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Summary Background Recent randomised controlled trials (RCTs) in primary Sjogren's syndrome used the European League Against Rheumatism (EULAR) Sjogren's Syndrome Disease Activity Index (ESSDAI) as their primary endpoint. Given the heterogeneous and complex nature of primary Sjogren's syndrome, it might be more appropriate to also assess other clinically relevant disease features. We aimed to develop a novel composite endpoint for assessing treatment efficacy in patients with primary Sjogren's syndrome: the Composite of Relevant Endpoints for Sjogren's Syndrome (CRESS). Methods A multidisciplinary expert team selected clinically relevant items and candidate measurements for inclusion in the composite score. For each measurement, cutoff points for response to treatment were chosen based on expert opinion, previously published data on minimal clinically important improvements, and trial data, primarily the week-24 data of the single-centre ASAP-III trial of abatacept versus placebo. CRESS was validated using data from three independent RCTs: one trial of rituximab (TRACTISS), one of abatacept (multinational trial), and one of tocilizumab (ETAP). We calculated the number and percentage of patients who were responders in the separate CRESS items, and the percentage of responders based on the total CRESS at the primary endpoint visits (week 48 for TRACTISS, week 24 for the other two trials). Patients with fewer than three items available for evaluating CRESS response were imputed as non-responders. Findings Based on expert opinion, five complementary items were selected to assess response: (1) systemic disease activity by Clinical ESSDAI (less than 5 points); (2) patient-reported symptoms by EULAR Sjogren's Syndrome Patient Reported Index, assessed by a decrease of at least 1 point or at least 15% from baseline; (3) tear gland item by Schirmer's test and ocular staining score, assessed by an increase of at least 5 mm or decrease of at least 2 points, respectively, in patients with abnormal Schirmer's test or ocular staining score findings at baseline, or, in patients with normal baseline values, assessed by no change to abnormal for both; (4) salivary gland item, assessed by unstimulated whole saliva secretion (increase of at least 25%) and salivary gland ultrasonography (decrease of at least 25%); and (5) serology, assessed by rheumatoid factor (decrease of at least 25%) and IgG (decrease of at least 10%). Total CRESS response is defined as response on at least three of five items. Post-hoc assessment of phase 3 trial data showed that CRESS response rates at the primary endpoint visits were 60% (24 of 40) for abatacept versus 18% (seven of 39) for placebo (p Interpretation The CRESS is a feasible, well-balanced, composite endpoint for use in trials of primary Sjogren's syndrome. As a next step, the CRESS will require validation in a prospective RCT. Funding None. Translation For the Dutch translation of the abstract see Supplementary Materials section.

Primary Sjögren’s syndrome (pSS) is often considered a B cell-mediated disease, yet the precise role of B cells in the pathogenesis is not fully understood. This is exemplified by the failure of multiple clinical trials directed at B cell depletion or inhibition. To date, most prognostic markers for severe disease outcomes are autoantibodies, but the underlying mechanisms by which B cells drive diverse disease presentations in pSS likely extend beyond autoantibody production. Here we outline an expanded role of B cells in disease pathogenesis drawing on examples from animal models of SS, and from other autoimmune diseases that share similar clinical or immunological abnormalities. We focus on recent findings from the detailed analysis of pathogenic B cells in patients with pSS to propose strategies for patient stratification to improve clinical trial outcomes. We conclude that an integrated cellular, molecular and genetic analysis of patients with pSS will reveal the underlying pathogenic mechanisms and guide precision medicine.

Primary Sjögren’s syndrome (pSS) is often considered a B cell-mediated disease, yet the precise role of B cells in the pathogenesis is not fully understood. This is exemplified by the failure of multiple clinical trials directed at B cell depletion or inhibition. To date, most prognostic markers for severe disease outcomes are autoantibodies, but the underlying mechanisms by which B cells drive diverse disease presentations in pSS likely extend beyond autoantibody production. Here we outline an expanded role of B cells in disease pathogenesis drawing on examples from animal models of SS, and from other autoimmune diseases that share similar clinical or immunological abnormalities. We focus on recent findings from the detailed analysis of pathogenic B cells in patients with pSS to propose strategies for patient stratification to improve clinical trial outcomes. We conclude that an integrated cellular, molecular and genetic analysis of patients with pSS will reveal the underlying pathogenic mechanisms and guide precision medicine.

OBJECTIVE: To conduct a systematic review of studies exploring potential biomarkers for development, course and efficacy of treatment of lymphomas in salivary glands of patients with Sjögren's syndrome.MATERIAL AND METHODS: Eligible studies were identified through a comprehensive search of two databases, i.e. PubMed and EMBASE. Quality of included articles was assessed with the 'Quality In Prognosis Studies' (QUIPS) tool. The 'CHecklist for critical Appraisal and data extraction for systematic Reviews of prediction Modelling Studies' (CHARMS) was used to facilitate data extraction.RESULTS: Fifty-eight studies met the inclusion criteria. Only one study assessed the progression of lymphoma. Moderate risk of bias was detected in 'outcome measurement', 'study participation' and 'study confounding' domains. Parotid gland enlargement, mixed monoclonal cryoglobulins and low C4 levels represented strongest predictors of lymphoma development. The role of histological biomarkers, and specifically germinal centers, remains controversial. Clinical and methodological heterogeneity across studies precluded conduct of a meta-analysis.CONCLUSIONS: Specific biomarkers in combination with clinical manifestations represent potential candidates for advancing precision medicine approaches to lymphoma prediction in patients with Sjögren's syndrome. Current focus has increasingly been on genetic and epigenetic markers as candidate predictors. Predictive accuracy of key biomarker candidates remains to be tested in well-designed prospectively-followed Sjögren's syndrome cohorts. This article is protected by copyright. All rights reserved.

A major complication of primary Sjögren's syndrome (pSS) is development of mucosa associated lymphoid tissue (MALT) B-cell lymphoma, particularly in salivary glands. These lymphomas express FcRL4 and are characteristically associated with lymphoepithelial lesions. Neoplastic B-cells may be derived from non-neoplastic glandular intraductal B-cells, also virtually all expressing FcRL4. A characteristic feature of MALT lymphomas is the production of rheumatoid factors (RFs), which are largely encoded by stereotypic immunoglobulin variable heavy chain (IGHV) sequences. The aim of this study was to examine whether there is a relationship between the intraductal and periductal B-cells and whether the intraductal B-cells are selected for RF. RNA was extracted from laser-microdissected infiltrated ductal areas and periductal infiltrates from frozen parotid gland tissue sections of 5 pSS patients. PCR amplified IGHV transcripts were cloned into pCR™4-TOPO vector and subsequently sequenced. Microdissected ducts yielded 96 unique IGHV sequences derived from intraductal B-cells, while 119 unique IGHV sequences were obtained from periductal infiltrates. No major difference in VH-gene usage was observed between intraductal and periductal B-cells. Nearly all (>90%) IGHV sequences derived from both intraductal and periductal B-cells were mutated. Clonal expansions as defined by shared VDJ rearrangements were also present among both intraductal and periductal B-cells: in total 32 clones were found, from which 12 were located within ducts, 15 in periductal areas, and five clones shared members in both areas. We observed 12 IGHV rearrangements encoding for RF sequences from which two were derived from intraductal B-cells and 10 from periductal B-cells. Nine RF sequences were part of a clone. Together these findings indicate that intraductal and periductal B-cells are closely related to each other. Intraductal B-cells are most likely derived from periductal B-cells. We did not obtain evidence that RF-specific B-cells are enriched within the striated ducts. We speculate that in principle any activated B-cell can enter the striated ducts from the periductal infiltrate, irrespective of its antigenic specificity. Within the ducts, these B-cells may receive additional activation and proliferation signals, to further expand at these sites and by acquisition of driver-mutations develop toward lymphoma.

Standardisation of immuno-assays for autoantibodies is a major challenge. Although multiple organisations participate in the generation of internationally accepted standards, adequate standardisation of assays has not yet been achieved. Harmonisation may offer an alternative approach to better align requesting, testing, reporting and interpretation of autoimmune diagnostics. The European Autoimmunity Standardisation Initiative (EASI) was founded to facilitate both standardisation as well as harmonisation of autoantibody tests, but over the years the focus has drifted away from standardisation in favour of harmonisation. In the current paper the options for harmonisation are highlighted.

Standardisation of immuno-assays for autoantibodies is a major challenge. Although multiple organisations participate in the generation of internationally accepted standards, adequate standardisation of assays has not yet been achieved. Harmonisation may offer an alternative approach to better align requesting, testing, reporting and interpretation of autoimmune diagnostics. The European Autoimmunity Standardisation Initiative (EASI) was founded to facilitate both standardisation as well as harmonisation of autoantibody tests, but over the years the focus has drifted away from standardisation in favour of harmonisation. In the current paper the options for harmonisation are highlighted.

Objectives. Serum immunoglobulin free light chains (FLCs) are frequently elevated in B-cell-mediated autoimmune diseases, including primary SS (pSS). The objective of this study was to assess if serum FLCs can contribute to classification, mucosa-associated lymphoid tissue (MALT) lymphoma detection, monitoring of disease activity and treatment response in pSS.Methods. Serum samples of 100 consecutive patients suspected of pSS were included. Forty-five patients fulfilled ACR-EULAR criteria for pSS. Additionally, samples of 17 pSS patients with MALT lymphoma and longitudinal samples of pSS patients treated with rituximab (n = 20), placebo (n = 10) or abatacept (n = 15) were included. Serum FLC kappa/FLC lambda. was measured by nephelometry or turbidimetry.Results. At diagnosis, FLC kappa and FLC lambda serum levels were significantly higher in pSS compared with non-SS sicca patients. The FLC kappa/FLC lambda ratio was abnormal in 11% of pSS patients. In established MALT-pSS patients, without recent rituximab treatment (n = 12), 50% had abnormal FLC kappa/FLC lambda ratios. FLC measurement had no additional value for pSS classification, compared with IgG and anti-SSA. FLC levels correlated significantly with systemic disease activity, assessed by EULAR SS Disease Activity Index (ESSDAI) and clinical ESSDAI, both cross-sectionally and longitudinally following treatment. Treatment with rituximab or abatacept significantly lowered FLC levels. FLCs show a large sensitivity to change and relative changes induced by treatment were higher compared with IgG.Conclusion. Serum FLCs are elevated in pSS, and abnormal FLC kappa/FLC lambda. ratios may be indicative for the presence of MALT lymhoma. FLC levels can be used as a biomarker for systemic disease activity and monitoring treatment responses. FLCs are sensitive to change and have more favorable kinetics than IgG.

Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1 × 10(-3) to 1.7 × 10(-4 )mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins.

A population of B cells with characteristics of chronic lymphocytic leukaemia was found in the peripheral blood of four patients who presented with cutaneous infiltration of atypical CD4+ T cells with cerebriform nuclei. The B cells had a low density of immunoglobulin on their surface membrane, expressed CD5-positivity, and showed monoclonality based on the restriction to either kappa or lambda light chains. In one patient with tumourous pleiomorphic CD4+CD30- T-cell lymphoma of the skin, it was the first manifestation of a concomitant B-cell non-Hodgkin's lymphoma of low-grade malignancy. In three other patients with reactive atypical T-cell erythroderma, there was no evidence for the coexistence of a B-cell malignancy. The number of CD5+ B cells decreased in two erythroderma patients with clinical remission of the cutaneous lesions. It is speculated that the presence of a monoclonal B cell population in patients with T-cell disorders of the skin is due either to a reactive process possibly conferring some protective effect, or a response to an unknown stimulus produced by the T cells.

A population of B cells with characteristics of chronic lymphocytic leukaemia was found in the peripheral blood of four patients who presented with cutaneous infiltration of atypical CD4+ T cells with cerebriform nuclei. The B cells had a low density of immunoglobulin on their surface membrane, expressed CD5-positivity, and showed monoclonality based on the restriction to either kappa or lambda light chains. In one patient with tumourous pleiomorphic CD4+CD30- T-cell lymphoma of the skin. it was the first manifestation of a concomitant B-cell non-Hodgkin's lymphoma of low-grade malignancy. In three other patients with reactive atypical T-cell erythroderma. there was no evidence for the coexistence of a B-cell malignancy. The number of CD5+ B cells decreased in two erythroderma patients with clinical remission of the cutaneous lesions. It is speculated that the presence of a monoclonal B cell population in patients with T-cell disorders of the skin is due either to a reactive process possibly conferring some protective effect. or a response to an unknown stimulus produced by the T cells.

The distribution of IgE FcR (Fc-epsilon-R)-positive and -negative B cells was examined in normal adult mice. Using three-color flow cytometry, the expression of the Fc-epsilon-R was analyzed on various B-cell subsets present in the peritoneum and spleen. The results demonstrate that in the peritoneal cavity, the Fc-epsilon-R is not expressed on the large majority of Ly 1+ B cells and Ly 1-, Mac 1+ sister B cells. The receptor is present, however, on the small number of conventional B cells residing in the peritoneum. Although interleukin 4 (IL-4) can increase the levels of the Fc-epsilon-R on conventional B cells, incubation of Ly 1 and sister B cells with IL-4 did not result in the expression of the Fc-epsilon-R. When examining B cells present in the spleen, a small subset of B cells was consistently found to be Fc-epsilon-R-. These Fc-epsilon-R- cells were IgM-bright, IgD-dull and largely Ly 1- and Mac 1-negative. Staining of splenic tissue sections revealed that the Fc-epsilon-R- B cells were primarily localized to the marginal zones, whereas the Fc-epsilon-R+ B cells were found in the follicles. Taken together, the results indicate that the Fc-epsilon-R may be a useful marker in delineating the various B-cell subsets. In the peritoneum, the Fc-epsilon-R appears to discriminate conventional B cells from those of the Ly 1/sister lineage, and in the spleen it is likely to distinguish resting follicular B cells from Ly 1/sister and marginal zone B cells.

The distribution of IgE FcR (Fc-epsilon-R)-positive and -negative B cells was examined in normal adult mice. Using three-color flow cytometry, the expression of the Fc-epsilon-R was analyzed on various B-cell subsets present in the peritoneum and spleen. The results demonstrate that in the peritoneal cavity, the Fc-epsilon-R is not expressed on the large majority of Ly 1+ B cells and Ly 1-, Mac 1+ sister B cells. The receptor is present, however, on the small number of conventional B cells residing in the peritoneum. Although interleukin 4 (IL-4) can increase the levels of the Fc-epsilon-R on conventional B cells, incubation of Ly 1 and sister B cells with IL-4 did not result in the expression of the Fc-epsilon-R. When examining B cells present in the spleen, a small subset of B cells was consistently found to be Fc-epsilon-R-. These Fc-epsilon-R- cells were IgM-bright, IgD-dull and largely Ly 1- and Mac 1-negative. Staining of splenic tissue sections revealed that the Fc-epsilon-R- B cells were primarily localized to the marginal zones, whereas the Fc-epsilon-R+ B cells were found in the follicles. Taken together, the results indicate that the Fc-epsilon-R may be a useful marker in delineating the various B-cell subsets. In the peritoneum, the Fc-epsilon-R appears to discriminate conventional B cells from those of the Ly 1/sister lineage, and in the spleen it is likely to distinguish resting follicular B cells from Ly 1/sister and marginal zone B cells.