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Squamous cell carcinoma antigen-IgM (SCCA-IgM) is associated with interstitial lung disease in systemic sclerosis
Joint Bone Spine 87 (2020) 331–335
Available online at
ScienceDirect www.sciencedirect.com
Original article
Squamous cell carcinoma antigen-IgM (SCCA-IgM) is associated with interstitial lung disease in systemic sclerosis Elisabetta Zanatta a,1 , Andrea Martini b,1 , Elena Scarpieri a , Alessandra Biasiolo b , Augusta Ortolan a , Francesco Benvenuti a , Franco Cozzi a , Patrizia Pontisso b , Andrea Doria a,∗ a b
Division of Rheumatology, Department of Medicine-DIMED, University of Padova„ 35128 Padova, Italy Unit of Internal Medicine and Hepatology (UIMH), Department of Medicine-DIMED, University of Padova, 35128 Padova, Italy
a r t i c l e
i n f o
Article history: Received 29 September 2019 Accepted 12 February 2020 Available online 19 February 2020 Keywords: Systemic sclerosis Interstitial lung disease SCCA-IgM Serpinb3
a b s t r a c t Objective: Interstitial lung disease (ILD) is the major determinant of prognosis in patients with systemic sclerosis (SSc). Squamous Cell Carcinoma Antigen (SCCA1) is a serin protease inhibitor which plays a pivotal role in inflammation and fibrosis. SCCA1 is overexpressed in pulmonary tissue of patients with idiopathic pulmonary fibrosis and can be detectable in serum as circulating immune complex bound to IgM (SCCA-IgM). We aimed to investigate the association between SCCA-IgM and clinical features of patients with SSc. Methods: Ninety-seven patients with SSc (ACR/EULAR criteria) were consecutively enrolled in the study. Clinical and serological variables and organ involvement were recorded. Pulmonary involvement was investigated by high-resolution CT (HRCT) and respiratory function tests. SCCA-IgM serum levels were measured by a validated ELISA assay (Hepa-IC, Xeptagen, Venice, Italy). We set the cut-off value for serum levels of SCCA-IgM > 200 AU/ml, calculated as mean + 3 standard deviations in 100 healthy subjects. Results: Forty-one (42.3%) patients were affected with ILD. SCCA-IgM values were significantly higher in patients with ILD than in those without: 218 (80-402) vs. 87.5 (59-150) AU/mL, P = 0.003. Patients with positive SCCA-IgM had more frequently ILD (69.7% vs. 28.1%, P ≤ 0.0001) and a lower total lung capacity (TLC) (P = 0.024) compared with negative ones. No differences were found in any other clinical and serological features. At multivariate analysis, SCCA-IgM was found to be associated with ILD diagnosis (OR 10.6, IC 2.9-38.4, P = 0.001). Conclusion: SCCA-IgM is associated with interstitial lung disease in scleroderma patients and might be used in the assessment of SSc-ILD. ´ e´ franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved. © 2020 Societ
1. Introduction Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial dysfunction and autoimmune dysregulation which can lead to fibrosis of skin and internal organs (heart, kidneys, gut and lungs) [1]. Interstitial lung disease (ILD) is frequently detectable and is the leading cause of death in SSc patients, accounting for 35% of disease-related deaths in the European Scleroderma Trial and Research (EUSTAR) database [2]. However, ILD can be totally asymptomatic in the early stage, thus making its diagnosis challenging [3]. To date only a few biomarkers have been identified in SSc-ILD (e.g. Serum Krebs von den Lungen-6, KL-6) and their clin-
∗ Corresponding author. E-mail address: [email protected] (A. Doria). 1 These two authors contributed equally to the article.
ical meaning is still under evaluation [4], as discussed in a recent systematic literature review performed by Bonhomme et al. [5]. Squamous Cell Carcinoma Antigen 1 (SCCA1, also known as SerpinB3) is a serin protease inhibitor, expressed by dysplastic and neoplastic cells of epithelial origin (including lung cancer) and in cirrhotic liver, which plays an important role in the regulation of inflammation, programmed cell death and fibrosis [6]. In a murine model of pulmonary fibrosis, SCCA1 transgenic mice showed higher expression of transforming grow factor beta 1 (TGF-1) and more extended fibrosis along with a significant gain of epithelial proliferation [7]. In line with these results, SCCA1 was found overexpressed in the pulmonary tissue of patients with idiopathic pulmonary fibrosis (IPF) [8], and it positively correlates with the extension of fibroblastic foci, expression of TGF-1 and carbon monoxide decline after 9 months of follow-up. Calabrese et al. [8] hypothesized two mode of action of SCCA1 in IPF: stimulation of epithelial
https://doi.org/10.1016/j.jbspin.2020.02.003 ´ e´ franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved. 1297-319X/© 2020 Societ
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proliferation (autocrine action) or activation of fibroblast proliferation/fibrosis via increased TGF-1 secretion (paracrine action). SCCA1 can be detectable in serum as circulating immune complex coupled to IgM (SCCA-IgM) and was found to correlate closely with the extent of liver fibrosis in patients with liver disease [9]. Only one study has investigated SCCA-IgM in SSc so far, showing high levels of this biomarker especially in patients with ILD [10]. The aim of our study was to assess the relation between SCCA-IgM serum levels and demographic and clinical features of SSc patients. 2. Methods 2.1. Patients Ninety-seven patients affected with SSc according to ACR/EULAR criteria [11] were consecutively enrolled in the study which was part of a national Project (FIRB Prot. RBLA03S4SP 005), and approved by the local Ethics Committee. All patients provided written informed consent. Demographic, clinical and serological data of the entire study population are reported in Appendix A, Table S1 [See the supplementary material associated with this article online]. Organ involvement was defined as follows: • cardiac: ventricular arrhythmia, conduction defects, systolic (ejection fraction < 55%) or diastolic dysfunction (according to the ASE criteria, [12]), significant pericardial effusion (> 10 mm and diffuse); • gastrointestinal (GI): gastroesophageal reflux, dysphagia, objective findings of esophageal dysmotility, hiatal hernia, esophagitis and gastritis; bowel obstruction and sub-occlusion, constipation, diarrhea; • pulmonary involvement: interstitial lung disease (ILD) was diagnosed using high-resolution computed tomography (HRCT), in presence of reticular abnormalities, ground-glass opacities and/or honeycombing. According to best practice in SSc-ILD [13], all patients underwent HRCT at diagnosis and then according to symptoms, presence of pulmonary crackles, annual pulmonary function tests (PTFs) and standard chest X-Ray radiography; • pulmonary arterial hypertension (PAH): defined as mean pulmonary pressure ≥ 25 mmHg at right heart catheterization with a pulmonary wedge pressure ≤ 15 mmHg. The modified Rodnan skin score (mRSS), presence of digital ulcers and C-reactive protein (CRP) were also evaluated. Disease activity was measured by EUSTAR activity score [14]. As for ILD, we also considered: • ILD duration = defined from the first evidence of ILD on HRCT; • Pulmonary function tests (PFTs) carried out no later than 6 months prior to the SCCA-IgM assay. The following pulmonary function tests (PFTs) (expressed as the percentage of observed/theoretic values) were considered: forced vital capacity (FVC), total lung capacity (TLC), diffuse capacity for carbon monoxide (DLCO), carbon monoxide transfer coefficient (KCO) calculated as the ratio between DLCO and alveolar volume (Va). According to the literature [15], values of FVC, TLC, DLCO and KCO below 80% were considered pathological. Immunosuppressive and corticosteroids therapy were also recorded. 2.2. SCCA-IgM assay In all patients SCCA-IgM serum levels were measured by a validated ELISA assay (Hepa-IC, Xeptagen, Venice, Italy), according to
the manufacturer’s instructions. Values were expressed in arbitrary units/milligram (AU/ml) and, according to our laboratory cut-off, was considered abnormal a value of SCCA-IgM > 200 AU/mL. This value was obtained by analyzing the sera from 100 healthy subjects (mean value + 3 standard deviations). 2.3. Statistical analysis Continuous variables were expressed as means ± standard deviation (SD) or medians (interquartile range) and categorical variables as frequency and percentage. Comparison between groups was carried out using the Student’s t-test and unpaired t-test with Welch’s correction, or the Mann–Whitney U-test for continuous variables, and the chi-squared-test or Fisher’s exact probability test for categorical data, where appropriate. Multiple comparisons were adjusted by Bonferroni correction. Variables which were found to be different (P < 0.05) between patients with and without ILD at univariate analysis, were then included into a multivariate logistic regression model, adjusted for age and gender. Only pathological FVC, anti-topoisomerase I (anti-Scl70), immunosuppressants and SCCA-IgM were included in the model to avoid multicollinearity. Results are reported as OR with a 95% confidence interval. Sensibility, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy and negative likelihood ratio (NLR) were calculated. Two-sided P < 0.05 was considered statistically significant. The statistical analysis was performed using the SPSS statistical package, version 22.0. 3. Results Among the 97 patients included in the study, 82 (84.5%) were female, mean age 55.4 ± 12.4 years and median disease duration 12 (6–20) years. According to Le Roy et al. [16], 35 patients (36.1%) were classified as affected with diffuse cutaneous form (dcSSc) and 62 (63.9%) with the limited one (lcSSc). Forty-one patients had ILD (42.3%). Demographic, clinical and serologic features in patients with and without ILD are reported in Table 1. The two groups of patients were similar in terms of demographic features and disease duration. Patients with ILD were more frequently affected with dcSSc (P < 0.0001), had higher values of mRSS (P = 0.002) and a higher frequency of anti-Scl70 (P = 0.001) and anti-RNA pol III (P = 0.047) autoantibodies compared with patients without ILD. Anti-centromere antibody was inversely associated with the presence of ILD (P < 0.0001). As expected, patients with ILD had lower values of FVC and TLC than patients without ILD (P < 0.0001 in both cases). In addition, DLCO were lower in the former group (P = 0.002). Conversely, no differences between the two groups in terms of digital ulcers and cardiac or gastrointestinal involvement were observed. EUSTAR score was higher in patients with ILD than in those without (P = 0.029). Median SCCA-IgM values were significantly higher in patients with ILD than in those without: 218 (80-402) vs. 87.5 (59-150) AU/mL, P = 0.004 (Fig. 1A) and in patients with abnormal TLC values (P = 0.03, Fig. 1B). Therefore, we subdivided the study population into two groups according to the positive/negative SCCA-IgM status (cut off: 200 AU/mL). The demographic and clinical features in these two groups of patients are reported in Table 2. Patients with positive SCCA-IgM were more frequently affected with ILD (69.7% vs. 28.1%, P < 0.0001) and had more frequently a TLC < 80% (34.6% vs. 12.5%, P = 0.024); moreover, they tended to have a pathological DLCO (P = 0.058). No differences were found between the two groups regarding other PFTs (FVC and KCO), which, however, were more frequently abnormal in patients with positive SCCA-IgM. Patients treated with steroids or immunosuppressants showed similar serum level of
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Table 1 Demographic and clinical features in the entire study population and according to the presence of ILD. Variable
All patients (n = 97) ILD (n = 41)
NO ILD (n = 56)
Age (yrs) Sex (Female) Disease duration, (yrs) dcSSc mRSS Cardiac involvement PAPs (mmHg) PAH GI involvement Digital ulcers ANA Anti-Scl70 Anti-centromere Anti-RNA polymerase III FVC TLC DLCO KCO FVC < 80% TLC < 80% DLCO < 80% KCO <80% CRP (mg/L) EUSTAR Score Steroids Immunosuppressants
55.4 (± 12.4) 82 (84.5) 12 (6-20) 35 (36.1) 7 (4-15) 20 (21.1) 27.5 (± 10) 6 (6.2) 62 (64.6) 30 (30.9) 97 (100) 26 (28) 50 (53.8) 3 (3.2) 95,9 (± 22.2) 92,9 (± 17) 73,5 (± 21.4) 74.7 (± 18) 19 (20) 15 (20.3) 60 (65.2) 47(63.5) 2.5 (2.0-6.9) 1.3 (± 1.2) 18 (18.8) 33 (34)
56.5 (± 12.5) 49 (87.5) 13.5 (4-21) 11 (19.6)* 4.5 (3.7-10.5)** 9 (16.1) 27.4 (± 10.4) 4 (7.1) 34 (60.7) 15 (26.8) 56 (100) 20 (35.7)** 23 (41.1)* 0 (0)*** 104.5 (± 18.1)* 101.2 (± 13.8)* 79,3 (± 20.8)** 75.8 (± 17.5) 4 (7.1)** 3 (5.4)* 30 (53.6)*** 25 (44.6) 3 (5.8-7) 1.1 (± 1.1)*** 9 (16.1) 13 (23.2)***
53.9 (± 12.4) 33 (80.5) 11 (6.7-16) 24 (58.5) 13.4 (7.5-17.5) 11 (26.8) 27.6 (± 9.9) 2 (4.9) 28 (68.3) 15 (36.6) 41 (100) 30 (73.2) 2 (4.9) 3 (7.3) 84.5 (± 22.1) 82,2 (± 15.1) 65.5 (± 19.7) 73.2 (± 20.2) 15 (36.6) 15 (36.5) 30 (73.2) 22 (53.6) 2.5 (2-10.4) 1.6 (± 1.3) 9 (22) 20 (48.8)
Values are reported as n (%) or mean (± SD) and median (range). ANA: antinuclear antibodies; yrs: years; CRP: C-reactive protein; dcSSc: diffuse cutaneous form; FVC: forced vital capacity; ILD: interstitial lung disease; TLC: total lung capacity; DLCO: diffuse capacity for carbon monoxide; KCO: carbon monoxide transfer coefficient; mRSS: modified Rodnan skin score; GI: gastrointestinal; PAPs: systolic pulmonary artery pressure; PAH: pulmonary arterial hypertension; Scl70: anti-topoisomerase I. For continuous and dichotomous variables significance values at P ≤ 0.005 and P ≤ 0.003, respectively, were obtained after Bonferroni correction for multiple comparisons. * P < 0.001. ** P < 0.01. *** P < 0.05.
SCCA-IgM, in comparison to those untreated (P = 0.435). The two groups were similar in all the other clinical variables, e.g. mRSS, cardiac and gastrointestinal involvement, digital ulcers, EUSTAR score. Considering only patients with SSc-ILD, those with TLC < 80% and FVC < 80% have more frequently SCCA-IgM positivity than patients with these PFTs within normal range (in the 84.2% vs. 15.8% and 76.2% vs. 23.8% of cases, P = 0.041 and P = 0.064, respectively). At the multivariate logistic regression analysis (Table 3) the independent predictors of ILD were anti-Scl70, FVC < 80% and SCCAIgM positivity (OR = 10.6, P = 0.001). Interestingly, patients with an ILD duration ≤ 3 years showed higher levels of SCCA-IgM: 436 (174–1058) vs. 86 (35-329) AU/ml, P = 0.003 (Fig. 1C). To improve the identification of patients with a very low risk of ILD, we combined the main PFTs with SCCA-IgM status (Suppl. Table 1). Among different associations, the one between SCCA-IgM and TLC showed the highest accuracy (80%) and NPV (83%) with the lowest NLR (0.26), performing better than any single variable.
Fig. 1. Squamous cell carcinoma antigen (SCCA)-IgM values and presence of Interstitial Lung Disease (ILD) (Panel A), TLC < 80% (Panel B) and ILD duration (Panel C) in patients with systemic sclerosis. The box indicates the lower and the upper quartile, and the middle line indicates the median. Bars indicate the range of values distribution. Round circles represent outliers. ILD: interstitial lung disease; TLC: total lung capacity; Squamous cell carcinoma antigen (SCCA)-IgM.
4. Discussion ILD is the major determinant of poor prognosis in SSc patients [2], therefore the identification of biomarkers of pulmonary involvement is paramount in the clinical assessment of these patients [4]. The pro-fibrogenic action of SCCA1 is supported by its ability to induce TGF-1 and by the evidence that in activated hepatic stellate cells it is able to exert a pro-fibrogenic response [17]. These findings were also confirmed in an animal model of idiopathic pulmonary
fibrosis [7]. SCCA1 is detectable in serum as bound to IgM (SCCAIgM) and its levels have been shown to be closely related with the extent of liver fibrosis in patients with chronic liver disease [9]. Our results showed that the levels of SCCA-IgM are higher in SSc patients with ILD than in those without. Moreover, at multivariate analysis patients with positive SCCA-IgM had a 10-fold greater risk of being affected with ILD. Importantly, this association was independent from the autoantibody profile, FVC values and immunosuppressive therapy, which are classical features closely
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Table 2 Demographic and clinical features in SSc patients according to the positive/negative SCCA-IgM status. Variable
SCCA-IgM > 200 AU/ml (n = 33)
SCCA-IgM < 200 AU/ml (n = 64)
Age (yrs) Female Disease duration (yrs) dcSSc mRSS ILD Cardiac involvement PAPs (mmHg) PAH GI involvement Digital ulcers ANA Anti-Scl70 Anti-centromere Anti-RNA polymerase III FVC < 80% TLC < 80% DLCO < 80% KCO < 80% CRP (mg/L) EUSTAR Score Steroids Immunosuppressants
56.2 (± 12.5) 30 (90.9) 14 (8-20) 12 (36.4) 10 (4-15) 23 (69.7) 7 (21.2) 28.1 (± 9) 2 (6.1) 20 (60.6) 10 (30.3) 33 (100) 19 (57.6) 9 (27.3) 0 (0)
55 (± 12.5) 52 (81.3) 10 (6-19.5) 23 (35.9) 7 (4-14.7) 18 (28.1)* 13 (21) 27.1 (± 10.6) 4 (6.3) 42 (66.7) 20 (31.3) 64 (100) 31 (51.7) 17 (28.3) 3 (5)
9 (27.3) 9 (34.6) 23 (74.2) 20 (74.1) 3.7 (2.4-11.3) 1.5 (± 1.3) 9 (27.3) 9 (27.3)
10 (16.1) 6 (12.5)** 37 (60.7) 27 (57.4) 2.5 (1.7-6.8) 1.2 (± 1.1) 9 (14.3) 24 (37.5)
Values are reported as n (%) or mean (± SD) and median (range). ANA: antinuclear antibodies; yrs: years; CRP: C-reactive protein; dcSSc: diffuse cutaneous form; FVC: forced vital capacity; ILD: interstitial lung disease; TLC: total lung capacity; DLCO: diffuse capacity for carbon monoxide; KCO: carbon monoxide transfer coefficient; mRSS: modified Rodnan skin score; GI: gastrointestinal; PAPs: systolic pulmonary artery pressure; PAH: pulmonary arterial hypertension; SCCA: squamous cell carcinoma antigen; Scl70: anti-topoisomerase I. For continuous and dichotomous variables significance values at P ≤ 0.008 and P ≤ 0.003, respectively, were obtained after Bonferroni correction for multiple comparisons. * P < 0.001. ** P < 0.05.
Table 3 Multivariate analysis for the diagnosis of ILD. Variable
OR (CI 95%)
Anti-Scl70 FVC < 80% SCCA-IgM (+) Immunosuppressants
3.3 (1.04–10.7)* 9.9 (1.8–54.8)** 10.6 (2.9–38.4)*** 3.149 (0.9–10.7)
Model adjusted for sex and age. OR, odds ratio, CI confidence interval 95%; dcSSC, diffuse cutaneous form; FVC, forced vital capacity; SCCA-IgM (+), Squamous cell carcinoma antigen-IgM > 200 AU/ml; Scl70, anti-topoisomerase I; yrs: years. * P < 0.05. ** P < 0.01. *** P < 0.001.
the pathogenesis of SSc, SCCA1 could be involved in this process, at least in the lung, becoming a potential target of new therapeutic approaches. Moreover, SCCA1 is able to induce epithelial-to mesenchymal transition [18], a process strongly involved in the fibrogenesis of SSc-ILD [19]. In our study, SCCA-IgM was not associated with any other clinical or serological disease features. Notably, SCCA-IgM was not associated with skin fibrosis (both dcSSc and mRSS), and cardiac or gastrointestinal involvement. It has been suggested that SCCA is upregulated in the skin of patients with cutaneous inflammatory diseases (i.e. psoriasis and atopic dermatitis). Inflammatory skin changes occur early in scleroderma, and are quickly replaced by fibrotic and atrophic lesions. Thus, the lack of association between SCCA1 and skin involvement in our patients could be due to their quite long mean disease duration. Moreover, the role of SCCA1 in inducing a pro-fibrogenic response is closely dependent on the profibrogenic environment [17], which may have different features in the skin and lungs of SSc patients. We did not find any association between positive SCCA-IgM and vascular manifestations of SSc (PAPs, digital ulcers) or KCO, which is an index of pulmonary vasculopathy in SSc [20]; overall, these findings strongly support SCCA-IgM as a potential biomarker for pulmonary fibrosis in SSc. Moreover, our results suggest that the addition of negative SCCA-IgM status to normal values of TLC seems helpful in the identification of scleroderma patients with a very low risk of ILD. The extreme variability of normal values of TLC (ranging from 80 to 120%) limits the usefulness of pulmonary function tests in excluding the presence of ILD. Therefore, the addition of serum SCCA-IgM assessment to routine PFTs may be useful in clinical practice for the identification of those patients who may not require CT-scan. Our study has some limitations. First of all, the sample size is relative small; however, it must be taken into account that scleroderma is a rare disease. Second, the lack of a radiological score evaluation does not allow to define the severity of radiological extension, in addition to functional impairment. However, the stratification of patients according to the ILD severity was not an aim of our study. By contrast, one strength of our study is that all patients considered are followed in a third referral center with a great expertise in the management of scleroderma and therefore the possibility that some patients have not been diagnosed with SSc-ILD is very unlikely. In conclusion, our data suggest that SCCA-IgM may be considered a candidate biomarker in SSc-ILD assessment. Disclosure of interest The authors declare that they have no competing interest. Funding
associated with ILD. All these data suggest that SCCA1 might be involved in the pulmonary fibrosis observed in SSc and that SCCAIgM might become a potential biomarker in SSc-ILD. Compared with a previous report [10], our study included a larger number of patients, with a comprehensive functional pulmonary characterization. Besides the association with ILD diagnosis, in the whole population and in the subgroup of patients with ILD we found a significant association between SCCA-IgM positivity and pathological values of TLC, an index of pulmonary restriction. We also observed higher levels of SCCA-IgM in patients with ILD duration ≤ 3 years. Taken together, these data suggest that SCCA-IgM could be useful in the early diagnosis of ILD along with the identification of severe forms. The increased levels of SCCA-IgM in the early stage of ILD could be explained by the evidence that SCCA1 can inhibit inflammation while favoring fibrogenesis [7]. Since the transition from an inflammatory to a fibrotic phase is one of the crucial step in
No funds were received for this study. Appendix A. Supplementary data Supplementary data (Table S1) associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jbspin.2020.02.003. References [1] Zanatta E, Codullo V, Avouac J, et al. Systemic sclerosis: recent insight in clinical management. Joint Bone Spine 2019 [pii: S1297-319X(19)30145-9]. [2] Tyndall AJ, Bannert B, Vonk M, et al. Causes and risk factors for death in systemic sclerosis: a study from the EULAR Scleroderma Trials and Research (EUSTAR) database. Ann Rheum Dis 2010;69:1809–15.
E. Zanatta et al. / Joint Bone Spine 87 (2020) 331–335 [3] Guarnieri G, Zanatta E, Mason P, et al. Determinants of impairment in lung diffusing capacity in patients with systemic sclerosis. Clin Exp Rheumatol 2015;33:S80–6. [4] Elhai M, Hoffmann-Vold AM, Avouac J, et al. Performance of Candidate Serum Biomarkers for Systemic Sclerosis-Associated Interstitial Lung Disease. Arthritis Rheumatol 2019;71:972–82. [5] Bonhomme O, André B, Gester F, et al. Biomarkers in systemic sclerosisassociated interstitial lung disease: review of the literature. Rheumatology (Oxford) 2019;58:1534–46. [6] Pontisso P. Role of SERPINB3 in hepatocellular carcinoma. Ann Hepatol 2014;13:722–7. [7] Lunardi F, Villano G, Perissinotto E, et al. Overexpression of SERPIN B3 promotes epithelial proliferation and lung fibrosis in mice. Lab Invest 2011;91:945–54. [8] Calabrese F, Lunardi F, Giacometti C, et al. Overexpression of squamous cell carcinoma antigen in idiopathic pulmonary fibrosis: clinicopathological correlations. Thorax 2008;63:795–802. [9] Biasiolo A, Chemello L, Quarta S, et al. Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis. J Viral Hepat 2008;15:246–9. [10] Giannelli G, Iannone F, Fransvea E, et al. Squamous cellular carcinoma immunocomplexed is increased in scleroderma patients with lung fibrosis. Clin Exp Rheumatol 2007;25:794–5. [11] Van den Hoogen F, Khanna D, Fransen J, et al. 2013 classification criteria for systemic sclerosis: an American college of rheumatology/European league against rheumatism collaborative initiative. Ann Rheum Dis 2013;72:1747–55.
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Original article
Squamous cell carcinoma antigen-IgM (SCCA-IgM) is associated with interstitial lung disease in systemic sclerosis Elisabetta Zanatta a,1 , Andrea Martini b,1 , Elena Scarpieri a , Alessandra Biasiolo b , Augusta Ortolan a , Francesco Benvenuti a , Franco Cozzi a , Patrizia Pontisso b , Andrea Doria a,∗ a b
Division of Rheumatology, Department of Medicine-DIMED, University of Padova„ 35128 Padova, Italy Unit of Internal Medicine and Hepatology (UIMH), Department of Medicine-DIMED, University of Padova, 35128 Padova, Italy
a r t i c l e
i n f o
Article history: Received 29 September 2019 Accepted 12 February 2020 Available online 19 February 2020 Keywords: Systemic sclerosis Interstitial lung disease SCCA-IgM Serpinb3
a b s t r a c t Objective: Interstitial lung disease (ILD) is the major determinant of prognosis in patients with systemic sclerosis (SSc). Squamous Cell Carcinoma Antigen (SCCA1) is a serin protease inhibitor which plays a pivotal role in inflammation and fibrosis. SCCA1 is overexpressed in pulmonary tissue of patients with idiopathic pulmonary fibrosis and can be detectable in serum as circulating immune complex bound to IgM (SCCA-IgM). We aimed to investigate the association between SCCA-IgM and clinical features of patients with SSc. Methods: Ninety-seven patients with SSc (ACR/EULAR criteria) were consecutively enrolled in the study. Clinical and serological variables and organ involvement were recorded. Pulmonary involvement was investigated by high-resolution CT (HRCT) and respiratory function tests. SCCA-IgM serum levels were measured by a validated ELISA assay (Hepa-IC, Xeptagen, Venice, Italy). We set the cut-off value for serum levels of SCCA-IgM > 200 AU/ml, calculated as mean + 3 standard deviations in 100 healthy subjects. Results: Forty-one (42.3%) patients were affected with ILD. SCCA-IgM values were significantly higher in patients with ILD than in those without: 218 (80-402) vs. 87.5 (59-150) AU/mL, P = 0.003. Patients with positive SCCA-IgM had more frequently ILD (69.7% vs. 28.1%, P ≤ 0.0001) and a lower total lung capacity (TLC) (P = 0.024) compared with negative ones. No differences were found in any other clinical and serological features. At multivariate analysis, SCCA-IgM was found to be associated with ILD diagnosis (OR 10.6, IC 2.9-38.4, P = 0.001). Conclusion: SCCA-IgM is associated with interstitial lung disease in scleroderma patients and might be used in the assessment of SSc-ILD. ´ e´ franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved. © 2020 Societ
1. Introduction Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial dysfunction and autoimmune dysregulation which can lead to fibrosis of skin and internal organs (heart, kidneys, gut and lungs) [1]. Interstitial lung disease (ILD) is frequently detectable and is the leading cause of death in SSc patients, accounting for 35% of disease-related deaths in the European Scleroderma Trial and Research (EUSTAR) database [2]. However, ILD can be totally asymptomatic in the early stage, thus making its diagnosis challenging [3]. To date only a few biomarkers have been identified in SSc-ILD (e.g. Serum Krebs von den Lungen-6, KL-6) and their clin-
∗ Corresponding author. E-mail address: [email protected] (A. Doria). 1 These two authors contributed equally to the article.
ical meaning is still under evaluation [4], as discussed in a recent systematic literature review performed by Bonhomme et al. [5]. Squamous Cell Carcinoma Antigen 1 (SCCA1, also known as SerpinB3) is a serin protease inhibitor, expressed by dysplastic and neoplastic cells of epithelial origin (including lung cancer) and in cirrhotic liver, which plays an important role in the regulation of inflammation, programmed cell death and fibrosis [6]. In a murine model of pulmonary fibrosis, SCCA1 transgenic mice showed higher expression of transforming grow factor beta 1 (TGF-1) and more extended fibrosis along with a significant gain of epithelial proliferation [7]. In line with these results, SCCA1 was found overexpressed in the pulmonary tissue of patients with idiopathic pulmonary fibrosis (IPF) [8], and it positively correlates with the extension of fibroblastic foci, expression of TGF-1 and carbon monoxide decline after 9 months of follow-up. Calabrese et al. [8] hypothesized two mode of action of SCCA1 in IPF: stimulation of epithelial
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proliferation (autocrine action) or activation of fibroblast proliferation/fibrosis via increased TGF-1 secretion (paracrine action). SCCA1 can be detectable in serum as circulating immune complex coupled to IgM (SCCA-IgM) and was found to correlate closely with the extent of liver fibrosis in patients with liver disease [9]. Only one study has investigated SCCA-IgM in SSc so far, showing high levels of this biomarker especially in patients with ILD [10]. The aim of our study was to assess the relation between SCCA-IgM serum levels and demographic and clinical features of SSc patients. 2. Methods 2.1. Patients Ninety-seven patients affected with SSc according to ACR/EULAR criteria [11] were consecutively enrolled in the study which was part of a national Project (FIRB Prot. RBLA03S4SP 005), and approved by the local Ethics Committee. All patients provided written informed consent. Demographic, clinical and serological data of the entire study population are reported in Appendix A, Table S1 [See the supplementary material associated with this article online]. Organ involvement was defined as follows: • cardiac: ventricular arrhythmia, conduction defects, systolic (ejection fraction < 55%) or diastolic dysfunction (according to the ASE criteria, [12]), significant pericardial effusion (> 10 mm and diffuse); • gastrointestinal (GI): gastroesophageal reflux, dysphagia, objective findings of esophageal dysmotility, hiatal hernia, esophagitis and gastritis; bowel obstruction and sub-occlusion, constipation, diarrhea; • pulmonary involvement: interstitial lung disease (ILD) was diagnosed using high-resolution computed tomography (HRCT), in presence of reticular abnormalities, ground-glass opacities and/or honeycombing. According to best practice in SSc-ILD [13], all patients underwent HRCT at diagnosis and then according to symptoms, presence of pulmonary crackles, annual pulmonary function tests (PTFs) and standard chest X-Ray radiography; • pulmonary arterial hypertension (PAH): defined as mean pulmonary pressure ≥ 25 mmHg at right heart catheterization with a pulmonary wedge pressure ≤ 15 mmHg. The modified Rodnan skin score (mRSS), presence of digital ulcers and C-reactive protein (CRP) were also evaluated. Disease activity was measured by EUSTAR activity score [14]. As for ILD, we also considered: • ILD duration = defined from the first evidence of ILD on HRCT; • Pulmonary function tests (PFTs) carried out no later than 6 months prior to the SCCA-IgM assay. The following pulmonary function tests (PFTs) (expressed as the percentage of observed/theoretic values) were considered: forced vital capacity (FVC), total lung capacity (TLC), diffuse capacity for carbon monoxide (DLCO), carbon monoxide transfer coefficient (KCO) calculated as the ratio between DLCO and alveolar volume (Va). According to the literature [15], values of FVC, TLC, DLCO and KCO below 80% were considered pathological. Immunosuppressive and corticosteroids therapy were also recorded. 2.2. SCCA-IgM assay In all patients SCCA-IgM serum levels were measured by a validated ELISA assay (Hepa-IC, Xeptagen, Venice, Italy), according to
the manufacturer’s instructions. Values were expressed in arbitrary units/milligram (AU/ml) and, according to our laboratory cut-off, was considered abnormal a value of SCCA-IgM > 200 AU/mL. This value was obtained by analyzing the sera from 100 healthy subjects (mean value + 3 standard deviations). 2.3. Statistical analysis Continuous variables were expressed as means ± standard deviation (SD) or medians (interquartile range) and categorical variables as frequency and percentage. Comparison between groups was carried out using the Student’s t-test and unpaired t-test with Welch’s correction, or the Mann–Whitney U-test for continuous variables, and the chi-squared-test or Fisher’s exact probability test for categorical data, where appropriate. Multiple comparisons were adjusted by Bonferroni correction. Variables which were found to be different (P < 0.05) between patients with and without ILD at univariate analysis, were then included into a multivariate logistic regression model, adjusted for age and gender. Only pathological FVC, anti-topoisomerase I (anti-Scl70), immunosuppressants and SCCA-IgM were included in the model to avoid multicollinearity. Results are reported as OR with a 95% confidence interval. Sensibility, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy and negative likelihood ratio (NLR) were calculated. Two-sided P < 0.05 was considered statistically significant. The statistical analysis was performed using the SPSS statistical package, version 22.0. 3. Results Among the 97 patients included in the study, 82 (84.5%) were female, mean age 55.4 ± 12.4 years and median disease duration 12 (6–20) years. According to Le Roy et al. [16], 35 patients (36.1%) were classified as affected with diffuse cutaneous form (dcSSc) and 62 (63.9%) with the limited one (lcSSc). Forty-one patients had ILD (42.3%). Demographic, clinical and serologic features in patients with and without ILD are reported in Table 1. The two groups of patients were similar in terms of demographic features and disease duration. Patients with ILD were more frequently affected with dcSSc (P < 0.0001), had higher values of mRSS (P = 0.002) and a higher frequency of anti-Scl70 (P = 0.001) and anti-RNA pol III (P = 0.047) autoantibodies compared with patients without ILD. Anti-centromere antibody was inversely associated with the presence of ILD (P < 0.0001). As expected, patients with ILD had lower values of FVC and TLC than patients without ILD (P < 0.0001 in both cases). In addition, DLCO were lower in the former group (P = 0.002). Conversely, no differences between the two groups in terms of digital ulcers and cardiac or gastrointestinal involvement were observed. EUSTAR score was higher in patients with ILD than in those without (P = 0.029). Median SCCA-IgM values were significantly higher in patients with ILD than in those without: 218 (80-402) vs. 87.5 (59-150) AU/mL, P = 0.004 (Fig. 1A) and in patients with abnormal TLC values (P = 0.03, Fig. 1B). Therefore, we subdivided the study population into two groups according to the positive/negative SCCA-IgM status (cut off: 200 AU/mL). The demographic and clinical features in these two groups of patients are reported in Table 2. Patients with positive SCCA-IgM were more frequently affected with ILD (69.7% vs. 28.1%, P < 0.0001) and had more frequently a TLC < 80% (34.6% vs. 12.5%, P = 0.024); moreover, they tended to have a pathological DLCO (P = 0.058). No differences were found between the two groups regarding other PFTs (FVC and KCO), which, however, were more frequently abnormal in patients with positive SCCA-IgM. Patients treated with steroids or immunosuppressants showed similar serum level of
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Table 1 Demographic and clinical features in the entire study population and according to the presence of ILD. Variable
All patients (n = 97) ILD (n = 41)
NO ILD (n = 56)
Age (yrs) Sex (Female) Disease duration, (yrs) dcSSc mRSS Cardiac involvement PAPs (mmHg) PAH GI involvement Digital ulcers ANA Anti-Scl70 Anti-centromere Anti-RNA polymerase III FVC TLC DLCO KCO FVC < 80% TLC < 80% DLCO < 80% KCO <80% CRP (mg/L) EUSTAR Score Steroids Immunosuppressants
55.4 (± 12.4) 82 (84.5) 12 (6-20) 35 (36.1) 7 (4-15) 20 (21.1) 27.5 (± 10) 6 (6.2) 62 (64.6) 30 (30.9) 97 (100) 26 (28) 50 (53.8) 3 (3.2) 95,9 (± 22.2) 92,9 (± 17) 73,5 (± 21.4) 74.7 (± 18) 19 (20) 15 (20.3) 60 (65.2) 47(63.5) 2.5 (2.0-6.9) 1.3 (± 1.2) 18 (18.8) 33 (34)
56.5 (± 12.5) 49 (87.5) 13.5 (4-21) 11 (19.6)* 4.5 (3.7-10.5)** 9 (16.1) 27.4 (± 10.4) 4 (7.1) 34 (60.7) 15 (26.8) 56 (100) 20 (35.7)** 23 (41.1)* 0 (0)*** 104.5 (± 18.1)* 101.2 (± 13.8)* 79,3 (± 20.8)** 75.8 (± 17.5) 4 (7.1)** 3 (5.4)* 30 (53.6)*** 25 (44.6) 3 (5.8-7) 1.1 (± 1.1)*** 9 (16.1) 13 (23.2)***
53.9 (± 12.4) 33 (80.5) 11 (6.7-16) 24 (58.5) 13.4 (7.5-17.5) 11 (26.8) 27.6 (± 9.9) 2 (4.9) 28 (68.3) 15 (36.6) 41 (100) 30 (73.2) 2 (4.9) 3 (7.3) 84.5 (± 22.1) 82,2 (± 15.1) 65.5 (± 19.7) 73.2 (± 20.2) 15 (36.6) 15 (36.5) 30 (73.2) 22 (53.6) 2.5 (2-10.4) 1.6 (± 1.3) 9 (22) 20 (48.8)
Values are reported as n (%) or mean (± SD) and median (range). ANA: antinuclear antibodies; yrs: years; CRP: C-reactive protein; dcSSc: diffuse cutaneous form; FVC: forced vital capacity; ILD: interstitial lung disease; TLC: total lung capacity; DLCO: diffuse capacity for carbon monoxide; KCO: carbon monoxide transfer coefficient; mRSS: modified Rodnan skin score; GI: gastrointestinal; PAPs: systolic pulmonary artery pressure; PAH: pulmonary arterial hypertension; Scl70: anti-topoisomerase I. For continuous and dichotomous variables significance values at P ≤ 0.005 and P ≤ 0.003, respectively, were obtained after Bonferroni correction for multiple comparisons. * P < 0.001. ** P < 0.01. *** P < 0.05.
SCCA-IgM, in comparison to those untreated (P = 0.435). The two groups were similar in all the other clinical variables, e.g. mRSS, cardiac and gastrointestinal involvement, digital ulcers, EUSTAR score. Considering only patients with SSc-ILD, those with TLC < 80% and FVC < 80% have more frequently SCCA-IgM positivity than patients with these PFTs within normal range (in the 84.2% vs. 15.8% and 76.2% vs. 23.8% of cases, P = 0.041 and P = 0.064, respectively). At the multivariate logistic regression analysis (Table 3) the independent predictors of ILD were anti-Scl70, FVC < 80% and SCCAIgM positivity (OR = 10.6, P = 0.001). Interestingly, patients with an ILD duration ≤ 3 years showed higher levels of SCCA-IgM: 436 (174–1058) vs. 86 (35-329) AU/ml, P = 0.003 (Fig. 1C). To improve the identification of patients with a very low risk of ILD, we combined the main PFTs with SCCA-IgM status (Suppl. Table 1). Among different associations, the one between SCCA-IgM and TLC showed the highest accuracy (80%) and NPV (83%) with the lowest NLR (0.26), performing better than any single variable.
Fig. 1. Squamous cell carcinoma antigen (SCCA)-IgM values and presence of Interstitial Lung Disease (ILD) (Panel A), TLC < 80% (Panel B) and ILD duration (Panel C) in patients with systemic sclerosis. The box indicates the lower and the upper quartile, and the middle line indicates the median. Bars indicate the range of values distribution. Round circles represent outliers. ILD: interstitial lung disease; TLC: total lung capacity; Squamous cell carcinoma antigen (SCCA)-IgM.
4. Discussion ILD is the major determinant of poor prognosis in SSc patients [2], therefore the identification of biomarkers of pulmonary involvement is paramount in the clinical assessment of these patients [4]. The pro-fibrogenic action of SCCA1 is supported by its ability to induce TGF-1 and by the evidence that in activated hepatic stellate cells it is able to exert a pro-fibrogenic response [17]. These findings were also confirmed in an animal model of idiopathic pulmonary
fibrosis [7]. SCCA1 is detectable in serum as bound to IgM (SCCAIgM) and its levels have been shown to be closely related with the extent of liver fibrosis in patients with chronic liver disease [9]. Our results showed that the levels of SCCA-IgM are higher in SSc patients with ILD than in those without. Moreover, at multivariate analysis patients with positive SCCA-IgM had a 10-fold greater risk of being affected with ILD. Importantly, this association was independent from the autoantibody profile, FVC values and immunosuppressive therapy, which are classical features closely
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Table 2 Demographic and clinical features in SSc patients according to the positive/negative SCCA-IgM status. Variable
SCCA-IgM > 200 AU/ml (n = 33)
SCCA-IgM < 200 AU/ml (n = 64)
Age (yrs) Female Disease duration (yrs) dcSSc mRSS ILD Cardiac involvement PAPs (mmHg) PAH GI involvement Digital ulcers ANA Anti-Scl70 Anti-centromere Anti-RNA polymerase III FVC < 80% TLC < 80% DLCO < 80% KCO < 80% CRP (mg/L) EUSTAR Score Steroids Immunosuppressants
56.2 (± 12.5) 30 (90.9) 14 (8-20) 12 (36.4) 10 (4-15) 23 (69.7) 7 (21.2) 28.1 (± 9) 2 (6.1) 20 (60.6) 10 (30.3) 33 (100) 19 (57.6) 9 (27.3) 0 (0)
55 (± 12.5) 52 (81.3) 10 (6-19.5) 23 (35.9) 7 (4-14.7) 18 (28.1)* 13 (21) 27.1 (± 10.6) 4 (6.3) 42 (66.7) 20 (31.3) 64 (100) 31 (51.7) 17 (28.3) 3 (5)
9 (27.3) 9 (34.6) 23 (74.2) 20 (74.1) 3.7 (2.4-11.3) 1.5 (± 1.3) 9 (27.3) 9 (27.3)
10 (16.1) 6 (12.5)** 37 (60.7) 27 (57.4) 2.5 (1.7-6.8) 1.2 (± 1.1) 9 (14.3) 24 (37.5)
Values are reported as n (%) or mean (± SD) and median (range). ANA: antinuclear antibodies; yrs: years; CRP: C-reactive protein; dcSSc: diffuse cutaneous form; FVC: forced vital capacity; ILD: interstitial lung disease; TLC: total lung capacity; DLCO: diffuse capacity for carbon monoxide; KCO: carbon monoxide transfer coefficient; mRSS: modified Rodnan skin score; GI: gastrointestinal; PAPs: systolic pulmonary artery pressure; PAH: pulmonary arterial hypertension; SCCA: squamous cell carcinoma antigen; Scl70: anti-topoisomerase I. For continuous and dichotomous variables significance values at P ≤ 0.008 and P ≤ 0.003, respectively, were obtained after Bonferroni correction for multiple comparisons. * P < 0.001. ** P < 0.05.
Table 3 Multivariate analysis for the diagnosis of ILD. Variable
OR (CI 95%)
Anti-Scl70 FVC < 80% SCCA-IgM (+) Immunosuppressants
3.3 (1.04–10.7)* 9.9 (1.8–54.8)** 10.6 (2.9–38.4)*** 3.149 (0.9–10.7)
Model adjusted for sex and age. OR, odds ratio, CI confidence interval 95%; dcSSC, diffuse cutaneous form; FVC, forced vital capacity; SCCA-IgM (+), Squamous cell carcinoma antigen-IgM > 200 AU/ml; Scl70, anti-topoisomerase I; yrs: years. * P < 0.05. ** P < 0.01. *** P < 0.001.
the pathogenesis of SSc, SCCA1 could be involved in this process, at least in the lung, becoming a potential target of new therapeutic approaches. Moreover, SCCA1 is able to induce epithelial-to mesenchymal transition [18], a process strongly involved in the fibrogenesis of SSc-ILD [19]. In our study, SCCA-IgM was not associated with any other clinical or serological disease features. Notably, SCCA-IgM was not associated with skin fibrosis (both dcSSc and mRSS), and cardiac or gastrointestinal involvement. It has been suggested that SCCA is upregulated in the skin of patients with cutaneous inflammatory diseases (i.e. psoriasis and atopic dermatitis). Inflammatory skin changes occur early in scleroderma, and are quickly replaced by fibrotic and atrophic lesions. Thus, the lack of association between SCCA1 and skin involvement in our patients could be due to their quite long mean disease duration. Moreover, the role of SCCA1 in inducing a pro-fibrogenic response is closely dependent on the profibrogenic environment [17], which may have different features in the skin and lungs of SSc patients. We did not find any association between positive SCCA-IgM and vascular manifestations of SSc (PAPs, digital ulcers) or KCO, which is an index of pulmonary vasculopathy in SSc [20]; overall, these findings strongly support SCCA-IgM as a potential biomarker for pulmonary fibrosis in SSc. Moreover, our results suggest that the addition of negative SCCA-IgM status to normal values of TLC seems helpful in the identification of scleroderma patients with a very low risk of ILD. The extreme variability of normal values of TLC (ranging from 80 to 120%) limits the usefulness of pulmonary function tests in excluding the presence of ILD. Therefore, the addition of serum SCCA-IgM assessment to routine PFTs may be useful in clinical practice for the identification of those patients who may not require CT-scan. Our study has some limitations. First of all, the sample size is relative small; however, it must be taken into account that scleroderma is a rare disease. Second, the lack of a radiological score evaluation does not allow to define the severity of radiological extension, in addition to functional impairment. However, the stratification of patients according to the ILD severity was not an aim of our study. By contrast, one strength of our study is that all patients considered are followed in a third referral center with a great expertise in the management of scleroderma and therefore the possibility that some patients have not been diagnosed with SSc-ILD is very unlikely. In conclusion, our data suggest that SCCA-IgM may be considered a candidate biomarker in SSc-ILD assessment. Disclosure of interest The authors declare that they have no competing interest. Funding
associated with ILD. All these data suggest that SCCA1 might be involved in the pulmonary fibrosis observed in SSc and that SCCAIgM might become a potential biomarker in SSc-ILD. Compared with a previous report [10], our study included a larger number of patients, with a comprehensive functional pulmonary characterization. Besides the association with ILD diagnosis, in the whole population and in the subgroup of patients with ILD we found a significant association between SCCA-IgM positivity and pathological values of TLC, an index of pulmonary restriction. We also observed higher levels of SCCA-IgM in patients with ILD duration ≤ 3 years. Taken together, these data suggest that SCCA-IgM could be useful in the early diagnosis of ILD along with the identification of severe forms. The increased levels of SCCA-IgM in the early stage of ILD could be explained by the evidence that SCCA1 can inhibit inflammation while favoring fibrogenesis [7]. Since the transition from an inflammatory to a fibrotic phase is one of the crucial step in
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