Myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) are a feature of the idiopathic inflammatory myopathies (IIM), but are also seen in other rheumatic diseases, and in individuals with no clinical symptoms. The aim of this study was to assess the clinical utility of MSA and MAA and in particular the clinical relevance of weakly positive results. We included all patients at our institution who had at least one positive result on the Immunoblot EUROLINE myositis panel over a 6-year period (2015–2020). Associations with clinical features and final diagnosis were evaluated. Eighty-seven of 225 (39%) myositis panel tests met the inclusion criteria. There were 52 strong positives and 35 weak positives for one or more MSA/MAAs. Among the strong positive group, 15% (8/52) were diagnosed with IIM, 34.6% (18/52) with interstitial lung disease, 7.7% (4/52) with anti-synthetase syndrome, 25% (13/52) with connective tissue disease, and others accounted for 25% (13/52). In weak-positive cases, only 14% (5/35) had connective tissue disease and none had IIM. 60% (21/35) of weak-positive cases were not associated with a specific rheumatic disease. A significant number of positive myositis panel results, particularly weak positives, are not associated with IIM or CTD.
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Introduction
The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of autoimmune rheumatic diseases characterized by proximal muscle weakness and frequent involvement of other organ systems [1]. The prevalence of IIM can be estimated between 2.4 and 33.8 per 100,000 persons [2].
Historically, the Bohan and Peter criteria were used for IIM, until 2017 when the European League Against Rheumatism and American College of Rheumatology (EULAR/ACR) proposed new classification criteria [3, 4]. These new classification criteria reflect the advances of medicine in the last 40 years as well as providing higher performance (sensitivity/specificity, 93%/88% with biopsies, 87%/82% without biopsies). The new criteria are based primarily on clinical history, examination, and biopsy results. Only one antibody, Anti-Jo-1, is included. The criteria are in the form of a calculator which gives a probability score of the patient having myositis. A classification tree is then used to help determine the subcategory (polymyositis (PM), dermatomyositis (DM), inclusion body myositis, and juvenile dermatomyositis) [4].
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However, autoantibodies have been reported in more than 80% of patients with IIM. These autoantibodies can be myositis-specific antibodies (MSA), or myositis-associated antibodies (MAA) which are also seen in a host of other connective tissue diseases (CTD). MSA have a 90% diagnostic specificity, while MAA are noted in up to 50% of myositis patients. These antibodies can help anticipate the clinical course and disease prognosis [5, 6].
MSA include anti-ARS (aminoacyl-tRNA synthetases) antibodies; (histidyl (Jo-1), threonyl (PL-7), alanyl (PL-12), glycyl (EJ), isoleucyl (OJ), asparaginyl (KS), tyrosyl (Ha), and phenylalanyl (Zo)), anti-Mi2 (nucleosome-remodeling deacetylase complex), anti-SRP (signal recognition particle), anti-TIF1 (transcription intermediary factor 1) and anti-NXP-2 (nuclear matrix protein 2), anti-MDA5 (melanoma differentiation-associated protein 5), and anti-SAE (small ubiquitin-like modifier activating enzyme). MAA include anti-PM-Scl, U1RNP, Ku, and Ro52 [7‐9].
Autoantibodies are a feature of the subclinical phase of systemic rheumatic diseases and can be present for many years before the onset of clinical symptoms [10, 11]. MSA and MAA are associated with IIM; however, only anti Jo-1 is included in the EULAR/ACR criteria. Weak-positive MSA/MAA are frequently seen and of uncertain clinical significance. Therefore, the aim of the study is to assess the clinical utility of MSA and MAA and in particular the clinical relevance of weakly positive results.
Materials and methods
Study design and setting
This study is a single-center retrospective observational study, performed over a 6-year period (2015–2020). All patients who had an extended myositis antibody panel in this period were assessed for eligibility. Those over age 18 with at least one positive MSA/MAA were included and patients who were followed up in other institutions were excluded. IIM patients with positive MSA/MAA were compared to weak-positive MSA/MAA patients. The study was approved by the St. James’ Hospital (SJH)/Tallaght University Hospital (TUH) Joint Research Ethics Committee under protocol number 2020–04 List 15, in May 2020.
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Determination/procedure
Myositis antibody testing was performed using the Immunoblot EUROLINE myositis panel, according to the manufacturer’s specifications. This assay allows the detection of human IgG autoantibodies to a range of different antigens. This includes 12 MSA (Mi-2a, Mi-2b, TIF1, MDA5, NXP2, SAE1, SRP, Jo-1, PL-7, PL-12, EJ, and OJ), in addition to 4 MAA (Ku, PM-Scl100, PM-Scl75, and Ro/SSA-52). Our immunology lab reports PM-Scl100 and PM-Scl75 separately. Some consider both anti-PM-Scl100 and anti-PM-Scl75 antibodies as one, since they target two closely related isoforms of the same protein. For the purpose of this study, we have included those who were positive for PM-Scl75 and/or PM-Scl100 under the one result. The same applies for Mi-2a and Mi-2b [12]. Anti-nuclear antibody (ANA) screening by indirect immunofluorescence (IIF) on HEp-2 cells is performed in tandem with each myositis panel to improve specificity, as some myositis antibodies have a distinct ANA staining pattern [13]. The assay was performed according to the manufacturer’s recommendations, using a screening dilution of 1:80. Comments are on the presence or absence of antibodies, in addition to the pattern.
Measurement
Immunoblot strips were analyzed using the EuroBlotOne Analyzer/Euroline Scan. This assay provides a semi-qualitative result based on signal intensity of each measured antibody. Results are reported as: negative, weak positive, and strong positive. According to the manufacturer’s recommendations, an antibody is considered negative if the signal is < 11. Low positivity is a signal between 11 and 25, and strong positivity beyond 25. The turnover time for the assay is 21 days.
Clinical features
Clinical features were defined as follows. Interstitial lung disease was diagnosed by a respiratory physician. Other features were identified by a rheumatologist and/or immunologist. Arthritis was defined as swelling and tenderness of one or more joints, arthralgia as joint pain with no evidence of arthritis, myositis as muscle weakness supported by relevant investigations, Raynaud’s phenomenon as recurrent events of sharply demarcated pallor and/or cyanosis of the skin of the digits with or without reactive hyperaemia, and cutaneous manifestations as Gottron’s papules or sign, heliotrope rash, photosensitive rash, calcinosis, digital ulceration, psoriasis, livedo reticularis, or sclerodactyly. Malignancy was defined as any cancer within 5 years of the index study.
Statistical analysis
Statistical analysis was performed using SPSS v26. Descriptive statistics were reported, with results given as frequency and percentages. Categorical variables were compared using Chi-square tests. p ≤ 0.05 was considered statistically significant throughout.
Results
Patients and demographics
A total of 225 myositis panels were performed in the 6-year study period. 87/225 (39%) patients had positive myositis panel results and met the inclusion criteria, 39% were male and 61% female, with a mean (SD) age of 58 (+ -16) years. Of the positive results, 60% (52/87) were strong positive for and 40% (35/87) weak positive for one or more MSA/MAAs. Full demographic data are shown seen in Table 1 (strong positive cohort) and Table 2 (weak-positive cohort).
Progressive pulmonary fibrosis (post COVID, ARDS and recurrent aspiration)
Esophageal Ca T1N2M0 s/p esophagectomy
Antibiotics + supportive care
Remission/stable
25
C
66M
Ro52
+
+
+
RA-ILD
Prednisolone + Rituximab
Remission/stable
26
−
74F
Ro52
+
+
Sjogren -ILD
Prednisolone + AZA + HCQ
Remission/stable
Connective tissue disease
26
+ S
60F
Ro52
SLE
HCQ
Lost follow-up
27
+ H
75F
Ro52
+
+
Sjogren
HCQ
Remission/stable
28
+ S
69F
Ro52
Sjogren
HCQ
Remission/stable
29
+ S
53F
Ro52
+
+
+
Sjogren
Breast cancer
No medication
Surgery + Radiotherapy + Hormonal
Remission/stable
30
+ S
18F
Ro52
EJ
+
Sjogren
HCQ
Remission/stable
31
−
33M
Ro52
+
Sjogren with neuropsychiatry manifestation
AZA
Remission/stable
32
+
73F
Ro52
+
Sjogren
HCQ
Remission/stable
34
+ `
66F
Ku/Ro52
+
+
Undifferentiated CTD
No medication
Lost follow-up
35
+ S
19 F
U1snRNP
+
+
+
Undifferentiated CTD
Prednisolone
MTX + HCQ
Remission/stable
36
−
70M
Ro52
+
+
Undifferentiated CTD query paraneoplastic on background melanoma and eosinophilia
Nifedipine
Remission/stable
37
+ S
48F
U1snRNP/ Ro52
OJ
+
+
+
+
MCTD
Autoimmune hepatitis
Prednisolone + AZA + HCQ
Remission/stable
38
+ Ce
52F
SRP
+
+
Limited cutaneous scleroderma
Nifedipine
Remission/stable
39
+
54F
PMscl100/75
+
+
Scleroderma
Scleroderma renal crisis
HCQ and ramipril
Remission/stable
Others
40
−
72M
NXP2
+
Polymyalgia rheumatica
Prednisolone
Remission/stable
41
+ S
61M
ku
Mi2b
+
large vessel vasculitis
Prednisolone
Tocilizumab
Remission/stable
42
−
35F
PL12
+
+
+
PsA
MTX
Remission/stable
43
+ S
49F
Mi2b
PBC
Ursodeoxycholic acid
Remission/stable
44
+ N
50F
Ro52
Liver cirrhosis
No medication
Remission/stable
45
+ S
53 F
Ro52
Autoimmune limbic encephalitis
IV methylpred + IVIG + plasma exchange + cyclophosphamide
Died
46
+ N
46F
Ro52
+
Fibromyalgia
No medication
Remission/stable
47
−
37F
PMscl100/75
Ro52
+
Fibromyalgia
No treatment
Remission/stable
48
C
45F
Ku/Ro52
Chronic spontaneous urticaria Hypothyroidism
Anti-histamine
Levothyroxine
Remission/stable
49
−
71F
Ro52
+
High grade serous ovarian carcinoma with metastasis
Surgery and chemotherapy
Remission/stable
50
+ H
62F
Ro52
Uterine fibroid
No treatment
Remission/stable
51
+ H
64F
Ro52
+
Rheumatoid arthritis
MTX
Remission/stable
52
−
73F
PMscl100/75
+
Extranodal NK/T lymphoma
-
Died
S speckled, H homogenous, C cytoplasmic, Ce centromere, N nucleolar
Table 2
Weak-positive myositis panel characteristics
Age/gender
ANA
MAA
MSA
ILD
Arthritis
Arthralgia
Myositis
Raynaud
Cutaneous
Malignancy
Final diagnosis
Medications
Outcome
1
19M
–
PMscl
100/75
EJ
OJ
+
+
IBD-related spondyloarthropathy
Adalimumab
Remission/stable
2
29F
–
TIF1
MDR TB and neuropathy
Intrauterine fibroid
Antibiotic
Pregabalin
Remission/stable
3
32M
–
Mi2b
+
Psoriasis
No medication
Remission/stable
4
63F
–
Mi2b
+
Asymptomatic idiopathic bi-apical fibrosis
No medication
Remission/stable
5
57M
–
SRP
+
Sarcoidosis
Bilateral interstitial pulmonary fibrosis
Nintedanib
Lost follow-up
6
54M
–
Mi2
+
+
+
Scleroderma, psoriatic arthritis
Prednisolone + MTX
Remission/stable
7
79F
–
SRP
+
IPF query RA related
Prednisolone
Died
8
52F
C
Mi2b
Fatty liver along with hepatosplenomegaly
Hypothyroidism
No medication
Remission/stable
9
54M
–
Ro52
+
NSCLC-adenocarcinoma T2N1M0 + antiphospholipid syndrome and VTE history
Prednisolone + chemotherapy
Remission/stable
10
59F
C
Ro52
+
Idiopathic livedo vs erythema ab igne
No medication
Remission/stable
11
71M
+ S
U1snRNP
PL12
+
Poorly controlled Myasthenia Gravis
Coeliac disease Hypothyroidism
IVIG + steroid + pyridostigmine
Remission/stable
12
43F
+ H
Ro52
PL7
+
+
UCTD
HCQ + MMF
Remission/stable
13
56M
–
PMscl
100/75
Ro52
+
+
+
Scleroderma/pulmonary fibrosis
IgA deficiency
Prednisolone + Rituximab + MTX
Remission/stable
14
73F
–
MDA5/SAE1
Degenerative lumbosacral spine
–
Remission/stable
15
66M
+ H
PmScl
100/75
+
No Unclear diagnosis—paroxysms of inflammation
cause unclear
No medications
Remission/stable
16
41F
–
PmScl
100/75
+
Raynaud phenomenon
Supportive
Remission/stable
17
67F
+ H
Ro52
MGUS
–
Remission/stable
18
18F
–
Mi2a/b
SRP
Chilblains likely secondary to anorexia nervosa
–
Remission/stable
19
61M
–
Mi2a
SRP
+
Idiopathic pulmonary fibrosis
Nintedanib
Remission/stable
20
32F
–
Ro52
+
+
Peripheral SpA
Certilizumab
Remission/stable
21
78F
–
SAE1
Autoimmune hepatitis
Remission/stable
22
33F
–
PmScl
100/75
+
+
Livedo-reticularis and previous peteacheal vasculitis rash in LL
Remission/stable
23
53F
+ H
Ro52
+
+
Diffuse systemic sclerosis
Steroid + MMF + Rituximab + Nintendinib
Remission/stable
24
62F
–
Mi2a
Pontine stroke and under workup for MS
Clopidogrel
Remission/stable
25
72M
–
Mi2b
AML and organizing pneumonia
Chemo + steroid taper for OP
Remission/stable
26
89M
–
Mi2a
+
UIP-ILD / IPF
Remission/stable
27
60F
–
Mi2b
SAE1
SRP
+
IPF query RA related
Died
28
64F
–
Ro52
+
+
Discoid lupus
Was on steroid, HCQ + MMF
Remission/stable
29
60F
–
Ro52
MDA5
+
IPF query RA related
o2
Remission/stable
30
69M
–
Mi2b
COPD and asthma
Inhalers + on/off steroid
Remission/stable
31
76M
–
Ku
Hospital Acquired Pneumonia with parapneumonic effusions
Remission/stable
32
64M
–
PL-12
+
IPF
Remission/stable
33
79M
–
SAE1/PL-7
IPF
Nintedanib
Remission/stable
34
67M
–
NXP2
+
IPAF ILD secondary to CTD
Steroid +
Remission/stable
35
46F
+ S
U1snRNP
Ro52
+
MCTD
HCQ
Remission/stable
S speckled, H homogenous, C cytoplasmic
Clinical features
Tables 1 and 2 summarize the clinical features, ANA results, medication, and outcome of included cases. A creatine kinase (CK) level was performed in 52% of patients, with a median result of 69 (IQR 44.5–277, p = 0.57). Respiratory medicine accounted for the highest number of test requests (33%, 29/87), followed by rheumatology and immunology (24%, 21/87 each).
Strong-positive MSA/MAA
Anti-PL12 was the most frequent strong positive MSA and anti-Ro52 the most common strong positive MAA (Table 3). The most frequently observed clinical features were arthralgia in 38% (20/52), ILD in 35% (18/52), and cutaneous manifestations in 29% (15/52). Arthritis was seen in 15% (8/52), Raynaud’s phenomenon in 15% (8/52), myositis in 13% (7/52), and malignancy in 12% (6/52). Thirteen percent (8/52) were diagnosed with dermatomyositis and 8% (4/52) with anti-synthetase syndrome.
Table3
The results of the antibodies for both positive and weakly positive
Antibody
Positive
Weakly positive
MSA
Anti-PL-12
4
2
Anti-SAE1
3
3
Anti-Mi2
3
12
Anti-NXP2
2
1
Anti-Jo
2
1
Anti-SRP
2
5
Anti-PL7
2
2
Anti-EJ
2
1
Anti-OJ
2
1
Anti-MDA5
1
2
MAA
Anti-Ro52
29
10
Anti-PMScl
7
5
Anti-Ku
3
–
Anti-U1RNP
2
2
Weak-positive MSA/MAA
Anti-Mi2 was the most frequent weak-positive MSA and anti-Ro52 the most frequent weak-positive MAA (Table 3). The most common clinical manifestations were ILD in 34% (12/35), cutaneous manifestations in 20% (7/35), and arthralgia in 17% (6/35), with Raynaud’s phenomenon and arthritis in 11% each (4/35) and myositis and malignancy in 3% (1/35) each. No patients were diagnosed with IIM or anti-synthetase syndrome.
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Clinical correlates of positive MSA/MAA
A statistically significant association between arthralgia and a positive myositis panel was identified (p = 0.033) (Table 4). There were numerical differences for presentations of ILD (p = 0.975), myositis (p = 0.093), and cutaneous (p = 0.140) manifestations, but these did not reach statistical significance. A diagnosis of IIM was associated with a strong positive panel (p = 0.008). Symptom duration < 1 year was associated with a weakly positive panel (p = 0.022).
Table 4
Chi-square analysis between weak-positive and positive myositis panel
Type
p value
Weak-positive myositis panel
Positive myositis panel
Count
Column N %
Count
Column N %
ILD
12
34.3
18
34.6
0.975
Arthritis
4
11.4
8
15.4
0.600
Arthralgia
6
17.1
20
38.5
0.033*
Myositis
1
2.9
7
13.5
0.093
Raynaud
4
11.4
8
15.4
0.600
Cutaneous
7
20.0
18
34.6
0.140
Malignancy
1
2.9
6
11.5
0.144
Final diagnosis
Inflammatory myositis
0
0.0
8
15.4
0.008*
Interstitial lung disease
12
34.3
18
34.6
Connective tissue disease
5
14.3
14
26.9
Others
18
51.4
12
23.1
Management
Corticosteroid
3
8.6
5
9.6
0.115
Corticosteroid + immunosuppression
7
20.0
17
32.7
Immunosuppression
3
8.6
12
23.1
No treatment
11
31.4
9
17.3
Others
11
31.4
9
17.3
Outcome
Died
2
5.7
3
5.8
0.773
Remission/stable
32
91.4
45
86.5
Worsening
0
0.0
1
1.9
Lost follow-up
1
2.9
3
5.8
Duration
= < 1 year
23
65.7
22
42.3
0.022*
2 years
6
17.1
14
26.9
3 years
6
17.1
5
9.6
4 years
0
0.0
8
15.4
5 years
0
0.0
3
5.8
*p < 0.05
Details of clinical features and diagnosis by individual MSA and MAA are shown in Supplementary Tables 1–7. There was no evident difference between single MSA/MAA positivity and positivity for more than one MSA/MAA and clinical features or diagnosis.
Discussion
Our study shows that those with a strong positive myositis panel were more likely to be diagnosed with an IIM and were more likely to present with arthralgia. There were no diagnoses of IIM in the weakly positive myositis panel group.
A review of the literature shows variations of clinical presentation and serology across different populations. It is felt that genetic factors and environmental triggers may be responsible for this disparity [14]. For example, a study of a Greek population found that the most frequently detected MAA was anti-Ro-52 (30%), while the most frequently detected MSA was anti-Jo-1 (22%) [15]. In our total population, only 3% tested positive for anti-Jo-1.
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Our study shows the association of MSA and MAA with IIM, ILD, and CTD are much higher at the strong positive antibody level when compared with the weak positive. However, the diagnostic yield of MSA was generally lower than previously reported studies [16, 17]. This may be because of a relatively short follow-up in our population compared to other published studies or may be due to testing in patients with a lower pre-test probability.
The American thoracic society/European respiratory society/Japanese respiratory society/Latin American thoracic society diagnostic guidelines recommend serial antibody testing in ILD to identify seroconversion and differentiate idiopathic pulmonary fibrosis (IPF) from CTD-ILD. In our study, 34% of all patients were diagnosed with ILD and respiratory having the highest number of requests. This shows the value of MSA testing in ILD as it may present with no or minimal symptoms suggestive of CTD [18]. As CTD- ILD confers a better prognosis and different treatment approach than IPF, it is of paramount importance to detect this subset at an early stage [19].
In our study, MSA were detected in many other inflammatory and non-inflammatory diseases. This finding is in contrast to the majority of prior studies. For instance, Vulseteke et al. reported positive MSA in half of patients with IIM compared to only 3.5% of patients with systemic inflammatory diseases and none in healthy controls [20]. This could suggest that MSA sensitivity and specificity vary from one testing lab to another [15, 16]. It may also be the case that there are differences in the populations being tested, with resultant variation in the pre-test probability.
We perform ANA in conjunction with the myositis panel to improve diagnostic performance [13]. 83% of weakly positive myositis panels in our cohort were ANA negative compared to 46% of strong positive panels (~ 93% correctly matched the non-ANA staining in the positive panel). A false-positive test should be considered if the autoantibody staining/pattern does not correlate with the ANA result and clinical context [9]. However, some MSA exhibit negative ANA testing due to cytoplasmic localisation, and as such negative ANA does not necessarily imply autoantibody negativity in IIM.
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This study was not without its limitations. Our power to detect significant differences was impacted by a relatively small sample size and low number of IIM diagnoses. This highlights the need for larger collaborative studies to evaluate these rare conditions. This was a single-center study and our findings require confirmation in other settings to confirm external validity. Given the significant mortality and morbidity burden of IIM, early and accurate diagnosis should be a primary goal in all cases. Based on the above, we have proposed an algorithm to guide the interpretation of myositis antibody panel results, Fig. 1. This highlights our findings and suggests that weak-positive panels should be repeated to confirm the result.
Fig. 1
A proposed algorithm to guide interpretation of myositis antibody panel results
×
The current EULAR/ACR guidelines suggest that clinical assessment and biopsy are the core components of the diagnostic approach to IIM. Our expanding knowledge of the importance of MSA/MAA suggests a key adjunctive role in diagnosis. Our study found that positive panels are more likely to be associated with IIM; however, a significant number of cases had no clinical features suggestive of CTD or IIM. A combined clinical and serological framework may be useful in IIM diagnosis.
Declarations
Conflict of interest
The authors declare that they have no conflict of interst.
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