Clinical experience in anti-synthetase syndrome: a monocentric retrospective analytical study

ABSTRACT OBJECTIVES: Anti-synthetase syndrome (ASS) is a rare autoimmune disorder combining autoantibodies and specific clinical manifestations. One of the particularities of ASS is the pleiomorphic radiological presentation seen at the initial work-up. Evaluating treatment response can also be challenging and requires specific clinical, functional, biological and radiological monitoring. For these reasons, it is fundamental to identify specific radiological and clinical features of ASS for improved diagnosis and therapeutic approaches. METHODS: We retrospectively studied all patients suffering from ASS in the CHU of Liège from 2008 to 2019. We analysed the clinical features, pulmonary function tests (PFTs), computed tomography (CT), and longitudinal evolution with regard to patient treatment. RESULTS: In the whole cohort of 30 patients, we identified 19 with anti-JO1 antibodies, 5 with anti-PL12 antibodies and 6 with anti-PL7 antibodies. The sex ratio was slightly in favour of males. Interestingly, PL-12 syndrome was more likely to be present in younger patients than those associated with other antibodies. Overall, 77% of the overall cohort exhibited specific pulmonary involvement without any significant difference with regard to the severity assessed by PFT at diagnosis. In contrast, the radiological presentation was pleomorphic for anti-JO1 syndrome, and anti-PL12 syndrome exhibited mainly ground-glass opacities (GGOs) and reticular abnormalities, while those with anti-PL7 antibodies showed reticulations and bronchiectasis. Longitudinal CT analysis mainly showed a reduction in consolidations and GGOs with specific therapies. CONCLUSION: In our single-centre retrospective study, we found different profiles for different autoantibodies according to age and radiological appearance.


Background
Anti-synthetase syndrome (ASS) is a rare autoimmune disorder that was described for the first time in 1990 [1]. The clinical expression varies and includes inflammatory myopathy (IM), interstitial lung disease (ILD), polyarthritis, mechanic's hands, Raynaud's phenomenon and unexplained fever [2]. The symptomatic presentation is highly variable, as is the clinical course of the disease [3]. Pulmonary manifestations mainly drive the overall prognosis and need to be carefully assessed in all patients. The severity of the initial presentation ranges from asymptomatic forms to acute respiratory failure, which can be responsible for acute respiratory distress syndrome in a small number of patients [4].
A systematic blood analysis focusing on specific antibody assessments must be considered in the presence of any ILD. Each specific autoantibody is associated with a variable clinical onset as well as clinical evolution [5]. Similarly, treatment strategies must be considered in an integrative way, considering the immunological patterns, clinical onset and disease severity. The diagnosis of ASS is associated with positive serologic testing for an anti-aminoacyl-transfer-RNA synthetase (anti-ARS) autoantibody and at least one pathognomonic clinical manifestation (Table 1) [6]. Ten specific antibodies are currently described in the scientific literature. Anti-JO1 (anti-histidyl), anti-PL7 (anti-threonyl), anti-PL12 (anti-alanyl), anti-OJ (antiisoleucyl) and anti-EJ (anti-glycyl) antibodies represent more than 90% of the detected antibodies [7]. They are now routinely tested for in practice, but clinicians must consider specific analyses in cases of positive anticytoplasmic nuclear antibodies in patients suffering from ILD [8].
Many tools have been developed to assess respiratory disease severity. It is recommended to systematically perform pulmonary function tests (PFTs) and thoracic high-resolution CT (HRCT) and evaluate potential desaturation occurring during exercises. Additionally, the results of blood gas analysis, bronchoalveolar lavage and exercise testing [9,10] have been used as relevant indicators. ASS linked to interstitial lung disease is typically associated with restrictive lung function impairment and a reduced diffusing capacity of the lung for carbon monoxide (DLCO). DLCO is commonly reduced in patients with ILD, often at an earlier stage of the disease than total lung capacity (TLC) and forced vital capacity (FVC). PFT provides important data to evaluate severity, as the results only partially correlate with HRCT scores and pattern. In addition, PFT is more sensitive to changes after therapy than HRCT [11]. However, HRCT remains essential for the evaluation of lung involvement. The main CT findings of anti-ARS-ILD are areas of ground-glass attenuation and reticulation, predominantly distributed as lower and peribronchovascular lesions, which can be compatible with a fibrosing nonspecific interstitial pneumonia (NSIP) pattern. A combination of patchy areas of consolidation and reticulation is common and may suggest the presence of myositis in subjects with ILD, especially with a subacute or an acute onset. Honeycombing (areas of small cystic spaces with thickened walls), traction bronchiectasis (bronchial dilation due to traction by fibrous tissue) and subpleural bands are less frequently observed [12].
The treatment of patients with ASS is assessed on the presence or absence of ILD, its severity and/or its initial response to treatment [13]. The available medications are limited, and corticosteroids are generally regarded as the most effective choice. The usual recommendations highlight the benefit of the combination of corticosteroids at the lowest efficient dose with another adjunctive immunosuppressive agent [14]. Currently, the most commonly used treatments are azathioprine, mycophenolate mofetil, calcineurin inhibitors, rituximab, intravenous cyclophosphamide or intravenous immunoglobulin [15]. The choice of the immunosuppressive drug regimen and when it should be started is left to the doctor's discretion. Despite these treatments, the overall mortality rate for treated patients is significantly higher than that for a standardized population of the same age and sex [16].
The aim of the present retrospective study was to analyse the ten-year clinical experience with ASS at CHU Liège (Belgium) and to confirm whether our clinical data are consistent with the literature.

Methods
Patients were selected for a retrospective data analysis focusing on positive laboratory results for an anti-ARS antibody at CHU of Liège from 1 January 2008 to 1 January 2019. The diagnosis of ASS was made according to the international recommendations of the European Respiratory Society (ERS) using major and minor criteria [17]. All clinical, imaging and spirometry data presented in this article were extracted from our electronic health records.

Pulmonary function tests
We performed PFTs in the routine respiratory laboratory of CHU of Liège. All spirometry tests in this study were performed using the pneumotachograph JaegerMasterlab system (Erich Jaeger GmbH, Wuzburg, Germany). The FEV1 and FVC were measured in accordance with the recommendations of the ERS. The results are expressed in millilitres (mL) and as a percentage of the predicted value (% pred). The Tiffeneau index or FEV1/FVC is expressed in percent (%). TLC was measured by body plethysmography according to ERS recommendations. The DLCO and the reported DLCO/VA were measured by the singlebreath carbon monoxide gas transfer method and are expressed as a percentage of the predicted value (% pred).

HRCT evaluation
The initial HRCT evaluation was first performed by a specialized thoracic radiologist, then a specific review of each case was performed by 2 members of the multidisciplinary ILDs team. The CT findings were interpreted according to the recommendations of the Fleischner Society [18] and assessed for honeycombing, reticulations, ground-glass opacities (GGOs), consolidations and traction bronchiectasis. The readers also analysed the presence of cysts, nodules, pleural or pericardial effusion and lymphadenopathy. The extent of honeycombing, reticulations, GGOs and consolidations was quantified for the whole lung using the method from Akira et al. [19], dividing the lungs into six zones, three for each lung (upper zone: above the level of the carina; middle zone: between the level of the carina and the level of the inferior pulmonary veins; and lower zone: under the level of the inferior pulmonary veins). The overall percentage of lung involvement for each sign was calculated by averaging the six lung zone values and then graded from 0 to 4, following the Evidence of overt or hypomyopathic myositis (elevated CPK levels, myalgia, proximal muscular weakness, positive muscular biopsy, electromyographic triad of myositis or MRI muscular oedema) Evidence of ILD according to the ATS criteria Evidence of articular involvement (symmetrical inflammatory arthralgia or overt arthritis) Or two minor involvements: Unexplained, persistent fever Raynaud's phenomenon Mechanic's hands Silva grading system [20]: 0, absent; 1, 1% to 4%; 2, 5% to 25%; 3, 26% to 50%; and 4, >50%. Traction bronchiectasis was graded from 0 to 3: 0, none; 1, minimal; 2, moderate; and 3, severe. The grades were dichotomized into two grade categories to enable statistical analysis. For each individual sign, grade A included grades 0-1, and grade B included grades 2-4.

Outcome
Outcome was arbitrarily defined with a multimodal approach (clinical signs, PFT, HRCT, blood testing) and was categorized as resolution, improvement/stabilization or deterioration. Resolution was defined as complete remission of pulmonary symptoms associated with the disappearance of radiographic signs of ILD and normalization of standard PFT values. Improvement was defined when any of the former pulmonary alterations improved without returning to normal value, according to an international consensus statement of the American Thoracic Society on idiopathic pulmonary fibrosis [21]. Changes ≥ 10% in FVC and/or ≥15% in DLCO were considered significant and were used to assess improvement or deterioration.

Statistics
The results are presented as the mean and standard deviation (SD) and range or as frequency tables. Comparisons between groups were performed by analysis of variance and the chi-square test. Survival was represented by a Kaplan-Meier curve, and groups were compared by the log-rank test. The results were considered significant at the 5% level (P < 0.05). Calculations were performed in SAS version 9.4, and the figures were created in R version 3.6.1.

Clinical and biological characteristics
The subject characteristics are presented in Table 2. In the whole cohort of 30 patients, we identified 19 with anti-JO1 antibodies, 5 with anti-PL12 antibodies and 6 with anti-PL-7 antibodies. The sex ratio was slightly in favour of males, with all patients being males in the anti-PL7 group (p = 0.021). Interestingly, PL-12 syndrome was significantly more likely to be present in younger patients than those associated with other antibodies (mean age 39,8 vs 53 (JO1) and 73 (PL7) (p = 0.0095)). Overall, 77% of the overall cohort exhibited specific pulmonary involvement. The prevalence of ILD was higher in anti-PL7/PL12-positive patients than in anti-JO1-positive patients but not with a statically significant difference. The former group also exhibited rheumatological signs less frequently. We found no significant difference between ASS patients with anti-JO1 antibodies and those with anti-PL7/PL12 antibodies regarding smoking status, dermatologic signs, malignancy development or blood test results. Anti-JO1-positive patients tended to more commonly exhibit gastroesophageal reflux disease (21% vs. 0%), although the difference was not significant.

Pulmonary function tests
Twenty-six subjects (87%) had available baseline PFT data. The mean data are shown in Table 3. Anti-JO1 and anti-PL7 patients presented a more restrictive pattern (TLC 73% pred and 75% pred, respectively) than the anti-PL12 group (TLC 81%). All three anti-ARS categories presented a sharp reduction in both DLCO and FVC.

Treatments, outcome and survival
The treatment characteristics are presented in Table  4. During the follow-up, 25 out of 30 patients benefited from systemic corticosteroids (CS). The CS were associated with corticoid-sparing immunosuppressive agents in the majority of cases (25). The most commonly used was methotrexate for anti-JO1-positive patients (13 cases), followed by azathioprine (8 cases). In the anti-PL12 group, the first-line corticoidsparing agent was azathioprine (4 cases), while there was no predominant therapy in the anti-PL7 group. CS and methotrexate were mainly used for anti-JO1positive patients (p = 0.039 and p = 0.041, respectively). After a one-year follow-up, half of the subjects had a favourable evolution according to our criteria (see Methods -Outcomes). The clinical evolution of ILD after one year is summarized in Table 5. Overall, four patients (17%) had clinical degradation of the disease under treatment, whereas six (26%) remained stable. Anti-PL7-positive subjects had proportionally the worst evolution, with 50% of them presenting clinical deterioration. Conversely, patients with anti-PL7 had better evolution, with improvements in 100% of the cases at one year.
The survival curves are presented in Figure 1. The median survival was longer than 132 months. The survival probability at 12 months was approximately 86%, while the 5-year survival rate was around 70%: there were no statistically significant differences among the three anti-ARS autoantibodies (p = 0.61). However, two subjects with anti-PL7 antibodies rapidly died after their diagnosis.

HRCT findings
The radiological features are presented in Table 6. The radiological presentation was pleomorphic in the anti-JO1 group. Patients suffering from anti-PL12 syndrome exhibited mainly ground-glass opacities (GGOs) (67%) and reticular abnormalities (67%), while those with anti-PL7 showed reticulations (75%) and bronchiectasis (75%). The longitudinal CT analysis from 6 to 12 months mainly showed a reduction in consolidations and GGOs (33% and 67%, respectively) with specific therapies (Table 7). To illustrate these findings, two HRCT scans for anti-PL12 and anti-PL7 patients are shown in Figure 2.

Discussion
The present study reports our experience with ASS at the University Hospital of Liege, highlighting the differences in the clinical and radiological aspects. Our analysis focused on the association between antisynthetase antibodies and ILD. Furthermore, longterm outcomes and prognostic factors were also analysed. The most prevalent anti-ARS autoantibodies found were anti-JO1 antibodies (59% vs 23% for PL7 and 18% for PL12). The epidemiological characteristics are partially similar to those seen in the literature [3]. Surprisingly, in our cohort, we encountered significantly more males than females in the PL12 and PL7 patient groups, contrary to what is generally seen for such disease [22]. The overall mean age is in accordance with that of other cohorts. In our experience, anti-PL12 subjects were significantly younger than anti-PL7 subjects. Interstitial lung disease was the most frequent presentation form for all groups (70% for JO1, 87% for PL7 and 83% for PL12) and can vary in its severity at diagnosis evaluation. Early identification of ILD in ASS is fundamental for these patients, as the fibroinflammatory lung process mainly drives the mortality rate [23]. In its most severe forms, ILD can evolve into severe respiratory failure [4]. Some patients present with acute respiratory distress syndrome at clinical onset. Therefore, increasing the global awareness of clinicians, especially those used to managing patients in the ICU, is critical for the overall outcomes    of patients with severe ASS. Likewise, lung involvement in PL7 and PL12 is usually described as most severe and associated with a worse prognosis than anti-JO1 but was not evidenced in this study due to the small number of patients. Concerning PFTs, it is widely known that DLCO and, to a lesser extent, FVC are the most affected values in ASS. In this context, PFT has to be evaluated at diagnosis and during follow-up to reduce the risk of underestimating the severity of the disease. In our opinion, PFTs must become the keystone for ASS-induced ILD management and should be performed at least every 3 months during the initial follow-up.
Currently, treatment management is still challenging in ASS [9]. There have been some recent studies comparing available immunosuppressive therapeutics and their efficacy on PFT, HRCT findings and corticosteroids sparing's efficiency [14,24]. Our study did not identify significant differences between immunosuppressive drugs or their regimens. Trying to find the best therapy for such diseases should be the main objective for further multicentric prospective longitudinal trials. Historically, corticosteroids have been the first-line therapy for idiopathic inflammatory myopathy (IIM). When corticosteroid tapering is used in monotherapy in ILD-IIM, the risk of lung disease persistence is high. Moreover, the severity of ILD presentation frequently requires the rapid use of aggressive immunosuppressive agents (e.g. cyclophosphamide, rituximab, etc.) [25]. Azathioprine is used as first-line therapy when an ILD is present, while mycophenolate mofetil is used by rheumatologists as the first-line to treat IIM without lung involvement. Other therapeutics, such as tacrolimus, rituximab, intravenous immunoglobulins and cyclophosphamide, should be considered second-line or rescue therapy in cases of refractory situations, such as acute respiratory distress syndrome. These specific therapies need to be closely monitored in their administration and require an expert multidisciplinary team to assess their efficacy and side effects.     Considering the HRCT findings, our data are in line with the literature. We found that the HRCT features of ASS are mainly suggestive of NSIP at presentation. Anti-PL12 disease showed predominant reticulation and GGOs, whereas the appearance of anti-JO1 disease was more pleiomorphic. The most significant HRCT findings for follow-up seem to be GGOs and consolidation. As expected, traction bronchectasis and honeycombing were stable during follow-up. This can be linked to the stabilization of the inflammatory process, reducing progressive fibrosing lung disease. Currently, the use of specific antifibrotic therapies in selected patients for progressive fibrosing lung diseases is gathering considerable attention. A recently published study identified nintedanib as an effective therapy to significantly reduce FVC over time in progressive fibrosing lung diseases [26].

Conclusion
In conclusion, our single-centre retrospective study highlights some similarities to the literature about the clinical and radiological features of ASS. Some differences are probably due to the disease's prevalence and to the small amount of available follow-up data. Heterogeneity in the clinical course and in the response to treatment make diagnosis, treatment and follow-up particularly complex. Therefore, dedicated multicentric prospective longitudinal studies are highly recommended in the specific context of ASS-ILD.

Ethics approval and consent to participate
The protocol was approved by the ethics committee of CHU of Liège, and all subjects gave written consent before their enrolment (Belgian number: B707201422832; ref: 2014/302).

Disclosure statement
No potential conflict of interest was reported by the author(s).