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Myeloid-related proteins 8/14 failed to act as theragnostic biomarker in axial spondyloarthritis patients on combination disease-modifying anti-rheumatic drugs therapy


1 Department of Clinical Immunology and Rheumatology, Christian Medical College and Hospital, Vellore, Tamil Nadu, India
2 Department of Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission14-Aug-2020
Date of Acceptance22-Sep-2020

Correspondence Address:
Debashish Danda,
Department of Clinical Immunology and Rheumatology, Christian Medical College and Hospital, Ida Scudder Road, Vellore - 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/injr.injr_223_20

  Abstract 


Background: Reports signifying the utility of myeloid-related proteins (MRP) 8/14 in axial spondyloarthritis (AxSpA) as a theragnostic biomarker are scarce.
Objectives: Evaluating the utility of serum MRP 8/14 (baseline levels and change from baseline to 3 months) in AxSpA as a predictor of Assessment of SpondyloArthritis International Society (ASAS) 20 response at 6 months post combination disease-modifying anti-rheumatic drugs (DMARD) therapy.
Methods: Serum MRP 8/14 was assayed using enzyme-linked immunosorbent assay platforms (R&D systems, USA) at baseline in 83 AxSpA patients satisfying ASAS 2009 criteria meeting the predefined eligibility criteria; treated with a combination of methotrexate, sulfasalazine at optimum tolerated doses with on-demand nonsteroidal anti-inflammatory agents and 30 healthy age-matched controls. Repeat measurement was done at 3 months in 60 patients.
Results: Median MRP 8/14 levels in AxSpA patients was 3.00 (3.96) μg/ml compared to 2.3 (3.29) μg/ml in controls (P = 0.2). Median baseline MRP 8/14 levels in ASAS 20 responders at 6 months (n = 36) was 3.6 (4.1) μg/ml compared to 2.4 (4.8) μg/ml in nonresponders (n = 35) (P = 0.4). Median △ (baseline to 3 months) MRP 8/14 levels in ASAS 20 responders at 6 months (n = 33) was – 1 (−2.7) μg/ml compared to − 0.2 (−3.8) μg/ml for nonresponders (n = 27) (P = 0.5). Among the 83 patients with 138 disease activity assessments at baseline and 3 months post therapy, median MRP 8/14 in active disease (bath ankylosing spondylitis disease activity index [BASDAI] ≥4) (n = 112) was 2.2 (3.8) μg/ml, compared to 2 (2.3) μg/ml (P = 0.5) in inactive disease (BASDAI < 4) (n = 26).
Conclusion: Serum MRP 8/14 did not serve as a theragnostic biomarker in our cohort of AxSpA patients treated with combination DMARDs and on-demand NSAIDs.

Keywords: Axial spondyloarthritis, calprotectin, biomarker, disease-modifying anti-rheumatic drugs, myeloid-related proteins 8/14



How to cite this URL:
Ganapati A, Kabeerdoss J, Gowri M, Antonisamy B, Danda D. Myeloid-related proteins 8/14 failed to act as theragnostic biomarker in axial spondyloarthritis patients on combination disease-modifying anti-rheumatic drugs therapy. Indian J Rheumatol [Epub ahead of print] [cited 2020 Dec 4]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=298857




  Introduction Top


Common applications of biomarkers in rheumatology are in disease activity assessment and prediction of therapeutic response (theragnostic), wherein they can supplement clinical assessments and guide in decision-making.[1] There is a dearth of good clinical biomarkers in axial spondyloarthritis (AxSpA). Even C-reactive protein (CRP), the most frequently and widely used biomarker, is elevated in only up to 40%–60% of active AxSpA patients.[2] Novel biomarkers in clinical disease activity assessment are being studied and validated in AxSpA;[3] however, theragnostic markers are scarce and validated reference standards for the same do not exist. Myeloid-related proteins (MRP) 8/14 are one among the many biomarkers being evaluated in AxSpA lately.

MRP 8/14 constitutes calcium-binding heterodimeric complexes of S100A8 and S100A9. Being endogenous ligands for toll-like receptor 4 (TLR4),[4] they are involved in the pathogenesis of AxSpA through innate immune activation.[5] TLR4 engagement can cause downstream activation of the MyD88 pathway with resultant release of nuclear factor-kappa B, tumor necrosis factor (TNF) alpha, and interleukin-17 (IL-17).[4]

MRP 8/14 proteins are produced by activated neutrophils and monocytes which migrate to the sites of inflammation and also released locally at the sites of peripheral arthritis in AxSpA. MRP 8/14 levels and MRP 8/14 positive staining cells have been shown to reduce with effective treatment of peripheral SpA.[6] However, expression of MRP 8/14 in AxSpA is less clearly elucidated in literature, except for a study showing the upregulation of MRP 8/14 in inflamed axial entheseal sites in SKG mice model.[7]

MRP 8/14 has been reported to be beneficial as theragnostic markers for treatment with (a) nonsteroidal anti-inflammatory agents (NSAIDs) in ankylosing spondylitis (AS),[8] (b) Anti-TNF therapy in AxSpA (although in small samples in proof-of-concept trial),[9] and (c) NSAIDs/disease-modifying anti-rheumatic drugs (DMARDs)/biological therapy in a recent large study from China.[10]

Furthermore, the theragnostic role of MRP 8/14 levels in AxSpA treated with combined conventional synthetic DMARDs has not been explored till date. Thus, we studied MRP 8/14 (baseline levels and change from baseline to 3 months) with the objective of evaluating its utility in AxSpA as a predictor of Assessment of SpondyloArthritis International Society (ASAS) 20 response at 6 months, post combined DMARD therapy.


  Methods Top


The study was undertaken at the Department of Clinical Immunology and Rheumatology, Christian Medical College Hospital, Vellore, India, from August 2016 to December 2017. Institutional review board and ethics committee (Min. No. 10207 dated August 8, 2016) approval was duly obtained and additional funding was also received from Indian Rheumatology Association research grant 2017-2018 dated July 15, 2017. The study was conducted in accordance with the principles of the Declaration of Helsinki.

Study population

Following written informed consent, 83 consecutive patients of AxSpA ≥18 years were recruited, satisfying ASAS 2009 criteria[11] with bath AS disease activity index (BASDAI)≥4 or ASDAS ≥2.1, initiated on the combination of methotrexate (MTX) and sulfasalazine (SSZ) along with need-based NSAIDs, as per treating physician discretion at our center (due to financial constraints related to biological usage). Patients with proven infection within a 6-week period of recruitment (n = 7) at assessment and those with secondary causes (n = 14) of AxSpA (psoriatic, enteropathic, reactive, and juvenile onset) were excluded.

Following informed consent, 30 healthy age-matched controls who did not suffer any acute illness at the time of assessment or in the preceding 6 weeks and were free from malignancies were also recruited.

Baseline serum samples were collected from all recruits. Repeat serum samples were collected from 60 of the total 83 patients after 3 months of therapy (19 patients were lost to follow-up at 3 months, one patient discontinued SSZ due to hypersensitivity within 3 months of initiation, one patient escaped to biological within 3 months of initiation of combined DMARDs and in two patients, there was the history of recent infection). Serum samples were stored at − 80°C, till the time of enzyme-linked immunosorbent assay (ELISA) assay.

Patient evaluation

Clinical examination and disease activity parameter assessment was performed at baseline and subsequent visits. Erythrocyte sedimentation rate (ESR) and CRP values were noted when available.

Definition of treatment response

Patients who attained and did not attain ASAS20 response at 3 and 6 months, posttherapy, were defined as responders and nonresponders, respectively. In addition, patients who escaped to biologicals during the study period or discontinued conventional synthetic DMARDs due to a serious adverse event or self-discontinuation were also defined as nonresponders.

Definition of active disease

BASDAI score of ≥4 and <4, were defined as active and inactive disease, respectively.

Myeloid related proteins 8/14 assay

Serum MRP 8/14 levels were measured using commercial ELISA kit (R&D Systems, USA), as per the manufacturer's instructions. The lower limit of detection was 0.215ng/ml.

Statistical analysis

The measurement data were expressed as mean (± standard deviation) or median (with inter-quartile range) based on normality of distribution of values (checked by Shapiro–Wilk test). Comparison of means for MRP 8/14 between AxSpA patients versus controls, ASAS 20 responders versus nonresponders and active versus inactive disease was made using two-sample Wilcoxon rank-sum (Mann–Whitney) test (STATA IC 16 software StataCorp LLC, Texas, USA).


  Results Top


Characteristics of patients and controls

The median age of patients and controls were 35 (14) and 34 (16) years, respectively, where the proportion of males was 89% and 77%, respectively. The median age at presentation of patients was 27 (11) years, with their median disease duration being 49 (81) months. The mean BASDAI was 4.9 ± 1.8 and 25 (30.1%) had peripheral arthritis at baseline. Before recruitment, 76 (91.5%) patients were on NSAIDs, 62 (74.7%) were on DMARDs (SSZ [n = 33], MTX [n = 26], leflunomide [n = 1], and combination DMARD [n = 2]). Median prescribed doses of MTX, SSZ, and ASAS NSAID index at baseline were 15 (5) mg/week, 2 (0) g/day, and 17.5 (43.3), respectively. Median doses of MTX, SSZ and ASAS NSAID index used by patients at 3 months were 15 (5) mg/week, 2 (0.5) g/day and 14.3 (37.2), respectively. Median doses of MTX, SSZ, and ASAS NSAID index used by patients at 6 months were 20 (5) mg/week, 2.5 (1) g/day, and 5 (20.3), respectively. ASAS20 response at 6 months was attained in 36 out of 71 patients (50.7%) who were followed up. Among the 35 ASAS nonresponders, four patients escaped to biologicals during the study period and one patient discontinued SSZ within 3 months of initiation due to hypersensitivity.

Baseline serum myeloid related proteins 8/14 levels in patients and controls

Median MRP 8/14 levels in patients was 3.0 (3.96) μg/ml compared to 2.3 (3.29) μg/ml in healthy controls (P = 0.2), at baseline.

Baseline serum myeloid-related proteins 8/14 levels as theragnostic marker of combination therapy disease-modifying anti-rheumatic drugs therapy

Median baseline MRP 8/14 levels in ASAS20 responders (n = 36) were 3.6 (4.1) μg/ml compared to 2.4 (4.8) μg/ml in nonresponders [n = 35, P = 0.4, [Figure 1]a. Median △ (baseline to 3 months) MRP 8/14 levels from baseline to 3 months [Figure 1]b in ASAS20 responders (n = 33) were −1 (−2.7) μg/ml compared to −0.2 (−3.8) μg/ml for nonresponders (n = 27) (P = 0.5).
Figure 1: (a) showing the baseline myeloid related proteins 8/14 levels in Assessment of SpondyloArthritis International Society 20 responders versus nonresponders at 6 months (b) showing the △ myeloid related proteins 8/14 value (baseline to post 3 months disease-modifying anti-rheumatic drugs therapy) in Assessment of SpondyloArthritis International Society 20 responders versus nonresponders at 6 months

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  Discussion Top


In this small, proof-of-concept study evaluating the theragnostic ability of MRP 8/14 in AxSpA patients treated with combination DMARD therapy for 6 months, neither baseline median MRP 8/14 levels nor median △ MRP 8/14 level from baseline to 3 months were able to differentiate between ASAS20 responders from nonresponders. These results contradict previous reports wherein baseline as well as the change in MRP 8/14 levels correlated with response to therapy to NSAIDs in 23 AS patients,[8] Anti-TNF agents in 49 patients[9] and also in a recent large study involving 142 patients of AS treated with NSAIDs/DMARDs/Biologicals.[10] Hu et al.[10] showed that change in MRP 8/14 levels during the first month of treatment could predict patients achieving ASAS 40 with an area-under-the curve of 0.69. Exploration of the theragnostic ability of MRP 8/14 in the AxSpA cohort treated with combination DMARD has never been reported till date and serves as the novelty of our study. However, the lack of serial MRP 8/14 (baseline and posttherapy) sample estimation in all recruited patients remained a challenge. In the post hoc analysis, higher median baseline CRP (n = 71) and higher median △ CRP from baseline to 3 months (n = 58) was seen in ASAS20 responders in comparison to nonresponders at 6 months posttherapy (P < 0.001). ESR measurements failed to differentiate the ASAS20 responders from nonresponders at 6 months.

Studies of MRP 8/14 with respect to utility as a disease activity biomarker have shown contrasting results, as many reports document good discriminative ability;[8],[9],[12],[13] however, a few reports demonstrate not only poor differentiation between AxSpA cases and controls[14],[15] but also active and inactive disease.[12] Evaluation of disease activity assessment of MRP 8/14 among the 83 patients with 138 assessments at baseline and 3 months posttherapy was performed, but being a post hoc analysis, the sample size was not powered to conclude on this aspect. Median MRP 8/14 in active disease (n = 112) was 2.2 (3.8) μg/ml, compared to 2 (2.3) μg/ml in inactive disease (n = 26) (P = 0.5). CRP, on the other hand, was able to differentiate between active and inactive disease in our cohort (P < 0.01), however, ESR failed do the same. The lack of discriminative ability of MRP 8/14 between patients and controls observed in the current study is in contrast to a previous study from India.[8] and could be due to two reasons: (a) lesser proportion of AxSpA patients having peripheral arthritis in our cohort (30% as opposed to ~60% in the previous study), (b) higher proportion (91%) of AxSpA patients being on background NSAIDs at the time of recruitment in our cohort compared to the previous study (75%).


  Conclusion Top


Serum MRP 8/14 did not serve as a theragnostic biomarker in AxSpA patients treated with combination DMARDs and on-demand NSAIDs, whereas traditional biomarker CRP displayed theragnostic utility.


  Acknowledgments Top


The authors would like to thank Dr. Ruchika Goel for intellectual input and Mr. Karthi for technical support. The authors would also like to thank the institutional review board and ethics committee of Christian Medical College, Vellore and Indian Rheumatology Association.

Financial support and sponsorship

Intramural fluid research of Christian Medical College (IRB No 10207 dated August 8, 2016). Extramural funding was also received from the Indian Rheumatology Association research grant 2017-2018 (July 15, 2017).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Robinson WH, Lindstrom TM, Cheung RK, Sokolove J. Mechanistic biomarkers for clinical decision making in rheumatic diseases. Nat Rev Rheumatol 2013;9:267-76.  Back to cited text no. 1
    
2.
Benhamou M, Gossec L, Dougados M. Clinical relevance of C-reactive protein in ankylosing spondylitis and evaluation of the NSAIDs/coxibs' treatment effect on C-reactive protein. Rheumatol Oxf Engl 2010:49:536-41.  Back to cited text no. 2
    
3.
Maksymowych WP. Biomarkers for diagnosis of axial spondyloarthritis, disease activity, prognosis, and prediction of response to therapy. Front Immunol 2019;10:305.  Back to cited text no. 3
    
4.
Ehrchen JM, Sunderkötter C, Foell D, Vogl T, Roth J. The endogenous toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J Leukoc Biol 2009;86:557-66.  Back to cited text no. 4
    
5.
Assassi S, Reveille JD, Arnett FC, Weisman MH, Ward MM, Agarwal SK, et al. Whole-blood gene expression profiling in ankylosing spondylitis shows upregulation of toll-like receptor 4 and 5. J Rheumatol 2011;38:87-98.  Back to cited text no. 5
    
6.
Baeten D, Kruithof E, De Rycke L, Boots AM, Mielants H, Veys EM, et al. Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy. Arthritis Res Ther 2005;7:R359-69.  Back to cited text no. 6
    
7.
Calprotectin is Highly Upregulated in Inflamed Axial Entheses in SKG Mice. In: ACR Meet. Abstr. Available form: https://acrabstracts.org/abstract/calprotectin-is-highly-upregulated-in-inflamed-axial-entheses-in-skg-mice/. [Last accessed on 2019 Dec 28].  Back to cited text no. 7
    
8.
Gupta L, Bhattacharya S, Agarwal V, Aggarwal A. Elevated levels of serum MRP 8/14 in ankylosing spondylitis: Associated with peripheral arthritis and active disease. Clin Rheumatol 2016;35:3075-9.  Back to cited text no. 8
    
9.
Turina MC, Yeremenko N, Paramarta JE, De Rycke L, Baeten D. Calprotectin (S100A8/9) as serum biomarker for clinical response in proof-of-concept trials in axial and peripheral spondyloarthritis. Arthritis Res Ther 2014;16:413.  Back to cited text no. 9
    
10.
Hu H, Du F, Zhang S, Zhang W. Serum calprotectin correlates with risk and disease severity of ankylosing spondylitis and its change during first month might predict favorable response to treatment. Mod Rheumatol 2019;29:836-42.  Back to cited text no. 10
    
11.
Rudwaleit M, van der Heijde D, Landewé R, Listing J, Akkoc N, Brandt J, et al. The development of Assessment of SpondyloArthritis International Society classification criteria for axial spondyloarthritis (part II): Validation and final selection. Ann Rheum Dis 2009;68:777-83.  Back to cited text no. 11
    
12.
Oktayoglu P, Bozkurt M, Mete N, Caglayan M, Em S, Nas K. Elevated serum levels of calprotectin (myeloid-related protein 8/14) in patients with ankylosing spondylitis and its association with disease activity and quality of life. J Investig Med Off Publ Am Fed Clin Res 2014;2:880-4.  Back to cited text no. 12
    
13.
Huang J, Yin Z, Song G, Cui S, Jiang J, Zhang L. Discriminating Value of Calprotectin in Disease Activity and Progression of Nonradiographic Axial Spondyloarthritis and Ankylosing Spondylitis. Dis Markers. 2017;2017:7574147. doi: 10.1155/2017/7574147. Epub 2017 May 24. PMID: 28630526; PMCID: PMC5463132.  Back to cited text no. 13
    
14.
Klingberg E, Carlsten H, Hilme E, Hedberg M, Forsblad-d'Elia H. Calprotectin in ankylosing spondylitis--frequently elevated in feces, but normal in serum. Scand J Gastroenterol 2012;47:435-44.  Back to cited text no. 14
    
15.
Genre F, Rueda-Gotor J, Remuzgo-Martínez S, Corrales A, Mijares V, Expósito R, et al. Association of circulating calprotectin with lipid profile in axial spondyloarthritis. Sci Rep 2018;8:13728.  Back to cited text no. 15
    


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