Synovial Predictors of Differentiation to Definite Arthritis in Patients With Seronegative Undifferentiated Peripheral Inflammatory Arthritis: microRNA Signature, Histological, and Ultrasound Features

Abstract

Objectives: To examine synovial tissue (ST) predictors of clinical differentiation in patients with seronegative undifferentiated peripheral inflammatory arthritis (UPIA). Methods: Fourty-two patients with IgA/IgM-Rheumatoid Factor and anti-citrullinated peptide antibodies negative UPIA, naive to Disease-Modifying Anti-Rheumatic Drugs, underwent Gray Scale (GSUS) and power Doppler (PDUS) evaluation and Ultrasound (US) guided ST biopsy. CD68, CD3, CD21, CD20, and CD31 synovial expression was evaluated by immunohistochemistry. Whole ST microRNA expression was assessed using miScript miRNA PCR Array. Peripheral blood (PB) and synovial fluid (SF) IL-6, VEGF-A, and VEGF-D levels were measured by ELISA and ST TNF expression was assessed by RT-PCR. Each patient was prospectively monitored and classified at baseline and within 1 year as UPIA, Rheumatoid Arthritis (RA), Spondyloarthritis (SpA) or Psoriatic Arthritis (PsA), respectively. Results: At baseline, CD68⁺ cells were the most common cells within the lining layer (p < 0.001) in seronegative UPIA, directly correlating with GSUS (R = 0.36; p = 0.02) and PDUS (R = 0.55; p < 0.001). Synovial CD31⁺ vessels count directly correlated with GSUS (R = 0.41; p = 0.01) and PDUS (R = 0.52; p < 0.001). During the follow-up, 6 (14.3%) UPIA reached a definite diagnosis (2 RA, 2 SpA and 2 PsA, respectively). At baseline, UPIA who differentiated had higher GSUS (p = 0.01), PDUS scores (p = 0.02) and higher histological scores for CD68⁺ (p = 0.005 and p = 0.04 for lining and sublining respectively), sublining CD3⁺ cells (p = 0.002), CD31⁺ vessels count (p < 0.001) and higher IL-6 PB levels (p = 0.01) than patients who remained as UPIA. MiRNA PCR Array showed that among the 86 tested miRNA species, at baseline, miR-346 and miR-214 were significantly down-regulated (p = 0.02 for both) in ST of UPIA who differentiated than in patients who remained as UPIA, inversely correlating with the lining CD68⁺ cells IHC score (R = -0.641; p = 0.048) and CD31⁺ vessels count (R = -0.665; p = 0.036) and with higher baseline ST expression of TNF (p = 0.014). Finally, logistic regression analysis demonstrated that baseline GSUS and PDUS scores ≥1.5 [OR:22.93 (95%CI:0.98-534.30)] and CD31⁺ vessels count ≥24.3 [OR:23.66 (95%CI:1.50-373.02)] were independent factors associated with the development of definite arthritis. Conclusions: MiRNA signature, histological and US features of ST may help in the identification of seronegative UPIA with high likelihood of clinical differentiation toward definite seronegative arthritis.

Keywords: microRNA; predictor; synovial tissue biopsy; ultrasonography; undifferentiated peripheral inflammatory arthritis.

Figure 1

(A–E) Synovial tissue immunohistochemical scores for CD68⁺, CD21⁺, CD20⁺, CD3⁺ cells of enrolled seronegative UPIA patients and synovitis pathotype distribution. (A,B) Baseline lining and sublining IHC scores for CD68⁺, CD20⁺ and CD3⁺ cells of synovial tissue of UPIA patients cohort (**p < 0.001 ANOVA Test); Mann-Whitney test for all comparisons: lining CD68⁺ vs lining CD20⁺ cells (1.57 ± 0.52 vs. 0.12 ± 0.40; p < 0.001) and lining CD68⁺ cells vs lining CD3⁺ cells (1.57 ± 0.52 vs. 0.25 ± 0.42; p < 0.001); CD20⁺ cells vs lining CD3⁺ cells (0.12 ± 0.40 vs. 0.25 ± 0.42; p = 0.13) (Data on graphs represent mean ± SD); (C) Correlation between lining and sublining ST IHC scores of CD68⁺ cells and CD31⁺ vessels count in the whole cohort of seronegative UPIA patients; (D) Correlation between lining and sublining ST IHC scores of CD3⁺ cells and CD31⁺ vessels count in the whole cohort of seronegative UPIA patients; (E) Example photos of H&E staining of ST of UPIA patients with follicular (f) or diffuse (d) synovitis pattern (magnification 20x). UPIA, Undifferentiated Peripheral Inflammatory Arthritis; CD, Cluster designation; IHC, Immunohistochemistry; ST, Synovial Tissue; L, Lining; SL, Sublining; H&E, Haematoxylin and Eosin; SD, Standard Deviation.

Figure 2

(A–P) Ultrasound assessment and immunohistochemistry for CD68⁺, CD21⁺, CD20⁺, CD3⁺ cells and CD31⁺ vessels of synovial tissue of enrolled UPIA patients based on the clinical differentiation. (A) Example photo of the ultrasound assessment of knee joint of UPIA patient who remained as UPIA afterwards; (B) Example photo of the ultrasound assessment of knee joint of UPIA patient who differentiated into RA afterwards; (C) Example photo of the ultrasound assessment of knee joint of UPIA patient who differentiated into PsA afterwards; (D) Example photo of the ultrasound assessment of knee joint of UPIA patient who differentiated into SpA afterwards; Example photos of CD68 (Red)/CD21 (Brown) staining of ST biopsies from UPIA patients who (E) remained as UPIA, (F) differentiated into RA, (G) differentiated into PsA or (H) differentiated into SpA afterwards (Magnification 20X); Example photos of CD3 (Red)/CD20 (Brown) staining of ST biopsies from UPIA patient who (I) remained as UPIA, (J) differentiated into RA, (K) differentiated into PsA or (L) differentiated into SpA afterwards (Magnification 20X); Example photos of CD31 (Brown) staining of ST biopsies from UPIA patient who (M) remained as UPIA, (N) differentiated into RA, (O) differentiated into PsA or (P) differentiated into SpA afterwards (Magnification 20X); US picture with PD scale of the knee used for ST biopsy is shown next to the corresponding patient; ST, synovial tissue; UPIA, Undifferentiated Peripheral Inflammatory Arthritis; CD, Cluster designation; RA, Rheumatoid Arthritis; SpA, Spondyloarthritis; PsA, Psoriatic Arthritis.

Figure 3

(A–D) Immunohistochemistry scores of synovial tissue of UPIA patients based on the differentiation during the follow-up. (A) Lining IHC scores for CD68⁺, CD21⁺, CD3⁺, and CD20⁺ cells in ST of UPIA patients based on the differentiation during the follow-up; *p = 0.005 lining CD68⁺ cells IHC score comparing UPIA patients who differentiated compared to patients who remained as UPIA during the follow-up; (B) Sublining IHC scores for CD68⁺, CD21⁺, CD3⁺, and CD20⁺ cells in ST of UPIA patients based on the differentiation during the follow-up; *p = 0.04 sublining CD68⁺ cells IHC score comparing UPIA patients who differentiated vs. patients who remained as UPIA during the follow-up; **p = 0.002 sublining CD3⁺ cells IHC score comparing UPIA patients who differentiated vs. patients who remained as UPIA during the follow-up; (C) IHC scores for CD31⁺ vessels in ST of UPIA patients based on the differentiation during the follow-up; *p < 0.001 CD31⁺ vessels count comparing UPIA patients who differentiated vs patients who remained as UPIA during the follow-up; (D) Baseline GSUS and PDUS scores in seronegative UPIA patients who reached a definite diagnosis during the follow-up compared to UPIA patients who remained as UPIA (*p < 0.001 for GSUS and *p = 0.01 for PDUS respectively); (All data on graphs represent mean ± SD); IHC, Immunohistochemistry; CD, Cluster Designation; UPIA, Undifferentiated Peripheral Inflammatory Arthritis; SD, Standard Deviation.

Figure 4

(A–F) microRNA panel of the whole synovial tissue of UPIA patients based on the clinical differentiation during the follow-up and its correlations with TNF tissue expression and histological features. (A) Volcano plot showing expression of miRNAs in the whole synovial tissue of UPIA patients; Color scheme: green decreased expression; Red, Increased expression; Black, Unchanged. The miRNAs that are significantly altered between the two groups are located above the horizontal black line corresponding to p < 0.05; (B) miR-346 and miR-214 expression in whole synovial tissue of seronegative UPIA stratified based on the clinical differentiation during the follow-up (*p = 0.02; **p = 0.02) (Data on graphs represent mean ± SD); (C) Correlation between miR-346 expression in the whole synovial tissue of UPIA patients and lining CD68⁺ cells IHC semi-quantitative score; Red, UPIA patients who differentiated during the follow-up; Gray, UPIA patients who remained as UPIA during the follow-up; (D) Correlation between miR-346 expression in the whole synovial tissue of UPIA patients and CD31⁺ vessels count (mean); Red, UPIA patients who differentiated during the follow-up; Gray, UPIA patients who remained as UPIA during the follow-up; (E) TNF expression in whole synovial tissue of seronegative UPIA stratified based on the clinical differentiation during the follow-up (*p = 0.01) (Data on graphs represent mean ± SD); (F) Correlation between TNF expression in the whole synovial tissue of UPIA patients and CD31⁺ vessels count (mean); Red, UPIA patients who differentiated during the follow-up; Gray, UPIA patients who remained as UPIA during the follow-up; SD, Standard Deviation; CD, Cluster Designation; UPIA, Undifferentiated Peripheral Inflammatory Arthritis.

Figure 5

(A,B) Baseline GSUS, PDUS and immunohistochemistry cut-off values for differentiation into seronegative definite arthritis in UPIA patients. (A) Distribution of GSUS and PDUS cut-off values in UPIA patients based on the differentiation during the follow-up; 5(83.3%) UPIA patients who reached a defined diagnosis during the follow-up had baseline GSUS score ≥1.5 vs. 9(25.0%) patients who remained as UPIA during the follow-up had baseline GSUS score ≥1.5, *p = 0.01; 4(66.7%) UPIA patients who reached a defined diagnosis during the follow-up had baseline PDUS score ≥1.5 vs. 2(5.6%) patients who remained as UPIA during the follow-up had baseline PDUS score ≥1.5, *p = 0.002; (B) Distribution of IHC cut-off values in UPIA patients based on the differentiation during the follow-up; 3(50.0%) UPIA patients who reached a defined diagnosis during the follow-up had baseline lining CD68⁺ cells score ≥2.16 vs. 2(5.6%) patients who remained as UPIA during the follow-up had baseline lining CD68⁺ cells score ≥2.16, *p = 0.01; 5(83.3%) UPIA patients who reached a defined diagnosis during the follow-up had baseline sublining CD68⁺ cells score ≥1.16 vs. 11(30.6%) patients who remained as UPIA during the follow-up had baseline lining CD68⁺ cells score ≥1.16, **p = 0.02; 5(83.3%) UPIA patients who reached a defined diagnosis during the follow-up had baseline sublining CD3⁺ cells score ≥1.16 vs. 6(16.7%) patients who remained as UPIA during the follow-up had baseline sublining CD3⁺ cells score ≥1.16, ***p = 0.003; 5(83.3%) UPIA patients who reached a defined diagnosis during the follow-up had baseline CD31⁺ vessels count ≥24.3 vs. 3(8.3%) patients who remained as UPIA during the follow-up had baseline CD31⁺ vessels count ≥24.3, ****p < 0.001. Data are shown as percentage of UPIA patients fulfilling the different cut-off values of US parameters or IHC scores, based on their clinical differentiation during the follow-up (all data on graphs represent mean ± SD). GSUS, Gray Scale Ultrasonography; PDUS, Power Doppler Ultrasonography; IHC, Immunohistochemistry; CD, Cluster Designation; UPIA, Undifferentiated Peripheral Inflammatory Arthritis; SD, Standard Deviation.