Martin Cornillet

Autoantibodies to citrullinated fibrinogen compared with anti-MCV and anti-CCP2 antibodies in diagnosing rheumatoid arthritis at an early stage: data from the French ESPOIR cohort

Objectives To compare the performance of anticitrullinated peptides/protein antibodies (ACPA) detected by three immunoassays in the French ESPOIR cohort of patients with early rheumatoid arthritis (RA) and undifferentiated arthritis (UA) and to study the relationship between ACPA and disease activity. Methods A diagnosis of RA (1987 American College of Rheumatology (ACR) criteria) was established at baseline in 497 patients and after a 2-year follow-up in 592 patients. At baseline, antibodies to citrullinated fibrinogen (AhFibA), antimutated citrullinated vimentin (anti-MCV) and anticyclic citrullinated peptide (anti-CCP2) were assayed and the individual and combined diagnostic sensitivities and predictive values of the tests were determined. Relationships between ACPA positivity and the 28-joint disease activity score and Health Assessment Questionnaire scores were analysed. Results At a diagnostic specificity of at least 98%, the three tests exhibited similar diagnostic sensitivities (47–48.5%). When considering as positive patients with at least one positive test, the sensitivity increased to 53.5% with a probable loss of specificity. Among the patients classified as having UA at baseline, 30% were positive for one ACPA, the positive predictive values for RA of the three tests ranging from 73% to 80% but increasing when two tests were associated. Whatever the test used, the addition of ACPA positivity to the 1987 criteria enhanced their sensitivity by 6%, close to that of the 2010 ACR/European League Against Rheumatism (EULAR) criteria. Conclusions In early arthritis, AhFibA, anti-MCV and anti-CCP2 showed similar diagnostic sensitivity with a high diagnostic specificity and a similar high positive predictive value for RA. Adding ACPA to the 1987 ACR criteria significantly increased the number of patients classified as having RA, confirming the validity of the recent inclusion of the serological criterion in the ACR/EULAR criteria.

In Black Africans with rheumatoid arthritis, ACPA recognize citrullinated fibrinogen and the derived peptides α36-50Cit38,42 and β60-74Cit60,72,74, like in Caucasians

Well documented in Caucasians and Asians, the diagnostic value of anti-CCP2 antibodies has been confirmed in Black African populations. However, autoantibodies to other citrullinated peptides/proteins and their fine specificities have not yet been studied. Here, we show that in Cameroonian patients, anti-citrullinated fibrinogen autoantibodies (AhFibA) are sensitive (73%) diagnostic markers for RA. We also determine that autoantibodies directed to α36-50Cit38,42 or β60-74Cit60,72,74 peptides which bear the immunodominant epitopes of citrullinated fibrin, are present in similar proportions in Black Africans and Caucasians with 25/56 (45%) and 41/56 (73%) positive RA-sera in Cameroonians, respectively. They also account for almost all the AhFibA reactivities since 38/41 (93%) AhFibA-positive sera contain anti-α36-50Cit38,42 and/or anti-β60-74Cit60,72,74 autoantibodies. Finally, HLA-DRB1 SE alleles were associated with higher titres of AhFibA and anti-β60-74Cit60,72,74 autoantibodies. In the genetic and environmental backgrounds of Black Africans, AhFibA are a hallmark of RA like in Caucasians, moreover they recognize the same fibrin epitopes.

Anticitrullinated protein/peptide antibody multiplexing defines an extended group of ACPA-positive rheumatoid arthritis patients with distinct genetic and environmental determinants

Introduction The second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients. Methods We investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array. Results The prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset. Conclusions Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.

1.60 Autoantibodies to human citrullinated fibrinogen (AhFibA) and their subfamilies directed to the fibrin peptides α36-50Cit38,42 and β60-7460,72,74 are prognostic markers of radiographic damage in the very early arthritides of the French ESPOIR cohort

Background and Objective In established RA, autoantibodies (AAB) to human citrullinated fibrinogen (AhFibA) were demonstrated to be mainly composed of two subfamilies of AAB directed to immunodominant epitopes borne by the fibrin peptides α36-50Cit38,42 and β60-74Cit60,72,74,respectively. Serum reactivity toward those peptides defines subgroups of patients. In the present study, we investigated whether AhFibA, anti-α36-50Cit38,42 and β60-74Cit60,72,74 AAB, have different predictive values for disease outcome in very early RA. We also analysed whether these AAB differentially associate with SE and tobacco exposure. Materials and Methods The French ESPOIR cohort is comprised of very early RA and of undifferentiated arthritides (UA) of less than 6-month duration. AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB were assayed by ELISA at baseline. After 3-year follow up, 701 patients were diagnosed RA according to the ACR/EULAR 2010 criteria. Relationships between SE HLA-DR alleles, tobacco exposure and the 3 AAB were analysed on those patients. Disease activity (DAS28, HAQ) and radiographic damage (SHS) were evaluated at baseline, and after 2- or 3-year follow up. Associations with clinical parameters and predictive value of each AAB were investigated. Results AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB were detected in 349/701 (50%), 203/701 (29%), and 257/701 (37%), RA sera, respectively. Their positive predictive values for RA (72%, 82%, and 79%, respectively) were not significantly different. The presence and titre of each AAB were associated with SE HLA-DR alleles without significant additional effect of tobacco exposure. When 2 vs 0 copies of SE alleles were present, the odds ratios for AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB presence reached 8.0, 6.1 and 9.5, respectively. Neither the presence nor the titres of AhFibA, α36-50Cit38,42 and β60-74Cit60,72,74 AAB were associated with DAS28 or HAQ at baseline and after 2 years. However, for the 3 AAB, patients whose sera contained one or several AAB had a progression of SHS during the first 3 years (medians from 6 to 8 depending on the subgroup), significantly higher than the AhFibA-negative patients (median = 4). Nevertheless, no significant correlation was observed between the titres of AAB and radiographic progression. Conclusion Not only AhFibA but also their anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB subfamilies, are prognostic markers for bone erosion in RA. Moreover, AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB are similarly associated to HLA-DR and to tobacco exposure.

Immunomodulation of RA Patients’ PBMC with a Multiepitope Peptide Derived from Citrullinated Autoantigens

Citrullinated peptides are used for measuring anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA). Accumulation of citrullinated proteins in the inflamed synovium suggests that they may be good targets for inducing peripheral tolerance. In view of the multiplicity of citrullinated autoantigens described as ACPA targets, we generated a multiepitope citrullinated peptide (Cit-ME) from the sequences of major citrullinated autoantigens: filaggrin, β-fibrinogen, vimentin, and collagen type II. We assessed the ability of Cit-ME or the citrullinated β60-74 fibrinogen peptide (β60-74-Fib-Cit) which bears immunodominant citrullinated epitopes (i) to modify cytokine gene expression and (ii) to modulate Treg and Th17 subsets in PBMC derived from newly diagnosed untreated RA patients. RA patients PBMC incubated with Cit-ME or β60-74-Fib-Cit, showed upregulation of TGF-β expression (16% and 8%, resp.), and increased CD4+Foxp3+ Treg (22% and 19%, resp.). Both peptides were shown to downregulate the TNF-α and IL-1β expression; in addition, Cit-ME reduced CD3+IL17+ T cells. We showed that citrullinated peptides can modulate the expression of anti- and proinflammatory cytokines in PBMC from RA patients as well as the proportions of Treg and Th17 cells. These results indicate that citrullinated peptides could be active in vivo and therefore might be used as immunoregulatory agents in RA patients.

SAT0065 Acpa against different citrullinated peptides identify specific phenotypes of rheumatoid arthritis

Background Anti-citrullinated protein/peptide antibodies (ACPA) have been suggested to identify a more severe phenotype of rheumatoid arthritis (RA). Objectives In this study in an inception cohort of early RA we have analysed a number of antibodies against different citrullinated and/or mutated peptides using a multiplex platform in relation to the patients disease inflammation and radiological destruction Methods Patients with early RA (≤12 m of symptoms) fulfilling the 1987 ARA criteria (n=1022, 692f/330m, mean age56.7±14.0 years) were sampled at the time of diagnosis and assessed using disease activity score (DAS28) at baseline, 6, 12, 18 and 24 months. Radiographs were graded using Larsen score (baseline and at 24 m). Plasma sampled at baseline was analysed for presence of antibody reactivities against 21 different citrullinated peptides/proteins; Fibrinogen (Fib) α36–50, Fibα573, Fibα591, Fibα621–635, Fibβ36–52, Fibβ60–74, Fibβ62–78 (72), Fibβ62–78 (74), Filaggrin (Fil307–324), α-Enolase peptide 5–21 (CEP-1), Vimentin (Vim) 2–17, Vim60–75, F4-R-Cit, F4-Cit-Cit, F4- Cit-R), or mutated proteins (Bla26, Pept1, Pept5, PeptZ1, PeptZ2) and type II Collagen citrullinated or not using a custom-made microarray assay based on the ImmunoCAP ISAC system (Phadia AB, Sweden). Cut-off levels were at the 98th percentile of controls (n=477). Anti-CCP2 was analysed using ELISA (Euro Diagnostica, Sweden). Results The most frequent appearing ACPA were; Fibβ60–74 (63%), Vim60–75 (56.6%), Fibβ36–52 (55.1%), Fil307–324 (54.9%), CEP-1 (53.7%) and Pept5 (52.0%) besides CCP2 (67.5%). Adding all ACPAs gave additional 13.1% of positivity in the anti-CCP2 negative group, yielding a positivity of 77.5%. The median (IQR) number of positive ACPA-peptide was 8 (11). There was a high degree of correlation between the antibodies, e.g., anti-Fibβ60–74 vs. -Vim60–75, -Fibβ36–52 or anti-CCP2 antibodies and also anti-Fil307–324 vs. -Fibβ36–52, -F4 R-Cit or anti-CCP2 antibodies (rs 0.692–0.79). Positivity for all antibodies was associated with higher ESR (baseline and AUC24). A number of antibodies were associated with both high DAS28 (baseline and AUC24) and radiological findings/progression (anti-CCP2, - Fil307–324, -Vim60–75 and Vim 2–17, and -CEP1 antibodies), whilst some others were more associated with inflammation (DAS28, baseline and AUC24) (anti-Fibβ60–74, -Pept5 and -F4R-cit antibodies) and others more with radiological destruction/progression (anti-Fibβ36–52, -Fibβ74, -PeptZ1, -F4 Cit-R antibodies). Partial least squares regression analyses confirmed the results with significant correlation between radiological progression and antibodies against Vim2–17, Fibβ36–52, CEP1, Fibα621–635, and CCP2 and between DAS28AUC24 and Vim60–75, Vim2–17, Fibα621–635 and F4-R-Cit. Patients treated with biologics during the first 24 months (11.2%) were significantly more frequent positive for anti-CCP2, -Vim60-75, -Fibα36–50, -PeptZ1 and -PeptZ2 antibodies vs. being negative. Conclusions Analyses at baseline, of the ACPA specificity profiles allowed different patterns of disease activity and radiological progression during the first 24 months of the disease to be identified. Disclosure of Interest None declared

A6.6 In RA patients, antibodies to the citrullinated peptide EBNA35-58Cit from epstein-barr nuclear antigen-1 crossreact with the citrullinated fibrin peptide β60-74Cit60,72,74

Background Among the Rheumatoid Arthritis (RA)-specific antibodies to citrullinated proteins/peptides (ACPA), the autoantibodies to citrullinated fibrinogen (AhFibA) target 2 immunodominant epitopes borne by the peptides α36-50Cit38,42 and β60-74Cit 60,72,74. RA sera also contain antibodies to the citrullinated peptide EBNA35-58Cit derived from EBNA-1, a protein of Epstein Barr Virus (EBV). Objectives To analyse the diagnostic value of anti-EBNA35-58Cit antibodies and their relationships with AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 autoantibodies. Methods A large representative series of 181 established RA and 436 non-RA rheumatic diseases was tested by ELISA for AhFibA, anti-CCP2, anti-α36-50Cit38,42, anti-β60-74Cit60,72,74 and anti-EBNA35-58Cit antibodies. Diagnostic indexes, correlations and concordances between tests were analysed. In addition, cross-reactivity of anti-EBNA35-58Cit toward β60-74Cit60,72,74 and α36-50Cit38,42 was evaluated in competition assays. Results At a diagnostic specificity of 98.5%, the diagnostic sensitivity of anti-EBNA35-58Cit antibodies was 36%, similar to that of anti-α36-50Cit38,42 (41%) but significantly lower than that of anti-β60-74Cit60,72,74 (62%) autoantibodies. Moreover, almost all sera containing anti-EBNA35-58 antibodies were reactive to citrullinated fibrinogen, 91% to β60-74Cit60,72,74, and 43% to α36-50Cit38,42. Anti-EBNA35-58 antibodies titres significantly correlated with those of anti-β60-74Cit60,72,74 (r = 0.482), AhFibA (r = 0.504) and anti-CCP2 (r = 0.479) but not with those of anti-α36-50Cit38,42 (r = 0.051). Competition assays revealed that anti-EBNA35-58Cit antibodies are highly crossreactive with anti-β60-74Cit60,72,74 but not with anti-α36-50Cit38,42 autoantibodies. Conclusions Anti-EBNA35-58Cit antibodies are present in 36% of AhFibA-positive patients with established RA. They are highly crossreactive with the autoantibodies to the immunodominant fibrin peptide β60-74Cit60,72,74. That suggests that the two antibodies have the same fine specificity and therefore correspond to a same subpopulation of ACPA.

Diagnostic performance of three assays for anticitrullinated protein antibodies in the very early arthritis ‘ESPOIR’ cohort

Background and objectives Anticitrullinated protein antibodies (ACPA) are recognised as the most specific markers for rheumatoid arthritis (RA). Their detection can be performed with various ELISAs using either the autoantigenic targets (human mutated vimentin: MCV and human fibrinogen: AhFibA) or cyclic peptides (CCP2). We investigated the diagnostic value of these ELISAs in a French cohort of undifferentiated arthritis of less than 6-month duration (ESPOIR cohort). Material and methods 685 patients were followed until 2 years after inclusion. Antibodies were determined at baseline. The cut-off of each assay was previously determined in order to obtain a similar 98% diagnostic specificity. Diagnostic sensitivity was determined for patients classified as RA at 2 years, according to the 1987 American College of Rheumatology (ACR) criteria. Results 592 (86.4 %) patients were classified as RA at 2 years. 47–48.5 % of RA patients were positive for one single test at baseline without any significant differences between the 3 assays, and 317 (53.5 %) were positive for at least one. Discrepant results were observed for eight patients positive only for anti-CCP2, 17 for anti-MCV antibodies, and 16 for AhFibA. 95 patients classified as RA at 2 years were not classified as RA at baseline: 30 of them (31.5%) were positive for at least one assay at baseline. If the positivity of at least one assay is included in the diagnostic criteria, 543 patients are classified as RA (79.3%) at inclusion, instead of 497 (72.5%) without it. Diagnostic sensitivities of ACPA assays Sensitivity (%) Anti-CCP 47 Anti-MCV 47.3 AhFibA 48.5 Anti-CCP or anti-MCV 50.8 Anti-CCP or AhFibA 50.7 Anti-MCV or AhFibA 52.2 At least 1 ACPA assay 53.5 Conclusions The performance of the three ACPA assays is similar but the tests do not totally overlap. In very early undifferentiated arthritis, the detection of the three ACPA subfamilies increased the sensitivity of 6% as compared with the assays of only 1 marker with the risk of a slight decrease in specificity. Moreover, the positivity of one ACPA assay 2 years before the diagnosis was predictive for RA in 30% of the 95 patients without a diagnosis at inclusion. When ACPA were included among the diagnostic criteria, 7% more patients were classified as RA at inclusion. These results are in perfect agreement with the new ACR/EULAR classification criteria for RA which now include ACPA.