- Email: [email protected]
Sensitivity and clinical utility of the anti-cytosolic 5′-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: Report of 40 patients from a single neuromuscular center
Accepted Manuscript
Sensitivity and clinical utility of the anti-cytosolic 5’-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: report of 40 patients from a single neuromuscular center Kevin J. Felice D.O. , Charles H. Whitaker M.D. , Qian Wu M.D. , Daniel T. Larose Ph.D. , Guo Shen Ph.D. , Allan L. Metzger M.D. , Randall W. Barton Ph.D. PII: DOI: Reference:
S0960-8966(18)30186-X 10.1016/j.nmd.2018.06.005 NMD 3564
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
9 March 2018 16 May 2018 10 June 2018
Please cite this article as: Kevin J. Felice D.O. , Charles H. Whitaker M.D. , Qian Wu M.D. , Daniel T. Larose Ph.D. , Guo Shen Ph.D. , Allan L. Metzger M.D. , Randall W. Barton Ph.D. , Sensitivity and clinical utility of the anti-cytosolic 5’-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: report of 40 patients from a single neuromuscular center, Neuromuscular Disorders (2018), doi: 10.1016/j.nmd.2018.06.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1 Highlights
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We report the sensitivity and clinical utility of the anti-cN1A antibody test in sporadic inclusion body myositis (IBM). This is a retrospective review of 40 patients with clinico-pathologically defined or clinically defined IBM. The anti-cN1A antibody test has limited diagnostic utility in IBM.
ACCEPTED MANUSCRIPT 2 Sensitivity and clinical utility of the anti-cytosolic 5’-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: report of 40 patients from a single neuromuscular center
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Kevin J. Felice, D.O.a Charles H. Whitaker, M.D. a Qian Wu, M.D.b Daniel T. Larose, Ph.D.c Guo Shen, Ph.D.d Allan L. Metzger, M.D.d Randall W. Barton, Ph.D. e a
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Muscular Dystrophy Association Care Center, Department of Neuromuscular Medicine, Hospital for Special Care, New Britain, Connecticut, USA b Department of Pathology, University of Connecticut School of Medicine, Farmington, Connecticut, USA c Department of Mathematical Sciences, Central Connecticut State University, New Britain, Connecticut, USA d RDL Reference Laboratory, Inc., 10755 Venice Blvd., Los Angeles, California, USA e Department of Research, Hospital for Special Care, New Britain, Connecticut, USA
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Corresponding author: Dr. Kevin Felice Muscular Dystrophy Association (MDA) Care Center Department of Neuromuscular Medicine Hospital for Special Care New Britain, Connecticut 06053, USA Phone: (860) 612-6305 Fax: (860) 612-6304 E-mail: [email protected]
ACCEPTED MANUSCRIPT 3 Abstract Sporadic inclusion body myositis (IBM) is the most common acquired myopathy affecting patients over age 50. The discovery of an autoantibody directed against a 43-44 kD protein (anti-cytosolic-5’-nucleotidase 1A or anti-cN1A) has provided support for the hypothesis
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of an immune-mediated pathogenesis. Previous studies have reported variable test sensitivity and specificity, and inconsistent results on the predictive value. In our cohort of 40 patients with clinico-pathologically or clinically defined IBM, we found the sensitivity of the anti-cN1A
antibody test to be 50%. Comparing characteristics for test positive and test negative groups, we
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found that patients in our cohort testing positive for the anti-cN1A antibody were significantly more likely to be older than age 60 years at symptom onset. We found no positive association between anti-cN1A reactivity and other clinical, laboratory, and muscle histopathologic findings.
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Based on all clinical studies published to date including the present, the anti-cN1A antibody test shows high diagnostic specificity, moderate sensitivity, and a low predictive value in regards to
Key Words:
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Inclusion body myositis
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age of onset, disease severity and other associated clinicopathological findings.
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Inflammatory myopathy Myopathy
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Anti-cytosolic-5’-nucleotidase 1A antibody Rimmed vacuoles Funding:
This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.
ACCEPTED MANUSCRIPT 4 1. Introduction Sporadic inclusion body myositis (IBM) is the most common acquired myopathy affecting patients over age 50 [1,2]. Prevalence rates are variable, ranging from 5 per million in the Netherlands to 117 per million in the Republic of Ireland [2,3,4]. IBM is a primary skeletal
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myopathy manifesting with slowly progressive weakness. Characteristic initial clinical findings include weakness and atrophy of long finger flexors and quadriceps muscles. Symptom onset may be difficult for patients to pinpoint given the slow, insidious and painless progression. Facial weakness, dysphagia, postural weakness, and gait difficulties are additional features for many
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patients. As the disease progresses and certainly when longstanding, muscular involvement in IBM becomes more generalized, necessitating the need for walking aids, powered mobility and transfer devices, and, in some patients with severe dysphagia, gastric feeding tubes. Reported
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rates of decline range from 3.5% to 14.9% per year [4,5]. Life expectancy is usually not altered given the absence of cardiac and symptomatic pulmonary involvement. Unfortunately, disease-
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modifying treatments are unavailable. Multiple clinical trials since 1993 – mostly designed to suppress or modulate potential immune mechanisms in IBM – have been universally ineffective
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in slowing or reversing disease progression [2]. Disease monitoring, rehabilitative support, and
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symptomatic treatments are the mainstay of care for IBM patients. In the US, Muscular Dystrophy Association (MDA) Care Centers are particularly suitable for such care.
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Although the etiology of IBM remains uncertain, the histopathologic findings of lymphocytic invasion, filamentous inclusions, and autophagic vacuoles have implicated both immune-mediated and degenerative mechanisms for muscle cell injury [6]. The discovery of an autoantibody directed against a 43-44 kD protein has provided additional support for the hypothesis of an immune-mediated pathogenesis [7]. Subsequently, the target protein has been
ACCEPTED MANUSCRIPT 5 identified as cytoplasmic 5’-nucleotidase 1A (cN1A) [8], an enzyme highly expressed in skeletal muscle that catalyzes the dephosphorylation of adenosine monophosphate and may be important in muscle contraction [9]. Several small or multi-site studies have reported test sensitivity for the anti-cN1A
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antibody ranging from 33 to 76% (Table 1) [7-16]. In addition, anti-cN1A autoantibodies have been reported in some patients with other inflammatory myopathies (e.g., polymyositis,
dermatomyositis), other autoimmune diseases (e.g., systemic lupus erythematosus, Sjögren syndrome), and motor neuron disease [13,14,17,18] – with a specificity ranging from 87 to
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100%. Based on pooled data from these multiple studies, anti-cN1A antibody, now a
commercially-available diagnostic test, has a sensitivity of 44% and specificity of 93%. The predictive value of the anti-cN1A antibody test is less certain. Published studies report
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inconsistent differences between anti-cN1A positive and negative IBM cohorts relative to age of onset, disease duration, clinical features and severity, and pathological findings. The aim of this
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present study was to retrospectively review the clinical and pathological data on a cohort of 40 IBM patients – all examined, diagnosed and followed at a single MDA Care Center in
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Connecticut – comparing and correlating characteristics of anti-cN1A positive and negative
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patients, and, ultimately, providing further data on the sensitivity and predictive value of the anticN1A antibody test in IBM.
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2. Material and methods 2.1 Subject Characteristics This was a retrospective review of the medical records and muscle histopathology of all
patients with the diagnosis of sporadic IBM who had been evaluated at our MDA Care Center from 2007 until 2017 and had undergone serum testing for the commercially-available anti-
ACCEPTED MANUSCRIPT 6 cN1A antibody test (RDL Reference Lab Inc., Los Angeles, California, USA). All subjects had clinico-pathologically or clinically defined IBM based on European Neuromuscular Centre (ENMC) 2011 research diagnostic criteria [5]. The data abstracted from the medical records included age, gender, age of disease onset, disease duration, anti-cN1A antibody positivity and
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units, creatine phosphokinase (CK) total and times upper normal limit, forced vital capacity (FVC) in liters and percent of predicted (for age, height and sex), hand grip dynamometry in newtons, Medical Research Council (MRC) grading system total, MRC for bilateral quadriceps muscles, and concurrent medical illnesses. Total quantitative MRC scores were obtained by two
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experienced neuromuscular neurologists (KJF, CHW) by adding the MRC score for the
following muscle groups on each side: arm abductors, elbow flexors, elbow extensors, wrist flexors, wrist extensors, finger extensors, hip flexors, hip adductors, knee flexors, knee
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extensors, foot plantar flexors, and foot dorsiflexors. For a subject with completely normal strength, the total MRC score would be 120. Hand grip strength was measured using a Jamar
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dynamometer (Sammons Preston Rolyan, Chicago, Illinois, USA). FVC was obtained by a certified respiratory therapist. Muscle biopsies were reviewed by a neuromuscular neurologist
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and neuropathologist (KJF, QW). We recorded muscle histopathological features, including
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presence or absence of inflammation and rimmed vacuoles [RV] on light microscopy, and tubulofilamentous inclusions [TFI] on electron microscopy. Degree of inflammation was graded
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as severe if lymphocyte aggregates were noted in 3 or more areas on low-powered light microscopy, mild for 1-2 aggregates, and none if inflammatory cells were not observed. This study was approved by the Human Research Advisory Committee at Hospital for Special Care. 2.2 Serological testing for anti-cN1A autoantibodies
ACCEPTED MANUSCRIPT 7 Testing for anti-cN1A autoantibodies was performed at Rheumatology Diagnostic Laboratory, Inc. (RDL). The enzyme-linked immunosorbent (ELISA) method (Euroimmun, Lubeck, Germany) has been reported previously [19] and is a unique laboratory-developed test (LDT). Briefly, microtiter wells were coated using Euroimmun full-length cN1A antigens
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(Microplate Immunoassay Kit) via DYNEX, incubated with diluted patient sera, and the bound antibodies were detected colorimetrically with horse radish peroxidase-conjugated with goat anti-human IgG (Fab-specific). Positive serum (~43-kd NT5C1A protein) samples were related to the level of measurement signal proportional to the concentration of cN1A antibodies and can
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be determined using a single calibrator with known cN1A concentrations. Intra-assay, inter-assay linearity, analytical sensitivity, and normal range were excellent, and correlation between Euroimmun and RDL LDT had an excellent value (R2=0.92). Anti-cN1A antibody results were
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reported as follows: <20 units, negative; 20-39 units, weak positive; 40-80 units, moderate
2.3 Statistical analyses
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positive; and >80 units, strong positive.
We compared the results of anti-cN1A antibody positive and negative patients. The three
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positive groups (weak, moderate, or strong) were collapsed into a single group, so that an
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investigation could be performed comparing the characteristics of those patients who were anticN1A antibody positive and those who were negative. There were two types of variables to be
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tested, continuous variables (such as age), and indicator variables (such as whether hypertension was present). The Fisher’s exact test was utilized to compare variables and a p-value of less than 0.05 was required for statistical significance. The results are shown in Table 3 for the continuous variables and Table 4 for the indicator variables. 3. Results
ACCEPTED MANUSCRIPT 8 We reviewed the medical records and muscle histopathology of 40 patients including 15 with clinico-pathologically defined IBM and 25 with clinically defined IBM. Summary of patient characteristics are shown in Table 2. In our cohort, 20 of 40 (50%) of patients tested positive for anti-cN1A, and, of these, antibodies were strong positive in 12 (60%), moderate positive in 5
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(25%), and weak positive in 3 (15%). This indicates an assay sensitivity of 0.5 with 95%
confidence intervals, 0.35 – 0.65. Subgroup analysis showed a test sensitivity of 33% in clinicopathologically defined IBM and 60% in clinically defined IBM.
Comparing characteristics for test positive and test negative groups, we found that
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patients in our cohort testing positive for the anti-cN1A antibody were significantly more likely to be older than age 60 years at symptom onset (Figure 1). However, further analyses found no significant differences in regards to age of onset, disease duration, gender, FVC in liters and
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percent predicted, MRC scores total and quadriceps only, hand grip forces, CK values, dysphagia symptoms, ambulation status, use of devices, muscle histopathology features, and concurrent
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medical illnesses including connective tissue diseases, hyperlipidemia or coronary artery disease
4. Discussion
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(Tables 3 and 4).
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Our aim was to assess the sensitivity and clinical utility of the anti-cN1A antibody test in clinico-pathologically or clinically defined IBM patients who were followed at a single site and
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whose antibody testing was performed by a single reference lab. We found the sensitivity of this test to be 50% which is similar to other studies and slightly higher than the mean value of 44% when pooling all published studies. Excluding our patients with weak positive antibody testing, the sensitivity of anti-cN1A antibody testing in our cohort drops to 43% and approaches the mean pooled value from published studies. Of interest, test sensitivity was higher in our IBM
ACCEPTED MANUSCRIPT 9 patients who were clinically defined versus the group who were clinico-pathologically defined. Comparing characteristics for test positive and test negative groups, patients in our cohort testing positive for the anti-cN1A antibody test were more likely to be older than age 60 years at
clinical, laboratory, and muscle histopathological findings.
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symptom onset. We found no significant differences between the two groups in regards to other
Previous studies assessing the predictive value of the anti-cN1A antibody test have reported variable, inconsistent and, generally, negative results [8,10-16]. For most studies
comparing cohorts testing positive versus negative for anti-cN1A antibodies, age of onset, age at
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testing, and disease duration have shown no significant differences [8,10-16]. Females had a higher odds of being seropositive in one study [12]; otherwise, no significant gender differences were noted. Regarding clinical findings and disease severity, most studies have reported no
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significant differences between the two groups [8,10,14,15,16]. In their study of 25 IBM patients, Goyal et al. found that anti-cN1A antibody positive patients took significantly longer to
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stand from a chair, demonstrated lower total MRC scores and forced vital capacities, and were more likely to have facial weakness, dysphagia symptoms, and use walking aids or wheelchairs
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compared to negative patients [12]. The IBM functional rating scale (IBMFRS) and distance
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covered on the 6-minute walk test were not significantly different, however. In the European study of 311 patients by Lilleker et al., anti-cN1A antibody negative patients were more likely to
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have proximal upper extremity weakness at disease onset whereas antibody positive patients were more likely to have facial weakness at last review [16]. However in this study and the Japanese study of 67 IBM patients, other disease characteristics including distribution of weakness, dysphagia, axial involvement, and concurrent polyneuropathy were not significantly different between the two groups [15,16].
ACCEPTED MANUSCRIPT 10 Several studies also compared laboratory and antibody tests, muscle histopathology, and concurrent medical illnesses in anti-cN1A antibody positive and negative patients. Creatine kinase values were not significantly different between the two groups in several reports [12,1416]. Of clinical importance, blood samples obtained from two patients initially diagnosed with
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idiopathic hyperCKemia tested positive for high anti-cN1A reactivity 5 and 8 years before the clinicopathological diagnosis of IBM was confirmed, suggesting a role for antibody testing in the evaluation of idiopathic myopathies [8]. Anti-HCV antibodies were significantly more prevalent in anti-cN1A antibody positive patients in one study [15]. Anti-La antibodies were more
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prevalent in one study [16] but not in another [8]. In these studies – antinuclear antibody, doublestranded DNA, anti-HTLV1, anti-Ro, myositis-associated, and myositis-specific antibodies – showed no significant association with anti-cN1A antibody status. Similarly, an association was
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not noted between the anti-cN1A antibody and risk for malignancy, degree of denervation on EMG, and positive response to treatment with intravenous immunoglobulin or corticosteroids
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[11,15]. The latter observation is especially interesting given that IBM clinical trials employing immunosuppressive medications have not documented efficacy.
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In addition to the current study, three prior studies have evaluated the association of anti-
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cN1A reactivity with muscle histopathological findings [14-16]. In the study by Lloyd et al., antibody positive IBM patients had a lower prevalence of rimmed vacuoles, but no association
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was noted in degree of focal invasion or degree of endomysial inflammation on pathological specimens [14]. Tawara et al. found type 2 myofibers to be significantly smaller in the anticN1A antibody positive compared to negative group [15]. However, no association was noted in regards to degree of inflammatory cells, congophilic material, co-localization with p62 protein, or perinuclear localization of cN1A protein. Finally, Lilleker et al. found significantly more
ACCEPTED MANUSCRIPT 11 COX-deficient fibers in anti-cN1A positive compared to the negative group [16]. Many other histopathological features – ragged red fibers, atrophic fibers, inflammation, MHC1 upregulation, necrosis, mononuclear infiltrate, invasion of non-necrotic fibers, rimmed vacuoles, protein deposits, and microfilaments – showed no association with anti-cN1A antibody
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reactivity. These and other inconsistent clinicopathological observations are difficult to explain, and may be attributed to the different assay methods used, sample sizes, or patients’ background [15].
Based on all clinical studies published to date including the present, the anti-cN1A
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antibody test shows low predictive value in regards to disease severity and associated
clinicopathological findings. Despite this limitation, the anti-cN1A antibody test may be clinically useful in certain situations. Given the high test specificity, a positive test provides
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strong diagnostic support for patients with clinically defined IBM who are refusing a muscle biopsy or those patients for whom a muscle biopsy may impose a degree of increased risk or
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morbidity (e.g. treatment with anti-coagulation therapy). Another potential use is as a screening test for patients with mild or partial IBM clinical features (e.g., isolated dysphagia or quadriceps
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weakness) and nonspecific or non-diagnostic muscle biopsy findings. However, for patients with
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clinico-pathologically IBM, the anti-cN1A test appears to provide no additional clinically useful information.
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In summary, based on all clinical studies published to date including the present, the anticN1A antibody test shows high diagnostic specificity, moderate sensitivity, and a low predictive value in regards to age of onset, disease severity and other associated clinicopathological findings. It is hopeful that further refinement of the anti-cN1A antibody test or development of another noninvasive test will provide improved clinical utility.
ACCEPTED MANUSCRIPT 12 References [1] Needham M, Mastaglia FL. Sporadic inclusion body myositis: a review of recent clinical advances and current approaches to diagnosis and treatment. Clin Neurophysiol 2016;127:176473. [2] Greenberg SA. Inclusion body myositis. Continnum 2016;22:1871-88.
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[3] Felice KJ, North WA. Inclusion myositis in Connecticut: observations in 35 patients during an 8-year period. Medicine 2001;80:320-7. [4] Lefter S, Hardiman O, Ryan AM. A population-based epidemiologic study of adult neuromuscular disease in the Republic of Ireland. Neurology 2017;88:304-13.
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[5] Rose MR. 188th ENMC international workshop: inclusion body myositis, 2-4 December 2011, Naarden, The Netherlands. Neuromuscul Disord 2013;23:1044-55. [6] Weihl CC, Mammen AL. Sporadic inclusion body myositis – a myodegenerative disease or an inflammatory myopathy. Neuropathol Appl Neurobiol 2017;43:82-91. [7] Salajegheh M, Lam T, Greenberg SA. Autoantibodies against a 43 KDa muscle protein in inclusion body myositis. PLoS One 2011;6:e20266.
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[8] Larman HB, Salajegheh M, Nazareno, et al. Cytosolic 5’-nucleotidase 1A autoimmunity in sporadic inclusion body myositis. Ann Neurol 2013;73:408-18.
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[9] Pluk H, van Hoeve BJ, van Dooren SH, et al. Autoantibodies to cytosolic 5’-nucleosidase 1A in inclusion body myositis. Ann Neurol 2013;73:397-407.
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[10] Greenberg SA. Cytoplasmic 5’-nucleotidase autoantibodies in inclusion body myositis: isotypes and diagnostic utility. Muscle Nerve 2014:50:488-92.
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[11] Limaye VS, Lester S, Blumbergs P, Greenberg SA. Anti-CN1A antibodies in South Australian patients with inclusion body myositis. Muscle Nerve 2016;53:654-5.
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[12] Goyal NA, Cash TM, Alam U, et al. Seropositivity for NT5c1A antibody in sporadic inclusion body myositis predicted more serve motor, bulbar and respiratory involvement. J Neurol Neurosurg Psychiatry 2016;87:373-8. [13] Herbert MK, Stammen-Vogelzangs J, Verbeek MM, et al. Disease specificity of antibodies to cystolic 5’nucleotidase 1A in sporadic inclusion body myositis versus known autoimmune diseases. Ann Rheum Dis 2016;75:696-701. [14] Lloyd TE, Christopher-Stine L, Pinal-Fernandez, I, et al. Cytosolic 5’-nucleotidase 1A as a target of circulating autoantibodies in autoimmune diseases. Arthritis Care Res 2016;68:66-71.
ACCEPTED MANUSCRIPT 13 [15] Tawara N, Yamashita S, Zhang X, et al. Pathomechanisms of anti-cytosolic 5’-nucleotidase 1A autoantibodies in sporadic inclusion body myositis. Ann Neurol 2017;81:512-25. [16] Lilleker JB, Rietveld A, Pye SR, et al. Cytosolic 5’-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis. Ann Rheum Dis 2017;76:862-8.
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[17] Herbert MK, Pruijn GJ. Novel serology testing for sporadic inclusion body myositis: disease-specificity and diagnostic utility. Curr Opin Rheumatol 2015;27:595-600. [18] Liewluck T. Anti-cytosolic 5’-nucleotidase 1A (cN1A) autoantibodies in motor neuron disease. Neurology 2017;89:2017-8.
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[19] Kramp SL, Karayev D, Shen G, et al. Development and evaluation of a standardized ELISA for the determination of autoantibodies against cN-1A (Mup44, NT5C1A) in sporadic inclusion body myositis. Auto Immun Highlights 2016;7:16.
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Figure 1. Dotplot comparing age of onset and anti-cN1A antibody positivity.
ACCEPTED MANUSCRIPT 15 Table 1. Sensitivity and specificity of the cN1A antibody in IBM. Sensitivity (%) 52 70 60 76 35 72 37 61 36 33 50
Specificity (%) 100 92 89 91 NA NA 96 87 92 NA NA
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IBM patients (n) 25 47 56 50 69 25 238 117 67 311 40
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Author [reference] Salajegheh [7] Larman [8] Pluk [9] Greenberg [10] Limaye [11] Goyal [12] Herbert [13] Lloyd [14] Tawara [15] Lilleker [16] Current study
ACCEPTED MANUSCRIPT 16 Table 2. Summary of IBM clinical and pathological findings. Mean (range) 70.6 years (47-88) 59.0 years (41-72) 11.6 years (2-30) 104 units (22-222) 74% (46-122) 642 unit/L (138-2344) 3.1 (0.5-8.7) 94.1 (38-118) 5 (0-10) 226.8 N (0-427)
Male cN1A positive total cN1A positive in clinico-pathologically defined (n=15) cN1A positive in clinically defined (n=25) Dysphagia Ambulatory Connective tissue disease Muscle inflammation severe Muscle inflammation mild Rimmed vacuoles Tubulofilamentous inclusions
Number (%) 28 (70) 20 (50) 5 (33) 15 (60) 28 (70) 32 (80) 5 (12%) 17 (42) 23 (58) 35 (87) 17 (42)
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Finding Age of presentation Age of onset Duration of symptoms cN1A titer (n=20) FVC %predicted CPK total (n=32) CPK xUNL (n=32) MRC total MRC quadriceps Grip force (n=31)
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Table 3. Means of continuous variables. Mean, cN1A56.9 69.7 12.8 3.133 75.0 91.95 4.65 237 678.8 3.207
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p-value 0.100 0.523 0.251 0.335 0.767 0.551 0.476 0.671 0.697 0.860
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Mean, cN1A+ 61.0 71.5 10.4 2.817 73.45 96.3 5.35 216 605.7 3.066
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Age onset Test age Duration FVC L FVC % MRC Total MRC Quad Grips Force CPK u/l CPK xULN
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Table 4. Percentages for indicator variables. Percent, cN1A25% 60%
p-value 0.010 0.053
50% 95% 40% 80% 65% 40% 15% 70% 45% 25%
80% 80% 20% 84% 70% 45% 10% 70% 60% 25%
0.096 0.342 0.301 1.000 1.000 1.000 1.000 1.000 0.527 1.000
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Percent, cN1A+ 70% 25%
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Age of onset >60 Tubulofilamentous inclusions+ Hypertension+ RV+ Sex Female Amb+ Device Inflam Grade Severe CTD+ Dysphagia+ Hyperlipidemia+ CAD+
Sensitivity and clinical utility of the anti-cytosolic 5’-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: report of 40 patients from a single neuromuscular center Kevin J. Felice D.O. , Charles H. Whitaker M.D. , Qian Wu M.D. , Daniel T. Larose Ph.D. , Guo Shen Ph.D. , Allan L. Metzger M.D. , Randall W. Barton Ph.D. PII: DOI: Reference:
S0960-8966(18)30186-X 10.1016/j.nmd.2018.06.005 NMD 3564
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
9 March 2018 16 May 2018 10 June 2018
Please cite this article as: Kevin J. Felice D.O. , Charles H. Whitaker M.D. , Qian Wu M.D. , Daniel T. Larose Ph.D. , Guo Shen Ph.D. , Allan L. Metzger M.D. , Randall W. Barton Ph.D. , Sensitivity and clinical utility of the anti-cytosolic 5’-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: report of 40 patients from a single neuromuscular center, Neuromuscular Disorders (2018), doi: 10.1016/j.nmd.2018.06.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1 Highlights
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We report the sensitivity and clinical utility of the anti-cN1A antibody test in sporadic inclusion body myositis (IBM). This is a retrospective review of 40 patients with clinico-pathologically defined or clinically defined IBM. The anti-cN1A antibody test has limited diagnostic utility in IBM.
ACCEPTED MANUSCRIPT 2 Sensitivity and clinical utility of the anti-cytosolic 5’-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: report of 40 patients from a single neuromuscular center
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Kevin J. Felice, D.O.a Charles H. Whitaker, M.D. a Qian Wu, M.D.b Daniel T. Larose, Ph.D.c Guo Shen, Ph.D.d Allan L. Metzger, M.D.d Randall W. Barton, Ph.D. e a
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Muscular Dystrophy Association Care Center, Department of Neuromuscular Medicine, Hospital for Special Care, New Britain, Connecticut, USA b Department of Pathology, University of Connecticut School of Medicine, Farmington, Connecticut, USA c Department of Mathematical Sciences, Central Connecticut State University, New Britain, Connecticut, USA d RDL Reference Laboratory, Inc., 10755 Venice Blvd., Los Angeles, California, USA e Department of Research, Hospital for Special Care, New Britain, Connecticut, USA
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Corresponding author: Dr. Kevin Felice Muscular Dystrophy Association (MDA) Care Center Department of Neuromuscular Medicine Hospital for Special Care New Britain, Connecticut 06053, USA Phone: (860) 612-6305 Fax: (860) 612-6304 E-mail: [email protected]
ACCEPTED MANUSCRIPT 3 Abstract Sporadic inclusion body myositis (IBM) is the most common acquired myopathy affecting patients over age 50. The discovery of an autoantibody directed against a 43-44 kD protein (anti-cytosolic-5’-nucleotidase 1A or anti-cN1A) has provided support for the hypothesis
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of an immune-mediated pathogenesis. Previous studies have reported variable test sensitivity and specificity, and inconsistent results on the predictive value. In our cohort of 40 patients with clinico-pathologically or clinically defined IBM, we found the sensitivity of the anti-cN1A
antibody test to be 50%. Comparing characteristics for test positive and test negative groups, we
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found that patients in our cohort testing positive for the anti-cN1A antibody were significantly more likely to be older than age 60 years at symptom onset. We found no positive association between anti-cN1A reactivity and other clinical, laboratory, and muscle histopathologic findings.
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Based on all clinical studies published to date including the present, the anti-cN1A antibody test shows high diagnostic specificity, moderate sensitivity, and a low predictive value in regards to
Key Words:
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Inclusion body myositis
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age of onset, disease severity and other associated clinicopathological findings.
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Inflammatory myopathy Myopathy
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Anti-cytosolic-5’-nucleotidase 1A antibody Rimmed vacuoles Funding:
This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.
ACCEPTED MANUSCRIPT 4 1. Introduction Sporadic inclusion body myositis (IBM) is the most common acquired myopathy affecting patients over age 50 [1,2]. Prevalence rates are variable, ranging from 5 per million in the Netherlands to 117 per million in the Republic of Ireland [2,3,4]. IBM is a primary skeletal
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myopathy manifesting with slowly progressive weakness. Characteristic initial clinical findings include weakness and atrophy of long finger flexors and quadriceps muscles. Symptom onset may be difficult for patients to pinpoint given the slow, insidious and painless progression. Facial weakness, dysphagia, postural weakness, and gait difficulties are additional features for many
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patients. As the disease progresses and certainly when longstanding, muscular involvement in IBM becomes more generalized, necessitating the need for walking aids, powered mobility and transfer devices, and, in some patients with severe dysphagia, gastric feeding tubes. Reported
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rates of decline range from 3.5% to 14.9% per year [4,5]. Life expectancy is usually not altered given the absence of cardiac and symptomatic pulmonary involvement. Unfortunately, disease-
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modifying treatments are unavailable. Multiple clinical trials since 1993 – mostly designed to suppress or modulate potential immune mechanisms in IBM – have been universally ineffective
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in slowing or reversing disease progression [2]. Disease monitoring, rehabilitative support, and
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symptomatic treatments are the mainstay of care for IBM patients. In the US, Muscular Dystrophy Association (MDA) Care Centers are particularly suitable for such care.
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Although the etiology of IBM remains uncertain, the histopathologic findings of lymphocytic invasion, filamentous inclusions, and autophagic vacuoles have implicated both immune-mediated and degenerative mechanisms for muscle cell injury [6]. The discovery of an autoantibody directed against a 43-44 kD protein has provided additional support for the hypothesis of an immune-mediated pathogenesis [7]. Subsequently, the target protein has been
ACCEPTED MANUSCRIPT 5 identified as cytoplasmic 5’-nucleotidase 1A (cN1A) [8], an enzyme highly expressed in skeletal muscle that catalyzes the dephosphorylation of adenosine monophosphate and may be important in muscle contraction [9]. Several small or multi-site studies have reported test sensitivity for the anti-cN1A
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antibody ranging from 33 to 76% (Table 1) [7-16]. In addition, anti-cN1A autoantibodies have been reported in some patients with other inflammatory myopathies (e.g., polymyositis,
dermatomyositis), other autoimmune diseases (e.g., systemic lupus erythematosus, Sjögren syndrome), and motor neuron disease [13,14,17,18] – with a specificity ranging from 87 to
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100%. Based on pooled data from these multiple studies, anti-cN1A antibody, now a
commercially-available diagnostic test, has a sensitivity of 44% and specificity of 93%. The predictive value of the anti-cN1A antibody test is less certain. Published studies report
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inconsistent differences between anti-cN1A positive and negative IBM cohorts relative to age of onset, disease duration, clinical features and severity, and pathological findings. The aim of this
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present study was to retrospectively review the clinical and pathological data on a cohort of 40 IBM patients – all examined, diagnosed and followed at a single MDA Care Center in
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Connecticut – comparing and correlating characteristics of anti-cN1A positive and negative
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patients, and, ultimately, providing further data on the sensitivity and predictive value of the anticN1A antibody test in IBM.
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2. Material and methods 2.1 Subject Characteristics This was a retrospective review of the medical records and muscle histopathology of all
patients with the diagnosis of sporadic IBM who had been evaluated at our MDA Care Center from 2007 until 2017 and had undergone serum testing for the commercially-available anti-
ACCEPTED MANUSCRIPT 6 cN1A antibody test (RDL Reference Lab Inc., Los Angeles, California, USA). All subjects had clinico-pathologically or clinically defined IBM based on European Neuromuscular Centre (ENMC) 2011 research diagnostic criteria [5]. The data abstracted from the medical records included age, gender, age of disease onset, disease duration, anti-cN1A antibody positivity and
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units, creatine phosphokinase (CK) total and times upper normal limit, forced vital capacity (FVC) in liters and percent of predicted (for age, height and sex), hand grip dynamometry in newtons, Medical Research Council (MRC) grading system total, MRC for bilateral quadriceps muscles, and concurrent medical illnesses. Total quantitative MRC scores were obtained by two
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experienced neuromuscular neurologists (KJF, CHW) by adding the MRC score for the
following muscle groups on each side: arm abductors, elbow flexors, elbow extensors, wrist flexors, wrist extensors, finger extensors, hip flexors, hip adductors, knee flexors, knee
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extensors, foot plantar flexors, and foot dorsiflexors. For a subject with completely normal strength, the total MRC score would be 120. Hand grip strength was measured using a Jamar
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dynamometer (Sammons Preston Rolyan, Chicago, Illinois, USA). FVC was obtained by a certified respiratory therapist. Muscle biopsies were reviewed by a neuromuscular neurologist
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and neuropathologist (KJF, QW). We recorded muscle histopathological features, including
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presence or absence of inflammation and rimmed vacuoles [RV] on light microscopy, and tubulofilamentous inclusions [TFI] on electron microscopy. Degree of inflammation was graded
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as severe if lymphocyte aggregates were noted in 3 or more areas on low-powered light microscopy, mild for 1-2 aggregates, and none if inflammatory cells were not observed. This study was approved by the Human Research Advisory Committee at Hospital for Special Care. 2.2 Serological testing for anti-cN1A autoantibodies
ACCEPTED MANUSCRIPT 7 Testing for anti-cN1A autoantibodies was performed at Rheumatology Diagnostic Laboratory, Inc. (RDL). The enzyme-linked immunosorbent (ELISA) method (Euroimmun, Lubeck, Germany) has been reported previously [19] and is a unique laboratory-developed test (LDT). Briefly, microtiter wells were coated using Euroimmun full-length cN1A antigens
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(Microplate Immunoassay Kit) via DYNEX, incubated with diluted patient sera, and the bound antibodies were detected colorimetrically with horse radish peroxidase-conjugated with goat anti-human IgG (Fab-specific). Positive serum (~43-kd NT5C1A protein) samples were related to the level of measurement signal proportional to the concentration of cN1A antibodies and can
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be determined using a single calibrator with known cN1A concentrations. Intra-assay, inter-assay linearity, analytical sensitivity, and normal range were excellent, and correlation between Euroimmun and RDL LDT had an excellent value (R2=0.92). Anti-cN1A antibody results were
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reported as follows: <20 units, negative; 20-39 units, weak positive; 40-80 units, moderate
2.3 Statistical analyses
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positive; and >80 units, strong positive.
We compared the results of anti-cN1A antibody positive and negative patients. The three
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positive groups (weak, moderate, or strong) were collapsed into a single group, so that an
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investigation could be performed comparing the characteristics of those patients who were anticN1A antibody positive and those who were negative. There were two types of variables to be
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tested, continuous variables (such as age), and indicator variables (such as whether hypertension was present). The Fisher’s exact test was utilized to compare variables and a p-value of less than 0.05 was required for statistical significance. The results are shown in Table 3 for the continuous variables and Table 4 for the indicator variables. 3. Results
ACCEPTED MANUSCRIPT 8 We reviewed the medical records and muscle histopathology of 40 patients including 15 with clinico-pathologically defined IBM and 25 with clinically defined IBM. Summary of patient characteristics are shown in Table 2. In our cohort, 20 of 40 (50%) of patients tested positive for anti-cN1A, and, of these, antibodies were strong positive in 12 (60%), moderate positive in 5
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(25%), and weak positive in 3 (15%). This indicates an assay sensitivity of 0.5 with 95%
confidence intervals, 0.35 – 0.65. Subgroup analysis showed a test sensitivity of 33% in clinicopathologically defined IBM and 60% in clinically defined IBM.
Comparing characteristics for test positive and test negative groups, we found that
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patients in our cohort testing positive for the anti-cN1A antibody were significantly more likely to be older than age 60 years at symptom onset (Figure 1). However, further analyses found no significant differences in regards to age of onset, disease duration, gender, FVC in liters and
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percent predicted, MRC scores total and quadriceps only, hand grip forces, CK values, dysphagia symptoms, ambulation status, use of devices, muscle histopathology features, and concurrent
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medical illnesses including connective tissue diseases, hyperlipidemia or coronary artery disease
4. Discussion
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(Tables 3 and 4).
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Our aim was to assess the sensitivity and clinical utility of the anti-cN1A antibody test in clinico-pathologically or clinically defined IBM patients who were followed at a single site and
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whose antibody testing was performed by a single reference lab. We found the sensitivity of this test to be 50% which is similar to other studies and slightly higher than the mean value of 44% when pooling all published studies. Excluding our patients with weak positive antibody testing, the sensitivity of anti-cN1A antibody testing in our cohort drops to 43% and approaches the mean pooled value from published studies. Of interest, test sensitivity was higher in our IBM
ACCEPTED MANUSCRIPT 9 patients who were clinically defined versus the group who were clinico-pathologically defined. Comparing characteristics for test positive and test negative groups, patients in our cohort testing positive for the anti-cN1A antibody test were more likely to be older than age 60 years at
clinical, laboratory, and muscle histopathological findings.
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symptom onset. We found no significant differences between the two groups in regards to other
Previous studies assessing the predictive value of the anti-cN1A antibody test have reported variable, inconsistent and, generally, negative results [8,10-16]. For most studies
comparing cohorts testing positive versus negative for anti-cN1A antibodies, age of onset, age at
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testing, and disease duration have shown no significant differences [8,10-16]. Females had a higher odds of being seropositive in one study [12]; otherwise, no significant gender differences were noted. Regarding clinical findings and disease severity, most studies have reported no
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significant differences between the two groups [8,10,14,15,16]. In their study of 25 IBM patients, Goyal et al. found that anti-cN1A antibody positive patients took significantly longer to
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stand from a chair, demonstrated lower total MRC scores and forced vital capacities, and were more likely to have facial weakness, dysphagia symptoms, and use walking aids or wheelchairs
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compared to negative patients [12]. The IBM functional rating scale (IBMFRS) and distance
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covered on the 6-minute walk test were not significantly different, however. In the European study of 311 patients by Lilleker et al., anti-cN1A antibody negative patients were more likely to
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have proximal upper extremity weakness at disease onset whereas antibody positive patients were more likely to have facial weakness at last review [16]. However in this study and the Japanese study of 67 IBM patients, other disease characteristics including distribution of weakness, dysphagia, axial involvement, and concurrent polyneuropathy were not significantly different between the two groups [15,16].
ACCEPTED MANUSCRIPT 10 Several studies also compared laboratory and antibody tests, muscle histopathology, and concurrent medical illnesses in anti-cN1A antibody positive and negative patients. Creatine kinase values were not significantly different between the two groups in several reports [12,1416]. Of clinical importance, blood samples obtained from two patients initially diagnosed with
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idiopathic hyperCKemia tested positive for high anti-cN1A reactivity 5 and 8 years before the clinicopathological diagnosis of IBM was confirmed, suggesting a role for antibody testing in the evaluation of idiopathic myopathies [8]. Anti-HCV antibodies were significantly more prevalent in anti-cN1A antibody positive patients in one study [15]. Anti-La antibodies were more
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prevalent in one study [16] but not in another [8]. In these studies – antinuclear antibody, doublestranded DNA, anti-HTLV1, anti-Ro, myositis-associated, and myositis-specific antibodies – showed no significant association with anti-cN1A antibody status. Similarly, an association was
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not noted between the anti-cN1A antibody and risk for malignancy, degree of denervation on EMG, and positive response to treatment with intravenous immunoglobulin or corticosteroids
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[11,15]. The latter observation is especially interesting given that IBM clinical trials employing immunosuppressive medications have not documented efficacy.
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In addition to the current study, three prior studies have evaluated the association of anti-
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cN1A reactivity with muscle histopathological findings [14-16]. In the study by Lloyd et al., antibody positive IBM patients had a lower prevalence of rimmed vacuoles, but no association
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was noted in degree of focal invasion or degree of endomysial inflammation on pathological specimens [14]. Tawara et al. found type 2 myofibers to be significantly smaller in the anticN1A antibody positive compared to negative group [15]. However, no association was noted in regards to degree of inflammatory cells, congophilic material, co-localization with p62 protein, or perinuclear localization of cN1A protein. Finally, Lilleker et al. found significantly more
ACCEPTED MANUSCRIPT 11 COX-deficient fibers in anti-cN1A positive compared to the negative group [16]. Many other histopathological features – ragged red fibers, atrophic fibers, inflammation, MHC1 upregulation, necrosis, mononuclear infiltrate, invasion of non-necrotic fibers, rimmed vacuoles, protein deposits, and microfilaments – showed no association with anti-cN1A antibody
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reactivity. These and other inconsistent clinicopathological observations are difficult to explain, and may be attributed to the different assay methods used, sample sizes, or patients’ background [15].
Based on all clinical studies published to date including the present, the anti-cN1A
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antibody test shows low predictive value in regards to disease severity and associated
clinicopathological findings. Despite this limitation, the anti-cN1A antibody test may be clinically useful in certain situations. Given the high test specificity, a positive test provides
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strong diagnostic support for patients with clinically defined IBM who are refusing a muscle biopsy or those patients for whom a muscle biopsy may impose a degree of increased risk or
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morbidity (e.g. treatment with anti-coagulation therapy). Another potential use is as a screening test for patients with mild or partial IBM clinical features (e.g., isolated dysphagia or quadriceps
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weakness) and nonspecific or non-diagnostic muscle biopsy findings. However, for patients with
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clinico-pathologically IBM, the anti-cN1A test appears to provide no additional clinically useful information.
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In summary, based on all clinical studies published to date including the present, the anticN1A antibody test shows high diagnostic specificity, moderate sensitivity, and a low predictive value in regards to age of onset, disease severity and other associated clinicopathological findings. It is hopeful that further refinement of the anti-cN1A antibody test or development of another noninvasive test will provide improved clinical utility.
ACCEPTED MANUSCRIPT 12 References [1] Needham M, Mastaglia FL. Sporadic inclusion body myositis: a review of recent clinical advances and current approaches to diagnosis and treatment. Clin Neurophysiol 2016;127:176473. [2] Greenberg SA. Inclusion body myositis. Continnum 2016;22:1871-88.
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[3] Felice KJ, North WA. Inclusion myositis in Connecticut: observations in 35 patients during an 8-year period. Medicine 2001;80:320-7. [4] Lefter S, Hardiman O, Ryan AM. A population-based epidemiologic study of adult neuromuscular disease in the Republic of Ireland. Neurology 2017;88:304-13.
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[5] Rose MR. 188th ENMC international workshop: inclusion body myositis, 2-4 December 2011, Naarden, The Netherlands. Neuromuscul Disord 2013;23:1044-55. [6] Weihl CC, Mammen AL. Sporadic inclusion body myositis – a myodegenerative disease or an inflammatory myopathy. Neuropathol Appl Neurobiol 2017;43:82-91. [7] Salajegheh M, Lam T, Greenberg SA. Autoantibodies against a 43 KDa muscle protein in inclusion body myositis. PLoS One 2011;6:e20266.
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[8] Larman HB, Salajegheh M, Nazareno, et al. Cytosolic 5’-nucleotidase 1A autoimmunity in sporadic inclusion body myositis. Ann Neurol 2013;73:408-18.
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[9] Pluk H, van Hoeve BJ, van Dooren SH, et al. Autoantibodies to cytosolic 5’-nucleosidase 1A in inclusion body myositis. Ann Neurol 2013;73:397-407.
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[10] Greenberg SA. Cytoplasmic 5’-nucleotidase autoantibodies in inclusion body myositis: isotypes and diagnostic utility. Muscle Nerve 2014:50:488-92.
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[11] Limaye VS, Lester S, Blumbergs P, Greenberg SA. Anti-CN1A antibodies in South Australian patients with inclusion body myositis. Muscle Nerve 2016;53:654-5.
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[12] Goyal NA, Cash TM, Alam U, et al. Seropositivity for NT5c1A antibody in sporadic inclusion body myositis predicted more serve motor, bulbar and respiratory involvement. J Neurol Neurosurg Psychiatry 2016;87:373-8. [13] Herbert MK, Stammen-Vogelzangs J, Verbeek MM, et al. Disease specificity of antibodies to cystolic 5’nucleotidase 1A in sporadic inclusion body myositis versus known autoimmune diseases. Ann Rheum Dis 2016;75:696-701. [14] Lloyd TE, Christopher-Stine L, Pinal-Fernandez, I, et al. Cytosolic 5’-nucleotidase 1A as a target of circulating autoantibodies in autoimmune diseases. Arthritis Care Res 2016;68:66-71.
ACCEPTED MANUSCRIPT 13 [15] Tawara N, Yamashita S, Zhang X, et al. Pathomechanisms of anti-cytosolic 5’-nucleotidase 1A autoantibodies in sporadic inclusion body myositis. Ann Neurol 2017;81:512-25. [16] Lilleker JB, Rietveld A, Pye SR, et al. Cytosolic 5’-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis. Ann Rheum Dis 2017;76:862-8.
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[17] Herbert MK, Pruijn GJ. Novel serology testing for sporadic inclusion body myositis: disease-specificity and diagnostic utility. Curr Opin Rheumatol 2015;27:595-600. [18] Liewluck T. Anti-cytosolic 5’-nucleotidase 1A (cN1A) autoantibodies in motor neuron disease. Neurology 2017;89:2017-8.
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[19] Kramp SL, Karayev D, Shen G, et al. Development and evaluation of a standardized ELISA for the determination of autoantibodies against cN-1A (Mup44, NT5C1A) in sporadic inclusion body myositis. Auto Immun Highlights 2016;7:16.
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Figure 1. Dotplot comparing age of onset and anti-cN1A antibody positivity.
ACCEPTED MANUSCRIPT 15 Table 1. Sensitivity and specificity of the cN1A antibody in IBM. Sensitivity (%) 52 70 60 76 35 72 37 61 36 33 50
Specificity (%) 100 92 89 91 NA NA 96 87 92 NA NA
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IBM patients (n) 25 47 56 50 69 25 238 117 67 311 40
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Author [reference] Salajegheh [7] Larman [8] Pluk [9] Greenberg [10] Limaye [11] Goyal [12] Herbert [13] Lloyd [14] Tawara [15] Lilleker [16] Current study
ACCEPTED MANUSCRIPT 16 Table 2. Summary of IBM clinical and pathological findings. Mean (range) 70.6 years (47-88) 59.0 years (41-72) 11.6 years (2-30) 104 units (22-222) 74% (46-122) 642 unit/L (138-2344) 3.1 (0.5-8.7) 94.1 (38-118) 5 (0-10) 226.8 N (0-427)
Male cN1A positive total cN1A positive in clinico-pathologically defined (n=15) cN1A positive in clinically defined (n=25) Dysphagia Ambulatory Connective tissue disease Muscle inflammation severe Muscle inflammation mild Rimmed vacuoles Tubulofilamentous inclusions
Number (%) 28 (70) 20 (50) 5 (33) 15 (60) 28 (70) 32 (80) 5 (12%) 17 (42) 23 (58) 35 (87) 17 (42)
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Finding Age of presentation Age of onset Duration of symptoms cN1A titer (n=20) FVC %predicted CPK total (n=32) CPK xUNL (n=32) MRC total MRC quadriceps Grip force (n=31)
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Table 3. Means of continuous variables. Mean, cN1A56.9 69.7 12.8 3.133 75.0 91.95 4.65 237 678.8 3.207
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p-value 0.100 0.523 0.251 0.335 0.767 0.551 0.476 0.671 0.697 0.860
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Mean, cN1A+ 61.0 71.5 10.4 2.817 73.45 96.3 5.35 216 605.7 3.066
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Age onset Test age Duration FVC L FVC % MRC Total MRC Quad Grips Force CPK u/l CPK xULN
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Table 4. Percentages for indicator variables. Percent, cN1A25% 60%
p-value 0.010 0.053
50% 95% 40% 80% 65% 40% 15% 70% 45% 25%
80% 80% 20% 84% 70% 45% 10% 70% 60% 25%
0.096 0.342 0.301 1.000 1.000 1.000 1.000 1.000 0.527 1.000
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Percent, cN1A+ 70% 25%
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Age of onset >60 Tubulofilamentous inclusions+ Hypertension+ RV+ Sex Female Amb+ Device Inflam Grade Severe CTD+ Dysphagia+ Hyperlipidemia+ CAD+