Identification of connective tissue disease autoantibodies and a novel autoantibody anti-annexin A11 in patients with "idiopathic" interstitial lung disease.

BACKGROUND: Autoantibodies are a hallmark feature of Connective Tissue Diseases (CTD). Their presence in patients with idiopathic interstitial lung disease (ILD) may suggest covert CTD. We aimed to determine the prevalence of CTD autoantibodies in patients diagnosed with idiopathic ILD. METHODS: 499 patient sera were analysed: 251 idiopathic pulmonary fibrosis (IPF), 206 idiopathic non-specific interstitial pneumonia (iNSIP) and 42 cryptogenic organising pneumonia (COP). Autoantibody status was determined by immunoprecipitation. RESULTS: 2.4% of IPF sera had a CTD-autoantibody compared to 10.2% of iNSIP and 7.3% of COP. 45% of autoantibodies were anti-synthetases. A novel autoantibody targeting an unknown 56 kDa protein was found in seven IPF patients (2.8%) and two NSIP (1%) patients. This was characterised as anti-annexin A11. CONCLUSION: Specific guidance on autoantibody testing and interpretation in patients with ILD could improve diagnostic accuracy. Further work is required to determine the clinical significance of anti-annexin A11.

International Guideline for Idiopathic Inflammatory Myopathy-Associated Cancer Screening: an International Myositis Assessment and Clinical Studies Group (IMACS) initiative.

Adult-onset idiopathic inflammatory myopathy (IIM) is associated with an increased cancer risk within the 3 years preceding and following IIM onset. Evidence- and consensus-based recommendations for IIM-associated cancer screening can potentially improve outcomes. This International Guideline for IIM-Associated Cancer Screening provides recommendations addressing IIM-associated cancer risk stratification, cancer screening modalities and screening frequency. The international Expert Group formed a total of 18 recommendations via a modified Delphi approach using a series of online surveys. First, the recommendations enable an individual patient's IIM-associated cancer risk to be stratified into standard, moderate or high risk according to the IIM subtype, autoantibody status and clinical features. Second, the recommendations outline a 'basic' screening panel (including chest radiography and preliminary laboratory tests) and an 'enhanced' screening panel (including CT and tumour markers). Third, the recommendations advise on the timing and frequency of screening via basic and enhanced panels, according to risk status. The recommendations also advise consideration of upper or lower gastrointestinal endoscopy, nasoendoscopy and 18F-FDG PET-CT scanning in specific patient populations. These recommendations are aimed at facilitating earlier IIM-associated cancer detection, especially in those who are at a high risk, thus potentially improving outcomes, including survival.

A Commercial Anti-TIF1γ ELISA Is Superior to Line and Dot Blot and Should Be Considered as Part of Routine Myositis-Specific Antibody Testing.

Objectives: Anti-TIF1γ is an important autoantibody in the diagnosis of cancer-associated dermatomyositis and the most common autoantibody in juvenile onset dermatomyositis. Its reliable detection is important to instigate further investigations into underlying malignancy in adults. We previously showed that commercial assays using line and dot blots do not reliably detect anti-TIF1γ. We aimed to test a new commercial ELISA and compare with previously obtained protein immunoprecipitation. Methods: Radio-labelled immunoprecipitation had previously been used to determine the autoantibody status of patients with immune-mediated inflammatory myopathies and several healthy controls. ELISA was undertaken on healthy control and anti-TIF1γ sera and compared to previous immunoprecipitation data. Results: A total of 110 serum samples were analysed: 42 myositis patients with anti- TIF1γ and 68 autoantibody negative healthy control sera. Anti-TIF1γ was detected by ELISA in 41 out of 42 of the anti-TIF1γ-positive samples by immunoprecipitation, and in none of the healthy controls, giving a sensitivity of 97.6% and specificity of 100%. The false negative rate was 2%. Conclusion: ELISA is an affordable and time-efficient method which is accurate in detecting anti-TIF1γ.

Identification and prediction of novel classes of long-term disease trajectories for patients with juvenile dermatomyositis using growth mixture models.

OBJECTIVES: Uncertainty around clinical heterogeneity and outcomes for patients with JDM represents a major burden of disease and a challenge for clinical management. We sought to identify novel classes of patients having similar temporal patterns in disease activity and relate them to baseline clinical features. METHODS: Data were obtained for n = 519 patients, including baseline demographic and clinical features, baseline and follow-up records of physician's global assessment of disease (PGA), and skin disease activity (modified DAS). Growth mixture models (GMMs) were fitted to identify classes of patients with similar trajectories of these variables. Baseline predictors of class membership were identified using Lasso regression. RESULTS: GMM analysis of PGA identified two classes of patients. Patients in class 1 (89%) tended to improve, while patients in class 2 (11%) had more persistent disease. Lasso regression identified abnormal respiration, lipodystrophy and time since diagnosis as baseline predictors of class 2 membership, with estimated odds ratios, controlling for the other two variables, of 1.91 for presence of abnormal respiration, 1.92 for lipodystrophy and 1.32 for time since diagnosis. GMM analysis of modified DAS identified three classes of patients. Patients in classes 1 (16%) and 2 (12%) had higher levels of modified DAS at diagnosis that improved or remained high, respectively. Patients in class 3 (72%) began with lower DAS levels that improved more quickly. Higher proportions of patients in PGA class 2 were in DAS class 2 (19%, compared with 16 and 10%). CONCLUSION: GMM analysis identified novel JDM phenotypes based on longitudinal PGA and modified DAS.

The promise, perceptions, and pitfalls of immunoassays for autoantibody testing in myositis.

BACKGROUND: A myositis-specific autoantibody can now be identified in the majority of patients with myositis. They identify homogeneous patient subgroups and are key tools in developing a personalized approach to disease management. There is substantial clinical interest in exploiting myositis autoantibodies as biomarkers, and consequently, a large number of commercial assays have been developed for their detection. These assays are already in widespread clinical use. In order to better understand perceived concerns from the international myositis community in relation to the reliability of these assays and how they are being used, we conducted a survey of international myositis experts, all of whom were members of the International Myositis Assessment and Clinical Studies group. RESULTS: We collected data on the types of assay used, manufacturers, and the nature of the report provided by different laboratories and received 111 complete responses. Respondents also provided information on how they used the different assays, their confidence in the results, and how this influenced their clinical practice. Enzyme immunoassay/ELISA was the most popular assay method used worldwide followed by line blot. Line blot was the most popular method used in Europe. Despite concerns from over 80% of respondents regarding false-positive and false-negative results with the assay used by their laboratory, over 80% reported that the identification of a myositis autoantibody influenced their diagnostic confidence, the information they provided to a patient, and their recommended treatment. CONCLUSIONS: In spite of ongoing concerns from the majority of users regarding the reliability of the results, myositis-specific autoantibody testing, using commercial immunoassays, is being used globally to inform clinical decision-making. These findings highlight the need for urgent guidance on the use of myositis autoantibody testing and on the interpretation of results. Knowledge of the reliability of currently available assays is essential given the importance already placed on myositis-specific autoantibodies as clinical decision-making tools.

The reliability of immunoassays to detect autoantibodies in patients with myositis is dependent on autoantibody specificity.

OBJECTIVES: In order to address the reliability of commercial assays to identify myositis-specific and -associated autoantibodies, we aimed to compare the results of two commercial immunoassays with the results obtained by protein immunoprecipitation. METHODS: Autoantibody status was determined using radio-labelled protein immunoprecipitation for patients referred to our laboratory for myositis autoantibody characterization. For each autoantibody of interest, the sera from 25 different patients were analysed by line blot (Euroline Myositis Antigen Profile 4, EuroImmun, Lübeck, Germany) and dot blot (D-Tek BlueDiver, Diagnostic Technology, Belrose, NSW, Australia). Sera from 134 adult healthy controls were analysed. RESULTS: Overall commercial assays performed reasonably well, with high agreement (Cohen's κ >0.8). Notable exceptions were the detection of rarer anti-synthetases with κ < 0.2 and detection of anti-TIF1γ, where κ was 0.70 for the line blot and 0.31 for dot blot. Further analysis suggested that the proportion of patients with anti-TIF1γ may recognize a conformational epitope, limiting the ability of blotting-based assays that utilize denatured antigen to detect this clinically important autoantibody. A false-positive result occurred in 13.7% of samples analysed by line blot and 12.1% analysed by dot blot. CONCLUSION: The assays analysed do not perform well for all myositis-specific and -associated autoantibodies and overall false positives are relatively common. It is crucial that clinicians are aware of the limitations of the methods used by their local laboratory. Results must be interpreted within the clinical context and immunoprecipitation should still be considered in selected cases, such as apparently autoantibody-negative patients where anti-synthetase syndrome is suspected.

The myositis clinical phenotype associated with anti-Zo autoantibodies: a case series of nine UK patients.

OBJECTIVES: It has been over 10 years since the first report of autoantibodies directed against phenylalanyl tRNA synthetase (anti-Zo) in a patient with features of the anti-synthetase syndrome. In that time no further cases have been published. Here we aim to characterize more fully the clinical phenotype of anti-Zo-associated myositis by describing the clinical features of nine patients. METHODS: Anti-Zo was identified by protein-immunoprecipitation in patients referred for extended spectrum myositis autoantibody testing at our laboratory. Results were confirmed by immunodepletion using a reference serum. Medical records were retrospectively reviewed to provide detailed information of the associated clinical phenotype for all identified patients. Where possible, HLA genotype was imputed using Illumina protocols. RESULTS: Nine patients with anti-Zo were identified. The median age at disease onset was 51 years, and six patients were female. Seven patients had evidence of inflammatory muscle disease, seven of interstitial lung disease and six of arthritis. The reported pattern of interstitial lung disease varied with usual interstitial pneumonia, non-specific interstitial pneumonia and organizing pneumonia all described. Other features of the anti-synthetase syndrome such as RP and mechanics hands were common. HLA data was available for three patients, all of whom had at least one copy of the HLA 8.1 ancestral haplotype. CONCLUSION: Patients with anti-Zo presenting with features of the anti-synthetase syndrome and interstitial lung disease is a common finding. Like other myositis autoantibodies, there is likely to be a genetic association with the HLA 8.1 ancestral haplotype.

Autoantibodies in connective tissue disease.

Autoimmune connective tissue diseases are heterogeneous rheumatic diseases with the potential to affect multiple body systems. Autoantibodies are a characteristic feature of these diseases and are typically highly disease specific. In addition to aiding diagnosis, many autoantibodies have established associations with clinically important disease complications including internal organ involvement. In this chapter, we review the autoantibodies relevant to autoimmune connective tissue diseases, excluding systemic lupus erythematosus, with particular reference to the associated clinical features and how identification of such an autoantibody may inform prognosis and clinical management. We also discuss the practicalities of testing for autoantibodies along with potential difficulties and pitfalls.

Juvenile Dermatomyositis-Clinical Phenotypes.

PURPOSE OF REVIEW: Juvenile dermatomyositis is a heterogeneous disease with variable clinical outcomes. Here, we describe the recognised subtypes of idiopathic inflammatory myositis which occur in children, with particular reference to disease-associated autoantibodies. RECENT FINDINGS: Large cohort studies have demonstrated that myositis autoantibodies are common in juvenile dermatomyositis and can be found in the majority of patients. They identify homogenous clinical subgroups and inform prognosis, particularly the risks of developing interstitial lung disease. Descriptions of immune-mediated necrotising myositis in juvenile patients have highlighted a rare but important clinical subset typically associated with severe muscle disease and treatment resistance. It is increasingly apparent that autoantibodies can provide detailed information on prognosis and the likely disease associations in those with juvenile dermatomyositis. Further work is needed to establish how this knowledge should influence our approach to treatment.

Focused HLA analysis in Caucasians with myositis identifies significant associations with autoantibody subgroups.

OBJECTIVES: Idiopathic inflammatory myopathies (IIM) are a spectrum of rare autoimmune diseases characterised clinically by muscle weakness and heterogeneous systemic organ involvement. The strongest genetic risk is within the major histocompatibility complex (MHC). Since autoantibody presence defines specific clinical subgroups of IIM, we aimed to correlate serotype and genotype, to identify novel risk variants in the MHC region that co-occur with IIM autoantibodies. METHODS: We collected available autoantibody data in our cohort of 2582 Caucasian patients with IIM. High resolution human leucocyte antigen (HLA) alleles and corresponding amino acid sequences were imputed using SNP2HLA from existing genotyping data and tested for association with 12 autoantibody subgroups. RESULTS: We report associations with eight autoantibodies reaching our study-wide significance level of p<2.9×10-5. Associations with the 8.1 ancestral haplotype were found with anti-Jo-1 (HLA-B*08:01, p=2.28×10-53 and HLA-DRB1*03:01, p=3.25×10-9), anti-PM/Scl (HLA-DQB1*02:01, p=1.47×10-26) and anti-cN1A autoantibodies (HLA-DRB1*03:01, p=1.40×10-11). Associations independent of this haplotype were found with anti-Mi-2 (HLA-DRB1*07:01, p=4.92×10-13) and anti-HMGCR autoantibodies (HLA-DRB1*11, p=5.09×10-6). Amino acid positions may be more strongly associated than classical HLA associations; for example with anti-Jo-1 autoantibodies and position 74 of HLA-DRB1 (p=3.47×10-64) and position 9 of HLA-B (p=7.03×10-11). We report novel genetic associations with HLA-DQB1 anti-TIF1 autoantibodies and identify haplotypes that may differ between adult-onset and juvenile-onset patients with these autoantibodies. CONCLUSIONS: These findings provide new insights regarding the functional consequences of genetic polymorphisms within the MHC. As autoantibodies in IIM correlate with specific clinical features of disease, understanding genetic risk underlying development of autoantibody profiles has implications for future research.

Autoantibodies in myositis.

The discovery of novel autoantigen systems related to idiopathic inflammatory myopathies (collectively referred to as myositis) in adults and children has had major implications for the diagnosis and management of this group of diseases across a wide range of medical specialties. Traditionally, autoantibodies found in patients with myositis are described as being myositis-specific autoantibodies (MSAs) or myositis-associated autoantibodies (MAAs), depending on their prevalence in other, related conditions. However, certain MSAs are more closely associated with extramuscular manifestations, such as skin and lung disease, than with myositis itself. It is very rare for more than one MSA to coexist in the same individual, underpinning the potential to use MSAs to precisely define genetic and disease endotypes. Each MSA is associated with a distinctive pattern of disease or phenotype, which has implications for diagnosis and a more personalized approach to therapy. Knowledge of the function and localization of the autoantigenic targets for MSAs has provided key insights into the potential immunopathogenic mechanisms of myositis. In particular, evidence suggests that the alteration of expression of a myositis-related autoantigen by certain environmental influences or oncogenesis could be a pivotal event linking autoantibody generation to the development of disease.

Autoantibodies in juvenile-onset myositis: Their diagnostic value and associated clinical phenotype in a large UK cohort.

OBJECTIVES: Juvenile myositis is a rare and heterogeneous disease. Diagnosis is often difficult but early treatment is important in reducing the risk of associated morbidity and poor outcomes. Myositis specific autoantibodies have been described in both juvenile and adult patients with myositis and can be helpful in dividing patients into clinically homogenous groups. We aimed to explore the utility of myositis specific autoantibodies as diagnostic and prognostic biomarkers in patients with juvenile-onset disease. METHODS: Using radio-labelled immunoprecipitation and previously validated ELISAs we examined the presence of myositis specific autoantibodies in 380 patients with juvenile-onset myositis in addition to, 318 patients with juvenile idiopathic arthritis, 21 patients with juvenile-onset SLE, 27 patients with muscular dystrophies, and 48 healthy children. RESULTS: An autoantibody was identified in 60% of juvenile-onset myositis patients. Myositis specific autoantibodies (49% patients) were exclusively found in patients with myositis and with the exception of one case were mutually exclusive and not found in conjunction with another autoantibody. Autoantibody subtypes were associated with age at disease onset, key clinical disease features and treatment received. CONCLUSIONS: In juvenile patients the identification of a myositis specific autoantibody is highly suggestive of myositis. Autoantibodies can be identified in the majority of affected children and provide useful prognostic information. There is evidence of a differential treatment approach and patients with anti-TIF1γ autoantibodies are significantly more likely to receive aggressive treatment with IV cyclophosphamide and/or biologic drugs, clear trends are also visible in other autoantibody subgroups.

Anti-HMGCR Autoantibodies in Juvenile Idiopathic Inflammatory Myopathies Identify a Rare but Clinically Important Subset of Patients.

OBJECTIVE: We aimed to establish the prevalence and clinical associations of anti-HMG-CoA-reductase (anti-HMGCR) in a large UK cohort with juvenile myositis. METHODS: There were 381 patients investigated for anti-HMGCR using ELISA. RESULTS: Anti-HMGCR autoantibodies were detected in 4 patients (1%). These children had no or minimal rash and significant muscle disease. Muscle biopsies were considered distinctive, with widespread variation in fiber size, necrotic fibers, and chronic inflammatory cell infiltrates; all had prolonged elevation of creatine kinase and all ultimately received biologic therapies. CONCLUSION: Anti-HMGCR in UK children with myositis are associated with severe disease that is poorly responsive to standard treatment.

Antibodies in juvenile-onset myositis.

PURPOSE OF REVIEW: Juvenile-onset myositis is a highly heterogeneous disease. Myositis-specific and associated autoantibodies provide a potential means of subdividing patients into clinically homogenous subgroups. Given the increasing availability of autoantibody testing, this review explores the phenotypes associated with different autoantibodies in juvenile-onset myositis and the potential clinical utility of autoantibody testing. RECENT FINDINGS: Autoantibodies can be identified in 60-70% of children with myositis and the recent discovery of novel myositis-associated autoantibodies in adult patients suggests this may increase in the near future. Detailed phenotype descriptions are now known for several autoantibodies commonly identified in juvenile-onset disease. Whilst there is insufficient evidence to recommend a differential treatment approach based on autoantibody status, it is becoming increasingly clear that some autoantibody subgroups are often treatment resistant and may benefit from a more aggressive approach. SUMMARY: The validation of nonspecialised methods for myositis-specific autoantibody detection should lead to more widely available testing. In juvenile-onset disease, this will provide detailed prognostic information and in the future may also influence approach.

Muscle Biopsy Findings in Combination With Myositis-Specific Autoantibodies Aid Prediction of Outcomes in Juvenile Dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (DM) is a rare and severe autoimmune condition characterized by rash and proximal muscle weakness. While some patients respond to standard treatment, others do not. This study was carried out to investigate whether histopathologic findings and myositis-specific autoantibodies (MSAs) have prognostic significance in juvenile DM. METHODS: Muscle biopsy samples (n = 101) from patients in the UK Juvenile Dermatomyositis Cohort and Biomarker Study were stained, analyzed, and scored for severity of histopathologic features. In addition, autoantibodies were measured in the serum or plasma of patients (n = 90) and longitudinal clinical data were collected (median duration of follow-up 4.9 years). Long-term treatment status (on or off medication over time) was modeled using generalized estimating equations. RESULTS: Muscle biopsy scores differed according to MSA subgroup. When the effects of MSA subgroup were accounted for, increased severity of muscle histopathologic features was predictive of an increased risk of remaining on treatment over time: for the global pathology score (histopathologist's visual analog scale [hVAS] score), 1.48-fold higher odds (95% confidence interval [95% CI] 1.12-1.96; P = 0.0058), and for the total biopsy score (determined with the standardized score tool), 1.10-fold higher odds (95% CI 1.01-1.21; P = 0.038). A protective effect was identified in patients with anti-Mi-2 autoantibodies, in whom the odds of remaining on treatment were 7.06-fold lower (95% CI 1.41-35.36; P = 0.018) despite muscle biopsy scores indicating more severe disease. In patients with anti-nuclear matrix protein 2 autoantibodies, anti-transcription intermediary factor 1γ autoantibodies, or no detectable autoantibody, increased histopathologic severity alone, without adjustment for the effect of MSA subtype, was predictive of the risk of remaining on treatment: for the hVAS global pathology score, 1.61-fold higher odds (95% CI 1.16-2.22; P = 0.004), and for the total biopsy score, 1.13-fold higher odds (95% CI 1.03-1.24; P = 0.013). CONCLUSION: Histopathologic severity, in combination with MSA subtype, is predictive of the risk of remaining on treatment in patients with juvenile DM and may be useful for discussing probable treatment length with parents and patients. Understanding these associations may identify patients at greater risk of severe disease.

Serological subsets of juvenile idiopathic inflammatory myopathies--an update.

In this review we explore the different characteristics of the serological phenotypes identified in juvenile-onset myositis and consider how the serological sub-classification of patients with juvenile myositis can be advantageous both in terms of reaching what can be a difficult diagnosis and informing on prognosis. Recent studies have described the autoantibody associated disease phenotypes and outcome for those with juvenile-onset disease and include analyses of large juvenile-onset myositis cohorts. Here we describe the autoantibody associated disease features for patients within juvenile-onset myositis in detail and discuss the expanding opportunities and strategies for myositis specific autoantibody testing in clinical practice.