of SCCHN cell lines, we observed a significant correlation between the activity of PI3K/AKT inhibitors and their ability to induce autophagy. More specifically, resistance to these inhibitors was associated with accumulation of p62/SQSTM1, a pleotropic protein that is consumed during autophagy, while loss of autophagy was, for the first time, found to be due to silencing of an essential autophagy gene, ATG7. Moreover, modulating ATG7 and p62/SQSTM1 could regulate sensitivity to PI3K/AKT inhibitors, underscoring a mechanistic link between autophagy and drug sensitivity. Analysis of human tissues revealed progressive accumulation of p62/SQSTM1 in a significant proportion of cancer samples compared to normal tissue, suggesting that defective autophagy has relevance to SCCHN. These findings are further validated by analysis of TCGA data confirming homozygous deletion and mRNA down-regulation of ATG7 in 10.0% of SCCHN samples. Taken together, these data indicate that p62/SQSTM1 levels modulate sensitivity to PI3K/AKT inhibitors; cancers vary in their capacity to undergo autophagy through epigenetic modification and, when deficient, accumulate p62/SQSTM1; and expression of autophagy-related proteins may serve as markers for resistance to PI3K/AKT inhibitors in SCCHN.
    PLoS ONE 03/2014; 9(3):e90171. DOI:10.1371/journal.pone.0090171 · 3.23 Impact Factor
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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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    ABSTRACT: Background Inclusion body myositis (IBM) is a slowly progressive inflammatory myopathy of the elderly that does not show significant clinical improvement in response to steroid therapy. Distinguishing IBM from polymyositis (PM) is clinically important since PM is steroid-responsive; however, the two conditions can show substantial histologic overlap. Results We performed quantitative immunohistochemistry for (1) autophagic markers LC3 and p62 and (2) protein aggregation marker TDP-43 in 53 subjects with pathologically diagnosed PM, IBM, and two intermediate T cell-mediated inflammatory myopathies (polymyositis with COX-negative fibers and possible IBM). The percentage of stained fibers was significantly higher in IBM than PM for all three immunostains, but the markers varied in sensitivity and specificity. In particular, both LC3 and p62 were sensitive markers of IBM, but the tradeoff between sensitivity and specificity was smaller (and diagnostic utility thus greater) for LC3 than for p62. In contrast, TDP-43 immunopositivity was highly specific for IBM, but the sensitivity of this test was low, with definitive staining present in just 67% of IBM cases. Conclusions To differentiate IBM from PM, we thus recommend using a panel of LC3 and TDP-43 antibodies: the finding of <14% LC3-positive fibers helps exclude IBM, while >7% of TDP-43-positive fibers strongly supports a diagnosis of IBM. These data provide support for the hypothesis that disruption of autophagy and protein aggregation contribute to IBM pathogenesis.
    07/2013; 1(1). DOI:10.1186/2051-5960-1-29
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