Michel W. P. Tsang-A-Sjoe

Fc-gamma receptor polymorphisms differentially influence susceptibility to systemic lupus erythematosus and lupus nephritis

OBJECTIVE To determine relevant Fc-gamma receptor (FcγR) polymorphisms in relation to susceptibility to SLE and LN, and to determine the functional consequences of genetic associations found. METHODS Using multiplex ligation-dependent probe amplification, copy number regions (CNRs) and relevant known functional single nucleotide polymorphisms of FcγRII and FcγRIII were determined in a LN-enriched cohort of 266 Dutch Caucasian SLE patients and 919 healthy Caucasian controls. Expression of FcγRs on leukocytes was assessed using flow cytometry. RESULTS In multivariable analysis, low copy number of CNR1 (including FCGR3B; odds ratio (OR) 2.04; 95% CI: 1.29, 3.23), FCGR2A-131RR (OR 2.00; 95% CI: 1.33, 2.99), and the 2B.4 haplotype of FCGR2B (OR 1.59; 95% CI: 1.13, 2.24), but not FCGR2C open reading frame, were significantly (all P < 0.01) and independently associated with susceptibility to SLE. The 2B.4 haplotype was negatively associated with LN and led to surface expression of FcγRIIb on neutrophils and monocytes. CONCLUSION This study is the first to investigate the most relevant and functional single nucleotide polymorphisms and copy number variations of FcγRII and FcγRIII polymorphisms in one study population, enabling the determination of the individual contribution of each polymorphism in multivariable analysis. Three polymorphisms were shown to be independently associated with susceptibility to SLE. The novel findings of a negative association of the 2B.4 haplotype with LN, and increased expression of FcγRIIb on neutrophils and monocytes as a result of this 2B.4 haplotype warrant future research in the role of these cells and FcγRs in the pathogenesis of SLE and LN.

Both prolonged remission and Lupus Low Disease Activity State are associated with reduced damage accrual in systemic lupus erythematosus

Objectives. To identify predictors of organ damage and specifically the relationship between prolonged disease remission or low disease activity and damage accrual in a longitudinal cohort of SLE patients. Methods. Data were prospectively assessed including the occurrence of minor/major flares. Once a year remission and Lupus Low Disease Activity State (LLDAS) were determined retrospectively. A prediction model for damage accrual during follow-up was constructed with backward logistic regression analyses. Secondly, odds ratios (ORs) for damage accrual (SLICC damage index increase of ⩾ 1 during follow-up) were calculated for patients with or without prolonged remission during 5 years, and with or without LLDAS in ⩾ 50% of observations. Results. Data from 183 patients with a median follow-up duration of 5.0 years were analysed. The most significant predictors for damage accrual were: occurrence of ⩾ 1 major flare, mean daily prednisone dose during follow-up and nephrological manifestations at baseline. Prolonged remission was present in 32.5% (38/117) and LLDAS in ⩾ 50% of observations in 64.5% (118/183) of patients. Both the presence of prolonged remission during 5 years and LLDAS in ⩾ 50% of observations were associated with a reduced risk of damage accrual (OR = 0.20, 95% CI: 0.07, 0.53, P = 0.001 and OR = 0.52, 95% CI: 0.28, 0.99, P = 0.046, respectively). Conclusion. This cohort study shows that prolonged remission and LLDAS were associated with an improved outcome, as determined by yearly assessments. In order to improve the outcome in SLE patients, future studies should investigate whether these targets can be reached actively with therapeutic strategies.

Anti-Hinge Antibodies Recognize IgG Subclass– and Protease-Restricted Neoepitopes

Anti-hinge Abs (AHAs) target neoepitopes exposed after proteolytic cleavage of IgG. In this study, we explored the diversity of protease- and IgG subclass–restricted AHAs and their potential as immunological markers in healthy donors (HDs) and patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). AHA reactivity against IgG-degrading enzyme of Streptococcus pyogenes (IdeS)– or pepsin-generated F(ab′)2 fragments of all four human IgG subclasses was determined. AHA reactivity against one or more out of eight F(ab′)2 targets was found in 68% (68 of 100) of HDs, 69% (68 of 99) of SLE patients, and 81% (79 of 97) of RA patients. Specific recognition of hinge epitopes was dependent on IgG subclass and protease used to create the F(ab′)2 targets, as confirmed by inhibition experiments with F(ab′)2 fragments and hinge peptides. Reactivity against IdeS-generated F(ab′)2 targets was found most frequently, whereas reactivity against pepsin-generated F(ab′)2 targets better discriminated between RA and HDs or SLE, with significantly higher AHA levels against IgG1/3/4. In contrast, AHA levels against pepsin-cleaved IgG2 were comparable. No reactivity against IdeS-generated IgG2-F(ab′)2s was detected. The most discriminatory AHA reactivity in RA was against pepsin-cleaved IgG4, with a 35% prevalence, ≥5.8-fold higher than in HDs/SLE, and significantly higher levels (p < 0.0001). Cross-reactivity for F(ab′)2s generated from different IgG subclasses was only observed for subclasses having homologous F(ab′)2 C termini (IgG1/3/4). For IgG2, two pepsin cleavage sites were identified; anti-hinge reactivity was restricted to only one of these. In conclusion, AHAs specifically recognize IgG subclass– and protease-restricted hinge neoepitopes. Their protease-restricted specificity suggests that different AHA responses developed under distinct inflammatory or infectious conditions and may be markers of, and participants in, such processes.

A multi-parameter response prediction model for rituximab in rheumatoid arthritis

OBJECTIVES To validate the IFN response gene (IRG) set for the prediction of non-response to rituximab in rheumatoid arthritis (RA) and assess the predictive performance upon combination of this gene set with clinical parameters. METHODS In two independent cohorts of 93 (cohort I) and 133 (cohort II) rituximab-starting RA patients, baseline peripheral blood expression of eight IRGs was determined, and averaged into an IFN score. Predictive performance of IFN score and clinical parameters was assessed by logistic regression. A multivariate prediction model was developed using a forward stepwise selection procedure. Patients with a decrease in disease activity score (ΔDAS28)≥1.8 after 6 months of therapy were considered responders. RESULTS The mean IFN score was higher in non-responders compared to responders in both cohorts, but this difference was most pronounced in patients who did not use prednisone, as described before. Univariate analysis in cohort I showed that baseline DAS28, IFN score, DMARD use and negativity for IgM-RF and/or ACPA were associated with rituximab non-response. The multivariate model consisted of DAS28, IFN score and DMARD use, which showed an area under the curve (AUC) of 0.82. In cohort II, this model revealed a comparable AUC in PREDN-negative patients (0.78), but AUC in PREDN-positive patients was significantly lower (0.63), which seemed due to effect modification of the IFN score by prednisone. CONCLUSIONS Combination of predictive parameters provided a promising model for the prediction of non-response to rituximab, with possibilities for optimization via definition of the exact interfering effect of prednisone on IFN score. TRIAL REGISTRATION (COHORT II, SMART TRIAL) NCT01126541, registered 18 May 2010.

FRI0377 The relationship between remission and health related quality of life in a cohort of sle patients

Background a treat-to-target approach for SLE was suggested by an international board of experts to further improve outcome in SLE. Remission was specifically identified as a suitable target. The Definition of Remission in SLE (DORIS) task force recently achieved international consensus on criteria for remission. Objectives to investigate the relationship between remission and health-related quality of life (HRQoL) in patients with systemic lupus erythematosus (SLE) in a longitudinal observational cohort. Methods retrospective analysis of prospectively obtained data. HRQoL was assessed using the physical and mental component score (PCS and MCS, respectively) of the Short Form 36 (SF-36) questionnaire. DORIS remission categories (no remission/remission on therapy/remission off therapy) were applied. Determinants of PCS and MCS were identified with simple linear regression analyses. Association between remission and HRQoL was assessed using Generalised Estimating Equation (GEE) models. Results Data from 154 patients with 2 years of follow-up were analysed. At baseline 70/154 (45.5%) of patients were in either form of remission. Patients in remission had higher SF-36 scores in all subdomains compared to patients not in remission (figure 1). PCS was positively associated with remission and having employment and negatively associated with erythrocyte sedimentation rate, patient global assessment, SLICC damage index, prednisone use, immunosuppressant use, and body mass index. MCS was positively associated with Caucasian ethnicity and negatively associated with patient global assessment. In GEE analysis, a gradual and significant increase of PCS was observed from patients not in remission (mean PCS 36.0) to remission on therapy (41.8) to remission off therapy (44.8) (table 1). No significant difference in MCS was found between remission states.Abstract FRI0377 – Table 1 GEE analysis of the association between PCS or MCS and remission in patients with SLE A. PCS Unadjusted Adjusted* Mean PCS (±SD) B (95% CI) p-value B (95% CI) p-value No remission 36.0 (10.9) Ref. Ref. Remission on therapy 41.8 (10.0) 6.3 (3.2–9.3) <0.001 6.2 (3.3–9.0) <0.001 Remission off therapy 44.8 (10.4) 8.2 (5.3–11.2) <0.001 8.3 (5.4–11.1) <0.001 *Adjusted for age and SDI B. MCS Unadjusted Adjusted** Mean MCS (±SD) B (95% CI) p-value B (95% CI) p-value No remission 46.1 (10.6) Ref. Ref. Remission on therapy 49.3 (10.5) 2.9 (0.1–5.7) 0.041 2.3 (−0.5–5.1) 0.112 Remission off therapy 46.8 (10.1) 0.8 (−1.7–3.4) 0.52 0.4 (−2.1–3.0) 0.739 **Adjusted ethnicityAbstract FRI0377 – Figure 1 Mean SF-36 subdomain scores in 154 patients at baseline, categorised between SLE patients in remission (n=60) or not in remission (n=94). Patients in remission at baseline have higher mean scores in all SF-36 subdomains compared to patients not in remission. Conclusions we show a strong and persistent association between remission and PCS, but not MCS. These results support the relevance (construct validity) of the DORIS remission definitions and the further development of a treat-to-target approach in SLE. Disclosure of Interest None declared

07.01 A multi-parameter response prediction model for rituximab in rheumatoid arthritis

Background Elevated expression of type I IFN response genes (IRGs) was previously described to be associated with a poor response to rituximab in rheumatoid arthritis (RA). In the present study, we aimed to validate this association and assessed the predictive performance upon combination of the predictive IRG gene set with clinical parameters. Materials and methods In two independent cohorts of 93 (Cohort I) and 133 (Cohort II) rituximab-starting RA patients, baseline peripheral blood expression of eight selected IRGs was determined, and averaged into an IFN score. Individual predictive performance of the IFN score and clinical parameters was assessed by logistic regression. A multivariate prediction model was developed using a forward stepwise selection procedure. Patients with a decrease in disease activity score (ΔDAS28)≥1.8 after 6 months of therapy were considered responders. Results A higher IFN score was observed in RTX non-responders compared to RTX responders from both Cohort I and II, but this difference was most pronounced in patients who did not use prednisone (PREDN-), as described before. Univariate analysis showed that baseline DAS28, IFN score and DMARD use were associated with non-response to rituximab, whereas positivity for IgM-RF and ACPA was associated with a good response to RTX. The multivariate model consisted of DAS28, IFN score and DMARD use, which showed an area under the curve (AUC) of 0.82. Validation of this multivariate model in Cohort II revealed a comparable AUC in PREDN- patients (0.78), but the AUC in PREDN+ patients was significantly lower (0.63), which seemed due to effect modification of the IFN score by prednisone. Multivariate analysis of the PREDN+ subgroup of Cohort II revealed a model containing DAS28 and positivity for IgM-RF and ACPA, which showed an AUC of 0.75. Conclusions The combination of predictive parameters, including IFN score, provided a promising model for the prediction of non-response to rituximab, with possibilities for optimisation via definition of the exact interfering effect of prednisone on IFN score.

A2.09 Longitudinal EBV antibody profiling in sle patients reveals patients with lupus nephritis

Background and objectives The cause of systemic lupus erythematosus (SLE), a severe autoimmune disease, remains unclear. Genetic predisposition cannot be the only explanation for the emergence of SLE and environmental factors could contribute to the complex aetiology of this disease. Epstein-Barr virus (EBV) infection has been implied as a possible factor in early SLE pathogenesis. This study aimed to obtain anti-EBV antibody characteristics in a longitudinal SLE serum cohort, to find correlations between anti-EBV reactivity and disease activity, including lupus nephritis (LN), and to show their clinical utility as a diagnostic marker in SLE. Materials and methods Blood samples were taken yearly from patients included in the Amsterdam SLE cohort (start 2007). Longitudinal characteristics of the anti-EBV antibody profile in SLE patient serum (n = 30), were tested blind with the immunoblot technique, using 3 mm nitrocellulose strips produced with SDS page and electrophoretic transfer of EBV lytic phase antigens. EBV antigens were obtained from nuclear extract of HH514.c16 cells induced with TPA/sodium butyrate for expression of EBV lytic phase. Patterns were compared to clinical characteristics, including SLEDAI (SLE disease activity index) and occurrence of lupus nephritis. Results We found that immune-reactivity against EBV antigens in the serum of SLE is highly perturbed when compared to healthy individuals. Virtually all patients had positive reactivity of IgG antibodies against latent (EBNA1) and lytic (EAd, Zebra, VCA) EBV associated antigens. The patterns of reactivity changed overtime and are possibly related to disease activity (SLEDAI). Strikingly, SLE patients that develop lupus nephritis seem to have an altered serum reactivity for currently unknown antigens, up to two years before clinical diagnosis. Conclusions Our results strongly suggest associations of EBV antibody reactivity in SLE patients. This suggests aberrant EBV control in all SLE patients but particular in those with renal involvement. The results warrant larger studies measuring EBV serum reactivity as a possible prognostic biomarker for renal involvement in SLE and response to treatment.

FRI0374 Inflammatory Exosomes Implicate a Role for Epstein Barr Virus Infection in the Pathophysiology of Systemic Lupus Erythematosus

Background The pathogenesis of Systemic Lupus Erythematosus (SLE) is incompletely understood. Low concordance rates in monozygotic twins and geographical differences in disease risk, suggest involvement of environmental factors in the etiology of SLE. Virtually all patients with SLE have increased antibody titers against EBV antigens, chronically amplified infection frequencies, and 99-100% of young SLE patients seroconvert against Epstein-Barr virus, compared to only 70% of controls. However, a specified role for EBV in SLE pathogenesis remains unclear. Recently, we discovered that EBV-infected B cells secrete highly inflammatory viral RNAs (EBERs) via extracellular vesicles (EVs). Objectives We aimed to investigate the extent of anti-EBV AB responses in SLE patients and study the potential pro-inflammatory role of EBV-secreted, EBER1-laden extracellular vesicles and assess EBV-RNAs as biomarkers in SLE tissues. Methods We explored the molecular basis of altered anti-EBV antibody responses at the epitope level, by immunoblot and peptide-based ELISA in serial samples of SLE patients. We determined EBERs with RT-PCR and in situ hybridization in blood, urine and affected tissue (renal, skin) of SLE patients (with or without nephritis). The inflammatory properties of EBV-EVs were studied in vitro using primary cultures. Results We demonstrate that aberrant antibodies against EBV-antigens and presence of extracellular EBV small RNA (EBER1) distinguish SLE patient sera from rheumatoid arthritis patients and healthy individuals. SLE patients with renal involvement have a distinct pattern of anti-EBV antibody reactivity and lack detectable EBER1 in circulating plasma exosomes. However, surprisingly high levels of EBER1 were detected in lupus nephritis (LN) biopsy material, but not in IgA nephritis tissue. This is compatible with an exogenous (blood-born) source of EBER1, because EBV DNA was near absent and similar observations were made in SLE-associated skin lesions. In situ hybridization showed EBER1 positivity in tubular epithelial cells (TECs). Purified EVs from latent EBV-infected cells are internalized by both primary TEC and plasmacytoid dendritic cells in a phosphatidylserine receptor-dependent manner, causing pro-inflammatory IL-6 production upon EBER1 recognition by viral sensors. Conclusions EBV-infection is deregulated in SLE patients based on anti-EBV Ab responses and elevated levels of extracellular EBER1 in circulation that targets the renal epithelium and skin. Our findings in vitro confirm strong pro-inflammatory functions of EBER1-containing exosomes that target specific cell types, shedding new light on EBV as co-factor in the pathophysiology of SLE and the role of EVs in autoimmune disease. Disclosure of Interest None declared

OP0222 Non-Invasive Pet Imaging of B-Cells in RA Patients Initiating Rituximab Treatment

Background In last decade rituximab (RTX), a B-cell targeted monoclonal antibody, has been introduced to treat rheumatoid arthritis (RA) patients but with variable response rates (30-50%). To increase treatment efficacy and reduce costs, treatment should be individualized to match the spectrum of RA. B-cell targeted therapy has been hypothesized to be most effective in a more B-cell mediated RA disease, possibly correlated to serological status (1). Whole body Positron Emission Tomography (PET) is capable to show B-cells targeting with high specificity in lymphoma patients using 89Zr-RTX (2). In addition, our group previously demonstrated the potential of PET for non-invasive visualization of immunological targets in RA (3). Objectives To investigate the feasibility of non-invasive imaging of B-cells in RA patients who initiate RTX treatment by [89Zr]RTX PET-CT. Methods Anti-B cell therapy naïve RA patients (n=20; female18/20; age 53±11; 65% IgM RF/a-CCP +) with clinical arthritis in at least two hand joints, who were eligible for RTX treatment, were included. Directly after the first therapeutic RTX infusion (1000 mg, without methylprednisolone), 18MBq [89Zr]-10 mg RTX was administered. Whole body PET-CT and detailed images of wrists and hands (22 joints/patient) were acquired at 3, and in addition, in a subpopulation at 6 days post-injection (p.i.). Thereafter, RTX treatment was continued according to standard clinical protocol. Areas of enhanced uptake on PET were defined and quantified as maximum standardized uptake values (SUVmax). Wrist and hand joints were clinically assessed for swelling and tenderness. Results Visually, all patients showed at least one hand joint with increased focal tracer uptake (87/440 joints; mean/patient ± SD 4.4±4.9) (Figure 1) with distribution: metacarpophalangeal (n=44/200), proximal interphalangeal (n=22/200) and wrist joints (n=21/40). Interestingly, 66% of PET-positive joints (57/87) corresponded to clinical findings of arthritis, while PET additionally displayed possible subclinical disease activity in another 30 joints (34%). Quantitative analysis showed high mean SUVmax values of hotspots in hand joints (2.98±1.46) which were up to 4 times higher than maximum background uptake, but varying between patients (range SUVmax 1.2-8.0), regardless of serological status. Stability of joint uptake was found over time (3-6 days p.i.) while the tracer cleared from circulation, pointing at specific binding in joints. Whole body PET-CT also demonstrated tracer uptake in extra-articular tissues especially in liver, spleen and in 5/20 patients slightly enhanced uptake in at least one peripheral lymph node. Conclusions [89Zr]RTX PET-CT seems to be a sensitive tool for in vivo identification of B-cells in arthritic joints and extra-articular tissues in RA patients. Whether quantitative differences in uptake (articular and body distribution) correlate to clinical and RTX response data is currently investigated in a prospective study. References Isaacs JD et al; Ann Rheum Dis 2013. Muylle K et al; Ann Oncol 2008. van der Laken CJ et al; Arthritis Rheum 2008. Acknowledgements This study was financially supported by Hoffmann-La-Roche,The Netherlands Disclosure of Interest S. Bruijnen: None declared, M. Tsang-A-Sjoe: None declared, H. Raterman: None declared, T. Ramwadhdoebe: None declared, D. Vugts: None declared, G. Van Dongen: None declared, M. Huisman: None declared, O. Hoekstra: None declared, P. Tak Grant/research support from: P.P. Tak participated in this study from his position at the Academic Medical Center, Amsterdam, The Netherlands. GSK did neither sponsor this study nor supported this study otherwise. P.P.Tak also has affiliations at Cambridge and Ghent but his activities at these locations were not involved in the current study., A. Voskuyl: None declared, C. van der Laken: None declared

FRI0377 Fc-Gamma Receptor Polymorphisms Differentially Influence Susceptibility to Systemic Lupus Erythematosus and Disease-Related Manifestations

Background Genetic polymorphisms on the locus that encodes low-affinity Fc-gamma receptors (FcγRs) have been associated with systemic lupus erythematosus (SLE). Due to complexity of the locus, the individual contribution of a certain polymorphism to SLE remained largely unclear. Objectives To determine relevant FcγR polymorphisms in relation to susceptibility to and disease-related manifestations of SLE. Secondly, to determine functional consequences of found genetic associations. Methods Using multiplex ligation-dependent probe amplification, copy number regions (CNRs) and all known functional single nucleotide polymorphisms (SNPs) of FcγRII and FcγRIII were determined in a lupus nephritis-enriched cohort of 266 Dutch Caucasian SLE patients and 919 healthy Caucasian controls. Expression of FcγRs on leukocytes was assessed using flow-cytometry. Results In multivariable analysis, low copy number of CNR1 including FCGR3B (OR 2.04, 95% CI 1.29 – 3.23, p=0.002 for 1 vs 2 copies), FCGR2A-131RR (OR 2.00, 95% CI 1.33 – 2.99, p=0.001 for HH vs RR), and the 2B.4 haplotype of FCGR2B (OR 1.59, 95% CI 1.13 – 2.24, p=0.008 for 2B.1/2B.4 vs 2B.4/2B.4) but not FCGR2C-ORF, were significantly and independently associated with susceptibility to SLE. The 2B.4 haplotype was negatively associated with lupus nephritis (OR 0.51, 95% CI 0.28 – 0.91). The 2B.4 haplotype led to surface expression of FcγRIIb on neutrophils and monocytes, but not to increased expression on B-cells, T-cells and NK cells (figure 1). Conclusions In a comprehensive study, we show that a low gene copy number encoding FcgRIIIb, a decrease in FcgRIIa affinity, and expression of the inhibitory FcgRIIb on myeloid cells, independently contribute to SLE susceptibility. Interestingly, while the 2B.4 haplotype was associated with susceptibility to SLE, it was also negatively associated with lupus nephritis, one of the major SLE manifestations. Future studies are needed to confirm and further elucidate these findings. Disclosure of Interest None declared