Molecular characterisation of lupus low disease activity state (LLDAS) and DORIS remission by whole-blood transcriptome-based pathways in a pan-European systemic lupus erythematosus cohort.

OBJECTIVES: To unveil biological milieus underlying low disease activity (LDA) and remission versus active systemic lupus erythematosus (SLE). METHODS: We determined differentially expressed pathways (DEPs) in SLE patients from the PRECISESADS project (NTC02890121) stratified into patients fulfilling and not fulfilling the criteria of (1) Lupus LDA State (LLDAS), (2) Definitions of Remission in SLE remission, and (3) LLDAS exclusive of remission. RESULTS: We analysed data from 321 patients; 40.8% were in LLDAS, and 17.4% in DORIS remission. After exclusion of patients in remission, 28.3% were in LLDAS. Overall, 604 pathways differed significantly in LLDAS versus non-LLDAS patients with an false-discovery rate-corrected p (q)<0.05 and a robust effect size (dr)≥0.36. Accordingly, 288 pathways differed significantly between DORIS remitters and non-remitters (q<0.05 and dr≥0.36). DEPs yielded distinct molecular clusters characterised by differential serological, musculoskeletal, and renal activity. Analysis of partially overlapping samples showed no DEPs between LLDAS and DORIS remission. Drug repurposing potentiality for treating SLE was unveiled, as were important pathways underlying active SLE whose modulation could aid attainment of LLDAS/remission, including toll-like receptor (TLR) cascades, Bruton tyrosine kinase (BTK) activity, the cytotoxic T lymphocyte antigen 4 (CTLA-4)-related inhibitory signalling, and the nucleotide-binding oligomerization domain leucine-rich repeat-containing protein 3 (NLRP3) inflammasome pathway. CONCLUSIONS: We demonstrated for the first time molecular signalling pathways distinguishing LLDAS/remission from active SLE. LLDAS/remission was associated with reversal of biological processes related to SLE pathogenesis and specific clinical manifestations. DEP clustering by remission better grouped patients compared with LLDAS, substantiating remission as the ultimate treatment goal in SLE; however, the lack of substantial pathway differentiation between the two states justifies LLDAS as an acceptable goal from a biological perspective.

Large vessel vasculitis is a risk factor for relapse only in giant cell arteritis patients without polymyalgia rheumatica.

OBJECTIVES: To evaluate differences in presentation and outcome of giant cell arteritis (GCA) patients with and without large vessel vasculitis (LVV) and according to the extent and severity of LVV. METHODS: Consecutive patients diagnosed with GCA between 2003 and 2020 who have had FDG PET imaging at diagnosis ≤3 days after initiation of glucocorticoids and followed for ≥12 months at the University Hospitals Leuven (Belgium), were included retrospectively. PET scans were visually scored (0-3) in 7 vascular areas and a total vascular score (TVS) was calculated. LVV was defined as FDG uptake ≥2 in any large vessel. RESULTS: We included 238 GCA patients, of which 169 (71%) had LVV. LVV patients were younger (69 vs 74 years, p< 0.001) and more frequently female (72% vs 49%, p= 0.001). In patients without PMR symptoms, the presence of LVV was associated with relapse (aOR 3.05 [95%CI 1.32-7.43], p= 0.011) and with a lower probability of stopping glucocorticoids (aHR 0.59 [95%CI 0.37-0.94], p= 0.025). However, in those with PMR symptoms, there was no difference in relapse risk (aOR 1.20 [95%CI 0.53-2.66], p= 0.657) and in the probability of stopping glucocorticoids (aHR 1.25 [95%CI 0.75-2.09], p= 0.394) between patients with and without LVV. A higher TVS was associated with an increased risk of relapse (aOR 1.09 [95%CI 1.04-1.15], p= 0.001] in patients without PMR symptoms, but not in those with PMR symptoms (aOR 1.01 [95%CI 0.96-1.07], p= 0.693). CONCLUSION: LVV is a risk factor for relapse in GCA patients without PMR symptoms with a higher relapse risk in those with higher TVS.

Prevalence, characteristics, and outcome of subclinical vasculitis in polymyalgia rheumatica: a retrospective cohort study.

OBJECTIVES: Two recent meta-analyses reported subclinical vasculitis in 22-23% of patients with polymyalgia rheumatica (PMR). We aimed to evaluate the prevalence, characteristics, and outcome of subclinical vasculitis among our PMR patients. METHODS: Consecutive patients with GCA/PMR spectrum disease with isolated PMR symptoms who underwent FDG PET imaging between 2003-2020 and who were followed for ≥6 months, were included retrospectively. Vasculitis was defined as FDG uptake ≥ grade 2 in any vessel. RESULTS: We included 337 patients, of whom 31 (9%) with subclinical vasculitis. Among those with subclinical vasculitis, 21 (58%) had isolated large vessel vasculitis, 3 (10%) had isolated cranial vasculitis and 7 (23%) had both cranial and large vessel vasculitis. The glucocorticoid (GC) starting dose and GC doses during follow-up were higher in those with subclinical vasculitis until 12 months after diagnosis (p< 0.001). There was no difference in the duration of GC treatment (25 vs 20 months, p= 0.187). Cox proportional hazard regression analyses showed no difference in the proportion of patients able to stop GC (HR 0.78 [95% CI 0.49-1.25], p= 0.303) and in the proportion of patients with relapse (HR 0.82 [95%CI 0.50-1.36], p= 0.441). CONCLUSION: Only 9% of our PMR patients had subclinical vasculitis with a predilection for large vessel vasculitis. There were no differences in relapse rate and duration of GC treatment, however those with subclinical vasculitis received higher GC doses until 12 months after diagnosis. Prospective interventional trials are needed to evaluate the outcome of PMR patients with and without subclinical vasculitis treated with similar GC protocol.

Extracorporeal Membrane Oxygenation for Acute Lung Injury in Idiopathic Inflammatory Myopathies-A Potential Lifesaving Intervention.

OBJECTIVES: Idiopathic inflammatory myopathies (IIM) can present with acute IIM-related lung injury and respiratory failure, leading to a high mortality risk in intensive care units (ICU). Extracorporeal membrane oxygenation (ECMO) in acute respiratory distress syndrome can be lifesaving. We aimed to report a case series of IIM patients that received ECMO. METHODS: Patients with IIM from tertiary care centers in Belgium, Canada, Denmark, United States, and Sweden who underwent ECMO were reviewed to describe clinical characteristics, disease outcomes and hospitalization course. Clinical characteristics at admission and during ICU stay including ECMO complications and mortality causes were summarized. RESULTS: The study included 22 patients (50% female, mean±SD age at admission 47 ± 12 years) with anti-MDA5 positive dermatomyositis (68%), anti-synthetase syndrome (14%), polymyositis (9%), overlap myositis (5%) and non-MDA5 dermatomyositis (5%). Patients had low comorbidity scores and 46% had received immunosuppression before their ICU admission. Eight (36%) patients died in the ICU, six (27%) were bridged to recovery and eight (36%) were bridged to transplant. When comparing patients bridged to recovery and those who died in the ICU, those who died were older (p= 0.03) and had higher median Charlson comorbidity index scores (p= 0.05). Both groups had similar frequencies of ECMO-related complications (33% vs 50%, p= 0.94). CONCLUSION: In the patients exposed to ECMO in this case series, 14 were successfully bridged to recovery or transplant, while 8 died in the ICU. Large studies are needed to collect data on clinical outcomes in patients with IIM-ILD exposed to ECMO to identify the best candidates for the intervention.

Biomarker analysis from the phase 2b randomized placebo-controlled trial of riociguat in early diffuse cutaneous systemic sclerosis.

OBJECTIVE: To examine disease and target engagement biomarkers in the RISE-SSc trial of riociguat in early diffuse cutaneous systemic sclerosis and their potential to predict the response to treatment. METHODS: Patients were randomized to riociguat (n = 60) or placebo (n = 61) for 52 weeks. Skin biopsies and plasma/serum samples were obtained at baseline and week 14. Plasma cyclic guanosine monophosphate (cGMP) was assessed using radio-immunoassay. Alpha smooth muscle actin (αSMA) and skin thickness were determined by immunohistochemistry, mRNA markers of fibrosis by qRT-PCR in skin biopsies, and serum CXC motif chemokine ligand 4 (CXCL-4) and soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1) by enzyme-linked immunosorbent assay. RESULTS: By week 14, cGMP increased by 94 ± 78% with riociguat and 10 ± 39% with placebo (p < 0.001, riociguat vs placebo). Serum sPECAM-1 and CXCL-4 decreased with riociguat vs placebo (p = 0.004 and p = 0.008, respectively). There were no differences in skin collagen markers between the 2 groups. Higher baseline serum sPECAM-1 or the detection of αSMA-positive cells in baseline skin biopsies were associated with a larger reduction of modified Rodnan skin score from baseline at week 52 with riociguat vs placebo (interaction P-values 0.004 and 0.02, respectively). CONCLUSION: Plasma cGMP increased with riociguat, suggesting engagement with the nitric oxide-soluble guanylate cyclase-cGMP pathway. Riociguat was associated with a significant reduction in sPECAM-1 (an angiogenic biomarker) vs placebo. Elevated sPECAM-1 and the presence of αSMA-positive skin cells may help to identify patients who could benefit from riociguat in terms of skin fibrosis. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02283762.

Mass spectrometry-based identification of new anti-Ly and known antisynthetase autoantibodies.

OBJECTIVES: To discover new and detect known antisynthetase autoantibodies (ASAs) through protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS). METHODS: IP-MS was performed using sera of individuals showing features of antisynthetase syndrome (ASyS) without (n=5) and with (n=12) previously detected ASAs, and healthy controls (n=4). New candidate aminoacyl-tRNA-synthetase (ARS) autoantigens identified through unbiased IP-MS were confirmed by IP-western blot. A targeted IP-MS assay for various ASA specificities was developed and validated with sera of patients with known ASAs (n=16), disease controls (n=20) and healthy controls (n=25). The targeted IP-MS assay was applied in an additional cohort of patients with multiple ASyS features or isolated myositis without previously detected ASAs (n=26). RESULTS: Autoantibodies to cytoplasmic cysteinyl-tRNA-synthetase (CARS1) were identified by IP-MS and confirmed by western blot as a new ASA specificity, named anti-Ly, in the serum of a patient with ASyS features. Rare ASAs, such as anti-OJ, anti-Zo and anti-KS, and common ASAs could also be identified by IP-MS. A targeted IP-MS approach for ASA detection was developed and validated. Application of this method in an additional cohort identified an additional patient with anti-OJ autoantibodies that were missed by line and dot immunoassays. DISCUSSION: CARS1 is the dominant cognate ARS autoantigen of the newly discovered anti-Ly ASA specificity. Rare and common ASA specificities could be detected by both unbiased and targeted IP-MS. Unbiased and targeted IP-MS are promising methods for discovery and detection of autoantibodies, especially autoantibodies that target complex autoantigens.

Autoantibodies against the NineTeen complex and U5 RNP in systemic sclerosis.

ObjectiveMultiple spliceosome components are known autoantigens in systemic sclerosis (SSc). Here we aim to identify new and characterize rare anti-spliceosomal autoantibodies in patients with SSc without known autoantibody specificity. MethodsSera that precipitated spliceosome subcomplexes, as detected by immunoprecipitation-mass spectrometry (IP-MS), were identified from a database of 106 patients with SSc without known autoantibody specificity. New autoantibody specificities were confirmed with immunoprecipitation-western blot. The IP-MS pattern of new anti-spliceosomal autoantibodies was compared with anti-U1 RNP-positive sera of patients with different systemic autoimmune rheumatic diseases and anti-SmD-positive sera of patients with systemic lupus erythematosus (n = 24). ResultsThe NineTeen Complex (NTC) was identified and confirmed as new spliceosomal autoantigen in one patient with SSc. U5 RNP, as well as additional splicing factors, were precipitated by the serum of another patient with SSc. The IP-MS patterns of anti-NTC and anti-U5 RNP autoantibodies were distinct from those of anti-U1 RNP- and anti-SmD-positive sera. Furthermore, there was no difference in IP-MS patterns between a limited number of anti-U1 RNP-positive sera of patients with different systemic autoimmune rheumatic diseases. ConclusionAnti-NTC autoantibodies are a new anti-spliceosomal autoantibody specificity, here first identified in a patient with SSc. Anti-U5 RNP autoantibodies are a distinct but rare anti-spliceosomal autoantibody specificity. All major spliceosomal subcomplexes have now been described as target of autoantibodies in systemic autoimmune diseases.

Identification of new telomere- and telomerase-associated autoantigens in systemic sclerosis.

PURPOSE: In up to 20% of patients with systemic sclerosis (SSc) no known autoantibody specificity can be identified. Recently discovered autoantigens, such as telomeric repeat binding factor 1 (TERF1), as well as established autoantigens, like RuvBL1/2, are associated with telomere and telomerase biology. We aimed to identify new telomere- and telomerase-associated autoantigens in patients with SSc without known autoantibody specificity. METHODS: Unlabelled protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS) was performed with sera of 106 patients with SSc from two tertiary referral centres that had a nuclear pattern on HEp-2 indirect immunofluorescence without previously identified autoantibody. Telomere- or telomerase-associated proteins or protein complexes precipitated by individual sera were identified. Candidate autoantigens were confirmed through immunoprecipitation-western blot (IP-WB). A custom Luminex xMAP assay for 5 proteins was evaluated with sera from persons with SSc (n = 467), other systemic autoimmune rheumatic diseases (n = 923), non-rheumatic disease controls (n = 187) and healthy controls (n = 199). RESULTS: Eight telomere- and telomerase-associated autoantigens were identified in a total of 11 index patients, including the THO complex (n = 3, all with interstitial lung disease and two with cardiac involvement), telomeric repeat-binding factor 2 (TERF2, n = 1), homeobox-containing protein 1 (HMBOX1, n = 2), regulator of chromosome condensation 1 (RCC1, n = 1), nucleolar and coiled-body phosphoprotein 1 (NOLC1, n = 1), dyskerin (DKC1, n = 1), probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2, n = 1) and nuclear valosin-containing protein-like (NVL, n = 2). A Luminex xMAP assay for THO complex subunit 1 (THOC1), TERF2, NOLC1, NOP2 and NVL revealed high reactivity in all index patients, but also in other patients with SSc and disease controls. However, the reactivity by xMAP assay in these other patients was not confirmed by IP-WB. CONCLUSION: IP-MS revealed key telomere- and telomerase-associated proteins and protein complexes as autoantigens in patients with SSc.

Autoantibodies against the melanoma differentiation-associated protein 5 in patients with dermatomyositis target the helicase domains.

OBJECTIVES: Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation-associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein. METHODS: We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n = 8) and serum (n = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using Western Blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains. RESULTS: All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein. CONCLUSIONS: Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade.

Clinical spectrum time course in non-Asian patients positive for anti-MDA5 antibodies.

OBJECTIVES: To define the clinical spectrum time-course and prognosis of non-Asian patients positive for anti-MDA5 antibodies. METHODS: We conducted a multicentre, international, retrospective cohort study. RESULTS: 149 anti-MDA5 positive patients (median onset age 53 years, median disease duration 18 months), mainly females (100, 67%), were included. Dermatomyositis (64, 43%) and amyopathic dermatomyositis (47, 31%), were the main diagnosis; 15 patients (10%) were classified as interstitial pneumonia with autoimmune features (IPAF) and 7 (5%) as rheumatoid arthritis. The main clinical findings observed were myositis (84, 56%), interstitial lung disease (ILD) (108, 78%), skin lesions (111, 74%), and arthritis (76, 51%). The onset of these manifestations was not concomitant in 74 cases (50%). Of note, 32 (21.5%) patients were admitted to the intensive care unit for rapidly progressive-ILD, which occurred in median 2 months from lung involvement detection, in the majority of cases (28, 19%) despite previous immunosuppressive treatment. One-third of patients (47, 32% each) was ANA and anti-ENA antibodies negative and a similar percentage was anti-Ro52 kDa antibodies positive. Non-specific interstitial pneumonia (65, 60%), organising pneumonia (23, 21%), and usual interstitial pneumonia-like pattern (14, 13%) were the main ILD patterns observed. Twenty-six patients died (17%), 19 (13%) had a rapidly progressive-ILD. CONCLUSIONS: The clinical spectrum of the anti-MDA5 antibodies-related disease is heterogeneous. Rapidly-progressive ILD deeply impacts the prognosis also in non-Asian patients, occurring early during the disease course. Anti-MDA5 antibody positivity should be considered even when baseline autoimmune screening is negative, anti-Ro52 kDa antibodies are positive, and radiology findings show a NSIP pattern.

Phenotypical and Functional Characterization of Neutrophils in Two Pyrin-Associated Auto-inflammatory Diseases.

PURPOSE: Familial Mediterranean Fever (FMF) and Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) are clinically distinct autoinflammatory disorders caused by mutations in the pyrin-encoding gene MEFV. We investigated the transcriptional, phenotypical, and functional characteristics of patient neutrophils to explore their potential role in FMF and PAAND pathophysiology. METHODS: RNA sequencing was performed to discover transcriptional aberrancies. The phenotypical features, degranulation properties, and phagocytic capacity of neutrophils were assessed by flow cytometry. Production of reactive oxygen species (ROS), myeloperoxidase (MPO) release, and chemotactic responses were investigated via chemiluminescence, ELISA, and Boyden chamber assays, respectively. RESULTS: Neutrophils from PAAND and FMF patients showed a partially overlapping, activated gene expression profile with increased expression of S100A8, S100A9, S100A12, IL-4R, CD48, F5, MMP9, and NFKB. Increased MMP9 and S100A8/A9 expression levels were accompanied by high plasma concentrations of the encoded proteins. Phenotypical analysis revealed that neutrophils from FMF patients exhibited an immature character with downregulation of chemoattractant receptors CXCR2, C5aR, and BLTR1 and increased expression of Toll-like receptor 4 (TLR4) and TLR9. PAAND neutrophils displayed an increased random, but reduced CXCL8-induced migration. A tendency for enhanced random migration was observed for FMF neutrophils. PAAND neutrophils showed a moderately but significantly enhanced phagocytic activity as opposed to neutrophils from FMF patients. Neutrophils from both patient groups showed increased MPO release and ROS production. CONCLUSIONS: Neutrophils from patients with FMF and PAAND, carrying different mutations in the MEFV gene, share a pro-inflammatory phenotype yet demonstrate diverse features, underscoring the distinction between both diseases.

Phenotypic analysis of pyrin-associated autoinflammation with neutrophilic dermatosis patients during treatment.

OBJECTIVE: In 2016 specific heterozygous gain-of-function mutations in the Mediterranean fever gene MEFV were reported as causal for a distinct autoinflammatory disease coined pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). We sought to provide an extended report on clinical manifestations in PAAND patients to date and evaluate the efficacy and safety of treatment with the IL-1-blocking agent anakinra. METHODS: We undertook an open-label pilot study with anakinra. Three patients were recruited in a preliminary phase of the study with the intention to expand the treatment cohort in case of a favourable response. Acute-phase reactants and plasma cytokine levels were monitored throughout. Skin biopsies at baseline and at week 12 were stained for relevant cytokines. Available clinical data on treatment responses were retrospectively collected on additional patients. RESULTS: The three patients from the preliminary phase of the study [patients 1-3 (P1-P3)] demonstrated one failed and two partial treatment responses, where one patient opted to continue treatment with anakinra and the other favoured adalimumab. While a partial systemic response was observed, there was no appreciable effect of anakinra on the prominent cutaneous manifestations, reflected in residual local inflammatory cytokine expression in lesional skin. These observations did not warrant further expansion of the treatment cohort. Clinical data was retrospectively collected on an additional eight patients (P4-P11), highlighting both dominant and recessive inheritance with variable penetrance in PAAND and common gastrointestinal involvement that was not previously appreciated. CONCLUSION: In our experience, while anakinra appears safe, it was not superior to biologicals targeting TNF-α in PAAND despite evidence directly implicating dysregulated IL-1ß signalling.

The clinical phenotype of systemic sclerosis patients with anti-PM/Scl antibodies: results from the EUSTAR cohort.

OBJECTIVE: To evaluate clinical associations of anti-PM/Scl antibodies in patients with SSc in a multicentre international cohort, with particular focus on unresolved issues, including scleroderma renal crisis (RC), malignancies, and functional outcome of interstitial lung disease (ILD). METHODS: (1) Analysis of SSc patients from the EUSTAR database: 144 anti-PM/Scl+ without SSc-specific autoantibodies were compared with 7202 anti-PM/Scl-, and then to 155 anti-Pm/Scl+ with SSc-specific antibodies. (2) Case-control study: additional data were collected for 165 anti-PM/Scl+ SSc patients (85 from the EUSTAR registry) and compared with 257 anti-PM/Scl- SSc controls, matched for sex, cutaneous subset, disease duration and age at SSc onset. RESULTS: Patients with isolated anti-PM/Scl+, as compared with anti-Pm/Scl-, had higher frequency of muscle involvement, ILD, calcinosis and cutaneous signs of DM, but similar frequency of SRC and malignancies (either synchronous with SSc onset or not). The presence of muscle involvement was associated with a more severe disease phenotype. Although very frequent, ILD had a better functional outcome in cases than in controls. In patients with both anti-PM/Scl and SSc-specific antibodies, a higher frequency of typical SSc features than in those with isolated anti-PM/Scl was observed. CONCLUSION: The analysis of the largest series of anti-PM/Scl+ SSc patients so far reported helps to delineate a specific clinical subset with muscle involvement, cutaneous DM, calcinosis and ILD characterized by a good functional outcome. SRC and malignancies do not seem to be part of this syndrome.

Can We Expect Any Effect of Rituximab on Fatigue in Primary Sjögren Syndrome?: A Systematic Review and Critical Appraisal.

INTRODUCTION: Fatigue is a major determinant of impaired quality of life in primary Sjögren syndrome (pSS) patients. Effective therapeutic strategies are lacking. OBJECTIVES: To review the potential benefit of rituximab, a chimeric anti-CD20 antibody, in the treatment of fatigue in pSS. METHODS: A systematic review on the effect of rituximab on fatigue-related outcome measures was conducted, retrieving evidence from CENTRAL (Cochrane Central Register of Controlled Trials), MEDLINE (via PubMed), EMBASE, and Scopus. RESULTS: No benefit of rituximab over placebo on any fatigue-related outcome measure could be demonstrated in the included trials. Significant effects were only observed when compared with baseline, but not when compared with placebo. CONCLUSIONS: The use of rituximab for the treatment of pSS-related fatigue cannot be supported by the currently available evidence.

Genome-wide whole blood transcriptome profiling in a large European cohort of systemic sclerosis patients.

OBJECTIVES: The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. METHODS: Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. RESULTS: Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. CONCLUSIONS: We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc.

Riociguat in patients with early diffuse cutaneous systemic sclerosis (RISE-SSc): randomised, double-blind, placebo-controlled multicentre trial.

OBJECTIVES: Riociguat is approved for pulmonary arterial hypertension and has antiproliferative, anti-inflammatory and antifibrotic effects in animal models of tissue fibrosis. We evaluated the efficacy and safety of riociguat in patients with early diffuse cutaneous systemic sclerosis (dcSSc) at high risk of skin fibrosis progression. METHODS: In this randomised, double-blind, placebo-controlled, phase IIb trial, adults with dcSSc of <18 months' duration and a modified Rodnan skin score (mRSS) 10-22 units received riociguat 0.5 mg to 2.5 mg orally three times daily (n=60) or placebo (n=61). The primary endpoint was change in mRSS from baseline to week 52. RESULTS: At week 52, change from baseline in mRSS units was -2.09±5.66 (n=57) with riociguat and -0.77±8.24 (n=52) with placebo (difference of least squares means -2.34 (95% CI -4.99 to 0.30; p=0.08)). In patients with interstitial lung disease, forced vital capacity declined by 2.7% with riociguat and 7.6% with placebo. At week 14, average Raynaud's condition score had improved ≥50% in 19 (41.3%)/46 patients with riociguat and 13 (26.0%)/50 patients with placebo. Safety assessments showed no new signals with riociguat and no treatment-related deaths. CONCLUSIONS: Riociguat did not significantly benefit mRSS versus placebo at the predefined p<0.05. Secondary and exploratory analyses showed potential efficacy signals that should be tested in further trials. Riociguat was well tolerated.

Anti-TIF1-γ autoantibodies: warning lights of a tumour autoantigen.

Anti-transcription intermediary factor 1 (TIF1)-γ autoantibodies are robustly linked with cancer-associated DM in adults. This review aims to give an overview of the physiological context of TIF1-γ and to determine whether there is a pathophysiological link between anti-TIF1-γ autoantibodies and the occurrence of cancer. Detection of anti-TIF1-γ autoantibodies has a high sensitivity and specificity for cancer-associated DM in adults and is therefore useful for both diagnosis and cancer risk stratification. The function of the autoantigen, TIF1-γ, may provide insight into the mechanism behind this association. TIF1-γ is a ubiquitously present protein involved in various biological pathways, including TGF-ß signalling. In cancer, it can act either as a tumour suppressor or promoter, depending on the cellular context and cancer stage. Evolving data provide pathophysiological insights, linking anti-TIF1-γ autoantibodies to both the anti-tumour response and to muscle and skin damage. TIF1-γ expression is increased in muscle and skin tissue of patients with DM. Mutations or loss-of-heterozygosity in TIF1-γ alleles in malignant tissue may result in the expression of tumour-specific neo-antigens stimulating autoantibody production. The newly formed autoantibodies are hypothesized to cross-react with antigens in muscle and skin, driving the development of DM. Based on the current evidence, anti-TIF1-γ autoantibodies should be considered warning lights of a potential tumour autoantigen and should alert the physician to the possibility of an underlying cancer.

Revised European Scleroderma Trials and Research Group Activity Index is the best predictor of short-term severity accrual.

BACKGROUND: The European Scleroderma Trials and Research Group (EUSTAR) recently developed a preliminarily revised activity index (AI) that performed better than the European Scleroderma Study Group Activity Index (EScSG-AI) in systemic sclerosis (SSc). OBJECTIVE: To assess the predictive value for short-term disease severity accrual of the EUSTAR-AI, as compared with those of the EScSG-AI and of known adverse prognostic factors. METHODS: Patients with SSc from the EUSTAR database with a disease duration from the onset of the first non-Raynaud sign/symptom ≤5 years and a baseline visit between 2003 and 2014 were first extracted. To capture the disease activity variations over time, EUSTAR-AI and EScSG-AI adjusted means were calculated. The primary outcome was disease progression defined as a Δ≥1 in the Medsger's severity score and in distinct items at the 2-year follow-up visit. Logistic regression analysis was carried out to identify predictive factors. RESULTS: 549 patients were enrolled. At multivariate analysis, the EUSTAR-AI adjusted mean was the only predictor of any severity accrual and of that of lung and heart, skin and peripheral vascular disease over 2 years. CONCLUSION: The adjusted mean EUSTAR-AI has the best predictive value for disease progression and development of severe organ involvement over time in SSc.

Outcomes of patients with systemic sclerosis treated with rituximab in contemporary practice: a prospective cohort study.

OBJECTIVE: To assess the safety and efficacy of rituximab in systemic sclerosis (SSc) in clinical practice. METHODS: We performed a prospective study including patients with SSc from the European Scleroderma Trials and Research (EUSTAR) network treated with rituximab and matched with untreated patients with SSc. The main outcomes measures were adverse events, skin fibrosis improvement, lung fibrosis worsening and steroids use among propensity score-matched patients treated or not with rituximab. RESULTS: 254 patients were treated with rituximab, in 58% for lung and in 32% for skin involvement. After a median follow-up of 2 years, about 70% of the patients had no side effect. Comparison of treated patients with 9575 propensity-score matched patients showed that patients treated with rituximab were more likely to have skin fibrosis improvement (22.7 vs 14.03 events per 100 person-years; OR: 2.79 [1.47-5.32]; p=0.002). Treated patients did not have significantly different rates of decrease in forced vital capacity (FVC)>10% (OR: 1.03 [0.55-1.94]; p=0.93) nor in carbon monoxide diffusing capacity (DLCO) decrease. Patients having received rituximab were more prone to stop or decrease steroids (OR: 2.34 [1.56-3.53], p<0.0001). Patients treated concomitantly with mycophenolate mofetil had a trend for better outcomes as compared with patients receiving rituximab alone (delta FVC: 5.22 [0.83-9.62]; p=0.019 as compared with controls vs 3 [0.66-5.35]; p=0.012). CONCLUSION: Rituximab use was associated with a good safety profile in this large SSc-cohort. Significant change was observed on skin fibrosis, but not on lung. However, the limitation is the observational design. The potential stabilisation of lung fibrosis by rituximab has to be addressed by a randomised trial.