Decreased serpin C1 in extracellular vesicles predicts response to methotrexate treatment in patients with pulmonary sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic granulomatous disease of unknown etiology primarily affecting the lungs. Treatment is needed when disease symptoms worsen and organ function deteriorates. In pulmonary sarcoidosis, prednisone and methotrexate (MTX) are the most common anti-inflammatory therapies. However, there is large inter-patient variability in response to treatment, and predictive response markers are currently lacking. OBJECTIVE: In this study, we investigated the predictive potential of biomarkers in extracellular vesicles (EVs) isolated from biobanked serum of patients with pulmonary sarcoidosis stored prior to start of therapy. METHODS: Protein concentrations of a four-protein test panel of inflammatory proteins were measured in a discovery (n = 16) and replication (n = 129) cohort of patients with sarcoidosis and 47 healthy controls. Response to therapy was defined as an improvement of the absolute score of > 5% forced vital capacity (FVC) and/or > 10% diffusion lung of carbon monoxide (DLCO) after 24 weeks compared to baseline (before treatment). RESULTS: Serum protein levels differed between EV fractions and serum, and between sarcoidosis cases and controls. Serpin C1 concentrations in the low density lipid particle EV fraction were lower at baseline in the group of patients with a good response to MTX treatment in both the discovery cohort (p = 0.059) and in the replication cohort (p = 0.032). EV Serpin C1 showed to be a significant predictor for response to treatment with MTX (OR 0.4; p = 0.032). CONCLUSION: This study shows that proteins isolated from EVs harbor a distinct signal and have potential as new predictive therapy response biomarkers in sarcoidosis.

Methotrexate Polyglutamate Concentrations as a Possible Predictive Marker for Effectiveness of Methotrexate Therapy in Patients with Sarcoidosis: A Pilot Study.

INTRODUCTION: Methotrexate (MTX), a folate antagonist, is often used as second-line treatment in patients with sarcoidosis. Effectiveness of MTX has large inter-patient variability and at present therapeutic drug monitoring (TDM) of MTX is not possible. Upon administration, MTX is actively transported into cells and metabolized to its active forms by adding glutamate residues forming MTXPG(n=1-5) resulting in enhanced cellular retention. In this study we address the question whether different MTXPG(n) concentrations in red blood cells (RBC) of patients with sarcoidosis after 3 months of MTX therapy correlate with response to treatment. METHODS: We retrospectively included patients with sarcoidosis that had started on MTX therapy and from whom blood samples and FDG-PET/CT were available 3 and 6-12 months after MTX initiation, respectively. FDG-uptake was measured by SUVmax in the heart, lungs and thoracic lymph nodes. Changes in SUVmax was used to determine anti-inflammatory response after 6-12 months of MTX therapy. MTXPG(n) concentrations were measured from whole blood RBC using an LC-MS/MS method. Pearson correlation coefficients were calculated to evaluate the relationship between changes in the SUVmax and MTXPG(n) concentrations. RESULTS: We included 42 sarcoidosis patients treated with MTX (15 mg/week); 31 with cardiac sarcoidosis and 11 with pulmonary sarcoidosis. In MTXPG3 and MTXPG4 a significant negative relation between the absolute changes in SUVmax and MTXPG(n) was found r = - 0.312 (n = 42, p = 0.047) for MTXPG3 and r = - 0.336 (n = 42, p = 0.031 for MTXPG4). The other MTXPG(n) did not correlate to changes in SUVmax. CONCLUSION: These results suggest a relation between MTXPG(n) concentrations and the anti-inflammatory effect in patients with sarcoidosis. Further prospective validation is warranted, but if measuring MTXPG concentrations could predict treatment effect of MTX this would be a step in the direction of personalized medicine.

Prevalence and clinical associations of myositis antibodies in a large cohort of interstitial lung diseases.

BACKGROUND: Serologic testing for autoantibodies is recommended in interstitial lung diseases (ILDs), as connective tissue diseases (CTDs) are an important secondary cause. Myositis antibodies are associated with CTD-ILD, but clinical associations with other ILDs are unclear. In this study, associations of myositis antibodies in various ILDs were evaluated. METHODS: 1463 ILD patients and 116 healthy subjects were screened for myositis antibodies with a line-blot assay on serum available at time of diagnosis. Additionally, bronchoalveolar lavage fluid (BALf) was analysed. RESULTS: A total of 394 patients demonstrated reactivity to at least one antibody, including anti-Ro52 (36.0%), anti-Mi-2ß (17.3%) and anti-Jo-1 (10.9%). Anti-Jo-1 (OR 6.4; p<0.100) and anti-Ro52 (OR 6.0; p<0.001) were associated with CTD-ILD. Interestingly, anti-Mi-2ß was associated with idiopathic pulmonary fibrosis (IPF; OR 5.3; p = 0.001) and hypersensitivity pneumonitis (HP; OR 5.9; p<0.001). Furthermore, anti-Mi-2ß was strongly associated with a histological usual interstitial pneumonia (UIP) pattern (OR 6.5; p < 0.001). Moreover, anti-Mi-2ß reactivity was identified in BALf and correlated with serum anti-Mi-2ß (r = 0.64; p = 0.002). No differences were found in survival rates between ILD patients with and without serum Mi-2ß reactivity (hazard ratio 0.835; 95% CI 0.442-1.575; p = 0.577). CONCLUSION: In conclusion, novel associations of antibody Mi-2ß with fibrotic ILD were found. Furthermore, serum anti-Mi-2ß was associated with a histological UIP pattern and presence of anti-Mi-2ß in BALf. Possibly, anti-Mi-2ß could be implemented as a future diagnostic biomarker for fibrotic ILD.

Design of a randomized controlled trial to evaluate effectiveness of methotrexate versus prednisone as first-line treatment for pulmonary sarcoidosis: the PREDMETH study.

BACKGROUND: Treatment of pulmonary sarcoidosis is recommended in case of significant symptoms, impaired or deteriorating lung function. Evidence-based treatment recommendations are limited and largely based on expert opinion. Prednisone is currently the first-choice therapy and leads to short-term improvement of lung function. Unfortunately, prednisone often has side-effects and may be associated with impaired quality of life. Methotrexate is presently considered second-line therapy, and appears to have fewer side-effects. OBJECTIVE: The primary objective of this trial is to investigate the effectiveness and tolerability of methotrexate as first-line therapy in patients with pulmonary sarcoidosis compared with prednisone. The primary endpoint of this study will be the change in hospital-measured Forced Vital Capacity (FVC) between baseline and 24 weeks. Secondary objectives are to gain more insights in response to therapy in individual patients by home spirometry and patient-reported outcomes. Blood biomarkers will be examined to find predictors of response to therapy, disease progression and chronicity, and to improve our understanding of the underlying disease mechanism. METHODS/DESIGN: In this prospective, randomized, non-blinded, multi-center, non-inferiority trial, we plan to randomize 138 treatment-naïve patients with pulmonary sarcoidosis who are about to start treatment. Patients will be randomized in a 1:1 ratio to receive either prednisone or methotrexate in a predefined schedule for 24 weeks, after which they will be followed up in regular care for up to 2 years. Regular hospital visits will include pulmonary function assessment, completion of patient-reported outcomes, and blood withdrawal. Additionally, patients will be asked to perform weekly home spirometry, and record symptoms and side-effects via a home monitoring application for 24 weeks. DISCUSSION: This study will be the first randomized controlled trial comparing first-line treatment of prednisone and methotrexate and provide valuable data on efficacy, safety, quality of life and biomarkers. If this study confirms the hypothesis that methotrexate is as effective as prednisone as first-line treatment for sarcoidosis but with fewer side-effects, this will lead to improvement in care and initiate a change in practice. Furthermore, insights into the immunological mechanisms underlying sarcoidosis pathology might reveal new therapeutic targets. TRIAL REGISTRATION: The study was registered on the 19th of March 2020 in the International Clinical Trial Registry, www.clinicaltrials.gov; ID NCT04314193 .

Prevalence of Novel Myositis Autoantibodies in a Large Cohort of Patients with Interstitial Lung Disease.

Connective tissue diseases (CTDs) are an important secondary cause of interstitial lung disease (ILD). If a CTD is suspected, clinicians are recommended to perform autoantibody testing, including for myositis autoantibodies. In this study, the prevalence and clinical associations of novel myositis autoantibodies in ILD are presented. A total of 1194 patients with ILD and 116 healthy subjects were tested for antibodies specific for Ks, Ha, Zoα, and cN1A with a line-blot assay on serum available at the time of diagnosis. Autoantibodies were demonstrated in 63 (5.3%) patients and one (0.9%) healthy control (p = 0.035). Autoantibodies were found more frequently in females (p = 0.042) and patients without a histological and/or radiological usual interstitial pneumonia (UIP; p = 0.010) and a trend towards CTD-ILDs (8.4%) was seen compared with other ILDs (4.9%; p = 0.090). The prevalence of antibodies specific for Ks, Ha, Zoα, and cN1A was, respectively, 1.3%, 2.0%, 1.4%, and 0.9% in ILD. Anti-Ha and Anti-Ks were observed in males with unclassifiable idiopathic interstitial pneumonia (unclassifiable IIP), hypersensitivity pneumonitis (HP), and various CTD-ILDs, whereas anti-cN1A was seen in females with antisynthetase syndrome (ASS), HP, and idiopathic pulmonary fibrosis (IPF). Anti-Zoα was associated with CTD-ILD (OR 2.5; 95%CI 1.11-5.61; p = 0.027). In conclusion, a relatively high prevalence of previously unknown myositis autoantibodies was found in a large cohort of various ILDs. Our results contribute to the awareness that circulating autoantibodies can be found in ILDs with or without established CTD. Whether these antibodies have to be added to the standard set of autoantibodies analysed in conventional myositis blot assays for diagnostic purposes in clinical ILD care requires further study.

Biomarkers in the Diagnosis and Prognosis of Sarcoidosis: Current Use and Future Prospects.

Sarcoidosis is a heterogeneous disease in terms of presentation, duration, and severity. Due to this heterogeneity, it is difficult to align treatment decisions. Biomarkers have proved to be useful for the diagnosis and prognosis of many diseases, and over the years, many biomarkers have been proposed to facilitate diagnosis, prognosis, and treatment decisions. Unfortunately, the ideal biomarker for sarcoidosis has not yet been discovered. The most commonly used biomarkers are serum and bronchoalveolar lavage biomarkers, but these lack the necessary specificity and sensitivity. In sarcoidosis, therefore, a combination of these biomarkers is often used to establish a proper diagnosis or detect possible progression. Other potential biomarkers include imaging tools and cell signaling pathways. Fluor-18-deoxyglucose positron emission tomography and high-resolution computed tomography have been proven to be more sensitive for the diagnosis and prognosis of both pulmonary and cardiac sarcoidosis than the serum biomarkers ACE and sIL-2R. There is an upcoming role for exploration of signaling pathways in sarcoidosis pathogenesis. The JAK/STAT and mTOR pathways in particular have been investigated because of their role in granuloma formation. The activation of these signaling pathways also proved to be a specific biomarker for the prognosis of sarcoidosis. Furthermore, both imaging and cell signaling biomarkers also enable patients who might benefit from a particular type of treatment to be distinguished from those who will not. In conclusion, the diagnostic and prognostic path of sarcoidosis involves many different types of existing and new biomarker. Research addressing biomarkers and disease pathology is ongoing in order to find the ideal sensitive and specific biomarker for this disease.