Clinical Utility of LC3 and p62 Immunohistochemistry in
Diagnosis of Drug-Induced Autophagic Vacuolar
Myopathies: A Case-Control Study
Han S. Lee1, Brianne H. Daniels1,2, Eduardo Salas1, Andrew W. Bollen1, Jayanta Debnath1,
1Department of Pathology, University of California San Francisco, San Francisco, California, United States of America, 2College of Osteopathic Medicine, Touro University
California, Vallejo, California, United States of America
Background: 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.
Methodology: 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.
Principal Findings: 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
Significance: 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.
Citation: Lee HS, Daniels BH, Salas E, Bollen AW, Debnath J, et al. (2012) Clinical Utility of LC3 and p62 Immunohistochemistry in Diagnosis of Drug-Induced
Autophagic Vacuolar Myopathies: A Case-Control Study. PLoS ONE 7(4): e36221. doi:10.1371/journal.pone.0036221
Editor: Konradin Metze, University of Campinas, Brazil
Received December 22, 2011; Accepted April 3, 2012; Published April 27, 2012
Copyright: ? 2012 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Marta.Margeta@ucsf.edu
Macroautophagy (hereafter called autophagy) is an evolution-
arily conserved mechanism for degradation of cytoplasmic
components, which at baseline contributes to cellular homeostasis
by enabling routine protein and organelle turnover (reviewed in
[1–3]). During periods of increased nutrient requirements,
autophagy induction is critical for survival [4,5]. Autophagy can
also be regulated by non-metabolic factors such as oxidative stress,
infection, and accumulation of aggregated proteins. Recently, it
has been recognized that autophagy plays an important role in the
pathogenesis of diverse diseases, including neurodegenerative,
neoplastic, infectious, inflammatory, and neuromuscular condi-
Distinct from the ubiquitin-proteasome system, the process of
autophagy occurs through a multi-step mechanism regulated by
ATG (AuTophagy Gene) proteins [1–3]. Early steps include the
formation of an isolation membrane (the phagophore), which
engulfs proteins and organelles destined for degradation. Closure
of the phagophore produces a membrane-bound vacuole (the
autophagosome), which moves along microtubules and fuses with
the lysosome to form the autolysosome (where the material is
ultimately degraded). 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]. Normally
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
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with a phosphatidylethanolamine group. This modified form,
termed LC3-II, is associated with autophagic membranes and is
preferentially detected by LC3 immunohistochemistry. LC3-II
also binds p62/SQSTM1, an adapter protein that targets
ubiquitinated protein aggregates for lysosomal degradation and
is selectively degraded via autophagy . Because LC3-II and p62
are both degraded in the autolysosome, the lysosomal-dependent
turnover of these proteins has emerged as a measure of bona fide
autophagic proteolysis, which is commonly termed autophagic
flux. Specifically, the accumulation of LC3-II-labeled autophago-
somes and/or p62 aggregates is a robust marker of autophagic flux
inhibition at any point beyond autophagosome formation .
Autophagy inhibition plays a key role in the pathogenesis of
several inherited myopathies including Danon disease, X-linked
myopathy with excessive autophagy (XMEA), and infantile
autophagic vacuolar myopathy [9,10], all of which are character-
ized by accumulation of autophagic vacuoles. While pathogenesis
of these disorders is still being elucidated, the overarching defect
seems to involve lysosomal function. For example, Danon disease
mutations involve the gene encoding lysosome-associated mem-
brane protein 2 (LAMP2), a protein thought to play a role in
autophagosome - lysosome fusion . Similarly, XMEA is caused
by mutations in VMA21 (a chaperone for lysosomal V-ATPase)
that result in the impairment of lysosomal acidification (Berge
Minassian, personal communication). While inherited autophagic
vacuolar myopathies are quite rare, autophagic vacuolar myop-
athies caused by autophagy-inhibiting drugs (such as chloroquine,
its analog hydroxychloroquine, and colchicine) are much more
common [12–15]. Chloroquine and hydroxychloroquine accumu-
late within lysosomes and are thought to block autophagy through
elevation of intralysosomal pH and inhibition of lysosomal
enzymes [16,17]. In addition, decreased autophagosome -
lysosome fusion has been observed in cell culture models of
chloroquine toxicity . Colchicine, on the other hand, is a well-
established microtubule-disrupting agent. Hence, colchicine likely
blocks autophagy by disrupting the movement of autophagosomes
and lysosomes along microtubules . Consistent with this idea,
reduced exocytosis of lysosomal contents has been observed
following colchicine administration . At the present, autoph-
agy-inhibiting drugs are clinically used for treatment of malaria
(chloroquine), rheumatologic disease (hydroxychloroquine), and
gout (colchicine). In addition, multiple ongoing clinical trials are
assessing the effectiveness of autophagy inhibitors as adjuvant
cancer chemotherapy (reviewed in [20–23]). Thus, the incidence
of drug-induced autophagic vacuolar myopathy can only be
expected to rise in the future.
Currently, definitive diagnosis of autophagic vacuolar myopa-
thies requires ultrastructural demonstration of autophagic vacuoles
in a muscle biopsy, thus necessitating use of electron microscopy –
a time-consuming and costly technique with a large possibility for
sampling error. Development of an immunohistochemical method
for detection of autophagosome accumulation thus has the
potential to increase both the speed and accuracy of diagnosis,
resulting in significant improvement in clinical care. Here, we
show that immunohistochemistry for LC3 and p62 can be used to
diagnose drug-induced autophagic vacuolar myopathies, obviating
the need for electron microscopy in the great majority of clinically
Study design was reviewed and approved by the University of
California San Francisco (UCSF) Committee on Human Research
(CHR). Given the non-invasive nature of the study and a minimal
potential for harm to study participants, the informed consent
requirement was waived by the CHR. No individually identifiable
patient data is presented in this report.
The objective of this study was to determine whether
immunohistochemistry for LC3 and/or p62 can be used as a
diagnostic tool for the diagnosis of drug-induced autophagic
To identify cases of autophagic vacuolar myopathy related to
colchicine, chloroquine or hydroxychloroquine use, we performed
a computerized search of the UCSF neuropathology case database
spanning the interval between 1990 and 2010; potential drug-
treated control cases within the same time span were identified
based on the history of the relevant drug use and the absence of
autophagic vacuolar myopathy diagnosis (but were allowed to
have other pathologic findings; see Table 1 for details). All subjects
had a history of either colchicine or hydroxychloroquine treatment
(no chloroquine-treated patients were identified in the database
search). Normal controls were selected from a larger pool of
muscle biopsies characterized by (1) lack of pathologic findings and
(2) no history of autophagy inhibitor use. Availability of the
archival formalin-fixed, paraffin-embedded (FFPE) and glutaral-
dehyde-fixed, Epon-embedded material were additional criteria
for inclusion in the study. To ensure accurate classification of
patients into ‘‘autophagic myopathy’’ and ‘‘drug-treated control’’
groups, blinded review of electron microscopy images was
performed by two Board-certified neuropathologists (MM and
AB) for all drug-treated cases; if ultrastructural analysis was not a
part of the original diagnostic work-up, electron microscopy was
performed on the original material as part of the study (see below).
A minimum of 10 electron micrographs, taken by trained electron
microscopy technicians, was reviewed for each case. Autophagic
vacuolar myopathy was diagnosed based on ultrastructural
identification of at least 15 definitive autophagic vacuoles in the
image set (although many cases had more than 50); drug-treated
control cases showed either none or very few autophagic vacuoles,
with largest number (4) identified in specimen #12. Only the cases
with consensus diagnosis were included in the study (1 case with
borderline findings and 2 cases without consensus diagnosis were
excluded). For the 7 drug-treated control subjects, pathologic
diagnoses reported in Table 1 were made following the review of
original light microscopy slides including the following: hematox-
ylin and eosin (H&E) stain of the FFPE material; H&E, modified
trichrome, ATPase (pH 9.2), NADH reductase, and SDH stains of
the frozen material; and Toluidine Blue stain of the glutaralde-
hyde-fixed material. Given that case and control group assignment
was based solely on history of drug use and morphologic criteria,
no attempt was made to match participants by age, sex, or other
Microscopy Core Lab. Sections were subsequently examined in
a JEOL 1400 transmission electron microscope at 120 kV. Images
were recorded with a Gatan SC1000 CCDE camera.
Immunoperoxidase staining for
LC3 (mouse monoclonal antibody, clone 5F10, Nanotools) and
Ultrathin (80 nm) sections of the
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Biotechnik) was performed using Ventana Benchmark XT
automated slide preparation system at the UCSF Brain Tumor
Research Center Tissue Core. Immunoperoxidase staining was
performed on the FFPE tissues of all subjects and on frozen tissue
from 11 representative subjects (2 normal controls, 3 drug-treated
controls, and 6 autophagic myopathy cases). Tissue sections (4–
5 mm thickness) were deparaffinized (EZ-Prep, Ventana Medical
Systems, at 75uC) followed by antigen-retrieval (Cell Conditioning
1, Ventana Medical Systems, at 95–100uC). Antibodies were
incubated at room temperature for 2 h, at 1:100 dilutions.
Antibody staining was developed using the UltraView Universal
DAB detectionsystem (Ventana
accompanied by hematoxylin counterstain. Frozen sections were
fixed in ice cold 100% acetone at 220uC for 5 min prior to
immunostaining as described above; no antigen retrieval was
required for frozen sections.
immunostained sections of FFPE material using a bright-field
light microscope, with the investigator blinded to group
assignment of each subject. Prior to counting, each slide was
viewed at low (26–206) and high power (406) to determine
whether positive fibers were present scarcely or in abundance.
Muscle fibers containing the characteristic central inclusion,
rimmed vacuole, or punctate staining pattern were counted as
positive, while fibers lacking such staining were counted as
negative. A total of 200 fibers/slide was counted in specimens
with abundant positivity, while a total of 600 fibers/slide was
counted in specimens with scarce or patchy positivity (to reduce
the sampling error). Tissue on the slide was divided into quadrants
Table 1. Study subject characteristics.
Subject IDAgeSexDrug Treatment Duration Pathologic Diagnosis
Normal Control Group
1 52F NoneNA Normal0.0 0.0
2 67F None NA Normal0.0 0.0
83FNoneNA Normal0.0 0.0
56F NoneNA Normal0.00.0
5 53M NoneNA Normal0.0 0.0
6 57M NoneNA Normal 0.00.0
7 60M NoneNANormal 0.00.0
64F NoneNANormal0.0 1.0
9 48M NoneNA Normal 0.0 0.0
1032MNone NANormal0.0 0.0
Drug-Treated Control Group
76M Colchicine UnknownNecrotizing myopathy 2.03.1
12 32F Hydroxychloroquine3 yearsNecrotizing myopathy* 13.0 10.5
$20 days Inflammatory myopathy2.5 4.3
33M Hydroxychloroquine1.5 years; stopped; then 1
$2 months Neurogenic changes4.02.0
44F ColchicineUnknown Neurogenic changes**1.81.5
#1 yearNormal0.8 0.3
Autophagic Myopathy Group
18 73M Colchicine
#2 yearsAutophagic myopathy64.0 86.0
58MColchicine UnknownAutophagic myopathy22.5 14.8
20 81F ColchicineUnknown Autophagic myopathy78.0 83.5
2184M Colchicine10 daysAutophagic myopathy 58.565.0
22 80M Colchicine ‘‘Chronic’’ Autophagic myopathy 13.0 21.0
2372F HydroxychloroquineUnknown Autophagic myopathy 56.553.0
2433F Hydroxychloroquine 1 monthAutophagic myopathy 18.012.5
2579M Colchicine ‘‘Chronic’’Autophagic myopathy 79.079.5
2679M Colchicine UnknownAutophagic myopathy 95.093.0
.1 year Autophagic myopathy12.5 25.5
# 1To minimize sampling error due to scant (#) or patchy (1) positivity, a total of 600 (rather than 200) fibers was counted.
*Scattered basophilic cores were present.
**Multiple rimmed vacuoles were present.
NA Not applicable.
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and randomly selected non-overlapping fields were counted at
high power in each quadrant until the total count was reached.
The results were recorded as a percentage (the number of positive
fibers divided by the total number of fibers counted).
Images were taken with a DP72 digital camera on a
BX41 bright-field light microscope using cellSens Entry 1.4
software (all by Olympus Corp) and were edited with Adobe
Photoshop CS5 Version 12.0.3.
ATG72/2 mouse embryonic fibroblasts , a gift from Dr.
Masaaki Komatsu (Tokyo Metropolitan Institute), were cultured
in DMEM containing 25 mM glucose (Invitrogen) supplemented
with 10% FBS, penicillin, and streptomycin. Following 8 h
treatment with 30 mM chloroquine (dissolved directly in growth
medium), fibroblasts were lysed in RIPA lysis buffer (Sigma-
Aldrich; catalog number R0278) plus 10 mM NaF, 10 mM b-
glycerophosphate, 1 mM Na3VO3, 10 nM calyculin A, and
protease inhibitors. Snap-frozen human muscle biopsy tissue was
thawed on ice in RIPA lysis buffer with aforementioned
supplements and lysosomal inhibitors E-64d and pepstatin A
(10 mg/ml each); subsequently, samples were sonicated at 2 W on
ice using a Fisher Scientific 60 Sonic Dismembrator until
centrifugation at 14,000 rpm for 15 min at 4uC. Following
measurement of protein concentration with a BCA protein assay
(Thermo), cleared homogenates were boiled in SDS sample buffer,
resolved using SDS- PAGE (15–50 mg of total protein per lane),
and transferred topolyvinylidene
Membranes were blocked in PBS +0.1% Tween 20 with 5%
nonfat dry milk, incubated with the primary antibodies overnight
at 4uC, washed, incubated with horseradish peroxidase-conjugated
chemiluminescence. Primary antibodies included anti-LC3 rabbit
polyclonal antibody generated against a peptide corresponding to
the N-terminus common to human, mouse, and rat LC3 (Fung et
al., 2008), guinea pig polyclonal anti-p62/SQSTM1 antibody
(Progen Biotechnik), andmouse
Data were analyzed with GraphPad Prism 5 statistical software.
For between-group comparison of the demographic and treatment
variables, we used one-way ANOVA with post-hoc Bonferroni test
(age) or chi-square test (sex and drug treatment). LC3 and p62
positivity data were not normally distributed; thus, between-group
comparison for all three study groups was performed by Kruskal-
Wallis one-way ANOVA on ranks. To calculate diagnostic
threshold values with optimal sensitivity and specificity, receiver
operating characteristic (ROC) analysis was performed on the two
drug-treated groups (autophagic myopathy vs. drug-treated
control group). All tests were 2-tailed with a=0.05.
Histologic and ultrastructural findings
On light microscopy, we identified several histologic patterns
suggestive (although not diagnostic) of autophagic vacuolar
myopathy. The most common finding was the presence of sharply
delineated areas of central dark staining, which we termed
‘‘basophilic cores’’. Basophilic cores were best visualized on
H&E (Fig. 1A), trichrome (Fig. 1B), and NADH reductase stains
(supplemental Fig. S1-A) of frozen material; their vacuolated
nature was highlighted by Toluidine Blue stain of Epon-embedded
material (supplemental Fig. S1-B). Basophilic core-like structures
have been observed in both human and animal muscle following
colchicine or chloroquine treatment and have been described as
areas of myofibrillar disarray [24,25], NADH-positive alterations
in intermyofibrillar network , and central basophilia .
Interestingly, similar ‘‘hematoxylin-positive structures’’ were seen
in autophagy deficient (ATG72/2) murine muscle, but only
following denervation . In the current study, basophilic cores
were seen in 5 of 10 autophagic myopathy cases and in 1 of 7
drug-treated controls (subject #12). Another relatively common
finding was the presence of classic rimmed vacuoles (arrowheads,
Figs. 1C and 1D), which were indistinguishable from rimmed
vacuoles seen in inclusion body myositis or inherited inclusion
body myopathy. In the current study, this pattern was seen in 3 of
10 autophagic myopathy cases and in 1 of 7 drug-treated controls.
Finally, some autophagic myopathy cases showed entirely non-
specific myopathic features, with vague vacuolization difficult to
distinguish from processing and/or preservation artifacts (not
shown). There was no correlation between the type of drug
treatment and the histologic pattern seen. On electron microscopy,
we identified autophagic vacuoles containing either membrane-
bound (Fig. 1E) or ‘‘free-floating’’ (Fig. 1F) cellular debris; there
was no correlation between the presence of either of these
ultrastructural patterns and histologic findings described above or
the type of drug treatment the subject received. Curvilinear bodies
(Fig. 1G) have been documented in many cases of hydroxychlor-
oquine myopathy [28,29]; in our autophagic myopathy group,
Figure 1. Histologic and ultrastructural patterns in autophagic
vacuolar myopathy. A and B. Sharply defined, central areas of dark
staining (‘‘basophilic cores’’; arrows) seen on H&E- (A) and trichrome- (B)
stained sections of frozen material (subject #22). C and D. Rimmed
vacuoles (arrowheads) seen on H&E- (C) and trichrome- (D) stained
sections of frozen material (subject #23) E–G. Autophagic vacuoles
with membrane-bound (E; subject #22) or ‘‘free-floating’’ (F; subject
#27) cellular debris on electron microscopy. Curvilinear bodies in a
hydroxychloroquine-treated subject (G; subject #23). Scale bars: A and
B, 50 mM; C and D, 20 mM; E–G, 1 mm.
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they were present in 2 of 3 subjects treated with hydroxychlor-
oquine and in 0 of 7 subjects treated with colchicine.
LC3 and p62 immunohistochemistry
Immunohistochemistry for LC3 and p62 was performed on
FFPE tissue. In normal control samples, there was no sarcoplasmic
staining (Figs. 2A and 2B); nuclear positivity, seen in p62-stained
sections, is typical for the specific anti-p62 antibody used and was
previously observed in human brain sections . In samples from
the drug-treated control group, LC3 and p62 positivity was seen in
rare fibers that typically showed a finely punctate staining pattern
(Figs. 2C and 2D). Occasionally, we observed coarser staining
coalescing around a proto-vacuole (supplemental Fig. S2). In
contrast, samples from the autophagic myopathy group showed
moderate to frequent LC3- and p62-positive fibers characterized
by coarsely punctate staining pattern (Figs. 2G and 2I) that tended
to coalesce, resulting in zones of increased staining running
linearly along the longitudinal axis of the fiber and most
commonly located in the fiber center (Figs. 2E and 2F). LC3
and p62 staining was also seen lining the rimmed vacuoles
(Figs. 2H and 2J) and, less frequently, at the periphery of fibers or
under the sarcolemma (not shown). When LC3 and p62
immunohistochemistry was performed on the frozen tissue, similar
staining patterns were observed (supplemental Fig. S3).
To statistically compare the degree of LC3 and p62 positivity
between the three experimental groups, we quantified the
percentage of LC3- and p62-positive muscle fibers in FFPE
sections; data for individual subjects are shown in Table 1. The
percentage of LC3-positive fibers was significantly higher in the
autophagic myopathy group (median 57.5%, SD 30.8%) com-
pared to either the normal control group (median 0.0%, SD 0.0%;
p,0.001) or the drug-treated control group (median 1.8%, SD
4.5%; p,0.05) (Fig. 3A; Kruskal-Wallis one-way ANOVA on
ranks). Similar results were seen with p62 staining: the percentage
of p62-positive fibers was significantly higher in the autophagic
myopathy group (median 59.0%, SD 32.2%) compared to either
the normal control group (median 0.0%, SD 0.3%; p,0.001) or
the drug-treated control group (median 2.0%, SD 3.6%; p,0.05)
(Fig. 3B; Kruskal-Wallis one-way ANOVA on ranks). To assess the
diagnostic value of LC3 and p62 immunohistochemistry, we
performed ROC (receiver-operator characteristic) curve analysis
using the data from the two drug-treated groups (Figs. 3C and 3D).
ROC analysis showed that either test can effectively distinguish
autophagic myopathy from drug-treated control specimens (p-
value for area under ROC curve: LC3, p=0.001; p62, p,0.001).
For LC3, there was a small trade-off between sensitivity and
specificity, with a threshold value of 8.3% resulting in 100%
sensitivity and 85.7% specificity, and a threshold value of 15.5%
resulting in 80% sensitivity and 100% specificity. For p62, the
optimal threshold value was 11.5% (100% sensitivity and 100%
LC3 and p62 immunoblotting
To confirm that LC3 staining in autophagic myopathy samples
reflects an increase in the autophagosome-bound LC3-II rather
than the cytoplasmic LC3-I, we performed immunoblotting for
LC3 on a representative subset of muscle biopsies; ATG7 +/+ and
2/2 mouse embryonic fibroblasts (MEFs) were used as a positive
control. Immunoblotting for p62 was performed in parallel on
both sets of samples (Fig. 4). As expected, chloroquine treatment of
ATG7+/+ MEFs resulted in an increase in LC3-II and p62
protein level. In contrast, autophagy-deficient ATG72/2 MEFs
showed no LC3-II formation and a large increase in the level of
p62 protein, both of which were independent of chloroquine
treatment (Fig. 4A). Immunoblotting of human muscle samples
showed results similar to wt (ATG7+/+) MEFs: protein level of
LC3-II and p62 was higher in subjects from the autophagic
myopathy group than in subjects from either the normal control or
drug-treated control groups (Fig. 4B). Interestingly, LC3-II and
p62 protein level was lower in samples #19 and #22 (which
showed LC3 and p62-positive fibers in 13.0–22.5% range) than in
sample #18 (which had 64.0% LC3-positive and 86.0% p62-
positive fibers). Thus, there is a good correspondence between
LC3-II and p62 protein level on immunoblotting and the
percentage of positive fibers on immunostaining, confirming both
the specificity and diagnostic utility of LC3 and p62 immunohis-
tochemistry in this clinical context.
Participant assignment to normal control, drug-treated control,
and autophagic myopathy study groups was based solely on
morphologic criteria and history of autophagy inhibitor use (see
Methods for details). While it is never possible to definitively prove
Figure 2. LC3 and p62 immunohistochemistry on FFPE
material. A and B. Lack of sarcoplasmic staining in a normal control
subject (#8). With p62, there was background nuclear positivity. C and
D. Rare fibers (asterisks) with finely punctate staining in a drug-treated
control subject (#11). E–J. Moderate to frequent LC3- and p62-positive
fibers in autophagic myopathy subjects. On longitudinal sections (E and
F; subject #23), the staining was linear, aligned with the longitudinal
axis of the fiber, and often centrally located. On cross sections, the
staining was coarsely punctate (G and I; subject #22) or confluent,
often lining vacuole rims (H and J; subject #23). Scale bars, 50 mM.
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the drug etiology of autophagic vacuolar myopathy, drug toxicity
is the most likely explanation for changes seen in this group of
subjects given the (1) positive history of relevant drug use, (2)
negative family history, (3) very low prevalence of inherited
autophagic vacuolar myopathies in the general population, (4)
subject age and sex, and (5) the lack of histologic features (such as
glycogen accumulation) that would suggest a different etiology.
There was no statistically significant difference in the mean age
(57.2613.3 [SD] vs. 50.7616.4 vs. 66.8620.2 years, respectively;
p=0.16) or sex distribution (50% vs. 71% vs. 30% female,
respectively; p=0.24) between the three study groups. Similarly,
the type of drug treatment was not significantly different between
the two drug-treated groups (29% colchicine for drug-treated
control group vs. 70% colchicine for autophagic myopathy group;
p=0.09). However, this analysis is limited by the relatively small
sample size; a larger study will be required to establish whether a
trend for preponderance of male, colchicine-treated subjects in the
autophagic myopathy relative to the drug-treated control group is
biologically meaningful and whether sex and drug treatment are
independent or dependent variables.
The length of drug treatment duration prior to biopsy was
available in the clinical record of 5 of 7 drug-treated control
subjects and 6 of 10 autophagic myopathy subjects (Table 1).
Given the incompleteness of the dataset and variations in the
reporting precision, statistical analysis of this parameter was not
possible; however, both acutely- (3 months or less) and chronically-
(1 year or more) treated subjects were present in each group.
No pathologic features were present in muscle biopsies from
subjects included in the normal control group. In contrast, several
different pathologies were present in the drug-treated control
group (no pathologic findings [2 subjects]; necrotizing myopathy,
not otherwise specified [2 subjects]; neurogenic change [2
subjects]; and inflammatory myopathy [1 subject]). Demographic,
Figure 3. Quantification of LC3- and p62-positive fibers in FFPE material. A and B. The percentage of LC3- (A) and p62-positive (B) fibers
was significantly higher in autophagic myopathy group (squares) than in either normal control (circles) or drug-treated control group (diamonds).
Each study subject is represented with a symbol; the lines indicate group means. ***, p,0.001; *, p,0.05. C and D. ROC analysis indicates that
quantitative immunohistochemistry for either LC3 (C) or p62 (D) can successfully differentiate autophagic myopathy from control cases among drug-
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diagnostic, and drug treatment information for all study subjects is
provided in Table 1.
Currently, definitive pathologic diagnosis of autophagic vacu-
olar myopathy requires electron microscopic identification of
moderate to frequent well-developed autophagic vacuoles. In this
study, we demonstrate that immunohistochemistry for LC3 and/
or p62 can be used to detect autophagosome accumulation by light
microscopy, thus providing a valuable diagnostic tool for this
group of disorders.
In both autophagic myopathy and drug-treated control muscle,
LC3 and p62 staining was largely punctate in nature. The
punctate pattern of LC3 staining reflects the association of LC3-II
with the membranes of early autophagosomes, whereas p62
puncta correspond to the accumulation of protein aggregates
within early autophagic (LC3-positive) vesicles; hence, the
increased punctate staining seen with these markers corresponds
to autophagosome buildup [7,31]. In drug-treated control
specimens, the puncta were largely small and distributed evenly
throughout the sarcoplasm (Figs. 2C and 2D). In autophagic
myopathy specimens, on the other hand, the puncta were larger
and primarily (although not exclusively) located in the center of the
fiber, creating a linear structure aligned with the fiber’s long axis.
This linear pattern of staining may be related to the linear
intermyofibrillar organization of microtubules (along which
autophagosomes propagate) , and likely corresponds to the
centrally-located zone of autophagic buildup that was detected in
autophagy-deficient (ATG52/2) murine skeletal muscle . In
addition to these qualitative differences in the pattern of staining
between autophagic myopathy and drug-treated control samples,
there was a significant difference in the percentage of LC3 and
p62-positive fibers between the two drug-treated groups (no
staining was observed in the normal control group). In autophagic
myopathy subjects, staining was present in a large fraction of
muscle fibers (12.5% to 95% for LC3; 12.5% to 93% for p62). In
contrast, the majority of drug-treated control samples showed
staining in less than 5% of muscle fibers, with only 1 of 7
specimens showing staining in up to 13% of fibers.
Based on the ROC analysis of our data, 100% specificity can be
achieved by setting a diagnostic threshold at 15% LC3-positive
muscle fibers. With this threshold, sensitivity is 80% (meaning that
20% of autophagic myopathies would be missed). In contrast,
threshold of 8% LC3-positive fibers would achieve 100%
sensitivity and 86% specificity (i.e., 14% of non-specific cases
would be falsely diagnosed as autophagic vacuolar myopathy).
Thus, using LC3 immunohistochemistry alone, a subset of cases
with 8% to 15% of LC3-positive fibers would fall in the diagnostic
‘‘gray zone’’. For cases in this range, diagnostic accuracy can be
improved by using additional diagnostic modalities, such as p62
immunohistochemistry and electron microscopy, and integrating
the information gleaned from the standard histology and
histochemistry stains (for example, the presence or absence of
basophilic cores). In this context, it is worth noting that a single
specimen from the drug-treated control group (#12, Table 1)
showed a degree of staining (13% for LC3, 10.5% for p62) that
overlapped with the range generally seen in the autophagic
myopathy group. This specimen was designated a drug-treated
control because only rare well-developed autophagic vacuoles
were identified on ultrastructural examination. However, light
microscopy showed scattered basophilic cores in the context of a
mild necrotizing myopathy. Thus, it is possible that this case
represents an instance of early and/or mild autophagic vacuolar
myopathy that was missed on electron microscopy. (Ultrastruc-
tural examination has high specificity but low sensitivity, resulting
in sampling bias and significant possibility of false negative results.)
When this case is excluded from the ROC analysis, the optimal
(100% sensitivity and 100% specificity) threshold value for
diagnosis of autophagic vacuolar myopathy is 8% positive fibers
on either LC3 or p62 immunohistochemistry.
In rodent models of chloroquine toxicity, concurrent denerva-
tion greatly contributes to the development of vacuolar pathology
[33,34]. Similarly, basophilic core-like structures were not present
in the autophagy deficient (ATG72/2) murine muscle at
baseline, developing only after muscle denervation . These
findings raise the possibility that a concurrent neurogenic process
contributes to the development of autophagic vacuolar myopathy
in patients treated with chloroquine, hydroxychloroquine, or
colchicine. Our data do not support this possibility: the drug-
treated control group included two specimens (#15 and #16) with
well-developed neurogenic changes, both of which showed a very
Figure 4. Immunoblotting confirms the specificity of LC3 and p62 immunohistochemistry. A. Wt (ATG7+/+) and autophagy-deficient
(ATG72/2) mouse embryonic fibroblasts (MEFs) were used as positive control. In wt MEFs, 8 h treatment with 30 mM chloroquine (CQ) increased the
level of LC3-II and p62 proteins compared to the untreated control (UT); LC3-I was barely detectable in either sample. In autophagy-deficient MEFs,
the level of LC3-I and p62 was high at baseline and did not change following CQ treatment; LC3-II was undetectable in both samples. GAPDH was
used as a loading control. B. In subjects from the autophagic myopathy group, LC3-II and p62 protein level was increased relative to subjects from
either normal or drug-treated control groups. LC3-I protein level was equally high in all samples, suggesting that this isoform is not detected by LC3
immunohistochemistry. Each lane contains sample from a different study subject, with subject ID numbers indicated on top. GAPDH was used as a
LC3 and p62 as Markers of Autophagic Myopathy
PLoS ONE | www.plosone.org7April 2012 | Volume 7 | Issue 4 | e36221
low degree of LC3 and p62 labeling. However, a separate study
will be required to definitively answer this question.
Rimmed vacuoles were detected in a subset of autophagic
myopathy specimens both by standard histology (Figs. 1C and 1D)
and on LC3 or p62 immunohistochemistry (Figs. 2H and 2J).
However, rimmed vacuoles are not specific for autophagic
vacuolar myopathies and can be seen in other disorders of skeletal
muscle such as inclusion body myositis and several subtypes of
muscular dystrophy. Indeed, positivity for either LC3 or p62 has
already been noted in inclusion body myositis [35–37], although
careful quantification and determination of proper diagnostic
thresholds still needs to be done. Similarly, inherited autophagic
vacuolar myopathies (such as Danon disease or XMEA) would be
expected to be highly LC3 and p62 positive, but were not included
in the current study because our archive does not include a
sufficient number of well documented cases. While diagnostically
helpful, neither LC3 nor p62 positivity is therefore pathognomonic
for drug-induced autophagic vacuolar myopathies. To establish
the correct diagnosis, positive LC3 and/or p62 staining needs to
be correlated with the remainder of histologic findings and with
full clinical history (including age at presentation, family history,
and medication history).
The current study was not designed to detect differences in age,
sex, or drug treatment distribution between the two drug-treated
study groups. Nonetheless, we found that male, colchicine-treated
subjects were more common in the autophagic myopathy group,
while female, hydroxychloroquine-treated subjects were more
common in the drug-treated control group. The differences we
observed were not statistically significant; however, a study with
95% power to detect the effects of magnitude we observed (with a
significance level of 0.05) would have needed to have 40 subjects in
each group. Therefore, a larger study – ideally with a prospective
design – will need to be performed in the future to establish
whether certain types of patients are more vulnerable to
development of drug-induced autophagic vacuolar myopathy.
In summary, we used specimens from human subjects treated
with either hydroxychloroquine or colchicine to establish that
immunohistochemical stains for LC3 and/or p62 are useful
ancillary tools in pathologic diagnosis of drug-induced autophagic
vacuolar myopathies. By limiting the need for electron microsco-
py, use of LC3 and/or p62 immunohistochemistry will decrease
both the time required to establish the diagnosis and the false
negative rate resulting from sampling bias, thus resulting in
significantly improved clinical care.
The major strengths of the current study are (1) the use of
pathologically well characterized subjects, (2) the inclusion of two
control groups (normal controls and drug-treated controls), (3) the
inclusion of drug-treated control subjects with abnormal muscle,
mimicking clinically relevant scenarios for diagnostic test use, (4)
the concordance of results across several experimental modalities
(immunostaining of FFPE tissue vs. immunostaining of frozen
tissue vs. immunoblotting of frozen tissue), and (5) the quantitative
study design, which enabled calculation of sensitivity and
specificity values for different diagnostic thresholds. The major
limitations are (1) the under-sampling of the drug-treated control
group, as patients with no symptoms are unlikely to undergo a
muscle biopsy, and (2) a non-negligible probability that one (or
more) drug-treated subjects were miss-assigned to the control
group given the significant false negative rate of electron
microscopy (the current ‘‘gold standard’’ method for diagnosis of
autophagic vacuolar myopathies). As discussed above, these
limitations would be expected to result in an elevated diagnostic
threshold and artificial widening of the diagnostic ‘‘gray zone’’.
Given that alternative treatments exist for both rheumatologic
diseases and gout, it would thus be reasonable to use a fairly low
diagnostic threshold (5% of LC3- or p62-positive fibers) for
recommendation to discontinue hydroxychloroquine or colchicine
therapy in a symptomatic patient with otherwise equivocal
subject (#22). A. On NADH histochemistry, basophilic cores
(arrows) are centrally located, sharply demarcated, and darker
than the rest of the fiber (in contrast to the central cores and mini-
cores, which are lighter than the surrounding sarcoplasm). B.
Toluidine Blue stain of the glutaraldehyde-fixed, Epon-embedded
tissue shows that basophilic cores (arrows) contain numerous
vacuoles. Scale bar, 50 mM.
Basophilic cores in an autophagic myopathy
sections from a drug-treated control subject (#16).
Coarsely punctate staining rims a proto-vacuole; this staining
pattern was rare in drug-treated control subjects. Scale bar,
LC3 and p62 immunohistochemistry on FFPE
frozen material. A and B. No sarcoplasmic staining is seen in a
drug-treated control subject (#14). C and D. Coarsely punctate
sarcoplasmic staining is seen in an autophagic myopathy subject
(#21). Focal sarcolemmal p62 positivity is seen in both control (B)
and myopathy (D) subjects; given that no corresponding staining is
present in LC3-stained sections (A and C), this likely represents a
cryosection-specific staining artifact of the p62 antibody. Scale bar,
LC3 and p62 immunohistochemistry on the
We thank Mr. King Chu (UCSF Brain Tumor Research Center Tissue
Core) for immunohistochemistry support, Mr. Larry Ackerman (UCSF
Electron Microscopy Core Lab) for electron microscopy support, and Ms.
Christine Lin for help with figure preparation. In addition, we are grateful
to Dr. Masaaki Komatsu (Tokyo Metropolitan Institute) for generous gift of
immortalized ATG7 +/+ and ATG7 2/2 mouse embryonic fibroblasts
and to Dr. Anne Hiniker (UCSF Department of Pathology) for valuable
comments on the manuscript.
Conceived and designed the experiments: HL JD MM. Performed the
experiments: HL BD ES. Analyzed the data: HL MM. Contributed
reagents/materials/analysis tools: JD. Wrote the paper: HL BD ES MM.
Classified subjects based on review of ultrastructural findings: AB MM.
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