Autoimmunity Reviews 13 (2014) 1008–1012
Contents lists available at ScienceDirect
Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev
Review
Identification of a novel myositis-associated antibody directed against cortactin Moisés Labrador-Horrillo a,⁎, Maria Angeles Martínez b, Albert Selva-O'Callaghan a, Ernesto Trallero-Araguás a, Josep M. Grau-Junyent c, Miquel Vilardell-Tarrés a, Candido Juarez b a b c
Internal Medicine Department, Vall d'Hebron General Hospital, Universitat Autonoma de Barcelona, Barcelona, Spain Immunology Department, Hospital de la Santa Creu i Sant Pau, Universitat Autónoma de Barcelona, Barcelona, Spain Muscle Research Unit, Internal Medicine Service, Hospital Clínic de Barcelona, Universitat de Barcelona, Fundación Cellex, Barcelona, Spain
a r t i c l e
i n f o
Article history: Accepted 25 June 2014 Available online 27 August 2014 Keywords: Anti-CTTN antibody Myositis-associated autoantibody MAA Cortactin
a b s t r a c t Objective: The aim of this study is to describe a novel myositis-associated autoantibody (anti-cortactin antibody) and assess related clinical and immunological manifestations and its clinical significance. Methods: Adult patients with myositis (dermatomyositis, polymyositis, immune-mediated necrotizing myopathy, and inclusion body myositis), as well as patients with other autoimmune diseases and non-inflammatory myopathies were analyzed for the presence of anti-cortactin antibody using in-house developed ELISA and immunoblotting techniques with a commercial source of purified cortactin. The cut-off for positive status was determined in a group of healthy volunteers. Results: Antibody against cortactin was positive in 7/34 (20%) polymyositis patients, 9/117 (7.6%) dermatomyositis, 2/7 (26%) immune-mediated necrotizing myopathy, and none of the 4 patients with inclusion body myositis. The antibody also tested positive in 3/101 patients with other autoimmune diseases (2 systemic sclerosis and 1 systemic lupus erythematosus), and in 1/29 patients with non-inflammatory myopathy. No relevant association with specific clinical features was found in patients with these antibodies. Anti-cortactin antibody was more frequently positive in patients with polymyositis and immune-mediated necrotizing myopathy than in the remaining myositis patients, and was the only myositis autoantibody found in sera of 3 patients from these groups. Conclusions: Our data indicate that cortactin is a novel target antigen in patients with autoimmune diseases, especially patients with polymyositis or immune-mediated necrotizing myopathy. Anti-cortactin can be considered a new myositis-associated antibody. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patients and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Patient population . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Laboratory tests and serological assay . . . . . . . . . . . . . . . . . . . 2.3. Anti-CTTN antibody detected by ELISA . . . . . . . . . . . . . . . . . . . 2.4. Anti-CTTN antibody detected by immunoblotting . . . . . . . . . . . . . . 2.5. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Clinical and serologic characteristics of patients with inflammatory myopathies 4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author at: Internal Medicine Department, Vall d' Hebron General Hospital, Passeig Vall d' Hebron, 119-129, 08035 Barcelona, Spain. Tel./fax: +34 93 2746169. E-mail address: [email protected] (M. Labrador-Horrillo).
http://dx.doi.org/10.1016/j.autrev.2014.08.038 1568-9972/© 2014 Elsevier B.V. All rights reserved.
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012
1. Introduction Autoimmune myopathies comprise a spectrum of acquired diseases characterized by skeletal muscle inflammation leading to chronic dysfunction and disability. Dermatomyositis (DM) and polymyositis (PM) are the main clinical forms, in which immune-mediated pathways of muscle injury are prominent; immune-mediated necrotizing myopathies (IMNM) and inclusion body myositis (IBM) are also considered members of this group [1–5]. One of the hallmark characteristics of autoimmune myopathies is the presence of serum autoantibodies. These autoantibodies are detected in around 60% to 70% of myositis patients and are traditionally classified into two groups based on their diagnostic accuracy: myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs), the latter mainly occurring in myositis-overlap syndrome patients, but also in connective tissue diseases with no evidence of myositis [6]. During the process of identifying autoantibodies in our myositis patients, we came across a new autoantibody reactive with a human protein. We noticed that some patients who were anti-MDA5 or antiHMGCR-positive using our in-house developed ELISA were negative for these antigens on confirmatory immunoblotting, but unexpectedly, we observed a 68-kD band. The protein was identified as a contaminant in two commercial recombinant proteins supplied by different companies, regularly used for diagnosing myositis. By in-gel digestion and mass spectrometry analysis of the band, we were able to identify the protein as cortactin (CTTN). In humans, cortactin is encoded by the CTTN gene (previously EMS1) on the long arm of chromosome 11. Cortactin was first identified as a prominent substrate of the oncogene Src tyrosine kinase and has a recognized association with progression of cancer [7,8]. Our objective was to determine the prevalence of this new autoantibody in a large series of patients with myositis from a single center and to investigate the existence of a characteristic associated clinical profile. 2. Patients and methods 2.1. Patient population The study included 162 white, adult myositis patients (121 women), 117 diagnosed with DM, 34 with PM, 7 with IMNM, and 4 with IBM. In addition, we included 101 patients with other systemic autoimmune diseases (41 systemic sclerosis [SSc)], 25 systemic lupus erythematosus [SLE], 25 rheumatoid arthritis [RA] and 10 primary Sjögren syndrome [pSS]), and 29 with non-inflammatory myopathies (18 limb girdle muscle dystrophies [LGMD]: 14 dysferlinopathy [LGMD 2B], and 4 calpainopathy [LGMD 2A]); 10 with other types of dystrophies: 3 Becker muscular dystrophy, 5 facioscapulohumeral dystrophy, 2 myotonic dystrophy; and 1 Pompe disease). Twenty-five healthy controls were studied to determine the cut-off value for establishing positive status for anti-cortactin (anti-CTTN) antibody by ELISA. All the myositis patients included belong to a historical cohort diagnosed with idiopathic inflammatory myopathy at Vall d'Hebron General Hospital in Barcelona (Spain) between 1983 and 2014. Our center is a single teaching hospital with approximately 700 acute care beds, attending a population of nearly 450,000 inhabitants. Serum samples from these patients are routinely collected at diagnosis and during follow-up in our outpatient clinic, and stored at − 80 °C. Patients and controls included in the study gave informed consent for the use of their serum for research purposes. The institutional review board of our hospital approved the study. The diagnosis of DM and PM was based on the criteria of Bohan and Peter [1,2].
Only patients with definite or probable disease were included. The Sontheimer criteria were used to diagnose amyopathic DM [9]. Distinctive pathological features enable the diagnosis of IMNM [3], and IBM was diagnosed according to established clinical and histological criteria [10]. Clinical data were obtained retrospectively by review of the patients' medical records. 2.2. Laboratory tests and serological assay Serum samples from each patient were screened by indirect immunofluorescence for antinuclear antibodies (ANA) using HEp-2 cells, and by a commercial ELISA used in our routine laboratory setting for antibodies against extractable nuclear antigens (Ro, La, RNP, Sm) and anti-histidyl-tRNA synthetase (anti-Jo-1). Anti-TIF1γ and anti-MDA5 antibodies were detected by an in-house ELISA and confirmed by immunoblotting [11,12]. In addition, all samples were tested by protein and RNA immunoprecipitation [13], which confirmed the ELISA results and enabled detection of other synthetases and myositis-specific and myositis-associated antibodies (anti-Mi-2, anti-SRP, anti-Ro52, anti-Ro60, anti-La, anti-PM/Scl, anti-p155, and anti-U1RNP) that may have been overlooked by ELISA. 2.3. Anti-CTTN antibody detected by ELISA Briefly, 96-well ELISA plates (Costar, Corning, New York) were coated with 2 μg/mL of purified recombinant CTTN (OriGene, Rockville, MD) diluted in phosphate buffered saline (PBS) and left to stand overnight at 4 °C. Wells were incubated for 1 h at room temperature (RT) with blocking buffer (10% nonfat dry milk in PBS). Plates were then washed in PBS, and human serum samples diluted 1:100 in HRP Sample Diluent, (INOVA Diagnostic Inc., San Diego, CA) were added in triplicate: two to CTTN-coated wells and one to a PBS-coated well (without antigen) to determine the background absorbance of each sample. Plates were incubated at RT for 1 h. After washing with HRP Wash (INOVA Diagnostic Inc., San Diego, CA), HRP-labeled goat anti-human IgG antibody (INOVA Diagnostic Inc., San Diego, CA) was added to each well, and plates were incubated for 1 h at RT and washed again. Color development was performed with peroxidase reagent TMB Chromogen (INOVA Diagnostic Inc., San Diego, CA) and absorbances at 450 nm were determined. For each sample, the background absorbance from the PBS-coated well was subtracted from the average absorbance of the two CTTN-coated wells. Sample absorbance was expressed as optical density units. The same positive serum was used as the reference in each assay. 2.4. Anti-CTTN antibody detected by immunoblotting Briefly, 5 μg of purified recombinant CTTN (OriGene, Rockville, MD) or 5 μg of purified recombinant MDA5 (OriGene, Rockville, MD) or 5 μg of HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase, catalytic domain human, recombinant GST fusion protein expressed in E. coli) (Sigma-Aldrich, St Louis, MO) was run on 4% to 12% polyacrylamideSDS minigels with MOPS running buffer, and western blot was performed on a nitrocellulose membrane using the Invitrogen NuPAGE (Carlsbad, CA) electrophoresis system, as previously described [11,12]. CTTN-transferred nitrocellulose was vertically cut into several strips and incubated for 1 h at RT in PBS containing 3% nonfat dry milk (blocking buffer). Each strip was then incubated with the corresponding human serum sample diluted 1:100 in blocking buffer for 1 hour at RT. After washing, phosphatase alkaline-labeled goat anti-human immunoglobulin antibody (Invitrogen Frederick, MD) was added to each strip
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Fig. 1. Immunoblots showing reactivity of IgG antibodies from myositis patients against commercially available purified recombinant CTTN (panel A), MDA5 contaminated with CTTN (panel B), and HMGCR contaminated with CTTN (panel C). Lane 1 corresponds to the same negative serum, lane 2 to the same positive serum against CTTN, and lane 3 to specific positive sera against MDA5 and HMGCR, respectively.
and strips were incubated for 1 h at RT. Color development was performed in phosphatase reagent (BCIP/NBT, Promega, Madison, WI). Based on signal intensity, the results were classified into negative or positive (Fig. 1, panels A, B and C, respectively).
2.5. Statistical analysis Associations between anti-CTTN antibody and qualitative variables were evaluated with the chi-square test and Fisher exact test. The strength of the associations between variables was measured using odds ratios (ORs) with 95% confidence intervals (CIs). The Mann– Whitney U test and Student t test were used for comparisons of median and mean values, respectively, when appropriate. Cumulative survival rates were estimated by the Kaplan–Meier test. The log-rank test was also used to compare survival rates. All tests were two-sided, and probability (P) values of b0.05 were considered statistically significant. All analyses were performed with SPSS, version 19.0 (SPSS, Chicago, IL).
3. Results One hundred sixty-two adult myositis patients (117 DM, 34 PM, 7 IMNM, and 4 IBM) were analyzed for the presence of anti-CTTN antibody, determined by our in-house ELISA and immunoblot techniques using a commercially available recombinant CTTN. The cut-off value for a positive ELISA result was established at 0.300 absorbance units, which corresponded to 5 standard deviations above the mean value obtained for the 25 healthy controls. We found excellent correlations between immunoblot and ELISA results above 0.4 OD (positive) and below 0.3 OD (negative), but there were some discrepancies with ELISA between 0.3 and 0.4 OD. Thus, we considered that all positive ELISA results had to be confirmed by immunoblotting. CTTN antibody tested positive in 7 of 34 (20%) PM, 9 of 117 (7.6%) DM, 2 of 7 (28%) IMNM patients, and none of the 4 IBM patients. In the autoimmune systemic diseases group, anti-CTTN antibody was positive in only 2 of 41 (4.8%) patients with SSc, and 1 of 25 (4%) SLE patients. A single patient with dysferlinopathy in the group of 29 with non-inflammatory myopathy had a low positive result on ELISA,
which was confirmed by blotting. All the RA and pSS patients had negative testing. ELISA values in the groups studied are shown in Fig. 2. 3.1. Clinical and serologic characteristics of patients with inflammatory myopathies Anti-CTTN antibody was more prevalent in patients with PM than in those with DM, but the difference did not reach statistical significance (20.6% vs. 7.7%; p = 0.052). When we analyzed other clinical characteristics (sex, age, arthritis, fever, interstitial lung disease, Raynaud phenomenon, and incidence of cancer, whether paraneoplastic or not) only male sex was significantly associated with anti-CTTN antibody (9 of 18, 50% vs. 32 of 110, 29.1%; p = 0.03). Clinical and immunological data of anti-CTTN-positive patients are summarized in Table 1. The presence of MAAs was more frequent in patients with anti-CTTN antibody as compared to the remainder of the cohort (10 of 14, 71% vs. 52 of 135, 38%; p = 0.02). Anti-Ro (in 3 patients) and anti-Ro52 (in 6 patients) were the most common MAAs. MSAs were positive in 9 patients: 5 with anti-Jo1, 1 with anti-SRP, 1 with anti-EJ, 1 with anti-Mi2, and 1 with anti-TIF1γ. In serum samples from 3 patients, 2 with PM and 1 with IMNM, anti-CTTN was the only myositis-related autoantibody detected. The mortality analysis disclosed no significant differences between anti-CTTN-positive and -negative patients in the overall patient series (3 of 18, 16% vs 40 of 144, 27%; p = 0.3) or when only patients with paraneoplastic myositis (cancer occurring within 3 years of the myositis diagnosis) were analyzed (3 of 3 cases, 100% vs. 14 of 23 cases, 60%; p = 0.1). Furthermore, no significant differences were found in the survival analysis between positive and negative patients (p = 0.3). Nonetheless, patients with paraneoplastic myositis and anti-CTTN antibody had poorer survival (although non-significant) than those with this condition testing anti-CTTN-negative (17.4 vs. 65.9 months). 4. Discussion In this study, we describe a novel myositis-associated autoantibody: anti-CTTN. Although no clinical or biological characteristics were found to be related with this antibody, at least in our cohort, the fact that anti-
M. Labrador-Horrillo et al. / Autoimmunity Reviews 13 (2014) 1008–1012
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Fig. 2. A. Representation of the anti-CTTN ELISA test results in patients with polymyositis (PM) (n = 34), dermatomyositis (DM) (n = 117), immune-mediated necrotizing myopathy (IMNM) (n = 7), inclusion body myositis (IBM) (n = 4), and non-inflammatory myopathies (NIM) (n = 29). The cut-off value for a positive result was established at 0.300 absorbance units, which corresponded to 5 standard deviations above the mean value obtained for the 25 healthy controls (dashed line). B. Representation of the anti-CTTN ELISA results in 101 patients with systemic autoimmune diseases: systemic sclerosis (SSc) (n = 41); systemic lupus erythematosus (SLE) (n = 25), rheumatoid arthritis (RA) (n = 25), and primary Sjögren syndrome (pSS) (n = 10). Healthy volunteers (n=25) were included to determine the cut-off value.
CTTN was strongly associated with adult PM patients (20% versus b 1% in controls) could be relevant. It is well known that PM remains a diagnosis of exclusion, and that other myopathies can mimic this muscle disorder [14]. Thus, detection of this autoantibody may be of help in
establishing the diagnosis of true autoimmune polymyositis. The fact that anti-CTTN was rarely present in non-inflammatory myopathies that are known to mimic polymyositis is also relevant and merits consideration.
Table 1 Clinical characteristics and immunologic profile in a series of 18 myositis patients with anti-CTTN antibodies. Patient
Age, y/Sex
Muscle biopsy
MSA/MAA
Anti-CTTN (ELISA/IB)
CK (UI/L)
ILD
Arthritis
Raynaud phenomenon
Skin manifestations
Fever
Dysphagia
Cancer
HLA-DRB
1 2 3 4
56/M 48/M 48/M 43/F
PM PM PM PM
U1RNP Jo1/Ro52 U1RNP Ro52
0.957/+++ 1.138/+++ 0.390/+ 0.309/+
1390 14,765 4450 345
Yes Yes Yes Yes
No Yes Yes No
Yes No Yes Yes
No No Yes Yes
Yes Yes No Yes
No No No No
DRB1*14,15 DRB1*11 N/A DRB1*03, 08
5 6 7
38/M 22/F 50/F
IMNM PM DM
SRP EJ Jo1/Ro52
0.573/++ 0.366/+ 0.329/+
2686 476 271
No No Yes
No No No
No Yes Yes
Yes No No
No No No
No No No
DRB1*03, 01 DRB1* 07, 15 DRB1* 03, 04
8 9 10
63/M 74/M 52/F
PM PM DM
Mi2/Ro
0.505/++ 0.413/++ 0.479/++
451 1200 902
Yes No Yes
No No No
No No No
No No Yes
No No No
Lung No No
N/A N/A DRB1* 03, 04
11
60/F
DM
PM/Scl
0.970/+++
558
Yes
Yes
No
No
No
Breast
DRB1* 11, 13
12 13
78/F 42/F
DM DM
Ro Jo1/Ro/Ro52
0.451/++ 0.329/+
234 2500
No Yes
Yes Yes
No No
No Yes
No Yes
Pancreas No
DRB1*07, 14 DRB1* 03, 08
14
63/F
DM
Jo1/Ro52
0.482/++
2000
Yes
Yes
Yes
No
No
No
DRB1*03, 04
15 16
38/M 22/F
DM DM
Jo1/Ro52 PM/Scl
0.331/+ 0.404/++
27,000 2096
Yes Yes
Yes Yes
No Yes
Yes No
Yes Yes
No No
DRB1*03, 11 DRB1*03, 01
17 18
77/M 42/M
IMNM DM
TIF1γ
0.343/+ 0.345/+
3000 300
No No
Yes No
Yes No
No No No Telangiectasia Poikiloderma Mechanic's hand No Mechanic's hand Gottron papules Heliotrope rash No No Gottron sign Heliotrope rash Mechanic's hand Gottron sign Gottron sign Mechanic's hand Heliotrope rash Mechanic's hand Gottron sign Mechanic's hand Mechanic's hand Gottron sign Calcinosis No Gottron sign
No No
Yes Yes
No Lung
DRB1*04, 11 N/A
Age at diagnosis; MSA, muscle-specific autoantibodies; MAA, muscle-associated autoantibodies; IMNM, immune-mediated necrotizing myopathy; anti-CTTN, anti-cortactin antibodies, detected by in-house ELISA and confirmed by immunoblot; ILD, interstitial lung disease. M, male; F, female. N/A, Not available. Cancer was only paraneoplastic in patient 18.
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Testing of sera from patients with other autoimmune disorders yielded positive results in a few SSc and SLE cases. This suggests that anti-CTTN antibody is not an absolutely specific myositis autoantibody, but can be considered a new myositis-associated antibody (MAA). Myositis-specific and myositis-associated autoantibodies are found in up to 50% of myositis patients and are useful for assisting the diagnosis and perhaps, the prognosis of these heterogeneous disorders. Although we were not able to ascribe a phenotypic association with the presence of anti-CTTN in the myositis patients studied, the strong association with the PM subgroup could be valuable from the clinical viewpoint. Considering that the percentage of various myositis-specific or -associated autoantibodies ranges from 5% in the case of anti-SRP to 35% in the case of antisynthetase antibody [15,16], the 20% positivity for anti-CTTN antibody in patients with PM is not negligible and could be of diagnostic value. Cortactin is a protein recognized for its association with progression of cancer [8,17–19]; the role of antibodies against this protein remains to be elucidated. In the paraneoplastic myositis patients in our study, those who were anti-CTTN-positive had poorer survival than the antiCTTN-negative cases. Although this association did not reach statistical significance, perhaps because of the small sample size, this finding seems to concur with the above-mentioned association with cancer progression. A possible role of anti-CTTN antibody in the prognosis of patients with paraneoplastic myositis must be confirmed in other patient cohorts. The results of this study prove the feasibility of detecting antibodies against CTTN in adult patients with autoimmune diseases by in-house ELISA and immunoblot techniques using commercially available recombinant CTTN as the antigen. These techniques can be reliably performed in a standard laboratory setting. Our findings indicate that laboratory technicians should be aware that when anti-MDA5 or anti-HMCGR are being determined by ELISA, confirmation of positive results by immunoblotting is mandatory because of possible false-positives in patients with anti-CTTN antibody. Although further studies are needed to better delineate the role of anti-CTTN antibody in patients with autoimmune disease, our data suggest that this novel target antigen can be considered a new myositis-associated antibody with a potential for application in clinical practice. Funding This study was funded in part by grants from the Spanish Ministry of Health and Consumer Affairs (PI12-01320 and PI10-01871). Contributors MLH (guarantor) and MAM had the original idea, and together with ASOC and ETA, designed the study. MLH, MAM, and CJ performed the laboratory techniques (ELISA, immunoblot, and immunoprecipitation). ASOC, ETA, JMGJ, and MVT managed the patients with myositis. All authors contributed to, and approved, the final manuscript. Conflict of interest statement This is an original work, and all authors meet the criteria for authorship, including acceptance of responsibility for the scientific content of the manuscript. There are no conflicts of interest.
Take-home messages • In this study, we describe a novel myositis-associated autoantibody: anti- cortactin (CTTN). • Anti-CTTN antibody was found in the 20% of patients with polymyositis and could be of diagnostic value in these patients. • In patients with anti-CTTN antibody, false positive results can be obtained for determination of ELISA method of anti-MDA5 or antiHMCGR antibodies. In this case, confirmation of positive results by immunoblotting is mandatory.
Acknowledgments We would like to thank Dr. Isabel Illa and Dr. Jordi Diaz-Manera from the Neuromuscular Diseases Unit at Hospital de la Sta Creu i Sant Pau for providing sera from patients with muscle diseases and also to Dr. Roser Solans, Dr. Carmen Pilar Simeon and Dr. Josep Ordi from the Internal Medicine Department at Vall d'Hebron Hospital for providing sera from patients with pSS, SSc and SLE respectively.
References [1] Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two parts). N Engl J Med 1975;292:344–7. [2] Bohan A, Peter JB. Polymyositis and dermatomyositis (second of two parts). N Engl J Med 1975;292:403–7. [3] Mammen AL. Autoimmune myopathies: autoantibodies, phenotypes and pathogenesis. Nat Rev Neurol 2011;7:343–54. [4] Catalán M, Selva-O'Callaghan A, Grau JM. Diagnosis and classification of sporadic inclusion body myositis (sIBM). Autoimmun Rev 2014;13:363–6. [5] Hamann PD, Cooper RG, McHugh NJ, Chinoy H. Statin-induced necrotizing myositis —a discrete autoimmune entity within the “statin-induced myopathy spectrum”. Autoimmun Rev 2013;12:1177–81. [6] Ghirardello A, Bassi N, Palma L, Borella E, Domeneghetti M, Punzi L, et al. Autoantibodies in polymyositis and dermatomyositis. Curr Rheumatol Rep 2013;15:335. [7] Schuuring E, Verhoeven E, Mooi WJ, Michalides RJ. Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas. Oncogene 1992;7:355–61. [8] Buday L, Downward J. Roles of cortactin in tumor pathogenesis. Biochim Biophys Acta 2007;1775:263–73. [9] Sontheimer RD. Cutaneous features of classic dermatomyositis and amyopathic dermatomyositis. Curr Opin Rheumatol 1999;11:475–82. [10] Griggs RC, Askanas V, DiMauro S, Engel A, Karpati G, Mendell JR, et al. Inclusion body myositis and myopathies. Ann Neurol 1995;38:705–13. [11] Labrador-Horrillo M, Martínez MA, Selva-O'Callaghan A, Trallero-Araguás E, Balada E, Vilardell-Tarrés M, et al. Anti-TIF1γ antibodies (anti-p155) in adult patients with dermatomyositis: comparison of different diagnostic assays. Ann Rheum Dis 2012;71:993–6. [12] Labrador-Horrillo M, Martínez MA, Selva-O'Callaghan A, Trallero-Araguás E, Balada E, Vilardell-Tarrés M, et al. Anti-MDA5 antibodies in a large mediterranean population of adults with dermatomyositis. J Immunol Res 2014;2014:290797. [13] Selva-O'Callaghan A, Labrador-Horrillo M, Solans-Laque R, Simeon-Aznar CP, Martínez-Gómez X, Vilardell-Tarrés M. Myositis-specific and myositis-associated antibodies in a series of eighty-eight Mediterranean patients with idiopathic inflammatory myopathy. Arthritis Rheum 2006;55:791–8. [14] Milisenda JC, Selva-O'Callaghan A, Grau JM. The diagnosis and classification of polymyositis. J Autoimmun 2014;48–49:118–21. [15] Tansley SL, Betteridge ZE, McHugh NJ. The diagnostic utility of autoantibodies in adult and juvenile myositis. Curr Opin Rheumatol 2013;25:772–7. [16] Mahler M, Miller FW, Fritzler MJ. Idiopathic inflammatory myopathies and the antisynthetase syndrome: a comprehensive review. Autoimmun Rev 2014;13:367–71. [17] Weaver AM. Cortactin and tumor invasiveness. Cancer Lett 2008;265:157–66. [18] MacGrath SM, Koleske AJ. Cortactin in cell migration and cancer at a glance. J Cell Sci 2012;125:1621–6. [19] Kirkbride KC, Sung BH, Sinha S, Weaver AM. Cortactin: a multifunctional regulator of cellular invasiveness. Cell Adh Migr 2011;5:187–98.
Contents lists available at ScienceDirect
Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev
Review
Identification of a novel myositis-associated antibody directed against cortactin Moisés Labrador-Horrillo a,⁎, Maria Angeles Martínez b, Albert Selva-O'Callaghan a, Ernesto Trallero-Araguás a, Josep M. Grau-Junyent c, Miquel Vilardell-Tarrés a, Candido Juarez b a b c
Internal Medicine Department, Vall d'Hebron General Hospital, Universitat Autonoma de Barcelona, Barcelona, Spain Immunology Department, Hospital de la Santa Creu i Sant Pau, Universitat Autónoma de Barcelona, Barcelona, Spain Muscle Research Unit, Internal Medicine Service, Hospital Clínic de Barcelona, Universitat de Barcelona, Fundación Cellex, Barcelona, Spain
a r t i c l e
i n f o
Article history: Accepted 25 June 2014 Available online 27 August 2014 Keywords: Anti-CTTN antibody Myositis-associated autoantibody MAA Cortactin
a b s t r a c t Objective: The aim of this study is to describe a novel myositis-associated autoantibody (anti-cortactin antibody) and assess related clinical and immunological manifestations and its clinical significance. Methods: Adult patients with myositis (dermatomyositis, polymyositis, immune-mediated necrotizing myopathy, and inclusion body myositis), as well as patients with other autoimmune diseases and non-inflammatory myopathies were analyzed for the presence of anti-cortactin antibody using in-house developed ELISA and immunoblotting techniques with a commercial source of purified cortactin. The cut-off for positive status was determined in a group of healthy volunteers. Results: Antibody against cortactin was positive in 7/34 (20%) polymyositis patients, 9/117 (7.6%) dermatomyositis, 2/7 (26%) immune-mediated necrotizing myopathy, and none of the 4 patients with inclusion body myositis. The antibody also tested positive in 3/101 patients with other autoimmune diseases (2 systemic sclerosis and 1 systemic lupus erythematosus), and in 1/29 patients with non-inflammatory myopathy. No relevant association with specific clinical features was found in patients with these antibodies. Anti-cortactin antibody was more frequently positive in patients with polymyositis and immune-mediated necrotizing myopathy than in the remaining myositis patients, and was the only myositis autoantibody found in sera of 3 patients from these groups. Conclusions: Our data indicate that cortactin is a novel target antigen in patients with autoimmune diseases, especially patients with polymyositis or immune-mediated necrotizing myopathy. Anti-cortactin can be considered a new myositis-associated antibody. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patients and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Patient population . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Laboratory tests and serological assay . . . . . . . . . . . . . . . . . . . 2.3. Anti-CTTN antibody detected by ELISA . . . . . . . . . . . . . . . . . . . 2.4. Anti-CTTN antibody detected by immunoblotting . . . . . . . . . . . . . . 2.5. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Clinical and serologic characteristics of patients with inflammatory myopathies 4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author at: Internal Medicine Department, Vall d' Hebron General Hospital, Passeig Vall d' Hebron, 119-129, 08035 Barcelona, Spain. Tel./fax: +34 93 2746169. E-mail address: [email protected] (M. Labrador-Horrillo).
http://dx.doi.org/10.1016/j.autrev.2014.08.038 1568-9972/© 2014 Elsevier B.V. All rights reserved.
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012
1. Introduction Autoimmune myopathies comprise a spectrum of acquired diseases characterized by skeletal muscle inflammation leading to chronic dysfunction and disability. Dermatomyositis (DM) and polymyositis (PM) are the main clinical forms, in which immune-mediated pathways of muscle injury are prominent; immune-mediated necrotizing myopathies (IMNM) and inclusion body myositis (IBM) are also considered members of this group [1–5]. One of the hallmark characteristics of autoimmune myopathies is the presence of serum autoantibodies. These autoantibodies are detected in around 60% to 70% of myositis patients and are traditionally classified into two groups based on their diagnostic accuracy: myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs), the latter mainly occurring in myositis-overlap syndrome patients, but also in connective tissue diseases with no evidence of myositis [6]. During the process of identifying autoantibodies in our myositis patients, we came across a new autoantibody reactive with a human protein. We noticed that some patients who were anti-MDA5 or antiHMGCR-positive using our in-house developed ELISA were negative for these antigens on confirmatory immunoblotting, but unexpectedly, we observed a 68-kD band. The protein was identified as a contaminant in two commercial recombinant proteins supplied by different companies, regularly used for diagnosing myositis. By in-gel digestion and mass spectrometry analysis of the band, we were able to identify the protein as cortactin (CTTN). In humans, cortactin is encoded by the CTTN gene (previously EMS1) on the long arm of chromosome 11. Cortactin was first identified as a prominent substrate of the oncogene Src tyrosine kinase and has a recognized association with progression of cancer [7,8]. Our objective was to determine the prevalence of this new autoantibody in a large series of patients with myositis from a single center and to investigate the existence of a characteristic associated clinical profile. 2. Patients and methods 2.1. Patient population The study included 162 white, adult myositis patients (121 women), 117 diagnosed with DM, 34 with PM, 7 with IMNM, and 4 with IBM. In addition, we included 101 patients with other systemic autoimmune diseases (41 systemic sclerosis [SSc)], 25 systemic lupus erythematosus [SLE], 25 rheumatoid arthritis [RA] and 10 primary Sjögren syndrome [pSS]), and 29 with non-inflammatory myopathies (18 limb girdle muscle dystrophies [LGMD]: 14 dysferlinopathy [LGMD 2B], and 4 calpainopathy [LGMD 2A]); 10 with other types of dystrophies: 3 Becker muscular dystrophy, 5 facioscapulohumeral dystrophy, 2 myotonic dystrophy; and 1 Pompe disease). Twenty-five healthy controls were studied to determine the cut-off value for establishing positive status for anti-cortactin (anti-CTTN) antibody by ELISA. All the myositis patients included belong to a historical cohort diagnosed with idiopathic inflammatory myopathy at Vall d'Hebron General Hospital in Barcelona (Spain) between 1983 and 2014. Our center is a single teaching hospital with approximately 700 acute care beds, attending a population of nearly 450,000 inhabitants. Serum samples from these patients are routinely collected at diagnosis and during follow-up in our outpatient clinic, and stored at − 80 °C. Patients and controls included in the study gave informed consent for the use of their serum for research purposes. The institutional review board of our hospital approved the study. The diagnosis of DM and PM was based on the criteria of Bohan and Peter [1,2].
Only patients with definite or probable disease were included. The Sontheimer criteria were used to diagnose amyopathic DM [9]. Distinctive pathological features enable the diagnosis of IMNM [3], and IBM was diagnosed according to established clinical and histological criteria [10]. Clinical data were obtained retrospectively by review of the patients' medical records. 2.2. Laboratory tests and serological assay Serum samples from each patient were screened by indirect immunofluorescence for antinuclear antibodies (ANA) using HEp-2 cells, and by a commercial ELISA used in our routine laboratory setting for antibodies against extractable nuclear antigens (Ro, La, RNP, Sm) and anti-histidyl-tRNA synthetase (anti-Jo-1). Anti-TIF1γ and anti-MDA5 antibodies were detected by an in-house ELISA and confirmed by immunoblotting [11,12]. In addition, all samples were tested by protein and RNA immunoprecipitation [13], which confirmed the ELISA results and enabled detection of other synthetases and myositis-specific and myositis-associated antibodies (anti-Mi-2, anti-SRP, anti-Ro52, anti-Ro60, anti-La, anti-PM/Scl, anti-p155, and anti-U1RNP) that may have been overlooked by ELISA. 2.3. Anti-CTTN antibody detected by ELISA Briefly, 96-well ELISA plates (Costar, Corning, New York) were coated with 2 μg/mL of purified recombinant CTTN (OriGene, Rockville, MD) diluted in phosphate buffered saline (PBS) and left to stand overnight at 4 °C. Wells were incubated for 1 h at room temperature (RT) with blocking buffer (10% nonfat dry milk in PBS). Plates were then washed in PBS, and human serum samples diluted 1:100 in HRP Sample Diluent, (INOVA Diagnostic Inc., San Diego, CA) were added in triplicate: two to CTTN-coated wells and one to a PBS-coated well (without antigen) to determine the background absorbance of each sample. Plates were incubated at RT for 1 h. After washing with HRP Wash (INOVA Diagnostic Inc., San Diego, CA), HRP-labeled goat anti-human IgG antibody (INOVA Diagnostic Inc., San Diego, CA) was added to each well, and plates were incubated for 1 h at RT and washed again. Color development was performed with peroxidase reagent TMB Chromogen (INOVA Diagnostic Inc., San Diego, CA) and absorbances at 450 nm were determined. For each sample, the background absorbance from the PBS-coated well was subtracted from the average absorbance of the two CTTN-coated wells. Sample absorbance was expressed as optical density units. The same positive serum was used as the reference in each assay. 2.4. Anti-CTTN antibody detected by immunoblotting Briefly, 5 μg of purified recombinant CTTN (OriGene, Rockville, MD) or 5 μg of purified recombinant MDA5 (OriGene, Rockville, MD) or 5 μg of HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase, catalytic domain human, recombinant GST fusion protein expressed in E. coli) (Sigma-Aldrich, St Louis, MO) was run on 4% to 12% polyacrylamideSDS minigels with MOPS running buffer, and western blot was performed on a nitrocellulose membrane using the Invitrogen NuPAGE (Carlsbad, CA) electrophoresis system, as previously described [11,12]. CTTN-transferred nitrocellulose was vertically cut into several strips and incubated for 1 h at RT in PBS containing 3% nonfat dry milk (blocking buffer). Each strip was then incubated with the corresponding human serum sample diluted 1:100 in blocking buffer for 1 hour at RT. After washing, phosphatase alkaline-labeled goat anti-human immunoglobulin antibody (Invitrogen Frederick, MD) was added to each strip
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Fig. 1. Immunoblots showing reactivity of IgG antibodies from myositis patients against commercially available purified recombinant CTTN (panel A), MDA5 contaminated with CTTN (panel B), and HMGCR contaminated with CTTN (panel C). Lane 1 corresponds to the same negative serum, lane 2 to the same positive serum against CTTN, and lane 3 to specific positive sera against MDA5 and HMGCR, respectively.
and strips were incubated for 1 h at RT. Color development was performed in phosphatase reagent (BCIP/NBT, Promega, Madison, WI). Based on signal intensity, the results were classified into negative or positive (Fig. 1, panels A, B and C, respectively).
2.5. Statistical analysis Associations between anti-CTTN antibody and qualitative variables were evaluated with the chi-square test and Fisher exact test. The strength of the associations between variables was measured using odds ratios (ORs) with 95% confidence intervals (CIs). The Mann– Whitney U test and Student t test were used for comparisons of median and mean values, respectively, when appropriate. Cumulative survival rates were estimated by the Kaplan–Meier test. The log-rank test was also used to compare survival rates. All tests were two-sided, and probability (P) values of b0.05 were considered statistically significant. All analyses were performed with SPSS, version 19.0 (SPSS, Chicago, IL).
3. Results One hundred sixty-two adult myositis patients (117 DM, 34 PM, 7 IMNM, and 4 IBM) were analyzed for the presence of anti-CTTN antibody, determined by our in-house ELISA and immunoblot techniques using a commercially available recombinant CTTN. The cut-off value for a positive ELISA result was established at 0.300 absorbance units, which corresponded to 5 standard deviations above the mean value obtained for the 25 healthy controls. We found excellent correlations between immunoblot and ELISA results above 0.4 OD (positive) and below 0.3 OD (negative), but there were some discrepancies with ELISA between 0.3 and 0.4 OD. Thus, we considered that all positive ELISA results had to be confirmed by immunoblotting. CTTN antibody tested positive in 7 of 34 (20%) PM, 9 of 117 (7.6%) DM, 2 of 7 (28%) IMNM patients, and none of the 4 IBM patients. In the autoimmune systemic diseases group, anti-CTTN antibody was positive in only 2 of 41 (4.8%) patients with SSc, and 1 of 25 (4%) SLE patients. A single patient with dysferlinopathy in the group of 29 with non-inflammatory myopathy had a low positive result on ELISA,
which was confirmed by blotting. All the RA and pSS patients had negative testing. ELISA values in the groups studied are shown in Fig. 2. 3.1. Clinical and serologic characteristics of patients with inflammatory myopathies Anti-CTTN antibody was more prevalent in patients with PM than in those with DM, but the difference did not reach statistical significance (20.6% vs. 7.7%; p = 0.052). When we analyzed other clinical characteristics (sex, age, arthritis, fever, interstitial lung disease, Raynaud phenomenon, and incidence of cancer, whether paraneoplastic or not) only male sex was significantly associated with anti-CTTN antibody (9 of 18, 50% vs. 32 of 110, 29.1%; p = 0.03). Clinical and immunological data of anti-CTTN-positive patients are summarized in Table 1. The presence of MAAs was more frequent in patients with anti-CTTN antibody as compared to the remainder of the cohort (10 of 14, 71% vs. 52 of 135, 38%; p = 0.02). Anti-Ro (in 3 patients) and anti-Ro52 (in 6 patients) were the most common MAAs. MSAs were positive in 9 patients: 5 with anti-Jo1, 1 with anti-SRP, 1 with anti-EJ, 1 with anti-Mi2, and 1 with anti-TIF1γ. In serum samples from 3 patients, 2 with PM and 1 with IMNM, anti-CTTN was the only myositis-related autoantibody detected. The mortality analysis disclosed no significant differences between anti-CTTN-positive and -negative patients in the overall patient series (3 of 18, 16% vs 40 of 144, 27%; p = 0.3) or when only patients with paraneoplastic myositis (cancer occurring within 3 years of the myositis diagnosis) were analyzed (3 of 3 cases, 100% vs. 14 of 23 cases, 60%; p = 0.1). Furthermore, no significant differences were found in the survival analysis between positive and negative patients (p = 0.3). Nonetheless, patients with paraneoplastic myositis and anti-CTTN antibody had poorer survival (although non-significant) than those with this condition testing anti-CTTN-negative (17.4 vs. 65.9 months). 4. Discussion In this study, we describe a novel myositis-associated autoantibody: anti-CTTN. Although no clinical or biological characteristics were found to be related with this antibody, at least in our cohort, the fact that anti-
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Fig. 2. A. Representation of the anti-CTTN ELISA test results in patients with polymyositis (PM) (n = 34), dermatomyositis (DM) (n = 117), immune-mediated necrotizing myopathy (IMNM) (n = 7), inclusion body myositis (IBM) (n = 4), and non-inflammatory myopathies (NIM) (n = 29). The cut-off value for a positive result was established at 0.300 absorbance units, which corresponded to 5 standard deviations above the mean value obtained for the 25 healthy controls (dashed line). B. Representation of the anti-CTTN ELISA results in 101 patients with systemic autoimmune diseases: systemic sclerosis (SSc) (n = 41); systemic lupus erythematosus (SLE) (n = 25), rheumatoid arthritis (RA) (n = 25), and primary Sjögren syndrome (pSS) (n = 10). Healthy volunteers (n=25) were included to determine the cut-off value.
CTTN was strongly associated with adult PM patients (20% versus b 1% in controls) could be relevant. It is well known that PM remains a diagnosis of exclusion, and that other myopathies can mimic this muscle disorder [14]. Thus, detection of this autoantibody may be of help in
establishing the diagnosis of true autoimmune polymyositis. The fact that anti-CTTN was rarely present in non-inflammatory myopathies that are known to mimic polymyositis is also relevant and merits consideration.
Table 1 Clinical characteristics and immunologic profile in a series of 18 myositis patients with anti-CTTN antibodies. Patient
Age, y/Sex
Muscle biopsy
MSA/MAA
Anti-CTTN (ELISA/IB)
CK (UI/L)
ILD
Arthritis
Raynaud phenomenon
Skin manifestations
Fever
Dysphagia
Cancer
HLA-DRB
1 2 3 4
56/M 48/M 48/M 43/F
PM PM PM PM
U1RNP Jo1/Ro52 U1RNP Ro52
0.957/+++ 1.138/+++ 0.390/+ 0.309/+
1390 14,765 4450 345
Yes Yes Yes Yes
No Yes Yes No
Yes No Yes Yes
No No Yes Yes
Yes Yes No Yes
No No No No
DRB1*14,15 DRB1*11 N/A DRB1*03, 08
5 6 7
38/M 22/F 50/F
IMNM PM DM
SRP EJ Jo1/Ro52
0.573/++ 0.366/+ 0.329/+
2686 476 271
No No Yes
No No No
No Yes Yes
Yes No No
No No No
No No No
DRB1*03, 01 DRB1* 07, 15 DRB1* 03, 04
8 9 10
63/M 74/M 52/F
PM PM DM
Mi2/Ro
0.505/++ 0.413/++ 0.479/++
451 1200 902
Yes No Yes
No No No
No No No
No No Yes
No No No
Lung No No
N/A N/A DRB1* 03, 04
11
60/F
DM
PM/Scl
0.970/+++
558
Yes
Yes
No
No
No
Breast
DRB1* 11, 13
12 13
78/F 42/F
DM DM
Ro Jo1/Ro/Ro52
0.451/++ 0.329/+
234 2500
No Yes
Yes Yes
No No
No Yes
No Yes
Pancreas No
DRB1*07, 14 DRB1* 03, 08
14
63/F
DM
Jo1/Ro52
0.482/++
2000
Yes
Yes
Yes
No
No
No
DRB1*03, 04
15 16
38/M 22/F
DM DM
Jo1/Ro52 PM/Scl
0.331/+ 0.404/++
27,000 2096
Yes Yes
Yes Yes
No Yes
Yes No
Yes Yes
No No
DRB1*03, 11 DRB1*03, 01
17 18
77/M 42/M
IMNM DM
TIF1γ
0.343/+ 0.345/+
3000 300
No No
Yes No
Yes No
No No No Telangiectasia Poikiloderma Mechanic's hand No Mechanic's hand Gottron papules Heliotrope rash No No Gottron sign Heliotrope rash Mechanic's hand Gottron sign Gottron sign Mechanic's hand Heliotrope rash Mechanic's hand Gottron sign Mechanic's hand Mechanic's hand Gottron sign Calcinosis No Gottron sign
No No
Yes Yes
No Lung
DRB1*04, 11 N/A
Age at diagnosis; MSA, muscle-specific autoantibodies; MAA, muscle-associated autoantibodies; IMNM, immune-mediated necrotizing myopathy; anti-CTTN, anti-cortactin antibodies, detected by in-house ELISA and confirmed by immunoblot; ILD, interstitial lung disease. M, male; F, female. N/A, Not available. Cancer was only paraneoplastic in patient 18.
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Testing of sera from patients with other autoimmune disorders yielded positive results in a few SSc and SLE cases. This suggests that anti-CTTN antibody is not an absolutely specific myositis autoantibody, but can be considered a new myositis-associated antibody (MAA). Myositis-specific and myositis-associated autoantibodies are found in up to 50% of myositis patients and are useful for assisting the diagnosis and perhaps, the prognosis of these heterogeneous disorders. Although we were not able to ascribe a phenotypic association with the presence of anti-CTTN in the myositis patients studied, the strong association with the PM subgroup could be valuable from the clinical viewpoint. Considering that the percentage of various myositis-specific or -associated autoantibodies ranges from 5% in the case of anti-SRP to 35% in the case of antisynthetase antibody [15,16], the 20% positivity for anti-CTTN antibody in patients with PM is not negligible and could be of diagnostic value. Cortactin is a protein recognized for its association with progression of cancer [8,17–19]; the role of antibodies against this protein remains to be elucidated. In the paraneoplastic myositis patients in our study, those who were anti-CTTN-positive had poorer survival than the antiCTTN-negative cases. Although this association did not reach statistical significance, perhaps because of the small sample size, this finding seems to concur with the above-mentioned association with cancer progression. A possible role of anti-CTTN antibody in the prognosis of patients with paraneoplastic myositis must be confirmed in other patient cohorts. The results of this study prove the feasibility of detecting antibodies against CTTN in adult patients with autoimmune diseases by in-house ELISA and immunoblot techniques using commercially available recombinant CTTN as the antigen. These techniques can be reliably performed in a standard laboratory setting. Our findings indicate that laboratory technicians should be aware that when anti-MDA5 or anti-HMCGR are being determined by ELISA, confirmation of positive results by immunoblotting is mandatory because of possible false-positives in patients with anti-CTTN antibody. Although further studies are needed to better delineate the role of anti-CTTN antibody in patients with autoimmune disease, our data suggest that this novel target antigen can be considered a new myositis-associated antibody with a potential for application in clinical practice. Funding This study was funded in part by grants from the Spanish Ministry of Health and Consumer Affairs (PI12-01320 and PI10-01871). Contributors MLH (guarantor) and MAM had the original idea, and together with ASOC and ETA, designed the study. MLH, MAM, and CJ performed the laboratory techniques (ELISA, immunoblot, and immunoprecipitation). ASOC, ETA, JMGJ, and MVT managed the patients with myositis. All authors contributed to, and approved, the final manuscript. Conflict of interest statement This is an original work, and all authors meet the criteria for authorship, including acceptance of responsibility for the scientific content of the manuscript. There are no conflicts of interest.
Take-home messages • In this study, we describe a novel myositis-associated autoantibody: anti- cortactin (CTTN). • Anti-CTTN antibody was found in the 20% of patients with polymyositis and could be of diagnostic value in these patients. • In patients with anti-CTTN antibody, false positive results can be obtained for determination of ELISA method of anti-MDA5 or antiHMCGR antibodies. In this case, confirmation of positive results by immunoblotting is mandatory.
Acknowledgments We would like to thank Dr. Isabel Illa and Dr. Jordi Diaz-Manera from the Neuromuscular Diseases Unit at Hospital de la Sta Creu i Sant Pau for providing sera from patients with muscle diseases and also to Dr. Roser Solans, Dr. Carmen Pilar Simeon and Dr. Josep Ordi from the Internal Medicine Department at Vall d'Hebron Hospital for providing sera from patients with pSS, SSc and SLE respectively.
References [1] Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two parts). N Engl J Med 1975;292:344–7. [2] Bohan A, Peter JB. Polymyositis and dermatomyositis (second of two parts). N Engl J Med 1975;292:403–7. [3] Mammen AL. Autoimmune myopathies: autoantibodies, phenotypes and pathogenesis. Nat Rev Neurol 2011;7:343–54. [4] Catalán M, Selva-O'Callaghan A, Grau JM. Diagnosis and classification of sporadic inclusion body myositis (sIBM). Autoimmun Rev 2014;13:363–6. [5] Hamann PD, Cooper RG, McHugh NJ, Chinoy H. Statin-induced necrotizing myositis —a discrete autoimmune entity within the “statin-induced myopathy spectrum”. Autoimmun Rev 2013;12:1177–81. [6] Ghirardello A, Bassi N, Palma L, Borella E, Domeneghetti M, Punzi L, et al. Autoantibodies in polymyositis and dermatomyositis. Curr Rheumatol Rep 2013;15:335. [7] Schuuring E, Verhoeven E, Mooi WJ, Michalides RJ. Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas. Oncogene 1992;7:355–61. [8] Buday L, Downward J. Roles of cortactin in tumor pathogenesis. Biochim Biophys Acta 2007;1775:263–73. [9] Sontheimer RD. Cutaneous features of classic dermatomyositis and amyopathic dermatomyositis. Curr Opin Rheumatol 1999;11:475–82. [10] Griggs RC, Askanas V, DiMauro S, Engel A, Karpati G, Mendell JR, et al. Inclusion body myositis and myopathies. Ann Neurol 1995;38:705–13. [11] Labrador-Horrillo M, Martínez MA, Selva-O'Callaghan A, Trallero-Araguás E, Balada E, Vilardell-Tarrés M, et al. Anti-TIF1γ antibodies (anti-p155) in adult patients with dermatomyositis: comparison of different diagnostic assays. Ann Rheum Dis 2012;71:993–6. [12] Labrador-Horrillo M, Martínez MA, Selva-O'Callaghan A, Trallero-Araguás E, Balada E, Vilardell-Tarrés M, et al. Anti-MDA5 antibodies in a large mediterranean population of adults with dermatomyositis. J Immunol Res 2014;2014:290797. [13] Selva-O'Callaghan A, Labrador-Horrillo M, Solans-Laque R, Simeon-Aznar CP, Martínez-Gómez X, Vilardell-Tarrés M. Myositis-specific and myositis-associated antibodies in a series of eighty-eight Mediterranean patients with idiopathic inflammatory myopathy. Arthritis Rheum 2006;55:791–8. [14] Milisenda JC, Selva-O'Callaghan A, Grau JM. The diagnosis and classification of polymyositis. J Autoimmun 2014;48–49:118–21. [15] Tansley SL, Betteridge ZE, McHugh NJ. The diagnostic utility of autoantibodies in adult and juvenile myositis. Curr Opin Rheumatol 2013;25:772–7. [16] Mahler M, Miller FW, Fritzler MJ. Idiopathic inflammatory myopathies and the antisynthetase syndrome: a comprehensive review. Autoimmun Rev 2014;13:367–71. [17] Weaver AM. Cortactin and tumor invasiveness. Cancer Lett 2008;265:157–66. [18] MacGrath SM, Koleske AJ. Cortactin in cell migration and cancer at a glance. J Cell Sci 2012;125:1621–6. [19] Kirkbride KC, Sung BH, Sinha S, Weaver AM. Cortactin: a multifunctional regulator of cellular invasiveness. Cell Adh Migr 2011;5:187–98.
