Computer vision applied to dual-energy computed tomography images for precise calcinosis cutis quantification in patients with systemic sclerosis.

BACKGROUND: Although treatments have been proposed for calcinosis cutis (CC) in patients with systemic sclerosis (SSc), a standardized and validated method for CC burden quantification is necessary to enable valid clinical trials. We tested the hypothesis that computer vision applied to dual-energy computed tomography (DECT) finger images is a useful approach for precise and accurate CC quantification in SSc patients. METHODS: De-identified 2-dimensional (2D) DECT images from SSc patients with clinically evident lesser finger CC lesions were obtained. An expert musculoskeletal radiologist confirmed accurate manual segmentation (subtraction) of the phalanges for each image as a gold standard, and a U-Net Convolutional Neural Network (CNN) computer vision model for segmentation of healthy phalanges was developed and tested. A validation study was performed in an independent dataset whereby two independent radiologists manually measured the longest length and perpendicular short axis of each lesion and then calculated an estimated area by assuming the lesion was elliptical using the formula long axis/2 × short axis/2 × π, and a computer scientist used a region growing technique to calculate the area of CC lesions. Spearman's correlation coefficient, Lin's concordance correlation coefficient with 95% confidence intervals (CI), and a Bland-Altman plot (Stata V 15.1, College Station, TX) were used to test for equivalence between the radiologists' and the CNN algorithm-generated area estimates. RESULTS: Forty de-identified 2D DECT images from SSc patients with clinically evident finger CC lesions were obtained and divided into training (N = 30 with image rotation × 3 to expand the set to N = 120) and test sets (N = 10). In the training set, five hundred epochs (iterations) were required to train the CNN algorithm to segment phalanges from adjacent CC, and accurate segmentation was evaluated using the ten held-out images. To test model performance, CC lesional area estimates calculated by two independent radiologists and a computer scientist were compared (radiologist 1 vs. radiologist 2 and radiologist 1 vs. computer vision approach) using an independent test dataset comprised of 31 images (8 index finger and 23 other fingers). For the two radiologists', and the radiologist vs. computer vision measurements, Spearman's rho was 0.91 and 0.94, respectively, both p < 0.0001; Lin's concordance correlation coefficient was 0.91 (95% CI 0.85-0.98, p < 0.001) and 0.95 (95% CI 0.91-0.99, p < 0.001); and Bland-Altman plots demonstrated a mean difference between radiologist vs. radiologist, and radiologist vs. computer vision area estimates of - 0.5 mm2 (95% limits of agreement - 10.0-9.0 mm2) and 1.7 mm2 (95% limits of agreement - 6.0-9.5 mm2, respectively. CONCLUSIONS: We demonstrate that CNN quantification has a high degree of correlation with expert radiologist measurement of finger CC area measurements. Future work will include segmentation of 3-dimensional (3D) images for volumetric and density quantification, as well as validation in larger, independent cohorts.

Large-Scale Characterization of Systemic Sclerosis Serum Protein Profile: Comparison to Peripheral Blood Cell Transcriptome and Correlations With Skin/Lung Fibrosis.

OBJECTIVE: To provide a large-scale assessment of serum protein dysregulation in diffuse cutaneous systemic sclerosis (dcSSc) and to investigate serum protein correlates of SSc fibrotic features. METHODS: We investigated serum protein profiles of 66 participants with dcSSc at baseline who were enrolled in the Scleroderma: Cyclophosphamide or Transplant Trial and 66 age- and sex-matched healthy control subjects. A panel of 230 proteins, including several cytokines and chemokines, was investigated. Whole blood gene expression profiling in concomitantly collected samples was performed. RESULTS: Among the participants with dcSSc, the mean disease duration was 2.3 years. All had interstitial lung disease (ILD), and none were being treated with immunosuppressive agents at baseline. Ninety proteins were differentially expressed in participants with dcSSc compared to healthy control subjects. Similar to previous global skin transcript results, hepatic fibrosis, granulocyte and agranulocyte adhesion, and diapedesis were the top overrepresented pathways. Eighteen proteins correlated with the modified Rodnan skin thickness score (MRSS). Soluble epidermal growth factor receptor was significantly down-regulated in dcSSc and showed the strongest negative correlation with the MRSS, being predictive of the score's course over time, whereas α1 -antichymotrypsin was significantly up-regulated in dcSSc and showed the strongest positive correlation with the MRSS. Furthermore, higher levels of cancer antigen 15-3 correlated with more severe ILD, based on findings of reduced forced vital capacity and higher scores of disease activity on high-resolution computed tomography. Only 14 genes showed significant differential expression in the same direction in serum protein and whole blood RNA gene expression analyses. CONCLUSION: Diffuse cutaneous SSc has a distinct serum protein profile with prominent dysregulation of proteins related to fibrosis and immune cell adhesion/diapedesis. The differential expression for most serum proteins in SSc is likely to originate outside the peripheral blood cells.

Performance Characteristics of Pulmonary Function Tests for the Detection of Interstitial Lung Disease in Adults With Early Diffuse Cutaneous Systemic Sclerosis.

OBJECTIVE: Interstitial lung disease (ILD) is the leading cause of death in patients with systemic sclerosis (SSc). Although pulmonary function tests (PFTs) are commonly used to screen for ILD in patients with SSc, studies have shown that they lack sensitivity for the detection of ILD in general SSc cohorts. This study was undertaken to assess the performance characteristics of PFTs for the detection of ILD in patients with early diffuse cutaneous SSc (dcSSc), a population at high risk for the development of ILD. METHODS: We performed a retrospective cohort study of patients enrolled in the Prospective Registry of Early Systemic Sclerosis at 11 sites in the US between April 2012 and January 2019. Patients were included if they underwent spirometry and high-resolution computed tomography (HRCT) of the chest. We calculated the performance characteristics of PFTs for the detection of ILD on HRCT. RESULTS: The study included 212 patients, 54% of whom had radiographic ILD. For the detection of ILD on HRCT imaging, a forced vital capacity (FVC) <80% predicted had a sensitivity of 63%. The combination of FVC <80% predicted or diffusing capacity for carbon monoxide (DLco) <80% predicted improved the sensitivity to 85%. An FVC <80% predicted had a negative predictive value (NPV) of 61%, while the combination of FVC <80% predicted or DLco <80% predicted had an NPV of 70%. CONCLUSION: PFTs alone are an inadequate screening tool for the diagnosis of ILD in patients with early dcSSc. HRCT should be part of the ILD screening algorithm in patients with dcSSc.

Global skin gene expression analysis of early diffuse cutaneous systemic sclerosis shows a prominent innate and adaptive inflammatory profile.

OBJECTIVES: Determine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease. METHODS: Skin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic Sclerosis (PRESS) cohort (mean disease duration 1.3 years) and 33 matched healthy controls was examined by next-generation RNA sequencing. Data were analysed for cell type-specific signatures and compared with similarly obtained data from 55 previously biopsied patients in Genetics versus Environment in Scleroderma Outcomes Study cohort with longer disease duration (mean 7.4 years) and their matched controls. Correlations with histological features and clinical course were also evaluated. RESULTS: SSc patients in PRESS had a high prevalence of M2 (96%) and M1 (94%) macrophage and CD8 T cell (65%), CD4 T cell (60%) and B cell (69%) signatures. Immunohistochemical staining of immune cell markers correlated with the gene expression-based immune cell signatures. The prevalence of immune cell signatures in early diffuse SSc patients was higher than in patients with longer disease duration. In the multivariable model, adaptive immune cell signatures were significantly associated with shorter disease duration, while fibroblast and macrophage cell type signatures were associated with higher modified Rodnan Skin Score (mRSS). Immune cell signatures also correlated with skin thickness progression rate prior to biopsy, but did not predict subsequent mRSS progression. CONCLUSIONS: Skin in early diffuse SSc has prominent innate and adaptive immune cell signatures. As a prominently affected end organ, these signatures reflect the preceding rate of disease progression. These findings could have implications in understanding SSc pathogenesis and clinical trial design.

Myeloablation followed by autologous stem cell transplantation normalises systemic sclerosis molecular signatures.

OBJECTIVE: In the randomised scleroderma: Cyclophosphamide Or Transplantation (SCOT trial) (NCT00114530), myeloablation, followed by haematopoietic stem cell transplantation (HSCT), led to improved clinical outcomes compared with monthly cyclophosphamide (CYC) treatment in systemic sclerosis (SSc). Herein, the study aimed to determine global molecular changes at the whole blood transcript and serum protein levels ensuing from HSCT in comparison to intravenous monthly CYC in 62 participants enrolled in the SCOT study. METHODS: Global transcript studies were performed at pretreatment baseline, 8 months and 26 months postrandomisation using Illumina HT-12 arrays. Levels of 102 proteins were measured in the concomitantly collected serum samples. RESULTS: At the baseline visit, interferon (IFN) and neutrophil transcript modules were upregulated and the cytotoxic/NK module was downregulated in SSc compared with unaffected controls. A paired comparison of the 26 months to the baseline samples revealed a significant decrease of the IFN and neutrophil modules and an increase in the cytotoxic/NK module in the HSCT arm while there was no significant change in the CYC control arm. Also, a composite score of correlating serum proteins with IFN and neutrophil transcript modules, as well as a multilevel analysis showed significant changes in SSc molecular signatures after HSCT while similar changes were not observed in the CYC arm. Lastly, a decline in the IFN and neutrophil modules was associated with an improvement in pulmonary forced vital capacity and an increase in the cytotoxic/NK module correlated with improvement in skin score. CONCLUSION: HSCT contrary to conventional treatment leads to a significant 'correction' in disease-related molecular signatures.

Risk Factors for Mortality and Cardiopulmonary Hospitalization in Systemic Sclerosis Patients At Risk for Pulmonary Hypertension, in the PHAROS Registry.

OBJECTIVE: We sought to identify predictors of mortality and cardiopulmonary hospitalizations in patients at risk for pulmonary hypertension (PH) and enrolled in PHAROS, a prospective cohort study to investigate the natural history of PH in systemic sclerosis (SSc). METHODS: The at-risk population for PH was defined by the following entry criteria: echocardiogram systolic pulmonary arterial pressure > 40 mmHg, or DLCO < 55% predicted or ratio of % forced vital capacity/%DLCO > 1.6, measured by pulmonary function testing. Baseline clinical measures were evaluated as predictors of hospitalization and death between 2005 and 2014. Cox proportional hazards models were censored at date of PH onset or latest study visit and adjusted for age, sex, race, and disease duration. RESULTS: Of the 236 at-risk subjects who were followed for a median of 4 years (range 0.4-8.5 yrs), 35 developed PH after entering PHAROS (reclassified as PH group). In the at-risk group, higher mortality was strongly associated with male sex, low %DLCO, exercise oxygen desaturation, anemia, abnormal dyspnea scores, and baseline pericardial effusion. Risks for cardiopulmonary hospitalization were associated with increased dyspnea and pericardial effusions, although PH patients with DLCO < 50% had the highest risk of cardiopulmonary hospitalizations. CONCLUSION: Risk factors for poor outcome in patients with SSc who are at risk for PH were similar to others with SSc-PH and SSc-pulmonary arterial hypertension, including male sex, DLCO < 50%, exercise oxygen desaturation, and pericardial effusions. This group should undergo right heart catheterization and receive appropriate intervention if PH is confirmed.

Diffuse cardiac fibrosis quantification in early systemic sclerosis by magnetic resonance imaging and correlation with skin fibrosis.

PURPOSE: To evaluate the utility of cardiac magnetic resonance (CMR) T1 mapping in early systemic sclerosis (SSc) and its association with skin score. METHODS: Twenty-four consecutive patients with early SSc referred for cardiovascular evaluation and 12 controls without SSc were evaluated. All patients underwent cine, T1 mapping, and late gadolinium enhanced (LGE) CMR imaging. T1 mapping indices were compared between SSc patients and controls (extracellular volume fraction [ECV], gadolinium partition coefficient [λ], pre-contrast T1, and post-contrast T1). The association between T1 mapping parameters and the modified Rodnan skin score (mRSS) was determined. RESULTS: There were no significant differences in cardiac structure/function between SSc patients and controls on cine imaging, and 8/24 (33%) SSc patients had evidence of LGE (i.e., focal myocardial fibrosis). Of the T1 mapping parameters (indices indicative of diffuse myocardial fibrosis), ECV differentiated SSc patients from controls the best, followed by λ, even when the eight SSc patients with LGE were excluded. ECV had a sensitivity and specificity of 75% and 75% for diffuse myocardial fibrosis (optimal abnormal cut-off value of >27% [area under ROC curve=0.85]). In the 16 patients without evidence of LGE, each of the 4 CMR T1 mapping parameters (ECV, λ, Pre-T1 and Post-T1) correlated with mRSS (R=0.51-0.65, P=0.007-0.043), indicating a correlation between SSc cardiac and skin fibrosis. CONCLUSIONS: The four T1 mapping indices are significantly correlated with mRSS in patients with early SSc. Quantification of diffuse myocardial fibrosis using ECV should be considered as a marker for cardiac involvement in SSc clinical studies.

A novel multi-network approach reveals tissue-specific cellular modulators of fibrosis in systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a multi-organ autoimmune disease characterized by skin fibrosis. Internal organ involvement is heterogeneous. It is unknown whether disease mechanisms are common across all involved affected tissues or if each manifestation has a distinct underlying pathology. METHODS: We used consensus clustering to compare gene expression profiles of biopsies from four SSc-affected tissues (skin, lung, esophagus, and peripheral blood) from patients with SSc, and the related conditions pulmonary fibrosis (PF) and pulmonary arterial hypertension, and derived a consensus disease-associate signature across all tissues. We used this signature to query tissue-specific functional genomic networks. We performed novel network analyses to contrast the skin and lung microenvironments and to assess the functional role of the inflammatory and fibrotic genes in each organ. Lastly, we tested the expression of macrophage activation state-associated gene sets for enrichment in skin and lung using a Wilcoxon rank sum test. RESULTS: We identified a common pathogenic gene expression signature-an immune-fibrotic axis-indicative of pro-fibrotic macrophages (MØs) in multiple tissues (skin, lung, esophagus, and peripheral blood mononuclear cells) affected by SSc. While the co-expression of these genes is common to all tissues, the functional consequences of this upregulation differ by organ. We used this disease-associated signature to query tissue-specific functional genomic networks to identify common and tissue-specific pathologies of SSc and related conditions. In contrast to skin, in the lung-specific functional network we identify a distinct lung-resident MØ signature associated with lipid stimulation and alternative activation. In keeping with our network results, we find distinct MØ alternative activation transcriptional programs in SSc-associated PF lung and in the skin of patients with an "inflammatory" SSc gene expression signature. CONCLUSIONS: Our results suggest that the innate immune system is central to SSc disease processes but that subtle distinctions exist between tissues. Our approach provides a framework for examining molecular signatures of disease in fibrosis and autoimmune diseases and for leveraging publicly available data to understand common and tissue-specific disease processes in complex human diseases.

Mycophenolate mofetil versus oral cyclophosphamide in scleroderma-related interstitial lung disease (SLS II): a randomised controlled, double-blind, parallel group trial.

BACKGROUND: 12 months of oral cyclophosphamide has been shown to alter the progression of scleroderma-related interstitial lung disease when compared with placebo. However, toxicity was a concern and without continued treatment the efficacy disappeared by 24 months. We hypothesised that a 2 year course of mycophenolate mofetil would be safer, better tolerated, and produce longer lasting improvements than cyclophosphamide. METHODS: This randomised, double-blind, parallel group trial enrolled patients from 14 US medical centres with scleroderma-related interstitial lung disease meeting defined dyspnoea, pulmonary function, and high-resolution CT (HRCT) criteria. The data coordinating centre at the University of California, Los Angeles (UCLA, CA, USA), randomly assigned patients using a double-blind, double-dummy, centre-blocked design to receive either mycophenolate mofetil (target dose 1500 mg twice daily) for 24 months or oral cyclophosphamide (target dose 2·0 mg/kg per day) for 12 months followed by placebo for 12 months. Drugs were given in matching 250 mg gel capsules. The primary endpoint, change in forced vital capacity as a percentage of the predicted normal value (FVC %) over the course of 24 months, was assessed in a modified intention-to-treat analysis using an inferential joint model combining a mixed-effects model for longitudinal outcomes and a survival model to handle non-ignorable missing data. The study was registered with ClinicalTrials.gov, number NCT00883129. FINDINGS: Between Sept 28, 2009, and Jan 14, 2013, 142 patients were randomly assigned to either mycophenolate mofetil (n=69) or cyclophosphamide (n=73). 126 patients (mycophenolate mofetil [n=63] and cyclophosphamide [n=63]) with acceptable baseline HRCT studies and at least one outcome measure were included in the primary analysis. The adjusted % predicted FVC improved from baseline to 24 months by 2·19 in the mycophenolate mofetil group (95% CI 0·53-3·84) and 2·88 in the cyclophosphamide group (1·19-4·58). The course of the % FVC did not differ significantly between the two treatment groups based on the prespecified primary analysis using a joint model (p=0·24), indicating that the trial was negative for the primary endpoint. However, in a post-hoc analysis of the primary endpoint, the within-treatment change from baseline to 24 months derived from the joint model showed that the % FVC improved significantly in both the mycophenolate mofetil and cyclophosphamide groups. 16 (11%) patients died (five [7%] mycophenolate mofetil and 11 [15%] cyclophosphamide), with most due to progressive interstitial lung disease. Leucopenia (30 patients vs four patients) and thrombocytopenia (four vs zero) occurred more often in patients given cyclophosphamide than mycophenolate mofetil. Fewer patients on mycophenolate mofetil than on cyclophosphamide prematurely withdrew from study drug (20 vs 32) or met prespecified criteria for treatment failure (zero vs two). The time to stopping treatment was shorter in the cyclophosphamide group (p=0·019). INTERPRETATION: Treatment of scleroderma-related interstitial lung disease with mycophenolate mofetil for 2 years or cyclophosphamide for 1 year both resulted in significant improvements in prespecified measures of lung function over the 2 year course of the study. Although mycophenolate mofetil was better tolerated and associated with less toxicity, the hypothesis that it would have greater efficacy at 24 months than cyclophosphamide was not confirmed. These findings support the potential clinical effectiveness of both cyclophosphamide and mycophenolate mofetil for progressive scleroderma-related interstitial lung disease, and the present preference for mycophenolate mofetil because of its better tolerability and toxicity profile. FUNDING: National Heart, Lung and Blood Institute, National Institutes of Health; with drug supply provided by Hoffmann-La Roche and Genentech.

Esophageal dilatation and interstitial lung disease in systemic sclerosis: A cross-sectional study.

OBJECTIVE: A patulous esophagus on high-resolution computed tomography (HRCT) of the thorax is frequently observed in patients with systemic sclerosis (SSc). Microaspiration has been purported to play a role in the development and progression of SSc interstitial lung disease (ILD), but studies examining the role of microaspiration in SSc ILD have yielded conflicting results. This study was conducted to determine the association between esophageal diameter and SSc ILD. METHODS: A cross-sectional study of Northwestern Scleroderma Registry patients with available HRCT exams was conducted. The predictor variable was the widest esophageal diameter (WED) on HRCT, and the primary and secondary outcome variables were radiographic ILD and pulmonary function tests respectively. The degree of radiographic ILD was assessed using a semi-quantitative score adapted from published methods. Estimated regression coefficients adjusted for age, sex, race, body mass index, smoking; SSc disease subtype, serum autoantibodies, and disease duration; modified Rodnan skin score, proton pump inhibitor, and immune suppressant medication use and erythrocyte sedimentation rate were calculated. RESULTS: A total of 270 subjects were studied. In the adjusted analyses, there were positive associations between WED and total ILD score (ß = 0.27; 95% CI: 0.09-0.41), fibrosis (ß = 0.15; 95% CI: 0.07-0.23), and ground glass opacities (ß = 0.12; 95% CI: 0.04-0.20); there were negative associations between WED and FVC % predicted (ß = -0.42; 95% CI: -0.69 to -0.13), and adjusted DLCO % predicted (ß = -0.45; 95% CI: -0.80 to -0.09) after adjusting for potential confounders. CONCLUSIONS: Increasing esophageal diameter on HRCT in patients with SSc is associated with more severe radiographic ILD, lower lung volumes, and lower DLCO % predicted. Longitudinal studies are needed to determine if esophageal dilatation is associated with the incidence and/or progression of ILD in patients with SSc.

The Histone Deacetylase Sirtuin 1 Is Reduced in Systemic Sclerosis and Abrogates Fibrotic Responses by Targeting Transforming Growth Factor ß Signaling.

OBJECTIVE: Persistent fibroblast activation underlies skin fibrosis in systemic sclerosis (SSc), but the transcriptional and epigenetic mechanisms controlling this process are not well understood. In view of the potent influence of acetylation status governing tissue fibrosis, we undertook this study to investigate the expression of the antiaging deacetylase enzyme sirtuin 1 (SIRT1) in SSc and its effects on fibrotic responses in vitro and in vivo. METHODS: Tissue expression of SIRTs was interrogated from publicly available genome-wide expression data sets and by immunohistochemistry. The effects of SIRT1 on modulating fibrotic responses, as well as the underlying mechanisms, were examined in human and mouse fibroblasts in culture and in an experimental fibrosis model in the mouse. RESULTS: Analysis of transcriptome data revealed a selective reduction of SIRT1 messenger RNA (mRNA) levels in SSc skin biopsy samples as well as a negative correlation of SIRT1 mRNA with the skin score. Cellular SIRT1 levels were suppressed in normal fibroblasts exposed to hypoxia or platelet-derived growth factor and were constitutively down-regulated in SSc fibroblasts. Activation of SIRT1 attenuated fibrotic responses in skin fibroblasts and skin organ cultures, while genetic or pharmacologic inhibition of SIRT1 had profibrotic effects. The antifibrotic effects of SIRT1 were due in part to decreased expression and function of the acetyltransferase p300. In mice, experimentally induced skin fibrosis was accompanied by reduced SIRT1 expression in lesional tissue fibroblasts, and both fibrosis and loss of SIRT1 in these mice were mitigated by treatment with a SIRT1 activator. CONCLUSION: SIRT1 has antifibrotic effects, and its reduced tissue expression in patients with SSc might have a direct causal role in progression of fibrosis. Pharmacologic modulation of SIRT1 in these patients therefore might represent a potential treatment strategy.

Systems level analysis of systemic sclerosis shows a network of immune and profibrotic pathways connected with genetic polymorphisms.

Systemic sclerosis (SSc) is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited) are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6-12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA) genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected and related to a patients underlying genetic risk.

Experimentally-derived fibroblast gene signatures identify molecular pathways associated with distinct subsets of systemic sclerosis patients in three independent cohorts.

Genome-wide expression profiling in systemic sclerosis (SSc) has identified four 'intrinsic' subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. Here we examine experimentally derived gene expression signatures in dermal fibroblasts for thirteen different signaling pathways implicated in SSc pathogenesis. These data show distinct and overlapping sets of genes induced by each pathway, allowing for a better understanding of the molecular relationship between profibrotic and immune signaling networks. Pathway-specific gene signatures were analyzed across a compendium of microarray datasets consisting of skin biopsies from three independent cohorts representing 80 SSc patients, 4 morphea, and 26 controls. IFNα signaling showed a strong association with early disease, while TGFß signaling spanned the fibroproliferative and inflammatory subsets, was associated with worse MRSS, and was higher in lesional than non-lesional skin. The fibroproliferative subset was most strongly associated with PDGF signaling, while the inflammatory subset demonstrated strong activation of innate immune pathways including TLR signaling upstream of NF-κB. The limited and normal-like subsets did not show associations with fibrotic and inflammatory mediators such as TGFß and TNFα. The normal-like subset showed high expression of genes associated with lipid signaling, which was absent in the inflammatory and limited subsets. Together, these data suggest a model by which IFNα is involved in early disease pathology, and disease severity is associated with active TGFß signaling.

Longitudinal evaluation of PROMIS-29 and FACIT-dyspnea short forms in systemic sclerosis.

OBJECTIVE: To assess the sensitivity of the Patient-Reported Outcomes Measurement Information System 29-item Health Profile (PROMIS-29) and the Functional Assessment of Chronic Illness Therapy-Dyspnea 10-item short form (FACIT-Dyspnea) for measuring change in health status and dyspnea in systemic sclerosis (SSc). METHODS: One hundred patients with SSc completed the PROMIS-29, FACIT-Dyspnea, and traditional instruments [Medical Research Council Dyspnea Score, St. George's Respiratory Questionnaire (SGRQ), Health Assessment Questionnaire-Disability Index (HAQ-DI), and Medical Outcomes Study Short Form-36 (SF-36)] at baseline and 1-year visits. PROMIS-29, FACIT-Dyspnea, and traditional instrument change scores were compared across composite modified Medsger Disease Severity and modified Rodnan Skin score (mRSS) change groups. RESULTS: Moderately high Spearman correlation coefficients were observed between FACIT-Dyspnea and SGRQ (r = 0.57), FACIT-Dyspnea functional limitations and SF-36 physical component summary (PCS; r = 0.51), PROMIS-29 physical functioning and HAQ-DI (r = 0.50), and SF-36 PCS (r = 0.52) change scores. In most validity comparisons, PROMIS-29, FACIT-Dyspnea, HAQ-DI, and SF-36 scores performed similarly. While PROMIS-29 covers more content areas than SF-36 (e.g., sleep), it may do so at the expense of responsiveness of its 4-item physical function scale as compared to the multiitem-derived SF-36 PCS. Statistically significant increases in SF-36 role physical (p = 0.01) and physical component scale (p = 0.016), but not PROMIS-29, were observed in patients with mRSS improvement. CONCLUSION: PROMIS-29 and FACIT-Dyspnea are valid instruments to measure health status and dyspnea in patients with SSc. In physical function assessment, longer PROMIS short forms or computer adaptive testing should be considered to improve responsiveness to the effect of skin disease changes on physical function in patients with SSc.

Development of pulmonary hypertension in a high-risk population with systemic sclerosis in the Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma (PHAROS) cohort study.

OBJECTIVES: PHAROS registry is a prospective longitudinal cohort study to understand the natural history of pulmonary hypertension (PH) in systemic sclerosis (SSc). METHODS: "At-risk" pulmonary arterial hypertension (PAH) is defined by these entry criteria: echocardiogram (echo) systolic pulmonary arterial pressure (sPAP) >40 mmHg, diffusion lung capacity of carbon monoxide (DLco) <55% predicted, or ratio of percentage forced vital capacity (FVC)/percentage DLco >1.6, as measured by pulmonary function testing (PFT). Patients were followed up annually and right heart catheterization (RHC) performed if PH was suspected. We used descriptive statistics and Kaplan-Meier estimate of time to PH diagnosis. RESULTS: A total of 251 "at-risk" subjects were enrolled between 2005 and 2012 and followed up for mean of 2.5 ± 1.2 years. The mean age at entry was 56.7 ± 11.0 and disease duration was 9.9 ± 8.7 years. Overall, 82 patients had RHC, and 35 were confirmed to have new PH. There were no differences in age, gender, SSc subtypes, antibodies, and disease duration between the "at-risk" and new PH groups. Using Kaplan-Meier survival, the time to PH was 10% at 2 years, 13% at 3 years, and 25% at 5 years. Most new PH patients at entry met the PFT criteria (76%), had significantly higher sPAP (p = 0.013), had shorter 6-min walk distance, and had exercise-induced hypoxia (p = 0.003) than "at-risk" PAH group. CONCLUSIONS: A low DLco, high FVC/DLco, exercise-induced hypoxia and entry echo sPAP > 40 were strongly associated with future PH, though RHC was necessary to confirm PH. This ongoing prospective study confirms that these high-risk factors do predict future PH.

Immunochip analysis identifies multiple susceptibility loci for systemic sclerosis.

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci.

Early growth response 3 (Egr-3) is induced by transforming growth factor-ß and regulates fibrogenic responses.

Members of the early growth response (Egr) gene family of transcription factors have nonredundant biological functions. Although Egr-3 is implicated primarily in neuromuscular development and immunity, its regulation and role in tissue repair and fibrosis has not been studied. We now show that in normal skin fibroblasts, Egr-3 was potently induced by transforming growth factor-ß via canonical Smad3. Moreover, transient Egr-3 overexpression was sufficient to stimulate fibrotic gene expression, whereas deletion of Egr-3 resulted in substantially attenuated transforming growth factor-ß responses. Genome-wide expression profiling in fibroblasts showed that genes associated with tissue remodeling and wound healing were prominently up-regulated by Egr-3. Notably, <5% of fibroblast genes regulated by Egr-1 or Egr-2 were found to be coregulated by Egr-3, revealing substantial functional divergence among these Egr family members. In a mouse model of scleroderma, development of dermal fibrosis was accompanied by accumulation of Egr-3-positive myofibroblasts in the lesional tissue. Moreover, skin biopsy samples from patients with scleroderma showed elevated Egr-3 levels in the dermis, and Egr-3 mRNA levels correlated with the extent of skin involvement. These results provide the first evidence that Egr-3, a functionally distinct member of the Egr family with potent effects on inflammation and immunity, is up-regulated in scleroderma and is necessary and sufficient for profibrotic responses, suggesting important and distinct roles in the pathogenesis of fibrosis.

Treatment of early diffuse systemic sclerosis skin disease.

Diffuse systemic sclerosis carries a high morbidity and mortality. The Prospective Registry of Early Systemic Sclerosis (PRESS), a multicentre incident cohort study of patients with early diffuse cutaneous systemic sclerosis, has the goal of advancing the understanding of disease pathogenesis and identifying novel biomarkers. In this review, PRESS investigators discuss the evidence pertaining to the more commonly used treatments for early diffuse SSc skin disease including methotrexate, mycophenolate, cyclophosphamide, azathioprine, and intravenous immunoglobulin. This review highlights the unmet need for effective treatment in early diffuse SSc as well as its more rigorous study. Nonetheless, the PRESS investigators aim to decrease intra- and inter-institutional variability in prescribing in order to improve the understanding of the clinical course of early diffuse SSc skin disease.

The pulmonary fibrosis-associated MUC5B promoter polymorphism does not influence the development of interstitial pneumonia in systemic sclerosis.

BACKGROUND: More than 80% of patients with systemic sclerosis (SSc) develop lung involvement, most commonly interstitial pneumonia (IP). We recently identified a common variant in the promoter region of MUC5B (rs35705950) that has a significant effect on the risk of developing both familial and sporadic forms of IP. We hypothesized that this MUC5B promoter polymorphism is also associated with IP in subjects with SSc. METHODS: We examined the minor allele frequency of the MUC5B polymorphism among 231 subjects with SSc, 109 with IP, and 122 without IP. IP diagnosis was confirmed by HRCT imaging and defined as the presence of reticular infiltrates and/or honeycomb cysts. FVC and diffusing capacity of the lung for carbon monoxide (Dlco) were also assessed. RESULTS: We found no association between IP and the MUC5B polymorphism among subjects with SSc (OR = 1.1, P = .80). The frequencies of the MUC5B polymorphism among subjects with SSc with IP (10.6%) and without IP (9.4%) were similar to the frequency observed in a population of unaffected control subjects (9.0%). In secondary analyses, we found the MUC5B polymorphism was not significantly associated with either FVC (P = .42) or Dlco (P = .06). No association with SSc-associated IP was found even when we used a more conservative definition of IP (FVC ≤ 70% and evidence of reticulations or honeycombing vs SSc FVC > 70% and no evidence of reticulation or honeycombing). CONCLUSIONS: Although SSc-associated IP is clinically, radiologically, and histologically similar to other forms of IP, it appears to have distinct genetic risk factors. This study highlights the genetic and phenotypic heterogeneity of IP in general.