Exploring the complexity of systemic sclerosis etiology by trio whole genome sequencing.

Systemic sclerosis (SSc) is a heterogeneous rare autoimmune fibrosing disorder affecting connective tissue. The etiology of systemic sclerosis is largely unknown and many genes have been suggested as susceptibility loci of modest impact by genome-wide association study (GWAS). Multiple factors can contribute to the pathological process of the disease, which makes it more difficult to identify possible disease-causing genetic alterations. In this study, we have applied whole genome sequencing (WGS) in 101 indexed family trios, supplemented with transcriptome sequencing on cultured fibroblast cells of four patients and five family controls where available. Single nucleotide variants (SNVs) and copy number variants (CNVs) were examined, with emphasis on de novo variants. We also performed enrichment test for rare variants in candidate genes previously proposed in association with systemic sclerosis. We identified 42 exonic and 34 ncRNA de novo SNV changes in 101 trios, from a total of over 6000 de novo variants genome wide. We observed higher than expected de novo variants in PRKXP1 gene. We also observed such phenomenon along with increased expression in patient group in NEK7 gene. Additionally, we also observed significant enrichment of rare variants in candidate genes in the patient cohort, further supporting the complexity/multi-factorial etiology of systemic sclerosis. Our findings identify new candidate genes including PRKXP1 and NEK7 for future studies in SSc. We observed rare variant enrichment in candidate genes previously proposed in association with SSc, which suggest more efforts should be pursued to further investigate possible pathogenetic mechanisms associated with those candidate genes.

Longitudinal global transcriptomic profiling of preclinical systemic sclerosis reveals molecular changes associated with disease progression.

OBJECTIVE: To investigate peripheral blood cell (PBCs) global gene expression profile of SSc at its preclinical stage (PreSSc) and to characterize the molecular changes associated with progression to a definite disease over time. MATERIAL AND METHODS: Clinical data and PBCs of 33 participants with PreSSc and 16 healthy controls (HCs) were collected at baseline and follow-up (mean 4.2 years). Global gene expression profiling was conducted by RNA sequencing and a modular analysis was performed. RESULTS: Comparison of baseline PreSSc to HCs revealed 2889 differentially expressed genes. Interferon signalling was the only activated pathway among top over-represented pathways. Moreover, 10 modules were significantly decreased in PreSSc samples (related to lymphoid lineage, cytotoxic/NK cell, and erythropoiesis) in comparison to HCs. At follow-up, 14 subjects (42.4%) presented signs of progression (evolving PreSSc) and 19 remained in stable preclinical stage (stable PreSSc). Progression was not associated with baseline clinical features or baseline PBC transcript modules. At follow-up stable PreSSc normalized their down-regulated cytotoxic/NK cell and protein synthesis modules while evolving PreSSc kept a down-regulation of cytotoxic/NK cell and protein synthesis modules. Transcript level changes of follow-up vs baseline in stable PreSSc vs evolving PreSSc showed 549 differentially expressed transcripts (336 up and 213 down) with upregulation of the EIF2 Signalling pathway. CONCLUSIONS: Participants with PreSSc had a distinct gene expression profile indicating that molecular differences at a transcriptomic level are already present in the preclinical stages of SSc. Furthermore, a reduced NK signature in PBCs was related to SSc progression over time.

Peripheral blood gene expression profiling shows predictive significance for response to mycophenolate in systemic sclerosis-related interstitial lung disease.

OBJECTIVES: To characterise the peripheral blood cell (PBC) gene expression changes ensuing from mycophenolate mofetil (MMF) or cyclophosphamide (CYC) treatment and to determine the predictive significance of baseline PBC transcript scores for response to immunosuppression in systemic sclerosis (SSc)-related interstitial lung disease (ILD). METHODS: PBC RNA samples from baseline and 12-month visits, corresponding to the active treatment period of both arms in Scleroderma Lung Study II, were investigated by global RNA sequencing. Joint models were created to examine the predictive significance of baseline composite modular scores for the course of forced vital capacity (FVC) per cent predicted measurements from 3 to 12 months. RESULTS: 134 patients with SSc-ILD (CYC=69 and MMF=65) were investigated. CYC led to an upregulation of erythropoiesis, inflammation and myeloid lineage-related modules and a downregulation of lymphoid lineage-related modules. The modular changes resulting from MMF treatment were more modest and included a downregulation of plasmablast module. In the longitudinal analysis, none of the baseline transcript module scores showed predictive significance for FVC% course in the CYC arm. In contrast, in the MMF arm, higher baseline lymphoid lineage modules predicted better subsequent FVC% course, while higher baseline myeloid lineage and inflammation modules predicted worse subsequent FVC% course. CONCLUSION: Consistent with the primary mechanism of action of MMF on lymphocytes, patients with SSc-ILD with higher baseline lymphoid module scores had better FVC% course, while those with higher myeloid cell lineage activation score had poorer FVC% course on MMF.

Large-scale analysis of longitudinal skin gene expression in systemic sclerosis reveals relationships of immune cell and fibroblast activity with skin thickness and a trend towards normalisation over time.

OBJECTIVES: Determine relationships between skin gene expression and systemic sclerosis (SSc) clinical disease features, and changes in skin gene expression over time. METHODS: A total of 339 forearm skin biopsies were obtained from 113 SSc patients and 44 matched healthy controls. 105 SSc patients had a second biopsy, and 76 had a third biopsy. Global gene expression profiling was performed, and differentially expressed genes and cell type-specific signatures in SSc were evaluated for relationships to modified Rodnan Skin Score (mRSS) and other clinical variables. Changes in skin gene expression over time were analysed by mixed effects models and principal component analysis. Immunohistochemical staining was performed to validate conclusions. RESULTS: Gene expression dysregulation was greater in SSc patients with affected skin than in those with unaffected skin. Immune cell and fibroblast signatures positively correlated with mRSS. High baseline immune cell and fibroblast signatures predicted higher mRSS over time, but were not independently predictive of longitudinal mRSS after adjustment for baseline mRSS. In early diffuse cutaneous SSc, immune cell and fibroblast signatures declined over time, and overall skin gene expression trended towards normalisation. On immunohistochemical staining, most early diffuse cutaneous SSc patients with high baseline T cell and macrophage numbers had declines in these numbers at follow-up. CONCLUSIONS: Skin thickness in SSc is related to dysregulated immune cell and fibroblast gene expression. Skin gene expression changes over time in early diffuse SSc, with a tendency towards normalisation. These observations are relevant for understanding SSc pathogenesis and could inform treatment strategies and clinical trial design.

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.

A Prospective Observational Study of Disease Severity and Mortality in Hispanic American Patients With Systemic Sclerosis.

OBJECTIVE: To characterize disease manifestations in Hispanic American patients with systemic sclerosis (SSc) in comparison with non-Hispanic White and Black patients. METHODS: Longitudinal clinical characteristics were collected prospectively in the Genetics versus Environment in Scleroderma Outcome Study cohort. All patients fulfilled the classification criteria for SSc and had a disease duration less than five years at enrollment. RESULTS: A cohort of 427 patients, consisting of 124 Hispanic, 220 non-Hispanic White, and 83 non-Hispanic Black participants were examined. At enrollment, Hispanic patients were significantly younger but had longer disease duration, higher frequency of U1-RNP positivity as well as concurrent systemic lupus erythematosus (SLE) diagnosis, and lower income and educational levels in comparison to non-Hispanic White patients. Compared with non-Hispanic Black patients, Hispanic patients had more frequently limited cutaneous involvement and anticentromere antibodies. In the longitudinal analysis, Hispanic patients had significantly lower forced vital capacity percents predicted (point estimate, -9.3%; P < 0.001) than non-Hispanic White but not Black patients. Hispanic patients had similar longitudinal modified Rodnan Skin Scores like non-Hispanic White patients but lower measurements than non-Hispanic Black patients (point estimate, -3.2; P = 0.029). Hispanic patients had significantly higher serially obtained perceived functional disability scores than White patients (point estimate, 0.29; P < 0.001). Hispanic patients also had higher mortality rates than White Americans even after adjustment for age, gender, and socioeconomic statuses. CONCLUSION: Hispanic patients have higher likelihood of having U1-RNP positivity and SLE overlap, more severe restrictive lung disease, as well as higher rate of mortality than non-Hispanic White patients.

Deletion of adipocyte Sine Oculis Homeobox Homolog 1 prevents lipolysis and attenuates skin fibrosis.

Background: The cardinal feature of systemic sclerosis (SSc) is skin thickening and tightening. Targetable mechanisms for skin features remain elusive. Drugs successful in treating internal organ manifestations have failed efficacy in skin. Dermal white adipose tissue (DWAT) is amongst the understudied contributors to skin manifestations. This study proposes the role of sine oculis homeobox homolog 1 (SIX1), a gene previously unrecognized as a contributor to dermal lipoatrophy characteristic of early skin fibrosis in SSc. Methods: Skin gene expression of SIX1 was analyzed in the GENISOS and PRESS SSc cohorts. Correlation analysis was performed with Spearman rank analysis. Novel mouse models were developed using the Cre-loxp system to knock out Six1 in all cells and mature adipocytes. Subcutaneous bleomycin was used to model early DWAT atrophy and dermal fibrosis characteristic of SSc. Findings: SIX1 was upregulated in SSc skin, the expression of which correlates with adipose-associated genes and molecular pathways. Genetic deletion of Six1 in all cells in mice challenged with bleomycin abrogated end-stage fibrotic gene expression and dermal adipocyte shrinkage. Adipocyte specific Six1 deletion was able to attenuate the early increase in skin thickness, a hallmark of experimental skin fibrosis. Further studies revealed a link between elevated SIX1 and increased expression of SERPINE1 and its protein PAI-1 which are known pro-fibrotic mediators. Interpretation: This work identifies SIX1 as an early marker of skin fibrosis in SSc. We also demonstrate a causative role of Six1 in skin fibrosis by promoting adipocyte loss and show that deletion of Six1 in adipocytes has the potential of impacting early disease progression.

Myeloablation Followed by Hematopoietic Stem Cell Transplantation and Long-Term Normalization of Systemic Sclerosis Molecular Signatures.

OBJECTIVE: In the randomized Scleroderma: Cyclophosphamide or Transplantation (SCOT) trial, myeloablation, followed by hematopoietic stem cell transplantation (HSCT), led to the normalization of systemic sclerosis (SSc) peripheral blood cell (PBC) gene expression signature at the 26-month visit. Herein, we examined long-term molecular changes ensuing 54 months after randomization for individuals receiving an HSCT or 12 months of intravenous cyclophosphamide (CYC). METHODS: Global PBC transcript studies were performed in study participants at pretreatment baseline and at 38 months and 54 months after randomization, as well as in healthy controls using Illumina HT-12 arrays. RESULTS: Thirty (HSCT = 19 and CYC = 11) participants had 38-month samples available, and 26 (HSCT = 16 and CYC = 11) had 54-month samples available. In the paired comparison to baseline, a significant down-regulation of interferon modules and an up-regulation of cytotoxic/natural killer module were observed at the 38-month and 54-month visits in the HSCT arm, indicating a long-term normalization of baseline SSc gene expression signature. No differentially expressed modules were detected in the CYC arm. In comparison to samples from healthy controls, 38-month visit samples in the HSCT arm showed an up-regulation of B cell and plasmablast modules and a down-regulation of myeloid and inflammation modules. Importantly, 54-month HSCT samples did not show any differentially expressed modules compared to healthy control samples, suggesting completion of immune reconstitution. Participants in the CYC arm continued to show an SSc transcript signature in comparison to controls at both time points. CONCLUSION: Paralleling the observed clinical benefit, HSCT leads to durable long-term normalization of the molecular signature in SSc, with completion of immune resetting to 54 months after HSCT.

Fibroblast Subpopulations in Systemic Sclerosis: Functional Implications of Individual Subpopulations and Correlations with Clinical Features.

Fibroblasts constitute a heterogeneous population of cells. In this study, we integrated single-cell RNA-sequencing and bulk RNA-sequencing data as well as clinical information to study the role of individual fibroblast populations in systemic sclerosis (SSc). SSc skin demonstrated an increased abundance of COMP+, COL11A1+, MYOC+, CCL19+, SFRP4/SFRP2+, and PRSS23/SFRP2+ fibroblasts signatures and decreased proportions of CXCL12+ and PI16+ fibroblast signatures in the Prospective Registry of Early Systemic Sclerosis and Genetics versus Environment in Scleroderma Outcome Study cohorts. Numerical differences were confirmed by multicolor immunofluorescence for selected fibroblast populations. COMP+, COL11A1+, SFRP4/SFRP2+, PRSS23/SFRP2+, and PI16+ fibroblasts were similarly altered between normal wound healing and patients with SSc. The proportions of profibrotic COMP+, COL11A1+, SFRP4/SFRP2+, and PRSS23/SFRP2+ and proinflammatory CCL19+ fibroblast signatures were positively correlated with clinical and histopathological parameters of skin fibrosis, whereas signatures of CXCL12+ and PI16+ fibroblasts were inversely correlated. Incorporating the proportions of COMP+, COL11A1+, SFRP4/SFRP2+, and PRSS23/SFRP2+ fibroblast signatures into machine learning models improved the classification of patients with SSc into those with progressive versus stable skin fibrosis. In summary, the profound imbalance of fibroblast subpopulations in SSc may drive the progression of skin fibrosis. Specific targeting of disease-relevant fibroblast populations may offer opportunities for the treatment of SSc and other fibrotic diseases.

Predictors of Perceived Functional Status in Early Systemic Sclerosis: A Prospective Longitudinal Study of an Early Disease Cohort.

OBJECTIVE: The modified Health Assessment Questionnaire (M-HAQ) is a well-established patient-reported outcome measure in systemic sclerosis (SSc) studies that reflects how a patient functions in several categories of activities of daily living. This study analyzed clinical, demographic, and socioeconomic factors that predict M-HAQ scores over time. METHODS: This study included 388 patients with baseline M-HAQ scores from the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) early disease cohort with a mean disease duration of 2.5 years, mean follow-up time of 3.9 years, and median follow-up of 7.2 years. A total of 1,950 M-HAQ measurements were analyzed. Baseline disease characteristics were recorded, and the association of these characteristics with the M-HAQ score was analyzed at baseline and longitudinally. RESULTS: Lower income and education levels, older age, and more severe skin involvement at enrollment were independent predictors of worse perceived functional disability over time (i.e., higher longitudinal M-HAQ levels). Higher longitudinal modified Rodnan skin scores correlated with higher M-HAQ scores, whereas higher longitudinal forced vital capacity percentage predicted values correlated with lower M-HAQ scores over time (P < 0.001 for both univariable and multivariable analyses). Moreover, higher baseline M-HAQ scores predicted higher mortality (hazard ratio 1.29, 95% confidence interval 1.09-1.52, P = 0.003). CONCLUSION: This large, longitudinal study of early disease SSc demonstrates that severity of skin disease and socioeconomic factors such as educational level and income are important predictors of perceived functional disability in SSc.

Proteomic aptamer analysis reveals serum markers that characterize preclinical systemic sclerosis (SSc) patients at risk for progression toward definite SSc.

BACKGROUND: The study of molecular mechanisms characterizing disease progression may be relevant to get insights into systemic sclerosis (SSc) pathogenesis and to intercept patients at very early stage. We aimed at investigating the proteomic profile of preclinical systemic sclerosis (PreSSc) via a discovery/validation two-step approach. METHODS: SOMAcan aptamer-based analysis was performed on a serum sample of 13 PreSSc (discovery cohort) according to 2001 LeRoy and Medsger criteria (characterized solely by Raynaud phenomenon plus a positive nailfold capillaroscopy and SSc-specific antibodies without any other sign of definite disease) and 8 healthy controls (HCs) age, gender, and ethnicity matched. Prospective data were available up to 4±0.6 years to determine the progression to definite SSc according to the EULAR/ACR 2013 classification criteria. In proteins with relative fluorescence units (RFU) > |1.5|-fold vs HCs values, univariate analysis was conducted via bootstrap aggregating models to determine the predicting accuracy (progression vs non-progression) of categorized baseline protein values. Gene Ontologies (GO terms) and Reactome terms of significant proteins at the adjusted 0.05 threshold were explored. Significant proteins from the discovery cohort were finally validated via ELISAs in an independent validation cohort of 50 PreSSc with clinical prospective data up to 5 years. Time-to-event analysis for interval-censored data was used to evaluate disease progression. RESULTS: In the discovery cohort, 286 out of 1306 proteins analyzed via SomaScan, were differentially expressed versus HCs. Ten proteins were significantly associated with disease progression; analysis through GO and Reactome showed differentially enriched pathways involving angiogenesis, endothelial cell chemotaxis, and endothelial cell chemotaxis to fibroblast growth factor (FGF). In the validation cohort, endostatin (HR=10.23, CI95=2.2-47.59, p=0.003) was strongly associated with disease progression, as well as bFGF (HR=0.84, CI95=0.709-0.996, p=0.045) and PAF-AHß (HR=0.372, CI95=0.171-0.809, p=0.013) CONCLUSIONS: A distinct protein profile characterized PreSSc from HCs and proteins associated with hypoxia, vasculopathy, and fibrosis regulation are linked with the progression from preclinical to definite SSc. These proteins, in particular endostatin, can be regarded both as markers of severity and molecules with pathogenetic significance as well as therapeutic targets.

Reactome pathway analysis from whole-blood transcriptome reveals unique characteristics of systemic sclerosis patients at the preclinical stage.

Objective: This study aims to characterize differential expressed pathways (DEP) in subjects with preclinical systemic sclerosis (PreSSc) characterized uniquely by Raynaud phenomenon, specific autoantibodies, and/or capillaroscopy positive for scleroderma pattern. Methods: Whole-blood samples from 33 PreSSc with clinical prospective data (baseline and after 4 years of follow-up) and 16 matched healthy controls (HC) were analyzed for global gene expression transcriptome analysis via RNA sequencing. Functional Analysis of Individual Microarray Expression method annotated Reactome individualized pathways. ANOVA analysis identified DEP whose predictive capability were tested in logistic regression models after extensive internal validation. Results: At 4 years, 42.4% subjects progressed (evolving PreSSc), while the others kept stable PreSSc clinical features (stable PreSSc). At baseline, out of 831 pathways, 541 DEP were significant at a false discovery rate <0.05, differentiating PreSSc versus HC with an AUROC = 0.792 ± 0.242 in regression models. Four clinical groups were identified via unsupervised clustering (HC, HC and PreSSc with HC-like features, PreSSc and HC with PreSSc-like features, and PreSSc). Biological signatures changed with disease progression while remaining unchanged in stable subjects. The magnitude of change was related to the baseline cluster, yet no DEP at baseline was predictive of progression. Disease progression was mostly related to changes in signal transduction pathways especially linked to calcium-related events and inositol 1,4,5-triphosphate metabolism. Conclusion: PreSSc had distinguished Reactome pathway signatures compared to HC. Progression to definite SSc was characterized by a shift in biological fingertips. Calcium-related events promoting endothelial damage and vasculopathy may be relevant to disease progression.

Blood Neutrophil Count and Neutrophil-to-Lymphocyte Ratio for Prediction of Disease Progression and Mortality in Two Independent Systemic Sclerosis Cohorts.

OBJECTIVE: To assess the predictive significance of blood neutrophil count and the ratio between neutrophil and lymphocyte count (neutrophil-to-lymphocyte ratio [NLR]) for disease severity and mortality in systemic sclerosis (SSc). METHODS: Neutrophil and lymphocyte counts were prospectively measured in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) and the Scleroderma Lung Study II (SLS II). Forced vital capacity percent predicted (FVC%) and modified Rodnan skin thickness score (MRSS) were used as surrogate measures for disease severity. Longitudinal analyses were performed using generalized linear mixed models. Cox proportional hazards models evaluated the predictive significance of these cell counts for mortality. RESULTS: Of the 447 SSc patients in the GENISOS cohort at the time of analysis, 377 (84.3%) had available baseline blood neutrophil and lymphocyte counts. Higher baseline neutrophil count and NLR predicted lower serially obtained FVC% (b = -4.74, P = 0.009 and b = -2.68, P = 0.028, respectively) and higher serially obtained MRSS (b = 4.07, P < 0.001 and b = 2.32, P < 0.001, respectively). Longitudinal neutrophil and NLR measurements also significantly correlated with lower concurrently obtained FVC% measurements and higher concurrently obtained MRSS. Baseline neutrophil count and NLR predicted increased risk of long-term mortality, even after adjustment for baseline demographic and clinical factors (hazard ratio [HR] 1.42, P = 0.02 and HR 1.48, P < 0.001, respectively). The predictive significance of higher baseline neutrophil count and NLR for declining FVC% and increased long-term mortality was confirmed in the SLS II. CONCLUSION: Higher blood neutrophil count and NLR are predictive of more severe disease course and increased mortality, indicating that these easily obtainable laboratory studies might be a reflection of pathologic immune processes in SSc.

False positive anti-Topoisomerase I (Scl-70) antibody results in clinical practice: A case series from a scleroderma referral center.

PURPOSE: To determine if some patients who tested positive for anti-Scl-70 antibody in clinical practice, but did not have classifiable systemic sclerosis, were negative for anti-Scl-70 antibody by the more specific immunodiffusion method of testing. METHODS: Patients evaluated by a rheumatologist at a Scleroderma referral center who had tested positive for anti-Scl-70 antibody prior to referral, but did not have classifiable SSc based on clinical criteria, were invited to undergo testing for anti-Scl-70 antibody by immunodiffusion. Patient demographics and clinical features were recorded at the time of their evaluation, and diagnostic testing results were reviewed using the medical records. RESULTS: 52 patients were enrolled over an 8-year period, with 48 (92.3%) testing negative and 4 (7.7%) testing positive for anti-Scl-70 antibody by immunodiffusion. Of the 48 patients who tested negative, 18 (37.5%) tested negative for ANA by indirect immunofluorescence, 33 (68.8%) did not have Raynaud's phenomenon, and 43 (89.6%) had ≤1 clinical criteria items based on the 2013 ACR/EULAR SSc classification criteria. Nevertheless, 21 (43.8%) patients who were negative for anti-Scl-70 antibody by immunodiffusion had undergone a chest CT and 14 (29.2%) had undergone an echocardiogram. A total of 23 patients had at least one follow up clinic visit. 3 out of 4 patients who were positive for anti-Scl-70 antibody by immunodiffusion, but none of the 19 patients who tested negative by immunodiffusion, developed sufficient criteria during follow up to be classified as SSc. CONCLUSION: Assays for anti-Scl-70 antibody in commercial laboratories that are commonly utilized in clinical practice can produce false positive results. These results can lead to angst for patients, as well as unnecessary referrals and diagnostic evaluations.

The Effect of Anti-Scl-70 Antibody Determination Method on Its Predictive Significance for Interstitial Lung Disease Progression in Systemic Sclerosis.

OBJECTIVE: The objective of this study was to assess the predictive significance of anti-Scl-70 (anti-topoisomerase I) antibodies, as determined by three different methods, for decline in forced vital capacity (FVC) within the first year of follow-up in patients with systemic sclerosis (SSc)-related interstitial lung disease (ILD). METHODS: Patients in the Genetics Versus Environment in Scleroderma Outcome Study cohort who had ILD (verified by imaging) and available FVC% at enrollment, plus 12 to 18 months thereafter, were examined. All patients had a disease duration of 5 years or less at enrollment. The annualized percentage change in FVC% at 1 year follow-up was the outcome variable. Anti-Scl-70 antibodies were determined by passive immunodiffusion (ID) against calf thymus extract, chemiluminescent immunoassay (CIA), and line blot immunoassay (LIA). RESULTS: Ninety-one patients with a mean disease duration of 2.36 years were included. Anti-Scl-70 antibodies by ID predicted a faster rate of FVC% decline (b = -0.06, P = 0.04). None of the other clinical or serological variables significantly predicted ILD progression. Interestingly, anti-Scl-70 antibodies as determined by CIA and LIA were not significant predictors of FVC decline (P = 0.26 and 0.64, respectively). The observed level of agreement between ID and LIA was moderate (κ = 0.568), whereas it was good between ID and CIA (κ = 0.66). CONCLUSION: Anti-Scl-70 antibodies determined by ID predicted faster FVC decline in patients with SSc-related ILD. Notably, both CIA and LIA for the same antibody did not predict rate of FVC decline at their current cutoffs of positivity. The discrepancy observed between anti-Scl-70 antibody assays can have relevant implications for clinical care and trial enrichment strategies in SSc-ILD.

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.