Nurhan Sutcliffe

The TRACTISS Protocol: a randomised double blind placebo controlled clinical TRial of Anti-B-Cell Therapy In patients with primary Sjögren's Syndrome

BackgroundPrimary Sjögren’s Syndrome (PSS) mainly affects women (9:1 female:male ratio) and is one of the commonest autoimmune diseases with a prevalence of 0.1 – 0.6% of adult women. For patients with PSS there is currently no effective therapy that can alter the progression of the disease. The aim of the TRACTISS study is to establish whether in patients with PSS, treatment with rituximab improves clinical outcomes.Methods/designTRACTISS is a UK multi-centre, double-blind, randomised, controlled, parallel group trial of 110 patients with PSS. Patients will be randomised on a 1:1 basis to receive two courses of either rituximab or placebo infusion in addition to standard therapy, and will be followed up for up to 48 weeks. The primary objective is to assess the extent to which rituximab improves symptoms of fatigue and oral dryness. Secondary outcomes include ocular dryness, salivary flow rates, lacrimal flow, patient quality of life, measures of disease damage and disease activity, serological and peripheral blood biomarkers, and glandular histology and composition.DiscussionThe TRACTISS trial will provide direct evidence as to whether rituximab in patients with PSS leads to an improvement in patient symptoms and a reduction in disease damage and activity.Trial registrationUKCRN Portfolio ID:9809 ISRCTN65360827.

Randomized controlled trial of rituximab and cost-effectiveness analysis in treating fatigue and oral dryness in primary Sjogren's Syndrome

To investigate whether rituximab, an anti–B cell therapy, improves symptoms of fatigue and oral dryness in patients with primary Sjögrens syndrome (SS).

[Accepted Manuscript] Randomized Controlled Trial of Rituximab and cost-effectiveness analysis in treating fatigue and oral dryness in primary Sjogren's Syndrome.

To investigate whether rituximab, an anti–B cell therapy, improves symptoms of fatigue and oral dryness in patients with primary Sjögrens syndrome (SS).

Subjective and Objective Measures of Dryness Symptoms in Primary Sjögren’s Syndrome – Capturing the discrepancy

To develop a novel method for capturing the discrepancy between objective tests and subjective dryness symptoms (a sensitivity scale) and to explore predictors of dryness sensitivity.

Effect of rituximab on a salivary gland ultrasound score in primary Sjögren’s syndrome: results of the TRACTISS randomised double-blind multicentre substudy

Objectives To compare the effects of rituximab versus placebo on salivary gland ultrasound (SGUS) in primary Sjögren’s syndrome (PSS) in a multicentre, multiobserver phase III trial substudy. Methods Subjects consenting to SGUS were randomised to rituximab or placebo given at weeks 0, 2, 24 and 26, and scanned at baseline and weeks 16 and 48. Sonographers completed a 0–11 total ultrasound score (TUS) comprising domains of echogenicity, homogeneity, glandular definition, glands involved and hypoechoic foci size. Baseline-adjusted TUS values were analysed over time, modelling change from baseline at each time point. For each TUS domain, we fitted a repeated-measures logistic regression model to model the odds of a response in the rituximab arm (≥1-point improvement) as a function of the baseline score, age category, disease duration and time point. Results 52 patients (n=26u2009rituximab and n=26u2009placebo) from nine centres completed baseline and one or more follow-up visits. Estimated between-group differences (rituximab-placebo) in baseline-adjusted TUS were −1.2 (95% CI −2.1 to −0.3; P=0.0099) and −1.2 (95% CI −2.0 to −0.5; P=0.0023) at weeks 16 and 48. Glandular definition improved in the rituximab arm with an OR of 6.8 (95% CI 1.1 to 43.0; P=0.043) at week 16 and 10.3 (95% CI 1.0 to 105.9; P=0.050) at week 48. Conclusions We demonstrated statistically significant improvement in TUS after rituximab compared with placebo. This encourages further research into both B cell depletion therapies in PSS and SGUS as an imaging biomarker. Trial registration number 65360827, 2010-021430-64; Results.

THU0008 A Negative High-Resolution Salivary Gland Ultrasound is Highly Predictive of Negative Labial Gland Biopsy in Patients with SICCA Symptoms

Background Sjögrens syndrome (SS) is a chronic autoimmune disease that affects salivary and lacrimal glands and results in xerostomia and xerophtalmia. Current criteria require the presence of either anti-Ro/La antibodies (ENA) or a positive focus score at labial salivary gland biopsy (LSGB) for SS classification [1]. Salivary gland biopsy is a highly specific marker for the diagnosis of SS but as an invasive procedure it is often indicated only in ENA- patients with sicca symptoms. Objectives Here we investigated the capacity of high-resolution salivary gland ultrasound (US) to predict the result of a LSGB biopsy in a consecutive cohort of patient with sicca. Methods Eighty-five consecutive patients attending the Combined Oral Medicine/Rheumatology SS Clinic at Barts and The London NHS Trust who underwent US and LSGB were recruited in the study. All patients displayed subjective and objective symptoms and signs of sicca. US imaging was scored as described by Salaffi et al. [2] while LSGB were scored following immunohistology, including CD3/CD20/CD138/CD21 markers [3]. US and LSGB were scored blindly with the radiologist and the pathologist unaware of the clinical data. Results Of the 85 patients recruited, 36 fulfilled SS criteria (15 LSGB+ENA+, 16 LSGB+ENA−, 5 LSGB−ENA+) while 49 were classified as sicca (LSGB−ENA−). Within the whole cohort, abnormal US findings were observed in 34 patients, of these 29 displayed a positive LSGB. Concordance between US and LSGB was 91.76% (Kappa=0.826). Irrespectively of diagnosis and ENA status, the positive predictive value of having a positive LSGB with abnormal US findings was 85.29% whilst a negative US gave a negative LSGB predictive value of 96.08%. Conclusions Our results demonstrate that high-resolution salivary gland US has a high concordance with LSGB. In particular, a negative US result is highly predictive of a negative labial gland biopsy in patients with sicca symptoms. These data suggests that, particularly in ENA− patients, the role of US is to guide whether or not LSGB is indicated, thus avoiding an invasive procedure in patients with an extremely low chance of a positive result. Thus, we propose that US should be considered in the SS diagnostic algorithm as a screening test before LSGB is performed. References Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, et al. Classification criteria for Sjogrens syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis. 2002 Salaffi F, Carotti M, Iagnocco A, Luccioli F, Ramonda R, Sabatini E, et al. Ultrasonography of salivary glands in primary Sjogrens syndrome: a comparison with contrast sialography and scintigraphy. Rheumatology (Oxford). 2008 Barone F, Bombardieri M, Rosado MM, Morgan PR, Challacombe SJ, De Vita S, et al. CXCL13, CCL21, and CXCL12 expression in salivary glands of patients with Sjogrens syndrome and MALT lymphoma: association with reactive and malignant areas of lymphoid organization. J Immunol. 2008 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.3114

OP0300 Enrichment of T follicular-helper cells (TFH) and exclusion of t follicular-regulatory cells (TFR) from ectoPIC germinal centers in salivary glands of sjogren's syndrome patients

Background B/T cell aggregates in the salivary glands (SG) of Sjögrens syndrome (SS) can give rise to ectopic lymphoid structures (ELS) forming ectopic germinal centers (GCs), which has been linked to the development of MALT lymphoma (MALT-L). T follicular-helper cells (Tfh) and T follicular-regulatory cells (Tfr) are specialized CD4+ T-cells that positively and negatively regulate, respectively, the magnitude of the GCs response and the onset of autoimmunity. Objectives To characterize the infiltration of Tfh and Tfr in the SG infiltrates of patients with SS in the context of the presence/absence of ectopic GCs and in subjects with MALT-L. Methods SG biopsies with matching histology and RNA from 37 SS and 38 non-specific chronic sialadenitis (NSCS) patients were stratified as ELS-/ELS+ based on CD3/CD20/CD21/CD138 immunostaining (IHC). Histological samples and mRNA from 12 parotid MALT-L were also studied. Gene expression was measured by Taqman rt-PCR. Multicolor immunofluorescence/confocal microscopy for CD3, CD4, CD45RO, ICOS, PD1, BCL6 and FoxP3 was used to identify Tfh and Tfr. Results Tfh (CD4+CD45RO+PD1+ICOS+FoxP3-) cells and Tfr (CD4+CD45RO+PD1+ICOS+FoxP3+) cells were strongly enriched in ELS+ vs ELS- SS samples. The Tfh:Tfr ratio in ELS+ SG was approximately 2:1. Interestingly, while in tonsils Tfr were routinely detected within GCs, in ELS+ SG Tfr were predominantly excluded from the B cell follicles and accumulated in the T cell rich areas at the periphery of the lymphoid aggregates. Conversely, Tfh densely infiltrated the B cell rich areas and, within ectopic GCs, acquired BCL6. Furthermore, Tfh infiltration closely correlated with SG IL-21 mRNA expression, which in turn was strongly correlated with CD3, CD20 and CD138 IHC scores and with CXCL13, LTb, BAFF, AID and Pax5 gene expression. Finally, MALT-L samples displayed 10-fold higher IL-21 mRNA and twice as much PD1+ICOS+BCL6+ Tfh-cells/field compared to ELS+ SS samples. Conclusions Within the SG of SS patients Tfh cells closely segregate with lesional IL-21 expression, localize within ELS and are strongly enriched during MALT-L development. Conversely, although Tfr cells are also recruited to ELS+ SG in SS patients, we consistently demonstrated follicular exclusion of this subset from ectopic GCs. This suggests that Tfr in SS SG fail to exert their physiological immunoregulatory properties in controlling the magnitude of the GCs response and B cell autoreactivity, as observed in tonsils. Acknowledgements This work was supported by project grants from the Medical Research Council (MR/N003063/1 to MB) and Arthritis Research UK (grant 20089 to MB). Disclosure of Interest E. Pontarini: None declared, W. Murray-Brown: None declared, C. Croia: None declared, E. Astorri: None declared, D. Lucchesi: None declared, N. Lepse: None declared, N. Sutcliffe: None declared, A. Tappuni: None declared, C. Pitzalis: None declared, M. Bombardieri Consultant for: Amgen/Medimmune, GSK and UCB

[OP.8D.02] FIRST IN MAN TREATMENT OF SEVERE BP VARIABILITY WITH BAROREFLEX ACTIVATION THERAPY.

Objective: Profound BP variability (BPV) is a major cause of cardiovascular morbidity and poor quality of life as there are no optimal pharmacological strategies to help patients. We hypothesised that in a patient with baroreflex dysfunction and preserved efferent baroreflex pathway, carotid sinus stimulation may help control BP, BPV and heart rate variability (HRV). Design and method: A 52 year old man was referred with profound HR and BPV. Home SBPs were in a range of 60–250u200ammHg and DBPs were 40–130u200ammHg and heart rate (HR) of 60–200 bpm (confirmed with ABPM, see Figure) despite multiple medications including felodipine 30u200amg daily, terazosin 16u200amg daily, doxazosin 8u200amg daily, bisoprolol 20u200amg daily and butrans patch 17.5u200amcg/hr. After extensive multi-disciplinary investigations the diagnosis was progressive central and peripheral dysautonomia consequent upon immune-mediated neuropathy secondary to undifferentiated connective tissue disease with Sjogrens syndrome. It was not possible to improve BP control with use of clonidine patches and he had frequent severe epistaxes due to hypertensive surges and blackouts due to hypotension and was therefore retired from work on medical grounds. Results: Autonomic function tests confirmed widespread dysautonomia with preserved but attenuated vasodepressor response to carotid sinus massage. Baroreflex activation therapy (BAT) was undertaken after numerous in-patient attempts to control BPV pharmacologically had failed. The Barostim Neo® device was implanted with a right carotid sinus electrode in March 2015 and subsequently device settings were reprogrammed on several occasions to optimise BP control. The patients BP profile improved considerably following BAT but significant hypotensive episodes continued and thus all antihypertensives were stopped with substantial improvement in HR and BP and halving of BPV and concomitant reduction in epistaxes and syncopal episodes. Figure. No caption available. Conclusions: Severe BPV is uncommon and challenging to manage when caused by baroreflex failure. Some antihypertensive drugs can increase BPV and elevate sympathetic tone which could further impair BP control in patients with this diagnosis. Use of BAT in this setting may be of benefit as long as the carotid sinus nerve and vasodepressor component of the baroreflex still function.

First-in-man treatment of severe blood pressure variability with baroreflex activation therapy

a William Harvey Research Institute, Barts NIHR Cardiovascular Biomedical Research Unit, Queen Mary University of London, UK b Barts BP Centre of Excellence, Barts Heart Centre, St Bartholomews Hospital, Barts Health NHS Trust, London, UK c Department of Cardiology, Galway University Hospital, Galway, Republic of Ireland d Swedish National Rett Center, Frösön, Sweden e Neuroscience Clinical Academic Unit, Barts Health NHS Trust, London, UK f Department of Rheumatology, Barts Health NHS Trust, London, UK g Clinical Research Facility, National University of Ireland, Galway, Republic of Ireland

Next-Generation Sequencing Demonstrates Dynamic Recirculation of B Cell Clones in Ectopic Lymphoid Structures of Sjogren's Syndrome

Background Sjögrens syndrome (SS) is characterised by immune cell infiltration in the salivary glands (SG) leading to xerostomia (dry mouth) and exocrine dysfunction1. B cells play a central role in SS pathogenesis whereby autoreactive B-cells populate ectopic germinal centres (EGC) in SS-SG and undergo somatic hypermutation and class-switch recombination of the immunoglobulin (Ig) genes2. However, the capacity of specific B cell clones to seed ECG in different SG and undergo clonal diversification is unclear. Objectives To unravel the dynamics of recirculation of B cell clones among different minor salivary gland (mSG) biopsies, we investigated immunoglobulin heavy chain (IgH) gene rearrangements and patterns of somatic hypermutation (SHM) using a high-throughput next-generation sequencing approach. Methods IgH gene usage and SHM were investigated in 4 pairs of mSG biopsies from 4 pSS patients with high B cell infiltration and EGC. Retro-transcription of 100ng total RNA using Ig-μ, -γ and -α specific primers yielded Ig-specific cDNA for use as template in producing 36 PCR libraries (12 forward primers for each Ig-C) per sample3. Modified primers3 allow for multiplexing of amplicons and assist in bioinformatic filtering of reads. Sequencing was performed using the Roche GS-FLX titanium platform that allows for bi-directional sequencing producing full length reads of ∼500bp. Raw data was processed using IGMT4 and IgAT5, and lineage trees were produced using IgTree© 6. Results We generated ∼166,000 reads for the 8 SS mSG samples. Ig isotype usage was similar between paired samples; patients 1-3 presented with ∼60% μ- and ∼30% α-gene usage; and patient 4 contained a predominance of γ-gene usage (∼60%) and approximately equal usage of μ and α (∼20%). In total, we detected 1631 clonotypes (defined as reads with same IgHV and IgHJ gene usage and equal CDR3 length). Between 5 and 9 shared clonotypes were observed among paired SG biopsies, demonstrating B cells can recirculate between glands. Lineage tree analysis revealed 3 patterns of B cell circulation: a) unidirectional circulation of B cell clones from 1 biopsy to another; b) clones circulating between both glands; c) an undefined pattern with less-mutated and unidentified precursors migrating from one site to the other. Finally, mRNA expression of AICDA, the enzyme required for SHM, correlated with the number of SHMs present in the shared clonotypes studied, supporting the notion that functional EGCs support clonal selection in SS. Conclusions We show that B cells recirculate between mSG in SS and undergo further rounds of SHM in adjacent glands. The level of B cell recirculation appears to be related to the Ig isotype, as Gamma rich samples appeared to recirculate less often. Although further study is needed to confirm these observations, these findings demonstrate the dynamic nature of B cell affinity maturation in SS within ECGs. References Voulgarelis & Tzioufas. Nat Rev Rheumatol (2010). Bombardieri, et al. J Immunol (2007). Tiller, Busse & Wardemann. J Immunol Methods (2009). Brochet, Lefranc & Giudicelli. Nucleic Acids Res (2008). Rogosch, et al. Front. Immun (2012). Barak, et al. J Immunol Methods (2008). Disclosure of Interest None declared