Identification of Definitive Serum Biomarkers Associated With Disease Activity in Primary Sjögren's Syndrome

Ayumi Nishikawa; Katsuya Suzuki; Yoshiaki Kassai; Yuumi Gotou; Maiko Takiguchi; Takahiro Miyazaki; Keiko Yoshimoto; Hidekata Yasuoka; Kunihiro Yamaoka; Rimpei Morita; Akihiko Yoshimura; Tsutomu Takeuchi

Disclosures

Arthritis Res Ther. 2016;18(106)

Abstract and Introduction

Abstract

Background: In this study, we sought to identify definitive biomarkers associated with disease activity in primary Sjögren's syndrome (pSS).

Methods: Serum protein concentrations in pSS patients and healthy controls (HCs) were comprehensively screened using high-throughput proteomic analysis, and differentially expressed proteins were extracted. Correlation between differentially expressed proteins and European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI) scores was analyzed and disease activity-associated biomarkers were identified. These biomarkers were validated by enzyme-linked immunosorbent assay (ELISA) in a separate pSS cohort.

Results: The serum concentrations of 1100 proteins were compared between 30 pSS patients and 30 HCs, with 82 differentially expressed proteins identified as pSS-associated proteins. Of these 82 proteins, 9 were identified as disease activity-associated biomarkers. These nine biomarkers underwent validation by ELISA in a separate pSS validation cohort (n = 58), with five proteins (CXCL13, TNF-R2, CD48, B-cell activating factor (BAFF), and PD-L2) subsequently being confirmed as candidate biomarkers. Of these five candidate biomarkers, CXCL13 exhibited the most significant correlation with the lymphadenopathy, glandular, and pulmonary domains of the ESSDAI. CXCL13, TNF-R2 and CD48 exhibited a positive correlation with the biological domain of the ESSDAI. TNF-R2 exhibited the most negative correlation with uptake in the submandibular gland on technetium 99m-pertechnetate salivary gland scintigraphy.

Conclusions: Our approach successfully identified serum biomarkers associated with disease activity in pSS patients. These markers might be potential therapeutic targets in pSS patients.

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Abstract and Introduction
Background
Methods
Results
Discussion
Conclusions
References

Table 1. Characteristics of patients and controls
Characteristic	Healthy controls (n = 30)	Patients with pSS (n = 30)
Demographic
Female, n (%)	30 (100)	29 (97)
Age, mean (SD), years*	39.9 (9.4)	61.1 (10.8)
Disease duration, mean (SD), years*	NA	4.7 (6.6)
Clinical manifestation, n (%)
Ocular symptoms	NA	18 (60)
Oral symptoms	NA	21 (70)
Objectively assessed dryness	NA	24 (80)
Anti-SSA positivity	NA	24 (80)
Anti-SSB positivity	NA	14 (48)
Lymphocytic sialadenitis with focus score ≥1	NA	12 (40)
ESSDAI, mean (SD)*	NA	2.6 (4.2)
Treatment
Corticosteroid	NA	1 (3)
Immunosuppressant	NA	0 (0)

*Data are presented as mean ± standard deviation (SD) or number (%). pSS primary Sjögren's syndrome, ESSDAI the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index, NA not applicable

Table 2. Serum proteins positively correlated with ESSDAI score in patients with pSS in first cohort ( n = 30)
Protein	Spearman's ρ	P value
EPHB-2	0.65	<0.0001
CXCL13	0.60	0.0005
CD48	0.56	0.0014
β2MG	0.55	0.0018
BAFF	0.52	0.0029
TNF-R2	0.51	0.0039
LAG-3	0.49	0.0058
CD163	0.49	0.0062
PD-L2	0.47	0.0081

Spearman's rho correlation coefficient test was used to test correlation between 82 differentially expressed protein concentrations and the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI) scores. EPHB-2, ephrin type-B receptor 2, CXCL13, C-X-C motif chemokine 13, β2MG β2-microglobulin, BAFF B-cell activating factor, TNF-R2 TNF receptor 2, LAG-3, lymphocyte activation gene-3, PD-L2 programmed cell death protein 1 ligand 2A P value <0.01 was considered significant

Table 3. Characteristics of patients in the validation and initial cohorts
Characteristic	Validation cohort (n = 58)	Initial cohort (n = 30)
Demographic
Female, n (%)	57 (98)	29 (97)
Age, mean (SD), years*	60.3 (13.3)	61.1 (10.8)
Disease duration, mean (SD), years*	3.5 (5.8)	4.7 (6.6)
Clinical manifestation, n (%)
Ocular symptoms	39 (67)	18 (60)
Oral symptoms	53 (91)	21 (70)
Objectively assessed dryness	48 (83)	24 (80)
Anti-SSA positivity	43 (74)	24 (80)
Anti-SSB positivity	24 (41)	14 (48)
Lymphocytic sialadenitis with focus score ≥1	10 (17)	12 (40)
ESSDAI, mean score (SD)*	2.6 (4.9)	2.6 (4.2)
Treatment
Corticosteroid	1 (1.7)	1 (3)
Immunosuppressant	0 (0)	0 (0)

*Data are presented as mean ± SD or number (%). Anti-SSA anti-Sjögren's syndrome antigen A antibodies, Anti-SSB anti-Sjögren's syndrome antigen B antibodies, ESSDAI European League Against Rheumatism Sjögren's Syndrome Disease Activity Index

Table 4. Clinical variables associated with five disease activity-associated biomarkers
Variable	N	CXCL13		TNF-R2		CD48		BAFF		PD-L2
Variable	N	r	P	r	P	r	P	r	P	r	P
ESSDAI score	58	0.7	<0.0001	0.59	<0.0001	0.54	<0.0001	0.43	0.001	0.39	0.002
Constitutional		NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Lymphadenopathy		0.69	<0.0001	0.54	<0.0001	0.58	<0.0001	0.58	<0.0001	0.5	<0.0001
Glandular		0.44	0.001	0.37	0.004	0.33	0.01	0.43	0.001	0.3	0.02
Articular		0.06	0.62	0.14	0.3	−0.03	0.8	0.11	0.39	−0.08	0.57
Cutaneous		NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Pulmonary		0.65	<0.0001	0.52	<0.0001	0.52	<0.0001	0.38	0.003	0.34	0.01
Renal		NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Muscular		NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Peripheral nervous system		NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Central nervous system		−0.001	0.1	0.01	0.94	−0.04	0.79	0.07	0.59	0.1	0.41
Hematological		0.17	0.2	0.1	0.47	0.02	0.9	−0.17	0.21	−0.13	0.32
Biological		0.5	<0.0001	0.42	0.001	0.45	0.0004	0.11	0.4	0.2	0.13
IgG	55	0.62	<0.0001	0.5	<0.0001	0.57	<0.0001	0.16	0.25	0.12	0.38
C3	52	−0.12	0.39	−0.06	0.69	−0.24	0.09	0.04	0.77	0.01	0.96
C4	52	0.13	0.37	0.24	0.08	0.18	0.2	0.2	0.15	0.13	0.37
Schirmer test (mm/5 min)	21	0.03	0.9	−0.28	0.23	−0.12	0.6	−0.38	0.09	0.23	0.32
RB test (score)	14	−0.04	0.9	0.18	0.55	0.17	0.57	0.05	0.86	−0.16	0.58
Fluorescein (score)	17	0.19	0.45	0.23	0.38	−0.12	0.65	0.3	0.23	0.2	0.44
Gum test (ml)	28	−0.18	0.35	−0.4	0.03	−0.28	0.15	−0.2	0.3	−0.02	0.91
GS grade (score)	23	0.08	0.72	0.13	0.54	0.23	0.29	0.28	0.2	0.23	0.3
Scintigraphy	40
PG uptake ratio (%)		−0.18	0.26	−0.27	0.1	−0.01	0.95	0.01	0.94	−0.13	0.44
SMG uptake ratio (%)		−0.48	0.004	−0.52	0.001	−0.28	0.1	−0.43	0.01	−0.15	0.4
PG excretion ratio (%)		0.12	0.45	0.06	0.7	0.02	0.9	−0.05	0.77	−0.08	0.64
SMG excretion ratio (%)		0.05	0.79	0.26	0.18	0.09	0.62	−0.49	0.01	0.26	0.17

Candidate biomarkers and clinical variables are shown in the header row and left columns, respectively. Data are presented as Pearson's correlation coefficients (r) and P values (P). A P value <0.05 was considered significant. TNFR TNF receptor, BAFF B-cell activating factor, PD-L2 programmed cell death protein 1 ligand 2, ESSDAI score the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI) score, Schirmer's test Schirmer's I test (mm/5 min), RB test Rose Bengal staining (score: van Bijsterveld score), Fluorescein fluorescein staining (score: van Bijsterveld score), GS grade Greenspan's grade, Scintigraphy technetium 99m-pertechnetate salivary gland scintigraphy, PG parotid gland, SMG submandibular gland, N number of patients with primary Sjögren's syndrome, NA not applicable