ABSTRACT

Conclusion:

SAA could be proposed as a more potential biomarker than S100A12. Both discriminative value for active vasculitis and correlation with disease activity of SAA were more significant than S100A12. Our results point to the need of further longitudinal researches regarding utility of SAA and SAA-like proteins in the assessment of vasculitis.

Results:

Patients with active vasculitis had significantly higher levels of CRP, S100A12, and SAA, and higher ratios of NLR and PLR compared to HCs. S100A12 did not correlate with disease activity and other biomarkers. SAA had the most significant correlation with BVAS (r=0.63, p<0.05). All biomarkers have the capability of effectively discriminating patients with vasculitis from HCs. The area under the curve (AUC) of CRP, SAA, S100A12, NLR and PLR was 0.95 (95% confidence interval 0.88-0.99), 0.82 (0.65-0.98), 0.77 (0.60-0.95), 0.90 (0.79-0.99) and 0.89 (0.76-0.99), respectively. SAA had better AUC compared to S100A12. A level of 7.5 mg/L (sensitivity 78%, specificity 83%) for SAA and 24 ng/mL (sensitivity 89%, specificity 67%) for S100A12 were found as the optimum cut-off points for defining active vasculitis.

Methods:

A total of 18 clinically active vasculitis patients [giant cell arteritis (n=6), Takayasu arteritis (n=6), and granulomatosis with polyangiitis (n=6)] and 12 healthy controls (HCs) were enrolled. Disease activity was assessed using the Birmingham disease activity score (BVAS). The correlation between SAA, S100A12, C-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and BVAS was analyzed. Receiver operating characteristics analysis was used to evaluate the diagnostic utility of each marker.

Objective:

There are still unmet needs for ideal biomarkers for management of vasculitis. This study aimed to analyze the potential utilities of the S100A12 and Serum Amyloid A (SAA) as diagnostic or activity-specific biomarkers of vasculitis.

Keywords:

Serum amyloid A, S100A12, vasculitis, biomarker

Introduction

Vasculitides are a heterogeneous group of diseases, characterized by inflammation of blood vessels, causing severe tissue destruction and organ/system failure. Given the variability of the location and size of the involved vessels, there is a great diversity of clinical presentation and severity. This heterogeneity and insidious natural history make the management of vasculitis challenging.^[1] Vasculitis related damage causes significant mortality, morbidity and economic burden.^[2] The prompt and accurate treatment of patients regarding their organ involvement is crucial to prevent damage. Therefore, identifying the disease and relapse is quite important. Despite our increasing knowledge about vasculitis, there are still unmet needs for ideal biomarkers. Available markers, including antineutrophil cytoplasmic antibodies,^[3] erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are unreliable and inaccurate for diagnosis, assessing disease activity and defining relapse. For instance ESR and CRP can be quite normal in some clinically active Takayasu and histopathologically active giant cell arteritis (GCA) patients.^[4-6]

Serum amyloid A (SAA) is an acute-phase protein that belongs to the apolipoprotein family. SAA is synthesized primarily in the liver by activated monocytes and macrophages in response to proinflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor-a (TNF-a).^[7] The S100 protein family is a unique class of calcium-binding proteins which are expressed and released at local sites of inflammation.^[8-10] Members of the S100 protein family are the most abundant danger-associated molecular patterns (DAMPs), which participated in an inflammatory reaction during stress, and associated with various receptors (e.g. Toll-like receptor 4) and advanced glycosylation end products (RAGE).^[11] S100A12 is more restricted to granulocytes with several important extracellular roles including activating the NF-kB pathway, which results in the expression of the proinflammatory cytokines and adhesion molecules.^[12,13] The neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) have emerged as a markers of inflammation and are found to be a useful indices for estimating the current inflammatory burden and disease activity of vasculitis.^[14,15]

The capability of neither SAA nor S100A12 in the assessment of vasculitis has not been fully elucidated. In particular, we are not aware of any study that compares S100A12 and SAA in that field. In light of this background, this study analyzed the plasma levels of SAA and S100A12 in patients with active vasculitis and healthy controls (HCs) and we aimed to investigate their correlation with disease activity and other biomarkers, and to identify the potential utilities as identifying diagnosis or relapse in patients with GCA, Takayasu arteritis, and granulomatosis with polyangiitis (GPA).

Materials and Methods

Eighty-five consecutive vasculitis patients, whether newly diagnosed or relapsing, were admitted to the clinic during the study period. The inclusion criterion was clinically active disease. Exclusion criteria were refusal to participate, having inactive disease, other autoimmune conditions or malignancies, being under 18 years of age and being pregnant. Sixty-seven of them were excluded because they met the exclusion criteria or reached the target number per group. Total of eighteen consecutive active vasculitis patients (six patients for each) who fulfilled the American College of Rheumatology classification criteria for GCA,^[16] Takayasu arteritis^[17] and GPA^[18] were enrolled in this cross-sectional study. Twelve healthy controls (HCs), who had no signs or symptoms that direct an inflammatory disease, participated in this study (Figure 1). The study was conducted after the ethical approval of the local ethics committee (approval number: 98, date: 27/01/2020) complied with the declaration of Helsinki. Informed consent was obtained from all patients and HCs before the initiation of the study.

Demographic data were collected. The disease activity was assessed by the Birmingham Vasculitis Activity Score (BVAS v3), which is composed of nine major items.^[19] BVAS>0 was accepted as active disease. Conventional inflammatory biomarkers included CRP, NLR and PLR analyzed by use of in-house method. CRP was studied with the nephelometric method and under 5 milligrams per liter (mg/L) is considered normal. Peripheral venous samples were obtained; after clothing, the tubes were centrifuged at 3000x for 5 min, and sera were stored at -80⁰C for further analyses. S100A12 and SAA were analyzed by enzyme-linked immunosorbent assay (ELISA) methods. Human S100A12 was studied with a commercially available ELISA kit (Boster Immunoleader En Rage PicoKineTMCA, USA). The assay detection range was 31.2 pg/mL-2000 pg/mL and sensitivity was <10 pg/mL. SAA was studied with commercially available SAA ELISA Kit (Assay Pro, MO, USA). The assay detection range was 0.125-2 ug/mL and sensitivity was 6 ug/mL.

Statistical Analyses

We conducted all statistical analyses with SPSS software (version 15 for Windows; SPSS INC., Chicago, IL, USA). The variables were investigated using visual (histograms, probability plots) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk’s test) to determine the distribution of data. Continuous variables were expressed as a median values with minimum-maximum (min-max). Categorical data are summarized as absolute frequencies and percentages. We compared the biomarker levels across different subgroups by either Kruskal-Wallis (>2 groups) or Mann-Whitney U (2 groups) tests, when appropriate. Spearman’s correlations were calculated to define the association between biomarkers and disease activity. Receiver operating characteristic (ROC) curve analysis was performed to establish the optimal discriminatory threshold for identifying patients with vasculitis from HCs. A two-tailed p-value <0.05 was considered statistically significant.

Results

Table 1 details the characteristics and biomarker levels of both groups. Patients were significantly older than HCs [52 (28-80) vs. 36 (26-56), respectively, p=0.01]. Gender distribution and body mass index (BMI) were comparable. Median BVAS of the patients was 4 (min-max, 1-14). CRP, SAA, S100A12, NLR and PLR were significantly higher in patients than in HCs [61.4 (3-306) vs. 2.2 (1.24-4.15), p<0.001; 26.0 (0.07-42) vs. 2.0 (0.49-15.62), p=0.004; 60.8 (17.1-125.3) vs. 21.4 (16.1-82.2), p=0.013; 5.1 (1.3-20.8) vs. 1.6 (1.2-4.5) p<0.001; and 304.5 (72.2-1322.5) vs. 107.0 (67.8-230.8), p<0.001, respectively].

The patient population comprised six patients with GCA, Takayasu arteritis and GPA. Further subgroup analyses showed a statistical difference between these groups in terms of CRP, SAA, NLR, and PLR, unlike S100A12 (p=0.083) (Table 2). Correlation analysis between biomarkers and BVAS were performed separately for the patients and HCs. There was no correlation between biomarkers in the HCs (p>0.05, data not shown). SAA has a high positive correlation with CRP, whereas NLR and PLR have a moderate positive correlations (Table 3, r=0.8, p<0.001; r=0.5, p=0.035; and r=0.49, p=0.038, respectively). S100A12 was highly correlated with CRP only in GCA patients (r=0.83, p<0.05,). Moreover, CRP, SAA and NLR were moderately correlated with BVAS for all patients (r=0.57, p=0.013; r=0.63, p=0.005; and r=0.52 p=0.028 respectively). There was no correlation between BVAS and S100A12.

The area under the curve (AUC) of CRP, SAA, S100A12, NLR and PLR was found to be significantly greater than the reference line [p<0.05, AUC (95% CI): 0.95 (0.88-0.99), 0.82 (0.65-0.98), 0.77 (0.60-0.95), 0.90 (0.79-0.99) and 0.89 (0.76-0.99), respectively]. A level of 7.5 mg/L (sensitivity 78%, speciﬁcity 83%) for SAA and 24 ng/mL (sensitivity 89%, specificity 67%) for S100A12 were found as the optimum cut-off points for diagnosing vasculitis (Figure 2). There were two patients with active vasculitis who had normal CRP levels. Both of them had GCA and their BVAS was found 2 and 3. SAA was higher than the defined cut off limit in one patient (25.44 mg/L) while S100A12 was higher than the defined cut off limit (81.44 and 37.22 ng/mL) in both patients.

Discussion

In view of challenges in the management of vasculitis and not having an ideal biomarker to diagnose, and to determine the disease activity and relapses, it is important to study new biomarkers. Especially, biomarkers, which can assess disease activity reliably and detect relapse, are urgently needed in clinical practice. S100A12 and SAA have not been fully analyzed for this purpose. In particular, this is the first study that compares SAA and S100A12. Here, we report a preliminary study, which analyzed the utility of SAA and S100A12 on discriminating patients with active vasculitis, whether at initial presentation or relapse, from healthy controls, and investigated the correlation between disease activity and S100A12 and SAA.

S100A12 did not correlate with disease activity and had a significant but a lower capability of discriminating active vasculitis from HCs among all biomarkers. This is noteworthy since S100 proteins have received much attention recently. The S100 protein family is a unique class of calcium-binding proteins which are expressed and released at local sites of inflammation.^[7-9] Besides S100A12 and S100A8/9 proteins have been shown to be useful biomarkers of disease activity in some rheumatic diseases in many studies.^[20-25] However, there are some conflicts about S100A12. On one hand, S100A12 was found to be elevated in patients with active MPO-ANCA-associated glomerulonephritis, Behçet’s disease and GCA compared to vasculitis patients or/and HCs. Moreover, S100A12 was correlated with disease activity and pathologic activity scores^[26] and was strongly expressed in the pathological examination of GCA, and Behçet’s disease.^[26-28] On the second hand, S100A12 was found similar in active and inactive Takayasu patients.^[29] Above all, S100A12 was found to be related to aortic aneurysms and wall repair itself, irrespective of inflammatory diseases.^[30,31] The major concern is the association between S100A12 and vessel wall damage itself, since this makes it impossible to discriminate between the active and inactive patients, which is an urgent need in clinical practice

Both the discriminative capability of active vasculitis from HCs and the correlation with disease activity of SAA was more significant than S100A12. SAA may be a reliable biomarker than S100A12 for assessing patients with vasculitis. This is somehow expected since all available data about SAA, even very few, supported our results; SAA might be related to the pathogenesis of GCA^[32] and was a more reliable biomarker than CRP in patients with active GCA and MPO-ANCA associated vasculitis.^[33,34] Moreover, SAA was elevated in Takayasu patients^[35] and decreased in treatment responders,^[36] unlike S100A12. Moreover, this study supported the knowledge that CRP can be quite normal in some clinically active Takayasu and histopathologically active GCA patients,^[4-6] as our two clinically active GCA patients had normal CRP levels. Though SAA was highly correlated with CRP, SAA effectively differentiated one of these patients from healthy controls. Although the knowledge regarding SAA and vasculitis is more enlightening and precise then S100A12, SAA fell out of favor. However, according to our results, this should be reconsidered and further research should be conducted.

Study Limitations

A small sample size and lack of follow-up assessment were the major limitations of our study. The main limitations were the lack of diseased controls (mimickers) and inactive vasculitis patients. Moreover, BVAS is not the perfect-activity assessment measure for patients with GCA and Takayasu arteritis.

Conclusion

Consequently, this is the first study that compares SAA and S100A12 as biomarkers in vasculitis patients and has results to widen the research fields. Both discriminative capability of active vasculitis and correlation with disease activity of SAA were more significant than S100A12. Our results draw attention to the SAA as a biomarker of vasculitis and point to the need of further longitudinal research regarding the diagnostic and assessment utility of SAA and SAA-like proteins for vasculitis.

Ethics

Ethics Committee Approval: The study was conducted after the ethical approval of the local ethics committee
(Gazi University Ethics Committee, approval number: 98, date: 27/01/2020) complied with the declaration of Helsinki.

Informed Consent: Informed consent was obtained from all patients and HCs before the initiation of the study.

Peer-review: Externally and peer reviewed.

Conflict of Interest: No conflict of interest was declared by the author.

Financial Disclosure: The author declare that they have no relevant financial disclosures.

References

Fauci AS, Haynes B, Katz P. The spectrum of vasculitis: clinical, pathologic, immunologic and therapeutic considerations. Ann Intern Med 1978;89:660-76.

Trieste L, Palla I, Baldini C, et al. Systemic vasculitis: how little we know about their societal and economic burden. Clin Exp Rheumatol 2012;30:S154-6.

Zena-Huancas PA, Iparraguirre-Lopez H, Gamboa-Cardenas RV, et al. Homocysteine levels are independently associated with damage accrual in systemic lupus erythematosus patients from a Latin-American cohort. Clin Rheumatol 2019;38:1139-46.

Dagna L, Salvo F, Tiraboschi M, et al. Pentraxin-3 as a marker of disease activity in Takayasu arteritis. Ann Intern Med 2011;155:425-33.

Kermani TA, Schmidt J, Crowson CS, et al. Utility of erythrocyte sedimentation rate and C-reactive protein for the diagnosis of giant cell arteritis. Semin Arthritis Rheum 2012;41:866-71.

Kerr GS, Hallahan CW, Giordano J, et al. Takayasu arteritis. Ann Intern Med 1994;120:919-29.

Urieli-Shoval S, Linke RP, Matzner Y. Expression and function of serum amyloid A, a major acute-phase protein, in normal and disease states. Curr Opin Hematol 2000;7:64-9.

Donato R, Cannon BR, Sorci G, et al. Functions of S100 proteins. Curr Mol Med 2013;13:24-57.

Moore BW. A soluble protein characteristic of the nervous system. Biochem Biophys Res Commun 1965;19:739-44.

Donato R. Intracellular and extracellular roles of S100 proteins. Microsc Res Tech 2003;60:540-51.

Foell D, Wittkowski H, Roth J. Mechanisms of disease: a ‘DAMP’ view of inflammatory arthritis. Nat Clin Pract Rheumatol 2007;3:382-90.

Foell D, Wittkowski H, Vogl T, Roth J. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol 2007;81:28-37.

Hofmann MA, Drury S, Fu C, et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 1999;97:889-901.

Pan L, Du J, Li T, Liao H. Platelet-to-lymphocyte ratio and neutrophil-to-lymphocyte ratio associated with disease activity in patients with Takayasu’s arteritis: a case-control study. BMJ Open 2017;7:e014451.

Abaza NM, El-Latif EMA, Gheita TA. Clinical Significance of Neutrophil/lymphocyte Ratio in Patients With Granulomatosis With Polyangiitis. Reumatol Clin (Engl Ed) 2019;15:363-7.

Hunder GG, Bloch DA, Michel BA, et al. The American College of Rheumatology 1990 criteria for the classification of giant cell arteritis. Arthritis Rheum 1990;33:1122-8.

Arend WP, Michel BA, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis. Arthritis Rheum 1990;33:1129-34.

Leavitt RY, Fauci AS, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of Wegener’s granulomatosis. Arthritis Rheum 1990;33:1101-7.

Mukhtyar C, Lee R, Brown D, et al. Modification and validation of the Birmingham Vasculitis Activity Score (version 3). Ann Rheum Dis 2009;68:1827-32.

Chen YS, Yan W, Geczy CL, Brown MA, Thomas R. Serum levels of soluble receptor for advanced glycation end products and of S100 proteins are associated with inflammatory, autoantibody, and classical risk markers of joint and vascular damage in rheumatoid arthritis. Arthritis Res Ther 2009;11:R39.

Foell D, Kane D, Bresnihan B, et al. Expression of the pro-inflammatory protein S100A12 (EN-RAGE) in rheumatoid and psoriatic arthritis. Rheumatology (Oxford) 2003;42:1383-9.

Frosch M, Strey A, Vogl T, et al. Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis. Arthritis Rheum 2000;43:628-37.

Lugering N, Stoll R, Schmid KW, et al. The myeloic related protein MRP8/14 (27E10 antigen)--usefulness as a potential marker for disease activity in ulcerative colitis and putative biological function. Eur J Clin Invest 1995;25:659-64.

Schulze zur Wiesch A, Foell D, Frosch M, Vogl T, Sorg C, Roth J. Myeloid related proteins MRP8/MRP14 may predict disease flares in juvenile idiopathic arthritis. Clin Exp Rheumatol 2004;22:368-73.

Soyfoo MS, Roth J, Vogl T, Pochet R, Decaux G. Phagocyte-specific S100A8/A9 protein levels during disease exacerbations and infections in systemic lupus erythematosus. J Rheumatol 2009;36:2190-4.

Komatsuda A, Ohtani H, Wakui H, et al. Increased serum levels of S100A12 in patients with MPO-ANCA-associated glomerulonephritis. Clin Nephrol 2006;66:315-21.

Foell D, Hernandez-Rodriguez J, Sanchez M, Vogl T, Cid MC, Roth J. Early recruitment of phagocytes contributes to the vascular inflammation of giant cell arteritis. J Pathol 2004;204:311-6.

Han EC, Cho SB, Ahn KJ, et al. Expression of Pro-inflammatory Protein S100A12 (EN-RAGE) in Behcet’s Disease and Its Association with Disease Activity: A Pilot Study. Ann Dermatol 2011;23:313-20.

Springer JM, Monach P, Cuthbertson D, et al. Serum S100 Proteins as a Marker of Disease Activity in Large Vessel Vasculitis. J Clin Rheumatol 2018;24:393-5.

Hofmann Bowman M, Wilk J, Heydemann A, et al. S100A12 mediates aortic wall remodeling and aortic aneurysm. Circ Res 2010;106:145-54.

Daugherty A, Rateri DL, Lu H. S100A12 links to thoracic aortic aneurysms. Circ Res 2010;106:13-5.

O’Neill L, Rooney P, Molloy D, et al. Regulation of Inflammation and Angiogenesis in Giant Cell Arteritis by Acute-Phase Serum Amyloid A. Arthritis Rheumatol 2015;67:2447-56.

Hachulla E, Saile R, Parra HJ, et al. Serum amyloid A concentrations in giant-cell arteritis and polymyalgia rheumatica: a useful test in the management of the disease. Clin Exp Rheumatol 1991;9:157-63.

Shikama Y, Kuriu K, Shibuya Y, et al. [Serum amyloid protein A was a useful marker for steroid tapering in a case of MPO-ANCA-associated vasculitis]. Nihon Kokyuki Gakkai Zasshi 2005;43:588-94.

Koga T, Nishino Y, Makiyama J, et al. Serum amyloid A is a useful marker to evaluate the disease activity of Takayasu’s arteritis. Rheumatol Int 2010;30:561-3.

Nair AM, Goel R, Hindhumati M, et al. Serum amyloid A as a marker of disease activity and treatment response in Takayasu arteritis. Rheumatol Int 2017;37:1643-9.

Serum amyloid A may be a potential biomarker for vasculitis