Maria DiCicco

Angiogenic gene expression and vascular density are reflected in ultrasonographic features of synovitis in early rheumatoid arthritis: an observational study

IntroductionNeovascularization contributes to the development of sustained synovial inflammation in the early stages of Rheumatoid Arthritis. Ultrasound (US) provides an indirect method of assessing synovial blood flow and has been shown to correlate with clinical disease activity in patients with Rheumatoid Arthritis. This study examines the relationship of US determined synovitis with synovial vascularity, angiogenic / lymphangiogenic factors and cellular mediators of inflammation in a cohort of patients with early Rheumatoid Arthritis (RA) patients prior to therapeutic intervention with disease modifying therapy or corticosteroids.MethodsAn ultrasound guided synovial biopsy of the supra-patella pouch was performed in 12 patients with early RA prior to treatment. Clinical, US and biochemical assessments were undertaken prior to the procedure. Ultrasound images and histological samples were obtained from the supra-patella pouch. Histological samples were stained for Factor VIII and a-SMA (a-smooth muscle actin). Using digital imaging analysis a vascular area score was recorded. QT-PCR (quantitative-PCR) of samples provided quantification of angiogenic and lymphangiogenic gene expression and immunohistochemistry stained tissue was scored for macrophage, T cell and B cell infiltration using an existing semi-quantitative score.ResultsPower Doppler showed a good correlation with histological vascular area (Spearman r - 0.73) and angiogenic factors such as vascular endothelial growth factor- A (VEGF-A), Angiopoietin 2 and Tie-2. In addition, lymphangiogenic factors such as VEGF-C and VEGF-R3 correlated well with US assessment of synovitis. A significant correlation was also found between power Doppler and synovial thickness, pro-inflammatory cytokines and sub-lining macrophage infiltrate. Within the supra-patella pouch there was no significant difference in US findings, gene expression or inflammatory cell infiltrate between any regions of synovium biopsied.ConclusionUltrasound assessment of synovial tissue faithfully reflects synovial vascularity. Both grey scale and power Doppler synovitis in early RA patients correlate with a pro-angiogenic and lymphangiogenic gene expression profile. In early RA both grey scale and power Doppler synovitis are associated with a pro-inflammatory cellular and cytokine profile providing considerable validity in its use as an objective assessment of synovial inflammation in clinical practice.

Use of ultrasound-guided small joint biopsy to evaluate the histopathologic response to rheumatoid arthritis therapy: recommendations for application to clinical trials.

To examine in a cohort of rheumatoid arthritis (RA) patients undergoing serial ultrasound (US)–guided biopsies of small joints in the context of clinical trials whether sufficient synovial tissue could be obtained at both baseline and second biopsy to: 1) accurately evaluate the synovial immune phenotype, 2) permit adequate RNA extraction to determine molecular signatures, and 3) sensitively detect change in the number of synovial sublining macrophages (CD68+) following effective therapy.

OP0240 Synovial Lymphocytic Aggregates Associate with Highly Active RA and Predict Erosive Disease Progression at 12 Months: Results from The Pathobiology of Early Arthritis Cohort

Background The synovial cellular infiltrate in RA has for some time been recognised to organise into lymphocytic aggregates with a number observations suggesting they are immunologically competent and can support chronic inflammation. However, their clinical significance has been controversial with conflicting publications reporting diverse associations with disease outcome. Objectives The aim of this study was to examine in a cohort of therapy naïve, early RA patients whether baseline synovial pathotype: i)associated with specific clinical phenotypes, ii)predicted response to DMARD therapy, and iii)predicted joint damage progression Methods 135 DMARD-naïve early RA patients were recruited as part of the Pathobiology of Early Arthritis Cohort at Barts Health NHS Trust. Following pre-treatment synovial biopsy clinical data was collected at baseline and 12 months. Following immunohistochemical staining the degree of synovial infiltration by CD20+B cells, CD3+T cells, CD68+macrophages and CD138+plasma cells was determined. Patients were then categorised into synovial pathotypes: lymphoid, myeloid or fibroid according to the degree of immune cell infiltration. Samples also underwent nanostring analysis of 238 genes. Significant differences in clinical parameters at baseline and progression in radiographic damage at 12 months between synovial pathotypes was determined. Results At baseline a lymphoid pathotype significantly associated with ACPA+ve (0.017) and highly active disease (DAS28, CRP, ESR and swollen joint count, p<0.01). Furthermore a significantly higher number of patients with a baseline lymphoid pathotype (vs myeloid/fibroid) developed radiographic progression (9/26 vs 5/53, p=0.026). A cohort of 79 genes were identifed from the nanostring analysis that were differentially upregulated in patients with joint damage progression. Logistic regression analysis was used to develop a clinical model for predicting radiographic progression (final model ESR, RF and ACPA) with an AUC of 0.86. Integration of synovial gene expression profiles into the clinical model improved the AUC to 0.96. Conclusions The demonstration in this early RA cohort of a significant association between a lymphoid pathotype and a severe clinical phenotype/sero positivity for ACPA supports a direct role for synovial lymphoid structures in disease pathogenesis. Furthermore patients with a lymphoid pathotype were significantly more likely to develop radiographic damage. Integration of synovial gene expression profiles into a clinical model of radiographic progression enhanced performance. This data strongly supports a role for integration of synovial biomarkers into clinical prediction models of radiographic progression in early RA with critical implications for future patient stratification. Disclosure of Interest None declared

FRI0157 A Baseline Prediction Model for Response To Certolizumab-Pegol: Role of Synovial Histopathology

Background Rheumatoid Arthritis (RA) is a severe joint disease characterized by chronic inflammation of synovial tissue. The inflammatory infiltrate within the rheumatoid synovium may be differently arranged, defining three distinct histopathotypes: myeloid (prevalent macrophage infiltration), lymphoid (B/T lymphocyte aggregates), and fibroid (fibroblastic infiltration with paucity of immune cells)1. Despite enabling a remarkable improvement of the diseases outcome, biologics are not effective in around 30–40% of treated patients. To date, no reliable pre-therapy predictors of response have been identified. The histological characterization of synovitis may represent a valuable tool in predicting response to biologics prior to treatment. Objectives To evaluate the role of synovial pathology in predicting clinical response to certolizumab-pegol combined with DMARD. Methods 32 RA patients fulfilling UK NICE criteria for starting TNF-inhibitors were enrolled at Barts Health Trust. Patients underwent a baseline US-guided biopsy prior to commencing certolizumab-pegol and a second biopsy after 12 weeks of treatment. Synovial immune infiltrate was assessed through immunohistochemical staining for CD3/CD20/CD68/CD138 and quantified using a semi-quantitative scoring system (0–4); accordingly, patients were classified as lymphoid (B cells aggregates grade ≥2), myeloid (sub-lining macrophages >2, +/− grade 1 aggregates) and fibroid (sub-lining macrophages ≤2). Therapeutic response was evaluated at 3 months according to EULAR criteria. Logistic regression model was performed to identify factors associated with good EULAR response at 3 months; the factors included clinical and biochemical parameters (ESR, CRP) as well as histopathology. The model selection was implemented using forward processes by AIC with 5-fold cross-validation (error=0.25). Results Out of 32 baseline biopsies, 53% were classified as lymphoid, 19% myeloid and 28% fibroid. Decrease in the number of sub-lining CD68-positive macrophages significantly correlated with clinical response (p<0.05). Baseline demographic and clinical parameters were comparable between the three histological groups; the CRP level was significantly higher in the myeloid compared to fibroid. The histological pathotype adjusted for baseline VAS pain and patient global health score, ESR, HAQ score and concomitant steroid treatment, was identified as a significant predictor of good response to certolizumab-pegol by forward selection (AUC 0.97, CI 0.92–1). The chance of achieving a good EULAR response to certolizumab-pegol was 91% lower in patients classified as a fibroid pathotype at baseline compared to myeloid/lymphoid pathotypes (OR 0.09, CI 0, 1.75). Conclusions Our study shows that patients with a fibroid pathotype at baseline are less likely to respond to Certolizumab-pegol. Moreover, we identified a novel prediction model of response based on clinical parameters and synovial histopathological classification. Histological assessment of synovial tissue may be of critical importance for developing personalised treatment for RA. References Pitzalis C et al, New learnings on the pathophysiology of RA from synovial biopsies. Curr Opin Rheumatol. 2013 Disclosure of Interest None declared

A7.6 Clinical and pathological differences of elderly- and younger-onset rheumatoid arthritis in an early arthritis cohort

Background and objectives Elderly-onset rheumatoid arthritis (EORA), starting after the age of 60, is a subset of disease with different characteristics from the classic younger-onset RA (YORA). We aimed to compare clinical and pathological features between these two disease subsets in the Barts Early Arthritis Cohort (BEAC). Material and methods We included BEAC patients fulfilling ACR 2010 criteria and assessed clinical and pathological variables at baseline and at 6 months of DMARD therapy. The primary outcome was the achievement of low disease activity (LDA) according to DAS28 at 6 months. We used multivariate logistic regression to determine predictors of LDA at 6 months. Results We included 140 patients, 99 YORA and 41 EORA. EORA patients were more frequently male (51.2 vs. 26.3, p = 0.004) and Caucasian (68.3 vs. 39.4, p = 0.02), had more proximal joint involvement at presentation (55.0 vs. 35.1, p = 0.031), more common abrupt (27.5 vs. 4.1, p < 0.001) and polymyalgia rheumatica (PMR)-like onset (23.7 vs. 0, p < 0.001), weight loss (15.0 vs. 4.1, p = 0.026), anaemia (48.6 vs. 28.4, p = 0.033), extra-articular manifestations (27.5 vs. 6.2, p = 0.004) and cardiovascular comorbidity (73.2 vs. 29.3, p < 0.001). At baseline, disease activity parameters, including ESR, CRP, joint counts, and composite scores (DAS28, CDAI and SDAI), were similar between both groups. Synovial pathotype classification (ectopic lymphoid-like structures positive (ELS+); diffuse myeloid infiltrate or non-inflammatory/pauci-immune) did not reveal differences between groups as a whole (p = 0.12). However, EORA patients presented pauci-immune pathotype less frequently (25.0 vs. 44.3, p = 0.045). Macrophage, B-cell, T-cell and plasmocyte semi-quantitative scores were also similar. At 6 months (n = 105), EORA (OR = 0.28, p = 0.047), female gender (OR = 0.23, p = 0.016), current smoking (OR = 0.11, p = 0.002) and high baseline DAS28 (OR = 0.41, p < 0.001) were negatively associated with reaching DAS28 LDA. Contrastingly, PMR-like onset was strongly predictive of DAS28 LDA (OR = 63.9, p = 0.004). Conclusions In an early arthritis cohort, EORA patients presented different clinical features at presentation, but similar synovial pathological characteristics. Adjusting for other significant factors such as gender, smoking, baseline DAS28 and PMR-like onset, EORA was negatively associated with the achievement of LDA at 6 months according to DAS28.

OP0032 Baseline Synovial B-Cell Status Predicts Response to Rituximab Therapy in RA

Background B-cells are key mediators in RA pathogenesis through the initiation of several pathways that lead to the perturbation of the immune system. Rituximab, an anti-CD20 monoclonal antibody is recommended for use after failure with traditional disease modifying agents and anti-TNF therapy. Despite using peripheral blood B-cells as a marker to ensure depletion, the clinical response to Rituximab remains variable, highlighting an unmet need for further biomarkers of response. Mechanisms that underpin the variation in clinical outcome are yet to be fully elucidated but may relate to distinct differences in cellular infiltrate and target expression within the synovium. Objectives The aim of this open-labelled pilot study was to test the hypothesis that patients with a B-cell rich versus B-cell poor synovial pathotype have an enhanced response to B-cell depletion following therapy with Rituximab. Methods Synovial biopsy (Ultrasound guided n=34, arthroscopic n=6) was performed at baseline in 40 patients with active RA (as defined by a DAS-28 score of >5.1) who had failed treatment with standard disease modifying therapy. 35 patients had received prior anti-TNF therapy, a subset of 5 patients were anti-TNF naïve. Rituximab was administered, with appropriate pre-medication, as two intravenous infusions at a dose of 1g 14 days apart. Synovial tissue was fixed in formalin, paraffin-embedded and cut to serial sections (3μm). Histological grading and lymphoid organization of the synovium was assessed by immunohistochemical analysis. After staining for CD20, the presence or absence of B-cells was determined by a semi-quantitative score (0-4)1. CD21 staining was carried out on samples with large aggregates to determine the presence of FDC in germinal centres (GC) within the synovium. Disease response to treatment (improvement in DAS-28>1.2) at 16 weeks was correlated with the presence or absence of B-cells and GC using Fishers exact test and Chi-squared testing, respectively. Results IHC for CD20 results were available from 39 patients. 29 [74%] patients were females, mean age 58.5 [29-80], 36 [92%] were anti-CCP positive and 32 [82%] were RF positive. 12 [30%] showed a response (DAS-28 >1.2) improvement at 3 months. 24 [62%] patients had little or no B-cell infiltrate within the synovium. In this subgroup, 20 [83%] did not exhibit a significant clinical response. (p=0.031). In B-cell rich non-responders, the presence of GC was noted in 5 out of 6 individuals [83%]; only 1 patient who was GC positive responded to Rituximab. (p=0.01, n=37) Conclusions This open-labeled pilot study strongly suggests that response to Rituximab therapy is determined by the presence or absence of B-cells within the synovium. In patients with B-cell rich synovium, the presence of GC appears to be an important marker of resistance to Rituximab therapy. This pilot study also highlights the potential to utilize synovial pathotype as a biomarker to predict disease outcomes and response. References Kraan, M.C., Haringman, J.J., Post W.J., Versendaal, J., Breedveld, F.C., Tak. P.P. Immunohistological analysis of synovial tissue for differential diagnosis in early arthritis. Rheumatology 1999; 38:1074–1080. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5609